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Rui Wang, Jie Hong, Ruixin Liu, Maopei Chen, Min Xu, Wiqiong Gu, Yifei Zhang, Qinyun Ma, Feng Wang, Juan Shi, Jiqiu Wang, Weiqing Wang, and Guang Ning

WNT/β-catenin signalling is involved in regulating adipogenesis, and its dysregulation occurs in obesity. Secreted frizzled-related protein 5 (SFRP5) is a WNT protein inhibitor; however, its role in adipogenesis and obesity is controversial. In this study, we observed that SFRP5 mRNA levels were increased in the fat tissues of obese humans and mice. Sfrp5 expression was gradually induced during differentiation of white and brown adipocytes and was highly increased in mature adipocytes rather than preadipocytes. However, the effects of the exogenous overexpression of Sfrp5 indicated that Sfrp5 may not directly regulate adipogenesis in vitro under the conditions studied. Moreover, SFRP5 did not inhibit the canonical WNT/β-catenin signalling pathway in preadipocytes. Subsequently, we measured the levels of circulating SFRP5 in obese patients and non-obese subjects using ELISA and did not find any significant difference. Collectively, these findings indicate that Sfrp5 represents a candidate for a mature adipocyte marker gene. Our data provide new evidence concerning the role of SFRP5 in adipogenesis of white and brown adipocytes and obesity.

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Gang Liu, Feng Luo, Qiang Song, Limin Wu, Yongxiu Qiu, Hongjuan Shi, Deshou Wang, and Linyan Zhou

In vitro studies have indicated that the maturation-inducing hormone 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP, DHP), probably through nuclear progestin receptor (Pgr), might be involved in the proliferation of spermatogonial cells and the initiation of meiosis in several fish species. However, further in vivo evidence is required to elucidate the role of DHP in spermatogenesis during sexual differentiation in teleosts. In this study, we cloned pgr and analyzed its expression in Nile tilapia (Oreochromis niloticus) and treated XY fish with RU486 (a synthetic Pgr antagonist) from 5 days after hatching (dah) to determine the role of DHP in spermatogenesis. Sequence and phylogenetic analyses revealed that the Pgr identified in tilapia is a genuine Pgr. Pgr was found to be expressed in the Sertoli cells surrounding spermatogonia and spermatids in the testis of tilapia. Real-time PCR analysis revealed that the expression of pgr in the testis was significantly upregulated from 10 dah, further increased at 50 dah, and persisted until adulthood in fish. In the testis of RU486-treated fish, the transcript levels of germ cell markers and a meiotic marker were substantially reduced. However, the expression of markers in Sertoli cells remained unchanged. Moreover, the production of 11-ketotestosterone and the expression of genes encoding various steroidogenic enzymes were also not altered. In contrast, the expression of cyp17a2, encoding one of the critical steroidogenic enzymes involved in DHP biosynthesis, declined significantly, possibly indicating the inhibition of DHP production by RU486. RU486 treatment given for 2 months did not affect spermatogenesis; however, treatment given for more than 3 months resulted in a decrease in spermatogonial cell numbers and depletion of later-phase spermatogenic cells. Simultaneous excessive DHP supplementation restored spermatogenesis in RU486-treated XY fish. Taken together, our data further indicated that DHP, possibly through Pgr, might be essential for spermatogonial cell proliferation and spermatogenesis in fish.

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Feng Wang, Xianfeng Zhang, Jiqiu Wang, Maopei Chen, Nengguang Fan, Qinyun Ma, Ruixin Liu, Rui Wang, Xiaoying Li, Mingyao Liu, and Guang Ning

The circadian clock plays an important role in the liver by regulating the major aspects of energy metabolism. Currently, it is assumed that the circadian clock regulates metabolism mostly by regulating the expression of liver enzymes at the transcriptional level, but the underlying mechanism is not well understood. In this study, we showed that L gr4 homozygous mutant (L gr4 m/m) mice showed alteration in the rhythms of the respiratory exchange ratio. We further detected impaired plasma triglyceride rhythms in L gr4 m/m mice. Although no significant changes in plasma cholesterol rhythms were observed in the L gr 4 m/m mice, their cholesterol levels were obviously lower. This phenotype was further confirmed in the context of ob/ob mice, in which lack of LGR4 dampened circadian rhythms of triglyceride. We next demonstrated that Lgr 4 expression exhibited circadian rhythms in the liver tissue and primary hepatocytes in mice, but we did not detect changes in the expression levels or circadian rhythms of classic clock genes, such as C lock, Bmal1 (Arntl), P ers, Rev-erbs, and C rys, in L gr 4 m/m mice compared with their littermates. Among the genes related to the lipid metabolism, we found that the diurnal expression pattern of the M ttp gene, which plays an important role in the regulation of plasma lipid levels, was impaired in L gr 4 m/m mice and primary L gr 4 m/m hepatocytes. Taken together, our results demonstrate that LGR4 plays an important role in the regulation of plasma lipid rhythms, partially through regulating the expression of microsomal triglyceride transfer protein. These data provide a possible link between the peripheral circadian clock and lipid metabolism.

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Feng Wang, Lu Wang, Yifeng Wang, Dai Li, Tianpeng Hu, Manyi Sun, and Ping Lei

Insulin-like growth factor-1 (IGF-1) improves cognitive function, but its mechanism has not been elucidated. The aim of the study was to explore whether IGF-1 exerted its protective effect on cognitive function and anxiety behavior through the activation of PI3K/Akt/CREB pathway in high-fat diet rats. Neuronal cells HT22 were treated with nothing, IGF-1, IGF-1 + LY294002 or IGF-1 + 666-15. Expressions of p-PI3K, p-Akt and p-CREB were measured using Western blot analysis. Thirty C57BL/6J rats were used. After feeding with high-fat diet, normal saline, PEG-IGF-1, PEG-IGF-1 + LY294002 or PEG-IGF-1 + 666-15 was treated. Cognitive function and anxiety behavior were assessed by Morris water maze and open field test. Several inflammation and oxidative stress biomarkers were measured using recognized methods. Expressions of p-PI3K and p-CREB were also measured using Western blot analysis. After IGF-1 treatment in cells, expressions of p-PI3K, p-Akt and p-CREB were increased. Furthermore, LY294002 downregulated the expressions of these three proteins, but 666-15 only inhibited the expression of CREB in the cells. Compared with the control rats, we found abnormalities of cognitive function and anxiety behavior, inhibition of PI3K/Akt/CREB pathway and increase of oxidative stress and inflammation in high-fat diet rats. After PEG-IGF-1 treatment, the changes in high-fat diet rats were reversed. Then, we blocked the pathway and found that these blockers attenuated the protective effects of PEG-IGF-1. In conclusion, IGF-1 improved cognitive function and anxiety behavior in high-fat diet rats and inhibited inflammation and oxidative stress in hippocampus tissue through the activation of PI3K/Akt/CREB pathway.

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Hui Juan Zhu, Hui Pan, Xu Zhe Zhang, Nai Shi Li, Lin Jie Wang, Hong Bo Yang, and Feng Ying Gong

Myostatin is a critical negative regulator of skeletal muscle development, and has been reported to be involved in the progression of obesity and diabetes. In the present study, we explored the effects of myostatin on the proliferation and differentiation of 3T3-L1 preadipocytes by using 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide spectrophotometry, intracellular triglyceride (TG) assays, and real-time quantitative RT-PCR methods. The results indicated that recombinant myostatin significantly promoted the proliferation of 3T3-L1 preadipocytes and the expression of proliferation-related genes, including Cyclin B2, Cyclin D 1, Cyclin E1, Pcna, and c-Myc, and IGF1 levels in the medium of 3T3-L1 were notably upregulated by 35.2, 30.5, 20.5, 33.4, 51.2, and 179% respectively (all P<0.01) in myostatin-treated 3T3-L1 cells. Meanwhile, the intracellular lipid content of myostatin-treated cells was notably reduced as compared with the non-treated cells. Additionally, the mRNA levels of Ppar γ, Cebp α, Gpdh, Dgat, Acs1, Atgl, and Hsl were significantly downregulated by 22–76% in fully differentiated myostatin-treated adipocytes. Finally, myostatin regulated the mRNA levels and secretion of adipokines, including Adiponectin, Resistin, Visfatin, and plasminogen activator inhibitor-1 (PAI-1) in 3T3-L1 adipocytes (all P<0.001). Above all, myostatin promoted 3T3-L1 proliferation by increasing the expression of cell-proliferation-related genes and by stimulating IGF1 secretion. Myostatin inhibited 3T3-L1 adipocyte differentiation by suppressing Ppar γ and Cebp α expression, which consequently deceased lipid accumulation in 3T3-L1 cells by inhibiting the expression of critical lipogenic enzymes and by promoting the expression of lipolytic enzymes. Finally, myostatin modulated the expression and secretion of adipokines in fully differentiated 3T3-L1 adipocytes.

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Ke-Hung Tsui, Li-Chuan Chung, Shyi-Wu Wang, Tsui-Hsia Feng, Phei-Lang Chang, and Horng-Heng Juang

Hypoxia induces metabolic alteration in cancer cells by stabilizing hypoxia-inducible factor 1α (HIF-1α (HIF1A)), which regulates the bioenergetic genes of glycolysis and lipid metabolic pathways. However, the target genes of hypoxia-induced metabolic alterations in the prostate remain uncertain. Mitochondrial aconitase (mACON) (ACONM) is an enzyme that is central to carbohydrate and energy metabolism and is responsible for the interconversion of citrate to isocitrate as part of the citric acid cycle in the human prostate. We evaluated the effects of the molecular mechanisms of hypoxia on mACON gene expression in PC-3 and LNCaP human prostate carcinoma cells. Immunoblotting assays revealed that hypoxia modulated mACON and lactate dehydrogenase A (LDHA) protein expression, while these effects were attenuated when HIF-1α was knocked down. Hypoxia induced fatty acid synthase (FASN) in PC-3 cells while hypoxia blocked FASN gene expression in LNCaP cells after 24-h incubation. Results of real-time RT-qPCR, immunoblotting, and transient gene expression assays revealed that hypoxia treatment or co-transfection with H IF-1α expression vector enhanced gene expression of mACON, implying that hypoxia modulated mACON at the transcriptional level. Hypoxia-induced mACON promoter activity is dependent on the DNA fragment located at −1013 to −842 upstream of the translation initiation site. l-mimosine, an iron chelator, stabilized HIF-1α but downregulated mACON gene expression, suggesting that iron chelation blocked the hypoxia-induced mACON gene expression. These results suggest that hypoxia dysregulates the expressions of LDHA, FASN, and mACON genes, and the hypoxia-induced mACON gene expression is via the HIF-1α-dependent and iron-dependent pathways in prostate carcinoma cells.

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Yu-Guang Ma, Liang Liang, Yin-Bin Zhang, Bao-Feng Wang, Yun-Gang Bai, Zhi-Jun Dai, Man-Jiang Xie, and Zhong-Wei Wang

Hyperglycemia and hypertension are considered to be the two leading risk factors for vascular disease in diabetic patients. However, few pharmacologic agents could provide a combinational therapy for controlling hyperglycemia and hypertension at the same time in diabetes. The objectives of this study are to investigate whether berberine treatment could directly reduce blood pressure and identify the molecular mechanism underlying the vascular protection of berberine in diabetic rats. Berberine was intragastrically administered with different dosages of 50, 100 and 200 mg/kg/day to diabetic rats for 8 weeks since the injection of streptozotocin. The endothelium-dependent/-independent relaxation in middle cerebral arteries was investigated. The activity of large-conductance Ca2+-activated K+ channel (BKCa) was investigated by recording whole-cell currents, analyzing single-channel activities and assessing the expressions of α- and β1-subunit at protein or mRNA levels. Results of the study suggest that chronic administration of 100 mg/kg/day berberine not only lowered blood glucose but also reduced blood pressure and improved vasodilation in diabetic rats. Furthermore, berberine markedly increased the function and expression of BKCa β1-subunit in cerebral vascular smooth muscle cells (VSMCs) isolated from diabetic rats or when exposed to hyperglycemia condition. The present study provided initial evidences that berberine reduced blood pressure and improved vasodilation in diabetic rats by activation of BKCa channel in VSMCs, which suggested that berberine might provide a combinational therapy for controlling hyperglycemia and blood pressure in diabetes. Furthermore, our work indicated that activation of BKCa channel might be the underlying mechanism responsible for the vascular protection of berberine in diabetes.

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Zhiyu Ma, Ying Zhang, Juan Su, Sheng Yang, Wenna Qiao, Xiang Li, Zhihai Lei, Ling Cheng, Na An, Wenshao Wang, Yanyan Feng, and Jinlong Zhang

Neuromedin B (NMB), a mammalian bombesin-related peptide, has numerous physiological functions, including regulating hormone secretions, cell growth, and reproduction, by binding to its receptor (NMBR). In this study, we investigated the effects of NMB on testosterone secretion, steroidogenesis, cell proliferation, and apoptosis in cultured primary porcine Leydig cells. NMBR was mainly expressed in the Leydig cells of porcine testes, and a specific dose of NMB significantly promoted the secretion of testosterone in the primary Leydig cells; moreover, NMB increased the expression of mRNA and/or proteins of NMBR and steroidogenic mediators (steroidogenic acute regulatory (STAR), CYP11A1, and HSD3B1) in the Leydig cells. In addition, specific doses of NMB promoted the proliferation of Leydig cells and increased the expression of proliferating cell nuclear antigen and Cyclin B1 proteins, while suppressing Leydig cell apoptosis and decreasing BAX and Caspase-3 protein expression. These results suggest that the NMB/NMBR system might play an important role in regulating boar reproductive function by modulating steroidogenesis and/or cell growth in porcine Leydig cells.

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Yu-Feng Zhao, Li Wang, Dingjun Zha, Li Qiao, Lianjun Lu, Jun Yu, Ping Qu, Qiang Sun, Jianhua Qiu, and Chen Chen

GW9508 is an agonist of G protein-coupled receptor 40 (GPR40) that is expressed in pancreatic β-cells and is reported to regulate insulin secretion. However, the effects of GW9508 on pancreatic β-cells in primary culture have not been well investigated. This study measured the acute effects of GW9508 on insulin secretion from rat pancreatic islets in primary culture, and the insulin secretion-related events such as the changes in membrane potential, ATP-sensitive potassium currents (KATP currents), and intracellular Ca2 + concentrations ([Ca2 +]i) of rat islet β-cells were also recorded. GW9508 (10–40 μM) did not influence basal insulin levels at 2 mM glucose, but it (above 20 μM) significantly inhibited 5 and 15 mM glucose-stimulated insulin secretion (GSIS). GW9508 did not inhibit insulin secretion stimulated by tolbutamide, the closer of KATP channels. GW9508 activated KATP channels and blocked the membrane depolarization and the increase in [Ca2 +]i that were stimulated by glucose. GW9508 itself stimulated a transient increase in [Ca2 +]i, which was fully blocked by depletion of intracellular Ca2 + stores with thapsigargin or by inhibition of phospholipase C (PLC) activity with U73122. GW9508-induced activation of KATP channels was only partly inhibited by U73122 treatment. In conclusion, although it stimulates a transient release of Ca2 + from intracellular Ca2 + stores via activation of PLC, GW9508 inhibits GSIS by activating KATP channels probably in a distal step to GPR40 activation in rat β-cells.

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Rubab Akbar, Kamran Ullah, Tanzil Ur Rahman, Yi Cheng, Hai-Yan Pang, Lu-Yang Jin, Qi-Jing Wang, He-Feng Huang, and Jian-Zhong Sheng

Receptive endometrium is a prerequisite for successful embryo implantation, and it follows that poor endometrial receptivity is a leading cause of implantation failure. miRNAs play important roles as epigenetic regulators of endometrial receptivity and embryo implantation through post-transcriptional modifications. However, the mechanisms of action of many miRNAs are poorly understood. In this study, we investigated the role of the miR-183 family, comprising three miRNAs (miR-183-5p, miR-182-5p, and miR-96-5p) in endometrial receptivity and embryo implantation. The miR-183 family shows estrogen-dependent upregulation in endometrial Ishikawa (IK) cells. The miR-183 family also has a positive role in migration and proliferation of IK cells. Furthermore, JAr spheroid attachment experiments show that attachment rates were significantly decreased after treatment of IK cells with inhibitors for miR-183-5p and miR-182-5p and increased after treatment with miR-183-5p-mimic and miR-96-5p-mimic, respectively. The downstream analysis shows that catenin alpha 2 (CTNNA2) is a potential target gene for miR-183-5p, and this was confirmed in luciferase reporter assays. An in vivo mouse pregnancy model shows that inhibition of miR-183-5p significantly decreases embryo implantation rates and increases CTNNA2 expression. Downregulation of CTNNA2 in endometrial cells by miR-183-5p may be significant in mediating estrogenic effects on endometrial receptivity. In conclusion, miR-183-5p and the CTNNA2 gene may be potential biomarkers for endometrial receptivity and may be useful diagnostic and therapeutic targets for successful embryo implantation.