Although poor growth in diabetes and malnutrition is attributed to decreased nutrient utilization, the mechanism of the defect is not known. Our studies indicate that use of fuels for growth reflects modulation through the balance of circulating insulin-like growth factor-1 (IGF-1) and IGF binding protein-1 (BP-1). In diabetes, BP-1 rises and IGF-1 falls, apparently regulated by the liver according to its nutritional and hormonal status. However, despite the apparent critical role of IGF-1 and BP-1 in diabetes and malnutrition, there is limited understanding of molecular mechanisms which lead to abnormal production of IGF-1 and BP-1. Recent critical findings by the P.I.: (1) When metabolic status is altered, circulating levels and hepatic release of IGF-1 and BP-1 closely reflect underlying changes in hepatic messenger RNA; (2) Rat hepatocytes in primary culture exhibit regulation of IGF-1 and BP-1 mRNA responsive to physiologic concentrations of insulin and amino acids; (3) Both IGF-1 and BP-1 mRNA appear to be regulated at the level of gene transcription; (4) In vivo and in vitro expression of IGF-1 promoter regions is responsive to metabolic status, indicating regulation by altered transcription factor mass or activity; (5) Footprinting studies of the IGF gene suggest the presence of putative metabolic regulatory elements. 1. To delineate the critical role of transcription, we will quantify both transcription rate and mRNA stability in normal, diabetic, and lysine-deficient animals, and in hepatocytes cultured with or without insulin or lysine -- conditions which lead to reproducible changes in mRNA. 2. To characterize regulatory regions IGF-1 and BP-1 genes will be studied via DNase I hypersensitivity analysis to identify areas likely to be involved in transcriptional control, the activity of probable promoter and enhancer regions will be assessed via in vitro transcription and transient transfection, and target sites for putative metabolically-regulated DNA- binding protein will be assessed by gel mobility shift assays and footprinting analysis in vitro and in vivo. 3. To isolate metabolically-regulated transcription factors, expression libraries will be screened via """"""""southwestern blotting"""""""" with catenated probes reflecting critical DNA-binding sites, and the identified cDNAs cloned and sequenced; conventional and DNA affinity chromatography can also be utilized if needed. 4. To develop antibodies to transcription- regulating factors, we will attempt to raise antisera to synthetic oligopeptides reflecting cDNA-predicted hydrophilic sequences, using bacterial expression and immunization with full-length molecules as an alternative if necessary. 5. To test the role of transcription factors in regulation of IGF-1, transcription factor mass and activity will be compared to IGF-1 mRNA in animal and hepatocyte models of altered insulin and nutrition status -- measuring transcription factor mass by Western blotting, and activity via gel mobility shift and in vitro transcription assays, using templates containing metabolism-sensitive elements. These studies should provide improved understanding of the regulation of normal growth, insight into the pathophysiology of impaired growth, and new concepts for therapy of growth failure in diabetes and malnutrition.
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