Lee Tran

A new method to measure muscle protein synthesis in humans by endogenously introduced d9-leucine and using blood for precursor enrichment determination

Enrichment from the easily accessible blood amino acid pool is commonly used as precursor enrichment to calculate rates of muscle protein fractional synthesis in relevant human studies in lieu of the less accessible muscle fluid amino acid pool. However, the accuracy of this approach depends largely on the extent to which there is low discrepancy in free amino acid enrichment between blood and muscle. Steady‐state gradient (i.e., ratio) of amino acid enrichment between blood and muscle fluid in the basal state and in response to amino acid infusion were determined in five healthy subjects, and in association with two separate tracers: d9‐leucine, introduced endogenously by the metabolism of d10‐leucine (i.e., l‐[2,3,3,4,5,5,5,6,6,6‐2H10]leucine) infused in blood, and 13C6‐phenylalanine introduced/infused in blood. The blood‐to‐muscle fluid amino acid enrichment ratio was lower (P < 0.05) for d9‐leucine compared to 13C6‐phenylalanine both before (1.5 ± 0.1 vs. 2.5 ± 0.1) and during (1.1 ± 0.1 vs. 1.2 ± 0.1) amino acid infusion. Importantly, the decrease in this ratio in association with the amino acid infusion was considerably less for the d9‐leucine than the 13C6‐phenylalanine (−0.38 ± 0.03 vs. −1.29 ± 0.07; P < 0.05). In conclusion, blood d9‐leucine enrichment introduced endogenously by intravenous infusion of d10‐leucine provides a closer estimate of the muscle fluid amino acid enrichment, and its associated changes, than blood phenylalanine enrichment to calculate rates of muscle protein synthesis in humans.

Insulin does not stimulate muscle protein synthesis during increased plasma branched-chain amino acids alone but still decreases whole body proteolysis in humans.

Insulin stimulates muscle protein synthesis when the levels of total amino acids, or at least the essential amino acids, are at or above their postabsorptive concentrations. Among the essential amino acids, branched-chain amino acids (BCAA) have the primary role in stimulating muscle protein synthesis and are commonly sought alone to stimulate muscle protein synthesis in humans. Fourteen healthy young subjects were studied before and after insulin infusion to examine whether insulin stimulates muscle protein synthesis in relation to the availability of BCAA alone. One half of the subjects were studied in the presence of postabsorptive BCAA concentrations (control) and the other half in the presence of increased plasma BCAA (BCAA). Compared with that prior to the initiation of the insulin infusion, fractional synthesis rate of muscle protein (%/h) did not change (P > 0.05) during insulin in either the control (0.04 ± 0.01 vs 0.05 ± 0.01) or the BCAA (0.05 ± 0.02 vs. 0.05 ± 0.01) experiments. Insulin decreased (P < 0.01) whole body phenylalanine rate of appearance (μmol·kg-1·min-1), indicating suppression of muscle proteolysis, in both the control (1.02 ± 0.04 vs 0.76 ± 0.04) and the BCAA (0.89 ± 0.07 vs 0.61 ± 0.03) experiments, but the change was not different between the two experiments (P > 0.05). In conclusion, insulin does not stimulate muscle protein synthesis in the presence of increased circulating levels of plasma BCAA alone. Insulins suppressive effect on proteolysis is observed independently of the levels of circulating plasma BCAA.

Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated with Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase β Subunit.

Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of β-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of β-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with β-F1-ATPase protein translation efficiency in humans (r = – 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased β-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish impaired β-F1-ATPase translation as an important consequence of obesity.

Lower Fasted-State but Greater Increase in Muscle Protein Synthesis in Response to Elevated Plasma Amino Acids in Obesity

Obesity alters protein metabolism in skeletal muscle, but consistent evidence is lacking. This study compared muscle protein synthesis in adults with obesity and in lean controls in the fasted state and during an amino acid infusion.Objective Obesity alters protein metabolism in skeletal muscle, but consistent evidence is lacking. We compared muscle protein synthesis in adults with obesity to that in lean controls in the fasted state and during an amino acid infusion. Methods Ten subjects with obesity (age 36 ± 3 years; BMI 34 ± 1 kg/m2) and ten controls (age 35 ± 3 years; BMI 23 ± 1 kg/m2) received an infusion of L-[2,3,3,4,5,5,5,6,6,6-2H10]leucine (0.15 μmol/kg FFM/min) to measure muscle protein synthesis after an overnight fast and during amino acid infusion. Results Despite greater muscle mTOR phosphorylation (P ≤ 0.05), fasted-state mixed-muscle and mitochondrial protein synthesis were lower in subjects with obesity (P ≤ 0.05). However, the change in mixed-muscle protein synthesis during the amino acid infusion was 2.7-fold greater in subjects with obesity (P ≤ 0.05), accompanied by a greater change in S6K1 phosphorylation (P ≤ 0.05). The change in mitochondrial protein synthesis did not differ between groups (P > 0.05). Conclusions Adults with obesity have reduced muscle protein synthesis in the fasted state, but this response is compensated for by a greater change in overall muscle protein synthesis during amino acid infusion.

Mitochondrial ATP Synthase Beta Subunit production rate and ATP Synthase Specific Activity are reduced in skeletal muscle of humans with obesity

What is the central question of this study? Humans with obesity have lower ATP synthesis in muscle along with lower content of the β‐subunit of the ATP synthase (β‐F1‐ATPase), the catalytic component of the ATP synthase. Does lower synthesis rate of β‐F1‐ATPase in muscle contribute to these responses in humans with obesity? What is the main finding and its importance? Humans with obesity have a lower synthesis rate of β‐F1‐ATPase and ATP synthase specific activity in muscle. These findings indicate that reduced production of subunits forming the ATP synthase in muscle may contribute to impaired generation of ATP in obesity.