From the Department of Biochemistry and Canadian
Institutes for Health Research Group on the Molecular and Cell Biology
of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada and
the
Department of Biochemistry, Memorial University of
Newfoundland, St. John's, Newfoundland A1B 3X9, Canada
Received for publication, December 2, 2002
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ABSTRACT |
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Mild hyperhomocysteinemia is an
independent risk factor for cardiovascular disease. Homocysteine, a
non-protein amino acid, is formed from
S-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 phosphatidylethanolamine
N-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 phosphatidylethanolamine N-methyltransferase secrete more homocysteine than
wild-type cells. Thus, phosphatidylethanolamine
N-methyltransferase is an important source of plasma
homocysteine and a potential therapeutic target for hyperhomocysteinemia.
Mild hyperhomocysteinemia is an independent risk factor for
cardiovascular (1, 2) and atherosclerotic disease (3). Total plasma
homocysteine (Hcy)1 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
(3). 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 (1, 2). In
addition, elevated plasma Hcy has recently been linked to Alzheimer's
disease and cognitive impairment in the elderly (4, 5).
Hcy is a non-protein amino acid derived from the catabolism of
S-adenosylhomocysteine (AdoHcy), an immediate product of
trans-methylation reactions that utilize
S-adenosylmethionine (AdoMet) (6). Hcy has three possible
fates: 1) methylation to methionine with
N-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 (6). 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) (7). PEMT accounts for the formation of
~30% PC made in liver (8, 9). 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 the
Pemt Materials--
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.
In Vivo Experiments--
The Pemt Primary Hepatocyte Cultures--
Male
Pemt Culture and Transfection of McArdle RH-7777 Rat Hepatoma
Cells--
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.
Measurement of Amino Acid Concentrations and Enzyme
Activities--
Livers were removed from mice and flash-frozen. The
protein content of the liver samples was determined by the Biuret
method (13). Thawed liver samples were homogenized in 50 mM
phosphate-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
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 (12).
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 Benson
D-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
(19).
Pemt
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 Decreased Secretion of Homocysteine from Hepatocytes Derived from
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 (20). These results demonstrate that altered flux through another major methyltransferase can also affect homocysteine metabolism.
Expression of PEMT Stimulates Secretion of Homocysteine from
Hepatoma Cells--
McArdle RH-7777 hepatoma cells have negligible
PEMT activity (19). 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 (22), and the PEMT pathway is an important source for PC
in the bile (23). Approximately, one-third of the PC in murine liver is
derived from the PEMT pathway (8, 9). 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 (24), 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 (7), 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 (25, 26)) 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 Mudd
et al. (27, 28) 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. (27, 28)
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 (29, 30) have demonstrated that
hyperhomocysteinemia is detrimental to normal vascular endothelial
function. Furthermore, elevated Hcy levels stimulate vascular smooth
muscle cell growth and migration as well as the recruitment of
monocytes to atherosclerotic lesions (31-34). Although Hcy enhances
the development of atherosclerotic lesions, it has not been
demonstrated that Hcy is a cause of atherosclerosis. Studies in
patients with mild hyperhomocysteinemia but without any other
cardiovascular disease risk factors were shown to be at no greater risk
for the disease compared with healthy individuals (35). Also, neither
cystathionine Regardless of whether or not mild hyperhomocysteinemia causes or merely
correlates with the development of cardiovascular disease, it is a
strong predictor of mortality in individuals (37). It is now shown that
the PEMT reaction in the liver is a major source of plasma Hcy. A
previous study (38) with hepatocytes derived from
Pemt
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
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
/
and
Pemt+/+ mice were fed ad libitum either
chow or the high fat/high cholesterol diet for 3 weeks (10).
/
and Pemt+/+ mice
(12-20-weeks old) that were fed chow were used. Primary hepatocytes
were isolated by collagenase perfusion (11) and plated on 60-mm
collagen-coated dishes at a density of 2.0 × 106
cells/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.
-synthase (14, 15), AdoMet synthase (15), methionine synthase (16),
5,10-methylenetetrahydrofolate reductase (17), and betaine:homocysteine
methyltransferase (18). The final product of betaine:homocysteine
methyltransferase, methionine, was measured by reverse-phase high
performance liquid chromatography.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
Mice Have Decreased Plasma Levels of
Homocysteine--
Female and male Pemt+/+ and
Pemt
/
mice were fed chow or a high
fat/cholesterol diet for 3 weeks. Fig. 1
shows 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.
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Fig. 1.
Plasma Hcy levels are lower in
Pemt /
mice than in
Pemt+/+ mice. Plasma Hcy levels were
measured in Pemt+/+ and Pemt
/
mice that were fed chow or a high fat/high cholesterol (HF/HC) diet for
3 weeks. Results are the means ± S.D. from at least four mice. *,
p < 0.001 for Pemt
/
compared
with Pemt+/+ mice based on a one-way ANOVA followed
by a Newman-Keuls post-test.
/
mice, we
assayed five enzymes listed in Table
I. The activities were not
statistically different between Pemt
/
and
Pemt+/+ 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.
Specific activities of enzymes involved in methionine and homocysteine
metabolism
/
mice were
analyzed using ANOVA followed by Newman-Keuls test, but no significant
differences were found.
/
Mice--
We measured the secretion of Hcy from
primary hepatocytes isolated from Pemt+/+ and
Pemt
/
mice. Fig. 2
shows 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 (20). 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 both
Pemt+/+ 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+/+ and
Pemt
/
mice (21). 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.
View larger version (31K):
[in a new window]
Fig. 2.
Less Hcy is secreted from
Pemt /
than from
Pemt+/+ hepatocytes. Hepatocytes were
isolated from Pemt+/+ and
Pemt
/
mice. After a 12-h incubation ± guanidinoacetate (GA), the media were collected and frozen, and the
amount of Hcy was determined. The differences among all of the four
variables were significant (p < 0.001) by ANOVA
followed by a Newman-Keuls post-test.
View larger version (12K):
[in a new window]
Fig. 3.
Hcy secretion is elevated in hepatoma cells
expressing PEMT. McArdle RH7777 hepatoma cells were stably
transfected with cDNA encoding human PEMT or vector alone
(pCI-MH) and incubated for 24 h with 100 µM ethanolamine. A, PEMT activity in cell
homogenates. B, the amount of Hcy secreted into the medium.
Values are the means ± S.D. of three independent experiments. *,
p < 0.05 based on an ANOVA analysis followed by a
Newman-Keuls post-test.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-synthase nor methylene tetrahydrofolate reductase-deficient mice developed atherosclerosis until old age, even
though the mice had severe homocysteinuria (36).
/
mice demonstrates that PEMT is also
important in regulation of the secretion of apolipoprotein B-containing
lipoproteins, high levels of which leads to cardiovascular disease.
Therefore, continued research on the role of PEMT in the generation of
plasma Hcy will lead to a better understanding of the possible
relationship between mild hyperhomocysteinemia and other cardiovascular
risk factors.
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ACKNOWLEDGEMENTS |
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We thank Sandra Ungarian, Susanne Lingrell, and Laura Hargreaves for excellent technical assistance and Dr. René Jacobs, Dr. David Wood, Dr. Martin Raabe, and Dr. Jean Vance for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by grants from the Heart and Stroke Foundation of Alberta, Innuvit and the Northwest Territories, the Heart and Stroke Foundation of Canada, the Canadian Institutes for Health Research, and the Canadian Diabetes Association and a contract from the Bayer Corporation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Recipient of a studentship award from the Canadian Heart and Stroke Foundation.
** Recipient of a K. M. Hunter/Canadian Institutes of Health Research Doctoral Research Award.
Senior Investigator of the Canadian Institutes of Health Research.
§§ Canada Research Chair in the Molecular and Cell Biology of Lipids and Heritage Medical Scientist of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed: Dept. of Biochemistry, 328 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. Tel.: 780-492-8286; Fax: 780-492-3383; E-mail: dennis.vance@ualberta.ca.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M212194200
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ABBREVIATIONS |
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The abbreviations used are: Hcy, homocysteine; AdoHcy, adenosylhomocysteine; AdoMet, S-adenosylmethionine; DMEM, Dulbecco's modified Eagle's medium; PC, phosphatidylcholine; PEMT, phosphatidylethanolamine N-methyltransferase; ANOVA, analysis of variance.
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