Expressions of leptin and insulin-like growth factor-I are highly correlated and region-specific in adipose tissue of growing rats.

OBJECTIVE: Anatomically distinct adipose tissue regions differ in their predominant modality of growth (i.e., cellular hypertrophy vs. hyperplasia). We examined site-specific patterns of expression of two genes whose products, leptin and insulin-like growth factor-I (IGF-I), could be involved in mediating differential growth and metabolism of white adipose tissue. We also related these patterns of expression to measures of adipose depot cellularity. RESEARCH METHODS AND PROCEDURES: Male Wistar rats were fed ad libitum and studied from ages 7 weeks to approximately 12 months. Terminal measures of body weights; weights, composition, and cellularity of four white adipose depots; circulating leptin and IGF-I; and adipose depot-specific expression levels of leptin and IGF-I were measured in subsets of rats at 7, 12, 22, 42, and 46 weeks of age. RESULTS: Both leptin and IGF-I mRNAs are quantitatively expressed in a depot-specific manner, in the following order: retroperitoneal approximately equals epididymal > mesenteric > subcutaneous inguinal. Furthermore, there is a marked correlation between the expressions of these hormones in the various regions of adipose tissue of rats during the first year of life. The mechanisms that underlie the parallel expressions of leptin and IGF-I appear to be related to fat-cell volume. DISCUSSION: Because both leptin and IGF-I have been implicated in the regulation of energy homeostasis and are both expressed in adipose tissue, the depot-specific linkage between the two genes suggests interaction at the autocrine level. This interaction may have an important role in determining functional properties particular to individual adipose depots.

Molecular regulation of insulin-like growth factor-I and its principal binding protein, IGFBP-3.

The insulin-like growth factors (IGFs) have diverse anabolic cellular functions, and structure similar to that of proinsulin. The distribution of IGFs and their receptors in a wide variety of organs and tissues enables the IGFs to exert endocrine, paracrine, and autocrine effects on cell proliferation and differentiation, caloric storage, and skeletal elongation. IGF-I exhibits particular metabolic responsiveness, and circulating IGF-I originates predominantly in the liver. Hepatic IGF-I production is controlled at the level of gene transcription, and transcripts are initiated largely in exon 1. Hepatic IGF-I gene transcription is reduced in conditions of protein malnutrition and diabetes mellitus, and our laboratory has used in vitro transcription to study mechanisms related to diabetes. We find that the presence of sequences downstream from the major transcription initiation sites in exon 1 is necessary for the diabetes-induced decrease in IGF-I transcription. Six nuclear factor binding sites have been identified within the exon 1 downstream region, and footprint sites III and V appear to be necessary for metabolic regulation; region V probes exhibit a decrease in nuclear factor binding with hepatic nuclear extracts from diabetic animals. IGFs in biological fluids are associated with IGF binding proteins, and IGFs circulate as a 150-kDa complex that consists of an IGF, an IGFBP-3, and an acid-labile subunit. Circulating IGFBP-3 originates mainly in hepatic nonparenchymal cells, where IGF-I increases IGFBP-3 mRNA stability, but insulin increases IGFBP-3 gene transcription. Regulation of IGFBP-3 gene transcription by insulin appears to be mediated by an insulin-responsive element, which recognizes insulin-responsive nuclear factors in both gel mobility shift assays and southwestern blots. Studies of mechanisms underlying the modulation of IGF-I and IGFBP-3 gene transcription, and identification of critical nuclear proteins involved, should lead to new understanding of the role and regulation of these important growth factors in diabetes mellitus and other metabolic disorders.

Identification of an insulin-responsive element in the rat insulin-like growth factor-binding protein-3 gene.

The hepatic expression and serum levels of insulin-like growth factor-binding protein-3 (IGFBP-3) are decreased in insulin-dependent and insulin-resistant diabetes. Insulin increases hepatic IGFBP-3 expression by enhancing gene transcription. This report identifies sequences within the IGFBP-3 promoter that are necessary and sufficient for the response to insulin in hepatic nonparenchymal cells. By transient transfection, we mapped the insulin response element to the -1150 to -1124 base pair (bp) region of the rat IGFBP-3 promoter. Three tandem repeats of the -1150 to -1117 bp region conferred insulin responses in a heterologous promoter. Gel shift analyses revealed a 3-fold increase in DNA-protein complex formation with nuclear extracts obtained from insulin-stimulated nonparenchymal cells compared with cells incubated without insulin and revealed 3-4-fold decrease in complex formation with nuclear extracts obtained from the livers of streptozotocin-diabetic rats compared with control rats. Mutational analysis of this 34-bp region showed a core sequence of 10 bp (-1148 to -1139) that is critical for interaction with insulin-induced trans-acting factors. Southwestern blotting revealed a approximately 90-kDa protein that was increased 2-3-fold by the addition of insulin. Thus, we have identified cis-acting DNA sequences that are responsible for regulation of IGFBP-3 transcription by insulin and essential for binding of insulin-responsive nuclear factors.

Transcription initiation of the rat insulin-like growth factor-I gene in hepatocyte primary culture.

Transcription initiation in the insulin-like growth factor-I (IGF-I) gene is complex, involving multiple sites in two exons. While most transcripts are initiated in exon 1 in vivo, critical regulatory mechanisms are difficult to assess in intact animals. To examine the impact of insulin and growth hormone (GH) under more controlled conditions, we have studied the utilization of different exon 1 and exon 2 transcription-initiation sites in normal rat hepatocytes in primary culture. Normal rat hepatocytes were cultured for 48 h in serum-free medium, with insulin at 10(-6) or 10(-11) M, and with or without human GH 200 ng/ml. Relative abundance of IGF-I transcripts was evaluated by the RNase-protection assay, using a probe which permitted identification of initiation in exon 1 (site 1 (-380 bp from the 3' end of exon 1), site 2 (-343 bp), site 3 (-242 bp), sites 1 and 2 spliced, and site 4 (-32 bp)), as well as in exon 2. After normalization of signal intensity to adjust for differences in length of protected probe, the utilization of initiation sites in vitro was remarkably similar to that in vivo: 1, 14, 6, 23, 19 and 37% for sites 1, 2, 3, 1 and 2 spliced, 4 and exon 2 respectively in the cultured hepatocytes, compared with 1, 12, 18, 21, 18 and 40% for these sites in normal liver. Insulin alone increased transcripts initiated from exon 1, site 2 by over 3 times, and both sites 1 and 2 spliced and exon 2 transcripts by about 5 times. GH alone had similar effects, producing a 4-5 times increase in transcripts from these initiation sites. Addition of both insulin and GH had additive effects, increasing transcripts from exon 1, sites 2, 3 and 4 by 4-6 times, and from exon 1, sites 1 and 2 spliced, and exon 2 by over 8 times. Of the total IGF-I mRNA transcripts, 37% were initiated from sites 2 and/or sites 1 and 2 spliced, and 37% from exon 2. Analysis of the relative contribution of individual initiation sites revealed hormone-induced increases which were statistically significant only for exon 2, in the presence of insulin alone and in combination with GH. In conclusion, in cultured hepatocytes, insulin or GH alone produced a coordinated increase in all exon 1 transcripts, and the effect of the combination of insulin and GH was additive for these transcripts. Exon 2 appeared to be more sensitive to insulin alone, and to GH in the presence of insulin, than exon 1. Since utilization of initiation sites in hepatocytes mimics that found in liver, this in vitro system should be useful for examining underlying transcriptional regulatory mechanisms.

Insulin and insulin-like growth factor-I regulate hepatic insulin-like growth factor binding protein-3 by different mechanisms.

UNLABELLED: Insulin-dependent diabetes mellitus is associated with decreased levels of circulating insulin-like growth factor binding protein-3 (IGFBP-3), which are restored toward normal by treatment with insulin and/or infusion of insulin-like growth factor-I (IGF-I). To understand underlying mechanisms, we studied IGFBP-3 production in cocultures of parenchymal and nonparenchymal cells from the livers of normal rats. Release of IGFBP-3 was measured by ligand blotting and was increased 1.9- and 15-fold by 10(-8) and 10(-8) M Insulin compared with 10(-10) M (P < 0.05 for 10(-6) vs. 10(-10) M). Expression of IGFBP-3 mRNA was increased concomitantly by 23 and 226% (P < 0.05 for 10(-6) M vs. 10(-10) M), consistent with regulation in part at pretranslational levels. To evaluate mRNA stability, transcription was inhibited with 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB): IGFBP-3 mRNA t1/2 was estimated at 13 h and 17 h with addition of 10(-5) M and 10(-10) M insulin, respectively, ruling out regulation at the level of mRNA turnover. IGFBP-3 gene transcription rates were evaluated by nuclear run-on assays and were increased 2,9-fold with the addition of 10(-6) M insulin, as compared with 10(-10) M insulin, comparable to stimulation of expression. Addition of IGF-I at 2.6 x 10(-8) M and 5.3 x 10(-8) M increased IGFBP-3 release by 5.2- and 8.2-fold (both P < 0.05 vs. no IGF-I), with concomitant increase in IGFBP-3 mRNA expression by 14- and 29-fold (both P < 0.05 vs. no IGF-I), suggesting regulation at a pretranslational level. Further studies showed that IGF-I did not have a significant effect on transcription initiation rates but prolonged the apparent half-life of IGFBP-3 mRNA about 2-fold. Stimulation of IGFBP-3 via type 1 IGF-I receptors was evaluated by studies with [QAYL] IGF-I; the analog increased IGFBP-3 mRNA expression 220 +/- 27% above the level obtained without IGF-I (vs. 133 +/- 9% with wild type IGF-I, P < 0.05), suggesting involvement of receptor-mediated synthesis. CONCLUSION: Insulin stimulates IGFBP-3 gene transcription but provides proportionally greater increases in IGFBP-3 release, consistent with regulation at both transcriptional and posttranslational levels; in contrast, IGF-I alters IGFBP-3 expression by decreasing IGFBP-3 mRNA degradation, consistent with regulation at pretranslational and posttranscriptional levels. Decreased IGFBP-3 levels in conditions of diabetes mellitus may be due to decreased hepatic IGFBP-3 release, and secondary both to decreased gene transcription (caused by insulin deficiency), as well as to decreased IGFBP-3 mRNA half-life (caused by low levels of IGF-I).

Glucocorticoid regulation of insulin-like growth factor-binding protein-3.

Short stature and decreased growth velocity are prominent features of endogenous and pharmacological glucocorticoid excess in children. Underlying processes may involve direct cellular effects or defective generation of insulin-like growth factors (IGFs) and/or IGF-binding proteins (IGFBPs), which modulate IGF-stimulated events and regulate growth. To evaluate potential mechanisms, we investigated the impact of dexamethasone (dex) on hepatic expression of IGFBP-3, the major carrier protein for IGFs. Using cocultured hepatic parenchymal and nonparenchymal cells, dex at 10(-8) and 10(-6) M decreased IGFBP-3 secretion by 67 +/- 9% and 73 +/- 9%, respectively (both P < 0.05 vs. no dex). In a separate experiment, IGFBP-3 messenger RNA (mRNA) expression was decreased by 84 +/- 2% and 75 +/- 2% (both P < 0.05 vs. no dex). In combined studies, levels of IGFBP-3 protein in conditioned medium were strongly correlated with the abundance of IGFBP-3 mRNA (r = 0.75; P < 0.01), consistent with regulation at a pretranslational level. After inhibition of transcription, levels of IGFBP-3 mRNA decreased 85% and 86% over 24 h in cells cultured in 10(-6) M and no dex, respectively; the t1/2 was 13.6 h at 10(-6) M and 12.6 h with no dex, indicating that dex had no effect on IGFBP-3 mRNA stability. To evaluate transcriptional effects, the rate of IGFBP-3 gene transcription was measured by incorporation of [alpha-32P]UTP into preinitiated message in isolated nuclei and fell 78% after the addition of 10(-6) M dex for 48 h (compared to cells cultured in 10(-9) M dex), an inhibition of a magnitude similar to the effects on protein release and mRNA abundance. We conclude that dex may reduce the production of IGFBP-3 by inhibiting IGFBP-3 gene transcription.

Coculture of primary rat hepatocytes and nonparenchymal cells permits expression of insulin-like growth factor binding protein-3 in vitro.

In biological fluids, the insulin-like growth factors (IGFs) are associated with binding proteins (IGFBPs), which modify IGF distribution and action. Circulating IGFs are bound predominantly to IGFBP-3, of apparent hepatic origin, but regulation of IGFBP-3 has been difficult to dissect because of the lack of systems suitable for examining hepatic production of IGFBP-3 in vitro. In the present studies, IGFBP-3 expression was identified primarily in hepatic nonparenchymal cells, particularly Kupffer and sinusoidal endothelial cells. Coculture with hepatocytes enhanced the stability of nonparenchymal cells to express IGFBP-3 in vitro. IGFBP-3 in conditioned medium had apparent mol wt of 150-300 kilodaltons, suggesting formation of a ternary complex with IGFs and the acid-labile subunit. Expression and secretion of IGFBP-3 were hormonally responsive and strongly correlated (r = 0.79; P < 0.001), with 2- to 3-fold stimulation by added insulin or IGF-I (both P < 0.05), but not by added GH alone. Our findings suggest that GH may act indirectly to promote IGFBP-3 generation in vivo via increasing both the secretion of insulin and the hepatic production of IGF-I; in patients with diabetes mellitus, reduced circulating levels of IGFBP-3 despite high levels of GH may result from both insulin deficiency and inadequate hepatic production of IGF-I. Coculture of hepatic nonparenchymal and parenchymal cells should be useful for further analysis of the mechanism of IGFBP-3 regulation.

Regulation of insulin-like growth factor-I (IGF-I) and IGF-binding protein 1 gene transcription by hormones and provision of amino acids in rat hepatocytes.

Synthesis of insulin-like growth factor-I (IGF-I) and IGF binding protein-1 (IGFBP-1) is altered in diabetes and malnutrition, but underlying processes are poorly understood. To study molecular mechanisms, we examined regulation of IGF-I and IGFBP-1 gene transcription in primary cultures of rat hepatocytes. Transcription of the IGF-I and IGFBP-1 genes was measured as incorporation of [alpha-32P]UTP into preinitiated message in isolated nuclei. IGFBP-1 gene transcription was not sensitive to reduction in amino acid concentration from 5x to 0.5x rat arterial plasma levels. However, IGF-I gene transcription fell 60-70% in response to reduced provision of amino acids. Culture with 10(-9) M insulin lowered IGFBP-1 gene transcription 50% below control levels (10-11 M) but did not affect IGF-I gene transcription; 10(-6) M insulin raised IGF-I gene transcription 2-fold. After an acute reduction in insulin concentration, IGFBP-1 transcription began to rise within 30 min, but IGF-I gene transcription was unchanged over 120 min. Similarly, 3-6 h were required for stimulation of IGF-I gene transcription by insulin, but a 40% decrease in IGFBP-1 gene transcription could be detected within 15 min after adding 10(-6) M insulin, and suppression of IGFBP-1 transcription by insulin was unaffected by the presence of cycloheximide. Effects of insulin on IGFBP-1 gene transcription were not mimicked or antagonized by phorbol ester.(ABSTRACT TRUNCATED AT 250 WORDS)

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 less than 0.01; increases in IGFBP-1 protein (ligand blot) and IGFBP-1 mRNA (Northern blot) were highly correlated (r = 0.62, P less than 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 approximately 10(-10) M is within the physiological range of insulin concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Nutrition and somatomedin. XXV. Regulation of insulinlike growth factor binding protein 1 in primary cultures of normal rat hepatocytes.

Although mRNAs encoding insulinlike growth factor (IGF) binding proteins (BPs) are present in adult rat liver and IGF BP-1 circulates at elevated levels in diabetic animals, there is little knowledge of the metabolic regulation of IGF BPs in normal tissues. We examined the release of IGF BPs by adult rat hepatocytes maintained in primary culture. When cultured for 2 days in the absence of added insulin, hepatocytes released a BP identified as BP-1 on the basis of approximately 30,000-Mr on ligand blotting and reactivity with antiserum to human BP-1 in immunoblotting and immunoprecipitation studies. Release of BP-1 was sensitive to insulin with suppression of 24 +/- 4, 73 +/- 5, and 64 +/- 14% at 10(-10), 10(-8), and 10(-6) M insulin, respectively; ED50 was approximately 1.7 x 10(-9) M, which is within the physiological range. Suppression by insulin was reversible and began within 3 h. Because normal hepatocytes in primary culture exhibit insulin-responsive release of both BP-1 and IGF-1, this system may be an ideal model for studies of molecular mechanisms of metabolic regulation.