O-GlcNacylation Links TxNIP to Inflammasome Activation in Pancreatic β Cells

Thioredoxin interacting protein (TxNIP), which strongly responds to glucose, has emerged as a central mediator of glucotoxicity in pancreatic β cells. TxNIP is a scaffold protein interacting with target proteins to inhibit or stimulate their activity. Recent studies reported that high glucose stimulates the interaction of TxNIP with the inflammasome protein NLRP3 (NLR family, pyrin domain containing 3) to increase interleukin-1 β (IL1β) secretion by pancreatic β cells. To better understand the regulation of TxNIP by glucose in pancreatic β cells, we investigated the implication of O-linked β-N-acetylglucosamine (O-GlcNAcylation) in regulating TxNIP at the posttranslational level. O-GlcNAcylation of proteins is controlled by two enzymes: the O-GlcNAc transferase (OGT), which transfers a monosaccharide to serine/threonine residues on target proteins, and the O-GlcNAcase (OGA), which removes it. Our study shows that TxNIP is subjected to O-GlcNAcylation in response to high glucose concentrations in β cell lines. Modification of the O-GlcNAcylation pathway through manipulation of OGT or OGA expression or activity significantly modulates TxNIP O-GlcNAcylation in INS1 832/13 cells. Interestingly, expression and O-GlcNAcylation of TxNIP appeared to be increased in islets of diabetic rodents. At the mechanistic level, the induction of the O-GlcNAcylation pathway in human and rat islets promotes inflammasome activation as evidenced by enhanced cleaved IL1β. Overexpression of OGT in HEK293 or INS1 832/13 cells stimulates TxNIP and NLRP3 interaction, while reducing TxNIP O-GlcNAcylation through OGA overexpression destabilizes this interaction. Altogether, our study reveals that O-GlcNAcylation represents an important regulatory mechanism for TxNIP activity in β cells

COUP-TFII Controls Mouse Pancreatic β-Cell Mass through GLP-1-β-Catenin Signaling Pathways

Background: The control of the functional pancreatic beta-cell mass serves the key homeostatic function of releasing the right amount of insulin to keep blood sugar in the normal range. It is not fully understood though how beta-cell mass is determined

O-GlcNacylation Links TxNIP to Inflammasome Activation in Pancreatic β Cells

International audienceThioredoxin interacting protein (TxNIP), which strongly responds to glucose, has emerged as a central mediator of glucotoxicity in pancreatic β cells. TxNIP is a scaffold protein interacting with target proteins to inhibit or stimulate their activity. Recent studies reported that high glucose stimulates the interaction of TxNIP with the inflammasome protein NLRP3 (NLR family, pyrin domain containing 3) to increase interleukin-1 β (IL1β) secretion by pancreatic β cells. To better understand the regulation of TxNIP by glucose in pancreatic β cells, we investigated the implication of O-linked β-N-acetylglucosamine (O-GlcNAcylation) in regulating TxNIP at the posttranslational level. O-GlcNAcylation of proteins is controlled by two enzymes: the O-GlcNAc transferase (OGT), which transfers a monosaccharide to serine/threonine residues on target proteins, and the O-GlcNAcase (OGA), which removes it. Our study shows that TxNIP is subjected to O-GlcNAcylation in response to high glucose concentrations in β cell lines. Modification of the O-GlcNAcylation pathway through manipulation of OGT or OGA expression or activity significantly modulates TxNIP O-GlcNAcylation in INS1 832/13 cells. Interestingly, expression and O-GlcNAcylation of TxNIP appeared to be increased in islets of diabetic rodents. At the mechanistic level, the induction of the O-GlcNAcylation pathway in human and rat islets promotes inflammasome activation as evidenced by enhanced cleaved IL1β. Overexpression of OGT in HEK293 or INS1 832/13 cells stimulates TxNIP and NLRP3 interaction, while reducing TxNIP O-GlcNAcylation through OGA overexpression destabilizes this interaction. Altogether, our study reveals that O-GlcNAcylation represents an important regulatory mechanism for TxNIP activity in β cells

COUP-TFII expression is activated by TCF7L2 in β-cells.

<p>(<b>A</b>) Fixed healthy human islets were triple-stained for COUP-TFII in red, for insulin in green and DAPI in blue. (B) 832/13 INS-1 β-cells were electroporated with scrambled (control) or specific TCF7L2 siRNA and cultured for 48 h. Relative TCF7L2, COUP-TFII and cyclin D1 mRNA levels were determined by RT-qPCR normalized to housekeeping gene cyclophilin. Results are means ± SEM of data from between 4 independent electroporation experiments. Significant differences between TCF7L2 knockdown and control values are shown by asterisks at the <i>P</i> value indicated. *<i>P</i><0.05.</p

Five-day-old pdx1^CRE/-, COUP-TFII^Fl/Fl mice have reduction of β-cell neogenesis and COUP-TFII knockdown increases 832/13 INS-1 β-cells apoptosis.

<p>In pancreatic sections of 5-day-old mice (A) β-cell proliferation was assessed by double staining insulin/Ki67 (B) β-cell neogenesis activation was evaluated through quantification of duct-associated insulin+ β-cells per unit of total tissue area. 832/13 INS-1 β-cells were electroporated with scrambled or specific COUP-TFII siRNA and cultured in INS-1 medium for 24 h or 48 h. (C) Relative COUP-TFII mRNA levels determined by RT-qPCR at 48 h. (D) Effect of COUP-TFII siRNA on cell viability analyzed by MTT assay (n = 5 electroporations). (E) Effect of COUP-TFII siRNA on β-cell proliferation. 832/13 INS-1 β-cells were cultured in INS-1 medium for 48 h and stained with BrdU. Cells were analyzed by flow cytometry (n = 4 electroprations). (F) Effect of COUP-TFII siRNA on β-cells apoptosis and comparison of the apoptotic rate with cell treated with a rat cytokine mix containing 25 ng/ml TNF-α, 10 ng/ml IL-1β and 10 ng/ml INF-γ during 24 h. 832/13 INS-1 β-cells were electroporated with scrambled or COUP-TFII siRNA and 48 h later they were stained with DiOC6(3) and analyzed by flow cytometry (n = 4 electroporations). * Significant difference between values linked by brackets at <i>P</i><0.03.</p

COUP-TFII controls GLP-1R expression in islet.

<p>RT-qPCR analysis of IGF-1R and GLP-1R mRNA from islets from 5-week-old pdx1^CRE/-, COUP-TFII^Fl/Fl and pdx1^CRE/- COUP-TFII^Fl/Fl mice normalized to housekeeping gene cyclophilin. Results are mean ± SEM (error bars) for 5 animals per genotype. *P<0.05 for pdx1^CRE/- COUP-TFII^Fl/Fl versus COUP-TFII^Fl/Fl and pdx1^CRE/- control mice.</p

COUP-TFII regulates β-catenin signaling target genes in mouse islets and a β-cell line.

<p>(A) 832/13 INS-1 β-cells were electroporated with scrambled (control) or COUP-TFII specific siRNA and cultured for 48 h. Levels of cyclin D1, c-myc, glutamine synthase, axin 2, and β-catenin mRNA were determined by RT-qPCR. Effects on Wnt/β-catenin signaling were assessed by the luciferase activity of a TOP/FOP-flash reporter. Results are means ± SEM of data from between 3 and 12 (mRNA levels) independent electroporation experiments. Significant differences between COUP-TFII knockdown and control values are shown by asterisks at the <i>P</i> value indicated. *<i>P</i><0.05; **<i>P</i><0.03. (B) A representative immunoblot of different experiments of 832/13 INS-1 β-cells that were electroporated with scrambled or COUP-TFII specific siRNA. 50 µ g of total protein extract was subjected to immunoblot analysis with antibodies against cyclin D1, CDK4 or β-actin. (C) RT-qPCR analysis of β-catenin and cyclin D1 mRNA levels of 832/13 INS-1 cells infected with Ad-GFP or Ad-hCOUP-TFII at 2 or 5 PFU per cell as indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030847#pone.0030847-Perilhou1" target="_blank">[4]</a> normalized to housekeeping gene cyclophilin. Results are means ± SEM of data from 3 independent infection experiments. *Significant difference between between COUP-TFII overexpression and control values at <i>P</i><0.05 (D) RT-qPCR analysis of β-catenin, cyclin D1, axin 2, pdx1 and proinsulin I-II mRNA from islets from 5-week-old pdx1^CRE/-, COUP-TFII^Fl/Fl and pdx1^CRE/- COUP-TFII^Fl/Fl mice. Results are mean ± SEM (error bars) for 5 animals per genotype. *P<0.05 for pdx1^CRE/- COUP-TFII^Fl/Fl versus COUP-TFII^Fl/Fl and pdx1^CRE/- control mice.</p

Pdx1^CRE/- COUP-TFII^Fl/Fl mice have defective glucose homeostasis.

<p>(A) Immunostaining of pancreatic sections from COUP-TFII^Fl/Fl control and pdx1^CRE/- COUP-TFII^Fl/Fl adult mice using mouse monoclonal antibodies against COUP-TFII. Scale bars =  100 µm (B) Fasting plasma insulin of 4 month and 6- week-old mice littermates. (C) Intraperitoneal glucose tolerance tests were performed in 4-month-old male pdx1^CRE/-, COUP-TFII^Fl/Fl and pdx1^CRE/- COUP-TFII^Fl/Fl mice that had been fasted for 14 h. Blood glucose levels, left panel. Rate of glucose disappearance (K), inset panel. Plasma insulin levels, right panel. Results are mean ± SEM (error bars) for 8 to 14 animals per genotype. Values for pdx1^CRE/- COUP-TFII^Fl/Fl are significantly different from values for COUP-TFII^Fl/Fl and pdx1^CRE/- control mice at <i>P</i><0.05 (*) or <i>P</i><0.03 (**).</p

Pdx1^CRE/- COUP-TFII^Fl/Fl mice have fewer β-cells at birth.

<p>(A) Pancreatic sections from adult mice were immunostained for pancreatic β-cells and α-cells using respectively anti-insulin and anti-glucagon antibodies. Scale bars =  100 µm (B) Estimation of the relative β-cell area and β-cell mass in the pancreatic sections of pdx1^CRE/-, COUP-TFII^Fl/Fl and pdx1^CRE/-COUP-TFII^Fl/Fl in 4-month-old mice. Beta-cell mass was estimated by morphometric analysis. Relative β-cell size (mean of ≥ 1500 cells counted). (C) Estimation of β-cell number and β-cell mass in pancreatic sections of 3-week-old mice. (D) Ratio of Insulin-positive cross-sectional area relative to pancreas (DAPI-positive) cross-sectional area in one-day-old neonate animals. # The reduction of pdx1^CRE/-, COUP-TFII^Fl/Fl over the cumulated controls (pdx1^CRE/- and COUP-TFII^Fl/Fl) is already quite important (23%) and is significant with student <i>t</i>-test and non-parametric tests (2-tailed Mann-Whitney U test, which does not assume normal distribution: p = 0.0476). It is not significant if referred individually to the two control populations. Data are means ± SEM (error bars) of values from 4 animals of each genotype. *Values for pdx1^CRE/-, COUP-TFII^Fl/Fl mice is significantly different from the corresponding values for control mice (pdx1^CRE/- and COUP-TFII^Fl/Fl) at <i>P</i><0.05.</p