Diabetes-induced alterations in the expression, functioning and phosphorylation state of the inhibitory guanine nucleotide regulatory protein Gi-2 in hepatocytes

Abstract

Levels of the G-protein alpha-subunits alpha-Gi-2, alpha-Gi-3 and the 42 kDa, form of alpha-Gs were markedly decreased in hepatocyte membranes from streptozotocin-diabetic animals as compared with normals. In contrast, no detectable changes in alpha-Gi subunits were seen in liver plasma membranes of streptozotocin-diabetic animals, although levels of the 45 kDa form of Gs were increased. G-protein beta subunits in plasma membranes were unaffected by diabetes induction. Analysis of whole-liver RNA indicated that the induction of diabetes had little effect on transcript levels of Gi-3, caused an increase in Gs transcripts and decreased transcript number for Gi-2, albeit to a much lesser extent than was observed upon analysis of hepatocyte RNA. In both hepatocyte and liver plasma membranes, immunoblot analysis showed that levels of the catalytic unit of adenylate cyclase were increased upon induction of diabetes. Under basal conditions, alpha-Gi-2 from hepatocytes of diabetic animals was found to be both phosphorylated to a greater extent than alpha-Gi-2 isolated from hepatocytes of normal animals, and furthermore was resistant to any further phosphorylation upon challenge of hepatocytes with angiotensin, vasopressin or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. Treatment of isolated plasma membranes from normal, but not diabetic, animals with purified protein kinase C caused the phosphorylation of alpha-Gi-2. Treatment of membranes from diabetic animals with alkaline phosphatase caused the dephosphorylation of alpha-Gi-2 and rendered it susceptible to subsequent phosphorylation with protein kinase C. Low concentrations of the non-hydrolysable GTP analogue guanylyl 5'-imidodiphosphate inhibited adenylate cyclase activity in both hepatocyte and liver plasma membranes from normal, but not diabetic, animals.

Diabetes induces selective alterations in the expression of protein kinase C isoforms in hepatocytes

“…In hepatocytes of streptozotocin-induced diabetic rats [25,26] there is a loss of Gi function which appears to result from both a reduction in the expression of Gi-2 and also its increased phosphorylation by protein kinase C. Treatment of a variety of different cell types with tumour promoting phorbol esters has been shown to lead to the loss of guanine nucleotide-elicited, G~-mediated inhibition of adenylate cyclase activity [13,37]. Similar observations of crippled G~ function have also been seen in cells challenged with ligands which stimulate phospholipid metabolism or with DAG [13], suggesting that it is the activation of protein kinase C which provides the molecular basis of this phenomenon.…”

“…As well as this, activation of protein kinase C also causes the loss of 'tonic' GTP-mediated inhibition of adenylyl cyclase through Gi [17]. This apparently physiological 'crosstalk' mechanism appears, however, to be inappropriately activated in streptozotocin-induced diabetes [25,26] where we have noted the loss of such 'tonic' GTP-elicited inhibition of adenylyl cyclase [25,26]. This appears to be due in part to the reduced expression of the inhibitory G-protein ~-Gi-2 but also to the increased phosphorylation, through the action of PKC, of ~-Gi-2 itself [18,26].…”

“…It is possible that abnormalities in PKC expression might occur in hepatocytes from streptozotocin-induced diabetes as reflecting the altered functioning of this enzyme activity deduced from analyses done on Gi function and phosphorylation [25,26]. We here describe the use of a panel of sub-type specific antisera to probe alterations in the expression and distribution, between membrane and cytosol compartments, of various protein kinase C isoforms.…”

GTP‐binding protein‐activator sequences in the insulin receptor

“…Another line of evidence comes from the altered function of Gi in diabetes, where abolished effects of insulin on Grlike proteins have been documented [33]. It has also been reported that the Gi function is impaired in liver membranes of animals with experimentally induced diabetes [34][35][36][37]. These suggest that some metabolic effects of insR may be mediated by Grlike proteins.…”

Purification of A1 adenosine receptor–G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling

“…G i α2, but not G i α3 from hepatocytes, was found to be phosphorylated on serine residues by protein kinase C both in i o and in itro [37][38][39]. Elevated phosphorylation of G i α2 was correlated with decreased ability to inhibit adenylate cyclase [40,41]. Tyrosine phosphorylation of G q α [42], G s α [43] and functional consequences have also been reported.…”