Supplementary Material for: Dietary Restriction Suppresses Steatosis-Associated Hepatic Tumorigenesis in Hepatitis C Virus Core Gene Transgenic Mice

<b><i>Background and Aims:</i></b> Dietary restriction (DR) is a preventive strategy for obesity, metabolic syndrome, cardiovascular disease, and diabetes. Although an interconnection between obesity, metabolic syndrome, fatty liver, and hepatocellular carcinoma has been documented, the mechanism and impact of DR on steatosis-derived hepatocarcinogenesis are not fully understood. This study aimed to evaluate whether DR can prevent hepatic tumorigenesis. <b><i>Methods:</i></b> Male hepatitis C virus core gene transgenic (HCVcpTg) mice that develop spontaneous age-dependent insulin resistance, hepatic steatosis, and ensuing liver tumor development without apparent hepatic fibrosis, were fed with either a control diet ad libitum (control group) or 70% of the same control diet (DR group) for 15 months, and liver phenotypes were investigated. <b><i>Results:</i></b> DR significantly reduced the number and volume of liver tumors. DR attenuated hepatic oxidative and endoplasmic reticulum stress and markedly suppressed nuclear factor-κB, signal transducer and activator of transcription 3 (STAT3) and STAT5, and phosphorylation of extracellular signal-regulated kinase, leading to downregulation of several pro-oncogenic mediators, such as cyclin D1. Serum insulin and insulin-like growth factor 1 levels, as well as hepatic expression of insulin receptor substrate 1/2, phosphatidylinositol-3 kinase, and serine/threonine-protein kinase AKT, were downregulated by DR. A transcriptome analysis revealed that STAT3 signaling and lipogenesis were the most suppressed hepatocarcinogenic pathways affected by DR. Additionally, DR stimulated autophagy and p62/sequestosome 1 degradation, enhanced phosphorylation of AMP-activated protein kinase α, increased fibroblast growth factor 21 expression, and attenuated expression of senescence-associated secretory phenotypes. <b><i>Conclusion:</i></b> DR suppressed steatosis-associated hepatic tumorigenesis in HCVcpTg mice, mainly due to attenuation of pathways involved in inflammation, cellular stress, cell proliferation, insulin signaling, and senescence. These findings support the notion that persistent 30% reduction of daily food intake is beneficial for preventing steatosis-associated hepatocarcinogenesis caused by HCV core protein.

<b><i>研究背景与目的：</i></b> 饮食限制（Dietary restriction）是肥胖、代谢综合征、心血管疾病及糖尿病的预防策略。尽管已有研究证实肥胖、代谢综合征、脂肪肝与肝细胞癌（hepatocellular carcinoma）之间存在关联，但饮食限制对脂肪变性诱导肝细胞癌变的作用机制与影响尚未完全阐明。本研究旨在评估饮食限制是否能够抑制肝脏肿瘤发生。<b><i>研究方法：</i></b> 雄性丙型肝炎病毒核心基因转基因（hepatitis C virus core gene transgenic, HCVcpTg）小鼠会随年龄增长自发出现胰岛素抵抗（insulin resistance）、肝脏脂肪变性（hepatic steatosis），并在无明显肝纤维化（hepatic fibrosis）的情况下进展为肝脏肿瘤。本实验将该类小鼠随机分为两组：一组自由进食对照饲料（对照组），另一组进食同配方饲料的70%（饮食限制组，DR组），持续干预15个月，随后对其肝脏表型开展分析。<b><i>研究结果：</i></b> 饮食限制显著降低了肝脏肿瘤的数量与体积。其可减轻肝脏氧化应激（oxidative stress）与内质网应激（endoplasmic reticulum stress），并显著抑制核因子κB（nuclear factor-κB）、信号转导与转录激活因子3（signal transducer and activator of transcription 3, STAT3）及STAT5的活性，以及细胞外调节蛋白激酶（extracellular signal-regulated kinase）的磷酸化水平，进而下调细胞周期蛋白D1（cyclin D1）等多种促癌介质的表达。饮食限制可降低血清胰岛素与胰岛素样生长因子1（insulin-like growth factor 1）的水平，并下调肝脏中胰岛素受体底物1/2（insulin receptor substrate 1/2）、磷脂酰肌醇3激酶（phosphatidylinositol-3 kinase）以及丝氨酸/苏氨酸蛋白激酶AKT（serine/threonine-protein kinase AKT）的表达。转录组分析（transcriptome analysis）显示，STAT3信号通路与脂肪生成（lipogenesis）是饮食限制抑制最为显著的肝细胞癌变相关通路。此外，饮食限制可激活自噬（autophagy）并促进p62/sequestosome 1的降解，增强腺苷酸活化蛋白激酶α（AMP-activated protein kinase α）的磷酸化水平，提升成纤维细胞生长因子21（fibroblast growth factor 21）的表达，并抑制衰老相关分泌表型（senescence-associated secretory phenotypes）的表达。<b><i>研究结论：</i></b> 饮食限制可抑制HCVcpTg小鼠体内脂肪变性相关的肝脏肿瘤发生，其主要机制为减轻炎症、细胞应激、细胞增殖、胰岛素信号通路及衰老相关通路的激活。本研究结果证实，每日进食量持续减少30%，可有效预防丙型肝炎病毒核心蛋白诱导的脂肪变性相关肝细胞癌变。