A role and mechanism for redox sensing by SENP1 in ß-cell responses to high fat feeding.

Pancreatic ß-cells respond to metabolic stress by upregulating insulin secretion, however the underlying mechanisms remain unclear. Here we show, in ß-cells from overweight humans without diabetes and mice fed a high-fat diet for 2 days, insulin exocytosis and secretion are enhanced without increased Ca2+ influx. RNA-seq of sorted ß-cells suggests altered metabolic pathways early following high fat diet, where we find increased basal oxygen consumption and proton leak, but a more reduced cytosolic redox state. Increased ß-cell exocytosis after 2-day high fat diet is dependent on this reduced intracellular redox state and requires the sentrin-specific SUMO-protease-1. Mice with either pancreas- or ß-cell-specific deletion of this fail to up-regulate exocytosis and become rapidly glucose intolerant after 2-day high fat diet. Mechanistically, redox-sensing by the SUMO-protease requires a thiol group at C535 which together with Zn+-binding suppresses basal protease activity and unrestrained ß-cell exocytosis, and increases enzyme sensitivity to regulation by redox signals.

Glucose-stimulated insulin secretion depends on FFA1 and Gq in neonatal mouse islets.

AIMS/HYPOTHESIS: After birth, the neonatal islets gradually acquire glucose-responsive insulin secretion, a process that is subjected to maternal imprinting. Although NEFA are major components of breastmilk and insulin secretagogues, their role for functional maturation of neonatal beta cells is still unclear. NEFA are the endogenous ligands of fatty acid receptor 1 (FFA1, encoded by Ffar1 in mice), a Gq-coupled receptor with stimulatory effect on insulin secretion. This study investigates the role of FFA1 in neonatal beta cell function and in the adaptation of offspring beta cells to parental high-fat feeding. METHODS: Wild-type (WT) and Ffar1-/- mice were fed high-fat (HFD) or chow diet (CD) for 8 weeks before mating, and during gestation and lactation. Blood variables, pancreas weight and insulin content were assessed in 1-, 6-, 11- and 26-day old (P1-P26) offspring. Beta cell mass and proliferation were determined in P1-P26 pancreatic tissue sections. FFA1/Gq dependence of insulin secretion was evaluated in isolated islets and INS-1E cells using pharmacological inhibitors and siRNA strategy. Transcriptome analysis was conducted in isolated islets. RESULTS: Blood glucose levels were higher in CD-fed Ffar1-/- P6-offspring compared with CD-fed WT P6-offspring. Accordingly, glucose-stimulated insulin secretion (GSIS) and its potentiation by palmitate were impaired in CD Ffar1-/- P6-islets. In CD WT P6-islets, insulin secretion was stimulated four- to fivefold by glucose and five- and sixfold over GSIS by palmitate and exendin-4, respectively. Although parental HFD increased blood glucose in WT P6-offspring, it did not change insulin secretion from WT P6-islets. In contrast, parental HFD abolished glucose responsiveness (i.e. GSIS) in Ffar1-/- P6-islets. Inhibition of Gq by FR900359 or YM-254890 in WT P6-islets mimicked the effect of Ffar1 deletion, i.e. suppression of GSIS and of palmitate-augmented GSIS. The blockage of Gi/o by pertussis toxin (PTX) enhanced (100-fold) GSIS in WT P6-islets and rendered Ffar1-/- P6-islets glucose responsive, suggesting constitutive activation of Gi/o. In WT P6-islets, FR900359 cancelled 90% of PTX-mediated stimulation, while in Ffar1-/- P6-islets it completely abolished PTX-elevated GSIS. The secretory defect of Ffar1-/- P6-islets did not originate from insufficient beta cells, since beta cell mass increased with the offspring's age irrespective of genotype and diet. In spite of that, in the breastfed offspring (i.e. P1-P11) beta cell proliferation and pancreatic insulin content had a genotype- and diet-driven dynamic. Under CD, the highest proliferation rate was reached by the Ffar1-/- P6 offspring (3.95% vs 1.88% in WT P6), whose islets also showed increased mRNA levels of genes (e.g. Fos, Egr1, Jun) typically high in immature beta cells. Although parental HFD increased beta cell proliferation in both WT (4.48%) and Ffar1-/- (5.19%) P11 offspring, only the WT offspring significantly increased their pancreatic insulin content upon parental HFD (5.18 µg under CD to 16.93 µg under HFD). CONCLUSIONS/INTERPRETATION: FFA1 promotes glucose-responsive insulin secretion and functional maturation of newborn islets and is required for adaptive offspring insulin secretion in the face of metabolic challenge, such as parental HFD.

Co-transplantation of pancreatic islets and microvascular fragments effectively restores normoglycemia in diabetic mice.

Insufficient revascularization of pancreatic islets is one of the major obstacles impairing the success of islet transplantation. To overcome this problem, we introduce in the present study a straightforward strategy to accelerate the engraftment of isolated islets. For this purpose, we co-transplanted 250 islets and 20,000 adipose tissue-derived microvascular fragments (MVF) from donor mice under the kidney capsule as well as 500 or 1000 islets with 40,000 MVF into the subcutaneous space of diabetic mice. We found that the co-transplantation of islets and MVF markedly accelerates the restoration of normoglycemia in diabetic recipients compared with the transplantation of islets alone. In fact, the transplantation of 250 islets with 20,000 MVF under the kidney capsule reversed diabetes in 88% of mice and the subcutaneous transplantation of 500 or 1000 islets with 40,000 MVF restored normoglycemia in 100% of mice. Moreover, diabetic mice receiving islets and MVF exhibited plasma insulin levels similar to nondiabetic control animals. Additional immunohistochemical analyses of the grafts revealed a significantly higher number of islet cells and microvessels in the co-transplantation groups. These findings demonstrate that the co-transplantation of islets and MVF is a promising strategy to improve the success rates of islet transplantation, which could be easily implemented into future clinical practice.

Pro-oxidative priming but maintained cardiac function in a broad spectrum of murine models of chronic kidney disease.

AIMS: Patients with chronic kidney disease (CKD) have an increased risk of cardiovascular events and exhibit myocardial changes including left ventricular (LV) hypertrophy and fibrosis, overall referred to as 'uremic cardiomyopathy'. Although different CKD animal models have been studied for cardiac effects, lack of consistent reporting on cardiac function and pathology complicates clear comparison of these models. Therefore, this study aimed at a systematic and comprehensive comparison of cardiac function and cardiac pathophysiological characteristics in eight different CKD models and mouse strains, with a main focus on adenine-induced CKD. METHODS AND RESULTS: CKD of different severity and duration was induced by subtotal nephrectomy or adenine-rich diet in various strains (C57BL/6J, C57BL/6 N, hyperlipidemic C57BL/6J ApoE-/-, 129/Sv), followed by the analysis of kidney function and morphology, blood pressure, cardiac function, cardiac hypertrophy, fibrosis, myocardial calcification and inflammation using functional, histological and molecular techniques, including cardiac gene expression profiling supplemented by oxidative stress analysis. Intriguingly, despite uremia of variable degree, neither cardiac dysfunction, hypertrophy nor interstitial fibrosis were observed. However, already moderate CKD altered cardiac oxidative stress responses and enhanced oxidative stress markers in each mouse strain, with cardiac RNA sequencing revealing activation of oxidative stress signaling as well as anti-inflammatory feedback responses. CONCLUSION: This study considerably expands the knowledge on strain- and protocol-specific differences in the field of cardiorenal research and reveals that several weeks of at least moderate experimental CKD increase oxidative stress responses in the heart in a broad spectrum of mouse models. However, this was insufficient to induce relevant systolic or diastolic dysfunction, suggesting that additional "hits" are required to induce uremic cardiomyopathy. TRANSLATIONAL PERSPECTIVE: Patients with chronic kidney disease (CKD) have an increased risk of cardiovascular adverse events and exhibit myocardial changes, overall referred to as 'uremic cardiomyopathy'. We revealed that CKD increases cardiac oxidative stress responses in the heart. Nonetheless, several weeks of at least moderate experimental CKD do not necessarily trigger cardiac dysfunction and remodeling, suggesting that additional "hits" are required to induce uremic cardiomyopathy in the clinical setting. Whether the altered cardiac oxidative stress balance in CKD may increase the risk and extent of cardiovascular damage upon additional cardiovascular risk factors and/or events will be addressed in future studies.

The loss of pancreatic islet NADPH oxidase (NOX)2 improves islet transplantation.

Islet transplantation is a promising treatment strategy for type 1 diabetes mellitus (T1DM) patients. However, oxidative stress-induced graft failure due to an insufficient revascularization is a major problem of this therapeutic approach. NADPH oxidase (NOX)2 is an important producer of reactive oxygen species (ROS) and several studies have already reported that this enzyme plays a crucial role in the endocrine function and viability of ß-cells. Therefore, we hypothesized that targeting islet NOX2 improves the outcome of islet transplantation. To test this, we analyzed the cellular composition and viability of isolated wild-type (WT) and Nox2-/- islets by immunohistochemistry as well as different viability assays. Ex vivo, the effect of Nox2 deficiency on superoxide production, endocrine function and anti-oxidant protein expression was studied under hypoxic conditions. In vivo, we transplanted WT and Nox2-/- islets into mouse dorsal skinfold chambers and under the kidney capsule of diabetic mice to assess their revascularization and endocrine function, respectively. We found that the loss of NOX2 does not affect the cellular composition and viability of isolated islets. However, decreased superoxide production, higher glucose-stimulated insulin secretion as well as expression of nuclear factor erythroid 2-related factor (Nrf)2, heme oxygenase (HO)-1 and superoxide dismutase 1 (SOD1) was detected in hypoxic Nox2-/- islets when compared to WT islets. Moreover, we detected an early revascularization, a higher take rate and restoration of normoglycemia in diabetic mice transplanted with Nox2-/- islets. These findings indicate that the suppression of NOX2 activity represents a promising therapeutic strategy to improve engraftment and function of isolated islets.

Targeting Pancreatic Islet NLRP3 Improves Islet Graft Revascularization.

Hypoxia-induced islet cell death, caused by an insufficient revascularization of the grafts, is a major obstacle for successful pancreatic islet transplantation. Recently, it has been reported that the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is expressed in pancreatic islets and that its loss protects against hypoxia-induced cell death. Therefore, we hypothesized that the inhibition of NLRP3 in islets improves the survival and endocrine function of the grafts. The transplantation of Nlrp3-/- islets or wild-type (WT) islets exposed to the NLRP3 inhibitor CY-09 into mouse dorsal skinfold chambers resulted in an improved revascularization compared with controls. An increased insulin release after NLRP3 inhibition caused the enhanced angiogenic response. Moreover, the inhibition of NLRP3 in hypoxic ß-cells triggered insulin gene expression by inducing the shuttling of MafA and pancreatic and duodenal homeobox-1 into the nucleus. This was mediated by a reduced interaction of NLRP3 with the thioredoxin-interacting protein (TXNIP). Transplantation of Nlrp3-/- islets or WT islets exposed to CY-09 under the kidney capsule of diabetic mice markedly improved the restoration of normoglycemia. These findings indicate that the inhibition of NLRP3 in isolated islets represents a promising therapeutic strategy to improve engraftment and function of the islets.

Unspecific CTL Killing Is Enhanced by High Glucose via TNF-Related Apoptosis-Inducing Ligand.

TNF-related apoptosis inducing ligand (TRAIL) is expressed on cytotoxic T lymphocytes (CTLs) and TRAIL is linked to progression of diabetes. However, the impact of high glucose on TRAIL expression and its related killing function in CTLs still remains largely elusive. Here, we report that TRAIL is substantially up-regulated in CTLs in environments with high glucose (HG) both in vitro and in vivo. Non-mitochondrial reactive oxygen species, NFκB and PI3K/Akt are essential in HG-induced TRAIL upregulation in CTLs. TRAILhigh CTLs induce apoptosis of pancreatic beta cell line 1.4E7. Treatment with metformin and vitamin D reduces HG-enhanced expression of TRAIL in CTLs and coherently protects 1.4E7 cells from TRAIL-mediated apoptosis. Our work suggests that HG-induced TRAILhigh CTLs might contribute to the destruction of pancreatic beta cells in a hyperglycemia condition.

A systematic review and meta-analysis of murine models of uremic cardiomyopathy.

Chronic kidney disease (CKD) triggers the risk of developing uremic cardiomyopathy as characterized by cardiac hypertrophy, fibrosis and functional impairment. Traditionally, animal studies are used to reveal the underlying pathological mechanism, although variable CKD models, mouse strains and readouts may reveal diverse results. Here, we systematically reviewed 88 studies and performed meta-analyses of 52 to support finding suitable animal models for future experimental studies on pathological kidney-heart crosstalk during uremic cardiomyopathy. We compared different mouse strains and the direct effect of CKD on cardiac hypertrophy, fibrosis and cardiac function in "single hit" strategies as well as cardiac effects of kidney injury combined with additional cardiovascular risk factors in "multifactorial hit" strategies. In C57BL/6 mice, CKD was associated with a mild increase in cardiac hypertrophy and fibrosis and marginal systolic dysfunction. Studies revealed high variability in results, especially regarding hypertrophy and systolic function. Cardiac hypertrophy in CKD was more consistently observed in 129/Sv mice, which express two instead of one renin gene and more consistently develop increased blood pressure upon CKD induction. Overall, "multifactorial hit" models more consistently induced cardiac hypertrophy and fibrosis compared to "single hit" kidney injury models. Thus, genetic factors and additional cardiovascular risk factors can "prime" for susceptibility to organ damage, with increased blood pressure, cardiac hypertrophy and early cardiac fibrosis more consistently observed in 129/Sv compared to C57BL/6 strains.

Lipotoxicity and ß-Cell Failure in Type 2 Diabetes: Oxidative Stress Linked to NADPH Oxidase and ER Stress.

A high caloric intake, rich in saturated fats, greatly contributes to the development of obesity, which is the leading risk factor for type 2 diabetes (T2D). A persistent caloric surplus increases plasma levels of fatty acids (FAs), especially saturated ones, which were shown to negatively impact pancreatic ß-cell function and survival in a process called lipotoxicity. Lipotoxicity in ß-cells activates different stress pathways, culminating in ß-cells dysfunction and death. Among all stresses, endoplasmic reticulum (ER) stress and oxidative stress have been shown to be strongly correlated. One main source of oxidative stress in pancreatic ß-cells appears to be the reactive oxygen species producer NADPH oxidase (NOX) enzyme, which has a role in the glucose-stimulated insulin secretion and in the ß-cell demise during both T1 and T2D. In this review, we focus on the acute and chronic effects of FAs and the lipotoxicity-induced ß-cell failure during T2D development, with special emphasis on the oxidative stress induced by NOX, the ER stress, and the crosstalk between NOX and ER stress.

Loss of Mitochondrial Ca2+ Uniporter Limits Inotropic Reserve and Provides Trigger and Substrate for Arrhythmias in Barth Syndrome Cardiomyopathy.

BACKGROUND: Barth syndrome (BTHS) is caused by mutations of the gene encoding tafazzin, which catalyzes maturation of mitochondrial cardiolipin and often manifests with systolic dysfunction during early infancy. Beyond the first months of life, BTHS cardiomyopathy typically transitions to a phenotype of diastolic dysfunction with preserved ejection fraction, blunted contractile reserve during exercise, and arrhythmic vulnerability. Previous studies traced BTHS cardiomyopathy to mitochondrial formation of reactive oxygen species (ROS). Because mitochondrial function and ROS formation are regulated by excitation-contraction coupling, integrated analysis of mechano-energetic coupling is required to delineate the pathomechanisms of BTHS cardiomyopathy. METHODS: We analyzed cardiac function and structure in a mouse model with global knockdown of tafazzin (Taz-KD) compared with wild-type littermates. Respiratory chain assembly and function, ROS emission, and Ca2+ uptake were determined in isolated mitochondria. Excitation-contraction coupling was integrated with mitochondrial redox state, ROS, and Ca2+ uptake in isolated, unloaded or preloaded cardiac myocytes, and cardiac hemodynamics analyzed in vivo. RESULTS: Taz-KD mice develop heart failure with preserved ejection fraction (>50%) and age-dependent progression of diastolic dysfunction in the absence of fibrosis. Increased myofilament Ca2+ affinity and slowed cross-bridge cycling caused diastolic dysfunction, in part, compensated by accelerated diastolic Ca2+ decay through preactivated sarcoplasmic reticulum Ca2+-ATPase. Taz deficiency provoked heart-specific loss of mitochondrial Ca2+ uniporter protein that prevented Ca2+-induced activation of the Krebs cycle during ß-adrenergic stimulation, oxidizing pyridine nucleotides and triggering arrhythmias in cardiac myocytes. In vivo, Taz-KD mice displayed prolonged QRS duration as a substrate for arrhythmias, and a lack of inotropic response to ß-adrenergic stimulation. Cellular arrhythmias and QRS prolongation, but not the defective inotropic reserve, were restored by inhibiting Ca2+ export through the mitochondrial Na+/Ca2+ exchanger. All alterations occurred in the absence of excess mitochondrial ROS in vitro or in vivo. CONCLUSIONS: Downregulation of mitochondrial Ca2+ uniporter, increased myofilament Ca2+ affinity, and preactivated sarcoplasmic reticulum Ca2+-ATPase provoke mechano-energetic uncoupling that explains diastolic dysfunction and the lack of inotropic reserve in BTHS cardiomyopathy. Furthermore, defective mitochondrial Ca2+ uptake provides a trigger and a substrate for ventricular arrhythmias. These insights can guide the ongoing search for a cure of this orphaned disease.

Early Cytokine-Induced Transient NOX2 Activity Is ER Stress-Dependent and Impacts ß-Cell Function and Survival.

In type 1 diabetes (T1D) development, proinflammatory cytokines (PIC) released by immune cells lead to increased reactive oxygen species (ROS) production in ß-cells. Nonetheless, the temporality of the events triggered and the role of different ROS sources remain unclear. Isolated islets from C57BL/6J wild-type (WT), NOX1 KO and NOX2 KO mice were exposed to a PIC combination. We show that cytokines increase O2•- production after 2 h in WT and NOX1 KO but not in NOX2 KO islets. Using transgenic mice constitutively expressing a genetically encoded compartment specific H2O2 sensor, we show, for the first time, a transient increase of cytosolic/nuclear H2O2 in islet cells between 4 and 5 h during cytokine exposure. The H2O2 increase coincides with the intracellular NAD(P)H decrease and is absent in NOX2 KO islets. NOX2 KO confers better glucose tolerance and protects against cytokine-induced islet secretory dysfunction and death. However, NOX2 absence does not counteract the cytokine effects in ER Ca2+ depletion, Store-Operated Calcium Entry (SOCE) increase and ER stress. Instead, the activation of ER stress precedes H2O2 production. As early NOX2-driven ROS production impacts ß-cells' function and survival during insulitis, NOX2 might be a potential target for designing therapies against early ß-cell dysfunction in the context of T1D onset.

High Glucose Enhances Cytotoxic T Lymphocyte-Mediated Cytotoxicity.

Cytotoxic T lymphocytes (CTLs) are key players to eliminate tumorigenic or pathogen-infected cells using lytic granules (LG) and Fas ligand (FasL) pathways. Depletion of glucose leads to severely impaired cytotoxic function of CTLs. However, the impact of excessive glucose on CTL functions still remains largely unknown. Here we used primary human CD8+ T cells, which were stimulated by CD3/CD28 beads and cultured in medium either containing high glucose (HG, 25 mM) or normal glucose (NG, 5.6 mM). We found that in HG-CTLs, glucose uptake and glycolysis were enhanced, whereas proliferation remained unaltered. Furthermore, CTLs cultured in HG exhibited an enhanced CTL killing efficiency compared to their counterparts in NG. Unexpectedly, expression of cytotoxic proteins (perforin, granzyme A, granzyme B and FasL), LG release, cytokine/cytotoxic protein release and CTL migration remained unchanged in HG-cultured CTLs. Interestingly, additional extracellular Ca2+ diminished HG-enhanced CTL killing function. Our findings suggest that in an environment with excessive glucose, CTLs could eliminate target cells more efficiently, at least for a certain period of time, in a Ca2+-dependent manner.

Erythropoietin exposure of isolated pancreatic islets accelerates their revascularization after transplantation.

AIMS: The exposure of isolated pancreatic islets to pro-angiogenic factors prior to their transplantation represents a promising strategy to accelerate the revascularization of the grafts. It has been shown that erythropoietin (EPO), a glycoprotein regulating erythropoiesis, also induces angiogenesis. Therefore, we hypothesized that EPO exposure of isolated islets improves their posttransplant revascularization. METHODS: Flow cytometric, immunohistochemical and quantitative real-time (qRT)-PCR analyses were performed to study the effect of EPO on the viability, cellular composition and gene expression of isolated islets. Moreover, islets expressing a mitochondrial or cytosolic H2O2 sensor were used to determine reactive oxygen species (ROS) levels. The dorsal skinfold chamber model in combination with intravital fluorescence microscopy was used to analyze the revascularization of transplanted islets. RESULTS: We found that the exposure of isolated islets to EPO (3 units/mL) for 24 h does not affect the viability and the production of ROS when compared to vehicle-treated and freshly isolated islets. However, the exposure of islets to EPO increased the number of CD31-positive cells and enhanced the gene expression of insulin and vascular endothelial growth factor (VEGF)-A. The revascularization of the EPO-cultivated islets was accelerated within the initial phase after transplantation when compared to both controls. CONCLUSION: These findings indicate that the exposure of isolated islets to EPO may be a promising approach to improve clinical islet transplantation.

Interleukin-1α Is a Central Regulator of Leukocyte-Endothelial Adhesion in Myocardial Infarction and in Chronic Kidney Disease.

BACKGROUND: Cardiovascular diseases and chronic kidney disease (CKD) are highly prevalent, aggravate each other, and account for substantial mortality. Both conditions are characterized by activation of the innate immune system. The alarmin interleukin-1α (IL-1α) is expressed in a variety of cell types promoting (sterile) systemic inflammation. The aim of the present study was to examine the role of IL-1α in mediating inflammation in the setting of acute myocardial infarction (AMI) and CKD. METHODS: We assessed the expression of IL-1α on the surface of monocytes from patients with AMI and patients with CKD and determined its association with atherosclerotic cardiovascular disease events during follow-up in an explorative clinical study. Furthermore, we assessed the inflammatory effects of IL-1α in several organ injury models in Il1a-/- and Il1b-/- mice and investigated the underlying mechanisms in vitro in monocytes and endothelial cells. RESULTS: IL-1α is strongly expressed on the surface of monocytes from patients with AMI and CKD compared with healthy controls. Higher IL-1α surface expression on monocytes from patients with AMI and CKD was associated with a higher risk for atherosclerotic cardiovascular disease events, which underlines the clinical relevance of IL-1α. In mice, IL-1α, but not IL-1ß, mediates leukocyte-endothelial adhesion as determined by intravital microscopy. IL-1α promotes accumulation of macrophages and neutrophils in inflamed tissue in vivo. Furthermore, IL-1α on monocytes stimulates their homing at sites of vascular injury. A variety of stimuli such as free fatty acids or oxalate crystals induce IL-1α surface expression and release by monocytes, which then mediates their adhesion to the endothelium via IL-1 receptor-1. IL-1α also promotes expression of the VCAM-1 (vascular cell adhesion molecule-1) on endothelial cells, thereby fostering the adhesion of circulating leukocytes. IL-1α induces inflammatory injury after experimental AMI, and abrogation of IL-1α prevents the development of CKD in oxalate or adenine-fed mice. CONCLUSIONS: IL-1α represents a key mediator of leukocyte-endothelial adhesion and inflammation in AMI and CKD. Inhibition of IL-1α may serve as a novel anti-inflammatory treatment strategy.

Improvement of islet transplantation by the fusion of islet cells with functional blood vessels.

Pancreatic islet transplantation still represents a promising therapeutic strategy for curative treatment of type 1 diabetes mellitus. However, a limited number of organ donors and insufficient vascularization with islet engraftment failure restrict the successful transfer of this approach into clinical practice. To overcome these problems, we herein introduce a novel strategy for the generation of prevascularized islet organoids by the fusion of pancreatic islet cells with functional native microvessels. These insulin-secreting organoids exhibit a significantly higher angiogenic activity compared to freshly isolated islets, cultured islets, and non-prevascularized islet organoids. This is caused by paracrine signaling between the ß-cells and the microvessels, mediated by insulin binding to its corresponding receptor on endothelial cells. In vivo, the prevascularized islet organoids are rapidly blood-perfused after transplantation by the interconnection of their autochthonous microvasculature with surrounding blood vessels. As a consequence, a lower number of islet grafts are required to restore normoglycemia in diabetic mice. Thus, prevascularized islet organoids may be used to improve the success rates of clinical islet transplantation.

Transient NADPH oxidase 2-dependent H2O2 production drives early palmitate-induced lipotoxicity in pancreatic islets.

Modern lifestyles, including lack of physical activity and poor nutritional habits, are driving the rapidly increasing prevalence of obesity and type 2 diabetes. Increased levels of free fatty acids (FFAs), particularly saturated FFAs, in obese individuals have been linked to pancreatic ß-cell failure. This process, termed lipotoxicity, involves activation of several stress responses, including ER stress and oxidative stress. However, the molecular underpinnings and causal relationships between the disparate stress responses remain unclear. Here we employed transgenic mice, expressing a genetically-encoded cytosolic H2O2 sensor, roGFP2-Orp1, to monitor dynamic changes in H2O2 levels in pancreatic islets in response to chronic palmitate exposure. We identified a transient increase in H2O2 levels from 4 to 8 h after palmitate addition, which was mirrored by a concomitant decrease in cellular NAD(P)H levels. Intriguingly, islets isolated from NOX2 knock-out mice displayed no H2O2 transient upon chronic palmitate treatment. Furthermore, NOX2 knockout rescued palmitate-dependent impairment of insulin secretion, calcium homeostasis and viability. Chemical inhibition of NOX activity protected islets from palmitate-induced impairment in insulin secretion, however had no detectable impact upon the induction of ER stress. In summary, our results reveal that transient NOX2-dependent H2O2 production is a likely cause of early palmitate-dependent lipotoxic effects.

Evidence for NADPH oxidase activation by GPR40 in pancreatic ß-cells.

Objective: Investigate the involvement of the fatty acids receptor GPR40 in the assembly and activation of NADPH oxidase and the implications on pancreatic ß-cell function.Methods: BRIN-BD11 ß-cells were exposed to GPR40 agonist (GW9508) or linoleic acid in different glucose concentrations. Superoxide and H2O2 were analyzed, respectively, by DHE fluorescence and by fluorescence of the H2O2 sensor, roGFP2-Orp1. Protein contents of p47phox in plasma membrane and cytosol were analyzed by western blot. NADPH oxidase role was evaluated by p22phox siRNA or by pharmacological inhibition with VAS2870. NOX2 KO islets were used to measure total cytosolic calcium and insulin secretion.Results: GW9508 and linoleic acid increased superoxide and H2O2 contents at 5.6 and 8.3 mM of glucose. In addition, in 5.6 mM, but not at 16.7 mM of glucose, activation of GPR40 led to the translocation of p47phox to the plasma membrane. Knockdown of p22phox abolished the increase in superoxide after GW9508 and linoleic acid. No differences in insulin secretion were found between wild type and NOX2 KO islets treated with GW9508 or linoleic acid.Discussion: We report for the first time that acute activation of GPR40 leads to NADPH oxidase activation in pancreatic ß-cells, without impact on insulin secretion.

Chronic activation of GPR40 does not negatively impact upon BRIN-BD11 pancreatic ß-cell physiology and function.

BACKGROUND: Free fatty acids (FFAs) are known for their dual effects on insulin secretion and pancreatic ß-cell survival. Short-term exposure to FFAs, such as palmitate, increases insulin secretion. On the contrary, long-term exposure to saturated FFAs results in decreased insulin secretion, as well as triggering oxidative stress and endoplasmic reticulum (ER) stress, culminating in cell death. The effects of FFAs can be mediated either via their intracellular oxidation and consequent effects on cellular metabolism or via activation of the membrane receptor GPR40. Both pathways are likely to be activated upon both short- and long-term exposure to FFAs. However, the precise role of GPR40 in ß-cell physiology, especially upon chronic exposure to FFAs, remains unclear. METHODS: We used the GPR40 agonist (GW9508) and antagonist (GW1100) to investigate the impact of chronically modulating GPR40 activity on BRIN-BD11 pancreatic ß-cells physiology and function. RESULTS: We observed that chronic activation of GPR40 did not lead to increased apoptosis, and both proliferation and glucose-induced calcium entry were unchanged compared to control conditions. We also observed no increase in H2O2 or superoxide levels and no increase in the ER stress markers p-eIF2α, CHOP and BIP. As expected, palmitate led to increased H2O2 levels, decreased cell viability and proliferation, as well as decreased metabolism and calcium entry. These changes were not counteracted by the co-treatment of palmitate-exposed cells with the GPR40 antagonist GW1100. CONCLUSIONS: Chronic activation of GPR40 using GW9508 does not negatively impact upon BRIN-BD11 pancreatic ß-cells physiology and function. The GPR40 antagonist GW1100 does not protect against the deleterious effects of chronic palmitate exposure. We conclude that GPR40 is probably not involved in mediating the toxicity associated with chronic palmitate exposure.

Endothelial Notch signaling controls insulin transport in muscle.

The role of the endothelium is not just limited to acting as an inert barrier for facilitating blood transport. Endothelial cells (ECs), through expression of a repertoire of angiocrine molecules, regulate metabolic demands in an organ-specific manner. Insulin flux across the endothelium to muscle cells is a rate-limiting process influencing insulin-mediated lowering of blood glucose. Here, we demonstrate that Notch signaling in ECs regulates insulin transport to muscle. Notch signaling activity was higher in ECs isolated from obese mice compared to non-obese. Sustained Notch signaling in ECs lowered insulin sensitivity and increased blood glucose levels. On the contrary, EC-specific inhibition of Notch signaling increased insulin sensitivity and improved glucose tolerance and glucose uptake in muscle in a high-fat diet-induced insulin resistance model. This was associated with increased transcription of Cav1, Cav2, and Cavin1, higher number of caveolae in ECs, and insulin uptake rates, as well as increased microvessel density. These data imply that Notch signaling in the endothelium actively controls insulin sensitivity and glucose homeostasis and may therefore represent a therapeutic target for diabetes.

Nutrient Metabolism, Subcellular Redox State, and Oxidative Stress in Pancreatic Islets and ß-Cells.

Insulin-secreting pancreatic ß-cells play a critical role in blood glucose homeostasis and the development of type 2 diabetes (T2D) in the context of insulin resistance. Based on data obtained at the whole cell level using poorly specific chemical probes, reactive oxygen species (ROS) such as superoxide and hydrogen peroxide have been proposed to contribute to the stimulation of insulin secretion by nutrients (positive role) and to the alterations of cell survival and secretory function in T2D (negative role). This raised the controversial hypothesis that any attempt to decrease ß-cell oxidative stress and apoptosis in T2D would further impair insulin secretion. Over the last decade, the development of genetically-encoded redox probes that can be targeted to cellular compartments of interest and are specific of redox couples allowed the evaluation of short- and long-term effects of nutrients on ß-cell redox changes at the subcellular level. The data indicated that the nutrient regulation of ß-cell redox signaling and ROS toxicity is far more complex than previously thought and that the subcellular compartmentation of these processes cannot be neglected when evaluating the mechanisms of ROS production or the efficacy of antioxidant enzymes and antioxidant drugs under glucolipotoxic conditions and in T2D. In this review, we present what is currently known about the compartmentation of redox homeostatic systems and tools to investigate it. We then review data about the effects of nutrients on ß-cell subcellular redox state under normal conditions and in the context of T2D and discuss challenges and opportunities in the field.