Correlation of glioma cell regression with inhibition of insulin-like growth factor 1 and insulin-like growth factor-binding protein-2 expression.

To explore the antitumor effect of insulin-like growth factor 1 (IGF-I) antisense RNA and the interaction of IGF-I with insulin-like growth factor-binding proteins (IGFBPs) in glioma cells, a recombinant retrovirus expressing IGF-I antisense RNA was constructed and introduced into C6 glioma cells. IGF-I antisense RNA reverses the transformed phenotype in glioma cells and inhibits glioma cell growth by blocking overexpression of endogenous IGF-I. Expression of IGFBP-2 is increased in glioma cells as compared with normal adult glial cells. IGF-I antisense RNA also inhibits expression of IGFBP-2 in glioma cells, but does not influence expression of the other IGFBPs. Although IGFBP-2 in conditioned medium from wild-type C6 cell cultures itself does not directly influence glioma cell growth, it synergistically enhances exogenous IGF-I-mediated DNA synthesis in IGF-I-negative C6 cells. These findings indicate the inhibitory effect of IGF-I antisense RNA on growth and development of glioma cells. IGF-I-dependent glioma cell growth may, in some circumstances, require IGFBP-2 as a cofactor. The antitumor effect of IGF-I antisense RNA is also associated with inhibition of IGFBP-2 expression.

Antihypertensive agents that limit ventricular hypertrophy inhibit cardiac expression of insulin-like growth factor-I.

BACKGROUND: Left ventricular hypertrophy (LVH) is a generalized adaptation to altered myocardial load. Hypertension induces significant increases in ventricular IGF-I gene expression that occur coordinately with development of LVH. To test whether IGF-I promotes initiation of LVH, we examined ventricular IGF-I mRNA content in spontaneously hypertensive rats (SHRs) treated with antihypertensive drugs that limit or permit LVH. METHODS: Prehypertensive SHRs were left untreated or treated with enalapril, nifedipine, or hydralazine. Systolic blood pressure (SBP), hypertrophy index (ventricular weight/body weight), and ventricular IGF-I mRNA levels were examined 2, 4, and 6 weeks after beginning therapy in the experimental groups. RESULTS: Systolic blood pressure reached hypertensive levels after 2 weeks in untreated animals, and was controlled in the treated animals. The hypertrophy index in untreated animals was significantly elevated at 4 weeks. By 6 weeks, the hypertrophy indices of both the enalapril- and nifedipine-treated groups were significantly lower than that of the untreated group. In contrast, the hypertrophy index of the hydralazine-treated animals remained comparable to that of the untreated animals. By 4 weeks, IGF-I mRNA levels in the enalapril- and nifedipine-treated groups were significantly lower than those in the untreated and hydralazine-treated groups. CONCLUSIONS: We conclude that: (1) antihypertensive drugs that reduce LVH blunt ventricular IGF-I mRNA content; and (2) the hemodynamic effects of antihypertensives may be dissociated from their ability to promote or limit a hypertrophic response. The clear association of LVH with ventricular IGF-I mRNA content suggests that IGF-I is an important determinant of ventricular growth. Our data also suggest that angiotensin-converting enzyme inhibitors and calcium channel blockers may reduce LVH by inhibiting cardiac IGF-I gene expression.

Pre- and post-translational regulation of renal insulin-like growth factor binding protein-1 in insulin-deficient diabetes.

BACKGROUND: Renal size and production of insulin-like growth factor-I (IGF-I) increase rapidly after the onset of insulin-deficient diabetes, despite decreases in serum and hepatic levels of IGF-I and linear growth retardation in affected animals and humans. This increase in kidney IGF-I gene expression is mediated both by pre- and post-translational mechanisms, with the relative contributions of each locus of control varying with the severity and/or duration of diabetes. Since the actions of IGF-I are modified by specific circulating as well as locally produced IGF binding proteins (IGF BPs), and since kidney IGF BP1 content is increased in diabetes, we asked whether: 1) the time course of induction of increased BP1 expression paralleled that for induction of IGF-I; 2) severity and/or duration of diabetes affected pre- and post-translational renal expression of this protein as it does expression of IGF-I itself; and 3) insulin deficiency or hyperglycemia was responsible for this increase in kidney IGF BP1 content. METHODS: Adult rats were made diabetic by injection of streptozotocin (STZ), and kidney BP1 mRNA and protein were assessed by Northern and Western ligand blotting, respectively, in comparison with nondiabetic, insulin-treated diabetic, and phlorizin-treated diabetic animals. RESULTS: Rapid time- and STZ dose-dependent increases in both pre- and post-translational renal IGF BP1 expression were noted in the untreated diabetic animals. Comparison of the relative changes in kidney BP1 mRNA and protein contents suggested that with increasing severity of diabetes, at least 20% of this effect was mediated pre-translationally and, therefore, did not merely reflect trapping of circulating BP1. Treatment with insulin completely inhibited the pre-translational and potently inhibited the post-translational component of the response, while correction of hyperglycemia with phlorizin did not. These observations were specific for BP1, with renal IGF BP3 mRNA and protein contents noted to be low basally and unaffected by diabetes. CONCLUSIONS: These data suggest that insulin strongly regulates pre- and post-translational renal IGF BP1 gene expression and implicate BP1 as an important determinant of IGF-I activity in diabetic kidney. The similarity of the time course of BP1 induction to that of IGF-I in animals of the same age and severity of diabetes suggests that local IGF-I/BP1 interactions may potentiate kidney IGF-I activity and promote initiation of the early stages of diabetic renal hypertrophy.

Induction of myocardial insulin-like growth factor-I gene expression in left ventricular hypertrophy.

BACKGROUND: Left ventricular hypertrophy is a generalized adaptation to increased afterload, but the growth factors mediating this response have not been identified. To explore whether the hypertrophic response was associated with changes in local insulin-like growth factor-I (IGF-I) gene regulation, we examined the induction of the cardiac IGF-I gene in three models of systolic hypertension and resultant hypertrophy. METHODS AND RESULTS: The model systems were suprarenal aortic constriction, uninephrectomized spontaneously hypertensive rats (SHR), and uninephrectomized, deoxycorticosterone-treated, saline-fed rats (DOCA salt). Systolic blood pressure reached hypertensive levels at 3 to 4 weeks in all three systems. A differential increase in ventricular weight to body weight (hypertrophy) occurred at 3 weeks in the SHR and aortic constriction models and at 4 weeks in the DOCA salt model. Ventricular IGF-I mRNA was detected by solution hybridization/RNase protection assay. IGF-I mRNA levels increased in all three systems coincident with the onset of hypertension and the development of ventricular hypertrophy. Maximum induction was 10-fold over control at 5 weeks in the aortic constriction model, 8-fold at 3 weeks in the SHR, and 6-fold at 6 weeks in the DOCA salt model. IGF-I mRNA levels returned to control values by the end of the experimental period despite continued hypertension and hypertrophy in all three systems. In contrast, ventricular c-myc mRNA content increased twofold to threefold at 1 week and returned to control levels by 2 weeks. Ventricular IGF-I receptor mRNA levels were unchanged over the time course studied. The increased ventricular IGF-I mRNA content was reflected in an increased ventricular IGF-I protein content, as determined both by radioimmunoassay and immunofluorescence histochemistry. CONCLUSIONS: We conclude that (1) hypertension induces significant increases in cardiac IGF-I mRNA and protein that occur coordinately with its onset and early in the development of hypertrophy, (2) IGF-I mRNA levels normalize as the hypertrophic response is established, (3) in comparison to IGF-I, both c-myc and IGF-I receptor genes are differentially controlled in experimental hypertension. These findings suggest that IGF-I may participate in initiating ventricular hypertrophy in response to altered loading conditions. The consistency of these findings in models of high-, moderate-, and low-renin hypertension suggests that they occur independently of the systemic renin-angiotensin endocrine axis.

Discordant, organ-specific regulation of insulin-like growth factor-I messenger ribonucleic acid in insulin-deficient diabetes in rats.

Linear growth retardation is common in uncontrolled insulin-deficient diabetes, but individual organs such as kidney may hypertrophy. To explore whether this heterogeneity of response might be mediated by differential local insulin-like growth factor-I (IGF-I) gene regulation, we injected rats with ip saline, 65, 120, or 175 mg/kg streptozotocin (STZ). Diabetics were untreated or received daily insulin. Animals were killed 24, 48, or 72 h after documentation of diabetes, and liver, kidney, and lung messenger RNA (mRNA) content analyzed by solution hybridization/RNase protection assay. Untreated diabetics had 10- to 100-fold reductions in hepatic IGF-I mRNA apparent as early as 24 h, and the magnitude of these changes varied directly with the severity of diabetes. In contrast, kidney IGF-I mRNA content increased by 400-500% at 24 h in untreated diabetics given 175 mg/kg STZ, and by 100-200% at 48 h in those given 120 mg/kg STZ, with return to control levels by 72 h. Renal IGF-I mRNA levels actually decreased by 250-350% at 24 h in rats injected with 65 mg/kg STZ, returning to supranormal values by 72 h. These results suggest that severity and/or duration of the metabolic abnormality qualitatively and quantitatively affect this response in the kidney. Liver and kidney IGF-I mRNA levels approached normal with insulin therapy and were similar to controls in rats which received STZ but did not develop diabetes. Lung IGF-I mRNA levels were minimally altered in all experimental groups. At the time point and STZ dosage at which liver IGF-I mRNA changes were most dramatic, little change in liver alpha-tubulin mRNA was noted. At the time point and STZ dosages at which kidney IGF-I mRNA induction was most dramatic, renal IGF-I receptor mRNA was only minimally changed, and renal alpha-tubulin mRNA was modestly reduced. In summary: 1) hepatic IGF-I mRNAs are dramatically reduced, and renal IGF-I mRNAs are significantly increased soon after the onset of insulin-deficient diabetes in STZ-treated rats; 2) insulin therapy restores IGF-I mRNA levels toward normal; and 3) these changes in IGF-I mRNA content are specific and are not the result of hepatic or renal STZ toxicity. These data suggest that IGF-I gene expression is regulated in a discordant, organ-specific manner in diabetes, and that metabolic factors in addition to GH may differentially modulate the endocrine and paracrine effects of IGF-I on growth.

The human erythrocyte insulin-like growth factor I receptor: characterization and demonstration of ligand-stimulated autophosphorylation.

To characterize the insulin-like growth factor I (IGF-I) receptor on human erythrocytes, cells were purified from peripheral blood by Ficoll-Hypaque gradient centrifugation and incubated with [125I]IGF-I. Specific binding was maximal at pH 8.0 after 24 h at 4 C and increased linearly with cell number to 3.9 +/- 0.2% (+/- SEM) for 3.0 X 10(9) cells/ml. The Scatchard plot of the binding data was linear, with 7 fmol [125I]IGF-I bound/10(9) cells and an affinity constant (K) of 1.8 X 10(9) M-1. Unlabeled IGF-I inhibited tracer binding half-maximally at 6 ng/ml. Multiplication-stimulating activity (or rat IGF-II) was 40% as potent (ED50, 15 ng/ml), whereas insulin and proinsulin were 30- to 500-fold less potent. A monoclonal antibody to the IGF-I receptor (alpha IR-3) inhibited IGF-I binding by 50% at a 1:1000 dilution and by 80% at a 1:250 dilution. Insulin binding was unaffected by the same dilutions. IGF-I receptor phosphorylation was studied in erythrocyte ghosts prepared by hypotonic lysis and solubilized in 1% Triton. The extract was preincubated with and without 100 ng/ml IGF-I or porcine insulin and incubated with [gamma-32P]ATP in the presence of Mn2+, and the receptor was identified by immunoprecipitation with alpha IR-3 antibody and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IGF-I stimulated 4-fold the incorporation of 32P into a protein of 95,000 mol wt, which was immunoprecipitated by alpha IR-3; insulin produced a 2-fold stimulation of this protein. This protein corresponds to the beta-subunit of the IGF-I receptor. These data demonstrate that human erythrocytes have specific receptors for IGF-I, and that this IGF-I receptor, like the insulin receptor, undergoes ligand-stimulated autophosphorylation. Thus, analysis of erythrocyte IGF-I binding and receptor phosphorylation may be useful tools for the study of patients with a variety of growth disorders.