K. Sreekumaran Nair

Effects of aging on in vivo synthesis of skeletal muscle myosin heavy-chain and sarcoplasmic protein in humans

A decline in muscle mass and contractile function are prominent features of the sarcopenia of old age. Because myosin heavy chain is an important contractile protein, it was hypothesized that synthesis of this protein decreases in sarcopenia. The fractional synthesis rate of myosin heavy chain was measured simultaneously with rates of mixed muscle and sarcoplasmic proteins from the increment of [13C]leucine in these proteins purified from serial needle biopsy samples taken from 24 subjects (age: from 20 to 92 yr) during a primed continuous infusion of L-[1-(13)C]leucine. A decline in synthesis rate of mixed muscle protein (P < 0.01) and whole body protein (P < 0.01) was observed from young to middle age with no further change with advancing age. An age-related decline of myosin heavy-chain synthesis rate was also observed (P < 0.01), with progressive decline occurring from young, through middle, to old age. However, sarcoplasmic protein synthesis did not decline with age. Myosin heavy-chain synthesis rate was correlated with measures of muscle strength (P < 0.05), circulating insulin-like growth factor I (P < 0.01), and dehydroepiandrosterone sulfate (P < 0.05) in men and women and free testosterone levels in men (P < 0.01). A decline in the synthesis rate of myosin heavy chain implies a decreased ability to remodel this important muscle contractile protein and likely contributes to the declining muscle mass and contractile function in the elderly.A decline in muscle mass and contractile function are prominent features of the sarcopenia of old age. Because myosin heavy chain is an important contractile protein, it was hypothesized that synthesis of this protein decreases in sarcopenia. The fractional synthesis rate of myosin heavy chain was measured simultaneously with rates of mixed muscle and sarcoplasmic proteins from the increment of [13C]leucine in these proteins purified from serial needle biopsy samples taken from 24 subjects (age: from 20 to 92 yr) during a primed continuous infusion ofl-[1-13C]leucine. A decline in synthesis rate of mixed muscle protein ( P < 0.01) and whole body protein ( P < 0.01) was observed from young to middle age with no further change with advancing age. An age-related decline of myosin heavy-chain synthesis rate was also observed ( P < 0.01), with progressive decline occurring from young, through middle, to old age. However, sarcoplasmic protein synthesis did not decline with age. Myosin heavy-chain synthesis rate was correlated with measures of muscle strength ( P < 0.05), circulating insulin-like growth factor I ( P < 0.01), and dehydroepiandrosterone sulfate ( P < 0.05) in men and women and free testosterone levels in men ( P < 0.01). A decline in the synthesis rate of myosin heavy chain implies a decreased ability to remodel this important muscle contractile protein and likely contributes to the declining muscle mass and contractile function in the elderly.

Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32–42%) in healthy humans (P < 0.01) i.v. infused with insulin (1.5 milliunits/kg of fat-free mass per min) while clamping glucose, amino acids, glucagon, and growth hormone. Increased ATP production occurred in association with increased mRNA levels from both mitochondrial (NADH dehydrogenase subunit IV) and nuclear [cytochrome c oxidase (COX) subunit IV] genes (164–180%) encoding mitochondrial proteins (P < 0.05). In addition, muscle mitochondrial protein synthesis, and COX and citrate synthase enzyme activities were increased by insulin (P < 0.05). Further studies demonstrated no effect of low to high insulin levels on muscle mitochondrial ATP production for people with type 2 diabetes mellitus, whereas matched nondiabetic controls increased 16–26% (P < 0.02) when four different substrate combinations were used. In conclusion, insulin stimulates mitochondrial oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels.

Endurance Exercise as a Countermeasure for Aging

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.

A PGC-1α isoform induced by resistance training regulates skeletal muscle hypertrophy

PGC-1α is a transcriptional coactivator induced by exercise that gives muscle many of the best known adaptations to endurance-type exercise but has no effects on muscle strength or hypertrophy. We have identified a form of PGC-1α (PGC-1α4) that results from alternative promoter usage and splicing of the primary transcript. PGC-1α4 is highly expressed in exercised muscle but does not regulate most known PGC-1α targets such as the mitochondrial OXPHOS genes. Rather, it specifically induces IGF1 and represses myostatin, and expression of PGC-1α4 in vitro and in vivo induces robust skeletal muscle hypertrophy. Importantly, mice with skeletal muscle-specific transgenic expression of PGC-1α4 show increased muscle mass and strength and dramatic resistance to the muscle wasting of cancer cachexia. Expression of PGC-1α4 is preferentially induced in mouse and human muscle during resistance exercise. These studies identify a PGC-1α protein that regulates and coordinates factors involved in skeletal muscle hypertrophy.

Sarcopenia of Aging and Its Metabolic Impact

Sarcopenia contributes significantly to the morbidity, decrease in quality of life, and health care costs in the elderly. It is characterized by a decrease in muscle mass and strength, starting as early as the fourth decade of life in humans. Distinct muscle changes include a decrease in type 2 muscle fibers and a decrease in myosin heavy chains IIa and IIx mRNA levels. In addition, a decrease in whole body protein turnover, mixed muscle protein synthesis, myosin heavy chain synthesis, and mitochondrial protein synthesis have been reported. Different tissues and organs display different responses to aging, with more oxidative tissue generally having more age-related changes. Exercise has been shown to increase strength, aerobic capacity, and muscle protein synthesis, as well as to increase muscle mitochondrial enzyme activity in both young and older people; however, exercise does not reverse all age-related changes. The metabolic effects of sarcopenia include a decrease in resting metabolic rate secondary to decreased fat-free mass and decreased physical activity, leading to a higher prevalence of insulin resistance, type 2 diabetes mellitus, dyslipidemia, and hypertension. The way in which age-related changes in hormone levels affect muscle remains to be fully understood. The effect of replacing those hormones on sarcopenia has led to some conflicting results, but further investigations are ongoing.

Quantitative Metabolomics by 1H-NMR and LC-MS/MS Confirms Altered Metabolic Pathways in Diabetes

Insulin is as a major postprandial hormone with profound effects on carbohydrate, fat, and protein metabolism. In the absence of exogenous insulin, patients with type 1 diabetes exhibit a variety of metabolic abnormalities including hyperglycemia, glycosurea, accelerated ketogenesis, and muscle wasting due to increased proteolysis. We analyzed plasma from type 1 diabetic (T1D) humans during insulin treatment (I+) and acute insulin deprivation (I-) and non-diabetic participants (ND) by 1H nuclear magnetic resonance spectroscopy and liquid chromatography-tandem mass spectrometry. The aim was to determine if this combination of analytical methods could provide information on metabolic pathways known to be altered by insulin deficiency. Multivariate statistics differentiated proton spectra from I- and I+ based on several derived plasma metabolites that were elevated during insulin deprivation (lactate, acetate, allantoin, ketones). Mass spectrometry revealed significant perturbations in levels of plasma amino acids and amino acid metabolites during insulin deprivation. Further analysis of metabolite levels measured by the two analytical techniques indicates several known metabolic pathways that are perturbed in T1D (I-) (protein synthesis and breakdown, gluconeogenesis, ketogenesis, amino acid oxidation, mitochondrial bioenergetics, and oxidative stress). This work demonstrates the promise of combining multiple analytical methods with advanced statistical methods in quantitative metabolomics research, which we have applied to the clinical situation of acute insulin deprivation in T1D to reflect the numerous metabolic pathways known to be affected by insulin deficiency.

Asian Indians Have Enhanced Skeletal Muscle Mitochondrial Capacity to Produce ATP in Association with Severe Insulin-Resistance

OBJECTIVE— Type 2 diabetes has become a global epidemic, and Asian Indians have a higher susceptibility to diabetes than Europeans. We investigated whether Indians had any metabolic differences compared with Northern European Americans that may render them more susceptible to diabetes. RESEARCH DESIGN AND METHODS— We studied 13 diabetic Indians, 13 nondiabetic Indians, and 13 nondiabetic Northern European Americans who were matched for age, BMI, and sex. The primary comparisons were insulin sensitivity by hyperinsulinemic-euglycemic clamp and skeletal muscle mitochondrial capacity for oxidative phosphorylation (OXPHOS) by measuring mitochondrial DNA copy number (mtDNA), OXPHOS gene transcripts, citrate synthase activity, and maximal mitochondrial ATP production rate (MAPR). Other factors that may cause insulin resistance were also measured. RESULTS— The glucose infusion rates required to maintain identical glucose levels during the similar insulin infusion rates were substantially lower in diabetic Indians than in the nondiabetic participants (P < 0.001), and they were lower in nondiabetic Indians than in nondiabetic Northern European Americans (P < 0.002). mtDNA (P < 0.02), OXPHOS gene transcripts (P < 0.01), citrate synthase, and MAPR (P < 0.03) were higher in Indians irrespective of their diabetic status. Intramuscular triglyceride, C-reactive protein, interleukin-6, and tumor necrosis factor-α concentrations were higher, whereas adiponectin concentrations were lower in diabetic Indians. CONCLUSIONS— Despite being more insulin resistant, diabetic Indians had similar muscle OXPHOS capacity as nondiabetic Indians, demonstrating that diabetes per se does not cause mitochondrial dysfunction. Indians irrespective of their diabetic status had higher OXPHOS capacity than Northern European Americans, although Indians were substantially more insulin resistant, indicating a dissociation between mitochondrial dysfunction and insulin resistance.

Skeletal muscle mitochondrial functions, mitochondrial DNA copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic subjects at equal levels of low or high insulin and euglycemia.

We investigated whether previously reported muscle mitochondrial dysfunction and altered gene transcript levels in type 2 diabetes might be secondary to abnormal blood glucose and insulin levels rather than an intrinsic defect of type 2 diabetes. A total of 13 type 2 diabetic and 17 nondiabetic subjects were studied on two separate occasions while maintaining similar insulin and glucose levels in both groups by 7-h infusions of somatostatin, low- or high-dose insulin (0.25 and 1.5 mU/kg of fat-free mass per min, respectively), and glucose. Muscle mitochondrial DNA abundance was not different between type 2 diabetic and nondiabetic subjects at both insulin levels, but the majority of transcripts in muscle that are involved mitochondrial functions were expressed at lower levels in type 2 diabetes at low levels of insulin. However, several gene transcripts that are specifically involved in the electron transport chain were expressed at higher levels in type 2 diabetic patients. After the low-dose insulin infusion, which achieved postabsorptive insulin levels, the muscle mitochondrial ATP production rate (MAPR) was not different between type 2 diabetic and nondiabetic subjects. However, increasing insulin to postprandial levels increased the MAPR in nondiabetic subjects but not in type 2 diabetic patients. The lack of MAPR increment in response to high-dose insulin in type 2 diabetic patients occurred in association with reduced glucose disposal and expression of peroxisome proliferator–activated receptor-γ coactivator 1α, citrate synthase, and cytochrome c oxidase I. In conclusion, the current data supports that muscle mitochondrial dysfunction in type 2 diabetes is not an intrinsic defect, but instead a functional defect related to impaired response to insulin.

Detection and Quantitation of Circulating Human Irisin by Tandem Mass Spectrometry.

Exercise provides many health benefits, including improved metabolism, cardiovascular health, and cognition. We have shown previously that FNDC5, a type I transmembrane protein, and its circulating form, irisin, convey some of these benefits in mice. However, recent reports questioned the existence of circulating human irisin both because human FNDC5 has a non-canonical ATA translation start and because of claims that many human irisin antibodies used in commercial ELISA kits lack required specificity. In this paper we have identified and quantitated human irisin in plasma using mass spectrometry with control peptides enriched with heavy stable isotopes as internal standards. This precise state-of-the-art method shows that human irisin is mainly translated from its non-canonical start codon and circulates at ∼ 3.6 ng/ml in sedentary individuals; this level is increased to ∼ 4.3 ng/ml in individuals undergoing aerobic interval training. These data unequivocally demonstrate that human irisin exists, circulates, and is regulated by exercise.

Age, Obesity, and Sex Effects on Insulin Sensitivity and Skeletal Muscle Mitochondrial Function

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.