PPARɑ variant V227A reduces plasma triglycerides through enhanced lipoprotein lipolysis

Figures

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  Fig. 1. Generation of Ppara V227A mouse model. A: Schematic representation of the gRNA target site in the Ppara gene locus at exon 6. The protospacer sequence is shown with activity window nucleotides and PAM sequence indicated. Wild-type adenine being targeted is blue, and base-edited change corresponding to indicated human SNP is red. B: Clonal populations derived from Hepa1-6 cells transfected with base editing plasmids (n = 8) were sequenced with activity window mutations and frequency shown. C: Adult mice derived from mouse zygotes microinjected with base editing mRNAs (n = 45) were sequenced, and total substitution frequency along protospacer sequence was calculated. D: Activity window mutations and frequency from (C) is shown. E: Representative Sanger sequencing traces from V227A colony mice with indicated codons and corresponding amino acids. F: Genotyping strategy using SrfI restriction enzyme site generated following successful base editing. G: Representative agarose gel with indicated genotypes is shown.

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  Fig. 2. V227A decreases plasma triglycerides. A–D: Plasma triglyceride levels following a 6-h fast were quantified from chow-fed male (n = 13–16 per group) (A), chow-fed female (n = 15–16 per group) (B), Western diet-fed male (n = 10–11 per group) (C), and Western diet-fed female mice (n = 6–8 per group) (D). E: Plasma total cholesterol levels were quantified from mice in (A–D). F: Plasma total non-esterified fatty acid levels were quantified from mice in (A–D). G: 16-h fasted male mice were injected with poloxamer-407 (1 g/kg) followed by blood collection and plasma triglyceride quantitation at indicated times. n = 5 per group. ∗ = P < 0.05, ∗∗ = P < 0.01 as assessed by two-sided Student’s t test. Error bars are SEM.

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  Fig. 3. V227A does not alter body mass or liver lipid accumulation. A: Chow-fed male (n = 13–16 per group), chow-fed female (n = 15–16 per group), Western diet-fed male (n = 10–11 per group) and Western diet-fed female mice (n = 6–8 per group) were assessed for body weight, liver weight, and liver-to-body weight ratio following a 6-h fast. B and C: Liver triglycerides, liver total cholesterol and non-esterified fatty acids were quantified from liver tissue harvested from chow-fed mice (B) and Western diet-fed mice (C) indicated in (A). Error bars are SEM.

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  Fig. 4. V227A enhances hepatic PPARɑ target gene expression. A: Gene set enrichment analysis with Wikipathways curated gene sets using differential transcript abundance between V227A liver tissue relative to wild-type. Liver tissue subjected to RNA sequencing was obtained from 6-h fasted adult male mice. Gene sets with FDR < 0.05 are shown. B: PPAR signaling pathway gene set enrichment from (A). C: Volcano plot of differentially expressed genes in V227A liver tissue relative to wild-type. Deregulated genes whose P < 0.05 and log2 fold-change either > 1 or < −1 are blue. Number of deregulated genes are shown in corners based on direction of change. D-F: Log2 fold-change of top 500 differentially expressed genes were compared between V227A relative to wild-type (x-axis) and indicated comparison (y-axis) using gene expression of 6-h fasted mouse liver tissues from GSE8396. Comparisons include (D) fenofibrate treatment relative to vehicle, (E) liver-specific Ppara knock out mice relative to wild-type mice, and (F) Wy-14643-treated liver-specific Ppara knock out mice relative to Wy-14643-treated wild-type mice. Linear regression (black line) with 95% CI (grey area) are shown. Pearson…

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  Fig. 5. V227A increases Lpl transcript abundance in liver. A: Genes that account for GSEA enrichment of lipoprotein catabolism gene set used in Fig. 4I. B: qRT-PCR of PPARɑ target genes using liver tissue from 6-h fasted chow-fed male (n = 11–15 per group), chow-fed female (n = 12 per group), Western diet-fed male (n = 10–11 per group), and Western diet-fed female mice (n = 6–8 per group). C: Lpl transcript abundance in liver tissue from male mice with indicated fasting duration as assessed by qRT-PCR (n = 4–9 per group). D: Gene expression in liver tissue from 6-h fasted male mice gavaged with PPARɑ agonist GW7647 (10 mg/kg) as assessed by qRT-PCR (n = 8). E: Ppara binding to Lpl enhancer (Enh) and promoter genomic regions in 6-h fasted V227A liver tissue (n = 5) as assessed by ChIP-qPCR. Enrichment is normalized to Rpl35 promoter region that does not exhibit known PPARɑ binding (27). ∗ = P < 0.05, ∗∗ = P < 0.01, ∗∗∗ = P < 0.001 as assessed by Student’s t test. Error bars are SEM.

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  Fig. 6. V227A increases cardiac Lpl transcript abundance and lipase activity. A: Ppara and Lpl mRNA expression from indicated tissues obtained from three wild-type mice as assessed by qRT-PCR. Expression is normalized to liver abundance. B: Heart (n = 25–40), (C) liver (n = 13–27), (D) brown adipose tissue (BAT) (n = 25–40) and kidney (n = 9–13) gene expression from 6-h fasted wild-type and V227A mice fed chow diet. E: Heart gene expression from 6-h fasted wild-type and V227A female mice fed Western diet for 4 weeks (n = 9–11). F: LPL activity as assessed using heart and liver tissue harvested from 16-h fasted female mice fed Western diet for 4 weeks (n = 6–7). G: 3H disintegrations per minute (DPM) from heart tissue and liver tissue harvested from 16-h fasted male mice 15 min after tail vein inoculation of 3H-trigylceride chylomicrons. CPM normalized to kidney CPM. (n = 6). ∗ = P < 0.05, ∗∗∗∗ = P < 0.0001 as assessed by two-sided Student’s t test. Error bars are SEM.