Leprdb/db Mice with Senescence Marker Protein-30 Knockout (Leprdb/dbSmp30Y/−) Exhibit Increases in Small Dense-LDL and Severe Fatty Liver Despite Being Fed a Standard Diet

Background/Aims The senescence marker protein-30 (SMP30) is a 34 kDa protein originally identified in rat liver that shows decreased levels with age. Several functional studies using SMP30 knockout (Smp30Y/−) mice established that SMP30 functions as an antioxidant and protects against apoptosis. To address the potential role of SMP30 in nonalcoholic fatty liver disease (NAFLD) pathogenesis, we established Smp30Y/− mice on a Leprdb/db background (Leprdb/dbSmp30Y/− mice). Research Design/Principal Findings Male Leprdb/dbSmp30Y/− mice were fed a standard diet (340 kcal/100 g, fat 5.6%) for 16 weeks whereupon the lipid/lipoprotein profiles, hepatic expression of genes related to lipid metabolism and endoplasmic reticulum stress markers were analyzed by HPLC, quantitative RT-PCR and western blotting, respectively. Changes in the liver at a histological level were also investigated. The amount of SMP30 mRNA and protein in livers was decreased in Leprdb/dbSmp30Y/+ mice compared with Leprdb/+Smp30Y/+ mice. Compared with Leprdb/dbSmp30Y/+ mice, 24 week old Leprdb/dbSmp30Y/− mice showed: i) increased small dense LDL-cho and decreased HDL-cho levels; ii) fatty liver accompanied by numerous inflammatory cells and increased oxidative stress; iii) decreased mRNA expression of genes involved in fatty acid oxidation (PPARα) and lipoprotein uptake (LDLR and VLDLR) but increased CD36 levels; and iv) increased endoplasmic reticulum stress. Conclusion Our data strongly suggest that SMP30 is closely associated with NAFLD pathogenesis, and might be a possible therapeutic target for NAFLD.


Introduction
Metabolic syndrome has been described as the association of insulin resistance, hypertension, hyperlipidemia and obesity. Its prevalence has increased dramatically, mainly in developed countries. The hepatic manifestations of metabolic syndrome include nonalcoholic fatty liver disease (NAFLD) and its progressive variant, nonalcoholic steatohepatitis (NASH) [1,2]. Several animal models have been proposed for NAFLD and NASH research [3]. Since leptin plays a major role in food intake and energy expenditure, total leptin deficiency or leptin resistance can lead to massive obesity, type 2 diabetes, dyslipidemia and fatty liver. Therefore, many investigations pertaining to NAFLD/ NASH have been carried out in genetic leptin-deficient ob/ob mice or leptin-resistant db/db mice that were fed a high fat diet (HFD) or the methionine/choline deficiency diet [3][4][5]. However, these models differ significantly from the human NAFLD/NASH phenotype in a number of pathogenically important ways.
The senescence marker protein-30 (SMP30) is a 34 kDa protein that was originally identified in rat liver and its levels decrease with age [6]. We previously reported that SMP30 participates in Ca 2+ efflux by activating the calmodulin-dependent Ca 2+ -pump that confers resistance to cell injury caused by high intracellular Ca 2+ concentrations [7]. We identified SMP30 as a gluconolactonase (GNL) that is involved in L-ascorbic acid biosynthesis in mammals, and have established SMP30-knockout (KO) mice [8]. The livers of SMP30-KO mice are highly susceptible to tumor necrosis factor-a (TNF-a) and Fas-mediated apoptosis, indicating that SMP30 has an anti-apoptotic effect [9]. SMP30-KO mice showed mitochondrial damage and abnormal accumulation of triglycerides, cholesterol, and phospholipids in the liver [10]. In addition, we reported that decreased SMP30 levels contribute to lowered glucose tolerance [11]. These results are in agreement with several functional studies, which also established that SMP30 functions as an antioxidant and anti-apoptotic protein [12][13][14][15].
To address the potential role of SMP30 in NAFLD/NASH pathogenesis, we generated SMP30-KO mice on a Lepr db/db background (Lepr db/db Smp30 Y/2 ) and investigated the lipid/lipoprotein profiles, hepatic expression of genes relevant to lipid metabolism and histological changes in the livers of Lepr db/ db Smp30 Y/2 mice fed a standard diet. Here we show that despite being fed a standard diet, Lepr db/db Smp30 Y/2 mice have altered lipoprotein components and severe fatty liver accompanied by increased inflammation and oxidative stress induced by mitochondrial and endoplasmic reticulum dysfunction.

Animal crossing and genotyping, and experimental protocol
We used type 2 diabetic obese Lepr db/db mice with a C57BLKS/J background. Male Lepr db/+ mice were obtained from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). SMP30-knockout (KO) mice with a C57BL/6 background were established and maintained as described previously [8,9]. Heterozygous SMP30-KO male mice do not exist, because the Smp30 gene is located on the X chromosome. SMP30-KO mice cannot synthesize vitamin C in vivo, because in mammals SMP30 is the penultimate enzyme in the vitamin C biosynthetic pathway [8]. To maintain vitamin C levels in tissues that were similar to that of wild type mice, and to eliminate any possible confounding influences of vitamin C deficiency, these mice were given free access to water supplemented with 1.5 g/L vitamin C and 10 mM ethylenediaminetetraacetic acid (EDTA) to avoid the effects of vitamin C deficiency [16]. As schematically illustrated in Figure 1A, male Lepr db/+ mice were first crossed with female Smp30 2/2 mice to produce male Lepr db/+ Smp30 Y/2 mice and female Lepr db/+ Smp30 +/2 mice. The SMP30 mutant mice genotypes were determined as described previously [9]. Next, male Lepr db/+ Smp30 Y/2 and female Lepr db/+ Smp30 +/2 mice were interbred to produce homozygous Lepr db/db Smp30 Y/+ and Lepr db/db Smp30 Y/2 mice and heterozygote control Lepr db/+ Smp30 Y/2 and Lepr db/+ Smp30 Y/+ mice. The mutant Lepr db gene was identified by restriction enzyme digestion of PCR products. In brief, Lepr db gene PCR products were amplified by PCR using genomic DNA and forward (59-AGAACGGA-CACTCTTTGAAGTCTC-39) and reverse (59-CATTCAAAC-CATAGTTTAGGTTTGTGT-39) primers. PCR products were then digested by AfaI (Takara Bio Inc., Shiga, Japan) and analyzed by agarose gel electrophoresis. The mutant Lepr db gene showed two bands of 108 bp and 27 bp while the wild type allele showed one 135 bp band.
We prepared four groups of five eight week old male mice, with each group having four genotypes: Lepr db/+ Smp30 Y/+ , Lepr db/+ Smp30 Y/2 , Lepr db/db Smp30 Y/+ , and Lepr db/db Smp30 Y/2 . All mice were fed a vitamin C free-standard diet (CL-2; 340 kcal/100 g, fat 5.6%, CLEA Japan, Tokyo, Japan) for 16 weeks. Lepr db/+ Smp30 Y/2 mice and Lepr db/db Smp30 Y/2 mice had free access to 1.5 g/L vitamin C water containing 10 mM EDTA, whereas Lepr db/+ Smp30 Y/+ and Lepr db/db Smp30 Y/+ mice had 10 mM EDTA water. Mice were maintained on a 12 h light/dark cycle in a controlled environment. All experimental procedures using laboratory animals were approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute of Gerontology (Permit Number: 12016).

Blood and liver tissue collection
All mice were fasted for 16 h and anesthetized at the age of 24 weeks. Blood was obtained from the inferior vena cava, anticoagulated with EDTA, and subsequently centrifuged at 8806g for 15 min at 4uC. Mice were systemically perfused with ice-cold phosphate buffered saline through the hepatic portal vein to wash out remaining blood cells and then the livers were removed. The whole body subcutaneous fat was collected and the weight measured. The livers were immersed in RNAlaterH (Life Technologies Corp., Carlsbad, CA, USA) for RNA extraction and fixed with 10% neutral buffered formalin for histological analysis or frozen in liquid nitrogen for biochemical analysis. All samples were stored at 280uC until use.

Western blotting analysis
Livers were homogenized in ice-cold homogenization buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM phenyl methanesulfonyl fluoride, and a protease inhibitor cocktail (cOmplete, EDTA-free; Roche Diagnostics GmbH, Mannheim, Germany)) for 30 seconds using a high speed homogenizer (POLYTRONH PT-MR 2100; Kinematica AG, Switzerland). The homogenate was then centrifuged at 21,0006g for 10 min at 4uC. The supernatants were boiled for 5 min with a lysis buffer containing 0.125 M Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.2% bromophenol blue at a ratio of 1:1. Total protein equivalents for each sample were separated on a 14% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The membrane was incubated with the primary antibody, followed by incubation with a horseradish peroxidase-linked goat anti-rabbit IgG (Bio-Rad Laboratories, Tokyo, Japan). The primary antibodies used were: anti-rat SMP30 rabbit polyclonal antibody (Cosmo Bio Co., Ltd., Tokyo, Japan), anti-phosphorylated eukaryotic initiation factor-2a (p-eIF2a) rabbit monoclonal antibody (#3597, Cell Signaling Technology, Beverly, MA) and anti-C/EBP homologous protein (CHOP) rabbit polyclonal antibody (sc-575, Santa Cruz Biotechnology, Santa Cruz, CA) and b-actin (Cell Signaling Technology, Beverly, MA) as a loading control. After washing, immunoreactivity was detected using ECL chemiluminescence reagents (ECL plus, GE Healthcare Japan, Tokyo, Japan). Chemiluminescence signals were quantified with a LAS-3000 imaging system (Fujifilm, Tokyo, Japan). The mean signals from five Lepr db/+ Smp30 Y/+ mice were assigned a relative value of 1.0. The total protein concentration was determined with a PierceH BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA) using bovine serum albumin as a standard.

Measurement of vitamin C levels in the liver
Livers were homogenized in 14 vol 5.4% metaphosphate and 1 mM EDTA, and the homogenate was then centrifuged at 21,0006g for 15 min at 4uC. Vitamin C was analyzed by highperformance liquid chromatography (HPLC) using an Atlantis dC18 5 mm column (4.66150 mm; Nihon Waters, Tokyo, Japan) [17]. The mobile phase was 50 mM phosphate buffer (pH 2.8), 0.2 g/L EDTA, and 2% methanol at a flow rate of 1.3 ml/min, and electrical signals were recorded using an electrochemical detector equipped with a glassy carbon electrode at +0.6 V.

Measurement of T-cho, TG and PL in the liver
Liver tissues were homogenized with 2 vol. of water using a handy homogenizer (Moji-mojikun; Nippon Genetics, Tokyo, Japan). Homogenates were added to a chloroform-methanol (2:1; v/v) mixture, and centrifuged at 21,0006g for 10 min at 4uC. The supernatant organic phase was then collected, dried under nitrogen gas and resolubilized in 2-propanol. T-cho, TG and PL concentrations in total lipid extracts were determined using commercial enzymatic kits (Wako Pure Chemical Industries, Osaka, Japan).

Thiobarbituric acid reactive substances (TBARS) assay
Lipid peroxidation was estimated by the amounts of TBARS in the liver that were determined according to the method of Ohkawa et al. [18]. The livers were first homogenized in ice-cold 0.1 M phosphate buffer (pH 7.4). The homogenates were then centrifuged at 15,0006g for 30 min at 4uC and the supernatant was used for further assays. One volume of sample was mixed thoroughly with two volumes of stock solution (15% (w/v) trichloroacetic acid, 0.375% (w/v) thiobarbituric acid, and 0.25 mM HCl). The mixture was then heated for 30 min in a boiling water bath. After cooling, the flocculate precipitate was removed by centrifugation at 1,0006g for 10 min and the absorbance (OD 532 nm) of the sample was measured. The TBARS levels are expressed as the equivalent amounts of malondialdehyde produced from 1,1,3,3-tetramethoxypropane.
The H&E-stained specimens were anonymized, and five different areas per mouse were randomly selected by a researcher. These specimens were scored by two independent investigators blinded to sample identity according to the NASH activity score (NAS) [20] for the degree of steatosis (0-3), lobular inflammation (0-3) and hepatocellular ballooning (0-2). The average of the two investigators' scores was regarded as the score for each mouse.
RNA isolation, first-strand cDNA synthesis, and gene expression analysis Liver tissue was finely ground with a liquid nitrogen-cooled mortar and pestle and homogenized in ice-cold TRIzol reagent (Life Technologies, Carlsbad, CA, USA) before isolation of total RNA according to the manufacturer's instructions. Total RNA (0.5 mg) was reverse-transcribed using PrimeScript RT Master Mix (TaKaRa Bio Inc., Shiga, Japan) for first-strand cDNA synthesis with an oligonucleotide dT primer and random hexamer priming according to the manufacturer's recommendations.
The mRNA expression levels of the following proteins: acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), sterol regulatory element-binding protein 1c (SREBP1c), SREBP2, 3hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR), peroxisome proliferator-activated receptor-a (PPARa), medium-chain acyl-CoA dehydrogenase (MCAD), microsomal triglyceride transfer protein (MTP), apolipoprotein-B100 (ApoB100), LDL receptor (LDLR) and VLDL receptor (VLDLR), CD36 and spliced X-box binding protein 1 (sXBP1), all of which are involved in lipid and lipoprotein metabolism, were quantitated using real-time reverse transcription polymerase chain reaction (RT-PCR). RT-PCR was performed using a Thermal Cycler Dice Real Time System II (TaKaRa Bio Inc.) and real-time SYBRH Premix Ex Taq TM (TaKaRa Bio Inc.) according to the manufacturer's instructions. The specific primers for the target genes and b-actin are described in Table 1. The following PCR conditions were used: 1 cycle for 30 s at 95uC, followed by 40 cycles for 5 s at 95uC, and 30 s at 60uC. The product specificity generated for each primer set was examined for each fragment using a melting curve and gel electrophoresis. The relative expression levels of each targeted gene were normalized to b-actin threshold cycle (CT) values and quantified using the comparative threshold cycle 2 2DDCT method as previously described [21]. Signals from Lepr db/+ Smp30 Y/+ mice were assigned a relative value of 1.0. Five mice from each group were examined, and real time RT-PCR was run in duplicate for each sample.

Statistical analysis
Data are expressed as means 6 SEM. Statistical differences between groups were determined by one-way analysis of variance (ANOVA) with Scheffe's post hoc test. A P value,0.05 was considered to be statistically significant.

Results
Generation of Lepr db/db Smp30 Y/2 mice As shown in Fig. 1A and B, we established Lepr db/db Smp30 Y/2 mice, which were born at the expected Mendelian ratios and by 24 weeks of age had appearances that were indistinguishable from obese Lepr db/db Smp30 Y/+ mice. Western blot analysis of liver tissue from Lepr db/+ Smp30 Y/2 and Lepr db/db Smp30 Y/2 mice demonstrated that these animals lacked SMP30 protein (Fig. 1C). There were no significant differences found in the liver vitamin C concentration among any of the experimental groups (Fig. 1D).
Given the difference in SMP30 protein levels observed for Lepr db/+ Smp30 Y/+ and Lepr db/db Smp30 Y/+ mice (Fig. 1C), we next quantified the amounts of SMP30 mRNA and protein and found that they were decreased by 25% and 47%, respectively, in Lepr db/db Smp30 Y/+ mice as compared to Lepr db/+ Smp30 Y/+ mice (both P,0.01) (Fig. 2).
Lepr db/db Smp30 Y/2 mice had manifestations of metabolic syndrome The physiological and blood biochemical parameters of 24 week old animals of the four groups are presented in Table 2. Compared with Lepr db/+ Smp30 Y/+ and Lepr db/+ Smp30 Y/2 mice, the food intake of Lepr db/db Smp30 Y/2 mice during the experimental period was significantly increased by 33% and 40%, respectively (P,0.001). Meanwhile, there were no significant differences between Lepr db/db Smp30 Y/2 and Lepr db/db Smp30 Y/+ mice (P = 0.07). The body weight of Lepr db/db Smp30 Y/2 mice was 79% and 87% higher than Lepr db/+ Smp30 Y/+ and Lepr db/+ Smp30 Y/2 mice, respectively (P,0.001). There were no significant differences in body weight between Lepr db/db Smp30 Y/2 and Lepr db/db Smp30 Y/+ mice. Likewise, the epididymal and subcutaneous fat weight in Lepr db/db Smp30 Y/2 mice were significantly higher than those of Lepr db/+ Smp30 Y/+ and Lepr db/+ Smp30 Y/2 mice (P,0.001), although no significant differences in epididymal or subcutaneous fat weight were observed between Lepr db/db Smp30 Y/2 and Lepr db/db Smp30 Y/+ mice.
Although there was a non-significant increase in plasma TG levels in the two groups of Lepr db/db mice (Lepr db/db Smp30 Y/2 and Lepr db/db Smp30 Y/+ ) compared with the two groups of Lepr db/+ mice (Lepr db/+ Smp30 Y/2 and Lepr db/+ Smp30 Y/+ ), there were no significant differences in plasma TG levels among any of the experimental groups. Both plasma T-cho and PL concentrations in Lepr db/db Smp30 Y/2 mice were significantly higher than those in Lepr db/+ Smp30 Y/+ and Lepr db/+ Smp30 Y/2 mice (both P,0.001). There were no significant differences in plasma T-cho and PL concentrations between Lepr db/db Smp30 Y/2 and Lepr db/db Smp30 Y/+ mice.
Lepr db/db Smp30 Y/2 mice show fatty liver accompanied by inflammatory cells and oxidative stress despite being fed a standard diet As shown in Fig. 5A, hepatic histological examination revealed increased steatosis in Lepr db/db Smp30 Y/+ mice and increased steatosis accompanied by inflammatory cells in Lepr db/db Smp30 Y/2 mice. In contrast, no histological abnormalities were observed for both Lepr db/+ Smp30 Y/+ and Lepr db/+ Smp30 Y/2 animals. Fibrosis was not observed in any of the groups.
MTP and ApoB100 are key genes for VLDL secretion. Both MTP and ApoB100 mRNA levels were significantly lower in Lepr db/db Smp30 Y/2 mice than in Lepr db/+ Smp30 Y/+ and Lepr db/+ Smp30 Y/2 mice (P,0.001). Compared with Lepr db/db Smp30 Y/+ mice, MTP mRNA expression levels in Lepr db/db Smp30 Y/2 mice were decreased, although the change did not reach statistical significance. There was no significant difference in ApoB100 mRNA levels between Lepr db/db Smp30 Y/+ and Lepr db/db Smp30 Y/2 mice.

Discussion
The two-hit theory proposed by Day and James [22], in which the initial trigger is the hepatic accumulation of excessive fat, followed by the second hit of oxidative stress, is widely advocated as a pathogenic mechanism for NASH. Therefore, it is of great interest to study in greater detail the role of SMP30 in relation to the pathogenic mechanism for NAFLD/NASH in SMP30-KO mice on a Lepr db/db background. Interestingly, we observed first that SMP30 levels in Lepr db/db Smp30 Y/+ mice were significantly lower than in Lepr db/+ Smp30 Y/+ mice, which suggests that this decrease is related to the development of obesity and obesityrelated disorders in Lepr db/db Smp30 Y/+ mice. The mechanism(s) of SMP30 reduction in Lepr db/db Smp30 Y/+ mouse livers is unknown. Liver SMP30 protein levels were reported to decrease following liver injury in animals treated with carbon tetrachloride [23], lipopolysaccharide (LPS) [24] or D-galactosamine/LPS [25]. Furthermore, we recently reported that 17b-estradiol attenuates saturated fatty acid diet-induced apoptotic liver injury in ovariectomized mice by up-regulating hepatic SMP30 [14]. Thus, the lipid deposition accompanied by increased oxidative and ER stress in Lepr db/db Smp30 Y/2 mice might result from decreased liver SMP30 levels and in turn exacerbate liver damage via decreased Ca 2+ -pumping activity and anti-oxidative effects of SMP30. Further study will be required to reveal the mechanisms of SMP30 suppression in obesity and obesity-related diseases.
A noteworthy finding of this study is that plasma levels of LDLcho, in particular the smaller sized particles of Fr.no. 12 and Fr.no. 13 that correspond to small dense LDL-cho (sdLDL-cho) and the LDL-cho/HDL-cho ratio in Lepr db/db Smp30 Y/2 mice, were significantly higher than those in Lepr db/db Smp30 Y/+ mice, although plasma levels of T-cho in Lepr db/db Smp30 Y/2 and Lepr db/db Smp30 Y/+ mice were similar. A significantly increased LDL-cho/HDL-cho ratio was also found in Lepr db/+ Smp30 Y/2 mice compared with Lepr db/+ Smp30 Y/+ mice. These results indicate that the SMP30 deficiency contributes to increases in plasma sdLDL-cho and decreases in HDL-cho regardless of leptin receptor mutation followed by hyperphagia and obesity. In a human study an association between fatty liver and increased sdLDL-cho was reported [26][27][28]. Furthermore, we recently reported that in patients with histologically diagnosed NAFLD/NASH, serum sdLDL-cho levels in patients with NAS $5 were significantly higher than those in patients with NAS#2, and sdLDL-cho was significantly and inversely correlated with hepatic SMP30 levels [29]. However, we do not presently have an explanation for the observed increase in sdLDL-cho in Lepr db/db Smp30 Y/2 mice. In humans, TG-rich VLDL (large VLDL1) can be a precursor of sdLDL-cho, i.e., large VLDL1 particles are converted to sdLDL particles by cholesteryl ester transport protein (CETP) and hepatic lipase (HL), the levels of which are commonly increased in type 2 diabetes [30]. Unlike humans, however, mice do not express CETP, and as such, cholesterol is mainly present in HDL. Qiu et al. reported that HL-deficient mice have sdLDLs, but TG enrichment was not observed in these mice [31]. In this study, no difference was observed in the mRNA expression of HL between Lepr db/db Smp30 Y/2 and Lepr db/db Smp30 Y/+ mice or between Lepr db/+ Smp30 Y/2 and Lepr db/+ Smp30 Y/+ mice (data not shown). Recently, we demonstrated that testosterone-deficient mice fed a high-fat diet showed markedly decreased serum TG and TG-VLDL levels and markedly increased serum sdLDL-cho levels, likely due to altered expression of genes involved in hepatic assembly and lipid secretion [32]. Further work will be required to elucidate the molecular mechanism for this increase in sdLDL-cho in SMP30-KO mice.
An additional notable finding of this study was that although Lepr db/db Smp30 Y/2 mice showed no advanced stage NASH including fibrosis, compared with Lepr db/db Smp30 Y/+ mice Lepr db/db Smp30 Y/2 mice had increased NAS activity and inflammation scores as well as enhanced oxidative stress. Hepatic steatosis results from an imbalance in lipid homeostasis in the liver, where fat uptake, de novo lipogenesis, fatty acid oxidation and fat export occur. Compared with Lepr db/db Smp30 Y/+ mice, RT-PCR of lipid homeostasis-related genes in the liver revealed that the expression levels of these genes, in particular PPARa and SREBP-1c (but not CD36), are reduced in Lepr db/db Smp30 Y/2 mice. CD36, a member of the class B scavenger receptor family of cell surface proteins, is abundantly expressed in monocytes/macrophages. Therefore, compared with Lepr db/db Smp30 Y/+ mice, the increase in CD36 mRNA seen in Lepr db/db Smp30 Y/2 mice is likely a partial reflection of the increase in the number of inflammatory cells. PPARa mRNA levels were significantly lower not only in Lepr db/+ Smp30 Y/2 mice compared to Lepr db/+ Smp30 Y/+ mice, but also in Lepr db/db Smp30 Y/2 mice compared to Lepr db/db Smp30 Y/+ mice, suggesting that decreased hepatic SMP30 mRNA expression is associated with mitochondrial and peroxisomal fatty acid b-oxidation.
Mitochondrial dysfunction is known to cause increases in oxidative stress, and indeed we showed that the levels of TBARS and 4-HNE rose in the livers of Lepr db/db Smp30 Y/2 mice. When SMP30-KO mice are fed a vitamin C-deficient diet they do not thrive and display symptoms of scurvy such as bone fractures and rachitic rosary before dying around three months after beginning the deficient diet. As such, Lepr db/+ Smp30 Y/2 and Lepr db/db Smp30 Y/2 mice were given vitamin C-supplemented water in this study. Vitamin C is known to play an important role in the structure and function of mitochondria and endoplasmic reticulum [33]. Harrison et al. reported that vitamin E and vitamin C treatment improves fibrosis but not necroinflammation in NASH patients [34]. Therefore, vitamin C supplementation would be one reason that fibrosis was not observed in Lepr db/db Smp30 Y/2 mice in this study.
In this study, Lepr db/+ Smp30 Y/2 mice exhibited no phenotypes for lipid accumulation and mitochondrial damage in the liver as was previously reported [10]. However, in the previous study, mice were fed an autoclaved CRF-1 diet containing ,55 mg of vitamin C per kg and tap water, and thus the liver vitamin C level in Smp30 Y/2 mice was about 6% that of Smp30 Y/+ mice [8]. Although lipid accumulation and mitochondrial damage were observed in livers of Smp30 Y/2 mice at 12 months [10], it is unclear whether the phenotypes were caused by SMP30 deficiency (loss of unknown function except for vitamin C biosynthesis) and/ or by vitamin C deficiency. Thus, the lack of steatosis in Lepr db/+ Smp30 Y/2 mice might result from their younger age (24 weeks) or the vitamin C supplementation in the current study.
Although further studies will be required to define the exact molecular mechanism of the altered lipid homeostasis and liver damage caused by decreases in SMP30 levels, our data strongly suggest that SMP30 is closely associated with NAFLD pathogenesis and might be a possible therapeutic target for NAFLD.