Nutrition and Somatomedin XXIX Molecular Regulation of IGFBP-1 in Hepatocyte Primary Culture B.C. VILLAFUERTE, S. GOLDSTEIN, D.G. ROBERTSON, C.-l. PAO, L J . MURPHY, AND LS. PHILLIPS
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; ED 50 of ~10~ 1 0 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
From the Division of Endocrinology and Metabolism, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and the University of Manitoba, Manitoba, Canada. Address correspondence and reprint requests to Lawrence S. Phillips, MD, Department of Medicine, Emory University School of Medicine, 69 Butler Street, SE, Atlanta, GA 30303. Received for publication 12 December 1991 and accepted in revised form 20 February 1992.
DIABETES, VOL. 41, JULY 1992
expression at 30 min, but suppressed IGFBP-1 mRNA 37% at 90 min and 97% at 180 min (t 1/2 - 1 1 0 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. Diabetes 41:835-42,1992
T
he insulinlike growth factor binding proteins (IGFBPs) have diverse actions, but there is incomplete understanding of their regulation. The IGFBPs prolong the half-life of insulinlike growth factor I (IGF-I); enhance and attenuate IGF-1 mediated mitogenesis; and prevent IGF-I binding to insulin receptors (1,2,3). Six distinct IGFBPs have been identified in animals and human subjects: IGFBP-1 is a 28,000-34,000 Mr nonglycosylated protein that is present at low levels in normal adult serum, and rises under conditions of diabetes or nutritional deprivation; IGFBP-2 is a -30,000 Mr form that is abundant in fetal and neonatal serum, decreases in adult serum, and increases during fasting; IGFBP-3 is a -43,000 Mr glycosylated form that carries most of the circulating IGF-1 and insulinlike growth factor II, and falls during fasting or diabetes; and IGFBP-4 is a -24,000 Mr form that recently has been purified from human cerebrospinal fluid (4,5,6,7,8,9,10). Regulation of IGFBP-5 and 6 is poorly understood. Although consistent changes in circulating binding protein levels have been found in different pathological states, the simultaneous fluctuations of hormones and fuels that occur in fasting or diabetes make it difficult to delineate specific regulation, and study of control in vitro is required for insight into underlying mechanisms. Our laboratory and others have examined the regulation of IGFBP synthesis in the liver, which is a target organ for glucoregulatory hormones. The liver also processes the
835
MOLECULAR REGULATION OF IGFBP-1
bulk of nutritional substrates, and is a major source of circulating IGFs and the IGFBPs. Although IGFBP synthesis has been examined in several liver cell lines, relatively little is known concerning the regulation of IGFBPs in normal liver cells (11,12). We have shown that adult rat hepatocytes maintained in primary culture release large amounts of IGFBP-1 (13). Because the pretranslational regulation of IGFBP-1 in normal liver cells is poorly understood, we used the hepatocyte primary culture system to investigate the molecular regulation of hepatic IGFBP-1 according to the availability of both hormones and nutrients. RESEARCH DESIGN AND METHODS Recombinant IGF-I was purchased from AMGen Biologicals (Thousand Oaks, CA); collagenase type I from Worthington Biomedical (Freehold, NJ); type I rat tail collagen, human transferrin, fetal bovine serum, and Williams-E medium from Sigma (St. Louis, MO); goat antirabbit IgG and normal rabbit serum from Cambridge Medical Diagnostics (Billerica, MA); GeneScreen Plus from NEN Products (Boston, MA); amino acid- and glucose-free Dulbeco's-modified Eagle and Ham's F12 media from JRH Bioscience (Lenexa, KS); 32P-dCTP from DuPont (Wilmington, DE); dexamethasone from LyphoMed (Melrose Park, II); penicillin/streptomycin from Mediatech (Washington, DC); bovine growth hormone from USDA Reproduction Laboratory (Beltsville, MD); and Matrigel from Collaborative Research, (Lexington, MA). Porcine insulin was a generous gift from Eli Lilly (Indianapolis, IN). Hepatocytes were isolated from 150-200 g male Sprague-Dawley rats, using a two-stage collagenase perfusion technique as described previously (14). An estimated 3.6 x 106 viable cells were plated in 60 mm collagen- or Matrigel-coated dishes and incubated at 37°C in 5% CO2/95% air in William's E medium containing 10% fetal calf serum until cells attached (-30 min). After decanting nonattached cells (largely nonparenchymal), cultures were then maintained with daily changes of serum-free medium (DMEM/F12,1:1), with hormones and substrates added as indicated. Generally, hepatocyte conditioned medium was collected for analysis of IGFBP-1 protein and hepatocyte RNA extracted after 48 h of culture. IGF-I was separated from IGF binding proteins by size-exclusion high-performance liquid chromotography on a Toyo Soda TSK 2000 SW column (960 x 750 mm), eluted at 0.71 ml/min with 0.1 M ammonium acetate (acidified to pH 3.9 with acetic acid). IGFs were eluted between 29.76 and 35.23 min, and - 9 0 % of added [125I] IGF-I was recovered in this fraction, with
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; ED 50 of ~10~ 1 0 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
From the Division of Endocrinology and Metabolism, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and the University of Manitoba, Manitoba, Canada. Address correspondence and reprint requests to Lawrence S. Phillips, MD, Department of Medicine, Emory University School of Medicine, 69 Butler Street, SE, Atlanta, GA 30303. Received for publication 12 December 1991 and accepted in revised form 20 February 1992.
DIABETES, VOL. 41, JULY 1992
expression at 30 min, but suppressed IGFBP-1 mRNA 37% at 90 min and 97% at 180 min (t 1/2 - 1 1 0 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. Diabetes 41:835-42,1992
T
he insulinlike growth factor binding proteins (IGFBPs) have diverse actions, but there is incomplete understanding of their regulation. The IGFBPs prolong the half-life of insulinlike growth factor I (IGF-I); enhance and attenuate IGF-1 mediated mitogenesis; and prevent IGF-I binding to insulin receptors (1,2,3). Six distinct IGFBPs have been identified in animals and human subjects: IGFBP-1 is a 28,000-34,000 Mr nonglycosylated protein that is present at low levels in normal adult serum, and rises under conditions of diabetes or nutritional deprivation; IGFBP-2 is a -30,000 Mr form that is abundant in fetal and neonatal serum, decreases in adult serum, and increases during fasting; IGFBP-3 is a -43,000 Mr glycosylated form that carries most of the circulating IGF-1 and insulinlike growth factor II, and falls during fasting or diabetes; and IGFBP-4 is a -24,000 Mr form that recently has been purified from human cerebrospinal fluid (4,5,6,7,8,9,10). Regulation of IGFBP-5 and 6 is poorly understood. Although consistent changes in circulating binding protein levels have been found in different pathological states, the simultaneous fluctuations of hormones and fuels that occur in fasting or diabetes make it difficult to delineate specific regulation, and study of control in vitro is required for insight into underlying mechanisms. Our laboratory and others have examined the regulation of IGFBP synthesis in the liver, which is a target organ for glucoregulatory hormones. The liver also processes the
835
MOLECULAR REGULATION OF IGFBP-1
bulk of nutritional substrates, and is a major source of circulating IGFs and the IGFBPs. Although IGFBP synthesis has been examined in several liver cell lines, relatively little is known concerning the regulation of IGFBPs in normal liver cells (11,12). We have shown that adult rat hepatocytes maintained in primary culture release large amounts of IGFBP-1 (13). Because the pretranslational regulation of IGFBP-1 in normal liver cells is poorly understood, we used the hepatocyte primary culture system to investigate the molecular regulation of hepatic IGFBP-1 according to the availability of both hormones and nutrients. RESEARCH DESIGN AND METHODS Recombinant IGF-I was purchased from AMGen Biologicals (Thousand Oaks, CA); collagenase type I from Worthington Biomedical (Freehold, NJ); type I rat tail collagen, human transferrin, fetal bovine serum, and Williams-E medium from Sigma (St. Louis, MO); goat antirabbit IgG and normal rabbit serum from Cambridge Medical Diagnostics (Billerica, MA); GeneScreen Plus from NEN Products (Boston, MA); amino acid- and glucose-free Dulbeco's-modified Eagle and Ham's F12 media from JRH Bioscience (Lenexa, KS); 32P-dCTP from DuPont (Wilmington, DE); dexamethasone from LyphoMed (Melrose Park, II); penicillin/streptomycin from Mediatech (Washington, DC); bovine growth hormone from USDA Reproduction Laboratory (Beltsville, MD); and Matrigel from Collaborative Research, (Lexington, MA). Porcine insulin was a generous gift from Eli Lilly (Indianapolis, IN). Hepatocytes were isolated from 150-200 g male Sprague-Dawley rats, using a two-stage collagenase perfusion technique as described previously (14). An estimated 3.6 x 106 viable cells were plated in 60 mm collagen- or Matrigel-coated dishes and incubated at 37°C in 5% CO2/95% air in William's E medium containing 10% fetal calf serum until cells attached (-30 min). After decanting nonattached cells (largely nonparenchymal), cultures were then maintained with daily changes of serum-free medium (DMEM/F12,1:1), with hormones and substrates added as indicated. Generally, hepatocyte conditioned medium was collected for analysis of IGFBP-1 protein and hepatocyte RNA extracted after 48 h of culture. IGF-I was separated from IGF binding proteins by size-exclusion high-performance liquid chromotography on a Toyo Soda TSK 2000 SW column (960 x 750 mm), eluted at 0.71 ml/min with 0.1 M ammonium acetate (acidified to pH 3.9 with acetic acid). IGFs were eluted between 29.76 and 35.23 min, and - 9 0 % of added [125I] IGF-I was recovered in this fraction, with
