Regulation of Glycogen Synthase in Rat Hepatocytes

Abstract

We examined the signaling pathways regulating glycogen synthase (GS) in primary cultures of rat hepatocytes. The activation of GS by insulin and glucose was completely reversed by the phosphatidylinositol 3-kinase inhibitor wortmannin. Wortmannin also inhibited insulin-induced phosphorylation and activation of protein kinase B/Akt (PKB/Akt) as well as insulin-induced inactivation of GS kinase-3 (GSK-3), consistent with a role for the phosphatidylinositol 3-kinase/PKB-Akt/ GSK-3 axis in insulin-induced GS activation. Although wortmannin completely inhibited the significantly greater level of GS activation produced by the insulinmimetic bisperoxovanadium 1,10-phenanthroline (bpV-(phen)), there was only minimal accompanying inhibition of bpV(phen)-induced phosphorylation and activation of PKB/Akt, and inactivation of GSK-3. Thus, PKB/Akt activation and GSK-3 inactivation may be necessary but are not sufficient to induce GS activation in rat hepatocytes. Rapamycin partially inhibited the GS activation induced by bpV(phen) but not that effected by insulin. Both insulin-and bpV(phen)-induced activation of the atypical protein kinase C (/) (PKC (/)) was reversed by wortmannin. Inhibition of PKC (/) with a pseudosubstrate peptide had no effect on GS activation by insulin, but substantially reversed GS activation by bpV(phen). The combination of this inhibitor with rapamycin produced an additive inhibitory effect on bpV-(phen)-mediated GS activation. Taken together, our results indicate that the signaling components mammalian target of rapamycin and PKC (/) as well as other yet to be defined effector(s) contribute to the modulation of GS in rat hepatocytes.Much current work has focused on defining the nature of the downstream effectors mediating key effects of insulin. The regulation of glycogen synthase (GS), 1 the rate-limiting enzyme in glycogen synthesis, by insulin has received increasing attention. GS is regulated by a complex interplay of diurnal, nutritional, and hormonal factors (reviewed in Refs. 1-3) that ultimately modulate the phosphorylation state and hence the activity of the enzyme. Whereas there have been numerous studies of GS activation in skeletal muscle, the modulators of GS in liver have been less completely evaluated. Studies to date indicate that glucose and insulin are the major physiologic effectors of hepatic GS activation (1, 2). It appears that glucose and/or its metabolite glucose 6-phosphate activate hepatic GS by physically associating with critical regulatory enzymes upstream of GS or with GS itself (2, 4, 5), although other mechanisms have been suggested (6). The mechanisms by which insulin effects activation of GS and stimulation of glycogen synthesis in liver are poorly defined, although data have been obtained indicating that modulation of PP1-G (1, 7) as well as PKB/Akt (8) and GSK-3 (9) may be involved. Insulin activation of the insulin receptor kinase (IRK) is followed by tyrosine phosphorylation of insulin receptor substrates (IRSs), the two major ones in liver being IRS-1 and -2 ...

Cross-talk between Protein Kinases Cζ and B in Cyclic AMP-mediated Sodium Taurocholate Co-transporting Polypeptide Translocation in Hepatocytes

“…4). PKC PS (80 M) has been shown to inhibit insulin-stimulated PKC activity in hepatocytes (17). PKC PS (100 M, but not 10 M) also inhibited cAMP-induced increases in TC uptake (Fig.…”

“…The activation of PKC, like that of PKB, requires phosphoinositides (12) and phosphorylation by PDK1 (13,14). Recent studies indicate that insulin also stimulates PI3K-dependent PKC in various cell types (15,16), including hepatocytes (17), and the PI3K/PKC signaling pathway is involved in insulin-stimulated GLUT4 translocation (16,18) and NHE1 activity in human erythrocytes (19). In hepatocytes, PKC has been reported to have an antiapoptotic effect (20) and to mediate peroxovanadium-induced activation of glycogen synthase (17).…”

Insulin Receptor Substrate-2 Phosphorylation Is Necessary for Protein Kinase Cζ Activation by Insulin in L6hIR Cells

“…Insulin-dependent inactivation of GSK3 has been known to be dependent on Akt/ PKB (also known as related to the A and C kinase kinase) (18,19). Akt/PKB in turn was shown to be phosphorylated in response to insulin at Thr-308 and Ser-473 (20 -22), and these phosphorylation events can be blocked by inhibitors of PI 3-ki-nase (10,13,23,24). PDK-1, a phosphatidylinositol 3,4,5-trisphosphate-dependent Akt/PKB kinase, was proved to phosphorylate Akt/PKB at Thr-308, which leads to a substantial but incomplete activation of Akt (21,25).…”

“…In addition, in liver PKC is not involved in insulin signaling to glycogen synthase (10). Also, the expression of the KRLB peptide in mouse liver cells inhibits insulin activation of Akt/PKB despite the unchanged induction occurring in the L6hIR muscle cells.…”

The role of protein kinase B/Akt in insulin-induced inactivation of phosphorylase in rat hepatocytes

“…The stimulation of glycogen synthesis [3,4], like insulin action in other cell types, is thought to occur downstream from the activation of the lipid kinase phosphoinositide-3 (PI-3)-kinase [5,6]. The lipid products of PI-3-kinase activate a member of the AGC class of protein kinases termed phosphoinositide-dependent protein kinase 1 (PDK-1), which itself can phosphorylate and activate other protein kinases of the AGC family.…”