Mitochondria-to-plasma-membrane H2O2 Signaling Induced by Branched-Chain Keto Acid Oxidation Essentially Stimulates Insulin Secretion in Pancreatic Β-Cells.

Oxidation (β-like) of branched-chain keto acids (BCKAs) α-ketoisocaproate (KIC), α-ketoisovalerate (KIV), and α-ketomethylvalerate (KMV), yields FADH2, NADH (in order of KIC < KMV < KIV), and acetyl-CoA (KIC, KMV) or succinyl-CoA (KIV, KMV). Here, we examined whether BCKA-oxidation contributes to H2O2-dependent redox signaling, studied mechanism(s) of its generation, and investigated whether such H2O2 signal is required for BCKA-stimulated insulin secretion (BCKA-SIS) in pancreatic β-cells and islets. Using Amplex UltraRed, we detected BCKA-induced H2O2 release to the exterior of INS-1E cells and pancreatic islets (PIs) upon BCKA-SIS. This H2O2 signal determined closure of ATP-sensitive K+ channels and enabled Ca2+oscillations. It was inhibited by the mitochondrial antioxidant SkQ1; partially by S1QEL, S3QEL (Complex I, III) superoxide-suppressors and by 80-90% after silencing of electron-transfer flavoprotein (ETF) ubiquinone (Q) oxidoreductase (ETFQOR). The H2O2 (redox) signal is generated i) due to the excessive ETFQOR QH2 input, which retards respiratory chain electron transport, providing surplus superoxide at Complex I site IQ (i.e., reversed electron transfer, representing an effective product inhibition of the Complex I QH2 output) and ii) due to the excessive incoming QH2 to the Complex III site IIIQo (minimum for KIV). 13C-incorporation from U-13C-KIC/KIV into various metabolites confirmed β-like oxidation and characterized auxiliary reactions. A causal dependence of BCKA-SIS in PIs on H2O2 generation was found at both phases, evidenced by non-constant correlations of insulin release vs. H2O2 release rates, similarly to glucose-stimulated insulin secretion. Thus, BCKA-stimulated insulin secretion requires coordinated peri-plasma-membrane elevations of ATP/ADP and H2O2, both arising from mitochondrial BCKA β-like oxidation.