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. 2013 May;62(5):1623-33.
doi: 10.2337/db12-0981. Epub 2013 Feb 22.

UCP2 regulates the glucagon response to fasting and starvation

Emma M Allister  1 , Christine A Robson-Doucette, Kacey J Prentice, Alexandre B Hardy, Sobia Sultan, Herbert Y Gaisano, Dong Kong, Patrick Gilon, Pedro L Herrera, Bradford B Lowell, Michael B Wheeler
Affiliations

UCP2 regulates the glucagon response to fasting and starvation

Emma M Allister et al. Diabetes. 2013 May.

Abstract

Glucagon is important for maintaining euglycemia during fasting/starvation, and abnormal glucagon secretion is associated with type 1 and type 2 diabetes; however, the mechanisms of hypoglycemia-induced glucagon secretion are poorly understood. We previously demonstrated that global deletion of mitochondrial uncoupling protein 2 (UCP2(-/-)) in mice impaired glucagon secretion from isolated islets. Therefore, UCP2 may contribute to the regulation of hypoglycemia-induced glucagon secretion, which is supported by our current finding that UCP2 expression is increased in nutrient-deprived murine and human islets. Further to this, we created α-cell-specific UCP2 knockout (UCP2AKO) mice, which we used to demonstrate that blood glucose recovery in response to hypoglycemia is impaired owing to attenuated glucagon secretion. UCP2-deleted α-cells have higher levels of intracellular reactive oxygen species (ROS) due to enhanced mitochondrial coupling, which translated into defective stimulus/secretion coupling. The effects of UCP2 deletion were mimicked by the UCP2 inhibitor genipin on both murine and human islets and also by application of exogenous ROS, confirming that changes in oxidative status and electrical activity directly reduce glucagon secretion. Therefore, α-cell UCP2 deletion perturbs the fasting/hypoglycemic glucagon response and shows that UCP2 is necessary for normal α-cell glucose sensing and the maintenance of euglycemia.

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Figures

FIG. 1.
FIG. 1.
UCP2 is efficiently deleted specifically from islet α-cells of UCP2AKO mice. A: Schematic diagram of the UCP2-targeting construct. B: Cre and UCP2 tissue expression measured by standard PCR. C: Immunostaining for UCP2 protein (red) and glucagon (green) in dispersed islet cells from Gcg-cre and UCP2AKO mice. UCP2AKO islet cells were also stained for Cre (red) and glucagon (green) or insulin (green). Colocalization is indicated by yellow and arrows. N = 3.
FIG. 2.
FIG. 2.
α-Cell UCP2 deletion reduces glucagon secretion in vivo. A: Blood glucose during an intraperitoneal insulin tolerance test (ipITT). *P < 0.05. N = 12–15. B: Plasma glucagon levels at 0 and 30 min during the ipITT. N = 6–8 mice/group. *P < 0.05; **P < 0.01.
FIG. 3.
FIG. 3.
UCP2AKO mice display normal glucose tolerance and GLP-1 secretion. A and B: Oral glucose tolerance test; mice were gavaged with 3 g/kg glucose after a 4-h fast (N = 7–8 mice/genotype). Blood glucose (A) and plasma active GLP-1 (B) were measured. The inset panel in A shows the incremental area under the glucose curve (iAUC).
FIG. 4.
FIG. 4.
α-Cell UCP2 deletion reduces the gluconeogenic response of the liver and switches fatty acid usage during a prolonged fast. Blood glucose (A), plasma glucagon (B), and plasma insulin (C) measured in fed, 24-h fasted (fasting), and 12-h re-fed mice. *P < 0.05. D–H: Hepatic expression of the enzymes Pepck (D), G6pc (E), Fbp1 (F), Gck (G), and Fasn (H) genes. N = 4–8/group. *P < 0.05 or **P < 0.01 compared with Gcg-cre. I: Hepatic glycogen levels. N = 4–6 mice/condition. *P < 0.05. J: Metabolomic analyses of MCFA and long-chain fatty acid (LCFA) plasma levels. Data are relative to Gcg-cre levels. N = 8 mice/genotype. *P < 0.05 genotype/fed-state interaction.
FIG. 5.
FIG. 5.
UCP2 expression is increased after nutrient depletion and glucagon secretion from UCP2AKO islets was impaired. Human (left panel) and mouse (right panel). A: UCP2 (i) and GCG (ii) expression in human islets exposed to low serum (0.01% FBS) and low glucose (2.8 mmol/L) RPMI (low serum and low glucose [LSLG]) for 6 h. N = 6 individual donors. B: Ucp2 (i) and Gcg (ii) expression in control CD1 mouse islets exposed to LSLG for 6 h. N = 4–5/condition. C: Glucose-inhibited glucagon secretion from human islets preincubated with or without genipin (50 µmol/L) during a half-hour static incubation. N = 6–7 donors. D: Glucose-inhibited glucagon secretion was measured from fresh-isolated islets incubated with high glucose (HG) (20 mmol/L), low glucose (LG) (1 mmol/L), or low glucose plus arginine (LG + Arg) (10 mmol/L). N = 8–9 mice/genotype. E: Glucose-inhibited glucagon secretion measured from islets preincubated with or without genipin (50 µmol/L). N = 7–9 mice/genotype. All mouse data are normalized to DNA, but the human data are per islet. *P < 0.05.
FIG. 6.
FIG. 6.
UCP2AKO α-cells display enhanced hyperpolarization of ΔΨm and increased superoxide levels. A: Representative traces of ΔΨm (Mmp) relative fluorescent unit (RFU) measurements in YFP+ α-cells. B: ΔΨm (Mmp) was measured in high glucose (20 mmol/L) (Δ1) and NaN3 (5 mmol/L) (Δ2) and normalized to basal fluorescence in the presence of low glucose (1 mmol/L). N = 28–29 cells from 6 mice/genotype. *P < 0.05. C and D: Fresh-isolated islets were cultured with or without H2O2 (16 µmol/L) or genipin (50 µmol/L) before measurement of H2O2 (CM-H2-DCFDA) (C) or superoxide (MitosoxRed) (D). N = 20–28 islets, 6–8 mice/genotype. *P < 0.05, **P < 0.01. E: Islets were cultured overnight, and then intracellular H2O2 (CM-H2-DCFDA) was measured. N = 19–27 islets, 5–7 mice/genotype. *P < 0.05. F: Islet Sod2 expression. N = 4–5 mice/genotype. *P < 0.05. G: Glucose-inhibited glucagon secretion with or without H2O2 (16 µmol/L). Secretion is per microgram of DNA, normalized to Gcg-cre high glucose (HG) conditions. LG, low glucose. N = 3–11 mice/genotype. *P < 0.05, **P < 0.01. H and I: Human islets were incubated with 50 µmol/L genipin for 2 h before measurement of H2O2 (CM-H2-DCFDA) (H) and superoxide (MitosoxRed) (I) levels. N = 4 individual donors. *P < 0.05, **P < 0.01.
FIG. 7.
FIG. 7.
UCP2AKO α-cells have more depolarized plasma membranes and reduced intracellular calcium. A: Representative traces of plasma membrane potential in YFP+ α-cells incubated in high (20 mmol/L) and low (1 mmol/L) glucose. B: Average membrane potential in YFP+ α-cells. N = 5–8 cells, 3 mice/genotype. *P < 0.05. C: Average plasma membrane potential measured in YFP+ α-cells (N = 8–13) with or without genipin (50 µmol/L) (N = 5 cells), and at least 3 mice/genotype. *P < 0.05, **P < 0.01. D: Glucose-inhibited glucagon secretion from islets incubated with or without diazoxide (1, 10, and 100 µmol/L) in the presence of high glucose (20 mmol/L) (i) or low glucose (1 mmol/L) (ii). *P < 0.05. N = 3 mice/genotype. E: Representative traces of cytosolic calcium uptake in YFP+ α-cells switched from high glucose (HG) (20 mmol/L) to low glucose (LG) (1 mmol/L) and then in the presence of arginine (Arg) (10 mmol/L), KCl (30 mmol/L), and the l-type calcium blocker nifedipine (Nif) (10 µmol/L). F: Incremental area under the curve (iAUC) for cytosolic calcium, normalized to high glucose plus arginine levels. Representative traces are shown in Supplementary Fig. 8. *P < 0.05. N = 19–40 cells/treatment, 3 mice/genotype.
FIG. 8.
FIG. 8.
UCP2 is required for normal glucagon secretion in response to hypoglycemia. A: UCP2AKO islets displayed increased glucose-induced hyperpolarization of ΔΨm, which caused increased ROS and perhaps ATP production. The altered coupling and ROS status resulted in greater depolarization of the plasma membrane, and although still responsive to changes in glucose, the UCP2AKO α-cells showed reduced calcium entry and lower glucagon secretion. B: UCP2 expression is normally increased in islets deprived of nutrients. Therefore, lack of UCP2 in α-cells results in impaired glucagon secretion during hypoglycemia. ITT, insulin tolerance test; TCA, tricarboxylic acid.
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