In this study, two novel GHRHR receptor splice variants, named chicken GHRHR-v1 (cGHRHR-v1) and cGHRHR-v2 respectively, were identified from chicken pituitary using RT-PCR assay. cGHRHR-v1 is characterized by an N-terminal deletion of 36 amino acid residues, including an aspartate at position 56 (Asp56) conserved in G protein-coupled receptor B-I subfamily. cGHRHR-v2 is a carboxyl-terminal truncated receptor variant with four putative transmembrane domains, which arose from alternative use of a splice acceptor site on intron 8. Using the pGL3-CRE-luciferase reporter system, the functionality of the two variants was examined in Chinese hamster ovary cells. cGHRHR-v1 was shown to be capable of transmitting signal upon agonist stimulation, but cGHRHR-v2 could not. Both GHRH and pituitary adenylate cyclase-activating peptide (PACAP) could activate cGHRHR-v1 at high dosages (GHRH ≥10−8 M; PACAP ≥10−6 M) and GHRH was much more potent than PACAP, suggesting that cGHRHR-v1 is a functional membrane-spanning receptor with an impairment in high-affinity ligand binding, rather than in receptor activation and ligand-binding specificity. This finding also points out the possibility that Asp56 is not a critical determinant for receptor activation and direct ligand–receptor interaction. To substantiate this hypothesis, using site-directed mutagenesis, two receptor mutants with replacement of Asp56 by Ala or Gly were generated. Expectedly, chicken or human GHRH could still activate both receptor mutants with reduced potencies (about 2- to 14-fold less potent). Taken together, our findings not only suggest that cGHRHR variants may play a role in controlling normal pituitary functions, but also support that Asp56 is nonessential for receptor activation and direct ligand–receptor interaction.
Crystal Ying Wang, Yajun Wang, Amy Ho Yan Kwok and Frederick C Leung
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.
Xiaonan Yan, Xiaonan Dai, Jing Wang, Nannan Zhao, Yugui Cui and Jiayin Liu
Owing to the heterogeneity in the clinical symptoms of polycystic ovary syndrome (PCOS), the early pathophysiological mechanisms of PCOS remain unclear. Clinical, experimental, and genetic evidence supports an interaction between genetic susceptibility and the influence of maternal environment in the pathogenesis of PCOS. To determine whether prenatal androgen exposure induced PCOS-related metabolic derangements during pubertal development, we administrated 5α-dihydrotestosterone (DHT) in pregnant rats and observed their female offspring from postnatal 4 to 8 weeks. The prenatally androgenized (PNA) rats exhibited more numerous total follicles, cystic follicles, and atretic follicles than the controls. Fasting glucose, insulin, leptin levels, and homeostatic model assessment for insulin resistance were elevated in the PNA rats at the age of 5–8 weeks. Following intraperitoneal glucose tolerance tests, glucose and insulin levels did not differ between two groups; however, the PNA rats showed significantly higher 30- and 60-min glucose levels than the controls after insulin stimulation during 5–8 weeks. In addition, prenatal DHT treatment significantly decreased insulin-stimulated phosphorylation of AKT in the skeletal muscles of 6-week-old PNA rats. The abundance of IR substrate 1 (IRS1) and IRS2 was decreased in the skeletal muscles and liver after stimulation with insulin in the PNA group, whereas phosphorylation of insulin-signaling proteins was unaltered in the adipose tissue. These findings validate the contribution of prenatal androgen excess to metabolic derangements in pubertal female rats, and the impaired insulin signaling through IRS and AKT may result in the peripheral insulin resistance during pubertal development.
Pearl P Y Lie, Weiliang Xia, Claire Q F Wang, Dolores D Mruk, Helen H N Yan, Ching-hang Wong, Will M Lee and C Yan Cheng
In adult rat testes, blood–testis barrier (BTB) restructuring facilitates the migration of preleptotene spermatocytes from the basal to the adluminal compartment that occurs at stage VIII of the epithelial cycle. Structural proteins at the BTB must utilize an efficient mechanism (e.g. endocytosis) to facilitate its transient ‘opening’. Dynamin II, a large GTPase known to be involved in endocytosis, was shown to be a product of Sertoli and germ cells in the testis. It was also localized to the BTB, as well as the apical ectoplasmic specialization (apical ES), during virtually all stages of the epithelial cycle. By co-immunoprecipitation, dynamin II was shown to associate with occludin, N-cadherin, zonula occludens-1 (ZO-1), β-catenin, junctional adhesion molecule-A, and p130Cas, but not nectin-3. An in vivo model in rats previously characterized for studying adherens junction (AJ) dynamics in the testes by adjudin (formerly called AF-2364, 1-(2,4-dichlorobenzyl)-1H-indazole-3-car-hohydrizide) treatment was used in our studies. At the time of germ cell loss from the seminiferous epithelium as a result of adjudin-induced AJ restructuring without disrupting the BTB integrity, a significant decline in the steady-state dynamin II protein level was detected. This change was associated with a concomitant increase in the levels of two protein complexes at the BTB, namely occludin/ZO-1 and N-cadherin/β-catenin. Interestingly, these changes were also accompanied by a significant increase in the structural interaction of dynamin II with β-catenin and ZO-1. β-Catenin and ZO-1 are adaptors that structurally link the cadherin- and occludin-based protein complexes together at the BTB in an ‘engaged’state to reinforce the barrier function in normal testes. However, β-catenin and ZO-1 were ‘disengaged’ from each other but bound to dynamin II during adjudin-induced AJ restructuring in the testis. The data reported herein suggest that dynamin II may assist the ‘disengagement’ of β-catenin from ZO-1 during BTB restructuring. Thus, this may permit the occludin/ZO-1 complexes to maintain the BTB integrity when the cadherin/catenin complexes are dissociated to facilitate germ cell movement.
Yan-Guang Wu, David H Barad, Vitaly A Kushnir, Emanuela Lazzaroni, Qi Wang, David F Albertini and Norbert Gleicher
Why IVF pregnancy rates decline sharply after age 43 is unknown. In this study, we compared granulosa cell (GC) function in young oocyte donors (n=31, ages 21–29), middle-aged (n=64, ages 30–37) and older infertile patients (n=41, ages 43–47). Gene expressions related to gonadotropin activity, steroidogenesis, apoptosis and luteinization were examined by real-time PCR and western blot in GCs collected from follicular fluid. FSH receptor (FSHR), aromatase (CYP19A1) and 17β-hydroxysteroid dehydrogenase (HSD17B) expression were found down regulated with advancing age, while LH receptor (LHCGR), P450scc (CYP11A1) and progesterone receptor (PGR) were up regulated. Upon in vitro culture, GCs were found to exhibit lower proliferation and increased apoptosis with aging. While FSH supplementation stimulated GCs growth and prevented luteinization in vitro. These observations demonstrate age-related functional declines in GCs, consistent with premature luteinization. To avoid premature luteinization in women above age 43, we advanced oocyte retrieval by administering human chorionic gonadotropin at maximal leading follicle size of 16 mm (routine 19–21 mm). Compared to normal cycles in women of similar age, earlier retrieved patients demonstrated only a marginal increase in oocyte prematurity, yet exhibited improved embryo numbers as well as quality and respectable clinical pregnancy rates. Premature follicular luteinization appears to contribute to rapidly declining IVF pregnancy chances after age 43, and can be avoided by earlier oocyte retrieval.
Michelle W M Li, Weiliang Xia, Dolores D Mruk, Claire Q F Wang, Helen H N Yan, Michelle K Y Siu, Wing-yee Lui, Will M Lee and C Yan Cheng
The timely restructuring of the blood–testis barrier (BTB) that facilitates the migration of preleptotene and leptotene spermatocytes from the basal to the adluminal compartment in the seminiferous epithelium of adult rat testes, which occurs at late stage VII through early stage VIII of the epithelial cycle, is a crucial cellular event of spermatogenesis. However, the regulation of BTB dynamics at the biochemical level remains elusive. In this study, tumor necrosis factor α (TNFα), a secretory product of Sertoli and germ cells in rat testes, was shown to affect junction dynamics in vivo. Following an acute administration of recombinant TNFα directly to adult rat testes in vivo at 0.5 and 2 μg/testis (with a body weight ~300 g), this treatment significantly and transiently disrupted the BTB. It also transiently inhibited the steady-state protein levels of occludin, zonula occludens-1, and N-cadherin, but not junction adhesion molecule-A, α-, and β-catenin in testes at the BTB site as illustrated by immunoblottings, immunohistochemistry, electron microscopy, and fluorescent microscopy. This transient disruption of the BTB integrity induced by TNFα treatment was further demonstrated by a functional test to assess the passage of a fluorescent dye (e.g. fluorescein-5-isothiocyanate) from the systemic circulation to the adluminal compartment. Additionally, both the phosphorylated-Ser/Thr protein kinase activated by MAP kinase kinase (p-p38) and phosphorylated-externally regulated kinase (p-ERK) mitogen -activated protein kinase-signaling pathways were transiently activated. Collectively, these data coupled with the recently published in vitro studies have illustrated that the BTB is likely utilizing a novel mechanism in which localized production of TNFα by Sertoli and germ cells into the microenvironment at the basal compartment facilitates the timely restructuring (‘opening’?) of the BTB during spermatogenesis to facilitate germ cell migration.
Dong-Xu Han, Chang-Jiang Wang, Xu-Lei Sun, Jian-Bo Liu, Hao Jiang, Yan Gao, Cheng-Zhen Chen, Bao Yuan and Jia-Bao Zhang
Circular RNAs (circRNAs) are a new class of RNA that have a stable structure characterized by covalently closed circular molecules and are involved in invasive pituitary adenomas and recurrent clinically nonfunctioning pituitary adenomas. However, information on circRNAs in the normal pituitary, especially in rats, is limited. In this study, we identified 4123 circRNAs in the immature (D15) and mature (D120) rat anterior pituitary using the Illumina platform, and 32 differentially expressed circRNAs were found. A total of 150 Gene Ontology terms were significantly enriched, and 16 KEGG pathways were found to contain differentially expressed genes. Moreover, we randomly selected eight highly expressed circRNAs and detected their relative expression levels in the mature and immature rat pituitary by qPCR. In addition, we predicted 90 interactions between 53 circRNAs and 57 miRNAs using miRanda. Notably, circ_0000964 and circ_0001303 are potential miRNA sponges that may regulate the Fshb gene. The expression profile of circRNAs in the immature and mature rat anterior pituitary may provide more information about the roles of circRNAs in the development and reproduction in mammals.
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.
Shu-Fang Xia, Xiao-Mei Duan, Xiang-Rong Cheng, Li-Mei Chen, Yan-Jun Kang, Peng Wang, Xue Tang, Yong-Hui Shi and Guo-Wei Le
The study was designed to investigate the possible mechanisms of hepatic microRNAs (miRs) in regulating local thyroid hormone (TH) action and ultimately different propensities to high-fat diet (HFD)-induced obesity. When obesity-prone (OP) and obesity-resistant (OR) mice were fed HFD for 7 weeks, OP mice showed apparent hepatic steatosis, with significantly higher body weight and lower hepatic TH receptor b (TRb) expression and type 1 deiodinase (DIO1) activity than OR mice. Next-generation sequencing technology revealed that 13 miRs in liver were dysregulated between the two phenotypes, of which 8 miRs were predicted to target on Dio1 or TRb. When mice were fed for 17 weeks, OR mice had mild hepatic steatosis and increased Dio1 and TRb expression than OP mice, with downregulation of T3 target genes (including Srebp1c, Acc1, Scd1 and Fasn) and upregulation of Cpt1α, Atp5c1, Cox7c and Cyp7a1. A stem-loop qRT-PCR analysis confirmed that the levels of miR-383, miR-34a and miR-146b were inversely correlated with those of DIO1 or TRb. Down-regulated expression of miR-383 or miR-146b by miR-383 inhibitor (anti-miR-383) or miR-146b inhibitor (anti-miR-146b) in free fatty acid-treated primary mouse hepatocytes led to increased DIO1 and TRb expressions, respectively, and subsequently decreased cellular lipid accumulation, while miR-34a inhibitor (anti-miR-34a) transfection had on effects on TRb expression. Luciferase reporter assay illustrated that miR-146b could directly target TRb 3′untranslated region (3′UTR). These findings suggested that miR-383 and miR-146b might play critical roles in different propensities to diet-induced obesity via targeting on Dio1 and TRb, respectively.
Meng Guo, Yuna Li, Yan Wang, Zhenkun Li, Xiaohong Li, Peikun Zhao, Changlong Li, Jianyi Lv, Xin Liu, Xiaoyan Du and Zhenwen Chen
Recent studies raise the possibility that eukaryotic translation elongation factor 1 alpha (eEF1A) may play a role in metabolism. One isoform, eEF1A2, is specifically expressed in skeletal muscle, heart and brain. It regulates translation elongation and signal transduction. Nonetheless, eEF1A2’s function in skeletal muscle glucose metabolism remains unclear. In the present study, suppression subtractive hybridisation showed a decrease in Eef1a2 transcripts in the skeletal muscle of diabetic Mongolian gerbils. This was confirmed at mRNA and protein levels in hyperglycaemic gerbils, and in db/db and high-fat diet-fed mice. Further, this downregulation was independent of Eef1a2 promoter methylation. Interestingly, adeno-associated virus-mediated eEF1A2 overexpression in skeletal muscle aggravated fasting hyperglycaemia, hyperinsulinaemia and glucose intolerance in male diabetic gerbils but not in female gerbil models. The overexpression of eEF1A2 in skeletal muscle also resulted in promoted serum glucose levels and insulin resistance in male db/db mice. Up- and downregulation of eEF1A2 by lentiviral vector transfection confirmed its inhibitory effect on insulin-stimulated glucose uptake and signalling transduction in C2C12 myotubes with palmitate (PA)-induced insulin resistance. Furthermore, eEF1A2 bound PKCβ and increased its activation in the cytoplasm, whereas suppression of PKCβ by an inhibitor attenuated eEF1A2-mediated impairment of insulin sensitivity in insulin-resistant myotubes. Endoplasmic reticulum (ER) stress was elevated by eEF1A2, whereas suppression of ER stress or JNK partially restored insulin sensitivity in PA-treated myotubes. Additionally, eEF1A2 inhibited lipogenesis and lipid utilisation in insulin-resistant skeletal muscle. Collectively, we demonstrated that eEF1A2 exacerbates insulin resistance in male murine skeletal muscle via PKCβ and ER stress.