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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 C/EBPβ-LIP represses expression of PEPCK and FABP4, but not asparagine synthetase in adipose tissue in vivo. Real-time PCR measurement of levels of mRNA encoding (A) PEPCK, (B) FABP4 and (C) asparagine synthetase in adipose tissue of wild-type control mice (Con, white bars), C/EBPβ(+/L) (+/L, black bars) and C/EBPβΔuORF (ΔuORF, grey bars). C/EBPβ(+/L) mice are heterozygous for an allele of C/EBPβ in which the normal gene has been replaced by C/EBPβ-LIP (a ‘knock-in’; Smink et al. 2009) and C/EBPβΔuORF mice have a homozygous disruption of the upstream ORF (Wethmar et al. 2010). Adipose tissue mRNA levels, normalised to TBP, are expressed relative to levels in control mice (arbitrarily set to 100%) and are mean±s.e.m.; n=6–9/group. *P≤0.05.
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fig4: Elevated C/EBPβ-LIP represses expression of PEPCK and FABP4, but not asparagine synthetase in adipose tissue in vivo. Real-time PCR measurement of levels of mRNA encoding (A) PEPCK, (B) FABP4 and (C) asparagine synthetase in adipose tissue of wild-type control mice (Con, white bars), C/EBPβ(+/L) (+/L, black bars) and C/EBPβΔuORF (ΔuORF, grey bars). C/EBPβ(+/L) mice are heterozygous for an allele of C/EBPβ in which the normal gene has been replaced by C/EBPβ-LIP (a ‘knock-in’; Smink et al. 2009) and C/EBPβΔuORF mice have a homozygous disruption of the upstream ORF (Wethmar et al. 2010). Adipose tissue mRNA levels, normalised to TBP, are expressed relative to levels in control mice (arbitrarily set to 100%) and are mean±s.e.m.; n=6–9/group. *P≤0.05.

Mentions: To test the relevance of the 3T3-L1 pSLIK-C/EBPβ-LIP adipocyte model to the in vivo situation, we measured the levels of mRNA encoding PEPCK, FABP4 and asparagine synthetase in the adipose tissue of mice genetically engineered to produce altered levels of C/EBPβ-LIP, which we have previously shown to regulate adipose tissue 11β-HSD1 mRNA levels (Esteves et al. 2012). C/EBPβ(+/L) mice, with a ‘knock-in’ of C/EBPβ-LIP (L allele) replacing the normal C/EBPβ gene, have increased C/EBPβ-LIP levels and therefore increased C/EBPβ-LIP:LAP ratio (Smink et al. 2009). In contrast, in C/EBPβΔuORF mice, deletion of the upstream open reading frame (ΔuORF allele) prevents translation of C/EBPβ-LIP (Wethmar et al. 2010), resulting in decreased C/EBPβ-LIP:LAP ratio (Esteves et al. 2012). In agreement with the results obtained in the 3T3-L1 pSLIK-C/EBPβ-LIP adipocytes, levels of mRNA encoding PEPCK were lower in the adipose tissue of C/EBPβ(+/L) mice compared with C/EBPβΔuORF (Fig. 4A). However, there was no significant decrease in PEPCK mRNA levels in C/EBPβ(+/L) mice compared with WT controls, suggesting a larger effect of C/EBPβ-LIP in vitro than in vivo or that compensation has occurred in vivo (e.g. through tissues other than adipose). FABP4 levels were lower in the adipose of C/EBPβ(+/L) mice compared with control and C/EBPβΔuORF mice, while asparagine synthetase was unchanged (Fig. 4B and C). Taken together, these results show that the 3T3-L1 pSLIK-C/EBPβ-LIP adipocytes mimic the in vivo regulation.


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 C/EBPβ-LIP represses expression of PEPCK and FABP4, but not asparagine synthetase in adipose tissue in vivo. Real-time PCR measurement of levels of mRNA encoding (A) PEPCK, (B) FABP4 and (C) asparagine synthetase in adipose tissue of wild-type control mice (Con, white bars), C/EBPβ(+/L) (+/L, black bars) and C/EBPβΔuORF (ΔuORF, grey bars). C/EBPβ(+/L) mice are heterozygous for an allele of C/EBPβ in which the normal gene has been replaced by C/EBPβ-LIP (a ‘knock-in’; Smink et al. 2009) and C/EBPβΔuORF mice have a homozygous disruption of the upstream ORF (Wethmar et al. 2010). Adipose tissue mRNA levels, normalised to TBP, are expressed relative to levels in control mice (arbitrarily set to 100%) and are mean±s.e.m.; n=6–9/group. *P≤0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Elevated C/EBPβ-LIP represses expression of PEPCK and FABP4, but not asparagine synthetase in adipose tissue in vivo. Real-time PCR measurement of levels of mRNA encoding (A) PEPCK, (B) FABP4 and (C) asparagine synthetase in adipose tissue of wild-type control mice (Con, white bars), C/EBPβ(+/L) (+/L, black bars) and C/EBPβΔuORF (ΔuORF, grey bars). C/EBPβ(+/L) mice are heterozygous for an allele of C/EBPβ in which the normal gene has been replaced by C/EBPβ-LIP (a ‘knock-in’; Smink et al. 2009) and C/EBPβΔuORF mice have a homozygous disruption of the upstream ORF (Wethmar et al. 2010). Adipose tissue mRNA levels, normalised to TBP, are expressed relative to levels in control mice (arbitrarily set to 100%) and are mean±s.e.m.; n=6–9/group. *P≤0.05.
Mentions: To test the relevance of the 3T3-L1 pSLIK-C/EBPβ-LIP adipocyte model to the in vivo situation, we measured the levels of mRNA encoding PEPCK, FABP4 and asparagine synthetase in the adipose tissue of mice genetically engineered to produce altered levels of C/EBPβ-LIP, which we have previously shown to regulate adipose tissue 11β-HSD1 mRNA levels (Esteves et al. 2012). C/EBPβ(+/L) mice, with a ‘knock-in’ of C/EBPβ-LIP (L allele) replacing the normal C/EBPβ gene, have increased C/EBPβ-LIP levels and therefore increased C/EBPβ-LIP:LAP ratio (Smink et al. 2009). In contrast, in C/EBPβΔuORF mice, deletion of the upstream open reading frame (ΔuORF allele) prevents translation of C/EBPβ-LIP (Wethmar et al. 2010), resulting in decreased C/EBPβ-LIP:LAP ratio (Esteves et al. 2012). In agreement with the results obtained in the 3T3-L1 pSLIK-C/EBPβ-LIP adipocytes, levels of mRNA encoding PEPCK were lower in the adipose tissue of C/EBPβ(+/L) mice compared with C/EBPβΔuORF (Fig. 4A). However, there was no significant decrease in PEPCK mRNA levels in C/EBPβ(+/L) mice compared with WT controls, suggesting a larger effect of C/EBPβ-LIP in vitro than in vivo or that compensation has occurred in vivo (e.g. through tissues other than adipose). FABP4 levels were lower in the adipose of C/EBPβ(+/L) mice compared with control and C/EBPβΔuORF mice, while asparagine synthetase was unchanged (Fig. 4B and C). Taken together, these results show that the 3T3-L1 pSLIK-C/EBPβ-LIP adipocytes mimic the in vivo regulation.

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