Ford, G. C.

Skeletal muscle protein synthesis rates decline with age. PURPOSE: We sought to determine if DHEA supplementation in elderly men (EM) and elderly women (EW) and low dose testosterone (T) supplementation in EM would raise protein synthesis rates. METHODS: 87 EM (with low bioavailable T and DHEAS) and 57 EW (with low DHEAS) were studied before and after 1 year in which EM received either T (5 mg/d), DHEA (75 mg/d), or placebo (P) and EW received either DHEA (50 mg/d) or P. To assess age effects, 30 young men and 32 young women were studied on a single occasion as controls. [ 15 N]phenylalanine and [ 2 H 4 ]tyrosine tracers were infused and isotopic enrichment was measured in plasma and v. lateralis muscle. Whole body amino acid kinetics were normalized to fat free mass, and mixed muscle protein fractional synthesis rate (FSR) was measured using plasma phenylalanine as the precursor. RESULTS: Despite induction of elevated androgen levels, no significant differences were seen between treated groups and the P group in either EM or EW. Whole body protein synthesis and muscle FSR were lower in elderly than in young (P < 0.05) and were higher in women than men (P < 0.05), with no age‐by‐gender interaction. CONCLUSIONS: Neither DHEA nor T supplementation for one year had any significant impact on age‐related decline in protein synthesis, while both age and gender do impact protein synthesis rates. NIH grants P01 AG14283 and UL1‐RR‐024150‐01.

Aging is associated with decreasing muscle mass and protein synthesis. Amino acid administration is reported to increase muscle protein synthesis and balance. We determined whether increasing dietary protein content from adequate (1.5g/kgFFM/d, AP) to high (3g/kgFFM/d, HP) had beneficial or adverse effects on whole body and muscle protein metabolism, renal function and insulin sensitivity in healthy young (24±1y) and older (70±2y) men and women. Subjects were randomized to a crossover design of 10 d of AP or HP protein diets, without changing caloric intake. Measurements of postabsoptive whole body leucine appearance rate (protein breakdown), oxidation, and non‐oxidative leucine disposal (protein synthesis) rates were performed using L‐[1‐ 13 C]‐leucine and fractional synthesis rate (FSR) of mixed muscle proteins was done using [ 15 N]‐lysine as tracers. Glomerular filtration rate (GFR) and insulin sensitivity were determined by iodothalamate and intravenous glucose tolerance test, respectively. HP diet increased protein breakdown in both young and old (p<0.05) and increased leucine oxidation in both age groups with greater increase in young (p<0.005). WBPS, muscle protein FSR and insulin sensitivity were unaffected by diet. GFR was lower (p<0.01) in older than in young during both diets. HP diet increased GFR in young (p<0.01) but decreased GFR in older people (p<0.01). In conclusion, increasing dietary protein content had no detectable beneficial effects on muscle or whole body protein metabolism in healthy older people and may cause deleterious effects on renal function. Support: NIH R01AG09531.

It has been shown ingestion of whey protein (WP) results in rapid systemic appearance rates of amino acids (AA) but in higher amino acid (AA) oxidation and whole body protein synthesis than in with casein (cas) ingestion which inhibits protein breakdown. To determine whether combined ingestion of both cas and WP in a meal containing carbohydrate has any disparate effects on protein economy in splanchnic and muscle beds we studied 12 healthy volunteers who ingested intrinsically labeled WP and Cas protein fractions. Following ingestion of 0.625 g·kg FFM −1 each of [ 15 N]WP and [ 13 C]Cas and 0.9 g·kg FFM −1 of lactose; or 0.625 g·kg FFM −1 each of [ 13 C]WP and [ 15 N]CAS and 0.9 g·kg FFM −1 of lactose blood samples were collected from femoral artery and vein and hepatic vein at frequent intervals. At 80–120 min, the rate of appearance (PheRa) of Phe at hepatic vein from WP and Cas were similar (5.5 (0.9) vs 5.2 (0.7) μmol, min −1 , but at 220–260 min, PheRa from WP was slower than PheRa from Cas (2.4 (0.5) vs 5.3 (1.2) μmol, min −1 , P<0.05). Retention rates of Phe in muscle (PheRd) absorbed from WP and Cas were similar at 80–120 min (0.8 (0.1) vs 0.9 (0.2) μmol, min −1 , ns) but was significantly lower for WP than Cas at 220–260 min (0.5 (0.1) vs 1.1 (0.3) μmol, min −1 , P<0.05). In conclusion Cas has more sustained systemic effects and muscle protein accretion than WP. The different metabolic effects of co‐ingested protein fractions have potential implications in designing appropriate nutritional support.

Muscle protein synthesis was measured by infusion ofl-[2H5]phenylalanine in two groups of anesthetized dogs, before and during infusion of insulin with euaminoacidemia, and with differing concentrations of unlabeled phenylalanine (tracee). With the infusion of insulin, muscle protein synthesis increased 39 ± 12% based on phenylalanyl-tRNA. Calculation with plasma phenylalanine enrichment overestimated insulin stimulation by 40% (56 ± 12 vs. 39 ± 12%). Raising the concentration of plasma phenylalanine twofold during infusion of insulin further increased the apparent stimulation of muscle protein synthesis based on plasma relative to phenylalanyl-tRNA by 225% (65 ± 19 vs. 20 ± 14%, P < 0.001). In both experiments, the stimulation of synthesis rates calculated from phenylalanine enrichment within the muscle was closer to that from phenylalanyl-tRNA (48 ± 19%, experiment 1; 30 ± 14%, experiment 2). Results indicate that the enrichment of a labeled amino acid within plasma and tissue amino acid pools is affected by the concentration of tracee infused. Increasing the concentration of tracee overestimates the insulin-mediated stimulation of muscle protein synthesis when amino acid pools other than aminoacyl-tRNA are used as the precursor enrichment.

Muscle protein synthesis in dogs measured by flooding withl-[2H5]phenylalanine (70 mg/kg) was significantly stimulated by infusion of insulin with amino acids. The stimulation of muscle protein synthesis was similar when calculated from the enrichment of phenylalanyl-tRNA (61 ± 10%, P< 0.001), plasma phenylalanine (61 ± 10%, P < 0.001), or tissue fluid phenylalanine (54 ± 10%, P < 0.001). The time course for changes in enrichment ofl-[2H5]phenylalanine throughout the flooding period was determined for plasma, tissue fluid, and phenylalanyl-tRNA in the basal state and during the infusion of insulin with amino acids. Enrichments of plasma free phenylalanine and phenylalanyl-tRNA were equalized between 20 and 45 min, although the enrichment of phenylalanyl-tRNA was lower at early time points. Rates of muscle protein synthesis obtained with the flooding method and calculated from plasma phenylalanine enrichment were comparable to those calculated from phenylalanyl-tRNA and also to those obtained previously with a continuous infusion of phenylalanine with phenylalanyl-tRNA as precursor. This study confirms that, with a bolus injection of labeled phenylalanine, the enrichment of aminoacyl-tRNA, the true precursor pool for protein synthesis, can be assessed from more readily sampled plasma phenylalanine.

Precise measurement of low enrichment of stable isotope labeled amino‐acid tracers in tissue samples is a prerequisite in measuring tissue protein synthesis rates. The challenge of this analysis is augmented when small sample size is a critical factor. Muscle samples from human participants following an 8 h intravenous infusion of L‐[ring‐13C6]phenylalanine and a bolus dose of L‐[ring‐13C6]phenylalanine in a mouse were utilized. Liquid chromatography tandem mass spectrometry (LC/MS/MS), gas chromatography (GC) MS/MS and GC/MS were compared to the GC‐combustion‐isotope ratio MS (GC/C/IRMS), to measure mixed muscle protein enrichment of [ring‐13C6]phenylalanine enrichment. The sample isotope enrichment ranged from 0.0091 to 0.1312 molar percent excess. As compared with GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS showed coefficients of determination of R2 = 0.9962 and R2 = 0.9942, and 0.9217 respectively. However, the precision of measurements (coefficients of variation) for intra‐assay are 13.0%, 1.7%, 6.3% and 13.5% and for inter‐assay are 9.2%, 3.2%, 10.2% and 25% for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. The muscle sample sizes required to obtain these results were 8 µg, 0.8 µg, 3 µg and 3 µg for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. We conclude that LC/MS/MS is optimally suited for precise measurements of L‐[ring‐13C6]phenylalanine tracer enrichment in low abundance and in small quantity samples. Copyright © 2013 John Wiley & Sons, Ltd.

When consumed separately, whey protein (WP) is more rapidly absorbed into circulation than casein (Cas), which prompted the concept of rapid and slow dietary protein. It is unclear whether these proteins have similar metabolic fates when coingested as in milk. We determined the rate of appearance across the splanchnic bed and the rate of disappearance across the leg of phenylalanine (Phe) from coingested, intrinsically labeled WP and Cas. Either [15N]Phe or [13C- ring C6]Phe was infused in lactating cows, and the labeled WP and Cas from their milk were collected. To determine the fate of Phe derived from different protein sources, 18 healthy participants were studied after ingestion of one of the following: 1) [15N]WP, [13C]Cas, and lactose; 2) [13C]WP, [15N]Cas, and lactose; 3) lactose alone. At 80–120 min, the rates of appearance (Ra) across the splanchnic bed of Phe from WP and Cas were similar [0.068 ± 0.010 vs. 0.070 ± 0.009%/min; not significant (ns)]. At time 220–260 min, Phe appearance from WP had slowed (0.039 ± 0.008%/min, P < 0.05) whereas Phe appearance from Cas was sustained (0.068 ± 0.013%/min). Similarly, accretion rates across the leg of Phe absorbed from WP and Cas were not different at 80–120 min (0.011 ± 0.002 vs. 0.012 ± 0.003%/min; ns), but they were significantly lower for WP (0.007 ± 0.002%/min) at 220–260 min than for Cas (0.013 ± 0.002%/min) at 220–260 min. Early after meal ingestion, amino acid absorption and retention across the leg were similar for WP and Cas, but as rates for WP waned, absorption and assimilation into skeletal muscle were better retained for Cas.

Insulin regulates many cellular processes, but the full impact of insulin deficiency on cellular functions remains to be defined. Applying a mass spectrometry–based nontargeted metabolomics approach, we report here alterations of 330 plasma metabolites representing 33 metabolic pathways during an 8-h insulin deprivation in type 1 diabetic individuals. These pathways included those known to be affected by insulin such as glucose, amino acid and lipid metabolism, Krebs cycle, and immune responses and those hitherto unknown to be altered including prostaglandin, arachidonic acid, leukotrienes, neurotransmitters, nucleotides, and anti-inflammatory responses. A significant concordance of metabolome and skeletal muscle transcriptome–based pathways supports an assumption that plasma metabolites are chemical fingerprints of cellular events. Although insulin treatment normalized plasma glucose and many other metabolites, there were 71 metabolites and 24 pathways that differed between nondiabetes and insulin-treated type 1 diabetes. Confirmation of many known pathways altered by insulin using a single blood test offers confidence in the current approach. Future research needs to be focused on newly discovered pathways affected by insulin deficiency and systemic insulin treatment to determine whether they contribute to the high morbidity and mortality in T1D despite insulin treatment.

We investigated age and sex effects and determined whether androgen replacement in elderly individuals (≥60 yr) could augment protein synthesis. Thirty young men and 32 young women (18‐31 yr) were studied once, whereas 87 elderly men were studied before and after 1 yr of treatment with 5 mg/day testosterone (T), 75 mg/day dehydroepiandrosterone (DHEA), or placebo (P);and 57 elderly women were studied before and after 1 yr of treatment with 50 mg/day DHEA or P. [ 15 N]Phenylalanine and [ 2 H 4 ]tyrosine tracers were infused, with measurements in plasma and vastus lateralis muscle. Whole‐body protein synthesis per fat‐free mass and muscle protein fractional synthesis rate (FSR) were lower in elderly than in young individuals (P< 0.001), not significantly affected by hormone treatments, and higher in women than in men (P<0.0001), with no sex × age interaction. In regression analyses, peak O 2 consumption (VO 2peak ), resting energy expenditure (REE), and sex were independently associated with muscle FSR, as were VO 2peak , REE, and interactions of sex with insulin‐like growth factor‐II and insulin for whole‐body protein synthesis. Women maintain higher protein synthesis than men across the lifespan as rates decline in both sexes, and neither full replacement of DHEA (in elderly men and women) nor partial replacement of bioavailable T (in elderly men) is able to amend the age‐related declines.— Henderson, G. C., Dhatariya, K., Ford, G. C., Klaus, K. A., Basu, R., Rizza, R. A., Jensen, M. D., Khosla, S., O'Brien, P., Nair, K. S. Higher muscle protein synthesis in women than men across the lifespan, and failure of androgen administration to amend age‐related decrements. FASEB J. 23, 631–641 (2009)