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Mitochondrial Respiration in Insulin-Producing β-Cells: General Characteristics and Adaptive Effects of Hypoxia.

Hals IK, Bruerberg SG, Ma Z, Scholz H, Björklund A, Grill V - PLoS ONE (2015)

Bottom Line: Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion.Such effects are accompanied by up-regulation of mitochondrial complexes also in pancreatic islets, highlighting adaptive capacities of possible importance in an islet transplantation setting.Results also indicate idiosyncrasies of β-cells that do not respire in response to a standard inclusion of malate in SUIT protocols.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.

ABSTRACT

Objective: To provide novel insights on mitochondrial respiration in β-cells and the adaptive effects of hypoxia.

Methods and design: Insulin-producing INS-1 832/13 cells were exposed to 18 hours of hypoxia followed by 20-22 hours re-oxygenation. Mitochondrial respiration was measured by high-resolution respirometry in both intact and permeabilized cells, in the latter after establishing three functional substrate-uncoupler-inhibitor titration (SUIT) protocols. Concomitant measurements included proteins of mitochondrial complexes (Western blotting), ATP and insulin secretion.

Results: Intact cells exhibited a high degree of intrinsic uncoupling, comprising about 50% of oxygen consumption in the basal respiratory state. Hypoxia followed by re-oxygenation increased maximal overall respiration. Exploratory experiments in peremabilized cells could not show induction of respiration by malate or pyruvate as reducing substrates, thus glutamate and succinate were used as mitochondrial substrates in SUIT protocols. Permeabilized cells displayed a high capacity for oxidative phosphorylation for both complex I- and II-linked substrates in relation to maximum capacity of electron transfer. Previous hypoxia decreased phosphorylation control of complex I-linked respiration, but not in complex II-linked respiration. Coupling control ratios showed increased coupling efficiency for both complex I- and II-linked substrates in hypoxia-exposed cells. Respiratory rates overall were increased. Also previous hypoxia increased proteins of mitochondrial complexes I and II (Western blotting) in INS-1 cells as well as in rat and human islets. Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion.

Conclusions: Exposure of INS-1 832/13 cells to hypoxia, followed by a re-oxygenation period increases substrate-stimulated respiratory capacity and coupling efficiency. Such effects are accompanied by up-regulation of mitochondrial complexes also in pancreatic islets, highlighting adaptive capacities of possible importance in an islet transplantation setting. Results also indicate idiosyncrasies of β-cells that do not respire in response to a standard inclusion of malate in SUIT protocols.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0138558.g001: 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.

Mentions: Four different HRR protocols were used (Fig 1A–1D). One was designed for intact cells to provide information on the effect of hypoxia on basal and uncoupled respiratory rates, together with the enzymatic capacity of ETS. The three substrate-uncoupler-inhibitor titration (SUIT) protocols were designed for permeabilized cells to probe mitochondrial function by the addition of TCA cycle metabolites in saturating concentrations in different combinations, together with an uncoupler and ETS inhibitors. Using flux ratios derived from measured oxygen flux enabled internal normalization and relative contributions of substrate conditions, in particular coupling states, to be compared.


Mitochondrial Respiration in Insulin-Producing β-Cells: General Characteristics and Adaptive Effects of Hypoxia.

Hals IK, Bruerberg SG, Ma Z, Scholz H, Björklund A, Grill V - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581632&req=5

pone.0138558.g001: 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.
Mentions: Four different HRR protocols were used (Fig 1A–1D). One was designed for intact cells to provide information on the effect of hypoxia on basal and uncoupled respiratory rates, together with the enzymatic capacity of ETS. The three substrate-uncoupler-inhibitor titration (SUIT) protocols were designed for permeabilized cells to probe mitochondrial function by the addition of TCA cycle metabolites in saturating concentrations in different combinations, together with an uncoupler and ETS inhibitors. Using flux ratios derived from measured oxygen flux enabled internal normalization and relative contributions of substrate conditions, in particular coupling states, to be compared.

Bottom Line: Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion.Such effects are accompanied by up-regulation of mitochondrial complexes also in pancreatic islets, highlighting adaptive capacities of possible importance in an islet transplantation setting.Results also indicate idiosyncrasies of β-cells that do not respire in response to a standard inclusion of malate in SUIT protocols.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.

ABSTRACT

Objective: To provide novel insights on mitochondrial respiration in β-cells and the adaptive effects of hypoxia.

Methods and design: Insulin-producing INS-1 832/13 cells were exposed to 18 hours of hypoxia followed by 20-22 hours re-oxygenation. Mitochondrial respiration was measured by high-resolution respirometry in both intact and permeabilized cells, in the latter after establishing three functional substrate-uncoupler-inhibitor titration (SUIT) protocols. Concomitant measurements included proteins of mitochondrial complexes (Western blotting), ATP and insulin secretion.

Results: Intact cells exhibited a high degree of intrinsic uncoupling, comprising about 50% of oxygen consumption in the basal respiratory state. Hypoxia followed by re-oxygenation increased maximal overall respiration. Exploratory experiments in peremabilized cells could not show induction of respiration by malate or pyruvate as reducing substrates, thus glutamate and succinate were used as mitochondrial substrates in SUIT protocols. Permeabilized cells displayed a high capacity for oxidative phosphorylation for both complex I- and II-linked substrates in relation to maximum capacity of electron transfer. Previous hypoxia decreased phosphorylation control of complex I-linked respiration, but not in complex II-linked respiration. Coupling control ratios showed increased coupling efficiency for both complex I- and II-linked substrates in hypoxia-exposed cells. Respiratory rates overall were increased. Also previous hypoxia increased proteins of mitochondrial complexes I and II (Western blotting) in INS-1 cells as well as in rat and human islets. Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion.

Conclusions: Exposure of INS-1 832/13 cells to hypoxia, followed by a re-oxygenation period increases substrate-stimulated respiratory capacity and coupling efficiency. Such effects are accompanied by up-regulation of mitochondrial complexes also in pancreatic islets, highlighting adaptive capacities of possible importance in an islet transplantation setting. Results also indicate idiosyncrasies of β-cells that do not respire in response to a standard inclusion of malate in SUIT protocols.

No MeSH data available.


Related in: MedlinePlus