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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 upregulate 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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Daniel Patiño-García, Leonor Cruz-Fernandes, Julio Buñay, Renán Orellana, and Ricardo D Moreno

Reproductive hormone imbalance in infertile women is correlated to high levels of phthalates and alkylphenols, which are among endocrine-disrupting chemicals (EDCs). Previous studies have shown that they interfere with gene expression by deregulating levels of microRNAs (miRs), small non-coding RNAs targeting mRNAs encoding enzymes in the hormone biosynthesis pathway. However, this effect depends on the target organ, dose and whether or not they are alone or in mixtures. Our goal was to study whether the biosynthesis, and a specific group of miRs targeting mRNAs encoding enzymes in steroid hormone biosynthesis, are deregulated in the ovaries of female mice chronically exposed to a mixture of three phthalates (DEHP+DBP+BBP) and two alkylphenols (NP+OP) at a human environmentally relevant dose. We performed qPCR and Western blot assays along with a bioinformatics approach and found that this mixture modified the biogenesis machinery of miRs, inducing an increase in the mRNA levels of Drosha and Dicer1 and DROSHA protein levels. In addition, we found changes in the precursor and mature forms of miR-96-5p, miR-200b-3p, miR-365-3p, miR-378a-3p and miR-503-5p which target steroidogenic pathway enzymes. Finally, using primary granulosa cell culture, we confirmed that miR-200b-3p targets Cyp19a1, transcript encoding CYP19A1, the enzyme that produces estradiol (E2). These results indicate that chronic exposure to phthalates and alkylphenols mixture alters the biogenesis of ovary miRs and increases the expression of miRs implicated in the control of steroidal hormone synthesis in female mice, thus contributing to reproductive pathologies.

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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.

Free access

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.

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Shuai Shao, Hui Wang, Wei Shao, and Na Liu

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and one of the most common causes of infertility in women. PCOS patients have been found with dysregulated miRNA, which is indicative of their roles as noninvasive biomarkers and novel therapeutic targets in PCOS. Herein, this study sets out to explore the mechanism of action of miR-199a-5p in PCOS in relation to the janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway via Wilms’ tumor 1 (WT1) regulation in a rat model of PCOS. The expression of miR-199a-5p was highly expressed in ovarian cortical tissues and serum of PCOS patients, as examined by RT-qPCR. Ovarian granulosa cells (GCs) were harvested from PCOS rat model, followed by subsequent purification. Gain- and loss-of-function experiments of miR-199a-5p were performed to determine its functions in PCOS. Cell viability, cell apoptosis and serum hormone levels were assessed, the results of which showed that downregulation of miR-199a-5p contributed to the promotion of GC viability and inhibition of apoptosis, while simultaneously inducing the elevation of serum E2 level and reduction of serum AMH, PG, LH and FSH levels in the PCOS rat model. WT1 was identified as a target gene of miR-199a-5p by dual-luciferase reporter gene assay, and inhibition of miR-199a-5p resulted in the activation of WT1-mediated JAK/STAT3 pathway. The activated JAK/STAT3 pathway suppressed the development of PCOS by miR-199a-5p, indicating a mechanism by which miR-199a-5p could potentially prevent PCOS through the WT1-mediated JAK/STAT3 pathway.

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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.

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Ruiqi Ma, Lu Gan, Hui Ren, Andrew Harrison, and Jiang Qian

The study aimed to investigate the role of pyruvate dehydrogenase kinase (PDK) in regulating glycolysis and proliferation of perimysial orbital fibroblasts (pOFs) during the pathogenesis of thyroid-associated ophthalmopathy (TAO). EdU and BrdU incorporation assays were performed to examine cell proliferation. Lactate production and oxygen consumption assays were conducted to evaluate glycolysis. Real-time PCR was adapted to quantify PDK mRNA levels. Capillary Western immunoassay was adapted to quantify PDK2, Akt, pAkt308 and GAPDH in protein samples. The TAO pOFs exhibited stronger proliferation activity, higher intracellular lactate concentration, and lower oxygen consumption rate than the control pOFs. The PDK inhibitor dichloroacetic acid (DCA) dose-dependently suppressed the proliferation of both TAO and control pOFs. DCA reduced lactate production and promoted oxygen consumption in the TAO pOFs but showed no significant effects on glycolysis in the control pOFs. Among four PDK isotypes, PDK2 was overexpressed in the TAO pOFs. The potential PDK signaling mediator, cytoplasmic Akt, was more abundant in TAO pOFs than control pOFs. Knockdown of PDK2 resulted in lower lactate production, stronger oxygen consumption, weaker proliferation activity, and less cytoplasmic Akt in the TAO pOFs but showed no significant effects in the control pOFs. The Akt inhibitor MK2206 suppressed proliferation in both TAO and control pOFs, and lactate production was inhibited by MK2206 in the TAO OFs but not the control pOFs. To conclude, PDK2 overexpression enhances glycolysis and promotes proliferation via Akt signaling in the TAO pOFs. These findings yield insights that PDK2 is a potential therapeutic target for TAO treatment.

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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.

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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.