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

Esteves CL, Kelly V, Bégay V, Lillico SG, Leutz A, Seckl JR, Chapman KE - J. Mol. Endocrinol. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Endocrinology Unit, Queen's Medical Research Institute, University/BHF Centre for Cardiovascular Science, The University of Edinburgh, Edinburgh, UK. cristina.esteves@ed.ac.uk

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 (C/ebpβ (Cebpb)) (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.

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Elevated adipocyte C/EBPβ-LIP represses expression of C/EBPβ target genes but not of asparagine synthetase. Real-time PCR measurement of levels of mRNAs encoding (A) 11β-HSD1, (B) PEPCK, (C) FABP4 and (D) asparagine synthetase in 3T3-L1 pSLIK-C/EBPβ-LIP adipocytes (black bars) compared with pSLIK control adipocytes (white bars), following 1–4 days of induction with DOX. Preadipocytes were differentiated into adipocytes in DOX-free medium and DOX was added to mature adipocytes to induce C/EBPβ-LIP expression. Levels of specific mRNAs, normalised to TBP are expressed relative to levels in pSLIK adipocytes (arbitrarily set to 100%). Data are mean±s.e.m. of two independent adipocyte differentiations, each carried out in triplicate. *Significantly different from pSLIK control adipocytes. *P≤0.05, **P<0.001 and ***P<0.0001.
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fig3: Elevated adipocyte C/EBPβ-LIP represses expression of C/EBPβ target genes but not of asparagine synthetase. Real-time PCR measurement of levels of mRNAs encoding (A) 11β-HSD1, (B) PEPCK, (C) FABP4 and (D) asparagine synthetase in 3T3-L1 pSLIK-C/EBPβ-LIP adipocytes (black bars) compared with pSLIK control adipocytes (white bars), following 1–4 days of induction with DOX. Preadipocytes were differentiated into adipocytes in DOX-free medium and DOX was added to mature adipocytes to induce C/EBPβ-LIP expression. Levels of specific mRNAs, normalised to TBP are expressed relative to levels in pSLIK adipocytes (arbitrarily set to 100%). Data are mean±s.e.m. of two independent adipocyte differentiations, each carried out in triplicate. *Significantly different from pSLIK control adipocytes. *P≤0.05, **P<0.001 and ***P<0.0001.

Mentions: The transcriptional impact of increased C/EBPβ-LIP in adipocytes was examined following induction with DOX only once 3T3-L1 adipocytes were fully differentiated. Concurrent with the increase in C/EBPβ-LIP expression, and consequently C/EBPβ-LIP:LAP ratio, DOX reduced 11β-HSD1 and PEPCK mRNA levels in 3T3-L1 pSLIK-C/EBPβ-LIP adipocytes compared with control cells (Fig. 3A and B). FABP4 mRNA levels were decreased following 4 days of DOX treatment but asparagine synthetase mRNA levels were unaffected (Fig. 3C and D).


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

Esteves CL, Kelly V, Bégay V, Lillico SG, Leutz A, Seckl JR, Chapman KE - J. Mol. Endocrinol. (2013)

Elevated adipocyte C/EBPβ-LIP represses expression of C/EBPβ target genes but not of asparagine synthetase. Real-time PCR measurement of levels of mRNAs encoding (A) 11β-HSD1, (B) PEPCK, (C) FABP4 and (D) asparagine synthetase in 3T3-L1 pSLIK-C/EBPβ-LIP adipocytes (black bars) compared with pSLIK control adipocytes (white bars), following 1–4 days of induction with DOX. Preadipocytes were differentiated into adipocytes in DOX-free medium and DOX was added to mature adipocytes to induce C/EBPβ-LIP expression. Levels of specific mRNAs, normalised to TBP are expressed relative to levels in pSLIK adipocytes (arbitrarily set to 100%). Data are mean±s.e.m. of two independent adipocyte differentiations, each carried out in triplicate. *Significantly different from pSLIK control adipocytes. *P≤0.05, **P<0.001 and ***P<0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672996&req=5

fig3: Elevated adipocyte C/EBPβ-LIP represses expression of C/EBPβ target genes but not of asparagine synthetase. Real-time PCR measurement of levels of mRNAs encoding (A) 11β-HSD1, (B) PEPCK, (C) FABP4 and (D) asparagine synthetase in 3T3-L1 pSLIK-C/EBPβ-LIP adipocytes (black bars) compared with pSLIK control adipocytes (white bars), following 1–4 days of induction with DOX. Preadipocytes were differentiated into adipocytes in DOX-free medium and DOX was added to mature adipocytes to induce C/EBPβ-LIP expression. Levels of specific mRNAs, normalised to TBP are expressed relative to levels in pSLIK adipocytes (arbitrarily set to 100%). Data are mean±s.e.m. of two independent adipocyte differentiations, each carried out in triplicate. *Significantly different from pSLIK control adipocytes. *P≤0.05, **P<0.001 and ***P<0.0001.
Mentions: The transcriptional impact of increased C/EBPβ-LIP in adipocytes was examined following induction with DOX only once 3T3-L1 adipocytes were fully differentiated. Concurrent with the increase in C/EBPβ-LIP expression, and consequently C/EBPβ-LIP:LAP ratio, DOX reduced 11β-HSD1 and PEPCK mRNA levels in 3T3-L1 pSLIK-C/EBPβ-LIP adipocytes compared with control cells (Fig. 3A and B). FABP4 mRNA levels were decreased following 4 days of DOX treatment but asparagine synthetase mRNA levels were unaffected (Fig. 3C and D).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Endocrinology Unit, Queen's Medical Research Institute, University/BHF Centre for Cardiovascular Science, The University of Edinburgh, Edinburgh, UK. cristina.esteves@ed.ac.uk

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 (C/ebpβ (Cebpb)) (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.

Show MeSH
Related in: MedlinePlus