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.
Liting Wu, Li Xiong, Jin Li, Zishan Peng, Luyao Zhang, Peijie Shi, Yingying Gong, and Haipeng Xiao
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 upregulate expression of the target gene Fshb and facilitate FSH secretion. These findings provide evidence for a novel regulatory role of circRNAs in pituitary.
Kathryn L Garner
All living cells are sensors of their environment: they sense signals, hormones, cytokines, and growth factors, among others. Binding of these signals to cell surface receptors initiates the transmission of messages along intracellular signalling pathways through protein–protein interactions, enzymatic modifications and conformational changes. Typically, the activation of signalling pathways are monitored in whole populations of cells, giving population average measures, often using experimental methods that destroy and homogenise the cell population. High content imaging is an automated, high-throughput fluorescence microscopy method that enables measurements of signal transduction pathways to be taken from live cells. It can be used to measure signalling dynamics, how the abundance of particular proteins of interest change over time, or to record how particular proteins move and change their localisation in response to a signal from their environment. Using this, and other single cell methods, it is becoming increasingly clear that cells are in fact very variable in their response to a given stimulus and in the quantities of cellular components they express, even in clonal (isogenic) cell lines. This review will discuss how high content imaging has contributed to our growing understanding of cellular heterogeneity. It will discuss how data generated has been combined with information theoretic approaches to quantify the amount of information transferred through noisy signalling pathways. Lastly, the relevance of heterogeneity to our understanding and treatment of disease will be considered, highlighting the importance of single cell measurements.
Frank A 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.
Rachel Njeim, William S Azar, Angie H Fares, Sami T Azar, Hala Kfoury Kassouf, and Assaad A Eid
NETosis, a novel form of neutrophil-related cell death, acts as a major regulator of diabetes and diabetes-associated complications. In this review, we show that the extrusion of neutrophil extracellular traps, termed NETs, plays an important role in the pathogenesis of type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), and diabetes-induced complications. In T1DM, β-cell death induces the sequestration of neutrophils in the pancreas and seems to be correlated with increased NETosis. In T2DM patients, products of NETs release are significantly elevated. Increased levels of dsDNA are correlated with the presence of cardiovascular disease and diabetic kidney disease, further supporting the role of NETosis in the pathogenesis of other diabetes-induced complications such as impaired wound healing and diabetic retinopathy. NETosis is induced by high glucose through incompletely understood mechanisms, but it also appears to be elevated in patients with diabetes who have tightly controlled glucose levels. We hypothesize that hyperglycemia worsens the already elevated baseline of NETosis in diabetic patients to further increase its detrimental effects.
Juan Carlos Arapa-Diaz, Wender do Nascimento Rouver, Jéssyca Aparecida Soares Giesen, Marcela Daruge Grando, Lusiane Maria Bendhack, and Roger Lyrio dos Santos
Physiological or supraphysiological levels of testosterone appear to be associated with the development of risk factors for cardiovascular diseases such as hypertension, as this hormone modulates the release of endothelial factors. However, its actions are still controversial, especially in the coronary circulation of hypertensive animals. This study was designed to assess the effects of testosterone treatment (T) on endothelium-dependent coronary vascular reactivity in orchiectomized SHR. The animals were divided into SHAM, orchiectomized (ORX), ORX+T and ORX+T+aromatase inhibitor (AI). All treatments lasted 15 days. Blood pressure (BP) was measured. Dose–response curves to bradykinin (BK) were constructed using the Langendorff technique, followed by inhibition of endothelium mediators (NO, prostanoids, EETs) and potassium channels. The intensity of eNOS, COX-1, COX-2, Akt, and gp91phox protein expression was quantified by Western blotting. BP was elevated in SHAM, ORX+T, and ORX+T+AI groups. However, we did not observe differences in the ORX group. Baseline coronary perfusion pressure (CPP) remained unaffected. Orchiectomy did not change the BK-induced relaxation compared to the SHAM group, whereas testosterone treatment increased it. This response was diminished in the absence of NO, prostanoids, and EETs in the SHAM and ORX groups, while in ORX+T group the relaxation was diminished only in the absence of NO and EETs. There was no difference in eNOS, COX-1, COX-2, and gp91phox protein expression, though Akt expression was increased in ORX and ORX+T groups. These results show that testosterone treatment can modulate endothelial function, especially in the coronary circulation under hypertension conditions, via NO and EETs pathways.
Vincent Giguere and Mathieu Vernier
Aging is a degenerative process that results from the accumulation of cellular and tissue lesions, leading progressively to organ dysfunction and death. Although the biological basis of human aging remains unclear, a large amount of data points to deregulated mitochondrial function as a central regulator of this process. Mounting years of research on aging support the notion that the engendered age-related decline of mitochondria is associated with alterations in key pathways that regulate mitochondrial biology. Particularly, several studies in the last decade have emphasized the importance of the estrogen-related receptor (ERR) family of nuclear receptors, master regulators of mitochondrial function, and their transcriptional coactivators PGC-1s in this context. In this review, we summarize key discoveries implicating the PGC-1/ERR axis in age-associated mitochondrial deregulation and tissue dysfunction. Also, we highlight the pharmacological potential of targeting the PGC-1/ERR axis to alleviate the onset of aging and its adverse effects.
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.
Ting Yan, Wangwang Qiu, Jianlu Song, You-ben Fan, and Zhili Yang
The diagnosis and treatment of recurrence and metastasis in papillary thyroid carcinoma (PTC) are still clinical challenges. One of the key factors is the lack of specific diagnostic markers and therapeutic targets for recurrence and metastasis. Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful approach to find specific biomarkers by dissecting expression profiling in human cancers at the resolution of individual cells. Here, we investigated cell profiles of the primary tumor and lymph node metastasis and paracancerous normal tissues in one PTC patient using scRNA-seq, and compared individual cell gene expression differences. The transcriptomes of 11,805 single cells were profiled, and malignant cells exhibited a profound transcriptional overlap between primary and metastatic lesions, but there were differences in the composition and quantity of non-malignant cells. ARHGAP36 was one of the genes that were highly expressed in almost all of the primary and metastatic malignant cells without non-malignant or normal follicular cells and was then confirmed by immunostaining in a sample cohort. Compared with the paracancerous normal tissue, the expression of ARHGAP36 in primary and metastatic carcinoma tissues was significantly higher as assayed by qRT-PCR. ARHGAP36 knockdown significantly inhibited the proliferation and migration of PTC cells in vitro and involved several proliferation and migration-associated signaling pathways by RNA seq. Our study demonstrated that ARHGAP36 is exclusively expressed in the malignant cells of primary PTC, as well as metastatic lesions, and regulates their proliferation and migration, meaning it can be used as a potential diagnostic marker and therapeutic target molecule.
Claire V Harper, Anne V McNamara, David G Spiller, Jayne C Charnock, Michael RH White, and Julian RE Davis
Pituitary cells have been reported to show spontaneous calcium oscillations and dynamic transcription cycles. To study both processes in the same living cell in real-time, we used rat pituitary GH3 cells stably expressing human prolactin-luciferase or prolactin-EGFP reporter gene constructs loaded with a fluorescent calcium indicator and measured activity using single cell time-lapse microscopy. We observed heterogeneity between clonal cells in the calcium activity and prolactin transcription in unstimulated conditions. There was a significant correlation between cells displaying spontaneous calcium spikes and cells showing spontaneous bursts in prolactin expression. Notably, cells showing no basal calcium activity showed low prolactin expression but elicited a significantly greater transcriptional response to BayK8644 compared to cells showing basal calcium activity. This suggested the presence of two subsets of cells within the population at any one time. Fluorescence-activated cell sorting was used to sort cells into two populations based on the expression level of prolactin-EGFP however, the bimodal pattern of expression was restored within 26h. Chromatin immunoprecipitation showed that these sorted populations were distinct due to the extent of histone acetylation. We suggest that maintenance of a heterogeneous bimodal population is a fundamental characteristic of this cell type and that calcium activation and histone acetylation at least in part, drive prolactin transcriptional competence.