Glucose acutely reduces cytosolic and mitochondrial H2O2 in rat pancreatic beta-cells.

Aims: Whether H2O2 contributes to the glucose-dependent stimulation of insulin secretion by pancreatic β-cells is highly controversial. We used two H2O2-sensitive probes, roGFP2-Orp1 and HyPer with its pH-control SypHer, to test the acute effects of glucose, monomethylsuccinate, leucine with glutamine, and α-ketoisocaproate, on β-cell cytosolic and mitochondrial H2O2 concentrations. We then tested the effects of low H2O2 and menadione concentrations on insulin secretion. Results: RoGFP2-Orp1 was more sensitive than HyPer to H2O2 (response at 2-5 vs. 10µM) and less pH-sensitive. Under control conditions, stimulation with glucose reduced mitochondrial roGFP2-Orp1 oxidation without affecting cytosolic roGFP2-Orp1 and HyPer fluorescence ratios, except for the pH-dependent effects on HyPer. However, stimulation with glucose decreased the oxidation of both cytosolic probes by 15µM exogenous H2O2. The glucose effects were not affected by overexpression of catalase, mitochondrial catalase or superoxide dismutase 1 and 2. They followed the increase in NAD(P)H autofluorescence, were maximal at 5mM glucose in the cytosol and 10mM glucose in the mitochondria, and were partly mimicked by the other nutrients. Exogenous H2O2 (1-15µM) did not affect insulin secretion. By contrast, menadione (1-5µM) did not increase basal insulin secretion but reduced the stimulation of insulin secretion by 20mM glucose. Innovation: Subcellular changes in β-cell H2O2 levels are better monitored with roGFP2-Orp1 than HyPer/SypHer. Nutrients acutely lower mitochondrial H2O2 levels in β-cells and promote degradation of exogenously supplied H2O2 in both cytosolic and mitochondrial compartments. Conclusion: The glucose-dependent stimulation of insulin secretion occurs independently of a detectable increase in β-cell cytosolic or mitochondrial H2O2 levels

Biomarkers of tumor redox status in response to modulations of glutathione and thioredoxin antioxidant pathways.

The ability of certain cancer cells to maintain a highly reduced intracellular environment is correlated with aggressiveness and drug resistance. Since the gluthathione (GSH) and thioredoxin (TRX) systems cooperate to a tight regulation of ROS in cell physiology, and to a stimulation of tumor initiation and progression, modulation of the GSH and TRX pathways are emerging as new potential targets in cancer. In vivo methods to assess changes in tumor redox status are critically needed to assess the relevance of redox-targeted agents. The current study assesses in vitro and in vivo biomarkers of tumor redox status in response to treatments targeting the GSH and TRX pathways, by comparing cytosolic and mitochondrial redox nitroxide Electron Paramagnetic Resonance (EPR) probes, and cross-validation with redox dynamic fluorescent measurement. For that purpose, the effect of the GSH modulator buthionine sulfoximine (BSO) and of the TRX reductase inhibitor auranofin were measured in vitro using both cytosolic and mitochondrial EPR and roGFP probes in breast and cervical cancer cells. In vivo, mice bearing breast or cervical cancer xenografts were treated with the GSH or TRX modulators and monitored using the mito-TEMPO spin probe. Our data highlight the importance of using mitochondria targeted spin probes to assess changes in tumor redox status induced by redox modulators. Further in vivo validation of the mito-tempo spin probe with alternative in vivo methods should be considered, yet the spin probe used in vivo in xenografts demonstrated sensitivity to the redox status modulators

Identification d’une altération glucotoxique de la fonction des cellules β pancréatiques unique à l’espèce humaine

Introduction La culture prolongée d’îlots pancréatiques de rongeurs en présence de concentrations élevées de glucose s’accompagne d’une augmentation de l’apoptose des cellules β et d’altérations majeures de la sécrétion d’insuline en réponse au glucose (SSIG) avec abaissement du seuil de stimulation et diminution de la SSIG maximale. Le but de cette étude était de caractériser les altérations glucotoxiques de la survie et de la fonction des cellules β dans des îlots humains en culture. Matériels et Méthodes Les îlots ont été isolés du pancréas de 14 donneurs non-diabétiques entre 2011 et 2016. Après une à deux semaines de préculture en milieu CMRL et/ou RPMI complet contenant 5mM de glucose et 10% de sérum de veau fœtal, les îlots ont été cultivés 7 jours en RPMI contenant 5, 8 ou 20mM glucose (G5, G8, G20). L’apoptose des cellules β a été mesurée sur coupes d’îlots (TUNEL/insuline double positivité). La fonction des cellules β a été évaluée en mesurant la concentration cytosolique de calcium ([Ca2+]c)(fura2-LR), et la sécrétion d’insuline lors d’une stimulation aiguë par des paliers de glucose (îlots périfusés à 0,5-2-5-10-30mM glucose). La sécrétion et le contenu en insuline ont été mesurés par dosage radio-immunologique et rapportés au contenu en ADN des îlots. Résultats Caractéristiques des donneurs: 11 hommes/3 femmes ; âge 17-74 (médiane 61) ans; indice de masse corporelle 21-33 (médiane 27,7) kg/m². Pendant la culture d'une semaine, la sécrétion était faible en G5 (~20 pg/îlot par heure) et stimulée 25x en G8 et 35x en G20. Après la culture, la proportion de cellules β dans les îlots (~70%), le pourcentage de cellules β apoptotiques (<1%), et le contenu en ADN des îlots (~250ng/îlot) étaient semblables dans les trois groupes, mais le rapport insuline/ADN des îlots était significativement réduit par la culture en G20 (103-72-21 pg insuline/ng ADN après culture en G5-G8-G20, respectivement). Du point de vue fonctionnel, les îlots cultivés en G5 présentaient une augmentation de la [Ca2+]c et de la sécrétion d’insuline en réponse à une stimulation par 10 et 30mM glucose. Après culture en G8, la stimulation survenait déjà au palier de 5mM glucose et la SSIG maximale aux paliers de 10 et 30mM glucose était plus élevée qu’après culture en G5. Après culture en G20, les îlots n’étaient que faiblement stimulés, et ce de façon maximale, à 5 et 10mM glucose, puis étaient inhibés de façon réversible lors d’une stimulation par 30mM glucose. Cette inhibition paradoxale n’est pas observée dans les îlots de rongeurs après culture en conditions glucotoxiques. Conclusions La culture d'îlots humains pendant une semaine à 8 au lieu de 5mM glucose augmente la sensibilité et l’amplitude de la réponse des cellules β au glucose. La culture d'une semaine à 20 mM glucose n’augmente pas l’apoptose des cellules β humaines mais réduit de 80% leur contenu en insuline et conduit à une inhibition paradoxale de la sécrétion d’insuline audessus de 10mM glucose unique à l'espèce humaine. Ces altérations fonctionnelles pourraient jouer un rôle dans l’adaptation puis la décompensation de la sécrétion d’insuline lors de l’intolérance au glucose et au début du diabète de type 2

Pancreatic beta-cell tRNA hypomethylation and fragmentation link TRMT10A deficiency with diabetes

Transfer RNAs (tRNAs) are non-coding RNA molecules essential for protein synthesis. Post-transcriptionally they are heavily modified to improve their function, folding and stability. Intronic polymorphisms in CDKAL1, a tRNA methylthiotransferase, are associated with increased type 2 diabetes risk. Loss-of-function mutations in TRMT10A, a tRNA methyltransferase, are a monogenic cause of early onset diabetes and microcephaly. Here we confirm the role of TRMT10A as a guanosine 9 tRNA methyltransferase, and identify tRNA(Gln) and tRNA(iMeth) as two of its targets. Using RNA interference and induced pluripotent stem cell-derived pancreatic beta-like cells from healthy controls and TRMT10A-deficient patients we demonstrate that TRMT10A deficiency induces oxidative stress and triggers the intrinsic pathway of apoptosis in beta-cells. We show that tRNA guanosine 9 hypomethylation leads to tRNA(Gln) fragmentation and that 5'-tRNA(Gln) fragments mediate TRMT10A deficiency-induced beta-cell death. This study unmasks tRNA hypomethylation and fragmentation as a hitherto unknown mechanism of pancre-atic beta-cell demise relevant to monogenic and polygenic forms of diabetes.Peer reviewe

Prolonged culture of human pancreatic islets under glucotoxic conditions changes their acute beta cell calcium and insulin secretion glucose response curves from sigmoid to bell-shaped

Aims/hypothesis: The rapid remission of type 2 diabetes by a diet very low in energy correlates with a marked improvement in glucose-stimulated insulin secretion (GSIS), emphasising the role of beta cell dysfunction in the early stages of the disease. In search of novel mechanisms of beta cell dysfunction after long-term exposure to mild to severe glucotoxic conditions, we extensively characterised the alterations in insulin secretion and upstream coupling events in human islets cultured for 1–3 weeks at ~5, 8, 10 or 20 mmol/l glucose and subsequently stimulated by an acute stepwise increase in glucose concentration. Methods: Human islets from 49 non-diabetic donors (ND-islets) and six type 2 diabetic donors (T2D-islets) were obtained from five isolation centres. After shipment, the islets were precultured for 3–7 days in RPMI medium containing ~5 mmol/l glucose and 10% (vol/vol) heat-inactivated FBS with selective islet picking at each medium renewal. Islets were then cultured for 1–3 weeks in RPMI containing ~5, 8, 10 or 20 mmol/l glucose before measurement of insulin secretion during culture, islet insulin and DNA content, beta cell apoptosis and cytosolic and mitochondrial glutathione redox state, and assessment of dynamic insulin secretion and upstream coupling events during acute stepwise stimulation with glucose [NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)]. Results: Culture of ND-islets for 1–3 weeks at 8, 10 or 20 vs 5 mmol/l glucose did not significantly increase beta cell apoptosis or oxidative stress but decreased insulin content in a concentration-dependent manner and increased beta cell sensitivity to subsequent acute stimulation with glucose. Islet glucose responsiveness was higher after culture at 8 or 10 vs 5 mmol/l glucose and markedly reduced after culture at 20 vs 5 mmol/l glucose. In addition, the [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were no longer sigmoid but bell-shaped, with maximal stimulation at 5 or 10 mmol/l glucose and rapid sustained inhibition above that concentration. Such paradoxical inhibition was, however, no longer observed when islets were acutely depolarised by 30 mmol/l extracellular K+. The glucotoxic alterations of beta cell function were fully reversible after culture at 5 mmol/l glucose and were mimicked by pharmacological activation of glucokinase during culture at 5 mmol/l glucose. Similar results to those seen in ND-islets were obtained in T2D-islets, except that their rate of insulin secretion during culture at 8 and 20 mmol/l glucose was lower, their cytosolic glutathione oxidation increased after culture at 8 and 20 mmol/l glucose, and the alterations in GSIS and upstream coupling events were greater after culture at 8 mmol/l glucose. Conclusions/interpretation: Prolonged culture of human islets under moderate to severe glucotoxic conditions markedly increased their glucose sensitivity and revealed a bell-shaped acute glucose response curve for changes in [Ca2+]c and insulin secretion, with maximal stimulation at 5 or 10 mmol/l glucose and rapid inhibition above that concentration. This novel glucotoxic alteration may contribute to beta cell dysfunction in type 2 diabetes independently from a detectable increase in beta cell apoptosis

Inhibition of aquaporin-1 prevents myocardial remodeling by blocking the transmembrane transport of hydrogen peroxide.

Pathological remodeling of the myocardium has long been known to involve oxidant signaling, but strategies using systemic antioxidants have generally failed to prevent it. We sought to identify key regulators of oxidant-mediated cardiac hypertrophy amenable to targeted pharmacological therapy. Specific isoforms of the aquaporin water channels have been implicated in oxidant sensing, but their role in heart muscle is unknown. RNA sequencing from human cardiac myocytes revealed that the archetypal is a major isoform. expression correlates with the severity of hypertrophic remodeling in patients with aortic stenosis. The AQP1 channel was detected at the plasma membrane of human and mouse cardiac myocytes from hypertrophic hearts, where it colocalized with NADPH oxidase-2 and caveolin-3. We show that hydrogen peroxide (HO), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of HO through its water pore, resulting in activation of oxidant-sensitive kinases in cardiac myocytes. Structural analysis of the amino acid residues lining the water pore of AQP1 supports its permeation by HO Deletion of or selective blockade of the AQP1 intrasubunit pore inhibited HO transport in mouse and human cells and rescued the myocyte hypertrophy in human induced pluripotent stem cell-derived engineered heart muscle. Treatment of mice with a clinically approved AQP1 inhibitor, Bacopaside, attenuated cardiac hypertrophy. We conclude that cardiac hypertrophy is mediated by the transmembrane transport of HO by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies

Inhibition of aquaporin-1 prevents myocardial remodeling by blocking the transmembrane transport of hydrogen peroxide

Pathological remodeling of the myocardium has long been known to involve oxidant signaling, but strategies using systemic antioxidants have generally failed to prevent it. We sought to identify key regulators of oxidant-mediated cardiac hypertrophy amenable to targeted pharmacological therapy. Specific isoforms of the aquaporin water channels have been implicated in oxidant sensing, but their role in heart muscle is unknown. RNA sequencing from human cardiac myocytes revealed that the archetypal AQP1 is a major isoform. AQP1 expression correlates with the severity of hypertrophic remodeling in patients with aortic stenosis. The AQP1 channel was detected at the plasma membrane of human and mouse cardiac myocytes from hypertrophic hearts, where it colocalized with NADPH oxidase-2 and caveolin-3. We show that hydrogen peroxide (H2O2), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of H2O2 through its water pore, resulting in activation of oxidant-sensitive kinases in cardiac myocytes. Structural analysis of the amino acid residues lining the water pore of AQP1 supports its permeation by H2O2 Deletion of Aqp1 or selective blockade of the AQP1 intrasubunit pore inhibited H2O2 transport in mouse and human cells and rescued the myocyte hypertrophy in human induced pluripotent stem cell-derived engineered heart muscle. Treatment of mice with a clinically approved AQP1 inhibitor, Bacopaside, attenuated cardiac hypertrophy. We conclude that cardiac hypertrophy is mediated by the transmembrane transport of H2O2 by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies.status: publishe