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Abstract

Modern lifestyle characterized by an obesogenic environment including consumption of energy-dense foods combined with a lack of exercise or physical activity is driving the epidemic of obesity. Obesity induced metabolic dysfunction paves a way for development of non-alcoholic fatty liver disease (NAFLD) which is a spectrum of liver disease ranging from a reversible condition to a progressive state of liver damage. Non-alcoholic steatohepatitis (NASH), characterized by fat infiltration in hepatocytes together with inflammation, has become one of the leading causes of chronic liver disease with the potential to develop into end-stage liver disease and hepatocellular carcinoma (HCC) which is the fastest rising cancer across the World. Nevertheless, there is currently no approved pharmacological intervention for NASH, and its increasing prevalence in the Western world is posing a huge socioeconomic burden. Therefore, there is an urgent need to find therapeutic targets to treat NASH and HCC. Intermittent fasting (IF), voluntary abstinence from intake of food and calorie dense liquids for two non-consecutive days a week (termed the 5:2 regimen), has recently been shown to improve metabolic disorders and various cancers. Multiple studies have shown that IF not only induces weight loss but also attenuates inflammation leading to an overall improvement in metabolism and health. Therefore, in this study, I investigated the role of IF in prevention against NASH development and assessed its therapeutic utility against limiting NASH-HCC transition using diet-induced preclinical mouse models of NASH and NASH-HCC. C57BL6/J mice fed with NASH inducing high-caloric diets, such as choline-deficient high-fat diet (CD-HFD) and western diet (WD) develop obesity and NASH. A subset of these mice subsequently leads to development of HCC offering an opportunity to investigate NASH-HCC transition. In the context of NASH, I tested a series of different dietary regimens comprising of variations in timing (fasting onset in either active phase or inactive phase), intensity (length of the fasting cycle) and duration (total fasting period per week) to unravel the regimen that could most effectively prevent development of NASH, resolve an existing NASH, improve overall fibrosis, and attenuate NASH-HCC by lowering HCC incidence. IF 5:2 regimen comprising of two non-consecutive fasting days per week with each fasting cycle beginning and ending at the start of the active phase of the mice i.e., 7pm proved out to be the most effective dietary intervention against NASH and NASH-HCC. C57BL6/J mice undergoing IF 5:2 in the context of a NASH diet showed resistance against diet-induced obesity and subsequent attenuation of NASH development with significant improvements in hepatomegaly, steatosis, fibrosis, and hepatic immune cell infiltration compared to control mice. Although IF 5:2 regimen consisted of food deprivation for two non-consecutive days in a week, mice under IF 5:2 ate significantly higher food on the non-fasting days so overall cumulative calorie intake per week remained unaffected compared to the control mice. Interestingly, fasting bouts were accompanied with physiological, cellular and molecular alterations rendering mice metabolically flexible compared to the control mice. Briefly, fasting cycles induced key metabolic pathways such as fatty acid oxidation, gluconeogenesis, ketogenesis and mitochondrial biogenesis in the fasted state. In contrast, signalling pathways involved in proliferation, inflammation and pro-carcinogenesis were dampened in the fasted state. Proteomic and transcriptomic data revealed Peroxisome Proliferator-Activated Receptor alpha (PPARα) and Phosphoenolpyruvate Carboxyinase 1 (PCK1) to be hepatic executors of fasting response in mice. Hepatic knockdown of Pparα and/or Pck1 abrogated the fasting mediated activation of hepatic transcriptional machinery and led to increased liver damage in the context of fasting. In the end, I investigated whether IF 5:2 could be used as a therapeutic intervention against established NASH and/or NASH-HCC transition in two distinct diet-induced mouse models of NASH and HCC. Surprisingly, IF 5:2 not only ameliorated established NASH but also lowered fibrosis and HCC incidence compared to control mice fed NASH diets. Collectively, these results suggest that IF 5:2 can be used in a prophylactic and therapeutic manner to attenuate NASH and NASH-HCC transition, respectively.