Hsp72 preserves muscle function and slows progression of severe muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca(2+), which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca(2+)) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.

Oxidative stress-induced insulin resistance in skeletal muscle cells is ameliorated by gamma-tocopherol treatment.

BACKGROUND: Oxidative stress-induced reactive oxygen species are associated with the clinical manifestation of insulin resistance. Evidence suggests that antioxidant treatment may reduce this incidence. AIM OF THE STUDY: This study determined whether glucose oxidase (GO)-induced insulin resistance in cultured skeletal muscle cells could be ameliorated by pre-treatment with gamma-tocopherol (GT). METHODS: Insulin sensitivity in L6 myotubes was assessed by 2-deoxy-D: -[(3)H]-glucose uptake. The phosphorylation of distal insulin signaling proteins Akt and the Akt substrate AS160 were determined by western blot. RESULTS: One hour treatment with 100 mU/ml GO decreased insulin-stimulated glucose uptake (P < 0.001). Pre-treatment with GT either partially (100 microM) or completely (200 microM) restored insulin-stimulated glucose uptake in cells after GO-induced insulin resistance. GO-induced oxidative stress did not impair insulin stimulated phosphorylation of Akt or AS160, but 200 microM GT increased insulin-stimulated phosphorylation of these key signaling proteins (P < 0.05). CONCLUSIONS: High-dose (200 microM) GT treatment ameliorated oxidative stress-induced insulin resistance in cultured rat L6 skeletal muscle cells.

HSP72 protects against obesity-induced insulin resistance.

Patients with type 2 diabetes have reduced gene expression of heat shock protein (HSP) 72, which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signaling proteins such as c-jun amino terminal kinase (JNK), inhibitor of kappaB kinase, and tumor necrosis factor-alpha, can induce insulin resistance, but HSP 72 can block the induction of these molecules in vitro. Accordingly, we examined whether activation of HSP72 can protect against the development of insulin resistance. First, we show that obese, insulin resistant humans have reduced HSP72 protein expression and increased JNK phosphorylation in skeletal muscle. We next used heat shock therapy, transgenic overexpression, and pharmacologic means to overexpress HSP72 either specifically in skeletal muscle or globally in mice. Herein, we show that regardless of the means used to achieve an elevation in HSP72 protein, protection against diet- or obesity-induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance was observed. This protection was tightly associated with the prevention of JNK phosphorylation. These findings identify an essential role for HSP72 in blocking inflammation and preventing insulin resistance in the context of genetic obesity or high-fat feeding.

Tissue-specific effects of rosiglitazone and exercise in the treatment of lipid-induced insulin resistance.

Both pharmacological intervention (i.e., thiazolidinediones [TZDs]) and lifestyle modification (i.e., exercise training) are clinically effective treatments for improving whole-body insulin sensitivity. However, the mechanism(s) by which these therapies reverse lipid-induced insulin resistance in skeletal muscle is unclear. We determined the effects of 4 weeks of rosiglitazone treatment and exercise training and their combined actions (rosiglitazone treatment and exercise training) on lipid and glucose metabolism in high-fat-fed rats. High-fat feeding resulted in decreased muscle insulin sensitivity, which was associated with increased rates of palmitate uptake and the accumulation of the fatty acid metabolites ceramide and diacylglycerol. Impairments in lipid metabolism were accompanied by defects in the Akt/AS160 signaling pathway. Exercise training, but not rosiglitazone treatment, reversed these impairments, resulting in improved insulin-stimulated glucose transport and increased rates of fatty acid oxidation in skeletal muscle. The improvements to glucose and lipid metabolism observed with exercise training were associated with increased AMP-activated protein kinase alpha1 activity; increased expression of Akt1, peroxisome proliferator-activated receptor gamma coactivator 1, and GLUT4; and a decrease in AS160 expression. In contrast, rosiglitazone treatment exacerbated lipid accumulation and decreased insulin-stimulated glucose transport in skeletal muscle. However, rosiglitazone, but not exercise training, increased adipose tissue GLUT4 and acetyl CoA carboxylase expression. Both exercise training and rosiglitazone decreased liver triacylglycerol content. Although both interventions can improve whole-body insulin sensitivity, our results show that they produce divergent effects on protein expression and triglyceride storage in different tissues. Accordingly, exercise training and rosiglitazone may act as complementary therapies for the treatment of insulin resistance.

Ciliary neurotrophic factor suppresses hypothalamic AMP-kinase signaling in leptin-resistant obese mice.

We examined the actions of a second-generation ciliary neurotrophic factor analog (CNTF(Ax15)) on AMP-activated protein kinase (AMPK), a known regulator of food intake. Unlike leptin CNTF(Ax15) has been shown to reduce food intake in obese rodents and humans. Intraperitoneal injection of CNTF(Ax15) acutely (45 min) reduced hypothalamic AMPKalpha2 activity, AMPKalpha2Thr172 phosphorylation, and acetyl-coenzyme A carboxylase phosphorylation, effects not observed 2 or 6 h after injection. Intracerebroventricular CNTF(Ax15) reduced food intake, increased arcuate nucleus (ARC) signal transducer and activator of transcription 3 phosphorylation, and reduced AMPK signaling but not in the paraventricular nucleus (PVN), posterior hypothalamus, or cortex. To compare the effects of leptin and CNTF(Ax15) in a diet-induced model of obesity, mice were fed a control carbohydrate or high-fat diet (HFD) for 12 wk. Leptin treatment ip reduced food intake in control mice but not in mice fed a HFD. In contrast, ip CNTF markedly reduced food intake in both control and HFD animals. Both leptin and CNTF reduced AMPK activity and acetyl-coenzyme A carboxylase phosphorylation in the ARC and PVN of control-fed mice. A HFD blunted leptin but not CNTF effects on AMPK signaling in the ARC and PVN. In summary, these data demonstrate that CNTF(Ax15) bypasses diet-induced leptin resistance to reduce hypothalamic AMPK activity.

Vitamin E isoform-specific inhibition of the exercise-induced heat shock protein 72 expression in humans.

Increased levels of reactive oxygen and nitrogen species, as seen in response to exercise, challenge the cellular integrity. Important protective adaptive changes include induction of heat shock proteins (HSPs). We hypothesized that supplementation with antioxidant vitamins C (ascorbic acid) and E (tocopherol) would attenuate the exercise-induced increase of HSP72 in the skeletal muscle and in the circulation. Using randomization, we allocated 21 young men into three groups receiving one of the following oral supplementations: RRR-alpha-tocopherol 400 IU/day + ascorbic acid (AA) 500 mg/day (CEalpha), RRR-alpha-tocopherol 290 IU/day + RRR-gamma-tocopherol 130 IU/day + AA 500 mg/day (CEalphagamma), or placebo (Control). After 28 days of supplementation, the subjects performed 3 h of knee extensor exercise at 50% of the maximal power output. HSP72 mRNA and protein content was determined in muscle biopsies obtained from vastus lateralis at rest (0 h), postexercise (3 h), and after a 3-h recovery (6 h). In addition, blood was sampled for measurements of HSP72, alpha-tocopherol, gamma-tocopherol, AA, and 8-iso-prostaglandin-F2alpha (8-PGF2alpha). Postsupplementation, the groups differed with respect to plasma vitamin levels. The marker of lipid peroxidation, 8-iso-PGF2alpha, increased from 0 h to 3 h in all groups, however, markedly less (P < 0.05) in CEalpha. In Control, skeletal muscle HSP72 mRNA content increased 2.5-fold (P < 0.05) and serum HSP72 protein increased 4-fold (P < 0.05) in response to exercise, whereas a significant increase of skeletal muscle HSP72 protein content was not observed (P = 0.07). In CEalpha, skeletal muscle HSP72 mRNA, HSP72 protein, and serum HSP72 were not different from Control in response to exercise. In contrast, the effect of exercise on skeletal muscle HSP72 mRNA and protein, as well as circulating HSP72, was completely blunted in CEalphagamma. The results indicate that gamma-tocopherol comprises a potent inhibitor of the exercise-induced increase of HSP72 in skeletal muscle as well as in the circulation.

Interleukin-6 stimulates lipolysis and fat oxidation in humans.

Although IL-6 is a key modulator of immune function, it also plays a role in regulating substrate metabolism. To determine whether IL-6 affects lipid metabolism, 18 healthy men were infused for 3 h with saline (Con; n = 6) or a high dose (High-rhIL6; n = 6) or a low dose (Low-rhIL6; n = 6) of recombinant human IL-6 (rhIL-6). The IL-6 concentration during Con, Low-rhIL6, and High-rhIL6 was at a steady state after 30 min of infusion at approximately 4, 140, and 320 pg/ml, respectively. Either dose of rhIL-6 was associated with a similar increase in fatty acid (FA) concentration and endogenous FA rate of appearance (R(a)) from 90 min after the start of the infusion. The FA concentration and FA R(a) continued to increase until the cessation of rhIL-6 infusion, reaching levels approximately 50% greater than Con values. The elevated levels reached at the end of rhIL-6 infusion persisted at least 3 h postinfusion. Triacylglycerol concentrations were unchanged during rhIL-6 infusion, whereas whole body fat oxidation increased after the second hour of rhIL-6 infusion. Of note, during Low-rhIL6, the induced elevation in FA concentration and FA R(a) occurred in the absence of any change in adrenaline, insulin, or glucagon, and no adverse side effects were observed. In conclusion, the data identify IL-6 as a potent modulator of fat metabolism in humans, increasing fat oxidation and FA reesterification without causing hypertriacylglyceridemia.