Plasma Homocysteine Is Regulated by Phospholipid Methylation

Mild hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine, a non-protein amino acid, is formed fromS-adenosylhomocysteine and partially secreted into plasma. A potential source for homocysteine is methylation of the lipid phosphatidylethanolamine to phosphatidylcholine by phosphatidylethanolamine N-methyltransferase in the liver. We show that mice that lack phosphatidylethanolamineN-methyltransferase have plasma levels of homocysteine that are ∼50% of those in wild-type mice. Hepatocytes isolated from methyltransferase-deficient mice secrete ∼50% less homocysteine. Rat hepatoma cells transfected with phosphatidylethanolamineN-methyltransferase secrete more homocysteine than wild-type cells. Thus, phosphatidylethanolamineN-methyltransferase is an important source of plasma homocysteine and a potential therapeutic target for hyperhomocysteinemia. Mild hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine, a non-protein amino acid, is formed fromS-adenosylhomocysteine and partially secreted into plasma. A potential source for homocysteine is methylation of the lipid phosphatidylethanolamine to phosphatidylcholine by phosphatidylethanolamine N-methyltransferase in the liver. We show that mice that lack phosphatidylethanolamineN-methyltransferase have plasma levels of homocysteine that are ∼50% of those in wild-type mice. Hepatocytes isolated from methyltransferase-deficient mice secrete ∼50% less homocysteine. Rat hepatoma cells transfected with phosphatidylethanolamineN-methyltransferase secrete more homocysteine than wild-type cells. Thus, phosphatidylethanolamineN-methyltransferase is an important source of plasma homocysteine and a potential therapeutic target for hyperhomocysteinemia. Mild hyperhomocysteinemia is an independent risk factor for cardiovascular (1Robinson K. Carmel R. Jacobson D.W. Homocysteine in Health and Disease. Cambridge University Press, Cambridge, UK2001: 371-383Google Scholar, 2Refsum H. Ueland P.M. Nygård O. Vollset S.E. Annu. Rev. Med. 1998; 49: 31-62Google Scholar) and atherosclerotic disease (3Boushey C.J. Beresford S.A.A. Omenn G.S. Motulsky A.G. J. Am. Med. Assoc. 1995; 274: 1049-1057Google Scholar). Total plasma homocysteine (Hcy) 1The abbreviations used are: Hcy, homocysteine; AdoHcy, adenosylhomocysteine; AdoMet, S-adenosylmethionine; DMEM, Dulbecco's modified Eagle's medium; PC, phosphatidylcholine; PEMT, phosphatidylethanolamineN-methyltransferase; ANOVA, analysis of variance 1The abbreviations used are: Hcy, homocysteine; AdoHcy, adenosylhomocysteine; AdoMet, S-adenosylmethionine; DMEM, Dulbecco's modified Eagle's medium; PC, phosphatidylcholine; PEMT, phosphatidylethanolamineN-methyltransferase; ANOVA, analysis of variance values of ∼10 μm for men and ∼8 μm for women are in the normal range. However, even a small increase (∼5 μm) in total plasma Hcy is associated with a 60% increased risk of coronary artery disease for men and 80% for women (3Boushey C.J. Beresford S.A.A. Omenn G.S. Motulsky A.G. J. Am. Med. Assoc. 1995; 274: 1049-1057Google Scholar). Moreover, a number of studies have demonstrated that smoking, excessive drinking of alcohol, obesity, type II diabetes, and an unhealthy diet contribute to mild hyperhomocysteinemia (1Robinson K. Carmel R. Jacobson D.W. Homocysteine in Health and Disease. Cambridge University Press, Cambridge, UK2001: 371-383Google Scholar, 2Refsum H. Ueland P.M. Nygård O. Vollset S.E. Annu. Rev. Med. 1998; 49: 31-62Google Scholar). In addition, elevated plasma Hcy has recently been linked to Alzheimer's disease and cognitive impairment in the elderly (4Clarke R. Smith A.D. Jobst K.A. Refsum H. Sutton L. Ueland P.M. Arch. Neurol. 1998; 55: 1449-1455Google Scholar, 5Selhub J. Bagely L.C. Miller J. Rosenberg I.H. Am. J. Clin. Nutr. 2000; 71: S614-S620Google Scholar). Hcy is a non-protein amino acid derived from the catabolism ofS-adenosylhomocysteine (AdoHcy), an immediate product of trans-methylation reactions that utilizeS-adenosylmethionine (AdoMet) (6Clarke S. Banfield K. Carmel R. Jacobson D.W. Homocysteine in Health and Disease. Cambridge University Press, Cambridge, UK2001: 63-78Google Scholar). Hcy has three possible fates: 1) methylation to methionine withN-5-methyltetrahydrofolate or betaine as the methyl donor, 2) conversion to cysteine via the trans-sulfuration pathway, and 3) release into extracellular fluids (e.g. plasma and urine). AdoMet-dependent methyltransferases catalyze many critical reactions including methylation of RNA, DNA, proteins, and small molecules such as guanidinoacetate and glycine (6Clarke S. Banfield K. Carmel R. Jacobson D.W. Homocysteine in Health and Disease. Cambridge University Press, Cambridge, UK2001: 63-78Google Scholar). The potential of methyltransferases to regulate plasma Hcy is not well defined. Phosphatidylethanolamine N-methyltransferase (PEMT) is a liver-specific enzyme that generates AdoHcy during the conversion of one membrane lipid, phosphatidylethanolamine, into another membrane lipid, phosphatidylcholine (PC) (7Vance D.E. Ridgway N.D. Prog. Lipid. Res. 1988; 27: 61-79Google Scholar). PEMT accounts for the formation of ∼30% PC made in liver (8DeLong C.J. Shen Y.-J. Thomas M.J. Cui Z. J. Biol. Chem. 1999; 274: 29683-29688Google Scholar, 9Reo N.V. Adinehzadeh M. Foy B.D. Biochim. Biophys. Acta. 2002; 1580: 171-188Google Scholar). With this large capacity for PC synthesis and the generation of three AdoHcy molecules for each PC molecule synthesized, we hypothesized that the PEMT reaction might contribute significantly to Hcy in plasma. We have utilized thePemt −/− mouse, hepatocytes derived from these mice, and overexpression of PEMT in McArdle RH7777 (rat hepatoma) cells to test this hypothesis. The results show that PEMT expression enhances plasma Hcy levels and the secretion of Hcy from hepatocytes. For the high fat/high cholesterol diet, a semi-purified diet lacking a fat source was purchased from Teklad (catalog number 84712, Madison, WI) and supplemented with 19% (w/w) olive oil, 1% (w/w) linseed oil (a source of essential fatty acids), and 1% (w/w) cholesterol. Rodent chow was from LabDiet (PICO Laboratory Rodent Diet 20). Hanks' balanced salt solution, Dulbecco's Modified Eagle's medium (DMEM), and fetal bovine serum were from Invitrogen. All other chemicals were from Sigma unless noted otherwise. The Pemt −/− mouse colony had a mixed genetic background of 129/J and C57BL/6 mice and was maintained by homozygous breeding in a reversed 12-h light/dark cycle. At the age of 12–14 weeks, Pemt −/− andPemt +/+ mice were fed ad libitum either chow or the high fat/high cholesterol diet for 3 weeks (10Cheema S.K. Cikaluk D. Agellon L.B. J. Lipid Res. 1997; 38: 157-165Google Scholar). MalePemt −/− and Pemt +/+ mice (12–20-weeks old) that were fed chow were used. Primary hepatocytes were isolated by collagenase perfusion (11Davis R.A. Engelhorn S.C. Pangburn S.H. Weinstein D.B. Steinberg D. J. Biol. Chem. 1979; 266: 497-509Google Scholar) and plated on 60-mm collagen-coated dishes at a density of 2.0 × 106cells/dish in DMEM containing 17% fetal bovine serum and 0.01 mg/ml insulin. After 7.5 h, the primary cultures were rinsed twice in serum-free DMEM over a 1-h period and then incubated in serum-free DMEM for 12 h ± 200 μm guanidinoacetate. Medium and cells were collected and frozen at −70 °C until analysis. The cells were re-suspended and sonicated in homogenization buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, and 0.1 mm phenylmethylsulfonylfluoride). Protein analysis was performed with the Coomassie Blue Plus protocol from Bio-Rad. McArdle RH-7777 rat hepatoma cells stably expressing human PEMT cDNA or vector alone (pCI, Promega) were maintained in DMEM containing 20% fetal bovine serum, 200 μg/ml gentamycin, and 100 μm ethanolamine. The ethanolamine was added to ensure that there was sufficient substrate for phosphatidylethanolamine biosynthesis so that this lipid would not be limiting for phospholipid methylation. Following a 24-h incubation period, the medium was removed, centrifuged to remove cell debris, and frozen until analysis. Livers were removed from mice and flash-frozen. The protein content of the liver samples was determined by the Biuret method (13Gornall A.G. Bardawill C.J. David M.M. J. Biol. Chem. 1949; 177: 751-766Google Scholar). Thawed liver samples were homogenized in 50 mmphosphate-buffered saline (pH 6.9). The homogenate was centrifuged at 18,000 × g for 30 min at 4 °C, and the following enzyme activities were measured in the supernatant: cystathionine β-synthase (14Mudd S.H. Finkelstein J.D. Irreverre F. Laster L. J. Biol. Chem. 1965; 240: 4382-4392Google Scholar, 15Taoka S. Ohja S. Shan X. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Google Scholar), AdoMet synthase (15Taoka S. Ohja S. Shan X. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Google Scholar), methionine synthase (16Koblin D.D. Watson J.E. Deady J.E. Stokstad E.L.R. Eger E.I. Anesthesiology. 1981; 54: 318-324Google Scholar), 5,10-methylenetetrahydrofolate reductase (17Engbersen A.M.T. Franken D.G. Boers G.H.J. Trijbels E.M.B. Blom H.J. Am. J. Hum. Genet. 1994; 56: 142-150Google Scholar), and betaine:homocysteine methyltransferase (18Wang J.A. Dudman N.P. Lynch J. Wilcken D.E. Clin. Chem. Acta. 1991; 204: 239-249Google Scholar). The final product of betaine:homocysteine methyltransferase, methionine, was measured by reverse-phase high performance liquid chromatography. Total Hcy content in plasma and media was measured by reverse-phase high performance liquid chromatography and fluorescence detection of ammonium 7-fluoro 2-oxa-1,3-diazole-4-sulfonate thiol adducts (12Vester B. Rasmussen K. Eur. J. Clin. Chem. Clin. Biochem. 1991; 29: 549-554Google Scholar). The amounts of methionine, total cysteine, serine, and glycine in media were measured in samples that had been deproteinized by treatment with 10% sulfosalicylic acid. The protein was removed by centrifugation, and the pH of the supernatant was adjusted to 2.2. The amino acids were analyzed on a Beckman 121 MB amino acid analyzer using Bensond-X 0.25 Cation Xchange Resin according to Beckman 121MB-TB-O17 application notes and quantitated using a Hewlett Packard Computing Integrator Model 3395A following post-column derivatization with ninhydrin. PEMT activity was measured as described previously (19Cui Z. Vance J.E. Chen M.H. Voelker D.R. Vance D.E. J. Biol. Chem. 1993; 268: 16655-16663Google Scholar). Female and male Pemt +/+ andPemt −/− mice were fed chow or a high fat/cholesterol diet for 3 weeks. Fig. 1shows that the plasma content of Hcy in Pemt −/−mice was ∼50% less than in Pemt +/+ mice. Neither gender nor diet influenced the levels of plasma Hcy. To determine whether alterations of activities of other key hepatic enzymes of homocysteine production and removal might have caused the decreased plasma Hcy levels in Pemt −/− mice, we assayed five enzymes listed in TableI. The activities were not statistically different between Pemt −/− andPemt +/+ mice. Hence, the 50% lower level of plasma Hcy in the Pemt −/− mice is probably because of the absence of PEMT activity in the liver.Table ISpecific activities of enzymes involved in methionine and homocysteine metabolismEnzymePemt −/−Pemt +/+nmol/min/mg proteinCystathionine β-synthaseMales11.1 ± 2.411.7 ± 2.4Females13.5 ± 2.713.4 ± 1.2AdoMet synthaseMales1.0 ± 0.11.0 ± 0.3Females0.92 ± 0.10.78 ± 0.2Methionine synthaseMales0.063 ± 0.0040.054 ± 0.015Females0.026 ± 0.0050.029 ± 0.002Methylenetetrahydrofolate reductaseMales0.027 ± 0.0060.031 ± 0.004Females0.037 ± 0.0080.033 ± 0.008Betaine:Hcy methyltransferaseMales3.7 ± 0.43.9 ± 0.3Females3.8 ± 0.33.6 ± 0.6The results are mean values ± S.D. for 3–5 animals that were fed a chow diet. Similar values were obtained from mice that were fed a high fat/high cholesterol diet. Differences betweenPemt +/+ and Pemt −/− mice were analyzed using ANOVA followed by Newman-Keuls test, but no significant differences were found. Open table in a new tab The results are mean values ± S.D. for 3–5 animals that were fed a chow diet. Similar values were obtained from mice that were fed a high fat/high cholesterol diet. Differences betweenPemt +/+ and Pemt −/− mice were analyzed using ANOVA followed by Newman-Keuls test, but no significant differences were found. We measured the secretion of Hcy from primary hepatocytes isolated from Pemt +/+ andPemt −/− mice. Fig. 2shows that hepatocytes from Pemt −/− mice secrete ∼50% less Hcy than hepatocytes from Pemt +/+ mice. It was conceivable that the depletion of intracellular AdoMet decreased Hcy secretion from Pemt −/− hepatocytes. To test this possibility, we performed a parallel experiment in which hepatocytes were incubated with guanidinoacetate. Methylation of guanidinoacetate to form creatine is an important contributor to the exported Hcy pool (20Stead L.M. Au K.P. Jacobs R.L. Brosnan M.E. Brosnan J.T. Am. J. Physiol. 2001; 281: E1095-E1100Google Scholar). Therefore, if AdoMet was sufficient in the hepatocytes from Pemt −/− mice, we should observe enhanced Hcy secretion. Fig. 2 indicates that guanidinoacetate-stimulated Hcy secretion from bothPemt +/+ and Pemt −/− hepatocytes to a similar degree. Thus, apparently a lack of AdoMet seems unlikely. These results concur with previous measurements of the levels of AdoMet (∼130 pmol/mg liver) and AdoHcy (∼40 pmol/mg liver) that were similar in the livers of Pemt +/+ andPemt −/− mice (21Walkey C.J. Importance of Phospholipid Methylation.Ph.D. Thesis. University of Alberta, Edmonton, Alberta, Canada1997Google Scholar). We also established at the end of the incubation period that methionine, cysteine, serine, and glycine had not been depleted from the medium and therefore were not limiting Hcy secretion. Thus, AdoMet was not limiting for Hcy formation, and the decreased secretion of Hcy from Pemt −/−hepatocytes was attributed to the lack of PEMT. Plasma homocysteine is also increased in rats that were provided with dietary guanidinoacetate as is the production of homocysteine by hepatocytes incubated with guanidinoacetate (20Stead L.M. Au K.P. Jacobs R.L. Brosnan M.E. Brosnan J.T. Am. J. Physiol. 2001; 281: E1095-E1100Google Scholar). These results demonstrate that altered flux through another major methyltransferase can also affect homocysteine metabolism. McArdle RH-7777 hepatoma cells have negligible PEMT activity (19Cui Z. Vance J.E. Chen M.H. Voelker D.R. Vance D.E. J. Biol. Chem. 1993; 268: 16655-16663Google Scholar). We reasoned that if Hcy secretion were dependent upon PEMT activity, stable expression of PEMT in these cells would stimulate Hcy secretion. Fig. 3 confirms the low PEMT activity in parental McArdle cells and indicates that the secretion of Hcy from cells that expressed human PEMT was enhanced. We also demonstrated that methionine, cysteine, serine, and glycine had not been depleted from the medium at the end of the incubation period. If ∼50% plasma Hcy was derived from the PEMT reaction as suggested by our results, PEMT must generate significant amounts of AdoHcy in the liver. A 20-g mouse secretes ∼30 μmol (23 mg) PC into bile each day (22Walkey C.J. Yu L. Agellon L.B. Vance D.E. J. Biol. Chem. 1998; 273: 27043-27046Google Scholar), and the PEMT pathway is an important source for PC in the bile (23Agellon L.B. Walkey C.J. Vance D.E. Kuipers F. Verkade H.J. Hepatology. 1999; 30: 725-729Google Scholar). Approximately, one-third of the PC in murine liver is derived from the PEMT pathway (8DeLong C.J. Shen Y.-J. Thomas M.J. Cui Z. J. Biol. Chem. 1999; 274: 29683-29688Google Scholar, 9Reo N.V. Adinehzadeh M. Foy B.D. Biochim. Biophys. Acta. 2002; 1580: 171-188Google Scholar). Therefore, ∼10 μmol biliary PC should be produced via PEMT in 24 h. Each phosphatidylethanolamine molecule methylated to PC produces three molecules of AdoHcy. Hence, to satisfy the export of PC into bile, the murine liver produces ∼30 μmol AdoHcy in 24 h from the PEMT reaction. This estimate does not take into account the presumed sizeable requirement of PC biosynthesis for hepatocyte membranes or for export with lipoproteins. From Fig. 2, we estimated the amount of Hcy secreted from 1 g of liver over a 24-h period. Murine hepatocytes secreted 15 nmol Hcy/mg protein over a 12-h period. Assuming that a 1-g liver yields 175 mg of protein (24Vance J.E. Vance D.E. J. Biol. Chem. 1988; 263: 5898-5909Google Scholar), a 1-g liver would produce 5.25 μmol Hcy/day. Although these calculations are approximations, it is clear that hepatic PEMT generates significantly more AdoHcy (>30 μmol) than Hcy (∼5 μmol) secreted from the liver each day. In addition to defining a role for PEMT in the regulation of plasma Hcy, the work also argues for a primary role of the liver in producing Hcy. All of the nucleated cells have the capacity to generate AdoHcy and probably Hcy; hence, it is difficult to determine the quantitative source of plasma Hcy. Because PEMT is quantitatively abundant only in the liver (7Vance D.E. Ridgway N.D. Prog. Lipid. Res. 1988; 27: 61-79Google Scholar), our data demonstrate that alterations in plasma Hcy in mice are largely mediated by the liver. Moreover, the hepatic methylation of guanidinoacetate to creatine to replace urinary creatinine loss (estimated at 3–15 μmol/day in mice (25Loo G. Goodman P.J. Hill K.A. Smith J.T. J. Nutr. 1986; 116: 2403-2408Google Scholar, 26Murray C.E. Warnes D.M. Ballard F.J. Tomas F.M. Clin. Sci. 1981; 61: 737-741Google Scholar)) is also considered a source of plasma Hcy. Much of what is known concerning AdoMet consumption in trans-methylation reactions is derived from the elegant studies of Muddet al. (27Mudd S.H. Poole J.R. Metabolism. 1975; 24: 721-735Google Scholar, 28Mudd S.H. Ebert M.H. Scriver C.R. Metabolism. 1980; 29: 707-720Google Scholar) on the balance of methyl groups in humans. These experiments estimated that creatine synthesis in the liver consumes ∼75% of available AdoMet. Of the remaining 25% AdoMet, ∼15% was estimated to be used for PC synthesis from phosphatidylethanolamine and the remaining ∼10% was estimated to be used for other trans-methylations and polyamine synthesis. In those studies, the measurement of AdoMet consumption via PEMT involved the oxidation of choline to sarcosine and the recovery of this compound in urine. When sarcosine was administered to a patient deficient in sarcosine dehydrogenase, only 60–80% was recovered in urine. Therefore, the recovery of sarcosine in the urine in these calculations underestimated its formation and, thus, the contribution of the PEMT reaction. Moreover, because many destinations require PEMT-derived PC (e.g. cellular membranes, bile secretion, lipoprotein secretion, sphingomyelin synthesis), the measurement of the oxidation of choline to sarcosine significantly underestimates the consumption of AdoMet in the PEMT reaction. Our results suggest that PC synthesis from phosphatidylethanolamine consumes substantially more AdoMet than was previously thought. Mudd et al. (27Mudd S.H. Poole J.R. Metabolism. 1975; 24: 721-735Google Scholar, 28Mudd S.H. Ebert M.H. Scriver C.R. Metabolism. 1980; 29: 707-720Google Scholar) note that their estimates must be revised as more data become available. It appears that such revision is now appropriate. The results demonstrate that the PEMT reaction in liver is a major source of plasma Hcy, an independent risk factor for cardiovascular disease. Several studies (29Tawakol A. Omland T. Gerhard M. Wu J.T. Creager M.A. Circulation. 1997; 95: Scholar, Circulation. 2000; Scholar) have demonstrated that hyperhomocysteinemia is to normal elevated Hcy levels stimulate cell and as well as the of to atherosclerotic J. 1997; Scholar, A. Biol. 1997; Scholar, R. P.M. K. D.W. Circulation. 2001; Scholar, A. 2001; Scholar). Although Hcy enhances the of atherosclerotic it has not been demonstrated that Hcy is a of in with mild hyperhomocysteinemia but other cardiovascular disease risk were to be at no risk for the disease with F.J. Blom H.J. Refsum H. Ueland P.M. 1997; Scholar). cystathionine β-synthase nor mice until even the mice had M. J. K. R. Sci. S. A. 1995; Scholar). of whether or not mild hyperhomocysteinemia or with the of cardiovascular it is a of in Z. S. S. Chen A. Smith T. Bagely J. M.A. R. Hum. Genet. 2001; Scholar). It is now that the PEMT reaction in the liver is a major source of plasma Hcy. A previous Vance D.E. J. Biol. Chem. 2002; Scholar) with hepatocytes derived −/− mice that PEMT is also important in regulation of the secretion of high levels of which to cardiovascular disease. Therefore, on the role of PEMT in the generation of plasma Hcy to a of the possible between mild hyperhomocysteinemia and other cardiovascular risk We and for and David and Vance for Plasma homocysteine is by phospholipid of Fig. The on the should have Hcy The is Open

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