Protective effect of glutamine from glucocorticoid-induced muscle atrophy occurs without alterations in circulating insulin-like growth factor (IGF)-I and IGF-binding protein levels.

We investigated whether the prevention of glucocorticoid-induced muscle atrophy by glutamine infusion is associated with alterations in serum levels of insulin-like growth factor (IGF)-I and its binding proteins (IGFBPs). Hormone (cortisol acetate [CA], 100 mg/kg body wt/day) and vehicle (carboxymethyl cellulose [CMC])-treated female rats were infused with either saline or glutamine (240 mM, 0.75 ml/hr) for a 7-day period. Glutamine infusion prevented over 70% of the skeletal muscle mass loss due to the glucocorticoid injections. Serum IGF-I concentrations, which were measured by radioimmunoassay (RIA) after acid solid-phase extraction of IGFBPs, were not significantly different among groups (range of means: 373-395 ng/ml). Saline/CA treatment resulted in a 2-fold increase in circulating levels of IGFBP-3 (38- to 50-kDa bands from ligand blotting measurements) versus the saline/CMC group. Levels of 30- to 32-kDa bands were increased by approximately 3-fold in the CA-treated rats. Immunoprecipitation studies suggested that the increase in the 30- to 32-kDa binding proteins were not due to elevated levels of IGFBP-1, -2, or -5. None of the treatments significantly modified circulating levels of IGFBP-4 (24 kDa). Glutamine infusion did not reverse the effects of glucocorticoids on circulating levels of 38- to 50- and 30- to 32-kDa IGFBPs. We conclude that the attenuation of glucocorticoid-induced muscle atrophy by glutamine infusion is not associated with changes in circulating levels of IGF-I or IGFBPs.

Effects of glucocorticoids on circulating levels and hepatic expression of insulin-like growth factor (IGF)-binding proteins and IGF-I in the adrenalectomized streptozotocin-diabetic rat.

Circulating levels and hepatic expression of insulin-like growth factor-binding protein-1 (IGFBP-1) are increased in insulin-deficient streptozotocin (STZ)-diabetic rats. Glucocorticoids stimulate and insulin suppresses hepatocellular expression of IGFBP-1 in vitro. We asked whether increased IGFBP-1 expression in STZ-diabetic animals is due to an effect of insulin deficiency per se or whether insulin deficiency represents a permissive state where glucocorticoids may play an important role in the regulation of IGFBP-1 and other circulating peptides involved in the modulation of IGF bioactivity. Intact female Sprague-Dawley-derived rats and rats undergoing bilateral adrenalectomy (ADNX) were injected with STZ (140 mg/kg) or buffer. Corticosterone acetate (50 mg/kg) or vehicle was administered to diabetic and nondiabetic animals immediately after ADNX and 24 h later. All rats were killed 48 h after surgery and/or STZ administration. Serum [125I]IGF-I-binding activity was increased 4-fold (P < 0.01), and Western ligand and immunoblotting demonstrated that levels of IGFBP-1 were high in intact STZ-diabetic animals. ADNX prevented these effects of STZ-diabetes, and corticosterone treatment restored serum IGF-binding activity and IGFBP-1 to intact diabetic levels. Similarly, Northern analysis demonstrated that the abundance of hepatic IGFBP-1 mRNA was increased 6-fold in intact STZ-diabetic animals (P < 0.01), but not in adrenalectomized diabetic animals. Corticosterone treatment restored hepatic IGFBP-1 mRNA to intact diabetic levels, and serum concentrations of corticosterone correlated with the abundance of IGFBP-1 mRNA (r = 0.475; P < 0.01), indicating that glucocorticoids play an important role in the regulation of expression of IGFBP-1 in insulin-deficient animals. In contrast, neither ADNX nor corticosterone altered the abundance of hepatic IGFBP-1 mRNA levels in nondiabetic animals. This pattern of regulation appeared to be specific; serum levels of immunoreactive IGFBP-2 and -4 tended to rise in adrenalectomized animals, and levels of IGFBP-3 were not affected by either ADNX or corticosterone treatment. Of note, serum levels of IGF-I by RIA were reduced in STZ-diabetic animals compared to control values (168 +/- 16 vs. 587 +/- 55 ng/ml, respectively; P < 0.01), were partially restored toward control values with ADNX (320 +/- 22 ng/ml), and were reduced again by corticosterone treatment (195 +/- 26 ng/ml), indicating that glucocorticoids also contribute to the regulation of IGF-I levels in insulin-deficient animals. The abundance of IGF-I mRNA was reduced in STZ-diabetic animals, and ADNX also partially prevented this effect of diabetes.(ABSTRACT TRUNCATED AT 400 WORDS)

Divergent effects of phorbol esters and insulin on insulin-like growth factor binding protein-1 (IGFBP-1) production and mRNA in rat H4IIE hepatoma cells.

125I-IGF-I binding assay, western ligand and immunoblotting, and northern analysis of total RNA reveal that phorbol ester agonists of protein kinase C rapidly enhance IGFBP-1 production and increase the abundance of IGFBP-1 mRNA in rat H4IIE hepatoma cells. In combination with insulin, a potent inhibitor of IGFBP-1 gene transcription, this early effect of phorbol esters is dominant. These results demonstrate divergent regulation of IGFBP-1 by phorbol esters and insulin and indicate that protein kinase C may play a critical role in the regulation of IGFBP-1 and modulation IGF bioactivity in metabolic disease.

Multihormonal regulation of insulin-like growth factor-binding protein-1 in rat H4IIE hepatoma cells: the dominant role of insulin.

Circulating levels of insulin-like growth factor-binding protein-1 (IGFBP-1) and the abundance of hepatic IGFBP-1 mRNA are increased in streptozotocin-diabetic rats and are regulated in accordance with insulin and metabolic status. We recently purified rat IGFBP-1 from medium conditioned by well differentiated rat H4IIE hepatoma cells. Since this cell line provides a useful model for examining the effects of hormones on hepatocellular function, we used H4IIE cells to examine the relative role that insulin and other factors may play in the regulation of IGFBP-1 production. H4IIE cells were stabilized in serum-free medium, then treated with specific hormones. The availability of IGFBPs in conditioned medium was estimated by [125I]IGF-I binding assay, and specific BPs were assessed by Western ligand and immunoblot analyses. The abundance of IGFBP-1 mRNA was determined by Northern and slot blot analysis. Initial studies revealed that [125I]IGF-I-binding activity in conditioned medium was reduced after 24-h incubation with 100 nM insulin (52 +/- 4% of control; P less than 0.001). In contrast, binding activity was increased after only 4 h of incubation with 75 microM 8-(4-chlorophenylthio)cAMP (8-CPT-cAMP) or 1 microM dexamethasone (P less than 0.001 vs. control for each), but these effects were prevented by insulin. Ligand and immunoblotting demonstrated that insulin decreased the production of 32K and 34K forms of IGFBP-1, while both 8-CPT-cAMP and dexamethasone increased the production of IGFBP-1; again, insulin prevented the effects of 8-CPT-cAMP and dexamethasone. Of note, 1 microM rat GH, testosterone, progesterone, or 17 beta-estradiol had no effect on either IGF-binding activity or IGFBP-1 production. Northern and slot blot analyses revealed that 100 nM insulin profoundly lowered the abundance of IGFBP-1 mRNA in H4IIE cells (4 +/- 0.6% of control at 4 h; P less than 0.001), while IGFBP-1 mRNA was increased 2-fold during incubation with 75 microM 8-CPT-cAMP (P less than 0.001) and 9-fold with 1 microM dexamethasone (P less than 0.001). Once again, the effect of insulin was dominant; insulin both prevented and reversed the effects of maximally effective concentrations of 8-CPT-cAMP and dexamethasone. To determine whether this effect of insulin reflected altered generation or stability of IGFBP-1 mRNA, H4IIE cells were incubated with 2.5 micrograms/ml actinomycin-D with or without insulin, and mRNA was quantitated by Northern blot.(ABSTRACT TRUNCATED AT 400 WORDS)

Production of the rat type 1 insulin-like growth factor-binding protein by well differentiated H4EIIC3 hepatoma cells: identification, purification, and N-terminal amino acid analysis.

We recently identified a 32 K mol wt insulin-like growth factor (IGF)-binding protein (BP) which is markedly increased in the serum of streptozotocin-diabetic rats and recognized by antiserum against the human amniotic fluid IGFBP (hIGFBP-1). In the present study we sought to confirm that this protein represents the rat homolog of IGFBP-1 (rIGFBP-1), and that rIGFBP-1 may, therefore, play an important role in the regulation of IGF bioactivity in experimental diabetes. Since the abundance of related hepatic mRNA is high in diabetic rats, we asked whether well differentiated H4EIIC3 rat hepatoma cells produce rIGFBP-1 and provide sufficient amounts of this protein for purification and further characterization. Specific IGF-binding activity in hepatoma conditioned medium was detected initially by incubation with 125I-labeled recombinant human IGF-II and precipitation with polyethylene glycol. Ligand blotting demonstrated a 32 K BP, identical in size to the major low mol wt IGFBP found in diabetic rat serum. Affinity labeling and immunoprecipitation confirmed that this BP is related to human IGFBP-1 and is distinct from the fetal rat IGFBP, rIGFBP-2. Incorporation of [35S]methionine into 32 K BPs confirmed synthesis by hepatoma cells. For purification of BPs, conditioned medium was collected in roller culture, and BPs were purified by ammonium sulfate precipitation, Sephadex G-75 chromatography, and reverse phase HPLC. Partial amino acid sequencing of purified protein demonstrated 68% identity with the human IGFBP-1 and distinguished this BP from previously characterized rat IGFBPs. Purified protein bound both IGF-I and IGF-II with high affinity. We conclude that the 32 K IGFBP produced by H4EIIC3 hepatoma cells in culture represents the rat form of IGFBP-1 (rIGFBP-1). Regulation of rIGFBP-1 may play an important role in the modulation of IGF bioactivity in experimental animals with metabolic disease. The availability of purified rIGFBP-1 and identification of a cell line that produces this BP will greatly facilitate future studies of IGFBP-1 in the rat model.

Regulation of low molecular weight insulin-like growth factor binding proteins in experimental diabetes mellitus.

Circulating insulin-like growth factor (IGF) bioactivity is reduced in animals and patients with diabetes mellitus. We sought to determine whether the availability and levels of specific IGF binding proteins (BPs) are altered in animals with experimental diabetes, and might contribute to changes in circulating IGF bioactivity in experimental diabetes. Female Sprague-Dawley rats were administered streptozotocin or citrate buffer iv, and then killed either 3 days later, or else after 4-day insulin treatment (7.5 U/kg human NPH twice daily), or 2 days after insulin was discontinued. Serum [125I]IGF-I binding activity was markedly increased in diabetic animals compared to controls when analyzed by Sephacryl S-200 chromatography, dot blot, and affinity labeling techniques, due to increased binding to low mol wt BPs (81 +/- 4% of ligand eluting with low mol wt BPs in diabetic serum vs. 22 +/- 3% in control, P less than 0.001). In contrast, activated charcoal removed ligand from these BPs and underestimated the availability of BPs in diabetes. Serum binding activity fell toward control levels during insulin therapy, then rose again after insulin was withdrawn, corresponding to changes in metabolic status. To distinguish changes in specific BPs, serum proteins were separated by 13% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to nitrocellulose. Ligand blotting with [125I]IGF-I demonstrated that serum levels of a 32 K mol wt IGF BP are markedly increased in diabetic rats and decline during insulin therapy. Levels of this 32 K IGF BP rose again after insulin was discontinued, demonstrating regulation in accordance with changes in insulin and metabolic status. Western analysis and affinity labeling with immunoprecipitation revealed that this 32 K protein is distinct from the 34 K fetal rat BP, and is immunologically related to the type 1 human IGF BP. We conclude that circulating [125I]IGF-I binding activity is markedly increased in animals with acute streptozotocin-induced diabetes, due to changes in low mol wt proteins, including a 32 K type 1 IGF BP that is regulated by changes in insulin and/or metabolic status. Regulation of low mol wt IGF BPs by insulin, and perhaps other factors, may play an important role in the modulation of tissue growth factor bioactivity in metabolic disease.

Identification of a type 1 insulin-like growth factor binding protein (IGF BP) in serum from rats with diabetes mellitus.

Circulating insulin-like growth factor binding protein (IGF BP) activity is increased in animals with streptozotocin-induced diabetes. Separation of BPs by SDS/PAGE for ligand and immunoblot analysis revealed that a 32,000 molecular weight BP is present and increased in diabetic serum. This BP is immunologically distinct from the low molecular weight fetal rat BP (rBP2) and is related to the human amniotic fluid BP (hBP1) that is increased in patients with insulin dependent diabetes mellitus.