Resveratrol regulates blood pressure by enhancing AMPK signaling to downregulate a Rac1-derived NADPH oxidase in the central nervous system
Abstract
Resveratrol is a polyphenol with pleiotropic effects against oxidative damage that has been widely implicated in lowering hypertension risk. The purpose of this study was to determine whether improve nitric oxide (NO) release in the brain, either through the activation of AMP-activated protein kinase (AMPK) or reduced Ras-related C3 botulinum toxin substrate 1 (Rac1)-induced reactive oxygen species (ROS) generation, thereby reducing blood pressure (BP) in rats with fructose-induced hypertension. The rats were fed with 10% fructose or Crestor (rosuvastatin; 1.5 mg·kg−1·day−1) and resveratrol (10 mg·kg−1·day−1) treatment for 1 wk, then the systolic blood pressure of the rats was measured by tail-cuff method. Endogenous in vivo superoxide radical production in the nucleus tractus solitarii (NTS) was determined with dihydroethidium. Immunoblotting analyses were used to quantify protein expression levels. Oral resveratrol treatment for 1 wk decreased BP and increased NO production in the NTS of fructose-fed rats but not in the control Wistar-Kyoto rats. The effect of Crestor is opposite that of resveratrol. Fructose induced hypertension by inactivating AMPK, which in turn enhanced the generation of ROS and reduced manganese superoxide dismutase by increasing the activity of Rac1-induced NADPH oxidase, abolishing the activity of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and ribosomal protein S6 kinase (RSK) and neuronal nitric oxide synthase (nNOS) phosphorylation signaling pathway in the brain. However, resveratrol had the opposite effect in the fructose-fed rats. Overall, we show that the resveratrol decreased BP better than Crestor, abolished ROS generation, and enhanced the ERK1/2-RSK-nNOS pathway by activating AMPK to downregulate Rac1-induced NADPH oxidase levels in the NTS during oxidative stress–associated hypertension.
NEW & NOTEWORTHY 1) Evidence showed that the Ras-related C3 botulinum toxin substrate 1 (Rac1) augmented by Crestor (rosuvastatin) did not result in a significant change in blood pressure (BP) in fructose-induced hypertension. 2) Fructose induced hypertension by inactivating AMP-activated protein kinase (AMPK), which in turn enhanced the generation of reactive oxygen species (ROS) and reduced manganese superoxide dismutase in the brain. 3) Resveratrol decreased BP better than Crestor, abolished ROS generation, and enhanced the ERK1/2-ribosomal protein S6 kinase-neuronal nitric oxide synthase pathway by activating AMPK to negatively regulate Rac1-induced NADPH oxidase levels in the nucleus tractus solitarii during oxidative stress–associated hypertension.
INTRODUCTION
The World Health Organization recommends that the daily intake of sugar should not exceed 10% of total energy. However, fructose is a very common sugar additive for general food merchandise such as corn syrup, soft drinks, desserts, pastries, and processed foods (32). High fructose consumption poses a global threat to human health and is an important risk factor for various metabolic diseases, such as type 2 diabetes mellitus, hypertension, and metabolic syndrome, and has outpaced various infectious diseases (1, 26). A better understanding of hypertension mechanisms and related metabolic comorbidities is necessary to improve human health by targeted treatment.
The nucleus tractus solitarii (NTS) is located in the dorsal medulla of the brain stem, which is the primary integrating center for cardiovascular regulation and other autonomic functions of the central nervous system (CNS). Previous studies have shown that fructose causes oxidative stress and sympathetic overactivity. Clinically, the derangements of the peripheral endothelial nitric oxide (NO) system have been related to the development of hypertension (20). The development of insulin-resistance hypertension may be linked to overactivity of sympathetic system. Sympathetic overactivity may be pathogenic for sympathetic nervous activity regulation in the NTS as a result of imbalanced NO and superoxide production (4, 40).
There is increasing evidence showing that fructose promotes reactive oxygen species (ROS) production and downregulates the key antioxidant enzymes copper-zinc superoxide dismutase (SOD1) and manganese superoxide dismutase (SOD2) (16). Ras-related C3 botulinum toxin substrate 1 (Rac1; small G protein) is a critical factor that triggers ROS production by increasing nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase expression (23). The cytosolic regulatory subunits of NADPH (p40phox, p47phox, p67phox, and rac1) are translocated to the plasma membrane for oxidase activation and ROS production (30). By inhibiting NADPH oxidase activity and expression, cardiac dysfunction may be alleviated (19). An et al. demonstrated that atorvastatin alleviates cardiac function of heart failure by inhibiting Rac1 and regulatory subunits of NADPH (P47phox, P67phox) mediated ROS release (2).
Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a stilbenoid, a type of natural phenol that has potential to impact various human diseases such as type 2 diabetes, obesity, Alzheimer's disease, and cancer (12). Furthermore, resveratrol is neuroprotective and is a potent AMP-activated protein kinase (AMPK) activator in primary neurons and the brain (13). AMPK is a serine/threonine protein kinase that functions as an energy sensor regulating cellular metabolism. AMPK isoforms α1 and α2 consist of a catalytic α subunit and non-catalytic β- and γ-subunits (28). AMPK responds to changes of the AMP-ATP ratio due to energy stress by phosphorylation of Thr172 in the catalytic α-subunit activation loop (17). Wang et al. (35) showed that AMPKα2 deletion enhanced NADPH oxidase subunits expression and oxidative stress in vascular endothelial cells. Consistently, studies have shown that AMPK inhibitor increased the activities of both Rac1 and NADPH oxidase and decreased SOD2 expression in human aortic endothelial cells (7, 37). In line with this finding, our previous study showed that Rac1 increased NADPH oxidase subunits p22-phox activity and reduced the activity of AMPK in the fructose-fed rats. The purpose of this study was to determine whether improve NO release in the brain either through the. Our results suggest that in the NTS, fructose induced hypertension by inactivating AMPK, which in turn enhanced the generation of ROS and reduced SOD2 via increasing the activity of Rac1-induced NADPH oxidase, abolishing the activity of the ERK1/2-ribosomal protein S6 kinase (RSK)-neuronal nitric oxide synthase (nNOS) signaling pathway in the brain. However, resveratrol had the opposite effect in the fructose-fed rats. Overall, we show that the resveratrol decreased blood pressure (BP) better than Crestor, abolished ROS generation, and enhanced the ERK1/2-RSK-nNOS pathway by activating AMPK to negatively regulate Rac1-induced NADPH oxidase levels in the NTS during oxidative stress–associated hypertension.
MATERIALS AND METHODS
Reagents and chemicals.
The drugs, including urethane, fructose, Crestor, resveratrol, compound C, and dimethyl sulfoxide, and the mouse anti-actin, goat anti-rabbit, and goat anti-mouse IgG secondary antibodies were purchased from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO). Anti-p-AMPKT172, anti-AMPK, anti-acetyl-CoA caroboxylase(ACC)S79, anti-p-ERKT202/Y204, anti-ERK, anti-nNOSS1416, anti-nNOS, anti-endothelial nitric oxide synthase(eNOS)S177, anti-eNOS, anti-inducible nitric oxide synthase, anti-p-RSKT359/S363, and anti-RSK antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-p22-phox was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p47-phox and anti-p67-phox were purchased from Millipore (Bedford, MA). Anti-Cu/Zn-SOD and anti-Mn-SOD were obtained from StressGen Biotechnologies (La Jolla, CA) and Abcam (Cambridge, United Kingdom), respectively.
Animals.
Sixteen-week-old male Wistar Kyoto rats were obtained from the National Science Council Animal Facility (Taipei, Taiwan) and housed in the animal room of Kaohsiung Veterans General Hospital (Kaohsiung, Taiwan). The rats were kept in individual cages in a light-controlled room (12-h light/12-h dark cycle), and the temperature was maintained between 23°C and 24°C. The rats were given normal rat chow (Purina, St. Louis, MO) and tap water ad libitum. All of the animal research protocols were approved by the Animal Research Committee. The institutional review board at Kaohsiung Veterans General Hospital approved all study procedures. Animal studies are reported in compliance with the ARRIVE guidelines (18a).
The rats were acclimated to the housing conditions for 1 wk. They were then habituated to the indirect BP measurement procedure for 1 wk. The rats were then divided randomly into five groups (control; fed with 10% fructose; fed with 10% fructose and Crestor 1.5 mg·kg−1·day−1; fed with 10% fructose and resveratrol 10 mg·kg−1·day−1; and fed with 10% fructose, resveratrol 10 mg·kg−1·day−1, and compound C 40 μM/kg/day) with six rats in each group. The fructose solution was prepared every two days by dissolving fructose into tap water. Ordinary tap water was provided to the control animals throughout the experimental period.
BP measurement.
The systolic blood pressure (SBP) of the rats was measured before the start of the fructose or Crestor, resveratrol, and compound C treatments (week 0) using a tail-cuff monitor (Noninvasive Blood Pressure System, SINGA, Taipei, Taiwan). The rats were placed in the fixer for 30 min at a constant temperature of 37°C. During the measurement, six individual readings were obtained in rapid sequence. The highest and the lowest readings were discarded, and the average of the remaining four readings was calculated. The SBP of the rats was measured daily.
Determination of NO in NTS.
The NTS (10 mg) were deproteinized using a Microcon YM-30 (Millipore). The total amount of NO in the samples was determined via a modified chemiluminescence-based procedure using the Sievers Nitric Oxide Analyzer (NOA 280i, Sievers Instruments, Boulder, CO) purge system (20a). The sample (10 mL) was injected into a reflux column containing 0.1 mol/l of VCl3 in 1 mol/l of HCl at 90°C to reduce any nitrates and nitrites into NO. The NO was then combined with the O3 produced by the analyzer to form NO2. The emission resulting from the excited NO2 was detected by a photomultiplier tube and digitally recorded (mV). The values were then interpolated to a standard curve of concurrently determined NaNO2 concentrations. The measurements were recorded in triplicate for each sample. The measured NO levels were corrected for the NTS of the studied rats.
ROS production in the NTS.
The endogenous in vivo produced in the NTS was determined by staining NTS slices with dihydroethidium (DHE, Invitrogen, Carlsbad, CA). The NTS dissected out of the studied rats was placed in optimal cutting temperature (OCT) compound (Shandon Cryomatrix, Thermo Electron Co., Pittsburgh, PA), flash-frozen in a methylbutane-chilled bath, and then placed in liquid nitrogen. Cryostat slices (10 μm) were stained in the dark for 20 min at 37°C in a humidified 5% CO2 incubator with 1 μM DHE. The samples were analyzed using fluorescence microscopy and Zeiss LSM Image software (Carl Zeiss MicroImaging, Jena, Germany).
Immunoblot analysis.
The NTS was dissected by micro punch (1-mm inner diameter) from a 1-mm-thick brain stem slice at the level of the obex under a microscope. Total protein extract was prepared by homogenizing the NTS in lysis buffer with protease inhibitor cocktail and phosphatase inhibitor cocktail, followed by incubation for 1 h at 4°C. The protein extracts (20 g per sample, as assessed by BCA protein assay; Pierce Biotechnology, USA) were subjected to 7.5%–10% SDS-Tris glycine gel electrophoresis and transferred to a polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, United Kingdom). The membrane was blocked with 5% nonfat milk in TBS/Tween-20 buffer (10 mmol/l Tris, 150 mmol/l NaCl, and 0.1% Tween-20, pH 7.4) and incubated with anti-p22-phox, anti-p47-phox, anti-p67-phox, anti-Cu/Zn-SOD, anti-Mn-SOD, anti-p-AMPKT172, anti-AMPK, anti-p-ACCS79, anti-p-ERKT202/Y204, anti-ERK, anti-p-RSKT359/S363, anti-RSK, anti-p-nNOSS1416 (Abcam), or anti-nNOS antibodies at 1:1,000 in PBST with 5% BSA at 4°C overnight. Peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5,000) were used. The specific bands were detected with an ECL-Plus detection kit (GE Healthcare) and exposed to film. The developed films were scanned using a photo scanner (4490, Epson, Long Beach, CA) and analyzed with NIH Image densitometry analysis software (National Institutes of Health, Bethesda, MD).
Immunofluorescent staining analysis.
The rats were perfused with saline, followed by a solution containing 4% formaldehyde and 30% sucrose. A 20-μm section of the brain stem was stained with cresyl violet, and the placement of the pipette tip in the NTS was verified by examining the sections under the microscope. The brain stem sections were incubated in a rabbit-anti-phospho-AMPKT172 antibody (1:100). After washing with PBS, the sections were incubated with green-fluorescent Alexa Fluor 488 donkey anti-rabbit IgG (1:200; Invitrogen) at 25°C for 2 h. The sections were analyzed using fluorescence microscopy and Zeiss LSM Image software (Carl Zeiss MicroImaging).
Rac1 activation assay.
Rac1 activity was measured using a Rac1 Activation ELISA Assay Kit (Cytoskeleton, Denver, CO) according to the manufacturer’s instructions. The Rac1 G-LISA kit contains a Rac-GTP-binding protein immobilized in the wells of a 96-well plate. Active, GTP-bound Rac1 in cell lysates binds to these wells, whereas inactive GDP-bound Rac1 is removed during the washing steps. The bound, active Rac1 is then detected with a luminescent Rac1-specific antibody. The degree of Rac1 activation is determined by comparing readings from activated cell lysates with readings from nonactivated cell lysates; Rac1 inactivation in tissue culture is generally achieved via serum starvation. Following the addition of the chemiluminescent substrate, the signals can be measured using a luminometer. The total lysate loaded into each well was limited to 10–100 μg in 200 μl.
Statistical analysis.
One-way analysis of variance with Scheffe’s post hoc comparison was performed to compare groups. Differences with P values <0.05 were considered significant. All of the data are expressed as the means ± SE.
RESULTS
Resveratrol improves NO release in the NTS through AMPK activation to reduce ROS generation in rats with fructose-induced hypertension.
Resveratrol (AMPK activator) or Crestor (Rac1 inhibitor) was used to investigate the ROS generation in the brain either through the inactivation of AMPK or upregulation of Rac1 activity causing BP to increase in rats with fructose-induced hypertension. We measured the SBP and nitrate levels in animals receiving both fructose and Crestor or resveratrol. SBP was significantly higher and the nitrate levels in the NTS were significantly decreased in the fructose-fed rats compared with the control groups (Fig. 1, A and B, lanes 1 and 2). Interestingly, treatment with resveratrol markedly enhanced the NO levels in the NTS and attenuated the SBP in the fructose-fed rats (Fig. 1, A and B, lanes 2 and 3). However, Crestor did not reduce BP in rats with fructose-induced hypertension. DHE fluorescence was used to estimate the superoxide levels in the NTS of animals fed with fructose for 1 wk. Representative images are shown in Fig. 1C. The levels of DHE fluorescence in the NTS sections were significantly higher in the fructose-fed group than in the control groups. Furthermore, the DHE fluorescence level in the NTS was significantly attenuated in the animals that received both fructose and resveratrol (Fig. 1C). These results indicate that the elimination of ROS may be required for the AMPK-induced release of NO and the depressor response in fructose-modulated hypertension.
Fig. 1.Resveratrol prevents superoxide-induced impairment of NO production and elevated SBP in the NTS of diet-induced hypertensive rats. A: graph reveals the effects of Crestor and resveratrol on SBP in the fructose groups after one wk. B: quantification of the NO concentrations in the NTS of rats. Bar graph displays the NO concentration (as micromoles nitrate per microgram of NTS protein) with the group treated with fructose exhibiting significantly decreased NO levels in the NTS compared with the fructose + resveratrol group. C: immunofluorescence image displays the dihydroethidium-treated brain sections. Representative red fluorescence images indicate superoxide-positive cells in the NTS with or without the administration of resveratrol. Nuclei of the cells in the NTS were counterstained with DAPI, displaying blue fluorescence. Images were photographed at ×280 magnification. Bar graph is representative of the ROS intensity in the NTS of the indicated groups. Values shown are the means ± SEM, n = 6. *P < 0.05; **P < 0.01. NO, nitric oxide; NTS, nucleus tractus solitarii; ROS, reactive oxygen species; SBP, systolic blood pressure; SEM, standard error of the mean.
Resveratrol attenuates Rac1-induced NADPH oxidase subunit p67 generation and impairment of SOD2 production in the NTS of rats with fructose-induced hypertension.
A recent study showed that deletion of AMPKα2 increased the activities of both Rac1 and NADPH oxidase and decreased SOD2 expression in human aortic endothelial cells (7, 37). Therefore, we investigated the in situ level of Rac1, NADPH oxidase subunits, and SOD in the NTS to establish a potential association between fructose and superoxide production.
Rac1 activation in the NTS was significantly enhanced in the fructose-fed group compared with the control group (Fig. 2A, lane 1 and lane 2). However, resveratrol treatment reduced Rac1 activation in the NTS of the fructose-fed rats (Fig. 2A, lane 2 and 3), whereas no significant difference was observed between resveratrol and controls (Figs. 2A and 3B, lanes 1 and 3). We then investigated which NADPH oxidase subunits involved in Rac1 trigger ROS production in the NTS. The immunoblot analysis showed that fructose intake significantly increased relative p67-phox protein expression levels in the NTS, and the coadministration of resveratrol prevented these increases (Fig. 2B). Conversely, after resveratrol treatment, the fructose-fed rats displayed a significant increase in SOD2 expression compared with the fructose-fed rats (Fig. 2C).
Fig. 2.Resveratrol ameliorates the fructose-induced Rac1 increases in NADPH oxidase subunits (p67-phox) and impairment of SOD2 production in the NTS of fructose-induced hypertensive rats. A: administration of fructose results in high levels of Rac1 activation. Systemic treatment with resveratrol leads to significantly lowered Rac1 levels in the NTS of the fructose group compared with the control group. B and C: quantitative immunoblotting analysis revealed that resveratrol treatment decreased the levels of NADPH oxidase subunit p67-phox and increased the SOD2 expression in NTS of diet-induced hypertensive rats. Values shown are the means ± SEM, n = 6. *P < 0.05; **P < 0.01. NTS, nucleus tractus solitarii; Rac1, Ras-related C3 botulinum toxin substrate 1; RLU, reactive light units; SEM, standard error of the mean; SOD1, copper-zinc superoxide dismutase; SOD2, manganese superoxide dismutase.
Fig. 3.AMPK overexpression abolished the Rac1-induced increases in Rac1 and NADPH oxidase activities and reduced the activities of SOD2 in the NTS of fructose-induced hypertensive rats. A: immunofluorescence image displays the green fluorescence used to estimate p-AMPKT172 levels in the NTS after treatment with resveratrol and compound C. Representative green fluorescence images indicate p-AMPKT172-positive cells in the NTS with or without the administration of resveratrol and compound C. Nuclei of the cells in the NTS were counterstained with DAPI, displaying blue fluorescence. Images were photographed at ×280 magnification. B: confocal microscopy analysis displays the red fluorescence images used to estimate superoxide-positive cells in the NTS with or without the administration of resveratrol. Nuclei of the cells in the NTS were counterstained with DAPI, displaying blue fluorescence. Images were photographed at ×280 magnification. C: quantitative immunoblotting analysis revealed that resveratrol treatment increased the phosphorylation levels of P-ACCS79 and P-AMPKT172 in the NTS of diet-induced hypertensive rats. Densitometric analysis of the P-ACCS79 and P-AMPKT172 levels after administration of resveratrol with or without compound C. D: bar graph showing the activation ratio of Rac1 after treatment with resveratrol and compound C. Rac1 activation in the NTS after treatment with resveratrol was significantly inhibited by treatment with compound C. E: quantitative immunoblotting analysis revealed that resveratrol treatment increased the protein levels of p67-phox and SOD2 in the NTS of diet-induced hypertensive rats. Densitometric analysis of the p67-phox and SOD2 levels after administration of resveratrol with or without compound C. Values are shown as the means ± SEM, n = 6. *P < 0.05; **P < 0.01. ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; NTS, nucleus tractus solitarii; Rac1, Ras-related C3 botulinum toxin substrate 1; RLU, reactive light units; SEM, standard error of the mean; SOD2, manganese superoxide dismutase.
These results indicate that the reactivation of AMPK by resveratrol treatment may abolish Rac1 triggers of p67-phox–induced ROS production in the NTS during fructose-induced hypertension.
Resveratrol abolishes Rac1-induced NADPH oxidase through activation of AMPK in the NTS of rats with diet-induced hypertension.
We investigated whether reactivation of AMPK by resveratrol treatment may prevent Rac1 from triggering p67-phox–induced ROS production in the NTS, thereby reducing BP in rats with fructose-induced hypertension. We used an AMPK activator (resveratrol) and inhibitor (compound C) to demonstrate the in situ level of superoxide in the NTS to establish a potential association between AMPKT172 and Rac1-NADPH oxidase activity. Immunofluorescence staining against phosphorylated AMPKT172 in the NTS revealed that fructose reduced the number of phosphorylated AMPKT172-positive cells compared with the control group (Fig. 3A, lanes 1 and 2). We performed immunofluorescence analyses to determine whether resveratrol treatment could restore AMPKT172 phosphorylation in the NTS of fructose-fed rats (Fig. 3A, lanes 2 and 3). However, treatment with compound C attenuated resveratrol-induced AMPKT172 phosphorylation in the NTS (Fig. 3A, lanes 3 and 4). Similarly, the DHE fluorescence levels in the NTS were significantly attenuated by resveratrol; treatment with compound C abolished resveratrol-reduced ROS generation (Fig. 3B). Immunoblot analysis showed that fructose intake with coadministration of resveratrol significantly increased relative ACCS79 and AMPKT172 phosphorylation levels in the NTS; the addition of an AMPK inhibitor (compound C) reversed the resveratrol-induced ACCS79 and AMPKT172 phosphorylation in the NTS (Fig. 3C). Moreover, Rac1 activation in the NTS was significantly attenuated by resveratrol; treatment with compound C abolished resveratrol-reduced Rac1 activation (Fig. 3D). Our studies showed that resveratrol abolished the Rac1-induced increases in the expression of NADPH oxidase subunits p67-phox and reduced the levels of SOD2 in the NTS, but treatment with compound C reversed the resveratrol-induced effects on Rac1/NADPH oxidases and enhanced the expression of SOD2 in the NTS (Fig. 3E). These results indicate that the AMPK pathway inhibited Rac1-induced NADPH oxidase expression and enhanced SOD2 expression.
Resveratrol attenuates fructose-induced ERK1/2-RSK-nNOS signaling defects in the NTS of fructose-induced hypertensive rats.
Our study demonstrates that reactivation of AMPK by resveratrol treatment improves impairment of NO production, and the depressor response may eliminate Rac1-induced NADPH oxidase in the NTS of rats with fructose-induced hypertension. However, the mechanism underlying how the oxidative stress causes NO dysfunction in the NTS during fructose-induced hypertension is still unclear. Interestingly, NO production in the NTS was found to increase significantly after fructose and resveratrol coadministration, and treatment with compound C restored the SBP and resulted in the recovery of the resveratrol-induced increase of NO production in the NTS (Fig. 4, A and B). Furthermore, immunoblotting analysis revealed significantly decreased ERK1/2T202/Y204, RSKT359/S363, and nNOSS1416 phosphorylation in the NTS of fructose-fed rats compared with controls (Fig. 4, C and D, lanes 1 and 2). After the administration of resveratrol, a significant increase in ERK1/2T202/Y204, RSKT359/S363, and nNOSS1416 phosphorylation was observed in the fructose-fed rats (Fig. 4, C and D, lanes 2 and 3), whereas the level in the compound C group was reversed these changes (Fig. 4, C and D, lanes 4).
Fig. 4.Resveratrol regulates blood pressure through enhancing the activity of the AMPK-ERK1/2-RSK-nNOS pathway in the NTS of fructose-induced hypertensive rats. A: graph showing the effects of resveratrol on SBP with or without the administration of compound C. SBP after treatment with resveratrol was significantly increased by compound C. B: quantification of the NO concentrations in the NTS of rats. Bar graph displays the NO concentration (as micromoles nitrate per microgram of NTS protein) with the group treated with fructose exhibiting significantly decreased NO levels in the NTS compared with the fructose + resveratrol group. Levels of NO in the NTS after the administration of compound C. Bar graph shows that the concentration of NO after treatment with resveratrol was significantly reduced by compound C. C and D: quantitative immunoblotting analysis revealed that resveratrol treatment increased the phosphorylation levels of ERK1/2, RSK, and nNOS in the NTS of diet-induced hypertensive rats. Densitometric analysis of the P-ERK1/2T202/Y204, P-RSKT359/S363, and nNOSS1416 levels after administration of resveratrol with or without compound C. Values shown are the means ± SEM, n = 6. *P < 0.05; **P < 0.01. AMPK, AMP-activated protein kinase; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NTS, nucleus tractus solitarii; RSK, ribosomal protein S6 kinase; SBP, systolic blood pressure; SEM, standard error of the mean.
DISCUSSION
Hypertension is a major public health problem associated with increased risk for heart disease and stroke, which are leading causes of death worldwide. Despite recent treatment advancement, approximately one-third of adults in the United States have hypertension (24). Studies in yeast have suggested that high fructose consumption is a risk factor for several metabolic diseases in humans through the resultant increase in ROS levels (26). To investigate whether higher ROS levels in fructose-fed rats would lead to downregulation of NO release and induce hypertension, we used fructose-fed rats as animal models in our current study to investigate the signaling mechanisms involved in ROS-mediated central BP regulation in the NTS. For several years, a number of laboratories have focused on the interplay between NO and ROS in the cardiovascular system and brain, organs that play critical roles in BP regulation. Evidence indicated that resveratrol treatment from 3–4 wk to 11–12 wk of age reduced H2O2 content, elevated SOD activity, and significantly attenuated the rise in BP of spontaneously hypertensive rats (3). Our previous data demonstrated that treatment with resveratrol attenuated the increase in SBP caused by fructose when administered together from week 1 to week 4 and when rats were fed with fructose only for 2 wk before the administration of resveratrol from week 3 to week 4 (6). Therefore, early treatment with resveratrol lowers oxidative stress, preserves SOD function, and attenuates development of hypertension.
Rac1 is a small GTPase essential for the assembly and activation of NADPH oxidase and the production of superoxide (39). 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), in addition to inhibiting cholesterol synthesis, downregulate Rac1-GTPase activity by reducing isoprenylation and translocation of Rac1 to the cell membrane (29). Recent studies have reported that the activation of AMPK suppressed oxidative stress (35). However, our previous results demonstrated that AMPKT172 phosphorylation was significantly attenuated in the animals that consumed fructose (7). Therefore, this study was to determine fructose enhanced ROS generation in the brain whether through the inactivation of AMPK expression or upregulated Rac1 activity, that leads to BP reduction in rats with fructose-induced hypertension. Increasing evidence has shown that the Rac1 augmented by Crestor (rosuvastatin) did not have a significant effect on BP in fructose-induced hypertension. There is interesting evidence showing that activation of AMPK expression by resveratrol not only attenuated the NTS levels of NADPH-P67-phox, Rac1 activity, and superoxide and decreased the BP, but also increased the NTS antioxidant capacity in hypertension. In addition, our findings demonstrated that the activation of AMPK abolished the ROS generation and enhanced the ERK1/2-RSK-nNOS pathway by negatively regulating Rac1-induced NADPH oxidase levels in the NTS during oxidative stress–induced hypertension (Fig. 5). Theodotou et al. investigated the effect of resveratrol in patients with primary hypertension in a clinical trial. Following the trial, which lasted two years (October 2010 to October 2012), the mean BP of both groups [antihypertensive therapy (Dapril) plus resveratrol, and resveratrol only] was within the normal range, indicating that BP was efficiently controlled. By clinical standard treatment, resveratrol 20 mg is antihypertensive and efficiently controls BP. This indicates that the addition of resveratrol to standard antihypertensive therapy is sufficient to reduce BP to normal levels without the need for additional antihypertensive drugs, which are commonly prescribed for many patients (31). Our studies also found that resveratrol is better than Crestor in lowering BP in rats with fructose-induced hypertension (Fig. 1).
Fig. 5.Resveratrol-activated, AMPK-mediated signaling pathway involved in the regulation of BP in the NTS of fructose-induced hypertensive rats. Fructose not only increased the generation of reactive oxygen species and decreased the activity of the ERK1/2-RSK-nNOS pathway by positively regulating Rac1-induced NADPH oxidase levels in the NTS of rats with oxidative stress-induced hypertension but also abolished the SOD2 and AMPK expression (black line). However, treatments with resveratrol (AMPK activator) and compound C (AMPK inhibitor) demonstrated that resveratrol acts as an important regulator of BP by downregulating Rac1-NADPH oxidase activity and reversing the inactivity of the ERK1/2-RSK-nNOS pathway (red line). AMPK, AMP-activated protein kinase; BP, blood pressure; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NTS, nucleus tractus solitarii; Rac1, Ras-related C3 botulinum toxin substrate 1; RSK, ribosomal protein S6 kinase; SBP, systolic blood pressure; SOD2, manganese superoxide dismutase.
Fructose disrupts the balance of oxidation state by enhancing ROS production and downregulating key antioxidant enzymes such as SOD1 and SOD2 (16). Nozoe et al. reported that the transfection of SOD1, which scavenges ROS in the NTS, decreased BP and HR (23). Furthermore, the silencing of either AMPKα1 or AMPKα2 elevated oxidative stress by downregulating genes involved in antioxidant defense, including SOD2, catalase, γ-glutamylcysteine synthase, and thioredoxin (10, 14, 15). However, the opposite effect was observed after treatment with AMPK agonists such as A769662 or AICAR (5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide)), which inhibited hyperglycemia-induced intracellular and mitochondrial ROS production and increased the expression of peroxisome proliferator-activated response-γ coactivator-1α and SOD2. Resveratrol can cross the blood–brain barrier to carry out neuroprotective effects on the CNS (25). Importantly, Vingtdeux et al. (34) demonstrated the presence of resveratrol in the brain after oral treatment, indicating that resveratrol has a direct effect on neurological disorders. Resveratrol has become a popular compound to test as treatment for a range of neurological disorders. AICAR is orally effective in mice and has been shown to increase endurance performance in sedentary mice by increasing expression of oxidative metabolism genes (22). Unfortunately, AICAR has a very poor ability to penetrate the blood–brain barrier, which limits its use to peripheral tissues (21). A769662 has beneficial effects on metabolic parameters in vivo and has a lead compound for drug development, but it is tempered by its poor oral absorption (11).
Resveratrol is thought to be useful in the control of BP when added to a standard antihypertensive therapy by increasing the production of NO, an endogenous and potent vasodilator. NO is synthesized in cardiac myocytes and plays key roles in modulating cardiovascular signaling. NO inhibits the activation of the sympathetic nervous system in the brain to modulate BP (33). The nNOS is present in neurons found in specific regions of the brain, including the NTS, rostral ventrolateral medulla (RVLM), paraventricular nucleus, and the caudal ventrolateral medulla (18). Our previous study revealed that ERK1/2-RSK signaling was involved in the regulation of nNOS in the NTS to modulate BP (8). Moreover, Ang II may modulate central BP effects via ROS to downregulate ERK1/2, RSK, and nNOS (9). Our data also showed that compound C (via intracerebroventricular injection) restored the SBP in animals fed with both fructose and resveratrol and abolished the resveratrol-induced increases in the NO levels in the NTS (Fig. 4, A and B). However, we demonstrated that the overexpression of AMPK affected nNOS but not eNOS or inducible nitric oxide synthase during the depressor response mediated by the ERK1/2-RSK signaling pathway in the NTS of rats with fructose-induced hypertension (Fig. 4). Boyle and Logan et al. indicated that rosiglitazone stimulates NO synthesis in human aortic endothelial cells via AMPK. They also found infection of endothelial cells with a virus encoding a dominant negative AMPK mutant abrogated rosiglitazone-stimulated Ser-1177 phosphorylation and NO production (5). The present study provides evidence that the tissue level of ROS was increased and NO was decreased in the RVLM of high-fructose diet–fed rats. They silenced a protein inhibitor of nNOS mRNA in the RVLM using lentivirus carrying small hairpin RNA inhibited protein inhibitor of nNOS expression, increased the ratio of nNOS dimer/monomer, restored NO content, and alleviated oxidative stress in the RVLM of high-fructose diet–fed rats, which was accompanied with reduced sympathoexcitation and hypertension. Our results suggest that nNOS decrease is involved in a vicious cycle that sustains ROS production in the RVLM, resulting in sympathoexcitation and hypertension associated with metabolic syndrome (38). Thus, we propose that AMPK decreases BP, abolishes the generation of ROS, and enhances the activity of the ERK1/2-RSK-nNOS pathway by negatively regulating Rac1-induced NADPH oxidase levels in the NTS during oxidative stress–associated hypertension (Fig. 5). Increasing evidence shows that NADPH oxidase is the source of ROS in the brain and has been identified as an underlying pathogenic mechanism linked to hypertension. The beneficial effects of resveratrol may be mediated through the activation of AMPK, which downregulates Rac1, NADPH oxidases, and SOD2 to protect the CNS. These novel findings suggest that resveratrol, an AMPK activator, is a potential pharmacological candidate for treatment of hypertension.
GRANTS
This work was supported by funding from the National Science Council (MOST106–2320-B075B-001, MOST104–2320-B-075B-003-MY3, and NSC102–2320-B-075B-002) and Kaohsiung Veterans General Hospital (VGHKS106–157, VGHKS107–175, VGHKS105-G03–1, VGHKS105-G03–2, VGHKS105-G03–3, and VGHKS105–131; to P.-W. Cheng, T.-C. Yeh and C.-J. Tsing).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
T.-C.Y., C.-S.S., and P.-W.C. conceived and designed research; T.-C.Y., C.-S.S., G.-C.S., C.-J.T., and P.-W.C. performed experiments; P.-W.C. analyzed data; H.-H.C., C.-C.L., C.-J.T., and P.-W.C. interpreted results of experiments; C.-J.T. and P.-W.C. prepared figures; P.-W.C. drafted manuscript; T.-C.Y. and P.-W.C. edited and revised manuscript; T.-C.Y. and P.-W.C. approved final version of manuscript.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the technical assistance, valuable input, and support from Bo-Rong Chen.
REFERENCES
- 1. . Starches, sugars and obesity. Nutrients 3: 341–369, 2011. doi:10.3390/nu3030341.
Crossref | PubMed | Web of Science | Google Scholar - 2. . Atorvastatin improves cardiac function of rats with chronic cardiac failure via inhibiting Rac1/P47phox/P67phox-mediated ROS release. Eur Rev Med Pharmacol Sci 19: 3940–3946, 2015.
PubMed | Web of Science | Google Scholar - 3. . Resveratrol prevents endothelial nitric oxide synthase uncoupling and attenuates development of hypertension in spontaneously hypertensive rats. Eur J Pharmacol 667: 258–264, 2011. doi:10.1016/j.ejphar.2011.05.026.
Crossref | PubMed | Web of Science | Google Scholar - 4. . Insulin reduces excitation in gastric-related neurons of the dorsal motor nucleus of the vagus. Am J Physiol Regul Integr Comp Physiol 303: R807–R814, 2012. doi:10.1152/ajpregu.00276.2012.
Link | Web of Science | Google Scholar - 5. . Rosiglitazone stimulates nitric oxide synthesis in human aortic endothelial cells via AMP-activated protein kinase. J Biol Chem 283: 11210–11217, 2008. doi:10.1074/jbc.M710048200.
Crossref | PubMed | Web of Science | Google Scholar - 6. . Resveratrol decreases fructose-induced oxidative stress, mediated by NADPH oxidase via an AMPK-dependent mechanism. Br J Pharmacol 171: 2739–2750, 2014. doi:10.1111/bph.12648.
Crossref | PubMed | Web of Science | Google Scholar - 7. . Resveratrol inhibition of Rac1-derived reactive oxygen species by AMPK decreases blood pressure in a fructose-induced rat model of hypertension. Sci Rep 6: 25342, 2016. doi:10.1038/srep25342.
Crossref | PubMed | Web of Science | Google Scholar - 8. . Central hypotensive effects of neuropeptide Y are modulated by endothelial nitric oxide synthase after activation by ribosomal protein S6 kinase. Br J Pharmacol 167: 1148–1160, 2012. doi:10.1111/j.1476-5381.2012.02077.x.
Crossref | PubMed | Web of Science | Google Scholar - 9. . Angiotensin II inhibits neuronal nitric oxide synthase activation through the ERK1/2-RSK signaling pathway to modulate central control of blood pressure. Circ Res 106: 788–795, 2010. doi:10.1161/CIRCRESAHA.109.208439.
Crossref | PubMed | Web of Science | Google Scholar - 10. . AMPKalpha1 regulates the antioxidant status of vascular endothelial cells. Biochem J 421: 163–169, 2009. doi:10.1042/BJ20090613.
Crossref | PubMed | Web of Science | Google Scholar - 11. . Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metab 3: 403–416, 2006. doi:10.1016/j.cmet.2006.05.005.
Crossref | PubMed | Web of Science | Google Scholar - 12. . Resveratrol bioavailability and toxicity in humans. Mol Nutr Food Res 54: 7–16, 2010. doi:10.1002/mnfr.200900437.
Crossref | PubMed | Web of Science | Google Scholar - 13. . Resveratrol stimulates AMP kinase activity in neurons. Proc Natl Acad Sci USA 104: 7217–7222, 2007. doi:10.1073/pnas.0610068104.
Crossref | PubMed | Web of Science | Google Scholar - 14. . Reduction of AMP-activated protein kinase alpha2 increases endoplasmic reticulum stress and atherosclerosis in vivo. Circulation 121: 792–803, 2010. doi:10.1161/CIRCULATIONAHA.109.900928.
Crossref | PubMed | Web of Science | Google Scholar - 15. . Activation and signaling by the AMP-activated protein kinase in endothelial cells. Circ Res 105: 114–127, 2009. doi:10.1161/CIRCRESAHA.109.201590.
Crossref | PubMed | Web of Science | Google Scholar - 16. . Changes induced by a fructose-rich diet on hepatic metabolism and the antioxidant system. Life Sci 86: 965–971, 2010. doi:10.1016/j.lfs.2010.05.005.
Crossref | PubMed | Web of Science | Google Scholar - 17. . AMP-activated protein kinase: an ultrasensitive system for monitoring cellular energy charge. Biochem J 338: 717–722, 1999. doi:10.1042/bj3380717.
Crossref | PubMed | Web of Science | Google Scholar - 18. . nNOS-containing neurons in the hypothalamus and medulla project to the RVLM. Brain Res 1037: 25–34, 2005. doi:10.1016/j.brainres.2004.11.032.
Crossref | PubMed | Web of Science | Google Scholar - 18a. ,
Group NCRRGW . Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol 160: 1577–1579, 2010.
Crossref | PubMed | Web of Science | Google Scholar - 19. . NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing heart. Proc Natl Acad Sci USA 107: 15565–15570, 2010. doi:10.1073/pnas.1002178107.
Crossref | PubMed | Web of Science | Google Scholar - 20. . Increased vascular endothelin-1 gene expression with unaltered nitric oxide synthase levels in fructose-induced hypertensive rats. Metabolism 50: 74–78, 2001. doi:10.1053/meta.2001.19527.
Crossref | PubMed | Web of Science | Google Scholar - 20a. . Gene transfer of neuronal nitric oxide synthase to carotid body reverses enhanced chemoreceptor function in heart failure rabbits. Circ Res 97: 260–267, 2005.
Crossref | PubMed | Web of Science | Google Scholar - 21. . Adenosinergic modulation of homocysteine-induced seizures in mice. Epilepsia 31: 239–246, 1990. doi:10.1111/j.1528-1157.1990.tb05371.x.
Crossref | PubMed | Web of Science | Google Scholar - 22. . AMPK and PPARdelta agonists are exercise mimetics. Cell 134: 405–415, 2008. doi:10.1016/j.cell.2008.06.051.
Crossref | PubMed | Web of Science | Google Scholar - 23. . Inhibition of Rac1-derived reactive oxygen species in nucleus tractus solitarius decreases blood pressure and heart rate in stroke-prone spontaneously hypertensive rats. Hypertension 50: 62–68, 2007. doi:10.1161/HYPERTENSIONAHA.107.087981.
Crossref | PubMed | Web of Science | Google Scholar - 24. . Hypertension among adults in the United States: National Health and Nutrition Examination Survey, 2011-2012. NCHS Data Brief 133: 1–8, 2013.
PubMed | Google Scholar - 25. . Resveratrol modulates astroglial functions: neuroprotective hypothesis. Ann N Y Acad Sci 1215: 72–78, 2011. doi:10.1111/j.1749-6632.2010.05857.x.
Crossref | PubMed | Web of Science | Google Scholar - 26. . Way back for fructose and liver metabolism: bench side to molecular insights. World J Gastroenterol 18: 6552–6559, 2012. doi:10.3748/wjg.v18.i45.6552.
Crossref | PubMed | Web of Science | Google Scholar - 28. . Mammalian AMP-activated protein kinase subfamily. J Biol Chem 271: 611–614, 1996. doi:10.1074/jbc.271.2.611.
Crossref | PubMed | Web of Science | Google Scholar - 29. . Statins as antioxidant therapy for preventing cardiac myocyte hypertrophy. J Clin Invest 108: 1429–1437, 2001. doi:10.1172/JCI13350.
Crossref | PubMed | Web of Science | Google Scholar - 30. . Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases. J Biol Chem 278: 25234–25246, 2003. doi:10.1074/jbc.M212856200.
Crossref | PubMed | Web of Science | Google Scholar - 31. . The effect of resveratrol on hypertension: A clinical trial. Exp Ther Med 13: 295–301, 2017. doi:10.3892/etm.2016.3958.
Crossref | PubMed | Web of Science | Google Scholar - 32. . Fructose and sucrose intake increase exogenous carbohydrate oxidation during exercise. Nutrients 9: 167, 2017. doi:10.3390/nu9020167.
Crossref | PubMed | Web of Science | Google Scholar - 33. . Cardiovascular effects of nitric oxide in the brain stem nuclei of rats. Hypertension 27: 36–42, 1996. doi:10.1161/01.HYP.27.1.36.
Crossref | PubMed | Web of Science | Google Scholar - 34. . AMP-activated protein kinase signaling activation by resveratrol modulates amyloid-beta peptide metabolism. J Biol Chem 285: 9100–9113, 2010. doi:10.1074/jbc.M109.060061.
Crossref | PubMed | Web of Science | Google Scholar - 35. . AMPKalpha2 deletion causes aberrant expression and activation of NAD(P)H oxidase and consequent endothelial dysfunction in vivo: role of 26S proteasomes. Circ Res 106: 1117–1128, 2010. doi:10.1161/CIRCRESAHA.109.212530.
Crossref | PubMed | Web of Science | Google Scholar - 37. . Normal IgG downregulates the intracellular superoxide level and attenuates migration and permeability in human aortic endothelial cells isolated from a hypertensive patient. Hypertension 60: 818–826, 2012. doi:10.1161/HYPERTENSIONAHA.112.199281.
Crossref | PubMed | Web of Science | Google Scholar - 38. . Role of nitric oxide synthase uncoupling at rostral ventrolateral medulla in redox-sensitive hypertension associated with metabolic syndrome. Hypertension 64: 815–824, 2014. doi:10.1161/HYPERTENSIONAHA.114.03777.
Crossref | PubMed | Web of Science | Google Scholar - 39. . Mixture of peanut skin extract and fish oil improves memory in mice via modulation of anti-oxidative stress and regulation of BDNF/ERK/CREB signaling pathways. Nutrients 8: 256, 2016. doi:10.3390/nu8050256.
Crossref | PubMed | Web of Science | Google Scholar - 40. . Caffeine intake improves fructose-induced hypertension and insulin resistance by enhancing central insulin signaling. Hypertension 63: 535–541, 2014. doi:10.1161/HYPERTENSIONAHA.113.02272.
Crossref | PubMed | Web of Science | Google Scholar
