Penny Y T Wang | Search PubMed

PubMed2006

Oleate-induced decrease in hepatocyte insulin binding is mediated by PKC-delta.

We have previously shown that free fatty acids (FFA) impair hepatic insulin extraction in vivo and thus generate hyperinsulinemia, a suspected risk factor for atherosclerosis and cancer. Hepatic insulin extraction is a receptor-mediated event, which is initiated by hepatocyte insulin binding. In the present study, we investigated the effect of FFA on insulin binding in freshly isolated rat hepatocytes maintained at 10 mM glucose. Hepatocyte insulin binding decreased after 1 h exposure to oleate in a concentration-dependent manner reaching a maximum (35-40%) at 125 microM. Inhibition of FFA oxidation by >90% with the carnitine palmitoyltransferase I (CPT-I) inhibitor methylpalmoxirate (MP, 30 microM) did not prevent the effect of oleate. However, when hepatocytes were treated with the PKC inhibitor bisindolylmaleimide (BIM, 1 microM) the effect of oleate was abolished. Subcellular fractionation and immunoblotting of specific PKC isoforms revealed that oleate-induced hepatic PKC-delta membrane translocation, but did not translocate-epsilon, -theta, -alpha, -betaI and -betaII. These results indicate that PKC-delta activation mediated the FFA-induced decrease in hepatocyte insulin binding under our conditions, and thus provides a mechanistic basis for FFA-induced hyperinsulinemia.

Animals↗

PubMed2006

Modulation of hepatocellular swelling-activated K+ currents by phosphoinositide pathway-dependent protein kinase C.

K(+) channels participate in the regulatory volume decrease (RVD) accompanying hepatocellular nutrient uptake and bile formation. We recently identified KCNQ1 as a molecular candidate for a significant fraction of the hepatocellular swelling-activated K(+) current (I(KVol)). We have shown that the KCNQ1 inhibitor chromanol 293B significantly inhibited RVD-associated K(+) flux in isolated perfused rat liver and used patch-clamp techniques to define the signaling pathway linking swelling to I(KVol) activation. Patch-electrode dialysis of hepatocytes with solutions that maintain or increase phosphatidylinositol 4,5-bisphosphate (PIP(2)) increased I(KVol), whereas conditions that decrease cellular PIP(2) decreased I(KVol). GTP and AlF(4)(-) stimulated I(KVol) development, suggesting a role for G proteins and phospholipase C (PLC). Supporting this, the PLC blocker U-73122 decreased I(KVol) and inhibited the stimulatory response to PIP(2) or GTP. Protein kinase C (PKC) is involved, because K(+) current was enhanced by 1-oleoyl-2-acetyl-sn-glycerol and inhibited after chronic PKC stimulation with phorbol 12-myristate 13-acetate (PMA) or the PKC inhibitor GF 109203X. Both I(KVol) and the accompanying membrane capacitance increase were blocked by cytochalasin D or GF 109203X. Acute PMA did not eliminate the cytochalasin D inhibition, suggesting that PKC-mediated I(KVol) activation involves the cytoskeleton. Under isotonic conditions, a slowly developing K(+) current similar to I(KVol) was activated by PIP(2), lipid phosphatase inhibitors to counter PIP(2) depletion, a PLC-coupled alpha(1)-adrenoceptor agonist, or PKC activators and was depressed by PKC inhibition, suggesting that hypotonicity is one of a set of stimuli that can activate I(KVol) through a PIP(2)/PKC-dependent pathway. The results indicate that PIP(2) indirectly activates hepatocellular KCNQ1-like channels via cytoskeletal rearrangement involving PKC activation.

Animals↗