In this study, we investigated how colonic epithelial cells maintained pyridine nucleotide (NADH/NAD+) redox homeostasis upon acute metabolic variation imposed by glucose deprivation or supplementation with mitochondrial substrates, succinate and malate/glutamate (M/G). Our results showed that low glucose caused cellular NADH/NAD+ redox imbalance that diminished lactate dehydrogenase (LDH) activity and resulted in lower lactate contents. The concurrent activation of malic enzyme (ME) suggested a role for malate in preserving cellular pyruvate that remained unchanged at low glucose. Mitochondrial substrates restored cellular NADH/NAD+ redox homeostasis at low glucose in association with specific compartmental catabolism of mitochondrial substrates. As compared with normal glucose, M/G and low glucose promoted glycolytic ATP production but inhibited mitochondrial-derived ATP generation in association with decreased glucose availability for mitochondrial respiration. At normal glucose, succinate and M/G enhanced mitochondrial respiratory activity, but had minimal impact on mitochondrial-derived ATP production. Collectively, these results are consistent with low glucose-induced NADH/NAD+ redox imbalance in association with decreased aerobic glycolysis that is reversed by supplementation with M/G but not succinate.

Previously we have shown that the redox cycler menadione (MQ) induced cellular pyridine nucleotide redox imbalance that was linked to a decrease in aerobic glycolysis and perturbation of the mitochondrial respiratory activity due to the redox cycling of the compound; these processes were potentiated by low glucose. In this study, we investigated how colonic epithelial cells maintained pyridine nucleotide (NAD+/NADH and NADP+/NADPH) redox homeostasis upon acute metabolic variation and exposure to the redox cycling diquat (DQ). Our results show that DQ challenge disrupted cellular NADH/NAD+ redox status and enhanced cellular NADPH generation. Notably, DQ-induced NADH decrease was associated with enhanced lactate production, a process that was potentiated by glucose availability, but not by the mitochondrial substrates, succinate or malate/glutamate. In addition, DQ increased glucose 6-phoshate dehydrogenase (G6PDH) activity consistent with glucose diversion towards pentose phosphate pathway. As a consequence, steady-state NADPH levels were maintained during MQ challenge at normal glucose. In contrast and despite increased G6PDH and malic enzyme (ME) activities, DQ induced cellular NADPH-to-NADP+ shift at low glucose, a situation that was reversed by mitochondrial substrates. Collectively, these results are consistent with increased aerobic glycolysis by DQ and specific metabolic changes leading to enhanced NADPH generation upon oxidative challenge.

We previously demonstrated that in normal glucose (5 mM), methylglyoxal (MG, a model of carbonyl stress) induced brain microvascular endothelial cell (IHEC) dysfunction that was associated with occludin glycation and prevented by N-acetylcysteine (NAC). Herein, we investigated the impact of high glucose and low GSH, conditions that mimicked the diabetic state, on MG-induced IHEC dysfunction. MG-induced loss of transendothelial electrical resistance (TEER) was potentiated in IHECs cultured for 7 or 12 days in 25 mM glucose (hyperglycemia); moreover, barrier function remained disrupted 6 h after cell transfer to normal glucose media (acute glycemic fluctuation). Notably, basal occludin glycation was elevated under these glycemic states. TEER loss was exaggerated by inhibition of glutathione (GSH) synthesis and abrogated by NAC, which corresponded to GSH decreases and increases, respectively. Significantly, glyoxalase II activity was attenuated in hyperglycemic cells. Moreover, hyperglycemia and GSH inhibition increased MG accumulation, consistent with a compromised capacity for MG elimination. alpha-Oxoaldehydes (MG plus glyoxal) levels were elevated in streptozotocin-induced diabetic rat plasma. Immunohistochemistry revealed a prevalence of MG-positive, but fewer occludin-positive microvessels in the diabetic brain in vivo, and Western analysis confirmed an increase in MG-occludin adducts. These results provide the first evidence that hyperglycemia and acute glucose fluctuation promote MG-occludin formation and exacerbate brain microvascular endothelial dysfunction. Low occludin expression and high glycated-occludin contents in diabetic brain in vivo are factors that would contribute to the dysfunction of the cerebral microvasculature during diabetes. (C) 2015 The Authors. Published by Elsevier B. V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

We recently demonstrated that methylglyoxal (MG) induced apoptosis of brain microvascular endothelial cells (IHECs) that was preceded by glutathione (GSH) depletion. Here, we test the hypothesis that MG induces occludin glycation and disrupts IHEC barrier function, which is prevented by GSH-dependent MG metabolism. Exposure of IHECs to MG decreased transendothelial electrical resistance (TEER) in association with MG-adduct formation. A 65-kDa MG-glycated protein corresponded to occludin, which was confirmed by immunoprecipitation. Moreover, immunofluorescence staining showed that MG disrupted the architectural organization of ZO-1. Occludin glycation and ZO-1 disruption were prevented by N-acetylcysteine (NAC). Accordingly, TEER loss was abrogated by NAC (via GSH synthesis) and exacerbated by buthionine sulfoximine (BSO; GSH synthesis inhibitor). BSO treatment attenuated D-lactate production, consistent with a role for GSH in glyoxalase I-catalyzed MG elimination. Although MG increased reactive oxygen species (ROS) generation, the ROS scavengers tempol and tiron did not block barrier disruption. This suggests that endogenously generated ROS were unlikely to be a major cause of or did not reach a threshold to elicit barrier failure as elicited by exogenous hydrogen peroxide (300-400 μM). Immunohistochemistry revealed a lower percentage of microvessels stained with anti-occludin, but a higher percentage stained with anti-MG in diabetic rat brain compared to controls. Western analyses confirmed the decrease in diabetic brain occludin expression, but an increase in glycated occludin levels. These results provide novel evidence that reactive carbonyl species can mediate occludin glycation in cerebral microvessels and in microvascular endothelial cells that contribute to barrier dysfunction, a process that was prevented by GSH through enhanced MG catabolism.

We recently demonstrated that menadione (MQ), a redox cycling quinone, mediated the loss of mitochondrial glutathione/glutathione disulfide redox balance. In this study, we showed that MQ significantly disrupted cellular pyridine nucleotide (NAD(+)/NADH, NADP(+)/NADPH) redox balance that compromised cellular ATP, mitochondrial respiratory activity, and NADPH-dependent reducing capacity in colonic epithelial cells, a scenario that was exaggerated by low glucose. In the cytosol, MQ induced NAD(+) loss concurrent with increased NADP(+) and NAD kinase activity, but decreased NADPH. In the mitochondria, NADH loss occurred in conjunction with increased nicotinamide nucleotide transhydrogenase activity and NADP(+), and decreased NADPH. These results are consistent with cytosolic NAD(+)-to-NADP(+) and mitochondrial NADH-to-NADPH shifts, but compromised NADPH availability. Thus, despite the sacrifice of NAD(+)/NADH in favor of NADPH generation, steady-state NADPH levels were not maintained during MQ challenge. Impairments of cellular bioenergetics were evidenced by ATP losses and increased mitochondrial O(2) dependence of pyridine nucleotide oxidation-reduction; half-maximal oxidation (P(50)) was 10-fold higher in low glucose, which was lowered by glutamate or succinate supplementation. This exaggerated O(2) dependence is consistent with increased O(2) diversion to nonmitochondrial O(2) consumption by MQ-semiquinone redox cycling secondary to decreased NADPH-dependent MQ detoxication at low glucose, a situation that was corrected by glucose-sparing mitochondrial substrates.