Stable conditional expression and effect of C/EBPβ-LIP in adipocytes using the pSLIK system

Cristina L Esteves,Achim Leutz,Simon G Lillico,Val Kelly,Karen E Chapman,Jonathan R Seckl,Valérie Bégay

doi:10.1530/jme-13-0029

Abstract

Murine 3T3-L1 adipocytes are widely used as a cellular model of obesity. However, whereas transfection of 3T3-L1 preadipocytes is straightforward, ectopic gene expression in mature 3T3-L1 adipocytes has proved challenging. Here, we used the pSLIK vector system to generate stable doxycycline-inducible expression of the liver-enriched inhibitor protein isoform of CCAAT/enhancer binding protein β (CEPB (C/EBPβ-LIP)) in fully differentiated 3T3-L1 adipocytes. Because overexpression of C/EBPβ-LIP impairs adipocyte differentiation, the C/EBPβ-LIP construct was first integrated in 3T3-L1 preadipocytes but expression was induced only when adipocytes were fully differentiated. Increased C/EBPβ-LIP in mature adipocytes down-regulated C/EBPβ target genes including 11β-hydroxysteroid dehydrogenase type 1, phosphoenolpyruvate carboxykinase and fatty acid binding protein 4 but had no effect on asparagine synthetase, demonstrating that transcriptional down-regulation by C/EBPβ-LIP in 3T3-L1 adipocytes is not a general effect. Importantly, these genes were modulated in a similar manner in adipose tissue of mice with genetically increased C/EBPβ-LIP levels. The use of the pSLIK system to conditionally express transgenes in 3T3-L1 cells could be a valuable tool to dissect adipocyte physiology.

Highlights

First developed almost 40 years ago from Swiss mouse embryo cells, 3T3-L1 adipocytes (Otto & Lane 2005, Rosen & MacDougald 2006) have proved to be an invaluable tool, advancing our understanding of cellular mechanisms underlying obesity-related disorders
We have recently shown that genetic manipulation of levels of C/EBPb-LIP in vivo changes adipose tissue expression of the C/EBPb target gene that encodes the glucocorticoid amplifying enzyme 11b-hydroxysteroid dehydrogenase type 1 (Hsd11b1 (11b-HSD1); Seckl et al 2004, Esteves et al 2012)
The use of the pSLIK system to conditionally express C/EBPb-LIP in fully differentiated 3T3-L1 mature adipocytes allowed investigating its impact on the transcriptional levels of 11b-HSD1, PEPCK, FABP4 and asparagine synthetase in these cells

Summary

C L ESTEVES and others

Stable conditional expression and effect of C/EBPb-LIP in adipocytes using the pSLIK system. Cristina L Esteves, Val Kelly, Valerie Begay, Simon G Lillico, Achim Leutz, Jonathan R Seckl and Karen E Chapman. Endocrinology Unit, Queen’s Medical Research Institute, University/BHF Centre for Cardiovascular Science, The University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK 1Max Delbrueck Centre for Molecular Medicine, Robert-Rossle-Straße 10, 13125 Berlin, Germany 2Division of Developmental Biology, The Roslin Institute, The University of Edinburgh, Easter Bush Campus, Midlothian EH25 9RG, UK

Introduction

Ethics statement

Results

Discussion