<p>(A) Cell death of INS-1E cells cultured without stress at 11.1 mM glucose concentration ... more <p>(A) Cell death of INS-1E cells cultured without stress at 11.1 mM glucose concentration (G11, control) or exposed to different culture conditions listed in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082364#pone-0082364-g001" target="_blank">figure 1</a>. Data are shown as means ± SEM of 4 independent experiments. (B) Representative immunoblotting showing levels of the cleaved CASPASE 3, TUBULIN and ACTIN from treated INS-1E cells. (C) Quantitative analysis of band densities normalized to TUBULIN from immunoblots as shown in (B) is presented as means ± SEM of 4 independent experiments. Results are expressed as protein levels normalized to the control value of G11. (D) Transcript levels normalized to those of 18S and expressed as changes <i>versus</i> value of G11. The <i>BclxL</i> and <i>Bcl2</i> genes encode antiapoptotic proteins; the <i>Hspa5</i>, <i>Ddit3</i> and <i>Xbp1</i> genes encode the endoplasmic reticulum stress related proteins BIP, CHOP and XBP1, respectively; the cytosolic isoform <i>Sod1</i>, the matrix isoform <i>Sod2</i> (superoxide dismutase) and <i>Cat</i> (catalase) genes encode antioxidative enzymes. Results are means ± SEM of 2 independent experiments done in triplicate. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.005 <i>versus</i> G11 controls.</p
<p>(A) Cell death of INS-1E cells cultured without stress at 11.1 mM glucose concentration ... more <p>(A) Cell death of INS-1E cells cultured without stress at 11.1 mM glucose concentration (G11, control) or exposed to different culture conditions listed in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082364#pone-0082364-g001" target="_blank">figure 1</a>. Data are shown as means ± SEM of 4 independent experiments. (B) Representative immunoblotting showing levels of the cleaved CASPASE 3, TUBULIN and ACTIN from treated INS-1E cells. (C) Quantitative analysis of band densities normalized to TUBULIN from immunoblots as shown in (B) is presented as means ± SEM of 4 independent experiments. Results are expressed as protein levels normalized to the control value of G11. (D) Transcript levels normalized to those of 18S and expressed as changes <i>versus</i> value of G11. The <i>BclxL</i> and <i>Bcl2</i> genes encode antiapoptotic proteins; the <i>Hspa5</i>, <i>Ddit3</i> and <i>Xbp1</i> genes encode the endoplasmic reticulum stress related proteins BIP, CHOP and XBP1, respectively; the cytosolic isoform <i>Sod1</i>, the matrix isoform <i>Sod2</i> (superoxide dismutase) and <i>Cat</i> (catalase) genes encode antioxidative enzymes. Results are means ± SEM of 2 independent experiments done in triplicate. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.005 <i>versus</i> G11 controls.</p
Bien que la consommation du fructose soit associée à un stockage accru de graisses par l’action d... more Bien que la consommation du fructose soit associée à un stockage accru de graisses par l’action de l’insuline, le fructose seul n’induit pas la sécrétion de l’insuline par la cellule bêta-pancréatique, contrairement au glucose. Nous avons étudié les effets d’une exposition chronique au fructose sur la fonction des cellules bêta. Nos résultats révèlent que le fructose potentialise la sécrétion de l’insuline stimulée par le glucose en activant la voie de signalisation de l’adénosine triphosphate (ATP) extracellulaire. Cet effet est médié par l’activation des récepteurs purinérgiques P2Y1 et est associé à la libération d’ATP cellulaire par les canaux pannexines-1. En conséquence, l’interaction entre les canaux pannexines et les récepteurs purinérgiques via l’ATP extracellulaire représente une nouvelle cible cellulaire, offrant de potentielles implications thérapeutiques
The mechanism by which the β-cell transcription factor Pax4 influences cell function/mass was stu... more The mechanism by which the β-cell transcription factor Pax4 influences cell function/mass was studied in rat and human islets of Langerhans. Pax4 transcripts were detected in adult rat islets, and levels were induced by the mitogens activin A and betacellulin. Wortmannin suppressed betacellulin-induced Pax4 expression, implicating the phosphatidylinositol 3-kinase signaling pathway. Adenoviral overexpression of Pax4 caused a 3.5-fold increase in β-cell proliferation with a concomitant 1.9-, 4-, and 5-fold increase in Bcl-xL (antiapoptotic), c-myc, and Id2 mRNA levels, respectively. Accordingly, Pax4 transactivated the Bcl-xL and c-myc promoters, whereas its diabetes-linked mutant was less efficient. Bcl-xL activity resulted in altered mitochondrial calcium levels and ATP production, explaining impaired glucose-induced insulin secretion in transduced islets. Infection of human islets with an inducible adenoviral Pax4 construct caused proliferation and protection against cytokine-evok...
While the use of fructose as a sweetener and its consumption are associated with increased fat st... more While the use of fructose as a sweetener and its consumption are associated with increased fat storage prompted by the action of insulin, fructose alone does not acutely stimulate insulin exocytosis from the pancreatic beta-cell, as opposed to the chief secretagogue glucose. We investigated the effects of chronic exposure to fructose on beta-cell function. Our results reveal that chronic fructose induces extracellular ATP signaling in the beta-cell, resulting in the potentiation of glucose-stimulated insulin secretion. This effect is mediated by the activation of the purinergic P2Y1 receptors and is associated with the release of cellular ATP through pannexin-1 channels. Consequently, the interplay between pannexin channels and purinergic receptors, through ATP signaling, represents a novel cellular target with potential therapeutic implications.
Chronic exposure of pancreatic β-cells to elevated nutrient levels impairs their function and pot... more Chronic exposure of pancreatic β-cells to elevated nutrient levels impairs their function and potentially induces apoptosis. Like in other cell types, AMPK is activated in β-cells under conditions of nutrient deprivation, while little is known on AMPK responses to metabolic stresses. Here, we first reviewed recent studies on the role of AMPK activation in β-cells. Then, we investigated the expression profile of AMPK pathways in β-cells following metabolic stresses. INS-1E β-cells and human islets were exposed for 3 days to glucose (5.5–25 mM), palmitate or oleate (0.4 mM), and fructose (5.5 mM). Following these treatments, we analyzed transcript levels of INS-1E β-cells by qRT-PCR and of human islets by RNA-Seq; with a special focus on AMPK-associated genes, such as the AMPK catalytic subunits α1 (Prkaa1) and α2 (Prkaa2). AMPKα and pAMPKα were also evaluated at the protein level by immunoblotting. Chronic exposure to the different metabolic stresses, known to alter glucose-stimulate...
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2019
Chronic exposure to elevated levels of glucose and free fatty acids impairs beta-cell function, l... more Chronic exposure to elevated levels of glucose and free fatty acids impairs beta-cell function, leading to insulin secretion defects and eventually beta-cell failure. Using a semi-high throughput approach applied to INS-1E beta-cells, we tested multiple conditions of chronic exposure to basal, intermediate and high glucose, combined with saturated versus mono- and polyunsaturated fatty acids in order to assess cell integrity, lipid metabolism, mitochondrial function, glucose-stimulated calcium rise and secretory kinetics. INS-1E beta-cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mM) without or with BSA-complexed 0.4 mM saturated (C16:0 palmitate), monounsaturated (C18:1 oleate) or polyunsaturated (C18:2 linoleate, C18:3 linolenate) fatty acids, resulting in 0.1-0.5 μM unbound fatty acids. Accumulation of triglycerides in cells exposed to fatty acids was glucose-dependent, oleate inducing the strongest lipid storage and protecting against glucose-induced cytotoxicity. The combined chronic exposure to both high glucose and either palmitate or oleate altered mitochondrial function as well as glucose-induced calcium rise. This pattern did not directly translate at the secretory level since palmitate and oleate exhibited distinct effects on the first and the second phases of glucose-stimulated exocytosis. Both fatty acids changed the activity of kinases, such as the MODY-associated BLK. Additionally, chronic exposure to fatty acids modified membrane physicochemical properties by increasing membrane fluidity, oleate exhibiting larger effects compared to palmitate. Chronic fatty acids differentially and specifically exacerbated some of the glucotoxic effects, without promoting cytotoxicity on their own. Each of the tested fatty acids functionally modified INS-1E beta-cell, oleate inducing the strongest effects.
American Journal of Physiology-Endocrinology and Metabolism, 2019
Fructose is widely used as a sweetener in processed food and is also associated with metabolic di... more Fructose is widely used as a sweetener in processed food and is also associated with metabolic disorders, such as obesity. However, the underlying cellular mechanisms remain unclear, in particular, regarding the pancreatic β-cell. Here, we investigated the effects of chronic exposure to fructose on the function of insulinoma cells and isolated mouse and human pancreatic islets. Although fructose per se did not acutely stimulate insulin exocytosis, our data show that chronic fructose rendered rodent and human β-cells hyper-responsive to intermediate physiological glucose concentrations. Fructose exposure reduced intracellular ATP levels without affecting mitochondrial function, induced AMP-activated protein kinase activation, and favored ATP release from the β-cells upon acute glucose stimulation. The resulting increase in extracellular ATP, mediated by pannexin1 (Panx1) channels, activated the calcium-mobilizer P2Y purinergic receptors. Immunodetection revealed the presence of both ...
Mitochondria play a central role in pancreatic beta-cells by coupling metabolism of the secretago... more Mitochondria play a central role in pancreatic beta-cells by coupling metabolism of the secretagogue glucose to distal events of regulated insulin exocytosis. This process requires transports of both metabolites and nucleotides in and out of the mitochondria. The molecular identification of mitochondrial carriers and their respective contribution to beta-cell function have been uncovered only recently. In type 2 diabetes, mitochondrial dysfunction is an early event and may precipitate beta-cell loss. Under diabetogenic conditions, characterized by glucotoxicity and lipotoxicity, the expression profile of mitochondrial carriers is selectively modified. This review describes the role of mitochondrial carriers in beta-cells and the selective changes in response to glucolipotoxicity. In particular, we discuss the importance of the transfer of metabolites (pyruvate, citrate, malate, and glutamate) and nucleotides (ATP, NADH, NADPH) for beta-cell function and dysfunction. This article is ...
In pancreatic ß-cells, mitochondria play a central role in coupling glucose metabolism to insulin... more In pancreatic ß-cells, mitochondria play a central role in coupling glucose metabolism to insulin secretion. Chronic exposure of ß-cells to metabolic stresses impairs their function and potentially induces apoptosis. Little is known on mitochondrial adaptation to metabolic stresses, i.e. high glucose, fatty acids, or oxidative stress; being all highlighted in the pathogenesis of type 2 diabetes. Here, human islets were exposed for 3 days to 25 mM glucose, 0.4 mM palmitate, 0.4 mM oleate, and transiently to H2O2. Culture at physiological 5.6 mM glucose served as no-stress control. Expression of mitochondrion-associated genes was quantified, including the transcriptome of mitochondrial inner membrane carriers. Targets of interest were further evaluated at the protein level. Three days after acute oxidative stress, no significant alteration in ß-cell function or apoptosis was detected in human islets. Palmitate specifically increased expression of the pyruvate carriers MPC1 and MPC2, w...
<p>(A) Cell death of INS-1E cells cultured without stress at 11.1 mM glucose concentration ... more <p>(A) Cell death of INS-1E cells cultured without stress at 11.1 mM glucose concentration (G11, control) or exposed to different culture conditions listed in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082364#pone-0082364-g001" target="_blank">figure 1</a>. Data are shown as means ± SEM of 4 independent experiments. (B) Representative immunoblotting showing levels of the cleaved CASPASE 3, TUBULIN and ACTIN from treated INS-1E cells. (C) Quantitative analysis of band densities normalized to TUBULIN from immunoblots as shown in (B) is presented as means ± SEM of 4 independent experiments. Results are expressed as protein levels normalized to the control value of G11. (D) Transcript levels normalized to those of 18S and expressed as changes <i>versus</i> value of G11. The <i>BclxL</i> and <i>Bcl2</i> genes encode antiapoptotic proteins; the <i>Hspa5</i>, <i>Ddit3</i> and <i>Xbp1</i> genes encode the endoplasmic reticulum stress related proteins BIP, CHOP and XBP1, respectively; the cytosolic isoform <i>Sod1</i>, the matrix isoform <i>Sod2</i> (superoxide dismutase) and <i>Cat</i> (catalase) genes encode antioxidative enzymes. Results are means ± SEM of 2 independent experiments done in triplicate. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.005 <i>versus</i> G11 controls.</p
<p>(A) Cell death of INS-1E cells cultured without stress at 11.1 mM glucose concentration ... more <p>(A) Cell death of INS-1E cells cultured without stress at 11.1 mM glucose concentration (G11, control) or exposed to different culture conditions listed in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082364#pone-0082364-g001" target="_blank">figure 1</a>. Data are shown as means ± SEM of 4 independent experiments. (B) Representative immunoblotting showing levels of the cleaved CASPASE 3, TUBULIN and ACTIN from treated INS-1E cells. (C) Quantitative analysis of band densities normalized to TUBULIN from immunoblots as shown in (B) is presented as means ± SEM of 4 independent experiments. Results are expressed as protein levels normalized to the control value of G11. (D) Transcript levels normalized to those of 18S and expressed as changes <i>versus</i> value of G11. The <i>BclxL</i> and <i>Bcl2</i> genes encode antiapoptotic proteins; the <i>Hspa5</i>, <i>Ddit3</i> and <i>Xbp1</i> genes encode the endoplasmic reticulum stress related proteins BIP, CHOP and XBP1, respectively; the cytosolic isoform <i>Sod1</i>, the matrix isoform <i>Sod2</i> (superoxide dismutase) and <i>Cat</i> (catalase) genes encode antioxidative enzymes. Results are means ± SEM of 2 independent experiments done in triplicate. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.005 <i>versus</i> G11 controls.</p
Bien que la consommation du fructose soit associée à un stockage accru de graisses par l’action d... more Bien que la consommation du fructose soit associée à un stockage accru de graisses par l’action de l’insuline, le fructose seul n’induit pas la sécrétion de l’insuline par la cellule bêta-pancréatique, contrairement au glucose. Nous avons étudié les effets d’une exposition chronique au fructose sur la fonction des cellules bêta. Nos résultats révèlent que le fructose potentialise la sécrétion de l’insuline stimulée par le glucose en activant la voie de signalisation de l’adénosine triphosphate (ATP) extracellulaire. Cet effet est médié par l’activation des récepteurs purinérgiques P2Y1 et est associé à la libération d’ATP cellulaire par les canaux pannexines-1. En conséquence, l’interaction entre les canaux pannexines et les récepteurs purinérgiques via l’ATP extracellulaire représente une nouvelle cible cellulaire, offrant de potentielles implications thérapeutiques
The mechanism by which the β-cell transcription factor Pax4 influences cell function/mass was stu... more The mechanism by which the β-cell transcription factor Pax4 influences cell function/mass was studied in rat and human islets of Langerhans. Pax4 transcripts were detected in adult rat islets, and levels were induced by the mitogens activin A and betacellulin. Wortmannin suppressed betacellulin-induced Pax4 expression, implicating the phosphatidylinositol 3-kinase signaling pathway. Adenoviral overexpression of Pax4 caused a 3.5-fold increase in β-cell proliferation with a concomitant 1.9-, 4-, and 5-fold increase in Bcl-xL (antiapoptotic), c-myc, and Id2 mRNA levels, respectively. Accordingly, Pax4 transactivated the Bcl-xL and c-myc promoters, whereas its diabetes-linked mutant was less efficient. Bcl-xL activity resulted in altered mitochondrial calcium levels and ATP production, explaining impaired glucose-induced insulin secretion in transduced islets. Infection of human islets with an inducible adenoviral Pax4 construct caused proliferation and protection against cytokine-evok...
While the use of fructose as a sweetener and its consumption are associated with increased fat st... more While the use of fructose as a sweetener and its consumption are associated with increased fat storage prompted by the action of insulin, fructose alone does not acutely stimulate insulin exocytosis from the pancreatic beta-cell, as opposed to the chief secretagogue glucose. We investigated the effects of chronic exposure to fructose on beta-cell function. Our results reveal that chronic fructose induces extracellular ATP signaling in the beta-cell, resulting in the potentiation of glucose-stimulated insulin secretion. This effect is mediated by the activation of the purinergic P2Y1 receptors and is associated with the release of cellular ATP through pannexin-1 channels. Consequently, the interplay between pannexin channels and purinergic receptors, through ATP signaling, represents a novel cellular target with potential therapeutic implications.
Chronic exposure of pancreatic β-cells to elevated nutrient levels impairs their function and pot... more Chronic exposure of pancreatic β-cells to elevated nutrient levels impairs their function and potentially induces apoptosis. Like in other cell types, AMPK is activated in β-cells under conditions of nutrient deprivation, while little is known on AMPK responses to metabolic stresses. Here, we first reviewed recent studies on the role of AMPK activation in β-cells. Then, we investigated the expression profile of AMPK pathways in β-cells following metabolic stresses. INS-1E β-cells and human islets were exposed for 3 days to glucose (5.5–25 mM), palmitate or oleate (0.4 mM), and fructose (5.5 mM). Following these treatments, we analyzed transcript levels of INS-1E β-cells by qRT-PCR and of human islets by RNA-Seq; with a special focus on AMPK-associated genes, such as the AMPK catalytic subunits α1 (Prkaa1) and α2 (Prkaa2). AMPKα and pAMPKα were also evaluated at the protein level by immunoblotting. Chronic exposure to the different metabolic stresses, known to alter glucose-stimulate...
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2019
Chronic exposure to elevated levels of glucose and free fatty acids impairs beta-cell function, l... more Chronic exposure to elevated levels of glucose and free fatty acids impairs beta-cell function, leading to insulin secretion defects and eventually beta-cell failure. Using a semi-high throughput approach applied to INS-1E beta-cells, we tested multiple conditions of chronic exposure to basal, intermediate and high glucose, combined with saturated versus mono- and polyunsaturated fatty acids in order to assess cell integrity, lipid metabolism, mitochondrial function, glucose-stimulated calcium rise and secretory kinetics. INS-1E beta-cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mM) without or with BSA-complexed 0.4 mM saturated (C16:0 palmitate), monounsaturated (C18:1 oleate) or polyunsaturated (C18:2 linoleate, C18:3 linolenate) fatty acids, resulting in 0.1-0.5 μM unbound fatty acids. Accumulation of triglycerides in cells exposed to fatty acids was glucose-dependent, oleate inducing the strongest lipid storage and protecting against glucose-induced cytotoxicity. The combined chronic exposure to both high glucose and either palmitate or oleate altered mitochondrial function as well as glucose-induced calcium rise. This pattern did not directly translate at the secretory level since palmitate and oleate exhibited distinct effects on the first and the second phases of glucose-stimulated exocytosis. Both fatty acids changed the activity of kinases, such as the MODY-associated BLK. Additionally, chronic exposure to fatty acids modified membrane physicochemical properties by increasing membrane fluidity, oleate exhibiting larger effects compared to palmitate. Chronic fatty acids differentially and specifically exacerbated some of the glucotoxic effects, without promoting cytotoxicity on their own. Each of the tested fatty acids functionally modified INS-1E beta-cell, oleate inducing the strongest effects.
American Journal of Physiology-Endocrinology and Metabolism, 2019
Fructose is widely used as a sweetener in processed food and is also associated with metabolic di... more Fructose is widely used as a sweetener in processed food and is also associated with metabolic disorders, such as obesity. However, the underlying cellular mechanisms remain unclear, in particular, regarding the pancreatic β-cell. Here, we investigated the effects of chronic exposure to fructose on the function of insulinoma cells and isolated mouse and human pancreatic islets. Although fructose per se did not acutely stimulate insulin exocytosis, our data show that chronic fructose rendered rodent and human β-cells hyper-responsive to intermediate physiological glucose concentrations. Fructose exposure reduced intracellular ATP levels without affecting mitochondrial function, induced AMP-activated protein kinase activation, and favored ATP release from the β-cells upon acute glucose stimulation. The resulting increase in extracellular ATP, mediated by pannexin1 (Panx1) channels, activated the calcium-mobilizer P2Y purinergic receptors. Immunodetection revealed the presence of both ...
Mitochondria play a central role in pancreatic beta-cells by coupling metabolism of the secretago... more Mitochondria play a central role in pancreatic beta-cells by coupling metabolism of the secretagogue glucose to distal events of regulated insulin exocytosis. This process requires transports of both metabolites and nucleotides in and out of the mitochondria. The molecular identification of mitochondrial carriers and their respective contribution to beta-cell function have been uncovered only recently. In type 2 diabetes, mitochondrial dysfunction is an early event and may precipitate beta-cell loss. Under diabetogenic conditions, characterized by glucotoxicity and lipotoxicity, the expression profile of mitochondrial carriers is selectively modified. This review describes the role of mitochondrial carriers in beta-cells and the selective changes in response to glucolipotoxicity. In particular, we discuss the importance of the transfer of metabolites (pyruvate, citrate, malate, and glutamate) and nucleotides (ATP, NADH, NADPH) for beta-cell function and dysfunction. This article is ...
In pancreatic ß-cells, mitochondria play a central role in coupling glucose metabolism to insulin... more In pancreatic ß-cells, mitochondria play a central role in coupling glucose metabolism to insulin secretion. Chronic exposure of ß-cells to metabolic stresses impairs their function and potentially induces apoptosis. Little is known on mitochondrial adaptation to metabolic stresses, i.e. high glucose, fatty acids, or oxidative stress; being all highlighted in the pathogenesis of type 2 diabetes. Here, human islets were exposed for 3 days to 25 mM glucose, 0.4 mM palmitate, 0.4 mM oleate, and transiently to H2O2. Culture at physiological 5.6 mM glucose served as no-stress control. Expression of mitochondrion-associated genes was quantified, including the transcriptome of mitochondrial inner membrane carriers. Targets of interest were further evaluated at the protein level. Three days after acute oxidative stress, no significant alteration in ß-cell function or apoptosis was detected in human islets. Palmitate specifically increased expression of the pyruvate carriers MPC1 and MPC2, w...
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