Establishment of the 3T3-L1 adipocyte model for investigation of GLUT4 translocation

Name: Thanarat Salahong

Organization : Mahidol University. Faculty of Medicine Siriraj Hospital

Name: Chutima Chanprasert

Organization : Mahidol University. Faculty of Medicine Siriraj Hospital

Name: Ladawan Khowawisetsut

Organization : Mahidol University. Faculty of Medicine Siriraj Hospital

Name: Pa-thai Yenchitsomanus

Organization : Mahidol University. Faculty of Medicine Siriraj Hospital

Name: Nattachet Plengvidhya

Organization : Mahidol University. Faculty of Medicine Siriraj Hospital

Name: Prapaporn Jungtrakoon Thamtarana

Organization : Mahidol University. Faculty of Medicine Siriraj Hospital

Email : prapaporn.jun@mahidol.ac.th

keyword: GLUT4 protein

LCSH: Fat cells

LCSH: Cell coculture

LCSH: Cell differentiation

LCSH: Flow cytometry

Abstract: The translocation of glucose transporter type 4 (GLUT4) containing vesicles from cytoplasm to plasma membrane in response to insulin stimulus is crucial for maintenance of glucose homeostasis. Failure in transporting GLUT4 to plasma membrane hinders peripheral glucose uptake and, thus, leads to hyperglycemia. Here, by tagging of GLUT4 extracellular domain with hemagglutinin (HA) epitope, we aim to establish an adipocyte model for investigation of GLUT4 translocation. Lentiviral particles carrying GLUT4-HA was generated and transduced into 3T3-L1 pre-adipocytes. Cells stably expressing GLUT4-HA were validated using Western blotting analysis (WB) and immunofluorescence staining assay (IFA). After differentiation to adipocyte, the level of GLUT4-HA expressed on plasma membrane was immunofluorescence-stained, without cell permeabilization, and quantified by flow cytometry. The result showed that, as determined by WB, the established pre-adipocyte model expressed the recombinant GLUT4-HA. IFA by skipping the process of cell permeabilization successfully demonstrated GLUT4-HA expressed on cell surface. However, once the pre-adipocytes were undergone differentiation process, the percentage of mature adipocytes, as determined by cellular size and granularity using flow cytometry, did not conform to what was observed under inverted microscopy. The fluorescent intensity of the immunostained adipocytes under basal and insulin-stimulated conditions was not different. Adipocyte-specific marker should be applied for discriminating the differentiated cells by means of flow cytometry before validating insulin-stimulated GLUT4 translocation in the established model

King Mongkut's University of Technology North Bangkok. Central Library

Address: BANGKOK

Email: library@kmutnb.ac.th

Created: 2023

Modified: 2024-04-29

Issued: 2024-04-29

บทความ/Article

application/pdf

BibliograpyCitation : In Rangsit University. The 8th RSU International Research Conference on Sciences and Technology 2023 (RSUCON 2023) (pp.179-186). Pathum Thani : Rangsit University

eng

©copyrights King Mongkut's University of Technology North Bangkok

ลำดับที่.	ชื่อแฟ้มข้อมูล	ขนาดแฟ้มข้อมูล	จำนวนเข้าถึง	วัน-เวลาเข้าถึงล่าสุด
1	RSUCON 2023pp.179-186.pdf	889.56 KB

ใช้เวลา

0.059168 วินาที