Lentiviral short hairpin ribonucleic acid-mediated knockdown of GLUT4 in 3T3-L1 adipocytes.
Adipose tissue is an important insulin target organ, and 3T3-L1 cells are a model cell line for adipocytes. In this study, we have used lentivirus-mediated short hairpin RNA (shRNA) for functional gene knockdown in 3T3-L1 adipocytes to assess the molecular mechanisms of insulin signaling. We chose to target GLUT4 to validate this approach. We showed that lentiviruses efficiently delivered transgenes and small interfering RNA (siRNA) into fully differentiated 3T3-L1 adipocytes. We established a strategy for identifying efficient siRNA sequences for gene knockdown by transfecting 293 cells with the target gene fluorescent fusion protein plasmid along with a plasmid that expresses shRNA. Using these methods, we identified highly efficient siGLUT4 sequences. We demonstrated that lentivirus-mediated shRNA against GLUT4 reduced endogenous GLUT4 expression to almost undetectable levels in 3T3-L1 adipocytes. Interestingly, insulin-stimulated glucose uptake was only reduced by 50-60%, suggesting that another glucose transporter mediates part of this effect. When siGLUT1 was introduced into GLUT4-deficient adipocytes, insulin-stimulated glucose uptake was essentially abolished, indicating that both GLUT4 and GLUT1 contribute to insulin-stimulated glucose transport in 3T3-L1 adipocytes. We also found that GLUT4 knockdown led to impaired insulin-responsive aminopeptidase protein expression that was dependent on whether GLUT4 was knocked down in the differentiating or differentiated stage. We further found that GLUT4 expression was not required for adipogenic differentiation but was necessary for full lipogenic capacity of differentiated adipocytes. These studies indicate that lentiviral shRNA constructs provide an excellent approach to deliver functional siRNAs into 3T3-L1 adipocytes for studying insulin signaling and adipocyte biology.
Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, 92093, USA.
Wei
W
Liao
M T Audrey
MT
Nguyen
Takeshi
T
Imamura
Oded
O
Singer
Inder M
IM
Verma
Jerrold M
JM
Olefsky
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D001391:Azo Compounds / Q000494:pharmacology; D001692:Biological Transport; D002454:Cell Differentiation; D002460:Cell Line; D003001:Cloning, Molecular; D018274:Electroporation; D005456:Fluorescent Dyes / Q000494:pharmacology; D020868:Gene Silencing; D005947:Glucose / Q000378:metabolism / Q000493:pharmacokinetics; D051275:Glucose Transporter Type 4 / Q000235:genetics* / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D000097573:Red Fluorescent Protein
D001391:Azo Compounds; D005456:Fluorescent Dyes; D005947:Glucose; D051275:Glucose Transporter Type 4; D049452:Green Fluorescent Proteins; D007328:Insulin; D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D000097573:Red Fluorescent Protein; C495693:Slc2a4 protein, mouse; C011049:oil red O