Acute and chronic regulation of phosphoenolpyruvate carboxykinase mRNA by insulin and glucose.

Using the well differentiated rat hepatoma Fao we have studied the regulation of phosphoenolpyruvate carboxykinase (PEPCK) mRNA by insulin and glucose and compared these results to glucose production as estimated by glucose release into the medium. Fao cells possess an active gluconeogenic pathway and, when grown in glucose-free medium, release glucose for over 8 h. Addition of the cAMP analog, 8-(4-chlorophenyl-thio) cAMP (8-CTP-cAMP) or increasing the concentration of dihydroxyacetone and oxaloacetate results in an increase in glucose release which can be suppressed by insulin at concentrations between 1 and 100 nM. These effect of cAMP and insulin are associated with parallel changes in the level of mRNAPEPCK. Insulin treatment reduces mRNAPEPCK levels in these cells by 80%; this effect is transient reaching a maximum at 2-4 h. Addition of glucose to cells grown in glucose-free (G-) medium produces a decrease in mRNAPEPCK which is similar in magnitude and kinetics to that produced by insulin. Conversely, when cells grown in normal medium are placed in G- medium mRNAPEPCK levels triple over a period of 8 h, then return toward the basal value. Cells grown in G- medium or in G- medium plus 10nM insulin for 1 yr exhibit only slightly increased levels of mRNAPEPCK and respond to both 8-CTP-cAMP, and insulin, although the response to 8-CTP-cAMP is slightly blunted. These data indicate that glucose and insulin can play independent roles in regulation of PEPCK gene expression, and that these regulatory effects are usually transient.

Insulin receptor function in fibroblasts from patients with leprechaunism. Differential alterations in binding, autophosphorylation, kinase activity, and receptor-mediated internalization.

Insulin receptor function was examined in cultured skin fibroblasts from three patients with leprechaunism (Ark-1, Minn-1, and Can-1), a rare syndrome of severe insulin resistance and neonatal growth retardation. All three patients cell lines demonstrated insulin binding less than 15% of control. This was primarily due to reduced affinity of the receptor in Can-1 and due to reduced number of receptors in the other two cell lines (Ark-1 and Minn-1). When expressed as a fraction of total insulin bound, the percentage of cell-associated insulin internalized and degraded did not differ between the patient cell lines and the controls. However, chloroquine, which inhibited degradation by 50% in the control cells, had no effect in the cells from the patients. When normalized to insulin binding, insulin receptor autophosphorylation was normal in cells from Can-1, but reduced in those of Ark-1 and Minn-1. In contrast, the receptor-associated tyrosine kinase activity toward exogenous substrates was decreased in all three patient cell lines. These results suggest that leprechaunism is a biochemically heterogenous disease associated with a variety of alterations in receptor function. Cells from Ark-1 and Minn-1 exhibit parallel alterations in receptor autophosphorylation and kinase activity. Cells from Can-1 demonstrate normal receptor autophosphorylation but reduced kinase activity, thus displaying a unique form of a mutant insulin receptor. Despite reduced kinase activity, all three cell lines exhibit normal rates of insulin internalization, but decreased lysosomal-mediated degradation. Our data imply that receptor autophosphorylation and kinase activity may be regulated separately and that kinase activity may be linked to insulin degradation, but not necessarily internalization.

Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity.

Tyrosyl phosphorylation is implicated in the mechanism of insulin action. Mutation of the beta-subunit of the insulin receptor by substitution of tyrosyl residue 960 with phenylalanine had no effect on insulin-stimulated autophosphorylation or phosphotransferase activity of the purified receptor. However, unlike the normal receptor, this mutant was not biologically active in Chinese hamster ovary cells. Furthermore, insulin-stimulated tyrosyl phosphorylation of at least one endogenous substrate (pp185) was increased significantly in cells expressing the normal receptor but was barely detected in cells expressing the mutant. Therefore, beta-subunit autophosphorylation was not sufficient for the insulin response, and a region of the insulin receptor around Tyr-960 may facilitate phosphorylation of cellular substrates required for transmission of the insulin signal.

Phorbol esters modulate insulin receptor phosphorylation and insulin action in cultured hepatoma cells.

The effect of the tumor-promoting agent phorbol 12-myristate 13-acetate (PMA) on insulin receptors and insulin action was studied in rat hepatoma cells in culture. PMA (0.1-1.0 micrograms/ml) did not affect insulin binding either acutely or chronically but inhibited insulin stimulation of glycogen synthase and tyrosine aminotransferase. PMA (1 microgram/ml) stimulated the phosphorylation of the beta subunit of insulin receptor purified from [32P]phosphate-labeled Fao cells by 1.3-fold in the absence of insulin. In contrast, insulin-stimulated phosphorylation in the presence of PMA was reduced. Phosphoamino acid analysis of the beta subunit after PMA stimulation revealed an increase of both phosphoserine and phosphothreonine residues, whereas insulin stimulated primarily phosphorylation of tyrosine and serine residues. Insulin stimulation of cells after PMA treatment revealed a decrease in phosphotyrosine when compared to cells stimulated by insulin alone. Tryptic peptide mapping of the beta subunit by a two-dimensional chromatographic/electrophoretic separation revealed nine phosphopeptides from the cells treated with PMA. Insulin stimulated phosphorylation at six new sites in the receptor, three of which appeared to be similar to those in PMA-treated cells. This report shows that phorbol esters stimulate insulin receptor phosphorylation, inhibit insulin-induced receptor phosphorylation and insulin action, and suggest a physiologic relation between insulin action and the calcium-activated and phospholipid-dependent protein kinase C.

Abnormality of insulin binding and receptor phosphorylation in an insulin-resistant melanoma cell line.

The insulin receptor possesses an insulin-stimulated tyrosine-kinase activity; however, the significance of receptor phosphorylation in terms of the binding and signaling function of the receptor is unclear. To help clarify this problem, we have studied insulin binding and receptor phosphorylation in a Cloudman S91 melanoma cell line and two of its variants: the wild type (1A) in which insulin inhibits cell growth, an insulin-resistant variant (111) in which insulin neither stimulates or inhibits growth, and a variant (46) in which insulin stimulates cell growth. 125I-insulin binding to intact cells was similar for the wild-type 1A and insulin-stimulated variant 46. The insulin-resistant variant 111, in contrast, showed approximately 30% decrease in insulin binding. This was due to a decrease of receptor affinity with no major difference in receptor number. When the melanoma cells were solubilized in 1% Triton X-100 and the insulin receptor was partially purified by chromatography on wheat germ agglutinin-agarose, a similar pattern of binding was observed. Phosphorylation was studied by incubation of the partially purified receptor with insulin and [gamma-32P]ATP, and the receptor was identified by immunoprecipitation and NaDodSO4 PAGE. Insulin stimulated phosphorylation of the 95,000-mol-wt beta-subunit of the receptor in all three cells types with similar kinetics. The amount of 32P incorporated into the beta-subunit in the insulin-resistant cell line 111 was approximately 50% of that observed with the two other cell lines. This difference was reflected throughout the entire dose-response curve (10(-9) M to 10(-6) M). Qualitatively similar results were obtained when phosphorylation was studied in the intact cell. Peptide mapping of the beta-subunit using tryptic digestion and reverse-phase high-performance liquid chromatography column separation indicated three sites of phosphorylation in receptor from the wild type and variant 46, but only two major sites of phosphorylation of variant 111. These data suggest that the insulin-resistant variant melanoma 111 possesses a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties, and also suggests a possible role of receptor phosphorylation in both the binding and the signaling function of the insulin receptor.

Expression of insulin receptors and insulin action in cell hybrids and cybrids.

The expression of insulin receptors and insulin action was studied in cell hybrids and cybrids produced by fusion of the BWIJ mouse hepatoma cell line with nucleated and enucleated mouse L-cells (LEA-2A) respectively. The BWIJ parent and the cybrids expressed high numbers of insulin receptors, whereas the hybrids resembled the L-cell parent with low numbers of receptors. Likewise, the hybrids resembled the LEA-2A cells with high levels of glycogen synthase, whereas the BWIJ cells and cybrids had much lower levels. Both parents, the cybrids, and the hybrids, expressed insulin stimulation of alpha-aminoisobutyric acid influx, but the dose-response curves indicated an increased insulin sensitivity in the cells with the higher receptor concentration. Insulin also stimulated 86Rb+ uptake in the hepatoma parent, hybrids and cybrids, but not in the L-cell parent. These data suggest that insulin receptors, like other hepatoma-specific properties, behave as a 'luxury function' of the hepatoma cell line and are extinguished when the hepatoma cell is fused with a less differentiated cell type. The biological activities associated with insulin action, on the other hand, are much more complex in their expression and probably the result of the interaction of multiple factors that vary in their expression in cell hybrids and cybrids.

Biological activity of rat proinsulin synthesized by Escherichia coli.

Fused proteins possessing insulin antigenic determinants are synthesized and secreted by several strains of Escherichia coli K-12 bearing plasmids containing cDNA copies of rat proinsulin mRNA. Small amounts of the secreted, fused protein from E. coli strains chi 1776 and HB101 bearing plasmid p147 were partially purified and assayed for biological activity by determining stimulation of [1-14C]glucose oxidation to 14CO2 in the rat epididymal fat pad assay. Guinea pig anti-insulin serum (GPAIS) was used to suppress glucose oxidation stimulated by insulin. After mild treatment with trypsin, fused protein stimulated 14CO2 production, and over 90% of this activity was suppressible by GPAIS. Untrypsinized fused protein demonstrated no GPAIS-suppressible 14CO2 production. Larger amounts of fused protein were obtained by transformation of strain PR13 of E. coli in which the yield of eukaryotic proteins is increased approx. 50-fold. A single intravenous injection of trypsinized fused protein from E. coli PR13 containing plasmid p287.47 resulted in a rapid decline in the plasma glucose levels of fasted mice, and the hypoglycemic effect persisted for at least 120 min.

Serially transplantable chemically induced rat islet cell tumor.

A serially transplantable, chemically induced pancreatic islet cell tumor was developed in Lewis rats. The original tumor was induced by the administration of streptozotocin and nicotinamide. It was subsequently maintained by ip or sc transplantation of tissue fragments into recipient animals. Tumors generally grew to 0.5--2.0 cm in diameter within 3--4 months of transplantation. They were well encapsulated, without gross evidence of metastasis. Peroxidase immunocytochemical staining revealed a predominance of insulin-positive cells. Somatostatin-positive cells were also present and varied widely in numbers between different tumors. In addition, small numbers of glucagon-positive cells were observed in all of the tumors. On electron microscopy, cells containing secretory granules, indistinguishable from nonneoplastic beta-cells, were most abundant. Other granulated cells were also observed, but the granule morphology was not identical to that of any of the other classically described islet cell types. Tumor extracts contained an average of 3260 micrograms insulin, 22.6 micrograms somatostatin, and 0.84 micrograms glucagon per g wet wt of tissue. Tumors caused marked, progressive hypoglycemia in recipients, with plasma glucose levels frequently falling below 30 mg/dl before death. Furthermore, the recipients' islets were markedly reduced in size due to a decreased beta-cell volume.

Continuous, clonal, insulin- and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor.

Continuous cell lines that secrete both insulin and somatostatin were established by two cooperating groups of investiagtors from a serially transplantable, radiation-induced, rat islet cell tumor. The cell lines, named RIN-r and RIN-m, were initiated from tumors maintained in inbred rats or in athymic nude mice, respectively. The cultured cells are epithelioid, free of fibroblast contamination, and can be cloned. They have a hypodiploid chromosome number, are tumorigenic, and posses amine-handling properties, including high levels of L-dopa decarboxylase and formaldehyde-induced fluorescence. Preliminary analysis of clones revealed a spectrum of insulin secretion from undetectable to relatively high. These clonal cell lines provide important systems to study the biology of insulin and somatostatin.

Artificial pancreas using living beta cells:. effects on glucose homeostasis in diabetic rats.

An artificial pancreas consisting of beta cells cultured on synthetic semipermeable hollow fibers was tested in rats with alloxan-induced diabetes. When implanted ex vivo as arteriovenous shunts in the circulatory system these devices lowered concentrations of plasma glucose from 533 to between 110 and 130 milligrams per 100 milliliters, increased concentrations of plasma insulin, and restored intravenous glucose tolerance tests essentially to normal.

A transplantable insulinoma in the rat.

A transplantable insulinoma was developed in inbred albino rats of the NEDH strain. The original tumor, 1 cm in diameter, was removed from the pancreas of a male parabiont 566 days folowing 1000 rads (10J/kg) of total body x-irradiation. The time required for implanted fragments to grow to 0.5-1.5 cm in diameter decreased from 5-8 months in the first generation to 2-5 months in the seventh generation. Successful transplantation in male animals followed for 4 or more months after transplantation was significantly greater than in female animals followed for a similar period of time (96% versus 69%). Light and electron microscopy revealed that the tumors consisted predominantly of well-granulated beta cells. Ultrastructural studies also showed small numbers of D-cells. Tumor extracts contained an average of 223 units of immunoreactive insulin and 25.9 mug of immunoreactive somato-statin per gram wet weight of tissue. Tumors generally produced increasingly profound hypoglycemia within 2-4 months following transplantation, with plasma glucose levels frequently falling to 40 mg/100 ml or lower prior to death. Removal of tumors from chronically hypoglycemic animals resulted in transient rebound hyperglycemia with plasma glucose levels above 300mg/100 ml within the first 24 hr and a gradual decline to normal levels of 129 mg/100ml in 2-4 days. These observations correlated with findings of marked atropy and degranulation of the beta cells in the pancreata of tumor-bearing animals, and with gradual return of normal light microscopic morphology following tumor removal.

Studies of insulin release and rat pancreatic islet metabolism with diastereomers of D-glucose.

Studies of insulin release with diastereomers and other analogues of D-glucose demonstrated that only sugars which undergo oxidation to CO2 stimulated insulin release by the pancreatic islet. None of the non-metabolizable diastereomers of glucose stimulated insulin release in the presence of a sub-stimulatory concentration of glucose for fuel. Although 5.5 mM glucose formed 77% as much CO2 as 16.7 mM mannose and twice that of 16.7 mM fructose, 5.5 mM glucose did not stimulate insulin release whereas 16.7 mM mannose and fructose did stimulate insulin release. These results indicate that the important stimulus for glucose-induced insulin release involves metabolism of glucose, but that the stimulus does not involve solely a fuel function of glucose.

Beta cell culture on synthetic capillaries: an artificial endocrine pancreas.

Beta cells from neonatal rats were cultured on bundles of artificial capillaries perfused with tissue culture medium. Cells continued to release insulin and remained responsive to changes in glucose concentration. The quantity of insulin released was similar to that of conventional flask cultures.

Pancreatic beta cell culture: preparation of purified monolayers.

Procedures were developed for preparing partially purified beta cell monolayer cultures. Neonatal rat pancreases were dissociated with a trypsin-collagenase solution. Beta cells were separated from denser acinar cells by centrifuging the initial suspension in a two layer discontinous Ficoll gradient (20, 25%). Resultant cultures, highly enriched in beta cells, were further purified by incubation with cystine-free medium. This caused necrosis of the majority of fibroblastoid cells within two days, while beta cells were considerably less affected. The resultant cultures contained an average of 72% beta cells compared to 10% in untreated control cultures. Insulin release by purified monolayers remained responsive to changes in the glucose concentration of the culture medium.