Grease on-Perspectives in lipid droplet biology.

Lipid droplets (LDs) are now recognized as omnipresent and dynamic subcellular organelles of amazing morphological and functional diversity. Beyond the obvious benefit of having molecules full of chemical energy stored in a dedicated structural entity, LDs may also be viewed as a safe harbor for potentially damaging metabolites. This protective function might in many cases even supersede the relevance of lipid storage for eventual energy gain and membrane biogenesis. Furthermore, the LD surface constitutes a unique membrane environment, creating a platform for hosting specific proteins and thus enabling their interactions. These metabolic hotspots would contribute decisively to compartmentalized metabolism in the cytosol. LDs are also communicating extensively with other subcellular organelles in directing and regulating lipid metabolism. Deciphering the relevance of LD storage and regulation at the organismic level will be essential for the understanding of widespread and serious metabolic complications in humans. Increasing attention is also devoted to pathogens appropriating LDs for their own benefit. LD biology is still considered an emerging research area in rapid and vibrant development, attracting scientists from all disciplines of the life sciences and beyond, which is mirrored by the accompanying review collection. Here, we present our personal views on areas we believe are especially exciting and hold great potential for future developments. Particularly, we address issues relating to LD biogenesis and heterogeneity, required technological advances, and the complexity of human physiology.

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.

Deficiency of acyl-CoA synthetase 5 is associated with a severe and treatable failure to thrive of neonatal onset.

Failure to thrive (FTT) causes significant morbidity, often without clear etiologies. Six individuals of a large consanguineous family presented in the neonatal period with recurrent vomiting and diarrhea, leading to severe FTT. Standard diagnostic work up did not ascertain an etiology. Autozygosity mapping and whole exome sequencing identified homozygosity for a novel genetic variant of the long chain fatty acyl-CoA synthetase 5 (ACSL5) shared among the affected individuals (NM_203379.1:c.1358C>A:p.(Thr453Lys)). Autosomal recessive genotype-phenotype segregation was confirmed by Sanger sequencing. Functional in vitro analysis of the ACSL5 variant by immunofluorescence, western blotting and enzyme assay suggested that Thr453Lys is a loss-of-function mutation without any remaining activity. ACSL5 belongs to an essential enzyme family required for lipid metabolism and is known to contribute the major activity in the mouse intestine. Based on the function of ACSL5 in intestinal long chain fatty acid metabolism and the gastroenterological symptoms, affected individuals were treated with total parenteral nutrition or medium-chain triglyceride-based formula restricted in long-chain triglycerides. The patients responded well and follow up suggests that treatment is only required during early life.

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.

Long-chain acyl-CoA synthetase 1 interacts with key proteins that activate and direct fatty acids into niche hepatic pathways.

Fatty acid channeling into oxidation or storage modes depends on physiological conditions and hormonal signaling. However, the directionality of this channeling may also depend on the association of each of the five acyl-CoA synthetase isoforms with specific protein partners. Long-chain acyl-CoA synthetases (ACSLs) catalyze the conversion of long-chain fatty acids to fatty acyl-CoAs, which are then either oxidized or used in esterification reactions. In highly oxidative tissues, ACSL1 is located on the outer mitochondrial membrane (OMM) and directs fatty acids into mitochondria for ß-oxidation. In the liver, however, about 50% of ACSL1 is located on the endoplasmic reticulum (ER) where its metabolic function is unclear. Because hepatic fatty acid partitioning is likely to require the interaction of ACSL1 with other specific proteins, we used an unbiased protein interaction technique, BioID, to discover ACSL1-binding partners in hepatocytes. We targeted ACSL1 either to the ER or to the OMM of Hepa 1-6 cells as a fusion protein with the Escherichia coli biotin ligase, BirA*. Proteomic analysis identified 98 proteins that specifically interacted with ACSL1 at the ER, 55 at the OMM, and 43 common to both subcellular locations. We found subsets of peroxisomal and lipid droplet proteins, tethering proteins, and vesicle proteins, uncovering a dynamic role for ACSL1 in organelle and lipid droplet interactions. Proteins involved in lipid metabolism were also identified, including acyl-CoA-binding proteins and ceramide synthase isoforms 2 and 5. Our results provide fundamental and detailed insights into protein interaction networks that control fatty acid metabolism.

ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition.

Ferroptosis is a form of regulated necrotic cell death controlled by glutathione peroxidase 4 (GPX4). At present, mechanisms that could predict sensitivity and/or resistance and that may be exploited to modulate ferroptosis are needed. We applied two independent approaches-a genome-wide CRISPR-based genetic screen and microarray analysis of ferroptosis-resistant cell lines-to uncover acyl-CoA synthetase long-chain family member 4 (ACSL4) as an essential component for ferroptosis execution. Specifically, Gpx4-Acsl4 double-knockout cells showed marked resistance to ferroptosis. Mechanistically, ACSL4 enriched cellular membranes with long polyunsaturated ω6 fatty acids. Moreover, ACSL4 was preferentially expressed in a panel of basal-like breast cancer cell lines and predicted their sensitivity to ferroptosis. Pharmacological targeting of ACSL4 with thiazolidinediones, a class of antidiabetic compound, ameliorated tissue demise in a mouse model of ferroptosis, suggesting that ACSL4 inhibition is a viable therapeutic approach to preventing ferroptosis-related diseases.

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.

Generation and functional characterization of epithelial cells with stable expression of SLC26A9 Cl- channels.

Recent studies identified the SLC26A9 Cl(-) channel as a modifier and potential therapeutic target in cystic fibrosis (CF). However, understanding of the regulation of SLC26A9 in epithelia remains limited and cellular models with stable expression for biochemical and functional studies are missing. We, therefore, generated Fisher rat thyroid (FRT) epithelial cells with stable expression of HA-tagged SLC26A9 via retroviral transfection and characterized SLC26A9 expression and function using Western blotting, immunolocalization, whole cell patch-clamp, and transepithelial bioelectric studies in Ussing chambers. We demonstrate stable expression of SLC26A9 in transfected FRT (SLC26A9-FRT) cells on the mRNA and protein level. Immunolocalization and Western blotting detected SLC26A9 in different intracellular compartments and to a lesser extent at the cell surface. Whole cell patch-clamp recordings demonstrated significantly increased constitutive Cl(-) currents in SLC26A9-FRT compared with control-transduced FRT (Control-FRT) cells (P < 0.01). Similar, transepithelial measurements showed that the basal short circuit current was significantly increased in SLC26A9-FRT vs. Control-FRT cell monolayers (P < 0.01). SLC26A9-mediated Cl(-) currents were increased by cAMP-dependent stimulation (IBMX and forskolin) and inhibited by GlyH-101, niflumic acid, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), as well as RNAi knockdown of WNK1 implicated in epithelial osmoregulation. Our results support that these novel epithelial cells with stable expression of SLC26A9 will be a useful model for studies of pharmacological regulation including the identification of activators of SLC26A9 Cl(-) channels that may compensate deficient cystic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) secretion and serve as an alternative therapeutic target in patients with CF and potentially other muco-obstructive lung diseases.

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.

Adipocyte-specific inactivation of Acyl-CoA synthetase fatty acid transport protein 4 (Fatp4) in mice causes adipose hypertrophy and alterations in metabolism of complex lipids under high fat diet.

Fatp4 exhibits acyl-CoA synthetase activity and is thereby able to catalyze the activation of fatty acids for further metabolism. However, its actual function in most tissues remains unresolved, and its role in cellular fatty acid uptake is still controversial. To characterize Fatp4 functions in adipocytes in vivo, we generated a mouse line with adipocyte-specific inactivation of the Fatp4 gene (Fatp4(A-/-)). Under standard conditions mutant mice showed no phenotypical aberrance. Uptake of radiolabeled palmitic and lignoceric acid into adipose tissue of Fatp4(A-/-) mice was unchanged. When exposed to a diet enriched in long chain fatty acids, Fatp4(A-/-) mice gained more body weight compared with control mice, although they were not consuming more food. Pronounced obesity was accompanied by a thicker layer of subcutaneous fat and greater adipocyte circumference, although expression of genes involved in de novo lipogenesis was not changed. However, the increase in total fat mass was contrasted by a significant decrease in various phospholipids, sphingomyelin, and cholesteryl esters in adipocytes. Livers of Fatp4-deficient animals under a high fat diet exhibited a higher degree of fatty degeneration. Nonetheless, no evidence for changes in insulin sensitivity and adipose inflammation was found. In summary, the results of this study confirm that Fatp4 is not crucial for fatty acid uptake into adipocytes. Instead, under the condition of a diet enriched in long chain fatty acids, adipocyte-specific Fatp4 deficiency results in adipose hypertrophy and profound alterations in the metabolism of complex lipids.

Rer1p competes with APH-1 for binding to nicastrin and regulates gamma-secretase complex assembly in the early secretory pathway.

The gamma-secretase complex, consisting of presenilin, nicastrin, presenilin enhancer-2 (PEN-2), and anterior pharynx defective-1 (APH-1) cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. The regulatory mechanisms governing the multistep assembly of this "proteasome of the membrane" are unknown. We characterize a new interaction partner of nicastrin, the retrieval receptor Rer1p. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 substantially increased binding of nicastrin to Rer1p, demonstrating the competitive nature of these interactions. Moreover, Rer1p expression levels control the formation of gamma-secretase subcomplexes and, concomitantly, total cellular gamma-secretase activity. We identify Rer1p as a novel limiting factor that negatively regulates gamma-secretase complex assembly by competing with APH-1 during active recycling between the endoplasmic reticulum (ER) and Golgi. We conclude that total cellular gamma-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value.

Phosphatidylcholine as a constituent in the colonic mucosal barrier--physiological and clinical relevance.

Phosphatidylcholine (PC) is an important constituent of the gastrointestinal tract. PC molecules are not only important in intestinal cell membranes but also receiving increasing attention as protective agents in the gastrointestinal barrier. They are largely responsible for establishing the hydrophobic surface of the colon. Decreased phospholipids in colonic mucus could be linked to the pathogenesis of ulcerative colitis, a chronic inflammatory bowel disease. Clinical studies revealed that therapeutic addition of PC to the colonic mucus of these patients alleviated the inflammatory activity. This positive role is still elusive, however, we hypothesized that luminal PC has two possible functions: first, it is essential for surface hydrophobicity, and second, it is integrated into the plasma membrane of enterocytes and it modulates the signaling state of the mucosa. The membrane structure and lipid composition of cells is a regulatory component of the inflammatory signaling pathways. In this perspective, we will shortly summarize what is known about the localization and protective properties of PC in the colonic mucosa before turning to its evident medical importance. We will discuss how PC contributes to our understanding of the pathogenesis of ulcerative colitis and how reinforcing the luminal phospholipid monolayer can be used as a therapeutic concept in humans.

Silybin and dehydrosilybin decrease glucose uptake by inhibiting GLUT proteins.

Silybin, the major flavonoid of Silybum marianum, is widely used to treat liver diseases such as hepatocellular carcinoma and cirrhosis-associated insulin resistance. Research so far has focused on its anti-oxidant properties. Here, we demonstrate that silybin and its derivative dehydrosilybin inhibit glucose uptake in several model systems. Both flavonoids dose-dependently reduce basal and insulin-dependent glucose uptake of 3T3-L1 adipocytes, with dehydrosilybin showing significantly stronger inhibition. However, insulin signaling was not impaired, and immunofluorescence and subcellular fractionation showed that insulin-induced translocation of GLUT4 to the plasma membrane is also unchanged. Likewise, hexokinase activity was not affected suggesting that silybin and dehydrosilybin interfere directly with glucose transport across the PM. Expression of GLUT4 in CHO cells counteracted the inhibition of glucose uptake by both flavonoids. Moreover, treatment of CHO cells with silybin and dehydrosilybin reduced cell viability which was partially rescued by GLUT4 expression. Kinetic analysis revealed that silybin and dehydrosilybin inhibit GLUT4-mediated glucose transport in a competitive manner with K(i)=60 and 116 µM, respectively. We conclude that silybin and dehydrosilybin inhibit cellular glucose uptake by directly interacting with GLUT transporters. Glucose starvation offers a novel explanation for the anti-cancer effects of silybin.

FATP4 contributes as an enzyme to the basal and insulin-mediated fatty acid uptake of C2C12 muscle cells.

The function of membrane proteins in long-chain fatty acid transport is controversial. The acyl-CoA synthetase fatty acid transport protein-4 (FATP4) has been suggested to facilitate fatty acid uptake indirectly by its enzymatic activity, or directly by transport across the plasma membrane. Here, we investigated the function of FATP4 in basal and insulin mediated fatty acid uptake in C(2)C(12) muscle cells, a model system relevant for fatty acid metabolism. Stable expression of exogenous FATP4 resulted in a twofold higher fatty acyl-CoA synthetase activity, and cellular uptake of oleate was enhanced similarly. Kinetic analysis demonstrated that FATP4 allowed the cells to reach apparent saturation of fatty acid uptake at a twofold higher level compared with control. Short-term treatment with insulin increased fatty acid uptake in line with previous reports. Surprisingly, insulin increased the acyl-CoA synthetase activity of C(2)C(12) cells within minutes. This effect was sensitive to inhibition of insulin signaling by wortmannin. Affinity purified FATP4 prepared from insulin-treated cells showed an enhanced enzyme activity, suggesting it constitutes a novel target of short-term metabolic regulation by insulin. This offers a new mechanistic explanation for the concomitantly observed enhanced fatty acid uptake. FATP4 was colocalized to the endoplasmic reticulum by double immunofluorescence and subcellular fractionation, clearly distinct from the plasma membrane. Importantly, neither differentiation into myotubes nor insulin treatment changed the localization of FATP4. We conclude that FATP4 functions by its intrinsic enzymatic activity. This is in line with the concept that intracellular metabolism plays a significant role in cellular fatty acid uptake.

Overexpression of CD36 and acyl-CoA synthetases FATP2, FATP4 and ACSL1 increases fatty acid uptake in human hepatoma cells.

BACKGROUND: Understanding the mechanisms of long chain fatty acid (LCFA) uptake in hepatic cells is of high medical importance to treat and to prevent fatty liver disease (FLD). ACSs (Acyl-CoA synthetases) are a family of enzymes that catalyze the esterification of fatty acids (FA) with CoA. Recent studies suggest that ACS enzymes drive the uptake of LCFA indirectly by their enzymatic activity and could promote special metabolic pathways dependent on their localization.The only protein located at the plasma membrane which has consistently been shown to enhance FA uptake is CD36. AIMS: The current study investigated whether ACSs and CD36 could regulate hepatic LCFA uptake. METHODS AND RESULTS: FATP2 and FATP4 were both localized to the ER of HuH7 and HepG2 cells as shown by double immunofluorescence in comparison to marker proteins. ACSL1 was located at mitochondria in both cell lines. Overexpression of FATP2, FATP4 and ACSL1 highly increased ACS activity as well as the uptake of [3H]-oleic acid and fluorescent Bodipy-C12 (B12) fatty acid. Quantitative FACS analysis showed a correlation between ACS expression levels and B12 uptake. FATP2 had the highest effect on B12 uptake of all proteins tested. CD36 was mainly localized at the plasma membrane. Whereas [3H]-oleic acid uptake was increased after overexpression, CD36 had no effect on B12 uptake. CONCLUSION: Uptake of LCFA into hepatoma cells can be regulated by the expression levels of intracellular enzymes. We propose that ACS enzymes drive FA uptake indirectly by esterification. Therefore these molecules are potential targets for treatment of nonalcoholic fatty liver disease (NAFLD) or steatohepatitis (NASH).

Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells.

Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)-binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.

Genetic analysis of BIRC4/XIAP as a putative modifier gene of Wilson disease.

Wilson disease (WD) is an autosomal-recessive copper overload disorder caused by mutations in the copper-transporting adenosine triphosphatase (ATPase) ATP7B. It presents with a highly variable clinical phenotype ranging from asymptomatic to fulminant hepatic failure or progressive neurological involvement. No clear genotype-phenotype correlation has been established. Thus, variants in modifier genes could have an impact on WD manifestation and severity. Recently, the antiapoptotic protein baculoviral IAP repeat-containing protein 4 BIRC4/XIAP has been suggested as a regulator of copper-induced cell death. With the aim of investigating a putative role of BIRC4/XIAP as modifier gene in individuals with copper overload, we analyzed a WD patient cohort (n = 98) for sequence variants at the BIRC4/XIAP locus. When compared with clinical data, the previously described coding single nucleotide polymorphisms (SNPs) at the BRIC4/XIAP locus (rs28382721, rs28382722, rs28382723, rs5956583, rs28382740, rs12838858, rs28382741) did not correlate with age of onset or clinical presentation in our collective. However, three previously unreported variants in the BIRC4/XIAP gene were identified (c.1-26 T > G; c.1408A > T; p.T470S; c.1019A > G; p.N340S). The two patients with variants leading to amino acid exchanges in the BIRC4/XIAP protein showed a remarkably early disease onset at the age of 5 years. Furthermore, one of these patients was only heterozygous for disease-causing mutations in the ATP7B gene. In summary, these data emphasize the need to further elucidate a role of BIRC4/XIAP variants as putative pathogenetic factors in copper overload disorders.