Stard 3 (Steroidogenic Acute Regulatory-Related Lipid Transfer Domain-Containing Protein 3) Play Important Role in Preadipocyte Differentiation

2022 ◽  
Vol 12 (4) ◽  
pp. 827-833
Author(s):  
Zhonge Chen ◽  
Yanhua Tang ◽  
Wenyong Jiang ◽  
Xiaoqian Zhou

Aim: To evaluate Stard 3’s effects and relative mechanisms in preadipocyto differentiation by vitro study. Materials and Methods: The 3T3-L1 cell were divided into 5 groups as NC, si-Stard 3, ROS agonist, ROS inhibitor and si-Stard 3+ROS agonist groups. The cell of different groups were evaluated by Oil red O staining and Triglyceride. Evaluating ROS production by DHE and NBT assay. Using RT-qPCR and WB methods to evaluate gene and protein expressions. Results: Compared with NC group, Triglyceride, DHE fluorescence intensity and NBT positive rate were significantly down-regulation in si-Stard 3 and ROS inhibitor groups (P < 0.001, respectively), and were significantly up-regulation in ROS agonist group (P < 0.001, respectively); However, with si-Stard 3 transfection and ROS agonist treatment, compared with si-Stard 3 group, Triglyceride, DHE fluorescence intensity and NBT positive rate were significantly increased in si-Stard 3+ROS agonist group (P < 0.001, respectively). With RT-qPCR and WB assay, Compared with NC group, Stard 3 gene and protein expressions of si-Stard 3 and si-Stard 3+ROS agonist group were significantly depressed (P < 0.001, respectively), AMPK, PPARγ, CEBPα and FABP4 gene expressions were significantly differences in si-Stard 3, ROS agonist and ROS inhibitor groups (P < 0.001, respectively) and p-AMPK, PPARγ, CEBPα and FABP4 protein expressions were significantly differences in si-Stard 3, ROS agonist and ROS inhibitor groups (P < 0.001, respectively), with si-Stard 3 transfection and ROS agonist the relative gene and protein expressions were significantly resumed compared with si-Stard 3 group (P < 0.001, respectively). Conclusion: Stard 3 knockdown had effects to suppress 3T3-L1 cells transformation into adipocytes in vitro study.

2019 ◽  
Vol 51 (11) ◽  
pp. 741-748
Author(s):  
Mengxi Wang ◽  
Yaoyao Guo ◽  
Yumeng Zhou ◽  
Wanwan Yuan ◽  
Huixia Li ◽  
...  

AbstractOsteopontin (OPN), a secreted glycoprotein, is involved in various pathophysiological processes including immune response, inflammation, tumor formation, and metabolism. OPN exists in 2 forms, secreted-OPN (sOPN) and intracellular-OPN (iOPN). While they might have different biological activities, it remains largely unknown whether sOPN and iOPN induce the differentiation of brown adipocytes. To test this possibility, 3T3-L1 cells were induced by DMI induction with or without recombinant human OPN (rhOPN, 10, 50, 100, 200 μM), respectively. Meanwhile, another batch of 3T3-L1 cells were infected with Ad-GFP-ap2-OPN and followed by DMI differentiation. Subsequently, the infected cells were treated with either anti-CD44 antibody or immunoglobulin G (Ig G). Accumulation of lipid droplets was visualized by Oil red O staining and protein levels were assayed by western blotting analysis. The results showed that sOPN and not rhOPN, notably increased the accumulation of lipid droplets and the expression of brown adipocyte-related genes. Moreover, neutralization of CD44 partially abrogated the effects induced by sOPN. These data demonstrate that sOPN and not rhOPN has the capacity to induce the differentiation of white preadipocytes into brown adipocytes through a CD44-dependent mechanism. The findings might provide a potential target for sOPN to combat obesity.


2020 ◽  
Author(s):  
Noor Alicezah Mohd Kasim ◽  
Nurul Ain Abu Bakar ◽  
Radzi Ahmad ◽  
Iman Nabilah Abd Rahim ◽  
Thuhairah Hasrah Abdul Rahman ◽  
...  

AbstractCrocus sativus L. or saffron has been shown to have anti-atherogenic effects. However, its effects on key events in atherogenesis such as endothelial activation and monocyte-endothelial cell binding in lipolysaccharides (LPS)-stimulated in vitro model have not been extensively studied.ObjectivesTo investigate the effects of saffron and its bioactive derivative crocin on the gene and protein expressions of biomarkers of endothelial activation in LPS stimulated human coronary artery endothelial cells (HCAECs).MethodologyHCAECs were incubated with different concentrations of aqueous ethanolic extracts of saffron and crocin together with LPS. Protein and gene expressions of endothelial activation biomarkers were measured using ELISA and qRT-PCR, respectively. Adhesion of monocytes to HCAECs was detected by Rose Bengal staining. Methyl-thiazol-tetrazolium assay was carried out to assess cytotoxicity effects of saffron and crocin.ResultsSaffron and crocin up to 25.0 and 1.6 μg/ml respectively exhibited >85% cell viability. Saffron treatment reduced sICAM-1, sVCAM-1 and E-selectin proteins (concentrations: 3.13, 6.25, 12.5 and 25.0 μg/ml; 3.13, 12.5 and 25.0 μg/ml; 12.5 and 25.0, respectively) and gene expressions (concentration: 12.5 and 25.0μg/ml; 3.13, 6.25 and 25.0 μg/ml; 6.25, 12.5 25.0; respectively). Similarly, treatment with crocin reduced protein expressions of sICAM-1, sVCAM-1 and E-selectin (concentration: 0.2, 0.4, 0.8 and 1.6 μg/ml; 0.4, 0.8 and 1.6 μg/ml; 0.8 and 1.6 μg/ml; respectively] and gene expression (concentration: 0.8 and 1.6 μg/ml; 0.4, 0.8 and 1.6 μg/ml; and 1.6 μg/ml, respectively). Monocyte-endothelial cell interactions were reduced following saffron treatment at concentrations 6.3, 12.5 and 25.00 μg/ml. Similarly, crocin also suppressed cellular interactions at concentrations 0.04, 0.08, 1.60 μg/ml.ConclusionSaffron and crocin exhibits potent inhibitory action for endothelial activation and monocyte-endothelial cells interaction suggesting its potential anti-atherogenic properties.


Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 890
Author(s):  
Jiahao Shao ◽  
Ting Pan ◽  
Jie Wang ◽  
Tao Tang ◽  
Yanhong Li ◽  
...  

microRNAs (miRNAs) play an important role in gene regulation in animals by pairing with target gene mRNA. Many miRNAs are differentially expressed in the adipose tissue, often with conserved expression. In our study, we found that miR-208b expression was observed differently in the preadipocyte differentiation model. When miR-208b was overexpressed in the preadipocyte differentiation model, the overexpressed group displayed higher expression of PPARγ and FABP4—the markers of preadipocyte differentiation. Oil Red O staining revealed that the count of lipid droplets was increased in the overexpressed group. When the expression of miR-208b was inhibited, the above indicators showed an opposite trend. Moreover, results from both 5-ethynyl-2’-deoxyuridine (EDU) and cell counting kit (CCK) analysis showed that miR-208b promoted the proliferation of preadipocyte. Expression of gene CSNK2A2, a direct miR-208b target, was downregulated in the overexpressed group, providing a possible link to multiple signal pathways. Overall, our data indicate that miR-208b play a positive regulatory effect on the proliferation and differentiation of rabbit preadipocyte.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Pengyu Hong ◽  
Xiaoyang Xu ◽  
Xin Hu ◽  
Hao Yang ◽  
Yue Wu ◽  
...  

Abstract Objective To explore the adipogenic effects of the small extracellular vesicles derived from the lipoma tissues (sEV-LT), and to find a new cell-free therapeutic approach for adipose tissue regeneration. Methods Adipose tissue-derived stem cells (ADSCs) and small extracellular vesicles derived from the adipose tissues (sEV-AT) were isolated from human adipose tissue, while sEV-LT were isolated from human lipomatous tissue. ADSCs were characterized by using flow cytometric analysis and adipogenic and osteogenic differentiation assays. sEV was identified by electron microscopy, nanoparticle tracking, and western blotting. ADSCs were treated with sEV-LT and sEV-AT, respectively. Fluorescence confocal microscopy was used to investigate whether sEV-LT and sEV-AT could be taken by ADSCs. The proliferation and migration abilities and adipogenic differentiation assay of ADSCs were evaluated by CCK-8 assays, scratch test, and oil red O staining test, and the expression levels of adipogenic-related genes C/EBP-δ, PPARγ2, and Adiponectin in ADSCs were assessed by real-time quantitative PCR (RT-PCR). The sEV-LT and sEV-AT transplantation tubes were implanted subcutaneously in SD rats, and the neotissues were qualitatively and histologically evaluated at 2, 4, 8, and 12 weeks after transplantation. Hematoxylin and eosin (H&E) staining was subsequently used to observe and compare the adipogenesis and angiogenesis in neotissues, while immunohistochemistry was used to examine the expression and the distribution of C/EBP-α, PPARγ, Adiponectin, and CD31 at the 4th week. Results The in vitro experiments showed that both sEV-LT and sEV-AT could be taken up by ADSCs via endocytosis. The scratch experiment and CCK-8 experiment showed that the migration area and proliferation number of ADSCs in sEV-LT group and sEV-AT group were significantly higher than those in the non-sEV group (p < 0.05). Compared with sEV-AT group, sEV-LT group had larger migration area and proliferation number of ADSCs (p < 0.05). Oil red O staining and RT-PCR experiments showed that, compared with the non-sEVs group, the lipid droplets and the mRNA expression levels of adipogenesis-related genes PPARγ2 and Adiponectin of ADSCs in sEV-LT group and sEV-AT group were significantly upregulated (p < 0.05); however, there was no statistical significance in the expression level of C/EBP-δ (p > 0.05). In addition, no significant difference in the amount of lipid droplets and adipogenesis-related genes between the sEV-LT groups and sEV-AT was seen (p > 0.05). At 2, 4, 8, and 12 weeks, the adipocyte area and the number of capillaries in neotissues in the sEV-LT groups and sEV-AT groups were significantly increased compared with the Matrigel group (p < 0.05); however, there was no dramatic difference between sEV-LT groups and sEV-AT groups (p > 0.05). At the 4th week, neotissues in the sEV-LT groups and sEV-AT groups all showed upregulated expression of C/EBP-α, PPARγ, Adiponectin, and CD31 protein, while neotissues in the Matrigel group only showed positive expression of CD31 protein. Conclusions This study demonstrated that sEV-LT exerted promotion effects on adipose tissue regeneration by accelerating the proliferation, migration, and adipogenic differentiation of ADSCs in vitro and recruiting adipocytes and promoting angiogenesis in vivo. The sEV-LT could serve as an alternative cell-free therapeutic strategy for generating adipose tissue, thus providing a promising application prospect in tissue engineering.


2021 ◽  
Author(s):  
Yangge Du ◽  
Yunsong Liu ◽  
Yongsheng Zhou ◽  
Ping Zhang

Abstract Background: Bone is a rigid organ that provides support and physical protection to vital organs of the body. Several bone loss disorders are commonly associated with increased bone marrow adipose tissue. Bone marrow mesenchymal stromal/stem cells (BMSCs) are multipotent progenitors differentiating into osteoblasts, adipocytes, and chondrocytes. CDC20 is a co-activator of APC/C, required for full ubiquitin ligase activity. In our previous study, CDC20 promoted the osteogenic commitment of BMSCs and Cdc20 conditional knockout mice suggested a decline in bone mass. In this study, we investigated the function of CDC20 in the adipogenic differentiation of BMSCs and provided a new clue between adipogenesis and osteogenesis. Methods: Lentivirus containing CDC20 shRNA was used for CDC20 knockdown in hBMSCs. Primary mBMSCs were isolated from Cdc20f/f and Sp7-Cre;Cdc20f/f mice. Adipogenesis was examined by qRT-PCR and western blot analysis of adipogenic regulators, Oil Red O staining and transplantation into nude mice. The CDC20 knockout efficiency was determined through immunochemistry, qRT-PCR and western blot of bone marrow. Accumulation of adiposity was measured through histology and staining of bone sections. Results: CDC20 expression in hBMSCs was significantly decreased during adipogenic differentiation. Knockdown of CDC20 enhanced adipogenic differentiation of hBMSCs in vitro. CDC20-knockdown hBMSCs showed more adipose tissue–like constructs in H&E staining and Oil Red O staining. Sp7-Cre;Cdc20f/f mice presented increased adipocytes in bone marrow compared with control mice. mBMSCs from Sp7-Cre;Cdc20f/f mice exerted upregulated adipogenic differentiation. Conclusions: Our findings showed that knockdown of CDC20 enhanced adipogenesis of h(m)BMSCs in vitro and in vivo. Overall, CDC20 regulated both adipogenesis and osteogenesis of BMSCs, and may lead to the development of new therapeutic target for “fatty bone” and osteoporosis.


2007 ◽  
Vol 4 (3) ◽  
pp. 229-232
Author(s):  
Wan Rong ◽  
Ding Jian ◽  
Zhou Zhen-Ming ◽  
Ren Li-Ping ◽  
Meng Qing-Xiang

AbstractThree Luxi adult Yellow steers were used to isolate and culture intramuscular pre-adipocytes in vitro as well as to examine factors influencing their proliferation and differentiation. The intramuscular pre-adipocytes were taken from adipose tissues within muscles between the sixth and seventh rib and cultured after digestion with collagenase I. The results showed that the separated cell populations were highly homogeneous, proliferative and doubled within 62 h. When the confluent pre-adipocytes were treated with 10 μg/ml insulin and 0.25 μmol/l dexamethasone, small lipid droplets appeared on day 2 and the number of lipid droplets rapidly increased around the nuclei on day 6. Their dynamic morphological changes, growth curve, Oil Red O staining, and reaction to insulin and dexamethasone all verified their pre-adipocyte identity. Under controlled conditions, the intramuscular pre-adipocytes resumed proliferating and differentiating in vitro. Interestingly, the proportion of cultured diploid pre-adipocytes reached more than 90% after six repeated cultures. This study confirms the existence of functionally active pre-adipocytes within the muscles of Chinese adult local breed cattle. These cell strains are a potentially useful model for understanding further the mechanism of intramuscular adipose deposition in tissues, in order to improve beef quality based on Chinese local breed beef cattle.


2008 ◽  
Vol 5 (3) ◽  
pp. 263-268
Author(s):  
Yu Fei ◽  
Ge Jian-Hui ◽  
Ni Li-Gang ◽  
He Xian-Hong ◽  
Xu Qi ◽  
...  

AbstractSpermatogonial stem cells (SSCs), which were isolated from chicken (Gallus gallus) embryo testes 16 days after laying, were cultured, subcultured, and induced into adipocytes in vitro. The differentiated cells were identified by oil red-O staining. Dexamethasone, insulin and 3-isobutyl-1-methylxanthine (IBMX) were tested for their induction potential. About 7–21 days after induction, SSCs differentiated into adipocytes, and the resulting adipocytes strongly expressed peroxisome proliferation activation receptor-γ (PPAR-γ). The assay outcome showed that an optimal treatment consisted of dexamethasone, insulin and IBMX application for 3 days and insulin for 1 day (3 cycles), then insulin for 21 days. The differentiation ratio was 85%, better than the combined use of dexamethasone, insulin and IBMX (P<0.01). However, the combination of the three derivatives triggered a stronger induction than any of them used alone (P<0.01). This study has demonstrated the potential of chicken embryonic SSCs to differentiate in vitro into adipocytes.


2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Abhilasha Singh ◽  
Ashok Kumar Srinivasan ◽  
Lakshmi Narasimhan Chakrapani ◽  
Periandavan Kalaiselvi

Background. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is the major receptor for oxidized low-density lipoprotein (Ox-LDL) in the aorta of aged rats. Ox-LDL initiates LOX-1 activation in the endothelium of lipid-accumulating sites of both animal and human subjects of hypercholesterolemia. Targeting LOX-1 may provide a novel diagnostic strategy towards hypercholesterolemia and vascular diseases. Hypothesis. This study was planned to address whether aegeline (AG) could bind to LOX-1 with a higher affinity and modulate the uptake of Ox-LDL in hypercholesterolemia. Study Design. Thirty-six Wistar rats were divided into six groups. The pathology group rats were fed with high-cholesterol diet (HCD) for 45 days, and the treatment group rats were fed with HCD and aegeline/atorvastatin (AV) for the last 30 days. In vivo and in vitro experiments were carried out to assay the markers of atherosclerosis like Ox-LDL and LOX-1 levels. Histopathological examination was performed. Oil Red O staining was carried out in the IC-21 cell line. Docking studies were performed. Results. AG administration effectively brought down the lipid levels induced by HCD. The lowered levels of Ox-LDL and LOX-1 in AG-administered rats deem it to be a potent antihypercholesterolemic agent. Compared to AV, AG had a pronounced effect in downregulating the expression of lipids evidenced by Oil Red O staining. AG binds with LOX-1 at a higher affinity validated by docking. Conclusion. This study validates AG to be an effective stratagem in bringing down the lipid stress induced by HCD and can be deemed as an antihypercholesterolemic agent.


2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Linqin Ma ◽  
Jingchun Zhang ◽  
Yu Qiao ◽  
Xinli Sun ◽  
Ting Mao ◽  
...  

Objective. The aim of this study was to establish a 3T3-L1 adipocyte model and ApoE−/− mouse model of intermittent hypoxia (IH) composite abnormal glucose metabolism (AGM) in vitro and in vivo and explore their synergistic damage effect leading to atherosclerosis (AS) and the influence of SREBP-1 signaling molecule-related mechanisms. Methods. Mature 3T3-L1 adipocytes were cultured with complete culture medium containing DEX 1×106 mol/L for 96 h to establish an AGM-3T3-L1 adipocyte model. Then, AGM-3T3-L1 adipocytes were treated with IH for 0 cycles, 2 cycles, 4 cycles, 8 cycles, 16 cycles, and 32 cycles and sustained hypoxia (SH). ApoE−/− mice were treated with high-fat diet and injection of STZ solution to establish an AGM-ApoE−/− mouse model. A total of 16 AGM-ApoE−/− mice were randomly and averagely divided into the normoxic control group (NC) and model group (CIH). AGM-ApoE−/− mice of the CIH group were treated with IH, which meant that the oxygen concentration fell to 10±0.5% in the first 90 seconds of one cycle and then increased to 21±0.5% in the later 90 seconds, continuous for eight hours per day (09 : 00-17 : 00) with a total of eight weeks. Eight C57BL/6J mice were used as the blank control group (Con) which was fed with conventional diet. qPCR and Western blotting were used to detect the expression level of SREBP-1c, FAS, and IRS-1. Oil Red O staining was used to compare the plaque of the aorta among each mouse group. Results. As a result, within 32 cycles of IH, mRNA and protein expression levels of SREBP-1c and FAS in AGM-3T3-L1 adipocytes were elevated with the increase in IH cycles; the mRNA expression of IRS-1 was decreased after IH 32 cycles and lower than that of the SH group. For the study in vivo, Oil Red O staining showed a more obvious AS aortic plaque in the CIH group. After CIH treatment of 4 w and 8 w, fasting blood glucose (FBG) of the NC group and CIH group was higher than that of the Con group, and the insulin level of the CIH group was higher than that of the Con group after IH treatment of 8 w. The expressions of the IRS-1 mRNA level in the aorta, skeletal muscle, and liver of the CIH group were lower than those in the Con group. The mRNA and protein expression of SREBP-1c and its downstream molecule FAS in the aorta, skeletal muscle, and liver significantly enhanced in the CIH group in contrast with those in the Con group. Conclusion. The CIH composite AGM could promote the progress of AS, which might be related to the modulation of the expression of SREBP-1-related molecular pathways.


2011 ◽  
Vol 22 (21) ◽  
pp. 4004-4015 ◽  
Author(s):  
Bruno Mesmin ◽  
Nina H. Pipalia ◽  
Frederik W. Lund ◽  
Trudy F. Ramlall ◽  
Anna Sokolov ◽  
...  

Nonvesicular transport of cholesterol plays an essential role in the distribution and regulation of cholesterol within cells, but it has been difficult to identify the key intracellular cholesterol transporters. The steroidogenic acute regulatory-related lipid-transfer (START) family of proteins is involved in several pathways of nonvesicular trafficking of sterols. Among them, STARD4 has been shown to increase intracellular cholesteryl ester formation and is controlled at the transcriptional level by sterol levels in cells. We found that STARD4 is very efficient in transporting sterol between membranes in vitro. Cholesterol levels are increased in STARD4-silenced cells, while sterol transport to the endocytic recycling compartment (ERC) and to the endoplasmic reticulum (ER) are enhanced upon STARD4 overexpression. STARD4 silencing attenuates cholesterol-mediated regulation of SREBP-2 activation, while its overexpression amplifies sterol sensing by SCAP/SREBP-2. To analyze STARD4's mode of action, we compared sterol transport mediated by STARD4 with that of a simple sterol carrier, methyl-β-cyclodextrin (MCD), when STARD4 and MCD were overexpressed or injected into cells. Interestingly, STARD4 and cytosolic MCD act similarly by increasing the rate of transfer of sterol to the ERC and to the ER. Our results suggest that cholesterol transport mediated by STARD4 is an important component of the cholesterol homeostasis regulatory machinery.


Sign in / Sign up

Export Citation Format

Share Document