Figure 11.

Substrate and inhibitor effects on the NADH/NAD redox state. Octanoate is metabolized in mitochondria by β-oxidation, generating NADH and FADH, which are oxidized in the electron transport chain, and the final end products: Acac and HOB. The ratio of HOB/Acac reflects the mitochondrial NADH/NAD redox state. It is increased by high metformin and rotenone (Complex I inhibitor) and decreased by low metformin and uncoupler, DNP (Fig. 1). It is increased by overexpression of mGPDH, which catalyzes oxidation of G3P with transfer of electrons to the respiratory chain (Fig. 6). DHA is metabolized to either glucose or pyruvate. The latter results in production of NADH (at GAPDH), which is coupled to either formation of lactate by lactate dehydrogenase or malate, which is oxidized in mitochondria by the malate–aspartate shuttle (MAS) (Figs. 3 and 7). Xylitol metabolism generates NADH during oxidation to xylulose and at GAPDH during formation of pyruvate (Figs. 3 and 8). Glycerol metabolism generates G3P, which is converted to DHAP (dihydroxyacetone phosphate) by either mGPDH, with transfer of electrons to the respiratory chain, or by cGPDH (cytoplasmic glycerol-3-phosphate dehydrogenase), generating NADH in the cytoplasm, which reoxidized via the MA-shuttle (Fig. 8). AOA inhibits the MA-shuttle at the transaminase reaction, increases the lactate/pyruvate ratio and G3P, and inhibits total xylitol metabolism (Figs. 3, 7, and 8). The MA-shuttle is also inhibited at the aspartate transport step by mitochondrial depolarization (↓ψ). GPi (80 μm, mGPDH inhibitor) increases G3P (Fig. 5). mGPDH overexpression lowers G3P, increases total glycerol metabolism, and favors DHA metabolism to pyruvate and lactate relative to glucose (Figs. 6–8). Octanoate, ATA (PFK1 inhibitor), and fructose 2,6-P₂ depletion promote DHA metabolism to glucose relative to pyruvate and lactate (Figs. 9 and 10). Metformin (100 μm or <2 nmol/mg of cell protein) promotes decreased DHA metabolism to glucose relative to pyruvate plus lactate and moderately increases the lactate/pyruvate ratio and decreases cell G3P (Figs. 3, 5, 9, and 10). It lowers G6P in conditions of restrained G6P entry into the endoplasmic reticulum with a transport inhibitor, S4048 (Fig. 10).