Nicotinamide nucleotide transhydrogenase and the glucose stimulation of insulin secretion

Nicotinamide nucleotide transhydrogenase (NNT) couples proton influx to NADPH production in the mitochondrial matrix. Its spontaneous inactivating truncation in C57Bl/6J mice reduces glucose-stimulated insulin secretion (GSIS), but the mechanisms involved are not clear. Here, we studied the role of NNT in β cell stimulus-secretion coupling by comparing islets from female C57Bl/6J mice (J) with truncated NNT to islets from genetically close C57Bl/6N mice (N) with wildtype NNT. Islets were cultured up to 4 days in RPMI and incubated for 30-60 min or perifused at 0.5, 15 and 30 mM glucose in Krebs containing 4.8 or 30 mM K+ (K4.8/K30). At K4.8, glucose acutely increased NADH/NAD+, mitochondrial membrane potential, ATP/ADP and cytosolic Ca2+ to the same extent in J and N islets. In contrast, an acute increase in NADPH/NADP+ and reduction in mitochondrial glutathione redox potential (mt-EGSH) were observed in N but not J islets, unless the latter were infected 2-4 days earlier with an adenovirus coding NNT. GSIS (expressed per ng of islet DNA) was ~66% lower in J vs. N islets, despite their similar insulin content. Insulin secretion was also ~66% lower in J vs. N islets incubated at K30 despite similar rises in cytosolic Ca2+, but the amplifying action of glucose on Ca2+-induced secretion was similar in both types of islets. Again, adenovirus-mediated expression of NNT in J islets restored GSIS and K30-induced secretion in proportion to the percentage of infected β cells (~25%). Finally, addition of 10 μM acetylcholine (ACh), but not 20 nM exenatide, much better amplified GSIS in J than N islets, and thereby reduced their relative defect in insulin secretion. In conclusion, the spontaneous inactivating truncation of NNT in C57Bl/6J mice is responsible for a lack of glucose-induced changes in NADPH/NADP+ and mt-EGSH in their pancreatic islets. It does not alter the triggering pathway of GSIS but markedly reduces the efficacy of Ca2+ on exocytosis by a mechanism that is largely corrected by ACh

Lack Of NADPH Oxidase-2 Does Not Improve Pancreatic β-cell Survival And Function In Male C57BL/6J Mouse Islets Cultured For 3 Weeks At High Glucose

High glucose-induced oxidative stress may contribute to the progressive loss of functional β-cell mass in type 2 diabetes. To test whether superoxide production by NADPH oxidase-2 (NOX2) is involved in β-cell dysfunction and apoptosis under glucotoxic conditions, we characterized the acute and long-term effects of glucose on cytosolic glutathione/thiol oxidation (GRX1-roGFP2 and roGFP1 redox-sensitive probes), H2O2 concentration (roGFP2-Orp1 H2O2-sensitive probe), β-cell stimulus-secretion coupling events and apoptosis in islets from male NOX2 knockout (NOX2-KO) and wild-type (WT) C57BL/6J mice cultured for up to 3 weeks in RPMI medium containing 10 or 30 mmol/L glucose (G10 or G30). After 1-2 days of culture at G10, the acute glucose stimulation of insulin secretion (GSIS) was ~1.7-fold higher in NOX2-KO vs. WT islets despite similar rises in NAD(P)H autofluorescence and intracellular calcium ([Ca2+]i) and no differences in cytosolic glutathione oxidation. After long-term culture at G10, cytosolic thiol oxidation, H2O2 concentration and β-cell apoptosis remained low in both islet types. Also, the glucose-induced rises in NAD(P)H autofluorescence, [Ca2+]i and GSIS were similar to the results obtained after 1-2 days of culture, except for a progressive reduction of the difference in GSIS between NOX2-KO and WT islets. After prolonged culture at G30, cytosolic thiol oxidation and H2O2 concentration increased in parallel with β-cell apoptosis, the glucose sensitivity of the NAD(P)H, [Ca2+]i and insulin secretion responses increased, the maximal [Ca2+]i response decreased, but maximal GSIS (at G15 and G30) was preserved. These responses were almost identical in both islet types. In conclusion, NOX2 is confirmed as a negative regulator of GSIS in C57BL/6J mouse islets, but it does not detectably contribute to the long-term glucotoxic alterations of β-cell function and survival in vitro

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

Glucolipotoxic conditions induce beta-cell iron import, cytosolic ROS formation and apoptosis

Type 2 diabetes (T2D) arises when the pancreatic beta-cell fails to compensate for increased insulin needs due to insulin resistance. Glucolipotoxicity has been proposed to induce beta-cell dysfunction in T2D by formation of reactive oxygen species (ROS). Here we examined if modelling glucolipotoxic conditions by high glucose-high free fatty acid (FFA) exposure (GLT) regulate beta-cell iron transport, thereby increasing the cytosolic labile iron pool and iron-catalyzed ROS formation. We show that GLT -induced ROS production is regulated by an increased labile iron pool (LIP) associated with elevated expression of genes regulating iron import. Using pharmacological and transgenic approaches, we show that iron reduction and decreased iron import protects from GLT-induced ROS production, prevents impairment of the mitochondrial membrane potential, and inhibits apoptosis. This study identifies a novel pathway underlying GLT-induced apoptosis involving increased iron import, generation of hydroxyl radicals from hydrogen peroxide through the Fenton reaction in the cytosolic compartment associated with dissipation of the mitochondrial membrane potential and beta cell apoptosis

Cytosolic ROS Formation Promoted by Metabolic Stress-induced Iron Transport Causes Secondary Beta-Cell Mitochondrial Dysfunction and Apoptosis

Metabolic stress has been proposed to induce β cell-damage by iron-catalyzed formation of reactive oxygen species (ROS). We have shown that inducible β cell-specific knock-out (KO) of divalent metal transporter 1(DMT1) protects against high-fat diet (HFD) induced diabetes (Cell Metab 2012), but the molecular mechanisms are unknown. Here we show that glucolipotoxic conditions (GLT) doubled DMT1 and transferrin receptor, but not ferritin heavy/light chain, ferroportin, pancreatic duodenum homeobox 1 or insulin 1 mRNA expression in MIN6 cells, correlating with a two-fold increase in the the labile iron pool (LIP) in MIN6 cells, mouse and human islets, and in islets from mice with HFD induced obesity. Iron chelation reduced LIP, apoptosis, cytosolic ROS, IL-6 and monocyte chemotactic factor expression in GLT-exposed mouse islets, and alleviated FFA-mediated inhibition in glucose- and KCl-induced mouse and human islet insulin secretion. DMT-1 KO prevented GLT induced increases in islet LIP, ROS and apoptosis, associated with improved mitochondrial membrane potential, and alleviated GLT induced inhibition of insulin secretion from perifused DMT-1KO islets. This study identifies a novel pathway underlying metabolic stress-induced apoptosis involving increased iron import and ROS formation in the cytosolic compartment causing dissipation of the mitochondrial membrane potential. We conclude that iron transport via DMT-1 is a novel central mechanism relaying metabolic stress with ROS production, cell dysfunction and death. Taken together with the accumulating epidemiological evidence suggesting that iron overload leads to diabetes and that patients with diabetes have increased tissue iron accumulation, our findings highlight control of iron levels as an attractive novel treatment option for the prevention of diabetes. Clinical trials are warranted to validate this translation of our preclinical findings