Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and IkappaB-alpha.

The possibility that lipid-induced insulin resistance in human muscle is related to alterations in diacylglycerol (DAG)/protein kinase C (PKC) signaling was investigated in normal volunteers during euglycemic-hyperinsulinemic clamping in which plasma free fatty acid (FFA) levels were increased by a lipid/heparin infusion. In keeping with previous reports, rates of insulin-stimulated glucose disappearance (G(Rd)) were normal after 2 h but were reduced by 43% (from 52.7 +/- 8.2 to 30.0 +/- 5.3 micromol. kg(-1). min(-1), P < 0.05) after 6 h of lipid infusion. No changes in PKC activity or DAG mass were seen in muscle biopsy samples after 2 h of lipid infusion; however, at approximately 6 h, PKC activity and DAG mass were increased approximately fourfold, as were the abundance of membrane-associated PKC-betaII and -delta. A threefold increase in membrane-associated PKC-betaII was also observed at approximately 2 h but was not statistically significant (P = 0.058). Ceramide mass was not changed at either time point. To evaluate whether the fatty acid-induced insulin activation of PKC was associated with a change in the IkB kinase (IKK)/nuclear factor (NF)-kappaB pathway, we determined the abundance in muscle of IkappaB-alpha, an inhibitor of NF-kappaB that is degraded after its phosphorylation by IKK. In parallel with the changes in DAG/PKC, no change in IkappaB-alpha mass was observed after 2 h of lipid infusion, but at approximately 6 h, IkappaB-alpha was diminished by 70%. In summary, the results indicated that the insulin resistance observed in human muscle when plasma FFA levels were elevated during euglycemic-hyperinsulinemic clamping was associated with increases in DAG mass and membrane-associated PKC-betaII and -delta and a decrease in IkappaB-alpha. Whether acute FFA-induced insulin resistance in human skeletal muscle is caused by the activation of these specific PKC isoforms and the IKK-beta/IkappaB/NFkappaB pathway remains to be established.

Glucose autoregulates its uptake in skeletal muscle: involvement of AMP-activated protein kinase.

Preexposure to a low concentration of glucose upregulates glucose transport into skeletal muscle, whereas exposure to a high concentration of glucose has the opposite effect. This autoregulatory process occurs independently of insulin, and the mechanism by which it operates is incompletely understood. Activation of the energy-sensing enzyme AMP-activated protein kinase (AMPK) has been shown to increase insulin-independent glucose transport into skeletal muscle in response to such stimuli as exercise and hypoxia. In the present study, we examined whether AMPK could also mediate glucose autoregulation. The activity of the alpha2 isoform of AMPK and 2-deoxyglucose uptake were assessed in incubated rat extensor digitorum longus muscle after preincubation for 4 h in media containing 0, 3, 6, or 25 mmol/l glucose. The principal findings were as follows. First, AMPK activity was highest in muscles incubated with no added glucose, and it decreased as the concentration of glucose was increased. In keeping with these findings, the concentration of malonyl CoA was increased, and acetyl CoA carboxylase phosphorylation at serine 79 was decreased as the medium glucose concentration was raised. Second, decreases in AMPK activity at the higher glucose concentrations correlated closely with decreases in glucose transport (2-deoxyglucose uptake), measured during a subsequent 20-min incubation at 6 mmol/l glucose (r(2) = 0.93, P < 0.001). Third, the decrease in AMPK activity at the higher glucose concentrations was not associated with changes in whole-tissue concentrations of creatine phosphate or adenine nucleotides; however, it did correlate with increases in the rate of glycolysis, as estimated by lactate release. The results suggest that glucose autoregulates its own transport into skeletal muscle by a mechanism involving AMPK. They also suggest that this autoregulatory mechanism is not paralleled by changes in whole-tissue concentrations of creatine phosphate ATP, or AMP, but they leave open the possibility that alterations in a cytosolic pool of these compounds play a regulatory role.

Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation.

gACRP30, the globular subunit of adipocyte complement-related protein of 30 kDa (ACRP30), improves insulin sensitivity and increases fatty acid oxidation. The mechanism by which gACRP30 exerts these effects is unknown. Here, we examined if gACRP30 activates AMP-activated protein kinase (AMPK), an enzyme that has been shown to increase muscle fatty acid oxidation and insulin sensitivity. Incubation of rat extensor digitorum longus (EDL), a predominantly fast twitch muscle, with gACRP30 (2.5 micro g/ml) for 30 min led to 2-fold increases in AMPK activity and phosphorylation of both AMPK on Thr-172 and acetyl CoA carboxylase (ACC) on Ser-79. Accordingly, concentration of malonyl CoA was diminished by 30%. In addition, gACRP30 caused a 1.5-fold increase in 2-deoxyglucose uptake. Similar changes in malonyl CoA and ACC were observed in soleus muscle incubated with gACRP30 (2.5 micro g/ml), although no significant changes in AMPK activity or 2-deoxyglucose uptake were detected. When EDL was incubated with full-length hexameric ACRP30 (10 micro g/ml), AMPK activity and ACC phosphorylation were not altered. Administration of gACRP30 (75 micro g) to C57 BL6J mice in vivo led to increased AMPK activity and ACC phosphorylation and decreased malonyl CoA concentration in gastrocnemius muscle within 15-30 min. Both in vivo and in vitro, activation of AMPK was the first effect of gACRP30 and was transient, whereas alterations in malonyl CoA and ACC occurred later and were more sustained. Thus, gACRP30 most likely exerts its actions on muscle fatty acid oxidation by inactivating ACC via activation of AMPK and perhaps other signal transduction proteins.