Table 1.
Reagents used in the different high-resolution respirometry protocols.
Fig 1.
Oxygraphic example output of protocols with intact and permeabilized INS-1 832/13 cells (at normoxia).
(A) Protocol with intact cells, (B) SUITCI protocol with permeabilized cells with glutamate as only reducing substrate, (C) SUITCII protocol with permeabilized cells with succinate as only reducing substrate, (D) SUITCI+II protocol with glutamate and succinate as reducing substrates. The blue line represents the oxygen concentration (nmol O2/mL) in the experimental chamber. The red line represents oxygen flux (pmol O2/s/106 cells), the negative time derivate calculated from the measured oxygen concentration, normalized to the number of cells. ROUTINE: ROUTINE respiratory state of basal respiration, LEAK: LEAK respiratory state of uncoupled respiration, OXPHOS: OXPHOS respiratory state of maximum phosphorylative capacity, ETS: ETS respiratory state of the maximum capacity of the ETS, ROX: ROX respiratory state of residual oxygen consumption, Dig: digitonin, G: glutamate, D: ADP, S: succinate, c: cytochrome c, F: FCCP, Rot: rotenone, Ama: antimycin A.
Table 2.
Definitions of the coupling control ratios (CCRs) used.
Table 3.
Definitions of the substrate control ratios (SCRs) used.
Fig 2.
Previous hypoxia (8 hours) increased basal but not stimulated insulin secretion in INS-1 832/13 cells.
Insulin release (final 60–90 min incubations) with 3.3, 11 and 27 mM glucose (G). Protein content was estimated from a mean of three measurements in each experiment. Data are mean ± SEM, n = 5, *P < 0.05.
Fig 3.
Previous hypoxia (8 hours) failed to affect levels of ATP in INS-1 832/13 cells.
Cells were cultured in RPMI with 11 mM glucose. Data are mean ± SEM based on five separate experiments (three parallels per experimental condition).
Fig 4.
Oxygen flux in different respiratory states in intact INS-1 832/13 cells.
Sequential respiratory states in the ProtocolInt protocol, comparing hypoxia with normoxia ROUTINE: basal respiration measured at physiological substrate conditions, before any reagent additions, LEAK: uncoupled respiration after addition of oligomycin, ETS: flux capacity of the electron transfer system, induced by FCCP, ROX: residual oxygen consumption, a measure of oxidative side reactions obtained by adding rotenone and antimycin A to inhibit complex I and II, respectively. Fluxes are expressed as means ± SEM of five experiments (comprising totally 12 single measurements),*P < 0.03.
Table 4.
Coupling control ratios (CCRs) derived from the protocol with intact cells.
Fig 5.
Phosphorylative capacities of different NADH-linked substrate combinations.
The graph shows the ability of different substrate combinations of pyruvate (5 mM), glutamate (10 mM) and malate (0.5 mM) to stimulate respiration in the presence of ADP (2.5 mM). State 1 (eN) is used as a control state. No difference was found between state 1 respiration and the OXPHOS states of the different substrate conditions. eN: endogenous respiration without reducing substrates or ADP (six measurements), P: pyruvate (three measurements), GM: glutamate and malate (six measurements), PM: pyruvate and malate (three measurements), PMG: pyruvate, malate and glutamate (three measurements). Fluxes are expressed as means ± SEM.
Table 5.
Coupling and substrate control ratios derived from the SUIT protocols.
Fig 6.
Effects of hypoxia on mitochondrial complexes I and II.
Immunoblotting of subunits of the complexes were performed on cells and islets that were harvested after hypoxia followed by re-oxygenation (H) or continuous normoxia (N). Representative Western blots are shown.
Table 6.
Effect of hypoxia on mitochondrial complex protein levels.