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Originally published In Press as doi:10.1074/jbc.M603422200 on August 20, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31713-31719, October 20, 2006
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CuZn-SOD Deficiency Causes ApoB Degradation and Induces Hepatic Lipid Accumulation by Impaired Lipoprotein Secretion in Mice*

Satoshi Uchiyama{ddagger}1, Takahiko Shimizu{ddagger}§, and Takuji Shirasawa{ddagger}§||2

From the {ddagger}Research Team for Molecular Biomarkers, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, §Applied Biological Chemistry, United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo 183-8509, ||Biological Science, Graduate School of Science, Tokyo Metropolitan University, Tokyo 113-8421, and Anti-Aging Science Inc., Tokyo 100-0011, Japan

Received for publication, April 10, 2006 , and in revised form, August 18, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Elevated hepatic reactive oxygen species play an important role in pathogenesis of liver diseases, such as alcohol-induced liver injury, hepatitis C virus infection, and nonalcoholic steatohepatitis. In the present study, we investigated and compared the hepatic lipid metabolisms of liver-specific Sod2 (superoxide dismutase 2) knock-out (Sod2 KO), Sod1 knock-out (Sod1 KO), and Sod1/liver-specific Sod2 double knock-out mice (double KO). We observed significant increases in lipid peroxidation and triglyceride (TG) in the liver of Sod1 KO and double KO mice but not in the liver of Sod2 KO mice. We also found that high fat diet enhanced fatty changes of the liver in Sod1 KO and double KO mice but not in Sod2 KO mice. These data indicated that CuZn-SOD deficiency caused lipid accumulation in the liver. To investigate the molecular mechanism of hepatic lipid accumulation in CuZn-SOD-deficient mice, we measured TG secretion rate from liver using Triton WR1339. We found significant decrease of TG secretion in CuZn-SOD-deficient mice. Furthermore, we observed marked degradation of apolipoprotein B (apoB) in the liver and plasma of CuZn-SOD-deficient mice, indicating that degradation of apoB impairs secretion of lipoprotein from the liver. Our data suggest that oxidative stress enhances hepatic lipid accumulation by impaired lipoprotein secretion due to the degradation of apoB in liver.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nonalcoholic fatty liver disease (NAFLD)3 is mostly associated with obesity and diabetes. NAFLD ranges from steatosis to nonalcoholic steatohepatitis (NASH), which can progress to cirrhosis of the liver (1-3). The prevalence of NAFLD and NASH are estimated to be ~20 and ~3% of the general population in the United States, respectively (4). NASH is generally characterized by lipid accumulation and inflammation, which is occasionally associated with fibrosis. Histology of NASH is similar to that of alcoholic steatohepatitis, suggesting that these two diseases have some common pathogenesis (5). Recent studies suggest that steatosis may progress to NASH by oxidative stress (1-3). The most popular hypothesis for the pathogenesis of NASH is a "two-hit theory", consisting of hepatic fat accumulation as the first hit and oxidative stress in liver as the second hit (1-3). Hepatic lipid content is increased by several pathways, such as increased uptake of fatty acid from peripheral tissues, enhanced de novo fatty acid synthesis, decreased beta-oxidation in mitochondria, and decreased secretion of apoli-poprotein B-containing lipoprotein (6). Reactive oxygen species (ROS) in fatty liver cells are reported to generate at three different intracellular sites, microsomes, peroxisomes, and mitochondria (7). Videla et al. (8) reported that malondialdehyde (MDA) was significantly increased in the livers of patients with steatosis and NASH. In addition, previous studies reported the up-regulation of microsomal hepatic cytochrome P450 2E1 (CYP2E1) in patients with NASH (9). Furthermore, Nieto et al. (10) reported that co-culture of hepatic stellate cells with HepG2 cells expressing CYP2E1 stimulated lipid peroxidation and the production of type I collagen, suggesting that ROS may trigger fibrosis of the liver. These studies suggest that oxidative stress plays a pivotal role in the progression of NAFLD.

SODs are major antioxidant enzymes that are classified into three groups, cytosolic CuZn-SOD (Sod1), mitochondrial Mn-SOD (Sod2), and extracellular EcSOD (Sod3) (11). Several studies also reported that SOD activities decreased in the liver of patients with NAFLD (8, 12). Laurent et al. (13) reported that SOD activities markedly decreased in the liver of ob/ob mice that develop experimental NASH. In addition, SOD mimics, manganese [III] tetrakis (5,10,15,20 benzoic acid) and copper [II] diisopropyl salicylate, significantly ameliorated the steatotic lesion in ob/ob mice (13). Mn-SOD-deficient mice showed neonatal death with dilated cardiomyopathy, massive lipid accumulation in liver, and lactic acidosis (14, 15). In a previous study, we reported the generation of liver-specific Sod2 knockout mice (Sod2 KO) using the Cre-loxp system (16). In Sod2 KO mice, no obvious morphological and biochemical abnormality was observed in the physiological condition of the mice, suggesting that the physiology of Mn-SOD in the liver is still to be defined (16). Recently, Elchuri et al. (17) reported that CuZn-SOD-deficient mice (Sod1 KO) showed increased incidence of neoplastic changes in the liver, which is concomitant with increase of 8-oxo dG and F2-isoprostanes. In addition, Kessova et al. (18) reported that long-term consumption of ethanol enhanced lipid peroxidation and protein carbonylation associated with impaired mitochondrial antioxidant defense systems in the liver of Sod1 KO mice, suggesting that CuZn-SOD plays an important physiological role in liver homeostasis.

To investigate the pathological role of intracellular superoxide in liver lipid metabolism, we examined lipid accumulation in the livers of Sod2 KO, Sod1 KO, and Sod1/liver-specific Sod2 double KO mice (double KO). We discuss here the pathological role of oxidative stress in the pathogenesis of NAFLD.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Genotyping—The generation of Mn-SOD flox (Sod2flox/flox) mice was described previously (16). The mice were backcrossed to C57BL/6CrSlc (Japan SLC, Shizuoka, Japan) mice for 5 or 6 generations. The cross-breeding of Sod2flox/flox with Alb-Cre transgenic mice (The Jackson Laboratory, Bar Harbor, ME) gave rise to Sod2 KO. Sod1 KO mice were purchased from The Jackson Laboratory. Sod1 KO mice were backcrossed to Sod2flox/flox or Sod2 KO mice for 2 or 3 generations. To obtain Sod1 KO and double KO mice, Sod1 KO male mice were mated to Sod1+/-/Sod2 KO females.

All experiments were carried out using 8-10-week-old male mice, except for the determination of total amount of MDA and 4-hydroxyalkenals (HAE). All genotypings of Sod1, Sod2, and Cre transgene were performed by PCR using genomic DNA isolated from the tail tip. The primers for identifying carriers of the Cre transgene, 5'-AGG TTC GTT CAC TCA TGG A-3' and 5'-TCG ACC AGT TTA GTT ACC C-3', were used under the following conditions: 1 cycle of 94 °C for 1.5 min, and 30 cycles of 94 °C for 0.5 min, 58 °C for 0.5 min, and 72 °C for 0.5 min followed by 1 cycle of 72 °C for 5 min. The primers for the Sod2 genotyping, 5'-TTA GGG CTC AGG TTT GTC CAG AA-3', 5'-CGA GGG GCA TCT AGT GGA GAA-3', and 5'-AGC TTG GCT GGA CGT AA-3', were used under the following conditions: 1 cycle of 94 °C for 3 min, and 35 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min followed by 1 cycle of 72 °C for 10 min. The primers for the Sod1 genotyping, 5'-GTT CTC CTC TTC CTC ATC TCC-3', 5'-ACC CTT TCC AAA TCC TCA GC-3', 5'-TGA ACC AGT TGT GTT GTC AGG-3', and 5'-TCC ATC ACT GGT CAC TAG CC-3', were used under the following conditions: 1 cycle of 94 °C for 3 min, 12 cycles of 94 °C for 20 s, 64 °C for 30 s, and 72 °C for 35 s, and 25 cycles of 94 °C for 20 s, 58 °C for 30 s, and 72 °C for 35 s followed by 1 cycle of 72 °C for 2 min. Mice were maintained and studied according to protocols approved by the Animal Care Committee of the Tokyo Metropolitan Institute of Gerontology.

High Fat Diet—4- or 5-week-old mice were fed high fat diets containing 20% (w/w) lard and 80% CRF-1 (Oriental Yeast, Tokyo, Japan) for 4 weeks. After feeding the high fat diet, we collected plasma and liver of mice for the determination of TG contents and stored at -80 °C until use. Liver was fixed in formalin or frozen in Tissue Tek O.C.T. compound (Sakura Fine-technical Co. Ltd., Tokyo, Japan) for histological staining.

Western Blot Analysis—Western blot analysis of CuZn-SOD, Mn-SOD, and beta-actin was performed with liver homogenates as previously described (16). Briefly, the liver was homogenized with Potter-type homogenizers in 10 volumes of phosphate-buffered saline (-), pH 7.4, containing Complete protease inhibitor mixture (Roche Applied Science). Homogenates were sonicated on ice and then centrifuged at 15,000 x g for 30 min. Equal amounts (5 µg) of total protein were subjected to 12.5% SDS-PAGE, electroblotted onto an Immobilon-P membrane (Millipore, Bedford, MA). The membranes were blocked in Block Ace (Daiichi Seiyaku, Tokyo, Japan) and probed with antibodies against Mn-SOD (1:10,000; SOD-111; StressGen, Victoria, Canada), CuZn-SOD (1:7000; SOD-100; StressGen), and actin (1:100; Sigma). Immunoreactive CuZn-SOD, Mn-SOD, and actin were visualized with the ECL system (Amersham Biosciences) and a luminoimage analyzer LAS-1000 (Fuji Film, Tokyo, Japan).

We used liver microsome fractions for Western blot analysis of apoB48, apoB100, microsomal triglyceride transfer protein (MTP), and glucose-regulated protein 78kDa (GRP78). Briefly, livers were homogenized with Potter-type homogenizers in 10 volumes of 20 mM Tris-HCl, pH 7.4, containing 0.25 M sucrose and Complete protease inhibitor mixture. Homogenates were centrifuged at 8,000 x g for 10 min, and the supernatant was further centrifuged at 100,000 x g for 1 h. The pellets were suspended in the same buffer. Equal amounts (50 µg for apoB, and 10 µg for MTP and GRP78) of total protein were subjected to 4.0% SDS-PAGE for apoB48 and apoB100 and 7.5% SDS-PAGE for MTP and GRP78. The proteins were electroblotted onto Protran BA83 nitrocellulose membrane (Schleicher & Schuell BioScience). The membranes were blocked in Block Ace and probed with antibodies against apoB100 (1:500; H-15; Santa Cruz Biotechnology, Santa Cruz, CA), apoB48 (1:500; S-18; Santa Cruz Biotechnology), MTP (1:2500; BD Bioscience), and GRP78 (1:1000; BD Bioscience). Immunoreactive apoB100, MTP, and GRP78 were visualized with ECL and LAS-3000 (Fuji Film). Immunoreactive apoB48 was visualized with Immobilon Western detection reagents (Millipore) and LAS-3000. The same procedure was performed with plasma for apoB100. Two µl of plasma were subjected to 4.0% SDS-PAGE. The proteins were electroblotted onto Protran BA83 nitrocellulose membrane. The membrane was blocked in Block Ace and probed with antibodies against apoB100 (1:500; H-15). Protein concentration was determined using a DC Protein Assay kit (Bio-Rad).

Histological Studies—For histological observation, we stained formalin-fixed, paraffin-embedded liver sections (5 µm thickness) with hematoxylin and eosin and frozen liver sections (5 µm thickness) with Oil Red O using standard protocols.

Lipid Peroxidation—Lipid peroxidation byproducts, MDA and HAE, in livers of 50-week-old-mice were measured with a Colorimetric assay kit for lipid peroxidation (Bioxyteck LPO-586; Oxis Research, Portland, OR) according to the manufacturer's protocol.

Assessment of Lipid Secretion Rate—We measured the lipid secretion rate in vivo as described by Stiles et al. (19) with slight modification. Briefly, 4-h-fasted mice were injected with 10% Triton WR1339 (5 µl/g body weight) (tyloxapol; Sigma) through the tail vein to inhibit TG degradation by lipoprotein lipase. Plasma samples were collected through the tail vein at 0 and 90 min after Triton WR1339 injection. TG in plasma was measured using Triglyceride E-Test Wako (Wako, Osaka, Japan) according to the manufacturer's protocol.


Figure 1
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  		FIGURE 1.
Lipid accumulation in the livers of control, Sod2 KO, Sod1 KO, and double KO mice. A, Western blot analysis of CuZn-SOD, Mn-SOD, and beta-actin and RT-PCR analysis of Sod1, Sod2, and Gapdh in the livers of 8-week-old control, Sod2 KO, Sod1 KO, and double KO mice. B, the total amount of MDA and HAE in the livers from 50-week-old control, Sod2 KO, Sod1 KO, and double KO mice (n = 5, 6, 7, and 8, respectively). C, Oil Red O staining of the liver sections. Bar indicates 100 µm. Values are mean ± S.D. ##, p < 0.01, compared with control mice by Student's t test.

 
Determination of Alanine Aminotransferase, Total Cholesterol, and Albumin of Serum and Liver TG—Alanine aminotransferase activities, total cholesterol, and albumin in serum were measured by Transaminase CII-Test Wako, Cholesterol E-Test Wako, and Albumin B-Test Wako (Wako), respectively, according to the manufacturer's protocol. TG in livers was extracted with 9 volumes of isopropanol and measured using Triglyceride E-Test Wako.

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)—Total RNA was extracted from livers of each of three 8-week-old mice with an RNeasy mini kit (Qiagen, Valencia, CA). For the RT-PCR analysis, cDNAs were synthesized using an avian myeloblastosis virus reverse transcriptase first-strand cDNA synthesis kit (Takara Bio, Shiga, Japan). The following primers used in RT-PCR were 5'-AAG AGC CTT CCA GTT GGC AAC A-3' and 5'-TTT TCC AAC CAG CGC TCC AAG T-3' for Apob;5'-TCG GTG TGA ACG GAT TTG GC-3' and 5'-ATT TCT CGT GGT TCA CAC CC-3' for Gapdh;5'-ATG AAA GCG GTG TGC GTG CTG-3' and 5'-AAT CAC TCC ACA GGC CAA GCG-3' for Sod1. The transcripts of Sod2 gene were amplified using a forward primer located in exon 2, 5'-GAC CTG CCT TAC GAC TAT GG-3', and a reverse primer located in exon 4, 5'-GAC CTT GCT CCT TAT TGA AGC-3' for Sod2. RT-PCR was performed for 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 60 s. PCR products were then subjected to gel electrophoresis on a 2% agarose gel containing ethidium bromide.

Degradation of ApoB by Cu2+—We collected plasma from C57BL/6 mice and stored it at -80 °C until use. Oxidation was initiated at 37 °C by the addition of 40 µl of 2x phosphate-buffered saline and 20 µl of 8 mM CuSO4 to 20 µl of plasma. Samples for immunoblot analysis were collected at 0, 3, and 6 h. The samples were denatured by boiling in SDS-PAGE sample buffer containing 0.5 M dithiothreitol and 25 mM EDTA. Ten µl of samples were subjected to 2-15% gradient gel. The proteins were electroblotted onto Protran BA83 nitrocellulose membrane and Immobilon-P membrane for detection of apoB100 and transferrin, respectively. The membrane was blocked in 5% skim milk and probed with antibodies against apoB100 (1:500; H-15) and anti-transferrin antibody (1:2000; Biomeda, Foster City, CA). Immunoreactive apoB and transferrin were visualized with ECL and LAS-3000.

Statistical Analysis—We analyzed data using the unpaired t test and one-way ANOVA followed by the Tukey-Kramer test. When needed, data were log transformed for homogeneity of variance in Figs. 2 and 3. We considered p values < 0.05 to be statistically significant. Results are expressed as mean values ± S.D.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Lipid Accumulation in CuZn-SOD-deficient Mouse Liver—To investigate the effect of oxidative stress on liver homeostasis, we used Sod2 KO, Sod1 KO, and double KO mice. As shown in Fig. 1A, exon 3-deleted transcripts of Sod2 were detected in the livers of Sod2 KO and double KO mice. We failed to detect Mn-SOD proteins in the livers of Sod2 KO and double KO by immunoblot analysis (Fig. 1A). We also confirmed CuZn-SOD deficiency in Sod1 KO and double KO mice by immunoblot analysis and RT-PCR (Fig. 1A).

To confirm the effect of CuZn-SOD and Mn-SOD deficiency on livers, we measured the byproducts of lipid peroxidation, MDA and HAE (Fig. 1B). In Sod1 KO and double KO mice, we observed significant increases in the total amount of MDA and HAE in the livers (145.7 and 151.0%) of control mice, respectively (Fig. 1B). In contrast, there was no difference in the total amount of MDA and HAE between the livers of control and Sod2 KO mice (Fig. 1B).

To assess the hepatic lipid accumulation, we stained liver sections and measured the levels of liver TG in Sod2 KO, Sod1 KO, and double KO mice. As shown in Fig. 1C, Oil Red O staining showed many small lipid droplets in the livers of Sod1 KO and double KO mice but not in Sod2 KO mice. In addition, liver TG of double KO mice significantly increased to 233.4% of control mice (p < 0.001, ANOVA with Tukey-Kramer test) (Fig. 2A). On the other hand, liver TG was not increased in Sod2 KO mice (Fig. 2A). However, ANOVA with Tukey-Kramer test failed to reveal a significant difference in liver TGs between Sod1 KO and control mice (Fig. 2A). Our data indicated that Mn-SOD and CuZn-SOD deficiency enhanced hepatic lipid accumulation in mice. We also observed significant increase of alanine aminotransferase activity and liver weights in Sod1 KO and double KO (Fig. 2, B and C). On the other hand, there was no significant difference in TG (ANOVA, p = 0.109), cholesterol (ANOVA, p = 0.520), and albumin (ANOVA, p = 0.148) in serum between these mutant mice and control mice (Fig. 2, D-F). The data in Figs. 1 and 2 suggest that CuZn-SOD deficiency causes hepatic lipid accumulation and liver dysfunction in mice.


Figure 2
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  		FIGURE 2.
Increased liver weight and increased liver TG content in Sod1 KO and double KO mice. Liver TG content (n = 8, 10, 8, and 10) (A), alanine aminotransferase activity (n = 14, 20, 11, and 12) (B), liver weight (n = 9, 17, 12, and 15) (C), serum TG content (n = 14, 17, 13, and 12) (D), serum total cholesterol (n = 9, 9, 7, and 8) (E), and serum albumin (n = 5, 6, 6, and 7) (F) in 8-week-old control, Sod2 KO, Sod1 KO, and double KO mice. Data are expressed as mean ± S.D. Data were analyzed by one-way ANOVA with Tukey-Kramer test; ***, p < 0.001.

 
Enhancement of Lipid Accumulation in CuZn-SOD-deficient Mice on High Fat Diet—We fed Sod2 KO, Sod1 KO, and double KO with a high fat diet containing 20% lard for 4 weeks, which corresponds to 38% of total calories. After feeding the high fat diet, we observed larger lipid droplets in the livers of Sod1 KO and double KO. In addition, lipid droplets of double KO mice were larger than those of Sod1 KO mice (Fig. 3, A and B). In contrast, high fat diet failed to increase the size of lipid droplets in the livers of control and Sod2 KO mice (Fig. 3, A and B). To confirm the increase of lipid level in the livers of Sod1 KO and double KO mice, we measured the TG contents in the livers of these mice. After feeding the high fat diet, the liver TG contents were significantly increased in Sod1 KO and double KO (ANOVA with Tukey-Kramer, p < 0.001 and p < 0.01) but not in control and Sod2 KO mice, indicating that CuZn-SOD deficiency and high fat diet synergistically enhanced hepatic lipid accumulation in mice (Fig. 3C).

Although we could not observe the increase of serum TG contents in Sod2 KO, Sod1 KO, and double KO mice, serum TG content was significantly increased in control mice on high fat diet (Fig. 3D). These results indicated that CuZn-SOD-deficient mice on high fat diet developed steatosis without developing hypertriglyceridemia, implying that cytosolic superoxide plays a pathological role in the development of liver steatosis.

Suppressed Lipoprotein Secretion from CuZn-SOD-deficient Mouse Liver—To confirm whether the lipoprotein secretion from livers is impaired in Sod1 KO and double KO mice, we measured TG secretion rates from livers using a lipoprotein lipase inhibitor, Triton WR1339. Surprisingly, as shown in Fig. 4A, we observed a marked suppression of TG secretion from the livers of Sod1 KO and double KO mice (5.5 and 6.9% of control mice, respectively). In contrast, we hardly detected any difference in TG secretion rates between Sod2 KO and control mice (Fig. 4A). These results suggest that cytosolic superoxide suppresses lipoprotein secretion from the livers of Sod1 KO and double KO mice.

Because apoB plays an essential role in lipoprotein secretion from the liver (20), we analyzed apoB levels in the plasma and the livers of Sod2 KO, Sod1 KO, and double KO mice. As shown in Fig. 4B, plasma apoB100 was markedly down-regulated in Sod1 KO and double KO mice. Furthermore, apoB48 and apoB100 were both markedly down-regulated in the liver microsome fractions from Sod1 KO and double KO mice (Fig. 4B), suggesting that cytosolic superoxide may suppress apoB protein in the livers by transcriptional or posttranslational down-regulation. To confirm the transcriptional levels of apoB in the livers of these mice, we assessed the expression levels of apoB mRNA by RT-PCR. As shown in Fig. 4C, we failed to detect obvious differences in the amounts of apoB transcripts between CuZn-SOD-deficient mice and control mice. These results suggest that apoB protein is suppressed or degraded by posttranslational mechanism in the livers of Sod1 KO and double KO mice. We also analyzed the expression of MTP in liver microsome fractions, because down-regulation of MTP leads to intracellular degradation of apoB (21, 22). The result showed that CuZn-SOD deficiency did not influence MTP levels in liver microsomal fractions, suggesting that apoB suppression may be independent of MTP in the liver of Sod1 KO and double KO mice (Fig. 4B). These results indicate that CuZn-SOD deficiency caused apoB suppression, which resulted in the impaired lipoprotein secretion from the liver.

Time-dependent Decrease of Plasma ApoB by Incubation with Cu2+—Previous studies reported that apoB protein was degraded by incubation with Cu2+ (23). To confirm the reactivity of apoB protein to ROS, we incubated plasma of control mice with 2 mM Cu2+. As shown in Fig. 5, transferrin was not decreased after incubation with Cu2+. In contrast, incubation of plasma with 2mM Cu2+ extensively decreased apoB protein in a time-dependent manner, suggesting that apoB is highly vulnerable to ROS generated by Cu2+ (Fig. 5).


Figure 3
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  		FIGURE 3.
Enhanced lipid accumulation in the livers of Sod1 KO and double KO mice by high fat diet. A and B, 4- or 5-week-old control, Sod2 KO, Sod1 KO, and double KO mice were fed normal diet or high fat diet for 4 weeks. Liver sections were stained with hematoxylin and eosin (upper panels) and Oil Red O (lower panels). Liver TG content (C) and serum TG content (D) in mutant and control mice were measured. The same data of TG content in mice on normal diet were used in Fig. 2, A and D. Data are expressed as mean ± S.D. Data were analyzed by one-way ANOVA with Tukey-Kramer test; *, p < 0.05, **, p < 0.01, and ***, p < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, our results suggested that CuZn-SOD deficiency causes lipid accumulation in the liver of mice. We hardly observed the increase of TG content and lipid peroxidation in the liver of Sod2 KO mice, although we confirmed the marked reduction of Mn-SOD mRNA and protein in Sod2 KO mice liver (Fig. 1A). However, it has been reported that total knockout or ubiquitous knockdown of the Sod2 gene in mice increases lipid accumulation in livers at early neonatal stage (14, 24). Taken together with the results presented in this report, we suggest that other antioxidant systems might compensate for the loss of Sod2 in liver or that hepatic lipid accumulation may be systemically induced by the oxidative stress of other organs. Huang and co-workers (17, 25) reported that Sod1 KO mice had an increased incidence of neoplastic changes and increased levels of 8-oxo-dG, F2-isoprostanes, and DNA mutations in the liver. In the present study, on the contrary, there was no significant difference in the total amount of MDA and HAE between Sod1 KO liver and double KO liver (Fig. 1B). Taken together with previous data and our data, we suggest that CuZn-SOD plays an important role in the antioxidant defense system of the liver, whereas Mn-SOD has a less important physiological role than CuZn-SOD in liver.

We also suggest that Mn-SOD deficiency enhanced hepatic lipid accumulation in CuZn-SOD-deficient mice, but not in Sod2 KO mice (Fig. 3). Our results implied that CuZn-SOD and Mn-SOD may play distinct but overlapping roles in the intracellular defense system against superoxide.

In Fig. 4, A and B, we show the suppressed apoB-lipoprotein secretion from the liver of CuZn-SOD-deficient mice. ApoB-lipoprotein secretion from liver is mainly regulated by apoB and MTP. ApoB-containing lipoproteins assemble cotranslationally in endoplasmic reticulum, where MTP plays a pivotal role in apoB-lipoprotein assembly and secretion (20, 21). Liao et al. (22) reported that blocking MTP function by liver-specific disruption of the Mttp gene and an MTP inhibitor, 8aR, significantly reduced apoB in plasma and liver microsomes. As shown in Fig. 4B, we failed to detect the difference in MTP protein level between control mice liver and CuZn-SOD-deficient mice liver, indicating that the reduced TG secretion is independent of MTP level in the CuZn-SOD-deficient mouse liver.


Figure 4
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  		FIGURE 4.
Impaired TG secretion from livers of Sod1 KO and double KO mice. A, TG secretion rate from livers (n = 5, 5, 5, and 3) of control, Sod2 KO, Sod1 KO, and double KO mice. B, immunoblot analysis of apoB100 in plasma and of apoB100, apoB48, MTP, and GRP78 in liver microsome fraction of control, Sod2 KO, Sod1 KO, and double KO mice. C, RT-PCR for Apob and Gapdh in mutant and control mice. ##, p < 0.01 compared with control mice by Student's t test.

 


Figure 5
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  		FIGURE 5.
Time-dependent decrease of plasma apoB by incubation with Cu2+. Plasma of control mice was treated with 2 mM Cu2+ at 37 °C. Samples were collected at 0, 3, and 6 h after the addition of Cu2+. ApoB and transferrin contents were analyzed by Western blot.

 
In this study, we have shown the decreased apoB level in the plasma and the livers of CuZn-SOD-deficient mice. In contrast, we hardly detected any difference in apoB mRNA expression levels between control mice and CuZn-SOD-deficient mice. Our results indicate that the decreased apoB level in CuZn-SOD-deficient mice is caused by posttranslational mechanisms such as degradation and modification. Several studies focused on the relationship between oxidative stress and apoB-lipoprotein secretion from hepatocytes (26, 27). In mice, phospholipid transfer protein deficiency caused decreased levels of vitamin E, increased lipid peroxide content in the liver, and degraded apoB via post-endoplasmic reticulum pre-secretory proteolysis pathway in hepatocyte (27). Pan et al. (26) also reported that docosahexaenoic acid and eicosapentaenoic acid stimulated apoB100 degradation in rat hepatoma or primary rodent hepatocytes via post-endoplasmic reticulum pre-secretory proteolysis. However, administration of docosahexaenoic acid and eicosapentaenoic acid with the iron chelator desferrioxamine or vitamin E resulted in reduced lipid peroxidation, restoration of apoB100 levels, and very low density lipoprotein secretion (26). In addition, it has been reported that the incubation of low density lipoprotein with dihydroxy-fumaric acid and Fe3+-ADP, a free radical-generating system, in the absence of cells resulted in the non-enzymatic cleavage of low density lipoprotein-associated apoB (28). These studies and our data suggest that oxidative stress may decrease apoB-lipoprotein secretion due to the accelerated degradation of apoB in hepatocytes.

In this report, we have shown no difference in plasma albumin levels between control mice and CuZn-SOD-deficient mice, suggesting that the general protein synthesis and secretion in the liver of CuZn-SOD-deficient mice are considered normal. We also observed that incubation of plasma with Cu2+ decreased apoB protein whereas transferrin was not decreased under the same condition, suggesting that apoB is highly sensitive to ROS (Fig. 5). Elchuri et al. (17) reported that cytosolic aconitase was markedly reduced in the liver of CuZn-SOD-deficient mice. In contrast, mitochondrial aconitase level in Sod1 KO liver remained unchanged, suggesting that excess ROS in CuZn-SOD-deficient mice may attack specific target molecules in cells (17). Our data suggest that apoB protein is one of the target molecules of ROS in the cytosol of liver.

Previous studies reported the involvement of oxidative stress in NAFLD. Laurent et al. (13) reported that manganese [III] tetrakis (5,10,15,20 benzoic acid) and copper [II] diisopropyl salicylate limited the extension of histological liver steatosis in ob/ob mice. As shown in Fig. 3, liver TG contents in CuZn-SOD-deficient mice were significantly increased by feeding a high fat diet. This result suggests that oxidative stress and high fat diet synergistically stimulate the progression of hepatic steatosis. The lifespan of ROS is extremely short, and they quickly react with other molecules, such as lipid and DNA (29). Lipid peroxides have a longer lifespan than ROS, and they can react with their target molecules (29). Because increased lipid storage in liver provides the substrate for lipid peroxidation, excess fat storage may contribute to amplifying oxidative stress (6).

Several studies focused on the relationship between apoB secretion and NASH. Charlton et al. (30) reported that synthesis rate of apoB100 in patients with NASH was significantly lower than that of lean controls and body mass index-matched obese controls without NASH. Musso et al. (31) reported that oral fat load failed to increase plasma apoB48 and apoB100 in patients with NASH. The suppression of apoB synthesis may increase lipid retention in the livers of patients with NASH, suggesting that suppressed apoB may be an important factor in the progression of NAFLD.

Our data suggest the possibility that apoB protein is a target molecule of ROS in liver. Our results imply that generation and removal of ROS have a close relationship with the pathogenesis of NAFLD.


   FOOTNOTES
 
* This work was supported by grants from Comprehensive Research on Aging and Health, Ministry of Health, Labor, and Welfare and by grants-in-aid for Scientific Research from the Ministry of Education, Science, Culture, Sports, and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Research resident of the Japan Foundation for Aging and Health. Back

2 To whom correspondence should be addressed: Research Team for Molecular Biomarkers, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan. Tel.: 81-3-3964-3241; Fax: 81-3-3579-4776; E-mail: sirasawa{at}tmig.or.jp.

3 The abbreviations used are: NAFLD, nonalcoholic fatty liver disease; apoB, apolipoprotein B; Grp78, glucose-regulated protein 78kDa; HAE, 4-hydroxyalkenal; MDA, malondialdehyde; MTP, microsomal triglyceride transfer protein; NASH, nonalcoholic steatohepatitis; ROS, reactive oxygen species; RT-PCR, reverse transcriptase PCR; SOD, superoxide dismutase; Sod1 KO, Sod1 knock-out mice; Sod2 KO, liver-specific Sod2 knock-out; double KO, Sod1/liver-specific Sod2 double knock-out mice; TG, triglyceride; ANOVA, analysis of variance. Back


   ACKNOWLEDGMENTS
 
We thank Drs. H. Saito and K. Tomita (Keio University) for discussions and Drs. M. Takahashi, E. Moriizumi, M. Yamaguchi, S. Kawakami, and T. Ikegami (Tokyo Metropolitan Institute of Gerontology) for technical support. We also thank W. Zhou for manuscript preparation.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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