Aminoguanidine normalizes ET-1-induced aortic contraction in type 2 diabetic Otsuka Long–Evans Tokushima Fatty (OLETF) rats by suppressing Jab1-mediated increase in ETA-receptor expression

Peptides 33 (2012) 109–119

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Aminoguanidine normalizes ET-1-induced aortic contraction in type 2 diabetic Otsuka Long–Evans Tokushima Fatty (OLETF) rats by suppressing Jab1-mediated increase in ETA -receptor expression Shingo Nemoto, Kumiko Taguchi, Takayuki Matsumoto, Katsuo Kamata, Tsuneo Kobayashi ∗ Department of Physiology and Morphology, Institute of Medicinal Chemistry, Hoshi University, Shinagawa-ku, Tokyo 142-8501, Japan

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Article history: Received 30 September 2011 Received in revised form 18 November 2011 Accepted 18 November 2011 Available online 28 November 2011 Keywords: Diabetes Endothelin-1 ETA -R Jab1 O-GlcNAc Otsuka Long–Evans Tokushima Fatty rat

a b s t r a c t Circulating levels of endothelin (ET)-1 are increased in the diabetic state, as is endogenous ETA -receptormediated vasoconstriction. However, the responsible mechanisms remain unknown. We hypothesized that ET-1-induced vasoconstriction is augmented in type 2 diabetes with hyperglycemia through an increment in advanced glycation end-products (AGEs). So, we investigated whether treatment with aminoguanidine (AG), an inhibitor of AGEs, would normalize the ET-1-induced contraction induced by ET-1 in strips of thoracic aortas isolated from OLETF rats at the chronic stage of diabetes. In such aortas (vs. those from age-matched genetic control LETO rats): (1) the ET-1-induced contraction was enhanced, (2) the levels of HIF1␣/ECE1/plasma ET-1 and plasma CML-AGEs were increased, (3) the ET-1-stimulated ERK phosphorylation mediated by ETA -R was increased, (4) the expression level of Jab1-modified ETA -R protein was reduced, and (5) the expression level of O-GlcNAcylated ETA -R protein was increased. Aortas isolated from such OLETF rats that had been treated with AG (50 mg/kg/day for 10 weeks) exhibited reduced ET-1-induced contraction, suppressed ET-1-stimulated ERK phosphorylation accompanied by down-regulation of ETA -R, and increased modification of ETA -R by Jab1. Such AG-treated rats exhibited normalized plasma ET-1 and CML-AGE levels, and their aortas exhibited decreased HIF1␣/ECE1 expression. However, such AG treatment did not alter the elevated levels of plasma glucose or insulin, or systolic blood pressure seen in OLETF rats. These data from the OLETF model suggest that within the timescale studied here, AG normalizes ET-1-induced aortic contraction by suppressing ETA -R/ERK activities and/or by normalizing the imbalance between Jab1 and O-GlcNAc in type 2 diabetes. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Diabetes mellitus is a major risk factor for all manifestations of atherosclerotic vascular disease [10,27]. Many of the vascular complications seen in type 1 and type 2 diabetes arise from hyperglycemia that cannot be completely prevented using the methods of blood glucose control that are at present available

Abbreviations: AG, aminoguanidine; AGEs, advanced glycation end-products; CML, N epsilon-(carboxymethyl) lysine; ECE1, endothelin converting enzyme; ERK, extracellular signal-regulated kinase; ET-1, endothelin-1; ETA -R, endothelin type A receptor; ETB -R, endothelin type B receptor; GPCRs, G protein-coupled receptors; HDL, high-density lipoprotein cholesterol; HIF1␣, hypoxia-inducible factor 1 alpha; HIF1␤, hypoxia-inducible factor 1 beta; IRS1, insulin receptor substrate 1; Jab1, Jun activation domain-binding protein; LDL, low-density lipoprotein cholesterol; LETO, Long–Evans Tokushima Otsuka; MAPK, mitogen-activated protein kinase; NEFA, non-esterified fatty acid; OLETF rat, Otsuka Long–Evans Tokushima Fatty rat; O-GlcNAc, O-glycoside-linked ␤-N-acetylglucosamine; pVHL, von Hippel–Lindau tumor suppressor protein; RAGE, receptor for AGEs. ∗ Corresponding author. Tel.: +81 3 5498 5849; fax: +81 3 5498 5849. E-mail address: [email protected] (T. Kobayashi). 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.11.018

[11,42,45]. Type 2 diabetes is known to be associated with a markedly increased incidence of cardiovascular diseases [54]. However, the exact relationships among type 2 diabetes, obesity, and cardiovascular diseases are not completely understood and have been the subject of some dispute. Type 2 diabetes is often part of an array of complex abnormalities referred to as the metabolic syndrome, which is frequently accompanied by an elevated systemic blood pressure [55]. Concerning the macrovascular complications of the metabolic abnormalities associated with diabetes, the in vivo relevance of the Maillard reaction and the subsequent production and accumulation of advanced glycation end-products (AGEs) was first emphasized in studies conducted using an inhibitor of advanced glycation, aminoguanidine (AG) [4]. Although several AGE structures have been reported [15,48], it has been demonstrated that N epsilon-(carboxymethyl) lysine (CML) is a major antigenic AGE structure. Subsequent studies, in which exogenous administration of AGEs was employed to mimic the diabetic serum concentration, indicated that AGEs could induce atherosclerosis [61]. Furthermore, AGEs interact with endothelial cells to induce expressions

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of atherogenic adhesion molecules implicated in atherogenesis [37,52]. Recently, Chen et al. indicated that AGE-modified albumin increased the O-glycoside-linked ␤-N-acetylglucosamine (O-GlcNAc) modification of protein [6]. Glucose flux through the hexosamine biosynthetic pathway leads to the post-translational modification of cytoplasmic and nuclear proteins by O-GlcNAc (O-GlcNAcylation) [62]. It is known that O-GlcNAcylation of proteins plays an important role in transcription, translation, nuclear transport, and cytoskeletal assembly [9,63]. Alongside the increasing realization that O-GlcNAcylation represents a new regulatory signal, it is emerging that abnormalities in the regulation of O-GlcNAc protein modification mediate some of the pathogenetic effects of type 2 diabetes. For instance, increased O-GlcNAcylation of insulin signal-pathway intermediates, such as IRS-1 and Akt, reportedly reduces the insulin-stimulated phosphorylation of IRS-1 at a tyrosine residue and of Akt at a serine residue in rat primary adipocytes [64], and increases the phosphorylation of IRS-1 at a serine residue in monkey kidney [67], with consequent insulin resistance. Very recently, it has been shown that ET-1 induces O-GlcNAcylation of vascular proteins and that this modification mediates some of the vascular effects of that peptide [25]. Endothelin-1 (ET-1), a potent vasoconstrictor peptide [35,66], contains 21 amino acids and is synthesized by and released from the endothelial cells of blood vessels [66]. ET-1 also stimulates cell growth [50,56]. Therefore, ET-1 is considered to play important roles in the physiological control of blood pressure and cardiac function, and also in the genesis and development of cardiovascular diseases such as atherosclerosis [20], the cardiac remodeling that accompanies chronic heart failure [50], and pulmonary hypertension [12]. There are two types of receptors for ET-1, endothelin type A receptor (ETA -R) and ETB -R, both of which are G protein-coupled receptors (GPCRs) [2,51]. Abnormal ET-1 signaling has been reported to be related to diabetic vasculopathy [8,14,21,31,46]. Interestingly, the levels of ET receptors, as well as of ET-1, have been reported to be elevated within cardiac muscle in chronic heart failure [50], in diabetic states [21,30], and in the infiltrating cells of atherosclerotic lesions, such as smooth muscle cells and macrophages [20]. The mechanism responsible for the elevation of the expression levels of ET receptors in these diseases is not completely understood. Recently, Nishimoto et al. reported that the amount of Jun activation domain-binding protein (Jab)-1 bound to ET-R regulates the degradation rate of ETA -R and ETB R by modulating the ubiquitination of these receptors, leading to changes in ETA -R and ETB -R levels [41]. Moreover, Jab1 increases the protein level of hypoxia-inducible factor (HIF) 1␣ by enhancing HIF1␣ stability [3]. The ␣ subunit of HIF1 is the hypoxia-responsive component of the dimer, while HIF1␤ is expressed constitutively. Under normoxic conditions, HIF1␣ is rapidly degraded by the ubiquitin–proteasome pathway [18]. Ubiquitination of HIF1␣ is mediated by interactions with pVHL (von Hippel–Lindau tumor suppressor protein) [36,43] and p53 [1,47]. HIF1␣ is targeted for VHL E3 ligase complex-mediated destruction by proline hydroxylation of its oxygen-dependent degradation region [16]. Moreover, HIF1␣ induces production of endothelin-1 through endothelin-converting enzyme (ECE) in rat cardiomyocytes overexpressing HIF1␣ [58]. Furthermore, Jesmin et al. found that ECE/ET-1 expression was increased in a type 2 diabetic model, the Otsuka Long–Evans Tokushima Fatty (OLETF) rat [17]. OLETF rats manifest stable clinical and pathological features that resemble human type 2 diabetes; indeed, they exhibit hypertension, obesity, hyperglycemia, and hyperlipidemia [19,65]. We hypothesized that ET-1-induced vasoconstriction is augmented in type 2 diabetic as a results of Jab1-regulated ET-1/ETA -R pathway alteration via AGEs-induced O-GlcNAcylation.

The first aim of the present study was to investigate whether the enhancing effect of ET-1 on the contractile responsiveness of the aorta is indeed altered in type 2 diabetic OLETF rats. We then focused on the regulation of ETA -R signal transduction by Jab1 and/or O-GlcNAcylation, and whether this correlated with alterations in the plasma ET-1 level. We also examined whether aortas from Long–Evans Tokushima Otsuka (LETO) rats, a genetic control for OLETF, differ from those of OLETF rat in their ETA -R expression profile and/or in the effects of AG, an inhibitor of AGE production, on ET-1-induced contractile responses in the aorta. 2. Materials and methods 2.1. Reagents Cyclo (d-␣-aspartyl-l-propyl-d-valyl-l-leucyl-d-tryptophyl) (BQ-123), phenylephrine, and monoclonal ␤-actin antibody were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). Echinomycin was from Enzo Life Sciences Inc. (St. Farmingdale, NY, USA). Endothelin (ET)-1, IRL1620, and prostaglandin E2 were from Peptide Institute, Inc. (Osaka, Japan). All drugs were dissolved in saline, unless otherwise noted. All concentrations are expressed as the final molar concentration of the base in the organ bath. Horseradish-peroxidase-linked secondary anti-mouse or anti-rabbit antibody was purchased from Promega (Madison, WI, USA), while antibodies against extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 (pT202/pY204) were obtained from BD Biosciences (San Jose, CA, USA). Antibodies against ECE1, ETA -R, ETB -R, HIF1␣, HIF2␣, Jab1, and O-GlcNAc were all obtained from Abcam (Cambridge, MA, USA), and those against RAGE were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). 2.2. Animals and experimental design Five-week-old male OLETF rats and Long–Evans Tokushima Otsuka (LETO) rats, a genetic control for OLETF, were supplied by the Tokushima Research Institute (Otsuka Pharmaceutical, Tokushima, Japan). Food and water were given ad libitum in a controlled environment (room temperature 21–22 ◦ C, room humidity 50 ± 5%) until the rats were 37–42 weeks old. OLETF and LETO rats were randomly allocated to receive either 50 mg/kg/day [57] of aminoguanidine (AG; Wako Pure Chemical Co. Ltd., Osaka, Japan) in the 5% HCl drinking water for 10 weeks starting at 27–32 weeks old, or to receive the 5% HCl drinking water for a similar term. Thus, we studied four groups: AG-untreated LETO and OLETF groups and AG-treated LETO and OLETF groups. This study was approved by the Hoshi University Animal Care and Use Committee, and all studies were conducted in accordance with “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health, and “Guide for the Care and Use of Laboratory Animals” adopted by the Committee on the Care and Use of Laboratory Animals of Hoshi University (which is accredited by the Ministry of Education, Culture, Sports, Science, and Technology, Japan). At the ages of 37–42 weeks, groups of rats were killed by decapitation under diethyl ether anesthesia. 2.3. Measurement of blood parameters and blood pressure Plasma or serum parameters and blood pressure were measured as described previously [21–24,40]. Briefly, plasma glucose, cholesterol, triglyceride, and high-density lipoprotein cholesterol (HDL), and serum non-esterified fatty acid (NEFA) levels were each determined by the use of a commercially available enzyme kit (Wako Chemical Company, Osaka, Japan). Plasma insulin was measured by enzyme-immunoassay (EIA) (Shibayagi, Gunma, Japan). Plasma

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ET-1 was eluted with methanol using C-18 columns (Cayman Chemical, MI, USA) and measured by quantitative sandwich EIA using a commercially available ET-1 quantiGlo chemiluminescent assay kit (R&D Systems, Minneapolis, MN). Plasma CML-AGEs were measured as described previously [34] using semi-quantitative EIA (CycLex Co. Ltd., Nagano, Japan). After a given rat had been in a constant-temperature box at 37 ◦ C for a few minutes, its blood pressure was measured by the tail-cuff method using a blood pressure analyzer (BP-98A; Softron, Tokyo, Japan). 2.4. Measurement of isometric force Vascular isometric force was recorded as in our laboratory’s previous papers [21–24,30,31,35,40]. At 37–42 weeks of age, rats were anesthetized with diethyl ether, and then euthanized by decapitation. The thoracic aorta was rapidly removed and immersed in oxygenated, modified Krebs–Henseleit solution (KHS). This solution consisted of (in mM) 118.0 NaCl, 4.7 KCl, 25.0 NaHCO3 , 1.8 CaCl2 , 1.2 NaH2 PO4 , 1.2 MgSO4 , and 11.0 dextrose. The artery was carefully cleaned of all fat and connective tissue, and aortic strips 3 mm in width and 20 mm in length were suspended by a pair of stainless-steel pins in a well-oxygenated (95% O2 –5% CO2 ) bath containing 10 mL of KHS at 37 ◦ C. The strips were stretched until an optimal resting tension of 1.0 g was loaded, and then allowed to equilibrate for at least 60 min. Force generation was monitored by means of an isometric transducer (model TB-611T; Nihon Kohden, Tokyo, Japan). For the contraction studies, ET-1 (10−10 –10−7 M), phenylephrine (10−9 –10−5 M), or prostaglandin E2 (10−9 –10−5 M) solutions were added cumulatively to the bath until a maximal response was achieved. To investigate the effects of BQ123 (10−6 M) on the ET-1-induced contractile response, the strips were incubated for 30 min in BQ123-containing medium before the cumulative addition of agonist. For the relaxation studies, strips were precontracted with phenylephrine (0.5–2 ␮M being used to permit amplitudematching of the precontractions). When the phenylephrineinduced contraction had reached a plateau level, IRL1620 (selective ETB -receptor agonist; 10−10 –10−7 M) was added in a cumulative manner. After the addition of sufficient aliquots of the agonist to produce the chosen concentration, a plateau response was allowed to develop before the addition of the next dose of the same agonist. 2.5. Western blotting Aortic strips were incubated with 10 nM ET-1 for 20 min, and for inhibitor experiments, tissues were pretreated with 10 ␮M BQ123 for 30 min or with 10 nM echinomycin for 16 h before the addition of ET-1. They were then frozen in liquid N2 before being physically crushed to a fine powder in liquid N2 using a Cryo-Press (Microtech Nichion, Chiba, Japan). Aortic tissues were homogenized in ice-cold lysis buffer containing 50 mM Tris–HCl (pH 7.2), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS containing protease- and phosphatase-inhibitor cocktails (Complete Protease Inhibitor Cocktail and PhosSTOP; Roche Diagnostics, Indianapolis, IN, USA). The lysate was cleared by centrifugation at 16,000 × g for 10 min at 4 ◦ C. The supernatant was collected, and the proteins were solubilized in Laemmli’s buffer containing mercaptoethanol. Protein concentrations were determined by means of a bicinchoninic acid (BCA) protein assay reagent kit (Pierce, Rockford, IL, USA). Samples (24 ␮g/lane) were resolved by electrophoresis on 10% SDSPAGE gels, then transferred onto polyvinylidene difluoride (PVDF) membranes. Briefly, after blocking the residual protein sites on the membrane with ImmunoBlock (Dainippon-Pharm., Osaka, Japan) or PVDF blocking reagent (Toyobo, Osaka, Japan), the membrane was incubated with anti-ERK1/2 (1:1000), anti-phospho-ERK1/2 (pT202/pY204) (1:1000), anti-ECE1 (1:500), anti-ETA -R (1:1000),

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anti-ETB -R (1:1500), anti-HIF1␣ (1:1000), anti-HIF2␣ (1:1000), anti-RAGE (1:100), or anti-Jab1 (1:1000) antibodies in blocking solution. HRP-conjugated, anti-mouse or anti-rabbit antibody was used at a 1:10,000 dilution in Tween PBS, followed by detection using SuperSignal (Thermo Fisher Scientific Inc., St. Waltham, MA, USA). Band intensity was quantified by densitometry.

2.6. Immunoprecipitation with anti-Jab1, anti-O-GlcNAc, or anti-ETA -R antibody The immunoprecipitation procedure involved a method described elsewhere [26]. Polyclonal ETA -R (1:100) antibody was cross-linked to Dynabeads protein A (Dynal Biotech, Oslo, Norway) according to the manufacturer’s protocol. Cell lysates were precleared with IgG Dynabeads-protein A for 30 min and then incubated with ETA -R-Dynabeads overnight at 4 ◦ C. The ETA -R-immunoprecipitated complexes were washed five times with immunoprecipitation buffer (10 mM Tris/HCl, pH 7.8, 1 mM EDTA, 150 mM NaCl, 1 mM NaF, 0.5% Nonidet P-40, 0.5% glucopyranoside, 1 g/mL aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride). Proteins were resuspended in Laemmli’s buffer containing mercaptoethanol, eluted by boiling in loading buffer, and then processed for Western blot analysis. Briefly, after blocking of the residual protein sites on the membrane with ImmunoBlock (Dainippon-Pharm., Osaka, Japan), the membrane was incubated with anti-Jab1 (1:1000), O-GlcNAc (1:1000), or ETA -R (1:1000), in blocking solution. Horseradish peroxidase-conjugated antibody was used at a 1:10,000 dilution in Tween PBS, followed by detection using SuperSignal.

2.7. Statistical analysis The contractile force developed by aortic strips is expressed in mg tension mg−1 tissue. Data are expressed as means ± S.E. Statistical differences were assessed using Dunnett’s test for multiple comparisons after a one-way ANOVA, a probability level of P < 0.05 being regarded as significant. Statistical comparisons between concentration–response curves were made using a twoway ANOVA, with a Bonferroni correction performed post hoc to correct for multiple comparisons (P < 0.05 being considered significant).

3. Results 3.1. General parameters As shown in Table 1, the non-fasted blood parameters examined here (plasma glucose, insulin, cholesterol, triglyceride, and LDL/HDL ratio, and serum NEFA concentration) of OLETF rats were all significantly higher than those of LETO control rats (also nonfasted). Treatment with aminoguanidine (AG) improved some of the above parameters (cholesterol, triglyceride, LDL/HDL ratio, and NEFA) in OLETF, but not in LETO rats.

3.2. Effects of aminoguanidine treatment on various values As shown in Fig. 1, the body weight, systolic blood pressure, and plasma CML-AGEs of OLETF rats were significantly greater than those of LETO rats. AG-treatment significantly decreased both body weight and plasma CML-AGEs in OLETF rats, but not in LETO rats (Fig. 1A and C). In contrast, neither systolic blood pressure nor RAGE (receptor for AGEs) was altered by AG-treatment in OLETF or in LETO (Fig. 1B and D).

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Table 1 Values of various blood parameters in aminoguanidine-treated and -untreated LETO and OLETF rats. Parameters

LETO (16)

Glucose (mg/dL) Insulin (ng/mL) Cholesterol (mg/dL) Triglyceride (mg/dL) LDL/HDL ratio NEFA (mequiv./L)

160.5 2.6 166.6 104.0 0.87 0.26

± ± ± ± ± ±

OLETF (16)

5.4 0.2 10.0 7.8 0.05 0.01

407.1 4.2 210.5 305.5 1.22 0.45

± ± ± ± ± ±

Aminoguanidine-treated OLETF (16)

Aminoguanidine-treated LETO (16)

16.0*** 0.2*** 6.3** 11.0*** 0.05*** 0.01***

159.4 2.4 168.5 105.7 0.80 0.25

± ± ± ± ± ±

4.2 0.2 10.5 3.2 0.03 0.01

407.0 4.0 193.1 239.1 1.01 0.35

± ± ± ± ± ±

7.6††† 0.1††† 3.9# , † 25.0# , ††† 0.03# , ††† 0.01### , †††

Values are means ± S.E. Number of determinations is shown in parenthesis. ** P < 0.01 vs. LETO. *** P < 0.001 vs. LETO. # P < 0.05 vs. OLETF. ### P < 0.001 vs. OLETF. † P < 0.05 vs. aminoguanidine-treated LETO. ††† P < 0.001 vs. aminoguanidine-treated LETO.

Fig. 1. Time-dependent changes in (A) body weight and (B) systolic blood pressure during 10 weeks’ administration of aminoguanidine (AG). Levels of (C) plasma CML-AGEs and (D) RAGE protein expression in AG-treated or -untreated LETO and OLETF rats at the end of the 10-week administration period. Data are means ± S.E. from 8 (D) or 16 (A–C) experiments. ***P < 0.001 – OLETF vs. LETO group. # P < 0.05, ### P < 0.001 – AG-treated OLETF vs. OLETF group.

Table 2 Maximal responses and −log EC50 values for ET-1-, phenylephrine-, or prostaglandin E2 -induced contractions of aortas from aminoguanidine-treated or -untreated LETO and OLETF rats. Reagent

LETO (8)

OLETF (8)

Max. response ET-1 BQ123 + ET-1 Phenylephrine Prostaglandin E2

150.89 144.96 170.73 114.16

± ± ± ±

6.00 4.01 17.44 10.61

−log EC50 7.98 7.19 6.50 4.37

± ± ± ±

0.08 0.06 0.15 0.75

Aminoguanidine-treated LETO (8)

Max. response 224.39 128.63 199.38 137.11

± ± ± ±

6.57*** 9.00 14.75 11.02

−log EC50 8.19 6.66 6.41 4.52

± ± ± ±

Values are means ± S.E. Number of determinations is shown in parenthesis. −log EC50 (Emax ), maximal agonist-induced response expressed as percent of 80 mM K+ . *** P < 0.001 vs. LETO. ### P < 0.001 vs. OLETF.

0.06 0.15 0.20 0.91

Max. response 154.19 122.83 185.04 122.82

± ± ± ±

8.57 8.41 10.68 7.70

−log EC50 8.09 7.22 6.32 4.41

± ± ± ±

0.10 0.06 0.28 0.70

Aminoguanidine-treated OLETF (8) Max. response 162.45 132.01 175.89 138.09

± ± ± ±

1.62### 7.14 17.51 6.43

−log EC50 7.97 6.30 6.32 3.84

± ± ± ±

0.07 0.73 0.21 0.93

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3.4. Effects of aminoguanidine treatment on ETB -R expression and IRL1620-induced relaxation NO plays important roles in dilation and in the modulation of contractile responses in blood vessels, and our laboratory previously reported [30] that NO contributes to the modulation of ET-1-induced contraction in mesenteric arteries from Goto–Kakizaki (non-obese type 2 diabetic) rats. ET-1 is widely known to stimulate ETA -R on vascular smooth muscle cells to produce vasoconstriction and ETB -R on endothelial cells to produce vasodilation [13,44,49]. Therefore, we added IRL1620 (selective ETB -receptor agonist; 10−10 –10−7 M) cumulatively to strips precontracted by phenylephrine (Fig. 3A). The IRL1620-induced relaxation was significantly weaker in strips from OLETF rats than in those from age-matched LETO rats. AG-treatment of LETO or OLETF rats caused no significant alteration in the relaxation to IRL1620 (vs. that seen in the AG-untreated group). ETB -R expression was greater in aortas from OLETF than in those from LETO, and AG-treatment did not change this relationship (Fig. 3B). Our laboratory previously reported that in 36–42-week OLETF rats, levels of anti-oxidants (e.g. superoxide dismutase, catalase, glutathione peroxidase, and relevant macro- and/or small-molecules) were reduced (we consider that this will lead to enhanced reactive oxygen species) [32,33]. So, we think that the above may be the cause of the decreased IRL1620-induced NO production/aortic relaxation. 3.5. Effects of aminoguanidine treatment on ET-1-stimulated ERK activation

Fig. 2. Concentration–response curves for (A) ET-1-, (B) phenylephrine- and (C) prostaglandin E2 -induced contractions of thoracic aortas obtained from aminoguanidine (AG)-treated or -untreated LETO and OLETF rats. Details are given under Section 2. Data are means ± S.E. from 8 experiments. *P < 0.05, ***P < 0.001 – OLETF vs. LETO group. ## P < 0.01 – AG-treated OLETF vs. OLETF group.

3.3. Effects of aminoguanidine treatment on contractions induced by ET-1, phenylephrine, and prostaglandin E2 Cumulative administration of ET-1 (10−10 –10−7 M; Fig. 2A), phenylephrine (10−9 –10−4 M; Fig. 2B), or prostaglandin E2 (10−9 –10−5 M; Fig. 2C) induced a concentration-dependent contraction in aortic strips from aminoguanidine (AG)-treated or -untreated LETO and OLETF rats. The ET-1-induced contractile response was significantly greater (3 × 10−9 –10−7 M) in strips from OLETF rats than in those from LETO rats (Fig. 2A and Table 2). Following AG treatment of OLETF rats, but not of LETO rats, the ET1-induced contraction was significantly weaker within the above concentration-ranges (Fig. 2A). Neither the phenylephrine- nor the prostaglandin E2 -induced contractions differed among the four groups (Fig. 2B and C and Table 2).

ET-1 modulates vascular tone through activation of MAPK pathways, including the ERK pathway [22,28]. To investigate whether such ERK-pathway activation might be altered by AG-treatment, we measured ET-1-stimulated ERK phosphorylation (activation) in the aorta (Fig. 4B). ET-1-stimulated ERK phosphorylation and ETA R expression were significantly elevated in the OLETF group (vs. the LETO group), and such elevations were significantly prevented by AG-treatment. In contrast, total ERK expression did not differ among the three groups (Fig. 4B). We also investigated the effect of 1 ␮M BQ123 (a selective ETA -R antagonist) pretreatment on the ET-1-induced contraction, ERK activation, and ETA -R expression. Treatment with BQ123 tended to cause a rightward shift in the concentration–response curve for the ET-1-induced aortic contraction in both OLETF and LETO (Fig. 4A; cf. Fig. 2A). Moreover, the increase in ERK phosphorylation seen in OLETF (vs. LETO) rats was significantly suppressed by BQ123 treatment, but such treatment did not affect the level in the LETO or AG-treated OLETF groups (Fig. 4B). The presence of BQ123 did not significantly alter the ETA -R expression level in aortas from any of the three groups (Fig. 4C). 3.6. Effects of aminoguanidine treatment on ET-1-stimulated Jab1 or O-GlcNAc binding to ETA -R The above results led us to speculate that the observed enhancement of ET-1-induced aortic contraction in the OLETF group might be caused by abnormal regulation of ETA -R expression. Nishimoto et al. reported that binding of Jun activation domain-binding protein (Jab)-1 to ETA -R regulates the degradation of ETA -R [41]. So, to investigate whether such ETA -R expression was altered by diabetes or by aminoguanidine (AG) treatment, we isolated aortas from LETO and from AG-treated or -untreated OLETF rats, and we measured Jab1 expression using Western blot (WB), and Jab1 or O-GlcNAc binding to ETA -R using immunoprecipitation (IP). As shown in Fig. 5A, Jab1 expression tended (but not significantly) to be decreased in the OLETF group. The WB method provides measurements that may include not only Jab1-containing ETA R protein, but also other Jab1-containing proteins. To establish

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Fig. 3. Analysis of ETB -R protein expression (B), and concentration–response curves for the relaxations induced by the selective ETB -receptor agonist IRL1620 (A) in aortic strips from aminoguanidine (AG)-treated or -untreated OLETF and LETO rats. (B) Protein expression normalized to the ␤-actin expression level. Data are means ± S.E. from 8 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 – OLETF vs. LETO group.

whether Jab1 really was present on ETA -R protein in the OLETF aorta, immunoprecipitates (IP) of aortic proteins obtained using anti-ETA -R antibody were stained using anti-Jab1 antibody. Using such IP and WB, we found that the amount of Jab1 protein present in the ETA -R immunoprecipitate was significantly decreased in OLETF aortas (vs. LETO; Fig. 5B). Despite the presence of similar expressions of ETA -R protein among the three groups, the AG-treated OLETF aortas exhibited markedly increased Jab1 protein within the ETA -R immunoprecipitate (vs. AG-untreated OLETF; Fig. 5B). Recently, Chen et al. found that AGE-modified albumin increased O-GlcNAc modification of protein [6], and in the present study we found that plasma CML-AGEs were significantly increased in OLETF rats (vs. LETO; Fig. 1C). So, we next determined the amount of O-GlcNAc protein binding to ETA -R using the IP and WB techniques. Interestingly, we found that the amount of O-GlcNAc protein present within the ETA -R immunoprecipitate was significantly increased in OLETF aortas (vs. LETO; Fig. 5C). Moreover, despite the presence of similar expressions of ETA -R protein among the three groups, AG-treated OLETF aortas exhibited markedly decreased O-GlcNAc protein within the ETA -R immunoprecipitate (vs. AG-untreated OLETF; Fig. 5C). These results suggest that in aortas from OLETF rats, CMLAGEs cause an enhancement of O-GlcNAcylated ETA -R expression through a mechanism that involves a reduction of Jab1 formation in ETA -R protein, and they also indicate that 10 days’ AG-treatment normalizes the above diabetic abnormalities seen in OLETF rats (see Graphical abstract). 3.7. Effects of aminoguanidine treatment on HIF1˛, HIF2˛, and ECE1 expressions Next, we examined HIF1␣ (Fig. 6A), HIF2␣ (Fig. 6B), and ECE1 (Fig. 6C) expressions using the WB method, because it has been reported that HIF1␣ produces endothelin-1 through endothelin-converting enzyme (ECE), and that ECE/ET-1 expression is increased in OLETF rats [13,16,58]. HIF1␣, HIF2␣, and ECE1 expressions were each significantly higher in aortas from OLETF than in those from LETO rats. AG-treatment of OLETF rats decreased the HIF1␣ and ECE1 expressions, but not that of HIF2␣ (Fig. 6). Furthermore, 16-h treatment with echinomycin (a small-molecule inhibitor of HIF1 DNA-binding activity) significantly suppressed the enhancement of ECE1 expression seen in aortas from OLETF rats (Fig. 6C).

3.8. Effects of aminoguanidine treatment on plasma ET-1 levels ET-1 levels were significantly higher in plasma samples from OLETF than in those from LETO rats. Treatment with AG significantly reduced (i.e., normalized) the ET-1 level in OLETF rats (Fig. 7). 4. Discussion In the present study, we examined whether the abnormal ET1-mediated contractile response seen in thoracic aortas isolated from established diabetic (OLETF) rats might be normalized by aminoguanidine (AG) treatment. For this, and to address the possible association between Jab1 or O-GlcNAc modification and ETA -R, we chose OLETF rats as our chronic type 2 diabetic model. We did this because we and others have demonstrated that: (1) OLETF rats manifest stable clinical and pathological features that resemble human type 2 diabetes mellitus [19] and (2) abnormalities of vascular function are present in several vessels in OLETF rats [32]. To evaluate the effects of sustained inhibition of advanced glycation end-product (AGE) signaling on ET-1-induced responses, we evaluated such responses in aortas isolated from either AG-treated or -untreated groups of rats (diabetic OLETF rats or age-matched non-diabetic LETO rats). We found that in such aortas: (1) the ET-1-induced contraction was enhanced in the OLETF group (vs. the LETO group); (2) in the OLETF group, but not in the LETO group, AG-treatment reduced the ET-1-induced contraction; (3) both the phenylephrine-induced and prostaglandin E2 -induced contractions were similar between the OLETF and LETO groups, and these contractions were not altered by AG-treatment in either group. To judge from these results, treatment with AG (which effectively inhibits AGE production) may specifically normalize ET1-mediated aortic contraction in established type 2 diabetic rats. Previous studies have noted that the vasculoprotective effects of AG in the type 2 diabetic state are exemplified by improved functioning of mesenteric arteries [53] and/or the thoracic aorta [5]. The present data suggest that the AG-induced improvement in the ET-1-mediated contractile response seen in the OLETF (type 2 diabetic) thoracic aorta might be due to decreased production of AGEs (Fig. 2A). Moreover, AG-treatment improved various parameters [namely, blood cholesterol, triglyceride, LDL/HDL ratio, and non-esterified fatty acid (NEFA)], but these were not completely normalized to the control levels (vs. LETO rats; Table 1 and Fig. 1B). In contrast, body weight and plasma CML-AGEs, but not RAGE

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Fig. 4. Contractions induced by ET-1, with the selective ETA -R antagonist BQ123 (1 ␮M), in aortic strips obtained from aminoguanidine (AG)-treated or -untreated LETO and OLETF rats (A). Analysis of p-ERK (B) and ETA -R (C) protein expressions under stimulation by 10 nM endothelin-1, with or without BQ123 (1 ␮M), in aortic strips obtained from AG-untreated LETO or AG-treated or -untreated OLETF rats. Protein expressions normalized to the total -ERK (B) or ␤-actin (C) expressions. Data are means ± S.E. from 6 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 – OLETF vs. LETO group. # P < 0.05, ## P < 0.01 – AG-treated OLETF vs. OLETF group. ††† P < 0.001 – OLETF (pretreated with BQ123) vs. BQ123-untreated OLETF group.

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Fig. 5. Analysis of Western blots for Jab1 (A) and of immunoprecipitation (IP) of Jab1 (B) or O-GlcNAc (C) on ETA -R protein in aortic strips obtained from LETO and aminoguanidine (AG)-treated or -untreated OLETF rats. (A) Expression of Jab1 as assayed by immunoblotting (IB). (B and C) For detection of Jab1 or O-GlcNAc on ETA R protein, an IP was obtained using anti-ETA -R antibody, and then IB was performed using anti-Jab1 or anti-O-GlcNAc antibody. (B and C) Protein expressions normalized to untreated LETO group as reference. Data are means ± S.E. from 6 experiments. *P < 0.05, **P < 0.01 – OLETF vs. LETO group. # P < 0.05 – AG-treated OLETF vs. OLETF group.

(receptor for AGEs), levels were normalized in the AG-treated OLETF group (Fig. 1A, C and D). These results led us to the interpretation that such normalization by AG-treatment of ET-1-induced diabetic abnormalities correlated with the reductions of plasma CML-AGEs and body weight, rather than with the improvements in the other parameters. In blood vessels, ETA -R is mainly located on the smooth muscle cells, and its activation by ET-1 leads to vasoconstriction [29,38]. In contrast, ETB -R exists on both smooth muscle and endothelial cells [29,38]. While the ETB -R present on the smooth muscle mediates vasoconstriction [29,38,60], those on the endothelial cells cause vasodilation via the release of endothelium-dependent relaxing factors, such as nitric oxide (NO) [28,39,59]. In the present study, we detected a significant increase in total ETB -R abundance in the OLETF aortas compared to the LETO ones. It might be argued that the changes observed in the OLETF aorta (vs. LETO) in ETB -R protein expression (increased) and IRL1620-induced relaxation (decreased) were inconsistent with each other. However, our immunoblotting was performed using a total vessel homogenate, and therefore could not differentiate between endothelial ETB -R and those on the vascular smooth muscle. Moreover, AG-treatment had no effects on ETB -R expression or on IRL1620-induced relaxation in those experiments. These results lead us to speculate that the OLETF aortic ETB -R mediates not only endothelium-dependent relaxation, but also smooth muscle contraction, with the net result being increased systemic blood pressure. However, we are not sure of this, and we have no more supporting data, so this hypothesis will need to be tested in future studies. Our laboratory previously reported that an enhancement of ET-1-induced vasoconstriction is caused by increased ETA R/ERK pathway activity in diabetic rats [21,30]. We therefore examined the ET-1-induced aortic contractile response, ERK phosphorylation (activity), and ETA -R expression with or without BQ123-pretreatment (Fig. 4). In the OLETF group, ERK phosphorylation and ETA -R expression levels were significantly increased (vs. LETO rats), and AG-treatment normalized these levels (Fig. 4B and C). Moreover, BQ123-pretreatment significantly decreased both the ET-1-induced contraction and ERK phosphorylation, but not ETA -R expression, in the OLETF group (Fig. 4A; cf. Fig. 2A). These results indicate that the enhanced ET-1-induced aortic contraction in OLETF rats may be caused by upregulation of ETA -R accompanied by ERK activation. ETA -R overexpression in the diabetic state was previously reported by our laboratory [21,30], and another laboratory recently found that ETA -R expression is regulated by Jab1 [41]. As shown in Fig. 5B, we detected Jab1 in the ETA -R immunoprecipitates derived from the rat aorta, and found that OLETF aortas exhibited a reduced ETA -R-associated Jab1 level. Moreover, the ETA -R level was enhanced in the OLETF (vs. LETO) group (Fig. 4C). Treatment with AG for 10 weeks normalized the above abnormalities seen in the OLETF rat (Figs. 4C and 5B). Recently, Nishimoto et al. reported that the amount of Jab-1 bound to ET-R regulates the degradation rate of ETA -R by modulating the ubiquitination of that receptor [41]. We therefore suggest that a reduction of Jab1-modified ETA -R leads to an upregulation of ETA -R protein (suppressed degradation of ETA R) in the diabetic OLETF aorta, and we propose that AG may have beneficial effects against obese-diabetic vascular complications. We also detected O-GlcNAc within the ETA -R immunoprecipitates derived from the rat aorta. We speculate that regulation of the O-GlcNAcylation of proteins might be a therapeutic target against diabetic vasocomplications [25,64,67]. As shown in Fig. 5C, the OLETF aorta exhibited an enhanced ETA -R-associated O-GlcNAc level, a result in accord with the plasma level of CML-AGEs in OLETF rats (Fig. 1C). AG-treatment normalized both elevated levels (Figs. 1C and 5C). In view of the results mentioned above, we consider that the increment in plasma CML-AGEs may have led to an

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Fig. 6. Analysis of (A) hypoxia-inducible factor (HIF) 1␣, (B) HIF2␣, and (C) endothelin-converting enzyme (ECE)-1 protein expressions under stimulation by 10 nM endothelin1 in thoracic aortas from LETO and aminoguanidine (AG)-treated or -untreated OLETF rats. Data in (C) were obtained with or without echinomycin (10 nM, 16 h), a smallmolecule inhibitor of HIF1 DNA-binding activity. (A–C) Protein expressions normalized to the ␤-actin expression level. Data are means ± S.E. from 6 experiments. *P < 0.05 – OLETF vs. LETO group. # P < 0.05 – AG-treated OLETF vs. OLETF group. † P < 0.05 – OLETF (treated with echinomycin) vs. echinomycin-untreated OLETF group.

enhancement of O-GlcNAc formation (O-GlcNAcylation) on ETA -R protein, and that an upregulation of ETA -R may follow a reduction of Jab1 binding. Indeed, the estrogen receptor ␤ (ER-␤) is modified by phosphorylation or O-GlcNAcylation at Ser16. It is promptly degraded as a target protein when the above site is phosphorylated, whereas decomposition of ER-␤ is suppressed by O-GlcNAcylation (it is then only very slowly degraded) [7]. What should be noted is that many regular catalytic subunits, including those of the proteasome, can be modified by O-GlcNAc [67]. Consequently, O-GlcNAc may exert a transitory influence on a lot of cell-activity processes by regulating the half-life of a given protein. The above results, which are consistent with the effect of ETA -R upregulation on ERK phosphorylation (Fig. 4B) and on the contraction induced by ET-1 under BQ123 pretreatment (Fig. 4A), are the first to suggest that in the diabetic OLETF aorta, O-GlcNAcylated ETA -R may inhibit Jab1 binding to ETA -R, and that this may in turn trigger contractile dysfunction in the thoracic aorta. We have no further data concerning the details of O-GlcNAcylation of ETA -R (e.g. whether there is competitive block/inhibition of the Jab1-binding site on ETA -R in a corresponding region or in the neighborhood of the O-GlcNAcylation site on ETA -R). Such issues remain to be examined in future studies. In the present study, we also determined the HIF (1␣, 2␣), ECE1, and plasma ET-1 levels because it was previously reported that levels of ET-1 are elevated in the diabetic state [21,30]. We found that HIF, ECE1 expression, and plasma ET-1 levels were all significantly higher in OLETF than in LETO (Figs. 6 and 7). Moreover, AG-treatment of OLETF rats normalized the HIF1␣, ECE1, and ET-1 levels, but not that of HIF2␣ (Figs. 6 and 7). We also asked whether ECE1 expression might be altered by echinomycin

(a small-molecule inhibitor of HIF1 DNA-binding activity). Preincubation with echinomycin significantly inhibited ET-1-induced ECE1 expression in aortas from OLETF, but not from LETO or AGtreated OLETF, rats (Fig. 6C). Taking all the present data together, we suggest that the augmentation of the ET-1-induced aortic contractile response seen in the long-term type 2 diabetic state may be attributable to increased levels of components of the ETA R/ERK pathway and to increased HIF1␣/ECE1/ET-1 levels, and that

Fig. 7. Plasma endothelin (ET)-1 levels in LETO and aminoguanidine (AG)-treated or -untreated OLETF rats. Data are means ± S.E. from 16 experiments. **P < 0.01 – OLETF vs. LETO group. # P < 0.05 – AG-treated OLETF vs. OLETF group.

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sustained suppression of AGEs might partially or completely prevent the progression of this enhancement of the response to ET-1. In conclusion, our study is the first to demonstrate that AGtreatment of type 2 diabetic OLETF rats normalizes ET-1-induced contraction in the thoracic aorta via both a suppressive effect on the O-GlcNAcylated ETA -R/ERK pathway and improved plasma ET-1 levels. We believe that our findings should stimulate further interest in manipulation of ET-1 signaling as a potential therapeutic target in the fight against diabetes-associated vascular diseases.

Acknowledgments This study was supported in part by the Ministry of Education, Culture, Sports, Science and Technology, and by the Open Research Center Project (Japan).

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