Posttranscriptional Control of the Expression and Function of Diacylglycerol Acyltransferase-1 in Mouse Adipocytes

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 ϳ90-fold following differentiation of 3T3-L1 into mature adipocytes, a change that was accompanied by ϳ7-fold increase in DGAT1 mRNA. On the other hand, forced overexpression of DGAT1 mRNA by >20-fold via a recombinant adenovirus only resulted in ϳ2-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 ϳ2-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-␥, 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. Adiposity, or the amount of triglyceride (TG)1 stored in adipocytes, is fundamentally a net result of two opposing metabolic processes, i.e. lipogenesis and lipolysis. These are highly regulated processes influenced by various hormones, cytokines, nutrients, and other factors (see Ref. 1) that may either serve as substrates or function as signaling molecules for one or more specific metabolic pathways. Acyl-CoA:diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) is thought to be a key enzyme in controlling the synthetic rate of TG in adipocytes (2). It catalyzes the last step in the de novo TG synthetic pathway employed by various types of cells, producing TG from its two substrates, diacylglycerol and fatty acyl-CoA (for a review, see Refs. 3 and 4). Two DGAT genes have been identified....

Paraoxonase 2 attenuates macrophage triglyceride accumulation via inhibition of diacylglycerol acyltransferase 1

“…Indeed, the DGAT1 inhibitor oleanolic acid (35) significantly decreased microsomal DGAT1 activity and, in parallel, inhibited the extent of triglyceride biosynthesis in PON2-deficient MPM. Previous studies indicated that DGAT1 is predominantly regulated posttranscriptionally (37)(38)(39)(40). Our results clearly suggest that in PON2-deficient macrophages, although DGAT1 mRNA and protein levels were similar to those observed in control MPM, DGAT1 is activated.…”

Thematic Review Series: Glycerolipids. DGAT enzymes and triacylglycerol biosynthesis

“…Levels of DGAT1 and DGAT2 mRNA increase markedly during the differentiation of NIH 3T3-L1 fibroblasts into adipocytes, corresponding to a large increase in DGAT activity (22,36,87,88). These findings indicate a robust upregulation of DGAT1 and DGAT2 during adipogenesis and suggest the involvement of C/EBPa or PPARg, transcription factors that control the expression of many genes in adipogenesis.…”

“…However, when this site was mutated in human DGAT1 (a T316F mutation) and expressed in cells, no effects on in vitro DGAT activity or TG synthesis were seen (88). Additional evidence that DGAT activity may be posttranscriptionally regulated comes from observations in 3T3-L1 adipocytes, in which levels of DGAT1 or DGAT2 mRNA are disproportionate to changes in DGAT activity during adipogensis (22,36,88). Allosteric regulation is found in the DGAT1-related ACAT enzymes (107) but has not been demonstrated for either DGAT.…”

Mitochondrial glycerol-3-phosphate acyltransferase-1 directs the metabolic fate of exogenous fatty acids in hepatocytes

“…Smaller changes were observed with stable mtGPAT1 overexpression in CHO cells, where a 3.8-fold increase in mtGPAT specific activity resulted in a 16% increase in total labeled glycerolipids (25). Fatty acid entry into cellular lipids also increases with overexpression of other acyltransferases, such as AGPAT1 activity in 3T3-L1 adipocytes and C 2 C 12 myotubes (39), and DGAT-1 in 3T3-L1 adipocytes (51). The only exception to this pattern of increased fatty acid uptake is a study in which stable overexpression of DGAT-1 activity in SV40-transformed human lung fibroblasts caused a marked decrease in cell proliferation and thus phospholipid synthesis (4).…”