TK-NOG mice, in which a herpes simplex virus type 1 thymidine kinase (TK) transgene under a mouse albumin promoter is expressed within the liver of highly immune-deficient NOG mice, were obtained from Taconic Biosciences. The TK converts an antiviral medication ganciclovir (GCV) into a toxic product that allows selective elimination of TK-positive cells in vivo. The cryopreserved primary human hepatocytes were obtained from Lonza. The humanized TK-NOG mice were prepared as previously described (29). Briefly, the TK-NOG mice at 8–9 weeks old received an i.p. injection of GCV at a dose of 25 mg/kg. One week later, 50 μL volume of 1 × 106 human primary hepatocytes suspended in HBSS solution were transplanted via intrasplenic injection. The serum human albumin in the mice was measured as an index of the extent of human hepatocyte replacement 8–12 weeks after transplantation. Humanized TK-NOG mice with serum human albumin levels above 0.5 mg/mL were used for experiments in which human hepatic genes could be reliably detected by qPCR. For the fasting-refeeding study, humanized mice were produced, and the experiment was carried out at Central Institute for Experimental Animals (CIEA). Humanized mice for the rest of the study were produced and analyzed at National Heart, Lung, and Blood Institute (NHLBI). For the fasting-refeeding study, humanized TK-NOG mice were allowed free access to food (fed, harvested at around 9–10 am) or subjected to a 24-hour food withdrawal (fasting, from ~9:00 am to 9:00 am), or subjected to a 24-hour food withdrawal followed by a 4-hour refeeding (refeeding from ~9:00 am to 1:00 pm) before tissue harvest. Animal data were excluded from experiments based on preestablished criteria of visible abnormal liver structure during sample harvest or other health issues such as fighting wounds or infections. According to the variability of metabolic parameters, group size was determined based on previous studies using similar assays within the laboratory and pilot experiments. Experimenters were not blinded to treatment group.

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