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Beta-cell uncoupling protein 2 regulates reactive oxygen species production, which influences both insulin and glucagon secretion.

Robson-Doucette CA, Sultan S, Allister EM, Wikstrom JD, Koshkin V, Bhattacharjee A, Prentice KJ, Sereda SB, Shirihai OS, Wheeler MB - Diabetes (2011)

Bottom Line: UCP2BKO islets had elevated intracellular ROS levels that associated with enhanced GSIS.UCP2 does not behave as a classical metabolic uncoupler in the β-cell, but has a more prominent role in the regulation of intracellular ROS levels that contribute to GSIS amplification.In addition, β-cell UCP2 contributes to the regulation of intraislet ROS signals that mediate changes in α-cell morphology and glucagon secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT

Objective: The role of uncoupling protein 2 (UCP2) in pancreatic β-cells is highly debated, partly because of the broad tissue distribution of UCP2 and thus limitations of whole-body UCP2 knockout mouse models. To investigate the function of UCP2 in the β-cell, β-cell-specific UCP2 knockout mice (UCP2BKO) were generated and characterized.

Research design and methods: UCP2BKO mice were generated by crossing loxUCP2 mice with mice expressing rat insulin promoter-driven Cre recombinase. Several in vitro and in vivo parameters were measured, including respiration rate, mitochondrial membrane potential, islet ATP content, reactive oxygen species (ROS) levels, glucose-stimulated insulin secretion (GSIS), glucagon secretion, glucose and insulin tolerance, and plasma hormone levels.

Results: UCP2BKO β-cells displayed mildly increased glucose-induced mitochondrial membrane hyperpolarization but unchanged rates of uncoupled respiration and islet ATP content. UCP2BKO islets had elevated intracellular ROS levels that associated with enhanced GSIS. Surprisingly, UCP2BKO mice were glucose-intolerant, showing greater α-cell area, higher islet glucagon content, and aberrant ROS-dependent glucagon secretion under high glucose conditions.

Conclusions: Using a novel β-cell-specific UCP2KO mouse model, we have shed light on UCP2 function in primary β-cells. UCP2 does not behave as a classical metabolic uncoupler in the β-cell, but has a more prominent role in the regulation of intracellular ROS levels that contribute to GSIS amplification. In addition, β-cell UCP2 contributes to the regulation of intraislet ROS signals that mediate changes in α-cell morphology and glucagon secretion.

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Effective deletion of Ucp2 specifically from pancreatic β-cells using a Cre-lox recombination strategy. A: Targeting construct for the Ucp2 gene shows the floxed region (flanked by loxP sites) and the Ucp2 allele after Cre recombinase excision. Homozygote loxUCP2 mice that express Cre undergo recombination of the DNA between loxP sites, deleting exons 3 and 4, which contains the start codon. B: Typical PCR results from mouse genotyping for loxUCP2 (floxing) and Cre expression. C: Quantitative PCR results show reduced Ucp2 mRNA expression in isolated UCP2BKO islets compared with RIPCre islets. Ucp2 mRNA expression was calculated as a percentage of β-actin. The error bar shows the SEM. D: Standard PCR of various tissues shows full-length UCP2 transcripts and truncated UCP2 (Δ) where exons 3 and 4 have been removed by Cre recombinase activity. Wt, wild type. E, Two left panels: Dispersed pancreatic islets isolated from RIPCre and UCP2BKO mice immunostained for UCP2 (red) and insulin (green). Two right panels: UCP2BKO dispersed islet cells stained for Cre (red) and insulin (green) show nuclear localization of Cre recombinase. The yellow coloring represents colocalization of red and green fluorescence. n = 5–7. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 1: Effective deletion of Ucp2 specifically from pancreatic β-cells using a Cre-lox recombination strategy. A: Targeting construct for the Ucp2 gene shows the floxed region (flanked by loxP sites) and the Ucp2 allele after Cre recombinase excision. Homozygote loxUCP2 mice that express Cre undergo recombination of the DNA between loxP sites, deleting exons 3 and 4, which contains the start codon. B: Typical PCR results from mouse genotyping for loxUCP2 (floxing) and Cre expression. C: Quantitative PCR results show reduced Ucp2 mRNA expression in isolated UCP2BKO islets compared with RIPCre islets. Ucp2 mRNA expression was calculated as a percentage of β-actin. The error bar shows the SEM. D: Standard PCR of various tissues shows full-length UCP2 transcripts and truncated UCP2 (Δ) where exons 3 and 4 have been removed by Cre recombinase activity. Wt, wild type. E, Two left panels: Dispersed pancreatic islets isolated from RIPCre and UCP2BKO mice immunostained for UCP2 (red) and insulin (green). Two right panels: UCP2BKO dispersed islet cells stained for Cre (red) and insulin (green) show nuclear localization of Cre recombinase. The yellow coloring represents colocalization of red and green fluorescence. n = 5–7. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: The loxUCP2 mice were a gift from Dr. Bradford Lowell (32). β-Cell–specific UCP2 deletion was accomplished by crossing loxUCP2 mice with rat insulin promoter–driven Cre recombinase (RIPCre) mice (The Jackson Laboratory, Bar Harbor, ME). Mice were genotyped using standard PCR of ear notch DNA (Fig. 1B). RIPCre mice were chosen as controls for experimentation because RIPCre and floxed mice (mice that express the floxed Ucp2 gene without Cre) gave similar results (Supplementary Fig. 1). All mice (10–13 weeks old) were age- and sex-matched and maintained on a 129J-C57BL/6-mixed background. Unless otherwise indicated, male mice were used. All animal experiments were approved by the University of Toronto Animal Care Committee, and animals were handled according to the guidelines of the Canadian Council of Animal Care. Human islets from healthy donors were isolated using the Edmonton protocol and provided by the ABCC Human Islet Distribution Program (University of Alberta, Edmonton, AB, Canada). Donation was approved by the local institutional review board.


Beta-cell uncoupling protein 2 regulates reactive oxygen species production, which influences both insulin and glucagon secretion.

Robson-Doucette CA, Sultan S, Allister EM, Wikstrom JD, Koshkin V, Bhattacharjee A, Prentice KJ, Sereda SB, Shirihai OS, Wheeler MB - Diabetes (2011)

Effective deletion of Ucp2 specifically from pancreatic β-cells using a Cre-lox recombination strategy. A: Targeting construct for the Ucp2 gene shows the floxed region (flanked by loxP sites) and the Ucp2 allele after Cre recombinase excision. Homozygote loxUCP2 mice that express Cre undergo recombination of the DNA between loxP sites, deleting exons 3 and 4, which contains the start codon. B: Typical PCR results from mouse genotyping for loxUCP2 (floxing) and Cre expression. C: Quantitative PCR results show reduced Ucp2 mRNA expression in isolated UCP2BKO islets compared with RIPCre islets. Ucp2 mRNA expression was calculated as a percentage of β-actin. The error bar shows the SEM. D: Standard PCR of various tissues shows full-length UCP2 transcripts and truncated UCP2 (Δ) where exons 3 and 4 have been removed by Cre recombinase activity. Wt, wild type. E, Two left panels: Dispersed pancreatic islets isolated from RIPCre and UCP2BKO mice immunostained for UCP2 (red) and insulin (green). Two right panels: UCP2BKO dispersed islet cells stained for Cre (red) and insulin (green) show nuclear localization of Cre recombinase. The yellow coloring represents colocalization of red and green fluorescence. n = 5–7. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198081&req=5

Figure 1: Effective deletion of Ucp2 specifically from pancreatic β-cells using a Cre-lox recombination strategy. A: Targeting construct for the Ucp2 gene shows the floxed region (flanked by loxP sites) and the Ucp2 allele after Cre recombinase excision. Homozygote loxUCP2 mice that express Cre undergo recombination of the DNA between loxP sites, deleting exons 3 and 4, which contains the start codon. B: Typical PCR results from mouse genotyping for loxUCP2 (floxing) and Cre expression. C: Quantitative PCR results show reduced Ucp2 mRNA expression in isolated UCP2BKO islets compared with RIPCre islets. Ucp2 mRNA expression was calculated as a percentage of β-actin. The error bar shows the SEM. D: Standard PCR of various tissues shows full-length UCP2 transcripts and truncated UCP2 (Δ) where exons 3 and 4 have been removed by Cre recombinase activity. Wt, wild type. E, Two left panels: Dispersed pancreatic islets isolated from RIPCre and UCP2BKO mice immunostained for UCP2 (red) and insulin (green). Two right panels: UCP2BKO dispersed islet cells stained for Cre (red) and insulin (green) show nuclear localization of Cre recombinase. The yellow coloring represents colocalization of red and green fluorescence. n = 5–7. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: The loxUCP2 mice were a gift from Dr. Bradford Lowell (32). β-Cell–specific UCP2 deletion was accomplished by crossing loxUCP2 mice with rat insulin promoter–driven Cre recombinase (RIPCre) mice (The Jackson Laboratory, Bar Harbor, ME). Mice were genotyped using standard PCR of ear notch DNA (Fig. 1B). RIPCre mice were chosen as controls for experimentation because RIPCre and floxed mice (mice that express the floxed Ucp2 gene without Cre) gave similar results (Supplementary Fig. 1). All mice (10–13 weeks old) were age- and sex-matched and maintained on a 129J-C57BL/6-mixed background. Unless otherwise indicated, male mice were used. All animal experiments were approved by the University of Toronto Animal Care Committee, and animals were handled according to the guidelines of the Canadian Council of Animal Care. Human islets from healthy donors were isolated using the Edmonton protocol and provided by the ABCC Human Islet Distribution Program (University of Alberta, Edmonton, AB, Canada). Donation was approved by the local institutional review board.

Bottom Line: UCP2BKO islets had elevated intracellular ROS levels that associated with enhanced GSIS.UCP2 does not behave as a classical metabolic uncoupler in the β-cell, but has a more prominent role in the regulation of intracellular ROS levels that contribute to GSIS amplification.In addition, β-cell UCP2 contributes to the regulation of intraislet ROS signals that mediate changes in α-cell morphology and glucagon secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT

Objective: The role of uncoupling protein 2 (UCP2) in pancreatic β-cells is highly debated, partly because of the broad tissue distribution of UCP2 and thus limitations of whole-body UCP2 knockout mouse models. To investigate the function of UCP2 in the β-cell, β-cell-specific UCP2 knockout mice (UCP2BKO) were generated and characterized.

Research design and methods: UCP2BKO mice were generated by crossing loxUCP2 mice with mice expressing rat insulin promoter-driven Cre recombinase. Several in vitro and in vivo parameters were measured, including respiration rate, mitochondrial membrane potential, islet ATP content, reactive oxygen species (ROS) levels, glucose-stimulated insulin secretion (GSIS), glucagon secretion, glucose and insulin tolerance, and plasma hormone levels.

Results: UCP2BKO β-cells displayed mildly increased glucose-induced mitochondrial membrane hyperpolarization but unchanged rates of uncoupled respiration and islet ATP content. UCP2BKO islets had elevated intracellular ROS levels that associated with enhanced GSIS. Surprisingly, UCP2BKO mice were glucose-intolerant, showing greater α-cell area, higher islet glucagon content, and aberrant ROS-dependent glucagon secretion under high glucose conditions.

Conclusions: Using a novel β-cell-specific UCP2KO mouse model, we have shed light on UCP2 function in primary β-cells. UCP2 does not behave as a classical metabolic uncoupler in the β-cell, but has a more prominent role in the regulation of intracellular ROS levels that contribute to GSIS amplification. In addition, β-cell UCP2 contributes to the regulation of intraislet ROS signals that mediate changes in α-cell morphology and glucagon secretion.

Show MeSH
Related in: MedlinePlus