Mutation in the distal NPxY motif of LRP1 alleviates dietary cholesterol-induced dyslipidemia and tissue inflammation

Figures

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  Fig. 1. LRP1 distal NPxY motif mutation reduces dietary cholesterol-induced adiposity and adipose inflammation. A: Body weight and (B) adiposity measured as % body fat of wild-type (WT, filled bars) and LRP1 NPxY mutant mice (open bars) after feeding a low-fat chow, HF, or HFHC diet for 16 weeks (11–18 mice per group). C: Leptin mRNA levels in epididymal adipose tissue. D: Leptin protein levels in plasma. E: Adiponectin mRNA levels in epididymal adipose tissue. F: Representative histological images of HF diet-fed WT and mutant mice (left panel), quantification of adipocyte size (middle panel), and crown-like structures (right panel) from four mice per genotype. G: Representative histological images of HFHC diet-fed WT and mutant mice (left panel), quantification of adipocyte size (middle panel), and crown-like structures (right panel) from four mice per genotype. Scale bar = 100 μm. H: Expression levels of EMR1, MCP1/CCL2, and MIP1α/CCL3. Data represent mean ± SEM from eight mice per genotype. I: AKT and AKT phosphorylation in adipose tissues of HFHC-fed mice after insulin injection. Differences between groups in each experiment were evaluated by one-way ANOVA with the…

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  Fig. 2. LRP1 distal NPxY motif mutation impairs insulin signaling in isolated hepatocytes but does not exacerbate diet-induced hyperglycemia and hyperinsulinemia. Plasma glucose (A) and insulin levels (B) of wild-type (WT, filled bars) and LRP1 NPxY mutant mice (open bars) after feeding a low-fat chow, HF, or HFHC diet for 16 weeks were determined after an overnight fast (5–15 per group). C: Homeostatic assessment of insulin resistance (HOMA-IR) index calculated from the data shown in panels (A) and (B). Differences between groups in each experiment were evaluated by one-way ANOVA with the Student-Newman-Keuls test for multiple group comparisons. Bars with different letters denote differences at P < 0.01. Glucose tolerance tests were performed by intraperitoneal injection of glucose (2 g/kg body weight) and measuring blood glucose levels over a 2-h period in wild-type (solid symbols) and LRP1 NPxY mutant (open symbols) mice after feeding chow (D), HF (E), or HFHC diets (F). G: Primary hepatocytes from WT and LRP1 NPxY mutant mice were stimulated with 100 nM insulin for the indicated time points. Phosphorylation and expression of AKT1, AKT2, GSK3β, and Foxo1 were analyzed by Western blot.…

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  Fig. 3. LRP1 distal NPxY motif mutation reduces dietary cholesterol-induced hypercholesterolemia. Plasma triglyceride (A) and cholesterol levels (B) levels of WT (filled bars) and LRP1 NPxY mutant mice (open bars) after feeding a low-fat chow, HF, or HFHC diet for 16 weeks were determined after an overnight fast (6–11 mice per group). Differences between groups in each experiment were evaluated by one-way ANOVA with the Student-Newman-Keuls test for multiple group comparisons. Bars with different letters denote differences at P < 0.01. C: Pooled plasma samples from fasting WT (filled symbols) and LRP1 NPxY mutant mice (open symbols) fed low-fat chow diet, HF diet (D) or HFHC diet (E) were fractionated by FPLC for lipoprotein analysis. F: Western Blot analysis of ApoB, ApoE, and ApoA-I distribution in plasma samples fractionated by FPLC from WT and LRP1 NPxY mutant mice after feeding an HFHC diet for 16 weeks.

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  Fig. 4. LRP1 distal NPxY motif mutation reduces dietary cholesterol-induced liver steatosis. A: Liver weight, (B) hepatic triglycerides, (C) hepatic total cholesterol and (D) hepatic free cholesterol of WT (filled bars) and LRP1 NPxY mutant mice (open bars) after feeding a low-fat chow, HF, or HFHC diet for 16 weeks (7–9 mice per group). Differences between groups in each experiment were evaluated by one-way ANOVA with the Student-Newman-Keuls test for multiple group comparisons. Bars with different letters denote differences at P < 0.01.

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  Fig. 5. LRP1 distal NPxY motif mutation reduces dietary cholesterol-induced liver inflammation. A: The percentage of CD68+ cells (left panel) and CD11b+ cells (right panel) in livers of WT (filled bars) and LRP1 NPxY mutant mice (open bars) after feeding an HFHC diet for 16 weeks was quantified by flow cytometry (seven mice per genotype). B, C: Expression levels of EMR1, CD68, CD11b, MCP1, Mip1α, ΤΝFα, and IL-1β in livers of WT (filled bars) and LRP1 NPxY mutant mice (open bars) after feeding a low-fat chow, HF, or HFHC diet for 16 weeks. Data represent mean ± SEM from 7–10 mice per group. Differences between groups in each experiment were evaluated by one-way ANOVA with the Student-Newman-Keuls test for multiple group comparisons. Bars with different letters denote differences at P < 0.01.

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  Fig. 6. LRP1 distal NPxY motif mutation does not affect VLDL synthesis but improves plasma lipid clearance via elevated LDL receptor (LDLR) levels in the liver. A: VLDL synthesis/secretion and (B) plasma lipid clearance in WT (filled symbols) and LRP1 NPxY mutant mice (open symbols) fed HFHC diet for 16 weeks (5–9 mice per group). C: Representative Western blot analysis of hepatic LDLR and cadherin levels in WT and LRP1 NPxY knock-in mutant mice (KI) fed chow, HF, or HFHC diets. LDLR levels from four mice in each group were presented in the bar graph as mean ± SEM. Bars with different letters denote differences at P < 0.01. The mRNA levels of LDLR and PCSK9 in the livers of wild-type (filled bars) and LRP1 NPxY mutant mice (filled bars) after feeding chow, HF, or HFHC diets are shown in panels D and E, respectively. Differences in mRNA expression levels were evaluated by one-way ANOVA with the Student-Newman-Keuls test for multiple group comparisons. Bars with different letters denote differences at P < 0.01.

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  Fig. 7. LRP1 distal NPxY motif mutation alleviates cholesterol-induced LXR activation in the liver. Expression levels of (A) SREBP2, (B) HMGCS, (C) HMGCR, (D) SREBP1, (E) FASN, (F) SCD1, (G) LXRα, and (H) ABCA1 in livers of WT (filled bars) and LRP1 NPxY mutant mice (open bars) after feeding a low-fat chow, HF, or HFHC diet for 16 weeks. Data represent mean ± SEM from 7–10 mice per group. Differences between groups in each experiment were evaluated by one-way ANOVA with the Student-Newman-Keuls test for multiple group comparisons. Bars with different letters denote differences at P < 0.01.

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  Fig. 8. LRP1 distal NPxY motif mutation reduces dietary cholesterol-induced plasma cytokine levels. Plasma levels of TNFα and IL-6 were measured in wild-type (filled bars) and LRP1 NPxY mutant (open bars) mice after feeding chow-, HF-, or HFHC diets. Data represent mean ± SEM from six mice in each group. Differences between groups in each experiment were evaluated by one-way ANOVA with the Student-Newman-Keuls test for multiple group comparisons. Bars with different letters denote differences at P < 0.01. N.D., not detectable.

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  Fig. 9. LRP1 distal NPxY motif mutation reduces cholesterol-induced neuroinflammation. A: Expression levels of TNFα, IL1β, and TGFβ mRΝΑ. B: Western blot analysis of LRP1, PSD-95, ARC, and Iba1 proteins in brains of WT (filled bars) and LRP1 NPxY mutant mice (open bars) after feeding a low-fat chow or HFHC diet for 16 weeks. Data represent mean ± SEM from 7–8 mice per group. Differences between groups in each experiment were evaluated by one-way ANOVA with the Student-Newman-Keuls test for multiple group comparisons. Bars with different letters denote differences at P < 0.01.