Our preliminary studies have demonstrated a marked decrease in pertussis toxin-sensitive G-proteins in adipocytes from obese mice. Thus, a major objective is to see whether this phenomenon is a general feature of diabetes. This study will examine the hypothesis that expression and/or function of pertussis toxin (PTX) sensitive G-proteins in adipocytes form animal models of Type I and Type II diabetes are abnormal. Expression and function of PTX sensitive G-proteins will be assessed on cells from the two types of animals using the following strategy: a) Adipocyte membranes will be solubilized and the extract subjected to in vitro ADP-ribosylation with pertussis toxin and [32P]-NAD, followed by SDS-PAGE and autoradiography. The autoradiograms will be subjected to scanning laser densitometry to quantitate the resolved 40,000 and 41,000 kDa G-proteins in each phenotype. b) The relative amount of G-protein that is coupled to adenylate cyclase inhibition and an assessment of communication between the components of this pathway will be examined by comparing the ability of the GTP analogue, Gpp(NH)p, to inhibit forskolin-stimulated adenylate cyclase in membranes from each phenotype. c) Functioning of the inhibitory G-protein circuit from hormone receptor to effector system will be assessed in membranes from each phenotype by comparing the ability of phenylisopropyladenosine (PIA) to inhibit isoproterenol-stimulated adenylate cyclase, and in cells by comparing the ability of insulin to activate the particulate low Km phosphodiesterase (PDE). The significance of using PIA inhibition of cyclase and insulin activation of PDE is that the pathways are independent and both involve G-proteins which are pertussis toxin substrates. The present approach is unique in that functional assessments will test for changes in receptor-G protein interactions and G-protein-adenylate cyclase interactions. Overall, this study will assess whether changes in PTX- sensitive G-protein expression or function are involved in altered hormonal signalling in models of Type I and Type II diabetes. The long term goal of this work is to clarify the interrelationships among several events involved in the insulin signal transduction sequence in adipocytes and improve understanding of the pathophysiology of diabetes.
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