Publications by authors named "Jocelyn E Manning Fox"

50 Publications

Cryopreservation and post-thaw characterization of dissociated human islet cells.

PLoS One 2022 26;17(1):e0263005. Epub 2022 Jan 26.

Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada.

The objective of this study is to optimize the cryopreservation of dissociated islet cells and obtain functional cells that can be used in single-cell transcriptome studies on the pathology and treatment of diabetes. Using an iterative graded freezing approach we obtained viable cells after cooling in 10% dimethyl sulfoxide and 6% hydroxyethyl starch at 1°C/min to -40°C, storage in liquid nitrogen, rapid thaw, and removal of cryoprotectants by serial dilution. The expression of epithelial cell adhesion molecule declined immediately after thaw, but recovered after overnight incubation, while that of an endocrine cell marker (HPi2) remained high after cryopreservation. Patch-clamp electrophysiology revealed differences in channel activities and exocytosis of various islet cell types; however, exocytotic responses, and the biophysical properties of voltage-gated Na+ and Ca2+ channels, are sustained after cryopreservation. Single-cell RNA sequencing indicates that overall transcriptome and crucial exocytosis genes are comparable between fresh and cryopreserved dispersed human islet cells. Thus, we report an optimized procedure for cryopreserving dispersed islet cells that maintained their membrane integrity, along with their molecular and functional phenotypes. Our findings will not only provide a ready source of cells for investigating cellular mechanisms in diabetes but also for bio-engineering pseudo-islets and islet sheets for modeling studies and potential transplant applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0263005PLOS
January 2022

β-Cell Knockout of SENP1 Reduces Responses to Incretins and Worsens Oral Glucose Tolerance in High-Fat Diet-Fed Mice.

Diabetes 2021 11 30;70(11):2626-2638. Epub 2021 Aug 30.

Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada

SUMOylation reduces oxidative stress and preserves islet mass at the expense of robust insulin secretion. To investigate a role for the deSUMOylating enzyme sentrin-specific protease 1 (SENP1) following metabolic stress, we put pancreas/gut-specific SENP1 knockout (pSENP1-KO) mice on a high-fat diet (HFD). Male pSENP1-KO mice were more glucose intolerant following HFD than littermate controls but only in response to oral glucose. A similar phenotype was observed in females. Plasma glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) responses were identical in pSENP1-KO and wild-type littermates, including the HFD-induced upregulation of GIP responses. Islet mass was not different, but insulin secretion and β-cell exocytotic responses to the GLP-1 receptor agonist exendin-4 (Ex4) and GIP were impaired in islets lacking SENP1. Glucagon secretion from pSENP1-KO islets was also reduced, so we generated β-cell-specific SENP1 KO mice. These phenocopied the pSENP1-KO mice with selective impairment in oral glucose tolerance following HFD, preserved islet mass expansion, and impaired β-cell exocytosis and insulin secretion to Ex4 and GIP without changes in cAMP or Ca levels. Thus, β-cell SENP1 limits oral glucose intolerance following HFD by ensuring robust insulin secretion at a point downstream of incretin signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db20-1235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8564408PMC
November 2021

Genetic variant effects on gene expression in human pancreatic islets and their implications for T2D.

Nat Commun 2020 09 30;11(1):4912. Epub 2020 Sep 30.

Department of Genetics, University of North Carolina, Chapel Hill, NC, 27599, USA.

Most signals detected by genome-wide association studies map to non-coding sequence and their tissue-specific effects influence transcriptional regulation. However, key tissues and cell-types required for functional inference are absent from large-scale resources. Here we explore the relationship between genetic variants influencing predisposition to type 2 diabetes (T2D) and related glycemic traits, and human pancreatic islet transcription using data from 420 donors. We find: (a) 7741 cis-eQTLs in islets with a replication rate across 44 GTEx tissues between 40% and 73%; (b) marked overlap between islet cis-eQTL signals and active regulatory sequences in islets, with reduced eQTL effect size observed in the stretch enhancers most strongly implicated in GWAS signal location; (c) enrichment of islet cis-eQTL signals with T2D risk variants identified in genome-wide association studies; and (d) colocalization between 47 islet cis-eQTLs and variants influencing T2D or glycemic traits, including DGKB and TCF7L2. Our findings illustrate the advantages of performing functional and regulatory studies in disease relevant tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-18581-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7528108PMC
September 2020

A role for alternative splicing in circadian control of exocytosis and glucose homeostasis.

Genes Dev 2020 08 2;34(15-16):1089-1105. Epub 2020 Jul 2.

Department of Medicine, Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.

The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic β cells that are perturbed in and β-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant β cells, including transcripts encoding () and (). Depletion of THRAP3 restores expression of the long isoforms of and , and mimicking exon skipping in these transcripts through antisense oligonucleotide delivery in wild-type islets reduces glucose-stimulated insulin secretion. Finally, we identify shared networks of alternatively spliced exocytic genes from islets of rodent models of diet-induced obesity that significantly overlap with clock mutants. Our results establish a role for pre-mRNA alternative splicing in β-cell function across the sleep/wake cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.338178.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397853PMC
August 2020

Vitamin-D-Binding Protein Contributes to the Maintenance of α Cell Function and Glucagon Secretion.

Cell Rep 2020 06;31(11):107761

Institute of Metabolism and Systems Research (IMSR), University of Birmingham, Birmingham B15 2TT, UK; Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham B15 2TT, UK; Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Midlands, UK. Electronic address:

Vitamin-D-binding protein (DBP) or group-specific component of serum (GC-globulin) carries vitamin D metabolites from the circulation to target tissues. DBP is highly localized to the liver and pancreatic α cells. Although DBP serum levels, gene polymorphisms, and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates α cell morphology, α cell function, and glucagon secretion. Deletion of DBP leads to smaller and hyperplastic α cells, altered Na channel conductance, impaired α cell activation by low glucose, and reduced rates of glucagon secretion both in vivo and in vitro. Mechanistically, this involves reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects are also seen in β cell and δ cell function. Immunostaining of human pancreata reveals generalized loss of DBP expression as a feature of late-onset and long-standing, but not early-onset, type 1 diabetes. Thus, DBP regulates α cell phenotype, with implications for diabetes pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.107761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302426PMC
June 2020

Improved glucose tolerance with DPPIV inhibition requires β-cell SENP1 amplification of glucose-stimulated insulin secretion.

Physiol Rep 2020 04;8(8):e14420

Department of Pharmacology and Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada.

Pancreatic islet insulin secretion is amplified by both metabolic and receptor-mediated signaling pathways. The incretin-mimetic and DPPIV inhibitor anti-diabetic drugs increase insulin secretion, but in humans this can be variable both in vitro and in vivo. We examined the correlation of GLP-1 induced insulin secretion from human islets with key donor characteristics, glucose-responsiveness, and the ability of glucose to augment exocytosis in β-cells. No clear correlation was observed between several donor or organ processing parameters and the ability of Exendin 4 to enhance insulin secretion. The ability of glucose to facilitate β-cell exocytosis was, however, significantly correlated with responses to Exendin 4. We therefore studied the effect of impaired glucose-dependent amplification of insulin exocytosis on responses to DPPIV inhibition (MK-0626) in vivo using pancreas and β-cell specific sentrin-specific protease-1 (SENP1) mice which exhibit impaired metabolic amplification of insulin exocytosis. Glucose tolerance was improved, and plasma insulin was increased, following either acute or 4 week treatment of wild-type (βSENP1 ) mice with MK-0626. This DPPIV inhibitor was ineffective in βSENP1 or βSENP1 mice. Finally, we confirm impaired exocytotic responses of β-cells and reduced insulin secretion from islets of βSENP1 mice and show that the ability of Exendin 4 to enhance exocytosis is lost in these cells. Thus, an impaired ability of glucose to amplify insulin exocytosis results in a deficient effect of DPPIV inhibition to improve in vivo insulin responses and glucose tolerance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.14814/phy2.14420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185381PMC
April 2020

GLP-1 receptor agonists synergize with DYRK1A inhibitors to potentiate functional human β cell regeneration.

Sci Transl Med 2020 02;12(530)

Diabetes, Obesity and Metabolism Institute and Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Glucagon-like peptide-1 receptor (GLP1R) agonists and dipeptidyl peptidase 4 inhibitors are widely prescribed diabetes drugs due to their ability to stimulate insulin secretion from remaining β cells and to reduce caloric intake. Unfortunately, they fail to increase human β cell proliferation. Small-molecule inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) are able to induce adult human β cell proliferation, but rates are modest (~2%), and their specificity to β cells is limited. Here, we provide evidence that combining any member of the GLP1R agonist class with any member of the DYRK1A inhibitor class induces a synergistic increase in human β cell replication (5 to 6%) accompanied by an actual increase in numbers of human β cells. GLP1R agonist-DYRK1A inhibitor synergy required combined inhibition of DYRK1A and an increase in cAMP and did not lead to β cell dedifferentiation. These beneficial effects on proliferation were seen in both normal human β cells and β cells derived from individuals with type 2 diabetes. The ability of the GLP1R agonist-DYRK1A inhibitor combination to enhance human β cell proliferation, human insulin secretion, and blood glucose control extended in vivo to studies of human islets transplanted into euglycemic and streptozotocin-diabetic immunodeficient mice. No adverse events were observed in the mouse studies during a 1-week period. Because of the relative β cell specificity of GLP1R agonists, the combination provides an improved, although not complete, degree of human β cell specificity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aaw9996DOI Listing
February 2020

A glucose-dependent spatial patterning of exocytosis in human β-cells is disrupted in type 2 diabetes.

JCI Insight 2019 05 14;5. Epub 2019 May 14.

Alberta Diabetes Institute and Department of Pharmacology and.

Impaired insulin secretion in type 2 diabetes (T2D) is linked to reduced insulin granule docking, disorganization of the exocytotic site, and an impaired glucose-dependent facilitation of insulin exocytosis. We show in β-cells from 80 human donors that the glucose-dependent amplification of exocytosis is disrupted in T2D. Spatial analyses of granule fusion, visualized by total internal reflection fluorescence (TIRF) microscopy in 24 of these donors, demonstrate that these are non-random across the surface of β-cells from donors with no diabetes (ND). The compartmentalization of events occurs within regions defined by concurrent or recent membrane-resident secretory granules. This organization, and the number of membrane-associated granules, is glucose-dependent and notably impaired in T2D β-cells. Mechanistically, multi-channel Kv2.1 clusters contribute to maintaining the density of membrane-resident granules and the number of fusion 'hotspots', while SUMOylation sites at the channel N- (K145) and C-terminus (K470) determine the relative proportion of fusion events occurring within these regions. Thus, a glucose-dependent compartmentalization of fusion, regulated in part by a structural role for Kv2.1, is disrupted in β-cells from donors with type 2 diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.127896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629118PMC
May 2019

Type 2 diabetes risk alleles in PAM impact insulin release from human pancreatic β-cells.

Nat Genet 2018 08 27;50(8):1122-1131. Epub 2018 Jul 27.

Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, Oxford, UK.

The molecular mechanisms underpinning susceptibility loci for type 2 diabetes (T2D) remain poorly understood. Coding variants in peptidylglycine α-amidating monooxygenase (PAM) are associated with both T2D risk and insulinogenic index. Here, we demonstrate that the T2D risk alleles impact negatively on overall PAM activity via defects in expression and catalytic function. PAM deficiency results in reduced insulin content and altered dynamics of insulin secretion in a human β-cell model and primary islets from cadaveric donors. Thus, our results demonstrate a role for PAM in β-cell function, and establish molecular mechanisms for T2D risk alleles at this locus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-018-0173-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237273PMC
August 2018

Kv2.1 Clustering Contributes to Insulin Exocytosis and Rescues Human β-Cell Dysfunction.

Diabetes 2017 07 12;66(7):1890-1900. Epub 2017 Jun 12.

Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada

Insulin exocytosis is regulated by ion channels that control excitability and Ca influx. Channels also play an increasingly appreciated role in microdomain structure. In this study, we examine the mechanism by which the voltage-dependent K (Kv) channel Kv2.1 () facilitates depolarization-induced exocytosis in INS 832/13 cells and β-cells from human donors with and without type 2 diabetes (T2D). We find that Kv2.1, but not Kv2.2 (), forms clusters of 6-12 tetrameric channels at the plasma membrane and facilitates insulin exocytosis. Knockdown of Kv2.1 expression reduces secretory granule targeting to the plasma membrane. Expression of the full-length channel (Kv2.1-wild-type) supports the glucose-dependent recruitment of secretory granules. However, a truncated channel (Kv2.1-ΔC318) that retains electrical function and syntaxin 1A binding, but lacks the ability to form clusters, does not enhance granule recruitment or exocytosis. Expression of appears reduced in T2D islets, and further knockdown of does not inhibit Kv current in T2D β-cells. Upregulation of Kv2.1-wild-type, but not Kv2.1-ΔC318, rescues the exocytotic phenotype in T2D β-cells and increases insulin secretion from T2D islets. Thus, the ability of Kv2.1 to directly facilitate insulin exocytosis depends on channel clustering. Loss of this structural role for the channel might contribute to impaired insulin secretion in diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db16-1170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482075PMC
July 2017

N-acyl Taurines and Acylcarnitines Cause an Imbalance in Insulin Synthesis and Secretion Provoking β Cell Dysfunction in Type 2 Diabetes.

Cell Metab 2017 Jun;25(6):1334-1347.e4

Research Unit Analytical Pathology, Helmholtz Zentrum München, Neuherberg 85764, Germany. Electronic address:

The processes contributing to β cell dysfunction in type 2 diabetes (T2D) are uncertain, largely because it is difficult to access β cells in their intact immediate environment. We examined the pathophysiology of β cells under T2D progression directly in pancreatic tissues. We used MALDI imaging of Langerhans islets (LHIs) within mouse tissues or from human tissues to generate in situ-omics data, which we supported with in vitro experiments. Molecular interaction networks provided information on functional pathways and molecules. We found that stearoylcarnitine accumulated in β cells, leading to arrest of insulin synthesis and energy deficiency via excessive β-oxidation and depletion of TCA cycle and oxidative phosphorylation metabolites. Acetylcarnitine and an accumulation of N-acyl taurines, a group not previously detected in β cells, provoked insulin secretion. Thus, β cell dysfunction results from enhanced insulin secretion combined with an arrest of insulin synthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cmet.2017.04.012DOI Listing
June 2017

STEAP4 expression in human islets is associated with differences in body mass index, sex, HbA1c, and inflammation.

Endocrine 2017 Jun 12;56(3):528-537. Epub 2017 Apr 12.

Department of Biomedical Sciences, Ohio University, Athens, OH, USA.

Objective: STEAP4 (six-transmembrane epithelial antigen of the prostate 4) is a metalloreductase that has been shown previously to protect cells from inflammatory damage. Genetic variants in STEAP4 have been associated with numerous metabolic disorders related to obesity, including putative defects in the acute insulin response to glucose in type 2 diabetes.

Purpose: We examined whether obesity and/or type 2 diabetes altered STEAP4 expression in human pancreatic islets.

Methods: Human islets were isolated from deceased donors at two medical centers and processed for quantitative polymerase chain reaction. Organ donors were selected by status as non-diabetic or having type 2 diabetes. Site 1 (Edmonton): N = 13 type 2 diabetes donors (7M, 6F), N = 20 non-diabetic donors (7M, 13F). Site 2 (Virginia): N = 6 type 2 diabetes donors (6F), N = 6 non-diabetic donors (3M, 3F).

Results: STEAP4 showed reduced islet expression with increasing body mass index among all donors (P < 0.10) and non-diabetic donors (P < 0.05) from Site 1; STEAP4 showed reduced islet expression among type 2 diabetes donors with increasing hemoglobin A1c. Islet STEAP4 expression was also marginally higher in female donors (P < 0.10). Among type 2 diabetes donors from Site 2, islet insulin expression was reduced, STEAP4 expression was increased, and white blood cell counts were increased compared to non-diabetic donors. Islets from non-diabetic donors that were exposed overnight to 5 ng/ml IL-1β displayed increased STEAP4 expression, consistent with STEAP4 upregulation by inflammatory signaling.

Conclusions: These findings suggest that increased STEAP4 mRNA expression is associated with inflammatory stimuli, whereas lower STEAP4 expression is associated with obesity in human islets. Given its putative protective role, downregulation of STEAP4 by chronic obesity suggests a mechanism for reduced islet protection against cellular damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12020-017-1297-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166871PMC
June 2017

SUMOylation and calcium control syntaxin-1A and secretagogin sequestration by tomosyn to regulate insulin exocytosis in human ß cells.

Sci Rep 2017 03 21;7(1):248. Epub 2017 Mar 21.

Alberta Diabetes Institute and Department of Pharmacology, University of Alberta, Edmonton, Alberta, T6G 2R3, Canada.

Insulin secretion from pancreatic ß cells is a multistep process that requires the coordination of exocytotic proteins that integrate diverse signals. These include signals derived from metabolic control of post-translational SUMOylation and depolarization-induced rises in intracellular Ca. Here we show that tomosyn, which suppresses insulin exocytosis by binding syntaxin1A, does so in a manner which requires its SUMOylation. Glucose-dependent de-SUMOylation of tomosyn1 at K298 releases syntaxin1A and controls the amplification of exocytosis in concert with a recently-identified tomosyn1-interacting partner; the Ca-binding protein secretagogin, which dissociates from tomosyn1 in response to Ca-raising stimuli and is required for insulin granule trafficking and exocytosis downstream of Ca influx. Together our results suggest that tomosyn acts as a key signaling hub in insulin secretion by integrating signals mediated by metabolism-dependent de-SUMOylation and electrically-induced entry of Ca to regulate the availability of exocytotic proteins required for the amplification of insulin secretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-00344-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428262PMC
March 2017

Decreased STARD10 Expression Is Associated with Defective Insulin Secretion in Humans and Mice.

Am J Hum Genet 2017 02 26;100(2):238-256. Epub 2017 Jan 26.

Section of Cell Biology and Functional Genomics, Department of Medicine, Imperial College London, Hammersmith Hospital, du Cane Road, London W12 0NN, UK. Electronic address:

Genetic variants near ARAP1 (CENTD2) and STARD10 influence type 2 diabetes (T2D) risk. The risk alleles impair glucose-induced insulin secretion and, paradoxically but characteristically, are associated with decreased proinsulin:insulin ratios, indicating improved proinsulin conversion. Neither the identity of the causal variants nor the gene(s) through which risk is conferred have been firmly established. Whereas ARAP1 encodes a GTPase activating protein, STARD10 is a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer protein family. By integrating genetic fine-mapping and epigenomic annotation data and performing promoter-reporter and chromatin conformational capture (3C) studies in β cell lines, we localize the causal variant(s) at this locus to a 5 kb region that overlaps a stretch-enhancer active in islets. This region contains several highly correlated T2D-risk variants, including the rs140130268 indel. Expression QTL analysis of islet transcriptomes from three independent subject groups demonstrated that T2D-risk allele carriers displayed reduced levels of STARD10 mRNA, with no concomitant change in ARAP1 mRNA levels. Correspondingly, β-cell-selective deletion of StarD10 in mice led to impaired glucose-stimulated Ca dynamics and insulin secretion and recapitulated the pattern of improved proinsulin processing observed at the human GWAS signal. Conversely, overexpression of StarD10 in the adult β cell improved glucose tolerance in high fat-fed animals. In contrast, manipulation of Arap1 in β cells had no impact on insulin secretion or proinsulin conversion in mice. This convergence of human and murine data provides compelling evidence that the T2D risk associated with variation at this locus is mediated through reduction in STARD10 expression in the β cell.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2017.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5294761PMC
February 2017

Urea impairs β cell glycolysis and insulin secretion in chronic kidney disease.

J Clin Invest 2016 09 15;126(9):3598-612. Epub 2016 Aug 15.

Disorders of glucose homeostasis are common in chronic kidney disease (CKD) and are associated with increased mortality, but the mechanisms of impaired insulin secretion in this disease remain unclear. Here, we tested the hypothesis that defective insulin secretion in CKD is caused by a direct effect of urea on pancreatic β cells. In a murine model in which CKD is induced by 5/6 nephrectomy (CKD mice), we observed defects in glucose-stimulated insulin secretion in vivo and in isolated islets. Similarly, insulin secretion was impaired in normal mouse and human islets that were cultured with disease-relevant concentrations of urea and in islets from normal mice treated orally with urea for 3 weeks. In CKD mouse islets as well as urea-exposed normal islets, we observed an increase in oxidative stress and protein O-GlcNAcylation. Protein O-GlcNAcylation was also observed in pancreatic sections from CKD patients. Impairment of insulin secretion in both CKD mouse and urea-exposed islets was associated with reduced glucose utilization and activity of phosphofructokinase 1 (PFK-1), which could be reversed by inhibiting O-GlcNAcylation. Inhibition of O-GlcNAcylation also restored insulin secretion in both mouse models. These results suggest that insulin secretory defects associated with CKD arise from elevated circulating levels of urea that increase islet protein O-GlcNAcylation and impair glycolysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI86181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004963PMC
September 2016

PI3 kinases p110α and PI3K-C2β negatively regulate cAMP via PDE3/8 to control insulin secretion in mouse and human islets.

Mol Metab 2016 Jul 11;5(7):459-471. Epub 2016 May 11.

Department of Pharmacology, and the Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada.

Objectives: Phosphatidylinositol-3-OH kinase (PI3K) signalling in the endocrine pancreas contributes to glycaemic control. However, the mechanism by which PI3K modulates insulin secretion from the pancreatic beta cell is poorly understood. Thus, our objective was two-fold; to determine the signalling pathway by which acute PI3K inhibition enhances glucose-stimulated insulin secretion (GSIS) and to examine the role of this pathway in islets from type-2 diabetic (T2D) donors.

Methods: Isolated islets from mice and non-diabetic or T2D human donors, or INS 832/13 cells, were treated with inhibitors of PI3K and/or phosphodiesterases (PDEs). The expression of PI3K-C2β was knocked down using siRNA. We measured insulin release, single-cell exocytosis, intracellular Ca(2+) responses ([Ca(2+)]i) and Ca(2+) channel currents, intracellular cAMP concentrations ([cAMP]i), and activation of cAMP-dependent protein kinase A (PKA) and protein kinase B (PKB/AKT).

Results: The non-specific PI3K inhibitor wortmannin amplifies GSIS, raises [cAMP]i and activates PKA, but is without effect in T2D islets. Direct inhibition of specific PDE isoforms demonstrates a role for PDE3 (in humans and mice) and PDE8 (in mice) downstream of PI3K, and restores glucose-responsiveness of T2D islets. We implicate a role for the Class II PI3K catalytic isoform PI3K-C2β in this effect by limiting beta cell exocytosis.

Conclusions: PI3K limits GSIS via PDE3 in human islets. While inhibition of p110α or PIK-C2β signalling per se, may promote nutrient-stimulated insulin release, we now suggest that this signalling pathway is perturbed in islets from T2D donors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molmet.2016.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4921792PMC
July 2016

A Glycine-Insulin Autocrine Feedback Loop Enhances Insulin Secretion From Human β-Cells and Is Impaired in Type 2 Diabetes.

Diabetes 2016 08 3;65(8):2311-21. Epub 2016 May 3.

Department of Pharmacology and Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada.

The secretion of insulin from pancreatic islet β-cells is critical for glucose homeostasis. Disrupted insulin secretion underlies almost all forms of diabetes, including the most common form, type 2 diabetes (T2D). The control of insulin secretion is complex and affected by circulating nutrients, neuronal inputs, and local signaling. In the current study, we examined the contribution of glycine, an amino acid and neurotransmitter that activates ligand-gated Cl(-) currents, to insulin secretion from islets of human donors with and without T2D. We find that human islet β-cells express glycine receptors (GlyR), notably the GlyRα1 subunit, and the glycine transporter (GlyT) isoforms GlyT1 and GlyT2. β-Cells exhibit significant glycine-induced Cl(-) currents that promote membrane depolarization, Ca(2+) entry, and insulin secretion from β-cells from donors without T2D. However, GlyRα1 expression and glycine-induced currents are reduced in β-cells from donors with T2D. Glycine is actively cleared by the GlyT expressed within β-cells, which store and release glycine that acts in an autocrine manner. Finally, a significant positive relationship exists between insulin and GlyR, because insulin enhances the glycine-activated current in a phosphoinositide 3-kinase-dependent manner, a positive feedback loop that we find is completely lost in β-cells from donors with T2D.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db15-1272DOI Listing
August 2016

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

Endocrinology 2016 Feb 11;157(2):560-9. Epub 2015 Dec 11.

Alberta Diabetes Institute IsletCore (J.L., J.E.M.F., P.E.M.) and Departments of Pharmacology (J.E.M.F., A.F.S., R.K., N.S., P.E.M.) and Surgery (D.O., T.K., A.M.J.S., R.V.R.), University of Alberta, Edmonton, Canada T6G 2E1.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/en.2015-1562DOI Listing
February 2016

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.

PLoS Genet 2015 Dec 1;11(12):e1005694. Epub 2015 Dec 1.

Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, Oxford, United Kingdom.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1005694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666611PMC
December 2015

Antiaging Glycopeptide Protects Human Islets Against Tacrolimus-Related Injury and Facilitates Engraftment in Mice.

Diabetes 2016 Feb 18;65(2):451-62. Epub 2015 Nov 18.

Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada Canadian National Transplant Research Program, University of Alberta, Edmonton, Alberta, Canada Clinical Islet Transplant Program, University of Alberta, Edmonton, Alberta, Canada

Clinical islet transplantation has become an established treatment modality for selected patients with type 1 diabetes. However, a large proportion of transplanted islets is lost through multiple factors, including immunosuppressant-related toxicity, often requiring more than one donor to achieve insulin independence. On the basis of the cytoprotective capabilities of antifreeze proteins (AFPs), we hypothesized that supplementation of islets with synthetic AFP analog antiaging glycopeptide (AAGP) would enhance posttransplant engraftment and function and protect against tacrolimus (Tac) toxicity. In vitro and in vivo islet Tac exposure elicited significant but reversible reduction in insulin secretion in both mouse and human islets. Supplementation with AAGP resulted in improvement of islet survival (Tac(+) vs. Tac+AAGP, 31.5% vs. 67.6%, P < 0.01) coupled with better insulin secretion (area under the curve: Tac(+) vs. Tac+AAGP, 7.3 vs. 129.2 mmol/L/60 min, P < 0.001). The addition of AAGP reduced oxidative stress, enhanced insulin exocytosis, improved apoptosis, and improved engraftment in mice by decreasing expression of interleukin (IL)-1β, IL-6, keratinocyte chemokine, and tumor necrosis factor-α. Finally, transplant efficacy was superior in the Tac+AAGP group and was similar to islets not exposed to Tac, despite receiving continuous treatment for a limited time. Thus, supplementation with AAGP during culture improves islet potency and attenuates long-term Tac-induced graft dysfunction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db15-0764DOI Listing
February 2016

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

J Clin Invest 2015 Oct 21;125(10):3847-60. Epub 2015 Sep 21.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI82498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4607115PMC
October 2015

Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose-Stimulated Insulin Secretion.

Mol Endocrinol 2015 Jul 10;29(7):988-1005. Epub 2015 Jun 10.

BIOLOG Life Science Institute (F.S., H.-G.G.), 28199 Bremen, Germany; Departments of Medicine (O.G.C., C.A.L., G.G.H.) and Pharmacology (G.G.H.), State University of New York, Upstate Medical University, Syracuse, New York 13210; Department of Biochemistry (M.K., D.B., F.W.H.), University of Kassel, 34132 Kassel, Germany; Eli Lilly and Company (O.C.), Indianapolis, Indiana 46225; Department of Integrative Biology and Pharmacology (Y.Z., F.M., X.C.), Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center, Houston, Texas 77030; Department of Pharmacology and the Alberta Diabetes Institute (J.E.M.F., P.E.M.), University of Alberta, Edmonton, Canada AB T6G 2E1.

cAMP-elevating agents such as the incretin hormone glucagon-like peptide-1 potentiate glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. However, a debate has existed since the 1970s concerning whether or not cAMP signaling is essential for glucose alone to stimulate insulin secretion. Here, we report that the first-phase kinetic component of GSIS is cAMP-dependent, as revealed through the use of a novel highly membrane permeable para-acetoxybenzyl (pAB) ester prodrug that is a bioactivatable derivative of the cAMP antagonist adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In dynamic perifusion assays of human or rat islets, a step-wise increase of glucose concentration leads to biphasic insulin secretion, and under these conditions, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, 4-acetoxybenzyl ester (Rp-8-Br-cAMPS-pAB) inhibits first-phase GSIS by up to 80%. Surprisingly, second-phase GSIS is inhibited to a much smaller extent (≤20%). Using luciferase, fluorescence resonance energy transfer, and bioluminescence resonance energy transfer assays performed in living cells, we validate that Rp-8-Br-cAMPS-pAB does in fact block cAMP-dependent protein kinase activation. Novel effects of Rp-8-Br-cAMPS-pAB to block the activation of cAMP-regulated guanine nucleotide exchange factors (Epac1, Epac2) are also validated using genetically encoded Epac biosensors, and are independently confirmed in an in vitro Rap1 activation assay using Rp-cAMPS and Rp-8-Br-cAMPS. Thus, in addition to revealing the cAMP dependence of first-phase GSIS from human and rat islets, these findings establish a pAB-based chemistry for the synthesis of highly membrane permeable prodrug derivatives of Rp-cAMPS that act with micromolar or even nanomolar potency to inhibit cAMP signaling in living cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/me.2014-1330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4484784PMC
July 2015

Human islet function following 20 years of cryogenic biobanking.

Diabetologia 2015 Jul 1;58(7):1503-12. Epub 2015 May 1.

Alberta Diabetes Institute, University of Alberta, LKS Centre, Edmonton, AB, Canada, T6G 2R3.

Aims/hypothesis: There are potential advantages to the low-temperature (-196 °C) banking of isolated islets, including the maintenance of viable islets for future research. We therefore assessed the in vitro and in vivo function of islets cryopreserved for nearly 20 years.

Methods: Human islets were cryopreserved from 1991 to 2001 and thawed between 2012 and 2014. These were characterised by immunostaining, patch-clamp electrophysiology, insulin secretion, transcriptome analysis and transplantation into a streptozotocin (STZ)-induced mouse model of diabetes.

Results: The cryopreservation time was 17.6 ± 0.4 years (n = 43). The thawed islets stained positive with dithizone, contained insulin-positive and glucagon-positive cells, and displayed levels of apoptosis and transcriptome profiles similar to those of freshly isolated islets, although their insulin content was lower. The cryopreserved beta cells possessed ion channels and exocytotic responses identical to those of freshly isolated beta cells. Cells from a subset of five donors demonstrated similar perifusion insulin secretion profiles pre- and post-cryopreservation. The transplantation of cryopreserved islets into the diabetic mice improved their glucose tolerance but did not completely normalise their blood glucose levels. Circulating human insulin and insulin-positive grafts were detectable at 10 weeks post-transplantation.

Conclusions/interpretation: We have demonstrated the potential for long-term banking of human islets for research, which could enable the use of tissue from a large number of donors with future technologies to gain new insight into diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00125-015-3598-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472956PMC
July 2015

Insulin secretion induced by glucose-dependent insulinotropic polypeptide requires phosphatidylinositol 3-kinase γ in rodent and human β-cells.

J Biol Chem 2014 Nov 6;289(46):32109-32120. Epub 2014 Oct 6.

Department of Pharmacology, and the Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta T6G 2E1, Canada. Electronic address:

PI3Kγ, a G-protein-coupled type 1B phosphoinositol 3-kinase, exhibits a basal glucose-independent activity in β-cells and can be activated by the glucose-dependent insulinotropic polypeptide (GIP). We therefore investigated the role of the PI3Kγ catalytic subunit (p110γ) in insulin secretion and β-cell exocytosis stimulated by GIP. We inhibited p110γ with AS604850 (1 μmol/liter) or knocked it down using an shRNA adenovirus or siRNA duplex in mouse and human islets and β-cells. Inhibition of PI3Kγ blunted the exocytotic and insulinotropic response to GIP receptor activation, whereas responses to the glucagon-like peptide-1 or the glucagon-like peptide-1 receptor agonist exendin-4 were unchanged. Downstream, we find that GIP, much like glucose stimulation, activates the small GTPase protein Rac1 to induce actin remodeling. Inhibition of PI3Kγ blocked these effects of GIP. Although exendin-4 could also stimulate actin remodeling, this was not prevented by p110γ inhibition. Finally, forced actin depolymerization with latrunculin B restored the exocytotic and secretory responses to GIP during PI3Kγ inhibition, demonstrating that the loss of GIP-induced actin depolymerization was indeed limiting insulin exocytosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.577510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231687PMC
November 2014

SUMOylation protects against IL-1β-induced apoptosis in INS-1 832/13 cells and human islets.

Am J Physiol Endocrinol Metab 2014 Oct 19;307(8):E664-73. Epub 2014 Aug 19.

Department of Pharmacology and Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada

Posttranslational modification by the small ubiquitin-like modifier (SUMO) peptides, known as SUMOylation, is reversed by the sentrin/SUMO-specific proteases (SENPs). While increased SUMOylation reduces β-cell exocytosis, insulin secretion, and responsiveness to GLP-1, the impact of SUMOylation on islet cell survival is unknown. Mouse islets, INS-1 832/13 cells, or human islets were transduced with adenoviruses to increase either SENP1 or SUMO1 or were transfected with siRNA duplexes to knockdown SENP1. We examined insulin secretion, intracellular Ca²⁺ responses, induction of endoplasmic reticulum stress markers and inducible nitric oxide synthase (iNOS) expression, and apoptosis by TUNEL and caspase 3 cleavage. Surprisingly, upregulation of SENP1 reduces insulin secretion and impairs intracellular Ca²⁺ handling. This secretory dysfunction is due to SENP1-induced cell death. Indeed, the detrimental effect of SENP1 on secretory function is diminished when two mediators of β-cell death, iNOS and NF-κB, are pharmacologically inhibited. Conversely, enhanced SUMOylation protects against IL-1β-induced cell death. This is associated with reduced iNOS expression, cleavage of caspase 3, and nuclear translocation of NF-κB. Taken together, these findings identify SUMO1 as a novel antiapoptotic protein in islets and demonstrate that reduced viability accounts for impaired islet function following SENP1 up-regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpendo.00168.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200309PMC
October 2014

SUMO1 enhances cAMP-dependent exocytosis and glucagon secretion from pancreatic α-cells.

J Physiol 2014 Sep 6;592(17):3715-26. Epub 2014 Jun 6.

Department of Pharmacology, University of Alberta, Edmonton, AB, Canada Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada

Post-translational modification by the small ubiquitin-like modifier-1 (SUMO1) limits insulin secretion from β-cells by inhibiting insulin exocytosis and glucagon-like peptide-1 (GLP-1) receptor signalling. The secretion of glucagon from α-cells is regulated in a manner opposite to that of insulin; it is inhibited by elevated glucose and GLP-1, and increased by adrenergic signalling. We therefore sought to determine whether SUMO1 modulates mouse and human α-cell function. Action potentials (APs), ion channel function and exocytosis in single α-cells from mice and humans, identified by glucagon immunostaining, and glucagon secretion from intact islets were measured. The effects of SUMO1 on α-cell function and the respective inhibitory and stimulatory effects of exendin 4 and adrenaline were examined. Upregulation of SUMO1 increased α-cell AP duration, frequency and amplitude, in part as a result of increased Ca(2+) channel activity that led to elevated exocytosis. The ability of SUMO1 to enhance α-cell exocytosis was cAMP-dependent and resulted from an increased L-type Ca(2+) current and a shift away from exocytosis dependent on non-L-type channels, an effect that was mimicked by knockdown of the deSUMOylating enzyme sentrin/SUMO-specific protease-1 (SENP1). Finally, although SUMO1 prevented GLP-1 receptor-mediated inhibition of α-cell Na(+) channels and single-cell exocytosis, it failed to prevent the exendin 4-mediated inhibition of glucagon secretion. Consistent with its cAMP dependence, however, SUMO1 enhanced α-cell exocytosis and glucagon secretion stimulated by adrenaline. Thus, by contrast with its inhibitory role in β-cell exocytosis, SUMO1 is a positive regulator of α-cell exocytosis and glucagon secretion under conditions of elevated cAMP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1113/jphysiol.2014.274084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4192699PMC
September 2014

Mitochondrial metabolism of pyruvate is essential for regulating glucose-stimulated insulin secretion.

J Biol Chem 2014 May 27;289(19):13335-46. Epub 2014 Mar 27.

From the School of Pharmacy, University of Waterloo, Waterloo, 10A Victoria Street South, Ontario N2G 1C5, Canada and.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.521666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036342PMC
May 2014

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

Proc Natl Acad Sci U S A 2013 Oct 24;110(41):16480-5. Epub 2013 Sep 24.

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.

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 Ca(2+), 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 Ca(2+) 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1214312110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3799350PMC
October 2013

Triton X-100 inhibits L-type voltage-operated calcium channels.

Can J Physiol Pharmacol 2013 Apr 15;91(4):316-24. Epub 2013 Jan 15.

Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.

Triton X-100 (TX-100) is a nonionic detergent frequently used at millimolar concentrations to disrupt cell membranes and solubilize proteins. At low micromolar concentrations, TX-100 has been reported to inhibit the function of potassium channels. Here, we have used electrophysiological and functional techniques to examine the effects of TX-100 on another class of ion channels, L-type voltage-operated calcium channels (VOCCs). TX-100 (30 nmol·L(-1) to 3 μmol·L(-1)) caused reversible concentration-dependent inhibition of recombinant L-type VOCC (CaV 1.2) currents and of native L-type VOCC currents recorded from rat vascular smooth muscle cells and cardiac myocytes, and murine and human pancreatic β-cells. In functional studies, TX-100 (165 nmol·L(-1) to 3.4 μmol·L(-1)) caused concentration-dependent relaxation of rat isolated mesenteric resistance arteries prestimulated with phenylephrine or KCl. This effect was independent of the endothelium. TX-100 (1.6 μmol·L(-1)) inhibited depolarization-induced exocytosis in both murine and human isolated pancreatic β-cells. These data indicate that at concentrations within the nanomolar to low micromolar range, TX-100 significantly inhibits L-type VOCC activity in a number of cell types, an effect paralleled by inhibition of cell functions dependent upon activation of these channels. This inhibition occurs at concentrations below those used to solubilize proteins and may compromise the use of solutions containing TX-100 in bioassays.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1139/cjpp-2012-0257DOI Listing
April 2013
-->