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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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Islets are highly uncoupled; mice islets more than human.A) Uncoupling of wild type mouse islets, INS-1 cells, C2C12 myotubes and human islets. The graph presents a summary of multiple experiments where OCR under oligomycin (defined as uncoupling) was measured until stable. Note that uncoupled OCR is significantly higher in the mouse islets and that the C2C12 myotubes show the lowest uncoupled OCR. N = 29 (INS-1), 20 (C2C12), 35 (C57Bl6/J islets), 14 (FVB/N islets), 8 (Human islets). **indicates p<0.01. B) Comparison of total ATP levels in INS-1 cells vs. C2C12 myotubes.
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pone-0033023-g003: Islets are highly uncoupled; mice islets more than human.A) Uncoupling of wild type mouse islets, INS-1 cells, C2C12 myotubes and human islets. The graph presents a summary of multiple experiments where OCR under oligomycin (defined as uncoupling) was measured until stable. Note that uncoupled OCR is significantly higher in the mouse islets and that the C2C12 myotubes show the lowest uncoupled OCR. N = 29 (INS-1), 20 (C2C12), 35 (C57Bl6/J islets), 14 (FVB/N islets), 8 (Human islets). **indicates p<0.01. B) Comparison of total ATP levels in INS-1 cells vs. C2C12 myotubes.

Mentions: To date there are no reports on the level of uncoupling in intact islets. In this study we examined islets from mice of two different genetic backgrounds; C57Bl6/J and FVB/N. Islets from C57Bl6/J mice exhibited a basal level of uncoupling (under oligomycin) of 59% as compared to basal respiration (100%, no oligomycin) (Fig. 3A). In contrast, FVB/N islets showed slightly lower levels of uncoupling: 49%. In clonal INS-1 cells and C2C12 myotubes uncoupling was measured to 38% and 23% respectively. Moreover, islets from a cohort of non diabetic donors were also examined. Interestingly, the level of uncoupling was significantly lower in the human islets as compared to the rodent islets.


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)

Islets are highly uncoupled; mice islets more than human.A) Uncoupling of wild type mouse islets, INS-1 cells, C2C12 myotubes and human islets. The graph presents a summary of multiple experiments where OCR under oligomycin (defined as uncoupling) was measured until stable. Note that uncoupled OCR is significantly higher in the mouse islets and that the C2C12 myotubes show the lowest uncoupled OCR. N = 29 (INS-1), 20 (C2C12), 35 (C57Bl6/J islets), 14 (FVB/N islets), 8 (Human islets). **indicates p<0.01. B) Comparison of total ATP levels in INS-1 cells vs. C2C12 myotubes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351473&req=5

pone-0033023-g003: Islets are highly uncoupled; mice islets more than human.A) Uncoupling of wild type mouse islets, INS-1 cells, C2C12 myotubes and human islets. The graph presents a summary of multiple experiments where OCR under oligomycin (defined as uncoupling) was measured until stable. Note that uncoupled OCR is significantly higher in the mouse islets and that the C2C12 myotubes show the lowest uncoupled OCR. N = 29 (INS-1), 20 (C2C12), 35 (C57Bl6/J islets), 14 (FVB/N islets), 8 (Human islets). **indicates p<0.01. B) Comparison of total ATP levels in INS-1 cells vs. C2C12 myotubes.
Mentions: To date there are no reports on the level of uncoupling in intact islets. In this study we examined islets from mice of two different genetic backgrounds; C57Bl6/J and FVB/N. Islets from C57Bl6/J mice exhibited a basal level of uncoupling (under oligomycin) of 59% as compared to basal respiration (100%, no oligomycin) (Fig. 3A). In contrast, FVB/N islets showed slightly lower levels of uncoupling: 49%. In clonal INS-1 cells and C2C12 myotubes uncoupling was measured to 38% and 23% respectively. Moreover, islets from a cohort of non diabetic donors were also examined. Interestingly, the level of uncoupling was significantly lower in the human islets as compared to the rodent islets.

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