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Cold Exposure & Other Mild Stressors for Increased Health & Longevity

Cold Exposure Exercise Fasting UCPs UCP1 UCP3 FGF21

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#121 Michael R

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Posted 11 March 2016 - 07:24 PM

Hi Dean,

Yeah, certainly cogent points — but in the end, not a chain of reasoning on which I think much stock can be laid. I've followed this thread with substantial interest, and haven't wanted to weigh in too firmly because to do so would require some weed botany for which I regret to say I haven't been able to find the time. But let me (finally ...) explain my reasons for being very skeptical of two of the main kinds of data on which the argument is based. I'm going to take this slightly out of your original order for clarity:
 

...we have extensive evidence that CR in rodents retards aging and extends maximum lifespan


shouldn't you really say "we have extensive evidence that CR in rodents retards aging and extends maximum lifespan when rodents are exposed to cold conditions"?

... PMID 9032756 yoked the weights of two groups of CR mice together so as to be identical, and kept one at thermal-neutrality (86°F) and the other at a "normal" (i.e. chilly-for-rodent) temperature of 72°F. It was only the chilly CR mice that lived longer than controls. In fact, despite eating fewer calories than the chilly CR mice (and a heck of a lot less than AL-fed controls), the thermally-neutral CR mice didn't live any longer on average than AL-fed controls. This would seem to suggest cold exposure is a critical component of the "CR magic", despite requiring animals to eat more calories.


I broadly speaking agree with what you say here, except that I think you have probably been led to greatly exaggerate in your mind as to just how "chilly" the mice kept at normal room temperature were. Your argument rests on the argument in (1) below (and op cit in this thread) that mice at room temperature (20-22°C) are really substantially chilly critters, whereas humans in clothing at room temperature are close to thermoneutrality, and therefore (you very reasonably deduce) more like the 30°C warm-housed animals in PMID 9032756 which got drastically-reduced anticancer benefits and no increase in median lifespan (tho' you neglect to highlight, as you did in an earlier post, the fact that while the first half of the survival curve of the warm-housed CR animals is almost isomorphic with that of the controls, there remains a substantial albeit reduced effect on max LS, evident in a quite sudden and dramatic break point in the survival curve at around 850 days. This is a critical distinction: the implication is a blunting of some of the specific health benefits of CR in the warm-housed mice (in this case, evidently, a blunting of protection against tumorigenesis), while still preserving much of the effect on aging per se).

However, the core premise behind this line of argument does not seem to be on strong footing, and indeed seems (to my admittedly somewhat superficial (for lack of time) reading) to be mistaken.

(2) takes on the argument in (1) directly:
 

It has been argued that mice should be housed at 30 °C to best mimic the thermal conditions experienced by humans, and that the current practice of housing mice at 20–22 °C impairs the suitability of mice as a model for human physiology and disease. In the current paper we challenge this notion. ...

The thermoneutral zone is the region where basal energy expenditure generates more than enough heat to balance heat losses due to the difference between ambient (Ta) and body temperatures (Tb). At the lower margin of this region (called the lower critical temperature or Tlc) heat requirements to maintain Tb and basal metabolic rate are exactly matched. At higher temperatures basal metabolic rate provides too much heat [MR emphasis] and this needs to be dissipated. Generally this is achieved by elevating evaporative water loss. At some upper critical temperature (Tuc) however other mechanisms need to be recruited and these paradoxically require an elevation of metabolism. Below Tlc metabolic rates must be higher than basal levels to balance heat loss, hence metabolic rate increases linearly as temperature declines and the gradient of this relationship reflects the degree of external insulation. ...

The thermoregulatory curve for naked humans has a lower critical temperature averaging about 28 °C ... Light clothing such as would typically be worn indoors consisting of a long sleeved shirt or blouse (Clo=0.2) and light trousers (Clo=0.25) provide together 0.45 Clo units of insulation [54]. The effect of such clothing would be to lower the lower critical temperature by 5.1 °C ... [to] a lower critical temperature of 22.9 °C.

Humans, however, seldom operate at basal metabolic rate. Studies using doubly labelled water show that routinely our energy expenditure is about 1.6–1.8× basal requirements. If we lived at our lower critical temperature defined from basal metabolism, we would be under continuous mild heat stress, so we normally seek out cooler temperatures than thermoneutral, where our routine heat production is balanced by a thermal gradient that generates an equivalent heat loss. This is why buildings are regulated at 19–21 °C (about 3 °C below the lower critical temperature) rather than within the thermoneutral zone (which for a lightly clothed individual is 23–27 °C). ...

Mouse Tlc is dependent on body weight and about 26–28 °C for adult mice weighing >25 g. The equivalent temperature to that normally experienced by humans for most single housed adult mice is therefore 23–25°C. Group housing or providing the mice with bedding and nesting material might lower this to about 20–22 °C, close to current standard practice.


The mice in PMID 9032756 were singly-housed at 20–22°C; the paper does not discuss bedding arrangements, but the use of bedding (usually softwood shavings) in government and academic animal housing protocols is routine (though not dictated by animal welfare guidelines).

It seems clear, therefore, that the mice housed at 30°C in PMID 9032756 are really rather toasty, and those at room temperature are only very modestly cooler-housed than a human in normal room temperatures. This merits CR folk bearing a certain amount of temperature discomfort, but doesn't imply that the benefits are abolished if we aren't either exposed to the autumn elements naked or wearing ice vests. And again: even 30°C housing didn't abolish the benefits of CR, and very few humans with modestly healthy lifestyles and good access to medical care die before the population median life expectancy.
 
If you wish to do a deep dive into this particular subject, and the arguments in (1), you will have a fine opportunity to discuss them at the Ninth Conference of the Calorie Restriction Society International, which I am pleased to know that you are attending: the renowned Dr. John Speakman, author of numerous provocative studies on CR and related subjects and the lead author of (1), will be one of our many exciting scientific presenters.
 

On the other hand, despite [its] depleting effects on BAT, we have extensive evidence that CR in rodents retards aging and extends maximum lifespan


Regarding CR's "depleting effects on BAT" as reported in PMID 18593277. In the quote you include, I point to the discussion of that study in this post. If you look at that discussion, you'll see that Valle et al did indeed see lower amounts of BAT in CRed rodents. But they also found the BAT the CR rodents did retain was much more active i.e. had a much higher expression of UPC1 as a result of mitochondrial biogenesis. The authors conclude (my emphasis):

As happens in liver or skeletal muscle, CR was also shown to promote mitochondrial biogenesis in BAT. On the whole, this conservation of BAT thermogenic capacity may confer several advantages, such as a higher ability to respond to cold exposure or to control body weight when food supply is restored, and, therefore, be part of the rejuvenation mechanisms underlying the life-span extension induced by CR.


Yeah. The problem with this is twofold. The first is unwittingly revealed by the authors' statement that "CR was also shown to promote mitochondrial biogenesis in BAT ... As happens in liver or skeletal muscle." The problem is that this finding, which is widely cited, is actually quite uncertain: most of the studies reporting this rely entirely on increased expression of genes involved in mitochondrial biogenesis (nitric oxide synthesis, TFAM, and above all PGC-1α); and the studies that actually count mitochondrial number are inconsistent, with most (even when they find confirm the increased expression of biogenesis-involved genes) finding no effect.(3) A substantial increase in mitochondrial numbers, activity, oxygen consumption, and possibly BAT-driven thermogenesis also seems to be in contradiction with the very well-established finding of a lack of effect of long-term CR on specific metabolic rate — ie, the amount of metabolic activity per unit metabolically-active mass.

So while I have no specific criticisms to level against the methods used or laboratory competence of Valle et al, I retain a substantial skepticism that mitochondrial biogenesis is actually occurring in these mice's BAT (or muscle or liver, which they don't of course report).
 

Regarding your statement that there is no interventional evidence for BAT's benefits in rodents not attributable to obesity-avoidance:

Obviously PMID 9032756 just discussed flat-out contradicts your assertion, since CRed mice housed at what for them is an uncomfortably cool temperature ate 20% more, weighted the same, and lived 40% longer on average than equivalently-skinny CRed mice housed at a comfortably temperature.



It doesn't really, both for the reason given and because there was no measurement of BAT quantity or activity in the PMID 9032756 study, let alone a linkage made to its effect on LS. That is, even if we agree (as I certainly do, and have advocated for many years as you know) that this study provides substantial reason to remain "uncomfortably cool" (albeit not to the extreme that the chain of reasoning you've been following suggests), there is no specific link being made to its effects on BAT, which (for reasons given) are unclear in CR rodents, as is their causal relationship to the anti-aging effects of CR or the role of cool temperatures in realizing its full benefits. (I will emphasize again that even at 30°C, the key effect on maximum lifespan, and the late effect on the survival curve, remains substantially intact).

Additionally, I just can't mechanistically see how increasing BAT quantity or activity would exert an effect on aging per se. When Valle et al say,
 

On the whole, this conservation of BAT thermogenic capacity may confer several advantages, such as a higher ability to respond to cold exposure or to control body weight when food supply is restored, and, therefore, be part of the rejuvenation mechanisms underlying the life-span extension induced by CR.


... that appears to me to be a complete, bald-faced non sequitur. What in the world does "a higher ability to respond to cold exposure or to control body weight when food supply is restored" have to do with "rejuvenation [sic!] mechanisms underlying the life-span extension induced by CR" ??

It's not an overwhelming counterpoint, but several studies find that overexpression of uncoupling proteins has no substantial effect on mean LS, and none at all on maximum — notably (4):
 

In the present investigation we describe the life span characteristics and phenotypic traits of ad libitum-fed mice that overexpress UCP2/3 (Positive-TG), their non-overexpressing littermates (Negative-TG), mice that do not expression [sic] UCP2 (UCP2KO) or UCP3 (UCP3KO), and wild-type C57BL/6J mice (WT-Control). We also included a group of C57BL/6J mice calorie-restricted to 70% of ad libitum-fed mice in order to test partially the hypothesis that UCPs contribute to the life extension properties of CR.
 
Mean survival was slightly, but significantly, greater in Positive-TG, than that observed in Negative-TG or WT-Control; mean life span did not significantly differ from that of the UCP3KO mice. Maximal life span did not differ among the ad libitum-fed groups. Genotype did not significantly affect body weight, food intake, or the type of pathology at time of death.
 
Calorie restriction increased significantly mean and maximal life span, and the expression of UCP2 and UCP3. The lack of difference in maximal life spans among the Positive-TG, Negative-TG, and UCP3KO suggests that UCP3 does not significantly affect longevity in mice.


By contrast, I can certainly see (as you have highlighted elsewhere) plenty of mechanisms whereby reduced body temperature — when not counteracted by overly-warm ambient temperature — could be responsible for the impairment of CR's effects on tumorigenesis in PMID 9032756, particularly since by far the greatest effect is on lymphomas: cancer is a disease of cell proliferation, and restricted energy by any means impairs cell proliferation (hence the modest effect of exercise against some cancers). It is notable in this regard that CR humans have typical signs thereof, consistent with the rodent model: very low WBC (particularly relevant for an effect on lymphomas); slow wound healing; slow-growing, weak or soft nails, etc.

This could also affect some of the other proliferative lesions that CR is known to retard, and as you've also noted, the sheer slowing of chemical reactions could even somewhat contribute to the anti-aging effect per se.

And, again, there is the health benefit of obesity-avoidance from the increased energy expenditure imposed by cool temperature, irrespective of the tissue-specific mechanisms thereof.

References
1: Translating animal model research: does it matter that our rodents are cold?
Maloney SK, Fuller A, Mitchell D, Gordon C, Overton JM.
Physiology (Bethesda). 2014 Nov;29(6):413-20. doi: 10.1152/physiol.00029.2014. Review.
PMID: 25362635 Free Article
http://physiologyonl...t/29/6/413.long

2: Speakman JR, Keijer J. Not so hot: Optimal housing temperatures for mice to mimic the thermal environment of humans. Mol Metab. 2012 Nov 8;2(1):5-9. doi: 10.1016/j.molmet.2012.10.002. Review. PubMed PMID: 24024125; PubMed Central PMCID: PMC3757658.
http://dx.doi.org/10...met.2012.10.002

3: Gouspillou G, Hepple RT. Facts and controversies in our understanding of how caloric restriction impacts the mitochondrion. Exp Gerontol. 2013 Oct;48(10):1075-84. doi: 10.1016/j.exger.2013.03.004. Epub 2013 Mar 21. Review. PubMed PMID: 23523973.
https://www.research...a15be000000.pdf

4: McDonald RB, Walker KM, Warman DB, Griffey SM, Warden CH, Ramsey JJ, Horwitz BA. Characterization of survival and phenotype throughout the life span in UCP2/UCP3 genetically altered mice. Exp Gerontol. 2008 Dec;43(12):1061-8. doi: 10.1016/j.exger.2008.09.011. Epub 2008 Sep 27. PubMed PMID: 18854208.

#122 AlPater

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Posted 12 March 2016 - 04:40 PM

Thanks Michael,

 

i once got a Chinese fortune cookie, whose fortune was:

 

'THERE IS A TENDENCY TO CARRY ACTIVITIES TOO FAR'.

 

To me it seemed to me that the below paper has not yet been thrown into the fray and may contribute.

 

If it matters, mice were kept at 24°C.
 
In conclusion, UCP3 overexpression and/or its associated phenotypes appear to delay some aspects of the CR response. This finding of delayed adaptation in UCP3Tg mice raises the general question of how pharmacologic uncoupling would impact other adaptive responses. While uncoupling may be beneficial in some cases such as with the ability of muscle to adapt to a high fat diet, the present study suggests that the impact could be to delay adaptive changes to some challenges. In addition to the primary goal of our study, we also present strong evidence that short term CR, as with longer term CR, is not associated with an extensive remodelling of the mitochondria nor an increase in mitochondrial content in mouse skeletal muscle; thus, the remodelling reported to occur in other tissues does not reflect a fundamental response to CR in mice.
 
 
Exp Gerontol. 2012 May;47(5):361-71. doi: 10.1016/j.exger.2012.02.008. Epub 2012 Mar 3.
Calorie restriction in mice overexpressing UCP3: evidence that prior mitochondrial uncoupling alters response.
Estey C1, Seifert EL, Aguer C, Moffat C, Harper ME.
PMID: 22406134 [PubMed - indexed for MEDLINE] PMCID: PMC4203376 Free PMC Article
 
Abstract
 
Calorie restriction (CR) without malnutrition is the only intervention to consistently increase lifespan in all species tested, and lower age-related pathologies in mammals including humans. It has been suggested that uncoupling of mitochondrial oxidative phosphorylation, using chemical uncouplers, mimics CR, and that overlapping mechanisms underlie the phenotypic changes induced by uncoupling and CR. We aimed to critically assess this using a unique mouse model of skeletal muscle-targeted UCP3-induced uncoupling (UCP3Tg), and focused our studies mainly on skeletal muscle mitochondria. Compared to ad libitum fed Wt mice, skeletal muscle mitochondria from ad libitum fed UCP3Tg mice showed higher basal uncoupling and lower H(2)O(2) emission, with unchanged maximal oxidative phosphorylation, and mitochondrial content. UCP3Tg CR mice showed some tendency for differential adaptation to CR, with lowered H(+) leak conductance and evidence for higher H(2)O(2) emission from skeletal muscle mitochondria following 2 weeks CR, and failure to lower H(2)O(2) emission after 1 month CR. Differential adaptation was also apparent at the whole body level: while UCP3Tg CR mice lost as much weight as Wt CR mice, the proportion of muscle lost was higher in UCP3Tg mice. However, a striking outcome of our studies was the absence of change with CR in many of the parameters of mitochondrial function and content that we measured in mice of either genotype. Overall, our study raises the question of whether CR can consistently modify skeletal muscle mitochondria; alterations with CR may only be apparent under certain conditions such as during the 2 wk CR intervention in the UCP3Tg mice.


#123 Dean Pomerleau

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Posted 14 March 2016 - 03:04 PM

All,

 

I'm in the process of putting together a big post in response to Michael's thoughtful contribution above. But in the meantime, I thought this study [1] pointed to by Al on the CR email list can serve as a useful addition to the list of BAT stimulators. It found that in mice, a diet supplemented with gluten reduces UCP1 expression in BAT, and promoted obesity despite no increase in energy intake. The obesogenic effect of gluten was particularly strong when the diet was high in fat.

 

I'll once again observe how uncanny it is that diet & lifestyle practices that are considered health-promoting, almost invariably seem to also promote BAT quantity and/or activity.

 

Here is the latest full list of modifiable and [non-modifiable] factors associated with increased BAT quantity and/or activity:
  • Cold exposure - by far the best BAT inducer/activator
  • Methionine restriction - Reduce animal protein. Soy is low in methionine and high in arginine (see below).
  • Spicy / pungent foods, herbs & supplements - capsaicin / chilli peppers, curcumin / turmeric root, menthol/mint/camphor, oregano, cloves, mustard, horseradish/wasabi, garlic, onions
  • Arginine-rich foods - Good vegan sources include seeds (esp. sesame, sunflower & pumpkin), nuts (esp. almonds and walnuts) and legumes (esp. soy, lupin & fava beans and peas)
  • Other foods - green tea, roasted coffee, cacao beans / chocolate
  • Avoiding gluten
  • Drugs - metformin, caffeine
  • Exercise
  • Fasting
  • Low protein diet
  • Avoid obesity/overweight
  • [Being naturally thin - high metabolic rate]
  • [Being younger]
  • [Being female]
  • [Ethnicity - having cold-climate ancestors]

 

----------

[1] Wheat gluten intake increases weight gain and adiposity associated with

reduced thermogenesis and energy expenditure in an animal model of obesity.
 
Freire RH, Fernandes LR, Silva RB, Coelho BS, de Ara?jo LP, Ribeiro LS,
Andrade JM, Lima PM, Ara?jo RS, Santos SH, Coimbra CC, Cardoso VN,
Alvarez-Leite JI.
Int J Obes (Lond). 2015 Oct 7. doi: 10.1038/ijo.2015.204. [Epub ahead of
print]
PMID: 26443339
 
Abstract
 
BACKGROUND/OBJECTIVES:
 
The association between gluten and body weight is inconsistent. Previously,
we showed that a gluten-free diet reduces weight gain without changing food
intake in mice fed high-fat diets. In the present study, we investigated the
effects of gluten intake on fat metabolism, thermogenesis and energy
expenditure in mice fed a standard or high-fat diet.
 
METHODS:
 
Mice were fed four different experimental diets during 8 weeks: a
control-standard diet (CD), a CD added with 4.5% of wheat gluten (CD-G), a
high-fat diet (HFD) and a HFD added with 4.5% of wheat gluten (HFD-G). After
8 weeks, the mice received 99mTc-radiolabeled gluten orally to study gluten
absorption and biodistribution or they underwent indirect calorimetry. After
killing, subcutaneous and brown adipose tissues (SAT and BAT) were collected
to assess thermogenesis-related protein expression. Lipid metabolism was
studied in adipocyte cultures from the four groups.
 
RESULTS:
 
Despite having had the same energy intake, CD-G and HFD-G mice exhibited
increased body weight and fat deposits compared with their respective
controls. 99mTc-GLU or its peptides were detected in the blood, liver and
visceral adipose tissue, suggesting that gluten can even reach
extraintestinal organs. Uncoupling protein-1 expression was reduced in the
BAT of HFD-G and in the SAT of CD-G and HFD-G mice. Indirect calorimetry
showed lower oxygen volume consumption in CD-G and HFD-G groups compared
with their controls. In HFD mice, daily energy expenditure was reduced with
gluten intake. Gluten also reduced adiponectin, peroxisome
proliferator-activated receptor (PPAR)-Alpha and PPARGamma and
hormone-sensitive lipase in cultures of isolated adipocytes from HFD mice,
whereas in the CD-G group, gluten intake increased interleukin-6 expression
and tended to increase that of tumor necrosis factor.
 
CONCLUSIONS:
 
Wheat gluten promotes weight gain in animals on both HFD and CD, partly by
reducing the thermogenic capacity of adipose tissues.

There will never be peace in the world while there are animals in our bellies.

#124 Michael R

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Posted 14 March 2016 - 06:44 PM

 

Brown Adipose Tissue Exhibits a Glucose-Responsive Thermogenic Biorhythm in Humans

Paul Lee, Ron Bova, Lynne Schofield, Wendy Bryant, William Dieckmann, Anthony Slattery, Matt A. Govendir, Louise Emmett, Jerry R. Greenfield

Cell Metabolism. Publication stage: In Press Corrected Proof

[No PMID yet]

 

Highlights
  • Brown fat utilizes glucose as substrate fuel to produce heat in humans
  • Human brown fat exhibits a thermogenic circadian rhythm
  • Brown fat circadian rhythm is glucose responsive
  • Low brown fat abundance is associated with greater glycaemic fluctuations
Summary

High abundance of brown adipose tissue (BAT) is linked to lower glycaemia in humans [(Lee et al., 2010, Matsushita et al., 2014), stimulation of glucose uptake in hBAT by cold and insulin (Orava et al., 2011), and improvement of insulin sensitivity following hBAT recruitment (Chondronikola et al., 2014, Lee et al., 2014b)], leading to the belief that BAT may protect against diabetes. The relationship between BAT glucose utilization and systemic glucose homeostasis has not been defined. 

 

In this paper we have characterized glycaemic excursions and BAT thermogenic responses in human brown adipocytes, BAT explants, and healthy adults through supraclavicular temperature profiling, revealing their circadian coupling in vivo and in vitro, orchestrated by UCP1, GLUT4, and Rev-erbα biorhythms. ...

 

gr1.jpg

 

During oral glucose tolerance test (OGTT), BAT temperature progressively increased and mean temperature was significantly greater than baseline after 60 min (E). Supraclavicular temperature response (gSTR)  [a surrogate for BAT activity] correlated positively with glucose-induced thermogenesis (GIT) (F). p < 0.05, ∗∗p < 0.01.

 

We explored this possibility [that the human BAT circadian rhythm might modulate systemic glucose homeostasis] by performing simultaneous continuous STR and subcutaneous glucose monitoring over a 12 hr period in 15 volunteers (27 ± 4 years old, 12 men, BMI 23 ± 3 kg/m2). They were stratified into three groups based on BAT activity and differed only by BAT status ... Among individuals with the highest BAT abundance (BAThigh), a negative correlation (R2 = 0.31, p < 0.0001) was detected between glucose and STR (Figure 3B), with STR leading glycaemic changes by three time periods (Figure 3D). In contrast, glucose correlated positively with STR (R2 = 0.10, p < 0.001) among individuals with low BAT abundance (BATlow) (Figure 3C), and glycaemic changes preceded STR evolution among these individuals (Figure 3F). No relationships were detected between STR or glucose excursions among volunteers devoid of any BAT (BATneg) (Figures S2A–S2D). BAT activity did not relate to maximal or mean glycaemia (Table S1) but correlated negatively with overall glycaemic variability (Figure 3E), which was greatest among BATneg individuals ...

 

gr3.jpg

 

Both BAThigh (B) and BATlow © individuals manifested significant cross-correlations between STR and glucose. (D) and (F) are cross-correlation plots between glucose excursion and STR. A positive lag indicates that the first series (i.e., glucose) leads the second series (i.e., STR), while a negative lag indicates that the first series (i.e., glucose) follows the second (i.e., STR). In this regard, STR tracked glucose excursions among BATlow volunteers as signified by weakly positive correlative coefficients (F). In contrast, STR led changes in glycaemia among BAThigh subjects, with strongly negative correlation coefficients (D). In other words, STR is a leading indicator among BAThigh individuals and predicts glucose level best three periods later. Total BAT activity correlated negatively with glucose variability (E).

 

Extent of glycated haemoglobin also correlated positively with environmental temperature among community-dwelling patients. These data uncover potential crosstalk between BAT and glucose regulatory pathways, evident on cellular, tissue, individual, and population levels, and provide impetus to search for BAT harnessing strategies for therapeutic purposes.

 



#125 Dean Pomerleau

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Posted 15 March 2016 - 06:58 PM

Michael wrote:

Brown Adipose Tissue Exhibits a Glucose-Responsive Thermogenic Biorhythm in Humans

Paul Lee, Ron Bova, Lynne Schofield, Wendy Bryant, William Dieckmann, Anthony Slattery, Matt A. Govendir, Louise Emmett, Jerry R. Greenfield

Cell Metabolism. Publication stage: In Press Corrected Proof

[Big cut-and-paste from full text of paper]

 

Thanks Michael, you beat me to it. But no commentary on your part to accompany it?  I was in the middle of incorporating this paper into my big post in reply to your earlier post, but I guess I'll address it here now rather than wait to include it in my big post. 

 

I was going to use this Lee et al paper [1],  in response to your previous comment:

 I just can't mechanistically see how increasing BAT quantity or activity would exert an effect on aging per se

 

First off, I'm never quite sure what you mean by "aging per se" (see this thread for discussion). But I'm going to assume you mean "accumulation of damage resulting from metabolic processes". By this definition it seems that [1] suggests just such a mechanism.

 

There is a lot going on in [1], and I'm still trying to digest it all, especially some of the biochemistry. But I'm going to do my best to highlight some of the most important stuff I got out of it.

 

First, the authors brough 15 relatively young (~27 years), mostly male (13/15),  healthy and relatively thin (BMI ~23) volunteers into their lab and measured their BAT volume and BAT activity in response to cold exposure. Not surprisingly, there was a lot of variation in the amount and activity of BAT across the subjects. They decided to divide them into three groups, BAThigh, BATlow  and BATneg, representing people with high, low and undetectable levels of BAT, respectively. Here is part of the table from the supplemental material (pdf) representing important measurements of the three groups. I've highlighted a few differences between them: 

Pv7iFLS.png

 

First off, notice from the very first line "Number (M/F)", that all three of the BATneg group are men, and despite there being only 2/15 women in the study, one of the women had BAT that was among the three highest in the study (the other women was in the middle group). This jibes with what others have observed, namely that women are more likely to have significant amounts of BAT than men.

 

Next, notice from the top two highlighted rows just how large the variation in BAT mass and BAT activity was between the three groups. The folks in the BAThigh group had 6.5x the BAT volume and 4x the BAT activity of the BATlow group. Not surprisingly, the BATneg group had no detectable BAT or BAT activity, by definition. 

 

You'll notice that none of the other regular anthropometric measures differed between the groups. What the authors mean by "Mean exposed environmental temperature", was the mean temperatures (presumably in °C) inside their clothes during the 'circadian' part of the study, during which they measured BAT activity, glucose and a few other things continuously for 12 hours. The fact that all three groups were near 31 °C for this measure means none were systematically exposed to more cold than the others during the testing.

 

Besides BAT volume and activity, the other two measurements that varied substantially between the groups were Glucose Induced Thermogenesis (GIT) and supraclavicular temperature response (STR, i.e. their measure of BAT activity) during the oral glucose tolerance test (OGTT) that is, the row labelled "%STR during OGTT". Basically, what these two rows show is that when the subjects were fed a big standardized bolus of glucose, the BAT tissue of those in the BATlow and especially the BAThigh group kicked in, increasing its metabolic rate, burning calories and warming up the body, especially in the area around the shoulder blades where BAT is most concentrated in humans. 

 

It is this responsiveness of BAT to glucose that seems to be at the heart of this study's results. In short, BAT kicks in around the time that glucose spikes. I think two of the graphs that best illustrate the significance of this responsiveness of BAT to glucose actually come from Figure S2 in the supplemental material:

 

iovdkC5.png

 

What you can see is that both as a groups (i.e. high, low and negative in the graph on the left), and on a per-individual basis (scatter plot on the right), there was a consistent decrease in "glucose variability" with increase in BAT activity. What do they mean by glucose variability? They basically measured BAT activity and blood glucose levels continuously for 12 hours in the 15 subjects (which spanned a dinner meal) and found those people with the most BAT had the smallest excursions from their mean glucose during that 12h period. Look at the guy (or gal?) with the most BAT activity in the lower right of the graph on the right. His glucose excursion was very small and he had a LOT of BAT activity. In contrast, the three people who had no BAT in the BATneg group (blue triangles against the vertical axis) had very large glucose excursions / variability.

 

I'll discuss the implications of this for health shortly. But first, one other, more subtle finding that seems worth mentioning. In the BATlow group (i.e. those with some, but not too much, BAT), they found that BAT kicked in (i.e. got warmer) shortly after blood glucose went up (e.g. shortly after a meal). This makes intuitive sense - when glucose levels go up after a meal, BAT-thermogenesis kicks in to burn calories and thereby use up and lower glucose. The authors suggest that in the BATlow group, BAT may be serving as a "glucose sink" - sucking up glucose when it's high to (modestly) reduce glucose excursions (and therefore glucose variability) relative to the BATneg group.

 

The really interesting thing is that in the BAThigh group, BAT activity was a leading indicator of glucose level, by about 15 minutes. And BAT activity was positively correlated with glucose level, but shifted 15 minutes earlier. In other words, 15 minutes before glucose went up, BAT activity tended to go up in the BAThigh group. Conversely, 15 minutes before glucose went down, BAT activity tended to go down in the  BAThigh group. It's as if the "superactive BAT" (the authors' phrase) in these BAThigh folks was anticipating the upcoming glucose variation, and responding appropriately to blunt the excursion (e.g. the BAT kicked in when glucose was about to spike). This is consistent with the dramatically lower glucose variability observed in the BAThigh group, although the authors can only speculate about what the mechanism for this effect might be:

 

Since BAT activity was more than doubled in these volunteers with ‘‘superactive BAT’’ compared to BATlow individuals, it is tempting to speculate an activity-dependent BAT threshold potentiating glucose-clearing dynamics. One cannot exclude the possibility that a yet-to-be-defined systemic signal mediates glycaemic changes in BAThigh individuals.

 

But whatever the mechanism by which BAT activity reduces glucose, the authors found that BAT kicks in around the time glucose spikes, and the more BAT subjects had, the lower their glucose variability - i.e. the smaller their glucose excursions. 

 

The reason this study is so interesting and important is that this reduction in glucose excursions is likely to be a very good think from a health / longevity perspective. As Michael has pointed out in the past (on the old email list), it is the large post-meal glucose spikes that are most damaging - i.e. that result in the the most glycation, which is a type of what the SENS folks call "extracellular cross linking", one of the seven forms of damage that characterizes aging per se, at least as far as I understand it.

 

In addition, large glucose spikes are often accompanied by large releases of insulin in an attempt to clear the glucose, leading to insulin resistance and other bad effects of elevated insulin. The large insulin spike often overcompensates, causing a large drop in glucose (i.e. hypoglycemia), i.e. a large glucose excursion in the negative direction. Big glucose excursions result in glycation, and so the amount of glycated hemoglobin (HbA1c) in the blood is a measure of long-term (~1month) average glucose control, and is a strong predictor of diabetes risk.

 

In short, smaller glucose excursions & variability is a good thing for health & longevity, and this study seems to show that having active BAT is a way to reduce glucose excursions & variability.

 

Which brings me to the second cool finding in this paper. If active BAT reduces glucose excursions, and glycated hemoglobin (HbA1c) is a long-term measure of glucose excursions, and BAT is activated by cold, might it be possible to see the impact of bat activity in a large population by looking for a correlation between HbA1c levels with seasonal temperature variations? In other words, if BAT activity helps with glucose control, than HbA1c levels should be lower in the winter, when people are exposed to colder temperatures and will therefore have more (active) BAT.

 

This is exactly what the authors found, as they describe in this passage:

 

So does BAT abundance affect glycaemia over time? Because BAT is known to be most abundant in winter (Saito et al., 2009), we probed this question by examining the relationship between glycaemia and outdoor temperature in 65,535 patients who had blood tests throughout a 1-year period. Environmental temperature correlated positively with glycated haemoglobin, a measurement of overall glucose control (Figure S3).

 

Here are the two relevant graphs from Figure S3:

 

WniF1xw.png

 

As you can see, although the mean temperature doesn't vary too much in Sidney (and their temperature minimum is in the middle of the year, when it's summer here in the northern hemisphere), there is a clear positive correlation between ambient temperature and glycated hemoglobin, as measured across a large population, as shown both visually in the graph on the left, and via the positively sloping best-fit curve relating temperature to HbA1c in the graph on the right. This is obviously not definitive - there could be other explanations for the positive correlation between ambient temperature and glycated hemoglobin than reduction in BAT activity when it's warmer.  But coupled with the other results from this study, it is a very intriguing possibility.

 

What I find most interesting though is the possible implications for human CR practitioners of the beneficial effects of increased BAT activity on glucose control. As some of you may recall, there was one distinct and troubling finding that came out of Luigi Fontana's testing of a bunch of us serious CR practitioners a few years ago. A sizeable fraction of us CRers (including me), showed terrible glucose control after an oral glucose tolerance test. And those who were the most severely CRed showed the worst ability to bring glucose levels down during the OGTT [2]. Here is the graph. I am the point highlighted by the orange circle :-( :

 

JcvbEPT.png

 

My (and our) insulin sensitivity was quite high - I (we) just couldn't seem to bring my (our) glucose down when given 75g (300kcal) of glucose in a quick dose. At the two-hour mark after drinking the glucose, my serum glucose was still at 199 mg/dL, as can be seen at the bottom of my blood test spreadsheet in the column labelled 11-19-02. 

 

In summary Luigi in [2] found that we hardcore CR folks had low fasting and circulating insulin, low IGF-1 and low testosterone levels, which are thought to all be good markers from a longevity perspective. But they (especially the low insulin) may have impaired our ability to deal with glucose, especially big doses of it. This has been a nagging concern for some of us ever since Luigi's study.

 

Now we may finally have an explanation, and a possible solution, for this impaired glucose tolerance in CRers.

 

It's very likely that we older, skinny, chilly but often cold-exposure-avoiding, male CR practitioners have zero BAT, like the BATneg group in [1] who had much higher glucose variability, apparently because they lack BAT to help blunt their glucose excursions. I recall quite distinctly I was very cold-sensitive at the time of the WUSTL testing, and therefore avoided cold by bundling up. In fact, I was quite cold in the metabolic ward they housed us in while at WUSTL despite it being normal room temperature, to the point where they had to bring in a warming box for my arm so they could draw my blood for the study (an incident which I recently recounted here).

 

Fast forward to today. I'm 5 lbs skinnier today than I was during the WUSTL OGTT test (118 vs. 123) and my most recent fasting insulin level was if anything lower that it was during my visit to WUSTL (1.6 vs. 1.8 uIU/ml), and of course I'm 14 years older (meaning I should have a lot less BAT). But I'm not longer a wimp about cold.

 

So how is my glucose control?

 

I haven't done an official OGTT test since that one at WUSTL, but for kicks I measured my glucose several times after my single, really big (~3400kcal) meal this morning. I started eating at 6am and finished at 8am. Fifteen minutes after the end of the meal, I measured by glucose twice in rapid succession - the readings were 126 and 121 mg/dL. I then went for a 1.5 mile run. It was 48 °F and lightly drizzling. I ran in shorts and no shirt. Quite invigorating. When I got back, after warming my hands up enough to extract a drop of blood, I measured my glucose. It was 30min after finishing my meal. My glucose was 105 mg/dL. Pretty darn good.

 

While it's not exactly an "apples to apples" comparison, my glucose control appears to be a heck of a lot better these days than it used to be. Can I be sure this is a result of my deliberate practice of cold exposure? Clearly not. But this study [1], along with Gordon's sundae experiments provide quite a bit of support for this being the explanation. In that post, Gordon also provides further evidence besides his own anecdotal evidence (via an ice cream sundae OGTT!) about the benefits for BAT on glucose control, referencing study [3]. Quoting from the abstract of [3]:

 

We studied 7 BAT positive (BAT+) men and 5 BAT negative (BAT-) men under thermoneutral conditions and after prolonged (5-8 h) cold exposure (CE). The two groups were similar in age, body mass index, and adiposity. CE significantly increased resting energy expenditure, whole-body glucose disposal, plasma glucose oxidation, and insulin sensitivity in the BAT+ group only. These results demonstrate a physiologically significant role of BAT in whole-body energy expenditure, glucose homeostasis, and insulin sensitivity in humans and support the notion that BAT may function as an anti-diabetic tissue in humans.

 

Here is a very dramatic graph from the full text of the paper showing the increase in the rate of glucose clearance under cold conditions in BAT- and BAT+ subjects:

 

YveKrCP.png

 

That is a darn impressive improvement in glucose clearance for those who've got BAT!

 

In summary, multiple lines of evidence suggest that in humans, BAT helps one to kick butt in the glucose control department. The best and perhaps the only reliable way to build up BAT, particularly in skinny, old guys like us, is via cold exposure.

 

--Dean

 

--------------------------

[1] Cell Metabolism Available online 10 March 2016, In Press, Corrected Proof

doi:10.1016/j.cmet.2016.02.007

 
Brown Adipose Tissue Exhibits a Glucose-Responsive Thermogenic Biorhythm in Humans
 
Paul Lee1, 2, 7, , , Ron Bova3, Lynne Schofield1, 2, Wendy Bryant5, William Dieckmann6, Anthony Slattery4, Matt A. Govendir1, Louise Emmett4, Jerry R. Greenfield1, 2, 5, 7
 
 
Highlights
• Brown fat utilizes glucose as substrate fuel to produce heat in humans
• Human brown fat exhibits a thermogenic circadian rhythm
• Brown fat circadian rhythm is glucose responsive
• Low brown fat abundance is associated with greater glycaemic fluctuations
 
Summary
 
High abundance of brown adipose tissue (BAT) is linked to lower glycaemia in humans, leading to the belief that BAT may protect against diabetes. The relationship between BAT glucose utilization and systemic glucose homeostasis has not been defined. In this paper we have characterized glycaemic excursions and BAT thermogenic responses in human brown adipocytes, BAT explants, and healthy adults through supraclavicular temperature profiling, revealing their circadian coupling in vivo and in vitro, orchestrated by UCP1, GLUT4, and Rev-erbα biorhythms. Extent of glycated haemoglobin also correlated positively with environmental temperature among community-dwelling patients. These data uncover potential crosstalk between BAT and glucose regulatory pathways, evident on cellular, tissue, individual, and population levels, and provide impetus to search for BAT harnessing strategies for therapeutic purposes.
 

---------------

[2] Age (Dordr). 2010 Mar;32(1):97-108. doi: 10.1007/s11357-009-9118-z. Epub 2009 Nov

11.
 
Effects of long-term calorie restriction and endurance exercise on glucose
tolerance, insulin action, and adipokine production.
 
Fontana L(1), Klein S, Holloszy JO.
 
Author information: 
(1)Washington University School of Medicine, St. Louis, MO 63110, USA.
lfontana@dom.wustl.edu
 
 
Calorie restriction (CR) slows aging and is thought to improve insulin
sensitivity in laboratory animals. In contrast, decreased insulin signaling
and/or mild insulin resistance paradoxically extends maximal lifespan in various 
genetic animal models of longevity. Nothing is known regarding the long-term
effects of CR on glucose tolerance and insulin action in lean healthy humans. In 
this study we evaluated body composition, glucose, and insulin responses to an
oral glucose tolerance test and serum adipokines levels in 28 volunteers, who had
been eating a CR diet for an average of 6.9 +/- 5.5 years, (mean age 53.0 +/- 11 
years), in 28 age-, sex-, and body fat-matched endurance runners (EX), and 28
age- and sex-matched sedentary controls eating Western diets (WD). We found that 
the CR and EX volunteers were significantly leaner than the WD volunteers.
Insulin sensitivity, determined according to the HOMA-IR and the Matsuda and
DeFronzo insulin sensitivity indexes, was significantly higher in the CR and EX
groups than in the WD group (P = 0.001). Nonetheless, despite high serum
adiponectin and low inflammation, approximately 40% of CR individuals exhibited
an exaggerated hyperglycemic response to a glucose load. This impaired glucose
tolerance is associated with lower circulating levels of IGF-1, total
testosterone, and triiodothyronine, which are typical adaptations to
life-extending CR in rodents.
 
PMCID: PMC2829643
PMID: 19904628
 
---------------
[3] Diabetes. 2014 Dec;63(12):4089-99. doi: 10.2337/db14-0746. Epub 2014 Jul 23.
 
Brown adipose tissue improves whole-body glucose homeostasis and insulin
sensitivity in humans.
 
Chondronikola M(1), Volpi E(2), Børsheim E(3), Porter C(3), Annamalai P(4),
Enerbäck S(5), Lidell ME(5), Saraf MK(3), Labbe SM(6), Hurren NM(3), Yfanti C(7),
Chao T(8), Andersen CR(3), Cesani F(9), Hawkins H(10), Sidossis LS(11).
 
 
Abstract
Brown adipose tissue (BAT) has attracted scientific interest as an antidiabetic
tissue owing to its ability to dissipate energy as heat. Despite a plethora of
data concerning the role of BAT in glucose metabolism in rodents, the role of BAT
(if any) in glucose metabolism in humans remains unclear. To investigate whether 
BAT activation alters whole-body glucose homeostasis and insulin sensitivity in
humans, we studied seven BAT-positive (BAT(+)) men and five BAT-negative (BAT(-))
men under thermoneutral conditions and after prolonged (5-8 h) cold exposure
(CE). The two groups were similar in age, BMI, and adiposity. CE significantly
increased resting energy expenditure, whole-body glucose disposal, plasma glucose
oxidation, and insulin sensitivity in the BAT(+) group only. These results
demonstrate a physiologically significant role of BAT in whole-body energy
expenditure, glucose homeostasis, and insulin sensitivity in humans, and support 
the notion that BAT may function as an antidiabetic tissue in humans.
 
 
PMCID: PMC4238005
PMID: 25056438

There will never be peace in the world while there are animals in our bellies.

#126 Gordo

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Posted 15 March 2016 - 10:08 PM

I continue to be fascinated with the possible practical applications of all of this research, particularly with respect to CR practice.  Like Dean, I am particularly interested in optimizing glucose control / glucose homeostasis.  The study Dean highlighted above, where they found that high BAT individuals had an almost immediate glucose lowering or stabilizing BAT activation upon feeding makes me want to try the following experiment, this time with a control:

 

  • Fast for 18 hours (this is my normal daily routine), take glucose measurement, eat some specific, well documented/weighed meal.  Take glucose measurements every 30 minutes for 3 hours.  Keep room temp constant and documented.
  • Repeat the above, with same exact fasting period, meal, and room temp but start wearing the cooling vest X minutes before the meal begins, to activate BAT "preemptively".  Continue wearing cooling vest for Y minutes.
  • Repeat but vary X or Y.

 

However there is FAR more going on with BAT than simply glucose control.  It has become apparent through the research that a host of cell signaling pathways associated with longevity are involved.

 

Regarding the points being made about mice and room/lab temps.  I got the sense from the back and forth as well as from reading some of the related publications, that there is quite a bit of uncertainty and/or opposing views on the matter even among researchers.  My thoughts are that perhaps the focus is misplaced - if you want to compare mice to humans, we should be looking at and comparing the temps at which mice and humans achieve some level of BAT activation as the benchmark for comparison - this would provide a reproducible and more definitive answer to the debate in a way that is directly relevant to the science most of us are interested in (longevity).  Also every scenario could be accounted for (bedding materials or lack thereof, clothing or lack thereof, singly or group housed, BMI, genetics, etc).

 

Regards,

Gordo


Edited by Gordo, 16 March 2016 - 12:13 PM.


#127 AlPater

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Posted 16 March 2016 - 06:49 PM

I really need to thank you for letting me know that your data from November 2002 was also used in the reference (2) paper Dean, since I was also in the group tested at that time.  And for your results for your glucose versus testosterone in Fig 3 of (2) were in contracts to mine with glucose AUC of 120, testosterone of 16.5 and IGF01 of 76.5 (I have not T3 value) were exceptional and greatly different from my values.  I was 97 pounds at the time.



#128 Dean Pomerleau

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Posted 17 March 2016 - 06:45 PM

I'm still working on my "magnum opus" (or should I call it my 'Albatross' ) in response to Michael's skepticism. Hopefully I'll get it finished tomorrow...

 

In the meantime here is a nice popular press story about the Lee et al. paper (doi:10.1016/j.cmet.2016.02.007) that Michael posted and that I discussed a couple posts earlier in this thread. The article focuses on the potential to use brown fat to prevent  diabetes.

 

Here are a few highlights and quotes from the lead author (Lee):

 

"We think brown fat may be functioning as a glucose buffer, smoothing out the fluctuations of glucose," endocrinologist Dr Paul Lee of Sydney's Garvan Institute of Medical Research said.

 

Interestingly, the researchers found brown fat activity rose at dawn just as people awoke.
 
"We speculate this may have an evolutionary origin because our ancestors had to go out and hunt in the mornings, which tended to be cold," Dr Lee said. "This brown fat and temperature boost may have helped prepare them."
 
Previous research has shown that the amount of brown fat we have increases with prolonged exposure to mildly cold temperatures (19 degrees Celsius) and decreases with prolonged exposure to warmer temperatures.
 
Dr Lee said that meant modern humans could actually be reducing their brown fat.
 
"Perhaps by not exposing ourselves to cold because of widespread central heating and clothing, this may also be contributing to diabetes."
 

That last one hits the nail on the head, as far as I'm concerned!

 

Its funny, the author uses the same old excuse used to motivate CR mimetic research, but in this case in the context of CE (Cold Exposure) rather than CR:

 

Dr Lee said it was not practical to boost brown fat by exposing ourselves to the cold, but that he and his colleagues were now trying to find out how the body itself switches on brown fat.
 
"Whichever signals that switch on this brown fat biorhythm may become a drug target in the future," he said.

 

It seems CE and CR are in the same boat in at least this one respect - both require too much discipline to expect the general population to engage in them!

 

--Dean


There will never be peace in the world while there are animals in our bellies.

#129 Dean Pomerleau

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Posted 18 March 2016 - 07:00 PM

Cold Exposure Albatross (Albatross-CE)To make it easy to search for this post in the future...

 

Michael,

 

First off, I want to thank you for engaging in this discussion. I really appreciate your challenges to my interpretation of the scientific data surrounding cold exposure (CE), which is really getting quite extensive. It is, paradoxically, a really hot area, it would seem. It's only through such challenges that we can identify and get past our implicit assumptions and biases, and hopefully get closer to the truth.

 

Before getting into a detailed response to your skepticism, let me see if I can summarize my hypothesis about cold exposure & BAT, so you have something more specific to express reservations about, and to focus my response in the remainder of this post. 

 

Cold Exposure Hypothesis:

 

Cold exposure in mammals (including humans) promotes health and longevity by several mechanisms, including, but not limited to, simply burning extra calories to create a net calorie deficit that helps the organism avoid obesity and hence avoid the negative health consequences that accompany it. If combined with eating limited calories and/or exercise, CE can make significant contributions to producing a net calorie deficit that is sufficiently large to cause the body to kick into a CR-like 'hunkered down' metabolic state of heightened repair and maintenance. At least some of these benefits are likely to result from an increased amount of brown adipose tissue (BAT) or more generally, an increased expression of uncoupling proteins (UCPs) in organs, fats and muscle tissues that cause mitochondria to burn calories in order to generate heat rather than make ATP for useful energy, and in the process potentially make the mitochondria operate more 'cleanly' - i.e. without generating as many ROSs (free radicals).  But like the multiple evolutionary adaptations that kick in as a result of the metabolic stress of CR, CE is likely to trigger many different hormonal / physiological / biochemical responses that together allow the organism to survive the metabolic stress of CE.

 

There is a stronger speculation that I'm not ready to fully endorse because there isn't enough evidence either way. Namely that at least some of the benefits observed for CR will only manifest if the animal also exposed to cold housing conditions, and perhaps only if those CR animals have BAT or some other mechanism for active thermogenesis. Put the other way around, this speculation is that housing mammals at a comfortable, thermoneutral temperature so as not to require thermogenesis will blunt or erase at least some of CR's benefits.

 

With that on the table, let me now try to summarize your perspective, and especially your reservations / skepticism about this hypothesis, and see if I can provide sufficient evidence to overcome your skepticism.

 

First, something we appear to agree on - the obesity-prevention effects of increased energy expenditure as a result of CE will in all likelihood have health benefits, independent of any more direct benefits from CE-induced metabolic/hormonal/biochemical adaptations. Given what I understand to be the SENS perspective on aging, namely that aging simple is the accumulation of damage which largely results from the so-called "diseases of aging" (several of which in turn are brought on by obesity),  it continues to confuse me how in your estimation delaying the onset and progression of the diseases of aging remains just a benefit to health, and isn't also conveying longevity benefits (i.e. slowing the aging process). For example, the 7th Day Adventists eat well & exercise, and thereby avoid obesity, CVD, diabetes, sarcopenia, cognitive decline, by slowing down accumulation of the damage resulting from a subset of what SENS characterizes as the Seven Causes of Aging, which manifest themselves symptomatically as the diseases of aging just listed. So as a result of this slowed accumulation of damage, the 7DAs live longer. Seems straightforward to me. But that is a topic for another thread where I hope you will indeed respond soon as promised, but in which one of your recent posts suggests you don't see quite as sharp a division between health-promoting and longevity-promoting interventions as I'd previously thought you did.

 

You also seem to acknowledge that colder ambient temperatures will result in cooler body temperatures, which will in turn likely slow down the onset and progression of cancer, something that rodents are particularly prone to and that CR in rodents does a good job of preventing, as long as housing temperatures are below thermoneutrality (more on that below).

 

Now on to where we seem to disagree, at least in part. 

 

You wrote:

It seems clear, therefore, that the mice housed at 30°C in PMID 9032756 are really rather toasty, and those at room temperature are only very modestly cooler-housed than a human in normal room temperatures. 

 

We could quibble over what the appropriate housing temperatures for mice should be, as Speakman & Keijer do with their critics in this series of back and forth and back again letters-to-the-editor about the their paper (PMID 24024125). Even Speakman acknowledges that cool-housed mice (20-22 °C) are thermally stressed:

 

At no point in our paper do we state that mice are ‘most comfortable’ at 20–22 °C, and the contention of our article was not, and was never stated to be, that ‘there is no distressing impact of cold temperatures on mice’

 

Your use of the colloquial "rather toasty" is ambiguous. By toasty do you mean "comfortably warm" or "heat stressed"? From a metabolic perspective, the evidence strongly favors the former interpretation (toasty = comfortably warm). Check out these two figures from [1], a huge review on rodent metabolic response to temperature variations:

 

QY2gL9I.png

 

As is apparent, across various strains of mice, their metabolic rate is minimized at around 30°C. Similarly, as can be seen from this graph also from [1], that the amount of BAT mass mice possess is minimized at 30 °C, and climbs steadily as temperature is reduced.

 

 

 

96Vf9Ep.png

 

The author of [1] addresses the weird apparent uptick in BAT above 30 °C with the observation that it doesn't actually appear to be BAT that is increasing at the high temperatures:

 

The weight of BAT increased in a near linear fashion as temperature decreased below 30 °C. Interestingly, BAT weight also increased in mice exposed to extremely warm temperatures of 35 and 37.5 °C, environments that are well above the limits of normothermy for mice and must have certainly been stressful. It was noted that histologically, the BAT in warm adapted mice differed markedly from cold acclimated mice. BAT of cold exposed mice was highly developed and multilocular. BAT of the warm acclimated mice was unilocular, looking more like white adipose tissue.

 

To put this mice data in perspective, here is a helpful graphic from [4] illustrating the change in metabolic rate of mice, naked humans and lightly clothed humans as a function of temperature:

 

iPxUnjF.png

 

According to the above graphic, a mouse has a metabolic rate approximately 250% of it's baseline at a "normal" indoor temperature (20-22 °C). With a little extrapolation, you can see that the same boost in metabolic rate for a human would require exposure to 10°C (50°F), and that's if they are naked. Clothed it would be much lower...

 

To summarize, from a metabolic perspective, it appears that thermal stress for mice is minimized at around 30 °C, and is increased dramatically when mice are housed at normal lab temperatures (20-22 °C), as reflected in both a higher metabolic rate and higher BAT content. As a result, at "normal" indoor temperatures (20-22 °C), mice are quite thermally stressed, while humans are not, which isn't surprising, since we are the ones who get to define what "normal" is.

 

So, getting back to your post, the bulk of the evidence seems to contradict you statement:

It seems clear, therefore, that the mice housed at 30°C in PMID 9032756 are really rather toasty, and those at room temperature are only very modestly cooler-housed than a human in normal room temperatures. This merits CR folk bearing a certain amount of temperature discomfort, but doesn't imply that the benefits are abolished if we aren't either exposed to the autumn elements naked or wearing ice vests. 

 

I'm glad to see we're on the same page that some amount of temperature discomfort is likely to be beneficial for CR folks. That seems like an important takeaway message that we both agree on. It seems like now we're just haggling over price.

 

We saw above, mice living at 20-22 °C are metabolically quite challenged relative to living at 30 °C. But what about subjectively? The interesting question you pose is what amount of thermal discomfort might be the human equivalent of what mice experience at "normal" lab temperatures?

 
Obviously you can't ask mice directly "how cold are you?" but fortunately there have been behavioral studies that address this very issue - thermal preferences in mice. Specifically, if given their druthers (i.e. if they can move freely between cages of different temperatures), mice of several strains preferred a cage with a temperature of 26-29°C even with bedding material and two cage mates to huddle with [2]. So the typical 20-22°C housing temperature used in PMID 9032756 and virtually all CR experiments is a full 6.5°C (12°F) cooler than the temperature that mice prefer. On top of that, mice in CR experiments (including PMID 9032756) are housed singly - so no huddling or cuddling  :-( as the mice in [2] were able to do to keep warm, and which (i.e. huddling) was shown in [1] to result in a nearly 50% reduction in BAT activity when mice were housed with just one other mouse rather than singly.
 
Michael, I'm not sure what temperature you find most comfortable when dressed in light clothing, but if you are like most people, it's probably in the neighborhood of 21°C (70°F). Subtract 6.5°C (12°F) from that and you get about 14.5°C (58°F). Try hanging out at 14.5°C (58°F) in light clothing for a few hours (to say nothing of 24/7 for your entire life) before suggesting that the mice aren't very thermally stressed at "normal" lab temperatures.
 
So the evidence is really quite solid - rodents housed at normal lab temperatures are quite thermally stressed, both metabolically and subjectively. The authors of [11] express it amusingly in the title of their paper - "Why We Should Put Clothes on Mice" and [12] even includes a helpful graphic illustrating how to improve the human-relevance of rodent lab experiments :
 
yktMQoq.png
 
But I have a different solution. Rather than warming up mice (with clothing or otherwise) so they have crappy health & longevity like we warm humans, why not cool ourselves down so we have good health & longevity like the thermally-challenged CR mice in PMID 9032756, who ate 20% more food, weighed the same, but lived longer (both median and max lifespan) than warm-housed CR mice? 
 
Then you wrote:

... the renowned Dr. John Speakman, author of numerous provocative studies on CR and related subjects and the lead author of (1), will be one of our many exciting scientific presenters. 

That is great news, especially since Dr. Speakman is the lead author of another, even more interesting and relevant paper I'll discuss below... [inserted later - actually to be discussed in an upcoming post...]

 
Next you quibble over whether mitochondrial biogenesis (i.e. formation of new mitochondria) is actually occurring in the BAT of CRed mice in PMID 18593277, as the authors suggest. This too seems like a red herring. Does it really matter whether the mechanism for increased BAT thermogenic capacity in CRed mice results from an increase in the number of mitochondria, the size of mitochondria or the enrichment of existing mitochondria with UCP1? Regardless of the mechanism, what the study showed was that CRed mice had ~60% less BAT than controls, but the mitochondria of the BAT they did retain was highly enriched (by about a factor of 2x) with heat-producing UCP1 protein. Hence the authors reference to "conservation of BAT thermogenic capacity" in CRed mice despite dramatic reduction in their total BAT tissue mass. 
 
Just to translate this into layman's terms - in order to keep their skinny little butts warm in their cold, lonely cages (I can picture Sthira is seething now...), the CR mice in PMID 18593277 were able to cold-adapt by boosting the ability of the what little BAT they retained to generate even more heat than usual.
 
You then complain (ok, observe) that CR doesn't increase metabolic rate (i.e. metabolic activity per unit metabolically-active mass), and so an increase in burning of calories through BAT-driven thermogenesis appears ruled out. If you look at the table of tissue masses in my post about PMID 18593277, you'll see that while the change in BAT mass between controls and CR mice is dramatic in relative terms (i.e. CR mice have 60% less BAT than control mice), the fraction of the total body weight that BAT represents is tiny - 0.1% in CR mice vs. 0.15% in control mice. A very little amount of BAT can go a long way, especially when your BAT is supercharged like the CRed mice in this study.
 
So consider the following and tell me if it doesn't fit the data (it may not, I'm just asking):
  1. CR mice have little body fat and a higher surface-to-volume ratio than control mice, meaning if anything they'll have to work (i.e. burn calories) at least as hard (and probably harder) to stay warm in their cool cages.
  2. Fortunately per PMID 18593277, they have a small amount of supercharged BAT to do just that.
  3. But since, as you observe, their overall metabolic rate remains relatively constant, they must be reducing metabolic expenditure elsewhere - either via reductions in other metabolic processes or in physical activity, in order to burn calories via thermogenesis in BAT or muscle cells.
In other words, mightn't the heretofore paradoxical lack of decrease in body-mass-normalized metabolic rate in CR mice (despite reduced body temperature) be explained by a shift in energy expenditure from "dirty" (ROS-producing) metabolic activities in other tissues to "clean-burning" uncoupled mitochondrial respiration in their tiny amount of supercharged BAT or in their muscles, in a desperate and only partially successful attempt by the CR mice to keep warm?
 
Put another way, mightn't part of the metabolic program that kicks in with CR in cold housing conditions be a relative increase in calorie expenditure devoted to clean-burning thermogenesis in order to keep warm? Seems pretty plausible to me.
 
Then you wrote:

I just can't mechanistically see how increasing BAT quantity or activity would exert an effect on aging per se.

This suggests to me a lack of imagination, or a reluctance to think very hard about it.

 
Here are a few prefatory remarks before diving into the details that constitute the bulk of this post.
 
First, as I mentioned above and in my last post, I'm never quite sure what you mean by "aging per se" (see this thread for discussion). But I'm going to assume you mean "accumulation of damage resulting from metabolic processes". 
 
Next, as my Cold Exposure Hypothesis at the top makes explicit, it is the body's metabolic response to CE in general that I postulate to convey health and longevity benefits. Increased BAT and BAT activity is just one of many metabolic responses that could be involved (more on those below). It's like CR in this regard; many evolutionarily conserved pathways may be involved in the benefits of CE. This brings to mind another pro-survival stress response involving cold exposure - the mammalian dive reflex (MDR), the evolutionarily conserved ability of many mammals (including humans) to conserve oxygen as a result of cold water contacting the face and thereby increase survival.  If we've preserved the MDR adaptation to cold water exposure, it seems pretty plausible to me that we'd preserve a highly-tuned, multifaceted metabolic response to an even more common, temperature-related and potentially life-threatening environmental stress, cold exposure. Parallels between the MDR and the body's response to more modest cold exposure will have to wait for another post...
 
What I'll do now is attempt to counter your skepticism about the anti-aging potential of CE & BAT by enumerating some of the mechanisms by which the research suggests they may indeed have health and longevity-promoting effects.
 
Obesity Avoidance - In your previous post you seem to poo-poo CE's (and specifically BAT's) ability to help prevent obesity/overweight as not a legitimate mechanism for slowing aging. This seems strange to me, and again gets back to your squirrelly definition of aging. It seems pretty clear that white adipose tissue, particularly abdominal WAT, is a very active endocrine organ, spewing out pro-inflammatory proteins that result in quite a bit of downstream aging-related damage. No? On top of that, the evidence in rodents (discussed herehere and here), dogs (discussed here), monkeys (discussed herehere and here), and humans (e.g. these discussions of Adventists and postmenopausal women), suggests that most of CR's benefits result from quite mild calorie restriction - i.e. avoiding obesity/overweight on a healthy diet. So if that's what the evidence says is happening with CR, than CE & BAT could very well have the same positive effect on aging. In short, BAT's ability to help people avoid obesity, and so avoid the negative health & aging consequences that accompany obesity, could by itself serve as a legitimate mechanism by which having extra BAT impacts aging.
 
Improved Glucose Metabolism I won't dwell on this one, since I addressed it in depth here yesterday in my response to your posting of Lee et al (doi:10.1016/j.cmet.2016.02.007). Suffice it to say that it appears that in humans, increased BAT levels is associated with improved glucose control, and effect that probably accounts for the reduced glycated hemoglobin (HbA1c) that occurs population-wide in winter months.
 
Improved Gut Microbiome - I won't dwell on this one either, since I addressed it in depth here and here. The basic upshot is that based on the results of PMID 26638070, chronic cold exposure alters the bacterial strains in the gut microbiome so as to make the digestive system more efficient at extracting calories, as well as improve insulin sensitivity and turn white fat into browning/beige fat, at least in rats. Several of us cold-exposed human CR practitioners show a similar gut bacteria profile to these cold-adapted rats, based on our uBiome.com results, as discussed here and here.
 
Reduced Core Temperature - One mechanism by which CR is thought to extend lifespan is through reduction in body temperature ([15] is a good review of this evidence). Study [9] found that reducing core body temperature of ad lib fed mice via genetic mutation that modulates hypothalamus temperature (the body's thermostat) and thereby reduces core body temperature, increases lifespan without changes in calorie intake. Depending on its severity, CE can easily reduces core body temperature even more than CR, and so is likely to extend lifespan along this same pathway.
 
Reduced Cancer ProliferationRelatedly, I think we're in agreement on this one, although I agree it may not be a direct result of BAT per se. Consistent CE reduces body temperature (even with BAT working to keep warm), which appears associated with a reduction in the rate of cancer proliferation, as observed in [3] (full text). As you suggest, this is probably the way that cool-housed CR mice lived longer than warm-housed CR mice in PMID 9032756, and possibly [9] as well. But see the post referenced in the next item (immunocompetence) for another pathway actively upregulated by CE that dramatically reduces (by 60%) the rate of cancer grown.
 
Improved Immunocompetence - I won't dwell on this one here, since in this post (written after the one you're reading now), I go into great detail on research showing that CE (alone or in combination with exercise) appears to improves the immune system's ability to fight off invaders, including cancer, by elevating norepinephrine and interleukin-6, which in turn boost natural killer (NK) immune cells.
 
Reduced Inflammation - In another striking strike against thermoneutrality, this very recent study [7], found that housing mice at thermoneutrality (30°C) resulted in a dramatic increase in systemic inflammation and atherosclerosis relative to cool-housed (22°C) mice:
 
 Mice housed at thermoneutrality develop metabolic inflammation in adipose tissue and in the
vasculature at an accelerated rate. Unexpectedly, this increased inflammatory response contributes
to the progression of atherosclerosis but not insulin resistance. These findings not only suggest that
metabolic inflammation can be uncoupled from obesity-associated insulin resistance, but also point
to how thermal stress might limit our ability to faithfully model human diseases in mice.
 
In other words, simply living in thermally-neutral, comfortable-for-a-mouse conditions resulted in increased inflammation which promoted atherosclerosis independent of obesity and insulin sensitivity.
 
Reduced Atherosclerosis - Speaking of CE's impact on atherosclerosis, Al and I went back and forth on this topic for a while earlier in this thread. I feel pretty confident I won that one . Here is the post where I summarize the debate and point to this 2016 review [22] which summarizes the relationship between BAT and atherosclerosis as follows:
 
BAT activation reduces plasma triglyceride and cholesterol levels and attenuates diet-induced atherosclerosis development. 
 
Improved Bone Health - Bone loss and the resulting increase in fracture risk is a serious concern as one ages. One troubling side effect of CR is a reduction in bone mineral density. Although there is some hope CR bones are lighter but not more fracture-prone, bone health remains a serious concern for CR practitioners. Fortunately increased levels of BAT are associated with increased bone mineral density, independent body weight. In other words, you can be thin with good bone density, as long as you've got BAT, as I discuss in detail here.
 
Reduced Oxidative Damage - Cold exposure reduces coupling in mitochondrial respiration, not just in BAT (via UCP1), but also in other tissues/organs, including muscles, via several different uncoupling proteins. Modest reductions in mitochondrial coupling is known to reduce the proton gradient substantially, which in turns results in reduced production of damaging ROSs (free radicals). So it seems to me a pretty clear story for aging-related benefits of CE accruing via this mechanism, although again it isn't specifically a result of BAT, but CE more generally. Suggestively, I talk in this post about two studies that found several genetic mutations that result in increased expression of several UCPs (including UCP1) are associated with extreme longevity in humans. In that same post I talk about how one of the few genetic mutations that set the long-lived naked mole rat apart from other rodents, and other mammals, is a mutation that upregulates expression of UCP1. Coincidence? Maybe, but maybe not, especially in light of a couple other studies by Speakman, that I was going to include here but that are important enough to merit their own post, especially considering how long this post is getting already. Stay tuned...
 
FGF-21 Production - I'll repeat and expand a bit on what I said in my post to which you are responding about the significance of FGF-21 as a pathway towards increased longevity.  Cold exposure upregulates circulating fibroblast growth factor 21 (FGF21) in people [23] by 37%, and FGF21 in mice BAT by a factor of 40x [5]. Here is a a very nice graph from [6] that shows that in mice, not just BAT but plasma levels of FGF21 is unaffected by only 6h of cold exposure, but doubles after 24h, and triples after 30 days of CE:
 
7dryQem.png
 
So what good is FGF21 you ask? PMID 23066506 found that transgenic mice that overexpress FGF21 lived 40% longer than controls without the mutation, despite the FGF21-mutant mice eating a bit more than the normal mice. It's an argument in several steps (cold exposure → ↑ BAT → ↑ FGF21 → ↑ longevity), but it shows obesity-independent benefits of cold exposure & BAT.  See this post for more discussion.
 
Interestingly, in addition to (or by way of) extending lifespan, it is well known that FGF21 improves insulin sensitivity & glucose metabolism [5]. And as we saw above, [6] shows in rats that cold exposure increased plasma FGF21, which helped with glucose control. We saw from Lee et al that in BAThigh humans, changes in glucose clearance occur subsequent to increased BAT activity, rather than proceeding it, and the authors were at a loss to say why/how. This results suggests that what could be going on in BAThigh humans is that FGF21 release by active BAT into the bloodstream improves glucose clearance a few minutes later. An intriguing possibility. 
 
Adiponectin Production - Like CR [10], cold exposure increases adiponectin levels. Two hours of cold exposure resulted in a 70% increase in circulating adiponectin in adult men [36]. Study [8] found centenarians and their offspring had genetic mutations that boost adiponectin, and had higher circulating adiponectin, suggesting to the authors "their [i.e. adiponectin-promoting gene mutations] may promote increased lifespan through the regulation of adiponectin production and/or secretion." Study [35] found the same thing in a group of centenarian women - "As compared to BMI-matched [young, ~28 year-old] female controls, female centenarians had significantly higher plasma adiponectin concentrations. In addition, high concentrations of plasma adiponectin in centenarians was associated with favorable metabolic indicators, and with lower levels of C-reactive protein and E-selectin". For those of us who aren't lucky enough to have adiponectin-boosting genes, we can increase adiponectin levels via CR, cold exposure, or both. This video illustrates how wearing the Cool Fat Burner for two hours raises a cold-adapted person's adiponectin level by a whopping 62%!
 
Irisin Production - As discussed here and here, both cold exposure and exercise increase circulating irisin [23]. Irisin improves insulin sensitivity, increases bone quality and quantity [24], is involved in the building of lean muscle mass, and helps reduce obesity by converting white fat to brown fat. In short, it appears CE provides many of the same benefits of exercise, including raising irisin, without the sweating . As a bonus, [25] found that:
 
healthy centenarians are characterized by increased serum irisin levels, whereas levels of this molecule were found to

be significantly lower in young patients with myocardial infarction. Our findings may prompt further research into the role

played by irisin not only in vascular disorders but also in life span modulation.

 
SIRT1 Pathway Activation - SIRT1 is upregulated by CR, and is thought to be one of the important pathways through which CR extends lifespan [27]. While the lifespan effects of SIRT1 in mammals are somewhat controversial, transgenic mice that overexpress SIRT1 have increased lifespan [30] and genetic mutations that result in elevated SIRT1 levels in people are associated with increased human longevity [31]. SIRT1 (and the other sirtuins) have many metabolic effects, but an important one for improving health and longevity is the fact that SIRT1 increases insulin sensitivity and glucose control in skeletal muscles [28], triggers the browning of white fat [32] and increases BAT activity [29], which is fully reversed by thermoneutral housing [29]. Mice partially lacking SIRT1 due to a genetic mutation experience BAT degeneration, reduced thermogenesis, increased inflammation and develop obesity and insulin resistance as a result [34].  Guess what - it's not just CR that upregulates SIRT1. Cold exposure increases SIRT1 phosphorylation/activity in both skeletal muscle and BAT, increasing thermogenesis and insulin sensitivity [33]. In short, both CR and CE upregulate SIRT1, which (may) increase lifespan, at least in part by improving insulin sensitivity in muscles and increasing BAT & BAT activity.
 
Improved Heat Tolerance / Increased Heat Shock Protein Expression - While it's not clear heat shock proteins (HSPs) increase lifespan in mammals like they do in lower organisms (like C elegans), HSPs are definitely important for stress resistance [18], and in particularly for the ability to cope with heat stress, hence the name. CR preserves HSP induction in response to thermal stress in aging mice [20], and prevent the age-related decline in several heat shock protein [21], and especially HSP70 [19] and HSP90 [21].  So might the cold of CE reduce one's heat shock protein levels, and ruin one's ability to cope with high temperatures? Nope. Quite the opposite in fact. 
 
Study [13] found that the induction of three important heat shock proteins, Hsp70, Hsp90 and Hsp110 was higher in tissues of mice housed at 22 °C than mice housed at thermoneutrality (30°C) following 6 h of heat stress (i.e. high temperatures) which elevated core body temperature to 39.5 °C. So it looks like relative to living at comfortable, thermoneutral temperatures, CE helps with thermal tolerance at both extremes - hot and cold, by improving heat shock protein induction. Interestingly, [14] found increased expression of one of Hsp70 is associated with improved insulin sensitivity in monkeys and humans - "higher levels of [...] HSP70 protect against insulin resistance development during healthy aging." Review article [16] is a good discussion of heat shock proteins and lifespan in general, and [17] is a study that shows genetic mutations in heat shock proteins may be associated with improved human survival.
 
FInally, since this section is about the association of CE and BAT with longevity, I'd be remiss if I didn't mention the fact that three of the longest-lived small mammals, grey squirrels (24 yrs), bats (30 yrs), and naked mole rats (32 yrs), all have remarkably high levels of BAT and BAT activity, which is suggestive, but obviously not conclusive, evidence that BAT promotes health & longevity.
 
To wrap up, this diagram from [15]:
Z30YLA3.png
 
is missing a few important pathways discussed above, and suggests too strongly that reduced body temperature is the sole mechanism by which CE operates. Nevertheless, it illustrates how CR and CE activate many of the same health and longevity-promoting pathways. In fact it shows how CR piggybacks on many of the same temperature / thermogenic pathways that are more directly activated via CE. It also clearly illustrates how exposure to warm conditions could interfere with many CR benefits, by raising body temperature and counteracting CR's beneficial modulation of many different pathways.
 
I think the evidence summarized in this diagram, and the more comprehensive evidence presented in the entirety of this post (and the rest of the 129 posts in this thread!), illustrate that cold exposure is a legitimate alternative/adjunct to CR for potentially improving health and extending human lifespan.
 
Frostily yours,
 
--Dean
 

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[1] Journal of Thermal Biology Volume 37, Issue 8, December 2012, Pages 654–685

Review
Thermal physiology of laboratory mice: Defining thermoneutrality
C.J. Gordon
 
Abstract
 
In terms of total number of publications, the laboratory mouse (Mus musculus) has emerged as the most popular test subject in biomedical research. Mice are used as models to study obesity, diabetes, CNS diseases and variety of other pathologies. Mice are classified as homeotherms and regulate their core temperature over a relatively wide range of ambient temperatures. However, researchers find that the thermoregulatory system of mice is easily affected by drugs, chemicals, and a variety of pathological conditions, effects that can be exacerbated by changes in ambient temperature. To this end, a thorough review of the thermal physiology of mice, including their sensitivity and regulatory limits to changes in ambient temperature is the primary focus of this review. Specifically, the zone of thermoneutrality for metabolic rate and how it corresponds to that for growth, reproduction, development, thermal comfort, and many other variables is covered. A key point of the review is to show that behavioral thermoregulation of mice is geared to minimize energy expenditure. Their zone of thermal comfort is essentially wedged between the thresholds to increase heat production and heat loss; however, this zone is above the recommended guidelines for animal vivariums. Future work is needed to better understand the behavioral and autonomic thermoregulatory responses of this most popular test species.
 
----------------

[2]  PLoS ONE 7(3): (2012)  e32799. doi:10.1371/journal.pone.0032799

 
Heat or Insulation: Behavioral Titration of Mouse Preference for Warmth or Access to a Nest.
 

Gaskill BN, Gordon CJ, Pajor EA, Lucas JR, Davis JK, et al. 

 

 
Abstract:
 
In laboratories, mice are housed at 20–24uC, which is below their lower critical temperature (<30uC). This increased thermal stress has the potential to alter scientific outcomes. Nesting material should allow for improved behavioral thermoregulation and thus alleviate this thermal stress. Nesting behavior should change with temperature and material, and the choice between nesting or thermotaxis (movement in response to temperature) should also depend on the balance of these factors, such that mice titrate nesting material against temperature. Naı¨ve CD-1, BALB/c, and C57BL/6 mice (36 male and 36 female/strain in groups of 3) were housed in a set of 2 connected cages, each maintained at a different temperature using a water bath. One cage in each set was 20uC (Nesting cage; NC) while the other was one of 6 temperatures (Temperature cage; TC: 20, 23, 26, 29, 32, or 35uC). The NC contained one of 6 nesting provisions (0, 2, 4, 6, 8, or 10g), changed daily. Food intake and nest scores were measured in both cages. As the difference in temperature between paired cages increased, feed consumption in NC increased. Nesting provision altered differences in nest scores between the 2 paired temperatures. Nest scores in NC increased with increasing provision. In addition, temperature pairings altered the difference in nest scores with the smallest difference between locations at 26uC and 29uC. Mice transferred material from NC to TC but the likelihood of transfer decreased with increasing provision. Overall, mice of different strains and sexes prefer temperatures between 26–29°C and the shift from thermotaxis to nest building is seen between 6 and 10 g of material. Our results suggest that under normal laboratory temperatures, mice should be provided with no less than 6 grams of nesting material, but up to 10 grams may be needed to alleviate thermal distress under typical temperatures.
 
----------
[3] Mech Ageing Dev. 1996 Nov 29;92(1):67-82.
 
A tumor preventive effect of dietary restriction is antagonized by a high housing
temperature through deprivation of torpor.
 
Koizumi A(1), Wada Y, Tuskada M, Kayo T, Naruse M, Horiuchi K, Mogi T, Yoshioka
M, Sasaki M, Miyamaura Y, Abe T, Ohtomo K, Walford RL.
 
Author information: 
(1)Department of Hygiene, Akita University School of Medicine, Japan.
koizumi@med.akita-u.ac.jp
 
 
Energy restriction (ER) has proven to be the only effective means of retarding
aging in mice. The mechanisms of multiplicity of effects of ER on aging remain,
however, fragmentary. ER induces daily torpor, the induction of which is reduced 
by increasing the ambient temperature to 30 degrees C. The effects of preventing 
hypothermia in ER animals were studied in terms of the expected consequences of
ER on survival, disease pattern and a number of physiological parameters in
autoimmune prone MRL/lpr mice and lymphoma prone C57BL, 6 mice. The results
demonstrate that torpor plays a crucial role in the prevention of lymphoma
development but does not have an affect on other aspects of ER, such as
prevention of autoimmune diseases.
 
PMID: 9032756
 
------------
[4] Physiology (Bethesda). 2014 Nov;29(6):413-20. doi: 10.1152/physiol.00029.2014.
 
Translating animal model research: does it matter that our rodents are cold?
 
Maloney SK(1), Fuller A(2), Mitchell D(1), Gordon C(3), Overton JM(4).
 
Author information: 
(1)School of Anatomy Physiology and Human Biology, The University of Western
Australia, Stirling Highway, Crawley, Australia; Brain Function Research Group,
School of Physiology, Faculty of Health Sciences, University of the
Witwatersrand, Parktown, South Africa; (2)Brain Function Research Group, School
of Physiology, Faculty of Health Sciences, University of the Witwatersrand,
Parktown, South Africa; (3)Toxicity Assessment Division, U.S. Environmental
Protection Agency, Research Triangle Park, North Carolina; and. (4)Department of 
Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, 
Florida.
 
Does it matter that rodents used as preclinical models of human biology are
routinely housed below their thermoneutral zone? We compile evidence showing that
such rodents are cold-stressed, hypermetabolic, hypertensive, sleep-deprived,
obesity-resistant, fever-resistant, aging-resistant, and tumor-prone compared
with mice housed at thermoneutrality. The same genotype of mouse has a very
different phenotype and response to physiological or pharmacological intervention
when raised below or at thermoneutrality.
 
©2014 Int. Union Physiol. Sci./Am. Physiol. Soc.
 
PMID: 25362635
 
------------
[5] Endocrinology. 2013 Sep;154(9):3099-109. doi: 10.1210/en.2013-1191. Epub 2013 Jun
13.
 
Cellular mechanisms by which FGF21 improves insulin sensitivity in male mice.
 
Camporez JP(1), Jornayvaz FR, Petersen MC, Pesta D, Guigni BA, Serr J, Zhang D,
Kahn M, Samuel VT, Jurczak MJ, Shulman GI.
 
Author information: 
(1)Department of Internal Medicine, Yale University School of Medicine, New
Haven, Connecticut 06536-9812, USA.
 
Fibroblast growth factor 21 (FGF21) is a potent regulator of glucose and lipid
metabolism and is currently being pursued as a therapeutic agent for insulin
resistance and type 2 diabetes. However, the cellular mechanisms by which FGF21
modifies insulin action in vivo are unclear. To address this question, we
assessed insulin action in regular chow- and high-fat diet (HFD)-fed wild-type
mice chronically infused with FGF21 or vehicle. Here, we show that FGF21
administration results in improvements in both hepatic and peripheral insulin
sensitivity in both regular chow- and HFD-fed mice. This improvement in insulin
responsiveness in FGF21-treated HFD-fed mice was associated with decreased
hepatocellular and myocellular diacylglycerol content and reduced protein kinase 
Cε activation in liver and protein kinase Cθ in skeletal muscle. In contrast,
there were no effects of FGF21 on liver or muscle ceramide content. These effects
may be attributed, in part, to increased energy expenditure in the liver and
white adipose tissue. Taken together, these data provide a mechanism by which
FGF21 protects mice from lipid-induced liver and muscle insulin resistance and
support its development as a novel therapy for the treatment of nonalcoholic
fatty liver disease, insulin resistance, and type 2 diabetes.
 
PMCID: PMC3749479
PMID: 23766126
 
-----------
[6] J Biol Chem. 2011 Apr 15;286(15):12983-90. doi: 10.1074/jbc.M110.215889. Epub

2011 Feb 13.

Thermogenic activation induces FGF21 expression and release in brown adipose
tissue.

Hondares E(1), Iglesias R, Giralt A, Gonzalez FJ, Giralt M, Mampel T, Villarroya
F.

 

Free full text: http://www.jbc.org/c...6/15/12983.full

FGF21 is a novel metabolic regulator involved in the control of glucose
homeostasis, insulin sensitivity, and ketogenesis. The liver has been considered
the main site of production and release of FGF21 into the blood. Here, we show
that, after thermogenic activation, brown adipose tissue becomes a source of
systemic FGF21. This is due to a powerful cAMP-mediated pathway of regulation of
FGF21 gene transcription. Norepinephrine, acting via β-adrenergic, cAMP-mediated,
mechanisms and subsequent activation of protein kinase A and p38 MAPK, induces
FGF21 gene transcription and also FGF21 release in brown adipocytes. ATF2 binding
to the FGF21 gene promoter mediates cAMP-dependent induction of FGF21 gene
transcription. FGF21 release by brown fat in vivo was assessed directly by
analyzing arteriovenous differences in FGF21 concentration across interscapular
brown fat, in combination with blood flow to brown adipose tissue and assessment
of FGF21 half-life. This analysis demonstrates that exposure of rats to cold
induced a marked release of FGF21 by brown fat in vivo, in association with a
reduction in systemic FGF21 half-life. The present findings lead to the
recognition of a novel pathway of regulation the FGF21 gene and an endocrine role
of brown fat, as a source of FGF21 that may be especially relevant in conditions
of activation of thermogenic activity.

PMCID: PMC3075644
PMID: 21317437

 

-------

[7] Cell Metab. 2016 Jan 12;23(1):165-78. doi: 10.1016/j.cmet.2015.10.003. Epub 2015 

Nov 5.
 
Thermoneutral Housing Accelerates Metabolic Inflammation to Potentiate
Atherosclerosis but Not Insulin Resistance.
 
Tian XY(1), Ganeshan K(1), Hong C(2), Nguyen KD(1), Qiu Y(1), Kim J(2), Tangirala
RK(3), Tonotonoz P(4), Chawla A(5).
 
Chronic, low-grade inflammation triggered by excess intake of dietary lipids has 
been proposed to contribute to the pathogenesis of metabolic disorders, such as
obesity, insulin resistance, type 2 diabetes, and atherosclerosis. Although
considerable evidence supports a causal association between inflammation and
metabolic diseases, most tests of this link have been performed in cold-stressed 
mice that are housed below their thermoneutral zone. We report here that
thermoneutral housing of mice has a profound effect on the development of
metabolic inflammation, insulin resistance, and atherosclerosis. Mice housed at
thermoneutrality develop metabolic inflammation in adipose tissue and in the
vasculature at an accelerated rate. Unexpectedly, this increased inflammatory
response contributes to the progression of atherosclerosis but not insulin
resistance. These findings not only suggest that metabolic inflammation can be
uncoupled from obesity-associated insulin resistance, but also point to how
thermal stress might limit our ability to faithfully model human diseases in
mice.
 
PMCID: PMC4715491 [Available on 2017-01-12]
PMID: 26549485
 
-------------
[8] J Gerontol A Biol Sci Med Sci. 2008 May;63(5):447-53.
 
Adiponectin levels and genotype: a potential regulator of life span in humans.
 
Atzmon G(1), Pollin TI, Crandall J, Tanner K, Schechter CB, Scherer PE, Rincon M,
Siegel G, Katz M, Lipton RB, Shuldiner AR, Barzilai N.
 
Author information: 
(1)Institute for Aging Research, Diabetes Research and Training Center,
Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, NY 10461,
USA.
 
Although caloric restriction in numerous models extends life, longevity in humans
is suggested to be limited by the increased prevalence of obesity. Adiponectin, a
fat-derived peptide, has a protective role against age-related disease, and thus 
is an excellent candidate gene for longevity. We studied adiponectin levels in
centenarians (n = 118), their offspring (n = 228), and unrelated participants <95
(n = 78). Adiponectin levels were significantly greater in participants older
than 95 years (p =.01), an effect that was independent of sex and body mass index
(BMI). Adiponectin levels in the offspring were higher (following adjustment for 
age, sex, and BMI) compared to controls (p =.02), suggesting that inherited
factors play a role in determining adiponectin levels. Over-representation of two
common variants in Adiponectin gene (ADIPOQ) in male long-lived individuals
combined with their independent association with elevated plasma adiponectin
levels (in men and women) suggests that their presence may promote increased life
span through the regulation of adiponectin production and/or secretion.
 
PMCID: PMC4507412
PMID: 18511746
 
----------
[9] Science. 2006 Nov 3;314(5800):825-8.
 
Transgenic mice with a reduced core body temperature have an increased life span.
 
Conti B(1), Sanchez-Alavez M, Winsky-Sommerer R, Morale MC, Lucero J, Brownell S,
Fabre V, Huitron-Resendiz S, Henriksen S, Zorrilla EP, de Lecea L, Bartfai T.
 
Author information: 
(1)Harold L. Dorris Neurological Research Center, Scripps Research Institute, La 
Jolla, CA 92037, USA. bconti@scripps.edu
 
 
Reduction of core body temperature has been proposed to contribute to the
increased life span and the antiaging effects conferred by calorie restriction
(CR). Validation of this hypothesis has been difficult in homeotherms, primarily 
due to a lack of experimental models. We report that transgenic mice engineered
to overexpress the uncoupling protein 2 in hypocretin neurons (Hcrt-UCP2) have
elevated hypothalamic temperature. The effects of local temperature elevation on 
the central thermostat resulted in a 0.3 degrees to 0.5 degrees C reduction of
the core body temperature. Fed ad libitum, Hcrt-UCP2 transgenic mice had the same
caloric intake as their wild-type littermates but had increased energy efficiency
and a greater median life span (12% increase in males; 20% increase in females). 
Thus, modest, sustained reduction of core body temperature prolonged life span
independent of altered diet or CR.
 
PMID: 17082459 
 
----------
[10] Exp Gerontol. 2007 Aug;42(8):733-44. Epub 2007 Jun 6.
 
Adipogenic signaling in rat white adipose tissue: modulation by aging and calorie
restriction.
 
Zhu M(1), Lee GD, Ding L, Hu J, Qiu G, de Cabo R, Bernier M, Ingram DK, Zou S.
 
Author information: 
(1)Laboratory of Experimental Gerontology, National Institute on Aging, 6200
Seaforth Street, Baltimore, MD 21224, USA.
 
Alterations in adipogenesis could have significant impact on several aging
processes. We previously reported that calorie restriction (CR) in rats
significantly increases the level of circulating adiponectin, a distinctive
marker of differentiated adipocytes, leading to a concerted modulation in the
expression of key transcription target genes and, as a result, to increased fatty
acid oxidation and reduced deleterious lipid accumulation in other tissues. These
findings led us to investigate further the effects of aging on adipocytes and to 
determine how CR modulates adipogenic signaling in vivo. CR for 2 and 25 months, 
significantly increased the expression of PPARgamma, C/EBPbeta and Cdk-4, and
partially attenuated age-related decline in C/EBPalpha expression relative to
rats fed ad libitum (AL). As a result, adiponectin was upregulated at both mRNA
and protein levels, resulting in activation of target genes involved in fatty
acid oxidation and fatty acid synthesis, and greater responsiveness of adipose
tissue to insulin. Moreover, CR significantly decreased the ratio of C/EBPbeta
isoforms LAP/LIP, suggesting the suppression of gene transcription associated
with terminal differentiation while facilitating preadipocytes proliferation.
Morphometric analysis revealed a greater number of small adipocytes in CR
relative to AL feeding. Immunostaining confirmed that small adipocytes were more 
strongly positive for adiponectin than the large ones. Overall these results
suggest that CR increased the expression of adipogenic factors, and maintained
the differentiated state of adipocytes, which is critically important for
adiponectin biosynthesis and insulin sensitivity.
 
PMCID: PMC1978194
PMID: 17624709
 
----------
[11] Cell Metab. 2009 Feb;9(2):111-2. doi: 10.1016/j.cmet.2009.01.004.
 
Why we should put clothes on mice.
 
Lodhi IJ(1), Semenkovich CF.
 
Author information: 
(1)Division of Endocrinology, Metabolism, and Lipid Research, Department of
Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
 
Comment on
    Cell Metab. 2009 Feb;9(2):203-9.
 
Mitochondrial uncoupling protein 1 (UCP1) is a key regulator of adaptive
thermogenesis and energy expenditure. Mice lacking UCP1 are cold sensitive, but
surprisingly not obese at room temperature. In this issue of Cell Metabolism,
Feldmann et al. (2009) unmask an obesogenic phenotype by simply maintaining these
mice at thermoneutrality.
 
PMID: 19187768 
 
---------------
[12] J Exp Med. 2012 Jun 4;209(6):1069-74. doi: 10.1084/jem.20120988.
 
Unstressing intemperate models: how cold stress undermines mouse modeling.
 
Karp CL(1).
 
Author information: 
(1)Division of Molecular Immunology, Cincinnati Children's Hospital Research
Foundation, Cincinnati, Ohio 45229, USA.
 
Mus musculus enjoys pride of place at the center of contemporary biomedical
research. Despite being the current model system of choice for in vivo
mechanistic analysis, mice have clear limitations. The literature is littered
with examples of therapeutic approaches that showed promise in mouse models but
failed in clinical trials. More generally, mice often provide poor mimics of the 
human diseases being modeled. Available data suggest that the cold stress to
which laboratory mice are ubiquitously subjected profoundly affects mouse
physiology in ways that impair the modeling of human homeostasis and disease.
Experimental attention to this key, albeit largely ignored, environmental
variable is likely to have a broad transformative effect on biomedical research.
 
PMCID: PMC3371737
PMID: 22665703
 
---------
[13] Int J Hyperthermia. 2014 Dec;30(8):540-6. doi: 10.3109/02656736.2014.981300.
 
Housing temperature influences the pattern of heat shock protein induction in
mice following mild whole body hyperthermia.
 
Eng JW(1), Reed CB, Kokolus KM, Repasky EA.
 
Author information: 
(1)Department of Immunology, Roswell Park Cancer Institute , Buffalo, New York , 
USA.
 
PURPOSE: Researchers studying the murine response to stress generally use mice
housed under standard, nationally mandated conditions as controls. Few
investigators are concerned whether basic physical aspects of mouse housing could
be an additional source of stress, capable of influencing the subsequent impact
of an experimentally applied stressor. We have recently become aware of the
potential for housing conditions to impact important physiological and
immunological properties in mice.
MATERIALS AND METHODS: Here we sought to determine whether housing mice at
standard temperature (ST; 22 °C) vs. thermoneutral temperature (TT; 30 °C)
influences baseline expression of heat shock proteins (HSPs) and their typical
induction following a whole body heating.
RESULTS: There were no significant differences in baseline expression of HSPs at 
ST and TT. However, in several cases, the induction of Hsp70, Hsp110 and Hsp90 in
tissues of mice maintained at ST was greater than at TT following 6 h of heating 
(which elevated core body temperature to 39.5 °C). This loss of HSP induction was
also seen when mice housed at ST were treated with propranolol, a β-adrenergic
receptor antagonist, used clinically to treat hypertension and stress.
CONCLUSIONS: Taken together, these data show that housing temperature
significantly influences the expression of HSPs in mice after whole body heating 
and thus should be considered when stress responses are studied in mice.
 
PMCID: PMC4340593
PMID: 25430986
 
-----------
[14] J Gerontol A Biol Sci Med Sci. 2015 Feb;70(2):155-62. doi: 10.1093/gerona/glu015.
Epub 2014 Feb 14.
 
Muscle heat shock protein 70 predicts insulin resistance with aging.
 
Chichester L(1), Wylie AT(1), Craft S(2), Kavanagh K(3).
 
Author information: 
(1)Departments of Pathology and. (2)Internal Medicine, Wake Forest School of
Medicine, Winston-Salem, North Carolina. (3)Departments of Pathology and
kkavanag@wakehealth.edu.
 
Heat shock protein 70 (HSP70) protects cells from accumulating damaged proteins
and age-related functional decline. We studied plasma and skeletal muscle (SkM)
HSP70 levels in adult vervet monkeys (life span ≈ 25 years) at baseline and after
4 years (≈10 human years). Insulin, glucose, homeostasis model assessment scores,
triglycerides, high-density lipoprotein and total plasma cholesterol, body
weight, body mass index, and waist circumference were measured repeatedly, with
change over time estimated by individual regression slopes. Low baseline SkM
HSP70 was a proximal marker for developing insulin resistance and was seen in
monkeys whose insulin and homeostasis model assessment increased more rapidly
over time. Changes in SkM HSP70 inversely correlated with insulin and homeostasis
model assessment trajectories such that a positive change in SkM level was
beneficial. The strength of the relationship between changes in SkM HSP70 and
insulin remained unchanged after adjustment for all covariates. Younger monkeys
drove these relationships, with HSP70 alone being predictive of insulin changes
with aging. Plasma and SkM HSP70 were unrelated and HSP70 release from peripheral
blood mononuclear cells was positively associated with insulin concentrations in 
contrast to SkM. Results from aged humans confirmed this positive association of 
plasma HSP70 and insulin. In conclusion, higher levels of SkM HSP70 protect
against insulin resistance development during healthy aging.
 
© The Author 2014. Published by Oxford University Press on behalf of The
Gerontological Society of America. All rights reserved. For permissions, please
e-mail: journals.permissions@oup.com.
 
PMCID: PMC4311181
PMID: 24532784
 
-------------
[15] Ageing Res Rev. 2011 Jan;10(1):153-62. doi: 10.1016/j.arr.2010.10.001. Epub 2010 
Oct 20.
 
Caloric restriction and longevity: effects of reduced body temperature.
 
Carrillo AE(1), Flouris AD.
 
Author information: 
(1)FAME Laboratory, Institute of Human Performance and Rehabilitation, Centre for
Research and Technology Thessaly, Trikala, Greece.
 
Caloric restriction (CR) causes a reduction in body temperature (T(b)) which is
suggested to contribute to changes that increase lifespan. Moreover, low T(b) has
been shown to improve health and longevity independent of CR. In this review we
examine the connections between CR, T(b) and mechanisms that influence longevity 
and ageing. Recent findings regarding the overlapping mechanisms of CR and T(b)
that benefit longevity are discussed, including changes in body composition,
hormone regulation, and gene expression, as well as reductions in low-level
inflammation and reactive oxygen species-induced molecular damage. This
information is summarized in a model describing how CR and low T(b), both
synergistically and independently, increase lifespan. Moreover, the nascent
notion that the rate of ageing may be pre-programmed in response to environmental
influences at critical periods of early development is also considered. Based on 
current evidence, it is concluded that low T(b) plays an integral role in
mediating the effects of CR on health and longevity, and that low T(b) may exert 
independent biological changes that increase lifespan. Our understanding of the
overlap between CR- and T(b)-mediated longevity remains incomplete and should be 
explored in future research.
 
Copyright © 2010 Elsevier B.V. All rights reserved.
 
PMID: 20969980
 
-------------
[16] Nat Rev Mol Cell Biol. 2010 Aug;11(8):545-55. doi: 10.1038/nrm2938. Epub 2010 Jul
14.
 
Heat shock factors: integrators of cell stress, development and lifespan.
 
Akerfelt M(1), Morimoto RI, Sistonen L.
 
Author information: 
(1)Department of Biosciences, Abo Akademi University, BioCity, 20520 Turku,
Finland.
 
Heat shock factors (HSFs) are essential for all organisms to survive exposures to
acute stress. They are best known as inducible transcriptional regulators of
genes encoding molecular chaperones and other stress proteins. Four members of
the HSF family are also important for normal development and lifespan-enhancing
pathways, and the repertoire of HSF targets has thus expanded well beyond the
heat shock genes. These unexpected observations have uncovered complex layers of 
post-translational regulation of HSFs that integrate the metabolic state of the
cell with stress biology, and in doing so control fundamental aspects of the
health of the proteome and ageing.
 
PMCID: PMC3402356
PMID: 20628411

 

-----------

[17] Age (Dordr). 2013 Aug;35(4):1367-76. doi: 10.1007/s11357-012-9417-7. Epub 2012

May 4.
 
Association of heat shock proteins with all-cause mortality.
 
Broer L(1), Demerath EW, Garcia ME, Homuth G, Kaplan RC, Lunetta KL, Tanaka T,
Tranah GJ, Walter S, Arnold AM, Atzmon G, Harris TB, Hoffmann W, Karasik D, Kiel 
DP, Kocher T, Launer LJ, Lohman KK, Rotter JI, Tiemeier H, Uitterlinden AG,
Wallaschofski H, Bandinelli S, Dörr M, Ferrucci L, Franceschini N, Gudnason V,
Hofman A, Liu Y, Murabito JM, Newman AB, Oostra BA, Psaty BM, Smith AV, van Duijn
CM.
 
Author information: 
(1)Department of Epidemiology, Erasmus Medical Center, Dr. Molewaterplein 50,
PO-Box 2040, 3000, CA, Rotterdam, The Netherlands.
 
Experimental mild heat shock is widely known as an intervention that results in
extended longevity in various models along the evolutionary lineage. Heat shock
proteins (HSPs) are highly upregulated immediately after a heat shock. The
elevation in HSP levels was shown to inhibit stress-mediated cell death, and
recent experiments indicate a highly versatile role for these proteins as
inhibitors of programmed cell death. In this study, we examined common genetic
variations in 31 genes encoding all members of the HSP70, small HSP, and heat
shock factor (HSF) families for their association with all-cause mortality. Our
discovery cohort was the Rotterdam study (RS1) containing 5,974 participants aged
55 years and older (3,174 deaths). We assessed 4,430 single nucleotide
polymorphisms (SNPs) using the HumanHap550K Genotyping BeadChip from Illumina.
After adjusting for multiple testing by permutation analysis, three SNPs showed
evidence for association with all-cause mortality in RS1. These findings were
followed in eight independent population-based cohorts, leading to a total of
25,007 participants (8,444 deaths). In the replication phase, only HSF2
(rs1416733) remained significantly associated with all-cause mortality. Rs1416733
is a known cis-eQTL for HSF2. Our findings suggest a role of HSF2 in all-cause
mortality.
 
PMCID: PMC3705092
PMID: 22555621
 
---------
[18] Sørensen, J. G., Kristensen, T. N. and Loeschcke, V. (2003), The evolutionary and ecological role of heat shock proteins. Ecology Letters, 6: 1025–1037. doi:10.1046/j.1461-0248.2003.00528.x
 
------------
[19] Dev Genet. 1996;18(2):114-24.
 
Effect of caloric restriction on the expression of heat shock protein 70 and the 
activation of heat shock transcription factor 1.
 
Heydari AR(1), You S, Takahashi R, Gutsmann A, Sarge KD, Richardson A.
 
Author information: 
(1)Geriatric Research, Education and Clinical Center, Audie L. Murphy Memorial
Veterans Hospital, San Antonio, TX 78284, USA.
 
The regulation of heat shock protein 70 (hsp70) expression is an excellent
example of a cellular mechanism that has evolved to protect all living organisms 
from various types of physiological stresses; therefore, the reported age-related
alterations in the ability of cells to express hsp70 in response to stress could 
seriously compromise the ability of a senescent organism in respond to changes in
its environment. Because caloric restriction (CR) is the only experimental
manipulation known to retard aging and increase the survival of rodents, it was
of interest to analyze the effect of CR on the age-related alteration in the
induction of hsp70 expression in rat hepatocytes. The effect of CR on the nuclear
transcription of hsp70 gene in rat hepatocytes in response to various levels of
heat shock was determined, and it was found that the age-related decline in the
transcription of hsp70 at all temperatures studied was reversed by CR. Because
the heat shock transcription factor (HSF) mediates the heat-induced transcription
of hsp70, the effect of CR on the induction of HSF binding activity by heat shock
was studied and found to arise from HSF1, which has been shown to be involved in 
the induction of HSF binding activity in other cell types. The age-related
decrease in the induction of HSF1 binding activity in rat hepatocytes was
reversed by CR, and did not appear to be due to an accumulation of inhibitory
molecules with age. Interestingly, the level of HSF1 protein was significantly
higher in hepatocytes isolated from old rats fed ad libitum compared to
hepatocytes obtained from rats fed the CR diet even though the levels of HSF1
binding activity were lower for hepatocytes isolated from the old rats fed ad
libitum. The levels of the mRNA transcript for HSF1 was not significantly altered
by age or CR. Thus, the changes in HSF1 binding activity with age and CR do not
arise from changes in the level of HSF1 protein available for activation.
 
PMID: 8934873
 
---------
[20] FASEB J. 2000 Jan;14(1):78-86.
 
Caloric restriction improves thermotolerance and reduces hyperthermia-induced
cellular damage in old rats.
 
Hall DM(1), Oberley TD, Moseley PM, Buettner GR, Oberley LW, Weindruch R, Kregel 
KC.
 
Author information: 
(1)Department of Exercise Science, The Free Radical Research Institute, and.
Radiation Research Laboratory, The University of Iowa, Iowa City, Iowa 52242,
USA.
 
Adult-onset, long-term caloric restriction (CR) prolongs maximum life span in
laboratory rodents. However, the effect of this intervention on an organism's
ability to cope with a physical challenge has not been explored. We investigated 
the influence of CR and aging on stress tolerance in old rats exposed to an
environmental heating protocol on two consecutive days. We hypothesized that CR
would increase heat tolerance by reducing cellular stress and subsequent accrual 
of oxidative injury. All calorically restricted rats survived both heat exposures
compared with only 50% of their control-fed counterparts. CR also decreased
heat-induced radical generation, stress protein accumulation, and cellular injury
in the liver. In addition, heat stress stimulated marked induction of the
antioxidant enzymes manganese-containing superoxide dismutase and catalase, along
with strong nuclear catalase expression in liver samples from rats subjected to
CR. In contrast, stress-related induction of antioxidant enzymes was blunted, and
nuclear catalase expression was unchanged from euthermic conditions in the
control-fed group. These data suggest that CR reduces cellular injury and
improves heat tolerance of old animals by lowering radical production and
preserving cellular ability to adapt to stress through antioxidant enzyme
induction and translocation of these proteins to the nucleus.
 
PMID: 10627282
 
------------
[21] Exp Gerontol. 2005 Jan-Feb;40(1-2):37-42.
 
Life long calorie restriction increases heat shock proteins and proteasome
activity in soleus muscles of Fisher 344 rats.
 
Selsby JT(1), Judge AR, Yimlamai T, Leeuwenburgh C, Dodd SL.
 
Author information: 
(1)Muscle Physiology Laboratory, Department of Applied Physiology and
Kinesiology, University of Florida, P.O. Box 118205, Gainesville, FL 32611, USA.
 
Heat shock proteins (HSP's) closely interact with 20S proteasome and have been
shown to maintain catalytic activity, responsible for the prevention of protein
aggregation. A decrease in both proteasome activity and heat shock proteins
(HSP's) has been observed with age. We investigated whether life-long calorie
restriction (CR), a natural intervention, which prolongs life span, could prevent
the age-associated decline in HSP's and restore the proteolytic activity of the
20S proteasome in skeletal muscle. Hence, we investigated HSP's and proteasome
activity in the soleus muscle from 12-mo-old (Adult) and 26-28 mo old ad libitum 
fed (Old), and 26-28 mo old CR (Old-CR; fed 40% of ad libitum for their lifespan)
male Fisher 344 rats. Trypsin-like proteasome activity in Old rats was
significantly less than both Adult and Old-CR rats. Furthermore, no significant
changes where found in chymotrypsin-like proteasome activity due to age or diet. 
Levels of HSP 72 and 25 were significantly less in Old animals when compared to
both Adult and Old-CR rats. In contrast, HSP 90 was elevated in Old rats by 220% 
compared to adult animals and life-long calorie restriction caused a significant 
induction (150%) compared to age-matched ad libitum fed animals. Protein
carbonyls were significantly elevated in Old when compared to Adult rats, but
showed no significant decline due to life long CR. This study shows that HSP's
may be largely responsible for the restoration of the trypsin-like activity of
the 20S proteasome with age. The large increase in HSP 90 is intriguing and
further studies are required to elucidate its role in maintaining 20S proteasome 
function.
 
PMID: 15664730
 
--------
[22] Circ Res. 2016 Jan 8;118(1):173-82. doi: 10.1161/CIRCRESAHA.115.306647.
 
Role of Brown Fat in Lipoprotein Metabolism and Atherosclerosis.
 
Hoeke G(1), Kooijman S(1), Boon MR(1), Rensen PC(1), Berbée JF(1).
 
Author information: 
(1)From the Department of Medicine, Division of Endocrinology and Einthoven
Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 
Leiden, The Netherlands.
 
Atherosclerosis, for which hyperlipidemia is a major risk factor, is the leading 
cause of morbidity and mortality in Western society, and new therapeutic
strategies are highly warranted. Brown adipose tissue (BAT) is metabolically
active in human adults. Although positron emission tomography-computed tomography
using a glucose tracer is the golden standard to visualize and quantify the
volume and activity of BAT, it has become clear that activated BAT combusts fatty
acids rather than glucose. Here, we review the role of brown and beige adipocytes
in lipoprotein metabolism and atherosclerosis, with evidence derived from both
animal and human studies. On the basis of mainly data from animal models, we
propose a model in which activated brown adipocytes use their intracellular
triglyceride stores to generate fatty acids for combustion. BAT rapidly
replenishes these stores by internalizing primarily lipoprotein
triglyceride-derived fatty acids, generated by lipoprotein lipase-mediated
hydrolysis of triglycerides, rather than by holoparticle uptake. As a
consequence, BAT activation leads to the generation of lipoprotein remnants that 
are subsequently cleared via the liver provided that an intact apoE-low-density
lipoprotein receptor pathway is present. Through these mechanisms, BAT activation
reduces plasma triglyceride and cholesterol levels and attenuates diet-induced
atherosclerosis development. Initial studies suggest that BAT activation in
humans may also reduce triglyceride and cholesterol levels, but potential
antiatherogenic effects should be assessed in future studies.
 
© 2016 American Heart Association, Inc.
 
PMID: 26837747
 
----------
[23] Cell Metab. 2014 Feb 4;19(2):302-9. doi: 10.1016/j.cmet.2013.12.017.
 
Irisin and FGF21 are cold-induced endocrine activators of brown fat function in
humans.
 
Lee P(1), Linderman JD(1), Smith S(1), Brychta RJ(1), Wang J(1), Idelson C(1),
Perron RM(1), Werner CD(1), Phan GQ(2), Kammula US(2), Kebebew E(3), Pacak K(4), 
Chen KY(1), Celi FS(5).
 
Comment in
    Cell Metab. 2014 Mar 4;19(3):352-4.
 
Rediscovery of cold-activated brown adipose tissue (BAT) in humans has boosted
research interest in identifying BAT activators for metabolic benefits. Of
particular interest are cytokines capable of fat browning. Irisin, derived from
FNDC5, is an exercise-induced myokine that drives brown-fat-like thermogenesis in
murine white fat. Here we explored whether cold exposure is an afferent signal
for irisin secretion in humans and compared it with FGF21, a brown adipokine in
rodents. Cold exposure increased circulating irisin and FGF21. We found an
induction of irisin secretion proportional to shivering intensity, in magnitude
similar to exercise-stimulated secretion. FNDC5 and/or FGF21 treatment
upregulated human adipocyte brown fat gene/protein expression and thermogenesis
in a depot-specific manner. These results suggest exercise-induced irisin
secretion could have evolved from shivering-related muscle contraction, serving
to augment brown fat thermogenesis in concert with FGF21. Irisin-mediated
muscle-adipose crosstalk may represent a thermogenic, cold-activated endocrine
axis that is exploitable in obesity therapeutics development.
 
Copyright © 2014 Elsevier Inc. All rights reserved.
 
PMID: 24506871
 
-------------
[24] Proc Natl Acad Sci U S A. 2015 Sep 29;112(39):12157-62. doi:
10.1073/pnas.1516622112. Epub 2015 Sep 15.
 
The myokine irisin increases cortical bone mass.
 
Colaianni G(1), Cuscito C(1), Mongelli T(1), Pignataro P(1), Buccoliero C(1), Liu
P(2), Lu P(2), Sartini L(3), Di Comite M(1), Mori G(4), Di Benedetto A(4),
Brunetti G(1), Yuen T(2), Sun L(2), Reseland JE(5), Colucci S(1), New MI(6),
Zaidi M(6), Cinti S(3), Grano M(7).
 
Erratum in
    Proc Natl Acad Sci U S A. 2015 Oct 20;112(42):E5763.
 
Comment in
    Nat Rev Endocrinol. 2015 Dec;11(12):689.
 
It is unclear how physical activity stimulates new bone synthesis. We explored
whether irisin, a newly discovered myokine released upon physical activity,
displays anabolic actions on the skeleton. Young male mice were injected with
vehicle or recombinant irisin (r-irisin) at a low cumulative weekly dose of 100
µg kg(-1). We observed significant increases in cortical bone mass and strength, 
notably in cortical tissue mineral density, periosteal circumference, polar
moment of inertia, and bending strength. This anabolic action was mediated
primarily through the stimulation of bone formation, but with parallel notable
reductions in osteoclast numbers. The trabecular compartment of the same bones
was spared, as were vertebrae from the same mice. Higher irisin doses (3,500 µg
kg(-1) per week) cause browning of adipose tissue; this was not seen with
low-dose r-irisin. Expectedly, low-dose r-irisin modulated the skeletal genes,
Opn and Sost, but not Ucp1 or Pparγ expression in white adipose tissue. In bone
marrow stromal cell cultures, r-irisin rapidly phosphorylated Erk, and
up-regulated Atf4, Runx2, Osx, Lrp5, β-catenin, Alp, and Col1a1; this is
consistent with a direct receptor-mediated action to stimulate osteogenesis. We
also noted that, although the irisin precursor Fndc5 was expressed abundantly in 
skeletal muscle, other sites, such as bone and brain, also expressed Fndc5,
albeit at low levels. Furthermore, muscle fibers from r-irisin-injected mice
displayed enhanced Fndc5 positivity, and irisin induced Fdnc5 mRNA expression in 
cultured myoblasts. Our data therefore highlight a previously unknown action of
the myokine irisin, which may be the molecular entity responsible for muscle-bone
connectivity.
 
PMCID: PMC4593131 [Available on 2016-03-29]
PMID: 26374841
 
------------
[25] Am J Med. 2014 Sep;127(9):888-90. doi: 10.1016/j.amjmed.2014.04.025. Epub 2014
May 9.
 
Serum irisin levels, precocious myocardial infarction, and healthy exceptional
longevity.
 
Emanuele E(1), Minoretti P(2), Pareja-Galeano H(3), Sanchis-Gomar F(3),
Garatachea N(4), Lucia A(5).
 
BACKGROUND: Skeletal muscles produce irisin. Growing controversy exists on the
association between this myokine and chronic disease risk. On the basis of the
potential protective effects that irisin could exert on both vascular function
and skeletal muscle mass, we hypothesized that an elevated level of this molecule
may contribute to successful aging.
METHODS: Serum irisin levels were measured using enzyme-linked immunosorbent
assay in disease-free centenarians, young healthy controls, and patients with
precocious acute myocardial infarction.
RESULTS: We found the highest levels of serum irisin in disease-free centenarians
(35.3 ± 5.5 ng/mL) compared with young healthy controls (20.7 ± 6.3 ng/mL) and
especially with young patients with acute myocardial infarction (15.1 ± 5.4
ng/mL).
CONCLUSIONS: Our study demonstrates that healthy centenarians are characterized
by increased serum irisin levels, whereas levels of this molecule were found to
be significantly lower in young patients with myocardial infarction. Our findings
may prompt further research into the role played by irisin not only in vascular
disorders but also in life span modulation.
 
Copyright © 2014 Elsevier Inc. All rights reserved.
 
PMID: 24813865
 
---------
[26] Mol Metab. 2014 Dec 18;4(2):118-31. doi: 10.1016/j.molmet.2014.12.008.
eCollection 2015.
 
SIRT1 enhances glucose tolerance by potentiating brown adipose tissue function.
 
Boutant M(1), Joffraud M(1), Kulkarni SS(1), García-Casarrubios E(2),
García-Roves PM(3), Ratajczak J(4), Fernández-Marcos PJ(5), Valverde AM(2),
Serrano M(5), Cantó C(1).
 
OBJECTIVE: SIRT1 has been proposed to be a key signaling node linking changes in 
energy metabolism to transcriptional adaptations. Although SIRT1 overexpression
is protective against diverse metabolic complications, especially in response to 
high-fat diets, studies aiming to understand the etiology of such benefits are
scarce. Here, we aimed to identify the key tissues and mechanisms implicated in
the beneficial effects of SIRT1 on glucose homeostasis.
METHODS: We have used a mouse model of moderate SIRT1 overexpression, under the
control of its natural promoter, to evaluate glucose homeostasis and thoroughly
characterize how different tissues could influence insulin sensitivity.
RESULTS: Mice with moderate overexpression of SIRT1 exhibit better glucose
tolerance and insulin sensitivity even on a low fat diet.
Euglycemic-hyperinsulinemic clamps and in-depth tissue analyses revealed that
enhanced insulin sensitivity was achieved through a higher brown adipose tissue
activity and was fully reversed by housing the mice at thermoneutrality. SIRT1
did not influence brown adipocyte differentiation, but dramatically enhanced the 
metabolic transcriptional responses to β3-adrenergic stimuli in differentiated
adipocytes.
CONCLUSIONS: Our work demonstrates that SIRT1 improves glucose homeostasis by
enhancing BAT function. This is not consequent to an alteration in the brown
adipocyte differentiation process, but as a result of potentiating the response
to β3-adrenergic stimuli.
 
PMCID: PMC4314542
PMID: 256856994
 
-----------------
[27] Trends Endocrinol Metab. 2009 Sep;20(7):325-31. doi: 10.1016/j.tem.2009.03.008.
Epub 2009 Aug 25.
 
Caloric restriction, SIRT1 and longevity.
 
Cantó C(1), Auwerx J.
 
Author information: 
(1)Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
 
More than 70 years after its initial report, caloric restriction stands strong as
the most consistent non-pharmacological intervention increasing lifespan and
protecting against metabolic disease. Among the different mechanisms by which
caloric restriction might act, Sir2/SIRT1 (Silent information regulator 2/Silent 
information regulator T1) has been the focus of much attention because of its
ability to integrate sensing of the metabolic status with adaptive
transcriptional outputs. This review focuses on gathered evidence suggesting that
Sir2/SIRT1 is a key mediator of the beneficial effects of caloric restriction and
addresses the main questions that still need to be answered to consolidate this
hypothesis.
 
PMCID: PMC3627124
PMID: 19713122 
 
 
-------------
[28] J Clin Invest. 2011 Nov;121(11):4281-8. doi: 10.1172/JCI58554. Epub 2011 Oct 10.
 
Sirt1 enhances skeletal muscle insulin sensitivity in mice during caloric
restriction.
 
Schenk S(1), McCurdy CE, Philp A, Chen MZ, Holliday MJ, Bandyopadhyay GK, Osborn 
O, Baar K, Olefsky JM.
 
Author information: 
(1)Division of Endocrinology and Metabolism, Department of Medicine, University
of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673,
USA.
 
Skeletal muscle insulin resistance is a key component of the etiology of type 2
diabetes. Caloric restriction (CR) enhances the sensitivity of skeletal muscle to
insulin. However, the molecular signals within skeletal muscle linking CR to
improved insulin action remain largely unknown. Recently, the mammalian ortholog 
of Sir2, sirtuin 1 (Sirt1), has been identified as a potential transducer of
perturbations in cellular energy flux into subsequent metabolic adaptations,
including modulation of skeletal muscle insulin action. Here, we have
demonstrated that CR increases Sirt1 deacetylase activity in skeletal muscle in
mice, in parallel with enhanced insulin-stimulated phosphoinositide 3-kinase
(PI3K) signaling and glucose uptake. These adaptations in skeletal muscle insulin
action were completely abrogated in mice lacking Sirt1 deacetylase activity.
Mechanistically, Sirt1 was found to be required for the deacetylation and
inactivation of the transcription factor Stat3 during CR, which resulted in
decreased gene and protein expression of the p55α/p50α subunits of PI3K, thereby 
promoting more efficient PI3K signaling during insulin stimulation. Thus, these
data demonstrate that Sirt1 is an integral signaling node in skeletal muscle
linking CR to improved insulin action, primarily via modulation of PI3K
signaling.
 
PMCID: PMC3204844
PMID: 21985785
 
------------
[29] Mol Metab. 2014 Dec 18;4(2):118-31. doi: 10.1016/j.molmet.2014.12.008.
eCollection 2015.
 
SIRT1 enhances glucose tolerance by potentiating brown adipose tissue function.
 
Boutant M(1), Joffraud M(1), Kulkarni SS(1), García-Casarrubios E(2),
García-Roves PM(3), Ratajczak J(4), Fernández-Marcos PJ(5), Valverde AM(2),
Serrano M(5), Cantó C(1).
 
OBJECTIVE: SIRT1 has been proposed to be a key signaling node linking changes in 
energy metabolism to transcriptional adaptations. Although SIRT1 overexpression
is protective against diverse metabolic complications, especially in response to 
high-fat diets, studies aiming to understand the etiology of such benefits are
scarce. Here, we aimed to identify the key tissues and mechanisms implicated in
the beneficial effects of SIRT1 on glucose homeostasis.
METHODS: We have used a mouse model of moderate SIRT1 overexpression, under the
control of its natural promoter, to evaluate glucose homeostasis and thoroughly
characterize how different tissues could influence insulin sensitivity.
RESULTS: Mice with moderate overexpression of SIRT1 exhibit better glucose
tolerance and insulin sensitivity even on a low fat diet.
Euglycemic-hyperinsulinemic clamps and in-depth tissue analyses revealed that
enhanced insulin sensitivity was achieved through a higher brown adipose tissue
activity and was fully reversed by housing the mice at thermoneutrality. SIRT1
did not influence brown adipocyte differentiation, but dramatically enhanced the 
metabolic transcriptional responses to β3-adrenergic stimuli in differentiated
adipocytes.
CONCLUSIONS: Our work demonstrates that SIRT1 improves glucose homeostasis by
enhancing BAT function. This is not consequent to an alteration in the brown
adipocyte differentiation process, but as a result of potentiating the response
to β3-adrenergic stimuli.
 
PMCID: PMC4314542
PMID: 25685699
 
--------
[30] Cell Metab. 2013 Sep 3;18(3):416-30. doi: 10.1016/j.cmet.2013.07.013.
 
Sirt1 extends life span and delays aging in mice through the regulation of Nk2
homeobox 1 in the DMH and LH.
 
Satoh A(1), Brace CS, Rensing N, Cliften P, Wozniak DF, Herzog ED, Yamada KA,
Imai S.
 
 
The mammalian Sir2 ortholog Sirt1 plays an important role in metabolic
regulation. However, the role of Sirt1 in the regulation of aging and longevity
is still controversial. Here we demonstrate that brain-specific
Sirt1-overexpressing (BRASTO) transgenic mice show significant life span
extension in both males and females, and aged BRASTO mice exhibit phenotypes
consistent with a delay in aging. These phenotypes are mediated by enhanced
neural activity specifically in the dorsomedial and lateral hypothalamic nuclei
(DMH and LH, respectively), through increased orexin type 2 receptor (Ox2r)
expression. We identified Nk2 homeobox 1 (Nkx2-1) as a partner of Sirt1 that
upregulates Ox2r transcription and colocalizes with Sirt1 in the DMH and LH.
DMH/LH-specific knockdown of Sirt1, Nkx2-1, or Ox2r and DMH-specific Sirt1
overexpression further support the role of Sirt1/Nkx2-1/Ox2r-mediated signaling
for longevity-associated phenotypes. Our findings indicate the importance of
DMH/LH-predominant Sirt1 activity in the regulation of aging and longevity in
mammals.
 
Copyright © 2013 Elsevier Inc. All rights reserved.
 
PMCID: PMC3794712
PMID: 24011076
 
-----------
[31] PLoS One. 2015 Mar 18;10(3):e0117954. doi: 10.1371/journal.pone.0117954.
eCollection 2015.
 
A remarkable age-related increase in SIRT1 protein expression against oxidative
stress in elderly: SIRT1 gene variants and longevity in human.
 
Kilic U(1), Gok O(1), Erenberk U(2), Dundaroz MR(2), Torun E(2), Kucukardali
Y(3), Elibol-Can B(4), Uysal O(5), Dundar T(6).
 
Aging is defined as the accumulation of progressive organ dysfunction.
Controlling the rate of aging by clarifying the complex pathways has a
significant clinical importance. Nowadays, sirtuins have become famous molecules 
for slowing aging and decreasing age-related disorders. In the present study, we 
analyzed the SIRT1 gene polymorphisms (rs7895833 A>G, rs7069102 C>G and rs2273773
C>T) and its relation with levels of SIRT1, eNOS, PON-1, cholesterol, TAS, TOS,
and OSI to demonstrate the association between genetic variation in SIRT1 and
phenotype at different ages in humans. We observed a significant increase in the 
SIRT1 level in older people and found a significant positive correlation between 
SIRT1 level and age in the overall studied population. The oldest people carrying
AG genotypes for rs7895833 have the highest SIRT1 level suggesting an association
between rs7895833 SNP and lifespan longevity. Older people have lower PON-1
levels than those of adults and children which may explain the high levels of
SIRT1 protein as a compensatory mechanism for oxidative stress in the elderly.
The eNOS protein level was significantly decreased in older people as compared to
adults. There was no significant difference in the eNOS level between older
people and children. The current study is the first to demonstrate age-related
changes in SIRT1 levels in humans and it is important for a much better molecular
understanding of the role of the longevity gene SIRT1 and its protein product in 
aging. It is also the first study presenting the association between SIRT1
expression in older people and rs7895833 in SIRT1 gene.
 
PMCID: PMC4365019
PMID: 25785999
 
---------------
[32] Cell. 2012 Aug 3;150(3):620-32. doi: 10.1016/j.cell.2012.06.027.
 
Brown remodeling of white adipose tissue by SirT1-dependent deacetylation of
Pparγ.
 
Qiang L(1), Wang L, Kon N, Zhao W, Lee S, Zhang Y, Rosenbaum M, Zhao Y, Gu W,
Farmer SR, Accili D.
 
Author information: 
(1)Naomi Berrie Diabetes Center, Department of Medicine, College of Physicians
and Surgeons of Columbia University, New York, NY 10032, USA.
 
Comment in
    Circ Res. 2013 Feb 1;112(3):411-4.
 
Brown adipose tissue (BAT) can disperse stored energy as heat. Promoting BAT-like
features in white adipose (WAT) is an attractive, if elusive, therapeutic
approach to staunch the current obesity epidemic. Here we report that gain of
function of the NAD-dependent deacetylase SirT1 or loss of function of its
endogenous inhibitor Deleted in breast cancer-1 (Dbc1) promote "browning" of WAT 
by deacetylating peroxisome proliferator-activated receptor (Ppar)-γ on Lys268
and Lys293. SirT1-dependent deacetylation of Lys268 and Lys293 is required to
recruit the BAT program coactivator Prdm16 to Pparγ, leading to selective
induction of BAT genes and repression of visceral WAT genes associated with
insulin resistance. An acetylation-defective Pparγ mutant induces a brown
phenotype in white adipocytes, whereas an acetylated mimetic fails to induce
"brown" genes but retains the ability to activate "white" genes. We propose that 
SirT1-dependent Pparγ deacetylation is a form of selective Pparγ modulation of
potential therapeutic import.
 
Copyright © 2012 Elsevier Inc. All rights reserved.
 
PMCID: PMC3413172
PMID: 22863012
 
-------------
[33] Mol Cell. 2011 Dec 23;44(6):851-63. doi: 10.1016/j.molcel.2011.12.005.
 
The cAMP/PKA pathway rapidly activates SIRT1 to promote fatty acid oxidation
independently of changes in NAD(+).
 
Gerhart-Hines Z(1), Dominy JE Jr, Blättler SM, Jedrychowski MP, Banks AS, Lim JH,
Chim H, Gygi SP, Puigserver P.
 
 
Comment in
    Mol Cell. 2012 Jan 13;45(1):9-11.
 
The NAD(+)-dependent deacetylase SIRT1 is an evolutionarily conserved metabolic
sensor of the Sirtuin family that mediates homeostatic responses to certain
physiological stresses such as nutrient restriction. Previous reports have
implicated fluctuations in intracellular NAD(+) concentrations as the principal
regulator of SIRT1 activity. However, here we have identified a cAMP-induced
phosphorylation of a highly conserved serine (S434) located in the SIRT1
catalytic domain that rapidly enhanced intrinsic deacetylase activity
independently of changes in NAD(+) levels. Attenuation of SIRT1 expression or the
use of a nonphosphorylatable SIRT1 mutant prevented cAMP-mediated stimulation of 
fatty acid oxidation and gene expression linked to this pathway. Overexpression
of SIRT1 in mice significantly potentiated the increases in fatty acid oxidation 
and energy expenditure caused by either pharmacological β-adrenergic agonism or
cold exposure. These studies support a mechanism of Sirtuin enzymatic control
through the cAMP/PKA pathway with important implications for stress responses and
maintenance of energy homeostasis.
 
Copyright © 2011 Elsevier Inc. All rights reserved.
 
PMCID: PMC3331675
PMID: 22195961
 
---------
[34] Obesity (Silver Spring). 2016 Mar;24(3):634-42. doi: 10.1002/oby.21393.
 
Diet-induced obesity and insulin resistance are associated with brown fat
degeneration in SIRT1-deficient mice.
 
Xu F(1,)(2), Zheng X(1,)(2), Lin B(1,)(2), Liang H(1,)(2), Cai M(1,)(2), Cao
H(1,)(2), Ye J(3), Weng J(1,)(2).
 
Author information: 
(1)Department of Endocrinology and Metabolism, the Third Affiliated Hospital of
Sun Yat-Sen University, Guangzhou, Guangdong, China. (2)Guangdong Provincial Key 
Laboratory of Diabetology, Guangzhou, Guangdong, China. (3)Antioxidant and Gene
Regulation Laboratory, Pennington Biomedical Research Center, Louisiana State
University System, Baton Rouge, Louisiana, USA.
 
OBJECTIVE: Recent studies have revealed that SIRT1 gain-of-function could promote
adipose tissue browning for the adaptive thermogenesis under normal diet. This
study investigated the role of SIRT1 loss-of-function in diet-induced obesity and
insulin resistance and the mechanism involved in adipose tissue thermogenesis.
METHODS: Male SIRT1(+/-) and wild-type (WT) mice were fed with a high-fat diet
(HFD) for 16 weeks to induce obesity and insulin resistance, while mice on a chow
diet were used as lean controls. The phenotype data were collected, and different
adipose tissue depots were used for mechanism research.
RESULTS: Compared with WT mice, SIRT1(+/-) mice exhibited increased adiposity and
more severe insulin resistance with less thermogenesis under HFD challenge.
Strikingly, SIRT1(+/-) mice displayed an exacerbated brown adipose tissue (BAT)
degeneration phenotype, which was characterized by lower thermogenic activity,
aggravated mitochondrial dysfunction, and more mitochondrial loss. In addition,
SIRT1(+/-) mice showed aggravated inflammation and dysfunction in epididymal
adipose tissue after HFD intervention, which also contributed to the systemic
insulin resistance.
CONCLUSIONS: Diet-induced obesity and insulin resistance are associated with BAT 
degeneration in SIRT1-deficient mice, which further underlined the beneficial
role of SIRT1 in obesity-associated metabolic disorders.
 
© 2016 The Obesity Society.
 
PMID: 26916242
 
--------------
[35] Geriatrics & Gerontology International  Volume 6, Issue 1, pages 32–39, March 2006
DOI: 10.1111/j.1447-0594.2006.00304.x
 
High adiponectin concentration and its role for longevity in female centenarians
 
Yasumichi Arai1, Susumu Nakazawa1,Toshio Kojima3, Michiyo Takayama1,Yoshinori Ebihara1, Ken-ichirou Shimizu5,
Ken Yamamura1, Satoki Homma1,Yasunori Osono6, Yasuyuki Gondo2, Yukie Masui2, Hiroki Inagaki2, 
Kohji Kitagawa4andNobuyoshi Hirose
  1.  
  2.  
  3.  
Background:  Evidence from experimental models of longevity indicates that maintenance of energy homeostasis could be indispensable for longevity across various species. In humans, it has been reported that maintenance of glucose homeostasis and vascular stability is one biomedical feature of centenarians, who have reached the maximum life-span. We hypothesized that adiponectin, a novel anti-inflammatory adipocytokine, could be a protective factor against age-related metabolic alteration and atherogeneity in centenarians.
 
Methods:  We measured plasma adiponectin concentration in 66 female centenarians and body mass index (BMI)-matched female controls (mean age 28.3 ± 6.3 years), followed by a genetic analysis of adiponectin locus.
 
Results:  As compared to BMI-matched female controls, female centenarians had significantly higher plasma adiponectin concentrations. In addition, high concentrations of plasma adiponectin in centenarians was associated with favorable metabolic indicators, and with lower levels of C-reactive protein and E-selectin. In contrast, genetic analysis of 10 single nucleotide polymorphism (SNP) at adiponectin locus did not show significant association between the adiponectin gene variation and longevity.
 
Conclusions:  Our results suggested that hyperadiponectinemia in centenarians could play a role in maintenance of energy homeostasis and vascular stability, and may contribute to longevity.
 
Keywords: adiponectin;centenarians;inflammation marker;leptin;single nucleotide polymorphism (SNP)
 
-------------
[36] Metabolism. 2009 Apr;58(4):552-9. doi: 10.1016/j.metabol.2008.11.017.
 
Cold exposure increases adiponectin levels in men.
 
Imbeault P(1), Dépault I, Haman F.
 
Author information: 
(1)Behavioural and Metabolic Research Unit (Montfort Hospital), School of Human
Kinetics, Faculty of Health Sciences, University of Ottawa, Ontario, Canada K1N
6N5. imbeault@uottawa.ca
 
Sympathetic nerve activation is recognized at the adipose tissue level during
cold exposure. Adiponectin is a key protein produced by adipose tissue, but its
acute modulation remains unknown in humans exposed to cold. The aim of this study
were (1) to examine the acute effects of cold exposure on circulating adiponectin
and (2) to determine whether the changes are modulated by (a) an acute glucose
ingestion as well as (b) a short-term modulation in carbohydrate (CHO)
availability. Using a random crossover design, 6 healthy men were exposed to cold
for 120 minutes with ingestion of beverages containing low (Control, 0.04 g/min) 
or high (High, 0.8 g/min) amounts of glucose during the course of the experiment 
(study 1). In study 2, 6 healthy men were exposed twice to cold for 120 minutes
after equicaloric low-CHO diet and exercise and high-CHO diet without exercise.
Plasma adiponectin concentrations were quantified before and during cold
exposure. In study 1, adiponectin levels did not change during High, whereas a
20% rise was observed during Control (condition x time interaction, P = .06). In 
study 2, adiponectin levels increased by approximately 70% during cold exposure
after both low- and high-CHO diets (effect of time, P < .05). A 120-minute period
of cold exposure is accompanied by a significant increase in adiponectin levels
in young healthy men. The rise in adiponectin levels observed during shivering is
inhibited with glucose ingestion but not after diets varying in CHO content.
 
PMID: 19303978

There will never be peace in the world while there are animals in our bellies.

#130 Dean Pomerleau

Dean Pomerleau
  • Lifetime Member
  • 2,458 posts

Posted 20 March 2016 - 03:41 PM

Cold Exposure Reduces Exercise-Induced Inflammation and Elevation in Testosterone / IGF-1

 

After that huge post, while waiting for Michael's response, I figured I'd share a short and relatively straightforward study [1], on the effects of combining cold exposure (CE) and exercise  in humans.

 

I think it's safe to say that virtually every CRer, and in fact everyone seriously concerned with their health, engages in some form of exercise for its CVD-preventing, muscle-preserving and bone-building benefits. But some of us worry about the effects of the inflammation and the 'wear-and-tear' associated with exercise, particularly as we get older.  And we aren't really interested in the getting big muscles, or raising our growth- and age-promoting testosterone & IGF-1 levels. 

 

In addition, some of us (well, maybe just me) who nearly continuously expose ourselves to cold, combine it with nearly continuous very modest exercise for several reasons: to keep the blood circulating in the cold, to burn calories to maintain a net calorie deficit, and for all the wonderful benefits of CE described in my previous post and elsewhere in this thread.

 

But I've been a bit worried about the possible deleterious impact of combining CE and exercise. Might the extra physiological stress resulting from CE + exercise magnify the exercise-induced inflammation, and result in the accumulation of the kind of systemic damage that is the hallmark of aging - or more accurately, according to the SENS perspective, that is aging?

 

Fortunately, it looks like this is not the case - in fact quite the opposite. Study [1] found that relative to exercising in at room temperature, exercising at 0°C in shorts and a t-shirt (that's my kind of experiment!) resulted in the reduction of a wide range of inflammatory and growth promoting markers, including many interleukins and a bunch of others cytokines, several of which I've never heard of, including IFN-γ, Rantes, Eotaxin, IP-10, MIP-1β, MCP-1, VEGF, PDGF, and G-CSF [see Note 1].

 

Adding CE to exercise also blunted the exercise-induced increase in lymphocytes (white blood cells), which is also considered an inflammatory response. Reduced lymphocytes is a well known side effect of CR as well, although there is some reason to be concerned that this could negatively impact one's ability to fight off an established illness, as discussed here

 

Adding CE to exercise also blunted the exercise-induced increase in testosterone and IGF-1. This too resembles the testosterone and IGF-1 lowering effects of long-term CR.

 
Interestingly, if they really tortured the subjects, by exposing them to a "pre-exercise low-intensity shivering protocol" (having them sit idle for 40-120min in 0°C in only shorts & t-shirt!)  plus continued cold exposure during the subsequent exercise session, the combination reversed some of the beneficial reduction in inflammation and exacerbated the reduction in lymphocytes induced by exercising in the cold. It seems that bringing subjects to the point of shivering and then having them exercise in the cold was going too far to be beneficial. In a similar fashion, [2] found that chronic cold water exposure (without exercise, and to the point of inducing shivering) resulted in a modest elevation in markers of immune system activity, and a trend towards increased lymphocytes also. This result is corroborated by [3], which found that exposing men to 2 hours of a 5°C environment with a breeze blowing on them while wearing just shorts and socks boosted immune system activity, especially if the CE is preceded by exercise. So if you are concerned about suppressed immunity as a result of CR or some other malady, and you want to increase immune system activity, CE to the point of shivering, perhaps in association with exercise, might be a way to do it.
 
Here is the summary from the authors of [1]:
 
This study demonstrated that exercising in the cold can diminish the exercise-induced systemic inflammatory response seen in a thermoneutral environment. Nonetheless, prolonged cooling inducing shivering thermogenesis prior to exercise, may induce an immuno-stimulatory response following moderate intensity exercise. Performing exercise in cold environments can be a useful strategy in partially inhibiting the acute systemic inflammatory response from exercise but oppositely, additional body cooling may reverse this benefit.
 
In short, it appears the combination of non-shivering cold exposure + exercise is a win relative to exercise-alone when it comes to inflammation and pro-aging growth factors like testosterone and IGF-1. If you want to boost your immune system, cold exposure to the point of shivering looks to be an option.
 
--Dean
 
Note 1: The reduction of VEGF by cold exposure is interesting since elevated levels are implicated in a variety of cancers as well as macular degeneration, the latter of which is of particular concern for me.
 
----------
[1] PLoS One. 2014 Oct 22;9(10):e110774. doi: 10.1371/journal.pone.0110774.
eCollection 2014.
 
The effects of cold exposure on leukocytes, hormones and cytokines during acute
exercise in humans.
 
Gagnon DD(1), Gagnon SS(2), Rintamäki H(3), Törmäkangas T(4), Puukka K(5), Herzig
KH(6), Kyröläinen H(7).
 
 
The purpose of the study was to examine the effects of exercise on total
leukocyte count and subsets, as well as hormone and cytokine responses in a
thermoneutral and cold environment, with and without an individualized
pre-cooling protocol inducing low-intensity shivering. Nine healthy young men
participated in six experimental trials wearing shorts and t-shirts. Participants
exercised for 60 min on a treadmill at low (LOW: 50% of peak VO2) and moderate
(MOD: 70% VO2peak) exercise intensities in a climatic chamber set at 22°C (NT),
and in 0°C (COLD) with and without a pre-exercise low-intensity shivering
protocol (SHIV). Core and skin temperature, heart rate and oxygen consumption
were collected continuously. Blood samples were collected before and at the end
of exercise to assess endocrine and immunological changes. Core temperature in NT
was greater than COLD and SHIV by 0.4±0.2°C whereas skin temperature in NT was
also greater than COLD and SHIV by 8.5±1.4°C and 9.3±2.5°C respectively in MOD.
Total testosterone, adenocorticotropin and cortisol were greater in NT vs. COLD
and SHIV in MOD. Norepinephrine was greater in NT vs. other conditions across
intensities. Interleukin-2, IL-5, IL-7, IL-10, IL-17, IFN-γ, Rantes, Eotaxin,
IP-10, MIP-1β, MCP-1, VEGF, PDGF, and G-CSF were elevated in NT vs. COLD and/or
SHIV. Furthermore, IFN-γ, MIP-1β, MCP-1, IL-10, VEGF, and PDGF demonstrate
greater concentrations in SHIV vs. COLD, mainly in the MOD condition. This study 
demonstrated that exercising in the cold can diminish the exercise-induced
systemic inflammatory response seen in a thermoneutral environment. Nonetheless, 
prolonged cooling inducing shivering thermogenesis prior to exercise, may induce 
an immuno-stimulatory response following moderate intensity exercise. Performing 
exercise in cold environments can be a useful strategy in partially inhibiting
the acute systemic inflammatory response from exercise but oppositely, additional
body cooling may reverse this benefit.
 
PMCID: PMC4206434
PMID: 25338085
 
-----------
[2] Eur J Appl Physiol Occup Physiol. 1996;72(5-6):445-50.
 
Immune system of cold-exposed and cold-adapted humans.
 
Janský L(1), Pospísilová D, Honzová S, Ulicný B, Srámek P, Zeman V, Kamínková J.
 
Author information: 
(1)Department of Comparative Physiology, Faculty of Science, Charles University
Vinicná 7, Prague, Czech Republic.
 
The aim of this study was to investigate whether or not the human immune system
can be activated by a noninfectious stimulus, thereby improving the physiological
status of the individual. The effect of a single cold water immersion (14 degrees
C for 1 h) on the immune system of athletic young men, monitored immediately
after immersion, was minimal. With the continuation of the cold water immersions 
(three times a week for a duration of 6 weeks) a small, but significant, increase
in the proportions of monocytes, lymphocytes with expressed IL2 receptors (CD25) 
and in plasma tumour necrosis factor alpha content was induced. An increase in
the plasma concentrations of some acute phase proteins, such as haptoglobin and
haemopexin, was also observed. After 6 weeks of repeated immersions a trend
towards an increase in the plasma concentrations of IL6 and the amount of total T
lymphocytes (CD3), T helper cells (CD4), T suppressor cells (CD8), activated T
and B lymphocytes (HLA-DR) and a decrease in the plasma concentration of alpha
1-antitrypsin was observed. Concentrations of IL1 beta, neopterin, C-reactive
protein, orosomucoid, ceruloplasmin, macroglobulin, immunoglobulins (IgG, IgM,
IgA) and C3, C4 components of the complement, as well as the total number of
erythrocytes, leucocytes, granulocytes and neutrophils showed no significant
changes after the repeated cold water immersions. It was concluded that the
stress-inducing noninfectious stimuli, such as repeated cold water immersions,
which increased metabolic rate due to shivering the elevated blood concentrations
of catecholamines, activated the immune system to a slight extent. The biological
significance of the changes observed remains to be elucidated.
 
PMID: 8925815
 
--------
[3] J Appl Physiol (1985). 1999 Aug;87(2):699-710.
 
Immune changes in humans during cold exposure: effects of prior heating and
exercise.
 
Brenner IK(1), Castellani JW, Gabaree C, Young AJ, Zamecnik J, Shephard RJ, Shek 
PN.
 
Author information: 
(1)Defence and Civil Institute of Environmental Medicine, Toronto, Ontario M3M
3B9.
 
 
This study examined the immunological responses to cold exposure together with
the effects of pretreatment with either passive heating or exercise (with and
without a thermal clamp). On four separate occasions, seven healthy men [mean age
24.0 +/- 1.9 (SE) yr, peak oxygen consumption = 45.7 +/- 2.0 ml. kg(-1). min(-1)]
sat for 2 h in a climatic chamber maintained at 5 degrees C. Before exposure,
subjects participated in one of four pretreatment conditions. For the
thermoneutral control condition, subjects remained seated for 1 h in a water bath
at 35 degrees C. In another pretreatment, subjects were passively heated in a
warm (38 degrees C) water bath for 1 h. In two other pretreatments, subjects
exercised for 1 h at 55% peak oxygen consumption (once immersed in 18 degrees C
water and once in 35 degrees C water). Core temperature rose by 1 degrees C
during passive heating and during exercise in 35 degrees C water and remained
stable during exercise in 18 degrees C water (thermal clamping). Subsequent cold 
exposure induced a leukocytosis and granulocytosis, an increase in natural killer
cell count and activity, and a rise in circulating levels of interleukin-6.
Pretreatment with exercise in 18 degrees C water augmented the leukocyte,
granulocyte, and monocyte response. These results indicate that acute cold
exposure has immunostimulating effects and that, with thermal clamping,
pretreatment with physical exercise can enhance this response. Increases in
levels of circulating norepinephrine may account for the changes observed during 
cold exposure and their modification by changes in initial status.
 
PMID: 10444630

There will never be peace in the world while there are animals in our bellies.

#131 Dean Pomerleau

Dean Pomerleau
  • Lifetime Member
  • 2,458 posts

Posted 23 March 2016 - 04:01 PM

Olive Oil Increases Uncoupling Protein Gene Expression in BAT & Skeletal Muscles

 

Here is an interesting one to tide people over while I finish up a big post on Speakman's work on cold exposure, BAT, metabolic rate & longevity. 

 

In this study [1], researchers fed rats ad lib diets high in fat - 40% of calories, which not far from typical human consumption. They divided the rats into four groups, feeding them four different fats - olive oil (OO - MUFA-rich), sunflower oil (SO - PUFA-rich), palm oil (PO - SFA-rich), and beef tallow (BT - SFA-rich).  No mention is made of the OO being "extra virgin", and it came from one of Spain's biggest olive oil companies (Koipe SA) so I think it's probably safe to assume it was run-of-the-mill olive oil rather than high-polyphenol EVOO. All the mice were housed at "normal" lab temperature (22 °C) for the duration of the experiment, which is chilly for rats.

 

 After four weeks on each of the diets, all four groups had gained about the same amount of weight - about 50% of their initial body weight! Weight of BAT tissue wasn't significantly different between the groups. 

 

What was interesting was the amount of gene expression of the three important uncoupling proteins, UCP-1, UCP-2 and UCP-3 in various tissues, including brown adipose tissue (BAT), white adipose tissue (WAT) and skeletal muscles. Here are graphs comparing UCP expression in BAT and muscle tissue for the four diets:

 

v9FN2W0.png

 

The letters (a, b) above the bars represent which groups were significantly different  As you can see, olive oil significantly boosted UCP1, UCP2, and UCP3 messenger RNA expression in BAT, and UCP3 mRNA expression in muscle tissue relative to the other three fat sources.

 

In terms of actual UCP content in the various tissues on the various diets (as opposed to just messenger RNA expression shown above), only UCP2 was higher in BAT and UPC3 was higher in muscle tissue in the OO group relative to the other diets, particularly the SFA-rich diets:

 

2KGgA2z.png

 

As we'll see in my next post, both UCP2 and UCP3 are associated with increased longevity, so it is interesting to see them elevated in BAT and muscle tissue by OO.

 

Finally, while the OO diet increased total body oxygen consumption per gram of body weight (i.e. metabolic rate) relative to the other diets, oxygen consumption by BAT wasn't any different between groups. I suspect the rats weren't cold-stressed enough for BAT to majorly kick in, since they were feeding ad lib and gained over 50% of their initial body weight during the four weeks of the study (240g → ~370g), presumably mostly as highly insulating white fat.

 

The authors checked for a bunch of different possible causes for why OO might boost UCP gene expression including differential changes to circulating hormones, glucocorticoids, or insulin. Nope - none of them were significantly different across the diets. They suggest instead that it may be the oleic acid in OO being incorporated into cell membranes and increasing the responsiveness of cells to UPC-stimulating adrenaline:

 

The explanation of the effects of olive oil is not clear. It seems that these effects are not mediated by systemic metabolic changes, but rather may be related to a local effect produced by oleic acid on IBAT and gastrocnemius muscle. In our laboratory, we observed that, after a 4-wk olive oil feeding period, oleic acid concentrations were increased in the stored triacylglycerols (45) and also incorporated into the plasma membrane phospholipids (46) in both perirenal and subcutaneous WAT. Although not measured, it can be expected that the same would be true for the triacylglycerols stored in IBAT, skeletal muscle, and mitochondrial membranes. This potential increase in oleic acid might modify the response of IBAT and gastrocnemius muscle to adrenaline. Furthermore, the modifications in membrane phospholipids could lead to modifications of the membrane-receptor interactions of the transduction of the hormonal signal (23, 47–49).

 

So while olive oil didn't increase BAT activity per se, it did increase UCP gene expression and whole body oxygen consumption - perhap through UCP3-mediated mitochondrial uncoupling in skeletal muscles. If challenged by cooler temperatures and/or less food, I strongly suspect these increases in UCP gene activity would have also resulted in increased BAT activity. So I'm going to take the liberty of adding olive oil to the master list of BAT promoters.

 

Here is the latest full list of modifiable and [non-modifiable] factors associated with increased BAT quantity and/or activity:
  • Cold exposure - by far the best BAT inducer/activator
  • Spicy / pungent foods, herbs & supplements - capsaicin / chilli peppers, curcumin / turmeric root, menthol/mint/camphor, oregano, cloves, mustard, horseradish/wasabi, garlic, onions
  • Arginine-rich foods - Good vegan sources include seeds (esp. sesame, sunflower & pumpkin), nuts (esp. almonds and walnuts) and legumes (esp. soy, lupin & fava beans and peas)
  • Other foods - green tea, roasted coffee, cacao beans / chocolate
  • Drugs - metformin, caffeine
  • Avoiding gluten
  • Olive Oil / MUFA-rich diet
  • Methionine restriction - Reduce animal protein. Soy is low in methionine and high in arginine (see below).
  • Low protein diet
  • Fasting
  • Exercise
  • Avoid obesity/overweight
  • [Being naturally thin - high metabolic rate]
  • [Being younger]
  • [Being female]
  • [Ethnicity - having cold-climate ancestors]

 

Once again we see a food or behavior, in this case consumption of olive oil / MUFA, which is known to promote health / longevity also be a (likely) promoter of BAT activity - perhaps when coupled with cold exposure. You can judge for yourself whether all of these are coincidence or not...

 

P.S. I've edited the cold exposure and immunity post by adding this study (PMID: 10444630), which basically reinforces the conclusion that very serious cold exposure in humans (sitting wearing just shorts in 5°C with slightly breeze for 2 hours) elevates immune system activity.

 

P.P.S. I'm happy to report that this thread is #6 on the first page of results when you (or at least when I) do a Google search for "cold exposure longevity".

 

--Dean

 

------------

[1] Am J Clin Nutr. 2002 Feb;75(2):213-20.

 
Olive oil feeding up-regulates uncoupling protein genes in rat brown adipose
tissue and skeletal muscle.
 
Rodríguez VM(1), Portillo MP, Picó C, Macarulla MT, Palou A.
 
Author information: 
(1)Department of Nutrition and Food Science, the University of País Vasco,
Vitoria, Spain.
 
 
BACKGROUND: Some nutrients, such as carotenoids, retinoic acid, and certain types
of fatty acids, increase thermogenic capacity.
OBJECTIVE: The influence of 4 dietary lipid sources (olive oil, sunflower oil,
palm oil, and beef tallow) on the content of uncoupling proteins 1, 2, and 3
(UCP1, UCP2, and UCP3) and their messenger RNA (mRNA) expression in several
tissues of rats was compared.
DESIGN: Wistar rats were randomly divided into 4 groups and fed ad libitum diets 
containing 40% of energy as fat. UCP1, UCP2, and UCP3 mRNA and protein were
assessed by Northern blot and Western blot, respectively. Oxygen consumption in
tissues was measured by polarography. Total-body oxygen consumption was assessed 
in an open-circuit chamber system. Circulating fuels (fatty acids and glucose)
and hormones (triiodothyronine, thyroxine, corticosterone, and insulin) were
measured.
RESULTS: Olive oil feeding induced the highest UCP1, UCP2, and UCP3 mRNA
expression in interscapular brown adipose tissue. An analogous effect was
observed in gastrocnemius muscle UCP3 mRNA. No significant differences were
observed in perirenal white adipose tissue UCP2 mRNA. Changes in mRNAs were not
accompanied by close changes in the protein content of UCPs and were not
associated with changes in adipose tissue oxygen consumption. Nevertheless,
total-body oxygen consumption was higher in rats fed olive oil than in those fed 
the other 3 diets. No significant differences were found in body and tissue
weights or in serum indexes.
CONCLUSION: Olive oil induced an up-regulating effect on UCP mRNA that was
probably not mediated by systemic metabolic changes, but rather related to a
local effect on interscapular brown adipose tissue and skeletal muscle.
 
PMID: 11815310

There will never be peace in the world while there are animals in our bellies.

#132 Gordo

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Posted 24 March 2016 - 07:39 AM

Not sure if this adds anything to the topic, but i noticed a study just published a few days ago with some conclusions I don't remember reading about elsewhere:

pubmed (PMID 26993316)

 

Full Text

 

They used one of these Blanketrol III circulating water cooler machines from Cincinnati Sub-Zero for rapidly cooling the research subjects:

koolKit.jpg?t=1458765103352

 

We conclude that cold stimulation in humans increases BATassociated EE and whole-body EE; however, BAT is minor contributor to this whole-body cold-induced thermogenesis, while deeper centrally located neck muscles, along with the pectoral muscles are among the major contributors to thermogenesis. Moreover, in BAT, both during RT and cold stress, oxygen consumption is interlinked with the circulatory uptake of NEFA

 

 

I'd like to know more about this "deep neck and pectoral" muscle contribution to cold induced thermogenesis.  Not sure if this has anything to do with shivering?  Or something else.  Study sounded like they cooled down to the shiver point then raised the temps presumably so there was no more shivering.  This study also notes the link between BAT activity and non-esterified fatty acid uptake.


Edited by Gordo, 24 March 2016 - 07:44 AM.


#133 Michael R

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Posted 24 March 2016 - 11:30 AM

 

pubmed (PMID 26993316)

 

Full Text

 

We conclude that cold stimulation in humans increases BAT-associated EE and whole-body EE; however, BAT is minor contributor to this whole-body cold-induced thermogenesis, while deeper centrally located neck muscles, along with the pectoral muscles are among the major contributors to thermogenesis.

 

 

Now we know the secret!

 

 
(I wish I could find it, but there is a great video of Schwarzenegger, still wearing his golf shirt, doing this "threateningly" to a talk show host ...


#134 Gordo

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Posted 25 March 2016 - 09:08 AM

 


 Now we know the secret!

 

Haha, I would have gone with:

 

But seriously, I had been wondering at first if test subjects were flexing muscles to stay warm kind of like people in this thread reported having to keep moving when cold?  But it appears that this BAT to "nearest muscle" interaction is involuntary, and in fact the muscle based thermogenesis was directly correlated with BAT quantity in test subjects.  So there may be an interesting relationship between BAT and the muscle that surrounds it.  This suggests that building up neck and pec muscles might be beneficial if not essential to one's personal "BAT building regimen".  I've been doing short duration HIIT workouts for the last 2 years with a particular focus on pull ups and hanging crunches, which has resulted in pretty significant pectoral and neck muscle development, so perhaps that is one more thing contributing to my apparently easy ability to activate BAT/cold induced thermogenesis.  I only work out for 5 minutes or less per day and eat a superfood rich, low protein plant based whole food diet (with no protein supplementation) which seems to be adequate:

leanpic.jpg

This may also partially explain why cold induced thermogenesis is so low or non-existent in severely CR'ed (anorexic) subjects with no muscle mass (and may also have something to do with a similar lack of cold induced thermogenesis in obese subjects?).


Edited by Gordo, 25 March 2016 - 11:27 AM.


#135 Dean Pomerleau

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Posted 25 March 2016 - 11:50 AM

Michael and Gordo,

 

Thanks for the pointer to this interesting, and it possibly quite important study (PMID 26993316). It has sent me down a two-day (very interesting and productive!) rabbit hole investigating muscle-related thermogenesis. I'm in the midst of a post about it now (putting my Speakman post on hold...).

 

But two quick responses to points you two have brought up. First, regarding the suggestion that the heat production by muscles is either voluntary (i.e. flexing/clinching) or involuntary but nonetheless contraction-related (i.e. shivering). It appears from the evidence that I'll present that non-shivering thermogenesis (NST) is the primary way muscles generate heat unless it's really cold and you start shivering - which the researchers took explicit steps in PMID 26993316 to avoid. I'll talk about the mechanism of NST in muscle tissue and its significance in my upcoming post.

 

Second, the reason PMID 26993316 only looked at the upper body Gordo is that the scanner they used has a narrow field of view. They wanted to look at BAT, so they pointed it at the part of the body where BAT is concentrated, and so only imaged muscles in the chest and neck region. BAT may indeed be signally nearby muscles to generate heat, but this study doesn't help to determine that, since it only looked at muscles that are in the vicinity of BAT pockets.

 

--Dean


There will never be peace in the world while there are animals in our bellies.

#136 Kenton

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Posted 25 March 2016 - 07:13 PM

In this post I discussed study [1], which found that cold exposure not only increases BAT in mice so they can burn more calories to stay warm, but it also shifts their gut microbiome ... As I discussed in this post, I appear to have 2x more Firmicutes relative to Bacteroidetes in my latest tests, and be enriched in Firmicutes relative to the general population. ... these shifts (i.e. an ↑ in Firmicutes and ↓ in Verrucomicrobia) are in the same direction as the cold-exposed mice. Interestingly, as of the date of those two 2015 tests, I wasn't yet intentionally engaged in my cold exposure experiments....Does anyone else, cold-adapted or not, have uBiome data on Firmicutes and Verrucomicrobia they'd be willing to share?

 

 My ubiome numbers from Nov. 2013 when I was surfing in cold water 1hr/day were Firmicutes 19.6% above the reference group and Verrucomicrobia 1/3rd of the reference group.  



#137 Gordo

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Posted 26 March 2016 - 01:47 PM

Dean -- thank you for putting together all that research and thoughtful analysis - great information. This thread is now the #2 web result link that comes up from a search on "cold exposure longevity" from BOTH bing.com and google.

I do think cold exposure has likely been a confounding factor in past DR/CR studies in rodents. I have even contacted the researcher doing this ongoing study of DR in mice which may be of interest to people here. He was nice enough to respond, but didn't seem to think CE was a factor. Professor Arlan Richardson says:

Hollozy’s study showed that cold exposure did not change lifespan and in a study done at San Antonio (Ikeno et al.) they showed housing mice 1 vs 4 had the same lifespan whether fed AL or DR (in fact the DR mice group housed lived a little, but not significant, longer)

 

I have not tracked down those references.

I have been continuing my personal observations/anecdotes with CE and blood glucose and have been seeing very positive results so far. I'll post more details after doing more rigorous experiments with multiple data points. I feel like CE opens the door to a LOT of foods with well documented health benefits that happen to also be higher glycemic index foods including most whole grains and rice, honey, maple syrup, most fruit especially things like apples, bananas, etc. Apples are of interest to me since I grow them, and their (organic) skins especially are top superfoods with many beneficial properties. Maple syrup is another personal interest not only because I have a maple grove behind my house that I tap for personal use, but because pure maple syrup has been shown in various research to have anti-cancer, anti-inflammatory, and anti-diabetic properties (oddly enough), plus loads of phytonutrients/compounds not found in any other foods. More recently it has even been shown to prevent the misfolding of brain cell proteins (related to aging/alzheimers/dimentia) See:
http://www.scienceco...discovers_54...
http://www.lifeexten...lefdailynews...
http://www.scienceda...00321182924.htm
http://www.medicalda...n-health-377846

Many of the same types of health benefits have been associated with raw pure honey as well.

But how can you eat all these great, health promoting foods without sending your blood sugar soaring? Some longevists take metformin for this purpose, but that always seemed like a bad idea to me, and has many side effects and drawbacks. Cold exposure is a better, completely natural, alternative.

Another personal anecdote - last night I had a huge bowl of porridge consisting of barley and oats (half cup dry of each) plus almond milk, tart cherries (Aldi's dark morello), high GI dried cranberries, walnuts, blueberries, flax, chia, lemon, a tablespoon of maple syrup and a tablespoon of buckwheat honey. I think I am going to make this my reference meal for more rigorous future experiments. I will more carefully measure/weigh everything next time and plug it into software for analysis. Anyway this is a meal that I'd expect to send blood sugar soaring. Unfortunately I again only took one postprandial measurement after CE so that doesn't say a whole lot, but I was pleased to see a 77 blood glucose reading (I have taken measurements with the same device just before and after official lab testing and it was spot on).

I think there is a better way than the type of CR practice some follow that results in difficulty recoverying from infections, osteoporosis, and eating only low GI foods all the time.  


Edited by Gordo, 10 April 2016 - 07:47 AM.
Cleaning Gordo's annoying tracker redirect links ;)


#138 Dean Pomerleau

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Posted 26 March 2016 - 05:15 PM

Skeletal Muscle Thermogenesis & Sarcolipin

 

Michael (and Gordo),

 

I really must thank you for presenting challenges to the "Cold Exposure Hypothesis" like this new study [1] (PMID 26993316). In short, it used what appears to be a relatively new imaging technique to assess the metabolic activity of human upper body BAT and muscle tissue during cold exposure. In agreement with many previous studies we've discussed, it found that BAT activity was increased by cold exposure in humans (by 50%), and that BAT mass and BAT activity correlated with increased cold-induced thermogenesis and energy expenditure.

 

But, in fascinating twist, it found that the actual contribution of BAT activity to thermogenesis and increased energy expenditure (as measured directly by oxygen consumption) in response to cold appeared to be quite modest in humans - on the order of 10 kcal out of the total increase of around 350 kcal per day. Further, they found that the upper body skeletal muscles (including Michael's pectoral muscles - nice video!) contributed far more to cold-induced thermogenesis than did BAT. This finding would seem to be corroborated by [2], which found that even in people with a relatively large amount of BAT, the actual contribution of BAT activity to cold-induced thermogenesis is small compared to the total size of the thermogenic response. The imaging technique the researchers employed in both these studies is pretty new and novel, so it's hard to say just how definitive these results are concerning the magnitude of BAT's role in human thermogenesis. But they can't be easily dismissed, since previous human studies have focused on the correlation of BAT mass and BAT activity with overall cold-induced thermogenesis, without showing that BAT was directly responsible for most or all of the observed increase in energy expenditure.

 

The authors of [1] are uncertain just how the muscles are generating heat. Subjects were told to relax their muscles and were lying down in a comfortable position, so voluntary muscle flexion (as so amusingly demonstrate in Michael's and Gordo videos) was unlikely, although can't be ruled out. Or possibly they could have been clenching their muscles involuntarily to cope with the cold. This too can't be ruled out, but seems unlikely since you would think subjects would have reported it. Similarly, they could been shivering imperceptibly, and generating heat that way. But the researchers took steps to prevent shivering by decreasing the severity of the cold when they or the subjects detected shivering.

 

Alternatively, the authors of [1] suggest that the increased energy expenditure in muscle cells could result from non-shivering thermogenesis (which I'll refer to as smNST for "skeletal muscle non-shivering thermogenesis"), and that this could result from increased expression of uncoupling proteins in muscles, like what occurs with UCP-1 in BAT. In particular, there is pretty good evidence that short-term (24h) cold exposure upregulates UPC-3 in mitochondria of rat muscle cells, and a lower mitochondrial membrane potential, increasing heat production and also potentially lowering ROS production [4]. But [5] and others have found that UCP-3 expression in muscle cells is transiently elevated by cold, but returns to baseline after chronic cold exposure (15 days). Or smNST could be increased by expression of UCP-2 in muscles as a result of cold exposure, which was observed in mice [21] and humans [20]. Alternatively, study [5] suggests that free fatty acids rather than uncoupling proteins, may be the cause of increased proton leakage and heat production in skeletal muscle mitochondria. 

 

Tantalizingly, quite a number of studies suggest mitochondrial uncoupling might be associated with increased longevity. Studies [22][23] found that increased UCP-2 expression results in increased longevity in rodents, and this may be associated with body temperature regulation [25], although this result is controversial [24]. Similarly suggestive, [26] found that mice chronically treated with a mild mitochondrial uncoupling agent that works independently of the UCPs (a chemical called 2,4-dinitrophenol or DPN) resulted in mice who ate more, weighed less, had lower measures of fasting glucose, triglycerides, insulin and oxidative damage to DNA, and most importantly, lived longer than controls - mean and maximum (oldest 10%) lifespan were extended by 7% and ~11%, respectively.  And in this post, I talk about the evidence that increased expression of UCP proteins is associated with greater human longevity.

 

Regardless of the mechanism of mitochondrial uncoupling, and regardless of whether uncoupling is occurring in BAT, skeletal muscle, other organs, or some combination, it appears to be happening as a result of cold exposure, and have the potential to be health and lifespan promoting. The importance of mitochondrial uncoupling for thermogenesis in humans in response to cold exposure is demonstrated by [19], in which resting metabolic rate was increased by 76kcal/day as a result of mild cold exposure (80 hours @ 16 °F), and about half of that increase appeared to result from mitochondrial uncoupling.

 

But there is another, very recently discovered (only since 2012 - more recently than human BAT!) mechanism for mammalian smNST that appears quite significant. In fact I'm surprised the authors of [1] didn't mention it. 

 

But before getting into the specific mechanism, a word about the significance of smNST for cold adaptation in humans and rodents. We've (mostly I've ) focused mainly on BAT-thermogenesis in response to cold. But apparently even in rodents, smNST is a very important response to cold adaptation, on par with BAT. Study [3] found that mice whose BAT had been removed and who were given a drug to prevent shivering, were still able to maintain body temperature via smNST. And no, they weren't clenching their muscles or increasing their activity level to generate heat. Instead, they were generating heat via smNST using a pathway I'll describe in detail below. How did the authors know this? Mice with a knockout mutation for an important protein involved in this new form of smNST, whose BAT had been removed and shivering inhibited, were unable to maintain their body temperature in response to cold, in contrast with WT mice without the knockout mutation given the same treatment. In fact, the poor mice lacking the ability for smNST either died or had to be removed from the cold conditions. So the point is that it's not just humans where smNST can play a very important role in cold-induced thermogenesis, but rodents as well.

 

The protein alluded to above that facilitates smNST is called Sarcolipin. I found the way it works to be very interesting, but required me to bone up on my muscle biochemistry. Bear with me, or skip this and the next two paragraph if you aren't interested in the mechanism, only the result. This is highly simplified, but skeletal muscles contract as a result of calcium ions (Ca++) flooding the muscle cell cytoplasm, causing a sequence of reactions that result in muscle fiber shortening. Where does the flood of Ca++ ions come from? It is stored up in a cellular organelle called the sacroplasmic reticulum (SR), which is related to, but differs in structure and function from, the endoplasmic reticulum that people have probably heard of. The SR's primary role in muscle cells is to serve as the reservoir for Ca++ ions. How do the Ca++ ions get into the SR? Through active transport by a pump called SERCA (I'll spare you what SERCA stands for). Basically when ATP and two Ca++ ions bind to the SERCA pump on the outside of the SR membrane, the ATP undergoes hydrolysis (conversion to ADP) and the two Ca++ ions get transported through the SERCA complex across the SR membrane to the interior of the SR.  This creates a strong gradient, with a much greater concentration of Ca++ ions on the inside of the SR than on the outside. When a motor neuron signals to the muscle cell that it's time to contract, what basically happens is that the SR gets the message and suddenly releases all it's built-up Ca++ ions, causing the muscle fibers to contract. And that, in a very simplified nutshell, is how muscles contract.

 

OK you might be saying, but what does the fact that SERCA pumps Ca++ ions to enable our muscles to contract and relax have to do with thermogenesis? Well, because this calcium ion pumping is a very energy hungry, heat generating process. According to [15] (a great review of all aspects of mammalian thermogenesis by pioneers of the research I'm describing), SERCA activity comprises approximately 42% to 48% of resting metabolic rate in slow and fast-twitch muscles. Even more significant, SERCA activity can account for approximately 12% to 15% of whole-body resting energy expenditure and approximately 15% to 20% of total daily energy expenditure [15]. So unlike BAT which varies dramatically between species, SERCA is known to play a big role in metabolism and energy expenditure across all mammalian species.

 

So you might ask, where does sarcolipin come in to generate smNST out of all this? Sarcolipin binds to the SERCA pump, causing it to go through the motion of hydrolyzing ATP to ADP like usual, but instead of actually transferring it's captured Ca++ ions into the interior of the SR, the SERCA pump simple releases its Ca++ ions back into the muscle cell cytoplasm. This is known as 'futile cycling' - because ATP is being reduced to ADP, thereby expending energy and generating heat, but no work is getting done (i.e. no Ca++ ions are getting transported from the cytoplasm across the SR membrane to the SR interior). In fact, more heat is generated per ATP molecule hydrolyzed when sarcolipin causes SERCA to drop its Ca++ ions early and not actually pump them across the SR membrane. And because the SERCA pump is trying to increase the reservoir of Ca++ ions inside the SR so the muscle can get ready to fire again, it will keep trying (futilely) to pump Ca++ ions, expending more energy and generating more heat. 

 

So how significant is this sarcolipin-mediated smNST for burning calories and generating heat in real life? Study [3] found that sarcolipin knockout mice were prone to diet-induced obesity compared with normal mice. Even more dramatically, study [6] studied mice in which sarcolipin was either knocked out or overexpressed. They found that when the knockout and overexpressing mice were fed the same amount of food, the knockout mice gained 22% of their body weight (as fat) and the sarcolipin overexpressing mice lost 23% of their body weight. In the end the sarcolipin-knockout mice weighted ~50% more than the sarcolipin-overexpressing mice, while eating the exact same amount of food. When fed ad lib, the sarcolipin overexpressing mice ate ~37% more food, but gained ~66% less weight and weighed half as much as the obese sarcolipin knockout mice; these effects were not a result of increased physical activity - the researchers ruled that out. Here is the dramatic difference in weight trajectories for the sarcolipin knockout mice (Sln-/-), sarcolipin overexpressing mice (SlnOE) and wild-type (WT) mice when fed a high fat diet ad lib from [6]:

 

wZ8GjeT.png

 

So in rodents at least, sarcolipin-induced smNST can have a really big effect on calorie expenditure and body weight, even at the thermoneutral temperature employed in this study (29 °C) to suppress BAT thermogenesis that might have masked the effects of sarcolipin-induced smNST. Interestingly, the tendency of sarcolipin-knockout mice to become obese when fed ad lib occurred despite a "comparable increase in BAT mass and UCP-1 content to that of wild-type mice" and despite eating the same amount of food and being no less active than wild-type mice. "This is in stark contrast to UCP-1 ablation, which is not obesogenic under standard animal housing conditions" [15]. In other words, even in rodents where BAT is prevalent and important for thermogenesis, sarcolipin-induced smNST appears more important for maintaining energy balance. As the authors of [15] put it:

 

These findings suggest that inefficient SERCA pumping [via attachment of sarcolipin to it] is induced during caloric excess to regulate energy balance and prevent excessive weight gain.

 

And it's not just in these mutant mice that variations in sarcolipin are associated with changes in energy balance.  In wild-type mice, sarcolipin expression increases by a factor of 3-5x as a result of high fat feeding [15]. Unlike BAT, which kicks in mostly as a result of cold exposure and only modestly to compensate for excess calories, sarcolipin (SLN in this diagram) can have important effects on metabolism across a range of energy demands, as illustrated in this diagram from [15]:

 

71iVNb2.png

 

This "gaining less weight while eating more food" as a result of extra sarcolipin brings to mind the "constitutionally lean" (CL) women from PMID 23393181 discussed in this post. Like the sarcolipin-overexpressing mice, the CL women had a naturally higher metabolic rate and were resistant to weight gain when overfed. And both the obesity-resistant, sarcolipin-overexpressing mice in [6] and the the CL women in PMID 23393181 had lower respiratory quotient, suggesting increased burning of fat relative to carbohydrates. The CL women were also shown to be the only ones with significant BAT, while anorexics, recovered anorexics, or normal-weight women who had none. But could their extra BAT be just part the metabolic program that enables them to stay slim? In particular, could it be that their muscles were also burning more calories, perhaps through this sarcolipin-induced smNST? And could it be what's going on in the muscles of the cold-exposed men in the study that triggered this discussion (PMID 26993316), whose muscles were burning a lot of calories apparently without shivering or flexing/contracting? It seems like a real possibility to me, and to many of the authors of the recent flurry of sarcolipin papers, such as this quote from [15]:

 

Although active BAT now has been confirmed in adult humans in small quantity, it is not detected in all individuals (ref) and only accounts for a small fraction of energy expenditure during physiological stress (ref). Thus, an alternative means of adaptive thermogenesis may lie within skeletal muscle of certain individuals and may involve [sarcolipin], considering it is expressed abundantly in human skeletal muscle (ref).

 

and this one from the model proposed in [16]:

 

In adult non-hibernating mammals, including humans, where UCP1 and BAT are limited, [sarcolipin] is the dominant source of thermogenesis. By contrast, in species where UCP1 and BAT are abundant, the contribution of [sarcolipin] to thermogenesis is secondary to that of BAT.

 

It seems to me like sarcolipin may play an important role in human thermogenesis. But unfortunately there appears to be little research so far on sarcolipin-induced smNST in humans. Sarcolipin has long been known to be an important protein for regulating heart muscle tissue in humans, and sarcolipin has recently been shown to be abundant in human skeletal muscle fibers as well, in fact in much more abundance than in the muscles of rodents [16], suggesting it may be playing the thermogenic role in humans that BAT/UCP-1 plays in small mammals [14][16] - unless the rodents have their BAT/UCP-1 knocked out, in which case they apparently rely on upregulation of sarcolipin-induced smNST to cope with thermal stress.  But definitive evidence of sarcolipin's involvement in human thermogenesis does not appear to yet be available. About all I could find was [7] which found sarcolipin-induced respiration (i.e. futile cycling & heat generation) was seriously reduced in morbidly obese humans (BMI around 50!) compared with lean humans.

 

But does sarcolipin-mediated smNST really kick in in response to cold exposure?  As we saw in [3], it can be quite significant in rodents, on par with the amount of heat produced by BAT, and the difference between life and death for cold-exposed BAT-less rodents lacking the gene to produce sarcolipin. Study [11] found cold-reared mice (4 °C) expressed between 3x and 10x as much sarcolipin (depending on which muscle was tested) than did warm-reared mice. Even more impressive is this study [8] in rabbits, which don't have any BAT but which are quite good at cold adaptation. Researchers kept rabbits either at normal room temperature or 4 °C for 10 days. The cold rabbits maintained normal body temperature, ate about 15% more, and gained 66% less weight as the warm rabbits when fed ad lib. So the cold rabbits were expending a lot more energy, but they didn't have BAT and weren't shivering or running around to keep warm, so how were they doing it? Apparently via futile cycling of the SERCA calcium channel discussed above. Muscle cells isolated from the cold-adapted rabbits showed a dramatic increase SERCA pump concentration and activity. As a result, the muscles of the cold-adapted rabbits produced twice as heat as the warm rabbits. Interestingly, this paper was from 2008 (4 years before the thermogenic role of sarcolipin was discovered), so they weren't quite sure how SERCA activity was resulting in so much heat generation. We now know the mechanism is likely to be the uncoupling of ATP hydrolysis from the transport of Ca++ ions - i.e. futile cycling in the SERCA pumps induced by sarcolipin.

 

But will upregulation of sarcolipin and SERCA pumps to support smNST compromise muscle performance? In other words, if sarcolipin is attached to all the SERCA pumps and so causing futile cycling, will the pumps be unable to restore the Ca++ ion gradient to allow effective muscle performance? To find out, study [9] looked at muscle performance in mice after 5 weeks of cold exposure (4 °C), compared to muscles from mice housed at 24 °C. They chose a muscle that did not participate in shivering to avoid the impact of shivering on their results. Consistent with the studies above, cold exposed mice showed 4x as much Ca++ ion leakage from the sarcoplasmic reticulum (SR) as the warm mice - a sign that futile cycling of the SERCA calcium pumps in the SR, almost certainly induced by sarcolipin (although this was from 2010 before sarcolipin's role was discovered), was playing a big role in keeping the cold-adapted mice warm. So what about muscle performance - were the cold-adapted mice weaklings because of all that Ca++ ion leakage? Nope - quite the opposite in fact, as shown in the figure below:

 

zLm3Fys.png

 

As you can see, the muscle fibers from the cold-adapted mice (right panel) didn't get fatigued nearly as quickly as the fibers from the warm-adapted mice (left panel). The authors conclude (my emphasis):

 

[M]uscle fibres from cold-acclimated mice showed significant increases in basal [Ca2+]i (∼50%), tetanic [Ca2+]i (∼40%), and sarcoplasmic reticulum (SR) Ca2+ leak (∼fourfold) as compared to fibres from room-temperature mice. Muscles of cold-acclimated mice showed increased expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and increased citrate synthase activity (reflecting increased mitochondrial content). Fibres of cold-acclimated mice were more fatigue resistant with higher tetanic [Ca2+]i and less force loss during fatiguing stimulation. In conclusion, cold exposure induces changes in FDB muscles similar to those observed with endurance training and we propose that increased [Ca2+]i is a key factor underlying these adaptations.

 

Similarly, and in support of the hypothesis that the cold-induced muscle performance improvements seen in [9] were indeed mediated by sarcolipin, study [10] found that muscles were more fatigue resistant in mice genetically altered to overexpress sarcolipin than the muscles of wild-type mice. Those same sarcolipin-upregulated mice were able to run 16% farther than WT-mice before exhaustion in an progressively more difficult treadmill endurance test. 

 

Based on [9] and [10] together, it appears cold-exposure boosted mitochondrial size and/or number ("increased mitochondrial content") and enabled the SR in muscle fibers to release 40% more calcium ions in response to stimulation ("higher tetanic [Ca2+]i") enabling stronger & more prolonged contraction during repeated stimulation, the same way endurance exercise improves muscle fiber performance. I know Michael will probably be skeptical about the claim about increased mitochondria. But data from [6] shows pretty definitively that the over or underexpression of sarcolipin (via genetic manipulation) results in large changes in both the number of mitochondria and the total amount of mitochondrial DNA (relatively to nuclear DNA) in two different mice muscles, the Tibialis Anterior (TA) and Extensor Digitorum Longus (EDL). Here is the graph comparing the mitochondria count and DNA content in sarcolipin knockout mice (Sln-/-), sarcolipin overexpressing mice (SlnOE) and wild-type (WT) mice:

 

kcVGEu6.png  vDbVcOI.png

 

How cool is that? What this strongly suggests is that cold exposure can not only burn calories via non-shivering thermogenesis in skeletal muscles, it also conditions muscles to perform better in the same way endurance exercise conditions them. In other words, you can burn calories and get in better shape via cold exposure without the wear and tear, or the pain of exercise. As a bonus, increased mitochondrial content in muscle cells may increase longevity. For example, CR boosts the number of mitochondria in skeletal muscles of rats [17] and humans in the CALERIE study [18], and this is thought to be a means by which CR reduces oxidative damage and possibly how it improves longevity i.e. spreading the workload between more mitochondria is thought to reduce free-radical damage.

 

Thanks for anyone who've stuck with me this far (and even those who've skipped to the bottom to see the upshot). I'll try to summarize what all this likely means:

  • It looks like BAT may not be the only game in town when it comes to cold-induced thermogenesis. In particular, non-shivering thermogenesis in skeletal muscles looks like it may be quite important, particularly in large mammals like humans that don't have as much BAT as small mammals like rodents.
  • Uncoupling proteins might be part of the mechanism by which humans generate extra heat in response to cold.
  • But it seems more likely that increased cold-induced thermogenesis results from an increase in the expression of the protein sarcolipin, which generates heat and expends more energy by uncoupling calcium ion pumping in skeletal muscle sarcoplasmic reticulum.
  • Sarcolipin-induced changes in calcium ion exchange and mitochondria biogenesis appears to improve muscle performance (reduce fatigue) in the same way endurance exercise improves muscle performance, and may reduce oxidative damage in muscle cells as well, perhaps improving health and longevity.
  • While sarcolipin's role in human thermogenesis is still speculative, I predict we'll see a lot of study of this very soon, given its therapeutic potential for treatment of obesity. Unlike BAT (which people have been excited about for obesity avoidance for a while now), which is minimal in most people and requires generating lots of new cells/tissue to have an effect, sarcolipin is just a protein, the action of which might be mimicked or bolstered directly via drugs rather than cold exposure, making it a much more promising avenue than boosting BAT or BAT activity for pharmaceutical companies to explore as a way to treat obesity.

Finally, I can hear the critics & skeptics saying:

 

But Dean, you've been harping on BAT all along as the likely mechanism by which cold exposure is beneficial. Are you now changing you tune? What about all those other studies you've been posting about the importance of BAT?"

 

First I'll note that my "Cold Exposure Hypothesis" is that cold exposure is beneficial, and BAT may be one mechanism by which those benefits are generated. The fact that there may be other ways as well, like sarcolipin-induced non-shivering thermogenesis in skeletal muscles, provides additional evidence to support, rather than refute, my hypothesis. Moreover, all this stuff about cold exposure influencing muscle thermogenesis doesn't negate the BAT-related evidence I've presented previously. It's clear that BAT is elevated by cold exposure in humans, does generate heat and burn calories, and is likely to be associated with beneficial health & longevity effects. Even if BAT's direct contribution to energy expenditure is relatively modest in humans, it is very actively involved in endocrine signalling, and may even upregulate glucose and/or fatty acid metabolism in skeletal muscles via FGF-21 or adiponectin release as suggested by [12] and [13]. So BAT and skeletal muscles appear to work synergistically to adapt to the stress of cold exposure.

 

Finally, skeletal muscles make up around 40% of a healthy person's body mass and the maintenance of muscle function is critically important for healthy aging. The fact that cold exposure appears to impact muscle directly, and in a way similar to endurance exercise and calorie restriction, shows yet another pathway by which cold exposure may improve health and longevity. This new mechanism, and particularly its apparent effects on muscle mitochondria, might even expand Michael's imagination about the way cold exposure could beneficially impact aging.

 

--Dean

 

----------

[1] Eur J Nucl Med Mol Imaging. 2016 Mar 19. [Epub ahead of print]

 
Human brown adipose tissue [(15)O]O2 PET imaging in the presence and absence of
cold stimulus.
 
U Din M(1,)(2), Raiko J(1,)(2), Saari T(1,)(2), Kudomi N(3), Tolvanen T(1,)(2),
Oikonen V(1,)(2), Teuho J(1,)(2), Sipilä HT(1,)(2), Savisto N(1,)(2), Parkkola
R(4), Nuutila P(1,)(2), Virtanen KA(5,)(6).
 
 
PURPOSE: Brown adipose tissue (BAT) is considered a potential target for
combatting obesity, as it produces heat instead of ATP in cellular respiration
due to uncoupling protein-1 (UCP-1) in mitochondria. However, BAT-specific
thermogenic capacity, in comparison to whole-body thermogenesis during cold
stimulus, is still controversial. In our present study, we aimed to determine
human BAT oxygen consumption with [(15)O]O2 positron emission tomography (PET)
imaging. Further, we explored whether BAT-specific energy expenditure (EE) is
associated with BAT blood flow, non-esterified fatty acid (NEFA) uptake, and
whole-body EE.
METHODS: Seven healthy study subjects were studied at two different scanning
sessions, 1) at room temperature (RT) and 2) with acute cold exposure.
Radiotracers [(15)O]O2, [(15)O]H2O, and [(18)F]FTHA were given for the
measurements of BAT oxygen consumption, blood flow, and NEFA uptake,
respectively, with PET-CT. Indirect calorimetry was performed to assess
differences in whole-body EE between RT and cold.
RESULTS: BAT-specific EE and oxygen consumption was higher during cold stimulus
(approx. 50 %); similarly, whole-body EE was higher during cold stimulus (range
2-47 %). However, there was no association in BAT-specific EE and whole-body EE. 
BAT-specific EE was found to be a minor contributor in cold induced whole-body
thermogenesis (almost 1 % of total whole-body elevation in EE). Certain deep
muscles in the cervico-thoracic region made a major contribution to this
cold-induced thermogenesis (CIT) without any visual signs or individual
perception of shivering. Moreover, BAT-specific EE associated with BAT blood flow
and NEFA uptake both at RT and during cold stimulus.
CONCLUSION: Our study suggests that BAT is a minor and deep muscles are a major
contributor to CIT. In BAT, both in RT and during cold, cellular respiration is
linked with circulatory NEFA uptake.
 
PMID: 26993316
 
-----------
[2] J Nucl Med. 2013 Apr;54(4):523-31. doi: 10.2967/jnumed.112.111336. Epub 2013 Jan 
29.
 
15O PET measurement of blood flow and oxygen consumption in cold-activated human 
brown fat.
 
Muzik O(1), Mangner TJ, Leonard WR, Kumar A, Janisse J, Granneman JG.
 
Author information: 
(1)Department of Pediatrics, Wayne State University, Detroit, MI 48201, USA.
otto@pet.wayne.edu
 
Although it has been believed that brown adipose tissue (BAT) depots disappear
shortly after the perinatal period in humans, PET imaging using the glucose
analog (18)F-FDG has shown unequivocally the existence of functional BAT in adult
humans, suggesting that many humans retain some functional BAT past infancy. The 
objective of this study was to determine to what extent BAT thermogenesis is
activated in adults during cold stress and to establish the relationship between 
BAT oxidative metabolism and (18)F-FDG tracer uptake.METHODS: Twenty-five healthy
adults (15 women and 10 men; mean age ± SD, 30 ± 7 y) underwent triple-oxygen
scans (H2(15)O, C(15)O, and (15)O2) as well as measurements of daily energy
expenditure (DEE; kcal/d) both at rest and after exposure to mild cold (15.5°C
[60°F]) using indirect calorimetry. The subjects were divided into 2 groups (high
BAT and low BAT) based on the presence or absence of (18)F-FDG tracer uptake
(standardized uptake value [SUV] > 2) in cervical-supraclavicular BAT. Blood flow
and oxygen extraction fraction (OEF) were calculated from dynamic PET scans at
the location of BAT, muscle, and white adipose tissue. Regional blood oxygen
saturation was determined by near-infrared spectroscopy. The total energy
expenditure during rest and mild cold stress was measured by indirect
calorimetry. Tissue-level metabolic rate of oxygen (MRO2) in BAT was determined
and used to calculate the contribution of activated BAT to DEE.
RESULTS: The mass of activated BAT was 59.1 ± 17.5 g (range, 32-85 g) in the
high-BAT group (8 women and 1 man; mean age, 29.6 ± 5.5 y) and 2.2 ± 3.6 g
(range, 0-9.3 g) in the low-BAT group (9 men and 7 women; mean age, 31.4 ± 10 y).
Corresponding maximal SUVs were significantly higher in the high-BAT group than
in the low-BAT group (10.7 ± 3.9 vs. 2.1 ± 0.7, P = 0.01). Blood flow values were
significantly higher in the high-BAT group than in the low-BAT group for BAT
(12.9 ± 4.1 vs. 5.9 ± 2.2 mL/100 g/min, P = 0.03) and white adipose tissue (7.2 ±
3.4 vs. 5.7 ± 2.3 mL/100 g/min, P = 0.03) but were similar for muscle (4.4 ± 1.9 
vs. 3.9 ± 1.7 mL/100 g/min). Moreover, OEF in BAT was similar in the 2 groups
(0.51 ± 0.17 in high-BAT group vs. 0.47 ± 0.18 in low-BAT group, P = 0.39).
During mild cold stress, calculated MRO2 values in BAT increased from 0.97 ± 0.53
to 1.42 ± 0.68 mL/100 g/min (P = 0.04) in the high-BAT group and were
significantly higher than those determined in the low-BAT group (0.40 ± 0.28 vs. 
0.51 ± 0.23, P = 0.67). The increase in DEE associated with BAT oxidative
metabolism was highly variable in the high-BAT group, with an average of 3.2 ±
2.4 kcal/d (range, 1.9-4.6 kcal/d) at rest, and increased to 6.3 ± 3.5 kcal/d
(range, 4.0-9.9 kcal/d) during exposure to mild cold. Although BAT accounted for 
only a small fraction of the cold-induced increase in DEE, such increases were
not observed in subjects lacking BAT.
CONCLUSION: Mild cold-induced thermogenesis in BAT accounts for 15-25 kcal/d in
subjects with relatively large BAT depots. Thus, although the presence of active 
BAT is correlated with cold-induced energy expenditure, direct measurement of
MRO2 indicates that BAT is a minor source of thermogenesis in humans.
 
PMCID: PMC3883579
PMID: 23362317
 
---------
[3] Nat Med. 2012 Oct;18(10):1575-9. doi: 10.1038/nm.2897. Epub 2012 Sep 9.
 
Sarcolipin is a newly identified regulator of muscle-based thermogenesis in
mammals.
 
Bal NC(1), Maurya SK, Sopariwala DH, Sahoo SK, Gupta SC, Shaikh SA, Pant M,
Rowland LA, Bombardier E, Goonasekera SA, Tupling AR, Molkentin JD, Periasamy M.
 
 
The role of skeletal muscle in nonshivering thermogenesis (NST) is not well
understood. Here we show that sarcolipin (Sln), a newly identified regulator of
the sarco/endoplasmic reticulum Ca(2+)-ATPase (Serca) pump, is necessary for
muscle-based thermogenesis. When challenged to acute cold (4 °C), Sln(-/-) mice
were not able to maintain their core body temperature (37 °C) and developed
hypothermia. Surgical ablation of brown adipose tissue and functional knockdown
of Ucp1 allowed us to highlight the role of muscle in NST. Overexpression of Sln 
in the Sln-null background fully restored muscle-based thermogenesis, suggesting 
that Sln is the basis for Serca-mediated heat production. We show that ryanodine 
receptor 1 (Ryr1)-mediated Ca(2+) leak is an important mechanism for
Serca-activated heat generation. Here we present data to suggest that Sln can
continue to interact with Serca in the presence of Ca(2+), which can promote
uncoupling of the Serca pump and cause futile cycling. We further show that loss 
of Sln predisposes mice to diet-induced obesity, which suggests that Sln-mediated
NST is recruited during metabolic overload. These data collectively suggest that 
SLN is an important mediator of muscle thermogenesis and whole-body energy
metabolism.
 
PMCID: PMC3676351
PMID: 22961106
 
---------
[4] Biochim Biophys Acta. 2001 Jun 1;1505(2-3):271-9.

Cold-induced changes in the energy coupling and the UCP3 level in rodent skeletal
muscles.

Simonyan RA(1), Jimenez M, Ceddia RB, Giacobino JP, Muzzin P, Skulachev VP.

Author information:
(1)Department of Bioenergetics, AN Belozersky Institute of Physico-Chemical
Biology, Moscow State University, Russia.

The mechanism of thermoregulatory uncoupling of respiration and phosphorylation
in skeletal muscles has been studied. It is found that 24 h cold exposure results
in (i) a 3-fold increase in the amount of UCP3 protein in rat skeletal muscle
mitochondria, and (ii) pronounced lowering of the membrane potential in isolated
rat or mouse skeletal muscle mitochondria.
The decrease in membrane potential is
reversed by adding bovine serum albumin. Cold exposure is also found to sensitize
the membrane potential to the uncoupling action of added fatty acid (laurate).
After laurate addition, the recoupling effects of GDP and carboxyatractylate
decrease whereas that of albumin increases in mitochondria from cold-treated rats
or mice. Changes similar to those induced by cold can be initiated by the in vivo
addition of thyroxine. Cold exposure does not affect energy coupling in liver
mitochondria. The possible involvement of UCP3 isoforms in nucleotide-sensitive
and -insensitive uncoupling is discussed.

PMID: 11334791

 

---------

[5] FEBS Lett. 2005 Mar 28;579(9):1978-82.

 
Cold exposure differently influences mitochondrial energy efficiency in rat liver
and skeletal muscle.
 
Mollica MP(1), Lionetti L, Crescenzo R, Tasso R, Barletta A, Liverini G, Iossa S.
 
Author information: 
(1)Department of Biological Sciences, Section of Physiology, University of Naples
"Federico II", Via Mezzocannone 8, I-80134 Naples, Italy.
 
This study deals with mitochondrial energy efficiency in liver and skeletal
muscle mitochondria in 15 days cold exposed rats. Cold exposure strongly
increases the sensitivity to uncoupling by added palmitate of skeletal muscle but
not liver mitochondria, while mitochondrial energy coupling in the absence of
fatty acids is only slightly affected by cold in liver and skeletal muscle. In
addition, uncoupling protein 3 content does not follow changes in skeletal muscle
mitochondrial coupling. It is therefore concluded that skeletal muscle could play
a direct thermogenic role based on fatty acid-induced mild uncoupling of
mitochondrial oxidative phosphorylation.
 
PMID: 15792806
 
-------
[6] J Biol Chem. 2015 Apr 24;290(17):10840-9. doi: 10.1074/jbc.M115.636878. Epub 2015
Feb 24.
 
Sarcolipin Is a Key Determinant of the Basal Metabolic Rate, and Its
Overexpression Enhances Energy Expenditure and Resistance against Diet-induced
Obesity.
 
Maurya SK(1), Bal NC(2), Sopariwala DH(2), Pant M(2), Rowland LA(2), Shaikh
SA(2), Periasamy M(3).
 
Author information: 
(1)the Sanford Burnham Medical Research Institute at Lake Nona, Orlando, Florida 
32827. (2)From the Department of Physiology and Cell Biology, The Ohio State
University, Columbus, Ohio 43210 and. (3)the Sanford Burnham Medical Research
Institute at Lake Nona, Orlando, Florida 32827 periasamy.1@osu.edu
mperiasamy@sanfordburnham.org.
 
Sarcolipin (SLN) is a novel regulator of sarcoplasmic reticulum Ca(2+) ATPase
(SERCA) in muscle. SLN binding to SERCA uncouples Ca(2+) transport from ATP
hydrolysis. By this mechanism, SLN promotes the futile cycling of SERCA,
contributing to muscle heat production. We recently showed that SLN plays an
important role in cold- and diet-induced thermogenesis. However, the detailed
mechanism of how SLN regulates muscle metabolism remains unclear. In this study, 
we used both SLN knockout (Sln(-/-)) and skeletal muscle-specific SLN
overexpression (Sln(OE)) mice to explore energy metabolism by pair feeding (fixed
calories) and high-fat diet feeding (ad libitum). Our results show that, upon
pair feeding, Sln(OE) mice lost weight compared with the WT, but Sln(-/-) mice
gained weight. Interestingly, when fed with a high-fat diet, Sln(OE) mice
consumed more calories but gained less weight and maintained a normal metabolic
profile in comparison with WT and Sln(-/-) mice. We found that oxygen consumption
and fatty acid oxidation were increased markedly in Sln(OE) mice. There was also 
an increase in both mitochondrial number and size in Sln(OE) muscle, together
with increased expression of peroxisome proliferator-activated receptor δ (PPARδ)
and PPAR γ coactivator 1 α (PGC1α), key transcriptional activators of
mitochondrial biogenesis and enzymes involved in oxidative metabolism. These
results, taken together, establish an important role for SLN in muscle metabolism
and energy expenditure. On the basis of these data we propose that SLN is a novel
target for enhancing whole-body energy expenditure.
 
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
 
PMCID: PMC4409248 [Available on 2016-04-24]
PMID: 25713078
 
--------
[7] Obesity (Silver Spring). 2015 Jul;23(7):1440-9. doi: 10.1002/oby.21123. Epub 2015
May 13.
 
Reduced efficiency of sarcolipin-dependent respiration in myocytes from humans
with severe obesity.
 
Paran CW(1,)(2,)(3), Verkerke AR(1,)(2), Heden TD(1,)(2), Park S(1,)(2), Zou
K(1,)(2), Lawson HA(4), Song H(5), Turk J(5), Houmard JA(1,)(2), Funai
K(1,)(2,)(3).
 
 
Author information: 
(1)East Carolina Diabetes and Obesity Institute, East Carolina University,
Greenville, North Carolina, USA. (2)Department of Kinesiology, East Carolina
University, Greenville, North Carolina, USA. (3)Department of Physiology, East
Carolina University, Greenville, North Carolina, USA. (4)Department of Genetics, 
Washington University School of Medicine, St. Louis, Missouri, USA. (5)Medicine
Mass Spectrometry Facility, Washington University School of Medicine, St. Louis, 
Missouri, USA.
 
OBJECTIVE: Sarcolipin (SLN) regulates muscle energy expenditure through its
action on sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump. It is unknown 
whether SLN-dependent respiration has relevance to human obesity, but
whole-transcriptome gene expression profiling revealed that SLN was more highly
expressed in myocytes from individuals with severe obesity (OB) than in lean
controls (LN). The purpose of this study was to examine SLN-dependent cellular
respiratory rates in LN and OB human muscles.
METHODS: Primary myocytes were isolated from muscle biopsy from seven LN and OB
Caucasian females. Cellular respiration was assessed with and without
lentivirus-mediated SLN knockdown in LN and OB myocytes.
RESULTS: SLN mRNA and protein abundance was greater in OB compared to LN cells.
Despite elevated SLN levels in wild-type OB cells, respiratory rates among
SLN-deficient cells were higher in OB compared to LN. Obesity-induced reduction
in efficiency of SLN-dependent respiration was associated with altered
sarcoplasmic reticulum phospholipidome.
CONCLUSIONS: SLN-dependent respiration is reduced in muscles from humans with
severe obesity compared to lean controls. Identification of the molecular
mechanism that affects SLN efficiency might lead to interventions that promote an
increase in skeletal muscle energy expenditure.
 
© 2015 The Obesity Society.
 
PMCID: PMC4483165 [Available on 2016-07-01]
PMID: 25970801
 
-------
[8] Endocrinology. 2008 Dec;149(12):6262-71. doi: 10.1210/en.2008-0564. Epub 2008 Aug
14.
 
Cold tolerance in hypothyroid rabbits: role of skeletal muscle mitochondria and
sarcoplasmic reticulum Ca2+ ATPase isoform 1 heat production.
 
Arruda AP(1), Ketzer LA, Nigro M, Galina A, Carvalho DP, de Meis L.
 
Author information: 
(1)Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Cidade
Universitária, Rio de Janeiro 21941-590, Brazil.
 
 
Brown adipose tissue (BAT) is involved in rat and mice thermoregulation, and heat
produced by BAT depends on the concerted action of thyroid hormones and
catecholamines. Little is known about cold-induced thermogenesis in mammals that 
have little or no BAT, such as rabbits. In these animals, thermogenesis primarily
occurs in skeletal muscle. In this work, we have studied the effect of cold
acclimation (4 C for 10 d) in normal and hypothyroid rabbits. It is known that
hypothyroid rats die after a few hours of cold exposure. We now show that,
different from rats, hypothyroid rabbits sustain their body temperature and
survive after 10 d cold exposure. When compared with rabbits kept at room
temperature, the muscles of cold-exposed rabbits showed a dark red color
characteristic of oxidative muscle fibers. According to this pattern, we observed
that in both normal and hypothyroid rabbits, cold exposure promotes an increase
in oxygen consumption by skeletal muscle mitochondria. Moreover, in red muscle,
cold acclimation induces an increase in the expression and activity of
sarcoplasmic reticulum Ca(2+) ATPase isoform 1 (SERCA1), one of the muscle
enzymes involved in heat production. We conclude that rabbit cold tolerance is
probably related to increased muscle oxidative metabolism and heat production by 
SERCA1 and that these changes are not completely dependent on normal thyroid
function.
 
PMID: 18703625
 
-------------
[9] J Physiol. 2010 Nov 1;588(Pt 21):4275-88. doi: 10.1113/jphysiol.2010.198598.

Increased fatigue resistance linked to Ca2+-stimulated mitochondrial biogenesis
in muscle fibres of cold-acclimated mice.

Bruton JD(1), Aydin J, Yamada T, Shabalina IG, Ivarsson N, Zhang SJ, Wada M, Tavi
P, Nedergaard J, Katz A, Westerblad H.

Free full text: http://www.ncbi.nlm....les/PMC3002456/

Mammals exposed to a cold environment initially generate heat by repetitive
muscle activity (shivering). Shivering is successively replaced by the
recruitment of uncoupling protein-1 (UCP1)-dependent heat production in brown
adipose tissue. Interestingly, adaptations observed in skeletal muscles of
cold-exposed animals are similar to those observed with endurance training. We
hypothesized that increased myoplasmic free [Ca2+] ([Ca2+]i) is important for
these adaptations. To test this hypothesis, experiments were performed on flexor
digitorum brevis (FDB) muscles, which do not participate in the shivering
response, of adult wild-type (WT) and UCP1-ablated (UCP1-KO) mice kept either at
room temperature (24°C) or cold-acclimated (4°C) for 4-5 weeks. [Ca2+]i (measured
with indo-1) and force were measured under control conditions and during fatigue
induced by repeated tetanic stimulation in intact single fibres. The results show
no differences between fibres from WT and UCP1-KO mice. However, muscle fibres
from cold-acclimated mice showed significant increases in basal [Ca2+]i (∼50%),
tetanic [Ca2+]i (∼40%), and sarcoplasmic reticulum (SR) Ca2+ leak (∼fourfold) as
compared to fibres from room-temperature mice. Muscles of cold-acclimated mice
showed increased expression of peroxisome proliferator-activated receptor-γ
coactivator-1α (PGC-1α) and increased citrate synthase activity (reflecting
increased mitochondrial content). Fibres of cold-acclimated mice were more
fatigue resistant with higher tetanic [Ca2+]i and less force loss during
fatiguing stimulation. In conclusion, cold exposure induces changes in FDB
muscles similar to those observed with endurance training and we propose that
increased [Ca2+]i is a key factor underlying these adaptations.

PMCID: PMC3002456
PMID: 20837639
 
-----
[10] J Appl Physiol (1985). 2015 Apr 15;118(8):1050-8. doi:

10.1152/japplphysiol.01066.2014. Epub 2015 Feb 19.

Sarcolipin overexpression improves muscle energetics and reduces fatigue.

Sopariwala DH(1), Pant M(1), Shaikh SA(1), Goonasekera SA(2), Molkentin JD(3),
Weisleder N(1), Ma J(4), Pan Z(4), Periasamy M(5).

Sarcolipin (SLN) is a regulator of sarcoendoplasmic reticulum calcium ATPase in
skeletal muscle. Recent studies using SLN-null mice have identified SLN as a key
player in muscle thermogenesis and metabolism. In this study, we exploited a SLN
overexpression (Sln(OE)) mouse model to determine whether increased SLN level
affected muscle contractile properties, exercise capacity/fatigue, and metabolic
rate in whole animals and isolated muscle. We found that Sln(OE) mice are more
resistant to fatigue and can run significantly longer distances than wild-type
(WT). Studies with isolated extensor digitorum longus (EDL) muscles showed that
Sln(OE) EDL produced higher twitch force than WT. The force-frequency curves were
not different between WT and Sln(OE) EDLs, but at lower frequencies the
pyruvate-induced potentiation of force was significantly higher in Sln(OE) EDL.
SLN overexpression did not alter the twitch and force-frequency curve in isolated
soleus muscle. However, during a 10-min fatigue protocol, both EDL and soleus
from Sln(OE) mice fatigued significantly less than WT muscles. Interestingly,
Sln(OE) muscles showed higher carnitine palmitoyl transferase-1 protein
expression, which could enhance fatty acid metabolism. In addition, lactate
dehydrogenase expression was higher in Sln(OE) EDL, suggesting increased
glycolytic capacity. We also found an increase in store-operated calcium entry
(SOCE) in isolated flexor digitorum brevis fibers of Sln(OE) compared with WT
mice. These data allow us to conclude that increased SLN expression improves
skeletal muscle performance during prolonged muscle activity by increasing SOCE
and muscle energetics.

Copyright © 2015 the American Physiological Society.

PMCID: PMC4398885

 

-------
[11] J Exp Biol. 2015 Aug;218(Pt 15):2321-5. doi: 10.1242/jeb.119164. Epub 2015 May
29.
 
Cold adaptation overrides developmental regulation of sarcolipin expression in
mice skeletal muscle: SOS for muscle-based thermogenesis?
 
Pant M(1), Bal NC(1), Periasamy M(2).
 
Author information: 
(1)Department of Physiology and Cell Biology, The Ohio State University,
Columbus, OH 43210, USA. (2)Department of Physiology and Cell Biology, The Ohio
State University, Columbus, OH 43210, USA mperiasamy@sbpdiscovery.org.
 
Neonatal mice have a greater thermogenic need than adult mice and may require
additional means of heat production, other than the established mechanism of
brown adipose tissue (BAT). We and others recently discovered a novel mediator of
skeletal muscle-based thermogenesis called sarcolipin (SLN) that acts by
uncoupling sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA). In addition, we have
shown that SLN expression is downregulated during neonatal development in rats.
In this study we probed two questions: (1) is SLN expression developmentally
regulated in neonatal mice?; and (2) if so, will cold adaptation override this?
Our data show that SLN expression is higher during early neonatal stages and is
gradually downregulated in fast twitch skeletal muscles. Interestingly, we
demonstrate that cold acclimation of neonatal mice can prevent downregulation of 
SLN expression. This observation suggests that SLN-mediated thermogenesis can be 
recruited to a greater extent during extreme physiological need, in addition to
BAT.
 
© 2015. Published by The Company of Biologists Ltd.
 
PMCID: PMC4528705 [Available on 2016-08-01]
PMID: 26026037
 
-----------------
[12] Am J Physiol Endocrinol Metab. 2013 Sep 1;305(5):E567-72. doi:

10.1152/ajpendo.00250.2013. Epub 2013 Jul 9.

An endocrine role for brown adipose tissue?

Villarroya J(1), Cereijo R, Villarroya F.

Author information:
(1)Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; and.

White adipose tissue is recognized as both a site of energy storage and an
endocrine organ that produces a myriad of endocrine factors called adipokines.
Brown adipose tissue (BAT) is the main site of nonshivering thermogenesis in
mammals. The amount and activity of brown adipocytes are associated with
protection against obesity and associated metabolic alterations. These effects of
BAT are traditionally attributed to its capacity for the oxidation of fatty acids
and glucose to sustain thermogenesis. However, recent data suggest that the
beneficial effects of BAT could involve a previously unrecognized endocrine role
through the release of endocrine factors. Several signaling molecules with
endocrine properties have been found to be released by brown fat, especially
under conditions of thermogenic activation. Moreover, experimental BAT
transplantation has been shown to improve glucose tolerance and insulin
sensitivity mainly by influencing hepatic and cardiac function. It has been
proposed that these effects are due to the release of endocrine factors by brown
fat, such as insulin-like growth factor I, interleukin-6, or fibroblast growth
factor-21. Further research is needed to determine whether brown fat plays an
endocrine role and, if so, to comprehensively identify which endocrine factors
are released by BAT. Such research may reveal novel clues for the observed
association between brown adipocyte activity and a healthy metabolic profile, and
it could also enlarge a current view of potential therapeutic tools for obesity
and associated metabolic diseases.

PMID: 23839524

 

----------

[13] Nat Med. 2002 Nov;8(11):1288-95. Epub 2002 Oct 7.

Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating
AMP-activated protein kinase.

Yamauchi T(1), Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S, Yamashita S, Noda
M, Kita S, Ueki K, Eto K, Akanuma Y, Froguel P, Foufelle F, Ferre P, Carling D,
Kimura S, Nagai R, Kahn BB, Kadowaki T.

Author information:
(1)Department of Internal Medicine, Graduate School of Medicine, University of
Tokyo, Tokyo, Japan.

Adiponectin (Ad) is a hormone secreted by adipocytes that regulates energy
homeostasis and glucose and lipid metabolism. However, the signaling pathways
that mediate the metabolic effects of Ad remain poorly identified. Here we show
that phosphorylation and activation of the 5'-AMP-activated protein kinase (AMPK)
are stimulated with globular and full-length Ad in skeletal muscle and only with
full-length Ad in the liver. In parallel with its activation of AMPK, Ad
stimulates phosphorylation of acetyl coenzyme A carboxylase (ACC), fatty-acid
oxidation, glucose uptake and lactate production in myocytes, phosphorylation of
ACC and reduction of molecules involved in gluconeogenesis in the liver, and
reduction of glucose levels in vivo. Blocking AMPK activation by
dominant-negative mutant inhibits each of these effects, indicating that
stimulation of glucose utilization and fatty-acid oxidation by Ad occurs through
activation of AMPK. Our data may provide a novel paradigm that an
adipocyte-derived antidiabetic hormone, Ad, activates AMPK, thereby directly
regulating glucose metabolism and insulin sensitivity in vitro and in vivo.

PMID: 12368907

 

----------

[14] Fajardo VA, Bombardier E, Vigna C, et al. Co-expression of SERCA isoforms, phospholamban and sarcolipin in human skeletal muscle fibers. PLoS One. 2013; 8(12):e84304.

 
---------
[15] Exerc Sport Sci Rev. 2014 Jul;42(3):136-42. doi: 10.1249/JES.0000000000000016.

Sarcolipin provides a novel muscle-based mechanism for adaptive thermogenesis.

Gamu D(1), Bombardier E, Smith IC, Fajardo VA, Tupling AR.

Author information:
(1)Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada.

 

Full text: https://sci-hub.io/1...000000000000016

The sarco(endo)plasmic reticulum Ca-ATPase (SERCA) transports Ca into the
sarcoplasmic reticulum lumen and contributes significantly to skeletal muscle
metabolic rate. Sarcolipin (SLN) has been shown recently to uncouple Ca transport
from adenosine triphosphate hydrolysis by SERCA. We have hypothesized that SLN
provides a novel mechanism of adaptive thermogenesis within skeletal muscle and
protects against diet-induced obesity.

PMID: 24949847

 

---------

[16] Biol Rev Camb Philos Soc. 2015 Nov;90(4):1279-97. doi: 10.1111/brv.12157. Epub

2014 Nov 25.

The role of skeletal-muscle-based thermogenic mechanisms in vertebrate
endothermy.

Rowland LA(1), Bal NC(1), Periasamy M(1).

Author information:
(1)Department of Physiology and Cell Biology, College of Medicine, The Ohio State
University, Columbus, OH 43210, U.S.A.

 

Full text: https://sci-hub.io/10.1111/brv.12157

Thermogenesis is one of the most important homeostatic mechanisms that evolved
during vertebrate evolution. Despite its importance for the survival of the
organism, the mechanistic details behind various thermogenic processes remain
incompletely understood. Although heat production from muscle has long been
recognized as a thermogenic mechanism, whether muscle can produce heat
independently of contraction remains controversial. Studies in birds and mammals
suggest that skeletal muscle can be an important site of non-shivering
thermogenesis (NST) and can be recruited during cold adaptation, although
unequivocal evidence is lacking. Much research on thermogenesis during the last
two decades has been focused on brown adipose tissue (BAT). These studies clearly
implicate BAT as an important site of NST in mammals, in particular in newborns
and rodents. However, BAT is either absent, as in birds and pigs, or is only a
minor component, as in adult large mammals including humans, bringing into
question the BAT-centric view of thermogenesis. This review focuses on the
evolution and emergence of various thermogenic mechanisms in vertebrates from
fish to man. A careful analysis of the existing data reveals that muscle was the
earliest facultative thermogenic organ to emerge in vertebrates, long before the
appearance of BAT in eutherian mammals. Additionally, these studies suggest that
muscle-based thermogenesis is the dominant mechanism of heat production in many
species including birds, marsupials, and certain mammals where BAT-mediated
thermogenesis is absent or limited. We discuss the relevance of our recent
findings showing that uncoupling of sarco(endo)plasmic reticulum Ca(2+)-ATPase
(SERCA) by sarcolipin (SLN), resulting in futile cycling and increased heat
production, could be the basis for NST in skeletal muscle. The overall goal of
this review is to highlight the role of skeletal muscle as a thermogenic organ
and provide a balanced view of thermogenesis in vertebrates.

© 2014 Cambridge Philosophical Society.

PMID: 25424279

 

----------

[17] Proc Natl Acad Sci U S A. 2006 Feb 7;103(6):1768-73. Epub 2006 Jan 30.

 
Calorie restriction induces mitochondrial biogenesis and bioenergetic efficiency.
 
López-Lluch G(1), Hunt N, Jones B, Zhu M, Jamieson H, Hilmer S, Cascajo MV,
Allard J, Ingram DK, Navas P, de Cabo R.
 
Author information: 
(1)Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, 41013
Sevilla, Spain.
 
Age-related accumulation of cellular damage and death has been linked to
oxidative stress. Calorie restriction (CR) is the most robust, nongenetic
intervention that increases lifespan and reduces the rate of aging in a variety
of species. Mechanisms responsible for the antiaging effects of CR remain
uncertain, but reduction of oxidative stress within mitochondria remains a major 
focus of research. CR is hypothesized to decrease mitochondrial electron flow and
proton leaks to attenuate damage caused by reactive oxygen species. We have
focused our research on a related, but different, antiaging mechanism of CR.
Specifically, using both in vivo and in vitro analyses, we report that CR reduces
oxidative stress at the same time that it stimulates the proliferation of
mitochondria through a peroxisome proliferation-activated receptor coactivator 1 
alpha signaling pathway. Moreover, mitochondria under CR conditions show less
oxygen consumption, reduce membrane potential, and generate less reactive oxygen 
species than controls, but remarkably they are able to maintain their critical
ATP production. In effect, CR can induce a peroxisome proliferation-activated
receptor coactivator 1 alpha-dependent increase in mitochondria capable of
efficient and balanced bioenergetics to reduce oxidative stress and attenuate
age-dependent endogenous oxidative damage.
 
PMCID: PMC1413655
PMID: 16446459
 
-----------
[18] PLoS Med. 2007 Mar;4(3):e76.

Calorie restriction increases muscle mitochondrial biogenesis in healthy humans.

Civitarese AE(1), Carling S, Heilbronn LK, Hulver MH, Ukropcova B, Deutsch WA,
Smith SR, Ravussin E; CALERIE Pennington Team.

Author information:
(1)Pennington Biomedical Research Center, Baton Rouge, Louisiana, United States
of America. CivitaAE@pbrc.edu

BACKGROUND: Caloric restriction without malnutrition extends life span in a range
of organisms including insects and mammals and lowers free radical production by
the mitochondria. However, the mechanism responsible for this adaptation are
poorly understood.
METHODS AND FINDINGS: The current study was undertaken to examine muscle
mitochondrial bioenergetics in response to caloric restriction alone or in
combination with exercise in 36 young (36.8 +/- 1.0 y), overweight (body mass
index, 27.8 +/- 0.7 kg/m(2)) individuals randomized into one of three groups for
a 6-mo intervention: Control, 100% of energy requirements; CR, 25% caloric
restriction; and CREX, caloric restriction with exercise (CREX), 12.5% CR + 12.5%
increased energy expenditure (EE). In the controls, 24-h EE was unchanged, but in
CR and CREX it was significantly reduced from baseline even after adjustment for
the loss of metabolic mass (CR, -135 +/- 42 kcal/d, p = 0.002 and CREX, -117 +/-
52 kcal/d, p = 0.008). Participants in the CR and CREX groups had increased
expression of genes encoding proteins involved in mitochondrial function such as
PPARGC1A, TFAM, eNOS, SIRT1, and PARL (all, p < 0.05). In parallel, mitochondrial
DNA content increased by 35% +/- 5% in the CR group (p = 0.005) and 21% +/- 4% in
the CREX group (p < 0.004), with no change in the control group (2% +/- 2%).
However, the activity of key mitochondrial enzymes of the TCA (tricarboxylic
acid) cycle (citrate synthase), beta-oxidation (beta-hydroxyacyl-CoA
dehydrogenase), and electron transport chain (cytochrome C oxidase II) was
unchanged. DNA damage was reduced from baseline in the CR (-0.56 +/- 0.11
arbitrary units, p = 0.003) and CREX (-0.45 +/- 0.12 arbitrary units, p = 0.011),
but not in the controls. In primary cultures of human myotubes, a nitric oxide
donor (mimicking eNOS signaling) induced mitochondrial biogenesis but failed to
induce SIRT1 protein expression, suggesting that additional factors may regulate
SIRT1 content during CR.
CONCLUSIONS: The observed increase in muscle mitochondrial DNA in association
with a decrease in whole body oxygen consumption and DNA damage suggests that
caloric restriction improves mitochondrial function in young non-obese adults.

PMCID: PMC1808482
PMID: 17341128

 
--------
[19] PLoS One. 2008 Mar 12;3(3):e1777. doi: 10.1371/journal.pone.0001777.

Human skeletal muscle mitochondrial uncoupling is associated with cold induced
adaptive thermogenesis.

Wijers SL(1), Schrauwen P, Saris WH, van Marken Lichtenbelt WD.

Author information:
(1)Department of Human Biology, Nutrition and Toxicology Research Institute
Maastricht (NUTRIM), Maastricht University, The Netherlands.
S.Wijers@hb.unimaas.nl

 

Free full text: http://www.ncbi.nlm....les/PMC2258415/

BACKGROUND: Mild cold exposure and overfeeding are known to elevate energy
expenditure in mammals, including humans. This process is called adaptive
thermogenesis. In small animals, adaptive thermogenesis is mainly caused by
mitochondrial uncoupling in brown adipose tissue and regulated via the
sympathetic nervous system. In humans, skeletal muscle is a candidate tissue,
known to account for a large part of the epinephrine-induced increase in energy
expenditure. However, mitochondrial uncoupling in skeletal muscle has not
extensively been studied in relation to adaptive thermogenesis in humans.
Therefore we hypothesized that cold-induced adaptive thermogenesis in humans is
accompanied by an increase in mitochondrial uncoupling in skeletal muscle.
METHODOLOGY/PRINCIPAL FINDINGS: The metabolic response to mild cold exposure in
11 lean, male subjects was measured in a respiration chamber at baseline and mild
cold exposure. Skeletal muscle mitochondrial uncoupling (state 4) was measured in
muscle biopsies taken at the end of the respiration chamber stays. Mild cold
exposure caused a significant increase in 24h energy expenditure of 2.8% (0.32
MJ/day, range of -0.21 to 1.66 MJ/day, p<0.05). The individual increases in
energy expenditure correlated to state 4 respiration (p<0.02, R(2) = 0.50).
CONCLUSIONS/SIGNIFICANCE: This study for the first time shows that in humans,
skeletal muscle has the intrinsic capacity for cold induced adaptive
thermogenesis via mitochondrial uncoupling under physiological conditions. This
opens possibilities for mitochondrial uncoupling as an alternative therapeutic
target in the treatment of obesity.

PMCID: PMC2258415
PMID: 18335051

 

----------

[20] FEBS Lett. 1997 Jul 21;412(1):111-4.

Tissue-dependent upregulation of rat uncoupling protein-2 expression in response
to fasting or cold.

Boss O(1), Samec S, Dulloo A, Seydoux J, Muzzin P, Giacobino JP.

Author information:
(1)Department of Medical Biochemistry, Faculty of Medicine, University of Geneva,
Switzerland. Olivier.Boss@medecine.unige.ch

The control of uncoupling protein-2 (UCP2) mRNA expression in rat brown adipose
tissue (BAT), heart and skeletal muscles was examined. Cold exposure (48 h)
increased UCP2 mRNA in BAT, heart and soleus muscle by 2.4-, 4.3- and 2.6-fold,
respectively.
Fasting (48 h) had no effect on UCP2 mRNA expression neither in BAT
nor in heart, but markedly increased it in skeletal muscles. While the
upregulation of UCP2 mRNA in response to cold exposure is in line with a putative
uncoupling role for this protein in thermoregulatory thermogenesis, the
unexpected upregulation of UCP2 in skeletal muscles in response to fasting seems
inconsistent with its role as an uncoupling protein involved in dietary
regulation of thermogenesis.

PMID: 9257701

 

-----------

[21] Methods Enzymol. 2009;457:395-404. doi: 10.1016/S0076-6879(09)05022-8.

Methods for assessing and modulating UCP2 expression and function.

Velloso LA(1), Degasperi GR, Vercesi AE, Saad MA.

Author information:
(1)Department of Internal Medicine, University of Campinas, Campinas, SP, Brazil.

Uncoupling protein 2 (UCP2) is a member of the uncoupling protein family. It is
expressed in the inner mitochondrial membrane and plays a role in the control of
free radical production, oxidative damage, insulin secretion, and fatty-acid
peroxide exportation. Although UCP2 expression occurs in several tissues, some of
its most remarkable functions are exerted in organs of difficult experimental
access, such as the central nervous system, particularly the hypothalamus and the
pancreatic islets. In addition, due to its low levels of expression in the
mitochondrial membrane, studying UCP2 expression and function depends on
specific- and well-established methods. This chapter describes methods for
directly assessing UCP2 expression and function in different tissues. Purified
mitochondria preparations are used for enhancing the capacity of detection of
UCP2 protein or for evaluating the role of UCP2 in mitochondria respiration.
Exposure of experimental animals to cold environment leads to increased UCP2
expression,
while reduction of its expression can be achieved directly by
targeting its mRNA with antisense oligonucleotides, or indirectly by targeting
PGC-1alpha expression with antisense oligonucleotides.

PMID: 19426880

 

---------

[22] Curr Aging Sci. 2010 Jul;3(2):102-12.

Uncoupling protein-2 and the potential link between metabolism and longevity.

Andrews ZB(1).

Author information:
(1)Department of Physiology, Monash University, Clayton, VIC 3183 Australia.
zane.andrews@med.monash.edu.au

The discovery of novel uncoupling proteins (UCP2 and UCP3) over 10 years ago
heralded a new era of research in mitochondrial uncoupling in a diverse range of
tissues. Despite the research vigor, debate stills surrounds the exact function
of these uncoupling proteins. For example, the level of uncoupling, the mechanism
and mode of action are all under-appreciated at this point in time. Our recent
work has used genetic mouse models to focus on the physiological relevance of
UCP2. We have used these mouse models to better appreciate the role UCP2 in human
health and disease. In this review we focus on new research showing that UCP2
promotes longevity by shifting a given cell towards fatty acid fuel utilization.
This metabolic hypothesis underlying UCP2-dependent longevity suggests that UCP2
is critically positioned to maintain fatty acid oxidation and restrict subsequent
oxidative damage allowing sustained mitochondrial oxidative capacity and
mitochondrial biogenesis. These mechanisms converge within the cell to boost cell
function and metabolism and the net result promotes healthy aging and increased
lifespan. Finally, UCP2 is a useful dietary and therapeutic target to promote
lifespan and is an important mitochondrial protein connecting longevity to
metabolism.

PMID: 20158496 [PubMed - indexed for MEDLINE]

------

[23] Am J Physiol Endocrinol Metab. 2009 Apr;296(4):E621-7. doi:

10.1152/ajpendo.90903.2008. Epub 2009 Jan 13.

Uncoupling protein-2 regulates lifespan in mice.

Andrews ZB(1), Horvath TL.

Author information:
(1)Section of Comparative Medicine, Department of Obstretics, Yale University
School of Medicine, 375 Congress Ave., LSOG 117, New Haven, CT 06519, USA.
zane.andrews@med.monash.edu.au

Comment in
Am J Physiol Endocrinol Metab. 2009 Apr;296(4):E619-20.

The long-term effects of uncoupled mitochondrial respiration by uncoupling
protein-2 (UCP2) in mammalian physiology remain controversial. Here we show that
increased mitochondrial uncoupling activity of different tissues predicts longer
lifespan of rats compared with mice. UCP2 reduces reactive oxygen species (ROS)
production and oxidative stress throughout the aging process in different tissues
in mice. The absence of UCP2 shortens lifespan in wild-type mice, and the level
of UCP2 positively correlates with the postnatal survival of superoxide
dismutase-2 mutant animals. Thus UCP2 has a beneficial influence on cell and
tissue function leading to increased lifespan.

PMCID: PMC2670629
PMID: 19141680 [PubMed - indexed for MEDLINE]
 

------

[24] Exp Gerontol. 2008 Dec;43(12):1061-8. doi: 10.1016/j.exger.2008.09.011. Epub 2008
Sep 27.

Characterization of survival and phenotype throughout the life span in UCP2/UCP3
genetically altered mice.

McDonald RB(1), Walker KM, Warman DB, Griffey SM, Warden CH, Ramsey JJ, Horwitz
BA.

Author information:
(1)Department of Nutrition, One Shields Avenue, University of California, Davis,
CA 95616, USA. rbmcdonald@ucdavis.edu

In the present investigation we describe the life span characteristics and
phenotypic traits of ad libitum-fed mice that overexpress UCP2/3 (Positive-TG),
their non-overexpressing littermates (Negative-TG), mice that do not expression
UCP2 (UCP2KO) or UCP3 (UCP3KO), and wild-type C57BL/6J mice (WT-Control). We also
included a group of C57BL/6J mice calorie-restricted to 70% of ad libitum-fed
mice in order to test partially the hypothesis that UCPs contribute to the life
extension properties of CR. Mean survival was slightly, but significantly,
greater in Positive-TG, than that observed in Negative-TG or WT-Control; mean
life span did not significantly differ from that of the UCP3KO mice. Maximal life
span did not differ among the ad libitum-fed groups. Genotype did not
significantly affect body weight, food intake, or the type of pathology at time
of death. Calorie restriction increased significantly mean and maximal life span,
and the expression of UCP2 and UCP3. The lack of difference in maximal life spans
among the Positive-TG, Negative-TG, and UCP3KO suggests that UCP3 does not
significantly affect longevity in mice.

PMID: 18854208 [PubMed - indexed for MEDLINE]

 

------

[25] Science. 2006 Nov 3;314(5800):825-8.

Transgenic mice with a reduced core body temperature have an increased life span.

Conti B(1), Sanchez-Alavez M, Winsky-Sommerer R, Morale MC, Lucero J, Brownell S,
Fabre V, Huitron-Resendiz S, Henriksen S, Zorrilla EP, de Lecea L, Bartfai T.

Author information:
(1)Harold L. Dorris Neurological Research Center, Scripps Research Institute, La
Jolla, CA 92037, USA. bconti@scripps.edu

Comment in
Science. 2006 Nov 3;314(5800):773-4.

Reduction of core body temperature has been proposed to contribute to the
increased life span and the antiaging effects conferred by calorie restriction
(CR). Validation of this hypothesis has been difficult in homeotherms, primarily
due to a lack of experimental models. We report that transgenic mice engineered
to overexpress the uncoupling protein 2 in hypocretin neurons (Hcrt-UCP2) have
elevated hypothalamic temperature. The effects of local temperature elevation on
the central thermostat resulted in a 0.3 degrees to 0.5 degrees C reduction of
the core body temperature. Fed ad libitum, Hcrt-UCP2 transgenic mice had the same
caloric intake as their wild-type littermates but had increased energy efficiency
and a greater median life span (12% increase in males; 20% increase in females).
Thus, modest, sustained reduction of core body temperature prolonged life span
independent of altered diet or CR.

PMID: 17082459

 

---------

[26] Aging Cell. 2008 Aug;7(4):552-60. doi: 10.1111/j.1474-9726.2008.00407.x. Epub

2008 Jul 10.
 
Mild mitochondrial uncoupling in mice affects energy metabolism, redox balance
and longevity.
 
Caldeira da Silva CC(1), Cerqueira FM, Barbosa LF, Medeiros MH, Kowaltowski AJ.
 
Author information: 
(1)Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo,
São Paulo, SP, Brazil.
 
 
Caloric restriction is the most effective non-genetic intervention to enhance
lifespan known to date. A major research interest has been the development of
therapeutic strategies capable of promoting the beneficial results of this
dietary regimen. In this sense, we propose that compounds that decrease the
efficiency of energy conversion, such as mitochondrial uncouplers, can be caloric
restriction mimetics. Treatment of mice with low doses of the protonophore
2,4-dinitrophenol promotes enhanced tissue respiratory rates, improved
serological glucose, triglyceride and insulin levels, decrease of reactive oxygen
species levels and tissue DNA and protein oxidation, as well as reduced body
weight. Importantly, 2,4-dinitrophenol-treated animals also presented enhanced
longevity. Our results demonstrate that mild mitochondrial uncoupling is a highly
effective in vivo antioxidant strategy, and describe the first therapeutic
intervention capable of effectively reproducing the physiological, metabolic and 
lifespan effects of caloric restriction in healthy mammals.
 
PMID: 18505478

There will never be peace in the world while there are animals in our bellies.

#139 Michael R

Michael R
  • Moderator
  • 493 posts
  • Website URL:http://www.sens.org/

Posted 26 March 2016 - 05:16 PM

Dean -- thank you for putting together all that research and thoughtful analysis - great information.


Hear, hear! I, too, want to once again express my gratitude for the large amount of in-depth digging as well as valuable lighter dips into CR- and health-related science on the Forums. Your return to the CR community has been even more productive and valuable than it was during your first tenure, and we should all be grateful for your contributions.

I have even contacted [Arlan Richardson,] the researcher doing this ongoing study of DR in mice which may be of interest to people here. He was nice enough to respond, but didn't seem to think CE was a factor. Professor Arlan says:

Hollozy’s study showed that cold exposure did not change lifespan and in a study done at San Antonio (Ikeno et al.) they showed housing mice 1 vs 4 had the same lifespan whether fed AL or DR (in fact the DR mice group housed lived a little, but not significant, longer)

I have not tracked down those references.


Huh ...

"Holloszy's study" refers to the classic "rats with cold feet" experiment, with which Dean opened this thread. But "Ikeno et al" is quite another matter — and quite a surprise!

A quick search reveals (1):

This study examined the effect of housing density on the longevity-extending and disease-delaying actions of calorie restriction (CR). Singly or multiply housed (four per cage) mice were either fed ad libitum (AL) or were on CR beginning at 2 months. All CR mice were fed 40% less food than were multiply housed AL mice [not, NB, 40% less food than matching CRS mice -MR. To wit:]mice were randomly distributed into four groups: ALM, fed AL and housed four mice per cage; ALS, fed AL and housed one mouse per cage; CRM, fed 60% of the food intake of ALM mice and housed four mice per cage; and CRS, fed 60% of the food intake of ALM mice and housed one mouse per cage. To measure the amount of food consumption, the amount of chow removed from the cage hopper and the spillage (the chow on the bottom of the cage) were weighed weekly ... ... Thus, when compared to their housing density-appropriate control group, CRS mice received only 42% of the food intake of ALS mice, whereas CRM mice received 60% of the food intake of ALM mice [as, in absolute terms, did CRS mice -MR]. ...Averaged across the life span, ALS mice consumed approximately 40% more food than did ALM mice. ... Month-to-month variation in food intake was considerable, but was similar in both housing groups. This variation was probably due to variation in temperature of the colony room, because food intake was inversely related to room temperature (data not shown). ... CRS mice weighed about 20% less than CRM mice ... despite the fact that CRS and CRM mice consumed the same amount of food .. indicat[ing] that CRS mice were unable to store in body mass as many of the calories they consumed as were the CRM mice. Thus, proportionately more of the calories consumed by the CRS mice were expended in metabolism and maintenance of body temperature than were in CRM mice.

CR increased median longevity by 19%, and housing density had no effect on this increase. [Not quite true: see below]. CR also reduced neoplastic lesions in both housing groups, but ... A striking observation was that singly housed CR mice showed a greater reduction in the incidence of presumptively fatal neoplasms and in the severity of neoplasms than did CR mice housed multiply. There are several possible explanations for this difference. First, previous studies have shown a graded and proportionate reduction in induced cancers and the degree of CR (refs). CRS mice were more restricted in relation to their singly housed AL counterparts than were CRM mice in relation to their AL counterparts. [MR: I don't really buy this, but there it is]. Furthermore, the fact that CRS mice weighed 20% less than CRM mice indicates that CRS mice were unable to store in body mass as many of the calories they consumed as were the CRM mice. Thus, proportionately more of the calories consumed by the CRS mice were expended in metabolism and maintenance of body temperature than were in CRM mice ... [so] it is possible that cell division was reduced more in the CRS group or that the partitioning of energy utilization between growth and heat generation favored heat generation more in the CRS group than in the CRM group. ...

Despite their greater reduction in cancer incidence, CRS mice did not live longer than CRM mice. [Again, this rather understates the actual data:]


F3.medium.gif


[Larger Figure]

[Can't get the survivorship Table to reproduce, but the numbers are, for median and maximum (tenth-decile) survivorship (95% CIs in brackets):


ALS: 925 (839, 73) --> 1034 (1002, 1099)
ALM 935 (861, 983) --> 1062 (992, 1135)
CRS 1080 (957, 1135) --> 1252 (1209, 1362)
CRM 1133 (931, 1284) --> 1368 (1326, 1452)

[Surprisingly, then, the warmer CRM mice, who weighed more and were slightly less protected from cancer than the CRS mice actually lived slightly longer! (yes, yes, the confidence intervals overlap — but just barely, and there were only 30 mice per subgroup). This is even more surprising, as group housing in CR mice can be dangerous, due to fighting over food. if you look at the survival curves, they were in lockstep until ≈900 d, similar to both cohorts' median LSs: this is consistent with the lesser cancer protection and apparently slower aging of the warmer CRS mice -MR]

Singly housed AL mice ate 40% more food than did multiply housed AL mice, but weighed the same and lived as long as multiply housed AL mice. These results indicate that CR can extend life span as effectively in multiply as in singly housed mice, even though housing density can differentially affect the cancer-reducing effect of CR.

The finding that the multiply housed CR mice that gained weight more slowly lived the longest is consistent with them ingesting fewer calories than their shorter lived cohabitants who gained weight more rapidly. In contrast, the finding that singly housed mice that gained weight the most rapidly lived longest would suggest that factors that enabled them to store a limited and fixed amount of energy more efficiently enabled them to outlive their counterparts who were less efficient at energy storage. Nevertheless, the fact that neither the median longevity nor its variance differed between the singly and the multiply housed CR groups indicates that, whatever the reason for the different relationships between weight gain and longevity in the two groups, this relationship did not impact the overall longevity-extending effect of CR.

Wow, that came up with a remarkable, relevant, and highly surprising study, Gordo! How fortunate you asked. That's a real puzzler under any "Keep Cool, CRONie!" hypothesis, BAT or otherwise. However, the broad fact of Calories trumping temperature on maximum LS despite a better anti-cancer benefit in cold mice is broadly consistent with the Koizumi & Walford study, where the warmer mice got more cancer (again, largely lymphoma), had a shorter median LS, but similar maximum: absolute Calories are the key to the anti-aging effect.

Reference
1: Ikeno Y, Hubbard GB, Lee S, Richardson A, Strong R, Diaz V, Nelson JF. Housing density does not influence the longevity effect of calorie restriction. J Gerontol A Biol Sci Med Sci. 2005 Dec;60(12):1510-7. PubMed PMID: 16424282.

Edited by Michael R, 26 March 2016 - 05:42 PM.
Survivalship Table


#140 Saul

Saul
  • Supporting
  • 472 posts

Posted 26 March 2016 - 06:29 PM

Hi Dean, Michael and Gordo!

I guess I might add that, when I was tested by Luigi, I recovered almost instantly from the glucose overload during the glucose tolerance test; so, I don't seem to
have the problem that some have had.

(However, at the wedding of one of my nieces, in the redwood forest of California, I suffered a sugar overload,
and nearly passed out. My tolerance for glucose overload is limited.)

I tend to be in the cold often -- my wife and I like the bedroom cold at night, and the house is at a fairly cool
temperature most often. And I take very cold daily showers; and walk around campus at the University of Rochester in the (usually)
cold weather for most of the year.

It will be interesting to see the material at CR IX on metabolic effects of cold exposure.

It might be nice if some sort of study were conducted to measure relationship between BAT, glucose tolerance, in both CR and Ad Lib humans.

I suspect the relation will be complex.

-- Saul





Also tagged with one or more of these keywords: Cold Exposure, Exercise, Fasting, UCPs, UCP1, UCP3, FGF21