A 6-month randomized, double-blind, placebo-controlled trial of weekly exenatide in adolescents with obesity.

BACKGROUND: Pharmacological treatment options for adolescents with obesity are very limited. Glucagon-like-peptide-1 (GLP-1) receptor agonist could be a treatment option for adolescent obesity. OBJECTIVE: To investigate the effect of exenatide extended release on body mass index (BMI)-SDS as primary outcome, and glucose metabolism, cardiometabolic risk factors, liver steatosis, and other BMI metrics as secondary outcomes, and its safety and tolerability in adolescents with obesity. METHODS: Six-month, randomized, double-blinded, parallel, placebo-controlled clinical trial in patients (n = 44, 10-18 years, females n = 22) with BMI-SDS > 2.0 or age-adapted-BMI > 30 kg/m2 according to WHO were included. Patients received lifestyle intervention and were randomized to exenatide extended release 2 mg (n = 22) or placebo (n = 22) subcutaneous injections given once weekly. Oral glucose tolerance tests (OGTT) were conducted at the beginning and end of the intervention. RESULTS: Exenatide reduced (P < .05) BMI-SDS (-0.09; -0.18, 0.00), % BMI 95th percentile (-2.9%; -5.4, -0.3), weight (-3 kg; -5.8, -0.1), waist circumference (-3.2 cm; -5.8, -0.7), subcutaneous adipose tissue (-552 cm3 ; -989, -114), 2-hour-glucose during OGTT (-15.3 mg/dL; -27.5, -3.1), total cholesterol (11.6 mg/dL; -21.7, -1.5), and BMI (-0.83 kg/m2 ; -1.68, 0.01) without significant change in liver fat content (-1.36; -3.12, 0.4; P = .06) in comparison to placebo. Safety and tolerability profiles were comparable to placebo with the exception of mild adverse events being more frequent in exenatide-treated patients. CONCLUSIONS: Treatment of adolescents with severe obesity with extended-release exenatide is generally well tolerated and leads to a modest reduction in BMI metrics and improvement in glucose tolerance and cholesterol. The study indicates that the treatment provides additional beneficial effects beyond BMI reduction for the patient group.

Basal hypersecretion of glucagon and insulin from palmitate-exposed human islets depends on FFAR1 but not decreased somatostatin secretion.

In obesity fasting levels of both glucagon and insulin are elevated. In these subjects fasting levels of the free fatty acid palmitate are raised. We have demonstrated that palmitate enhances glucose-stimulated insulin secretion from isolated human islets via free fatty acid receptor 1 (FFAR1/GPR40). Since FFAR1 is also present on glucagon-secreting alpha-cells, we hypothesized that palmitate simultaneously stimulates secretion of glucagon and insulin at fasting glucose concentrations. In addition, we hypothesized that concomitant hypersecretion of glucagon and insulin was also contributed by reduced somatostatin secretion. We found basal glucagon, insulin and somatostatin secretion and respiration from human islets, to be enhanced during palmitate treatment at normoglycemia. Secretion of all hormones and mitochondrial respiration were lowered when FFAR1 or fatty acid ß-oxidation was inhibited. The findings were confirmed in the human beta-cell line EndoC-ßH1. We conclude that fatty acids enhance both glucagon and insulin secretion at fasting glucose concentrations and that FFAR1 and enhanced mitochondrial metabolism but not lowered somatostatin secretion are crucial in this effect. The ability of chronically elevated palmitate levels to simultaneously increase basal secretion of glucagon and insulin positions elevated levels of fatty acids as potential triggering factors for the development of obesity and impaired glucose control.

Functional differences between aggregated and dispersed insulin-producing cells.

AIMS/HYPOTHESIS: Beta cells situated in the islet of Langerhans respond more vigorously to glucose than do dissociated beta cells. Mechanisms for this discrepancy were studied by comparing insulin-producing MIN6 cells aggregated into pseudoislets with MIN6 monolayer cells and mouse and human islets. METHODS: MIN6 monolayers, pseudoislets and mouse and human islets were exposed to glucose, α-ketoisocaproic acid (KIC), pyruvate, KIC plus glutamine and the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin. Insulin secretion (ELISA), cytoplasmic Ca(2+) concentration ([Ca(2+)]c; microfluorometry), glucose oxidation (radiolabelling), the expression of genes involved in mitochondrial metabolism (PCR) and the phosphorylation of insulin receptor signalling proteins (western blotting) were measured. RESULTS: Insulin secretory responses to glucose, pyruvate, KIC and glutamine were higher in pseudoislets than monolayers and comparable to those of human islets. Glucose oxidation and genes for mitochondrial metabolism were upregulated in pseudoislets compared with single cells and monolayers, respectively. Phosphorylation at the inhibitory S636/639 site of IRS-1 was significantly higher in monolayers and dispersed human and mouse cells than pseudoislets and intact human and mouse islets. PI3K inhibition only slightly attenuated glucose-stimulated insulin secretion from monolayers, but substantially reduced that from pseudoislets and human and mouse islets without suppressing the glucose-induced [Ca(2+)]c response. CONCLUSIONS/INTERPRETATION: We propose that islet architecture is critical for proper beta cell mitochondrial metabolism and IRS-1 signalling, and that PI3K regulates insulin secretion at a step distal to the elevation of [Ca(2+)]c.

Islet protein profiling.

Islet protein profiling is defined as generation of extended protein expression data sets from islets or islet cells. Islets from rodent control and animal models of type 1 and type 2 diabetes mellitus and healthy humans and insulin- and glucagon-producing cell lines have been used. Protein profiling entails separation, differential expression determination, identification and expression analysis. Protein/peptide separation is either gel-based or by chromatography. Differential expression is based on comparison of visualized spots/proteins between gels or by sample labelling in gel-free systems. Identification of proteins is made by tryptic fragmentation of proteins, fragment mass determination and mass comparison with protein databases. Analysis of expression data sets interprets the complex protein changes into cellular mechanisms to generate hypotheses. The importance of such protein expression sets to elucidate islet cellular events is evidenced by the observation that only about 50% of the differentially expressed proteins and transcripts showed concordance when measured in parallel. Using protein profiling, different areas related to islet dysfunction in type 1 and type 2 diabetes mellitus have been addressed, including dysfunction induced by elevated levels of glucose and fatty acids and cytokines. Because islets from individuals with type 1 or type 2 diabetes mellitus have not yet been protein profiled, islets from rat (BB-DP) and mouse (NOD, ob/ob, MKR) models of the disease have been used, and mechanisms responsible for islet dysfunction delineated offering avenues of intervention.

Glucose-induced de novo synthesis of fatty acyls causes proportional increases in INS-1E cellular lipids.

Raised concentrations of glucose for extended periods of time have detrimental effects on the insulin-producing beta-cell. As de novo synthesis of lipids has been observed under such conditions, it was hypothesized that newly formed lipids may preferentially contain saturated fatty acids, which in particular have been associated with impaired beta-cell function. Glucose-induced de novo synthesis of fatty acids in INS-1E cells cultured in 5.5, 11, 20 or 27 mM glucose for 5 days was assessed by high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy and gas chromatography-mass spectrometry (GC-MS). The glucose origin of the increase in fatty acyls was verified by replacing glucose with [1-13C]glucose during culture followed by analysis with two-dimensional 1H-13C NMR spectroscopy. The composition of the fatty acyls was determined by GC-MS. Fatty acyls determined by HR-MAS (1)H NMR spectroscopy were increased fivefold in INS-1E cells cultured in 20 or 27 mM glucose compared with cells cultured in 5.5 mM glucose. The five most abundant fatty acids with their relative percentages in INS-1E cells cultured in 5.5 mM glucose were oleate (33%), palmitate (25%), stearate (19%), octadecenoate (13%) and palmitoleate (4.4%). These proportions were not affected by glucose- induced de novo synthesis in INS-1E cells cultured in 11, 20 or 27 mM glucose. It is concluded that glucose-induced de novo lipid synthesis results in accumulation of both saturated and unsaturated fatty acids in specific proportions that are identical with those present under control conditions.

Glucose-induced changes of multiple mouse islet proteins analysed by two-dimensional gel electrophoresis and mass spectrometry.

AIMS/HYPOTHESIS: The aim of this study was to investigate molecular mechanisms of glucose-induced changes in islets of Langerhans by analysing global changes in protein patterns of islets exposed to elevated glucose concentrations. METHODS: Islets were isolated from C57BL/6J mice and used either directly or after exposure to 11 mmol/l glucose for 24 h. Islet protein profiles were obtained by two-dimensional gel electrophoresis, and protein spots were identified by peptide mass fingerprinting using mass spectrometry. RESULTS: Two-dimensional gels of freshly isolated islets and islets exposed to 11 mmol/l glucose contained 1,074 and 1,254 spots, respectively. The number of differentially expressed spots was 379, with 20 spots appearing as new proteins in islets exposed to 11 mmol/l glucose. We identified 124 spots corresponding to 77 protein entries and generated a reference map from freshly isolated islets. Actin, alpha enolase, cytokeratin 8, endoplasmin, glucose-regulated proteins, heat shock proteins, peroxiredoxins, prohormone convertase 2, protein disulphide isomerase, superoxide dismutase, tubulin, and V-type H+ -ATPase (V1 subunit A) were upregulated in islets exposed to 11 mmol/l glucose. In contrast, exocrine proteins and secretagogin were downregulated in these islets compared with in freshly isolated islets. CONCLUSIONS/INTERPRETATION: The islet proteome approach revealed simultaneous changes in protein patterns of islets exposed to elevated glucose concentrations, indicating enhanced insulin synthesis, granular mobilisation and maturation, and increased stress response. The changes may be of relevance for the understanding of altered islet function in the hyperglycaemic state. It is expected that the islet reference map will become an important tool for dissecting multifactorial islet processes.

Observers for Takagi-Sugeno fuzzy systems.

We focus on the analysis and design of two different sliding mode observers for dynamic Takagi-Sugeno (TS) fuzzy systems. A nonlinear system of this class is composed of multiple affine local linear models that are smoothly interpolated by weighting functions resulting from a fuzzy partitioning of the state space of a given nonlinear system subject to observation. The Takagi-Sugeno fuzzy system is then an accurate approximation of the original nonlinear system. Our approach to the analysis and design of observers for Takagi-Sugeno fuzzy systems is based on extending sliding mode observer schemes to the case of interpolated multiple local affine linear models. Thus, our main contribution is nonlinear observer analysis and design methods that can effectively deal with model/plant mismatches. Furthermore, we consider the difficult case when the weighting functions in the Takagi-Sugeno fuzzy system depend on the estimated state.

Phenotyping of individual pancreatic islets locates genetic defects in stimulus secretion coupling to Niddm1i within the major diabetes locus in GK rats.

The major diabetes quantitative trait locus (Niddm1), which segregates in crosses between GK rats affected with spontaneous type 2-like diabetes and normoglycemic F344 rats, encodes at least two different diabetes susceptibility genes. Congenic strains for the two subloci (Niddm1f and Niddm1i) have been generated by transfer of GK alleles onto the genome of F344 rats. Whereas the Niddm1f phenotype implicated insulin resistance, the Niddm1i phenotype displayed diabetes related to insulin deficiency. Individual islets from 16-week-old congenic rats were characterized for insulin release and oxygen tension (pO(2)). In the presence of 3 mmol/l glucose, insulin release from Niddm1f and Niddm1i islets was approximately 5 pmol. g(-1). s(-1) and pO(2) was 120 mmHg. Similar recordings were obtained from GK and F344 islets. When glucose was raised to 11 mmol/l, insulin release increased significantly in Niddm1f and F344 islets but was essentially unchanged in islets from GK and Niddm1i. The high glucose concentration lowered pO(2) to the same extent in islets from all strains. Addition of 1 mmol/l tolbutamide to the perifusion medium further increased pulsatile insulin release threefold in all islets. The pulse frequency was approximately 0.4 min(-1). alpha-Ketoisocaproate (11 mmol/l) alone increased pulsatile insulin release eightfold in islets from Niddm1f, Niddm1i, and control F344 rats but had no effect on insulin release from GK islets. These secretory patterns in response to alpha-ketoisocaproate were paralleled by reduction of pO(2) in Niddm1f, Niddm1i, and control F344 islets and no change of pO(2) in GK islets. The results demonstrate that Niddm1i carries alleles of gene(s) that reduce glucose-induced insulin release and that are amenable to molecular identification by genetic fine mapping.

Glucose metabolism and pulsatile insulin release from isolated islets.

The effects of metabolic inhibition on insulin release and the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) were studied in individually perifused pancreatic islets from ob/ob mice. The modest basal secretion in the presence of 3 mmol/l glucose was pulsatile with a frequency of approximately 0.2/min, although [Ca(2+)](i) was stable at approximately 100 nmol/l. Introduction of 11 mmol/l glucose resulted in large amplitude oscillations of [Ca(2+)](i) and almost 20-fold stimulation of average secretion manifested as increased amplitude of the insulin pulses without change in frequency. Inhibition of glycolysis with iodoacetamide or mitochondrial metabolism with dinitrophenol or antimycin A reduced glucose-stimulated secretion back to basal levels with maintained pulsatility. The [Ca(2+)](i) responses to the metabolic inhibitors were more complex, but in general there was an initial peak and eventually sustained elevation without oscillations. When introduced in the presence of 3 mmol/l glucose, the metabolic inhibitors tended to increase the amplitude of the insulin pulses, although the simultaneous elevation in [Ca(2+)](i) occurred without oscillations. The data indicate that pulsatile secretion is regulated by factors other than [Ca(2+)](i) under basal conditions and after metabolic inhibition. Although pulsatile secretion can be driven by oscillations in metabolism when [Ca(2+)](i) is stable, it was not possible from the present data to determine whether insulin pulses have a glycolytic or mitochondrial origin.

Glucose-regulated pulsatile insulin release from mouse islets via the K(ATP) channel-independent pathway.

OBJECTIVE: Regulation of insulin release by glucose involves dual pathways, including or not inhibition of ATP-sensitive K(+) channels (K(ATP) channels). Whereas the K(ATP) channel-dependent pathway produces pulsatile release of insulin it is not clear whether the independent pathway also generates such kinetics. DESIGN AND METHODS: To clarify this matter, insulin secretion and cytoplasmic Ca(2+) ([Ca(2+)](i)) were studied in perifused pancreatic islets from ob/ob mice. Insulin release was measured by ELISA technique and [Ca(2+)](i) by dual-wavelength fluorometry. RESULTS: Insulin secretion was pulsatile (0.2--0.3/min) at 3 mmol/l glucose when [Ca(2+)](i) was low and stable. Stimulation with 11 mmol/l of the sugar increased the amplitude of the insulin pulses with maintained frequency and induced oscillations in [Ca(2+)](i). Permanent opening of the K(ATP) channels with diazoxide inhibited glucose-stimulated insulin secretion back to basal levels with maintained pulsatility despite stable and basal [Ca(2+)](i) levels. Increase of the K(+) concentration to 30.9 mmol/l in the continued presence of diazoxide and 11 mmol/l glucose restored the secretory rate with maintained pulsatility and caused stable elevation in [Ca(2+)](i). Simultaneous introduction of diazoxide and elevation of K(+) augmented average insulin release almost 30-fold in 3 mmol/l glucose with maintained pulse frequency. Subsequent elevation of the glucose concentration to 11 and 20 mmol/l increased the release levels. After prolonged exposure to diazoxide, elevated K(+) and 20 mmol/l glucose, the pulse frequency decreased significantly. CONCLUSIONS: Not only glucose signaling via the K(ATP) channel-dependent but also that via the independent pathway generates amplitude-modulated pulsatile release of insulin from isolated islets.

Amplitude modulation of pulsatile insulin secretion by intrapancreatic ganglion neurons.

Neuron activity and insulin release were measured simultaneously from 33 preparations of intrapancreatic canine ganglia and pancreatic parenchyma adjacent to the ganglia. The electrical activity of single neurons of the ganglia was recorded with intracellular microelectrodes, and insulin release from the attached islets was determined with an enzyme-linked immunosorbent assay. Insulin release was 62 +/- 18 fmol preparation/min in the presence of 10 mmol/l glucose and pulsatile (3.7 +/- 0.4 min/pulse). Corresponding measurements of neuronal electrical activity showed a stable membrane potential of -53.5 +/- 0.6 mV. Short, high-frequency (20 Hz) preganglionic nerve stimulation evoked action potentials and, in 46% of the preparations, a threefold rise in the insulin secretory rate associated with increased amplitude of the insulin pulses. The effects were blocked by 10 micromol/l tetrodotoxin (TTX). In other preparations, continuous low-frequency (0.05-0.5 Hz) preganglionic nerve stimulation evoked action potentials and, in 50% of the preparations, a gradual increase of insulin release associated with augmentation of insulin pulse amplitude without alteration of the duration. The effects were blocked by 50 micromol/l hexamethonium (HEX). In the remaining preparations, no change in insulin release was observed during nerve stimulation. In the absence of stimulation, neither TTX nor HEX affected the membrane potential or insulin secretion. These first simultaneous measurements of intrapancreatic ganglion activity and insulin secretion are consistent with amplitude modulation of pulsatile insulin secretion induced by changes in electrical activity in a population of intrapancreatic ganglion neurons.

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 x g(-1) x 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 (r2 = 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.

Pathophysiology of impaired pulsatile insulin release.

Plasma insulin displays 5-10 min oscillations. In Type 2 diabetes the regularity of the oscillations disappears, which may lead to insulin receptor down-regulation and glucose intolerance and explain why pulsatile delivery of the hormone has a greater hypoglycemic effect than continuous delivery. The rhythm is intrinsic to the islet. Variations in metabolism, cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), other hormones, neuronal signaling and possibly beta-cell insulin receptor expression have been implicated in the regulation of plasma insulin oscillations. Most of these factors are important for amplitude-regulation of the insulin pulses. Although evidence exists supporting a role of both metabolism and [Ca(2+)](i) as pacemakers of the pulses, metabolic oscillations probably have a primary role and [Ca(2+)](i) oscillations a permissive role. Results from islets from animal models of diabetes suggest that altered plasma insulin pattern could be due to lowering of pulse amplitude of insulin oscillations rather than alterations in their frequency. Supporting a role of metabolism, altered plasma insulin oscillations were found in MODY2, MIDD and glycogenosis Type VII, which are linked to alterations in glucokinase, mitochondrial tRNALeu(UUR) and phosphofructokinase. Plasma insulin oscillations require coordination of islet secretory activities in the pancreas. The intrapancreatic ganglia have been suggested as coordinators. The diabetes-associated neuropathy may contribute to the deranged pattern as indicated by glucose intolerance in chagasic patients. Continued investigation of the role and regulation of pulsatile insulin release will lead to better understanding of the pathophysiology of impaired pulsatile insulin release, which could lead to new approaches to restore normal plasma insulin oscillations in diabetes and related diseases.

Preserved pulsatile insulin release from prediabetic mouse islets.

During the development of type I diabetes, the plasma insulin pattern changes. Because the islet secretory pattern has been implicated in this phenomenon, insulin release was measured from female nonobese diabetic (NOD) mouse islets isolated at different ages. Islets from 5-week-old mice were used as controls because they had no infiltrating mononuclear cells and insulin release rose almost 9-fold with maintained oscillatory frequency when the glucose concentration was raised from 3 to 11 mM. Islets isolated from 13- and 25-week-old mice were infiltrated with mononuclear cells. In these islets, increase in the glucose concentration from 3 to 11 mM only doubled insulin release. However, despite the cellular infiltration, insulin release was pulsatile. Islets from 13-week-old mice had reduced glucose oxidation rate. Culture of such islets for 7 days at 11.1 mM glucose causes a decrease in the number of mononuclear cells infiltrating the islets, which in the present study was accompanied by a normalization of both glucose oxidation and glucose-induced insulin release. In the presence of the mitochondrial substrate alpha-keto-isocaproate (5 mM) both control and infiltrated islets responded with pronounced insulin pulses with similar amplitudes. The results suggest that the deranged plasma insulin pattern observed during the development of type I diabetes may be related to decrease in the insulin pulse amplitude rather than loss of the pulsatile release from the islets.

Appearance of glucose-induced insulin release in fetal rat beta-cells.

Fetal rat pancreatic cells were isolated from pancreatic primordia on days 12-14 of pregnancy and cultured for 48 h in the presence of 5 mmol/l glucose. Insulin accumulation in the medium over the next 24 h was measured. Cultured cells from day 12 fetuses secreted about 1 fmol insulin per pancreas in response to 5 or 15 mmol/l glucose irrespective of whether 1 mmol/l tolbutamide, 400 mumol/l diazoxide, 5 mmol/l theophylline or 10 mmol/l mannoheptulose was present. In contrast, insulin released from day 13 cultured cells increased significantly from 3.0 +/- 0.6 to 6.2 +/- 2.2 fmol per pancreas, when the glucose concentration was raised. Tolbutamide increased, diazoxide and mannoheptulose decreased and theophylline had no effect on insulin release. Even more pronounced effects were found on insulin release from day 14 cultured cells, in which theophylline also increased the release. In addition, insulin release from cells from pregnancy day 14 was 75 +/- 16 amol/min per pancreas when the cells were perifused for 15-20 min in the presence of 5 mmol/l glucose within 3 h of isolation. Increasing the glucose concentration to 15 mmol/l or adding tolbutamide increased, whereas diazoxide decreased, insulin release in the freshly isolated cells. The insulin content of rat pancreata from pregnancy day 13 was 0.06 +/- 0.01 pmol per pancreas and increased approximately 10-fold every second day up to 6.7 +/- 0.9 pmol on day 17 of pregnancy. Between day 17 and 19 the pancreatic insulin content increased about fivefold to 39 +/- 2 pmol. The present data suggest that critical components of the insulin-secretory machinery, including ATP-regulated K+ channels, glucokinase and adenylate cyclase activities, are present in the developing beta-cell earlier than hitherto thought.

Glucose-induced pulsatile insulin release from single islets at stable and oscillatory cytoplasmic Ca2+.

The cytoplasmic Ca2+ concentration ([Ca2+]i) and insulin release were measured simultaneously in mouse pancreatic islets cultured overnight. [Ca2+]i was 105 nM and insulin release 3 pmol.g-1.s-1 at 3 mM glucose. An increase to 7 mM glucose reduced [Ca2+]i transiently, whereas insulin release doubled and was pulsatile with a frequency of 0.47 min-1. [Ca2+]i oscillations with similar frequency appeared at 11 mM glucose associated with increased amplitude of the insulin oscillations, raising the secretory rate 10-fold. In the presence of 16 and 20 mM glucose [Ca2+]i was > 300 nM and showed no oscillations apart from two islets, which demonstrated [Ca2+]i oscillations with small amplitude at 16 mM glucose. Insulin release with maintained frequency increased by 46 and 31%, respectively. When the glucose concentration was increased from 3 to 11 mM, [Ca2+]i decreased with a nadir that appeared significantly earlier than when the glucose concentration was raised from 3 to 7 mM. Glucose-induced insulin release from the isolated islet is pulsatile both at stable and oscillatory [Ca2+]i, with changes in secretory rate caused by the sugar also when [Ca2+]i is unchanged.

Pulsatile insulin release: role of cytoplasmic Ca2+ oscillations.

Oscillations of plasma insulin are essential for the hypoglycaemic effect of the hormone. Disturbance and partial loss of these oscillations occur during the development of Type 2 diabetes, in association with down-regulation of insulin receptors and insulin resistance. Oscillations with a frequency similar to that of plasma insulin have been observed in the cytoplasmic Ca2+ concentration ([Ca2+]i) of pancreatic beta cells, indicating that the ion plays a role in generating insulin pulses. Studies of individual islets have revealed that oscillations of [Ca2+]i and insulin release are synchronous. However, insulin release is also pulsatile under conditions in which [Ca2+]i is stable. These results support the notion that variations in the ATP/ADP ratio are sufficient to induce pulsatile insulin release. Under physiological conditions, this pulsatility may depend on the synergistic effects of ATP/ADP and [Ca2+]i oscillations.

Pulsatile insulin release from pancreatic islets with nonoscillatory elevation of cytoplasmic Ca2+.

The relationship between insulin release and cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in isolated pancreatic islets from ob/ob mice. Although [Ca2+]i was low and stable in the presence of 3 mM glucose, basal insulin release exhibited low amplitude pulsatility, with a frequency of 0.32 +/- 0.04 min-1. Depolarization by raising K+ from 5.9 to 30.9 mM or by the addition of 1 mM tolbutamide caused a pronounced initial insulin pulse followed by declining pulses, but there was no change in frequency. This decline in amplitude of the insulin pulses was prevented in similar experiments performed in the presence of 11 mM glucose. Corresponding measurements of [Ca2+]i in islets exposed to tolbutamide or the high K+ concentration revealed stable elevations without oscillations. Although the [Ca2+]i level is an important determinant for the rate of secretion, the results indicate that pulsatile insulin release does not always depend on [Ca2+]i oscillations. It is suggested that cyclic generation of ATP may fuel pulsatile release under conditions when [Ca2+]i remains stable.