Isolation and in vitro culture of intramuscular pre-adipocytes from Luxi adult Yellow cattle

2007 ◽  
Vol 4 (3) ◽  
pp. 229-232
Author(s):  
Wan Rong ◽  
Ding Jian ◽  
Zhou Zhen-Ming ◽  
Ren Li-Ping ◽  
Meng Qing-Xiang
Keyword(s):  
Lipid Droplets ◽  
Morphological Changes ◽  
Local Breed ◽  
Chinese Adult ◽  
Yellow Cattle ◽  
Proliferation And Differentiation ◽  
Cell Strains ◽  
Oil Red O Staining ◽  
Oil Red O

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.

Secreted-Osteopontin Contributes to Brown Adipogenesis In Vitro via a CD44-Dependent Pathway

2019 ◽  
Vol 51 (11) ◽  
pp. 741-748
Author(s):  
Mengxi Wang ◽  
Yaoyao Guo ◽  
Yumeng Zhou ◽  
Wanwan Yuan ◽  
Huixia Li ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Biological Activities ◽  
Tumor Formation ◽  
Brown Adipocyte ◽  
Brown Adipocytes ◽  
Protein Levels ◽  
Infected Cells ◽  
Oil Red O Staining ◽  
Oil Red O

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.

scholarly journals miR-208b Regulates Rabbit Preadipocyte Proliferation and Differentiation

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 890
Author(s):  
Jiahao Shao ◽  
Ting Pan ◽  
Jie Wang ◽  
Tao Tang ◽  
Yanhong Li ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Target Gene ◽  
Cell Counting ◽  
Signal Pathways ◽  
Preadipocyte Differentiation ◽  
Proliferation And Differentiation ◽  
Oil Red O Staining ◽  
Oil Red O ◽  
Differentiation Model ◽  
Gene Mrna

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.

scholarly journals Therapeutic potential of small extracellular vesicles derived from lipoma tissue in adipose tissue regeneration—an in vitro and in vivo study

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Pengyu Hong ◽  
Xiaoyang Xu ◽  
Xin Hu ◽  
Hao Yang ◽  
Yue Wu ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Extracellular Vesicles ◽  
Tissue Regeneration ◽  
Lipid Droplets ◽  
Adipogenic Differentiation ◽  
Rt Pcr ◽  
Oil Red O Staining ◽  
Oil Red O

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.

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
Keyword(s):  
Fluorescence Intensity ◽  
Lipid Transfer ◽  
Gene Expressions ◽  
Preadipocyte Differentiation ◽  
Protein Expressions ◽  
Positive Rate ◽  
Steroidogenic Acute Regulatory ◽  
Oil Red O Staining ◽  
Oil Red O

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.

scholarly journals ANTIHYPERGLYCEMIC ACTIVITY OF TINOCRISPOSIDE BY STIMULATING 3T3-L1 ADIPOCYTE CELL DIFFERENTIATION

2018 ◽  
Vol 11 (11) ◽  
pp. 494
Author(s):  
Adek Zamrud Adnan ◽  
Muhammad Taher ◽  
Annisa Fauzana ◽  
Tika Afriani ◽  
Dewi Imelda Roesma ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Lipid Droplets ◽  
Negative Control Group ◽  
Negative Control ◽  
Control Group ◽  
Antihyperglycemic Activity ◽  
Significant Difference ◽  
Adipocyte Cell ◽  
Oil Red O Staining ◽  
Oil Red O

Objective: The aim of this study is to investigate the antihyperglycemic activity of tinocrisposide by stimulating 3T3-L1 adipocyte cell differentiation. Tinocrisposide is a furanoditerpene glycoside that was isolated from Tinospora crispa (Menispermaceae).Methods: Adipocyte cell differentiation activity of tinocrisposide in interval concentrations of 50, 25, 12.5, and 6.25 μg/ml has been investigated on 3T3-L1 cell line using insulin of 1 μg/ml as a positive and Dulbecco’s modified Eagle media (DMEM) as a negative control group. The effect of tinocrisposide was quantified with oil red O staining method by measuring an absorbance of lipid solution in isopropanol at a wavelength (λ) of 520 nm.Results: Tinocrisposide in the concentrations of 50, 25, 12.5, and 6.25 μg/ml insulin of 1 μg/ml and DMEM groups showed absorbance value of 0.7669, 0.7253, 0.6563, 0.6481, 0.954, and 0.2653, respectively. It was found that there was a significant difference statistically in lipid droplets accumulation among all groups (p<0.05) and tinocrisposide at a concentration of 50 μg/ml showed the highest lipid droplets accumulation in 3T3-L1 adipocyte cells.Conclusion: From the study, it could be concluded that tinocrisposide was able to stimulate the differentiation of adipocyte cell and had antihyperglycemic activity.

scholarly journals Korean Traditional Fermented Soybean Paste (Doenjang) Regulate Renin-Angiotensin System (RAS) in 3T3-L1 Adipocytes

2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 791-791
Author(s):  
Hayoung Woo ◽  
Jung Eun Park ◽  
Youn-Soo Cha
Keyword(s):  
Blood Pressure ◽  
Lipid Accumulation ◽  
Lipid Droplets ◽  
Renin Angiotensin System ◽  
Angiotensin System ◽  
Renin Angiotensin ◽  
Fermented Soybean ◽  
Soybean Paste ◽  
Oil Red O Staining ◽  
Oil Red O

Abstract Objectives Doenjang, the Korean traditional fermented soybean paste, contains much salt. There is a concern that cardiovascular disease may occur due to such high salinity. Nevertheless, previous studies have demonstrated functional properties of doenjang anti-obesity and anti-cancer effects. Furthermore, in our recent studies, we showed that the anti-hypertensive effect of doenjang through renin-angiotensin system (RAS) regulation. Doenjang regulated the RAS to improve lipid metabolism in adipose tissue, which had a positive effect on blood pressure control. Therefore, we expected to find the exact mechanism of action or target point of doenjang in adipocyte using 3T3-L1 cells. Methods In this study, 3T3-L1 cells were treated with doenjang and RAS blockers, Losartan (10−4 M), and Captopril (10−4 M), were treated as positive control which suppresses AT1R and ACE, respectively. Non-cytotoxic concentrations of samples were selected as per MTT assay and added with induction media, harvested after 4 days for RNA extraction. Lipid droplets were detected by Oil Red O staining. Results Doenjang downregulated mRNA levels of peroxisome proliferator-activated receptor-γ (Pparg), RAS related genes such as angiotensinogen (Agt), Renin (Ren), and aldosterone-releasing factors (P &lt; 0.05). Especially, angiotensin convert enzyme (Ace) and angiotensin II receptor 2 (Agtr2) levels were decreased by doenjang treatment. Doenjang reduced the lipid accumulation, which was confirmed from the Oil Red O staining of lipid droplets. As a result, it is revealed that doenjang not only inhibits lipid accumulation in adipocytes but also may inhibit ACE in 3T3-L1 adipocytes through a mechanism similar to the effect of Captopril. Conclusions These data are consistent with our animal study. It have been shown to regulate blood pressure through lipid improvement and ACE inhibition despite high salt content in doenjang. Funding Sources This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. 2018R1A2B6006477).

scholarly journals Upregulated microRNA-106a Promotes Porcine Preadipocyte Proliferation and Differentiation by Targeting Different Genes

Genes ◽  
2019 ◽  
Vol 10 (10) ◽  
pp. 805 ◽  
Author(s):  
Kuilong Huang ◽  
Xin’e Shi ◽  
Jie Wang ◽  
Ying Yao ◽  
Ying Peng ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Lipid Droplets ◽  
Luciferase Reporter ◽  
Cell Cycles ◽  
Lipogenic Genes ◽  
Proliferation And Differentiation ◽  
Luciferase Reporter Vector ◽  
Membrane Bound ◽  
Porcine Adipocytes ◽  
Oil Red O

Adipose tissue is one of the main organs for the energy storage and supply of organisms. Adipose deposition and metabolism are controlled by a cascade of transcription factors and epigenetic regulatory mechanisms. Previous studies have also shown that miR-106a plays a considerable role in the development of organisms. The regulatory mechanism of miR-106a on porcine preadipocytes is still not clear. In this study, preadipocytes were isolated from the neck subcutaneous deposits of 3–5-day old Chinese native Guanzhong black pigs using 5-ethynyl-20-deoxyuridine (EdU) staining and a CCK-8 assay to detect the number of proliferous cells and real-time qPCR (RT-qPCR) and western blot analysis to detect gene expression, as well as Oil Red O and BODIPY staining dye lipid droplets and flow cytometry (FCM) to detect cell cycles. We also used the double luciferase method to detect the relative luciferase activities. Upregulated miR-106a increased the number of proliferous cells and enhanced the expression of cell proliferation-related genes in porcine adipocytes. The double luciferase reporter vector confirmed that p21 was a target gene of miR-106a in the cell proliferation phase. miR-106a upregulation increased the number of lipid droplets and the expression of lipogenic genes and directly targeted BMP and activin membrane-bound inhibitor (BAMBI) in the process of differentiation. Our results indicated that miR-106a promotes porcine preadipocyte proliferation and differentiation by targeting p21 and BAMBI.

scholarly journals Perfluorooctane sulfonate affects proliferation and differentiation of pluripotent human teratocarcinoma cells

2016 ◽  
Author(s):  
Mina Popovic ◽  
Brett A Neilan ◽  
Francesco Pomati
Keyword(s):  
Stem Cell ◽  
Cell Model ◽  
Morphological Changes ◽  
Perfluorooctane Sulfonate ◽  
Stem Cell Marker ◽  
Marker Genes ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Proliferation And Differentiation

Perfluorinated compounds have raised concern due to their potential association with detrimental postnatal outcomes in animals and humans. We tested the effects of perfluorooctane sulfonate (PFOS) on a human pluripotent teratocarcinoma (known as NCCIT) cells as an in vitro prototype for developmental toxicity in mammals. NCCIT contains stem-cells able to differentiate into endoderm, mesoderm and ectoderm. We tested our cell model using a teratogenic compound, retinoic acid (RA), a cytotoxin, nocodazole (ND), and PFOS. We assayed cells proliferation, morphology and expression of stem cell and germ layer marker genes. PFOS reduced NCCIT cell proliferation in a concentration-dependent manner and induced morphological changes in cell cultures that resembled ectodermal phenotypes. A tendency towards a differentiated state in NCCIT was confirmed by real-time gene expression. PFOS triggered up-regulation of the gene nestin, indicative of ectodermal lineage differentiation, and interfered with the expression of the pluripotency stem-cell marker TERT. PFOS produced effects on both cells proliferation and differentiation, although not as severe as those observed for RA and ND, at levels that fall within the range of concentrations found in animal and human plasma. We discuss our findings in the context of possible interference of PFOS with the processes governing the early development of mammalian tissues.

scholarly journals Embryonic cardiospecific knockout of α-E-catenin gene leads to alteration of energy metabolism in adult heart

2017 ◽  
Vol 23 (2) ◽  
pp. 65-69
Author(s):  
V. Balatskyy ◽  
L. Macewicz ◽  
O. Piven
Keyword(s):  
Lipid Metabolism ◽  
Energy Metabolism ◽  
Transgenic Mice ◽  
Lipid Droplets ◽  
Metabolic Disorders ◽  
Conditional Knockout ◽  
Heart Hypertrophy ◽  
Oil Red O Staining ◽  
Oil Red O

Previously we have shown that the α-E-catenin knockout in the embryonic heart leads to hypertrophy in adult and activation of canonical Wntsignaling. Heart hypertrophy is also accompanied by metabolic disorders, but role of the α-E-catenin in these processes is not known. Aim of our work is to study the effect of α-E-catenin deletion on the lipid metabolism in the heart. Methods. In our experiment we have used α-Е-catenin conditional knockout and αMHC-Cre transgenic mice. We have utilized histological (Oil Red O staining) and molecular biological (Western blot) methods. Results. α-Е-catenin deletion leads to accumulation of lipid droplets in myocardium, and to violation of expression and phosphorylation of key regulators of lipid metabolism (Ampk, Pparα, Acc, Hsl). Conclusions. Ous results suggest that α-Е-catenin deletion leads to inhibition of lipid metabolism in the heart.

scholarly journals Evaluation on the phagocytosis of apoptotic spermatogenic cells by Sertoli cells in vitro through detecting lipid droplet formation by Oil Red O staining

Reproduction ◽  
2006 ◽  
Vol 132 (3) ◽  
pp. 485-492 ◽  
Author(s):  
Huizhen Wang ◽  
Haikun Wang ◽  
Weipeng Xiong ◽  
Yongmei Chen ◽  
Quanhong Ma ◽  
...  
Keyword(s):  
Lipid Droplet ◽  
Sertoli Cells ◽  
Lipid Droplets ◽  
Apoptotic Cell ◽  
Practical Method ◽  
Droplet Formation ◽  
Spermatogenic Cells ◽  
Lipid Droplet Formation ◽  
Oil Red O

During spermatogenesis, more than half of the differentiating spermatogenic cells undergo apoptosis before they mature into spermatozoa. Ultrastructure studies showed that the formation of lipid droplets in Sertoli cells was associated with phagocytosis of residual bodies and apoptotic germ cells by Sertoli cells. Here, a relationship between the phagocytosis of apoptotic spermatogenic cells and lipid droplet formation in Sertoli cells was studiedin vitroby Oil Red O (ORO) staining. The results confirmed that the formation of lipid droplets was a result of phagocytosis of apoptotic spermatogenic cells in Sertoli cells. By comparing phagocytosis of apoptotic spermatogenic cells and thymocytes by Sertoli cells to that by macrophages, we demonstrated that the lipid droplets accumulation in phagocytes depended on phagocytosed apoptotic cell type, but not phagocyte type. However, the size of lipid droplets was related to the type of phagocytes. By this approach, we found that Sertoli cells at different postnatal stages of development had a similar phagocytic ability. These results suggested that the detection of lipid droplets by ORO staining was a practical method to evaluate the phagocytic functions of Sertoli cellsin vitro. This approach could also be considered as anin vitromodel to study the lipid formation, metabolism, and function in Sertoli cells.

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