Obesity appears to be an independent risk factor for cardiovascular disease and to be interrelated with hypercholesterolemia. While much progress has been made in elucidating the pathways regulating cholesterol uptake and storage in most cells, adipocytes have not been well investigated. Adipocytes have many unusual features of cholesterol homeostasis that make them unique cells to study. For instance, most of the cholesterol in adipocytes is found as non-membrane associated free cholesterol. In addition, adipocyte stores of cellular cholesterol are modulated not only by circulating lipoproteins but also by the nutritional and metabolic status of the organism. The cellular steps controlling cholesterol homeostasis in adipocytes are poorly understood. The overall goal of this proposal is to define the underlying mechanisms regulating low density lipoprotein (LDL) receptor expression in adipocytes. The first objective is to demonstrate that LDL receptors have a different subcellular localization in adipocytes than other cells; specifically, adipocytes contain a large intracellular pool of LDL receptors. This will be examined by studying primary rat adipocytes, the Ob1771 and 3T3-L1 adipocyte cell lines, and primary rat hepatocytes for comparison. LDL receptor content in subcellular fractions isolated by differential centrifugation will be quantitated using immunoblotting with specific anti-LDL receptor antibodies, LDL receptors in subcellular fractions will be quantitated using binding assays, and LDL receptor subcellular distribution will be analyzed immunohistochemically. Furthermore, in order to determine whether the existence of this intracellular pool of receptors is a function of the terminal differentiation of the adipocyte, the distribution and expression of LDL receptors in Ob1771 and in 3T3-L1 cells during growth, confluence, and differentiation will be examined. The second objective is to demonstrate that LDL receptor activity in adipocytes is predominantly mediated by alterations in the intracellular pool of receptors, i.e. the intracellular pool is metabolically active. This will be studied by culturing adipocytes under conditions that should up- or down-regulate LDL receptor expression and then by measuring changes in LDL receptor synthesis, degradation, mRNA levels, subcellular distribution, and overall expression. Furthermore, changes in the synthesis, degradation, mRNA levels, subcellular distribution, and overall expression of LDL receptors will be determined in adipocytes isolated from rats which have been fed or fasted or which have been made hypo- or hypercholesterolemic. The results of this proposal will increase the understanding of cholesterol metabolism in adipocytes and will contribute to the overall understanding of the cell biology of lipoprotein transport by exploring novel mechanisms for the regulation of LDL receptor expression.
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