Insulin-like growth factor-I (IGF-I) is a major anabolic regulator. Our studies indicate that use of fuels for growth reflects modulation via IGF-I, and that poor growth in diabetes mellitus may be attributed to reduced hepatic production of IGF-I. However, there is limited understanding of underlying mechanisms. Recent findings by the P.I.: (a) Reduced IGF-I expression in diabetic animals is due to a fall in IGF-I gene transcription. (b) Downstream cis-regulatory regions in exon 1 are necessary for the fall in IGF-I gene transcription. (c) Within these regions, diabetes reduces DNA-protein interactions in DNase I footprint sites III and V. (d) Deletion of site V largely normalized IGF-I gene transcription with nuclear extracts from diabetic animals. (e) A role for the accessory factor Sp1 is shown by antibody-induced supershifts with site V oligonucleotides, and decreased IGF-I transcription. (f) A critical role for another factor -- HpTF, a putative homeodomain protein is shown by persistence of diabetes-sensitive gel shifts despite mutation of Sp1 sites, and loss of diabetes responsive in vitro transcription with templates mutated in an AT-rich region of site V.
Specific Aims are: (1) To delineate the involvement of Sp1, the investigators will use gel shifts, methylation interference analysis, and in vitro transcription to assess both DNA-binding and transcription-modulating interactions. (2) To clone HpTF, the factor binding to the AT-rich region, a rat liver GAL4 activation domain cDNA library will be screened using the yeast one-hybrid system; RACE-PCR will be used to obtain a full-length clone. We will also attempt to clone the factor binding to core sequences in region III, and to study it along with HpTF. (3) To obtain functional HpTF, the cDNA will be expressed as a His-tagged, highly soluble thioredoxin fusion protein--permitting affinity isolation; antibodies will be developed with protein fractions and/or oligopeptides. (4) To characterize the physiologic regulation of HpTF, the investigators will use models of insulin deficiency, both in animals and in hepatocyte primary culture: HpTF mRNA will be measured by RNase protection or primer extension, and protein by Western blotting. (5) To test essential mechanisms, in vitro transcription will be used to determine if HpTF is sufficient to normalize IGF-I transcriptional activity in diabetic nuclear extracts, and whether availability of HpTF is necessary to increase transcriptional activity in extracts from normal compared to diabetic animals. The contribution of Sp1 will also be evaluated in vitro, while antisense approaches will be used to explore the impact of HpTF in vivo, in cultured hepatocytes. These studies should aid understanding of the metabolic regulation of IGF-1, and may lead to new anabolic therapy for patients with diabetes mellitus.
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