An alternative membrane topology permits lipid droplet localization of peroxisomal fatty acyl-CoA reductase 1.

Fatty acyl-CoA reductase 1 (Far1) is a ubiquitously expressed peroxisomal membrane protein that generates the fatty alcohols required for the biosynthesis of ether lipids. Lipid droplet localization of exogenously expressed and endogenous human Far1 was observed by fluorescence microscopy under conditions of increased triglyceride synthesis in tissue culture cells. This unexpected finding was supported further by correlative light electron microscopy and subcellular fractionation. Selective permeabilization, protease sensitivity and N-glycosylation tagging suggested that Far1 is able to assume two different membrane topologies, differing in the orientation of the short hydrophilic C-terminus towards the lumen or the cytosol, respectively. Two closely spaced hydrophobic domains are contained within the C-terminal region. When analyzed separately, the second domain was sufficient for the localization of a fluorescent reporter to lipid droplets. Targeting of Far1 to lipid droplets was not impaired in either Pex19 or ASNA1 (also known as TRC40) CRISPR/Cas9 knockout cells. In conclusion, our data suggest that Far1 is a novel member of the rather exclusive group of dual topology membrane proteins. At the same time, Far1 shows lipid metabolism-dependent differential subcellular localizations to peroxisomes and lipid droplets.

Lipid droplet quantification based on iterative image processing.

Lipid droplets (LDs) are ubiquitous and highly dynamic subcellular organelles required for the storage of neutral lipids. LD number and size distribution are key parameters affected not only by nutrient supply but also by lipotoxic stress and metabolic regulation. Current methods for LD quantification lack general applicability and are either based on time consuming manual evaluation or show limitations if LDs are high in numbers or closely clustered. Here, we present an ImageJ-based approach for the detection and quantification of LDs stained by neutral lipid dyes in images acquired by conventional wide-field fluorescence microscopy. The method features an adjustable preprocessing procedure that resolves LD clusters. LD identification is based on their circular edges and central fluorescence intensity maxima. Adaptation to different cell types is mediated by a set of interactive parameters. Validation was done for three different cell lines using manual evaluation of LD numbers and volume measurement by 3D rendering of confocal datasets. In an application example, we show that overexpression of the acyl-CoA synthetase, FATP4/ACSVL5, in oleate-treated COS7 cells increased the size of LDs but not their number.

The metabolic capacity of lipid droplet localized acyl-CoA synthetase 3 is not sufficient to support local triglyceride synthesis independent of the endoplasmic reticulum in A431 cells.

ACSL3 is the only long chain fatty acyl-CoA synthetase consistently found on growing and mature lipid droplets (LDs), suggesting that this specific localization has biological relevance. Current models for LD growth propose that triglycerides are synthesized by enzymes at the LD surface, with activated fatty acids provided by LD localized ACSL3, thus allowing growth independent of the ER. Here, we tested this hypothesis by quantifying ACSL3 on LDs from human A431 cells. RNAi of ACSL3 reduced the oleoyl-CoA synthetase activity by 83%, suggesting that ACSL3 is by far the dominant enzyme of A431 cells. Molar quantification revealed that there are 1.4 million ACSL3 molecules within a single cell. Metabolic labeling indicated that each ACSL3 molecule contributed a net gain of 3.1 oleoyl-CoA/s. 3D reconstruction of confocal images demonstrated that 530 individual lipid droplets were present in an average oleate fed A431 cell. A representative single lipid droplet with a diameter of 0.66 µm contained 680 ACSL3 molecules on the surface. Subcellular fractionation showed that at least 68% of ACSL3 remain at the ER even during extensive fatty acid supplementation. High resolution single molecule microscopy confirmed the abundance of cytoplasmic ACSL3 outside of LDs. Model calculations for triglyceride synthesis using only LD localized ACSL3 gave significant slower growth of LDs as observed experimentally. In conclusion, although ACSL3 is an abundant enzyme on A431 LDs, the metabolic capacity is not sufficient to account for LD growth solely by the local synthesis of triglycerides.

Differentially localized acyl-CoA synthetase 4 isoenzymes mediate the metabolic channeling of fatty acids towards phosphatidylinositol.

The acyl-CoA synthetase 4 (ACSL4) has been implicated in carcinogenesis and neuronal development. Acyl-CoA synthetases are essential enzymes of lipid metabolism, and ACSL4 is distinguished by its preference for arachidonic acid. Two human ACSL4 isoforms arising from differential splicing were analyzed by ectopic expression in COS cells. We found that the ACSL4_v1 variant localized to the inner side of the plasma membrane including microvilli, and was also present in the cytosol. ACSL4_v2 contains an additional N-terminal hydrophobic region; this isoform was located at the endoplasmic reticulum and on lipid droplets. A third isoform was designed de novo by appending a mitochondrial targeting signal. All three ACSL4 variants showed the same specific enzyme activity. Overexpression of the isoenzymes increased cellular uptake of arachidonate to the same degree, indicating that the metabolic trapping of fatty acids is independent of the subcellular localization. Remarkably, phospholipid metabolism was changed by ACSL4 expression. Labeling with arachidonate showed that the amount of newly synthesized phosphatidylinositol was increased by all three ACSL4 isoenzymes but not by ACSL1. This was dependent on the expression level and the localization of the ACSL4 isoform. We conclude that in our model system exogenous fatty acids are channeled preferentially towards phosphatidylinositol by ACSL4 overexpression. The differential localization of the endogenous isoenzymes may provide compartment specific precursors of this anionic phospholipid important for many signaling processes.

Protein mediated fatty acid uptake: synergy between CD36/FAT-facilitated transport and acyl-CoA synthetase-driven metabolism.

The mechanism of cellular fatty acid uptake is highly relevant for basic and clinical research. Previous work has demonstrated that fatty acid uptake is facilitated by cell surface membrane proteins as well as by intracellularly localized enzymes. Here, the exogenous expression of the CD36/FAT glycoprotein and the acyl-CoA synthetases FATP4 and ACSL1 in MDCK cells was quantified by comparison to recombinant proteins, and related to the corresponding increases of fatty acid uptake. At the molecular level, CD36/FAT was 30-fold more efficient than either FATP4 or ACSL1 in enhancing fatty acid uptake. Remarkably, co-expression of CD36/FAT with FATP4 or ACSL1 led to a higher increase of fatty acid uptake than expected from the combined individual contributions, whereas co-expression of FATP4 and ACSL1 did not. Immunofluorescence microscopy confirmed the plasma membrane localization of CD36/FAT and the intracellular localization of FATP4 to the endoplasmic reticulum, and of ACSL1 to mitochondria. Concluding, we suggest that fatty acid uptake in our model system is organized by two spatially distinct but synergistic mechanisms: the cell surface protein CD36/FAT directly facilitates fatty acid transport across the plasma membrane, whereas the intracellular acyl-CoA synthetases FATP4 and ACSL1 enhance fatty acid uptake indirectly by metabolic trapping.

Acyl-CoA synthetase expression in human skeletal muscle is reduced in obesity and insulin resistance.

Upon intramuscular entry, fatty acids are converted to amphiphatic fatty acyl-CoAs by action of the acyl-CoA synthetase (ACS) enzymes. While it has been reported that insulin resistant skeletal muscle shows an accumulation of fatty acyl-CoAs, the role of the enzymes which catalyze their synthesis is still sparsely studied in human muscle, in particular the influence of obesity, and insulin resistance. We analyzed muscle biopsies obtained from normal weight controls (n = 7, average BMI 24), males/females with obesity (n = 7, average BMI 31), and males/females with obesity and type 2 diabetes (T2D) (n = 7, average BMI 34), for relevant ACS (long-chain acyl-CoA synthetase 1 (ACSL1), -3 (ACSL3) and - 4 (ACSL4), fatty acid transport protein 1 (FATP1) and - 4 (FATP4)). The mRNA expression was determined by real-time PCR, and total oleoyl-CoA synthetase activity was measured. In the males/females with obesity and T2D, the response to 16 weeks of exercise training with minor weight loss was evaluated. ACSL1 is the dominantly expressed ACS isoform in human skeletal muscle. The content of total ACS mRNA, as well as ACSL1 mRNA, were lower in muscle of males/females with obesity and T2D. Exercise training in the males/females with obesity and T2D increased the total ACS enzyme activity, along with a lowering of the HOMA-IR index. The capacity for synthesis of fatty acyl-CoAs is lower in skeletal muscle of obese males/females with T2D. This suggests a decreased ability to convert fatty acids to fatty acyl-CoAs, which in turn may affect their entry into storage or metabolic pathways in muscle. Thus, the accumulation of fatty acyl-CoAs in the obese or insulin resistant state that has been shown in previous reports is not likely to result from increased fatty acid acylation. The upregulation of ACS activity by exercise training appears beneficial and occurred concomitantly with increased insulin sensitivity.

The N-terminal region of acyl-CoA synthetase 3 is essential for both the localization on lipid droplets and the function in fatty acid uptake.

Cytosolic lipid droplets (LDs) are storage organelles for neutral lipids derived from endogenous metabolism. Acyl-CoA synthetase family proteins are essential enzymes in this biosynthetic pathway, contributing activated fatty acids. Fluorescence microscopy showed that ACSL3 is localized to the endoplasmic reticulum (ER) and LDs, with the distribution dependent on the cell type and the supply of fatty acids. The N-terminus of ACSL3 was necessary and sufficient for targeting reporter proteins correctly, as demonstrated by subcellular fractionation and confocal microscopy. The N-terminal region of ACSL3 was also found to be functionally required for the enzyme activity. Selective permeabilization and in silico analysis suggest that ACSL3 assumes a hairpin membrane topology, with the N-terminal hydrophobic amino acids forming an amphipathic helix restricted to the cytosolic leaflet of the ER membrane. ACSL3 was effectively translocated from the ER to nascent LDs when neutral lipid synthesis was stimulated by the external addition of fatty acids. Cellular fatty acid uptake was increased by overexpression and reduced by RNA interference of ACSL3. In conclusion, the structural organization of ACSL3 allows the fast and efficient movement from the ER to emerging LDs. ACSL3 not only esterifies fatty acids with CoA but is also involved in the cellular uptake of fatty acids, presumably indirectly by metabolic trapping. The unique localization of the acyl-CoA synthetase ACSL3 on LDs suggests a function in the local synthesis of lipids.

Measurement of Long-Chain Fatty Acyl-CoA Synthetase Activity.

Long-chain fatty acyl-CoA synthetases (ACS) are a family of essential enzymes of lipid metabolism, activating fatty acids by thioesterification with coenzyme A. Fatty acyl-CoA molecules are then readily utilized for the biosynthesis of storage and membrane lipids, or for the generation of energy by ß-oxidation. Acyl-CoAs also function as transcriptional activators, allosteric inhibitors, or precursors for inflammatory mediators. Recent work suggests that ACS enzymes may drive cellular fatty acid uptake by metabolic trapping, and may also regulate the channeling of fatty acids towards specific metabolic pathways. The implication of ACS enzymes in widespread lipid associated diseases like type 2 diabetes has rekindled interest in this protein family. Here, we describe in detail how to measure long-chain fatty acyl-CoA synthetase activity by a straightforward radiometric assay. Cell lysates are incubated with ATP, coenzyme A, Mg(2+), and radiolabeled fatty acid bound to BSA. Differential phase partitioning of fatty acids and acyl-CoAs is exploited to quantify the amount of generated acyl-CoA by scintillation counting. The high sensitivity of this assay also allows the analysis of small samples like patient biopsies.

Outlook: membrane junctions enable the metabolic trapping of fatty acids by intracellular acyl-CoA synthetases.

The mechanism of fatty acid uptake is of high interest for basic research and clinical interventions. Recently, we showed that mammalian long chain fatty acyl-CoA synthetases (ACS) are not only essential enzymes for lipid metabolism but are also involved in cellular fatty acid uptake. Overexpression, RNAi depletion or hormonal stimulation of ACS enzymes lead to corresponding changes of fatty acid uptake. Remarkably, ACS are not localized to the plasma membrane where fatty acids are entering the cell, but are found instead at the endoplasmic reticulum (ER) or other intracellular organelles like mitochondria and lipid droplets. This is in contrast to current models suggesting that ACS enzymes function in complex with transporters at the cell surface. Drawing on recent insights into non-vesicular lipid transport, we suggest a revised model for the cellular fatty acid uptake of mammalian cells which incorporates trafficking of fatty acids across membrane junctions. Intracellular ACS enzymes are then metabolically trapping fatty acids as acyl-CoA derivatives. These local decreases in fatty acid concentration will unbalance the equilibrium of fatty acids across the plasma membrane, and thus provide a driving force for fatty acid uptake.

Overexpressed FATP1, ACSVL4/FATP4 and ACSL1 increase the cellular fatty acid uptake of 3T3-L1 adipocytes but are localized on intracellular membranes.

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.