Kazuhiko Higashida

Normal adaptations to exercise despite protection against oxidative stress

It has been reported that supplementation with the antioxidant vitamins C and E prevents the adaptive increases in mitochondrial biogenesis and GLUT4 expression induced by endurance exercise. We reevaluated the effects of these antioxidants on the adaptive responses of rat skeletal muscle to swimming in a short-term study consisting of 9 days of vitamins C and E with exercise during the last 3 days and a longer-term study consisting of 8 wk of antioxidant vitamins with exercise during the last 3 wk. The rats in the antioxidant groups were given 750 mg·kg body wt(-1)·day(-1) vitamin C and 150 mg·kg body wt(-1)·day(-1) vitamin E. In rats euthanized immediately after exercise, plasma TBARs were elevated in the control rats but not in the antioxidant-supplemented rats, providing evidence for an antioxidant effect. In rats euthanized 18 h after exercise there were large increases in insulin responsiveness of glucose transport in epitrochlearis muscles mediated by an approximately twofold increase in GLUT4 expression in both the short- and long-term treatment groups. The protein levels of a number of mitochondrial marker enzymes were also increased about twofold. Superoxide dismutases (SOD) 1 and 2 were increased about twofold in triceps muscle after 3 days of exercise, but only SOD2 was increased after 3 wk of exercise. There were no differences in the magnitudes of any of these adaptive responses between the control and antioxidant groups. These results show that very large doses of antioxidant vitamins do not prevent the exercise-induced adaptive responses of muscle mitochondria, GLUT4, and insulin action to exercise and have no effect on the level of these proteins in sedentary rats.

Effects of resveratrol and SIRT1 on PGC-1α activity and mitochondrial biogenesis: a reevaluation

Feeding resveratrol to rodents has no effect on mitochondrial biogenesis, and deacetylation of PGC-1α results in a decrease, not an increase, in its coactivator activity.

Does calorie restriction induce mitochondrial biogenesis? A reevaluation

It has been reported that 30% calorie restriction (CR) for 3 mo results in large increases in mitochondrial biogenesis in heart, brain, liver, and adipose tissue, with concomitant increases in respiration and ATP synthesis. We found these results surprising, and performed this study to determine whether 30% CR does induce an increase in mitochondria in heart, brain, liver, adipose tissue, and/or skeletal muscle. To this end, we measured the levels of a range of mitochondrial proteins, and mRNAs. With the exception of long‐chain acyl‐CoA dehydrogenase protein level, which was increased ~60% in adipose tissue, none of the mitochondrial proteins or mRNAs that we measured were increased in rats subjected to 30% CR for 14 wk. There was also no increase in citrate synthase activity. Because it is not possible to have an increase in mitochondria without any increase in key mitochondrial proteins, we conclude that 30% CR does not induce an increase in mitochondria in heart, brain, liver, adipose tissue, or skeletal muscle in laboratory rodents.—Hancock, C. R., Han, D.‐H., Higashida, K., Kim, S. H., Holloszy, J. O. Does calorie restriction induce mito‐chondrial biogenesis? A reevaluation. FASEB J. 25, 785–791 (2011). www.fasebj.org

Deficiency of the Mitochondrial Electron Transport Chain in Muscle Does Not Cause Insulin Resistance

Background It has been proposed that muscle insulin resistance in type 2 diabetes is due to a selective decrease in the components of the mitochondrial electron transport chain and results from accumulation of toxic products of incomplete fat oxidation. The purpose of the present study was to test this hypothesis. Methodology/Principal Findings Rats were made severely iron deficient, by means of an iron-deficient diet. Iron deficiency results in decreases of the iron containing mitochondrial respiratory chain proteins without affecting the enzymes of the fatty acid oxidation pathway. Insulin resistance was induced by feeding iron-deficient and control rats a high fat diet. Skeletal muscle insulin resistance was evaluated by measuring glucose transport activity in soleus muscle strips. Mitochondrial proteins were measured by Western blot. Iron deficiency resulted in a decrease in expression of iron containing proteins of the mitochondrial respiratory chain in muscle. Citrate synthase, a non-iron containing citrate cycle enzyme, and long chain acyl-CoA dehydrogenase (LCAD), used as a marker for the fatty acid oxidation pathway, were unaffected by the iron deficiency. Oleate oxidation by muscle homogenates was increased by high fat feeding and decreased by iron deficiency despite high fat feeding. The high fat diet caused severe insulin resistance of muscle glucose transport. Iron deficiency completely protected against the high fat diet-induced muscle insulin resistance. Conclusions/Significance The results of the study argue against the hypothesis that a deficiency of the electron transport chain (ETC), and imbalance between the ETC and β-oxidation pathways, causes muscle insulin resistance.

The AMPK β2 Subunit Is Required for Energy Homeostasis during Metabolic Stress

ABSTRACT AMP activated protein kinase (AMPK) plays a key role in the regulatory network responsible for maintaining systemic energy homeostasis during exercise or nutrient deprivation. To understand the function of the regulatory β2 subunit of AMPK in systemic energy metabolism, we characterized β2 subunit-deficient mice. Using these mutant mice, we demonstrated that the β2 subunit plays an important role in regulating glucose, glycogen, and lipid metabolism during metabolic stress. The β2 mutant animals failed to maintain euglycemia and muscle ATP levels during fasting. In addition, β2-deficient animals showed classic symptoms of metabolic syndrome, including hyperglycemia, glucose intolerance, and insulin resistance when maintained on a high-fat diet (HFD), and were unable to maintain muscle ATP levels during exercise. Cell surface-associated glucose transporter levels were reduced in skeletal muscle from β2 mutant animals on an HFD. In addition, they displayed poor exercise performance and impaired muscle glycogen metabolism. These mutant mice had decreased activation of AMPK and deficits in PGC1α-mediated transcription in skeletal muscle. Our results highlight specific roles of AMPK complexes containing the β2 subunit and suggest the potential utility of AMPK isoform-specific pharmacological modulators for treatment of metabolic, cardiac, and neurological disorders.

Prolonged exercise training induces long-term enhancement of HSP70 expression in rat plantaris muscle.

Skeletal muscle may develop adaptive molecular chaperone enhancements as a potential defense system through repeated daily exercise stimulation. The present study investigated whether prolonged exercise training alters the expression of molecular chaperone proteins for the long term in skeletal muscle. Mature male Wistar rats were subjected for 8 wk to either a single bout of acute intermittent treadmill running (30 m/min, 5 min x 4, 5 degrees grade) or prolonged treadmill running training (15-40 m/min, 5 min x 4, 5-7 degrees grade). Levels of five molecular chaperone proteins [heat shock protein (HSP)25, HSP60, glucose-regulated protein (GRP)78, HSP70, and heat shock cognate (HSC)70] were measured in response to acute exercise and prolonged training. HSP70 levels were increased 6 and 24 h after acute exercise, but expression returned to control level within 2 days. In contrast, prolonged training had a long-term effect on HSP70 expression. Levels of HSP70 were notably increased by 4.5-fold over control 2 days after prolonged training; the enhancement was maintained for at least 14 days after training ended. However, other molecular chaperone proteins did not show adaptive changes in response to prolonged training. In addition, HSP70 enhancement by prolonged exercise training was not accompanied by transcription of HSP70 mRNA. These findings demonstrate that prolonged training can induce long-term enhancement of HSP70 expression without change at the mRNA level in skeletal muscle.

Mixed lactate and caffeine compound increases satellite cell activity and anabolic signals for muscle hypertrophy

We examined whether a mixed lactate and caffeine compound (LC) could effectively elicit proliferation and differentiation of satellite cells or activate anabolic signals in skeletal muscles. We cultured C2C12 cells with either lactate or LC for 6 h. We found that lactate significantly increased myogenin and follistatin protein levels and phosphorylation of P70S6K while decreasing the levels of myostatin relative to the control. LC significantly increased protein levels of Pax7, MyoD, and Ki67 in addition to myogenin, relative to control. LC also significantly increased follistatin expression relative to control and stimulated phosphorylation of mTOR and P70S6K. In an in vivo study, male F344/DuCrlCrlj rats were assigned to control (Sed, n = 10), exercise (Ex, n = 12), and LC supplementation (LCEx, n = 13) groups. LC was orally administered daily. The LCEx and Ex groups were exercised on a treadmill, running for 30 min at low intensity every other day for 4 wk. The LCEx group experienced a significant increase in the mass of the gastrocnemius (GA) and tibialis anterior (TA) relative to both the Sed and Ex groups. Furthermore, the LCEx group showed a significant increase in the total DNA content of TA compared with the Sed group. The LCEx group experienced a significant increase in myogenin and follistatin expression of GA relative to the Ex group. These results suggest that administration of LC can effectively increase muscle mass concomitant with elevated numbers of myonuclei, even with low-intensity exercise training, via activated satellite cells and anabolic signals.

PGC‐1α mediates a rapid, exercise‐induced downregulation of glycogenolysis in rat skeletal muscle

Long‐term endurance exercise training results in a reduction in the rates of muscle glycogen depletion and lactic acid accumulation during submaximal exercise; this adaptation is mediated by an increase in muscle mitochondria. There is evidence suggesting that short‐term training induces adaptations that downregulate glycogenolysis before there is an increase in functional mitochondria. We discovered that a single long bout of exercise induces decreases in expression of glycogenolytic and glycolytic enzymes in rat skeletal muscle; this adaptation results in slower rates of glycogenolysis and lactic acid accumulation in muscle during contractile activity. Two additional days of training amplified the adaptive response, which appears to be mediated by PGC‐1α; this adaptation is biologically significant, because glycogen depletion and lactic acid accumulation are major causes of muscle fatigue.

PPARβ Is Essential for Maintaining Normal Levels of PGC-1α and Mitochondria and for the Increase in Muscle Mitochondria Induced by Exercise

The objective of this study was to evaluate the specific mechanism(s) by which PPARβ regulates mitochondrial content in skeletal muscle. We discovered that PPARβ increases PGC-1α by protecting it from degradation by binding to PGC-1α and limiting ubiquitination. PPARβ also induces an increase in nuclear respiratory factor 1 (NRF-1) expression, resulting in increases in mitochondrial respiratory chain proteins and MEF2A, for which NRF-1 is a transcription factor. There was also an increase in AMP kinase phosphorylation mediated by an NRF-1-induced increase in CAM kinase kinase-β (CaMKKβ). Knockdown of PPARβ resulted in large decreases in the levels of PGC-1α and mitochondrial proteins and a marked attenuation of the exercise-induced increase in mitochondrial biogenesis. In conclusion, PPARβ induces an increase in PGC-1α protein, and PPARβ is a transcription factor for NRF-1. Thus, PPARβ plays essential roles in the maintenance and adaptive increase in mitochondrial enzymes in skeletal muscle by exercise.

Effects of alternate-day fasting on high-fat diet-induced insulin resistance in rat skeletal muscle.

AIMS The purpose of this study was to investigate the effects of alternate-day fasting (ADF) on insulin-stimulated glucose transport activity in skeletal muscle in rats fed a high-fat diet. MAIN METHODS Male Wistar rats were placed on a high-fat diet (n=24) or standard chow diet (Chow, n=12) for 10weeks. Rats fed the high-fat diet were separated into two groups after 4weeks. One group was subjected to ADF for the subsequent 6weeks (HF-ADF, n=12), and the other group was maintained on an ad libitum diet (HF-AL, n=12). After the 10-week dietary intervention, measurements of insulin-stimulated glucose uptake and insulin tolerance test (ITT) were performed. KEY FINDINGS Whereas the total intra-abdominal fat mass in the HF-AL group was significantly higher than in the Chow and HF-ADF groups, there was no significant difference between the Chow and HF-ADF groups. However, insulin-stimulated glucose uptake in skeletal muscles was significantly lower in both high-fat fed groups than in the Chow group. Muscle GLUT-4 protein content in HF-AL is significantly lower (~30%) than in Chow, and further reduction (~42%) was observed in the HF-ADF group rats. The HF-ADF and HF-AL group rats had less reduction in glycemia than did the Chow group rats during ITT. SIGNIFICANCE ADF was unable to eliminate high-fat diet-induced muscle insulin resistance, despite a substantial decrease in total intra-abdominal fat mass. This might have resulted from a reduction in GLUT-4 protein in both HF-AL and HF-ADF rats compared to the Chow group.