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,
USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, USC Research Center for Liver Diseases,
* Department of Pathology,
Division of Gastrointestinal and Liver Diseases,
* Department of Medicine, Keck School of Medicine of USC, Los Angeles, California 90033, USA
Department of Medicine, Keck School of Medicine of USC, Los Angeles, California 90033, USA
1Correspondence: Keck School of Medicine of the University of Southern California, 1333 San Pablo St., MMR-412, Los Angeles, CA 90033, USA. E-mail: htsukamo{at}hsc.usc.edu
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ABSTRACT |
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 TOP ABSTRACT CONCEPTUAL APPROACH TO THE...CELL TYPE-SPECIFIC MECHANISMS OF...GENETIC BASIS OF ALDPOTENTIAL NEW TREATMENTS OF...REFERENCES |
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Key Words: mitochondria • ALD • ethanol • antioxidants
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CONCEPTUAL APPROACH TO THE PATHOGENESIS OF ALCOHOLIC LIVER DISEASE (ALD) |
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TOPABSTRACTCONCEPTUAL APPROACH TO THE...CELL TYPE-SPECIFIC MECHANISMS OF...GENETIC BASIS OF ALDPOTENTIAL NEW TREATMENTS OF...REFERENCES |
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2
3
4
5
6
7
8)
. At the
same time, Lieber and colleagues show that progressive ALD proceeds
despite adequate nutrition (9
, 10)
. The latter hypothesis
was based primarily on the observation that baboons fed a nutritionally
adequate liquid diet containing ethanol at 50% calories developed
nearly the whole spectrum of ALD including cirrhosis, a finding that
remains to be reproduced by other laboratories.
The intragastric ethanol infusion technique, which allows maximal
ethanol consumption and absolute control over nutrient intake by
rodents, has depicted the critical role of nutrition in determining
sensitization and priming of the liver to ethanol-induced liver injury.
Studies demonstrated profound effects on ethanol-induced liver injury
by intake of nutrients such as polyunsaturated fat and iron in
quantities that were never thought to be important. The concept of
‘sensitization’ and ‘priming’ is currently considered fundamental
to our pursuit for elucidation of pathogenetic mechanisms of ALD. We
define sensitization as a conditioning that makes the target cells,
hepatocytes, more vulnerable to harmful effects triggered by ethanol
and priming as the effect that promotes specific injurious mechanisms.
The sensitizing and priming are rendered by the complex interactions of
primary mechanistic factors and secondary risk factors (Fig. 1
). Thus, in addition to independent in-depth studies focused on one
mechanistic aspect, a more comprehensive and interactive approach is
required to understand how sensitization and priming work together to
determine individual predisposition to ALD and to precipitate the
disease process. For example, intake of polyunsaturated fat in
ethanol-fed rats, but not in pair-fed controls, results in a
synergistic priming effect on induction of cytochrome P4502E1 (CYP2E1)
with consequent oxidative injury to the liver (11)
.
Conversely, saturated fat prevents this priming effect and abrogates
depletion of a mitochondrial pool of glutathione (GSH)
(12)
, one of the most crucial sensitization effects of
ethanol on hepatocytes (13)
. Iron is another example.
Whereas a slight increase in hepatic iron content by dietary iron
supplementation is harmless in control rats, it exacerbates alcoholic
liver injury via accentuation of oxidative stress (14)
.
Further, increased iron storage in hepatic macrophages is a potential
priming mechanism for enhanced expression of tumor necrosis factor 
(TNF-) in experimental ALD (15)
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Besides nutritional factors, female gender, age, concomitant intake of
other drugs that can induce CYP2E1, hepatitis virus infection, and
genetic predisposition are all considered risk factors. Even among the
primary mechanistic factors that include acetaldehyde, oxidative
stress, immune response, hypoxia, and membrane alterations, there are
cross-interactive relationships to render sensitization or priming
effects. For instance, acetaldehyde, a potent toxic metabolite of
ethanol, induces liver injury via its covalent binding to structural or
functional proteins of the cells (16)
while promoting
oxidative stress via consumption of GSH. In turn, deleterious effects
of acetaldehyde-protein adduct formation may be accentuated by
oxidative stress since malondialdehyde, a lipid peroxidation end
product, can increase the binding affinity of acetaldehyde by 13-fold
(17)
. The resulting novel hybrid adducts are highly
immunogenic and may incite immune response-mediated liver injury
(18
, 19)
. Although cellular immune response and
inflammation are regarded as independent mechanisms of ALD, they can
also lead to oxidative stress via the release of reactive oxygen
species (ROS) by NADPH oxidase or action of TNF- at the electron
transport chain in target cells.
The multifactorial nature and complex interactions among primary mechanistic factors and between primary and secondary factors appear to be the basis for the heterogeneous response that alcoholics exhibit for ALD. Elucidation of the sensitization and priming mechanisms involving cross-interactions of these factors should allow us to gain insight into the most fundamental question, which is why only a small fraction of alcoholics develop advanced ALD. How, then, do we effectively approach the complicated and interactive mechanisms? First, investigators need experimental models or systems by which they can perform controlled deletion and addition analyses in order to identify what primary and secondary factors are required for the expression of a particular aspect or whole spectrum of experimental ALD. Second, investigation itself has to become interactive and multidisciplinary; experts in various disciplines need to work together to provide cutting-edge science for elucidating the precise nature and mechanisms that underlie interactions.
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CELL TYPE-SPECIFIC MECHANISMS OF ALD PATHOGENESIS |
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TOPABSTRACTCONCEPTUAL APPROACH TO THE...CELL TYPE-SPECIFIC MECHANISMS OF...GENETIC BASIS OF ALDPOTENTIAL NEW TREATMENTS OF...REFERENCES |
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).
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Hepatocytes
How ethanol primes and sensitizes the hepatocytes is a topic of
primary importance. After all, hepatocytes are the site of ethanol
oxidation and ethanol-induced injury. For the past 20 years, much
interest has been generated for the pathogenetic role of CYP2E1 in ALD.
Alterations in methionine metabolism have also been well described in
ALD, and several key changes in this metabolic pathway can sensitize
and prime hepatocytes to injury. Changes in mitochondrial function have
also received increasing attention as potential explanations for
sensitization and priming mechanisms for hepatocellular injury.
Role of CYP2E1
CYP2E1 catalyzes the production of ROS (20)
and lipid
peroxidation of microsomal membranes in vitro (21
, 22)
. It
is well known that CYP2E1’s expression and activity are induced after
ethanol consumption (23
, 24)
. Studies using cell lines
stably transfected with a CYP2E1 expression vector have made a major
contribution to research on this topic, confirming the role of CYP2E1
in ethanol-induced ROS production and cell death (25)
and
GSH depletion in both cytosol and mitochondria (26)
.
However, the in vivo role of CYP2E1 in experimental ALD is
controversial. Although there is a close correlation between induction
of CYP2E1 and experimental ALD (11)
and inhibitors for
CYP2E1 ameliorates alcoholic liver injury (27)
, CYP2E1
knockout mice are just as susceptible to ethanol-induced liver injury
and exhibit similar induction of other CYP families (CYP1A, CYP2A,
CYP2B, and CYP3A) in response to ethanol feeding as wild-type mice
(28)
. Furthermore, gadolinium chloride blocks experimental
alcoholic liver injury despite induction of CYP2E1 (29)
,
demonstrating dissociation of CYP2E1 induction from alcoholic liver
injury. However, these latter two studies only examined the early stage
of experimental ALD; whether CYP2E1 plays a role in progression of
experimental ALD remains to be tested. It also has to be pointed out
that CYP2E1 activity is inherently much lower in mice than in rats or
humans. Thus, the findings in mice may need to be interpreted with
caution with respect to the role of CYP2E1 in ALD (M.
Ingleman-Sundberg, personal communication). To this end, the effects of
CYP2E1 overexpression on alcoholic liver injury in mice need to be
evaluated.
Role of abnormal methionine metabolism
The liver plays a central role in methionine metabolism, as half
of the daily methionine intake is catabolized here (Fig. 3
). The first step in methionine metabolism is the formation of
S-adenosylmethionine (SAM) catalyzed by methionine adenosyltransferase
(MAT) (30
, 31)
. In this reaction, the adenosyl moiety of
ATP is transferred to methionine, forming a sulfonium ion that is a
high-energy reagent and can easily transfer its methyl group to a large
variety of acceptor substrates including nucleic acids, proteins,
phospholipids, biological amines, and a long list of small molecules
(31)
. SAM is the principal biological methyl donor, the
precursor of aminopropyl groups used in polyamine biosynthesis and the
liver, the precursor of glutathione (GSH) through its conversion to
cysteine via the trans-sulfuration pathway (30
, 31)
. Under
normal conditions, most of the 6–8 gm of SAM generated per day is used
in transmethylation reactions in which methyl groups are added to
compounds and SAM is converted to S-adenosylhomocysteine (SAH)
(31
, 32)
. SAH is a potent competitive inhibitor of
transmethylation reactions; both an increase in SAH level as well as a
decrease in the SAM to SAH ratio are known to inhibit transmethylation
reactions (31
, 33)
. For this reason, the removal of SAH is
essential. The reaction that converts SAH to homocysteine and adenosine
is reversible and catalyzed by SAH hydrolase (30
, 31)
. In
vivo, the reaction proceeds in the direction of hydrolysis only if the
products, adenosine and homocysteine, are rapidly removed (30
, 31
, 33)
.
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There are three pathways that metabolize homocysteine. One is the
trans-sulfuration pathway, which converts homocysteine to cysteine.
This is a unique pathway present only in the liver and lens
(34)
that condenses homocysteine with serine to form
cystathionine in a reaction catalyzed by cystathionine ß-synthase
(CBS) with vitamin B6 as a cofactor (32
, 34)
. Cleavage of cystathionine, catalyzed by another vitamin
B6-dependent enzyme, 
-cystathionase, then
releases free cysteine, the rate-limiting precursor for GSH synthesis
(34)
. The other two pathways that metabolize homocysteine
resynthesize methionine from homocysteine. One is catalyzed by
methionine synthase (MS), which requires normal levels of folate and
vitamin B12; the other is catalyzed by
betaine-homocysteine methyltransferase (BHMT), which requires betaine,
a metabolite of choline (30
, 32)
. If homocysteine
metabolism is impaired in these pathways, homocysteine may be released
into sinusoidal blood (resulting in hyperhomocystinemia) and the ratio
of hepatic SAM to SAH may decrease (leading to inhibition of
transmethylation reactions). Given the critical role of methylation in
determining various cellular processes, these changes have far-reaching
effects. Hyperhomocystinemia is associated with increased risk for
arteriosclerosis (35)
and was recently shown to induce
tissue inhibitor of metalloproteinases-1 (TIMP-1) and collagen gene
expression in a hepatic stellate cell (HSC) line (36)
. As
discussed below, ALD affects many of these enzymatic steps in
methionine metabolism. It is therefore vital to have a better
understanding of the abnormalities in the hepatic methionine metabolism
for both the pathogenesis of the disease and the design of therapy
against alcoholic liver injury.
Methionine adenosyltransferase MAT is a critical
cellular enzyme because it catalyzes the only reaction that generates
SAM. The MAT gene is one of 482 genes required for survival of an
organism (32)
. In mammals, the genes MAT1A and MAT2A
encode for two homologous MAT catalytic subunits, 1 and 2
(37
, 38)
. MAT1A is expressed only in the liver and encodes
the 1 subunit found in two native MAT isozymes, which are either a
dimer (MAT III) or tetramer (MAT I) of this single subunit
(38)
. MAT2A encodes for a catalytic subunit ( 2) found
in a native MAT isozyme (MAT II), which is widely distributed
(38)
. MAT2A and its gene product also predominate in the
fetal liver and are progressively replaced by MAT1A during development
(39)
. In adult liver (40)
, MAT1A is primarily
expressed. However, an intriguing switch in the gene expression from
MAT1A to MAT2A is demonstrated in liver cancer (41
, 42)
,
liver regeneration after partial hepatectomy (42)
, and
hepatotoxicity induced by thioacetamide (43)
. Using a cell
line model that differs only in the type of MAT expressed, the type of
MAT expressed by the cell significantly influences the rate of cell
growth (44)
. The mechanism is likely via a change in the
steady-state SAM level and methylation status. This is because MAT
isozymes differ in kinetic parameters and regulatory properties, so a
switch in MAT expression can affect the steady-state SAM level and
methylation (45)
.
Non-liver-specific MAT (MAT II) has the lowest
Km for methionine but is tightly regulated by SAM
with an IC50 of 60 µM, which is close to the
normal intracellular SAM concentration (30
, 47
, 48)
. In
contrast, SAM has a minimal inhibitory effect on MAT I
(IC50=400 µM) and stimulates MAT III (up to
eightfold at 500 µM concentration of SAM) (47)
. Thus,
the SAM level in cells that express only the non-liver-specific MAT
isoform is relatively unaffected by fluctuations in methionine
availability because of the negative feedback inhibition. In contrast,
the rate of SAM synthesis and the SAM level increase with increasing
methionine availability in cells that express mostly liver-specific MAT
(30)
. Consistent with this, we found that cells that
express MAT1A have much higher levels of SAM and DNA methylation but
lower rates of cell growth than cells that express MAT2A
(44)
. A caveat to this is the recently described
regulatory subunit (ß) associated with the MAT enzyme in lymphocytes
(48
, 49)
. The ß subunit was shown to lower the
Km of MAT II for methionine and render the enzyme
more susceptible to feedback inhibition by SAM (49)
.
Whether it exists in other cells is unknown. In liver, the current data
suggest the relative expression of MAT isozymes is likely to influence
the rate of liver growth and possibly hepatocarcinogenesis.
It has long been realized that patients with alcoholic
cirrhosis often have hypermethioninemia and delayed plasma clearance of
methionine after intravenous injection (50
, 51)
.
Subsequent studies showed that hypermethioninemia in cirrhotic patients
is attributed to a 50–60% decrease in the activity of the
liver-specific MAT (52
, 53)
. This likely contributes to a
decreased hepatic GSH level in patients with ALD, as SAM administration
prevented the fall in GSH (54)
. The fall in hepatic GSH
sensitizes the liver to oxidative injury and sets up a vicious cycle
since it can further contribute to inactivation of liver-specific MAT.
A 30% reduction in hepatic GSH levels results in a 60% reduction in
hepatic MAT activity and a 40% reduction in SAM level
(55)
. The decrease in MAT activity is thought to occur
primarily by post-translational mechanisms (37)
. A fall in
MAT activity can be a result of a change in the oligomeric equilibrium
of the liver-specific MAT (decrease in the ratio of tetramer to dimer)
or covalent modification of the enzyme. The liver-specific MAT contains
several critical cysteine residues, modifications of which can
inactivate the enzyme by direct interference with the substrate binding
site(s) or by causing dissociation of the oligomers
(56
57
58
59)
In end-stage liver disease, the decrease in MAT activity
affects the tetramer selectively (52)
. Since the
intracellular GSH/GSSG ratio is known to be an important modulator of
the oligomeric equilibrium of the hepatic MAT isozymes
(56)
, part of the selective loss of the tetramer is due to
a reduction in the GSH/GSSG ratio (31)
. The cysteine at
position 121, conserved in rat and human liver-specific MAT and absent
in non-liver-specific MAT, is a target of covalent modification
(58
59
60)
. Although this cysteine is not essential for
activity, as substitution of serine for this residue has no effect on
MAT activity, when cysteine is modified either by oxidation or by the
formation of a nitrosothiol, the enzyme is inactivated
(57
58
59
60)
. The inactivation could be reversed by GSH and
other thiol-reducing agents. The amount of GSH required to reverse the
inactivation is 3 mM for the dimer and 25 mM for the tetramer
(59)
. Since normal hepatic GSH concentration is 5–10 mM,
the difference in reversibility of inactivation by GSH may also
contribute to the selective loss of the tetramer in liver disease.
Nitrosylation of Cys 121 of liver-specific MAT and its inactivation
have been demonstrated both in vitro and in vivo in animals treated
with lipopolysaccharide (LPS) (58
, 60)
. Both oxidative
stress and endotoxemia occur in ALD and contribute to the liver injury.
One of the mechanisms may well be inactivation of the liver-specific
MAT.
Although liver-specific MAT is inactivated in end-stage
ALD in humans, changes in MAT expression have been largely unknown.
Finkelstein et al. showed induction of hepatic MAT activity in rats
given 50% ethanol by gavage and a low protein diet for up to 10 days
(61)
. Thus, changes in MAT may depend on the stage of
ethanol-induced liver disease. Since a change in hepatic MAT expression
can affect the steady-state SAM level, methylation status, and cell
growth, we examined MAT expression and SAM homeostasis using the
Tsukamoto-French intragastric ethanol feeding model (62)
.
In this model, fatty liver develops by the 4th week, centrilobular
steatonecrosis, mononuclear cell infiltration, and increased TNF-
gene expression occur by the 9th week, and early fibrosis by the 16th
week (62)
. We found a twofold increase in the mRNA level
of both MAT1A and MAT2A 9 wk after ethanol feeding (63)
.
However, the protein level of MAT1A was essentially unchanged whereas
that of the MAT2A was greatly induced. Hepatic levels of methionine,
SAM, and DNA methylation fell by 
40%. The question is whether these
changes are of pathogenetic importance.
Reduced SAM levels and methylation can affect gene
expression, membrane fluidity, and GSH levels in liver (32
, 64)
. Effects on membrane fluidity (32
, 65
, 66)
and
GSH levels (32
, 64)
have been well studied. How changes in
DNA methylation affect ethanol-induced liver injury is unclear. ALD is
associated with increased risk of liver cancer, but the molecular
mechanism is unclear. Global DNA hypomethylation is observed in the
development of many types of cancer, although there is often regional
hypermethylation (67)
. A common hypothesis is that
hypomethylation of growth-promoting proto-oncogenes and/or
hypermethylation of tumor suppressor genes will alter transcription
factor binding and expression of these genes to promote a selective
growth advantage for the initiated cell (68)
.
Alternatively, DNA hypomethylation may promote malignant transformation
by inducing regional alterations in DNA conformation and chromatin
structure, rendering affected regions more accessible to DNA-damaging
agents (68)
. We found hypomethylation and increased
expression of c-myc in the ethanol-fed livers
(63)
. We also detected increased genome-wide DNA strand
break accumulation (63)
. Thus, even at the prefibrotic
stage of ethanol-induced liver injury, there are already changes that
may predispose the liver to malignant degeneration.
In summary, available data show in ALD a relative switch in MAT expression due primarily to inactivation of liver-specific MAT and increased expression of non-liver-specific MAT. As a result, there is depletion of hepatic SAM and GSH levels and decreased transmethylation. Important consequences include impairment in antioxidant defense, altered phospholipid composition and membrane fluidity, gene expression, and DNA stability. Further studies should define when these changes occur in the course of ALD and whether they can be corrected or prevented by SAM administration (see Treatment section for a discussion). Genes whose expression are affected by a change in methylation status should be further defined to improve our understanding of the pathological consequence of altered DNA methylation in this chronic liver disease.
Resynthesis of methionine In addition to changes
in MAT, decreased MS activity and increased BHMT activity occur in
animals fed ethanol (61
, 69
70
71)
. These changes can also
affect the availability of SAM and homocysteine metabolism. In rats
given 50% ethanol by gavage and a low protein diet for up to 10 days,
Finkelstein et al. showed decreased MS activity and increased hepatic
BHMT activity (61)
. Other investigators also showed the
same changes in MS and BHMT activities in rats fed a Lieber-DeCarli
ethanol liquid diet for 2–4 wk (69
, 70)
. In these models,
the methionine pool is conserved at the expense of betaine. Betaine
supplementation greatly increased the hepatic SAM level and protected
against fatty infiltration of the liver (69)
. However,
whether this is true for other animal models or in rats with more
advanced ethanol-induced liver disease is unclear. In fact, hepatic
methionine level depended on the stage of liver injury in rats fed
ethanol intragastrically. Our previous study using a lower amount of
fat (25%) combined with intragastric ethanol feeding found increased
hepatic methionine level after 5 wk but a 50% reduction in methionine
level after 16 wk (72)
. Our recent study used a diet
regimen that contains additional fat, which is known to result in
accentuated ethanol-induced oxidative liver injury (73)
.
The most likely explanation for the fall in hepatic methionine level is
decreased resynthesis of methionine from homocysteine. MS has already
been shown to be inactivated (61
, 69
70
71)
. It is likely
that the induction in BHMT was not sufficient to maintain the
methionine pool. Another possibility to be examined is decreased
methionine uptake as ethanol-induced liver injury progresses. Although
the changes in MS and BHMT activity have been described, the mechanisms
are not clear. Thus, although folate and B12
deficiency contribute to the fall in MS activity, the enzyme is also
postulated to be inactivated by possibly forming adducts with
acetaldehyde or nitrosylated (69
, 71
, 74)
. Recently we
found the mRNA levels of three key enzymes for homocysteine
metabolism—MS, BHMT, and CBS—to be markedly lower in 10 unselected
patients with alcoholic cirrhosis (75)
. Thus, multiple
mechanisms likely contribute to decreased resynthesis of methionine and
homocysteine catabolism.
How would impaired methionine resynthesis contribute to
the live injury in ALD? One mechanism is decreased SAM availability
with the possible consequences discussed in the preceding section.
Another mechanism is impaired homocysteine metabolism, resulting in
increased homocysteine release by hepatocytes. This may contribute to
alcoholic liver fibrogenesis (see the Hepatic Stellate Cells section).
Role of the mitochondria
An important cellular organelle that contributes to both priming
and sensitizing effects of ethanol is the mitochondria. Mitochondria
isolated from ethanol-fed rats produce more ROS (76)
.
Ceramide, an intracellular signaling molecule for TNF-, acts on
mitochondrial electron transport chain to generate ROS
(77)
and represents a key mechanism of TNF--induced
oxidative stress in mitochondria. Depletion of mitochondrial GSH is the
most important sensitizing mechanism rendered by ethanol feeding
(78
, 79)
to TNF--induced cell death (13)
.
This ethanol-induced defect is caused by impaired transport of GSH into
mitochondria and corrected by administration of SAM but not
N-acetylcysteine (66)
. It is not known what molecular
mechanisms underlie the ethanol-induced impairment in mitochondrial GSH
transport except for an observation that fluidization of mitochondria
with a fatty acid derivative restored their ability to transport GSH
(66)
. This finding and the demonstrated efficacy of SAM
suggest the possibility that ethanol-feeding induced alterations in
mitochondrial membrane phospholipid and fatty acid compositions with
resulting impairment in GSH uptake. It is well known that phospholipid
methylation is required for maintenance of normal membrane fluidity and
function (64)
. Thus, reduced SAM level as a result of
abnormalities in the methionine metabolism can affect mitochondrial GSH
transport and sensitize the cell to oxidative stress-induced injury by
this mechanism. We do not know why the depletion in GSH occurs
selectively in mitochondria and more profoundly in perivenular
hepatocytes. Nor do we know whether it requires oxidative stress
mediated by CYP2E1, acetaldehyde, or TNF-. It remains to be
determined how significant this defect is in the mitochondrion’s
regulatory role in determining the type of cell death
(13)
.
Chronic ethanol consumption causes decreases in cellular ATP content
and mitochondrial membrane potential, particularly in perivenular
hepatocytes (77)
, and these functional impairments are
corrected by repletion of mitochondrial GSH by administration of SAM
(80)
. Ethanol feeding also results in increased oxidative
modification and single-strand breaks of mitochondrial DNA
(81)
. It is not known whether these changes can be
corrected by repletion of mitochondrial GSH. Ethanol was shown to
induce apoptosis in human primary hepatocytes and HepG2 cells in
culture (82)
, although this effect has been difficult to
reproduce by others. Mitochondria from rats chronically fed ethanol are
more sensitive to induction of the mitochondrial permeability
transition by ceramide, GD3 ganglioside, and Bax (83)
.
Whether and how ethanol sensitizes hepatocytes for apoptosis need to be
pursued. Reasons for the discrepancy in ethanol-induced apoptosis of
hepatocytes in vitro may be related to different cellular conditions in
culture resulting in differential sensitivity to apoptotic and/or
anti-apoptotic signaling. What determines the type of cell death
(apoptosis vs. necrosis) in ALD is another important question. The
extent of ATP depletion may be critical. Ethanol-fed animals are
vulnerable to hepatocellular ATP depletion and necrosis caused by
ischemia and hypoxia (84)
. The role of uncoupling
protein-2 (UCP-2) in ALD also needs to be addressed. UCP2 expression is
induced in hepatocytes of obese mice; their livers have reduced
mitochondrial membrane potential and ATP stores and are sensitive to
necrosis caused by ischemia (85)
. LPS induces UCP-2
expression in hepatocytes via a TNF--dependent mechanism
(86)
. Thus, UCP-2 is expected to be up-regulated in ALD,
which may make hepatocytes more vulnerable to cell death by
compromising the mitochondrial membrane potential and ATP generation.
Cell biology related to the role of mitochondria in hepatocellular cell
death is an exciting area of science. Further research on how ethanol
perturbs the underlying mechanisms should serve as an insightful model
to generate basic information in this important area of research.
Effects of ethanol on hepatocyte growth
The effect of ethanol on hepatocyte growth is an interesting and
complex topic. It is well known that animals with ALD exhibit impaired
liver regeneration (87)
. Ethanol feeding has been shown to
inhibit calcium mobilization and DNA synthesis in hepatocytes induced
by HGF, the most potent mitogen for hepatocytes (88)
and
TGF-stimulated receptor autophosphorylation (89)
.
Ethanol intake also inhibits a TNF--dependent increase in nuclear
factor 
B (NF-B) binding in partially hepatectomized rat livers
but does not affect IL-6-induced Stat-3 phosphorylation and DNA binding
(90)
. Administration of HGF improved recovery from
alcohol-induced fatty liver (91)
. This illustrates the
importance of the liver’s regenerative state in opposing progression
of degenerative processes. It is important to understand how ethanol
affects signaling of growth factors and comitogens in hepatocytes,
which future investigations should help to achieve.
Although the regenerative capacity after partial hepatectomy of
ethanol-fed animals is impaired, chronic alcoholic liver disease is
associated with an increased risk of cancer. This seemingly opposing
effect of ethanol illustrates the importance of differentiating normal
regenerative growth from abnormal malignant transformation. In
micropigs fed ethanol, both increased apoptosis and regenerative
proliferation occur in the same liver specimen (71)
. We
have also reported increased expression of proto-oncogene
c-myc in ethanol-fed rats with prefibrotic liver injury
(63)
. A common finding is the development of decreased
hepatic SAM level and global DNA hypomethylation after ethanol feeding
(63
, 70
, 71)
. We and others speculate that this change in
methylation status may be causally related to the propensity for this
chronic liver disease to undergo malignant transformation. Future
studies to define changes in gene expression as a result of the change
in DNA methylation status may help us understand the molecular mechanism(s) responsible for increased risk of malignant
degeneration. Studies to examine whether preventing the fall in SAM
level will prevent induction in the expression of proto-oncogene should
also be insightful.
Hepatic macrophages
Some of the best support for the essential role of hepatic
macrophages in the pathogenesis of ALD is offered in a study by
Thurman’s group on Kupffer cells (92)
, which demonstrates
amelioration of early alcoholic liver injury in the intragastric
ethanol infusion model by depletion of Kupffer cells with gadolinium
chloride. Even though this method of Kupffer cell elimination is not
without confounding effects on the liver, such as enhanced NF-B
activation and increased TNF- expression by bile duct epithelium and
vascular endothelium (93
, 94)
, the study highlights the
direct regulatory role of the Kupffer cells in hepatocyte injury caused
by ethanol. The role of Kupffer cells is also supported by studies in
which a similar protective effect is seen in ethanol-fed animals given
polymyxin B and neomycin (95)
or lactobacillus
(96)
, which theoretically reduce portal endotoxin levels
and Kupffer cell activation. In fact, administration of antibodies
against TNF-, the cytokine predominantly expressed by Kupffer cells,
attenuates alcoholic liver injury (97)
, and the importance
of TNF- is confirmed by the absence of alcoholic liver injury in TNF
receptor 1 knockout mice (98)
. These findings collectively
support the pathogenetic role played by Kupffer cell-derived TNF- in
experimental ALD. However, we need to be more precise with respect to
the source of TNF-. Is it resident macrophages (Kupffer cells),
newly recruited monocyte-derived macrophages, or both that serve as a
primary effector cell type for expression of this proinflammatory
cytokine? Since the latter is a possibility in any liver disease
including ALD, we should use the term hepatic macrophages. The next
question has to be why TNF- expression by hepatic macrophages is
induced and sustained in ALD. Macrophages are arrayed with the control
mechanisms for their immune functions, including negative feedback
regulation for expression of TNF-. As postulated for other chronic
inflammatory diseases such as rheumatoid arthritis and Crohn’s
disease, there may be defective regulation of cytokine expression in
hepatic macrophages in ALD. Such possibilities include defective
release of IL-10 (99)
, which may be associated with
polymorphism of IL-10 promoter (100)
, or increased TNF-
expression due to polymorphism of its promoter (101)
.
Alternatively, hepatic macrophages may acquire phenotypic alterations
due to intracellular changes. If so, what are they? Suppression of
NF-B and TNF- expression by antioxidants in monocytes from
alcoholic hepatitis patients suggests dysregulation of TNF- gene
transcription driven by NF-B (102)
. Increased iron
storage by hepatic macrophages may prime NF-B activation in
experimental ALD (15)
. Altered methionine metabolism may
also play a role in dysregulation of TNF- gene expression in hepatic
macrophages as described below.
Until recently it was believed that the expression of MAT1A and
liver-specific MAT isozymes was restricted to hepatocytes.
Shimizu-Saito and colleagues showed that both hepatic macrophages and
endothelial cells express the liver-specific and non-liver-specific
MATs whereas HSC express only non-liver-specific MAT
(103)
. The relative contribution of the two MAT isoforms
to the overall MAT activity is unknown in hepatic macrophages. Since
endotoxin and oxidative stress both selectively inactivate
liver-specific MAT, a relative switch in MAT expression is conceivable
in hepatic macrophages of alcoholic livers that could affect the SAM
level, which in turn can influence the expression of TNF-. This idea
is supported by studies from Chawla et al. showing that rats with
decreased hepatic SAM levels are predisposed to liver injury caused by
LPS, which was prevented with exogenous SAM treatment (104
, 105)
. Rats with deficient hepatic SAM levels had much higher
levels of basal serum TNF- and secreted more TNF- after LPS
challenge (105)
; treatment of murine macrophage cells (RAW
cells) with SAM in the culture medium down-regulated TNF- mRNA and
protein synthesis upon stimulation by LPS (106)
. These
findings emphasize the importance of understanding changes in MAT and
SAM in hepatic macrophages during the course of ethanol-induced injury.
A potential role for leptin in regulating proinflammatory immune
response has also been demonstrated by recent studies from Diehl’s
group (107)
. The circulating level of leptin is increased
in alcoholic patients (108)
. Thus, this ob/ob
gene product that regulates appetite, metabolic rate, and fat depot
size may modulate the phenotype of hepatic macrophages in ALD. Diehl
and colleagues draw an intriguing analogy between pathophysiology of
fatty livers caused by obesity and ethanol feeding. Notably, the livers
in both conditions are susceptible to injury induced by LPS
(109)
. Underlying mechanisms for this resemblance may
include induction of UCP-2 and proinflammatory cytokine expression as
well as mitochondrial oxidative stress (110
, 111)
. Further
cell type-specific analysis of hepatic macrophages should provide more
insight into this interesting proposal. Elucidation of priming
mechanisms for TNF- expression by hepatic macrophages is a critical
prerequisite for potential development of antiinflammatory therapeutic
modalities for ALD. It is also important to recognize that TNF-
serves as a comitogen for liver regeneration (112)
. Thus,
future research must address how to differentially suppress
proinflammatory signaling of TNF- while preserving its
pro-regenerative effect by understanding the kinetic of TNF-
induction and intracellular signaling induced by this cytokine in
different target cell types in the liver.
Liver-associated T lymphocytes
It is important to note that ethanol also sensitizes the liver for
concanavalin A (Con A) -mediated injury (113)
. This T
lymphocyte-mediated injury accompanies accentuated increases in plasma
levels of TNF- and IL-6 and the percentage of activated CD25+ T
cells in both blood and liver. Ethanol feeding increases TNF-
production ex vivo by CD4+ T cells isolated from the liver, and
ConA-induced liver necrosis is associated with infiltration of CD4+ T
cells. These potentiating effects of ethanol on Con A-mediated TNF-
production and liver injury are reproducible in naive normal rats after
adoptive transfer of liver-associated T cells obtained from ethanol-fed
rats and Con A administration (114)
. These two studies
demonstrate that ethanol primes liver-associated T lymphocytes for Con
A-mediated TNF- production and suggest that this priming mechanism
may be sufficient to induce liver injury in the normal liver. These
provocative findings deserve further research because alcoholic
hepatitis is associated with an increased ratio of Th1/Th2 cytokine
production and characterized by infiltration of not only neutrophils,
but also T lymphocytes. Remaining questions include 1) how
ethanol feeding primes T lymphocytes; 2) what polyclonal
mitogens can be implicated in alcoholic hepatitis; and 3)
how ethanol affects cross-talk between hepatic macrophages and T
lymphocytes.
Sinusoidal endothelial cells
In contrast to the research on hepatic macrophages, only a limited
number of studies have investigated the role of sinusoidal endothelial
cells in ALD. Ethanol and LPS induce similar morphological and
functional changes in sinusoidal endothelial cells typically
characterized by reduced fenestration and hyaluronan uptake (115
, 116)
, both of which are preventable by elimination of Kupffer
cells, suggesting a role in mediating the effects of ethanol and LPS.
Chronic ethanol intake impairs receptor-mediated endocytosis by
sinusoidal endothelial cells at the level of internalization
(117)
. It remains to be determined whether these cells
experience two chronological stages of ethanol-induced abnormalities in
receptor endocytosis (receptor inactivation, followed by reduced
receptor expression), as shown for asialoglycoprotein receptors on
hepatocytes (118)
. Studies of sinusoidal endothelial cells
are important for understanding the mechanisms underlying
ethanol-induced hepatic inflammation. Sinusoidal endothelium is the
site of inflammatory cell adhesion and transmigration. Intracellular
adhesion molecule 1 (ICAM-1) expression by endothelial cells is
up-regulated in experimental ALD and correlates with plasma endotoxin,
hepatic TNF- mRNA, and liver inflammation and injury
(119)
. Expression of a receptor component for the adhesion
molecule such as CD18 (ß 2-integrin) is up-regulated on neutrophils
of ethanol-fed rats (120)
. However, ethanol-induced
neutrophilic inflammation in the liver, the hallmark of alcoholic
hepatitis, has not been successfully reproduced in experimental
animals, suggesting the models are missing a critical factor(s)
required for this pathological change. Endotoxin challenge to
ethanol-sensitized livers results in coagulative necrosis and
neutrophilic inflammation, but most critical changes appear to occur
initially at the sinusoidal endothelium such as cellular swelling,
blood cell aggregation, and microcirculation disturbance
(121)
. Elucidation of biochemical and molecular basis for
sensitizing and priming sinusoidal endothelium for initiation of
hepatic inflammation appears to be a pivotal prerequisite for
understanding what controls progression of ALD, namely, alcoholic
hepatitis and liver fibrosis. The potential importance of cross-talk
between sinusoidal endothelial cells and hepatic stellate cells in the
evolution of ALD needs to be emphasized.
Hepatic stellate cells
Research on HSC has seen exponential growth in recent years. This
is partly because the mechanisms of HSC activation in liver
fibrogenesis have been increasingly recognized as a clinically relevant
and important area. Growing interest also stems from the fact that the
biology of HSC is now known to be involved in many different aspects of
liver homeostasis including vitamin A storage, regulation of sinusoidal
blood flow, communication with hepatocytes, maintenance of hepatocyte
phenotype, matrix remodeling and deposition, and regulation of local
inflammation. Although development of the HSC isolation technique was
largely responsible for advancement in this field, our knowledge of the
mechanisms of HSC activation in ALD is limited because of difficulties
in both inducing diffuse liver fibrosis in ethanol-fed animals and
isolating sufficient numbers of HSC from ethanol-fed rats. HSC from the
intragastric ethanol infusion model of ALD show the expected changes of
cellular activation such as increased collagen and DNA synthesis
(122)
, induced expression of -smooth muscle actin, and
depletion of retinyl palmitate (123)
. HSC isolated from
rats fed a diet high in polyunsaturated fat with or without ethanol
show increased responsiveness to hepatic macrophage-derived factors for
stimulation of either collagen or DNA synthesis, providing the
potential cellular basis for increased alcoholic liver fibrogenesis
under the high polyunsaturated fat diet regimen (122)
.
Besides the paracrine mode of HSC stimulation involving soluble
factors, oxidative stress may be particularly relevant to the
mechanisms of HSC activation in alcoholic liver fibrogenesis. Human HSC
collagen synthesis is induced by 4-hydroxynonenal, one of the common
lipid peroxidation by-products, via its interaction with JNK and AP-1
activation (124)
. In contrast, manipulation of GSH content
in cultured rat HSC does not affect collagen synthesis despite its
theoretical influence on endogenous lipid peroxidation and aldehyde
adduct formation (125)
. Stable transfection of a rat HSC
line with CYP2E1 cDNA results in increased production of ROS, collagen
type I mRNA synthesis, and stability, effects that can be abrogated by
treatment with antioxidants but are exacerbated by reduction of
cellular GSH with L-buthionine sulfoximine (126)
. Thus,
although the role of oxidative stress in hepatocellular injury in ALD
is convincing, the direct involvement of oxidative stress in HSC
activation is still controversial. Central questions now being
addressed in the field of HSC biology can be directly applicable to
alcoholic liver fibrogenesis. They include 1) do
acetaldehyde and lipid aldehydes have direct and major roles in
activation of HSC in vivo and 2) do extracellular or
intracellular ROS serve as pivotal signals for expression of HSC
activation phenotype? A potential mechanistic link between vitamin A
depletion and HSC activation needs to be further investigated.
Recent studies suggest hyperhomocystinemia, a consequence of altered
methionine metabolism, may also contribute to activation of HSC.
Hyperhomocystinemia, a well-recognized independent risk factor for
atherosclerosis (35)
, can be a result of genetic factors
such as CBS deficiency, nutritional deficiencies in folate, vitamin
B6 or B12, and impaired
liver function (127
, 128)
. Although homocysteine is
metabolized in the liver efficiently under normal conditions, this
amino acid may also be protein-bound and its excess may be released by
hepatocytes into the extracellular medium (32
, 129)
.
Plasma homocysteine is believed to be derived largely from the
sinusoidal release of homocysteine via a mechanism that is poorly
understood. Increased plasma levels of this amino acid occur in
patients and animals with impaired liver function (71
, 130
131
132
133)
and after a methionine load (129)
. This
attests to the central role of the liver in the metabolism of
methionine and the subsequent catabolism of homocysteine
(134)
. An elevated plasma homocysteine level in patients
with ALD is likely to be a consequence of deficiencies in vitamin
B6, B12, or folate
(132
, 135)
as well as decreased activities of enzymes that
metabolize homocysteine as described above. Homocysteine exerts
multiple effects on a variety of cells. It caused endothelial injury,
leading to impaired platelet-modulating activity (136
, 137)
. In vascular smooth muscle cells (VSMCs), homocysteine
promoted DNA synthesis and enhanced collagen production
(138
139
140
141)
possibly via activation of ERK2. These results
suggest that homocysteine may serve as a direct fibrogenic mediator for
vascular atherosclerosis. Since the role of smooth muscle cells in
atherosclerosis is considered analogous to that of HSC in liver
fibrosis (142)
, whether homocysteine also exerts
stimulatory effects on HSC is an intriguing question. Indeed, Torres
and co-workers demonstrated induction of 1(I) procollagen and
TIMP-1 mRNA expression by a HSC line and TIMP-1 mRNA and AP-1
activation by cultured hepatocytes (36)
. These findings
support the potential role of homocysteine in liver fibrogenesis. A
plausible hypothesis is that increased homocysteine release in
ethanol-induced liver injury by hepatocytes due to abnormal methionine
metabolism can exert paracrine effects on stellate cells to induce
fibrogenesis. This remains to be examined.
|
GENETIC BASIS OF ALD |
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, 144)
. In fact, alcoholism is believed to result from
interactions between genetic predisposition and environmental factors.
To this end, the genes encoding enzymes involved in alcohol metabolism
have been studied. Both alcohol dehydrogenase (ADH) and aldehyde
dehydrogenase (ALDH) exhibit functional polymorphism. ADH2*2, ADH3*1,
and ALDH2*2 alleles are suggested to confer protection from developing
alcoholism presumably via faster production or slower elimination of
acetaldehyde, a metabolite that induces unpleasant biological effects
(145
, 146)
. Thus, these polymorphisms should theoretically
protect individuals from developing ALD. An association of functional
polymorphisms of CYP2E1 and alcoholism or ALD has so far been negative
(147
, 148)
. Since cytokines are considered to be central
mediators of ALD, as discussed earlier, polymorphisms in cytokines
genes have been studied for their association with ALD. Polymorphism of
TNF promoter was reported to be associated with alcoholic
steatohepatitis (101)
, but the functional significance of
the polymorphism and confirmation of the association in larger and
diverse populations need to be assessed. Recently, the single base pair
substitution at -627 (C
A) in IL-10 promoter, which results in
reduced IL-10 expression, was shown to be associated with an increased
risk of developing advanced ALD (100)
; this important
observation awaits confirmation. Genetic studies of ALD are just
beginning to identify potential associations. Further exploration is
needed especially for alcoholic hepatitis; recent developments in
genetic epidemiology and microarray techniques may promote this area of
research in the future. |
POTENTIAL NEW TREATMENTS OF ALD |
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. However,
in animal models of alcoholic liver disease and carbon tetrachloride
hepatotoxicity, exogenous administration of SAM prevented the depletion
of SAM and GSH levels and significantly ameliorated liver injury,
including fibrosis (32
, 64
, 149
, 150)
. It has been
proposed that SAM’s protective effect is via increased GSH levels. SAM
has also been used to prevent the development of hepatocarcinogenesis.
Decreased hepatic SAM levels and hypomethylation of certain
proto-oncogenes have correlated with the development of
experimental hepatocarcinogenesis, which were completely prevented by
exogenous administration of SAM (151)
. Here, the mechanism
of protective action was proposed to be via a change in the DNA
methylation status. Impaired mitochondrial uptake of GSH has been
postulated to be an important pathogenetic factor in alcoholic liver
injury, and Colell et al. showed that SAM administration prevented
impaired GSH uptake into mitochondria in chronically ethanol fed rats
by preventing changes in mitochondrial membrane fluidity
(66)
. Another important protective mechanism is
SAM-mediated inhibition of TNF release from macrophages (105
, 106)
. It is clear from these studies that SAM exerts numerous
effects, from increasing membrane fluidity to altering gene expression.
Recently a randomized double-blind trial using SAM in patients with
alcoholic liver disease reported improved survival in SAM-treated
patients with less advanced liver disease (152)
. However,
the mechanism of protection was not investigated. With the wide
availability of SAM, it is important to examine the molecular
mechanisms of SAM’s protective effect at different stages of liver
disease.
Phosphatidylcholine
Depletion of SAM may also lead to hypomethylation of
phosphatidylethanolamine and a consequent reduction in the hepatic
level of phosphatidylcholine, the methylation product. This condition
is aggravated by known inhibition in ALD of the activity of
phosphatidylethanolamine methyltransferase (153)
, an
enzyme that catalyzes this methylation process. Administration of
phosphatidylcholine has been advocated for prevention and treatment of
ALD. Polyenylphosphatidylcholine is shown to protect the baboon against
alcohol-induced fibrosis and cirrhosis (154)
. Underlying
mechanisms for the observed anti-fibrotic effects may include induction
of collagenase activity (155)
or suppressed
platelet-derived growth factor (PDGF) -induced proliferation of hepatic
stellate cells (156)
, protection against oxidant stress
(157)
, and suppression of CYP2E1 inducibility
(158)
; a clinical trial is under way. Additional
experimental and clinical studies are needed to fully understand the
mechanisms and specificity of the therapeutic effects of
polyenylphosphatidylcholine on ALD.
Antioxidants
Antioxidants represent a potential group of therapeutic agents for
ALD. They likely provide beneficial effects on hepatocytes via
desensitization against oxidant stress while inhibiting priming
mechanisms for expression of proinflammatory and cytotoxic mediators
via suppression of NF-B (102
, 159
)
. The question
remains as to how one can achieve antioxidant-mediated,
antiinflammatory effects without suppressing the anti-apoptotic
effects of NF-B. Potential approaches may include cell type-specific
targeting of antioxidant therapy and development of modalities for more
specific and selective regulation of NF-B-mediated signaling.
Insulin sensitization
Insulin resistance is a common finding in ALD (160)
.
As discussed above, the livers of alcohol-fed animals exhibit features
similar to obese mice with insulin resistance, including induced TNF
expression, hepatocellular ATP depletion, susceptibility to necrosis
induced by ischemia, and hypoxia (85)
. These findings
suggest that insulin resistance may be of pathogenetic importance. A
recent work demonstrates striking beneficial effects of metformin, an
insulin-sensitizing agent, on fatty liver disease in insulin-resistant,
obese mice including reversal of hepatomegaly, hepatic steatosis,
plasma aminotransferase levels, hepatic ATP depletion, and TNF
induction (161)
. This suggests the potential therapeutic
effects of insulin-sensitizing agents such as PPAR ligands on
ALD.
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ACKNOWLEDGMENTS |
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REFERENCES |
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B activation in alcoholic liver injury. Am. J. Physiol. 277,G1240-G1250
SAM). SAM depletion causes reduced synthesis of glutathione (GSH)
and may impair transport of GSH to mitochondria, causing a selective
decrease in the mitochondrial pool of GSH (
ROS) by CYP2E1 or mitochondria.
Acetaldehyde (Ach) and lipid peroxidation aldehydes such as
malondialdehyde (MA) may impair transport via protein adduct formation
while giving rise to a new hybrid adduct, MAA. This adduct is highly
immunogenic and may incite both humoral and cellular immune responses.
Endotoxin-mediated activation of hepatic macrophages (HM) appears to be
central to the ALD pathogenesis. Critical paracrine effects of
activated HM include ROS or TNF-