Prolonged interleukin-6 administration enhances glucose tolerance and increases skeletal muscle PPARalpha and UCP2 expression in rats.

Chronic elevations in interleukin (IL)-6 have been associated with insulin resistance, but acute IL-6 administration can enhance insulin sensitivity. Our aim was to exogenously administer IL-6 to rats to elicit either chronic or repeated acute elevations in systemic IL-6. We hypothesized that a continuous elevation of IL-6 would inhibit glucose tolerance and insulin sensitivity while acute intermittent elevations would improve it. Male Wistar rats were treated for 14d with recombinant human IL-6 (2.4 microy) or saline administered either by miniosmotic pump (continuous IL-6) or via twice-daily injection (intermittent IL-6). Glucose and insulin tolerance tests were performed following 14-d treatment and 24 h later rats were administered a bolus of insulin (150 mU/g) or saline intraperitoneally. Approximately, 10 min after insulin injection soleus, gastrocnemius and liver were excised and rapidly frozen in liquid nitrogen for subsequent metabolic measures. Irrespective of the mode of delivery, IL-6 treatment increased basal insulin sensitivity, as measured by the homeostatic model assessment of insulin resistance, and enhanced glucose clearance during an i.p. glucose tolerance test. IL-6 increased circulating fatty acids, but did not increase triglyceride accumulation in either skeletal muscle or liver, while it increased the protein expression of both PPARalpha and UCP2 in skeletal muscle, suggesting that IL-6 can enhance fat oxidation via mitochondrial uncoupling. These data demonstrate that, irrespective of the mode of delivery, IL-6 administration over 2 weeks enhances glucose tolerance. Our results do not support the notion that prolonged chronically elevated IL-6 impairs insulin action in vivo.

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.

FOXO1 regulates the expression of 4E-BP1 and inhibits mTOR signaling in mammalian skeletal muscle.

The mammalian target of rapamycin (mTOR) is regulated by growth factors to promote protein synthesis. In mammalian skeletal muscle, the Forkhead-O1 transcription factor (FOXO1) promotes catabolism by activating ubiquitin-protein ligases. Using C2C12 mouse myoblasts that stably express inducible FOXO1-ER fusion proteins and transgenic mice that specifically overexpress constitutively active FOXO1 in skeletal muscle (FOXO(++/+)), we show that FOXO1 inhibits mTOR signaling and protein synthesis. Activation of constitutively active FOXO1 induced the expression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) mRNA via binding to the promoter. This resulted in an increased total 4E-BP1 abundance and a reduced 4E-BP1 (Thr-37/46) phosphorylation. The reduction in 4E-BP1 phosphorylation was associated with a reduction in the abundance of Raptor and mTOR proteins, Raptor-associated mTOR, reduced phosphorylation of the downstream protein p70S6 kinase, and attenuated incorporation of [(14)C]phenylalanine into protein. The FOXO(++/+) mice, characterized by severe skeletal muscle atrophy, displayed similar patterns of mRNA expression and protein abundance to those observed in the constitutively active FOXO1 C2C12 myotubes. These data suggest that FOXO1 may be an important therapeutic target for human diseases where anabolism is impaired.

PGC-1alpha gene expression is down-regulated by Akt- mediated phosphorylation and nuclear exclusion of FoxO1 in insulin-stimulated skeletal muscle.

There are multiple binding domains on the promoter region of the peroxisome proliferator activator receptor gamma coactivator-1 alpha (PGC-1alpha) gene, including a trio of insulin responsive elements that are activated by the forkhead box class-O (FoxO1) winged helix transcription factor, which is known to be regulated by acute transforming retrovirus thymoma (Akt). Here we show that in skeletal muscle biopsy specimens from healthy humans and cultured human skeletal myotubes, insulin phosphorylates Akt (Ser473) and FoxO1 (Thr24, Ser256), leading to reduced nuclear abundance of FoxO1 total protein. This is associated with an insulin-mediated repression of the mRNA expression PGC-1alpha and downstream genes associated with oxidative phosphorylation. In contrast, in muscle taken from insulin resistant humans or in palmitate-treated insulin resistant myotubes, neither Akt nor FoxO1 was phosphorylated by insulin, resulting in a failure for nuclear exclusion of FoxO1 total protein, and an inability for insulin to repress the mRNA expression of PGC-1alpha and down-stream genes. To determine whether the regulation of FoxO1 was Akt dependent, we next treated Akt2 -/- and wild-type mice with or without insulin. Insulin phosphorylated Akt and FoxO1 (Thr24, Ser256) resulting in a reduced nuclear expression of FoxO1 total protein in wild-type but not Akt2 -/- skeletal muscle. We conclude that insulin decreases the expression of genes involved in oxidative metabolism in healthy but not insulin resistant muscle, due to a decrease in FoxO1 phosphorylation and nuclear exclusion secondary to reduced Akt activity.

Failure to repeatedly supercompensate muscle glycogen stores in highly trained men.

PURPOSE: It is not known whether it is possible to repeatedly supercompensate muscle glycogen stores after exhaustive exercise bouts undertaken within several days. METHODS: We evaluated the effect of repeated exercise-diet manipulation on muscle glycogen and triacylglycerol (IMTG) metabolism and exercise capacity in six well-trained subjects who completed an intermittent, exhaustive cycling protocol (EX) on three occasions separated by 48 h (i.e., days 1, 3, and 5) in a 5-d period. Twenty-four hours before day 1, subjects consumed a moderate (6 g.kg)-carbohydrate (CHO) diet, followed by 5 d of a high (12 g.kg.d)-CHO diet. Muscle biopsies were taken at rest, immediately post-EX on days 1, 3, and 5, and after 3 h of recovery on days 1 and 3. RESULTS: Compared with day 1, resting muscle [glycogen] was elevated on day 3 but not day 5 (435+/-57 vs 713+/-60 vs 409+/-40 mmol.kg, P<0.001). [IMTG] was reduced by 28% (P<0.05) after EX on day 1, but post-EX levels on days 3 and 5 were similar to rest. EX was enhanced on days 3 and 5 compared with day 1 (31.9+/-2.5 and 35.4+/-3.8 vs 24.1+/-1.4 kJ.kg, P<0.05). Glycogen synthase activity at rest and immediately post-EX was similar between trials. Additionally, the rates of muscle glycogen accumulation were similar during the 3-h recovery period on days 1 and 3. CONCLUSION: We show that well-trained men cannot repeatedly supercompensate muscle [glycogen] after glycogen-depleting exercise and 2 d of a high-CHO diet, suggesting that the mechanisms responsible for glycogen accumulation are attenuated as a consequence of successive days of glycogen-depleting exercise.