SEMA7AR148W mutation promotes lipid accumulation and NAFLD progression via increased localization on the hepatocyte surface

Figures

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  Figure 1. The Sema7aR145W heterozygous mutation promotes the progression of NAFLD in mice following HFD feeding. Male WT and Sema7aR145W heterozygous (HE) mice were fed with normal chow diet (NCD) or HFD for 26 weeks. (A) The dynamic changes in the gains of body weights. (B) Liver weights. (C) The liver/body weight ratios. NCD-WT (n = 5), NCD-HE (n = 6), HFD-WT (n = 5), HFD-HE (n = 6). (D) Representative images (original magnification, ×200) of H&E, Oil Red O, and Sirius red staining of liver sections. (E) Quantitative analysis of the NAFLD activity scores (NASs). (F) Hepatic TG and Tch levels. The data were analyzed by 1-way ANOVA with Tukey’s post hoc tests or by Kruskal-Wallis test with Dunn’s post hoc test analysis. *P < 0.05 versus the WT mice with NCD; #P < 0.05 versus the Sema7aR145W heterozygous mice with NCD; $P < 0.05 versus the WT mice with HFD. TG, triglycerides; Tch, total cholesterol.

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  Figure 2. The Sema7aR145W mutation causes intrahepatic accumulation of small lipid droplets in male mice at 10 weeks of age. (A) Representative images (original magnification, ×200) of H&E staining, Sirius red staining, and Oil Red O staining in WT and Sema7aR145W heterozygous and homozygous male mice at the age of 10 weeks. (B) Analysis of lipid droplets in the Oil Red O–stained liver sections of WT (n = 6) and Sema7aR145W heterozygous (n = 9) and homozygous male mice (n = 9). (C) The levels of serum ALT and AST in Sema7aR145W WT (n = 6), heterozygous (n = 9), and homozygous male mice (n = 9). The data were analyzed by 1-way ANOVA with Tukey’s post hoc tests or by Kruskal-Wallis test with Dunn’s post hoc test analysis.

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  Figure 3. The Sema7aR145W mutation increases hepatic FA and TG concentrations in mouse livers. Male WT and Sema7aR145W homozygous (HO) mice at 10 weeks old (n = 4 per group) were euthanized and their liver samples were prepared. (A) Gas chromatography tandem mass spectrometry (GC/MS) analysis of total saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) levels (μg/g of mouse liver) in WT and Sema7aR145W homozygous mouse livers. FFA, free fatty acid. (B) Quantification of hepatic long chain FA (FA μg/g of mouse liver) in WT and Sema7aR145W homozygous mice. (C) Score scatterplot corresponding to a principal component analysis of the lipidomic data in the livers of WT and Sema7aR145W homozygous mice. (D) Quantitative analysis of lipid ion (μg/g of mouse liver) in WT and Sema7aR145W homozygous mouse livers. (E) Heatmap analysis of the TG number of carbons and double bond contents in the livers of WT and Sema7aR145W homozygous mice. (F) Proteomic analysis in the livers of WT and Sema7aR145W heterozygous and homozygous mice (n = 5 per group). (G) Volcano plot of the quantified proteins from WT and Sema7aR145W homozygous mouse livers (n = 5 per…

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  Figure 4. The Sema7aR145W mutation increases FA and TG synthesis and FA uptake in mouse livers. (A) Proteomic heatmap analysis of the NAFLD pathway in the livers of WT and Sema7aR145W homozygous mice. The liver extracts from the WT2 and 3, WT4 and 5, HO2 and 3, and HO4 and 5 male mice were combined for proteomics analysis. (B) The relative levels of mRNA transcripts of the genes for FA and TG synthetic enzymes, FA uptake transporters, and FA partitioning in WT mice (n = 6), Sema7aR145W heterozygous mice (n = 5), and Sema7aR145W homozygous mice (n = 7). (C) Western blot analysis of the relative levels of Acc1, Fasn, Scd1, Gpat, Lipin2, Cd36, Fatp5, Caveolin1, Plin3, Plin5, and Cpt1β protein expression in the livers of WT (n = 4), Sema7aR145W heterozygous (n = 5), and Sema7aR145W homozygous (n = 5) mice. The data were analyzed by 1-way ANOVA with Tukey’s post hoc tests or by Kruskal-Wallis test with Dunn’s post hoc test analysis. *P < 0.05 versus the WT mice, #P < 0.05 versus the Sema7aR145W heterozygous mice.

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  Figure 5. The Sema7aR145W mutation enhances hepatic FA and TG synthesis and FA uptake by enhancing PKC-α signaling–stimulated expression of transcription factors Srebp1 and Chrebp and nuclear receptor Lxr. (A) Western blot analysis of the relative levels of Srebp1, Chrebp, Lxr, phosphorylated PKC-α, and PKC-α protein expression in 10-week-old male WT mice (n = 4), Sema7aR145W heterozygous mice (n = 5), and Sema7aR145W homozygous mice (n = 5). Western blot analysis of the relative levels of nuclear Srebp1, Chrebp, and Lxr proteins in primary hepatocytes from WT and Sema7aR145W homozygous mice (B) and in human hepatoma HepG2 cells (C) after transfection with the plasmid for the expression of SEMA7A_WT and SEMA7A_R148W proteins. (D) Representative Western blot of the relative levels of nuclear Srebp1, Chrebp, and Lxr proteins in nuclear extracts and (E) Fasn, Acc1, and Cd36 proteins in whole-cell lysates of primary mouse hepatocytes after transfection with empty vector (CTR) or the plasmid for the expression of PKCα_WT or PKCα_dominant negative (DN) mutant, respectively. All primary mouse hepatocytes were isolated from 12-week-old male WT and Sema7aR145W homozygous mice. Data are…

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  Figure 6. The Sema7aR145W mutation does not alter total Sema7a expression but increases Sema7a and integrin β1 in the cell surface membrane and activates PKC-α signaling in hepatocytes. Relative levels of Sema7a mRNA transcripts (A) and protein expression (B) in 10-week-old male WT, Sema7aR145W heterozygous, and homozygous mice. One-way ANOVA with post hoc analysis. (C) Immunofluorescence and (D) IHC analyses of Sema7a protein in the livers of WT and Sema7aR145W homozygous mice. Normal rabbit IgG was used as the negative control. Original magnification, ×200. (E) Relative expression levels of Sema7a and its receptors integrin β1 and plexin C1 in whole-cell lysates (left) and membrane fractions (right) extracted from primary mouse hepatocytes. (F) N-glycosylated-Sema7a protein (~130 kDa) was detected in primary mouse hepatocytes. Western blot revealed the deglycosylated Sema7a (black arrow). (G) Relative levels of Sema7a and integrin β1 proteins in whole-cell lysates (left) and membrane fractions (right) from human hepatoma HepG2 cells after transfection with the plasmid for SEMA7A_WT or SEMA7A_R148W. (H) Co-immunoprecipitation analysis of protein interactions among Sema7a, PKCα, and…

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  Figure 7. The SEMA7A mutation activates PKC-α signaling in hepatocytes. HepG2 cells were transfected with the plasmid for the expression of SEMA7A_WT or SEMA7A_V334I, _P302K, _P74L, _R66Q, _N559Y, or _T2M mutant, and the relative levels of ACC1, FASN, CD36, phosphorylated PKC-α (T638), and PKC-α expression in each group of cells were determined by Western blot. (A) Representative images of Western blot analyses. (B) Quantitative analysis of each mutant protein and (C) the relative levels of T638 and PKC-α in HepG2 cells from 3 separate experiments. The levels of each protein in the SEMA7A_WT–transfected cells were designated as 1. The data were analyzed by 1-way ANOVA with Tukey’s post hoc tests or by Kruskal-Wallis test with Dunn’s post hoc test analysis. *P < 0.05 versus the SEMA7A_WT cells.

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  Figure 8. The potential mechanism by which the SEMA7AR148W mutation causes lipid accumulation in hepatocytes. First, the mutation increases SEMA7A and its receptor integrin β1 proteins on the surface of cell membranes to promote PKC-α activation in hepatocytes. Second, the activated PKC-α signaling enhances the expression of transcriptional factors SREBP1 and ChREBP and nuclear receptor LXR, increasing FA and TG synthesis and FA uptake in hepatocytes. Finally, these increased the accumulation of small lipid droplets in the liver, leading to the development and progression of NAFLD.