OBJECTIVE—Recent evidence suggests that the AMP-activated protein kinase (AMPK) is an important therapeutic target for diabetes. The present study was conducted to determine how AMPK activation suppressed tyrosine nitration of prostacyclin synthase in diabetes.
RESEARCH DESIGN AND METHODS—Confluent human umbilical vein endothelial cells (HUVECs) or mice were treated with 5-amino-4-imidazole carboxamide riboside (AICAR) for the detection of AMPK phosphorylation and the expression of mitochondrial uncoupling protein (UCP)-2.
RESULTS—Exposure of HUVECs to high glucose (30 mmol/l) increased superoxide anions (O2·−) and prostacyclin synthase nitration. In addition, overexpression of constitutively active AMPK (Ad-CA-AMPK) or the addition of AICAR reduced both O2·− and prostacyclin synthase nitration caused by high glucose, whereas adenoviral overexpression of dominant-negative AMPK mutants (Ad-DN-AMPK) enhanced the latter effects of high glucose. Exposure of HUVECs to either AICAR or metformin caused AMPK-dependent upregulation of both UCP-2 mRNA and UCP-2 protein. Furthermore, overexpression of UCP-2 significantly ablated both O2·− and prostacyclin synthase nitration triggered by high glucose. Furthermore, overexpression of Ad-CA-AMPK increased, whereas overexpression of Ad-DN-AMPK inhibited AICAR-induced phosphorylation of p38 kinase at Thr180/Tyr182. Inhibition of p38 kinase with SB239063, which had no effect on AICAR-induced AMPK-Thr172 phosphorylation, dose dependently suppressed AICAR-induced upregulation of UCP-2, suggesting that AMPK lies upstream of p38 kinase. Finally, AICAR markedly increased UCP-2 expression and reduced both O2·− and prostacyclin synthase nitration in diabetic wild-type mice but not in their AMPKα2-deficient counterparts in vivo.
CONCLUSIONS—We conclude that AMPK activation increases UCP-2, resulting in the inhibition of both O2·− and prostacyclin synthase nitration in diabetes.
AMP-activated protein kinase (AMPK) is a heterotrimer made up of α-, β-, and γ-subunits, each of which has at least two isoforms (1–3). Increases in the AMP-to-ATP ratio activate AMPK by a number of mechanisms, including direct allosteric activation and α-subunit phosphorylation (at Thr172) by at least two AMPK kinases (i.e., LKB1 and calcium calmodulin–dependent kinase kinase [caMKK]) (4). AMPK is ubiquitous and is activated in a variety of cell types by inhibition of ATP production (i.e., anoxia and ischemia) or acceleration of ATP consumption (i.e., muscle contraction and fasting). As first noted by Hardie and Carling (1), AMPK activation appears to be a fundamental component of cellular responses to stresses that threaten cell viability. AMPK is phosphorylated and activated in various tissues by hormones acting through Gq receptors (5), adiponectin (6,7), leptin (8,9), α- and β-adrenoreceptor agonists (10), metformin (11), thiazolidinediones (12), and oxidants, such as peroxynitrite (ONOO−) (13,14) and H2O2 (15). Activation of AMPK leads to the phosphorylation of a number of target molecules, resulting in, among other things, increased fatty acid oxidation and muscle glucose transport (to generate more ATP) and inhibition of various biosynthetic processes (to conserve ATP) (16). Increasing evidence suggests that the functions of AMPK are beyond energy metabolism. For example, both endothelial nitric oxide (NO) synthase (eNOS) and neuronal NO synthase (nNOS) are targets of AMPK in the endothelium and muscle (17,18). Winder and colleagues (19,20) have shown that treatment of rats with 5-amino-4-imidazole carboxamide riboside (AICAR) increases the expression of a wide variety of proteins in muscle, including the GLUT-4 glucose transporter and several mitochondrial oxidative enzymes. AMPK activation has also been shown to increase the expression of mitochondrial uncoupling protein (UCP)-2 in liver after infection with constitutively active AMPK (Ad-CA-AMPK) (21). Similar effects of AMPK on UCP2 and UCP3 have been reported in skeletal muscle (22).
Strong accumulating evidence suggests that oxidative stress, defined as increased formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and/or decreased antioxidant potentials, plays an important role in the development of diabetic complications (23–27). This hypothesis is supported by the finding that many biochemical pathways strictly associated with hyperglycemia (glucose auto-oxidation, polyol pathway, prostanoid synthesis, and protein glycation) increase the production of free radicals and oxidants (27). The functions of many proteins are likely affected by increased oxidant levels. We have found (24–26) that prostacyclin synthase, an enzyme releasing vasoprotective prostacyclin, is particularly susceptible to tyrosine nitration by RNS, including ONOO−. In cultured endothelial cells, hyperglycemic medium increases the levels of nitrated prostacyclin synthase and decreases prostacyclin synthase activity (20,23). Tyrosine nitration of prostacyclin synthase and consequent thromboxane receptor activation are thought to be important mechanisms contributing to the initiation and progression of vascular complications in diabetes (rev. in 23). This is because of the downregulation of the protective actions of NO and prostacyclin and accumulation of nonmetabolized prostaglandin H2, which promotes platelet aggregation, atheroma accumulation, and thrombus formation (23).
Emerging data support a role for ROS and RNS in cell signaling. Lee and Griendling (28) found that angiotensin II augments O2·− production in smooth muscle cells via NADH/NADPH oxidase-like enzymatic activity. This enzymatic system now appears to be involved in a number of “maladaptive” characteristics of atherosclerosis, such as PDGF-induced cell proliferation (29), smooth muscle cell hypertrophy (30), diabetic retinopathy (31), and impaired NO bioactivity (32). Our earlier results had also demonstrated that pathologically relevant concentrations of ONOO− are capable of activating AMPK independently of changes in AMP/ATP and that ONOO−-dependent AMPK activation occurs during hypoxia reoxygenation (13) and in metformin-treated endothelial cells (33). However, the consequences of AMPK activation on cellular oxidative stress remain to be determined. In the present study, we provide evidence that AMPK prevents oxidative stress associated with diabetes, in part, by upregulating mitochondrial UCP-2.
RESEARCH DESIGN AND METHODS
A full description of the research design and methods, including adenoviral infection, RT-PCR of UCP-2 mRNA, prostacyclin synthase activity assays, NO bioactivity, immunocytochemistry, and high-performance liquid chromatography (HPLC) detection of 3-nitrotyrosine can be found in an online appendix available at http://dx.doi.org/10.2337/db08-0610.
The animal protocol was reviewed and approved by the institutional animal care and use committee. Male AMPKα2 knockout (KO) (AMPK−/−) mice, which had been cross-bred with C57BL6 mice, were bred at the animal house of the University of Oklahoma Health Sciences Center. Their littermates, C57BL6 mice, were obtained from The Jackson Laboratories (Bar Harbor, ME). Mice were housed in temperature-controlled cages with a 12-h light/dark cycle and given free access to water and normal chow. Mice aged 10 weeks were randomly divided into control and treated groups.
AMPKα2 KO mice and age-matched C57BL/6 mice were used to study whether AICAR attenuates diabetes-enhanced UCP-2 expression and prostacyclin synthase nitration. The mice were made diabetic after five consecutive injections of 50 mg/kg streptozotocin (STZ) in citrate buffer, pH 4.5. Nondiabetic mice were injected with a comparable volume of citrate buffer. Glucose levels were measured in tail blood by a Free Style blood glucose monitoring system (TheraSense, Alameda, CA). Hyperglycemia was confirmed by nonfasting blood glucose >200 mg/dl (11 mmol/l) 1 week after the initial STZ injection. The nondiabetic and diabetic mice were randomly divided into three groups: control, STZ/untreated, and STZ but treated with AICAR (250 mg · kg−1 · day−1 s.c.). Six weeks later, the animals were killed, and the aorta was collected for analysis.
Human umbilical vein endothelial cells (HUVECs) (American Type Culture Collection, Rockville, MD) were cultured in endothelial basal media (EBM). When they reached confluence, the cells were maintained in 1% fetal calf serum and exposed to normal glucose (5.5 mmol/l) or high glucose (30 mmol/l) for 3–7 days, during which the medium was changed every 2 days. Control groups (to account for media hyperosmolarity) were exposed to mannitol (24.5 mmol/l) in normal medium containing glucose (5.5 mmol/l). After incubation, the media were collected, and assays were conducted as described below. For measurements of O2·− and cyclic GMP, the growth medium was replaced by PBS containing no glucose. Unless otherwise noted, these measurements were performed on cells or the PBS bathing cells, which were stimulated by the calcium ionophore A23187 (10−5 mol/l, 2 h).
Measurement of intracellular ROS.
Intracellular O2·− was measured using the dihydroethidium (DHE) fluorescence/HPLC assay (34) with minor modification. Briefly, HUVECs were incubated with 0.5 μmol/l DHE for 30 min, harvested, and then methanol extracted. Oxyethidium (a product of DHE and O2·−) and ethidium (a product of DHE auto-oxidation) were separated and quantified using a C-18 HPLC column (mobile phase: gradient of acetonitrile and 0.1% trifluoroacetic acid). O2·− production was determined by the conversion of DHE into oxyethidium. ROS production in aorta was assayed using 5 μmol/l lucigenin chemiluminescence as described previously (24–26).
Statistical comparison was performed using a one- or two-way ANOVA, and intergroup differences were determined using the Bonferroni inequality. Values are expressed as means ± SE. P < 0.05 was considered significant.
AICAR reduces high glucose–induced oxidative stress in HUVECs. Our earlier studies (24,25) demonstrated that hyperglycemia not only increases O2·− production and tyrosine nitration of prostacyclin synthase but also reduces NO bioactivity, as determined by cyclic GMP levels. A subsequent study (32) revealed that the AMPK activator, metformin, dramatically attenuates the latter effect. To understand whether the beneficial effects of AMPK activation may be attributable to its ability to reduce oxidative stress, we tested the effect of AICAR on markers of oxidative stress in HUVECs. Confluent HUVECs were exposed to 5 mmol/l d-glucose (normal glucose), 30 mmol/l d-glucose (high glucose), or 5 mmol/l d-glucose plus 25 mmol/l mannitol (OG) for 72 h with or without 0.5 mmol/l AICAR. High glucose but not the osmotic control (i.e., 5 mmol/l d-glucose plus 25 mmol/l mannitol) caused a threefold increase of O2·− in HUVECs (Fig. 1A). Administration of 0.5 mmol/l AICAR had no effect on basal O2·− production but attenuated high glucose–enhanced O2·− release. We also examined the effect of AICAR on NO bioactivity, which depends on the overall production and/or depletion of NO by O2·−. In line with elevated O2·− release, high glucose significantly reduced the levels of cyclic GMP, and AICAR prevented this effect (Fig. 1B). Furthermore, AICAR increased the phosphorylation of eNOS-Ser1177, whereas it had no effects on the total amount of eNOS protein in high glucose–exposed HUVECs (data not shown). These results suggest that AICAR might maintain NO bioactivity under high glucose conditions by increasing NO release and/or counteracting oxidative stress.
Further analysis of the antioxidant effects of AICAR was performed by measuring 8-iso-prostaglandin F2α, a marker of lipid peroxidation. As shown in Fig. 1C, high glucose markedly increased 8-iso-prostaglandin F2α levels. Although AICAR had no effect on basal levels of 8-iso-prostaglandin F2α, it partially but significantly reduced high glucose–induced increases in 8-iso-prostaglandin F2α. AICAR did not completely abolish high glucose–enhanced 8-iso-prostaglandin F2α in HUVECs (P < 0.05, normal glucose vs. high glucose plus AICAR). To understand whether AICAR acts as an oxidant scavenger, we exposed HUVECs to chemically synthesized ONOO−. ONOO− significantly increased 8-iso-prostaglandin F2α levels; however, cotreatment with AICAR did not alter this effect (Fig. 1C). Additional experiments revealed that AICAR had no effect on the reduction of cytochrome C caused by xanthine/xanthine oxidase (data not shown). Thus, the reduction of oxidative stress by AICAR cannot be attributed to its ability to directly scavenge O2·− or ONOO−.
AICAR inhibits high glucose–induced nitration and inactivation of prostacyclin synthase.
Our earlier studies (24,25) had demonstrated that high glucose increases tyrosine nitration of prostacyclin synthase, which inhibits prostacyclin synthase activity. Analysis of 6-keto-PGF1α concentrations revealed that high glucose, but not mannitol, significantly suppressed prostacyclin synthase activity in HUVECs (Fig. 1D). Interestingly, 1 mmol/l AICAR significantly attenuated high glucose–induced reduction of 6-keto-PGF1α but had no effect on the levels of 6-keto-PGF1α in cells exposed to mannitol (Fig. 1D).
We next determined whether increased prostacyclin synthase activity was due to increased prostacyclin synthase expression. Under normal glucose conditions, 1 mmol/l AICAR (1- to 72-h exposure) altered neither prostacyclin synthase protein levels (Fig. 2A) nor prostacyclin synthase activity (Fig. 2B). These results suggest AICAR had no effect on prostacyclin synthase activity or prostacyclin synthase expression in HUVECs.
Because high glucose exposure increased prostacyclin synthase nitration and inactivation, we next determined whether the protective effects of AICAR on prostacyclin synthase activity were due to the reduction of prostacyclin synthase nitration caused by high glucose. Consistent with the idea that AMPK reduces oxidative stress, 0.5 mmol/l AICAR prevented prostacyclin synthase nitration in HUVECs exposed to high glucose (Fig. 2C and D).
AMPK activation is required for AICAR-induced reduction of oxidative stress.
Incubation of HUVECs with AICAR resulted in time-dependent AMPK activation, as determined by Thr172 phosphorylation of AMPK (Fig. 3A). Similarly, a 24-h incubation with 1 mmol/l metformin markedly increased Thr172-AMPK phosphorylation (Fig. 3A). Because either AICAR (Fig. 1A) or metformin (data not shown) significantly reduced ROS production under high glucose conditions, we tested whether AMPK activation was required for this antioxidant effect. Under normal glucose conditions, inhibition of AMPK by adenoviral overexpression of dominant-negative AMPK (Ad-DN-AMPK) significantly increased ROS in HUVECs, whereas GFP overexpression had no effect (Fig. 3B). Moreover, Ad-DN-AMPK overexpression significantly accentuated high glucose–induced ROS production (Fig. 3B). These results suggest that AMPK functions as an endogenous protector against ROS in endothelial cells.
AMPK attenuates high glucose–induced oxidative stress and prostacyclin synthase nitration through upregulation of UCP-2.
To further investigate the mechanism by which AMPK activation reduces oxidative stress, we analyzed UCP-2 expression in HUVECs. Under normal conditions, UCP-2 mRNA was abundant, but very low levels of UCP-2 protein were detected (Fig. 3C). However, exposure of HUVECs to AICAR significantly increased both UCP-2 mRNA (Fig. 3C) and UCP-2 protein expression (Fig. 3D). Importantly, inhibition of AMPK with Ad-DN-AMPK, but not control Ad-GFP, significantly attenuated AICAR-induced UCP-2 expression, implying that AMPK upregulates UCP-2 expression in HUVECs (Fig. 3D).
Next, we tested whether adenoviral overexpression of UCP-2 in HUVECs altered high glucose–induced ROS production. Overexpression of Ad-UCP-2 markedly increased UCP-2 protein in HUVECs (Fig. 4A). In addition, adenoviral overexpression of UCP-2 significantly reduced high glucose–induced increases in ROS (Fig. 4B) and 3-nitrotyrosine (Fig. 4C), with GFP overexpression having no effect on either parameter. Consistent with this result, UCP-2 overexpression markedly inhibited high glucose–induced prostacyclin synthase nitration (Fig. 4D) and prostacyclin synthase inactivation (Fig. 4E).
Activation of p38 kinase is required for AMPK-dependent UCP-2 expression.
The fact that both AMPK and p38 kinase are activated by extracellular stresses (e.g., hypoxia/reoxygenation and osmotic stress) (34,35) prompted us to investigate whether p38 kinase is required for AMPK-dependent UCP-2 expression. As shown in Fig. 5A, the phosphorylation of p38 kinase at Thr180/Tyr182 was markedly increased for up to 4 h after AICAR treatment. In parallel, AICAR increased the phosphorylation of c-Jun, a downstream enzyme of p38 kinase. Adenoviral overexpression of constitutively active AMPK for 24 h before AICAR treatment (2 mmol/l for 2 h) resulted in an increase in c-Jun and p38 kinase phosphorylation that was even greater than that elicited by AICAR alone (Fig. 5B). Conversely, overexpression of Ad-DN-AMPK inhibited AICAR-induced phosphorylation of p38 kinase and c-Jun (Fig. 5B).
To determine whether p38 kinase is required for AMPK-dependent UCP-2 expression, we treated HUVECs with SB239063, a potent p38 kinase inhibitor. Incubation of HUVECs with SB239063 abolished AICAR-induced phosphorylation of both p38 kinase (Fig. 5C) and c-Jun (data not shown). Importantly, SB239063 also suppressed AICAR-induced upregulation of UCP-2 in a dose-dependent manner (Fig. 5D). When used at concentrations up to 20 μmol/l, SB239063 did not alter AICAR-induced AMPK-Thr172 phosphorylation (Fig. 5E), suggesting that AMPK lies upstream of p38 kinase.
Chronic stimulation of AMPK with AICAR attenuates diabetes-induced oxidative stress and prostacyclin synthase nitration.
To extend our in vitro findings, we investigated the effect of AICAR on prostacyclin synthase nitration and UCP-2 expression associated with STZ-induced diabetes. Ten-week-old C57BL6 mice or AMPKα2 KO mice were subjected to five consecutive injections of 50 mg/kg STZ in citrate buffer, pH 4.5, or an equivalent volume of citrate buffer. Two weeks after STZ injection, animals received daily subcutaneous injections of 250 mg/kg AICAR (n = 7) or a corresponding volume of 0.9% NaCl for 6 weeks. One week after injection, mice administered STZ had significantly higher serum glucose levels than their control counterparts (wild type, 488 ± 25 vs. 119 ± 7 mg/dl, P < 0.001, n = 11; AMPKα2 KO, 493 ± 27 vs. 129 ± 11 mg/dl, P < 0.001, n = 9). In addition, diabetic animals had significantly lower body weights (wild type, 16.0 ± 0.8 vs. 18.8 ± 1 g, −14.9%, P < 0.05, n = 11; AMPKα2 KO, 15.8 ± 0.9 vs. 17.9 ± 0.7 g, P < 0.05, n = 10) and plasma insulin concentrations (wild type, 0.3 ± 0.1 vs. 1.2 ± 0.2 ng/ml, P < 0.01, n = 11; AMPKα2 KO, 0.4 ± 0.2 vs. 1.0 ± 0.2 ng/ml) than controls. Diabetic and nondiabetic animals had a similar heart weight (wild type, 70 ± 3.5 vs. 75 ± 2.6 mg). Comparison of AICAR and non-AICAR–treated diabetic mice revealed that both groups had similar blood glucose levels (wild type, 488 ± 25 vs. 461 ± 28 mg/dl; AMPKα2 KO, 493 ± 27 vs. 509 ± 27 mg/dl, P < 0.001, n = 9), water consumption (16.1 ± 0.7 vs. 15.1 ± 0.5 ml/day), and body weight (17.5 ± 0.8 vs. 19.1 ± 0.1 g).
Next, isolated aortas were analyzed for O2·− levels, prostacyclin synthase nitration, and UCP-2 expression. A comparison of aortas from nondiabetic C57BL6 mice and AMPKα2 KO mice revealed that AMPKα2 KO aortas exhibited higher O2·− levels and prostacyclin synthase nitration but had lower levels of prostacyclin synthase activity (Fig. 6A–C). Compared with aortas from nondiabetic mice, those from diabetic mice had markedly increased O2·− levels (Fig. 6A), increased prostacyclin synthase nitration (Fig. 6C), and decreased prostacyclin synthase activity (Fig. 6C) (P < 0.01, n = 7). STZ injection exacerbated aortic O2·− levels and prostacyclin synthase nitration in AMPKα2 KO mice. Activation of AMPK with AICAR significantly inhibited O2·− release, prostacyclin synthase nitration, and prostacyclin synthase inactivation in diabetic wild-type mice (P < 0.01, n = 7), but AICAR administration had no effect in AMPKα2 KO mice. Taken together, these results suggest that AMPK activation is required for the suppression of aortic O2·− formation and prostacyclin synthase nitration by AICAR in vivo.
Finally, to test whether UCP-2 participates in AMPK-dependent reduction in oxidative stress associated with diabetes, we performed immunohistochemical staining for UCP-2 in aortas from wild-type and AMPKα2 KO mice. UCP-2 staining was very weak in aortic tissue from wild-type mice, but was greatly increased by STZ injection, suggesting that diabetes increases UCP-2 expression (Fig. 6D). Similar to the aortas from nondiabetic wild-type mice, nondiabetic AMPKα2 KO aortas had barely detectable levels of UCP-2 (Fig. 6D). However, UCP-2 expression was still barely detectable in AMPKα2 KO mice with STZ-induced diabetes (Fig. 6D). In accord with these results, AICAR significantly increased UCP-2 staining in both nondiabetic and diabetic wild-type mice but had no effect in AMPKα2 KO mice, suggesting that AMPK is required for UCP-2 expression in vivo.
Administration of catalase does not alter UCP-2 expression enhanced by high glucose. Because several cellular types exhibited reduced AMPK activity in the presence of high glucose and high glucose increases ROS production, we next determined whether the addition of catalase altered UCP-2 expression in HUVECs. As shown in Fig. 7, short exposure (2 h) of HUVECs to high glucose significantly increased the phosphorylation of AMPK Thr172 along with increased detection of UCP-2 in HUVECs. Administration of catalase altered neither AMPK phosphorylation nor UCP-2 expression in HUVECs exposed to high glucose.
In the present study, we have found that AICAR, an activator of AMPK, reduces oxidative stress (O2·− and 3-nitrotyrosine) and increases UCP-2 expression in cultured endothelial cells and in aorta from diabetic mice. In high glucose–exposed HUVECs, AMPK inhibition of O2·− formation and prostacyclin synthase nitration was accompanied by increased NO bioactivity. These protective effects of AMPK were confirmed by the finding that AMPK gene deletion not only exacerbated STZ-induced O2·− production and prostacyclin synthase nitration but also rendered AICAR incapable of protecting against increased O2·− and prostacyclin synthase nitration. The ability of AICAR to upregulate UCP-2 expression in diabetic C57BL6 mice but not in AMPKα2 KO mice suggests that AMPK-dependent UCP-2 expression is essential for reduction of oxidative stress by AMPK.
UCPs are mitochondrial transporters that are present in the inner mitochondrial membrane and belong to a family of mitochondrial anion carriers, which includes adenine nucleotide transporters (36,37). Mild uncoupling of respiration is known to diminish mitochondrial ROS formation by complex I and II (37). Recent evidence implies that the basic role of all UCPs is to prevent oxidative tissue injury by reducing oxidative stress (37). A role for UCP-2 in the downregulation of mitochondrial ROS production is plausible, because available evidence suggests that this protein is expressed in numerous mammalian tissues (37). Macrophages of leptin-deficient ob/ob mice have low UCP-2 levels compared with those of normal mice, and these low UCP-2 levels are associated with increased mitochondrial ROS production (38). In addition, ROS levels in macrophages of UCP-2−/− mice (39) and muscle tissue of UCP-3−/− mice (40,41) exceed wild-type levels. UCP-2 has the ability to reduce ROS not only in mitochondria, but also within the remainder of the cell and even in the extracellular space (36,41,42). Duval et al. (44) have recently shown that UCP-2–mediated uncoupling in endothelial cells decreases extracellular ROS. Lee et al. (45) have demonstrated that adenoviral transfer of the UCP-2 gene into human airway epithelial cells profoundly suppresses ROS generation, decreases NF-κB activity, enhances eNOS transcription, and improves endothelium-dependent vascular relaxation. Nevertheless, the molecular mechanisms underlying UCP-2 expression remain poorly defined. We have provided the first evidence that AMPK is essential for UCP-2 expression in endothelial cells in vivo. In line with this hypothesis, we have found that activation of AMPK with AICAR prevents O2·− formation, NO inactivation, and prostacyclin synthase nitration that accompanies prolonged exposure of HUVECs to high glucose. Also in line with this hypothesis, pharmacological or genetic inhibition of AMPK abolished the ability of AICAR to not only reduce oxidative stress but also to upregulate UCP-2 expression. The finding that STZ amplified O2·− production and prostacyclin synthase nitration in AMPKα2 KO mice strongly suggests that AMPK suppresses oxidative stress. However, the most conclusive evidence for this idea is provided by the finding that AICAR failed to alter markers of oxidative stress or UCP-2 expression in the AMPK-α 2 KO mice. Consistent with our results, AMPK activation has also been shown to increase the expression of UCP-2 in liver and in skeletal muscle (21,22). In pre-diabetic (impaired glucose tolerance) subjects, a 1-year lifestyle diabetes prevention program involving increased physiological exercise improves metabolic control and increases UCP-3 levels by twofold (45,46). Physiological exercise is known to lead to AMPK activation (47,48). Activation of AMPK leads to a reduction of oxidative stress and vascular function.
AMPK is activated by multiple stimuli, including oxidants such as ONOO− and H2O2. Thus, the production of oxidants might be required for AMPK-dependent UCP-2 expression. This idea is supported by the fact that UCP-2 was weakly expressed in endothelial cells from both wild-type and AMPKα2 KO mice (Fig. 6D). The ability of STZ to induce aortic O2·− formation, prostacyclin synthase nitration, and UCP-2 expression in wild-type mice, taken with its inability to induce UCP-2 expression in AMPKα2 KO mice (Fig. 6D), strongly suggests that intracellular ROS activates AMPK, which then stimulates transcription of the UCP-2 gene. Consistent with this hypothesis, recent studies (49,50) suggested that O2·− itself activates UCP-2 within the matrix by an unspecified mechanism. We propose that ROS-activated AMPK, in return, limits ROS production by increasing mitochondrial UCP-2 expression. That is, AMPK-dependent UCP-2 upregulation is a compensatory mechanism aimed at counteracting intracellular oxidative stress. Collectively, our findings suggest that AMPK is a physiological regulator of ROS that protects endothelial cells against the adverse effects of hyperglycemia by inhibiting the processes that generate oxidants.
Published ahead of print at http://diabetes.diabetesjournals.org on 3 October 2008.
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
M.-H.Z. has received National Institutes of Health Grants HL079584, HL080499, HL074399, and HL089920; a research award from the American Diabetes Association; a research award from the Juvenile Diabetes Research Foundation; a grant from the Oklahoma Center for the Advancement of Science and Technology; and Paul H. Doris Eaton Travis Chair Funds in Endocrinology of the University of Oklahoma Health Sciences Center.
Parts of this study were presented at the 66th Scientific Session of the American Diabetes Association, Washington, DC, 9–13 June 2006.