Alanyl-Glutamine or Alanine + Glutamine? Dipeptide or Free Form Aminos? What Offer Maximal Muscle Protection?

"Wouldn't have happened if she'd used alanyl-glutamine instead of regular that cheap alanine + glutamine combo!" - True or False? Recent study says: False!

If you combine your liver's favorite gluconeogenic amino acids, i.e. alanine and glutamine, into a single peptide the result is called alanyl-glutamine and marketed as the ueber-potent alternative to regular l-glutamine supplements. It goes without saying that a comparison like this is about as stupid as comparing french fries with mayo to regular french fries and saying that the former are worse because they contain more fat, or whatever. Even if we didn't care about the physiological significance of the effects of alanyl-glutamine, we would obviously have to compare the purported cryogenic effects of this "innovative" dipeptide to those of a simple combination of free form amino acids to deserve the bragging rights for having created an advanced form of glutamine.

Alanine + glutamine vs. alanyl-glutamine - fight!

By now you are probably asking yourselves why I am bothering you with things like this. Right? Well, the reason is that Éder Ricardo Petry and his colleagues from the University of Sao Paulo must recently have been pondering the same question. To answer it, they conducted an experiment that would allow them to verify if the oral supplementation with l-glutamine and l-alanine as dipeptide has more pronounced muscle protective effects than a simple mixture of l-glutamine and l-alanine (GLN+ALA, both in their free forms) in a group of Wistar rats that are subjected to intense aerobic training (treadmill).

I know what you are thinking now: "Not another rodent study...", but think about it: How many people are willing to pay $50 and more on supplements without any in vivo evidence of their efficacy let alone long-term safety? Against that background Petry's rodent is a major advancement - isn't it?

True or False: You can (ab-)use glutamine to replenish your glycogen stores!? True! It sounds strange, but according to a study from the late 20th century glutamine is a pretty effective glycogen replenisher, even in the absence of your bodies favorite nitrous glucose precursor alanine | learn more

Don't get me wrong, there are a few alanyl-glutamine studies in humans, but there is not a single one that would compare the dipeptide to a reasonable placebo in an exercise scenario. I mean, who tells me that the basketball players in the 2012 study by Hoffman et al. wouldn't have experience the same beneficial effects on basketball skill performance and visual reaction time if their rehydration solution had contained alanine and glutamine or even glutamine alone? Yes, I know... the increased absorption: Well, let's just look at a fair comparison, i.e. the study at hand, and see what happens when the dreams of supplement formulators and reality meet ;-)

Ok, back to the facts - the exercise & supplementation protocol

The male Wistar rats, the researchers used in their experiment were exercised 5x per week - at increasing intensities: Starting with 30 and 45 min of treadmill running (incline 3°) at 20 and 22.5 m/min in the first three weeks, the speed and duration of their treadmill runs increased to 60 min at a speed of 25 m/min in week four and remained like that for the rest of the 8-week study period.

The supplements were administered via oral gavage in the course of the last 3 weeks, only. The daily doses for the animals in the dipeptide (DIP) and free form amino acid groups (GLN+ALA) were...

• 1.5g/kg alanyl-glutamine in the DIP group,
• 0.67g/kg l-alanine + 1.0g/kg l-glutamine in the GLN+ALA group, and
• plain water in the control group

The amount of of alanyl-glutamine the scientists used was calculated in such a way that the total amount of l-glutamine was the same as that of l-glutamine administered in its free form.

Changes? YES! Dipeptide benefits? Not really...

The gavage was provided 1 h after the end of each session of exercise, after which the animals had with free access to water and chow. To make sure that the results of the examinations on the last day of exercise would not reflect the acute effects of a single dose of the supplements, the animals were killed 10 h after the last exercise session.

Figure 1: Plasma glutamine, glutamate, ammonium, malondialdehyde, myoglobin, and creatine kinase activity in Wistar rats supplemented with alanyl-glutamine (DIP) or regular glutamine + alanine; data expressed rel. to control (Petry. 2013)

The virtually identical increases in l-glutamine and l-glutamate, you see in Figure 1 should thus represent the baseline and not the 'immediately post supplementation level' of these amino acids. For the exercise-induced accumulation of ammonium, malondialdehyde (MDA; indicates lower lipid oxidation), myoglobin and creatine kinase (both indicate lower muscle damage) the timing is not that important, anyway. What is important, however, is the fact that there were no physiologically relevant advantages for the "super glutamine".

DHEA & estrogen are alternative muscle protectors. Despite the fact that estrogen has repeatedly been shown to have muscle protective-effects, I would not suggest you steel your granny's HRT medication. DHEA on the other hand, may be something to consider - specifically if you are about to overreach, like the male subjects in a 2012 study by Liao et al. (learn more)

If we take a closer look at the p-values and the statistical significance of these changes, it turns out that, the minor increase in glutamate aside, all of the difference to the placebo group were statistically significant. The DIP vs. GLN+ALA differences, on the other hand, were marginal and reached statistical significance only in the case of the marker of myoglobin. Where the dipeptide has a physiologically probably irrelevant edge of 9% over the GLN + ALA combination.

Figure 2: Glutathione (GSH) and glutathione disulfide (GSSG = used glutathione) levels in soleus and gastrocnemius skeletal muscles of the rodents; data expressed relaitve to control (Petry. 2013)

For the muscular GSH levels, it does not look much different. In this case, there is however not even a statistical difference between alanyl-glutamine and the simple l-alanine + l-glutamine mix - neither for the universal anti-oxidant glutathione (GSH), nor for its "used form" glutathione disulfide (GSSG).

Does that mean that alanyl-glutamine is another supplemental rip-off?I would say that it's too early to use such harsh words. There was after all one statistically, and maybe even physiologically relevant difference between the two groups I didn't mention, yet: The dipeptide group presented with a different heat-shock protein response: They had higher HSP-70 and lower HSF-1 levels in the soleus and lower HSP-70 and lower HSF-1 levels in the gastrocnemius.

"Will training your biceps, heal your heart & protect your brain!?" - a study on the effects of exercise induced HSP increases suggests so | more

In view of the fact that the subsequent "deficit in HSP70 expression" is supposed to "impair recovery from these injuries" Petry et al. are probably right to point out that

"one cannot discard the possibility that part of the beneficial effects of high-intensity exercise training may be due to the enhancement of HSP70 expression which is exacerbated by glutamine supplementation."

In view of the fact that the total amount of proteins from the HSP70 and HSF1 family was increased in both groups, and the differences appear random, it is impossible to tell, whether the slight differences in HSP expression actually matter and whether this is an advantage for alanyl-glutamine or rather for the cheap free form amino acids.

Before future studies provide additional data based on which we can decide whether these differences are relevant and why they differ between slow- (soleus) and fast-twitch (gastrocnemius) skeletal muscle fibers, I'd say that the study at hand would suggest that alanine and glutamine have muscle protective effects irrespective of whether they are bound or not, when you ingest them.

References:

• Cruzat VF, Rogero MM, Tirapegui J. Effects of supplementation with free glutamine and the dipeptide alanyl-glutamine on parameters of muscle damage and inflammation in rats submitted to prolonged exercise. Cell Biochem Funct. 2010 Jan;28(1):24-30. 
• Cruzat VF, Tirapegui J. Effects of oral supplementation with glutamine and alanyl-glutamine on glutamine, glutamate, and glutathione status in trained rats and subjected to long-duration exercise. Nutrition. 2009 Apr;25(4):428-35.
• Hoffman JR, Williams DR, Emerson NS, Hoffman MW, Wells AJ, McVeigh DM, McCormack WP, Mangine GT, Gonzalez AM, Fragala MS. L-alanyl-L-glutamine ingestion maintains performance during a competitive basketball game. J Int Soc Sports Nutr. 2012 Mar 7;9(1):4.
• Petry ER, Cruzat VF, Heck TG, et al. Alanyl-glutamine and glutamine plus alanine supplements improve skeletal redox status in trained rats: Involvement of heat shock protein pathways. Life Sciences. 20 November 2013 [ahead of print]
• Rogero MM, Tirapegui J, Pedrosa RG, Castro IA, Pires IS. Effect of alanyl-glutamine supplementation on plasma and tissue glutamine concentrations in rats submitted to exhaustive exercise. Nutrition. 2006 May;22(5):564-71.

Whey Beyond Brawn: 10+ Things You Probably Didn't Know Whey & Peptides That Form During its Digestion Can Do: From A as in Vitamin A Uptake to Z as in CanZer Protection

If you've got brawn and brain you will realize that whey is much more than a potent muscle builder.

As a SuppVersity reader you are well familiar with the pluripotent benefits whey protein has to offer to the average and extra-ordinary gymrat. You will also be aware that it can promote weight loss and help you maintain lean muscle mass, when you're dieting.

If you've read almost all ~2,000 SuppVersity articles, you will even know about the GLUT4 and thus glucose uptake promoting effects isoleucine containing dipeptides in whey protein hyrolysates, but I guess that some of the other benefits whey protein owes to its complex mixture of proteins and peptides are going to be news for you.

Learn more about the effects of your diet on your body composition at the SuppVersity

Only Whey, Not Soy Works for Wheytloss

Minimal Carb Reduction, Max. Results?

Dairy Protein Satiety Shoot-Out: Casein vs. Whey

How Much Carbs Before Fat is Unhealthy?

5 Tips to Improve & Maintain Insulin Sensitivity

Carbohydrate Shortage in Paleo Land

In their latest paper in the Austin Journal of Nutrition and Food Science Rie Tsutsumi and Yasuo M. Tsutsumi provide a concise overview of the biological effects of a range of peptides and proteins in whey protein. The latter include...

• 

  Amino acid composition of whey, casein and breast milk - whey excells in terms of pro-anabolic BCAAs (McDonough. 1974)

  whey is the richest natural source of BCAA -- I don't have to tell you that, but I thought maybe there is someone who has never heard of it before ;-)
• whey protein has the highest biological value (indicative of the most balanced EAA profile) of all dairy proteins (the  biological  value  is  the  ratio  of  the amount of nitrogen that is consumed to the amount of nitrogen that is absorbed, and this value is 74 for soy protein, 71 for casein, and 104 for whey protein)
• whey protein has the highest protein efficacy ratio, i.e. the body weight increase associated with an intake of 1 g protein is 3.0 (vs. 2.0 for soy protein and 2.5 for casein protein)

Whey proteins are however far more than building blocks / muscle builders, the  proteins  in  whey  have a variety of roles and immune-related functions:

• b-lactoglobulin binds retinol (vitamin A) and promotes uptake of retinol via gut; by a similar mechanism it may also facilitate the uptake of long-chain fatty acids
• a-lactoalbumin kills tumour cells (in vitro) and exerts anti-bacterial effects in the upper respiratory systems; it has also been shown to have protective effects on gastric mucosa.

• 

  Table 1: Content of minor bioactive proteins in whey concentrates (levels are probably lower in iso- and hydrolysates | values from Smithers. 2008) and the lactalbumin fractions. Since the major proteins in whey (not listed here) can form bonds with albumin (Havea. 2001), whey is another case, where an increase in processing may lead to a decrease in beneficial biological activity.

  lactoferrin regulates the absorption of iron via gut; it will inhibit the growth of various bacteria and regulates immunological response of immunocomponent cells
• serum albumin binds and carries fatty acids and bile pigment
• immuno-globulin G involves with bactericidal (anti-bacterial) effects with complements and prevents bacteria from adhering to tissues; neutralizes toxins and viruses
• immuno-globulin A inhibits growth of various bacteria by condensing them; prevents bacteria from adhering to the surface of mucosa; neutralizes toxins produced by viruses and bacteria.
• immuno-globulin M has the same effects as IgG, but its bioactivity is stronger
• lactoperoxidase catalyzes the reaction of producing cyanogen ion with strong bactericidal power from cyanic ion and hydrogen peroxide in the body
• lysozyme kills bacteria by destroying cell walls

As the Japanese researchers point out, the mechanisms relating to the whey functions are varied. The antioxidant and detoxifying activity of whey is most likely linked to its contribution to GSH synthesis.

"Cysteine, which contains an antioxidant thiol group, combines with glycine and glutamate to form GSH. GSH is the major endogenous antioxidant produced by cells, providing production for RNA, DNA, and proteins via its redox cycling from the reduced form, GSH, to the oxidized form, GSSH. Though direct conjugation, GSH detoxifies a host of endogenous and exogenous toxins including toxic metals, petroleum distillates, lipid peroxides, bilirubin, and prostaglandins." (Tsutsumi. 2014)

The antioxidant and antimicrobial effects of lactoferrin have already been mentioned above. In addition, lactoferrin demonstrates an ability to stimulate immune responses involving natural killer cells, neutrophils, and macrophage cytotoxicity. Furthermore, a mouse study concluded that lactoferrin acts as an anti-inflammatory by regulating the levels of tumor necrosis factor and interleukin-6.

"Owing to its ability to chelate iron, organisms requiring iron for replication appear to be particularly vulnerable to the effects of lactoferrin. The protein beta-lactoglobulin contains anti-hypertensive peptides, which lower blood pressure as significantly as angiotensin converting enzyme (ACE) inhibitors. Cholesterol-lowering effects have also been noted as a result of changes in micellar cholesterol solubility in the intestine." (Tsutsumi. 2014)

Moreover, the  formation of peptides through the hydrolysis of whey proteins in your tummy is a rather novel, but very interesting effect that may well contribute to the beneficial health efects of whey proteins. In fact, whey peptide is one of the major peptides that inhibit ACE (FitzGerald. 2004), which induces blood-pressure regulating effects.

It is very likely that peptides are also responsible for many of the metabolic benefits

Pal et al. demonstrated a decrease in fasting plasma concentrations of triacylglycerols after long-term whey protein intake (12 weeks) in overweight and obese individuals (Pal. 2010a,b,c). And while the mechanisms behind the effects of whey protein on triacylglycerols are not understood, Mortensen et al. proposed that a meal containing whey might have resulted in reduced production of chylomicrons and accelerated chylomicron clearance resulting from the stimulation of lipoprotein lipase by whey.

Figure 2: Changes in insulin and HOMA-IR (insulin resistance) i response to 12 weeks on 27g of whey vs. casein (vs. control) in overweight / obese subjects (Pal. 2010b)

This would yet not explain the significant improvements in glucose management evidenced by reduced insulin and HOMA-IR values in the whey group of Pal et al.'s 12-week intervention with 27g of whey (vs. glucose vs. casein; cf. Figure 2).

Pal et al. are obviously not the only ones, who observed significant beneficial effects on glucose management in response to the ingestion of whey protein supplements. As Tsutsumi & Tsutsumi point out "[t]he majority of these studies reported that whey protein intake decreases blood glucose and insulin levels" (Tsutsumi. 2014)

Whey a source of bioactive anti-diabetic, pro-satiety peptides?

The latter is interesting, because we know that in type II diabetics, whey protein will increase not decrease the insulin response. I that, the acute effects of whey protein on postprandial blood glucose are comparable to sulfonylureas and other insulin secretagogues used for the pharmaceutical management of hyperglycemia in type 2 diabetes. A benefit that is probably related to bioactive peptides and amino acids that are generated during gastrointestinal digestion and enhance the release of several hormones (including insulin) which are able to reduce the food intake and increased satiety (e.g. cholecystokinin, peptide YY, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1)). How exactly this works is still being researched, but both, ...

• 

  Figure 3: Effects of 48 g casein (full circles) or whey (open squres) on GLP-1 (Hall. 2003)

  the production of bioactive peptides that serve as endogenous inhibitors of dipeptidyl peptidase 4 in the proximal gut, preventing the degradation of the insulinotropic incretins GLP-1 and GIP, as well as ...
• a mechanism that involves BCAAs, specifically leucine, which activates the mTOR signaling pathway and protein synthesis leading to elevated hormone expression and secretion and increased thermogenesis

...have been brought forward and supported by research. In the end, it does not really matter how whey does it. The resulting increases in satiety, thermogenesis, and reduction of blood glucose, which is comparable to pharmaceutical treatment, support the use of whey protein in the management of type 2 diabetes and obesity, anyway.

I won't bore you with the muscle building effects of whey: You just have to click here to see previous articles on whey protein, if you actually feel you need to know more about the muscle building prowess of whey protein.

If whey protein helps prevent or even cure diabesity it will also help to prevent a hell lot of the side effects of being an overweight type II diabetic. And still, researchers believe that there may be a more direct link than diabesity prevention to the following health benefits of whey protein that are listed in Tsutsumi & Tsutsumi's latest review (the following bulletpoints are in large parts direct quotes from Tsutsumi. 2014):

• cancer  -- A number of animal studies have examined the anti-cancer potential of whey, believed to be primarily associated with the antioxidizing, detoxifying, and immune-enhancing effects of GSH and lactoferrin.
  
  A few clinical trials have been undertaken, proposing that high levels of GSH in tumor cells confer resistance to chemotherapeutic agents. One of these studies showed that 20 patients with stage IV malignancies were treated daily with 40 g whey in combination with supplements such as ascorbic acid and a multi-vitamin/mineral formulation (See. 2002). The 16 survivors demonstrated increased levels of natural killer cell function, GSH, hemoglobin, and hematocrit 6 months later. An aggressive combination of immunoactive nutraceuticals was effective in significantly increasing natural killer function, other immune parameters, and plasma hemoglobin in patients with late stage cancers.
• hepatitis B & C -- The results of trials for the hepatitis B virus have been positive,  particularly  those  from  an  open  study  that  included  8  patients administered 12 g non-heated whey/day. The patients demonstrated improved liver function markers, decreased serum lipid peroxidase levels, and increased  interleukin-2  and  natural  killer  cell  activity (Watanabe. 1999)
  
  Regarding hepatitis C, several trials have proved inconclusive, although an initial in vitro study found that bovine lactoferrin prevented the hepatitis C virus in a human hepatocyte line (Ikeda. 1998)

• 

  Figure 4: Next to reductions in blood pressure, whey induced reductions in blood lipids are a likely mechanism behind the reduced CVD risk with whey (illustration from Pal. 2013)

  cardiovascular disease -- According to the results of a number of studies, intake of milk and milk products can lower blood pressure and reduce the risk of hypertension (Marshall. 2004). Kawase et al. performed an 8-week trial in which 20 healthy men were given a combination of fermented milk and whey protein concentrate and examined the effect on serum lipids and blood pressure (Kawase. 2000). After the 8 weeks, the fermented milk group demonstrated comparatively higher high-density lipoproteins, lower triglycerides, and lower systolic blood pressure. 
• hypertension --Various investigators have hypothesized that certain bioactive peptides formed through the hydrolysis of food proteins have the ability to inhibit ACE, and this subject has been comprehensively reviewed in a number of studies. In general, it has been claimed that a diet rich in foods containing anti-hypertensive peptides is effective for the prevention and treatment of hypertension. ACE-inhibitory peptides may be obtained from precursor food proteins via enzymatic hydrolysis, the use of viable or lysed microorganisms, or specific proteases.
  
  However, studies relating to whey peptides with ACE inhibitory activities are more limited; this may be due to the rigid structure of beta-lactoglobulin, which makes it particularly resistant to digestive enzymes. 
• osteoporosis -- Milk basic protein (MBP) is a component of whey that demonstrates the ability to not only suppress bone resorption but also stimulate proliferation and differentiation of osteoblastic cells (Marshall. 2004).
  
  The role of calcium intake in determining bone mineral mass is well recognized to be the most critical nutritional factor to achieve optimal peak bone mass; milk protein is also important for preventing osteoporosis. A number of clinical trials support milk protein’s positive effects in both men and women, the latter ranging in age from young to postmenopausal. Daily doses of 40 mg MBP (equivalent to 400–800 mL milk) appear to be sufficient to significantly increase bone mineral density and reduce bone resorption.

• 

  Figure 5: Mean (±SD) reaction time in the high stress–vulnerable (▪) and low stress–vulnerable (□) groups after consumption of a diet containing protein as sodium casein (control diet) and a diet containing α-lactalbumin–enriched whey protein (α-lactalbumin diet | Markus. 2002)

  stress adaptation -- Whey enriched with the protein alpha-lactalbumin has been shown to improve cognitive performance and mood in stress vulnerable subjects (Markus. 2002). Alpha-lactalbumin is particularly high in tryptophan, and the authors proposed that this acts as a substrate to increase serotonin levels, which may be vulnerable to depletion by chronic stress. At the completion of the studies, all of the participants had higher ratios of plasma Tryp-LNAA (the ratio of plasma tryptophan to the sum of the other large neutral amino acids), believed to be an indirect indication of brain serotonin function.
  
  Recently, de Moura et al. evaluated the effects of whey protein intake on the expression of heat shock protein HSP70 (de Moura. 2013). HSP70 confers cellular tolerance against stressors, and there was a greater increase in the HSP70 expression in the soleus, gastrocnemius, and lungs of the whey protein hydrolysate-fed rats than in the casein-fed rats.

Tsutsumi & Tsutsumi also mention the battle against sarcopenia, where whey is about as useful as it is as a muscle builder in athletes and the support of the gastrointestinal integrity that is mediated by the glutamic acid content of whey which is converted to glutamine and serves as fuel for the intestinal mucosa among the proven health benefits of whey protein, before they conclude their review by stating that we still need studies investigating the mechanisms underlying the effects of whey protein.

Stop protein wheysting!

I have to admit, I am a bit ashamed of how extensively I used the excellent review by Rie and Yasuo M. Tsutsumi when I compiled today's SuppVersity Article. What came out of it, though, is an article with so many facts about the health benefits of whey that I am confident that it contains at least one surprising study result you have not heard of before for each of you... true?

In case it didn't, stay tuned for reports on the future studies investigating the mechanisms underlying the effects of whey protein, Tsutsumi & Tsutsumi demand in the conclusion of their review: I bet you won't have to wait long for the next SuppVersity Whey Protein Article | Comment on Facebook!

References:

• de Moura, Carolina Soares, et al. "Whey protein hydrolysate enhances the exercise-induced heat shock protein (HSP70) response in rats." Food chemistry 136.3 (2013): 1350-1357.
• FitzGerald, Richard J., Brian A. Murray, and Daniel J. Walsh. "Hypotensive peptides from milk proteins." The Journal of Nutrition 134.4 (2004): 980S-988S.
• Hall, W. L., et al. "Casein and whey exert different effects on plasma amino acid profiles, gastrointestinal hormone secretion and appetite." British Journal of Nutrition 89.02 (2003): 239-248. 
• Havea, Palatasa, Harjinder Singh, and Lawrence K. Creamer. "Characterization of heat-induced aggregates of β-lactoglobulin, α-lactalbumin and bovine serum albumin in a whey protein concentrate environment." Journal of Dairy Research 68.03 (2001): 483-497.
• Ikeda, Masanori, et al. "Lactoferrin markedly inhibits hepatitis C virus infection in cultured human hepatocytes." Biochemical and biophysical research communications 245.2 (1998): 549-553. 
• Markus, C. Rob, Berend Olivier, and Edward HF de Haan. "Whey protein rich in α-lactalbumin increases the ratio of plasma tryptophan to the sum of the other large neutral amino acids and improves cognitive performance in stress-vulnerable subjects." The American journal of clinical nutrition 75.6 (2002): 1051-1056.
• Marshall, Keri N. D. "Therapeutic applications of whey protein." Alternative Medicine Review 9.2 (2004): 136-156.
• Pal, Sebely, and Vanessa Ellis. "The chronic effects of whey proteins on blood pressure, vascular function, and inflammatory markers in overweight individuals." Obesity 18.7 (2010a): 1354-1359.
• Pal, Sebely, Vanessa Ellis, and Satvinder Dhaliwal. "Effects of whey protein isolate on body composition, lipids, insulin and glucose in overweight and obese individuals." British journal of nutrition 104.05 (2010b): 716-723.
• Pal, Sebely, Vanessa Ellis, and Suleen Ho. "Acute effects of whey protein isolate on cardiovascular risk factors in overweight, post-menopausal women." Atherosclerosis 212.1 (2010c): 339-344. 
• Pal, Sebely, and Simone Radavelli‐Bagatini. "The effects of whey protein on cardiometabolic risk factors." Obesity Reviews 14.4 (2013): 324-343.
• See D, Mason S, Roshan R. "Increased tumor necrosis factor alpha (TNFalpha) and natural killer cell (NK) function using an integrative approach in late stage cancers." Immunol Invest 21 (2002):137-153.
• Smithers, Geoffrey W. "Whey and whey proteins—from ‘gutter-to-gold’." International Dairy Journal 18.7 (2008): 695-704.
• Tsutsumi, R., and Y. M. Tsutsumi. "Peptides and proteins in whey and their benefits for human health." Austin J Nutri Food Sci 1.1 (2014): 9.
• Watanabe, Akiharu, et al. "Nutritional therapy of chronic hepatitis by whey protein (non-heated)." Journal of medicine 31.5-6 (1999): 283-302.

Confirmed: All Wheys, Not Just Hydro Whey Boost Glucose Uptake And Liver + Muscle Glycogen Supercompensation. Plus: How Could Taurine Be Involved in This Benefits?

Do it or don't? If the question is about consuming whey protein, the answer is clear: Do it! Use whey!

As a diligent student of the SuppVersity you will remember my previous article "The Glucose Repartioning Effects of Isoleucine: Falsely Underappreciated BCAA and Its Dipeptides Maximize GLUT-4 Expression and Ramp Up Muscular Glucose Uptake" (read more). If you don't let me briefly bring you up to speed in back in February, I told you about the beneficial effects of a class of isoleucine peptides in whey protein hydrosylate [as the study at hand goes to show you, this is important, see bottom line] on glucose transporter (GLUT-4) expression and thus glucose uptake in skeletal muscle.

Today I am pleased to be able to continue and expand on this discussion based on the results of the latest study from the same group of researchers from Sao Paulo, Brazil (Morato. 2013).

Whey, an anti-diabetic glycogen supercompensation tool

As Morato et al. point out, their own study is by no means the only one that supports the very special insulin sensitizing activity of whey proteins. In fact, whey is already touted as potential anti-diabetic. If the medical orthodoxy or rather it's "legislative" arm was not trapped by its own dogmas WPH [whey protein hydrolysate] would already be a central part of the dietary recommendation for type II diabetics. With the current study being the first to show that a whey protein based diet will lead to chronically increased GLUT-4 expression and thus help to lower blood glucose and improve glycogen storage, the study at hand is albeit similarly interesting for the average musclehead and his obese type II diabetic neighbor.

Figure 1: Effects of casein, whey and whey hydro(lysate) diets on GLUT-4 expression, baseline insulin, liver glycogen and muscle glycogen levels (g/100g tissue; Moreto. 2013)

Apropos obese neighbor. You should go and convince him to go to the gym with you. After all, the rodent data in figure 1 clearly shows that WPH leads its trumps only when it is combined with training - in this case treadmill running for 60 minutes at 15 m/min (the exercise took place 16 h before the sacrifice; so the increases are not in response to the exercise! they are just amplified by chronic endurance during).

Suggested read: "The Overlooked Glucose Repartioning Effects of Isoleucine" (read more)

While exercise alone is well known to boost GLUT-4 expression and subsequent glucose uptake significantly (Christ-Roberts. 2004; Kuo. 2004; note. GLUT-4 activity correlates with the degree of muscular clycogen depletion, so no "5 min rest, 2 sets all out and go home workouts!"), the addition of a whey protein hydrosylate with a pre-hydrolysation level of 12.5% (think of it as being enzymatically "pre-digested) as the sole protein component of the baseline diet (15% protein total, 7% fat from vegetable oil, 68% carbs from sugar and corn starches) of the 48 male Wistar rats in the experiment at hand did turn the +100% increase from exercise alone into a  +160% increase.

I have to admit, the increased GLUT-4 uptake per se may not be news, but this is in fact the first chronic feeding study where it was observed in conjunction with higher glycogen levels - ca. 90%, 70% and a whopping 400% in the heart, the musclulature and the liver in the sedentary state for both WPH and regular whey protein. That's certainly impressive, but you got to remember that this is a result of combining whey with a high carbohydrate diet (69% of the diet vs. 7% fat) which provides the necessary readily available substrate for optimal glycogen super-saturation (=packing in more glycogen than you usually could).

You will and can very well live with the insulin spike!

Though it may not look like it in figure 1, you got to be aware that the values were not taken right after the ingestion of a meal, let alone a protein shake. In other words, it is almost certain that the whey protein groups will have had higher insulin levels immediately after a meal (note: Casein is still way more insulinogenic than meat or eggs).

I've gone into quite some detail on why insulin spikes (in the presence of glucose) are not a problem, but rather a vital necessity in a previous post (read it)

"One of the primary means to increase the concentration of GLUT-4 in the plasma membrane is through insulin-regulated trafficking (Zorzano. 2005). However, in the present experiment, no increase was noted in serum insulin levels in the groups consuming WPH.

The experimental design of the study focused on the moment of greatest mobilization of glucose transporter-4, and the animals were sacrificed 2 h after consuming the meal; this was too long an interval to observe the maximum plasma insulin response." (Morato. 2013)

It is thus a given that the  GLUT-4 translocation was at least supported by profound and temporary (at least in the presence of an adequate carbohydrate intake, their temporary nature is what makes the whey induced insulin spikes physiologic and beneficial vs. pathological and detrimental as chronic elevations would be; learn more). According to Morato et al. this is however not the only way the ingestion of whey affected the translocation of GLUT-4 (upstream) and the subsequent uptake of glucose into the muscle and liver (downstream):

Translocation of GLUT-4 to the PM [plasma membrane] can also be stimulated in an insulinindependent manner. Carneiro et al. (2009) accomplished this through taurine activation of the insulin pathway, thus raising the GLUT-4 concentration in the plasma membrane independent of insulin. However, the molecular mechanism behind this effect has still not been elucidated (Carneiro. 2009).

There is actually evidence that would suggest that whey protein hydrolysate is not simply not superior, but actually inferior to regular whey proteins when it comes to improvements in body composition in athletes (read more)

In the exercised animals of the WP and WPH groups, the plasma concentrations of taurine (Table 1) were greater (p,0.05) than those in the control group consuming CAS. This could explain, at least in part, the greater translocation of GLUT-4 in the WP and WPH groups.

After investigating the amino acid composition of the WP and WPH, it was found they were rich in sulfur amino acids (Table 2), and methionine and cysteine are endogenous precursors of taurine. Thus, the consumption of WP or WPH provided a greater amount of substrate for the endogenous production of taurine than casein, and the presence of this amino acid may have facilitated activation of the insulin pathway and cell capture of glucose, as indicated in the literature." (Morato. 2013; my emphases)

That's quite a surprising insight, isn't it? I mean, as a SuppVersity reader you have long known about the anti-diabetic prowess of taurine, but who would have suspected that it could be #3 alongside the active isoleucine dipeptides and the insulin release among the mechanisms behind the profound beneficial effects whey has on glucose? I mean, there is basically no taurine in whey.

SuppVersity readers have known for years, that whey is far superior to an amino acid (AA) mixture with the same AA make-up (read more)

Bottom line: Yep, this is support for a previous advice I've gicen: You better never run out of whey protein for both, health and performance reasons. Personally, I am yet most fascinated by the potential involvement of endogenous (=your body's own) taurine synthesis. That 's certainly going to be a topic in Sunday's 2nd installment on supplements to improve and maintain insulin sensitivity (read part I on lifestlye modifications here).

Pratically speaking the most important and eventually less surprising message of the study at hand is however that it does not necessarily have to be whey protein hydrolysate. The regular whey protein did an outstanding job, as well, and the "real-world" = visible / noticeable differences are propably non-significant.

In this context, some of you may also remember the results from another recently published study by Lollo et al. (read it) which did in fact suggest that the muscle building and body recompositioning effects of whey hydrolysate are inferior and not superior to those of regular whey.

References:

• Carneiro EM, Latorraca MQ, Araujo E, Beltra M, Oliveras MJ, et al. Taurine supplementation modulates glucose homeostasis and islet function. J Nutr Biochem. 2009; 20: 503–511.
• Christ-Roberts CY, Mandarino LJ. Glycogen synthase: key effect of exercise on insulin action. Exerc Sport Sci Rev. 2004; 32: 90–94.
• Kuo CH, Hwang H, Lee MC, Castle AL, Ivy JL. Role of insulin on exercise-induced GLUT-4 protein expression and glycogen supercompensation in rat skeletal muscle. J Appl Physiol.  2004; 96: 621–627.
• Morato PN, Lollo PC, Moura CS, Batista TM, Carneiro EM, Amaya-Farfan J. A dipeptide and an amino acid present in whey protein hydrolysate increase translocation of GLUT-4 to the plasma membrane in Wistar rats. Food Chem. 2013 Aug 15;139(1-4):853-9.
• Zorzano A, Palacín M,Gumá A. Mechanisms regulating GLUT 4 glucose transporter expression and glucose transport in skeletal muscle. Acta Physiol Scand. 2006. 183: 43–58.

True or False? Glycine & Proline Supplements Ramp Up Collagen Synthesis & Improve Joint Health. Plus: The Tripeptide Advantage of Collagen Hydrolysates

The "Paleo" cult has repopularized eating and preparing your own (Chicken) bone broth, but will this also help with bone and cartilage health?

Although you're probably thinking of collagen as the stuff that's important for joint health, its implications in human health are more far-reaching than most of us believe.

In fact, collagens are the most abundant group of organic macro-molecules in human and animal body. Because of their tensile strength, they perform numerous structural functions within the body - specifically in connective tissues which include among other tissue also organs as your heart, your intestines, your lungs and the parenchymal organs like the liver and the kidneys and even the fibrous matrix of skin and blood vessels.

As I already said, collagens are yet by far best known as structural components of the protein matrix of the skeleton and its related structures, like bones, teeth, tendons, cartilage and ligament, which bring us back to the original question that bothered me after assuring Chris who emailed me asking about the necessity of taking glycine and proline supplements in the absence of any other protein (my answer was "that's bullocks"): Do glycine and problem supplements even help with collagen synthesis and joint health? Or is the supplement vendor next door the only person who benefits?

You can find more True or False articles at the SuppVersity

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We do have evidence (from rodent studies) that the ingestion of low molecular weight (=small peptides) collagen hydrolysates with intact glycyl-prolyl-hydroxyproline tripeptides that actually make it through the gut into the bloodstream and will increasee the organic substance content and decreased the water content of the left femur (Watanabe-Kamiyama. 2009). Previous studies had already shown hat the content of an orally administered gelatin hydrolysate will be incorporated into the cartilage tissue of rats (Oesser. 1999). Similar observations have been made by Iwai et al. for human volunteers and porcine gelatine hydrolysate, as well.

"After the oral ingestion, the peptide form of Hyp significantly increased and reached a maximum level (20-60 nmol/mL of plasma) after 1-2 h and then decreased to half of the maximum level at 4 h after the ingestion. Major constituents of food-derived collagen peptides in human serum and plasma were identified as Pro-Hyp. In addition, small but significant amounts of Ala-Hyp, Ala-Hyp-Gly, ProHyp-Gly, Leu-Hyp, Ile-Hyp, and Phe-Hyp were contained." (Iawai. 2005)

If we assume a similar physiological effect as it was observed by Watanabe-Kamiyama in rodents, the ingestion of (large) quantities of gelatine could thus very well, after it's hydrolysation in the gut, have similar effects on human cartilage tissue as the collagen hydrolysate that was used in the Watanabe-Kamiyama study.

Table 1: Summary of Structure and Recovery of Food-Derived Collagen Peptide in Human Serum or Plasma after Oral Ingestion of Gelatin Hydrolysates (Iawai. 2005).

With respect to the occurence of glycyl-polyl-hydroxproline tripeptides, of which the Watanabe-Kamiyama study suggests that they may be responsible for the beneficial effects on cartilage synthesis it should yet be said that it occurred in human plasma only after the ingestion of chicken, but not in porcine collagen in the Iawai study (see Table 1). If that's no coincidence, HARIBO, which is usually made with porcine gelatine is no "collagen builder", a real chicken soup, cooked with bone, on the other hand, could be.

Given that your stomach is working properly a nice paleo bone broth (preferably from chicken bone) could thus produce similar results as a collagen hydrolysate of which a recent review in Current Medical Research and Opinion says that its ingestion stimulates a statistically significant increase in synthesis of extracellular matrix macromolecules by chondrocytes.

There is more to collagen hydrolysates than joint health: In 2009 Saito et al. were able to show that fish collagen hydrolysates affect lipid absorption and metabolism in rats and may be useful in suppressing the transient increase of plasma triglycerides (Saito. 2009). Moreover, Spanish researchers showed that the daily dietary intake of hydrolyzed collagen seems to have a potential role in enhancing bone remodeling at key stages of growth and development in 60 children (9.42±1.31 years) who had been randomly assigned to either placebo or collagen (+ calcium) supplementation. In spite of these benefits, the ingestion of corresponding supplements is not necessary for people with healthy collagen metabolism who exercise regularly and eat clean.

Figure 1: Physician rated (top) and subject-rated (bottom) improvement in joint pain walking (left) and standing (right) in the Clark study (Clark. 2008).

The authors, researchers from the University of Illinois College of Medicine at Chicago and the University of Kiel in Germany add:

"These findings suggest mechanisms that might help patients affected by joint disorders such as OA. Four open-label and three double-blind studies were identified and reviewed; although many of these studies did not provide key information – such as the statistical significance of the findings – they showed collagen hydrolysate to be safe and to provide improvement in some measures of pain and function in some men and women with OA or other arthritic conditions." (Bello. 2006)

Subsequent studies such as Benito-Ruiz et al. (2009) or Clark et al. who evaluated data from 97 athletes from a varsity team or a club sport in Pennsylvania support Bello's conclusion (see Figure 1).

Similar beneficial effects were also observed by  et al. in a more recent study with "normal" subjects with articular pain in response to 1,200mg/day of collagen hydrolysate (Bruyère. 2012). When we're looking into the effects of single amino acids, however, things look different. If they're ingested separately, glycine and proline are not going to form a tripeptide in the course of the digestive process. And while they may still serve as a raw material for the endogenous synthesis of such peptides the chance that they actively promote the synthesis of new collagen is slim.

Biologically active tripeptides, not just glycine & proline is what you want!

Bottom line: Collagen hydrolysates with intact tripeptides seem to have a beneficial effect on collagen synthesis. Classic broth and gelatine, both best made from chicken bones (absorption data on beef is not available), could have beneficial effects on collagen synthesis. In view of the chance that and rate at which the physiologically relevant  glycyl-prolyl-hydroxyproline tripeptides (see image to the right) are produced during the natural digestion process it does yet appear certain that you would have to garble down tons of it on a daily basis to actually trigger collagen synthesis and not just to do what individual amino acids could probably do as well: provide the necessary substrates without actually accelerating collagen synthesis.

Chris' original question whether you'd have to take glycine and proline supplement on their own and in the absence of any other proteins and amino acids would thus actually be obsolete (you shouldn't take them at all), but I guess it may be worth mentioning that doing that, i.e. taking them on their own will only increase the "risk" of both being used by the liver as a substrate for glyconeogenesis (proline for example has the 3rd highest potential for gluconeogenesis 75% of the most glycogenic amino acid, i.e alanine; cf. Ross. 1967) - especially if you top "taking them on their own" with "taking them during a fast".

References:

• Bello, Alfonso E., and Steffen Oesser. "Collagen hydrolysate for the treatment of osteoarthritis and other joint disorders: a review of the literature." Current Medical Research and Opinion® 22.11 (2006): 2221-2232.
• Benito-Ruiz, P., et al. "A randomized controlled trial on the efficacy and safety of a food ingredient, collagen hydrolysate, for improving joint comfort." International journal of food sciences and nutrition 60.S2 (2009): 99-113. 
• Bruyère, Olivier, et al. "Effect of collagen hydrolysate in articular pain: a 6-month randomized, double-blind, placebo controlled study." Complementary therapies in medicine 20.3 (2012): 124-130.
• Iwai, Koji, et al. "Identification of food-derived collagen peptides in human blood after oral ingestion of gelatin hydrolysates." Journal of agricultural and food chemistry 53.16 (2005): 6531-6536.
• Oesser, Steffen, et al. "Oral administration of 14C labeled gelatin hydrolysate leads to an accumulation of radioactivity in cartilage of mice (C57/BL)." The Journal of nutrition 129.10 (1999): 1891-1895. 
• Ross, B. D., R. Hems, and H. A. Krebs. "The rate of gluconeogenesis from various precursors in the perfused rat liver." Biochem. J 102 (1967): 942-951.
• Saito, Masataka, et al. "Effect of collagen hydrolysates from salmon and trout skins on the lipid profile in rats." Journal of agricultural and food chemistry 57.21 (2009): 10477-10482.
• Watanabe-Kamiyama, Mari, et al. "Absorption and effectiveness of orally administered low molecular weight collagen hydrolysate in rats." Journal of agricultural and food chemistry 58.2 (2009): 835-841.

There is More To Glucose Control Than Carbohydrates (1/?): Non-Carbohydrate Nutrients And Their Effects On Blood Glucose Management ➲ Amino Acids, Proteins, Peptides

This is part I of a multipart series, you will be able to navigate by clicking on the pictures in the box below.

While it appears to be obvious that eating a low-to-no-carbohydrate diet would be the easiest way to manage your blood glucose levels, carbs are by far not the only nutrient that will have an effect on your blood glucose levels. In a recent overview article, Martina Heer and Sarah Egert from the Department of Nutrition and Food Science at the University of Bonn provide a decent overview of the multiple ways by which "other nutrients, such as dietary protein and amino acids, the supply of  fat, vitamin D, and vitamin K, and sodium intake seem to affect glucose homeostasis." (Heer. 2014).

In the coming weeks I will use their review as a starting point for my own overview of the effects of non-carbohydrate and "almost cabohydrate" nutrients  on glucose metabolism. And for today, I decided, to conclude this week that was full of exciting protein news on Monday ("Protein Power" | read more) and Saturday ("Dieting, High Protein, Testosterone & IGF-1" | read more) with - what else could it be - a summary of a the anti-diabetic effects of peptides, proteins and amino acids.

You can learn more about this topic at the SuppVersity

Proteins, Peptides & Blood Glucose

SFA, MUFA, PUFA & Blood Glucose

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Protein, the glucose repartitioner?! Due to its insulinogenic effects protein increases the non-oxidative glucose disposal. In contrast to whey proteins, turkey, beef, eggs and co., i.e. "slow digesting proteins", will induce a significantly reduced insulin surge and have a correspondingly less pronounced effect on blood glucose.

It does not even take whole proteins. Single amino acids and dipeptides (=2-amino acids) can have glucose repoartitioning trick, too | learn more

Insulin is yet probably not the major, or primary agent behind these effects. More recent studies appear to suggest that the mechanism of the blood glucose lowering-effect of whey protein seems to be mediated primarily via increases in glucagon-like peptide 1 (GLP-1). This "satiety hormone" will then, in turn, lead to increased insulin secretion (Lan-Pidhainy. 2010; Maier. 2012). Combined with its ability to decrease gastric emptying and the correspondingly reduced influx of glucose through / from the portal vein (Maier. 2012; Bendtsen. 2013), GLP-1 makes the perfect anti-diabetes "drug" - no wonder that Ligratude, a synthetic analogue is already used very successfully in diabetes and obesity treatment (Astrup. 2012).

In its effects on GLP-1 whey is pretty unique. Even similarly fast absorbing protein sources, such as soy, or other dairy proteins such as casein, do not cause such a pronounced effect on GLP-1 and insulin secretion. A recent study from the Iran University of Medical Sciences and Health Services, for example, compared the effects of the pre-ingestion of additional 65/60g of whey protein concentrate (WPC) and soy protein isolate (ISP) before a meal on a hole host of metabolic markers in 45 healthy overweight and obese men.

Hypoglycemia warning: If experience fatigue, agitation, sweating, shivering, feeling cold, a having really bad temper and/or other symptoms of low blood sugar, after having a bolus of whey protein, it may be a good idea to (a) check your blood glucose levels and (b) consume your whey with a source of readily available carbohydrates in the future.

The first improvements in a hole host of parameters were observed after only two weeks and at the end of the 12-week study period, the consumption of additional 65 gr WPC or 60 gr ISP in 500 ml water 30 min before lunch in a non-restricted diet scenario had brought about significant improvements in

Figure 1: Effects of 12 week of WPC vs. ISP supplementation on fasting blood glucose (Tahavorgar. 2014)

• systolic and diastolic blood pressure
  ⤷ improved heart health, 
• apo lipoprotein A-I and apo B
  ⤷ improved cholesterol metabolism, 
• malondialdehyde
  ⤷ reduced lipid oxidation,
• HDL, LDL and triglycerides
  ⤷ improved lipid metabolism,
• high sensitive C- reactive protein
  ⤷ reduced inflammation

What the scientists did not observe, though, was an improvement in fasting blood glucose levels in the subjects in the soy protein group. The latter was - and that's in line with what we've said before about the effects of different protein sources on GLP-1 - "whey exclusive".

Figure 2: Glucose and insulin release levels after 50g glucose load w/ 30g whey or 30g canola oil (Lan-Pidhainy. 2010)

Similar beneficial effects have been observed, among others, by Lan-Pidhainy, whose study was the first to prove that the insulinotropic effects of whey protein are not attenuated by insulin resistance (Lan-Pidhainy. 2010). In contrast to Tahavorgar et al., the researchers from the University of Toronto measured the acute effects on co-ingesting 30g of whey protein with a standardized 50g glucose load - a study that's obviously of lower real-world significance than the chronic administration scheme in the more recent study by Tahavorgar et al. (2014) or the 60-day bedrest study Martin Heer et al. conducted for the NASA. In this tightly controlled study, the provision of a high protein diet (1.45g/kg body mass/d dietary protein plus 7.2g branched chain amino acids per day) with an "animal:vegetable protein ratio" of 60:40, almost fully compensated for the bed rest-induced 35% reduction in insulin sensitivity during 60-day bed rest.

How does protein work?

The ameliorative, yet not significant effect of soy protein isolate on the blood sugar levels of the subjects in Tahavorgar study, as well as the observations the researchers from University of Bonn made when they studied the effect of bed-rest, confirm that it does not always have to be whey protein to benefit from the anti-diabetic effects of  the chains of amino acid residues we know as "proteins".

Ok, the GLP-1 inducing effects of whey protein appears to be particularly pronounced, and partly related to the presence of certain functional dairy peptides, which may, as the data from a 2009 study by Chen et al. suggests, be even more pronounced for casein than whey (see Figure 3).

Figure 3: Relative GLP-1 production in intestinal cell culture exposed to BCAAs, skim milk or casein (Chen. 2009)

The Chen clearly supports a hypothesis Heer & Egert form in their previously cited review of the contemporary evidence of the involvement of nutrients other than carbohydrates in blood glucose management. Interestingly, though, the evidence the German researchers cite involves yet another non-BCAA amino acid - alanine, which is also the #1 substrate for hepatic de novo glucogenesis:

"Although the mechanism is not well understood, some in vitro studies show how the insulinotropic effect might be induced (Dunne, 1990; Brennan. 2005; Cunningham. 2005). In cell experiments with the application of L-alanine, the increase in insulin secretion might be caused by an increased intracellular oxidation of amino acids, which raises the ATP content of the cell. Increase in intracellular ATP content leads to closure of the ATP-sensitive potassium channels, and this channel closure leads to depolarization of the cell membrane and activation of the calcium channels. Activation of the calcium channels then causes an exocytosis of insulin from the cells (Brennan. 2005; Cunningham. 2005)." (Heer. 2014)

Another possibility could be that amino acids are co-transported into the cell together with
sodium, as shown in further cell experiments [26]. This could also lead to a depolarization of
the plasma membrane in the pancreas and finally to an exocytosis (=pumping process) of insulin.

While 200mcg of chromium are essential, consuming way more can have pro-diabetic effects | learn more.

You can learn more about the other nutrients in the next installment(s): I know that this is not exactly fair, but let's be honest - Aren't there better ways to spend a Sunday afternoon than writing SuppVersity articles? I personally feel the answer is "YES!" The discussion of the effects of dietary fat, in general, (one of the things that's going to be mentioned may already be inferred from Figure 2) and individual fatty acids in particular, as well as the influence of vitamin D, vitamin K, calcium, magnesium, chromium, zinc, sodium, and a couple of other nutrients will thus have to wait until next week (some even longer ;-).

References:

• Astrup, Arne, et al. "Safety, tolerability and sustained weight loss over 2 years with the once-daily human GLP-1 analog, liraglutide." International journal of obesity 36.6 (2012): 843-854. 
• Bendtsen, Line Q., et al. "Effect of dairy proteins on appetite, energy expenditure, body weight, and composition: A review of the evidence from controlled clinical trials." Advances in Nutrition: An International Review Journal 4.4 (2013): 418-438.
• Brennan, Lorraine, et al. "A nuclear magnetic resonance-based demonstration of substantial oxidative L-alanine metabolism and L-alanine-enhanced glucose metabolism in a clonal pancreatic β-cell line metabolism of L-alanine is important to the regulation of insulin secretion." Diabetes 51.6 (2002): 1714-1721.
• Chen, Qixuan, and Raylene A. Reimer. "Dairy protein and leucine alter GLP-1 release and mRNA of genes involved in intestinal lipid metabolism in vitro." Nutrition 25.3 (2009): 340-349. 
• Cunningham, GA, et al. "L-Alanine induces changes in metabolic and signal transduction gene expression in a clonal rat pancreatic β-cell line and protects from pro-inflammatory cytokine-induced apoptosis." Clinical science 109 (2005): 447-455.
• Dunne, M. J., et al. "Effects of alanine on insulin-secreting cells: Patch-clamp and single cell intracellular Ca ²⁺ measurements." Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1055.2 (1990): 157-164.
• Heer, Martina, et al. "High Protein Intake Improves Insulin Sensitivity but Exacerbates Bone Resorption in Immobility (WISE Study)." (2012).
• Lan-Pidhainy, Xiaomiao, and Thomas MS Wolever. "The hypoglycemic effect of fat and protein is not attenuated by insulin resistance." The American journal of clinical nutrition 91.1 (2010): 98-105.
• Meier, Juris J. "GLP-1 receptor agonists for individualized treatment of type 2 diabetes mellitus." Nature Reviews Endocrinology 8.12 (2012): 728-742.
• Tahavorgar, Atefeh, et al. "Effects of whey protein concentrate consumption compared with isolated soy protein on metabolic indices, inflammatory and oxidative stress factors in healthy overweight and obese men." Razi Journal of Medical Sciences 20.115 (2014): 17-30.