Multi-omics integrative analysis to access role of coiled-coil domain-containing 80 in lipid metabolism.

Coiled-coil domain-containing 80 (Ccdc80) is closely linked to energy homeostasis. However, the molecular mechanism remains unclear. This study aims to uncover the potential mechanism of Ccdc80 in modulating lipid metabolism by accessing the metabolic and transcriptional consequences of removing Ccdc80. We established a Ccdc80 knockout model (Ccdc80-/-) in C57BL/6 mouse. Serum and liver samples from Ccdc80+/+ (n = 8) and Ccdc80-/- (n = 8) male mice were obtained at the age of week 10. The serum metabolites and lipids were analyzed by gas chromatography-mass spectrometry and ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, respectively. RNA expression microarray was performed in the livers of the same mice. Results showed that a total of 58 metabolites and 30 lipids were altered between the Ccdc80+/+ and Ccdc80-/- mice. A total of 873 hepatic differentially expressed genes (DEGs) were identified. The enrichment analysis of discriminant metabolites and lipids reflected alterations in α-linolenic acid and linoleic acid metabolism. Reactome pathway analysis of DEGs revealed a decreased hydroxylation of arachidonic acid in Ccdc80-/- mice. The Kyoto Encyclopedia of Genes and Genomes pathway result suggested a decrease of PPAR signaling and fatty acid degradation by Ccdc80-knockout. The joint pathway analysis integrating metabolomics, lipidomics and transcriptomics indicated that Ccdc80-knockout could down-regulate arachidonic acid and α-linolenic acid metabolism. These results provide new insights into the role of Ccdc80 in fatty acid metabolism.

Deficiency of coiled-coil domain containing 80 increases plasma cholesterol by decreasing fecal sterols excretion in hypercholesterolemic mice.

Disorders in cholesterol and bile acid metabolism have been acknowledged as critical in pathogenesis of hypercholesterolemia. Coiled-coil domain containing 80 (CCDC80) has been closely linked to lipid homeostasis in mice, with its role in cholesterol metabolism yet to be fully elucidated. This study aims to uncover the regulatory mechanisms of CCDC80 in diet-induced hypercholesterolemia. We generated a CCDC80 knockout (CCDC80-/-) model in C57BL/6 mouse. The initial transcriptional and metabolic consequences of removing CCDC80 were accessed at baseline by gene expression microarrays and gas chromatography-mass spectrometry / ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, respectively. The hepatic cholesterol was investigated in both CCDC80+/+ and CCDC80-/- male mice at baseline and after feeding a high-cholesterol diet for 12 weeks. The regulatory effects of CCDC80 on gene expressions and protein masses were measured by RT-qPCR and western blot, respectively. At baseline, the KEGG pathway enrichment analysis combining metabolomics, lipidomics and transcriptomics, revealed a down-regulation of hepatic bile acid biosynthesis by CCDC80-knockout, especially for primary bile acids. In the hypercholesterolemic models, our results showed that deficiency of CCDC80 increased plasma and liver cholesterol levels, but decreased fecal neutral and acidic sterols excretion in mice. Mechanistically, we found that such effects were partly mediated by attenuating the alternative pathway of bile acid synthesis catalyzed by oxysterol 7-alpha-hydroxylase (CYP7B1). In conclusion, our results suggest CCDC80 as a novel modulator of cholesterol homeostasis in male mice. Deficiency of CCDC80 could further impair fecal sterols excretion in diet-induced hypercholesterolemia.

The Influence of LepR Tyrosine Site Mutations on Mouse Ovary Development and Related Gene Expression Changes.

Leptin exerts many biological functions, such as in metabolism and reproduction, through binding to and activating the leptin receptor, LepRb, which is expressed in many regions of the brain. To better understand the roles of LepR downstream signaling pathways, Y123F mice, which expressed mutant leptin receptors with phenylalanine (F) substituted for three tyrosines (Y) (Tyr985, Tyr1077 and Tyr1138), were generated. The body weight and abdominal fat deposits of Y123F homozygous mice (HOM) were higher than those of wild-type mice (WT). HOM ovaries were atrophic and the follicles developed abnormally; however, the HOM ovaries did not exhibit polycystic phenotypes. Moreover, Y123F HOM adults had no estrous cycle and the blood estrogen concentration remained stable at a low level below detection limit of 5 pg/ml. LepR expression in HOM ovaries was higher than in WT ovaries. Using cDNA Microarrays, the mRNA expressions of 41 genes were increased, and 100 were decreased in HOM vs. WT ovaries, and many signaling pathways were evaluated to be involved significantly. The expressions of 19 genes were validated by real-time quantitative PCR, most of which were consistent with the microarray results. Thus, Y123F HOM mice were suggested as a new animal model of PCOS for research that mainly emphasizes metabolic disorders and anovulation, but not the polycystic phenotype. Meanwhile, using the model, we found that JAK-STAT and hormone biosynthesis pathways were involved in the follicular development and ovulation disorders caused by LepR deficiency in ovaries, although we could not exclude indirect actions from the brain.

Downregulation of SPARC expression inhibits the invasion of human trophoblast cells in vitro.

Successful pregnancy depends on the precise regulation of extravilloustrophoblast (EVT) invasion into the uterine decidua. SPARC (secreted protein acidic and rich in cysteine) is a matricellular glycoprotein that plays critical roles in the pathologies associated with obesity and diabetes, as well as tumorigenesis. The objective of this study was to investigate the role of SPARC in the process of trophoblast invasion which shares many similarities with tumor cell invasion. By Western blot, higher expression of SPARC was observed in mouse brain, ovary and uterus compared to other mouse tissues. Immunohistochemistry analysis revealed a spatio-temporal expression of SPARC in mouse uterus in the periimplantation period. At the implantation site of d8 pregnancy, SPARC mainly accumulated in the secondary decidua zone (SDZ), trophoblast cells and blastocyst. The expression of SPARC was also detected in human placental villi and trophoblast cell lines. In a Matrigel invasion assay, we found SPARC-specific RNA interference significantly reduced the invasion of human extravilloustrophoblast HTR8/SVneo cells. Microarray analysis revealed that SPARC depletion upregulated the expression of interleukin 11 (IL11), KISS1, insulin-like growth factor binding protein 4 (IGFBP4), collagen type I alpha 1 (COLIA1), matrix metallopeptidase 9 (MMP9), and downregulated the expression of the alpha polypeptide of chorionic gonadotropin (CGA), MMP1, gap junction protein alpha 1 (GJA1), et al. The gene array result was further validated by qRT-PCR and Western blot. The present data indicate that SPARC may play an important role in the regulation of normal placentation by promoting the invasion of trophoblast cells into the uterine decidua.

Winter hibernation and UCHL1-p34cdc2 association in toad oocyte maturation competence.

Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1) is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor) controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34(cdc2), a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13(suc1) was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34(cdc2) protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte's dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34(cdc2) and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.