The effects of exogenous insulin on somatostatin secretion from the isolated perfused rat pancreas have been investigated in the presence of 5.6 mM glucose and when somatostatin secretion was stimulated by either glucose (16.7 mM) or arginine (20 mM). Insulin (15 mU/ml) significantly and rapidly suppressed glucoseand arginine-stimulated somatostatin release. However, at 5.6 mM glucose and, in the absence of other stimulators of somatostatin release, insulin had no effect on the somatostatin secretion rate. GERBER, Pietro, et al. Effect of insulin on glucoseand arginine-stimulated somatostatin secretion from the isolated perfused rat pancreas. Endocrinology, 1981, vol. 109, no. 1, p.

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This chapter contains section titled: Materials and Methods Results Discussion Summary References Discussion

The gastro-intestinal mucosa of the rat has been systematically studied with the electron-microscope at the following levels: gastric fundus, pylorus, duodenum, jejunum and ileum. Two types of non-digestive epithelial cells have been observed, both exhibiting electron-dense granulations, both candidates for endocrine activity. The first type of cells contains round granules bearing remarkable similarity to the granules of pancreatic endocrine A cells. The second cell type is characterised by strikingly polymorphic granulations and would appear to correspond to the so-called enterochromaffine cells of light-microscopy. On the basis of this morphological criterion, and of the biochemical evidence suggesting the existence of a substance in the intestine which greatly resembles glucagon, biologically and immunologically, it is proposed that this substance is secreted by the first type of cells which resemble the pancreatic A cells.

The low-dose streptozocin (STZ) model of diabetes has been reported to involve direct STZ beta-cytotoxicity and/or immunologically mediated β-cell destruction. Because the T-lymphocyte dependency of such a model is controversial, we further assessed the role of T-lymphocytes by determining the occurrence and magnitude of hyperglycemia as well as the pancreatic insulin contents in both STZ-injected nude C57BL/6J male mice and STZ-injected euthymic C57BL/6J male mice selectively depleted in helper and/or cytotoxic T-lymphocytes with monoclonal antibodies (MoAbs). The effectiveness of MoAb treatment was assessed in lymph node cells by flow-microfluorometry analysis and in spleen cells by concanavalin A stimulation, allospecific cytotoxic T-lymphocyte activity, and T-lymphocyte lymphokine production. Sixteen days after the first STZ injection, hyperglycemia (plasma glucose > 200 mg/dl) occurred in significantly fewer helper T-lymphocyte-depleted mice ( P < .005) or helper and cytotoxic T-lymphocyte–depleted mice ( P < .001) than in non–MoAb-treated mice. However, a progressive increase in the number of mice with hyperglycemia ensued in all MoAb-treated groups, and 2 mo after STZ was administered, the prevalence of hyperglycemia, mean plasma glucose levels, and pancreatic insulin contents did not differ significantly from the values obtained in the non–MoAb-treated animals. Similarly, STZ-injected C57BL/6J male nude mice developed hyperglycemia that was associated with a marked decrease in pancreatic insulin contents on a time course comparable with that of STZ-injected euthymic C57BL/6J male mice depleted in helper or in helper and cytotoxic T-lymphocytes by MoAbs. Thus, selective helper T-lymphocyte depletion, either genetic or induced by MoAb treatment, although able to temporarily protect mice from the development of hyperglycemia, did not prevent the ultimate loss of the β-cell mass initiated by the direct toxic effect of STZ.

The tissue distribution of glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was examined by protein blotting analysis. Antibodies raised against rat liver glucokinase recognized a single protein subunit with an apparent Mr of 56,500 on nitrocellulose blots of cytosol protein from liver, separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A protein of identical electrophoretic mobility was detected by immunoblotting of cytosol protein from pancreatic islets. Hepatic glucokinase and the immunoreactive islet product bound to and were eluted from DEAE-cellulose at the same ionic strength. Glucokinase was displayed as a set of two spots with apparent pI values of 5.54 and 5.64 by immunoblotting after two-dimensional gel electrophoresis. The two isoforms appeared equally abundant in liver extract, whereas the component with a pI of 5.64 was predominant in islets. By quantitative immunoblotting, glucokinase was estimated to represent 0.1% of total cytosol protein in liver and 1/20th as much in islets. The glucokinase activity of both liver and islet cytosols was suppressed by the antibodies to hepatic glucokinase. Immunoblotting of cytosol protein from intestinal mucosa, exocrine pancreas, epididymal adipose tissue, kidney, brain, and spleen failed to reveal the glucokinase protein. Thus, significant expression of the glucokinase gene appears restricted to the liver and pancreatic islets.

1. An insulin-producing cell line, RINm5F, derived from a rat insulinoma was studied. 2. The cellular content of immunoreactive insulin was 0.19 pg/cell, which represents approx. 1% of the insulin content of native rat beta-cells, whereas that of immunoreactive glucagon and somatostatin was five to six orders of magnitude less than that of native alpha- or delta-cells respectively. 3. RINm5F cells released 7-12% of their cellular immunoreactive-insulin content at 2.8 mM-glucose during 60 min in Krebs-Ringer bicarbonate buffer. 4. Glucose utilization was increased by raising glucose from 2.8 to 16.7 mM. There was, however, no stimulation of immunoreactive-insulin release even when glucose was increased from 2.8 to 33.4 mM. A small stimulation of release was, however, found when glucose was raised from 0 to 2.8 mM. 5. Glyceraldehyde stimulated the release of immunoreactive insulin in a dose-dependent manner. 6. At 20 mM, leucine or arginine stimulated release at 2.8 mM-glucose. 7. Raising intracellular cyclic AMP by glucagon or 3-isobutyl-1-methylxanthine stimulated release at 2.8 mM-glucose with no additional stimulation at 16.7 mM-glucose. 8. Stimulation of immunoreactive-insulin release by K+ was dose-related between 2 and 30 mM. Another depolarizing agent, ouabain, also stimulated release. 9. Adrenaline (epinephrine) inhibited both basal (2.8 mM-glucose) release and that stimulated by 30 mM-K+. 10. Raising Ca2+ from 1 to 3 mM stimulated immunoreactive-insulin release, whereas a decrease from 1 to 0.3 or to 0.1 mM-Ca2+ lowered the release. 11. These findings could reflect a relatively specific impairment in glucose handling by RINm5F cells, contrasting with the preserved response to other modulators of insulin release.

A B S T RA C T Calcium and cyclicAMP are important in the stimulation of insulin release. The phosphodiesterase inhibitor 3-isobutyl- l-methylxanthine (IBMX) raises islet cAMP levels and causes insulin release at nonstimulatory glucose concentrations. In isolated rat pancreatic islets maintained for 2 d in tissue culture, the effects of IBMX on insulin release and 45Ca++ fluxes were compared with those of glucose. During perifusion at 1 mM Ca++, 16.7 mM glucose elicited a biphasic insulin release, whereas 1 mM IBMX in the presence of2.8mM glucose caused a monophasic release. Decreasing extracellular Ca++ to 0.1 mM during stimulation reduced the glucose effect by 80% but did not alter IBMX-induced release. Both glucose and IBMX stimulated 45Ca++ uptake (5 min). 45Ca++ efflux from islets loaded to isotopic equilibrium (46 h) was increased by both substances. IBMX stimulation of insulin release, of 45Ca++ uptake, and of efflux were not inhibited by blockade of Ca++ uptake with verapamil, whereas glucose-induced changes are known to be inhibited. Because IBMX-induced insulin release remained unaltered at 0.1 mM calcium, it appears that cAMP-stimulated

The involvement of Ca2+ in the impaired insulin release of diabetic C57BL/KsJ-db/db mice was studied. Twenty-week-old severely hyperglycemic mice were compared to nondiabetic C57BL/KsJ mice as controls. Collagenase-isolated islets were maintained for 46 h in tissue culture allowing for equilibration at the same glucose concentration (8.3) mM). The insulin content of both types of islets was similar. In control islets preloaded during culture with 45Ca2+ glucose-induced insulin release was associated with increased 45Ca2+ effux. Islets from diabetic mice showed markedly reduced insulin response to glucose and a smaller increase in 45Ca2+ efflux. Because insulin release was strikingly potentiated by 3-isobutyl-1-methylxanthine (IBMX), even more than in control islets, there was no generalized release defect. In both types of islets, IBMX potentiation was accompanied by a further enhanced 45Ca2+ efflux, possibly suggesting that cAMP effects are associated with increased cytosol Ca2+% concentrations. As Ca2+ uptake was stimulated by glucose in both types of islets, a defect may lie in the mechanism by which glucose uses cellulr calcium to raise cytosol Ca2+ in the beta-cell of these diabetic mice.

The feasibility of infection and transformation by SV40 (simian virus 40) of primary cell cultures derived from newborn-rat pancreas was investigated. As judged by the presence of intranuclear SV40 T-antigen, exposure to the virus resulted specifically in infection and transformation of epithelioid (predominantly endocrine) cells. The transformed cells were subcultured (more than 64 passages) and cloned. Culture medium and acid/ethanol extracts of the cells did not contain detectable amounts of immunoreactive insulin after the third subculture. However, inoculation of such SV40-transformed pancreatic cells into immunodeficient rats results in tumours in which insulin production was partially restored through the passage in vivo, since the tumour cells contained and synthesized small amounts of immunoreactive insulin which co-migrated with an insulin marker on gel chromatography. Interestingly, the transformed cells maintained under tissue-culture conditions produced a protein immunologically related to insulin, soluble in aqueous buffer but insoluble in acid/ethanol. This 3000-dalton protein is too large to be a translation product of the rat preproinsulin 9S mRNA. SV40-transformed pancreatic cells might prove useful in the investigation of the factors controlling and maintaining insulin biosynthesis.