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Biomedical subjects

L A Bach

Publications and source records attributed to L A Bach.

At least 37 records · Page 2Linked to original sources
PubMed1999

Differential dissociation kinetics explain the binding preference of insulin-like growth factor binding protein-6 for insulin-like growth factor-II over insulin-like growth factor-I.

Insulin-like growth factor binding protein-6 binds insulin-like growth factor-II with a marked preferential affinity over insulin-like growth factor-I. The kinetic basis of this binding preference was studied using surface plasmon resonance. Binding of insulin-like growth factor-I and insulin-like growth factor-II to immobilized insulin-like growth factor binding protein-6 fitted a two-site binding kinetic model. Insulin-like growth factor-I and insulin-like growth factor-II association rates were similar whereas the dissociation rate was approximately 60-fold lower for insulin-like growth factor-II, resulting in a higher equilibrium binding affinity for insulin-like growth factor-II. The equilibrium binding affinities of a series of insulin-like growth factor-II mutants were also explained by differential dissociation kinetics. O-glycosylation had a small effect on the association kinetics of insulin-like growth factor binding protein-6. The insulin-like growth factor binding properties of insulin-like growth factor binding protein-6 are explained by differential dissociation kinetics.

Binding, Competitive↗
PubMed1999

Induction of insulin-like growth factor binding protein expression by ICI 182,780 in a tamoxifen-resistant human breast cancer cell line.

Earlier studies in our laboratory demonstrated that the steroidal antiestrogen ICI 182,780 is very effective in abolishing the tamoxifen-resistant proliferation of MCF 7/5-23 cells. In addition, preliminary binding studies showed that ICI 182,780 increased the binding of insulin-like growth factor (IGF)-I to the MCF 7/5-23 cells, although this finding was not the result of an increase in the expression of the insulin-like growth factor-I receptor (IGF-IR). Hence, we reasoned that the inhibition of tamoxifen-resistant cell growth by ICI 182,780 might have been due to increased expression of insulin-like growth factor binding proteins (IGFBPs). We observed the up-regulation of non-insulin-suppressible IGF-I binding in both the tamoxifen-sensitive MCF 7/5-21 cell line (1.5-fold) and the tamoxifen-resistant MCF 7/5-23 cell line (2.5-fold) after 5 days of treatment with ICI 182,780 (10(-7) M) in serum-free medium, suggesting a role for cell-associated IGFBPs. Affinity cross-linking experiments confirmed the presence of an IGF-I:IGFBP complex of approximately 38-kDa in tamoxifen or ICI 182,780-treated cells. Western ligand blots showed higher levels of a soluble 30-kDa IGFBP in media conditioned by either of the subclones that had been treated with ICI 182,780, an effect consistently opposed by estrogen (E2: 10(-9) M). RT-PCR showed higher levels of IGFBP-5 mRNA than any of the other known IGFBPs, suggesting that this was the major IGFBP subtype. The protein was subsequently identified by Western immunoblotting as IGFBP-5. In conclusion, we postulate that this may be a mechanism contributing to the greater potency of ICI 182,780 in the growth inhibition of the MCF 7/5-23, tamoxifen-resistant cell line.

Antineoplastic Agents↗
PubMed1999

HaCaT human keratinocytes express IGF-II, IGFBP-6, and an acid-activated protease with activity against IGFBP-6.

The insulin-like growth factor (IGF) system plays an important role in skin. HaCaT human keratinocytes proliferate in response to IGFs and synthesize IGF-binding protein-3 (IGFBP-3). Recently, IGFBP-6 was also identified by NH2-terminal sequencing, but it has not been identified by Western ligand blotting. In the present study, IGFBP-6 was detected in HaCaT-conditioned medium by use of immunoblotting and Western ligand blotting with 125I-labeled IGF-II. Proteolytic activity against IGFBPs, an important mechanism for regulation of their activity, was then studied. An acid-activated, cathepsin D-like protease that cleaved both IGFBP-6 and IGFBP-3 was detected. Although proteolysis did not substantially reduce the size of immunoreactive IGFBP-6, it greatly reduced the ability of IGFBP-6 to bind 125I-IGF-II as determined by Western ligand blotting and solution assay. HaCaT keratinocytes do not express IGF-I mRNA, but IGF-II mRNA and protein expression was detected. These observations suggest the possibility of an autocrine IGF-II loop that is regulated by the relative expression of IGF-II, IGFBP-3, and IGFBP-6, and IGFBP proteases in these keratinocytes, although demonstration of this loop requires further study.

Acids↗
PubMed1999

Localization of the insulin-like growth factor system in a rat model of heart failure induced by myocardial infarction.

Although cardiac effects of growth hormone (GH) and insulin-like growth factor (IGF)-I have been reported in experimental models of heart failure and in human dilated cardiomyopathy, the IGF system has not been comprehensively assessed in the failing heart. We therefore localized the IGF system in the left ventricle during congestive heart failure after myocardial infarction (MI) in the rat. The left anterior descending coronary artery was ligated in adult female Sprague-Dawley rats and hearts were examined after 6 months when congestive heart failure had developed. In situ hybridization histochemistry was used to localize mRNA for the components of the IGF system in the left ventricle of sham and congestive heart failure animals. We were able to detect changes in the spatial distribution of mRNA for IGF-I and IGF binding proteins 3, 4, 5, and 6 in the left ventricle during congestive heart failure after MI. IGF-I and the binding proteins were predominantly increased in the infarct/peri-infarct area of the left ventricle. Other components of the IGF system were indistinguishable from the low to undetectable levels in sham-operated rats. These results demonstrate that the IGF system is altered in the failing heart and suggest that the IGF system plays an important role in the response of the heart to MI and consequent failure.

Animals↗
PubMed1999

Basic fibroblast growth factor induces proteolysis of secreted and cell membrane-associated insulin-like growth factor binding protein-2 in human neuroblastoma cells.

Insulin-like growth factor (IGF) action in the brain is modulated by IGF-binding proteins (IGFBPs) whose abundance can be altered by other locally expressed growth factors. However, the mechanisms involved are unclear. We here employed the neuroblastoma cell line SK-N-MC as a model to define the mechanisms involved in modulation of IGFBPs in neuronal cells. Western ligand blotting analysis and immunoprecipitation of conditioned media (CM) from SK-N-MC cells showed that in these cells, as in the brain, the most abundantly expressed IGFBP was IGFBP-2. However, IGFBP-2 was barely detectable in CM from cells treated with basic fibroblast growth factor (bFGF) without a change in IGFBP-2 messenger RNA (mRNA) abundance. These CM contained specific IGFBP-2 proteolytic activity, resulting in two IGFBP-2 fragments of 14 and 22 kDa. The activity was inhibited by EDTA/phenylmethylsulfonyl fluoride or aprotinin. Competitive binding studies indicated that IGFBP-2 fragments had reduced binding affinity for IGF-I. bFGF induced IGFBP-3 mRNA and protein. Affinity cross-linking of [125I]IGF-I to neuroblastoma cell membranes followed by immunoprecipitation revealed a approximately 38 kDa [125I]IGF-I/IGFBP-2 complex. Cell surface-associated IGFBP-2 was also susceptible to bFGF-induced proteolysis, with the appearance of a single cross-linked 21-kDa complex with low affinity for IGF-I. These findings indicate that intact IGFBP-2 and the 14-kDa, but not the 22-kDa fragment, bind to the cell surface. Our data suggest that induction of IGFBP-2 proteolysis on neuronal cell surface is a novel mechanism whereby IGF availability is modulated by the local growth factor bFGF.

Binding Sites↗
PubMed1999

Increased renal expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in experimental diabetes.

It has been suggested that the cytokine vascular endothelial growth factor (VEGF) has an important role in the pathogenesis of diabetic retinopathy, but its role in nephropathy has not been clearly demonstrated. Assessment of VEGF, 125I-VEGF binding, and vascular endothelial growth factor receptor-2 (VEGFR-2) in the kidney was performed after 3 and 32 weeks of streptozotocin-induced diabetes. Gene expression of both VEGF and VEGFR-2 was assessed by Northern blot analysis and the localization of the ligand and receptor was examined by in situ hybridization. VEGF and VEGFR-2 protein were also evaluated by immunohistochemistry. Binding of the radioligand 125I-VEGF was evaluated by in vitro and in vivo autoradiography. Diabetes was associated with increased renal VEGF gene expression. VEGF mRNA and protein were localized to the visceral epithelial cells of the glomerulus and to distal tubules and collecting ducts in both diabetic and nondiabetic rats. Renal VEGFR-2 mRNA was increased after 3 weeks of diabetes but not in long-term diabetes. In situ hybridization and immunohistochemical studies revealed that glomerular endothelial cells were the major site of VEGFR-2 expression. In addition, VEGFR-2 gene expression was detected in cortical and renomedullary interstitial cells and on endothelial cells of peritubular capillaries. There was an increase in 125I-VEGF binding sites after 3 but not 32 weeks of diabetes. The major VEGF binding sites were in the glomeruli. 125I-VEGF binding was also observed in medullary rays and in the renal papillae. These studies indicate an early and persistent increase in renal VEGF gene expression in association with experimental diabetes. In addition, an early and transient increase in renal VEGF receptors was also observed in diabetic rats. These findings are consistent with a role for VEGF in mediating some of the changes observed in the diabetic kidney.

Animals↗
PubMed1998

Identification of O-glycosylation sites and partial characterization of carbohydrate structure and disulfide linkages of human insulin-like growth factor binding protein 6.

The actions of insulin-like growth factors (IGFs) are modulated by a family of high-affinity binding proteins (IGFBPs), including IGFBP-6, which preferentially binds IGF-II and is O-glycosylated. Glycosylated and nonglycosylated recombinant human IGFBP-6, expressed in Chinese hamster ovary cells and Escherichia coli, respectively, were purified using IGF-II affinity chromatography and reverse-phase medium-pressure chromatography. Electrospray ionization mass spectrometry (ESMS) of glycosylated IGFBP-6 revealed considerable heterogeneity of carbohydrate composition. Major glycoforms contained 8-16 monosaccharides, including N-acetylhexosamine, hexose, and N-acetylneuraminic acid. Glycosylation sites of IGFBP-6 were identified as Thr126, Ser144, Thr145, Thr146, and Ser152 by using a combination of ESMS and Edman sequencing of tryptic fragments separated by reverse-phase high-pressure liquid chromatography. One oligosaccharide chain contained 5-6 monosaccharides, whereas the others contained 2-4 monosaccharides. Glycosylated IGFBP-6 exhibited greater resistance to proteolysis by chymotrypsin and trypsin than nonglycosylated IGFBP-6. Native disulfide bond positions in IGFBP-6 were localized by means of observed disulfide-linked tryptic fragments, revealing that there are two disulfide-linked subdomains within each of the N- and C-terminal regions and confirming a previous suggestion that the latter regions are not interconnected. A model of IGFBP-6 is developed in which these distinct domains are separated by a central region which is O-glycosylated.

Amino Acid Sequence↗
PubMed1998

Cyclic AMP agonists increase levels of insulin-like growth factor (IGF) binding protein-6 in PC12 rat phaeochromocytoma cells.

The predominant insulin-like growth factor binding protein (IGFBP) synthesized by PC12 rat phaeochromocytoma cells is IGFBP-6. Since cAMP agonists regulate IGFBP-6 in other cells, and they may increase neurite outgrowth and catecholaminergic enzyme expression in PC12 cells, we studied regulation of IGFBP-6 by these agents. After 72 h incubation, forskolin and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) both increased IGFBP-6 protein levels in conditioned media to maximum levels of 231 +/- 40 and 275 +/- 30%, respectively. Incubation with forskolin resulted in a small, transient rise in IGFBP-6 mRNA levels which was insufficient to account for the increased protein levels. The increased protein levels also could not be attributed to increased cell number, protection of IGFBP-6 from proteolysis or release of IGFBP-6 from a cell-associated reservoir. These findings suggest that increased protein levels may have been due to increased translation of mRNA. Co-incubation of forskolin with dexamethasone (which decreases IGFBP-6 protein and mRNA) demonstrated that the effects of the latter were dominant. The effects of cAMP agonists and IGF-II, which increases IGFBP-6 protein but not mRNA levels, were not inhibited by rapamycin, suggesting that p70 S6 kinase is not involved. The effects of cAMP agonists on IGFBP-6 levels were not directly correlated with neurite outgrowth. These findings extend our knowledge of the molecular basis of the regulation of IGFBP-6 by cAMP agonists, and indicate a novel action of these agents in PC12 cells.

Animals↗
PubMed1997

Insulin-like growth factor binding protein-2 binds to cell surface proteoglycans in the rat brain olfactory bulb.

A family of six insulin-like growth factor binding proteins (IGFBPs) bind IGF-I and modulate its biological activity. IGFBPs may bind to macromolecules on the cell surface or pericellular extracellular matrix, and this interaction may modulate their effect on IGF activity. To date, little is known about the specificity of IGFBPs in the regulation of IGF action in the brain. We therefore explored whether IGFBPs were associated with cell membrane or extracellular matrix components in the rat brain. IGF-I binding sites with the characteristics of an IGFBP were found in the olfactory bulb mitral cell layer. This IGFBP was identified as IGFBP-2 by immunoprecipitation of both solubilized membrane preparations and cross-linked 125I-IGF: IGFBP complexes. While binding of IGFBP-2 to cell membranes was unaffected by RGD-containing peptide, it was inhibited by high salt concentration, suggesting interaction with proteoglycans. IGFBP-2 bound in vitro to the glycosaminoglycans chondroitin-4 and -6-sulfate, keratan sulfate, and heparin. IGFBP-2 also bound the proteoglycan aggrecan, an effect reduced by digestion of its glycosaminoglycans. Binding of IGFBP-2 to chondroitin-6-sulfate decreased the binding affinity of IGFBP-2 for IGF-I approximately 3-fold. Finally, an IGFBP-2 antibody coimmunoprecipitated IGFBP-2 and an approximately 200 kDa proteoglycan containing chondroitin-sulfate side chains from the rat olfactory bulb, providing definitive evidence for IGFBP-2 binding to olfactory bulb proteoglycans. These findings indicate that IGFBP-2 binds to proteoglycans in cell membranes of the rat olfactory bulb. Because we have previously shown that IGFs are highly expressed in the rat olfactory bulb, cell associated IGFBP-2 may have an important role in directing IGFs to specific sites in this brain region.

Aggrecans↗
PubMed1997

Cell-specific regulation of mRNAs for IGF-I and IGF-binding proteins-4 and -5 in streptozotocin-diabetic rat kidney.

Streptozotocin (STZ)-induced diabetes in the rat causes early renal enlargement preceded by a transient elevation in IGF-I content and an increase in IGF-I tissue binding. The effects of IGF-I are mainly mediated through the IGF-I receptor (IGF-IR) and modulated by six specific IGF-binding proteins (IGFBPs). We investigated the gene expression of IGF-I, IGF-IR and IGFBPs at a cellular level within the kidney using in situ hybridisation techniques in short-term (7 day) STZ-diabetic, insulin-treated euglycaemic and normal rats. In diabetes, IGFBP-1 mRNA showed markedly increased expression in distal tubules, collecting ducts and thick ascending limbs of Henle (TALs). IGF-I, and IGFBP-4 and -5 mRNAs showed site-specific tubular changes whilst remaining unchanged in other parts of the kidney normally expressing the genes: IGF-I and IGFBP-4 mRNAs were reduced in TALs and proximal tubules respectively; IGFBP-5 mRNA was reduced in most distal tubular cells but strongly expressed in a few of these cells. IGF-IR mRNA and the mRNAs for IGFBP-2, -3 and -6 were unchanged in STZ diabetes. There was no difference between control and insulin-treated kidneys. These complex changes suggest possible involvement of the IGF/IGFBP system in the early stages of diabetic renal hypertrophy.

Animals↗
PubMed1997

Regulation of IGF-binding protein-6 by dexamethasone and IGFs in PC12 rat phaeochromocytoma cells.

PC12 rat phaeochromocytoma cells are widely used as a model of neuronal differentiation. They express IGF receptors and are responsive to IGFs. The main IGF-binding protein synthesized by these cells is IGFBP-6. Glucocorticoids induce differentiation of PC12 cells towards a chromaffin phenotype. The effect of dexamethasone on IGFBP-6 levels was therefore studied. Dexamethasone (500 nM) decreased IGFBP-6 protein in conditioned media and mRNA levels to 61 +/- 5% (P < 0.0001) and 34 +/- 14% (P = 0.03) of control levels respectively. Incubation of PC12 cells with IGF-II (100 ng/ml) for 72 h increased IGFBP-6 protein levels in media to 217 +/- 19% of control (P < 0.0001). IGFBP-6 mRNA levels, however, were unchanged. IGF-I had similar effects on IGFBP-6 protein and mRNA levels. IGFs increased cell number by 50-60%, but this was insufficient to explain the increases in protein levels. IGFBP-6 was not released from a cell-associated reservoir or protected from proteolysis by IGFs, excluding these post-translational mechanisms as explanations for the IGF effects on IGFBP-6 levels. The effects of IGF-II and dexamethasone on IGFBP-6 levels were independent. These results indicated that (1) dexamethasone decrease IGFBP-6 at the mRNA level, and (2) IGFs stimulate IGFBP-6 levels by a post-transcriptional mechanism.

Animals↗
PubMed1996

Measurement of insulin-like growth factor (IGF)-II binding to purified IGF binding proteins 1-6: comparison of charcoal adsorption and high performance size exclusion chromatography.

Insulin-like growth factor (IGF) binding to IGF binding proteins is commonly assessed by adsorbing free IGF to albumin-coated charcoal and quantitating bound IGF in the supernatant, but the validity of this technique has been questioned and many variations have been described. We compared the measurement of binding affinity and capacity of purified IGFBPs 1-6 for IGF-II using charcoal adsorption and Superdex G75 high performance size exclusion chromatography (HPSEC) to separate free and bound 125I-IGF-II. Optimal HPSEC recovery and resolution was obtained for IGFBP-1 and IGFBP-6 with low salt buffer (0.1 M sodium phosphate, pH 7.4), whereas phosphate buffer supplemented with 0.5 M NaCl was optimal for IGFBPs 2-5. Measurement of binding of 125I-IGF-II to IGFBPs 3-5 using the charcoal assay was also increased by the use of high salt buffer. Under optimal conditions, charcoal measurements of 125I-IGF-II binding to IGFBPs 1-5 were consistently lower than HPSEC measurements. By competitive binding using unlabeled IGF-II, the binding affinity of each of the IGFBPs for IGF-II was the same using both methods. Similarly, binding affinities as measured by charcoal assay were not affected by buffer composition. Differences in total binding obtained using the two methods and under different conditions were therefore due to differences in binding capacity. Charcoal adsorbs 15% of cross-linked 125I-IGF-II:IGFBP complexes which may partially explain the lower binding capacity for IGFBPs 1-5 determined by charcoal adsorption. Charcoal adsorption and HPSEC, therefore, are both valid methods for the measurement of binding affinities of IGFBPs for IGF-II, but assay conditions must be validated prior to measurement of binding capacity.

Adsorption↗
PubMed1995

Cell membrane association of insulin-like growth factor binding protein-2 (IGFBP-2) in the rat brain olfactory bulb.

Identification of sites of expression of IGF, IGF receptors and IGFBPs in the olfactory bulb of the rat brain suggested the presence of a paracrine IGF system. Since cell association of IGFBPs has been suggested as an important factor in their modulation of IGF action, we investigated whether IGFBPs are cell associated in olfactory bulb (OB). This was supported by des(1-3)IGF-I only partially competing for [125I] IGF-I binding to rat OB membrane, suggesting the presence of a cell associated IGFBP. Affinity cross-linking of [125I]IGF-I to rat OB membrane demonstrated a 39-kDa complex which was reduced by IGF-I and IGF-II, but not by des(1-3)IGF-I or insulin. Western ligand blotting of solubilised membrane showed a 38-kDa IGFBP which was immunoprecipitated by anti-IGFBP-2 antiserum but not by anti-IGFBP-5 antiserum. We conclude that in the rat IGFBP-2 is associated with membranes from OB. Whether the cell membrane association is due to integrin binding via its RGD sequence or glycosaminoglycan binding is currently under investigation. Cell associated IGFBP-2 may modulate IGF action in the neonatal rat OB.

Animals↗
PubMed1995

Localization of mRNAs for insulin-like growth factor binding proteins 1 to 6 in rat kidney.

Insulin-like growth factor-I (IGF-I) is a peptide growth factor whose activity is modulated by interaction with the family of six IGF binding proteins (IGFBPs). IGF-I is detected in rat kidney and has metabolic and growth effects. We have used in situ hybridization to localize mRNAs for the IGFBPs in rat kidney. Messenger RNAs for all six IGFBPs were detected, each with a distinctive distribution. IGFBP-1 mRNA was expressed in the distal nephron, from the thick ascending limb of the loop of Henle to the cortical collecting ducts. IGFBP-2 mRNA expression was confined to epithelial cells of the glomeruli and the thin limbs of the loop of Henle. IGFBP-3 mRNA was localized to the cortical interstitium while IGFBP-4 was the only IGFBP mRNA found in the proximal tubule. IGFBP-5 mRNA, the most abundant and widely distributed of the IGFBP mRNAs in the kidney, occurred in the glomerular mesangium and the medullary interstitium as well as in the epithelial cells of the distal nephron. IGFBP-6 mRNA, the least abundant, was expressed mainly in fibroblasts associated with renal blood vessels and the ureter. This heterogeneous distribution of the IGFBPs may enable IGF action to be regulated by multiple factors in a site-specific manner.

Animals↗
PubMed1995

Roles of insulin-like growth factor (IGF) receptors and IGF-binding proteins in IGF-II-induced proliferation and differentiation of L6A1 rat myoblasts.

Insulin-like growth factor II (IGF-II) stimulates the proliferation and differentiation of rat myoblasts. Previous studies suggest that these response are mediated by the IGF-I receptor, but the IGF-II/mannose 6-phosphate receptor was recently implicated in differentiation of mouse myoblasts. L6A1 myoblasts synthesize IGF-binding protein-4 (IGFBP-4), IGFBP-5, and IGFBP-6, which modulate IGF action. We studied the roles of IGF receptors and IGFBPs in L6A1 myoblast proliferation and differentiation by comparing the effects of IGF-II and a number of IGF-II mutants with decreased affinities for IGF receptors and/or IGFBPs. IGF-II induced concentration-dependent proliferation with a maximum increase of 47%; half-maximal proliferation was seen with approximately 50 ng/ml. [Arg54, Arg55]IGF-II bound to the IGF-I receptor with slightly lower affinity than IGF-II, did not bind to the IGF-II/mannose 6-phosphate receptor, and bound to IGFBPs secreted by myoblasts with approximately 16-fold decreased affinity. It induced proliferation with equal potency to IGF-II. [Leu27]IGF-II, which did not bind to the IGF-I receptor but bound to the IGF-II/mannose 6-phosphate receptor and IGFBPs with slightly lower affinity than IGF-II, had a markedly impaired proliferative effect, inducing proliferation only at high concentrations. [Thr48, Ser49, Ile50]IGF-II, which bound to the IGF-I receptor with slightly lower affinity than IGF-II but did not substantially bind to the IGF-II/mannose 6-phosphate receptor or IGFBPs, induced proliferation with approximately 5-fold greater potency than IGF-II. The order of potency in inducing myoblast differentiation was the same, although there was less difference in the relative potencies of IGF-II and mutants. Coincubation of recombinant human (rh) IGFBP-6 in molar excess with IGF-II inhibited myoblast proliferation and differentiation. rhIGFBP-6 was slightly less potent did not inhibit proliferation or proliferation or differentiation induced by [Thr48,Ser49,Ile50]IGF-II. These results suggest that 1) IGF-II-induced proliferation and differentiation of L6A1 myoblasts are predominantly mediated by the IGF-I receptor; 2) the IGF-II/mannose 6-phosphate receptor is not required for these actions of IGF-II; 3) nevertheless, the IGF-II/mannose 6-phosphate receptor may be capable of mediating these actions; and 4) IGFBPs secreted by myoblasts inhibit IGF actions.

Animals↗
PubMed1994

Hypertension and cardiac hypertrophy in growth hormone-deficient rats.

1. Growth hormone may influence cardiac growth during post-natal maturation or in response to hypertension, and the growth-hormone deficient dwarf rat model offers an opportunity to study this question. 2. We compared the blood pressure and heart weight of dwarf rats and Fischer (F344) control rats in early adulthood, after two hypertensive stimuli: unilateral renal ischaemia (two-kidney, one-clip) or the administration of deoxycorticosterone acetate and saline drinking fluid. 3. In untreated animals at 13 weeks of age the body weight of dwarf rats was significantly less than that of F344 rats, but the mean arterial pressure was similar. Although the hearts of dwarf rats were smaller than those of F344 rats, the heart weight/body weight ratio was significantly greater in dwarf rats. 4. Both dwarf and F344 rats developed similar hypertensive mean arterial pressures 5 weeks after left renal artery clipping or treatment with deoxycorticosterone acetate salt. The heart weights of hypertensive dwarf and F344 rats were equivalent, indicating a proportionally greater increase in cardiac size in dwarf rats for the same rise in blood pressure. 5. The plasma insulin-like growth factor-I level was markedly lower in dwarf than in F344 rats, and hypertension did not have any significant effects on these levels. 6. These findings indicate that the developmental increase in blood pressure and heart size in growing animals and the adaptive cardiac hypertrophy accompanying hypertension are not affected by growth hormone deficiency.

Animals↗
PubMed1994

Recombinant human insulin-like growth factor (IGF)-binding protein-6 inhibits IGF-II-induced differentiation of L6A1 myoblasts.

Insulin-like growth factor-binding protein-6 (IGFBP-6) is an O-linked glycoprotein that binds insulin-like growth factor-II (IGF-II) with marked preferential affinity over IGF-I. Recombinant human IGFBP-6 (rhIGFBP-6) was synthesized by COS-7 monkey kidney cells that were transiently transfected with a eukaryotic expression vector into which a complementary DNA for IGFBP-6 modified for optimal translation had been inserted. rhIGFBP-6 was similar to IGFBP-6 purified from human cerebrospinal fluid with respect to IGF binding and O-glycosylation. The effect of rhIGFBP-6 on IGF-induced L6A1 myoblast differentiation was studied using creatine kinase activity as an index of differentiation. rhIGFBP-6 inhibited differentiation initiated by IGF-II in a dose-dependent manner, inhibition was complete when rhIGFBP-6 was present in a slight molar excess. In contrast, rhIGFBP-6 had no effect on IGF-I-induced differentiation, even when coincubated in a 5-fold molar excess. These results are consistent with the preferential affinity of IGFBP-6 for IGF-II. As cell association and proteolysis have been associated with the potentiation, rather than the inhibition, of IGF action by IGFBPs, we investigated whether they occurred in the L6A1 myoblast system. After incubation of L6A1 myoblasts with rhIGFBP-6, IGFBP-6 was recovered from the medium, but not from cell lysates or extracellular matrix. In addition, [125I]IGFBP-6 did not bind to myoblast monolayers, and there was no evidence that proteolysis had occurred. Together, these results indicate that rhIGFBP-6 remains intact and soluble and, hence, inhibits IGF-II-induced differentiation. The fidelity of the IGFBP-6 expression system used for these studies will enable us to use this system to determine how structural modifications of the protein affect the modulation of IGF action by IGFBP-6.

Animals↗