Human interleukin-1β induced stimulation of insulin release from rat pancreatic islets is accompanied by an increase in mitochondrial oxidative events

Summary Acute exposure of pancreatic islets to interleukinl-1 results in an increase in insulin release, while an extension of the exposure time induces a functional suppression and eventually, destruction of the B-cells. We have recently suggested that the interleukin-1 induced inhibition of islet function is mediated through an impairment in oxidative metabolism. The aim of the current study was to investigate if the acute, stimulatory effects of interleukin-1 on islet function could also be related to changes in the substrate metabolism. For this purpose, rat islets were exposed for 90–120 min to 30 pmol/l human recombinant interleukin-1 (biological activity of 2.5 U/ml) and their function and metabolism characterized during this period. The cytokine did not increase insulin release in the presence of 1.7 or 5.5 mmol/l glucose but in both the presence of 16.7 mmol/l glucose or 10 mmol/l leucine + 2 mmol/l glutamine there was a 50% increase in insulin release. Interleukin-1 exposure increased the oxidation of D-[U-¹⁴C]glucose at 5.5 mmol/l glucose by 25% and at 16.7 mmol/l glucose by 60%. Carbohydrate and amino acid metabolism were further examined in the presence of D-[5-³H] glucose, D-[6-¹⁴C]glucose, [1-¹⁴C]pyruvate, L-[U-¹⁴C]glutamine, L-[U-¹⁴C]leucine and L-[1-¹⁴C]leucine. There was no difference between control islets and interleukin-1 exposed islets in terms of D-[5-³H]glucose utilization or [1-¹⁴C]pyruvate decarboxylation, but the oxidation of D-[6-¹⁴C]glucose was increased by 64% in the interleukin-1 exposed islets. There was also an interleukin-1 induced 45–60% increase in the decarboxylation of L-[1-¹⁴C]leucine and oxidation of L-[U-¹⁴C]leucine and L-[U-¹⁴C]glutamine, all intramitochrondrial events. The stimulation of insulin release by interleukin-1 in the presence of 16.7 mmol/l glucose was abolished in islets incubated in Ca²⁺ depleted medium, but the rate of D-[6-¹⁴C] glucose oxidation remained elevated (47% increase at 16.7 mmol/l glucose). These data indicate an increase in substrate metabolism at the mitochondrial level during acute exposure of rat pancreatic islets to interleukin-1. The increase in oxidative events can explain the observed interleukin-1 induced increase in insulin release during glucose stimulation. Furthermore, these findings raise the possibility that mitochondria are primary targets of interleukin-1 action in the B-cells.     

Different effects of glucose and glyburide on insulin secretion in rat pancreatic islets pre-exposed to interleukin-1β. Possible involvement of K+ and Ca2+ channels

Summary In vitro islet exposure to interleukin 1 inhibits the beta-cell response to glucose. We have studied whether a similar inhibition also occurs in response to the sulphonylurea glyburide. Rat pancreatic islets were cultured for 24 h in the presence or absence of 50 U/ml interleukin 1 and then stimulated with either glucose or glyburide for 1 h at 37 °C. In control islets basal insulin secretion was 117±32 pg · islet^–1 · h^–1 (mean ± SEM, n=7) and greatly increased in response to 16.7 mmol/l glucose (2140±293) or 10 mol/l glyburide (1464±234). When islets were pre-exposed to interleukin 1, insulin release was significantly reduced in response to glucose (323±80, p<0.001) but not in response to glyburide (1316±185). Since both glucose and glyburide influence beta-cell K⁺ and Ca²⁺ efflux, to further investigate this different response in islets exposed to interleukin 1 we measured both Rb⁺ efflux (as index of the ATP-sensitive K⁺ channel activity) and Ca²⁺ uptake. In control islets, the increased insulin secretion in response to 16.7 mmol/l glucose or 10 mol/l glyburide was associated with a reduction of ⁸⁶Rb efflux (decrement of –50±1.2 % and –49±2.3 %, respectively, mean ± SEM, n=5). In contrast, in interleukin 1pre-exposed islets both glucose and glyburide stimulation only slightly modified ⁸⁶Rb efflux (decrement of –19±1.9% and –5.3±3.1 %, respectively, n=5, p<0.001). ⁴⁵Ca²⁺ uptake in control islets was 2.6±0.4 pmol · islet^–1 · 20 min^–1 under basal conditions (at 2.8 mmol/l glucose), and increased to 16.8±3.2 and 10.7±2.1 pmol · islet^–1 · 20 min^–1 in islets stimulated with 16.7 mmol/l glucose or 10 mol/l glyburide, respectively (mean ± SEM, n=6). ⁴⁵Ca²⁺ uptake in interleukin 1 treated islets was higher than in control islets under basal conditions (4.6±0.6 pmol · islet^–1 · 20 min^–1 at 2.8 mmol/l glucose, p<0.05), but was significantly reduced in response to glucose 16.7 mmol/l (7.1±1.1, p<0.01 with respect to control islets). In contrast to glucose, 10 mol/l glyburide was able to stimulate calcium uptake in interleukin 1 treated islets in a similar way to control islets (12.8±2.5). The present data demonstrate that rat pancreatic islets treated with interleukin 1 for 24 h lose their responsivity to glucose, but not to glyburide. The difference between the two secretagogues is associated with the persistent ability of glyburide to influence Ca²⁺ uptake even in islets with impaired K⁺-channel function.     

B cell adrenoceptors and sulphonylurea-induced insulin release in mouse islets

Summary Interactions of tolbutamide and glibenclamide with B cell adrenoceptors have been reported. This study evaluated the possible role of such interactions in the stimulation of insulin release. Mouse islets were incubated in the presence of 10 mmol/l glucose alone or with tolbutamide (10 mol/l) or glibenclamide (0.02 mol/l). At 0.01–10 mol/l, blockers of ₂-adrenoceptors (yohimbine, idazoxan) or ₁-adrenoceptors (prazosin) had practically no effect on glucose-induced insulin release and did not affect its potentiation by sulphonylureas, except for a slight increase by 10 mol/l prazosin and idazoxan. Nonspecific -blockers (phentolamine, dihydroergotamine) increased control release at 10 mol/l, but only the latter amplified the response to tolbutamide. Blockers of -adrenoceptors were tested at 0.1–100 mol/l: propranolol (₁, ₂), metoprolol (₁) and compound ICI 118-551 (₂). They increased glucose-induced insulin release at 100 mol/l but variably altered the effect of sulphonylureas. Blockers of adrenoceptors have, thus, no effect on insulin release in vitro at therapeutic concentrations. At high concentrations, they non-specifically affect the action of sulphonylureas. We conclude that an interaction with B cell adrenoceptors is not involved in the insulinotropic action of sulphonylureas.     

Palmitate dependence of insulin secretion, “de novo” phospholipid synthesis and 45Ca2+-turnover in glucose stimulated rat islets

Summary Palmitate ability to modify D-[U-¹⁴C]glucose incorporation into different lipids (de novo synthesis), as well as sugar-stimulation of insulin release and ⁴⁵Ca²⁺-fluxes, was investigated in islets of fed and 48-h starved rats. The fatty-acid induced dose-dependent, correlative increments of insulin secretion, ⁴⁵Ca²⁺-influx and the de novo synthesis of each phospholipid fraction analysed at 20 mmol/l (but not 3 mmol/l) glucose. Omission of calcium reduced drastically (p<0.001) insulin release and the de novo synthesis of neutral glycerolipids, leaving unaltered that of acidic phospholipids (phosphatidate and phosphoinositides). The increased synthesis of the latter is therefore not the consequence of stimulated secretion. It could initiate or contribute to maintain an increased turnover of islet phosphoinositides, thus generating some mediators of the calcium signalling system (inositol phosphates). Starvation led to a drastic reduction (p<0.001) of both insulin secretion, de novo synthesis of each lipid fraction, and ⁴⁵Ca²⁺-influx in response to glucose and palmitate. The presence of a fatty-acid oxidation inhibitor (2-bromostearate or 2-tetradecylglycidate) prevented the effect of starvation on ⁴⁵Ca²⁺-influx, as it has been shown to do on insulin secretion and palmitate incorporation into islet lipids. It is finally suggested that palmitate might amplify the insulin secretory response of islets to glucose, through the stimulation of the de novo synthesis of phosphoinositides and the subsequent generation of inositol phosphates, which would contribute to accelerated calcium turnover.     

High glucose and hydrogen peroxide increase c-Myc and haeme-oxygenase 1 mRNA levels in rat pancreatic islets without activating NFκB

Aims/hypothesis Hyperglycaemia and the pro-inflammatory cytokine IL-1 induce similar alterations of beta cell gene expression, including up-regulation of c-Myc and haeme-oxygenase 1. These effects of hyperglycaemia may result from nuclear factor-kappa B (NFB) activation by oxidative stress. To test this hypothesis, we compared the effects of IL-1, high glucose, and hydrogen peroxide, on NFB DNA binding activity and target gene mRNA levels in cultured rat islets.Methods Rat islets were pre-cultured for 1 week in serum-free RPMI medium containing 10 mmol/l glucose, and further cultured in glucose concentrations of 5–30 mmol/l plus various test substances. Islet NFB activity was measured by ELISA and gene mRNA expression was measured by RT-PCR.Results IL-1 consistently increased islet NFB activity and c-Myc, haeme-oxygenase 1, inducible nitric oxide synthase (iNOS), Fas, and inhibitor of NFB alpha (IB) mRNA levels. In comparison, 1- to 7-day culture in 30 mmol/l instead of 10 mmol/l glucose stimulated islet c-Myc and haeme-oxygenase 1 expression without affecting NFB activity or iNOS and IB mRNA levels. Fas mRNA levels only increased after 1 week in 30 mmol/l glucose. Overnight exposure to hydrogen peroxide mimicked the effects of 30 mmol/l glucose on haeme-oxygenase 1 and c-Myc mRNA levels without activating NFB. On the other hand, the antioxidant N-acetyl-l-cysteine inhibited the stimulation of haeme-oxygenase 1 and c-Myc expression by 30 mmol/l glucose and/or hydrogen peroxide.Conclusions/interpretation In contrast to IL-1, high glucose and hydrogen peroxide do not activate NFB in cultured rat islets. It is suggested that the stimulation of islet c-Myc and haeme-oxygenase 1 expression by 30 mmol/l glucose results from activation of a distinct, probably oxidative-stress-dependent signalling pathway.     

Decreased cyclic AMP and Insulin responses to glucose in pancreatic islets of diabetic chinese hamsters

Summary The dose as well as the time kinetics of insulin and adenosine-3,5-monophosphate (cyclic AMP) responses to glucose were compared in pancreatic islets isolated from normal and diabetic Chinese hamsters. The insulin content in diabetic islets was about one-half that in normal islets. Insulin release in diabetic islets incubated for 10 min with glucose 60–1000 mg/l00 ml was from one-third to one-half that in normal islets. Glucose 1000 mg/l00 ml stimulated three-fold increases in insulin release without increasing the accumulation of [³H] cyclic AMP in either normal or diabetic islets prelabelled with [³H] adenine. However, in the presence of 1.0 mM of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), glucose 150 mg/l00 ml elicited significant increases of insulin release (+ 134%) and [³H] cyclic AMP accumulation in islets (+ 44%) and incubation medium (+ 48%) of islets of normal but not diabetic hamsters. Also, in perifusion experiments with 0.1 mM IBMX, glucose 500 mg/l00 ml produced threefold greater increases in insulin release and two-fold greater increases in efflux of cyclic AMP in normal than diabetic islets. By contrast with the lesser effects of glucose in diabetic islets, 1.0 mM IBMX increased islet and medium cyclic AMP, as well as insulin release, similarly in normal and diabetic islets. It is suggested that the impairment of glucose induced insulin release in islets of the diabetic Chinese hamster may be due to a defective interaction of glucose with the adenylate cyclase-cyclic AMP system in the pancreatic B cell.This work was presented in part at a meeting of the European Association for the Study of Diabetes, Sept. 1975, Munich, Germanyon leave from the Department of Endocrinology, Karolinska Hospital, Stockholm     

Heat shock protein induction in rat pancreatic islets by recombinant human interleukin 1β

Summary Interleukin 1, potentiated by tumour necrosis factor , is cytotoxic to pancreatic Beta cells in vitro. We have hypothesized that interleukin 1 induces oxygen free radicals in Beta cells. Since cytotoxicity induced by free radicals and by heat may activate the same cellular repair mechanism (the heat shock response), the aim of this study was to investigate the pattern of protein synthesis in isolated islets after exposure to interleukin 1 (150 pg/ml, 24 h), tumour necrosis factor (50 ng/ml, 24 h), heat shock (43°C, 30 min) and H₂O₂ (0.1 mmol/l, 20 min). By polyacrylamide gel electrophoresis, autoradiography, Western-blot analysis and partial peptide mapping of ³⁵S-methionine labelled islets, interleukin 1 was found to induce a 73 kilodalton protein belonging to the heat shock protein family heat shock protein 70, a heat shock protein 90, and haem oxygenase. A minor induction of heat shock protein 73 and haem oxygenase was seen after H₂O₂. Interleukin 1 did not induce heat shock proteins in rat thyroid cells, rat mesangial cells or in human monocytes. Tumour necrosis factor did not induce selective protein synthesis. Pre-exposure of islets to heat, tumour necrosis factor , or H₂O₂ did not prevent the impairment of glucose-stimulated insulin release seen after 24 h of interleukin 1 exposure. The data are compatible with free radical induction by interleukin 1. However, the heat shock response is not specific for oxidative injury, and previous studies have shown discrepant effects as to a protective effect of free radical scavengers against interleukin 1-mediated beta-cytotoxicity. Thus, a role for free radicals in this context is not definitely proven.     

α2-Adrenergic stimulation counteracts glucose-induced rise of sodium in pancreatic islets exposed to ouabain

The effects of ₂-adrenergenic activation by clonidine on sodium handling were analysed in beta-cell-rich pancreatic mouse islets. In the steady-state situation, clonidine (1 M) amplified lowering of sodium induced by 20 mM glucose, while the content remained unchanged in 3 mM glucose. The loss of sodium in Na⁺-deficient medium was stimulated by glucose but was not affected by clonidine. This agonist also did not influence the ouabain-induced uptake of sodium at 3 mM glucose but partially counteracted additional uptake in response to 20 mM glucose. Although lacking effects of its own, 5 M yohimbine completely counteracted the action of clonidine. The glucose amplification of the ouabain-induced uptake of sodium was suppressed also by 10 M of the Ca²⁺-channel blockers methoxyverapamil and diltiazem. Both tolbutamide (100 M) and dibutyryl cyclic AMP (1 mM) mimicked the action of glucose by promoting clonidine-sensitive uptake of sodium in the presence of ouabain. It is concluded that activation of ₂-adrenoceptors has profound effects on the sodium handling of pancreatic beta-cells exposed to glucose and other stimulators of insulin release.     

The effect of monooleoylglycerol on insulin secretion from isolated perifused rat islets

Summary The effect of monooleoylglycerol on cholecystokinin-and tolbutamide-induced insulin secretion was examined in isolated perifused rat islets. In the presence of 5.5 mmol/l glucose, addition of 10 nmol/l cholecystokinin or 50 mol/l tolbutamide had practically no effect on insulin secretion. Combined tolbutamide and cholecystokinin led to a biphasic insulin secretory response which was significantly enhanced by addition of 50 mol/l monooleoylglycerol, an inhibitor of diacylglycerol kinase. Monooleoylglycerol (50 mol/l) alone had a minimal stimulatory effect on insulin release in the presence of 5.5 mmol/l glucose. Perifusion of islets with 1 mol/l forskolin had no significant effect on basal insulin secretion in the presence of 5.5 mmol/l glucose, but markedly enhanced the responses to both cholecystokinin plus tolbutamide, and to the combination of cholecystokinin, tolbutamide and monooleoylglycerol. Lowering the glucose level to 2.75 mmol/l abolished the profound stimulatory effect to these agonist combinations on insulin release. Finally, monooleoylglycerol also enhanced the first and second phase insulin secretory responses induced by 20 mmol/l glucose. These results are discussed in relationship to the possible role of protein kinase C in mediating insulin secretion.     

Short-term inhibition of peroxisome proliferator-activated receptor-γ coactivator-1α expression reverses diet-induced diabetes mellitus and hepatic steatosis in mice

Aims/hypothesis The coactivator of nuclear receptors, peroxisome proliferator-activated receptor- coactivator-1 (PGC-1) has been implicated in a series of events that contribute to the control of glucose metabolism. We have recently reported the use of a PGC-1 antisense oligonucleotide (PGC-1AS) that inhibits up to 60% of PGC-1 expression in pancreatic islets, leading to increased insulin secretion. This oligonucleotide was used in this study to try to ameliorate diet-induced type 2 diabetes in a genetically predisposed mouse strain (Swiss mice).Materials and methods Glucose and insulin tolerance tests, euglycaemic–hyperinsulinaemic clamp, immunoprecipitation assays, immunoblotting assays and immunohistochemistry were used in this investigation.Results Swiss mice became obese and overtly diabetic after 8 weeks of feeding with chow containing 24% saturated fat. One daily dose (1.0 nmol) of PGC-1AS significantly reduced glucose and increased insulin blood levels without affecting food intake and body weight. These effects were accompanied by a reduced area under the glucose curve during an intraperitoneal glucose tolerance test, an increased constant of glucose decay (K_itt) during an insulin tolerance test, and an increased glucose consumption rate during a euglycaemic–hyperinsulinaemic clamp. Moreover, mice treated with PGC-1AS presented an outstanding reduction of macroscopic and microscopic features of hepatic steatosis. These effects were accompanied by reduced expression or function of a series of proteins involved in lipogenesis.Conclusions/interpretation PGC-1 is an attractive target for pharmacological therapeutics in type 2 diabetes mellitus and diet-induced hepatic steatosis.     

Interactive influences of peroxisome proliferator-activated receptor α activation and glucocorticoids on pancreatic beta cell compensation in insulin resistance induced by dietary saturated fat in the rat

Aims/hypothesis We sought to elucidate whether excess glucocorticoids and increased dietary lipids act synergistically to impair glucose tolerance and, if so, whether activation of peroxisome proliferator-activated receptor (PPAR) has an adverse or beneficial effect on glucose tolerance.Methods Dexamethasone (100 g kg^–1 body weight day^–1; 5 days) was administered to insulin-resistant rats fed a high-saturated-fat (HF) diet for 4weeks. The PPAR agonist WY14643 was administered (50 mg kg^–1 body weight intraperitoneally) 24 h before sampling. Glucose-stimulated insulin secretion (GSIS) was assessed in vivo after an acute glucose bolus injection, and in vitro using step-up and step-down islet perifusions.Results Although neither PPAR activation nor dexamethasone alone affected fasting glycaemia in the HF group, dexamethasone in combination with PPAR activation elicited marked postabsorptive hyperglycaemia. Dexamethasone treatment of HF rats had little effect on GSIS after an acute glucose challenge in vivo, but induced glucose intolerance. PPAR activation augmented GSIS in dexamethasone-treated HF rats in vivo, restoring glucose tolerance. Contrasting with data obtained in vivo, greatly enhanced peak rates of GSIS were observed ex vivo in perifusions of islets from dexamethasone-treated HF rats compared with those from untreated HF rats, an effect attenuated by antecedent PPAR activation.Conclusions/interpretation The study demonstrates that glucocorticoid excess precipitates the development of glucose intolerance in rats maintained on a high-saturated-fat diet. It does this by interrupting the negative feedback loop between insulin sensitivity and secretion in vivo, such that further enhancement of compensatory insulin secretion is not possible. PPAR activation restores the coupling between insulin secretion and action.     

Diminished inhibitory effect of noradrenaline on insulin release from mouse islets transplanted to kidney

Insulin release is inhibited by adrenergic alpha-2 agonism in normal beta-cells. To test whether the inhibitory response to noradrenaline is modified by transplantation, we studied insulin release from freshly isolated islets and from syngeneic islets transplanted under the kidney capsule of non-diabetic C57BL/6 mice. When perifused in vitro, fresh islets, as well as grafts harvested 1 or 3 weeks after transplantation, reacted to 2.5 mol/l noradrenaline with a complete inhibition of insulin release induced by 16.7 mmol/ld-glucose. In contrast, islet grafts harvested after 6, 12, or 21 weeks exhibited a conspicuous insulin secretory response to 16.7 mmol/l glucose in the presence of 2.5 mol/l noradrenaline. Also a concentration of 0.25 mol/l, noradrenaline inhibited the glucose-induced insulin release from fresh islets but not from 6-week-old islet grafts. It is concluded that transplantation under the kidney capsule induces a decreased inhibitory responsiveness to noradrenaline in islet grafts.     

Prolonged exposure of pancreatic islets isolated from “pre-diabetic” non-obese diabetic mice to a high glucose concentration does not impair Beta-cell function

Summary In the early stages of Type 1 (insulin-dependent) diabetes mellitus patients present a deficient insulin response to glucose. The reasons for this defective response are unknown, but it has been suggested that it reflects a deleterious effect of excessive glucose stimulation on a reduced Beta-cell mass. Female non-obese diabetic (NOD) mice from our colony, at the age of 12–13 weeks, have a normal basal glycaemia but an impaired intravenous glucose tolerance test, insulitis and a defective insulin response to glucose. In order to characterize the potential effect of glucose on the Beta cells at that pre-diabetic stage, pancreatic islets were isolated from 12–13 week old female NOD mice. Immediately after isolation (day 0) the NOD islets displayed a defective insulin response to an acute stimulation with 16.7 mmol/l glucose. After seven days in culture at both 11 and 28 mmol/l glucose these islets showed an increased insulin release in response to an acute glucose stimulation. This increase was more pronounced in the islets cultured at 28 mmol/l glucose. Experiments performed in parallel, using islets obtained from a non-diabetes prone strain of mice (Naval Medical Research Institute, NMRI) showed that these islets had a similar insulin release in response to glucose both on day 0 and after seven days in culture at 11 mmol/l glucose. The insulin mRNA levels of NOD islets did not change over one week in culture at 11 or 28 mmol/l glucose, but culture at the high glucose concentration induced a decrease in the islet insulin content. The present data show that culture at high glucose concentrations does not impair the function of islets isolated from NOD mice. These observations make excessive glucose stimulation, as a single factor, an unlikely explanation for the defective insulin release observed in NOD islets in the prediabetic period.     

Functional and morphological effects of interleukin-1β on the perfused rat pancreas

Summary We recently reported a potentiating effect of recombinant human interleukin-1 on glucose-stimulated insulin release from the isolated perfused pancreas. With the aim of determining whether the stimulatory effect of recombinant interleukin-1 on the B cell in the intact gland was modulated by varying the concentration, time of exposure to recombinant interleukin-1 or B-cell activity, and to elucidate a possible mechanism of action, we measured in the perfused rat pancreas the release of insulin, glucagon and/or prostaglandin E₂ according to the following three different protocols: (1) perfusion with 20 ng/ml of recombinant interleukin-1 for 92 min at 5 and 20 mmol/1 D-glucose (2) perfusion with varying concentrations of recombinant interleukin-1 ranging from 0.1×10^–3 ng/ml to 100 ng/ml at 5 and 20 mmol/l D-glucose (3) perfusion with 20 ng/ml of recombinant interleukin-1 at 5,11 or 20 mmol/l D-glucose. Furthermore, in a separate set of experiments we examined the influence of the cytokine on the morphology of the endocrine pancreas. Interleukin-1 stimulated insulin secretion at 11 and 20 mmol/l D-glucose and potentiated first as well as second phase insulin release in a dose-dependent fashion, with decreasing effect at higher concentrations. Glucagon secretion was also stimulated by recombinant interleukin-1, irrespective of increasing glucose (5, 11, 20 mmol/l) and insulin concentrations. The potentiating effect of recombinant interleukin-1 on insulin secretion was evident even after discontinued perfusion with the cytokine, suggesting a priming effect on B-cell function. Furthermore, we did not observe any relation between the recombinant interleukin-1 mediated insulin and glucagon release and prostaglandin E₂. Electron microscopy of the pancreata perfused with recombinant interleukin-1 revealed significant B cell and to a lesser extent A-cell lysis as well as induction of cell protrusions (blebs) in B cells only, accompanied by peripheral degranulation and rearrangement of rough endoplasmatic reticulum. We suggest that in addition to a paracrine effect of locally produced interleukin-1 systemic interleukin-1 may have an endocrine effect on A- and B-cell function and viability. Interleukin-1 should be considered to be a physiological modulator of insulin and glucagon secretion e.g. during the acute phase response, but also as a pathogenetic factor in Type 1 (insulin-dependent) diabetes mellitus.     

A possible role of plasma glutathione in glucose-mediated insulin secretion: in vitro and in vivo studies in rats

Summary In isolated rat pancreatic islets exogenous glutathione which is not able to penetrate into cells, augmented glucose (11.1 mmol/l)-mediated insulin release. In the presence of a non-stimulatory glucose concentration (2.8 mmol/l) glutathione had no effect. The half-maximal synergistic action of glutathione on insulin secretion was observed at approximately 8.0 mol/l. This concentration of glutathione is similar to that found in the plasma of non-fasted anaesthetised rats (6.5 mol/l). Oral ingestion of glucose increased the arterial plasma glutathione in rats. Our data provide for the first time indirect evidence for a modulating effect of plasma glutathione in postprandial glucose-mediated insulin secretion which appears to be located at the extracellular site of islet cells.     

Immunolocalization of Transforming Growth Factor-α and Epidermal Growth Factor Receptor in the Rat Gastroduodenal Area

The maintenance of gastrointestinal epitheliumintegrity requires a fine balance between proliferationand differentiation as well as protection againstgastric acid secretion. Transforming growthfactor- (TGF-) regulates these functions bybinding to epidermal growth factor receptor (EGF-R).This study was designed to identify the localization ofTGF- and EGF-R in the rat gastroduodenal region. In the stomach, the surface and gastric pitcells showed staining for TGF- antibodies in thecytoplasm and basolateral and apical membranes.TGF- and EGF-R were observed in the supranuclearregion of the cells lining the gland. In the duodenum,the enterocytes coexpressed both TGF- and EGF-Rin the supranuclear area. The EGF-R was also observed inthe apical membrane. Brunner's glands were positive for both TGF- and EGF-R antibodies. Ourresults demonstrate the coexpression of TGF- andEGF-R in the rat gastroduodenal area, which suggests afunctional role for them in the establishment and maintenance of the epithelialrenewal.     

Effect of a hypoglycaemic agent M&B 39890A on glucagon secretion in isolated rat islets of Langerhans

Summary Administration of the compound M&B 39890A lowered serum glucose levels significantly (p<0.001) in genetically obese mice, while no effect on serum insulin levels was observed. In in vitro experiments with isolated rat islets of Langerhans M&B 39890A inhibited arginine-stimulated glucagon release at all concentrations tested (0.5, 5.0 and 50 mol/l). Insulin secretion was not inhibited by M&B 39890A (0.5 and 5.0 mol/l), but was slightly decreased at 50 mol/l. M&B 39890A (5 mol/l) also inhibited glucagon secretion in vitro in the presence of 2 mmol/l, 6 mmol/l and 20 mmol/l glucose, while exerting no effect on insulin secretion. These results suggest that the hypoglycaemic action of M&B 39890A may be due to its direct and selective effect on glucagon secretion; this appears to operate by a mechanism different to that of glucose.     

The effects of parabiosis on serum and kidney glycosidase activities in spontaneously diabetic mice

Summary Spontaneously diabetic non-obese mice of the ICR strain were newly inbred in Shionogi laboratory, Japan. Animals became diabetic suddenly, more frequently and severely in females. Blood glucose levels were 452±73 mg/100 ml with serum insulin levels of < 1.0 U/ml in the fed state. Parabiosis with normal control ICR mice for 2 weeks decreased the blood glucose level to 260±51 mg/ 100ml (P<0.01) and resulted in serum insulin levels of 46.0±18.0 U/ml (P<0.01). Kidney homogenate -N-acetylglucosaminidase and -galactosidase activities were reduced in diabetic mice (42% and 44% decrease respectively) (P<0.025 and P<0.001), and restored almost to normal after 2 weeks of parabiosis. Renal -mannosidase activity was decreased 43% (P<0.001) in the diabetic mice but unaffected by parabiosis. Serum -N-acetylglucosaminidase, -galactosidase and -glucosidase activities were significantly increased in diabetic mice (179%; 233% and 58% increase respectively) (P<0.005, P<0.001 and P<0.001), and returned to normal with parabiosis.     

Effects of an enkephalin analogue (DAMME) on insulin release from cultured rat islets of langerhans

Summary Rat islets of Langerhans were maintained for 2 days in tissue culture. Following the culture period, the insulin secretory responses of the islets on incubation in bicarbonate medium were measured. The enkephalin analogue D-ala², MePhe⁴, Met(0)-ol (DAMME), 8.3×10^-8mol/l, augmented insulin release stimulated by glucose (5 or 7 mmol/l) by 76% and 47% respectively; increased insulin release stimulated by -ketoisocaproate (7.5 mmol/l) by 23%; and enhanced insulin release in the presence of glibenclamide (10 g/ml) plus glucose (3.3 mmol/l) by 38%. Insulin release in the presence of glucose at 2 or 12 mmol/l was not affected by DAMME (8.3×10^-8mol/l). The potentiatory effect of DAMME on insulin release in the presence of glucose (5 mmol/l) was blocked by naloxone (11 mol/l): naloxone alone did not affect glucose-stimulated insulin release. A high concentration (3.3×10^-6mol/l) of DAMME did not modify glucose-stimulated insulin release. Inhibition of glucose-stimulated insulin release by trifluoperazine, an inhibitor of calmodulin, was not overcome by DAMME. Insulin secretory responses were not enhanced by exposure of the islets to DAMME (8.3×10^-8mol/l) during the culture period. It is concluded that insulin release from isolated islets is capable of being influenced by an opioid peptide.     

γ/δ Receptor-expressing T-cell clones from a cutaneous T-cell lymphoma suppress hematopoiesis

Summary Recently we described a cutaneous T-cell lymphoma expressing the / T-cell receptor [5]. The patient suffering from this lymphoma showed low numbers of myeloid and T cells in peripheral blood, while B and NK cells were relatively increased. In vitro culture of the patient's bone marrow (BM) cells revealed a significant suppression of myeloid/monocyte colony formation (GM-CFU) compared with normal controls. This was not due to infiltration of the BM with lymphoma cells. We speculated that a soluble factor either secreted or induced by the lymphoma cells might be responsible for the marked suppression of hematopoiesis in this patient. From a skin biopsy with infiltrating / T-lymphoma cells we established T-cell clones bearing the / T-cell receptor and resembling the phenotype of the lymphoma cells. The supernatant (SN) of these / T-cell clones reduced the number of colonies in a CFU-GM assay (using normal control BM) in comparison to SN of / T-cell clones established from the same biopsy. This suppression was seen mainly on day 7 of culture and was not neutralized by the addition of placenta-CM. The main mediator of this suppression seems to be IFN-,since it was detectable in high amounts in the SN of these / T-cell tumor clones as well as in the serum of the patient. In addition, anti-IFN- antibodies can reverse the T-cell SN-mediated suppression of CFU-GM. We conclude that high serum levels of interferon-, which is secreted in high amounts from / T-cells grown from a biopsy of a cutaneous lymphoma, can suppress hematopoiesis.Abbreviations TCR T-cell receptor - IFN- interferon- - SN supernatant - placenta CM placenta conditioned medium - BM bone marrow - CFU-GM myeloid/monocyte colony formation - NK cells natural killer cells - Ab antibody M. Wilhelm was supported by theDeutsche Forschungsgemeinschaft (DFG Wi 728-2)