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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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Glucose-induced mitochondrial membrane hyperpolarization is mildly increased in UCP2BKO β-cells, while islet ATP content and uncoupled respiration rates remain unchanged. A: Representative traces of ΔΨm measurements using rhodamine123 in selected large cells (β-cells) from dispersed islets isolated from UCP2BKO and RIPCre mice. ΔΨm was measured initially in the presence of a low (2.8 mmol/L) glucose concentration. Membrane hyperpolarization was induced by the addition of high (20 mmol/L) glucose concentration, and 5 mmol/L sodium azide (NaN3) was used to completely depolarize the mitochondrial membrane to ensure membrane integrity. n = 5 mice/genotype. RFU, relative fluorescence units. B: Measurements of ΔΨm were normalized to basal fluorescence in the presence of low (2.8 mmol/L) glucose concentration and then expressed relative to RIPCre control ΔΨm levels. Δ1, the change in mitochondrial membrane hyperpolarization in response to increased glucose concentration. Δ2, the change in mitochondrial membrane depolarization in response to NaN3 above basal mitochondrial membrane potential. (20–30 cells/coverslip with 3 coverslips per animal were measured.) n = 5 mice/genotype. ***P < 0.001. C: Uncoupled OCR is unchanged in UCP2BKO islets. OCR was measured under saturating concentrations of oligomycin (5 µmol/L) with 3 or 20 mmol/L glucose. Measurements shown are after steady state was achieved. n = 3 separate experiments, with 13–15 measurements per condition. D: Basal OCR is increased in UCP2BKO islets. Measurements are steady state in 3 mmol/L glucose. **P < 0.01. n = 3 separate experiments with 27 (control) and 30 (UCP2BKO) measurements per condition. E: Glucose-stimulated oxygen consumption (expressed as a percentage of the basal OCR) is similar between UCP2BKO and RIPCre islets. Both genotypes experience a 1.5-fold increase in OCR after stimulation with glucose. OCRs were measured in 3 and 20 mmol/L glucose. Measurements were taken after steady state was achieved. n = 4 separate experiments with 18 (control) and 20 (UCP2BKO) measurements per condition. F: Islet ATP content in RIPCre and UCP2BKO islets cultured overnight and incubated in low (2.8 mmol/L) or high (16.7 mmol/L) glucose for 30 min. n = 3 mice/genotype; *P < 0.05. LG, low glucose; HG, high glucose. The error bars show the SEM.
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Figure 2: Glucose-induced mitochondrial membrane hyperpolarization is mildly increased in UCP2BKO β-cells, while islet ATP content and uncoupled respiration rates remain unchanged. A: Representative traces of ΔΨm measurements using rhodamine123 in selected large cells (β-cells) from dispersed islets isolated from UCP2BKO and RIPCre mice. ΔΨm was measured initially in the presence of a low (2.8 mmol/L) glucose concentration. Membrane hyperpolarization was induced by the addition of high (20 mmol/L) glucose concentration, and 5 mmol/L sodium azide (NaN3) was used to completely depolarize the mitochondrial membrane to ensure membrane integrity. n = 5 mice/genotype. RFU, relative fluorescence units. B: Measurements of ΔΨm were normalized to basal fluorescence in the presence of low (2.8 mmol/L) glucose concentration and then expressed relative to RIPCre control ΔΨm levels. Δ1, the change in mitochondrial membrane hyperpolarization in response to increased glucose concentration. Δ2, the change in mitochondrial membrane depolarization in response to NaN3 above basal mitochondrial membrane potential. (20–30 cells/coverslip with 3 coverslips per animal were measured.) n = 5 mice/genotype. ***P < 0.001. C: Uncoupled OCR is unchanged in UCP2BKO islets. OCR was measured under saturating concentrations of oligomycin (5 µmol/L) with 3 or 20 mmol/L glucose. Measurements shown are after steady state was achieved. n = 3 separate experiments, with 13–15 measurements per condition. D: Basal OCR is increased in UCP2BKO islets. Measurements are steady state in 3 mmol/L glucose. **P < 0.01. n = 3 separate experiments with 27 (control) and 30 (UCP2BKO) measurements per condition. E: Glucose-stimulated oxygen consumption (expressed as a percentage of the basal OCR) is similar between UCP2BKO and RIPCre islets. Both genotypes experience a 1.5-fold increase in OCR after stimulation with glucose. OCRs were measured in 3 and 20 mmol/L glucose. Measurements were taken after steady state was achieved. n = 4 separate experiments with 18 (control) and 20 (UCP2BKO) measurements per condition. F: Islet ATP content in RIPCre and UCP2BKO islets cultured overnight and incubated in low (2.8 mmol/L) or high (16.7 mmol/L) glucose for 30 min. n = 3 mice/genotype; *P < 0.05. LG, low glucose; HG, high glucose. The error bars show the SEM.

Mentions: To determine the contribution of UCP2 to mitochondrial uncoupling, the total OCR and OCR in the presence of saturating amounts of oligomycin were measured, the latter representing the uncoupled fraction of respiration. UCP2BKO and RIPCre islets exhibited similar uncoupled OCRs (Fig. 2C) under both basal and glucose-stimulated conditions, suggesting that UCP2 deficiency in the β-cell does not increase mitochondrial coupling. Interestingly, UCP2BKO islets showed higher total OCR compared with RIPCre islets (Fig. 2D), with both genotypes experiencing approximately 1.5-fold increased OCR upon stimulation with 20 mmol/L glucose (Fig. 2E). No difference in islet size was observed between the two groups (Supplementary Fig. 5).


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)

Glucose-induced mitochondrial membrane hyperpolarization is mildly increased in UCP2BKO β-cells, while islet ATP content and uncoupled respiration rates remain unchanged. A: Representative traces of ΔΨm measurements using rhodamine123 in selected large cells (β-cells) from dispersed islets isolated from UCP2BKO and RIPCre mice. ΔΨm was measured initially in the presence of a low (2.8 mmol/L) glucose concentration. Membrane hyperpolarization was induced by the addition of high (20 mmol/L) glucose concentration, and 5 mmol/L sodium azide (NaN3) was used to completely depolarize the mitochondrial membrane to ensure membrane integrity. n = 5 mice/genotype. RFU, relative fluorescence units. B: Measurements of ΔΨm were normalized to basal fluorescence in the presence of low (2.8 mmol/L) glucose concentration and then expressed relative to RIPCre control ΔΨm levels. Δ1, the change in mitochondrial membrane hyperpolarization in response to increased glucose concentration. Δ2, the change in mitochondrial membrane depolarization in response to NaN3 above basal mitochondrial membrane potential. (20–30 cells/coverslip with 3 coverslips per animal were measured.) n = 5 mice/genotype. ***P < 0.001. C: Uncoupled OCR is unchanged in UCP2BKO islets. OCR was measured under saturating concentrations of oligomycin (5 µmol/L) with 3 or 20 mmol/L glucose. Measurements shown are after steady state was achieved. n = 3 separate experiments, with 13–15 measurements per condition. D: Basal OCR is increased in UCP2BKO islets. Measurements are steady state in 3 mmol/L glucose. **P < 0.01. n = 3 separate experiments with 27 (control) and 30 (UCP2BKO) measurements per condition. E: Glucose-stimulated oxygen consumption (expressed as a percentage of the basal OCR) is similar between UCP2BKO and RIPCre islets. Both genotypes experience a 1.5-fold increase in OCR after stimulation with glucose. OCRs were measured in 3 and 20 mmol/L glucose. Measurements were taken after steady state was achieved. n = 4 separate experiments with 18 (control) and 20 (UCP2BKO) measurements per condition. F: Islet ATP content in RIPCre and UCP2BKO islets cultured overnight and incubated in low (2.8 mmol/L) or high (16.7 mmol/L) glucose for 30 min. n = 3 mice/genotype; *P < 0.05. LG, low glucose; HG, high glucose. The error bars show the SEM.
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Figure 2: Glucose-induced mitochondrial membrane hyperpolarization is mildly increased in UCP2BKO β-cells, while islet ATP content and uncoupled respiration rates remain unchanged. A: Representative traces of ΔΨm measurements using rhodamine123 in selected large cells (β-cells) from dispersed islets isolated from UCP2BKO and RIPCre mice. ΔΨm was measured initially in the presence of a low (2.8 mmol/L) glucose concentration. Membrane hyperpolarization was induced by the addition of high (20 mmol/L) glucose concentration, and 5 mmol/L sodium azide (NaN3) was used to completely depolarize the mitochondrial membrane to ensure membrane integrity. n = 5 mice/genotype. RFU, relative fluorescence units. B: Measurements of ΔΨm were normalized to basal fluorescence in the presence of low (2.8 mmol/L) glucose concentration and then expressed relative to RIPCre control ΔΨm levels. Δ1, the change in mitochondrial membrane hyperpolarization in response to increased glucose concentration. Δ2, the change in mitochondrial membrane depolarization in response to NaN3 above basal mitochondrial membrane potential. (20–30 cells/coverslip with 3 coverslips per animal were measured.) n = 5 mice/genotype. ***P < 0.001. C: Uncoupled OCR is unchanged in UCP2BKO islets. OCR was measured under saturating concentrations of oligomycin (5 µmol/L) with 3 or 20 mmol/L glucose. Measurements shown are after steady state was achieved. n = 3 separate experiments, with 13–15 measurements per condition. D: Basal OCR is increased in UCP2BKO islets. Measurements are steady state in 3 mmol/L glucose. **P < 0.01. n = 3 separate experiments with 27 (control) and 30 (UCP2BKO) measurements per condition. E: Glucose-stimulated oxygen consumption (expressed as a percentage of the basal OCR) is similar between UCP2BKO and RIPCre islets. Both genotypes experience a 1.5-fold increase in OCR after stimulation with glucose. OCRs were measured in 3 and 20 mmol/L glucose. Measurements were taken after steady state was achieved. n = 4 separate experiments with 18 (control) and 20 (UCP2BKO) measurements per condition. F: Islet ATP content in RIPCre and UCP2BKO islets cultured overnight and incubated in low (2.8 mmol/L) or high (16.7 mmol/L) glucose for 30 min. n = 3 mice/genotype; *P < 0.05. LG, low glucose; HG, high glucose. The error bars show the SEM.
Mentions: To determine the contribution of UCP2 to mitochondrial uncoupling, the total OCR and OCR in the presence of saturating amounts of oligomycin were measured, the latter representing the uncoupled fraction of respiration. UCP2BKO and RIPCre islets exhibited similar uncoupled OCRs (Fig. 2C) under both basal and glucose-stimulated conditions, suggesting that UCP2 deficiency in the β-cell does not increase mitochondrial coupling. Interestingly, UCP2BKO islets showed higher total OCR compared with RIPCre islets (Fig. 2D), with both genotypes experiencing approximately 1.5-fold increased OCR upon stimulation with 20 mmol/L glucose (Fig. 2E). No difference in islet size was observed between the two groups (Supplementary Fig. 5).

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