Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD⁺Ratio

Abstract

Aims: Glucose-stimulated insulin secretion (GSIS) in pancreatic b cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria. Results: Unexpectedly, INS-1E cells at transitions from 3 (11 mM; pancreatic islets from 5 mM) to 25 mM glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H 2 O 2 (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADH F, indicated its *20% decrease. Matrix NAD + F increased on GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD + F. The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPH F (iNAP1 fluorescence monitoring) at the expense of matrix NADH F , was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. 13 Cincorporation from 13 C-L-glutamine into 13 C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPH F (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I I F site contribution, but a lower superoxide fraction ascribed to the Complex III site III Qo. Thus, the diminished matrix NADH F /NAD + F decreased Complex I flavin site I F superoxide formation on GSIS. Innovation: Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic b cells on GSIS, due to the decreasing matrix NADH F /NAD + F (NADPH F /NADP + F) at increasing cytosolic NADPH F levels. The developed innovative methods enable real-time NADH/NAD + and NADPH/ NADP + monitoring in any distinct cell compartment. Conclusion: The export of reducing equivalents from mitochondria adjusts lower mitochondrial superoxide production on GSIS, but it does not prevent oxidative stress in pancreatic b cells. Antioxid. Redox Signal. 33, 789-815.

The Pancreatic β-Cell: The Perfect Redox System

“…We also documented that the redox signaling upon GSIS is provided by elevations of cytosolic H 2 O 2 , whereas ROS in the mitochondrial matrix (both H 2 O 2 and superoxide release) are diminished due to the enhanced operation of the redox shuttles upon GSIS [ 37 ] (see Section 4 ). Since the silencing of glucose-6-phosphate (G6P) dehydrogenase (G6PDH) in INS-1E cells abolished approximately half of the GSIS rate [ 4 ], one may conclude that a portion of cytosolic NADPH as the substrate for NOX4 is provided by G6PDH and downstream 6-phosphogluconate dehydrogenase within the PPP, while the second portion is generated due to the operation of redox shuttles, i.e., due to NADPH increasingly produced by isocitrate dehydrogenate 1 (IDH1) and malic enzyme 1 (ME1) in the cytosol upon glucose intake [ 37 ]. The essential role of PPP was emphasized elsewhere [ 38 ].…”

“…Even without knowledge of NOX4 participation in GSIS, it has long been known that an increase in cytosolic NADPH facilitates insulin secretion responding to glucose [ 16 , 37 , 290 , 291 , 292 , 293 ]. The three major metabolic shuttles were revealed, which are activated at higher glucose and export reducing equivalents of NADH from the mitochondrial matrix to the cytosol during GSIS [ 290 ].…”

“…Upon a higher glucose intake, the forward (oxidative) mode is reversed to the counter-Krebs cycle direction. This reductive carboxylation mode requires CO 2 and enables the operation of the pyruvate/citrate cycle [ 37 ]. At the same time, the aconitase reaction and isocitrate formation is slow, thus allowing the reverse IDH2 reaction, despite the CO 2 requirement.…”

“…To our knowledge, the direct observation of the H + flux direction by NNT has never been attempted. Our preliminary data concerning the comparison of experimentally determined Δ Ψ m , using fluorescent indicators of membrane potential, showed instead increases in Δ Ψ m in NNT-silenced INS-1E cells [ 37 ]. This suggests that NNT acts in the forward mode, producing NADPH.…”

“…A major role has been suggested for the malate/aspartate shuttle in pancreatic β-cells [ 279 , 307 ]. However, one must recognize that the active malate/aspartate shuttle operation upon GSIS excludes the operation of the pyruvate/malate and pyruvate/isocitrate shuttles [ 37 ], the existence of which was documented by numerous experiments [ 16 , 275 , 276 , 277 , 278 ]. The malate/aspartate shuttle could not effectively function under high glucose conditions, since the 2-oxoglutarate carrier would have to translocate malate in the opposite direction to the malate export, which is active during the pyruvate/malate and pyruvate/isocitrate redox shuttles.…”