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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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Higher intracellular ROS levels and antioxidant gene expression in UCP2BKO islets. A: Fluorescent microscopy was used to image intracellular H2O2 levels in islets isolated from RIPCre and UCP2BKO mice that were cultured overnight and incubated with the ROS-sensitive fluorescent dye CM-H2-DCFDA, in the presence of 11 mmol/L glucose. Islet intracellular H2O2 levels were manipulated by incubation of RIPCre islets with (+) or without (−) the pro-oxidant DEM (5 mmol/L) or UCP2BKO islets with (+) or without (−) the antioxidant NAC (0.2 mmol/L). Data shown are expressed as the fold-change over RIPCre islets. Representative fluorescent microscopy images of each condition are shown above each bar. n = 11–16 islets from 4 mice/genotype. *P < 0.05, ***P < 0.0001. B: Measurement of intracellular H2O2 in β-cells selected from dispersed islet cells. H2O2 was measured as in A. A total of 20–30 larger cells (i.e. β-cells) were selected for each coverslip. n = 5–7. C: Quantitative PCR was used to quantify the expression of several antioxidant genes as well as the oxidative stress-responsive HO-1 in isolated islets. Data shown are expressed as a percentage of β-actin mRNA expression levels for n = 3–7. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 3: Higher intracellular ROS levels and antioxidant gene expression in UCP2BKO islets. A: Fluorescent microscopy was used to image intracellular H2O2 levels in islets isolated from RIPCre and UCP2BKO mice that were cultured overnight and incubated with the ROS-sensitive fluorescent dye CM-H2-DCFDA, in the presence of 11 mmol/L glucose. Islet intracellular H2O2 levels were manipulated by incubation of RIPCre islets with (+) or without (−) the pro-oxidant DEM (5 mmol/L) or UCP2BKO islets with (+) or without (−) the antioxidant NAC (0.2 mmol/L). Data shown are expressed as the fold-change over RIPCre islets. Representative fluorescent microscopy images of each condition are shown above each bar. n = 11–16 islets from 4 mice/genotype. *P < 0.05, ***P < 0.0001. B: Measurement of intracellular H2O2 in β-cells selected from dispersed islet cells. H2O2 was measured as in A. A total of 20–30 larger cells (i.e. β-cells) were selected for each coverslip. n = 5–7. C: Quantitative PCR was used to quantify the expression of several antioxidant genes as well as the oxidative stress-responsive HO-1 in isolated islets. Data shown are expressed as a percentage of β-actin mRNA expression levels for n = 3–7. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: To further investigate the function of UCP2 in β-cells, a cell-permeable fluorescent H2O2 indicator (CM-H2-DCFDA), was used to measure intracellular ROS levels. Similar to that shown in islets from whole-body KO mice (26–28), UCP2BKO isolated islets (Fig. 3A) and β-cells (Fig. 3B) displayed significantly higher intracellular H2O2 levels compared with RIPCre control mice, demonstrating that UCP2 plays a role in the regulation of intracellular ROS levels in the β-cell. Subsequently, basal intracellular ROS levels were correlated with the expression of several ROS-responsive genes by quantitative PCR (Fig. 3C). In UCP2BKO islets, upregulated expression of the H2O2-scavenging glutathione peroxidase 1 (Gpx1) and Gpx4 genes was observed (Fig. 3C). In addition, Gpx2, Gpx3, and catalase (Cat) all showed a trend toward higher gene expression in UCP2BKO islets, but these values did not reach statistical significance. Interestingly, the expression of heme oxygenase-1 (HO-1), which is induced by oxidative stress and confers cytoprotection to limit tissue damage (36), was unchanged in UCP2BKO islets (Fig. 3C). These findings suggest that long-term elevation of ROS in UCP2BKO islets induces changes in signal transduction pathways that lead to altered antioxidant gene expression; however, these ROS levels are not high enough to induce oxidative stress/damage that requires cytoprotection by HO-1.


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)

Higher intracellular ROS levels and antioxidant gene expression in UCP2BKO islets. A: Fluorescent microscopy was used to image intracellular H2O2 levels in islets isolated from RIPCre and UCP2BKO mice that were cultured overnight and incubated with the ROS-sensitive fluorescent dye CM-H2-DCFDA, in the presence of 11 mmol/L glucose. Islet intracellular H2O2 levels were manipulated by incubation of RIPCre islets with (+) or without (−) the pro-oxidant DEM (5 mmol/L) or UCP2BKO islets with (+) or without (−) the antioxidant NAC (0.2 mmol/L). Data shown are expressed as the fold-change over RIPCre islets. Representative fluorescent microscopy images of each condition are shown above each bar. n = 11–16 islets from 4 mice/genotype. *P < 0.05, ***P < 0.0001. B: Measurement of intracellular H2O2 in β-cells selected from dispersed islet cells. H2O2 was measured as in A. A total of 20–30 larger cells (i.e. β-cells) were selected for each coverslip. n = 5–7. C: Quantitative PCR was used to quantify the expression of several antioxidant genes as well as the oxidative stress-responsive HO-1 in isolated islets. Data shown are expressed as a percentage of β-actin mRNA expression levels for n = 3–7. *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 3: Higher intracellular ROS levels and antioxidant gene expression in UCP2BKO islets. A: Fluorescent microscopy was used to image intracellular H2O2 levels in islets isolated from RIPCre and UCP2BKO mice that were cultured overnight and incubated with the ROS-sensitive fluorescent dye CM-H2-DCFDA, in the presence of 11 mmol/L glucose. Islet intracellular H2O2 levels were manipulated by incubation of RIPCre islets with (+) or without (−) the pro-oxidant DEM (5 mmol/L) or UCP2BKO islets with (+) or without (−) the antioxidant NAC (0.2 mmol/L). Data shown are expressed as the fold-change over RIPCre islets. Representative fluorescent microscopy images of each condition are shown above each bar. n = 11–16 islets from 4 mice/genotype. *P < 0.05, ***P < 0.0001. B: Measurement of intracellular H2O2 in β-cells selected from dispersed islet cells. H2O2 was measured as in A. A total of 20–30 larger cells (i.e. β-cells) were selected for each coverslip. n = 5–7. C: Quantitative PCR was used to quantify the expression of several antioxidant genes as well as the oxidative stress-responsive HO-1 in isolated islets. Data shown are expressed as a percentage of β-actin mRNA expression levels for n = 3–7. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: To further investigate the function of UCP2 in β-cells, a cell-permeable fluorescent H2O2 indicator (CM-H2-DCFDA), was used to measure intracellular ROS levels. Similar to that shown in islets from whole-body KO mice (26–28), UCP2BKO isolated islets (Fig. 3A) and β-cells (Fig. 3B) displayed significantly higher intracellular H2O2 levels compared with RIPCre control mice, demonstrating that UCP2 plays a role in the regulation of intracellular ROS levels in the β-cell. Subsequently, basal intracellular ROS levels were correlated with the expression of several ROS-responsive genes by quantitative PCR (Fig. 3C). In UCP2BKO islets, upregulated expression of the H2O2-scavenging glutathione peroxidase 1 (Gpx1) and Gpx4 genes was observed (Fig. 3C). In addition, Gpx2, Gpx3, and catalase (Cat) all showed a trend toward higher gene expression in UCP2BKO islets, but these values did not reach statistical significance. Interestingly, the expression of heme oxygenase-1 (HO-1), which is induced by oxidative stress and confers cytoprotection to limit tissue damage (36), was unchanged in UCP2BKO islets (Fig. 3C). These findings suggest that long-term elevation of ROS in UCP2BKO islets induces changes in signal transduction pathways that lead to altered antioxidant gene expression; however, these ROS levels are not high enough to induce oxidative stress/damage that requires cytoprotection by HO-1.

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