Respiratory Capacity Of Skeletal Muscle Research Articles

Exercise Training Differentially Affects Skeletal Muscle Mitochondria in Rats with Inherited High or Low Exercise Capacity.

Exercise capacity has been related to morbidity and mortality. It consists of an inherited and an acquired part and is dependent on mitochondrial function. We assessed skeletal muscle mitochondrial function in rats with divergent inherited exercise capacity and analyzed the effect of exercise training. Female high (HCR)- and low (LCR)-capacity runners were trained with individually adapted high-intensity intervals or kept sedentary. Interfibrillar (IFM) and subsarcolemmal (SSM) mitochondria from gastrocnemius muscle were isolated and functionally assessed (age: 15 weeks). Sedentary HCR presented with higher exercise capacity than LCR paralleled by higher citrate synthase activity and IFM respiratory capacity in skeletal muscle of HCR. Exercise training increased exercise capacity in both HCR and LCR, but this was more pronounced in LCR. In addition, exercise increased skeletal muscle mitochondrial mass more in LCR. Instead, maximal respiratory capacity was increased following exercise in HCRs' IFM only. The results suggest that differences in skeletal muscle mitochondrial subpopulations are mainly inherited. Exercise training resulted in different mitochondrial adaptations and in higher trainability of LCR. HCR primarily increased skeletal muscle mitochondrial quality while LCR increased mitochondrial quantity in response to exercise training, suggesting that inherited aerobic exercise capacity differentially affects the mitochondrial response to exercise training.

Localized Heat Therapy Improves Mitochondrial Respiratory Capacity but Not Fatty Acid Oxidation.

AIM: Mild heat stress can improve mitochondrial respiratory capacity in skeletal muscle. However, long-term heat interventions are scarce, and the effects of heat therapy need to be understood in the context of the adaptations which follow the more complex combination of stimuli from exercise training. The purpose of this work was to compare the effects of 6 weeks of localized heat therapy on human skeletal muscle mitochondria to single-leg interval training. METHODS: Thirty-five subjects were assigned to receive sham therapy, short-wave diathermy heat therapy, or single-leg interval exercise training, localized to the quadriceps muscles of the right leg. All interventions took place 3 times per week. Muscle biopsies were performed at baseline, and after 3 and 6 weeks of intervention. Mitochondrial respiratory capacity was assessed on permeabilized muscle fibers via high-resolution respirometry. RESULTS: The primary finding of this work was that heat therapy and exercise training significantly improved mitochondrial respiratory capacity by 24.8 ± 6.2% and 27.9 ± 8.7%, respectively (p < 0.05). Fatty acid oxidation and citrate synthase activity were also increased following exercise training by 29.5 ± 6.8% and 19.0 ± 7.4%, respectively (p < 0.05). However, contrary to our hypothesis, heat therapy did not increase fatty acid oxidation or citrate synthase activity. CONCLUSION: Six weeks of muscle-localized heat therapy significantly improves mitochondrial respiratory capacity, comparable to exercise training. However, unlike exercise, heat does not improve fatty acid oxidation capacity.

Skeletal muscle mitochondrial function and exercise capacity are not impaired in mice with knockout of STAT3.

Signal transducer and activator of transcription 3 (STAT3) was recently found to be localized to mitochondria in a number of tissues and cell types, where it modulates oxidative phosphorylation via interactions with the electron transport proteins, complex I and complex II. Skeletal muscle is densely populated with mitochondria although whether STAT3 contributes to skeletal muscle oxidative capacity is unknown. In the present study, we sought to elucidate the contribution of STAT3 to mitochondrial and skeletal muscle function by studying mice with muscle-specific knockout of STAT3 (mKO). First, we developed a novel flow cytometry-based approach to confirm that STAT3 is present in skeletal muscle mitochondria. However, contrary to findings in other tissue types, complex I and complex II activity and maximal mitochondrial respiratory capacity in skeletal muscle were comparable between mKO mice and floxed/wild-type littermates. Moreover, there were no genotype differences in endurance exercise performance, skeletal muscle force-generating capacity, or the adaptive response of skeletal muscle to voluntary wheel running. Collectively, although we confirm the presence of STAT3 in skeletal muscle mitochondria, our data establish that STAT3 is dispensable for mitochondrial and physiological function in skeletal muscle.NEW & NOTEWORTHY Whether signal transducer and activator of transcription 3 (STAT3) can regulate the activity of complex I and II of the electron transport chain and mitochondrial oxidative capacity in skeletal muscle, as it can in other tissues, is unknown. By using a mouse model lacking STAT3 in muscle, we demonstrate that skeletal muscle mitochondrial and physiological function, both in vivo and ex vivo, is not impacted by the loss of STAT3.

Inhibition of Mitochondrial Fission Improves Mitochondrial Respiratory Capacity in Primary Myotubes Derived from Obese Humans

Obesity induces mitochondrial fragmentation in skeletal muscle causing reduced mitochondrial respiratory capacity, which has been linked with the development of insulin resistance and type 2 diabetes. Recent evidence suggests that excessive mitochondrial fission is the culprit behind the mitochondrial fragmentation in skeletal muscle from obese humans. The purpose of this study was to determine if inhibiting mitochondrial fission in human skeletal muscle cells derived from severely obese individuals reduces mitochondrial fragmentation and improves mitochondrial respiratory capacity. Human skeletal muscle cells were isolated from muscle biopsies obtained from severely obese, insulin‐resistant subjects (n=6, BMI = 47.3 ± 2.8 kg/m2, HOMA‐IR = 3.4 ± 0.4), pooled and differentiated into myotubes. On day 6 of differentiation, myotubes were treated with either mitochondrial fission inhibitor Mdivi‐1 (20μM) or DMSO for 12 hrs. Mitochondrial respiratory capacity was measured using Seahorse XFp Analyzer. Live cells were stained with MitoTracker, and mitochondrial morphology was examined by confocal microscopy. Mitochondrial fission, fusion, and OXPHOS protein expressions were assessed by immunoblot. All experiments were repeated on three different occasions. Preliminary data indicate that mitochondrial fission protein dynamin related protein (Drp1) was reduced (20.9%, P = 0.093), with no changes in mitochondrial content or mitochondrial fusion proteins. Inhibiting mitochondrial fission resulted in a reduction of mitochondrial networks (9.7%) while increasing mitochondrial branch length (14.7%). Reserved mitochondrial respiratory capacity, as measured by the mitochondrial spare capacity, was significantly increased (31.4%, P &lt; 0.05) with Mdivi‐1 treatment. These results demonstrate that inhibition of mitochondrial fission improves mitochondrial morphology and respiratory capacity in skeletal muscle from obese humans, suggesting mitochondrial fission may be a novel therapeutic target for treating metabolic disorders that are typically linked to mitochondrial dysfunction.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Influence of exercise amount and intensity on long-term weight loss maintenance and skeletal muscle mitochondrial ROS production in humans

Sustaining a weight loss after a lifestyle intervention is challenging. The objective of the present study was to investigate if mitochondrial function is associated with the ability to maintain a weight loss. Sixty-eight former participants in an 11-12-week lifestyle intervention were recruited into 2 groups; weight loss maintenance (WLM; body mass index (BMI): 32 ± 1 kg/m2) and weight regain (WR; BMI: 43 ± 2 kg/m2) based on weight loss measured at a follow-up visit (WLM: 4.8 ± 0.4; WR: 7.6 ± 0.8 years after lifestyle intervention). Maximal oxygen consumption rate, physical activity level, and blood and muscle samples were obtained at the follow-up experiment. Mitochondrial respiratory capacity and reactive oxygen species (ROS) production were measured. Fasting blood samples were used to calculate glucose homeostasis index. WR had impaired glucose homeostasis and decreased maximal oxygen uptake and physical activity level compared with WLM. The decreased physical activity in WR was due to a lower activity level at vigorous and moderate intensities. Mitochondrial respiratory capacity and citrate synthase (CS) activity was higher in WLM, but intrinsic mitochondrial respiratory capacity (mitochondrial respiratory capacity corrected for mitochondrial content (CS activity)) was similar. ROS production was higher in WR compared with WLM, which was accompanied by a decreased content of antioxidant proteins in WR. Intrinsic mitochondrial respiratory capacity in skeletal muscle is not associated with the ability to maintain a long-term weight loss. WLM had a higher maximal oxygen uptake, physical activity level, mitochondrial respiratory capacity and CS activity compared with WR. The reduced glucose tolerance was concurrent with increased ROS production per mitochondria in WR, and could also be associated with the lower physical activity level in this group.

Exercise Is Required to Improve Mitochondrial Respiratory Capacity in Skeletal Muscle of Older Obese Subjects during Calorie Restriction–Induced Weight Loss to Improve Insulin Sensitivity

Calorie restriction has been shown to have “anti-aging” effects in animal models, and calorie restriction-induced weight loss (CRWL) and exercise can both improve insulin sensitivity. The aim of this study was to investigate the effect of 6-month diet-induced weight loss intervention with or without the addition of exercise training on skeletal muscle mitochondrial respiratory capacity and IS. Methods: Forty-four older adults with obesity were randomized to one of the following 6-month intervention: Health education (CON: n=14, age=70±4 years, BMI=34±5kg/m2), Diet induced weight-loss (DIWL: n=14, age=70±4 years, BMI=36±5kg kg/m2), or Weight-loss and exercise (WLEX: n=16, age=66±3 years, BMI=37±5kg/m2). CRWL and WLEX participants had a goal of 10% weight-loss through calorie restriction. Subjects in the WLEX group completed a supervised exercise program. Mitochondrial respiratory capacity of permeabilized myofibers from biopsies was evaluated by high-resolution respirometry. Complex I+II supported maximal oxidative phosphorylation (OXPHOS), and maximal uncoupled respiration were evaluated. IS was determined by hyperinsulinemic euglycemic clamps. Results: IS improved in both CRWL (10.4%) and WLEX (50%) compared to CON (0.7%). However, while mitochondrial respiration was unchanged in the CON and CRWL groups after the 6-month intervention (p&amp;gt;0.05), subjects in the WLEX group presented an increased maximal OXPHOS (PRE: 50.7±10.1 vs. POST: 72.6±17.9 pmol/s*mg) and uncoupled respiration (PRE: 57.2±10.5 vs. POST: 79.6±19.3 pmol/s*mg) after the exercise training. Conclusions: Despite weight loss and exercise both improving insulin sensitivity, exercise is required to improve mitochondrial respiratory capacity in skeletal muscle of older obese human subjects undergoing calorie restriction-induced weight loss. Disclosure G. Distefano: None. R. Standley: None. E.A. Carnero: None. P.M. Coen: None. R.B. Vega: None. B.H. Goodpaster: None.

Elevated Nicotinamide Phosphoribosyl Transferase in Skeletal Muscle Augments Exercise Performance and Mitochondrial Respiratory Capacity Following Exercise Training.

Mice overexpressing NAMPT in skeletal muscle (NamptTg mice) develop higher exercise endurance and maximal aerobic capacity (VO2max) following voluntary exercise training compared to wild-type (WT) mice. Here, we aimed to investigate the mechanisms underlying by determining skeletal muscle mitochondrial respiratory capacity in NamptTg and WT mice. Body weight and body composition, tissue weight (gastrocnemius, quadriceps, soleus, heart, liver, and epididymal white adipose tissue), skeletal muscle and liver glycogen content, VO2max, skeletal muscle mitochondrial respiratory capacity (measured by high-resolution respirometry), skeletal muscle gene expression (measured by microarray and qPCR), and skeletal muscle protein content (measured by Western blot) were determined following 6 weeks of voluntary exercise training (access to running wheel) in 13-week-old male NamptTg (exercised NamptTg) mice and WT (exercised WT) mice. Daily running distance and running time during the voluntary exercise training protocol were recorded. Daily running distance (p = 0.51) and running time (p = 0.85) were not significantly different between exercised NamptTg mice and exercised WT mice. VO2max was higher in exercised NamptTg mice compared to exercised WT mice (p = 0.02). Body weight (p = 0.92), fat mass (p = 0.49), lean mass (p = 0.91), tissue weight (all p > 0.05), and skeletal muscle (p = 0.72) and liver (p = 0.94) glycogen content were not significantly different between exercised NamptTg mice and exercised WT mice. Complex I oxidative phosphorylation (OXPHOS) respiratory capacity supported by fatty acid substrates (p < 0.01), maximal (complex I+II) OXPHOS respiratory capacity supported by glycolytic (p = 0.02) and fatty acid (p < 0.01) substrates, and maximal uncoupled respiratory capacity supported by fatty acid substrates (p < 0.01) was higher in exercised NamptTg mice compared to exercised WT mice. Transcriptomic analyses revealed differential expression for genes involved in oxidative metabolism in exercised NamptTg mice compared to exercised WT mice, specifically, enrichment for the gene set related to the SIRT3-mediated signaling pathway. SIRT3 protein content correlated with NAMPT protein content (r = 0.61, p = 0.04). In conclusion, NamptTg mice develop higher exercise capacity following voluntary exercise training compared to WT mice, which is paralleled by higher mitochondrial respiratory capacity in skeletal muscle. The changes in SIRT3 targets suggest that these effects are due to remodeling of mitochondrial function.

Mitochondrial respiratory capacity remains stable despite a comprehensive and sustained increase in insulin sensitivity in obese patients undergoing gastric bypass surgery.

It has been proposed, but not yet demonstrated by convincing evidence in published articles, that insulin resistance and mitochondrial respiratory function are causally related physiological phenomena. Here, we tested the prediction that weight loss-induced increase in insulin sensitivity will correlate with a corresponding change in mitochondrial respiratory capacity over the same time period. Insulin sensitivity was evaluated using the hyperinsulinaemic-euglycaemic clamp technique, and skeletal muscle mitochondrial respiratory capacity was evaluated by high-resolution respirometry in 26 patients with obesity. Each experiment was performed ~2months and 1-2weeks before, and ~4 and ~19months after Roux-en-Y gastric bypass (RYGB) surgery. A substantial weight loss was observed in all patients, and insulin sensitivity increased in all patients over the 21-months time period of the study. In contrast, skeletal muscle mitochondrial respiratory capacity, intrinsic mitochondrial respiratory capacity and mitochondrial content remained unchanged over the same time period. Among obese patients with and without type 2 diabetes undergoing RYGB surgery, intrinsic mitochondrial respiratory capacity in skeletal muscle is not correlated with insulin sensitivity before or after the surgical intervention. Mitochondrial respiratory function may not be germane to the pathophysiology and/or aetiology of obesity and/or type 2 diabetes.

Increased intrinsic mitochondrial respiratory capacity in skeletal muscle from rats with streptozotocin-induced hyperglycemia.

Type I diabetes mellitus (T1DM) is a chronic disorder, characterized by an almost or complete insulin deficiency. Widespread tissue dysfunction and deleterious diabetes-complications are associated with long-term elevations of blood glucose. The aim of this study was to investigate the effects of type I diabetes, as induced by streptozotocin, on the mitochondria in skeletal muscles that predominantly consist of either slow or fast twitch fibers. Soleus (primarily slow twitch fiber type) and the plantaris muscle (mainly fast twitch fiber type) were removed in order to measure mitochondrial protein expression and integrated mitochondrial respiratory function. Mitochondrial capacity for oxidative phosphorylation (OXPHOS) was found to be higher in the slow (more oxidative) soleus muscle from STZ rats when evaluating lipid and complex I linked OXPHOS capacity, whereas no difference was detected between the groups when evaluating the more physiological complex I and II linked OXPHOS capacity. These findings indicate that chronic hyperglycemia results in an elevated intrinsic mitochondrial respiratory capacity in both soleus and, at varying degree, plantaris muscle, findings that are consistent with human T1DM patients.

Skeletal muscle respiratory capacity is enhanced in rats consuming an obesogenic Western diet

Obesity-induced lipid oversupply promotes skeletal muscle mitochondrial biogenesis. Previous investigations have utilized extreme high-fat diets (HFD) to induce such mitochondrial perturbations despite their disparity from human obesogenic diets. Here, we evaluate the effects of Western diet (WD)-induced obesity on skeletal muscle mitochondrial function. Long-Evans rats were given ad libitum access to either a WD [40% energy (E) from fat, 17% protein, and 43% carbohydrate (30% sucrose); n = 12] or a control diet (CON; 16% of E from fat, 21% protein, and 63% carbohydrate; n = 12) for 12 wk. Rats fed the WD consumed 23% more E than CON (P = 0.0001), which was associated with greater increases in body mass (23%, P = 0.0002) and adiposity (17%, P = 0.03). There were no differences in fasting blood glucose concentration or glucose tolerance between diets, although fasting insulin was increased by 40% (P = 0.007). Fasting serum triglycerides were also elevated in WD (86%, P = 0.001). The maximal capacity of the electron transfer system was greater following WD (37%, P = 0.02), as were the maximal activities of several mitochondrial enzymes (citrate synthase, β-hydroxyacyl-CoA dehydrogenase, carnitine palmitoyltransferase). Protein expression of citrate synthase, UCP3, and individual respiratory complexes was greater after WD (P < 0.05) despite no differences in the expression of peroxisome proliferator-activated receptor (PPAR)α, PPARδ, or PPARγ coactivator-1 mRNA or protein abundance. We conclude that the respiratory capacity of skeletal muscle is enhanced in response to the excess energy supplied by a WD. This is likely due to an increase in mitochondrial density, which at least in the short term, and in the absence of increased energy demand, may protect the tissue from lipid-induced impairments in glycemic control.

Modulation of mitochondrial respiratory capacity by carrier-mediated transfer of RNA in vivo

Genetic dysfunction of mitochondria is pathological, but an effective method of nucleic acid delivery to mitochondria in vivo is lacking. Injection into rodents of tagged polycistronic RNAs (pcRNAs) encoding parts of the organelle genome and bound to a carrier complex, resulted in rapid uptake and concentration of the RNA in many tissues. The delivered RNA was localized to mitochondria. A pan-genomic cocktail of pcRNAs restored mRNA levels, stimulated mitochondrial translation and respiratory capacity of skeletal muscle of middle-aged and old rats. Thus, the carrier-based protocol may be suitable for delivery of functional RNAs to mitochondria in vivo.

CytochromecOxidase Rather than Cytochromecis a Major Determinant of Mitochondrial Respiratory Capacity in Skeletal Muscle of Aged Rats: Role of Carnitine and Lipoic Acid

The release of mitochondrial cytochrome c followed by activation of caspase cascade has been reported with aging in various tissues, whereas little is known about the caspase-independent pathway involved in mitochondrial dysfunction. To determine the functional impact of cytochrome c loss on mitochondrial respiratory capacity, we monitored NADH redox transitions and oxygen consumption in isolated skeletal muscle mitochondria of 4- and 24-month-old rats in the presence and absence of exogenous cytochrome c; and assessed the efficacy of cosupplementation of carnitine and lipoic acid on age-related alteration in mitochondrial respiration. The loss of mitochondrial cytochrome c with age was accompanied with alteration in respiratory transition, which in turn was not rescued by exogenous addition of cytochrome c to isolated mitochondria. The analysis of mitochondrial and nuclear-encoded cytochrome c oxidase subunits suggests that the decreased levels of cytochrome c oxidase may be attributed for the irresponsiveness to exogenously added cytochrome c on mitochondrial respiratory transitions, possibly through reduction of upstream electron carriers. Oral supplementation of carnitine and lipoic acid to aged rats help to maintaining the mitochondrial oxidative capacity by regulating the release of cytochrome c and improves cytochrome c oxidase transcript levels. Thus, carnitine and lipoic acid supplementation prevents the loss of cytochrome c and their associated decline in cytochrome c oxidase activity; thereby, effectively attenuating any putative decrease in cellular energy and redox status with age.

Mitochondrial enzymes increase in muscle in response to 7-10 days of cycle exercise.

Endurance exercise training induces a significant increase in the respiratory capacity of skeletal muscle. This is reflected by a training-induced increase in mitochondrial enzyme activity. One consequence of this adaptation is that there is a decreased reliance on carbohydrate utilization with a concomitant increase in fat utilization, resulting in an improvement in endurance capacity. Recently it has been reported that 7-14 days of cycle ergometer exercise training does not induce an increase in mitochondrial enzyme levels in skeletal muscle but, nevertheless, results in smaller decreases in phosphocreatine and glycogen and smaller increases in Pi and lactate in muscle in response to the same exercise after compared with before training. However, previous studies in rats have shown that an adaptive increase in mitochondrial enzymes is already evident after only 2 days of exercise training. In view of this discrepency, the present study was performed to reevaluate the effect of short-term training (7-10 days) on mitochondrial enzymes in skeletal muscle of humans. Twelve subjects [6 men and 6 women, 27 +/- 5 (SE) yr old] underwent 7 (n = 5) or 10 days (n = 7) of cycle ergometer exercise for 2h/day at 60-70% of peak O2 consumption. Peak O2 consumption was increased by 9% (from 2.97 +/- 0.16 to 3.24 +/- 0.17 l/min) in response to training. Blood lactate levels were lower at the same absolute work rates after than before training. The activities of citrate synthase, beta-hydroxyacyl-CoA dehydrogenase, mitochondrial thiolase, and carnitine acetyltransferase were increased by approximately 30% in response to training. The results of the present study provide evidence that in humans, as in rats, the adaptive increase in mitochondrial enzymes in skeletal muscle occurs fairly rapidly in response to exercise training. They provide no support for the claim that this adaptive response is delayed for > 2 wk after the onset of training.

Biochemical adaptation of cardiac and skeletal muscle to physical activity

1. 1. Female Wistar rats were randomly assigned to control (C) or exercising (T) groups and subsequently portioned into 1, 3, 5 and 10 day T and C groups. The T groups completed a progressive endurance running program. Biochemical indices of adaptation were measured in cardiac muscle and in plantaris and soleus muscles of C and T animals after their last exercise bout. 2. 2. In cardiac muscle, myofibrillar ATPase activity was significantly elevated in the 3T (0.241 ± 0.031) and 5T (0.242 ± 0.013) groups ( P ⩽ 0.05) compared to their respective controls (3C = 0.187 ± 0.015 and 5C = 0.190 ± 0.007). 3. 3. After 10 days of training cardiac myofibrillar ATPase activity was elevated by 17% but this was not significant ( P ⩽ 0.05). 4. 4. No changes in myofibrillar ATPase activity were seen in skeletal muscle ( P ⩾ 0.05), however, hexokinase activity progressively increased and was significantly elevated in the 3T, 5T and 10T soleus and plantaris muscles of rats over controls ( P ⩽ 0.05). 5. 5. Minimal nonsignificant changes were noted in the hexokinase activity of the hearts of all T groups ( P ⩾ 0.05). 6. 6. These results indicate that metabolic adaptation of the heart and skeletal muscles takes place after as little as three training sessions. 7. 7. Although the adaptation of the skeletal muscles continually progresses, the adaptation of the heart appears to be transitory. 8. 8. The reason for the decline in the cardiac adaptation could be related to the increased respiratory capacity of skeletal muscle and/or to a decrease in the stimulus specific to the myocardium.

The effect of high-intensity exercise on the respiratory capacity of sceletal muscle

The effect of high-intensity exercise on the respiratory capacity of skeletal muscle was studied in horses which ran five 600-m bouts on a track with 2 min of rest between exercise bouts, or once to fatigue on a treadmill at an intensity that elicited the maximal oxygen uptake. Venous blood and biopsy samples of the middle gluteal muscle were collected at rest, after each exercise bout, and 30 and 60 min post-exercise. Blood samples were analyzed for lactate concentration and pH and muscle samples for metabolites, pH, and respiratory capacity. Venous blood and muscle pH declined to 6.91 +/- 0.02 and 6.57 +/- 0.02, respectively, after the fifth track run and to 6.98 +/- 0.02 and 6.71 +/- 0.07, respectively, after treadmill running. Muscle metabolite changes were consistent with the metabolic response to high-intensity exercise. Muscle respiratory capacity declined greater than 20% (P less than 0.05) after a single exercise bout and was 45% of the control value after the fifth track run. Tissue respiration was depressed 60 min post-exercise but was normal 24 h later. These observations suggest that high-intensity exercise impairs the respiratory capacity of the working muscle. Although this occurred in parallel with reductions in pH, other factors could be responsible for this response.

Effects of thyroid hormone administration on skeletal muscle mitochondria

The effects of thyroid hormone administration on the levels of a number of mitochondrial markers were measured in skeletal muscle and liver of normal rats. Injection of 18 mug of L-thyroxine (T4) per 100 g body wt every 4th day for 3 wk had no effect on the concentrations of cytochrome c, on citrate synthase activity, or on respiratory capacity of skeletal muscle. Injection of 200 mug of L-triiodothyronine (T3) daily for 5 days, or feeding 23 mg T4 and 7 mg T3/kg of diet for 2 wk, resulted in thyrotoxicosis and large increases in the activity of hepatic alpha-glycerophosphate dehydrogenase and other mitochondrial markers; however, the levels of activity of mitochondrial marker enzymes in gastrocnemius and quadriceps muscles were not significantly changed. Only when rats were fed 3 mg T4 and 1 mg T3/kg diet for a 6-wk period did we observe an increase in skeletal muscle mitochondrial markers. Thus, thyroxine treatment must be sufficiently prolonged if it is to be used as a tool for studying skeletal muscle mitochondrial biogenesis.