Circulating interleukin-6 (IL-6), insulin, and free fatty acid (FFA) concentrations are associated with impaired insulin action in obese and type 2 diabetic individuals. However, a causal relationship between elevated plasma FFAs and IL-6 has not been shown. Because skeletal muscle represents a major target of impaired insulin action, we studied whether FFAs may affect IL-6 expression in human myotubes. We demonstrate that specifically saturated FFAs, e.g. palmitate (0.25 mM), induce IL-6 mRNA expression and protein secretion by a proteasome-dependent mechanism that leads to a rapid and chronic activation of nuclear factor-B. Insulin, high glucose concentrations, or unsaturated FFAs did not activate IL-6 expression. In fact, the unsaturated FFA linoleate inhibited palmitate-induced IL-6 production. Because inhibition of palmitate metabolism by the acyl-CoA synthetase inhibitor triacsin C did not abolish IL-6 expression, it appears that the palmitate molecule per se exerts the observed effects. Furthermore, we show that in human myotubes, IL-6 activates the phosphorylation of signal transducer and activator of transcription 3 in concentrations similar to hepatocytes. However, no inhibitory effect of IL-6 on insulin action, determined as phosphatidylinositol 3-kinase association with insulin receptor substrate-1, Akt phosphorylation, and glycogen synthesis, was detected. We conclude that IL-6 expression may be modulated by the composition of circulating FFA, e.g. by diet, and that skeletal muscle cells could be target cells for IL-6.

The exercise-induced interleukin (IL)-6 production and secretion within skeletal muscle fibers has raised the question of a putative tissue-specific function of IL-6 in the energy metabolism of the muscle during and after the exercise. In the present study, we followed the hypothesis that IL-6 signaling may directly interact with insulin receptor substrate (IRS)-1, a keystone in the insulin signaling cascade. We showed that IL-6 induces a rapid recruitment of IRS-1 to the IL-6 receptor complex in cultured skeletal muscle cells. Moreover, IL-6 induced a rapid and transient phosphorylation of Ser-318 of IRS-1 in muscle cells and in muscle tissue, but not in the liver of IL-6-treated mice, probably via the IL-6-induced co-recruitment of protein kinase C-␦. This Ser-318 phosphorylation improved insulin-stimulated Akt phosphorylation and glucose uptake in myotubes since transfection with an IRS-1/Glu-318 mutant simulating a permanent phospho-Ser-318 modification increased Akt phosphorylation and glucose uptake. Noteworthily, two inhibitory mechanisms of IL-6 on insulin action, phosphorylation of the inhibitory Ser-307 residue of IRS-1 and induction of SOCS-3 expression, were only found in liver but not in muscle of IL-6-treated mice. Thus, the data provided evidence for a possible molecular mechanism of the physiological metabolic effects of IL-6 in skeletal muscle, thereby exerting short term beneficial effects on insulin action.

Aims/hypothesis. Activation of the renal renin-angiotensin system has been implicated in the pathogenesis of diabetic nephropathy. Because previous in vitro studies demonstrated the angiotensin II (ang II)-mediated up-regulation of the prosclerotic transforming growth factor β1 (TGF) we studied the molecular mechanism of ang II-induced TGF-β1 gene activation. Methods. Mesangial cells were stimulated with 100 nmol/l ang II with or without inhibitors of protein kinase C (PKC) and p38 MAPK and the TGF-β1 promoter activity was determined by promoter-reporter assays. The effect of ang II on the binding of nuclear proteins to the regulatory AP-1 site B, previously shown to mediate the high glucose-response of the TGF-β1 promoter, was studied by electrophoretic mobility shift assays. Results. Ang II enhanced the activity of the TGF-β1 promoter fragment -453/+11 approximately 1.6-fold.Mutation of each of two AP-1 binding sites or inhibition of the PKC-and p38 MAPK-dependent pathways blocked the ang II-stimulated activity completely. Furthermore, ang II activated the binding of nuclear proteins to the AP-1 box B of the TGF-β1 promoter. These effects were similar to those previously observed with high glucose. Co-incubation with ang II and high glucose had no additive effect on TGF-β1 promoter activity, protein binding to the AP-1 box B or activation of p38 MAPK. Conclusion/interpretation. The findings indicate that ang II and hyperglycaemia stimulate the TGF-β1 gene activation through the same PKC-and p38 MAPK-dependent pathways by the same regulatory elements of the TGF-β1 promoter. Our data could also be relevant for e.g. hypertension-induced glomerulosclerosis. [Diabetologia (2002) 45:890-898]

Previous studies showed an insulin-"desensitizing" action of IL-6 on glycogen synthesis in hepatocytes. We recently found no inhibition of the proximal steps of the insulin signal cascade in human skeletal muscle cells. Because these data indicate a possible tissue-specific effect of IL-6, we investigated the influence of IL-6 on insulin-stimulated glycogen synthesis in these cells. At first, we found that incubation of the cells with 20 ng/ml IL-6 alone induced phosphorylation of Ser473 of Akt, but not of Thr308 time dependently and we observed that IL-6 augments insulin-induced Ser473 and Thr308 phosphorylation in the low nanomolar range of insulin. Moreover, IL-6 increased insulin-stimulated phosphorylation of glycogen synthase kinase-3. Accordingly, IL-6 enhanced glycogen synthesis in the presence of 3 and 10 nM insulin, whereas IL-6 alone had only a marginal effect. IL-6 treatment of C57Bl/6 mice readily stimulated phosphorylation of Ser473 in skeletal muscle. Our result that IL-6 did not induce Ser473 phosphorylation in the liver of these mice suggests a tissue-specific effect. Together, our data demonstrate a novel insulin-sensitizing function of IL-6 on glycogen synthesis in skeletal muscle cells and indicate that IL-6 exerts cell/tissue-specific effects on insulin action.

The hyperglycemia-enhanced flux through the hexosamine biosynthetic pathway (HBP) has been implicated in the up-regulated gene expression of transforming growth factor-␤1 (TGF-␤1) in mesangial cells, thus leading to mesangial matrix expansion and diabetic glomerulosclerosis. Since the ؊1013 to ؊1002 region of the TGF-␤1 promoter shows high homology to glucose-response elements (GlRE) formerly described in genes involved in glucose metabolism, we studied the function of the GlRE in the high glucose-induced TGF-␤1 gene activation in mesangial cells. We found that high glucose concentrations enhanced the nuclear amount of upstream stimulatory factors (USF) and their binding to this sequence. Fusion of the GlRE to the thymidine kinase promoter resulted in glucose responsiveness of this promoter construct. Overexpression of either USF-1 or USF-2 increased TGF-␤1 promoter activity 2-fold, which was prevented by mutation or deletion of the GlRE. The high glucose-induced activation of the GlRE is mediated by the HBP; increased flux through the HBP induced by high glucose concentrations, by glutamine, or by overexpression of the rate-limiting enzyme glutamine:fructose-6-phosphate aminotransferase (GFAT) particularly activated USF-2 expression. GFAT-overexpressing cells showed higher USF binding activity to the GlRE and enhanced promoter activation via the GlRE. Increasing O-GlcNAc modification of proteins by streptozotocin, thereby mimicking HBP activation, also resulted in increased mRNA and nuclear protein levels of USF-2, leading to enhanced DNA binding activity to the GlRE. USF proteins themselves were not found to be O-GlcNAcmodified. Thus, we have provided evidence for a new molecular mechanism linking high glucose-enhanced HBP activity with increased nuclear USF protein levels and DNA binding activity and with up-regulated TGF-␤1 promoter activity.The adverse effects of hyperglycemia in human and experimental diabetic nephropathy have been linked to the enhanced expression and bioactivity of the prosclerotic cytokine transforming growth factor-␤1 (TGF-␤1) 1 (1-3). The hyperglycemiainduced TGF-␤1 stimulates the production of extracellular matrix proteins in mesangial cells and other renal cells (4 -7), thus leading to the thickening of glomerular and tubular basement membranes and the progressive expansion of the glomerular mesangium and the tubulointerstitium (8, 9). Increased renal expression of TGF-␤1 has been found in experimental and human diabetes (2, 3) and has also been demonstrated in high glucose-treated renal mesangial and tubular cells (5, 6). The molecular mechanism of up-regulated human TGF-␤1 gene expression involves protein kinase C-and p38 MAPK-dependent pathways (10 -12) leading to AP-1 activation (13, 14) and subsequently enhanced TGF-␤1 promoter activity via two adjacent AP-1 binding sites located at Ϫ418/Ϫ412 and Ϫ371/ Ϫ363, respectively (13).Moreover, increased synthesis of amino sugars through the hexosamine biosynthetic pathway (HBP) has also been implicated in hyperglycemia-induced TGF-␤1 s...