Abstract
Acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) catalyzes the final step of triglyceride synthesis in mammalian cells. Data obtained from DGAT1-knockout mice have indicated that this enzyme plays an important role in energy homeostasis. We investigated the regulation of the expression and function of DGAT1 in mouse 3T3-L1 cell as a model for mammalian adipocytes. We demonstrated that the DGAT1 protein level increased by approximately 90-fold following differentiation of 3T3-L1 into mature adipocytes, a change that was accompanied by approximately 7-fold increase in DGAT1 mRNA. On the other hand, forced overexpression of DGAT1 mRNA by >20-fold via a recombinant adenovirus only resulted in approximately 2-fold increase in DGAT1 protein in mature adipocytes and little increase in preadipocytes. These results indicated that gene expression of DGAT1 in adipocytes is subjected to rigorous posttranscriptional regulation, which is modulated significantly by the differentiation status of 3T3-L1 cells. Protein stability is not a significant factor in the control of DGAT1 expression. The steady-state levels of DGAT1 were unaffected by blockage of proteolytic pathways by ALLN. However, translational control was suggested by sequence analysis of the 5'-untranslated region of human DGAT1 (hDGAT1) mRNA. We found that the level of DGAT1 activity was predominantly a function of the steady-state level of DGAT1 protein. No significant functional changes were observed when the conserved tyrosine phosphorylation site in hDGAT1 was mutated by a single base pair substitution. Despite only a approximately 2-fold increase in DGAT1 protein caused by recombinant viral transduction, a proportionate increase in cellular triglyceride synthesis resulted without affecting the triglyceride lipolysis rate, leading to >2-fold increase in intracellular triglyceride accumulation. No change in adipocyte morphology or in the expression levels of lipoprotein lipase, proxisomal proliferation-activating receptor-gamma, and aP2 was evident secondary to DGAT1 overexpression at different stages in 3T3-L1 differentiation. These data suggest that dysregulation of DGAT1 may play a role in the development of obesity, and manipulation of the steady-state level of DGAT1 protein may offer a potential means to treat or prevent obesity.
Highlights
§ To whom correspondence should be addressed: Division of Preventive Medicine and Nutrition, Dept. of Medicine, Columbia University College of Physicians and Surgeons, 630 W. 168th St., New York, NY 10032
DGAT1 Expression Is Regulated at the Posttranscriptional Level, and the Protein Level of DGAT1 Is Determined Predominantly by the Differentiation Status of 3T3-L1 Cells—To investigate how the expression and function of DGAT1 are regulated in mammalian adipocytes, we used a well characterized mouse cell line, 3T3-L1. 3T3-L1 cells are preadipocytes, which undergo adipocyte differentiation when induced by insulin/dexamethasone
A diacylglycerol transacylase pathway that bypasses DGAT and appears to operate in oilseed maturation may play a limited role in mammalian gastrointestinal system [4, 29]. Another major TG biosynthetic pathway has been identified in yeast mediated by Lro1, a homolog of human lecithin cholesterol acyltransferase, which esterifies diacylglycerol using phosphatidylcholine as the acyl donor, bypassing DGAT [30]. It is unclear if this type of TG biosynthesis plays any significant role in mammalian cells
Summary
§ To whom correspondence should be addressed: Division of Preventive Medicine and Nutrition, Dept. of Medicine, Columbia University College of Physicians and Surgeons, 630 W. 168th St., New York, NY 10032. These results indicated that gene expression of DGAT1 in adipocytes is subjected to rigorous posttranscriptional regulation, which is modulated significantly by the differentiation status of 3T3-L1 cells.
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