imaging-calcium-dynamics-subpopulations-mouse-pancreatic-islet

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

University of Oxford

Biomedical Research Centre

University of Hertfordshire

ATP-sensitive potassium (K(ATP)) channels couple cell metabolism to electrical activity by regulating K(+) fluxes across the plasma membrane. Channel closure is facilitated by ATP, which binds to the pore-forming subunit (Kir6.2). Conversely, channel opening is potentiated by phosphoinositol bisphosphate (PIP(2)), which binds to Kir6.2 and reduces channel inhibition by ATP. Here, we use homology modelling and ligand docking to identify the PIP(2)-binding site on Kir6.2. The model is consistent with a large amount of functional data and was further tested by mutagenesis. The fatty acyl tails of PIP(2) lie within the membrane and the head group extends downwards to interact with residues in the N terminus (K39, N41, R54), transmembrane domains (K67) and C terminus (R176, R177, E179, R301) of Kir6.2. Our model suggests how PIP(2) increases channel opening and decreases ATP binding and channel inhibition. It is likely to be applicable to the PIP(2)-binding site of other Kir channels, as the residues identified are conserved and influence PIP(2) sensitivity in other Kir channel family members.

Identification of the PIP2-binding site on Kir6.2 by molecular modelling and functional analysis.

Los iones de zinc son esenciales para la formaci&oacute;n de un hex&aacute;mero de insulina y hormona de cristalizaci&oacute;n. Un rs13266634 de nonsynonymous de un solo nucle&oacute;tido polimorfismo en el gen SLC30A8, codificaci&oacute;n del transportador de cinc de gr&aacute;nulos secretores ZnT8, se asocia con diabetes tipo 2. Describimos los efectos de eliminar el gen de la ZnT8 en ratones y explorar la acci&oacute;n del alelo en riesgo.

Homeostasis de glucosa y almacenamiento de insulina en ratones nulas para el gr&aacute;nulo de zinc transportador ZnT8 y estudios de las variantes asociadas a diabetes de tipo 2.

&#x950C;&#x79BB;&#x5B50;&#x5BF9;&#x516D;&#x6FC0;&#x7D20;&#x548C;&#x80F0;&#x5C9B;&#x7D20;&#x7ED3;&#x6676;&#x7684;&#x5F62;&#x6210;&#x81F3;&#x5173;&#x91CD;&#x8981;&#x3002;2 &#x578B;&#x7CD6;&#x5C3F;&#x75C5;&#x76F8;&#x5173;&#x7684; SLC30A8 &#x57FA;&#x56E0;&#xFF0C;&#x7F16;&#x7801;&#x5206;&#x6CCC;&#x9897;&#x7C92;&#x950C;&#x8F6C;&#x8FD0;&#x86CB;&#x767D; ZnT8&#xFF0C;&#x7A81;&#x53D8;&#x7684;&#x5355;&#x6838;&#x82F7;&#x9178;&#x591A;&#x6001;&#x6027; rs13266634&#x3002;&#x6211;&#x4EEC;&#x63CF;&#x8FF0;&#x5220;&#x9664; ZnT8 &#x57FA;&#x56E0;&#x5728;&#x5C0F;&#x9F20;&#x4E2D;&#x7684;&#x5F71;&#x54CD;&#xFF0C;&#x5E76;&#x63A2;&#x8BA8;&#x5904;&#x5883;&#x5371;&#x9669;&#x7B49;&#x4F4D;&#x57FA;&#x56E0;&#x7684;&#x4F5C;&#x7528;&#x3002;

&#x80F0;&#x5C9B;&#x7D20;&#x50A8;&#x5B58;&#x548C;&#x8461;&#x8404;&#x7CD6;&#x7A33;&#x6001;&#x7684;&#x5C0F;&#x9F20;&#x7A7A;&#x9897;&#x7C92;&#x950C;&#x8F6C;&#x8FD0;&#x86CB;&#x767D; ZnT8 &#x548C; 2 &#x578B;&#x7CD6;&#x5C3F;&#x75C5;&#x76F8;&#x5173;&#x53D8;&#x5F02;&#x7684;&#x7814;&#x7A76;&#x3002;

Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele.

Insulin storage and glucose homeostasis in mice null for the granule zinc transporter ZnT8 and studies of the type 2 diabetes-associated variants.

Les ions de zinc sont essentielles pour la formation de la cristallisation de l&#39;insuline et l&#39;hormone hexam&eacute;rique. Un rs13266634 de polymorphisme non-synonymes nucl&eacute;otide dans le g&egrave;ne SLC30A8, codant le transporteur de zinc des granules s&eacute;cr&eacute;teurs ZnT8, est associ&eacute;e au diab&egrave;te de type 2. Nous d&eacute;crire les effets de la suppression du g&egrave;ne ZnT8 chez les souris et &eacute;tudier l&#39;action de l&#39;all&egrave;le &agrave; risque.

Hom&eacute;ostasie du glucose et de stockage insuline chez la souris null pour les granules de zinc transporteur ZnT8 et &eacute;tudes sur les variantes associ&eacute;s au diab&egrave;te de type 2.

Zinkionen sind essentiell f&uuml;r die Bildung von Hexameric Insulin und Hormon-Kristallisation. Ein nonsynonymous single Nucleotide Polymorphismus rs13266634 im SLC30A8 gen, den sekretorischen Granulat-Zink-Transporter ZnT8, ist Typ-2-Diabetes zugeordnet. Wir beschreiben die Auswirkungen des L&ouml;schens des ZnT8-Gens bei M&auml;usen und erkunden Sie die Aktion des gef&auml;hrdeten Allels.

Insulin Speicher- und Glukose-Hom&ouml;ostase bei M&auml;usen null f&uuml;r das Untermodul Zink Transporter ZnT8 und Studien der Typ 2 Diabetes-assoziierten Varianten.

Gli ioni di zinco sono essenziali per la formazione di esamerica insulina e ormone della cristallizzazione. Un singolo nucleotide nonsynonymous polimorfismo rs13266634 nel gene SLC30A8, che codifica il trasportatore di zinco granuli secretori ZnT8, &egrave; associato con il diabete di tipo 2. Si descrivono gli effetti dell&#39;eliminazione del gene ZnT8 nei topi ed Esplora l&#39;azione dell&#39;allele a rischio.

Omeostasi di archiviazione e glucosio insulina nei topi null per il granello di zinco transporter ZnT8 e studi delle varianti associato al diabete di tipo 2.

&#x4E9C;&#x925B;&#x30A4;&#x30AA;&#x30F3;&#x306F; 6 &#x91CF;&#x4F53;&#x30DB;&#x30EB;&#x30E2;&#x30F3;&#x3068;&#x30A4;&#x30F3;&#x30B9;&#x30EA;&#x30F3;&#x7D50;&#x6676;&#x306E;&#x5F62;&#x6210;&#x306E;&#x305F;&#x3081;&#x306B;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3059;&#x3002;&#x55C5;&#x899A;&#x306E;&#x5358;&#x4E00;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9;&#x591A;&#x578B; rs13266634 &#x306E;&#x5206;&#x6CCC;&#x9846;&#x7C92;&#x4E9C;&#x925B;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30FC;&#x30BF;&#x30FC; ZnT8&#x3001;&#x30A8;&#x30F3;&#x30B3;&#x30FC;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x306E; SLC30A8 &#x907A;&#x4F1D;&#x5B50;&#x306F;&#x30BF;&#x30A4;&#x30D7; 2 &#x7CD6;&#x5C3F;&#x75C5;&#x3068;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306E;&#x30DE;&#x30A6;&#x30B9;&#x306B;&#x304A;&#x3051;&#x308B; ZnT8 &#x907A;&#x4F1D;&#x5B50;&#x306E;&#x524A;&#x9664;&#x306E;&#x5F71;&#x97FF;&#x3092;&#x8AAC;&#x660E;&#x3057;&#x3001;&#x30A2;&#x30C3;&#x30C8;&#x30EA;&#x30B9;&#x30AF;&#x306E;&#x5BFE;&#x7ACB;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x4F5C;&#x7528;&#x3092;&#x63A2;&#x308B;&#x3002;

&#x30A4;&#x30F3;&#x30B9;&#x30EA;&#x30F3;&#x306E;&#x30B9;&#x30C8;&#x30EC;&#x30FC;&#x30B8;&#x3068;&#x30B0;&#x30EB;&#x30B3;&#x30FC;&#x30B9;&#x306E;&#x6052;&#x5E38;&#x6027;&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x9846;&#x7C92;&#x306E; null &#x4E9C;&#x925B;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30FC;&#x30BF;&#x30FC; ZnT8 &#x3068; 2 &#x578B;&#x7CD6;&#x5C3F;&#x75C5;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x305F;&#x30D0;&#x30EA;&#x30A2;&#x30F3;&#x30C8;&#x306E;&#x7814;&#x7A76;&#x3002;

&#xC544;&#xC5F0; &#xC774;&#xC628; hexameric &#xC778;&#xC290;&#xB9B0;&#xACFC; &#xD638;&#xB974;&#xBAAC; &#xACB0;&#xC815; &#xD654; &#xD615;&#xC131;&#xC744; &#xC704;&#xD574; &#xD544;&#xC218;&#xC801;&#xC785;&#xB2C8;&#xB2E4;. &#xC778;&#xCF54;&#xB529; &#xBD84;&#xACFC; &#xB9BD; &#xC544;&#xC5F0; &#xC804;&#xC1A1; ZnT8, SLC30A8 &#xC720;&#xC804;&#xC790;&#xC758; nonsynonymous &#xB2E8;&#xC77C; &#xC5FC;&#xAE30; &#xB2E4;&#xD615;&#xC131; rs13266634&#xC740; 2 &#xD615; &#xB2F9;&#xB1E8;&#xBCD1;&#xACFC; &#xAD00;&#xB828; &#xB41C;. &#xC6B0;&#xB9AC; &#xC950;&#xC5D0; ZnT8 &#xC720;&#xC804;&#xC790; &#xC0AD;&#xC81C;&#xC758; &#xD6A8;&#xACFC; &#xC124;&#xBA85; &#xD558; &#xACE0; &#xC704;&#xD5D8; &#xB300;&#xB9BD; &#xC720;&#xC804;&#xC790;&#xC758; &#xD589;&#xB3D9;&#xC744; &#xD0D0;&#xAD6C;.

&#xC0DD;&#xC950;&#xB294;&#xACFC; &#xB9BD;&#xC5D0; &#xB300; &#xD55C; null&#xC5D0;&#xC11C; &#xC778;&#xC290;&#xB9B0; &#xC800;&#xC7A5; &#xBC0F; &#xD3EC;&#xB3C4; &#xB2F9; &#xD56D;&#xC0C1;&#xC131; &#xC544;&#xC5F0; &#xC804;&#xC1A1; ZnT8 &#xBC0F; &#xD0C0;&#xC785;-2 &#xB2F9;&#xB1E8;&#xBCD1;&#xC5D0; &#xAD00;&#xB828; &#xB41C; &#xBCC0;&#xD615; &#xC5F0;&#xAD6C;.

Incretins such as glucagon-like peptide 1 (GLP-1) are released from the gut and potentiate insulin release in a glucose-dependent manner. Although this action is generally believed to hinge on cAMP and protein kinase A signaling, up-regulated beta cell intermediary metabolism may also play a role in incretin-stimulated insulin secretion. By employing recombinant probes to image ATP dynamically in situ within intact mouse and human islets, we sought to clarify the role of GLP-1-modulated energetics in beta cell function. Using these techniques, we show that GLP-1 engages a metabolically coupled subnetwork of beta cells to increase cytosolic ATP levels, an action independent of prevailing energy status. We further demonstrate that the effects of GLP-1 are accompanied by alterations in the mitochondrial inner membrane potential and, at elevated glucose concentration, depend upon GLP-1 receptor-directed calcium influx through voltage-dependent calcium channels. Lastly, and highlighting critical species differences, beta cells within mouse but not human islets respond coordinately to incretin stimulation. Together, these findings suggest that GLP-1 alters beta cell intermediary metabolism to influence ATP dynamics in a species-specific manner, and this may contribute to divergent regulation of the incretin-axis in rodents and man. 

Incretin-modulated beta cell energetics in intact islets of Langerhans.

ATP is not only recognized as the universal energy "currency" in most cells but also plays a less well-known role as an intracellular and extracellular messenger. Here, we review novel approaches for measuring free ATP (or ATP/ADP ratios) in living mammalian cells by using genetically encoded sensors. We also discuss the key technical aspects of routine real-time ATP/ADP monitoring using as a model one of the last-generation fluorescent probes, a fusion protein commonly known as "Perceval." Finally, we present detailed guidelines for the simultaneous measurement of cytosolic ATP/ADP ratios and Ca(2+) concentrations alongside electrical parameters in individual pancreatic β cells, in which energy metabolism is tightly linked to plasma membrane excitability to control the secretion of insulin. With appropriate variations, this approach can be adapted to the study of cytosolic ATP/ADP ratios and Ca(2+) concentrations in malignant cells, two important aspects of oncometabolism. 

Use of genetically encoded sensors to monitor cytosolic ATP/ADP ratio in living cells.

Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the KATP channel blocker tolbutamide, and the L-type Ca(2+) channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na(+). The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na(+)-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca(2+) from thapsigargin-sensitive Ca(2+) stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by β cells.

GLP-1 stimulates insulin secretion by PKC-dependent TRPM4 and TRPM5 activation.

How is β-cell mass adjusted to changes in the functional insulin requirements? The answer to this question is central to the understanding of the causes and (potentially) the therapy of type 2 diabetes. In this issue of Cell Metabolism, El Ouaamari et al. (2016) report that increased production of the protease inhibitor SerpinB1 in the liver links insulin resistance to stimulation of β-cell proliferation.

Dramatis Personae in β-Cell Mass Regulation: Enter SerpinB1.

The transcription factor Sox4 has been proposed to underlie the increased type 2 diabetes risk linked to an intronic single nucleotide polymorphism in CDKAL1 In a mouse model expressing a mutant form of Sox4, glucose-induced insulin secretion is reduced by 40% despite normal intracellular Ca(2+) signaling and depolarization-evoked exocytosis. This paradox is explained by a fourfold increase in kiss-and-run exocytosis (as determined by single-granule exocytosis measurements) in which the fusion pore connecting the granule lumen to the exterior expands to a diameter of only 2 nm, which does not allow the exit of insulin. Microarray analysis indicated that this correlated with an increased expression of the exocytosis-regulating protein Stxbp6. In a large collection of human islet preparations (n = 63), STXBP6 expression and glucose-induced insulin secretion correlated positively and negatively with SOX4 expression, respectively. Overexpression of SOX4 in the human insulin-secreting cell EndoC-βH2 interfered with granule emptying and inhibited hormone release, the latter effect reversed by silencing STXBP6 These data suggest that increased SOX4 expression inhibits insulin secretion and increased diabetes risk by the upregulation of STXBP6 and an increase in kiss-and-run exocytosis at the expense of full fusion. We propose that pharmacological interventions promoting fusion pore expansion may be effective in diabetes therapy.

Increased Expression of the Diabetes Gene SOX4 Reduces Insulin Secretion by Impaired Fusion Pore Expansion.

Scanning ion conductance microscopy (SICM) is a super-resolution live imaging technique that uses a glass nanopipette as an imaging probe to produce three-dimensional (3D) images of cell surface. SICM can be used to analyze cell morphology at nanoscale, follow membrane dynamics, precisely position an imaging nanopipette close to a structure of interest, and use it to obtain ion channel recordings or locally apply stimuli or drugs. Practical implementations of these SICM advantages, however, are often complicated due to the limitations of currently available SICM systems that inherited their design from other scanning probe microscopes in which the scan assembly is placed right above the specimen. Such arrangement makes the setting of optimal illumination necessary for phase contrast or the use of high magnification upright optics difficult. Here, we describe the designs that allow mounting SICM scan head on a standard patch-clamp micromanipulator and imaging the sample at an adjustable approach angle. This angle could be as shallow as the approach angle of a patch-clamp pipette between a water immersion objective and the specimen. Using this angular approach SICM, we obtained topographical images of cells grown on nontransparent nanoneedle arrays, of islets of Langerhans, and of hippocampal neurons under upright optical microscope. We also imaged previously inaccessible areas of cells such as the side surfaces of the hair cell stereocilia and the intercalated disks of isolated cardiac myocytes, and performed targeted patch-clamp recordings from the latter. Thus, our new, to our knowledge, angular approach SICM allows imaging of living cells on nontransparent substrates and a seamless integration with most patch-clamp setups on either inverted or upright microscopes, which would facilitate research in cell biophysics and physiology.

Angular Approach Scanning Ion Conductance Microscopy.

We explored the role of the Krebs cycle enzyme fumarate hydratase (FH) in glucose-stimulated insulin secretion (GSIS). Mice lacking Fh1 in pancreatic β cells (Fh1βKO mice) appear normal for 6-8 weeks but then develop progressive glucose intolerance and diabetes. Glucose tolerance is rescued by expression of mitochondrial or cytosolic FH but not by deletion of Hif1α or Nrf2. Progressive hyperglycemia in Fh1βKO mice led to dysregulated metabolism in β cells, a decrease in glucose-induced ATP production, electrical activity, cytoplasmic [Ca] elevation, and GSIS. Fh1 loss resulted in elevated intracellular fumarate, promoting succination of critical cysteines in GAPDH, GMPR, and PARK 7/DJ-1 and cytoplasmic acidification. Intracellular fumarate levels were increased in islets exposed to high glucose and in islets from human donors with type 2 diabetes (T2D). The impaired GSIS in islets from diabetic Fh1βKO mice was ameliorated after culture under normoglycemic conditions. These studies highlight the role of FH and dysregulated mitochondrial metabolism in T2D.

Fumarate Hydratase Deletion in Pancreatic β Cells Leads to Progressive Diabetes.

Spatial control of G-protein-coupled receptor (GPCR) signaling, which is used by cells to translate complex information into distinct downstream responses, is achieved by using plasma membrane (PM) and endocytic-derived signaling pathways. The roles of the endomembrane in regulating such pleiotropic signaling via multiple G-protein pathways remain unknown. Here, we investigated the effects of disease-causing mutations of the adaptor protein-2σ subunit (AP2σ) on signaling by the class C GPCR calcium-sensing receptor (CaSR). These AP2σ mutations increase CaSR PM expression yet paradoxically reduce CaSR signaling. Hypercalcemia-associated AP2σ mutations reduced CaSR signaling via Gα and Gα pathways. The mutations also delayed CaSR internalization due to prolonged residency time of CaSR in clathrin structures that impaired or abolished endosomal signaling, which was predominantly mediated by Gα. Thus, compartmental bias for CaSR-mediated Gα endomembrane signaling provides a mechanistic basis for multidimensional GPCR signaling.

AP2σ Mutations Impair Calcium-Sensing Receptor Trafficking and Signaling, and Show an Endosomal Pathway to Spatially Direct G-Protein Selectivity.

Adrenaline is a powerful stimulus of glucagon secretion. It acts by activation of β-adrenergic receptors, but the downstream mechanisms have only been partially elucidated. Here, we have examined the effects of adrenaline in mouse and human α-cells by a combination of electrophysiology, imaging of Ca and PKA activity, and hormone release measurements. We found that stimulation of glucagon secretion correlated with a PKA- and EPAC2-dependent (inhibited by PKI and ESI-05, respectively) elevation of [Ca] in α-cells, which occurred without stimulation of electrical activity and persisted in the absence of extracellular Ca but was sensitive to ryanodine, bafilomycin, and thapsigargin. Adrenaline also increased [Ca] in α-cells in human islets. Genetic or pharmacological inhibition of the Tpc2 channel (that mediates Ca release from acidic intracellular stores) abolished the stimulatory effect of adrenaline on glucagon secretion and reduced the elevation of [Ca] Furthermore, in Tpc2-deficient islets, ryanodine exerted no additive inhibitory effect. These data suggest that β-adrenergic stimulation of glucagon secretion is controlled by a hierarchy of [Ca] signaling in the α-cell that is initiated by cAMP-induced Tpc2-dependent Ca release from the acidic stores and further amplified by Ca-induced Ca release from the sarco/endoplasmic reticulum.

Adrenaline Stimulates Glucagon Secretion by Tpc2-Dependent Ca Mobilization From Acidic Stores in Pancreatic α-Cells.

High-content real-time imaging of hormone secretion in tissues or cell populations is a challenging task, which is unlikely to be resolved directly, despite immense translational value. We approach this problem indirectly, using compensatory endocytosis, a process that closely follows exocytosis in the cell, as a surrogate read-out for secretion. The tissue is immobilized in an open-air perifusion chamber and imaged using a two-photon microscope. A fluorescent polar tracer, perifused through the experimental circuit, gets trapped into the cells via endocytosis, and is quantified using a feature-detection algorithm. The signal of the tracer that accumulates into the endocytotic system reliably reflects stimulated exocytosis, which is demonstrated via co-imaging of the latter using existing reporters. A high signal-to-noise ratio and compatibility with multisensor imaging affords the real-time quantification of the secretion at the tissue/population level, whereas the cumulative nature of the signal allows imprinting of the "secretory history" within each cell. The technology works for several cell types, reflects disease progression and can be used for human tissue.

Monitoring real-time hormone release kinetics via high-content 3-D imaging of compensatory endocytosis.

Diabetes is a bihormonal disorder resulting from combined insulin and glucagon secretion defects. Mice lacking fumarase (Fh1) in their β cells (Fh1βKO mice) develop progressive hyperglycemia and dysregulated glucagon secretion similar to that seen in diabetic patients (too much at high glucose and too little at low glucose). The glucagon secretion defects are corrected by low concentrations of tolbutamide and prevented by the sodium-glucose transport (SGLT) inhibitor phlorizin. These data link hyperglycemia, intracellular Na accumulation, and acidification to impaired mitochondrial metabolism, reduced ATP production, and dysregulated glucagon secretion. Protein succination, reflecting reduced activity of fumarase, is observed in α cells from hyperglycemic Fh1βKO and β-V59M gain-of-function K channel mice, diabetic Goto-Kakizaki rats, and patients with type 2 diabetes. Succination is also observed in renal tubular cells and cardiomyocytes from hyperglycemic Fh1βKO mice, suggesting that the model can be extended to other SGLT-expressing cells and may explain part of the spectrum of diabetic complications.

Dysregulation of Glucagon Secretion by Hyperglycemia-Induced Sodium-Dependent Reduction of ATP Production.

Limited access to human islets has prompted the development of human beta cell models. The human beta cell lines EndoC-βH1 and EndoC-βH2 are increasingly used by the research community. However, little is known of their electrophysiological and secretory properties. Here, we monitored parameters that constitute the glucose-triggering pathway of insulin release. Both cell lines respond to glucose (6 and 20 mM) with 2- to 3-fold stimulation of insulin secretion which correlated with an elevation of [Ca], membrane depolarisation and increased action potential firing. Similar to human primary beta cells, K channel activity is low at 1 mM glucose and is further reduced upon increasing glucose concentration; an effect that was mimicked by the K channel blocker tolbutamide. The upstroke of the action potentials reflects the activation of Ca channels with some small contribution of TTX-sensitive Na channels. The repolarisation involves activation of voltage-gated Kv2.2 channels and large-conductance Ca-activated K channels. Exocytosis presented a similar kinetics to human primary beta cells. The ultrastructure of these cells shows insulin vesicles composed of an electron-dense core surrounded by a thin clear halo. We conclude that the EndoC-βH1 and -βH2 cells share many features of primary human β-cells and thus represent a useful experimental model.

Electrophysiological properties of human beta-cell lines EndoC-βH1 and -βH2 conform with human beta-cells.

Diabetes is a global health problem caused primarily by the inability of pancreatic β-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of β-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic βV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 β-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in β-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of β-cells in diabetes.

Andrei I. Tarasov

Oxford Centre for Diabetes,
Endocrinology and Metabolism,
Oxford Centre for Diabetes, Endocrinology and Metabolism

University of Oxford

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Andrei I. Tarasov

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Oxford Centre for Diabetes,Endocrinology and Metabolism,Oxford Centre for Diabetes, Endocrinology and Metabolism

University of Oxford

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Oxford Centre for Diabetes,
Endocrinology and Metabolism,
Oxford Centre for Diabetes, Endocrinology and Metabolism