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J Chen, L Wang, YS Boeg, B Xia, and J Wang

Secreted by white adipose tissue as a hormone, resistin was identified as a possible link between obesity and insulin resistance. High circulating resistin levels were observed to correlate with obesity. Administration of resistin lowered the glucose tolerance threshold and impaired insulin activity; whereas anti-resistin antibodies had the opposite effects. However, contradictory data were subsequently reported in regard to the correlation between resistin expression level and obesity or type 2 diabetes. Two additional proteins that share a highly homologous C-terminus with resistin have been identified in mouse, and one in human, forming a resistin-related protein family. Resistin was shown to dimerize through a disulfide bond formed by the N-terminal-most cysteine (Cys26). Here we demonstrate that while Cys26 is both necessary and sufficient for homodimer formation, all three resistin family members can also interact with one another regardless of the presence of Cys26 through non-covalent interactions. Furthermore, protein crosslinking analysis indicated that resistin and resistin beta, but not resistin alpha, exist as multimers, probably with a dimer as the subunit. The multiple protein complex formation is obviously at a level higher than the Cys26 disulfide bonding. These results suggest the potential importance of considering intermolecular interactions among resistin family members in studying their functions.

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Tianxue Zhao, Qian Li, Qianyun Mao, Kaida Mu, and Chen Wang

Neuronal nitric oxide synthase (nNOS) interacts with its adaptor protein NOS1AP through its PZD domain in the neurons. Previously, we had reported that NOS1AP enhanced hepatic insulin sensitivity through its PZD-binding domain, which suggested that nNOS might mediate the effect of NOS1AP. This study aimed to examine the role and underlying mechanisms of nNOS in regulating hepatic insulin sensitivity. nNOS co-localized with NOS1AP in mouse liver. The overexpression of NOS1AP in mouse liver decreased the level of phosphorylated nNOS (p-nNOS (Ser1417)), the active form of nNOS. Conversely, the liver-specific deletion of NOS1AP increased the level of p-nNOS (Ser1417). The overexpression of nNOS in the liver of high-fat diet-induced obese mice exacerbated glucose intolerance, enhanced intrahepatic lipid accumulation, decreased glycogen storage, and blunted insulin-induced phosphorylation of IRβ and Akt in the liver. Similarly, nNOS overexpression increased triglyceride production, decreased glucose utilization, and downregulated insulin-induced expression of p-IRβ, p-Akt, and p-GSK3β in the HepG2 cells. In contrast, treatment with Nω-propyl-L-arginine (L-NPA), a selective nNOS inhibitor, improved glucose tolerance and upregulated insulin-induced phosphorylation of IRβ and Akt in the liver of ob/ob mice. Furthermore, overexpression of nNOS increased p38MAPK phosphorylation in the HepG2 cells. In contrast, inhibition of p38MAPK with SB203580 significantly reversed the nNOS-induced inhibition of insulin signaling activity (all P < 0.05). This indicated that hepatic nNOS inhibited the insulin-signaling pathway through the activation of p38MAPK. These findings suggest that nNOS is involved in the development of hepatic insulin resistance and that nNOS might be a potential therapeutic target for diabetes.

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Cuili Wang, Dongteng Liu, Weiting Chen, Wei Ge, Wanshu Hong, Yong Zhu, and Shi X Chen

Our previous study showed that the in vivo positive effects of 17α,20β-dihydroxy-4-pregnen-3-one (DHP), the major progestin in zebrafish, on early spermatogenesis was much stronger than the ex vivo ones, which may suggest an effect of DHP on the expression of gonadotropins. In our present study, we first observed that fshb and lhb mRNA levels in the pituitary of male adult zebrafish were greatly inhibited by 3 weeks exposure to 10nM estradiol (E2). However, an additional 24h 100nM DHP exposure not only reversed the E2-induced inhibition, but also significantly increased the expression of fshb and lhb mRNA. These stimulatory effects were also observed in male adult fish without E2 pretreatment, and a time course experiment showed that it took 24h for fshb and 12h for lhb to respond significantly. Because these stimulatory activities were partially antagonized by a nuclear progesterone receptor (Pgr) antagonist mifepristone, we generated a Pgr-knockout (pgr –/–) model using the TALEN technique. With and without DHP in vivo treatment, fshb and lhb mRNA levels of pgr –/– were significantly lower than those of pgr +/+. Furthermore, ex vivo treatment of pituitary fragments of pgr –/– with DHP stimulated lhb, but not fshb mRNA expression. Results from double-colored fluorescent in situ hybridization showed that pgr mRNA was expressed only in fshb-expressing cells. Taken together, our results indicated that DHP participated in the regulation of neuroendocrine control of reproduction in male zebrafish, and exerted a Pgr-mediated direct stimulatory effect on fshb mRNA at pituitary level.

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Xiaoli Wang, Fei Chang, Yinyang Bai, Fang Chen, Jun Zhang, and Ling Chen

Bisphenol-A (BPA), an environmental estrogen, adversely affects female reproductive health. However, the underlying mechanisms remain largely unknown. We found that oral administration (p.o.) of BPA (20 μg/kg) to adult female mice at proestrus, but not at estrus or diestrus, significantly increased the levels of plasma E2, LH and FSH, and Gnrh mRNA within 6 h. The administration of BPA at proestrus, but not at diestrus, could elevate the levels of Kiss1 mRNA and kisspeptin protein in anteroventral periventricular nucleus (AVPV) within 6 h. In contrast, the level of Kiss1 mRNA in arcuate nucleus (ARC) was hardly altered by BPA administration. In addition, at proestrus, a single injection (i.c.v.) of BPA dose-dependently enhanced the AVPV-kisspeptin expression within 6 h, this was sensitive to E2 depletion by ovariectomy and an estrogen receptor α (ERα) antagonist. Similarly, the injection of BPA (i.c.v.) at proestrus could elevate the levels of plasma E2, LH, and Gnrh mRNA within 6 h in a dose-dependent manner, which was blocked by antagonists of GPR54 or ERα. Injection of BPA (i.c.v.) at proestrus failed to alter the timing and peak concentration of LH-surge generation. In ovariectomized mice, the application of E2 induced a dose-dependent increase in the AVPV-Kiss1 mRNA level, indicating ‘E2-induced positive feedback’, which was enhanced by BPA injection (i.c.v.). The levels of Erα (Esr1) and Erβ (Esr2) mRNAs in AVPV and ARC did not differ significantly between vehicle-and BPA-treated groups. This study provides in vivo evidence that exposure of adult female mice to a low dose of BPA disrupts the hypothalamic–pituitary–gonadal reproductive endocrine system through enhancing AVPV-kisspeptin expression and release.

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Xiaohui Wang, Yuxia Chen, Yan Wang, Xiaoyan Zhu, Yuanyuan Ma, Shimin Zhang, and Jian Lu

Although glucocorticoid (GC) has been reported to inhibit macrophage killing activity and cytokine production in response to proinflammatory stimuli, the effect of GC on macrophage proliferation is controversial. In our previous study, we found that inhibition of glucocorticoid receptor (GR) expression in murine macrophage cell line RAW264.7 cells (RAW-GR(−) cells) by RNAi significantly promoted cell proliferation. In the present study, we provide the evidence that the expression of Rhob, a member of Rho GTPases with anti-cancer character, remarkably decreased in RAW-GR(−) and RAW264.7 cells transiently transfected with GR-RNAi vector. Overexpression or constitutive activation of Rhob in RAW-GR(−) and RAW264.7 cells by transfection with wild-type Rhob expression vector (Rhob-wt) or constitutively activated Rhob plasmid (Rhob-V14) resulted in decreased proliferation of the two cell lines. Oppositely, the proliferation of RAW264.7 cells was significantly increased when the expression of Rhob by RNA interference technique or the activity of Rhob by transfection with dominant negative Rhob mutant that is defective in nucleotide binding (Rhob-N19) was inhibited. In addition, enhanced activity of Akt, but not MAPK3/1 or MAPK14, was found in RAW-GR(−) cells. Blocking the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt with the specific inhibitor LY294002 decreased the proliferation and elevated RHOB protein level, indicating that PI3K/Akt signal plays its role of proliferation modulation upstream of RHOB protein. In conclusion, these results demonstrate that Rhob plays an important role in the antiproliferative effect of GR on RAW264.7 cells by GR→Akt→Rhob signaling and Rhob negatively regulates the proliferation of RAW264.7 cells.

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Weixia Han, Chen Wang, Zhifen Yang, Lin Mu, Ming Wu, Nan Chen, Chunyang Du, Huijun Duan, and Yonghong Shi

Renal fibrosis is the major pathological characteristic of diabetic nephropathy (DN). Reportedly, increased SIRT1 expression played a renal protective role in animal models of DN. This study was designed to elucidate the molecular mechanisms underlying the protective effects of SRT1720, an SIRT1 activator, against diabetes-induced renal fibrosis. Type 2 diabetic mice (db/db) were treated with SRT1720 (50 mg/kg/day) by gavage for 10 weeks. Renal proximal tubular epithelial cells (HK-2 cells) were treated with high glucose (HG, 30 mM) in the presence or absence of SRT1720 (2.5 µM) for 48 h. We observed that impaired SIRT1 expression and activity were restored by SRT1720 administration in db/db mice as well as in HG-treated HK-2 cells. Moreover, SRT1720 administration improved the renal function, attenuated glomerular hypertrophy, mesangial expansion, glomerulosclerosis and interstitial fibrosis and inhibited TGFB1 and CTGF expressions and nuclear factor κB (NF-KB) activation in db/db mice. Similarly, HG-induced epithelial-to-mesenchymal transformation (EMT) and collagen IV and fibronectin expressions were inhibited in SRT1720-treated HK-2 cells. Mechanistic studies demonstrated that SRT1720 suppressed HIF1A, GLUT1 and SNAIL expressions both in vivo and in vitro. Furthermore, HIF1A or GLUT1 knockdown effectively abrogated HG-induced EMT and collagen IV and fibronectin expressions in HK-2 cells. These findings suggest that SRT1720 prevented diabetes-induced renal fibrosis via the SIRT1/HIF1A/GLUT1/SNAIL pathway.

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Li Wang, Yufeng Zhao, Baosong Gui, Rongguo Fu, Feng Ma, Jun Yu, Ping Qu, Lei Dong, and Chen Chen

The role of free fatty acids (FFAs) in glucagon secretion has not been well established, and the involvement of FFA receptor GPR40 and its downstream signaling pathways in regulating glucagon secretion are rarely demonstrated. In this study, it was found that linoleic acid (LA) acutely stimulated glucagon secretion from primary cultured rat pancreatic islets. LA at 20 and 40 μmol/l dose-dependently increased glucagon secretion both at 3 mmol/l glucose and at 15 mmol/l glucose, although 15 mmol/l glucose reduced basal glucagon levels. LA induced an increase in cytoplasmic free calcium concentrations ([Ca2 +]i) in identified rat α-cells, which is reflected by increased Fluo-3 intensity under confocal microscopy recording. The increase in [Ca2 +]i was partly inhibited by removal of extracellular Ca2 + and eliminated overall by further exhaustion of intracellular Ca2 + stores using thapsigargin treatment, suggesting that both Ca2 + release and Ca2 + influx contributed to the LA-stimulated increase in [Ca2 +]i in α-cells. Double immunocytochemical stainings showed that GPR40 was expressed in glucagon-positive α-cells. LA-stimulated increase in [Ca2 +]i was blocked by inhibition of GPR40 expression in α-cells after GPR40-specific antisense treatment. The inhibition of phospholipase C activity by U73122 also blocked the increase in [Ca2 +]i by LA. It is concluded that LA activates GPR40 and phospholipase C (and downstream signaling pathways) to increase Ca2 + release and associated Ca2 + influx through Ca2 + channels, resulting in increase in [Ca2 +]i and glucagon secretion.

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Shu-Ju Chang, Tao-Yeuan Wang, Yi-Hsuan Lee, and Chen-Jei Tai

ATP has been shown to activate the mitogen-activated protein kinase (MAPK) signaling pathway in various systems. However, little is known about the signaling events and the effects in human endometrial stromal cells (hESCs). The present study examined the effect of ATP on activating MAPKs and its subsequent events in hESCs. This study demonstrated the expression of the P2U/P2Y2 receptor in hESCs by reverse transcription-PCR (RT-PCR). A PCR product with a sequence identical to the reported 599 bp P2U/P2Y2 receptor cDNA was obtained. Western blot analysis, using a monoclonal antibody against the phosphorylated forms of ERK1/2, demonstrated that ATP activated MAPK in a dose- and time-dependent manner. Confocal microscopy showed an evident nuclear translocation of phosphorylated ERKs after 10 μM ATP treatment, but this effect was blocked by PD98059. To study the gene(s) induced by exogenous ATP, mRNA was extracted from hESCs in the presence or absence of 10 μM ATP. The gene array for 96 genes associated with members of human matrix metalloproteinases (MMPs) and adhesion molecules revealed that the expression of MMP-2, -3, -10, and -24 genes was increased and the effect was attenuated by PD98059. Furthermore the effects of ATP on the expression of MMP genes were confirmed by semiquantitative RT-PCR. To our knowledge, this is the first demonstration of the ATP-induced nuclear translocation of phospho-ERK1/2 that mediates MMPs gene expression in human endometrial cells. These results support the notion that the ERK1/2 signaling pathway is involved in mediating ATP actions in the human reproductive system.

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Hongjuan Shi, Tian Gao, Zhilong Liu, Lina Sun, Xiaolong Jiang, Lili Chen, and Deshou Wang

Induction of sex reversal of XY fish has been restricted to the sex undifferentiated period. In the present study, differentiated XY tilapia were treated with trilostane (TR), metopirone (MN) and glycyrrhetinic acid (GA) (inhibitor of 3β-HSD, Cyp11b2 and 11β-HSD, respectively) alone or in combination with 17β-estradiol (E2) from 30 to 90 dah (days after hatching). At 180 dah, E2 alone resulted in 8.3%, and TR, MN and GA alone resulted in no secondary sex reversal (SSR), whereas TR + E2, MN + E2 and GA + E2 resulted in 88.3, 60.0 and 46.7% of SSR, respectively. This sex reversal could be rescued by simultaneous administration of 11-ketotestosterone (11-KT). Compared with the control XY fish, decreased serum 11-KT and increased E2 level were detected in SSR fish. Immunohistochemistry analyses revealed that Cyp19a1a, Cyp11b2 and Dmrt1 were expressed in the gonads of GA + E2, MN + E2 and TR + E2 SSR XY fish at 90 dah, but only Cyp19a1a was expressed at 180 dah. When the treatment was applied from 60 to 120 dah, TR + E2 resulted in 3.3% of SSR, MN + E2 and GA + E2 resulted in no SSR. These results demonstrated that once 11-KT was synthesized, it could antagonize E2-induced male-to-female SSR, which could be abolished by simultaneous treatment with the inhibitor of steroidogenic enzymes. The upper the enzyme was located in the steroidogenic pathway, the higher SSR rate was achieved when it was inhibited as some of the precursors, such as androstenedione, testosterone and 5α-dihydrotestosterone, could act as androgens. These results highlight the key role of androgen in male sex maintenance.

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Hong Liu, Jian Guo, Lin Wang, Ning Chen, Andrew Karaplis, David Goltzman, and Dengshun Miao

To assess the roles of 1,25-dihydroxyvitamin D (1,25(OH)2D) and parathyroid hormone (PTH) in hard tissue formation in oro-facial tissues, we examined the effect of either 1,25(OH)2D or PTH deficiency on dentin and dental alveolar bone formation and mineralization in the mandibles, and osteoblastic bone formation in long bones of 1α-hydroxylase knockout (1α(OH)ase−/−) mice. Compared with wild-type mice, the mineral density was decreased in the teeth and mandibles, and unmineralized dentin (predentin and biglycan immunopositive dentin) and unmineralized bone matrix in the dental alveolar bone were increased in 1α(OH)ase−/− mice. The dental volume, reparative dentin volume, and dentin sialoprotein immunopositive areas were reduced in 1α(OH)ase−/− mice. The cortical thickness, dental alveolar bone volume, and osteoblast number were all decreased significantly in the mandibles; in contrast, the osteoblast number and surface were increased in the trabecular bone of the tibiae in 1α(OH)ase−/− mice consistent with their secondary hyperparathyroidism. The expression of PTH receptor and IGF1 was reduced slightly in mandibles, but enhanced significantly in the long bones in the 1α(OH)ase−/− mice. To control for the role of secondary hyperparathyroidism, we also examined teeth and mandibles in 6-week-old PTH−/− mice. In these animals, dental and bone volumes in mandibles were not altered when compared with their wild-type littermates. These results suggest that 1,25(OH)2D3 plays an anabolic role in both dentin and dental alveolar bone as it does in long bones, whereas PTH acts predominantly in long bones rather than mandibular bone.