Apolipoprotein D Transgenic Mice Develop Hepatic Steatosis through Activation of PPARγ and Fatty Acid Uptake

Abstract

Transgenic mice (Tg) overexpressing human apolipoprotein D (H-apoD) in the brain are resistant to neurodegeneration. Despite the use of a neuron-specific promoter to generate the Tg mice, they expressed significant levels of H-apoD in both plasma and liver and they slowly develop hepatic steatosis and insulin resistance. We show here that hepatic PPARγ expression in Tg mice is increased by 2-fold compared to wild type (WT) mice. Consequently, PPARγ target genes Plin2 and Cide A/C are overexpressed, leading to increased lipid droplets formation. Expression of the fatty acid transporter CD36, another PPARgamma target, is also increased in Tg mice associated with elevated fatty acid uptake as measured in primary hepatocytes. Elevated expression of AMPK in the liver of Tg leads to phosphorylation of acetyl CoA carboxylase, indicating a decreased activity of the enzyme. Fatty acid synthase expression is also induced but the hepatic lipogenesis measured in vivo is not significantly different between WT and Tg mice. In addition, expression of carnitine palmitoyl transferase 1, the rate-limiting enzyme of beta-oxidation, is slightly upregulated. Finally, we show that overexpressing H-apoD in HepG2 cells in presence of arachidonic acid (AA), the main apoD ligand, increases the transcriptional activity of PPARγ. Supporting the role of apoD in AA transport, we observed enrichment in hepatic AA and a decrease in plasmatic AA concentration. Taken together, our results demonstrate that the hepatic steatosis observed in apoD Tg mice is a consequence of increased PPARγ transcriptional activity by AA leading to increased fatty acid uptake by the liver.

Fig 1. PPARγ and C/EBP expression in the liver of H-apoD Tg mice.

Semi- quantitative RT-PCR (A) and Western blot (B) analysis of PPARγ expression in liver and skeletal muscle of WT and H-apoD Tg mice. A- Graphs represent the mRNA expression level normalized by HPRT. A representative gel is presented above. B- The graph represents the level of PPARγ protein expression standardized by amidoblack staining. Muscle tissue was used for PPARγ1/PPARγ2 positive control. Semi-quantitative RT-PCR analysis of C/EBPα (C) and C/EBPβ (D) mRNA expression. The graphs represent the level of mRNA expressions normalized by HPRT. Values are expressed relatively to the WT mice and are the means ± SD of 4 mice per group. *P<0.05 and **P<0.01 vs WT mice.

Fig 2. Lipid droplets formation in the liver of H-apoD Tg mice.

A- Western blot analysis of Plin2 expression. The graph represents the level of Plin2 protein expression standardized by amidoblack staining. A representative gel is presented. Semi-quantitative RT-PCR analysis of Cide A (B), Cide B (C) and Cide C (D) mRNA expression. The graphs represent the level of mRNA expressions normalized by HPRT. Values are expressed relatively to the WT mice and are the means ± SD of 4 mice per group. E-Confocal analysis of lipid droplets in liver tissues of WT and H-apoD Tg mice. Lipid droplets are stained with bodipy (in green) and nucleus with propidium iodide (in red). Graphs represent the quantification of 18 images. *P<0.05, **P<0.01, P<0.001 vs WT mice.

Fig 3. FFA uptake in the liver of H-apoD Tg mice.

A- Semi-quantitative RT-PCR analysis of CD36 expression in liver tissue from WT and H-apoD Tg mice. Graph represents the mRNA expression levels normalized by HPRT. Representative gels are presented. Values are expressed relatively to the WT mice and are the means ± SD of 4 mice per group. B- ³H-oleate uptake was evaluated in primary hepatocytes prepared from WT and Tg mice. Results are expressed as CPM of ³H per mg of hepatic protein and represent the mean of 3 independent experiments. **P<0.01 vs WT mice.

Fig 4. Lipogenesis in the liver of H-apoD Tg mice.

Western blot analysis of total and phospho-AMPKα (A), total and phospho-ACC (B) and FAS (C) protein expression in the liver of WT and H-apoD Tg mice. The graphs represent the levels of protein expressions standardized by amidoblack staining. Representative gels are presented. D- Semi-quantitative RT-PCR analysis of ACC, SCD1, DGAT and LXRα mRNA expression. The graph represents the level of mRNA normalized by HPRT. Representative gels are presented. E- In vivo lipogenesis measured in 1 year old mice. The values represent the amount of ³H₂O incorporated into triglycerides. Values are expressed relatively to the WT mice and are the means ± SD of 4 mice per group. *P<0.05, **P<0.01 vs WT mice.

Fig 5. Analysis of genes involved in β-oxidation in the liver of H-apoD Tg mice.

A- Western blot analysis of PPARα protein expression. The graph represents the level of PPARα protein expression standardized by amidoblack staining. A representative gel is presented. Semi-quantitative RT-PCR analysis of PGC-1α (B) and CPT1 (C) expression in liver of WT and H-apoD Tg mice. PGC1α and CPT1 gene expression was normalized by HPRT. For each graph, the H-apoD Tg values were normalized by the WT values and are the means ± SD of 4 mice per group. *P<0.05 and **P<0.01 vs WT mice.

Fig 6. PPARγ transcriptional activity in presence of AA and/or apoD.

A- HepG2 cells were either non transfected (NT) or transfected with a myc-Tag apoD-cDNA or empty vector (EV) construct and incubated with BSA or arachidonic acid (AA). The level of H-apoD expression was evaluated by Western blot using a specific H-apoD antibody. B- HepG2 cells were transfected with UAS-Luc, GAL4-PPARγ, β-galactosidase and with either an empty vector or a myc-Tag apoD-cDNA construct. After transfection, cells were treated with 7 μM AA for 4h. Luciferase activity represents data normalized by β-galactosidase activity. The data represent the mean ± SD (n = 3). *P<0.05 and **P<0.01 vs the non-stimulated control without apoD. The gel presented below showed the expression of apoD in transfected cells using a myc antibody.

Fig 7. Concentration of plasmatic and hepatic AA in WT and H-apoD mice.

The concentration of arachidonic acid was evaluated by isotope dilution gas chromatography-mass spectrometry and reported in A) plasma as absolute values (left panel: μM) and relative to total fatty acid content (right panel: %); and in B) liver as absolute values (left panel: nmol/mg tissue wet weight) and relative to total fatty acid content (%). The data represent the mean ± SD (n = 3 for plasma and 3 for liver). *P<0.05 vs WT mice.