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. 2012) - 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. 2012); 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. 2012)

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. 2012)

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. 2012)

• 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. 2012)

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). 2012 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.

High Fat vs. High Carb and How the Tiny Word "Synthetic" May Turn Out to Be an Epic Game Changer That Puts a HUGE Question Mark Behind "If It Fits Your Macros"

Even if both "Fit Your Macros" you should know better than to select the "fruit" loops (where is the "fruit" in those loops anyway?).

A war is raging! Well, at least in the Internet the "battle" between the low-carb revolutionists and the low-fat veterans does sometimes in fact resemble an epic battle between god and evil... at least that's what the combatants believe.

As of late it seems as if the rebels, i.e. the low-carbers were getting the upper hand and that despite the constant supplies the low fat veterans are getting from their friends in the medical establishment - friends who pay for studies the veterans to agrue how stupid and off base the rebels' assumption that "low carbing" would help us to solve the obesity epidemic actually actually were.

Don't worry this post is not a war epic ;-)

I know, up to this point, all this may not have sounded much like a SuppVersity article, but this will change now that we are about to take a look at a soon-to-be-published paper by Bérengère Benoit et al. from the Lyon University (Benoit. 2013).

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. Whether you will be able to get a whopping amount of 10% inulin in your diet w/out the use of supplements or "enriched" foods, is yet as questionable as how beneficial this actually is for friends of physical culture (read more)

The French researchers started out with the hypothesis that the metabolic effects of a diet does not simply depend on its macro-nutrient composition. Instead, they made the SuppVersity standard assumption that the food quality is about as important as food quantity and assumed that negative effect of one of the standardized synthetic high fat rodent diets (sy-HFD) consumption would be

"[...]impacted by the choice of the control diet using 2 control diets that are typically used in HFD-induced obesity protocols. The specific research objectives were to test the impact of diets on typical parameters studied in HFD-induced obesity protocols: body weight gain, fat accumulation in tissues, alteration of insulin sensitivity, markers of inflammation, and markers of plasma endotoxemia." (Benoit. 2013)

And this means? Well, think about it as if you wanted to analyze the quality of the shiny new iPhone. Obviously, it will be all the shinier the trashier your baseline comparison is - right?

The French scientists assumed something very similar may apply for the high fat diets. Depending on whether you compare them to a "healthy" or a trashy diet, the outcome of your study and the message "Low carb is..." will be different. The same obviously goes the other way around. If you have a "high carb" diet that's all fructose and nothing else, your outcome will be different and you will draw different conclusions and may make diametrically opposed recommendation.

Ok, I got to be honest with you...

... this is not - at least not yet - the study that compares what you probably understand when I say "low carb" to what you probably think of, when I say "low fat". So this is not something like 25/5/70 (protein, carbs, fats) vs. 25/70/5 (protein carbs, fats). Rather than that, Benoit et al. used those "high fat" diets you see in 99% of the corresponding rodent studies. Diets that are high in both carbohydrates (45%) and fats (40%) - values expressed in weight units - and would thus imho qualify as "low protein" diets.

Figure 1: Differential effects of synthetic low fat (sy-LFD) and high fat diet (sy-HFD) on lean mass, fat mass, insulin, triglycerides, cholesterol and the amount of free fatty acids in the blood of the rodents; all data expressed relative to the corresponding values of the rodents in the regular chow group (Benoit. 2013)

What the researchers did though was to give their analysis a twist by including not one, but two control diets. One containing a synthetic and one containing natural low fat chow - both of them with identical macro-nutrient composition (18, 70, 12% - protein, carbs, fats), but with the former, the synthetic low carb chow being based on the same artificial (in this case this just means that the combination of nutrients was artificial and would not occur like that in nature) ingredients as the synthetic high fat diet.

---

This is hilarious, right? Identical macro make, up totally different results? Yep, that's right and it should be reason enough for us to look into the differences. So let's see what we've got

• 

  According to Wood et al. (1988) black pepper contains between 3-8% piperine. This means that 4 teaspoons of it should help you combat abdominal fat (-16% on cornstarch diet in rodent study) - at least according to these previously reported results.

  Barley, wheat, corn, those are the main carbohydrate sources in the regular low fat chow. In the synthetic diet, however it's corn starch, succrose and a tiny bit of lactose.
• Soybean (not oil, but whole!) and fatty fish solubles, those are the main fat sources in the natural diet. The synthetic diet on the other hand contains soy bean oil, lard and milk fat.
• Wheat bran that's the major source of fiber in the natural diet. It's synthetic counter-part on the other hand features pure wood aka cellulose. 
• Inert proteins, i.e. those which are already present in the plant material in the regular chow diet are the only protein sources in the regular diet. The synthetic diet on the other hand features casein of which numerous previous studies have shown that it appears to promote the mass (lean and fat) accrual in rodents.

It is thus quite obvious: Hitting your macros is not all that counts. It's not all that counts for rodents and you can bet that it ain't all that counts for human beings.

So what's the real take home message then?

• From a science perspective: At least the rodent-part of the (hi-)story about the good and bad effects of low fat and/or low carb diets does have to be rewritten, now that we know that we have been fooling ourselves with figures that are worthless unless you know exactly what (which foods) they represent. Ever since the late 1990s we know for example that a "natural" high fat diets with a high amount of whole eggs will delay (not accelerate) the natural decline in insulin sensitivity and that in rats (Berdanier. 1998)!
  
  So, have the synthetic rodent diets fooled us? Probably yes. And what about the synthetic Standard American Diet? Well, it may not have fooled, but certainly killed us - at least several millions of us.
   
• From a real world perspective: Actually I already said, it, but I guess I will simply repeat that quality counts and macros are nice numbers with little meaning without at least a broad guideline (like: "Eat whole foods only!") on where you get them from.

Personally, I am curious whether anyone will actually take the results of this study at heart and finally throw the synthetic rodent chow away... you don't think so? Well, me neither.

References: 

• Benout B, et al. High-fat diet action on adiposity, inflammation, and insulin sensitivity depends on the control low-fat diet. Nutrition Research. 2013 [epub ahead of print]
• Berdanier CD, Kras KM, Wickwire K, Hall DG. Whole-egg diet delays the age-related impaired glucose tolerance of BHE/Cdb rats. Proc Soc Exp Biol Med. 1998 Oct;219(1):28-36.
• Wood AB, Barrow ML, James DJ.  Piperine determination in pepper (Piper nigrum L.) and its oleoresins - a reversed-phase high-performance liquid chromatographic method. Flavour Fragr. J., 3: 55–64.

Inulin & Beta Glucan Reduce Body Fat Gain By -50% & -33%! Both Have Similar Effects on the Gut Microbiome, But Only Inulin Appears to Be More Than An Appetite Suppressant

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. Whether you will be able to get a whopping amount of 10% inulin in your diet w/out the use of supplements or "enriched" foods, is yet as questionable as how beneficial this actually is for friends of physical culture.

The gut microbiome is not just one of the hottest topics in the (health-)blogosphere, it is also a subject of ongoing research. Research, however, that is, if we are honest, still very much in its infancy. As impressive as the results from the latest studies into the metabolic downstream effects of the administration of fermentable fiber to rodents may be and as obvious as their relation to certain changes in the gut microbiome of the animals may appear - in the end, our understanding of the underlying mechanisms does not allow any reliable prognoses like "double the amount of lactobacilli and you will eventually be able to lose that pouch of body fat you've been carrying around for years now". And yet, if the results from the latest rodent experiments at the Imperial College in London, could be reproduced in humans, I can already foresee that both, the consumption and use of the foods I listed in the caption of the image to the right, as well as related products, extracts and supplements, which contain more or less significant amounts of the naturally occurring polysaccharides, we usually refer to as inulin, will increase in the months and years to come.

Fermentable fiber and the gut-brain-axes: The key to lifelong leanness?

If this is not your first visit to the SuppVersity, you will certainly be aware that the idea of a magic pill (or fiber) that will allow you to eat whatever, whenever and in whichever amounts without having to cope with the metabolic consequences is illusive. When the addition of 10% inulin (or beta glucan) to the diets of 36 male C57BL/6 mice had an "anti-obesogenic" effect, this does not mean that the poor critters who were kept on a hypercaloric high fat (41.8%) diet for 8 weeks did not get obese. What it does mean, though, is that the addition of 10% fermentable (=being food for certain gut bacteria) fiber in the form of

*the producers of these products did not fund or support the study (at least the scientists don't mention that in the respective disclosure ;-)

• inulin from Synergy(TM)*, a fructan based preparation containing both long and short chain
  fructooligosaccharides, or
• beta-glucan from Glucagel(TM)* a highly rich (,80%) barley derived b-glucan preparation

to their otherwise iso-caloric diet (the HFD control contained cellulose) was not without helped to mitigate the negative effects of this diet - a fact the majority of you, of whom I would expect that they are not on a fast-food diet should keep in mind, before they head over to their favorite online supplement vendor and type "Synergy inulin" into the search box.

Figure 1: Effect of addition of 10% fermentable fiber as inulin or beta glucan to the high fat diet of male mice on cumulative weight gain (left), body composition and fatness (middle) and food intake (right) over the course of 8 weeks (data adapted from Arora. 2012)

In spite of that, the results are simply too impressive not to think about their implications in otherwise healthy and even more so previously obese individuals. This is particularly true, because the same microbial changes about which the authors write in a previously published paper from May 2012 that the ...

"[...] increases in both Bifidobacteria and Lactobacillius and a significant increase in short chain fatty acids (SCFA) [went hand in hand with] increase in neuronal activation within the arcuate nucleus (ARC) of animals that received In [inulin] supplementation" (Anastasovska. 2012)

do not (and this is a result of the researchers very latest experiments) simply blunt the rodents appetite. If that was the case, the rodents that received the beta glucan supplemented chow and consumed 12% less energy should have had the most favorable body composition. A cursory glance at figure 1 will yet tell you that this was not the case, though.

Inulin beats beta glucan when it comes to body fat reduction / repression

If we take a closer look a the differential effects of inulin and beta glucan, there yet only one figure that really sticks out and that's the accumulation of fat within the musculature of the animals. The "beautiful marbling" people are looking for in their steaks, however, usually is a harbinger of impeding or even existing skeletal muscle insulin resistance. A muscle fat content above the high fat control (it's certainly a weakness that we don't have a "real" control group on standard rodent chow, here) as Arora et al. observed it in the tissue samples of the beta glucan group, does thus tell you something about its potential usefulness, or rather uselessness of this specific type of fermentable fiber.

Figure 2: Effects of the different types of fermentable fiber on cecal microflora groups (figures are in scientific notation, this means "1E+6" equals 1mio, "1E+9" would be 1 billion etc.; data based on Arora. 2012)

In conjunction with the information about the corresponding changes in the gut microbiome (see figure 2), which appear virtually identical in both groups (specifically the extreme increasesin in both Bifidobacteria (BIF) and Lactobacillius (LAB) really stick out), this does however suggest that the modulatory effect on the composition of the gut flora, or at least the part of it the scientists evaluated in the study at hand, cannot be the only driving force behind the beneficial metabolic effects of inulin.

Inulin or beta glucan? This is not a question... 

While the latter, i.e. inulin, which has by the way been found to directly suppress lipogenesis in a 2011 study by Belgian scientists in a similar HFD rodent model (Dewulf. 2011), appears to be promising for everyone, regardless of whether he or she is poisoning him- or herself with the standard American diet (which is, with its high fat and high carbohydrate content de facto an identical twin of the so-called "high fat diet" in rodent studies), the ingestion of larger amounts of the former, i.e. beta glucan, does at least appear questionable.

If you want to use inulin to your metabolic advantage, you better make sure you get your self a more comfortable place to answer the call of nature - it could call thrice as often! Moreover, large amounts of inulin and other fermentable fiber can induce gastrointestinal distress-

The question is therefore not so much whether it's worth supplementing (it's certainly worth to incorporate some of the initially mentioned foods into your diet, as most of them contain a whole list of other advantageous micronutrients) with inulin or beta glucan - the answer would obviously be inulin - but rather whether it's worth adding larger amounts of inulin to an already healthy diet. And while we cannot answer this question based on the results of the previously cited rodent studies, we could argue that Marwa Zenhom and her colleagues from the Christian Albrecht University in Kiel have already supplied relevant evidence that this would be the case (Zenhom. 2011). After all, the German researchers have been able to show that the PPAR-gamma related anti-inflammatory effects (significant reductions IL-12 secretion in Caco-2 cells and gene expression of IL-12p35, IL-8, and TNFa as well as NF-kB) of oligosaccharides are not (exclusively) brought about by their effects on the gut microbiome, because bacteria simply were not present in their in-vitro study with human Caco-2 cells (cells from the gut lining). Bassaganya-Riera et al. even argue that this effect could be beneficial for IBS patients (Bassaganya-Riera. 2011).

Whether having 10% of your diet in form of inulin, or to make this more conceivable, having 1 tablespoon of plain inulin for every 9 tablespoons of whatever else you eat is either feasible or reasonable, is a whole different story (to put that into perspective: The average inulin intake of Westerners is 1-10g per day (van Loo. 1995). Even 10g would yet only be enough if you ate only 100g of food within 24h!)... and I must forewarn you, if you go by the fecal volume of the mice in the Arora study, it is possible that you will spend >3x more time on the toilette than usual ;-)

References:

• Arora T, Loo RL, Anastasovska J, Gibson GR, Tuohy KM, Sharma RK, Swann JR, Deaville ER, Sleeth ML, Thomas EL, Holmes E, Bell JD, Frost G. Differential effects of two fermentable carbohydrates on central appetite regulation and body composition. PLoS One. 2012;7(8):e43263.
• Anastasovska J, Arora T, Sanchez Canon GJ, Parkinson JR, Touhy K, Gibson GR, Nadkarni NA, So PW, Goldstone AP, Thomas EL, Hankir MK, Van Loo J, Modi N, Bell JD, Frost G. Fermentable carbohydrate alters hypothalamic neuronal activity and protects against the obesogenic environment. Obesity (Silver Spring). 2012 May;20(5):1016-23.
• Astegiano M, Pellicano R, Terzi E, Simondi D, Rizzetto M. Treatment of irritable bowel syndrome. A case control experience. Minerva Gastroenterol Dietol. 2006 Dec;52(4):359-63.
• Bassaganya-Riera J, DiGuardo M, Viladomiu M, de Horna A, Sanchez S, Einerhand AW, Sanders L, Hontecillas R. Soluble fibers and resistant starch ameliorate disease activity in interleukin-10-deficient mice with inflammatory bowel disease. J Nutr. 2011 Jul;141(7):1318-25.
• Dewulf EM, Cani PD, Neyrinck AM, Possemiers S, Van Holle A, Muccioli GG, Deldicque L, Bindels LB, Pachikian BD, Sohet FM, Mignolet E, Francaux M, Larondelle Y, Delzenne NM. Inulin-type fructans with prebiotic properties counteract GPR43 overexpression and PPARγ-related adipogenesis in the white adipose tissue of high-fat diet-fed mice. J Nutr Biochem. 2011 Aug;22(8):712-22.  
• van Loo J, Coussement P, de Leenheer L, Hoebregs H, Smits G. On the presence of inulin and oligofructose as natural ingredients in the western diet. Crit Rev Food Sci Nutr. 1995 Nov;35(6):525-52.
• Zenhom M, Hyder A, de Vrese M, Heller KJ, Roeder T, Schrezenmeir J. Prebiotic oligosaccharides reduce proinflammatory cytokines in intestinal Caco-2 cells via activation of PPARγ and peptidoglycan recognition protein 3. J Nutr. 2011 May;141(5):971-7.

Amorfrutins: Plant-Derived Selective PPAR-Modulator Outperforms Regular Diabetes Drug and Exhibit Significant Weight Loss, Insulin and Leptin Sensitizing Effects

Image 1: Amorpha fruticosa (photo R. Ott) is a deciduous shrub growing to 4.5 m, the fruits of which contain about 500mg of amorfrutins per 1kg raw material.

One major argument I have been bringing forward against the use of diabetes "medications" for quite some time, now, is that most of them are not "treatment" strategies, in the sense that they help people lose body fat to naturally restore insulin sensitivity and get off their drugs, but rather the opposite. Drugs like rosiglitazone, for example, allow for the further expansion of the adipose tissue and reduce blood glucose levels by storing the excess glucose in those new or expanding fat cells. A recent paper by Weidner et al. does now suggest that there may be a natural alternative (Weidner. 2012), which - despite acting on the same PPAR pathways lacks the fattening effects of synthetic PPAR-gamma ligands (molecules in drugs that interact with the peroxisome pro-liferator-activated receptor gamma) - lacks those highly undesirable fattening effects of thiazolidinediones.

Glycyrrhiza foetida & Amorpha fruticosa amorfrutins - the future of blood sugar management?

With the so-called "amorfrutins" from the edible parts of the two legumes Glycyrrhiza foetida (roots) and Amorpha fruticosa (fruits), the former being related to the "licorice plant" Glycyrrhiza glaba and the latter a brush that is native to the east of the USA, a group of scientists from Germany and the UK have thus identified yet another potent plant-component that outperforms its synthetic competition pretty easily.

Figure 1: Body weight (in g; left) and phosphorylated / non-phosphorylated PPAR-gamma (data from densitometric analyisis) of diet-induced obese mice on high fat diet (HFD) + placebo (vehicle), HFD + rosiglitazone or HFD + amorfrutin 1 (data adapted from Weidner. 2012)

Administered at a dosage of 100mg/kg per day (human equivalent 8mg/kg), the amorfrutins, which have a 2x higher binding affinity for the PPARγ receptor (236 to 354nM) than the aforementioned diabetes drug rosiglitazone (aka Avandia, one of the commonly prescribed thiazolidinediones), had a much more favorable effect on the phosphorylated to unphosporylated PPAR-gamma ratio and lead to statistically highly significant reductions in body weight over the 23-day supplementation period (cf. figure 1).

A note on the significance of PPAR-gamma phospohorylation: The phosphorylation of the peroxisome proliferator-receptor gamma is associated with a profound dysregulation of a large number of genes whose expression is altered in obesity. Its prevention is thus currently regarded as one of the most promising treatment strategies for insulin resistance; one that comes without the negative side-effect of increases in body weight for which all the other thiazolidinediones are notorious. It is therefore not surprising that the blockade of the phosphorylation of PPAR-gamma by amorfrutin 1 (the one denotes the first of the 4 amorfrutins the biological activity of which was investigated in the study) was "significantly correlated with improved insulin sensitivity" in the study at hand (Weidner. 2012).

Other than in most synthetic selective PPAR-modulators, such as bezafibrate, for example, the weight loss was also not mediated by a reduction in food intake, but, as Weidner et al. speculate, a direct results of an increase in energy expenditure - a hypothesis that would certainly be supported by the slight, yet likewise statistically significant increase in thyroid hormone concentration (T4) in the amorfrutin group:

Because the complex effects of PPARγagonism on various endocrine systems and downstreamphysiological changes (e.g., change in thermogenesis, fatty acid oxidation, or activity) are not fully understood, it is difficult to probe all potential mechanisms by which the amorfrutins may affect weight regulation. For example, recent studies suggest that complex interaction of brain PPARγ-signaling with peripheral organs may contribute to the physio-logical regulation of energy balance (30, 31). Presumably, the amorfrutins as partial agonists may act on neuronal PPARγby an-tagonising diet-derived endogenous agonists such as fatty acids, thereby leading to relative weight loss.

Against the background that we are apparently dealing with the PPAR equivalent to SERMs (selective estrogen receptor modulators such as clomid or tamoxifen) and SARMs (selective androgen receptor modulators), it appears prudent to mention that Weidner et al. did not find any cross-activities with other receptors, such as the estrogen receptors alpha and beta, the liver x receptor alpha, the constitutive androstane receptor, and the pregnane receptor. Cross-reactions like these are quite common with other xenobiotics (exogenous substances with biological effects that are produced by other organisms) and can lead to unexpected and mostly undesirable side effects (e.g. anti-androgen activity).

Leptin resistant? No problem for amorfrutins!

Contrary to rosgliatazone, the amorfrutins work their antidiabetic magic even in the presence of full-blown leptin resistance. While the former does not just fail to reduce, but actually promotes weight gain in leptin receptor-deficient db/db mice, treatment with amorfrutin 1...

[...] had no significant effects on mouse body weigh [... but] reduced plasma insulin concentrations more strongly than rosiglitazone (36% vs 19% decrease after 24 d) . Amorfrutin 1 treatment also decreased plasma concentrations of glucose, triglycerides, and free fatty acids. Possibly as a result of enhanced insulin sensitivity, amorfrutin 1 also appeared to prevent deterioration of pancreatic function in insulin-resistant mice, as pancreatic insulin levels improved compared to nontreated control mice.

In genetically non-disadvantaged, normal diet-induced obese mice (DIO) and thus purportedly in most obese humans, treatment with amorfrutin 1 lead to identical reductions in the areas under the glucose and insulin curve in an intraperitoneal insulin sensitivity test (IPIST; is similar to an oral test, but the injection into the intraperitoneal cave ensures that 100% of the glucose actually hits the blood stream) and reduced the basal leptin levels to the same extend as rosiglitazone did (cf. figure 2).

Figure 2: Areas under the glucose and insulin curve in an intraperitoneal insulin sensitivity test (left), basal leptin levels and photographs of the livers of the diet-induced obese mice at the end of the 24-day study period (data and images adapted from Weidner. 2012)

Despite almost identical i improvements in insulin and leptin sensitivity,u>only the amorfrutins, not the thiazolidinedione, rosiglitazone, were able to reduce the diet-induced triglyceride accumulation in in the liver of the treated animals (cf. figure 2, upper right corner) and could thusly help prevent, maybe even revert non-alcoholic fatty liver disease; an effect, by the way, that may be ascribed to

• an increase of the PPAR-alpha dependent purported "anti liver-fat" co-factor Tbl1, and
• reduced inflammation in both the liver, as well as the white visceral adipose tissue of the rodents.

In the end, it does thusly come down to the usual suspects, inflammation + insulin resistance and while the amorfrutins from the roots of a certain variety of licorice (Glycyrrhiza foetida) and the fruits of an American shrub (Amorpha fruticosa) share their ability to reduce the former and increase the latter, they don't to it at the expense of further increases in body and organ fat and could therefore help to actually resolve - not just manage and perpetuate - the current diabesity epidemic.

"When are we going to see those amor-thingies on the market?"

Image 2: Amorfrutilean could be a weight loss adjuvant that works.

Yet while they could, just as their inferior, since fattening synthetic counterparts, rosiglitazone (Avandia), pioglitazone (Actos) & co be combined with metformin (which works via a totally different mechanism), it appears questionable that the next generation of Avandamedm which combines metformin and rosiglitazone in one pill, is going to have amorfrutins in it. Not because we still need human trials (which is obviously the case), but rather due to the fact that no pharma company will be willing to pay those trials, when the outcome, a "drug" based on a naturally occurring substance that is easily extracted from a common plant, would not be patentable and may seriously compromise the sales of their current "antidiabetic" (actually I should write "pro-obesity") drugs.

If independent future human trials were yet able to confirm the previously discussed results, I am still pretty confident that we are soon going to see the supplement industry jumping aboard. With Amorfrutiburn, Amorfrutilean or Amorfrutibol being the most likely candidates for the BB.COM topselling "fat burners", weight loss adjuvants or "nutrient partitioners", respectively. And you know what, combined with a couple of lifestyle changes, this stuff could actually work - at least  for the chubbier one's among the soon-to-be physical culturist. Whether leaner folks or even bodybuilders will benefit to the same extend does yet remain to be seen.

Leucine Supplementation Exemplifies Potential Downsides of Non-Specific Insulin Sensitizers on a Hypercaloric Diet / Bulk

Adelfo's latest progress pics speak for themselves. Maybe that consoles you for the missing update!?

Another Thursday and ... no post from your's truly Adelfo Cerame. In fact, Adelfo shot me an email earlier today, that he won't be able to submit a blogpost this week if he wanted to get some sleep this night (remember we are living in different time zones) And while this was in fact a problem for me, since I am pretty pressed on time, today and did actually allocate the time I usually spend writing these blogposts, I agreed to cover his a** and step into the breach (so please ignore the surplus of typos, mistakes an missing words, please ;-). I mean, what could I say? We all know about the importance of sleep to bring in your best physique possible and I don't want to be the one who could be blamed in the very unlikely case that he won't finally take away his pro-card on his upcoming competition in March, right?

SuppVersity Science Round-Up Sneak Peak

Apropos sleep, I guess sleep is going to be one of the topics Carl and I are going to talk about on today's SuppVersity Science Round-Up (check out all previous installments and the respective Seconds, here), other topics I've got on my list here, are the Aspartame causes cancer study that resurfaced as of late on the pertinent "science websites" as if it had been released yesterday, and a couple of other news, e.g.

• The thrifty phenotype - Is it an effect of "healthy calorie restriction" during gestation?
• Fish protein & glucose metabolism - More evidence that a little can go a long way
• Eat more fruits & vegetables! - Why "more" is not specific enough
• Vitamin D - Convincing evidence that the obesity connection is a one-way street
• BPA and prostate cancer - Changes in aromatase and 5α-reductase increase malignancy
• Night shift & breast cancer - Meta analysis finds 30%+ increased risk

Even if you are not interested in any of these (btw. there will be more on obesity & healthy eating than the first two ;-), you should know by now that there are usually side-tracks, follow up an "on the other hands" that often lead the discussion into a completely new direction. So make sure you don't forget to tune in live - 1PM (EST) on the Super Human Radio Network. As usual, a podcast and the Seconds with everything that did not make it into the show will be available tomorrow.

Chronic leucine supplementation + hypercaloric diet = ???

The subheading actually summarizes pretty well, what the scientists from the School of Public Health at the Huazhong University of Science and Technology, the Department of Nutrition at the University of North Carolina at Chapel Hill and one colleague from the Hubei University of Medicine in the People's Republic of China wanted to find out, when they bought a group of male Sprague-Dawley rats, and kept them in eight groups of 10 animals for 24 weeks on one of the following dietary regimen

normal chow diet (3.78 kcal/g)	hypercaloric "high fat"(54%) diet  (5.20 kcal/g)
+ 0%, 1.5%, 3.0% or 4.5% leucine	+ 0%, 1.5%, 3.0% or 4.5% leucine

The animals had free access to food and water. The body weight and average food intake were recorded once a week. And if you take a peek at the graph on the left hand side of figure 1 it does not take a rocket scientists to recognize that any effects the supplementation may have had was "negligible" at best, with higher weight gain in the high leucine (3% an 4.5%) and minimally lower weight gain in the medium leucine supplementation group (1.5%) for the normal diet and higher weight gain for all leucine supplemented diets in the HFD group (1.5% > 3.0% > 4.5%)

Figure 1: Body weight development (remember the food intake was identical) and perirenal fat depot weight at the end of the study (Lee. 2013)

In view of what leucine is hailed for in the fitness and bodybuilding community, the explanation should be obvious: "Of course are those glutenous rodents simply gaining more muscles." The anabolic prowess of leucine makes it possible. They are ..." I am not going to repeat the superlatives with which the supplement industry is going head over heels to explain why they have just ramped up their BCAA product from a 2:1:1 to a  5:1:1 and from there to whatever ratio, as you will by now already have realize that what the rodents gained was not mere muscle, but a significant amount of body fat. So much in fact that the total boy weight to fat ratio was skewed (in other words, the body fat % increased):

[T]he perirenal white adipose tissue was −1.20% of the total body weight in animals on ND [nromal diet]. HFD alone increased the perirenal fat to 1.62% (p<0.05). Chronic supplementation of leucine (1.5 and 3.0%) increased the percentage of the perirenal white adipose tissue to 2.01–2.03% in animals on HFD (p<0.05). [...] These results show that chronic supplementation of leucine increases the ratio of white fat over total body weight in rats on HFD." (Li. 2013)

Interestingly, this increase in adiposity went hand in hand with a decrease in the expression of TNF-alpha and various inflammatory cytokines in the adipose tissue of the rodents. The picture of leucine that emerges here is therefore one of a "healthy growth factor". unfortunately one that obviously does not make a difference between muscle and fat tissue.

Leucine as a non-selective growth promoter and insulin sensitizer

Whether the decrease in inflammatory cytokines is the chicken or the egg here cannot be said for sure. What is pretty certain though is that the combination of decreased inflammation → increased adipose tissue insulin sensitivity → increase energy uptake by the fat tissue may lead to a healthier, but certainly not smaller adipose organ and does thus go against what the usual muscle head would be looking for, when he or she buys a product that claims to provide lean mass gains.

Figure 3: HOMA-IR and Area Under the Curve (AUC) of the glucose response in a glucose challenge (Lee. 2013)

In fact, the data in figure 2 hints at a classic dilemma I have alluded to in various contexts before. Many of the purported insulin mimetics or insulin sensitizers are non-tissue specific, which means that the increase in insulin sensitivity happens in the adipose tissue as well. This allows for lower blood glucose levels, but only because the energy can be stashed away in the fat cells. For a diabetic that probably does not matter much, as his or her first concern would be to get the blood glucose levels back in check. If that happens at the cost of yet another midriff bulge that may be unaesthetic, but better than the progression from type II to type III diabetes aka Alzheimer's or the advent of other side effects of chronically elevated blood glucose levels.

Figure 3: Plasma insulin response to the ingestion of 0.7g/kg CHO, 0.7g/kg CHO + 0.3g/kg WPH, and the former with additional 0.1g/kg leucine in type II diabetics and healthy controls (Manders. 2006)

What's more from previous human trials in type II diabetics, we already know that the addition of leucine to a whey protein hydrolysate does not augment its beneficial effects on postprandial glucose clearance. While statistically non-significant, the data from a 2006 study by Manders et al. rather suggests that it has a negative effect on the benefits of the 0.3g/kg whey protein hydrolysateof which the scientists were able to show that it reduced the plasma glucose response by 15% (vs. only 12% with 0.3g/kg whey + 0.1g/kg leucine; cf. Manders. 2006). Moreover, this was no diabetes specific "problem". In fact, the unnecessary insulin overshoot from the added leucine was even more pronounced in the healthy controls of the Manders study (see data in figure 3).

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Bottom line: You could in fact argue that leucine works much like the diabetes drug rosiglitazone. While the underlying mechanism it totally different, the outcome is very similar. Both increase the insulin sensitivity by reducing adipose tissue inflammation and allowing for greater energy storage in the fat tissue. Good or bad thing? Well, I guess for the majority of SuppVersity readers of whom I hope that they are not type II diabetics an only battling with one or another unaesthetic, but totally healthy pound of body fat, this is bad news.

Even if it was not for the non-specific and potentially obesogenic insulin sensitizing effects of leucine, the increased protein anabolic response to whey hydrosolate compared to free form amino acids is another thing that speaks against the use of leucine or other free-form amino acis in isolation. After all, you would be missing out on the anabolic effects of the peptides in whole proteins (read more).

When you are dieting and there is no energy surplus to stored, you don't have to bother. When you are bulking, on the other hand, and trying to do yourself a favor by adding some "highly anabolic" leucine to each and every meal, you better watch out what it is you are "building" here - is it going to be the intended lean mass or is it going to be a pot belly? I obviously don't have an answer to that question, but in view of the fact that previous trials with "leucine only" supplementation (Balage. 2010) and the provision of leucine as part of an already leucine rich protein shake (Koopman. 2008) did fail to produce superior gains, it's actually not worth trying.

If you also take into consideration the latest SuppVersity post on the non-insulin dependent increase in skeletal-muscle glucose uptake from isoleucine, the #3 in the original branch-chain amino acid concert (leucine, valine, isoleucine; read more), you better keep away from bulk supplies of l-leucine and "superior high leucine BCAA powders" and stick to the tried and proven.

References:

• Balage M, Dardevet D. Long-term effects of leucine supplementation on body composition. Curr Opin Clin Nutr Metab Care. 2010 May;13(3):265-70.
• Koopman R, Verdijk LB, Beelen M, Gorselink M, Kruseman AN, Wagenmakers AJ, Kuipers H, van Loon LJ. Co-ingestion of leucine with protein does not further augment post-exercise muscle protein synthesis rates in elderly men. Br J Nutr. 2008 Mar;99(3):571-80. Epub 2007 Aug 13.
• Li X, Wang X, Liu R, Ma Y, Guo H, Hao L, Yao P, Liu L, Sun X, He K, Cao W, Yang X. Chronic leucine supplementation increases body weight and insulin sensitivity in rats on high-fat diet likely by promoting insulin signaling in insulin-target tissues. Mol Nutr Food Res. 2013 Feb 13.
• Manders RJ, Koopman R, Sluijsmans WE, van den Berg R, Verbeek K, Saris WH, Wagenmakers AJ, van Loon LJ. Co-ingestion of a protein hydrolysate with or without additional leucine effectively reduces postprandial blood glucose excursions in Type 2 diabetic men. J Nutr. 2006 May;136(5):1294-9.