Integrated hepatic transcriptome and proteome analysis of mice with high-fat diet-induced nonalcoholic fatty liver disease

Abstract

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease in the US and refers to a wide spectrum of liver damage, including simple steatosis, steatohepatitis, fibrosis and cirrhosis. The goal of the present study was to achieve a more detailed understanding of the molecular changes in response to high fat-induced liver steatosis through the identification of a differentially expressed liver transcriptome and proteome. Male C57/BL6 mice fed a high-fat lard diet for 8 weeks developed visceral obesity and hepatic steatosis characterized by significantly increased liver and plasma free fatty acid and triglyceride levels and plasma alanine aminotransferase activities. Transcriptome analysis demonstrated that, compared to the control diet (CD), high-fat diet changed the expression of 309 genes (132 up- and 177 down-regulated; by a twofold change and more, P<.05). Multiple genes encoding proteins involved in lipogenesis were down-regulated, whereas genes involved in fatty acid oxidation were up-regulated. Proteomic analysis revealed 12 proteins which were differentially expressed. Of these, glutathione S-transferases mu1 and pi1 and selenium-binding protein 2 were decreased at both the gene and protein levels. This is the first study to perform a parallel transcriptomic and proteomic analysis of diet-induced hepatic steatosis. Several key pathways involving xenobiotic and lipid metabolism, the inflammatory response and cell-cycle control were identified. These pathways provide targets for future mechanistic and therapeutic studies as related to the development and prevention of NAFLD.

Fig. 1

Liver histology (H&E staining) of CD-fed mice (A) and HFLD-fed mice (B). Fat accumulation in the hepatocyte has the shape of macrovacuole. (Magnification: 200x).

Fig. 2

2D-DIGE analysis of the mice liver fed HFLD vs. CD. Analysis of the resulting 2D-DIGE gel images showed 18 protein spots to be differentially expressed between the HFLD and CD groups. Of these, six protein spots were found to be up-regulated and 12 protein spots down-regulated. Differentially expressed protein spots were excised from preparative gels, in-gel-digested with trypsin and analyzed using MALDI-TOF/MS-MS. A number of proteins were represented by several spots. (A) Liver lysates of CD-fed mice were labeled with Cy3, HFLD-fed mice with Cy5; a mixture of both (1:1) was used as internal standard labeled with Cy2. (B) Representative image of a 2D-DIGE gel (an overlay of the three dye scan images) showing spot identification numbers (refer to those in table) for proteins found differentially expressed in HFLD-fed vs. CD-fed mice. (C) 3D view of MATI, SBP2, GSTm1, GSTp1.

Fig. 3

Validation of cDNA microarray analysis by qRT-PCR. Selenbp2, Gstp1 and Gstm1 were selected to validate cDNA microarray gene expression by qRT-PCR. qRT-PCR was performed in duplicate for each mouse (four mice per group). 18S was used as an endogenous control. Results are presented as the fold changes in mRNA expression of a given gene in HFLD-fed vs. CD-fed mice.

Fig. 4

Conformation of the 2D-DIGE results by Western blot analysis. (A) ApoE, GSTp1 and GSTmu1 proteins were selected to confirm 2D-DIGE results by Western blot analysis. (B) The intensity of protein bands was quantified by densitometry using the NIH Image software (NIH, Bethesda, MD, USA). For changes in protein level, ratios of the respective protein to GAPDH, as a housekeeping protein, and densitometric values were compared between HFLD- and CD-fed mice by Student’s t test.

Fig. 5

(A) Representative Western blots show no significant changes in Vanin-1 and lipoprotein lipase (LPL) at the protein level. Vanin-1 and LPL were selected as differentially expressed genes identified by cDNA microarray, but changes in their expression at the protein level were not detected by 2D-DIGE proteomic analysis. (B) The intensity of protein bands was quantified by densitometry using the NIH Image software (NIH, Bethesda, MD, USA). For changes in protein level, ratios of the respective protein to GAPDH, as a housekeeping protein, and densitometric values were compared between HFLD- and CD-fed mice by Student’s t test (*P<.05).