SUN-033 RNA-Seq Analysis of Ovariectomy-Induced Changes in Mouse Liver Reveals New Targets for Menopause-Associated Metabolic Derangement

Abstract

Menopause is associated with significant adverse metabolic changes and an increased cardiovascular risk profile. Though well described, the underlying molecular mechanisms for these changes remain unclear. The liver is both mediator and target of metabolic derangement. In this study we used an unbiased RNA-sequencing approach to discover differently expressed genes in the oophorectomized mouse liver as well as response to treatment with 17-β-estradiol (E2). 5 groups of 9-week-old C57BL/6J female mice (10 per group) were subjected to ovariectomy (OVX) or sham operation and left untreated for 6 weeks to allow for development of metabolic changes. At this point 2 groups were sacrificed and tissues harvested. The rest of the mice were treated with E2 or vehicle for 6 weeks and then sacrificed. RNA-sequencing was performed on liver samples 6 weeks post surgery. Differential gene expression was assessed using DESeq2 and changes in gene sets using Gene set enrichment analyses (GSEA). Quantitative Real Time PCR was used to validate select differently expressed genes, as well as to assess changes with E2 treatment at 12 weeks post surgery. We found a total of 103 genes (35 up and 68 downregulated) to be differently expressed (fold change ≥ 2, adjusted P ≤ 0.05, max count ≥ 30) between OVX and SHAM at 6 weeks post surgery. GSEA showed significant upregulation (FDR < 0.01) in gene sets for: fatty acid metabolism (normalized enrichment score = 5.13), oxidative phosphorylation (3.91), myc targets (3.81), peroxisome (2.94), adipogenesis (2.82), androgen response (2.56), UV response up (2.49) and TNF-α signaling via NF-Kβ (2.17). Significant downregulated gene sets were: mitotic spindle (-2.88) and G2M checkpoint (-2.4). Specific differently expressed genes, known to be related to metabolic pathways, were validated using real-time PCR and analyzed at 6- and 12-weeks post-surgery with or without E2 treatment. Surprisingly, ENHO, encodes for Adropin, a secreted peptide and known regulator of fat metabolism, associated with cardiovascular health, was found to be significantly decreased at 6 weeks (fold change -2.1, adjusted p = 7.2e-9) and 12 weeks after OVX (-1.42, p=0.008), though it wasn’t changed following treatment with E2. Importantly, reduced ENHO expression was found in cardiomyocytes 12 weeks post-surgery (relative mRNA expression for OVX vs SHAM = 0.44, P=0.03). Our results show that ovariectomy induces transcriptional changes in liver expression of gene sets related to metabolism and inflammation. The role of ENHO encoding for adropin as a biomarker and therapeutic target in menopause-associated metabolic derangement remains to be investigated.