Nutrition and Somatomedin XXIX: Molecular Regulation of IGFBP-1 in Hepatocyte Primary Culture

The insulinlike growth factors (IGFs) circulate in association with insulinlike growth factor binding proteins (IGFBPs) that modulate IGF action, but mechanisms of IGFBP regulation are poorly understood. We investigated the regulation of IGFBPs in primary cultures of rat hepatocytes, measuring the appearance of export proteins by ligand blotting after separation via SDS/PAGE, and evaluating mRNA with cDNA probes. Northern blotting studies revealed that IGFBP-1 was expressed at high levels in cultured hepatocytes, in which sustained release of both insulinlike growth factor I and albumin marks preservation of differentiated status. In contrast, transcripts of IGFBP-3 and IGFBP-2 were not detected. Release of IGFBP-1 was unaffected by exposure to glucose (20–500 mg/dl) or to provision of amino acids (0.25'6.25 times normal rat arterial plasma levels). Hormonal studies revealed little effect of glucagon, inhibition by insulin, stimulation by dexamethasone, and blunting of dexamethasone effects by added insulin. Adding dexamethasone provided progressive stimulation: 5-, 11-, and 26-fold at 10⁻⁹, 10⁻⁸, and 10⁻⁷ M, all P < 0.01; increases in IGFBP-1 protein (ligand blot) and IGFBP-1 mRNA (Northern blot) were highly correlated (r = 0.62, P < 0.001). In contrast, adding insulin resulted in progressive suppression of both IGFBP-1 protein and IGFBP-1 mRNA, 43% at 10⁻¹⁰ M, 74% at 10⁻⁹ M, and 83% (maximal) at 10⁻⁸ M; ED₅₀ of ∼ 10⁻¹⁰ M is within the physiological range of insulin concentrations. Directly adding growth hormone and insulinlike growth factor I had little effect, whereas adding insulinlike growth factor I attenuated the effect of insulin to decrease IGFBP-1 release. Exposure to insulin at 10⁻⁶ M did not change IGFBP-1 gene expression at 30 min, but suppressed IGFBP-1 mRNA 37% at 90 min and 97% at 180 min (t_1/2 ∼110 min). IGFBP-1 release by normal rat hepatocytes is stimulated by dexamethasone and inhibited by insulin, apparently modulated at pretranslational levels. This system should be useful for further studies of biological regulation and underlying molecular mechanisms.