Palmitate, but not unsaturated fatty acids, induces the expression of interleukin-6 in human myotubes through proteasome-dependent activation of nuclear factor-kappaB.

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-kappaB. 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.

Palmitate-induced activation of the hexosamine pathway in human myotubes: increased expression of glutamine:fructose-6-phosphate aminotransferase.

The nutrient sensing capacity of the hexosamine biosynthetic pathway (HBP) has been implicated in the development of insulin resistance of skeletal muscle. To study the molecular mechanism of the free fatty acid (FFA)-induced activation of the HBP myotubes obtained from muscle biopsies of metabolically characterized, subjects were stimulated with different fatty acids for 20 h. Incubation with the saturated fatty acids palmitate and stearate (0.5 mmol/l) resulted in a three- to fourfold increase in mRNA expression of glutamine:fructose-6-phosphate aminotransferase (GFAT), the key and rate-limiting enzyme of the hexosamine pathway. Unsaturated fatty acids or 30 mmol/l glucose had little or no effect. Palmitate increased the amount of GFAT protein nearly two-fold, and subsequently, the concentration of UDP-N-acetylglucosamine, the end product of the HBP, was 1.3-fold enhanced in the palmitate-stimulated myotubes. The nonmetabolized fatty acid bromopalmitate had no effect. The DNA binding activity of the transcription factor Sp1, a target downstream of the HBP, was increased by palmitate and completely lost after enzymatic removal of O-GlcNAc. No correlation was found between the palmitate-induced increase in GFAT protein and the insulin resistance in the respective subjects. The findings reveal a new mechanism for how FFAs induce the activation of the HBP.

Effect of glimepiride on insulin-stimulated glycogen synthesis in cultured human skeletal muscle cells: a comparison to glibenclamide.

OBJECTIVE: To examine the effect of glimepiride on insulin-stimulated glycogen synthesis in cultured human skeletal muscle cells in comparison with glibenclamide. RESEARCH DESIGN AND METHODS: Myotubes derived from glucose-tolerant subjects were incubated with glimepiride or glibenclamide (0-100 micro mol/l) for 4 h and with or without insulin (100 nmol/l) for 2 h, and subsequently glycogen synthesis was determined. RESULTS: Glimepiride had no significant effect on basal glycogen synthesis; in contrast, glimepiride caused a dose-dependent increase of insulin-stimulated glycogen synthesis, with a maximal effect of 39.97 +/- 8.4% (mean +/- SEM, n = 4, P < 0,02). The time course of this glimepiride effect on insulin-stimulated glycogen synthesis showed a peak after 12 h incubation with a half maximal effect after 4 h. Preincubation of the myotubes with wortmannin (100 nmol/l), an inhibitor of phosphatidylinositol (PI)- 3 kinase, caused an inhibition of this glimepiride effect on insulin-stimulated glycogen synthesis. In contrast to glimepiride, incubation of myotubes with glibenclamide (0-100nmol/l), a second generation sulfonylurea, had no significant effect on basal or insulin-stimulated glycogen synthesis. CONCLUSIONS: Incubation of cultured human skeletal muscle cells derived from glucose-tolerant subjects with glimepiride caused a dose-dependent increase of insulin-stimulated glycogen synthesis using therapeutic glimepiride concentrations. This glimepiride effect seems to be mediated via the PI3 kinase pathway. In contrast to glimepiride, glibenclamide had no significant effect on basal or insulin-stimulated glycogen synthesis. These results suggest that glimepiride, beside its well-known effect to stimulate insulin secretion, possess an insulin-sensitizing action in cultured human skeletal muscle cells in support of the concept of an extrapancreatic action of glimepiride.

Troglitazone downregulates delta-6 desaturase gene expression in human skeletal muscle cell cultures.

Delta-6 Desaturase, one of the rate-limiting enzymes, catalyzes the conversion of linoleic acid (C18:2 omega6) into gamma-linolenic acid (C18:3 omega6), arachidonic acid (C20:4 omega6), and further metabolites. Recently, it has been shown that human Delta-6 desaturase is expressed not only in liver but in a variety of human tissues, including muscle. Skeletal muscle is a major site of insulin action, and insulin sensitivity may be related to the fatty acid composition of muscle lipids. We examined the effects of troglitazone on the regulation of Delta-6 desaturase gene expression in human muscle cell cultures obtained from muscle biopsies (n = 15). Delta-6 Desaturase mRNA and peroxisome proliferator-activated receptor gamma2 (PPARgamma2) mRNA were quantified by two-step RT-PCR, and the activity of the Delta-6 desaturase enzyme was estimated by gas chromatographic analysis of the omega 6-C18:3/C18:2 fatty acids ratio. In cells treated with 11.5 micromol troglitazone for 4 days, PPARgamma2 mRNA levels were significantly increased (301.0 +/- 51.5%, P < 0.05) and Delta-6 desaturase mRNA levels were significantly decreased (41.7 +/- 5.9%, P < 0.0005) compared with the untreated controls. In accordance with the decrease of Delta-6 desaturase mRNA, there was a significant decrease in the omega6-C18:3/C18:2 ratio down to 47.4 +/- 7.5% in cholesterol esters, 54.2 +/- 7.4% in phospholipids, 56.7 +/- 6.5% in nonesterified fatty acids, and 67.7 +/- 5.9% in triglycerides. The troglitazone-induced decrease in Delta-6 desaturase mRNA is associated with a change in the unsaturated fatty acid composition of the muscle cells. These results add new aspects to the known thiazolidinedione effects on lipid metabolism.