RNAi-based gene silencing in primary mouse and human adipose tissues.
Cultured adipocyte cell lines are a model system widely used to study adipose function, but they exhibit significant physiological differences compared with primary cells from adipose tissue. Here we report short interfering RNA-based methodology to selectively attenuate gene expression in mouse and human primary adipose tissues as a means of rapidly validating findings made in cultured adipocyte cell lines. The method is exemplified by depletion of the PTEN phosphatase in white adipose tissue (WAT) from mouse and humans, which increases Akt phosphorylation as expected. This technology is also shown to silence genes in mouse brown adipose tissue. Previous work revealed upregulation of the mitochondrial protein UCP1 in adipose cells from mice lacking the gene for the transcriptional corepressor RIP140, whereas in cultured adipocytes, loss of RIP140 has a little effect on UCP1 expression. Application of our method to deplete RIP140 in primary mouse WAT elicited markedly increased oxygen consumption and expression of UCP1 that exactly mimics the phenotype observed in RIP140-null mice. This ex-vivo method of gene silencing should be useful in rapid validation studies as well as in addressing the depot- and species-specific functions of genes in adipose biology.
Puri
Vishwajeet
V
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Chakladar
Abhijit
A
Virbasius
Joseph V
JV
Konda
Silvana
S
Powelka
Aimee M
AM
Chouinard
My
M
Hagan
G Nana
GN
Perugini
Richard
R
Czech
Michael P
MP
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D052436:Adipose Tissue, White / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D005947:Glucose / Q000378:metabolism; D051272:Glucose Transporter Type 1 / Q000235:genetics; D051275:Glucose Transporter Type 4 / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D000078723:Nuclear Receptor Interacting Protein 1; D051059:PTEN Phosphohydrolase / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
D048868:Adaptor Proteins, Signal Transducing; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; C000627127:NRIP1 protein, human; D009687:Nuclear Proteins; D000078723:Nuclear Receptor Interacting Protein 1; D012333:RNA, Messenger; C495677:SLC2A1 protein, human; C495692:SLC2A4 protein, human; D051057:Proto-Oncogene Proteins c-akt; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; D005947:Glucose