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Sulfur amino acid metabolism in Zucker diabetic fatty rats Hui Chan Kwak a, Young-Mi Kim b, Soo Jin Oh c, Sang Kyum Kim a,* a

College of Pharmacy, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 305-764, Republic of Korea College of Pharmacy, Hanyang University, Ansan, Gyeonggido 426-791, Republic of Korea c Bio-Evaluation Center, KRIBB, Ochang, Chungbuk, Republic of Korea b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 31 March 2015 Accepted 27 May 2015 Available online xxx

The present study was aimed to investigate the metabolomics of sulfur amino acids in Zucker diabetic fatty (ZDF) rats, an obese type 2 diabetic animal model. Plasma levels of total cysteine, homocysteine and methionine, but not glutathione (GSH) were markedly decreased in ZDF rats. Hepatic methionine, homocysteine, cysteine, betaine, taurine, spermidine and spermine were also decreased. There are no significant difference in hepatic S-adenosylmethionine, S-adenosylhomocysteine, GSH, GSH disulfide, hypotaurine and putrescine between control and ZDF rats. Hepatic SAH hydrolase, betainehomocysteine methyltransferase and methylene tetrahydrofolate reductase were up-regulated while activities of gamma-glutamylcysteine ligase and methionine synthase were decreased. The area under the curve (AUC) of methionine and methionine-d4 was not significantly different in control and ZDF rats treated with a mixture of methionine (60 mg/kg) and methionine-d4 (20 mg/kg). Moreover, the AUC of the increase in plasma total homocysteine was comparable between two groups, although the homocysteine concentration curve was shifted leftward in ZDF rats, suggesting that the plasma total homocysteine after the methionine loading was rapidly increased and normalized in ZDF rats. These results show that the AUC of plasma homocysteine is not responsive to the up-regulation of hepatic BHMT in ZDF rats. The present study suggests that the decrease in hepatic methionine may be responsible for the decreases in its metabolites, such as homocysteine, cysteine, and taurine in liver and consequently decreased plasma homocysteine levels. ß 2015 Elsevier Inc. All rights reserved.

Keywords: ZDF rat Sulfur amino acids Type 2 diabetes

1. Introduction Although a recent study suggested that elevated plasma homocysteine is a marker, rather than a cause, of atherosclerotic disease [1], elevated plasma homocysteine levels may be a risk factor for cardiovascular disease [2]. A recent meta-analysis and Mendelian randomization analysis performed among 4011 cases

Abbreviations: HHcy, hyperhomocysteinemia; ZDF, Zucker diabetic fatty; CbS, cystathionine b-synthase; BHMT, betaine-homocysteine methyltransferase; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; MS, methionine synthase; GSH, glutathione; CDO, cysteine dioxygenase; GCL, g-glutamylcysteine ligase; GSSG, glutathione disulfide; MPG, N-(2-mercaptopropionyl)-glycine; EDTA, ethylenediaminetetraacetic acid; TCEP, tris-(2-carboxyethyl)-phosphine hydrochloride; SBD-F, ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid; DTNB, 5,5dithiobis-2-nitrobenzoic acid; MAT, adenosyltransferase; MTHFR, methylenetetrahydrofolate reductase; SAHH, S-adenosylhomocysteine hydrolase; GCLC, gglutamylcysteine ligase catalytic subunit; GCLM, g-glutamylcysteine ligase modifier subunit; LC, liquid chromatography; ESI, electrospray ionization; MS, mass spectrometry; HPLC, high performance liquid chromatography; SD, standard deviation. * Corresponding author. Tel.: +82 42 821 5930; fax: +82 42 823 6566. E-mail address: [email protected] (S.K. Kim).

and 4303 controls provided a strong evidence for a causal association of homocysteine levels with the development of type 2 diabetes [3]. Homocysteine is an intermediate of the sulfur amino acid metabolic pathway. Altered homocysteine metabolism, particularly in the liver, is a major cause of hyperhomocysteinemia (HHcy). It has been demonstrated that total homocysteine in blood is either lower or normal in type 2 diabetes patients, whereas an increased homocysteine level was observed only in the presence of nephropathy [4]. Zucker diabetic fatty (ZDF) rats, an experimental type 2 diabetic model, have a defective leptin receptor and a decreased plasma total homocysteine level, which appears to be due to increased homocysteine clearing enzymes, such as cystathionine betasynthase (CbS) and betaine-homocysteine methyltransferase (BHMT) [5]. Our previous studies showed that changes in plasma homocysteine level in obese or diabetic conditions were dependent on the type of experimental animal model used [6–8]. Plasma total homocysteine concentration increased by 1.6-fold in obese mice fed a high-fat diet for 12 weeks [7], but was 40% lower in db/ db mice, obese type 2 diabetic animals with a G-to-T point mutation of the leptin receptor [6]. In contrast, plasma homocysteine levels were similar between control rats and non-obese type

http://dx.doi.org/10.1016/j.bcp.2015.05.014 0006-2952/ß 2015 Elsevier Inc. All rights reserved.

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diabetic Goto–Kakizaki rats [8]. Interestingly, db/db mice exhibited decreases in hepatic and plasma sulfur amino acids and their metabolites, including methionine, homocysteine, cysteine, Sadenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and hypotaurine. These results raise the possibility that depletion of methionine, a precursor for all sulfur-containing intermediates in the sulfur amino acid metabolic pathway, may be responsible in part for decreased homocysteine, as well as decreased cysteine, SAM, SAH and hypotaurine. The liver plays a significant role in the regulation of plasma homocysteine levels because of its full complement of enzymes involved in sulfur amino acid metabolism [9]. Dynamic processes regulate sulfur amino acid homeostasis [10,11]. Homocysteine intersects two competitive metabolic pathways: (i) remethylation to methionine by two independent enzymes, methionine synthase (MS) or BHMT, and (ii) transsulfuration to cysteine via cystathionine by the consecutive actions of CbS and cystathionine g-lyase (Fig. 1). Cysteine is metabolized to taurine, glutathione (GSH), and inorganic sulfate. Cysteine dioxygenase (CDO) catalyzes the oxidation of cysteine to cysteine sulfinate, which is converted to hypotaurine by cysteine sulfinate decarboxylase. Hypotaurine is non-enzymatically transformed into taurine. Meanwhile, gglutamylcysteine ligase (GCL) and GSH synthetase consecutively mediate GSH synthesis. To expand on previous studies, the present study aimed to investigate the metabolomics of sulfur amino acids in ZDF rats, an obese type 2 diabetic animal model. Hepatic levels and activities of enzymes involved in sulfur amino acid metabolism were investigated to determine whether differences in sulfur amino acid metabolic profiles reflected changes in the activities of their metabolizing enzymes. Moreover, we performed plasma kinetic analysis of methionine and homocysteine in ZDF rats intravenously treated with a mixture of methionine and methionined4. The present study suggests that the decrease in hepatic methionine may be responsible for the decreases in its metabolites, such as homocysteine, cysteine, and taurine in liver and consequently decreased plasma homocysteine levels in ZDF rats. 2. Materials and methods 2.1. Chemicals and antibodies Chemicals including amino acid standard, DL-homocysteine, Lcysteine, L-methionine, GSH, GSH disulfide (GSSG), GSH reductase, SAH, SAM, taurine, hypotaurine, L-serine, putrescine, spermidine, spermine, cystathionine, betaine hydrochloride, hexamethylenediamine, adenosine, homocysteine thiolactone, N-(2-mercaptopropionyl)-glycine (MPG), O-phthaldehyde, dansyl chloride, ATP, MgCl2, ethylenediaminetetraacetic acid (EDTA), pyridoxal-5-phosphate, 5-methyl-tetrahydrofolate, hydroxocobalamin, 1-heptanesulfonic acid, trichloroacetic acid, tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP), ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid (SBD-F), 2-mercaptoethanol, dithiothreitol, chloroform, KCl, HCl, sodium acetate, methanol, acetonitrile, ferrous ammonium sulfate, 5,5-dithiobis-2-nitrobenzoic acid (DTNB), hydroxylamine, NAD, 2-vinyl pyridine, sodium hydroxide, sodium carbonate and boric acid were purchased from Sigma– Aldrich (St. Louis, MO). Perchloric acid, sodium dihydrogenphosphate and disodium hydrogenphosphate were purchased from Junsei Chemical (Tokyo, Japan). Deuterium labeled betaine-d9 hydrochloride was purchased from CDN isotopes Inc. (Quebec, Canada). Deuterium labeled methionine-d3 and methionine-d4 were purchased from Cambridge Isotope Laboratories (Andover, MA). Antibodies against methionine adenosyltransferase (MAT)I/ III, methylene tetrahydrofolate reductase (MTHFR) and a-tubulin

were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and the anti-SAH hydrolase (SAHH), anti-MS and anti-CDO antibodies were purchased from Abcam (Cambridge, UK). The antiBHMT antibody was purchased from Everest Biotech (Oxfordshire, UK). The anti-GCL catalytic subunit (GCLC) and anti-GCL modifier subunit (GCLM) antibodies were purchased from NeoMarker Inc. (Fremont, CA). The anti-CbS antibody was kindly provided by Dr. Matherly (Wayne State University School of Medicine, Detroit, MI). Horseradish peroxidase-conjugated goat anti-rabbit and rabbit anti-goat antibodies were obtained from BioRad Laboratories (Hercules, CA). Enhanced chemiluminescence detection reagent solutions were purchased from Thermo scientific (Rockford, IL). All chemicals and solvents used in this study were reagent grade or higher. 2.2. Animal experiments This study used male ZDF rats (ZDF/Gmi fa/fa) aged 5 and 11 weeks and male lean rats (ZDF/Gmi fa/?) of the same ages. All animals were maintained at Korea Research Institute of Bioscience and Biotechnology (Ochang, Korea). The rats were housed at 22  2 8C and 50  5% humidity controlled rooms with a 12-h light/ dark cycle for at least 1 week prior to experimentation. Laboratory chow and tap water were allowed ad libitum. The rats were euthanized using CO2. All animal experiments were approved by the Institutional Animal Care and Use Committee, and were performed in accordance with institutional guidelines. 2.3. Preparation of plasma and hepatic samples Following euthanasia, blood samples were obtained from the abdominal aorta. Plasma was obtained by centrifugation of blood at 10,000 g for 15 min at 4 8C. The supernatant fraction and remaining plasma were stored at 70 8C until analysis. The liver was rapidly removed and homogenized in a 3-fold volume of icecold buffer consisting of 0.154 M KCl, 50 mM Tris–HCl, and 1 mM EDTA (pH 7.4). All subsequent steps were performed at 0–4 8C. The liver homogenates were deproteinized in a 3-fold volume of icecold methanol to measure methionine, hypotaurine and taurine, or in an equal volume of 10% PCA to measure SAM, SAH, homocysteine, cysteine, GSH, GSSG, putrescine, spermidine and spermine. After centrifugation at 10,000 g for 20 min, the supernatant fraction was collected and refrigerated in 70 8C. The liver homogenates were centrifuged at 10,000 g for 20 min. The supernatant fraction was further centrifuged at 104,000  g for 65 min. The supernatant fraction was collected and refrigerated in 70 8C (the cytosol samples). The total protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Scientific, IL). 2.4. Determination of sulfur amino acids and their metabolites in liver and plasma The methionine concentrations were measured using liquid chromatography (LC) triple quadruple electrospray ionization (ESI)/mass spectrometry (MS)/MS and methionine-d3 as an internal standard. The LC-ESI/MS/MS system consisted of a Shimadzu LC-20AD XR high performance liquid chromatography (HPLC) system (Shimadzu, Tokyo, Japan) and an API 3200 QTRAP1 LC–MS/MS system (AB Sciex, Framingham, MA) equipped with a Turbo VTM Ion Source (Applied Biosystems, Foster City, CA) operated in the positive multiple reaction monitoring ion mode with the following transition: methionine m/z 150 ! 104; methionine-d3 153 ! 107. Plasma samples were deproteinized with 10-fold methanol and diluted in 50% acetonitrile containing methionine-d3 (200 nM). The sample

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(10 mL) was injected into a Waters Atlantis hydrophilic interaction liquid chromatography silica (2.1 mm  150 mm, 3 mm) column with a mobile phase composed of water (solution A) and acetonitrile (solution B). The column was eluted at a flow rate of 0.25 mL/min, and developed with a gradient elution as follows: 0–2 min, 10% A; 4 min, 60% A; 9 min, 40% A; 10.5 min, 10% A. The lower limit for the quantification of methionine was less than 1 nM. An HPLC method was used to determine the hepatic SAM and SAH concentration [8]. The supernatant obtained after 10% PCA treatment was applied to an HPLC system (Shimadzu, SCL-10A) equipped with an ultraviolet detector (Shimadzu, SPD-10Avp, 254 nm) and a TSK-GEL ODS-80TM column (4.6 mm  250 mm, 5 mm; Tosoh Co., Tokyo, Japan). Betaine concentrations were determined using LC triple quadruple ESI/MS/MS and d9-betaine, as an internal standard. Analytical method was developed according to Holm et al. [12]. The LC ESI/MS/MS was operated in the positive multiple reaction monitoring ion mode with following transition: betaine m/z 118 ! 58; betaine-d9 127 ! 68. Sample (5 mL) was injected into a Waters Atlantis HILIC silica (2.1 mm  150 mm, 3 mm) column with a mobile phase composed of 0.1% formic acid (solution A) and acetonitrile containing 0.1% formic acid (solution B). The column was eluted at a flow rate of 0.25 mL/min and developed with gradient elution as follows: 0– 3 min, 5% A; 14 min, 40% A; 15 min, 5% A. All gradient steps were linear. The lower limit of quantification of betaine was less than 50 pM. Homocysteine, GSH, and cysteine in the plasma and liver were quantified using the SBD-F method [6]. For sample preparation, 10 mL of 10 mM MPG was used as an internal standard, and was added to 50 mL sample. Following the addition of 6 mL TCEP (100 g/ L), tubes were incubated at room temperature for 30 min. Then, 54 mL trichloroacetic acid (100 g/L with 1 mM EDTA) was added to each sample and centrifuged at 13,000 g for 10 min. The supernatant (50 mL) was added to another tube containing 10 mL 1.55 M NaOH, 125 mL 0.125 M borate buffer (pH 9.5) with 4 mM EDTA, and 50 mL 1 g/L SBD-F in a borate buffer. The samples were incubated for 1 h at 60 8C, and then a 10 mL aliquot was injected into the HPLC system equipped with a fluorescence detector for analysis (Shimadzu, RF-10AXL; Ex 385 nm and Em 515 nm). The chromatographic separation was achieved using a Phenomenex Luna C18 column (4.6 mm  150 mm, 5 mm). Measurement of the total GSH and GSSG concentration was based on enzymatic reactions using a DTNB-GSSG reductase-recycling assay [13]. Hypotaurine and taurine were derivatized with O-phthalaldehyde/2-mercaptoethanol and quantified using an HPLC system with a fluorescence detector. An Eclipse XDB-C18 column (4.6 mm  150 mm, 3.5 mm; Agilent Technologies, Palo Alto, CA) was used for analyses of hypotaurine and taurine [14]. Polyamines (putrescine, spermine, and spermidine) were quantified using the dansyl-derivative method utilizing hexamethylenediamine as an internal standard [15]. Following the addition of 20 mL of 3 M sodium carbonate and 20 mL of dansyl chloride (4 mg/ml), samples were vortexed and incubated for 30 min at 50 8C (adjusted to pH 9.0). Then, a 10 mL aliquot was injected into the HPLC system using a fluorescence detector for analysis (Shimadzu, RF-10AXL; Ex 370 nm and Em 506 nm). The chromatographic separation was performed using a Phenomenex Luna C18 column (4.6 mm  150 mm, 5 mm) with a mobile phase composed of water (solution A) and methanol (solution B). The column was eluted at a flow rate of 1 mL/min and developed with a gradient elution as follows: 0 min, 20% A; 0–11 min, 0% A; 11– 12 min, 20% A; 14 min, 20% A.

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2.5. Immunoblot analyses To determine the expression of b-actin, MATI/III, SAHH, BHMT, MS, MTHFR, CbS, CDO, GCLC and GCLM, the cytosol samples were diluted in a loading buffer to 2 mg/mL. Lysates (10 mL) were separated using a 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose and blocked in 5% non-fat dry milk in PBS-T (0.1% Tween 20 in phosphate buffer solution). The blots were incubated with antibodies overnight at 4 8C. Antibodies were diluted with PBS-T containing 3–5% bovine serum albumin: bactin (1:10,000), MATI/III (1:3000), SAHH (1:500), BHMT (1:5000), MS (1:200), MTHFR (1:200), CbS (1:2000), CDO (1:1000), GCLC (1:3000) and GCLM (1:2000). The primary antibody incubation was followed by incubation with horseradish peroxidase-conjugated with the secondary antibody (1:10,000 in PBS-T containing 5% non-fat dry milk). Proteins were detected using enhanced chemiluminescence on the Fusion SL2 chemiluminescence system (Vilber Loumat, Eberhardzell, Germany) and quantified using the Fusion Capt Advance (Vilber Loumat) and the Multi Gauge (Fuji Photo Film Co., Tokyo, Japan). 2.6. Enzyme assays MAT activity was estimated by quantifying the SAM production using a method by Kim et al. [16]. Reaction mixtures (final volume 0.5 ml) consisted of 80 mM Tris buffer containing 50 mM KCl (pH 7.4), 5 mM ATP, 40 mM MgCl2, 5 mM methionine and 1 mg protein/mL. After preincubation for 3 min, methionine was added to initiate the enzyme reaction. The samples were incubated at 37 8C for 30 min, and the incubation was terminated by adding 0.25 mL ice-cold 6% PCA. SAM, a metabolite of the MAT enzyme assay, was quantified as described above. The SAHH enzyme assay was performed using a modification of a method by Isa et al. [17]. The reaction mixtures (final volume 500 mL) consisted of 70 mM Tris buffer containing 35 mM KCl, 35 mM MgCl2 (pH 8.0), 1.4 mM dithiothreitol, 10 mM homocysteine, 1 mM adenosine, and 0.8 mg protein/mL. After preincubation for 5 min, homocysteine was added to initiate the enzyme reaction. The samples were incubated at 37 8C for 60 min, and the incubation was terminated by adding 50 mL ice-cold 4 M PCA. SAH, the metabolite of the SAHH enzyme assay, was quantified as described above. Both BHMT and MS activities were estimated by quantifying the formation of methionine [6]. For BHMT, the reaction mixtures (final volume 150 mL) consisted of 50 mM potassium phosphate buffer (PPB, pH 7.4) containing 2.5 mM betaine, 7 mM homocysteine and 1 mg protein/mL. After preincubation for 5 min, homocysteine was added to initiate the enzyme reaction. The samples were incubated at 37 8C for 30 min, and the incubation was terminated by adding 450 mL ice-cold methanol. In MS enzyme assay, the reaction mixtures (final volume 300 mL) consisted of 60 mM PPB (pH 7.4), containing 0.05 mM hydroxocobalamin, 125 mM 2-mercaptoethanol, 0.3 mM SAM, 0.5 mM 5-methyltetrahydrofolate, 0.4 mM homocysteine and 1 mg protein/mL. The samples were incubated at 37 8C for 30 min in dark, after 5 min preincubation. For reaction stop, 900 mL ice-cold methanol was used. Methionine, the metabolite of the BHMT and MS enzyme assays, was quantified as described above. The CbS enzyme assay was performed using a method by Costa et al. [18]. The reaction mixtures (final volume 300 mL) consisted of 0.1 M PPB (pH 7.8) containing 20 mM homocysteine, 50 mM serine, 5 mM pyridoxal 5-phosphate, 5 mM EDTA and 1 mg protein/mL. The incubation continued at 37 8C for 60 min, and was terminated by adding 600 mL ice-cold methanol. Cystathionine, formed during the incubation, was derivatized with Ophthalaldehyde/2-mercaptoethanol and quantified using a HPLC

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system with a fluorescence detector (Shimadzu RF-10AXL; Ex 340 nm and Em 450 nm). The supernatant (20 mL) was injected into a Phenomenex Luna (4.6 mm  150 mm, 5 mm) column with a mobile phase composed of 40 mM sodium dihydrogen phosphate buffer with 0.5% THF (pH 7.8, solution A) and acetonitrile:methanol:distilled water (45:45:10, solution B). The column was eluted at a flow rate of 1 mL/min and developed with gradient elution as follows: 0 min, 90% A; 4 min, 84% A; 4–7 min, 84% A; 8 min, 0% A; 8–11 min, 0% A; 12 min, 90% A; 12–16 min, 90% A. The CDO activity was estimated by quantifying the formation of cysteine sulfinic acid [19]. The reaction mixtures (final volume 200 mL) consisted of 0.1 M PPB buffer (pH 6.3) containing 0.5 mM ferrous ammonium sulfate, 5 mM hydroxylamine, 1.4 mM NAD hydrate, 5 mM cysteine and 1 mg protein/mL. The incubation continued at 37 8C for 16 min and the incubation was stopped by adding 500 mL ice-cold methanol. Cysteine sulfinic acid, formed during the incubation, was derivatized with O-phthalaldehyde/2mercaptoethanol and quantified using a HPLC system with a fluorescence detector (Shimadzu RF-10AXL; Ex 338 nm and Em 425 nm). The supernatant (20 mL) was injected into a Phenomenex Luna (4.6 mm  150 mm, 5 mm) column with a mobile phase composed of 0.1 M sodium acetate buffer (pH 7.2, solution A) and methanol (solution B). The column was eluted at a flow rate of 1 mL/min and developed with gradient elution as follows: 0 min, 90% A; 4 min, 84% A; 4–7 min, 84% A; 8 min, 0% A; 8–11 min, 0% A; 12 min, 90% A; 12–16 min, 90% A. The GCL activity was estimated by quantifying the formation of g-glutamylcysteine [20]. The reaction mixtures (final volume 200 mL) consisted of 0.1 M Tris buffer contained 0.15 M KCl and 2 mM EDTA (pH 8.2), 200 mM MgCl2, 100 mM glutamate, 50 mM cysteine, 100 mM ATP and 0.5 mg protein/mL. The incubation continued at 37 8C for 15 min and the incubation was stopped by adding 100 mL ice-cold 6% PCA. Gamma-glutamylcysteine, formed during the incubation, was derivatized with O-phthalaldehyde/2mercaptoethanol and quantified using a HPLC system with a fluorescence detector (Shimadzu RF-10AXL; Ex 340 nm and Em 420 nm). Sample (20 mL) was injected into a Phenomenex Luna (4.6 mm  150 mm, 5 mm) column with a mobile phase composed of 50 mM sodium acetate buffer (pH 6.2, solution A) and acetonitrile (solution B). The column was eluted at a flow rate of 1 mL/min and developed with gradient elution as follows: 0– 2 min, 100% A; 11 min, 85% A; 12 min, 0% A; 12–15 min, 0% A; 16 min, 100% A; 16–20 min, 100% A. 2.7. Methionine and homocysteine plasma kinetics in control and ZDF rats intravenously treated with a mixture of methionine and methionine-d4 Jugular vein catheter (Braintree Scientific Inc., Braintree, MA) was surgically implanted in the right jugular vein under Ketamine– Xylazine anesthesia. The animals were then allowed to recover for 4 days. Male ZDF and lean rats aged 11 weeks were treated with a single i.v. injection of a mixture of 0.6 mol/kg of L-methionine and 0.2 mol/kg of methionine-d4. Blood was collected before and after 10, 30, 60, 120, 210, 300 and 420 min of injection. Blood samples (400 mL each) were collected into BD MicrotainerTM plasma separator tubes kept on ice at predetermined time points through the right jugular vein cannula. The blood samples were centrifuged under refrigeration at 6000  g for 5 min to separate the plasma and then kept frozen at 70 8C until analysis. Plasma methionine concentrations were determined using LC triple quadruple ESI/MS/MS system (AB Sciex, Framingham, MA) equipped with a Turbo VTM Ion Source (Applied Biosystems, Foster City, CA) operated in the positive multiple reaction monitoring ion mode with following transition: methionine m/z 150 ! 104; methionine-d3 153 ! 107; methionine-d4 154 ! 108. Plasma

samples were deproteinized with 10-fold methanol and diluted in 50% acetonitrile containing internal standard methionine-d3 (200 nM). Plasma homocysteine concentrations were quantified as described above. 2.8. Statistical analyses Significant differences between groups were determined using a two-tailed Student’s t-test (P < 0.05). All data are presented as the mean  standard deviation (SD) for five to six samples. Linear regression was calculated using GraphPad Prism, version 4.0 (GraphPad Software Inc., San Diego, CA). 3. Results 3.1. Changes in body weight, liver weight and liver to body weight ratio in ZDF rats ZDF rat body weight and absolute liver weight increased to 125% and 183% of control rats, respectively. Consequently, liver weight per unit body weight was also greater in ZDF rats compared with control rats (data not shown). 3.2. Changes in serum biochemical parameters in control and ZDF rats Serum biochemical data are shown in Table 1. Blood glucose and glycated hemoglobin (HbA1c), a diabetes marker, were 3.3- and 1.7-fold greater, respectively, than those of control rats. Serum insulin levels were markedly increased in ZDF rats and ketone bodies were decreased, similar to a previous study [21]. These results indicate that ZDF rats become overtly diabetic within 11 weeks after birth. Liver injury was not observed in ZDF rats, as evaluated by serum enzyme activities of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. The blood urea nitrogen (BUN) levels, but not creatinine, were increased in ZDF rats, but the BUN levels were within the normal range (15– 22 mg/dL) [22]. Total cholesterol and triglyceride levels were 1.3and 8.5-fold greater than those of control rats. 3.3. Changes in plasma and hepatic levels of sulfur amino acids and their metabolites in control and ZDF rats Total homocysteine, cysteine, GSH and methionine plasma concentrations were determined in control and ZDF rats (Table 2). There were no significant differences in plasma total GSH levels in rats. Plasma total cysteine, homocysteine, and methionine levels were markedly decreased to 60, 61 and 69% in ZDF rats, respectively, relative to control rats. In addition, changes in various amino acid levels in plasma were determined in control Table 1 Serum biochemical parameters in control and ZDF rats.

Blood glucose (mg/dL) HbA1c (%) Insulin (ng/ml) Ketone bodies (mmol/L) AST (IU/L) ALT (IU/L) ALP (IU/L) BUN (mg/dL) CRE (mg/dL) Cholesterol (mg/dL) Triglyceride (mg/dL)

Control

ZDF

139  14 5.23  0.39 0.20  0.03 1.25  0.10 91.0  7.3 63.3  1.0 629  52 16.6  1.0 0.525  0.050 132  7 113  56

457  60*** 9.04  1.15*** 3.12  0.77*** 0.30  0.07*** 142.4  53.7 80.0  25.0 1080  608 20.2  1.6** 0.600  0.141 177  15*** 964  132***

Each value represents the mean  SD of 5–6 rats. ** Significantly different from control rats at P < 0.01 (Student’s t-test). *** Significantly different from control rats at P < 0.001 (Student’s t-test).

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Cysteine (mM) Homocysteine (mM) GSH (mM) Methionine (mM)

Control

ZDF

162.5  29.4 1.83  0.26 35.9  6.5 13.53  2.16

97.2  30.2** 1.11  0.23** 44.2  11.9 9.33  1.53**

Each value represents the mean  SD of 5–6 rats. ** Significantly different from control rats at P < 0.01 (Student’s t-test).

and ZDF rats. Valine, leucine, glutamic acid, and alanine were slightly but significantly increased by less than 20% of the control level in ZDF rats. In contrast, threonine, lysine, arginine, tyrosine, and glycine were 80, 79, 74, 84, and 63% of the control level, respectively. Isoleucine, phenylalanine, histidine, aspartic acid, serine, and proline were similar between the two groups (data not shown). Thus, changes in sulfur amino acids were dominant in ZDF rats compared with other amino acids; however, altered plasma amino acids were not limited to sulfur amino acids. Total homocysteine, cysteine, GSH and methionine in plasma were determined in 5-week-old ZDF rats and the same age control rats. The 5-week-old ZDF rats were characterized by insulin resistance without elevated blood glucose or HbA1c (data not shown). The plasma levels of total homocysteine, cysteine, GSH and methionine were comparable between control and ZDF rats aged five weeks (data not shown). Thus, decreased sulfur amino acid in plasma was accompanied by the development of diabetes. In addition branched-chain amino acids including leucine, isoleucine and valine in plasma were increased to 134, 122 and 126% of the control level, respectively. In contrast, glycine, alanine and

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Table 3 Hepatic concentrations of sulfur amino acids and their metabolites in control and ZDF rats.

Methionine (nmol/g liver) SAM (nmol/g liver) SAH (nmol/g liver) SAM/SAH Homocysteine (nmol/g liver) Cysteine (nmol/g liver) GSH (mmol/g liver) GSSG (nmol/g liver) GSSG/GSH (%) Betaine (mmol/g liver) Hypotaurine (nmol/g liver) Taurine (mmol/g liver) Putrescine (nmol/g liver) Spermidine (nmol/g liver) Spermine (nmol/g liver)

Control

ZDF

22.4  2.0 141  20 22.7  4.1 6.41  1.48 13.9  2.6 153  13 9.48  0.62 112  18 1.49  0.16 2.63  0.43 272  92 0.932  0.357 1.37  0.30 416  96 1229  208

12.8  4.8** 165  40 21.3  7.9 8.47  3.29 6.4  0.7*** 107  17** 9.14  0.49 142  123 1.87  1.56 1.53  0.22*** 473  163 0.465  0.106* 1.23  0.25 289  34* 866  191*

Each value represents the mean  SD of 5–6 rats. * Significantly different from control rats at P < 0.05 (Student’s t-test). ** Significantly different from control rats at P < 0.01 (Student’s t-test). *** Significantly different from control rats at P < 0.001 (Student’s t-test).

aspartate were decreased to 80, 61 and 77% in 5-week-old ZDF rats, respectively. The hepatic concentrations of sulfur amino acids and their metabolites were monitored in control and ZDF rats (Table 3). Hepatic methionine, homocysteine, and cysteine levels were 57, 46, and 70% of control values in ZDF rats, respectively, which is a similar pattern to that observed in plasma. ZDF rats had hepatic betaine and taurine levels that were 58 and 50% of those in control rats, respectively. There were no significant differences in hepatic

Fig. 1. Hepatic metabolic pathways of sulfur amino acids. MAT, methionine adenosyltransferase; SAHH, SAH hydrolase; BHMT, betaine-homocysteine methyltransferase; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; CbS, cystathionine b-synthase; CgL, cystathionine g-lyase; CDO, cysteine dioxygenase; CDC, cysteine sulfinate decarboxylase; GCL, g-glutamylcysteine ligase and THF, tetrahydrofolate.

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SAM, SAH, GSH, GSSG or hypotaurine levels. The SAM/SAH ratio, which serves as an index of transmethylation potential, was also unchanged. In polyamine synthesis, SAM is a source of a propylamine group for spermidine and spermine, following its decarboxylation by SAM decarboxylase [23]. Hepatic spermidine and spermine levels were 69 and 70%, respectively, of control values in ZDF rats, but the hepatic putrescine level was similar between the two groups. 3.4. Changes in hepatic sulfur amino acid-metabolizing enzymes in ZDF rats Levels of hepatic enzymes involved in sulfur amino acid metabolism were determined using immunoblot analysis with specific antibodies in control and ZDF rats (Fig. 2). There were no significant differences in hepatic protein levels of a-tubulin, which was used as a loading control (data not shown). Hepatic MAT I/III, MS, GCLC and CDO were not significantly changed in ZDF rats (Fig. 2A, D, G, and I). However, hepatic SAHH, BHMT and MTHFR were upregulated to 152, 194 and 385%, respectively, of the levels observed in control rats (Fig. 2B, C, and E). In contrast, CbS and GCLM were downregulated in ZDF rats (Fig. 2F and H). Enzymes involved in sulfur amino acid metabolism are subjected to post-translational modification and allosteric regulation [24]. Thus, hepatic activities, including MAT, SAHH, BHMT, MS, CbS, GCL and CDO, were measured using an HPLC system equipped with a fluorescence detector, an ultraviolet detector, or a tandem mass spectrometry (MS/MS) system (Fig. 3). There was no significant difference in the activity of MAT or CDO between the two groups (Fig. 3A and G). SAHH and BHMT activities were significantly greater than the control levels, 113 and 147%, respectively (Fig. 3B and C). Thus, the changes in hepatic activities of these enzymes reflected the results from immunoblot assay. MS and GCL hepatic activities were 83 and 46% of the control levels, respectively (Fig. 3D and F). Hepatic CbS activity was comparable between the two groups, despite the decrease in CbS protein levels (Fig. 3E). 3.5. Methionine and homocysteine plasma kinetics in control and ZDF rats intravenously treated with a mixture of methionine and methionine-d4 The liver plays a central role in sulfur amino acid metabolism and elevation of metabolic clearance of methionine in liver can cause to depletion of hepatic methionine levels. To investigate involvement of methionine clearance in decreased sulfur amino acid levels and their metabolites, plasma methionine and homocysteine levels were kinetically determined to calculate their AUC in control and ZDF rats intravenously treated with a mixture of methionine (60 mg/kg) and methionine-d4 (20 mg/kg) (Fig. 4). The area under the plasma methionine concentration curve from 0 to 420 min (AUC0!420) was 102  9 or 103  23 mM min in control or ZDF rats, respectively. The AUC0!420 of methionine-d4 was 20.3  0.6 mM min in control rats and 21.8  1.4 mM min in ZDF rats. The plasma kinetic patterns of methionine and methionine-d4 were similar. The AUC0!420 of methionine and methionine-d4 did not differ significantly between the two groups (Fig. 4A). Fig. 4B shows the plasma homocysteine concentration increments achieved after methionine loading. The DAUC0!420 of the increase in homocysteine was 1885  145 mM min in control rats and 1721  399 mM min in ZDF rats, which was not significantly different. However, ZDF rats exhibited a greater increase in homocysteine at 30 min (early phase) than control rats, while homocysteine was lower in ZDF rats at 210 and 300 min (late phase) than in control rats. The maximal plasma total homocysteine concentration increment was observed at 120 min in control rats

and at 60 min in ZDF rats. Thus, the homocysteine concentration curve was shifted leftward in ZDF rats, suggesting that the plasma total homocysteine concentrations in ZDF rats after methionine loading rapidly increased and then normalized relative to control rats. 4. Discussion In the present study, ZDF rats exhibited increased serum glucose, HbA1c, cholesterol, triglyceride, and insulin levels, as well as decreased plasma ketone bodies, which is consistent with previous studies [25,26]. ZDF rats had hypohomocysteinemia, hypomethioninemia, and hypocysteinemia. Hepatic methionine, homocysteine, cysteine, betaine, taurine, spermidine, and spermine levels were also lower in ZDF rats. There were no significant differences in hepatic SAM, SAH, GSH, GSSG, hypotaurine, or putrescine levels between control and ZDF rats. Hepatic protein levels of SAHH, BHMT, and MTHRF were upregulated, while CbS and GCLM were down-regulated in ZDF rats. MAT I/III, MS, GCLC and CDO protein levels were not significantly different between control and ZDF rats. Hepatic activities of SAHH and BHMT were greater in ZDF rats, while hepatic activities of MS and GCL were lower. The observed changes in hepatic and plasma sulfur amino acids and their metabolites are similar to our previous findings in db/db mice [6], with the exception of hepatic levels of SAM, SAH, and polyamines. We observed lower SAM and SAH and greater putrescine in db/db mice. Interestingly, both ZDF rats and db/db mice exhibited lower levels of methionine, homocysteine and cysteine in liver and plasma [6]. Hepatic sulfur amino acid maintenance is a dynamic process that is regulated by a balance of hepatic transport and metabolism [14,16]. One of the main roles of hepatic sulfur amino acid metabolism is to maintain methionine levels. Thus, these results show that the regulation of hepatic sulfur amino acid metabolism is impaired in ZDF rats. Furthermore, decreased homocysteine and cysteine in plasma and liver may be due to depleted hepatic methionine. Although the pathophysiological role of methionine in diabetes state is unknown, supplementation of methionine and choline improved heart function in diabetic rats induced by streptozotocine [27]. In mammals, the liver plays a central role in sulfur amino acid metabolism, where almost 50% of methionine metabolism and up to 85% of methylation reactions occur [28]. Hepatic SAM serves as a regulator of hepatic sulfur amino acid metabolism. SAM regulates MAT via feedback activation [28,29]. SAM is also an allosteric CbS activator and MTHFR inhibitor [11]. Moreover, SAM inactivates BHMT in rat liver [30] and down-regulates BHMT expression in HepG2 cells [31]. Thus, elevated hepatic SAM can accelerate methionine consumption to produce cysteine via increased homocysteine transsulfuration and decreased homocysteine remethylation. In the present study, hepatic SAM levels were not significantly different between control and ZDF rats. Hepatic BHMT protein levels and activities were upregulated in ZDF rats; consequently, the hepatic betaine level was markedly decreased, suggesting that homocysteine remethylation by BHMT may be greater in ZDF rats than in controls. Moreover, the methionineloading test demonstrated that the AUC0!420 of methionine and methionine-d4 did not differ significantly between control and ZDF rats, although plasma methionine was increased to supraphysiological level in this test. These results are supported by previous studies showing that transsulfuration flux of 0.1 mM L-[1-14C] methionine was not significantly different between hepatocytes isolated from control and ZDF rats [5]. These results suggest that changes in hepatic methionine metabolism may be not responsible for the decreased hepatic methionine levels. The hepatic methionine level is regulated by its hepatic uptake from blood, as well as by the balance of homocysteine metabolism.

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Fig. 2. Changes in the protein level of hepatic sulfur-amino-acid-metabolising enzymes in ZDF rats. Each value represents the mean  SD of 5–6 rats. **Significantly different from control rats at P < 0.01 (Student’s t-test).

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Fig. 3. Changes in the activity of hepatic sulfur-amino-acid-metabolising enzymes in ZDF rats. Each value represents the mean  SD of 5–6 rats. *,**,***Significantly different from control rats at P < 0.05, 0.01, or 0.001, respectively (Student’s t-test).

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Fig. 4. Plasma methionine concentrations and homocysteine concentration increments in control and ZDF rats after a single i.v. injection of methionine (100 mM in saline). Control and ZDF rats were treated with a single i.v. injection of methionine (0.8 mmol/kg, L-methionine:L-methionine-d4 = 3:1). Blood was collected before and 10, 30, 60, 120, 210, 300 and 420 min after methionine loading. Each value represents the mean  SD for 5–6 rats. *,*** Significantly different from control rats at P < 0.05 or 0.001 (Student’s ttest).

Based on arterial–venous methionine differences, methionine is released into the blood by the splanchnic organs and removed by the liver [32]. Methionine is transported mainly by amino acid transport system L in hepatocytes [33]. System L consists of a high affinity component I, with a Km < 200 mM for methionine, and a low affinity component II, with a Km > 2 mM. Although system L was not affected in hepatocytes isolated from diabetic rats treated with streptozotocin, decreased methionine in plasma may cause a decrease in its hepatic uptake, based on the kinetic parameters of system L. In a previous study, a decreased methionine level in plasma but not liver was observed in 11-week-old ZDF rats [34]. Differences in the hepatic methionine level may reflect differences associated with feeding and diabetic conditions. Elevated plasma insulin levels were observed in ZDF rats in this study, but not in a previous study [34]. Interestingly, the hepatic uptake of L-[methyl-11C] methionine was decreased in patients with diabetes mellitus [35]. Although, the mechanism(s) for the decreased in plasma methionine remains to be determined, decrease in splanchnic efflux of methionine into blood and/or intestinal absorption of methionine from diet can be speculated an casual factors. The hepatic metabolism of homocysteine is a major determinant of plasma homocysteine concentration [36,37]. In the liver, there are three homocysteine-clearing enzymes: CbS, BHMT and MS. In this study, the hepatic protein levels of BHMT and MTHFR were up-regulated, and the hepatic betaine concentration was decreased. MTHFR catalyzes the reduction of N5,N10-methylenetetrahydrofolate to N5-methyl-tetrahydrofolate, which is a methyl donor for homocysteine methylation by MS. This reduction is the rate-limiting step in folic acid biosynthesis [38]. The hepatic upregulation of BHMT and decrease in betaine level are consistent with previous results reported in db/db mice [6] and ZDF rats [5,39]. Up-regulation of hepatic BHMT plays an important role in hepatic methionine maintenance during periods of inadequate intake of this amino acid [40]. These results raise the possibility that the induction of BHMT is an adaptive response involved in maintaining hepatic methionine levels. Despite decreased plasma homocysteine levels in ZDF rats, the methionine-loading test results showed that the DAUC of homocysteine in plasma did not significantly differ between control and ZDF rats. The methionine-loading test is necessary to diagnose moderate HHcy, as a considerable number of HHcy

patients have normal fasting homocysteine levels [41]. An abnormal increase in plasma homocysteine after methionine loading primarily reflects homocysteine transsulfuration defects, and, to a lesser degree, methionine remethylation defects [42]. An abnormal increase in homocysteine levels after methionine loading depends not only on CbS activity, but also on pyridoxine, folate, and cobalamin levels, as well as MTHFR activity [41,43]. In fact, the plasma homocysteine DAUC in the methionine-loading test was significantly decreased in rats acutely or chronically treated with betaine [44,45]. Furthermore, the rate of elevation in plasma homocysteine concentrations was significantly lower in betaine-treated rats after methionine loading than in control rats. These results are in sharp contrast with the present findings, indicating that plasma homocysteine concentrations rapidly increase in ZDF rats. Thus, the methionine-loading test results suggest that the DAUC of plasma homocysteine may be not responsive to hepatic BHMT upregulation in ZDF rats. In this study, the plasma homocysteine concentration curve was shifted leftward in ZDF rats. The upregulation of SAHH, multiple methyl transferases, and homocysteine-producing enzymes in diabetic conditions [5,46] may be involved in the rapid increase in plasma homocysteine in ZDF rats. The decrease in hepatic methionine levels was reflected in the hepatic cysteine levels. Hepatic uptake from plasma and generation via the transsulfuration pathway are two major sources of cysteine supply. Cysteine is a metabolic intermediate of GSH and taurine synthesis. In this study, ZDF rats exhibited up-regulated BHMT and MTHFR, decreased taurine, and maintenance of liver GSH, indicating that changes in hepatic sulfur amino acid metabolizing enzymes do not explain the decrease in hepatic cysteine. Thus, the decrease in hepatic cysteine can be attributed to the decreased availability of methionine. In addition, cysteine is mainly transported by the sodium-dependent amino acid transport system ASC (preferred substrates are alanine, cysteine, serine and threonine) in rat hepatocytes. The ASC system is not responsive to insulin or glucagon. Cysteine is unstable and extracellular, where it is easily oxidized to cystine, a cysteine disulfide, the major disulfide form found in plasma [47,48]. Cystine transport is mediated by the sodium-independent amino acid transport system, Xc, in hepatocytes. System Xc activity is markedly increased by electrophilic agents in cultured rat hepatocytes [47]. Although an extensive literature survey failed

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to reveal the effect of diabetes on hepatic uptake of cysteine/ cystine, system Xc was up-regulated by insulin in cultured rat hepatocytes via de novo synthesis [49]. These results raise the possibility that hepatic cystine uptake may be decreased in diabetic conditions. The hepatic taurine level was decreased in ZDF rats. Neither CDO protein level nor activity differed between control and ZDF rats. The CDO Km value for cysteine is approximately 0.45 mM, and hepatic cysteine concentration ranges from 0.1 to 0.2 mM/g in liver [50]. Thus, CDO can respond to changes in hepatic cysteine levels. These results suggest that the decrease in hepatic cysteine levels may be associated with the change in hepatic taurine level. The hepatic GSH levels were maintained in ZDF rats despite decreases in both the hepatic cysteine level and GCL activity. The plasma GSH concentrations were also maintained in ZDF rats, suggesting that changes in hepatic efflux of GSH into blood may be not responsible for the maintenance of hepatic GSH. Thus, the underlying mechanism involved in hepatic GSH regulation in ZDF rats remains to be determined. The hepatic SAM level was also maintained in ZDF rats despite the decrease in hepatic methionine, which may be associated with the decrease in hepatic polyamines, such as spermidine and spermine. In our previous study, non-obese type 2 diabetic Goto–Kakizaki rats exhibited the increased hepatic methionine, cysteine, GSH, hypotaurine and taurine levels and the decreased hepatic SAM levels [8]. Homocysteine levels in liver and plasma were comparable between normal and Goto–Kakizaki rats. These results in conjunction with the present results suggest that hepatic sulfur amino acid metabolism may be differentially regulated depending on the types of experimental diabetic conditions. Neither insulin nor triglyceride in blood was increased in Goto–Kakizaki rats, which is sharply different from ZDF rats. The difference in regulation of hepatic sulfur amino acid metabolism between Goto–Kakizaki rats and ZDF rats may reflect the differences associated with lipid metabolism, insulin level and leptin signaling. In conclusion, the methionine loading test results showed that the AUC of homocysteine, methionine and methionine-d4 in plasma did not significantly differ between control and ZDF rats, despite the decrease in methionine, homocysteine, and cysteine in plasma and liver of ZDF rats. These results suggest that the DAUC of plasma homocysteine is not responsive to hepatic BHMT upregulation in ZDF rats; however, these data do not rule out a possible role of BHMT in the decreased homocysteine levels observed in ZDF rats. The present study suggests that decreased hepatic methionine may be responsible for the decreases in its metabolites, such as homocysteine, cysteine, and taurine, in liver, and consequently decreased plasma homocysteine levels. Acknowledgments This work was supported by the Priority Research Centers Program (2009-0093815) through the Research Foundation of Korea (NRF) grant, funded by the Korean Government (MEST).

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Please cite this article in press as: Kwak HC, et al. Sulfur amino acid metabolism in Zucker diabetic fatty rats. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.05.014