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Open access

Tien-Chun Yang, Mei-Hua Lu, Wei-Jie Wang, and Jang-Yi Chen

The pathogenesis of hypertension is not fully understood; endothelin 1 (EDN1) is involved in developing essential hypertension. EDN1 can promote vascular smooth muscle cell (VSMC) proliferation or hypertrophy through autocrine and paracrine effects. Proliferating smooth muscle cells in the aorta are 'dedifferentiated' cells that cause increased arterial stiffness and remodeling. Male SHRs had higher aortic stiffness than normal control male WKY rats. Male SHR VSMCs expressed high levels of the EDN1 gene, but endothelial cells did not. Therefore, it is necessary to understand the molecular mechanism of enhanced EDN1 expression in SHR VSMCs. We identified POU2F2 and CEBPB as the main molecules that enhance EDN1 expression in male SHR VSMCs. A promoter activity analysis confirmed that the enhancer region of the Edn1 promoter in male SHR VSMCs was from −1309 to −1279 bp. POU2F2 and CEBPB exhibited an additive role in the enhancer region of the EdnET1 promoter. POU2F2 or CEBPB overexpression sufficiently increased EDN1 expression, and co-transfection with the CEBPB and POU2F2 expression plasmids had additive effects on the activity of the Edn1 promoter and EDN1 secretion level of male WKY VSMCs. In addition, the knockdown of POU2F2 also revealed that POU2F2 is necessary to enhance EDN1 expression in SHR VSMCs. The enhancer region of the Edn1 promoter is highly conserved in rats, mice, and humans. POU2F2 and CEBPB mRNA levels were significantly increased in remodeled human VMSCs. In conclusion, the novel regulation of POU2F2 and CEBPB in VSMCs will help us understand the pathogenesis of hypertension and support the development of future treatments for hypertension.

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Francesco Paolo Zummo, Alexandre Berthier, Céline Gheeraert, Manjula Vinod, Marie Bobowski-Gérard, Olivier Molendi-Coste, Laurent Pineau, Matthieu Jung, Loic Guille, Julie Dubois-Chevalier, David Dombrowicz, Bart Staels, Jérôme Eeckhoute, and Philippe Lefebvre

The functional versatility of the liver is paramount for organismal homeostasis. Adult liver functions are controlled by a tightly regulated transcription factor network including nuclear receptors (NRs), which orchestrate many aspects of hepatic physiology. NRs are transcription factors sensitive to extracellular cues such as hormones, lipids, xenobiotics, etc. and are modulated by intracellular signaling pathways. While liver functional zonation and adaptability to fluctuating conditions rely on a sophisticated cellular architecture, a comprehensive knowledge of NR functions within liver cell populations is still lacking. As a step toward the accurate mapping of NR functions in the liver, we characterized their levels of expression in the whole liver from C57Bl6/J male mice as a function of time and diet. Nr1d1 (Rev-erba), Nr1d2 (Rev-erbb), Nr1c2 (Pparb/d), and Nr1f3 (Rorg) exhibited a robust cyclical expression in ad libitum-fed mice which was, like most cyclically expressed NRs, reinforced upon time-restricted feeding. In a few instances, cyclical expression was lost or gained as a function of the feeding regimen. NR isoform expression was explored in purified hepatocytes, cholangiocytes, Kupffer cells, hepatic stellate cells, and liver sinusoidal cells. The expression of some NR isoforms, such as Nr1h4 (Fxra) and Nr1b1 (Rara) isoforms, was markedly restricted to a few cell types. Leveraging liver single-cell RNAseq studies yielded a zonation pattern of NRs in hepatocytes, liver sinusoidal cells, and stellate cells, establishing a link between NR subtissular localization and liver functional specialization. In summary, we provide here an up-to-date compendium of NR expression in mouse liver in space and time.

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Beibei Wu, Ruojun Qiu, Shuo Wang, Yingzi He, Jing Wang, Zhiye Xu, Xihua Lin, Hong Li, and Fenping Zheng

Liver transthyretin (TTR) synthesis and release are exacerbated in insulin-resistant states but are decreased by exercise training, in relation to the insulin-sensitizing effects of exercise. We hypothesized that TTR knockdown (TTR-KD) may mimic this exercise-induced metabolic improvement and skeletal muscle remodeling. Adeno-associated virus-mediated TTR-KD and control mice were trained for 8 weeks on treadmills. Their metabolism status and exercise capacity were investigated and then compared with sedentary controls. After treadmill training, the mice showed improved glucose and insulin tolerance, hepatic steatosis, and exercise endurance. Sedentary TTR-KD mice displayed metabolic improvements comparable to the improvements in trained mice. Both exercise training and TTR-KD promoted the oxidative myofiber compositions of MyHC I and MyHC IIa in the quadriceps and gastrocnemius skeletal muscles. Furthermore, training and TTR-KD had an additive effect on running performance, accompanied by substantial increases in oxidative myofiber composition, Ca2+-dependent Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, and the downstream expression of PGC1α as well as the unfolded protein response (UPR) segment of PERK-p-eIF2a pathway activity. Consistent with these findings, electrical pulse stimulation of an in vitro model of chronic exercise (with differentiated C2C12 myoblasts) showed that exogenous TTR protein was internalized and localized in the endoplasmic reticulum, where it disrupted Ca2+ dynamics; this led to decreases in intracellular Ca2+ concentration and downstream pathway activity. TTR-KD may function as an exercise/Ca2+-dependent CaMKII-PGC1α-UPR regulator that upregulates the oxidative myofiber composition of fast-type muscles; it appears to mimic the effect of exercise training on insulin sensitivity-related metabolic improvement and endurance capacity.

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Huixia Li, Hui Meng, Min Xu, Xin Gao, Xulei Sun, Xinxin Jin, and Hongzhi Sun

Bone mass declines with age and its maintenance is tightly linked to osteoblasts (crucial bone-building cells). Although disruption of the peripheral circadian clock is involved in various pathologies including aging-related diseases, evidence regarding how the peripheral clock regulates bone mass remains elusive. In the present study, we aimed to elucidate the effects of Bmal1 (the key activator of the peripheral circadian clock system) knockdown by lentivirus-mediated shRNA on osteoblast differentiation and its related mechanisms. We found that the expression of osteogenic markers, alkaline phosphatase activity, and mineralization were decreased, whereas apoptosis and inflammatory response were increased in Bmal1 knockdown osteoblasts. In addition, Bmal1 knockdown promoted ERK and JNK phosphorylation, as well as mTOR activity, whereas mTOR inhibition by rapamycin abrogated Bmal1 knockdown-mediated effects on osteoblast differentiation and mineralization capacity. Remarkably, Bmal1 knockdown in osteoblasts inhibited GSK3β/β-catenin signaling with decreased β-catenin expression and GSK-3β phosphorylation at serine 9, while GSK3β inhibition with TDZD-8, but not WNT3a or SKL2001, rescued Bmal1 knockdown-induced defects in osteoblast differentiation. Moreover, rapamycin partly nullified the suppression of Bmal1 knockdown on β-catenin expression and GSK-3β phosphorylation. Collectively, overall data indicated that circadian gene Bmal1 regulated osteoblast differentiation and inflammatory response in an mTOR/GSK3β/β-catenin-dependent manner, and thereby may contribute to the mineralization process and bone modeling/remodeling.

Open access

Hong Chen, Weiyu Li, Suping Zhang, Yunteng Sun, Yiping Shen, and Ruimin Chen

Pathogenic variants in the transcription factor CCCTC-binding factor (CTCF) are associated with mental retardation, autosomal dominant 21 (MRD21, MIM#615502). Current studies supported the strong relationship between CTCF variants and growth, yet the mechanism of CTCF mutation leading to short stature is not known. Clinical information, treatment regimens, and follow-up outcomes of a patient with MRD21 were collected. The possible pathogenic mechanisms of CTCF variants leading to short stature were investigated using immortalized lymphocyte cell lines (LCLs), HEK-293T, and immortalized normal human liver cell lines (LO2). This patient received long-term treatment with recombinant human growth hormone (rhGH) which resulted in an increased height of 1.0 SDS. She had low serum insulin-like growth factor 1 (IGF1) before the treatment and the IGF1 level was not significantly increased during the treatment (−1.38 ± 0.61 SDS). The finding suggested that the CTCF R567W variant could have impaired IGF1 production pathway. We further demonstrated that the mutant CTCF had a reduced ability to bind to the promoter region of IGF1, consequently significantly reducing the transcriptional activation and expression of IGF1. Our novel results demonstrated a direct positive regulation of CTCF on the transcription of the IGF1 promoter. The impaired IGF1 expression due to CTCF mutation may explain the substandard effect of rhGH treatment on MRD21 patients. This study provided novel insights into the molecular basis of CTCF-associated disorder.

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Shima Mohammadi, Maryam Zahmatkesh, Yazdan Asgari, Samaneh Aminyavari, and Gholamreza Hassanzadeh

Arylalkylamine N-acetyltransferase (AANAT), a rate-limiting enzyme in melatonin synthesis, is present in extra-pineal tissues such as the hippocampus. The hippocampal AANAT activity in amyloid β (Aβ) neurotoxicity has not been exactly defined. Adult male rats received bilateral intra-CA1 Aβ administration. The hippocampus tissue sampling was performed 2, 12, and 24 hours after Aβ injection in the morning and night. The inflammation was monitored using TNF-α immunohistochemistry. The AANAT enzyme activity and melatonin levels were measured using western blotting and HPLC. The sampling in the morning versus night showed no significant differences in the AANAT activity. The Aβ increased the area of TNF-α positive staining 24 hours after injection, which indicated the induction of an inflammatory context. It was accompanied by a significant reduction in AANAT activity and hippocampal melatonin. A reverse correlation was also detected between TNF-α and AANAT activity in the 24-hours group. The TNF-α positive area was significantly increased in the 24-hours group as compared to the 12-hours group. Data showed that inflammatory processes began 12 hours after the Aβ injection and augmented 24 hours later. In the second experiment, the impact of Aβ injection on hippocampus AANAT activity was examined in the pinealectomized (PIN×) animals. The PIN× per se did not affect the hippocampal AANAT and melatonin level. However, there was a significant decrease in hippocampal melatonin in the PIN×+Aβ group. The findings suggest the accompanying of hippocampal inflammatory context and AANAT enzyme activity reduction in early stages after Aβ administration. Understanding the underlying mechanism of the decreased AANAT activity may suggest new treatment strategies.

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Xiangnan Wu, Yiqiao Wang, Hang Wang, Meirui Ma, Zhichao Hao, and Yuanyuan Ma

Neuropeptide Y (NPY) is a widespread hormone in the central and peripheral nervous systems that maintains body homeostasis. Central actions of hypothalamic NPY have been identified in bone metabolism. Osteocytes are the main source of NPY in bone tissue, indicating that osteocytic NPY could be a local alternative pathway for hypothalamic-mediated regulation of bone and bone cells. Here, we show that osteocytic NPY induces cell viability and proliferation. Osteocyte-derived factors are also closely associated with changes in cellular NPY mRNA levels. Furthermore, osteoblast mineralization was significantly induced by conditioned medium collected from NPY-overexpressing osteocytes (P < 0.05). Importantly, the NPY-AHNAK interaction was identified for the first time by Co-Immunoprecipitation, and significant inactivation of p-Smad1/5/9 was found in osteocytes with NPY or AHNAK insufficiency (P < 0.05). The activation of p-Smad1/5/9 reversed NPY insufficiency-caused decreases in the expression of osteocytic PCNA and osteoblast markers including osteocalcin and Runx2 (P < 0.05); these findings showed an additional molecular mechanism by which NPY acts on cells through AHNAK-mediated Smad1/5/9 signalling. Collectively, our findings provide novel insights into the function of NPY in regulating osteocyte phenotype and function and provide new insights for further investigation into osteocytic NPY-mediated therapy.

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Shinjini C Spaulding, Vivek Choudhary, and Wendy B Bollag

Aldosterone is considered to be a link between hypertension and obesity; obese individuals have high serum levels of very low-density lipoprotein (VLDL). VLDL has been shown to induce aldosterone production in multiple adrenal zona glomerulosa models, mediated in part by phospholipase D (PLD). PLD is an enzyme that hydrolyzes phosphatidylcholine to produce phosphatidic acid (PA), a lipid second messenger that can also be dephosphorylated by lipin to yield diacylglycerol (DAG), yet another lipid signal. However, it is unclear which of the two lipid second messengers, PA or DAG, underlies PLD’s mediation of aldosterone production. We hypothesized that the key signal produced by PLD (indirectly) is DAG such that PLD mediates VLDL-induced aldosterone production via lipin-mediated metabolism of PA to DAG. To assess the role of lipin in VLDL-induced aldosterone production, lipin-1 was overexpressed (using an adenovirus) or inhibited (using propranolol) in HAC15 cells followed by treatment with or without VLDL. Lipin-1 overexpression enhanced the VLDL-stimulated increase in CYP11B2 expression (by 75%), and lipin-1 inhibition decreased the VLDL-stimulated increase in CYP11B2 expression (by 66%). Similarly, the VLDL-stimulated increase in aldosterone production was enhanced by lipin-1 overexpression (182%) and was decreased by lipin inhibition (80%). Our results are suggestive of DAG being the key lipid signal since manipulating lipin-1 levels/activity affects VLDL-stimulated steroidogenic gene expression and ultimately, aldosterone production. Our study warrants further investigation into VLDL-stimulated steroidogenic signaling pathways which may lead to the identification of novel therapeutic targets, such as lipin-1 and its downstream pathways, to potentially treat obesity-associated hypertension.

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Xiaodan Li, Xiaolei Yao, Yongjin Bao, Kaiping Deng, Mingtian Deng, Fan Yang, Xuan Sun, Peihua You, Qingxian Cai, and Feng Wang

The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme complex that plays a vital role in regulating male reproductive activities. However, as an essential member of the PP2A family, the physiological functions of PP2A regulatory subunit B55α (PPP2R2A) in testis remain inconclusive. Hu sheep are noted for their reproductive precocity and fertility, and are ideal models for the study of male reproductive physiology. Here, we analyzed the expression patterns of PPP2R2A in the male Hu sheep reproductive tract at different developmental stages and further investigated its role in testosterone secretion and its underlying mechanisms. In this study, we found that there were temporal and spatial differences in PPP2R2A protein expression in the testis and epididymis, especially the expression abundance in the testis at 8 months old (8M) was higher than that at 3 months old (3M). Interestingly, we observed that PPP2R2A interference reduced the testosterone levels in the cell culture medium, which is accompanied by a reduction in Leydig cell proliferation and an elevation in Leydig cell apoptosis. The level of reactive oxygen species in cells increased significantly, while the mitochondrial membrane potential (ΔΨm) decreased significantly after PPP2R2A deletion. Meanwhile, the mitochondrial mitotic protein DNM1L was significantly upregulated, while the mitochondrial fusion proteins MFN1/2 and OPA1 were significantly downregulated after PPP2R2A interference. Furthermore, PPP2R2A interference suppressed the AKT/mTOR signaling pathway. Taken together, our data indicated that PPP2R2A enhanced testosterone secretion, promoted cell proliferation, and inhibited cell apoptosis in vitro, all of which were associated with the AKT/mTOR signaling pathway.

Open access

Aqfan Jamaluddin and Caroline M Gorvin

G protein-coupled receptors (GPCRs) have a critical role in energy homeostasis, contributing to food intake, energy expenditure and glycaemic control. Dysregulation of energy expenditure can lead to metabolic syndrome (abdominal obesity, elevated plasma triglyceride, LDL cholesterol and glucose, and high blood pressure), which is associated with an increased risk of developing obesity, diabetes mellitus, non-alcoholic fatty liver disease and cardiovascular complications. As the prevalence of these chronic diseases continues to rise worldwide, there is an increased need to understand the molecular mechanisms by which energy expenditure is regulated to facilitate the development of effective therapeutic strategies to treat and prevent these conditions. In recent years, drugs targeting GPCRs have been the focus of efforts to improve treatments for type-2 diabetes and obesity, with GLP-1R agonists a particular success. In this review, we focus on nine GPCRs with roles in energy homeostasis that are current and emerging targets to treat obesity and diabetes. We discuss findings from pre-clinical models and clinical trials of drugs targeting these receptors and challenges that must be overcome before these drugs can be routinely used in clinics. We also describe new insights into how these receptors signal, including how accessory proteins, biased signalling, and complex spatial signalling could provide unique opportunities to develop more efficacious therapies with fewer side effects. Finally, we describe how combined therapies, in which multiple GPCRs are targeted, may improve clinical outcomes and reduce off-target effects.