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Nonalcoholic fatty liver (NAFL) and steatohepatitis (NASH) may accompany obesity, diabetes, parenteral nutrition, jejeuno-ileal bypass, and chronic inflammatory bowel disease. Currently there is no FDA approved and effective therapy available. We investigated the potential efficacy of those agents that stimulate glutathione (GSH) biosynthesis on the development of experimental steatohepatitis. Rats fed (ad libitum) amino acid based methionine-choline deficient (MCD) diet were further gavaged with (1) vehicle (MCD), (2) S-adenosylmethionine (SAMe), or (3) 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA). Results: MCD diet significantly reduced hematocrit, and this abnormality improved in the treated groups (p < 0.01). Serum transaminases were considerably elevated (AST: 5.8-fold; ALT: 3.22-fold) in MCD rats. However, administration of GSH-enhancing agents significantly suppressed these abnormal enzyme activities. MCD rats developed severe liver pathology manifested by fatty degeneration, inflammation, and necrosis, which significantly improved with therapy. Blood levels of GSH were significantly depleted in MCD rats but normalized in the treated groups. Finally, RT-PCR measurements showed a significant upregulation of genes involved in tissue remodeling and fibrosis (matrix metalloproteinases, collagen-α1), suppressor of cytokines signaling1, and the inflammatory cytokines (IL-1β, IL-6, TNF-α, and TGF-β) in the livers of rats fed MCD. GSH-enhancing therapies significantly attenuated the expression of deleterious proinflammatory and fibrogenic genes in this dietary model. This is the first report that oral administration of SAMe and PTCA provide protection against liver injury in this model and suggests therapeutic applications of these compounds in NASH patients.
In 1980, the term “nonalcoholic steatohepatitis (NASH)” was coined to describe a new syndrome occurring in patients who usually were obese females who had a liver biopsy picture consistent with alcoholic hepatitis, but who denied alcohol use . The cause(s) of this syndrome were unknown, and there was no defined therapy. Over two decades later, this clinical syndrome is somewhat better understood, but there remains no FDA-approved or even generally accepted drug therapy. Patients with “primary” NASH usually have the metabolic syndrome which is characterized by obesity, hyperlipidemia, hypertension, and in some instances, other metabolic abnormalities such as polycystic ovary disease . The etiology of NASH remains elusive, but most investigators agree that a baseline of steatosis requires a second “hit” capable of inducing inflammation, fibrosis, or necrosis in order to develop NASH. Oxidative stress, with the accumulation of lipid peroxidation products, mitochondrial dysfunction [3-5], endotoxins derived from the gut, and inflammatory cytokines in the liver have been implicated in the initiation of liver injury in NASH [5-8]. Glutathione (GSH) is a major endogenous antioxidant, and GSH prodrugs are attractive therapy for many forms of liver injury. Methionine, a sulfur amino acid which is converted to cysteine, serves as a precursor for the biosynthesis of GSH. Reactive oxygen species (ROS) promote oxidative damage in varying cellular constituents, including amino acids, lipids, and nucleic acids, and play a critical role in the development of liver injury [9,10]. S-adenosylmethionine (also termed AdoMet, SAMe, and SAM); SAMe will be used to denote both the natural metabolite and therapeutic agent in this article is an over-the-counter complementary and alternative medicine (CAM) agent. SAMe is an important methyl donor and antioxidant/glutathione-enhancing agent [9,11]. SAMe can be produced in all cells, but the liver is the primary organ responsible for the conversion of dietary methionine to SAMe. Another GSH enhancer, 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA), serves as a prodrug for l-cysteine in GSH biosynthesis, and has been shown to be an effective therapeutic agent for the treatment of acetaminophen hepatotoxicity and in a murine model of colitis [12,13]. The aim of the present study was to evaluate the potential efficacy of two agents (SAMe and PTCA) that stimulate GSH biosynthesis in a dietary-induced model of NASH.
2(RS)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA) was synthesized and kindly provided by Dr. H. T. Nagasawa . PTCA is a prodrug of l-cysteine with masked sulfhydryl groups to stabilize it against air oxidation in the form of thiazolidine-4-carboxylic acids. S-adenosylmethionine (SAMe), as its 1,4-butanedisulfonate salt, was provided by Knoll Pharmaceuticals, Piscataway, NJ. Methionine-choline deficient diet (MCD) and methionine-choline sufficient diet (MCS) were prepared from amino acids purchased from ICN Biochemicals (Cleveland, OH). The MCD diet was deficient only in choline and methionine and contained sufficient amounts of all other nutrients similar to MCS diet. These diets were stored at 4°C when not in use. RT-PCR reagents were obtained from Invitrogen (Carlsbad, CA). Liver enzymes kits and other chemicals were purchased from Sigma Chemical Co (St. Louis, MO).
Specific pathogen-free Sprague Dawley male rats 4–6 weeks of age (100 g) were purchased from Sprague Dawley (Indianapolis, IN) and housed in microfilter top cages at the University of Louisville Animal Research Resources Center. Animals were maintained at 22°C with a 12:12-h light/dark cycle and fed rodent chow and water ad libitum. This experimental study was approved and performed in accordance with the guidelines for Institutional Animal Care and Use Committee (IACUC) of the University of Louisville Research Resource Center, Louisville, KY, which is certified by the American Association of Accreditation of Laboratory Animal Care.
After an initial three days of acclimation, the rats were randomly fed (ad libitum) one of the following amino acid based diets: (A) methionine choline deficient (MCD) or (B) methionine choline sufficient diet (MCS). Three weeks after initiation of the study, rats on the MCD diet were further divided into three groups (9/each), and gavaged intragastrically with (1) vehicle/sucrose (MCD), (2) S-adenosylmethionine (SAMe), a GSH-enhancing agent that is an obligatory intermediate in the conversion of methionine to cysteine in the hepatic transmethylation pathway, or (3) 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA) which is involved in GSH biosynthesis following the liberation of l-cysteine in vivo. Each animal was orally administered (once a day) with the assigned regimen for two additional weeks at a concentration of 1% of body weight. The animals received food and water ad libitum.
Treatments were continued for 2 weeks until animals were killed by halothane overexposure.
Immediately after euthanizing rats with an overdose of halothane inhalation, blood was collected from the right ventricle of the heart into a syringe containing a minute amount of heparin and placed on ice. Plasma was separated by centrifugation at 5000×g for 5 min at 4°C. Samples were kept at −80°C until further analysis.
Liver was perfused with phosphate buffer saline (PBS). Samples from liver and intestines were collected and fixed in formalin for histopathology, and the remaining tissue was immediately snap-frozen in liquid nitrogen and stored at −70°C for analysis of GSH, oxidized glutathione (GSSG), SAMe concentrations, and molecular analysis.
Blood samples were collected in heparinized tubes, and a 20% homogenate in 5% metaphosphoric acid was prepared. After standing for 30 min on ice, the homogenate was centrifuged for 10 min (10,000×g) and the acid-soluble fraction was collected for measurement of sulfhydryl and disulfide compounds. Tissue homogenates (10%, w/v) were prepared in 5% (w/v) metaphosphoric acid, using all-glass Tenbroeck homogenizers, and kept on ice. After standing for 20–40 min, the homogenates were centrifuged for 1 min (10,000×g) and the acid soluble fractions collected for measurement of free thiol-disulfides.
GSH, GSSG, cysteine, and cystine were simultaneously quantified by high-performance liquid chromatography with dual electrochemical detection (HPLC-DEC) according to the method of Chen et al. . In brief, 20-μL samples were injected onto a 250 × 4.6 mm, 5 μm, C-18 column (Val-U-Pak HP, fully end capped ODS, 5 μm, 250 × 4.6 mm; Chrom Tech Inc., Apple Valley, MN). Samples (20 μL) were injected onto the column and eluted isocratically with a mobile phase consisting of 0.1 M monochloroacetic acid, 2 mM heptane sulfonic acid, and 2% acetonitrile at pH 2.8 and delivered at a flow rate of 1 mL/min. The compounds were detected in the eluant with a Bioanalytical Systems model LC4B dual electrochemical detector using two Au-Hg electrodes in series with potentials of −1.2 V and 0.15 V for the upstream and downstream electrodes, respectively. Current (nA) was measured at the downstream electrode. Analytes were quantified from peak area measurements using authentic external standards.
Deproteinized tissue extracts (4% metaphosphoric acid) and blood were prepared, and SAMe was determined by an HPLC method, using a 5-μm Hypersil C-18 column (250 × 4.6 mm). The mobile phase consisted of 40 mM ammonium phosphate, 8 mM heptane sulfonic acid (ion-pairing reagent, pH 5.0), and 6% acetonitrile and was delivered at a flow rate of 1.0 mL/min. SAMe was detected using a Waters 740 UV detector at 254 nm. An internal standard, S-adenosylethionine (SAE), was added to all samples and standard solutions to a concentration of 100 nmol/mL. Protein concentrations were measured by protein assay kit from Bio-Rad (Bio-Rad Laboratories, Hercules, CA) in accordance with the manufacturer’s instructions.
Serum transaminase activities (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) were measured colorimetrically using a diagnostic kit (procedure no. 505, Sigma Chemical Co., St. Louis, MO) according to the instructions provided.
A small portion of the right lobe from liver tissues was placed in cassettes and fixed with 10% neutral formalin. The specimens were dehydrated and embedded in paraffin, and tissue sections of 5 μm were stained by hematoxylin and eosin (H&E). Each slide was coded and evaluated by the two of the coauthors using a light microscopy. Hepatic lesions were graded on a scale of 0 to 4+, based on fatty degeneration, inflammation, and necrosis as follows:
Total RNA was extracted from 500 mg of liver tissue using TRIzol reagent (Invitrogen), and the homogenate was centrifuged. The supernatant was mixed with 300 μL of bromochlorophenol (BCP), and the mixture was centrifuged for 20 min. The upper aqueous phase containing cellular total RNA was extracted, and BCP step was repeated. The aqueous phase was then mixed with 750 μL of isopropanol and equal amount of the high salt solution and incubated overnight at −20°C. The mixture was centrifuged, and the total RNA pellet collected. The integrity of the RNA was confirmed by visualizing the 18S and 28S ribosomal fragments on an agarose gel.
The total extracted RNA (2 μg) was reverse transcribed using oligo (dT) which served as a primer for the first strand complementary DNA (cDNA) synthesis with the addition of 1× PCR buffer (5 mM MgCl2, 1 M each of dNTPs, 1U/μL of RNAse inhibitor) and 2.5U/μL of ThermoScript reverse transcriptase RT-PCR system (Invitrogen, Carlsbad, CA). The sequences for the primers and the conditions for their use are summarized in Table 1. Each RT sample was assessed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. Genomic DNA was included for the PCR to ensure that there was no genomic DNA contamination in the total RNA samples. The reaction was performed at 42°C for 60 min followed by heat inactivation of the enzymes at 95°C for 5 min, using an Eppendorf mastercycler gradient thermal cycler (Eppendorf Scientific, Westbury, NY). The cDNA was then subjected to amplification by Taq polymerase (Universal PCR, Invitrogen). The reaction mixture was then denatured at 95°C for 2 min, followed by a cyclic procedure of denaturation at 95°C for 45 s, annealing for 30 s, and elongation for 45 s. The annealing temperature, calculated for each pair of sense and antisense primers, is shown in Table 1. This procedure was repeated for 25–35 cycles. Following these amplification steps, a final extension period was carried out at 72°C for 7 min. The PCR products were then resolved on a 1.8% agarose gel prepared in 100 mM Tris, 90 mM boric acid, and 1 mM EDTA buffer, and the size of the PCR products was determined using an appropriate DNA size marker. The bands were visualized by ethidium bromide staining and UV transillumination. Integrated density values for the genes in question were normalized to the GAPDH values to yield a semiquantitative assessment.
All data are expressed as mean ± SEM. Statistical analysis was performed using ANOVA. The data were further analyzed by post hoc test for statistical difference (Tukey–Kramer multiple comparison test). Differences between groups were considered to be statistically significant at P < 0.05.
Young rats (4–6 weeks old, weighing approximately 100 g) fed amino acid based MCD diet for five consecutive weeks showed lack of weight gain (MCD 68% of MCS) (Table 2), otherwise, the rats appeared normal. GSH-enhancing agents (SAMe or PTCA) administered for two consecutive weeks did not significantly correct this abnormality. Hematocrit was significantly decreased in MCD rats (MCD vs. MCS p < 0.01), and this abnormality improved in the treated groups. The serum transaminases, aspartate aminotransferase (AST: 5.8-fold) and alanine aminotransferase (ALT: 3.2-fold), were considerably elevated in MCD animals (MCD vs. MCS p < 0.001). However, GSH-enhancing agents significantly suppressed these abnormal enzyme levels (MCD vs. treated p < 0.001) (Figure 1). MCD rats developed severe liver lesions manifested by fatty degeneration, inflammation, and necrosis (MCD vs. MCS p < 0.001) which was improved with GSH-enhancing therapy (treated vs. MCD p < 0.05, MCD vs. MCS p < 0.01) (Figure 2, panels A–D). The blood level of GSH was significantly decreased in MCD rats (848 ± 55 nmol/mL) compared to MCS (1482 ± 90 nmol/mL), and levels were restored to normal with therapies. There was a modest decrease in liver-reduced GSH level and increased level of GSSG in MCD rats (MCD vs. MCS p < 0.05). The endogenous liver SAMe was significantly decreased in rats fed MCD diet (MCD 7 ± 0.8; MCS 59.2 ± 4.5, nmol/g; p < 0.001). Therapies partially but significantly improved this abnormality (treated vs. MCD p < 0.05) (Table 2). Livers from rats on normal diet (MCS) showed minimal expression of inflammatory cytokines and genes involved in tissue remodeling and fibrosis. RT-PCR analysis showed significant upregulation of matrix metalloproteinase, MMP-9 (8.5-fold), MMP-13 (3.4-fold), collagen-α1 (9.2-fold), and suppressor of cytokines signaling 1 (SOCS1, 2.1-fold) expression in MCD animals, (Figures 3 and and4).4). These findings were consistent with the increased expression of genes for the inflammatory/fibrotic cytokines, (IL-1β (7-fold), IL-6 (17-fold), TNF-α (6-fold)), and TGF-β (3-fold) in MCD rats. Treatment with GSH-enhancing agents significantly improved expression of some of these proinflammatory and fibrogenic genes (Figures 5–7). The results of this study are summarized in Tables 2 and and33.
Reactive oxygen species (ROS) promote oxidative damage and contribute to tissue destruction in a wide variety of diseases , including steatohepatitis . GSH, a potent endogenous antioxidant which neutralizes free radicals, is severely depleted in a variety of forms of liver injury, acetaminophen hepatotoxicity serving as the prototype . GSH deficiency may lead to severe injuries such as sepsis, organ failure, and death .
NASH develops in patients with the obesity-hypertriglyceridemia-insulin resistance syndrome, and can be simulated by chronic chemical intoxication  as well as by the MCD dietary model. It is postulated that both lipid peroxidation and ROS-induced cytokine release (TGF-β, TNF-α, IL-8) may contribute to NASH . In this study, young rats maintained on diets containing sufficient amounts of amino acids (MCS) had normal liver histology with low levels of serum transaminases and inflammatory gene expression. All animals on the MCD diet for 5 weeks showed lack of weight gain. MCD diet also caused anemia with decreased hematocrits (MCD vs. MCS p < 0.01), severe hepatocyte injury (steatosis, neutrophil infiltration) and elevation in serum aspartate transaminase (AST, 5.8-fold), and alanine transaminase (ALT, 3.2-fold) enzyme levels. Animals fed the MCD diet developed marked depletion of blood GSH (848 ± 55 nmol/mL) when compared to those fed the MCS diet (1482 ± 90 nmol/mL), and MCD rats had decreased liver GSH and endogenous SAMe concentrations (Table 1).
Oral administration of SAMe and PTCA significantly attenuated liver damage and improved hematocrit levels. PTCA significantly reduced the serum levels of AST and ALT to normal levels, and had a modests beneficial effect on liver SAMe concentrations compared to MCD animals. Oral administration of SAMe similarly reduced the abnormal liver enzyme activity. SAMe corrected the blood deficiency of GSH to normal level and partially increased the hepatic SAMe concentration.
SAMe deficiency is often associated with GSH deficiency [3,8], since SAMe enhances hepatic GSH. Endogenous liver SAMe is also a substrate for the de novo biosynthesis of choline. SAMe deficiency occurs in liver injuries such as alcoholic  and acetaminophen overdoses , and these injuries may be attenuated by the administration of SAMe.
We observed significantly increased (7.7- fold) gene expression of TNF-α with RT-PCR in the livers of MCD animals consistent with increased TNF-α previously reported by us in MCD rats . TNF-α is mainly released from sensitized Kupffer cells and damages hepatocytes. TNF-α cytotoxicity is mediated, at least in part, through mitochondrial dysfunction and oxidative stress . PTCA and SAMe administration significantly blunted TNF-α (2.8-fold vs. MCS) genes expressed in MCD animals in this study. These findings in general support the biological antioxidant effects of oral administration of these agents in acetaminophen-induced hepatotoxicity  and inflammatory bowel disease in a DSS induced colitis model  as well as alcohol-induced liver injury  by improving GSH levels and reducing TNF-α concentration. SAMe also may attenuate TNF-α cytotoxicity by correcting the mitochondrial abnormalities , especially mitochondrial GSH depletion in alcoholic liver disease.
There was a significant upregulation of other genes involved in inflammation and fibrosis such as, IL-6 (17-fold), IL-1β (7-fold), and TGF-β (3-fold) in rats on MCD diet analyzed by RT-PCR. TGF-β is an important profibrogenic cytokine. TGF-β, an important profibrogenic cytokine, is an activator and survival factor for HSCs, and alters gene expression to favor extracellular matrix deposition. Elevated levels of TGF-β have been detected in fibrotic human liver, cirrhotic rat liver, and in the livers of rats fed the MCD diet  (where TGF-β likely plays an important role in the development of hepatic fibrosis . Simultaneously, expression of genes involved in expression of matrix structure such as MMP-9 (gelatinase-B, 8.5-fold), MMP-13 (4-fold), collagen-α1 (9.2-fold), and SOCS1 were upregulated in deficient animals. Moreover, inflammatory/fibrotic cytokine gene expression was significantly improved in PTCA and SAMe treated rats.
In our study, GSH-enhancing therapy provided protection against liver injury induced by deficient diet and significantly inhibited the expression of genes involved in fibrosis. Matrix metalloproteinases (MMPs), Zn2+-containing endopeptidases, are the mediators of tissue damage in inflammatory diseases and play a key role in degrading extracellular matrix (ECM) components. MMP activation has been implicated in pathological tissue remodeling, including inflammation, cancer invasion, and fibrosis . Activated MMPs trigger the degradation of ECM components such as collagens, gelatin, and fibronectin. MMP-9 (gelatinases B) expression is induced by proinflammatory stimuli including cytokines, chemokines, and bacterial wall components [25,26], and data suggest a role for MMPs, especially MMP-9, in the development of NASH. Steatohepatitis may lead to hepatic fibrosis. Hepatic fibrosis, which involves excess deposition of extracellular connective tissue, predominantly collagen type I fibers, may progress to cirrhosis. During liver inflammation, IL-1, produced by the Kupffer cells, plays a pivotal role in the remodeling of liver fibrosis by regulation and expression of MMP-9 and MMP-13 mRNA and collagen degradation that leads to apoptosis. Hepatic fibrosis results from excess extracellular matrix produced primarily by hepatic stellate cells (HSC). Normally, MMP expression is regulated by inflammatory mediators such as growth factors and proinflammatory cytokines . MMP-9 attacks and degrades basement membrane collagens and gelatins of all types [28,29]. Polymorphonuclear leukocytes (PMNs) and endothelial cells contain large amounts of MMP-9 in secretory vesicles , which enable the rapid release of MMP-9 following specific stimuli . MMP-9 has been shown to cleave inactive TNF-α and release activated TNF-α . In addition, MMP-9 gene expression by macrophages and PMNs can be induced by TNF-α and IL-1β [28,30]. MMPs may play a role both in matrix degradation with cellular infiltration as well as active TNF-α release in mediating hepatotoxicity.
SOCS1 gene (the suppressor of the cytokine signaling-1) is a negative regulator of cytokine signaling by DNA methylation, and has been implicated in the development or progression of HCC and fibrosis. The severity of liver fibrosis in patients is reported to strongly correlate with SOCS1 gene methylation in this study. SOCS1 expression was significantly (2.1-fold) upregulated in the livers from MCD animals. However, GSH-enhancing (SAMe and PTCA) therapy did not meaningfully affect SOCS1 gene expression. These findings suggested that SOCS1 contributes to protection against hepatic injury and fibrosis, and may also protect against hepatocarcinogenesis.
Our previous studies  also indicated that GSH and/or cysteine deficiency occurs in aging tissues as well as following acetaminophen administration. PTCA protected against GSH deficiency caused by aging . SAMe and PTCA also protected against GSH deficiency due to acetaminophen hepatotoxicity  and dextran sodium sulfate (DSS) induced colitis  when orally supplemented into diets. These studies support the concept that the protective effect of SAMe and PTCA (GSH prodrugs) are mediated by prompting the biosynthesis of hepatic GSH. Finally orally administered SAMe  as well as PTCA  have been reported to be well tolerated and safe.
Indeed, SAMe is sold as an over-the-counter (complementary and alternative therapy) compound in the United States, and not requiring a prescription. New drugs are required, as life style changes such as weight management to treat NASH are frequently unsuccessful.
This study has some limitation that findings provide an initial observation that GSH-enhancing therapies improve this diet-induced NASH, but mechanisms need to be further defined (e.g., do GSH-enhancing agents influence transcription factors?). Animals were not long enough to develop major fibrosis, and more extended studies are required with evaluation of stellate cell cultivation. How GSH-enhancing agents provide protection also requires further research (necrosis, apoptosis, mitochondrial function, etc.). However, these findings clearly indicate a need for further research concerning GSH-enhancing agents in NASH.
In conclusion, this is the first report that oral administration of GSH-enhancing agents protects against liver injury in this dietary model of NASH.
PTCA was synthesized and kindly provided by Dr. Herbert T. Nagasawa from the Department of Medicinal Chemistry, University of Minnesota. Prasuna Muddasani, MS, Seth L. Stephens, BS, and Marcia C. Liu provided technical assistance.
Contract Grant Sponsor: Martha H. Stege Endowment Fund.
Contract Grant Number: 423574.
Contract Grant Sponsor: NCCAM.
Contract Grant Number: AT1490-01A1.
Contract Grant Sponsor: NIH.
Contract Grant Numbers: RO1AA010496, R37AA010762.
Contract Grant Sponsor: VAMC.
This research was partially presented at AASLD, DDW 2005, in Chicago IL (Gastroenterology Suppl 2,128:4 A-686, 183), and the 7th International Cytokines Chemokines Symposium, Montreal Canada (2005) and granted NIAAA travel award.