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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

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.
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pone.0138558.g002: 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.

Mentions: Exposure to hypoxia for 18 hours reduced numbers of viable INS-1 832/13 cells to 1.74 ± 0.15 million cells, compared to controls (6.11 ± 0.37 million cells). After 20 hours of re-oxygenation, the cell number increased to 3.32 ± 0.23 and 8.12 ± 0.26 million cells for hypoxia-treated (n = 17) and controls (n = 13), respectively. This increase was more marked in hypoxia treated cells (45 ± 7%) than in control cells (21 ± 4%). All cell suspensions used in the respiratory experiments showed a viability of > 97%, given by the trypan blue parameter. Exposure to hypoxia for only 8 hours led to lesser damage with a 25.8 ± 4.8% loss of viability when measured after re-oxygenation (P < 0.02, n = 7). Parallel measurements of insulin secretion (basal and stimulated) in five separate experiments showed an increase in basal secretion in hypoxia (8 hours) exposed cells: 0.194 ± 0.034 μU/μg protein for hypoxia vs. 0.131 ± 0.015 μU/μg protein for normoxia, P < 0.05. However, glucose-induced insulin secretion post hypoxia was not reduced. Results are shown in Fig 2.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4581632&req=5

pone.0138558.g002: 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.
Mentions: Exposure to hypoxia for 18 hours reduced numbers of viable INS-1 832/13 cells to 1.74 ± 0.15 million cells, compared to controls (6.11 ± 0.37 million cells). After 20 hours of re-oxygenation, the cell number increased to 3.32 ± 0.23 and 8.12 ± 0.26 million cells for hypoxia-treated (n = 17) and controls (n = 13), respectively. This increase was more marked in hypoxia treated cells (45 ± 7%) than in control cells (21 ± 4%). All cell suspensions used in the respiratory experiments showed a viability of > 97%, given by the trypan blue parameter. Exposure to hypoxia for only 8 hours led to lesser damage with a 25.8 ± 4.8% loss of viability when measured after re-oxygenation (P < 0.02, n = 7). Parallel measurements of insulin secretion (basal and stimulated) in five separate experiments showed an increase in basal secretion in hypoxia (8 hours) exposed cells: 0.194 ± 0.034 μU/μg protein for hypoxia vs. 0.131 ± 0.015 μU/μg protein for normoxia, P < 0.05. However, glucose-induced insulin secretion post hypoxia was not reduced. Results are shown in Fig 2.

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