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Cheng
Ji, Neil Kaplowitz, Gastroenterology/Liver Division, Keck School
of Medicine, University of Southern California, Los Angeles, CA
90033, USA
Supported by the U.S. National Institute of Alcohol Abuse and
Alcoholism, R01 AA014428 and by the Robert E. and May R. Wright
Foundation, No. 263
Correspondence to: Cheng Ji, Ph.D., Faculty of Medicine,
Gastroenterology/Liver Division, Keck School of Medicine, University
of Southern California, HMR-101, 2011 Zonal Avenue, Los Angeles, CA
90033, USA. chengji@usc.edu
Telephone: +1-323-442-3452
Fax: +1-323-442-5425
Received: 2004-04-20
Accepted: 2004-05-06
Abstract
Deficiencies in vitamins or other factors (B6, B12, folic acid,
betaine) and genetic disorders for the metabolism of the non-protein
amino acid-homocysteine (Hcy) lead to hyperhomocysteinemia (HHcy).
HHcy is an integral component of several disorders including
cardiovascular disease, neurodegeneration, diabetes and alcoholic
liver disease. HHcy unleashes mediators of inflammation such as NFkB,
IL-1b,
IL-6, and IL-8, increases production of intracellular superoxide
anion causing oxidative stress and reducing intracellular level of
nitric oxide (NO), and induces endoplasmic reticulum (ER) stress
which can explain many processes of Hcy-promoted cell injury such as
apoptosis, fat accumulation, and inflammation. Animal models have
played an important role in determining the biological effects of
HHcy. ER stress may also be involved in other liver diseases such as
a1-antitrypsin
(a1-AT)
deficiency and hepatitis C and/or B virus infection. Future research
should evaluate the possible potentiative effects of alcohol and
hepatic virus infection on ER stress-induced liver injury, study
potentially beneficial effects of lowering Hcy and preventing ER
stress in alcoholic humans, and examine polymorphism of Hcy
metabolizing enzymes as potential risk-factors for the development
of HHcy and liver disease.
Ji C, Kaplowitz N.
Hyperhomocysteinemia, endoplasmic reticulum stress, and alcoholic
liver injury. World J Gastroenterol
2004; 10(12): 1699-1708
http://www.wjgnet.com/1007-9327/10/1699.asp
INTRODUCTION
Homocysteine (Hcy) is a toxic non-protein sulfur containing
amino acids in humans. It is formed exclusively upon demethylation
of the essential amino acid- methionine. Hcy is metabolized either
through remethylation or transsulfuration pathways and is
nutritionally regulated. Normal concentrations of total homocysteine
in plasma are in the range of 5 to 16 mmol/L
and the desired upper limit for Hcy concentration should be 10 mmol/L.
An elevated plasma Hcy level is denoted hyperhomocysteinemia (HHcy).
Three ranges of HHcy are defined: moderate (16 to 30 mmol/L),
intermediate (31 to 100 mmol/L),
and severe (>100 mmol/L).
Individuals who consume a large amount of food rich in animal
protein may ingest two to three grams of methionine, resulting in
postprandial Hcy concentrations greater than 20 mmol/L.
Clinical HHcy was first described more than 40 years ago in children
with learning difficulties[1-3], and it has since been
estimated that moderate HHcy occurs in 5-7% of the general
population. Evidence now indicates that moderate HHcy is an
important and independent risk factor for several disorders,
including atherosclerosis, diabetes, fatty liver, immune activation,
and neurodegenerations such as Alzheimer's and Parkinson's diseases[3-9]
.
Readers are referred to
recent reviews on HHcy and functions of Hcy[10,11]. The
main goal of this article is to provide information on major causes
of HHcy, potential mechanisms of Hcy toxicity, with emphasis on
endoplasmic reticulum (ER) stress mechanism, and animal models for
the study of biological effects of HHcy. We would also summarize our
ongoing work on ethanol-induced HHcy and liver injury in an
intragastric ethanol fed murine model.
HCY METABOLISM AND HHCY
Figure 1(PDF)
Homocysteine metabolism. Homocysteine has three main
metabolic fates: to be remethylated to methionine, to enter the
cysteine biosynthetic pathway, and to be released into the
extracellular medium. CBS, cystathionine -synthase; MS, methionine
synthase; THF, tetrahydrofolate; MTHFR, 5,
10-methylenetetrahydrofolate reductase; BHMT, betaine-homocysteine
methyltransferase; DMG, dimethylglycine; MAT, methionine
adenosyltransferase; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine.
Hcy
was formed from methionine after removal of the methyl group on S-adenosylmethionine
(SAM) (Figure 1). Hcy metabolism involves reversible formation of S-adenosylhomocysteine
(SAH), remethylation to methionine by betaine-homocysteine
methyltransferase (BHMT) (liver and kidney restricted), which is
vitamin-independent, and by the ubiquitous methionine synthase (MS),
which is dependent on vitamin B12 and methylenetetrahydrofolate (MTHF)
production via 5, 10-methylenetetrahydrofolate reductase (MTHFR).
Hcy can also be converted through transsulfuration to cystathionine
for the formation of cysteine and glutathione (GSH). The
transsulfuration is catalyzed by cystathionine-b-synthase
(CBS) and is dependent on vitamin B6. In addition, Hcy can be
converted to Hcy-tRNA. Although it was not incorporated into protein
due to editing mechanisms, nitroso-Hcy-tRNA is stable and might play
a role in Hcy-induced protein misfolding along with the formation of
Hcy-protein-SH mixed disulfides and Hcy thiolactone covalent binding
to lysine amino groups[11]. Hcy-t-RNA is edited through
the action of methionyl-t-RNA synthetase (ATP consuming) by
formation of a thioester thiolactone which could covalently bind to
protein amino groups. Thus, homocysteinylation of proteins depends
on the formation of thiolactone[12,13].
Tight regulation of Hcy
metabolism depends on different affinities of MS, BHMT, and CBS for
Hcy. MS and BHMT show low Km values for Hcy (<0.1 mmol/L), and
CBS has high Km values for Hcy (>1 mmol/L). At low Hcy
concentrations, methionine conservation was favored; and at high Hcy
concentrations, immediate and long-term drainage of Hcy via the
transsulfuration pathway was ensured[14]. SAM could play
a key regulatory role by allosterically inhibiting MTHFR and BHMT
and activating CBS[15-18]. Thus, SAM may be a regulatory
switch in Hcy metabolism: low SAM favors remethylation and
conservation of Hcy for methionine synthesis, whereas high levels
favor transsulfuration. High Hcy levels can decrease the SAM/SAH
ratio, since most methyltransferases bind to SAH with higher
affinity than SAM, elevated SAH inhibits methylation. In vitro,
under "physiological" conditions of concentrated 27 000
g postmitochondrial supernatant with 8 mmol/L GSH, 0.3 mmol/L
serine, 2 mmol/L betaine, 60 mmol/L
methionine, 50 mmol/L
methyl THF, 60 mmol/L
SAM and 10 mmol/L
SAH, transsulfuration accounted for 46% of Hcy metabolism and the
remainder was equally contributed to by MS and BHMT. The need to
conserve methionine (e.g. low protein diet) resulted in decreased
cystathionine production and increased Hcy remethylation.
Conversely, in the presence of excess methionine, SAM
activated the cystathionine pathway.
HHcy results from
increased levels of intracellular Hcy that is readily released into
the extracellular medium: plasma or body fluid. Kidney might be a
major site for the removal and metabolism of Hcy primarily through
the transsulfuration pathway[19]. Renal impairment often
causes HHcy, reflecting a role of kidney in Hcy clearance from
plasma. This fact might contribute to the high incidence of vascular
complications in patients with chronic renal failure[20].
Genetic abnormalities, age, sex and various nutritional and hormonal
determinants contribute to HHcy. However, genetic and nutritional
disorders are the major factors. Genetic disorders involve
polymorphism in the genes coding for MTHFR and CBS. The most common
genetic defect associated with mild HHcy is a point mutation,
namely, a C to T substitution at nucleotide 677 (C677→T)
in the open reading frame of the gene for MTHFR. This point mutation
could cause a substitution of valine for alanine in the functional
enzyme[21], resulting in a thermolabile variant of the
enzyme with decreased total activity. This is an autosomal recessive
mutation, and the frequency of the C677→T
polymorphism varied among racial and ethnic groups, with 13% of T/T
homozygous and 50% C/T heterozygous among Caucasian and Asian
populations, and very low incidence among African-Americans[21-27].
Premature atherosclerosis and thrombotic disease were observed in
MTHFR deficiency[23-28]. The most common genetic cause associated with severe HHcy is
homozygous CBS deficiency, which resulted in plasma Hcy
concentrations of up to 400 mmol/L,
compared to normal plasma levels of 10 mmol/L[28-30].
Homozygous CBS deficiency, T833→C
and G919→A
mutations, were inherited as an autosomal recessive disorder with
pleiotropic clinical manifestations, including mental retardation,
ectopia lentis, osteoporosis, skeletal abnormalities and hepatic
steatosis[28-30]. Patients were usually at higher risk
for premature atherosclerosis and thrombotic disease, which is the
major cause of death[31-33]. CBS deficiency has a
worldwide incidence of 1:344 000 live births, ranging from 1:58 000
to 1:1 000 000 in countries that perform newborn screening[31].
While homozygous CBS deficiency is rare, heterozygous CBS deficiency
occurs in approximately 1% of the general population and is
associated with premature atherosclerosis and thrombotic disease in
phenotypically normal individuals[31-33].
Nutritional disorders
that potentially lead to HHcy include deficiencies in vitamin B12,
folate and vitamin B6, as the de novo synthesis of methionine methyl
groups requires both vitamin B12 and folate cofactors whereas the
synthesis of cystathionine requires pyridoxal 5-phosphate (vitamin
B6). Although it has been shown that deficiencies of vitamin B12 and
folate are related to increased plasma Hcy concentrations[32-35],
the relationship of Hcy levels to vitamin B6 status is less clear[36,37].
In addition, excess dietary methionine in normal mice has been shown
to induce HHcy[38]. Under normal conditions, several
methylation reactions in the liver contribute to the bulk (90%) of
SAM utilization and Hcy production via SAH. For example,
phosphatidylethanolamine to phosphatidylcholine is mediated by
phosphatidylethanolamine N-methyltransferase (PEMT).
PEMT-/- mice had 50% decreased plasma Hcy despite being
choline and betaine deficient[39]. PEMT null mice
exhibited fatty liver and apoptosis but this was not prevented by
betaine administration, impaired lipoprotein secretion rather than
methyl donor deficiency appeared to be the dominant effect of
choline deficiency[40]. The other major source of Hcy is
the activity of hepatic guanidinoacetate (GAA) N-methyltransferase (NMT).
GAA is produced in the kidney by L-arginine:glycine
amidinotransferase. GAA is then converted to creatine in the liver
by GAA-NMT, utilizing SAM and generating SAH.
Creatine is exported to muscle and also represses the kidney
enzyme which produces GAA. GAA supplementation could induce HHcy and
creatine feeding lowers Hcy[41].
HCY TOXICITY
Possible cellular mechanisms by which elevated Hcy promotes
liver disease are oxidative stress, endoplasmic reticulum (ER)
stress and the activation of pro-inflammatory factors (Figure 2).
Hcy enhances the production of several pro-inflammatory cytokines.
Expression of monocyte chemoattractant protein 1 (MCP-1) was
increased in cultured human vascular endothelial cells, smooth
muscle cells and monocytes treated with Hcy[42-44]. Hcy
has also been shown to increase expression of IL-8[42], a
T-lymphocyte and neutrophil chemoattractant, in cultured endothelial
cells. Hcy-induced expression of MCP-1 and IL-8 in monocytes and
endothelial cells has been shown to occur through activation of NF-kB,
a transcription factor involved in mediating downstream inflammatory
processes[44,45]. Active NF-kB
could stimulate production of cytokines, chemokines, interferons,
leukocyte adhesion molecules, hemopoietic growth factors and major
histocompatibility (MHC) class I molecules- all of which are thought
to influence inflammation[45,46].
Hcy can generate a
procoagulant state, which may be related to its proclivity to
auto-oxidize, generating H2O2. Various in
vitro studies using vascular tissues have implicated Hcy in causing
abnormal vascular relaxation responses by enhancing the
intracellular production of superoxide anion (O2-)[47-54].
O2-
is believed to react with and decrease the availability of
endothelial nitric oxide (NO) and yield peroxynitrite, thereby
limiting normal vasodilation responses[55,56]. Deceased
GSH peroxidase transcription (reduction of peroxides protects NO)
may play a role in this process[49,57], since
overexpression of GSH peroxidase could restore the NO response[57].
O2-
and peroxynitrite are also known to contribute to the oxidative
modification of tissues, resulting in the formation of lipid
peroxides and nitrosated end products such as 3-nitrotyrosine.
The observations that Hcy decreased the expression of a wide
range of antioxidant enzymes[57-59] and impaird
endothelial NO bioavailability by inhibiting glutathione peroxidase
activity raise the possibility that Hcy sensitizes cells to the
cytotoxic effects of agents or conditions known to generate ROS.
Decreased NO bioavailability has also been shown in vitro to
increase the expression of MCP-1, which may enhance intravascular
monocyte recruitment and lead to accelerated lesion formation[60].
Figure
2(PDF)
Cellular mechanisms by which homocysteine promotes cell
injury. Homocysteine causes activation of necrosis factor-kB
(NF-kB)
and enhances production of cytokines (IL-1b,
IL-6, and IL-8) resulting in inflammatory reactions, increases
intracellular levels of superoxide anion causing oxidative stress,
and induces endoplasmic reticulum (ER) stress by causing misfolding
of proteins traversing the ER. Homocysteinyl-tRNA increases
production of highly reactive derivative homocysteine thiolactone
which damages enzymes and DNA. IRE1, type 1 ER transmembrane protein
kinase; ATF6, the activating transcription factor 6; PERK, the PKR
like ER kinase; SREBP, sterol regulatory element binding protein,
PON, paraoxonase.
Intracellular
Hcy can be converted by methionyl tRNA synthase into an Hcy-AMP
complex, which is subsequently catabolised to Hcy thiolactone,
thereby preventing the incorporation of Hcy into nascent polypeptide
chains. Hcy thiolactone has unique reactive properties that can lead
to the homocysteinylation of lysine residues and free amine groups
on numerous cellular proteins, thereby resulting in decreased
biological activity and premature degradation[61]. In
addition, Hcy thiolactone secreted into the circulation may induce
widespread modifications of plasma proteins that could potentially
contribute to the development of liver and cardiovascular diseases.
Recent studies have demonstrated that Hcy thiolactone decreases
paraoxonase activity associated with HDL, thereby rendering HDL less
protective against oxidative damage or against toxicity of Hcy
thiolactone[62].
HCY-INDUCED ER STRESS
ER is a principal site for protein synthesis and folding,
calcium storage and calcium signaling. It also serves as a site of
biosynthesis for steroids, cholesterol and other lipids. The
physiological roles of the ER include regulation of protein
synthesis, folding and targeting and maintenance of cellular calcium
homeostasis. The ER has a high concentration of numerous resident
chaperone proteins such as glucose regulated protein-78 (GRP78) and
GRP94, a high level of calcium and an oxidative environment to carry
out these functions efficiently. Proteins that were translocated
into the ER lumen underwent post-translational modifications and the
folding required for optimal function. Properly folded proteins were
allowed to reach their destiny via the secretory pathway, whereas
unfolded and misfolded proteins were exported or dislocated from the
ER and degraded by cytoplasmic proteasomes[63-68]. ER
stress is a condition under which unfolded and misfolded proteins
accumulate (Figure 3). ER stress triggers unfolded protein response
(UPR), which is an intracellular signaling pathway and is mediated
via three ER-resident sensors in mammalian cells: a type-I ER
transmembrane protein kinase (IRE-1), the activating transcription
factor 6 (ATF-6) and the PKR like ER kinase (PERK). Activation of
these three pathways is mediated by GRP78, which is associated with
each sensor in the absence of ER stress. As unfolded proteins
accumulated in the ER, GRP78 dissociated from and thereby activating
IRE-1, ATF-6 and PERK[68-70]. Activation of both IRE-1
and ATF-6 increases the expression of ER-resident chaperones. IRE-1
is a stress-activated transmembrane protein kinase having
endoribonuclease activity. Following ER stress, IRE-1 dimerized and
was autophosphorylated, thereby allowing IRE-1 to act as an
endoribonuclease in the alternative splicing of XBP-1 mRNA. The
removal of a 26 base pair intron resulted in a translation
frameshift that permits XBP-1 to act as a transcriptional activator
of genes containing upstream ER stress response elements (ERSE).
Upon ER stress, ATF-6 was transported to the Golgi where the
cytosolic transactivation domain of ATF-6 is cleaved from the
membrane by specific proteases (S1P and S2P) that also recognize,
cleave and activate sterol regulatory element-binding proteins (SREBPs)
leading to increased lipids needed for ER membrane synthesis.
Following release, the transactivation domain of ATF-6 localized to
the nucleus where it interacts with ERSE, thereby activating
transcription of numerous UPR-responsive genes, including GRP78,
GADD153 (CHOP), XBP-1, ERp72, and Hcy-induced ER protein (Herp). ER
stress could also lead to a rapid attenuation in protein synthesis,
a cellular process mediated by the transmembrane protein kinase,
PERK. Activation of PERK could cause phosphorylation of eukaryotic
initiation factor-2a
(eIF-2a),
which blocks mRNA translation initiation to help relieve the
unfolded protein burden on the ER. Recent studies have also
demonstrated that PERK-dependent eIF-2a
phosphorylation is required for transcriptional activation of a wide
range of UPR-responsive genes[71,72]. The early UPR
co-coordinately enhances cell survival by ensuring that the adverse
effects of ER stress are dealt with in a timely and efficient
manner. However, prolonged UPR following ER stress has severe
consequences. It can lead to activation of the tumor necrosis factor
receptor associated facter 2 (TRAF2), which activates caspases (e.g.
caspase-12 in mice) and JNK resulting in programmed cell death.
Over-expression of CHOP, a basic region leucine zipper transcription
factor, could also promote cell death[71]. Overproduction
of lipids by SREBP can lead to fat accumulation. In addition, ER
stress is associated with release of ER Ca2+ stores which
can trigger oxidative stress via effects on mitochondria and NF-kB
activation leading to inflammatory reactions[73]. NF-kB
activation could be blocked by calcium chelators and antioxidants[19].
Increased cytosol calcium also activates calpains which
proteolytically cleave Bcl-XL (inactivation) and caspase 12
(activation). ER stress could contribute to the pathogenesis of a
number of human diseases, including diabetes, Alzheimer's disease,
Parkinson's disease and cancer[72].
Hcy induced ER stress
response has recently received much attention[6,74-79].
Hcy causes ER stress by disrupting disulphide bond formation and
causing misfolding of proteins traversing the ER. Elevated levels of
intracellular Hcy could increase the expression of several ER stress
response genes, including GRP78, GRP94, Herp and RTP[6,58,74,77,78,80-82].
Hcy could induce expression of GADD153[58,78,79] involved
in ER stress-induced cell death[83]. Hcy-induced ER
stress could cause dysregulation of lipid biosynthesis by activating
the SREBPs[6,76-79], ER resident transcription factors
are responsible for the induction of genes in the cholesterol/triglyceride
biosynthesis and uptake pathways[6]. Hcy-induced cell
death was mimicked by other ER stress agents and was dependent on
IRE-1 signaling. Activation of IRE-1 by Hcy could lead to a rapid
and sustained activation of JNK protein kinases[84,85], a
result consistent with the finding that activation of JNK by ER
stress involved binding of IRE-1 to TRAF2[86]. Because
persistent activation of JNK correlated with cell death[87],
these studies could provide further support for a mechanism
involving Hcy-induced programmed cell death.
Figure 3(PDF)
Consequences of endoplasmic reticulum (ER) stress response.
In the early phase, unfolded proteins cause dissociation of
chaperones such as Bip/GRP78 from ER resident kinases-IRE1 and PERK
and transcription factor-ATF6. Activated PERK phosphorylates eIF2
resulting in translational attenuation. Activated IRE1 and ATF6
up-regulate genes encoding ER chaperone proteins such as GRP78/94
leading to increased protein-folding capacity. Overall, the unfolded
protein response (UPR) goes down.
In the late phase, IRE1 interacts with TRAF2 (tumor necrosis
factor receptor associated factor 2) which activates caspases and
JNK (cJUN NH2-terminal kinase) leads to apoptosis. ATF6 and PERK
upregulate CHOP (C/EBP homologous protein) promoting cell death.
SREBP upregulates lipid synthesis. Prolonged UPR leads to Ca2+
release from ER causing production of reactive oxygen intermediates
which may lead to activation of NF-kB.
APPROACHES FOR STUDY OF HHCY
Cell and animal models with altered plasma Hcy are among the
most useful approaches in determining the biological effects of HHcy.
However, cell and transgenic animal models expressing Hcy
metabolism-related genes/enzymes are not available. Nevertheless,
diet- and, especially, genetic-induced animal models of HHcy have
been developed. The gene knockout animals have significantly
enhanced the status of Hcy as an independent risk factor for several
disorders.
Homozygous and
heterozygous CBS-deficient mice were generated in 1995[88].
Homozygous mutants completely lacking CBS were born at the expected
frequency from mating of heterozygotes, but they suffered from
severe growth retardation and a majority of them died within 5 wk
after birth. Histological examination showed that the hepatocytes of
homozygotes were enlarged, multinucleated, and filled with
microvesicular lipid droplets. Plasma Hcy levels of the homozygotes
(203.6±65.3 mmol/L)
were 33 times higher than normal (6.1±0.8 mmol/L).
The homozygous CBS deficient mice represented a model for severe
HHcy. Heterozygous CBS deficient mice had 50% reduction in CBS mRNA
and enzyme activity in the liver and had twice normal plasma Hcy
levels (13.5± 3.2 mmol/L).
The CBS knockouts significantly help elucidate the in vivo role of
elevated levels of Hcy in the etiology of several HHcy-related
disorders and in the cellular mechanisms by which Hcy promote cell
injury. The CBS-deficient mice were predisposed to HHcy during
dietary folate deficiency, and moderate HHcy was associated with
marked impairment of endothelial function in mice[89].
Results from a subsequent study indicated that endothelial
dysfunction occurred in HHcy mice even in the absence of folate
deficiency[90]. Endothelial dysfunction in CBS (+/-) mice
was associated with increased tissue levels of SAH, which suggests
that altered SAM-dependent methylation may contribute to vascular
dysfunction in HHcy[91]. Further studies with the CBS
deficient mice revealed the importance of intracellular redox
balance for nitric oxide bioactivity and endothelial function, and
the importance of ER stress in abnormal hepatic accumulation of
lipid[92,93]. Expression of several genes analyzed by DNA
microarray was found to be reproducibly abnormal in the livers of
heterozygous and homozygous CBS-deficient mice[94]. These
genes encode ribosomal protein S3a and methylthioadenosine
phosphorylase, suggesting cellular growth and proliferation
perturbations may occur in homozygous CBS-deficient mice liver.
MTHFR-deficient mice have
been recently developed to examine the effects of HHcy resulting
from genetic deficiencies in the remethylation pathway[95].
MTHFR-deficient mice shared basic phenotypic similarities with
CBS-deficient mice. However, they were unique in that they developed
mild HHcy and atherosclerosis. Recent study has demonstrated the
importance of choline metabolism in HHcy in this model[96].
Comparison study by administrating the alternate choline-derived
methyl donor, betaine, to wild-type mice and MTHFR deficient mice
revealed that plasma Hcy and liver choline metabolite levels were
strongly dependent on the MTHFR genotype. Betaine supplementation
decreased Hcy in all three genotypes, restored liver betaine and
phosphocholine pools, and prevented severe steatosis in MTHFR-deficient
mice. Since there was a significant negative correlation between
plasma betaine and Hcy concentrations in humans with cardiovascular
disease, the results emphasize the strong interrelationship between
Hcy, folate, and choline metabolism. MTHFR-compromised mice with
HHcy appeared to be much more sensitive to changes of choline/betaine
intake than wild-type animals. HHcy, in the range of that associated
with folate deficiency or with homozygosity for the 677T MTHFR
variant, may be associated with disturbed choline metabolism.
MS could directly
catalyze the remethylation pathway and inactivation of this gene has
been attempted recently[97]. Heterozygous MS knockout
mice from an outbred background had slightly elevated plasma Hcy
(6.1 mol/L) and methionine compared to wild-type mice (4.1 mmol/L)
but seemed to be otherwise indistinguishable. Homozygous knockout
embryos survived through implantation but died soon thereafter.
Nutritional supplementation during pregnancy was unable to rescue
embryos that were completely deficient in MS. This study indicated
that MS activity was essential for early embryonic development of
mice. Although the MS knockout mouse has not provided an immediately
obvious animal model of human disease, heterozygotes with 50%
reduction of MS activity may be useful. It is likely that MS
heterozygote knockouts are more susceptible to dietary deficiencies
than wild type mice and thus having merits as a model in which
interactions between genetic status and nutritional status can be
studied.
The animal models are
valuable in vivo tools to further examine potential therapeutic
approaches in lowering plasma Hcy while decreasing the prevalence of
HHcy-induced disorders. However, the animal models neither have
tissue or organ specificity nor exclude potential compensatory
pathways of Hcy metabolism. Conditional disruption of Hcy
metabolism-related genes and crossing between animal models that are
deficient in different genes should be the future directions in the
effort of creating animal models for study of HHcy.
ETHANOL-INDUCED HHCY AND
LIVER INJURY
The pathogenesis of the pathologic features of alcoholic liver
injury, namely steatosis, apoptosis, necrosis, inflammation and
fibrosis, is an area of intense interest. Although much progress has
been made over the past decade, we still do not have a complete
understanding of this process[98]. We recently found that
in a murine model of intragastric ethanol there was an upregulation
of genes associated with endoplasmic reticulum (ER) stress response,
including GRP78 and 94, CHOP and SREBP. The expression of these
genes was associated with protein malfolding as well as apoptosis
and lipid synthesis[78,79].
Since alcoholism and alcohol-related diseases constitute a
severe health problem in the world and ER stress has been linked to
Hcy in the pathogenesis of several disorders such as atherosclerosis,
Alzheimer's disease, and liver steatosis, we would direct the
readers' attention to ethanol-induced alterations in Hcy metabolism.
Alcoholic patients have
been shown to have elevated plasma Hcy (average two-fold) which
rangeed from 10-120 mol/L (normal 5-15 mol/L)[99-101].
Total folate, B12 and B6 levels were normal. However, Hcy levels
correlated with folate levels and blood alcohol levels. Well
nourished alcoholics exhibited markedly lower levels of serum
pyridoxal-phosphate (PLP) and mildly lower red cell folate[102].
Even "social" drinking (30 g/d×6 wk) caused 20% increased
Hcy and decreased folate[99,103]. Heavy alcohol
consumption is a risk factor for stroke and brain atrophy[103-105].
Rats fed ethanol exhibited a doubling of plasma Hcy despite normal
levels of folate, PLP and B12[106]. We have observed a 7
fold increase of plasma Hcy levels (22.3±2.8 mmol/L
vs pair-fed control 3.0±0.9 mmol/L)
in mice fed ethanol intragastrically for 4 wk[78].
With alcohol feeding of
rats intragastrically for 9 wk liver specific MAT1A protein
expression did not change, whereas MAT2A increased in conjunction
with -40% decreased hepatic levels of methionine and SAM[107].
However, shorter exposure of rats and minipigs to ethanol was not
associated with a decrease in methionine or SAM in most studies.
Depending on route, ethanol dose and duration, variable
changes in SAM and SAH have been described[107-110].
Ethanol feeding has been
known to lower MS[111], leading to increased accumulation
of 5-methyl THF and to increased BHMT leading to utilization-induced
decreased betaine levels[111]. These effects depended on
increased blood ethanol. Golden
Syrian hamsters with high ADH fed a 360 mL/L ethanol diet did not
develop increased blood ethanol levels or changes in Hcy metabolism
unless ADH was inhibited[112]. Of note, the SAM levels
were maintained by the utilization of betaine.
However prolonged ethanol feeding eventually could lead to
depletion of SAM. Chronic alcohol could increase choline uptake[113]
and mitochondrial oxidation to betaine[114] suggesting
compensation for increased demand for betaine. Feeding betaine
(0.5%) raised SAM levels which was accentuated in alcohol fed
animals (minimal to begin with) and prevented fatty liver[78,108,111]. Raised SAM was initially assumed to contribute to betaine's
ameliorative effect on fatty liver. It may be equally important that
the protective role of betaine was due to lowering Hcy directly
through BHMT and indirectly by raised SAM leading to activation of
CBS.
The mechanism of the
ethanol induced decrease in MS is not well understood. Kenyon et
al.[110] showed that the enzyme was inhibited by high
concentrations of acetaldehyde, whereas we have found decreased mRNA.
Increase in BHMT activity appeared to be a compensatory phenomenon
to maintain methionine and was seen after 2 wk in ethanol fed rats
and after 4 wk in ethanol fed mice.
In the chronic (12 mo)
ethanol fed micropigs with adequate folate, MS activity decreased by
20% which was associated with slightly decreased serum methionine,
20% increased serum Hcy, and increased hepatic SAH but no change in
SAM. These small changes due to ethanol were not associated with
increased ALT or fatty liver but were associated with increased
scattered apoptosis[115]. Interestingly addition of
folate deficient diet to ethanol feeding of the castrated minipig
accentuated plasma Hcy and liver injury[116] although ER
stress was not considered in this study.
Betaine lowered Hcy and
prevented ER stress and alcoholic liver injury in alcohol fed mice[78].
However, we need to be cognizant of other actions of betaine.
Feeding rats betaine in drinking water (1.5 g/kg) blunted the TNF
response to LPS and decreased concomitant liver injury[117].
Importantly, however, taurine was equally effective. Earlier work
has suggested an indirect protective role of choline
supplementation, suggesting choline oxidation to betaine could
protect against Kupffer cell activation[118-120]. It has
been suggested that betaine and taurine serve as organic osmolytes
which are critical in regulating Kupffer cell function[121].
Hyperosmotic conditions induce Na+ betaine transporter mRNA while
hypoosmotic conditions do the opposite, this occurred in Kupffer
cells but not hepatocytes. Betaine or taurine protects the liver
against warm ischemia-reperfusion. Recently, betaine pretreatment
was shown to protect the hepatocyte from bile acid induced
apoptosis. The mechanism is not certain and high concentrations of
betaine (mmol/L) were required[122,123]. We observed that
increased gene expression of TNF and CD 14 was indicative of the
alcohol-induced Kupffer cell activation[78]. However,
betaine treatment did not significantly attenuate these changes,
suggesting that betaine either acts downstream of alcohol-induced
Kupffer cell activation or acts via an independent pathway.
The effect of SAM feeding
is of interest since it was reported to decrease fatty liver and
mitochondrial abnormalities[115].
SAM might be expected to inhibit re-methylation and promote
transsulfuration of Hcy. It is unclear as to what the overall effect
on Hcy would be. Severe SAM deficiency in MAT1A knockout did not
alter Hcy but was associated with increased expression of BHMT and
CBS[124]. SAM could transcriptionally activate MAT1A and
suppress MAT2A[125].
Overall chronic ethanol
exposure seemed to cause a modest decrease in SAM and increase in
SAH along with early-decreased MS and late-increased BHMT. All these
changes were accompanied with increased Hcy which occurs despite
adequate dietary folate, B12, B6 and choline. Thus there are
possible contributions of decreased MS, unknown effects on CBS, and
decreased SAM (decreased activation of CBS) leading to HHcy. The
decrease in SAM levels was accompanied with increased SAH levels.
Since both SAM and SAH activated CBS, it is doubtful that changes in
levels of these metabolites exerted a significant regulatory role on
transulfuration[11,16]. The increase in BHMT appeared
insufficient to lower Hcy due to limitation on the availability of
betaine and already-impaired cell function. Although high dietary
choline might generate sufficient betaine in rodents, the choline
oxidase pathway is normally low in primates. Thus, providing excess
dietary betaine rather than choline would seem to be an approach
more applicable to the human situation. Since betaine corrects
hyperhomocysteinemia, fatty liver injury and ER stress, and
homocysteine is known to cause all these changes, it is reasonable
to state that an important mechanism by which betaine protects
against alcoholic liver disease is the correction of
hyperhomocysteinemia and proof of this hypothesis requires further
work.
POSSIBLE ROLE OF ER STRESS IN
OTHER LIVER DISEASES
ER stress may also be involved in liver injury caused by a1-antitrypsin (a1-AT) deficiency and hepatitis C virus (HCV) or
hepatitis B virus (HBV) infection. a1-antitrypsin (a1-AT) deficiency was caused by a point mutation
encoding a substitution of lysine for glutamate-342[126].
Aggregated mutant a1-AT was retained in ER rather than secreted in the
blood and body fluids where its function is to inhibit neutrophil
proteases. Individuals with this deficiency had a markedly increased
risk of developing emphysema by a loss of function mechanism by
which reduced levels of a1-AT in the lung inhibit connective tissue breakdown
by neutrophil elastase, cathepsin G, and proteinase 3. Some
individuals with a1-AT deficiency developed liver injury and
hepatocellular carcinoma by a gain of function mechanism, i.e.,
accumulation of aggregated mutant a1-AT within the ER which is toxic to liver cells.
However, the exact mechanism by which ER retention of this
aggregated mutant protein leads to cellular injury is still unknown.
Recent studies have demonstrated that ER retention of mutant a1-AT induces a marked autophagic response in cell
culture and transgenic mouse models of a1-AT deficiency as well as in the liver of patients
with a1-AT deficiency[127]. The autophagic
response is a general mechanism whereby cytosol and intracellular
organelles, such as ER, are first sequestered from the rest of the
cytoplasm within unique vacuoles and then degraded by fusion with
lysosomes to clear the cells of senescent constituents. Under a
fasting condition, a marked increase in fat accumulation was
observed in a1-AT-containing globules in the liver of a1-AT deficient mice[128], suggesting a
malfunction of ER caused by accumulation of mutant a1-AT. Investigations of ER stress markers such as
GRP78, CHOP, SREBP, XBP1, and ATF6 are needed to assess the direct
involvement of ER stress in a1-AT deficiency-induced liver injury.
Evidence of ER stress in
HBV or HCV infection is emerging. HBV codes for three forms of
surface protein. The minor and large forms are translated from
transcripts specified by the preS1 promoter, while the middle and
small forms are translated from transcripts specified by the
downstream S promoter. Overexpression of the large surface protein
of HBV could lead to a 10-fold activation of the S promoter but not
of an unrelated promoter[129]. The large surface protein
could also activate the cellular grp78 and grp94 promoters. Neither
the middle nor the small surface protein, nor a secretable form of
the large surface protein, could activate the S promoter, but agents
that induced endoplasmic reticulum (ER) stress had an effect similar
to that of the large surface protein, suggesting that HBV may evolve
a feedback mechanism, such that ER stress induced by accumulation of
the large surface protein increases the synthesis of the middle and
small surface proteins, which in combination with the large surface
protein can form mixed, secretable particles. HCV-induced ER stress
was more evident. HCV replicates from a ribonucleoprotein (RNP)
complex that is associated with ER membrane. The replication
activities of the HCV subgenomic replicon have been shown to induce
ER stress[130]. HCV replicons induce the UPR which is
paralleled by the proteolytic cleavage of ATF6. The HCV
non-structural protein 5A (NS5A) can bind to and inactivate the
cellular double-stranded RNA-activated protein kinase, PKR. NS5A has
recently been demonstrated to engage ER-nucleus signal transduction
pathway[131]. Expression of NS5A in the ER could induce
an ER stress leading to the activation of STAT-3 and NF-kB,
which is sensitive to inhibitors of Ca2+ uptake. The
NS5A-induced ER stress signaling has also been shown in the context
of an HCV subgenomic replicon[131]. Another HCV
component, the HCV envelope protein E2, is an ER-bound protein that
contains a region of sequence homology with the PKR and its
substrate, the eIF2b.
E2 could modulate global translation by inhibition of the
interferon-induced PKR through its PKR-eIF2a
phosphorylation site homology domain (PePHD)[132]. E2
could also bind to and inhibit PERK[132]. At low
expression levels, E2 induced ER stress, but at high expression
levels, E2 inhibited PERK kinase activity in vitro. Mammalian cells
that stably expressed E2 were refractory to the
translation-inhibitory effects of ER stress inducers, and E2
relieved general translation inhibition induced by PERK. The PePHD
of E2 was required for the rescue of translation that was inhibited
by activated PERK. These findings may explain why the virus promotes
persistent infection by overcoming the cellular ER stress response.
In addition, HCV-induced ER stress resulted in a decline in protein
glycosylation. Decreasing protein glycosylation could disrupt the
proper protein folding of MHC class I molecules, preventing the
assembly of MHC class I molecules. Cells expressing HCV subgenomic
replicons had a lower MHC class I cell surface expression[133].
HCV-infected cells may thus be undetectable in the immune system by
suppressing MHC class I antigen presentation to cytotoxic T
lymphocytes. Therefore, the persistence and pathogenesis of HCV may
depend upon the ER stress-mediated interference of MHC class I
assembly and cell surface expression. Finally, HCV infection may
suppress the degradation of misfolded proteins while stimulating the
synthesis of its viral proteins in the ER. In the ER, IRE1-XBP1
pathway directs both protein refolding and degradation in response
to ER stress. It was demonstrated that XBP1 expression was elevated
in cells carrying HCV subgenomic replicons, but XBP1 transactivating
activity was repressed[134]. This prevents the IRE1-XBP1
transcriptional induction of EDEM (ER degradation-enhancing -mannosidase-like
protein), which is required for the degradation of misfolded
proteins. Consequently, misfolded proteins are stable in cells
expressing HCV replicons. Study with a cell line with a defective
IRE1-XBP1 pathway showed elevated levels of HCV internal ribosome
entry site-mediated translation[134]. This study
indicated that the HCV suppression of misfolded protein degradation
in the ER not only promoted HCV expression but also contributed to
the persistence of the virus in infected hepatocytes.
HCV infection is common
in alcoholic patients presenting with liver disease. Heavy alcohol
intake would worsen the outcome of HCV infection[135-137],
which has directed much attention to the interaction between alcohol
and HCV infection. Alcohol plays an important role in HCV infection
resulting in increased viral replication, enhanced HCV quasispecies
complexity, increased liver-cell death, suppression of immune
responses, and iron overload[138]. However, the
pathogenic mechanisms underlying the alcohol-HCV interactions are
not fully understood. Based on the above evidence that both HCV and
homocysteine could induce endoplasmic reticulum (ER) stress response[6,78,130]
and that there was a link between alcohol-induced significant
elevation of homocysteine level, ER stress, and the pathogenesis of
liver injury[78], it is reasonable to hypothesize that a
locus of the potentiative interaction between alcohol and HCV in
accelerating the progression of liver disease is at the level of ER
stress. In the case of both HBV and HCV infection, it is widely
recognized that severely immunosuppressed patients may develop a
paradoxically severe and rapidly progressive liver disease. This has
been seen in the post-OLT setting and in patient with AIDS.
Therefore, the loss of immune detection of viral-infected
hepatocytes may lead to an unopposed massive overload of hepatocytes
with viral proteins triggering ER stress. Future studies should
examine the contribution of ER stress to these pathologic
conditions.
CONCLUSION
HHcy is an integral component of several disorders including
cardiovascular and cerebrovascular diseases, neurodegeneration,
liver steatosis, diabetes, and cancer. HHcy can result from
deficiencies of vitamin cofactors (B6, B12, folic acid) required for
Hcy metabolism and/or from genetic disorders of its metabolism. Hcy
unleashes inflammation mediators such as NFkB,
IL-1b,
IL-6, and IL-8. Hcy increases production of intracellular superoxide
anion causing oxidative stress. Hcy-induced misfolding or
malfunctioning of numerous intracellular proteins are increasingly
important and attract much attention because the Hcy-induced ER
stress mechanism can explain many processes of cell injury. Animal
model creation and integral investigation of available animal models
will certainly play important role in determining precisely the
biological effects of HHcy. Our observations with the murine
intragastric ethanol fed model have suggested a link between Hcy
metabolism, ER stress, and the pathogenesis of alcohol induced liver
injury. Figure 4 demonstrates our hypothesis in which ethanol
feeding causes HHcy which then induces the ER stress response in
parenchymal and nonparenchymal cells in the liver leading to fatty
liver, apoptosis and possibly inflammation. The potential beneficial effects of lowering Hcy and
preventing ER stress in alcoholic humans needs to be studied. In
addition, since a minority of alcoholics develop liver disease and a
wide range of Hcy levels are seen in alcoholics, it will be
important to examine polymorphism of Hcy metabolizing enzymes as
potential risk-factors for the development of HHcy and liver
disease.
Figure 4(PDF)
Hypothesis for the role of ethanol-induced HHcy in the
pathogenesis of alcoholic liver disease.
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