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Am J Physiol Gastrointest Liver Physiol 290: G1170-G1176, 2006. First published February 2, 2006; doi:10.1152/ajpgi.00402.2005
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LIVER AND BILIARY TRACT

A targeted apoB38.9 mutation in mice is associated with reduced hepatic cholesterol synthesis and enhanced lipid peroxidation

Xiaobo Lin,1 Zhouji Chen,1 Pin Yue,1 Maurizio R. Averna,1 Richard E. Ostlund, Jr.,1 Mark A. Watson,2 and Gustav Schonfeld1

1Department of Medicine and 2Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri

Submitted 29 August 2005 ; accepted in final form 29 January 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Familial hypobetalipoproteinemia (FHBL) due to truncation-specifying mutations of apolipoprotein B (apoB), which impair hepatic lipid export in very low-density lipoprotein (VLDL) particles, is associated with fatty liver. In an FHBL-like mouse with the apoB38.9 mutation, fatty liver develops despite reduced hepatic fatty acid synthesis. However, hepatic cholesterol contents in apoB38.9 mice are normal. We found that cholesterogenic enzymes (3-hydroxy-3-methylglutaryl-coenzyme A reductase, sterol-C5-desaturase, and 7-dehydrocholesterol reductase) were consistently downregulated in two separate expression-profiling experiments using a total of 19 mice (n = 7 each for apob+/+ and apob+/38.9, and n = 5 for apob38.9/38.9) and Affymetrix Mu74Av2 GeneChip microarrays. Results were confirmed by real-time PCR. Cholesterol synthesis rates in cultured hepatocytes were reduced by 35% and 25% in apob38.9/38.9 and apob+/38.9, respectively, vs. apob+/+. Hepatic triglycerides and lipid peroxides, the latter measured by thiobarbituric acid-reactive substances (TBARS) assay, were significantly elevated in apob+/38.9 (117%) and apob38.9/38.9 (132%) vs. apob+/+ (100%), as were mRNA expression of the microsomal lipid peroxidizing enzymes Cyp4A10 and Cyp4A14. Hepatic lipid peroxide levels were positively correlated with triglyceride contents (r = 0.601, P = 0.0065). Thus the fatty liver due to a VLDL secretion defect is associated with insufficient adaptation to triglyceride accumulation and with increased lipid peroxidation. In contrast, apoB38.9 mice effectively maintain cholesterol homeostasis in the liver, at least in part, by reducing hepatic cholesterol synthesis.

nonalcoholic fatty liver; familial hypobetalipoproteinemia; oxidative stress

NONALCOHOLIC FATTY LIVER DISEASE (NAFLD) includes a spectrum of histopathological changes in the liver ranging from steatosis (fatty liver) to nonalcoholic steatohepatitis (NASH) (3). NASH resembles alcoholic steatohepatitis histopathologically (13) and is an important cause of chronic liver disease worldwide (1, 4), but it develops in the absence of alcohol. The most common form of fatty liver in humans is associated with obesity, insulin resistance, and/or type 2 diabetes (31, 32, 41). Fatty liver is also present in mouse models of diabetes mellitus, due to increased hepatic fatty acid synthesis, which, in turn, is due to elevated levels of nuclear sterol regulatory element-binding protein (SREBP)-1c, the transcription factor that activates the expression of the genes of the lipogenic pathway (43). The liver adapts to the resulting lipid overload by the production of greater quantities of triglyceride-enriched VLDL. However, the adaptation is insufficient because fatty liver still develops as the overload of lipids exceeds the capacity of a normally functioning VLDL export system (42).

Another form of fatty liver is associated with a genetic subset of familial hypobetalipoproteinemia (FHBL), which is caused by truncation-producing mutations in the apolipoprotein B (apoB) gene (28, 38, 39). FHBL is an autosomal codominant disorder in humans characterized by low levels (<5th percentile) of plasma apoB and LDL cholesterol (28, 38). ApoB is an indispensable structural protein in VLDL formation and secretion (11). ApoB truncation-producing mutations cause decreased hepatic apoB100 production, and the truncated apoB molecules have impaired lipid-transporting capacities resulting in an overall reduced capacity of the VLDL export system and hence an increased susceptibility to the development of fatty liver (9, 40, 44).

Recently, we generated a human FHBL mouse model carrying an apoB38.9-specifying mutation using embryonic stem cell gene-targeting and the Cre-loxP system (12). These mice develop fatty liver due to the reduced secretion rate of apoB100 (25% of normal, instead of the 50% expected from one normally functioning allele) (2, 12, 16, 48) and the impaired capacity of the truncated apoB to secrete triglycerides (TG) from the liver (12). In contrast to the enhanced hepatic lipogenesis seen in mouse models of insulin resistance or type 2 diabetes, the apoB-truncated mice have reduced lipogenesis in the liver caused by lower expression of hepatic SREBP-1c, fatty acid synthase (FAS), and sterol-CoA desaturase-1 (27). Thus the FHBL mouse develops NAFLD despite a reduced synthesis of fatty acids, indicating insufficient adaptation to chronic liver TG accumulation resulting from the impaired VLDL export system.

The dysfunctional VLDL system in apoB38.9 mice indicates that cholesterol secretion from the liver through apoB ought to be impaired since cholesterol is secreted as an integral component of VLDL. Hepatic cholesterol content was normal in apoB38.9 mice (12), suggesting effective adaptation in hepatic cholesterol homeostasis in response to the apoB defect in secreting lipids in VLDL. However, the adaptive mechanisms remain unknown.

Fatty liver may progress to NASH, fibrosis, cirrhosis, and end-stage liver disease (3, 30, 45). The pathophysiological mechanisms by which NASH develops remain unclear, but steatosis and oxidative stress have been suggested to be essential (14). Oxidative stress results from an imbalance between prooxidant and antioxidant forces. Recently, reactive oxygen species (ROS)-mediated oxidative stress has been found in experimental models of NASH (24, 47), in alcoholic fatty liver, and in livers of humans with steatosis of different etiologies as well (26, 35, 36). Thus oxidative stress has been postulated to be of critical importance as a "second hit" in the pathogenesis of NASH (13, 14).

Microsomal monooxygenases, cytochromes P450, are important sources of lipid peroxidation in NAFLD. P450s are hemoproteins that catalyze the oxidation of various endogenous and exogenous hydrophobic compounds and therefore play an essential role in detoxification. Ethanol-inducible cytochrome P450 2E1 (Cyp2E1) metabolizes lipophilic molecules such as alcohol. It plays a key role in alcoholic liver disease by stimulating lipid peroxidation (26). Three Cyp4A genes are expressed in mice: 4a10, 4a12, and 4a14 (5, 20). Like Cyp2E1, Cyp4A enzymes are also involved in microsomal fatty acid metabolism, catalyzing the {omega} and {omega}-1 hydroxylation of medium-chain fatty acids. Cyp2E1 protein or mRNA is unchanged or reduced in ob/ob leptin-deficient mice, a model of obesity and type 2 diabetes (17, 46). In contrast, mRNA levels for Cyp4A10 and Cyp4A14 are greatly increased (17). Cyp2E1 was induced together with an 100-fold increase in lipid peroxides in the liver when wild-type mice were fed a methionine- and choline-deficient (MCD) diet (24). The MCD diet, but not the control diet, increased mRNA expression of Cyp4A10 and Cyp4A14 in the liver of Cyp2E1-1- mice (24). Furthermore, hepatic microsomal lipid peroxidation was substantially inhibited by anti-mouse Cyp4A10 antibody in vitro (24). Thus Cyp4A enzymes are important initiators of oxidative stress in the liver.

In the current investigation, we hypothesized that in the mice with apoB38.9 defects and chronic TG accumulation 1) the cholesterogenic pathway would be downregulated, as we have previously reported for fatty acid synthetic pathway, and 2) the lipid peroxidation could be increased.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Animals. Heterozygotes (apob+/38.9) and homozygotes (apob38.9/38.9) (12) have a mixed genetic background of 50% 129/SVJ and 50% C57BL/6J. Male wild-type controls (apob+/+), together with apob+/38.9 and apob38.9/38.9, were fed a standard mouse diet (5053, Purina Mills) containing 4.5% fat, 20.0% protein, and 54.8% carbohydrate (LabDiet) and housed in a pathogen-free barrier facility with a 12-h light and 12-h dark cycle (6:00 AM to 6:00 PM). Mice were killed between 10:00 AM to 12:00 PM. For the first set of microarray experiments, a total of nine mice with three from each genotype were used. In the second study, a total of 10 (4 apob+/+, 4 apob+/38.9, and 2 apob38.9/38.9) were used. Mice were killed without fasting, and the order of death was alternated by genotype. Primary hepatocytes from male mice similar in age to those for microarray analyses (3 from each genotype) were prepared for the determination of hepatic cholesterol synthesis. The animal protocol was reviewed and approved by Washington University's Animal Studies Committee.

Expression profiling. Frozen livers were placed immediately into TRIzol reagent (Invitrogen, Carlsbad, CA) and homogenized. Total RNA was isolated from TRIzol homogenates according to the manufacturer's protocol.

To minimize false-positive changes in gene expression due to biological and random technical variability, we performed two independent microarray experiments using different groups of mice for each experiment. For both microarray analyses, extracted RNA was then further purified using RNeasy spin columns (Qiagen, Valencia, CA) following the manufacturer's protocol. Purified RNA was quantitated by UV absorbance at 260 and 280 nm and assessed qualitatively using an RNA LabChip and Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Equal quantities of purified total RNA from each animal of the same genotype were pooled. Ten micrograms of pooled total RNA was converted to cDNA, purified, and then used as a template for in vitro transcription of biotin-labeled antisense RNA (aRNA). Target synthesis and microarray hybridization was performed by the Siteman Cancer Center GeneChip Facility. All protocols were performed as recommended by the manufacturer (Affymetrix, Santa Clara, CA) and have been described elsewhere (29). Fifteen micrograms of each biotinylated aRNA preparation was fragmented, assessed by gel electrophoresis, and placed in hybridization cocktail containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Samples were hybridized to Affymetrix Mu74Av2 GeneChip microarrays for 16 h. Microarrays were washed and stained using the instrument's standard "eukaryotic GE wash 2" protocol, utilizing antibody-mediated signal amplification. The images from the scanned microarrays were processed using Affymetrix Microarray Analysis Suite 5.0. The image from each GeneChip was scaled such that the average intensity value for all arrays was adjusted to a target intensity of 1,500. Signal and detection metrics from each GeneChip were exported as flat text files and used for further analysis. The detection metric is a qualitative assessment generated by Affymetrix software and indicates whether the hybridization signal intensity from the oligonucleotide probe pair set is sufficiently robust to be reliably scored as detected. The signal data is the quantitative hybridization signal value obtained from the probe pair set. Comparison-expression analysis between genotypes was also performed by directly comparing matching cells on two arrays so that any inherent differences in the hybridization efficiency of these cells were cancelled out. Gene annotation data (http://www.affymetrix.com/analysis/index.affx) was appended to expression data, and the resulting flat text file was imported into DecisionSite 7.1 and Array Explorer software (Spotfire, Somerville, MA) for further data visualization and analysis. A twofold or greater change in apob+/38.9 or apob38.9/38.9 relative to apob+/+ was used as the criterion to select genes for further examination.

RT-PCR and real-time quantitative PCR analyses. Gene expression for cholesterogenic enzyme genes was confirmed by an independent method using real-time PCR. Total RNA was treated with RNase-free DNase (Promega) and isolated by phenol-chloroform followed by purification with RNeasy mini kit (Qiagen). First-strand cDNA was synthesized with SuperScript II RNase H– reverse transcriptase (Invitrogen) on total RNA (0.5 µg) in a volume of 20 µl using oligo(dT) as the primer. Aliquots (2 µl) of the reverse transcription were then subjected to PCR (50°C for 2 min, 95°C for 10 min, 95°C for 15 s followed by 60°C for 1 min for 40 cycles) using gene-specific primers (Table 3). Real-time quantitative PCR analyses were performed with SYBR green and a GeneAmp 5700 sequence detector from Applied Biosystems (Foster City, CA) in a volume of 25 µl, each reaction using AmpliTag Gold DNA polymerase. Real-time PCR products were electrophoresed on 1.2% agarose gels to verify that the primer pairs amplified a single product of the predicted size. The GeneAmp 5700 sequence detection system software was used to analyze the data, and threshold cycle numbers were calculated for different genotypes. GAPDH RNA levels were used as an internal control. A relative standard curve was constructed. Known amounts of total RNA were analyzed for both gene of interest and GAPDH. For each unknown sample, relative amount was calculated using linear regression analysis from their respective standard curve. A relative expression value for each gene of interest was obtained by division of its value by the GAPDH value, which was also calculated from its own standard curve. The mRNA levels are presented in arbitrary units where the wild-type control quantity was assigned a value of 1.0.


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  		Table 3. Primers for real-time quantitative RT-PCR

 
Rates of cholesterol synthesis determined in primary hepatocytes. Hepatocytes were isolated as described (12). Viability of the cells (~80%) was determined by Trypan exclusion. Cells were then plated onto wells of 6-well plates coated with poly-D-lysine (Sigma, St. Louis, MO) and incubated at 37°C under 5% CO2 in DMEM containing 10% fetal bovine serum. After 2 h of attachment, the cells were washed twice and incubated in the same medium for 6 h. To determine rates of cholesterol synthesis, 6 µCi of [1-14C]acetate (0.2 µCi/µl) was added to each well and incubated for 1.5, 3, and 6 h. Cells were harvested, and labeled sterols were digitonin precipitated (15). Rates of cholesterol synthesis are reported as disintegrations per minute of 14C radioactivity per milligram cell protein.

Western blot analysis. To determine whether mature nuclear sterol regulatory element binding protein-2 (SREBP-2) was reduced in the apob+/38.9 and apob38.9/38.9 mice, nuclear extracts of mouse livers were prepared using the CelLytic NuCLEAR extraction kit (Sigma) according to the manufacturer's protocol, except that calpain inhibitor 1 (25 µg/ml) and leupeptin (50 µg/ml) were added in addition to the protease inhibitor mix provided. Aliquots of nuclear extracts (100 µg) were mixed with SDS loading buffer, subjected to SDS-PAGE on an 8% gel, transferred, and immobilized on Immobilon-P transfer membrane. After blocking with 5% nonfat milk in Tris-buffered saline buffer (pH 8.0) plus Tween 20 at room temperature for 1 h, the membrane was washed and incubated with rabbit polyclonal anti-mouse SREBP-2 as primary antibody (courtesy of Dr. Jay Horton, Dallas, TX) and a horseradish peroxidase-labeled donkey anti-rabbit IgG as the secondary antibody. Visualization of the SREBP-2 protein was performed with Western blotting detection system kit (Pierce Chemical, Rockford, IL). The filter was exposed to film for 5 s at room temperature. Signal intensities were quantified by using Quantity One (Bio-Rad).

Total lipid peroxide measurement. To determine total lipoperoxides, a 100-mg aliquot of liver was homogenized in a 1.15% KCl solution containing 0.01% butylated hydroxytoluene (BHT) to prevent the endogenous peroxidation of lipids during the procedure. TBARS were measured in 200 µl liver homogenate according to the technique described (34).

Miscellaneous procedures. Lipids were extracted from liver (6). Commercial kits were used to measure concentrations of TG, total cholesterol, and phospholipids in liver lipid extracts as well as in plasma (Wako Chemicals, Richmond, VA). Cellular protein contents were determined as described (12).

Statistical analyses. ANOVA followed by the Tukey procedure was performed for comparison between genotypes. The Pearson correlation test and stepwise linear regression analyses were performed on results across genotypes using SAS Proc CORR and Proc REG (SAS/STAT version 8, 2000).


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Mice. To avoid potentially confounding differences in gene expression due to sex, only male mice were used in the present study (Table 1). Significantly lower plasma total cholesterol concentration was seen for apob38.9/38.9 mice.


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  		Table 1. Parameters of wild-type and apoB38.9-bearing mice

 
Hepatic mRNA levels from microarray analyses. The first microarray analysis showed that 160 transcripts were downregulated in apob38.9/38.9 and 86 were downregulated in apob+/38.9 compared with apob+/+. Seventy-five transcripts were upregulated in apob38.9/38.9, and 17 in apob+/38.9. Results were similar in the number of differentially expressed genes from the second expression profiling experiment (not shown). Of special interest for this work, several lipid-related transcripts were downregulated in both experiments (Table 2), including three cholesterogenic enzymes, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), sterol-C5-desaturase (SC5D), and 7-dehydrocholesterol reductase (DHCR7) (Table 2). mRNA levels for HMGR were reduced by 60% and 50% in apob38.9/38.9 and apob+/38.9, respectively. Respective values for DHCR7 mRNA levels were 60% and 50%; for SC5D mRNA, the values were 50% and 30% (Table 2). Consistent with our previous results, hepatic FAS mRNA levels were reduced in apoB38.9 mice (27).


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  		Table 2. Subsets of differentially regulated genes in apoB38.9 mice from microarray analyses

 
The mRNA levels for insulin-like growth factor binding protein 1 (IGFBP1) and cellular retinol binding protein 1 (CRBP1) were downregulated in apoB38.9 mice (Table 2). By contrast, the fatty acid peroxidizing enzyme, microsomal cytochrome P450 4A10, was upregulated.

Quantitative real-time PCR confirmed GeneChip results. The results of the microarray analysis were confirmed for the three cholesterogenic enzyme genes, HMGR, SC5D, and DHCR7, by quantitative real-time PCR performed in individual mice (Fig. 1A). Real-time PCR (Table 4) also showed reductions of mRNA levels for other enzyme genes in the cholesterol synthetic pathway such as mevalonate kinase (MK), squalene synthase, and squalene epoxidase, indicating that the entire cholesterol synthetic pathway was downregulated. However, the mRNA levels of SREBP-cleavage-activating protein (SCAP) and SREBP-2 were not altered (Table 4). The mRNA level for LDL receptor (LDLR) was also downregulated, whereas ABCG5 and ABCG8 were upregulated (Table 4), indicating that uptake of cholesterol might be decreased, whereas cholesterol efflux into bile may have been increased. These implied changes are consistent with a response to the accumulation of cholesterol in hepatocytes.


Figure 1
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  		Fig. 1. Effects of apoB38.9 mutation on hepatic mRNA levels of HMGR, DHCR7, and SC5D (A); hepatic cholesterol synthesis (B); and mature nuclear SREBP-2 level (C). A: levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), 7-dehydrocholesterol reductase (DHCR7), and sterol-C5-desaturase (SC5D) mRNA levels from individual male mice fed ad libitum (apob+/+, n = 7; apob+/38.9, n = 7; or apob38.9/38.9, n = 5) were determined by quantitative real-time PCR as described in METHODS. B: rate of cholesterol synthesis in primary hepatocytes (n = 3 for each genotype) using [14C]acetate as the label substrate was determined as described in METHODS. C: immunoblot analysis of SREBP-2 in nuclear extracts pooled from equal amounts of protein in each group (apob+/+, n = 7; apob+/38.9, n = 7; or apob38.9/38.9, n = 5) was performed as described in METHODS. Values represent the fold change of SREBP-2 nuclear protein relative to apob+/+. The bars (A) or time points (B) represent the mean and standard deviation of measurements in each genotype. The levels of statistical differences relative to apob+/+ are indicated by *P < 0.05 or **P < 0.01.

 

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  		Table 4. mRNA levels by real-time PCR analysis

 
Compared with apob+/+, mRNA for Cyp4A10 increased by 1.5- and 2.0-fold in apob+/38.9 and apob38.9/38.9, respectively, but Cyp2E1 mRNA remained unchanged. The mRNA for Cyp4A14 was also elevated in apoB38.9 mice as shown by real-time PCR analysis. The downregulation of genes coding for IGFBP1 and CRBP1 was also confirmed (Table 4).

Rates of cholesterol synthesis. Cholesterol synthesis in primary hepatocytes was linear for all genotypes up to 6 h (Fig. 1B). Rates of cholesterol synthesis at 6 h of incubation were reduced by 25% and 35% in apob+/38.9 and apob38.9/38.9, respectively. These data are compatible with the changes in the mRNA levels of cholesterogenic enzymes noted above (Fig. 1A).

Mature nuclear SREBP-2 protein level. Since SREBP-2 activates transcription of its regulated genes through its mature nuclear form in the nucleus, it is important to look at the level of the nuclear form of the protein. Although mRNA levels did not differ among the genotypes, the protein level of the active form was reduced by ~30% and 50%, respectively, in apob+/38.9 and apob38.9/38.9 compared with apob+/+ (Fig. 1C).

Liver TG and lipid peroxides. As expected, liver TG contents were increased by about twofold and threefold, respectively, in apob+/38.9 and apob38.9/38.9 vs. apob+/+ (Fig. 2A). Although the mean liver total cholesterol content appeared to be apoB38.9 gene dose dependent, the means were not significantly different, similarly for hepatic phospholipid concentrations as reported previously (12). Since the mRNA levels of Cyp4A10 and Cyp4A14, two microsomal fatty acid peroxidizing enzymes, were increased in the liver of apoB38.9-carrying mice (Table 4), we hypothesized that lipid peroxidation is increased in the liver. Indeed, lipid peroxide contents (TBARS) were significantly higher in apob+/38.9 (117%) and apob38.9/38.9 (132%) vs. apob+/+ (100%) (Fig. 2B).


Figure 2
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  		Fig. 2. A: elevated triglyceride content and enhanced lipid peroxidation in the liver of apoB38.9 mice under ad libitum feeding. A: Concentrations of liver triglycerides, phospholipids, and cholesterol were measured on 7 wild-type (apob+/+) mice, 7 heterozygous (apob+/38.9) mice, and 5 homozygous (apob38.9/38.9) mice, all of which were male. B: lipid peroxide levels were determined by the thiobarbituric acid-reactive substances (TBARS) assay as described in METHODS. Each bar represents the mean with standard deviation. The levels of statistical differences relative to apob+/+ are indicated by *P < 0.05 and **P < 0.01.

 
Statistical relationships among liver TG, lipid peroxides, and mRNA levels. A significant positive correlation was obtained between liver TG and lipid peroxides (r = 0.601, P = 0.0065). This suggests that chronic accumulation of TG in the liver may increase lipid peroxidation. A significant negative correlation was observed between the hepatic HMGR mRNA level and lipid peroxides in the liver (r = –0.582, P = 0.009). On multiple regression analysis, the hepatic HMGR mRNA level was predicted by peroxide level in the liver (R2 = 0.401, P = 0.0035).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The hepatic VLDL export system is impaired in apoB38.9-bearing mice, similar to humans (9, 40, 44), due to the reduced hepatic production rates of apoB100 and the reduced capacity of truncated apoB to export lipids via VLDL particles (2, 12, 16, 48). As a result, fatty liver is observed in apoB38.9 mice even on a normal diet despite reduced hepatic fatty acid synthesis, indicating ineffective adaptation to chronic TG accumulation. Since not only TG but also cholesterol and phospholipids are carried in VLDL, cholesterol secretion from the liver via apoB in VLDL ought to be impaired as well. However, hepatic cholesterol content was similar across genotypes, indicating sufficient adaptation in hepatic cholesterol homeostasis to the impaired VLDL export system. We thus hypothesized that hepatic cholesterol synthesis is reduced in apoB38.9 mice.

Consistent with our hypothesis, mRNA levels of three cholesterol synthetic enzyme genes, HMGR (the rate-limiting enzyme), SC5D, and DHCR7 (the last enzyme in the pathway), were consistently reduced from gene expression profiling experiments, which was confirmed by real-time PCR. Real-time PCR also revealed downregulation of mRNA levels of other cholesterol synthetic enzyme genes such as MK, squalene synthase, and squalene epoxidase, indicating that the whole cholesterol synthetic pathway was downregulated. Indeed, hepatic cholesterol synthesis, measured in primary hepatocytes, was reduced in apoB38.9 mice. Furthermore, the mRNA levels of LDLR, the mediator of hepatic cholesterol uptake, were downregulated, whereas mRNAs of ABCG5/8, the mediators of the secretion of hepatic cholesterol into bile, were upregulated, suggesting cholesterol uptake through LDLR may be reduced and cholesterol efflux through ABCG5/8 may be enhanced. These changes are consistent with increases of the cholesterol contents of the "metabolically active" pool, perhaps in the endoplasmic reticulum of hepatocytes.

SREBP-2, a transcription factor that preferentially activates transcription of cholesterol synthetic enzyme genes, plays a crucial role in maintaining cholesterol homeostasis in hepatocytes (21, 22). The levels of SCAP/SREBP-2 mRNAs did not change across the mouse apoB genotypes; however, the protein level of the biologically active nuclear form of SREBP-2 was reduced in the liver of apob+/38.9 and apob38.9/38.9 mice. This indicated that the downregulation of cholesterogenesis in the liver of apoB38.9 mice was mediated by reduced active SREBP-2 protein levels.

Cholesterol synthesis is regulated by cholesterol flux, either by cholesterol uptake of LDL or HDL or through cholesterol absorption from the small intestine (18). Neither LDL receptor-related protein 1 nor scavenger receptor 1 was altered in apoB38.9 mice, suggesting that hepatic cholesterol uptake may not change via these receptors. Cholesterol absorption determined by stable isotope methodology was reduced in apob38.9/38.9 (data not shown). These data strongly suggest that the downregulation of cholesterol synthesis is attributed to the impaired VLDL lipid secretion due to the apoB38.9 mutation.

Microsomal lipid peroxidizing enzymes, Cyp4A10 and Cyp4A14, are important initiators of oxidative stress in the liver of ob/ob mice and mouse models of NASH (17, 24). Microarray analyses revealed upregulation of the mRNA levels of Cyp4A10 in the livers of apoB38.9 mice. Consistent with the upregulation of oxidative stress initiators, the TBARS assay revealed a gene dose-dependent increment in lipid peroxidation. In addition, levels of hepatic TG and lipid peroxides were positively correlated (r = 0.601, P = 0.0065). These results strongly suggest that chronic TG accumulation in this unique mouse model of NAFLD could lead to lipid peroxidation and oxidative stress. Lipid peroxidation is detrimental to organelle function and cellular homeostasis, potentially leading to the induction of hepatocyte death and necrosis, inflammation, and liver fibrosis (7). Whether prolonged exposure of the livers of apoB38.9 animals results in irreversible damage is unknown.

Separate studies showed no differences in total hepatic lipid peroxides between younger (~13 wk of age for both genotypes) apob+/+ and apob+/38.9 mice (data not shown). TBARS results on younger apob38.9/38.9 mice were not available for comparison; nonetheless, this suggests that aging may play a role in enhanced lipid peroxidation in apoB38.9 mice with fatty liver. Several lines of evidence suggest that palmitate or FAS-related saturated fatty acids activate peroxisome proliferator-activated receptor-{alpha} (10, 19, 23), the transcriptional factor of fatty acid beta-oxidation. Reduced hepatic FAS mRNA and fatty acid synthesis in apoB38.9 mice might result in insufficient mitochondrial beta-oxidation in the liver over time, although normal fatty acid beta-oxidation was suggested from a single time point analysis (27). It is of interest to learn whether more severe fatty liver develops in apoB38.9 mice with aging and whether this is related to insufficient fatty acid beta-oxidation, which in turn may enhance hepatic lipid peroxidation in apoB38.9 mice.

Intriguingly, our results revealed a significant negative correlation between hepatic HMGR mRNA levels and lipid peroxide levels in the liver. Moreover, hepatic HMGR mRNA levels were predicted by lipid peroxide level in the liver by multiple regression analysis. It is interesting to note that iron/ascorbate-induced microsomal lipid peroxidation reduced HMGR enzyme activities in rat liver (8). Whether lipid peroxidation in the liver plays a possible role in the downregulation of hepatic cholesterol synthetic enzyme genes warrants further studies.

Gene expression profiling demonstrated reductions in mRNA levels of CRBP1 and IGFBP1. CRBP1 is highly expressed in the liver and is involved in vitamin A metabolism. Downregulation of CRBP1 contributes to tumor growth and progression via retinoid-mediated signaling and disruption of cellular vitamin A homeostasis (37). IGFBP1, on the other hand, is rapidly and highly induced in the regenerating liver (33). IGFBP1 has also been shown to be a critical hepatic survival factor by reducing the level of proapoptotic signals (25). Downregulation of IGFBP1 may therefore suggest impairment of hepatocyte proliferative and anti-apoptotic pathways, which remains to be confirmed by further experimentation.

In conclusion, results of the present study demonstrate that the mouse liver sufficiently adapts to the impaired VLDL lipid secretion caused by the well-defined apoB defect in maintaining hepatic cholesterol homeostasis, at least in part by downregulating hepatic cholesterol synthesis. This is in contrast with the ineffective adaptation of reduced hepatic fatty acid synthesis in response to chronic liver TG accumulation. The increased liver TG is associated with enhanced lipid peroxidation in the livers of these mice. Increased lipid peroxidation in turn may enhance the possible development of NASH in FHBL mice and apoB-defective humans with fatty liver. Our results also point to a possibly enhanced susceptibility of the liver to apoptosis and impaired hepatocyte regeneration that may also contribute to the development of NASH. Further studies are required to investigate whether fatty liver in FHBL subjects in fact do develop into NASH, advanced fibrosis, or cirrhosis over time.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant from The Alan and Edith Wolf Charitable Fund and National Heart, Lung, and Blood Institute Grants R37-HL-424460, RO1-HL-59515, RO1-HL-73939 (to Z. Chen), and R01-HL-50420 (to R. E. Ostlund).


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Jay Horton for the generous gift of SREBP-2 antibody used in these studies.

Present address of M. R. Averna: Institute of Internal Medicine and Geriatrics, Univ. of Palermo, School of Medicine, Via del vespro 141, 90127 Palermo, Italy.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Schonfeld, 660 S. Euclid, Campus Box 8046, St. Louis, MO 63110 (e-mail: gschonfe{at}wustl.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
 METHODS
 RESULTS
 DISCUSSION
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