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Am J Physiol Gastrointest Liver Physiol 291: G55-G62, 2006. First published January 26, 2006; doi:10.1152/ajpgi.00360.2005
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LIVER AND BILIARY TRACT

Roles of phosphatidylinositol 3-kinase and osteopontin in steatosis and aminotransferase release by hepatocytes treated with methionine-choline-deficient medium

Atul Sahai, Xiaomin Pan, Rachelle Paul, Padmini Malladi, Rohit Kohli, and Peter F. Whitington

Department of Pediatrics, Northwestern University Feinberg School of Medicine and Children's Memorial Research Center, Chicago, Illinois

Submitted 2 August 2005 ; accepted in final form 23 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Feeding mice a methionine and choline-deficient (MCD) diet serves as an experimental animal model for nonalcoholic steatohepatitis (NASH). In the present study we examined the effect of exposing AML-12 hepatocytes to MCD culture medium in regard to mechanisms of steatosis and alanine amino-transferase (ALT) release. Cells exposed to MCD medium developed significant and progressive steatosis from 6 to 24 h and also had significantly increased loss of ALT into the medium at 18 and 24 hours of incubation. No increased oxidative injury or cell death was observed. Osteopontin (OPN) mRNA in cells and protein expression in medium were significantly increased during 6–24 hours of incubation. MCD medium treatment also resulted in activation of PI3-kinase by 30 minutes and its downstream target p-Akt within 1hour of incubation. Steatosis was associated with increased expression of microsomal triglyceride transfer protein (MTTP) mRNA and increased ALT release with over expression of ALT mRNA, all of which were completely prevented by inhibition of PI3-kinase (LY294002). Blocking OPN signaling by treating with anti-OPN or anti-beta3-integrin antibody prevented the increased ALT release while only partially prevented the increased ALT mRNA expression, but had no effect on either steatosis or MTTP expression. In conclusion, incubation of cultured hepatocytes with MCD medium results in cellular steatosis and OPN dependent ALT release. PI3-kinase plays a central role in signaling the MCD medium-induced steatosis and increased OPN expression, whereas OPN appears to play a role in signaling hepatocyte ALT release but not steatosis.

nonalcoholic fatty liver; experimental steatosis

THE ADMINISTRATION OF A diet deficient in methionine and choline (MCD diet) to mice produces experimental steatohepatitis that simulates human nonalcoholic steatohepatitis (NASH) (14, 36). We have demonstrated in this model that osteopontin (OPN) plays an important role in the progression from steatosis to steatohepatitis and liver fibrosis (30). OPN is synthesized and secreted by immune cells as well as epithelial, endothelial, and smooth muscle cells (19) and has been shown to function as a Th1 cytokine in the pathogenesis of fibroinflammatory diseases in various organ systems including the liver (1, 8, 9, 12, 13, 16, 21, 24, 37). We have shown that hepatocytes in culture synthesize and secrete OPN and that its expression is upregulated by TNF-{alpha}, transforming growth factor (TGF)-beta, and leptin (30). We also found that hepatic OPN expression increases experimental murine steatohepatitis early in MCD diet and that the increased expression localizes mainly to hepatocytes (30). Moreover, OPN knockout mice fed MCD diet show lower serum alanine aminotransferase (ALT) activity and develop markedly less liver fibrosis than do wild-type mice but show similar degrees of hepatic steatosis (30). Finally, we have shown that MCD diet fed to obese and Type 2 diabetic db/db mice exhibits greater increases in OPN expression than do outbred db/m littermates, which parallels the accelerated NASH they express when fed the MCD diet (31). Taken together, these findings suggest a role for OPN as a hepatocyte-derived Th1 cytokine in the pathogenesis of NASH.

The cellular and molecular mechanisms involved in the evolution of experimental NASH in the MCD diet model are incompletely understood. With regard to human NASH, it is generally believed that obesity and insulin resistance result in hepatic steatosis, which provides the environment for increased oxidative stress and peroxidative injury (7, 27, 32). Similarly, there is evidence of oxidative stress in the MCD diet model and other models of experimental NASH (14, 18, 28, 38). In both instances, it is thought that the induction of oxidative stress results in liver injury and fibrosis that characterize NASH. We, however, found in the MCD model of experimental NASH that increased OPN expression preceded the induction of collagen 1 mRNA expression by days and preceded significant oxidative stress and fibrosis by weeks (30). In addition, intact leptin signaling is considered to be essential for hepatic fibrosis to occur (15). We showed that increased OPN expression in MCD diet-fed db/db mice is accompanied by parallel increases in serum leptin and in the hepatic expression of short-form leptin receptors (31). Furthermore, leptin stimulates OPN expression by cultured mouse hepatocytes, a signal that appears to involve short-form leptin receptors (31). Leptin has also been shown to activate phosphatidylinositol (PI)3-kinase in primary cultures of rat hepatocytes (23, 33, 35). Activation of PI3-kinase is implicated in the control of cell growth and apoptosis in several organ systems including the liver (10, 17, 25, 29, 34). Moreover, the activation of PI3-kinase has been suggested to mediate OPN expression in various cell systems including HepG2 cells (39). The possible role of PI3-kinase in the regulation of OPN expression and in the development of steatosis, steatohepatitis, and fibrosis in experimental NASH is undefined.

In the present study, we examined the short-term effect of exposing cultured hepatocytes to MCD culture medium with regard to the development of steatosis and release of ALT into the medium and determined whether oxidative injury and/or cell death resulted from exposure to MCD medium. Finally, we examined the expressions of OPN and PI3-kinase and assessed their roles in signaling the MCD medium-induced hepatocyte steatosis and ALT release.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. DMEM/Ham's F-12 medium and identical medium that was manufactured to be completely deficient of methionine and choline (MCD medium) were purchased from Invitrogen (Carlsbad, CA). The mouse hepatocyte cell line AML-12 was obtained from American Type Culture Collection (Bethesda, MD). Monoclonal anti-OPN antibody MPIIIB10 was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences (Iowa City, IA). Polyclonal antibody to PI3-kinase p85 subunit was purchased from Upstate Biotechnology (Lake Placid, NY). Phospho-Akt and Akt antibodies were from Cell Signaling Technology (Beverly, MA). beta-Actin antibody was obtained from Sigma (St. Louis, MO). Neutralizing monoclonal antibody to beta3-integrin (OPN receptor, CD-61) was purchased from BD Biosciences Pharmingen (Mountain View, CA), and a specific inhibitor of PI3-kinase (LY294002) was purchased from Calbiochem (La Jolla, CA). All other reagents were of high chemical grade from Sigma.

Hepatocyte cell culture. AML-12 cells were grown in 1:1 mixture of DMEM-Ham's F-12 medium supplemented with 10% fetal bovine serum and antibiotics (growth medium) as previously described (30). Cells were subcultured in growth medium until the cultures reached 70–80% confluence, at which time they were made quiescent by incubation for 24 h in serum-free DMEM-Ham's F-12 medium. Quiescent cells were then exposed either to control growth medium or MCD medium supplemented with identical glucose and serum concentrations at 37°C. In preliminary studies, we found that these cells show toxicity if maintained in MCD medium for 48–72 h. There is increased cell death beginning at 48 h and very limited survival at 72 h. Therefore, data are given for exposure up to 24 h. At the end of each incubation time, the conditioned medium was decanted from the cells and concentrated using Ultrafree-4 centrifugal filter units (Millipore, Billerica, MA). In every experimental condition, 4.0 ml of cell-free medium were concentrated to 500 µl final volume, which was used to assess ALT activity and the protein expression of OPN. Cells were harvested to assess triglyceride content or processed for total RNA isolation. Cell lysates were used to examine total and phosphorylated PI3-kinase and Akt expression. In some experiments, cultures were exposed to MCD medium in the absence or presence of a specific inhibitor of PI3-kinase (LY294002) and antibodies to OPN or beta3-integrin to examine the roles of PI3-kinase and OPN in affecting steatosis and ALT release.

Assessment of medium ALT, hepatocyte steatosis, cell viability, apoptosis, and oxidative injury. ALT activity was determined by a spectrophotometric kit procedure from Biotron Diagnostics (Hemet, CA) and expressed as international units per liter. Cellular triglyceride levels were measured in cultures that were washed with cold PBS, scraped, and centrifuged. The cell pellet was suspended in 20 mM Tris buffer, pH 7.5, and homogenized. Cell triglyceride content in the whole homogenates was measured using a spectrophotometric kit procedure from Thermo DMA (Arlington, TX) and expressed as micrograms of triglyceride per milligram of protein. Hepatocyte steatosis was also analyzed histologically using Oil Red O dye. Cultures were grown on a coverslip, and quiescent cells were exposed to MCD or control medium for 18 h followed by 30 min of staining with Oil Red O dye (0.5 g Oil Red O in 100 ml isopropanol). The cells were briefly washed and mounted for microscopic examination. Cell viability was determined by trypan blue method (26). AML-12 cells were grown, subcultured, and made quiescent as described previously. After exposure to control growth medium or MCD medium, cells were washed twice with prewarmed PBS, stained with 0.8 mM trypan blue for 20 min, and rewashed twice with prewarmed PBS. Cell viability was evaluated by standard microscopy wherein dead cells stained blue. Cells were scraped free of the plastic and suspended in 4.0 ml cold PBS for determining total cell number by hemocytometer. Apoptosis was assessed by DNA fragmentation method. AML-12 cells were cultured as described previously to confluence, brought to quiescence with serum-free medium exposure for 24 h, and then incubated for 18 h in MCD medium. DNA was isolated using "Easy- DNA" kit from Invitrogen. Samples were run on a 2% agarose gel with ethidium bromide and photographed under ultraviolet illumination. As a measure of oxidative injury, thiobarbituric acid-reactive substances (TBARs) were measured in cell homogenates by a routine spectrophotometric kit procedure (Zeptometrix, Buffalo, NY) and expressed as nanomoles of malondialdehyde (MDA) produced per milligram of protein.

Real-time PCR analysis. Total RNA was isolated from cultured hepatocytes using TRIzol reagent (GIBCO-BRL, Grand Island, NY). One microgram of total RNA was reverse transcribed using iSCRIPT cDNA synthesis kit from Bio-Rad Laboratories, Hercules, CA. Real-time PCR was performed using 4 µl total cDNA in a 50-µl reaction volume containing QuantiTect SYBR Green PCR Master Mix (Qiagen, Valencia, CA) with the specific primers for mouse OPN and the housekeeping gene GAPDH (Integrated DNA Technologies, Coralville, IA). The primer sequences used are as follows: OPN: forward 5'-CAG CCT GCA AGA TCC TA-3'; reverse 5'-GCG CAA GGA GAT TCT GCT TCT-3'; microsomal triglyceride transfer protein (MTTP): forward 5'-GTG GAG GAA TCC TGA TGG TGA-3'; reverse 5'-TGA TCT TAG GTG TAC TTT TGC CC-3'; ALT2: forward 5'-CAG ACC CAG ACA ACA TTT ACC TG-3'; reverse 5'-CGC GGA GTA CAA GGG ATA CTG-3'; GAPDH: forward 5'-GTC GTG GAT CTG ACG TGC C-3'; reverse 5'-TGC CTG CTT CAC CAC CTT C-3'. Amplification was performed in duplicate for each sample in an ABI Prism 7500 real-time PCR system (Applied Biosystems, Foster City, CA) with a hold for 2 min at 50°C, denaturation for 10 min at 95°C, followed by 40 PCR cycles of denaturation at 95°C for 15 s, and annealing at 60°C for 60 s. The relative amount of mRNA was calculated using GAPDH as the endogenous control.

Western blot analysis. Immunoreactive levels of PI3-kinase, p-Akt, and Akt in the cell lysates and of OPN in conditioned medium were assessed by Western blot analysis as previously described (30). Briefly, cells were scraped off the flask in cold PBS and centrifuged. The cell pellet was dissolved in a lysis buffer (50 mM Tris·HCl, pH 7.4, containing 150 mM NaCl, 25 mM EDTA, 5 mM EGTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, and 1 mM DTT) containing protease inhibitor cocktail (Calbiochem, La Jolla, CA). For gel electrophoresis, samples of cell homogenate containing 25–50 µg protein were applied. For measurement of protein in conditioned medium, in every experiment, 4.0 ml medium were concentrated to 0.5 ml (8-fold concentration), and samples consisting of 150 µl of concentrated medium were applied. Proteins were resolved by 10% SDS polyacrylamide gel electrophoresis under reducing conditions and then transferred overnight onto nitrocellulose membranes by electrophoresis. Specific primary antibodies to OPN, PI3-kinase p85 subunit, phospho-Akt, and Akt were used to assess their expressions. beta-Actin antibody was used to confirm equal protein loading among samples. The bound primary antibodies were detected with a respective horseradish peroxidase-conjugated secondary antibody (1:1,000 dilution, Amersham, Arlington Heights, IL) and visualized with an enhanced chemiluminescence method. Protein expression levels were determined by densitometry.

Statistical analysis. Comparison between groups was performed using Student's t-test for unpaired samples or by ANOVA. A P value of <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of MCD medium on hepatocyte steatosis, medium ALT, cell viability, apoptosis, and oxidative injury. We first examined the effect of MCD medium on hepatocyte steatosis. Incubation of cultured hepatocytes with MCD medium resulted in progressive increases in cell triglyceride content beginning at 6 h with a maximal threefold increase observed at 24 h (Fig. 1A). Oil Red O staining of MCD medium-treated hepatocytes for 18 h showed a marked increase in neutral fat within hepatocytes compared with cells incubated with control medium (Fig. 1B).


Figure 1
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  		Fig. 1. Effect of methionine-choline-deficient (MCD) medium on fat accumulation in cultured hepatocytes. Cultures were exposed to control medium or MCD medium for 6–24 h, and cell triglyceride content (A) was assessed. Each value is the mean ± SE of 5 separate determinations (A). *P < 0.01 vs. control. Histological steatosis assessed by Oil-Red O staining at 18 h shows neutral fat (arrows) within the hepatocytes (B).

 
ALT release was determined by measuring its activity in the concentrated conditioned medium. As shown in Fig. 2, treatment with MCD medium resulted in a twofold increase in medium ALT by 18 h, which then declined slightly by 24 h of examination. Cell viability, as determined by trypan blue exclusion, and number of adherent cells were not affected by incubating with MCD medium for 6–24 h (data not shown). Furthermore, no apoptosis could be detected by DNA laddering at any time point (data not shown). TBARs were measured in cells exposed to MCD or control medium for 24 h to assess oxidative injury. Incubating in MCD medium produced no significant difference in TBARs, 0.24 ± 0.10 vs. 0.14 ± 0.04 nmol/mg protein for control medium (P = not significant), indicating no significant increase in global oxidative injury induced by MCD medium.


Figure 2
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  		Fig. 2. Effect of MCD medium on alanine aminotransferase (ALT) release in cultured hepatocytes. Cultures were exposed to control medium or MCD medium for the indicated time, and ALT activity was measured in the concentrated conditioned medium. Each value is the mean ± SE of 5 separate determinations. *P < 0.01 vs. control.

 
Role of OPN in MCD medium-induced steatosis and ALT release. We examined the effect of MCD medium on OPN mRNA and protein expression. Exposure to MCD medium induced significant increases in OPN mRNA levels beginning at 6 h with an ~10-fold increase observed at 18–24 h of examination (Fig. 3). Similarly, significant increases in OPN protein expression were observed at 6 h of treatment and remained elevated for up to 24 h of incubation (Fig. 4).


Figure 3
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  		Fig. 3. Effect of MCD medium on osteopontin (OPN) mRNA expression in cultured hepatocytes. Cultures were exposed to control medium or MCD medium for 1–24 h followed by collection of cells and assessment of OPN mRNA levels by real-time PCR (mean ± SE, n = 4 for each analysis). *P < 0.01 vs. control.

 

Figure 4
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  		Fig. 4. Effect of MCD medium on OPN protein expression and secretion by cultured hepatocytes. Cultures were incubated with control medium or MCD medium for 6–24 h, and OPN protein expression in conditioned media was determined by Western blot with loading by volume at 150 µl per lane. Bottom shows the concentration of OPN as determined by densitometry expressed as fold increase vs. control (mean ± SE, n = 4 for each analysis). *P < 0.01 vs. control.

 
To assess the possible role of OPN in MCD medium-induced hepatocyte fat accumulation and ALT release, an anti-OPN antibody or a neutralizing antibody to OPN receptor beta3-integrin was used. Cultures were exposed to MCD or control medium for 18 h in the absence or presence of either anti-OPN antibody or beta3-integrin-neutralizing antibody followed by measurement of cell triglyceride content and medium ALT. Treatment with either anti-OPN or beta3-integrin receptor antibody completely prevented the increase in medium ALT induced by the MCD medium (Fig. 5A). In contrast, OPN or beta3-integrin receptor antibody had no significant effect on steatosis induced by the MCD medium (Fig. 5B). These antibodies had no significant effect on medium ALT or triglyceride content in cells incubated with control medium (data not shown).


Figure 5
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  		Fig. 5. Effect of neutralizing antibodies (ab) to OPN and its beta3-intregin (Int) receptors on MCD medium-induced ALT release (A) and cell triglyceride content (B) of cultured hepatocytes. Cultures were exposed to control medium or MCD medium for 18 h in the absence or presence of the antibodies, and ALT activity in concentrated medium and cell triglyceride content were determined (mean ± SE, n = 3 for each measurement). *P < 0.01 vs. control; **P < 0.01 vs. MCD; and P not significant (NS) vs. control.

 
Effect of MCD medium on PI3-kinase and p-Akt activities. We determined the effect of MCD medium on PI3-kinase and its downstream target p-Akt by Western blot analysis. As shown in Fig. 6A, MCD medium induced a significant increase in PI3-kinase beginning at 1 h of incubation, which remained elevated through 12 h of examination and declined to control levels at 18 h of examination. Treatment with MCD medium produced a sustained increase in p-Akt levels during 1–18 h of incubation (Fig. 6B). Immunoreactive levels of Akt protein were not altered during this incubation time, indicating the specificity of the changes in p-Akt induced by the MCD medium (Fig. 6B).


Figure 6
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  		Fig. 6. Effect of MCD medium on phosphatidylinositol (PI)3-kinase activation (A) and p-Akt (B) in cultured hepatocytes. Cultures were incubated in the MCD medium for the indicated time, and the immunoreactive levels of PI3-kinase p85 subunit and p-Akt were assessed by Western blot analysis, with beta-actin and total Akt used for comparison of loading, respectively. When densitometry was used to quantitate protein expression (n = 3 for each measurement), phosphorylated PI3-kinase was increased vs. control at 30 min through 12 h (P < 0.01 at each point) but not at 18 h, whereas Akt was increased vs. control from 1 through 18 h (P < 0.01 vs. control for each point).

 
Role of PI3-kinase in the regulation of OPN expression and in the development of fat accumulation and ALT release. Subsequent studies examined the role of PI3-kinase in MCD medium-induced stimulation of OPN expression, steatosis, and ALT release. Cultures were exposed to MCD or control medium for 18 h in the absence or presence of a specific inhibitor of PI3-kinase (LY294002, 10 or 25 µM), and OPN expression, medium ALT, and cell triglyceride content were assessed. PI3-kinase inhibitor blocked the MCD medium-induced increases of OPN mRNA expression in a dose-dependent manner, with 25 µM inhibitor completely preventing the effect (Fig. 7A). The inhibitor also blocked the increase in OPN protein expression induced by the MCD medium (Fig. 7B). Moreover, the PI3-kinase inhibitor prevented any MCD medium-induced increases in medium ALT (Fig. 8A) and cell triglyceride content (Fig. 8C). The inhibitor had no effect in cultures incubated with control medium (data not shown).


Figure 7
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  		Fig. 7. Effect of PI3-kinase inhibitor (Inh) in MCD medium-induced increase in OPN mRNA (A) and protein (B) expression by cultured hepatocytes. Cultures were exposed to MCD medium for 18 h with and without the inhibitor of PI3-kinase (LY294002). OPN mRNA was measured in cells by real-time PCR and expressed as fold change from control (mean ± SE), and OPN protein was measured in concentrated conditioned medium by Western blot with loading by volume at 150 µl per lane (n = 3 for each measurement). *P < 0.01 vs. control; **P < 0.01 vs. MCD.

 

Figure 8
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  		Fig. 8. Effect of PI3-kinase inhibitor on MCD medium-induced increase in ALT release (A) and cell triglyceride content (B) in cultured hepatocytes. Cultures were exposed to MCD medium for 18 h with and without the inhibitor of PI3-kinase (LY294002; 10 µM), and medium ALT and cell triglyceride levels were assessed (means ± SE, n = 3 for each measurement). *P < 0.01 vs. control; **P < 0.01 vs. MCD; and P = NS vs. control.

 
Relative roles of PI3-kinase and OPN signaling in expressions of MTTP and ALT mRNA. To determine whether MCD medium-induced activation of PI3-kinase may be working through sterol regulatory element-binding protein (SREBP-1) to cause increased transcription of MTTP, we measured MTTP mRNA in cells exposed to MCD medium for 18 h in the absence or presence of inhibitor. Compared with control medium, exposure to MCD medium resulted in an ~2.5-fold increase in MTTP mRNA expression, which was completely blocked by inhibition of PI3-kinase activation but not by blocking OPN signaling by inclusion of anti-OPN antibody or beta3-integrin receptor-neutralizing antibody (Fig. 9A). To examine the possibility that OPN was feeding back to the hepatocytes to induce ALT synthesis as a possible mechanism of increased ALT release, we measured ALT mRNA expression. As shown in Fig. 9B, incubation with MCD medium resulted in an approximately fourfold increase in ALT mRNA expression vs. control medium. Inhibition of PI3-kinase activation completely eliminated the MCD medium-induced overexpression of ALT mRNA, whereas incubating with either anti-OPN antibody or beta3-integrin-neutralizing antibody reduced ALT mRNA expression somewhat, to approximately threefold the values seen with control medium (Fig. 9B).


Figure 9
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  		Fig. 9. Effect of MCD medium with and without inhibition of PI3-kinase (LY294002; 10 µM) and OPN signaling (using OPN or beta3-integrin antibodies) on microsomal triglyceride transfer protein (MTTP) mRNA expression (A) and ALT mRNA expression (B). Cultures exposed to MCD medium for 18 h had increased expression of MTTP mRNA (A) and ALT mRNA (B). Inhibition of PI3-kinase activation completely blocked this effect, whereas blocking OPN signaling with antibodies had no effect on the MCD-induced overexpression of MTTP mRNA and only partial reduction in the expression of ALT mRNA (mean ± SE, n = 3 for each measurement). *P < 0.01 vs. control; **P < 0.01 vs. MCD.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our results demonstrate that incubating cultured mouse hepatocytes with MCD medium causes cellular steatosis and increased release of ALT. The MCD medium-induced increases in triglyceride formation and ALT release were accompanied by the activation of PI3-kinase and markedly increased OPN expression. An inhibitor of PI3-kinase prevented the increases in OPN expression, cellular steatosis, and ALT release. Antibodies to OPN or its receptor blunted the increase in ALT release but had no effect on steatosis induced by the MCD medium. Together, these findings indicate that the activation of PI3-kinase plays an important role in the development of hepatocyte steatosis and induction of OPN expression in cultured hepatocytes exposed to MCD medium. OPN, in turn, is involved in signaling increased ALT release by hepatocytes. As has been shown elsewhere (30), OPN expression is involved in fibrogenesis in the MCD diet model of NASH. OPN, therefore, is potentially involved in progression from steatosis to the phenotype of NASH in the MCD model.

Obesity and its complications including nonalcoholic fatty liver disease and NASH are enormous public health problems and the focus of intense research. Among the experimental models most successfully used in the study of NASH is feeding mice an MCD diet. This feeding regimen results in hepatic steatosis within a few days, elevated serum ALT by 1 wk, histological inflammation by 2 wk, and hepatic fibrosis by 4 wk (30). Here, we showed that cultured mouse hepatocytes exposed to MCD medium demonstrate the early effects observed in MCD diet-fed mice, namely, hepatocyte steatosis and release of ALT. This is potentially a worthy in vitro model to study the signaling mechanisms involved in fatty liver disease.

We have shown that hepatocyte-derived OPN plays an important role in the pathogenesis of experimental NASH. MCD diet induces a marked increase in the hepatic expression of OPN in A/J mice, which precedes increased serum ALT and liver collagen 1 expression (30). OPN knockout mice fed the MCD diet exhibit significantly lower hepatic collagen 1 expression than WT mice and develop essentially no liver fibrosis (30). Increased OPN protein expression in MCD diet-fed animals is localized mainly to hepatocytes, and TNF-{alpha}, TGF-beta, and leptin stimulate OPN expression in cultured hepatocytes (30). These findings suggested a paracrine role for hepatocyte-derived OPN in activating stellate cells to produce collagen and cause fibrosis in experimental NASH. In the current study, exposing cultured hepatocytes to MCD medium produced significant stimulation of OPN mRNA and protein expression. This effect was analogous to the OPN expression in MCD diet experimental NASH (30, 31). Moreover, neutralizing antibody to OPN or its beta3-integrin receptor was able to block the MCD medium-induced ALT release. These findings suggest that exposure to MCD medium induces the synthesis and secretion of OPN by hepatocytes, which then acts in an autocrine fashion to cause an increase in the hepatocyte release of ALT. This is also analogous to in vivo experimental NASH, in which OPN knockout mice show substantially lower serum ALT than WT littermates when fed the MCD diet (30). In this study, we showed that the MCD medium induced, OPN-dependent ALT release into the medium is not associated with increased oxidative injury, cell necrosis, or apoptosis. Although not examined in this study, hepatic fibrosis in experimental NASH is probably also effected by hepatocyte secretion of OPN, which then acts on stellate cells to increase collagen synthesis (16).

We sought to determine whether signaling through PI3-kinase was responsible for enhanced OPN expression in this model of NASH. We found that MCD medium increases the expression of PI3-kinase and its downstream target Akt. Moreover, a specific inhibitor of PI3-kinase completely prevented the MCD medium-induced increases in OPN expression. Our findings are consistent with recent observations where PI3-kinase has been shown to mediate the EGF-induced OPN expression by HepG2 cells (39), high glucose-induced increase in OPN expression in renal proximal tubular cells (11), and in the metastatic behavior of breast cancer cells (20). Together, these findings suggest a potential role of PI3-kinase-dependent OPN expression in the pathogenesis of NASH.

We found that PI3-kinase inhibition prevented MCD medium-induced hepatocyte steatosis and ALT release. In contrast, antibodies to either OPN or its receptor prevented the ALT release but had no effect on steatosis induced by the MCD medium. These results complement our in vivo findings where OPN knockout and WT mice on MCD diet developed similar degrees of steatosis, but the knockout mice exhibited significantly reduced serum ALT levels and no hepatic fibrosis (30). Of interest, citrus flavonoid naringenin causes decreased VLDL secretion via activation of PI3-kinase, which in turn enhances the expression of SREBP-1 (4). Increased expression of SREBP-1 causes reduced expression of MTTP, which reduces movement of triglyceride into microsomes leading to reduced lipidation and secretion of VLDL (3). Abnormal lipidation and synthesis of VLDL are considered to play a significant role in the development of steatosis (5, 6), and polymorphisms of MTTP have been implicated as risk factors in human NASH (2, 22). We found that MCD medium-induced increased expression of MTTP was eliminated by blocking PI3-kinase activation, suggesting that signaling through SREBP-1 is involved in the development of MCD-associated steatosis.

It appears that PI3-kinase-dependent increased OPN expression is involved in release of ALT from cultured hepatocytes exposed to MCD medium. Preventing increased ALT release by blocking OPN signaling is quite analogous to the in vivo model of experimental NASH (30). However, we found that treatment with MCD medium evoked increased expression of ALT mRNA. Blockade of PI3-kinase activation completely inhibited the increased expression of ALT mRNA and accumulation of ALT in the medium. Blocking OPN signaling completely blocked the MCD medium-induced accumulation of ALT in the medium but only partially reduced the overexpression of ALT mRNA. In addition, we could find no evidence of cellular oxidative injury, cell death, or apoptosis. These somewhat surprising findings suggest that increased PI3-kinase-dependent ALT transcription is a response to exposure to the MCD medium and is at least in part responsible for the increased accumulation of ALT in the medium. The differential effect of blocking OPN signaling on ALT mRNA expression and accumulation in the medium suggests that mechanisms other than increased synthesis are involved in the loss of ALT into the medium. Taken together, these provocative findings suggest that the increase in serum ALT in in vivo experimental NASH may not necessarily reflect hepatocyte injury or death but may be in part an adaptive response to metabolic challenge.

In summary, our studies show that treating cultured hepatocytes with MCD medium causes hepatocyte steatosis, induction of OPN synthesis, and increased ALT loss. Activation of PI3-kinase appears to be directly involved in all of these effects of exposure to MCD medium. PI3-kinase-dependent increases in OPN expression appear to play an important role in signaling release of ALT and potentially act as a hepatocyte-derived signal for fibrogenesis in experimental NASH. The model used in these studies should provide the opportunity to further dissect the pathogenic mechanisms of experimental fatty liver disease.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research work was supported by the Children's Memorial Research Center of Children's Memorial Hospital, The American Diabetes Association, The Children's Liver Research Fund of Children's Memorial Hospital, and The Liver Foundation for Kids (Lemont, IL).


   ACKNOWLEDGMENTS
 
Present address of A. Sahai: National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. F. Whitington, Dept. of Pediatrics, Children's Memorial Research Center, 2300 Children's Plaza, Chicago, IL 60614 (e-mail: p-whitington{at}northwestern.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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