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 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     

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     

Placental glucose transport and utilisation is altered at term in insulin-treated, gestational-diabetic patients

Aims/hypothesis: We have previously shown that placentae from patients with gestational diabetes mellitus who did not receive insulin had lower glucose transport and utilisation than non-diabetic control subjects. To assess the placental glucose handling characteristics of women with gestational diabetes mellitus receiving insulin, we examined glucose transport and utilisation in placentae from three groups of women after term delivery: those with gestational diabetes mellitus and receiving insulin (n = 9, insulin group); those with gestational diabetes mellitus and not receiving insulin (n = 10, no insulin group); and those with normal, non-diabetic pregnancies (n = 9, control group). Methods: Dual perfusion of an isolated placental lobule was done using maternal glucose concentrations of 4, 8, 16 and 24 mmol/l. Glucose and l-lactate concentrations in the maternal and fetal effluents were measured. Direct glucose transfer from the maternal to the fetal effluent was measured using ¹⁴C-d-glucose. Mean rates in μmol ming^–1 (wet tissue) at maternal glucose concentration of 8 mmol/l are shown. Results: Glucose uptake from the maternal perfusate (insulin group 0.57, no insulin group 0.30) and net glucose transfer to the fetal effluent (insulin group 0.41, no insulin group 0.20) both increased in the placentae of women receiving insulin compared with the diabetic group not receiving insulin. Both groups of patients had lower placental glucose utilisation than the control group (insulin group 0.16, no insulin group 0.10, control group 0.25). Conclusion/interpretation: These results suggest that materno-fetal glucose transport increases in the placentae of women with gestational diabetes mellitus who receive insulin compared with those women who do not receive insulin. [Diabetologia (2001) 44: 1133–1139] Received: 19 February 2001 and in revised form: 17 April 2001     

The effects of hyperglycaemia on isotopic measurement of glucose utilisation using [23H], [33H] and [614C] glucose in patients with Type 1 (insulin-dependent) diabetes mellitus

Summary To determine whether hyperglycaemia alters the accuracy with which [2³H] and [3³H]glucose reflect glucose turnover measured with [6¹⁴C]glucose in patients with Type 1 (insulin-dependent) diabetes mellitus, glucose utilisation rates were measured during a simultaneous infusion of [2³H], [3³H] and [6¹⁴C]glucose after maintenance of normoglycaemia overnight and when glucose concentrations were clamped at 5.3, 7.5 and 9.7 mmol/l while insulin and glucagon concentrations were held constant. Glucose utilisation rates determined with all three isotopes were comparable in the diabetic patients at all glucose concentrations studied. On the other hand, glucose utilisation rates in nondiabetic subjects determined with [6¹⁴C]glucose were greater (p<0.01) than those determined with [3³H]glucose and lower (p<0.04) than those determined with [2³H]glucose during the 5.3, 7.5 and 9.7 mmol/l clamps. Nevertheless, glucose utilisation rates in the diabetic patients were lower (p<0.05) than those in the nondiabetic subjects for each glucose isotope. We conclude that hyperglycaemia does not alter the pattern of metabolism of [2³H] or [3³H]glucose in patients with Type 1 (insulin-dependent) diabetes mellitus.     

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.     

Impairment of the mitochondrial oxidative response to D-glucose in pancreatic islets from adult rats injected with streptozotocin during the neonatal period

Summary Pancreatic islets removed from adult rats injected with streptozotocin during the neonatal period display an impaired secretory response to D-glucose and, to a lesser extent, to L-leucine. Despite normal to elevated hexokinase and glucokinase activities in the islets of these glucose-intolerant animals and despite normal mitochondrial binding of the hexokinase isoenzymes, the metabolic response to a high concentration of D-glucose is severely affected, especially in terms of D-[6-¹⁴C]glucose oxidation. Thus, the ratio in D-[6-¹⁴C]glucose oxidation/D-[5-³H]glucose utilization is much less markedly increased in response to a rise in hexose concentration and, at a high concentration of D-glucose (16.7 mmol/l), less markedly decreased by the absence of Ca²⁺ and presence of cycloheximide in diabetic than control rats. This metabolic defect contrasts with (1) a close-to-normal or even increased capacity of the islets of diabetic rats to oxidize D-[6-¹⁴C]glucose, [2-¹⁴C]pyruvate, L-[U-¹⁴C]glutamine and L-[U-¹⁴C]leucine at low, non-insulinotropic, concentrations of these substrates; (2) a lesser impairment of the oxidation of L-[U-¹⁴ C]leucine tested in high concentration (20 mmol/l), the effect of Ca²⁺ deprivation upon the latter variable being comparable in diabetic and control rats; (3) an unaltered transamination of either [2-¹⁴ C]pyruvate or L-[U-¹⁴C]leucine; and (4) a modest perturbation of glycolysis. The most obvious alteration in glycolysis consists in a lesser increase of the glycolytic flux in response to a rise of D-glucose concentration in diabetic than control rats, this coinciding with an apparent decrease in affinity of glucokinase for the hexose. It is speculated that the preferential impairment of the metabolic and secretory response to D-glucose may be mainly attributable to an altered coupling between calcium accumulation and the stimulation of oxidative events in Beta-cell mitochondria of diabetic rats.     

Can desensitization of the B-cell to D-glucose be simulated in cultured pancreatic islets?

Summary In order to investigate the phenomenon of B-cell desensitization to D-glucose, rat pancreatic islets were cultured for 20–44h in the presence of increasing concentrations of D-glucose in the 5.6 to 27.8 mM range, and then incubated for 30 to 120 min for measurement of secretory, metabolic and ionic variables. After culture in the presence of 5.6 mM D-glucose, the release of insulin evoked by D-glucose (16.7 mM) was less marked than that seen in islets cultured in the presence of 11.1 mM D-glucose. In the latter islets, the secretory response to D-glucose (8.3 mM or more) was still modest, especially over short periods of incubation, but was markedly enhanced by either theophylline or forskolin. The release of insulin evoked by D-glucose in the presence of theophylline was little affected by either Ca²⁺ concentration of the culture medium or length of culture period (20hvs 44h). The culture-induced alteration in the responsiveness to D-glucose coincided with a smaller relative increase of D-[5³H]glucose utilization, D-[U-¹⁴C]glucose oxidation or net⁴⁵Ca uptake at increasing concentrations of the hexose. It contrasted with a well-preserved secretory response to nonnutrient secretagogues. Although these findings could be interpreted as evidence of B-cell desensitization to D-glucose, the fact that the secretory behavior of the islets was not vastly different whether they were first cultured at physiological (8.3 mM) or higher (11.1 to 27.8 mM) concentrations of D-glucose suggests that this experimental design may not be an optimal model for the functional alteration of the B-cell in hyperglycemic non-insulin-dependent diabetic subjects.     

Alloxan-induced alteration of insulin release,rubidium efflux and glucose metabolism in rat islets stimulated by various secretagogues

Summary Insulin release and⁸⁶Rb efflux were studied in perifused rat islets exposedin vitro to alloxan (2 mmol/l) for 5 min. At a low glucose concentration, alloxan transiently increased⁸⁶Rb efflux. Alloxan immediately and completely abolished the secretory response to glucose (15 mmol/l) and markedly delayed the reduction in⁸⁶Rb efflux normally produced by the sugar. 3-O-methylglucose (20 mmol/l) provided complete protection against the alteration of⁸⁶Rb efflux and partial protection against the inhibition of insulin release. Immediately after alloxan treatment, glyceraldehyde,-ketoisocaproic acid and tolbutamide still induced a rapid release of insulin, but the late phase normally stimulated by glyceraldehyde and-ketoisocaproic acid was inhibited. If islets were exposed to glyceraldehyde or tolbutamide 15 min after alloxan treatment, the rapid insulin release was also markedly impaired. Alloxan failed, however, to affect the ability of these three stimuli to reduce⁸⁶Rb efflux from islet cells. Glucose oxidation and utilization were decreased in alloxan-treated islets and 3-O-methylglucose protected against this effect. The results show that the glucose recognition system in B-cells is the most rapidly and severely affected by alloxan. The drug also alters the response to other secretagogues, the insulin releasing properties of which can be impaired without alteration of their ability to reduce⁸⁶Rb efflux.     

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.     

Fat-induced changes in mouse pancreatic islet insulin secretion,insulin biosynthesis and glucose metabolism

Insulin secretion, insulin biosynthesis and islet glucose oxidation were studied in pancreatic islets isolated from fat-fed diabetic mice of both sexes. Insulin secretion from isolated islets was studied after consecutive stimulation with -ketoisocaproic acid + glutamine, glucose, forskolin, and 12-O-tetradecanoylphorbol 13-acetate. Glucose-induced insulin secretion was impaired in islets from fat-fed mice. This was associated with a reduction of approximately 50% in islet glucose oxidation. Islet insulin secretion stimulated by the non-carbohydrate secretagogues tended to be higher in the fat-fed mice, but a statistically significant effect was not observed. Pancreatic insulin content was reduced by 50%, whereas the islet insulin and DNA content was unchanged after fat feeding. Proinsulin mRNA was reduced by 35% in islets from fat-fed mice, and was associated with a reduction of approximately 50% in glucose-stimulated (pro)insulin biosynthesis. It is concluded that the insulin secretory response of islets isolated from fat-fed mice is similar to the secretory pattern known from human type 2, non-insulin-dependent diabetics, and that a defect in islet glucose recognition, resulting in decreased glucose oxidation, may be responsible for the observed insulin secretory and biosynthetic defects seen after glucose stimulation.     

Preserved initiatory and potentiatory effect of α-ketoisocaproate on insulin release in islets of glucose intolerant rats

Summary Insulin responses to glucose and non-glucose secretagogues were studied in short-term cultured pancreatic islets and perfused pancreata of the glucose intolerant F₁ hybrid rats of spontaneously diabetic Goto-Kakizaki and control Wistar rats. After culture at 5.5 mmol/l glucose, hybrid islet responses to 11.1, 16.7 and 27.0 mmol/l glucose were between 60 and 40 % of control islet responses. A combination of 1 mmol/l isobutylmethylxanthine and 16.7 mmol/l glucose induced a pronounced insulin release, which was of similar magnitude in hybrid and control rat islets. This response was not further augmented by addition of glibenclamide and arginine. The slope of potentiation of arginine (10 mmol/l)-stimulated insulin secretion by glucose (5.5–16.7 mmol/l) was greatly impaired in hybrid islets. In contrast to glucose, α-ketoisocaproate (KIC), which is metabolized in Krebs cycle, dose-dependently stimulated insulin secretion to similar levels in hybrid and control islets, cultured at 5.5 mmol/l glucose. Also in hybrid islets depolarized by potassium chloride (30 mmol/l) and with adenosine triphosphate-sensitive K⁺-channels kept open by diazoxide, insulin responses to glucose were greatly impaired but intact to KIC. Furthermore, KIC potentiated normally the insulin response to arginine in hybrid islets. In the isolated perfused pancreas, KIC induced similar insulin responses in hybrid rats and control rats. The potentiating effect by 5.5 mmol/l glucose on the KIC-stimulated insulin responses was, however, greatly reduced in isolated islets and absent in the perfused pancreata of hybrid rats. Taken together, these findings suggest an intact capacity for insulin release, although the initiating and potentiating effect by glucose on insulin release are defective in the Goto-Kakizaki-hybrid rats. An abnormal beta-cell glucose metabolism proximal to the Krebs cycle is likely to account for the impairment of insulin release. [Diabetologia (1998) 41: 1368–1373] Received: 23 March 1998 and in revised form: 23 April 1998     

Effect of 2-deoxyglucose on [32P] phosphate and insulin release from perifused rat pancreatic islets

Summary The effect of 2-deoxyglucose on glucose mediated insulin and [³²P]phosphate release was studied by perifusion of isolated rat pancreatic islets. When islets were perifused with media containing 2.8 mmol/l glucose and 20 mmol/l 2-deoxyglucose for 60 minutes and then exposed to media containing 8.3 or 16.7 mmol/l glucose and 20 mmol/l 2-deoxyglucose for the next 15 minutes, insulin release at either glucose concentration was prompt but blunted. Similarly, islets preincubated (90 min) with [³²P] orthophosphate, then perifused with 20 mmol/l 2 deoxyglucose for 75 min and stimulated by either 8.3 or 16.7 mmol/l glucose for the final 15 minutes of 2 deoxyglucose exposure demonstrated obtundation of [³²P]phosphate release. Perifusion of islets with 20 mmol/l 2-deoxyglucose alone induced no heightened³²P efflux. These studies suggest that 2-deoxyglucose affects initial events in stimulus-secretion coupling of glucose mediated insulin release.     

Influence of gestational diabetes on the long-term control of glucose tolerance

Aims/hypothesis Gestational diabetes (GDM) carries a high risk of subsequent diabetes. We asked what impact prior GDM has on beta cell function and insulin action in women who maintain normal glucose tolerance (NGT) for a long time. Methods Ninety-one women with NGT (aged 41 ± 8 years, mean±SD) were studied (by mathematical modelling of the C-peptide response to an OGTT) 7 [6] years (median [interquartile range]) after the index pregnancy, during which 52 had GDM (pGDM) and 39 had NGT (pNGT). In all women an OGTT had also been performed at 29 ± 3 weeks of the index pregnancy. Results Women with pGDM were matched with women with pNGT for age, familial diabetes, time and weight gain since index pregnancy, parity, BMI (25.4 ± 3.9 vs 26.8 ± 6.4 kg/m²), and fasting (4.64 ± 0.56 vs 4.97 ± 0.46 mmol/l) and 2 h plasma glucose levels (5.91 ± 1.14 vs 5.91 ± 1.21 mmol/l). Nonetheless, fasting (49 [29] vs 70 [45] pmol min⁻¹ m⁻², p < 0.001) and total insulin secretion (32 [17] vs 48 [21] nmol m⁻², p < 0.0001) and beta cell glucose sensitivity (slope of the insulin secretion/plasma glucose concentration–response function) (95 [71] vs 115 [79] pmol min⁻¹ m⁻² (mmol/l)⁻¹, p = 0.025) were reduced in the pGDM group compared with the pNGT group, while insulin sensitivity was preserved (424 [98] vs 398 [77] ml min⁻¹ m⁻²). At index pregnancy, women with pGDM and those with pNGT had similar age and BMI. However, both insulin sensitivity (359 [93] vs 417 [92] ml min⁻¹ m⁻², p = 0.0012) and the insulin/glucose incremental area ratio (an empirical index of beta cell function; 98 [74] vs 138 [122] pmol/mmol, p = 0.028) were reduced in women with pGDM. Conclusions Even in women who maintain normal insulin sensitivity, impaired beta cell function is carried over into the NGT status several years after a GDM pregnancy.     

Insulin resistant subjects lack islet adaptation to short-term dexamethasone-induced reduction in insulin sensitivity

Aims/hypothesis. To establish whether islet compensation to deterioration of insulin action depends on inherent insulin sensitivity. Methods. We examined insulin and glucagon secretion after iv arginine (5 g) at fasting, 14 and greater than 25 mmol/l glucose concentrations before and after lowering of insulin sensitivity by oral dexamethasone (3 mg twice daily for 2 1/2 days) in 10 women with normal glucose tolerance, aged 58 or 59 years. Five women had high insulin sensitivity as shown by euglycaemic, hyperinsulinaemic clamp (99 ± 12 nmol glucose · kg body weight^–1· min^–1/pmol insulin · l^–1; means ± SD) whereas five women had low insulin sensitivity (34 ± 15 nmol glucose · kg body weight^–1· min^–1/pmol insulin · l^–1). Results. Dexamethasone reduced insulin sensitivity in both groups. Fasting insulin concentration increased by dexamethasone in high insulin sensitivity (72 ± 10 vs 49 ± 9 pmol/l, p = 0.043) but not in low insulin sensitivity (148 ± 63 vs 145 ± 78 pmol/l) whereas the fasting glucose concentration increased in low insulin sensitivity (6.5 ± 0.8 vs 5.8 ± 0.6 mmol/l, p = 0.043) but not in high insulin sensitivity (5.3 ± 0.8 vs 5.3 ± 0.6 mmol/l). Fasting glucagon concentration was not changed. Plasma insulin concentrations after raising glucose to 14 and more than 25 mmol/l and the insulin response to arginine at more than 25 mmol/l glucose were increased by dexamethasone in high insulin sensitivity (p < 0.05) but not changed by dexamethasone in low insulin sensitivity. Furthermore, in high but not in low insulin sensitivity, dexamethasone reduced the glucagon response to arginine (p = 0.043). Conclusion/interpretation. The results show that adaptation in islets function to dexamethasone-induced short-term reduction in insulin sensitivity is lacking in subjects with low inherent insulin sensitivity. [Diabetologia (1999) 42: 936–943] Received: 26 January 1999 and in revised form: 1 March 1999     

Copper addition prevents the inhibitory effects of interleukin 1-β on rat pancreatic islets

Summary Since copper [Cu(II)] is a necessary cofactor for both intra-mitochondrial enzymes involved in energy production and hydroxyl scavenger enzymes, two hypothesised mechanisms for action of interleukin-Iβ (IL-1β), we studied whether CU(II) addition could prevent the inhibitory effect of IL-1β on insulin release and glucose oxidation in rat pancreatic islets. Islets were incubated with or without 50 U/ml IL-1β, in the presence or absence of various concentrations of Cu(II)-GHL (Cu(II) complexed with glycyl-l-histidyl-l-lysine, a tripeptide known to enhance copper uptake into cultured cells). CuSO₄ (1–1000 ng/ml) was used as a control for Cu(II) effect when present as an inorganic salt. At the end of the incubation period, insulin secretion was evaluated in the presence of either 2.8 mmol/l (basal insulin secretion) or 16.7 mmol/l glucose (glucose-induced release). In control islets basal insulin secretion was 92.0±11.4 pg · islet⁻¹ h⁻¹ (mean ± SEM,n=7) and glucose-induced release was 2824.0±249.0 pg · islet⁻¹ h⁻¹. In islets pre-exposed to 50 U/ml IL-1β, basal insulin release was not significantly affected but glucose-induced insulin release was greatly reduced (841.2±76.9,n=7,p<0.005). In islets incubated with IL-1β and Cu-GHL (0.4 μmol/l, maximal effect) basal secretion was 119.0±13.1 pg · islet⁻¹ h⁻¹ and glucose-induced release was 2797.2±242.2, (n=7,p<0.01 in respect to islets exposed to IL-1β alone). In contrast to data obtained with Cu(II)-GHL, increasing concentrations of CuSO4 (up to 10 μmol/l) did not influence the inhibitory effect of IL-1β on glucose-stimulated insulin release. Glucose oxidation (in the presence of 16.7 mmol/l glucose) was 31.5±2.4 pmol · islet⁻¹·90min⁻¹ in control islets and 7.0±0.9 (p<0.01) in IL-1β-exposed islets. In islets exposed to IL-1β and Cu-GHL glucose oxidation was similar to control islets (31.9±1.9). In contrast, Cu-GHL did not prevent the IL-1β-induced increase in nitric oxide production. Nitrite levels were 5±1.7, 26±5 and to 29±4 pmol · islet⁻¹·48 h⁻¹ (mean ± SEM,n=5) in the culture medium from control IL-1β and IL-1β+Cu-GHL exposed islets, respectively. These data indicate that the Cu(II) complexed to GHL is able to prevent the inhibitory effects of IL-1β on insulin secretion and glucose oxidation, but not on NO production. The mechanism of action of Cu-GHL is still unclear, but it might restore the activity of the enzymatic systems inhibited by IL-1β. [Diabetologia (1995) 38∶39–45]     

Insulin secretion profiles are modified by overexpression of glutamate dehydrogenase in pancreatic islets

Aims/hypothesis Glutamate dehydrogenase (GDH) is a mitochondrial enzyme playing a key role in the control of insulin secretion. However, it is not known whether GDH expression levels in beta cells are rate-limiting for the secretory response to glucose. GDH also controls glutamine and glutamate oxidative metabolism, which is only weak in islets if GDH is not allosterically activated by L-leucine or (+/–)-2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH).Methods We constructed an adenovirus encoding for GDH to overexpress the enzyme in the beta-cell line INS-1E, as well as in isolated rat and mouse pancreatic islets. The secretory responses to glucose and glutamine were studied in static and perifusion experiments. Amino acid concentrations and metabolic parameters were measured in parallel.Results GDH overexpression in rat islets did not change insulin release at basal or intermediate glucose (2.8 and 8.3 mmol/l respectively), but potentiated the secretory response at high glucose concentrations (16.7 mmol/l) compared to controls (+35%). Control islets exposed to 5 mmol/l glutamine at basal glucose did not increase insulin release, unless BCH was added with a resulting 2.5-fold response. In islets overexpressing GDH glutamine alone stimulated insulin secretion (2.7-fold), which was potentiated 2.2-fold by adding BCH. The secretory responses evoked by glutamine under these conditions correlated with enhanced cellular metabolism.Conclusions/interpretation GDH could be rate-limiting in glucose-induced insulin secretion, as GDH overexpression enhanced secretory responses. Moreover, GDH overexpression made islets responsive to glutamine, indicating that under physiological conditions this enzyme acts as a gatekeeper to prevent amino acids from being inappropriate efficient secretagogues.Abbreviations AUC Area under the curve - BCH (+/–)-2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid - _m mitochondrial membrane potential - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - GABA -aminobutyric acid - GDH glutamate dehydrogenase - HPLC high-pressure liquid chromatography - KRBH Krebs-Ringer bicarbonate HEPES buffer - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl tetrazolium bromide - RPMI Roswell Park Memorial Institute - TCA tricarboxylic acid     

Risk and mechanism of dexamethasone-induced deterioration of glucose tolerance in non-diabetic first-degree relatives of NIDDM patients

Summary We tested the hypothesis that glucose intolerance develops in genetically prone subjects when exogenous insulin resistance is induced by dexamethasone (dex) and investigated whether the steroid-induced glucose intolerance is due to impairment of beta-cell function alone and/or insulin resistance. Oral glucose tolerance (OGTT) and intravenous glucose tolerance tests with minimal model analysis were performed before and following 5 days of dex treatment (4 mg/day) in 20 relatives of non-insulin-dependent diabetic (NIDDM) patients and in 20 matched control subjects (age: 29.6 ± 1.7 vs 29.6 ± 1.6 years, BMI: 25.1 ± 1.0 vs 25.1 ± 0.9 kg/m²). Before dex, glucose tolerance was similar in both groups (2-h plasma glucose concentration (PG): 5.5 ± 0.2 [range: 3.2–7.0] vs 5.5 ± 0.2 [3.7–7.4] mmol/l). Although insulin sensitivity (Si) was significantly lower in the relatives before dex, insulin sensitivity was reduced to a similar level during dex in both the relatives and control subjects (0.30 ± 0.04 vs 0.34 ± 0.04 10^–4 min^–1 per pmol/l, NS). During dex, the variation in the OGTT 2-h PG was greater in the relatives (8.5 ± 0.7 [3.9–17.0] vs 7.5 ± 0.3 [5.7–9.8] mmol/l, F-test p < 0.05) which, by inspection of the data, was caused by seven relatives with a higher PG than the maximal value seen in the control subjects (9.8 mmol/l). These “hyperglycaemic” relatives had diminished first phase insulin secretion (?1) both before and during dex compared with the “normal” relatives and the control subjects (pre-dex ?1: 12.6 ± 3.6 vs 26.4 ± 4.2 and 24.6 ± 3.6 (p < 0.05), post-dex ?1: 22.2 ± 6.6 vs 48.0 ± 7.2 and 46.2 ± 6.6 respectively (p < 0.05) pmol · l^–1· min^–1 per mg/dl). However, Si was similar in “hyperglycaemic” and “normal” relatives before dex (0.65 ± 0.10 vs 0.54 ± 0.10 10−4 · min^–1 per pmol/l) and suppressed similarly during dex (0.30 ± 0.07 vs 0.30 ± 0.06 10−4 · min^–1 per pmol/l). Multiple regression analysis confirmed the unique importance of low pre-dex beta-cell function to subsequent development of high 2-h post-dex OGTT plasma glucose levels (R ² = 0.56). In conclusion, exogenous induced insulin resistance by dex will induce impaired or diabetic glucose tolerance in those genetic relatives of NIDDM patients who have impaired beta-cell function (retrospectively) prior to dex exposure. These subjects are therefore unable to enhance their beta-cell response in order to match the dex-induced insulin resistant state. [Diabetologia (1997) 40: 1439–1448] Received: 20 January 1997 and in final revised form: 17 July 1997     

Oscillations in oxygen tension and insulin release of individual pancreatic ob/ob mouse islets

Aims/hypothesis. The role of beta-cell metabolism for generation of oscillatory insulin release was investigated by simultaneous measurements of oxygen tension (pO₂) and insulin release from individual islets of Langerhans.¶Methods. Individual islets isolated from the ob/ob-mice were perifused. Insulin in the perifusate was measured with a sensitive ELISA and pO₂ with a modified Clark-type electrode inserted into the islets.¶Results. In the presence of 3 mmol/l d-glucose, pO₂ was 102 ± 9 mmHg and oscillatory (0.26 ± 0.04 oscillations/min). Corresponding insulin measurements showed oscillatory release with similar periodicity (0.25 ± 0.02 oscillations/min). When the d-glucose concentration was increased to 11 mmol/l, pO₂ decreased by 30 % to 72 ± 10 mmHg with maintained frequency of the oscillations. Corresponding insulin secretory rate rose from 5 ± 2 to 131 ± 16 pmol · g^–1· s^–1 leaving the frequency of the insulin pulses unaffected. The magnitude of glucose-induced change in pO₂ varied between islets but was positively correlated to the amount of insulin released (r ² = 0.85). When 1 mmol/l tolbutamide was added to the perifusion medium containing 11 mmol/l glucose no change in average oscillatory pO₂ was observed despite a doubling in the secretory rate. When 8 mmol/l 3-oxymethyl glucose was added to perifusion medium containing 3 mmol/l d-glucose, neither pO₂ nor insulin release of the islets were changed. Temporal analysis of oscillations in pO₂ and insulin release revealed that maximum respiration correlated to maximum or close to maximum insulin release.¶Conclusion/interpretation. The temporal relation between oscillations in pO₂ and insulin release supports a role for metabolic oscillations in the generation of pulsatile insulin release. [Diabetologia (2000) 43: 1313–1318]     

Glucagon-like peptide-1 stimulates insulin secretion by a Ca-independent mechanism in Zucker diabetic fatty rat islets of Langerhans

This study investigates the mechanisms responsible for glucagon-like peptide-1 (GLP-1)-induced insulin secretion in Zucker diabetic fatty (ZDF) rats and their lean control (ZLC) littermates. Glucose, and 100 nmol/L GLP-1 (7-37 hydroxide) in the presence of stimulatory glucose concentrations, induced insulin secretion in islets from ZLC animals. In contrast, ZDF islets hypersecreted insulin at low glucose (5 mmol/L) and were poorly responsive to 15 mmol/L glucose stimulation, but increased insulin secretion following exposure to GLP-1. The insulin secretory response to 100 nmol/L GLP-1 was reduced by 88% in ZLC islets exposed to exendin 9-39. The intracellular Ca²⁺ concentration ([Ca²⁺]_i) increased in fura-2-loaded ZLC islets following stimulation with 12 mmol/L glucose alone or GLP-1 in the presence of 12 mmol/L glucose. The increases in [Ca²⁺]_i and insulin secretion in ZLC islets induced by GLP-1 were attenuated by 1 μmol/L nitrendipine. In contrast, neither glucose nor GLP-1 substantially increased [Ca²⁺]_i in ZDF islets. Furthermore, insulin secretory responses to GLP-1 were not significantly inhibited in ZDF islets by nitrendipine. However, the insulin secretory response to GLP-1 in both ZLC and ZDF islets was ablated by cholera toxin. Our findings indicate that in ZLC islets, GLP-1 induces insulin secretion by a mechanism that depends on Ca²⁺ influx through voltage-dependent Ca²⁺ channels, whereas in ZDF islets, the action of GLP-1 is mediated by Ca²⁺-independent signaling pathways.