Total Protein Biosynthesis Research Articles

Functional and metabolic perturbations in isolated pancreatic islets from the GK rat, a genetic model of noninsulin-dependent diabetes

Spontaneously diabetic nonobese GK rats exhibit high basal plasma glucose and insulin levels and a poor insulin secretory response to glucose. We studied insulin biosynthesis, insulin release, and glucose metabolism in freshly isolated islets from GK rats and control Wistar rats. In GK rats, islet insulin content was decreased when expressed per islet but normal when related to DNA content. In the presence of a low concentration (2.8 mM) of glucose both (pro)insulin and total protein biosynthesis was doubled in islets from GK rats. As judged from the (pro)insulin/total protein synthesis ratio, (pro)insulin biosynthesis was normally stimulated by 16.7 mM glucose. In islets from diabetic rats both basal and glucose-stimulated insulin release were dramatically decreased. A reduced secretory response to 10 mM leucine or 10 mM leucine plus 10 mM glutamine and a lack of response to 10 mM monomethylsuccinate were observed. By contrast the insulinotropic capacity of nonnutrient secretagogues such as 62 microM gliclazide or the combination of 2 mM Ba2+ and 1.4 mM theophylline in the absence of extracellular Ca2+ remained normal. Glucose oxidation (estimated as the production of 14CO2 from D-[6-14C]glucose) was severely impaired, whereas no major alteration of glycolytic flux (as judged from the conversion of D-[5-3H]glucose to 3H2O) could be detected. Accordingly, the D-[6-14C]glucose oxidation/D-[5-3H]glucose use ratio was less markedly increased in response to a rise in glucose concentration in islets from GK rats than in islets from control rats. Thus, in islets from diabetic GK rats, glucose-induced insulin release but not insulin biosynthesis was impaired. This defect is associated with, and probably at least in part due to, a deficient oxidative metabolism of glucose in islet mitochondria.

Lack of long-term beta-cell glucotoxicity in vitro in pancreatic islets isolated from two mouse strains (C57BL/6J; C57BL/KsJ) with different sensitivities of the beta-cells to hyperglycaemia in vivo.

Previous studies have shown that 4 weeks after syngeneic transplantation of a suboptimal number of islets into either C57BL/6J (BL/6J) or C57BL/KsJ (BL/KsJ) diabetic mice there is an impaired insulin secretion by the perfused grafts. After normalization of the blood glucose level with a second islet graft, the BL/6J strain showed restored insulin secretion whilst that of the BL/KsJ strain remained impaired. The aim of the present work was to study the effects of glucose on the in-vitro function of islet beta-cells from these two mouse strains, with different sensitivities of their beta-cells to glucose in vivo. Isolated pancreatic islets from each strain were kept for 1 week in tissue culture at 5.6, 11, 28 or 56 mmol glucose/l and were subsequently analysed with regard to insulin release, (pro)-insulin and total protein biosynthesis, insulin, DNA and insulin mRNA contents and glucose metabolism. Islets from both strains cultured at 28 or 56 mmol glucose/l showed an increased accumulation of insulin in the culture medium and an enhanced glucose-stimulated insulin release compared with corresponding control islets cultured at 11 mmol glucose/l. After culture at either 5.6 or 56 mmol/l, rates of (pro)insulin biosynthesis were decreased in BL/KsJ islets in short-term incubations at 17 mmol glucose/l, whereas islets cultured at 56 mmol glucose/l showed a marked increase at 1.7 mmol glucose/l. In BL/6J islets, the (pro)insulin biosynthesis rates were similar to those of the BL/KsJ islets with one exception, namely that no decrease was observed at 56 mmol glucose/l. Islets of both strains showed a decreased insulin content after culture with 56 mmol glucose/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged exposure of human pancreatic islets to high glucose concentrations in vitro impairs the beta-cell function.

The aim of the present study was to clarify whether prolonged in vitro exposure of human pancreatic islets to high glucose concentrations impairs the function of these cells. For this purpose, islets isolated from adult cadaveric organ donors were cultured for seven days in RPMI 1640 medium supplemented with 10% fetal calf serum and containing either 5.6, 11, or 28 mM glucose. There was no glucose-induced decrease in islet DNA content or signs of morphological damage. However, islets cultured at 11 or 28 mM glucose showed a 45 or 60% decrease in insulin content, as compared to islets cultured at 5.6 mM glucose. Moreover, when such islets were submitted to a 60-min stimulation with a low (1.7 mM) followed by a high (16.7 mM) concentration of glucose, the islets cultured at 5.6 mM glucose showed a higher insulin response to glucose than those of the two other groups. Islets cultured at the two higher glucose concentrations showed increased rates of insulin release in the presence of low glucose, and a failure to enhance further the release in response to an elevated glucose level. Islets cultured at 28 mM glucose showed an absolute decrease in insulin release after stimulation with 16.7 mM glucose, as compared to islets cultured at 5.6 mM glucose. The rates of glucose oxidation, proinsulin biosynthesis, and total protein biosynthesis were similar in islets cultured at 5.6 or 11 mM glucose, but they were decreased in islets cultured at 28 mM glucose. These combined results suggest that lasting exposure to high glucose concentrations impairs the function of human pancreatic islets.

Biosynthesis of fibronectin by rabbit aorta.

The in vitro interactions between vascular cells and fibronectin have been shown to influence phenotypic expression of both cultured endothelial and smooth muscle cells. To more effectively assess the potential functional role of fibronectin in vivo in modulating vascular phenotypes, we have established methodology for studying fibronectin biosynthesis in the rabbit aorta using aortic rings that are morphologically and functionally intact and metabolically active. Aortic rings were incubated with 35S-labeled methionine in a supplemented physiological salt solution. The tissue was fractionated, and quantitative immunoprecipitation was performed using a polyclonal antibody directed against human plasma fibronectin. Newly synthesized fibronectin was most abundant in the fraction solubilized using 4% sodium dodecyl sulfate and in the incubation medium. In all fractions studied, fibronectin was present predominantly as a dimer with no detectable aggregates of fibronectin. Pulse-chase experiments showed that a substantial amount of newly synthesized fibronectin was found in the 4% sodium dodecyl sulfate extract after only 1 h, suggesting that fibronectin was rapidly incorporated into the extracellular matrix. The more soluble forms of newly synthesized fibronectin appeared to be the precursors for secreted fibronectin, and no precursor-product relationship between soluble and insoluble fibronectin was found. Dissection of aortic rings following incubation with labeled methionine showed that newly synthesized fibronectin was uniformally distributed in both intima-media and media-adventitia segments. Endothelial cell denudation caused only a 20% decrease of fibronectin biosynthesis concomitant with similar changes in total protein biosynthesis, consistent with the medial smooth muscle cell as the major source of newly synthesized fibronectin. Biosynthesis of fibronectin was increased following a 24-h preincubation of the aortic rings, and concomitant increases in steady state mRNA for fibronectin were found. These in vitro studies documented the utility of aortic rings for the general purpose of studying protein synthesis in vascular cells and provide new information on the characteristics of fibronectin biosynthesis by aortic tissue.

Propriétés antiélastasiques des propionylaminoacides

SynopsisAntielastase activity of derivatives like ‘propionylaminoacid’(C3 prolin, C3 hydroxyprolin, C3 collagen) was examined for pancreatic elastase, and fibroblastic elastase production. Essential metabolic variations of normal dermal fibroblasts were evaluated: adhesion, proliferation capacity, total protein biosynthesis and collagen type I and type III production. Possible other factors such as cellular nutrients were examined by oxygen consumption evaluation.Propionylaminoacid derivatives have antielastase activities. Pancreatic elastase showed dose related inhibition (20% to 50% inhibition for concentration from 5 to 80 mg ml−1.Moreover, fibroblastic elastase production was inhibited, cellular respiration was enhanced. A very good tolerance in vitro was observed for concentration 0–1 mg ml−1 range: adhesion, proliferation capacity and collagen (type I and type III) production were not altered, and oxygen consumption was enhanced.

Interleukin-1β Induces an Early Decrease in Insulin Release, (Pro)Insulin Biosynthesis and Insulin Mrna in Mouse Pancreatic Islets by a Mechanism Dependent on Gene Transcription and Protein Synthesis

In an attempt to further characterize the mechanisms of action of recombinant interleukin-1 beta (rIL-1 beta) on mouse pancreatic islets, islets were exposed for different periods of time (6, 12 and 24 h) to 50 U/ml rIL-1 beta. After 6 h there was already a significant decrease in glucose (16.7 mM)-induced insulin release. This was followed at 12 h by a decrease in insulin mRNA contents and (pro)insulin biosynthesis and, after 24 h, by a decrease in islet insulin contents. There was no decrease in total protein biosynthesis or DNA contents in any of the studied time points and the glucose oxidation rates were not affected by rIL-1 beta after 12 h of exposure. A similar inhibition of insulin release, (pro)insulin biosynthesis and insulin mRNA content was observed 12 h after a short (2 h) exposure of the islets to rIL-beta, suggesting that a brief exposure of mouse islets to the cytokine can modify their function for several hours. When islets were exposed for 12 h to 50 U/ml rIL-1 beta in the presence of either an inhibitor of gene transcription (actinomycin D) or an inhibitor of mRNA translation (cycloheximide) there was a complete protection against the suppressive effects of rIL-1 beta on insulin release, (pro)insulin biosynthesis and insulin mRNA contents. However, when islets were exposed for 2 h to rIL-1 beta in the presence of actinomycin D, and studied 12 h later, actinomycin counteracted the inhibitory effects of rIL-1 beta on insulin release, but not on (pro)insulin biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 beta increases the biosynthesis of the heat shock protein hsp70 and selectively decreases the biosynthesis of five proteins in rat pancreatic islets.

Prolonged exposure to high concentrations of human recombinant interleukin-1 beta (rIL-1 beta) has been reported to exert both suppressive and cytotoxic effects on pancreatic beta-cells during culture in vitro. In order to investigate the molecular mechanism(s) underlying the actions of rIL-1 beta on the beta-cell, we have exposed isolated rat pancreatic islets for 3 or for 24 h to 25 U/ml of rIL-1 beta. Subsequently the biosynthesis of heat shock proteins, as assessed by western blot analysis, and total protein biosynthesis patterns were studied, using one and two-dimensional gel electrophoresis of [35S]methionine labelled islet proteins from different subcellular compartments. It was found that rIL-1 beta exerted no specific effects on protein synthesis when added during a 3 h incubation period. However, after a 24 h incubation period, the synthesis of a group of acidic proteins with the approximate molecular weight of 35 kD was specifically inhibited in the rIL-1 beta treated islets. This alteration was predominantly associated with the endoplasmic reticulum fraction. The cytokine also inhibited the synthesis of four cytosolic proteins with the molecular weights 75, 85, 95 and 120 kD. In contrast, rIL-1 beta increased the expression of the heat shock protein hsp70 both in the microsomal and cytosolic fractions, in contrast to the islet nuclei in which no increase was found. These results show that exposure of pancreatic islets to rIL-1 beta is accompanied by specific alterations in the protein synthesis of the islet cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Short-term exposure of rat pancreatic islets to human interleukin-1β increases cellular uptake of calcium

Interleukin-1 (IL-1) may be one of the effector molecules involved in the destruction of the pancreatic islet B cells resulting in insulin-dependent diabetes mellitus. Isolated islets exposed to IL-1 show an acutely increased substrate metabolism and insulin release, which is followed by a metabolic and functional suppression. Since an increased cellular uptake of calcium in the islets may be associated with both nutrient-induced insulin release and cell damage, the effects of recombinant IL-1β (rIL-β) on net cellular calcium uptake by isolated rat pancreatic islets were investigated. In short-term experiments the islets were exposed to 25 U/ml rIL-1β for 120 min in the presence of 1.7 mM or 16.7 mM glucose, or 16.7 mM glucose plus 5 mM verapamil. In these experiments rIL-1β induced an increase both in net cellular uptake of calcium and in insulin release only in the presence of 16.7 mM glucose. The stimulatory effect of rIL-1β at 16.7 mM glucose was blocked by verapamil. By long-term experiments, under tissue culture conditions in the presence of 11.1 mM glucose, islet net calcium uptake, insulin release and glucose oxidation were measured at different time points over a 24-h period. During the first 2 h of incubation 25 U/ml rIL-1β effected a significant increase of net calcium uptake, insulin release and glucose oxidation. However, after 4 – 5 h of incubation with the cytokine no such stimulatory effects were seen. After longer incubations with rIL-1β all the islet functions studied were suppressed. To further elucidate the potential role of cellular calcium uptake via voltage-dependent channels for the long-term deleterious effects of rIL-1β on islet function, isolated islets were incubated for 24 h with 25 U/ml rIL-1β in the absence and presence of 5 mM verapamil. Except for a partial protection against a decrease in islet DNA content, there was no effect of verapamil on the suppressive action of the cytokine on islet insulin content, insulin release, glucose oxidation or proinsulin-insulin and total protein biosynthesis. The combined data suggest that rIL-1β acutely increases insulin release by an increased substrate metabolism and cellular calcium uptake via voltage-dependent channels. In the suppressive phase of rIL-1β action on the pancreatic islet B cells, calcium uptake and insulin biosynthesis and release are apparently decreased by a deprived substrate metabolism.

Amino Acids and Human Amniotic Fluid Increase DNA Biosynthesis in Pancreatic Islets of Adult Mouse, But This Effect is Lost Following Exposure to Streptozotocin

The present study describes the effects of growth hormone (GH), amino acids, and human amniotic fluid on the function and replication of normal and SZ-treated adult mouse pancreatic islets. Thus, mouse islets were exposed in vitro to SZ or vehicle only, and maintained in culture for 7 days in RPMI 1640 containing 11.1 mM glucose and the different supplements described above. Supplementation with amino acids increased the insulin accumulation in the medium, DNA biosynthesis, and polyamine contents in the control islets. In the SZ-treated islets, amino acids increased insulin accumulation in the medium and polyamine contents, but not DNA biosynthesis. Culture of control islets in the presence of 10% human amniotic fluid increased the insulin accumulation in the medium, islet insulin content, total protein and (pro)insulin biosynthesis, and DNA biosynthesis. However, in the SZ-treated islets, the effects of human amniotic fluid were limited to an increase in insulin content and insulin accumulation in the medium. GH significantly increased insulin accumulation in the medium in control, but not SZ-treated islets. In both groups of islets, GH failed to induce a significant increase in thymidine incorporation. It is concluded that amino acids and human amniotic fluid are potent stimulators of DNA biosynthesis in adult mouse pancreatic beta cells, perhaps due to an increase in cellular polyamine contents. However, following exposure to streptozotocin, the islets are not anymore responsive to these stimulators of DNA biosynthesis.

Interleukin-1β Depletes Insulin Messenger Ribonucleic Acid and Increases the Heat Shock Protein hsp70 in Mouse Pancreatic Islets Without Impairing the Glucose Metabolism*

In order to further characterize the actions of recombinant interleukin-1 beta (rIL-1 beta) on the function of insulin-producing cells, the effects of different concentrations of the cytokine were studied on islets obtained from four different mouse strains (NMRI, NOD, C57BL/6, and C57BL/Ks). For this purpose the islets were exposed to rIL-1 beta (25, 50, or 100 U/ml) for a 48-h period in medium RPMI 1640 containing 10% calf serum and 11.1 mM glucose. In all groups and at the various rIL-1 beta concentrations tested, there was a similar 30-50% inhibition in glucose-induced insulin release, a 70-80% decrease in islet insulin content, and no significant differences in islet DNA content or insulin accumulation in the culture medium. To clarify the mechanisms underlying the decreased islet insulin content, rates of (pro)insulin biosynthesis and insulin messenger RNA (mRNA) contents were determined. Exposure of NMRI and C57BL/6 islets to 50 U/ml rIL-1 beta reduced the (pro)insulin biosynthesis by 40-50% and the insulin mRNA contents by 80-90%. The cytokine also induced an increased cellular content of the heat shock protein hsp70, as measured by western blot analysis, and a decrease in DNA biosynthesis, as measured by [methyl-3H]thymidine incorporation. However, exposure to rIL-1 beta did not decrease islet total protein biosynthesis, glucose oxidation, ATP content, ATP/ADP ratio, cAMP content, or polyamine contents. In conclusion, these data suggest that exposure of mouse islets to rIL-1 beta reduces DNA synthesis, insulin mRNA levels, and the biosynthesis of (pro)insulin, without equally impairing other cellular functions. The mechanisms behind these reductions seem to be different from those observed in rat islets, where a rIL-1 beta-induced impairment of substrate metabolism at the mitochondrial level seems to be related to the decrease of several cellular functions.

Effects of a diabetogenic strain of encephalomyocarditis (EMC) virus on protein synthesis in mouse islets of Langerhans

The effects of a diabetogenic strain of encephalomyocarditis (EMC) virus on total protein and insulin biosynthesis in mouse islets of Langerhans have been studied in tissue culture. In dispersed mouse islets, the rates of protein biosynthesis were assessed by measuring the incorporation of [3H]leucine into proteins. In infected dispersed islets incubated in 20 mM-glucose, both insulin and total protein biosynthesis were decreased at 6 h; only insulin biosynthesis was significantly decreased at 3 h. In whole islets, EMC virus brought about a decrease in glucose-stimulated protein and insulin biosynthesis as early as 2 h after infection without concomitant effects on insulin release. This inhibition of protein biosynthesis was still apparent at 20 h post-infection, at which time insulin release was found to be markedly elevated, and the islet insulin content was moderately decreased. At 44 h post-infection, glucose-induced insulin biosynthesis was preferentially inhibited. Infected islets at this later time point also displayed elevated levels of insulin release, and a marked loss of islet insulin content. When insulin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were assessed by dot-blot hybridization using appropriate cDNA probes, levels of insulin mRNA were shown to decrease steadily during the first 20 h of infection, in contrast with the levels of GAPDH mRNA. At 44 h post-infection, both types of mRNA were markedly decreased. It is suggested that there is an initial early 'shut-off' of protein synthesis without other detectable changes in islet function. This is followed by a phase where both insulin mRNA levels and insulin synthesis are dramatically decreased.

Reversal of beta-cell suppression in vitro in pancreatic islets isolated from nonobese diabetic mice during the phase preceding insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is characterized by a progressive autoimmune destruction of the pancreatic beta-cells. One of the best-suited animal models for IDDM is the nonobese diabetic (NOD) mouse. In this investigation pancreatic islets were isolated from female NOD mice aged 5-7, 8-11, and 12-13 wk and examined immediately (day 0) or after 7 d of culture (day 7). The mice showed a progressive disturbance in glucose tolerance with age, and a correspondingly increased frequency of pancreatic insulitis. Islets isolated from the oldest mice often contained inflammatory cells on day 0, which resulted in an elevated islet DNA content. During culture these islets became depleted of infiltrating cells and the DNA content of the islets decreased on day 7. Islets of the eldest mice failed to respond with insulin secretion to high glucose, whereas a response was observed in the other groups. After culture all groups of islets showed a markedly improved insulin secretion. Islets from the 12-13-wk-old mice displayed a lower glucose oxidation rate at 16.7 mM glucose on day 0 compared with day 7. Islet (pro)insulin and total protein biosynthesis was essentially unaffected. In conclusion, islets obtained from 12-13-wk-old NOD mice exhibit an impaired glucose metabolism, which may explain the suppressed insulin secretion observed immediately after isolation. This inhibition of beta-cell function can be reversed in vitro. Thus, there may be a stage during development of IDDM when beta-cell destruction can be counteracted and beta-cell function restored, provided the immune aggression is arrested.

Interleukin-6 Affects Insulin Secretion and Glucose Metabolism of Rat Pancreatic Isletsin Vitro*

Recently it has been postulated that interleukin-1 (IL-1) locally released by infiltrating mononuclear cells may destroy the pancreatic B cells during the development of insulin-dependent diabetes mellitus. Since IL-1 is a potent inducer of interleukin-6 (IL-6) in various cells, it is conceivable that IL-6 is a second mediator of the IL-1 action. In the present study the effects of IL-6 alone or in combination with IL-1 were studied on pancreatic islet function in vitro after tissue culture and compared with the effects observed after exposure to IL-1 only. Rat pancreatic islets were cultured in medium RPMI 1640 + 10% calf serum with or without the addition of human recombinant IL-6 (500-5000 pg/ml) for 48 h. The medium insulin accumulation was increased by 40-50% after culture with 500-2000 pg/ml IL-6, but was similar to the controls at 5000 pg/ml. When islets were cultured for 18 h only, also 5000 pg/ml IL-6 stimulated the medium insulin accumulation. IL-6 did not affect the islet insulin content and the rates of islet (pro)insulin and total protein biosynthesis. It inconsistently decreased the islet DNA content. In short-term experiments after 48-h culture with IL-6, there was a dose-dependent inhibition of the glucose-stimulated insulin release. On the other hand, islets cultured with IL-6 (5000 pg/ml) exhibited an elevated glucose oxidation and oxygen uptake, but a lower ATP content at 16.7 mM glucose and an unaffected glucose utilization and glutamine oxidation compared to the controls. This raises the possibility that IL-6 had induced a condition with an increased energy expenditure, resulting in an enhanced mitochondrial metabolism of glucose. Islets cultured with human recombinant IL-1 beta (25 units/ml) showed a strong inhibition of the insulin accumulation in the culture medium and of glucose-stimulated insulin release and a marked decrease in the islet DNA and insulin content. A combination of IL-1 (25 U/ml) + IL-6 (1000 pg/ml) did not alter the inhibitory action of IL-1 alone. The present findings thus show that IL-6 induces a dissociation between insulin secretion and glucose oxidation in islets in vitro. This has not been observed in islets exposed to IL-1, which suggests that IL-6 does not solely mediate the inhibitory effects of IL-1 on islet function.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Enterosorption on Animal Lifespan

Experiments were performed on Wistar male rats, starting from the 28th month of age. The effect of dietary sorbent (non coated nitrogen-containing carbon administered as 10 day courses at 1 month intervals in dosage of 10 ml/kg) on lifespan and a number of biological indices were studied. Enterosorption resulted in the increase of mean and maximal lifespan by 43 and 34% respectively. Analysis of the effect of enterosorption on activity of microsomal enzymes, intensity of total RNA and protein biosynthesis, lipid metabolism, formation of free radicals etc. showed that it produced a positive influence on the functional state of the studied systems and increased the organism's adaptive capacities. Enterosorption was found to delay the rate of onset of age-related structural changes in the organs and tissues.

Tissue Culture of Human Fetal Pancreas; Effects of Nicotinamide on Insulin Production and Formation of Isletlike Cell Clusters

Human fetal pancreas (HFP) is a potential source of beta-cells for transplantation to insulin-dependent diabetic patients. We have previously described a method for tissue culture of HFP that results in the in vitro development of isletlike cell clusters (ICCs) containing a minority of insulin-positive cells. Recently we found that nicotinamide, an inhibitor of poly(ADP-ribose) synthetase, induces an increased islet cell DNA replication both in vivo and in vitro. In this study, this culture technique was used to evaluate the effects of addition of 10 mM nicotinamide on HFP explants cultured in RPMI-1640 medium plus 10% human serum. ICCs developed in 11 of 19 consecutive cultures with nicotinamide increased the yield of ICCs by 40%. Also, the insulin content of ICCs increased approximately 50% with nicotinamide supplementation, although measurements of DNA indicated an unchanged number of cells in each ICC. Neither the rates of insulin release in response to 16.7 mM glucose plus 5 mM theophylline nor the (pro)insulin or total protein biosynthesis rates were affected by nicotinamide addition. The combined results of this study suggest that nicotinamide is useful for stimulating the formation of ICCs from HFP.

Morphology, yield and functional integrity of islet-like cell clusters in tissue culture of human fetal pancreata obtained after different means of abortion.

Morphology, yield and function were studied in cultured islet-like cell clusters (ICC) from 140 human fetal pancreata obtained after abortions of different types performed at 11-23 weeks of gestation (12 by hysterotomy, 75 by mechanical dilation and extraction, and 53 induced with prostaglandin). After collagenase digestion and culture in medium supplemented with 10% human serum, up to 2000 free-floating ICC were formed from a single pancreas. Randomly scattered insulin- and glucagon-immunoreactive cells were found in the medullary part of the ICC. More than 100 ICC developed in 100% of the hysterotomies and 87% of the mechanical abortions, but in only 53% of the prostaglandin-induced abortions. Insulin and glucagon levels in the culture medium decreased rapidly during the first 7 days of culture, but then remained stable for at least 31 days. The hysterotomy-derived ICC responded to 10 mmol/l theophylline plus 20 mmol/l glucose by a 12.2 +/- 3.1 (SEM, N = 7) fold increase in insulin release, as compared with a 5.4 +/- 0.9 fold response of the prostaglandin ICC (N = 16; P less than 0.02). Despite the low proportion of B-cells, (pro)insulin biosynthesis accounted for 10% of the total protein biosynthesis in low (2 mmol/l) glucose. In conclusion, the yield and viability of the ICC were clearly better, if prostaglandin had not been used for the induction of the abortion.

Preferential Reduction of Insulin Production in Mouse Pancreatic Islets Maintained in Culture after Streptozotocin Exposure*

The ability of the pancreatic beta-cell to repair itself after a cytotoxic injury and reassume its functional activities may be a key issue in affording protection from insulin-dependent diabetes mellitus. The molecular mechanisms behind the functional responses of the beta-cell after cytotoxic damage are still largely unknown. The present study in an attempt to elucidate this issue. Mouse pancreatic islets were isolated with collagenase and, after overnight culture, exposed for 30 min at 37 C to 2.2 mM streptozotocin (SZ) or vehicle alone (controls). The islets were subsequently cultured for 6 days in medium RPMI-1640 plus 10% calf serum. After the culture they were subjected to light microscopical examinations or different functional tests during short term incubations. The SZ-treated islets showed markedly diminished insulin release after stimulation with the beta-cell nutrients glucose and leucine plus glutamine. Compounds known to increase intracellular cAMP [theophylline and (Bu)2-cAMP] were able to partially counteract the SZ-induced reduction of insulin release. Stimulation with arginine could also slightly restore the impaired insulin release. Glucose-stimulated oxygen uptake, proinsulin biosynthesis, and insulin and insulin mRNA contents were also decreased, with values at about 50% of the controls. However, the cellular contents of DNA and RNA and total protein biosynthesis rates were essentially normal. Besides mild degranulation in some islets, the morphological appearance of the SZ-treated islets did not reveal any obvious differences compared to the control islets. The present observations suggest that after a toxic injury there remains a population of partially damaged beta-cells, which are able to maintain most of their basal metabolic functions, but fail to maintain adequate insulin biosynthesis and release.

Islet cell growth and function: A reappraisal of the role of progesterone and prednisolone

The mechanism of the inhibitory effect of steroid hormones, progesterone and prednisolone on the incorporation of [ 3H]-thymidine into pancreatic islet cell DNA was investigated. Treatment with either hormone had no effect on the incorporation of 32P-orthophosphate into islet cell DNA. Both prednisolone (10 μM) and progesterone (3 μM) markedly stimulated the activity of the enzyme thymidylate synthetase of islet cells possibly leading to increased synthesis of endogenous thymidine which resulted in dilution of the [ 3H]-thymidine added to the islets in tissue culture. Prednisolone (10 μM) significantly increased both insulin biosynthesis and release, while at 5 μM it was effective in increasing only insulin release. In contrast, progesterone at the two concentrations employed did not affect insulin biosynthesis or release. The smaller doses of both hormones markedly stimulated the total protein biosynthesis.

Interleukin 1 dose-dependently affects the biosynthesis of (pro)insulin in isolated rat islets of Langerhans.

Human crude and recombinant interleukin 1 (IL-1) was found to dose- and time-dependently affect the biosynthesis of (pro)insulin in isolated rat islets of Langerhans. Incubation of rat islets with either 0.5 U/ml or 5 U/ml of crude IL-1 for 1 h had no detectable effect on (pro)insulin biosynthesis. After 24 hours of exposure 0.5 U/ml of crude or 0.6 ng/ml of recombinant IL-1 (beta) increased the (pro)insulin biosynthesis by 42% and 58%, respectively, whereas a 10-fold greater concentration of IL-1 decreased the (pro)insulin biosynthesis by 74% and 89%, respectively. The increase in (pro)insulin biosynthesis was accompanied by an increase in total protein biosynthesis indicating a nonspecific stimulatory action of low IL-1 concentrations. In contrast, high IL-1 concentrations caused a more selective decrease of the (pro) insulin biosynthesis when compared to the total protein biosynthesis. In addition, low IL-1 concentrations were found to increase and high concentrations to decrease the relative levels of pre-proinsulin mRNA suggesting that IL-1 may act both at a pre- and post-translational level of insulin biosynthesis.

Stimulation of pancreatic secretory process in the rat by low-molecular weight proteinase inhibitor

Oral application of a single dose of a new synthetic proteinase inhibitor Camostate (Foy-305) in male Wistar rats was carried out together with studies of in vitro amino acid incorporation followed by separation of proteins by two-dimensional gel electrophoresis. The aim of this experiment was to analyze changes produced by the inhibitor in total protein and individual enzyme biosynthesis. Administration of 100 mg/kg Foy-305 resulted in significant inhibition of total pancreatic protein synthesis, without changes in fractional rates for individual enzymes. 50 mg/kg Foy-305 induced a 10-fold elevation of cholecystokinin (CCK) levels in serum; this persisted for 3 h and led to a significant increase in the total rate of protein synthesis with peak values at 6 and 9 h (78% and 84% above control levels, respectively), returning to control by 15 h. Changes in fractional rates of synthesis occurred with a latency of 6 h and were restricted to amylase and the anionic form of trypsinogen and chymotrypsinogen. Amylase biosynthesis decreased by about 40% from control levels at 9 h to return to control levels by 15 h. Increased synthesis of trypsinogen and chymotrypsinogen was observed; this was also phasic. The results show similar enzyme-specific regulation as previously described for exogenous CCK stimulation and for the adaptation of the pancreas to diets enriched in protein. They demonstrate the effectiveness of pulsatory endogenous hormone release in the regulation of protein synthesis.