Abnormal insulin secretion and glucose metabolism in pancreatic islets from the spontaneously diabetic GK rat

Summary Insulin secretion and islet glucose metabolism were compared in pancreatic islets isolated from GK/Wistar (GK) rats with spontaneous Type 2 (non-insulin-dependent) diabetes mellitus and control Wistar rats. Islet insulin content was 24.5±3.1 U/ng islet DNA in GK rats and 28.8±2.5 U/ng islet DNA in control rats, with a mean (±SEM) islet DNA content of 17.3±1.7 and 26.5±3.4 ng (p < 0.05), respectively. Basal insulin secretion at 3.3 mmol/l glucose was 0.19±0.03 · ng islet DNA^–1· h^–1 in GK rat islets and 0.40±0.07 in control islets. Glucose (16.7 mmol/l) stimulated insulin release in GK rat islets only two-fold while in control islets five-fold. Glucose utilization at 16.7 mmol/l glucose, as measured by the formation of ³H₂O from [5-³ H]glucose, was 2.4 times higher in GK rat islets (3.1±0.7 pmol · ng islet DNA^–1 · h^–1) than in control islets (1.3±0.1 pmol · ng islet DNA^–1 · h^–1; p<0.05). In contrast, glucose oxidation, estimated as the production of ¹⁴CO₂ from [U-¹⁴C]glucose, was similar in both types of islets and corresponded to 15±2 and 30±3 % (p<0.001) of total glucose phosphorylated in GK and control islets, respectively. Glucose cycling, i. e. the rate of dephosphorylation of the total amount of glucose phosphorylated, (determined as production of labelled glucose from islets incubated with ³H₂O) was 16.4±3.4% in GK rat and 6.4±1.0% in control islets, respectively (p<0.01). We conclude that insulin secretion stimulated by glucose is markedly impaired in GK rat islets. Glucose metabolism is also altered in GK rat islets, with diminished ratio between oxidation and utilization of glucose, and increased glucose cycling, suggesting links between impaired glucose-induced insulin release and abnormal glucose metabolism.     

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.     

Time-dependent potentiation of insulin release induced by alpha-ketoisocaproate and leucine in rats: possible involvement of phosphoinositide hydrolysis

Summary The ability of the amino acid leucine and its keto acid, alpha-ketoisocaproate, to induce insulin release, to initiate phosphoinositide hydrolysis, and to amplify the subsequent insulin secretory response to glucose was assessed. In islets whose inositol-containing lipids were prelabelled with myo[2-³H]inositol, the addition of either compound resulted in an increase in insulin output, an increase in ³H efflux, rapid and significant increases in labelled inositol phosphate accumulation and a sustained increase in ³H efflux after removal of the stimulant. Direct measurements of labelled inositol phosphate accumulation in islets previously stimulated with alpha-ketoisocaproate demonstrate that this sustained increase in ³H efflux was the result of a persistent increase in phosphoinositide hydrolysis and was not simply a consequence of the hydrolysis of preformed inositol phosphates into more membrane permeable species. Prior exposure of islets to alpha-ketoisocaproate or leucine also resulted in an amplified secretory response to a subsequent glucose (10 mmol/l) stimulus. While peak first phase insulin release averaged 66±4 (mean±SEM, n=18) pg-islet^–1. min^–1 from control islets, this value increased to 204±14 and 246±11 pg·islet^–1· min^–1 in the leucine or alpha-ketoisocaproate pretreated islets respectively. The duration of this amplified response paralleled the duration of the persistent increase in ³H efflux. Prior alpha-ketoisocaproate exposure also amplified the subsequent insulin secretory response to tolbutamide and glyceraldehyde. While control (non-pretreated) islets in response to tolbutamide (200 mol/l) released insulin at a rate of 50±6pg·islet^–1·min^–1 (n = 3), this first phase response increased to 506±38 pg·islet^–1. min^–1 in prior alpha-ketoisocaproate treated islets. Peak first and second phase insulin responses to glyceraldehyde were increased 5-fold and 2-fold, respectively, by prior alpha-ketoisocaproate. These results suggest that events coupled to the hydrolysis of membrane inositol-containing phospholipids induced by leucine and alpha-ketoisocaproate participate not only in their acute insulin stimulatory action, but also in their ability to induce time-dependent potentiation (memory) in isolated islets.     

Increased glucose carbon recycling in severely insulin deficient Type 1 (insulin-dependent) diabetic subjects

Summary Six Type 1 (insulin-dependent) diabetic subjects were studied in order to determine the contribution of recycling of glucose carbon to the overproduction of glucose which is characteristic of the fasting hyperglycaemia produced by insulin withdrawal. The subjects were studied on two occasions, once after an overnight insulin infusion and once following 24 h of insulin withdrawal. The difference in turnover rates of 1-¹⁴C-glucose and 3-³H-glucose was used as a measure of glucose recycling. Insulin withdrawal caused a marked metabolic derangement with a rise in non-esterified fatty acids from 0.69±0.23 to 1.11±0.21 mmol/l (mean±SEM, p<0.05), total ketones from 0.27±0.06 to 2.06±0.51 mmol/l (p<0.01), cortisol from 341±43 to 479±31 nmol/l (p<0.05) and growth hormone from 1.1±0.3 to 19+5-mu/l (p<0.05). Glucose turnover rose from 13.8±2.3 mol·kg^–1·min^–1 at a glucose of 6.9±0.7 mmol/l in the insulin infused study to 25.8±4.4 mol·kg^–1·min^–1 (p<0.05) at a glucose of 16.4±0.7 mmol/l in the insulin withdrawn study. Recycling also rose from 3.0±0.4 mol· kg^–1·min^–1 to 9.4±2.2 mol·kg^–1·min^–1 (p<0.05) when insulin withdrawn, accounting for 23±3% and 36±3% of glucose turnover, respectively. We conclude that in the severely insulin deficient Type 1 diabetic subject recycling of glucose carbon is a major contributor to the excess glucose production.     

Glucose tolerance and plasma insulin response to intravenous glucose infusion and test meal in rats with microencapsulated islet allografts

Summary Albino Oxford rats made diabetic with 75 mg/kg streptozotocin were intraperitoneally transplanted with 2500–2900 alginate-polylysine microencapsulated Lewis islets (n=9, total islet tissue volume 8.0–11.0 l), or a similar volume of non-encapsulated Lewis islets (n=5). All rats with microencapsulated islets became normoglycaemic, and remained normoglycaemic for 5–16 weeks. In rats with non-encapsulated islet grafts, only a temporary decrease in blood glucose was observed, and all were again severely hyperglycaemic at 1 week after implantation. At 5–6 weeks after transplantation, glucose tolerance in rats with microencapsulated islets was tested by intravenous glucose infusion (10 mg/min over 20 min) and test meal administration (n=4). During glucose infusion, maximum glucose levels were 13.0±0.4 mmol/l in rats with microcapsules and 8.9±0.4 mmol/l in healthy control rats (p<0.01). Concomitant maximum plasma insulin levels were 215±17 pmol/l in rats with microcapsules and 715±85 pmol/l in controls (p<0.001). After the test meal, maximum blood glucose was 10.6±0.9 mmol/l in rats with microcapsules and 6.2±0.1 mmol/l in controls (p<0.001), with concomitant maximum plasma insulin levels of 247±11 pmol/l and 586±59 pmol/l, respectively (p<0.001). In conclusion, although the glucose tolerance is impaired and plasma insulin responses to intravenous glucose-load and test-meal are reduced, the alginate-polylysine membrane does provide adequate immunoisolation for the prolongation of allograft survival, resulting in prolonged normoglycaemia in streptozotocin diabetic rats.     

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     

Growth hormone regulation of DNA replication,but not insulin production,is partly mediated by somatomedin-C/insulin-like growth factor I in isolated pancreatic islets from adult rats

Summary We have investigated whether the previously demonstrated stimulatory actions of growth hormone on DNA synthesis and (pro)insulin biosynthesis and release of isolated adult rat islets of Langerhans are mediated by an autocrine release of somatomedin-C/insulin-like growth factor I (SM-C/IGF I). In medium containing 1% fetal calf serum, the presence of 16.7 mmol/l glucose, or 2.7 mmol/l glucose supplemented with a concentrate of essential amino acids, caused a significant increase in ³H-thymidine incorporation and insulin release compared to 2.7 mmol/l glucose alone but no increase in SM-C/IGFI release. Further supplementation with 1 g/ml growth hormone increased ³H-thymidine incorporation and SM-C/IGF I release within all groups, and insulin release in the 16.7 mmol/l glucose and 2.7 mmol/l plus amino acid groups. The ability of growth hormone to increase ³H-thymidine incorporation in the presence of 16.7 mmol/l glucose, but not its action on insulin release, was partly inhibited by a monoclonal antibody against SM-C/IGF I (control cultures 100%; growth hormone alone 261±27%, mean±SEM; growth hormone+anti-SM-C/IGFI 179±21%; p<0.05, n=18). Growth hormone, but not 100 ng/ml SM-C/IGF I, increased insulin biosynthesis assessed as immunoprecipitable ³H-labelled insulin by 45%, but this was accompanied by a similar increase in overall protein synthesis. Similarly growth hormone, but not SM-C/IGF I caused a 75% increase in glucose oxidation by islets. Both growth hormone and SM-C/IGF I failed to increase the cellular uptake of -aminoisobutyric acid or 3-O-methyl glucose over a 90 min period. The results suggest that while the stimulatory effect of growth hormone on islet cell insulin biosynthesis and release, glucose oxidation and general protein synthesis is probably direct, its action on B-cell replication is partly mediated by a paracrine release of SM-C/IGF I. This may provide a mechanism for increasing B-cell mass and consequently total insulin output during times of increased metabolic demands on insulin secretion.     

Stimulation of insulin release by an organic calcium agonist

Summary The calcium-agonist 4-[2-(difluoromethoxy)phenyl]-1,4,5,7-tetrahydro-2-methyl-5-oxo-furo[3,4-b]pyridine-3-carboxylic acid ethylester provoked, in the 1.0–100 mol/l range, a dose-related increase of glucose-stimulated insulin release by rat pancreatic islets. A fixed concentration of the drug (50 mol/l) caused a shift to the left of the sigmoidal curve relating insulin output to glucose concentration. The drug failed to affect insulin release evoked, in the absence of Ca²⁺, by the combination of Ba²⁺ and theophylline. The enhancing action of the calcium-agonist upon insulin release was rapid and sustained, and coincided with stimulation of both ⁴⁵Ca net uptake and ⁴⁵Ca efflux, the latter phenomenon being abolished in the absence of extracellular Ca²⁺. It is concluded that the gating of Ca-channels, as presumably provoked by the calcium-agonist, simulates the stimulant action of glucose upon both Ca influx into and insulin release from the pancreatic islets.     

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     

Effects of calcium and calcitonin on circulating levels of glucagon and glucose in diabetes mellitus

Summary The effects of Ca²⁺ and calcitonin infusions on circulating glucagon, glucose, C-peptide, Ca²⁺, and calcitonin were investigated in hyperglucagonaemic insulin-dependent diabetics. In 14 insulin-deprived diabetics and 12 healthy volunteers 2 h infusions of saline (0.154 mol/l), Ca²⁺ (0.375 mmol/kg body weight), and calcitonin (4.5 IU/kg body weight) were performed. There were no significant changes during saline infusion. In the diabetics, Ca²⁺ infusions induced a rise of plasma Ca²⁺ up to 3.2±0.1 mmol/l and a fall of circulating glucagon (-26.4±5.7%; p<0.001) and glucose (-23.3 ±3.6%; p<0.05). Plasma calcitonin rose to twice basal values (p<0.025). During calcitonin infusions plasma Ca²⁺ decreased slightly to 2.1±0.2 mmol/l; a fall was found in both glucose (— 24.4±4.0%; p<0.05) and circulating glucagon (-22.5±4.3%; p<0.001). Two groups of 6 healthy volunteers were subjected to saline and Ca²⁺, or to Ca²⁺ and calcitonin infusions. Both Ca²⁺ and calcitonin infusions induced a fall of serum insulin (— 30.1±6.6%; p< 0.05). Calcitonin depressed circulating glucagon by-18.6±4.4% (p<0.025), whereas during Ca²⁺ infusions glucagon decreased only by -6.5±1.9% (p>0.1). We conclude from our results that an increase of circulating calcitonin induced by Ca²⁺ infusions or by exogenous calcitonin administration appears to depress elevated circulating glucagon and glucose in insulin-dependent diabetics.     

Glucose regulates both glucose transport and the glucose transporter gene expression in a hamster-derived pancreatic Beta-cell line (HIT)

Summary We studied the effect of chronic exposure to high glucose on the glucose transport regulation in hamster pancreatic Beta cells in permanent culture (HIT). Cells were exposed to either 5.5 mmol/l or 16.7 mmol/l glucose for 48 h and then glucose transport was studied by measuring the (³H)-2-deoxyglucose uptake for 5 and 10 min at 37 °C. The 2- deoxyglucose uptake was lower in cells pre-exposed to glucose 16.7 mmol/l for 48 h compared to cells pre-exposed to glucose 5.5 (12.0±1.6 vs 19.1±1.2 nmol/0.1 mg after 5 min, and 22.2±2.6 vs 39.0±2.9 after 10 min respectively, mean ±SEM, n=5, p < 0.01). In order to investigate the mechanism(s) for glucose impairment of glucose transport, we studied the glucose carrier gene expression in the same cells by Northern and slot-blot analysis. When total RNA was extracted from HIT cells cultured at either 5.5 or 16.7 mmol/l glucose and then hybridized to ³²P-labelled cDNA probes for the glucose transporter 1, the glucose transporter 2 and -actin, a significant reduction of both glucose transporter 1 (–63.9±4.1%, mean±SEM, n=3) and glucose transporter 2 (–48.9±3.2%) mRNA was observed in HIT cells cultured with high glucose. In the same experiments no change of -actin mRNA was observed, suggesting that the effect of high glucose was specific on the glucose-transporter mRNAs. In HIT cells cultured at glucose 16.7 mmol/l the glucose-induced insulin release was also reduced compared to cells cultured at glucose 5.5 (715±19 U · h^–1 · mg^–1 vs 1301±28 U · h^–1 · mg^–1, respectively, mean ±SEM, n=3, p < 0.05). In conclusion, in hamster pancreatic Beta-cells, chronic exposure to high glucose concentrations impairs glucose transporter mRNA levels, glucose transport, and glucose-induced insulin secretion in a co-ordinate manner. Note: After this paper was submitted we became aware of two recent publications [9, 10] showing in animal models of non-insulin-dependent diabetes a decreased expression of the Beta-cell glucose transporter in pancreatic islets unresponsive to glucose, further supporting the view that this transporter may play a role in the glucosesensing mechanism.     

Culture duration and conditions affect the oscillations of cytoplasmic calcium concentration induced by glucose in mouse pancreatic islets

Summary The pattern of the increase in cytoplasmic Ca_i ²⁺ that glucose produces in beta cells has been reported to be highly variable. Here, we evaluated the influence of the culture duration (1–4 days) and conditions (5–10 mmol/l glucose) on Ca_i ²⁺ in normal mouse islets stimulated by glucose. After 1 day of culture in 10 mmol/l glucose, a rise of the glucose concentration from 3 to 15 mmol/l induced a triphasic change of Ca_i ²⁺ in the islets. A small initial decrease was followed by a large peak increase and then by regular fast oscillations (2.5/min). When the culture was prolonged to 2, 3 and 4 days, the initial decrease became inconsistent and the peak occurred earlier, whereas the oscillations decreased in frequency, increased in duration and eventually disappeared; on day 4 the Ca_i ²⁺ rise was sustained. After culture in 5 mmol/l glucose, the pattern of Ca_i ²⁺ changes induced by 15 mmol/l glucose was different. The initial decrease was very pronounced, the first peak was delayed and clearly separated from the subsequent oscillations. These were of a mixed type (fast Ca²⁺ transients on top of slow ones) after 1 day, and of a slow type only after 4 days. These alterations in the Ca_i ²⁺ oscillations triggered by glucose could not be ascribed to desynchronization of the signal between different regions of the islets. In conclusion, culturing normal mouse islets in 5 or 10 mmol/l glucose for 1–4 days, markedly alters the characteristics of the changes in Ca_i ²⁺ produced by glucose. This pitfall must be borne in mind when studying stimulus-secretion coupling in beta cells from normal or diabetic animals, or from human islets.Abbreviation Ca_i ²⁺ concentration of cytoplasmic calcium     

Crosstalk between membrane potential and cytosolic Ca2+ concentration in beta cells from Sur1 −/− mice

Aims/hypothesis Islets or beta cells from Sur1^–/– mice were used to determine whether changes in plasma membrane potential (V_m) remain coupled to changes in cytosolic Ca²⁺ ([Ca²⁺]_i) in the absence of K_ATP channels and thus provide a triggering signal for insulin secretion. The study also sought to elucidate whether [Ca²⁺]_i influences oscillations in V_m in sur1^–/– beta cells.Methods Plasma membrane potential and ion currents were measured with microelectrodes and the patch–clamp technique. [Ca²⁺]_i was monitored with the fluorescent dye fura-2. Insulin secretion from isolated islets was determined by static incubations.Results Membrane depolarisation of Sur1^–/– islets by arginine or increased extracellular K⁺, elevated [Ca²⁺]_i and augmented insulin secretion. Oligomycin completely abolished glucose-stimulated insulin release from Sur1^–/– islets. Oscillations in V_m were influenced by [Ca²⁺]_i as follows: (1) elevation of extracellular Ca²⁺ lengthened phases of membrane hyperpolarisation; (2) simulating a burst of action potentials induced a Ca²⁺-dependent outward current that was augmented by increased Ca²⁺ influx through L-type Ca²⁺ channels; (3) Ca²⁺ depletion of intracellular stores by cyclopiazonic acid increased the burst frequency in Sur1^–/– islets, elevating [Ca²⁺]_i and insulin secretion; (4) store depletion activated a Ca²⁺ influx that was not inhibitable by the L-type Ca²⁺ channel blocker D600.Conclusions/interpretation Although V_m is largely uncoupled from glucose metabolism in the absence of K_ATP channels, increased electrical activity leads to elevations of [Ca²⁺]_i that are sufficient to stimulate insulin secretion. In Sur1^–/– beta cells, [Ca²⁺]_i exerts feedback mechanisms on V_m by activating a hyperpolarising outward current and by depolarising V_m via store-operated ion channels.     

Insulin resistance in Type 2 (non-insulin-dependent) diabetic patients with hypertriglyceridaemia

Summary Hypertriglyceridaemia, which is frequently seen in Type 2 (non-insulin-dependent) diabetes mellitus, is associated with insulin resistance. The connection between hypertriglyceridaemia and insulin resistance is not clear, but could be due to substrate competition between glucose and lipids. To address this question we measured glucose and lipid metabolism in 39 Type 2 diabetic patients with hypertriglyceridaemia, i. e. mean fasting serum triglyceride level equal to or above 2 mmol/l (age 59±1 years, BMI 27.4±0.5 kg/m², HbA_1c8.0±0.2%, serum triglycerides 3.2±0.2 mmol/l) and 41 Type 2 diabetic patients with normotriglyceridaemia, i. e. mean fasting serum triglyceride level below 2 mmol/l (age 58±1 years, BMI 27.0±0.7 kg/m², HbA_1c7.8±0.2 %, serum triglycerides 1.4±0.1 mmol/l). Insulin sensitivity was assessed using a 340 pmol·(m²)^–1· min^–1 euglycaemic insulin clamp. Substrate oxidation rates were measured with indirect calorimetry and hepatic glucose production was estimated using a primed (25 Ci)-constant (0.25 Ci/min) infusion of [3-³H]-glucose. Suppression of lipid oxidation by insulin was impaired in patients with hypertriglyceridaemia vs patients with normal triglyceride levels (3.5±0.2 vs 3.0±0.2mol·kg^–1· min^–1; p<0.05). Stimulation of glucose disposal by insulin was reduced in hypertriglyceridaemic vs normotriglyceridaemic patients (27.0±1.3 vs 31.9±1.6 mol·kg^–1·min^–1; p<0.05) primarily due to impaired glucose storage (9.8±1.0 vs 14.6±1.4mol·kg^–1·min^–1; p<0.01). In contrast, insulinstimulated glucose oxidation was similar in patients with hypertriglyceridaemia and in patients with normal triglyceride concentrations (16.9±0.8 vs 17.2±0.7mol·kg^–1·min^–1). Hepatic glucose production in the basal state and during the clamp did not differ between the two groups. We conclude therefore that oxidative substrate competition between glucose and lipids does not explain insulin resistance associated with hypertriglyceridaemia in Type 2 diabetes. The question remains whether the reduced nonoxidative glucose disposal observed in the patients with hypertriglyceridaemia is genetically determined or a consequence of increased lipid oxidation.     

Dynamics of calcium-induced insulin release

Summary Extracellular Ca²⁺, at concentrations exceeding 10 mmol/l, causes a dose-related stimulation of insulin release. The dynamics of Ca²⁺-induced insulin release are characterized by a quick onset, a progressive build-up and a later return towards basal secretory rate. The release of insulin evoked by Ca²⁺ is inhibited in the presence of either Mg²⁺ (10 mmol/l) or the organic Ca²⁺-antagonist verapamil (81 mol/l), both of which are known to inhibit Ca²⁺ entry in the B-cell. Glucose and theophylline, which are thought to affect the net uptake or intracellular distribution of Ca²⁺ in the B-cell, both enhance Ca²⁺-induced insulin release. As little as 2.7 mmol/l glucose is sufficient to augment Ca²⁺-induced insulin secretion. Exposure of the pancreas to somatostatin significantly retards the secretory response to Ca²⁺. Cytochalasin B potentiates the insulin release evoked by Ca²⁺ (12 mmol/l) and lowers the threshold concentration of Ca²⁺ required to stimulate secretion. These data suggest that high extracellular Ca²⁺ concentrations may sufficiently increase the amount of Ca²⁺ accumulated in the B-cell to eventually trigger insulin release. Agents known to cause a remodelling of Ca²⁺ fluxes across membrane systems in the B-cell interfere with Ca²⁺-induced insulin release, a process also dependent on the integrity of the cytochalasin B-sensitive microfilamentous effector system.     

Effect of adrenalectomy on the development of a pancreatic islet lesion in fa/fa rats

Summary Adrenalectomy prevents development of obesity and hyperinsulinaemia in obese (fa/fa) Zucker rats, thereby implicating the hypothalamopituitary-adrenal axis in the pathogenesis of obesity. In this study glucose-induced insulin secretion and glucokinase activity were investigated in isolated islets from adrenalectomized and control obese and lean female rats. Islets from control fa/fa rats were more sensitive to glucose with a half-maximal effective concentration (EC₅₀) of 6.1±2.0 mmol · l^–1 compared with 10.6±2.7 mmol · l^–1 for adrenalectomized fa/fa rat islets. Adrenalectomy did not alter the islet sensitivity to glucose in the lean rats (EC₅₀ of 9.4±1.5 mmol · l^–1 and 9.3±2.0 mmol · l^–1 for adrenalectomized and control lean rats respectively). Mannoheptulose did not inhibit insulin secretion from control obese rats; however at concentrations of 1.0 mmol · l^–1 or more it significantly inhibited glucose-induced insulin secretion in adrenalectomized obese and lean, and control lean rat islets (p<0.05). In adrenalectomized fa/fa islets the glucokinase K_m was increased twofold compared with the control fa/fa rats (9.5±1.5 mmol · l^–1 vs 5.0±1.5 mmol · l^–1, respectively), but there was no significant change in glucokinase K_m in the lean rat islets after adrenalectomy. Mannoheptulose (10 mmol · l^–1) caused a significant reduction in glucose phosphorylation in disrupted islets of adrenalectomized fa/fa and lean, and of control lean rats, but not of control fa/fa rats. These data demonstrate that development of abnormal regulation of glycolysis in pancreatic islet beta cells of fa/ fa rats, as indicated by the insulin response to mannoheptulose and glucokinase activity, is dependent on an intact hypothalamo-pituitary-adrenal axis.Abbreviations ADX Adrenalectomy/adrenalectomized - CRH corticotrophin releasing hormone - DMEM Dulbecco's modified Eagle's medium - EC₅₀ half-maximal effective concentration - HPA hypothalamo-pituitary-adrenal - MH mannoheptulose - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid     

The permissive effect of glucose,tolbutamide and high K+ on arginine stimulation of insulin release in isolated mouse islets

Summary Mouse islets were used to study how glucose modulates arginine stimulation of insulin release. At 3 mmol/l glucose, arginine (20 mmol/l) decreased the resting membrane potential of B cells by about 10 mV, but did not evoke electrical activity. This depolarisation was accompanied by a slight but rapid acceleration of ⁸⁶Rb⁺ efflux and ⁴⁵Ca²⁺ influx. However, ⁴⁵Ca²⁺ efflux and insulin release increased only weakly and belatedly. When the membrane was depolarised by threshold (7 mmol/l) or stimulatory (10–15 mmol/l) concentrations of glucose, arginine rapidly induced or augmented electrical activity, markedly accelerated ⁸⁶Rb⁺ efflux, ⁴⁵Ca²⁺ influx and efflux, and triggered a strong and fast increase in insulin release. When glucose-induced depolarisation of the B-cell membrane was prevented by diazoxide, arginine lost all effects but those produced at low glucose. However, the delayed increase in release still exhibited some glucose-dependency. In contrast, depolarisation by tolbut amide, at low glucose, largely mimicked the permissive effect of high glucose. Depolarisation by high K⁺ also amplified arginine stimulation of insulin release, but did not accelerate it as did glucose or tolbutamide. Omission of extracellular Ca²⁺ abolished the releasing effect of arginine under all conditions. The results thus show that the permissive action of glucose mainly results from its ability to depolarise the B-cell membrane. It enables the small depolarisation by arginine itself to activate Ca channels more rapidly and efficiently. Changes in the metabolic state of B cells may also contribute to this permissive action by increasing the efficacy of the initiating signal triggered by arginine.     

Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance

Summary Plasma insulin levels in healthy subjects oscillate and non-insulin-dependent diabetic patients display an irregular pattern of such oscillations. Since an increase in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) in the pancreatic beta cell is the major stimulus for insulin release, this study was undertaken to investigate the dynamics of electrical activity, [Ca²⁺]_i-changes and insulin release, in stimulated islets from subjects of varying glucose tolerance. In four patients it was possible to investigate more than one of these three parameters. Stimulation of pancreatic islets with glucose and tolbutamide sometimes resulted in the appearance of oscillations in [Ca²⁺]_i, lasting 2–3 min. Such oscillations were observed even in some islets from patients with impaired glucose tolerance. In one islet from a diabetic patient there was no response to glucose, whereas that islet displayed [Ca²⁺]_i-oscillations in response to tolbutamide, suggesting that sulphonylurea treatment can mimic the complex pattern of glucose-induced [Ca²⁺]_i-oscillations. We also, for the first time, made patch-clamp recordings of membrane currents in beta-cells in situ in the islet. Stimulation with glucose and tolbutamide resulted in depolarization and appearance of action potentials. The islet preparations responded to stimulation with a number of different secretagogues with release of insulin. The present study shows that human islets can respond to stimulation with glucose and sulphonylurea with oscillations in [Ca²⁺]_i, which is the signal probably underlying the oscillations in plasma insulin levels observed in healthy subjects. Interestingly, even subjects with impaired glucose tolerance had islets that responded with oscillations in [Ca²⁺]_i upon glucose stimulation, although it is not known to what extent the response of these islets was representative of most islets in these patients.Abbreviations [Ca²⁺]_i Cytoplasmic free Ca²⁺ - NIDDM non-insulin-dependent diabetes mellitus - DMSO dimethylsulphoxide - PC pancreatic cancer     

Inhibition of the high-affinity glucose transporter GLUT 1 affects the sensitivity to glucose in a hamster-derived pancreatic beta cell line (HIT)

Summary HIT is a hamster-derived beta-cell line which in contrast to normal beta cells that only express the high K_m GLUT-2 glucose transporter, also expresses the low K_m glucose transporter GLUT 1. In HIT cells the abnormal glucose transport mechanism is associated with a marked shift to the left of the glucose-induced insulin release dose-response curve. We have used this cell model to investigate whether changes in glucose transport affect the glucose-induced insulin release. HIT cells were first incubated with a concentration of cytochalasin B (0.4 mol/l) that selectively inhibits the GLUT-1 but not the GLUT-2 transporter. The consequences of blocking glucose phosphorylation and insulin release were studied. Exposure to 0.4 mol/l cytochalasin B for 1 h caused a selective loss of the low K_m transport: the calculated V_max of GLUT 1 was reduced from 1726±98 to 184±14 pmol · mg protein^–1 5 min^–1 (mean±SEM, n=6, p<0.005), while no major difference in the high K_m (GLUT-2) transport was observed. In cytochalasin B exposed HIT cells the glucose phosphorylating activity (due to hexokinase and glucokinase) was unaffected. In these cells, however, the dose-response curve of glucose-induced insulin release was significantly shifted to the right: the 50% of maximal response (increment over baseline) was reached at an average glucose concentration of 2.9±0.2 mmol/l (vs 0.6±0.01 mmol/l in control HIT cells mean±SE, n=5, p<0.05) and the maximal effect was reached at 11.0 mmol/l glucose (vs 2.8 mmol/l in control HIT cells p<0.005). These results are consistent with the hypothesis that the affinity of the glucose transport system may contribute to determination of the glucose threshold concentration that triggers insulin secretion.     

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.