Propargylglycine Inhibits Hypotaurine/Taurine Synthesis and Elevates Cystathionine and Homocysteine Concentrations in Primary Mouse Hepatocytes
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CORE Metadata, citation and similar papers at core.ac.uk Provided by Springer - Publisher Connector Amino Acids (2015) 47:1215–1223 DOI 10.1007/s00726-015-1948-7 ORIGINAL ARTICLE Propargylglycine inhibits hypotaurine/taurine synthesis and elevates cystathionine and homocysteine concentrations in primary mouse hepatocytes Halina Jurkowska · Martha H. Stipanuk · Lawrence L. Hirschberger · Heather B. Roman Received: 29 December 2014 / Accepted: 18 February 2015 / Published online: 13 March 2015 © The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Our investigation showed that hepatocytes iso- methionine resulted in higher intracellular concentrations / lated from cysteine dioxygenase knockout mice (Cdo1− −) of homocysteine, which is a cosubstrate for cystathionine / had lower levels of hypotaurine and taurine than Cdo1+ + β-synthase (CBS). It seems that PPG increases CBS-medi- hepatocytes. Interestingly, hypotaurine accumulates in cul- ated desulfhydration by enhancing homocysteine levels in tured wild-type hepatocytes. DL-propargylglycine (PPG, hepatocytes. There were no overall effects of PPG or geno- inhibitor of cystathionine γ-lyase and H2S production) dra- type on intracellular or medium glutathione levels. matically decreased both taurine and hypotaurine levels in wild-type hepatocytes compared to untreated cells. Addi- Keywords Cysteine dioxygenase · Taurine · tion of 2 mM PPG resulted in the decrease of the intracel- Hypotaurine · Cystathionine · Homocysteine · Cysteine lular taurine levels: from 10.25 5.00 observed in control, desulfuration ± to 2.53 0.68 nmol/mg protein (24 h of culture) and from ± 17.06 9.40 to 2.43 0.26 nmol/mg protein (control vs. ± ± PPG; 48 h). Addition of PPG reduced also intracellular Introduction hypotaurine levels: from 7.46 3.55 to 0.31 0.12 nmol/ ± ± mg protein (control vs. PPG; 24 h) and from 4.54 3.20 In mammals, cysteine is catabolized by several nonoxida- ± to 0.42 0.11 nmol/mg protein (control vs. PPG; 48 h). tive cysteinesulfinate-independent desulfuration pathways ± The similar effects of PPG on hypotaurine and taurine lev- as well as by oxidative cysteinesulfinate-dependent path- els were observed in culture medium. PPG blocked hypo- ways or it may be used for protein and glutathione synthe- taurine/taurine synthesis in wild-type hepatocytes, suggest- sis (Fig. 1). Cysteine is catabolized by several desulfuration ing that it strongly inhibits cysteinesulfinate decarboxylase reactions that release sulfur in a reduced oxidation state, (pyridoxal 5′-phosphate-dependent enzyme) as well as generating sulfane sulfur or hydrogen sulfide (H2S), which cystathionine γ-lyase. In the presence of PPG, intracellu- can be further oxidized to sulfate. Cysteine desulfuration is lar and medium cystathionine levels for both wild-type and accomplished by alternate reactions catalyzed by cystathio- / Cdo1− − cells were increased. Addition of homocysteine or nine β-synthase (CBS), cystathionine γ-lyase (CTH), and 3-mercaptopyruvate sulfurtransferase (MPST) (Fig. 1). The oxidative pathway leads to production of taurine or sulfate Handling Editor: H. Jakubowski. (Stipanuk and Ueki 2011). Taurine (2-aminoethanesulfonic acid) is synthesized H. Jurkowska · M. H. Stipanuk · L. L. Hirschberger · endogenously from cysteine or via conversion from H. B. Roman Division of Nutritional Sciences, Cornell University, methionine, but also provided by diet, especially fish and Ithaca, NY 14853, USA seafood. The main pathway of taurine synthesis in mam- mals involves the oxidation of L-cysteine by the cysteine * H. Jurkowska ( ) dioxygenase (CDO) to produce cysteine sulfinic acid. Chair of Medical Biochemistry, Jagiellonian University Medical College, Kopernika 7 St., 31‑034 Kraków, Poland This acid is decarboxylated to hypotaurine by the cysteine e-mail: [email protected] sulfinic acid decarboxylase, which is finally oxidized to 1 3 1216 H. Jurkowska et al. Fig. 1 The production of taurine and desulfhydration Methionine pathways of cysteine L-Homocysteine Serine Cystathionine β-synthase L-Cystathionine Cystathionine γ-lyase Protein L-Cysteine Glutathione Cysteine dioksygenase Cysteine aminotransferase Cystathionine Cystathionine γ-lyase 3-mercaptopyruvate Cysteinesulfinate β-synthase sulfurtransferase Aspartate Cysteinesulfinate aminotransferase H S decarboxylase 2 2- Hypotaurine S03 Sulfidequinone reductase Sulfidedioxygenase Hypotaurine Sulfite dehydrogenase oxidase Sulfur transferase 2- 2- 2- Taurine S04 S03 SS03 taurine by hypotaurine dehydrogenase. The conversion of restricted by the low availability of cysteinesulfinate as the cysteinesulfinate to hypotaurine/taurine is minimized substrate for cysteinesulfinate decarboxylase, and taurine by the active transaminative catabolism of cysteinesulfinate production is increased when cysteinesulfinate increases in to pyruvate and inorganic sulfur (Rosa et al. 2014; Stipanuk response to an increase in the hepatic cysteine concentra- 2004; Hayes and Sturman 1981). tion and the associated increase in cysteine dioxygenase Taurine is the most abundant (~millimolar concentra- activity (Stipanuk 2004). tion) free amino acid that is involved in many fundamental Homocysteine and taurine are both sulfur-containing biological functions (Imae et al. 2014; Hansen et al. 2006; amino acids sharing the same biosynthetic pathway. As an Miyazaki and Matsuzaki 2014). In liver, the most estab- end product of the transsulfuration pathway in homocyst- lished role of taurine is its conjugation with bile acids for eine metabolism, taurine supplementation may play a cru- excretion into bile (Danielsson 1963; Sjovall 1959). How- cial role in methionine metabolism in liver (Deminice et al. ever, taurine has many other physiological and pharmaco- 2014). logical roles in liver and other tissues, including stabiliza- In this study, determination of taurine and hypotaurine tion of cellular plasma membrane (Pasantes et al. 1985), levels in the primary cultures of hepatocytes partially con- 2 Ca + transport regulation (Huxtable 1992; Schaffer et al. firmed previous results from the experiments performed on 2010), osmoregulation (Nieminen et al. 1988; Huxtable mice (Roman et al. 2013; Ueki et al. 2011), and added new 1992; Schaffer et al. 2010), antioxidant effects (Huxtable insights to our understanding of metabolism of the studied / 1992; Nakamura et al. 1993; Schaffer et al. 2010; Rosa metabolites. Taurine level in Cdo1− − hepatocytes is low, / et al. 2014), anti-inflammation (Rosa et al. 2014), and as was observed previously in the liver of Cdo1− − mice. detoxification (Huxtable1992 ). Taurine supplementation Taurine levels in the cultured hepatocytes isolated from / has shown protective effects—such as antioxidant proper- Cdo1− − mice ranged from 4.6 to 6.2 % of the correspond- ties—to improve insulin sensitivity and dyslipidemia, as ing wild-type levels (this study), whereas taurine levels in / well as fatty liver disease, in rat models of obesity, diabe- the liver of Cdo1− − mice fed on taurine-free diets were tes type 2, and steatohepatitis (Deminice et al. 2014; Imae found to be consistently less than 5 % of the wild-type lev- et al. 2014; Balkan et al. 2002; Rosa et al. 2014; Xiao et al. els (previously published study). Hypotaurine level is neg- / / 2008). ligible in Cdo1− − hepatocytes as in the liver of Cdo1− − In liver, taurine is abundantly maintained by endogenous mice, reflecting the block in conversion of cysteine to biosynthesis and exogenous transport systems (Miyazaki cysteinesulfinate which is a precursor in the process of and Matsuzaki 2014). Hepatic taurine synthesis is largely hypotaurine/taurine synthesis. 1 3 Effect of propargylglycine on taurine synthesis and cystathionine and homocysteine levels in hepatocytes 1217 Interestingly, we have found in this study that signifi- Sigma–Aldrich). 2 mM DL-propargylglycine (PPG, Sigma– cant differences in hypotaurine and taurine levels in wild- Aldrich), 0.2 mM L-homocysteine (Hcy, Sigma–Aldrich), type hepatocytes compared to liver of wild-type mice were or 0.5 mM L-methionine (Met, Research Products Interna- shown. We have shown that hypotaurine accumulates in tional, Inc) was added as indicated. cultured wild-type hepatocytes. Hepatocytes were cultured in the treatment medium for In the present study, we have also investigated the effect 24 and 48 h prior to harvest. For measurement of metabo- of propargylglycine (PPG; inhibitor of gamma-cystathion- lite levels in the medium, the medium was removed prior ase) on hepatic hypotaurine and taurine synthesis and the to harvesting the hepatocytes and immediately frozen and levels of cystathionine, total unbound homocysteine, and stored at 80 °C for later analysis. For measurement of − glutathione in hepatocytes isolated from wild-type and metabolite levels in cells, hepatocytes were harvested by Cdo1 knockout male mice. Interestingly, inhibitory effects treating with 0.25 % trypsin (Gibco #25200), washing with of PPG on hypotaurine/taurine synthesis were found in ice-cold PBS to remove trypsin, suspending released hepat- wild-type hepatocytes. In addition, this study proved that ocytes in ice-cold PBS, and centrifuging the cell suspen- PPG, as well as homocysteine and methionine, increase sion at 1600 g for 10 min at 4 °C to obtain the pelleted × cystathionine and homocysteine levels in the cells and in cells, which were immediately frozen and stored at 80 °C − the conditional medium, which might result in an intensifi- for later analysis.