The objective is to allow the candidate to develop into an independent biomedical investigator while studying rat adipose tissue lipoprotein lipase. Lipoprotein lipase (LPL) is the enzyme responsible for the rate-limiting step in plasma triglyceride removal. Although LPL participates in the anabolism or catabolism of every major plasma lipoprotein and may play some role in mediating coronary heart disease risk in humans, molecular characterization of the enzyme has been limited. LPL activity is known to be affected by insulin and a number of other hormones, but the molecular events responsible for hormonal regulation of LPL activity are unknow. The current proposal aims to characterize LPL by cloning the LPL cDNA and determining the nucleotide sequence of the cDNA. Hormonal regulation of LPL activity will be studied by determining LPL protein levels, LPL biosynthetic rate, LPL mRNA concentration and half-life, and nuclear transcription initiation rates in response to insulin treatment and other physiological manipulations. Methods will include antibody screening of a Lambdagt11 cDNA library, preparation of monoclonal antibodies, Western blotting for protein analysis, Northern blotting for RNA analysis, Southern blotting for DNA analysis, isolation of nuclei for transcription initiation analysis, dot-blot hybridization, and pulse labeling of LPL protein and mRNA. Experimental systems used include differentiated 3T3-L1 lines, rat adipocytes in culture and intact rats. Information generated in these studies will be important to our understanding of the hormonal regulation of LPL synthesis and activity, and the potential role of LPL in lipid derangements in diabetes mellitus and in the development of atherosclerosis.