Enhancing effects of long-term elevated glucose and palmitate on stored and secreted proinsulin-to-insulin ratios in human pancreatic islets.

Relative hypersecretion of proinsulin is a feature of type 2 diabetes. We investigated to what extent this feature can be induced in human pancreatic islets by elevated glucose or fatty acids, two major abnormalities of the diabetic state. A 48-h culture period with 27 mmol/l glucose increased the intraislet proinsulin-to-insulin (PI/I) ratio 5.0-fold, owing to preferential decrease of insulin. The PI/I ratio in culture medium was enhanced 1.9-fold versus islets cultured with 5.5 mmol/l glucose. This effect of elevated glucose persisted after normalization of glucose levels: during 60-min postculture incubations at a basal glucose concentration (3.3 mmol/l), the PI/I ratio of secretion increased 4.9-fold. The ratio was also increased (14-fold) after renewed postculture stimulation with 16.7 mmol/l glucose. Diazoxide was added to culture medium to block glucose-induced insulin secretion and thus investigate the importance of overstimulation. In cultures at 27 mmol/l glucose, the presence of diazoxide decreased the PI/I ratio of islet contents by 76%, the accumulated secretion to culture medium by 70%, and the release at 3.3 or 16.7 mmol/l glucose during postculture incubations by 85 and 86%, respectively. None of these PI/I-decreasing effects of diazoxide were reproduced during or after coculture with 5.5 mmol/l glucose. Culture with 0.2 mmol/l palmitate and 5.5 mmol/l glucose decreased islet contents of proinsulin and insulin and increased the secreted products in culture media without affecting PI/I ratios. During postculture conditions, however, prior palmitate culture enhanced the PI/I ratio of release at 3.3 mmol/l glucose (from 2.2 +/- 0.4 to 5.4 +/- 0.9%, P < 0.05). Culture with palmitate together with 27 mmol/l glucose decreased islet contents of proinsulin and insulin and further enhanced intraislet PI/I ratios (from 9.3 +/- 1.1 to 13.4 +/- 2.5%, P < 0.05). However, palmitate failed to affect PI/I ratios in culture medium. In contrast, in postculture incubations at 3.3 mmol/l glucose, prior palmitate culture further elevated the PI/I ratio of secretion (from 10.8 +/- 1.2 after previous 27 mmol/l glucose alone to 13.9 +/- 2.8% after palmitate and glucose, P < 0.05). We conclude that 1) long-term exposure of human islets to elevated glucose leads to preferential secretion of proinsulin, and this effect persists also after glucose normalization; 2) the glucose effect appears secondary to depletion of mature insulin granules; and 3) elevated fatty acids influence PI/I ratios of secretion by mechanisms that are, in part, incongruous with an over-stimulation effect.     

Differential patterns of glucose-induced electrical activity and intracellular calcium responses in single mouse and rat pancreatic islets

Although isolated rat islets are widely used to study in vitro insulin secretion and the underlying metabolic and ionic processes, knowledge on the properties of glucose-induced electrical activity (GIEA), a key step in glucose-response coupling, has been gathered almost exclusively from microdissected mouse islets. Using a modified intracellular recording technique, we have now compared the patterns of GIEA in collagenase-isolated rat and mouse islets. Resting membrane potentials of rat and mouse beta-cells were approximately -50 and -60 mV, respectively. Both rat and mouse beta-cells displayed prompt membrane depolarizations in response to glucose. However, whereas the latter exhibited a bursting pattern consisting of alternating hyperpolarized and depolarized active phases, rat beta-cells fired action potentials from a nonoscillating membrane potential at all glucose concentrations (8.4-22.0 mmol/l). This was mirrored by changes in the intracellular Ca2+ concentration ([Ca2+]i), which was oscillatory in mouse and nonoscillatory in rat islets. Stimulated rat beta-cells were strongly hyperpolarized by diazoxide, an activator of ATP-dependent K+ channels. Glucose evoked dose-dependent depolarizations and [Ca2+]i increases in both rat (EC50 5.9-6.9 mmol/l) and mouse islets (EC50 8.3-9.5 mmol/l), although it did not affect the burst plateau potential in the latter case. We conclude that there are important differences between beta-cells from both species with respect to early steps in the stimulus-secretion coupling cascade based on the following findings: 1) mouse beta-cells have a larger resting K+ conductance in 2 mmol/l glucose, 2) rat beta-cells lack the compensatory mechanism responsible for generating membrane potential oscillations and holding the depolarized plateau potential in mouse beta-cells, and 3) the electrical and [Ca2+]i dose-response curves in rat beta-cells are shifted toward lower glucose concentrations. Exploring the molecular basis of these differences may clarify several a priori assumptions on the electrophysiological properties of rat beta-cells, which could foster the development of new working models of pancreatic beta-cell function.     

Altered cAMP and Ca2+ signaling in mouse pancreatic islets with glucagon-like peptide-1 receptor null phenotype.

1-Cells from rodents and humans express different receptors recognizing hormones of the secretin-glucagon family, which--when activated--synergize with glucose in the control of insulin release. We have recently reported that isolated islets from mice homozygous for a GLP-1 receptor null mutation (GLP-1R(-/-)) exhibit a well-preserved insulin-secretory response to glucose. This observation can be interpreted in two different ways: 1) the presence of GLP-1R is not essential for the secretory response of isolated islets to glucose alone; 2) beta-cells in GLP-1R(-/-) pancreases underwent compensatory changes in response to the null mutation. To explore these possibilities, we studied islets from control GLP-IR(+/+) mice in the absence or presence of 1 pmol/l exendin (9-39)amide, a specific and potent GLP-1R antagonist. Exendin (9-39)amide (15-min exposure) reduced glucose-induced insulin secretion from both perifused and statically incubated GLP-1R(+/+) islets by 50% (P < 0.05), and reduced islet cAMP production in parallel (P < 0.001). Furthermore, GLP-1R(-/-) islets exhibited: 1) reduced cAMP accumulation in the presence of 20 mmol/l glucose (knockout islets versus control islets, 12 +/- 1 vs. 27 +/- 3 fmol x islet(-1) x 15 min(-1); P < 0.001) and exaggerated acceleration of cAMP production by 10 nmol/l glucose-dependent insulinotropic peptide (GIP) (increase over 20 mmol/l glucose by GIP in knockout islets versus control islets: 66 +/- 5 vs. 14 +/- 3 fmol x islet(-1) x 15 min(-1); P < 0.001); 2) increased mean cytosolic [Ca2+] ([Ca2+]c) at 7, 10, and 15 mmol/l glucose in knockout islets versus control islets; and 3) signs of asynchrony of [Ca2+]c oscillations between different islet subregions. In conclusion, disruption of GLP-1R signaling is associated with reduced basal but enhanced GIP-stimulated cAMP production and abnormalities in basal and glucose-stimulated [Ca2+]c. These abnormalities suggest that GLP-1R signaling is an essential upstream component of multiple beta-cell signaling pathways.     

Chronology of cellular events leading to derangements in function of pancreatic islets in chronic renal failure.

In chronic renal failure (CRF), a multitude of metabolic derangements occur in the pancreatic islets, resulting in impaired glucose-induced insulin secretion. These abnormalities include a rise in the basal level of cytosolic calcium ([Ca2+]i) in the islet, a decrease in their basal and stimulated ATP and ATP/ADP ratio, a reduction in the Vmax of Ca2+ATPase and Na(+)-K+ATPase, and an impaired glucose-induced calcium signal. The sequence of events that lead to these derangements and to the impairment in insulin secretion during the evolution of CRF are not defined. The study presented here examined this issue by measuring the metabolic profile of pancreatic islets weekly during the evolution of CRF over a period of 6 wk. The results show that serum levels of parathyroid hormone (PTH) begin to rise during the first week of CRF. The Vmax of Ca2+ATPase and Na(+)-K+ATPase increased during weeks 1 to 3 of CRF but fell to low levels thereafter. At week 3 of CRF, the basal level of [Ca2+]i began to rise, whereas basal and the stimulated ATP content and ATP/ADP ratio started to fall. Glucose-induced calcium signal, delta[Ca2+]i/basal [Ca2+]i, and insulin secretion became abnormally low between weeks 3 and 6 of CRF. The data allow the following formulation: as serum levels of PTH begins to rise, calcium entry into islets is augmented; this in turn will stimulate the activity of Ca2+ATPase and the Na(+)-Ca2+ exchanger, and hence, calcium extrusion out of the islets is increased. As a result, [Ca2+] remains normal during the first 2 wk of CRF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous ghrelin in pancreatic islets restricts insulin release by attenuating Ca2+ signaling in beta-cells: implication in the glycemic control in rodents

Ghrelin, isolated from the human and rat stomach, is the endogenous ligand for the growth hormone (GH) secretagogue receptor, which is expressed in a variety of tissues, including the pancreatic islets. It has been shown that low plasma ghrelin levels correlates with elevated fasting insulin levels and type 2 diabetes. Here we show a physiological role of endogenous ghrelin in the regulation of insulin release and blood glucose in rodents. Acylated ghrelin, the active form of the peptide, was detected in the pancreatic islets. Counteraction of endogenous ghrelin by intraperitoneal injection of specific GH secretagogue receptor antagonists markedly lowered fasting glucose concentrations, attenuated plasma glucose elevation, and enhanced insulin responses during the glucose tolerance test (GTT). Conversely, intraperitoneal exogenous ghrelin GH-independently elevated fasting glucose concentrations, enhanced plasma glucose elevation, and attenuated insulin responses during GTT. Neither GH secretagogue receptor antagonist nor ghrelin affected the profiles of the insulin tolerance test. In isolated islets, GH secretagogue receptor blockade and antiserum against acylated ghrelin markedly enhanced glucose-induced increases in insulin release and intracellular Ca2+ concentration ([Ca2+]i), whereas ghrelin at a relatively high concentration (10 nmol/l) suppressed insulin release. In single beta-cells, ghrelin attenuated glucose-induced first-phase and oscillatory [Ca2+]i increases via the GH secretagogue receptor and in a pertussis toxin-sensitive manner. Ghrelin also increased tetraethylammonium-sensitive delayed outward K+ currents in single beta-cells. These findings reveal that endogenous ghrelin in islets acts on beta-cells to restrict glucose-induced insulin release at least partly via attenuation of Ca2+ signaling, and that this insulinostatic action may be implicated in the upward control of blood glucose. This function of ghrelin, together with inducing GH release and feeding, suggests that ghrelin underlies the integrative regulation of energy homeostasis.     

Measurements of cytoplasmic Ca2+ in islet cell clusters show that glucose rapidly recruits beta-cells and gradually increases the individual cell response

The proportion of isolated single beta-cells developing a metabolic, biosynthetic, or secretory response increases with glucose concentration (recruitment). It is unclear whether recruitment persists in situ when beta-cells are coupled. We therefore measured the cytoplasmic free Ca2+ correction ([Ca2+]i) (the triggering signal of glucose-induced insulin secretion) in mouse islet single cells or clusters cultured for 1-2 days. In single cells, the threshold glucose concentration ranged between 6 and 10 mmol/l, at which concentration a maximum of approximately 65% responsive cells was reached. Only 13% of the cells did not respond to glucose plus tolbutamide. The proportion of clusters showing a [Ca2+]i rise increased from approximately 20 to 95% between 6 and 10 mmol/l glucose, indicating that the threshold sensitivity to glucose differs between clusters. Within responsive clusters, 75% of the cells were active at 6 mmol/l glucose and 95-100% at 8-10 mmol/l glucose, indicating that individual cell recruitment is not prominent within clusters; in clusters responding to glucose, all or almost all cells participated in the response. Independently of cell recruitment, glucose gradually augmented the magnitude of the average [Ca2+]i rise in individual cells, whether isolated or associated in clusters. When insulin secretion was measured simultaneously with [Ca2+]i, a good temporal and quantitative correlation was found between both events. However, beta-cell recruitment was maximal at 10 mmol/l glucose, whereas insulin secretion increased up to 15-20 mmol/l glucose. In conclusion, beta-cell recruitment by glucose can occur at the stage of the [Ca2+]i response. However, this type of recruitment is restricted to a narrow range of glucose concentrations, particularly when beta-cell association decreases the heterogeneity of the responses. Glucose-induced insulin secretion by islets, therefore, cannot entirely be ascribed to recruitment of beta-cells to generate a [Ca2+]i response. Modulation of the amplitude of the [Ca2+]i response and of the action of Ca2+ on exocytosis (amplifying actions of glucose) may be more important.     

Oscillations of insulin secretion can be triggered by imposed oscillations of cytoplasmic Ca2+ or metabolism in normal mouse islets

Glucose-induced insulin secretion depends on an acceleration of glucose metabolism, requires a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i), and is modulated by activation of protein kinases in beta-cells. Normal mouse islets were used to determine whether oscillations of these three signals are able and necessary to trigger oscillations of insulin secretion. The approach was to minimize or abolish spontaneous oscillations and to compare the impact of forced oscillations of each signal on insulin secretion. In a control medium, repetitive increases in the glucose concentration triggered oscillations in metabolism [NAD(P)H fluorescence], [Ca2+]i (fura-PE3 method), and insulin secretion. In the presence of diazoxide, metabolic oscillations persisted, but [Ca2+]i and insulin oscillations were abolished. When the islets were depolarized with high K+ with or without diazoxide, [Ca2+]i was elevated, and insulin secretion was stimulated. Forced metabolic oscillations transiently decreased or did not affect [Ca2+]i and potentiated insulin secretion with oscillations of small amplitude. These oscillations of secretion followed metabolic oscillations only when [Ca2+]i did not change. When [Ca2+]i fluctuated, these changes prevailed over those of metabolism for timing secretion. Repetitive depolarizations with high K+ in the presence of stable glucose (10 mmol/l) induced synchronous pulses of [Ca2+]i and insulin secretion with only small oscillations of metabolism. Continuous stimulation of protein kinase A (PKA) and protein kinase C (PKC) did not dissociate the [Ca2+]i and insulin pulses from the high K+ pulses. However, the amplitude of the insulin pulses was consistently increased, whereas that of the [Ca2+]i pulses was either increased (PKA) or decreased (PKC). In conclusion, metabolic oscillations can induce oscillations of insulin secretion independently of but with a lesser effectiveness than [Ca2+]i oscillations. Although oscillations in metabolism may cyclically influence secretion through an ATP-sensitive K+ channel (K+-ATP channel)-independent pathway, their regulatory effects are characterized by a hysteresis that makes them unlikely drivers of fast oscillations, unless they also involve [Ca2+]i changes through the K+-ATP channel-dependent pathway.     

Inhibition of purinoceptors amplifies glucose-stimulated insulin release with removal of its pulsatility

External ATP has been proposed to be an autocrine regulator of glucose-stimulated insulin secretion and responsible for the synchronization of the Ca2+ rhythmicity in the beta-cells required for a pulsatile release of insulin from the pancreas. The importance of external ATP for glucose-stimulated insulin release was evaluated in rats with the aid of 2-deoxy-N-methyladenosine-3,5-bisphosphate (MRS 2179), an inhibitor of the purinoceptors known to affect the Ca2+ signaling in beta-cells. The concentration of cytoplasmic Ca2+ was measured in single beta-cells and small aggregates with ratiometric fura-2 technique and the release of insulin recorded from isolated islets and the perfused pancreas. Addition of 1 micromol/l ATP induced premature cytoplasmic Ca2+ concentration ([Ca2+]i) oscillations similar to those found in beta-cells exposed to 20 mmol/l glucose. In most experiments, the presence of 10 micromol/l MRS 2179 did not remove the glucose-induced [Ca2+]i rhythmicity in single beta-cells or the synchronization seen in coupled cells. Nevertheless, the same concentration of MRS 2179 promptly interrupted the pulsatility (frequency 0.22 +/- 0.01/min) of insulin secretion, raising the total amounts released from the pancreas. Prolonged exposure of islets to 1 and 10 micromol/l MRS 2179 enhanced insulin secretion at 20 mmol/l glucose 33% (P < 0.05) and 63% (P < 0.01), respectively, without affecting the release at 3 mmol/l glucose. The results support the idea that neural ATP signals entrain the islets into a common rhythm resulting in pulsatile release of insulin and that glucose stimulation of the secretory activity is counteracted by accumulation of inhibitory ATP around the beta-cells.     

Impaired glucose-induced calcium signal in pancreatic islets in chronic renal failure.

Basal level of cytosolic calcium ([Ca2+]i) is elevated in islets of rats with chronic renal failure (CRF). The high [Ca2+]i level was implicated in the impaired insulin secretion of CRF, and its effect is due, in part, to a reduction in ATP content and impaired glucose metabolism by the islets. However, elevated [Ca2+]i may interfere with insulin secretion via another pathway. Exposure of the islets to glucose causes an acute rise in [Ca2+]i which generates events leading to insulin secretion. It is possible that a sustained rise in [Ca2+]i interferes with the magnitude of glucose-induced calcium signal and the ratio between this signal and basal [Ca2+]i. We examined this question in normal, CRF, normocalcemic CRF-PTX rats and in CRF rats treated with verapamil (CRF-V). Basal [Ca2+]i was higher (p less than 0.01) in CRF (130 +/- 7.0 nM) than in normal (82 +/- 5.5 nM), CRF-PTX (75 +/- 3.6 nM) and CRF-V rats (84 +/- 3.8 nM). Glucose-induced calcium signal (95 +/- 10.4 nM) and the ratio between this signal and basal [Ca2+]i (0.73 +/- 0.07) in CRF rats were lower (p less than 0.01) than in normal (153 +/- 14.4 nM; 1.90 +/- 0.24), CRF-PTX (130 +/- 16.7 nM; 1.75 +/- 0.25) and CRF-V (124 +/- 5.8 nM; 1.90 +/- 0.12) rats despite high PTH in the latter. The data indicate that a sustained rise in [Ca2+]i of islets interferes with the glucose-induced calcium signal which in turn plays a role in impaired insulin secretion.     

Mechanisms of glucose hypersensitivity in beta-cells from normoglycemic, partially pancreatectomized mice.

Increased beta-cell sensitivity to glucose precedes the loss of glucose-induced insulin secretion in diabetic animals. Changes at the level of beta-cell glucose sensor have been described in these situations, but it is not clear whether they fully account for the increased insulin secretion. Using a euglycemic-normolipidemic 60% pancreatectomized (60%-Px) mouse model, we have studied the ionic mechanisms responsible for increased beta-cell glucose sensitivity. Two weeks after Px (Px14 group), Px mice maintained normoglycemia with a reduced beta-cell mass (0.88 +/- 0.18 mg) compared with control mice (1.41 +/- 0.21 mg). At this stage, the dose-response curve for glucose-induced insulin release showed a significant displacement to the left (P < 0.001). Islets from the Px14 group showed oscillatory electrical activity and cytosolic Ca2+ ([Ca2+]i) oscillations in response to glucose concentrations of 5.6 mmol/l compared with islets from the control group at 11.1 mmol/l. All the above changes were fully reversible both in vitro (after 48-h culture of islets from the Px14 group) and in vivo (after regeneration of beta-cell mass in islets studied 60 days after Px). No significant differences in the input resistance and ATP inhibition of ATP-sensitive K+ (K(ATP)) channels were found between beta-cells from the Px14 and control groups. The dose-response curve for glucose-induced MTT (C,N-diphenyl-N'-4,5-dimethyl thiazol 2 yl tetrazolium bromide) reduction showed a significant displacement to the left in islets from the Px14 group (P < 0.001). These results indicate that increased glucose sensitivity in terms of insulin secretion and Ca2+ signaling was not due to intrinsic modifications of K(ATP) channel properties, and suggest that the changes are most likely to be found in the glucose metabolism.     

Different effects of tolbutamide and diazoxide in alpha, beta-, and delta-cells within intact islets of Langerhans

Interaction between the different types of cells within the islet of Langerhans is vital for adequate control of insulin release. Once insulin secretion becomes defective, as in type 2 diabetes, the most useful drugs to increase insulin release are sulfonylureas. It is well-known that sulfonylureas block K(ATP) channels, which results in depolarization of the membrane that provokes calcium influx and increases intracellular calcium concentration ([Ca2+]i), which thereby triggers insulin secretion. The sulfonamide diazoxide produces the opposite effect: it activates K(ATP) channels, resulting in a decreased insulin secretion. Despite such evidence, little is known about the effect of sulfonylureas and sulfonamides in non-beta-cells of the islet of Langerhans. In this article, we describe the effects of tolbutamide and diazoxide on [Ca2+]i in alpha-, beta-, and delta-cells within intact islets of Langerhans. Tolbutamide elicits an increase in [Ca2+li in beta- and delta-cells, regardless of glucose concentrations. Remarkably, tolbutamide is without effect in alpha-cells. When diazoxide is applied, glucose-induced [Ca2+]i oscillations in beta- and delta-cells are abolished, whereas [Ca2+]i oscillations in alpha-cells remain unaltered. Furthermore, the existence of sulfonylurea receptors is demonstrated in beta-cells but not in alpha-cells by using binding of glybenclamide-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) combined with immunostaining for insulin and glucagon.     

Metformin restores insulin secretion altered by chronic exposure to free fatty acids or high glucose: a direct metformin effect on pancreatic beta-cells

Because metformin affects glucose and free fatty acid (FFA) metabolism in peripheral insulin target tissues, we investigated the effect of this drug in restoring a normal secretory pattern in rat pancreatic islets whose function has been impaired by chronic exposure to elevated FFA or glucose concentrations. We cultured rat pancreatic islets with or without FFA (2 mmol/l oleate/palmitate 2:1) or high glucose (16.7 mmol/l) concentrations in the presence or absence of metformin (0.25-12.5 microg/ml) and then measured insulin release, glucose utilization, glucose, and FFA oxidation. When compared with control islets, islets exposed to high FFA or glucose concentrations showed an increased basal and a decreased glucose-induced insulin release. In islets cultured for an additional 24 h with FFA or glucose in the presence of metformin (2.5 microg/ml), both basal and glucose-induced insulin secretions were restored. Both glucose utilization and glucose oxidation were altered in islets pre-exposed to high FFA or glucose concentrations. In particular, regarding control islets, glucose utilization was increased at 2.8 mmol/l glucose and decreased at 16.7 mmol/l glucose; glucose oxidation was similar to control islets at 2.8 mmol/l glucose but decreased at 16.7 mmol/l glucose. In contrast, oleate oxidation was increased in islets pre-exposed to FFA. All of these abnormalities were reversed in islets cultured for an additional 24 h with high FFA or glucose concentrations in the presence of metformin (2.5 microg/ml). In conclusion, our data show that metformin is able to restore the intracellular abnormalities of glucose and FFA metabolism and to restore a normal secretory pattern in rat pancreatic islets whose secretory function has been impaired by chronic exposure to elevated FFA or glucose levels. These data raise the possibility that, in diabetic patients, metformin (in addition to its peripheral effects) may have a direct beneficial effect on the beta-cell secretory function.     

Increased intracellular calcium is required for spreading of rat islet beta-cells on extracellular matrix

Rat islet beta-cells spread in response to glucose when attached on the matrix produced by a rat bladder carcinoma cell line (804G). Furthermore, in a mixed population of cells, it has been observed previously that spread cells secrete more insulin acutely in response to glucose, compared with cells that remain rounded. These results suggest bi-directional signaling between the islet beta-cell and the extracellular matrix. In the present study, the role of increased intracellular free Ca2+ concentration [Ca2+]i as an intracellular step linking glucose stimulation and beta-cell spreading (inside-out signaling) was investigated. Purified rat beta-cells were attached to this matrix and incubated under various conditions known to affect [Ca2+]i. The effect of glucose on beta-cell spreading was mimicked by 25 mmol/l KCl (which induces calcium influx) and inhibited by diazoxide (which impairs depolarization and calcium entry) and by the L-type Ca2+ channel blocker SR-7037. When a 24-h incubation at 16.7 glucose was followed by 24 h at 2.8 mmol/l, beta-cells that had first spread regained a round phenotype. In the presence of thapsigargin, spreading progressed throughout the experiment, suggesting that capture of calcium by the endoplasmic reticulum is involved in the reversibility of spreading previously induced by glucose. Spreading was still observed in degranulated beta-cells and in botulinum neurotoxin E-expressing beta-cells when exocytosis was prevented. In summary, the results indicate that increased [Ca2+]i is required for the glucose-induced spreading of beta-cells on 804G matrix and that it is not a consequence of exocytotic processes that follow elevation of [Ca2+]i.     

Glucose dependence of imidazoline-induced insulin secretion: different characteristics of two ATP-Sensitive K+ channel-blocking compounds

The glucose dependence of the insulinotropic action of KATP channel-blocking imidazoline compounds was investigated. Administration of 100 micromol/l phentolamine, but not 100 micromol/l efaroxan, markedly increased insulin secretion of freshly isolated mouse islets when the perifusion medium contained 5 mmol/l glucose. When the glucose concentration was raised to 10 mmol/l in the continued presence of either imidazoline, a clear potentiation of secretion occurred as compared with 10 mmol/l glucose alone. In the presence of efaroxan, a brisk first-phase-like increase was followed by a sustained phase, whereas a more gradual increase resulted in the presence of phentolamine. Administration of 100 micromol/l phentolamine was somewhat more effective than 100 micromol/l efaroxan to inhibit KATP channel activity in intact cultured beta-cells (reduction by 96 vs. 83%). Both compounds were similarly effective to depolarize the beta-cells. When measured by the perforated patch-technique, the depolarization by efaroxan was often oscillatory, whereas that by phentolamine was sustained. In perifused cultured islets, both compounds increased the cytosolic calcium concentration ([Ca2+]c) in the presence of 5 and 10 mmol/l glucose. Efaroxan induced large amplitude oscillations of [Ca2+]c, whereas phentolamine induced a sustained increase. It appears that a KATP channel block by imidazolines is not incompatible with a glucose-selective enhancement of insulin secretion. The glucose selectivity of efaroxan may involve an inhibitory effect distal to [Ca2+]c increase and/or the generation of [Ca2+]c oscillations.     

Human anti-CD38 autoantibodies raise intracellular calcium and stimulate insulin release in human pancreatic islets

CD38 is involved in transmembrane signaling in many cell types; anti-CD38 autoantibodies have been described in diabetic patients. We tested whether human anti-CD38 antibodies possess signaling properties by measuring their ability to raise intracellular calcium ([Ca2+]i) using the fluo-3-acetoxymethyl ester method in a human-derived T-cell line (Jurkat T-cells, expressing high levels of surface CD38) and in dispersed human islet cells from normal donors. In Jurkat T-cells, 11 of 19 anti-CD38-positive sera raised [Ca2+]i (by > or =20% of baseline), whereas no [Ca2+]i-mobilizing activity was found in 27 anti-CD38-negative sera (chi2 = 20.5, P < 0.0001). In dispersed human islet cells, 5 of 11 anti-CD38-positive sera (and none of three anti-CD38-negative sera) raised [Ca2+]i significantly. When preincubated with Staphylococcus aureus protein A to remove IgG, anti-CD38-positive sera showed a 70 +/- 5% reduction in [Ca2+]i-mobilizing activity. Preincubation with CD38-transfected NIH-3T3 fibroblasts, but not with mock-transfected NIH-3T3 cells, abolished [Ca2+]i mobilization. In blocking experiments, preincubation with nonagonistic anti-CD38 monoclonal antibodies also prevented [Ca2+]i mobilization. In cultured human islets, anti-CD38-positive sera exhibiting [Ca2+]i-mobilizing activity in Jurkat T-cells (n = 6) significantly stimulated insulin release at 3.3 mmol/l glucose (median [interquartile range] 738 microU/ml [234], P = 0.0001 vs. 320 [52] microU/ml of control), whereas 6 anti-CD38-positive sera without [Ca2+]i-mobilizing activity and 10 anti-CD38-negative did not. In further incubations, the five anti-CD38-positive sera displaying [Ca2+]i-mobilizing activity in dispersed islet cells significantly stimulated insulin release at both 3.3 mmol/l glucose (2.2 +/- 0.3% of insulin islet content, P < 0.002 vs. 1.2 +/- 0.1% of control) and 16.7 mmol/l glucose (3.7 +/- 0.3 vs. 2.3 +/- 0.3%, P < 0.002). We conclude that human anti-CD38 autoantibodies with agonistic properties on the CD38 effector system occur in nature; in human islets, their [Ca2+]i-mobilizing activity is coupled with the ability to stimulate insulin release.     

Overstimulation and beta-cell function

Previous and present evidence ascribes an important role to overstimulation of beta-cells for the secretory abnormalities associated with type 2 diabetes. The abnormality most clearly linked to overstimulation is the elevated ratio of circulating proinsulin to insulin. Evidence obtained in human pancreatic islets suggests that aberrations in insulin oscillations that occur in type 2 diabetes could at least in part be linked to abnormalities in cytoplasmic Ca2+ oscillations induced by overstimulation. Furthermore, in a transplantation model, we have obtained evidence for long-lasting, perhaps irreversible, effects of overstimulation, implying that this is a causative factor for the well-recognized deterioration of insulin secretion with increasing duration of type 2 diabetes. The mechanisms behind the effects of overstimulation are only partly clarified, but it is clear that reduced insulin secretion after overstimulation is only partly explained by decreased insulin stores. In cultured human pancreatic islets, overstimulation by high glucose leads to a rise in cytoplasmic Ca2+ levels, which persists after normalization of the glucose levels. Persistent elevation of cytoplasmic Ca2+ may trigger apoptosis, thus participating in long-term irreversible deterioration of beta-cell function. These data provide sufficient rationale for clinical studies to test the beneficial effects of relative beta-cell rest in type 2 diabetic patients.     

In vivo and in vitro glucose-induced biphasic insulin secretion in the mouse: pattern and role of cytoplasmic Ca2+ and amplification signals in beta-cells

The mechanisms underlying biphasic insulin secretion have not been completely elucidated. We compared the pattern of plasma insulin changes during hyperglycemic clamps in mice to that of glucose-induced insulin secretion and cytosolic calcium concentration ([Ca(2+)](c)) changes in perifused mouse islets. Anesthetized mice were infused with glucose to clamp blood glucose at 8.5 (baseline), 11.1, 16.7, or 30 mmol/l. A first-phase insulin response consistently peaked at 1 min, and a slowly ascending second phase occurred at 16.7 and 30 mmol/l glucose. Glucose-induced insulin secretion in vivo is thus biphasic, with a similarly increasing second phase in the mouse as in humans. In vitro, square-wave stimulation from a baseline of 3 mmol/l glucose induced similar biphasic insulin secretion and [Ca(2+)](c) increases, with sustained and flat second phases. The glucose dependency (3-30 mmol/l) of both changes was sigmoidal with, however, a shift to the right of the relation for insulin secretion compared with that for [Ca(2+)](c). The maximum [Ca(2+)](c) increase was achieved by glucose concentrations, causing half-maximum insulin secretion. Because this was true for both phases, we propose that contrary to current concepts, amplifying signals are also implicated in first-phase glucose-induced insulin secretion. To mimic in vivo conditions, islets were stimulated with high glucose after being initially perifused with 8.5 instead of 3.0 mmol/l glucose. First-phase insulin secretion induced by glucose at 11.1, 16.7, and 30 mmol/l was decreased by approximately 50%, an inhibition that could not be explained by commensurate decreases in [Ca(2+)](c) or in the pool of readily releasable granules. Also unexpected was the gradually ascending pattern of the second phase, now similar to that in vivo. These observations indicated that variations in prestimulatory glucose can secondarily affect the magnitude and pattern of subsequent glucose-induced insulin secretion.     

Glucose-dependent modulation of insulin secretion and intracellular calcium ions by GKA50, a glucokinase activator

Because glucokinase is a metabolic sensor involved in the regulated release of insulin, we have investigated the acute actions of novel glucokinase activator compound 50 (GKA50) on islet function. Insulin secretion was determined by enzyme-linked immunosorbent assay, and microfluorimetry with fura-2 was used to examine intracellular Ca(2+) homeostasis ([Ca(2+)](i)) in isolated mouse, rat, and human islets of Langerhans and in the MIN6 insulin-secreting mouse cell line. In rodent islets and MIN6 cells, 1 micromol/l GKA50 was found to stimulate insulin secretion and raise [Ca(2+)](i) in the presence of glucose (2-10 mmol/l). Similar effects on insulin release were also seen in isolated human islets. GKA50 (1 micromol/l) caused a leftward shift in the glucose-concentration response profiles, and the half-maximal effective concentration (EC(50)) values for glucose were shifted by 3 mmol/l in rat islets and approximately 10 mmol/l in MIN6 cells. There was no significant effect of GKA50 on the maximal rates of glucose-stimulated insulin secretion. In the absence of glucose, GKA50 failed to elevate [Ca(2+)](i) (1 micromol/l GKA50) or to stimulate insulin release (30 nmol/l-10 micromol/l GKA50). At 5 mmol/l glucose, the EC(50) for GKA50 in MIN6 cells was approximately 0.3 micromol/l. Inhibition of glucokinase with mannoheptulose or 5-thioglucose selectively inhibited the action of GKA50 on insulin release but not the effects of tolbutamide. Similarly, 3-methoxyglucose prevented GKA50-induced rises in [Ca(2+)](i) but not the actions of tolbutamide. Finally, the ATP-sensitive K(+) channel agonist diazoxide (200 micromol/l) inhibited GKA50-induced insulin release and its elevation of [Ca(2+)](i.) We show that GKA50 is a glucose-like activator of beta-cell metabolism in rodent and human islets and a Ca(2+)-dependent modulator of insulin secretion.     

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.