Cellular lipid binding proteins as facilitators and regulators of lipid metabolism

Evidence is accumulating that cellular lipid binding proteins are playing central roles in cellular lipid uptake and metabolism. Membrane-associated fatty acid-binding proteins putatively function in protein-mediated transmembrane transport of fatty acids, likely coexisting with passive diffusional uptake. The intracellular trafficking of fatty acids, bile acids, and other lipid ligands, may involve their interaction with specific membrane or protein targets, which are unique properties of some but not of all cytoplasmic lipid binding proteins. Recent studies indicate that these proteins not only facilitate but also regulate cellular lipid utilization. For instance, muscle fatty acid uptake is subject to short-term regulation by translocation of fatty acid translocase (FAT)/CD36 from intracellular storage sites to the plasma membrane, and liver-type cytoplasmic fatty acid-binding protein (L-FABP_c) functions in long-term, ligand-induced regulation of gene expression by directly interacting with nuclear receptors. Therefore, the properties of the lipid-protein complex, rather than those of the lipid ligand itself, determine the fate of the ligand in the cell. Finally, there are an increasing number of reports that deficiencies or altered functioning of both membrane-associated and cytoplasmic lipid binding proteins are associated with disease states, such as obesity, diabetes and atherosclerosis. In conclusion, because of their central role in the regulation of lipid metabolism, cellular lipid binding proteins are promising targets for the treatment of diseases resulting from or characterised by disturbances in lipid metabolism, such as atherosclerosis, hyperlipidemia, and insulin resistance.     

Fatty acid metabolism of isolated mammalian cells

It is now clear that a wide variety of differentiated cells in culture exhibit essentially the full spectrum of mammalian fatty acid metabolism. These cells readily incorporate free fatty acids into membrane phosphoglycerides, modify exogenous fatty acids by desaturation and elongation, and store excess fatty acyl groups, primarily as triacylglycerols. Similarly, many different types of cells synthesize cyclooxygenase and lipoxygenase derivatives of long chain polyunsaturated fatty acids. Furthermore, although the fatty acid composition of cellular phospholipids can be modified by medium supplementation, cells in culture exhibit definite fatty acyl specificities for the various steps of fatty acid activation, transesterification and release. As the extensive repertoire of fatty acid metabolism in mammalian cells has been elucidated, and as the ability to grow differentiated cells in culture has increased, new questions have arisen. There is still much to be learned about the enzymes involved in synthesizing and maintaining the unique fatty acid composition of the different cellular phospholipids and the processes which regulate the desaturation, elongation and retroconversion of polyunsaturated fatty acids. Other areas of great current interest are the mechanisms by which certain long chain polyunsaturated fatty acids are made available for conversion to oxygenated, biologically-active derivatives, the metabolic interactions between different polyunsaturated fatty acids, particularly n-3 and n-6 fatty acids, the cellular roles of the C22 polyunsaturated fatty acids, and the functions of particular molecular species of phospholipids in membrane-mediated events. Further research in these areas will contribute to unravelling the role of fatty acids and fatty acid derivatives in the physiological processes of mammalian cells.     

Translocation of long chain fatty acids across the plasma membrane – lipid rafts and fatty acid transport proteins

Translocation of long chain fatty acids across the plasma membrane is achieved by a concert of co-existing mechanisms. These lipids can passively diffuse, but transport can also be accelerated by certain membrane proteins as well as lipid rafts. Lipid rafts are dynamic assemblies of proteins and lipids, that float freely within the two dimensional matrix of the membrane bilayer. They are receiving increasing attention as devices that regulate membrane function in vivo and play an important role in membrane trafficking and signal transduction. In this review we will discuss how lipid rafts might be involved in the uptake process and how the candidate proteins for fatty acid uptake FAT/CD36 and the FATP proteins interact with these domains. We will also discuss the functional role of FATPs in general. To our understanding FATPs are indirectly involved in the translocation process across the plasma membrane by providing long chain fatty acid synthetase activity.     

Comparative biochemical studies of the murine fatty acid transport proteins (FATP) expressed in yeast

The fatty acid transport protein (FATP) family is a group of proteins that are predicted to be components of specific fatty acid trafficking pathways. In mammalian systems, six different isoforms have been identified, which function in the import of exogenous fatty acids or in the activation of very long-chain fatty acids. This has led to controversy as to whether these proteins function as membrane-bound fatty acid transporters or as acyl-CoA synthetases, which activate long-chain fatty acids concomitant with transport. The yeast FATP orthologue, Fat1p, is a dual functional protein and is required for both the import of long-chain fatty acids and the activation of very long-chain fatty acids; these activities intrinsic to Fat1p are separable functions. To more precisely define the roles of the different mammalian isoforms in fatty acid trafficking, the six murine proteins (mmFATP1-6) were expressed and characterized in a genetically defined yeast strain, which cannot transport long-chain fatty acids and has reduced long-chain acyl-CoA synthetase activity (fat1Delta faa1Delta). Each isoform was evaluated for fatty acid transport, fatty acid activation (using C18:1, C20:4, and C24:0 as substrates), and accumulation of very long-chain fatty acids. Murine FATP1, -2, and -4 complemented the defects in fatty acid transport and very long-chain fatty acid activation associated with a deletion of the yeast FAT1 gene; mmFATP3, -5, and -6 did not complement the transport function even though each was localized to the yeast plasma membrane. Both mmFATP3 and -6 activated C20:4 and C20:4, while the expression of mmFATP5 did not substantially increase acyl-CoA synthetases activities using the substrates tested. These data support the conclusion that the different mmFATP isoforms play unique roles in fatty acid trafficking, including the transport of exogenous long-chain fatty acids.     

Fat targets for insulin signaling

Adipose cells control flux of fatty acids to peripheral tissues by storing and hydrolyzing triglyceride under hormonal control. New data reveal that insulin may regulate this process in part by promoting membrane trafficking of intracellular fatty acid transporters FATP1 and FATP4 to the plasma membrane.     

Human Fatty Acid Transport Protein 2a/Very Long Chain Acyl-CoA Synthetase 1 (FATP2a/Acsvl1) Has a Preference in Mediating the Channeling of Exogenous n-3 Fatty Acids into Phosphatidylinositol

The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (M_r 70,000) and FATP2b (M_r 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14–C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6). Using the same cells expressing FATP2a or FATP2b, electrospray ionization/MS was used to follow the trafficking of stable isotopically labeled n-3 fatty acids into phosphatidylcholine and phosphatidylinositol. The expression of FATP2a resulted in the trafficking of C18:3-CoA and C22:6-CoA into both phosphatidylcholine and phosphatidylinositol but with a distinct preference for phosphatidylinositol. Collectively these data demonstrate FATP2a functions in fatty acid transport and activation and provides specificity toward n-3 fatty acids in which the corresponding n-3 acyl-CoAs are preferentially trafficked into acyl-CoA pools destined for phosphatidylinositol incorporation.     

Good Fat, Essential Cellular Requirements for Triacylglycerol Synthesis to Maintain Membrane Homeostasis in Yeast

Storage triacylglycerols (TAG) and membrane phospholipids share common precursors, i.e. phosphatidic acid and diacylglycerol, in the endoplasmic reticulum. In addition to providing a biophysically rather inert storage pool for fatty acids, TAG synthesis plays an important role to buffer excess fatty acids (FA). The inability to incorporate exogenous oleic acid into TAG in a yeast mutant lacking the acyltransferases Lro1p, Dga1p, Are1p, and Are2p contributing to TAG synthesis results in dysregulation of lipid synthesis, massive proliferation of intracellular membranes, and ultimately cell death. Carboxypeptidase Y trafficking from the endoplasmic reticulum to the vacuole is severely impaired, but the unfolded protein response is only moderately up-regulated, and dispensable for membrane proliferation, upon exposure to oleic acid. FA-induced toxicity is specific to oleic acid and much less pronounced with palmitoleic acid and is not detectable with the saturated fatty acids, palmitic and stearic acid. Palmitic acid supplementation partially suppresses oleic acid-induced lipotoxicity and restores carboxypeptidase Y trafficking to the vacuole. These data show the following: (i) FA uptake is not regulated by the cellular lipid requirements; (ii) TAG synthesis functions as a crucial intracellular buffer for detoxifying excess unsaturated fatty acids; (iii) membrane lipid synthesis and proliferation are responsive to and controlled by a balanced fatty acid composition.     

Mechanisms and functions of membrane lipid remodeling in plants

Lipid remodeling, defined herein as post-synthetic structural modifications of membrane lipids, play crucial roles in regulating the physicochemical properties of cellular membranes and hence their many functions. Processes affected by lipid remodeling include lipid metabolism, membrane repair, cellular homeostasis, fatty acid trafficking, cellular signaling and stress tolerance. Glycerolipids are the major structural components of cellular membranes and their composition can be adjusted by modifying their head groups, their acyl chain lengths and the number and position of double bonds. This review summarizes recent advances in our understanding of mechanisms of membrane lipid remodeling with emphasis on the lipases and acyltransferases involved in the modification of phosphatidylcholine and monogalactosyldiacylglycerol, the major membrane lipids of extraplastidic and photosynthetic membranes, respectively. We also discuss the role of triacylglycerol metabolism in membrane acyl chain remodeling. Finally, we discuss emerging data concerning the functional roles of glycerolipid remodeling in plant stress responses. Illustrating the molecular basis of lipid remodeling may lead to novel strategies for crop improvement and other biotechnological applications such as bioenergy production.     

The relationship between the fatty acid composition of immune cells and their function

The immune system, including its inflammatory components, is fundamental to host defence against pathogenic invaders. It is a complex system involving interactions amongst many different cell types dispersed throughout the body. Central to its actions are phagocytosis of bacteria, processing of antigens derived from intracellular and extracellular pathogens, activation of T cells with clonal expansion (proliferation) and production of cytokines that elicit effector cell functions such as antibody production and killing cell activity. Inappropriate immunologic activity, including inflammation, is a characteristic of many common human disorders. Eicosanoids produced from arachidonic acid have roles in inflammation and regulation of T and B lymphocyte functions. Eicosapentaenoic acid (EPA) also gives rise to eicosanoids and these may have differing properties from those of arachidonic acid-derived eicosanoids. EPA and docosahexaenoic acid (DHA) give rise to newly discovered resolvins which are anti-inflammatory and inflammation resolving. Human immune cells are typically rich in arachidonic acid, but arachidonic acid, EPA and DHA contents can be altered through oral administration of EPA and DHA. This results in a changed pattern of production of eicosanoids and probably also of resolvins, although the latter are not well examined in the human context. Changing the fatty acid composition of immune cells also affects phagocytosis, T cell signaling and antigen presentation capability. These effects appear to mediated at the membrane level suggesting important roles of fatty acids in membrane order, lipid raft structure and function, and membrane trafficking. Thus, the fatty acid composition of human immune cells influences their function and the cell membrane contents of arachidonic acid, EPA and DHA are important. Fatty acids influence immune cell function through a variety of complex mechanisms and these mechanisms are now beginning to be unraveled.     

Dietary fatty acids and membrane protein function

In recent years, there has been growing public awareness of the potential health benefits of dietary fatty acids, and of the distinction between the effects of the omega6 and omega3 polyunsaturated fatty acids that are concentrated in vegetable and fish oils, respectively. A part of the biologic effectiveness of the two families of polyunsaturated fatty acids resides in their relative roles as precursors of the eicosanoids. However, we are also beginning to appreciate that as the major components of the hydrophobic core of the membrane bilayer, they can interact with and directly influence the functioning of select integral membrane proteins. Among the most important of these are the enzymes, receptors, and ion channels that are situated in the plasma membrane of the cell, since they carry out the communication and homeostatic processes that are necessary for normal cell function. This review examines current information regarding the effects of diet-induced changes in plasma membrane fatty acid composition on several specific enzymes (adenylate cyclase, 5'-nucleotidase, Na(+)/K(+)-ATPase) and cell-surface receptors (opiate, adrenergic, insulin). Dietary manipulation studies have demonstrated a sensitivity of each to a fatty acid environment that is variably dependent on the nature of the fatty acid(s) and/or source of the membrane. The molecular mechanisms appear to involve fatty acid-dependent effects on protein conformation, on the "fluidity" and/or thickness of the membrane, or on protein synthesis. Together, the results of these studies reinforce the concept that dietary fats have the potential to regulate physiologic function and to further our understanding of how this occurs at a membrane level.     

GTPases in intracellular trafficking: an overview

Small GTPases that belong to the ras sub-families of Rab, Arf, and Rho, and the large GTPase dynamin, regulate intracellular trafficking. This issue of Seminars of Cell and Developmental Biology highlights topics regarding mechanisms by which these GTPases regulate the different steps of vesicular transport: vesicle formation, scission, targeting and fusion. In addition, the emerging roles of GTPases in coordination of individual transport steps as well as coordination of intracellular trafficking with other cellular processes are reviewed. Finally, common structures and mechanisms underlying the function of the ras-like GTPases and the importance of their function to human health and disease are discussed.     

Fatty acids regulate pigmentation via proteasomal degradation of tyrosinase: a new aspect of ubiquitin-proteasome function

Fatty acids are common components of biological membranes that are known to play important roles in intracellular signaling. We report here a novel mechanism by which fatty acids regulate the degradation of tyrosinase, a critical enzyme associated with melanin biosynthesis in melanocytes and melanoma cells. Linoleic acid (unsaturated fatty acid, C18:2) accelerated the spontaneous degradation of tyrosinase, whereas palmitic acid (saturated fatty acid, C16:0) retarded the proteolysis. The linoleic acid-induced acceleration of tyrosinase degradation could be abrogated by inhibitors of proteasomes, the multicatalytic proteinase complexes that selectively degrade intracellular ubiquitinated proteins. Linoleic acid increased the ubiquitination of many cellular proteins, whereas palmitic acid decreased such ubiquitination, as compared with untreated controls, when a proteasome inhibitor was used to stabilize ubiquitinated proteins. Immunoprecipitation analysis also revealed that treatment with fatty acids modulated the ubiquitination of tyrosinase, i.e. linoleic acid increased the amount of ubiquitinated tyrosinase whereas, in contrast, palmitic acid decreased it. Furthermore, confocal immunomicroscopy showed that the colocalization of ubiquitin and tyrosinase was facilitated by linoleic acid and diminished by palmitic acid. Taken together, these data support the view that fatty acids regulate the ubiquitination of tyrosinase and are responsible for modulating the proteasomal degradation of tyrosinase. In broader terms, the function of the ubiquitin-proteasome pathway might be regulated physiologically, at least in part, by fatty acids within cellular membranes.     

The fatty acid transport function of fatty acid-binding proteins

The intracellular fatty acid-binding proteins (FABPs) comprise a family of 14-15 kDa proteins which bind long-chain fatty acids. A role for FABPs in fatty acid transport has been hypothesized for several decades, and the accumulated indirect and correlative evidence is largely supportive of this proposed function. In recent years, a number of experimental approaches which more directly examine the transport function of FABPs have been taken. These include molecular level in vitro modeling of fatty acid transfer mechanisms, whole cell studies of fatty acid uptake and intracellular transfer following genetic manipulation of FABP type and amount, and an examination of cells and tissues from animals engineered to lack expression of specific FABPs. Collectively, data from these studies have provided strong support for defining the FABPs as fatty acid transport proteins. Further studies are necessary to elucidate the fundamental mechanisms by which cellular fatty acid trafficking is modulated by the FABPs.     

Lipotoxicity: when tissues overeat

PURPOSE OF REVIEW: This review will provide the reader with an update on our understanding of the adverse effects of fatty acid accumulation in non-adipose tissues, a phenomenon known as lipotoxicity. Recent studies will be reviewed. Cellular mechanisms involved in the lipotoxic response will be discussed. Physiologic responses to lipid overload and therapeutic approaches to decreasing lipid accumulation will be discussed, as they add to our understanding of important pathophysiologic mechanisms. RECENT FINDINGS: Excess lipid accumulation in non-adipose tissues may arise in the setting of high plasma free fatty acids or triglycerides. Alternatively, lipid overload results from mismatch between free fatty acid import and utilization. Evidence from human studies and animal models suggests that lipid accumulation in the heart, skeletal muscle, pancreas, liver, and kidney play an important role in the pathogenesis of heart failure, obesity and diabetes. Excess free fatty acids may impair normal cell signaling, causing cellular dysfunction. In some circumstances, excess free fatty acids induce apoptotic cell death. SUMMARY: Recent studies provide clues regarding the cellular mechanisms that determine whether excess lipid accumulation is well tolerated or cytotoxic. Critical in this process are physiologic mechanisms for directing excess free fatty acids to specific tissues as well as cellular mechanisms for channeling excess fatty acid to particular metabolic fates. Insight into these mechanisms may contribute to the development of more effective therapies for common human disorders in which lipotoxicity contributes to pathogenesis.     

Mechanisms regulating membrane trafficking of G protein-coupled receptors in the endocytic pathway

Endocytic membrane trafficking plays multiple roles in GPCR signaling and regulation. In the past several years much has been learned about molecular mechanisms that mediate and regulate endocytic trafficking of cloned GPCRs expressed in transfected cell lines, and there is accelerating progress toward elucidating the membrane trafficking of GPCRs in native tissues. Current views regarding ligand-induced endocytosis of adrenergic catecholamine and opioid neuropeptide receptors will be reviewed, focusing on recent data suggesting the existence of additional machinery controlling the endocytosis of specific GPCRs via clathrin-coated pits. Evidence that GPCRs are selectively 'sorted' between divergent downstream pathways after endocytosis will be discussed, focusing on recent insight to mechanisms controlling receptor sorting between distinct recycling and non-recycling membrane pathways.     

Protein lipidation

Proteins are covalently modified with a variety of lipids, including fatty acids, isoprenoids, and cholesterol. Lipid modifications play important roles in the localization and function of proteins. The focus of this review is S-palmitoylation, the reversible addition of palmitate and other long-chain fatty acids to proteins at cysteine residues in a variety of sequence contexts. The functional consequences of palmitoylation are diverse. Palmitoylation facilitates the association of proteins with membranes, mediates protein trafficking, and more recently has been appreciated as a regulator of protein stability. Members of a family of integral membrane proteins that harbor a DHHC cysteine-rich domain mediate most cellular palmitoylation events. Here we focus on DHHC proteins that modify Ras proteins in yeast and mammalian cells.     

Cellular fatty acid uptake: the contribution of metabolism

PURPOSE OF REVIEW: The aim of this review is to highlight the importance of fatty acid metabolism as a major determinant in fatty acid uptake. In particular, we emphasize how the activation, intracellular transport and downstream metabolism of fatty acids influence their uptake into cells. RECENT FINDINGS: Studies examining fatty acid entry into cells have focused primarily on the roles of plasma membrane proteins or the question of passive diffusion. Recent studies, however, strongly suggest that a driving force governing fatty acid uptake is the metabolic demand for fatty acids. Both gain and loss-of-function experiments indicate that fatty acid uptake can be modulated by activation at both the plasma membrane and internal sites, by intracellular fatty acid binding proteins, and by enzymes in synthetic or degradative metabolic pathways. Although the mechanism is not known, it appears that converting fatty acids to acyl-CoAs and downstream metabolic intermediates increases cellular fatty acid uptake, probably by limiting efflux. SUMMARY: Altered fatty acid metabolism and the accumulation of triacylglycerol and lipid metabolites has been strongly associated with insulin resistance and diabetes, but we do not fully understand how the entry of fatty acids into cells is regulated. Future studies of cellular fatty acid uptake should consider the influence of fatty acid metabolism and the possible interactions between fatty acid metabolism or metabolites and fatty acid transport proteins.