Keywords: ACAT-1; Kupffer cells; Inflammation; Nonalcoholic steatohepatitis
K-604 inhibits ACAT-1, which mediates the conversion of FC into Cholesterol Ester (CE) (Figure S1). The IC50 values of K-604 for human ACAT-1 and ACAT-2 are 0.45 and 103 μmol/ L, respectively, indicating that K-604 is 229-fold more selective for ACAT-1 [8]. K-604 competitively inhibited the enzyme activity in the presence of oleoyl-CoA, with a Ki value of 0.38 μmol/ L. We previously demonstrated that K-604 directly prevented arterial macrophages from forming foam cells, without altering the plasma lipid levels in atherogenic F1B hamsters and apolipoprotein E-deficient mice [8,9]. We observed a remarkable reduction of Tumor Necrosis Factor-Α (TNF-α) gene expression in the liver of Low-Density Lipoprotein Receptor-Deficient (Ldlr(−/−)) mice fed an HFC diet for 12 weeks during our evaluations of anti-atherosclerotic actions of K-604. Both liver macrophage foam cell formation in the progression of NASH and the arterial macrophages in advanced atherogenic lesions may use a similar mechanism that involves ACAT. In support of this suggestion, the ACAT-1 mRNA is up-regulated, resulting in cholesterol accumulation in hepatocytes in NAFLD or NASH [10,11]. Therefore, we verified the hypothesis that ACAT-1 up-regulation plays a pivotal role in regulating cholesterol metabolism in the NASH liver by examining whether liver inflammation could be attenuated by ACAT-1 inhibition.
The first model is based on MCD diet-fed KK-Ay mice, one of the most common NASH models that are used to cause severe steatohepatitis, similar to human NASH [5,12]. We also compared the efficacy of bezafibrate, well-known as a broad-spectrum Peroxisome Proliferator-Activated Receptor (PPAR) agonist, to modulate fatty acid metabolism and to effectively ameliorate steatohepatitis in animal models and tamoxifen-induced human NASH [5,12,13]. The second model is based on the HFC dietfed Ldlr(−/−) mice that exhibit fatty liver and inflammatory cell infiltration. The Ldlr(−/−) mice are a promising model for investigating the onset of hepatic inflammation in NASH, and high levels of plasma cholesterol play a pivotal role in the formation foamy Kupffer cells and hepatic inflammation [2,14– 17]. We investigated whether the inhibition of ACAT-1 activity could affect the appearance of foamy Kupffer cells and liver inflammation when K-604 was administered to the HFC diet-fed Ldlr(−/−) mice. The third model uses Zucker fatty (ZF) rats, which exhibit severe obesity, hyperphagia, hyperlipidemia, severe fatty liver and hepatocyte hypertrophy, even when fed a normal diet [18]. We evaluated the efficacy of K-604 on steatosis as a model of NAFLD. Here, we revealed that the inhibition of ACAT-1 by K-604 suppressed the appearance of foamy Kupffer cells and ameliorated the inflammatory changes in the compromised liver. Our results suggest a crucial role for ACAT-1 in the progression of steatohepatitis, and ACAT-1 inhibition may be a novel therapeutic target for NAFLD and NASH treatments.
The five-week-old male Ldlr(−/−) mice (Jackson Laboratory, Bar Harbor, ME) were divided into five groups (6-8 mice/ group). Group 1 was fed a normal diet; group 2 was given a 1.25% cholesterol + 40 kcal% fat (HFC) diet (Research Diet, New Brunswick, NJ); and groups 3-5 received the HFC diet supplemented with 0.026% and 0.087% K-604 (30 and 100 mg/ kg, respectively) or 0.052% bezafibrate (60 mg/ kg) for 16 weeks. K-604 was also administered to the Ldlr (−/−) mice for 3 weeks to measure the K-604 concentrations in the plasma and liver (n = 4).
The eight-week-old male Zucker-fa/fa (ZF) rats and Zucker-?/ + (lean) rats (Charles River Laboratories Japan, Kanagawa, Japan) were divided into three groups (5-8 rats/ group). Groups 1 and 2 (lean and ZF rats) were fed a normal diet; group 3 (ZF rats) was given a normal diet supplemented with 0.133% K-604 (100 mg/ kg) for 12 weeks.
For immunohistochemistry, small pieces of the liver were embedded in OCT compound (Sakura Finetek Japan Co., Ltd., Tokyo, Japan). Cryosections (8 μm) of the liver were stained with a rat anti-mouse CD68 monoclonal antibody (clone: FA- 11, 1:50–1:100, Serotec, Oxford, UK) to detect the Kupffer cells/ macrophages. Histofine Simple Stain Mouse MAX PO (#414351, Nichirei Biosciences Inc., Tokyo, Japan) was used as a horseradish peroxidase-conjugated secondary antibody. The immune complexes were visualized with diaminobenzidine and the slides were counterstained with hematoxylin. The area of the CD68- positive cells was quantified by image analysis software (Win ROOF; Mitani Co., Tokyo, Japan) and calculated as the ratio of the area of the CD68-positive cells relative to that of the sections. The size of the CD68-positive cells was calculated by dividing the area of the CD68-positive cells by the number of CD68-positive cells counted with Win ROOF. ACAT-1 was stained with a rabbit antihuman ACAT-1 polyclonal antibody (#100028, 1:100, Cayman Chemical, Ann Arbor, MI). Double immunofluorescence staining was performed using the anti-ACAT-1 and anti-CD68 antibodies as primary antibodies. Alexa Fluor® 488 donkey anti-rabbit IgG (1:2000, Molecular Probes, Eugene, OR) and Alexa Fluor® 594 donkey anti-rat IgG (1:2000, Molecular Probes) were used as the secondary antibodies.
(A-F): Hematoxylin-eosin-stained liver sections from the KK-Ay mice. The arrows indicate the inflammatory foci.
(G-H): Steatosis score and inflammatory foci in the liver sections. The data are expressed as the means ± SEM (2–6 fields/mouse, n = 5-9).
(I-K): Hepatic gene expression in the KK-Ay mice (n = 5-9). The data are expressed as the means ± SEM. #P < 0.05, ###P < 0.001 compared to the normal diet-fed group. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group.
(E): Size of the CD68-positive cells (4–6 fields/ mouse, n = 5–9). The data are expressed as the means ± SEM. #P < 0.05, compared to the normal diet-fed group. *P < 0.05 compared to the control group.
To analyze hepatic fibrosis, we performed picrosirius red staining on the liver sections. Although it was not obvious, collagen deposition appeared in the MCD diet-fed group (S2 Figure A-B). Neither K-604 nor bezafibrate exerted significant effects on liver fibrosis (S2 Figure C-D).
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|
MCD diet |
||||
|
Normal diet |
Control |
K-604 30 mg/ kg |
K-604 100 mg// kg |
K-604 200 mg/ kg |
Bezafibrate 60 mg/ kg |
|
|
|
|
|
|
|
Body weight (g) |
51.2 ± 1.3 |
17.9 ± 0.6### |
17.9 ± 0.4 |
17.5 ± 0.4 |
16.4 ± 0.4 |
16.9 ± 0.7 |
ALT (U/ L) |
32.9 ± 5.5 |
34.3 ± 3.9 |
34.5 ± 3.2 |
38.3 ± 6.1 |
27.1 ± 2.5 |
14.7 ± 0.7 * |
TC (mg/ dL) |
84.5 ± 1.9 |
73.9 ± 3.4# |
76.3 ± 5.1 |
87.5 ± 7.8 |
81.7 ± 2.8 |
74.3 ± 1.6 |
TG (mg/ dL) |
242.0 ± 47.7 |
44.6 ± 4.4### |
50.7 ± 5.7 |
52.5 ± 9.1 |
55.8 ± 5.7 |
42.4 ± 5.5 |
Glucose (mg/ dL) |
384.0 ± 65.9 |
118.9 ± 5.6### |
129.7 ± 5.6 |
114.5 ± 6.7 |
108.4 ± 6.7 |
107.2 ± 7.2 |
Choline (μM) |
10.1 ± 0.3 |
6.0 ± 0.2### |
6.4 ± 0.2 |
6.4 ± 0.1 |
6.2 ± 0.1 |
5.5 ± 0.3 |
Hepatic TC (mg/ g) |
5.3 ± 0.7 |
5.6 ± 0.4 |
5.3 ± 0.2 |
5.4 ± 0.2 |
5.6 ± 0.2 |
4.2 ± 0.2** |
Hepatic CE (mg/ g) |
1.5 ± 0.6 |
1.3 ± 0.4 |
1.9 ± 0.1 |
1.4 ± 0.2 |
1.8 ± 0.1 |
1.1 ± 0.1 |
Hepatic FC (mg/ g) |
4.1 ± 0.5 |
4.4 ± 0.5 |
3.4 ± 0.2 |
3.9 ± 0.2 |
3.8 ± 0.1 |
3.2 ± 0.1 |
Hepatic CE/FC ratio |
0.45 ± 0.20 |
0.36 ± 0.09 |
0.56 ± 0.05 |
0.37 ± 0.06 |
0.47 ± 0.03 |
0.33 ± 0.03 |
Hepatic TG (mg/ g) |
84.1 ± 17.6 |
87.3 ± 17.4 |
58.7 ± 12.2 |
70.5 ± 9.0 |
47.6 ± 9.7 |
28.4 ± 5.6* |
The mice fed HFC diet exhibited elevated TC and TG contents in the liver (Table 2). K-604 decreased the hepatic TC levels (30 mg/ kg:–29%, 100 mg/ kg:–49%). Moreover, at doses of 30 and 100 mg/ kg, K-604 markedly decreased the hepatic CE levels by 45% and 86%, respectively, and bezafibrate decreased the levels by 65%. Despite the reduction of the hepatic CE levels, the K-604-treated mice exhibited the same level of FC as the controls, but bezafibrate increased the FC levels in the liver. Both agents significantly decreased the CE/FC ratio.
The picrosirius red-stained liver sections exhibited weak collagen deposition in the HFC diet group (S3 Figure A–B). Neither K-604 nor bezafibrate exerted significant effects on liver fibrosis (S3 Figure C–D).
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|
HFC diet |
|||
|
Normal diet |
Control |
K-604 30 mg/ kg |
K-604 100 mg/ kg |
Bezafibrate 60 mg/ kg |
Body weight (g) |
26.9 ± 0.4 |
31.4 ± 1.4# |
30.2 ± 0.7 |
34.1 ± 1.4 |
29.1 ± 0.7 |
Liver/ body weight ratio |
0.050 ± 0.001 |
0.049 ± 0.001 |
0.047 ± 0.001 |
0.047 ± 0.001 |
0.065 ± 0.001*** |
ALT (U/ L) |
18.7 ± 1.7 |
21.3 ± 5.5 |
20.2 ± 2.3 |
27.7 ± 2.7 |
54.0 ± 8.5 |
TC (mg/ dL) |
184.7 ± 7.0 |
1357.3 ± 86.5### |
1327.6 ± 85.1 |
929.3 ± 75.7* |
817.1 ± 57.6** |
TG (mg/ dL) |
71.4 ± 5.6 |
523.5 ± 40.0### |
659.8 ± 45.8 |
569.0 ± 74.6 |
253.4 ± 18.4 |
Glucose (mg/ dL) |
196.9 ± 7.8 |
198.1 ± 9.4 |
182.0 ± 5.2 |
211.2 ± 5.7 |
174.9 ± 8.0 |
Hepatic TC (mg/ g) |
3.2 ± 0.1 |
16.9 ± 1.0### |
12.0 ± 0.8*** |
8.7 ± 0.4*** |
13.8 ± 1.0* |
Hepatic CE (mg/ g) |
0.6 ± 0.1 |
9.9 ± 0.5### |
5.4 ± 0.3*** |
1.4 ± 0.4*** |
3.5 ± 0.7*** |
Hepatic FC (mg/ g) |
2.5 ± 0.1 |
7.1 ± 1.1## |
6.6 ± 0.5 |
7.4 ± 0.6 |
10.3 ± 1.1* |
Hepatic CE/ FC ratio |
0.25 ± 0.03 |
1.57 ± 0.24### |
0.85 ± 0.06** |
0.23 ± 0.07*** |
0.36 ± 0.08*** |
Hepatic TG (mg/ g) |
11.6 ± 0.8 |
69.1 ± 14.3## |
79.8 ± 8.2 |
103.7 ± 10.2 |
118.3 ± 8.9* |
The plasma and liver concentrations of K-604 in the Ldlr(−/−) mice were measured after a 3-week treatment with 30 and 100 mg/ kg K-604. While the concentrations of K-604 in the plasma from each treatment group were 0.12 ± 0.05 and 0.54 ± 0.21 μmol/ L, respectively, those in the liver were 0.58 ± 0.16 and 3.82 ± 1.17 μmol/ kg liver, respectively. Compared to the IC50 values for human ACAT-1 (0.45 μmol/ L) and the Ki value (0.38 μmol/ L), the concentration of K-604 (100 mg/ kg) in the liver would be sufficient to suppress ACAT-1.
(A-C): Hematoxylin-eosin-stained liver sections from the lean rats and the ZF rats.
(D-F): Hepatic gene expression in the lean rats and the ZF rats (n = 5–8). The data are expressed as the means ± SEM. ## P < 0.01 compared to the lean rats.
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|
ZF rats |
|
|
Lean |
Control |
K-604 100 mg/ kg |
Body weight (g) |
414.7 ± 11.2 |
670.4 ± 20.1### |
641.3 ± 19.3 |
Liver/ body weight ratio |
0.036 ± 0.000 |
0.057 ± 0.004# |
0.045 ± 0.002* |
ALT (U/ L) |
37.9 ± 3.7 |
176.8 ± 30.6## |
71.2 ± 10.4* |
TC (mg/ dL) |
51.9 ± 2.5 |
148.6 ± 17.9## |
230.6 ± 32.2* |
TG (mg/ dL) |
147.1 ± 11.9 |
1073.3 ± 195.1## |
1117.4 ± 376.4 |
Glucose (mg/ dL) |
191.8 ± 10.9 |
343.9 ± 30.1## |
228.6 ± 15.4** |
Hepatic TC (mg/ g) |
2.4 ± 0.1 |
5.4 ± 0.7## |
3.3 ± 0.1* |
Hepatic CE (mg/ g) |
0.4 ± 0.1 |
2.4 ± 0.6# |
1.2 ± 0.1 |
Hepatic FC (mg/ g) |
2.0 ± 0.1 |
3.0 ± 0.2## |
2.1 ± 0.1** |
Hepatic CE/ FC ratio |
0.21 ± 0.07 |
0.81 ± 0.21 |
0.57 ± 0.08 |
Hepatic TG (mg/ g) |
13.5 ± 1.9 |
119.3 ± 12.4### |
43.6 ± 4.6*** |
An analysis of the hepatic lipid levels revealed that the ZF rats exhibited increased hepatic TC and TG levels compared to the lean rats (Table 3). The K-604 treatment significantly decreased both the hepatic TC and TG levels. In addition, K-604 decreased both the hepatic CE and FC levels, resulting in a reduction of CE/ FC ratio, but the difference was not significant.
The HFC diet-fed Ldlr(−/−) mice represent a promising model for investigating the onset of hepatic inflammation and steatosis in two components of NASH pathophysiology [2,14- 17]. By comparing K-604 with the controls and bezafibrate, we demonstrated that the selective ACAT-1 inhibitor K-604 effectively suppressed inflammatory foci formation. The lipid analysis indicated that both K-604 and bezafibrate decreased the plasma TC and liver TC and CE levels while tending to increase the liver TG levels. While K-604 did not affect the plasma TG levels, bezafibrate decreased the plasma TG levels and significantly increased the liver TG levels compared to HFC diet-fed controls. Notably, bezafibrate was reported to significantly reduce the liver microsomal ACAT activities in rats [24]; thus, the reduction of the CE levels and an increase in the FC levels can be explained by ACAT inhibition. A question was raised of where to draw the line between the efficacies of K-604 and bezafibrate if both agents inhibit ACAT activities. To date, two ACAT isozymes have been identified. ACAT-1 is ubiquitously expressed in various human tissues and cells, such as various types of macrophages (including Kupffer cells), the adrenal glands and the liver while ACAT-2 is primarily expressed in the rodent intestine and liver and is involved in the absorption of cholesterol from the diet [25,26]. We presumed that bezafibrate might effectively inhibit ACAT in hepatocytes (ACAT-2), but not in Kupffer cells (ACAT-1).
The histopathological analysis indicated that K-604 also decreased the size of the CD68-positive Kupffer cells in a dosedependent manner, accompanied by suppressed inflammatory foci formation, but not the accumulation of lipid droplets. This observation prompted us to focus on the relationship between ACAT and Kupffer cells. Macrophages take up modified lowdensity lipoprotein, and the esterification of cholesterol by ACAT- 1 leads to foam cell formation. Foamy macrophages are thought to contribute to the development of atherosclerotic lesions, and the inhibition of ACAT is the basis for potential therapies targeting atherosclerosis [27,28]. To determine the correlation between ACAT-1 and Kupffer cells, we conducted double immunofluorescence analysis, which indicated that the ACAT-1 and CD68 proteins were co-localized in the liver (S3 Figure G). These observations suggest that K-604 inhibited ACAT-1 and thus modulated the cholesterol levels in the Kupffer cells. As previously reported, impaired Kupffer cell function is important in the pathogenesis of NASH [29-31]. Thus, within the paradigm of the NASH etiology, it appears plausible that the inhibition of ACAT-1 by K-604 may suppress foam cell formation and lead to the normalized phagocytic function and morphology of the Kupffer cells. Leroux, et al. [32] reported that fat-laden Kupffer cells are ‘primed’ to exhibit a proinflammatory phenotype and are sensitive to inflammatory stimuli, resulting in enhanced production of inflammatory cytokines, regardless of the presence or absence of LPS.
In contrast, bezafibrate showed no effects on liver inflammation or steatosis, despite the reductions in the plasma and liver cholesterol levels, which were similar to those observed with K-604. One of the differences between the K-604 and bezafibrate treatments was the hepatic FC levels (K-604 30 mg/ kg: 6.6, K-604 100 mg/ kg: 7.4 vs. bezafibrate: 10.3 mg/g, Table 2), and the increase in the hepatic FC levels (46% increase) in response to bezafibrate may lead to inflammatory reactions. Of note, Tomita, et al. [7] indicated that the FC accumulation in hepatic stellate cells results in enhanced Toll-like receptor 4 signaling and an exacerbation of liver fibrosis [7]. This theory might be plausible to support the behavior of bezafibrate. The dysregulation of cholesterol homeostasis also alters membrane fluidity and phagocytosis, which is disturbed in an animal model of NASH [33]. Kupffer cell dysfunction may promote steatohepatitis by sensitizing the hepatocytes to LPS [33]. These observations were in contrast with the reports on the suppression of fatty droplet formation and inflammatory reactions following activation of PPARα by bezafibrate or Wy-14,643 in the MCD diet-fed mice [34,35].
Considering the differences between the KK-Ay mice and Ldlr(−/−) mice in this study, bezafibrate significantly decreased the liver TG levels in the MCD diet-fed KK-Ay mice, but not in the HFC diet-fed Ldlr(−/−) mice, although the expression of the ACOX1 gene was induced in both mouse models. Based on the remarkable reduction in the hepatic TG levels (67% decrease, Table 1), bezafibrate suppressed fatty liver and resulted in a reduction in the steatosis score and inflammatory foci formation in the KK-Ay mice. In contrast, K-604 did not decrease the liver TG levels and fatty droplet accumulation as well as bezafibrate, but reduced the TNF-α and COL1A1 gene expression in the livers of the Ldlr(−/−) mice. We presumed that K-604 acts by directly preventing the formation of foamy Kupffer cells and exhibits antiinflammatory activity.
The result showing that the ZF rats exhibit severe steatosis by overfeeding as a model of NAFLD appear to elucidate the efficacy of ACAT inhibition [18]. K-604 ameliorated fatty liver and normalized the enlarged hepatocytes in the ZF rats. This indicates that a potent inhibition of ACAT-1 (100 mg/ kg) may promote favorable effects on lipid metabolism in the ZF rats. We noted that genetic deletion of ACAT-2 or treatment with antisense oligonucleotides of ACAT-2 could protect the mouse liver from dietary cholesterol-induced steatosis by facilitating TG secretion to the plasma [36,37]. In the mouse liver, ACAT-2 is primarily expressed in hepatocytes [7]. Although K-604 exhibits 229-fold selectivity in human CHO cell lines [8], it cannot be always reflected in rodent models. Furthermore, K-604 could inhibit ACAT activity in hepatocytes and in Kupffer cells because of the high concentrations of K-604 (100 mg/ kg) that were administered to the ZF rats.
Recently, Tomita, et al. [7] proposed that ACAT-1 deficiency leading to FC accumulation could promote liver fibrosis induced by bile duct ligation or carbon tetrachloride. This discrepancy between the previous report and our study may be attributed to the different experimental conditions, such as a complete genetic deficiency of ACAT-1 and its enzyme function. Additional extensive investigations are required to clarify the potential role of ACAT-1 in NAFLD/NASH pathophysiology. Here, ACAT- 1 inhibition resulted in the suppression of foamy Kupffer cell formation, with a subsequent reduction in inflammatory gene expression and an improvement of inflammatory cell infiltration, even when fatty liver had progressed. ACAT-1 plays a pivotal role in the formation of foamy Kupffer cells, followed by liver inflammation. Liver-specific inhibition of ACAT-1 may provide a useful basis for novel therapeutic strategies targeting steatosis and liver inflammation in NAFLD and NASH.
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