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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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Related in: MedlinePlus

Measuring islet oxygen consumption in a high throughput format.A) Islet plate development. The XF24 measures oxygen consumption from monolayers of adherent cells in a 24 well cell culture plate format. Oxygen consumption is measured with probe heads coated with oxygen sensitive fluorophores that are optically read (marked white and red). During the “measure” mode a volume of only a few micro-liters is formed. In this minute volume oxygen tension rapidly drops which enables OCR calculations. In “mix” mode, the probe head moves up and down, reoxygenating the cells and exposing them to injected compounds. The wells with flat bottoms, originally designed for cell monolayers, proved inadequate for islets as they gathered in the well periphery, too far from the probe head (left panel). To address this problem, an islet plate with a central depression covered by a screen was developed. This trapped the islets while still maintaining sufficient media access (right panel). The screen consists of a polycarbonate ring attached to a nylon net with 50 µm pore size. B) Image of islet plate well. After experiments each well of the 24-well plate was imaged and the images were used for calculating OCR per islet and to measure islet diameter. The islet images were then used to obtain an exact islet count per well and to measure islet diameter. C) Flat bottom (V7) plates are not suitable for islets. 70 islets were seeded per well. Traces recorded from separate wells in a plate are shown. Note the large data variation and that subsequent to 20 mM glucose injection the OCR only increase in one well out of four. D) Islet plate reduces data variability. As islet seeding could vary and thus the absolute OCR, data from each well was often normalized to its initial steady state values before any compound was injected. Traces from islets in 3 mM and 20 mM glucose are shown with absolute (left) and normalized rates (right); note the stability over 2.5 hours. Subsequent to 20 mM glucose injection (after 1st data point) the OCR increased in all the wells. Islets that stay in 3 mM glucose do not display any major change in OCR. E) Initial measurements are unstable. OCR in the islet plate showed an initial drift during the first 1–2 measurements (dashed square), therefore these data points where always omitted. F) Experimental set up – bioenergetic principles. Respiration recorded may be deciphered by using drugs acting on the mitochondrial inner membrane/complexes. Oligomycin blocks complex V and remaining respiration represents the proton leak. Rotenone/Myxothiazol blocks complexes I/III and remaining respiration is non-mitochondrial. FCCP stimulation shows maximal respiratory capacity. Nutrient stimulation (glucose) may be used in addition to these drugs to study the effect of nutrient metabolism.
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pone-0033023-g001: Measuring islet oxygen consumption in a high throughput format.A) Islet plate development. The XF24 measures oxygen consumption from monolayers of adherent cells in a 24 well cell culture plate format. Oxygen consumption is measured with probe heads coated with oxygen sensitive fluorophores that are optically read (marked white and red). During the “measure” mode a volume of only a few micro-liters is formed. In this minute volume oxygen tension rapidly drops which enables OCR calculations. In “mix” mode, the probe head moves up and down, reoxygenating the cells and exposing them to injected compounds. The wells with flat bottoms, originally designed for cell monolayers, proved inadequate for islets as they gathered in the well periphery, too far from the probe head (left panel). To address this problem, an islet plate with a central depression covered by a screen was developed. This trapped the islets while still maintaining sufficient media access (right panel). The screen consists of a polycarbonate ring attached to a nylon net with 50 µm pore size. B) Image of islet plate well. After experiments each well of the 24-well plate was imaged and the images were used for calculating OCR per islet and to measure islet diameter. The islet images were then used to obtain an exact islet count per well and to measure islet diameter. C) Flat bottom (V7) plates are not suitable for islets. 70 islets were seeded per well. Traces recorded from separate wells in a plate are shown. Note the large data variation and that subsequent to 20 mM glucose injection the OCR only increase in one well out of four. D) Islet plate reduces data variability. As islet seeding could vary and thus the absolute OCR, data from each well was often normalized to its initial steady state values before any compound was injected. Traces from islets in 3 mM and 20 mM glucose are shown with absolute (left) and normalized rates (right); note the stability over 2.5 hours. Subsequent to 20 mM glucose injection (after 1st data point) the OCR increased in all the wells. Islets that stay in 3 mM glucose do not display any major change in OCR. E) Initial measurements are unstable. OCR in the islet plate showed an initial drift during the first 1–2 measurements (dashed square), therefore these data points where always omitted. F) Experimental set up – bioenergetic principles. Respiration recorded may be deciphered by using drugs acting on the mitochondrial inner membrane/complexes. Oligomycin blocks complex V and remaining respiration represents the proton leak. Rotenone/Myxothiazol blocks complexes I/III and remaining respiration is non-mitochondrial. FCCP stimulation shows maximal respiratory capacity. Nutrient stimulation (glucose) may be used in addition to these drugs to study the effect of nutrient metabolism.

Mentions: To develop a higher throughput islet respirometry assay we built upon the existing respirometry platform originally designed for adherent cells, XF24 (Seahorse Bioscience, Billerica, MA). The XF24 measures oxygen consumption from monolayers of adherent cells in a 24-well cell culture plate format [11]. The plates used for cell monolayers proved inadequate for non-adherent islets as they gathered in the periphery of the well due to turbulence from pipetting and probe head movement (Fig. 1A). This produced data with high variability and unreliable response to stimuli, such as glucose (Fig. 1B). To solve this, we designed a plate that enclosed the islets during an experiment (Fig. 1A). The islets are kept in a depression in the middle of the well that keeps them in close proximity to the probe head and a screen is used to protect them from turbulence. The screen consists of a polycarbonate ring attached to a nylon net with a 50 µm pore size. After an experiment, which could last up to 5 h, the islet plate was imaged and the islets were harvested for protein or other downstream assays. The islet images were then used to obtain an exact islet count per well and to measure islet diameter for normalization.


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 islet oxygen consumption in a high throughput format.A) Islet plate development. The XF24 measures oxygen consumption from monolayers of adherent cells in a 24 well cell culture plate format. Oxygen consumption is measured with probe heads coated with oxygen sensitive fluorophores that are optically read (marked white and red). During the “measure” mode a volume of only a few micro-liters is formed. In this minute volume oxygen tension rapidly drops which enables OCR calculations. In “mix” mode, the probe head moves up and down, reoxygenating the cells and exposing them to injected compounds. The wells with flat bottoms, originally designed for cell monolayers, proved inadequate for islets as they gathered in the well periphery, too far from the probe head (left panel). To address this problem, an islet plate with a central depression covered by a screen was developed. This trapped the islets while still maintaining sufficient media access (right panel). The screen consists of a polycarbonate ring attached to a nylon net with 50 µm pore size. B) Image of islet plate well. After experiments each well of the 24-well plate was imaged and the images were used for calculating OCR per islet and to measure islet diameter. The islet images were then used to obtain an exact islet count per well and to measure islet diameter. C) Flat bottom (V7) plates are not suitable for islets. 70 islets were seeded per well. Traces recorded from separate wells in a plate are shown. Note the large data variation and that subsequent to 20 mM glucose injection the OCR only increase in one well out of four. D) Islet plate reduces data variability. As islet seeding could vary and thus the absolute OCR, data from each well was often normalized to its initial steady state values before any compound was injected. Traces from islets in 3 mM and 20 mM glucose are shown with absolute (left) and normalized rates (right); note the stability over 2.5 hours. Subsequent to 20 mM glucose injection (after 1st data point) the OCR increased in all the wells. Islets that stay in 3 mM glucose do not display any major change in OCR. E) Initial measurements are unstable. OCR in the islet plate showed an initial drift during the first 1–2 measurements (dashed square), therefore these data points where always omitted. F) Experimental set up – bioenergetic principles. Respiration recorded may be deciphered by using drugs acting on the mitochondrial inner membrane/complexes. Oligomycin blocks complex V and remaining respiration represents the proton leak. Rotenone/Myxothiazol blocks complexes I/III and remaining respiration is non-mitochondrial. FCCP stimulation shows maximal respiratory capacity. Nutrient stimulation (glucose) may be used in addition to these drugs to study the effect of nutrient metabolism.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0033023-g001: Measuring islet oxygen consumption in a high throughput format.A) Islet plate development. The XF24 measures oxygen consumption from monolayers of adherent cells in a 24 well cell culture plate format. Oxygen consumption is measured with probe heads coated with oxygen sensitive fluorophores that are optically read (marked white and red). During the “measure” mode a volume of only a few micro-liters is formed. In this minute volume oxygen tension rapidly drops which enables OCR calculations. In “mix” mode, the probe head moves up and down, reoxygenating the cells and exposing them to injected compounds. The wells with flat bottoms, originally designed for cell monolayers, proved inadequate for islets as they gathered in the well periphery, too far from the probe head (left panel). To address this problem, an islet plate with a central depression covered by a screen was developed. This trapped the islets while still maintaining sufficient media access (right panel). The screen consists of a polycarbonate ring attached to a nylon net with 50 µm pore size. B) Image of islet plate well. After experiments each well of the 24-well plate was imaged and the images were used for calculating OCR per islet and to measure islet diameter. The islet images were then used to obtain an exact islet count per well and to measure islet diameter. C) Flat bottom (V7) plates are not suitable for islets. 70 islets were seeded per well. Traces recorded from separate wells in a plate are shown. Note the large data variation and that subsequent to 20 mM glucose injection the OCR only increase in one well out of four. D) Islet plate reduces data variability. As islet seeding could vary and thus the absolute OCR, data from each well was often normalized to its initial steady state values before any compound was injected. Traces from islets in 3 mM and 20 mM glucose are shown with absolute (left) and normalized rates (right); note the stability over 2.5 hours. Subsequent to 20 mM glucose injection (after 1st data point) the OCR increased in all the wells. Islets that stay in 3 mM glucose do not display any major change in OCR. E) Initial measurements are unstable. OCR in the islet plate showed an initial drift during the first 1–2 measurements (dashed square), therefore these data points where always omitted. F) Experimental set up – bioenergetic principles. Respiration recorded may be deciphered by using drugs acting on the mitochondrial inner membrane/complexes. Oligomycin blocks complex V and remaining respiration represents the proton leak. Rotenone/Myxothiazol blocks complexes I/III and remaining respiration is non-mitochondrial. FCCP stimulation shows maximal respiratory capacity. Nutrient stimulation (glucose) may be used in addition to these drugs to study the effect of nutrient metabolism.
Mentions: To develop a higher throughput islet respirometry assay we built upon the existing respirometry platform originally designed for adherent cells, XF24 (Seahorse Bioscience, Billerica, MA). The XF24 measures oxygen consumption from monolayers of adherent cells in a 24-well cell culture plate format [11]. The plates used for cell monolayers proved inadequate for non-adherent islets as they gathered in the periphery of the well due to turbulence from pipetting and probe head movement (Fig. 1A). This produced data with high variability and unreliable response to stimuli, such as glucose (Fig. 1B). To solve this, we designed a plate that enclosed the islets during an experiment (Fig. 1A). The islets are kept in a depression in the middle of the well that keeps them in close proximity to the probe head and a screen is used to protect them from turbulence. The screen consists of a polycarbonate ring attached to a nylon net with a 50 µm pore size. After an experiment, which could last up to 5 h, the islet plate was imaged and the islets were harvested for protein or other downstream assays. The islet images were then used to obtain an exact islet count per well and to measure islet diameter for normalization.

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
Related in: MedlinePlus