However, no direct link has been provided yet. Studies that aim to link HCC and NAFLD are blunted by the lack of reliable animal models. The use of a choline-deficient L-aminoaciddefined diet in rats provided the most interesting results inducing steatohepatitis. Concerning this issue, a still unresolved question is however related to the potential of CDAA diet in inducing insulin resistance. The purpose of this study is to develop a mouse model of liver injury which mimics NASH features that lead to HCC. On the other hand, the hepatic expression of the Sterol Regulatory Element Binding Protein-1c, that is the transcription factor activating all genes required for lipogenesis, was slightly reduced by CDAA treatment without major modifications induced by CCl4 administration in our experimental model. Furthermore, the Carbohydrate-Responsive Element-Binding Protein, a key element of glucosemediated stimulation of lipogenesis progressively decreased in CDAA-treated animals, independently from CCl4 administration, starting at 1 month. These data indicate that the development of steatosis in CDAA model was not associated with an increased lipogenesis. Thus, we analyzed the pathways related to FA oxidation, finding a decreased expression of both ACOX-1 and CPT1A observed in CDAA+CCl4-treated animals at 1 months and in CDAA mice thereafter, indicates that reduced fatty acid oxidation could be one of the mechanisms of hepatic steatosis. In this study, we have provided evidences that CDAA diet induces peripheral insulin resistance already in the first month after treatment, and this was associated to the pathological spectrum of NAFLD, including NASH and HCC. Peripheral insulin resistance is a primary feature of NAFLD/NASH, and is probably one of the main co-factors involved in the worsening of the disease. Thus the novelty of our results is the demonstration that the CDAA+CCl4 model determines peripheral insulin resistance, NAFLD and its progression to HCC. Using this novel experimental approach we observed: a) development of peripheral insulin resistance already after 1 month; b) entire spectrum of lesions ranging from simple steatosis to NASH and HCC; c) development of HCC after 9 months of treatment in all mice; d) association of HCC development to increased extracellular matrix deposition; e) significant modification of oncogenic genes expression already after 3 months of treatment. Thus, this experimental model is able to guarantee in 9 months the development of HCC in almost 100% of animals and to early resemble the main features of the progression from NAFLD to NASH and HCC. In the majority of human cases, HCC arises in patients with advanced chronic liver injury and/or cirrhosis. NAFLD, which is present in up to 90% of all obese persons and in up to 70% of persons with type 2 diabetes, is a recognized risk factor for HCC, that may develop in NASH in the absence of cirrhosis. However, the study of the molecular mechanisms linking steatosis development to chronic liver injury and HCC is hampered by the lack of PF-4217903 adequate experimental models that often do not resemble the human situation, either are not associated to a significant development of chronic liver injury or lead to a cachectic phenotype that does not allow a long period of observation, as needed for carcinogenesis. In the CDAA model, mice develop steatosis in the absence of a high fat diet, mice continue to eat, do not reduce the appetite and the amount of calories introduced and weight changes are similar to control diet. Furthermore, in comparison to other existing rodent models, CDAA is able to drive the progression of steatosis toward a condition of inflammation and fibrosis. We have investigated the potential mechanisms that could explain the hepatic steatosis. First of all we demonstrated that CDAA-treated mice were more insulin resistant already at one month as compared to the control CSAA diettreated mice. Data in the literature show controversial results concerning the potential condition of insulin resistance in the course of CDAA treatment in rodents, mainly based on methods that provide only a partial and indirect measurement of insulin resistance, and related to the fasting glycemic and insulinemic state. Here insulin resistance was measured by the euglycemic-hyperinsulinemic clamp, which represents the gold standard for the evaluation of insulin sensitivity, and the results were confirmed by finding increased fasting insulin concentrations. This condition was even enhanced by the addition of CCl4 to the diet. The mechanisms by which CDAA diet induces insulin resistance are unknown but could be related to the gut microbiota metabolism of choline as shown in humans. Metabolomics data have indicated that reduced concentrations of lysophosphocholine, in particular reduced lyso-PC C18:2, and lyso-PC C16:0, are associated with peripheral insulin resistance and hepatic steatosis. Moreover, we found that CDAA diet increases Inflammasome components in the liver, which supports our hypothesis of a possible link between gut microbiota modifications, insulin resistance and progression of liver injury. The discovery that CDAA diet induces peripheral insulin resistance is important for the translation of this animal model to the human studies. To our knowledge, this is the first experimental model where a clear link between peripheral insulin resistance, NASH development and HCC formation has been established. In human patients with NAFLD/NASH, peripheral insulin resistance is a primary feature of the disease, even in lean subjects that do not present the characteristics of metabolic syndrome. Moreover, a worse peripheral insulin resistance state has been associated with the presence of fibrosis. To investigate if CDAA was worsening also hepatic insulin resistance, we have measured the expression of liver enzymes associated with glucose production, gluconeogenesis and de-novo lipogenesis as SREBP-1c and ChREBP.