CORE--ANIMAL PHYSIOLOGY

The Protein Chemistry/Animal Physiology Core will provide highly cost-effective services to the program project in the areas of protein chemistry, analytical biochemistry, and animal physiology. The Protein Chemistry component of this core will provide measurements of the amount of flux through the hexosamine pathway in tissues, and determinations of plasma levels of key proteins whose expression is likely to be regulated by the hexosamine pathway. In particular, the core will perform HPLC measurements of intracellular levels of the major hexosamine pathway products, which will indicate the degree of activation of this pathway in fat, muscle and endothelial tissue under various metabolic conditions (projects by Rossetti, Barzilai, Hawkins, Brownlee). As an additional, direct measurement of the degree of activation of this pathway, the core will use a highly specific technique ofimmtmoprecipitation and Western blotting to quantify the amount of glycosylation of a key intracellular protein, sp-1. This transcription factor, whose activation is known to be affected by its degree of glycosylation, regulates the transcription of many proteins associated with the metabolic syndrome, including many cytokines and acute phase reactants. Since this interaction is of central importance to all projects, the Core will measure plasma levels ofplasminogen activator inhibitor-1 (PAI- 1), angiotensinogen, TNF-alpha, Interleukins 1-beta and 6, Acrp30, resistin, serum amyloid A and leptin (projects by Rossetti, Barzilai, Hawkins, Shamoon), and PAI-1 levels in aortic extracts (project by Brownlee), by a variety of techniques including radioimmunoassays, ELISA assays, enzyme immunoassays and Western blotting. The Animal Physiology component of this core will provide a superlative service for animal preparation in performing indwelling arterial and venous catheterizations and surgical resections of visceral fat and aortas (projects by Rossetti, Barzilai, Brownlee).