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Elisa Maseroli, Ilaria Cellai, Sandra Filippi, Paolo Comeglio, Sarah Cipriani, Giulia Rastrelli, Martina Rosi, Flavia Sorbi, Massimiliano Fambrini, Felice Petraglia, Roberta Amoriello, Clara Ballerini, Letizia Lombardelli, Marie-Pierre Piccinni, Erica Sarchielli, Giulia Guarnieri, Annamaria Morelli, Mario Maggi, and Linda Vignozzi

Chronic inflammation is involved in the genitourinary syndrome of menopause (GSM) and beneficial effects of androgens in the vagina have been described. We investigated the potential involvement of human vagina smooth muscle cells (hvSMCs) in the inflammatory response and the immunomodulatory effect of androgen receptor (AR) agonist dihydrotestosterone (DHT). HvSMCs isolated from menopausal women were evaluated for sex steroids receptors and toll-like receptors mRNA expression, and left untreated or treated in vitro with lipopolysaccharide (LPS) or IFNγ, in the presence or absence of DHT. We evaluated mRNA expression (by RT-PCR) and secretion in cell culture supernatants (by a bead-based immunoassay) of pro-inflammatory markers. Nuclear translocation of NF-κB (by immunofluorescence) and cell surface HLA-DR expression (by flow cytometry) were also evaluated. Similar experiments were repeated in rat vSMCs (rvSMCs). In hvSMCs and rvSMCs, AR was highly expressed. DHT pre-treatment inhibited LPS-induced mRNA expression of several pro-inflammatory mediators (i.e. COX2, IL-6, IL-12A and IFNγ), effect significantly blunted by AR antagonist bicalutamide. DHT significantly counteracted the secretion of IL-1RA, IL-2, IL-5, IL-15, FGF, VEGF and TNFα. LPS-induced NF-κB nuclear translocation was significantly inhibited by DHT, an effect counteracted by bicalutamide. DHT pre-treatment significantly decreased IFNγ-induced expression of HLA-DR, mRNA expression of iNOS, COX2 and MCP1, and secretion of IL-1, IL-2, IL-5, IL-6, MCP1 and GCSF. Similar effects were observed in rvSMCs. The activation of AR suppresses the inflammatory response in hvSMCs, reducing their potential to be involved in the initiation and maintaining of inflammation, thus representing a therapeutic strategy in conditions, such as the GSM.

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Laurent Beck and Sarah Beck-Cormier

The critical role of phosphate (Pi) in countless biological processes requires the ability to control its concentration both intracellularly and extracellularly. At the body level, this concentration is finely regulated by numerous hormones, primarily parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). While this control of the body’s Pi homeostasis is now well documented, knowledge of the mechanisms that allow the cell and the body to detect extracellular Pi variations is much less known. These systems are well described in bacteria, yeasts and plants, but as will be discussed in this review, knowledge obtained from these organisms is not entirely relevant to the requirements of Pi biology in mammals. In this review, we present the latest findings on extracellular Pi sensing in mammals, and describe the mammalian Pi sensors identified to date, such as SLC20A1 (PIT1)/SLC20A2 (PIT2) heterodimers and the calcium-sensing receptor (CaSR). While there are many questions remaining to be resolved, a clarification of the Pi sensing mechanisms in mammals is critical to understanding the deregulation of Pi balance in certain life-threatening disease states, such as end-stage renal disease and associated vascular calcifications, and to proposing relevant therapeutic approaches.

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G Rosales-Soto, A Diaz-Vegas, M Casas, A Contreras-Ferrat, and E Jaimovich

Fibroblast growth factor 21 (FGF21) is a pleiotropic peptide hormone that is considered a myokine playing a role in a variety of endocrine functions, including regulation of glucose transport and lipid metabolism. Although FGF21 has been associated with glucose metabolism in skeletal muscle cells, its cellular mechanism in adult skeletal muscle fibers glucose uptake is poorly understood. In the present study, we found that FGF21 induced a dose−response effect, increasing glucose uptake in skeletal muscle fibers from the flexor digitorum brevis muscle of mice, evaluated using the fluorescent glucose analog 2-NBDG (300 µM) in single living fibers. This effect was prevented by the use of either cytochalasin B (5 µM) or indinavir (100 µM), both antagonists of GLUT4 activity. The use of PI3K inhibitors such as LY294002 (50 µM) completely prevented the FGF21-dependent glucose uptake. In fibers electroporated with the construct encoding GLUT4myc-eGFP chimera and stimulated with FGF21 (100 ng/mL), a strong sarcolemmal GLUT4 label was detected. This effect promoted by FGF21 was demonstrated to be dependent on atypical PKC-ζ, by using selective PKC inhibitors. FGF21 at low concentrations potentiated the effect of insulin on glucose uptake but at high concentrations, completely inhibited the uptake in the presence of insulin. These results suggest that FGF21 regulates glucose uptake by a mechanism mediated by GLUT4 and dependent on atypical PKC-ζ in skeletal muscle.

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David W Scoville, Artiom Gruzdev, and Anton M Jetten

Recent advances in high throughput RNA sequencing have revealed that, in addition to messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs) play an important role in the regulation of many cell functions and of organ development. While a number of lncRNAs have been identified in pancreatic islets, their function remains largely undetermined. Here, we identify a novel long ncRNA regulated by the transcription factor GLIS3, which we refer to as GLIS3 regulated 1 (G3R1). This lncRNA was identified for its significant loss of expression in GLIS3 knockout mouse pancreatic islets. G3R1 appears to be specifically expressed in mouse pancreatic β-cells and in a β-cell line (βTC-6). ChIP-seq analysis indicated that GLIS3 and other islet-enriched transcription factors bind near the G3R1 gene, suggesting they directly regulate G3R1 transcription. Similarly, an apparent human homolog of G3R1 displays a similar expression pattern, with additional expression seen in human brain. In order to determine the function of G3R1 in mouse pancreatic β-cells, we utilized CRISPR to develop a knockout mouse where ~80% of G3R1 sequence is deleted. Phenotypic analysis of these mice did not reveal any impairment in β-cell function or glucose regulation, indicating the complexity underlying the study of lncRNA function.

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Bo Li, Yue Zhou, Jing Chen, Tingting Wang, Zhijuan Li, Yili Fu, Aixia Zhai, and Changlong Bi

Diabetic foot ulcer (DFU) is a chronic and non-healing complication of diabetes that leads to high hospital costs and, in extreme cases, to amputation. Recent studies have reported that long non-coding RNAs (lncRNAs) are linked to various diabetes-related symptoms. Thus, we aim to explore the role of lncRNA H19 in the wound healing process following DFU. Fibroblasts were isolated from the ulcer margin tissues of DFU patients, with the expression of lncRNA H19, connective tissue growth factor (CTGF) or serum response factor (SRF) altered by lentivirus infection. Next, rat models of DFU induced by high glucose and lipid diet were established, which was also infected with the corresponding lentivirus. The interaction among lncRNA H19, SRF and CTGF was determined. Afterward, cell proliferation and apoptosis, angiogenesis, ECM remodeling and wound healing in DFU tissues were evaluated to explore the effects of lncRNA H19/SRF/CTGF and MAPK signaling pathway on DFU. CTGF was poorly expressed in ulcer tissues from DFU rats and patients. CTGF overexpression was shown to activate the MAPK signaling pathway to promote cell proliferation, ECM remodeling, angiogenesis and wound healing while inhibiting cell apoptosis. lncRNA H19 was validated to elevate CTGF expression by recruiting SRF to the promoter region of CTGF, thus accelerating cell proliferation, ECM remodeling and wound healing while repressing cell apoptosis. Furthermore, MAPK signaling pathway activation is confirmed to be the underlying mechanism behind lncRNA H19/CTGF/SRF-induced results. Thus, lncRNA H19 accelerated wound healing in DFU through elevation of CTGF and activation of the MAPK signaling pathway.

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Yuanyuan Huang, Hanlin Zhang, Meng Dong, Lei Zhang, Jun Lin, Rongcai Ye, Huiqiao Zhou, Xiaomeng Liu, and Wanzhu Jin

White adipose tissue (WAT) browning may have beneficial effects for treating metabolic syndrome. miRNA are important regulators of the differentiation, development, and function of brown and beige adipocytes. Here, we found that the cold-inducible miRNA17-92 cluster is enriched in brown adipose tissue (BAT) compared with WAT. Overexpression of the miR17-92 cluster in C3H10T1/2 cells, a mouse mesenchymal stem cell line, enhanced the thermogenic capacity of adipocytes. Furthermore, we observed a significant reduction in adiposity in adipose tissue-specific miR17-92 cluster transgenic (TG) mice. This finding is partly explained by dramatic increases in white fat browning and energy expenditure. Interestingly, the miR17-92 cluster stimulated WAT browning without altering BAT activity in mice. In addition, when we removed the intrascapular BAT (iBAT), the TG mice could maintain their body temperature well under cold exposure. At the molecular level, we found that the miR17-92 cluster targets Rb1, a beige cell repressor in WAT. The present study reveals a critical role for the miR17-92 cluster in regulating WAT browning. These results may be helpful for better understanding the function of beige fat, which could compensate for the lack of BAT in humans, and may open new avenues for combatting metabolic syndrome.

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Pablo Pánico, Marcia Hiriart, Patricia Ostrosky-Wegman, and Ana María Salazar

The calpain-10 (CAPN10) protease is implicated in the translocation of the glucose transporter 4 (GLUT4), which is retained in the Golgi matrix via the Tether containing a UBX domain for GLUT4 (TUG) protein. Insulin stimulation induces the proteolytic processing of TUG, which leads to the translocation of GLUT4 to the cell membrane. We tested whether TUG is a CAPN10 substrate. Proteolysis of TUG by calpains was assessed using a cell-free system containing calpain-1 and TUG. In situ proteolysis of TUG by calpains was demonstrated in 3T3-L1 adipocytes in the presence of insulin or calpain inhibitors to modulate calpain activity. Proteolysis of TUG by CAPN10 was confirmed using transient or stable silencing of CAPN10 in 3T3-L1 adipocytes. Calpains proteolyzed the C-terminus of TUG in vitro. In adipocytes, insulin-induced cleavage of TUG was correlated with the activation of calpains. Treatment with calpain inhibitors reduced TUG cleavage, resulting in impaired GLUT4 translocation without altering Akt phosphorylation. Furthermore, CAPN10 but not calpain-1 or calpain-2 colocalized with GLUT4 in the absence of insulin, and their colocalization was reduced after stimulation with insulin. Finally, we demonstrated that CAPN10 knockdown reduced the proteolysis of TUG without altering the phosphorylation of Akt or the expression of the Usp25m protease. Thus, our results provide evidence that the TUG protein is cleaved by CAPN10 to regulate GLUT4 translocation.

Open access

Liting Wu, Li Xiong, Jin Li, Zishan Peng, Luyao Zhang, Peijie Shi, Yingying Gong, and Haipeng Xiao

This study aimed to identify circular RNAs differentially expressed in the islets of type 2 diabetes (T2DM) models and clarify their roles in the control of β-cell functions. Circular RNAs dysregulated in the islets of diabetic db/db mice were identified by high-throughput RNA sequencing. Then, the expression level of the selected circular RNA circ-Tulp4 was confirmed by real-time PCR in the islets of diabetic models and Min6 cells. MTS, EdU, western blot, flow cytometric analysis, and luciferase assay were performed to investigate the impact of circ-Tulp4 on β-cell functions. This study identified thousands of circular RNAs in mouse pancreatic islets. The circ-Tulp4 level significantly decreased in the diabetic models and altered in the Min6 cells under lipotoxic condition. The modulation of circ-Tulp4 level in Min6 cells regulated cell proliferation. Furthermore, an interaction was demonstrated between circ-Tulp4 and miR-7222-3p, which suppressed the expression of cholesterol esterification related gene, sterol O-Acyltransferase 1 (SOAT1). The accumulation of soat1 activated cyclin D1 expression, thus promoting cell cycle progression. These findings showed that circ-Tulp4 regulated β-cell proliferation via miR-7222-3p/soat1/cyclin D1 signaling. Our research suggested that circ-Tulp4 might be a potential therapeutic intervention for T2DM. Besides, soat1 might be important for β-cell adaptation to lipotoxicity.

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Chang-Jiang Wang, Fei Gao, Yi-Jie Huang, Dong-Xu Han, Yi Zheng, Wen-Hua Wang, Hao Jiang, Yan Gao, Bao Yuan, and Jia-Bao Zhang

The pituitary gland functions as a prominent regulator of diverse physiologic processes by secreting multiple hormones. Circular RNAs (circRNAs) are an emerging novel type of endogenous noncoding RNA that have recently been recognized as powerful regulators participating in various biological processes. However, the physiological roles and molecular mechanisms of circRNAs in pituitary remain largely unclear. Herein, we concentrated on expounding the biological function and molecular mechanism of circRNA in rat pituitary. In this study, we identified a novel circRNA in pituitary tissue, circAkap17b, which was pituitary- and stage-specific. Then, we designed circAkap17b siRNA and constructed an overexpression plasmid to evaluate the effect of loss- and gain-of-circAkap17b function on FSH secretion. Interestingly, silencing circAkakp17b significantly inhibited FSH expression and secretion, while overexpression of circAkap17b enhanced FSH expression and secretion. Furthermore, dual luciferase reporter and RNA immunoprecipitation (RIP) assays confirmed that circAkap17b could serve as miR-7 sponge to regulate target genes. Additionally, miR-7b suppressed FSH expression and secretion by directly targeting FSHb through the dual luciferase reporter and RT-qPCR analysis. Additionally, rescue experiments showed that circAkap17b could regulate FSH secretion in pituitary cells through a circAkap17b-miR-7-FSHb axis. Collectively, we demonstrated that circAkap17b could act as a molecular sponge of miR-7 to up-regulate expression of the target gene FSHb and facilitate FSH secretion. These findings provide evidence for a novel regulatory role of circRNAs in pituitary.

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Frank Simmen, Iad Alhallak, and Rosalia C.m. Simmen

Malic enzyme 1 (ME1) is a cytosolic protein that catalyzes the conversion of malate to pyruvate while concomitantly generating NADPH from NADP. Early studies identified ME1 as a mediator of intermediary metabolism primarily through its participatory roles in lipid and cholesterol biosynthesis. ME1 was one of the first identified insulin-regulated genes in liver and adipose and is a transcriptional target of thyroxine. Multiple studies have since documented that ME1 is pro-oncogenic in numerous epithelial cancers. In tumor cells, the reduction of ME1 gene expression or the inhibition of its activity resulted in decreases in proliferation, epithelial-to-mesenchymal transition and in vitro migration, and conversely, in promotion of oxidative stress, apoptosis and/or cellular senescence. Here, we integrate recent findings to highlight ME1’s role in oncogenesis, provide a rationale for its nexus with metabolic syndrome and diabetes, and raise the prospects of targeting the cytosolic NADPH network to improve therapeutic approaches against multiple cancers.