AMPK and PPARß positive feedback loop regulates endurance exercise training-mediated GLUT4 expression in skeletal muscle.

The objective of this study is to determine whether AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), or peroxisome proliferator-activated receptor ß (PPARß) can independently mediate the increase of glucose transporter type 4 (GLUT4) expression that occurs in response to exercise training. We found that PPARß can regulate GLUT4 expression without PGC-1α. We also found AMPK and PPARß are important for maintaining normal physiological levels of GLUT4 protein in the sedentary condition as well following exercise training. However, AMPK and PPARß are not essential for the increase in GLUT4 protein expression that occurs in response to exercise training. We discovered that AMPK activation increases PPARß via myocyte enhancer factor 2A (MEF2A), which acted as a transcription factor for PPARß. Furthermore, exercise training increases the cooperation of AMPK and PPARß to regulate glucose uptake. In conclusion, cooperation between AMPK and PPARß via NRF-1/MEF2A pathway enhances the exercise training mediated adaptive increase in GLUT4 expression and subsequent glucose uptake in skeletal muscle.

The nuclear receptor PPARß/δ programs muscle glucose metabolism in cooperation with AMPK and MEF2.

To identify new gene regulatory pathways controlling skeletal muscle energy metabolism, comparative studies were conducted on muscle-specific transgenic mouse lines expressing the nuclear receptors peroxisome proliferator-activated receptor α (PPARα; muscle creatine kinase [MCK]-PPARα) or PPARß/δ (MCK-PPARß/δ). MCK-PPARß/δ mice are known to have enhanced exercise performance, whereas MCK-PPARα mice perform at low levels. Transcriptional profiling revealed that the lactate dehydrogenase b (Ldhb)/Ldha gene expression ratio is increased in MCK-PPARß/δ muscle, an isoenzyme shift that diverts pyruvate into the mitochondrion for the final steps of glucose oxidation. PPARß/δ gain- and loss-of-function studies in skeletal myotubes demonstrated that PPARß/δ, but not PPARα, interacts with the exercise-inducible kinase AMP-activated protein kinase (AMPK) to synergistically activate Ldhb gene transcription by cooperating with myocyte enhancer factor 2A (MEF2A) in a PPARß/δ ligand-independent manner. MCK-PPARß/δ muscle was shown to have high glycogen stores, increased levels of GLUT4, and augmented capacity for mitochondrial pyruvate oxidation, suggesting a broad reprogramming of glucose utilization pathways. Lastly, exercise studies demonstrated that MCK-PPARß/δ mice persistently oxidized glucose compared with nontransgenic controls, while exhibiting supranormal performance. These results identify a transcriptional regulatory mechanism that increases capacity for muscle glucose utilization in a pattern that resembles the effects of exercise training.

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

It has been reported that feeding mice resveratrol activates AMPK and SIRT1 in skeletal muscle leading to deacetylation and activation of PGC-1α, increased mitochondrial biogenesis, and improved running endurance. This study was done to further evaluate the effects of resveratrol, SIRT1, and PGC-1α deacetylation on mitochondrial biogenesis in muscle. Feeding rats or mice a diet containing 4 g resveratrol/kg diet had no effect on mitochondrial protein levels in muscle. High concentrations of resveratrol lowered ATP concentration and activated AMPK in C2C12 myotubes, resulting in an increase in mitochondrial proteins. Knockdown of SIRT1, or suppression of SIRT1 activity with a dominant-negative (DN) SIRT1 construct, increased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C2C12 cells. Expression of a DN SIRT1 in rat triceps muscle also induced an increase in mitochondrial proteins. Overexpression of SIRT1 decreased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C2C12 myotubes. Overexpression of SIRT1 also resulted in a decrease in mitochondrial proteins in rat triceps muscle. We conclude that, contrary to some previous reports, the mechanism by which SIRT1 regulates mitochondrial biogenesis is by inhibiting PGC-1α coactivator activity, resulting in a decrease in mitochondria. We also conclude that feeding rodents resveratrol has no effect on mitochondrial biogenesis in muscle.

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

KEY POINTS: 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. ABSTRACT: Endurance exercise training can increase the ability to perform prolonged strenuous exercise. The major adaptation responsible for this increase in endurance is an increase in muscle mitochondria. This adaptation occurs too slowly to provide a survival advantage when there is a sudden change in environment that necessitates prolonged exercise. In the present study, we discovered another, more rapid adaptation, a downregulation of expression of the glycogenolytic and glycolytic enzymes in muscle that mediates a slowing of muscle glycogen depletion and lactic acid accumulation. This adaptation, which appears to be mediated by PGC-1α, occurs in response to a single exercise bout and is further enhanced by two additional daily exercise bouts. It is biologically significant, because glycogen depletion and lactic acid accumulation are two of the major causes of muscle fatigue and exhaustion.

Association between specific adipose tissue CD4+ T-cell populations and insulin resistance in obese individuals.

BACKGROUND & AIMS: An increased number of macrophages in adipose tissue is associated with insulin resistance and metabolic dysfunction in obese people. However, little is known about other immune cells in adipose tissue from obese people, and whether they contribute to insulin resistance. We investigated the characteristics of T cells in adipose tissue from metabolically abnormal insulin-resistant obese (MAO) subjects, metabolically normal insulin-sensitive obese (MNO) subjects, and lean subjects. Insulin sensitivity was determined by using the hyperinsulinemic euglycemic clamp procedure. METHODS: We assessed plasma cytokine concentrations and subcutaneous adipose tissue CD4(+) T-cell populations in 9 lean, 12 MNO, and 13 MAO subjects. Skeletal muscle and liver samples were collected from 19 additional obese patients undergoing bariatric surgery to determine the presence of selected cytokine receptors. RESULTS: Adipose tissue from MAO subjects had 3- to 10-fold increases in numbers of CD4(+) T cells that produce interleukin (IL)-22 and IL-17 (a T-helper [Th] 17 and Th22 phenotype) compared with MNO and lean subjects. MAO subjects also had increased plasma concentrations of IL-22 and IL-6. Receptors for IL-17 and IL-22 were expressed in human liver and skeletal muscle samples. IL-17 and IL-22 inhibited uptake of glucose in skeletal muscle isolated from rats and reduced insulin sensitivity in cultured human hepatocytes. CONCLUSIONS: Adipose tissue from MAO individuals contains increased numbers of Th17 and Th22 cells, which produce cytokines that cause metabolic dysfunction in liver and muscle in vitro. Additional studies are needed to determine whether these alterations in adipose tissue T cells contribute to the pathogenesis of insulin resistance in obese people.

ß-Adrenergic stimulation does not activate p38 MAP kinase or induce PGC-1α in skeletal muscle.

There are reports that the ß-adrenergic agonist clenbuterol induces a large increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in skeletal muscle. This has led to the hypothesis that the increases in PGC-1α and mitochondrial biogenesis induced in muscle by endurance exercise are mediated by catecholamines. In the present study, we evaluated this possibility and found that injecting rats with clenbuterol or norepinephrine induced large increases in PGC-1α and mitochondrial proteins in brown adipose tissue but had no effect on PGC-1α expression or mitochondrial biogenesis in skeletal muscle. In brown adipocytes, the increase in PGC-1α expression induced by ß-adrenergic stimulation is mediated by activation of p38 mitogen-activated protein kinase (p38 MAPK), which phosphorylates and activates the cAMP response element binding protein (CREB) family member activating transcription factor 2 (ATF2), which binds to a cyclic AMP response element (CRE) in the PGC-1α promoter and mediates the increase in PGC-1α transcription. Phospho-CREB does not have this effect. Our results show that the reason for the lack of effect of ß-adrenergic stimulation on PGC-1α expression in muscle is that catecholamines do not activate p38 or increase ATF2 phosphorylation in muscle.

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.

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.

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

A potential link between muscle peroxisome proliferator- activated receptor-alpha signaling and obesity-related diabetes.

The role of the peroxisome proliferator-activated receptor-alpha (PPARalpha) in the development of insulin-resistant diabetes was evaluated using gain- and loss-of-function approaches. Transgenic mice overexpressing PPARalpha in muscle (MCK-PPARalpha mice) developed glucose intolerance despite being protected from diet-induced obesity. Conversely, PPARalpha null mice were protected from diet-induced insulin resistance in the context of obesity. In skeletal muscle, MCK-PPARalpha mice exhibited increased fatty acid oxidation rates, diminished AMP-activated protein kinase activity, and reduced insulin-stimulated glucose uptake without alterations in the phosphorylation status of key insulin-signaling proteins. These effects on muscle glucose uptake involved transcriptional repression of the GLUT4 gene. Pharmacologic inhibition of fatty acid oxidation or mitochondrial respiratory coupling prevented the effects of PPARalpha on GLUT4 expression and glucose homeostasis. These results identify PPARalpha-driven alterations in muscle fatty acid oxidation and energetics as a potential link between obesity and the development of glucose intolerance and insulin resistance.

Is "fat-induced" muscle insulin resistance rapidly reversible?

Elevated plasma free fatty acids (FFA) cause insulin resistance and are thought to play a key role in mediating insulin resistance in patients with the metabolic syndrome (MTS) and type 2 diabetes mellitus (DM). Two experimental models used to study the mechanisms responsible for insulin resistance in patients are high-fat diet-fed rodents and administration of triglycerides and heparin to raise plasma FFA. As evidence that insulin resistance in high-fat diet-fed rats is due to high FFA, it has been reported that the insulin resistance is rapidly reversed by an overnight fast, a high-glucose meal, and an exercise bout. If true, these findings would invalidate the high-fat diet-fed rodent as a model for MTS or type 2 DM, because insulin resistance is not rapidly reversed by these treatments in patients. The purpose of this study was to determine whether diet-induced insulin resistance is, in fact, rapidly reversible. Incubation of muscles in vitro rapidly reversed insulin resistance induced by administration of triglycerides and heparin, but not by a high-fat diet. An overnight fast and a high-glucose meal were followed by a large increase in insulin-stimulated muscle glucose transport. However, these are adaptive responses, rather than reversals of insulin resistance, because they also occurred in muscles of insulin-sensitive, chow-fed control rats. Our results show that insulin resistance induced by high FFA, i.e., Randle glucose-fatty acid cycle, is transient. In contrast, the insulin resistance induced by a high-fat diet does not reverse rapidly.

PET measurements of myocardial glucose metabolism with 1-11C-glucose and kinetic modeling.

UNLABELLED: The aim of this study was to investigate whether compartmental modeling of 1-(11)C-glucose PET kinetics can be used for noninvasive measurements of myocardial glucose metabolism beyond its initial extraction. METHODS: 1-(11)C-Glucose and U-(13)C-glucose were injected simultaneously into 22 mongrel dogs under a wide range of metabolic states; this was followed by 1 h of PET data acquisition. Heart tissue samples were analyzed for (13)C-glycogen content (nmol/g). Arterial and coronary sinus blood samples (ART/CS) were analyzed for glucose (mumol/mL), (11)C-glucose, (11)CO(2), and (11)C-total acidic metabolites ((11)C-lactate [LA] + (11)CO(2)) (counts/min/mL) and were used to calculate myocardial fractions of (a) glucose and 1-(11)C-glucose extractions, EF(GLU) and EF((11)C-GLU); (b) (11)C-GLU and (11)C-LA oxidation, OF((11)C-GLU) and OF((11)C-LA); (c) (11)C-glycolsysis, GCF((11)C-GLU); and (d) (11)C-glycogen content, GNF((11)C-GLU). On the basis of these measurements, a compartmental model (M) that accounts for the contribution of exogenous (11)C-LA to myocardial (11)C activity was implemented to measure M-EF(GLU), M-GCF(GLU), M-OF(GLU), M-GNF(GLU), and the fraction of myocardial glucose stored as glycogen M-GNF(GLU)/M-EF(GLU)). RESULTS: ART/CS data showed the following: (a) A strong correlation was found between EF((11)C-GLU) and EF(GLU) (r = 0.92, P < 0.0001; slope = 0.95, P = not significantly different from 1). (b) In interventions with high glucose extraction and oxidation, the contribution of OF((11)C-GLU) to total oxidation was higher than that of OF((11)C-LA) (P < 0.01). In contrast, in interventions in which glucose uptake and oxidation were inhibited, OF((11)C-LA) was higher than OF((11)C-GLU) (P < 0.05). (c) A strong correlation was found between GNF((11)C-GLU)/EF(GLU) and direct measurements of fractional (13)C-glycogen content, (r = 0.96, P < 0.0001). Model-derived PET measurements of M-EF(GLU), M-GCF(GLU), and M-OF(GLU) strongly correlated with EF(GLU) (slope = 0.92, r = 0.95, P < 0.0001), GCF((11)C-GLU) (slope = 0.79, r = 0.97, P < 0.0001), and OF((11)C-GLU) (slope = 0.70, r = 0.96, P < 0.0001), respectively. M-GNF(GLU)/M-EF(GLU) strongly correlated with fractional (13)C-content (r = 0.92, P < 0.0001). CONCLUSION: Under nonischemic conditions, it is feasible to measure myocardial glucose metabolism noninvasively beyond its initial extraction with PET using 1-(11)C-glucose and a compartmental modeling approach that takes into account uptake and oxidation of secondarily labeled exogenous (11)C-lactate.

PPARß 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 PPARß regulates mitochondrial content in skeletal muscle. We discovered that PPARß increases PGC-1α by protecting it from degradation by binding to PGC-1α and limiting ubiquitination. PPARß 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-ß (CaMKKß). Knockdown of PPARß 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, PPARß induces an increase in PGC-1α protein, and PPARß is a transcription factor for NRF-1. Thus, PPARß plays essential roles in the maintenance and adaptive increase in mitochondrial enzymes in skeletal muscle by exercise.

Calcineurin does not mediate exercise-induced increase in muscle GLUT4.

Exercise induces a rapid increase in expression of the GLUT4 isoform of the glucose transporter in skeletal muscle. One of the signals responsible for this adaptation appears to be an increase in cytosolic Ca(2+). Myocyte enhancer factor 2A (MEF2A) is a transcription factor that is involved in the regulation of GLUT4 expression. It has been reported that the Ca(2+)-regulated phosphatase calcineurin mediates the activation of MEF2 by exercise. It has also been shown that the expression of activated calcineurin in mouse skeletal muscle results in an increase in GLUT4. These findings suggest that increases in cytosolic Ca(2+) induce increased GLUT4 expression by activating calcineurin. However, we have obtained evidence that this response is mediated by a Ca(2+)-calmodulin-dependent protein kinase. The purpose of this study was to test the hypothesis that calcineurin is involved in mediating exercise-induced increases in GLUT4. Rats were exercised on 5 successive days using a swimming protocol. One group of swimmers was given 20 mg/kg body weight of cyclosporin, a calcineurin inhibitor, 2 h before exercise. A second group was given vehicle. GLUT4 protein was increased approximately 80%, GLUT4 mRNA was increased approximately 2.5-fold, MEF2A protein was increased twofold, and hexokinase II protein was increased approximately 2.5-fold 18 h after the last exercise bout. The cyclosporin treatment completely inhibited calcineurin activity but did not affect the adaptive increases in GLUT4, MEF2A, or hexokinase expression. We conclude that calcineurin activation does not mediate the adaptive increase in GLUT4 expression induced in skeletal muscle by exercise.

Ca2+ and AMPK both mediate stimulation of glucose transport by muscle contractions.

It is now generally accepted that activation of AMP-activated protein kinase (AMPK) is involved in the stimulation of glucose transport by muscle contractions. However, earlier studies provided evidence that increases in cytosolic Ca(2+) mediate the effect of muscle contractions on glucose transport. The purpose of this study was to test the hypothesis that both the increase in cytosolic Ca(2+) and the activation of AMPK are involved in the stimulation of glucose transport by muscle contractions. Caffeine causes release of Ca(2+) from the sarcoplasmic reticulum. Incubation of rat epitrochlearis muscles with a concentration of caffeine that raises cytosolic Ca(2+) to levels too low to cause contraction resulted in an approximate threefold increase in glucose transport. Caffeine treatment also resulted in increased phosphorylation of calmodulin-dependent protein kinase (CAMK)-II in epitrochlearis muscle. The stimulation of glucose transport by caffeine was blocked by the Ca(2+)-CAMK inhibitors KN62 and KN93. Activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) also resulted in an approximate threefold increase in glucose transport in the epitrochlearis. The increases in glucose transport induced by AICAR and caffeine were additive, and their combined effect was not significantly different from that induced by maximally effective contractile activity. KN62 and KN93 caused an approximately 50% inhibition of the stimulation of glucose transport by contractile activity. Our results provide evidence that both Ca(2+) and AMPK are involved in the stimulation of glucose transport by muscle contractions. They also suggest that the stimulation of glucose transport by Ca(2+) involves activation of CAMK.

A peroxovanadium compound stimulates muscle glucose transport as powerfully as insulin and contractions combined.

Stimulation of glucose transport by insulin involves tyrosine phosphorylation of the insulin receptor (IR) and IR substrates (IRSs). Peroxovanadates inhibit tyrosine phosphatases, also resulting in tyrosine phosphorylation of the IRSs. Muscle contractions stimulate glucose transport by a mechanism independent of the insulin-signaling pathway. We found that the peroxovanadate compound bis-peroxovanadium,1,10-phenanthrolene [bpV(phen)] stimulates glucose transport to the same extent as the additive effects of maximal insulin and contraction stimuli. Translocation of GLUT4 to the cell surface mediates stimulation of glucose transport. There is evidence suggesting there are separate insulin- and contraction-stimulated pools of GLUT4-containing vesicles. We tested the hypothesis that bpV(phen) stimulates both the insulin- and the contraction-activated pathways. Stimulation of glucose transport and GLUT4 translocation by bpV(phen) was completely blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin and LY294002. The combined effect of bpV(phen) and contractions was no greater than that of bpV(phen) alone. Activation of the IRS-PI 3-K signaling pathway was much greater with bpV(phen) than with insulin. Our results suggest that the GLUT4 vesicles that are normally translocated in response to contractions but not insulin can respond to the signal generated via the IRS-PI 3-K pathway if it is sufficiently powerful.

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.

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 delta-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 approximately twofold and delta-aminolevulinate synthase was increased approximately 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.

Ginsenoside Re rapidly reverses insulin resistance in muscles of high-fat diet fed rats.

OBJECTIVE: In a previous study, it was found that a ginseng berry extract with a high content of the ginsenoside Re normalized blood glucose in ob/ob mice. The objective of this study was to evaluate the effect of the ginsenoside Re on insulin resistance of glucose transport in muscles of rats made insulin resistant with a high-fat diet. MATERIAL/METHOD: Rats were fed either rat chow or a high-fat diet for 5 weeks. The rats were then euthanized, and insulin stimulated glucose transport activity was measured in epitrochlearis and soleus muscle strips in vitro. RESULTS: Treatment of muscles with Re alone had no effect on glucose transport. The high-fat diet resulted in ~50% decreases in insulin responsiveness of GLUT4 translocation to the cell surface and glucose transport in epitrochlearis and soleus muscles. Treatment of muscles with Re in vitro for 90 min completely reversed the high-fat diet-induced insulin resistance of glucose transport and GLUT4 translocation. This effect of Re is specific for insulin stimulated glucose transport, as Re treatment did not reverse the high-fat diet-induced resistance of skeletal muscle glucose transport to stimulation by contractions or hypoxia. CONCLUSIONS: Our results show that the ginsenoside Re induces a remarkably rapid reversal of high-fat diet-induced insulin resistance of muscle glucose transport by reversing the impairment of insulin-stimulated GLUT4 translocation to the cell surface.

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 ß-oxidation pathways, causes muscle insulin resistance.