Flutamide exacerbates steatosis and promotes early hepatocarcinogenesis in high-fat diet-fed non-obese steatotic rats: Insights from clustering analysis of mitophagy regulators AMBRA1 and LC3

Flutamide exacerbates steatosis and promotes early hepatocarcinogenesis in high-fat diet-fed non-obese steatotic rats: Insights from clustering analysis of mitophagy regulators AMBRA1 and LC3 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Flutamide exacerbates steatosis and promotes early hepatocarcinogenesis in high-fat diet-fed non-obese steatotic rats: Insights from clustering analysis of mitophagy regulators AMBRA1 and LC3 Emika Hara, Kanami Ohshima, Wen Zeng, Suzuka Uomoto, Mio Kobayashi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4371202/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Flutamide (FL), a non-steroidal drug used for its anti-androgenic, anticancer,and disrupting endocrine properties, induces mitochondrial toxicity and drug metabolism enzymesand promotes hepatocarcinogenesis. The inhibition of mitophagy, leading to the accumulation of damaged mitochondria, is implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). In this study, we investigated the effects of FL in high-fat diet (HFD)-induced non-obese steatosis rats, categorized into four groups: basal diet (BD), BD + FL, HFD, and HFD + FL. The introduction of FL exacerbated HFD-induced steatosis and marginally increased preneoplastic lesions. To analyzehepatic preneoplastic lesions, we divided them into clusters based on the expression ratios of the mitophagy regulators LC3 and AMBRA1. The expression rates of LC3 and AMBRA1 in these precancerous lesions were classified into three clusters using k -means clustering. The HFD group exhibited an increased ratio of mitophagy inhibition clusters, as indicated by decreased LC3 and increased AMBRA1 levels in background hepatocytes and preneoplastic lesions. FL counteracted HFD-mediated mitophagy inhibition, as indicated by increased LC3 and decreased AMBRA1 levels in background hepatocytes. Our clustering analysis revealed that FL-induced mitophagy induction relied on Parkin expression. The present study underscores the significance of cluster analysis in understanding the role of mitophagy within small preneoplastic lesions and suggests that FL may potentially exacerbate NAFLD-associated hepatocarcinogenesis by affecting mitophagy. Biological sciences/Cancer/Cancer models Health sciences/Diseases/Gastrointestinal diseases/Liver diseases/Non alcoholic fatty liver disease Biological sciences/Cell biology/Autophagy/Mitophagy AMBRA1 Flutamide LC3 Mitophagy NAFLD Figures Figure 1 Figure 2 Figure 3 Highlights Clustering analysis of mitophagy markers AMBRA1 and LC3 elucidates the role of mitophagy in preneoplastic lesions. A high-fat diet increases the clusters with high AMBRA1 but low LC3 expression, indicating suppressed mitophagy-induced steatosis elevated preneoplastic lesions. The mitochondrial toxin flutamide increases the number of clusters with low AMBRA1 and LC3 expression, exacerbating mitophagy-induced steatosis and marginally increasing preneoplastic hepatic lesions in high-fat diet-fed rats. Introduction Non-alcoholic fatty liver disease (NAFLD) is a severe liver disease, globally affecting approximately 25% of the population and imposing a significant health burden along with widespread social and economic consequences 1 . Metabolic dysfunction-related fatty liver disease (MAFLD) has been proposed as a treatment for NAFLD, because it is associated with metabolic abnormalities. Increased fatty acid oxidation within the mitochondria plays a crucial role in preventing fat accumulation 2 ; however, a chronic high-fat diet (HFD), excessive glucose intake, and genetic obesity lead to increased oxidation rates that exceed the reduction capacity of oxygen storage sites, resulting in increased ROS generation 3 . The ROS generated are not adequately removed by reducing antioxidants, such as manganese superoxide dismutase, glutathione peroxidase, and reduced glutathione, causing damage to mitochondrial DNA. Moreover, reduced hepatic ATP synthesis, lowered mitochondrial respiratory chain complex activity, and abnormal liver mitochondrial ultrastructure have all been reported in NAFLD patients 4-6 . Furthermore, mitochondrial dysfunction promotes ROS production and lipid peroxidation. It also promotes abnormal hepatic lipid homeostasis, insulin resistance, cell death, and cytokine release, thereby leading to progression from NAFLD to nonalcoholic steatohepatitis (NASH) and eventually cirrhosis 7 . As some patients face the risk of developing hepatocellular carcinoma (HCC) with or without cirrhosis, effective strategies to combat this disease have gained paramount importance in recent years 8 . Therefore, promoting the removal of dysfunctional mitochondria, which contribute to fatty liver development, may play a pivotal role in the treatment of NAFLD. Autophagy is a catabolic process that eliminates unwanted intracellular materials through lysosomal degradation, thereby furnishing nutrients and energy for cellular homeostasis 9 . Cells often activate autophagy to cope with various stressors, such as nutrient scarcity, protein misfolding and aggregation, and intracellular organelle damage to intracellular. Activated autophagy serves to clear lipid droplets and abnormal organelles. Notably, it has been demonstrated to selectively remove damaged intracellular organelles 10 . This selective process, known as mitophagy, focuses on the removal of impaired mitochondria and has been extensively studied over the past decade. Numerous molecules associated with mitophagy regulation have been identified during this period 11 . The autophagy-mediated elimination of damaged mitochondria involves two steps: induction of general autophagy and priming of damaged mitochondria for selective autophagy. Recent advances in mitophagy research have revealed that mitochondrial priming is mediated by phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1)-Parkin-dependent or -independent mechanisms 11-13 . These targeted mitochondria are assimilated into the sequestration membrane by the microtubule-associated protein 1A/1B-light chain 3 (LC3) and mitophagy-related receptors, which bind to the sequestration membrane to form autophagosomes. Subsequently, these autophagosomes fuse with lysosomes to form autolysosomes, where damaged mitochondria are degraded. In this process, the autophagy and Beclin 1 regulator 1 (AMBRA1) is defined to assume a pivotal role as a mitophagy regulator involved in both Parkin-dependent and-independent pathways 14 . In the Parkin-dependent mitophagy pathway, activated Parkin interacts with AMBRA1 expressed on the outer mitochondrial membrane, which, in turn, promotes mitophagy by activating the class III phosphatidylinositol 3-kinase (PI3K) complex, an essential contributor to autophagosome formation 15,16 . Additionally, AMBRA1 directly binds to LC3, further driving the progression of autophagosome formation 17 . Accumulating evidence suggests that the levels of mitophagy regulators decrease with the progression of steatosis, signifying the involvement of inhibited mitophagy in the pathogenesis of NAFLD. In a murine model of diet-induced NAFLD, hepatocyte autophagy was arrested, leading to mitochondrial dysfunction 18 . In the context of diet-induced NAFLD in mice, the progression of mitophagy was suppressed, evident in the accumulation of megamitochondria containing mitophagy intermediates such as p62/SQSTM1 and ubiquitin 19 . In primary hepatocytes from mice treated with palmitic acid to mimic high-fat stress, the expression levels of LC3 and Parkin in the mitochondria were notably reduced, indicating that mitophagy is inactivated in a Parkin-dependent manner under high-fat stress 20 . PINK1-Parkin-dependent mitophagy is inhibited in the livers of NAFLD mouse models generated by high-fat feeding as well as in patients with NAFLD 21 . Flutamide (FL) is a non-steroidal antiandrogenic and anticancer drug widely used to treat prostate cancer and recognized as an endocrine-disrupting environmental contaminant 22-24 . Moreover, FL inhibits mitochondrial respiratory chain complexes I, II, and V and is, therefore, known as a mitochondrial toxicant 25,26 . In a previous study conducted in our laboratory, FL was observed to enhance precancerous lesions in a medium-term liver carcinogenesis rat model; however, the underlying mechanism remains unclear 27 . In this study, we hypothesized that FL induces mitochondrial injury followed by mitophagy in an HFD-mediated early hepatocarcinogenesis model 28-31 . This model, based on the medium-term hepatocarcinogenesis assay 32 , served as a valuable tool for understanding the augmentation of autophagy in precancerous lesions facilitated by fatty liver conditions 28-31 . This model is particularly advantageous since hepatic precancerous lesions specifically express the autophagosome marker LC3 and the cargo receptor p62 29, 31, 33 . Using immunohistochemical techniques and cluster analysis using autophagy markers, we investigated the role of autophagy in small precancerous lesions in rat livers 34 . Further, we investigated the role of mitophagy in NAFLD-related hepatocarcinogenesis by clustering hepatic preneoplastic lesions according to the expression ratios of the autophagy marker LC3 and mitophagy indicators AMBRA1 and Parkin. Results Flutamide inhibits body weight growth, increases HFD-induced NAFLD score, and enhances preneoplastic hepatic lesions During the study period, FL suppressed body weight gain after 6 weeks, regardless of the diet type (Fig. 1 a). No discernible difference was observed in terms of food and water intake (Table 1 , Supplemental Tables 3 and 4). In the final autopsy, FL significantly reduced the final body weight and intra-abdominal fat weight, independent of the HFD (Table 1 ). Importantly, HFD-induced obesity was not observed under these experimental conditions (Fig. 1 a, Table 1 ). Table 1 Final body weight, organ weight, food intake, and water intake in rats§ Group BD BD + FL HFD HFD + FL No. of animals 6 6 6 6 Final body weight (g) 301.5 ± 15.15 a 273.7 ± 6.96 bc 293.4 ± 15.13 ab 271.8 ± 12.05 c Food intake (g/kg) 47.1 ± 16.8 46.0 ± 15.5 34.1 ± 15.0 33.3 ± 13.9 Water intake (g/kg) 66.6 ± 19.3 64.4 ± 17.3 53.8 ± 17.6 51.3 ± 16.5 Absolute liver weight (g) 7.68 ± 0.72 ab 7.91 ± 0.32 a 6.89 ± 0.86 b 6.90 ± 0.38 b Relative liver weight (%BW) 2.54 ± 0.13 a 2.89 ± 0.07 b 2.34 ± 0.19 a 2.54 ± 0.16 a Absolute intraperitoneal fat weight (g) 6.27 ± 1.47 a 4.40 ± 0.40 b 6.78 ± 0.86 a 5.04 ± 1.33 ab Relative intraperitoneal fat weight (%BW) 2.07 ± 0.46 ab 1.61 ± 0.13 b 2.30 ± 0.18 a 1.85 ± 0.46 ab Abbreviations: BD, basal diet; HFD, high fat diet; FL, flutamide, BW; body weight. § : All animals were subjected to two-thirds partial hepatectomy at week 3. Data are shown as the mean ± standard deviation. Different letters indicate significant differences between groups (p < 0.05, significantly different by Tukey's or Steel-Dwass test). Previous studies have shown that an HFD increases NAS and steatosis scores in the liver 28 – 31 . Similar results were obtained in the present study, where FL further increased these scores (Fig. 1 b- 1 d). Notably, the scores associated with ballooning degeneration and inflammatory foci increased in response to an HFD but remained unaffected by FL (Fig. 1 e, 1 f). FL caused a significant increase in relative liver weight in the BD + FL group, which might have been caused by CYP induction 27 , as is evident from the Cyp1a gene expression data presented below (Table 2 ). However, no statistically significant change in relative liver weight was observed in the HFD + FL group (Table 1 ). Table 2 Gene expression analysis of autophagy, mitochondria/mitophagy, lipid metabolism, drug metabolism, inflammation, and oxidative stress in liver samples. Group BD BD + FL HFD HFD + FL No. of animals 6 6 6 6 Autophagy-related genes Atg3 1.05 ± 0.34 1.69 ± 0.40 2.58 ± 1.23 1.60 ± 0.32 Atg5 1.10 ± 0.51 a 1.77 ± 0.72 ab 2.61 ± 0.91 b 2.27 ± 0.83 ab Atg7 1.03 ± 0.26 a 1.10 ± 0.23 a 2.08 ± 0.64 b 1.98 ± 1.02 ab Lamp1 1.06 ± 0.40 a 1.50 ± 0.41 ab 2.08 ± 0.72 b 1.54 ± 0.59 ab Lamp2 1.05 ± 0.35 a 1.57 ± 0.36 ab 2.13 ± 0.93 b 1.19 ± 0.39 a Lc3 1.00 ± 0.08 a 1.24 ± 0.38 ab 2.06 ± 0.83 b 1.58 ± 1.04 ab p62 1.03 ± 0.26 1.30 ± 0.57 1.82 ± 0.72 1.67 ± 1.02 Mitochondria/Mitophagy-related genes Ambta1 1.14 ± 0.66 1.98 ± 0.75 3.95 ± 3.18 4.64 ± 5.32 Parkin 1.17 ± 0.64 a 2.54 ± 0.99 ab 3.27 ± 2.12 ab 3.57 ± 1.59 b Pink1 1.03 ± 0.24 1.09 ± 0.22 1.81 ± 0.65 1.12 ± 0.29 Bnip3 1.01 ± 0.18 1.48 ± 0.41 1.43 ± 0.47 1.05 ± 0.34 Nadh 1.03 ± 0.26 1.32 ± 0.26 2.21 ± 0.97 1.17 ± 0.32 Sdhd 1.04 ± 0.30 a 1.67 ± 0.40 ab 2.17 ± 0.86 b 1.51 ± 0.51 ab ATP synthase 1.03 ± 0.29 1.47 ± 0.45 1.79 ± 0.75 1.24 ± 0.30 Lipid metabolism-related genes Abca1 1.09 ± 0.40 a 1.89 ± 0.79 ab 3.26 ± 1.07 c 2.74 ± 0.89 bc Acox1 1.14 ± 0.59 1.80 ± 0.54 2.74 ± 1.36 1.89 ± 0.34 Apob 1.05 ± 0.30 0.82 ± 0.21 1.49 ± 0.76 1.31 ± 0.32 Dgat2 1.06 ± 0.39 a 1.36 ± 0.41 ab 1.77 ± 0.51 b 1.70 ± 0.35 ab Fasn 1.27 ± 0.87 3.04 ± 1.50 1.72 ± 0.96 2.43 ± 2.25 Hmgcs1 1.09 ± 0.47 0.72 ± 0.36 1.51 ± 0.77 1.18 ± 0.36 Hsd3b1 1.10 ± 0.48 a 0.38 ± 0.40 a 2.17 ± 0.90 b 0.83 ± 0.60 a Lpl 1.06 ± 0.37 0.97 ± 0.27 1.50 ± 0.80 1.17 ± 0.46 Lss 1.11 ± 0.53 1.46 ± 0.72 1.69 ± 0.66 2.30 ± 1.19 Ppara 1.19 ± 0.69 a 1.81 ± 0.97 a 4.55 ± 1.44 b 2.08 ± 0.71 a Pparg 1.05 ± 0.36 1.16 ± 0.98 0.61 ± 0.26 1.91 ± 1.47 Scd1 1.03 ± 0.26 1.27 ± 0.38 1.69 ± 0.46 1.25 ± 0.62 Srebf1 1.17 ± 0.73 0.84 ± 0.43 1.59 ± 1.26 1.58 ± 1.02 Srebf2 1.08 ± 0.41 a 1.53 ± 0.44 ab 2.49 ± 0.95 b 2.33 ± 0.88 b Drug metabolism-related genes Cyp1a1 1.27 ± 1.09 a 73.20 ± 45.16 b 1.90 ± 0.94 a 96.14 ± 93.51 b Cyp2b1 1.64 ± 1.91 1.97 ± 0.95 1.84 ± 0.91 2.51 ± 1.10 Cyp3a1 1.05 ± 0.34 0.65 ± 0.25 1.40 ± 0.54 0.89 ± 0.79 Inflammation related genes Tnf-a 1.11 ± 0.51 1.29 ± 0.64 1.81 ± 1.11 3.33 ± 3.87 Oxidative stress-related genes Catalase 1.04 ± 0.33 a 1.75 ± 0.58 ab 2.70 ± 1.22 b 2.18 ± 0.98 ab Gpx1 1.16 ± 0.70 1.68 ± 0.67 1.92 ± 0.75 2.05 ± 0.95 Gpx2 1.08 ± 0.47 a 5.36 ± 1.91 b 3.44 ± 2.86 ab 4.98 ± 2.16 b Mn-SOD 1.04 ± 0.32 1.23 ± 0.38 1.65 ± 0.73 1.54 ± 0.56 Sod1 1.11 ± 0.53 1.32 ± 0.54 1.18 ± 0.55 1.00 ± 0.40 Sod2 1.13 ± 0.63 1.41 ± 0.23 2.07 ± 0.87 1.33 ± 0.35 Abbreviations: BD, basal diet; HFD, high-fat diet; FL, flutamide. §: All animals were subjected to two-thirds partial hepatectomy at week 3. Data are shown as the mean ± standard deviation. Different letters indicate significant differences between groups (p < 0.05, significantly different by Tukey's or Steel-Dwass test). The consumption of HFD induced a significant increase in the number and area of GST-P-positive foci (Fig. 1 g, 1 h, 1 k), as previously reported 28 – 31 . A previous study showed that that higher FL doses (1000 and 2000 ppm) enhanced hepatocarcinogenesis in rats 27 ; however, in the present study, a lower FL dose (500 ppm) caused a marginal increase in both the number and area of GST-P-positive foci (Fig. 1 g- 1 j). The area of GST-P-positive foci was significantly increased in the HFD + FL group compared to the BD group (Fig. 1 h- 1 j); however, the cumulative effects of HFD and FL on the area of GST-P-positive foci were marginal. The number of Ki-67-positive cells within hepatic precancerous lesions showed an increasing trend in the FL-treated groups (BD + FL and HFD + FL); however, no statistically significant change was detected (Supplemental Table 4). These findings suggest that FL exacerbated steatosis and marginally increased preneoplastic lesions in a rat model of non-obesity-related steatosis. Expression analysis of mitophagy markers in background hepatocytes and precancerous lesions FL exhibits mitochondrial toxicity 35 , while HFD inhibits mitophagy 21 , 36 . In light of these insights, we assessed the expression of the autophagy marker LC3 and the mitophagy indicator AMBRA1 in both background hepatocytes and precancerous lesions of rats treated with HFD and/or FL. LC3 and AMBRA1 showed positive granular signals in hepatocytes (Supplemental Figs. 2 and 3). Quantitative analysis showed that LC3-positive granules exhibited an increasing trend in the FL-treated groups (BD + FL and HFD + FL groups) compared to the non-FL-treated groups (BD and HFD groups) (Supplemental Fig. 2b, d), both in background hepatocytes (non-foci) and precancerous lesions (foci). Similarly, AMBRA1-positive granules tended to increase in number in the HFD group compared to the BD group. However, when combined with FL, a decreasing trend was observed, particularly in the BD + FL group, in both background hepatocytes (non-foci) and precancerous lesions (foci) (Supplemental Fig. 3b, 3d). These results indicate a potential suppression of mitophagy in the HFD group, as indicated by the increased expression of AMBRA1. Conversely, in combination with the FL treatment, mitophagy might be induced by the suppression of increased AMBRA1 expression; however, these effects on mitophagy were found to be marginal in this standard analysis. No statistically significant differences in Parkin expression were observed in either background hepatocytes or preneoplastic lesions in each group (Supplemental Fig. 4a-d). Cluster analysis of LC3, AMBRA1, and Parkin expression in background hepatocytes To further investigate the relationship between LC3 and AMBRA1 expression, we compared the expression ratios of both indicators in background hepatocytes using cluster analysis, as previously reported 34 , aiming to elucidate the dynamics of autophagy flux (Supplemental Fig. 1). Using the k -means, the clusters were divided into three groups (Fig. 2 a-c) and examined the cluster composition of each group (Fig. 2 d). These clusters were classified into three levels: C1, characterized by low expression of both LC3 and AMBRA1, indicative of a basal level; C2, characterized by low AMBRA1 expression and high LC3 expression, suggestive of mitophagy induction; and C3, characterized by low LC3 expression and high AMBRA1 expression, implying mitophagy inhibition (Fig. 2 b). In the BD group, the major constitutive cluster was C1, followed by C3, and lastly C2 in a descending order (Fig. 2 d). This pattern closely resembled that of the BD + FL group, where the primary constitutive cluster remained C1, similar to the BD group. However, the percentage of C2 clusters in the BD + FL group increased compared to the BD group, although this difference was not statistically significant. In contrast, in the HFD group, the prevalence of C3 composition significantly increased. In the HFD + FL group, the cluster composition was similar to that observed in the BD + FL group, with a significant increase in the rate of C2 composition. We analyzed LC3 and Parkin expression to evaluate whether mitophagy induction was Parkin-dependent in the FL-treated groups (Fig. 2 e- 2 h). The clusters were classified into four levels: C1, characterized by low LC3 expression and high Parkin expression, implying mitophagy inhibition; C2, with low Parkin expression and high LC3 expression, suggestive of Parkin-independent mitophagy induction; C3, a basal cluster with low expression of both LC3 and Parkin; and C4, featuring high expression of both LC3 and Parkin, indicative of Parkin-dependent mitophagy induction (Fig. 2 f). In the BD + FL group, the primary cluster was C3, while in the HFD + FL group, the C4 composition was significantly augmented (Fig. 2 h), suggesting that FL induced mitophagy in a Parkin-dependent manner in a population of preneoplastic lesions in HFD-fed rats. Cluster analysis of the GST-P-positive foci area and LC3, AMBRA1, and Parkin expression in hepatic precancerous lesions To determine whether AMBRA1 expression played a role in the development of hepatic preneoplastic lesions, we examined the relationship between the area of individual GST-P-positive foci and the expression rate of AMBRA1 in each lesion (Fig. 3 a-d). Subsequently, we classified cluster into four levels, namely C1 to C4, and observed that C4 exhibited the highest AMBRA1 expression (mitophagy inhibition cluster) among all four levels (Fig. 3 b). The percentage of C4 composition significantly increased in the HFD group, and this effect was attenuated when combined with FL (Fig. 3 d), suggesting that FL might cancel mitophagy inhibition in HFD-fed rats. We investigated the relationship between LC3 and AMBRA1 expression in hepatic precancerous lesions, which were divided into three clusters (Fig. 3 e- 3 h), and examined the cluster composition of each group (Fig. 3 h). These clusters were classified into three levels, namely C1–C3: C1 represented the basal level with low expression of both LC3 and AMBRA1; C2 indicated mitophagy induction with low expression of AMBRA1 and high expression of LC3; and C3 denoted mitophagy inhibition with high expression of AMBRA1 and low expression of LC3 (Fig. 3 f), similar to the categorization in background hepatocytes (Fig. 2 b). The percentage of C3 cells was significantly higher in the HFD group than in the BD group (Fig. 3 h). The composition of clusters in the HFD + FL group was similar to that in the BD + FL group. The results suggested that FL might cancel mitophagy inhibition in HFD-fed rats as well. Finally, we analyzed LC3 and Parkin expression in hepatic precancerous lesions to evaluate whether mitophagy induction was Parkin-dependent in the FL-treated groups (Fig. 3 i- 3 l). The clusters were classified into three levels: C1 represented the basal level with low expression of both LC3 and Parkin; C2 indicated a cluster with low LC3 expression and high Parkin expression, indicative of mitophagy inhibition; and C3 represented a level with high LC3 expression and low Parkin expression, suggestive of Parkin-independent mitophagy induction (Fig. 3 j). Unlike in background hepatocytes, we did not detect C4, a Parkin-dependent mitophagy inhibition cluster (Fig. 2 f). C3 was minimally detected in the FL-treated groups BD + FL and HFD + FL (Fig. 3 l). HFD and HFD combined with FL alters hepatic gene expression A comprehensive gene expression analysis was conducted on the liver samples to determine the contributions of autophagy, mitophagy, injured mitochondria, lipid metabolism, inflammation, drug-metabolizing enzymes, and oxidative stress-related genes, which might be related to NAFLD-related liver carcinogenesis. We analyzed autophagy-related genes and showed that the expression of Atg5, Atg7, Lamp1, Lamp2 , and Lc3 was significantly increased in the HFD group compared to the BD group. However, when HFD feeding was combined with FL administration, a significant decrease was observed in the expression of these genes (Table 2 ). These gene expression changes in the HFD group were considered a compensatory response to the suppression of mitophagy, as discussed earlier. Regarding mitophagy-related genes, Parkin expression was significantly elevated in the HFD + FL group compared to the BD group, along with higher levels of Ambra1 expression. Furthermore, a relatively higher increase in the expression of rkin and Ambra1 was observed in the BD + FL and/or HFD groups. This could potentially indicate the presence of Parkin-dependent mitophagy in background hepatocytes, aligning with the observations discussed earlier. FL, a known inhibitor of the mitochondrial respiratory chain complex I, has been reported to impair mitochondrial functions 25 . Moreover, 2-hydroxyflutamide, a major FL metabolite, impairs the functions of mitochondrial respiratory chain complexes II and V 26 . In this study, we analyzed the gene expression of complex I ( NADH : quinone oxidoreductase), complex II (SDHD: succinate dehydrogenase), and complex V ( ATP synthase ). We found that the expression level of Sdhd tended to increase in the BD + FL group and was significantly elevated in the HFD group compared to the BD group. However, this expression trend reversed with the context of combined HFD feeding and FL administration, indicating a decreasing pattern (Table 2 ). Although no evident differences in the expression of Nadh and ATP synthase were observed between the groups, the expression of Nadh tended to increase in the HFD group; however, it remained comparable to that in the BD group. It is important to note that FL induces significant upregulation of Cyp1a1 expression in rats 37 . In our study, the expression of Cyp1a1 was significantly elevated upon FL administration, regardless of the presence of HFD. Regarding the expression levels of genes related to lipid metabolism, Abca1 , which is responsible for the production of high-density lipoproteins, and Srebf2 , which is involved in cholesterol metabolism, were significantly increased in the HFD groups (HFD and HFD + FL) compared to the BD group (Table 2 ). In addition, the TG synthase gene Dgat2 , steroid hormone synthase Hsd3b1 , and transcription factor Ppara , which promotes β-oxidation, were significantly increased in the HFD group compared to the BD group, while they either showed a declining trend or a significant decrease in the HFD + FL group. No obvious differences in the expression of Tnf-α , one of the most effective cytokines, between the groups (Table 2 ). Regarding oxidative stress gene expression, the antioxidant enzyme Catalase exhibited a significant increase in the HFD group compared to the BD group. Furthermore, the expression level of Gpx2 significantly increased in the FL alone group and in the combination of HFD feeding and FL administration (Table 2 ). Thus, in the HFD group, antioxidant enzyme-related genes exhibited increased expression parallel to steatosis. However, the combined effect of HFD feeding and FL administration on gene expression was not detected. Discussion In recent years, the global incidence of NAFLD, also known as MAFLD, has been on the rise, and a subset of patients has been shown to develop liver cancer 8 . Because mitochondria are intricately involved in both ROS production and lipid metabolism, dysfunctional mitochondria have been implicated in the pathogenesis of NAFLD 7 . While healthy individuals effectively clear out abnormal mitochondria through mitophagy, this regulatory mechanism appears to be impaired in patients with NAFLD, potentially contributing to the persistence of abnormal mitochondria and excessive ROS generation, which in turn may exacerbate the fatty liver condition 19 – 21 . However, the precise role of mitophagy in NAFLD-related hepatocarcinogenesis remains unclear. To examine the role of mitophagy in the present non-obese rat model of NAFLD-related hepatocarcinogenesis, we chose to utilize FL as a mitochondrial toxicant. This is because of FL’s dual nature as an inhibitor of mitochondrial complexes I, II, and V 25 , 26 , as well as its ability to promote hepatic carcinogenesis in a medium-term hepatic carcinogenesis assay 27 . In the present study, the effects of 500 ppm FL in inducing mitochondrial toxicity and hepatocarcinogenesis were found to be marginal, as evidenced by limited alterations in mitochondria/mitophagy-related gene expression (Table 2 ), and a gradual increase in the area of hepatic precancerous lesions compared to the BD group (Fig. 1 h- 1 j). Consistent with a previous study involving 1000 or 2000 ppm FL within a medium-term liver initiation/promotion model 27 , we observed a noticeable expression of Cyp1a1 in the FL-treated liver (Table 2 ); however, CYP-mediated oxidative stress was not involved in hepatocarcinogenesis 27 , 38 . In this study, a significant increase in Gpx2 gene expression in the BD + FL group suggested that FL induced an oxidative stress response; however, the effect remained limited, as other antioxidant genes were largely unaffected. Under our study conditions, we assessed mitophagy flux using immunohistochemistry-based clustering analysis involving mitophagy markers in background hepatocytes and preneoplastic lesions, following a previously reported methodology 34 to understand the role of mitophagy in FL and HFD-mediated hepatocarcinogenesis. To understand the dynamics of mitophagy flux, it becomes essential to confirm the progression of mitophagy and identify various autophagy markers. In selective autophagy, the initial reaction involves the formation of a sequestration membrane that surrounds the cargo, ultimately giving rise to autophagosomes 9 , 10 , 36 . Subsequently, lysosomes merge with these autophagosomes to form autolysosomes, where lysosome-derived enzymes degrade the cargo, and the degraded products are reused within cells. This sequence of events constitutes the autophagic flux (Supplemental Fig. 1). To understand alterations in the expression of autophagosomes and cargo receptors, it is imperative to consider this autophagic flux, thereby ensuring a meticulous and informed interpretation of the outcomes 39 , 40 . In general, LC3 is expressed both inside and outside sequestration membranes and autophagosomes, making it a widely used marker for examining autophagy involvement 39 , 41 . In our study, LC3 was observed as granules in hepatocytes, suggesting that the accurate detection of autophagosomes (Supplemental Fig. 1c, 2a, 2c). LC3 includes two isoforms: LC3-I, which is ubiquitously expressed in autophagosomes, and LC3-II, whose elevation signifies increased autophagy. It is noteworthy that the anti-LC3 antibody used in our study does not discriminate between LC3-I and LC3-II. Therefore, when analyzing autophagic flux, it becomes essential to observe specific molecules expressed on cargo receptors and injured intracellular organelles that are taken up by autophagosomes. Escalation in the expression of cargo receptors and injured organelle markers indicates inhibition of autophagy 39 , 40 . In this study, tour approach involved analyzing mitophagy flux by combining LC3 with the mitophagy regulatory factors AMBRA1 and Parkin, which are expressed on the membranes of impaired mitochondria (Supplemental Fig. 1). Initially identified as a causative gene for Parkinson's disease 42 , Parkin plays a role beyond neurological conditions to encompass liver diseases as well 43 . In HFD-fed mice, an increased expression of the macrophage stimulating 1 ( Mst1 ) gene, a key mitophagy regulator, suppressed Parkin-dependent mitophagy and worsened steatosis. In contrast, Mst1 knockout mice exhibited a lifting of the suppression of Parkin-dependent mitophagy, leading to an amelioration of steatosis 20 . These data were further validated in vitro , where palmitate-treated hepatocytes showed a reduction in Parkin expression. The analysis of the dynamics of mitophagy flux using LC3, AMBRA1, and Parkin provided new insights into the relationship between Parkin-dependent or -independent mitophagy in an early HFD-mediated hepatocarcinogenesis. In the livers of patients with NAFLD, the suppression of autophagy has been linked to increased accumulation of fat droplets, as determined by analyzing the expression of the autophagosome markers LC3-I/LC3-II and cargo receptor p62 44, 45 . These findings are recapitulated in in vitro models stimulating NAFLD liver conditions. In studies employing hepatocytes and hepatocellular carcinoma cells, the inhibition of mitophagy has been shown to increase fat droplet deposition, as indicated by the altered expression patterns of mitophagy indicators such as AMBRA1, Parkin, and PINK1 20, 21 . In the present study, although no distinct differences were observed in the standard analysis of LC3-, AMBRA-, and Parkin-positive granules in background hepatocytes (Supplemental Fig. 2–4), our clustering analysis revealed that HFD increased the proportion of mitophagy inhibition clusters with high AMBRA1 expression (C3 in Fig. 2 b, 2 d). The higher expression of Atg5, Atg7, Lamp1, Lamp2 , and Lc3 in the HFD group (Table 2 ) could potentially reflect an adaptive response to the processing of excess fat droplet load caused by mitophagy inhibition. In contrast, the HFD + FL group showed an increased proportion of clusters indicative of mitophagy induction (C2 in Fig. 2 b, 2 d), a response that was dependent on Parkin (C4 in Fig. 2 f, 2 h). Remarkably, the increased expression of Atg5, Atg7, Lamp1, Lamp2 , and Lc3 in the HFD་FL group appeared to be somewhat obscured (Table 2 ), suggesting that FL-induced mitophagy was concurrently suppressing gene expression. Furthermore, the HFD + FL group showed a more pronounced increase in steatosis score than the HFD group (Fig. 1 d). In drug-induced NAFLD, it has been shown that the drugs contribute to the development of steatosis by inhibiting mitochondrial β-oxidation 46 . Therefore, it is plausible that the HFD-mediated steatosis observed in the HFD + FL group may have been further exacerbated by FL-mediated inhibition of β-oxidation, as FL has been reported to downregulate the expression of genes associated with mitochondrial β-oxidation 25 . Consistent with this, FL suppressed the HFD-induced increase in the expression of Ppara , a transcription factor known to induce the expression of enzymes involved in HFD-induced β-oxidation (Table 2 ). AMBRA1, expressed on the membranes of damaged mitochondria, plays a vital role in inducing mitophagy 14 . Moreover, it has been reported that AMBRA1 is explicitly expressed in tumorigenic lesions in a two-stage rat liver carcinogenesis model 47 . The escalation of hepatic precancerous lesions observed in the HFD group was concurrent with the increased proportion of clusters showing high levels of AMBRA1 (C4 in Fig. 3 b, 3 d) and mitophagy inhibition (C3 in Fig. 3 f, 3 h). However, these effects of HFD were counteracted upon co-administration with FL. Because the cluster showing Parkin-independent mitophagy induction was marginal in both the BD + FL and HFD + FL groups (C3 in Fig. 3 j, 3 l), we could not definitively conclude that FL-induced mitophagy was Parkin-independent in hepatic preneoplastic lesions in the present study. Mitophagy generally suppresses carcinogenesis in the early stages, when normal cells transition into transformed cells 48 . In contrast, during late carcinogenesis, when transformed cells progress towards further malignant transformation and metastasis, mitophagy enhances carcinogenesis by eliminating damaged mitochondria, preventing ROS production, and enhancing mitochondrial energy production 48 , 49 . In this study, we observed precancerous lesions, which represent the early stage of carcinogenic progression; therefore, it is possible that mitophagy inhibits the development of precancerous lesions, and that the HFD-induced inhibition of mitophagy increases the formation of hepatic precancerous lesions. This hypothesis is supported by the increased expression of LC3 and p62 that were previously observed in hepatic precancerous lesions 29 , 31 . Despite the marginal cumulative effects of FL and HFD on the generation of hepatic precancerous lesions (Fig. 1 h- 1 j), it is conceivable that FL-induced mitophagy could be involved in the maintenance of certain populations of precancerous lesions in this group. These results suggest that mitophagy induction might lead to a phenomenon similar to further malignant transformation of tumor cells. This result is consistent with an increase in the formation of tumorigenic lesions when the autophagy inducer amiodarone was applied to a two-stage rat liver carcinogenesis model 47 . In addition to regulating mitophagy, Ambra1 acts as a tumor suppressor gene in vivo ; it inhibits cell cycle progression into the G1-S phase by promoting the degradation of cyclin D 50 . Additionally, it interacts with protein phosphatase 2A (PP2A), a cell cycle regulator, independently of autophagy regulation, and modulates cell growth and tumorigenesis by promoting the PP2A-mediated dephosphorylation of the oncogene C-MYC 51 . Further evidence supporting its tumor suppressor function emerges from studies in which primary mouse embryonic fibroblasts from wild-type (AMBRA1 +/+) and deficient (AMBRA1 gt/gt) AMBRA1 loci were subcutaneously injected into mice, resulting in earlier tumor formation in AMBRA1-deficient mice than in wild-type counterparts. This underscores the role of AMBRA1 as a tumor suppressor gene. Notably, mutations in Ambra1 have also been identified in various human tumors. In the HFD + FL group, the downregulation of AMBRA1 may potentially contribute to the formation of precancerous lesions (C3 in Fig. 3 b, 3 d). In conclusion, we found that mitophagy was suppressed in the HFD group and that this suppression was reversed by FL administration, which led to enhanced precancerous lesion formation. Similar findings were observed in the analysis of background hepatocytes other than the precancerous lesions. While current research is focused on developing drugs that induce mitophagy for the treatment of NAFLD and cancer 52 – 55 , it is crucial to consider the potential risk of mitophagy inducers aggravating NAFLD. Analyzing mitophagy both inside and outside precancerous lesions can provide a clear understanding of its pathological significance in NAFLD models. As shown in the present study, employing cluster analysis using the autophagy marker LC3 and mitophagy indicators AMBRA1 and Parkin proves to be a valuable approach for studying mitophagy. This methodology can also be extended to the analysis of autophagy flux in preneoplastic lesions and micro-cancers, enabling a deeper exploration of the roles of mitophagy in malignant transformation and the development of novel therapeutic strategies. Methods Chemicals N-Nitrosodiethylamine (DEN; CAS No. 55-18-5, purity > 99%) and flutamide (FL; CAS No. 13311-84-7, purity > 98%) were purchased from Tokyo Kasei Kogyo (Tokyo, Japan) and Combi-Blocks (San Diego, CA, USA), respectively. Animals and treatments A total of 30 5-week-old male F344/DuCrlCrlj rats were purchased from Charles River Laboratories Japan (Kanagawa, Japan) and reared under the following conditions: temperature 23 ± 3°C, humidity 50 ± 20%, and 12 h light/12 h dark lighting cycle. The rats were fed a powdered diet (MF; Oriental Yeast Co., Japan) as the basal diet (BD) and provided with drinking water in a clean rack equipped with rat paper-type enrichment, with a maximum of three animals per cage. In a previous study, rats were treated with 1000 or 2000 ppm doses of FL, resulting in final body weights of 85% and 81%, respectively, at the end of the study (8 weeks), compared to the control group 27 . Preliminary analyses were conducted on six animals, wherein FL was administered at three different doses (250, 500, and 1000 ppm) over a span of two weeks. Based on evaluations of body and liver weights, the FL dose of 500 ppm was selected for the present study. Six days before the start of the study, 12 rats were fed a HFD (D12451, 45 kcal%; Research Diets Inc., New Brunswick, NJ, USA), while the remaining rats were provided with a BD. The study was conducted as previously reported 28 – 31 , according to an initiation and promotion model 32 . The rats were divided into four groups: BD-fed (BD, n = 6), BD-fed and FL-administered (BD + FL, n = 6), HFD-fed (HFD, n = 6), and HFD-fed and FL-administered (HFD + FL, n = 6). Throughout the study period, daily observations were performed to assess the general conditions of the animals. Additionally, the body weight and food and water intake measurements were performed weekly. At 13 weeks after study initiation, all animals were subjected to laparotomies under isoflurane anesthesia and humane euthanization through blood release. Subsequently, the liver and intraperitoneal fat were collected. The livers were fixed in 4% paraformaldehyde (PFA) for histopathological examination and immunohistochemical staining. A portion of the liver was frozen in liquid nitrogen and stored at − 80°C. The experimental plan involving animals was approved and reviewed by the Laboratory Animal Committee of Tokyo University of Agriculture and Technology (No. R03-149) prior to the commencement of the experiments. All animals were handled in accordance with the Guidelines for Animal Experimentation issued by the Japanese Association for Laboratory Animal Science ( https://www.jalas.jp/english/en_about.html ). The study was conducted in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Histopathology Following fixation in 4% PFA, liver specimens were subjected to thin sectioning to a thickness of approximately 3 µm. These sections were subsequently stained using hematoxylin and eosin (H&E) staining. In the liver tissue, we evaluated the NAFLD activity score (NAS), encompassing assessments from steatosis, ballooning, and inflammatory foci 30 , 56 . Immunohistochemistry and clustering analysis The PFA-fixed liver specimens were thinly sliced and subjected to immunohistochemistry for targeting specific markers, including glutathione S-transferase placental form (GST-P), which is positive for rat liver proliferative lesions; Ki-67, a marker of cell proliferative activity; LC3, an autophagy marker; and AMBRA1 and Parkin, both pivotal regulators of mitophagy. Detailed information about the antibodies used, antigen activation methods, and antibody dilution ratios is presented in (Supplemental Table 1). Signal detection was performed according to the protocol of VECTASTAIN® Elite ABC Kit (Vector Laboratories Inc, Burlingame, CA, USA), with subsequent immunoreaction carried out using 3,3′-diaminobenzidine/H 2 O 2 . After visualization, the cells were counterstained with hematoxylin. The number and area of the GST-P-positive foci were measured as previously described 28 , 32 . The positive rates of LC3, AMBRA1, and Parkin within each GST-P-positive focus were measured using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). The number of Ki-67-positive cells per GST-P-positive focus was defined as the labeling index (%) 31 . The GST-P-positive foci and the positivity rates for AMBRA1, LC3, and Parkin were collated for each individual and analyzed in groups, rather than individually, as previously reported 34 . For further analysis, the positive rates of LC3, AMBRA1, or Pink in GST-P-positive foci were plotted on the x- and y-axes, respectively. Subsequently, clustering was performed for analyzing each cluster using the k -means method. The positive rates of LC3, AMBRA1, and Parkin in background hepatocytes were also analyzed. Real-time reverse transcription-polymerase chain reaction analysis Expression analysis at the mRNA level was performed using the genes and primers listed in (Supplemental Table 2). RT-PCR was performed as described previously 28 . Statistical analyses Means and standard deviations were calculated for all data. Statistical analyses were performed using either the Tukey or Steel-Dwass test. A significance level of 5% or less indicated a significant difference. A test of mother proportions (Tukey–Kramer test) was performed to determine the proportion of clusters in each group. Declarations Acknowledgments: We thank Editage (www.editage.com) for the English language editing. Authors' contributions: EH and TY conceived the project, designed and conducted the animal experiments, analyzed and interpreted the data, and wrote the manuscript. EH, KO, WZ, SU, and MK performed the animal experiments and immunohistochemistry analysis. MS initiated the study and interpreted the results. All the authors have read and approved the final manuscript. Ethics approval: The clinical, operative, and experimental procedures were performed according to the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan, June 1, 2006). The study protocol was duly approved by the Animal Care and Use Committee of the Tokyo University of Agriculture and Technology, ensuring compliance with ethical standards and welfare of the animals involved (Approval number R03-149). Consent to participate: Not applicable. Consent for publication: Not applicable. Funding: This work was supported by a Grant-in-Aid for Scientific Research (Grant No. 20H03146) provided by the Ministry of Education, Culture, Sports, Science and Technology of Japan. Competing interests: The authors declare that they have no competing interests. Data availability: The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. References Lazarus, J.V. et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4371202","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":305127551,"identity":"08fa00df-d88d-424a-9e2c-da68ad0add88","order_by":0,"name":"Emika Hara","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Emika","middleName":"","lastName":"Hara","suffix":""},{"id":305127552,"identity":"179ead61-0275-4f56-b739-9244420b0b0e","order_by":1,"name":"Kanami Ohshima","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Kanami","middleName":"","lastName":"Ohshima","suffix":""},{"id":305127553,"identity":"a70f282e-9848-4e85-bcbb-450242d5e737","order_by":2,"name":"Wen Zeng","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Zeng","suffix":""},{"id":305127554,"identity":"777d9e6c-787c-40fc-ad63-9da03004a6aa","order_by":3,"name":"Suzuka Uomoto","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Suzuka","middleName":"","lastName":"Uomoto","suffix":""},{"id":305127555,"identity":"64c34072-2a55-47c1-9bb6-6a977c820ea4","order_by":4,"name":"Mio Kobayashi","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Mio","middleName":"","lastName":"Kobayashi","suffix":""},{"id":305127556,"identity":"f65b268d-801c-46f5-b23f-b5b353cf7fc2","order_by":5,"name":"Makoto Shibutani","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Makoto","middleName":"","lastName":"Shibutani","suffix":""},{"id":305127557,"identity":"71d5bbb0-691d-435f-a423-a4cf45218991","order_by":6,"name":"Toshinori Yoshida","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIie2Rv2rDMBCHzxjcxVTraWoeQRBQyOJnUTG0S2YTCMSZlLFr8hYZM6po8CI8e1QoeOqSLWMOQyGFVO5YqD40/PTnQ7oTQCTyN8lAZVC/fVvDXyjJfkPRfB0eVWgkB3OrhBDNtve+KtJpY3161sUaHqyH+TGgODcTqi0z6V4EvOsSIafAXUDpFhk+6zSXXQ6kpPSwBQDXIeW1J6XG6W5QagT2OaYoSYoVAgfFIuDILdw5iaptFFItxrUN19gLE6rlkTrGL9VKsa09+WW1YoyVpw8e6NjE3EyGTN8Elm9+Vp7u7iXngBKJRCL/jivfDE6AjLVrVQAAAABJRU5ErkJggg==","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":true,"prefix":"","firstName":"Toshinori","middleName":"","lastName":"Yoshida","suffix":""}],"badges":[],"createdAt":"2024-05-05 10:03:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4371202/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4371202/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57006011,"identity":"49578771-265e-42ad-80bd-cfe3132b60ed","added_by":"auto","created_at":"2024-05-23 10:03:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1262481,"visible":true,"origin":"","legend":"\u003cp\u003eBody weight change, NAFLD activity score and quantitative analysis of preneoplastic lesions in rats. Rats were initially fed a basal diet (BD) or high fat diet (HFD) at the beginning of the study, with flutamide (FL) treatment initiated after 2 weeks. The rats were subjected to partial hepatectomy at 3 weeks and sacrificed at 13 weeks. (a) Body weight changes in the BD, BD+FL, HFD, and HFD+FL groups during the study. * p\u0026lt;0.05 compared to the BD group, # p\u0026lt;0.05 compared to the BD and HFD groups (Tukey’s or Steel–Dwass multiple comparison test). (b) Representative images illustrating steatosis in the HFD and HFD+FL groups. (c) NAFLD activity score (NAS). Scores for (d) steatosis, (e) ballooning, and (f) inflammatory foci. Quantitative analysis of the (g) number and (h) area of GST-P-positive foci. (i) Box plot depicting GST-P-positive foci larger than 0.001 mm\u003csup\u003e2\u003c/sup\u003e. (j) Box plot illustrating GST-P-positive foci larger than 0.1 mm\u003csup\u003e2\u003c/sup\u003e. (k) Representative images of GST-P foci. Different letters indicate significant intergroup differences (p \u0026lt; 0.05, Tukey's or Steel–Dwass test). Abbreviations: NAFLD = non-alcoholic fatty liver disease; BD = basal diet; FL = flutamide; GST-P = glutathione S-transferase placental form; HFD = high-fat diet.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4371202/v1/9119f251552bab0ae9a49f6f.png"},{"id":57006008,"identity":"92408891-07c3-46c9-a79d-93d935266855","added_by":"auto","created_at":"2024-05-23 10:03:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":231209,"visible":true,"origin":"","legend":"\u003cp\u003eScatterplot and clustering analysis of AMBRA1, Parkin, and LC3 expression in background hepatocytes (N = 96). Comparison of AMBRA1 and LC3 expression (%) (a–d) and of Parkin and LC3 (e–h) expression in the BD, BD+FL, HFD, and HFD+FL groups. Scatterplot (a, e), clustering analysis (b, f), percentage distribution of clusters (c, g), distribution of clusters (d, h) in the BD, BD+FL, HFD, and HFD+FL groups (N = 24 in each group). (b) Clustering analysis of AMBRA1 and LC3 was conducted using \u003cem\u003ek\u003c/em\u003e-means analysis (\u003cem\u003ek\u003c/em\u003e=3), and clusters C1, C2, and C3 were identified as representing basal level, mitophagy induction, and mitophagy inhibition, respectively, depending on the expression levels of each marker (see Supplemental Fig. 1). (f) Clustering analysis of Parkin and LC3 was conducted using \u003cem\u003ek\u003c/em\u003e-means analysis (\u003cem\u003ek\u003c/em\u003e=4), and clusters C1, C2, C3, and C4 were identified as representative of mitophagy inhibition, Parkin-independent mitophagy induction, basal level, and Parkin-dependent mitophagy induction, respectively, depending on expression levels of each marker (see Supplemental Fig 1). (d, h) Bar chart showing the percentage distribution of clusters in each group. Different letters indicate significant intergroup differences (p\u0026lt;0.05, significantly different by Tukey–Kramer test (d, h). Abbreviations: NAFLD = non-alcoholic fatty liver disease; BD = basal diet; FL = flutamide; HFD = high-fat diet.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4371202/v1/5bc029d905f56aaa66c1f5c3.png"},{"id":57006010,"identity":"2a0e6727-e613-43d3-9b2a-cc15734147f1","added_by":"auto","created_at":"2024-05-23 10:03:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":365135,"visible":true,"origin":"","legend":"\u003cp\u003eScatterplot and clustering analysis of individual GST-P-positive focus areas and AMBRA1, Parkin, and LC3 expression rates in hepatic preneoplastic lesions (N = 240). Comparison of the areas of individual GST-P-positive foci and AMBRA1 expression (%) (a–d), LC3 and AMBRA1expression (%) (e–h), and LC3 and Parkin expression (%) (i–l). Scatterplot (a, e, i), clustering analysis (b, f, j), percentage distribution of clusters (c, g, k), distribution of clusters (d, h, l) in the BD, BD+FL, HFD, and HFD+FL groups (N = 60 in each group). (b, f, j) Clustering analysis was conducted using \u003cem\u003ek\u003c/em\u003e-means analysis (k=3 or 4). (f) Clustering analysis of LC3 and AMBRA1 reveals that clusters C1, C2, and C3 represent basal level mitophagy, mitophagy induction, and mitophagy inhibition, respectively, depending on expression levels of each marker (see Supplemental Fig. 1). (j) Clustering analysis of LC3 and Parkin reveals that clusters C1, C2, and C3 represented basal level mitophagy, mitophagy inhibition, and Parkin-independent mitophagy induction, respectively, depending on expression levels of each marker (see Supplemental Fig. 1). (d, h, l) Bar chart showing the percentage distribution of clusters in each group. Different letters indicate significant intergroup differences (p\u0026lt;0.05, significantly different by Tukey–Kramer test (d, h, l). BD = basal diet, FL = flutamide, GST-P = glutathione S-transferase placental form, HFD = high-fat diet.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4371202/v1/184f27069a5366a65990caff.png"},{"id":71315561,"identity":"3916fea5-0424-40af-8859-96e7518ec238","added_by":"auto","created_at":"2024-12-13 08:39:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2622570,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4371202/v1/fef0e5a3-238d-4159-9d7c-9b1035b8d4d8.pdf"},{"id":57006012,"identity":"f4a16c87-3924-4a3a-b209-8f1dac5f5a52","added_by":"auto","created_at":"2024-05-23 10:03:20","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":5577237,"visible":true,"origin":"","legend":"","description":"","filename":"TSOD40HaraetalsTableFigs20231109submitted.docx","url":"https://assets-eu.researchsquare.com/files/rs-4371202/v1/44551962543b04ba973533ad.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Flutamide exacerbates steatosis and promotes early hepatocarcinogenesis in high-fat diet-fed non-obese steatotic rats: Insights from clustering analysis of mitophagy regulators AMBRA1 and LC3","fulltext":[{"header":"Highlights","content":"\u003cul start=\"12\"\u003e\n \u003cli\u003eClustering analysis of mitophagy markers AMBRA1 and LC3 elucidates the role of mitophagy in preneoplastic lesions.\u003c/li\u003e\n \u003cli\u003eA high-fat diet increases the\u0026nbsp;clusters with\u0026nbsp;high AMBRA1\u0026nbsp;but\u0026nbsp;low LC3 expression, indicating\u0026nbsp;suppressed mitophagy-induced steatosis elevated preneoplastic lesions.\u003c/li\u003e\n \u003cli\u003eThe mitochondrial toxin flutamide increases the number of clusters with low AMBRA1 and LC3 expression, exacerbating mitophagy-induced steatosis and marginally increasing preneoplastic hepatic lesions in high-fat diet-fed rats.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eNon-alcoholic fatty liver disease (NAFLD) is a severe liver disease, globally affecting approximately 25% of the population and imposing a significant health burden along with widespread social and economic consequences\u003csup\u003e1\u003c/sup\u003e. Metabolic dysfunction-related fatty liver disease (MAFLD) has been proposed\u0026nbsp;as a treatment for NAFLD,\u0026nbsp;because it is associated with metabolic abnormalities.\u0026nbsp;Increased fatty acid oxidation within the mitochondria plays a crucial role in preventing fat accumulation\u003csup\u003e2\u003c/sup\u003e; however, a chronic high-fat diet (HFD), excessive glucose intake, and genetic obesity lead to increased oxidation rates that exceed the reduction capacity of oxygen storage sites, resulting in increased ROS generation\u003csup\u003e3\u003c/sup\u003e. The ROS generated are not adequately removed by reducing antioxidants, such as manganese superoxide dismutase,\u0026nbsp;glutathione peroxidase, and reduced\u0026nbsp;glutathione, causing damage to mitochondrial DNA. Moreover, reduced hepatic ATP synthesis, lowered mitochondrial respiratory chain complex activity, and abnormal liver mitochondrial ultrastructure have all been reported in NAFLD patients\u003csup\u003e4-6\u003c/sup\u003e. Furthermore, mitochondrial dysfunction promotes ROS production and lipid peroxidation. It also promotes abnormal hepatic lipid homeostasis, insulin resistance, cell death, and cytokine release, thereby leading to progression from NAFLD to\u0026nbsp;nonalcoholic steatohepatitis (NASH)\u0026nbsp;and eventually cirrhosis\u003csup\u003e7\u003c/sup\u003e.\u0026nbsp;As some patients face the risk of developing hepatocellular carcinoma (HCC) with or without cirrhosis, effective strategies to combat this disease have gained paramount importance in recent years\u003csup\u003e8\u003c/sup\u003e.\u0026nbsp;Therefore, promoting the removal of dysfunctional mitochondria, which contribute to fatty liver development, may play a pivotal role in the treatment of NAFLD.\u003c/p\u003e\n\u003cp\u003eAutophagy is a catabolic process that eliminates unwanted intracellular materials through lysosomal degradation, thereby furnishing nutrients and energy for cellular homeostasis\u003csup\u003e9\u003c/sup\u003e. Cells often activate autophagy to cope with various stressors, such as nutrient scarcity, protein misfolding and aggregation, and intracellular organelle damage to intracellular. Activated autophagy serves to clear lipid droplets and abnormal organelles. Notably, it has been demonstrated to selectively remove damaged intracellular organelles\u003csup\u003e10\u003c/sup\u003e. This selective process, known as mitophagy, focuses on the removal of impaired mitochondria and has been extensively studied over the past decade. Numerous molecules associated with mitophagy regulation have been identified during this period\u003csup\u003e11\u003c/sup\u003e. The autophagy-mediated elimination of damaged mitochondria involves two steps: induction of general autophagy and priming of damaged mitochondria for selective autophagy. Recent advances in mitophagy research have revealed that mitochondrial priming is mediated by phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1)-Parkin-dependent or -independent mechanisms\u003csup\u003e11-13\u003c/sup\u003e. These targeted mitochondria are assimilated into the sequestration membrane by the microtubule-associated protein 1A/1B-light chain 3 (LC3) and mitophagy-related receptors, which bind to the sequestration membrane to form autophagosomes. Subsequently, these autophagosomes fuse with lysosomes to form autolysosomes, where damaged mitochondria are degraded. In this process, the autophagy and Beclin 1 regulator 1 (AMBRA1) is defined to assume a pivotal role as a mitophagy regulator involved in both Parkin-dependent and-independent pathways\u003csup\u003e14\u003c/sup\u003e. In the Parkin-dependent mitophagy pathway, activated Parkin interacts with AMBRA1 expressed on the outer mitochondrial membrane, which, in turn, promotes mitophagy by activating the class III phosphatidylinositol 3-kinase (PI3K) complex, an essential contributor to autophagosome formation\u003csup\u003e15,16\u003c/sup\u003e. Additionally, AMBRA1 directly binds to LC3, further driving the progression of autophagosome formation\u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAccumulating evidence suggests that the levels of mitophagy regulators decrease with the progression of steatosis, signifying the involvement of inhibited mitophagy in the pathogenesis of NAFLD. In a murine model of diet-induced NAFLD, hepatocyte autophagy was arrested, leading to mitochondrial dysfunction\u003csup\u003e18\u003c/sup\u003e. In the context of diet-induced NAFLD in mice, the progression of mitophagy was suppressed, evident in the accumulation of megamitochondria containing mitophagy intermediates such as p62/SQSTM1 and ubiquitin\u003csup\u003e19\u003c/sup\u003e. In primary hepatocytes from mice treated with palmitic acid to mimic high-fat stress, the expression levels of LC3 and Parkin in the mitochondria were notably reduced, indicating that mitophagy is inactivated in a Parkin-dependent manner under high-fat stress\u003csup\u003e20\u003c/sup\u003e. PINK1-Parkin-dependent mitophagy is inhibited in the livers of NAFLD mouse models generated by high-fat feeding as well as in patients with NAFLD\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFlutamide (FL) is a non-steroidal antiandrogenic and anticancer drug widely used to treat prostate cancer and recognized as an endocrine-disrupting environmental contaminant\u003csup\u003e22-24\u003c/sup\u003e. Moreover, FL inhibits mitochondrial respiratory chain complexes I, II, and V and is, therefore, known as a mitochondrial toxicant\u003csup\u003e25,26\u003c/sup\u003e. In a previous study conducted in our laboratory, FL was observed to enhance precancerous lesions in a medium-term liver carcinogenesis rat model; however, the underlying mechanism remains unclear\u003csup\u003e27\u003c/sup\u003e. In this study, we hypothesized that FL induces mitochondrial injury\u0026nbsp;followed by mitophagy in an HFD-mediated early hepatocarcinogenesis model\u003csup\u003e28-31\u003c/sup\u003e. This model, based on the medium-term hepatocarcinogenesis assay\u003csup\u003e32\u003c/sup\u003e, served as a valuable tool for understanding the augmentation of autophagy in precancerous lesions facilitated by fatty liver conditions\u003csup\u003e28-31\u003c/sup\u003e. This model is particularly advantageous since hepatic precancerous lesions specifically express the autophagosome marker LC3 and the cargo receptor p62\u003csup\u003e29, 31, 33\u003c/sup\u003e. Using immunohistochemical techniques and cluster analysis using autophagy markers, we investigated the role of autophagy in small precancerous lesions in rat livers\u003csup\u003e34\u003c/sup\u003e. Further, we investigated the role of mitophagy in NAFLD-related hepatocarcinogenesis by clustering hepatic preneoplastic lesions according to the expression ratios of the autophagy marker LC3 and mitophagy indicators AMBRA1 and Parkin.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eFlutamide inhibits body weight growth, increases HFD-induced NAFLD score, and enhances preneoplastic hepatic lesions\u003c/h2\u003e \u003cp\u003eDuring the study period, FL suppressed body weight gain after 6 weeks, regardless of the diet type (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). No discernible difference was observed in terms of food and water intake (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplemental Tables\u0026nbsp;3 and 4). In the final autopsy, FL significantly reduced the final body weight and intra-abdominal fat weight, independent of the HFD (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Importantly, HFD-induced obesity was not observed under these experimental conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e Final body weight, organ weight, food intake, and water intake in rats\u0026sect;\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBD\u0026thinsp;+\u0026thinsp;FL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHFD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHFD\u0026thinsp;+\u0026thinsp;FL\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of animals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinal body weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e301.5\u0026thinsp;\u0026plusmn;\u0026thinsp;15.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e273.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.96\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e293.4\u0026thinsp;\u0026plusmn;\u0026thinsp;15.13\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e271.8\u0026thinsp;\u0026plusmn;\u0026thinsp;12.05\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFood intake (g/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.1\u0026thinsp;\u0026plusmn;\u0026thinsp;16.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.0\u0026thinsp;\u0026plusmn;\u0026thinsp;15.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.1\u0026thinsp;\u0026plusmn;\u0026thinsp;15.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e33.3\u0026thinsp;\u0026plusmn;\u0026thinsp;13.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater intake (g/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.6\u0026thinsp;\u0026plusmn;\u0026thinsp;19.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.4\u0026thinsp;\u0026plusmn;\u0026thinsp;17.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53.8\u0026thinsp;\u0026plusmn;\u0026thinsp;17.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51.3\u0026thinsp;\u0026plusmn;\u0026thinsp;16.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsolute liver weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRelative liver weight (%BW)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsolute intraperitoneal fat weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRelative intraperitoneal fat weight (%BW)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eAbbreviations: BD, basal diet; HFD, high fat diet; FL, flutamide, BW; body weight.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e\u0026sect;\u003c/sup\u003e: All animals were subjected to two-thirds partial hepatectomy at week 3.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eData are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eDifferent letters indicate significant differences between groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, significantly different by Tukey's or Steel-Dwass test).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have shown that an HFD increases NAS and steatosis scores in the liver\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Similar results were obtained in the present study, where FL further increased these scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Notably, the scores associated with ballooning degeneration and inflammatory foci increased in response to an HFD but remained unaffected by FL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). FL caused a significant increase in relative liver weight in the BD\u0026thinsp;+\u0026thinsp;FL group, which might have been caused by CYP induction\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, as is evident from the \u003cem\u003eCyp1a\u003c/em\u003e gene expression data presented below (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, no statistically significant change in relative liver weight was observed in the HFD\u0026thinsp;+\u0026thinsp;FL group (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGene expression analysis of autophagy, mitochondria/mitophagy, lipid metabolism, drug metabolism, inflammation, and oxidative stress in liver samples.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBD\u0026thinsp;+\u0026thinsp;FL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHFD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHFD\u0026thinsp;+\u0026thinsp;FL\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of animals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAutophagy-related genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAtg3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAtg5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAtg7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLamp1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLamp2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLc3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ep62\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMitochondria/Mitophagy-related genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmbta1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.95\u0026thinsp;\u0026plusmn;\u0026thinsp;3.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.64\u0026thinsp;\u0026plusmn;\u0026thinsp;5.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eParkin\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePink1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBnip3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eNadh\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSdhd\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eATP synthase\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLipid metabolism-related genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAbca1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAcox1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eApob\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eDgat2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eFasn\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHmgcs1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHsd3b1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLpl\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLss\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePpara\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePparg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eScd1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSrebf1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSrebf2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrug metabolism-related genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCyp1a1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e73.20\u0026thinsp;\u0026plusmn;\u0026thinsp;45.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e96.14\u0026thinsp;\u0026plusmn;\u0026thinsp;93.51\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCyp2b1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCyp3a1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInflammation related genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTnf-a\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxidative stress-related genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCatalase\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eGpx1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eGpx2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.86\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eMn-SOD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSod1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSod2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eAbbreviations: BD, basal diet; HFD, high-fat diet; FL, flutamide.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u0026sect;: All animals were subjected to two-thirds partial hepatectomy at week 3.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eData are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eDifferent letters indicate significant differences between groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, significantly different by Tukey's or Steel-Dwass test).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe consumption of HFD induced a significant increase in the number and area of GST-P-positive foci (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek), as previously reported\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. A previous study showed that that higher FL doses (1000 and 2000 ppm) enhanced hepatocarcinogenesis in rats\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e; however, in the present study, a lower FL dose (500 ppm) caused a marginal increase in both the number and area of GST-P-positive foci (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej). The area of GST-P-positive foci was significantly increased in the HFD\u0026thinsp;+\u0026thinsp;FL group compared to the BD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej); however, the cumulative effects of HFD and FL on the area of GST-P-positive foci were marginal. The number of Ki-67-positive cells within hepatic precancerous lesions showed an increasing trend in the FL-treated groups (BD\u0026thinsp;+\u0026thinsp;FL and HFD\u0026thinsp;+\u0026thinsp;FL); however, no statistically significant change was detected (Supplemental Table\u0026nbsp;4). These findings suggest that FL exacerbated steatosis and marginally increased preneoplastic lesions in a rat model of non-obesity-related steatosis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExpression analysis of mitophagy markers in background hepatocytes and precancerous lesions\u003c/h3\u003e\n\u003cp\u003eFL exhibits mitochondrial toxicity\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, while HFD inhibits mitophagy\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In light of these insights, we assessed the expression of the autophagy marker LC3 and the mitophagy indicator AMBRA1 in both background hepatocytes and precancerous lesions of rats treated with HFD and/or FL. LC3 and AMBRA1 showed positive granular signals in hepatocytes (Supplemental Figs.\u0026nbsp;2 and 3). Quantitative analysis showed that LC3-positive granules exhibited an increasing trend in the FL-treated groups (BD\u0026thinsp;+\u0026thinsp;FL and HFD\u0026thinsp;+\u0026thinsp;FL groups) compared to the non-FL-treated groups (BD and HFD groups) (Supplemental Fig.\u0026nbsp;2b, d), both in background hepatocytes (non-foci) and precancerous lesions (foci). Similarly, AMBRA1-positive granules tended to increase in number in the HFD group compared to the BD group. However, when combined with FL, a decreasing trend was observed, particularly in the BD\u0026thinsp;+\u0026thinsp;FL group, in both background hepatocytes (non-foci) and precancerous lesions (foci) (Supplemental Fig.\u0026nbsp;3b, 3d). These results indicate a potential suppression of mitophagy in the HFD group, as indicated by the increased expression of AMBRA1. Conversely, in combination with the FL treatment, mitophagy might be induced by the suppression of increased AMBRA1 expression; however, these effects on mitophagy were found to be marginal in this standard analysis.\u003c/p\u003e \u003cp\u003eNo statistically significant differences in Parkin expression were observed in either background hepatocytes or preneoplastic lesions in each group (Supplemental Fig.\u0026nbsp;4a-d).\u003c/p\u003e\n\u003ch3\u003eCluster analysis of LC3, AMBRA1, and Parkin expression in background hepatocytes\u003c/h3\u003e\n\u003cp\u003eTo further investigate the relationship between LC3 and AMBRA1 expression, we compared the expression ratios of both indicators in background hepatocytes using cluster analysis, as previously reported\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, aiming to elucidate the dynamics of autophagy flux (Supplemental Fig.\u0026nbsp;1). Using the \u003cem\u003ek\u003c/em\u003e-means, the clusters were divided into three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c) and examined the cluster composition of each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). These clusters were classified into three levels: C1, characterized by low expression of both LC3 and AMBRA1, indicative of a basal level; C2, characterized by low AMBRA1 expression and high LC3 expression, suggestive of mitophagy induction; and C3, characterized by low LC3 expression and high AMBRA1 expression, implying mitophagy inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In the BD group, the major constitutive cluster was C1, followed by C3, and lastly C2 in a descending order (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). This pattern closely resembled that of the BD\u0026thinsp;+\u0026thinsp;FL group, where the primary constitutive cluster remained C1, similar to the BD group. However, the percentage of C2 clusters in the BD\u0026thinsp;+\u0026thinsp;FL group increased compared to the BD group, although this difference was not statistically significant. In contrast, in the HFD group, the prevalence of C3 composition significantly increased. In the HFD\u0026thinsp;+\u0026thinsp;FL group, the cluster composition was similar to that observed in the BD\u0026thinsp;+\u0026thinsp;FL group, with a significant increase in the rate of C2 composition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe analyzed LC3 and Parkin expression to evaluate whether mitophagy induction was Parkin-dependent in the FL-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). The clusters were classified into four levels: C1, characterized by low LC3 expression and high Parkin expression, implying mitophagy inhibition; C2, with low Parkin expression and high LC3 expression, suggestive of Parkin-independent mitophagy induction; C3, a basal cluster with low expression of both LC3 and Parkin; and C4, featuring high expression of both LC3 and Parkin, indicative of Parkin-dependent mitophagy induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). In the BD\u0026thinsp;+\u0026thinsp;FL group, the primary cluster was C3, while in the HFD\u0026thinsp;+\u0026thinsp;FL group, the C4 composition was significantly augmented (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh), suggesting that FL induced mitophagy in a Parkin-dependent manner in a population of preneoplastic lesions in HFD-fed rats.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCluster analysis of the GST-P-positive foci area and LC3, AMBRA1, and Parkin expression in hepatic precancerous lesions\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine whether AMBRA1 expression played a role in the development of hepatic preneoplastic lesions, we examined the relationship between the area of individual GST-P-positive foci and the expression rate of AMBRA1 in each lesion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d). Subsequently, we classified cluster into four levels, namely C1 to C4, and observed that C4 exhibited the highest AMBRA1 expression (mitophagy inhibition cluster) among all four levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The percentage of C4 composition significantly increased in the HFD group, and this effect was attenuated when combined with FL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), suggesting that FL might cancel mitophagy inhibition in HFD-fed rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe investigated the relationship between LC3 and AMBRA1 expression in hepatic precancerous lesions, which were divided into three clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh), and examined the cluster composition of each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). These clusters were classified into three levels, namely C1\u0026ndash;C3: C1 represented the basal level with low expression of both LC3 and AMBRA1; C2 indicated mitophagy induction with low expression of AMBRA1 and high expression of LC3; and C3 denoted mitophagy inhibition with high expression of AMBRA1 and low expression of LC3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), similar to the categorization in background hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The percentage of C3 cells was significantly higher in the HFD group than in the BD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). The composition of clusters in the HFD\u0026thinsp;+\u0026thinsp;FL group was similar to that in the BD\u0026thinsp;+\u0026thinsp;FL group. The results suggested that FL might cancel mitophagy inhibition in HFD-fed rats as well.\u003c/p\u003e \u003cp\u003eFinally, we analyzed LC3 and Parkin expression in hepatic precancerous lesions to evaluate whether mitophagy induction was Parkin-dependent in the FL-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el). The clusters were classified into three levels: C1 represented the basal level with low expression of both LC3 and Parkin; C2 indicated a cluster with low LC3 expression and high Parkin expression, indicative of mitophagy inhibition; and C3 represented a level with high LC3 expression and low Parkin expression, suggestive of Parkin-independent mitophagy induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). Unlike in background hepatocytes, we did not detect C4, a Parkin-dependent mitophagy inhibition cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). C3 was minimally detected in the FL-treated groups BD\u0026thinsp;+\u0026thinsp;FL and HFD\u0026thinsp;+\u0026thinsp;FL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el).\u003c/p\u003e\n\u003ch3\u003eHFD and HFD combined with FL alters hepatic gene expression\u003c/h3\u003e\n\u003cp\u003eA comprehensive gene expression analysis was conducted on the liver samples to determine the contributions of autophagy, mitophagy, injured mitochondria, lipid metabolism, inflammation, drug-metabolizing enzymes, and oxidative stress-related genes, which might be related to NAFLD-related liver carcinogenesis.\u003c/p\u003e \u003cp\u003eWe analyzed autophagy-related genes and showed that the expression of \u003cem\u003eAtg5, Atg7, Lamp1, Lamp2\u003c/em\u003e, and \u003cem\u003eLc3\u003c/em\u003e was significantly increased in the HFD group compared to the BD group. However, when HFD feeding was combined with FL administration, a significant decrease was observed in the expression of these genes (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These gene expression changes in the HFD group were considered a compensatory response to the suppression of mitophagy, as discussed earlier. Regarding mitophagy-related genes, \u003cem\u003eParkin\u003c/em\u003e expression was significantly elevated in the HFD\u0026thinsp;+\u0026thinsp;FL group compared to the BD group, along with higher levels of \u003cem\u003eAmbra1\u003c/em\u003e expression. Furthermore, a relatively higher increase in the expression of \u003cem\u003erkin\u003c/em\u003e and \u003cem\u003eAmbra1\u003c/em\u003e was observed in the BD\u0026thinsp;+\u0026thinsp;FL and/or HFD groups. This could potentially indicate the presence of Parkin-dependent mitophagy in background hepatocytes, aligning with the observations discussed earlier.\u003c/p\u003e \u003cp\u003eFL, a known inhibitor of the mitochondrial respiratory chain complex I, has been reported to impair mitochondrial functions \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Moreover, 2-hydroxyflutamide, a major FL metabolite, impairs the functions of mitochondrial respiratory chain complexes II and V\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In this study, we analyzed the gene expression of complex I (\u003cem\u003eNADH\u003c/em\u003e: quinone oxidoreductase), complex II (SDHD: succinate dehydrogenase), and complex V (\u003cem\u003eATP synthase\u003c/em\u003e). We found that the expression level of \u003cem\u003eSdhd\u003c/em\u003e tended to increase in the BD\u0026thinsp;+\u0026thinsp;FL group and was significantly elevated in the HFD group compared to the BD group. However, this expression trend reversed with the context of combined HFD feeding and FL administration, indicating a decreasing pattern (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Although no evident differences in the expression of \u003cem\u003eNadh\u003c/em\u003e and \u003cem\u003eATP synthase\u003c/em\u003e were observed between the groups, the expression of \u003cem\u003eNadh\u003c/em\u003e tended to increase in the HFD group; however, it remained comparable to that in the BD group. It is important to note that FL induces significant upregulation of \u003cem\u003eCyp1a1\u003c/em\u003e expression in rats\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In our study, the expression of \u003cem\u003eCyp1a1 was\u003c/em\u003e significantly elevated upon FL administration, regardless of the presence of HFD.\u003c/p\u003e \u003cp\u003eRegarding the expression levels of genes related to lipid metabolism, \u003cem\u003eAbca1\u003c/em\u003e, which is responsible for the production of high-density lipoproteins, and \u003cem\u003eSrebf2\u003c/em\u003e, which is involved in cholesterol metabolism, were significantly increased in the HFD groups (HFD and HFD\u0026thinsp;+\u0026thinsp;FL) compared to the BD group (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, the TG synthase gene \u003cem\u003eDgat2\u003c/em\u003e, steroid hormone synthase \u003cem\u003eHsd3b1\u003c/em\u003e, and transcription factor \u003cem\u003ePpara\u003c/em\u003e, which promotes β-oxidation, were significantly increased in the HFD group compared to the BD group, while they either showed a declining trend or a significant decrease in the HFD\u0026thinsp;+\u0026thinsp;FL group.\u003c/p\u003e \u003cp\u003eNo obvious differences in the expression of \u003cem\u003eTnf-α\u003c/em\u003e, one of the most effective cytokines, between the groups (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding oxidative stress gene expression, the antioxidant enzyme \u003cem\u003eCatalase\u003c/em\u003e exhibited a significant increase in the HFD group compared to the BD group. Furthermore, the expression level of \u003cem\u003eGpx2\u003c/em\u003e significantly increased in the FL alone group and in the combination of HFD feeding and FL administration (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Thus, in the HFD group, antioxidant enzyme-related genes exhibited increased expression parallel to steatosis. However, the combined effect of HFD feeding and FL administration on gene expression was not detected.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, the global incidence of NAFLD, also known as MAFLD, has been on the rise, and a subset of patients has been shown to develop liver cancer\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Because mitochondria are intricately involved in both ROS production and lipid metabolism, dysfunctional mitochondria have been implicated in the pathogenesis of NAFLD\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. While healthy individuals effectively clear out abnormal mitochondria through mitophagy, this regulatory mechanism appears to be impaired in patients with NAFLD, potentially contributing to the persistence of abnormal mitochondria and excessive ROS generation, which in turn may exacerbate the fatty liver condition\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, the precise role of mitophagy in NAFLD-related hepatocarcinogenesis remains unclear. To examine the role of mitophagy in the present non-obese rat model of NAFLD-related hepatocarcinogenesis, we chose to utilize FL as a mitochondrial toxicant. This is because of FL\u0026rsquo;s dual nature as an inhibitor of mitochondrial complexes I, II, and V\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, as well as its ability to promote hepatic carcinogenesis in a medium-term hepatic carcinogenesis assay\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In the present study, the effects of 500 ppm FL in inducing mitochondrial toxicity and hepatocarcinogenesis were found to be marginal, as evidenced by limited alterations in mitochondria/mitophagy-related gene expression (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and a gradual increase in the area of hepatic precancerous lesions compared to the BD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej). Consistent with a previous study involving 1000 or 2000 ppm FL within a medium-term liver initiation/promotion model\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, we observed a noticeable expression of \u003cem\u003eCyp1a1\u003c/em\u003e in the FL-treated liver (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e); however, CYP-mediated oxidative stress was not involved in hepatocarcinogenesis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In this study, a significant increase in \u003cem\u003eGpx2\u003c/em\u003e gene expression in the BD\u0026thinsp;+\u0026thinsp;FL group suggested that FL induced an oxidative stress response; however, the effect remained limited, as other antioxidant genes were largely unaffected. Under our study conditions, we assessed mitophagy flux using immunohistochemistry-based clustering analysis involving mitophagy markers in background hepatocytes and preneoplastic lesions, following a previously reported methodology\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e to understand the role of mitophagy in FL and HFD-mediated hepatocarcinogenesis.\u003c/p\u003e \u003cp\u003eTo understand the dynamics of mitophagy flux, it becomes essential to confirm the progression of mitophagy and identify various autophagy markers. In selective autophagy, the initial reaction involves the formation of a sequestration membrane that surrounds the cargo, ultimately giving rise to autophagosomes\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Subsequently, lysosomes merge with these autophagosomes to form autolysosomes, where lysosome-derived enzymes degrade the cargo, and the degraded products are reused within cells. This sequence of events constitutes the autophagic flux (Supplemental Fig.\u0026nbsp;1). To understand alterations in the expression of autophagosomes and cargo receptors, it is imperative to consider this autophagic flux, thereby ensuring a meticulous and informed interpretation of the outcomes \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In general, LC3 is expressed both inside and outside sequestration membranes and autophagosomes, making it a widely used marker for examining autophagy involvement\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In our study, LC3 was observed as granules in hepatocytes, suggesting that the accurate detection of autophagosomes (Supplemental Fig.\u0026nbsp;1c, 2a, 2c). LC3 includes two isoforms: LC3-I, which is ubiquitously expressed in autophagosomes, and LC3-II, whose elevation signifies increased autophagy. It is noteworthy that the anti-LC3 antibody used in our study does not discriminate between LC3-I and LC3-II. Therefore, when analyzing autophagic flux, it becomes essential to observe specific molecules expressed on cargo receptors and injured intracellular organelles that are taken up by autophagosomes. Escalation in the expression of cargo receptors and injured organelle markers indicates inhibition of autophagy\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In this study, tour approach involved analyzing mitophagy flux by combining LC3 with the mitophagy regulatory factors AMBRA1 and Parkin, which are expressed on the membranes of impaired mitochondria (Supplemental Fig.\u0026nbsp;1). Initially identified as a causative gene for Parkinson's disease\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, Parkin plays a role beyond neurological conditions to encompass liver diseases as well\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In HFD-fed mice, an increased expression of the macrophage stimulating 1 (\u003cem\u003eMst1\u003c/em\u003e) gene, a key mitophagy regulator, suppressed Parkin-dependent mitophagy and worsened steatosis. In contrast, Mst1 knockout mice exhibited a lifting of the suppression of Parkin-dependent mitophagy, leading to an amelioration of steatosis\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. These data were further validated \u003cem\u003ein vitro\u003c/em\u003e, where palmitate-treated hepatocytes showed a reduction in Parkin expression. The analysis of the dynamics of mitophagy flux using LC3, AMBRA1, and Parkin provided new insights into the relationship between Parkin-dependent or -independent mitophagy in an early HFD-mediated hepatocarcinogenesis.\u003c/p\u003e \u003cp\u003eIn the livers of patients with NAFLD, the suppression of autophagy has been linked to increased accumulation of fat droplets, as determined by analyzing the expression of the autophagosome markers LC3-I/LC3-II and cargo receptor p62\u003csup\u003e44, 45\u003c/sup\u003e. These findings are recapitulated in \u003cem\u003ein vitro\u003c/em\u003e models stimulating NAFLD liver conditions. In studies employing hepatocytes and hepatocellular carcinoma cells, the inhibition of mitophagy has been shown to increase fat droplet deposition, as indicated by the altered expression patterns of mitophagy indicators such as AMBRA1, Parkin, and PINK1\u003csup\u003e20, 21\u003c/sup\u003e. In the present study, although no distinct differences were observed in the standard analysis of LC3-, AMBRA-, and Parkin-positive granules in background hepatocytes (Supplemental Fig.\u0026nbsp;2\u0026ndash;4), our clustering analysis revealed that HFD increased the proportion of mitophagy inhibition clusters with high AMBRA1 expression (C3 in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The higher expression of \u003cem\u003eAtg5, Atg7, Lamp1, Lamp2\u003c/em\u003e, and \u003cem\u003eLc3\u003c/em\u003e in the HFD group (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) could potentially reflect an adaptive response to the processing of excess fat droplet load caused by mitophagy inhibition. In contrast, the HFD\u0026thinsp;+\u0026thinsp;FL group showed an increased proportion of clusters indicative of mitophagy induction (C2 in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), a response that was dependent on Parkin (C4 in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Remarkably, the increased expression of \u003cem\u003eAtg5, Atg7, Lamp1, Lamp2\u003c/em\u003e, and \u003cem\u003eLc3\u003c/em\u003e in the HFD་FL group appeared to be somewhat obscured (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), suggesting that FL-induced mitophagy was concurrently suppressing gene expression. Furthermore, the HFD\u0026thinsp;+\u0026thinsp;FL group showed a more pronounced increase in steatosis score than the HFD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). In drug-induced NAFLD, it has been shown that the drugs contribute to the development of steatosis by inhibiting mitochondrial β-oxidation\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Therefore, it is plausible that the HFD-mediated steatosis observed in the HFD\u0026thinsp;+\u0026thinsp;FL group may have been further exacerbated by FL-mediated inhibition of β-oxidation, as FL has been reported to downregulate the expression of genes associated with mitochondrial β-oxidation\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Consistent with this, FL suppressed the HFD-induced increase in the expression of \u003cem\u003ePpara\u003c/em\u003e, a transcription factor known to induce the expression of enzymes involved in HFD-induced β-oxidation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAMBRA1, expressed on the membranes of damaged mitochondria, plays a vital role in inducing mitophagy\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Moreover, it has been reported that AMBRA1 is explicitly expressed in tumorigenic lesions in a two-stage rat liver carcinogenesis model\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. The escalation of hepatic precancerous lesions observed in the HFD group was concurrent with the increased proportion of clusters showing high levels of AMBRA1 (C4 in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) and mitophagy inhibition (C3 in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). However, these effects of HFD were counteracted upon co-administration with FL. Because the cluster showing Parkin-independent mitophagy induction was marginal in both the BD\u0026thinsp;+\u0026thinsp;FL and HFD\u0026thinsp;+\u0026thinsp;FL groups (C3 in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el), we could not definitively conclude that FL-induced mitophagy was Parkin-independent in hepatic preneoplastic lesions in the present study. Mitophagy generally suppresses carcinogenesis in the early stages, when normal cells transition into transformed cells\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In contrast, during late carcinogenesis, when transformed cells progress towards further malignant transformation and metastasis, mitophagy enhances carcinogenesis by eliminating damaged mitochondria, preventing ROS production, and enhancing mitochondrial energy production\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In this study, we observed precancerous lesions, which represent the early stage of carcinogenic progression; therefore, it is possible that mitophagy inhibits the development of precancerous lesions, and that the HFD-induced inhibition of mitophagy increases the formation of hepatic precancerous lesions. This hypothesis is supported by the increased expression of LC3 and p62 that were previously observed in hepatic precancerous lesions\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Despite the marginal cumulative effects of FL and HFD on the generation of hepatic precancerous lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej), it is conceivable that FL-induced mitophagy could be involved in the maintenance of certain populations of precancerous lesions in this group. These results suggest that mitophagy induction might lead to a phenomenon similar to further malignant transformation of tumor cells. This result is consistent with an increase in the formation of tumorigenic lesions when the autophagy inducer amiodarone was applied to a two-stage rat liver carcinogenesis model\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In addition to regulating mitophagy, \u003cem\u003eAmbra1\u003c/em\u003e acts as a tumor suppressor gene \u003cem\u003ein vivo\u003c/em\u003e; it inhibits cell cycle progression into the G1-S phase by promoting the degradation of cyclin D\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Additionally, it interacts with protein phosphatase 2A (PP2A), a cell cycle regulator, independently of autophagy regulation, and modulates cell growth and tumorigenesis by promoting the PP2A-mediated dephosphorylation of the oncogene \u003cem\u003eC-MYC\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Further evidence supporting its tumor suppressor function emerges from studies in which primary mouse embryonic fibroblasts from wild-type (AMBRA1 +/+) and deficient (AMBRA1 gt/gt) AMBRA1 loci were subcutaneously injected into mice, resulting in earlier tumor formation in AMBRA1-deficient mice than in wild-type counterparts. This underscores the role of AMBRA1 as a tumor suppressor gene. Notably, mutations in \u003cem\u003eAmbra1\u003c/em\u003e have also been identified in various human tumors. In the HFD\u0026thinsp;+\u0026thinsp;FL group, the downregulation of AMBRA1 may potentially contribute to the formation of precancerous lesions (C3 in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eIn conclusion, we found that mitophagy was suppressed in the HFD group and that this suppression was reversed by FL administration, which led to enhanced precancerous lesion formation. Similar findings were observed in the analysis of background hepatocytes other than the precancerous lesions. While current research is focused on developing drugs that induce mitophagy for the treatment of NAFLD and cancer\u003csup\u003e\u003cspan additionalcitationids=\"CR53 CR54\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, it is crucial to consider the potential risk of mitophagy inducers aggravating NAFLD. Analyzing mitophagy both inside and outside precancerous lesions can provide a clear understanding of its pathological significance in NAFLD models. As shown in the present study, employing cluster analysis using the autophagy marker LC3 and mitophagy indicators AMBRA1 and Parkin proves to be a valuable approach for studying mitophagy. This methodology can also be extended to the analysis of autophagy flux in preneoplastic lesions and micro-cancers, enabling a deeper exploration of the roles of mitophagy in malignant transformation and the development of novel therapeutic strategies.\u003c/p\u003e"},{"header":"Methods","content":" \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eN-Nitrosodiethylamine (DEN; CAS No. 55-18-5, purity\u0026thinsp;\u0026gt;\u0026thinsp;99%) and flutamide (FL; CAS No. 13311-84-7, purity\u0026thinsp;\u0026gt;\u0026thinsp;98%) were purchased from Tokyo Kasei Kogyo (Tokyo, Japan) and Combi-Blocks (San Diego, CA, USA), respectively.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and treatments\u003c/h2\u003e \u003cp\u003eA total of 30 5-week-old male F344/DuCrlCrlj rats were purchased from Charles River Laboratories Japan (Kanagawa, Japan) and reared under the following conditions: temperature 23\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C, humidity 50\u0026thinsp;\u0026plusmn;\u0026thinsp;20%, and 12 h light/12 h dark lighting cycle. The rats were fed a powdered diet (MF; Oriental Yeast Co., Japan) as the basal diet (BD) and provided with drinking water in a clean rack equipped with rat paper-type enrichment, with a maximum of three animals per cage. In a previous study, rats were treated with 1000 or 2000 ppm doses of FL, resulting in final body weights of 85% and 81%, respectively, at the end of the study (8 weeks), compared to the control group\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Preliminary analyses were conducted on six animals, wherein FL was administered at three different doses (250, 500, and 1000 ppm) over a span of two weeks. Based on evaluations of body and liver weights, the FL dose of 500 ppm was selected for the present study. Six days before the start of the study, 12 rats were fed a HFD (D12451, 45 kcal%; Research Diets Inc., New Brunswick, NJ, USA), while the remaining rats were provided with a BD. The study was conducted as previously reported\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, according to an initiation and promotion model\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The rats were divided into four groups: BD-fed (BD, n\u0026thinsp;=\u0026thinsp;6), BD-fed and FL-administered (BD\u0026thinsp;+\u0026thinsp;FL, n\u0026thinsp;=\u0026thinsp;6), HFD-fed (HFD, n\u0026thinsp;=\u0026thinsp;6), and HFD-fed and FL-administered (HFD\u0026thinsp;+\u0026thinsp;FL, n\u0026thinsp;=\u0026thinsp;6). Throughout the study period, daily observations were performed to assess the general conditions of the animals. Additionally, the body weight and food and water intake measurements were performed weekly. At 13 weeks after study initiation, all animals were subjected to laparotomies under isoflurane anesthesia and humane euthanization through blood release. Subsequently, the liver and intraperitoneal fat were collected. The livers were fixed in 4% paraformaldehyde (PFA) for histopathological examination and immunohistochemical staining. A portion of the liver was frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. The experimental plan involving animals was approved and reviewed by the Laboratory Animal Committee of Tokyo University of Agriculture and Technology (No. R03-149) prior to the commencement of the experiments. All animals were handled in accordance with the Guidelines for Animal Experimentation issued by the Japanese Association for Laboratory Animal Science (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.jalas.jp/english/en_about.html\u003c/span\u003e\u003cspan address=\"https://www.jalas.jp/english/en_about.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The study was conducted in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistopathology\u003c/h3\u003e\n\u003cp\u003eFollowing fixation in 4% PFA, liver specimens were subjected to thin sectioning to a thickness of approximately 3 \u0026micro;m. These sections were subsequently stained using hematoxylin and eosin (H\u0026amp;E) staining. In the liver tissue, we evaluated the NAFLD activity score (NAS), encompassing assessments from steatosis, ballooning, and inflammatory foci\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry and clustering analysis\u003c/h2\u003e \u003cp\u003eThe PFA-fixed liver specimens were thinly sliced and subjected to immunohistochemistry for targeting specific markers, including glutathione S-transferase placental form (GST-P), which is positive for rat liver proliferative lesions; Ki-67, a marker of cell proliferative activity; LC3, an autophagy marker; and AMBRA1 and Parkin, both pivotal regulators of mitophagy. Detailed information about the antibodies used, antigen activation methods, and antibody dilution ratios is presented in (Supplemental Table\u0026nbsp;1). Signal detection was performed according to the protocol of VECTASTAIN\u0026reg; Elite ABC Kit (Vector Laboratories Inc, Burlingame, CA, USA), with subsequent immunoreaction carried out using 3,3\u0026prime;-diaminobenzidine/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. After visualization, the cells were counterstained with hematoxylin. The number and area of the GST-P-positive foci were measured as previously described\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The positive rates of LC3, AMBRA1, and Parkin within each GST-P-positive focus were measured using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). The number of Ki-67-positive cells per GST-P-positive focus was defined as the labeling index (%)\u003csup\u003e31\u003c/sup\u003e. The GST-P-positive foci and the positivity rates for AMBRA1, LC3, and Parkin were collated for each individual and analyzed in groups, rather than individually, as previously reported\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. For further analysis, the positive rates of LC3, AMBRA1, or Pink in GST-P-positive foci were plotted on the x- and y-axes, respectively. Subsequently, clustering was performed for analyzing each cluster using the \u003cem\u003ek\u003c/em\u003e-means method. The positive rates of LC3, AMBRA1, and Parkin in background hepatocytes were also analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eReal-time reverse transcription-polymerase chain reaction analysis\u003c/h2\u003e \u003cp\u003eExpression analysis at the mRNA level was performed using the genes and primers listed in (Supplemental Table\u0026nbsp;2). RT-PCR was performed as described previously\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eMeans and standard deviations were calculated for all data. Statistical analyses were performed using either the Tukey or Steel-Dwass test. A significance level of 5% or less indicated a significant difference. A test of mother proportions (Tukey\u0026ndash;Kramer test) was performed to determine the proportion of clusters in each group.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments: We thank Editage (www.editage.com) for\u0026nbsp;the English language editing.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions: EH and TY conceived the project, designed and conducted\u0026nbsp;the animal experiments, analyzed and interpreted the data, and wrote the manuscript. EH, KO, WZ, SU, and MK performed the animal experiments and immunohistochemistry analysis. MS initiated the study and interpreted the results. All\u0026nbsp;the authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eEthics approval:\u0026nbsp;The clinical, operative, and experimental procedures were performed according to the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan, June 1, 2006). The study protocol was duly approved by the Animal Care and Use Committee of the Tokyo University of Agriculture and Technology, ensuring compliance with ethical standards and welfare of the animals involved (Approval number R03-149).\u003c/p\u003e\n\u003cp\u003eConsent to participate: \u0026nbsp; Not applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication:\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003eFunding:\u0026nbsp;This work was supported by a Grant-in-Aid for Scientific Research (Grant No. 20H03146) provided by the Ministry of Education, Culture, Sports, Science and Technology of Japan.\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eData availability: The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLazarus, J.V. \u003cem\u003eet al.\u003c/em\u003e NAFLD Consensus Consortium Advancing the global public health agenda for NAFLD: a consensus statement. 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Hepatology 41, 1313\u0026ndash;1321 (2005)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"AMBRA1, Flutamide, LC3, Mitophagy, NAFLD","lastPublishedDoi":"10.21203/rs.3.rs-4371202/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4371202/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFlutamide (FL), a non-steroidal drug used for its anti-androgenic, anticancer,and disrupting endocrine properties, induces mitochondrial toxicity and drug metabolism enzymesand promotes hepatocarcinogenesis. The inhibition of mitophagy, leading to the accumulation of damaged mitochondria, is implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). In this study, we investigated the effects of FL in high-fat diet (HFD)-induced non-obese steatosis rats, categorized into four groups: basal diet (BD), BD + FL, HFD, and HFD + FL. The introduction of FL exacerbated HFD-induced steatosis and marginally increased preneoplastic lesions. To analyzehepatic preneoplastic lesions, we divided them into clusters based on the expression ratios of the mitophagy regulators LC3 and AMBRA1. The expression rates of LC3 and AMBRA1 in these precancerous lesions were classified into three clusters using \u003cem\u003ek\u003c/em\u003e-means clustering. The HFD group exhibited an increased ratio of mitophagy inhibition clusters, as indicated by decreased LC3 and increased AMBRA1 levels in background hepatocytes and preneoplastic lesions. FL counteracted HFD-mediated mitophagy inhibition, as indicated by increased LC3 and decreased AMBRA1 levels in background hepatocytes. Our clustering analysis revealed that FL-induced mitophagy induction relied on Parkin expression. The present study underscores the significance of cluster analysis in understanding the role of mitophagy within small preneoplastic lesions and suggests that FL may potentially exacerbate NAFLD-associated hepatocarcinogenesis by affecting mitophagy.\u003c/p\u003e","manuscriptTitle":"Flutamide exacerbates steatosis and promotes early hepatocarcinogenesis in high-fat diet-fed non-obese steatotic rats: Insights from clustering analysis of mitophagy regulators AMBRA1 and LC3","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-23 10:03:15","doi":"10.21203/rs.3.rs-4371202/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"584c812b-468d-48f8-a7de-764a0a23297d","owner":[],"postedDate":"May 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":32216819,"name":"Biological sciences/Cancer/Cancer models"},{"id":32216820,"name":"Health sciences/Diseases/Gastrointestinal diseases/Liver diseases/Non alcoholic fatty liver disease"},{"id":32216821,"name":"Biological sciences/Cell biology/Autophagy/Mitophagy"}],"tags":[],"updatedAt":"2024-12-13T08:38:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-23 10:03:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4371202","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4371202","identity":"rs-4371202","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}