Overexpressed FATP1, ACSVL4/FATP4 and ACSL1 Increase the Cellular Fatty Acid Uptake of 3T3-L1 Adipocytes but Are Localized on Intracellular Membranes

Abstract

Long chain acyl-CoA synthetases are essential enzymes of lipid metabolism, and have also been implicated in the cellular uptake of fatty acids. It is controversial if some or all of these enzymes have an additional function as fatty acid transporters at the plasma membrane. The most abundant acyl-CoA synthetases in adipocytes are FATP1, ACSVL4/FATP4 and ACSL1. Previous studies have suggested that they increase fatty acid uptake by direct transport across the plasma membrane. Here, we used a gain-of-function approach and established FATP1, ACSVL4/FATP4 and ACSL1 stably expressing 3T3-L1 adipocytes by retroviral transduction. All overexpressing cell lines showed increased acyl-CoA synthetase activity and fatty acid uptake. FATP1 and ACSVL4/FATP4 localized to the endoplasmic reticulum by confocal microscopy and subcellular fractionation whereas ACSL1 was found on mitochondria. Insulin increased fatty acid uptake but without changing the localization of FATP1 or ACSVL4/FATP4. We conclude that overexpressed acyl-CoA synthetases are able to facilitate fatty acid uptake in 3T3-L1 adipocytes. The intracellular localization of FATP1, ACSVL4/FATP4 and ACSL1 indicates that this is an indirect effect. We suggest that metabolic trapping is the mechanism behind the influence of acyl-CoA synthetases on cellular fatty acid uptake.

Pathways commonly dysregulated in mouse and human obese adipose tissue: FAT/CD36 modulates differentiation and lipogenesis

“…42 This mitochondrial protein directs fatty acids toward b oxidation in adipocytes 43 synergistic to CD36-mediated plasma membrane transport. 44 In 3T3L1 adipocytes, ACSL1 increases fatty acid uptake 18 and promotes lipogenic gene expression. 45 The hormone sensitive lipase LIPE is the major mediator of lipolysis induced by b adrenergic stimulation in adipocytes 46 and gene polymorphisms are linked to abdominal obesity.…”

“…6 Both adipogenesis and lipogenesis are stimulated by insulin which increases adipocyte cell size 7 through transcriptional inhibition of apoptosis and activation of lipid droplet synthesis 8 but also controls the transcription and/or secretion of numerous secreted factors such as adiponectin. 9 Lipid uptake by adipocytes is performed through interaction with intracellular fatty acid binding proteins FABP1 and FABP4, with fatty acid translocase FAT/CD36, lipoprotein receptor heparan sulfate proteoglycans HSPG but also G-protein coupled receptors, then accumulation as triglycerides in lipid droplets, [10][11][12][13][14][15][16][17][18] however their respective functions in fatty acid storage and intracellular signaling need further investigations.…”

Acyl-CoA Metabolism and Partitioning

“…Alternatively, studies of FA uptake may be an exception to the idea that location dictates function, because changing the expression level of intracellular ACSLs or FATPs alters cellular FA retention (47, 133, 207). In 3T3-L1 cells, for example, overexpressing FATP1 or FATP4 on the ER or overexpressing ACSL1 on the mitochondria increases FA uptake and retention by 40% (207).…”

“…In 3T3-L1 cells, for example, overexpressing FATP1 or FATP4 on the ER or overexpressing ACSL1 on the mitochondria increases FA uptake and retention by 40% (207). This increase in FA uptake may have occurred because of the altered FA or acyl-CoA concentration gradient as intracellular FAs are converted to acyl-CoAs, or because the addition of the CoA has trapped the FAs within the cell.…”

“…Whether these proteins have separate transport and activation functions remains controversial (52, 77). FATPs that are not located on the plasma membrane are thought to enhance FA uptake into cells because the FAs become trapped intracellularly as acyl-CoAs, whose subsequent metabolism ensures that uptake is unidirectional (207). The outlier in this family is FATP5, a bile acid-CoA synthetase that does not activate fatty acids (70, 184).…”