Biomedical subjects
Publications and source records attributed to M M Rechler.
Total RNA extracted from a rat liver cell line (BRL 3A) that synthesizes rat insulin-like growth factor II (IGF-II) was translated in a reticulocyte lysate cell-free system. Incubation of the translation products with antisera to mature rat IGF-II (Mr 8700) selectively immunoprecipitated a Mr 21,600 protein. We propose that this protein represents pre-pro-IGF-II.
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The structure of receptors for insulin-like growth factors in rat liver plasma membranes and the BRL 3A2 rat liver cell line has been examined by chemical cross-linking with disuccinimidyl suberate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. Two receptor subtypes have been identified: (i) 125I multiplication-stimulating activity cross-linked to liver membranes or intact cells appeared in a complex of Mr = 260,000 (reduced) and 220,000 (nonreduced) and (ii) 125I-insulin-like growth factor I cross-linked to BRL 3A2 cells appeared predominantly in two bands of Mr greater than 300,000 without disulfide reduction and in a Mr = 130,000 complex following reduction. The two subtypes of insulin-like growth factor receptors identified by structural analysis correspond to previously observed differences in their specificity for insulin and insulin-like growth factors.
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The properties of multiplication stimulating activity (MSA), an insulin-like growth factor (somatomedin) purified from culture medium conditioned by the BRL 3A rat liver cell line are summarized. The relationship of MSA to somatomedins purified from human and rat plasma are considered. MSA appears to be the predominant somatomedin in fetal rat serum, but a minor component of adult rat somatomedin. In vitro biological effects of MSA and insulin in adipocytes, fibroblasts and chondrocytes are examined to determine whether they are mediated by insulin receptors or insulin-like growth factor receptors. The possible relationship of a primary defect of insulin receptors observed in fibroblasts from a patient with the rare genetic disorder, leprechaunism, to intrauterine growth retardation is discussed.
Fibroblasts cultured from an infant with leprechaunism and insulin resistance have been reported to exhibit profound, selective defect in insulin binding. We now examine the effect of this defect on two acute metabolic actions of insulin thought to be mediated by the insulin receptor, glucose incorporation and N-methyl-alpha-aminoisobutyric acid (Me-AiBu) uptake. In the patient's fibroblasts, maximal insulin-stimulated glucose incorporation was less than 25% of that in control fibroblasts, whereas stimulation by hydrogen peroxide, an insulinomimetic agent that acts distal to the insulin receptor, was normal. By contrast, insulin stimulated Me-AiBu uptake to the same extent in patient's and control fibroblasts. Impaired glucose incorporation and relatively normal Me-AiBu uptake also were observed in the patient's cells with multiplication-stimulating activity, an insulin-like growth factor, despite the fact that multiplication-stimulating activity appeared to stimulate both responses in normal fibroblasts via an insulin-like growth factor receptor. The divergent effects on two hormone-stimulated functions in the patient's cells suggests differences in the coupling of a receptor to different effectors. The same coupling mechanisms appear to be used by insulin receptors and receptors for insulin-like growth factors.
We previously have demonstrated that fibroblasts from a patient with leprechaunism exhibited markedly decreased insulin binding to insulin receptors and that the ability of insulin to stimulate glucose incorporation in the patient's cells was greatly impaired. In addition, the insulinlike growth factor, multiplication-stimulating activity (MSA), also exhibited an impaired ability to stimulate glucose incorporation in the patient's fibroblasts, although in normal fibroblasts this response appears to be mediated by an insulinlike growth factor receptor. The present study examines 125I-labeled insulinlike growth factor I (IGF-I) binding to patient's and control fibroblasts. 125I-labeled IGF-I binds to a specific IGF-I receptor in normal fibroblasts. At steady state, binding was inhibited by unlabeled IGF-I, IGF-II, MSA III-2, MSA II, insulin, and proinsulin, in order of potency, but not by high concentrations of epidermal growth factor and human growth hormone, chemically unrelated polypeptides 125I-labeled IGF-I binding to patient's cells was decreased by approximately 75%, whereas binding of epidermal growth factor to its cell surface receptors was unaffected. Computer curve-fitting of untransformed equilibrium binding data suggests that the decreased binding resulted from a decreased Ka for IGF-I. The ability of the patient's IGF-I receptor to recognize insulin also appears to be altered. Impaired IGF-I binding by the leprechaun patient's fibroblasts may contribute to the abnormal biological response to insulinlike growth factors observed in vitro and to the in utero growth retardation.
Primary cultures of endothelial cells were prepared from the arteries and veins of human umbilical cords. Both arterial and venous endothelial cells demonstrated specific receptors for the insulin-like growth factor MSA (Multiplication-Stimulating Activity). Insulin, at concentrations up to 10(-7) M, had little effect on 125I-MSA binding whereas MSA was congruent to 1% as potent as insulin in competing with 125I-insulin binding. Serum containing anti-insulin receptor antibodies blocked binding of 125I-insulin to the endothelial cells but had little effect on 125I-MSA binding. We conclude that human endothelial cells have specific and distinct receptors for both MSA and insulin.
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Insulin stimulates a 2-fold increase in the amount of tyrosine aminotransferase and a 5-10-fold increase in the rate of amino acid transport in dexamethasone-treated rat hepatoma cells. In order to determine whether these effects are mediated by insulin receptors or receptors for insulin-like growth factors, we have examined the binding of 125I-labeled insulin and 125I-labeled multiplication-stimulating activity, a prototype insulin-like growth factor, and compared the biological effects of these polypeptides. Insulin and multiplication-stimulating activity cause an identical increase in transaminase activity and transport velocity; half-maximal biological effects were observed at 35 ng/mg (5.5 nM) insulin and 140 ng/ml multiplication-stimulating activity. The hepatoma cells display typical insulin receptors of appropriate specificity; half-maximal displacement of tracer insulin binding occurred at 33 ng/ml unlabeled insulin, but only at 2500 ng/ml unlabeled multiplication-stimulating activity. Specific multiplication-stimulating activity receptors also were demonstrated with which insulin did not interact even at 10 micrograms/ml. Half-maximal displacement of tracer multiplication-stimulating activity occurred at 200 ng/ml unlabeled multiplication-stimulating activity. We conclude that insulin cannot act via the multiplication-stimulating activity receptor and presumably acts via typical insulin receptors. The effects of multiplication-stimulating activity on enzyme induction and amino acid transport are probably mediated primarily via the multiplication-stimulating activity receptor.
This report describes an infant with physical features typical of leprechaunism, including a characteristic facies, hirsutism, and decreased subcutaneous tissue and muscle mass. Intermittent hypoglycemia and severe hyperinsulinemia were documented. The patient's insulin was normal in molecular size and biological activity, but its binding to the patient's cultured fibroblasts was profoundly decreased. Insulin antibodies were not present. A literature review has been undertaken to clarify further the clinical, metabolic, and pathological characteristics of this condition.
Multiplication-stimulating activity (MSA), purified from medium conditioned by the BRL-3A rat liver cell line, previously has been shown to be closely related to the human somatomedins or insulin-like growth factors. A radioimmunoassay was utilized to measure MSA levels in sera from fetal, maternal, and young rats. A serum somatomedin-binding protein was found to interfere in the radioimmunoassay by competing with antibody for binding 125I-labeled MSA. Therefore, prior to radioimmunoassay, sera were filtered on Sephadex G-75 in 1 M acetic acid to dissociate and separate somatomedin activity from the binding protein. Concentrations of MSA by radioimmunoassay were 20- to 100-fold higher in feta rat sera (1.8-4.4 micrograms/ml) than in maternal sera. MSA levels gradually decreased after birth, reaching maternal levels by day 25 of extrauterine life. MSA concentrations in feta rat sera also were found to be correspondingly high by a rat liver membrane radioreceptor assay and a competitive binding protein assay using rat serum somatomedin-binding protein. The findings of higher levels of MSA in fetal than in maternal rat sera and the gradual decline in MSA serum concentrations after birth are in direct contrast to total somatomedin activities measured by biosassay. Thus, MSA may function as a growth factor in the fetal rat whereas other somatomedins may play a role in stimulating growth during extrauterine life.
Multiplication-stimulating activity (MSA) refers to a family of insulin-like growth factors that have been purified from serum-free medium conditioned by a Buffalo rat liver cell line (BRL-3A). Using Dowex ion-exchange chromatography, gel chromatography on Sephadex G-75, and preparative disc acrylamide gel electrophoresis, several polypeptides with the full biological multiplication-stimulating activity have been isolated. One of these polypeptides, designated MSA II-1, previously has been used to study the relationship of the activity to the insulin-like growth factors (somatomedins) purified from human plasma. Polypeptide II-1 is a single chain polypeptide of molecular weight 8700. Glycine is the COOH-terminal amino acid. Edman degradation of carboxymethylated MSA II-1 did not reveal a free NH2-terminus. A polypeptide of lower molecular weight than MSA II-1 has also been purified. This polypeptide (MSA III-2) has been shown to be more potent than MSA II-1 in the rat-liver-membrane radioreceptor assay and in a competitive binding assay utilizing the rat-serum somatomedin-binding protein(s). The relationship of these various polypeptides has been investigated by gel filtration in guanidine hydrochloride and by acrylamide gel electrophoresis of the reduced and native polypeptides.
The immunoreactivity of the multiple species of multiplication-stimulating activity (MSA) purified from medium conditioned by a rat liver cell line (BRL-3A) has been examined. Antibodies were raised in rabbits following immunization with MSA II polypeptides. Subpopulations of antibodies were purified from one antiserum using DEAE-cellulose chromatography. One antibody subpopulation recognized common antigenic determinants on MSA I and MSA II polypeptides; whereas a second antibody subpopulation recognized common determinants on MSA I, II, and III polypeptides. In a radioimmunoassay utilizing 125I-MSA III-2 and a purified antibody subpopulation, the human somatomedins (somatomedin A, insulin-like growth factor I and II) showed weak, but significant cross-reactivity: insulin-like growth factor II was 10% as potent as MSA II. By contrast, somatomedin partially purified from rat serum, insulin, growth hormone, epidermal growth factor, nerve factor, and fibroblast growth factor, showed no reactivity in the radioimmunoassay.
Insulin and such insulinlike growth factors as multiplication stimulating activity (MSA) are related polypeptides that have common biological activities. Both insulin and MSA produce acute metabolic responses (stimulation of glucose oxidation in isolated fat cells) as well as growth effects (stimulation of [(3)H]thymidine incorporation into DNA in cultured fibroblasts). In addition, most cells have separate receptors for insulin and insulinlike growth factors, and both peptides have weaker affinity for each other's specific receptors than for their own. To determine, therefore, whether these effects are mediated by receptors for insulin, insulinlike growth factors, or both, we have selectively blocked insulin receptors with a specific antagonist, namely Fab fragments derived from naturally occurring antibodies to the insulin receptor. In rat adipocytes, 10 mug/ml of antireceptor Fab inhibited insulin binding by 90%, whereas it inhibited MSA binding <5%. The anti-insulin receptor Fab is without intrinsic biological activity, but acts as a competitive inhibitor of insulin receptors. Blockade of insulin receptors with Fab fragments produced a 30-fold rightward shift in the dose response for stimulation of glucose oxidation by both insulin and MSA. The dose-response curves for stimulation of oxidation by vitamin K(5) and spermine, agents that stimulate glucose oxidation through noninsulin receptor pathways, were not affected by the blockade of insulin receptors with Fab antibody fragments. These data suggest that this acute metabolic effect of both insulin and MSA is mediated via the insulin receptor. In cultured human fibroblasts, 10 mug/ml of Fab inhibited insulin binding by 90% and MSA binding by 15%. In fibroblasts, however, blockade of the insulin receptor did not alter the dose response for stimulation of thymidine incorporation into DNA by either insulin or MSA. Furthermore, intact antireceptor antibody immunoglobulin (Ig)G, which produces multiple other insulinlike effects, and Fab fragments of antireceptor antibody did not stimulate thymidine incorporation. These data demonstrate directly that the insulin receptor mediates the metabolic effects of insulin and MSA, whereas the growth-promoting action of both peptides is mediated by the MSA receptor or other growth factors.
Insulin-like growth factors (IGFs) I and II, purified from human plasma, and multiplication-stimulating activity (MSA), purified from media conditioned by the BRL 3A rat liver cell line, are polypeptides with similar biological and biochemical properties. We have compared the interaction of 125I-labeled and unlabeled MSA, IGF-I, and IGF-II with four intact cell or cell membrane preparations previously shown to possess MSA receptors: rat liver plasma membranes, chick embryo fibroblasts, human fibroblasts, and BRL 3A2 cells. In each case, specific binding of 125I-labeled IGF-I and IGF-II was demonstrated. With each 125I-labeled peptide, significant inhibition of binding and parallel dose-response curves were observed with unlabeled IGF-I, IGF-II, and MSA. Striking differences were noted, however, in the relative potencies of the unlabeled peptides as competitive inhibitors of binding. We conclude that the different specificities of binding inhibition reflect a significant heterogeneity among IGF receptors. A similar heterogeneity appears to occur among somatomedin carrier proteins in rat and human sera.
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Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.