To detect intracellular lipid droplets accumulation, the cells were brought to room temperature and the medium was replaced with 200 μL PBS. Five μL of AdipoRed reagent
(Lonza, Walkersville, MD) were added in each well and the plates were incubated at room temperature for 10 minutes. The relative fluorescence was measured (λ excitation at 485 nm, λ emission at 572 nm) using a fluorescence spectrometer (HTS-7000 Plus-plaque-lecteur, BI 2536 Perkin Elmer). Each fluorescence value was normalized to DNA content. The analyses were performed on three independent cell isolates in sextuplicate. Methods describing biochemical serum analysis, immunohistochemistry, protein expression, vascular corrosion casting, quantitative real-time polymerase chain reaction (PCR) and statistics are provided in the Supporting Materials and Methods. αPlGF, antiplacental growth factor; αSMA, alpha-smooth muscle actin; αVEGFR2, antivascular endothelial growth factor receptor 2; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HCC, hepatocellular carcinoma; HSC, hepatic stellate cells; Il1b, interleukin 1b; L-fabp1, liver fatty acid binding protein 1; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis;
MCD, methionine choline-deficient; PlGF, placental growth factor; Scd1, stearoyl-CoA desaturase 1; TG, triglyceride; Tnf, tumor necrosis factor NVP-LDE225 alpha; VEGF, vascular endothelial growth factor; Vwf, Von Willebrand factor Both C56BL6/J and db/db mice on an MCD diet displayed significant weight loss after 3 days of MCD diet (P < 0.01) (Table 1). After 8 weeks of the MCD diet both mouse models lost 40% of their Clomifene initial body weight. Liver/body weight ratio significantly augmented after 3 days of MCD diet in C57/BL6 and after 2 weeks of MCD diet db/db mice (P < 0.05) (Table 1). These alterations can be taken into account for the onset of steatosis. Db/db mice had a significantly higher food consumption
compared to C57BL6/J mice. Nonetheless, there was no significant difference in food consumption between mice fed an MCD or a control diet (Table 1). Biochemical analysis of serum of C57BL6/J and db/db mice showed significantly increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels when fed a MCD diet (Table 1). Steatosis, inflammation, ballooning, and fibrosis were assessed histologically using H&E and Sirius Red staining. From 4 weeks onwards, liver sections of the C57BL6/J mice fed an MCD diet were scored as NASH (Fig. 1A). The liver of C57BL6/J mice fed the control diet were normal, whereas liver sections of mice on 8 weeks of the MCD diet showed large fat droplets, clusters of inflammatory cells, and ballooned hepatocytes (Fig. 1B,C). Liver histology of db/db mice fed a control diet showed steatosis and ballooning. Db/db mice on MCD diet developed NASH after 1 week of the MCD diet, which worsened visibly after 8 weeks (Fig. 1D-F).