Is Hypoglycemia Obesogenic? Is the GI Totally Worthless? Is Mild, But Chronic Stress Behind the Diabesity Pandemic? Is Leptin Obesogenic? And How Do You Calculate the Energy Requirements of Diabetics?

One of the mistakes researchers and dieters make time and again, using the scale as their only guide

Since I had a couple of interesting, but not earth-shatteringly exciting studies on obesity, body weight gain, the GI, leptin and a couple of other things lying around, I thought I'd compile a brief potpourri for you to get you on par with the helplessness with which researchers are still facing the diabesity pandemic. So don't expect any of the one-size-fits-it-all solutions the scientists still appear to be looking for from any of the following items. What you may find, however, is some inspiration when you read between the lines or follow up on the suggested reads, I mention. And if that's not the case, you can still browse previous articles on fat loss or simply go to the gym and try the fat loss example routine from the SuppVersity "Step By Step Guide for Your Own Workout Routine" or simply go to be early to preserve your circadian rhythm.

• Going "Hypo" time and again will make you fat (McNay. 2013) -- Usually you think of hyogylcemia as a sign of a lack of energy, yet despite the fact that this may well be the case this very lack in energy has recently been shown to exert obesogenic effects in a rodent model.
  
  

  

  Often a picture says more than 1000 words: Normal (left) and repeatedly hypoglycemic rodents after 8 months of weekly insulin injections (McNay. 2013)

  The weekly injections Ewan C McNay and his colleagues administered to their rodents and the subsequent episodes of hypoglycemia lead to profound weight gain in the absence of diabetes, hyperphagia, changes in hypothalamic NPY or POMC mRNA expression and  the other usual suspects that could explain this phenomenon. The one thing that's left is therefore what the researchers call a "multi-faceted deficit in metabolic regulation" (McNay. 2013) - interestingly enough the 69.5% higher body weight at 12 months went hand in hand with the usual laziness (-25% activity) of people whose brains are starving in abundance (e.g. type II diabetics).
  
  What remains to be seen, though, is whether similar effects would occur in response to "regular" non-insulin induced hypoglycemia. In view of the easy with which crash dieters and people with roller-coaster blood glucose levels gain weight, it is yet not unlikely that it is actually the avoidance of (reactive) hypoglycemia and not so so much the prevention of hyperglycemia that makes low GI diets successful for weight maintenance (for weight loss the picture is more complicated, since this will require a energy deficit and that's a game changer).

• Dietary glycemic index and load are not associated with type II diabetes risk in 12,403 Europeans (Sluijs. 2013) -- Apropos GI, scientists from the University Medical Center in Utrecht did not find statistical significant correlations dietary glycemic index and/or glycemic load and the risk to develop type II diabetes in in a subcohort of the European Prospective Investigation into Cancer and Nutrition Study (n = 12,403 participants).
  
  Even when they compared participants in the highest and lowest quantiles, the increase in risk was only 5% and 7% for GI and GL respectively. Since this is by no means the first study that suggests that the still propagated concept of the beneficial health effects "low GI diets" is faulty, I would suggest you rather watch the actual food items, than their respective glycemic indexes if you intend to ward off obesity and diabetes. Potatoes for example may have a high GI (including sweet potatoes, by the way), but their high potassium and overall mineral content, as well as the mere fact that you can hardly eat the same amount of total carbohydrates you can easily annihilate, when you are eating pasta still makes them one of the best sources of starchy carbs you have (learn more in the Potato Manifesto, Part I & II).

• 

  Figure 1: There were no statistically significant difference in terms of weight gain or loss, but the 2-week re-feed had a greater impact on blood glucose and insulin levels in the high GI group (Lagerpusch. 2013)

  There is use for the GI on a bulk or after a diet, but only if you are concerned about insulin sensitivity (Lagerpusch. 2013) -- While the general value of the GI as a means to distinguish good from bad carbohydrate sources is certainly questionable, the recently published results from a study that was conducted at the Institute of Human Nutrition and Food Science, of the -Albrechts University in Kiel, Germany, does suggest that monitoring the GI of your diet and adding additional fiber to reduce the insulin response to your meals can come quite handy, in phases, where you are particularly prone to store body fat. On a bulk, for example, or even more so when you have been dieting and are trying to return to a normal caloric intake.
  
  According to the results Lagerpusch et al. present in the November issue of the British Journal of Nutrition even healthy young men who were subjected to a 3-week diet phase (-50% in caloric intake) and subsequent overfeeding (+50% in caloric intake) the subjects in the high GI study arm had a 135% higher increase in fasting insulin levels during the refeed than those in the low GI group. In view of the fact that the glucose clearance (measured in an oral glucose tolerance test) was identical, it is not only no wonder that the weight gain did not differ either (see figure 1), but also unlikely that we would see significant differences as far as the fat gains are concerned (the latter were unfortunately not measured in the study at hand). At the same time, longer hyper-caloric high GI diets are certainly a risk factor for both insulin resistance and obesity, so that you are probably still at lower risk with 65g instead of 27g of fibre per day and a mean GI of 40 vs. 74.
  
  If you are interested in the influence of different diets on weight gain and health during overfeeding, I suggest you check out the following two SuppVersity posts: "194 Bananas in Three Weeks" and "A Tale of Macro- and Micronutrient Modifications".

• 

  Figure 2: Chronic mild stress leads to an overactivation of the HTPA and subsequen metabolic dysregulations (Takahashi. 2013)

  Further evidence that chronic mild stress is to blame for the obesity pandemic (Takahashi. 2013) -- As researchers from the Tohoku University Graduate School of Medicine in Japan report in the latest issue of the  American Journal of Physiology - Endocrinology & Metabolism, the localized re-setting of the clock genes in the liver, yet not the hypothalamic suprachiasmatic nuculeus (SCN), of BALB/c mice in response to chronic mild stress exposure elevated and phase-shifted serum corticosterone levels (see figure 2).
  
  Takahashi et al. argue that the observed changes are indicative of an overactivation of the HPA axis, which induced disturbances in the rhythmic expressions of core clock genes, e.g. Clock, Npas2, Bmal1, Per1 and Cry1 in the liver and subsequently circadian patterns of glucose and lipid metabolism-related genes such as the proliferator activated receptor (PPAR) family which favor the storage and hamper the oxidation of fatty acids.
  
  If you want to learn more about clock genes and how you can modify them, (re-)read the SuppVersity Circadian Rhythm Series!

• Scientists develop improved formula to calculate the resting energy expenditure of diabetics (Ikeda. 2013) -- While I would hope that you don't belong to the group who would have to use the new and improved formula scientists from the Department of Diabetes and Clinical Nutrition at the Kyoto University in Japan have now proposed, you may have clients or relatives who could benefit from its high predictive validity (78% +/- 103kcal vs. 50% for Harris-Benedict; 38% for Oxford, 42% for Liuand 63% for Ganpule):
  
  
  What you should keep in mind though, is that this equation was tested on Japanese individuals. Since we know from other studies that there are certain metabolic differences between people with different ethnic backgrounds I would remain a "healthy skeptic" as far as the outcomes of this equation are concerned - the same obviously goes for any other equation, e.g. the ones for athletes I provided in part III of the Female Athlete Triad series.

• 

  If you are interested in ways to modulate your leptin levels that may facilitate weight gain, I suggest you take a look at my second "Carbs Past 6PM Won't Make You Fat" post.

  Leptin induced weight gain? 13% more body fat in 2 weeks, when it hits the wrong part of the brain (Harris. 2013) -- With the mixed results from intervention trials, the enthusiasm around leptin has abated over the past months, the general consensus is yet still that leptin and leptin resistance loom large in the metabolic dysregulation that's at the heart of the diabesity pandemic. Against that background, the results Ruth B.S: Harris presents in her latest paper in the American Journal of Physiology - Endocrinology & Metabolism are unquestionably surprising.
  
  When Harris injected twice the amount of leptin (0.6 µg leptin/day) that had previously been shown to decreased 24 food intake, body fat and lean tissue, when it was injected into the third ventricle of the hindbrain, into the fourth ventricle of her lab rats, the rodents gained an almost incredible amount of 13% body fat within only 2 weeks! And that in the absence of statistically significant change in daily food intake, suggests an "increase in efficiency of energy utilization" (Harris. 2013). Fortunately, further experiments showed that the pro-obesogenic effects of leptin in the 4th ventricle was antagonized when both the 3rd and the 4th ventricle were exposed to leptin. In this scenario the leptin exposure of the 4th ventricle did even protect the lean mass of the rodents from the negative effects the exclusive exposure of the 3rd ventricle had. Overall, the study is yet somewhat chaotic and a clearcut message aside from "look people things are even more complex than we already thought", is probably not going to contribute to a solution of the obesity dilemma in the near future. 

There are, as usual more news on Facebook, some of them, such as the relation between hypothyroidism during pregnancy and the diabetes risk of the offspring later in life, are even related to the topic at hand. And if that's nothing you are interested, you may want to read about ...

• the non-existent effects of coffee consumption before bed on the sleep quality of habitual coffee drinkers (read more),
• the problem with inaccurate vitamin D tests and the absence of a reliable and scientifically sound definition of "vitamin D deficiency" (read more), or
• the strength promoting in-vitro effects of sodium bicarbonate, or in other words, a rather alkaline milieu on muscular force production (read more)

... and if neither of those can satisfy your thirst for more information from the realms of exercise and nutrition sciences, you can still wait for the next serving of facebook news or tomorrow's SuppVersity article :-)


References:

• Harris RB. Leptin-induced increase in body fat content of rats. Am J Physiol Endocrinol Metab. 2013 Dec 4.
• Ikeda K, et al. A new equation to estimate basal energy expenditure of patients with diabetes. Clinical Nutrition. 2013 [article in press] 
• Lagerpusch M, Enderle J, Later W, Eggeling B, Pape D, Müller MJ, Bosy-Westphal A. Impact of glycaemic index and dietary fibre on insulin sensitivity during the refeeding phase of a weight cycle in young healthy men. Br J Nutr. 2013 Nov 28:1-11.
• McNay EC, Teske JA, Kotz CM, Dunn-Meynell A, Levin BE, McCrimmon RJ, Sherwin RS. Long-term, intermittent, insulin-induced hypoglycemia produces obesity without hyperphagia or insulin resistance: a model for weight gain with insulin therapy. Am J Physiol Endocrinol Metab. 2013 Nov 20.
• Sluijs I, Beulens JW, van der Schouw YT, van der A DL, Buckland G, Kuijsten A, Schulze MB, Amiano P, Ardanaz E, Balkau B, Boeing H, Gavrila D, Grote VA, Key TJ, Li K, Nilsson P, Overvad K, Palli D, Panico S, Quirós JR, Rolandsson O, Roswall N, Sacerdote C, Sánchez MJ, Sieri S, Slimani N, Spijkerman AM, Tjønneland A, Tumino R, Sharp SJ, Langenberg C, Feskens EJ, Forouhi NG, Riboli E, Wareham NJ; on behalf of the InterAct consortium. Dietary Glycemic Index, Glycemic Load, and Digestible Carbohydrate Intake Are Not Associated with Risk of Type 2 Diabetes in Eight European Countries. J Nutr. 2013 Nov 28.
• Takahashi K, Yamada T, Tsukita S, Kaneko K, Shirai Y, Munakata Y, Ishigaki Y, Imai J, Uno K, Hasegawa Y, Sawada S, Oka Y, Katagiri H. Chronic mild stress alters circadian expressions of molecular clock genes in the liver. Am J Physiol Endocrinol Metab. 2013 Dec 4.

Chromium Picolinate Worsens Insulin Sensitivity in Healthy, Non-Diabetic, Non-Obese Individuals by Up to 25%

The more supplements you take the more likely you are to get way more than the 200mcg of chromium of which previous studies have shown that they are useless for healthy people, but at least not detrimental (cf. Lukaski 1996 & 2007; Vincent. 2007). Especially people who like the  'poly-supplementary' approach are yet at risk of getting so much of a this trace mineral that it will hamper not improve their insulin sensitivity.

I don't have to tell you that you would already be dead if you were following all the bro-scientific advice that's out there on the Internet and still I usually recognize a certain reluctance to give up on what X suggests and Y has tried an what has worked so well for Z. One of the instances, where I have hitherto been missing a 100% convincing argument to argue that this is just another instance where common wisdom would in fact be better called "common stupidity" is the "insulin mimetic" or "insulin sensitizer" (or whatever your favorite bro-expert may call it) chromium picolinate. With the recent publication of the result of a study on the effects of chromium supplementation in healthy individuals there is now finally a human study that confirms that chromium, which has never been an "insulin sensitizer", but rather an "insulin release amplifier" that reduced blood glucose in diabetics by simply having them produce even more insulin is not a supplement any healthy man or woman, let alone athlete should consider a staple of his or her regimen.

The long and short: Chromium hampers insulin sensitivity in normoglycemic individuals

For their experiment lead author Umesh Masharani and his colleagues from the UCSA recruited a group of 27 non-obese, non-diabetic, healthy subjects between the ages of 20 and 50 with a body mass index of less than 27 kg/m² and <24 kg/m² for subjects with Asian heritage (the cut-off limits were set so that they would be below a BMI that has not yet been shown to be an independent risk factor for insulin resistance; cf. Clausen. 1996; Newell-Morris. 1998).

To evaluate whether chromium picolinate (ChrPic) supplements, which contributed with $150,000,000 to the revenue of the supplement industry in 1996 (Nielsen. 1996), could come up to the claims that they would exert beneficial effects on glucose tolerance and insulin sensitivity, the study participants were randomized to take either a placebo or a high dose 500µg CrPic supplement twice daily for 4 months (the dosage was selected in view of previews studies reporting greater benefits of 1,000 vs. 200mcg of CrPic in - you already guessed it - diabetic subjects; cf. Morris. 2000).

Figure 1: Insulin sensitivity measured by euglycemic clamp before and after the 16 week intervention (left); change in insulin sensitivity of the individual subjects plotted against serum chromium levels at the end of the study (Masharani. 2013)

As the data in figure 1 goes to show, the results of the CrPic intervention were more or less the exact opposite of what the ~10 million US consumers of respective supplements probably expect from the pills many of them are taking almost religiously. Despite the fact that all subjects had very low chromium levels at the beginning of the study, the previously non-significant minimally benificial relation between both serum and urinary chromium, on the one hand, and insulin sensitivity (r = 0.24, p=0.1; r=0.08, p=0.79 respectively), on the other hand, had turned into a very significant negative correlation between high(er) urinary and serum chromium concentrations and lower insulin sensitivity at the end of the 16 week intervention period (figure 1, right).

"Due to the apparent variation in the degree of chromium absorption between subjects, we examined the relationship between serum chromium and change in insulin resistance. After controlling for baseline patient characteristics, results of a multiple regression analysis showed a strong association between serum chromium and worsening of insulin–mediated glucose disposal (β= -0.83, p<0.01), where subjects with the highest serum chromium had a decline in their insulin sensitivity. To further explore the association between chromium absorption and insulin resistance, patients within the chromium group were divided (based on  a medial split at 3.10 µg/L) into a high (n=6) and low (n=8) serum chromium group [...] There were no group differences at baseline; however, at post-assessment participants in the high serum chromium group (> 3.1  µg/L) were more insulin resistant than participants in the low serum chromium group (≤3.1  µg/L) or the placebo group (p=0.02, p=0.05 respectively) (Figure 3)." (my emphases in Masharani. 2013)

Due to the fact that the scientists did not observe any differences between the placebo and low serum  chromium groups (on a side note, contrary to many other studies insulin Masharani et al. measured the insulin sensitivity in a very reliable way with an euglycemic hyperinsulinemic clamp; cf. Defronzo. 1979), the scientists also conducted a post-hoc analysis to identify potential confounding factors that may have influenced the outcome of the trial. Neither changes in triglycerides levels LDL, BMI, or truncal fat were yet associated with the differences they observed between the supplemented and non-supplemented participants. Interactions that would reduce the significance of the observed correlations were likewise absent:

"Furthermore, when changes in triglycerides, LDL, BMI, and truncal fat were individually added to the model, none were independent significant predictors of change in insulin sensitivity, and chromium absorption remained a significant predictor of reduced insulin sensitivity in each model." (Masharani. 2013)

Against that background the scientists conclude that there must be a "direct effect of chromium on changes in insulin action". A mechanism, by the way, which is totally independent of classic markers of insulin resistance such as high serum lipids and abdominal / truncal adiposity .

Being healthy is a good predictor of increased chromium absorption and more pronounced negative effects, so if you are healthy and want to stay this way don't even think of taking high dose chromium supplements.

Despite the fact that the changes in insulin resistance did not depend on changes in serum lipids and other markers of metabolic health, Masharani and his colleagues were able to show that the increase in chromium levels in response to supplementation did. With the already mentioned statistically significant correlation between increases in serum chromium levels (higher response to supplementation = higher increase), on the one hand, and the worsening of insulin sensitivity, on the other hand, this means that the healthiest subjects, namely ...

"[...] subjects with lower triglycerides, and those with lower levels of homocysteine [who had] a greater likelihood of being in the high absorption group" (Masharani. 2013)

... were at the same time those who were at the greatest risk of the ill side-effects high dose chromium supplements exert on the insulin tolerance of healthy, non-diabetic, normal-weight individuals.

No matter if it may have helped you produce insulin back in your obese days, once you have accomplished this you better avoid high dose or multiple (hidden) sources of supplemental chromium like a plague - unless you can't afford new jeans, of course ;-)

Bottom line: Unless you are not a type II diabetic or feel the urgent desire to become one, you better steer clear of exuberant amounts of supplemental chromium the RDA is enough. This is particularly true, if you are already taking a multi (which is almost guaranteed to have 200mcg in it), or any BB-style supplements. After all, "broscience" wants it that chromium is in everything that's even remotely related to insulin / nutrient uptake or whatever. With the use of only one of these products and 200mcg of supplemental dietary chromium per day, you may still argue that it probably won't do much harm. When you add another 200mcg from your "nutrient partitioner" on top of the 200mcg you get from your multi and the 200mcg of which you probably did not even realize yet that they are part of your pre-workout supplement, however, you can hardly complain about simply not being able to tolerate carbohydrates - I mean, what's your body supposed to do if you are dumb enough to believe in the promises of fat loss and lean mass increases that have been debunked in the late 1990s, already (cf. Lukaski 1996 & 2007; Vincent. 2007), and simply chose to ignore the latest scientific evidence that chromium picolinate supplements are not just useless, but actually detrimental to your health?


References:

• Clausen JO, Borch-Johnsen K, Ibsen H, Bergman RN, Hougaard P, Winther K, Pedersen O. Insulin sensitivity index, acute insulin response, and glucose effectiveness in a population-based sample of 380 young healthy Caucasians. Analysis of  the impact of gender, body fat, physical fitness, and life-style factors.  J Clin Invest. 1996;  98(5):1195– 1209.
• Defronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol. 1979; 237:E214–E223. 
• Lukaski HC, Bolonchuk WW, Siders WA, Milne DB. Chromium supplementation and resistance training: effects on body composition, strength, and trace element status of men. Am J Clin Nutr. 1996 Jun;63(6):954-65.
• Lukaski HC, Siders WA, Penland JG.  Chromium picolinate supplementation in women: effects on body weight, composition, and iron status. Nutrition. 2007; 23(3):187– 195.
• Masharani U, Gjerde C, McCoy S, Maddux BA, Hessler D, Goldfine ID, Youngren JF. Chromium supplementation in non-obese non-diabetic subjects is associated with a decline in insulin sensitivity. BMC Endocr Disord. 2013 Nov 30;12(1):31.
• Morris BW, Kouta S, Robinson R, MacNeil S, Heller S. Chromium supplementation improves insulin resistance in patients with Type 2 diabetes mellitus.  DiabetMed. 2000; 17(9):684–685.
• Newell-Morris LL, Treder RP, Shuman WP, Fujimoto WY. Fatness, fat distribution, and glucose tolerance in second-generation Japanese-American (Nisei) men. Am J Clin Nutr. 1989; 50(1):9–18.
• Nielsen FH. Controversial Chromium: Does the superstar minearal of the mountebanks receive appropriate attention from clinicians and nutritionists?  Nutr Today. 1996; 31(6):226–233.
• Vincent JB: The nutritional biochemistry of chromium (III). Amsterdam, Boston: Elsevier. 2007.

Probiotics + Green Tea - Synergistic Superstack or Sciency Non-Sense? Green Tea Alone Totally Blunts HFD Induced Weight Gain, L. Plantarum Does Not Add to Its Effects

L. plantarum may metabolize green tea phenols, but don't add to their anti-diabesity effects 

Green tea has actually never seized being all the rage and probiotics are the sexy new kid on the block right around the corner of the supplement shops and and science laboratories of the western hemisphere. Against that background I guess that the title of a paper that's been published ahead of print on Monday will probably suffice to catch your interest: "Green tea powder and Lactobacillus plantarum affect gut microbiota, lipid metabolism and inflammation in high-fat fed C57BL/6J mice." (Axling. 2013) - and that despite the fact that "mice are no little men" ;-)

'1 + 1 =4' the synergism of green tea and probiotics could make it possible

I guess, the idea sounds logic: Take one thing that has been proven to ameliorate diet induced obesity, namely green tea, and combine that with another one, of which it appears as if it would also exhibit beneficial effects into an even more potent stack. In fact, the scientists' rationale was yet slightly different:

"The species Lactobacillus plantarum (L. plantarum) has the ability to metabolize phenolic acids  and to split up tannins. The metabolites are presumably more easily absorbed and distributed into the tissues where they can act as antioxidants and electron scavengers. Phenolic compounds can also have antimicrobial effects that may affect the composition of the gut microbiota, in favour of polyphenol-metabolizing components of the microbiota. Also, green tea extracts have been shown to selectively inhibit the growth of pathogenic bacteria while either enhancing or not affecting the growth of beneficial bacteria like lactic acid bacteria. To the best of our knowledge, the impact of green tea powder as a prebiotic compound to promote lactobacilli or other health promoting components of the microbiota has not previously been evaluated."

In other words, the expected benefits of providing both green tea and probiotics in conjunction were (1) an increased bioavailability of the phenols and tannins from the green tea that would be induced by the probiotics and (2) an increase in the probiotics' survival and ability to modify the gut microbiome that would be brought about by the addition of the green tea.

What looks good on paper does not necessarily work out in a complex organism

Figure 1: Ingredient total amount of Flavan-3-ol, Phenolic acid and Flavenol in water and methanol extracts from the green tea leaves that have been used in the study (Axling. 2013); as you can see the total quantity and the ratios of the bioactive ingredients of the extract actually depend on the extraction method.

Apropos green tea, you can see the exact ingredient profile of the green tea supplement that has been used in the study at hand in figure 1. In view of the fact that the C57BL/6J mice received no extract, but simply powdered green tea leaves, it may not be important in this context, but could be relevant for your future purchases that methanol and water extracts differ not only in terms of the total amount of Flavan-3-ol, Phenolic acid and Flavenol they contain, but also with respect to the ratio of the respective phytochemicals. I guess, those of you who have been around in September 2011, already, will remember that I have discussed the impact these ostensibly negligible differences can have more than a year ago in "-20% Reduction in Serum Testosterone by 5 Cups of Green Tea. Endocrine Effects Depend on Catechin Composition". In case you are one of the many newcomers or have simply forgotten (let alone missed ;-) this post, I suggest you go back and read that up, as it may help you get a better understanding of the underlying reasons due to which quality and quantity of the health effects of green tea (supplements) wary from study to study... but let's now get back to the experimental setup of the Axling study.

Green tea alone already blunts HFD induced weight gain

As mentioned before the extracts were simply mixed with the high fat diet, the mice were consuming in the course of the 22 week study period. With the probiotic supplement that was administered with the drinking water (L. plantarum at 1.5% (v/v) or roughly 3 × 10^9 cfu/ml) we are thus dealing with four different groups:

• Control: High fat chow + no supplement
• LP: High fat chow + L. plantarum
• GT: High fat chow + green tea
• GT + LP: High fat chow + green tea + L. plantarum

If you focus solely on the initially quoted hypothesis about the synergistic effects of green tea + L. plantarum, the actual study outcomes - at least as far as the blood markers in figure 2 are concerned  - are certainly disappointing.

Figure 2: Glucose insulin, fructosamine, cholesterol, triacylglycerol, non-esterified fatty acids and adiponectin levels in the blood of the mice in week 11 and week 22 of the study (Axling. 2013)

It's not like '1+1 would equal 4', but rather like '1 + 1' would just be sufficient to yield '1' not just '0.9' or even less. The in fact, the addition of the probiotics, alone, did very little within the first 11 weeks as far as it's ability to th reduce the diet-induced insulin resistance is concerned and it's addition to the green tea supplement did not improve blood glucose and lipid management, but did in fact diminish the impressive effects the green tea supplement brought about.

Figure 3: Relative change (compared to control) in bacterial diversity and lactobacilli count in response to the supplement regimen (Axling. 2013)

That the probiotic was basically useless, is actually no wonder if you take a closer look at the changes of gut microbiome in figure 3. Aside from an intermediate increase in lactobacilli, it could not boost the amount of these supposedly healthy bacteria in the long term. Rather than that it did induce an allegedly statistically non-significant decrease in the overall diversity (figure 3, left).

Minor differences with quasi-nonexistent real-world effects

At the mRNA level, the addition of L. planatrum counter-acted the anti-obesity effects of green tea, as evidenced by

Figure 4: Body weight and fat levels of the mice (Axling. 2013)

• 20% higher fatty acid synthase levels, an enzyme that's responsible for the synthesis of fatty acid
• the reversal of the statistically significant reduction in acetyl-CoA caroxylase (ACC), an enzyme that's one step ahead of FAS in the cascade of which you could say that it supplies the raw material for fatty acid synthesis, and
• minimally higher PPAR-gamma levels (responsible for fat storage) 

in the LP + GT vs. GT group, respectively. The net effects on body weight and fat mass, on the other hand were negligible. In essence the bulk of the beneficial effects of the green tea extract remained intact. Moreover, the addition of L. plantarum did have two distinct effects, that were not observed in the GT only group:

Figure 5: Liver cholesterol and HMG-CoA-R after 11 (top) and 12 (bottom) weeks (Axling. 2013)

1. a statistically non-significant -20% reduction in the mRNA expression of the inflammatory marker TNF-alpha, and
2. a whopping and surprising increase in HMG-CoA reductase of +50% and +70% increase in HMG-CoA reductase mRNA compared to the green tee only and the control group, respectively

And while there is nothing in the study that would suggest that there were any beneficial effects from the TNF-alpha reduction, the increase in HMG-CoA reductase is in fact an oddity. After all, despite statistically significant increases in the enzyme that's responsible for the synthesis of cholesterol and the main target of statin drugs (Stancu. 2001), the cholesterol levels dropped by 64% and 39% compared to the control group, in weeks 11 and 22, respectively.

What do these Jerusalem artichokes, agave, bananas, burdock, camas, chicory, coneflower, costus, dandelion, elecampane, garlic,jicama, Leopard's-bane, mugwort, onion, wild yams, yacon and a whole host of other foods have in common? Right! They contain inulin. which has only recently been shown to have the ability to ameliorate body weight gains by up to 50%! Intriguing? Go back to my previous post and learn more about inulin, beta-glucans and their anti-diabesity effects.

Bottom line: A non-statistically significant reduction in TNF-alpha and an elevation of cholesterol synthesis in the presence of lower liver cholesterol levels, which would be suggestive of an increased excretion of cholesterol (thus the increased synthesis to come up for the loss), are in my humble opinion nothing that would render the combination of green tea + L. plantarum superior to the provision of green tea alone. The latter on the other hand, appears to be a great tool to keep the damage of the energy-dense Western diet in check - with no added, let alone synergistic benefit of these particular probiotic.

Maybe the provision of another probiotic or even another strain of L. plantaris would yield at least '1 + 1' results. This would yet be a research question for another study (one I would by the way not be willing to finance ;-) and does not change the fact that the original research hypothesis that there would be a potentiating effect due to the synergism of the two supplements is - even if the scientists don't openly acknowledge that - debunked for L. plantaris DSM 15313 and green tea.

References:

• Axling U, Olsson C, Xu J, Fernandez C, Larsson S, Ström K, Ahrné S, Holm C, Molin G, Berger K. Green tea powder and Lactobacillus plantarum affect gut microbiota, lipid metabolism and inflammation in high-fat fed C57BL/6J mice. Nutr Metab (Lond). 2013 Nov 26;9(1):105.
• Stancu C, Sima A. Statins: mechanism of action and effects. J Cell Mol Med. 2001 Oct-Dec;5(4):378-87.

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.

A Higher Intake of CLA and Vaccenic Acid from Dairy, Beef, Veal and Lamp Could Prevent Subtle Weight Gain in Healthy Middle-Aged Individuals. Is 1.5g/day the Magic Number?

A dairy cow: Does her stomach hold the key to a leaner, healthier life or are CLA and vaccenic acid, the ruminant trans-fatty acids just as bad as their grainy cousins?

There are supplements that work and supplements that don't work and then there are those supplements, where nobody can actually tell, whether they belong to the former or the latter category. Conjugated linoleic acid, the ruminant omega-6 trans-fat you will find at particularly high concentrations in milk and meat products from grassfed dairy, unquestionably belongs to the latter category. While we do actually have plenty of in parts almost unsettlingly impressive rodent data (e.g. "CLA Destroys Body Fat & Increases Endurance! But at Which Costs?"), the outcomes of independent  controlled human studies are equivocal; with results ranging from "total failure", to "promising, but not half as impressive as we have expected based on previous rodent studies".

That being said, I was quite intrigued, when I hit onto a recently published study that takes a novel angle on the whole CLA for weight loss issue. One I usually don't like, as it involves a lot of statistical shenanigan, but still appears appropriate in this particular case, where the controlled small scale trials are failing us.

The Nordic Men (and women) love their full-fat dairy - rightly so?

If you are a loyal reader of the SuppVersity, who does not just read the detailed elaborations here on www.suppversity.com, but is also following the latest short news on the SuppVersity Facebook Wall, it probably won't surprise you that the study which is going to be published in the October issue of the European Journal of Clinical Nutrition has been conducted in Northern Europe. After all, you will have noticed that many of the interesting short news items relating to (larger scale) studies on the effects of one or another of the "bad fats" are conducted at universities and research centers in Sweden, Finland, Norway and, as in this case, Denmark - at the Aarhus University, to be precise, where Hansen and his colleagues datasets from the Diet, Cancer and Health study from December 1993 to May 1997. The participants, 160,725 men and women, aged 50–64 years, who were all born in Denmark and had been living in the greater Aarhus or Copenhagen areas, had all completed detailed food frequency questionnaire (FFQ) and a self-administered lifestyle questionnaire, before they underwent a physical examination and a follow up 5-6 years later.

How did the scientists know how much CLA and vaccinic acid the individual food items contained? unfortunately, they didn't. The way by which they calculated / estimated it,  i.e, by combining the content of r-TFAs in milk fat (data based on another Danish study) with the content of milk fat in dairy products given by the Danish food composition tables and using the values of r-TFA content in ruminant meat products representative of the supply in Denmark, does however make sense to me. The resulting averages should therefore be relatively reliable.

Based on the 77 food items of the food frequency questionnaires which contained ruminant trans-fatty acids R-TFA (this includes both CLA, as well as vaccinic acid which can be converted to CLA in the human body; cf. Turpeinen. 2002), i.e.

• dairy products (n=63), 
• ruminant meat products (beef, veal or lamb) (n=2), and
• composite recipes containing both dairy and ruminant meat (n=12) 

Hansen et al. calculated the average r-TFA intake of each of the 57053 subjects with complete datasets and correlated them with the participants changes in body weight and waist circumference (WC) over the 5-year period to the follow-up.

A massive amount of data suggest minimal amounts of r-TFA are necessary

As the subheading to this paragraph already reveals, the result of the all this statistical shenanigan suggest that the ruminant trans-fatty acid intake from foods, not supplements, does have a beneficial effect on the change in total body weight (an ameliorating effect on weight gain, to be precise).

Figure 1: Absolute intake of ruminant R-TFA (in g/day) and changes in weight; adjustment for sex, age, height, baseline weight, smoking, alcohol intake, education, weighted intake of foods containing high amounts of I-TFA (g/day) and in women, menopausal status and hormone replacement therapy (Hansen. 2013).

A brief glance at the graphs in figure 1 will yet also tell you that their effect on body fatness (as indicated by changes in visceral adipose tissue), is negligible, not to say non-existent. In a way you may say that this is a good thing, because the turning point at a daily r-TFA intake of >1.5g/day, where the restricted cubic spline (that's a statistical fit into the data; figure 1, solid lines) seems to indicate that r-TFA intakes of more than 1.5g/day would precipitate weight gain, is thus absent as well.

Figure 2: Relative intake of R-TFA (in % of total energy intake) and changes in waist circumference. Solid lines: restricted cubic spline with five knots; Dashed lines: 95% confidence interval; same adjustments as in figure 1(Hansen. 2013)

In addition, if we do also consider total energy consumption and the contribution of r-TFAs to the latter (see figure 2), it becomes obvious that we cannot neglect the profound widening of the 95% confidence interval in figure 1 (dashed lines), which tells us that some of the high r-TFA consumers did get even leaner, while others did gain a significant amount of weight. Adjusted for caloric intake and the other confounding variables this effect vanishes and a trend towards lower / even no body weight gain in high r-TFA consumers becomes visible (even within the higher intakes, where the confidence interval widens, due to the lower number of participants, but does not change the general trend). The beneficial effect on waist circumference, however, remains negligible.

So what, if anything, can we learn from these results?

At first sight, the results of the study at hand seem to stand in line with what you have read in "Fat Advantage: 61% Lower Rates of Metabolic Syndrome in High Fat Dairy Lovers", here at the SuppVersity exactly one week ago. It even appears to provide a mechanism by which the high fat dairy products could exert their highly desirable anti-obesity effects, if the high CLA + vaccenic acid (r-TFAs) consumers in the Hansen study were not just the subjects who gained the least weight (measured against their nutrient intake and adjusted for all sort of other confounding factors), but also those with the lowest increase in visceral adipose tissue.  

If that were the case, however, the graph on the right hand side of figure 2 should have at least some kind of slope. Since it hasn't, we must assume that the beneficial health effects of r-TFAs are either (a) not brought about by changes in visceral obesity, (b) the latter are not appropriately quantified by simply measuring the waist circumference or (c) in view of the fact that we are not talking about "weight loss", but rather a prevention of the (partly probably age induced) increase in weight gain a stable waist circumference has to be considered a "success", already.

The scale is an unreliable tool to judge visceral obesity. And even a measuring tape can be misleading, if you are really "skinny fat".

Personally, I tend towards a combination of all three. First of all even moderate weight gain has been shown to increase the risk of impeding metabolic syndrome. The weight stability over >5years in the high r-TFA consumers must therefore be considered to be prognostic of a lower risk of metabolic syndrome. Secondly, visceral does not necessarily equal abdominal fat. Especially in older individuals the gynoid fat areas contribute to visceral obesity, as well. Moreover, we all know the skinny fats, men and women with a relatively large amount of highly inflammatory visceral fat and normal or even low waist circumferences also known as "normal weight obese"; cf. Romero-Corral. 2010).

And thirdly and most importantly: Weight loss is mainly an issue for people who are already overweight or obese. For best-agers who are still in form (and in Denmark there are such people ;-), success is better defined by maintaining the muscle mass you have, not accumulating additional (visceral) body fat and leading an overall healthy lifestyle. That ruminant trans-fatty acids can, maybe even should be a part of the dietary side of this healthy life-style is therefore the main take home message of this study.

What should not be forgotten, however, is the fact that this study was at least in parts supported by the Danish Dairy Research Foundation, certainly not an organization with a particular interest in "bad news" on vaccenic acid, conjugated linoleic acid and dairy products in general, right?


References:

• Hansen CP, Berentzen TL, Halkjær J, Tjønneland A, Sørensen TI, Overvad K, Jakobsen MU. Intake of ruminant trans fatty acids and changes in body weight and waist circumference. Eur J Clin Nutr. 2013 Oct;66(10):1104-9. doi: 10.1038/ejcn.2013.87.
• Romero-Corral A, Somers VK, Sierra-Johnson J, Korenfeld Y, Boarin S, Korinek J, Jensen MD, Parati G, Lopez-Jimenez F. Normal weight obesity: a risk factor for cardiometabolic dysregulation and cardiovascular mortality. Eur Heart J. 2010 Mar;31(6):737-46.
• Turpeinen AM, Mutanen M, Aro A, Salminen I, Basu S, Palmquist DL et al. Bioconversion of vaccenic acid to conjugated linoleic acid in humans.Am J Clin Nutr2002;76: 504–510.

Leucine + Resveratrol - Synergistic Sirtuin Boosters: +118% Fatty Acid Oxidation, 60% Increase In Muscular Glucose Uptake, -30% Visceral Fat & More - To Good to be True?

Image 1: Can you really team up leucine (or HMB) and resveratrol to make tired mitochondria get a move on? NuSirt Sciences says "YES!" And in the dish and rodents it's actually already working.

What happens if you marry a well-known AMPK promoter and exercise mimetic, with an even more prominent exercise adjuvant and nutritional mTOR booster? Will they neutralize each other? Think about it.... ok, now gimme your answer: What happens if you put resveratrol and leucine together? At first it does not really make sense, does it? Right, it doesn't, at least not unless you follow the same train of thought, the researchers from NuSirt Sciences. NuSirt? That rings a bell, hah? Yeah those were the guys who did a study on their 250mg leucine + 30mg vitamin B6 proprietary blend NuFit (see "Testosterone - 12% Drop /W 75g Glucose? Fat Loss - Adzuki, Leucine + B6 or HiMaize & More") and actually, the leucine + resveratrol combination is sort of a spin-off of this initial research.

If you put Sirt1 & Sirt1 together, it suddenly makes sense!

In their latest study (and you bet a future product!) Bruckbauer et al. build on their previous research on the agonistic effects HMB, alpha-KIC or leucine have on skeletal muscle Sirt-1 activity (Bruckbauer. 2011) and rationalize that it seems legit to combine one Sirtuin portein promoter with another one in order to achieve an even more pronounced effect - makes sense, right? Resveratrol the proven AMPK-promoter and igniter of the longevity, gene transcription, cell survival and apoptosis regulating Sir2 proteins (=sirtuins) and leucine the mTOR promoting and, as of late, proven Sirt1 agonist, they could actually form a synergistic duo for fat oxidation, glucose management, the reduction of oxidative stress and inflammation and even longevity!

Figure 1: Effects on sirtuin & AMPK expression in muscle and fat cells upon incubation with leucine, HMB and resveratrol and the respective combinations (left) and effects fatty acid oxidation in isolated rat skeletal muscle upon incubation in low and high glucose conditions (data based on Bruckbauer. 2013)

Now, aside from Sirt1, which is mainly expressed in the nucleus of a cell, another one of the Sir2 proteins, Sirt3, which is expressed predominantly in the mitochondria has as of late gathered quite some attention, as mitochondrial dys- or malfunction is one, if not the common denominator of many of the pathological features of the metabolic and neuro-endocrine ailments the Western diabesity society is suffering from: insulin resistance, type II diabetes, Alzheimer's , you name them! No wonder the NuSirt guys (and girls) are striving to find a marketable way to set them both in full gear and if you take a closer look at the data in figure 1 their initially counter-intuitive approach to bath muscle and fat cells in resveratrol  + HMB / leucine solutions yields impressive results:

• resveratrol, leucine and HMB, alone, exerted only weak independent effects on Sirt1, Sirt 3 and AMPK
• resveratrol and leucine or HMB, combined, yielded Sirt1 and Sirt3 activity increases in the ~50% range (p < 0.05) and AMPK increases of +42% and +55% (p < 0.03); particularly noteworthy are the ~125-175% increases (p < 0.02) muscle cells (remember: Sirt3 is expressed in the mitochondria!)
• the ensuing increases in fatty acid oxidation in incubated muscle cells reached statistical significance in the presence of low (5 mM) glucose levels, only, when and 5 µM HMB or  0.5 mM leucine were co-incubated with 200 nM (~18%; p < 0.05), in the high glucose condition, however, all treatments broad about significant increases in fatty acid oxidation, of which those in the leucine- and HMB-resveratrol combination treatments were the most pronounced (118% and 91% stimulation, respectively; p < 0.005)

Especially the last finding, i.e. the increase in fatty acid oxidation in an in-vitro condition that resembles the hyperglycemic state the average type II diabetic who is not popping tons of metformin and/or injecting insulin is constantly in, makes these results particularly interesting, as it appears as if a "non-pharmacological" (what by the way is "pharmacological" and what isn't?) solution to the diabesity problem could already be hidden on the shelves of your GNC right next door (I assume they carry leucine and resveratrol products ;-)!

Outside of the box... ahh, I mean, ... the petri dish!

In view of the fact that 75% of the in-vitro high performers suck in the rodent model already and of those another 75% don't work in human trials you will be pleased to hear that NuScirt Sciences' resveratrol + leucine / HMB combination has already overcome the first of these hurdles: At least in DIO (diet-induced-obese) rodents who on a 6-week high fat diet regimen, the combination works.

Figure 2: Weight gain, visceral adipose volume, PET measured palmitate uptake, respiratory rate (lower levels = higher relative fat oxidation), heat production relative to body weight, food intake; all value expressed relative to DIO mice who were maintained on an unsupplemented control diet (data based on Bruckbauer. 2013)

Now, it's not as if the rodents would have made it to the Mr Olympia stage, but if you take a closer look at the pattern that's emerging here, it's quite clear that the sirtuin booster does its job in this rodent model. Aside from its ameliorative effect on weight gain, the combination of resveratrol and leucine, led to statistically significant improvements in glucose management and improvements in inflammatory markers (including the anti-inflammatory adipokine adiponectin, see figure 2).

Figure 2: Glucose, insulin and HOMA IR levels, muscular glucose uptake (left), C-reactive protein , IL-6, MCP-1 and adiponectin (right) ; all value expressed relative to DIO mice who were maintained on an unsupplemented control diet (data based on Bruckbauer. 2013)

Most importantly, however it effectively cut through the exuberant amount of visceral adipose tissue (>30% reduction), ramped up the palmitate (fatty acid) uptake, oxidation and heat production (=thermogenesis). Despite all these metabolic improvements which took place in the absence of a simple reduction in food intake, there are still a couple of things left to be desired:

What are the human equivalent doses, here? Since I know you would be asking I did the math for you and you will be pleasantly surprised (HED for 80kg humans)

• 12.5mg resv. = 9mg
• 225mg resv. = 136mg
• 2g HMB = 1.1-1.4g
• 10g HMB = 7.2g
• 24g leucine = 14.3g

I am well aware that it must look as if I had the typical poor arithmetic abilities of the average physicist who has totally forgotten how to calculate using figures instead of letters, but the reason for the discrepancies is that I calculated the exact HEDs based on body weight and food intake for each of the groups.

1. Supplementation with the respective human equivalent doses should yield the same astonishing results in humans as it did in the diet-induced obese mice.
2. The protocol should have effects not just in morbidly obese diabetic human beings, but also in overweight and ideally even lean individuals.
3. The supp must work if you don't put it into the chow, but pop it in separate doses (e.g. 3x/day) as a capsule or tablet.

The good news however is that if 1-3 apply, you could start benefiting from this "super supplement" right now! After all, the resveratrol dose of 12.5mg per kilogram of chow (the mice in the study did not consume more than max. 4g(!) per day) is so low that the 10g package I just saw for 20$ over at the webshop of a major bulk supplier would last you literally forever ...

Unfortunately, this is exactly why I don't believe that LeuResSirt, or whatever other stupid name the final product will be given, is going to work - I mean, come on, you can't tell me that there are not already people out there who get 15-20g of leucine everyday and pop resveratrol in 100x the necessary dose of 8-9mg everyday!? And did they turn into a beast, become fast-food resistant or lose fat magically? What? Yeah... that must be Phil Heath secret, right... how come I did not realize that before? ;-)

Bottom line: Regardless of the probably justified skepticism, I will still keep you posted on whether or not NuSirt knocks out another incredible (in the literal sense) human study like the one on NuFit (see "Testosterone - 12% Drop /W 75g Glucose? Fat Loss - Adzuki, Leucine + B6 or HiMaize & More"). So stay tuned, you all know that no supplement will ever more ergogenic than your daily dose of SuppVersity news!

References:

• Bruckbauer A, Zemel MB. Effects of dairy consumption on SIRT1 and mitochondrial biogenesis in adipocytes and muscle cells. Nutr Metab (Lond). 2011 Dec 20;8:91.
• Bruckbauer A, Zemel MB, Thorpe T, Akula MR, Stuckey AC, Osborne D, Martin EB, Kennel S, Wall JS. Synergistic effects of leucine and resveratrol on insulin sensitivity and fat metabolism in adipocytes and mice. Nutr Metab (Lond). 2013 Aug 22;9(1):77.

Tangeritin, Natural Metformin from the Rind of Mandarin Oranges Hits the OFF-Switch on Diet Induced Obesity

Image 1: Tell me the truth! How much "natural metformin" did you throw away with those mandarine rinds in your life? Update (08/11/2013) Scroll down to the red box on dosing, I added, to check out why you better start saving your mandarine rinds now to be able to do a "tangeritine cycle" in a couple of years ;-)

I guess it is about time for another longer post on the "Supp" part of SuppVersity and what would be better suited than providing my readers from inside the business to spice up their #1 selling product which has as of late lost yet two other "all natural" ingredients in the never-ending arsenal of funky herbs, spices, -amines and -ephrines. Luckily the supplies nature has in stock are endless and with 5, 6, 7, 8, 40-pentamethoxyflavone (I am not kidding, now that 1,3 DMAA is gone, you will have to remember a couple more numbers ;-) the "next big" thing that's ready to get out of the starting blocks could actually prove to be more than just a stim and an actual weight loss adjuvant! After all, the AMPK boosting effects of tangeretin, which is abundant in the rinds of citrus fruits, such as mandarin orange, rutaceae and yuhu in Korea, appear to be everything but "ordinary" and they are not even the most interesting effect this compound may have on your metabolism.

Tangeritin, is not be be confused with citrus aurantium, is no stim and could thus actually work!

In a paper that has recently been published in the Journal of Molecular and Cellular Endocrinology Kim et al. report that the administration of of 200mg/kg (human equivalent 1.3g) of tangeritin per day to mice on a high fat diet did not only slow down weight gain and the development of glucose intolerance and hypercholesterolemia, but also had beneficial modulatory effects on the secretion of the adipokines adiponectin, leptin and resistin, as well as the release of IL-6 and MCP-1.

Figure 1: Adipocytokine expression and body weight gain of mice after 27 and 55 days on control, high fat (HFD) or high fat diet with 200mg/kg tangeritin (HFD + 200Tan; left) and in vitro glucose uptake of isolated myocytes upon incubation with tangeritin at doses of 25, 50 and 100mMol (right; data adapted from Kim. 2013)

For Kim et al. the profound effects wich lower leptin, aidponectin, resistin and IL-6 expression in the HFD + Tangeritin group than in the control (I wish we had the body fat levels, I am curious if those were not lower with HFD + tangeritin than in the control group, as well) did yet not come as a surprise.

Additional figure: How much rind (in g) do you need to produce 1g of tangeritin by water extraction at different temperatures and extraction times (data calculated based on Xu. 2013)

Update (08/11/2013): In response to questions both on Facebook as well as in the comment section I have compiled the graph on the right, which shows you how much rind (in grams) you need if you wanted to water-extract your 1g daily dose of tangeritin from Satsuma mandarin, which is probably the type of mandarin that is most widely sold here in the West and the more exotic, but tangeritin-rich Ponkan. If you take a closer look at the data from Xu et al. you will yet have to acknowledge that it is probably not feasible to produce your own extract at home - even if you you managed to extract ALL the tangeritin from the Ponkan, which obviously won't work by simply cooking it up, you would still need 232g of rind per day!

In previous in vitro experiments on isolated muscle cells (C2C12 myotubes), the researchers had already established that tangeritin, when it is appropriately dosed (!), exerts extraordinary powerful effects on the expression of the energy sensor and metabolic switch AMPK, which has as of late gotten quite some attention in  the laypress as the target for "the exercise pill", the "ultimate antiobesity + antidiabetes pill", the "anticancer and longevity pill",... well basically everything that could make a respective drug a pharmacological "bockboster" (read more about AMPK in the SuppVersity Intermittent Thougths Series: "The AMPK/mTOR Seesaw".

Figure 2: Hand in hand with the beneficial effects on adipocytokine production, the addition of tangerine to the high fat diet also ameliorated the growth and necrosis of adipocytes (Kim. 2013)

They also knew from previous studies that aside from its role as an inducer of AMPK and GLUT-4 expression in the muscle tissue, tangeretin posses anti-cancer, anti-oxidant and anti-inflammatory properties, as well (Yoon. 2011; Xu. 2008). Kim et al. also point out that

In addition to its anti-oxidant effects, tangeretin has been reported to inhibit the growth of hepatocytes both in vitro and in vivo via inhibition of mTOR/p70S6 kinase (Cheng. 2011). Regarding tangeretin-mediated effects on neuronal disease, a number of studies have shown that tangeretin reduces dopaminergic neurotoxin-induced neuronal injury and prevents tunicamycin-induced cell death in mice through an increase in glucose-regulated protein (GRP)78 and heme oxygenase (HO)-1 expression in renal tubular epithelium (Takano. 2007).

Moreover, another recent study by Choi et al. reports that tangeritin also has protective effects against the lipopolysaccharide (LPS) induced nitric oxide (NO) expression in the digestive tract and could thus offer acute and chronic protection from LPS induced cell damage (Choi. 2007).

Awesome? Well, in a way it may be, but in the end most of what we see is a result of pushing the right switches and in this regards tangeritin appears to be outstandingly good - for a supplement to say the least; that you don't need exercise in a pill if you hit the gym three to five times a week and lead an active life is something I don't have to tell you anyway, right?

Figure 3: Unlike thiazolidinedione like Rosiglitazone and "harmless" health supplements as fish oil (Neschen. 2006) tangeritin does not work its antihyperlipidemic (=triglyceride lowering) and anti-hyperglycemic (=blood sugar lowering) effects effects by inducing PPAR-gamma and thus allowing your body to stash away even more energy as body fat. On the contrary, reduces ppar-gamma expression and consequently stops the expansion of adipose tissue even in the presence of a hypercaloric high fat diet (cf. figure 1, tissue samples) - the beneficial effects of PPAR-gamma blockade have only recently been investigated by Lohdi et al. who call it an "off switch" for diet induced obesity (Lohdi. 2013).

Implications: Now, we are certainly not dealing with the "supplemental reinvention of the wheel here", and still: The effect size in the Tan200 group was so pronounced that I felt that the news on tangeritin, which had originally been part of last weeks "On Short Notice" deserved it's own post - not the least, because  it will not have you pay dearly for the improved glucose tolerance and lower blood lipid levels as thiazolidinediones like Rosiglitazone and even fish oil, both of which have been shown to reduce serum glucose and triglyceride levels via PAR-gamma dependent mechanisms (figure 3; specifically for fish oil Neschen. 2006) and thusly promote not block fat loss. Tangeritin, on the other hand decreases the PPAR-gamma activity and therefore acts as an "off-switch" for dietary induced obesity (Lohdi. 2013) .

Moreover, the reduced obesity was not a mere side effect of a loss of appetite and subsequent anorexia. The animals in the HFD and the HFD + Tan200 group consumed the exact same amount of chow - with the one small but absolutely critical difference that the rodents in the Tan200 group did not store the superfluous energy as body fat. It is this "anti-fat" effect which is not borne by negative health effect as it is the case for conjugated linoleic acid (CLA), for example (cf. "CLA Destroys Body Fat") which makes tangeretin a very interesting candidate for a weight loss supplement for both obese and lean dieters and, when I come to think about it, probably even for people who want to bulk.

There are however a few potential downsides and questions that remain to be answered before we celebrate the advent of yet another "next big thing" that will then line up with the rest of the supplemental non-starters:

• First of all, the mechanism of action does remind me of alpha lipoic acid (ALA), which is still a very good supplement for obese or at least insulin resistant dieters, but turned out to be useless, even potentially counterproductive in exactly those lean selectively (muscle) insulin sensitive athletes it is currently heavily marketed to as "repartitioning agent" (see "Lean and Muscular With Alpha Lipoic Acid?"). So the question is: "Is tangeritin only another ALA?" Is it better or worse and will it work fr lean people as well as it did for the mice on the obesogenic diet in the study at hand?
• Secondly, despite the fact that tangeritin acts in an almost metformin-esque fashion via AMPK and PGC-1alpha, which is much more likely to work in humans as well than your usual beta-3 agonist (e.g. synephrine) or other thermogenic with promising rodent data and absolute no effects in human beings, we still need controlled human trials to see whether or not this nquestionably promising flavenoid does work in humans at all.
• And thirdly, in view of the fact that only the high dose tangeritin (100mMol) elicited a pronounced increase in glucose uptake in the in-vitro study (cf. figure 1), it will be of utmost importance that future supplements or pharmacological agents are appropriately dosed - and we all know that this is in 90% of the cases where the raw material is more expensive than caffeine anhydrous simply not the case. 

That said, I would venture the guess that (assuming one of the smaller supplement companies finds a bulk supplier in China) we are going to see "Tange(R)iburn" or a tangerine based substrate repartitioning agent (after all those sell pretty well, too) in the near future - before anyone has a clue what dosages will be necessary to see beneficial effects and probably with way less than the 1g+ of tangeritin in it than Kim et al.'s results would suggest as a minimal daily dose for an adult to see similarly outstanding results as our hairy friends in the study at hand.

References

• Cheng Z, Surichan S, Ruparelia K, Arroo R, Boarder MR. Tangeretin and its metabolite 4'-hydroxytetramethoxyflavone attenuate EGF-stimulated cell cycle progression in hepatocytes; role of inhibition at the level of mTOR/p70S6K. Br J Pharmacol. 2011 Apr;162(8):1781-91.
• Choi SY, Ko HC, Ko SY, Hwang JH. Correlation between flavonoid content and the NO production inhibitory activity of peel extracts from various citrus fruits. Biol. Pharm. Bull. Choi, S.Y., Ko, H.C., Ko, S.Y., Hwang, J.H., 2007. Correlation between flavonoid content and the NO production inhibitory activity of peel extracts from various citrus fruits. Biol. Pharm. Bull. 30, 772–778.; 30, 772–778. 
• Lodhi IJ, Yin L, Jensen-Urstad AP, Funai K, Coleman T, Baird JH, El Ramahi MK, Razani B, Song H, Fu-Hsu F, Turk J, Semenkovich CF. Inhibiting Adipose Tissue Lipogenesis Reprograms Thermogenesis and PPARγ Activation to Decrease Diet-Induced Obesity. Cell Metab. 2013 Aug 1.
• Neschen S, Morino K, Rossbacher JC, Pongratz RL, Cline GW, Sono S, Gillum M, Shulman GI. Fish oil regulates adiponectin secretion by a peroxisome proliferator-activated receptor-gamma-dependent mechanism in mice. Diabetes. 2006 Apr;55(4):924-8. 
• Kim MS, Hur HJ, Kwon DY, Hwang JT. Tangeretin stimulates glucose uptake via regulation of AMPK signaling pathways in C2C12 myotubes and improves glucose tolerance in high-fat diet-induced obese mice. Mol Cell Endocrinol. 2013 Jul 6;358(1):127-34. 
• Takano K, Tabata Y, Kitao Y, Murakami R, Suzuki H, Yamada M, Iinuma M, Yoneda Y, Ogawa S, Hori O. Methoxyflavones protect cells against endoplasmic reticulum stress and neurotoxin. Am J Physiol Cell Physiol. 2007 Jan;292(1):C353-61.
• Xu HG, Chen CJ, Liu DH, Minerals, phenolic compounds, and antioxidant capacity of citrus peel extract by hot water. J. Food Sci. 2008; 73, C11–C18. 
• Yoon JH, Lim TG, Lee KM. Tangeretin reduces ultraviolet B (UVB)-induced cyclooxygenase-2 expression in mouse epidermal cells by blocking mitogen-activated protein kinase (MAPK) activation and reactive oxygen species (ROS) generation. J. Agric. Food Chem. 2011; 59, 222–228