Identification of caveolin-1 as a fatty acid binding protein

In an attempt to identify high affinity, fatty acid binding proteins present in 3T3-L1 adipocytes plasma membranes, we labeled proteins in purified plasma membranes with the photoreactive fatty acid analogue, 11-m-diazirinophenoxy[11-3H]undecanoate. A single membrane protein of 22 kDa was covalently labeled after photolysis. This protein fractionated with caveolin-1 containing caveolae and was immunoprecipitated by an anti-caveolin-1 monoclonal antibody. Furthermore, 2D-PAGE analysis revealed that both the alpha and beta isoforms of caveolin-1 could be labeled by the photoreactive fatty acid upon photolysis, indicating that both bind fatty acids. The saturable binding of the photoreactive fatty acid suggests caveolin-1 has a lipid binding site that may either operate during intracellular lipid traffic or regulate caveolin-1 function.     

Inhibition of polyunsaturated fatty acid accumulation in plants expressing a fatty acid epoxygenase

Earlier, we described the isolation of a Crepis palaestina cDNA (Cpal2) which encoded a Delta12-epoxygenase that could catalyse the synthesis of 12,13-epoxy-cis-9-octadecenoic acid (18:1E) from linoleic acid (18:2). When the Cpal2 gene was expressed under the control of a seed-specific promoter in Arabidopsis, plants were able to accumulate small amounts 18:1E and 12,13-epoxy-cis-9,15-octadec-2-enoic acid in their seed lipids. In this report we give results obtained from a detailed analysis of transgenic Arabidopsis plants containing the Cpal2 gene. The seeds from these plants accumulate varying levels of 18:1E, but show a marked increase in 18:1 and equivalent decrease in 18:2 and 18:3. We further observed that the co-expression of a C. palaestina Delta12-desaturase in Arabidopsis appears to return the relative proportions of the C(18) seed fatty acids to normal levels and results in a 2-fold increase in total epoxy fatty acids.     

Methionine limitation results in increased hepatic FAS activity, higher liver 18:1 to 18:0 fatty acid ratio and hepatic TAG accumulation in Atlantic salmon, Salmo salar

The current experiment aimed to study whether interactions with lipid metabolism possibly might explain the relative increased liver weight obtained in fish fed sub-optimal methionine levels. A basal diet based on a blend of plant proteins which is low in methionine (1.6 g Met/16 g N) was compared to a methionine adequate diet (2.2 g Met/16 g N) prepared by adding dl-methionine (2.4 g/kg) to the basal diet in the expense of wheat grain. Fish oil was used as the lipid source. The diets were balanced in all nutrients except methionine. The diets were fed to Atlantic salmon (500 g BW) for a period of 3 months. Feed intake did not differ, rendering the intake of all nutrients except methionine equal. Fish fed the low methionine diet had an increased liver size relative to body weight, indicating fat deposition in the liver. Fish given the sub-optimal methionine diet showed about six times higher fatty acid synthase (FAS) activity as compared to the fish fed the adequate methionine diet, indicating a higher de novo lipogenesis. A significant rise in the liver 18:1 to 18:0 fatty acid ratios also supported storage of lipids over fatty acid oxidation. Indeed, methionine limitation resulted in significantly higher TAG concentrations in the liver. Sub-optimal dietary methionine also resulted in lower hepatic taurine concentrations and the total bile acids concentrations were reduced in faeces and tended to be reduced in plasma. Taken together, our data show that salmon fed sub-optimal methionine levels had increased relative liver weight and developed signs commonly described in the early stage of non-alcoholic fatty liver disease in rodent models (increased FAS activity, changed fatty acid ratios and TAG accumulation).     

Leaf lipids of Ribes nigrum: a plant containing 16:3, alpha-18:3, gamma-18:3 and 18:4 fatty acids

The glycerolipid composition of Ribes nigrum (blackcurrant) leaves was determined. The total fatty acid composition was unusual in that alpha-linolenic acid (alpha-18:3) occurred together with cis-7,10,13-hexadecatrienoic acid (16:3) and lower amounts of stearidonic acid (18:4) and gamma-linolenic acid (gamma-18:3). Monogalactosyldiacylglycerol contained the highest proportion of 16:3 with less in digalactosyldiacylglycerol. gamma-18:3 and 18:4 were present in all lipids and 18:4 was always greater than gamma-18:3. The highest percentages of gamma-18:3 and 18:4 were in phosphatidylcholine, but phosphatidylglycerol was particularly low in these acids. In summary, the lipid composition was largely typical of 16:3 plants but there was a minor contribution typical of 18:4 plants. The possibility of three pathways for glycolipid biosynthesis is discussed.     

Identification of fatty acid molecules in a Fasciola hepatica immunoprophylactic fatty acid-binding protein

Fasciola hepatica adult flukes have a native protein complex denoted nFh12 and consisting of fatty acid binding proteins that comprise at least 8 isoforms. It is a potent immunogen because in several animal hosts it induces an early antibody response to F. hepatica infection. It is also a potent cross-protective immunogen because it induces a protective immune response in mice to challenge infection with Schistosoma mansoni cercariae. The gene encoding this protein has been cloned and sequenced. It produces a polypeptide of 132 amino acids with a predicted molecular mass of 14.7 kDa and is denoted rFh15. It also has a significant homology to a 14-kDa S. mansoni fatty acid binding protein (Sm14). In the present study, nFh12 was delipidated with charcoal treatment and then studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Additionally, a lipid analysis of nFh12 was undertaken using gas chromatography-mass spectrometry to demonstrate that the nFh12 protein complex is, in fact, a complex of fatty acid binding proteins. Five long-chain saturated and unsaturated fatty acids were detected. The most abundant were palmitic acid (38%), stearic acid (24%), and oleic acid (13%). These fatty acid molecules do not have covalent bonds attached to the protein molecule. Because both nFh12 and Sm14 protect mice against challenge infection with F. hepatica and S. mansoni, it is possible that they have common protective epitopes in which fatty acids could be involved. Further studies are in progress to determine the chemical nature of these potential common epitopes.     

Identification of high affinity membrane-bound fatty acid-binding proteins using a photoreactive fatty acid

A photoaffinity labeling method was developed to identify and characterize high affinity fatty acid-binding proteins in membranes. The specific labeling of these sites requires the use of low concentrations (nanomolar) of the photoreactive fatty acid 11-m-diazirinophenoxy-[11-³H]undecanoate. It was delivered as a bovine serum albumin (BSA) complex which serves as a reservoir for fatty acid and thus allows precise control of unbound fatty acid concentrations. ThefadL protein ofE. coli, which is required for fatty acid permeation of its outer membrane, was labeled by the photoreactive fatty acid neither specifically nor saturably when the probe was added in the absence of BSA; however when a nanomolar concentration of the uncomplexed probe was maintained in the presence of BSA, the labeling of thefadL protein was highly specific and saturable. This photoaffinity labeling method was also used to characterize a 22 kDa, high affinity fatty acid-binding protein which we have recently identified in the plasma membrane of 3T3-L1 adipocytes. This protein bound the probe with a K_d of 216 nM. The approach described is easily capable of identifying membrane-bound fatty acid-binding proteins and can distinguish between those of high and low affinities for fatty acids. It represents a general method for the identification and characterization of fatty acid-binding proteins.Abbreviations BSA Bovine Serum Albumin - DAP m-Diazirinophenoxy - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis     

Identification of genes and pathways involved in the synthesis of Mead acid (20:3n − 9), an indicator of essential fatty acid deficiency

In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n − 9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1–6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n − 9, 20:1n − 9 and 20:2n − 9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n − 9 → (Fads2) → 18:2n − 9 → (Elovl5) → 20:2n − 9 → (Fads1) → 20:3n − 9 and pathway 2) 18:1n − 9 → (Elovl5) → 20:1n − 9 → (Fads2) → 20:2n − 9 → (Fads1) → 20:3n − 9.     

The effect of temperature on the degradation of triglycerides by a pseudomonad isolated from milk: free fatty acid accumulation as a balance between rates of triglyceride hydrolysis and fatty acid consumption

The lipolysis of triglyceride by a lipolytic pseudomonad, LS107d2, and the net levels of free fatty acids (FFA) in milk cultures of LS107d2 have been studied as a function of temperature. The metabolism of triolein results in the accumulation of oleic acid at 4°C but, at higher temperatures, triolein is hydrolysed without detectable oleate accumulation. Temperature also has a profound effect on the levels of FFA in whole milk cultures of LS107d2, and on the temporal pattern of changes in FFA levels. The results are consistent in demonstrating that FFA consumption plays a significant role in determining the net level of FFA in milk, even in the presence of very high rates of lipolysis. Thus the net levels of FFA as a result of the growth of LS107d2 in milk are the result of a dynamic balance between rates of FFA production (by lipolysis) and subsequent consumption. Temperature can dramatically affect the levels of FFA in milk cultures, and these changes are proposed to be due to opposing influences on FFA consumption and production, affecting the balance between them.     

Free fatty acid receptor 3 is a key target of short chain fatty acid

Nervous system (NS) activity participates in metabolic homeostasis by detecting peripheral signal molecules derived from food intake and energy balance. High quality diets are thought to include fiber-rich foods like whole grain rice, breads, cereals, and grains. Several studies have associated high consumption of fiber-enriched diets with a reduced risk of diabetes, obesity, and gastrointestinal disorders. In the lower intestine, anaerobic fermentation of soluble fibers by microbiota produces short chain fatty acids (SCFAs), key energy molecules that have a recent identified leading role in the intestinal gluconeogenesis, promoting beneficial effects on glucose tolerance and insulin resistance¹. SCFAs are also signaling molecules that bind to specific G-protein coupled receptors (GPCRs) named Free Fatty Acid Receptor 3 (FFA3, GPR41) and 2 (FFA2, GPR43). However, how SCFAs impact NS activity through their GPCRs is poorly understood.

Recently, studies have demonstrated the presence of FFA2 and FFA3 in the sympathetic NS of rat, mouse and human^{2, 3}. Two studies have showed that FFA3 activation by SCFAs increases firing and norepinephrine (NE) release from sympathetic neurons^{3, 4}. However, the recent study from the Ikeda Laboratory² revealed that activation of FFA3 by SCFAs impairs N-type calcium channel (NTCC) activity, which contradicts the idea of FFA3 activation leading to increased action potential evoked NE release. Here we will discuss the scope of the latter study and the putative physiological role of SCFAs and FFAs in the sympathetic NS.     

An alternative n-3 fatty acid elongation pathway utilising 18:3n-3 in barramundi (Lates calcarifer)

Desaturase and elongase are two key enzyme categories in the long-chain polyunsaturated fatty acid (LCPUFA) pathway that convert dietary α-linolenic acid (18:3n-3) to docosahexaenoic acid (22:6n-3). The Δ6 desaturase is considered as rate limiting in the conversion. In a previous study in barramundi we demonstrated that the desaturase had a low Δ6 activity but noted that the enzyme also possessed Δ8 ability that utilised 20-carbon fatty acids. This observation suggests that an alternative pathway may exist in the barramundi via elongases to form 20-carbon metabolites from 18:3n-3 to 20:3n-3 and then Δ6/8 desaturase to 20:4n-3. Cloning of the barramundi elongation of very long-chain fatty acid gene (ELOVL) and heterologous expression of the corresponding elongase were performed to examine activity with regard to time course, substrate concentration and substrate preference. Results revealed that the barramundi elongase showed a broad range of substrate specificity including 18-carbon PUFA (including 18:3n-3 and 18:2n-6), 20- and 22-carbon LCPUFA, with greater activity towards omega-3 (n-3) than n-6 fatty acids. The findings from this study provide molecular evidence for an alternative n-3 fatty acid elongation pathway utilising 18:3n-3 in barramundi.     

Expression of fatty acid synthesis genes and fatty acid accumulation in haematococcus pluvialis under different stressors

Background

Biofuel has been the focus of intensive global research over the past few years. The development of 4^th generation biofuel production (algae-to-biofuels) based on metabolic engineering of algae is still in its infancy, one of the main barriers is our lacking of understanding of microalgal growth, metabolism and biofuel production. Although fatty acid (FA) biosynthesis pathway genes have been all cloned and biosynthesis pathway was built up in some higher plants, the molecular mechanism for its regulation in microalgae is far away from elucidation.

Results

We cloned main key genes for FA biosynthesis in Haematococcus pluvialis, a green microalga as a potential biodiesel feedstock, and investigated the correlations between their expression alternation and FA composition and content detected by GC-MS under different stress treatments, such as nitrogen depletion, salinity, high or low temperature. Our results showed that high temperature, high salinity, and nitrogen depletion treatments played significant roles in promoting microalgal FA synthesis, while FA qualities were not changed much. Correlation analysis showed that acyl carrier protein (ACP), 3-ketoacyl-ACP-synthase (KAS), and acyl-ACP thioesterase (FATA) gene expression had significant correlations with monounsaturated FA (MUFA) synthesis and polyunsaturated FA (PUFA) synthesis.

Conclusions

We proposed that ACP, KAS, and FATA in H. pluvialis may play an important role in FA synthesis and may be rate limiting genes, which probably could be modified for the further study of metabolic engineering to improve microalgal biofuel quality and production.     

Identification of albumin-bound fatty acids as the major factor in serum-induced lipid accumulation by cultured cells

Factors responsible for the high lipogenic activity of rabbit serum were investigated using an assay procedure based on the gravimetric determination of the 24 hr increase in cell lipid. Cellular synthesis of fatty acids was inhibited by the presence of serum in the assay medium. Approximately 90% of the increase in cell lipid produced by serum fractions was due to triglyceride accumulation. Fractionation of rabbit serum by precipitation with ammonium sulfate or by ultracentrifugation in high density medium, both indicated that three-quarters of its lipogenic activity was associated with albumin. The lipoproteins prepared by ultracentrifugation also exhibited about one-half the activity of whole serum. The lipogenic activity of albumin was confirmed by the high potency of the albumin isolated in a nearly pure form from proteins of d>1.21 by precipitation with trichloroacetic acid and extraction with ethanol. As judged from chemical and isotopic analysis, neither the lipid content nor the lipid composition of the albumin was appreciably altered during its isolation. Of the albumin-bound lipids, only the free fatty acids, as determined by DEAE column chromatography, were present in an amount sufficient to account for the observed increase in cell triglycerides. In control experiments with horse serum of low lipogenic activity, the proteins of d>1.21 also possessed low activity in conjunction with a low content of free fatty acid. However, the albumin isolated from the latter preparation exhibited the high lipogenic activity of rabbit serum albumin. Chemical and isotopic analysis of the recovered horse serum albumin revealed that its free fatty acid content was the same as that of rabbit serum albumin. These results indicated that the isolation of horse serum albumin was attended by a substantial increase in its free fatty acid content. When the rabbit serum and horse serum content of media were adjusted to provide equivalent concentrations of albumin-bound fatty acids, the rabbit liver cells grown on the former media accumulated more lipid than cells grown on the latter media. This difference was shown to be due to the higher concentration of albumin per micro mole of fatty acid in horse serum as compared with rabbit serum. Consequently, the albumin to fatty acid ratio also controls the lipogenic activity of a serum. A linear relationship is presented which relates the cell lipid content to the molar ratio of albumin to free fatty acids and to the absolute concentration of free fatty acids in the medium.     

Identification and comparative analysis of a second runx3 promoter

Adenosine deaminase (ADA) catalyzes the hydrolysis of adenosine to inosine. Its lack determines severe combined immunodeficiency in mammals, possibly due to accumulation of extracellular adenosine, which induces apoptosis in lymphocytes (Franco et al., 1998). Thus, presence of normal levels of ADA leads to normal growth and proliferation of lymphocytes. Several vertebrate and microbial ADA amino-acid sequences are known, with substantial similarity to each other. On the other hand, there are invertebrate growth factors as well as a candidate gene for the human cat eye syndrome (CECR1) (Riazi et al., 2000. Genomics 64, 277-285), which share substantial similarity to each other, and also to ADA. In this study, we report the expression and ADA enzymatic activity of a cDNA from the salivary glands of Lutzomyia longipalpis, a blood-sucking insect, with substantial similarity to insect growth factors and to human CECR1. We also demonstrate the existence of a subfamily of the adenosine deaminase family characterized by their unique amino-terminal region. Both Drosophila melanogaster and humans have both types of adenosine deaminases. Results indicate that these invertebrate proteins previously annotated as growth factors, as well as the human CECR1 gene product, may exert their actions through adenosine depletion. The different roles played by each type of adenosine deaminase in humans and Drosophila remains to be fully investigated.