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Cold Exposure & Other Mild Stressors for Increased Health & Longevity

Dean Pomerleau

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Gordo wrote:

Brown Adipose Tissue Is Linked To A Distinct Thermoregulatory Response To Mild Cold In People

(Full/Preliminary article is downloadable from above, this is very recent work)


Great find Gordo! Very interesting study [1]. It is part of a clinical trial to study BAT in humans, and particularly its potential to combat insulin resistance and obesity. Here is the table from the full text of the study showing the characteristics of the BAT+ and BAT- subjects:




The only significant difference between groups was age - with the BAT+ group being almost 20 years younger on average than the BAT- group! Us oldsters are really at a disadvantage when it comes to BAT. The entire subject pool was pretty hefty, verging on obese, but the BAT+ group had a tendency to be a bit leaner.


As you observed, the BAT+ group was able to maintain their core body temperature during CE, while the BAT- folks saw a drop in core temperature despite being slightly less cold-challenged than the BAT+ group (the ambient temperature was adjusted for each subject to be just above their individual shivering threshold and the BAT+ group had a lower ambient temperature threshold). This again shows that it may not be possible for cold-acclimated humans to increase core temperature in response to cold like is observed in rodents, but instead to simply to maintain it.


It continues to amaze me how little BAT even BAT+ folks have - 67ml on average. That's only a little over 2oz. That's tiny. It's hard to believe so little tissue can be responsible for so much thermogenesis, and it makes me wonder whether BAT is working in concert with other thermogenic processes (sarcolipin-induced futile cycling in muscle sarcoplasmic reticulum perhaps?) in the BAT+ group to maintain body temperature during a cold challenge. Having measureable BAT may therefore be a marker for cold adaptation and/or genetic variations that heighten thermogenic capacity.


I hope these researchers publish a lot more detailed investigation of the metabolism and blood measures in these subjects, per their promise in the clinical trial description. 


Gordo wrote:

They found that supraclavicular skin/surface temperature measurements were a good method of measuring BAT activity.  The last such measurement I took on myself after CE showed 96.0F.  


Can you remind me how you measure your skin temperature? I might be interested in doing the same, so we could compare notes.







[1] Front. Physiol. | doi: 10.3389/fphys.2016.00129


Brown Adipose Tissue Is Linked To A Distinct Thermoregulatory Response To Mild Cold In People
 Maria Chondronikola1*, Elena Volpi1,  Elisabet Borsheim1, Tony Chao1,  Craig Porter1, Palam Annamalai1, Christina Yfanti1,  Sebastien M. Labbe2,  Nicholas M. Hurren1, Ioannis Malagaris1, Fernardo Cesani1 and  Labros S. Sidossis1
1University of Texas Medical Branch, USA
Brown adipose tissue (BAT) plays an important role in thermoregulation in rodents. Its role in temperature homeostasis in people is less studied. To this end, we recruited 18 men [8 individuals with no/minimal BAT activity (BAT-) and 10 with pronounced BAT activity (BAT+)]. Each volunteer participated in a 6 h, individualized, non-shivering cold exposure protocol. BAT was quantified using positron emission tomography/computed tomography. Body core and skin temperatures were measured using a telemetric pill and wireless thermistors, respectively. Core body temperature decreased during cold exposure in the BAT- group only (-0.34oC, 95% CI: -0.6 to -0.1, p = 0.03), while the cold-induced change in core temperature was significantly different between BAT+ and BAT- individuals (BAT+ vs. BAT-, 0.43oC, 95% CI: 0.20 to 0.65, p = 0.0014). BAT volume was associated with the cold-induced change in core temperature (p = 0.01) even after adjustment for age and adiposity. Compared to the BAT- group, BAT+ individuals tolerated a lower ambient temperature (BAT-: 20.6± 0.3oC vs. BAT+: 19.8 ± 0.3oC, p=0.035) without shivering. The cold-induced change in core temperature (r = 0.79, p = 0.001) and supraclavicular temperature (r = 0.58, p = 0.014) correlated with BAT volume, suggesting that these non-invasive measures can be potentially used as surrogate markers of BAT when other methods to detect BAT are not available or their use is not warranted. These results demonstrate a physiologically significant role for BAT in thermoregulation in people. This trial has been registered with Clinaltrials.gov: NCT01791114 https://clinicaltrials.gov/ct2/show/NCT01791114)
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Cold Exposure Upregulates Heat Shock Proteins


On a tangent, I was thinking about heat shock proteins. Those are released by all sorts of stress, including CE. But they got their name originally from heat stress. In this context, I wonder about saunas as a hormetic generator of HSP. Saunas are popular in Northern Europe, Russia etc. as a sort of folk "health therapy", though I suppose that is not meaningful in and of itself. A sauna seems about 180 degrees from CE. An interesting wrinkle is that in some of those cultures they like to combine both - first you cook in a sauna, then you plunge into ice cold water or snow - and in Russia, I think they even whip each other with nettle twigs/switches. I bet these various methods all generate HSP, though what the net effect is I have no idea.




More good questions. For those not familiar with Heat Shock Proteins (HSPs) they are a family of proteins the body produces in response to various stressful conditions, not just thermal extremes. Their primary role appears to be to act as a chaperone to prevent misfolding and aggregation of other proteins under stressful conditions. They also appear to serve as signalling molecules for cellular stress, that kick in a host of other  biochemical responses, including beneficial chances to the cardiovascular and immune systems. Both acute and chronic cold exposure trigger the induction of a variety of HSPs as a result of elevated norepinephrine [1].


Here is the section from the Cold Exposure Albatross post discussing heat shock proteins (follow link for references):


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.
Here is a recent study [2] that  I overlooked in the Albatross post, which found people stationed in Antarctica over the winter and subject to cold had higher circulating HSP65 (the only HSP they tested for) than a control group stationed in tropical India over the same period.
In summary, both acute and chronic cold exposure upregulates HSPs in all organisms tested, from flies to rodents to humans. Yet one more benefit of cold. As to your question about the potential benefits (or harm) of rapid thermal cycling (e.g. a plunge into cold water after a sauna), I haven't come across evidence pointing one way or the other.
[1] Am J Physiol. 1995 Jul;269(1 Pt 2):R38-47.
Characterization and regulation of cold-induced heat shock protein expression in 
mouse brown adipose tissue.
Matz JM(1), Blake MJ, Tatelman HM, Lavoi KP, Holbrook NJ.
Author information: 
(1)Department of Pharmacology and Toxicology, University of North Dakota, Grand
Forks 58202, USA.
The accumulation of heat shock proteins (HSPs) after the exposure of cells or
organisms to elevated temperatures is well established. It is also known that a
variety of other environmental and cellular metabolic stressors can induce HSP
synthesis. However, few studies have investigated the effect of cold temperature 
on HSP expression. Here we report that exposure of Institute of Cancer Research
(ICR) mice to cold ambient temperatures results in a tissue-selective induction
of HSPs in brown adipose tissue (BAT) coincident with the induction of
mitochondrial uncoupling protein synthesis. Cold-induced HSP expression is
associated with enhanced binding of heat shock transcription factors to DNA,
similar to that which occurs after exposure of cells or tissues to heat and other
metabolic stresses. Adrenergic receptor antagonists were found to block
cold-induced HSP70 expression in BAT, whereas adrenergic agonists induced BAT HSP
expression in the absence of cold exposure. These findings suggest that
norepinephrine, released in response to cold exposure, induces HSP expression in 
BAT. Norepinephrine appears to initiate transcription of HSP genes after binding 
to BAT adrenergic receptors through, as yet, undetermined signal transduction
pathways. Thermogenesis results from an increase in activity and synthesis of
several metabolic enzymes in BAT of animals exposed to cold challenge. The
concomitant increase in HSPs may function to facilitate the translocation and
activity of the enzymes involved in this process.
PMID: 7631901
[2] International Journal of Scientific and Research Publications, Volume 4, Issue 5, May 2014 1
ISSN 2250-3153
Heat Shock Protein Response to Chronic Cold Exposure in Antarctic Expedition Members
IB Udaya*
, CC Laxmi**
, AK Kavitha†
, TN Satyaprabha††
, TR Raju#
, Shripad Patil ##
 Abstract- The heat shock response is seen when cells are
exposed to extremes of thermal environment, which may be
acute or chronic. The heat shock response is featured by
increased expression of heat shock proteins (HSPs). One such
key stresses is extreme cold environment as seen in Antarctica.
The Antarctic continent on the planet Earth is full of
environmental challenges. It is considered as natural stress
model. The objective of this study was to study the effect of
chronic cold environment on HSP levels. Seventeen healthy men
of XXVI Indian Antarctic expedition with mean age of
39.7±1.95 years and age ranged from 29 to 56 years participated
in this study. Antibodies of IgG, IgA and IgM classes against
HSP65 were investigated by indirect ELISA method. Samples
were collected in 2 phases. In phase-1, pre-expedition samples
were collected before leaving to Antarctica at National Center for
Antarctic and ocean research (NCAOR), Goa. In phase-2, end
expedition samples were collected after 11 months of stay in
Antarctica in an Indian permanent station (Maitri) during polar
days. The raw data on analysis using statistical tool revealed that
the anti-HSP65 IgM antibody were significantly elevated (p=
<0.001). It was observed that the anti-HSP65 antibodies were
increased in expedition members compared with the control
group who stayed in India. The present study concluded that HSP
expression increased in Indian Antarctic expedition members
who were exposed to chronic cold stress in Antarctica.
 Index Terms- HSP65, Chronic cold stress, Antarctica, Immunological response.
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I hope these researchers publish a lot more detailed investigation of the metabolism and blood measures in these subjects, per their promise in the clinical trial description. 


I don't really like how they conducted this experiment, but you may find some more info related to the above in this other study that was just published:

A randomized trial of cold-exposure on energy expenditure and supraclavicular brown adipose tissue volume in humans

The nice thing about this one is that they did CE for 6 weeks (62.5 degrees F exposure for 2 hours a day).

If I'm reading it right, they found that level of CE resulted in burning about an extra 100 calories a day.

They also found that BAT volume increased by 23% in 6 weeks with this level of CE.


Can you remind me how you measure your skin temperature? I might be interested in doing the same, so we could compare notes.


Not sure how "legit" this is, but I took a standard digital oral thermometer, firmly pushed it into the supraclavicular:



so that the probe end was completely enveloped in skin (I leaned my head and neck over until it was covered).

Edited by Gordo
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Thanks Gordo,


I was considering purchasing a couple of these iButtons, along with the interface to download the data. This is the same hardware used to continuously monitor and log skin temperature in the study we're discussing (doi:10.3389/fphys.2016.00129), as well as several other human BAT studies (e.g. PMIDs 24922545 and 23867626). This study [1] is an evaluation of iButtons for human skin temperature monitoring. Here is what they look like taped to the skin around the supraclavicle region:




At $29 a pop (plus $100 for the interface kit), I'm not going to spring for as many as shown in the photo! But if you and/or anyone else would agree to purchase them as well for comparison (competition ), I'd be game...





[1] Physiol Behav. 2006 Jul 30;88(4-5):489-97. Epub 2006 Jun 23.

Evaluation of wireless determination of skin temperature using iButtons.
van Marken Lichtenbelt WD(1), Daanen HA, Wouters L, Fronczek R, Raymann RJ,
Severens NM, Van Someren EJ.
Author information: 
(1)Nutrition and Toxicology Institute Maastricht (NUTRIM), Department of Human
Biology, Maastricht University, Maastricht, The Netherlands.
Measurements of skin temperatures are often complicated because of the use of
wired sensors. This is so in field studies, but also holds for many laboratory
conditions. This article describes a wireless temperature system for human skin
temperature measurements, i.e. the Thermochron iButton DS1291H. The study deals
with validation of the iButton and its application on the human skin, and
describes clinical and field measurements. The validation study shows that
iButtons have a mean accuracy of -0.09 degrees C (-0.4 degrees C at most) with a 
precision of 0.05 degrees C (0.09 degrees C at most). These properties can be
improved by using calibration. Due to the size of the device the response time is
longer than that of conventional sensors, with a tau in water of 19 s. On the
human skin under transient conditions the response time is significantly longer, 
revealing momentary deviations with a magnitude of 1 degrees C. The use of
iButtons has been described in studies on circadian rhythms, sleep and cardiac
surgery. With respect to circadian rhythm and sleep research, skin temperature
assessment by iButtons is of significant value in laboratory, clinical and home
situations. We demonstrate that differences in laboratory and field measurements 
add to our understanding of thermophysiology under natural living conditions. The
advantage of iButtons in surgery research is that they are easy to sterilize and 
wireless so that they do not hinder the surgical procedure. In conclusion, the
application of iButtons is advantageous for measuring skin temperatures in those 
situations in which wired instruments are unpractical and fast responses are not 
PMID: 16797616
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I love the idea of the temperature loggers, I was looking at the same one earlier, there are a few listings on eBay but they are missing the interface/data reader part which kind of makes them worthless to me.  There are several do-it-yourself instructions on the web to build something similar.  You can buy the iButton temp sensor part alone for under $2 but obviously you need more than just that...

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If you can find an alternative temperature logging technology, let me know. Otherwise, I'm game for purchasing iButtons, if anyone else will too...



I wonder if this one could be made to work?  It has the same detection range and resolution as the iButton, but much better tech since it logs via bluetooth and you don't need to buy an interface for it. Only $30 to find out... if it doesn't work, maybe I'll just use it to track indoor or outdoor temps.

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That SensorBug looks pretty cool, and the price is better, since you don't in theory need a separate interface other than a Bluetooth 4.0 phone.


But it doesn't look like you can record the data, or even get the instantaneous temp data off the device except simply to view it in their App. It seems to me that what we want to do is have (at least) two temperature sensors at two different points on our body (e.g. one over BAT and one elsewhere) to see if the difference between them changes when BAT kicks in due to CE. With two (or more) sensors you can filter out changes due to ambient temperature, and see if the area over BAT is indeed differentially warmer compared to other parts of our body surface in response to cold.


I'm not sure that would be possible with the SensorBug at least without a lot of hacking.


What do you have in mind for usage of the device?



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Beige is the New Brown - Implications for CR and Cold Exposure




This is another one of those slightly complicated stories based on a new study [1] and related evidence. But I think it will be worth it to follow along for anyone practicing CR, either without or without CE. So I urge you to bear with me.


Rapamycin extends lifespan in mice [5], and was once considered a promising anti-aging therapy, possibly acting as a CR-mimetic. Like one of the effects of CR, rapamycin inhibits the anabolic protein complex called 'mTOR'. In fact, the 'R' in 'mTOR' stands for 'Rapamycin', as in "mammalian Target Of Rapamycin" (mTOR).


But a Michael points out here, Rapamycin has some nasty side effects. One of the biggies is by turning off mTOR, rapamycin results in immune system suppression as shown in the diagram below. Rapamycin is much worse in this regard than the immunosuppressive effects of CR that operates at least in part by the same mechanism, i.e. downregulation of mTOR by reducing circulating insulin and IGF-1. In fact rapamycin is used as an immunosuppressant drug given to organ transplant recipients to prevent organ rejection by turning down the vigilance of the immune system.




That side-effect alone is enough reason to avoid taking rapamycin, unless you're an organ transplant recipient. Unfortunately for transplantees, immunosuppression isn't the only adverse side effect of rapamycin. It also reduces insulin sensitivity and impairs both glucose and lipid metabolism, leading to hyperlipidemia, metabolic syndrome and diabetes. But apparently the mechanism that causes these additional negative consequences of rapamycin has not been well understood...


This new study [1] sheds some light on that mystery, and also has some really interesting implication for fans of CR and CE.


What they did in [1] was feed mice either an ad lib control diet or ad lib control diet + rapamycin. Then the tested their metabolism and brown fat characteristics both after thermoneutral housing for several weeks, or after several weeks of cold exposure. 


The important study findings for our purposes were:

  • Cold exposure & "simulated" cold exposure via β3-adrenergic (catecholamine) receptor activation causes the browning of White Adipose Tissue (WAT) - turning it "beige" by adding mitochondria to WAT and upregulating UPC-1 expression so the WAT turns "beige" and starts burning calories, improving both glucose and lipid metabolism in the cold-exposed control mice.
  • This happens through the CE → PKA → mTOR pathway discussed here, and graphically depicted below:



  • But by suppressing mTOR activity, rapamycin prevented the cold-induced browning of WAT, and as a result blocked the improvements in glucose and lipid metabolism normally associated with cold exposure, as depicted below:


  • Interestingly, rapamycin treatment didn't prevent the cold-induced increase of thermogenesis via expression of UCP-1 in existing BAT tissue, it just prevented WAT from turning brown/beige, apparently by suppressing mitochondrial biogenesis in WAT cells.
  • Nevertheless, the existing BAT wasn't enough, and the rapamycin-treated mice weren't able to maintain their body temperature like the control mice could when exposed to extreme cold (4 °C).

So what does all this mean, especially for people?


The most interesting thing is that rodents (and baby humans) have both true BAT cells (cells that were always brown, ever since they were created) and beige cells (cells that were born as WAT but have been turned brown through the addition of mitochondria and other cellular machinery). In contrast, it appears adult humans may have only beige cells [2], and no true BAT:


To our surprise, nearly all the human BAT abundantly expressed beige cell-selective genes, but the expression of classical brown fat-selective genes were nearly undetectable.


Beige or brown - so what? In fact, [3] found it doesn't make much difference thermogenically:


 When stimulated by such external cues [including cold exposure - DP], beige adipocytes express UCP1 protein at a similar level to classical brown adipocytes and exhibit UCP1-dependent thermogenic capacity.


So what difference does the pedigree of human thermogenic fat cells make?  Perhaps plenty of difference. In fact, this might explain several puzzling mysteries associated with cold exposure, and perhaps even CR.


First, from [3] the "browning" of white fat to beige can at least in theory happen to any normal white adipose cells if it receives the right signals, e.g. as a result of cold exposure. And, unlike true BAT cells which tend to form localized, homogeneous deposits/pads, beige cells are usually found mixed amongst white fat cells. Finally, [3] points out that "currently available devices do not have enough sensitivity and resolution to detect UCP1-positive adipocytes (i.e. BAT cells) that sporadically reside in subcutaneous WAT and other adipose depots." 


In other words, CE turns WAT cells to thermogenic 'beige' cells in adult humans, and these are what we call human BAT. And these beige cells may be forming anywhere in the body where WAT is deposited, not just in the neck, upper chest and upper back regions where existing PET & thermal imaging technology can detect BAT or BAT-related thermogenic activity. 


This could point to an answer to the mystery of how such small amounts of measurable BAT tissue (i.e. only a couple ounces in BAT+ people) can possibly account for the dramatic improvement in glucose clearance (discussed here) and increase metabolic rate (~200kcal/day, as discussed here) that is observed in people chronically exposed to cold. If beige adipose cells are more numerous than commonly believed in cold-exposed people and distributed around the body rather than concentrated only in detectable pockets of BAT, they could be contributing a lot more to thermogenesis and calorie-expenditure than seems possible based on the small amount of BAT that existing technology can detect around the neck region.  This could also explain Michael's now infamous "jiggling pecs" study [4], which found the BAT deposits near the neck account for only a small fraction of human thermogenesis in response to cold. Perhaps the neck BAT is just the tip of the iceberg... I still think sarcolipin-induced thermogenesis in skeletal muscles is the more likely explanation for where all the calories are going, but thermogenic "beige" BAT cells may be more numerous and distributed than previously believed, and therefore playing a bigger role in human cold-induced thermogenesis than anyone realizes.


Which brings me to the second set of puzzles that this study may solve.


Ever wonder why detectable human BAT tissue (I'm going to continue to use BAT, even those human BAT is really beige, not brown) has a sweet spot when it comes to BMI? On the one hand, very thin people, like anorexics and still-quite-thin recovered anorexics, have zero detectable BAT, as discussed here. But on the other hand, it is the leaner people in the normal/overweight range who have greater amounts of BAT than the really fat people, as discussed here. The upper end isn't too hard to explain via two possible mechanisms. First, obese people have more thermal insulation and so probably need less BAT than learner people to stay warm. Second, reverse causality. I.e people with BAT burn more calories, and hence remain thinner than people without BAT. So in the heavyweight range, BAT → greater thinness rather than greater thinness → BAT.


But why don't really skinny folks have any BAT, when they are in dire need of more thermogenesis? Recall, Speakman found that BAT was the only tissue which was increased (doubled no less!) in mass in CRed mice relative to controls [7], as discussed here. So why do skinny mice have lots of BAT, but skinny humans have none?


This study [1] may explain the paradox. If human BAT (really beige adipose tissue) is generated only through the 'browning' of white adipose tissue, it's no wonder it's entirely lacking in people who have extremely low levels of (white) body fat. Anorexics, and by implication, hard-core CR practitioners don't have enough WAT to convert into appreciable amounts of BAT, even when chronically cold exposed. In other words, unlike mice, humans have to have a bit of fat on their bones in order for it to be converted into BAT!  


And the strong apparent linkage between BAT and glucose control could explain why, in Luigi Fontana's study of human CR practitioners, those of us who were the most severely CRed paradoxically exhibited impaired glucose clearance in response to a glucose tolerance test as I discussed here. Since we have so little white fat and since we have such low Insulin/IGF-1 levels which suppresses mTOR and therefore suppressing browning of what little WAT we may have, seriously CRed humans just don't have enough BAT to help clear a large glucose load from our circulatory system during an OGTT.


But getting back to the study at hand [1] on rapamycin and cold exposure. It seems pretty well established that rapamycin extends lifespan in mice, perhaps by about 10% [5], by shutting down mTOR as shown in the diagram above. In striking contrast, rapamycin is pretty toxic to humans due to it's side effects as Michael discussed here, including compromised immune system and increased risk of metabolic syndrome / diabetes.


This discrepancy might at least in part be explained by the facts that a) mice can have both true BAT and beige adipose tissue and b) the mice in the rapamycin longevity studies (like virtually all rodent studies) were housed at temperatures that are chilly for mice. While rapamycin may have shut down the mice's ability to turn white fat into beige in [1], presumably the mice still likely had substantial true BAT deposits as a result of their cool housing conditions. And so the rapamycin-treated mice could benefit from the BAT-induced improvements in insulin sensitivity / glucose control, as well as improvements in immune system performance, and live a long time, since absent the negative side effects, mTOR suppression really is beneficial for longevity after all. The current study [1] supports this conjecture, since they found the thermogenic capacity of true BAT was not impaired by rapamycin treatment:


However, the respiratory capacity of BAT was 5-10 fold greater ... and was only
mildly impaired by rapamycin, suggesting that significant capacity for BAT mediated nonshivering
thermogenesis might still remain in cold-challenged, rapamycin-treated animals. 


In short shutting down mTOR (via rapamycin or CR) may not be so detrimental in mice because mice can maintain native BAT and BAT-thermogenesis even in the absence of mTOR activity, although not enough thermogenic capacity to keep them warm when subjected to the extreme 4°C cold challenge used in this study!  In contrast, adult humans don't have much (if any) native, true BAT, but only beige adipose tissue, and they can't even produce any of the beige fat from WAT if mTOR is shut down by CR or rapamycin. if BAT is as beneficial as I argue it is, and is indeed critical for CR to work it's magic (as I've argued herehere and here) it's no wonder rapamycin (and, heaven forbid CR...) is more toxic in humans than rodents, because only in humans does rapamycin (or severe CR) entirely prevent BAT formation.


Finally, this rapamycin study might explain one more seeming anomaly in the CR literature. Study [6] found that mice strains that retain the most fat when subjected to CR live the longest. Conversely, mice strains that lose the most fat have their lives cut short, rather than extended, by CR. Could it be that this correlation between CR lifespan benefits and the ability to retain some fat when CRed results from the chubbier CRed mice's ability to turn some of their remaining white fat to beneficial beige fat in the cool housing conditions of these lifespan experiments? Obviously this idea is quite speculative, but an intriguing possibility nonetheless...


Takeaway messages based on these studies:

  • Humans shouldn't take rapamycin unless you've had an organ transplant - in case you didn't know that already.
  • Rodents (and baby humans) have two types of thermogenic fat tissue - true BAT and "beige" adipose tissue which is white fat cells that has been "browned" - i.e. converted to a BAT-like profile by cold exposure or pharmacological means. But adult humans have only beige adipose tissue.
  • If you don't eat enough to have a little fat on your bones, you won't have sufficient WAT that can be browned, so you won't generate any BAT, even if you beat yourself up with extreme cold exposure.
  • (Speculative) CR, like rapamycin, extends lifespan in rodents at least if they are are cold-exposed, but CR may not work in humans for the following reason.  Both CR and rapamycin greatly depress mTOR activity in mice and men. Mice can still have BAT without mTOR activity, but mTOR activity is obligatory if humans are to have any BAT at all. If BAT, or more generally, the metabolic milieu created by BAT and CE, is a critical adjunct to CR as I've argued here, here and here, that means that in humans, no mTOR → no BAT → no life extension.

Fortunately, cold exposure can activate mTOR via the PKA pathway, as discussed here, so there is still hope for CR benefits in humans despite humans being so different from mice with respect to BAT formation and prevalence. But if this model is correct, for humans to benefit from CR they need to practice cold exposure and eat enough to allow mTOR to "do it's thing" of turning WAT into BAT.


For those of us playing around with cold exposure, it seems like that third point can't be stressed enough. And for anyone practicing CR, the last point, while speculative, seems well worth considering.





[1] Diabetes. 2016 Feb 8. pii: db150502. [Epub ahead of print]

Rapamycin blocks induction of the thermogenic program in white adipose tissue.
Tran CM(1), Mukherjee S(1), Ye L(2), Frederick DW(1), Kissig M(3), Davis JG(1),
Lamming DW(4), Seale P(3), Baur JA(5).
Rapamycin extends lifespan in mice, yet paradoxically causes lipid dysregulation 
and glucose intolerance through mechanisms that remain incompletely understood.
Whole body energy balance can be influenced by beige/brite adipocytes, which are 
inducible by cold and other stimuli via β-adrenergic signaling in white adipose
depots. Induction of beige adipocytes is considered a promising strategy to
combat obesity because of their ability to metabolize glucose and lipids,
dissipating the resulting energy as heat through uncoupling protein 1 (UCP1).
Here, we report that rapamycin blocks the ability of β-adrenergic signaling to
induce beige adipocytes and expression of thermogenic genes in white adipose
depots. Rapamycin enhanced transcriptional negative feedback on the β3-adrenergic
receptor. However, thermogenic gene expression remained impaired even when the
receptor was bypassed with a cell-permeable cAMP analogue, revealing the
existence of a second inhibitory mechanism. Accordingly, rapamycin-treated mice
are cold-intolerant, failing to maintain body temperature and weight when shifted
to 4 °: C. Adipocyte-specific deletion of the mTORC1 subunit Raptor recapitulated
the block in beta-adrenergic signaling. Our findings demonstrate a positive role 
for mTORC1 in the recruitment of beige adipocytes and suggest that inhibition of 
β-adrenergic signaling by rapamycin may contribute to its physiological effects.
© 2016 by the American Diabetes Association. Readers may use this article as long
as the work is properly cited, the use is educational and not for profit, and the
work is not altered.
PMCID: PMC4806661 [Available on 2017-04-01]
PMID: 26858361
[2] PLoS One. 2012;7(11):e49452. doi: 10.1371/journal.pone.0049452. Epub 2012 Nov 16.
Human BAT possesses molecular signatures that resemble beige/brite cells.
Sharp LZ(1), Shinoda K, Ohno H, Scheel DW, Tomoda E, Ruiz L, Hu H, Wang L,
Pavlova Z, Gilsanz V, Kajimura S.
Author information: 
(1)UCSF Diabetes Center and Department of Cell and Tissue Biology, University of 
California San Francisco, San Francisco, California, USA.
Brown adipose tissue (BAT) dissipates chemical energy and generates heat to
protect animals from cold and obesity. Rodents possess two types of UCP-1
positive brown adipocytes arising from distinct developmental lineages:
"classical" brown adipocytes develop during the prenatal stage whereas "beige" or
"brite" cells that reside in white adipose tissue (WAT) develop during the
postnatal stage in response to chronic cold or PPARγ agonists. Beige cells'
inducible characteristics make them a promising therapeutic target for obesity
treatment, however, the relevance of this cell type in humans remains unknown. In
the present study, we determined the gene signatures that were unique to
classical brown adipocytes and to beige cells induced by a specific PPARγ agonist
rosiglitazone in mice. Subsequently we applied the transcriptional data to humans
and examined the molecular signatures of human BAT isolated from multiple adipose
depots. To our surprise, nearly all the human BAT abundantly expressed beige
cell-selective genes, but the expression of classical brown fat-selective genes
were nearly undetectable. Interestingly, expression of known brown fat-selective 
genes such as PRDM16 was strongly correlated with that of the newly identified
beige cell-selective genes, but not with that of classical brown fat-selective
genes. Furthermore, histological analyses showed that a new beige cell marker,
CITED1, was selectively expressed in the UCP1-positive beige cells as well as in 
human BAT. These data indicate that human BAT may be primary composed of
beige/brite cells.
PMCID: PMC3500293
PMID: 23166672
[3] J Clin Invest. 2015 Feb;125(2):478-86. doi: 10.1172/JCI78362. Epub 2015 Feb 2.
Brown and beige fat in humans: thermogenic adipocytes that control energy and
glucose homeostasis.
Sidossis L, Kajimura S.
Brown adipose tissue (BAT), a specialized fat that dissipates energy to produce
heat, plays an important role in the regulation of energy balance. Two types of
thermogenic adipocytes with distinct developmental and anatomical features exist 
in rodents and humans: classical brown adipocytes and beige (also referred to as 
brite) adipocytes. While classical brown adipocytes are located mainly in
dedicated BAT depots of rodents and infants, beige adipocytes sporadically reside
with white adipocytes and emerge in response to certain environmental cues, such 
as chronic cold exposure, a process often referred to as "browning" of white
adipose tissue. Recent studies indicate the existence of beige adipocytes in
adult humans, making this cell type an attractive therapeutic target for obesity 
and obesity-related diseases, including type 2 diabetes. This Review aims to
cover recent progress in our understanding of the anatomical, developmental, and 
functional characteristics of brown and beige adipocytes and discuss emerging
questions, with a special emphasis on adult human BAT.
PMCID: PMC4319444
PMID: 25642708 
[4] 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
[5] Nature. 2009 Jul 16;460(7253):392-5. doi: 10.1038/nature08221. Epub 2009 Jul 8.
Rapamycin fed late in life extends lifespan in genetically heterogeneous mice.
Harrison DE(1), Strong R, Sharp ZD, Nelson JF, Astle CM, Flurkey K, Nadon NL,
Wilkinson JE, Frenkel K, Carter CS, Pahor M, Javors MA, Fernandez E, Miller RA.
Author information: 
(1)The Jackson Laboratory, Bar Harbor, Maine 04609, USA. david.harrison@jax.org
Comment in
    Nature. 2009 Jul 16;460(7253):331-2.
Inhibition of the TOR signalling pathway by genetic or pharmacological
intervention extends lifespan in invertebrates, including yeast, nematodes and
fruitflies; however, whether inhibition of mTOR signalling can extend lifespan in
a mammalian species was unknown. Here we report that rapamycin, an inhibitor of
the mTOR pathway, extends median and maximal lifespan of both male and female
mice when fed beginning at 600 days of age. On the basis of age at 90% mortality,
rapamycin led to an increase of 14% for females and 9% for males. The effect was 
seen at three independent test sites in genetically heterogeneous mice, chosen to
avoid genotype-specific effects on disease susceptibility. Disease patterns of
rapamycin-treated mice did not differ from those of control mice. In a separate
study, rapamycin fed to mice beginning at 270 days of age also increased survival
in both males and females, based on an interim analysis conducted near the median
survival point. Rapamycin may extend lifespan by postponing death from cancer, by
retarding mechanisms of ageing, or both. To our knowledge, these are the first
results to demonstrate a role for mTOR signalling in the regulation of mammalian 
lifespan, as well as pharmacological extension of lifespan in both genders. These
findings have implications for further development of interventions targeting
mTOR for the treatment and prevention of age-related diseases.
PMCID: PMC2786175
PMID: 19587680
[6] Aging Cell. 2011 Aug;10(4):629-39. doi: 10.1111/j.1474-9726.2011.00702.x. Epub
2011 Apr 25.
Fat maintenance is a predictor of the murine lifespan response to dietary
Liao CY(1), Rikke BA, Johnson TE, Gelfond JA, Diaz V, Nelson JF.
Author information: 
(1)Department of Physiology, University of Texas Health Science Center, San
Antonio, TX 78229, USA.
Dietary restriction (DR), one of the most robust life-extending manipulations, is
usually associated with reduced adiposity. This reduction is hypothesized to be
important in the life-extending effect of DR, because excess adiposity is
associated with metabolic and age-related disease. Previously, we described
remarkable variation in the lifespan response of 41 recombinant inbred strains of
mice to DR, ranging from life extension to life shortening. Here, we used this
variation to determine the relationship of lifespan modulation under DR to fat
loss. Across strains, DR life extension correlated inversely with fat reduction, 
measured at midlife (males, r= -0.41, P<0.05, n=38 strains; females, r= -0.63,
P<0.001, n=33 strains) and later ages. Thus, strains with the least reduction in 
fat were more likely to show life extension, and those with the greatest
reduction were more likely to have shortened lifespan. We identified two
significant quantitative trait loci (QTLs) affecting fat mass under DR in males
but none for lifespan, precluding the confirmation of these loci as coordinate
modulators of adiposity and longevity. Our data also provide evidence for a QTL
previously shown to affect fuel efficiency under DR. In summary, the data do not 
support an important role for fat reduction in life extension by DR. They suggest
instead that factors associated with maintaining adiposity are important for
survival and life extension under DR.
© 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society
of Great Britain and Ireland.
PMCID: PMC3685291
PMID: 21388497
[7] Mech Ageing Dev. 2005 Jun-Jul;126(6-7):783-93. Epub 2005 Mar 16.
Energy expenditure of calorically restricted rats is higher than predicted from
their altered body composition.
Selman C(1), Phillips T, Staib JL, Duncan JS, Leeuwenburgh C, Speakman JR.
Author information: 
(1)University of Florida, Department of Aging and Geriatric Research, College of 
Medicine, Gainesville, 32608, USA. c.selman@ucl.ac.uk
Debate exists over the impact of caloric restriction (CR) on the level of energy 
expenditure. At the whole animal level, CR decreases metabolic rates but in
parallel body mass also declines. The question arises whether the reduction in
metabolism is greater, smaller or not different from the expectation based on
body mass change alone. Answers to this question depend on how metabolic rate is 
normalized and it has recently been suggested that this issue can only be
resolved through detailed morphological investigation. Added to this issue is the
problem of how appropriate the resting energy expenditure is to characterize
metabolic events relating to aging phenomena. We measured the daily energy
demands of young and old rats under ad libitum (AD) food intake or 40% CR, using 
the doubly labeled water (DLW) method and made detailed morphological examination
of individuals, including 21 different body components. Whole body energy demands
of CR rats were lower than AD rats, but the extent of this difference was much
less than expected from the degree of caloric restriction, consistent with other 
studies using the DLW method on CR animals. Using multiple regression and
multivariate data reduction methods we built two empirical predictive models of
the association between daily energy demands and body composition using the ad
lib animals. We then predicted the expected energy expenditures of the CR animals
based on their altered morphology and compared these predictions to the observed 
daily energy demands. Independent of how we constructed the prediction, young and
old rats under CR expended 30 and 50% more energy, respectively, than the
prediction from their altered body composition. This effect is consistent with
recent intra-specific observations of positive associations between energy
metabolism and lifespan and theoretical ideas about mechanisms underpinning the
relationship between oxygen consumption and reactive oxygen species production in
PMID: 15888333
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Remarkable finds and very, very important in enlarging our knowledge of the effects of CR - and highly relevant to us, CRONies. I have personally noticed that at my lowest weight, when I practiced my most restricted CR, my QOL took a dip compared to slightly higher BMI, and more significantly, my blood panel numbers wrt. glucose also got worse. I found that very puzzling, as I read repeatedly how more CR is better (short of actual starvation), and mice on CR70 were doing better than f.ex. CR30. Now, at my strictest, I don't think I ever exceeded CR50, because at that point I was on 1060 cal/day - which is right about 50% of the recommended 2200 cal/day for a mildly active ad lib man. Which meant that I had plenty of room to CR70. And yet, my sense of well-being, and more crucially objective blood panel numbers were better on 1390 cal/day. At which point, I decided that linear extrapolation from rodents to humans probably is not warranted, or at least we don't have enough science to recommend going below that level. Ever since I've hovered between 1400 - 1500 a day - and felt best on it - though there is one wrinkle in that calorie number, as the 1400-1500 number is true for 5 days a week, because I've incorporated one half-fast day where I limit myself to 900 cal/day and one full day of 0 calories; this averages over a full week to some 1200 cal/day, but I feel somehow the dynamics of fasting don't translate straightforwardly to averaging of calories over fasted and non-fasted days.


That said, and acknowledging all the valuable information about rapa, that you have found so spectacularly, I feel that you have been a bit hasty in your conclusions about rapa. You state, as if uncontroversial, that rapa is immunosuppressive. You also link to a post by MR on some negatives of rapa for humans. You really should have been digging deeper. As you, of all people, must appreciate, sometimes knowing more leads you to the very opposite conclusion, which is why it can be dangerous to base one's actions on incomplete information - we might be doing more harm than good.


The issue is readily apparent when you cite the MR link - at the time of the original discussion of rapa in humans as an aid in longevity, I made a number of objections to MR's post - in the link you provide, he even references those objections, but again does not address them. Now it is possible that he has excellent answers to those objections, but short of stating them, we are still left at an impasse. Unfortunately my objections and the studies I used to bolster them have been lost to the mists of time and disappeared archives, but in the briefest of references, my objections were centered around the extremely fundamental fact that you cannot make blanket statements about the effect of a drug without carefully referencing the dosage. It's the oldest principle in medicine: "the dose makes the poison" (Paracelsus). This is so fundamental and uncontroversial that I find it astonishing that folks are not doing it for rapa, while discussing dosage at great length for every other drug, whether aspirin or alcohol. Of course, my objection was not centered around the mere principle, but actual research results with differing dosages - unfortunately, there is not much research and that's a tragic state of affairs, one wishes there were a lot more, but the absence of research should not be used to bolster the wobbly conclusions of the status quo ("rapa is immunosuppressive"). The immune system is highly dependent on these very effects of dosage - the entire hormetic effect rests on this, not to mention the principle of vaccination.


The idea that rapa is immunosuppressive is based on the experience of organ transplant patients having more cancer and generally exhibiting symptoms of a compromised immune system. Hence the conclusion - rapa is immunosuppressive. This actually qualifies as a genuinely comical example of circular reasoning. The goal was to repress the immune system so that a transplant organ would not be rejected, and in service of that goal we administered massive doses of a drug until the point where we did achieve the goal of immunosuppression - and then we turned around and disapprovingly point to that suppression as proof that the drug is deleterious because we've reached the goal! I nominate water as the latest dangerous substance to consume, based on those cases where people induced hyponatremia by quickly consuming massive quantities of water and then dying. Clearly, water is a killer, and so is alcohol when college kids drink absurd quantities in one sitting. Of course, if you try your darndest to reach a goal, you'll reach it, but then don't complain that you've reached it. So yes, with massive doses of rapa designed to compromise the immune system, we - DUH! - managed to compromise the immune system and the consequences were increased cancer among other effects of a compromised immune system. However, what if we lowered the dosage? There was a study showing rapa to be cancer-protective at lower dosages, although it was unclear as to whether that was a global effect of priming the immune system through a hormetic effect, or a more direct pathway - unfortunately I can't locate the study at the moment, but another study found this:




Transplant Proc. 2009 Jan-Feb;41(1):359-65. doi: 10.1016/j.transproceed.2008.10.090.


Effect of low-dose rapamycin on tumor growth in two human hepatocellular cancer cell lines.




Liver transplantation is the best treatment for patients with early hepatocellular carcinoma (HCC) and cirrhosis. A limiting factor for long-term survival remains posttransplant tumor recurrence. Thus, there is widespread discussion about the role of various immunosuppressive agents. The newly developed immunosuppressive drug rapamycin may aid to lower recurrence rates. We investigated the efficiency of rapamycin as compared with previous immunosuppressants in a tumor cell model.



We studied two HCC cell lines for cell-cycle and proliferation analyses after treatment with rapamycin or other immunosuppressants. To elucidate the underlying molecular signaling pathway, we performed Western blotting for phosphorylated p70 S6 kinase protein expression.



Low-dose rapamycin inhibited tumor cell growth at doses of 1, 5, and 10 ng/mL, while standard immunosuppressants stimulated growth. A rapamycin dose of 20 ng/mL showed a marked decrease in the growth inhibition of both HCC cell lines compared to low-dose administration.



Rapamycin in low doses inhibited the growth of two HCC cell lines in vitro. Inhibition of tumor cell growth was observed with a high dose of rapamycin (20 ng/mL), which appears to be the dividing line between growth and inhibition. We postulated that at higher doses the immunosuppressive effect of rapamycin is overrode by its antitumor effects.

PMID:19249557 [PubMed - indexed for MEDLINE]


There are of course other effects, such as on glucose metabolism and lipids etc., but again - people, for crying out loud - AT WHAT DOSAGE?? Any conclusions about rapa and morbidities and biomarkers such as diabetes, insulin response, hyperlipidemia - in this case all drawn from severely immunosuppressed transplant patients are by definition impossible to draw, except that at very high dosages, in addition to immunosuppression such massive dosages of rapa can result in diabetes and other problems. And water can kill too. How do we know that it is not the same as with cancer and rapa dosage? That at appropriate dosage rapa can boost the immune system, or cut down on cancer, or give us better glucose control and greater insulin sensitivity and a superior lipid profile etc.? Maybe low dose rapa give us *superior* glucose and lipid metabolism?


Without discussing the dosage - whether of rapa or water or aspirin - the resulting conclusions are going to be highly limited. All we can say is that at dosages high enough to suppress the immune system, rapa will suppress the immune system. And that when the dosage is so high as to suppress the immune system it can also result in other unfavorable effects including diabetes. 


The other aspect of this is that it is often unrealistic to affect a complex biological system for a specific goal with a single "silver bullet". It is a dream of humanity since ancient times that there should be a single silver bullet, an ambrosia drink of the gods, a fountain of youth, a philosopher's stone to accomplish extremely complex goals - health, longevity, wealth. It's not realistic from a dynamic systems point of view. It is therefore highly likely that rapa by itself may not result in appreciable (or any) extension of max life span. That may be true for any one substance (say, metformin). However, what if it were possible to combine multiple drugs with other interventions (the way it's possible to combine CR with CE) to accomplish such a complex goal in a dynamic system? There is a reason why doctors often prescribe multiple drugs which counteract various negative side effects - the hope is that the NET will be positive. Same here - it is really no different at all. Say rapa results in glucose intolerance - maybe combining with metformin could take care of that? In fact, when discussing this, I proposed a cocktail of drugs with rapa, metformin, melatonin, vaccination against pneumonia and so forth.


However, before we even get so far as designing a complex multi drug regiment combined with other interventions, we must establish threshold effects. At what precise dosage does rapa exhibit life extending effects? Before or after undesirable side effects (IN HUMANS!) of suppressed immune system, diabetes etc.? If before (i.e. there are doses low enough to prolong lifespan but not high enough to result in compromised immune function or diabetes etc.), then can we push this effect higher by combining with other drugs? So f.ex. we gain 5 years with a low dose of no side effects, but a higher dose would give us 10 years if we can mitigate the side effects with other drugs. This is why it is so important to establish threshold points.


Same with the whole rapa/BAT issue - at what dose? Is it really the case, as apparently with arsenic - any amount is bad - or maybe there are the well-known flip effects, lower dosage might result in *superior* engagement of BAT tissue (btw. once upon a time the consensus was that any amount of radiation is bad - today we believe that a small amount might be actively good, but in any case there is a threshold effect of harm). It would be useful to see the impact of rapa on BAT at varying dosages. And then maybe see if there are other drugs or therapies or interventions that may extend the benefits of rapa without impacting BAT negatively.


Bottom line, without a more nuanced look at rapa, we should not jump to conclusions. 

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Good point. I may have been too hasty - extrapolating from the effects of high-dose rapamycin in transplant patients to dismiss rapamycin as a potential life-extension drug at low enough doses. That'll teach me to listen to Michael's analysis .


But nevertheless I think the main point of my argument stands - rapamycin may have quite different effects wrt life extension in rodents vs. humans, since mice can maintain BAT while taking rapamycin while it doesn't look like people can.


So, when you say:

 At what precise dosage does rapa exhibit life extending effects? Before or after undesirable side effects (IN HUMANS!) of suppressed immune system, diabetes etc.?


I presume you mean "At what precise dosage does rapa exhibit life extending effects in rodents?" since we'll won't have human lifespan trials (with rapamycin or anything else) in our lifetime.


And my response would be - what gives you confidence that a life extending effect of rapamycin in rodents will carry over to humans, even if we factor out overt negative side effects (like immunosuppression) that may or may not occur at the human-equivalent of the life-extending dosage in rodents?


In other words, it looks like rapamycin will have a different impact on BAT expression in human vs rodents - rapamycin will probably eliminate BAT in humans, but it doesn't eliminate it (entirely) in mice. If BAT and/or thermogenesis is important for health/longevity as I've been arguing, rapamycin may be harmful (or neutral) for human lifespan even if it makes rodents live longer. This seems like an instance where it may be very difficult to extrapolate rodent results to humans.


Bottom line, without a more nuanced look at rapa, we should not jump to conclusions. 


Bottom line, until we can eliminate the hypothesis that BAT (or thermogenesis) is important for lifespan, or show rapamycin doesn't eliminate BAT in humans, I'd consider even low-dose rapamycin a non-starter, independent of any other negative side effects it may or may not have.


Note at this point I'd say the same thing about serious CR - which at 1200kcal per day you certainly qualify for!


CR suppresses mTOR, like rapamycin does. Until we can eliminate the hypothesis that BAT (or thermogenesis) is important for lifespan, or show rapamycin serious CR doesn't eliminate BAT in humans, I'd consider even low-dose rapamycin serious CR a non-starter, independent of any other negative side effects it may or may not have. 



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Bottom line, until we can eliminate the hypothesis that BAT (or thermogenesis) is important for lifespan, or show rapamycin doesn't eliminate BAT in humans, I'd consider even low-dose rapamycin a non-starter, independent of any other negative side effects it may or may not have.


Note at this point I'd say the same thing about serious CR, which suppresses mTOR like rapamycin does. Until we can eliminate the hypothesis that BAT (or thermogenesis) is important for lifespan, or show rapamycin serious CR doesn't eliminate BAT in humans, I'd consider even low-dose rapamycin serious CR a non-starter, independent of any other negative side effects it may or may not have. 




The thermogenesis hypothesis may be correct in that BAT is important for lifespan, but in the interest of precision if nothing else, I think one can qualify your conclusions somewhat. If, rapa negates the BAT pathway, but prolongs life along a different pathway to end up with a net extension over BAT, it may still be worth it, but there is no way of knowing at the moment. More interesting to me is the mechanism of action of rapa on BAT and BAT-like tissue. I mean in the same way that if f.ex. rapa were to extend human life except unfortunately it doesn't because, say, it causes diabetes as a side effect so the net effect on life extension is a wash, if f.ex. metformin would take care of that side effect, then we'd reap the benefit without the negative consequence and the net would be in favor of extension. Again, I come back to my instinctive belief that we are not going to impact aging appreciably with just ONE drug or intervention - in a dynamic system, that is not likely. There have to be multiple interventions - similar to how you don't go to repair a complex machine like a computer only equipped with a hammer (or worse, high dose hammer like a sledgehammer), a hammer is a fine tool, but you need more. What if we could modify the effect of rapa on BAT in humans (through some other drug or intervention - to speculate wildly f.ex. if we managed to modify fat cells to become TRUE brown BAT like in rodents) and thus even if BAT were critical to LE in humans, rapa might yet be a useful intervention. So I wouldn't make a blanket statement like the one of yours I quoted above.


Looking at the studies you cited, f.ex. PMID: 19587680 - I looked at the full study and to quote: "Rapamycin reduces function of the rapamycin target kinase TOR and has anti-neoplastic activities[...]" - as I stated above, if this is true for humans as it is for mice (and as I mentioned in my previous post there are reasons to suspect it's possible), then this is an example of "if rapa negates the BAT pathway, but prolongs life along a different pathway", as long as the net is higher LE than with BAT alone. What is the LE in mice due to? In PMID: 19587680 they say: "Rapamycin may extend lifespan by postponing death from cancer, by retarding mechanisms of ageing, or both." If we cut down on cancer and achieve LE in humans, is that valid? To what degree is there more or less LE based on the BAT pathway, or the cancer-inhibition pathway? Or is BAT activation also necessary to inhibit cancer (unlikely)? So that's my answer to: "what gives you confidence that a life extending effect of rapamycin in rodents will carry over to humans" - if rapa can cut down on cancer in humans (which is testable within our lifetimes), without negatively impacting other aspects of health, then through that mechanism alone it might lead to (modest) LE in humans


More properly then, one could position rapa as one of those interventions that is unfortunately not synergistic but antagonistic to LE through CE. So you can have LE through CE, or LE through rapa, but not LE through both. The example here would be ad lib people who are not practicing CE who might achieve a measure of LE through rapa alone without bothering to attempt any blocking of rapa action on the BAT pathway.


Before commenting further, I need to digest PMID: 26858361 - I want to know both mechanisms of rapa inhibition of thermogenic gene expression, seeing as one was bypassed; if one can successfully do that so that the entire effect is obviated, then I don't see why rapa would not be back in play for those who practice CE.

Edited by TomBAvoider
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Given that many LE drugs and interventions cannot be practically studied in humans, and all we are left with are studies in animals, the entire topic of the translatability of findings from animals to humans takes on a huge importance. How do you know when a study in rodents is applicable to humans (as you say, Dean wrt. rapa "what gives you confidence that a life extending effect of rapamycin in rodents will carry over to humans" - which can fairly be asked of the BAT effect just as well... what gives you confidence?). Especially when it comes to things lower than mammals, I simply tend to dismiss such studies from the point of view of actionable info for CRON practices. However, the question is valid: what translates? 


Perhaps a clue might be: if the intervention affects the same genes in both the study animal and the human (they share those particular genes), the chance of translatability might be higher. There are mapping efforts to see what is shared across the animal kingdom - here is a neat graphic:




"In wide range of species, longevity proteins affect dozens of the same genes


Scientists studying the biology of aging have found dozens of genes common to worms, flies, mice and humans that are all affected by the same family of proteins."

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A new study looking at the "Effects of L-Citrulline Supplementation on Arterial Stiffness, Pressure Wave Reflection, and Cardiac Autonomic Responses to Acute Cold Exposure with Isometric Exercise" may indicate eating more watermelon is in order (didn't know you should eat the rinds too by the way, I will try that)...


p.s. Dean to answer your question about temp logging - I know it wouldn't be ideal, but I think just a single point plot of body temps throughout the day might be useful during CE experimentation to see what activities elicit the largest thermogenesis (TG) response.  I'm less interested in temperature deltas between various organs although that would also be useful...

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You wrote:

I come back to my instinctive belief that we are not going to impact aging appreciably with just ONE drug or intervention - in a dynamic system, that is not likely. 


I agree with this belief 100%. No single, isolated tweak to metabolism is going to dramatically impact longevity, at least not positively. Metabolism is just too damn complicated


But where I start to part company with you is when you say stuff like:

There have to be multiple interventions - similar to how you don't go to repair a complex machine like a computer...  In fact, when discussing this, I proposed a cocktail of drugs with rapa, metformin, melatonin, vaccination against pneumonia and so forth.


The human body is (unfortunately) not much like a complex, human-created machine. Machines, even incredibly complex ones like the microprocessors designed by Intel (where I formerly worked for a time) or a Boeing 747, only work and are serviceable to the extent they have been engineered using modular, scalable, and well-documented design processes. The only thing of intentional human origin that even resembles human metabolism is legacy computer software, so call "spaghetti-code" in which not only the documentation, but the source code has been lost, and the original programmer is long dead. A good example is the air traffic control system for US airspace.  It is combination of hardware, software, and human best practices that has evolved and accreted over the last 50 years. It works reasonably well, but has definite limitations and threatens to hamper innovations, like civilian drones, because it just can't handle any increase in load. Unfortunately we're stuck with it because it's so "mission critical" we can't shut it down to do a major overhaul, and it is so complicated and fraught with interdependencies that improving it piecemeal threatens to bring the whole thing crashing down (literally and figuratively).


It seems like a hubristic pipe dream to me to think that we know enough about the interdependencies and hidden side effects of multi-drug cocktails to intervene effectively to increase human longevity, when even as simple and seemingly harmless cocktail of creatine + caffeine may have negative effects that neither exhibits on their own (PMID: 26366971).


FWIW, Aubrey de Grey seems to think the same way, as expressed in this Q&A:

Q: You comment in your talks that tinkering with metabolism is not a viable approach, because it is too complicated and impossible to modify without causing "more harm than good". However, it seems a number of anti aging companies, focused on drugs and genetic engineering, seem to be pursuing this route. Can you explain this disagreement?
Aubrey: Great question. The short answer is that there is one exception to my comment, but it’s an exception that doesn’t seem likely to have much practical significance for humans. The exception is calorie restriction. The drugs and other simple interventions (including genetic ones) that companies are looking at are almost all focused on making the body behave as if it is in a famine. The motivation, of course, is that famine (and these drugs) can greatly postpone aging in short-lived laboratory organisms like mice, rats and (even more so) worms. But it turns out - and for very obvious evolutionary reasons - that this doesn’t work nearly so well in long-lived species as in short-lived ones. The most that I think humans can possibly benefit by that kind of approach is a couple of years. 


I agree with Aubrey It's most likely futile to try to tweak metabolism using pharmacological interventions formulated as single drugs or multi-drug cocktails whose components we hope will make up for each other's shortcomings and negative side effects. It's like trying to patch spaghetti-code to mask a bug you don't really understand.


Unlike Aubrey however, I'm not so sure his SENS approach is a better answer, or is much of a step up from this strategy. It too tries to work around the edges of metabolism, trying to clean up the damage done by "bugs" we don't understand. It might work for a while, but it's only a patchwork solution to keep the system working until it can be redesigned from scratch to be more resilient and maintainable. And if the bugs are too numerous, or too interdependent, it will take forever to squash them individually. I've not heard much of an answer from Michael or Aubrey to this open question - namely, just how many metabolic bugs will need to be squashed before meaningful human life extension can be achieved? All I've heard from Aubrey on the topics is "To get more than about 15 years we'd definitely need to have at least reasonably effective repair of all seven categories" which seems to me like a tall order indeed!


Rather than betting on a magic drug cocktail or expecting Aubrey and Co. to engineer reasonably effective repair for all seven categories of aging damage within my healthy lifetime, it seems to me our best hope is to embrace and leverage what we've already got - namely our genetic endowment, spaghetti-code and all.


Aubrey dismisses the idea that humans have retained, or will benefit much from, the systemic "hunkered down" metabolic state that CR triggers. He says our ancestors didn't need it to the same extent that rodents needed it to survive and procreate effectively, so the "CR response" will have been jettisoned from the human genome. I think Aubrey's perspective on this is mistaken. Luigi Fontana has shown [1] that long-term human CR practitioners exhibit a similar gene expression profile and quite a few of the same changes in biomarkers that are associated with extended longevity in CRed rodents. For example, just like it does in rodents, CR in humans inhibits the IGF-1 → AKT → mTOR pathway we've been discussing on this thread a lot lately.  So there is hope for human CR doing something good...


BUT, based on all the evidence explored in this thread, I have a strong suspicion that calorie restriction alone will be insufficient to reproduce in humans the longevity benefits observed in rodents. The reason is simply, it's not just CR that researchers have been subjecting rodents too all these years with beneficial results - it's CR plus cold exposure. It makes sense from an evolutionary perspective, since CR and CE would have been frequently conjoined in our evolutionary past, as discussed here. And the metabolic adjustments made by CR and CE appear to be eerily complementary, both at the systemic and mitochondrial level. And most importantly, experiments testing one without the other have failed to extend lifespan in rodents (PMID: 9032756) as discussed herehere, and here.


I think about the "cocktail" of CR1 + CE + copious exercise this way. This combination is not like trying to poke around with the original source code to patch a bug you don't understand. And it's not like trying to play whack-a-mole with the damage created by a poorly designed system subjected to inputs it wasn't designed to handle - i.e. over-nutrition & under-exertion in a constantly warm environment. 


Instead, CR1 + CE + exercise is a cocktail designed to mimic the environmental input our genes have already been tuned to handle in order to help our starving mammalian ancestors vigorously and nearly-continuously forage for food in a cold environment so as to survive a few more weeks of winter. We see the benefits of this "cocktail" in action in the natural world all around us - within a species individuals living in colder climates live longer than those living in more temperate conditions, and this holds across the entire animal kingdom (PMID: 19666552).


So the idea is rather than trying to patch the spaghetti-code (via pharma cocktails), or clean up the mess it creates (via the SENS strategy), to instead get the inputs to the system right (CR + CE + EX) and hope that our spaghetti-code genetic program will do the rest, helping us to live long enough for a better solution to come alone...





1When I refer to "CR" in this context I mean net calorie restriction - i.e. "calories in - calories out", rather than absolute calorie restriction. Specifically, CR to the degree that it results in a low BMI and low glucose/insulin/IGF-1, while still providing enough energy to support BAT synthesis & thermogenesis, along with lots of exercise.



[1] Aging Cell. 2013 Aug;12(4):645-51. doi: 10.1111/acel.12088. Epub 2013 Jun 5.
Calorie restriction in humans inhibits the PI3K/AKT pathway and induces a younger
transcription profile.
Mercken EM(1), Crosby SD, Lamming DW, JeBailey L, Krzysik-Walker S, Villareal DT,
Capri M, Franceschi C, Zhang Y, Becker K, Sabatini DM, de Cabo R, Fontana L.
Author information:
(1)Laboratory of Experimental Gerontology, National Institute on Aging, National
Institutes of Health, Baltimore, MD 21224, USA.
Caloric restriction (CR) and down-regulation of the insulin/IGF pathway are the
most robust interventions known to increase longevity in lower organisms.
However, little is known about the molecular adaptations induced by CR in humans.
Here, we report that long-term CR in humans inhibits the IGF-1/insulin pathway in
skeletal muscle, a key metabolic tissue. We also demonstrate that CR induces
dramatic changes of the skeletal muscle transcriptional profile that resemble
those of younger individuals. Finally, in both rats and humans, CR evoked similar
responses in the transcriptional profiles of skeletal muscle. This common
signature consisted of three key pathways typically associated with longevity:
IGF-1/insulin signaling, mitochondrial biogenesis, and inflammation. Furthermore,
our data identify promising pathways for therapeutic targets to combat
age-related diseases and promote health in humans.
© 2013 John Wiley & Sons Ltd and the Anatomical Society.
PMCID: PMC3714316
PMID: 23601134
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There is much more in the below paper than that on cold exposure.



Nutrition and Energetics in Rodent Longevity Research.
Gibbs VK, Smith DL Jr.
Exp Gerontol. 2016 Apr 9. pii: S0531-5565(16)30093-6. doi: 10.1016/j.exger.2016.04.004. [Epub ahead of print]
PMID: 27073168
The impact of calorie amount on aging has been extensively described; however, variation over time and among laboratories in animal diet, housing condition, and strains complicates discerning the true influence of calories (energy) versus nutrients on lifespan. Within the dietary restriction field, single macronutrient manipulations have historically been researched as a means to reduce calories while maintaining adequate levels of essential nutrients. Recent reports of nutritional geometry, including rodent models, highlight the impact macronutrients have on whole organismal aging outcomes. However, other environmental factors (e.g., ambient temperature) may alter nutrient preferences and requirements revealing context specific outcomes. Herein we highlight factors that influence the energetic and nutrient demands of organisms which often times have underappreciated impacts on clarifying interventional effects on health and longevity in aging studies and subsequent translation to improve the human condition.
carbohydrate; dietary restriction; longevity; macronutrients; protein; thermogenesis
"Although typical room Ta of 20-24˚C is within the TNZ for a clothed human,
it is well below that of a mouse and is in fact a significant cold stress (Gonder & Laber,
2007; Cannon & Nedergaard, 2011). This results in a hypermetabolic state to offset the
chronic cold challenge endured by the animal, resulting in elevated food intake,
metabolic rate, heart rate, blood pressure, and circulating metabolites (e.g., blood
glucose, lipids) (Swoap et al., 2004; Overton & Williams, 2004). If a mouse living at a
thermoneutral (TN) Ta of 30˚C is transferred to the typical room temperature (RT) Ta
(22˚C), an immediate cold response is observed with increases in heat production
including shivering, food intake, heart rate, sympathetic nervous system tone, metabolic
rate, etc.(Cannon & Nedergaard, 2011). Prolonged exposure to the typical RT (22˚C) for
2 weeks or longer results in adaptive changes to meet the increased, chronic, thermal
demand (Cannon & Nedergaard, 2004). Therefore, mice in typical animal facilities show
few overt signs of cold stress unless directly compared to TN housed animals. Even
within the ILAR (Institute for Laboratory Animal Research) guidelines, a significant
increase in food intake is observed when comparing the lowest and highest
recommended Ta (19-26˚C personal observation). While this cold-induced
hypermetabolism is not unique to rodents and can be observed in humans with a large
enough cold stress (Johnson & Kark, 1947) – the chronic nature and magnitude of the
Ta exposure of mice in research facilities does not accurately reflect human exposures
in modern society. In fact, rats and mice from multiple strain backgrounds show a
reduction of caloric intake, VO2 (ml/min), mean arterial pressure and heart rate when
housed at 30˚C vs. 23˚C (Overton & Williams, 2004; Swoap et al., 2004). As discussed
above, metabolism and food intake are inversely related to Ta in mice when housed
below the TNZ. As such, a mouse housed at TN (30˚C) voluntarily reduces (~40-50%)
food intake compared with typical housing Ta (23˚C) (Overton & Williams, 2004; Swoap
et al., 2004; Cannon & Nedergaard, 2004; Cannon & Nedergaard, 2011), which is near
the maximal range of restriction normally practiced in CR/DR studies in rodents (30-
40%) (Merry, 2002; Weindruch & Walford, 1988). This raises the possibility that DR
suppresses the hypermetabolic state with elevated food intake in typical (cold) housing
conditions, returning energy intake to basal levels, and that thermoneutral housing
where energy intake is already voluntarily lowered could not be reduced a further 40-
50% without inducing malnutrition. Future studies should address this question,
particularly in light of the macronutrient requirements for long-term health and longevity
in the absence of chronic cold challenges, and the benefit (or detriment) of nutrient
restriction under basal intake conditions.
Even more to the point of macronutrient and caloric influences on lifespan,
Donhoffer and Vonotzky (1947) reported a choice experiment allowing white mice to
modulate both intake amount (calories) and preference (macronutrients) by offering
three diets with different macronutrient compositions (Donhoffer & Vonotzky, 1947).
Upon housing at typical room temperature for approximately 2-3 weeks, intake
stabilized with approximately 2/3 of calories chosen from the fat (lard) diet, with lower
amounts of protein (casein) and carbohydrate (cornstarch) diets. Lowering Ta (to 10-
11°C) resulted in an increase in caloric intake, albeit this was accounted for primarily
from additional carbohydrate consumption with protein intake remaining stable.
Furthermore, shifts from low to high Ta (29-33°C) primarily suppressed carbohydrate
intake, with again stable intake of protein and fat (Donhoffer & Vonotzky, 1947). These
intake responses are in contrast to a “no choice” scenario where lower Ta still induces
increased food intake, but proportionally across macronutrients based on the singular
diet composition (e.g., to normal ‘low fat’ chow or semi-purified rodent diets), while a
macronutrient choice scenario modulates caloric needs based on altered carbohydrate
intake (Donhoffer & Vonotzky, 1947; Leshner et al., 1971). Similar observations have
been reported with activity (exercise) preferentially modulating carbohydrate intake in
rodents (Collier et al., 1969). Considering most rodent research utilizes subthermoneutral
housing, including the GF dietary assessments related to longevity
discussed above (Solon-Biet et al., 2014), it would be important to know how
thermoneutral housing modifies the macronutrient ratio effect on health and longevity
outcomes. Similarly, the interaction of exercise or activity on macronutrient ratio
optimization could be further explored.
One example of a relatively recent gene by environment interaction in obesity
and metabolism is found in the uncoupling protein 1 (Ucp1) deletion mice. As mentioned
above, the typical housing condition in most rodent facilities requires a significant,
adaptive thermogenic response to maintain Tb. Brown adipose tissue (BAT) has been
shown to contribute to this thermogenic requirement through uncoupling metabolic
substrate utilization from ATP (adenosine triphosphate) production in the mitochondria
producing heat (i.e. a type of energy inefficiency/wasting) (Cannon & Nedergaard,
2004). Thus, it was expected the deletion of the Ucp1 gene would produce a more
“efficient” organism resulting in greater weight gain per calorie consumed. However,
early reports of Ucp1 gene knockout mice demonstrated reduced thermogenic capacity,
but with unexpected protection from diet-induced obesity (Enerback et al., 1997; Liu et
al., 2003). Follow-up studies with the same Ucp1 deletion strain performed under
housing conditions near the lower end of the TNZ (Ta 29°C) uncovered an obesogenic
phenotype of the Ucp1-/- mutant, with increased energetic efficiency (significantly
greater weight gain despite equivalent energy intake) on both low-fat and high-fat diet
protocols (Feldmann et al., 2009). In essence, correcting the thermally-induced, hypermetabolic
response by altering the Ta uncovered a metabolic phenotype in the mutant
mouse model which was expected based on the known biochemical and molecular
function, but not previously observed. How other diets with altered macronutrient
proportions might interact with this Ucp1-/- genotype and Ta for metabolic health remain
to be fully explored. The number of additional gene by environment (diet and Ta)
interactions related to metabolic health and/or longevity outcomes that are concealed in
rodent models due to standard operating procedures remains largely untested and
unknown, suggesting more research in this area may be warranted."
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A new study looking at the "Effects of L-Citrulline Supplementation on Arterial Stiffness, Pressure Wave Reflection, and Cardiac Autonomic Responses to Acute Cold Exposure with Isometric Exercise" may indicate eating more watermelon is in order (didn't know you should eat the rinds too by the way, I will try that)...


It should be noted that the cardiovascular effects observed in this study are the result of acute, extreme (4 °C) cold exposure in people who aren't cold-adapted. In such people under such conditions, it's not surprising to see a rise in blood pressure, arterial stiffness and heart rate. And there is definitely an increase in heart attacks in the winter, likely attributable at least in part to an acute, cold-induced cardiovascular stress response [1][2]. Fortunately, cold acclimation and/or light exercise mitigates these sorts of cardiovascular effects [3]. From [1]:


In cold-adapted humans, the reduced activity of the sympathetic nervous system, in response to cold stress (due to a gradual induced decline in autonomic stimulation) may decrease the physiological perturbation during cold exposure. Furthermore, cold adaptation may mitigate cold-stress-induced changes in serum lipids and haemostatic risk factors.


But watermelon can't hurt either!





[1] QJM. 1999 Dec;92(12):747-51.

Cold adaptation and the seasonal distribution of acute myocardial infarction.
De Lorenzo F(1), Sharma V, Scully M, Kakkar VV.
Author information: 
(1)Thrombosis Research Institute, London, UK. dlorenzo@tri-london.ac.uk
PMID: 10581338
[2]  Lancet. 1997 May 10;349(9062):1341-6.
Cold exposure and winter mortality from ischaemic heart disease, cerebrovascular 
disease, respiratory disease, and all causes in warm and cold regions of Europe. 
The Eurowinter Group.
[No authors listed]
BACKGROUND: Differences in baseline mortality, age structure, and influenza
epidemics confound comparisons of cold-related increases in mortality between
regions with different climates. The Eurowinter study aimed to assess whether
increases in mortality per 1 degree C fall in temperature differ in various
European regions and to relate any differences to usual winter climate and
measures to protect against cold.
METHODS: Percentage increases in deaths per day per 1 degree C fall in
temperature below 18 degrees C (indices of cold-related mortality) were estimated
by generalised linear modelling. We assessed protective factors by surveys and
adjusted by regression to 7 degrees C outdoor temperature. Cause-specific data
gathered from 1988 to 1992 were analysed by multiple regression for men and women
aged 50-59 and 65-74 in north Finland, south Finland, Baden-Württemburg, the
Netherlands, London, and north Italy (24 groups). We used a similar method to
analyse 1992 data in Athens and Palermo.
FINDINGS: The percentage increases in all-cause mortality per 1 degree C fall in 
temperature below 18 degrees C were greater in warmer regions than in colder
regions (eg, Athens 2.15% [95% CI 1.20-3.10] vs south Finland 0.27% [0.15-0.40]).
At an outdoor temperature of 7 degrees C, the mean living-room temperature was
19.2 degrees C in Athens and 21.7 degrees C in south Finland; 13% and 72% of
people in these regions, respectively, wore hats when outdoors at 7 degrees C.
Multiple regression analyses (with allowance for sex and age, in the six regions 
with full data) showed that high indices of cold-related mortality were
associated with high mean winter temperatures, low living-room temperatures,
limited bedroom heating, low proportions of people wearing hats, gloves, and
anoraks, and inactivity and shivering when outdoors at 7 degrees C (p < 0.01 for 
all-cause mortality and respiratory mortality; p > 0.05 for mortality from
ischaemic heart disease and cerebrovascular disease).
INTERPRETATION: Mortality increased to a greater extent with given fall of
temperature in regions with warm winters, in populations with cooler homes, and
among people who wore fewer clothes and were less active outdoors.
PMID: 9149695 
[3] Alaska Med. 2007;49(2 Suppl):29-31.
Human responses to cold.
Rintamäki H(1).
Author information: 
(1)Finnish Institute of Occupational Health, Oulu, Finland.
The thermoneutral ambient temperature for naked and resting humans is ca. 27
degrees C. Exposure to cold stimulates cold receptors of the skin which causes
cold thermal sensations and stimulation of the sympathetic nervous system.
Sympathetic stimulation causes vasoconstriction in skin, arms and legs.
Diminished skin and extremity blood flow increases the thermal insulation of
superficial tissues more than 300% corresponding to 0.9 clo (0.13 degrees C x
m(-2) x W(-1)). With thermoregulatory vasoconstriction/ vasodilatation the body
heat balance can be maintained within a range of ca. 4 degrees C, the middle of
the range being at ca. 21 degrees C when light clothing is used. Below the
thermoneutral zone metabolic heat production (shivering) is stimulated and above 
the zone starts heat loss by evaporation (sweating). Cold induced
vasoconstriction increases blood pressure and viscosity and decreases plasma
volume consequently increasing cardiac work. Cold induced hypertensive response
can be counteracted by light exercise, while starting heavy work in cold markedly
increases blood pressure. Under very cold conditions the sympathetic stimulation 
opens the anastomoses between arterioles and venules which increases skin
temperatures markedly but temporarily, especially in finger tips. Adaptation to
cold takes ca. 2 weeks, whereafter the physiological responses to cold are
attenuated and cold exposure is subjectively considered less stressful.
PMID: 17929604
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p.s. Dean to answer your question about temp logging - I know it wouldn't be ideal, but I think just a single point plot of body temps throughout the day might be useful during CE experimentation to see what activities elicit the largest thermogenesis (TG) response.  I'm less interested in temperature deltas between various organs although that would also be useful...


Temperature data from a single channel would certainly be better than nothing. But I worry that changes in ambient temperature would swamp the small effects of thermogenesis on skin temperature. That is why differences in skin temperatures between a region known to be above BAT deposits vs. a region nearby but not above BAT might be a lot more useful for assessing one's thermogenic response to cold exposure.  But this would require multiple simultaneous temperature channels.



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I pretty much agree with your points in your #215 post, Dean. Some small nuance, if you'll bear with me. There is true LE that works through slowing or temporarily suspending the aging process itself, and there is LE through squaring the survival curve by eliminating LE-shorteners (such as morbidity, accidents, lifestyle choices etc.). One may quibble whether the latter is true LE (since it does not in principle extend max LS). I think that while the odds of any single drug or intervention significantly affecting true LE are remote, it is evidently true that we can affect LS through lifestyle, nutrition, hygiene etc., and yes, through drugs. After all, there are drugs that successfully save lives and manage diseases. If we now think of the effects of aging as a disease state, then in principle we should be able to affect these disease states through pharmacological and other interventions (including gene therapy). 


To bring this home, we can ask: is there any drug that has shown true LE in mammals? I think the answer is "no" in keeping with the instinct that no single drug is likely to affect such a complex process as aging. The one *possible* exception is rapa (to date), as PMID: 19587680 indicates, although the authors of that study are somewhat equivocal as to whether the observed LE is due to slowing of the aging process or through frank disease suppression, or a combo of both. To quote from that paper:


"Rapamycin may extend lifespan by postponing death from cancer, by
retarding mechanisms of ageing, or both. To our knowledge, these are the first
results to demonstrate a role for mTOR signalling in the regulation of mammalian 
lifespan, as well as pharmacological extension of lifespan in both genders. These
findings have implications for further development of interventions targeting
mTOR for the treatment and prevention of age-related diseases."
Now, assuming the lesser of these claims, i.e. that rapa only extends LE through postponing death by suppressing cancer (or some other disease process), that still leaves open - at least in principle - the case for using pharmacological cocktails to suppress diseases of aging and bring us closer to max LS instead of dropping dead before that.
Obviously, a few things have to be assumed - are we even sure that there is any LE as observed by the authors of PMID: 19587680, and are we sure that's not an artifact of confounders, which seem to be horribly prevalent in almost all rodent studies, as we're reminded by Al P.'s just posted PMID: 27073168 above. This is not a trivial question - we've long known that the husbanding of rodents impacts f.ex. CR studies, but more and more we are seeing that this is a much bigger issue that encompases more variables yet (like temperature, chow composition etc.). So I think we should be cautious in simply accepting that rapa leads to LE in rodents without qualification. But assuming the best (that the answer is "yes"), we then face the dilemma you identified: what if using rapa leads to "x" time of LE, but precludes CR+CE benefits that would lead to a LE of "x+" - in which case, obviously, we would drop rapa like a hot potato and keep to CR+CE. But this is also the point at which I say we are obligated to explore the issue of using pharma interventions to augment CR/CE (my original interest in rapa) - whether it is possible, if not rapa, maybe something else or another cocktail. It is even possible that we may come up with one that displaces CR/CE and leads to greater LE. In other words, I don't think the door on pharmaceutical interventions is closed - or should be closed... after all, you must admit, Dean, that taking any supplements or indeed taking great care to consume plants that have speculative phytochemical benefits, nutraceuticals, is a form of polypharma intervention. If you are already doing that, then why not look to things like rapa, in principle? 
Edited by TomBAvoider
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It is even possible that we may come up with [a pharmacological intervention] that displaces CR/CE and leads to greater LE. In other words, I don't think the door on pharmaceutical interventions is closed - or should be closed... 


Agreed. I think we're on the same page. Recall I said:


ntil we can eliminate the hypothesis that BAT (or thermogenesis) is important for lifespan, or show rapamycin doesn't eliminate BAT in humans, I'd consider even low-dose rapamycin a non-starter, independent of any other negative side effects it may or may not have.


In other words, I'm not precluding the possibility that rapamycin alone or in combination might be beneficial, in practice or in principle.


I'm just saying I'd need to be convinced first that it "does no harm", especially when it comes to BAT in humans, before I'd consider taking it.


... after all, you must admit, Dean, that taking any supplements or indeed taking great care to consume plants that have speculative phytochemical benefits, nutraceuticals, is a form of polypharma intervention. If you are already doing that, then why not look to things like rapa, in principle? 


I give whole plants that humans have been consumed for millennia a bit more leeway when it comes to demonstrated benefits required before I'll consider consuming them, relative to rare, isolated and powerful pharmaceutical compounds that can profoundly influence core metabolic processes (like rapamycin influences mTOR activity). It's like playing with fire. You're bound to get burned if you're not really careful.


But speaking of playing with fire, I've recently started experimenting with taking a capsaicin supplement to boost thermogenic activity, so to some degree I'm talking out of both sides of my mouth on this one...



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Cold Exposure Shrinks and Browns Visceral Fat Deposits




Recall in the Beige is the New Brown post above, we learned that virtually all of what we understand to be brown adipose tissue (BAT) in humans is actually beige (otherwise known as brite) adipose tissue which is composed of cells that were formerly white fat (WAT) which has been "browned" through the catecholamine-mediated addition of mitochondria, UPC-1 receptors and other cellular machinery to enable them to burn calorie for heat rather than store calories for later.


With that background, I call your attention to [1], in which researchers subjected two groups of ad lib-fed, singly-housed male mice to 3 weeks of either thermoneutral temperature (30°C = 86°F) or to cold exposure (4°C = 39°F), a temperature which I would characterize as 'bone-crushingly' cold, even for a human, to say nothing of mice.


What they found can be summarized in the graphic below. It shows different fat deposits dissected from (unfortunate ☹) mice exposed to the two housing temperatures:



Ignore the green box around rpWAT fat deposit for now - that will become important in my next post, which focuses on a study even cooler than this one...


For now simply observe just how dramatically cold exposure changes the size and color of both BAT and visceral WAT deposits.  As can be seen from the tissue samples in the upper right corner, the various deposits of "native" or "real" BAT are generally bigger and browner in the 4 °C group (right) than the 30 °C group (left). This is especially evident in the classic interscapular BAT (iBAT) deposit from the mice's neck region - much bigger and browner at 4°C than 30°C. Of course this is to be expected after this sort of chronic, extreme cold exposure.


All the rest of the inserts are various visceral white adipose tissue (WAT) deposits. Remarkably, you can see is that virtually all of them are smaller and browner after 4°C housing compared to 30°C, representing increased mitochondrial content and therefore thermogenic potential. In short, as a result of (albeit pretty "bone-crushing") cold exposure, all these (unhealthy) visceral WAT deposits are shrunk and exhibit the type of browning characteristic of "beige" fat cells, which is the only type of thermogenic fat that adult humans possess - as discussed in the Beige is the New Brown post above.


Recall I speculated in that post about how the fact that adult humans don't have much if any true BAT, but only thermogenic beige fat, might explain (at least in part) the mystery of the missing calories - namely what's burning all the calories, and generating all the heat in humans if our total BAT deposits are only at most a few ounces? The solution - If lots of different white fat deposits get a little bit browner, they could all be engaged in thermogenesis, burning calories and generating heat.


While this study [1] was in mice and so can't definitively determine what's happening in humans, it supports this speculation - since it shows dramatic browning of lots of different, large visceral white fat deposits as a result of cold exposure.


Perhaps as importantly, I'm sure everyone knows that visceral fat (otherwise known as abdominal fat) is toxic to human health and longevity. Excess subcutaneous fat (the kind that forms "love handles") isn't that bad for you. But excess visceral fat is associated with systemic inflammation, insulin resistance, high cholesterol and triglycerides, cardiovascular disease and diabetes.


This browning of white visceral fat might explain another curious side effect of cold exposure - improved insulin sensitivity and glucose clearance, as observed by me (here and here), Gordo (here and here) and in controlled studies of rodents (hereherehere, here, and here) and people (here and here).


Again, while this was a study only in mice, it suggests a very direct way cold exposure in humans could both be burning extra calories and promoting health/longevity, independent of BAT, and independent of muscle thermogenesis or shivering (i.e. Michael's jiggling pecs), by shrinking and browning visceral fat deposits.



[1] Am J Physiol Endocrinol Metab. 2015 Jun 15;308(12):E1085-105. doi:
10.1152/ajpendo.00023.2015. Epub 2015 Apr 21.
A stringent validation of mouse adipose tissue identity markers.
de Jong JM(1), Larsson O(2), Cannon B(1), Nedergaard J(3).
The nature of brown adipose tissue in humans is presently debated: whether it is 
classical brown or of brite/beige nature. The dissimilar developmental origins
and proposed distinct functions of the brown and brite/beige tissues make it
essential to ascertain the identity of human depots with the perspective of
recruiting and activating them for the treatment of obesity and type 2 diabetes. 
For identification of the tissues, a number of marker genes have been proposed,
but the validity of the markers has not been well documented. We used established
brown (interscapular), brite (inguinal), and white (epididymal) mouse adipose
tissues and corresponding primary cell cultures as validators and examined the
informative value of a series of suggested markers earlier used in the discussion
considering the nature of human brown adipose tissue. Most of these markers
unexpectedly turned out to be noninformative concerning tissue classification
(Car4, Cited1, Ebf3, Eva1, Fbxo31, Fgf21, Lhx8, Hoxc8, and Hoxc9). Only Zic1
(brown), Cd137, Epsti1, Tbx1, Tmem26 (brite), and Tcf21 (white) proved to be
informative in these three tissues. However, the expression of the brite markers 
was not maintained in cell culture. In a more extensive set of adipose depots,
these validated markers provide new information about depot identity. Principal
component analysis supported our single-gene conclusions. Furthermore, Zic1,
Hoxc8, Hoxc9, and Tcf21 displayed anteroposterior expression patterns, indicating
a relationship between anatomic localization and adipose tissue identity (and
possibly function). Together, the observed expression patterns of these validated
marker genes necessitates reconsideration of adipose depot identity in mice and
Copyright © 2015 the American Physiological Society.
PMID: 25898951
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Cold Exposure Shrinks and Browns "Love Handles" (Subcutaneous Fat) Too!


OK, I lied in my last post when I said my next post would be about the visceral fat deposit called rpWAT. That will have to wait until my next post - since this study [1] is a natural follow-up.


Recall in this post we saw that adult humans have only thermogenic "beige" fat, rather than true "brown" fat, and in the last post we saw that chronic cold exposure can convert visceral white fat cells to beige, at least in mice.


But in that post, no mention was made of subcutaneous (SC) fat (the kind associated with "love handles") being influenced by cold exposure. This post remedies that omissions. The research in [1] has a ton of moving parts, with a bunch of mice with different mutations - all to nail down a pretty simple idea and solve a mystery. The simple idea is to prove that cold exposure does indeed turn subcutaneous white fat to thermogenic beige fat. The mystery is how it does it.


The reason the mechanism is a bit mysterious is summarized by the authors as follows:


In the textbook view of thermogenesis, the sensing of cold
by the neuronal system triggers the sympathetic efferents that
promote the biogenesis and activation of brown fat (Cannon
and Nedergaard, 2011; Lowell and Spiegelman, 2000). Although
this model works well for tissues that are densely innervated by
the sympathetic nerves (Morrison and Nakamura, 2011), such as
the interscapular BAT, it does not explain how cold exposure
results in the rapid remodeling of the poorly innervated scWAT
(Daniel and Derry, 1969; Slavin and Ballard, 1978; Trayhurn
and Ashwell, 1987). In these classic studies, adrenergic nerves
only innervated 2%–3% of all adipocytes in WAT, leading these
authors to conclude that the sympathetic nerves primarily innervate
blood vessels of the WAT (Daniel and Derry, 1969; Slavin
and Ballard, 1978). These older observations thus suggest that
the adrenergic tone of WAT must somehow be amplified during
cold stress to stimulate biogenesis of beige fat.


In short, regions where BAT (or the human equivalent, beige fat) is concentrated (e.g. around the neck and shoulders) have lots of "sympathetic efferents" - i.e. nerve fibers coming from temperature-regulating parts of the brain (i.e. hypothalamus [4]) that directly stimulate white fat cells with the neurotransmitter norepinephrine (NE), and this NE stimulation causes them to morph into thermogenic beige cells. The NE also causes the differentiation of adipose stem cells into 'native' BAT cells in rodents (but probably not humans).


But "love handle" fat (subcutaneous fat = SC fat = scWAT) doesn't have the advantage of many direct connections from the sympathetic nervous system, so where does the NE come from to turn SC fat to beige? It will turn out to be quite an interesting story, with tantalizing implications for humans. But I get ahead of myself.


First, it may not be too much of a big deal for us skinny folks, but burning off love handles is something that pudgy people care a lot about - to put it mildly. So is there really evidence that cold exposure can turn white SC fat to brown? Yes - in spades!


In the first of the experiments in [1], experimenters subjected mice to three different housing temperatures for only 48 hours - thermoneutral (30°C), standard lab temperature (22°C), and very cold (5°C). Then they looked at cells from subcutaneous fat deposits for sign of browning. And boy did they find it! Take a look at the first three bars of this graph from [1], which shows the expression of uncoupling protein 1 (UCP1) in SC fat of mice from the three different housing temperatures:




The first bar is the UCP1 expression in the thermoneutral mice, and is defined to be height=1 - it is the baseline. At a housing temperature of 22°C, the UCP1 expression in SC fat is 19x higher than at thermoneutrality. But look at the 5°C bar - it shows the cold mice had 550x higher expression of UCP1 in the SC fat cells than mice housed at thermoneutrality!


The bars with the three other colors are for a mutant mice strain we need not concern ourselves with. The other sets of bars represent the expression of different "thermogenic" genes responsible for either converting white fat to beige or generating heat in beige cells once created. For example "Ppargc1a" (peroxisome-proliferator-activated receptor γ co-activator-1α) is involved with mitochondrial biogenesis [2] crucial for white fat browning. What you can see is that they are all elevated by relatively brief cold exposure.


But seeing is believing - so here are pictures of subcutaneous fat deposits from mice at the three housing temperatures, as seen under a microscope:




The cells were stained to show the level of UCP1 expression. You can clearly see that as for UPC-1 expression, 5 °C > 22 °C > 30 °C.


And the new beige SC fat cells can be seen to be burning a ton of calories, as illustrated in this graph showing the oxygen consumption of isolated SC fat cells from mice housed at 22 °C and 5 °C (data for 30 °C not shown):




As you can see, the SC fat of the cold-housed WT mice consumed 4.5x as much oxygen as the SC fat from mice housed at standard lab temperature, as a result of all those extra mitochondria and UPC1 receptors burning calories to produce heat. But note both sets of cells were incubated at the same temperature for this test - so it wasn't simply an acute effect of the 5°C cells being colder during the test. Instead, the mice subcutaneous fat cells became ​intrinsically capable of generating more heat, and burning more calories, as a result of cold exposure and fat cell browning.


We could leave it there, with the nice tidy conclusion from this study that cold exposure turns subcutaneous white fat to calorie-burning beige fat. But the story gets even more interesting when we look at the "mystery" mechanism. The authors go through a litany of experiments with different mutant mice strains that overexpress or are knockouts for one gene or another.


What they found after a lot of hoop-jumping is summarized in this diagram:




Basically, it works as follows: Cold exposure causes eosinophils (EOS), a type of white blood cell (WBC) circulating in the bloodstream, to congregate in the neighborhood of subcutaneous fat cells. The EOS cells release Interleukin-4 (IL4), which attracts macrophage cells (M2), another type of WBC, to the same neighborhood and causes them to synthesize and release norepinephrine (NE) which in turn signals the WAT cells to turn beige and start burning calories.


Skipping all the mutant mice tests in-between, here is a really interesting test of this pathway the authors did to support their model's validity. They figured that if the above model is correct, then treating normal C57BL/6J mice with Interleukin-4 (IL-4) should stimulate the browning of white fat and alter mice metabolism. Boy did it ever! Here is a description of the experiment and results in the author's own words:


Because administration of IL-4 selectively increased beige fat mass in thermoneutral mice, we investigated whether this newly recruited beige fat can ameliorate metabolic dysfunction in the setting of pre-established obesity. For these studies, thermoneutral C57BL/6J mice were fed normal chow (NC) or high-fat diet (HFD) for 10 weeks (Figure 7A). After matching for adiposity (Figure S7A), mice on HFD were treated with vehicle or IL-4 complexes over a period of 14 days (Figure 7A). Remarkably, dual-energy X-ray absorptiometry (DXA) revealed that, compared to vehicle-treated animals, treatment with IL-4 decreased total body mass (∼5.7 g) and fat mass (∼13.5%) without significantly affecting lean body mass (Figures 7B, 7C, and S7B). This decrease in adiposity was also reflected in the smaller mass of scWAT and eWAT (Figures S7C and S7D). Immunoblotting analysis of adipose tissues revealed that HFD feeding decreased expression of TH (∼70%) and UCP1 (∼85%) proteins in the scWAT, which were restored after IL-4 therapy (Figure 7D). This was not limited to UCP1 because expression of the entire set of core thermogenic genes was restored by IL-4 therapy in the scWAT (Figure S7E). Similar increases in expression of TH and UCP1 proteins were observed in eWAT, but not in BAT, of mice treated with IL-4 (Figures 7D and S7F), findings that were confirmed by the histologic examination of scWAT and eWAT of treated animals.


Note - the mass of "native" BAT wasn't increased by IL-4 treatment - IL-4 seems to be involved only in the browning of white fat to form beige fat, not the synthesis of new "native" BAT cells. This is fine for humans, since we don't synthesize "native" BAT cells anyway.


Also notice, that 10 weeks of a high fat diet at thermoneutral temperatures resulted in obesity and 85% suppression of UCP1 expression in subcutaneous fat of the HFD mice relative to mice fed normal (low-fat) chow, which was reversed by treating the HFD mice with IL-4. In other words, a high fat diet without cold exposure (or injections of interleukin-4) makes mice fat and turns their subcutaneous fat even whiter than normal.


Take a look at these graphs showing body mass (left), fat mass (middle left), subcutaneous fat mass (middle right) and eWAT mass (right) in mice housed at 30 °F and fed normal chow (NC), a high fat diet (HFD) or a high fat diet with IL-4 treatment (HFD-IL4):




In short, the IL4-treated mice fed an ad lib, high-fat diet for only 14 days lost weight and fat mass overall, rather than gaining weight and fat like the control HFD mice. They especially lost weight in the form of subcutaneous fat and eWAT (more on what eWAT is below) . Not only that, but they also kicked butt in the metabolic health department, as illustrated by improved postprandial glucose control (left), lower total cholesterol (middle) and lower serum triglycerides (right) relative to both high-fat and even normal-chow controls (for triglycerides):




What caused all these benefits you ask? Apparently, their dramatically browner subcutaneous fat (scWAT), as well as browner eWAT, or "epididymal white adipose tissue", as can be seen visually in this micrograph of UCP1-stained cells from the subcutaneous and epididymal fat deposits of the three groups:




You should be asking, what's this new epididymal stuff you're talking about now Dean? Epididymal fat (eWAT) is another one of those unhealthy visceral fat deposits down near the gonads, present in both rodents and humans. You can see it in the lower left of this image I posted yesterday:



Note this graphic was from a different study (PMID: 25898951  - see post immediately above) that also observed shrinking and browning of eWAT in response to cold exposure. And they aren't the only one's to observe this cold-induced shrinkage and browning of eWAT [3].


So cold exposure browns the visceral fat deposit called eWAT too. And like subcutaneous fat, this browning of eWAT appears to be mediated by the pathway:


Cold → ↑ eosinophils →  ↑ macrophages →  ↑ norepinephrine → browning of white fat cells


Are you thinking what I'm thinking?


If so, then you are thinking "Hey now, we know something about our own eosinophil and macrophage levels!"  Eosinophil level in the blood is indicated by the EOS measurements as part of the breakdown of white blood cell counts on a standard blood test. And the precursor to macrophages are the "monocytes" measured on a standard blood test as well. 


So I checked out my own bloodwork. The second-to-last column is from last December, about a month before starting cold exposure and the last column is from just last month, two months after starting cold exposure. Sure enough, since starting cold exposure both my EOS and monocytes have gone up by a factor of ~3x, from (0.01 → 0.03) and (0.17 → 0.49), respectively. In fact, both my latest monocyte percentage and absolute count are the highest they've ever been since I started documenting my blood tests 16 years ago. Similarly, except for a couple anomalies while suffering from anemia a few years ago, my EOS count is the highest it's ever been as well. Note - neither my monocytes nor eosinophils are elevated in an absolute sense. My monocytes count is in the middle of the reference range now, and EOS count is still quite near the lower end of the RR. But these two types of white blood cells have both gone up a lot since starting cold exposure. Very interesting, and perhaps not a coincidence...


Back in this post, Al criticized my latest (post-CE) bloodwork on the grounds that my WBC count went up (to the bottom of the normal range), and that elevated WBC count is associated with adverse outcomes. We know how that discussion ended... But now we have even more direct evidence that Al may be off base on this one.


In short, extrapolating the evidence from this study [1], my observations since starting cold exposure of an increase in resting heart rate (suggesting elevated norepinephrine levels), coupled with an increase in EOS and monocyte counts, may reflect a shift towards a white blood cell profile that promotes the browning of white fat, likely both the visceral and subcutaneous varieties.


Of course this is making quite a few assumptions about the translatability of these results from mice to humans. But if these results do apply to humans, the metabolic impact of subcutaneous fat thermogenesis could be quite substantial:


[T]he UCP1-dependent and IL-4-induced beige fat mass could account for ∼15%–20% of total thermogenic capacity in mice, findings that are in agreement with the recent report that suggested beige fat respiration can account for ∼10%–37% of interscapular BAT thermogenic capacity (Shabalina et al., 2013).


Since unlike mice who have a lot of true BAT, humans have negligible true BAT deposits, making it likely that browned subcutaneous and visceral white fat deposits throughout the body are contributing a significantly higher percentage of cold-induced thermogenesis in humans than in rodents.


In fact, the authors are pretty optimistic about the potential for using pharmacological means to turn white fat to beige in order to burn fat and treat human obesity:


Not only do these findings provide strong experimental support for the therapeutic potential of beige fat in the setting of obesity, but they also outline a rigorous experimental strategy to systematically evaluate the potency and activity of other browning factors, such as irisin, Fgf21, and natriuretic peptides. Together, our results demonstrate that recruitment of new beige fat can ameliorate the established metabolic dysfunction resulting from diet-induced obesity, thereby providing strong support for the therapeutic targeting of beige fat for the treatment of human obesity and obesity-associated insulin resistance.


But it appears that there may be no free lunch - even if white fat is turned beige by pharmacological means (IL-4 injections) you still need cold exposure to burn extra calories:


[T]reatment of thermoneutral WT mice with IL-4 increases beige fat mass, but not oxygen consumption. However, upon exposure to progressively colder temperatures, these IL-4-treated animals have ∼8%–12% higher energy expenditure, reflecting their higher thermogenic capacity.


It goes without saying that the significant involvement of browned subcutaneous (and visceral) fat in human thermogenesis could go a long way towards explaining how cold exposure burns calories and improves measures of metabolic health (cholesterol, glucose, triglycerides). In fact, we can now add "subcutaneous fat" to the growing list of loci where cold-induced, non-shivering thermogenesis may be happening in people, a list which now includes:

  • Concentrated pockets of 'BAT' (actually beige fat) in the neck and shoulder regions
  • Browned visceral fat in the abdomen
  • Browned subcutaneous fat, aka "love handles", throughout the body
  • Sarcolipin-mediated futile cycling of calcium channels in skeletal muscles.

In summary:

  • Cold exposure turns subcutaneous white fat ("love handles") to thermogenic beige fat, at least in mice.
  • As a result of this browning of white fat, the mice showed dramatic improvements in measures of metabolic health relative to controls when both were fed an ad lib high fat diet.
  • The pathway responsible for browning of subcutaneous fat requires elevated levels of two types of immune system cells in the neighborhood of white fat deposits - eosinophils (EOSs) and macrophages (mature monocytes), the latter of which releases norepinephrine to turn the white fat cells to beige.
  • Speculatively, my increased heart rate (suggesting ↑ norepinephrine), along with increased EOS and monocyte levels since starting cold exposure may reflect a shift in my immune system cell population to promote the browning of white fat deposits through the same pathway observed in this study.




[1] Cell. 2014 Jun 5;157(6):1292-308. doi: 10.1016/j.cell.2014.03.066.

Eosinophils and type 2 cytokine signaling in macrophages orchestrate development
of functional beige fat.

Qiu Y(1), Nguyen KD(1), Odegaard JI(1), Cui X(1), Tian X(1), Locksley RM(2),
Palmiter RD(3), Chawla A(4).

Free full text: http://ac.els-cdn.com/S0092867414006011/1-s2.0-S0092867414006011-main.pdf?_tid=a7c10040-05be-11e6-86c7-00000aacb35e&acdnat=1461022889_bcfb2810b884e7b5e3064694a3383d65

Comment in
Cell. 2014 Jun 5;157(6):1249-50.
Nat Rev Endocrinol. 2014 Aug;10(8):443.

Beige fat, which expresses the thermogenic protein UCP1, provides a defense
against cold and obesity. Although a cold environment is the physiologic stimulus
for inducing beige fat in mice and humans, the events that lead from the sensing
of cold to the development of beige fat remain poorly understood. Here, we
identify the efferent beige fat thermogenic circuit, consisting of eosinophils,
type 2 cytokines interleukin (IL)-4/13, and alternatively activated macrophages.
Genetic loss of eosinophils or IL-4/13 signaling impairs cold-induced biogenesis
of beige fat. Mechanistically, macrophages recruited to cold-stressed
subcutaneous white adipose tissue (scWAT) undergo alternative activation to
induce tyrosine hydroxylase expression and catecholamine production, factors
required for browning of scWAT. Conversely, administration of IL-4 to
thermoneutral mice increases beige fat mass and thermogenic capacity to
ameliorate pre-established obesity. Together, our findings have uncovered the
efferent circuit controlling biogenesis of beige fat and provide support for its
targeting to treat obesity.

Copyright © 2014 Elsevier Inc. All rights reserved.

PMCID: PMC4129510
PMID: 24906148



[2] Jornayvaz, François R., and Gerald I. Shulman. “Regulation of Mitochondrial Biogenesis.” Essays in biochemistry 47 (2010): 10.1042/bse0470069. PMC. Web. 19 Apr. 2016.



[3] J Ultrastruct Mol Struct Res. 1988 Nov-Dec;101(2-3):109-22.

Epididymal white adipose tissue after cold stress in rats. I. Nonmitochondrial changes.
Loncar D1, Afzelius BA, Cannon B.
Epididymal adipose tissue in the rat is generally considered to be "pure" white adipose tissue (WAT) with a characteristic structure and function. Previous studies in cats have, however, indicated that adipose tissue with the morphological appearance of WAT could be converted into a tissue with the morphological appearance of brown adipose tissue (BAT) by intermittent cold stress. The present electron microscopic and morphometric study describes the effect of intermittent cold stress on the epididymal WAT of young rats. The tissue volume decreased markedly as did the lipid content. The mitochondrial volume increased dramatically. The extracellular matrix was vastly reduced as was the thickness of the plasma membrane, and the number of gap junctions between adipocytes increased markedly. Indications of neoinnervation and neovascularization were observed. A great abundance of preadipocytes indicated proliferative activity of the endothelium. The low amount of lipid droplets and a relative abundance of smooth and rough endoplasmic reticulum. Golgi apparatus, and lysosomes in the epididymal WAT of cold-stressed rats gave the cells the morphological appearance of young adipocytes or preadipocytes whereas the hypertrophic and hyperplastic mitochondria, the relative paucity of ribosomes on lipid droplet membranes, and the increased innervation and vascularization gave the cells the morphological characteristics of brown adipose tissue.
PMID: 3268608
[4] Front Syst Neurosci. 2015 Nov 3;9:150. doi: 10.3389/fnsys.2015.00150. eCollection
Hypothalamic control of brown adipose tissue thermogenesis.
Labbé SM(1), Caron A(1), Lanfray D(1), Monge-Rofarello B(1), Bartness TJ(2),
Richard D(1).
Author information: 
(1)Centre de Recherche de l'Institut Universitaire de Cardiologie et de
Pneumologie de Québec, Department of Medicine, Université Laval Québec, QC,
Canada. (2)Department of Biology, Center for Obesity Reversal (COR), Georgia
State University Atlanta, GA, USA.
It has long been known, in large part from animal studies, that the control of
brown adipose tissue (BAT) thermogenesis is insured by the central nervous system
(CNS), which integrates several stimuli in order to control BAT activation
through the sympathetic nervous system (SNS). SNS-mediated BAT activity is
governed by diverse neurons found in brain structures involved in homeostatic
regulations and whose activity is modulated by various factors including
oscillations of energy fluxes. The characterization of these neurons has always
represented a challenging issue. The available literature suggests that the
neuronal circuits controlling BAT thermogenesis are largely part of an autonomic 
circuitry involving the hypothalamus, brainstem and the SNS efferent neurons. In 
the present review, we recapitulate the latest progresses in regards to the
hypothalamic regulation of BAT metabolism. We briefly addressed the role of the
thermoregulatory pathway and its interactions with the energy balance systems in 
the control of thermogenesis. We also reviewed the involvement of the brain
melanocortin and endocannabinoid systems as well as the emerging role of
steroidogenic factor 1 (SF1) neurons in BAT thermogenesis. Finally, we examined
the link existing between these systems and the homeostatic factors that modulate
their activities.
PMCID: PMC4630288
PMID: 26578907
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Interleukin-33, Alzheimer's Disease, Cardiovascular Disease and Cold Exposure


Here is a brief post before I finally get to the bigger discussion rpWAT I've been promising.

Yesterday we saw that eosinophil (EOS) cells are critical for the browning of white fat especially subcutaneous WAT, and that they release the cytokine Interleukin 4 (IL-4) to orchestrate the browning process. 


It turns out that IL-4 isn't the only member of the interleukin family that is critical for browning of white fat. Two recent studies in the journals Cell [1] and Nature [2] found that another interleukin, IL-33, is important for the process. In fact, IL-33 may kick off the whole process, as illustrated in this graphical abstract from [1]:




IL-33 is a so-called "Alarmin" (alarm signal) released from cells when they get damaged or stressed [5]. Note how IL-33 is upstream of the eosinophils and IL-4 I discussed in yesterday's post, so it doesn't contradict the insight from yesterday that IL-4 can induce the browning of white fat.


Why do I single out IL-33 as of special interest, rather than IL-5 or IL-13 which are also part of the causal chain outlined above? Because yesterday while researching IL-4 and after reading [1] about how IL-33 is involved in WAT browning too, I happened to come across this article, talking about this study [3] on the beneficial effects of IL-33 for Alzheimer's disease.


The authors of [3] found that 2 days of injecting IP-33 into the bloodstream (not the brain as the popular press article claims) of mice resulted in a dramatic reduction of Amyloid-Beta plaque (Aβ) burden and reducing brain inflammation in a mouse model of Alzheimer's Disease (AD). As a result, the mice exhibited better memories in a fear-conditioning test (sorry Sthira...).  Like its role in the browning of white fat, the "alarm signal" from IL-33 reprograms the behavior of immune system cells, in this case the microglia in the brain, to more effectively degrade and "mop up" the Aβ.


In support of this IL-33 signalling mechanism being important in human AD, the researchers cite evidence that IL-33 expression is reduced in the brains of human AD patients, and they cite several studies showing people with a particular genetic polymorphism linked to IL-33 are more susceptible to AD. Also, like the mice in this study, AD patients have a blood-blood brain barrier which is permeable to IL-33, although I get the impression that normal blood-brain barriers (i.e. from non-AD people or mice) are not permeable to IL-33, but that it is synthesized endogenously by cells in the brain.


Furthermore, AD is not the only pathology it appears IL-33 may be able to mitigate. This review article [3] cites studies (e.g. [4]) showing IL-33 "has shown various protective effects in cardiovascular diseases such as atherosclerosis, obesity, type 2 diabetes and cardiac remodeling." These CVD benefits would be consistent with its triggering the conversion of pro-inflammatory white adipose tissue to beige adipose tissue.


But it's not all just rainbows and puppy dogs for IL-33 and it's effects: [3] also notes that IL-33 appears to be promote some diseases associated with an overactive immune system: 


"IL-33 strongly induces Th2 cytokine production from these cells and can promote the pathogenesis of Th2-related disease such as asthma, atopic dermatitis and anaphylaxis."


That's not too surprising - if IL-33 is an alarm signal, overexpressing it or overreacting to its presence, might be expected to cause the immune system to become too active, as it is in that set of diseases/conditions.


But in general, it appears that improved IL-33 signalling both in the peripheral bloodstream and in the brain may be beneficial. To that end, study [2] provides this intriguing positive (or negative, depending on how you look at it) feedback loop involving IL-33 and cold exposure:





As suggested above, obesity seems to block the signalling action of IL-33, which prevents it from recruiting EOS cells to white fat, which eventually prevents those white fat cells from turning brown, which results in greater obesity, in a viscous circle of ever increasing obesity, metabolic dysfunction, and less IL-33 signalling. 


Conversely, cold exposure has the potential to break this vicious cycle, and turn it virtuous. By browning white fat, cold exposure reduces obesity, unblocking IL-33 signalling, which facilitates further white fat browning, further reducing obesity, further unblocking IL-33 signalling, etc. - with an ultimate end result of white fat loss, improved metabolic function, reduced cardiovascular disease and very speculatively (due to blood-brain barrier issues), potentially reducing risk or progression of Alzheimer's disease.





[1] Cell. 2015 Jan 15;160(1-2):74-87. doi: 10.1016/j.cell.2014.12.011. Epub 2014 Dec 

Activated type 2 innate lymphoid cells regulate beige fat biogenesis.
Lee MW(1), Odegaard JI(1), Mukundan L(1), Qiu Y(1), Molofsky AB(2), Nussbaum
JC(3), Yun K(1), Locksley RM(4), Chawla A(5).
Free full text:
Type 2 innate lymphoid cells (ILC2s), an innate source of the type 2 cytokines
interleukin (IL)-5 and -13, participate in the maintenance of tissue homeostasis.
Although type 2 immunity is critically important for mediating metabolic
adaptations to environmental cold, the functions of ILC2s in beige or brown fat
development are poorly defined. We report here that activation of ILC2s by IL-33 
is sufficient to promote the growth of functional beige fat in thermoneutral
mice. Mechanistically, ILC2 activation results in the proliferation of
bipotential adipocyte precursors (APs) and their subsequent commitment to the
beige fat lineage. Loss- and gain-of-function studies reveal that ILC2- and
eosinophil-derived type 2 cytokines stimulate signaling via the IL-4Rα in
PDGFRα(+) APs to promote beige fat biogenesis. Together, our results highlight a 
critical role for ILC2s and type 2 cytokines in the regulation of adipocyte
precursor numbers and fate, and as a consequence, adipose tissue homeostasis.
Copyright © 2015 Elsevier Inc. All rights reserved.
PMCID: PMC4297518
PMID: 25543153
[2] Nature. 2015 Mar 12;519(7542):242-6. doi: 10.1038/nature14115. Epub 2014 Dec 22.
Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit
Brestoff JR(1), Kim BS(2), Saenz SA(2), Stine RR(3), Monticelli LA(1), Sonnenberg
GF(4), Thome JJ(5), Farber DL(6), Lutfy K(7), Seale P(3), Artis D(1).
Obesity is an increasingly prevalent disease regulated by genetic and
environmental factors. Emerging studies indicate that immune cells, including
monocytes, granulocytes and lymphocytes, regulate metabolic homeostasis and are
dysregulated in obesity. Group 2 innate lymphoid cells (ILC2s) can regulate
adaptive immunity and eosinophil and alternatively activated macrophage
responses, and were recently identified in murine white adipose tissue (WAT)
where they may act to limit the development of obesity. However, ILC2s have not
been identified in human adipose tissue, and the mechanisms by which ILC2s
regulate metabolic homeostasis remain unknown. Here we identify ILC2s in human
WAT and demonstrate that decreased ILC2 responses in WAT are a conserved
characteristic of obesity in humans and mice. Interleukin (IL)-33 was found to be
critical for the maintenance of ILC2s in WAT and in limiting adiposity in mice by
increasing caloric expenditure. This was associated with recruitment of
uncoupling protein 1 (UCP1)(+) beige adipocytes in WAT, a process known as
beiging or browning that regulates caloric expenditure. IL-33-induced beiging was
dependent on ILC2s, and IL-33 treatment or transfer of IL-33-elicited ILC2s was
sufficient to drive beiging independently of the adaptive immune system,
eosinophils or IL-4 receptor signalling. We found that ILC2s produce
methionine-enkephalin peptides that can act directly on adipocytes to upregulate 
Ucp1 expression in vitro and that promote beiging in vivo. Collectively, these
studies indicate that, in addition to responding to infection or tissue damage,
ILC2s can regulate adipose function and metabolic homeostasis in part via
production of enkephalin peptides that elicit beiging.
PMCID: PMC4447235
PMID: 25533952
[3] Proc Natl Acad Sci U S A. 2016 Apr 18. pii: 201604032. [Epub ahead of print]
IL-33 ameliorates Alzheimer's disease-like pathology and cognitive decline.
Fu AK(1), Hung KW(1), Yuen MY(1), Zhou X(1), Mak DS(1), Chan IC(1), Cheung TH(1),
Zhang B(2), Fu WY(1), Liew FY(3), Ip NY(4).
Alzheimer's disease (AD) is a devastating condition with no known effective
treatment. AD is characterized by memory loss as well as impaired locomotor
ability, reasoning, and judgment. Emerging evidence suggests that the innate
immune response plays a major role in the pathogenesis of AD. In AD, the
accumulation of β-amyloid (Aβ) in the brain perturbs physiological functions of
the brain, including synaptic and neuronal dysfunction, microglial activation,
and neuronal loss. Serum levels of soluble ST2 (sST2), a decoy receptor for
interleukin (IL)-33, increase in patients with mild cognitive impairment,
suggesting that impaired IL-33/ST2 signaling may contribute to the pathogenesis
of AD. Therefore, we investigated the potential therapeutic role of IL-33 in AD, 
using transgenic mouse models. Here we report that IL-33 administration reverses 
synaptic plasticity impairment and memory deficits in APP/PS1 mice. IL-33
administration reduces soluble Aβ levels and amyloid plaque deposition by
promoting the recruitment and Aβ phagocytic activity of microglia; this is
mediated by ST2/p38 signaling activation. Furthermore, IL-33 injection modulates 
the innate immune response by polarizing microglia/macrophages toward an
antiinflammatory phenotype and reducing the expression of proinflammatory genes, 
including IL-1β, IL-6, and NLRP3, in the cortices of APP/PS1 mice. Collectively, 
our results demonstrate a potential therapeutic role for IL-33 in AD.
PMID: 27091974
[3] J Inflamm (Lond). 2011 Aug 26;8(1):22. doi: 10.1186/1476-9255-8-22.
Role of IL-33 in inflammation and disease.
Miller AM(1).
Author information: 
(1)Institute of Infection, Immunity and Inflammation, College of Medical,
Veterinary and Life Sciences, GBRC, University of Glasgow, Glasgow G12 8TA, UK.
Interleukin (IL)-33 is a new member of the IL-1 superfamily of cytokines that is 
expressed by mainly stromal cells, such as epithelial and endothelial cells, and 
its expression is upregulated following pro-inflammatory stimulation. IL-33 can
function both as a traditional cytokine and as a nuclear factor regulating gene
transcription. It is thought to function as an 'alarmin' released following cell 
necrosis to alerting the immune system to tissue damage or stress. It mediates
its biological effects via interaction with the receptors ST2 (IL-1RL1) and IL-1 
receptor accessory protein (IL-1RAcP), both of which are widely expressed,
particularly by innate immune cells and T helper 2 (Th2) cells. IL-33 strongly
induces Th2 cytokine production from these cells and can promote the pathogenesis
of Th2-related disease such as asthma, atopic dermatitis and anaphylaxis.
However, IL-33 has shown various protective effects in cardiovascular diseases
such as atherosclerosis, obesity, type 2 diabetes and cardiac remodeling. Thus,
the effects of IL-33 are either pro- or anti-inflammatory depending on the
disease and the model. In this review the role of IL-33 in the inflammation of
several disease pathologies will be discussed, with particular emphasis on recent
PMCID: PMC3175149
PMID: 21871091 
[4] BMC Immunol. 2014 May 10;15:19. doi: 10.1186/1471-2172-15-19.
IL-33 is negatively associated with the BMI and confers a protective
lipid/metabolic profile in non-diabetic but not diabetic subjects.
Hasan A, Al-Ghimlas F, Warsame S, Al-Hubail A, Ahmad R, Bennakhi A, Al-Arouj M,
Behbehani K, Dehbi M, Dermime S(1).
Author information: 
(1)Immunology and Innovative Cell Therapy Unit, Dasman Diabetes Institute, Kuwait
City, Kuwait. sdermime@hotmail.com.
OBJECTIVE: Recent studies have demonstrated a protective role for IL-33 against
obesity-associated inflammation, atherosclerosis and metabolic abnormalities.
IL-33 promotes the production of T helper type 2 (Th2) cytokines, polarizes
macrophages towards a protective alternatively activated phenotype, reduces lipid
storage and decreases the expression of genes associated with lipid metabolism
and adipogenesis. Our objective was to determine the level of serum IL-33 in
non-diabetic and diabetic subjects, and to correlate these levels with clinical
(BMI and body weight) and metabolic (serum lipids and HbA1c) parameters.
METHODS: The level of IL-33 was measured in the serum of lean, overweight and
obese non-diabetic and diabetic subjects, and then correlated with clinical and
metabolic parameters.
RESULTS: Non-lean subjects had significantly (P = 0.01) lower levels of IL-33
compared to lean controls. IL-33 was negatively correlated with the BMI and body 
weight in lean and overweight, but not obese (non-diabetic and diabetic),
subjects. IL-33 is associated with protective lipid profiles, and is negatively
correlated with HbA1c, in non-diabetic (lean, overweight and obese) but not
diabetic subjects.
CONCLUSIONS: Our data support previous findings showing a protective role for
IL-33 against adiposity and atherosclerosis, and further suggest that reduced
levels of IL-33 may put certain individuals at increased risk of developing
atherosclerosis and insulin resistance. Therefore, IL-33 may serve as a novel
marker to predict those who may be at increased risk of developing
PMCID: PMC4053278
PMID: 24886535
[5] Curr Opin Immunol. 2014 Dec;31:31-7. doi: 10.1016/j.coi.2014.09.004. Epub 2014

Sep 29.

IL-33: an alarmin cytokine with crucial roles in innate immunity, inflammation
and allergy.

Cayrol C(1), Girard JP(2).

Author information:
(1)CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route
de Narbonne, F-31077 Toulouse, France; Université de Toulouse, UPS, IPBS, F-31077
Toulouse, France. (2)CNRS, IPBS (Institut de Pharmacologie et de Biologie
Structurale), 205 route de Narbonne, F-31077 Toulouse, France; Université de
Toulouse, UPS, IPBS, F-31077 Toulouse, France. Electronic address:

IL-33 is a nuclear cytokine from the IL-1 family constitutively expressed in
epithelial barrier tissues and lymphoid organs, which plays important roles in
type-2 innate immunity and human asthma. Recent studies indicate that IL-33
induces production of large amounts of IL-5 and IL-13 by group 2 innate lymphoid
cells (ILC2s), for initiation of allergic inflammation shortly after exposure to
allergens or infection with parasites or viruses. IL-33 appears to function as an
alarmin (alarm signal) rapidly released from producing cells upon cellular damage
or cellular stress. In this review, we discuss the cellular sources, mode of
action and regulation of IL-33, and we highlight its crucial roles in vivo with
particular emphasis on results obtained using IL33-deficient mice.

Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

PMID: 25278425



[5] Curr Opin Immunol. 2014 Dec;31:31-7. doi: 10.1016/j.coi.2014.09.004. Epub 2014

Sep 29.
IL-33: an alarmin cytokine with crucial roles in innate immunity, inflammation
and allergy.
Cayrol C(1), Girard JP(2).
IL-33 is a nuclear cytokine from the IL-1 family constitutively expressed in
epithelial barrier tissues and lymphoid organs, which plays important roles in
type-2 innate immunity and human asthma. Recent studies indicate that IL-33
induces production of large amounts of IL-5 and IL-13 by group 2 innate lymphoid 
cells (ILC2s), for initiation of allergic inflammation shortly after exposure to 
allergens or infection with parasites or viruses. IL-33 appears to function as an
alarmin (alarm signal) rapidly released from producing cells upon cellular damage
or cellular stress. In this review, we discuss the cellular sources, mode of
action and regulation of IL-33, and we highlight its crucial roles in vivo with
particular emphasis on results obtained using IL33-deficient mice.
Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.
PMID: 25278425  
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In support of this IL-33 signalling mechanism being important in human AD, the researchers cite evidence that IL-33 expression is reduced in the brains of human AD patients, and they cite several studies showing people with a particular genetic polymorphism linked to IL-33 are more susceptible to AD.


This study seems interesting: 




Transcriptomic and genetic studies identify IL-33 as a candidate gene for Alzheimer’s disease



The only recognised genetic determinant of the common forms of Alzheimer’s disease (AD) is the ε4 allele of the apolipoprotein E gene (APOE). To identify new candidate genes, we recently performed transcriptomic analysis of 2,741 genes in chromosomal regions of interest using brain tissue of AD cases and controls.

From 82 differentially expressed genes, 1,156 polymorphisms were genotyped in two independent discovery sub-samples (n=945). Seventeen genes exhibited at least one polymorphism associated with AD risk and following correction for multiple testing, we retained the IL-33 gene.

We first confirmed that the IL-33 expression was decreased in the brain of AD cases compared with that of controls. Further genetic analysis led us to select 3 polymorphisms within this gene, which we analysed in three independent case-control studies. These polymorphisms and a resulting protective haplotype were systematically associated with AD risk in non-APOE ε4 carriers. Using a large prospective study, these associations were also detected when analyzing the prevalent and incident AD cases together or the incident AD cases alone. These polymorphisms were also associated with less cerebral amyloid angiopathy (CAA) in the brain of non-APOE ε4 AD cases. Immunohistochemistry experiments finally indicated that the IL-33 expression was consistently restricted to vascular capillaries in the brain. Moreover, IL-33 overexpression in cellular models led to a specific decrease in secretion of the Aβ40 peptides, the main CAA component.

In conclusion, our data suggest that genetic variants in IL-33 gene may be associated with a decrease in AD risk potentially in modulating CAA formation.

As more and more such studies come out, it will be more and more interesting to see where one stands with one's own promethease analysis. In principle, it should be possible to see if you are a candidate for IL-33 over/under expression and what that might mean for all sorts of things including beige fat mobilization.
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I haven't seen this study referenced (at least in the last 6 pages of this thread) - PMC3335871 which allows for exploring your own 23andme data after some backtracking in the following sequence:


From PMC3335871:




Essential Role for miR-196a in Brown Adipogenesis of White Fat Progenitor Cells



The recent discovery of functional brown adipocytes in adult humans illuminates the potential of these cells in the treatment of obesity and its associated diseases. In rodents, brown adipocyte-like cells are known to be recruited in white adipose tissue (WAT) by cold exposure or β-adrenergic stimulation, but the molecular machinery underlying this phenomenon is not fully understood. Here, we show that inducible brown adipogenesis is mediated by the microRNA miR-196a. We found that miR-196a suppresses the expression of the white-fat gene Hoxc8 post-transcriptionally during the brown adipogenesis of white fat progenitor cells. In mice, miR-196a is induced in the WAT-progenitor cells after cold exposure or β-adrenergic stimulation. The fat-specific forced expression of miR-196a in mice induces the recruitment of brown adipocyte-like cells in WAT. The miR-196a transgenic mice exhibit enhanced energy expenditure and resistance to obesity, indicating the induced brown adipocyte-like cells are metabolically functional. Mechanistically, Hoxc8 targets and represses C/EBPβ, a master switch of brown-fat gene program, in cooperation with histone deacetylase 3 (HDAC3) through the C/EBPβ 3′ regulatory sequence. Thus, miR-196a induces functional brown adipocytes in WAT through the suppression of Hoxc8, which functions as a gatekeeper of the inducible brown adipogenesis. The miR-196a-Hoxc8-C/EBPβ signaling pathway may be a therapeutic target for inducing brown adipogenesis to combat obesity and type 2 diabetes.


Back through PMID: 25557604 (full text available):




[my bold TomBAvoider]


The rs11614913 is a common functional variant located in pre-miR-196a2 that has linked to several disorders and phenotypes. This variant contributes to the increased risk of breast cancer, lung cancer, and cancers of digestive system [28, 29]. A meta-analysis of GWAS on bone mineral density (BMD) demonstrates that the risk allele of rs11614913 is inversely associated with lumbar spine and femoral neck BMD [30]. Furthermore, rs11614913 has been reported to be associated with the risk of CVD in type 2 diabetes patients [31] and congenital heart disease [32]. However, despite using a large sample size (22,233 cases and 64,762 controls) from the CARDIOGRAM consortium, we did not find a significant association between rs11614913 and risk of CAD [19]. Our findings showed a significant association between the rs11614913 mutant allele and higher WHR. We found that there is a difference between MFE of the thermodynamic ensemble of pre-miR-196a2 with the mutant versus wild-type alleles which may affect the structure and processing of the pre-miRNA. In agreement with our conjecture, it has experimentally shown that rs11614913 affects the processing of pre-miR-196a2 and results in altered expression levels of the mature miRNA [27, 33]. For example, Hoffman et al. by delivering expression vectors containing either wild-type or mutant precursors of miR-196a2 have shown that mature miRNA levels in cells transfected with pre-miR-196a2 hosting the mutant allele (T) is significantly lower than cells transfected with the wild-type allele construct [27]. In addition, Hu et al. have demonstrated that rs11614913 wild-type allele © is associated with a significant increase in mature miR-196a2 expression [33]. Therefore, an altered expression level of mature miR-196a2 due to rs11614913 could serve as a functional mechanism underlying the observed association between the variant and WHR. MiRNAs regulate phenotypes through regulation of their target genes expression. Therefore, we sought to identify target genes that may mediate the effect miR-196a2 on WHR and found SFMBT1 and HOXC8. We showed these genes are bona fide targets of miR-196a2. Previous studies have provided evidence for co-expression of miR-196a2 and its two highlighted targets in adipose tissue which is a prerequisite for miRNA-mRNA interaction. MiR-196a2 has been identified as a regulator in brown adipogenesis of white fat progenitor cells [22]. Accordingly, HOXC8 is known to increase white fat cells and the risk of obesity [34]. SFMBT1 has reported to be associated with circulating adiponectin levels [35]. In addition, it has experimentally shown that decreasing HOXC8 expression by overexpression of miR-196a2 lead to an increased brown adipocyte [34]. Taken together, these findings suggest that depletion of miR-196a2 by rs11614913 mutant allele T elevate HOXC8 and SFMBT1 expression and subsequently contribute to higher WHR, potentially through an increase in white fat cells.


And of course, rs11614913 in snpedia:




is available from your 23andme raw data. I'm a CC FWIW. 

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