TRANSMEMBRANE SIGNALLING IN GENETIC OBESITY

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The objective of this study is to understand the mechanisms of body weight regulation. Adenosine receptors or the GTP binding proteins, which act as transducers for these receptors, may be important in normal regulation of lipid storage and may be altered in some forms of obesity. The genetically obese (fa/fa) Zucker rat, which appears to exhibit abnormalities in adenosine receptor signalling and Gi function, will be used as an animal model. Gi is one of a closely related family of heterotrimeric GTP binding proteins which function as intermediates in transmembrane signalling. Genetically obese animals and animals rendered obese by a variety of brain lesions exhibit a common array of symptoms. These include insulin resistance, varying degrees of hyperinsulinemia, impaired non-shivering thermogenesis, and slow norepinephrine turnover. Many of these symptoms might be ascribed to excessive agonist binding to the A1 adenosine receptor in muscle, brown fat, white fat and sympathetic nerve endings. The insulin resistance, typical of skeletal muscle strips from obese Zucker rats, can be reversed with an A1 adenosine receptor antagonist and by prior treatment with pertussis toxin. Recent studies in this laboratory show that impaired hormone stimulated lipolysis, typical of white adipocytes of Zucker rats can be reversed with an adenosine receptor antagonist. Isolated plasma membranes from obese animals exhibit enhanced GTP dependent and adenosine receptor agonist dependent inhibition of adenylate cyclase, compared to lean animals. However, recent studies reveal no differences in amounts of receptor or amounts of adenylate cyclase. Surprisingly low levels of Gi are found in obese membrane compared to lean. The aim of the proposed studies is to determine the structural basis for the difference between lean and obese animals in A1 adenosine receptor activity. Studies are designed to reconstitute receptors from obese animals with normal Gi and visa versa. Structural studies are planned for the isolated receptor and Gi which include assays for changes in phosphorylation, glycosylation, acylation or alterations of amino acid sequence, due to proteolysis or mutation. The cDNA of the receptor will be obtained to determine the original coded amino acid sequence. Adipocytes will be placed in culture to determine the effect of hormonal milieu on receptor function. To determine the effect of A1 adenosine receptor activity levels on whole body metabolism obese and lean Zucker rats will be chronically treated with either agonist or antagonists. Measurements of glucose tolerance, insulin levels, serum glycerol turnover as well as overall weight gain should provide an indication of the importance of the adenosine receptors as regulators of energy storage vs. utilization.