Patrick E. MacDonald

Glucose-sensing mechanisms in pancreatic beta-cells

The appropriate secretion of insulin from pancreatic β-cells is critically important to the maintenance of energy homeostasis. The β-cells must sense and respond suitably to postprandial increases of blood glucose, and perturbation of glucose-sensing in these cells can lead to hypoglycaemia or hyperglycaemias and ultimately diabetes. Here, we review β-cell glucose-sensing with a particular focus on the regulation of cellular excitability and exocytosis. We examine in turn: (i) the generation of metabolic signalling molecules; (ii) the regulation of β-cell membrane potential; and (iii) insulin granule dynamics and exocytosis. We further discuss the role of well known and putative candidate metabolic signals as regulators of insulin secretion.

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans.

Glucagon, secreted from pancreatic islet α cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring β cells, or to an intrinsic glucose sensing by the α cells themselves. We examined hormone secretion and Ca2+ responses of α and β cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn2+ signalling was blocked, but was reversed by low concentrations (1–20 μM) of the ATP-sensitive K+ (KATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 μM). Higher diazoxide concentrations (≥30 μM) decreased glucagon and insulin secretion, and α- and β-cell Ca2+ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 μM) stimulated glucagon secretion, whereas high concentrations (>10 μM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na+ (TTX) and N-type Ca2+ channels (ω-conotoxin), but not L-type Ca2+ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca2+ channels and α-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an α-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

Role of kinin B2 receptor signaling in the recruitment of circulating progenitor cells with neovascularization potential

Reduced migratory function of circulating angiogenic progenitor cells (CPCs) has been associated with impaired neovascularization in patients with cardiovascular disease (CVD). Previous findings underline the role of the kallikrein-kinin system in angiogenesis. We now demonstrate the involvement of the kinin B2 receptor (B2R) in the recruitment of CPCs to sites of ischemia and in their proangiogenic action. In healthy subjects, B2R was abundantly present on CD133+ and CD34+ CPCs as well as cultured endothelial progenitor cells (EPCs) derived from blood mononuclear cells (MNCs), whereas kinin B1 receptor expression was barely detectable. In transwell migration assays, bradykinin (BK) exerts a potent chemoattractant activity on CD133+ and CD34+ CPCs and EPCs via a B2R/phosphoinositide 3-kinase/eNOS-mediated mechanism. Migration toward BK was able to attract an MNC subpopulation enriched in CPCs with in vitro proangiogenic activity, as assessed by Matrigel assay. CPCs from cardiovascular disease patients showed low B2R levels and decreased migratory capacity toward BK. When injected systemically into wild-type mice with unilateral limb ischemia, bone marrow MNCs from syngenic B2R-deficient mice resulted in reduced homing of sca-1+ and cKit+flk1+ progenitors to ischemic muscles, impaired reparative neovascularization, and delayed perfusion recovery as compared with wild-type MNCs. Similarly, blockade of the B2R by systemic administration of icatibant prevented the beneficial effect of bone marrow MNC transplantation. BK-induced migration represents a novel mechanism mediating homing of circulating angiogenic progenitors. Reduction of BK sensitivity in progenitor cells from cardiovascular disease patients might contribute to impaired neovascularization after ischemic complications.

G protein-coupled receptor (GPR)40-dependent potentiation of insulin secretion in mouse islets is mediated by protein kinase D1

Aims/hypothesisActivation of the G protein-coupled receptor (GPR)40 by long-chain fatty acids potentiates glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells, and GPR40 agonists are in clinical development for type 2 diabetes therapy. GPR40 couples to the G protein subunit Gαq/11 but the signalling cascade activated downstream is unknown. This study aimed to determine the mechanisms of GPR40-dependent potentiation of GSIS by fatty acids.MethodsInsulin secretion in response to glucose, oleate or diacylglycerol (DAG) was assessed in dynamic perifusions and static incubations in islets from wild-type (WT) and Gpr40−/− mice. Depolymerisation of filamentous actin (F-actin) was visualised by phalloidin staining and epifluorescence. Pharmacological and molecular approaches were used to ascertain the roles of protein kinase D (PKD) and protein kinase C delta in GPR40-mediated potentiation of GSIS.ResultsOleate potentiates the second phase of GSIS, and this effect is largely dependent upon GPR40. Accordingly, oleate induces rapid F-actin remodelling in WT but not in Gpr40−/− islets. Exogenous DAG potentiates GSIS in both WT and Gpr40−/− islets. Oleate induces PKD phosphorylation at residues Ser-744/748 and Ser-916 in WT but not Gpr40−/− islets. Importantly, oleate-induced F-actin depolymerisation and potentiation of GSIS are lost upon pharmacological inhibition of PKD1 or deletion of Prkd1.Conclusions/interpretationWe conclude that the signalling cascade downstream of GPR40 activation by fatty acids involves activation of PKD1, F-actin depolymerisation and potentiation of second-phase insulin secretion. These results provide important information on the mechanisms of action of GPR40, a novel drug target for type 2 diabetes.

Investigation of Transport Mechanisms and Regulation of Intracellular Zn2+ in Pancreatic α-Cells

During insulin secretion, pancreatic α-cells are exposed to Zn2+ released from insulin-containing secretory granules. Although maintenance of Zn2+ homeostasis is critical for cell survival and glucagon secretion, very little is known about Zn2+-transporting pathways and the regulation of Zn2+ in α-cells. To examine the effect of Zn2+ on glucagon secretion and possible mechanisms controlling the intracellular Zn2+ level ([Zn2+]i), we employed a glucagon-producing cell line (α-TC6) and mouse islets where non-β-cells were identified using islets expressing green fluorescent protein exclusively in β-cells. In this study, we first confirmed that Zn2+ treatment resulted in the inhibition of glucagon secretion in α-TC6 cells and mouse islets in vitro. The inhibition of secretion was not likely via activation of KATP channels by Zn2+. We then determined that Zn2+ was transported into α-cells and was able to accumulate under both low and high glucose conditions, as well as upon depolarization of cells with KCl. The nonselective Ca2+ channel blocker Gd3+ partially inhibited Zn2+ influx in α-TC cells, whereas the L-type voltage-gated Ca2+ channel inhibitor nitrendipine failed to block Zn2+ accumulation. To investigate Zn2+ transport further, we profiled α-cells for Zn2+ transporter transcripts from the two families that work in opposite directions, SLC39 (ZIP, Zrt/Irt-like protein) and SLC30 (ZnT, Zn2+ transporter). We observed that Zip1, Zip10, and Zip14 were the most abundantly expressed Zips and ZnT4, ZnT5, and ZnT8 the dominant ZnTs. Because the redox state of cells is also a major regulator of [Zn2+]i, we examined the effects of oxidizing agents on Zn2+ mobilization within α-cells. 2,2′-Dithiodipyridine (-SH group oxidant), menadione (superoxide generator), and SIN-1 (3-morpholinosydnonimine) (peroxynitrite generator) all increased [Zn2+]i in α-cells. Together these results demonstrate that Zn2+ inhibits glucagon secretion, and it is transported into α-cells in part through Ca2+ channels. Zn2+ transporters and the redox state also modulate [Zn2+]i.

The phosphatidylinositol 3-kinase inhibitor LY294002 potently blocks KV currents via a direct mechanism

Voltage‐dependent K+ (Kv) channels negatively regulate Ca2+ entry into pancreatic β‐cells by repolarizing glucose‐stimulated action potentials. A role for phosphatidylinositol 3‐kinase (PI3K) modulation of Kv channel function was investigated using the PI3K inhibitors wortmannin and LY294002, and LY303511, a negative control compound with respect to PI3K activity. In MIN6 insulinoma cells, wortmannin (100 nM) had no effect on whole‐cell outward K+ currents, but LY294002 and LY303511 reversibly blocked currents in a dose‐dependent manner (IC50=9.0±0.7 µM and 64.6±9.1 µM, respectively). Western blotting confirmed the specific inhibitory effects of LY294002 and wortmannin on insulin‐stimulated PI3K activity. Kv currents in rat β‐cells at near physiological temperatures were inhibited 92% by 25 µM LY294002. Kv2.1 and Kv1.4 are highly expressed in β‐cells, and in Kv2.1‐transfected tsA201 cells, 50 µM LY294002 and 100 µM LY303511 reversibly inhibited currents by 99% and 41%, respectively. In Kv1.4‐transfected tsA201 cells, 50 µM LY294002 reduced the inactivation time constant from 73 to 18 ms. The insulinotropic properties of LY294002 and its effects in other excitable cells may be caused by inhibition of Kv currents rather than PI3K antagonism. Furthermore, LY294002 may represent a novel structure from which future Kv channel blockers may be developed.

KATP-channels and glucose-regulated glucagon secretion

Glucagon, secreted by the alpha-cells of the pancreatic islets, is the most important glucose-increasing hormone of the body. The precise regulation of glucagon release remains incompletely defined but has been proposed to involve release of inhibitory factors from neighbouring beta-cells (paracrine control). However, the observation that glucose can regulate glucagon secretion under conditions when insulin secretion does not occur argues that the alpha-cell is also equipped with its own intrinsic (exerted within the alpha-cell itself) glucose sensing. Here we consider the possible mechanisms involved with a focus on ATP-regulated K(+)-channels and changes in alpha-cell membrane potential.

Corelease and differential exit via the fusion pore of GABA, serotonin, and ATP from LDCV in rat pancreatic beta cells

The release of γ-aminobutyric acid (GABA) and ATP from rat β cells was monitored using an electrophysiological assay based on overexpression GABAA or P2X2 receptor ion channels. Exocytosis of LDCVs, detected by carbon fiber amperometry of serotonin, correlated strongly (∼80%) with ATP release. The increase in membrane capacitance per ATP release event was 3.4 fF, close to the expected capacitance of an individual LDCV with a diameter of 0.3 μm. ATP and GABA were coreleased with serotonin with the same probability. Immunogold electron microscopy revealed that ∼15% of the LDCVs contain GABA. Prespike “pedestals,” reflecting exit of granule constituents via the fusion pore, were less frequently observed for ATP than for serotonin or GABA and the relative amplitude (amplitude of foot compared to spike) was smaller: in some cases the ATP-dependent pedestal was missing entirely. An inward tonic current, not dependent on glucose and inhibited by the GABAA receptor antagonist SR95531, was observed in β cells in clusters of islet cells. Noise analysis indicated that it was due to the activity of individual channels with a conductance of 30 pS, the same as expected for individual GABAA Cl− channels with the ionic gradients used. We conclude that (a) LDCVs accumulate ATP and serotonin; (b) regulated release of GABA can be accounted for by exocytosis of a subset of insulin-containing LDCVs; (c) the fusion pore of LDCVs exhibits selectivity and compounds are differentially released depending on their chemical properties (including size); and (d) a glucose-independent nonvesicular form of GABA release exists in β cells.

SUMOylation regulates Kv2.1 and modulates pancreatic β-cell excitability

The covalent attachment of small ubiquitin-like modifier (SUMO) proteins regulates protein localization and function. SUMOylation has recently been shown to modulate ion-channel function; however, the extent to which this affects native currents and cellular excitability remains to be determined. The voltage-dependent K+ (Kv) channel Kv2.1 regulates pancreatic β-cell excitability and insulin secretion. We found that YFP-tagged SUMO1 (SUMO1-YFP) can be immunoprecipitated with Kv2.1 when these two proteins are coexpressed in HEK 293 cells. Furthermore, direct infusion of recombinant SUMO1 peptide or coexpression of SUMO1-YFP inhibited current through cloned Kv2.1 by 80% and 48%, respectively. Insulin-secreting cells express SUMO variants 1 and 3, and expression of the SUMO1-YFP in human β-cells or insulinoma cells inhibited native Kv currents (by 49% and 33%, respectively). Inhibition of the channel resulted from an acceleration of channel inactivation and an inhibition of recovery from inactivation, resulting in the widening of β-cell action potentials and a decreased firing frequency. Finally, these effects on channel function and excitability were augmented by the conjugating enzyme Ubc9 and rescued by the SUMO protease SENP1. Thus, protein SUMOylation can exert a strong inhibitory action on the voltage-dependent K+ channel Kv2.1 and can regulate cellular excitability in native β-cells.

Islet Cholesterol Accumulation Due to Loss of ABCA1 Leads to Impaired Exocytosis of Insulin Granules

OBJECTIVE The ATP-binding cassette transporter A1 (ABCA1) is essential for normal insulin secretion from β-cells. The aim of this study was to elucidate the mechanisms underlying the impaired insulin secretion in islets lacking β-cell ABCA1. RESEARCH DESIGN AND METHODS Calcium imaging, patch clamp, and membrane capacitance were used to assess the effect of ABCA1 deficiency on calcium flux, ion channel function, and exocytosis in islet cells. Electron microscopy was used to analyze β-cell ultrastructure. The quantity and distribution of proteins involved in insulin-granule exocytosis were also investigated. RESULTS We show that a lack of β-cell ABCA1 results in impaired depolarization-induced exocytotic fusion of insulin granules. We observed disturbances in membrane microdomain organization and Golgi and insulin granule morphology in β-cells as well as elevated fasting plasma proinsulin levels in mice in the absence of β-cell ABCA1. Acute cholesterol depletion rescued the exocytotic defect in β-cells lacking ABCA1, indicating that elevated islet cholesterol accumulation directly impairs granule fusion and insulin secretion. CONCLUSIONS Our data highlight a crucial role of ABCA1 and cellular cholesterol in β-cells that is necessary for regulated insulin granule fusion events. These data suggest that abnormalities of cholesterol metabolism may contribute to the impaired β-cell function in diabetes.