Contraction-induced interleukin-6 gene transcription in skeletal muscle is regulated by c-Jun terminal kinase/activator protein-1.

Exercise increases the expression of the prototypical myokine IL-6, but the precise mechanism by which this occurs has yet to be identified. To mimic exercise conditions, C2C12 myotubes were mechanically stimulated via electrical pulse stimulation (EPS). We compared the responses of EPS with the pharmacological Ca(2+) carrier calcimycin (A23187) because contraction induces marked increases in cytosolic Ca(2+) levels or the classical IκB kinase/NFκB inflammatory response elicited by H(2)O(2). We demonstrate that, unlike H(2)O(2)-stimulated increases in IL-6 mRNA, neither calcimycin- nor EPS-induced IL-6 mRNA expression is under the transcriptional control of NFκB. Rather, we show that EPS increased the phosphorylation of JNK and the reporter activity of the downstream transcription factor AP-1. Furthermore, JNK inhibition abolished the EPS-induced increase in IL-6 mRNA and protein expression. Finally, we observed an exercise-induced increase in both JNK phosphorylation and IL-6 mRNA expression in the skeletal muscles of mice after 30 min of treadmill running. Importantly, exercise did not increase IL-6 mRNA expression in skeletal muscle-specific JNK-deficient mice. These data identify a novel contraction-mediated transcriptional regulatory pathway for IL-6 in skeletal muscle.

IκB kinase ß (IKKß) does not mediate feedback inhibition of the insulin signalling cascade.

In the present study, we have examined whether IKKß [IκB (inhibitor of nuclear factor κB) kinase ß] plays a role in feedback inhibition of the insulin signalling cascade. Insulin induces the phosphorylation of IKKß, in vitro and in vivo, and this effect is dependent on intact signalling via PI3K (phosphoinositide 3-kinase), but not PKB (protein kinase B). To test the hypothesis that insulin activates IKKß as a means of negative feedback, we employed a variety of experimental approaches. First, pharmacological inhibition of IKKß via BMS-345541 did not potentiate insulin-induced IRS1 (insulin receptor substrate 1) tyrosine phosphorylation, PKB phosphorylation or 2-deoxyglucose uptake in differentiated 3T3-L1 adipocytes. BMS-345541 did not prevent insulin-induced IRS1 serine phosphorylation on known IKKß target sites. Secondly, adenovirus-mediated overexpression of wild-type IKKß in differentiated 3T3-L1 adipocytes did not suppress insulin-stimulated 2-deoxyglucose uptake, IRS1 tyrosine phosphorylation, IRS1 association with the p85 regulatory subunit of PI3K or PKB phosphorylation. Thirdly, insulin signalling was not potentiated in mouse embryonic fibroblasts lacking IKKß. Finally, insulin treatment of 3T3-L1 adipocytes did not promote the recruitment of IKKß to IRS1, supporting our findings that IKKß, although activated by insulin, does not promote direct serine phosphorylation of IRS1 and does not contribute to the feedback inhibition of the insulin signalling cascade.

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.

Skeletal myocytes are a source of interleukin-6 mRNA expression and protein release during contraction: evidence of fiber type specificity.

In this study, we aimed to determine whether skeletal muscle cells per se are a source of interleukin (IL)-6 during contraction and whether IL-6 production is fiber type specific. Muscle biopsy samples were collected from seven males before (PRE) and after (POST) completing 120 min of continuous bicycle ergometry. Biopsies were sectioned and analyzed for the following: IL-6 protein detected by immunohistochemistry (IHC), IL-6 mRNA content detected by in situ hybridization, fiber type measured by either IHC or myofibrillar ATPase activity stain, and glycogen content measured by periodic acid schiff (PAS) assay. Fibers were qualitatively categorized according to glycogen content to one of five groups (1-5), with 1 being very low (LOW) and 5 being very high (HIGH) glycogen. Total fluorescence (PRE vs. POST) and glycogen-dependent fluorescence (LOW vs. HIGH) of IL-6 protein were quantitated using Metamorph software. Total IL-6 protein was elevated from PRE to POST exercise (P<0.05). At PRE, IL-6 protein was evenly distributed across all fibers at low levels, consistent with glycogen distribution. At POST, IL-6 protein was greater (P<0.05) in HIGH compared with LOW glycogen fibers, which coincided with type 2 fibers. IL-6 mRNA was distributed peripherally in all fibers at PRE. At POST, however, IL-6 mRNA appeared predominantly in type 2 fibers, which also had higher glycogen content (P<0.05). These data demonstrate that myocytes per se are a source of IL-6 produced during contraction. Our data also suggest that type 2 fibers predominantly produce IL-6 during muscle contractile activity.

Altering dietary nutrient intake that reduces glycogen content leads to phosphorylation of nuclear p38 MAP kinase in human skeletal muscle: association with IL-6 gene transcription during contraction.

To determine the effect of glycogen availability and contraction on intracellular signaling and IL-6 gene transcription, eight males performed 60 min of exercise on two occasions: either with prior ingestion of a normal (Con) or low carbohydrate (LCHO) diet that reduced pre-exercise muscle glycogen content. Muscle biopsies were obtained and analyzed for IL-6 mRNA. In addition, nuclear proteins were isolated from the samples and analyzed for the mitogen- activated protein kinases (MAPK) c-jun amino-terminal kinase (JNK) 1 and 2 and p38 MAPK. Nuclear fractions were also analyzed for the phosphorylated forms of JNK (p-JNK) and p38 MAPK (p-p38 MAPK) and the abundance of the nuclear transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor kappa-beta (NF-kappabeta). No differences were observed in the protein abundance of total JNK 1/2, p38 MAPK, NFAT, or NF-kappabeta before exercise, but the nuclear abundance of p-p38 MAPK was higher (P<0.05) in LCHO. Contraction resulted in an increase (P<0.05) in nuclear p-JNK 1/2, but there were no differences when comparing CON with LCHO. The fold increase in IL-6 mRNA with contraction was potentiated (P<0.05) in LCHO. A correlation between pre-exercise nuclear phosphorylated p38 MAPK and contraction-induced fold increase in IL-6 mRNA was performed, revealing a highly significant correlation (r=0.96; P<0.01). We next incubated L6 myotubes in ionomycin (a compound known to induce IL-6 mRNA) with or without the pyridinylimidazole p38 MAPK inhibitor SB203580. Treatments did not affect total nuclear p38 MAPK, but ionomycin increased (P<0.05) both nuclear p-p38 MAPK and IL-6 mRNA. The addition of SB203580 to ionomycin decreased (P<0.05) nuclear p-p38 MAPK and totally abolished (P<0.05) the ionomycin- induced increase in IL-6 mRNA. These data suggest that reduced carbohydrate intake that results in low intramuscular glycogen leads to phosphorylation of p38 MAPK at the nucleus. Furthermore, phosphorylation of p38 MAPK in the nucleus appears to be an upstream target for IL-6, providing new insights into the regulation of IL-6 gene transcription.

Increasing leucine concentration stimulates mechanistic target of rapamycin signaling and cell growth in C2C12 skeletal muscle cells.

Leucine is a key amino acid for initiating translation in muscle cells, but the dose-dependent effects of leucine on intracellular signaling are poorly characterized. This study examined the effect that increasing doses of leucine would have on changes in mechanistic target of rapamycin (mTOR)-mediated signaling, rates of protein synthesis, and cell size in C2C12 cells. We hypothesized that a leucine "threshold" exists, which represents the minimum stimulus required to initiate mTOR signaling in muscle cells. Acute exposure to 1.5, 3.2, 5.0, and 16.1 mM leucine increased phosphorylation of mTOR(Ser2448) (~1.4-fold; P < .04), 4E-BP1 (Thr37/46) (~1.9-fold; P < .001), and rpS6(Ser235/6) (~2.3-fold; P < .001). However, only p70S6k(Thr389) exhibited a dose-dependent response to leucine with all treatments higher than control (~4-fold; P < .001) and at least 5 mM higher than the 1.5-mM concentration (1.2-fold; P < .02). Rates of protein synthesis were not altered by any treatment. Seven days of exposure to 0.5, 1.5, 5.0, and 16.5 mM leucine resulted in an increase in cell size in at least 5 mM treatments (~1.6-fold, P < .001 vs control). Our findings indicate that even at low leucine concentrations, phosphorylation of proteins regulating translation initiation signaling is enhanced. The phosphorylation of p70S6k(Thr389) follows a leucine dose-response relationship, although this was not reflected by the acute protein synthetic response. Nevertheless, under the conditions of the present study, it appears that leucine concentrations of at least 5 mM are necessary to enhance cell growth.

Cytokine gene expression in human skeletal muscle during concentric contraction: evidence that IL-8, like IL-6, is influenced by glycogen availability.

To determine the expression and induction of cytokines in human skeletal muscle during concentric contractions, eight males performed 60 min of bicycle exercise, with either a normal (Con) or reduced (Lo Gly) preexercise intramuscular glycogen content. Muscle biopsy samples were obtained before and after exercise and analyzed for glycogen and the mRNA expression of 13 cytokines. Resting muscle glycogen was higher (P < 0.05) in Con compared with Lo Gly and was reduced (P < 0.05) to 102 +/- 32 vs. 17 +/- 5 mmol U glycosyl/kg dry mass for Con and Lo Gly, respectively. We detected mRNA levels in human skeletal muscle for five cytokines, namely interleukin (IL)-1 beta, IL-6, IL-8, IL-15, and tumor necrosis factor-alpha. However, muscle contraction increased (P < 0.05) the mRNA expression of IL-6 and IL-8 alone. In addition, the fold change for both IL-8 and IL-6 was markedly higher (P < 0.05) in Lo Gly compared with Con. Given these results, we analyzed venous blood samples, obtained before and during exercise, for IL-6 and IL-8. Plasma IL-6 was not different at rest, and although the circulating concentration of this cytokine increased (P < 0.05) it increased to a greater extent (P < 0.05) throughout exercise in Lo Gly. In contrast, plasma IL-8 was not affected by exercise or treatment. These data demonstrate that cytokines are not ubiquitously expressed in skeletal muscle and that only IL-6 and IL-8 mRNA are increased during contraction of this mode and duration. Furthermore, the mRNA abundance of IL-6 and IL-8 appears to be influenced by glycogen availability in the contracting muscle.