Jocelyn E. Manning Fox

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.

Distinct myoprotective roles of cardiac sarcolemmal and mitochondrial KATP channels during metabolic inhibition and recovery

The protective roles of sarcolemmal (sarc) and mitochondrial (mito)KATP channels are un‐clear despite their apparent importance to ischemic preconditioning. We examined these roles by monitoring intracellular calcium ([Ca]int), using fura‐2 and fluo‐3, in enzymatically isolated rat right ventricular myocytes. Myocyte mortality, estimated using a trypan blue assay, changed approximately in parallel with changes in [Ca]int. Chemically induced hypoxia (CIH), induced by application of cyanide and 2‐deoxy‐glucose, caused a steady rise in [Ca]int. Calcium increased more rapidly on ‘reoxygenation’ by return to control solutions. The protein kinase C (PKC) activator PMA abolished both phases of calcium increase. The mitoKATP channel‐selective blocker 5‐hydroxydecanoate partially prevented the PMA‐induced protection during CIH, but not during reoxygenation. In contrast, HMR 1098, a sarcKATP channel‐selective blocker, abolished protection only during the reoxygenation. Adenosine (A1) receptor activation prevented or reduced increases in [Ca]int and improved cell viability via a PKC and mito/sarcKATP channel‐dependent mechanism. PKC‐dependent protection against cytoplasmic calcium increases was also observed in a human cell line (tsA201) transiently expressing sarcKATP channels. Protection was abolished only during the reoxygenation phase by the amino acid substitution (T180A) in the pore‐forming Kir6.2 subunit, a mutation previously shown to prevent PKC‐dependent modulation. Our data suggest that sarc and mitoKATP channel populations play distinct protective roles, triggered by PKC and/or adenosine, during chemically induced hypoxia/reoxygenation.—Light, P. E., Kanji, H. D., Manning Fox, J. E., French, R. J. Distinct myoprotective roles of cardiac sarcolem‐mal and mitochondrial KATP channels during metabolic inhibition and recovery. FASEB J. 15, 2586–2594 (2001)

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.

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.

Transcript Expression Data from Human Islets Links Regulatory Signals from Genome-Wide Association Studies for Type 2 Diabetes and Glycemic Traits to Their Downstream Effectors.

The intersection of genome-wide association analyses with physiological and functional data indicates that variants regulating islet gene transcription influence type 2 diabetes (T2D) predisposition and glucose homeostasis. However, the specific genes through which these regulatory variants act remain poorly characterized. We generated expression quantitative trait locus (eQTL) data in 118 human islet samples using RNA-sequencing and high-density genotyping. We identified fourteen loci at which cis-exon-eQTL signals overlapped active islet chromatin signatures and were coincident with established T2D and/or glycemic trait associations. ‎At some, these data provide an experimental link between GWAS signals and biological candidates, such as DGKB and ADCY5. At others, the cis-signals implicate genes with no prior connection to islet biology, including WARS and ZMIZ1. At the ZMIZ1 locus, we show that perturbation of ZMIZ1 expression in human islets and beta-cells influences exocytosis and insulin secretion, highlighting a novel role for ZMIZ1 in the maintenance of glucose homeostasis. Together, these findings provide a significant advance in the mechanistic insights of T2D and glycemic trait association loci.

Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells

Insulin secretion from β cells of the pancreatic islets of Langerhans controls metabolic homeostasis and is impaired in individuals with type 2 diabetes (T2D). Increases in blood glucose trigger insulin release by closing ATP-sensitive K+ channels, depolarizing β cells, and opening voltage-dependent Ca2+ channels to elicit insulin exocytosis. However, one or more additional pathway(s) amplify the secretory response, likely at the distal exocytotic site. The mitochondrial export of isocitrate and engagement with cytosolic isocitrate dehydrogenase (ICDc) may be one key pathway, but the mechanism linking this to insulin secretion and its role in T2D have not been defined. Here, we show that the ICDc-dependent generation of NADPH and subsequent glutathione (GSH) reduction contribute to the amplification of insulin exocytosis via sentrin/SUMO-specific protease-1 (SENP1). In human T2D and an in vitro model of human islet dysfunction, the glucose-dependent amplification of exocytosis was impaired and could be rescued by introduction of signaling intermediates from this pathway. Moreover, islet-specific Senp1 deletion in mice caused impaired glucose tolerance by reducing the amplification of insulin exocytosis. Together, our results identify a pathway that links glucose metabolism to the amplification of insulin secretion and demonstrate that restoration of this axis rescues β cell function in T2D.

Mitochondrial Metabolism of Pyruvate Is Essential for Regulating Glucose-stimulated Insulin Secretion

Background: Pyruvate metabolism plays an essential role in pancreatic β-cells. Results: Pharmacological and siRNA-mediated inhibition of mitochondrial pyruvate carrier-1 and -2 inhibit β-cell metabolism and insulin secretion. Conclusion: Pyruvate entry into β-cell mitochondria is critical for regulating insulin release. Significance: Mitochondrial metabolism of pyruvate plays a key role in generating signals in response to nutrients that control insulin release. It is well known that mitochondrial metabolism of pyruvate is critical for insulin secretion; however, we know little about how pyruvate is transported into mitochondria in β-cells. Part of the reason for this lack of knowledge is that the carrier gene was only discovered in 2012. In the current study, we assess the role of the recently identified carrier in the regulation of insulin secretion. Our studies show that β-cells express both mitochondrial pyruvate carriers (Mpc1 and Mpc2). Using both pharmacological inhibitors and siRNA-mediated knockdown of the MPCs we show that this carrier plays a key role in regulating insulin secretion in clonal 832/13 β-cells as well as rat and human islets. We also show that the MPC is an essential regulator of both the ATP-regulated potassium (KATP) channel-dependent and -independent pathways of insulin secretion. Inhibition of the MPC blocks the glucose-stimulated increase in two key signaling molecules involved in regulating insulin secretion, the ATP/ADP ratio and NADPH/NADP+ ratio. The MPC also plays a role in in vivo glucose homeostasis as inhibition of MPC by the pharmacological inhibitor α-cyano-β-(1-phenylindol-3-yl)-acrylate (UK5099) resulted in impaired glucose tolerance. These studies clearly show that the newly identified mitochondrial pyruvate carrier sits at an important branching point in nutrient metabolism and that it is an essential regulator of insulin secretion.

Cardioselectivity of the sulphonylurea HMR 1098: studies on native and recombinant cardiac and pancreatic KATP channels

In this study we investigated the effects of the putative cardioselective sulphonylurea derivative HMR 1098 on ATP‐sensitive potassium (KATP) channels from cardiac ventricular myocytes, the INS‐1 β‐cell line and from recombinant KATP channels composed of SUR2A/Kir6.2, SUR1/Kir6.2, SUR1/Kir6.1 an Kir6.2,ΔC26. Recombinant channels were expressed in tsA201 or COS‐1 cells. The effects of HMR 1098 on single channel and whole‐cell currents were recorded using the patch‐clamp technique. At the single channel level, using excised inside‐out membrane patches, HMR 1098 inhibited KATP channels from ventricular cells and INS‐1 cells with IC50s of 0.88 and 720 μM respectively. Similar results to those in cardiac cells were obtained using recombinant SUR2A/Kir6.2 KATP channels. HMR 1098 inhibition of SUR2A/Kir6.2 KATP channels was unaffected by the presence of internal ADP. In whole‐cell recordings, HMR 1098 inhibited SUR2A/Kir6.2 and SUR1/Kir6.2 currents with IC50s of 2.1 and 860 μM respectively. HMR 1098 was without effect on currents either from the Kir6.2,ΔC26 truncation mutant or from Kir2.1. Our results demonstrate that HMR 1098 is a selective inhibitor of cardiac KATP channels, showing a 400–800‐fold selectivity over β‐cell KATP channels. The non‐aromatic substitutions in the sulphonylurea moiety greatly increase the cardioselectivity of this compound while reducing the overall blocking potency of this sulphonylurea derivative.

Research-Focused Isolation of Human Islets From Donors With and Without Diabetes at the Alberta Diabetes Institute IsletCore

Recent years have seen an increased focus on human islet biology, and exciting findings in the stem cell and genomic arenas highlight the need to define the key features of mature human islets and β-cells. Donor and organ procurement parameters impact human islet yield, although for research purposes islet yield may be secondary in importance to islet function. We examined the feasibility of a research-only human islet isolation, distribution, and biobanking program and whether key criteria such as cold ischemia time (CIT) and metabolic status may be relaxed and still allow successful research-focused isolations, including from donors with type 1 diabetes and type 2 diabetes. Through 142 isolations over approximately 5 years, we confirm that CIT and glycated hemoglobin each have a weak negative impacts on isolation purity and yield, and extending CIT beyond the typical clinical isolation cutoff of 12 hours (to ≥ 18 h) had only a modest impact on islet function. Age and glycated hemoglobin/type 2 diabetes status negatively impacted secretory function; however, these and other biological (sex, body mass index) and procurement/isolation variables (CIT, time in culture) appear to make only a small contribution to the heterogeneity of human islet function. This work demonstrates the feasibility of extending acceptable CIT for research-focused human islet isolation and highlights the biological variation in function of human islets from donors with and without diabetes.

Intraislet SLIT–ROBO signaling is required for beta-cell survival and potentiates insulin secretion

Significance There is an unmet need for factors that can protect pancreatic islet beta cells from apoptosis and improve insulin secretion in the context of diabetes. There are many candidate factors produced locally in islets. We investigated the role of axon guidance factors and found that the SLIT–Roundabout receptors system is present, where it responds to stress. Full expression of SLIT ligands is essential for optimal beta-cell survival. Recombinant SLIT promotes survival and increases insulin secretion via mechanisms involving Ca2+ and actin. We previously cataloged putative autocrine/paracrine signaling loops in pancreatic islets, including factors best known for their roles in axon guidance. Emerging evidence points to nonneuronal roles for these factors, including the Slit–Roundabout receptor (Robo) family, in cell growth, migration, and survival. We found SLIT1 and SLIT3 in both beta cells and alpha cells, whereas SLIT2 was predominantly expressed in beta cells. ROBO1 and ROBO2 receptors were detected in beta and alpha cells. Remarkably, even modest knockdown of Slit production resulted in significant beta-cell death, demonstrating a critical autocrine/paracrine survival role for this pathway. Indeed, recombinant SLIT1, SLIT2, and SLIT3 decreased serum deprivation, cytokine, and thapsigargin-induced cell death under hyperglycemic conditions. SLIT treatment also induced a gradual release of endoplasmic reticulum luminal Ca2+, suggesting a unique molecular mechanism capable of protecting beta cells from endoplasmic reticulum stress-induced apoptosis. SLIT treatment was also associated with rapid actin remodeling. SLITs potentiated glucose-stimulated insulin secretion and increased the frequency of glucose-induced Ca2+ oscillations. These observations point to unexpected roles for local Slit secretion in the survival and function of pancreatic beta cells. Because diabetes results from a deficiency in functional beta-cell mass, these studies may contribute to therapeutic approaches for improving beta-cell survival and function.