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 insulin-mimetic 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 (zeta/lambda) (PKC (zeta/lambda)) was reversed by wortmannin. Inhibition of PKC (zeta/lambda) 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 (zeta/lambda) as well as other yet to be defined effector(s) contribute to the modulation of GS in rat hepatocytes.
Highlights
We examined the signaling pathways regulating glycogen synthase (GS) in primary cultures of rat hepatocytes
Whereas PD98059, an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase, the upstream activator of p44/42 MAP kinases, did not inhibit insulin’s stimulatory effect, wortmannin completely inhibited GS activation induced by insulin. bpV(phen), at a dose producing insulin receptor kinase (IRK) activation comparable with that observed with 100 nM insulin [22], stimulated GS to a significantly greater degree (250% of basal level (p Ͻ 0.001 compared with insulin)
We found that rapamycin had only a modest (17%), and statistically not significant, inhibitory effect on insulin-induced GS activation (Fig. 1B)
Summary
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. The IRSs recruit, on defined phosphotyrosine motifs, key molecules involved in signal transduction (reviewed in Ref. 12) In this fashion, PI 3-kinase associates with IRS-1/2 and is activated, resulting in the activation of downstream signaling molecules including PKB/Akt [13,14,15], p70s6k [16], and atypical PKC isoforms such as PKC (/)2 [17,18,19]. We sought to identify, in primary rat hepatocyte cultures, the molecular components and signal transduction pathways involved in the regulation of GS by both insulin and bpV(phen). Mitogen-activated protein; mTOR, mammalian target of rapamycin; DMEM, Dulbecco’s modified Eagle’s medium; Tricine, N-[2-hydroxy1,1-bis(hydroxymethyl)ethyl]glycine; MOPS, 4-morpholinepropanesulfonic acid; ANOVA, analysis of variance; PTEN, phosphatase and tensin homolog deleted on chromosome 10
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