Abstract
To study the effects of autophagy inducer carbamazepine (CBZ) in a high-fat diet (HFD)/streptozotocin (STZ)-related early hepatocarcinogenesis model, we determined autophagic flux by immunohistochemical analysis of autophagy marker expression in preneoplastic liver foci and compared that with the expression of the NADPH oxidase subunit. Male F344 rats were fed a basal diet or HFD and subjected to two-stage hepatocarcinogenesis; diabetes mellitus was induced via STZ administration. Several STZ-treated, HFD-fed rats were administered CBZ (a total of five doses every one or two days) at week 7 and 8. STZ-treated, HFD-fed rats decreased β cells in the islet of Langerhans and increased adipophilin-positive lipid droplets in the liver; moreover, they had a larger area of glutathione S-transferase placental form-immunopositive preneoplastic liver foci, which was associated with inhibition of autophagy and induction of the NADPH oxidase subunit, as demonstrated by increased immunohistochemical expression of an autophagosome receptor marker microtubule-associated protein light chain 3 (LC3)-binding protein p62, and of an NADPH oxidase subunit p22phox in the preneoplastic foci. An increased trend of an autophagy phagophore marker LC3 in preneoplastic foci was also detected. CBZ administration could induce autophagy and impair p22phox expression, as shown by altered expression of autophagy regulators (Atg5, Atg6, Lamp1, Lamp2, and Lc3), NADPH oxidase subunits (P22phox and P67phox), and antioxidant enzymes Gpx1 and Gpx2. These results suggest that inhibition of autophagy and induction of p22phox might contribute to HFD/STZ-related early hepatocarcinogenesis in rats; however, the effects of CBZ administration on the STZ/HFD-increased preneoplastic foci were marginal in this study.
INTRODUCTION
Nonalcoholic fatty liver disease (NAFLD) is expected to become the leading cause of chronic liver disease, including hepatocellular carcinoma (HCC) (Geh et al., 2021). The rising incidence of NAFLD has been associated with metabolic syndrome, obesity, and diabetes mellitus (DM) (Pinyopornpanish et al., 2021; Estes et al., 2018). The prevalence of NAFLD-related HCC is estimated to increase by 47% in Japan and 130% in the USA between 2016 and 2030 owing to the growing epidemic of obesity and DM (Estes et al., 2018). HCC-related mortality rates have been increasing along with the increasing number of elderly people with metabolic risk factors, including NAFLD and DM, with the prognosis in most cases being poor (Dyson et al., 2014).
In NAFLD rodent models, a high-fat diet (HDF) is reproducibly able to induce steatosis (Peng et al., 2020), while a combination of HFD feeding with the administration of the hepatocarcinogenesis initiator N-diethylnitrosamine (DEN) is useful in establishing rodent models of HCC (de Lima et al., 2008). We have previously reported a steatosis-related early hepatocarcinogenesis animal model using HFD-fed rats, based on a medium-term liver assay for 8 weeks (Ito et al., 1989) to understand the mechanism underlying the development of steatosis-related preneoplastic liver foci (Yoshida et al., 2017; Murayama et al., 2018; Nakamura et al., 2018). In DM rodent models, the administration of streptozotocin (STZ) causes injury to β cells in the pancreas of rats with or without HFD feeding and establishes type 1 DM or type 2 DM (Furman, 2021). Furthermore, by combining STZ and DEN administration in rats, the effects of DM on hepatocarcinogenesis promotion can be examined (Saha et al., 2001; Ichikawa et al., 2010). According to epidemiological analyses on NAFLD, DM, and HCC, it is necessary to develop an NAFLD/DM-related hepatocarcinogenesis animal model to understand the complex relationship between these metabolic disorders and HCC and to explore potential therapeutic targets.
In NAFLD, while steatosis inhibits the fusion of autophagosomes with lysosomes and increases autophagosomes and lipid droplets, autophagy plays a role in the degradation of excessive lipid droplets to protect hepatocytes from steatosis-related injury (Singh et al., 2009; Khambu et al., 2018). We have previously demonstrated that the selective autophagy markers LC3 and p62 were particularly expressed in preneoplastic liver foci in HFD-fed rats, and the results suggested that the inhibition of autophagy might play a role in the development preneoplastic lesions (Masuda et al., 2019; Eguchi et al., 2022). However, autophagy has anti- and pro-cancer effects and thus plays a dual role in cancer (Weiskirchen and Tacke, 2019). To understand the contribution of autophagy to the pathogenesis of preneoplastic lesions in rats with NAFLD and DM, we developed an HFD/STZ-related early hepatocarcinogenesis rat model and examined the effects of autophagy inducer carbamazepine (CBZ) by studying the expression of the selective autophagy markers LC3 and p62. We also compared the expression of the NADPH oxidase subunit p22phox with that of LC3 or p62, as we had previously demonstrated that p22phox was expressed in preneoplastic liver foci in HFD-fed rats (Murayama et al., 2018; Nakamura et al., 2018).
MATERIALS AND METHODS
Chemicals
CBZ (CAS No. 298-46-4; purity > 97.0%) and STZ (CAS No. 18883-66-4; purity > 98.0%) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan), and DEN (CAS No. 55-18-5; purity > 99%) was obtained from Tokyo Kasei Kogyo (Tokyo, Japan).
Animals and treatments
In total, 45 5-week-old male F344/N rats were purchased from Japan SLC, Inc. (Shizuoka, Japan), maintained in an air-conditioned room (room temperature, 22 ± 3°C; relative humidity, 50 ± 20%; 12-hr light/dark cycle), and given free access to a powdered diet (Oriental MF; Oriental Yeast, Tokyo, Japan) and tap water. After an acclimatization period of 1 week, an 8-week medium-term liver carcinogenesis bioassay (Ito et al., 1989) was conducted according to the procedure described next. The rats in the basal diet group (BD) received a control basal diet (D12450K; Research Diets, Lane, NJ, USA), while the rats in the HFD group received HFD (D12451; Research Diets) for 8 weeks. At the beginning of the liver carcinogenesis bioassay, all animals received an intraperitoneal (i.p). injection of DEN at a dose of 200 mg/kg body weight to initiate hepatocarcinogenesis (Ito et al., 1989) and were subsequently randomly assigned into the following five groups: BD (n = 8), BD + STZ (n = 8), HFD (n = 8), HFD + STZ (n = 9), and HFD + STZ + CBZ (n = 12). At 2 weeks after DEN initiation, the rats received an i.p. injection of STZ (30 mg/kg) or vehicle control (0.05 M citrate buffered solution, pH 4.5) as previously reported (Ichikawa et al., 2010). At 3 weeks after DEN injection, all animals were subjected to two-thirds partial hepatectomy (PH) under isoflurane anaesthesia to develop preneoplastic liver foci (Ito et al., 1989). During weeks 7 and 8, some HFD-fed rats received CBZ (200 mg/kg/day, p.o., a total of five doses every one or two days). CBZ is a typical autophagy inducer in mice (Lin et al., 2013) and rats (Zhang et al., 2018). CBZ hepatotoxicity was enhanced by concomitant use with a glutathione synthetase inhibitor in rats (Iida et al., 2015), and 400 mg/kg CBZ (p.o.) for 6 days to diabetic (SDT) rats causes hepatotoxicity along with a decrease in glutathione (Takahashi et al., 2019). Therefore, in the present study, 200 mg/kg was administered due to the concern of enhanced hepatic injury in the diabetes model. The animals were observed clinically during the study; body weight, and food and water consumption were measured every week, and their caloric intake was calculated. At the end of the 8-week experiment (the day after the last dose of CBZ), the rats were euthanized by exsanguination in a carbon-monoxide atmosphere after overnight fasting to reduce glycogen and induce autophagy in the liver. Blood was withdrawn from abdominal large vein, and serum was obtained by centrifugation. The livers, pancreases, and abdominal adipose tissues (surrounding the spermatic cord) were excised and weighed. The sliced liver samples were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered formalin (pH 7.4) for histopathology and immunohistochemistry. The pancreas was fixed in 10% neutral-buffered formalin for histopathology and immunohistochemistry. The liver pieces were frozen in liquid nitrogen and stored at −80°C until further analysis. All procedures in this study were conducted in compliance with the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan, June 1, 2006), and the protocol (Approval No. 30–76) was approved by the Animal Care and Use Committee of the Tokyo University of Agriculture and Technology.
Glucose tolerance test
A glucose tolerance test was conducted in rats (n = 5 per group) after overnight fasting during 8 weeks of study (one day and two days after two doses of CBZ administration). After overnight fasting, blood samples were collected from the tail vein under an anesthesia with isoflurane at each test day, and blood glucose levels were measured using a blood glucose meter GT-1830 (ARKRAY, Inc., Tokyo, Japan) (Nakamura et al., 2018). After rats received an intraperitoneal administration of glucose solution (2.0 g/kg), blood glucose levels were measured after 60 and 120 min in the same way. Blood glucose levels before administration of glucose solution were also measured as normal values at 0 min.
Blood biochemistry
Alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), alkaline phosphatase (ALP), glucose, total cholesterol (T.CHO), triglyceride (TG), and glucose (GLU) were measured in serum samples, which collected at the end of the study (ORIENTAL YEAST CO., LTD., Nagahama Biolaboratories, Shiga, Japan).
Histopathology and immunohistochemistry
Liver and pancreas sections were routinely dehydrated in graded ethanol, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for histopathological examination. The pathological changes in the liver, including the presence of steatosis, hepatocellular ballooning, and lobular inflammation, were graded by the NAFLD activity score (NAS) (Nakamura et al., 2018). In the pancreas, the number and area of islets of Langerhans per total area were measured using Scion Image (Scion Corp., Frederick, MD, USA) (Miranda-Osorio et al., 2016; Nna et al., 2018). The insulin-positive β cells were counted in five islets of Langerhans, and the results were divided by five to obtain the average number of β cells per islet of Langerhans as previously described (Miranda-Osorio et al., 2016). Immunohistochemical staining of the liver for adipophilin (ADFP), cleaved-caspase-3, glutathione S-transferase placental form (GST-P), glucagon, insulin, Ki-67, MAP1LC3A (LC3), p22phox, and p62/SQSTM1 (p62) was performed under the conditions indicated in Supplemental Table 1. The deparaffinized liver sections were treated as previously reported (Yoshida et al., 2017). The numbers and areas of GST-P-positive foci (> 0.2 mm in diameter) and the total areas of the liver sections were quantified using Scion Image (Scion Corp.) as previously described (Ito et al., 1989; Yoshida et al., 2017). Sometimes, the assay increased nodules diagnosed as hepatocellular adenomas (Ito et al., 1989); however, we did not observe any hepatocellular adenomas in the present study. Labeling indices (LIs, %) of p22phox-, LC3-, p62- Ki-67-, and active-caspase-3-positive cells were examined in over 1000 cells within GST-P-positive foci and surrounding hepatocytes (Yoshida et al., 2017; Nakamura et al., 2018). The LI of ADFP-positive cells was also examined in over 1000 cells within the foci, and the areas of ADFP-positive lipid droplets per liver section were measured in the surrounding hepatocytes using Scion Image (Scion Corp.).
Real-time reverse transcription-polymerase chain reaction analysis (RT-PCR)
Analysis of the mRNA levels of the genes listed in Supplemental Table 2 in liver tissues (n = 6 animals per group) was performed with RT-PCR as previously reported (Murayama et al., 2018). The relative differences in gene expression were calculated using the threshold cycle (CT) values that were first normalized to those of the hypoxanthine phosphoribosyl transferase 1 (Hprt1) gene, the endogenous control in the same sample, and then relative to a control CT value using the 2−ΔΔCT method.
Statistical analyses
All data are expressed as mean ± standard deviation (SD). The statistical significance of the differences between the control and the treated group(s) was determined by the Tukey or Steel-Dwass multiple comparison test. A p-value of < 0.05 was considered statistically significant.
RESULTS
HFD feeding and STZ injection promote obesity, DM, and steatosis, and the effects are at least partially recovered by applying CBZ
We previously reported that the final body weight and adipose tissue weight were significantly increased in HFD-fed F344 rats compared with those in BD-fed rats (Murayama et al., 2018). In this study, similar effects on obesity in the HFD group were observed for both parameters; the final body weight tended to increase, and the absolute adipose tissue weight was statistically significantly increased (Table 1). HFD-fed F344 rats became obese, but these rats were less susceptible to HFD-induced obesity than other strains of rats (Gheibi et al., 2017). There were no clear differences in food, calorie, and water intake among groups (Table 1). Injection of STZ cancelled obesity in HFD-fed rats (Table 1) and induced hyperglycemia in a glucose tolerance test (Figs. 1A and 1B), owing to atrophy of the islet of Langerhans with low numbers of β cell in the pancreas (Figs. 1C and 1D; Table 2), as previously reported (Miranda-Osorio et al., 2016; Nna et al., 2018; Srinivasan et al., 2005). Our previous studies demonstrated that HFD feeding histologically increased NAS (Murayama et al., 2018; Nakamura et al., 2018). In this study, HFD feeding and/or STZ injection increased the NAS and ballooning score of hepatocytes, which were accompanied with expression of ADFP at the margins of intracytoplasmic lipid droplets (Figs. 1E and 1F; Table 2), without an increase of ADFP LI in the foci (Fig. 1G). On the contrary, HFD feeding with or without STZ injection significantly decreased blood total cholesterol (T.CHO) and glucose (GLU), but not triglyceride (TG) (Fig. 1H; Supplemental Table 3). Hypocholesterolemia and hypoglycemia might be caused by cholesterol accumulation in the liver and hyperinsulinemia without insulin resistance (Furman, 2021). No liver injury was detected with HFD feeding with or without STZ injection as indicated by ALT, AST and ALP measurements (Supplemental Table 3).
Table 1. Final body weight, organ weight, food intake, and water intake in rats
§.
| Diet |
|
BD |
|
HFD |
| Diabetes induction |
|
- |
STZ |
|
- |
STZ |
STZ |
| Autophagy induction |
|
- |
- |
|
- |
- |
CBZ |
| No. of rats |
|
8 |
8 |
|
8 |
9 |
12 |
| Final body weight (g) |
|
264.5 |
± |
14.3 |
259.4 |
± |
22.7 |
|
282.2 |
± |
14.0 |
264.9 |
± |
14.7 |
247.0 |
± |
11.7 |
| Food intake (g/rat/day)† |
|
13.7 |
± |
4.0 |
12.9 |
± |
4.0 |
|
10.3 |
± |
4.1 |
9.7 |
± |
3.8 |
9.6 |
± |
4.0 |
| Calorie intake (kcal/rat/day)† |
|
52.9 |
± |
4.0 |
49.8 |
± |
3.8 |
|
48.8 |
± |
4.2 |
45.9 |
± |
3.5 |
45.5 |
± |
2.7 |
| Water intake (g/rat/day)† |
|
18.3 |
± |
3.5 |
18.0 |
± |
4.2 |
|
20.0 |
± |
3.0 |
18.5 |
± |
3.6 |
18.3 |
± |
4.5 |
| Absolute adipose tissue weight (g) |
|
8.09 |
± |
1.08 |
6.80 |
± |
1.83 |
|
9.14 |
± |
1.60b |
7.34 |
± |
1.16 |
5.84 |
± |
1.30ac |
| Relative adipose tissue weight (%BW) |
|
3.05 |
± |
0.38 |
2.59 |
± |
0.51 |
|
3.22 |
± |
0.47 |
2.77 |
± |
0.44 |
2.35 |
± |
0.42ac |
| Absolute liver weight (g) |
|
7.10 |
± |
0.60 |
7.00 |
± |
0.52 |
|
6.74 |
± |
0.64 |
6.72 |
± |
0.75 |
9.07 |
± |
0.52abcd |
| Relative liver weight (%BW) |
|
2.69 |
± |
0.20 |
2.71 |
± |
0.21 |
|
2.39 |
± |
0.21ab |
2.54 |
± |
0.18 |
3.68 |
± |
0.18abc |
Abbreviations: BD, basal diet; BW, body weight; CBZ, carbamazepine; HFD, high fat diet; STZ, streptozotocin.
§: All rats were fed BD or HFD, and subjected to two-thirds partial hepatectomy at week 3, and some received STZ at week 2. CBZ was treated at weeks 7 and 8.
†: Average intake throughout the study, calculated from the data at each week.
Data are shown as the mean ± standard deviation.
a p < 0.05 vs BD (Tukey’s or Steel-Dwass test).
b p < 0.05 vs BD + STZ (Tukey’s or Steel-Dwass test).
c p < 0.05 vs HFD (Tukey’s or Steel-Dwass test).
d p < 0.05 vs HFD + STZ (Tukey’s or Steel-Dwass test).
Table 2. Quantitative analyses of islets of Langerhans in the pancreas, and NAFLD activity score and ballooning of hepatocytes in the liver of rats
§.
| Diet |
|
BD |
|
HFD |
| Diabetes induction |
|
- |
STZ |
|
- |
STZ |
STZ |
| Autophagy induction |
|
- |
- |
|
- |
- |
CBZ |
| No. of rats |
|
8 |
8 |
|
8 |
9 |
12 |
| No. of islets per pancreas (No./mm2) |
|
0.42 |
± |
0.11 |
0.26 |
± |
0.09a |
|
0.37 |
± |
0.10 |
0.23 |
± |
0.08a |
0.28 |
± |
0.08a |
| Area of islets per pancreas (%) |
|
1.39 |
± |
0.36 |
1.03 |
± |
0.34 |
|
1.36 |
± |
0.34 |
0.85 |
± |
0.27a |
0.95 |
± |
0.22a |
| NAFLD activity score (NAS) (Score) |
|
2.9 |
± |
1.2 |
4.3 |
± |
0.9a |
|
3.4 |
± |
0.5b |
4.2 |
± |
1.0ac |
3.7 |
± |
1.2 |
| Hepatocyte ballooning (Score) |
|
0.8 |
± |
0.7 |
1.6 |
± |
0.5a |
|
1.4 |
± |
0.5 |
1.6 |
± |
0.7a |
0.4 |
± |
0.7bcd |
Abbreviations: BD, basal diet; CBZ, carbamazepine; HFD, high fat diet; STZ, streptozotocin.
§: All rats were fed BD or HFD, and subjected to two-thirds partial hepatectomy at week 3, and some received STZ at week 2. CBZ was treated at weeks 7 and 8.
Data are shown as the mean ± standard deviation.
a p < 0.05 vs BD (Tukey’s or Steel-Dwass test).
b p < 0.05 vs BD + STZ (Tukey’s or Steel-Dwass test).
c p < 0.05 vs HFD (Tukey’s or Steel-Dwass test).
d p < 0.058 vs HFD + STZ (Tukey’s or Steel-Dwass test).
CBZ treatment tended to decrease body weight and adipose tissue weight when compared with those in the BD and HFD group but significantly increased the liver weight when compared with those in the other group (Table 1). No liver injury was detected in blood biochemistry in the CBZ-treated group, but decreased ALP (Supplemental Table 3). Interestingly, CBZ improved HFD/STZ-induced hyperglycemia at 120 min in a glucose tolerance test (Figs. 1B) and the ballooning of hepatocytes, but not the expression of ADFP (Fig. 1E and 1F; Table 2). However, CBZ significantly increased ADFP LI in the foci as compared with that in the other group (Fig. 1G).
HFD feeding and STZ injection increase preneoplastic hepatocellular lesions associated with p62 and p22phox induction, and CBZ can marginally cancel these effects
Next, we examined whether HFD feeding and/or STZ injection could enhance hepatocarcinogenesis in this model. We found that the HFD feeding in combination with STZ injection significantly increased the area, but not the number, of preneoplastic GST-P-positive liver foci, compared with BD feeding (Figs. 2A and 2B; Supplemental Table 4). This effect was associated with an increasing trend in cell proliferation marker Ki-67 LI but not cleaved caspase-3 LI in the foci (Supplemental Table 4). We previously reported that the expression of a NADPH oxidase subunit p22phix was specifically increased in the preneoplastic liver foci in HFD-fed rats; NOX-generated reactive oxygen species (ROS) might enhance HFD-mediated hepatocarcinogenesis (Murayama et al., 2018; Nakamura et al., 2018). A similar effect was confirmed in HFD-fed rats with or without STZ injection (Figs. 2A and 2C).
Autophagy can be monitored by expression of autophagosome marker LC3, which is indicative of the induction or inhibition of autophagy, and by the increased or decreased expression of selective autophagosome receptor p62, which is indicative of the inhibition or induction of autophagy, respectively (Zhang et al., 2013; Yoshii and Mizushima, 2017; Ueno and Komatsu, 2020). HFD feeding in combination with STZ injection tended to increase LI of LC3 and significantly increase p62, when compared with the BD and BD + STZ groups (Figs. 2A, 2D, and 2E). The results suggested that HFD feeding in combination with STZ injection might inhibit autophagy in the preneoplastic foci, and the results was consistent with findings in HFD-fed rats in our previous studies (Masuda et al., 2019; Eguchi et al., 2022).
CBZ can reduce steatosis in HFD-fed mice, and protect liver injury against ischemia and reperfusion in mice through the induction of autophagy (Kim et al., 2013; Lin et al., 2013). CBZ treatment tended to cancel the HFD/STZ-mediated changes in GST-P, p22phox, and p62, while the drug preserved the changes in LC3 (Figs. 2A–2E); the results suggest that autophagy inhibition and increase of the NADPH subunit marginally reduce the preneoplastic foci. Autophagy induction by CBZ was supported by findings in the hepatocytes surrounding preneoplastic foci: the drug significantly increased LC3 LI and decreased p62 LI (Figs. 2F and 2G).
CBZ alters lipid metabolism-, oxidative stress-, and autophagy-related gene expression in the liver
To further explore the mechanism underlying HFD/STZ-mediated hepatocarcinogenesis, we determined the expression of genes involved in lipid metabolism, oxidative stress, and autophagy in liver samples of each group. Regarding the expression levels of lipid metabolism-related genes, we found that the HFD feeding in combination with STZ injection significantly decreased the expression of the fatty acid desaturation gene Scd1 and β-oxidation gene Acox1, compared with expression of these genes in the BD and BD + STZ, and HFD groups (Table 3). Surprisingly, CBZ treatment further significantly decreased expression of the cholesterol transporter gene Abca1, TG-binding protein gene ApoB, TG synthetase gene Dgat2, transcription factor gene for β-oxidation Ppara, fatty acid synthetase Srebf1, transcriptional factor for cholesterol synthesis and uptake Srebf2, as well as Scd1 and Acox1, compared with expression of these genes in the other group. These changes in both groups might be inversely associated with high NAS and ADFP expression (Fig. 1E and 1F; Table 2), implying adaptive responses to excessive fatty droplet deposition.
Table 3. Gene expression analysis of lipid metabolism, oxidative stress, and autophagy in liver samples of rats
§.
| Diet |
|
BD |
|
HFD |
| Diabetes induction |
|
- |
STZ |
|
- |
STZ |
STZ |
| Autophagy induction |
|
- |
- |
|
- |
- |
CBZ |
| No. of rats |
|
6 |
6 |
|
6 |
9 |
10 |
| Lipid metabolism-related genes |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
| Abca1 |
|
1.05 |
± |
0.41 |
1.03 |
± |
0.13 |
|
1.07 |
± |
0.24 |
0.85 |
± |
0.30 |
0.46 |
± |
0.09abc |
| Acox1 |
|
1.10 |
± |
0.62 |
1.03 |
± |
0.32 |
|
0.88 |
± |
0.06 |
0.65 |
± |
0.21c |
0.45 |
± |
0.09ac |
| Apob |
|
1.01 |
± |
0.18 |
1.08 |
± |
0.17 |
|
1.23 |
± |
0.14 |
0.91 |
± |
0.26 |
0.71 |
± |
0.29c |
| Dgat2 |
|
1.06 |
± |
0.43 |
0.92 |
± |
0.12 |
|
1.06 |
± |
0.09 |
0.96 |
± |
0.36 |
0.56 |
± |
0.10abc |
| Ppara |
|
1.08 |
± |
0.53 |
0.95 |
± |
0.33 |
|
0.97 |
± |
0.07 |
1.08 |
± |
0.66 |
0.66 |
± |
0.12c |
| Pparg |
|
1.04 |
± |
0.28 |
1.22 |
± |
0.75 |
|
1.71 |
± |
0.55 |
1.29 |
± |
0.32 |
1.08 |
± |
0.23 |
| Scd1 |
|
1.33 |
± |
1.37 |
1.14 |
± |
0.71 |
|
0.25 |
± |
0.26 |
0.11 |
± |
0.09ab |
0.01 |
± |
0.01abc |
| Srebf1 |
|
1.15 |
± |
0.77 |
0.97 |
± |
0.52 |
|
0.54 |
± |
0.23 |
0.37 |
± |
0.20 |
0.21 |
± |
0.13ab |
| Srebf2 |
|
1.05 |
± |
0.40 |
0.98 |
± |
0.12 |
|
0.92 |
± |
0.09 |
0.72 |
± |
0.26 |
0.63 |
± |
0.11abc |
| Oxidative stress-related genes |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
| P22phox (Cyba) |
|
1.02 |
± |
0.20 |
0.99 |
± |
0.24 |
|
1.12 |
± |
0.43 |
1.02 |
± |
0.43 |
0.54 |
± |
0.23c |
| Gpx1 |
|
1.04 |
± |
0.36 |
1.02 |
± |
0.12 |
|
1.07 |
± |
0.22 |
0.86 |
± |
0.30 |
0.36 |
± |
0.13abcd |
| Gpx2 |
|
1.08 |
± |
0.45 |
1.43 |
± |
0.63 |
|
1.74 |
± |
0.74 |
2.20 |
± |
0.76 |
19.0 |
± |
10.2abcd |
| Mn-SOD |
|
1.01 |
± |
0.20 |
0.93 |
± |
0.13 |
|
0.83 |
± |
0.11 |
0.66 |
± |
0.26a |
0.90 |
± |
0.14 |
| Nox4 |
|
1.07 |
± |
0.41 |
1.62 |
± |
0.97 |
|
1.64 |
± |
0.50 |
1.62 |
± |
0.59 |
0.75 |
± |
0.11 |
| P67phox |
|
1.03 |
± |
0.28 |
1.06 |
± |
0.26 |
|
1.11 |
± |
0.30 |
1.67 |
± |
0.74 |
0.83 |
± |
0.35d |
| Autophagy-related genes |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
| Atg3 |
|
1.01 |
± |
0.15 |
0.96 |
± |
0.12 |
|
1.00 |
± |
0.11 |
0.83 |
± |
0.17 |
0.84 |
± |
0.11 |
| Atg5 |
|
1.05 |
± |
0.38 |
1.07 |
± |
0.17 |
|
1.19 |
± |
0.20 |
0.94 |
± |
0.28 |
0.69 |
± |
0.06bc |
| Atg6 |
|
1.01 |
± |
0.16 |
0.97 |
± |
0.12 |
|
1.06 |
± |
0.11 |
1.00 |
± |
0.11 |
0.81 |
± |
0.10c |
| Atg7 |
|
1.03 |
± |
0.26 |
0.92 |
± |
0.19 |
|
0.93 |
± |
0.10 |
0.83 |
± |
0.31 |
0.74 |
± |
0.13 |
| Lamp1 |
|
1.01 |
± |
0.20 |
1.07 |
± |
0.14 |
|
1.17 |
± |
0.06 |
1.08 |
± |
0.23 |
0.68 |
± |
0.14abcd |
| Lamp2 |
|
1.02 |
± |
0.23 |
1.06 |
± |
0.13 |
|
1.04 |
± |
0.15 |
0.95 |
± |
0.27 |
0.58 |
± |
0.13abcd |
| Lc3 |
|
1.06 |
± |
0.47 |
0.99 |
± |
0.19 |
|
1.06 |
± |
0.13 |
0.80 |
± |
0.31 |
0.76 |
± |
0.05c |
| P62 |
|
1.04 |
± |
0.33 |
1.13 |
± |
0.29 |
|
0.99 |
± |
0.09 |
0.80 |
± |
0.19 |
1.19 |
± |
0.20 |
Abbreviations: BD, basal diet; CBZ, carbamazepine; HFD, high fat diet; STZ, streptozotocin.
§: All rats were fed BD or HFD, and subjected to two-thirds partial hepatectomy at week 3, and some received STZ at week 2. CBZ was treated at weeks 7 and 8.
Data are shown as the mean ± standard deviation.
a p < 0.05 vs BD (Tukey’s or Steel-Dwass test).
b p < 0.05 vs BD + STZ (Tukey’s or Steel-Dwass test).
c p < 0.05 vs HFD (Tukey’s or Steel-Dwass test).
d p < 0.05 vs HFD + STZ (Tukey’s or Steel-Dwass test).
Regarding the expression of oxidative stress-related genes, we found that the HFD feeding in combination with STZ injection significantly decreased expression of the antioxidant enzyme gene Mn-SOD, compared with that in the BD group (Table 3). CBZ treatment significantly decreased the expression of the NADPH oxidase genes, P22phox (Cyba) and P67phox and of the antioxidant enzyme gene Gpx1, and increased the expression of Gpx2, compared with expression of these genes in the BD, BD + STZ, HFD, and/or HFD + STZ groups These changes might be in response to steatosis-mediated oxidative stress.
Regarding the expression of autophagy-related genes, we found that CBZ treatment significantly decreased the expression of Atg5, Atg6, Lamp1, Lamp2, and Lc3, compared with expression of these genes in the BD, BD + STZ, HFD, and/or HFD + STZ groups (Table 3).
DISCUSSION
Non-alcoholic fatty liver disease and DM are known to be as two important metabolic disorders contributing to the pathogenesis of HCC (Pinyopornpanish et al., 2021; Estes et al., 2018). We developed a rat model of HFD/STZ-induced early hepatocarcinogenesis that can be employed to examine glucose tolerance and atrophy of pancreatic islet cells with the reduction in β cells as well as steatosis and preneoplastic liver foci. A previous study in a modified medium-term liver assay using a low STZ dose (30 mg/kg) did not find an increase in GST-P-positive preneoplastic liver foci in rats that received BD (Ichikawa et al., 2010). The present study, using a further modified medium-term liver assay with an STZ dose of 30 mg/kg in combination with HFD feeding, found an increased area of GST-P-positive preneoplastic liver foci, compared with the area in the BD group. Using this rat model to explore therapeutic targets, we demonstrated the expression of the autophagosome marker LC3 and the LC3-binding receptor p62 in the preneoplastic liver foci and compared their expression levels with those of NADPH oxidase subunit p22phox, which could generate ROS.
We previously reported that LC3 and p62 were specifically expressed in GST-P-positive preneoplastic liver foci in HFD-fed rats (Masuda et al., 2019; Eguchi et al., 2022). A mammalian homologue of yeast Atg8, LC3 is known to exist on autophagosomes, serving as a widely used autophagosome marker. An increased level of LC3 is not always indicative of autophagy as it could represent a blockade of autophagy maturation (Yoshii and Mizushima, 2017; Ueno and Komatsu, 2020). p62 plays a role in the delivery of polyubiquitinated proteins and organelles for autophagosomal-lysosomal degradation, resulting in p62-mediated degradation, which is attenuated by interference with autophagic flux (Zhang et al., 2013; Yoshii and Mizushima, 2017; Ueno and Komatsu, 2020). In this study, high protein expression of LC3 and p62 in GST-P-positive foci was confirmed in HFD-fed rats with STZ administration, compared with that in BD-fed rats with or without STZ administration, although a statistically significant difference was only found in p62 expression. Thus, the observed accumulation of p62 in preneoplastic liver foci suggests that autophagy interruption by HFD feeding in combination with STZ injection might be associated with early hepatocarcinogenesis in this model. The interpretation is supported by the findings that overexpression of p62 with Sirt1, a p62 binding protein, enhanced the incidence of HCC in DEN-treated mice (Feng et al., 2021). Autophagy plays a dual role in tumorigenesis; prior to transformation, autophagy can function to suppress tumor formation, while after transformation, autophagy can promote tumor formation (Weiskirchen and Tacke, 2019). Thus, we did not conclude but rather suggest that induction of autophagy plays a role in tumor suppression in this model at a stage prior to transformation. Our previous study also demonstrated that the autophagy inducer amiodarone could reduce the number of GST-P-positive preneoplastic liver foci in HFD-fed rats (Masuda et al., 2019).
We speculated that induction of autophagy could inhibit the development of preneoplastic foci as well as steatosis in this model; however, the effects of CBZ on the liver lesions were limited. We confirmed autophagy induction in the hepatocytes surrounding preneoplastic foci when treated with CBZ in HFD-fed rats with STZ administration, as shown by a decrease in p62 expression in highly preserved LC3. Carbamazepine is an anti-epileptic drug that can induce autophagy by decreasing intracellular calcium through the inhibition of inositol synthesis and reduction of inositol triphosphate in a mammalian target of rapamycin (mTOR)-independent manner (Fleming et al., 2011). The protective effects of CBZ in reducing steatosis and improving insulin sensitivity were previously demonstrated in HFD-induced non-alcoholic fatty liver in mice (Lin et al., 2013). We found that CBZ treatment improved hyperglycemia in the glucose tolerance test at 120 min after stimulation and reduced blood TG levels at the end of the study, but did not decrease the NAS or ADFP-positive lipid droplets in the liver. The limitation of CBZ treatment might be caused by the study design; however, the drug could affect lipid metabolism, as the drug significantly decreased the expression of genes involved in fatty acid and cholesterol metabolism, including Srebf1, Srebf2, Abca1, Acox1, ApoB, Dgat2 and Scd1. A similar effect on gene expression involved in lipogenesis and fatty acid oxidation was reported in HFD-fed mice (Lin et al., 2013). Further, in the NAFLD scores, hepatocellular ballooning, a marker of hepatocyte injury specific to NAFLD, was significantly reduced, suggesting an inhibitory effect on liver injury by CBZ in the present study. Induction of autophagy with CBZ administration in the liver has been performed mainly in mice (Kim et al., 2013; Lin et al., 2013), while hepatotoxicity of CBZ as a model of drug-induced liver injury has been investigated in rats (Iida et al., 2015; Takahashi et al., 2019); therefore, the study using CBZ in rats is challenging to understand induction of autophagy in HFD/STZ-related early rat hepatocarcinogenesis. The effects of CBZ, an autophagy inducer, on the size of preneoplastic foci is limited in this study. The foci are a region that initiates by DEN-mediated DNA damage and promotes precancerous changes by enhancing cell proliferation after PH (Ito et al., 1989) and suppressing autophagy (Masuda et al., 2019; Eguchi et al., 2022), and might be resistant to autophagy induction with CBZ as compared with surrounding hepatocytes. In fact, CBZ administration increased the expression of ADFP within the foci, and although the mechanism is unknown, it is possible that an increase in lipid droplets, i.e. lipophagy inhibition, may have occurred. In this study, LC3 and p62 were used as indicators of selective autophagy, but the status of autophagy inside and outside of foci may differ significantly, and analysis of cargo (mitochondria, endoplasmic reticulum, lipid droplets, etc.) that is incorporated into selective autophagy might be required to better understand the relationship between autophagy and precancerous growth in the future study (Uomoto et al., unpublished data).
Gene expression analysis focusing on autophagy-related substances showed no significant changes in the expression of autophagy-related genes, including Lc3 in the HFD + STZ group, and the unexpected effects of CBZ; expression of Atg5, Atg6, Lamp1, Lamp2, and Lc3 was significantly reduced, compared with those in the other group. The discrepancy between the protein and gene expression level of LC3 has been reported in training model studies (Gunadi et al., 2020), althoiugh the mechanism was not discussed. A possible mechanism to explain this is the inhibition of gene expression on autophagosome and lysosome formation was caused by negative feedback after induction of autophagy. Further, the differences between mRNA level and protein level may be influenced by the expression of transcription factors, the stabilities of mRNA and protein, and the protein degradation system as a whole. For example, Lc3 is regulated by FOXO3 and E2F1/NF-kB, Atg5 by E2F1/NF-kB, and Atg7 by CREB-CRTC2 and p53 for gene expression (Di Malta et al., 2019), the genome p62 has binding sites for SP-1, AP-1, NF-kB, and Ets-1 (Vadlamudi and Shin, 1998), and Lamp1 and Lamp2 for SP1 and AP1 (Sawada et al., 1993). These complex combinations of transcription factors regulate the mRNA expression of autophagy-related molecules. The ubiquitin-proteasome pathway as well as the autophagy-lysosome pathway are important intracellular proteolytic mechanisms, and abnormalities in the ubiquitin-proteasome can cause intracellular retention of autophagy-related molecules (Li et al., 2019). Therefore, further researchs are required to examine the activation of ubiquitin-proteasome as well as transcription factors for fluctuations in mRNA and protein of autophagy-related molecules.
Our previous study reported that p22phox was specifically expressed in preneoplastic liver foci in HFD-fed rats (Yoshida et al., 2017; Murayama et al., 2018), which was consistent with the finding in HFD-fed rats with or without STZ administration in the present study. NADPH oxidases comprise membrane-bound subunits, including p22phox, and can be divided into NOX1−5, DUOX1, and DUOX2 that generate ROS, superoxide or hydrogen peroxide (Kalyanaraman, 2013). NOX1 and NOX2 are expressed in hepatocytes and have been implicated in the progress of liver diseases (Gabbia et al., 2021); the membrane regulatory subunit p22phox plays a critical role in NOX activity (Gabbia et al., 2021). NOX activity might play a role in the development of GST-P-positive preneoplastic foci in rats, as a NOX inhibitor apocynin can reduce the liver foci as previously reported (Fuji et al., 2017; Yoshida et al., 2017). The interaction between autophagy and NOX is not fully understood. Several data demonstrated that Run/cysteine-rich-domain-containing Beclin1-interacting autophagy protein, Rubicon, positively regulates NOX activity, facilitating the stabilization and phagosome trafficking of the p22phox−NOX complex to induce a ROS generation, cytokine production, and thereby potent anti-microbial activities (Kim et al., 2020; Yang et al., 2012). We speculated that autophagy inhibition with p22phox expression, resulting in ROS generation, might contribute to the development of preneoplastic foci in this model. This was supported by evidence that an autophagy inducer CBZ treatment increased gene expression of Gpx2, which can convert H2O2 to H2O in a reduced glutathione-dependent manner (Kalyanaraman, 2013), and autophagy eliminates the sources of ROS generation such as damaged mitochondria and peroxisomes via mitophagy and pexophagy, respectively (Ornatowski et al., 2020).
In conclusion, we demonstrated specific expression of autophagy markers LC3 and p62 as well as a NOX subunit p22phox in the preneoplastic lesions in rats with NAFLD and DM, and the in vivo system would be useful to understand the role of autophagy and NOX on early hepatocarcinogenesis and to explore potential therapeutic targets. A major limitation in this study is a marginal effect of autophagy inducer CBZ in the development of preneoplastic lesions; therefore, modified dose levels and treatment periods of CBZ and combined administration of an autophagy inducer and NOX inhibitor are required in future studies. The additional insight into the role of autophagy and NOX status may help design chemotherapeutic drug strategies for hepatocarcinogenesis.
ACKNOWLEDGMENTS
We would like to thank Editage (www.editage.com) for the English language editing. This work was supported by a Grand-in-Aid for Scientific Research © (Grant No. 17K08075 and 20H03146) from the Ministry of Education, Culture, Sports, Science.
Conflict of interest
The authors declare that there is no conflict of interest.
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