Role of Nitric Oxide Synthase Uncoupling at Rostral Ventrolateral Medulla in Redox-Sensitive Hypertension Associated With Metabolic Syndrome Kay L.H. Wu, Yung-Mei Chao, Shiow-Jen Tsay, Chen Hsiu Chen, Samuel H.H. Chan, Ima Dovinova and Julie Y.H. Chan Hypertension. 2014;64:815-824; originally published online June 23, 2014; doi: 10.1161/HYPERTENSIONAHA.114.03777 Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 0194-911X. Online ISSN: 1524-4563
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://hyper.ahajournals.org/content/64/4/815
Data Supplement (unedited) at: http://hyper.ahajournals.org/content/suppl/2014/06/23/HYPERTENSIONAHA.114.03777.DC1.html
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Hypertension is online at: http://hyper.ahajournals.org//subscriptions/
Oxidative Stress Role of Nitric Oxide Synthase Uncoupling at Rostral Ventrolateral Medulla in Redox-Sensitive Hypertension Associated With Metabolic Syndrome Kay L.H. Wu, Yung-Mei Chao, Shiow-Jen Tsay, Chen Hsiu Chen, Samuel H.H. Chan, Ima Dovinova, Julie Y.H. Chan Abstract—Metabolic syndrome (MetS), which is rapidly becoming prevalent worldwide, is long known to be associated with hypertension and recently with oxidative stress. Of note is that oxidative stress in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons reside, contributes to sympathoexcitation and hypertension. This study sought to identify the source of tissue oxidative stress in RVLM and their roles in neural mechanism of hypertension associated with MetS. Adult normotensive rats subjected to a high-fructose diet for 8 weeks developed metabolic traits of MetS, alongside increases in sympathetic vasomotor activity and blood pressure. In RVLM of these MetS rats, the tissue level of reactive oxygen species was increased, nitric oxide (NO) was decreased, and mitochondrial electron transport capacity was reduced. Whereas the protein expression of neuronal NO synthase (nNOS) or protein inhibitor of nNOS was increased, the ratio of nNOS dimer/monomer was significantly decreased. Oral intake of pioglitazone or intracisternal infusion of tempol or coenzyme Q10 significantly abrogated all those molecular events in high-fructose diet–fed rats and ameliorated sympathoexcitation and hypertension. Gene silencing of protein inhibitor of nNOS mRNA in 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 RVLM of high-fructose diet–fed rats, alongside significantly reduced sympathoexcitation and hypertension. These results suggest that redoxsensitive and protein inhibitor of nNOS–mediated nNOS uncoupling is engaged in a vicious cycle that sustains the production of reactive oxygen species in RVLM, resulting in sympathoexcitation and hypertension associated with MetS. (Hypertension. 2014;64:815-824.) Online Data Supplement
•
Key Words: hypertension
■
metabolic cardiovascular syndrome ■ nitric oxide ■ sympathetic nervous system
M
etabolic syndrome (MetS), defined as a cluster of ≥3 disorders that include insulin resistance, dyslipidemia, hypertension, hypercholesterolemia, and abdominal obesity,1,2 is associated with the risk of developing type 2 diabetes mellitus, coronary heart disease, stroke, and cardiovascular-related mortality.3,4 MetS affects >20% of adults in Western populations. However, given the acculturation of Western diet in the developing world, the burden of MetS is highly prevalent worldwide. Although insulin resistance is generally accepted as the main underlying pathogenic mechanism, increasing evidence links excessive reactive oxygen species (ROS) production and tissue oxidative stress to the pathogenesis of MetS and the progression of its complications.5,6 A recent cross-sectional study reported that subjects with more MetS components exhibit higher levels
■
reactive oxygen species
of oxidative stress.7 Better understanding of the significance of the redox signaling in MetS is therefore warranted. The brain uses large amounts of oxygen and ATP for its normal functions and is therefore highly susceptible to oxidative stress. Reports on brain oxidative stress in the pathogenesis of MetS, however, are sporadic. Of the few studies reported, oxidative stress in the hypothalamus is involved in the progression of obesity-induced hypertension.8 In the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons reside,9 oxidative stress induces sympathoexcitation in rats with obesity-induced hypertension.10 ROS are products of normal cellular metabolism and are derived from many sources in different cellular compartments. In RVLM, we11,12 and others13,14 have demonstrated that ROS generated from nicotinamide adenine
Received April 21, 2014; first decision May 5, 2014; revision accepted May 26, 2014. From the Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan (K.L.H.W., Y.M.C., S.H.H.C., J.Y.H.C.); Institute of Biological Science, National Sun Yat-sen University, Kaohsiung, Taiwan (S.J.T.); Department of Anesthesiology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan (C.H.C.); and Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava, Slovakia (I.D.). The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA. 114.03777/-/DC1. Correspondence to Julie Y.H. Chan, Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung 83301, Taiwan. E-mail [email protected] or Ima Dovinova, Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava, Slovakia. E-mail [email protected] © 2014 American Heart Association, Inc. Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.114.03777
815
816 Hypertension October 2014 dinucleotide phosphate (NADPH) oxidase and mitochondrial respiratory enzymes play pivotal roles in neural mechanism of hypertension. In addition, uncoupling of nitric oxide synthase (NOS) contributes to the increased ROS production in cerebral microvessels.15 Intriguingly, NOS dysfunction is implicated in hypertension of individuals with MetS.16 The significance of the interplay between NOS and ROS in RVLM in MetS-associated hypertension is currently unknown. Normotensive rat fed a high-fructose diet (HFD) is a wellestablished rodent model for the study of human MetS. This model mimics several features, including hypertension, insulin resistance, and abnormal lipid profile, of patients with MetS induced by Western diet.17,18 Using this MetS model, we sought to identify the source of ROS and its interplay with NOS in RVLM on neural mechanisms of hypertension associated with MetS.
Materials and Methods All experimental procedures were performed in compliance with the guidelines of Institutional Animal Care and Use Committee in Kaohsiung Chang Gung Memorial Hospital or Kaohsiung Veterans General Hospital. They were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Detailed Materials and Methods are described in the onlineonly Data Supplement. Adult (8 weeks of age) male Sprague-Dawley rats (235–296 g; n=136) purchased from the Experimental Animal Center of the National Applied Research Laboratories, Taiwan, were used. Animals were acclimatized for 1 week in an Association for Assessment and Accreditation of Laboratory Animal Care International–accredited animal facility during which they had free access to commercial rat chow. Rats were subsequently divided randomly into 2 groups to receive either normal diet (ND; containing 46% complex carbohydrate, 3.4 kcal/g; Harlan Laboratories, Madison, WI) or HFD (60% fructose diet, 3.6 kcal/g; TD.89247, Harlan). The assigned diets were offered ad libitum for 8 weeks. Within the ND and HFD groups, animals were further divided randomly into the following groups to receive additional treatments for 2 weeks, beginning on week 6 after the initiation of ND or HFD: (1) oral intake of the insulin sensitizer,19 pioglitazone (20 mgkg−1day−1), or saline; and (2) intracisternal infusion via osmotic minipump of a superoxide dismutase (SOD) mimetic,20 tempol (100 pmolkg−1day−1), a mobile mitochondrial electron carrier,21 coenzyme Q10 (CoQ10, 50 pmolkg−1day−1), or artificial cerebrospinal fluid. A third group of animals received microinjection bilaterally into RVLM, nucleus tractus solitarii (NTS), or hypothalamic paraventricular nucleus (PVN) of a lentiviral vector (Lv) carrying small hairpin RNA (shRNA) on week 6 to silence protein inhibitor of neuronal NOS (nNOS; PIN; Lv-PIN shRNA, 1×106 pfu/80 nL). The dose of test agents used was adopted from previous studies12,22,23 or determined in the pilot study. In each experimental group, the levels of fasting plasma glucose, insulin, and triglycerides were determined before, every week, and at the end of 8 weeks of ND or HFD. The presence of insulin resistance was determined by calculating the homeostatic model assessment of insulin resistance indices, and glucose intolerance was determined by the oral glucose tolerance test. Arterial pressure was measured before and weekly after ND or HFD treatment under conscious condition by radiotelemetry. Sympathetic vasomotor outflow from RVLM was assessed by the power density of the low-frequency (LF) component of the systolic blood pressure spectrum.24 At various time intervals after the commencement of ND or HFD treatment, animals were killed with an overdose of pentobarbital sodium (100 mg/kg, IV), and both sides of RVLM were removed for assessment of tissue ROS, nitric/nitrite (NOx), or tetrahydrobiopterin levels; protein expression of insulin receptor (InsR), NADPH oxidase subunits, NOS isoforms, monomer or homodimer of nNOS, PIN, SOD isoforms, or GTP cyclohydrolase I (GTPCH I); as well as enzyme activity of the NADPH oxidase, mitochondrial electron transport chain (ETC) enzymes, SOD, or NOS.
Statistical Analysis All values are expressed as means±SEM. One-way or 2-way ANOVA with repeated measures was used to assess group means, as appropriate, to be followed by the Scheffé multiple-range test for post hoc assessment of individual means. The Student t test was used to compare the means of 2 groups. P
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://hyper.ahajournals.org/content/64/4/815
Data Supplement (unedited) at: http://hyper.ahajournals.org/content/suppl/2014/06/23/HYPERTENSIONAHA.114.03777.DC1.html
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Hypertension is online at: http://hyper.ahajournals.org//subscriptions/
Oxidative Stress Role of Nitric Oxide Synthase Uncoupling at Rostral Ventrolateral Medulla in Redox-Sensitive Hypertension Associated With Metabolic Syndrome Kay L.H. Wu, Yung-Mei Chao, Shiow-Jen Tsay, Chen Hsiu Chen, Samuel H.H. Chan, Ima Dovinova, Julie Y.H. Chan Abstract—Metabolic syndrome (MetS), which is rapidly becoming prevalent worldwide, is long known to be associated with hypertension and recently with oxidative stress. Of note is that oxidative stress in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons reside, contributes to sympathoexcitation and hypertension. This study sought to identify the source of tissue oxidative stress in RVLM and their roles in neural mechanism of hypertension associated with MetS. Adult normotensive rats subjected to a high-fructose diet for 8 weeks developed metabolic traits of MetS, alongside increases in sympathetic vasomotor activity and blood pressure. In RVLM of these MetS rats, the tissue level of reactive oxygen species was increased, nitric oxide (NO) was decreased, and mitochondrial electron transport capacity was reduced. Whereas the protein expression of neuronal NO synthase (nNOS) or protein inhibitor of nNOS was increased, the ratio of nNOS dimer/monomer was significantly decreased. Oral intake of pioglitazone or intracisternal infusion of tempol or coenzyme Q10 significantly abrogated all those molecular events in high-fructose diet–fed rats and ameliorated sympathoexcitation and hypertension. Gene silencing of protein inhibitor of nNOS mRNA in 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 RVLM of high-fructose diet–fed rats, alongside significantly reduced sympathoexcitation and hypertension. These results suggest that redoxsensitive and protein inhibitor of nNOS–mediated nNOS uncoupling is engaged in a vicious cycle that sustains the production of reactive oxygen species in RVLM, resulting in sympathoexcitation and hypertension associated with MetS. (Hypertension. 2014;64:815-824.) Online Data Supplement
•
Key Words: hypertension
■
metabolic cardiovascular syndrome ■ nitric oxide ■ sympathetic nervous system
M
etabolic syndrome (MetS), defined as a cluster of ≥3 disorders that include insulin resistance, dyslipidemia, hypertension, hypercholesterolemia, and abdominal obesity,1,2 is associated with the risk of developing type 2 diabetes mellitus, coronary heart disease, stroke, and cardiovascular-related mortality.3,4 MetS affects >20% of adults in Western populations. However, given the acculturation of Western diet in the developing world, the burden of MetS is highly prevalent worldwide. Although insulin resistance is generally accepted as the main underlying pathogenic mechanism, increasing evidence links excessive reactive oxygen species (ROS) production and tissue oxidative stress to the pathogenesis of MetS and the progression of its complications.5,6 A recent cross-sectional study reported that subjects with more MetS components exhibit higher levels
■
reactive oxygen species
of oxidative stress.7 Better understanding of the significance of the redox signaling in MetS is therefore warranted. The brain uses large amounts of oxygen and ATP for its normal functions and is therefore highly susceptible to oxidative stress. Reports on brain oxidative stress in the pathogenesis of MetS, however, are sporadic. Of the few studies reported, oxidative stress in the hypothalamus is involved in the progression of obesity-induced hypertension.8 In the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons reside,9 oxidative stress induces sympathoexcitation in rats with obesity-induced hypertension.10 ROS are products of normal cellular metabolism and are derived from many sources in different cellular compartments. In RVLM, we11,12 and others13,14 have demonstrated that ROS generated from nicotinamide adenine
Received April 21, 2014; first decision May 5, 2014; revision accepted May 26, 2014. From the Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan (K.L.H.W., Y.M.C., S.H.H.C., J.Y.H.C.); Institute of Biological Science, National Sun Yat-sen University, Kaohsiung, Taiwan (S.J.T.); Department of Anesthesiology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan (C.H.C.); and Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava, Slovakia (I.D.). The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA. 114.03777/-/DC1. Correspondence to Julie Y.H. Chan, Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung 83301, Taiwan. E-mail [email protected] or Ima Dovinova, Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava, Slovakia. E-mail [email protected] © 2014 American Heart Association, Inc. Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.114.03777
815
816 Hypertension October 2014 dinucleotide phosphate (NADPH) oxidase and mitochondrial respiratory enzymes play pivotal roles in neural mechanism of hypertension. In addition, uncoupling of nitric oxide synthase (NOS) contributes to the increased ROS production in cerebral microvessels.15 Intriguingly, NOS dysfunction is implicated in hypertension of individuals with MetS.16 The significance of the interplay between NOS and ROS in RVLM in MetS-associated hypertension is currently unknown. Normotensive rat fed a high-fructose diet (HFD) is a wellestablished rodent model for the study of human MetS. This model mimics several features, including hypertension, insulin resistance, and abnormal lipid profile, of patients with MetS induced by Western diet.17,18 Using this MetS model, we sought to identify the source of ROS and its interplay with NOS in RVLM on neural mechanisms of hypertension associated with MetS.
Materials and Methods All experimental procedures were performed in compliance with the guidelines of Institutional Animal Care and Use Committee in Kaohsiung Chang Gung Memorial Hospital or Kaohsiung Veterans General Hospital. They were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Detailed Materials and Methods are described in the onlineonly Data Supplement. Adult (8 weeks of age) male Sprague-Dawley rats (235–296 g; n=136) purchased from the Experimental Animal Center of the National Applied Research Laboratories, Taiwan, were used. Animals were acclimatized for 1 week in an Association for Assessment and Accreditation of Laboratory Animal Care International–accredited animal facility during which they had free access to commercial rat chow. Rats were subsequently divided randomly into 2 groups to receive either normal diet (ND; containing 46% complex carbohydrate, 3.4 kcal/g; Harlan Laboratories, Madison, WI) or HFD (60% fructose diet, 3.6 kcal/g; TD.89247, Harlan). The assigned diets were offered ad libitum for 8 weeks. Within the ND and HFD groups, animals were further divided randomly into the following groups to receive additional treatments for 2 weeks, beginning on week 6 after the initiation of ND or HFD: (1) oral intake of the insulin sensitizer,19 pioglitazone (20 mgkg−1day−1), or saline; and (2) intracisternal infusion via osmotic minipump of a superoxide dismutase (SOD) mimetic,20 tempol (100 pmolkg−1day−1), a mobile mitochondrial electron carrier,21 coenzyme Q10 (CoQ10, 50 pmolkg−1day−1), or artificial cerebrospinal fluid. A third group of animals received microinjection bilaterally into RVLM, nucleus tractus solitarii (NTS), or hypothalamic paraventricular nucleus (PVN) of a lentiviral vector (Lv) carrying small hairpin RNA (shRNA) on week 6 to silence protein inhibitor of neuronal NOS (nNOS; PIN; Lv-PIN shRNA, 1×106 pfu/80 nL). The dose of test agents used was adopted from previous studies12,22,23 or determined in the pilot study. In each experimental group, the levels of fasting plasma glucose, insulin, and triglycerides were determined before, every week, and at the end of 8 weeks of ND or HFD. The presence of insulin resistance was determined by calculating the homeostatic model assessment of insulin resistance indices, and glucose intolerance was determined by the oral glucose tolerance test. Arterial pressure was measured before and weekly after ND or HFD treatment under conscious condition by radiotelemetry. Sympathetic vasomotor outflow from RVLM was assessed by the power density of the low-frequency (LF) component of the systolic blood pressure spectrum.24 At various time intervals after the commencement of ND or HFD treatment, animals were killed with an overdose of pentobarbital sodium (100 mg/kg, IV), and both sides of RVLM were removed for assessment of tissue ROS, nitric/nitrite (NOx), or tetrahydrobiopterin levels; protein expression of insulin receptor (InsR), NADPH oxidase subunits, NOS isoforms, monomer or homodimer of nNOS, PIN, SOD isoforms, or GTP cyclohydrolase I (GTPCH I); as well as enzyme activity of the NADPH oxidase, mitochondrial electron transport chain (ETC) enzymes, SOD, or NOS.
Statistical Analysis All values are expressed as means±SEM. One-way or 2-way ANOVA with repeated measures was used to assess group means, as appropriate, to be followed by the Scheffé multiple-range test for post hoc assessment of individual means. The Student t test was used to compare the means of 2 groups. P
