Showing posts with label adipocyte size. Show all posts
Showing posts with label adipocyte size. Show all posts

Monday, November 11, 2013

Get Lean & Stay Lean with Emedin, Galangin & Antibiotics. Plus: Breakfast & Morning Glucose Metabolism. Diet Once, Never Eat to Satiety Again? Adipocyte Size & NAFLD

Instead of making excuses for posting yet another "short news" collection instead of the next installment of the Athlete's Triad series, I will honestly tell you that I simply wasn't in the mood. Moreover, I have the feeling that I have already outlined what is going to work, i.e. train less, eat more and don't get all psyched up about being lean and looking good. Live your life! Against that background my gut tells me that any further details would just get you off track and back into the viscous cycle of overtraining, overdieting and overthinking why things don't work out for you by evoking the impression that as long as you take supplement X you could get away with doing a little bit 'less less' and eat a little bit 'less more'.

This would be about as counter-productive as the eternal quest for the ultimate body fat blocker or fat burner of which today's Get lean and Stay Lean Quickie does actually feature three. While the temporary use of all of them as a crutch or 'afterburner' to a reasonably planned diet and workout regimen certainly makes sense, it's not like anyone of us got fat, because he or she was "fat burner deficient". A fat burner is not an essential nutrient and only an adjunct to diet and exercise! Keep that in mind not just when you read the following short news items, but also whenever you enter a supplement store (real or on the Internet) and find a new "revolutionary fat burner" on sale -- regardless of whether it has Dr. Oz or Mr. O on the packaging it won't actively, i.e. on its own and in the absence of a dialed in nutritional regimen, make you lose body fat.
  • Cassia tora (Leguminosae) seed, yet another "next big thing" to get rid of the blubber?  (Tzeng. 2013 --) The results the scientists from the Department of Internal Medicine, at the Pao Chien Hospital in  Ping Tung City will be publishing in the January 2013 issue of Food Chemistry do at at least look intriguing.  Although - and this goes to show you that SuppVersity readers always (well "almost always" ;-) are the first know first - at least one of the active ingredients in Cassia tora, which is also known as Senna tora and is, besides its use in Ayurveda medicine, also used in Sri Lankan cousin, is an old friend: Emodin! The stuff that gives rhubarb the fat burning prowess you read about in not  too long ago, here at the Suppversity.

    CSEE  had dose dependent ameliorative effects on body weight gain and visceral body fat levels that were - ad the highest dose - identical to those of the thiazolidinedione (TZD) drug pioglitazone (Tzeng. 2013)
    After fattening them for 2 weeks with the notorious high fat diet, the Koreans assigned their now obese lab rats to groups who received either
    • Cassia seed ethanol extract (CSEE) by oral gavage, once per day for 8 week with CSEE doses of 100, 200, and 300 mg/kg in a volume of 2 ml/kg distilled water,
    • the diabetes drug pioglitazone dosed at 20mg/kg/day, or
    • a placebo, containing just the distilled water.
    Without any effects on the amount of food the animals consumed, the Cassia seed ethanol extract totally blunted the HFD induced weight gain (weight gain was identical to control group on normal chow, see figure to the right).

    In that. the highest dosage had the greatest effect on both body weight gain, as well as plasma lipid levels and epididymal WAT sizes in HFD-fed rats. These effects were probably mediated by CSEE's beneficial effect on the phosphorylation of AMP-activated protein kinase (AMPK) and its primary downstream targeting enzyme, acetyl-CoA carboxylase. In addition, the researchers found that the cassia seed extract directly increased genes that are responsible for fatty acid oxidation and down-regulated their fat synthesizing counterparts in the visceral white adipose tissue of the animals.

    Whether CSEE is going to be a go-to supplement of the future cannot be said, now. What is certain, however, is that it constitutes yet another example of a potentially highly effective natural alternative to the established pharmacological 'treatment' (or rather management) of the diabesity epidemic.
  • Obese, once and forever, unless you diet for the rest of your life? (Kirchner. 2013) -- A paper that's been published in the latest issue of the Journal of the American Diabetes Association, clearly suggests that the ravenous appetite of "reduced-obese" individuals, i.e. people who have been dieting for weeks and months to shed they weight they have accumulated over years is not (solely) psychologically induced gluttony.

    Suggested read: "Longterm 5% Calorie Restriction & Longterm Dieting Make You Fat and Insulin Resistant." (read full article)
    When Kirchner et al. put their diet-induced obese mice were on a  food restricted for 5 weeks, they did in fact reach the same body fat levels as age-matched rodents who had never received anything but the standard chow. Their  blood glucose levels normalized and their insulin sensitivity increased, but the "reduced-obese" mice also showed markedly increased fasting-induced hyperphagia. In fact, when they given ad libitum access to their beloved high fat diet, they ate like there was no tomorrow and ended up gaining weight at a much faster pace than their never-obese peers, who were likewise allowed free access to the HFD.

    And it gets even worse, as the conclusion the scientists draw based on their results says that despite the fact that "caloric restriction on a HFD provides metabolic benefits", it may actually require a previously obese dieter to continue on the path of caloric restriction (i.e. never eat to 'satiety') for the rest of his/her life!
  • Morning to evening decline in insulin response to carbs suggests breakfast is the time where your body reacts most sensitive to carbs (Saad. 2013) -- Likewise published in the latest issue of Diabetes is a study by Ahmed Saad and colleagues from the Mayo College of Medicine in Rochester and the the University of Padova in Italy, which does at first not really sound like it was revolutionary new. Two definitive advantages of the study at hand were yet that the scientists used healthy individuals as subject and gave them regular mixed meals instead of a glucose solution in order to confirm the existence and identify the characteristic features of the diurnal pattern of glucose tolerance most people take for granted.

    The implications of this study for intermittent fasting are not as clear as you may think and certainly don't imply that you must break your fast in the morning (read more about breaking the fast, here)
    Overall 20 healthy volunteers with normal fasting glucose (4.8 ± 0.1 mmol/L) and HbA1c (5.2 ± 0.0%) participated in the study. They were provided with identical mixed meals during breakfast, lunch, or dinner at 0700, 1300, and 1900 h in a random order on 3 consecutive days. Physical activity was held constant so that e.g. muscle glycogen depletion and subsequent increases in AMPK induced GLUT-4 expression  would not skew the results.

    What Saad et al. fonud was that the postprandial glucose excursion was significantly lower (P < 0.01) at breakfast than lunch and dinner. At the same time the β-Cell responsivity to glucose was higher. This means there was more insulin released per unit of glucose, than during lunch or dinner.

    The time the hepatic insulin extraction was also lower at breakfast; although the difference reached statistical significance only in comparison to the dinner condition. Since the overall meal glucose appearance did not differ between meals and that the suppression of endogenous glucose production "tended to be lower (P < 0.01) and insulin sensitivity tended to be higher (P < 0.01) at breakfast than at lunch or dinner" (Saad. 2013), it is no wonder that the spike in blood glucose was largely augmented, when the subjects consumed the standardized meal for breakfast.
  • Adipocyte size is a determinant of non-alcoholic fatty liver disease (NAFLD) risk (Petäjä. 2013) -- One thing scientists still have not really understood is how some obese people seem to be way better off than others, although their BMIs, fat and lean mass appears to be identical. In view of the latest paper by a group of researchers from Finland and Sweden on the association between the average fat cell size and the occurrence of NAFLD, it could well be that ratio of the total adipose volume to the total fat cell number, which obviously is the adipocyte size, may be providing at least another piece to the puzzle that holds the answer to this question.

    In a previos post on the yoyo effect, I already discussed some aspects of adipocyte morphology - read more
    The scientists have studied 119 non-diabetic subjects in a cross-sectional study. The participants had a median age of 39 (26-53) years, and a mean BMI of 30.0±5.7kg/m2. Subcutaneous abdominal fat cell size, as well as the total amount of liver fat were measured by proton magnetic resonance spectroscopy, intra-abdominal (IA) and abdominal subcutaneous adipose tissue (SC) volumes by magnetic resonance imaging (MRI) and an additional gene analysis yielded information about the genotype (susceptible or not susceptble to metabolic syndrome) of the individuals.

    Simply based on a multiple linear regression analysis, age, gender, BMI, the intra-abdominal to subcutaneous fat ratio and the subject's PNPLA3 genotype, the results were only able to explain 42% of the variation of the liver fat. The inclusion of the adipocyte sizes increased the predictive value by 11%, so that "21% of the known variation in liver fat could be explained by adipocyte size alone" (Petäjä. 2013) This does yet also mean that once we are up to a 90% explanation  (which is unrealistic, by the way) the adipocyte size will only be able to explain "of the known variations".
  • Antibiotic that's commonly used in animal fattening kills body fat (Szkudlarek-Mikho, 2013) -- Reserachers from the College of Medicine at the University of Toledo in Ohio have found that polyether ionophoric antibiotics including monensin, salinomycin, and narasin, which are widely used in veterinary medicine and as food additives and growth promoters in animal husbandry including poultry farming have toxic effects on adipose cells.

    Whether eating the chicken that ate antibiotics is going to make  you lean does still have to be established. Based on the results of the study at hand, it does however appear likely that eating antibiotics could - I do however doubt that they will achieve that without potentially serious side effects.
    Although previous studies suggest that salinomycin has anti-carcinogenic effects (Huczyński. 2013), the sharp increase in poultry consumption over the last decade(s) and the increased use of these "growth promoting" antibiotics by veterinaries and poultry farmers has often been suspected to be involved in the increase in metabolic and autoimmune diseases.

    At least in view of the former, i.e. metabolic diseases in general and obesity, in particular, it may therefore be surprising that the scientists from the University of Toledo discovered that the tested ionophoric antibiotics did not just inhibit the differentiation of cancer, but also that of preadipocytes into adipocytes:
    "The block of differentiation is not due to the induction of apoptosis nor the inhibition of cell proliferation. In addition, salinomycin also suppresses the transcriptional activity of the CCAAT/enhancer binding proteins and the peroxisome proliferator-activated receptor γ." (Szkudlarek-Mikho. 2013)
    Now, I would fully subscribe to the scientists suggestion that these "ionophoric antibiotics can be exploited as novel anti-obesity therapeutics", but until that has been done and we know which other cells' differentiation they may inhibit, as well, I'd strongly discourage anyone from 'supplementing' with the antibiotics from his or her poultry farmer next door. After all, you may well end up not just with less body fat, but with less brain tissue, as well... what? You don't care? Oh I see. The doctor must have inserted the cannula into your ears instead of your belly on your last liposuction, right?
  • Alpinia officinarum, a plant in the ginger family, stops fat gains in its tracks (Jung. 2013) -- Jung, Jang, Ahn and the rest of the researchers from the Korea Food Research Institute in Seongnam, report in their latest paper that an ethanol extract from Alpinia officinarum, a plant in the ginger family that's cultivated in Southeast Asia and is also known as lesser galangal, is yet another mainstay of traditional medicine with significant anti-obesity effects.

    It looks almost like ginger and works almost like ginger, but A. officinarum contains galangin, not gingerol and works via the PPAR-gamma pathway, as well. That's something gingerol doesn't do (Huang. 2013)
    Originally used throughout Asia in curries and perfumes, A. officinarum contains a dietary flavenol called galangin, which has already been shown to exert profound anti-cancer effects (Kapoor. 2013), whether it is solely responsible for the in vitro and in vivo inhibitory effects on lipid accumulation during the differentation of 3T3-L1 adipocytes is not certain, but appears to be likely.

    Via its effects on the fat synthesis and breakdown and PPAR-gamma activity the A. officinarum extract (AOE) lead to dose-dependent decreases in body weight gains of mice who were fed a high fat diet. It also reduced the visceral and liver fat deposition and partially restored the abnormally elevated insulin and leptin levels of the rodents.
    "Collectively, these results suggest that AOE prevents obesity by suppressing adipogenic and lipogenic genes. AOE has potential for use as an antiobesity therapeutic agent that can function by regulating lipid metabolism." (Jung. 2013)
    Certainly another nice find, but let's be honest, what's the real value of all this herbs? I mean yeah they work almost as effectively (in some cases even better) than pharmacological drugs, but both share a detrimental downside, that's not mentioned under "side effects" on the package insert or supplement bottle: They will only manage a problem the root course of which is the net result of a totally messed up diet.
That's it and since you've gotten the bottom line in advance and another time, just to make sure nobody can over-read it, in the last paragraph of the last news item, I just want to remind everyone that there are a couple of other interesting science news and links, for example about ...
  • the pro-carcinogenic effects of shift work and to a lesser degree constantly working at night (read),
  • the connection between high GI carbs and prostate cancer (read), or
  • the idiocy of battling the high GI carb induced decline in cognitive performance with even more sugar (read)
waiting for you on Facebook. Have a nice day and get lean and stay lean ;-)

References
  • Huang TH, Teoh AW, Lin BL, Lin DS, Roufogalis B. The role of herbal PPAR modulators in the treatment of cardiometabolic syndrome. Pharmacol Res. 2009 Sep;60(3):195-206. Epub 2009 Apr 7.
  • Huczyński A, Janczak J, Antoszczak M, Wietrzyk J, Maj E, Brzezinski B. Antiproliferative activity of salinomycin and its derivatives. Bioorg Med Chem Lett. 2013 Dec 1;22(23):7146-50.
  • Jung CH, Jang SJ, Ahn J, Gwon SY, Jeon TI, Kim TW, Ha TY. Alpinia officinarum Inhibits Adipocyte Differentiation and High-Fat Diet-Induced Obesity in Mice Through Regulation of Adipogenesis and Lipogenesis. J Med Food. 2013 Nov;15(11):959-67.
  • Kapoor S. Galangin and its emerging anti-neoplastic effects. Cytotechnology. 2013 Oct 25.
  • Kirchner H, Hofmann SM, Fischer-Rosinsky A, Hembree J, Abplanalp W, Ottaway N, Donelan E, Krishna R, Woods SC, Müller TD, Spranger J, Perez-Tilve D, Pfluger PT, Tschöp MH, Habegger KM. Caloric restriction chronically impairs metabolic programming in mice. Diabetes. 2013 Nov;61(11):2734-42. doi: 10.2337/db11-1621.
  • Petäjä EM, Sevastianova K, Hakkarainen A, Orho-Melander M, Lundbom N, Yki-Järvinen H. Adipocyte size is associated with NAFLD independent of obesity, fat distribution and PNPLA3 genotype. Obesity. 2013. Ahead of Print.
  • Saad A, Dalla Man C, Nandy DK, Levine JA, Bharucha AE, Rizza RA, Basu R, Carter RE, Cobelli C, Kudva YC, Basu A. Diurnal pattern to insulin secretion and insulin action in healthy individuals. Diabetes. 2013 Nov;61(11):2691-700.
  • Szkudlarek-Mikho M, Saunders RA, Yap SF, Ngeow YF, Chin KV. Salinomycin, A Polyether Ionophoric Antibiotic, Inhibits Adipogenesis. Biochem Biophys Res Commun. 2013 Oct 31.
  • Tzeng TF, Lu HJ, Liou SS, Chang CJ, Liu IM. Reduction of lipid accumulation in white adipose tissues by Cassia tora (Leguminosae) seed extract is associated with AMPK activation. Food Chem. 2013 Jan 15;136(2):1086-94. doi: 10.1016/j.foodchem.2013.09.017.

Wednesday, October 16, 2013

Rhein, PPAR-Gamma Antagonist from Rhubarb, Blunts Diet Induced Weight Gain, Increases Thermogenesis, Blocks Fat Storage, Improves Glucose Metabolism & More!

With 2lbs of rhubarb in the self-made syrup base, this Rhubarb Mojito (img +recipe metropochris.com) could be a real "fat loss drink"... well, if you sweetened the syrup with stevia and used it all at once with just a tiny amount of alcohol  ;-)
Allegedly, autumn it is not exactly rhubarb season, but it is the season where we usually begin to assemble our share of winter fat and that is, as you will probably understand after having read today's SuppVersity post, actually reason enough to turn it into "rhubarb" season. Chinese rhubarb, to be precise. The same rhubarb that has been used 2700BC already and is still one of the mainstays of TCM that's being hailed for its purging effects, as well as its ability to suppress feverish conditions, to cure stomach ailments and as a “cathartic” (an agent used to relieve severe constipation). According to a review by Steven foster, da-huang, as it is called by TCM practicioners, also has antibacterial effects and has been used to treat shingles, fevers, hypertension, burns, acute appendicitis, acute infectious hepatitis, conjunctivitis, swelling and pain of gums, and sores of the mouth or tongue (Foster. 2006).

Yet though many of these effects could come handy for anyone who is looking to make it through the coming winter season in good health, none of them tailors so directly to the aforementioned nasty winter fat as one of the less known "side-effects" of da-huang: its ability to suppress diet induced weight gain!

Rhein from Chinese rhubarb could keep (seasonal) weight gain at bay

According to Zhang et al. the ability of Rhein, one out of six potentially bioactive constituents of Rheum palmatum (see figure in the bluish infobox at the end of this article for a detailed analysis), which has not only recently been shown to directly inhibit the differentiation of 3T3-L1 adipocyte in vitro (Liu. 2011), has...
Figure 1: Molecular structure of Rhein (Zhang. 2013)
"[...] also been reported to have pharmacological and biochemical effects on the inhibition of liver fibrosis and insulin sensitizing and prevent hepatic steatosis through LXR inhibition in a high-fat diet-induced obese mouse model." (Zhang. 2013)
What still has to be elucidated, though, is whether these effects of Rhein, many of which have been observed in the petri dish, only, can provide protection against diet induced obesity in the "real world" (in this case first of all in the real world of rodents).

To be assess the in-vivo efficacy of Rhein as an anti-obesity supplement, the scientists from the Shanghai University of Traditional Chinese Medicine conducted an 8-week experimental trial, in the course of which two groups of obesity prone DB/DB and normal mice were fed a high fat diet (20% protein, 20% carbs, 60% fats; relative to total energy) with or without 0.1% Rhein in it and compared that to the effects of a species-appropriate low fat diet containing 20%, 70% and 10% of the total energy from proteins, carbohydrates and fats, respectively.
Figure 2: Effect of control, high fat(HF) and high fat + Rhein (HF + RH) on body weight, adipocyte size, and body temperature after cold exposure (4°C) of C57BL/6 (=normal) mice. Mice were fed a high-fat diet for 8 weeks and Rhein was powdered and mixed in the diet at 0.1% (Zhang. 2013).
I guess, even if it was not for the data in figure 2, you probably won't be surprised, when I am telling you now that the results of the 8-week dietary intervention were more than just promising for both the obesity prone, but also the normal mice (I guess, otherwise the study would not have made it into the SuppVersity news, anyways, right?). In fact the addition of no more than 3mg of Rhein /day (see blueish infobox at the end of the article for a calculation of the human equivalent dose) effectively ...
  • reduced fat weight in db/db mice from 45.2 ± 1.4 g to 40.8 ± 1.4 g, while the lean and fluid weights remained unaffected between all groups (data not shown, since less relevant)
  • blocked the weight gain and increase the energy expenditure in normal mice on a high fat diet, who had similar food intake as the control mice, but still remained as lean as the mice on the regular diet (see figure 2)
Yet despite the fact that it's always nice to have a supplement help the "geneticall disadvantaged" among our hairy friends - in other words the leptin mutants, scientists refer to as "obesity proe DB/BD mice - for you (probably no DB/DB human, right?) the weight stability of the normal mice, which was brought about at least in part by an increase in thermogenesis and uncoupling protein expression in the brown adipose (BAT) tissue of the rodents are is probably of greater relevance.

The significance of BAT and UCP-activity measures in mice remains questionable

Figure 3: There is more to the weight loss effect of Rhein than its effects on UCP expression in BAT - mRNA levels of selected hepatic genes involved in the metabolism of fatty acids (top, right), and glucose response to intravenous glucose load (bottom, left; Zhang. 2013).
That said, it is unfortunately, whether and to which extend these effects will observed in human beings. After all, the amount of brown adipose we have on our frame appears to be highly very low, and way less active that that of the critters in the study at hand.

However, even if the direct fat burning effects won't translate 1:1 or even 10:1 from mice to men, we may still be left with improved triglycerides and LDL-C levels (data not shown) and, maybe even more importantly, an improved glucose disposal (see figure 3, top); two factors that certainly won't hurt your health and/or ability to lose body fat, specifically since they come hand in hand with increases in LPL (= fat breakdown), and decreases in FAS (= lipid synthesis) in the livers of the animals that consumed the Rhein enriched chow.

And if that's not yet reason enough to look with different eyes on the allegedly somewhat sour rhubarb stalks you have certainly seen at the super, farmers, or whatever market you are shopping, its
  • antagonistic effect on the PPAR-gamma receptor (data from test with rosiglitazone in white adipose tissue not shown), by which it blocks fat storage and induces weight loss (cf. Huang. 2006; Gong. 2009), as well as the ...
  • distinct drop in hepatic fatty acid translocase CD36 activity (see figure 3, top right) that could offer at least some protection against NAFLD and subsequent insulin resistance even in the presence of a the sugar and fat overload of the standard American diet (Miquilena-Colina. 2011), 
may eventually convince you that this stuff is not so bad - in the end, some stevia will turn even the sourest rhubarb shake or stew into a nightmarishly sweet treat ;-).
"I guess eating rhubarb won't suffice, right?" Wrong!


How much do I have to take? The human equivalent of 0.1% Rhein at a daily food intake of 3g/mouse and a mean body weight of 45g (at the beginning of the study) would be ~5.4mg/kg body weight. And what's best, with a Rhein content of 0.96 mg/g and 1.12mg/g in regular raw rhubarb and Mongolian rhubarb, respectively (see figure above with data from Shang. 2003). You could theoretically get your daily dose of 300-550mg of Rhein from 300-550g of rhubarb per day. Whether your digestive tract will like that, remains to be seen, though ;-)
Apropos, rhubarb, I guess you will probably be expecting that this is another instance, where the "fat burner" may be "naturally occuring", but only in so minuscule amounts that you will have to wait for some supplement producer to read this post and come up with a "standardized extract" in a product carrying an imaginative name like RhubaLean(TM).

Now the good news is: If we assume that (a) none of the six other ingredients interferes with the effects of Rhein (in the study at hand, Zhang et al. tested whether Emodin would have similar weight loss effects - it did not; however, that does not mean that it would negate the effects of Rhein) and (b) you'd simple need the dose equivalent of the ~3mg the mice in the study consumed, an extract is not really necessary. According to my calculations (see blueish info-box to the right), approximately 300-500g of rhubarb per day would be enough!

Well, I know, that's plenty, but if we assume that the Rhein in the rhubarb stalks is not extremely susceptible to heat, mechanical processing etc., there are countless ways for you to incorporate it into your diet. And once you are fed up of rhubarb shakes, cakes, salad dressing, ice cream, etc. you could theoretically still create your own extract.

An important note of caution: Making your own or buying an extract would also have the advantage of being able to avoid the potentially toxic oxalic acid overload, you could get if (a) your rhubarb is of the high oxalic acid variety (500-750mg/100g; the lower end would be 150-250mg/100g) and you consumed so much of it that you got in the "danger zone" of >5g/day of oxalic acid. That said, most of the oxalic acid is contained in the leaves which have once been recommended as a replacement for spinach, while the edible part, i.e. the petioles of rhubarb leaves are just that, i.e. edible, because of the low oxalate content (Barceloux. 2009)

References:
  • Barceloux DG. Rhubarb and oxalosis (Rheum species). Dis Mon. 2009 Jun;55(6):403-11.
  • Foster, Steven. Desk Reference to Nature's Medicine. Washington, D.C.: National Geographic Society. 2006. 104–105.
  • Gong Z, Huang C, Sheng X, Zhang Y, Li Q, Wang MW, Peng L, Zang YQ. The role of tanshinone IIA in the treatment of obesity through peroxisome proliferator-activated receptor gamma antagonism. Endocrinology. 2009 Jan;150(1):104-13.
  • Huang C, Zhang Y, Gong Z, Sheng X, Li Z, Zhang W, Qin Y. Berberine inhibits 3T3-L1 adipocyte differentiation through the PPARgamma pathway. Biochem Biophys Res Commun. 2006 Sep 22;348(2):571-8.
  • Liu Q, Zhang XL, Tao RY, Niu YJ, Chen XG, Tian JY, Ye F. Rhein, an inhibitor of adipocyte differentiation and adipogenesis. J Asian Nat Prod Res. 2011 Aug;13(8):714-23.
  • Miquilena-Colina ME, Lima-Cabello E, Sánchez-Campos S, García-Mediavilla MV, Fernández-Bermejo M, Lozano-Rodríguez T, Vargas-Castrillón J, Buqué X, Ochoa B, Aspichueta P, González-Gallego J, García-Monzón C. Hepatic fatty acid translocase CD36 upregulation is associated with insulin resistance, hyperinsulinaemia and increased steatosis in non-alcoholic steatohepatitis and chronic hepatitis C. Gut. 2011 Oct;60(10):1394-402.
  • Shang X, Yuan Z. Determination of hydroxyanthraquinoids in Rhubarb by cyclodextrin-modified micellar electrokinetic chromatography using a mixed micellar system of sodium dodecyl sulfate and sodium cholate. J Pharm Biomed Anal. 2003 Feb 5;31(1):75-81.
  • Zhang Y, Fan S, Hu N, Gu M, Chu C, Li Y, Lu X, Huang C. Rhein Reduces Fat Weight in db/db Mouse and Prevents Diet-Induced Obesity in C57Bl/6 Mouse through the Inhibition of PPARγ Signaling. PPAR Res. 2013;2013:374936. doi: 10.1155/2013/374936. Epub 2013 Sep 25.

Wednesday, August 21, 2013

Whey or Casein, Pulse or Spread Evenly Across the Day? Does it Even Make a Difference in Terms of Fat Loss and Lean Mass Retention on a Diet? New + Old Empirical Data!

Image 1: Instinctively right? Milk contains soluble (=whey) proteins and casein. Are we overthinking things, when we rip them apart and does it even make a difference? Or is timing all that counts?
It's funny "overthinking", right next to overtraining and overdieting, has become one of the most common problems among the health and fitness enthusiasts who spend equal (or even more) time online as in the gym. "Would it be better if I take my BCAAs at a 3:1:1 or 2:1:1 ratio?", "Does it matter if my protein powder is 10% hydrosolate, 50% isolate and 40% concentrate or has a 30/50/20 ratio?" All that may well make a difference, but let's be honest: Look at the things 80% of these people are eating day in and day out and the way they throw the weights around in the gym and contrast that to a question like "Will my post-workout protein synthesis be 5% greater, when I switch from concentrates to hydrosolates?" ... enough of the ranting, though. After all this post is actually about one of the more sensible among these world-shattering questions:

Q
Will it make a difference, whether I use casein or whey protein on a diet and... what's the significance of having my daily allotment of protein spread evenly across the day vs. mostly (80%) in one sitting, when I am dieting?

In order to find the answer to this question a group of French scientists recruited 41 healthy, but chubby subjects (BMI ~32kg/m²; age ~33y) and put them on a relatively moderate caloric deficit that was calculated based on their basal energy requirement (what you would need lying around all day). In all four arms of the study, the macro-nutrient composition (25% as proteins, 25% as lipids, and 50% as carbohydrates) and energy content per pound of lean body weight (average energy intake 5.87 MJ per day) of the meals, which were prepared according to personalized menus the subjects received from trained dietitians, were identical.
Figure 1: It did not make a difference if the protein was ingested either spread equally across the day or as a pulse mostly (80%) in one sitting (top), fat and weight loss after the 6 week study period were virtually identical (data based on Adechian. 2013)
The little information on the exact menu choices the scientists offers includes a list of stable foods, such as various proportions of spinaches, broccoli, lentils, or green beans, butter, bread, fruits, soy yogurt, rice cakes and gingerbread and suggest that we are dealing with the typical "your dietitian recommends diet", here. With one exception, of course, the main protein source of all four experimental diets were dairy proteins (~80g; >80% of total protein). Casein and whey aka "milk soluble protein"* (see red box above), which were to be ingested either spread equally across or in a "pulsed" fashion (see figure 1, left):
*Note: the scientists refer to whey as "milk soluble protein, I stuck to the terminology in the graphs, but in essence these are mainly β-lactoglobulin, α-lactalbumin, as well as serum albumin, immunoglobulins, lactoferrin, and other minor fractions and thus the same you would find in your average whey concentrate which is, as you may have notices "more soluble" than casein (cf. Lacroix. 2006)
  • casein spread- subjects consumed ~20g of a casein protein supplement 4x a day
  • milk spread - subjects consumed ~20g of milk protein supplement 4x a day
  • casein pulse - subjects consumed the lions share, i.e. 80% of their ~80g of casein, as part of their 2nd meal, so that the protein intake over the day was 6.4g / 64g / 3.2g / 6.4g (see figure 1)
  • milk pulse - same as above, but with milk instead of casein protein
In view of the overemphasisze nutrient timing has gotten as of late withing the physical culture and the assumption that you would expect to see profound differences based on when you consume how much of fast or slow, high (milk) or average (casein) leucine protein etc., it may be disappointing that the weight loss was absolutely identical in all four arms of the study (-7.5 ± 0.4 kg).

Differences are few and far between: Weight loss, fat loss, muscle loss - NOT different! 

What may yet surprise even you, a seasons SuppVersity veteran, who will probably already have expected the non-significant (in fact non-existent) differences in terms of weight loss, could be surprised that the changes in body composition (see figure 1, bottom), i.e. -5.1 ± 0.2 kg reduction in body fat mass and -2.2 ± 0.2 kg reduction in lean body mass, were identical.

Since the same goes for the changes in the fat "liberating" proteins lipoprotein lipase (LPL) and adipose triacylglycerol lipase (AGTL), the fat "forming" protein fatty acid synthase (FAS), and three of the usual subjects, i.e. leptin, the adipoQ gene which is responsible for encoding adiponectin, of which recent research suggests it may be even more important than leptin for your metabolic health (Li. 2013; Hickman. 2013), and the reduction in the pro-inflammatory monocyte chemotactic protein-1  (MPC-1), the slightly more pronounced meal-induced postprandial protein synthetic response in the casein group at the end of the study period is actually the only difference based on which you could argue for one over the other protein source:
Figure 2: While the changes in LPL, AGTL, FAS, leptin, AdipoQ and MCP expression were identical (left); the post 6-week protein synthetic response to identical meals was slightly more pronounced in the casein group (right), the overall significance of this finding is yet questionable in view of identical lean mass losses - it could yet become important on a diet + exercise regimen as in the Demling study discussed in the bottom line box  (data based on Adechian. 2013).
Whether the measurable advantage of casein during this test (the evaluation was carried out by leucine tracer infusion, by the way) is just an experimental artifact or
Adherence is the key to success: While there was no difference in terms of the hunger the subjects felt when they were on the diet, the fact that only 23 of the initially 41 subjects did make it through the 6- week on ~ 1,500kcal/day is quite telling, also in view of the perceived inability to lose weight - if you can't stick to a by no means crazy caloric restriction for 6 weeks, how can you expect to get lean and stay lean, when the inevitable prerequisite for the latter is that you totally revamp your dietary habits for the rest of your life not just six, eight, or twelve weeks.
  • maybe something like "leucine resistance" in response to the higher leucine concentrations after the ingestion of the milk protein supplement in the course of the study period, or
  • alternatively, the greater IGF-1 response to casein (cf. Hoppe. 2009, a study which compares whey vs. casein, but would obviously suggest an advantage of casein over milk = whey + casein, as well); unfortunately IGF-1 wasn't measured, but the insulin levels which were minimally higher in the casein group could support that hypothesis,
... is questionable. Since the same is true for the practical relevance of the ~10-13% larger leucine balance during the postprandial phase of the post-diet whole body protein metabolism test in week 6, I would not fret about this difference too much, though.

Maybe, just maybe, the adipocyte morphology could make a difference

What I would consider significant, though it did not reach that status (probably due to the low number of participant that actually made it to the end of the study, see red box on the right), is the slight but in my eyes potentially important superiority of the equally spread protein ingestion in terms with respect to the before vs. after adipocyte diameter in the casein group:
Figure 3: The difference did not reach statistical significance, but if we take for granted that greater reductions in adopcyte sizes are associated with healthier metabolic profiles, you would be better advised to take your casein protein equally spaced across (15% reduction in adipocyte size vs. 7%, only, for pulsed casein intake) the day... for whey, aka "milk soluble protein", on the other hand it does not seem to matter (data calculated base on Adechian. 2013)
Now, even if we assume that this made a difference and a greater reduction in adipocyte size was a significant advantage, which it probably is from a health perspective, as Skurk et al. state that there is
"[...] a differential expression of pro- and antiinflammatory factors with increasing adipocyte size resulting in a shift toward dominance of proinflammatory adipokines largely as a result of a dysregulation of hypertrophic, very large cells." (Skurk. 2006)
and a recently conducted human trial, by Rizkalla et al. the main message this study should be sending out is not that it does not make a difference whether you use casein or milk protein as your main protein source on a diet, but that a high protein diet with a mediocre caloric reduction of ~20-25% and supplemented with high quality dairy protein (whey or casein) works: After all, more than -1kg of weight loss per week, 68% of the weight loss from fat in the absence of exercise is more than your average celebrity XYZ diet will do for you ;-)
Whey or casein? It's high cysteine content that can help to replenish your glutathione (=the master antioxidant) pools would be another factor that speaks in favor of whey. Whether normal-weight individuals on an already optimized dietary regimen would benefit to the same extend as the obese young men in the 6-week whey supplementation trial, Vatani et al. describe in the August issue of Appetite, is however questionable. After all, the increases in HDL the total antioxidant capacity and glutathione is as questionable as any possible negative influence of the starchy placebo the researchers used in that study (some of you may have seen the link on the SuppVersity Facebook Wall, already).
Figure 4: Fat loss and lean mass gains in formerly overweight police officers after 12 weeks of training and dieting with or without casein / whey hydrosolate (Demling. 2000)
Moreover, one of the few long-term (=non acute protein synthesis) studies investigating the differential effects of concomitant whey vs. casein hydrosolate protein supplementation, found statistically significant higher body fat reductions and lean mass gains in those 33-34 year-old police officers who supplemented their 12-week diet + strength training regimen with 2x37g of casein hydrosolate (8h apart; for the exact data see figure 4; Demling. 2000).
Note: since both the whey (Pro-Score Champion Nutrition) and the casein protein (MET-Rx USA) in this study were hydrosolates the differences in lean mass gains and fat loss are depend primarily on the amino acid composition of the proteins, and not, as it would be with micelle casein vs. whey, the absorption kinetics!
Bottom Line: Against that background the study at hand supports previous findings of the importance of a threshold intake of protein. Interestingly, it did not confirm the notion that this threshold intake should be spread equally across the day, which is something most commenters (me included) read into the seminal paper by Loenneke et al., which found a statistically significant negative correlation not between total protein intake, but between the number of meals with 10g or more essential amino acids in them and abdominal obesity (Loenneke. 2013). So, does timing matter, or does it not? 
  1. It does matter, when you work out, there is ample evidence to support that the ingestion of protein in the vicinity of the workout cannot just amplify the protein synthetic response but will also results in an increase in real world muscle gains.
  2. It appears that it does not matter, when you are dieting (only), though; not just the study at hand, but also the success many people report on intermittent fasting regimen, would support the notion that the more sustained anabolism you may be able to achieve by ingesting say 4x25g of protein instead of 1x80 + 2x10g has, compared to the total amount of protein you eat, relatively little influence on the conservation of lean body mass, when you are dieting.
And as far as the choice between casein and milk soluble protein, aka whey (see first red box), is concerned (see box on the right, as well), it would appear prudent to assume that a combination of both - just like nature intended it - would be the best choice as a "standalone" protein source (cf. "Whey and Casein Work Hand in Hand for Protein Anabolism, but Scientists Overlook Fat, When They Reassemble Milk"), while the higher leucine content and faster digestibility render whey the better candidate for classic "supplementation", as in having an additional shake before you start preparing your whole-foods post-workout meal, which should - and I hope it's not really necessary that I say that - obviously include a significant amount of protein (fish, eggs, meats, and if you will even more dairy ;-), as well. The usefulness (again, not necessarily the superiority!)  of slow digesting protein is something you should be aware of, anyway, right? If not re-read the "3.2kg of Lean Mass Over Night W/ 40g of Slow Digesting Protein 30min Before Bed!?" post from February 22, 2013.

References:
  • Adechian S, Balage M, Remond D, Migné C, Quignard-Boulange A, Marset-Baglieri A, Rousset S, Boirie Y, Gaudichon C, Dardevet D, Mosoni L. Protein feeding pattern, casein feeding or milk soluble protein feeding did not change the evolution of body composition during a short-term weight loss program. Am J Physiol Endocrinol Metab. 2013 Aug 14.
  • Demling RH, DeSanti L. Effect of a hypocaloric diet, increased protein intake and resistance training on lean mass gains and fat mass loss in overweight police officers. Ann Nutr Metab. 2000;44(1):21-9.
  • Hickman IJ, Whitehead JP. Structure, signalling and physiologic role of adiponectin - dietary and exercise-related variations. Curr Med Chem. 2013 Aug 9.
  • Hoppe C, Mølgaard C, Dalum C, Vaag A, Michaelsen KF. Differential effects of casein versus whey on fasting plasma levels of insulin, IGF-1 and IGF-1/IGFBP-3: results from a randomized 7-day supplementation study in prepubertal boys. Eur J Clin Nutr. 2009 Sep;63(9):1076-83. 
  • Lacroix M, Bos C, Léonil J, Airinei G, Luengo C, Daré S, Benamouzig R, Fouillet H, Fauquant J, Tomé D, Gaudichon C. Compared with casein or total milk protein, digestion of milk soluble proteins is too rapid to sustain the anabolic postprandial amino acid requirement. Am J Clin Nutr. 2006 Nov;84(5):1070-9.
  • Li FY, Lam KS, Xu A. Therapeutic perspectives for adiponectin: an update. Curr Med Chem. 2013 Aug 9.
  • Loenneke JP, Wilson JM, Manninen AH, Wray ME, Barnes JT, Pujol TJ. Quality protein intake is inversely related with abdominal fat. Nutr Metab (Lond). 2013 Jan 27;9(1):5. 
  • Rizkalla SW, Prifti E, Cotillard A, Pelloux V, Rouault C, Allouche R, Laromiguière M, Kong L, Darakhshan F, Massiera F, Clement K. Differential effects of macronutrient content in 2 energy-restricted diets on cardiovascular risk factors and adipose tissue cell size in moderately obese individuals: a randomized controlled trial. Am J Clin Nutr. 2013 Jan;95(1):49-63.
  • Skurk T, Alberti-Huber C, Herder C, Hauner H. Relationship between adipocyte size and adipokine expression and secretion. J Clin Endocrinol Metab. 2007 Mar;92(3):1023-33.
  • Vatani DS, Golzar FA. Changes in Antioxidant Status and Cardiovascular Risk Factors of Overweight Young Men after Six Weeks Supplementation of Whey Protein Isolate and Resistance Training. Appetite. 2013 Aug 10.

Monday, January 28, 2013

How Working Out Changes the Morphology of Your Body Fat and Why This Explains that Intensity and Explosiveness Hold the Key to Getting and Staying Lean, Fit and Healthy

Can the guy on the right run away from the bloated macrophage coated fat cells on the left? Today's first post in the SuppVersity Exercise Science Week does hold the answer to this existential question.
Those of you who don't just read, but also think about the headlines of these posts will probably already have theorized about the remote possibility that I could have changed my mind and discarded the original plan to do an "exercise quickie" early this week (see SuppVersity post from Saturday). Now, before you are getting mad at me, let me give you the (as I would say good) reason for doing that: Some, if not almost all of the exercise related studies, I have piled up here are actually too interesting to end up as part of a mash-up. That's particularly true in view of the fact that my time budget during the week does not allow me to discuss them in appropriate detail. So, even if wanted to end up writing >10,000 word posts, this would only work on 48h days ;-)

The solution I came up with is easy and in my humble opinion actually quite cool: The world's first SuppVersity Exercise Science Week! In the course of the next days, I will serve you one or two of the latest studies from the realms of exercise science per day - with the rest (if there is one) being "mashed up" in the Short News on Saturday. Cool? No? Well, bad luck, then ;-)

We will kick off the week with the modulatory effect of exercise on adipose tissue

We all know that exercise is a good way to support and control diet induced weight loss. Specifically for those of us who are already on the lean(er) side of a "divide", where obese is soon going to be the new normal (see "Obese is Going About to Become the New Normal"), it's probably even obligatory, if it's not being skinny fat that's our diet goal. That being said, a recent review of Izawa et al. provides a couple of intriguing insights and links between physical activity and the structural changes our white adipose tissue is undergoing (Izawa . 2013). In this first post of the SuppVersity Exercise Science Week I am going to summarize and expand on some of these points and point towards the implications these more or less recent, in parts pretty geeky and abstract findings have on the way we could, should, or might train:
  • Training can increase lypolysis: Compared to subcutaneous fat (SAT), visceral fat (VAT) has a higher lipid turnover. Interestingly, the same high lypolytic rates which render people with high VAT so vulnerable to high levels of blood fats, are also responsible for the comparable ease with which you can shake those unhealthy VAT depots melt away, when you're working out and/or dieting. The fat around the organs is not just the easiest to store, it's also the easiest to access and liberate (Ross. 2000). That this is not a location-specific characteristic, has already been proven. What does yet still have to be elucidated, is when and due to which complex mechanisms the genetic differences between the subcutaneous and visceral fat cells are getting programmed and whether there may be a way to reverse them.
  • Training will modulate the growth environment: The maturation of stem cells into SAT, VAT, or BAT (brown adipose tissue) cells appears to be highly dependent the specific milieu in which they are in. The latter, in turn, is characterized by the presence of  various growth factors. The best known and allegedly most important ones belong to the transforming growth  factor (TGF) family. It is their presence that will control whether a stem cell turns into a "brown" = metabollically active fat cell with thermogenic abilities or a regular white one (SAT or VAT). While BMP2- and BMP-4 are responsible for the formation of white adipocytes, BMP-7 drives brown fat cell development. Together with FGF21 another of those growth factors, these proteins determine the fate of pre-adipocytes. Unfortunately, the research on the different ways by which exercise controls these factors is yet still in its infancy. We will take a closer look at what we know already in the next paragraph.
  • As you know from the "CLA Destroys Body Fat" post, PPAR-γ downregulation is also the main pathway by which conjugated linolic acid strips mice of almost all their body fat (read more). The exercise induced upregulation of the so-called hypoxia-inducible factor (HIF-1α) appears to do the exact same thing: Suppress PPAR-γ and thus hit the off switch on body fat storage.
    Training reduces WAT size and number: According to a 2004 review of the literature it appears as if exercise training (aerobic) specifically in early life reduces the number and size of WAT in rodent (Stallknecht. 2004). These results have been confirmed in a 5-week study designed that was conducted to elicit the underlying molecular mechanisms only recently (Sakurai. 2010); and the results of the Sakurai study suggest that it is one of our old acquaintances that is to "blame": The peroxisome proliferator-activated receptor-γ (PPAR-γ), a central regulator of adipogenesis! It's inhibition by physical activity is what does the trick. Now, as a diligent student of the SuppVersity you will certainly remember that this is also the main pathway by which CLA, rhein (from rhubarb) and other weight loss adjuvants work their "magic" - a blockade of the PPAR-γ receptor is like hitting the "off switch" on the body fat storage control panel. And who is it who hits that switch? Well, according to the current research it appears that this is the prerogative of the hypoxia-inducible factor (HIF-1α) which is in turn controlled by exercise induced WNT and AMPK signaling.
  • Training improves angiogenesis in WAT: While you hear about angiogenesis, i.e. the physiological process through which new blood vessels form from pre-existing vessels, oftentimes in the context of various endothelial pathologies, its induction within the white adipose tissue is actually highly desirable. Scientists have long been speculating that the insufficient wiring of the adipose organ with blood vessels and the subsequent hypoxia are at least partly to blame for the constant inflammation in the ever-expanding fat depots of the obese (Ye. 2009). Since HIF-1α (see previous paragraph) also promotes the expression of vascular endothelial growth factors and their receptors (VEGFRs / VEGFs), its downstream activation by preferably intense physical activity should improve the endothelial wiring of the fat tissue and thus help sooth the chronic inflammation, that's at the heart of many, if not all of the key-features of the "metabolic syndrome".
  • Training increases the adipocyte size depended release of adiponectin: In the past couple of months, the adipokine adiponectin turned out to do most of the good stuff (esp. improvements in glucose and fatty acid metabolism), of which scientists previously thought it was the prerogative of leptin. It is therefore important that exercise increases the rate at which a given increase in adipocyte size (obviously in response to fat storage) will increase the release of adiponectin (Miyazaki. 2010). If we use the classic notion of the adipokine as a signal the fat cells use to tell the brain and the rest of the body how much fat remains to fuel its energetic demands, you could say: Working out allows your body to see how much fat you actually got. The physiological consequences of this revelation are increases in lypolysis and fatty oxidation, as well as overall metabolic benefits.
So far for the stuff that will make you look smart, when you parrot it in front of your gymbros. In order to not just look smart, but also be smart -- and in this case train smart -- you will yet also have to know the implications of these revelations and this is exactly what the rest of this article is going to deal with:
  • Making HIIT a Hit! learn how in the SuppVersity  Special (read more) and use it to get lean & healthy and, more importantly, stay lean and healthy!
    HIF-1α <> PPAR-γ <> adipocyte crosstalk - an(-other) argument for high intensity exercise: Since the HIF-1α response to a given training stimulus decreases once the body has adapted to the stressor by increasing its exercise capacity (Lundby. 2005). The crosstalk between HIF-1α, PPAR-γ, and your fat cells provide another reason to work out in the higher range of the VO2max continuum and to never neglect the imperative of constant progression (even if it's only a progression of 0.1km/h during your sprints on the treadmill - adaptation means stagnation, if you don't raise the bar appropriately)
  • Wnt10B response to stretching - an argument for heavy eccentrics and/or statics to revamp your body (less fat, more muscle!): The findings of Akimoto et al. point towards the existence of another rather strength-training specific contributer to the fat loss and leanness promoting effects of exercise - the stretch-induced activation of the wingless-type (WNT) MMV integration site family member WNT10b, the quasi cousin of an upstream mediator of HIF-1α (Akimoto. 2005). The activation of the WTNs does actually get down to the root of the trouble and will not just inhibit the formation of new fat cells from pre-adipocytes, it will also divert the mesenchymal and not yet specialized stem cells to turn into osteroblasts (bone) or myoblasts (muscle). In fact, research has shown that WNT signaling is a major contributer to both the recruitment of new muscle progenitor cells from the aforementioned pool of yet unspecific stem cells and skeletal muscle hypertrophy (Polesskaya. 2005; Armstrong. 2005).
  • You will also benefit from integrating plyometrics into your existing routine - build the Jack of All Traits Workout
    The greater WNT response to power vs. strength training points towards the superiority of a plyometrics to get and stay lea: In view of the results of Leal et al., who report a 3x greater WNT gene response to power compared to strength training in their 2011 paper on the effect of different resistance-training regimens on the WNT-signaling pathway, plyometrics, which have way more in common with the power training protocol in the Leal study (40% lighter weights; faster, explosive contractions) than whatever powerlifting routine you may have been thinking of, should be a superior means to stay lean (Leal. 2011)
  • Exercise restores your body's fat gauge: If you wanted to pointedly summarize the exercise induced reductions in leptin expression, the associated restoration of leptin sensitivity in the obese, and the increased adiponectin release relative to the increase in fat cell diameter, you could actually say that exercise restores your body's fat gauge. It allows your brain and the other organs to see how much body fat you still got and have them react appropriately. Funnily this is also why you body will, clever as is is, pull the emergency break, whenever your body fat levels become too low (cf. "The Athlete Triad Series").
Did you know that 10% of the fat cells have to be renewed every year? I know this is speculative and we are not talking about ZERO adipocyte maturation here, but what do you think will happen when a fat cell is due and you just hit the off-switch on adipocyte maturation?
A final word of caution: I am well aware that some of you may take this article as justification for training themselves into the ground. So please(!) keep in mind that hypoxia induced WNT10 and stretch induced WNT10b signaling, as well as most of the other fancy stuff you have learned about in the previous paragraphs are stress responses that require adequate recovery periods for the metabolic and growth responses they induce to take effect. Sleep, Eat, Train, Rest, Sleep, Eat, Train, Rest, Sleep, Eat... do you notice something? Yeah, right that's a 3/1 ratio of non-stressful occupations, namely sleeping, eating and resting to a single stressor, i.e. training. In other words, 25% of your result are "made" in the gym, 75% in bed (don't make it too stressful there ;-), in the kitchen and even, when you spend time with friends and family or simply sprawl out on the couch. Think of that, when you're designing your next training routine.

References:
  • Armstrong DD, Esser KA. Wnt/beta-catenin signaling activates growth-control genes during overload-induced skeletal muscle hypertrophy. Am J Physiol Cell Physiol. 2005 Oct;289(4):C853-9. Epub 2005 May 11.
  • Akimoto T, Ushida T, Miyaki S, Akaogi H, Tsuchiya K, Yan Z, Williams RS, Tateishi T. Mechanical stretch inhibits myoblast-to-adipocyte differentiation through Wnt signal-ing. Biochem Biophys Res Commun. 2005; 329: 381-385
  • Izawa T, Ogasawara J, Sakurai T, Nomura S, Kizaji T, Ohno H. Recent advances in the adaptations of adipose tissue to physical activity: Morphology and adipose tissue cellularity. J Phys Fitness Sports Med. 2013:1(3): 381-387. 
  • Leal ML, Lamas L, Aoki MS, Ugrinowitsch C, Ramos MS,  Tricoli V, Moriscot AS. Effect of different resistance-training regimens on the WNT-signaling pathway. Eur J Appl  Physiol. 2011; 111: 2535-2545
  • Miyazaki S, Izawa T, Ogasawara JE, Sakurai T, Nomura S, Kizaki T, Ohno H, Komabayashi T.  Effect of exercise training on adipocyte-size-dependent expression of leptin and adiponectin. Life Sci. 2010; 86: 691-698.
  • Lundby C, Gassmann M, Pilegaard H. Regular endurance training reduces the exercise induced HIF-1alpha and HIF-2alpha mRNA expression in human skeletal muscle in normoxic conditions. Eur J Appl Physiol. 2006 Mar;96(4):363-9. Epub 2005 Nov 12.
  • Polesskaya A, Seale P, Rudnicki MA. Wnt signaling induces the myogenic specification of resident CD45+ adult stem cells during muscle regeneration. Cell. 2003 Jun 27;113(7):841-52.
  • Ross R, Dagnone D, Jones PJ, Smith H, Paddags A, Hudson R, Janssen I. Reduction in obesity and related comor-bid conditions after diet-induced weight loss or exercise-induced weight loss in men. A randomized, controlled trial. Ann Intern Med. 2000; 133: 92-103.
  • Sakurai T, Endo S, Hatano D, Ogasawara J, Kizaki T, Oh-ishi S, Izawa T, Ishida H, Ohno H. Effects of exercise training on adipogenesis of stromal-vascular fraction cells in rat epididymal white adipose tissue. Acta Physiol (Oxf). 2010; 200: 325-338.
  • Stallknecht B. 2004. Influence of physical training on adipose tissue metabolism -- with special focus on effects of insulin and epinephrine. Dan Med Bull. 2004; 51: 1-33.
  • Ye J. Emerging role of adipose tissue hypoxia in obesity and insulin resistance. Int J Obes (Lond). 2009 Jan;33(1):54-66.