Reduced insulin-like growth factor (IGF, or somatomedin) activity in diabetes may contribute to decreased growth, poor wound healing, and impaired anabolism. While blood IGF levels may be low, circulating levels of IGF inhibitors are high and may further reduce net IGF activity in diabetes. Because inhibitors are similar in size (molecular weight 20- 40kDa) to recently characterized IGF binding proteins (BPs), we asked whether changed in BPs may contribute to high circulating inhibitory activity in diabetes. Analysis by Sephacryl S-200 chromatography and affinity labeling techniques confirmed a marked increase sue to changes in BPs of appropriate molecular weight (20 to 50kDA) in streptozotocin- diabetic animals. We now have applied ligand blot, immunoblot, immunoprecipitation, and northern blot techniques to identify a specific 32kDa BP that is responsible for these changes in diabetes, and is distinct from the previously characterized low molecular weight IGF BP present in fetal rat serum (BP-3A). Since 32kDa BPs are normalized by insulin therapy, regulation of these BPS may represent one mechanism by which insulin, and other hormones, may modulate growth processes and anabolism in diabetes and other disease states. To further investigate this possibility, specific tools will now be developed to examine mechanisms of 32kDa BP regulation in vivo and in vitro. Our recent observation that the hormone-responsive, well differentiated H4IIE rat hepatoma cell line secretes this 32kDa protein in roller bottle culture will greatly facilitate BP purification and provide a model for initial in vitro studies of regulation. Purified BP will be used to prepare specific antiserum and to obtain amino acid sequence data for subsequent development of cDNA probes to investigate specific mechanisms of BP regulation in intact animals, and isolated cells. Since the liver appears to be an important site of IGF BP and inhibitor production, late hepatic 32kDA BP production, attention will be focused on identifying hormonal and/or metabolic factors that modulate hepatic 32kDa BP production, providing the basis for subsequent investigations regarding specific mechanisms of BP regulation. Since IGFs are growth regulators for many cell types, studies now proposed should provide better understanding of the regulation of cell growth and other anabolic processes in diabetes, and other metabolic diseases.
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