Short, Kevin R.

Rodent skeletal muscle mitochondrial DNA has been shown to be a potential site of oxidative damage during aging. Caloric restriction (CR) is reported to reduce oxidative stress and prolong life expectancy in rodents. Gene expression profiling and measurement of mitochondrial ATP production capacity were performed in skeletal muscle of male rats after feeding them either a control diet or calorie-restricted diet (60% of control diet) for 36 wk to determine the potential mechanism of the beneficial effects of CR. CR enhanced the transcripts of genes involved in reactive oxygen free radical scavenging function, tissue development, and energy metabolism while decreasing expression of those genes involved in signal transduction, stress response, and structural and contractile proteins. Real-time PCR measurments confirmed the changes in transcript levels of cytochrome- c oxidase III, superoxide dismutase (SOD)1, and SOD2 that were noted by the microarray approach. Mitochondrial ATP production and citrate synthase were unaltered by the dietary changes. We conclude that CR alters transcript levels of several genes in skeletal muscle and that mitochondrial function in skeletal muscle remains unaltered by the dietary intervention. Alterations in transcripts of many genes involved in reactive oxygen scavenging function may contribute to the increase in longevity reported with CR.

High-fat diets are reported to increase oxidative stress in a variety of tissues, whereas antioxidant supplementation prevents many diseases attributed to high-fat diet. Rodent skeletal muscle mitochondrial DNA has been shown to be a potential site of oxidative damage. We hypothesized that the effects of a high-fat diet on skeletal muscle DNA functions would be attenuated or partially reversed by antioxidant supplementation. Gene expression profiling and measurement of mitochondrial ATP production capacity were performed in skeletal muscle from male rats after feeding one of three diets (control, high-fat diet with or without antioxidants) for 36 wk. The high-fat diet altered transcript levels of 18 genes of 800 surveyed compared with the control-fed rats. Alterations included reduced expression of genes involved in free-radical scavenging and tissue development and increased expression of stress response and signal transduction genes. The magnitude of these alterations due to high-fat diet was reduced by antioxidant supplementation. Real-time PCR measurements confirmed the changes in transcript levels of cytochrome c oxidase subunit III and superoxide dismutase-1 and -2 noted by microarray approach. Mitochondrial ATP production was unaltered by dietary changes or antioxidant supplemention. It is concluded that the high-fat diet increases the transcription of genes involved in stress response but reduces those of free-radical scavenger enzymes, resulting in reduced DNA repair/metabolism (increased DNA damage). Antioxidants partially prevent these changes. Mitochondrial functions in skeletal muscle remain unaltered by the dietary intervention due to many adaptive changes in gene transcription.

Hyperthyroidism causes increased energy intake and expenditure, although anorexia and higher weight loss have been reported in elderly individuals with hyperthyroidism. To determine the effect of age on energy homeostasis in response to experimental hyperthyroidism, we administered 200 μg triiodothyronine (T3) in 7‐ and 27‐mo‐old rats for 14 d. T3 increased energy expenditure (EE) in both the young and the old rats, although the old rats lost more weight (147 g) than the young rats (58 g) because of the discordant effect of T3 on food intake, with a 40% increase in the young rats, but a 40% decrease in the old ones. The increased food intake in the young rats corresponded with a T3‐mediated increase in the appetite‐regulating proteins agouti‐related peptide, neuropeptide Y, and uncoupling protein 2 in the hypothalamus, but no increase occurred in the old rats. Evidence of mitochondrial biogenesis in response to T3 was similar in the soleus muscle and heart of the young and old animals, but less consistent in old plantaris muscle and liver. Despite the comparable increase in EE, T3's effect on mitochondrial function was modulated by age in a tissue‐specific manner. We conclude that older rats lack compensatory mechanisms to increase caloric intake in response to a T3‐induced increase in EE, demonstrating a detrimental effect of age on energy homeostasis.—Walrand, S., Short, K. R., Heemstra, L. A., Novak, C. M., Levine, J. A., Coenen‐Schimke, J. M., Nair, K. S. Altered regulation of energy homeostasis in older rats in response to thyroid hormone administration. FASEB J. 28, 1499–1510 (2014). www.fasebj.org

Systemic insulin administration causes hypoaminoacidemia by inhibiting protein degradation, which may in turn inhibit muscle protein synthesis (PS). Insulin enhances muscle mitochondrial PS and ATP production when hypoaminoacidemia is prevented by exogenous amino acid (AA) replacement. We determined whether insulin would stimulate mitochondrial PS and ATP production in the absence of AA replacement. Using l-[1,2-13C]leucine as a tracer, we measured the fractional synthetic rate of mitochondrial as well as sarcoplasmic and mixed muscle proteins in 18 participants during sustained (7-h) insulin or saline infusion ( n = 9 each). We also measured muscle ATP production, mitochondrial enzyme activities, mRNA levels of mitochondrial genes, and phosphorylation of signaling proteins regulating protein synthesis. The concentration of circulating essential AA decreased during insulin infusion. Mitochondrial, sarcoplasmic, and mixed muscle PS rates were also lower during insulin (2–7 h) than during saline infusions despite increased mRNA levels of selected mitochondrial genes. Under these conditions, insulin did not alter mitochondrial enzyme activities and ATP production. These effects were associated with enhanced phosphorylation of Akt but not of protein synthesis activators mTOR, p70S6K, and 4EBP1. In conclusion, sustained physiological hyperinsulinemia without AA replacement did not stimulate PS of mixed muscle or protein subfractions and did not alter muscle mitochondrial ATP production in healthy humans. These results support that insulin and AA act in conjunction to stimulate muscle mitochondrial function and mitochondrial protein synthesis.

Abstract Context: A reduction in maximal mitochondrial ATP production rate (MAPR) and mitochondrial DNA (mtDNA) abundance occurs with age in association with muscle weakness and reduced endurance in elderly people. Branched chain amino acids (BCAA) have been extensively used to improve physical performance. Objective: The objective was to determine whether an 8-h infusion of BCAA enhances MAPR equally in healthy young and elderly adults. Methods: Using a crossover study design, we compared the effect BCAA vs. saline infusion in 12 young (23.0 ± 0.8 yr) and 12 elderly (70.7 ± 1.1 yr) participants matched for sex and body mass index. Skeletal muscle MAPR and mtDNA abundance were measured in muscle biopsy samples obtained before and at the end of the 8-h infusion. Results: In young participants, MAPR with the substrates glutamate plus malate (supplying electrons to complex I) and succinate plus rotenone (complex II) increased in response to BCAA infusion, relative to a decline in MAPR in response to the saline infusion. In contrast, MAPR was unaffected by BCAA infusion in the elderly participants. Moreover, mtDNA abundance was lower in the elderly compared with the young participants but was unaffected by the BCAA infusion. Insulin and C-peptide concentrations declined over time during the saline infusion, but these declines were prevented by the BCAA infusion. Conclusions: BCAA increased skeletal muscle MAPR in the young participants in comparison with saline, but this effect was not seen in the elderly participants indicating, that unlike in the young, BCAA does not increase muscle mitochondrial function in the elderly.

OBJECTIVE Reductions in insulin sensitivity in conjunction with muscle mitochondrial dysfunction have been reported to occur in many conditions including aging. The objective was to determine whether insulin resistance and mitochondrial dysfunction are directly related to chronological age or are related to age-related changes in body composition. RESEARCH DESIGN AND METHODS Twelve young lean, 12 young obese, 12 elderly lean, and 12 elderly obese sedentary adults were studied. Insulin sensitivity was measured by a hyperinsulinemic-euglycemic clamp, and skeletal muscle mitochondrial ATP production rates (MAPRs) were measured in freshly isolated mitochondria obtained from vastus lateralis biopsy samples using the luciferase reaction. RESULTS Obese participants, independent of age, had reduced insulin sensitivity based on lower rates of glucose infusion during a hyperinsulinemic-euglycemic clamp. In contrast, age had no independent effect on insulin sensitivity. However, the elderly participants had lower muscle MAPRs than the young participants, independent of obesity. Elderly participants also had higher levels inflammatory cytokines and total adiponectin. In addition, higher muscle MAPRs were also noted in men than in women, whereas glucose infusion rates were higher in women. CONCLUSIONS The results demonstrate that age-related reductions in insulin sensitivity are likely due to an age-related increase in adiposity rather than a consequence of advanced chronological age. The results also indicate that an age-related decrease in muscle mitochondrial function is neither related to adiposity nor insulin sensitivity. Of interest, a higher mitochondrial ATP production capacity was noted in the men, whereas the women were more insulin sensitive, demonstrating further dissociation between insulin sensitivity and muscle mitochondrial function.

Glucocorticoids cause muscle atrophy and weakness, but the mechanisms for these effects are unclear. The purpose of this study was to test a hypothesis that prednisone (Pred) counteracts insulin's anabolic effects on muscle. A randomized, double-blind cross-over design was used to test the effects of 6 days either Pred (0.8 mg·kg−1·day−1) or placebo use in seven healthy young volunteers. Protein dynamics were measured across the leg using stable isotope tracers of leucine (Leu) and phenylalanine (Phe) after overnight fast and during a hyperinsulinemic (1.5 μU·min−1·kg FFM−1) euglycemic clamp with amino acid replacement. Fasting glucose, amino acids, insulin, and glucagon were higher ( P < 0.01) on Pred vs. placebo, whereas leg blood flow was 18% lower. However, basal whole body and leg kinetics of Leu and Phe were unaltered by Pred. Insulin infusion increased leg glucose uptake in both trials but was 65% lower with Pred than with placebo. Insulin in both trials similarly suppressed whole body flux of Leu and Phe. Importantly, insulin increased net Leu and Phe balance across the leg and the balance between muscle protein synthesis and breakdown, but these changes were 45–140% lower ( P < 0.03) in Pred than in placebo. The present study demonstrates that short-term Pred use in healthy people does not alter whole body or leg muscle protein metabolism during the postaborptive state but causes muscle insulin resistance for both glucose and amino acid metabolism, with a blunted protein anabolism. This interactive effect may lead to muscle atrophy with continued use of glucocorticoids.

OBJECTIVE— We determined whether reduced insulin sensitivity, mitochondrial dysfunction, and other age-related dysfunctions are inevitable consequences of aging or secondary to physical inactivity. RESEARCH DESIGN AND METHODS— Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp and ATP production in mitochondria isolated from vastus lateralis biopsies of 42 healthy sedentary and endurance-trained young (18–30 years old) and older (59–76 years old) subjects. Expression of proteins involved in fuel metabolism was measured by mass spectrometry. Citrate synthase activity, mitochondrial DNA (mtDNA) abundance, and expression of nuclear-encoded transcription factors for mitochondrial biogenesis were measured. SIRT3, a mitochondrial sirtuin linked to lifespan-enhancing effects of caloric restriction, was measured by immunoblot. RESULTS— Insulin-induced glucose disposal and suppression of endogenous glucose production were higher in the trained young and older subjects, but no age effect was noted. Age-related decline in mitochondrial oxidative capacity was absent in endurance-trained individuals. Although endurance-trained individuals exhibited higher expression of mitochondrial proteins, mtDNA, and mitochondrial transcription factors, there were persisting effects of age. SIRT3 expression was lower with age in sedentary but equally elevated regardless of age in endurance-trained individuals. CONCLUSIONS— The results demonstrate that reduced insulin sensitivity is likely related to changes in adiposity and to physical inactivity rather than being an inevitable consequence of aging. The results also show that regular endurance exercise partly normalizes age-related mitochondrial dysfunction, although there are persisting effects of age on mtDNA abundance and expression of nuclear transcription factors and mitochondrial protein. Furthermore, exercise may promote longevity through pathways common to effects of caloric restriction.