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Cold Exposure & Other Mild Stressors for Increased Health & Longevity

Dean Pomerleau

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One note on berberine. I have always wondered why LEF isn't selling a supplement with it, particularly since they have been heavily hyping metformin in their magazine in the past few years, and berberine seems to act in a similar fashion. By googling the LEF website I did find this note in one of their articles:





Although berberine has been studied in human clinical trials and shown to have several metabolic benefits, concerns about long-term use of berberine have been raised on the basis of certain preclinical studies (Kysenius 2014; Mikes 1985; Mikes 1983). Some evidence suggests that long-term berberine use, especially at high doses, may impair particular aspects of cellular metabolism in specific types of cells. The implications of this preclinical research are yet to be determined by long-term human clinical trials, therefore Life Extension currently recommends short-term use of berberine.

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Thanks for the cautionary note from LEF on the unproven benefits, and possible metabolic downsides of long-term use of berberine. Ironically, before I discovered that berberine may not work for skinny people (based on the rodent data I reviewed), I'd written out a paragraph along similar lines to LEF's caution. If berberine really does what it is appears to do, i.e. modulate AMPK, it's quite a powerful drug. I was going to say that I'd be hesitant to take berberine to boost BAT & thermogenesis simply because I'd be hesitant to mess with such fundamental metabolic processes through pharmacological means.


But you might ask, aren't we doing messing with fundamental metabolic processes already via cold exposure? Yes, but lifestyle interventions like cold exposure have the advantage that we've long been exposed to them naturally, and have had a chance to adapt to them via evolution. When you start going in with drugs (and drug-like plant compounds like berberine) which mess with core metabolic functions, all (or most) bets are off. Who knows what the safe and effective dosage might be, or what side effect it might have. Rapamycin is a good example of this - it has many apparent benefits at least in rodents, but it's side effects (e.g. immune system suppression), can be a killer - literally. That's why it's used in transplant patients as an immunosuppressant. Powerful stuff not to be taken lightly - literally or figuratively.


So what you posted is a good additional reason for us to steer clear of berberine for boosting thermogenesis, it seems to me.



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Exercise Not Required? - Cold Exposure May Prevent Sarcopenia by Boosting Mitofusin 2




I received my regular weekly digest from FightAging.org (a great resource BTW), and one particular post caught my attention, titled Mitofusin 2 in the Development of SarcopeniaIn the post, Reason talks about the problem of muscle loss (sarcopenia) in older people, and how the cause(s) aren't completely understood, nor is there a good treatment. This is (or should be) a particular concern for CR folks. While CR may slow the progression of age-associated sarcopenia in rodents, our relatively low muscle mass to start with, along with reduced levels of anabolic hormones like testosterone and IGF-1, could be problematic for maintaining healthy muscles deep into old age. We just don't know.


With that background in mind, Reason highlights a new study [2] (popular press account) which found:


[T]he loss of the protein Mitofusin 2 in the muscles of young mice speeds up aging and causes early sarcopenia, thus leading to the muscle quality of aged mice. Sarcopenia, which is muscle wastage and the accompanied loss of strength, is one of the most weakening conditions of old age and it has no treatment.
Mitofusin 2 (Mfn2), as the name implies, is involved in (among many other things) the fusion of mitochondria. In general, it appears to be a "mitochondria tuneup" protein - changing the mitochondria shape, structure and function in positive ways, and by doing so, reducing cell loss, especially in muscle cells where mitochondria need to work overtime.
Basically [2] found that in old wild-type mice, drop in Mfn2 in muscles was directly correlated with degree of sarcopenia. They also found that even when young, Mfn2-deficient mice (due to a genetic mutation) exhibited sarcopenia, as a result of "reduced autophagy and impaired mitochondrial quality, which contributed to an exacerbated age‐related mitochondrial dysfunction. "
In short, Mfn2 appears to trigger mitochondrial housekeeping, and without Mfn2 muscle mitochondria quickly go to hell in a handbasket, taking with them the muscle cells they inhabit.
So, on a lark, I did what I always do when I stumble across a new and interesting health-related compound. I did a search to see if it happens to be influenced by cold exposure. 
And wouldn't you know it, it sure is. And it won't come as a surprise for anyone who has been following along, Mfn2 is influenced in a good way by CE.
I won't belabor this one to keep from boring everybody with another long and technical post - [1] is a very dense paper. Instead I'll just share the highlights of [1]. Basically even brief (48h) exposure to cold (4 °C) resulted in an increase in Mfn2 in both BAT and skeletal (quadricep) muscles of normal rats by a factor of 2-3x. The authors conclude:
Here, we show that Mfn2 gene expression is induced in skeletal muscle and brown adipose tissue by conditions associated with enhanced energy expenditure, such as cold exposure or β3-adrenergic agonist treatment. 
The authors determined that the upregulation of Mfn2 was a result of higher induction of the gene master gene regulator "Peroxisome Proliferator–Activated Receptor-γ Coactivator-1α " (PGC-1α), which upregulates many genes involved in mitochondria synthesis and proper functioning.  In turn PGC-1α is upregulated by the elevated adrenergic signalling (e.g. norepinephrine) resulting from cold exposure. In other words:
Cold exposure → ↑ norepinephrine → ↑ PGC-1α → ↑ Mfn2 → [ ↑ mitochondria health → ↓ Sarcopenia]
The last two steps are in brackets because they come from [2], rather than [1]. But together these two studies suggest once again that cold exposure may provide a tremendous benefit that is synergistic with CR, namely preventing muscle loss without the need for an increase in potentially life-shortening anabolic hormones like IGF-1 and testosterone, thereby obviating one potential achilles heel of CR. 
Chalk another one up in the win column for cold exposure. But don't skimp on the exercise either, just to be safe and for its other, well-documented benefits.
[1] Diabetes. 2006 Jun;55(6):1783-91.
Evidence for a mitochondrial regulatory pathway defined by peroxisome
proliferator-activated receptor-gamma coactivator-1 alpha, estrogen-related
receptor-alpha, and mitofusin 2.
Soriano FX(1), Liesa M, Bach D, Chan DC, Palacín M, Zorzano A.
Author information: 
(1)Institute for Research in Biomedicine (IRB), Scientífic Park of Barcelona,
Departament of Biochemistry and Molecular Biology, Faculty of Biology, University
of Barcelona, Barelona, Spain.
Mitofusin 2 (Mfn2) is a mitochondrial membrane protein that participates in
mitochondrial fusion and regulates mitochondrial metabolism in mammalian cells.
Here, we show that Mfn2 gene expression is induced in skeletal muscle and brown
adipose tissue by conditions associated with enhanced energy expenditure, such as
cold exposure or beta(3)-adrenergic agonist treatment. In keeping with the role
of peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1 alpha on 
energy expenditure, we demonstrate a stimulatory effect of PGC-1 alpha on Mfn2
mRNA and protein expression in muscle cells. PGC-1 alpha also stimulated the
activity of the Mfn2 promoter, which required the integrity of estrogen-related
receptor-alpha (ERR alpha)-binding elements located at -413/-398. ERR alpha also 
activated the transcriptional activity of the Mfn2 promoter, and the effects were
synergic with those of PGC-1 alpha. Mfn2 loss of function reduced the stimulatory
effect of PGC-1 alpha on mitochondrial membrane potential. Exposure to cold
substantially increased Mfn2 gene expression in skeletal muscle from heterozygous
Mfn2 knock-out mice, which occurred in the presence of higher levels of PGC-1
alpha mRNA compared with control mice. Our results indicate the existence of a
regulatory pathway involving PGC-1 alpha, ERR alpha, and Mfn2. Alterations in
this regulatory pathway may participate in the pathophysiology of
insulin-resistant conditions and type 2 diabetes.
PMID: 16731843
[2] The EMBO Journal (22 June 2016) doi: 10.15252/embj.201593084
Mfn2 deficiency links age-related sarcopenia and impaired autophagy to activation of an adaptive mitophagy pathway
David Sebastián, Eleonora Sorianello, Jessica Segalés, Andrea Irazoki, Vanessa Ruiz-Bonilla, David Sala, Evarist Planet, Antoni Berenguer-Llergo, Juan Pablo Muñoz, Manuela Sánchez-Feutrie, Natàlia Plana, María Isabel Hernández-Álvarez, Antonio L. Serrano, Manuel Palacín, and Antonio Zorzano
Mitochondrial dysfunction and accumulation of damaged mitochondria are considered major contributors to aging. However, the molecular mechanisms responsible for these mitochondrial alterations remain unknown. Here, we demonstrate that mitofusin 2 (Mfn2) plays a key role in the control of muscle mitochondrial damage. We show that aging is characterized by a progressive reduction in Mfn2 in mouse skeletal muscle and that skeletal muscle Mfn2 ablation in mice generates a gene signature linked to aging. Furthermore, analysis of muscle Mfn2‐deficient mice revealed that aging‐induced Mfn2 decrease underlies the age‐related alterations in metabolic homeostasis and sarcopenia. Mfn2 deficiency reduced autophagy and impaired mitochondrial quality, which contributed to an exacerbated age‐related mitochondrial dysfunction. Interestingly, aging‐induced Mfn2 deficiency triggers a ROS‐dependent adaptive signaling pathway through induction of HIF1α transcription factor and BNIP3. This pathway compensates for the loss of mitochondrial autophagy and minimizes mitochondrial damage. Our findings reveal that Mfn2 repression in muscle during aging is a determinant for the inhibition of mitophagy and accumulation of damaged mitochondria and triggers the induction of a mitochondrial quality control pathway.
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More Evidence of Thermogenic Role for Muscle Sarcolipin


I've been highlighting futile Ca++ cycling in the sarcoplasmic reticulum of skeletal muscle cells as an important source of thermogenesis since Michael's famous quip about jiggling pecs on this thread many moons ago. For all the details about sarcolipin and thermogenesis, see herehere and most recently, here.


But in a nutshell, sarcolipin is a protein expressed in muscle cells that facilitates futile Ca++ cycling, which involves allowing calcium ions to leak (or be actively transported) across the membrane of the sarcoplasmic reticulum (SR), in which the calcium ions are usually sequestered to enable muscle contraction. This generates heat, and pumping the Ca++ back across the SR membrane to maintain the required Ca++ gradient generates more heat. 


As we saw here, this alternative form of thermogenesis can rival and substitute for the heat produced via the UCP1-induced leakage of protons across the mitochondrial membrane in BAT and beige fat. After shivering for a couple days when exposed to cold, BAT-less mice shift to sarcolipin-induced muscle thermogenesis to keep warm - getting beyond the need for Michael's "jiggling pecs". Just goes to show how clever the body really is, with backups to its backups, especially for vital functions like maintaining body temperature.


But that was a study of rodents who'd had their BAT surgically removed, and so had to fall back on sarcolipin-induced thermogenesis to keep warm. What about under normal conditions? Does sarcolipin-induced thermogenesis play a role in normal metabolism? Apparently yes, at least according to this new study [1] comparing normal, wild-type (WT) mice to sarcolipin-knockout (SKO) mice.


In it, the researchers fed both WT and SKO mice an obesity-inducing high-fat diet for 8 weeks (the human equivalent of a couple years) while housed at a cool-for-mice typical lab temperature of 22 °C. The two groups of mice ate the same amount, but the SKO mice gained twice as much weight, were more glucose intolerant and had higher circulating insulin than the WT mice.


Interestingly, the SKO also had larger amounts of BAT tissue, and higher circulating epinephrine/norephinephrine, indicating that the SKO mice were trying to burn off the excess calories via BAT thermogenesis, but not succeeding. I can hear Scotty in the engine room shouting to Kirk, 'Can't do it Captain! I'm givin' it all she's got!' ☺


Moreover, the SKO mice had higher levels of proinflammatory markers including IL-6, TNF-α, IL-1β, in visceral (but not subcutaneous or brown) fat deposits, as well as infiltration of visceral fat cells by macrophages. All of these are thought to be harbingers/contributors along the road to glucose intolerance (which the SKO mice were already exhibiting), insulin resistance (ditto) and diabetes.


In short, it looks like non-shivering thermogenesis in muscle cells induced by sarcolipin is an important means by which the body maintains metabolic health, avoids obesity & prevents metabolic syndrome, particularly in an state of positive energy balance, at least in mice, and at least when they are exposed to mild cold conditions (i.e. normal lab temperatures).


So we shouldn't think of cold exposure as inducing thermogenesis just in brown/beige adipose tissue. Non-shivering thermogenesis in skeletal muscles seems very important as well. This alternative thermogenic path is upregulated by cold exposure, as we saw in those earlier sarcolipin posts.


BTW - I just did a Google search from an "incognito" Chrome window (to avoid search personalization) for 'Cold Exposure Longevity'. This thread appears to have final passed Josh Mitteldorf's page on the topic to become the #1 resource on the internet for the combination of cold exposure and longevity. In mid March it was all the way down at #6. Pretty cool!





[1] Obesity (Silver Spring). 2016 Jul;24(7):1499-505. doi: 10.1002/oby.21521.

Sarcolipin knockout mice fed a high-fat diet exhibit altered indices of adipose
tissue inflammation and remodeling.
MacPherson RE(1), Gamu D(2), Frendo-Cumbo S(1), Castellani L(1), Kwon F(2),
Tupling AR(2), Wright DC(1).
Author information: 
(1)Department of Human Health and Nutritional Sciences, University of Guelph,
Guelph, Ontario, Canada. (2)Department of Kinesiology, University of Waterloo,
Waterloo, Ontario, Canada.
OBJECTIVE: To investigate indices of adipose tissue inflammation and remodeling
in high-fat diet (HFD) sarcolipin-knockout (SLN(-) (/-) ) mice. SLN regulates
muscle-based nonshivering thermogenesis and is up-regulated with HFD. SLN(-) (/-)
mice develop greater diet-induced obesity and glucose intolerance. This is
accompanied by increases in circulating catecholamines and fatty acids.
Catecholamines and fatty acids play a role in the pathology of adipose tissue
METHODS: Male mice (wild type and SLN(-) (/-) ) were fed a HFD (42% kcal from
fat) for 8 weeks.
RESULTS: SLN(-) (/-) mice displayed greater obesity and glucose intolerance. This
was accompanied by higher circulating epinephrine and nonesterified fatty acids. 
Epididymal but not inguinal subcutaneous adipose tissue from SLN(-) (/-) mice
displayed higher interleukin-6, suppressor of cytokine signaling 3,
interleukin-1β, and tumor necrosis factor-α mRNA expression, and this was
associated with increased markers of macrophage infiltration (F4/80 expression
and crown-like structures) and M1 polarization (higher CD11c expression and
CD11c/MGL1). Interestingly, this occurred despite SLN(-) (/-) mice having smaller
CONCLUSIONS: In conditions of nutrient excess, SLN(-) (/-) mice display
depot-specific increases in indices of adipose tissue inflammation and
remodeling. This could be a compensatory response to reductions in muscle-based
© 2016 The Obesity Society.
DOI: 10.1002/oby.21521 
PMID: 27345961
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There have been some reports lately that dairy fat blood markers are inversely related to diabetes incidence (note, this blog link is biased towards high fat diets, but still interesting I think):




Since it seems many things that are anti-diabetes seem to end up relating in part to BAT, I started wondering if there might be some link.

I ran across this abstract that mentions a possible link:



Cooperative action of bioactive components in milk fat with PPARs may explain its anti-diabetogenic properties.
"Animal studies demonstrated that most milk fat bioactive compounds induced uncoupling protein-1 expression in brown adipose tissue, which was associated with suppression of diet-induced obesity and improvement in insulin sensitivity."


Well I don't have the full paper, it sounds more like speculation than hard data at this point.

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Thanks for the pointer. That was a good idea to probe the possible relationship between dairy products and BAT - since dairy has long been thought associated with reduced weight gain and lower risk of diabetes. As you mention, the study you posted [1] was pretty speculative, in fact it was published in the notoriously flaky journal Medical Hypotheses, where researchers go to speculate.


The authors speculate that specific, rare fatty acids in dairy products stimulate PPARs, which do many things related to energy homeostasis, including upregulate UPC-1 expression in BAT. One of those dairy fats is Phytanic acid, which according to [2] does indeed cause adipose precursor cells to differentiate into BAT cells, at least in high concentrations in vitro.


A Pubmed search didn't turn up any in vivo studies linking specific dairy fats with BAT or thermogenesis. Whey protein from dairy products does appear to increase diet-induced thermogenesis in people [3], while simultaneously blocking the thermogenic effects of green tea. In contrast, dietary whey protein decreased BAT mass in both sedentary and exercised-trained (via swimming) mice relative to controls [4]. 


So it looks like the jury is out on just what effect dairy products will have on thermogenesis, especially cold thermogenesis.





[1] Med Hypotheses. 2016 Apr;89:1-7. doi: 10.1016/j.mehy.2015.12.028. Epub 2016 Jan

Cooperative action of bioactive components in milk fat with PPARs may explain its
anti-diabetogenic properties.
Parodi PW(1).
Author information: 
(1)Human Health and Nutrition Research, Dairy Australia, Melbourne, Australia.
Electronic address: peterparodi@bigpond.com.
Type 2 diabetes and its comorbidity insulin resistance is a major public health
problem in developed countries and those with the disorder have up to a fourfold 
increased risk of cardiovascular disease relative to those without the disease.
The cardiovascular complications are believed to be due largely to multiple
mechanisms relating to hyperglycemia that defines the disease. Overweight and
obesity are the predominant risk factors and lifestyle changes aimed at reducing 
weight and increasing physical activity are the basis for prevention and
treatment of type 2 diabetes. The role of diet in type 2 diabetes has been
investigated widely, but the results have been inconclusive. Recently two large
meta-analyses of prospective cohort studies found that dairy product consumption 
was inversely associated with the risk of type 2 diabetes. Numerous observational
studies including large prospective studies found that a high intake of dairy fat
or markers of dairy fat were inversely associated with the risk of type 2
diabetes. These observations suggest that dairy fat could contain components with
anti-diabetogenic properties. Candidates for the antidiabetic affect are rumenic 
and vaccenic acids, phytanic and pristanic acids vitamin A and β-carotene and
butyric acid. The role of these compounds in glucose homeostasis and energy
balance is discussed. A common feature is that all are agonists for one or more
of the three PPAR isoforms that are expressed in metabolically active tissue,
such as the liver, skeletal muscle and adipose tissue where they play a critical 
role in regulating energy balance and the metabolism of fatty acids and glucose, 
the main energy sources. Because PPARs have a larger ligand binding pocket than
other nuclear receptors they can be activated by a wide range of agonists.
Whereas individual components may not be present in sufficient concentration to
produce a physiological effect such an effect may be obtained by several
components acting in concert, and forms the basis of the hypothesis. PPAR
agonists such as anthocyanidins and resveratrol present in nondairy items may
also contribute to outcome. In addition, PPAR-α, -β and -γ are abundant in brown 
adipose tissue where agonists and cold exposure induce uncoupling protein-1
expression in the mitochondria where it acts to generate heat at the expense of
storing energy. Animal studies demonstrated that most milk fat bioactive
compounds induced uncoupling protein-1 expression in brown adipose tissue, which 
was associated with suppression of diet-induced obesity and improvement in
insulin sensitivity.
Copyright © 2016. Published by Elsevier Ltd.
DOI: 10.1016/j.mehy.2015.12.028 
PMID: 26968898
[2] Biochem J. 2002 Feb 15;362(Pt 1):61-9.
Phytanic acid, a novel activator of uncoupling protein-1 gene transcription and
brown adipocyte differentiation.
Schlüter A(1), Barberá MJ, Iglesias R, Giralt M, Villarroya F.
Author information: 
(1)Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona,
Facultat de Biologia, Avda Diagonal 645, Barcelona 08028, Spain.
Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a phytol-derived
branched-chain fatty acid present in dietary products. Phytanic acid increased
uncoupling protein-1 (UCP1) mRNA expression in brown adipocytes differentiated in
culture. Phytanic acid induced the expression of the UCP1 gene promoter, which
was enhanced by co-transfection with a retinoid X receptor (RXR) expression
vector but not with other expression vectors driving peroxisome
proliferator-activated receptor (PPAR)alpha, PPARgamma or a form of RXR devoid of
ligand-dependent sensitivity. The effect of phytanic acid on the UCP1 gene
required the 5' enhancer region of the gene and the effects of phytanic acid were
mediated in an additive manner by three binding sites for RXR. Moreover, phytanic
acid activates brown adipocyte differentiation: long-term exposure of brown
preadipocytes to phytanic acid promoted the acquisition of the brown adipocyte
morphology and caused a co-ordinate induction of the mRNAs for gene markers of
brown adipocyte differentiation, such as UCP1, adipocyte lipid-binding protein
aP2, lipoprotein lipase, the glucose transporter GLUT4 or subunit II of
cytochrome c oxidase. In conclusion, phytanic acid is a natural product of phytol
metabolism that activates brown adipocyte thermogenic function. It constitutes a 
potential nutritional signal linking dietary status to adaptive thermogenesis.
PMCID: PMC1222360
PMID: 11829740
[3] Nutrients. 2011 Aug;3(8):725-33. doi: 10.3390/nu3080725. Epub 2011 Jul 27.

Consumption of milk-protein combined with green tea modulates diet-induced

Hursel R(1), Westerterp-Plantenga MS.

Author information:
(1)Department of Human Biology, Nutrition and Toxicology Research Institute
Maastricht, Maastricht University, P.O. Box 616, Maastricht 6200 MD, The
Netherlands. rick.hursel@maastrichtuniversity.nl


Free full text: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257698/

Green tea and protein separately are able to increase diet-induced thermogenesis.
Although their effects on long-term weight-maintenance were present separately,
they were not additive. Therefore, the effect of milk-protein (MP) in combination
with green tea on diet-induced thermogenesis (DIT) was examined in 18 subjects
(aged 18-60 years; BMI: 23.0 ± 2.1 kg/m(2)). They participated in an experiment
with a randomized, 6 arms, crossover design, where energy expenditure and
respiratory quotient (RQ) were measured. Green tea (GT)vs. placebo (PL) capsules
were either given in combination with water or with breakfasts containing milk
protein in two different dosages: 15 g (15 MP) (energy% P/C/F: 15/47/38; 1.7
MJ/500 mL), and 3.5 g (3.5 MP) (energy% P/C/F: 41/59/0; 146.4 kJ/100 mL). After
measuring resting energy expenditure (REE) for 30 min, diet-induced energy
expenditure was measured for another 3.5 h after the intervention. There was an
overall significant difference observed between conditions (p < 0.001). Post-hoc,
areas under the curve (AUCs) for diet-induced energy expenditure were
significantly different (P ≤ 0.001) for GT + water (41.11 [91.72] kJ·3.5 h) vs.
PL + water (10.86 [28.13] kJ·3.5 h), GT + 3.5 MP (10.14 [54.59] kJ·3.5 h) and PL
+ 3.5 MP (12.03 [34.09] kJ·3.5 h), but not between GT + 3.5 MP, PL + 3.5 MP and
PL + water, indicating that MP inhibited DIT following GT. DIT after GT + 15 MP
(167.69 [141.56] kJ·3.5 h) and PL + 15 MP (168.99 [186.56] kJ·3.5 h) was
significantly increased vs. PL + water (P < 0.001), but these were not different
from each other indicating that 15 g MP stimulated DIT, but inhibited the GT
effect on DIT. No significant differences in RQ were seen between conditions for
baseline and post-treatment. In conclusion, consumption of milk-protein inhibits
the effect of green tea on DIT.

DOI: 10.3390/nu3080725
PMCID: PMC3257698
PMID: 22254119

[4] Med Sci Sports Exerc. 2014 Aug;46(8):1517-24. doi: 10.1249/MSS.0000000000000272.
Whey protein improves exercise performance and biochemical profiles in trained
Chen WC(1), Huang WC, Chiu CC, Chang YK, Huang CC.
Author information: 
(1)1Center for General Education, Chang Gung University of Science and
Technology, TAIWAN; 2Graduate Institute of Athletics and Coaching Science,
National Taiwan Sport University, TAIWAN; and 3Graduate Institute of Sports
Science, National Taiwan Sport University, TAIWAN.
PURPOSE: The objective of this study is to verify the beneficial effects of whey 
protein (WP) supplementation on health promotion and enhance exercise performance
in an aerobic-exercise training protocol.
METHODS: In total, 40 male Institute of Cancer Research mice (4 wk old) were
divided into four groups (n = 10 per group): sedentary control with vehicle (SC) 
or WP supplementation (4.1 g·kg, SC + WP), and exercise training with vehicle
(ET) or WP supplementation (4.1 g·kg, ET + WP). Animals in the ET and ET + WP
groups underwent swimming endurance training for 6 wk, 5 d·wk. Exercise
performance was evaluated by forelimb grip strength and exhaustive swimming time 
as well as by changes in body composition and biochemical parameters at the end
of the experiment.
RESULTS: ET significantly decreased final body and muscle weight and levels of
albumin, total protein, blood urea nitrogen, creatinine, total cholesterol, and
triacylglycerol. ET significantly increased grip strength; relative weight (%) of
liver, heart, and brown adipose tissue (BAT); and levels of aspartate
aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate
dehydrogenase, creatine kinase, and total bilirubin. WP supplementation
significantly decreased final body, muscle, liver, BAT, and kidney weight and
relative weight (%) of muscle, liver, and BAT as well as levels of aspartate
aminotransferase, lactate dehydrogenase, creatine kinase, and uric acid. In
addition, WP supplementation slightly increased endurance time and significantly 
increased grip strength and levels of albumin and total protein.
CONCLUSION: WP supplementation improved exercise performance, body composition,
and biochemical assessments in mice and may be an effective ergogenic aid in
aerobic exercise training.
DOI: 10.1249/MSS.0000000000000272 
PMCID: PMC4186725
PMID: 24504433
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Brown Adipose Tissue Increases Male Fertility


In this post, Michael pointed to a press release from a company developing a method to transplant BAT tissue into humans to help with weight control. This study [1] suggests that if/when they succeed, it might help with the growing problem of male infertility in developed nations, which appears to largely be a result of the obesity epidemic, according to this review article [2] published in the journal Spermatogenesis.


Study [1] found that transplanting brown fat from thin mice into obese mice resulted in the recipient obese mice experiencing a drop in weight, improved cholesterol, and increased sperm motility. 


Is there anything BAT can't do?!





[1] Obes Res Clin Pract. 2016 Jun 27. pii: S1871-403X(16)30037-0. doi:
10.1016/j.orcp.2016.06.001. [Epub ahead of print]

Brown adipose tissue transplantation ameliorates male fertility impairment caused
by diet-induced obesity.

Liu H(1), Liu X(2), Wang L(3), Sheng N(2).

Author information:
(1)Department of Laboratory Medicine, Bengbu Medical College, Bengbu 233030, PR
China. Electronic address: shooterlau1220@aliyun.com. (2)Key Laboratory of Animal
Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of
Sciences, Beijing 100101, PR China. (3)Department of Preventive Medicine, Bengbu
Medical College, Bengbu 233030, PR China.

Populations with obesity or overweight have a high incidence of infertility. We
hypothesised that brown adipose tissue (BAT) transplantation can attenuate the
impairment of male fertility caused by diet-induced obesity. BATs were
transplanted from male donor mice into age and sex matched recipient mice fed
high-fat diets (HFD). Sperm motility experiment was conducted after surgical
procedure. X-ray computed tomography scanning, biochemical assay, real-time PCR
and western blot analysis were performed. BAT transplantation reduced body fat
and epididymal fat mass, as well as triglycerides (TG) content in testis and
epididymis and total cholesterol (TCHO) contents in epididymis compared with the
HFD group. Sperm motility and progressiveness were recovered and mRNA and protein
levels of genes related to sperm motility such as cullin 3 (Cul3), peroxisome
proliferator activated receptor alpha (PPARα) and its down-stream genes were
significantly down-regulated post BAT transplantation. BAT transplantation
partially ameliorated impairment of male fertility caused by diet-induced

Copyright © 2016 Asia Oceania Association for the Study of Obesity. Published by
Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.orcp.2016.06.001
PMID: 27364235



[2] Spermatogenesis. 2012 Oct 1;2(4):253-263.

Impact of obesity on male fertility, sperm function and molecular composition.

Palmer NO(1), Bakos HW, Fullston T, Lane M.

Author information:
(1)School of Paediatrics and Reproductive Health; The Robinson Institute;
Discipline of Obstetrics and Gynaecology; The University of Adelaide; Adelaide,
SA Australia.

Male obesity in reproductive-age men has nearly tripled in the past 30 y and
coincides with an increase in male infertility worldwide. There is now emerging
evidence that male obesity impacts negatively on male reproductive potential not
only reducing sperm quality, but in particular altering the physical and
molecular structure of germ cells in the testes and ultimately mature sperm.
Recent data has shown that male obesity also impairs offspring metabolic and
reproductive health suggesting that paternal health cues are transmitted to the
next generation with the mediator mostly likely occurring via the sperm.
Interestingly the molecular profile of germ cells in the testes and sperm from
obese males is altered with changes to epigenetic modifiers. The increasing
prevalence of male obesity calls for better public health awareness at the time
of conception, with a better understanding of the molecular mechanism involved
during spermatogenesis required along with the potential of interventions in
reversing these deleterious effects. This review will focus on how male obesity
affects fertility and sperm quality with a focus on proposed mechanisms and the
potential reversibility of these adverse effects.

DOI: 10.4161/spmg.21362
PMCID: PMC3521747
PMID: 23248766

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Luigi Fontana - Missing the Obvious?


Our good friend Luigi Fontana and colleagues have just published a new paper [1] (thanks Al for pointing to it!). It investigates the metabolic benefits of diets that are either low in total protein or low in branched-chain amino acids (BCAAs) which include leucine, isoleucine, and valine.


Here is the nice graphical abstract of what they found, in both mice and humans:




Basically, reduction in either total protein or BCAAs reduced visceral white adipose tissue, improve glucose metabolism / insulin sensitivity and increased FGF21 in both humans and mice.


Those should sound like familiar effects to anyone who has been following along with this thread. In fact, anyone who has been following along carefully will already know the likely explanation - diets with reduced total protein, methionine or BCAAs (especially leucine) all induce BAT activity and/or the conversion of white fat to beige fat. This results in increased thermogenesis, a higher metabolic rate, improved insulin sensitivity and elevated FGF21 - all the effects Luigi observed in his mice and humans. It's the same beneficial feedback loop as shown in this picture that I discussed here, only with "Protein / BCAA restriction" substituted for "Cold Exposure":





But surprisingly, Luigi and company seems to have overlooked this likely explanation. In fact, from the discussion of the full text of [1], Luigi et al seem a bit baffled about the mechanism responsible for the metabolic improvements they observed (my emphasis):


Notably, mice on the Low AA and Low BCAA diets ate significantly

more than mice on the Control diet, yet gained less weight. In

mice placed on a Low AA diet, this may be explained in part by

an FGF21-mediated increase in energy expenditure (Laeger

et al., 2014a), but mice eating a diet specifically reduced in the

BCAAs do not have increased FGF21 or increased energy

expenditure; the mechanism for this remains to be determined...


However, the ultimate molecular mechanism that drives the effect of a

leucine reduced diet on white adipose tissue is as yet unknown. If

altered dietary levels of a single amino acid can also regulate adipose

mass in humans, it suggests that the obesity epidemic sweeping the

world could be impacted by relatively subtle changes in dietary

quality at the level of amino acid composition.


The explanation for all the metabolic benefits Luigi et al saw with protein and BCAA restriction seems pretty clear to me - increased BAT and beige fat thermogenesis.


Since he is a friend and quite accessible, I emailed Luigi about this possible explanation for his consideration. I'll let everyone know if/when/how he responds.





[1] Cell Rep. 2016 Jun 21. pii: S2211-1247(16)30733-1. doi:

10.1016/j.celrep.2016.05.092. [Epub ahead of print]


Decreased Consumption of Branched-Chain Amino Acids Improves Metabolic Health.


Fontana L(1), Cummings NE(2), Arriola Apelo SI(3), Neuman JC(4), Kasza I(5),

Schmidt BA(3), Cava E(6), Spelta F(7), Tosti V(7), Syed FA(3), Baar EL(3),

Veronese N(8), Cottrell SE(9), Fenske RJ(4), Bertozzi B(10), Brar HK(3), Pietka

T(10), Bullock AD(11), Figenshau RS(11), Andriole GL(11), Merrins MJ(12),

Alexander CM(5), Kimple ME(13), Lamming DW(14).


Free full text: http://linkinghub.elsevier.com/retrieve/pii/S2211-1247(16)30733-1


Protein-restricted (PR), high-carbohydrate diets improve metabolic health in

rodents, yet the precise dietary components that are responsible for these

effects have not been identified. Furthermore, the applicability of these studies

to humans is unclear. Here, we demonstrate in a randomized controlled trial that 

a moderate PR diet also improves markers of metabolic health in humans.

Intriguingly, we find that feeding mice a diet specifically reduced in

branched-chain amino acids (BCAAs) is sufficient to improve glucose tolerance and

body composition equivalently to a PR diet via metabolically distinct pathways.

Our results highlight a critical role for dietary quality at the level of amino

acids in the maintenance of metabolic health and suggest that diets specifically 

reduced in BCAAs, or pharmacological interventions in this pathway, may offer a

translatable way to achieve many of the metabolic benefits of a PR diet.


Copyright © 2016. Published by Elsevier Inc.


DOI: 10.1016/j.celrep.2016.05.092 

PMID: 27346343

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Thanks for the analysis.  But as shown by the cartoon figure of the effects of low BCAA, total amino acids or protein, FGF21 was not affected by BCAA.  See also Figure 5.  What is shown as well is that there was more heat generated by the use of low amino acids during the night, an effect not seen with low BCAA.

Edited by AlPater
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Thanks Al,


I wasn't claiming that the low BCAAs (or low leucine) explained the all of the metabolic benefits of low protein / amino acids. There is also e.g. the effect of low methionine, which boosts BAT activity and thermogenesis. But thanks for pointing to Figure 5 from Luigi's new paper (PMID 27346343):




It reminded me of another thing Luigi et al found in that points to BAT and thermogenesis being involved - elevated adioponectin (labelled D above).


Increased adiponectin is also associated with elevated BAT and thermogenesis. Remember cold-exposure and thermogenesis increase adiponectin (by 62% in just a couple hours in Eric's CoolFatBurner video), and remember this study [1] which connects Luigi's protein restriction results, adiponectin and BAT thermogenesis:


Dietary methionine restriction (MR) limits fat deposition and decreases plasma
leptin, while increasing food consumption, total energy expenditure (EE), plasma 
adiponectin, and expression of uncoupling protein 1 (UCP1) in brown and white
adipose tissue (BAT and WAT).


Even more reason to think BAT & thermogenesis were responsible for the effects Luigi et al saw from protein / AA restriction.





[1] Am J Physiol Regul Integr Comp Physiol. 2010 Sep;299(3):R740-50. doi:
10.1152/ajpregu.00838.2009. Epub 2010 Jun 16.
Role of beta-adrenergic receptors in the hyperphagic and hypermetabolic responses
to dietary methionine restriction.
Plaisance EP(1), Henagan TM, Echlin H, Boudreau A, Hill KL, Lenard NR, Hasek BE, 
Orentreich N, Gettys TW.
Author information: 
(1)Laboratory of Nutrient Sensing and Adipocyte Signaling, Pennington Biomedical 
Research Center, Baton Rouge, Louisiana, USA.
Dietary methionine restriction (MR) limits fat deposition and decreases plasma
leptin, while increasing food consumption, total energy expenditure (EE), plasma 
adiponectin, and expression of uncoupling protein 1 (UCP1) in brown and white
adipose tissue (BAT and WAT). beta-adrenergic receptors (beta-AR) serve as
conduits for sympathetic input to adipose tissue, but their role in mediating the
effects of MR on energy homeostasis is unclear. Energy intake, weight, and
adiposity were modestly higher in beta(3)-AR(-/-) mice on the Control diet
compared with wild-type (WT) mice, but the hyperphagic response to the MR diet
and the reduction in fat deposition did not differ between the genotypes. The
absence of beta(3)-ARs also did not diminish the ability of MR to increase total 
EE and plasma adiponectin or decrease leptin mRNA, but it did block the
MR-dependent increase in UCP1 mRNA in BAT but not WAT. In a further study,
propranolol was used to antagonize remaining beta-adrenergic input (beta(1)- and 
beta(2)-ARs) in beta(3)-AR(-/-) mice, and this treatment blocked >50% of the
MR-induced increase in total EE and UCP1 induction in both BAT and WAT. We
conclude that signaling through beta-adrenergic receptors is a component of the
mechanism used by dietary MR to increase EE, and that beta(1)- and beta(2)-ARs
are able to substitute for beta(3)-ARs in mediating the effect of dietary MR on
EE. These findings are consistent with the involvement of both UCP1-dependent and
-independent mechanisms in the physiological responses affecting energy balance
that are produced by dietary MR.
PMCID: PMC2944424
PMID: 20554934
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I don't think you fully acknowledged Al's comment -- which was about something that I had also noticed, but originally decided not to comment on.


Dean wrote:

Here is the nice graphical abstract of what they found, in both mice and humans:

. . .

Basically, reduction in either total protein or BCAAs reduced visceral white adipose tissue, improve glucose metabolism / insulin sensitivity and increased FGF21 in both humans and mice.

But the graphical abstract did not appear to show exactly what you stated. Specifically, the column for (BCAAs?) Leucine/Isoleucine/Valine had an '=' in the FGF21 row. I'm not certain why you didn't correct your original statement before moving on to Luigi's new paper in your reply post. [Feel free to correct the original statement and delete this comment if you like.]



Edited by Todd S
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But the graphical abstract did not appear to show exactly what you stated. Specifically, the column for (BCAAs?) Leucine/Isoleucine/Valine had an '=' in the FGF21 row.


Both you and Al are right - a low BCAAs (or low leucine) diet did not appear to increase FGF21 in Luigi's new study. This is in contrast with the Wanders et al's 2015 paper (PMID 26643647), which found leucine restriction does increase FGF21 by a factor of 3x in serum and 2x in the liver, as well as increase browning and UPC1 expression in white fat, as discussed here.


So Luigi's single new paper (there aren't two Fontana papers under discussion Todd as you seem to think) seems to be in conflict with PMID 26643647 regarding the effects of BCAA/Leucine restriction on FGF21.


But whether or not the FGF21 is involved or impacted by BCAA/Leucine restriction, both Luigi and PMID 26643647 observed increased energy expenditure, reduced weight and improved metabolic health as a result of a diet reduced in BCAAs/Leucine. PMID 26643647 appears to have gone further than Luigi by looking at the gene expression in adipose tissue, and observing that the likely cause of the metabolic improvements was the browning of white fat. I was surprised that Luigi et al didn't even cite the Wander et al 2015 paper, since it was highly relevant to their own experiment and results. 


If FGF21 isn't increased by BCAA/Leucine restriction as observed by Luigi (and contra PMID 26643647), the increased FGF21 resulting from protein restriction or overall amino acid restriction that Luigi observed is likely a result of restricting that other bad boy AA, methionine, as we've discussed before (e.g. [1] and other studies discussed here).


But in the end, we can quibble over the detail of which amino acids are best to reduce, and how their reduction impacts metabolism until the cows come home. But practically speaking, it's a pain in the a$$ to try to modulate down individual amino acids like leucine or methionine in one's diet, except (luckily) in broad stroke by a low-protein (and esp. low animal protein) diet. And one important mechanism by which a these dietary protein intervenes have benefit appears to be through boosting BAT, beige fat & thermogenesis, an explanation Luigi seems to me to be overlooking, although as you can see from Figure 5 from his paper, he did look at heat generation.





[1] Aging Cell. 2014 Oct;13(5):817-27. doi: 10.1111/acel.12238. Epub 2014 Jun 17.

Methionine restriction restores a younger metabolic phenotype in adult mice with 
alterations in fibroblast growth factor 21.
Lees EK(1), Król E, Grant L, Shearer K, Wyse C, Moncur E, Bykowska AS, Mody N,
Gettys TW, Delibegovic M.
Author information: 
(1)Institute of Medical Sciences, College of Life Sciences and Medicine,
University of Aberdeen, Aberdeen, AB25 2ZD, UK.
Methionine restriction (MR) decreases body weight and adiposity and improves
glucose homeostasis in rodents. Similar to caloric restriction, MR extends
lifespan, but is accompanied by increased food intake and energy expenditure.
Most studies have examined MR in young animals; therefore, the aim of this study 
was to investigate the ability of MR to reverse age-induced obesity and insulin
resistance in adult animals. Male C57BL/6J mice aged 2 and 12 months old were fed
MR (0.172% methionine) or control diet (0.86% methionine) for 8 weeks or 48 h.
Food intake and whole-body physiology were assessed and serum/tissues analyzed
biochemically. Methionine restriction in 12-month-old mice completely reversed
age-induced alterations in body weight, adiposity, physical activity, and glucose
tolerance to the levels measured in healthy 2-month-old control-fed mice. This
was despite a significant increase in food intake in 12-month-old MR-fed mice.
Methionine restriction decreased hepatic lipogenic gene expression and caused a
remodeling of lipid metabolism in white adipose tissue, alongside increased
insulin-induced phosphorylation of the insulin receptor (IR) and Akt in
peripheral tissues. Mice restricted of methionine exhibited increased circulating
and hepatic gene expression levels of FGF21, phosphorylation of eIF2a, and
expression of ATF4, with a concomitant decrease in IRE1α phosphorylation.
Short-term 48-h MR treatment increased hepatic FGF21 expression/secretion and
insulin signaling and improved whole-body glucose homeostasis without affecting
body weight. Our findings suggest that MR feeding can reverse the negative
effects of aging on body mass, adiposity, and insulin resistance through an FGF21
mechanism. These findings implicate MR dietary intervention as a viable therapy
for age-induced metabolic syndrome in adult humans.
PMCID: PMC4331744
PMID: 24935677
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For a researcher wanting to study cold exposure effects on humans, might the homeless population provide a suitable source of subjects? Sleeping on the streets of San Francisco, for example, could already have provided plenty of cold exposure over a long period of time.



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Interesting idea, but it seems to me the physical and psychiatric health conditions (incl. drug and alcohol addiction) that unfortunately plague many homeless people, coupled with their less-than-stable living arrangements would make them a difficult study population to work with or learn much from that is relevant for a health population. Intervention trials would be difficult too, as such experiments would be viewed as exploitative - using down-and-out homeless folks as 'guinea pigs' by subjecting them to unnecessary cold (and suffering), even if they were well-compensated. I can just see the clickbait headline now - "San Francisco Homeless Denied Blankets in the Name of Science".


But maybe the folks in the Coney Island Polar Bear Club would be a better option:


The Coney Island Polar Bear Club is the oldest winter bathing organization in the United States. We swim in the Atlantic Ocean at Coney Island every Sunday from October through April.


Now those are some folks we might really learn from!



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I had also thought of the cold-water swimming clubs (such as the local Dolphin Club), but my thinking is that the "pool" of potential subjects that are exposed daily to cold would be at least two orders of magnitude greater in the homeless population. Although there are likely many homeless folks without serious addiction or psychiatric issues, I agree that it would probably be a lot more complicated to select subjects from that pool.



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Two Genetic Polymorphisms That Impact BAT & Adiposity in Humans


A while back, prompted by Gordo IIRC, I promised to look into the genetics of BAT and thermogenesis. Well I've finally gotten around to at least taking a preliminary stab at it with this post.


There appear to be two main single nucleotide polymorphisms (SNPs) that are consistently associated with BAT activity, and a bunch of studies that seem to show pretty much the same thing - namely that specific alleles for these two SNPs alter BAT thermogenesis and are associated with visceral adiposity. 


One of the SNPs (rs1800592) is in the UCP1 gene. The G allele (reported as 'C' in 23andMe - see here for your own data if you are a 23andMe subscriber) in this SNP is associated with reduced prevalence of BAT, especially in older people [1]. I'm fortunate to be 'TT' according to 23andMe for this allele, so no C's which translates1 into no G's which translates into more BAT. Hurray for me!


The second SNP is rs4994 and it is part of the gene for the β3-adrenergic receptor (β3AR). This receptor is where norepinephrine binds to BAT cells to tell them to burn calories to generate heat. For this one, the 'C' allele is associated with reduced BAT [1]. 23andMe reports rs4994 as A/G rather than T/C, so on 23andMe the reduced-BAT variant for this allele is G1. It's all very confusing, but the bottom line is on 23andMe you want to be AA for rs4994 to have a better chance of building BAT. I'm AA, so again - bully for me! 23andMe subscribers can check there own results for rs4994 here.


In summary, after looking over a bunch of studies including most relevantly [1], you want to have the following two polymorphisms as reported by 23andMe (and as I do) to maximize your chance of building BAT, promoting thermogenesis, and avoiding visceral fat buildup as you age:


rs1800592:  TT

rs4994:       AA


Any other 23andMe subscribers care to share?


Edit: I've added these two SNPs to the list of BAT / beige fat / thermogenesis inducers below:





1Sometimes 23andMe reports SNP data from the mirror strand of DNA from what is reported in published studies. So for example, sometimes a study reports A vs. G for SNP but 23andMe reports T vs C for the same SNP, or visa versa. Here is the transformation to remember: C ≈ G and T ≈ A. In other words, if you see a 'C' allele in a study, it could be reported as a 'C' or a 'G' on 23andMe. Similarly if a study reports a 'T' allele that could be reported as a 'T' or an 'A' on 23andMe. The easy way I just thought of to remember this transformation is that the two 'curvy' letters (C & G) are interchangeable and the two 'straight' letters (T and A) are interchangeable. 


So for example, in this case [1] reported 'G' was the 'bad' (low BAT) allele for SNP rs1800592. 23andMe reports rs1800592 as either C or T. Remember Curly letters go together, so the 'bad' allele on 23andMe is 'C'. So you want to avoid C and instead be TT for rs1800592 to have a good chance of developing BAT.



Here is the latest full list of modifiable and [nonmodifiable] factors associated with increased brown/beige adipose tissue and/or thermogenesis, with the factors mentioned in this post highlighted in red:

  • Cold exposure - by far the best BAT inducer/activator
  • Spicy / pungent foods, herbs & supplements - capsaicin / chilli peppers, curcumin / turmeric root, menthol/mint/camphor, oregano, cloves, mustard, horseradish/wasabi, garlic, onions
  • Sulforaphane-rich foods - Broccoli, brussels sprouts, cabbage
  • Arginine-rich foods - Good vegan sources include seeds (esp. sesame, sunflower & pumpkin), nuts (esp. almonds and walnuts) and legumes (esp. soy, lupin & fava beans and peas)
  • Healthy Fats - DHA / EPA / fish-oil, MUFA-rich diet,  Extra Virgin Olive Oil
  • Olive Polyphenols - Extra Virgin Olive Oil / Olive Leaf Extract / Olive Leaf Tea
  • Nitrate-rich foods - beets, celery, arugula, and spinach
  • Other foods - Apples / apple peels / ursolic acid; Citrus fruit / citrus peels / limonene; Honey / chrysin
  • Beverages - green tea, roasted coffee, red wine, cacao beans / chocolate
  • Low gluten diet
  • Methionine restriction - Reduce animal protein. Soy is low in methionine and high in arginine, but also high in leucine.
  • Leucine restriction - Reduce animals protein. Leucine is highest in beef, fish, eggs, cheese and soy.
  • Low protein diet
  • Drugs / Supplements - metformin, berberine, caffeine, creatine, nicotinamide riboside (NAD), resveratrol, ginseng, cannabidiol / hemp oil / medicinal marijuana
  • Time Restricted Feeding - most calories at breakfast
  • Exercise
  • Acupuncture - locations Zusanli (foot - ST36) and Neiting (lower leg - ST44) 
  • Whole body vibration therapy
  • Avoid obesity/overweight
  • [being naturally thin - high metabolic rate]
  • [being younger]
  • [being female]
  • [Ethnicity - having cold-climate ancestors]
  • [being of genotype TT for rs1800592 and AA for rs4994 as reported by 23andMe]


[1] Int J Obes (Lond). 2013 Jul;37(7):993-8. doi: 10.1038/ijo.2012.161. Epub 2012 Oct


Impact of UCP1 and β3AR gene polymorphisms on age-related changes in brown
adipose tissue and adiposity in humans.

Yoneshiro T(1), Ogawa T, Okamoto N, Matsushita M, Aita S, Kameya T, Kawai Y,
Iwanaga T, Saito M.

Author information:
(1)Laboratory of Histology and Cytology, Department of Anatomy, Hokkaido
University Graduate School of Medicine, Sapporo, Japan.

BACKGROUND: Brown adipose tissue (BAT) is involved in the regulation of
whole-body energy expenditure and adiposity. The activity and prevalence of BAT
decrease with age in humans.
OBJECTIVE: To examine the effects of single nucleotide polymorphisms of the genes
for uncoupling protein 1 (UCP1) and β3-adrenergic receptor (β3AR), key molecules
of BAT thermogenesis, on age-related decline of BAT activity and accumulation of
body fat in humans.
METHODS: One hundred ninety-nine healthy volunteers (20-72 years old (y.o.))
underwent fluorodeoxyglucose-positron emission tomography (FDG-PET) and computed
tomography (CT) after 2-h cold exposure to assess BAT activity. The visceral and
subcutaneous fat areas at the abdominal level were estimated from the CT images.
They were genotyped for -3826 A/G polymorphism of the UCP1 gene and 64 Trp/Arg
mutation of the β3AR gene.
RESULTS: BAT was detected in 88 subjects out of 199 (44%), more in younger
(30 y.o., 55%) than older subjects (>40 y.o., 15%). BAT prevalence of older
subjects tended to be lower in the UCP1 G/G group than the A allele group (A/A
and A/G), and also in the β3AR Arg allele group (Trp/Arg and Arg/Arg) than the
Trp/Trp group. When compared subjects who had two or more base substitutions on
the two genes (the 2-4 allele group) with those who had less than two base
substitutions (the 0-1 allele group), BAT prevalence was comparable in younger
subjects (62% vs 50%) but lower in older subjects (0% vs 24%, P<0.05). Visceral
fat area of the 2-4 allele group was higher than that of the 0-1 allele group
(P<0.05) in older subjects, but not in younger subjects.
CONCLUSION: UCP1 -3826 A/G and β3AR 64 Trp/Arg substitutions accelerate
age-related decrease in BAT activity, and thereby may associate with visceral fat
accumulation with age.

DOI: 10.1038/ijo.2012.161
PMID: 23032405

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Alas, I'm CC on rs1800592 - no BAT for me :(... and AA for rs4994, however with no BAT, it seems hardly useful.


Just to clarify - I don't think being CC for rs1800592 necessarily means 'no BAT'. It appears to mean your UCP1 gene isn't quite as active/effective, and this gets reflected in older folks who have CC for that allele appearing to have little BAT activity on average. But being CC certainly doesn't preclude having BAT, since younger folks with CC have BAT according to PMID 23032405. So don't give up so easily!



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I wonder if there is a possibility of cultivating BAT when younger that might persist - with CE practices - into older age despite the fatal CC. Sort of like if you are naturally prone to sarcopenia, you might want to try to develop muscle mass when younger and then attempt to maintain it through a rigorous program of diet + exercise in older age when sarcopenia strikes.

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The Flavonoid Luteolin Browns Fat & Increases Thermogenesis via AMPK


Here is another BAT Rule1 example, this time involving the dietary flavonoid Luteolin (LU), which is high in the following foods (ranked highest to lowest per 100g) based on this list:

  • Fresh thyme
  • Dried parsley
  • Fresh peppermint
  • Hot peppers - hotter the better. chili > serrano > japaleno > green/red
  • Fresh Rosemary
  • Lemons (w/o peel) - oranges too
  • Celery
  • Kohlrabi
  • Fresh Parley
  • Spinach
  • Beets
  • Brussels sprouts
  • Chives
  • Cauliflower
  • Lettuce
  • Red cabbage
  • Green cabbage

In addition to these, other places (like the LU Wikipedia page) lists olive oil, dandelions, chamomile tea, and carrots as additional sources of LU not listed above.


How's that for a healthy list of foods!? Add a B12 supplement and I bet you could live off those and those alone.


So what's the evidence that LU boosts BAT, browning of white fat and thermogenesis? Several studies, starting with this one [1] which found low-dose LU fed to mice eating either a low-fat or an obesity-inducing high-fat diet resulted in less obesity, better insulin sensitivity, and less inflammation.


In this new follow-up study [2], the same group found that the beneficial effects of LU on metabolic health appear to result from, you guessed it, upregulation of BAT, browning of WAT and increased energy expenditure via thermogenesis. 


From the full text of [2], they fed male C57BL/6 mice either a low-fat diet (LFD), a high-fat diet (HFD) or a high-fat diet with 0.01% LU (HFD+LU). The experiment lasted 12 weeks during which the mice were housed at the usual, cool-for-mice temperature of 22°C.


Once again, as in [1], they saw that LU protected against HFD-induced weight gain, fat accumulation, insulin resistance, without reducing energy intake.


What they found new in [2] was about the mechanism. They found that LU dramatically increased the UPC1 gene expression and protein content in both BAT (left) and Subcutaneous Adipose Tissue (SAT - right), relative to a HFD w/o LU:




The LU also upregulated expression of a bunch of genes associated with both fat browning and thermogenesis in both BAT & SAT (data not shown). UPC1 and other thermogenic genes were upregulated in a group of mice they added, which was fed a LFD + LU. So the effect wasn't limited to mice eating only a high-fat diet.


LU also was associated with increased levels of AMPK and SIRT1 in both BAT and SAT - in fact the upregulation of these two master switches probably was responsible for the fat browning and increased thermogenesis. They verified this by showing in vitro that both BAT and SAT cells dripped with LU exhibited elevated levels of AMPK & SIRT1, along with all the other browning/thermogenesis genes they saw were elevated in their in vivo study. They have a long section in the paper with additional analysis to investigate the pathway involved, a section of the paper I won't belabor. In a nutshell, here is how they characterize their results:


Dietary luteolin → ↑ AMPK → ↑ SIRT1 → ↑ PGC1α → ↑ UPC1 & other browning/thermogenic genes


A few salient quotes from the discussion to drive these points home (and to prove I'm not making it up ☺):


Notably, dietary luteolin enhanced BAT thermogenic program in either
HFD-fed (Figures 2a–e) or LFD-fed (Figures 4a–d) mice, suggesting
luteolin can strengthen the functional activities of classic brown
In this study, both HFD and LFD supplement of luteolin promoted
browning and thermogenic program in [subcutaneous white adipose tissue].


Once again, in accord with the BAT Rule1, we see all things healthy also seem to upregulate BAT, browning of white fat and thermogenesis. I've updated the list of BAT, beige fat and thermogenesis inducers below to include the above list of luteolin-rich foods.


P.S. I've also included at the bottom the two gene variants discussed recently in this post as non-modifiable factors associated with brown/beige fat.





1BAT Rule - Virtually every dietary or lifestyle intervention that is known to be healthy is also associated with an increase in BAT activity, browning of white fat and/or thermogenesis.



Here is the latest full list of modifiable and [nonmodifiable] factors associated with increased brown/beige adipose tissue and/or thermogenesis, with the factors mentioned in this post highlighted in red:

  • Cold exposure - by far the best BAT inducer/activator
  • Spicy / pungent foods, herbs & supplements - capsaicin / chilli peppers, curcumin / turmeric root, menthol/mint/camphor, oregano, cloves, mustard, horseradish/wasabi, garlic, onions
  • Sulforaphane-rich foods - Broccoli, brussels sprouts, cabbage
  • Arginine-rich foods - Good vegan sources include seeds (esp. sesame, sunflower & pumpkin), nuts (esp. almonds and walnuts) and legumes (esp. soy, lupin & fava beans and peas)
  • Healthy Fats - DHA / EPA / fish-oil, MUFA-rich diet,  Extra Virgin Olive Oil
  • Olive Polyphenols - Extra Virgin Olive Oil / Olive Leaf Extract / Olive Leaf Tea
  • Luteolin-rich foods - Herbs (thyme, parsley, oregano, peppermint, rosemary), hot peppers, citrus fruit, celery, beets, spinach, cruciferous veggies, olive oil, carrots. 
  • Other foods - Apples / apple peels / ursolic acid; Citrus fruit / citrus peels / limonene; Honey / chrysin
  • Beverages - green tea, roasted coffee, red wine, cacao beans / chocolate
  • Low gluten diet
  • Methionine restriction - Reduce animal protein. Soy is low in methionine and high in arginine, but also high in leucine.
  • Leucine restriction - Reduce animals protein. Leucine is highest in beef, fish, eggs, cheese and soy.
  • Low protein diet
  • Drugs / Supplements - metformin, berberine, caffeine, creatine, nicotinamide riboside (NAD), resveratrol, ginseng, cannabidiol / hemp oil / medicinal marijuana
  • Time Restricted Feeding - most calories at breakfast
  • Exercise
  • Acupuncture - locations Zusanli (foot - ST36) and Neiting (lower leg - ST44) 
  • Whole body vibration therapy
  • Avoid obesity/overweight
  • [being naturally thin - high metabolic rate]
  • [being younger]
  • [being female]
  • [Ethnicity - having cold-climate ancestors]
  • [being of genotype TT for rs1800592 and AA for rs4994 as reported by 23andMe]


[1] Mol Nutr Food Res. 2014 Jun;58(6):1258-68. doi: 10.1002/mnfr.201300830. Epub 2014

Feb 24.
Low-dose diet supplement of a natural flavonoid, luteolin, ameliorates
diet-induced obesity and insulin resistance in mice.
Xu N(1), Zhang L, Dong J, Zhang X, Chen YG, Bao B, Liu J.
Author information: 
(1)School of Biotechnology & Food Engineering, Hefei University of Technology,
Hefei, P. R. China; School of Life Science, University of Anhui Science &
Technology, Fengyang, P. R. China.
SCOPE: Mast cells play important roles in diet-induced obesity and diabetes, and 
some synthetic mast cell stabilizers can improve related metabolic disturbances
in mice. Luteolin (LU) is a potent natural mast cell stabilizer. However, a
direct correlation between LU and these common metabolic diseases is not
METHODS AND RESULTS: Male C57BL/6 mice were fed low-fat diet, high-fat diet
(HFD), HFD with 0.002 and 0.01% LU for 12 wk, respectively. Dietary LU suppressed
HFD-induced body weight gain, fat deposition, and adipocyte hypertrophy.
Meanwhile, glucose intolerance and insulin sensitivity was also improved.
Interestingly, dietary LU ameliorated angiogenesis and associated cell apoptosis 
and cathepsin activity in epididymis adipose tissues, which is a critical
mechanism that mast cells are involved in diet-induced obesity and diabetes.
Further, we showed dietary LU reduced mast cell and macrophage infiltrations and 
inflammatory cytokine levels in epididymis adipose tissues. Finally, LU inhibited
mast cell-derived IL-6 expression, which is a key cytokine that contributes to
mast cell-associated metabolic derangements, and protein kinase C activator
phorbol myristoyl acetate reversed the inhibitory effects.
CONCLUSIONS: As a natural flavonoid, low-dose diet supplement of LU ameliorates
diet-induced obesity and insulin resistance in mice, suggesting a new therapeutic
and interventional approach for these diseases.
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
DOI: 10.1002/mnfr.201300830 
PMID: 24668788 
[2] Int J Obes (Lond). 2016 Jul 5. doi: 10.1038/ijo.2016.108. [Epub ahead of print]

Dietary luteolin activates browning and thermogenesis in mice through an
AMPK/PGC1α pathway-mediated mechanism.

Zhang X(1), Zhang QX(1), Wang X(1), Zhang L(1), Qu W(1), Bao B(1), Liu CA(1), Liu

Author information:
(1)Department of Biological Sciences, School of Biotechnology and Food
Engineering, Hefei University of Technology, Hefei, China.


Full text: http://www.nature.com.sci-hub.cc/ijo/journal/vaop/ncurrent/full/ijo2016108a.html

BACKGROUND: Two brown-like adipocytes, including classical brown adipocytes from
brown adipose tissues and beige cells from white adipose tissues, regulate
thermogenesis. The developmental and functional induction of brown-like cells
provides a defense against obesity and associated metabolic diseases. Our
previous study suggests dietary luteolin can improve diet-induced obesity and
insulin resistance in mice. Here we further elucidated the action of the natural
flavonoid on energy expenditure and adaptive thermogenesis.
METHODS: Five-week-old male C57BL/6 mice were fed low-fat diet (LFD), high-fat
diet (HFD) and HFD supplemented with 0.01% luteolin. After 12 weeks, their energy
expenditure were detected using a combined indirect calorimetry system. Moreover,
thermogenic program and associated molecular regulators were assessed in adipose
tissues. In another independent study, even-aged mice were fed LFD and
luteolin-containing LFD for 12 weeks, and their energy expenditure and
thermogenic program were also investigated. Finally, differentiated primary brown
and subcutaneous adipocytes were used to identify the critical participation of
AMPK/PGC1α signaling in luteolin-regulated browning and thermogenesis.
RESULTS: In mice fed either HFD or LFD, dietary luteolin supplement increased
oxygen consumption, carbon dioxide production and respiratory exchange ratio. The
enhancement in energy expenditure was accompanied by the upregulation of
thermogenic genes in brown and subcutaneous adipose tissues. Meanwhile, several
important AMPK/PGC1α signaling molecules were activated by dietary luteolin in
the tissues. Further, luteolin treatment directly elevated thermogenic gene
expressions and activated AMPK/PGC1α signaling in differentiated primary brown
and subcutaneous adipocytes, whereas AMPK inhibitor Compound C reversed the
CONCLUSIONS: Dietary luteolin activated browning and thermogenesis through an
AMPK/PGC1α pathway-mediated mechanism.International Journal of Obesity advance
online publication, 5 July 2016; doi:10.1038/ijo.2016.108.

DOI: 10.1038/ijo.2016.108
PMID: 27377953

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If nicotinamide riboside as an NAD precursor is likely to boost thermogenesis then nicotinic acid (the original vitamin B3) is likely to do so as well.  And considering their prices, NA is likely a more cost effective approach than NR.

Here's a review paper, NAD+ and Vitamin B3: From Metabolism to Therapies, http://jpet.aspetjournals.org/content/324/3/883.full with references to a lot of other papers on B3 and NAD which I found useful to learn about the benefits and risks of the various forms of B3.

Edited by Todd Allen
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Endurance Exercise & Lactate Increases Browning of Subcutaneous Fat


As I'm sure almost everyone reading this is painfully aware, I'm a big fan of endurance exercise, engaging it in nearly continuously throughout the day. In this post, Gordo questioned the wisdom of combining endurance exercise with cold exposure like I do, saying:


One difference between Dean and I might be that he exercises all day, to me this seems counter-productive, the cooling he is doing might be offset by the warming caused from continuous exercise.  By contrast, I do "HIIT" style exercise of minimal duration, and cooling mostly while sedentary - maximizing the impact of CE on my body.


I also recently reported that my latest bloodwork showed lactate dehydrogenase (LD or LDH) levels well above the reference range (284 vs. 121-224 U/L). As I discussed in that post, elevated levels of LDH are a sign of increased lactate (= lactic acid1) metabolism. This is commonly seen in endurance athletes, and is thought to be both a result of exercise and a reason for their endurance - i.e. changes to mitochondrial metabolism which increase their ability to effectively metabolize lactate to generate energy, as described in detail here. So I (speculatively) chalked by my elevated LDH level to increased lactate as a result of all the exercise do, and had done in the hours prior to that blood draw.


So it was with great interest that I read this brand new review article [1] discussing how exercise regulates adipose tissue. It discusses evidence that exercise increases BAT and the browning of subcutaneous white adipose tissue (scWAT) in both rodents and humans. The authors cite studies of rodents in their own lab and by others that have found exercise reprograms scWAT into thermogenic 'beige' adipose cells by increasing their mitochondria content and their expression of UCP1.


Interestingly in the full text of [1], the authors express the same befuddlement about the relationship between exercise and thermogenesis that Gordo does:


First, it is surprising that exercise training would lead to the induction of a thermogenic, heat-producing cell type such as beige cells, since the contracting muscles generate significant amounts of heat during exercise [refs] . Second, exercise is an energy-consuming process, so it is perplexing that exercise would induce the expression of cells that inherently increase energy expenditure, resulting in even greater fuel requirements for the organism. Finally, during exercise, skeletal muscle is supplied with energy from other tissues, specifically free fatty acids released from WAT; thus it is puzzling that exercise would result in adaptations to the scWAT that result in the burning of residual fat stores, potentially making less substrate available for the working skeletal muscle [ref] .


Here is one mechanism they say might explain it - but it remains speculative:


One hypothesis to explain the exercise-induced beiging of scWAT is that exercise training-induced decreases in cell size and lipid content in scWAT decreases insulation of the body, necessitating increased heat production and resulting in the beiging of scWAT [refs]. While this is an intriguing hypothesis, research aimed at directly addressing this question and other hypotheses will be necessary to determine the primary physiological function of exercise-induced beiging.
But whatever the reason, it does appear that exercise training increases browning of subcutaneous white fat, and not just in rodents, but in young healthy humans as well. The authors of [1] reference two studies showing this. The first found that the combination strength training and endurance exercise increased UCP1 expression in scWAT of both normal and pre-diabetic men [2]. The second was the authors' own study [3] which found:
...12 weeks of aerobic exercise training in young healthy male subjects significantly increased UCP1 mRNA expression by approximately 2-fold...

The authors discuss several pathways by which exercise may increase beiging of white fat. Most of these are ones we've discussed before, including myostatin inhibition (which we discussed here), increased circulating irisin (discussed here and here), and elevated BDNF (when coupled with environmental enrichment, as discussed here and here).


But the one beiging pathway that caught my attention for obvious reasons was lactate. Here is what the authors said about lactate and scWAT beiging:


A potential mechanism that has generated much interest in this field is that exercise results in the release of myokines from the contracting skeletal muscles that signal the scWAT to induce expression of beige adipocytes [refs] . Lactate is a well-established myokine that could potentially function in this regard, and most types of exercise increase circulating lactate concentrations. Although not directly measured as a response to exercise, increasing circulating lactate concentrations, by either cold exposure or exogenous administration, was shown to increase the beiging of scWAT as determined by increased Ucp1 and Cidea expression [4].


The details of study [4] which investigated the relationship between cold, lactate and thermogenesis are quite interesting.  Apparently, cold exposure increases expression of Mct1 in BAT and scWAT. Mct1 is the main protein responsible for lactate transport and uptake: 

Higher amount of Mct1 mRNA was found in BAT compared with WAT depots (Fig. 1B). ... Furthermore, in mice exposed to 4°C for 1–7 days, we observed, in addition to the increase in Ucp1 expression in both BAT and inguinal [scWAT] depots (Fig. 1D), an increase in the expression of the lactate-importing isoform Mct1 (Fig. 1E)... In addition, we found that plasma lactate levels significantly rose after 24 h and 72 h of cold exposure and returned to basal levels after 7 days of cold exposure (Fig. 1G). These data show that brown and white adipose depots display different profiles for MCT expression and that in vivo browning induced by cold acclimation is associated with an increased lactate import system in adipose depots together with transient modification of plasma lactate levels.


In short, [4] showed that cold exposure increases circulating lactate and lactate metabolism in BAT and subcutaneous inguinal WAT of mice. The authors of [4] also did studies in vitro using adipose cells from both mouse and human. When they cultured the cells in a lactate-rich medium they found:


 Acute 48-h treatment of differentiated adipocytes isolated from inguinal fat pad with lactate resulted in a robust increase of Ucp1 mRNA levels together with an upregulation of the expression of additional key thermogenic genes...


The increase of Ucp1 mRNA levels by lactate was associated with an increase in UCP1 content as shown by Western blot (Fig. 2B) (in a dose-dependent manner, with an effect starting at 10 mmol/L [data not shown]) and by immunofluorescence (Fig. 2C), which showed that lactate increased both the number of UCP1-positive cells and the intensity of UCP1 staining per cell...


Further highlighting the brown-like phenotype of lactate-treated adipocytes, we found higher glucose consumption rate in lactate-treated cells compared with control cells.


Here is the graph showing how UCP1 level in scWAT adipocytes increased as result of being cultured with varying concentrations of lactate:




That is a pretty dramatic beiging effect of lactate! Here is how the authors of [4] summarize their results:


Together, these data reveal that lactate is a strong inducer of brown adipose gene expression in white adipose cells, and this effect is conserved between species.


Since both cold exposure and exercise increase lactate level, this seems like pretty compelling support for my speculative explanation in this post for why my lactate dehydrogenase (LDH) level was elevated in my most recent blood work. And as a bonus, it looks like elevated lactate is good for you, resulting in the beiging of subcutaneous white fat!


The authors of review article [1] do point out that while exercise pretty clearly causes the beiging of subcutaneous white fat,  there is conflicting evidence in both rodents and humans regarding the influence of exercise on BAT and BAT activity. Depending on methodology, some studies show exercise can increase BAT, while others show no influence or even a decrease in BAT and BAT activity as a result of exercise training. The authors speculate that it may be a function of genetic or environmental factors (including temperature) that determine whether exercise boosts, inhibits or leaves BAT unchanged.


But we humans, unlike rodents, have very little true BAT anyway, and therefore likely rely more on beige fat than true BAT for thermogenesis. So even if exercise increases beige fat at the expense of true BAT, this is likely to have a net positive effect on thermogenic capacity in humans. 


As a results of this evidence, I'm adding "elevated lactate / lactic acid" to the line that's already there for exercise in the master list of BAT, beige fat and thermogenesis inducers, included below.




1Lactic acid and Lactate are basically the same molecule, except lactic acid has an extra hydrogen atom - i.e. CH3CH(OH)CO2H vs. CH3CH(OH)CO2-. I'll refer to it as "lactate" throughout the rest of this post, for simplicity, since that is how it is referenced in the research I'll discuss.



Here is the latest full list of modifiable and [nonmodifiable] factors associated with increased brown/beige adipose tissue and/or thermogenesis, with the factors mentioned in this post highlighted in red:


  • Cold exposure - by far the best BAT inducer/activator
  • Spicy / pungent foods, herbs & supplements - capsaicin / chilli peppers, curcumin / turmeric root, menthol/mint/camphor, oregano, cloves, mustard, horseradish/wasabi, garlic, onions
  • Sulforaphane-rich foods - Broccoli, brussels sprouts, cabbage
  • Arginine-rich foods - Good vegan sources include seeds (esp. sesame, sunflower & pumpkin), nuts (esp. almonds and walnuts) and legumes (esp. soy, lupin & fava beans and peas)
  • Healthy Fats - DHA / EPA / fish-oil, MUFA-rich diet,  Extra Virgin Olive Oil
  • Olive Polyphenols - Extra Virgin Olive Oil / Olive Leaf Extract / Olive Leaf Tea
  • Luteolin-rich foods - Herbs (thyme, parsley, oregano, peppermint, rosemary), hot peppers, citrus fruit, celery, beets, spinach, cruciferous veggies, olive oil, carrots. 
  • Other foods - Apples / apple peels / ursolic acid; Citrus fruit / citrus peels / limonene; Honey / chrysin
  • Beverages - green tea, roasted coffee, red wine, cacao beans / chocolate
  • Low gluten diet
  • Methionine restriction - Reduce animal protein. Soy is low in methionine and high in arginine, but also high in leucine.
  • Leucine restriction - Reduce animals protein. Leucine is highest in beef, fish, eggs, cheese and soy.
  • Low protein diet
  • Drugs / Supplements - metformin, berberine, caffeine, creatine, nicotinamide riboside (NAD), resveratrol, ginseng, cannabidiol / hemp oil / medicinal marijuana
  • Time Restricted Feeding - most calories at breakfast
  • Exercise & elevated lactate / lactic acid
  • Acupuncture - locations Zusanli (foot - ST36) and Neiting (lower leg - ST44) 
  • Whole body vibration therapy
  • Avoid obesity/overweight
  • [being naturally thin - high metabolic rate]
  • [being younger]
  • [being female]
  • [Ethnicity - having cold-climate ancestors]
  • [being of genotype TT for rs1800592 and AA for rs4994 as reported by 23andMe]



[1] Adipocyte. 2016 May 18;5(2):153-162.

Exercise regulation of adipose tissue.
Stanford KI(1), Goodyear LJ(2).
Exercise training results in adaptations to numerous organ systems and offers
protection against metabolic disorders including obesity and type 2 diabetes, and
recent reports suggest that adipose tissue may play a role in these beneficial
effects of exercise on overall health. Multiple studies have investigated the
effects of exercise training on both white adipose tissue (WAT) and brown adipose
tissue (BAT), as well as the induction of beige adipocytes. Studies from both
rodents and humans show that there are exercise training-induced changes in WAT
including decreased cell size and lipid content, and increased mitochondrial
activity. In rodents, exercise training causes an increased beiging of WAT.
Whether exercise training causes a beiging of human scWAT, as well as which
factors contribute to the exercise-induced beiging of WAT are areas of current
investigation. Studies investigating the effects of exercise training on BAT mass
and function have yielded conflicting data, and hence, is another area of
intensive investigation. This review will focus on studies aimed at elucidating
the mechanisms regulating exercise training induced-adaptations to adipose
DOI: 10.1080/21623945.2016.1191307 
PMID: 27386159
[2]  Norheim, F. et al. The effects of acute and chronic exercise on PGC-1alpha, irisin and
browning of subcutaneous adipose tissue in humans. The FEBS journal 281, 739-749,
doi:10.1111/febs.12619 (2014).
[3]  Stanford, K.I. et al. Exercise training alters subcutaneous white adipose tissue (scWAT)
from mice and humans [Abstract]. In: Proceedings of the 73rd Annual Meeting of the
American Diabetes Association; 2013 June 21-25, Chicago, IL, CT-OR14. 
[4] Diabetes. 2014 Oct;63(10):3253-65. doi: 10.2337/db13-1885. Epub 2014 May 1.
Browning of white adipose cells by intermediate metabolites: an adaptive
mechanism to alleviate redox pressure.
Carrière A(1), Jeanson Y(1), Berger-Müller S(1), André M(1), Chenouard V(1),
Arnaud E(1), Barreau C(1), Walther R(1), Galinier A(1), Wdziekonski B(2),
Villageois P(2), Louche K(3), Collas P(4), Moro C(3), Dani C(2), Villarroya F(5),
Casteilla L(6).
The presence of brown adipose tissue (BAT) in human adults opens attractive
perspectives to treat metabolic disorders. Indeed, BAT dissipates energy as heat 
via uncoupling protein (UCP)1. Brown adipocytes are located in specific deposits 
or can emerge among white fat through the so-called browning process. Although
numerous inducers have been shown to drive this process, no study has
investigated whether it could be controlled by specific metabolites. Here, we
show that lactate, an important metabolic intermediate, induces browning of
murine white adipose cells with expression of functional UCP1. Lactate-induced
browning also occurs in human cells and in vivo. Lactate controls Ucp1 expression
independently of hypoxia-inducible factor-1α and PPARα pathways but requires
active PPARγ signaling. We demonstrate that the lactate effect on Ucp1 is
mediated by intracellular redox modifications as a result of lactate transport
through monocarboxylate transporters. Further, the ketone body β-hydroxybutyrate,
another metabolite that impacts redox state, is also a strong browning inducer.
Because this redox-dependent increase in Ucp1 expression promotes an oxidative
phenotype with mitochondria, browning appears as an adaptive mechanism to
alleviate redox pressure. Our findings open new perspectives for the control of
adipose tissue browning and its physiological relevance.
© 2014 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.
DOI: 10.2337/db13-1885 
PMID: 24789919
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Lactate increases FGF21 which Turns White Fat to Beige


While reading a review article with the clever title What induces watts in WAT? I came across two studies that are quite relevant to my previous post about lactate, not to mention the topic we've discussed many times - namely the interaction of FGF21, cold exposure and BAT/Beige fat. 


The first [1] was from earlier this year. It studied both normal male C57Bl/6J mice as well as several gene knockouts (including most importantly FGF21-/- mice). The mice given free access to food and housed at the standard cool mouse temperature of 21 °C.


The first thing [1] found was confirmation of the in vitro result from PMID 24789919 discussed in my previous post - namely that culturing adipose cells in lactate dramatically upregulates UCP1 expression the fat cells from both wild type (WT) and FGF21-/- mice - indicating that FGF21 isn't strictly required for the lactate-induced browning of white fat, although as you can see there was a tendency of cells from FGF21 knockout mice to have lower levels of UCP1 than WT cells when cultured in lactate:




Nevertheless, they found that in adipose cells from WT mice, culturing them in lactate does result in rapid and dramatic increase (by 4x) in FGF21 levels:




This was obviously very nice to see, given my elevated lactate level, since as we've discussed many times before (including  here, herehere. and here), elevated FGF21 is associated with improved metabolic health and a 40% increase in mouse lifespan. FWIW, the authors of [1] determined that lactate upregulates FGF21 via p38-MAPK pathway.


The authors of [1] suggest an explanation for why elevated lactate would lead to increased FGF21:


The present study demonstrates that lactate, which is accumulated when the oxidative capacities of the cells are overwhelmed, signals to FGF21 which is a relevant activator of glucose consumption [ref] and carbohydrate and lipid oxidative processes [refs]. In addition to the up-regulation of Ucp1 expression, this constitutes an additional and different mechanism by which lactate would increase the oxidative capacity of adipocytes.


In short, elevated lactate acts as a signal that the body needs to be able to increase it's capacity for energy expenditure - either for work (i.e. physical activity / exercise) or heat generation. Boosting FGF21 is one means by which the body causes cells to increase their energy expenditure capacity.


The second study [2] also involved FGF21. It is from 2012, and I'm very surprised I haven't stumbled across it before. It found that in vivo infusion of exogenous FGF21 via an osmotic pump caused both a modest (2x) increase of UCP1 in BAT, but a 20x increase in UCP1 expression in subcutaneous WAT:


Taken together, these data demonstrate that FGF21 can induce thermogenic gene expression and augmentation of a brown fat-like phenotype in white adipose cells and tissues.


They then showed a similar increase in FGF21 and UCP1 in BAT and scWAT as a result of acute (2 days) of cold exposure in WT mice. But not only that, once scWAT cells were browned, they started synthesizing and secreting more FGF21. Here is the level of FGF21 at thermoneutral temperature (27 °F) and after 3 days of cold exposure in four different fat deposits, BAT, subcutaneous IWAT and PRWAT and visceral EWAT:




In short, cold induces FGF21 expression, which induces browning in subcutaneous fat deposits. The browned subcutaneous fat then expresses more FGF21, in a positive feedback loop. This is exactly the FGF21 positive feedback model I described with this diagram a couple weeks ago:




Here is how the authors of [2] describe it:


FGF21 has been described as an endocrine hormone with beneficial metabolic actions. We report here that adipose FGF21 is a critical paracrine/autocrine-acting component of the endogenous cold response and is required for normal adaptations to cold exposure. FGF21 expression is also necessary for the cold-induced recruitment of brown-like (beige) adipocytes in IWAT. In fact, pharmacologic doses of FGF21 are able to induce a thermogenic gene response in both BAT and thermogenically competent WAT depots... FGF21 is the only known cold-induced secreted protein that functions to increase the appearance of brown-like/brite adipocytes in WAT depots.


[W]e found that FGF21 can increase thermogenic gene expression, such as UCP1 and CIDEA, in BAT and primary classical brown adipocytes. Interestingly, however, we found that FGF21 had far more profound effects on thermogenic gene expression in specific [subcutaneous] WAT depots (IWAT and PRWAT) that are thermogenically competent. Indeed, FGF21 appeared to induce the expression of many genes associated with the function of the brown/beige adipocytes...


FGF21 not only has the ability to induce a brown-like phenotype in WAT pharmacologically, but is required for this physiological adaptation to occur during cold exposure...


When viewed in total, our data clearly show that FGF21 plays a far more important role in thermogenically competent WAT-containing tissue depots, such as inguinal subcutaneous and perirenal fat.


The beneficial affects [sic!] of FGF21 on glucose metabolism and body weight have evoked a substantial interest in FGF21 as a potential treatment for diseases such as obesity and diabetes (refs). Our data support a clear function for FGF21 in regulating chronic adaptive thermogenesis by increasing the transcription of thermogenic genes and enhancing the thermogenic capacity of the organism by “browning” white fat. It is tempting to speculate that the thermogenic effects of FGF21 on adipose tissue may underlie many of its beneficial effects observed in vivo. Thermogenesis is intimately linked with energy expenditure and may explain the weight loss effects of FGF21 in mice. Further work will be required to delineate the role of adipose tissue thermogenesis in FGF21-mediated weight loss and glycemic control, and whether FGF21 could be used in the therapy of obesity.


In short, FGF21 is well known for its ability to improve metabolic health. The authors of [2] suggest these benefits may be largely a result of FGF21's ability to turn white fat to beige. And both cold exposure [2] and elevated lactate [1] increase levels of FGF21.


I'm very curious whether anyone else has observed elevated lactate dehydrogenase (LD or LDH) in their own blood tests, and whether it correlates with increased exercise, cold exposure or both.





[1] Biochem J. 2016 Mar 15;473(6):685-92. doi: 10.1042/BJ20150808. Epub 2016 Jan 14.

Lactate induces FGF21 expression in adipocytes through a p38-MAPK pathway.
Jeanson Y(1), Ribas F(2), Galinier A(3), Arnaud E(1), Ducos M(1), André M(1),
Chenouard V(1), Villarroya F(2), Casteilla L(1), Carrière A(4).
FGF21 (fibroblast growth factor 21), first described as a main fasting-responsive
molecule in the liver, has been shown to act as a true metabolic regulator in
additional tissues, including muscle and adipose tissues. In the present study,
we found that the expression and secretion of FGF21 was very rapidly increased
following lactate exposure in adipocytes. Using different pharmacological and
knockout mice models, we demonstrated that lactate regulates Fgf21 expression
through a NADH/NAD-independent pathway, but requires active p38-MAPK (mitogen
activated protein kinase) signalling. We also demonstrated that this effect is
not restricted to lactate as additional metabolites including pyruvate and ketone
bodies also activated the FGF21 stress response. FGF21 release by adipose cells
in response to an excess of intermediate metabolites may represent a
physiological mechanism by which the sensing of environmental metabolic
conditions results in the release of FGF21 to improve metabolic adaptations.
© 2016 Authors; published by Portland Press Limited.
DOI: 10.1042/BJ20150808 
PMID: 26769382
[2] Genes Dev. 2012 Feb 1;26(3):271-81. doi: 10.1101/gad.177857.111.

FGF21 regulates PGC-1α and browning of white adipose tissues in adaptive

Fisher FM(1), Kleiner S, Douris N, Fox EC, Mepani RJ, Verdeguer F, Wu J,
Kharitonenkov A, Flier JS, Maratos-Flier E, Spiegelman BM.

Free full text: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278894/

Certain white adipose tissue (WAT) depots are readily able to convert to a
"brown-like" state with prolonged cold exposure or exposure to β-adrenergic
compounds. This process is characterized by the appearance of pockets of
uncoupling protein 1 (UCP1)-positive, multilocular adipocytes and serves to
increase the thermogenic capacity of the organism. We show here that fibroblast
growth factor 21 (FGF21) plays a physiologic role in this thermogenic recruitment
of WATs. In fact, mice deficient in FGF21 display an impaired ability to adapt to
chronic cold exposure, with diminished browning of WAT. Adipose-derived FGF21
acts in an autocrine/paracrine manner to increase expression of UCP1 and other
thermogenic genes in fat tissues. FGF21 regulates this process, at least in part,
by enhancing adipose tissue PGC-1α protein levels independently of mRNA
expression. We conclude that FGF21 acts to activate and expand the thermogenic
machinery in vivo to provide a robust defense against hypothermia.

DOI: 10.1101/gad.177857.111
PMCID: PMC3278894
PMID: 22302939

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