Proteomics Analysis to Identify and Characterize the Molecular Signatures of Hepatic Steatosis in Ovariectomized Rats as a Model of Postmenopausal Status

Abstract

Postmenopausal women are particularly at increased risk of developing non-alcoholic fatty liver disease (NAFLD). Here we aimed to determine the impact of postmenopausal-induced NAFLD (PM-NAFLD) in an ovariectomized rat model. Sixteen six-week-old Sprague-Dawley female rats were randomly divided into two groups (eight per group), for sham-operation (Sham) or bilateral ovariectomy (Ovx). Four months after surgery, indices of liver damage and liver histomorphometry were measured. Both serum aspartate aminotransferase (AST) and alanine aminotranferease (ALT) levels were significantly higher in the Ovx than Sham group. We performed quantitative LC-MS/MS-based proteomic profiling of livers from rats with PM-NAFLD to provide baseline knowledge of the PM-NAFLD proteome and to investigate proteins involved in PM-NAFLD by ingenuity pathways analysis (IPA) to provide corroborative evidence for differential regulation of molecular and cellular functions affecting metabolic processes. Of the 586 identified proteins, the levels of 59 (10.0%) and 48 (8.2%) were significantly higher and lower, respectively, in the Ovx group compared to the Sham group. In conclusion, the changes in regulation of proteins implicated in PM-NAFLD may affect other vital biological processes in the body apart from causing postmenopause-mediated liver dysfunction. Our quantitative proteomics analysis may also suggest potential biomarkers and further clinical applications for PM-NAFLD.

Keywords: ingenuity pathways analysis; liver; proteome.

Figure 1

Effect of Ovx on plasma estradiol (A), TG (B), TC (C), AST (D) and ALT (E) levels at four months after ovariectomy. Mean ± SEM (n = 8). * p < 0.05 and ** p < 0.0001 vs. Sham, respectively.

Figure 2

Effect of Ovx on liver gross and histopathology. (A) The body (B) liver weight and (C) liver mass/body weight ratio (relative liver weight, %) of Sham and Ovx rats at 4 months after ovariectomy. Mean ± SEM (n = 8). ** p < 0.005 versus Sham group; (D) Excised livers of Sham and Ovx rats; (E) Histological sections of the livers from Sham and Ovx rats with H&E staining. Specimens were photographed under a light microscope at 40×, 100×, and 200× magnification, and scale bars represent 500, 200, and 20 μm, respectively; (F) Histological sections of the livers from Sham and Ovx rats with Masson trichrome staining for collagenous scar tissue. Scale bars, 20 μm (magnification, 200×).

Figure 3

Venn diagrams comparing the common, only detected, or overlap of significantly regulated proteins in liver samples from Sham and Ovx rats. (A) Venn diagram of differentially expressed proteins; (B) Percentage distribution for the nine proteins detected only in the Sham group; (C) Percentage distribution for the 19 proteins detected only in the Ovx group; (D) Percentage distribution for the 40 proteins with significantly increased expression in the Ovx group compared to the Sham group; (E) Percentage distribution for the 39 proteins with significantly decreased expression in the Ovx group compared to the Sham group.

Figure 3

Venn diagrams comparing the common, only detected, or overlap of significantly regulated proteins in liver samples from Sham and Ovx rats. (A) Venn diagram of differentially expressed proteins; (B) Percentage distribution for the nine proteins detected only in the Sham group; (C) Percentage distribution for the 19 proteins detected only in the Ovx group; (D) Percentage distribution for the 40 proteins with significantly increased expression in the Ovx group compared to the Sham group; (E) Percentage distribution for the 39 proteins with significantly decreased expression in the Ovx group compared to the Sham group.

Figure 4

Classification of the differentially expressed proteins identified from Ovx and Sham groups. (A) Pie charts representing the distribution of the 88 differential proteins of the Sham group by their cellular location; (B) Pie charts representing the distribution of the 98 differential proteins of the Ovx group by their cellular location.

Figure 5

Comparison of canonical signaling pathways between the Ovx and Sham groups. The significantly enriched canonical pathways are displayed as a bar chart. Only the 14 most different pathways are shown, ranked by the significance in the Ovx group. The vertical line indicates a threshold of p < 0.05. Significantly enriched canonical pathways were determined by ingenuity systems pathways analyses and are displayed along the left y-axis. The x-axis displays the negative log of the p-value (blue bars), calculated by the right-tailed Fisher exact test.

Figure 6

Comparison of biologic function proteins between the Ovx and Sham groups. Only the 15 most different biologic functions are shown, ranked by the significance in the Ovx group. The vertical line indicates a threshold of p < 0.05.

Figure 7

Comparison of toxic function proteins between the Ovx and Sham groups. Only the 15 most different toxic functions are shown, ranked by the significance in the Ovx group. The vertical line indicates a threshold of p < 0.05.