Sequential Regulation of Diacylglycerol Acyltransferase 2 Expression by CAAT/Enhancer-binding Protein β (C/EBPβ) and C/EBPα during Adipogenesis

Victoria A Payne,Wo-Shing Au,Sarah L Gray,Edoardo Dalla Nora,Shaikh M Rahman,Rebecca Sanders,Dirk Hadaschik,Jacob E Friedman,Stephen O’Rahilly,Justin J Rochford

doi:10.1074/jbc.m702871200

Victoria A Payne, Wo-Shing Au + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m702871200

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Abstract

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triacylglycerol (TG) synthesis. Despite the existence of an alternative acyltransferase (DGAT1), mice lacking DGAT2 have a severe deficiency of TG in adipose tissue, indicating a nonredundant role for this enzyme in adipocyte TG synthesis. We have studied the regulation of DGAT2 expression during adipogenesis. In both isolated murine preadipocytes and 3T3-L1 cells the temporal pattern of DGAT2 expression closely mimicked that of genes whose expression is regulated by CAAT/enhancer-binding protein beta (C/EBPbeta). Inhibition of C/EBPbeta expression in differentiating preadipocytes reduced DGAT2 expression, and electrophoretic mobility shift assay and chromatin immunoprecipitation experiments identified a promoter element in the DGAT2 gene that is likely to mediate this effect. The importance of C/EBPbeta in adipocyte expression of DGAT2 was confirmed by the finding of reduced DGAT2 expression in the adipose tissue of C/EBPbeta-null animals. However, DGAT2 expression is maintained at high levels during the later stages of adipogenesis, when C/EBPbeta levels decline. We show that, at these later stages of differentiation, C/EBPalpha is capable of substituting for C/EBPbeta at the same promoter element. These observations provide novel insight into the transcriptional regulation of DGAT2 expression. Moreover, they further refine the complex and serial roles of the C/EBP family of transcription factors in inducing and maintaining the metabolic properties of mature adipocytes.

Highlights

The increased adipose tissue mass of obesity results from a combination of increased lipid storage in existing adipocytes and the generation of new adipocytes from precursors residing within the adipose tissue [1]
To determine the expression of DGAT1 and DGAT2 during adipogenesis, 3T3-L1 preadipocytes were induced to differentiate, and DGAT1 and DGAT2 expression was assayed by realtime PCR
Both DGAT1 and DGAT2 mRNA were induced with a similar time course, with a minor increase in expression in the first few hours following induction, and strong and sustained increase of mRNA occurring within 3 days

Summary

EXPERIMENTAL PROCEDURES

Preadipocyte Isolation and Culture—Murine preadipocyte isolation, culture, and differentiation were performed as described previously [23]. At various times following induction of differentiation, cells were harvested, and RNA was extracted. RNA Isolation, cDNA Synthesis, and Real-time PCR—Total RNA was extracted from cell cultures using an RNeasy kit (Qiagen). Primer Express, version 1.0 software (PerkinElmer Life Sciences, Applied Biosystems) was used to design the probes and primers for real-time quantitative PCR to determine DGAT2, DGAT1, C/EBP␤, and 11␤HSD1 mRNA expression. Two microliters of eluted DNA was used to assay the presence of DNA sequences associated with the immunoprecipitated proteins using specific primers amplifying DNA sequences, including the binding sites being assayed and SYBR green master mix according to the manufacturer’s instructions. Western Blotting—Protein samples were extracted by scraping in lysis buffer containing 1% Nonidet P-40, followed by sonication as described previously [25].

RESULTS

DISCUSSION

It is particularly difficult to discriminate the relative importance of