The mechanisms responsible for increased expression of TNF-alpha in skeletal muscle cells in diabetic states are not well understood. We examined the effects of the saturated acid palmitate on TNF-alpha expression. Exposure of C2C12 skeletal muscle cells to 0.75 mm palmitate enhanced mRNA (25-fold induction, P < 0.001) and protein (2.5-fold induction) expression of the proinflammatory cytokine TNF-alpha. This induction was inversely correlated with a fall in GLUT4 mRNA levels (57% reduction, P < 0.001) and glucose uptake (34% reduction, P < 0.001). PD98059 and U0126, inhibitors of the ERK-MAPK cascade, partially prevented the palmitate-induced TNF-alpha expression. Palmitate increased nuclear factor (NF)-kappaB activation and incubation of the cells with the NF-kappaB inhibitors pyrrolidine dithiocarbamate and parthenolide partially prevented TNF-alpha expression. Incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C (PKC), abolished palmitate-induced TNF-alpha expression, and restored GLUT4 mRNA levels. Palmitate treatment enhanced the expression of phospho-PKCtheta, suggesting that this PKC isoform was involved in the changes reported, and coincubation of palmitate-treated cells with the PKC inhibitor chelerythrine prevented the palmitate-induced reduction in the expression of IkappaBalpha and insulin-stimulated Akt activation. These findings suggest that enhanced TNF-alpha expression and GLUT4 down-regulation caused by palmitate are mediated through the PKC activation, confirming that this enzyme may be a target for either the prevention or the treatment of fatty acid-induced insulin resistance.

These results suggest that PPARbeta/delta activation inhibits PE-induced cardiac hypertrophy and LPS-induced NF-kappaB activation.

The mechanisms by which elevated levels of free fatty acids cause insulin resistance are not well understood. Previous studies have reported that insulin-resistant states are characterized by a reduction in the expression of peroxisome proliferator-activated receptor-␥ coactivator (PGC)-1, a transcriptional activator that promotes oxidative capacity in skeletal muscle cells. However, little is known about the factors responsible for reduced PGC-1 expression. The expression of PGC-1 mRNA levels was assessed in C2C12 skeletal muscle cells exposed to palmitate either in the presence or in the absence of several inhibitors to study the biochemical pathways involved. We report that exposure of C2C12 skeletal muscle cells to 0.75 mmol/l palmitate, but not oleate, reduced PGC-1␣ mRNA levels (66%; P < 0.001), whereas PGC-1␤ expression was not affected. Palmitate led to mitogen-activated protein kinase (MAPK)-extracellular signal-related kinase (ERK) 1/2 (MEK1/2) activation. In addition, pharmacological inhibition of this pathway by coincubation of the palmitate-exposed cells with the MEK1/2 inhibitors PD98059 and U0126 prevented the downregulation of PGC-1␣. Furthermore, nuclear factor-B (NF-B) activation was also involved in palmitatemediated PGC-1␣ downregulation, since the NF-B inhibitor parthenolide prevented a decrease in PGC-1␣ expression. These findings indicate that palmitate reduces PGC-1␣ expression in skeletal muscle cells through a mechanism involving MAPK-ERK and NF-B activation.

The mechanisms by which elevated levels of free fatty acids cause insulin resistance are not well understood. In addition, accumulating evidence suggests a link between inflammation and type 2 diabetes. Here, we report that exposure of C2C12 skeletal muscle cells to 0.5 mm palmitate results in increased mRNA levels (3.5-fold induction; P < 0.05) and secretion (control 375 +/- 57 vs. palmitate 1129 +/- 177 pg/ml; P < 0.001) of the proinflammatory cytokine IL-6. Palmitate increased nuclear factor-kappaB activation and coincubation of the cells with palmitate and the nuclear factor-kappaB inhibitor pyrrolidine dithiocarbamate prevented both IL-6 expression and secretion. Furthermore, incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C, and phorbol myristate acetate, that down-regulates protein kinase C in long-term incubations, abolished induction of IL-6 production. Finally, exposure of skeletal muscle cells to palmitate caused a fall in the mRNA levels of glucose transporter 4 and insulin-stimulated glucose uptake, whereas in the presence of anti-IL-6 antibody, which neutralizes the biological activity of mouse IL-6 in cell culture, these reductions were prevented. These findings suggest that IL-6 may mediate several of the prodiabetic effects of palmitate.

Type 2 diabetes has been related to a decrease of mitochondrial DNA (mtDNA) content. In this study, we show increased expression of the peroxisome proliferator-activated receptor-␣ (PPAR ␣ ) and its target genes involved in fatty acid metabolism in skeletal muscle of Zucker Diabetic Fatty (ZDF) ( fa/fa ) rats. In contrast, the mRNA levels of genes involved in glucose transport and utilization (GLUT4 and phosphofructokinase) were decreased, whereas the expression of pyruvatedehydrogenase kinase 4 (PDK-4), which suppresses glucose oxidation, was increased. The shift from glucose to fatty acids as the source of energy in skeletal muscle of ZDF rats was accompanied by a reduction of subunit 1