Tlr4-mutant mice are resistant to acute alcohol-induced sterol-regulatory element binding protein activation and hepatic lipid accumulation

Previous studies demonstrated that acute alcohol intoxication caused hepatic lipid accumulation. The present study showed that acute alcohol intoxication caused hepatic lipid accumulation in Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic sterol-regulatory element binding protein (SREBP)-1, a transcription factor regulating fatty acid and triglyceride (TG) synthesis, was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic Fas, Acc, Scd-1 and Dgat-2, the key genes for fatty acid and TG synthesis, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Additional experiment showed that hepatic MyD88 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic NF-κB was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Moreover, hepatic GSH content was reduced and hepatic MDA level was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic CYP2E1 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic p67phox and gp91phox, two NADPH oxidase subunits, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin-trapping agent, protected against alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. In conclusion, Tlr4-mutant mice are resistant to acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation.

LPS in plasma 22,23 . In addition, acute alcohol intoxication induced hepatic lipid accumulation 24,25 . Nevertheless, whether TLR4 is involved in acute alcohol-induced hepatic lipid accumulation is not fully understood.
In the present study, we analyzed the effects of acute alcohol intoxication on hepatic lipid accumulation in Tlr4-wild-type and Tlr4-mutant-type mice. Our results showed that Tlr4-mutant mice are resistant to acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. We demonstrate for the first time that TLR4-derived ROS is partially involved in acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation.

Acute alcohol intoxication induces hepatic TG accumulation in Tlr4-wild-type mice but not in
Tlr4-mutant mice. Biochemical parameters were measured 24 h after alcohol exposure. As shown in Table 1, acute alcohol exposure had little effect on serum TG, total cholesterol, total bilirubin, direct bilirubin and total bile acid. Of interest, the absolute liver weight was elevated in alcohol-treated Tlr4-wild-type (ICR and C3H/ HeN) mice but not in Tlr4-mutant-type (C3H/HeJ) mice (Table 1). In addition, liver/body weight ratio was elevated in alcohol-treated Tlr4-wild-type (ICR and C3H/HeN) mice but not in Tlr4-mutant-type (C3H/HeJ) mice (Fig. 1a). The effects of acute alcohol exposure on hepatic TG and cholesterol were then analyzed. Although acute alcohol exposure had no effect on hepatic cholesterol level (Table 1), it elevated hepatic TG content in Tlr4-wildtype (ICR and C3H/HeN) mice but not in Tlr4-mutant-type (C3H/HeJ) mice (Fig. 1b). Correspondingly, an obvious hepatic lipid accumulation was observed in alcohol-treated Tlr4-wild-type (ICR and C3H/HeN) mice but not in Tlr4-mutant-type (C3H/HeJ) mice (Fig. 1c).
Acute alcohol intoxication induces hepatic Akt activation independent insulin signaling. As shown in Fig. 3a, acute alcohol exposure had little effect on serum insulin level. Unexpectedly, serum glucose level was significantly reduced 6 h after alcohol exposure (Fig. 3b). To investigate whether acute alcohol intoxication alters hepatic insulin signaling, the expression of hepatic insulin receptor substrate (Irs)-1 and Irs-2 was measured. As shown in Fig. 3c,d, acute alcohol intoxication had no effect on hepatic Irs-1 and Irs-2 mRNA. The effects of acute alcohol intoxication on hepatic Akt phosphorylation were then analyzed. As shown in Fig. 3e,f, hepatic phosphorylated Akt level was elevated in alcohol-treated Tlr4-wild-type (C3H/HeN) mice but not in Tlr4-mutant-type (C3H/HeJ) mice.
PBN protects against acute alcohol-induced hepatic lipid accumulation. The effects of PBN, a free radical spin-trapping agent, on alcohol-induced hepatic lipid accumulation were analyzed. As expected, PBN alone did not affect hepatic TG content (Fig. 7a). In addition, PBN alone did not induce hepatic lipid accumulation (Fig. 7b). Of interest, alcohol-induced elevation of hepatic TG content was attenuated in mice pretreated with

Discussion
The present study showed that hepatic TG content was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. An obvious hepatic lipid accumulation, as determined by Oil red O staining, was observed in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Increasing evidence has demonstrated that de novo fatty acid and TG synthesis plays an important role in hepatic lipid accumulation 3 . SREBP-1c is an important transcription factor that regulates genes for hepatic fatty acid and TG synthesis 26 . The present study showed that hepatic SREBP-1 was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Several key genes for hepatic fatty acid and TG synthesis were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. These results suggest that Tlr4-mutant mice are resistant to acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. TLR4 activates two signaling pathways through different adapter molecules: MyD88 and TRIF 20 . The present study showed that hepatic NF-κ B binding activity was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic pIκ Bα was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Nuclear translocation of hepatic NF-κ B p65 subunit was observed in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. These results suggest that hepatic NF-κ B, a downstream molecule of TLR4 signaling, was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Of interest, acute alcohol exposure had no effect on hepatic Irf-3 and Irf-7, two downstream genes of TRIF signaling. Taken together, these results indicate that acute alcohol exposure activates hepatic MyD88-dependent but TRIF-independent TLR4 signaling.
Several studies indicate that excess ROS promote hepatic SREBP-1c activation and hepatic lipid accumulation 27,28 . Hepatic NADPH oxidase is the major source of ROS in alcohol-intoxicated mice 29 . According to an earlier report, hepatic p22phox, gp91phox, p47phox, and p67phox, four NADPH oxidase subunits, were up-regulated in chronic alcohol-exposed mice 21,30 . The present study investigated the effects of acute alcohol intoxication on hepatic NADPH oxidase subunits. Although acute alcohol intoxication had little effect on hepatic p22phox and p47phox expression, hepatic p67phox and gp91phox, two NADPH oxidase subunits, were up-regulated in alcohol-intoxicated Tlr4-wild-type mice but not in Tlr4-mutant-type mice. Thus, we hypothesize that NADPH oxidase-derived ROS may be involved in acute alcohol-intoxicated hepatic SREBP-1c activation and hepatic lipid accumulation. To demonstrate this hypothesis, we investigated the effect of PBN, a free radical spin-trapping agent, on alcohol-induced hepatic SREBP-1c activation and hepatic lipid accumulation. As expected, acute alcohol-evoked hepatic SREBP-1 activation was attenuated in PBN-pretreated mice. Acute alcohol-induced elevation of hepatic TG content was alleviated by PBN pretreatment. In addition, acute alcohol-induced hepatic lipid accumulation was blocked by PBN pretreatment. These results suggest that ROS, probably sourced from NADPH oxidase, a downstream molecule of TLR4 signaling, contribute to acute alcohol-evoked hepatic SREBP-1 activation and hepatic lipid accumulation. CYP2E1 is another major source of hepatic ROS production [31][32][33] . Several studies demonstrated that hepatic CYP2E1 was up-regulated in chronic alcohol-exposed mice 34,35 . The present study found that hepatic CYP2E1 was elevated in alcohol-treated Tlr4-wild-type mice. These results are in agreement with an earlier report 36 , in which CYP2E1 catalytic activity was elevated by 2-fold in alcohol-intoxicated mice. Of interest, the present study showed that hepatic CYP2E1 level was not elevated in alcohol-treated Tlr4-mutant-type mice. Therefore, the present study does not exclude the role of CYP2E1-sourced ROS in acute alcohol-evoked hepatic SREBP-1 activation and hepatic lipid accumulation.
The mechanism through which ROS mediates hepatic SREBP-1 activation and hepatic lipid accumulation remains obscure. According to an earlier report, excess ROS promoted hepatic phosphatase and tensin homolog deleted on chromosome ten (PTEN) oxidation and subsequent phosphoinositide 3-kinase (PI3K) activation 37 . Another study demonstrated that hepatic SREBP-1 maturation was tightly associated with PI3K activation and subsequent Akt phosphorylation 38 . Indeed, the present study showed that hepatic pAkt level was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant-type mice. It is well known that insulin not only elevates hepatic SREBP-1 expression but also promotes hepatic SREBP-1 maturation 9,39-42 . Unexpectedly, the present study showed that acute alcohol intoxication did not increase serum insulin level. Moreover, acute alcohol intoxication did not up-regulate hepatic Irs-1 and Irs-2 expression, indicating that acute alcohol-evoked hepatic Akt phosphorylation and SREBP-1 activation are independent of insulin signaling. These results suggest that ROS-mediated hepatic Akt phosphorylation may be associated with acute alcohol-evoked hepatic SREBP-1 activation and hepatic lipid accumulation.
In summary, the present study investigated the role of TLR4 on acute alcohol-induced hepatic lipid accumulation. Our results showed that acute alcohol intoxication caused hepatic lipid accumulation in Tlr4-wild-type mice but not in Tlr4-mutant-type mice. Moreover, acute alcohol intoxication induced hepatic SREBP-1c activation in Tlr4-wild-type mice but not in Tlr4-mutant-type mice. The present study demonstrates for the first time that TLR4-mediated excess ROS generation, probably sourced from NADPH oxidase, contribute, at least partially, to acute alcohol-evoked hepatic SREBP-1 activation and hepatic lipid accumulation.

Animals and treatments.
Male CD-1 (Tlr4-wild-type, 6~8 week-old; 24~28 g) and C3H/HeN (Tlr4-wild-type, 6~8 week-old; 20~22 g) mice were purchased from Beijing Vital River whose foundation colonies were all introduced from Charles River Laboratories, Inc. C3H/HeJ (Tlr4-mutant-type) mice were purchased from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). The animals were maintained on a 12-h light/dark cycle in a controlled temperature (20-25 °C) and humidity (50 ± 5%) environment for a period of 1 week before use. All mice were fed with regular diet. The food consisted of standard rodent chow (50.76% carbohydrate, 15.44% fat, 33.8% protein calories), which were purchased from Jiangsu Cooperative Medical Biological Engineering Company Limited (Nanjing, China). The present study consisted of two independent experiments. Experiment 1, to investigate the effects of acute alcohol exposure on hepatic lipid accumulation, thirty-six mice each strain (ICR, C3H/HeN and C3H/HeJ) were divided into six groups. For five alcohol-treated groups, mice were administered with a single dose of ethanol (4 g/kg) by gavage. Six mice each strain were sacrificed at each time point (0, 0.5, 2, 6 and 12 h after alcohol). Experiment 2, to investigate the effects of PBN on alcohol-induced hepatic SREBP-1c activation and hepatic lipid accumulation, twenty-four ICR mice were divided into four groups. In alcohol-treated group, mice were administered with a single dose of ethanol (4 g/kg) by gavage. In PBN and PBN + alcohol groups, mice were injected with two doses of PBN (100 mg/kg), one 30 min before alcohol and the other 4 h after alcohol. The alcohol dose used in the present study referred to Figure 5. Acute alcohol intoxication elevates hepatic NF-κB binding activity in Tlr4-wild-type mice but not in Tlr4-mutant-type mice. All mice except controls were administered with ethanol (4 g/kg) by gavage. Liver tissue was collected 2 h after ethanol. Hepatic NF-κ B binding activity was detected using EMSA. All experiments were repeated for six times. Data were expressed as means ± SEM. (N = 6) **P < 0.01. others with minor modification 43,44 . Mice were sacrificed 12 h after alcohol exposure. For all mice, blood serum was collected for measurement of biochemical parameters. Liver was collected and frozen immediately in liquid nitrogen for reverse transcription polymerse chain reaction (RT-PCR), immunoblot and hepatic TG measurement. Some liver tissues were frozen-fixed in optimum cutting temperature compound mounting media for Oil red O staining. All procedures on animals followed the guidelines for humane treatment set by the Association of Laboratory Animal Sciences and the Center for Laboratory Animal Sciences at Anhui Medical University.
Biochemical parameters. The levels of serum TG, total cholesterol, serum glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, direct bilirubin and total bile acid were measured using a commercially available kit. Hepatic TG measurement. Hepatic TG was extracted using method developed by Bligh with minor modification 45 . Briefly, liver samples were homogenized in ice-cold 2 × PBS. TG was extracted with methanol/chloroform (1:2), dried, and resuspended in 5% fat-free bovine serum albumin. Hepatic TG was measured using the TG reagent kit (Zhejiang Dongou Diagnostics Co., LTD) according to manufacturer's protocol. Hepatic TG content was expressed as μ mol/g liver.

Isolation of total RNA and real-time RT-PCR.
Total RNA was extracted from liver tissues using TRI reagent (Molecular Research Center). RNase-free DNase-treated total RNA (1.0 μ g) was reverse-transcribed with AMV (Promega). Real-time RT-PCR was performed with a LightCycler ® 480 SYBR Green I kit (Roche Diagnostics GmbH, Mannheim, Germany) using gene-specific primers as listed in Supplementary Table S1. According to a recent report, housekeeping gene variability was observed in the liver of alcoholic patients, in which β -actin and GAPDH tended to decrease with steatosis and to increase with alcoholic hepatitis 46 . In liver of alcoholic patients, the most constantly expressed housekeeping gene is 18S. Our preliminary experiments showed that hepatic β-actin and Gapdh mRNAs were up-regulated in alcohol-exposed mice. By contrary, hepatic 18S had the lowest coefficient of dispersion (Supplementary Table S2). Thus, 18S is an appropriate reference gene for normalization of real-time RT-PCR. The amplification reactions were carried out on a LightCycler ® 480 Instrument (Roche Diagnostics GmbH) with an initial hold step (95 °C for 5 minutes) and 50 cycles of a three-step PCR (95 °C for 15 seconds, 60 °C for 15 seconds, 72 °C for 30 seconds). The comparative CT-method was used to determine the amount of target, normalized to an endogenous reference (18S) and relative to a calibrator using the LightCycler 480 software (Roche, version 1.5.0) 47 . All RT-PCR experiments were performed in triplicate.
Immunoblots. Hepatic lysate was prepared by homogenizing 50 mg liver tissue in 300 μ l lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecylsylphate, 1 mM phenylmethylsulfonyl fluoride) supplemented with a cocktail of protease inhibitors (Roche). For nuclear protein extraction, hepatic lysate was suspended in hypotonic buffer and then kept on ice for 15 min. The suspension was then mixed with detergent and centrifuged for 30 s at 14,000 × g. The nuclear pellet obtained was resuspended in complete lysis buffer in the presence of the protease inhibitor cocktail, incubated on ice for 30 min, and centrifuged for 10 min at 14,000 × g. Protein concentrations were determined with BCA protein assay (Pierce, Rockford, IL, USA) according to manufacturer's instructions. For immunoblots, same amount of protein (40~80 μ g) was separated electrophoretically by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membranes were incubated for 2 h with the following antibodies: p-Akt (1:2000), Akt (1:3000), MyD88 (1:1000), p-Iκ B (1:1000), NF-κ B p65 (1:1000), SREBP-1 (1:1000), HO-1(1:1000) and CYP2E1 (1:2000). For total protein, β -actin (1:3000) was used as a loading control. For nuclear protein, lamin A/C (1:2000) was used as a loading control. After washes in DPBS containing 0.05% Tween-20 four times for 10 min each, the membranes were incubated with goat anti-rabbit IgG or goat anti-mouse antibody for 2 h. The membranes were then washed for four times in DPBS containing 0.05% Tween-20 for 10 min each, followed by signal development using an ECL detection kit.
Oil red O staining. To determine hepatic lipid accumulation, frozen sections of liver (10 μ m) were stained with Oil Red O for 10 min, washed, and counterstained with hematoxylin for 45 seconds. Representative photomicrographs were captured at 400x magnification using a system incorporated in the microscope.
Glutathione assay. The glutathione (GSH) in liver tissue homogenate was determined by the method of Griffith 49 . Briefly, 0.3 mL supernatant of 20% liver homogenate was added in 0.1 mL of 20% trichloroacetic acid, and samples were centrifuged at 4000 rpm for 15 min. Then, 0.1 mL supernatant was combined with 4.4 mL 0.3 M Na2HPO4 and 0.5 mL of 0.04% dithio-bis-nitrobenzoic acid (DTNB), and the absorbance of the solution was read at 412 nm. The content of GSH was expressed as micromole per gram protein.
Lipid peroxidation assay. Hepatic lipid peroxidation was evaluated by measuring malondialdehyde (MDA) as described previously 50 . To prepared 10% liver homogenate, 0.1 gram liver tissue was homogenized in 1 mL saline and samples were centrifuged at 4000 rpm for 15 min. After that, 0.1 mL supernatant was added into the reaction mixture containing 0.1 mL sodium dodecylsulfate (8.1%), 1.5 mL acetic acid (20%, v/v), 1.5 mL thiobarbituric acid (0.9%). All the tubes were placed in a boiling water bath for 1 hour. After cooling on ice, the reaction mixture centrifuged at 10,000 × g for 10 min. The amount of MDA per sample was assessed by determining the absorbance of the supernatant at 532 nm using tetraethoxypropane as standard. MDA contant was expressed as micromole per gram protein.
Statistical analysis. All data were expressed as means ± SEM. SPSS 13.0 statistical software was used for statistical analysis. All statistical tests were two-sided using an alpha level of 0.05. ANOVA and the Student-Newmann-Keuls post hoc test were used to determine differences among different groups. Student t test was used to determine differences between two groups.

Ethics statement. This study was approved by the Association of Laboratory Animal Sciences and the
Center for Laboratory Animal Sciences at Anhui Medical University (Permit Number: 12-0010). All procedures on animals followed the guidelines for humane treatment set by the Association of Laboratory Animal Sciences and the Center for Laboratory Animal Sciences at Anhui Medical University.