Dietary cholesterol reduces plasma triacylglycerol in apolipoprotein E-null mice: suppression of lipin-1 and -2 in the glycerol-3-phosphate pathway

Abstract

Background: Cholesterol metabolism is tightly regulated by both cholesterol and its metabolites in the mammalian liver, but the regulatory mechanism of triacylglycerol (TG) synthesis remains to be elucidated. Lipin, which catalyzes the conversion of phosphatidate to diacylglycerol, is a key enzyme involved in de novo TG synthesis in the liver via the glycerol-3-phosphate (G3P) pathway. However, the regulatory mechanisms for the expression of lipin in the liver are not well understood.

Methodology/principal findings: Apolipoprotein E-knock out (apoE-KO) mice were fed a chow supplemented with 1.25% cholesterol (high-Chol diet). Cholesterol and bile acids were highly increased in the liver within a week. However, the amount of TG in very low-density lipoprotein (VLDL), but not in the liver, was reduced by 78%. The epididymal adipose tissue was almost eradicated in the long term. DNA microarray and real-time RT-PCR analyses revealed that the mRNA expression of all the genes in the G3P pathway in the liver was suppressed in the high-Chol diet apoE-KO mice. In particular, the mRNA and protein expression of lipin-1 and lipin-2 was markedly decreased, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), which up-regulates the transcription of lipin-1, was also suppressed. In vitro analysis using HepG2 cells revealed that the protein expression of lipin-2 was suppressed by treatment with taurocholic acid.

Conclusions/significance: These data using apoE-KO mice indicate that cholesterol and its metabolites are involved in regulating TG metabolism through a suppression of lipin-1 and lipin-2 in the liver. This research provides evidence for the mechanism of lipin expression in the liver.

Figure 1. Accumulation of Chol in the liver and reduction of the plasma TG in the apoE-KO mice fed the high-Chol diet for 1 week.

After apoE-KO mice were fed normal chow (white bar) or the high-Chol diet (black bar) for 1 week, the liver and plasma were collected (n = 3–4 for each group). (a, b) Chol (a) and TG (b) contents in the liver tissue were determined. (c–f) Lipoproteins in the plasma from the apoE-KO mice were fractionated by ultracentrifugation. Chol content in the VLDL (c) and LDL (d) fractions and TG contents in the VLDL (e) and LDL (f) fractions was determined. *, p<0.05; ***, p<0.005.

Figure 2. Effects of the high-Chol diet on apoE-KO mice.

(a) The weights of the epididymal white adipose tissues (WAT) in apoE-KO mice fed normal chow (white bar) or the high-Chol diet (black bar) for up to 30 weeks were measured (n = 3–5 for each group). The data are expressed as the percent of epididymal WAT in the body weight (*p<0.05). (b,c) Epididymal tissues taken from mice fed normal chow (b) or the high-Chol (c) diet for 30 weeks. The brackets indicate the epididymal WAT. Note that WAT was diminished in the mice fed the high-Chol diet. (d) ApoE-KO mice (normal: n = 12, high-Chol: n = 14) were fed either the normal chow or high-Chol diet for up to 7 weeks. Statistical significance against the level at week 0 was calculated by ANOVA. *, P<0.05; **, P<0.01; ***, P<0.005. (e) The amounts of food consumed during each 7 day period in the cages were monitored (n = 2–4 for each cage, n = 10–12 mice in one group). The data are expressed as the amount of food consumed in a week per mouse.

Figure 3. Reduction of the expression of genes and proteins involved in the G3P pathway in the liver.

(a) The liver was collected from apoE-KO mice fed normal chow (white bar) or the high-Chol diet (black bar) for 1 week. The mRNA expression levels of genes involved in the G3P pathway (lipin-1α, lipin-1β, lipin-2, DGAT2, GPAT4 and AGPAT1) were analyzed by real-time RT-PCR (n = 7). (b) Changes in the protein levels of lipin-1 and lipin-2 in the liver of apoE-KO mice fed normal chow or the high-Chol diet for 1 week were analyzed by Western blotting. Quantitative analysis is indicated in the graph (n = 3). *, p<0.05; **, p<0.01; ***, p<0.005; ****, p<0.001.

Figure 4. Hepatic mRNA expression of lipogenic genes in apoE-KO mice fed normal chow (white bar) or the high-Chol diet (black bar) for 1 week.

The mRNA expression patterns of (a) SREBP-1c, FAS, SCD-1 and ME, (b) PGC-1α, PPARα and its target genes (ACOX-1 and CPT-1), (c) SREBP-2 and its target genes (HMG-CoA reductase and HMG-CoA synthase) and (d) FXR, in the liver were analyzed by real-time RT-PCR (n = 7). *, p<0.05; **, p<0.01; ***, p<0.005; ****, p<0.001.

Figure 5. The effect of bile acids on lipin-1 and -2 protein expression in HepG2 cells.

HepG2 cells were cultured in DMEM medium containing 10% lipoprotein-deficient serum , and TCA (0, 20, and 50 µM) was added for 24 h. Cells were lysed and analyzed by Western blotting. Quantitative analysis is shown with the graph (n = 3). *, p<0.05; **, p<0.01.