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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, p70(S6K), 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.

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 x kg(-1) x 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 microU x min(-1) x 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.

Skeletal muscle mitochondrial dysfunction occurs in many conditions including aging and insulin resistance, but the molecular pathways of the mitochondrial dysfunction remain unclear. Presently, no methodologies are available to measure synthesis rates of individual mitochondrial proteins, which limits our ability to fully understand the translational regulation of gene transcripts. Here, we report a methodology to measure synthesis rates of multiple muscle mitochondrial proteins, which, along with large-scale measurements of mitochondrial gene transcripts and protein concentrations, will enable us to determine whether mitochondrial alteration is due to transcriptional or translational changes. The methodology involves in vivo labeling of muscle proteins with l-[ring-(13)C(6)]phenylalanine, protein purification by two-dimensional gel electrophoresis of muscle mitochondrial fraction, and protein identification and stable isotope abundance measurements by tandem mass spectrometry. Synthesis rates of 68 mitochondrial and 23 nonmitochondrial proteins from skeletal muscle mitochondrial fraction showed a 10-fold range, with the lowest rate for a structural protein such as myosin heavy chain (0.16 +/- 0.04%/h) and the highest for a mitochondrial protein such as dihydrolipoamide branched chain transacylase E2 (1.5 +/- 0.42%/h). This method offers an opportunity to better define the translational regulation of proteins in skeletal muscle or other tissues.

Decline in muscle mass, protein synthesis, and mitochondrial function occurs with age, and amino acids are reported to enhance both muscle protein synthesis and mitochondrial function. It is unclear whether increasing dietary protein intake corrects postabsorptive muscle changes in aging. We determined whether a 10-day diet of high [HP; 3.0 g protein x kg fat-free mass (FFM)(-1) x day(-1)] vs. usual protein intake (UP; 1.5 g protein x kg FFM(-1) x day(-1)) favorably affects mitochondrial function, protein metabolism, and nitrogen balance or adversely affects insulin sensitivity and glomerular filtration rate (GFR) in 10 healthy younger (24+/-1 yr) and 9 older (70+/-2 yr) participants in a randomized crossover study. Net daily nitrogen balance increased equally in young and older participants, but postabsorptive catabolic state also increased, as indicated by higher whole body protein turnover and leucine oxidation with no change in protein synthesis. Maximal muscle mitochondrial ATP production rate was lower in older people, with no change occurring in diet. GFR was lower in older people, and response to HP was significantly different between the two groups, with a significant increase occurring only in younger people, thus widening the differences in GFR between the young and older participants. In conclusion, a short-term high-protein diet increased net daily nitrogen balance but increased the postabsorptive use of protein as a fuel. HP did not enhance protein synthesis or muscle mitochondrial function in either young or older participants. Additionally, widening differences in GFR between young and older patients is a potential cause of concern in using HP diet in older people.

We hypothesized that enhanced skeletal muscle mitochondrial function following aerobic exercise training is related to an increase in mitochondrial transcription factors, DNA abundance [mitochondrial DNA (mtDNA)], and mitochondria-related gene transcript levels, as well as spontaneous physical activity (SPA) levels. We report the effects of daily treadmill training on 12-wk-old FVB mice for 5 days/wk over 8 wk at 80% peak O(2) consumption and studied the training effect on changes in body composition, glucose tolerance, muscle mtDNA muscle, mitochondria-related gene transcripts, in vitro muscle mitochondrial ATP production capacity (MATPC), and SPA levels. Compared with the untrained mice, the trained mice had higher peak O(2) consumption (+18%; P < 0.001), lower percentage of abdominal (-25.4%; P < 0.02) and body fat (-19.5%; P < 0.01), improved glucose tolerance (P < 0.04), and higher muscle mitochondrial enzyme activity (+19.5-43.8%; P < 0.04) and MATPC (+28.9 to +32.4%; P < 0.01). Gene array analysis showed significant differences in mRNAs of mitochondria-related ontology groups between the trained and untrained mice. Training also increased muscle mtDNA (+88.4 to +110%; P < 0.05), peroxisome proliferative-activated receptor-gamma coactivator-1alpha protein (+99.5%; P < 0.04), and mitochondrial transcription factor A mRNA levels (+21.7%; P < 0.004) levels. SPA levels were higher in trained mice (P = 0.056, two-sided t-test) and significantly correlated with two separate substrate-based measurements of MATPC (P < 0.02). In conclusion, aerobic exercise training enhances muscle mitochondrial transcription factors, mtDNA abundance, mitochondria-related gene transcript levels, and mitochondrial function, and this enhancement in mitochondrial function occurs in association with increased SPA.

Hyperthyroidism increases metabolic rate, mitochondrial ATP production, and protein synthesis, but it remains to be determined whether all tissues and synthesis of specific protein pools are equally affected by hyperthyroidism. Previous studies showed that mitochondrial function was less responsive to elevated triiodothyronine (T(3)) levels in the low-oxidative plantaris muscle compared with other tissues in rats. We tested the hypothesis that in T(3)-treated animals mitochondrial protein synthesis would increase in oxidative but not glycolytic tissues. Male rats received either T(3) (200 mug/day, n = 10) or saline (controls, n = 9) by subcutaneous pump for 14 days, and then in vivo protein synthesis rates were measured using [(15)N]phenylalanine in liver, heart, plantaris, and red gastrocnemius (Red Gast). Mitochondrial protein synthesis rate in T(3)-treated rats was higher than in controls by 62% in Red Gast and plantaris and 89 and 115% in liver and heart, respectively (P < 0.01). Cytoplasmic protein synthesis rates in the T(3) group were 107-176% higher than control values (P < 0.01). There was also indirect evidence that protein breakdown was increased in all tissues of the T(3)-treated rats. Phosphorylation of selected regulators of protein synthesis in plantaris and Red Gast (mTOR, p70 S6 kinase, 4E-BP1), however, were not significantly affected by T(3). We conclude that T(3) infusion stimulates a general increase in mitochondrial and cytoplasmic protein synthesis rate among tissues and that this does not appear to explain the tissue-specific responses in mitochondrial oxidative capacity.

Aging is associated with reduced muscle strength and atrophy of type II muscle fibers. Muscle fiber type and contractile function are primarily determined by myosin heavy chain (MHC) isoforms. There are few data available on the effects of aging on MHC isoform expression in humans. In the present study, we tested the hypothesis that MHC isoform protein composition and mRNA abundance would favor a fast-to-slow isoform shift with aging and in response to endurance exercise training. Muscle biopsies were obtained from previously sedentary, healthy men and women, aged 21-87 yr before (n = 77) and after (n = 65) 16 wk of bicycle training (up to 45 min at 80% peak heart rate, 3-4 days/wk). At baseline, MHC I mRNA was unchanged with age, whereas IIa and IIx declined by 14 and 10% per decade, respectively (P < 0.001). MHC IIa and IIx protein declined by 3 and 1% per decade with a reciprocal increase in MHC I (P < 0.05). After training, MHC I and IIa mRNA increased by 61 and 99%, respectively, and IIx decreased by 50% (all P < 0.001). The increase in MHC I mRNA was positively associated with age, whereas the changes in MHC IIa and IIx mRNA were similar across age. MHC I protein increased by 6% and was positively related to age, whereas IIx decreased by 5% and was inversely related to age. These results suggest that the altered expression of MHC isoforms with aging is transcriptionally regulated. In response to endurance exercise, regulation of MHC isoform transcripts remains robust in older muscle, but this did not result in corresponding changes in MHC protein expression.

Proteomics is the comprehensive and systematic study of proteins, which are functional molecules. Although proteins are products of gene expression, there are more proteins than genes due to the posttranslational modifications of proteins, making the study of proteins difficult. Protein expression is tissue specific, and its function is modulated by variety of factors, including other proteins, phosphates, sulfates, carbohydrates, and lipids, as well as other metabolites. Because of the dynamic nature of protein expression and posttranslational modifications, identification and quantification of proteins alone are not sufficient to understand functional changes. Emerging technologies will allow investigators to perform a combination of metabolic labeling and identification as well as quantification and measurement of the synthesis rates of a large number of proteins in a tissue. This offers the opportunity to better understand the regulation of tissue functions. Rapid advances in mass spectrometry, protein purification techniques, isotope labeling of proteins, and bioinformatics are likely to improve our understanding of physiological states and altered functions in diseased states. Such mechanistic information will improve the ability to perform early diagnosis of tumors and other diseases and develop prognostic indexes and novel therapies.

Aging in humans is associated with loss of lean body mass, but the causes are incompletely defined. Lean tissue mass and function depend on continuous rebuilding of proteins. We tested the hypotheses that whole body and mixed muscle protein metabolism declines with age in men and women and that aerobic exercise training would partly reverse this decline. Seventy-eight healthy, previously untrained men and women aged 19-87 yr were studied before and after 4 mo of bicycle training (up to 45 min at 80% peak heart rate, 3-4 days/wk) or control (flexibility) activity. At the whole body level, protein breakdown (measured as [13C]leucine and [15N]phenylalanine flux), Leu oxidation, and protein synthesis (nonoxidative Leu disposal) declined with age at a rate of 4-5% per decade (P < 0.001). Fat-free mass was closely correlated with protein turnover and declined 3% per decade (P < 0.001), but even after covariate adjustment for fat-free mass, the decline in protein turnover with age remained significant. There were no differences between men and women after adjustment for fat-free mass. Mixed muscle protein synthesis also declined with age 3.5% per decade (P < 0.05). Exercise training improved aerobic capacity 9% overall (P < 0.01), and mixed muscle protein synthesis increased 22% (P < 0.05), with no effect of age on the training response for either variable. Fat-free mass, whole body protein turnover, and resting metabolic rate were unchanged by training. We conclude that rates of whole body and muscle protein metabolism decline with age in men and women, thus indicating that there is a progressive decline in the body's remodeling processes with aging. This study also demonstrates that aerobic exercise can enhance muscle protein synthesis irrespective of age.

Thyroid hormone excess is associated with increased energy expenditure. The contributions of increases in spontaneous physical activity and nonexercise activity thermogenesis (NEAT) to this effect have not been defined. To address the hypothesis that hyperthyroidism is associated with increased spontaneous physical activity and NEAT, we rendered rats hyperthyroid by using continuous infusion of high-dose triiodothyronine for 14 days and measured the effects on physical activity and NEAT. On day 14, in the hyperthyroid group the mean +/- SD triiodothyronine concentration was 755 +/- 137 (range 574-919) ng/dl and in the control group 59 +/- 0.5 (58-59) ng/dl. Over the 14-day treatment period, mean spontaneous physical activity increased in the hyperthyroid rats from 24 +/- 7 to 36 +/- 6 activity units (AU)/min; P < 0.001 but did not increase in the controls (23 +/- 7 vs. 22 +/- 4 AU/min). Also, over the 14-day period, daily NEAT increased in the hyperthyroid rats from 8.1 +/- 2.8 to 19.7 +/- 5.0 kcal/day (P < 0.001) but did not increase in the controls (8.7 +/- 3.5 cf 9.4 +/- 1.7 kcal/day; not significant). In conclusion, hyperthyroidism is associated with increased spontaneous physical activity and NEAT.

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 measurements 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 supplementation. 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.

Triiodothyronine (T(3)) increases O(2) and nutrient flux through mitochondria (Mito) of many tissues, but it is unclear whether ATP synthesis is increased, particularly in different types of skeletal muscle, because variable changes in uncoupling proteins (UCP) and enzymes have been reported. Thus Mito ATP production was measured in oxidative and glycolytic muscles, as well as in liver and heart, in rats administered T(3) for 14 days. Relative to saline-treated controls, T(3) rats had 80, 168, and 62% higher ATP production in soleus muscle, liver, and heart, respectively, as well as higher activities of citrate synthase (CS; 63, 90, 25%) and cytochrome c oxidase (COX; 119, 225, 52%) in the same tissues (all P < 0.01). In plantaris muscle of T(3) rats, CS was only slightly higher (17%, P < 0.05) than in controls, and ATP production and COX were unaffected. mRNA levels of COX I and III were 33 and 47% higher in soleus of T(3) rats (P < 0.01), but there were no differences in plantaris. In contrast, UCP2 and -3 mRNAs were 2.5- to 14-fold higher, and protein levels were 3- to 10-fold higher in both plantaris and soleus of the T(3) group. We conclude that T(3) increases oxidative enzymes and Mito ATP production and Mito-encoded transcripts in oxidative but not glycolytic rodent tissues. Despite large increases in UCP expression, ATP production was enhanced in oxidative tissues and maintained in glycolytic muscle of hyperthyroid rats.

Phenylalanine (Phe) kinetics are increasingly used in studies of amino acid kinetics, because the metabolic fate of Phe is limited to incorporation into protein (protein synthesis, Sp) and catabolism via hydroxylation (Qpt) to tyrosine (Tyr). Besides an infusion of labeled Phe to measure Phe flux (Qp), a priming dose of Tyr and an independent Tyr tracer are used to measure Tyr flux (Qt) and Qpt. Alternatively, Qt, Qpt, and Sp can be approximated by using equations, based on Phe and Tyr concentrations in body proteins, that eliminate the need for a Tyr tracer. To evaluate the accuracy of this approach, data were obtained from 12 type I diabetic patients and 24 nondiabetic control subjects who were studied with the full complement of tracers both with and without insulin infusion. Sp approximations closely matched measured values in both groups (mean difference <2%, all values <5%), but the agreement was poor for Qpt (error range = -8 to +43%) and Qt (error range -22 to +41%). Insulin status had no effect on these comparisons. The lower approximation error for Sp vs. Qpt is due to the small contribution ( approximately 10%) of Qpt to Qp. Approximation error for Qpt (r > 0.99) can be explained by variability in the ratio of Tyr to Phe coming from protein breakdown, (Qt - Qpt)/Qp. Ideally, all fluxes should be directly measured, but these data suggest that whole body Sp can be approximated with an acceptably small margin of error. However, the same equations do not yield reliably accurate values for Qpt or Qt.

The purpose of this study was to determine whether aerobic fitness level would influence measurements of excess postexercise oxygen consumption (EPOC) and initial rate of recovery. Twelve trained [Tr; peak oxygen consumption (VO2 peak) = 53.3 +/- 6.4 ml . kg-1 . min-1] and ten untrained (UT; VO2 peak = 37.4 +/- 3.2 ml . kg-1 . min-1) subjects completed two 30-min cycle ergometer tests on separate days in the morning, after a 12-h fast and an abstinence from vigorous activity of 24 h. Baseline metabolic rate was established during the last 10 min of a 30-min seated preexercise rest period. Exercise workloads were manipulated so that they elicited the same relative, 70% VO2 peak (W70%), or the same absolute, 1.5 l/min oxygen uptake (VO2) (W1.5), intensity for all subjects, respectively. Recovery VO2, heart rate (HR), and respiratory exchange ratio (RER) were monitored in a seated position until baseline VO2 was reestablished. Under both exercise conditions, Tr had shorter EPOC duration (W70% = 40 +/- 15 min, W1.5 = 21 +/- 9 min) than UT (W70% = 50 +/- 14 min; W1.5 = 39 +/- 14 min), but EPOC magnitude (Tr: W70% = 3.2 +/- 1.0 liters O2, W1.5 = 1.5 +/- 0.6 liters O2; UT: W70% = 3.5 +/- 0.9 liters O2, W1.5 = 2.4 +/- 0.6 liters O2) was not different between groups. The similarity of Tr and UT EPOC accumulation in the W70% trial is attributed to the parallel decline in absolute VO2 during most of the initial recovery period. Tr subjects had faster relative decline during the fast-recovery phase, however, when a correction for their higher exercise VO2 was taken. Postexercise VO2 was lower for Tr group for nearly all of the W1.5 trial and particularly during the fast phase. Recovery HR kinetics were remarkably similar for both groups in W70%, but recovery was faster for Tr during W1.5. RER values were at or below baseline throughout much of the recovery period in both groups, with UT experiencing larger changes than Tr in both trials. These findings indicate that Tr individuals have faster regulation of postexercise metabolism when exercising at either the same relative or same absolute work rate.