Intracellular calcium ion response to glucose in beta-cells of calbindin-D28k nullmutant mice and in betaHC13 cells overexpressing calbindin-D28k

This article describes studies on the glucose-induced responses of intracellular Ca²⁺ concentration ([Ca²⁺]_i), insulin release, and redistribution of calbindin-D_28k, a calcium-binding regulatory protein, in β-cells of pancreatic islets of calbindin-D_28k knockout (KO) and wild-type mice (C57BL6) as well as in βHC-13 control cells and βHC-13 CaBP40 cells (β-cell line overexpressing calbindin-D_28k). Upon increasing the glucose concentration from 2.8 to 30 mM, islets of KO mice showed a significantly greater increase in [Ca²⁺]_i (mean increase in [Ca²⁺]_i, i.e., Δ[Ca²⁺], was 296 nM) compared with wild-type mice (Δ[Ca²⁺]_i=97 nM). βHC-13 CaBP40 cells showed little change in [Ca²⁺]_i upon elevation of glucose from 5.5 to 32.7 mM, whereas βHC-13 control cells exhibited significant increases in [Ca²⁺]_i (Δ[Ca²⁺]_i=510 nM). Similarly, upon addition of 30 mM glucose, the rate of insulin release increased from 25.2 (basal rate) to 145.2 pg/mL/min in βHC-13 control cells, whereas in βHC-13 CaBP40 cells the rate of insulin release was only 27.5 pg/mL/min in high glucose. Thus, levels of calbindin-D_28k in β-cells affect both [Ca²⁺]_i and insulin secretion in response to glucose. The three-dimensional reconstruct of confocal immunofluorescent images showed that glucose caused redistribution of calbindin-D_28k resulting in co-localization in the region of L-type voltage-dependent calcium channels (VDCC). This colocalization may be an important regulatory function concerning Ca²⁺ influx via L-type VDCC and exocytosis of insulin granules.     

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.     

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

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

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

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

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

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

Glucose inhibits glucagon secretion by a direct effect on mouse pancreatic alpha cells

Aims/hypothesis The mechanisms by which glucose regulates glucagon release are poorly understood. The present study aimed to clarify the direct effects of glucose on the glucagon-releasing alpha cells and those effects mediated by paracrine islet factors. Materials and methods Glucagon, insulin and somatostatin release were measured from incubated mouse pancreatic islets and the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) recorded in isolated mouse alpha cells. Results Glucose inhibited glucagon release with maximal effect at 7 mmol/l. Since this concentration corresponded to threshold stimulation of insulin secretion, it is unlikely that inhibition of glucagon secretion is mediated by beta cell factors. Although somatostatin secretion data seemed consistent with a role of this hormone in glucose-inhibited glucagon release, a somatostatin receptor type 2 antagonist stimulated glucagon release without diminishing the inhibitory effect of glucose. In islets exposed to tolbutamide plus 8 mmol/l K⁺, glucose inhibited glucagon secretion without stimulating the release of insulin and somatostatin, indicating a direct inhibitory effect on the alpha cells that was independent of ATP-sensitive K⁺ channels. Glucose lowered [Ca²⁺]_i of individual alpha cells independently of somatostatin and beta cell factors (insulin, Zn²⁺ and γ-aminobutyric acid). Glucose suppression of glucagon release was prevented by inhibitors of the sarco(endo)plasmic reticulum Ca²⁺-ATPase, which abolished the [Ca²⁺]_i-lowering effect of glucose on isolated alpha cells. Conclusions/interpretation Beta cell factors or somatostatin do not seem to mediate glucose inhibition of glucagon secretion. We instead propose that glucose has a direct inhibitory effect on mouse alpha cells by suppressing a depolarising Ca²⁺ store-operated current.     

Loss of high-frequency glucose-induced Ca2+ oscillations in pancreatic islets correlates with impaired glucose tolerance in Trpm5?/? mice

Glucose homeostasis is critically dependent on insulin release from pancreatic β-cells, which is strictly regulated by glucose-induced oscillations in membrane potential (V_m) and the cytosolic calcium level ([Ca²⁺]_cyt). We propose that TRPM5, a Ca²⁺-activated monovalent cation channel, is a positive regulator of glucose-induced insulin release. Immunofluorescence revealed expression of TRPM5 in pancreatic islets. A Ca²⁺-activated nonselective cation current with TRPM5-like properties is significantly reduced in Trpm5^−/− cells. Ca²⁺-imaging and electrophysiological analysis show that glucose-induced oscillations of V_m and [Ca²⁺]_cyt have on average a reduced frequency in Trpm5^−/− islets, specifically due to a lack of fast oscillations. As a consequence, glucose-induced insulin release from Trpm5^−/− pancreatic islets is significantly reduced, resulting in an impaired glucose tolerance in Trpm5^−/− mice.     

Low doses of hydrogen peroxide impair glucose-stimulated insulin secretion via inhibition of glucose metabolism and intracellular calcium oscillations

The inhibitory effect of hydrogen peroxide (H₂O₂) on glucose-stimulated insulin secretion was previously reported. However, the precise mechanism involved was not systematically investigated. In this study, the effects of low concentrations of H₂O₂ (5-10 μmol/L) on glucose metabolism, intracellular calcium ([Ca²⁺]_i) oscillations, and dynamic insulin secretion in rat pancreatic islets were investigated. Low concentrations of H₂O₂ impaired insulin secretion in the presence of high glucose levels (16.7 mmol/L). This phenomenon was observed already after 2 minutes of exposure to H₂O₂. Glucose oxidation and the amplitude of [Ca²⁺]_i oscillations were dose-dependently suppressed by H₂O₂. These findings indicate that low concentrations of H₂O₂ reduce insulin secretion in the presence of high glucose levels via inhibition of glucose metabolism and consequent impairment in [Ca²⁺]_i handling.     

Regulation of insulin secretion: a matter of phase control and amplitude modulation

The consensus model of stimulus–secretion coupling in beta cells attributes glucose-induced insulin secretion to a sequence of events involving acceleration of metabolism, closure of ATP-sensitive K⁺ channels, depolarisation, influx of Ca²⁺ and a rise in cytosolic free Ca²⁺ concentration ([Ca²⁺]_c). This triggering pathway is essential, but would not be very efficient if glucose did not also activate a metabolic amplifying pathway that does not raise [Ca²⁺]_c further but augments the action of triggering Ca²⁺ on exocytosis. This review discusses how both pathways interact to achieve temporal control and amplitude modulation of biphasic insulin secretion. First-phase insulin secretion is triggered by the rise in [Ca²⁺]_c that occurs synchronously in all beta cells of every islet in response to a sudden increase in the glucose concentration. Its time course and duration are shaped by those of the Ca²⁺ signal, and its amplitude is modulated by the magnitude of the [Ca²⁺]_c rise and, substantially, by amplifying mechanisms. During the second phase, synchronous [Ca²⁺]_c oscillations in all beta cells of an individual islet induce pulsatile insulin secretion, but these features of the signal and response are dampened in groups of intrinsically asynchronous islets. Glucose has hardly any influence on the amplitude of [Ca²⁺]_c oscillations and mainly controls the time course of triggering signal. Amplitude modulation of insulin secretion pulses largely depends on the amplifying pathway. There are more similarities than differences between the two phases of glucose-induced insulin secretion. Both are subject to the same dual, hierarchical control over time and amplitude by triggering and amplifying pathways, suggesting that the second phase is a sequence of iterations of the first phase. Adapted from the A. E. Renold Lecture 2008.     

Effects of external ATP on Ca(2+) signalling in endothelial cells isolated from mouse islets

External ATP is believed to initiate and propagate Ca²⁺ signals co-ordinating the insulin release pulses within and among the different islets in the pancreas. The possibility that islet endothelial cells participate in this process was evaluated by comparing the effects on [Ca²⁺]_i of purinoceptor activation in these cells with those in β-cells. β-Cell-rich pancreatic islets were isolated from ob/ob mice and dispersed into single cells/aggregates. After culture with or without endothelial cell growth supplement (ECGS), the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was measured with ratiometric fura-2 technique. Presence of ECGS or prolongation of culture (>5 days) resulted in proliferation of endothelial cells and altered their phenotype from rounded to elongated. Endothelial cells, preliminarily identified by attachment of Dynabeads coated with the Bandeiraea simplicifolia 1 lectin (BS-1), responded in a similar way as those stained with CD31 antibodies after measurements of [Ca²⁺]_i. Spontaneous transients and oscillations of [Ca²⁺]_i were seen in β-cells, but not in endothelial cells exposed to 20 mM glucose. Addition of ATP (10 μM) resulted in pronounced and more extended rise of [Ca²⁺]_i in endothelial cells than in β-cells. The endothelial cells differed from the β-cells by also responding with a rise of [Ca²⁺]_i to 10 μM UTP, but not to equimolar ADP and acetylcholine. The results support the idea of mutual interactions between islet endothelium and β-cells based on ATP-induced Ca²⁺ signals. It is suggested that the endothelial cells have a tonic inhibitory action on β-cell P2 purinoceptors resulting in impaired synchronization of the insulin release pulses.     

A potent diazoxide analogue activating ATP-sensitive K+ channels and inhibiting insulin release

Aims/hypothesis. To characterise the effects of BPDZ 73 (7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide), a newly synthesised diazoxide analogue, on insulin secretory cells.¶Methods. Measurements of ⁸⁶Rb, ⁴⁵Ca outflow, membrane potential, [Ca²⁺]_i, insulin release in secretory cells as well as measurements of smooth muscle contractile activity and glycaemia were carried out.¶Results. The analogue BPDZ 73 induced a dose-dependent decrease in insulin output. The IC₅₀ value averaged 0.73 ± 0.05 μmol/l. The drug increased the rate of ⁸⁶Rb (⁴²K substitute) outflow from perifused rat pancreatic islets. This effect was inhibited by glibenclamide, a K_ATP channel blocker. Measurements of DiBAC₄(3) fluorescence further indicated that BPDZ 73 hyperpolarised the insulin secreting cells. It also decreased ⁴⁵Ca outflow from pancreatic islets perifused throughout in the presence of 16.7 mmol/l glucose and extracellular Ca²⁺. By contrast, the drug did not affect the increase in ⁴⁵Ca outflow mediated by K⁺ depolarisation. In single beta cells, BPDZ 73 inhibited the glucose-induced but not the K⁺-induced rise in [Ca²⁺]_i. Moreover, in Wistar rats, i. p. injection of BPDZ 73 provoked a considerable increase in blood glucose concentration whereas diazoxide induced a modest rise in glycaemia. Lastly, the vasorelaxant properties of BPDZ 73 were slightly less pronounced than those of diazoxide.¶Conclusion/interpretation. The inhibitory effect of BPDZ 73 on the insulin-releasing process results from the activation of K_ATP channels with subsequent decrease in Ca²⁺ inflow and [Ca²⁺]_i. The drug seems to be a K_ATP channel opener, more potent and more selective than diazoxide for insulin secreting cells. [Diabetologia (2000) 43: 723-732]     

A role for kisspeptin in islet function

Aims/hypothesis We investigated the production of kisspeptin (KISS1) and the KISS1 receptor, GPR54, in pancreatic islets and determined the effects of exogenous kisspeptin on insulin secretion.Methods RT-PCR and immunohistochemistry were used to detect expression of KISS1 and GPR54 mRNAs and the production of KISS1 and GPR54 in human and mouse islets and in beta (MIN6) and alpha- (alphaTC1) cell lines. The effects of KISS1 on basal and glucose-induced insulin secretion from mouse and human islets were measured in a perifusion system.Results KISS1 and GPR54 mRNAs were both detected in human and mouse islets, and GPR54 mRNA expression was also found in the MIN6 and alphaTC1 endocrine cell lines. In sections of mouse pancreas, KISS1 and GPR54 immunoreactivities were co-localised in both beta and alpha cells within islets, but were not detected in the exocrine pancreas. Exposure of mouse and human islets to KISS1 caused a stimulation of glucose-induced (20 mmol/l) insulin secretion, but had no effect on the basal rate of secretion at a sub-stimulatory concentration of glucose (2 mmol/l). In contrast, KISS1 inhibited insulin secretion from MIN6 cells at both 2 and 20 mmol/l glucose. KISS1 had no significant effect on glucagon secretion from mouse islets.Conclusions/interpretation This is the first report to show that the GPR54/KISS1 system is expressed in the endocrine pancreas, where it influences beta cell secretory function. These observations suggest an important role for this system in the normal regulation of islet function.     

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

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

Autocrine activation of P2Y1 receptors couples Ca2+ influx to Ca2+ release in human pancreatic beta cells

Aims/hypothesis

There is evidence that ATP acts as an autocrine signal in beta cells but the receptors and pathways involved are incompletely understood. Here we investigate the receptor subtype(s) and mechanism(s) mediating the effects of ATP on human beta cells.

Methods

We examined the effects of purinergic agonists and antagonists on membrane potential, membrane currents, intracellular Ca²⁺ ([Ca²⁺]_i) and insulin secretion in human beta cells.

Results

Extracellular application of ATP evoked small inward currents (3.4?±?0.7 pA) accompanied by depolarisation of the membrane potential (by 14.4?±?2.4 mV) and stimulation of electrical activity at 6 mmol/l glucose. ATP increased [Ca²⁺]_i by stimulating Ca²⁺ influx and evoking Ca²⁺ release via InsP₃-receptors in the endoplasmic reticulum (ER). ATP-evoked Ca²⁺ release was sufficient to trigger exocytosis in cells voltage-clamped at ?70 mV. All effects of ATP were mimicked by the P2Y_(1/12/13) agonist ADP and the P2Y₁ agonist MRS-2365, whereas the P2X_(1/3) agonist α,β-methyleneadenosine-5-triphosphate only had a small effect. The P2Y₁ antagonists MRS-2279 and MRS-2500 hyperpolarised glucose-stimulated beta cells and lowered [Ca²⁺]_i in the absence of exogenously added ATP and inhibited glucose-induced insulin secretion by 35%. In voltage-clamped cells subjected to action potential-like stimulation, MRS-2279 decreased [Ca²⁺]_i and exocytosis without affecting Ca²⁺ influx.

Conclusions/interpretation

These data demonstrate that ATP acts as a positive autocrine signal in human beta cells by activating P2Y₁ receptors, stimulating electrical activity and coupling Ca²⁺ influx to Ca²⁺ release from ER stores.     

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 Ca²⁺ concentration ([Ca²⁺]_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 [Ca²⁺]_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 [Ca²⁺]_c.     

Glutamate is an essential mediator in glutamine-amplified insulin secretion

Aims/IntroductionGlutamine is the most abundant amino acid in the circulation. In this study, we investigated cell signaling in the amplification of insulin secretion by glutamine.Materials and MethodsClonal pancreatic β‐cells MIN6‐K8, wild‐type B6 mouse islets, glutamate dehydrogenase (GDH) knockout clonal β‐cells (Glud1KOβCL), and glutamate‐oxaloacetate transaminase 1 (GOT1) knockout clonal β‐cells (Got1KOβCL) were studied. Insulin secretion from these cells and islets was examined under various conditions, and intracellular glutamine metabolism was assessed by metabolic flux analysis. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also measured.ResultsGlutamine dose‐dependently amplified insulin secretion in the presence of high glucose in both MIN6‐K8 cells and Glud1KOβCL. Inhibition of glutaminases, the enzymes that convert glutamine to glutamate, dramatically reduced the glutamine‐amplifying effect on insulin secretion. A substantial amount of glutamate was produced from glutamine through direct conversion by glutaminases. Glutamine also increased [Ca²⁺]_i at high glucose, which was abolished by inhibition of glutaminases. Glutamic acid dimethylester (dm‐Glu), a membrane permeable glutamate precursor that is converted to glutamate in cells, increased [Ca²⁺]_i as well as induced insulin secretion at high glucose. These effects of glutamine and dm‐Glu were dependent on calcium influx. Glutamine also induced insulin secretion in clonal β‐cells MIN6‐m14, which otherwise exhibit no insulin secretory response to glucose.ConclusionsGlutamate converted from glutamine is an essential mediator that enhances calcium signaling in the glutamine‐amplifying effect on insulin secretion. Our data also suggest that glutamine exerts a permissive effect on glucose‐induced insulin secretion.     

Subplasmalemmal Ca2+ measurements in mouse pancreatic beta cells support the existence of an amplifying effect of glucose on insulin secretion

Aims/hypothesis  

Glucose-induced insulin secretion is attributed to a rise of beta cell cytosolic free [Ca²⁺] ([Ca²⁺]_c) (triggering pathway) and amplification of the action of Ca²⁺. This concept of amplification rests on observations that glucose can increase Ca²⁺-induced insulin secretion without further elevating an imposed already high [Ca²⁺]_c. However, it remains possible that this amplification results from an increase in [Ca²⁺] just under the plasma membrane ([Ca²⁺]_SM), which escaped detection by previous measurements of global [Ca²⁺]_c. This was the hypothesis that we tested here by measuring [Ca²⁺]_SM.     

Relationships between dopamine-induced changes in cytosolic free calcium concentration ([Ca2+]i) and rate of prolactin secretion

This study was undertaken to investigate the relationship between dopamine (DA) induced changes in the cytosolic calcium concentration ([Ca²⁺]_i) and the rate of prolactin secretion using GH₄ZR₇, a rat pituitary cell line, which express only one subtype of D₂ receptor. GH₄ZR₇ cells were loaded with Fluo-3, a fluorescent Ca²⁺ indicator, and then perifused with two different doses of DA (10⁻⁷ mol/L and 5×10⁻⁴ mol/L). We monitored changes in [Ca²⁺]_i and rate of prolactin release simultaneously by attaching a spectrofluorometer to a dynamic perifusion system. DA has stimulatory and inhibitory effect on prolactin secretion in GH₄ZR₇ cells; 10⁻⁷ mol/L DA slightly increased [Ca²⁺]_i and stimulated prolactin release, whereas 5×10⁻⁴ mol/L DA decreased [Ca²⁺]_i and inhibited prolactin secretion. When the cells were pretreated with pertussis toxin (PTX), 10⁻⁷ mol/L DA had no significant change in [Ca²⁺]_i while stimulating prolactin release, and 5×10⁻⁴ mol/L DA reduced [Ca²⁺]_i without having any significant effect on the rate of prolactin secretion. The results of this study demonstrate that changes in [Ca²⁺]_i do not always correlate with the rate of prolactin release from lactotrophs. The dissociation between [Ca²⁺]_i and prolactin release is somewhat expected considering the diverse role of [Ca²⁺]_i and post-[Ca²⁺]_i events, which can change the rate of prolactin release.     

Calcium elevation in mouse pancreatic beta cells evoked by extracellular human islet amyloid polypeptide involves activation of the mechanosensitive ion channel TRPV4

Aims/hypothesis  To investigate the mechanism by which human islet amyloid polypeptide (hIAPP) fibril formation results in calcium influx across the plasma membrane of pancreatic beta cells, and its association with apoptosis. Methods  Cytoplasmic intracellular calcium concentrations ([Ca²⁺]_i) were monitored for 2 h as the 340/380 nm fluorescence ratio in fura-2 loaded cells of the MIN6 mouse pancreatic beta cell line. Cell morphology was evaluated by transmission electron microscopy, and viability by FACS. Results  hIAPP (10 μmol/l) increased [Ca²⁺]_i in 21% of MIN6 cells in standard buffer, and in 8% of cells in Na⁺-free buffer. Transient receptor potential (TRP) channel inhibitors (gadolinium and ruthenium red) prevented the [Ca²⁺]_i rise under both conditions, whilst nifedipine was only effective in the presence of Na⁺. hIAPP increased apoptosis in both insulinoma cells and islets in primary culture, and cell viability was partially rescued by ruthenium red (p < 0.001). By RT-PCR, we detected expression of the mechanosensitive TRP cation channel subfamily V member 4 (Trpv4) in MIN6 cells and mouse pancreas. Small interference RNA against Trpv4 prevented hIAPP-induced [Ca²⁺]_i rises, decreased hIAPP-triggered expression of the endoplasmic reticulum (ER) stress response, and reduced hIAPP-triggered cell death by 50% (p < 0.05). Conclusions/interpretation  Alterations in [Ca²⁺]_i play a key role in hIAPP-induced beta cell cytotoxicity. By electron microscopy, we detected extracellular hIAPP aggregates adjacent to irregular invaginated regions of the plasma membrane. We propose that TRPV4 channels may sense physical changes in the plasma membrane induced by hIAPP aggregation, enabling Ca²⁺ entry, membrane depolarisation and activation of L-type Ca²⁺ channels. Decreasing the activity of TRPV4 prevented hIAPP-induced [Ca²⁺]_i changes, reduced hIAPP-triggered ER stress and improved cell viability.     

Oscillations of sub-membrane ATP in glucose-stimulated beta cells depend on negative feedback from Ca2+

Aims/hypothesis

ATP links changes in glucose metabolism to electrical activity, Ca²⁺ signalling and insulin secretion in pancreatic beta cells. There is evidence that beta cell metabolism oscillates, but little is known about ATP dynamics at the plasma membrane, where regulation of ion channels and exocytosis occur.

Methods

The sub-plasma-membrane ATP concentration ([ATP]_pm) was recorded in beta cells in intact mouse and human islets using total internal reflection microscopy and the fluorescent reporter Perceval.

Results

Glucose dose-dependently increased [ATP]_pm with half-maximal and maximal effects at 5.2 and 9 mmol/l, respectively. Additional elevations of glucose to 11 to 20 mmol/l promoted pronounced [ATP]_pm oscillations that were synchronised between neighbouring beta cells. [ATP]_pm increased further and the oscillations disappeared when voltage-dependent Ca²⁺ influx was prevented. In contrast, K⁺-depolarisation induced prompt lowering of [ATP]_pm. Simultaneous recordings of [ATP]_pm and the sub-plasma-membrane Ca²⁺ concentration ([Ca²⁺]_pm) during the early glucose-induced response revealed that the initial [ATP]_pm elevation preceded, and was temporarily interrupted by the rise of [Ca²⁺]_pm. During subsequent glucose-induced oscillations, the increases of [Ca²⁺]_pm correlated with lowering of [ATP]_pm.

Conclusions/interpretation

In beta cells, glucose promotes pronounced oscillations of [ATP]_pm, which depend on negative feedback from Ca²⁺ _. The bidirectional interplay between these messengers in the sub-membrane space generates the metabolic and ionic oscillations that underlie pulsatile insulin secretion.