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Aim cAMP typically signals downstream of Gs -coupled receptors and regulates numerous cell functions. In β-cells, cAMP amplifies Ca2+ -triggered exocytosis of insulin granules. Glucose-induced insulin secretion is associated with Ca2+ - and metabolism-dependent increases of the sub-plasma-membrane cAMP concentration ([cAMP]pm ) in β-cells, but potential links to canonical receptor signalling are unclear. The aim of this study was to clarify the role of glucagon-like peptide-1 receptors (GLP1Rs) for glucose-induced cAMP signalling in β-cells. Methods Total internal reflection microscopy and fluorescent reporters were used to monitor changes in cAMP, Ca2+ and ATP concentrations as well as insulin secretion in MIN6 cells and mouse and human β-cells. Insulin release from mouse and human islets was also measured with ELISA. Results The GLP1R antagonist exendin-(9-39) (ex-9) prevented both GLP1- and glucagon-induced elevations of [cAMP]pm , consistent with GLP1Rs being involved in the action of glucagon. This conclusion was supported by lack of unspecific effects of the antagonist in a reporter cell-line. Ex-9 also suppressed IBMX- and glucose-induced [cAMP]pm elevations. Depolarization with K+ triggered Ca2+ -dependent [cAMP]pm elevation, an effect that was amplified by high glucose. Ex-9 inhibited both the Ca2+ and glucose-metabolism-dependent actions on [cAMP]pm . The drug remained effective after minimizing paracrine signalling by dispersing the islets and it reduced basal [cAMP]pm in a cell-line heterologously expressing GLP1Rs, indicating that there is constitutive GLP1R signalling. The ex-9-induced reduction of [cAMP]pm in glucose-stimulated β-cells was paralleled by suppression of insulin secretion. Conclusion Agonist-independent and glucagon-stimulated GLP1R signalling in β-cells contributes to basal and glucose-induced cAMP production and insulin secretion.

Although endoplasmic reticulum (ER) chaperone binding to mutant proinsulin has been reported, the role of protein chaperones in the handling of wild-type proinsulin is underinvestigated. Here, we have explored the importance of glucose-regulated protein 94 (GRP94), a prominent ER chaperone known to fold insulin-like growth factors, in proinsulin handling within b-cells. We found that GRP94 coimmunoprecipitated with proinsulin and that inhibition of GRP94 function and/or expression reduced glucose-dependent insulin secretion, shortened proinsulin half-life, and lowered intracellular proinsulin and insulin levels. This phenotype was accompanied by post-ER proinsulin misprocessing and higher numbers of enlarged insulin granules that contained amorphic material with reduced immunogold staining for mature insulin. Insulin granule exocytosis was accelerated twofold, but the secreted insulin had diminished bioactivity. Moreover, GRP94 knockdown or knockout in b-cells selectively activated protein kinase R–like endoplasmic reticulum kinase (PERK), without increasing apoptosis levels. Finally, GRP94 mRNA was overexpressed in islets from patients with type 2 diabetes. We conclude that GRP94 is a chaperone crucial for proinsulin handling and insulin secretion. Fil: Ghiasi, Seyed Mojtaba. Universidad de Copenhagen; Dinamarca Fil: Dahlby, Tina. Universidad de Copenhagen; Dinamarca Fil: Andersen, Caroline Hede. Universidad de Copenhagen; Dinamarca Fil: Haataja, Leena. University of Michigan; Estados Unidos Fil: Petersen, Sólrun. Universidad de Copenhagen; Dinamarca Fil: Omar-Hmeadi, Muhmmad. Uppsala Universitet; Suecia Fil: Yang, Mingyu. Uppsala Universitet; Suecia Fil: Pihl, Celina. Universidad de Copenhagen; Dinamarca Fil: Bresson, Sophie Emilie. Universidad de Copenhagen; Dinamarca Fil: Khilji, Muhammad Saad. Universidad de Copenhagen; Dinamarca Fil: Klindt, Kristian. Universidad de Copenhagen; Dinamarca Fil: Cheta, Oana. Universidad de Copenhagen; Dinamarca Fil: Perone, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad de Copenhagen; Dinamarca Fil: Tyrberg, Björn. Astrazeneca. IMED Biotech Unit; Suecia Fil: Prats, Clara. Universidad de Copenhagen; Dinamarca Fil: Barg, Sebastian. Uppsala Universitet; Suecia Fil: Tengholm, Anders. Uppsala Universitet; Suecia Fil: Arvan, Peter. University of Michigan; Estados Unidos Fil: Mandrup-Poulsen, Thomas. Universidad de Copenhagen; Dinamarca Fil: Marzec, Michal Tomasz. Universidad de Copenhagen; Dinamarca

The "second messenger" archetype cAMP is one of the most important cellular signalling molecules with central functions including the regulation of insulin and glucagon secretion from the pancreatic β- and α-cells, respectively. cAMP is generally considered as an amplifier of insulin secretion triggered by Ca2+ elevation in the β-cells. Both messengers are also positive modulators of glucagon release from α-cells, but in this case cAMP may be the important regulator and Ca2+ have a more permissive role. The actions of cAMP are mediated by protein kinase A (PKA) and the guanine nucleotide exchange factor Epac. The present review focuses on how cAMP is regulated by nutrients, hormones and neural factors in β- and α-cells via adenylyl cyclase-catalysed generation and phosphodiesterase-mediated degradation. We will also discuss how PKA and Epac affect ion fluxes and the secretory machinery to transduce the stimulatory effects on insulin and glucagon secretion. Finally, we will briefly describe disturbances of the cAMP system associated with diabetes and how cAMP signalling can be targeted to normalize hypo- and hypersecretion of insulin and glucagon, respectively, in diabetic patients.

Epac is a cAMP-activated guanine nucleotide exchange factor that mediates cAMP signaling in various types of cells, including β-cells, where it is involved in the control of insulin secretion. Upon activation, the protein redistributes to the plasma membrane, but the underlying molecular mechanisms and functional consequences are unclear. Using quantitative high-resolution microscopy, we found that cAMP elevation caused rapid binding of Epac2A to the β-cell plasma membrane, where it accumulated specifically at secretory granules and rendered them more prone to undergo exocytosis. cAMP-dependent membrane binding required the high-affinity cyclic nucleotide-binding (CNB) and Ras association domains, but not the disheveled–Egl-10–pleckstrin domain. Although the N-terminal low-affinity CNB domain (CNB-A) was dispensable for the translocation to the membrane, it was critical for directing Epac2A to the granule sites. Epac1, which lacks the CNB-A domain, was recruited to the plasma membrane but did not accumulate at granules. We conclude that Epac2A controls secretory granule release by binding to the exocytosis machinery, an effect that is enhanced by prior cAMP-dependent accumulation of the protein at the plasma membrane.

Aims/hypothesis Glucagon is critical for normal glucose homeostasis and aberrant secretion of the hormone aggravates dysregulated glucose control in diabetes. However, the mechanisms by which glucose controls glucagon secretion from pancreatic alpha cells remain elusive. The aim of this study was to investigate the role of the intracellular messenger cAMP in alpha-cell-intrinsic glucose regulation of glucagon release. Methods Subplasmalemmal cAMP and Ca2+ concentrations were recorded in isolated and islet-located alpha cells using fluorescent reporters and total internal reflection microscopy. Glucagon secretion from mouse islets was measured using ELISA. Results Glucose induced Ca2+-independent alterations of the subplasmalemmal cAMP concentration in alpha cells that correlated with changes in glucagon release. Glucose-lowering-induced stimulation of glucagon secretion thus corresponded to an elevation in cAMP that was independent of paracrine signalling from insulin or somatostatin. Imposed cAMP elevations stimulated glucagon secretion and abolished inhibition by glucose elevation, while protein kinase A inhibition mimicked glucose suppression of glucagon release. Conclusions/interpretation Glucose concentrations in the hypoglycaemic range control glucagon secretion by directly modulating the cAMP concentration in alpha cells independently of paracrine influences. These findings define a novel mechanism for glucose regulation of glucagon release that underlies recovery from hypoglycaemia and may be disturbed in diabetes. Electronic supplementary material The online version of this article (10.1007/s00125-019-4857-6) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- (Rapgef4-/-) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis., eLife digest Insulin is the hormone that signals to the body to take up sugar from the blood. Specialized cells in the pancreas – known as β-cells – release insulin after a meal. Before that, insulin molecules are stored in tiny granules inside the β-cells; these granules must fuse with the cells’ surface membranes to release their contents. The first step in this process creates a narrow pore that allows small molecules, but not the larger insulin molecules, to seep out. The pore then widens to release the insulin. Since the small molecules are known to act locally in the pancreas, it is possible that this “molecular sieve” is biologically important. Yet it is not clear how the pore widens. One of the problems for people with type 2 diabetes is that they release less insulin into the bloodstream. Two kinds of drugs used to treat these patients work by stimulating β-cells to release their insulin. One way to achieve this is by raising the levels of a small molecule called cAMP, which is well known to help prepare insulin granules for release. The cAMP molecule also seems to slow the widening of the pore, and Gucek et al. have now investigated how this happens at a molecular level. By observing individual granules of human β-cells using a special microscope, Gucek et al. could watch how different drugs affect pore widening and content release. They also saw that cAMP activated a protein called Epac2, which then recruited two other proteins – amisyn and dynamin – to the small pores. These two proteins together then closed the pore, rather than expanding it to let insulin out. Type 2 diabetes patients sometimes have high levels of amisyn in their β-cells, which could explain why they do not release enough insulin. The microscopy experiments also revealed that two common anti-diabetic drugs activate Epac2 and prevent the pores from widening, thereby counteracting their positive effect on insulin release. The combined effect is likely a shift in the balance between insulin and the locally acting small molecules. These findings suggest that two common anti-diabetic drugs activate a common mechanism that may lead to unexpected outcomes, possibly even reducing how much insulin the β-cells can release. Future studies in mice and humans will have to investigate these effects in whole organisms.

Regulated exocytosis establishes a narrow fusion pore as the initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of larger peptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in type-2 diabetes and neurodegenerative disease. Here we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation leads to pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- mice. Conversely, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.

Zinc finger BED domain containing protein 6 ( Zbed6) has evolved from a domesticated DNA transposon and encodes a transcription factor unique to placental mammals. The aim of the present study was to investigate further the role of ZBED6 in insulin-producing cells, using mouse MIN6 cells, and to evaluate the effects of Zbed6 knockdown on basal β-cell functions, such as morphology, transcriptional regulation, insulin content, and release. Zbed6-silenced cells and controls were characterized with a range of methods, including RNA sequencing, chromatin immunoprecipitation sequencing, insulin content and release, subplasma membrane Ca2+ measurements, cAMP determination, and morphologic studies. More than 700 genes showed differential expression in response to Zbed6 knockdown, which was paralleled by increased capacity to generate cAMP, as well as by augmented subplasmalemmal calcium concentration and insulin secretion in response to glucose stimulation. We identified >4000 putative ZBED6-binding sites in the MIN6 genome, with an enrichment of ZBED6 sites at upregulated genes, such as the β-cell transcription factors v-maf musculoaponeurotic fibrosarcoma oncogene homolog A and Nk6 homeobox 1. We also observed altered morphology/growth patterns, as indicated by increased cell clustering, and in the appearance of axon-like Neurofilament, medium polypeptide and tubulin β 3, class III-positive protrusions. We conclude that ZBED6 acts as a transcriptional regulator in MIN6 cells and that its activity suppresses insulin production, cell aggregation, and neuronal-like differentiation.-Wang, X., Jiang, L., Wallerman, O., Younis, S., Yu, Q., Klaesson, A., Tengholm, A., Welsh, N., Andersson, L. ZBED6 negatively regulates insulin production, neuronal differentiation, and cell aggregation in MIN6 cells.

Cytoplasmic ATP and Ca2+ are implicated in current models of glucose’s control of glucagon and insulin secretion from pancreatic α- and β-cells, respectively, but little is known about ATP and its relation to Ca2+ in α-cells. We therefore expressed the fluorescent ATP biosensor Perceval in mouse pancreatic islets and loaded them with a Ca2+ indicator. With total internal reflection fluorescence microscopy, we recorded subplasma membrane concentrations of Ca2+ and ATP ([Ca2+]pm; [ATP]pm) in superficial α- and β-cells of intact islets and related signaling to glucagon and insulin secretion by immunoassay. Consistent with ATP’s controlling glucagon and insulin secretion during hypo- and hyperglycemia, respectively, the dose-response relationship for glucose-induced [ATP]pm generation was left shifted in α-cells compared to β-cells. Both cell types showed [Ca2+]pm and [ATP]pm oscillations in opposite phase, probably reflecting energy-consuming Ca2+ transport. Although pulsatile insulin and glucagon release are in opposite phase, [Ca2+]pm synchronized in the same phase between α- and β-cells. This paradox can be explained by the overriding of Ca2+ stimulation by paracrine inhibition, because somatostatin receptor blockade potently stimulated glucagon release with little effect on Ca2+. The data indicate that an α-cell-intrinsic mechanism controls glucagon in hypoglycemia and that paracrine factors shape pulsatile secretion in hyperglycemia.—Li, J., Yu, Q., Ahooghalandari, P., Gribble, F. M., Reimann, F., Tengholm, A., Gylfe, E. Submembrane ATP and Ca2+ kinetics in α-cells: unexpected signaling for glucagon secretion.

In human pancreatic islets, the neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule synthesized by and released from the insulin-secreting β cells. The effective, physiological GABA concentration range within human islets is unknown. Here we use native GABAA receptors in human islet β cells as biological sensors and reveal that 100–1000 nM GABA elicit the maximal opening frequency of the single-channels. In saturating GABA, the channels desensitized and stopped working. GABA modulated insulin exocytosis and glucose-stimulated insulin secretion. GABAA receptor currents were enhanced by the benzodiazepine diazepam, the anesthetic propofol and the incretin glucagon-like peptide-1 (GLP-1) but not affected by the hypnotic zolpidem. In type 2 diabetes (T2D) islets, single-channel analysis revealed higher GABA affinity of the receptors. The findings reveal unique GABAA receptors signaling in human islets β cells that is GABA concentration-dependent, differentially regulated by drugs, modulates insulin secretion and is altered in T2D., Highlights • In human islets GABA (≤μM) activates β cell-specific GABAA receptors that become supersensitive to GABA in type 2 diabetes. • GABAA receptors activity in β cells is enhanced by diazepam, anesthetics, the incretin GLP-1 but not the hypnotic zolpidem. • GABA modulates rate of insulin granule exocytosis and glucose-stimulated insulin secretion. GABA is a signal molecule in the brain but is also secreted by the insulin-producing β cells in pancreatic islets. GABA has many roles in human islets that most aim at optimizing function and survival of β cells. In the report by Korol, Jin et al. the authors identify and characterize the molecular unit that GABA binds to in human β cells, the GABAA receptors. These receptors normally sensitive become super-sensitive to GABA in type 2 diabetes. The GABAA receptors regulate insulin secretion and can themselves be regulated by the anxiolytic diazepam, anesthetics, the incretin GLP-1 but not the hypnotic zolpidem. Targeting GABA signaling in human islets in diabetes mellitus is likely to be a part of the solution when curing diabetes.

The "second messenger" archetype cAMP is one of the most important cellular signalling molecules with central functions including the regulation of insulin and glucagon secretion from the pancreatic β- and α-cells, respectively. cAMP is generally considered as an amplifier of insulin secretion triggered by Ca

Chronic palmitate exposure impairs glucose-stimulated insulin secretion and other aspects of β-cell function, but the underlying mechanisms are not known. Using various live-cell fluorescence imaging approaches, we show here that long-term palmitate treatment influences cAMP signaling in pancreatic β-cells. Glucose stimulation of mouse and human β-cells induced oscillations of the subplasma-membrane cAMP concentration, but after 48 h exposure to palmitate, most β-cells failed to increase cAMP in response to glucose. In contrast, GLP-1–triggered cAMP formation and glucose- and depolarization-induced increases in cytoplasmic Ca2+ concentration were unaffected by the fatty acid treatment. Insulin secretion from control β-cells was pulsatile, but the response deteriorated after long-term palmitate exposure. Palmitate-treated mouse islets showed reduced expression of adenylyl cyclase 9, and knockdown of this protein in insulinoma cells reduced the glucose-stimulated cAMP response and insulin secretion. We conclude that impaired glucose-induced generation of cAMP is an important determinant of defective insulin secretion after chronic palmitate exposure.

Aims/hypothesis: Insufficient insulin release and hyperglucagonaemia are culprits in type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART, encoded by Cartpt) affects islet hormone secretion and beta cell survival in vitro in rats, and Cart−/− mice have diminished insulin secretion. We aimed to test if CART is differentially regulated in human type 2 diabetic islets and if CART affects insulin and glucagon secretion in vitro in humans and in vivo in mice. Methods: CART expression was assessed in human type 2 diabetic and non-diabetic control pancreases and rodent models of diabetes. Insulin and glucagon secretion was examined in isolated islets and in vivo in mice. Ca2+ oscillation patterns and exocytosis were studied in mouse islets. Results: We report an important role of CART in human islet function and glucose homeostasis in mice. CART was found to be expressed in human alpha and beta cells and in a subpopulation of mouse beta cells. Notably, CART expression was several fold higher in islets of type 2 diabetic humans and rodents. CART increased insulin secretion in vivo in mice and in human and mouse islets. Furthermore, CART increased beta cell exocytosis, altered the glucose-induced Ca2+ signalling pattern in mouse islets from fast to slow oscillations and improved synchronisation of the oscillations between different islet regions. Finally, CART reduced glucagon secretion in human and mouse islets, as well as in vivo in mice via diminished alpha cell exocytosis. Conclusions/interpretation: We conclude that CART is a regulator of glucose homeostasis and could play an important role in the pathophysiology of type 2 diabetes. Based on the ability of CART to increase insulin secretion and reduce glucagon secretion, CART-based agents could be a therapeutic modality in type 2 diabetes.

Insulin secretion from pancreatic β-cells is tightly regulated by glucose and other nutrients, hormones, and neural factors. The exocytosis of insulin granules is triggered by an elevation of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and is further amplified by cyclic AMP (cAMP). Cyclic AMP is formed primarily in response to glucoincretin hormones and other G(s)-coupled receptor agonists, but generation of the nucleotide is critical also for an optimal insulin secretory response to glucose. Nutrient and receptor stimuli trigger oscillations of the cAMP concentration in β-cells. The oscillations arise from variations in adenylyl cyclase-mediated cAMP production and phosphodiesterase-mediated degradation, processes controlled by factors like cell metabolism and [Ca(2+)](i). Protein kinase A and the guanine nucleotide exchange factor Epac2 mediate the actions of cAMP in β-cells and operate at multiple levels to promote exocytosis and pulsatile insulin secretion. The cAMP signaling system contains important targets for pharmacological improvement of insulin secretion in type 2 diabetes.

Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic β-cells involves ion channels and mobilization of Ca(2+) and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-β-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca(2+) currents, and attenuates cytoplasmic accumulation of Ca(2+) and cAMP in β-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity.

Pulsatile insulin release from glucose-stimulated beta-cells is driven by oscillations of the Ca(2+) and cAMP concentrations in the subplasma membrane space ([Ca(2+)](pm) and [cAMP](pm)). To clarify mechanisms by which cAMP regulates insulin secretion, we performed parallel evanescent wave fluorescence imaging of [cAMP](pm), [Ca(2+)](pm), and phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) in the plasma membrane. This lipid is formed by autocrine insulin receptor activation and was used to monitor insulin release kinetics from single MIN6 beta-cells. Elevation of the glucose concentration from 3 to 11 mm induced, after a 2.7-min delay, coordinated oscillations of [Ca(2+)](pm), [cAMP](pm), and PIP(3). Inhibitors of protein kinase A (PKA) markedly diminished the PIP(3) response when applied before glucose stimulation, but did not affect already manifested PIP(3) oscillations. The reduced PIP(3) response could be attributed to accelerated depolarization causing early rise of [Ca(2+)](pm) that preceded the elevation of [cAMP](pm). However, the amplitude of the PIP(3) response after PKA inhibition was restored by a specific agonist to the cAMP-dependent guanine nucleotide exchange factor Epac. Suppression of cAMP formation with adenylyl cyclase inhibitors reduced already established PIP(3) oscillations in glucose-stimulated cells, and this effect was almost completely counteracted by the Epac agonist. In cells treated with small interfering RNA targeting Epac2, the amplitudes of the glucose-induced PIP(3) oscillations were reduced, and the Epac agonist was without effect. The data indicate that temporal coordination of the triggering [Ca(2+)](pm) and amplifying [cAMP](pm) signals is important for glucose-induced pulsatile insulin release. Although both PKA and Epac2 partake in initiating insulin secretion, the cAMP dependence of established pulsatility is mediated by Epac2.

PKC signaling has been implicated in the regulation of many cell functions, including metabolism, cell death, proliferation, and secretion. Activation of conventional and novel PKC isoforms is associated with their Ca2+- and/or diacylglycerol (DAG)-dependent translocation to the plasma membrane. In 13 cells, exocytosis of insulin granules evokes brief (

In insulin-secreting beta-cells, activation of phosphatidylinositol 3'-OH-kinase with resulting formation of phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) has been implicated in the regulation of ion channels, insulin secretion, and gene transcription as well as in cell growth and survival, but the kinetics of PIP(3) signals following physiological stimulation of insulin secretion is unknown. Using evanescent wave microscopy and a green fluorescent protein-tagged PIP(3)-binding protein domain for real-time monitoring of plasma membrane PIP(3) concentration in single MIN6 beta-cells, we now demonstrate that glucose stimulation of insulin secretion results in pronounced PIP(3) oscillations via autocrine stimulation of insulin receptors. Glucose lacked effect when insulin secretion was prevented with the hyperpolarizing agent diazoxide, but the sugar dose dependently enhanced the PIP(3) response to maximal insulin stimulation without affecting the rate of PIP(3) degradation. We conclude that glucose is an important co-activator of phosphatidylinositol-3'-OH-kinase and that the plasma membrane PIP(3) concentration in beta-cells undergoes oscillations due to pulsatile release of insulin.

We previously reported that human growth hormone (hGH) increases cytoplasmic Ca2+concentration ([Ca2+]i) and proliferation in pancreatic β-cells (Sjöholm Å, Zhang Q, Welsh N, Hansson A, Larsson O, Tally M, and Berggren PO. J Biol Chem 275: 21033–21040, 2000) and that the hGH-induced rise in [Ca2+]iinvolves Ca2+-induced Ca2+release facilitated by tyrosine phosphorylation of ryanodine receptors (Zhang Q, Kohler M, Yang SN, Zhang F, Larsson O, and Berggren PO. Mol Endocrinol 18: 1658–1669, 2004). Here we investigated the tyrosine kinases that convey the hGH-induced rise in [Ca2+]iand insulin release in BRIN-BD11 β-cells. hGH caused tyrosine phosphorylation of Janus kinase (JAK)2 and c-Src, events inhibited by the JAK2 inhibitor AG490 or the Src kinase inhibitor PP2. Although hGH-stimulated rises in [Ca2+]iand insulin secretion were completely abolished by AG490 and JAK2 inhibitor II, the inhibitors had no effect on insulin secretion stimulated by a high K+concentration. Similarly, Src kinase inhibitor-1 and PP2, but not its inactive analog PP3, suppressed [Ca2+]ielevation and completely abolished insulin secretion stimulated by hGH but did not affect responses to K+. Ovine prolactin increased [Ca2+]iand insulin secretion to a similar extent as hGH, effects prevented by the JAK2 and Src kinase inhibitors. In contrast, bovine GH evoked a rise in [Ca2+]ibut did not stimulate insulin secretion. Neither JAK2 nor Src kinase inhibitors influenced the effect of bovine GH on [Ca2+]i. Our study indicates that hGH stimulates rise in [Ca2+]iand insulin secretion mainly through activation of the prolactin receptor and JAK2 and Src kinases in rat insulin-secreting cells.

Phospholipase C (PLC) regulates various cellular processes by catalyzing the formation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we have investigated the influence of Ca2+ on receptor-triggered PLC activity in individual insulin-secreting β-cells. Evanescent wave microscopy was used to record PLC activity using green fluorescent protein (GFP)-tagged PIP2/IP3-binding pleckstrin homology domain from PLCδ1, and the cytoplasmic Ca2+ concentration ([Ca2+]i) was simultaneously measured using the indicator Fura Red. Stimulation of MIN6 β-cells with the muscarinic-receptor agonist carbachol induced rapid and sustained PLC activation. By contrast, only transient activation was observed after stimulation in the absence of extracellular Ca2+ or in the presence of the non-selective Ca2+ channel inhibitor La3+. The Ca2+-dependent sustained phase of PLC activity did not require voltage-gated Ca2+ influx, as hyperpolarization with diazoxide or direct Ca2+ channel blockade with nifedipine had no effect. Instead, the sustained PLC activity was markedly suppressed by the store-operated channel inhibitors 2-APB and SKF96365. Depletion of intracellular Ca2+ stores with the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitors thapsigargin or cyclopiazonic acid abolished Ca2+ mobilization in response to carbachol, and strongly suppressed the PLC activation in Ca2+-deficient medium. Analogous suppressions were observed after loading cells with the Ca2+ chelator BAPTA. Stimulation of primary mouse pancreatic β-cells with glucagon elicited pronounced [Ca2+]i spikes, reflecting protein kinase A-mediated activation of Ca2+-induced Ca2+ release via IP3 receptors. These [Ca2+]i spikes were found to evoke rapid and transient activation of PLC. Our data indicate that receptor-triggered PLC activity is enhanced by positive feedback from Ca2+ entering the cytoplasm from intracellular stores and via store-operated channels in the plasma membrane. Such amplification of receptor signalling should be important in the regulation of insulin secretion by hormones and neurotransmitters.

Activation of phosphatidyl-inositol-3′-OH-kinase (PI3K) and the resulting production of phosphatidyl-inositol-3,4,5-trisphosphate (PIP3) are ubiquitous signaling steps that link various cell surface receptors to multiple intracellular targets [1]. In fat and muscle cells, the same PI3K pathway that regulates metabolic enzymes, proliferation, and differentiation has also been shown to be involved in insulin-triggered insertion of glucose transporter GLUT4 into the plasma membrane [2, 3]. The multiple PI3K functions raise the question of how the same PI3K pathway can be selectively used for different cell functions. Here we developed a dual-color evanescent wave microscopy method to simultaneously measure PIP3 production and GLUT4 insertion in individual 3T3L1 adipocytes. Activation of PI3K was found to be both necessary and sufficient for triggering GLUT4 insertion, but transporter insertion was markedly suppressed for small-amplitude, persistent PIP3 signals and for large-amplitude, short PIP3 signals. The rejection of these common PI3K signaling responses may explain the selective advantage of insulin over platelet-derived growth factor and other stimuli for inducing GLUT4 insertion. Our study suggests that the same PI3K pathway can control specific cell functions by relying on effector systems that respond to particular receptor-encoded time courses and amplitudes of PIP3 signals.

Pulsatile secretion is an inherent property of hormone-releasing pancreatic islet cells. This secretory pattern is physiologically important and compromised in diabetes. Neurotransmitters released from islet cells may shape the pulses in auto/paracrine feedback loops. Within islets, glucose-stimulated β-cells couple via gap junctions to generate synchronized insulin pulses. In contrast, α- and δ-cells lack gap junctions, and glucagon release from islets stimulated by lack of glucose is non-pulsatile. Increasing glucose concentrations gradually inhibit glucagon secretion by α-cell-intrinsic mechanism/s. Further glucose elevation will stimulate pulsatile insulin release and co-secretion of neurotransmitters. Excitatory ATP may synchronize β-cells with δ-cells to generate coinciding pulses of insulin and somatostatin. Inhibitory neurotransmitters from β- and δ-cells can then generate antiphase pulses of glucagon release. Neurotransmitters released from intrapancreatic ganglia are required to synchronize β-cells between islets to coordinate insulin pulsatility from the entire pancreas, whereas paracrine intra-islet effects still suffice to explain coordinated pulsatile release of glucagon and somatostatin. The present review discusses how neurotransmitters contribute to the pulsatility at different levels of integration.

The Src homology-2 domain containing protein B (SHB) has previously been shown to function as a pleiotropic adapter protein, conveying signals from receptor tyrosine kinases to intracellular signaling intermediates. The overexpression ofShbin β-cells promotes β-cell proliferation by increased insulin receptor substrate (IRS) and focal adhesion kinase (FAK) activity, whereasShbdeficiency causes moderate glucose intolerance and impaired first-peak insulin secretion. Using an array of techniques, including live-cell imaging, patch-clamping, immunoblotting, and semi-quantitative PCR, we presently investigated the causes of the abnormal insulin secretory characteristics inShb-knockout mice.Shb-knockout islets displayed an abnormal signaling signature with increased activities of FAK, IRS, and AKT. β-catenin protein expression was elevated and it showed increased nuclear localization. However, there were no major alterations in the gene expression of various proteins involved in the β-cell secretory machinery. Nor wasShbdeficiency associated with changes in glucose-induced ATP generation or cytoplasmic Ca2+handling. In contrast, the glucose-induced rise in cAMP, known to be important for the insulin secretory response, was delayed in theShb-knockout compared with WT control. Inhibition of FAK increased the submembrane cAMP concentration, implicating FAK activity in the regulation of insulin exocytosis. In conclusion,Shbdeficiency causes a chronic increase in β-cell FAK activity that perturbs the normal insulin secretory characteristics of β-cells, suggesting multi-faceted effects of FAK on insulin secretion depending on the mechanism of FAK activation.

Oscillatory signaling and insulin release were studied in isolated pancreatic islets and β-cells obtained from human cadaveric organ donors. Taking advantage of Sr2+ as an analog for Ca2+, it was possible to demonstrate glucose-induced rhythmic activity in individual β-cells identified by immunostaining. Glucose-induced slow oscillations of Sr2+ (frequency, 0.1–1.0/min) were sometimes seen at a sugar concentration as low as 3 mm. Addition of 20 nm glucagon resulted in a broadening of the oscillations or in their transformation into sustained elevation. Moreover, the presence of glucagon resulted in the appearance of short transients of Sr2+, which disappeared after exposure to the intracellular Ca2+-adenosine triphosphatase inhibitor thapsigargin. Digital image analyses indicated that slow oscillations can be synchronized among cells in small aggregates and intact islets. The rhythmic activity in the glucose-stimulated β-cell had its counterpart in pulsatile insulin release when single islets were perifus...

Diacylglycerol (DAG) controls numerous cell functions by regulating the localization of C1-domain-containing proteins, including protein kinase C (PKC), but little is known about the spatiotemporal dynamics of the lipid. Here, we explored plasma membrane DAG dynamics in pancreatic β cells and determined whether DAG signaling is involved in secretagogue-induced pulsatile release of insulin. Single MIN6 cells, primary mouse β cells, and human β cells within intact islets were transfected with translocation biosensors for DAG, PKC activity, or insulin secretion and imaged with total internal reflection fluorescence microscopy. Muscarinic receptor stimulation triggered stable, homogenous DAG elevations, whereas glucose induced short-lived (7.1 ± 0.4 s) but high-amplitude elevations (up to 109 ± 10% fluorescence increase) in spatially confined membrane regions. The spiking was mimicked by membrane depolarization and suppressed after inhibition of exocytosis or of purinergic P2Y₁, but not P2X receptors, reflecting involvement of autocrine purinoceptor activation after exocytotic release of ATP. Each DAG spike caused local PKC activation with resulting dissociation of its substrate protein MARCKS from the plasma membrane. Inhibition of spiking reduced glucose-induced pulsatile insulin secretion. Thus, stimulus-specific DAG signaling patterns appear in the plasma membrane, including distinct microdomains, which have implications for the kinetic control of exocytosis and other membrane-associated processes.

AIMS/HYPOTHESIS: It is not clear how small tyrosine kinase inhibitors, such as imatinib mesilate, protect against diabetes and beta cell death. The aim of this study was to determine whether imatinib, as compared with the non-cAbl-inhibitor sunitinib, affects pro-survival signalling events in the phosphatidylinositol 3-kinase (PI3K) pathway. METHODS: Human EndoC-βH1 cells, murine beta TC-6 cells and human pancreatic islets were used for immunoblot analysis of insulin receptor substrate (IRS)-1, Akt and extracellular signal-regulated kinase (ERK) phosphorylation. Phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] plasma membrane concentrations were assessed in EndoC-βH1 and MIN6 cells using evanescent wave microscopy. Src homology 2-containing inositol 5'-phosphatase 2 (SHIP2) tyrosine phosphorylation and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) serine phosphorylation, as well as c-Abl co-localisation with SHIP2, were studied in HEK293 and EndoC-βH1 cells by immunoprecipitation and immunoblot analysis. Gene expression was assessed using RT-PCR. Cell viability was measured using vital staining. RESULTS: Imatinib stimulated ERK(thr202/tyr204) phosphorylation in a c-Abl-dependent manner. Imatinib, but not sunitinib, also stimulated IRS-1(tyr612), Akt(ser473) and Akt(thr308) phosphorylation. This effect was paralleled by oscillatory bursts in plasma membrane PI(3,4,5)P3 levels. Wortmannin induced a decrease in PI(3,4,5)P3 levels, which was slower in imatinib-treated cells than in control cells, indicating an effect on PI(3,4,5)P3-degrading enzymes. In line with this, imatinib decreased the phosphorylation of SHIP2 but not of PTEN. c-Abl co-immunoprecipitated with SHIP2 and its binding to SHIP2 was largely reduced by imatinib but not by sunitinib. Imatinib increased total β-catenin levels and cell viability, whereas sunitinib exerted negative effects on cell viability. CONCLUSIONS/INTERPRETATION: Imatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00125-013-2903-3) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Specificity and versatility in cyclic AMP (cAMP) signalling are governed by the spatial localisation and temporal dynamics of the signal. Phosphodiesterases (PDEs) are important for shaping cAMP signals by hydrolyzing the nucleotide. In pancreatic beta-cells, glucose triggers sub-plasma-membrane cAMP oscillations, which are important for insulin secretion, but the mechanisms underlying the oscillations are poorly understood. Here, we investigated the role of different PDEs in the generation of cAMP oscillations by monitoring the concentration of cAMP in the sub-plasma-membrane space ([cAMP](pm)) with ratiometric evanescent wave microscopy in MIN6 cells or mouse pancreatic beta-cells expressing a fluorescent translocation biosensor. The general PDE inhibitor IBMX increased [cAMP](pm), and whereas oscillations were frequently observed at 50 mu M IBMX, 300 mu M-1 mM of the inhibitor caused a stable increase in [cAMP](pm). The [cAMP](pm) was nevertheless markedly suppressed by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine, indicating IBMX-insensitive cAMP degradation. Among IBMX-sensitive PDEs, PDE3 was most important for maintaining a low basal level of [cAMP](pm) in unstimulated cells. After glucose induction of [cAMP](pm) oscillations, inhibitors of PDE1, PDE3 and PDE4 inhibitors the average cAMP level, often without disturbing the [cAMP](pm) rhythmicity. Knockdown of the IBMX-insensitive PDE8B by shRNA in MIN6 cells increased the basal level of [cAMP](pm) and prevented the [cAMP](pm)-lowering effect of 2',5'-dideoxyadenosine after exposure to IBMX. Moreover, PDE8B-knockdown cells showed reduced glucose-induced [cAMP](pm) oscillations and loss of the normal pulsatile pattern of insulin secretion. It is concluded that [cAMP](pm) oscillations in beta-cells are caused by periodic variations in cAMP generation, and that several PDEs, including PDE1, PDE3 and the IBMX-insensitive PDE8B, are required for shaping the sub-membrane cAMP signals and pulsatile insulin release. (Less)

OBJECTIVE cAMP is a critical messenger for insulin and glucagon secretion from pancreatic β- and α-cells, respectively. Dispersed β-cells show cAMP oscillations, but the signaling kinetics in cells within intact islets of Langerhans is unknown. RESEARCH DESIGN AND METHODS The subplasma-membrane cAMP concentration ([cAMP]pm) was recorded in α- and β-cells in the mantle of intact mouse pancreatic islets using total internal reflection microscopy and a fluorescent translocation biosensor. Cell identification was based on the opposite effects of adrenaline on cAMP in α- and β-cells. RESULTS In islets exposed to 3 mmol/L glucose, [cAMP]pm was low and stable. Glucagon and glucagon-like peptide-1(7-36)-amide (GLP-1) induced dose-dependent elevation of [cAMP]pm, often with oscillations synchronized among β-cells. Whereas glucagon also induced [cAMP]pm oscillations in most α-cells, CONCLUSIONS Oscillatory [cAMP]pm signaling in secretagogue-stimulated β-cells is maintained within intact islets and depends on transmembrane AC activity. The discovery of glucose- and glucagon-induced [cAMP]pm oscillations in α-cells indicates the involvement of cAMP in the regulation of pulsatile glucagon secretion.

Many effects of insulin, insulin-like growth factors and other receptor stimuli are mediated via the phospholipid second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP(3)). PIP(3) is formed by the activity of phosphoinositide 3-kinases in the plasma membrane, where it serves to recruit signalling proteins. These proteins coordinate complex events leading to changes in cell metabolism, growth, movement and survival. Over the past decade, new techniques for measurements of PIP(3) in the plasma membrane of individual living cells have markedly improved our understanding of the role of this messenger in a variety of cellular processes. This review summarises the mechanisms involved in formation and degradation of PIP(3) in insulin-responsive cells, how PIP(3) can be measured in individual cells as well as accumulating evidence that the plasma membrane PIP(3) concentration undergoes complex spatio-temporal patterns in many types of cells, with particular emphasis on autocrine insulin-induced PIP(3) oscillations in pancreatic beta-cells.

Many effects of insulin, insulin-like growth factors and other receptor stimuli are mediated via the phospholipid second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 is formed by ...

Pancreatic beta-cells possess an inherent ability to generate oscillatory signals that trigger insulin release. Coordination of the secretory activity among beta-cells results in pulsatile insulin secretion from the pancreas, which is considered important for the action of the hormone in the target tissues. This review focuses on the mechanisms underlying oscillatory control of insulin secretion at the level of the individual beta-cell. Recent studies have demonstrated that oscillations of the cytoplasmic Ca(2+) concentration are synchronized with oscillations in beta-cell metabolism, intracellular cAMP concentration, phospholipase C activity and plasma membrane phosphoinositide lipid concentrations. There are complex interdependencies between the different messengers and signalling pathways that contribute to amplitude regulation and shaping of the insulin secretory response to nutrient stimuli and neurohormonal modulators. Several of these pathways may be important pharmacological targets for improving pulsatile insulin secretion in type 2 diabetes.

Cyclic AMP (cAMP) and Ca(2+) are key regulators of exocytosis in many cells, including insulin-secreting beta cells. Glucose-stimulated insulin secretion from beta cells is pulsatile and involves oscillations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), but little is known about the detailed kinetics of cAMP signaling. Using evanescent-wave fluorescence imaging we found that glucose induces pronounced oscillations of cAMP in the submembrane space of single MIN6 cells and primary mouse beta cells. These oscillations were preceded and enhanced by elevations of [Ca(2+)](i). However, conditions raising cytoplasmic ATP could trigger cAMP elevations without accompanying [Ca(2+)](i) rise, indicating that adenylyl cyclase activity may be controlled also by the substrate concentration. The cAMP oscillations correlated with pulsatile insulin release. Whereas elevation of cAMP enhanced secretion, inhibition of adenylyl cyclases suppressed both cAMP oscillations and pulsatile insulin release. We conclude that cell metabolism directly controls cAMP and that glucose-induced cAMP oscillations regulate the magnitude and kinetics of insulin exocytosis.

Cyclic AMP is a ubiquitous second messenger that transduces signals from a variety of cell surface receptors to regulate diverse cellular functions, including secretion, metabolism and gene transcription. In pancreatic beta-cells, cAMP potentiates Ca2+-dependent exocytosis and mediates the stimulation of insulin release exerted by the hormones glucagon and glucagon-like peptide-1 (GLP-1) (refs 4, 5-6). Whereas Ca2+ signals have been extensively characterized and shown to involve oscillations important for the temporal control of insulin secretion, the kinetics of receptor-triggered cAMP signals is unknown. Here we introduce a new ratiometric evanescent-wave-microscopy approach to measure cAMP concentration beneath the plasma membrane, and show that insulin-secreting beta-cells respond to glucagon and GLP-1 with marked cAMP oscillations. Simultaneous measurements of intracellular Ca2+ concentration revealed that the two messengers are interlinked and reinforce each other. Moreover, cAMP oscillations are capable of inducing rapid on-off Ca2+ responses, but only sustained elevation of cAMP concentration induces nuclear translocation of the catalytic subunit of the cAMP-dependent protein kinase. Our results establish a new signalling mode for cAMP and indicate that temporal encoding of cAMP signals might constitute a basis for differential regulation of downstream cellular targets.

Phospholipase C (PLC) is a ubiquitous enzyme involved in the regulation of a variety of cellular processes. Its dependence on Ca2+ is well recognized, but it is not known how PLC activity is affected by physiological variations of the cytoplasmic Ca2+ concentration ([Ca2+](i)). Here, we applied evanescent wave microscopy to monitor PLC activity in parallel with [Ca2+](i) in individual insulin-secreting INS-1 cells using the phosphatidylinositol 4,5-bisphosphate- and inositol 1,4,5-trisphosphate-binding pleckstrin homology domain from PLCdelta(1) fused to green fluorescent protein (PH(PLCdelta1)-GFP) and the Ca2+ indicator fura red. In resting cells, PH(PLCdelta1)-GFP was located predominantly at the plasma membrane. Activation of PLC by muscarinic or purinergic receptor stimulation resulted in PH(PLCdelta1)-GFP translocation from the plasma membrane to the cytoplasm, detected as a decrease in evanescent wave-excited PH(PLCdelta1)-GFP fluorescence. Using this translocation as a measure of PLC activity, we found that depolarization by raising extracellular [K+] triggered activation of the enzyme. This effect could be attributed both to a rise of [Ca2+](i) and to depolarization per se, because some translocation persisted during depolarization in a Ca2+-deficient medium containing the Ca2+ chelator EGTA. Moreover, oscillations of [Ca2+](i) resulting from depolarization with Ca2+ influx evoked concentration-dependent periodic activation of PLC. We conclude that PLC activity is under tight dynamic control of [Ca2+](i). In insulin-secreting beta-cells, this mechanism provides a link between Ca2+ influx and release from intracellular stores that may be important in the regulation of insulin secretion.

Epifluorescence microscopy is widely used for monitoring the cytoplasmic Ca2+ concentration ([Ca2+]i) in living cells. In our laboratory we have applied fluorometric techniques for measuring [Ca2+]i in insulin-releasing pancreatic β-cells using conventional microscopy with fura-2 and confocal laser scanning microscopy with visible wavelength indicators (fluo-3, calcium green-1 and fura red). The β-cells were found to have an intrinsic ability of oscillating in response to glucose, the physiological stimulus of insulin release. When exposed to stimulatory concentrations of glucose the pancreatic β-cells reacted with influx of Ca2+, resulting in large oscillations or a sustained elevation of [Ca2+]i (Graneneiesser et al., 1989).

Mechanisms of pulsatile insulin release in man were explored by studying the induction of oscillatory Ca2+ signals in individual beta cells and islets isolated from the human pancreas. Evidence was provided for a glucose-induced closure of ATP-regulated K+ channels, resulting in voltage-dependent entry of Ca2+. The observation of step-wise increases of capacitance in response to depolarizing pulses suggests that an enhanced influx of Ca2+ is an effective means of stimulating the secretory activity of the isolated human beta cell. Activation of muscarinic receptors (1-10 mumol/l carbachol) and of purinergic P2 receptors (0.01-1 mumol/l ATP) resulted in repetitive transients followed by sustained elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i). Periodic mobilisation of intracellular calcium was seen also when injecting 100 mumol/l GTP-gamma-S into beta cells hyperpolarized to -70 mV. Individual beta cells responded to glucose and tolbutamide with increases of [Ca2+]i, manifested either as large amplitude oscillations (frequency 0.1-0.5/min) or as a sustained elevation. Glucose regulation was based on sudden transitions between the basal and the two alternative states of raised [Ca2+]i at threshold concentrations of the sugar characteristic for the individual beta cells. The oscillatory characteristics of coupled cells were determined collectively rather than by particular pacemaker cells. In intact pancreatic islets the glucose induction of well-synchronized [Ca2+]i oscillations had its counterpart in 2-5 min pulses of insulin. Each of these pulses could be resolved into regularly occurring short insulin transients. It is concluded that glucose stimulation of insulin release in man is determined by the number of beta cells entering into a state with Ca(2+)-induced secretory pulses.

The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in single pancreatic mouse islets superfused in a system allowing concomitant recordings of insulin release. When glucose was raised from 3 to 11 mM, [Ca2+]i responded by a transient lowering followed by a rise to an average level of 192 +/- 11 nM. In 77% of the islets the rise was associated with the gradual appearance of oscillations, which were either fast (2-7/min), slow (0.3-0.9/min), or a combination of both types. The characteristics of the fast [Ca2+]i oscillations were those expected from a relationship with the electrical burst activity in islets. Accordingly, in most cases the fast oscillations were remarkably regular. The slow [Ca2+]i oscillations had characteristics similar to the large amplitude ones in individual beta-cells. Whereas glucagon and dibutyryl cAMP could transform slow islet oscillations into fast ones, the alpha 2-adrenergic agonist clonidine had the opposite effect. The rapid islet oscillations were also facilitated by elevated concentrations of extracellular Ca2+. Reinforcing the arguments for [Ca2+]i oscillations as responsible for a pulsatile insulin secretion it was possible to demonstrate that the release of the hormone from single islets is synchronized with the slow [Ca2+]i oscillations.

Increase of the glucose concentration from 3 to 11 mM resulted in a triphasic release of insulin from perifused ob/ob-mouse beta-cells. A slight inhibition was followed after 2 min by a marked peak and a less pronounced sustained response. At the lower glucose concentration glycine had only marginal effects. However, in the presence of 11 mM glucose, 1-10 mM glycine triggered an immediate and dose-dependent response with an initial peak of insulin release followed by sustained stimulation. In individual beta-cells, rise of the glucose concentration from 3 to 11 mM induced initial lowering of the cytoplasmic Ca2+ concentration ([Ca2+]i) followed by large amplitude oscillations from a level of 50-90 nM to peak values exceeding 300 nM. Already at a concentration of 1 mM, glycine transformed the oscillatory pattern into a sustained level with increase of time-average [Ca2+]i. This elevation became more pronounced in the presence of 10 mM glycine. The effects of glycine on insulin release and [Ca2+]i required extracellular Na+ and were reproduced with the N-methyl analogue sarcosine. It is suggested that glycine potentiation of secretion reflects the elevation of time-average [Ca2+]i both by increased entry and reduced elimination of the cation from the cytoplasm.

Data de publicació electrònica: 03-03-2022 Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes. We are grateful to the Nordic Network for Islet Transplantation (supported by the strategic grant consortium Excellence of Diabetes Research in Sweden, EXODIAB. This study was supported by the Academy of Finland grant 297466 and MetaStem Center of Excellence grant 312437 (to T.O., V.H., P.K.), the Sigrid Jusélius Foundation Grant (to T.O.) and the Helsinki University Hospital Research Funds (to T.O.) and an EMBO Long-Term Fellowship ALT295-2019 (to D.B.). Additional funding was provided by the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement 115797 (INNODIA) and 945268 (INNODIA HARVEST). This Joint Undertaking receives support from the Union’s Horizon 2020 research and innovation program and the European Federation of Pharmaceutical Industries and Associations

The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. beta- and alpha-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing delta-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca2+ and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca2+ signaling among alpha-cells. The measurements allowed comparison of the phase relationship of Ca2+ oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets.

Significance Here we report that intact islets and islet cells from type 2 diabetes (T2D) donors exhibit attenuated molecular oscillators bearing lower circadian amplitude and compromised synchronization capacity in vitro. Furthermore, we reveal that secretion profiles of insulin, proinsulin, and glucagon were circadian rhythmic under physiological conditions. The temporal coordination of the islet hormone secretion was perturbed in human T2D islets, concomitant with the islet molecular clock alterations. Strikingly, clock-deficient human islet cells exhibited disrupted insulin and glucagon granule docking and exocytosis. Treating the T2D islets with the clock modulator Nobiletin boosted circadian amplitude and insulin secretion. Our study uncovers a link between human molecular clockwork and T2D, thus considering clock modulators as putative pharmacological intervention to combat this disorder., Circadian clocks operative in pancreatic islets participate in the regulation of insulin secretion in humans and, if compromised, in the development of type 2 diabetes (T2D) in rodents. Here we demonstrate that human islet α- and β-cells that bear attenuated clocks exhibit strongly disrupted insulin and glucagon granule docking and exocytosis. To examine whether compromised clocks play a role in the pathogenesis of T2D in humans, we quantified parameters of molecular clocks operative in human T2D islets at population, single islet, and single islet cell levels. Strikingly, our experiments reveal that islets from T2D patients contain clocks with diminished circadian amplitudes and reduced in vitro synchronization capacity compared to their nondiabetic counterparts. Moreover, our data suggest that islet clocks orchestrate temporal profiles of insulin and glucagon secretion in a physiological context. This regulation was disrupted in T2D subjects, implying a role for the islet cell-autonomous clocks in T2D progression. Finally, Nobiletin, an agonist of the core-clock proteins RORα/γ, boosted both circadian amplitude of T2D islet clocks and insulin secretion by these islets. Our study emphasizes a link between the circadian clockwork and T2D and proposes that clock modulators hold promise as putative therapeutic agents for this frequent disorder.