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Post-Workout High Isoleucine AA+CHO Decreases Glucose Spikes, But Impairs Musclular Glyocogen Resynthesis - Reason Enough to Skip Amino Acids?

If you put any faith into the promises of the supplement industry, amino acid supplements are the solution to all your problems - including those you havent even known about, yet. Against that background its always interesting if scientists study the real world effects of amino acid supplements in a realistic scenario like after strenuous exercise.

In their latest study Wang and colleagues from the University of Texas at Austin and the Shanghai Research Institute of Sports Science did just that: They studied the effects isoleucine and four additional amino acids, on blood glucose homeostasis and glycogen synthesis after strenuous exercise.
As the scientists point out, the results of their study "could provide a practical and safe means of increasing the rate of muscle glycogen synthesis after exercise and enhancing the rate of recovery" (Wang. 2015).

Table 1:  Subjects’ characteristics (Wang. 2015).

Ten healthy active adults volunteered for the study. All subjects were accustomed to cycling for prolonged periods of 3–5 h during an exercise session. The ,aximum oxygen uptake (VO2max) was measured in all subjects on a cycle ergometer by using a TrueOne 2400 metabolic measurement system (ParvoMedics, Sandy, Utah) to verify adequate aerobic fitness levels (results see Table 1).

Figure 1: Basically the AA supplement contained almost exclusively isoleucine. It was administered in the dosage shown above and at twice that amount in the LAA and HAA trials (Wang. 2015)

"Two to three days after the VO2max test, the subjects reported to the laboratory to perform a practice ride to familiarize them with the laboratory environment and the experimental protocol. The practice ride was also used to adjust and verify appropriate workloads for the experimental trials. The practice rides simulated the protocol ride but without blood samples or muscle biopsies being taken. The ride consisted of cycling at 70 % VO2max for 2 h, which was followed by five 1-min sprints at 85 % VO2max. The sprints were separated by 1 min cycling at 45 % VO2max. During the first 15 min of each hour, oxygen uptake was measured for 5 min to verify workload.

Water (250 mL) was provided every 20 min of exercise. Heart rate (HR) was monitored and ratings of perceived exertion (RPE) on a Borg-scale (ranging from 6 to 20) were collected every 30 min of exercise. The practice ride and each of the following three experimental trials were separated by a minimum of 7 days and maximum of 12 days" (Wang. 2015).

The actual tests consisted of cycling on an ergometer to deplete muscle glycogen. Blood sampling and a muscle biopsy were performed immediately on cessation of exercise. After the muscle biopsy, subjects were given the first of two supplement doses. More specifically they received either...

• 1.2 g carbohydrate/kg body weight (CHO), 1.2 g carbohydrate/kg body weight plus 6.5 g AA (CHO/LAA) or 
• the same carbohydrate supplement plus 6.5g (CHO/LAA) or 13 g AA (CHO/HAA) 

immediately after the first muscle biopsy and at 120 min of recovery. The carbohydrate base consisted of simple dextrose dissolved at a ratio of 100g/296 mL in an orange flavored drink (SUN-DEX, Fisher Healthcare, Houston, Texas). The additional amino acids contained 0.046 g cystine 2HCl, 0.023 g methionine, 0.045 g valine, 6.342 g Isoleucine, and 0.044 g leucine per person, or twice that amount in the CHO/HAA trial. The amino acids were simply added to the dextrose drink.

Why would you even believe that there may be benefits from AA supplementation?

As Wang et al. point out, "this amino acid mixture was selected as it was previously reported to be more effective in lowering the blood glucose response to a glucose challenge than isoleucine alone" (Wang. 2015) by Bernard et al. (2011).

Figure 2: Blood glucose AUC during the oral glucose tolerance test (OGTT). Sprague-Dawley rats were gavaged with either glucose (CHO), glucose plus a 5-amino acid mixture (CHO-AA-1), glucose plus a 5-amino acid mixture with increased leucine concentration (CHO-AA-2), or placebo (PLA). Blood was taken from the tail immediately before the gavage and 15, 30, 60, and 120 min afterward (Bernard. 2011).

The three test beverages were similar in color, taste, and texture to allow a double-blinded and counter-balanced study design. All test drinks were randomly assigned and dispensed by a laboratory technician who was not involved in the data collection.

Figure 3: Blood glucose postexercise and during the 4-h recovery. Treatments were with CHO (circle), CHO/LAA (triangle), and CHO/HAA (filled circle) supplements provided immediately after and 2 h after exercise. Values are mean ± SE. CHO/HAA vs. CHO (*p < 0.05). CHO/LAA vs. CHO (# p < 0.05) - left; Blood glucose area under the curve (AUC) during the 4-h recovery. Treatments were CHO, CHO/LAA, and CHO/HAA supplements provided immediately after and 2 h after exercise. AUC was calculated with baseline (pre). Values are mean ± SE. CHO/HAA vs. CHO (*p < 0.05). CHO/LAA vs. CHO (# p < 0.05) - right (Wang. 2015).

As the data in Figure 3 indicates,There was a similar effect in humans as it has previously been observed in rodents. An effect of which you as a SuppVersity reader know that it is probably mostly ascribable to isoleucine (see "The Glucose-Repartitioning Effects of Isoleucine" | more).

Figure 4: Total muscle glycogen storage in the vastus lateralis during the 4-h recovery from intense cycling. Treatments were CHO, CHO/LAA, and CHO/HAA supplements provided immediately after and 2 h after exercise. Values are mean ± SE. CHO/HAA vs. CHO (*p < 0.05 | Wang. 2015)

What is a bit disappointing is the fact that the decrease in blood glucose did not come with an increase in glycogen storage.

As the data in Figure 4 shows, the exact opposite was the case. After 4h of recovery the muscle glycogen levels were not higher, but lower in the amino acid supplemented trials.

For diabetics this wouldnt be a problem. For athletes its yet clearly a disadvantage that the 4-g recovery glycogen levels were lower and significantly lower in the low and high dose amino acid supplement trials.

Eventually this result is surprising because specifically in the high amino acid group (a) the insulin levels, (b) the AS160, a protein that controls insulin mediated glucose uptake, (c) the mTOR & p-AKT levels, (d) the "exercise hormon" levels of serum irisin  and (e) the levels of glycogen synthase which stores carbs in forms of glycogen in the high dose AA trials were significantly elevated.
References:

• Armstrong, Jane L., et al. "Regulation of glycogen synthesis by amino acids in cultured human muscle cells." Journal of biological Chemistry 276.2 (2001): 952-956.
• Bernard, Jeffrey R., et al. "An amino acid mixture improves glucose tolerance and insulin signaling in Sprague-Dawley rats." American Journal of Physiology-Endocrinology and Metabolism 300.4 (2011): E752-E760.
• Doi, Masako, et al. "Isoleucine, a potent plasma glucose-lowering amino acid, stimulates glucose uptake in C2C12 myotubes." Biochemical and biophysical research communications 312.4 (2003): 1111-1117. 
• Ivy, John L., et al. "Early postexercise muscle glycogen recovery is enhanced with a carbohydrate-protein supplement." Journal of Applied Physiology 93.4 (2002): 1337-1344.
• Ivy, J. L., et al. "Post exercise carbohydrate–protein supplementation: phosphorylation of muscle proteins involved in glycogen synthesis and protein translation." Amino acids 35.1 (2008): 89-97.
• Morifuji, Masashi, et al. "Dietary whey protein increases liver and skeletal muscle glycogen levels in exercise-trained rats." British journal of nutrition 93.04 (2005): 439-445.
• Morifuji, Masashi, et al. "Post-exercise carbohydrate plus whey protein hydrolysates supplementation increases skeletal muscle glycogen level in rats." Amino acids 38.4 (2010): 1109-1115.
• Wang, Bei, et al. "Amino acid mixture acutely improves the glucose tolerance of healthy overweight adults." Nutrition Research 32.1 (2012): 30-38.
• Zawadzki, K. M., B. B. Yaspelkis, and J. L. Ivy. "Carbohydrate-protein complex increases the rate of muscle glycogen storage after exercise." J Appl Physiol 72.5 (1992): 1854-9.

Trp and its metabolite 5-HTP may be particularly useful for female sugar cravings and binges.

Can l-tryptophan help you lose body fat? If you look at the results of the latest study from the University for Health Sciences, Medical Informatics and Technology it would seem that the answer to this question may be "Possibly, yes, but..." Before we come to the implications I would yet like to take a closer look at said study which shows that a lack of tryptophan (Trp) during diets does not just affect the biosynthesis of serotonin, but may also be associated with increased susceptibility for mood disturbances and carbohydrate craving. Accordingly, "strategies to supplement Trp while dieting could be highly useful in treating uncontrolled weight gain or in preventing neuropsychiatric symptoms" (Strasser. 2014).
As Strasser et al. point out, both overweight and obesity go hand in hand with significant increases in low-grade inflammation. The latter is not just the reason that obesity increases the risk of cardiovascular disease, though. Recent evidence suggests that it is also associated with errors in the kynurenine (Kyn) pathway, in which tryptophan is broken down to kynurenine which in turn has been associated with increased risk of depressive symptoms, cognitive deficits in schizophrenia, Alzheimers and, as mentioned before, cardiovascular disease. Weight loss, on the other hand,

"[...] has been shown to improve or prevent many of the aforementioned conditions. Bariatric surgical intervention in patients with adiposity was found not to improve tryptophan breakdown rates and other signs of immune activation and inflammation [4], whereas caloric restriction is known to be a strong activator of protective metabolic pathways, thereby leading to lower blood pressure, improved blood lipids, and reduced inflammatory markers, including CRP [9]. Still, little is known about the effects of an extreme short-term hypocaloric diet on Trp metabolism and changes in inflammatory biomarkers" (Strasser. 2014).

The study Barbara Strasser, Ken Berger and Dietmar Fuchs conducted was thus designed to assess the effect of a 2-week caloric restriction weight loss diet on Trp breakdown, leptin, and inflammatory biomarkers in over weight adults.
The scientists randomized 27 overweight and 11 obese participants (22 men and 16 women, mean age 52.8 ± 9.1 years) from the health center Lanserhof, Innsbruck–Lans, into two diet groups:

• a very low kcal diet group (VLCD; Ø 600 kcal/ day) and 
• a low kcal diet group (LCD; Ø 1,200 kcal/day). 

Only healthy subjects with BMI [25 kg/m²] between the ages of 35 and 70 years were accepted for the study. A physician performed physical examinations on all subjects before the study. Subjects were excluded if they consume any anti-inflammatory drugs (e.g., ibuprofen or aspirin) or supplements (such as antioxidants or fish-oil capsules). None from either group was involved in regular training programs.

Figure 1: Changes in body composition pre- vs. post (Strasser. 2015).

As the measurements of body composition, which were just like the energy intake and biologic markers conducted in all subjects before and after the 2-week energy restriction intervention period, indicate, both diets lead to significant reductions in body mass - and that almost exclusively in form of body fat.

Table 1: Biologic markers before and after a 2-week very low kcal diet (VLCD) or low kcal die (LCD) in 38 overweight subjects (mean ± SD)

"Data for biologic markers are shown in Table [1]. Fasting blood glucose declined significantly (P < 0.05) in the LCD group with no significant changes in insulin sensitivity in both groups after 2 weeks of caloric restriction. Weight loss diet lowered leptin levels in both groups, although not reaching the level of significance. Inflammatory biomarkers were not significantly altered during the trial, although there was a tendency toward an increase in IL-6 and TNF-a in the LCD group" (Strasser. 2015).

In contrast to what the researchers expected, both the Trp and Kyn concentrations decreased significantly by 21 and 16 % for VLCD and by 15 and 17 % for the LCD group, respectively, with no significant difference between groups. Practically speaking, this means that the ratio of Kyn/Trp concentrations did not change significantly in both groups.
A significant reduction in Phe concentrations was only seen after VLCD. Neopterin and Tyr levels remained unchanged during the trial. Which leaves us with only one significant finding:

"Trp concentrations decreased significantly with a caloric restriction weight loss diet, and lowest Trp concentrations were observed in the group of individuals with the lowest calorie intake." (Strasser. 2015)

This reduction in Trp levels may well induce a disturbance in the biosynthesis of neurotransmitter 5-hydroxytryptamine (5-HT | Anderson. 1990), and appears to be associated with an increased susceptibility for depression (Widnet. 2002; Raison. 2009). Strasser et al. highlight:

Figure 2: The consumption of tryptophan-free amino acid supplements leads to highly significant increases in hunger ratings in healthy female subjects (Rieber. 2010).

"Because Trp is precursor in various biochemical pathways, e.g., it is hydroxylated by tryptophan-5-hydroxylase (T5H) into the intermediate product 5-hydroxy-tryptophan, which by decarboxylation is further converted to neurotransmitter 5-HT (serotonin), and because substrate saturation of T5H is only about 50 % (Dantzer. 2011), changes in plasma Trp levels may have an immediate impact on brain serotonin levels" (Strasser. 2014).

Experiments in which Trp was acutely depleted (in many studies by administering BCAAs | see red boy) support this assumption. Young et al. (2013), for example, confirmed that the acute depletion of tryptophan will lead to low serotonin and subsequently lower mood and increased aggression, although results vary somewhat between studies with similar participants.

Figure 3: Correlations between changes in tryp:LNAA ratio and appetite ratings (Gendall. 2000).

For the link to obesity, though, the correlation (r-values in Figure 3) between high Trp:LNAA (BCAAs, tyrosine, phenlylanine) and a reduction carbohydrate cravings, general hunger and binge eating is yet way more important - and that specifically for women, who appear more vulnerable than men both to the diet-induced reductions in Trp and to its consequences for brain serotonin function (Anderson. 1990).

Ah, and in case you are asking yourself why carbohydrate / sugar binges are a common consequence of low tryptophane:LNAA ratios, its important to know that increases in glucose and insulin in response to high carbohydrate meals will trigger an increase in brain tryptophan and serotonin synthesis (Benton. 2002). This is why the effects of low tryptophan or high LNAA (BCAA, tyrosine, phenylalanine) levels are more pronounced if you avoid dietary carbohydrates.
References:

• Anderson, I. M., et al. "Dieting reduces plasma tryptophan and alters brain 5-HT function in women." Psychological medicine 20.04 (1990): 785-791. 
• Benton, David. "Carbohydrate ingestion, blood glucose and mood." Neuroscience & Biobehavioral Reviews 26.3 (2002): 293-308.
• Cangiano, Carlo, et al. "Eating behavior and adherence to dietary prescriptions in obese adult subjects treated with 5-hydroxytryptophan." The American journal of clinical nutrition 56.5 (1992): 863-867.
• Cangiano, Carlos, et al. "Effects of oral 5-hydroxy-tryptophan on energy intake and macronutrient selection in non-insulin dependent diabetic patients." International journal of obesity and related metabolic disorders: journal of the International Association for the Study of Obesity 22.7 (1998): 648-654.
• Ceci, F., et al. "The effects of oral 5-hydroxytryptophan administration on feeding behavior in obese adult female subjects." Journal of neural transmission 76.2 (1989): 109-117.
• Cui, Y., T. F. Lee, and L. C. H. Wang. "Thermoregulatory responses following injection of 5-hydroxytryptamine into the septohippocampal complex in rats." Pharmacology Biochemistry and Behavior 45.4 (1993): 935-939.
• Dantzer, Robert, et al. "Inflammation-associated depression: from serotonin to kynurenine." Psychoneuroendocrinology 36.3 (2011): 426-436. 
• Fernstrom, John D. "Branched-chain amino acids and brain function." The Journal of nutrition 135.6 (2005): 1539S-1546S.
• Gendall, Kelly A., and Peter R. Joyce. "Meal-induced changes in tryptophan: LNAA ratio: effects on craving and binge eating." Eating behaviors 1.1 (2000): 53-62. 
• Le Feuvre, R. A., L. Aisenthal, and N. J. Rothwell. "Involvement of corticotrophin releasing factor (CRF) in the thermogenic and anorexic actions of serotonin (5-HT) and related compounds." Brain research 555.2 (1991): 245-250.
• Nieuwenhuizen, Arie G., et al. "Acute effects of breakfasts containing ?-lactalbumin, or gelatin with or without added tryptophan, on hunger,‘satiety’hormones and amino acid profiles." British journal of nutrition 101.12 (2009): 1859-1866.
• Raison, Charles L., et al. "CSF concentrations of brain tryptophan and kynurenines during immune stimulation with IFN-?: relationship to CNS immune responses and depression." Molecular psychiatry 15.4 (2009): 393-403.
• Rieber, N., et al. "Acute tryptophan depletion increases experimental nausea but also induces hunger in healthy female subjects." Neurogastroenterology & Motility 22.7 (2010): 752-e220.
• Strasser, Barbara, Ken Berger, and Dietmar Fuchs. "Effects of a caloric restriction weight loss diet on tryptophan metabolism and inflammatory biomarkers in overweight adults." European journal of nutrition (2014): 1-7.
• Widner, Bernhard, et al. "Neopterin production, tryptophan degradation, and mental depression—What is the link?." Brain, behavior, and immunity 16.5 (2002): 590-595.
• Young, Simon N. "The effect of raising and lowering tryptophan levels on human mood and social behaviour." Philosophical Transactions of the Royal Society B: Biological Sciences 368.1615 (2013): 20110375.