ORIGINAL ARTICLES Metformin reduces asymmetric dimethylarginine and prevents hypertension in spontaneously hypertensive rats CHIH-MIN TSAI, HSUAN-CHANG KUO, CHIEN-NING HSU, LI-TUNG HUANG, and YOU-LIN TAIN KAOHSIUNG, TAIWAN
Elevated asymmetric dimethylarginine (ADMA) levels and nitric oxide (NO) deficiency are associated with the development of hypertension. Metformin, an antidiabetic agent, is a structural analog of ADMA. We examined whether metformin can prevent the development of hypertension in spontaneously hypertensive rats (SHRs) by restoration of ADMA-NO balance. SHRs and control normotensive Wistar-Kyoto (WKY) rats were assigned to 4 groups (N 5 8 for each group): untreated SHRs and WKY rats, metformin-treated SHRs and WKY rats. Metformintreated rats received metformin 500 mg/kg per day via oral gavage for 8 weeks. All rats were sacrificed at the age of 12 weeks. We found an increase in the blood pressure of SHRs was prevented by metformin. ADMA levels in the plasma and lung were elevated in SHRs, which metformin prevented. Lung dimethylarginine dimethylaminohydrolase (DDAH, ADMA-metabolizing enzyme) activity was lower in SHRs than WKY rats. Next, metformin had no effect on protein arginine methyltransferase 1 (ADMA-synthesizing enzyme), DDAH-1, DDAH-2, NO synthase enzymes, and DDAH activity in the kidney. Moreover, metformin increased the levels of NO in kidney. Conclusively, the observed antihypertensive effect of metformin in SHRs is because of the restoration of the ADMA-NO pathway. Our findings support the consideration of metformin as an antihypertensive agent for diabetic patients with prehypertension. (Translational Research 2014;164:452–459) Abbreviations: AAR ¼ L-Arginine-to-ADMA ratio; ADMA ¼ asymmetric dimethylarginine; ASR ¼ ADMA-to-SDMA ratio; CAT ¼ cationic amino acid transporter; DDAH ¼ dimethylarginine dimethylaminohydrolase; NOS ¼ nitric oxide synthase; PRMT ¼ protein arginine methyltransferase; SDMA ¼ symmetric dimethylarginine; SHR ¼ spontaneously hypertensive rat; WKY ¼ Wistar Kyoto
From the Department of Pediatrics, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan; Chang Gung University, College of Medicine, Taoyuan, Taiwan; Department of Pharmacy, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan; School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan; Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan. Submitted for publication April 6, 2014; revision submitted July 15, 2014; accepted for publication July 16, 2014.
452
Reprint requests: You-Lin Tain, Department of Pediatrics, Kaohsiung Chang Gung Memorial Hospital, 123 Dabi Road, Niausung, Kaohsiung 833, Taiwan; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2014.07.005
Translational Research Volume 164, Number 6
AT A GLANCE COMMENTARY Tsai C-M, et al. Background
The imbalance between asymmetric dimethylarginine (ADMA) and nitric oxide (NO) is associated with the development of hypertension. Metformin is a structural analog of ADMA. We examined whether metformin can restore ADMA-NO to prevent hypertension in spontaneously hypertensive rats. Translational Significance
Metformin blocks the development of hypertension in spontaneously hypertensive rats by reduction in plasma ADMA and increases in renal NO production. Our findings support the consideration of metformin as an antihypertensive agent for diabetic patients with prehypertension and highlight that the restoration of ADMA-NO pathway might be a therapeutic target for prehypertension.
Tsai et al
453
properties.13 Previous human studies on the effect of metformin on BP have been varied,14,15 finding decreased or unaltered BP. Metformin has been reported to lower BP in SHRs.16-18 The protective effect of metformin on the development of hypertension in SHRs is unclear, as are the underlying mechanisms of its antihypertensive effect. Metformin and ADMA are structural analogs; it has been proposed that they have opposite effects at multiple signaling pathways.19 ADMA is mainly generated by type I protein arginine methyltransferase (PRMT) and metabolized by dimethylarginine dimethylaminohydrolase (DDAH).4 Plasma levels of ADMA are elevated in diabetic patients and positively correlated with insulin resistance.20 Given that metformin reduces ADMA levels in patients with diabetes21 and that ADMA-NO pathway is involved in the development of hypertension,6-9 we examined whether metformin reduces ADMA to prevent the development of hypertension in SHRs in this study. The second aim of this study was to elucidate whether an ADMA-lowering effect of metformin is associated with increased DDAH expression and activity, decreased PRMT-1 expression, or its transport.
INTRODUCTION
Hypertension is a highly prevalent disease. Emerging evidence indicates nitric oxide (NO)-reactive oxygen species (ROS) imbalance in the kidney is involved in the development of hypertension.1,2 Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS), which competes with L-arginine to inhibit NO production and induction of ROS.3 Balance between L-arginine and ADMA maintains NO homeostasis. Elevated ADMA levels have been shown in various experimental hypertensive models and human hypertension.4-7 We recently showed that ADMA level increase and an L-arginine-to-ADMA ratio (AAR) decrease in plasma and kidneys develop early on, even before the onset of hypertension in young spontaneously hypertensive rat (SHR).8 Additionally, our studies demonstrated that therapeutic approaches shifting disturbed ROS/NO balance in prehypertensive stage toward increase in NO lead to blood pressure (BP) lowering in young SHRs.9-11 These observations support that the restoration of ADMA-NO balance might be a therapeutic target for prehypertension. Metformin (dimethylbiguanide) is a first-line drug in the treatment of type 2 diabetic patients by its effect on insulin resistance.12 In addition to lowering blood glucose, metformin has been shown to have platelet antiaggregating effects to reduce advanced glycation end products and to decrease the cellular oxidative reactions, thus demonstrating its broad set of pharmacologic
METHODS Experimental design. This experiment was approved and performed under the Guidelines for Animal Experiments of Chang Gung Memorial Hospital and Chang Gung University. The treatment of animals conformed to the US National Institutes of Health guidelines. Male SHRs and control Wistar-Kyoto (WKY) rats at the age of 3 weeks were obtained (BioLASCO Taiwan Co, Ltd, Taipei, Taiwan) and maintained in an Association for Assessment and Accreditation of Laboratory Animal Care International accredited facility, with free access to tap water and standard rat chow. Rats aged 4 weeks were randomly assigned to 4 groups (N 5 8 for each group): group 1, WKY rats without treatment; group 2, SHRs without treatment; group 3, WKY rats received metformin 500 mg/kg per day via oral gavage (WKY 1 M); and group 4, SHRs received metformin treatment (SHR 1 M). The dose of metformin used here was based on the previous study conducted on SHRs.16 BP was measured in conscious rats by an indirect tail-cuff method (BP-2000; Visitech Systems, Inc, Apex, NC) after systematically trained at the of age 4, 6, 8, 10, and 12 weeks. To ensure accuracy and reproducibility, the rats were acclimated to restraint and tail-cuff inflation for 1 week before the experiment, and measurements were taken at 1:00 PM to 5:00 PM each day. Rats were placed on the specimen platform, and their tails were passed through tail cuffs
454
Translational Research December 2014
Tsai et al
Table I. Weights and functional parameters (N 5 8 for each group) Groups
WKY
SHR
WKY 1 M
SHR 1 M
Mortality Body weight (g) Lung weight (g) Lung weight/100 g body weight Left kidney weight (g) Left kidney weight/100 g body weight Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Mean arterial pressure (mm Hg)
0% 306 6 4 1.67 6 0.1 0.55 6 0.03 1.36 6 0.03 0.44 6 0.01 163 6 2 104 6 4 124 6 3
0% 313 6 3 1.92 6 0.25 0.61 6 0.08 1.4 6 0.02 0.43 6 0.01 193 6 5* 117 6 4* 143 6 4*
0% 310 6 5 1.39 6 0.09 0.45 6 0.03 1.35 6 0.02 0.42 6 0.01 161 6 2† 91 6 3† 114 6 2†
0% 301 6 4 2.07 6 0.19 0.69 6 0.06 1.28 6 0.03 0.43 6 0.01 172 6 1† 102 6 3† 125 6 1†
Abbreviations: M, metformin; SHR, spontaneously hypertensive rat; WKY, Wistar-Kyoto rat. *P , 0.05 vs WKY † P , 0.05 vs SHR
and secured in place with tape. After a 10-minute warmup period, 10 preliminary cycles were performed to allow the rats to adjust to the inflating cuff. For each rat, 5 cycles were recorded at each time point. Three stable measures were taken and averaged. All rats were sacrificed at the age of 12 weeks. Heparinized blood samples were collected. The kidney and lung were harvested and stored in 280 C freezer. Detection of L-arginine, L-citrulline, and dimethylarginines by high-performance liquid chromatography.
Plasma and kidney L-arginine, L-citrulline, ADMA, and symmetric dimethylarginine (SDMA, a stereoisomer of ADMA) levels were measured using highperformance liquid chromatography (HP series 1100; Agilent Technologies, Inc, Santa Clara, CA) with the o-phthaldialdehyde 3-mercaptopropionic acid derivatization reagent as described by us previously.6 Standards contained concentrations of L-arginine, L-citrulline, ADMA, and SDMA in the range of 1–100, 1–100, 0.5–5, and 0.5–5 mM, respectively. The recovery rate was approximately 90%. The tissue concentration was factored for protein concentration, which was represented as micromolar per milligram of protein. Western blot. Western blot analysis was performed as described by us previously.6 A mouse monoclonal antibody (Santa Cruz, Santa Cruz, CA) was used for the detection of neuronal NOS (nNOS). A mouse monoclonal antibody (1:250 dilution, 1-hour incubation; Transduction Laboratories), followed by a goat antimouse Immunoglobulin G-Horseradish Peroxidase secondary antibody, was used to detect endothelial NOS (eNOS). For PRMT-1, we used a rabbit anti-rat PRMT-1 antibody (1:2000; Millipore, Billerica, MA). For DDAH, we used a goat anti-rat DDAH-1 antibody (1:500 dilution, overnight incubation; Santa Cruz) and a goat anti-rat DDAH-2 antibody (1:100 dilution, overnight incubation; Santa Cruz), followed by a secondary donkey anti-goat antibody. Bands of interest were visualized using enhanced chemiluminescence reagents
(PerkinElmer, Waltham, MA) and quantified by densitometry (Quantity One Analysis software; Bio-Rad, Hercules, CA), as integrated optical density (IOD) after subtraction of background. The IOD was factored for Ponceau red staining to correct for any variations in total protein loading. The protein abundance was represented as IOD to PonS ratio. DDAH activity. DDAH activity was measured by a colorimetric assay measuring the rate of citrulline production, as optimized by us recently.22 Kidney cortex was homogenized by sodium phosphate buffer. Tissue homogenate was preincubated with urease for 15 minutes, then 100 mL (2 mg) of homogenate was incubated with 1 mm of ADMA for 45 minutes at 37 C. After deproteinization, supernatant was incubated with color mixture at 60 C for 110 minutes. Each sample was analyzed with a paired blank (which omitted ADMA) to prevent the citrulline interference effect. The absorbance was measured by spectrophotometry at 466 nm. The DDAH activity was represented as micromolar citrulline formation per gram of protein per minute at 37 C. Detection of NO by electron paramagnetic resonance. NO was detected by electron paramagnetic reso-
nance (EPR) with spin probe N-methyl-D-glucamine dithiocarbamate and FeSO4 as described by us previously.23 Samples were placed in a 50 mL glass capillary (Wilmad Glass, Buena, NJ). The EPR spectra were recorded using an EMXplus EPR spectrometer (Bruker) equipped with an EMX-m40X microwave bridge operating at 3.16 GHz. RESULTS
After 8 weeks of experiment, the body weight was no different among 4 groups (Table I). Similarly, kidney weight, lung weight, and their weights to body weight ratios were not different among 4 groups. The systolic and diastolic BPs of SHRs were significantly increased compared with age-matched WKY
Translational Research Volume 164, Number 6
Tsai et al
455
difference in nNOS and eNOS protein abundance among 4 groups (Fig 3E and F). As shown in Fig 4, DDAH activity in the kidney was not different among 4 groups. In the lung, DDAH activity was lower in SHRs compared with WKY rats (Fig 4B). Metformin significantly increased renal NO production in the WKY 1 M and SHR 1 M groups (Fig 4C). DISCUSSION
Fig 1. Effect of metformin on mean arterial pressure in 12-week-old Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). *P , 0.05 vs WKY; #P , 0.05 vs SHR.
rats at 12 weeks. As shown in Fig 1, the mean arterial BP was not different among 4 groups at the age of 4 weeks. The increases in BP during the development of SHRs from age 4 to 12 weeks were prevented by metformin therapy. As shown in Table II, plasma ADMA level was higher in SHRs than that in WKY rats. Metformin therapy significantly reduced the plasma ADMA level in SHRs. In addition, plasma SDMA level was lower in the WKY 1 M group compared with SHRs. In the kidney, L-citrulline, L-arginine, ADMA, and SDMA levels were no different among 4 groups. In the lung, ADMA level was lower in the WKY 1 M group compared with SHRs. Metformin reduced SMDA level in the SHR 1 M group compared with SHRs and WKY rats. As ADMA and L-arginine compete for NOS, the AAR has been used to represent NO bioavailability. As shown in Fig 2, metformin decreased AAR in the kidney in WKY rats and SHRs. The ADMA-to-SDMA ratio (ASR) may provide indirect information about the DDAH activity because only ADMA is metabolized by the enzyme. We found that metformin decreased ASR in the plasma, whereas it increased this ratio in the kidney in SHRs. In the lung, ASR is higher in the SHR 1 M group compared with WKY and WKY 1 M groups. We next studied the expression and activity of proteins involved in the ADMA-NO pathway. We found no difference in ADMA-synthesizing enzyme PRMT-1 protein levels in the kidney between WKY rats and SHRs. However, metformin increased renal protein level of PRMT-1 in the WKY 1 M group (Fig 3B). DDAH-1 and DDAH-2, the ADMA-metabolizing enzymes, abundance in the kidney was not different among 4 groups (Fig 3). Next, there was no significant
The main findings of this study are as follows: (1) metformin blocks the development of hypertension in SHRs; (2) metformin prevents the increases in ADMA levels in the plasma and lung; (3) metformin decreases AAR in the kidney in WKY rats and SHRs; (4) metformin reduces ASR in the plasma, whereas it increases this ratio in the kidney and lung in SHRs; and (5) metformin increased renal NO production in WKY rats and SHRs. To the best of our knowledge, this is the first report that provides a novel protective mechanism of metformin on the development of hypertension focused on the ADMA-NO pathway. In line with previous studies, metformin therapy decreases BP in SHRs.16,17 Although one report showed treatment of metformin at a dose of 500 mg/kg per day over a 2-week period did not affect BP in adult SHRs,18 we found early metformin therapy for 8 weeks can prevent the increase in BP in young SHRs. How does metformin prevent SHRs against the elevation of BP? Our recent observation showed that augmentation of NO in the prehypertensive stage can prevent the development of hypertension in young SHRs.11 In this study, metformin blocks the development of hypertension by reduction in plasma ADMA and increases in renal NO production. Free ADMA levels are dependent on PRMT activity, the rate of protein degradation, the DDAH activity, and the rate of transport in and out of the tissue. Free ADMA level was higher in the kidney compared with the lung as kidney is a major organ for ADMA degradation in the body. However, a higher level of proteinincorporated ADMA was found in the lung compared with other organs, including the kidney.24 We found no difference in renal protein levels of PRMT-1, DDAH-1, DDAH-2, eNOS, and nNOS, which are related to ADMA-NO metabolism, between SHR and SHR 1 M groups. In addition, DDAH activity in the kidney and lung was not different between SHR and SHR 1 M groups. These data suggest that the increased levels of ADMA in the plasma and lung are probably a result of interorgan ADMA transport. It is noteworthy that DDAH activity in lung is approximately 100-fold lower when compared with the activity in kidney. This may explain excessive circulating ADMA leads
456
Translational Research December 2014
Tsai et al
Table II. Plasma and tissue L-arginine and ADMA levels (N 5 8 for each group) Groups
WKY
Plasma (mM) L-Arginine L-Citrulline ADMA SDMA Kidney (mM/mg protein) L-Arginine L-Citrulline ADMA SDMA Lung (mM/mg protein) L-Arginine L-Citrulline ADMA SDMA
SHR
WKY 1 M
SHR 1 M
185.4 6 27.9 74.4 6 5.1 1.8 6 0.2 2.9 6 0.3
212.7 6 25.8 71.2 6 4.4 2.6 6 0.3* 3.4 6 0.2
171.2 6 19.3 70.6 6 4.3 2.2 6 0.1 2.9 6 0.1†
161.9 6 13.2 66.1 6 3.8 1.8 6 0.1†,‡ 3.1 6 0.2
204.4 6 15.4 12 6 0.9 3.5 6 0.5 2.3 6 0.2
206.4 6 14.5 13.5 6 1 4 6 0.4 2.3 6 0.2
181.3 6 12.2 11.2 6 0.8 3.8 6 0.3 1.8 6 0.2
187 6 22.6 11.9 6 1 4 6 0.4 1.8 6 0.3
4.8 6 0.1 8.3 6 1.4 0.112 6 0.004 0.025 6 0.001
6.1 6 0.5 6.2 6 0.7 0.144 6 0.008* 0.027 6 0.002
4 6 0.2 5 6 0.4 0.092 6 0.003† 0.02 6 0.001
4.5 6 0.2 3.7 6 0.2 0.095 6 0.004† 0.013 6 0.001*,†
Abbreviations: ADMA, asymmetric dimethylarginine; M, metformin; SDMA, symmetric dimethylarginine; SHR, spontaneously hypertensive rat; WKY, Wistar-Kyoto rat. *P , 0.05 vs WKY † P , 0.05 vs SHR ‡ P , 0.05 WKY 1 M vs SHR 1 M.
Fig 2. Effect of metformin on (A) L-arginine-to-ADMA ratio and (B) ADMA-to-SDMA ratio in plasma, kidney, and lung in 12-week-old Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). *P , 0.05 vs WKY; #P , 0.05 vs SHR; $P , 0.05 vs WKY 1 M.
to an increase in ADMA in lung but not kidney in SHRs. One report showed that metformin might compete with ADMA for same transporter.25 However, another study demonstrated that metformin has no effect on the activity of cationic amino acid transporter 1 (CAT-1), a transport for ADMA and L-arginine.26 As the kidney is a major organ in ADMA metabolism, as CAT-1 level and activity are decreased in SHRs, our previous report suggests that SHR kidney might protect against injury by decreasing the ADMA uptake from circulation into the kidneys,8 whereas plasma and pulmonary ADMA levels were decreased in response to metformin therapy, metformin might compete with ADMA to move in or out of cells to differentially regulate ADMA homeostasis in different
tissues. Another possibility is metformin competes with ADMA to inhibit NOS to restore NO production. This notion was supported by metformin increased NO production, despite no change in NOS protein levels in the kidney. Although AAR has been considered as a marker of NO bioavailability,27 there are reciprocal changes of renal AAR and NO level in response to metformin therapy in this study. Metformin significantly reduced AAR and had a tendency to decrease L-arginine and SDMA level in the kidney. Given that L-arginine and dimethylarginines (ADMA and SDMA) share the same CAT, SDMA may indirectly inhibit NO availability by competition with L-arginine for transporter uptake. Additional study is required to clarify whether metformin, as a structural analog of ADMA, can compete with
Translational Research Volume 164, Number 6
Tsai et al
Fig 3. Representative Western blots (A) show PRMT-1 (42 kDa), DDAH-1 (34 kDa), DDAH-2 (30 kDa), eNOS (150 kDa), and nNOS (160 kDa) in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) at the age of 12 weeks. Relative abundance of renal cortical (B) PRMT-1, (C) DDAH-1, (D) DDAH-2, (E) eNOS, and (F) nNOS. N 5 8 for each group, *P , 0.05 vs WKY; #P , 0.05 vs SHR. PRMT, protein arginine methyltransferase; DDAH, dimethylarginine dimethylaminohydrolase; eNOS, endothelial nitric oxide synthase; nNOS, neuronal NOS.
Fig 4. Effect of metformin on (A) renal and (B) pulmonary DDAH activity, and (C) renal NO production in 12-week-old Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). N 5 6 for each group, *P , 0.05 vs WKY; #P , 0.05 vs SHR. DDAH, dimethylarginine dimethylaminohydrolase; EPR, electron paramagnetic resonance; NO, nitric oxide.
457
458
Translational Research December 2014
Tsai et al
L-arginine and SDMA to regulate their homeostasis and consequent NO function. Next, ASR may provide indirect information about the DDAH activity because only ADMA is metabolized by the DDAH. However, it is not supported by our results as metformin did not affect DDAH activity but increased ASR in the kidney. Metformin significantly increased ASR and decreased SDMA level in the lung. As SDMA cannot be metabolized by DDAH, the decreased SDMA level reflects decreased PRMT activity, decreased protein turnover rate, and increased efflux out of the cell. Nevertheless, the relative importance of each component remains to be determined. One limitation in this study is that we did not measure blood glucose, insulin, and leptin levels. Although our study lacked these data, a previous study has reported that there are no differences in plasma glucose, insulin, and leptin levels between WKY rats and SHRs.28 This observation indicated that alterations in these parameters in response to metformin, if they did occur, were unlikely to explain the protective effects of metformin in the present study. A second limitation is that we did not examine all the other possible protective effects of metformin in this study. Basal adenosine monophosphate (AMP)-activated protein kinase (AMPK) activation was reduced in aorta of SHRs vs WKY rats.29 Metformin was reported to activate AMPK to cause vasodilatation.30 Therefore, it is possible that metformin may activate aortic AMPK to lower BP in SHRs. Next, elevated ADMA levels were reported to be associated with inflammation markers in cardiovascular disease.6,7 Given that metformin could be beneficial in kidney disease with attenuation of inflammation,31 additional study is required to clarify the protective effects of metformin is via the ADMA-NO pathway alone or via other mechanisms (eg, inflammation) to prevent the development of hypertension. Moreover, metformin was reported to induce NOS activity but not NOS expression.32 Whether metformin can regulate NOS activity by post-translational modifications (eg, phosphorylation), translocation, and protein-protein interactions, to prevent hypertension awaits further evaluation.
CONCLUSIONS
Several important mechanisms are involved in the protective actions of metformin on the ADMA-NO pathway to block the development of hypertension in SHRs, including a reduction in plasma ADMA and an increase in renal NO level. Our findings highlight that the restoration of ADMA-NO balance might be a therapeutic target for prehypertension and support the consideration of metformin as an antihypertensive agent for diabetic patients with prehypertension.
ACKNOWLEDGMENTS
Conflicts of Interest: All authors have read the journal’s authorship agreement. All authors have read the journal’s policy on conflicts of interest and have none to declare. This work was supported by grants CMRPG8C0331 and CMRPG8C0341 from Chang Gung Memorial Hospital, Kaohsiung, Taiwan. The authors thank Dr Samuel H.H. Chan and the Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, for providing space to support EPR. REFERENCES
1. Wilcox CS. Oxidative stress and nitric oxide deficiency in the kidney: a critical link to hypertension? Am J Physiol Regul Integr Comp Physiol 2005;289:R913–35. 2. Vaziri ND. Roles of oxidative stress and antioxidant therapy in chronic kidney disease and hypertension. Curr Opin Nephrol Hypertens 2004;13:93–9. 3. Sydow K, M€unzel T. ADMA and oxidative stress. Atheroscler Suppl 2003;4:41–51. 4. Matsuoka H, Itoh S, Kimoto M, et al. Asymmetrical dimethylarginine, an endogenous nitric oxide synthase inhibitor, in experimental hypertension. Hypertension 1997;29:242–7. 5. B€oger RH. Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, explains the ‘‘L-arginine paradox’’ and acts as a novel cardiovascular risk factor. J Nutr 2004;134: S2842–7. 6. Teerlink T, Luo Z, Palm F, Wilcox CS. Cellular ADMA: regulation and action. Pharmacol Res 2009;60:448–60. 7. Tain YL, Huang LT. Asymmetric dimethylarginine: clinical applications in pediatric medicine. J Formos Med Assoc 2011;110:70–7. 8. Hsu CN, Huang LT, Lau YT, Lin CY, Tain YL. The combined ratios of L-arginine and asymmetric and symmetric dimethylarginine as biomarkers in spontaneously hypertensive rats. Transl Res 2012;159:90–8. 9. Tain YL, Huang LT, Lin IC, Lau YT, Lin CY. Melatonin prevents hypertension and increased asymmetric dimethylarginine in young spontaneous hypertensive rats. J Pineal Res 2010;49: 390–8. 10. Tain YL, Hsu CN, Huang LT, Lau YT. Apocynin attenuates oxidative stress and hypertension in young spontaneously hypertensive rats independent of ADMA/NO pathway. Free Radic Res 2012; 46:68–76. 11. Chien SJ, Lin KM, Kuo HC, et al. Two different approaches to restore renal nitric oxide and prevent hypertension in young spontaneously hypertensive rats: L-citrulline and nitrate. Transl Res 2014;163:43–52. 12. Viollet B, Guigas B, Sanz Garcia N, Leclerc J, Foretz M, Andreelli F. Cellular and molecular mechanisms of metformin: an overview. Clin Sci (Lond) 2012;122:253–70. 13. Andujar-Plata P, Pi-Sunyer X, Laferrere B. Metformin effects revisited. Diabetes Res Clin Pract 2012;95:1–9. 14. De Leo V, Musacchio MC, Piomboni P, Di Sabatino A, Morgante G. The administration of metformin during pregnancy reduces polycystic ovary syndrome related gestational complications. Eur J Obstet Gynecol Reprod Biol 2011;157:63–6. 15. He H, Zhao Z, Chen J, et al. Metformin-based treatment for obesity-related hypertension: a randomized, double-blind, placebo-controlled trial. J Hypertens 2012;30:1430–9.
Translational Research Volume 164, Number 6
16. Verma S, Bhanot S, McNeill JH. Metformin decreases plasma insulin levels and systolic blood pressure in spontaneously hypertensive rats. Am J Physiol 1994;267:H1250–3. 17. Bhalla RC, Toth KF, Tan E, Bhatty RA, Mathias E, Sharma RV. Vascular effects of metformin. Possible mechanisms for its antihypertensive action in the spontaneously hypertensive rat. Am J Hypertens 1996;9:570–6. 18. Muntzel MS, Hamidou I, Barrett S. Metformin attenuates saltinduced hypertension in spontaneously hypertensive rats. Hypertension 1999;33:1135–40. 19. Bestermann WH Jr. The ADMA-metformin hypothesis: linking the cardiovascular consequences of the metabolic syndrome and type 2 diabetes. Cardiorenal Med 2011;1:211–9. 20. Sydow K, Mondon CE, Cooke JP. Insulin resistance: potential role of the endogenous nitric oxide synthase inhibitor ADMA. Vasc Med 2005;10:S35–43. 21. Asagami T, Abbasi F, Stuelinger M, et al. Metformin treatment lowers asymmetric dimethylarginine concentrations in patients with type 2 diabetes. Metabolism 2002;51:843–6. 22. Tain YL, Baylis C. Determination of dimethylarginine dimethylaminohydrolase activity in the kidney. Kidney Int 2007;72:886–9. 23. Tain YL, Lee WC, Hsu CN, et al. Asymmetric dimethylarginine is associated with developmental programming of adult kidney disease and hypertension in offspring of streptozotocin-treated mothers. PLoS ONE 2013;8:e55420. 24. Bulau P, Zakrzewicz D, Kitowska K, et al. Analysis of methylarginine metabolism in the cardiovascular system identifies the lung as a major source of ADMA. Am J Physiol Lung Cell Mol Physiol 2007;292:L18–24.
Tsai et al
459
25. Detaille D, Guigas B, Leverve X, Wiernsperger N, Devos P. Obligatory role of membrane events in the regulatory effect of metformin on the respiratory chain function. Biochem Pharmacol 2002;63:1259–72. 26. Strobel J, Mieth M, Endress B, et al. Interaction of the cardiovascular risk marker asymmetric dimethylarginine (ADMA) with the human cationic amino acid transporter 1 (CAT1). J Mol Cell Cardiol 2012;53:392–400. 27. Bode-Boger SM, Scalera F, Ignarro LJ. The L-arginine paradox: importance of the L-arginine/asymmetrical dimethylarginine ratio. Pharmacol Ther 2007;114:295–306. 28. Iwase M, Ichikawa K, Tashiro K, et al. Effects of monosodium glutamate-induced obesity in spontaneously hypertensive rats vs. Wistar Kyoto rats: serum leptin and blood flow to brown adipose tissue. Hypertens Res 2000;23:503–10. 29. Ford RJ, Teschke SR, Reid EB, Durham KK, Kroetsch JT, Rush JW. AMP-activated protein kinase activator AICAR acutely lowers blood pressure and relaxes isolated resistance arteries of hypertensive rats. J Hypertens 2012;30:725–33. 30. Sung JY, Choi HC. Metformin-induced AMP-activated protein kinase activation regulates phenylephrine-mediated contraction of rat aorta. Biochem Biophys Res Commun 2012;421:599–604. 31. Rocha A, Almeida M, Santos J, Carvalho A. Metformin in patients with chronic kidney disease: strengths and weaknesses. J Nephrol 2013;26:55–60. 32. Sartoretto JL, Melo GA, Carvalho MH, et al. Metformin treatment restores the altered microvascular reactivity in neonatal streptozotocin-induced diabetic rats increasing NOS activity, but not NOS expression. Life Sci 2005;77:2676–89.
Elevated asymmetric dimethylarginine (ADMA) levels and nitric oxide (NO) deficiency are associated with the development of hypertension. Metformin, an antidiabetic agent, is a structural analog of ADMA. We examined whether metformin can prevent the development of hypertension in spontaneously hypertensive rats (SHRs) by restoration of ADMA-NO balance. SHRs and control normotensive Wistar-Kyoto (WKY) rats were assigned to 4 groups (N 5 8 for each group): untreated SHRs and WKY rats, metformin-treated SHRs and WKY rats. Metformintreated rats received metformin 500 mg/kg per day via oral gavage for 8 weeks. All rats were sacrificed at the age of 12 weeks. We found an increase in the blood pressure of SHRs was prevented by metformin. ADMA levels in the plasma and lung were elevated in SHRs, which metformin prevented. Lung dimethylarginine dimethylaminohydrolase (DDAH, ADMA-metabolizing enzyme) activity was lower in SHRs than WKY rats. Next, metformin had no effect on protein arginine methyltransferase 1 (ADMA-synthesizing enzyme), DDAH-1, DDAH-2, NO synthase enzymes, and DDAH activity in the kidney. Moreover, metformin increased the levels of NO in kidney. Conclusively, the observed antihypertensive effect of metformin in SHRs is because of the restoration of the ADMA-NO pathway. Our findings support the consideration of metformin as an antihypertensive agent for diabetic patients with prehypertension. (Translational Research 2014;164:452–459) Abbreviations: AAR ¼ L-Arginine-to-ADMA ratio; ADMA ¼ asymmetric dimethylarginine; ASR ¼ ADMA-to-SDMA ratio; CAT ¼ cationic amino acid transporter; DDAH ¼ dimethylarginine dimethylaminohydrolase; NOS ¼ nitric oxide synthase; PRMT ¼ protein arginine methyltransferase; SDMA ¼ symmetric dimethylarginine; SHR ¼ spontaneously hypertensive rat; WKY ¼ Wistar Kyoto
From the Department of Pediatrics, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan; Chang Gung University, College of Medicine, Taoyuan, Taiwan; Department of Pharmacy, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan; School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan; Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan. Submitted for publication April 6, 2014; revision submitted July 15, 2014; accepted for publication July 16, 2014.
452
Reprint requests: You-Lin Tain, Department of Pediatrics, Kaohsiung Chang Gung Memorial Hospital, 123 Dabi Road, Niausung, Kaohsiung 833, Taiwan; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2014.07.005
Translational Research Volume 164, Number 6
AT A GLANCE COMMENTARY Tsai C-M, et al. Background
The imbalance between asymmetric dimethylarginine (ADMA) and nitric oxide (NO) is associated with the development of hypertension. Metformin is a structural analog of ADMA. We examined whether metformin can restore ADMA-NO to prevent hypertension in spontaneously hypertensive rats. Translational Significance
Metformin blocks the development of hypertension in spontaneously hypertensive rats by reduction in plasma ADMA and increases in renal NO production. Our findings support the consideration of metformin as an antihypertensive agent for diabetic patients with prehypertension and highlight that the restoration of ADMA-NO pathway might be a therapeutic target for prehypertension.
Tsai et al
453
properties.13 Previous human studies on the effect of metformin on BP have been varied,14,15 finding decreased or unaltered BP. Metformin has been reported to lower BP in SHRs.16-18 The protective effect of metformin on the development of hypertension in SHRs is unclear, as are the underlying mechanisms of its antihypertensive effect. Metformin and ADMA are structural analogs; it has been proposed that they have opposite effects at multiple signaling pathways.19 ADMA is mainly generated by type I protein arginine methyltransferase (PRMT) and metabolized by dimethylarginine dimethylaminohydrolase (DDAH).4 Plasma levels of ADMA are elevated in diabetic patients and positively correlated with insulin resistance.20 Given that metformin reduces ADMA levels in patients with diabetes21 and that ADMA-NO pathway is involved in the development of hypertension,6-9 we examined whether metformin reduces ADMA to prevent the development of hypertension in SHRs in this study. The second aim of this study was to elucidate whether an ADMA-lowering effect of metformin is associated with increased DDAH expression and activity, decreased PRMT-1 expression, or its transport.
INTRODUCTION
Hypertension is a highly prevalent disease. Emerging evidence indicates nitric oxide (NO)-reactive oxygen species (ROS) imbalance in the kidney is involved in the development of hypertension.1,2 Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS), which competes with L-arginine to inhibit NO production and induction of ROS.3 Balance between L-arginine and ADMA maintains NO homeostasis. Elevated ADMA levels have been shown in various experimental hypertensive models and human hypertension.4-7 We recently showed that ADMA level increase and an L-arginine-to-ADMA ratio (AAR) decrease in plasma and kidneys develop early on, even before the onset of hypertension in young spontaneously hypertensive rat (SHR).8 Additionally, our studies demonstrated that therapeutic approaches shifting disturbed ROS/NO balance in prehypertensive stage toward increase in NO lead to blood pressure (BP) lowering in young SHRs.9-11 These observations support that the restoration of ADMA-NO balance might be a therapeutic target for prehypertension. Metformin (dimethylbiguanide) is a first-line drug in the treatment of type 2 diabetic patients by its effect on insulin resistance.12 In addition to lowering blood glucose, metformin has been shown to have platelet antiaggregating effects to reduce advanced glycation end products and to decrease the cellular oxidative reactions, thus demonstrating its broad set of pharmacologic
METHODS Experimental design. This experiment was approved and performed under the Guidelines for Animal Experiments of Chang Gung Memorial Hospital and Chang Gung University. The treatment of animals conformed to the US National Institutes of Health guidelines. Male SHRs and control Wistar-Kyoto (WKY) rats at the age of 3 weeks were obtained (BioLASCO Taiwan Co, Ltd, Taipei, Taiwan) and maintained in an Association for Assessment and Accreditation of Laboratory Animal Care International accredited facility, with free access to tap water and standard rat chow. Rats aged 4 weeks were randomly assigned to 4 groups (N 5 8 for each group): group 1, WKY rats without treatment; group 2, SHRs without treatment; group 3, WKY rats received metformin 500 mg/kg per day via oral gavage (WKY 1 M); and group 4, SHRs received metformin treatment (SHR 1 M). The dose of metformin used here was based on the previous study conducted on SHRs.16 BP was measured in conscious rats by an indirect tail-cuff method (BP-2000; Visitech Systems, Inc, Apex, NC) after systematically trained at the of age 4, 6, 8, 10, and 12 weeks. To ensure accuracy and reproducibility, the rats were acclimated to restraint and tail-cuff inflation for 1 week before the experiment, and measurements were taken at 1:00 PM to 5:00 PM each day. Rats were placed on the specimen platform, and their tails were passed through tail cuffs
454
Translational Research December 2014
Tsai et al
Table I. Weights and functional parameters (N 5 8 for each group) Groups
WKY
SHR
WKY 1 M
SHR 1 M
Mortality Body weight (g) Lung weight (g) Lung weight/100 g body weight Left kidney weight (g) Left kidney weight/100 g body weight Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Mean arterial pressure (mm Hg)
0% 306 6 4 1.67 6 0.1 0.55 6 0.03 1.36 6 0.03 0.44 6 0.01 163 6 2 104 6 4 124 6 3
0% 313 6 3 1.92 6 0.25 0.61 6 0.08 1.4 6 0.02 0.43 6 0.01 193 6 5* 117 6 4* 143 6 4*
0% 310 6 5 1.39 6 0.09 0.45 6 0.03 1.35 6 0.02 0.42 6 0.01 161 6 2† 91 6 3† 114 6 2†
0% 301 6 4 2.07 6 0.19 0.69 6 0.06 1.28 6 0.03 0.43 6 0.01 172 6 1† 102 6 3† 125 6 1†
Abbreviations: M, metformin; SHR, spontaneously hypertensive rat; WKY, Wistar-Kyoto rat. *P , 0.05 vs WKY † P , 0.05 vs SHR
and secured in place with tape. After a 10-minute warmup period, 10 preliminary cycles were performed to allow the rats to adjust to the inflating cuff. For each rat, 5 cycles were recorded at each time point. Three stable measures were taken and averaged. All rats were sacrificed at the age of 12 weeks. Heparinized blood samples were collected. The kidney and lung were harvested and stored in 280 C freezer. Detection of L-arginine, L-citrulline, and dimethylarginines by high-performance liquid chromatography.
Plasma and kidney L-arginine, L-citrulline, ADMA, and symmetric dimethylarginine (SDMA, a stereoisomer of ADMA) levels were measured using highperformance liquid chromatography (HP series 1100; Agilent Technologies, Inc, Santa Clara, CA) with the o-phthaldialdehyde 3-mercaptopropionic acid derivatization reagent as described by us previously.6 Standards contained concentrations of L-arginine, L-citrulline, ADMA, and SDMA in the range of 1–100, 1–100, 0.5–5, and 0.5–5 mM, respectively. The recovery rate was approximately 90%. The tissue concentration was factored for protein concentration, which was represented as micromolar per milligram of protein. Western blot. Western blot analysis was performed as described by us previously.6 A mouse monoclonal antibody (Santa Cruz, Santa Cruz, CA) was used for the detection of neuronal NOS (nNOS). A mouse monoclonal antibody (1:250 dilution, 1-hour incubation; Transduction Laboratories), followed by a goat antimouse Immunoglobulin G-Horseradish Peroxidase secondary antibody, was used to detect endothelial NOS (eNOS). For PRMT-1, we used a rabbit anti-rat PRMT-1 antibody (1:2000; Millipore, Billerica, MA). For DDAH, we used a goat anti-rat DDAH-1 antibody (1:500 dilution, overnight incubation; Santa Cruz) and a goat anti-rat DDAH-2 antibody (1:100 dilution, overnight incubation; Santa Cruz), followed by a secondary donkey anti-goat antibody. Bands of interest were visualized using enhanced chemiluminescence reagents
(PerkinElmer, Waltham, MA) and quantified by densitometry (Quantity One Analysis software; Bio-Rad, Hercules, CA), as integrated optical density (IOD) after subtraction of background. The IOD was factored for Ponceau red staining to correct for any variations in total protein loading. The protein abundance was represented as IOD to PonS ratio. DDAH activity. DDAH activity was measured by a colorimetric assay measuring the rate of citrulline production, as optimized by us recently.22 Kidney cortex was homogenized by sodium phosphate buffer. Tissue homogenate was preincubated with urease for 15 minutes, then 100 mL (2 mg) of homogenate was incubated with 1 mm of ADMA for 45 minutes at 37 C. After deproteinization, supernatant was incubated with color mixture at 60 C for 110 minutes. Each sample was analyzed with a paired blank (which omitted ADMA) to prevent the citrulline interference effect. The absorbance was measured by spectrophotometry at 466 nm. The DDAH activity was represented as micromolar citrulline formation per gram of protein per minute at 37 C. Detection of NO by electron paramagnetic resonance. NO was detected by electron paramagnetic reso-
nance (EPR) with spin probe N-methyl-D-glucamine dithiocarbamate and FeSO4 as described by us previously.23 Samples were placed in a 50 mL glass capillary (Wilmad Glass, Buena, NJ). The EPR spectra were recorded using an EMXplus EPR spectrometer (Bruker) equipped with an EMX-m40X microwave bridge operating at 3.16 GHz. RESULTS
After 8 weeks of experiment, the body weight was no different among 4 groups (Table I). Similarly, kidney weight, lung weight, and their weights to body weight ratios were not different among 4 groups. The systolic and diastolic BPs of SHRs were significantly increased compared with age-matched WKY
Translational Research Volume 164, Number 6
Tsai et al
455
difference in nNOS and eNOS protein abundance among 4 groups (Fig 3E and F). As shown in Fig 4, DDAH activity in the kidney was not different among 4 groups. In the lung, DDAH activity was lower in SHRs compared with WKY rats (Fig 4B). Metformin significantly increased renal NO production in the WKY 1 M and SHR 1 M groups (Fig 4C). DISCUSSION
Fig 1. Effect of metformin on mean arterial pressure in 12-week-old Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). *P , 0.05 vs WKY; #P , 0.05 vs SHR.
rats at 12 weeks. As shown in Fig 1, the mean arterial BP was not different among 4 groups at the age of 4 weeks. The increases in BP during the development of SHRs from age 4 to 12 weeks were prevented by metformin therapy. As shown in Table II, plasma ADMA level was higher in SHRs than that in WKY rats. Metformin therapy significantly reduced the plasma ADMA level in SHRs. In addition, plasma SDMA level was lower in the WKY 1 M group compared with SHRs. In the kidney, L-citrulline, L-arginine, ADMA, and SDMA levels were no different among 4 groups. In the lung, ADMA level was lower in the WKY 1 M group compared with SHRs. Metformin reduced SMDA level in the SHR 1 M group compared with SHRs and WKY rats. As ADMA and L-arginine compete for NOS, the AAR has been used to represent NO bioavailability. As shown in Fig 2, metformin decreased AAR in the kidney in WKY rats and SHRs. The ADMA-to-SDMA ratio (ASR) may provide indirect information about the DDAH activity because only ADMA is metabolized by the enzyme. We found that metformin decreased ASR in the plasma, whereas it increased this ratio in the kidney in SHRs. In the lung, ASR is higher in the SHR 1 M group compared with WKY and WKY 1 M groups. We next studied the expression and activity of proteins involved in the ADMA-NO pathway. We found no difference in ADMA-synthesizing enzyme PRMT-1 protein levels in the kidney between WKY rats and SHRs. However, metformin increased renal protein level of PRMT-1 in the WKY 1 M group (Fig 3B). DDAH-1 and DDAH-2, the ADMA-metabolizing enzymes, abundance in the kidney was not different among 4 groups (Fig 3). Next, there was no significant
The main findings of this study are as follows: (1) metformin blocks the development of hypertension in SHRs; (2) metformin prevents the increases in ADMA levels in the plasma and lung; (3) metformin decreases AAR in the kidney in WKY rats and SHRs; (4) metformin reduces ASR in the plasma, whereas it increases this ratio in the kidney and lung in SHRs; and (5) metformin increased renal NO production in WKY rats and SHRs. To the best of our knowledge, this is the first report that provides a novel protective mechanism of metformin on the development of hypertension focused on the ADMA-NO pathway. In line with previous studies, metformin therapy decreases BP in SHRs.16,17 Although one report showed treatment of metformin at a dose of 500 mg/kg per day over a 2-week period did not affect BP in adult SHRs,18 we found early metformin therapy for 8 weeks can prevent the increase in BP in young SHRs. How does metformin prevent SHRs against the elevation of BP? Our recent observation showed that augmentation of NO in the prehypertensive stage can prevent the development of hypertension in young SHRs.11 In this study, metformin blocks the development of hypertension by reduction in plasma ADMA and increases in renal NO production. Free ADMA levels are dependent on PRMT activity, the rate of protein degradation, the DDAH activity, and the rate of transport in and out of the tissue. Free ADMA level was higher in the kidney compared with the lung as kidney is a major organ for ADMA degradation in the body. However, a higher level of proteinincorporated ADMA was found in the lung compared with other organs, including the kidney.24 We found no difference in renal protein levels of PRMT-1, DDAH-1, DDAH-2, eNOS, and nNOS, which are related to ADMA-NO metabolism, between SHR and SHR 1 M groups. In addition, DDAH activity in the kidney and lung was not different between SHR and SHR 1 M groups. These data suggest that the increased levels of ADMA in the plasma and lung are probably a result of interorgan ADMA transport. It is noteworthy that DDAH activity in lung is approximately 100-fold lower when compared with the activity in kidney. This may explain excessive circulating ADMA leads
456
Translational Research December 2014
Tsai et al
Table II. Plasma and tissue L-arginine and ADMA levels (N 5 8 for each group) Groups
WKY
Plasma (mM) L-Arginine L-Citrulline ADMA SDMA Kidney (mM/mg protein) L-Arginine L-Citrulline ADMA SDMA Lung (mM/mg protein) L-Arginine L-Citrulline ADMA SDMA
SHR
WKY 1 M
SHR 1 M
185.4 6 27.9 74.4 6 5.1 1.8 6 0.2 2.9 6 0.3
212.7 6 25.8 71.2 6 4.4 2.6 6 0.3* 3.4 6 0.2
171.2 6 19.3 70.6 6 4.3 2.2 6 0.1 2.9 6 0.1†
161.9 6 13.2 66.1 6 3.8 1.8 6 0.1†,‡ 3.1 6 0.2
204.4 6 15.4 12 6 0.9 3.5 6 0.5 2.3 6 0.2
206.4 6 14.5 13.5 6 1 4 6 0.4 2.3 6 0.2
181.3 6 12.2 11.2 6 0.8 3.8 6 0.3 1.8 6 0.2
187 6 22.6 11.9 6 1 4 6 0.4 1.8 6 0.3
4.8 6 0.1 8.3 6 1.4 0.112 6 0.004 0.025 6 0.001
6.1 6 0.5 6.2 6 0.7 0.144 6 0.008* 0.027 6 0.002
4 6 0.2 5 6 0.4 0.092 6 0.003† 0.02 6 0.001
4.5 6 0.2 3.7 6 0.2 0.095 6 0.004† 0.013 6 0.001*,†
Abbreviations: ADMA, asymmetric dimethylarginine; M, metformin; SDMA, symmetric dimethylarginine; SHR, spontaneously hypertensive rat; WKY, Wistar-Kyoto rat. *P , 0.05 vs WKY † P , 0.05 vs SHR ‡ P , 0.05 WKY 1 M vs SHR 1 M.
Fig 2. Effect of metformin on (A) L-arginine-to-ADMA ratio and (B) ADMA-to-SDMA ratio in plasma, kidney, and lung in 12-week-old Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). *P , 0.05 vs WKY; #P , 0.05 vs SHR; $P , 0.05 vs WKY 1 M.
to an increase in ADMA in lung but not kidney in SHRs. One report showed that metformin might compete with ADMA for same transporter.25 However, another study demonstrated that metformin has no effect on the activity of cationic amino acid transporter 1 (CAT-1), a transport for ADMA and L-arginine.26 As the kidney is a major organ in ADMA metabolism, as CAT-1 level and activity are decreased in SHRs, our previous report suggests that SHR kidney might protect against injury by decreasing the ADMA uptake from circulation into the kidneys,8 whereas plasma and pulmonary ADMA levels were decreased in response to metformin therapy, metformin might compete with ADMA to move in or out of cells to differentially regulate ADMA homeostasis in different
tissues. Another possibility is metformin competes with ADMA to inhibit NOS to restore NO production. This notion was supported by metformin increased NO production, despite no change in NOS protein levels in the kidney. Although AAR has been considered as a marker of NO bioavailability,27 there are reciprocal changes of renal AAR and NO level in response to metformin therapy in this study. Metformin significantly reduced AAR and had a tendency to decrease L-arginine and SDMA level in the kidney. Given that L-arginine and dimethylarginines (ADMA and SDMA) share the same CAT, SDMA may indirectly inhibit NO availability by competition with L-arginine for transporter uptake. Additional study is required to clarify whether metformin, as a structural analog of ADMA, can compete with
Translational Research Volume 164, Number 6
Tsai et al
Fig 3. Representative Western blots (A) show PRMT-1 (42 kDa), DDAH-1 (34 kDa), DDAH-2 (30 kDa), eNOS (150 kDa), and nNOS (160 kDa) in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) at the age of 12 weeks. Relative abundance of renal cortical (B) PRMT-1, (C) DDAH-1, (D) DDAH-2, (E) eNOS, and (F) nNOS. N 5 8 for each group, *P , 0.05 vs WKY; #P , 0.05 vs SHR. PRMT, protein arginine methyltransferase; DDAH, dimethylarginine dimethylaminohydrolase; eNOS, endothelial nitric oxide synthase; nNOS, neuronal NOS.
Fig 4. Effect of metformin on (A) renal and (B) pulmonary DDAH activity, and (C) renal NO production in 12-week-old Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). N 5 6 for each group, *P , 0.05 vs WKY; #P , 0.05 vs SHR. DDAH, dimethylarginine dimethylaminohydrolase; EPR, electron paramagnetic resonance; NO, nitric oxide.
457
458
Translational Research December 2014
Tsai et al
L-arginine and SDMA to regulate their homeostasis and consequent NO function. Next, ASR may provide indirect information about the DDAH activity because only ADMA is metabolized by the DDAH. However, it is not supported by our results as metformin did not affect DDAH activity but increased ASR in the kidney. Metformin significantly increased ASR and decreased SDMA level in the lung. As SDMA cannot be metabolized by DDAH, the decreased SDMA level reflects decreased PRMT activity, decreased protein turnover rate, and increased efflux out of the cell. Nevertheless, the relative importance of each component remains to be determined. One limitation in this study is that we did not measure blood glucose, insulin, and leptin levels. Although our study lacked these data, a previous study has reported that there are no differences in plasma glucose, insulin, and leptin levels between WKY rats and SHRs.28 This observation indicated that alterations in these parameters in response to metformin, if they did occur, were unlikely to explain the protective effects of metformin in the present study. A second limitation is that we did not examine all the other possible protective effects of metformin in this study. Basal adenosine monophosphate (AMP)-activated protein kinase (AMPK) activation was reduced in aorta of SHRs vs WKY rats.29 Metformin was reported to activate AMPK to cause vasodilatation.30 Therefore, it is possible that metformin may activate aortic AMPK to lower BP in SHRs. Next, elevated ADMA levels were reported to be associated with inflammation markers in cardiovascular disease.6,7 Given that metformin could be beneficial in kidney disease with attenuation of inflammation,31 additional study is required to clarify the protective effects of metformin is via the ADMA-NO pathway alone or via other mechanisms (eg, inflammation) to prevent the development of hypertension. Moreover, metformin was reported to induce NOS activity but not NOS expression.32 Whether metformin can regulate NOS activity by post-translational modifications (eg, phosphorylation), translocation, and protein-protein interactions, to prevent hypertension awaits further evaluation.
CONCLUSIONS
Several important mechanisms are involved in the protective actions of metformin on the ADMA-NO pathway to block the development of hypertension in SHRs, including a reduction in plasma ADMA and an increase in renal NO level. Our findings highlight that the restoration of ADMA-NO balance might be a therapeutic target for prehypertension and support the consideration of metformin as an antihypertensive agent for diabetic patients with prehypertension.
ACKNOWLEDGMENTS
Conflicts of Interest: All authors have read the journal’s authorship agreement. All authors have read the journal’s policy on conflicts of interest and have none to declare. This work was supported by grants CMRPG8C0331 and CMRPG8C0341 from Chang Gung Memorial Hospital, Kaohsiung, Taiwan. The authors thank Dr Samuel H.H. Chan and the Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, for providing space to support EPR. REFERENCES
1. Wilcox CS. Oxidative stress and nitric oxide deficiency in the kidney: a critical link to hypertension? Am J Physiol Regul Integr Comp Physiol 2005;289:R913–35. 2. Vaziri ND. Roles of oxidative stress and antioxidant therapy in chronic kidney disease and hypertension. Curr Opin Nephrol Hypertens 2004;13:93–9. 3. Sydow K, M€unzel T. ADMA and oxidative stress. Atheroscler Suppl 2003;4:41–51. 4. Matsuoka H, Itoh S, Kimoto M, et al. Asymmetrical dimethylarginine, an endogenous nitric oxide synthase inhibitor, in experimental hypertension. Hypertension 1997;29:242–7. 5. B€oger RH. Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, explains the ‘‘L-arginine paradox’’ and acts as a novel cardiovascular risk factor. J Nutr 2004;134: S2842–7. 6. Teerlink T, Luo Z, Palm F, Wilcox CS. Cellular ADMA: regulation and action. Pharmacol Res 2009;60:448–60. 7. Tain YL, Huang LT. Asymmetric dimethylarginine: clinical applications in pediatric medicine. J Formos Med Assoc 2011;110:70–7. 8. Hsu CN, Huang LT, Lau YT, Lin CY, Tain YL. The combined ratios of L-arginine and asymmetric and symmetric dimethylarginine as biomarkers in spontaneously hypertensive rats. Transl Res 2012;159:90–8. 9. Tain YL, Huang LT, Lin IC, Lau YT, Lin CY. Melatonin prevents hypertension and increased asymmetric dimethylarginine in young spontaneous hypertensive rats. J Pineal Res 2010;49: 390–8. 10. Tain YL, Hsu CN, Huang LT, Lau YT. Apocynin attenuates oxidative stress and hypertension in young spontaneously hypertensive rats independent of ADMA/NO pathway. Free Radic Res 2012; 46:68–76. 11. Chien SJ, Lin KM, Kuo HC, et al. Two different approaches to restore renal nitric oxide and prevent hypertension in young spontaneously hypertensive rats: L-citrulline and nitrate. Transl Res 2014;163:43–52. 12. Viollet B, Guigas B, Sanz Garcia N, Leclerc J, Foretz M, Andreelli F. Cellular and molecular mechanisms of metformin: an overview. Clin Sci (Lond) 2012;122:253–70. 13. Andujar-Plata P, Pi-Sunyer X, Laferrere B. Metformin effects revisited. Diabetes Res Clin Pract 2012;95:1–9. 14. De Leo V, Musacchio MC, Piomboni P, Di Sabatino A, Morgante G. The administration of metformin during pregnancy reduces polycystic ovary syndrome related gestational complications. Eur J Obstet Gynecol Reprod Biol 2011;157:63–6. 15. He H, Zhao Z, Chen J, et al. Metformin-based treatment for obesity-related hypertension: a randomized, double-blind, placebo-controlled trial. J Hypertens 2012;30:1430–9.
Translational Research Volume 164, Number 6
16. Verma S, Bhanot S, McNeill JH. Metformin decreases plasma insulin levels and systolic blood pressure in spontaneously hypertensive rats. Am J Physiol 1994;267:H1250–3. 17. Bhalla RC, Toth KF, Tan E, Bhatty RA, Mathias E, Sharma RV. Vascular effects of metformin. Possible mechanisms for its antihypertensive action in the spontaneously hypertensive rat. Am J Hypertens 1996;9:570–6. 18. Muntzel MS, Hamidou I, Barrett S. Metformin attenuates saltinduced hypertension in spontaneously hypertensive rats. Hypertension 1999;33:1135–40. 19. Bestermann WH Jr. The ADMA-metformin hypothesis: linking the cardiovascular consequences of the metabolic syndrome and type 2 diabetes. Cardiorenal Med 2011;1:211–9. 20. Sydow K, Mondon CE, Cooke JP. Insulin resistance: potential role of the endogenous nitric oxide synthase inhibitor ADMA. Vasc Med 2005;10:S35–43. 21. Asagami T, Abbasi F, Stuelinger M, et al. Metformin treatment lowers asymmetric dimethylarginine concentrations in patients with type 2 diabetes. Metabolism 2002;51:843–6. 22. Tain YL, Baylis C. Determination of dimethylarginine dimethylaminohydrolase activity in the kidney. Kidney Int 2007;72:886–9. 23. Tain YL, Lee WC, Hsu CN, et al. Asymmetric dimethylarginine is associated with developmental programming of adult kidney disease and hypertension in offspring of streptozotocin-treated mothers. PLoS ONE 2013;8:e55420. 24. Bulau P, Zakrzewicz D, Kitowska K, et al. Analysis of methylarginine metabolism in the cardiovascular system identifies the lung as a major source of ADMA. Am J Physiol Lung Cell Mol Physiol 2007;292:L18–24.
Tsai et al
459
25. Detaille D, Guigas B, Leverve X, Wiernsperger N, Devos P. Obligatory role of membrane events in the regulatory effect of metformin on the respiratory chain function. Biochem Pharmacol 2002;63:1259–72. 26. Strobel J, Mieth M, Endress B, et al. Interaction of the cardiovascular risk marker asymmetric dimethylarginine (ADMA) with the human cationic amino acid transporter 1 (CAT1). J Mol Cell Cardiol 2012;53:392–400. 27. Bode-Boger SM, Scalera F, Ignarro LJ. The L-arginine paradox: importance of the L-arginine/asymmetrical dimethylarginine ratio. Pharmacol Ther 2007;114:295–306. 28. Iwase M, Ichikawa K, Tashiro K, et al. Effects of monosodium glutamate-induced obesity in spontaneously hypertensive rats vs. Wistar Kyoto rats: serum leptin and blood flow to brown adipose tissue. Hypertens Res 2000;23:503–10. 29. Ford RJ, Teschke SR, Reid EB, Durham KK, Kroetsch JT, Rush JW. AMP-activated protein kinase activator AICAR acutely lowers blood pressure and relaxes isolated resistance arteries of hypertensive rats. J Hypertens 2012;30:725–33. 30. Sung JY, Choi HC. Metformin-induced AMP-activated protein kinase activation regulates phenylephrine-mediated contraction of rat aorta. Biochem Biophys Res Commun 2012;421:599–604. 31. Rocha A, Almeida M, Santos J, Carvalho A. Metformin in patients with chronic kidney disease: strengths and weaknesses. J Nephrol 2013;26:55–60. 32. Sartoretto JL, Melo GA, Carvalho MH, et al. Metformin treatment restores the altered microvascular reactivity in neonatal streptozotocin-induced diabetic rats increasing NOS activity, but not NOS expression. Life Sci 2005;77:2676–89.
