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

Jakob D. Wikstrom, Samuel B. Sereda, Linsey Stiles, Alvaro Elorza, Emma M. Allister, Andy Neilson, David A. Ferrick, Michael B. Wheeler, Orian S. Shirihai

Resultado de la investigación: Article

69 Citas (Scopus)

Resumen

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.

Idioma originalEnglish
Número de artículoe33023
PublicaciónPLoS ONE
Volumen7
N.º5
DOI
EstadoPublished - 14 may 2012

Huella dactilar

cell respiration
Oxygen Consumption
oligomycin
oxygen consumption
Oligomycins
Rodentia
Assays
Respiration
rodents
islets of Langerhans
Throughput
Oxygen
Nutrients
assays
Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone
Rotenone
Food
rotenone
H-transporting ATP synthase
Skeletal Muscle Fibers

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Citar esto

Wikstrom, J. D., Sereda, S. B., Stiles, L., Elorza, A., Allister, E. M., Neilson, A., ... Shirihai, O. S. (2012). A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets. PLoS ONE, 7(5), [e33023]. https://doi.org/10.1371/journal.pone.0033023
Wikstrom, Jakob D. ; Sereda, Samuel B. ; Stiles, Linsey ; Elorza, Alvaro ; Allister, Emma M. ; Neilson, Andy ; Ferrick, David A. ; Wheeler, Michael B. ; Shirihai, Orian S. / A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets. En: PLoS ONE. 2012 ; Vol. 7, N.º 5.
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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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Wikstrom, JD, Sereda, SB, Stiles, L, Elorza, A, Allister, EM, Neilson, A, Ferrick, DA, Wheeler, MB & Shirihai, OS 2012, 'A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets', PLoS ONE, vol. 7, n.º 5, e33023. https://doi.org/10.1371/journal.pone.0033023

A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets. / Wikstrom, Jakob D.; Sereda, Samuel B.; Stiles, Linsey; Elorza, Alvaro; Allister, Emma M.; Neilson, Andy; Ferrick, David A.; Wheeler, Michael B.; Shirihai, Orian S.

En: PLoS ONE, Vol. 7, N.º 5, e33023, 14.05.2012.

Resultado de la investigación: Article

TY - JOUR

T1 - A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets

AU - Wikstrom, Jakob D.

AU - Sereda, Samuel B.

AU - Stiles, Linsey

AU - Elorza, Alvaro

AU - Allister, Emma M.

AU - Neilson, Andy

AU - Ferrick, David A.

AU - Wheeler, Michael B.

AU - Shirihai, Orian S.

PY - 2012/5/14

Y1 - 2012/5/14

N2 - 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.

AB - 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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