Insulin in the extrapancreatic tissues of guinea pigs differs markedly from the insulin in their pancreas and plasma.
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Biomedical subjects
Publications and source records attributed to C C Yip.
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Dispersed cell preparations enriched in beta-cells were obtained by collagenase digestion of fetal bovine pancreas and separation by Ficoll gradient centrifugation. These cells actively incorporated [3H]leucine into proinsulin and insulin. Incubation of these cells in the presence of the arginine analogue, L-canavanine, resulted in the inhibition of conversion of newly formed proinsulin to insulin and the appearance of a radioactive component of molecular weight 11,000-12,000. Incorporation of [35S]methionine into this component was detected in the presence of canavanine, an event not observed in control incubation. Canavanine thus induced the formation of a component possessing molecular weight and compositional properties expected for preproinsulin. Further characterization of cellular products by polyacrylamide slab gel electrophoresis in sodium dodecyl sulfate showed a highly labeled band corresponding to molecular weight 18,000-20,000 which might be involved in insulin biosynthesis.
Cell lines derived from a Syrian hamster insulinoma have been cloned in agar and maintained continuously in culture in vitro for 1 1/2 years. The cell lines have average doubling times of 48 h, they have modal chromosome numbers between 38 and 39, and they retain the ability to form tumors when injected into Syrian hamsters. The cells do not produce immunoreactive insulin when grown in vitro (less than 0.5 ng/mg protein), but do produce immunoreactive insulin when grown in vivo as tumors (mean value from six determinations = 7.1 +/- 1.5 ng/mg protein).
A photosensitive insulin derivative was synthesized by reacting radioactive iodinated bovine insulin with N-hydroxysuccinimide ester of 4-azidobenzolic acid. The photo-sensitivity and specificity of this insulin derivative were established by its covalent nonspecific cross-link to albumin and its covalent specific cross-link to the heavy and light chains of anti-insulin immunoglobulin. Plasma membrane preparations of rate adipocytes were incubated with the photosensitive insulin derivative and irradiated with light. Sodium dodecyl sulfate gel electrophoresis of these plasma membrane preparations after solubilization with sodium dodecyl sulfate and reduction with beta-mercaptoethanol showed that a protein having a molecular weight of 130,000 was specifically labeled by the radioactive photosensitive insulin, suggesting that this protein may be the insulin receptor.
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Confluent fibroblasts were assayed for insulin binding to determine whether there was an inherent abnormality in receptor function to explain the insulin resistance of lipoatrophic diabetes (LD). Cells from three patients and four controls were compared for confluent density, receptor saturation, specific and non-specific binding and the concentration of unlabelled hormone producing 50% competition for binding with labelled ligand. Cells from patients with LD did not differ significantly in any of these characteristics from the controls. These findings indicate that there is not a basic defect in insulin receptors of LD patients. A secondary disruption of binding, e.g. by a circulating factor, remains possible.
Specific and nonspecific [125I]insulin binding and concentration of unlabeled hormone producing 50 percent competition with 1.0 nanomolar [125I]insulin for specific binding sites correlated positively with age of fibroblast donors. Cells from four children with precocious aging--three with progeria and one with Rothmund syndrome-resembled those from the chronologically old.
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Bovine des-B-Ala30,des-A-Asn21-insulin and guinea pig des-B-Asp30,des-A-Asn21-insulin were prepared from bovine and guinea pig insulin by digestion with carboxypeptidase A (EC 3.4.12.2). As reported by other investigators, the biological activity of bovine des-Ala30,des-Asn21-insulin was less than 10% that of bovine insulin. Contrary to theoretical consideration, removal of A-Asn21 and B-Asp30 from the carbosyl termini of guinea pig insulin resulted in a loss of more than 90% of the biological activity. Receptor binding studies of these insulin derivatives demonstrated a good correlation between the loss of biological activity and the decrease in binding affinity. It is suggested that the carboxyl terminal A-Asn21 of insulin may interact directly with the insulin receptor.
We have reported previously that increasing amounts of immunoreactive glucagon (IRG), measured by four specific antisera, appeared in plasma of depancreatized insulin-deficient dogs. It was therefore concluded that pancreatectomy was not accompanied by glucagon deficiency in the dog, but instead excessive amounts of extrapancreatic IRG could contribute to the diabetic syndrome. In order to locate the source of extrapancreatic glucagon, tissue extracts were assayed with anti-glucagon sera 30-K and K-44, which cross-react minimally with crude gut extracts. IRG was detected in all gastrointestinal tissues and in the salivary glands, but not in extracts of liver, kidney, brain, heart atrium, and adenohypophysis. Immunologic dilution curves of extracts from all gastrointestinal tissues were parallel to those of the pure pancreatic glucagon standard, and both antisera (30-K and K-44) measured the same concentrations. The highest concentration of gastrointestinal IRG was found in the fundus and corpus of the stomach. Presence of IRG in gastrointestinal tissues of depancreatized dogs indicates that gastrointestinal cells can not only secrete but also store large amounts of IRG. Extracts of mucosa of stomach fundus were further purified by gel filtration on Biogel P-30 columns. The immunoreactivity in the eluate was assayed by 30-K and a strongly crossreacting antibody, K-4023. One pooled fraction corresponding to marker pancreatic glucagon in its elution volume was found to contain the largest amount of IRG and the highest specific immunoreactivity (IRG/protein concentration). This fraction showed also the highest activity in a glucagon-receptor assay system. Disc gel electrophoresis in the presence of urea resolved this fraction into three immunoreactive components, one of which was identical to pancreatic glucagon in its electrophoretic mobility. It appears, therefore, that mucosa of the upper stomach in the dog contains a polypeptide similar to pancreatic glucagon. We conclude that (a) hyperglucagonemia in the dog can result from excessive secretion of IRG not only by the pancreatic alpha cells but also by cells of the gastrointestinal tract; (b) the highest IRG concentration was found in fundus and corpus of the stomach and lower concentrations throughout the gastrointestinal tract; (c) the IRG component in the stomach displayed immunologic and physical properties similar to pancreatic glucagon.
RNA preparations from isolated rat pancreatic islets and from human insulinomas were injected into oocytes of Xenopus laevis which were then incubated with [3H]leucine. Acid-ethanol extracts of the oocytes were immunoprecipitated with anti-insulin serum using the double antibody technique. Sodium dodecyl sulfate disc gel electrophoresis of the immunoprecipitates showed the presence of an insulin-displaceable immunoreactive material with a molecular weight of about 18,000 in extracts from oocytes injected with RNA of 9-11 S. This immunoreactive product was not detected in extracts from oocytes injected with buffer or 4-8S RNA or RNA heavier than 11 S. These observations suggest the involvement of a precursor larger than proinsulin in the biosynthesis of insulin.
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When rabbit reticulocytes were incubated in vitro with [(3)H]acetate, their ribosomal proteins were rapidly acetylated within 10 min. Polyacrylamide-urea gel electrophoresis showed that several major ribosomal protein fractions were highly acetylated. By the double-isotope labeling technique, the incorporation of [(3)H]acetate and [(14)C]aminoacid mixture into ribosomal proteins and nascent chains was found to be closely associated. Sodium fluoride abolished the acetylation of ribosomal proteins, whereas cycloheximide reduced the acetylation of ribosomal proteins to a much lower level. These findings suggest that acetylation of ribosomal proteins may be involved in the formation of the initiation complex during protein biosynthesis.