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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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UCP2BKO islets display ROS-dependent enhanced GSIS. A: Measurement of GSIS over time in RIPCre and UCP2BKO islets by perifusion with varying concentrations of glucose (2.8, 11, and 16.7 mmol/L). Areas under the curve (AUC) are shown for 11 mmol/L glucose (B) and 16.7 mmol/L glucose (C). n = 6. *P < 0.05. Measurement of GSIS after manipulation of intracellular ROS levels in RIPCre (D) and UCP2BKO (E) islets using the pro-oxidant DEM (5 mmol/L) or the antioxidant NAC (0.2 mmol/L) by static incubation. Islets were preincubated in 2.8 mmol/L glucose for 1 h (±5 mmol/L DEM or 0.2 mmol/L NAC), followed by 30-min incubation in 2.8 or 16.7 mmol/L glucose (±5 mmol/L DEM or 0.2 mmol/L NAC). Insulin secretion was measured in nanograms of insulin per islet. n = 4. *P < 0.05. F: Measurement of GSIS in human islets in the presence or absence of 50 µmol/L genipin. Human islets were subjected to a similar static secretion protocol as mouse islets. Acute preincubation in genipin, a known UCP2 inhibitor, stimulates insulin secretion from human islets. n = 3 independent experiments. **P < 0.01. LG, low glucose; HG, high glucose. The error bars show the SEM.
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Figure 4: UCP2BKO islets display ROS-dependent enhanced GSIS. A: Measurement of GSIS over time in RIPCre and UCP2BKO islets by perifusion with varying concentrations of glucose (2.8, 11, and 16.7 mmol/L). Areas under the curve (AUC) are shown for 11 mmol/L glucose (B) and 16.7 mmol/L glucose (C). n = 6. *P < 0.05. Measurement of GSIS after manipulation of intracellular ROS levels in RIPCre (D) and UCP2BKO (E) islets using the pro-oxidant DEM (5 mmol/L) or the antioxidant NAC (0.2 mmol/L) by static incubation. Islets were preincubated in 2.8 mmol/L glucose for 1 h (±5 mmol/L DEM or 0.2 mmol/L NAC), followed by 30-min incubation in 2.8 or 16.7 mmol/L glucose (±5 mmol/L DEM or 0.2 mmol/L NAC). Insulin secretion was measured in nanograms of insulin per islet. n = 4. *P < 0.05. F: Measurement of GSIS in human islets in the presence or absence of 50 µmol/L genipin. Human islets were subjected to a similar static secretion protocol as mouse islets. Acute preincubation in genipin, a known UCP2 inhibitor, stimulates insulin secretion from human islets. n = 3 independent experiments. **P < 0.01. LG, low glucose; HG, high glucose. The error bars show the SEM.

Mentions: To investigate the effect of β-cell–specific UCP2 deletion on insulin secretion, GSIS was measured in RIPCre and UCP2BKO isolated islets using an islet perifusion system (Fig. 4A and B). At 2.8 mmol/L glucose, RIPCre and UCP2BKO secreted similar amounts of insulin. At 11 mmol/L glucose, insulin secretion was more rapid and robust in the UCP2BKO islets (Fig. 4B). Similarly, insulin secretion was greater in UCP2BKO islets compared with RIPCre controls at 16.7 mmol/L glucose (Fig. 4C), demonstrating that UCP2 deficiency in β-cells enhances GSIS but has no effect on insulin secretion at low glucose concentrations. In addition, preincubation of human islets with genipin, a known UCP2 inhibitor (37), resulted in significantly enhanced GSIS (Fig. 4F), further proof that acute inhibition of UCP2 enhances GSIS. To our knowledge, this is the first demonstration that the role of UCP2 in mouse islets is shared with human islets.


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)

UCP2BKO islets display ROS-dependent enhanced GSIS. A: Measurement of GSIS over time in RIPCre and UCP2BKO islets by perifusion with varying concentrations of glucose (2.8, 11, and 16.7 mmol/L). Areas under the curve (AUC) are shown for 11 mmol/L glucose (B) and 16.7 mmol/L glucose (C). n = 6. *P < 0.05. Measurement of GSIS after manipulation of intracellular ROS levels in RIPCre (D) and UCP2BKO (E) islets using the pro-oxidant DEM (5 mmol/L) or the antioxidant NAC (0.2 mmol/L) by static incubation. Islets were preincubated in 2.8 mmol/L glucose for 1 h (±5 mmol/L DEM or 0.2 mmol/L NAC), followed by 30-min incubation in 2.8 or 16.7 mmol/L glucose (±5 mmol/L DEM or 0.2 mmol/L NAC). Insulin secretion was measured in nanograms of insulin per islet. n = 4. *P < 0.05. F: Measurement of GSIS in human islets in the presence or absence of 50 µmol/L genipin. Human islets were subjected to a similar static secretion protocol as mouse islets. Acute preincubation in genipin, a known UCP2 inhibitor, stimulates insulin secretion from human islets. n = 3 independent experiments. **P < 0.01. LG, low glucose; HG, high glucose. The error bars show the SEM.
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Figure 4: UCP2BKO islets display ROS-dependent enhanced GSIS. A: Measurement of GSIS over time in RIPCre and UCP2BKO islets by perifusion with varying concentrations of glucose (2.8, 11, and 16.7 mmol/L). Areas under the curve (AUC) are shown for 11 mmol/L glucose (B) and 16.7 mmol/L glucose (C). n = 6. *P < 0.05. Measurement of GSIS after manipulation of intracellular ROS levels in RIPCre (D) and UCP2BKO (E) islets using the pro-oxidant DEM (5 mmol/L) or the antioxidant NAC (0.2 mmol/L) by static incubation. Islets were preincubated in 2.8 mmol/L glucose for 1 h (±5 mmol/L DEM or 0.2 mmol/L NAC), followed by 30-min incubation in 2.8 or 16.7 mmol/L glucose (±5 mmol/L DEM or 0.2 mmol/L NAC). Insulin secretion was measured in nanograms of insulin per islet. n = 4. *P < 0.05. F: Measurement of GSIS in human islets in the presence or absence of 50 µmol/L genipin. Human islets were subjected to a similar static secretion protocol as mouse islets. Acute preincubation in genipin, a known UCP2 inhibitor, stimulates insulin secretion from human islets. n = 3 independent experiments. **P < 0.01. LG, low glucose; HG, high glucose. The error bars show the SEM.
Mentions: To investigate the effect of β-cell–specific UCP2 deletion on insulin secretion, GSIS was measured in RIPCre and UCP2BKO isolated islets using an islet perifusion system (Fig. 4A and B). At 2.8 mmol/L glucose, RIPCre and UCP2BKO secreted similar amounts of insulin. At 11 mmol/L glucose, insulin secretion was more rapid and robust in the UCP2BKO islets (Fig. 4B). Similarly, insulin secretion was greater in UCP2BKO islets compared with RIPCre controls at 16.7 mmol/L glucose (Fig. 4C), demonstrating that UCP2 deficiency in β-cells enhances GSIS but has no effect on insulin secretion at low glucose concentrations. In addition, preincubation of human islets with genipin, a known UCP2 inhibitor (37), resulted in significantly enhanced GSIS (Fig. 4F), further proof that acute inhibition of UCP2 enhances GSIS. To our knowledge, this is the first demonstration that the role of UCP2 in mouse islets is shared with human islets.

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