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...

Skeletal muscle cells have been established as significant producers of IL-6 during exercise. This IL-6 production is discussed as one possible mediator of the beneficial effects of physical activity on glucose and fatty acid metabolism. IL-6 itself could be the exercise-related factor that upregulates and maintains its own production. We investigated this hypothesis and the underlying molecular mechanism in cultured C(2)C(12) cells. IL-6 led to a rapid and prolonged increase in IL-6 mRNA, which was also found in human myotubes. Because IL-6 has been shown to activate AMP-activated kinase (AMPK), we studied whether, in turn, activated AMPK induces IL-6 expression. Pharmacological activation of AMPK with 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside upregulated IL-6 mRNA expression, which was blocked by knockdown of AMPK alpha(1) and alpha(2) using small, interfering RNA (siRNA) oligonucleotides. However, the effect of IL-6 was shown to be independent of AMPK, since the siRNA approach silencing the AMPK alpha-subunits did not reduce the upregulation of IL-6 induced by IL-6 stimulation. The self-stimulatory effect of IL-6 partly involves a Ca(2+)-dependent pathway: IL-6 increased intracellular Ca(2+), and intracellular blockade of Ca(2+) with a Ca(2+) chelator reduced the IL-6-mediated increase in IL-6 mRNA levels. Moreover, inhibition of Ca(2+)/calmodulin-dependent kinase kinase with STO-609 or the siRNA approach decreased IL-6 mRNA levels of control and IL-6-stimulated cells. A major, STO-609-independent mechanism is the IL-6-mediated stabilization of its mRNA. The data suggest that IL-6 could act as autocrine factor upregulating its mRNA levels, thereby supporting its function as an exercise-activated factor in skeletal muscle cells.

Recent experimental work indicates that the hyperglycemia-induced increase in mesangial matrix production, which is a hallmark in the development of diabetic nephropathy, is mediated by increased expression of GLUT1. Mesangial cells stably transfected with human GLUT1 mimic the effect of hyperglycemia on the production of the extracellular matrix proteins, particularly fibronectin, when cultured under normoglycemic conditions. Our investigation of the molecular mechanism of this effect has revealed that the enhanced fibronectin production was not mediated by the prosclerotic cytokine transforming growth factor (TGF)-␤1. We found markedly increased nuclear content in Jun proteins, leading to enhanced DNA-binding activity of activating protein 1 (AP-1). AP-1 inhibition reduced fibronectin production in a dosage-dependent manner. Moreover, inhibition of classic protein kinase C (PKC) isoforms prevented both the activation of AP-1 and the enhanced fibronectin production. In contrast to mesangial cells exposed to high glucose, no activation of the hexosamine biosynthetic, p38, or extracellular signal-related kinase 1 and 2 mitogen-activated protein kinase pathways nor any increase in TGF-␤1 synthesis could be detected, which could be explained by the absence of oxidative stress in cells transfected with the human GLUT1 gene. Our data indicate that increased glucose uptake and metabolism induce PKC-dependent AP-1 activation that is sufficient for enhanced fibronectin production, but not for increased TGF-␤1 expression.

The Ser/Thr phosphorylation of insulin receptor substrate 1 (IRS) is one key mechanism to stimulate and/or attenuate insulin signal transduction. Using a phospho-specific polyclonal antibody directed against phosphorylated Ser 318 of IRS-1, we found a rapid and transient insulin-stimulated phosphorylation of Ser 318 in human and rodent skeletal muscle cell models and in muscle tissue of insulin-treated mice. None of the investigated insulin resistanceassociated factors (e.g. high glucose, tumor necrosis factor-␣, adrenaline) stimulated the phosphorylation of Ser 318 . Studying the function of this phosphorylation, we found that replacing Ser 318 by alanine completely prevented both the attenuation of insulin-stimulated Akt/protein kinase B Ser 473 phosphorylation and glucose uptake after 60 min of insulin stimulation. Unexpectedly, after acute insulin stimulation, we observed that phosphorylation of Ser 318 is not inhibitory but rather enhances insulin signal transduction because introduction of Ala 318 led to a reduction of the insulinstimulated Akt/protein kinase B phosphorylation. Furthermore, replacing Ser 318 by glutamate, i.e. mimicking phosphorylation, improved glucose uptake after acute insulin stimulation. These data suggest that phosphorylation of Ser 318 is not per se inhibitory but is necessary to trigger the attenuation of the insulin-stimulated signal in skeletal muscle cells. Investigating the molecular mechanism of insulin-stimulated Ser 318 phosphorylation, we found that phosphatidylinositol 3-kinase-mediated activation of atypical protein kinase C-and recruitment of protein kinase C-to IRS-1 was responsible for this phosphorylation. We conclude that Ser 318 phosphorylation of IRS-1 is an early physiological event in insulin-stimulated signal transduction, which attenuates the continuing action of insulin.