Paul N. Black

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 (Mr 70,000) and FATP2b (Mr 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.

Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of FATP2 resulted in increases in all four classes of phospholipid, indicating little selectivity. In the case of C22:6, there were significant increases of this exogenous fatty acids being trafficking into PC and PI. Collectively, these data support the conclusion that FATP2 has a dual function in the pathways linking the transport and activation of exogenous fatty acids. We discuss the differential roles of FATP2 and its role in both fatty acid transport and fatty acid activation in the context of lipid homeostasis.

Identification and characterization of small compound inhibitors of human FATP2

Fatty acid transport proteins (FATPs) are bifunctional proteins, which transport long chain fatty acids into cells and activate very long chain fatty acids by esterification with coenzyme A. In an effort to understand the linkage between cellular fatty acid transport and the pathology associated with excessive accumulation of exogenous fatty acids, we targeted FATP-mediated fatty acid transport in a high throughput screen of more than 100,000 small diverse chemical compounds in yeast expressing human FATP2 (hsFATP2). Compounds were selected for their ability to depress the transport of the fluorescent long chain fatty acid analogue, C(1)-BODIPY-C(12). Among 234 hits identified in the primary screen, 5 compounds, each representative of a structural class, were further characterized in the human Caco-2 and HepG2 cell lines, each of which normally expresses FATP2, and in 3T3-L1 adipocytes, which do not. These compounds were effective in inhibiting uptake with IC(50)s in the low micromolar range in both Caco-2 and HepG2 cells. Inhibition of transport was highly specific for fatty acids and there were no effects of these compounds on cell viability, trans-epithelial electrical resistance, glucose transport, or long chain acyl-CoA synthetase activity. The compounds were less effective when tested in 3T3-L1 adipocytes suggesting selectivity of inhibition. These results suggest fatty acid transport can be inhibited in a FATP-specific manner without causing cellular toxicity.

Modeling of rhythmic behavior in neutral lipid production due to continuous supply of limited nitrogen: Mutual growth and lipid accumulation in microalgae

The relative effects of three precise nitrogen limitation regimes on green micro-algae were assessed using the Trebouxiophycean alga Coccomyxa subellipsoidea grown in a chemostatic bioreactor system. The data provides further evidence that growth and triglyceride (TAG) accumulation are concurrent and independently proportional to the degree of nitrogen limitation in algae. Additionally, TAG accumulation was observed to proceed via oscillations with respect to time and percent dry weight quantity. The predator-prey model was applied to fit the experimental data and to obtain the physiological significance of these oscillations. The results determine the conditions of maximum neutral lipid productivity with respect to nitrate stress and highlight an area of potential future research.

Methods to Monitor Fatty Acid Transport Proceeding Through Vectorial Acylation

The process of fatty acid transport across the plasma membrane occurs by several mechanisms that involve distinct membrane-bound and membrane-associated proteins and enzymes. Among these are the fatty acid transport proteins (FATP) and long-chain acyl CoA synthetases (Acsl). Previous studies in yeast and adipocytes have shown FATP and Acsl form a physical complex at the plasma membrane and are required for fatty acid transport, which proceeds through a coupled process-linking transport with metabolic activation termed vectorial acylation. At present, six isoforms of FATP and five isoforms of ACSL have been identified in mice and man. In addition, there are a number of splice variants of different FATP and Acsl isoforms. The different FATP and Acsl isoforms have distinct tissue expression profiles and different cellular locations suggesting they function in the channeling of fatty acids into discrete metabolic pools. The concerted activity of these proteins is proposed to allow cells to discriminate different classes of fatty acids and provides the mechanistic basis underpinning the selectivity and specificity of the fatty acid transport process.

Chemical inhibition of fatty acid absorption and cellular uptake limits lipotoxic cell death

Chronic elevation of plasma free fatty acid (FFA) levels is commonly associated with obesity, type 2 diabetes, cardiovascular disease and some cancers. Experimental evidence indicates FFA and their metabolites contribute to disease development through lipotoxicity. Previously, we identified a specific fatty acid transport inhibitor CB16.2, a.k.a. Lipofermata, using high throughput screening methods. In this study, efficacy of transport inhibition was measured in four cell lines that are models for myocytes (mmC2C12), pancreatic β-cells (rnINS-1E), intestinal epithelial cells (hsCaco-2), and hepatocytes (hsHepG2), as well as primary human adipocytes. The compound was effective in inhibiting uptake with IC50s between 3 and 6μM for all cell lines except human adipocytes (39μM). Inhibition was specific for long and very long chain fatty acids but had no effect on medium chain fatty acids (C6-C10), which are transported by passive diffusion. Derivatives of Lipofermata were evaluated to understand structural contributions to activity. Lipofermata prevented palmitate-mediated oxidative stress, induction of BiP and CHOP, and cell death in a dose-dependent manner in hsHepG2 and rnINS-1E cells, suggesting it will prevent induction of fatty acid-mediated cell death pathways and lipotoxic disease by channeling excess fatty acids to adipose tissue and away from liver and pancreas. Importantly, mice dosed orally with Lipofermata were not able to absorb (13)C-oleate demonstrating utility as an inhibitor of fatty acid absorption from the gut.

Dietary omega-3 and omega-6 polyunsaturated fatty acids modulate hepatic pathology

Recent evidence has suggested that dietary polyunsaturated fatty acids (PUFAs) modulate inflammation; however, few studies have focused on the pathobiology of PUFA using isocaloric and isolipidic diets and it is unclear if the associated pathologies are due to dietary PUFA composition, lipid metabolism or obesity, as most studies compare diets fed ad libitum. Our studies used isocaloric and isolipidic liquid diets (35% of calories from fat), with differing compositions of omega (ω)-6 or long chain (Lc) ω-3 PUFA that were pair-fed and assessed hepatic pathology, inflammation and lipid metabolism. Consistent with an isocaloric, pair-fed model we observed no significant difference in diet consumption between the groups. In contrast, the body and liver weight, total lipid level and abdominal fat deposits were significantly higher in mice fed an ω-6 diet. An analysis of the fatty acid profile in plasma and liver showed that mice on the ω-6 diet had significantly more arachidonic acid (AA) in the plasma and liver, whereas, in these mice ω-3 fatty acids such as eicosapentaenoic acid (EPA) were not detected and docosahexaenoic acid (DHA) was significantly lower. Histopathologic analyses documented that mice on the ω-6 diet had a significant increase in macrovesicular steatosis, extramedullary myelopoiesis (EMM), apoptotic hepatocytes and decreased glycogen storage in lobular hepatocytes, and hepatocyte proliferation relative to mice fed the Lc ω-3 diet. Together, these results support PUFA dietary regulation of hepatic pathology and inflammation with implications for enteral feeding regulation of steatosis and other hepatic lesions.

Identification and metabolite profiling of chemical activators of lipid accumulation in green algae

Small compound activators of lipid accumulation in algae are distinguished by differential effects on lipid and polar metabolite profiles. Microalgae are proposed as feedstock organisms useful for producing biofuels and coproducts. However, several limitations must be overcome before algae-based production is economically feasible. Among these is the ability to induce lipid accumulation and storage without affecting biomass yield. To overcome this barrier, a chemical genetics approach was employed in which 43,783 compounds were screened against Chlamydomonas reinhardtii, and 243 compounds were identified that increase triacylglyceride (TAG) accumulation without terminating growth. Identified compounds were classified by structural similarity, and 15 were selected for secondary analyses addressing impacts on growth fitness, photosynthetic pigments, and total cellular protein and starch concentrations. TAG accumulation was verified using gas chromatography-mass spectrometry quantification of total fatty acids, and targeted TAG and galactolipid measurements were performed using liquid chromatography-multiple reaction monitoring/mass spectrometry. These results demonstrated that TAG accumulation does not necessarily proceed at the expense of galactolipid. Untargeted metabolite profiling provided important insights into pathway shifts due to five different compound treatments and verified the anabolic state of the cells with regard to the oxidative pentose phosphate pathway, Calvin cycle, tricarboxylic acid cycle, and amino acid biosynthetic pathways. Metabolite patterns were distinct from nitrogen starvation and other abiotic stresses commonly used to induce oil accumulation in algae. The efficacy of these compounds also was demonstrated in three other algal species. These lipid-inducing compounds offer a valuable set of tools for delving into the biochemical mechanisms of lipid accumulation in algae and a direct means to improve algal oil content independent of the severe growth limitations associated with nutrient deprivation.

Carbon and Acyl Chain Flux during Stress-induced Triglyceride Accumulation by Stable Isotopic Labeling of the Polar Microalga Coccomyxa subellipsoidea C169

Deriving biofuels and other lipoid products from algae is a promising future technology directly addressing global issues of atmospheric CO2 balance. To better understand the metabolism of triglyceride synthesis in algae, we examined their metabolic origins in the model species, Coccomyxa subellipsoidea C169, using stable isotopic labeling. Labeling patterns arising from [U-13C]glucose, 13CO2, or D2O supplementation were analyzed by GC-MS and/or LC-MS over time courses during nitrogen starvation to address the roles of catabolic carbon recycling, acyl chain redistribution, and de novo fatty acid (FA) synthesis during the expansion of the lipid bodies. The metabolic origin of stress-induced triglyceride was found to be a continuous 8:2 ratio between de novo synthesized FA and acyl chain transfer from pre-stressed membrane lipids with little input from lipid remodeling. Membrane lipids were continually synthesized with associated acyl chain editing during nitrogen stress, in contrast to an overall decrease in total membrane lipid. The incorporation rates of de novo synthesized FA into lipid classes were measured over a time course of nitrogen starvation. The synthesis of triglycerides, phospholipids, and galactolipids followed a two-stage pattern where nitrogen starvation resulted in a 2.5-fold increase followed by a gradual decline. Acyl chain flux into membrane lipids was dominant in the first stage followed by triglycerides. These data indicate that the level of metabolic control that determines acyl chain flux between membrane lipids and triglycerides during nitrogen stress relies primarily on the Kennedy pathway and de novo FA synthesis with limited, defined input from acyl editing reactions.

Long-Chain Omega-3 Polyunsaturated Fatty Acids Modulate Mammary Gland Composition and Inflammation.

Studies in rodents have shown that dietary modifications as mammary glands (MG) develop, regulates susceptibility to mammary tumor initiation. However, the effects of dietary PUFA composition on MGs in adult life, remains poorly understood. This study investigated morphological alterations and inflammatory microenvironments in the MGs of adult mice fed isocaloric and isolipidic liquid diets with varying compositions of omega (ω)-6 and long-chain (Lc)-ω3FA that were pair-fed. Despite similar consumption levels of the diets, mice fed the ω-3 diet had significantly lower body-weight gains, and abdominal-fat and mammary fat pad (MFP) weights. Fatty acid analysis showed significantly higher levels of Lc-ω-3FAs in the MFPs of mice on the ω-3 diet, while in the MFPs from the ω-6 group, Lc-ω-3FAs were undetectable. Our study revealed that MGs from ω-3 group had a significantly lower ductal end-point density, branching density, an absence of ductal sprouts, a thinner ductal stroma, fewer proliferating epithelial cells and a lower transcription levels of estrogen receptor 1 and amphiregulin. An analysis of the MFP and abdominal-fat showed significantly smaller adipocytes in the ω-3 group, which was accompanied by lower transcription levels of leptin, IGF1, and IGF1R. Further, MFPs from the ω-3 group had significantly decreased numbers and sizes of crown-like-structures (CLS), F4/80+ macrophages and decreased expression of proinflammatory mediators including Ptgs2, IL6, CCL2, TNFα, NFκB, and IFNγ. Together, these results support dietary Lc-ω-3FA regulation of MG structure and density and adipose tissue inflammation with the potential for dietary Lc-ω-3FA to decrease the risk of mammary gland tumor formation.