Henderson, Gregory 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.

The effects of different modes of exercise on mixed muscle protein synthesis (MMPS) in young (Y) and older (O) adults are not fully understood. We determined the effects of ET, RT, and CT on MMPS in 33Y (25±1y) and 31O (70±1y) adults who were randomized to one of three 8wk interventions: (i) Control: no exercise, (ii) ET: cycling 1h @ 65% VO 2 peak, 5x/wk, (iii) RT: RT 4x/wk. After completing the control period, the controls completed an 8wk (iv) CT program (ET & RT). Subjects were studied at baseline and 48h following the last training bout. [ 13 C 6 ]Phe was infused for 8h, with serial v. lateralis biopsies. Isotopic enrichments (IE) were measured by LC/MS for free Phe from tissue fluid (TF) and from that incorporated into MMP. MMPS (%/h) = (ΔIE MMP )/(IE TF )/t*100. A rank sum test was used to test baseline differences between Y and O. 2‐way ANCOVA was used to test for differences between groups by treatment, with baseline values serving as the covariate. MMPS (%/h) tended to be higher in Y (0.060±0.003) compared to O (0.053±0.003) (P=0.06). ET [57%, P<0.0001], RT [21%, P=0.042] and CT [42%, P<0.0001] increased MMPS independently of age (P>0.4). The ET‐induced increase in MMPS was greater than the RT‐induced increase (P<0.05), although ET unlike RT does not increase muscle mass suggesting that muscle protein turnover was increased. In conclusion, ET, RT and CT were effective at increasing MMPS independently of age. R01AG09531, KL2RR024151

Diet-induced insulin resistance (IR) adversely affects human health and life span. We show that muscle-specific overexpression of human mitochondrial transcription factor A (TFAM) attenuates high-fat diet (HFD)–induced fat gain and IR in mice in conjunction with increased energy expenditure and reduced oxidative stress. These TFAM effects on muscle are shown to be exerted by molecular changes that are beyond its direct effect on mitochondrial DNA replication and transcription. TFAM augmented the muscle tricarboxylic acid cycle and citrate synthase facilitating energy expenditure. TFAM enhanced muscle glucose uptake despite increased fatty acid (FA) oxidation in concert with higher β-oxidation capacity to reduce the accumulation of IR-related carnitines and ceramides. TFAM also increased pAMPK expression, explaining enhanced PGC1α and PPARβ, and reversing HFD-induced GLUT4 and pAKT reductions. TFAM-induced mild uncoupling is shown to protect mitochondrial membrane potential against FA-induced uncontrolled depolarization. These coordinated changes conferred protection to TFAM mice against HFD-induced obesity and IR while reducing oxidative stress with potential translational opportunities.

Context: Skeletal muscle from sedentary older adults exhibits reduced mitochondrial abundance and oxidative capacity. Objective: The primary objective was to determine whether 8 weeks of combined training (CT) has a more robust effect than endurance training (ET) or resistance training (RT) on mitochondrial physiology in healthy young (18–30 years) and older (≥65 years) adults. Intervention: Thirty-four young and 31 older adults were randomly assigned to 8 weeks of ET, RT, and control/CT. Control subjects completed 8 weeks of no exercise (control) followed by 8 weeks of CT. Body composition, skeletal muscle strength, and peak oxygen uptake were measured before and after the intervention. Vastus lateralis muscle biopsy samples were obtained before and 48 hours after the intervention. Mitochondrial physiology was evaluated by high-resolution respirometry and expression of mitochondrial proteins and transcription factors by quantitative PCR and immunoblotting. Results: ET and CT significantly increased oxidative capacity and expression of mitochondrial proteins and transcription factors. All training modalities improved body composition, cardiorespiratory fitness, and skeletal muscle strength. CT induced the most robust improvements in mitochondria-related outcomes and physical characteristics despite lower training volumes for the ET and RT components. Importantly, most of the adaptations to training occurred independent of age. Conclusion: Collectively, these results demonstrate that both ET and CT increase muscle mitochondrial abundance and capacity although CT induced the most robust improvements in the outcomes measured. In conclusion, CT provides a robust exercise regimen to improve muscle mitochondrial outcomes and physical characteristics independent of age.

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.

OBJECTIVE The accumulation of old and damaged proteins likely contributes to complications of diabetes, but currently no methodology is available to measure the relative age of a specific protein alongside assessment of posttranslational modifications (PTM). To accomplish our goal of studying the impact of insulin deficiency and hyperglycemia in type 1 diabetes upon accumulation of old damaged isoforms of plasma apolipoprotein A-1 (ApoA-1), we sought to develop a novel methodology, which is reported here and can also be applied to other specific proteins. RESEARCH DESIGN AND METHODS To label newly synthesized proteins, [ring-13C6]phenylalanine was intravenously infused for 8 h in type 1 diabetic participants (n = 7) during both insulin treatment and 8 h of insulin deprivation and in nondiabetic participants (n = 7). ApoA-1 isoforms were purified by two-dimensional gel electrophoresis (2DGE) and assessment of protein identity, PTM, and [ring-13C6]phenylalanine isotopic enrichment (IE) was performed by tandem mass spectrometry. RESULTS Five isoforms of plasma ApoA-1 were identified by 2DGE including ApoA-1 precursor (pro-ApoA-1) that contained the relatively highest IE, whereas the older forms contained higher degrees of damage (carbonylation, deamidation) and far less IE. In type 1 diabetes, the relative ratio of IE of [ring-13C6]phenylalanine in an older isoform versus pro-ApoA-1 was higher during insulin deprivation, indicating that de novo synthesized pro-ApoA-1 more rapidly accumulated damage, converting to mature ApoA-1. CONCLUSIONS We developed a mass spectrometry–based methodology to identify the relative age of protein isoforms. The results demonstrated accelerated oxidative damage to plasma ApoA-1, thus offering a potential mechanism underlying the impact of poor glycemic control in type 1 diabetic patients that affects a patient's risk for vascular disease.

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)