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. 2009 Feb 26;150(7):3040–3048. doi: 10.1210/en.2008-1642

Persistent Oxidative Stress Due to Absence of Uncoupling Protein 2 Associated with Impaired Pancreatic β-Cell Function

Jingbo Pi 1, Yushi Bai 1, Kiefer W Daniel 1, Dianxin Liu 1, Otis Lyght 1, Diane Edelstein 1, Michael Brownlee 1, Barbara E Corkey 1, Sheila Collins 1
PMCID: PMC2703519  PMID: 19246534

Abstract

Uncoupling protein (UCP) 2 is a widely expressed mitochondrial protein whose precise function is still unclear but has been linked to mitochondria-derived reactive oxygen species production. Thus, the chronic absence of UCP2 has the potential to promote persistent reactive oxygen species accumulation and an oxidative stress response. Here, we show that Ucp2−/− mice on three highly congenic (N >10) strain backgrounds (C57BL/6J, A/J, 129/SvImJ), including two independently generated sources of Ucp2-null animals, all exhibit increased oxidative stress. Ucp2-null animals exhibit a decreased ratio of reduced glutathione to its oxidized form in blood and tissues that normally express UCP2, including pancreatic islets. Islets from Ucp2−/− mice exhibit elevated levels of numerous antioxidant enzymes, increased nitrotyrosine and F4/80 staining, but no change in insulin content. Contrary to results in Ucp2−/− mice of mixed 129/B6 strain background, glucose-stimulated insulin secretion in Ucp2−/− islets of each congenic strain was significantly decreased. These data show that the chronic absence of UCP2 causes oxidative stress, including in islets, and is accompanied by impaired glucose-stimulated insulin secretion.

Genetic background of mice lacking UCP2 has major influence on their results and interpretation; results show oxidative stress and impaired insulin secretion in all backcrossed strains.

Mitochondria are the primary source of ATP: the metabolic currency of the cell (1). They are also the source of most of the reactive oxygen species (ROS) that are generated in cells (2). Similar to the net rate of ATP synthesis in mitochondria, the production of superoxide (· O2−) from the mitochondrial matrix, such as at complex I, is highly sensitive to the proton motive force (2,3). Consequently, uncoupling or any other process that reduces the mitochondrial membrane potential can substantially decrease mitochondria-derived ROS. Because this, in turn, is believed to ameliorate oxidative damage to cell constituents (2,3), this mechanism could be particularly important for cell types that are not endowed with a robust antioxidant defense system, such as pancreatic β-cells (4).

Uncoupling protein (UCP) 2 is a widely expressed mitochondrial inner membrane carrier protein that was discovered through its sequence homology to the brown fat UCP1. UCP1 dissipates caloric energy as heat by uncoupling mitochondrial respiration from ATP production (5). Although the biochemical functions of UCP2 are still much debated (6), there is strong evidence that UCP2 is not a physiologically relevant “uncoupling protein” in the manner of UCP1 and does not contribute to adaptive thermogenesis (3,7). Instead, accumulating evidence supports the idea that UCP2 participates in the control of mitochondria-derived ROS (2,8). For example, the genetic absence of UCP2 results in increased ROS production in macrophages (9,10), whereas acute overexpression of UCP2 in vitro (immortalized β-cells) and in vivo (neurons) is protective against overt oxidative damage (11,12). More recently, a role has been presented for UCP2 in metabolic sensing through mitochondrial respiration in hypothalamic neuronal populations (13). In addition, as with most antioxidant responses, the transcription of the Ucp2 gene itself is highly inducible under conditions of oxidative stress. For example, agents such as H2O2 (11), lipopolysaccharide (14), TNFα (15), free fatty acids (16), irradiation (17), as well as high-fat diet challenge (18,19) have all increased UCP2 expression in vitro or in vivo. Thus, there is good evidence that a major physiological function of UCP2 is to attenuate mitochondrial production of ROS such that activation of UCP2 and/or its increased expression may function as an adaptive response to oxidative stress.

Although most often considered to be solely a wasteful and damaging by-product of metabolism, ROS have, in some instances, come to be appreciated as important factors in normal cellular signal transduction processes (20,21). In the case of β-cells, evidence is emerging that in addition to ATP and the ATP to ADP ratio, ROS derived from glucose metabolism, in particular H2O2, serve as metabolic signals to mediate glucose-stimulated insulin secretion (GSIS) (22,23,24) as well as neuronal glucose sensing (25). Nevertheless, excessive or sustained ROS production has been recognized as a chief culprit in the development of β-cell dysfunction in diabetes (26,27). To counteract oxidative stress most cells, including β-cells, are equipped with mechanisms to protect against oxidative damage by recruiting a suite of antioxidant enzymes and small molecules such as glutathione [reduced glutathione (GSH)] to scavenge and eliminate ROS. In response to intracellular ROS accumulation, the cellular ROS-scavenging capacity is bolstered in part by specifically increasing the cellular levels of many antioxidant enzymes (24,28). This innate adaptive response to oxidative stress is vital for proper oxidation/reduction (redox) homeostasis to protect cells from irreversible oxidative damage (28,29). However, a possible consequence of this augmented cellular ROS-scavenging ability could be the potential to blunt a normal signaling function of ROS. In support of this idea, our recent studies have shown not only a role for ROS as a “signal” in β-cells but that acute oxidative stress in islets or INS-1 (832/13) cells induces cellular antioxidants, and inhibits both glucose-stimulated ROS production and GSIS (24). Therefore, the signaling role of ROS in GSIS coupled with the negative regulatory effect of UCP2 on mitochondria-derived ROS supports the hypothesis that UCP2 is an endogenous suppressor of insulin secretion (30). Consistent with this hypothesis, overexpression of UCP2 in isolated β-cells has inhibited GSIS (31,32,33) and short-term “knockdown” of UCP2 in mouse islets, or inhibition of UCP2 activity with a small molecule has acutely increased GSIS (34,35).

Given the overwhelming evidence that UCP2 is a physiologically relevant negative regulator of ROS production (2,3,36,37), the chronic absence of this protein as achieved by targeted deletion methods (to date there are no reports of naturally occurring null mutants) has the potential to lead to persistent ROS accumulation and an adaptive oxidative stress response, in particular in those tissues with high UCP2 expression, including pancreatic β-cells. Despite intensive research focused on oxidative stress and UCP2, cellular adaptive responses to the chronic absence of UCP2 in β-cell dysfunction have not been addressed. In the work described here, we provide evidence from in vivo and in vitro studies that the chronic absence of UCP2 results in persistent oxidative stress and impairment of β-cell function.

Materials and Methods

Animals

One line of Ucp2−/− mice was originally created in our laboratory on a 129X1/SvJ/C57BL/6J mixed genetic background (36). At the F2 generation, backcrossing was initiated to C57BL/6J (B6), 129S1/SvImJ (129), and A/J for more than 12 generations each, using alternating male and female stock mice from The Jackson Laboratory (Bar Harbor, ME): B6 (JAX stock no. 000664), 129 (JAX stock no. 002448), and A/J (JAX stock no. 000646). Another independently generated line of Ucp2−/− mice (30) subsequently backcrossed to B6 for 10 generations was obtained from The Jackson Laboratory (JAX stock no. 005934: B6.129S4-Ucp2tm1Lowl/J, referred to hereafter as JAX B6 Ucp2−/−). For experiments, male age-matched (10–16 wk old) mice were used. Animals were housed in virus-free facilities on a 12-h light, 12-h dark cycle and were fed standard rodent food. Genotyping was performed by PCR using genomic DNA isolated from tail snips. All protocols for animal use were approved by the Institutional Animal Care and Use Committee of The Hamner Institutes, and were in accordance with National Institutes of Health guidelines.

Reagents

Fatty acid-free BSA, HISTOPAQUE-1077, Hanks’ balanced salt solution, β-mercaptoethanol, and glucose solution (45%) were obtained from Sigma-Aldrich Corp. (St. Louis, MO). Culture media, fetal bovine serum, and supplements were purchased from Invitrogen Corp. (Carlsbad, CA).

Measurement of glutathione (GSH) and glutathione disulfide [oxidized glutathione (GSSG)]

Levels of total glutathione (GSH plus GSSG) and GSSG in blood were measured immediately after collection using the BIOXYTECH GSH/GSSG-412 kit (OxisResearch, Portland, OR) according to the manufacturer’s protocols. Samples for GSSG measurement were immediately mixed with thiol-scavenging reagent 1-methyl-2-vinyl-pyridium trifluoromethane sulfonate after separation. The concentrations of GSH were calculated by the equation: GSH = total glutathione − (2 × GSSG). For tissue measurements of GSSG and total glutathione, animals were euthanized by CO2 asphyxiation, followed by cardiac puncture, and quickly perfused three times with ice-cold PBS. Tissues were homogenized in cold PBS immediately after isolation with or without 10% 1-methyl-2-vinyl-pyridium trifluoromethane sulfonate, followed by centrifugation at 12,000 × g for 5 min. The resulting supernatants were used for measurement of GSSG and total glutathione, respectively.

Immunohistochemistry

Pancreata from Ucp2+/+ and −/− littermates were immersion fixed in 10% neutral-buffered formalin for 24 h and then transferred to 70% ethanol. The tissues were embedded in paraffin, sectioned at 5 μm thickness, placed on slides, and stored at room temperature until processed. Slides were deparaffinized through three changes of xylene each for 3 min, then rehydrated through descending grades of ethanol to water. Antigen retrieval was performed using Target Retrieval Solution (Dako Corp., Carpinteria, CA) in a rice steamer for 20 min. After cooling at room temperature, endogenous peroxidases were blocked with 3% hydrogen peroxide for 5 min. The slides were then incubated overnight at 4 C with the following primary antibodies: nitrotyrosine (rabbit affinity isolated, 1:500; Sigma-Aldrich); F4/80 (rat antimouse IgG2b, 1:100; AbD Serotec, Raleigh, NC); and insulin (guinea pig antihuman IgG, 1:750; Millipore Corp., Billerica, MA) diluted in Antibody Diluent (Dako). After rinsing in PBS, slides were incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA), followed by incubation with Streptavidin Horseradish Peroxidase (Zymed Laboratories, Inc., South San Francisco, CA). Slides were rinsed in distilled water and reacted with AEC chromogen (Zymed Laboratories) for 5 min to observe color reaction. After counterstaining slides with hematoxylin (Vector Laboratories), slides were coverslipped using Crystal Mount (Electron Microscopy Sciences, Hatfield, PA).

Islet isolation, primary culture, and measurement of insulin secretion

Pancreatic islets were isolated from 9- to 12-wk-old mice by collagenase P (F. Hoffmann-La Roche Ltd., Basel, Switzerland) digestion and cultured as previously described (24). Before GSIS measurement, isolated islets were cultured overnight in RPMI 1640 media supplemented with 10 mm glucose, 10% fetal bovine serum, 2 mm l-glutamine, 100 U penicillin/ml, and 100 μg streptomycin/ml. Measurements of insulin secretion were either performed in static incubation or oscillatory perfusion conditions exactly as detailed previously (24,38).

ROS determination in isolated islets

ROS levels in individual islets were measured by confocal microscopy using the fluorescent probe 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA; Invitrogen) as described previously (24). In brief, islets were seeded onto 10% Matrigel (Invitrogen) in glass-bottomed culture dishes (MatTek, Ashland, MA). The fluorescence images were obtained using a laser scanning confocal microscope (LSM 510 NLO) mounted on an Axiovert 100M microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY), using a 488-nm laser for excitation, and LP 505 filter for emission. The software used for acquisition is Zeiss LSM510 version 3.2 (Carl Zeiss Microimaging). The final concentration of CM-H2DCFDA used was 2 μm, and the preloading time was 60 min. ROS levels in groups of islets were measured by spectrofluorometer. Briefly, 100 islets were incubated with 3 μm CM-H2DCFDA in Krebs buffer containing 3 or 16.7 mm glucose for 1 h and rinsed three times with cold BSA-free Krebs buffer. The islets were then sonicated immediately in 100 μl cold PBS and centrifuged at 5000 rpm for 5 min. The fluorescence in the supernatants was measured using a Wallace Victor3 1420 multilabel counter (PerkinElmer Analytical Life Sciences, Inc., Waltham, MA) (λex = 485 nm; λem = 540 nm).

Measurement of H2O2-scavenging activity

Isolated islets from five mice of each genotype were cultured in RPMI 1640 media supplemented with 10 mm glucose for 24 h. The islets were then washed three times with ice-cold PBS and lysed in the same by sonication, followed by centrifugation at 12,000 × g for 5 min. The resulting supernatants were used immediately for measurement of H2O2-scavenging activity. Briefly, 30 μmol/liter H2O2 in PBS was incubated with the islet lysates (3.8 μg protein/100 μl) for 30 min. The H2O2 remaining in the solutions was measured using the Amplex Red Hydrogen Peroxide Assay Kit (Invitrogen). The difference in H2O2 concentrations between lysate-treated and a PBS control represents the H2O2-scavenging activity. Protein concentrations were determined by Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA) using BSA as a standard.

Quantitative real-time RT-PCR analysis

Total RNA was isolated with TRIzol (Invitrogen) and then subjected to cleanup using ribonuclease-free deoxyribonuclease and the RNeasy Mini kit (QIAGEN, Inc., Valencia, CA). The resultant DNA-free RNA was quantitated by UV spectroscopy at 260 nm and stored in ribonuclease-free H2O at −70 C. Quantitative real-time PCR was performed as described previously (39). Briefly, total RNA from each sample was reverse transcribed with MuLV reverse transcriptase and oligo deoxythymidine primers (Applied Biosystems, Foster City, CA). The SYBR Green PCR Kit (Applied Biosystems) was used for quantitative real-time RT-PCR analysis. The primers were designed using Primer Express software (Applied Biosystems) and synthesized by MWG-BIOTECH Inc. (High Point, NC). Relative differences in gene expression between groups were determined from cycle time (Ct) values. These values were first normalized to 18S in the same sample (ΔCt) and expressed as fold over control. Real-time fluorescence detection was performed using an ABI PRISM 7900 Sequence Detector (Applied Biosystems). The forward and reverse primer sequences for selected genes were designed with the ABI Primer Express software (Applied Biosystems), and the sequences are shown in supplemental Table S1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org.

Statistical analyses

All statistical analyses were performed using GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA), with P < 0.05 taken as significant. More specific indices of statistical significance are indicated in the individual figure legends. Data are expressed as mean ± sem. For comparisons between groups, a Student’s t test was performed. Statistical analyses to evaluate the insulin secretion and H2O2 production in isolated islets of Ucp2+/+ and −/− mice in response to different concentrations of glucose were performed using two-way ANOVA with Bonferroni post hoc testing.

Results

Oxidative stress in Ucp2-null mice

Accumulating evidence supports a major role for UCP2 as a factor that modulates mitochondria-derived ROS generation in response to various stimuli (2,3). However, the possibility that a deficiency of UCP2 in vivo might cause oxidative stress in general has not been investigated. Therefore, levels of GSH and GSSG in blood and various tissues were measured. GSH is the most important, as well as abundant, redox buffer in cells (40). In its reaction to scavenge peroxides, GSH is converted by oxidation to GSSG, which can subsequently be reduced back to GSH by glutathione reductase (GR). The balance between GSH and GSSG has long been recognized as an important indicator of oxidative stress (40). As shown in Fig. 1A, left, in the B6 background, blood GSH levels showed a precipitous decrease in Ucp2−/− mice, with GSSG levels markedly increased (Fig. 1A, middle). As a result, the GSH to GSSG ratios in mice lacking UCP2 were dramatically decreased from 176 ± 57.0 to 9.4 ± 2.0 (Fig. 1A, right). This represents a difference of nearly 20-fold. Ucp2−/− congenic mice in the 129 (Fig. 1B) and the A/J (Fig. 1C) strain backgrounds also showed a decreased blood GSH, enhanced GSSG, and a reduced GSH to GSSG ratio. The GSH to GSSH ratio did not reach statistical significance in the A/J background. (It is difficult to obtain large numbers of A/J Ucp2−/− mice due to low-breeder performance of the heterozygous mating pairs.) Finally, Fig. 1D shows that the JAX B6 Ucp2−/− mice, originally generated elsewhere (30) and subsequently backcrossed to B6 exhibit the same dramatic increase in GSSG and reversal of the GSH to GSSG ratio as the B6 Ucp2−/− mice in Fig. 1A. Note also that the absolute values for GSH differ among strains; this will be discussed later. Therefore, this finding across strains is consistent with the interpretation that the absence of UCP2 evokes a state of oxidative stress. Because the B6 strain is commonly used for studies of diet-induced obesity, insulin resistance, and atherosclerosis, many of our studies were conducted in this strain background, with data from the other strains included as appropriate to illustrate the overall consistency of phenotype resulting from the absence of UCP2.

Figure 1.

Figure 1

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Blood levels of glutathione in its reduced (GSH) and oxidized (GSSG) states and the ratio of GSH to GSSG in Ucp2−/− and wild-type mice on three genetic backgrounds. B6 (generation N19-20) (A), 129 (N12-13) (B), and A/J (N13) (C) background. *, P < 0.05 vs. Ucp2+/+ (n = 3–5 of each genotype for each respective strain). D, JAX B6 background. *, P < 0.05 vs. Ucp2+/+ (n = 6–7).

Although blood levels of GSH and GSSG are sensitive in vivo biomarkers for oxidative stress, these measurements do not indicate the primary source of oxidative stress. Because the expression of UCP2 varies widely among tissues and cell types (36,41,42), GSH to GSSG ratios in various tissues of B6 were determined immediately after whole body perfusion and tissue isolation. As shown in Fig. 2, A–C, B6/Ucp2−/− mice exhibited a significantly decreased GSH to GSSG ratio in lung, spleen, and pancreatic islets: tissues in which levels of UCP2 in normal wild-type mice are relatively high (36,41,42). In contrast, in tissues with relatively low or no UCP2 expression, such as heart (Fig. 2D) and liver (Fig. 2E), there were no differences in GSH to GSSG ratios between the genotypes. These results suggest that in those tissues with high basal levels of UCP2, it serves an important role in the regulation of cellular redox status.

Figure 2.

Figure 2

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Ratios of GSH to GSSG in lung (A), spleen (B), pancreatic islets (C), heart (D), and liver (E) of B6/Ucp2+/+ and −/− mice. *, P < 0.05 vs. Ucp2+/+ (n = 5 for each genotype).

In addition to measuring GSH and GSSG, the existence of oxidative stress can also be monitored by various biomarkers such as nitrotyrosine (43). As shown in Fig. 3, immunohistochemical staining for nitrotyrosine in the pancreas revealed slightly elevated levels in islets of Ucp2−/− mice compared with wild-type animals. Furthermore, the macrophage marker F4/80 was also increased in Ucp2−/− islets, suggesting a heightened macrophage infiltration. From examining several different mice (n = 4 for each genotype) and four tissue sections per mouse, there were no significant differences in islet mass or insulin content between the two genotypes. These data from intact tissues provide additional evidence to suggest that the chronic absence of UCP2 results in mild but detectable oxidative stress in pancreatic islets.

Figure 3.

Figure 3

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Representative immunohistochemistry images of pancreas from B6/Ucp2+/+ and −/− mice. A–C, Insulin. D–F, Nitrotyrosine. G–I, F4/80. n = 4 mice for each genotype. Black arrows indicate islets.

Increased compensatory oxidative stress response in pancreatic islets of Ucp2−/− mice

In the islets from either B6/Ucp2−/− (Fig. 4A) or 129/Ucp2−/− (Fig. 4B) mice, we observed that transcripts for a broad array of antioxidant enzymes, including superoxide dismutases (SODs), glutathione peroxidase (GPx) 2 and 4, γ-glutamate cysteine ligase catalytic subunit (GCLC), GSH synthetase (GSH-S), catalase (CAT), thioredoxin (Trx), and heme oxygenase-1 (HO1), were all significantly elevated. Consistent with this pattern of elevated expression of antioxidant enzymes, total cellular H2O2-inactivating activity was also significantly higher in islets of Ucp2−/− mice compared with wild-type (supplemental Fig. S1).

Figure 4.

Figure 4

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Increased expression of antioxidant enzymes in isolated islets of Ucp2−/− mice. A, B6 strain background. B, 129 strain background. Islets were prepared, and mRNA was isolated for real-time RT-PCR measurements, normalized to levels of 18S RNA (n = 3 for each genotype). *, P < 0.05 vs. Ucp2+/+. NQO1, Reduced nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1; Prx1, peroxiredoxin 1.

Acute glucose-stimulated ROS production is decreased in islets of Ucp2−/− mice

As we reported previously (24), in response to an acute glucose challenge, there was a marked increase in H2O2 production in islets of wild-type B6 mice, as measured by CM-H2DCFDA-derived fluorescence (Fig. 5A, upper panels). However, in Ucp2−/− islets, there was no discernible glucose-stimulated H2O2 production, and basal H2O2 fluorescence at 3 mm glucose was actually higher than in wild-type B6 islets (Fig. 5A, lower panels). Similar decreases in glucose-stimulated H2O2 production coupled with an elevated basal level were also observed in 129/Ucp2−/− compared with 129/Ucp2+/+ islets (Fig. 5B).

Figure 5.

Figure 5

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Loss of acute glucose-stimulated H2O2 production in isolated islets of Ucp2−/− mice. A, Representative fluorescence images indicating H2O2 in isolated islets of B6/Ucp2+/+ and −/− mice (n = 3 mice each). H2O2 was determined by confocal microscopy using the H2O2-sensitive fluorescent probe CM-H2DCFDA. B, H2O2-derived fluorescence in isolated islets of B6/Ucp2+/+ and −/− and 129/Ucp2+/+ and −/− mice. At least 100 islets were used for each condition, and the quantification was by spectrofluorometry (n = 6 mice each). *, P < 0.05 vs. Ucp2+/+ at 3 mm glucose (G); #, P < 0.05 vs. Ucp2+/+ at 16.7 mm glucose. AU, Arbitrary units.

Impaired GSIS in islets of Ucp2−/− mice

In experiments that were originally performed in our Ucp2−/− mice of the B6/129 mixed background (36), we observed significantly increased GSIS in isolated islets from these mice (Fig. 6A), as had been reported by others (30). However, to our surprise, once the mice were backcrossed into inbred strain backgrounds, all four lines of Ucp2−/− mice now show a significantly decreased GSIS in Ucp2−/− islets compared with those from the wild-type mice (Fig. 6, B–F). This was observed by the eighth backcross generation into B6 (Fig. 6B), and the phenotype has persisted through subsequent backcrossing (Fig. 6C). An identical phenotype was observed in islets from Ucp2−/− mice that had been similarly backcrossed to the 129 (Fig. 6D) or A/J backgrounds (Fig. 6E), as well as from the JAX B6 Ucp2−/− mice (Fig. 6F). Consistent with the finding that there was decreased GSIS from Ucp2−/− islets, insulin secretion in response to the sulfhydryl depletor diethyl maleate (supplemental Fig. S2) or KCl (supplemental Fig. S3) was also lower in Ucp2−/− islets from each of the strains relative to wild-type islets. The consistent results of diminished GSIS we have obtained from three independently generated highly congenic lines, at different backcross generations, and from different original targeted deletion constructs, suggest that the chronic absence of UCP2 results in oxidative stress and impairs pancreatic β-cell function. However, although GSIS was significantly decreased in isolated islets of these mice, there was no overt hyperglycemia (supplemental Fig. S4), hypoinsulinemia (supplemental Table S2), or glucose intolerance (supplemental Fig. S4). An interesting possibility for these findings could be that because UCP2 is not expressed in the liver, and shows limited expression in skeletal muscle (two key organs for glucose disposal), the lack of UCP2 from these organs may not affect their oxidant/antioxidant status. Thus, there could be preserved or even increased insulin sensitivity in these organs, so that the GTT does not show abnormal glucose tolerance. Future experiments that include clamp studies, high-fat diet challenges, and β-cell specific disruption of the Ucp2 gene will likely shed light on the molecular basis of this impaired GSIS and its physiological consequences.

Figure 6.

Figure 6

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GSIS in isolated islets of Ucp2+/+ and −/− mice on different genetic backgrounds. A, 129/B6 mixed background (n = 5–6 mice per group, each measured in triplicate). B, B6 background (backcross generation N8) (n = 6 mice per group, each measured in triplicate). C, B6 background (N19–20) (n = 6–9 mice per group, each measured in triplicate). D, 129 background (N12–13) (n = 3–4 mice per group, each measured in triplicate). E, A/J background (N13) (n = 4 mice per group, each measured in triplicate). F, JAX B6 background (n = 5 mice per group, each measured in triplicate). *, P < 0.05 vs. Ucp2+/+ at 3 mm glucose; #, P < 0.05 vs. Ucp2+/+ at 16.7 mm glucose.

Discussion

UCP2 appears to function as part of a feedback loop regulating mitochondria-derived ROS, and an assortment of mechanisms have been proposed beyond bona fide uncoupling (3,7,44). As such, its chronic absence may lead to a net accumulation of ROS and/or an adaptive oxidative stress response, particularly in those tissues with a relatively high basal level of UCP2 expression. In the current study, we provide direct in vivo evidence that the absence of UCP2 in mice results in significant oxidative stress. In addition to a decreased GSH and/or elevated GSSG in islets of Ucp2−/− mice, immunohistochemistry staining revealed elevated levels of nitrotyrosine. Nitrotyrosine is considered a valid in vivo marker for peroxynitrite (43), which is a highly reactive species formed from nitric oxide and · O2− when both are locally increased (45). The accumulation of this marker in islets of Ucp2−/− mice suggests a persistent local oxidative stress. We also observed modestly increased staining for the macrophage cell surface marker F4/80 (46), suggesting that the absence of UCP2 might be promoting some macrophage infiltration and/or inflammation in the islets. These findings are consistent with our previous reports that Ucp2−/− mice exhibit increased ROS and nitric oxide production in spleen and macrophages (36,37).

Oxidative stress is a common denominator among the various mechanisms proposed for β-cell dysfunction and the progression to frank diabetes (47,48,49). In most tissues, cells exhibit an early “adaptive” response to oxidative stress that stimulates the cellular antioxidant system to protect them from oxidative damage. Such an acute compensatory response is generally appropriate to protect against ROS toxicity (28,29). However, over time the combination of chronic oxidative stress coupled with continuously elevated antioxidants perturbs normal homeostasis.

There are examples of normal homeostasis of ROS generation that might serve a positive role in cells (50,51). A relevant case in point is the recent proposal that transient ROS generation in β-cells in response to glucose serves as a “signal” for insulin secretion (22,23,24). In this context, one could envision that under normal acute, short-term circumstances, a well-controlled and buffered production of ROS could act as a signal for GSIS, but chronic uncontrolled ROS production (as in Ucp2−/− mice or even due to metabolic overload in uncontrolled type-2 diabetes) might lead to oxidative stress that then correlates with reduced GSIS and β-cell dysfunction. Although this scenario has not been directly examined, it may apply to GSIS in Ucp2−/− mice. For example, under low-glucose conditions, H2O2 levels were already elevated in islets from Ucp2−/− mice, and this was accompanied by increases of several antioxidant enzymes, including the H2O2-scavenging enzymes GPxs and CAT. However, in response to a glucose challenge, the net increase in H2O2 production seen in the Ucp2−/− islets was substantially lower than what we typically observe in wild-type mice (24), and was also accompanied by a reduced GSIS in Ucp2−/− islets. Therefore, one hypothesis stemming from these findings is that the adaptive response to chronic oxidative stress caused by the absence of UCP2 may have interfered with the “beneficial” aspects of ROS as a glucose-dependent signal for insulin secretion. A direct test of this concept will require the future generation and validation of additional models. Because elevated levels of nitrotyrosine and F4/80 were also observed in Ucp2−/− islets, other factors independent of impaired ROS signaling, such as oxidative damage or inflammation, might also contribute to the impaired β-cell function in Ucp2−/− mice. In addition, because the absence of UCP2 leads to the decreased GSH to GSSH ratio in several tissues, including lung, spleen, islet, and blood, it is possible that the β-cell dysfunction and increased expression of antioxidant genes may be secondary to the general oxidative stress.

The difference in phenotype between Ucp2−/− mice of 129/B6 mixed background vs. these congenic lines suggests that genetic background affects the phenotype of Ucp2−/− mice. Concerns regarding the contribution of genetic background to the interpretation of phenotypes in targeted knockout studies are, in fact, not new. Genetic background and the confounding effects of genes that flank the area of the targeted allele in genetically manipulated mice have been intensively investigated (52,53,54,55). As discussed by others (52,54,55), in “knockout” mice generated by the approach using embryonic stem cells derived from 129 substrains crossed with B6, selection for the null allele derived from 129 also includes a large number of flanking genes surrounding the targeted allele, whereas littermates with the wild-type allele will possess the equivalent regions from B6. In most cases this genetic bias is of limited concern. However, when the phenotype ascribed to the targeted disruption is already vastly different between the two inbred strains, it is prudent to consider the possibility of a confounding effect of genetic background. In such cases backcrossing to several inbred strains significantly reduces the flanking area that is linked to the selected targeted allele, in addition to achieving homozygosity at all other loci (52,53,54,55). It has already been established by others that B6 mice exhibit a remarkably lower glucose tolerance and defective GSIS from islets compared with other inbred mice, including 129 (56,57,58). It is interesting that isolated islets from 129 and B6 mice obtained from The Jackson Laboratory and compared side by side indicate a 15-fold higher GSIS in 129 mice than B6 mice, a slightly higher basal level of secretion from B6, but no differences in total islet insulin content (supplemental Fig. S5). This striking difference between 129 and B6 mice, coupled with the inconsistent phenotype of Ucp2−/− mice of 129/B6 mix generated in two different laboratories vs. these same mice in the B6, 129, or A/J backgrounds, together suggests that we cannot dismiss genetic background as a potential contributor to the widely reported heightened secretion observed in Ucp2−/− mice of mixed strain parentage (30,35). Indeed, as shown in Fig. 6A, we were able to replicate independently the phenotype of increased GSIS using our own 129/B6 Ucp2−/− mice. Unfortunately, once we were successful in generating the congenic lines of Ucp2−/− mice, there was no perceived need to retain these mixed background mice in our inventory, and so for cost reasons, they were eliminated.

It should be noted that a recent study has attributed the reduced GSIS in B6 mice to the deletion of several exons of the nicotinamide nucleotide transhydrogenase (Nnt) gene in this strain (from The Jackson Laboratory colony) (58). Although it was not measured directly in that study, this loss of Nnt function was proposed to result in decreased NADPH generation and, thus, less GSSG being reduced back to GSH, leading to H2O2 accumulation and subsequent “activation” of proton leak by UCP2. Consequently, in this situation increased UCP2 activity and proton leak would lower ATP production and GSIS. However, the noticeably higher GSH to GSSG ratio we observe (Fig. 1) in wild-type B6 mice (71 and 37.6 times higher than that in 129 and A/J, respectively) does not appear on the surface to support such a scenario. In addition, Aston-Mourney et al. (59) recently reported that replacement of the truncated B6 Nnt allele with the corresponding DBA/2 allele restored the expression and activity of full-length Nnt. However, this maneuver had no effect on the B6 phenotype in terms of either insulin secretion or overall glucose tolerance, suggesting that the truncated Nnt allele (58) in and of itself is not solely responsible for the lower insulin secretion observed in B6 mice. Indeed, different technical approaches were used that might contribute to this discrepancy. The study by Freeman et al. (58) introduced a large bacterial artificial chromosome clone from a 129-strain library into B6 embryos to provide a full-length Nnt allele, whereas Aston-Mourney et al. (59) used recombinant inbred and reciprocal congenic mice of the B6 and DBA/2 strains that contained the Nnt allele and a limited number of other annotated genes. Obviously, this subject needs to be investigated further, but it, nevertheless, highlights the fact that this issue is neither straightforward nor resolved.

Based upon our findings that disruption of the Ucp2 gene causes persistent oxidative stress in general and impairs β-cell function, it raises the question as to whether inhibiting expression or activity of UCP2 is an appropriate therapeutic approach for type 2 diabetes to improve GSIS, considering that other negative consequences of the absence of UCP2 include increased development of atherosclerotic plaque (60, 61) neurodegeneration (62) and a heightened propensity for colon tumors (63). Certainly, the chronic absence of UCP2 likely represents a significantly different physiology from any acute moment-to-moment manipulation of its level or activity. Therefore, given the apparent paradoxical roles for UCP2 in β-cell function that now exist, a better understanding of the role of UCP2 in the function of pancreatic β-cells and pathogenesis of diabetes is needed.

Supplementary Material

[Supplemental Data]
en.2008-1642_index.html (2.9KB, html)

Acknowledgments

We thank Drs. Claire Pecqueur, Ann Petro, Richard Surwit, and Qiang Zhang, Ms. Lisa M. Floering, and Mr. Steve Rockwood for discussions and technical contributions to this study. We also thank Drs. Ed Leiter, Michael Seldin, David Threadgill, and Alan Attie for advice and helpful discussions concerning mouse genetics and backcrossing. We also wish to commemorate Diane Edelstein, who passed away during the preparation of this manuscript. Her zest for life, friendship, and research made our collaboration rich and full of fun. She will remain in our memory.

Footnotes

This research was supported by the National Institutes of Health Grants DK35914 (to B.E.C.), DK54024 (to S.C.), DK76788 (to J.P.), and ES016005 (to J.P.).

Disclosure Summary: The content is solely the responsibility of the authors, and all authors affirm that they have nothing to disclose.

First Published Online February 26, 2009

For editorial see page 2994

Abbreviations: CAT, Catalase; CM-H2DCFDA, 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester; Ct, cycle time; GCLC, γ-glutamate cysteine ligase catalytic subunit; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSH-S, reduced glutathione synthetase; GSIS, glucose-stimulated insulin secretion; GSSG, oxidized glutathione; HO1, heme oxygenase 1; Nnt, nicotinamide nucleotide transhydrogenase; · O2−, superoxide; ROS, reactive oxygen species; SOD, superoxide dismutase; Trx, thioredoxin; UCP, uncoupling protein.

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Supplementary Materials

[Supplemental Data]
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en.2008-1642_1.pdf (10.4KB, pdf)
en.2008-1642_2.pdf (13.2KB, pdf)
en.2008-1642_3.pdf (10.8KB, pdf)
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