ILRUN, a Human Plasma Lipid GWAS Locus, Regulates Lipoprotein Metabolism in Mice

Abstract

Rationale: Single-nucleotide polymorphisms near the ILRUN (inflammation and lipid regulator with ubiquitin-associated-like and NBR1 [next to BRCA1 gene 1 protein]-like domains) gene are genome-wide significantly associated with plasma lipid traits and coronary artery disease (CAD), but the biological basis of this association is unknown.

Objective: To investigate the role of ILRUN in plasma lipid and lipoprotein metabolism.

Methods and results: ILRUN encodes a protein that contains a ubiquitin-associated-like domain, suggesting that it may interact with ubiquitinylated proteins. We generated mice globally deficient for Ilrun and found they had significantly lower plasma cholesterol levels resulting from reduced liver lipoprotein production. Liver transcriptome analysis uncovered altered transcription of genes downstream of lipid-related transcription factors, particularly PPARα (peroxisome proliferator-activated receptor alpha), and livers from Ilrun-deficient mice had increased PPARα protein. Human ILRUN was shown to bind to ubiquitinylated proteins including PPARα, and the ubiquitin-associated-like domain of ILRUN was found to be required for its interaction with PPARα.

Conclusions: These findings establish ILRUN as a novel regulator of lipid metabolism that promotes hepatic lipoprotein production. Our results also provide functional evidence that ILRUN may be the casual gene underlying the observed genetic associations with plasma lipids at 6p21 in human.

Keywords: cholesterol; hepatocytes; lipids; lipoproteins; metabolism; ubiquitin.

Figure 1.. Ilrun expression and Ilrun whole body knockout mice.

A, Relative Ilrun mRNA expression across different tissues (fold change from pancreas) in male C57BL/6 mice was determined by RT-PCR. n=3/group. B, Targeting strategy for generation of IlrunKO mice. C, Multiple tissues of female WT (left) and IlrunKO (right) mice were stained with X-gal. Images of representative stained tissues and cross sections (light eosin) are shown. Scale bars represent 50μm. n=4/group. Deletion of Ilrun was validated by (D) RT-PCR (n=3/group) and (E) western blot. Results from representative tissues are shown. Data are shown as the mean ± SEM. Results in D were compared between genotypes by Mann-Whitney test.

Figure 2.. Deletion of Ilrun decreases plasma cholesterol levels in mice.

Four-hour fasted plasma from chow-fed male control (n=15) and IlrunKO (n=9) mice were collected, plasma (A) TC and (B) HDL-C levels were measured. C, Non-HDL-C was calculated by subtracting HDL-C from TC. D, FPLC analysis of pooled plasma from each genotype. WT control (n=10) and IlrunKO (n=6) mice were switched from chow diet to WTD for 14wk. Time course plasma (E) TC, (F) HCL-C and (G) non-HDL-C were determined. Pooled plasma from these mice were fractionated by FPLC (H). Data are shown as the mean ± SEM. Results in A, C, and E-G (area under the curve) were analyzed by Student’s t test. Results in B were analyzed by Mann-Whitney test. Results in E-G were also compared with two-way ANOVA with Sidak’s multiple comparison test. P values indicate comparisons between genotypes.

Figure 3.. Reconstitution of ILRUN in livers of IlrunKO mice restores plasma lipids to WT levels.

Male mice on chow diet were i.p. injected with AAV-null or AAV-ILRUN (WT-AAV-null n=5, IlrunKO-AAVnull and IlrunKO-AAVILRUN n=4/group). Plasma lipid levels were monitored before (0wk), 2wk and 4.5wk after AAV injection. Plasma TC (A) and HDL-C (B) levels were measured. Pooled plasma from groups were fractionated by FPLC and TC at (C) baseline and (D) 4.5 wk after AAV injection were measured by enzymatic assays. Data are shown as the mean ± SEM.

Figure 4.. Ilrun deficiency reduces liver and plasma apoA-I levels.

Primary hepatocytes from chow-fed male mice were isolated for (A) mRNA (n=9/group) and (B) apoA-I protein analysis (n=3/group). C, ApoA-I levels in whole plasma (n=6/group) and (D) HDL fractions of male mice on chow diet (n=8/pool) were examined by western blot. ApoA-I expression in (E) liver and (F) FPLC fractions of pooled plasma from WTD-fed male mice were compared by western blot. n=6/group. Data are shown as the mean ± SEM. Results in A were analyzed by Student’s t test with (Ilrun) or without (Apoa1) Welch’s correction.

Figure 5.. Deletion of Ilrun reduces VLDL-TG secretion rate.

Poloxamer 407 and ³⁵S-Methionine/Cysteine were i.v. injected into WTD-fed 4h-fasted male control (n=5) and IlrunKO (n=7) mice. A, Plasma TG levels before and after injection were measured by enzymatic assays and the line of best fit was determined by linear regression analysis. B, TG secretion rate was calculated based on the slope of TG accumulation curve. Plasma samples from different time points were fractionated by SDS-PAGE and secretion rate of newly synthesized (C) apoB100 and (D) apoB48 were quantified by autoradiography. Data are shown as the mean ± SEM. Results in B-D were compared by Mann-Whitney test. P values indicate comparisons between genotypes.

Figure 6.. Ilrun deficiency alters liver transcriptome profiles and increases hepatic PPARα/RXRα protein levels.

Livers from chow-fed male IlrunKO and WT mice were used for microarray analysis (n=4/group). A, Summary of up and down regulated genes at the cut-off of FDR<10% and absolute FC>1.5. B, Ingenuity pathway analysis was performed to predict upstream transcriptional regulators. Total PPARα and RXRα protein (top) and mRNA expression (bottom) in (C) livers (n=4/group) and (D) primary hepatocytes from chow-fed mice (n=3/group). Representative western blot images are from one of the experiments and reflect typical expression pattern between genotypes for proteins of interest. Data are shown as the mean ± SEM. RT-PCR results in C-D were analyzed by Mann-Whitney test.

Figure 7.. ILRUN binds to ubiquitinated proteins and PPARα.

A, Binding of full length or mutant forms of ILRUN to ubiquitinylated proteins was examined. B, Co-immunoprecipitation of PPARα and different forms of ILRUN followed by western blot analysis. C, Representative images depicting features typical of ILRUN stained HuH7 cells and livers using fluorescence microscope with a 20x objective lens (left) and confocal microscopy (right) are shown. Scale bars represent 50μm (left), 10μm (HuH7 confocal), and 5μm (liver confocal) respectively.