Multiple defects occur in the guanine nucleotide regulatory protein system in liver plasma membranes of obese (fa/fa) but not lean (Fa/Fa) zucker rats: Loss of functional G 1 and abnormal G s function

• Published in: Cellular signalling Vol. 1; no. 1; pp. 9 - 22
• Main Authors: Houslay, Miles D., Gawler, Debra J., Milligan, Graeme, Wilson, Andrew
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 1989
• Subjects: adenylate cyclase; ADP-ribosylation; cholera and pertussis toxins; G i; G s; glucagon; guanine nucleotide regulatory proteins; Obesity; Obesity; G i; SDS-PAGE; guanine nucleotide regulatory proteins; cholera and pertussis toxins; TPA; adenylate cyclase; p[NH]ppG; glucagon; G s; ADP-ribosylation
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/0898-6568(89)90016-8

Hepatocyte membranes from both lean and obese Zucker rats exhibited adenylate cyclase activity that could be stimulated by glucagon, forskolin, NaF and elevated concentrations of p[NH]ppG. In membranes from lean animals, functional G i was detected by the ability of low concentrations of p[NH]ppG to inhibit forskolin-activated adenylate cyclase. This activity was abolished by treatment of hepatocytes with either pertussis toxin or the phorbol ester TPA, prior to making membranes for assay of adenylate cyclase activity. In hepatocyte membranes from obese animals no functional G i activity was detected. Quantitative immunoblotting, using an antibody able to detect the alpha subunit of G i, showed that hepatocyte plasma membranes from both lean and obese Zucker rats had similar amounts of G i-alpha subunit. This was 6.2 pmol/mg plasma membrane for lean and 6.5 pmol/mg plasma membrane for obese animals. Using thiol pre-activated pertussis toxin and [ 32P]-NAD +, similar degrees of labelling of the 40 kDa alpha subunit of G i were found using plasma membranes of both lean and obese Zucker rats. We suggest that liver plasma membranes from obese Zucker rats express an inactive G i alpha subunit. Thus lesions in liver G i functioning are seen in insulin-resistant obese rats and in alloxan- and streptozotocin-induced diabetic rats [9] which also show resistance as regards the acute actions of insulin [13]. Liver plasma membranes of obese animals also showed an impairment in the coupling of glucagon receptors to G s-controlled adenylate cyclase, with the K a values for activation by glucagon being 17.3 and 126 nM for lean and obese animals respectively. Membranes from obese animals also showed a reduced ability for high concentration of p[NH]ppG to activate adenylate cyclase. The use of [ 32P]-NAD + and thiol-preactivated cholera toxin to label the 43 kDa and 52 kDa forms of the alpha-subunit of G s showed that a reduced labelling occurred using liver plasma membranes from obese animals. It is suggested that abnormalities in the levels of expression of primarily the 52 kDa form of alpha-G s may give rise to the abnormal coupling between glucagon receptors and adenylate cyclase in liver membranes from obese (fa/fa) Zucker rats.