Molecular and Cellular Endocrinology

Altered miR-186 and miR-135a contribute to granulosa cell dysfunction by targeting ESR2: A possible role in polycystic ovary syndrome Publication date: 20 August 2019 Source: Molecular and Cellular Endocrinology, Volume 494 Author(s): Yuxia Song, Guo Yu, Yungai Xiang, Yan Li, Lijing Wan, Li Tan Abstract MicroRNAs (miRNAs) are a group of negative regulators of gene expression that function at the posttranscriptional level. Dysregulation of miRNAs is involved in many pathophysiological processes, including polycystic ovary syndrome (PCOS). In this study, we first detected the expression levels of 6 candidate miRNA in granulosa cells (GCs) from 63 women with PCOS and 20 healthy controls. We found that miR-186 and miR-135a were overexpressed in GCs from PCOS patients. Subsequently, the direct targets of miR-186 and miR-135a were predicted using bioinformatics analysis and verified by luciferase assays and immunoblotting. The present study determined that miR-186 and miR-135a repressed ESR2 expression in GCs, which further inhibited CDKN1A expression, promoted GC proliferation and repressed GC apoptosis. Meanwhile, the levels of miR-186 and miR-135a in GCs were found to positively correlate with serum estradiol levels in patients with PCOS. Furthermore, estradiol treatment directly increased miR-186 and miR-135a levels in KGN and primary GCs, which provides new insight into understanding the pathophysiology of PCOS.

Empagliflozin improves primary haemodynamic parameters and attenuates the development of atherosclerosis in high fat diet fed APOE knockout mice Publication date: 20 August 2019 Source: Molecular and Cellular Endocrinology, Volume 494 Author(s): Georgios K. Dimitriadis, Narjes Nasiri-Ansari, Georgios Agrogiannis, Ioannis D. Kostakis, Manpal S. Randeva, Nikolaos Nikiteas, Vanlata H. Patel, Gregory Kaltsas, Athanasios G. Papavassiliou, Harpal S. Randeva, Eva Kassi Abstract The effects of long-term treatment with empagliflozin on biochemical and immunohistochemical markers related to atherosclerosis and atherosclerosis development in the aorta of apolipoprotein E knockout [Apo-E (−/−)] mice were evaluated in this study. Empagliflozin-treated mice had lower total cholesterol (P < 0.05), fasting glucose (P < 0.01), heart rate (P < 0.01) and diastolic blood pressure (DBP) (P < 0.05) compared to controls. Histomorphometry revealed reduced atherosclerotic lesion progress approaching statistical significance (P = 0.06) and approximately 50% wider lumen area for the Empagliflozin treated mice group. Although empagliflozin significantly reduced Vcam-1 and Mcp-1 (P < 0.05, P < 0.01, respectively) and marginally induced Timp-1 and Timp-2 mRNA expression (P < 0.08, P = 0.1 respectively), immunohistochemistry revealed a marginal reduction in VCAM-1 and MMP-9 (P = 0.1) without affecting the expression of TIMP-2 and MCP-1 in atherosclerotic lesions. Empagliflozin improves primary haemodynamic parameters and attenuates the progression of atherosclerosis by reducing hyperlipidemia and hyperglycemia, while direct actions in aorta vessel mediated via SGLT-1 are strongly hypothesized.

ALK4-SMAD2/3-SMAD4 signaling mediates the activin A-induced suppression of PTX3 in human granulosa-lutein cells Publication date: 1 August 2019 Source: Molecular and Cellular Endocrinology, Volume 493 Author(s): Chang Liu, Hsun-Ming Chang, Yuyin Yi, Ying Fang, Feiyan Zhao, Peter C.K. Leung, Xiaokui Yang Abstract As one of the members of the transforming growth factor-β (TGF-β) superfamily, activin A plays an important role in regulating follicular development and oocyte maturation. Pentraxin 3 (PTX3) is the key component that promotes the process of cumulus expansion during mammalian ovulation. At present, the regulation of PTX3 expression in human granulosa cells remains largely unknown. This study aimed to examine the effects of activin A on the expression of PTX3 in human granulosa-lutein (hGL) cells and to investigate the underlying molecular mechanisms. Using an established immortalized hGL cell line (SVOG) and primary hGL cells as study models, we demonstrated that activin A significantly increased the phosphorylation of SMAD2 and SMAD3, which suppressed the expression of PTX3 at both the mRNA and protein levels. Additionally, these effects induced by activin A were completely reversed by pretreatment with the TGF-β type I receptor inhibitor SB431542 and knockdown of ALK4. Furthermore, knockdown of SMAD2, SMAD3, or SMAD4 completely reversed the activin A-induced suppressive effects on PTX3 expression. Notably, the ChIP analyses demonstrated that phosphorylated SMADs could bind to human PTX3 promoter. Collectively, our results showed that the ALK4-SMAD2/3-SMAD4 signaling pathway most likely mediates the suppressive effect of activin A on PTX3 expression in hGL cells.

Defective endoplasmic reticulum export causes proinsulin misfolding in pancreatic β cells Publication date: 1 August 2019 Source: Molecular and Cellular Endocrinology, Volume 493 Author(s): Ruimin Zhu, Xin Li, Jialu Xu, Cesar Barrabi, Dilini Kekulandara, James Woods, Xuequn Chen, Ming Liu Abstract Endoplasmic reticulum (ER) homeostasis is essential for cell function. Increasing evidence indicates that, efficient protein ER export is important for ER homeostasis. However, the consequence of impaired ER export remains largely unknown. Herein, we found that defective ER protein transport caused by either Sar1 mutants or brefeldin A impaired proinsulin oxidative folding in the ER of β-cells. Misfolded proinsulin formed aberrant disulfide-linked dimers and high molecular weight proinsulin complexes, and induced ER stress. Limiting proinsulin load to the ER alleviated ER stress, indicating that misfolded proinsulin is a direct cause of ER stress. This study revealed significance of efficient ER export in maintaining ER protein homeostasis and native folding of proinsulin. Given the fact that proinsulin misfolding plays an important role in diabetes, this study suggests that enhancing ER export may be a potential therapeutic target to prevent/delay β-cell failure caused by proinsulin misfolding and ER stress.

The development of the serotonergic and dopaminergic systems during chicken mid-late embryogenesis Publication date: 1 August 2019 Source: Molecular and Cellular Endocrinology, Volume 493 Author(s): Xiaohong Huang, Shihuan Kuang, Todd J. Applegate, Tsang-long Lin, Heng-wei Cheng Abstract Serotonin (5-HT) acts as a morphogen influencing embryonic brain development, and as a neurotransmitter regulating multiple biological functions with lifelong effects on animal physical, physiological and mental health, especially during the rapid growth phase prior to birth when embryos face many challenges to reach structural and functional completion. In this study, the development of the serotoninergic (5-HTergic) system and its modulatory effect on the dopaminergic (DAergic) system and related neural circuits were investigated during the mid-late embryogenesis, embryonic day (E)12-E20, in the chicken's brain. During 5-HTergic neuronal maturation, a growth-related anatomical and functional remodeling was highlighted: the 5-HT neurons continuously grew during E12-E20 except for a remarkable regression during E14-E16. Correspondingly, there was a time-dependent change in the 5-HT synthetic capacity. Specifically, 5-HT concentrations in the raphe nuclei increased from E12 to E14, reaching a first plateau during E14-E16, then continuously increased up to E19, and reaching a second plateau between E19-E20. The second plateau of the 5-HT concentration was in correspondence with the establishment of the 5-HTergic autoregulatory loop during E19-E20 and the development of the DAergic system. The DA concentrations remained unchanged from E12 to E16, then started to increase at E16, reaching a maximum at E19, and diminished before hatching. The unique developing time sequence between the 5-HTergic and DAergic systems suggests that the 5-HTergic system may play a critical role in forming the 5-HT – DA neural circuit during chicken embryogenesis. These results provide new insights for understanding the functional organization of the 5-HTergic system during embryonic development and raise the possibility that prenatally modulating the 5-HTergic system may lead to long-lasting brain structural and functional alterations. Graphical abstract

Correlation between renin-angiotensin system (RAS) related genes, type 2 diabetes, and cancer: Insights from metanalysis of transcriptomics data Publication date: 1 August 2019 Source: Molecular and Cellular Endocrinology, Volume 493 Author(s): Luciana Xavier Pereira, Laryssa Cristina Alves da Silva, Alexya de Oliveira Feitosa, Ricardo Jansen Santos Ferreira, Ana Kelly Fernandes Duarte, Valdemir da Conceição, Carolinne de Sales Marques, Amanda Karine Barros Ferreira Rodrigues, Bruna Del Vechio Koike, Aline Cavalcante de Queiroz, Talita Antunes Guimaraes, Carlos Dornels Freire de Souza, Carlos Alberto de Carvalho Fraga Abstract Although studies have provided significant evidence about the role of RAS in mediating cancer risk in type 2 diabetes mellitus (DM), conclusions about the central molecular mechanisms underlying this disease remain to be reached, because this type of information requires an integrative multi-omics approach. In the current study, meta-analysis was performed on type 2 diabetes and breast, bladder, liver, pancreas, colon and rectum cancer-associated transcriptome data, and reporter biomolecules were identified at RNA, protein, and metabolite levels using the integration of gene expression profiles with genome-scale biomolecular networks in diabetes samples. This approach revealed that RAS biomarkers could be associated with cancer initiation and progression, which include metabolites (particularly, aminoacyl-tRNA biosynthesis and ABC transporters) as novel biomarker candidates and potential therapeutic targets. We detected downregulation and upregulation of differentially expressed genes (DEGs) in blood, pancreatic islets, liver and skeletal muscle from normal and diabetic patients. DEGs were combined with 211 renin-angiotensin-system related genes. Upregulated genes were enriched using Pathway analysis of cancer in pancreatic islets, blood and skeletal muscle samples. It seems that the changes in mRNA are contributing to the phenotypic changes in carcinogenesis, or that they are as a result of the phenotypic changes associated with the malignant transformation. Our analyses showed that Ctsg and Ednrb are downregulated in cancer samples. However, by immunohistochemistry experiments we observed that EDNRB protein showed increased expression in tumor samples. It is true that alterations in mRNA expression do not always reflect alterations in protein expression, since post-translational changes can occur in proteins. In this study, we report valuable data for further experimental and clinical analysis, because the proposed biomolecules have significant potential as systems biomarkers for screening or for therapeutic purposes in type 2 diabetes and cancer-associated pathways.

Identification and functional characterization of microRNAs in rat Leydig cells during development from the progenitor to the adult stage Publication date: 1 August 2019 Source: Molecular and Cellular Endocrinology, Volume 493 Author(s): Hongxia Chen, Xiaoping Guo, Xue Xiao, Leping Ye, Yadong Huang, Chunbin Lu, Zhijian Su Abstract The aim of the present study was to identify microRNAs (miRNAs) that regulate the proliferation and differentiation of Leydig cells (LCs) of rat. Three small RNA libraries derived from progenitor LCs (PLCs), immature LCs (ILCs) and adult LCs (ALCs) were analyzed by microarrays. In total, 68 differentially expressed miRNAs (DEMs) were identified. Based on the trend of DEM expression from PLCs to ALCs, primary LCs were transfected with miRNA mimics or inhibitors. Five miRNAs (miR-30a-5p, miR-3585-5p, miR-212-3p, miR-369-5p and miR-434-3p) promoted PLC proliferation, and 3 miRNAs (miR-17-5p, miR-532-3p and miR-329-3p) activated caspase-3, which triggered LC apoptosis. For steroidogenesis, 18 miRNAs could elevate or inhibit androsterone release at the PLC stage. Eleven and 9 miRNAs inhibited the production of 5α-androstane-3α,17β-diol in ILCs and testosterone in ALCs, respectively. miR-17-5p, miR-29a-3p and miR-299a-5p decreased androgen production by LCs at all developmental stages. Furthermore, the miR-299a-5p-mediated decrease in androgen production by the LC lineage was primarily achieved by downregulating the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). These findings provide insights into the regulatory roles of miRNAs during the postnatal development of LCs and suggest potential strategies for the treatment of steroid-related disorders.

Adiponectin is required for pioglitazone-induced improvements in hepatic steatosis in mice fed a high-fat diet Publication date: 1 August 2019 Source: Molecular and Cellular Endocrinology, Volume 493 Author(s): Mariana de Mendonça, Bruna de Araújo Cardoso dos Santos, Érica de Sousa, Alice Cristina Rodrigues Abstract Pioglitazone has been used for the treatment of nonalcoholic fatty liver disease (NAFLD) related to diabetes. The role of adiponectin in pioglitazone-induced improvements in NAFLD was studied by using wild-type (adipoWT) and adiponectin knockout (adipoKO) mice. High-fat diet fed mice were insulin resistant, glucose intolerant and had increased hepatic lipid accumulation as evidenced by increased NAFLD activity score. Despite pioglitazone has improved insulin resistance in both genotypes, hepatic steatosis was only improved in adipoWT obese mice. Amelioration of NAFLD in adipoWT mice promoted by pioglitazone was associated with up-regulation of Pparg, Fgf21 and down-regulation of Pepck liver expression. On the other hand, resistance to pioglitazone treatment in adipoKO mice was associated with increased expression of miR-192 and Hsl, which was not followed by increased fatty acid oxidation. In conclusion, our data provides evidence that increased adiponectin production by pioglitazone is necessary for its beneficial action on NAFLD. Graphical abstract

Ubiquitin proteasome system (UPS) activation in the cardiac hypertrophy of hyperthyroidism Publication date: 1 August 2019 Source: Molecular and Cellular Endocrinology, Volume 493 Author(s): Caroline Antunes Lino, Marilene Demasi, Maria Luiza Barreto-Chaves Abstract Ubiquitin proteasome system (UPS) is the main proteolytic pathway in eukaryotic cells. Changes in proteasome expression and activity have been associated to cardiovascular diseases as cardiac hypertrophy. Considering that cardiac hypertrophy is commonly associated to hyperthyroidism condition, the present study aimed to investigate the contribution of UPS in cardiac hypertrophy induced by thyroid hormones. Hyperthyroidism was induced in male Wistar rats by intraperitoneal injections of triiodothyronine (T3; 7  μg/100 g of body weight) for 7 days and confirmed by raised levels of total T3 and decreased levels of total T4. In addition, systolic blood pressure and heart rate were significantly increased in hyperthyroid group. Cardiac hypertrophy was confirmed in hyperthyroid group by increased heart weight/tibia length ratio and by increased α-MHC/β-MHC relative expression. Both catalytic (20SPT) and regulatory subunits (19SPT) of the constitutive proteasome were upregulated in hyperthyroid hearts. In addition, the transcripts that encode immunoproteasome subunits were also elevated. Furthermore, ATP-dependent chymotrypsin-like activity (26SPT) was significantly increased in hyperthyroid group. Despite the upregulation and activation of UPS in hyperthyroid hearts, the content of polyubiquitinated proteins was unaltered in relation to control. Together, these results evidence the activation of cardiac proteasome by thyroid hormones, which possibly contribute to the maintenance of protein quality control and regulation of cardiac hypertrophy in response to thyroid hormones.

Temperature alters the hypothalamic transcription of photoperiod responsive genes in induction of seasonal response in migratory redheaded buntings Publication date: 1 August 2019 Source: Molecular and Cellular Endocrinology, Volume 493 Author(s): Amit Kumar Trivedi, Sayantan Sur, Aakansha Sharma, SK Tahajjul Taufique, Neelu Jain Gupta, Vinod Kumar Abstract We investigated the temperature effects on hypothalamic transcription of genes involved in the induction of photoperiodic response in redheaded buntings. Birds were exposed at 22 and 38 °C to 13-h long photoperiods (LP), with controls at 22 °C on 8-h short photoperiods (SP). At 22 °C, compared to SP, we found higher tshb, eya3 and dio2 and low dio3 and gnih mRNA expressions after a week of LP; concomitant with testis recrudescence this confirmed buntings’ responsiveness to LP-induced photostimulation. tshb, dio2 and gnrh mRNA levels were further increased by 2.5 weeks of LP at 38 °C. Temperature sensitive trpm8, but not trpv4, bdnf or adcyap1 also showed LP-induced expression at 22 °C. Concomitant changes in dnmt3b and tet2 mRNA expressions further suggested epigenetic modification of temperature influence on photoperiodic responses. These results demonstrate the role of temperature in hypothalamic molecular regulation of the photoperiodic gonadal response in seasonally breeding birds.