Extended Data Fig. 4: Characterization of L-Usp20−/− mice and the regulation of hepatic HMGCR by glucose and insulin.
From: Feeding induces cholesterol biosynthesis via the mTORC1–USP20–HMGCR axis
a, Tissue distribution of mouse USP20 protein. b, Schematic of L-Usp20−/− gene-targeting strategy. c, Immunoblotting analysis of different tissues of WT and L-Usp20−/− mice. d-f, Body weight (d), food intake (e) and serum AST (f) of male WT and L-Usp20−/− (n = 5 per group) mice fed chow diet ad libitum. g, Incorporation of tritium-labelled H2O into sterol in kidney of the mice (n = 4 per group) as in Fig. 1f. h, Eight-week-old male L-Usp20−/− and their male WT littermates were fed chow diet and subjected to fasting and refeeding treatment. The metabolic parameters were measured. Each value represents mean ± SEM of data from five mice. *P < 0.05, **P < 0.01, *** P < 0.001 for the level of statistical significance (unpaired two-tailed Student’s t-test) between WT and L-Usp20−/− mice under the same condition. The “a” means refed WT mice versus fasted WT mice; “b” means refed L-Usp20−/− versus fasted L-Usp20−/−; “c” means fasted L-Usp20−/− versus fasted WT mice; “d” means refed L-Usp20−/− versus refed WT mice. i, Relative amounts of mRNAs in the livers of the mice treated as in (h). Each value represents mean of data from three mice. j, Immunoblotting analysis of hepatic HMGCR protein upon glucose and insulin stimulation. Eight-week-old male WT mice were fasted overnight and intraperitoneally injected with 2 mg g-1 glucose or 0.75 mU g-1 insulin or both. After 3 h, liver samples were subjected to immunoblotting. k, qPCR analysis of the mRNAs in mice livers (n = 3 per group) treated as in (j). l, WT mouse primary hepatocytes were untreated (-) or treated (+) with 25.5 mM glucose or 10 nM insulin for 3 h or 5 h. m, qPCR analysis the mouse primary hepatocytes (n = 3 per group) treated as in (l). n, Inhibition of mTOR signalling antagonizes insulin-induced stabilization of HMGCR. WT primary hepatocytes were grown in M199 medium. On day 1, cells were transfected plasmids and depleted of cholesterol by incubating in M199 medium containing 5% LPDS, 1 μM lovastatin and 10 μM mevalonate for 16 h. Then, the cells were switched to the same medium with indicated inhibitor (Wort. wortmannin, 0.2 μM; AKTi, AKT1/2 kinase inhibitor 10 μM; Rap. Rapamycin, 0.1 μM). After 2 h, cells were treated with or without 1 μg ml-1 25-HC plus 10 mM mevalonate in the absence (-) or presence (+) of insulin and the indicated inhibitors for 5 h. Immunoblots were performed with the indicated antibodies. o, Effect of AMPK inhibition Dorsomorphin. WT primary hepatocytes were transfected, depleted of sterol, treated with 1 μM Dorsomorphin, 5.5 mM (-) or 25.5 mM (+) glucose combined with 1 μg ml-1 25-HC plus 10 mM mevalonate for 5 h. p, Effect of AMPK activation A-769662. WT primary hepatocytes were transfected, depleted of sterol, treated with 1 μM AMPK activator A-769662, 25.5 mM (+) glucose combined with 1 μg ml-1 25-HC plus 10 mM mevalonate for 5 h. Experiments in (a, c, l–p) were performed as indicated twice with similar results. Statistical significance was determined using unpaired two-tailed Student’s t-test (d–g, k, m). Bars represent mean ± SEM.
