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A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets.

Wikstrom JD, Sereda SB, Stiles L, Elorza A, Allister EM, Neilson A, Ferrick DA, Wheeler MB, Shirihai OS - PLoS ONE (2012)

Bottom Line: The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP.Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets.The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT

Background: The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR) may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets.

Methodology/principal findings: The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets.

Conclusions/significance: The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

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Measuring coupling efficacy.A) Oligomycin titration. The F1F0 ATP synthase inhibitor oligomycin was added after 20 mM glucose as indicated. Note that 5 µM oligomycin has a similar effect as 10 µM. B) Experimental setup to measure uncoupling. A few data points are recorded under basal conditions (3 mM glucose) followed by injection of oligomycin. The remaining OCR under oligomycin represents respiration that is not coupled to ATP synthesis (uncoupling). Data is shown both as absolute rates (left) and normalized to initial values (right). C) FCCP increase OCR under oligomycin. To verify the capacity of oligomycin to reveal uncoupling, islets were treated with FCCP (1 µM) prior to oligomycin. Note the increase in OCR under oligomycin representing maximal uncoupled respiration. D) Norepinephrine activated brown adipocytes show higher respiration under oligomycin. Basal measurement was followed by injection of control media or media containing 1 µM norepinephrine which activates uncoupling. Note the increase in OCR under oligomycin indicative of uncoupling. E) Oligomycin does not alter non-mitochondrial respiration. OCR was first measured with no drugs present followed by injection of only oligomycin (OM) or both oligomycin and rotenone/myxothiazol (Rot) (5 µM of both). Later, islets that were only injected with oligomycin at first also received rotenone/myxothiazol. Note that there is no difference in non mitochondrial respiration (under rotenone/myxothiazol) between the different treatments indicating that oligomycin does not alter non mitochondrial respiration.
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pone-0033023-g002: Measuring coupling efficacy.A) Oligomycin titration. The F1F0 ATP synthase inhibitor oligomycin was added after 20 mM glucose as indicated. Note that 5 µM oligomycin has a similar effect as 10 µM. B) Experimental setup to measure uncoupling. A few data points are recorded under basal conditions (3 mM glucose) followed by injection of oligomycin. The remaining OCR under oligomycin represents respiration that is not coupled to ATP synthesis (uncoupling). Data is shown both as absolute rates (left) and normalized to initial values (right). C) FCCP increase OCR under oligomycin. To verify the capacity of oligomycin to reveal uncoupling, islets were treated with FCCP (1 µM) prior to oligomycin. Note the increase in OCR under oligomycin representing maximal uncoupled respiration. D) Norepinephrine activated brown adipocytes show higher respiration under oligomycin. Basal measurement was followed by injection of control media or media containing 1 µM norepinephrine which activates uncoupling. Note the increase in OCR under oligomycin indicative of uncoupling. E) Oligomycin does not alter non-mitochondrial respiration. OCR was first measured with no drugs present followed by injection of only oligomycin (OM) or both oligomycin and rotenone/myxothiazol (Rot) (5 µM of both). Later, islets that were only injected with oligomycin at first also received rotenone/myxothiazol. Note that there is no difference in non mitochondrial respiration (under rotenone/myxothiazol) between the different treatments indicating that oligomycin does not alter non mitochondrial respiration.

Mentions: Using isolated mitochondria to examine uncoupling is common; however it is not feasible with islets. E.g. a standard mitochondrial isolation from 1×106 cells would require more than 6000 islets isolated from 30–40 mice (assuming each islet has 1500 cells). To address this issue we developed an approach to assess uncoupling of intact islets based on oligomycin, a specific inhibitor of the F1F0ATPsynthase [12]. The basic experimental algorithm was as follows (Fig. 2B). First, basal respiration levels were measured repeatedly until stable. Then, in order to determine the level of uncoupling, oligomycin was added at a saturating concentration (Fig. 2A). Oligomycin caused a decrease in respiration that reached a steady-state within 30–45 min (Fig. 2A). As absolute rates of oxygen consumption varied depending on islet seeding number, each well was normalized to its initial basal rates when only relative measurements were needed (Fig. 2B). To determine what fraction of total respiration is uncoupled, only relative values are needed.


A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets.

Wikstrom JD, Sereda SB, Stiles L, Elorza A, Allister EM, Neilson A, Ferrick DA, Wheeler MB, Shirihai OS - PLoS ONE (2012)

Measuring coupling efficacy.A) Oligomycin titration. The F1F0 ATP synthase inhibitor oligomycin was added after 20 mM glucose as indicated. Note that 5 µM oligomycin has a similar effect as 10 µM. B) Experimental setup to measure uncoupling. A few data points are recorded under basal conditions (3 mM glucose) followed by injection of oligomycin. The remaining OCR under oligomycin represents respiration that is not coupled to ATP synthesis (uncoupling). Data is shown both as absolute rates (left) and normalized to initial values (right). C) FCCP increase OCR under oligomycin. To verify the capacity of oligomycin to reveal uncoupling, islets were treated with FCCP (1 µM) prior to oligomycin. Note the increase in OCR under oligomycin representing maximal uncoupled respiration. D) Norepinephrine activated brown adipocytes show higher respiration under oligomycin. Basal measurement was followed by injection of control media or media containing 1 µM norepinephrine which activates uncoupling. Note the increase in OCR under oligomycin indicative of uncoupling. E) Oligomycin does not alter non-mitochondrial respiration. OCR was first measured with no drugs present followed by injection of only oligomycin (OM) or both oligomycin and rotenone/myxothiazol (Rot) (5 µM of both). Later, islets that were only injected with oligomycin at first also received rotenone/myxothiazol. Note that there is no difference in non mitochondrial respiration (under rotenone/myxothiazol) between the different treatments indicating that oligomycin does not alter non mitochondrial respiration.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351473&req=5

pone-0033023-g002: Measuring coupling efficacy.A) Oligomycin titration. The F1F0 ATP synthase inhibitor oligomycin was added after 20 mM glucose as indicated. Note that 5 µM oligomycin has a similar effect as 10 µM. B) Experimental setup to measure uncoupling. A few data points are recorded under basal conditions (3 mM glucose) followed by injection of oligomycin. The remaining OCR under oligomycin represents respiration that is not coupled to ATP synthesis (uncoupling). Data is shown both as absolute rates (left) and normalized to initial values (right). C) FCCP increase OCR under oligomycin. To verify the capacity of oligomycin to reveal uncoupling, islets were treated with FCCP (1 µM) prior to oligomycin. Note the increase in OCR under oligomycin representing maximal uncoupled respiration. D) Norepinephrine activated brown adipocytes show higher respiration under oligomycin. Basal measurement was followed by injection of control media or media containing 1 µM norepinephrine which activates uncoupling. Note the increase in OCR under oligomycin indicative of uncoupling. E) Oligomycin does not alter non-mitochondrial respiration. OCR was first measured with no drugs present followed by injection of only oligomycin (OM) or both oligomycin and rotenone/myxothiazol (Rot) (5 µM of both). Later, islets that were only injected with oligomycin at first also received rotenone/myxothiazol. Note that there is no difference in non mitochondrial respiration (under rotenone/myxothiazol) between the different treatments indicating that oligomycin does not alter non mitochondrial respiration.
Mentions: Using isolated mitochondria to examine uncoupling is common; however it is not feasible with islets. E.g. a standard mitochondrial isolation from 1×106 cells would require more than 6000 islets isolated from 30–40 mice (assuming each islet has 1500 cells). To address this issue we developed an approach to assess uncoupling of intact islets based on oligomycin, a specific inhibitor of the F1F0ATPsynthase [12]. The basic experimental algorithm was as follows (Fig. 2B). First, basal respiration levels were measured repeatedly until stable. Then, in order to determine the level of uncoupling, oligomycin was added at a saturating concentration (Fig. 2A). Oligomycin caused a decrease in respiration that reached a steady-state within 30–45 min (Fig. 2A). As absolute rates of oxygen consumption varied depending on islet seeding number, each well was normalized to its initial basal rates when only relative measurements were needed (Fig. 2B). To determine what fraction of total respiration is uncoupled, only relative values are needed.

Bottom Line: The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP.Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets.The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT

Background: The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR) may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets.

Methodology/principal findings: The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets.

Conclusions/significance: The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

Show MeSH