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  • Author: Yao Wang x
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Paul G Farnworth, Yao Wang, Pauline Leembruggen, Guck T Ooi, Craig Harrison, David M Robertson and Jock K Findlay

Inhibins are expressed in the adrenal cortex, but little is known of their binding or role in the adrenal. The aims of the present study were, first, to establish whether a mouse adrenocortical (AC) cell line expresses inhibins/activins and bone morphogenetic proteins (BMP), along with proteins required for inhibin to antagonise activin and BMP actions and, secondly, to characterise and compare inhibin binding sites and proteins in the rat adrenal gland and AC cells. AC cells were found to: (1) express mRNA for multiple BMPs (BMP-2, -3, -4, -6, -8a), growth/differentiation factors (GDF-1, -3, -5, -9), Lefty A and B, and the inhibin α, βA and βB subunits (2) secrete inhibin A and inhibin B and (3) express mRNA encoding the inhibin co-receptor, betaglycan, along with activin and BMP type I (ALK2–7) and type II (ActRII, ActRIIB, BMPRII) receptors, and binding proteins (follistatin, BAMBI, gremlin). When applied to sections of rat adrenal glands, [125I]inhibin A specifically bound to cells of the adrenal cortex, mainly in the zona reticularis. Scatchard analyses of in vitro [125I]inhibin A binding to dispersed rat adrenal cells and AC cells revealed sites of high affinity (Kd(1) of 0.18 and 0.15 nM, respectively) and low affinity (Kd(2) of 2.6 and 1.3 nM, respectively. Competition for [125I]inhibin A binding by activin A or B (30 nM) was negligible, whereas BMP-2, -6 and -7 competed for between 21 and 33% of specific inhibin A binding (IC50 between 0.2 and 0.3 nM). Inhibin B crossreaction with inhibin A binding sites was < 8%. Multiple binding protein complexes (molecular weight ranging from 35 to > 220 kDa) were affinity labelled by [125I]inhibin A on both the primary rat adrenal and AC cells. The species of > 220 kDa were shown by immunoprecipitation to include betaglycan, the species of 105 kDa is consistent in size with type II receptors for activin/BMP, and that of 62 kDa co-migrates with the inhibin-follistatin complex.

In summary, the results show that inhibin A binds selectively and with both high and low affinity to AC cells via multiple binding proteins, including a single betaglycan-like species. The results support the role of glycosylated betaglycan in the high affinity binding of inhibin A, but provide consistent evidence from two independent sources of adrenal cells that inhibin A interacts with several membrane proteins in addition to those currently understood to mediate the anti-activin/BMP actions of inhibin.

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Shou-Si Lu, Yun-Li Yu, Hao-Jie Zhu, Xiao-Dong Liu, Li Liu, Yao-Wu Liu, Ping Wang, Lin Xie and Guang-Ji Wang

Berberine (BBR), a hypoglycemic agent, has shown beneficial metabolic effects for anti-diabetes, but its precise mechanism was unclear. Glucagon-like peptide-1 (GLP-1) is considered to be an important incretin that can decrease hyperglycemia in the gastrointestinal tract after meals. The aim of this study was to investigate whether BBR exerts its anti-diabetic effects via modulating GCG secretion. Diabetes-like rats induced by streptozotocin received BBR (120 mg/kg per day, i.g) for 5 weeks. Two hours following the last dose, the rats were anaesthetized and received 2.5 g/kg glucose by gavage. At 15-minute and 30-minute after glucose load, blood samples, pancreas, and intestines were obtained to measure insulin and GCG using ELISA kit. The number of L cells in the ileum and β-cells in the pancreas were identified using immunohistology. The expression of proglucagon mRNA in the ileum was measured by RT-PCR. The results indicated that BBR treatment significantly increased GCG levels in plasma and intestine (P<0.05) accompanied with the increase of proglucagon mRNA expression and the number of L-cell compared with the controls (P<0.05). Furthermore, BBR increased insulin levels in plasma and pancreas as well as β-cell number in pancreas. The data support the hypothesis that the anti-diabetic effects of BBR may partly result from enhancing GCG secretion.

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Laura E Pascal, Khalid Z Masoodi, June Liu, Xiaonan Qiu, Qiong Song, Yujuan Wang, Yachen Zang, Tiejun Yang, Yao Wang, Lora H Rigatti, Uma Chandran, Leandro M Colli, Ricardo Z N Vencio, Yi Lu, Jian Zhang and Zhou Wang

Elongation factor, RNA polymerase II, 2 (ELL2) is an RNA Pol II elongation factor with functional properties similar to ELL that can interact with the prostate tumor suppressor EAF2. In the prostate, ELL2 is an androgen response gene that is upregulated in benign prostatic hyperplasia (BPH). We recently showed that ELL2 loss could enhance prostate cancer cell proliferation and migration, and that ELL2 gene expression was downregulated in high Gleason score prostate cancer specimens. Here, prostate-specific deletion of ELL2 in a mouse model revealed a potential role for ELL2 as a prostate tumor suppressor in vivo. Ell2-knockout mice exhibited prostatic defects including increased epithelial proliferation, vascularity and PIN lesions similar to the previously determined prostate phenotype in Eaf2-knockout mice. Microarray analysis of prostates from Ell2-knockout and wild-type mice on a C57BL/6J background at age 3 months and qPCR validation at 17 months of age revealed a number of differentially expressed genes associated with proliferation, cellular motility and epithelial and neural differentiation. OncoPrint analysis identified combined downregulation or deletion in prostate adenocarcinoma cases from the Cancer Genome Atlas (TCGA) data portal. These results suggest that ELL2 and its pathway genes likely play an important role in the development and progression of prostate cancer.

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Yao Wang, Peter K Nicholls, Peter G Stanton, Craig A Harrison, Mai Sarraj, Robert B Gilchrist, Jock K Findlay and Paul G Farnworth

Growth differentiation factor 9 (GDF9) produced within the ovary plays an essential role during follicle maturation through actions on granulosa cells, but extra-ovarian expression, signalling and actions of GDF9 are less well characterised. The present studies confirm GDF9 expression in the mouse testis, pituitary gland and adrenocortical cancer (AC) cells, and establish its expression in LβT2 gonadotrophs, and in mouse adrenal glands, particularly foetal and neonatal cortical cells. AC, LβT2, TM3 Leydig and TM4 Sertoli cells express the requisite GDF9 binding signalling components, particularly activin receptor-like kinase (ALK) 5 and the bone morphogenetic protein (BMP)/GDF type II receptor, BMPRII (BMPR2). We therefore compared GDF9 activation of these potential extra-ovarian target cell types with its activation of granulosa cells. Recombinant mouse GDF9 stimulated expression of activin/transforming growth factor-β-responsive reporters, pGRAS-luc or pAR3-lux, in TM4 and AC cells (IC50=145 ng/ml in the latter case), and two granulosa cell lines, KGN and COV434. The ALK4/5/7 inhibitor, SB431542, blocked GDF9 activity in each case. By contrast, GDF9 lacked specific effects on TM3 cells and rat primary pituitary and mouse LβT2 gonadotrophs. Our findings show that GDF9 regulates the expression of R-SMAD2/3-responsive reporter genes through ALK4, 5 or 7 in extra-ovarian (adrenocortical and Sertoli) cells with similar potency and signalling pathway to its actions on granulosa cells, but suggest that expression of BMPRII, ALK5 (TGFBR1) and R-SMADs 2 and 3 may not be sufficient for a cell to respond to GDF9.

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Xin-gang Yao, Xin Xu, Gai-hong Wang, Min Lei, Ling-ling Quan, Yan-hua Cheng, Ping Wan, Jin-pei Zhou, Jing Chen, Li-hong Hu and Xu Shen

Impaired glucose-stimulated insulin secretion (GSIS) and increasing β-cell death are two typical dysfunctions of pancreatic β-cells in individuals that are destined to develop type 2 diabetes, and improvement of β-cell function through GSIS enhancement and/or inhibition of β-cell death is a promising strategy for anti-diabetic therapy. In this study, we discovered that the small molecule, N-(2-benzoylphenyl)-5-bromo-2-thiophenecarboxamide (BBT), was effective in both potentiating GSIS and protecting β-cells from cytokine- or streptozotocin (STZ)-induced cell death. Results of further studies revealed that cAMP/PKA and long-lasting (L-type) voltage-dependent Ca2 + channel/CaMK2 pathways were involved in the action of BBT against GSIS, and that the cAMP/PKA pathway was essential for the protective action of BBT on β-cells. An assay using the model of type 2 diabetic mice induced by high-fat diet combined with STZ (STZ/HFD) demonstrated that BBT administration efficiently restored β-cell functions as indicated by the increased plasma insulin level and decrease in the β-cell loss induced by STZ/HFD. Moreover, the results indicated that BBT treatment decreased fasting blood glucose and HbA1c and improved oral glucose tolerance further highlighting the potential of BBT in anti-hyperglycemia research.

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Kok Lim Kua, Shanming Hu, Chunlin Wang, Jianrong Yao, Diana Dang, Alexander B Sawatzke, Jeffrey L Segar, Kai Wang and Andrew W Norris

Offspring exposed in utero to maternal diabetes exhibit long-lasting insulin resistance, though the initiating mechanisms have received minimal experimental attention. Herein, we show that rat fetuses develop insulin resistance after only 2-day continuous exposure to isolated hyperglycemia starting on gestational day 18. Hyperglycemia-induced reductions in insulin-induced AKT phosphorylation localized primarily to fetal skeletal muscle. The skeletal muscle of hyperglycemia-exposed fetuses also exhibited impaired in vivo glucose uptake. To address longer term impacts of this short hyperglycemic exposure, neonates were cross-fostered and examined at 21 days postnatal age. Offspring formerly exposed to 2 days late gestation hyperglycemia exhibited mild glucose intolerance with insulin signaling defects localized only to skeletal muscle. Fetal hyperglycemic exposure has downstream consequences which include hyperinsulinemia and relative uteroplacental insufficiency. To determine whether these accounted for induction of insulin resistance, we examined fetuses exposed to late gestational isolated hyperinsulinemia or uterine artery ligation. Importantly, 2 days of fetal hyperinsulinemia did not impair insulin signaling in murine fetal tissues and 21-day-old offspring exposed to fetal hyperinsulinemia had normal glucose tolerance. Similarly, fetal exposure to 2-day uteroplacental insufficiency did not perturb insulin-stimulated AKT phosphorylation in fetal rats. We conclude that fetal exposure to hyperglycemia acutely produces insulin resistance. As hyperinsulinemia and placental insufficiency have no such impact, this occurs likely via direct tissue effects of hyperglycemia. Furthermore, these findings show that skeletal muscle is uniquely susceptible to immediate and persistent insulin resistance induced by hyperglycemia.