May Chen

Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator PGC-1

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 ~ 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.—Baar, K., Wende, A. R., Jones, T. E., Marison, M., Nolte, L. A., Chen, M., Kelly, D. P., Holloszy, J. O. Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator PGC‐1. FASEB J. 16, 1879–1886 (2002)

High-fat diets cause insulin resistance despite an increase in muscle 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)δ by fatty acids, which results in a gradual, posttranscriptionally regulated increase in PPAR γ coactivator 1α (PGC-1α) protein expression. Similarly, overexpression of PPARδ results in a large increase in PGC-1α protein in the absence of any increase in PGC-1α mRNA. We interpret our findings as evidence that raising free fatty acids results in an increase in mitochondria by activating PPARδ, which mediates a posttranscriptional increase in PGC-1α. Our findings argue against the concept that insulin resistance is mediated by a deficiency of muscle mitochondria.

Skeletal muscle atrophy in old rats: Differential changes in the three fiber types

This study was undertaken to reevaluate the effects of ageing on skeletal muscle mass and on mitochondrial and glycolytic enzyme levels in the different types of skeletal muscle in rats. It was found that some muscles atrophy with ageing, while others do not, in male rats. Atrophy appears to occur in weight-bearing muscles, and is most marked in those with a high proportion of type IIb fibers. The muscles that did not atrophy are non-weight-bearing, and include the epitrochlearis (predominantly type IIb fibers) and the adductor longus (predominantly type I fibers). The average cross-sectional area of muscle fibers in the plantaris muscles of 28-30-month-old rats was approximately 30% smaller than that of 9-10-month-old animals, providing evidence that the approximately 30% lower weight of the plantaris in the old group was entirely due to fiber atrophy. The proportion of type IIa fibers was decreased and the proportion of type I fibers was increased in the plantaris of the old rats. The respiratory capacity of the soleus muscle (predominantly type I fibers), and the glycolytic capacity of the superficial, white (type IIb) and deep, red (predominantly type IIa) portions of the vastus lateralis, were reduced in the old rats. Our results provide evidence that ageing has differential effects on the three types of skeletal muscle fiber, and on weight-bearing and non-weight-bearing muscles, in the rat.

Raising Ca2+ in L6 myotubes mimics effects of exercise on mitochondrial biogenesis in muscle

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.

Raising plasma fatty acid concentration induces increased biogenesis of mitochondria in skeletal 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) δ 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 PPARδ.

Skeletal muscle overexpression of nuclear respiratory factor 1 increases glucose transport capacity

Nuclear respiratory factor 1 (NRF‐1) is a transcriptional activator of nuclear genes that encode a range of mitochondrial proteins including cytochrome c, various other respiratory chain subunits, and δ‐aminolevulinate synthase. Activation of NRF‐1 in fibroblasts has been shown to induce increases in cytochrome c expression and mitochondrial respiratory capacity. To further evaluate the role of NRF‐1 in the regulation of mitochondrial biogenesis and respiratory capacity, we generated transgenic mice overexpressing NRF‐1 in skeletal muscle. Cytochrome c expression was increased ∼twofold and δ‐aminolevulinate synthase was increased ∼50% in NRF‐1 transgenic muscle. The levels of some mitochondrial proteins were increased 50–60%, while others were unchanged. Muscle respiratory capacity was not increased in the NRF‐1 transgenic mice. A finding that provides new insight regarding the role of NRF‐1 was that expression of MEF2A and GLUT4 was increased in NRF‐1 transgenic muscle. The increase in GLUT4 was associated with a proportional increase in insulin‐stimulated glucose transport. These results show that an isolated increase in NRF‐1 is not sufficient to bring about a coordinated increase in expression of all of the proteins necessary for assembly of functional mitochondria. They also provide the new information that NRF‐1 overexpression results in increased expression of GLUT4.—Baar, K., Song, Z., Semenkovich, C. F., Jones, T. E,. Han, D.‐H., Nolte, L. A., Ojuka, E. O., Chen, M., Holloszy, J. O. Skeletal muscle overexpression of nuclear respiratory factor 1 increases glucose transport capacity. FASEB J. 17, 1666–1673 (2003)

DHEA protects against visceral obesity and muscle insulin resistance in rats fed a high-fat diet

Visceral obesity is frequently associated with muscle insulin resistance. Rats fed a high-fat diet rapidly develop obesity and insulin resistance. Dehydroepiandrosterone (DHEA) has been reported to protect against the development of obesity. This study tested the hypothesis that DHEA protects against the increase in visceral fat and the development of muscle insulin resistance induced by a high-fat diet in rats. Feeding rats a diet providing 50% of the energy as fat for 4 wk resulted in a twofold greater visceral fat mass and a 50% lower rate of maximally insulin-stimulated muscle 2-deoxyglucose (2-DG) uptake compared with controls. Rats fed the high-fat diet plus 0.3% DHEA were largely protected against the increase in visceral fat (+11.3 g in high fat vs. +2.9 g in high fat plus DHEA, compared with controls) and against the decrease in insulin-stimulated muscle 2-DG uptake (0.94 ± 0.15 μmol ⋅ ml-1 ⋅ 20 min-1, controls; 0.46 ± 0.06 μmol ⋅ ml-1 ⋅ 20 min-1, high-fat diet; 0.78 ± 0.07 μmol ⋅ ml-1 ⋅ 20 min-1, high fat + DHEA). DHEA did not affect food intake. These results show that DHEA has a protective effect against accumulation of visceral fat and development of muscle insulin resistance in rats fed a high-fat diet.Visceral obesity is frequently associated with muscle insulin resistance. Rats fed a high-fat diet rapidly develop obesity and insulin resistance. Dehydroepiandrosterone (DHEA) has been reported to protect against the development of obesity. This study tested the hypothesis that DHEA protects against the increase in visceral fat and the development of muscle insulin resistance induced by a high-fat diet in rats. Feeding rats a diet providing 50% of the energy as fat for 4 wk resulted in a twofold greater visceral fat mass and a 50% lower rate of maximally insulin-stimulated muscle 2-deoxyglucose (2-DG) uptake compared with controls. Rats fed the high-fat diet plus 0.3% DHEA were largely protected against the increase in visceral fat (+ 11.3 g in high fat vs. + 2.9 g in high fat plus DHEA, compared with controls) and against the decrease in insulin-stimulated muscle 2-DG uptake (0.94 +/- 0.15 mumol.ml-1.20 min-1, controls; 0.46 +/- 0.06 mumol.ml-1.20 min-1, high-fat diet; 0.78 +/- 0.07 mumol.ml-1.20 min-1, high fat + DHEA). DHEA did not affect food intake. These results show that DHEA has a protective effect against accumulation of visceral fat and development of muscle insulin resistance in rats fed a high-fat diet.

Transgenic Overexpression of Hexokinase II in Skeletal Muscle Does not Increase Glucose Disposal in Wild-Type or Glut1-Overexpressing Mice

Glut1 transgenic mice were bred with transgenic mice that overexpress hexokinase II in skeletal muscle in order to determine whether whole-body glucose disposal could be further augmented in mice overexpressing glucose transporters. Overexpression of hexokinase alone in skeletal muscle had no effect on glucose transport or metabolism in isolated muscles, nor did it alter blood glucose levels or the rate of whole-body glucose disposal. Expression of the hexokinase transgene in the context of the Glut1 transgenic background did not alter glucose transport in isolated muscles but did cause additional increases in steady-state glucose 6-phosphate (3.2-fold) and glycogen (7.5-fold) levels compared with muscles that overexpress the Glut1 transporter alone. Surprisingly, however, these increases were not accompanied by a change in basal or insulin-stimulated whole-body glucose disposal in the doubly transgenic mice compared with Glut1 transgenic mice, probably due to an inhibition of de novo glycogen synthesis as a result of the high levels of steady-state glycogen in the muscles of doubly transgenic mice (430 μmol/g versus 10 μmol/g in wild-type mice). We conclude that the hexokinase gene may not be a good target for therapies designed to counteract insulin resistance or hyperglycemia.