α‐Tocopherol suppresses hepatic steatosis by increasing CPT‐1 expression in a mouse model of diet‐induced nonalcoholic fatty liver disease

Abstract Aim Antioxidant therapy for with vitamin E appears to be effective for the treatment of nonalcoholic fatty liver disease　(NAFLD). However, the mechanism of action and optimal therapeutic dosage is unclear. The present study was undertaken to examine whether the effects of α‐tocopherol (α‐Toc) on NAFLD are dose‐dependent in a diet‐induced obese model. Methods Male mice were fed standard chow, high‐fat (HF) diet, HF diet with low‐dose, or with high dose of α‐Toc supplementation. Histological findings, triglyceride content, and the levels of protein expression related to fatty acid synthesis/oxidation such as carnitine palmitoyltransferase I (CPT‐1) of liver were evaluated. In addition, 2‐tetradecylglycidic acid (TDGA), a CPT‐1 inhibitor, was administered to mice fed HF diet with low‐dose of α‐Toc. Finally, HepG2 cells in fat‐loaded environment were treated with 0–50 μM α‐Toc. Results Treatment of low‐dose of α‐Toc decreased HF‐induced hepatic fat accumulation, but this finding was not observed in treatment of high dose of α‐Toc. HF‐induced reduction of CPT‐1 was attenuated with low‐dose of α‐Toc but not with high dose of α‐Toc. TDGA suppressed the improvement of histological findings in liver induced by low‐dose of α‐Toc treatment. CPT‐1 expression in HepG2 cells increased in response to low‐dose of α‐Toc, but not in high dose. Conclusions Dual action of α‐Toc on CPT‐1 protein levels was observed. The effect of vitamin E on NAFLD may be not be dose‐dependent.


| INTRODUCTION
Nonalcoholic fatty liver disease (NAFLD) is a clinicopathological diagnosis in which more than 5% of hepatocytes demonstrate macrovesicular steatosis in an individual without a significant history of alcohol intake. 1 NAFLD encompasses a broad spectrum of metabolic fatty liver disorders ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which can develop into cirrhosis and liver cancer. [2][3][4] NASH was originally interpreted with a "dual-hit" hypothesis, where steatosis ("first hit"), resulting from increased fat accumulation in the liver, predisposes to the initiation of NASH through downstream ("second hit") pro-inflammatory mediators. 5 Previous studies have demonstrated that an imbalance between fatty acid synthesis and oxidation induces fat accumulation in liver, indicating that fatty acid synthesis/oxidation mechanisms are regulated by different hepatic enzymes and transcription factors, which play a central role in controlling lipid homeostasis. 6,7 Carnitine palmitoyl transferase-1 (CPT-1), the mitochondrial gateway for fatty acid entry into the matrix, is a main controller of the hepatic mitochondrial β-oxidation flux. 8 In the liver, CPT-1 regulates ∼80% of fatty acid β-oxidation in physiological conditions. 9 The impairment of β-oxidation in mitochondria induced by the reduction of hepatic CPT-1 may be a crucial event in the pathogenesis of hepatic steatosis in mice. 10 In addition, oxidative stress has been implicated as a key factor contributing to hepatic injury in NASH patients. Fat accumulation within hepatocytes enhances mitochondrial reactive oxidative species (ROS) production, which in turn may cause oxidative stress. 11 The most important cellular damage caused by ROS is peroxidation of membrane lipids, resulting in a generalized alteration of membrane function. 12 Vitamin E is a potent antioxidant that protects organisms against ROS-mediated damage. 13 A previous study has shown that vitamin E can prevent free radicals from entering the cell membrane and helps to maintain the stability of the cell membrane. 14 In addition, vitamin E inhibits the generation of mitochondrial ROS by increasing the mitochondrial membrane potential and improving mitochondrial function. 15 Several clinical trials showed that antioxidant therapy with vitamin E was effective in preventing the development of NASH or NAFLD, suggesting that higher intake of vitamin E might be effective in counteracting the increase of oxidative stress found in patients with NASH or NAFLD. 16,17 On the other hand, a question regarding the safety of a high dose of vitamin E intake has been raised by several meta-analyses of randomized trials. 18,19 Taken together, we hypothesized that the therapeutic effect of NAFLD using vitamin E is not dose-dependent. Here, an in vivo study was conducted to determine the effects of vitamin E on hepatic fat accumulation and the expression of hepatic enzymes and transcription factors related to fatty acid synthesis/oxidation in high-fat (HF)-fed mice with different doses of the agent. In addition, in vitro study was performed whether vitamin E treatment also alters the expression of proteins related to hepatic fatty acid synthesis/oxidation in HepG2 cells cultured in fat loaded condition.

| Animals
Eight-week-old male C57BL/6J mice were purchased from KBT Oriental (Fukuoka, Japan). The mice were housed in a light-, temperature-, and humidity-controlled room (lights on/off at 7:00/ 19:00 h; 21 � 1°C; 55 � 5% relative humidity). The mice were allowed free access to chow pellets and water during the experiment. All animals were treated in accordance with the Oita University Guidelines for the Care and Use of Laboratory Animals.

| Serum alanine aminotransferase, triglyceride, alpha-tocopherol levels, and potential antioxidant assay
At the end of both experiments 1 and 2, the mice were anesthetized with sodium pentobarbital as above and perfused transcardially with isotonic phosphate buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 M PBS. The liver was removed immediately, it was frozen in liquid nitrogen, and then stored at À 80°C until protein extraction. Blood samples were withdrawn through cardiac puncture, and the serum was separated and immediately frozen at À 80°C until analysis. Serum alanine aminotransferase (ALT) levels and triglyceride (TG) levels were measured using an automatic analyzer (SRL).
Serum α-Toc levels were measured using the following kits in accordance with the method described by the manufacturer: alphatocopherol ELISA Kit (LifeSpan BioSource, Inc). The evaluation of antioxidant power in serum samples was determined by using the "potential antioxidant (PAO)" test kit (JaICA). This assay evaluated Cu þ levels derived by reduction of Cuþþ from the action of antioxidant present in the sample. The stable complex between Cuþ and bathocuproine is revealed at 490 nm.

| Histological analyses
Liver tissue samples were fixed with 4% paraformaldehyde and embedded in paraffin. Then, 5-µm-thick sections were cut, and liver sections were stained with hematoxylin and eosin and examined under a microscope (Olympus). The tissue was analyzed with scoring of the Histological Scoring System for Nonalcoholic Fatty Liver Disease Score and Fibrosis staging by the NASH clinical research network scoring system (NAFLD activity score: NAS). The NAS is defined as the unweighted sum of the scores for steatosis (0-3), lobular inflammation (0-3), and ballooning (0-2).

| TG contents in the liver
Liver tissue (100 mg) was homogenized in 2 mL of a solution containing 150 mM NaCl, 0.1% Triton X-100, and 10 mM Tris using a Polytron homogenizer (NS-310E; Micro Tech Nichion) for 1 min. The liver TG contents were measured using an automatic analyzer (SRL).
A 50-μL aliquot of the homogenate was removed for the protein assay (Bio-Rad).

| Western blotting
Frozen liver tissue preparations were homogenized with sodium dodecyl sulfate (SDS) sample buffer, clarified by centrifugation, and boiled. The total protein concentration in each tissue sample was quantified by the Bradford method, and 8 µg of total protein per sample was loaded onto 8% SDS-polyacrylamide gels for electrophoresis. The separated proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories). The membranes were incubated with primary antibody solution consisting of 5 g/L polyclonal antiserum with specificity for mouse carnitine palmitoyltransferase I (CPT-1), peroxisome proliferatoractivated receptor alpha (PPARα), uncoupling protein 2 (UCP-2), acetyl-CoA carboxylase α (ACACA), sterol regulatory elementbinding protein 1 (SREBP-1), fatty acid synthase (FAS), and β-actin.
These primary antibodies were purchased from Santa Cruz Biotechnology. Immunoreactive bands were detected by enhanced chemiluminescence (Amersham Life Science) and quantified using National Institutes of Health imaging software. FAS, CPT-1, PPARα, UCP-2, ACACA, SREBP-1, and β-actin of each specimen were measured and determined each protein level as a ratio to that of β-actin between each group.

| Statistics
All data are expressed as the means � SD. Statistical significance was evaluated using one-way analysis of variance followed by Tukey's test for post hoc comparisons. For all tests, the level of significance was set at p < 0.05. All statistical analyses were performed by using IBM SPSS Statistics version 22.0 for Windows.

| Effect of α-Toc supplementation on serum ALT levels
HF feeding increased serum ALT levels compared with standard feeding ( Figure S1). The supplementation of α-Toc at 50 mg/kg for HF diet suppressed the elevation of serum ALT levels induced by HF feeding. Furthermore, the supplementation of α-Toc at 200 mg/kg for HF diet increased serum ALT levels significantly compare to the supplementation of α-Toc at 50 mg/kg. Thus, the supplementations of α-Toc at 50 mg/kg as low-dose of α-Toc, and α-Toc at 200 mg/kg as high dose of α-Toc were defined.

| Low-dose but not high dose α-Toc improves hepatic damage and lipid accumulation by HF consumption
As shown in Table 1, total body weight, liver weight, and serum ALT levels were increased in HF-fed mice. The low-dose but not high dose α-Toc treatment reduced liver weight and serum ALT levels significantly, whereas both doses did not alter total body weight. Serum TG levels were not significantly different among groups. HF-fed mice developed moderate steatosis with fat accumulation and increased hepatic TG levels in the liver ( Figures 1A   and 1B).
Treatment with a low-dose of α-Toc decreased these HF-induced alterations in the liver, however, this improvement was not observed with a high dose of α-Toc treatment ( Figures 1A and 1B). Moreover, serum α-Toc levels were significantly increased in high dose of α-Toc more than low-dose of α-Toc, although both doses elevated serum α-Toc levels compared with Standard as well as HF groups ( Figure   1C). Similarly, PAO levels were significantly increased in high dose of α-Toc more than low-dose of α-Toc, which indicates that high dose of α-Toc has more antioxidant capacity than low-dose of α-Toc, although both doses elevated PAO levels compared with Standard as well as HF groups ( Figure 1D).
In line with hepatic fat accumulation and the hepatic TG content, HF-induced elevation of the NAS score was also suppressed with the low-dose but not with the high dose of α-Toc treatment ( Table 2).

| A low-dose of α-Toc enhances the expression of proteins involved in fatty acid oxidation pathways
Protein expression levels related to hepatic fatty acid synthesis/ oxidation, such as CPT-1, PPARα, UCP-2, ACACA, SREBP-1, and FAS, in liver tissues were assessed ( Figure 2). The levels of CPT-1 protein expression were significantly decreased in the HF group compared to those of the Standard group. Low-dose but not high dose α-Toc treatment increased the levels of CTP-1 protein (Figures 2A and 2B). However, there were no significant differences in PPARα, UCP-2, ACACA, SREBP-1, or FAS expression in the liver among all groups (Figure 2A, Figures 2C-2G). These data indicate that low-dose α-Toc selectively increased CPT-1 protein levels in the liver. Notes: Values are the means � SD. Standard, standard including vitamin E acetate-fed; HF, HF including vitamin E acetate-fed; HF þ low α-Toc, HF-fed with α-tocopherol (50 mg/kg) supplementation; HF þ high α-Toc, HF-fed with α-tocopherol (200 mg/kg) supplementation.

| TDGA, a specific CPT-1 inhibitor, suppressed the α-Toc-induced improvement in hepatic function and fat accumulation in the liver
To further evaluate the effect of CPT-1 on hepatic accumulation by HF consumption, a specific CPT-1 inhibitor was used. As shown in Figure 3A, TDGA treatment attenuated the α-Toc-induced upregulation of CPT-1 protein levels in the liver. In addition, TDGA also suppressed the α-Toc-induced improvement in fat accumulation ( Figure 3B), reduction in hepatic TG levels ( Figure 3C) and NAS (Table 3) in the liver although TDGA alone did not alter HF-induced hepatic fat accumulation.   Figures 1C and 1D, α-Toc supplementation increases serum α-Toc levels and has antioxidant effects in a dose-dependent manner. Therefore, the biphasic effects of α-Toc were hypothesized on hepatic fat accumulation as follows. The low-dose α-Toc ameliorates hepatic fat accumulation due to elevation of CPT-1 levels whereas high dose α-Toc reciprocally worsens hepatic changes due probably to toxic pharmacological effects of α-Toc as some papers have already reported. 38,39 A recent meta-analysis showed improvements in body mass index, ballooning, fibrosis and histology, but such improvements were not much significant to draw a firm conclusion. 40  These are often determined optionally and it is difficult to be determined adequately. 43 Therefore, the effectiveness and safety of using vitamin E as a form of treatment require further study.

| Dual mode of action of α -Toc on CPT-1 protein levels in HepG2 cells
In conclusion, we demonstrated that a low-dose of α-Toc protects against NAFLD-induced liver injury through an increase in CPT-1 protein, which regulates mitochondrial lipid metabolism in the liver, whereas a high dose of α-Toc suppresses these alterations.
Therefore, the useful effect of vitamin E on NAFLD might not be dose-dependent. In the future, large multicenter, double-blinded randomized controlled trials to investigate the appropriate dose of vitamin E administration on NAFLD are warranted.