Endurance exercise induces increases in mitochondria and the GLUT4 isoform of the glucose transporter in muscle. Although little is known about the mechanisms underlying these adaptations, new information has accumulated regarding how mitochondrial biogenesis and GLUT4 expression are regulated. This includes the findings that the transcriptional coactivator PGC-1 promotes mitochondrial biogenesis and that NRF-1 and NRF-2 act as transcriptional activators of genes encoding mitochondrial enzymes. We tested the hypothesis that increases in PGC-1, NRF-1, and NRF-2 are involved in the initial adaptive response of muscle to exercise. Five daily bouts of swimming induced increases in mitochondrial enzymes and GLUT4 in skeletal muscle in rats. One exercise bout resulted in approximately twofold increases in full-length muscle PGC-1 mRNA and PGC-1 protein, which were evident 18 h after exercise. A smaller form of PGC-1 increased after exercise. The exercise induced increases in muscle NRF-1 and NRF-2 that were evident 12 to 18 h after one exercise bout. These findings suggest that increases in PGC-1, NRF-1, and NRF-2 represent key regulatory components of the stimulation of mitochondrial biogenesis by exercise and that PGC-1 mediates the coordinated increases in GLUT4 and mitochondria.

It has been hypothesized that insulin resistance is mediated by a deficiency of mitochondria in skeletal muscle. In keeping with this hypothesis, high-fat diets that cause insulin resistance have been reported to result in a decrease in muscle mitochondria. In contrast, we found that feeding rats high-fat diets that cause muscle insulin resistance results in a concomitant gradual increase in muscle mitochondria. This adaptation appears to be mediated by activation of peroxisome proliferator-activated receptor (PPAR)␦ by fatty acids, which results in a gradual, posttranscriptionally regulated increase in PPAR ␥ coactivator 1␣ (PGC-1␣) protein expression. Similarly, overexpression of PPAR␦ results in a large increase in PGC-1␣ protein in the absence of any increase in PGC-1␣ mRNA. We interpret our findings as evidence that raising free fatty acids results in an increase in mitochondria by activating PPAR␦, which mediates a posttranscriptional increase in PGC-1␣. Our findings argue against the concept that insulin resistance is mediated by a deficiency of muscle mitochondria.I t has been hypothesized that insulin resistance in patients with impaired or diabetic glucose tolerance is mediated by a deficiency of mitochondria in skeletal muscle (1, 2). The mechanism by which a decrease in mitochondria is proposed to cause insulin resistance is accumulation of intramyocellular lipids caused by a decrease in the capacity to oxidize fat (2). This hypothesis is based on the finding that type 2 diabetics and insulin-resistant individuals with impaired glucose tolerance have Ϸ30% less mitochondria in their muscles than insulinsensitive control subjects (3-7). In support of this concept, recent studies have reported that raising serum free fatty acids (FFA) by a high-fat diet in humans (8), or by feeding mice or rats high-fat diets (8-10), results in decreases in skeletal muscle peroxisome proliferator-activated receptor ␥ coactivator-1␣ (PGC-1␣) mRNA (8-10) and the mRNA levels of various mitochondrial constituents (8). In contrast, a number of earlier studies provided evidence that high-fat diets induce increases in mitochondrial marker enzymes (11-14), and Turner et al. (15) recently reported that a high-fat diet resulted in increases in mitochondrial biogenesis and fatty acid oxidative capacity in skeletal muscle of mice.We have found that raising serum FFA in rats by feeding them a high-fat diet and giving them daily heparin injections results in an increase in muscle mitochondria (16). The initial purpose of the present study was to determine whether the more modest increase in FFA induced by a high-fat diet also results in increased mitochondrial biogenesis with an increase in the capacity of muscle to oxidize fat. We found that a high-fat diet does induce an increase in muscle mitochondria. This finding made it possible to evaluate whether a high-fat diet causes muscle insulin resistance despite increases in mitochondria and fat oxidative capacity.Overexpression of peroxisome proliferator-activated receptor (PPAR)␦ i...

Skeletal muscle adapts to endurance exercise with an increase in mitochondria. Muscle contractions generate numerous potential signals. To determine which of these stimulates mitochondrial biogenesis, we are using L6 myotubes. Using this model we have found that raising cytosolic Ca2+ induces an increase in mitochondria. In this study, we tested the hypothesis that raising cytosolic Ca2+ in L6 myotubes induces increased expression of PGC-1, NRF-1, NRF-2, and mtTFA, factors that have been implicated in mitochondrial biogenesis and in the adaptation of muscle to exercise. Raising cytosolic Ca2+ by exposing L6 myotubes to caffeine for 5 h induced significant increases in PGC-1 and mtTFA protein expression and in NRF-1 and NRF-2 binding to DNA. These adaptations were prevented by dantrolene, which blocks Ca2+ release from the SR. Exposure of L6 myotubes to caffeine for 5 h per day for 5 days induced significant increases in mitochondrial marker enzyme proteins. Our results show that the adaptive response of L6 myotubes to an increase in cytosolic Ca2+ mimics the stimulation of mitochondrial biogenesis by exercise. They support the hypothesis that an increase in cytosolic Ca2+ is one of the signals that mediate increased mitochondrial biogenesis in muscle.

A number of studies have reported that a high-fat diet induces increases in mitochondrial fatty acid oxidation enzymes in muscle. In contrast, in two recent studies raising plasma free fatty acids (FFA) resulted in a decrease in mitochondria. In this work, we reevaluated the effects of raising FFA on muscle mitochondrial biogenesis and capacity for fat oxidation. Rats were fed a high-fat diet and given daily injections of heparin to raise FFA. This treatment induced an increase in mitochondrial biogenesis in muscle, as evidenced by increases in mitochondrial enzymes of the fatty acid oxidation pathway, citrate cycle, and respiratory chain, with an increase in the capacity to oxidize fat, as well as an increase in mitochondrial DNA copy number. Raising FFA also resulted in an increase in binding of peroxisome proliferator-activated receptor (PPAR) ␦ to the PPAR response element on the carnitine palmitoyltransferase 1 promoter. We interpret our results as evidence that raising FFA induces an increase in mitochondrial biogenesis in muscle by activating PPAR␦.peroxisome proliferator-activated receptor (PPAR)␦ ͉ high-fat diet ͉ fatty acid oxidation I t has been reported that high-fat diets induce increased expression of mitochondrial fatty acid oxidation (FAO) enzymes in skeletal muscle, and it has been suggested that this adaptation results in an increase in the capacity to oxidize fatty acids (1-6). Similarly, exposure of cardiac myocytes to moderately high concentrations of fatty acids activates transcription of the muscle carnitine palmitoyltransferase (mCPT) gene (7). The peroxisome proliferator-activated receptors PPAR␣ and PPAR␤/␦ (PPAR␦) are ligand-activated nuclear receptors that activate transcription of genes encoding FAO enzymes as well as the uncoupling proteins (8-11). Both PPAR␣ and PPAR␦ are expressed in skeletal muscle, with PPAR␦ being the predominant isoform (12, 13). The PPARs are activated by long-chain fatty acids (14, 15). It, therefore, seemed likely that if raising plasma free fatty acids (FFA) does increase muscle FAO enzymes, this effect is mediated by PPAR␦ and ␣. However, in contrast to the earlier reports, two recent studies have reported that raising plasma FFA with a lipid emulsion (16) or a high-fat diet (17) results in decreases in muscle mitochondrial enzymes.In this context, the present study was undertaken to determine whether raising plasma FFA does induce an increase in expression of FAO enzymes in skeletal muscle and, if it does, to obtain evidence regarding whether the increase in FFA activates PPAR␦. A second aim, if raising FFA was found to increase FAO enzymes, was to determine whether an isolated increase in FAO enzymes results in an increase in the capacity to oxidize fatty acids or whether the citrate cycle and respiratory chain are rate-limiting. ResultsPlasma FFA Concentrations. Plasma FFA were markedly elevated in the rats fed the high-fat diet and given heparin (2.06 Ϯ 0.42 mM; range 1.22-3.84 mM) for six rats given heparin compared with 0.25 Ϯ 0.02 mM for six chow-f...

There is evidence suggesting that adaptive increases in GLUT4 and mitochondria in skeletal muscle occur in parallel. It has been reported that raising cytosolic Ca2+ in myocytes induces increases in mitochondrial enzymes. In this study, we tested the hypothesis that an increase in cytosolic Ca2+ induces an increase in GLUT4. We found that raising cytosolic Ca2+ by exposing L6 myotubes to 5 mM caffeine for 3 h/day for 5 days induced increases in GLUT4 protein and in myocyte enhancer factor (MEF)2A and MEF2D, which are transcription factors involved in regulating GLUT4 expression. The caffeine-induced increases in GLUT4 and MEF2A and MEF2D were partially blocked by dantrolene, an inhibitor of sarcoplasmic reticulum Ca2+ release, and completely blocked by KN93, an inhibitor of Ca2+-calmodulin-dependent protein kinase (CAMK). Caffeine also induced increases in MEF2A, MEF2D, and GLUT4 in rat epitrochlearis muscles incubated with caffeine in culture medium. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), which activates AMP-activated protein kinase (AMPK), also induced approximately twofold increases in GLUT4, MEF2A, and MEF2D in L6 myocytes. Our results provide evidence that increases in cytosolic Ca2+and activation of AMPK, both of which occur in exercising muscle, increase GLUT4 protein in myocytes and skeletal muscle. The data suggest that this effect of Ca2+ is mediated by activation of CAMK and indicate that MEF2A and MEF2D are involved in this adaptive response.