Overexpression of DGAT1 in 3T3-L1 Cells by Lentiviral Transduction

Abstract

Adipocytes function as the major storage site for triacylglyerol (TAG) and have important roles as active endocrine cells. It is widely assumed that the endocrine function of fat cells is determined by their size. As adipocytes increase in size, they tend to release more pro-inflammatory secretions leading to the recruitment of macrophages, setting up a vicious cycle of inflammation. The aim of this study was to genetically manipulate the size of fat cells in culture to determine the effect on inflammatory behaviour. The size of adipocytes may be manipulated in vitro by increasing TAG storage in 3T3-L1 cells. Acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) catalyses the final and only committed step of TAG synthesis. Therefore key strategies in this study were to achieve overexpression of DGAT1 in 3T3-L1 preadipocytes, assess the resulting phenotype following differentiation, and to examine the adipocyte response to inflammatory stimuli as well as interaction with RAW 264.7 macrophages. Interactions between the cells were simulated by incubating the 3T3- L1 adipocytes with secretions collected from RAW 264.7 macrophages and vice versa. DGAT1 was overexpressed in 3T3-L1 preadipocytes via recombinant lentivirus and resulted in a more than 16-fold increase in mRNA compared to control cells transduced with an “empty” reporter lentiviral vector. Prior to differentiation, the DGAT1 overexpressing preadipocytes showed elevated intracellular TAG accumulation and the formation of distinct lipid droplets, whereas the control cells did not demonstrate this behaviour. After differentiation, the increase in lipid was unexpectedly not maintained, characterised by a lower rate of lipogenesis and smaller adipocyte size. Assessment of adipocyte interactions with RAW 264.7 macrophages revealed both the recombinant DGAT1 and control vectors were associated with elevated inflammatory gene expression in response to macrophage secretions. Similarly, exposure to adipocyte secretions obtained from both populations of the transduced 3T3-L1 cells led to increased RAW 264.7 macrophage activation compared to secretory factors collected from non-transduced adipocytes. Genetic manipulation of 3T3-L1 cells by lentiviral transduction led to elevated inflammation which appeared to be due to activation of an immune response to the vector. Overexpression via the recombinant DGAT1 vector resulted in an unexpected reduction in adipocyte size which did not occur in the transduced control cells despite both populations having an impaired inflammatory profile. This loss of adipogenic behaviour was likely attributable to interference with the complex differentiation process due to an excessively high gene expression load, rather than the DGAT1 transgene itself. These results emphasise the need to exercise caution when using genetic manipulation techniques with the 3T3-L1 cell line, particularly because changes in inflammatory profile can occur without alterations in observable traits and may therefore be difficult to detect.