Endothelin-1 stimulates insulin secretion by direct action on the islets of Langerhans in mice

Summary Endothelin-1 (ET-1), a potent endothelium-derived vasoconstrictor peptide, is secreted in response to insulin. Elevated circulating ET-1 levels have been found in patients with diabetes mellitus and vascular dysfunction. The question arises whether ET-1 acts as a direct modulator of insulin secretion. To test this, we studied the effects of ET-1 on isolated mouse islets of Langerhans. ET-1 (1 nmol/l-1 mol/l) dose-dependently stimulated insulin secretion from islets incubated in the presence of 16.7 mmol/l glucose (p<0.05). The effect of ET-1 is glucose-dependent since no potentiation was found at 3.3 mmol/l glucose. Furthermore, ET-1 induced a large, transient increase in glucose-stimulated insulin secretion during islet perifusion in the presence (p<0.001), but not in the absence, of extracellular Ca²⁺. The rate of ⁴⁵Ca²⁺-efflux from ⁴⁵Ca²⁺-prelabelled islets was transiently stimulated by ET-1 during perifusion at 16.7 mmol/l glucose in the presence of extracellular Ca²⁺ (p<0.001). A short-lived increase in ⁴⁵Ca²⁺-efflux was also observed in the absence of extracellular Ca²⁺ (p<0.05). It is suggested that the effects of ET-1 on insulin secretion are critically dependent on influx via Ca²⁺-channels. In addition, ET-1 transiently enhanced ⁸⁶Rb⁺-efflux from ⁸⁶Rb⁺-prelabelled islets both in the presence (p<0.001) and in the absence (p<0.001) of extracellular Ca²⁺ suggesting that ET-1 does not elicit insulin secretion by inhibition of the potassium permeability. Our study provides evidence that ET-1 stimulates insulin secretion via a direct effect on the islets of Langerhans.Abbreviations ET-1 Endothelin-1     

Insulin secretion by fetal human pancreas in organ culture

Summary Whole fetal human pancreases of 12–22 weeks gestation, showed histological growth and differentiation in vitro over 3 weeks. At glucose concentrations of 1–4 g/l, there was no difference in insulin secretion into culture medium over 1 h. There was no stimulation of insulin release by D-glyceraldehyde, thus defective glucose-stimulated insulin release was probably not due to impairment of an early step in glycolysis. In the presence of 0.5 mmol/l dibutyryl cyclic AMP, insulin secretion was enhanced (0.188±0.030 versus 0.100±0.012 mU·mg tissue^-1·h^-1, p<0.001) independently of glucose concentrations. It thus appears that impairment of glucose-stimulated insulin release was unlikely to be due to insufficient intracellular cyclic AMP. Insulin release increased in response to tolbutamide and theophylline. Insulin secretion was stimulated in the presence of a fivefold increase in amino acid concentration (0.118±0.018 versus 0.031±0.008 mU·mg tissue ^-1·h^-1, p<0.001). There was a fourfold increase in basal insulin secretion from islets previously grown in high concentration of amino acids compared with standard culture medium, (0.284±0.052 versus 0.067±0.011 mU·mg tissue^-1·h^-1, p<0.001), emphasizing the important role of amino acids as substrates for B cell metabolism and development.     

The role of islet secretory function in the development of diabetes in the GK Wistar rat

Summary Insulin secretion and glucose metabolism were compared in islets isolated from GK Wistar rats (a non-obese, spontaneous model of non-insulin-dependent diabetes mellitus) and control Wistars aged 8 and 14 weeks. By 8 weeks of age, GK Wistar rats were clearly diabetic as indicated by non-fasting plasma glucose concentrations and impaired glucose tolerance. Islet insulin content was not significantly different to controls at either age. In islets from 14-week-old GK Wistar rats glucose-stimulated insulin release (6–16 mmol/l glucose) was significantly reduced to 25–50% of controls in static incubations (p<0.001). In perifusion, glucose-stimulated insulin release was reduced by 90% for first phase (p<0.01) and by 75% for second phase (p<0.05). The responses to arginine and 2 Ketoisocaproate in islets were similar to those in controls. In contrast, islets isolated from 8-week-old GK Wistar rats exhibited no significant reduction in glucose-stimulated insulin secretion in static incubations. In perifusion, although both first and second phases of glucose-stimulated insulin release were slightly reduced, these were not significantly different to controls. Islets from 8-week-old GK Wistar rats failed however to respond to stimulation by glyceraldehyde. Raising the medium glucose concentration to 16 mmol/l significantly increased rates of glucose utilisation ([³H] H₂O production from 5-[³H] glucose) and oxidation ([¹⁴C] CO₂ production from U-[¹⁴C] glucose) in islets isolated from 8-week-old control and GK Wistar rats, respectively. The rates of oxidation were not significantly different at stimulatory glucose concentrations whereas the rates of utilisation were significantly higher in islets from the diabetic animals (p<0.05). Production of [³H] H₂O from 2-[³H] glycerol metabolism was increased (p<0.05) at 2 mmol/l glucose but was not significantly different to controls at 16 mmol/l glucose in islets from 8-week-old GK Wistar rats. This data would suggest that abnormalities in islet function are present in 8-week-old diabetic animals although these do not seriously impair glucose-stimulated insulin release from isolated islets. This in turn would indicate that a defect in the glucose signalling pathway in beta cells is not a primary cause of the diabetes of GK Wistar rats and that deterioration of the secretory response is the consequence of some factor associated with the diabetic condition.Abbreviations KIC 2 Ketoisocaproate - BSA bovine serum albumin - GLUT glucose transporter     

Effect of GIP on the secretion of insulin and somatostatin and the accumulation of cyclic AMP in vitro in the rat

The effects of gastric inhibitory polypeptide (GIP) on insulin secretion as well as on the intra-islet accumulation of [3H]cyclic AMP were investigated in isolated pancreatic islets of the rat. In the presence of 6.7 mmol/l of glucose, 3.0 and 30 nmol/l of GIP induced both insulin and [3H]cyclic AMP responses, while lower and higher concentrations of the peptide were ineffective. A coupling of the two parameters was also found with regard to interaction between glucose and GIP. Thus while 30 nmol/l of GIP was stimulatory together with 6.7, 16.7 or 33.3 mmol/l of glucose, the peptide stimulated neither insulin release, nor the accumulation of [3H]cyclic AMP in the presence of a low concentration of glucose (3.3 mmol/l). The concomittant release of insulin and somatostatin was studied in the perfused pancreas in order to assess a possible influence by somatostatin on the dose-response pattern for GIP-induced insulin release. In this preparation 1.0 to 10 nmol/l of GIP stimulated insulin and somatostatin secretion; however while these concentrations were equipotent on insulin release, 10 nmol/l of GIP stimulated somatostatin release more than 1 nmol/l, indicating differences in dose-response curves for the GIP-induced stimulation of the two hormones. It is concluded that 1) modulation of GIP-induced insulin release is coupled to changes in cyclc AMP response in the islet, 2) GIP-induced somatostatin secretion may influence the concomittant insulin response.     

Hyperresponse in calcium-induced insulin release from electrically permeabilized pancreatic islets of diabetic GK rats and its defective augmentation by glucose

Summary In spontaneously diabetic GK rats, insulin secretion from pancreatic beta cells in response to glucose is selectively impaired, probably due to deficient intracellular metabolism of glucose and impaired closure of K_ATP channels during glucose stimulation. By using electrically permeabilized islets of GK rats, we explored the functional modulations in exocytotic steps distal to the rise in [Ca^{2 +} ]i in the diabetic condition. At 30 nmol/l Ca^{2 +} (basal conditions) insulin release was similar between GK and non-diabetic control Wistar rats. In response to 3.0 μmol/l Ca^{2 +} (maximum stimulatory conditions), insulin release was significantly augmented in permeabilized GK islets (p < 0.01). Raising glucose concentrations from 2.8 to 16.7 mmol/l further augmented insulin release induced by 3.0 μmol/l Ca^{2 +} from permeabilized control islets(p < 0.001), but had no effect on that from permeabilized GK islets. The stimulatory effect of glucose on insulin release from permeabilized control islets was partly inhibited by 2,4-dinitrophenol, an inhibitor of mitochondrial oxidative phosphorylation (p < 0.01). The hyperresponse to Ca^{2 +} in GK islets may play a physiologically compensatory role on the putative functional impairment both in [Ca^{2 +} ]i rise and energy state in response to glucose in diabetic β cells, and may explain the relative preservation of insulin release induced by non-glucose depolarizing stimuli, such as arginine, from pancreatic islets in non-insulin-dependent diabetes mellitus. [Diabetologia (1995) 38: 772–778] Received: 17 September 1994 and in revised form: 29 December 1994     

Properties of isolated human islets of langerhans: insulin secretion,glucose oxidation and protein phosphorylation

Summary In the present study, human islets were isolated by collagenase digestion from the pancreases of three kidney donors. Maintainance of the islets in tissue culture enabled insulin release, glucose oxidation and Ca²⁺-calmodulin-dependent protein phosphorylation to be determined using the same islets. Increasing glucose over a range 0–20 mmol/l resulted in a sigmoidal stimulation of insulin release (28.8±5.2 to 118.4±25.8 U-islet^–-h^–, n=10; threshold <4 mmol/l). There was a marked correlation between the insulin secretory response of the islets to glucose and their rate of glucose oxidation (5.9±0.3 at glucose 2 mmol/l up to 25.8±1.8 pmol-islet^–.h^– at 20 mmol/l, r = 0.98). N-acetylglucosamine (20 mmol/l) failed to elicit a secretory response from the islets. Stimulation of insulin secretion by glucose was dependent upon the presence of extracellular Ca²⁺. Extracts of the islets contained a Ca²⁺-calmodulin-dependent protein kinase which phosphorylated a 48-kdalton endogenous polypeptide. Myosin light-chain kinase activity was demonstrated in the presence of exogenous myosin light chains. This report demonstrates for the first time the sigmoidal nature of glucose-stimulated insulin release from isolated human islets, and its correlation with enhanced glucose oxidation. Furthermore, this is the first report of the presence of Ca²⁺-dependent protein kinases in human islets.     

The atypical antipsychotic clozapine impairs insulin secretion by inhibiting glucose metabolism and distal steps in rat pancreatic islets

Aims/hypothesis Diabetogenic effects of some atypical antipsychotic drugs have been reported, although the mechanisms are not fully understood. We investigated the long-term effects of culturing isolated rat pancreatic islets with atypical antipsychotic clozapine.Methods Glucose- and non-glucose-stimulated insulin secretion, glucose metabolism and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were measured in islets cultured with or without clozapine.Results Although acute incubation or 3-day culture with clozapine did not affect glucose-stimulated insulin secretion, clozapine suppressed glucose-stimulated insulin secretion by 53.2% at 1.0 μmol/l (therapeutic concentration) after 7 days of culture. Islet glucose oxidation and [Ca²⁺]_i elevation by high glucose were not affected after 3 days of culture, but clozapine significantly inhibited islet glucose oxidation, ATP production, and [Ca²⁺]_i elevation by high glucose after 7 days of culture. Moreover, 7 days of culture with clozapine inhibited insulin secretion stimulated by: (1) membrane depolarisation induced by high K⁺; (2) protein kinase C activation; and (3) mastoparan at 16.7 mmol/l glucose under stringent Ca²⁺-free conditions. Elevation of [Ca²⁺]_i by high K⁺-induced membrane depolarisation was similar in control and clozapine-treated islets. Clozapine, a muscarinic blocker, acutely inhibited carbachol-induced insulin secretion, as did atropine, whereas after 7 days of culture atropine did not have the inhibitory effect shown by clozapine after 7 days. The impairment of glucose-stimulated insulin secretion recovered 3 days after the removal of clozapine treatment.Conclusions/interpretation The present study demonstrated that the atypical antipsychotic drug clozapine directly impaired insulin secretion via multiple sites including glucose metabolism and the distal step in insulin exocytosis in a long-term culture condition. These mechanisms may be involved in the form of diabetes mellitus associated with atypical antipsychotic drugs.     

Possible participation of an islet B-cell calcium-sensing receptor in insulin release

The calcium-sensing receptor gene was recently shown to be expressed in rat pancreatic islets and purified islet B-cells. In this study, we investigated the possible role of this receptor in the regulation of insulin release from isolated rat pancreatic islets. Poly-l-arginine (0.2–0.3 μM) and poly-l-lysine (0.03–0.1 μM) increased insulin output evoked by d-glucose (8.3 mM). This positive effect faded out at higher concentrations of the basic peptides. Likewise, the release of insulin evoked by 8.3 mM d-glucose was significantly lower at high (1.0 mM) than low (0.05–0.1 mM) concentrations of neomycin. The insulinotropic action of Ba²⁺ in Ca²⁺-deprived islets was potentiated in rats pretreated with pertussis toxin. However, Gd³⁺ inhibited insulin release evoked by dd-glucose in islets prepared from normal rats or animals pretreated with pertussis toxin and incubated in the absence or presence of either theophylline or forskolin. Gd³⁺ (0.3 mM) failed to affect effluent radioactivity from islets prelabeled with myo-[2-³H]inositol and cyclic AMP net production in islets incubated in the absence or presence of forskolin. Gd³⁺ decreased, however, ⁴⁵Ca efflux from prelabeled islets perifused in the absence or presence of extracellular Ca²⁺. It is speculated that a negative insulinotropic action mediated by the calcium-sensing receptor, and possibly attributable to a fall in cytosolic Ca²⁺ concentration, may prevent excessive insulin secretion in pathological situations of hypercalcemia.     

Overexpression of mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase does not correct glucose-stimulated insulin secretion from diabetic GK rat pancreatic islets

Summary Glucose-stimulated insulin secretion is impaired in GK (Goto-Kakizaki) rats, perhaps because of abnormalities in glucose metabolism in pancreatic islet beta cells. The glycerol phosphate shuttle plays a major role in glucose metabolism by reoxidizing cytosolic NADH generated by glycolysis. In the pancreatic islets of GK rats, the activity of mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase (mGPDH), the key enzyme of the glycerol phosphate shuttle, is decreased and this abnormality may be responsible, at least in part, for impaired glucose-stimulated insulin secretion. To investigate this possibility, we overexpressed mGPDH in islets isolated from GK rats via recombinant adenovirus-mediated gene transduction, and examined glucose-stimulated insulin secretion. In islets isolated from diabetic GK rats at 8 to 10 weeks of age, glucose-stimulated insulin secretion was severely impaired, and mGPDH activity was decreased to 79 % of that in non-diabetic Wistar rats. When mGPDH was overexpressed in islets from GK rats, enzyme activity and protein content increased 2- and 6-fold, respectively. Basal (3 mmol/l glucose) and glucose-stimulated (20 mmol/l) insulin secretion from the Adex1CAlacZ-infected GK rat islets were, respectively, 4.4 ± 0.7 and 8.1 ± 0.7 ng · islet⁻¹· 30 min⁻¹, and those from mGPDH-overexpressed GK rat islets 4.7 ± 0.3 and 9.1 ± 0.8 ng · islet⁻¹· 30 min⁻¹, in contrast to those from the Adex1CAlacZ-infected non-diabetic Wistar rat islets (4.7 ± 1.6 and 47.6 ± 11.9 ng · islet⁻¹· 30 min⁻¹). Thus, glucose-stimulated insulin secretion is severely impaired in GK rats even in the stage when mGPDH activity is modestly decreased, and at this stage, overexpression of mGPDH cannot restore glucose-stimulated insulin secretion. We conclude that decreased mGPDH activity in GK rat islets is not the defect primarily responsible for impaired glucose-stimulated insulin secretion. [Diabetologia (1998) 41: 649–653] Received: 20 October 1997 and in revised form: 22 December 1997     

Mechanisms of octanoic acid potentiation of insulin secretion in isolated islets

ABSTRACT

A potentiating effect of medium-chain triglycerides on glucose-stimulated insulin secretion (GSIS) has been observed since the 1960s. Subsequent observations identified octanoic acid (OA), the main component of medium-chain triglyceride, as the potentiator of GSIS, but the mechanism was unclear. We used wild-type (WT), short-chain 3-hydroxyacyl-CoA dehydrogenase knockout (Hadh^-/-), and sulfonylurea receptor 1 knockout (Sur1^-/-) mouse islets to define the mechanism of OA potentiation of insulin secretion. Application of OA alone induced a 2- to 3- fold increase of insulin secretion with an apparent threshold of 3 mM in WT mouse islets, suggesting that OA itself is a weak insulin secretagogue. However, OA at 1 mM strongly potentiated fuel-stimulated insulin secretion, especially GSIS. The potentiating effect on fuel-stimulated insulin secretion by OA did not require fatty acid β-oxidation because OA also potentiated amino acid-stimulated insulin secretion in islets isolated from Hadh^-/- mice, which cannot fully oxidize OA. Measurements using Sur1^-/- islets indicated that the potentiating effect of OA on fuel-stimulated insulin secretion is Ca²⁺ dependent and is often accompanied by β-cell membrane potential depolarization, and may also involve the Ca²⁺/calmodulin complex. Experiments using DCPIB, an ethacrynic acid derivative, to inhibit volume-sensitive anion channels (VSACs) in Sur1^-/- islets demonstrated that the potentiation effects of OA on insulin secretion are in part medicated by activation of VSAC. In addition, inhibition of IP3 receptor also abolishes the OA-induced intracellular Ca²⁺ increase in Sur1^-/- islets.     

The metabolism of cyclic AMP and glucose in isolated islets from Acomys cahirinus

Summary Glucose-induced cyclic (³H) AMP accumulation, insulin secretory responses and the metabolism of glucose were studied in pancreatic islets from Acomys cahirinus. 27.7 mmol/l of glucose stimulated neither islet cyclic (³H) AMP accumulation nor insulin release during the first 5 min of incubation. Stimulation by glucose of cyclic (³H) AMP was observed after 15 min of incubation and insulin release was markedly stimulated between 15 and 30 min. The utilization of glucose, measured as the production of (³H)₂O from (5-³H) glucose was stimulated by glucose after 10 min and proceeded at an apparently linear rate during a 20 min incubation period. In incubations of 5 min, glibenclamide, glucagon or chloromercuribenzene-p-sulphonic acid failed to stimulate islet cyclic (³H) AMP accumulation. 3-isobutyl-1-methylxanthine in a concentration of 1.0 mmol/l was the only agent tested that elevated rapidly (1 min) islet cyclic (³H) AMP. None of the agents tested elicited an insulin secretory response in 5 min incubations. It is concluded that 1) no gross defect is apparent in the utilization of glucose by Acomys islets, 2) the secretory derangement of the Acomys is associated with a delayed cyclic AMP response to glucose, 3) however a decreased level of cyclic AMP cannot be the sole explanation for the delayed insulin secretion in the Acomys.     

Fuel-induced amplification of insulin secretion in mouse pancreatic islets exposed to a high sulfonylurea concentration: role of the NADPH/NADP+ ratio

Aims/hypothesis The aim of this study was to examine whether the cytosolic NADPH/NADP⁺ ratio of beta cells serves as an amplifying signal in fuel-induced insulin secretion and whether such a function is mediated by cytosolic α-ketoglutarate. Methods Pancreatic islets and islet cells were isolated from albino mice by collagenase digestion. Insulin secretion of incubated or perifused islets was measured by ELISA. The NADPH and NADP⁺ content of incubated islets was determined by enzymatic cycling. The cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in islets was measured by microfluorimetry and the activity of ATP-sensitive K⁺ channels in islet cells by patch-clamping. Results Both 30 mmol/l glucose and 10 mmol/l α-ketoisocaproate stimulated insulin secretion and elevated the NADPH/NADP⁺ ratio of islets preincubated in the absence of fuel. The increase in the NADPH/NADP⁺ ratio was abolished in the presence of 2.7 μmol/l glipizide (closing all ATP-sensitive K⁺ channels). However, α-ketoisocaproate, but not glucose, still stimulated insulin secretion. That glipizide did not inhibit α-ketoisocaproate-induced insulin secretion was not the result of elevated [Ca²⁺]_c, as glucose caused a more marked [Ca²⁺]_c increase. Insulin release triggered by glipizide alone was moderately amplified by dimethyl α-ketoglutarate (which is cleaved to produce cytosolic α-ketoglutarate), but there was no indication of a signal function of cytosolic α-ketoglutarate. Conclusions/interpretation The results strongly suggest that the NADPH/NADP⁺ ratio in the beta cell cytosol does not serve as an amplifying signal in fuel-induced insulin release. The study supports the view that amplification results from the intramitochondrial production of citrate by citrate synthase and from the associated export of citrate into the cytosol.     

Effect of human lymphoblastoid interferon on insulin synthesis and secretion in isolated human pancreatic islets

Summary Human islets of Langerhans were isolated from the pancreas removed from a 13-year-old female transplant donor. The islets were incubated in a culture medium for 24 h in the presence of human lymphoblastoid interferon (1000 units/ml). Insulin secretion, proinsulin biosynthesis, total protein biosynthesis and total insulin content were assessed at various concentrations of glucose in the presence of interferon. In interferon-treated islets glucose-stimulated insulin secretion was unaltered from that of control islets; however, glucose-stimulated proinsulin biosynthesis was specifically inhibited by interferon (48%, p<0.025). Total protein biosynthesis and total insulin content were not significantly affected by interferon.     

Nitric oxide and hydroperoxide affect islet hormone release and Ca(2+) efflux

We have investigated the influence of the intracellular free radical donors hydroxylamine (giving nitric oxide [NO]) and tert-butylhydroperoxide (giving hydroperoxide [“H₂O₂”]) on glucose- and cyclic adenosine monophosphate (cAMP)-induced transduction signaling in islet hormone release. Both donors dose dependently inhibited glucose-stimulated insulin release and induced modest (hydroxylamine) or profound (tert-butylhydroperoxide) suppression of ⁴⁵Ca²⁺-efflux from perifused islets. By contrast, both donors stimulated glucagon release. Similar effects on hormone release were displayed after K⁺-depolarization. Insulin and glucagon release stimulated by activation of the cAMP system through isobutylmethylxanthine (IBMX) at basal glucose was modestly potentiated by low concentrations of both donors. These effects were still observed, although less pronounced, in K⁺-depolarized islets. In vitro as well as in vivo, the NO-synthase inhibitor N^G-nitro-L-arginine methyl ester inhibited IBMX-induced glucagon release, but did not affect insulin release. The results suggest that NO and hydroperoxide inhibit glucose-stimulated insulin release by perturbing Ca²⁺ fluxes and probably acting through S-nitrosylation (NO) or oxidation (hydroperoxide) of thiol groups critical to the secretory process. These effects are largely independent of depolarization events. By contrast, both NO and hydroperoxide can potentiate cAMP-stimulated hormone release presumably at a distal site in the stimulus-secretion coupling.     

Mebendazole and insulin secretion from isolated rat islets

In a preliminary communication we reported that mebendazole, a vermicide, decreased plasma glucose and free fatty acid concentrations and increased plasma C peptide concentrations in both type II diabetic patients. Therefore, we suggested that mebendazole was an insulin secretagogue. However, these were uncontrolled studies, and improved metabolic control in these patients due to spontaneous remission rather than drug-induced insulin secretion was a possibility. To investigate the direct effect of mebendazole on insulin secretion we used intact islets isolated from normal rat pancreata. Mebendazole in concentrations as low as 10 to 20 mumol/L caused a twofold to threefold increase in acute-phase insulin release from isolated perifused rat islets. This heightened insulin release occurred in the presence of glucose-stimulated insulin secretion.     

Effects of phosphoenolpyruvate,other glycolytic intermediates and methylxanthines on calcium uptake by a mitochondrial fraction from rat pancreatic islets

Summary ⁴⁵Ca²⁺-accumulation by a mitochondrial fraction from isolated rat pancreatic islets was strongly stimulated by ATP. The ATP-dependent uptake was inhibited by phosphoenolpyruvate in a dose-dependent manner over a wide variety of conditions. Inhibition by phosphoenolpyruvate was noncompetitive with respect to calcium, competitive with respect to magnesium, and antagonised by high Mg-ATP^2– concentrations; fructose 1,6-diphosphate also decreased ⁴⁵Ca²⁺-uptake. Other glucose metabolites were either less effective or ineffective in diminishing mitochondrial ⁴⁵Ca²⁺-accumulation. The ATP-dependent uptake was also inhibited by xanthine derivatives (caffeine and 3-isobutyl-l-methylxanthine) which potentiate the effects of glucose on insulin secretion. Cyclic AMP had no effect. It is thought that the rate of insulin secretion is a function of the cytosolic calcium concentration in the B-cell. These data show that phosphoenolpyruvate, fructose 1,6-diphosphate and methylxanthines might influence exocytosis by direct effects on mitochondrial calcium accumulation, and thus the intracellular distribution of calcium.Abbreviations PEP phosphoenolpyruvate - BSA bovine albumin - EDTA ethylenediaminetetraacetic acid - IBMX 3-isobutyl-1-methylxanthine - cyclic AMP cyclic 3,5-adenosine monophosphate - ATP adenosine triphosphate - ATPase adenosine triphosphatase - Ca-ATPase calcium-activated adenosine triphosphatase - FCCP carbonylcyanide p-trifluoromethoxy-phenylhydrazone - DNP 2,4-dinitrophenol     

Regulation of86Rb outflow from pancreatic islets

Summary Theophylline (1.4 mM), cyclic AMP (1.0 mM) and dibutyryl-cyclic AMP (0.5 mM) decreased⁸⁶Rb fractional outflow rate from pancreatic islets perifused in the absence of glucose. In the presence of glucose (16.7 mM), however, the same drugs provoked a modest increase in⁸⁶Rb fractional outflow rate. The increase in⁸⁶Rb outflow evoked by theophylline in the presence of glucose was suppressed by quinine, suggesting that it may result from an increase in cytosolic Ca²⁺ concentration. It is proposed that changes in the cyclic AMP content of islet cells may participate in the regulation of K⁺ conductance by insulin secretagogues.     

The hypoglycaemic and insulinotropic activity of Tinospora crispa: studies with human and rat islets and HIT-T15 B cells

Summary In Malaysia, Tinospora crispa extract is taken orally by Type 2 (non-insulin-dependent) diabetic patients to treat hyperglycaemia. We have evaluated the claimed hypoglycaemic property by adding aqueous extract to the drinking water of normal and alloxan-diabetic rats. After one week, fasting blood glucose levels were significantly (p<0.01) lower and serum insulin levels were significantly (p<0.01) higher in treated diabetic animals (10.4±1.0 mmol/l and 12.8±1.1 U/ml respectively) compared to untreated diabetic controls (17.4±1.7 mmol/l and 8.0±0.7 U/ml respectively). The insulinotropic action of T. crispa was further investigated in vitro using isolated human or rat islets of Langerhans and HIT-T15 cells. In static incubations with rat islets and HIT-T15 B cells, the extract induced a dosage dependent stimulation and potentiation of basal and glucose-stimulated insulin secretion respectively. This insulinotropic effect was also evident in perifused human and rat islets and HIT-T5 B-cells. The observations that (i) in all three models insulin secretory rates rapidly returned to basal levels on removal of the extract and (ii) in rat islets, a second challenge with T. crispa induced an additional, stimulated response, are all consistent with physiological release of insulin by B cells. Moreover, the rate of HIT-T15 glucose utilisation was not affected by incubation with T. crispa, suggesting that the cells were viable throughout. These are the first studies to provide biochemical evidence which substantiates the traditional claims for an oral hypoglycaemic effect of Tinospora crispa, and which also show that the hypoglycaemic effect is associated with increased insulin secretion.     

Antagonistic effect of dibutyryl cyclic GMP on caerulein induced insulin secretion in isolated perifused rat islets

The effects of caerulein and dibutyryl cyclic GMP on isolated rat islets were studied using the perifusion system. One, 20,50 and 100nM caerulein stimulated insulin secretion in the perifused rat islets, in a dose dependent manner. Insulin secretion reached a maximal level in the presence of 50 nM caerulein. Caerulein-induced insulin secretion showed a predominant first and minimal second phase. After cessation of stimulation with 50nM caerulein, the insulin secretion level did not return to the basal value, but enhanced hormone secretion was sustained to a significant extent (p<0.01.). The simultane ous administration of 10^-3M dibutyryl cyclic GMP, a specific membrane antagonist against cholecysto kinin (CCK), inhibited the first and second phases of caerulein-induced insulin secretion. Moreover, with this nucleotide, “persistent” insulin secretion induced by 50 nM caerulein was inhibited. These data indicate that caerulein directly stimulates pancreatic B cells and that this peptide causes “persistent” insulin secretion. Dibutyryl cyclic GMP counteracts the effects of caerulein. Thus, both pancreatic B cells and pancreatic acinar cells possibly possess a CCK receptor.     

Beta cell compensation for insulin resistance in Zucker fatty rats: increased lipolysis and fatty acid signalling

Aims/hypothesis The aim of this study was to determine the role of fatty acid signalling in islet beta cell compensation for insulin resistance in the Zucker fatty fa/fa (ZF) rat, a genetic model of severe obesity, hyperlipidaemia and insulin resistance that does not develop diabetes.Materials and methods NEFA augmentation of insulin secretion and fatty acid metabolism were studied in isolated islets from ZF and Zucker lean (ZL) control rats.Results Exogenous palmitate markedly potentiated glucose-stimulated insulin secretion (GSIS) in ZF islets, allowing robust secretion at physiological glucose levels (5–8 mmol/l). Exogenous palmitate also synergised with glucagon-like peptide-1 and the cyclic AMP-raising agent forskolin to enhance GSIS in ZF islets only. In assessing islet fatty acid metabolism, we found increased glucose-responsive palmitate esterification and lipolysis processes in ZF islets, suggestive of enhanced triglyceride–fatty acid cycling. Interruption of glucose-stimulated lipolysis by the lipase inhibitor Orlistat (tetrahydrolipstatin) blunted palmitate-augmented GSIS in ZF islets. Fatty acid oxidation was also higher at intermediate glucose levels in ZF islets and steatotic triglyceride accumulation was absent.Conclusions/interpretation The results highlight the potential importance of NEFA and glucoincretin enhancement of insulin secretion in beta cell compensation for insulin resistance. We propose that coordinated glucose-responsive fatty acid esterification and lipolysis processes, suggestive of triglyceride–fatty acid cycling, play a role in the coupling mechanisms of glucose-induced insulin secretion as well as in beta cell compensation and the hypersecretion of insulin in obesity. Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users.