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Open access

Feng Zhang, Qi Xiong, Hu Tao, Yang Liu, Nian Zhang, Xiao-Feng Li, Xiao-Jun Suo, Qian-Ping Yang, and Ming-Xin Chen

Acyl-coenzyme A oxidase 1 (ACOX1) is the first and rate-limiting enzyme in peroxisomal fatty acid β-oxidation of fatty acids. Previous studies have reported that ACOX1 was correlated with the meat quality of livestock, while the role of ACOX1 in intramuscular adipogenesis of beef cattle and its transcriptional and post-transcriptional regulatory mechanisms remain unclear. In the present study, gain-of-function and loss-of-function assays demonstrated that ACOX1 positively regulated the adipogenesis of bovine intramuscular preadipocytes. The C/EBPα-binding sites in the bovine ACOX1 promoter region at −1142 to −1129 bp, −831 to −826 bp, and −303 to −298 bp were identified by promoter deletion analysis and site-directed mutagenesis. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) further showed that these three regions are C/EBPα-binding sites, both in vitro and in vivo, indicating that C/EBPα directly interacts with the bovine ACOX1 promoter and inhibits its transcription. Furthermore, the results from bioinformatics analysis, dual luciferase assay, site-directed mutagenesis, qRT-PCR, and Western blotting demonstrated that miR-25-3p directly targeted the ACOX1 3’UTR (3’UTR). Taken together, our findings suggest that ACOX1, regulated by transcription factor C/EBPα and miR-25-3p, promotes adipogenesis of bovine intramuscular preadipocytes via regulating peroxisomal fatty acid β-oxidation.

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Hiromi Nagata, Jingya Lyu, Hitomi Imachi, Kensaku Fukunaga, Seisuke Sato, Toshihiro Kobayashi, Takanobu Saheki, Kayoko Seo, Japar B Salimah, Hisakazu Iwama, Ryuichi Sakamoto, Yoshihiro Ogawa, and Koji Murao

Vascular complications are the main cause of morbidity and mortality in diabetic patients, and advanced glycation end products (AGEs) play a critical role in promoting diabetic vascular dysfunction. The human homolog of scavenger receptor class B type I (SR-BI), CD36, and LIMPII analog-1 (hSR-BI/CLA-1) facilitates the cellular uptake of cholesterol from HDL. In endothelial cells, HDL activates endothelial nitric oxide synthase (eNOS) via hSR-BI/CLA-1. In this study, we elucidated the effects of AGEs on hSR-BI/CLA-1 expression in human umbilical vein endothelial cells (HUVECs). HSR-BI/CLA-1 expression was examined by real-time PCR, western blot analysis, and reporter gene assay in HUVECs incubated with AGEs. eNOS activity was assessed by detecting the phosphorylation (Ser 1179) of eNOS. Our results showed that AGEs decreased the endogenous expression of hSR-BI/CLA-1. AGEs also inhibited the activity of the hSR-BI/CLA-1 promoter and its mRNA expression via receptor RAGE. We identified the binding site for Smad1 on the hSR-BI/CLA-1 promoter: Smad1 bound to its promoter. AGE treatment stimulated the transcriptional activity of Smad1, and mutation of the Smad1 binding site inhibited the effect of AGEs on the hSR-BI/CLA-1 promoter. HDL-treatment enhanced the phosphorylation of eNOS at Ser 1179, but pretreatment with AGEs inhibited the phosphorylation of eNOS Ser 1179. These results suggested that AGEs downregulate the expression of the endothelial hSR-BI/CLA-1 via the Smad1 pathway, which may be a therapeutic target for diabetic endothelial dysfunction.

Open access

Nathan Appanna, Hylton Gibson, Elena Gangitano, Niall J Dempster, Karen Morris, Sherly George, Anastasia Arvaniti, Laura L Gathercole, Brian Keevil, Trevor M Penning, Karl-Heinz Storbeck, Jeremy W Tomlinson, and Nikolaos Nikolaou

Steroid hormones, including glucocorticoids and androgens, exert a wide variety of effects in the body across almost all tissues. The steroid A-ring 5β-reductase (AKR1D1) is expressed in human liver and testes, and three splice variants have been identified (AKR1D1-001, AKR1D1-002, AKR1D1-006). Amongst these, AKR1D1-002 is the best described; it modulates steroid hormone availability and catalyses an important step in bile acid biosynthesis. However, specific activity and expression of AKR1D1-001 and AKR1D1-006 are unknown. Expression of AKR1D1 variants were measured in human liver biopsies and hepatoma cell lines by qPCR. Their three-dimensional (3D) structures were predicted using in silico approaches. AKR1D1 variants were overexpressed in HEK293 cells, and successful overexpression confirmed by qPCR and Western blotting. Cells were treated with either cortisol, dexamethasone, prednisolone, testosterone or androstenedione, and steroid hormone clearance was measured by mass spectrometry. Glucocorticoid and androgen receptor activation were determined by luciferase reporter assays. AKR1D1-002 and AKR1D1-001 are expressed in human liver, and only AKR1D1-006 is expressed in human testes. Following overexpression, AKR1D1-001 and AKR1D1-006 protein levels were lower than AKR1D1-002, but significantly increased following treatment with the proteasomal inhibitor, MG-132. AKR1D1-002 efficiently metabolised glucocorticoids and androgens and decreased receptor activation. AKR1D1-001 and AKR1D1-006 poorly metabolised dexamethasone, but neither protein metabolised cortisol, prednisolone, testosterone or androstenedione. We have demonstrated the differential expression and role of AKR1D1 variants in steroid hormone clearance and receptor activation in vitro. AKR1D1-002 is the predominant functional protein in steroidogenic and metabolic tissues. In addition, AKR1D1-001 and AKR1D1-006 may have a limited, steroid-specific role in the regulation of dexamethasone action.

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Haiqin Wang, Li Nian, Zhonghua Li, and Changhui Lu

Hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome is a life-threatening pregnancy complication. Though there are several medications widely used to treat HELLP syndrome, delivery is the only efficient treatment. The goal of the present study was to investigate the effects of platelet-derived growth factor-D (PDGF-D), a newly identified PDGF, in a rat model of HELLP syndrome which was accomplished by sFlt-1 and sEng injection. The expression levels of PDGF-D in pregnant women diagnosed with HELLP syndrome was determined. A HELLP rat model was established and the PDGF-D expression level in the plasma and the placenta tissue was evaluated. To evaluate the effects of PDGF-D in HELLP syndrome model, siPDGF-D was injected into the rats and the HELLP syndrome-related parameters were measured. The levels of inflammatory cytokines and PDGF-D were determined by ELISA. The oxidative stress activities in the plasma were also determined. Furthermore, the expression of PDGF-D/PDGFR-β/nuclear factor κB (NF-κB) p65 in placenta tissues was evaluated by Western blotting. Compared to the normal pregnant (NP) group, the levels of PDGF-D were augmented regardless of species. Knockdown of PDGF-D can result in the alleviation of HELLP syndrome development and progression in the HELLP rat model. Importantly, as a result of PDGF-D knockdown, the serum levels of inflammatory cytokines and oxidative stress activities were modulated, and the phosphorylation of PDGFR-β and NF-κB p65 in placenta tissue was inhibited. Taking together, our findings indicate that targeting PDGF-D could be used as a novel strategy to treat patients with HELLP syndrome.

Free access

Filipe De Vadder, Amélie Joly, and François Leulier

The worrying number of children suffering from undernutrition and consequent stunting worldwide makes the understanding of the relationship between nutritional status and postnatal growth crucial. Moreover, it is now well established that undernourished children harbor an altered microbiota, correlating with impaired growth. In this review, we describe how murine models have been used to explore the functional relationships between endocrine regulation of growth, nutrition and gut microbiota. In numerous Mammalian species, postnatal growth is mainly regulated by the conserved GH/IGF1 somatotropic axis that acts through endocrine and paracrine pathways, notably enabling longitudinal bone growth. Recent studies have demonstrated that the microbiota effects on growth could involve a modulation of GH and IGF1 circulating levels. Besides, the GH/IGF1 somatotropic axis may regulate the gut microbiota composition and diversity. Studying the bidirectional relationship between growth hormones and the gut microbiome could therefore help developing microbiota-targeting therapies in order to reduce the long-term consequences of stunting.

Open access

Zuo Zhang, Hongli Zhou, and Jiyin Zhou

Earlier, it was shown that reversing the downregulation of neuritin expression in the brain improves central neuropathy in diabetic rats. We investigated the protective mechanism of neuritin in diabetic cognitive dysfunction via astrocytes. Further, the impact of the overexpression of neuritin in the cortex and the hippocampus on diabetic cognitive dysfunction and astrogliosis in type 2 diabetic (db/db) mice was assessed. Antagonists were used to inhibit the JAK2/STAT3 signaling pathway in U-118MG, an astrocyte cell line. Immunofluorescence, western blotting, and real-time PCR were performed. Neuritin overexpression in the hippocampus of db/db mice significantly ameliorated cognitive dysfunction, hippocampal neuronal impairment, and synaptic plasticity deterioration, and inhibited astrogliosis and the JAK2/STAT3 signaling pathway in the hippocampus. Neuritin suppressed the JAK2/STAT3 signaling pathway to inhibit lipopolysaccharide-induced gliosis in U-118MG cells. It was observed that neuritin regulates the JAK2/STAT3 signaling pathway in astrocytes to inhibit astrogliosis and improve diabetic cognitive dysfunction.

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Jie Wang, Yuchao Zhang, Qi Shen, Jing Wu, and Jian-Xin Li

Obesity is a chronic disease that increases the risk of type II diabetes, heart diseases and nonalcoholic fatty liver disease. Unfortunately, to date, only a handful of drugs are approved for clinical use. This study aims at the discovery of anti-obesity agents based on naturally sourced oleanolic acid (OA) derivatives. 3T3-L1 preadipocytes were differentiated into mature adipocytes for in vitro assays, and a high-fat diet (HFD)-induced obesity mice model was established for in vivo studies. The screening of the OA derivatives was performed with 3T3-L1 cell, and resulted in a discovery of a novel compound HA-20 with a potent inhibitory activity on 3T3-L1 adipogenesis. In vitro data demonstrated that HA-20 markedly suppressed the adipogenesis in 3T3-L1 at the early stage without cytotoxicity. In vivo research using HFD mice revealed that HA-20 lowered the body weight, and possessed a lipid-lowering effect. Transcriptome analysis discovered that the mainly adipogenesis/lipogenesis genes regulated by HA-20 were Pparg, Cebpa, Fas, Acc, and Fabp4/aP2. Mechanism study revealed that HA-20 played its bioactive roles at least via downregulating PPARγ-FABP4/aP2 pathway in 3T3-L1, which was further confirmed in HFD-induced obesity mice. Our findings provided a new insight into fighting fat accumulation based on OA derivatives, and demonstrated that HA-20 may sever as a worthy leading compound for the further development of anti-obesity agents.

Open access

Shree Senthil Kumar, Marie-Louise Ward, and Kathleen Grace Mountjoy

The melanocortin-4-receptor (MC4R), a critical G-protein-coupled-receptor (GPCR) regulating energy homeostasis, activates multiple signalling pathways, including mobilisation of intracellular calcium ([Ca2+]i). However, very little is known about the physiological significance of MC4R-induced [Ca2+]i since few studies measure MC4R-induced [Ca2+]i. High-throughput, read-out assays for [Ca2+]i have proven unreliable for overexpressed GPCRs like MC4R, which exhibit low sensitivity mobilising [Ca2+]i. Therefore, we developed, optimised, and validated a robust quantitative high-throughput assay using Fura-2 ratio-metric calcium dye and HEK293 cells stably transfected with MC4R. The quantitation enables direct comparisons between assays and even between different research laboratories. Assay conditions were optimised step-by-step to eliminate interference from stretch-activated receptor increases in [Ca2+]i, and to maximise ligand-activated MC4R induced [Ca2+]i. Calcium imaging was performed using a PheraStar FS multi-well plate reader. Probenecid, included in the buffers to prevent extrusion of Fura-2 dye from cells, was found to interfere with the EGTA-chelation of calcium, required to determine Rmin for quantitation of [Ca2+]i. Therefore, we developed a method to determine Rmin in specific wells without probenecid, which was run in parallel with each assay. Validation of the assay was shown by reproducible -melanocyte stimulating hormone (-MSH) concentration-dependent activation of stably-expressed human MC4R (hMC4R) and mouse MC4R (mMC4R), inducing increases in [Ca2+]i, for three independent experiments. This robust, reproducible, high-throughput assay that quantitatively measures MC4R-induced mobilisation of [Ca2+]i in vitro, has potential to advance the development of therapeutic drugs, and understanding of MC4R signalling associated with human obesity.

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Romina Hermann, Victoria Evangelina Mestre Cordero, María de las Mercedes Fernández Pazos, Mailen Florencia Córdoba, Federico Joaquín Reznik, Débora Elisabet Vélez, Andrea Lorena Fellet, and María Gabriela Marina Prendes

Recent studies have provided evidence that triiodothyronine (T3) might play an effective role in the recovery of ischemic myocardium, through the preservation of mitochondrial function and the improvement of energy substrate metabolism. To this respect, it has been suggested that T3 could activate AMP-activated protein kinase (AMPK), the cellular ‘fuel-gauge’ enzyme, although its role has yet to be elucidated. The aim of the present study was to investigate the effects produced by acute treatment with T3 (60 nM) and the pharmacological inhibition of AMPK by compound C on isolated rat left atria subjected to 75 min simulated ischemia-75 min reperfusion. Results showed that T3 increased AMPK activation during simulated ischemia-reperfusion, while compound C prevented it. At the end of simulated reperfusion, acute T3 treatment increased contractile function recovery and cellular viability conservation. Mitochondrial ultrastructure was better preserved in the presence of T3 as well as mitochondrial ATP production rate and tissue ATP content. Calcium retention capacity, a parameter widely used as an indicator of the resistance of mitochondrial permeability transition pore (MPTP) to opening, and GSK-3β phosphorylation, a master switch enzyme that limits MPTP opening, were increased by T3 administration. All these beneficial effects exerted by T3 acute treatment were prevented when compound C was co-administrated. The present study provided original evidence that T3 enhances intrinsic activation of AMPK during myocardial ischemia-reperfusion, being this enzyme involved, at least in part, in the protective effects exerted by T3, contributing to mitochondrial structure and function preservation, post-ischemic contractile recovery and conservation of cellular viability.

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Jing Lu, Cheng Cheng, Zhen-Chao Cheng, Qian Wu, Han Shen, Ming-xia Yuan, Bo Zhang, and Jin-Kui Yang

RFX6 transcription factor is believed to play a central role in directing cell development of insulin-producing pancreatic islet. RFX6 homozygous mutations cause syndromic neonatal diabetes with hypoplastic pancreas. However, RFX6 heterozygous mutations cause maturity-onset diabetes of the young (MODY) with normal pancreas development. Here, we show that RFX6 may control islet cell development and insulin production in different manners. The rfx6 knockout zebrafish generated by CRISPR/Cas9 exhibited an overt diabetes phenotype. Pancreatic islet failed to form compact structures in the knockout fish. While endocrine pancreatic islet non-β-cells were absent, insulin-producing β-cells were present in the knockout fish. Although insulin mRNA level was normal in the β-cells of the knockout fish, insulin protein level was decreased. High-throughput RNA sequencing (RNAseq) showed that differentially expressed genes were enriched in the translation term in islet β-cells from the knockout fish. Chromatin immunoprecipitation sequencing (ChIPseq) of normally developed islet β-cells from mice demonstrated that rfx6 interacted with translation initiation factors and controlled insulin translation. Our data indicate that Rfx6 may act as a transcription factor regulating the transcription of genes involved in mRNA translation, which may represent a new mechanism and treatment strategy for diseases.