Effects of perilipin 2 antisense oligonucleotide treatment on hepatic lipid metabolism and gene expression - PubMed (original) (raw)

Effects of perilipin 2 antisense oligonucleotide treatment on hepatic lipid metabolism and gene expression

Yumi Imai et al. Physiol Genomics. 2012.

Abstract

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. We previously showed that Perilipin 2 (Plin2), a member of lipid droplet protein family, is markedly increased in fatty liver, and its reduction in the liver of diet-induced obese mice by antisense oligonucleotide (ASO) decreased steatosis and enhanced insulin sensitivity. Plin2-ASO treatment markedly suppressed lipogenic gene expression. To gain a better understanding of the biological role of Plin2 in liver, we performed microarray analysis to determine genes differentially regulated by Plin2-ASO compared with a control (scrambled) oligonucleotide (Cont). Male C57BL/6J mice on a high-fat diet were treated with Plin2- or Cont-ASO for 4 wk. Plin2-ASO decreased hepatic triglycerides, and this was associated with changes in expression of 1,363 genes. We analyzed the data for functional clustering and validated the expression of representative genes using real-time PCR. On the high-fat diet, Plin2-ASO decreased the expression of enzymes involved in fatty acid metabolism (acsl1, lipe) and steroid metabolism (hmgcr, hsd3b5, hsd17b2), suggesting that Plin2 affects hepatic lipid metabolism at the transcriptional level. Plin2-ASO also increased the expression of genes involved in regulation of hepatocyte proliferation (afp, H19), mitosis (ccna2, incenp, sgol1), and extracellular matrix (col1a1, col3a1, mmp8). Plin2-ASO had similar effects on gene expression in chow-fed mice. Together, these results indicate that Plin2 has diverse metabolic and structural roles in the liver, and its downregulation promotes hepatic fibrosis and proliferation.

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Figures

Fig. 1.

Fig. 1.

Mice on high-fat diet (HF) were treated for 4 wk with Plin2-ASO or control oligo (Cont). A: Plin2 mRNA in the liver was determined by qPCR. Enzymatic colorimetric assay comparing liver triglycerides (TG) (B) and liver cholesterol (Chol) (C) levels. Liver lipids were also separated and quantified by TLC. D: TG, E: monoglycerides (MG), F: cholesterol ester, and G: free cholesterol. Data are means ± SE, n = 5 per group. **P < 0.01 vs. Cont.

Fig. 2.

Fig. 2.

Expression of genes in the livers of HF mice treated with Plin2-ASO (ASO) and Cont was measured with qPCR. A: genes associated with fatty acid metabolism, i.e., acyl-CoA synthetase long-chain family member 4 (Acsl4), stearoyl-Coenzyme A desaturase 2 (SCD2), acyl-CoA synthetase long-chain family member 1 (Acsl1), and hormone-sensitive lipase (Lipe). B: genes associated with cholesterol metabolism, i.e., 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 5 (Hsd3b5), and hydroxysteroid (17-beta) dehydrogenase 2 (Hsd17b2). C: α-fetoprotein (Afp) and H19 fetal liver (H19). D: genes associated with mitosis/kinetochore, i.e., cycline A2 (Ccna2), inner centromere protein antigens 135/155kDa (Incenp), and shugoshin-like 1 (Sgol1). E: genes associated with extracellular matrix (ECM), i.e., type 1 α1 collagen (Col1a1), type 1 α2 collagen (Col1a2), type 3 α1 collagen (Col3a1), and matrix metallopeptidase 8 (Mmp8). Data are means ± SE, n = 4–5 per group. *P < 0.05 vs. Cont, **P < 0.01 vs. Cont, ***P < 0.005 vs. Cont. F: histological study of livers from Cont- and Plin2-ASO-treated mice. Top: immunofluorescence showing Plin2 (green) staining in the cytoplasm and DAPI (blue) in the nuclei. Bottom: Sirius red staining of collagen in Cont vs. Plin2-ASO liver. Scale bar, 25 μm.

Fig. 3.

Fig. 3.

A–E: effects of Plin2-ASO on hepatic gene expression in regular chow-fed mice. A: Plin2 and genes associated with fatty acid metabolism, i.e., SCD2, Acsl1, and Lipe. B: genes associated with cholesterol metabolism, i.e., Hmgcr, Hsd3b5, Hsd17b2. C: Afp and H19. D: genes associated with mitosis/kinetochore, i.e., Ccna2, Incenp, and Sgol1. E: genes associated with extracellular matrix, i.e., Col1a1, Col1a2, Col3a1, and Mmp8. F–H: hepatic gene expression in livers from regular chow (NC) and HF livers without Plin2-ASO treatment. F: genes associated with cholesterol metabolism, i.e., Hmgcr, Hsd3b5. G: Afp, H19, and Ccna2. H: ECM genes, i.e., Col1a1, Col1a2, and Col3a1. Data are means ± SE, n = 4 per group. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, and n.s. (no significant difference) between the groups.

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