Mitochondrial ALDH2 improves ß-cell survival and function against doxorubicin-induced apoptosis by targeting CK2 signaling.

AIMS: The aim of this study was to better understand how the chemotherapy drug doxorubicin contributes to the development of ß-cell dysfunction and to explore its relationship with mitochondrial aldehyde dehydrogenase-2 (ALDH2). MATERIALS AND METHODS: In order to investigate this hypothesis, doxorubicin was administered to INS-1 cells, a rat insulinoma cell line, either with or without several target protein activators and inhibitors. ALDH2 activity was detected with a commercial kit and protein levels were determined with western blot. Mitochondrial ROS, membrane potential, and lipid ROS were determined by commercial fluorescent probes. The cell viability was measured by CCK-assay. RESULTS: Exposure of INS-1 cells to doxorubicin decreased active insulin signaling resulting in elevated ALDH2 degradation, compared with control cells by the induction of acid sphingomyelinase mediated ceramide induction. Further, ceramide induction potentiated doxorubicin induced mitochondrial dysfunction. Treatment with the ALDH2 agonist, ALDA1, blocked doxorubicin-induced acid sphingomyelinase activation which significantly blocked ceramide induction and mitochondrial dysfunction mediated cell death. Treatment with the ALDH2 agonist, ALDA1, stimulated casein kinase-2 (CK2) mediated insulin signaling activation. CK2 silencing neutralized the function of ALDH2 in the doxorubicin treated INS-1 cells. CONCLUSIONS: Mitochondrial ALDH2 activation could inhibit the progression of doxorubicin induced pancreatic ß-cell dysfunction by inhibiting the acid sphingomyelinase induction of ceramide, by regulating the activation of CK2 signaling. Our research lays the foundation of ALDH2 activation as a therapeutic target for the precise treatment of chemotherapy drug induced ß-cell dysfunction.

Mitochondrial aldehyde dehydrogenase-2 coordinates the hydrogen sulfide - AMPK axis to attenuate high glucose-induced pancreatic ß-cell dysfunction by glutathione antioxidant system.

Progression of ß-cell loss in diabetes mellitus is significantly influenced by persistent hyperglycemia. At the cellular level, a number of signaling cascades affect the expression of apoptotic genes, ultimately resulting in ß-cell failure; these cascades have not been elucidated. Mitochondrial aldehyde dehydrogenase-2 (ALDH2) plays a central role in the detoxification of reactive aldehydes generated from endogenous and exogenous sources and protects against mitochondrial deterioration in cells. Here we report that under diabetogenic conditions, ALDH2 is strongly inactivated in ß-cells through CDK5-dependent glutathione antioxidant imbalance by glucose-6-phosphate dehydrogenase (G6PD) degradation. Intriguingly, CDK5 inhibition strengthens mitochondrial antioxidant defense through ALDH2 activation. Mitochondrial ALDH2 activation selectively preserves ß-cells against high-glucose-induced dysfunction by activating AMPK and Hydrogen Sulfide (H2S) signaling. This is associated with the stabilization and enhancement of the activity of G6PD by SIRT2, a cytoplasmic NAD+-dependent deacetylase, and is thereby linked to an elevation in the GSH/GSSG ratio, which leads to the inhibition of mitochondrial dysfunction under high-glucose conditions. Furthermore, treatment with NaHS, an H2S donor, selectively preserves ß-cell function by promoting ALDH2 activity, leading to the inhibition of lipid peroxidation by high-glucose concentrations. Collectively, our results provide the first direct evidence that ALDH2 activation enhances H2S-AMPK-G6PD signaling, leading to improved ß-cell function and survival under high-glucose conditions via the glutathione redox balance.

Perfluorooctane sulfonate-induced oxidative stress contributes to pancreatic ß-cell apoptosis by inhibiting cyclic adenosine monophosphate pathway: Prevention by pentoxifylline.

Endocrine-disrupting chemical perfluorooctane sulfonate (PFOS) acute exposure stimulates insulin secretion from pancreatic ß-cells. However, chronic exposure to PFOS on pancreatic ß-cells, its role in insulin secretion, and the underlying mechanisms have not been studied. We used rat insulinoma INS-1 and human 1.1b4 islet cells to investigate the chronic effects of PFOS on glucose-stimulated insulin secretion and toxicity implicated in the downregulation of ß-cell functionality. Chronic exposure of INS-1 cells or human pancreatic 1.1b4 ß-cells to PFOS stimulated the small G-protein RAC1-guanosine triphosphate-dependent nicotinamide adenine dinucleotide phosphate oxidase (NOX2/gp91phox) subunit expression and activation. Upregulated NOX2/gp91phox activation led to elevated reactive oxygen species (ROS) production with a decrease in the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway in both cell types. Inhibition of cAMP/PKA signaling induces ß-cell mitochondrial dysfunction and endoplasmic stress via the loss of PDX1-SERCA2B and glucose-stimulated insulin release. Inhibiting RAC1-NOX2/gp91phox activation or elevating cAMP by pentoxifylline, a Food and Drug Administration-approved phosphodiesterase inhibitor, significantly reduced PFOS-induced ROS production and restored insulin secretory function of pancreatic ß-cells. Enhanced secretory function in pentoxifylline-treated cells was associated with increased stability of PDX1-SERCA2B protein levels. Intriguingly, inhibition of cAMP/PKA signaling impaired pentoxifylline-induced insulin secretion caused by the activation of ROS production and mitochondrial dysfunction. Overall, our findings show that PFOS has a new and first-ever direct chronic effect on pancreatic ß-cell failure through increased RAC1-NOX2/gp91phox activation and pentoxifylline-induced cAMP/PKA signaling, which inhibits PFOS-mediated mitochondrial dysfunction.

Ferroptosis Signaling in Pancreatic ß-Cells: Novel Insights & Therapeutic Targeting.

Metabolic stress impairs pancreatic ß-cell survival and function in diabetes. Although the pathophysiology of metabolic stress is complex, aberrant tissue damage and ß-cell death are brought on by an imbalance in redox equilibrium due to insufficient levels of endogenous antioxidant expression in ß-cells. The vulnerability of ß-cells to oxidative damage caused by iron accumulation has been linked to contributory ß-cell ferroptotic-like malfunction under diabetogenic settings. Here, we take into account recent findings on how iron metabolism contributes to the deregulation of the redox response in diabetic conditions as well as the ferroptotic-like malfunction in the pancreatic ß-cells, which may offer insights for deciphering the pathomechanisms and formulating plans for the treatment or prevention of metabolic stress brought on by ß-cell failure.

c-Abl tyrosine kinase inhibition attenuate oxidative stress-induced pancreatic ß-Cell dysfunction via glutathione antioxidant system.

Chronic oxidative stress, which is caused by aberrant non-receptor tyrosine kinase (c-Abl) signaling, plays a key role in the progression of ß-cell loss in diabetes mellitus. Recent studies, however, have linked ferroptotic-like death to the ß-cell loss in diabetes mellitus. Here, we report that oxidative stress-driven reduced/oxidized glutathione (GSH/GSSG) loss and proteasomal degradation of glutathione peroxidase 4 (GPX4) promote ferroptotic-like cell damage through increased lipid peroxidation. Mechanistically, treatment with GNF2, a non-ATP competitive c-Abl kinase inhibitor, selectively preserves ß-cell function by inducing the orphan nuclear receptor estrogen-related receptor gamma (ERRγ). ERRγ-driven glutaminase 1 (GLS1) expression promotes the elevation of the GSH/GSSG ratio, and this increase leads to the inhibition of lipid peroxidation by GPX4. Strikingly, pharmacological inhibition of ERRγ represses the expression of GLS1 and reverses the GSH/GSSG ratio linked to mitochondrial dysfunction and increased lipid peroxidation mediated by GPX4 degradation. Inhibition of GLS1 suppresses the ERRγ agonist DY131-induced GSH/GSSG ratio linked to ferroptotic-like death owing to the loss of GPX4. Furthermore, immunohistochemical analysis showed enhanced ERRγ and GPX4 expression in the pancreatic islets of GNF2-treated mice compared to that in streptozotocin-treated mice. Altogether, our results provide the first evidence that the orphan nuclear receptor ERRγ-induced GLS1 expression augments the glutathione antioxidant system, and its downstream signaling leads to improved ß-cell function and survival under oxidative stress conditions.

CD36 Signal Transduction in Metabolic Diseases: Novel Insights and Therapeutic Targeting.

The cluster of differentiation 36 (CD36) is a scavenger receptor present on various types of cells and has multiple biological functions that may be important in inflammation and in the pathogenesis of metabolic diseases, including diabetes. Here, we consider recent insights into how the CD36 response becomes deregulated under metabolic conditions, as well as the therapeutic benefits of CD36 inhibition, which may provide clues for developing strategies aimed at the treatment or prevention of diabetes associated with metabolic diseases. To facilitate this process further, it is important to pinpoint regulatory mechanisms that are relevant under physiological and pathological conditions. In particular, understanding the mechanisms involved in dictating specific CD36 downstream cellular outcomes will aid in the discovery of potent compounds that target specific CD36 downstream signaling cascades.

NADPH Oxidase (NOX) Targeting in Diabetes: A Special Emphasis on Pancreatic ß-Cell Dysfunction.

In type 2 diabetes, metabolic stress has a negative impact on pancreatic ß-cell function and survival (T2D). Although the pathogenesis of metabolic stress is complex, an imbalance in redox homeostasis causes abnormal tissue damage and ß-cell death due to low endogenous antioxidant expression levels in ß-cells. Under diabetogenic conditions, the susceptibility of ß-cells to oxidative damage by NADPH oxidase has been related to contributing to ß-cell dysfunction. Here, we consider recent insights into how the redox response becomes deregulated under diabetic conditions by NADPH oxidase, as well as the therapeutic benefits of NOX inhibitors, which may provide clues for understanding the pathomechanisms and developing strategies aimed at the treatment or prevention of metabolic stress associated with ß-cell failure.

Pioglitazone-induced AMPK-Glutaminase-1 prevents high glucose-induced pancreatic ß-cell dysfunction by glutathione antioxidant system.

Prolonged hyperglycemia plays a major role in the progression of ß-cell loss in diabetes mellitus. Here we report an insulin sensitizer thiazolidinedione Pioglitazone selectively preserves the beta cells against high glucose-induced dysfunction by activation of AMPK and Glutaminase 1 (GLS1) axis. AMPK activation increases the stability of Glutaminase 1 by HSP90 family mitochondrial heat shock protein 75 (HSP75/TRAP1). This is associated with an elevation of GSH/GSSG ratio which leads to inhibition of mitochondrial dysfunction by induction of BCL2/BCL-XL in high glucose conditions. Pioglitazone was able to also protect against high glucose-induced elevations in maladaptive ER stress markers and increase the adaptive unfolded protein response (UPR) by inhibiting mTORC1-eEF2 protein translation machinery. Moreover, the pioglitazone effect on AMPK activation was not dependent on the PPARγ pathway. Strikingly, chemical inhibition of AMPK signaling or glutaminase-1 inhibition abrogates the pioglitazone effect on the TRAP1-GLS1 axis and GSH/GSSG ratio linked to mitochondrial dysfunction. Finally, inhibition of AMPK signaling enhanced maladaptive ER stress markers by mTORC1-eEF2 activation. Altogether, these results support the proposal that pioglitazone induced AMPK activation stabilizes a novel interaction of TRAP1/HSP75-GLS1 and its downstream signaling leads to improved ß-cell function and survival under high glucose conditions.

High glucose-induced PRDX3 acetylation contributes to glucotoxicity in pancreatic ß-cells: Prevention by Teneligliptin.

Chronic hyperglycemia has deleterious effects on pancreatic ß-cell function and survival in type 2 diabetes (T2D) due to the low expression level of endogenous antioxidants in the ß-cells. Peroxiredoxin-3 (PRDX3) is a mitochondria specific H202 scavenger and protects the cell from mitochondrial damage. However, nothing is known about how glucotoxicity influences PRDX3 function in the pancreatic beta cells. Exposure of rat insulinoma INS-1 cells and human beta cells (1.1B4) to high glucose conditions (30mM) stimulated acetylation of PRDX3 which facilitates its hyper-oxidation causing mitochondrial dysfunction by SIRT1 degradation. SIRT1 deficiency induces beta cell apoptosis via NOX-JNK-p66Shc signalosome activation. Herein we investigated the direct effect of Teneligliptin, a newer DPP-4 inhibitor on beta-cell function and survival in response to high glucose conditions. Teneligliptin treatment enhances SIRT1 protein levels and activity by USP22, an ubiquitin specific peptidase. Activated SIRT1 prevents high glucose-induced PRDX3 acetylation by SIRT3 resulted in inhibition of PRDX3 hyper-oxidation thereby strengthening the mitochondrial antioxidant defense. Notably, we identify PRDX3 as a novel SIRT3 target and show their physical interaction. Intriguingly, inhibition of SIRT1 activity by EX-527 or SIRT1 siRNA knockdown exacerbated the SIRT3 mediated PRDX3 deacetylation which leads to peroxiredoxin-3 hyper-oxidation and beta-cell apoptosis by the activation of NOX-JNK-p66Shc signalosome. Collectively, our results unveil a novel and first direct effect of high glucose on PRDX3 acetylation on beta-cell dysfunction by impaired antioxidant defense and SIRT1 mediated SIRT3-PRDX3 activation by Teneligliptin suppresses high glucose-mediated mitochondrial dysfunction.

Myricetin prevents thapsigargin-induced CDK5-P66Shc signalosome mediated pancreatic ß-cell dysfunction.

Chronic endoplasmic reticulum (ER) stress has deleterious effects on pancreatic ß-cell function and survival in type 2 diabetes (T2D). Cyclin-dependent kinase 5 (CDK5) plays a critical role in ß-cell failure under diabetic milieu conditions. However, little information is available on CDK5's ability to impair the function of ß-cells via a chemical ER stress inducer thapsigargin. Myricetin, a natural flavonoid, has therapeutic potential for the treatment of type 2 diabetes mellitus. Therefore, we examined the effect of CDK5 on thapsigargin-induced ß-cell apoptosis, and explored the relationship between myricetin and CDK5. Exposure of beta cells with thapsigargin, induced a Src-mediated redox signaling (VAV2-Rac1-NOX) formation and CDK5 activation. Activated CDK5 induced antiapoptotic protein myeloid cell leukemia sequence 1 (Mcl-1) degradation which was associated with p66Shc serine 36 phosphorylation, causing beta cell apoptosis via mitochondrial dysfunction. Exposure of beta cells to myricetin resulted in an acute inhibition of Src-mediated redox signaling (VAV2-Rac1-NOX) formation and CDK5 activation. Myricetin inhibited CDK5 activation by directly binding to its ATP-binding pocket. Treatment with myricetin also enhanced the stability of Mcl-1 after thapsigargin treatment. Inhibition of CDK5 with myricetin or roscovitine, a CDK5 inhibitor attenuates thapsigargin induced p66Shc serine 36 phosphorylation and also reduced mitochondrial dysfunction by decreasing mitochondrial ROS and caspase-3 activation. In addition, myricetin was observed to enhance PDX-1 and insulin mRNA expression and potentiate glucose stimulated insulin secretion (GSIS). Taken together, these findings indicate that thapsigargin-induced early molecular events lead to CDK5-p66Shc signalosome contributes to thapsigargin-induced pancreatic ß-cell dysfunction. Myricetin blocked thapsigargin induced CDK5-p66Shc signalosome formation and prevented pancreatic beta cell dysfunction. In this study, we demonstrated for the first time that thapsigargin initiated CDK5-p66Shc signalosome mediates the pancreatic beta cell dysfunction and myricetin protects the pancreatic beta cells through the inhibition of CDK5-p66Shc signalosome.

CD36 dependent redoxosomes promotes ceramide-mediated pancreatic ß-cell failure via p66Shc activation.

Altered metabolism is implicated in the pathogenesis of beta-cell failure in type 2 diabetes (T2D). Plasma and tissue levels of ceramide species play positive roles in inflammatory and oxidative stress responses in T2D. However, oxidative targets and mechanisms underlying ceramide signaling are unclear. We investigated the role of CD36-dependent redoxosome (redox-active endosome), a membrane-based signaling agent, in ceramide-induced beta-cell dysfunction and failure. Exposure of beta cells to C2-ceramide (N-acetyl-sphingosine) induced a CD36-dependent non-receptor tyrosine kinase Src-mediated redoxosome (Vav2-Rac1-NOX) formation. Activated Rac1-GTP-NADPH oxidase complex induced c-Jun-N-terminal kinase (JNK) activation and nuclear factor (NF)-kB transcription, which was associated with thioredoxin-interacting protein (TXNIP) upregulation and thioredoxin activity suppression. Upregulated JNK expression induced p66Shc serine36 phosphorylation and peroxiredoxin-3 hyperoxidation, causing beta-cell apoptosis via mitochondrial dysfunction. CD36 inhibition by sulfo-N-succinimidyl oleate (SSO) or CD36 siRNA blocked C2-ceramide-induced redoxosome activation, thereby decreasing JNK-dependent p66Shc serine36 phosphorylation. CD36 inhibition downregulated TXNIP expression and promoted thioredoxin activity via enhanced thioredoxin reductase activity, which prevented peroxiredoxin-3 oxidation. CD36 inhibition potentiated glucose-stimulated insulin secretion and prevented beta-cell apoptosis. Our results reveal a new role of CD36 during early molecular events that lead to Src-mediated redoxosome activation, which contributes to ceramide-induced pancreatic beta-cell dysfunction and failure.

Myricetin Protects Against High Glucose-Induced ß-Cell Apoptosis by Attenuating Endoplasmic Reticulum Stress via Inactivation of Cyclin-Dependent Kinase 5.

BACKGROUND: Chronic hyperglycemia has deleterious effects on pancreatic ß-cell function and turnover. Recent studies support the view that cyclin-dependent kinase 5 (CDK5) plays a role in ß-cell failure under hyperglycemic conditions. However, little is known about how CDK5 impair ß-cell function. Myricetin, a natural flavonoid, has therapeutic potential for the treatment of type 2 diabetes mellitus. In this study, we examined the effect of myricetin on high glucose (HG)-induced ß-cell apoptosis and explored the relationship between myricetin and CDK5. METHODS: To address this question, we subjected INS-1 cells and isolated rat islets to HG conditions (30 mM) in the presence or absence of myricetin. Docking studies were conducted to validate the interaction between myricetin and CDK5. Gene expression and protein levels of endoplasmic reticulum (ER) stress markers were measured by real-time reverse transcription polymerase chain reaction and Western blot analysis. RESULTS: Activation of CDK5 in response to HG coupled with the induction of ER stress via the down regulation of sarcoendoplasmic reticulum calcium ATPase 2b (SERCA2b) gene expression and reduced the nuclear accumulation of pancreatic duodenal homeobox 1 (PDX1) leads to ß-cell apoptosis. Docking study predicts that myricetin inhibit CDK5 activation by direct binding in the ATP-binding pocket. Myricetin counteracted the decrease in the levels of PDX1 and SERCA2b by HG. Moreover, myricetin attenuated HG-induced apoptosis in INS-1 cells and rat islets and reduce the mitochondrial dysfunction by decreasing reactive oxygen species production and mitochondrial membrane potential (ΔΨm) loss. CONCLUSION: Myricetin protects the ß-cells against HG-induced apoptosis by inhibiting ER stress, possibly through inactivation of CDK5 and consequent upregulation of PDX1 and SERCA2b.

Rac1-NADPH oxidase signaling promotes CD36 activation under glucotoxic conditions in pancreatic beta cells.

We recently reported that cluster determinant 36 (CD36), a fatty acid transporter, plays a pivotal role in glucotoxicity-induced ß-cell dysfunction. However, little is known about how glucotoxicity influences CD36 expression. Emerging evidence suggests that the small GTPase Rac1 is involved in the pathogenesis of beta cell dysfunction in type 2 diabetes (T2D). The primary objective of the current study was to determine the role of Rac1 in CD36 activation and its impact on ß-cell dysfunction in diabetes mellitus. To address this question, we subjected INS-1 cells and human beta cells (1.1B4) to high glucose conditions (30mM) in the presence or absence of Rac1 inhibition either by NSC23766 (Rac1 GTPase inhibitor) or small interfering RNA. High glucose exposure in INS-1 and human beta cells (1.1b4) resulted in the activation of Rac1 and induced cell apoptosis. Rac1 activation mediates NADPH oxidase (NOX) activation leading to elevated ROS production in both cells. Activation of the Rac1-NOX complex by high glucose levels enhanced CD36 expression in INS-1 and human 1.1b4 beta cell membrane fractions. The inhibition of Rac1 by NSC23766 inhibited NADPH oxidase activity and ROS generation induced by high glucose concentrations in INS-1 & human 1.1b4 beta cells. Inhibition of Rac1-NOX complex activation by NSC23766 significantly reduced CD36 expression in INS-1 and human 1.1b4 beta cell membrane fractions. In addition, Rac1 inhibition by NSC23766 significantly reduced high glucose-induced mitochondrial dysfunction. Furthermore, NADPH oxidase inhibition by VAS2870 also attenuated high glucose-induced ROS generation and cell apoptosis. These results suggest that Rac1-NADPH oxidase dependent CD36 expression contributes to high glucose-induced beta cell dysfunction and cell death.

Metformin prevents glucotoxicity by alleviating oxidative and ER stress-induced CD36 expression in pancreatic beta cells.

AIM/HYPOTHESIS: Cluster determinant 36 (CD36), a fatty acid transporter, was reported to have a pivotal role in glucotoxicity-induced beta cell dysfunction. However, little is known about how glucotoxicity influences CD36 expression, and it is unknown whether this action can be counteracted by metformin. In the present study, we showed that metformin counteracts glucotoxicity by alleviating oxidative and endoplasmic reticulum (ER) stress-induced CD36 expression. METHODS: We used primary rat islets as well as INS-1 cells for 72h to 24h with 30mM glucose, respectively. Thapsigargin was used as strong ER stressor, and Sulfo-N-succinimidyl oleate (SSO) and RNA interference were chosen for CD36 inhibition. Free fatty acid uptake was measured by radioisotope tracing technique. RESULTS: Exposure of isolated rat islets to high glucose (HG) for 3days decreased insulin and pancreatic duodenal homeobox1 (Pdx1) mRNA expression, with the suppression of glucose-stimulated insulin secretion (GSIS) along with elevation of reactive oxygen species (ROS) levels. Incubation with metformin restored insulin and Pdx1 mRNA expression with significant improvements in GSIS and decrease in ROS production. HG exposure in INS-1 cells increased free fatty acid uptake via induction of CD36 along with impaired insulin and Pdx1 mRNA expression. Moreover, thapsigargin also increased the induction of CD36 expression. Metformin blocked HG- and thapsigargin-induced CD36 expression. In addition, the simultaneous inhibition of intracellular ROS production by metformin or CD36 activation by SSO or CD36 siRNA significantly decreased the apoptotic response in HG-treated INS-1 cells. CONCLUSION/INTERPRETATION: In conclusion, metformin conferred protection against HG-induced apoptosis of pancreatic beta cells, largely by interfering with ROS production, and inhibited the CD36-mediated free fatty acid influx. This report provides evidence that the inhibition of CD36 may have potential therapeutic effects against hyperglycemia-induced beta cell damage in diabetes.