Fig 2: Effect of CM1 on the plasma lipid profiles and the expression of plasma apoAI, apoB and LPL (n = 5). A, plasma TC concentrations; B, plasma TG concentrations; C and D, plasma TC and TG profiles of the lipoproteins after ÄKTA-FPLC separation, respectively; E, F and G, plasma apoAI, apoB and LPL expression and densitometric quantification, respectively; H, plasma LPL activity. Data are presented as mean ± SD. Ezetimibe (Ezeti.): hamsters treated with ezetimibe at the dose of 25 mg/kg/d; CM1: LDLR(+/-) hamsters treated with CM1 at the dose of 100 mg/kg/d. All the abbreviations applied for the rest of the figures

Fig 3: Apolipoprotein B 100 secretion and SAR1. A, immunoblot of APOB in the top fraction of OptiPrep gradient from medium of WT and SAR1B-/- McArdle cells incubated with oleic acid for the indicated time. B, immunoblot of APOB in SAR1B-/- McArdle cells transformed by a lentivirus to overexpress the indicated SAR1 constructs after doxycycline induction. ß-Actin was used as a loading control. C, quantification of APOB secreted into medium (n = 3). D, proposed model of divergent roles of SAR1 paralogs in cells. SAR1A is proposed to homodimerize at the membrane for ER exit site remodeling, whereas SAR1B, which has a higher affinity for SEC23, leads to higher recruitment of the SEC23/24 heterodimer, enabling better secretion of large cargos that rely on tighter control of the kinetics of COPII vesicle formation.

Fig 4: Metformin alleviates non-obese NAFLD induced by GP73.a Microscale thermophoresis (MST) analysis of the interaction between metformin (Met) and GP73. The data were derived from the effect of metformin on the fluorescence decay of fluorescently labeled GP73. The half-maximum effective concentration (EC50) was determined by the Hill slope. n = 3 independent biological experiments. b, c Immunoprecipitation analysis of the interaction between GP73 and Rab23 (b) or TBC1D20 and Rab1b (c) in the presence or absence of Met. Data were repeated three times with similar results. d, e Kinetic analysis of GP73 activity toward Rab23 (d) or TBC1D20 activity toward Rab1b (e) in the presence of different concentrations of Met. n = 3 independent biological experiments. f, g ApoB (f) and ApoB100 (g) secretion efficiency in cells from Flag-vector- or Flag-GP73-transfected cells treated with Met. n = 3 independent biological experiments. Differences between the two groups were evaluated using one-way ANOVA and Bonferroni’s post hoc analysis. Data are presented as mean ± SEM. **P < 0.01; ***P < 0.001. h Schematic depicting the experimental setup. i–k Hepatic levels of TGs (i) and CHO (j); 6 h-fasted glucose levels (k) of AAV-V- or AAV-GP73-treated mice given normal drinking water (NW) or drinking water with metformin (3 g/L) (NW) for 4 months (n = 6 per group). Differences between the four groups were evaluated using one-way ANOVA and Bonferroni’s post hoc analysis. Data are presented as mean ± SEM. *P < 0.05.

Fig 5: GP73 harbors potent TBC-domain GAP activity that inhibits VLDLs’ secretion.a Comparison of amino acid sequences around R248 and Q310 in GP73 and equivalent residues in Gyp1p, VirA, EspG, and EspG2 with dual-finger catalytic motifs in the TBC domain. b GAP activity profiles of GP73 for a panel of 13 mammalian Rabs. The catalytic efficiency (kcat/KM) of GP73-catalyzed GTP hydrolysis relative to the intrinsic GTP hydrolysis rate constant was determined for each Rab. n = 3 independent biological experiments. Data are presented as mean ± SEM. c, d Kinetic analysis of GP73 GAP activity toward Rab23. The kcat/KM determined by using a Lineweaver-Burk plot is listed above the activity curves. n = 3 independent biological experiments. e Effects of R248K and Q310A mutations in GP73 (GP73-RQ) on GP73 GAP activity. n = 3 independent biological experiments. f, g ApoB (f) and ApoB100 (g) secretion efficiency of Huh-7 cells transfected with Flag-vector (Flag-V), Flag-GP73, or Flag-GP73-RQ mutant at the indicated time points after transfection. Secretion efficiency was calculated as the fraction secreted, defined as the ratio between the amounts of cargo that was secreted and the total amount of cargo (secreted plus cell-associated cargo) present in a well. n = 3 independent biological experiments. Differences between the two groups were evaluated using two-tailed Student’s t-test. Data are presented as mean ± SEM. ns, no statistical significance; **P < 0.01. h GP73 and ApoB100 protein levels in mice livers at week 3 after the injection of AAV-V or AAV-GP73 (n = 2 per group). a-Tubulin was used as the equal loading control. Relative expression was calculated as the fold change in expression relative to the expression in No. 1 control mice. i TG concentrations at the indicated time points after blood sampling of AAV-V-, AAV-GP73-, or AAV-GP73-RQ-injected mice fasted for 4 h and then intravenously administered tyloxapol (400 mg/kg; n = 6 per group). Differences between the three groups were evaluated using two-way ANOVA and Bonferroni’s post hoc analysis. Data are presented as mean ± SEM. ns, no statistical significance; ***P < 0.001.