Corticoadrenal activity in rat regulates betaine-homocysteine S-methyltransferase expression with opposite effects in liver and kidney 1,2,3, 1 2 1 OSVALDO FRIDMAN *, ANALÍA VMORALES , LAURA EBORTONI , PAULA CTURK-NOCETO and ELIO APRIETO1 11Centro de Altos Estudios en Ciencias Humanas y de la Salud, Universidad Abierta Interamericana (UAI), 2Instituto de Oncología Ángel H Roffo, 3Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, ArgentinaBuenos Aires, Argentina *Corresponding author (Email, [email protected]) Betaine-homocysteine S-methyltransferase (BHMT) is an enzyme that converts homocysteine (Hcy) to methionine using betaine as a methyl donor. Betaine also acts as osmolyte in kidney medulla, protecting cells from high extracellular osmolarity. Hepatic BHMT expression is regulated by salt intake. Hormones, particularly cortico- steroids, also regulate BHMT expression in rat liver. We investigated to know whether the corticoadrenal activity plays a role in kidney BHMT expression. BHMT activity in rat kidneys is several orders of magnitude lower than in rat livers and only restricted to the renal cortex. This study confirms that corticosteroids stimulate BHMT activity in the liver and, for the first time in an animal model, also up-regulate the BHMT gene expression. Besides, unlike the liver, corticosteroids in rat kidney down-regulate BHMT expression and activity. Given that the classical effect of adrenocortical activity on the kidney is associated with sodium and water re-absorption by the distal tubule leading to volume expansion, by promoting lesser use of betaine as a methyl donor, corticosteroids would preserve betaine for its other role as osmoprotectant against changes in the extracellular osmotic conditions. We conclude that corticosteroids are, at least in part, responsible for the inhibition of BHMT expression and activity in rat kidneys. [Fridman O, Morales AV, Bortoni LE, Turk-Noceto PC and Prieto EA 2012 Corticoadrenal activity in rat regulates betaine-homocysteine S-methyltransferase expression with opposite effects in liver and kidney. J. Biosci. 37 115–123] DOI 10.1007/s12038-011-9171-5 1. Introduction condensed with serine to form cystathionine. This latter reaction is catalysed by cystathionine β-synthase. Remethy- Homocysteine (Hcy), a sulphur-containing non-protein lation of Hcy to methionine occurs through two separate amino acid, is an intermediary in the metabolism of paths, one is catalysed by 5-methyltetrahydrofolate- methionine. The elevation of plasma Hcy levels, known as homocysteine methyltransferase (methionine synthase) and hyperhomocysteinemia, is a recognized risk factor for the other by betaine-homocysteine methyltransferase coronary and cerebrovascular atherosclerosis and venous (BHMT; E.C. 2.1.1.5). The first enzyme, widely distributed thrombosis (Adachi et al. 2002; Albert et al. 2002; in animal tissues, requires as a cofactor and methyl donor Sundström et al. 2003). Methionine is incorporated with 5′-methyltetrahydrofolate and vitamin B12 respectively. On the diet and is S-adenosylated, leading to S-adenosylme- the other hand, BHMT, the only known enzyme that uses thionine, which can transfer its methyl group to cellular betaine as a substrate, mediates the transfer of a methyl acceptors, becoming S-adenosyl homocysteine. S-adenosyl group from betaine (N,N,N-trimethylglycine) to Hcy, form- homocysteine is then converted in homocysteine. The ing methionine and N,N-dimethylglycine (Sunden et al. intracellular Hcy may be remethylated to methionine or 1997; Millian and Garrow 1998). Methionine synthase is Keywords. Betaine; homocysteine; methylation; osmolyte http://www.ias.ac.in/jbiosci J. Biosci. 37(1), March 2012, 115–123, * Indian Academy of Sciences 115 Published online: 30 December 2011 116 Osvaldo Fridman et al. ubiquitously expressed (Chen et al. 1997) but the distribu- Garrow 2005; Schäfer et al. 2007) and also various tion of BHMT is more restricted (Delgado-Reyes et al. hormones, including corticosteroids, insulin, estradiol and 2001). BHMT has been shown to be expressed at high testosterone (Finkelstein et al. 1971;Ratnamet al. 2006). In levels in the livers of all vertebrate species tested. In the rat hepatoma cell line (H4IIE cells), the synthetic mammalian liver, BHMT represents 1% or more of the total glucocorticoid triamcinolone increases the level and rate of soluble protein (Garrow 1996). In addition to being present BHMT mRNA synthesis (Ratnam et al. 2006). in the liver, BHMT activity also is present at significant In the present study, we investigated the role of the levels in the pancreas of ruminants and guinea pigs, and in corticoadrenal activity to regulate BHMT in the liver and the kidney cortex of guinea pigs, pigs and primates kidney of an in vivo rat model. Our aim was to determine (Delgado-Reyes et al. 2001; Delgado-Reyes and Garrow whether corticosteroids are involved in the regulation of 2005). In humans, pigs and guinea pigs, BHMT has been kidney BHMT expression. specifically localized to the proximal tubules of kidney. In contrast, BHMT activity in rat kidney has been reported to 2. Material and methods be either absent (McKeever et al. 1991) or very low (Finkelstein et al. 1971). Delgado-Reyes et al. (2001) and 2.1 Chemicals and reagents Delgado-Reyes and Garrow (2005) found that whole rat kidney homogenates have about 1–2% of the BHMT- Betaine hydrochloride, L-Hcy thiolactone hydrochloride, specific activity as that found in liver. Betaine, a body Dowex 1-X4 (OH−; 200–400 mesh) resin, protease inhibitor requirement, is found at high levels in certain foods cocktail and 3,3′-diaminodbenzidine were purchased from (De Zwart et al. 2003; Zeisel et al. 2003) and is absorbed 14 directly from the diet via non-specific transporters in the Sigma-Aldrich. [ C-methyl]-betaine (57 mCi/mmol) was gut. Betaine can also be endogenously produced in the liver obtained from Moravek Biochemicals. Scintillation cocktail and kidney cortex from its metabolic precursor choline Optiphase Hisafe 3 was obtained from Perkin Elmer. Anti- (Zhang et al. 1992; Zeisel and Blusztajn 1994; Fischer et al. BHMT polyclonal antibodies (rabbit) were kindly provided by 2005). In addition to its important role as a methyl donor in Dra. María A Pajares Instituto de Investigaciones Biomédicas one-carbon metabolism, betaine, together with myo-inositol, Alberto Sols (CSIC-UAM), Madrid, Spain. Goat anti-rabbit sorbitol, taurine and glycerophosphorylcholine, plays a vital IgG horseradish peroxidase, peroxidase-labelled goat anti- function as an organic osmolyte that is accumulated by the rabbit IgG secondary antibody, primers, Taq DNA polymerase cells of the inner medulla of animal and human kidneys to and SYBR Green PCR Master Mix, were supplied by maintain osmotic balance (Bagnasco et al. 1986; Garcia- Promega. All other chemicals were of the highest purity Perez and Burg 1991; Sizeland et al. 1995). Betaine available from commercial sources. regulates cell volume by counteracting changes in extracel- lular tonicity and stabilizing macromolecules against a 2.2 Animals variety of physiological perturbations (Häussinger 1996; Schliess and Häussinger 2002; Wehner et al. 2003). BHMT Adult male Sprague–Dawley rats weighing 210 to 230 g were expression and activity plays a key role in regulating betaine obtained from the Institute of Biology and Experimental concentrations (Finkelstein et al. 1982) and determines the Medicine Animal Facility (NIH Assurance Certificate # fate of betaine between its two divergent biological A5072-01) and were housed under controlled temperature functions: metabolized to provide a methyl group for (22°C) and humidity (50%) with 12 h/12 h light/dark cycles homocysteine methylation or stored to control cellular (lights on at 7:00 a.m.). Rats were fed with free access to osmolarity. In a recent work, Teng et al. (2011) showed that standard chow and tap water (or saline after adrenalectomy) deletion of BHMT in mice resulted in betaine accumulation and randomized into study and control groups. Animals were in most tissues. A number of nutritional factors were shown to play a role in the regulation of methyl group and Hcy 12 weeks old when sacrificed. All animal experiments metabolism by specifically altering the expression of key followed the NIH Guide for the Care and Use of Laboratory enzymes. BHMT activity and gene expression in rat liver are Animals and were approved by the Ethical Committee of the affected by the supply of dietary betaine, choline and Institute of Biology and Experimental Medicine. All efforts methionine (Finkelstein et al. 1982;Park et al. 1997;Park were made to minimize animal suffering and to reduce the and Garrow 1999). However, unlike the rat liver enzyme, rat number of rats used. Rats were euthanized by carbon dioxide kidney BHMT is refractory to treatment with low dietary asphyxiation (Slow and Garrow 2006) according to protocols methionine and choline (Delgado-Reyes et al. 2001;Slow for animal use, in agreement with NIH guidelines for care and Garrow 2006). In addition to diet, BHMT expression in and use of experimental animals and approved by the local liver is influenced by osmotic stress (Delgado-Reyes and ethical committee (IBYME-CONICET). J. Biosci. 37(1), March 2012 Corticosteroids down-regulate BHMT in rat kidney 117 2.3 Hormonal treatment and study procedure β-mercaptoethanol, 1 mg/mL lysozyme and (1 mL/20 g tissue) protease inhibitor cocktail, and centrifuged at 20,000g After 1 week of acclimatization, 20 rats were divided in two at 4°C for 30 min. After being heated to 95°C for 5 min, groups. Under Nembutol anaesthesia (60 mg/kg i.p.), one proteins were subjected to SDS-PAGE (30 and 100 μg group was bilaterally adrenalectomized (ADX) and the other protein/lane for liver and kidney respectively). Following was sham operated as adrenal-intact controls. The day after electrophoresis, gels were equilibrated with transfer buffer operation five rats of each group received i.m. injections of (39 mM glycine, 48 mM Tris-HCl, 0.03% SDS, and 20% methylprednisolone (MP) (Riedel-de Haën, Germany), methanol).