Growth hormone modulates insulin regulation of hepatic insulin-like growth factor binding protein-1 transcription

Hepatic transcription of insulin-like growth factor-binding protein-1 (IGFBP-1) is enhanced in hypophysectomized (hypox) rats and can be rapidly down-regulated by GH administration. Here we examined the effect of insulin on IGFBP-1 messenger RNA abundance in hypox rats and the effects of insulin and GH on IGFBP-1/chloramphenicol acetyltransferase (CAT) reporter plasmids transiently transfected into isolated hepatocytes from pituitary-intact and hypox rats. Unlike GH, administration of insulin to hypox rats in doses of 10 or 50 micrograms/100 g BW had no effect on hepatic IGFBP-1 messenger RNA abundance. Insulin at 10(-7) M resulted in a 42.1 +/- 9.8% suppression of CAT activity in hepatocytes from pituitary-intact animals transfected with a CAT reporter plasmid containing 1671 bp of the 5'-flanking region of the rat IGFBP-1 gene. In the same assay, GH at a concentration of 2.3 x 10(-8) M significantly reduced CAT activity. In contrast, insulin had no effect on CAT activity in hepatocytes from hypox rats, whereas GH resulted in comparable suppression of CAT activity in hepatocytes from hypox rats and pituitary-intact rats, 13.6 +/- 2.3% vs. 18.2 +/- 3.2%. Deletional analysis and mobility shift assays were used to identify the GH-responsive regions in the IGFBP-1 gene. GH suppression of CAT activity was lost when the IGFBP-1 5'-flanking region was deleted down to -277 bp, whereas insulin suppression was retained for all but the smallest fragment of the IGFBP-1 gene. Mobility shift assays were used to compare nuclear extracts from sham-operated, hypox, and GH-treated hypox rats. When hepatic nuclear extracts from hypox rats were incubated with the -277 to -82 and the -556 to -368 bp fragments, retarded bands were apparent that were not present in the extracts from sham-operated rats. GH treatment of hypox rats 15 or 30 min before death completely normalized the retardation pattern seen with the -277 to -82 bp fragment, but did not affect the pattern seen with the -556 to -368 bp fragment. A 20-bp fragment corresponding to the previously identified insulin response element, -108 to -89 bp, was also analyzed. An additional retarded band, not seen with nuclear extracts from sham-operated rats, was apparent when nuclear extracts of hypox rats or GH-treated hypox rats were used. These data provide the first in vitro evidence that GH directly regulates transcription of IGFBP-1 expression. In addition, our findings suggest that GH modulates insulin regulation of IGFBP-1 transcription, possibly by altering the milieu of trans-acting factors that interact with both the insulin response element and distinct upstream sites.