Distinct mitochondrial membrane potential, ATP-to-ADP ratio and NAD(P)H responses to glucose and methylsuccinate. Distinct mitochondrial membrane potential,

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Distinct mitochondrial membrane potential, ATP-to-ADP ratio and NAD(P)H responses to glucose and methylsuccinate. Distinct mitochondrial membrane potential, ATP-to-ADP ratio and NAD(P)H responses to glucose and methylsuccinate. (A) The mitochondrial membrane potential in response to 16.7 mM glucose (black circles) or 5 mM methylsuccinate (gray circles) was measured in INS-1E cells by using the lipophilic cation TMRM. Changes in Δψm are expressed as the ratio of the fluorescence in mitochondria divided by the cytosolic fluorescence (Fmito/Fcyto) measured in the same cells. At the end of the recording the protonophore FCCP (2 µM) was used to dissipate Δψm. The effect of the substrates on Δψm was measured on normalized responses, as a fraction of basal (set to 1) and after FCCP depolarization (set to 0). Data shown are the mean±s.e.m. from five (49 cells) and four (39 cells) independent experiments for glucose- and methylsuccinate-stimulated cells, respectively. (B) Glucose- and methylsuccinate-dependent ATP-to-ADP ratio responses in INS-1E cells. INS-1E cells were incubated for 20 min in standard KRBH at either resting (2.5 mM, white bars) or stimulatory (16.7 mM, black bars) glucose concentration or after stimulation with methylsuccinate (5 mM, gray bars). Inset: quantification of [ADP] after the indicated time for each condition. Shown is the mean±s.e.m. of nine measurements. The experiment was repeated three times. (C) Kinetics of NAD(P)H fluorescence changes were followed over time in standard KRBH medium containing 2.5 mM glucose. Glucose (raised from 2.5 to 16.7 mM, black circles) and methylsuccinate (5 mM, MetSucc, gray circles) was added as indicated (stimulation). Inset: statistical evaluation of the data shown in C. The half-time to reach a new steady-state of the NAD(P)H signal after stimulation was calculated. Shown is the mean±s.e.m. from five and three independent experiments for glucose- (black) and methylsuccinate-stimulated (gray) cells, respectively. *P<0.05; **P<0.001; ***P<0.0001; NS, not significant (Student's t-test). Umberto De Marchi et al. J Cell Sci 2017;130:1929-1939 © 2017. Published by The Company of Biologists Ltd