Quantitative Detection of DNA by Coamplification Polymerase Chain Reaction: A Wide Detectable Range Controlled by the Thermodynamic Stability of Primer Template Duplexes

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 ∼90-fold following differentiation of 3T3-L1 into mature adipocytes, a change that was accompanied by ∼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 ∼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 ∼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-γ, 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.