Glucose-Stimulated Insulin Secretion Fundamentally Requires H2O2 Signaling by NADPH Oxidase 4

Abstract

NADPH facilitates glucose-stimulated insulin secretion (GSIS) in pancreatic islets (PIs) of β-cells through an as yet unknown mechanism. We found NADPH oxidase isoform 4 (NOX4) to be the main producer of cytosolic H2O2, which is essential for GSIS; an increase in ATP alone was insufficient for GSIS. The fast GSIS phase was absent from PIs from NOX4-null, β-cell–specific knockout mice (NOX4βKO) (though not from NOX2 knockout mice) and from NOX4-silenced or catalase-overexpressing INS-1E cells. Lentiviral NOX4 overexpression or H2O2 rescued GSIS in PIs from NOX4βKO mice. NOX4 silencing suppressed Ca2+ oscillations, and the patch-clamped KATP channel opened more frequently when glucose was high. Mitochondrial H2O2, decreasing upon GSIS, provided alternative redox signaling when 2-oxo-isocaproate or fatty acid oxidation formed superoxides through electron-transfer flavoprotein:Q-oxidoreductase. Unlike GSIS, such insulin secretion was blocked with mitochondrial antioxidant SkQ1. Both NOX4 knockout and NOX4βKO mice exhibited impaired glucose tolerance and peripheral insulin resistance. Thus, the redox signaling previously suggested to cause β-cells to self-check hypothetically induces insulin resistance when it is absent. In conclusion, increases in ATP and H2O2 constitute an essential signal that switches on insulin exocytosis for glucose and branched-chain oxoacids as secretagogues (it does so partially for fatty acids). Redox signaling could be impaired by cytosolic antioxidants; hence, those targeting mitochondria should be preferred for clinical applications to treat (pre)diabetes at any stage.

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

“…Glucose sensing was considered to be exclusively coupled with insulin exocytosis by elevated ATP, stemming from augmented glucose metabolism and oxidative phosphorylation (OXPHOS). However, recently we revealed that, in addition to ATP, also a parallel redox signaling from NADPH oxidase 4 (NOX4) is essential for GSIS (48). In b cells, the insulin-independent glucose transporter GLUT2 (in rodents) allows cell glucose equilibration with plasma glucose levels.…”

“…However, we demonstrated that a parallel H 2 O 2 burst is essentially required to close the K ATP channel, together with ATP. Any single component (sole ATP or sole H 2 O 2 ) is not sufficient to stimulate insulin secretion (48). The resulting Ca 2+ influx elevates cytosolic Ca 2+ concentration and stimulates Ca 2+ -dependent exocytosis of insulin-containing granules.…”

“…The resulting H 2 O 2 may also serve as a signaling molecule (50). Indeed, insulin release is also stimulated due to the elevated superoxide formation by b-like oxidation of branched-chain ketoacids in mitochondria, wherein superoxide is converted to ''signaling'' H 2 O 2 , diffusing up to the K ATP channel to aid its closure (48). However, whether such matrix redox burst exists in glucose metabolism is not yet resolved.…”

“…The resulting elevated cytosolic NADPH has been reported to facilitate GSIS (52). Oxidative glucose metabolism leads to cytosolic ATP plus H 2 O 2 elevation (48), thus initiating K ATP channel-dependent GSIS (2,52,55). Glutamate dehydrogenase is depicted as producing glutamate (44); NNT is depicted to allow both reverse and forward mode (58).…”

“…that adds to the production of pentose-phosphate pathway (PPP) supplying NOX4 (48). For each molecule of malate or citrate/isocitrate exported from the mitochondrial matrix by the respective shuttle, one molecule of NADH should be missing for Complex I, since MDH or isocitrate dehydrogenase 3 (IDH3) (otherwise producing NADH) cannot use their substrates.…”