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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.

Show MeSH
Respiration of a small cohort of human Type 2 diabetic islets.Each graph represents islets from one patient (n = 4−5). Drugs added during the experiment are indicated. Type 2 diabetic donor: Male age 52, weight 100 kg, BMI 31.9, Caucasian. Non diabetic donor: Male age 32, weight 69 kg, BMI 24.7, Caucasian.
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pone-0033023-g004: Respiration of a small cohort of human Type 2 diabetic islets.Each graph represents islets from one patient (n = 4−5). Drugs added during the experiment are indicated. Type 2 diabetic donor: Male age 52, weight 100 kg, BMI 31.9, Caucasian. Non diabetic donor: Male age 32, weight 69 kg, BMI 24.7, Caucasian.

Mentions: Moreover, we examined the level of uncoupling of islets from three Type 2 diabetic patients (Fig. 4). These islets presented with similar levels of uncoupling as the healthy control islets (Fig. 3A) although the n was not high enough for a meaningful statistical comparison.


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)

Respiration of a small cohort of human Type 2 diabetic islets.Each graph represents islets from one patient (n = 4−5). Drugs added during the experiment are indicated. Type 2 diabetic donor: Male age 52, weight 100 kg, BMI 31.9, Caucasian. Non diabetic donor: Male age 32, weight 69 kg, BMI 24.7, Caucasian.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0033023-g004: Respiration of a small cohort of human Type 2 diabetic islets.Each graph represents islets from one patient (n = 4−5). Drugs added during the experiment are indicated. Type 2 diabetic donor: Male age 52, weight 100 kg, BMI 31.9, Caucasian. Non diabetic donor: Male age 32, weight 69 kg, BMI 24.7, Caucasian.
Mentions: Moreover, we examined the level of uncoupling of islets from three Type 2 diabetic patients (Fig. 4). These islets presented with similar levels of uncoupling as the healthy control islets (Fig. 3A) although the n was not high enough for a meaningful statistical comparison.

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