PNPLA3-I148M reduces hepatic triacylglycerol secretion and mitigates diet induced left ventricular diastolic dysfunction

Figures

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  Fig. 1. Whole animal metabolic phenotyping in mice at ~ 20 weeks of age, following 12 weeks of MASH diet at thermoneutrality. Bodyweights (A), food intake (B), energy expenditure (C), respiratory exchange ratio (RER) (D), VO2 (oxygen uptake). (E), intraperitoneal glucose tolerance (IP-GTT) after 12–13 weeks of MASH diet (F), and intraperitoneal insulin tolerance test (IP-ITT) after 13–14 weeks of MASH diet (G) (performed at ~ 20–22 weeks of age). Data represents mean +/− s.e.m. *P < 0.05 as indicated by one-way ANOVA (A-E) or *P < 0.05 and **P < 0.01 by two-way ANOVA with repeated measures with a Tukey’s post-hoc analysis for glucose curves or one-way ANOVA for area under the curve analysis (F–G). Sample sizes were n = 9–12 mice per group except for glucose tolerance (n = 18–21 mice per group).

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  Fig. 2. Biochemical analysis & histological examination of liver tissue following 16 weeks of MASH diet or ~ 24 weeks of age. Representative western blot for human PNPLA3 in mouse liver tissue (A), liver to bodyweight ratios (B), liver triacylglycerol (TAGs) (C), liver cholesterol (D), plasma cholesterol (E), and representative hematoxylin & eosin-stained liver sections (F). Data represents mean +/− s.e.m. *P < 0.05 as indicated by one-way ANOVA with Tukey’s post-hoc analysis. Sample sizes were n = 8–12 mice per group.

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  Fig. 3. Liver inflammation and fibrosis following 16 weeks of MASH diet at ~ 24 weeks of age. Representative F4/80 + immune cell staining (red color) of liver tissue (A), and quantification of F4/80 + immune cells/area (B). mRNA expression of Fibrosis genes by qPCR (C): Acta2, Col1a1, MMP2, Timp1, TGF-β1, TGF-β1receptor, and inflammatory genes (D): CCL2, CXCl-10, TNF-α, IL-6, IL-1β. Sample sizes were n = 4–5 mice per group for F4/80 + staining and n = 8–10 mice per group for fibrotic and inflammatory gene expression.

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  Fig. 4. Echocardiography analysis of cardiac morphology and left ventricular diastolic function in mice following 16 weeks of the MASH diet or ~ 26 weeks of age. Representative Power Doppler traces of blood flow through the mitral valve (A), Heart rates (beats per minute) at time of echocardiography (B), % fractional shortening (C), % ejection fraction (D), Left ventricular mass (E), Left ventricular anterior wall thickness during diastole (LVAWd) (F), Left ventricular relaxation E/A ratios (G). Data represents means +/− s.e.m. *p < 0.05 **p < 0.01 as indicated by Two-way ANOVA with Tukey’s post-hoc analysis. Sample sizes were n = 4–6 mice per group.

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  Fig. 5. Hepatic TAG secretion in standard chow-fed mice at ~ 12 weeks of age and again following a 2-week recovery and 4 weeks of MASH diet at ~ 18 weeks of age. Plasma TAG levels collected from chow fed mice following 500 mg/kg injection with Tyloxapol (A). Plasma TAG levels in mice fed MASH diet for 4 weeks, following 500 mg/kg injection with Tyloxapol (B). Data represents means +/− s.e.m. * WT-PNPLA3 vs. PNPLA3-I148M, &GFP vs. WT-PNPLA3, #GFP vs. PNPLA3-I148M, *,&,#p < 0.05, ***p < 0.001 for indicated timepoints as indicated by two-way ANOVA with repeated measures (RM) and Holm-Sidak post-hoc analysis. An overall difference was observed between WT-PNPLA3 and PNPLA3-I148M. **p < 0.01 by one-way ANOVA with RM. Sample sizes were n = 7–8 mice per group for chow diet and n = 5 mice per group for MASH diet.

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  Fig. 6. Hepatic TAG secretion in standard chow fed mice at ~ 12 weeks of age following injection with the βeta3-adrenergic agonist CL 316, 243. (A), Timeline for the CL 316,243 (CL) experiment. Mice expressing GFP, WT-PNPLA3 or PNPLA3-I148M in the liver were injected twice, 8 h apart with CL 316,243 (1 mg/kg) and examined for plasma TAG secretion (B) plasma TAGs 24 h following the first injection. *P < 0.05 as indicated by one-way ANOVA to detect an overall genotype difference between GFP and I148M. *p < 0.05 for indicated timepoint difference between GFP and I148M as indicated by two-way ANOVA with repeated measures (RM) and Holm–Sidak post-hoc analysis. n = 4 mice per group. (C), liver TAG content of mice from A and B, following CL 316,243 injection and the TAG secretion assay. *P < 0.05 and **P < 0.01 as indicated by one-way ANOVA. n = 4 mice per group. (D), representative H&E images of liver sections from mice expressing the indicated AAV construct. Scale bar: 50 μm.

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  Fig. 7. Untargeted complex lipidomic analysis of liver, plasma and heart from mice post 16 weeks of MASH diet at ~ 24 weeks of age. Hepatic lipidome (A), Plasma lipidome (B), Cardiac lipidome (C). The -log (p-value) of PNPLA3-I148M vs. WT-PNPLA3 was plotted against the log2 foldchange of the peak intensities for indicated lipid from liquid chromatography mass spectrometry data analysis. Comparison is between PNPLA3-I148M and WT-PNPLA3 expressing mice. Sample sizes were n = 4–6 mice per group.

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  Fig. 8. Quantification of atherosclerotic lesions following 24 weeks of MASH diet or ~ 30 weeks of age. Representative images of Oil-Red-O stained aortic roots for atherosclerotic lesion analysis (A). Aortic sinus lesion area (% of total area * 104) (B), Total aortic sinus area (arbitrary units) (C). Representative western blot images for plasma ApoB100 and ApoB48 (D). Plasma samples were blotted for ApoB and ApoB100 and ApoB48 were quantified. Graphs represent quantified expressions of ApoB100 (E) and ApoB48 (F) from western blot shown in (D), which were generated using ImageJ. Data represents mean +/− s.e.m. No Significance differences were observed by one-way ANOVA on GraphPad. Sample sizes were n = 4–6 mice per group.