Transportation of vitamin C [also called ascorbic acid (AA)], an important water-soluble antioxidant and cofactor in testis, requires glucose transporter family (GLUTs) and sodium/vitamin C cotransporter family (SVCT1 and SVCT2). There is so far scant information vis-à-vis the functional roles of SVCTs in testis, although they possess higher affinity for transportation of AA compared to GLUTs. To analyze the biological effects of SVCT2 in testis, we assessed testicular expression of SVCT2 in different experimental settings and the effect of SVCT2 ablation on spermatogenesis. Persistent expression of SVCT2 was shown in the mouse testis at different stages of postnatal development, demonstrated on day 14 of testicular development in mice consistent with appearance of pachytene spermatocytes during the first wave of spermatogenesis. Testicular expression of SVCT2 was enriched in the cytoplasm of murine Sertoli cells (SCs). We then showed that in vivo inhibition of SVCT2 in mouse testis significantly impaired male fertility by causing oligozoospermia and asthenospermia, which mainly stemmed from a deficiency in lactate production. By generating the TM4SVCT2-/- cells and by profiling TM4SVCT2-/- cells with a constitutively activated HIF-1α mutant, we demonstrated that SVCT2 deficiency led to impaired lactate synthesis and reduced expression of Ldha mRNA in SCs. Mechanistically, ablation of SVCT2 resulted in ubiquitination and subsequent degradation of HIF-1α protein in the FSH-stimulated SCs. Collectively, our data document a novel testicular site of action of SVCT2 in the control of lactate synthesis by SCs, probably via ubiquitination-dependent regulation of HIF-1α stability.
Guozheng Gao, Yong Zhao, Ke Wang, and Fang Wang
Lijuan Yin, Fang Fang, Xinglei Song, Yan Wang, Gaoxiang Huang, Jie Su, Ning Hui, and Jian Lu
Cell adhesion to extracellular matrix (ECM) is controlled by multiple signaling molecules and intracellular pathways, and is pivotal for survival and growth of cells from most solid tumors. Our previous works demonstrated that dexamethasone (DEX) significantly enhances cell adhesion and cell resistance to chemotherapeutics by increasing the levels of integrin β1, α4, and α5 in human ovarian cancer cells. However, it is unclear whether the components of ECM or other membrane molecules are also involved in the pro-adhesive effect of DEX in ovarian cancer cells. In this study, we demonstrated that the treatment of cells with DEX did not change the expression of collagens (I, III, and IV), laminin, CD44, and its principal ligand hyaluronan (HA), but significantly increased the levels of intracellular and secreted fibronectin (FN). Inhibiting the expression of FN with FN1 siRNA or blocking CD44, another FN receptor, with CD44 blocking antibody significantly attenuated the pro-adhesion of DEX, indicating that upregulation of FN mediates the pro-adhesive effect of DEX by its interaction with CD44 besides integrin β1. Moreover, DEX significantly enhanced cell resistance to the chemotherapeutic agent paclitaxel (PTX) by activating PI-3K-Akt pathway. Finally, we found that DEX also significantly upregulated the expression of MUC1, a transmembrane glycoprotein. Inhibiting the expression of MUC1 with MUC1 siRNA significantly attenuated the DEX-induced effects of pro-adhesion, Akt-activation, and pro-survival. In conclusion, these results provide new data that upregulation of FN and MUC1 by DEX contributes to DEX-induced pro-adhesion and protects ovarian cancer cells from chemotherapy.
Zhi Zhang, Fang Wang, Bing-jian Wang, Guang Chu, Qunan Cao, Bao-Gui Sun, and Qiu-Yan Dai
Vascular extracellular matrix (ECM) remodelling, which is the result of disruption in the balance of ECM synthesis and degradation, induces vessel fibrosis and thereby leads to hypertension. Leptin is known to promote tissue fibrosis, while adiponectin has recently been demonstrated to be anti-fibrogenic in tissue fibrosis. In this study, we aimed to evaluate the leptin-antagonist function of adiponectin and to further elucidate the mechanisms through which adiponectin dampens leptin signalling in vascular smooth muscle cells, thus preventing excess ECM production, in our already established 3D co-culture vessel models. Our 3D co-culture vessel model, which mimics true blood vessels, is composed of vascular endothelial cells, vascular smooth muscle cells and collagen type I. We validated the profibrogenic effects of leptin and analysed matrix metalloproteinase 2 (MMP2), MMP9, tissue inhibitor of metalloproteinase 1 (TIMP1) and collagen types II/IV secretion in 3D vessel models. The protective/inhibitory effects of adiponectin were re-analysed by inhibiting adiponectin receptor 1 (AdipoR) and AdipoR2 expression in endothelial cells using RNAi technology. In the 3D vessel models, adiponectin blocked the leptin-stimulated secretion of collagen types II/IV and TIMP1 while significantly increasing MMP2/9 activity. In endothelial cells, adiponectin induced phosphorylation of AMPK, thereby suppressing leptin-mediated STAT3 phosphorylation through induction of SOCS3 in smooth muscle cells. Our findings indicate that adiponectin disrupted the leptin-induced vascular ECM remodelling via AdipoR1 and enhanced AMPK signalling in endothelial cells, which, in turn, promoted SOCS3 up-regulation in smooth muscle cells to repress leptin-stimulated phosphorylation of STAT3.
X Fang, R Palanivel, X Zhou, Y Liu, A Xu, Y Wang, and G Sweeney
Adiponectin has been shown to regulate glucose and fatty acid uptake and metabolism in skeletal muscle. Here we investigated the role of the recently cloned adiponectin receptor (AdipoR) isoforms in mediating effects of both globular (gAd) and full-length (fAd) adiponectin, and their regulation by hyperglycemia (25 mM, 20 h) and hyperinsulinemia (100 nM, 20 h). We used L6 rat skeletal muscle cells, which were found to express both AdipoR1 and AdipoR2 mRNA in a ratio of over 6:1 respectively. Hyperglycemia and hyperinsulinemia both decreased AdipoR1 receptor expression by approximately 50%, while the latter induced an increase of approximately threefold in AdipoR2 expression. The ability of gAd to increase GLUT4 myc translocation, glucose uptake, fatty acid uptake and oxidation, as well as AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation, was decreased by both hyperglycemia and hyperinsulinemia. Interestingly, hyperinsulinemia induced the ability of fAd to elicit fatty acid uptake and enhanced fatty acid oxidation in response to fAd. In summary, our results suggest that both hyperglycemia and hyperinsulinemia cause gAd resistance in rat skeletal muscle cells. However, hyperinsulinemia induces a switch toward increased fAd sensitivity in these cells.
Wei-An Lai, Yi-Ting Yeh, Wei-Ling Fang, Leang-Shin Wu, Nobuhiro Harada, Peng-Hui Wang, Ferng-Chun Ke, Wen-Ling Lee, and Jiuan-Jiuan Hwang
Estrogens are essential for female reproduction and overall well-being, and estrogens in the circulation are largely synthesized in ovarian granulosa cells. Using primary cultures of ovarian granulosa cells from gonadotropin-primed immature rats, we have recently discovered that pituitary FSH and ovarian cytokine transforming growth factor beta 1 (TGFβ1) induce calcineurin-mediated dephosphorylation–activation of cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC2) to modulate the expression of Star, Cyp11a1, and Hsd3b leading to increased production of progesterone. This study explored the role of calcineurin and CRTC2 in FSH and TGFβ1 regulation of Cyp19a1 expression in granulosa cells. Ovarian granulosa cells treated with FSH displayed increased aromatase protein at 24 h post-treatment, which subsided by 48 h, while TGFβ1 acting through its type 1 receptor augmented the action of FSH with a greater and longer effects. It is known that the ovary-specific Cyp19a1 PII-promoter contains crucial response elements for CREB and nuclear receptor NR5A subfamily liver receptor homolog 1 (LRH1/NR5A2) and steroidogenic factor 1 (SF1/NR5A1), and that the Nr5a2 promoter also has a potential CREB-binding site. Herein, we demonstrate that FSH+TGFβ1 increased LRH1 and SF1 protein levels, and their binding to the Cyp19a1 PII-promoter evidenced, determined by chromatin immunoprecipitation analysis. Moreover, pretreatment with calcineurin auto-inhibitory peptide (CNI) abolished the FSH+TGFβ1-upregulated but not FSH-upregulated aromatase activity at 48 h, and the corresponding mRNA changes in Cyp19a1, and Nr5a2 and Nr5a1 at 24 h. In addition, FSH and TGFβ1 increased CRTC2 binding to the Cyp19a1 PII-promoter and Nr5a2 promoter at 24 h, with CREB bound constitutively. In summary, the results of this study indicate that calcineurin and CRTC2 have important roles in mediating FSH and TGFβ1 collateral upregulation of Cyp19a1 expression together with its transcription regulators Nr5a2 and Nr5a1 in ovarian granulosa cells.
Juan Liu, Xiaocen Kong, Long Wang, Hanmei Qi, Wenjuan Di, Xiao Zhang, Lin Wu, Xia Chen, Jing Yu, Juanmin Zha, Shan Lv, Aisen Zhang, Peng Cheng, Miao Hu, Yujie Li, Jianhua Bi, Yan Li, Fang Hu, Yi Zhong, Yong Xu, and Guoxian Ding
Brown adipose tissue (BAT) increases energy expenditure and is an attractive therapeutic target for obesity. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1), an amplifier of local glucocorticoid activity, has been shown to modulate white adipose tissue (WAT) metabolism and function. In this study, we investigated the roles of 11β-HSD1 in regulating BAT function. We observed a significant increase in the expression of BAT-specific genes, including UCP1, Cidea, Cox7a1, and Cox8b, in BVT.2733 (a selective inhibitor of 11β-HSD1)-treated and 11β-HSD1-deficient primary brown adipocytes of mice. By contrast, a remarkable decrease in BAT-specific gene expression was detected in brown adipocytes when 11β-HSD1 was overexpressed, which effect was reversed by BVT.2733 treatment. Consistent with the in vitro results, expression of a range of genes related to brown fat function in high-fat diet-fed mice treated with BVT.2733. Our results indicate that 11β-HSD1 acts as a vital regulator that controls the expression of genes related to brown fat function and as such may become a potential target in preventing obesity.