Blocking fatty acids' mystery tour: a therapy for metabolic syndrome?

Elevated circulating fatty acids are associated with impaired insulin action and inflammation. During intracellular transit, fatty acids use fatty acid-binding proteins (FABPs) as shuttles. A recent study (Furuhashi et al., 2007) explores inhibiting FABP4/aP2 as a strategy for treating atherosclerosis and type 2 diabetes.     

Comparative study of fatty-acid composition of table eggs from the Jeddah food market and effect of value addition in omega-3 bio-fortified eggs

Health consciousness has increased the desire of people around the world to consume functional foods. Omega-3 essential fatty acids are one among these beneficial and important health supplements without which a general predisposition to degenerative and stress related disorders can occur. Saudi Arabia has shown an alarming increase in obesity (Al-Nozha et al., 2005), diabetes (Alqurashi et al., 2011), and cardiovascular disease (Al-Nozha et al., 2004) in the last few decades mainly due to nutritional transitions and lifestyle alterations (Amuna and Zotor, 2008). Lack of nutrient dense foods and the prevailing food related disorder of obesity (Popkin, 2001; Prentice, 2014) especially render egg as a choice food to be value-added for attaining nutritional security in Saudi Arabia and in effect reverse the increasing incidences of lifestyle diseases. Nutritional intervention through a commonly consumed food product would be an important step in improving the health of the people, and reducing health care costs. As eggs are a frequently consumed food item in Saudi Arabia, enriching them with omega-3 fatty acids would be an excellent way to alleviate the existing problems. A significant deposition of omega-3 fatty acids in the eggs was observed when the diet of hens was supplemented with omega-3 fatty acids from either flaxseed or fish oil source. Inadequacy of omega-3 fatty acids could thus be rectified by producing omega-3 enriched eggs from hens supplemented with flaxseed or fish oil source, and thus contribute toward better health choice of the consumer.     

Fluxing the mitochondria to insulin resistance

Elevated fatty acids promote inflammation and insulin resistance. In this issue of Cell Metabolism, Koves et al. (2008) explore a novel paradigm suggesting that beta-oxidation of fatty acids exceeding the capacity of the tricarboxylic acid cycle yields incomplete fat oxidation and mitochondrial distress, obligatory events in the pathogenesis of insulin resistance.     

Percentage recovery of free fatty acids from bovine muscle lipids by nonchromatographic techniques

The inability of silicic acid to completely separate the neutral lipids from phospholipids has been reported by several investigators (1,2). Hornstein et al. (3) increased the polarity of the solvent system and reported a clean separation of the phospholipid fraction by adsorption on activated silicic acid. Studies on bovine lipids by Hood and Allen (2) utilized acid-washed Florisil to separate the lipid fractions claiming that silicic acid incompletely separates the free fatty acids from the phospholipids. Work performed in this laboratory (4) on bovine lipids confirmed that phospholipids could be effectively separated from free fatty acids by adsorption on silicic acid by incorporating the solvent system described by Hornstein et al. (3). The liquid-liquid partition procedure of Hamilton and McDonald (5) was also found to be sensitive enough to partition the extremely small amount of free fatty acids from the esterified fatty acids. This paper provides evidence for the effectiveness of these methods in separating the frec fatty acids by incorporating an internal standard [1-¹⁴C]palmitic acid.     

Purification of stearidonic acid (18:4(n-3)) and hexadecatetraenoic acid (16:4(n-3)) from algal fatty acid with lipase and medium pressure liquid chromatography

Stearidonic acid (18:4(n-3)) and hexadecatetraenoic acid (16:4(n-3)) are included in some edible marine algae such as Undaria pinnatifida and Ulva pertusa with relatively high compositions (up to 40%) of total fatty acids. In order to prepare 16:4(n-3) and 18:4(n-3) enriched fatty acid concentrates, we screened for a suitable lipase which concentrates these acids by the removal of other fatty acids in the selective esterification reaction reported by Shimada et al. (Shimada et al. (1997), J. Am. Oil Chem. Soc., 74, 1465-1470). In combination with the lipase reaction and reversed-phase medium pressure liquid chromatography, we purified 18:4(n-3) and 16:4(n-3) to more than 95% purity.     

Fatty acid synthase: A metabolic oncogene in prostate cancer?

In 1920, Warburg suggested that tumors consistently rely on anaerobic pathways to convert glucose to ATP even in the presence of abundant oxygen [Warberg, 1956] despite the fact that it is less efficient for energy supply than aerobic glycolysis. The reasons for this remain obscure to date. More often than not, the microenvironment of solid tumors contains regions of poor oxygenation and high acidity. In this context hypoxia can act in an epigenetic fashion, inducing changes in gene expression and in metabolism for survival. It is reasonable to assume that only the tumor cells capable of developing an unusual tolerance to limiting oxygen availability and to the acidosis resulting from excessive lactate production, can survive. In addition to the striking changes that occur in glucose metabolism, studies in human cancer patients suggest that there is often also an increase in free fatty acid turnover, oxidation and clearance [Legaspi et al., 1987; Hyltander et al., 1991]. For instance, a lipid mobilizing factor produced by tumor cells appears to be responsible for the increase in whole body fatty acid oxidation [Russell and Tisdale, 2002]. Fatty acids synthesis in tumor tissues also occurs at very high rates, as first demonstrated more than half a century ago [Medes et al., 1953]. Importantly, (14)C glucose studies have shown that in tumor cells almost all fatty acids derive from de novo synthesis despite adequate nutritional supply [Sabine and Abraham, 1967; Ookhtens et al., 1984; Weiss et al., 1986]. In addition, tumors overexpressing fatty acid synthase (FAS), the enzyme responsible for de novo synthesis of fatty acids, display aggressive biologic behavior compared to those tumors with normal FAS levels, suggesting that FAS overexpression confers a selective growth advantage. Here, we will review the roles that FAS plays in important cellular processes such as apoptosis and proliferation. In addition, speculations on the putative role of FAS in the altered metabolic pathways of prostate cancer cells will be explored. Because of the frequent overexpression of this enzyme prostate cancer, FAS constitutes a therapeutic target in this disease.     

Cytochrome P450 reductase (POR)as a ferroptosis fuel

Oxygen,iron,and polyunsaturated fatty acids (PUFAs;fatty acids containing more than one double bond) are all bene-ficial to our cellular lives.Incorporation of these components into cellular processes,however,comes at a cost:the bis-allylic structure of PUFAs and the enrichment of cellular environments with iron and oxygen render PUFA-containing phospholipids (PUFA-PLs) particularly susceptible to per-oxidation (Yang and Stockwell,2016).Accumulation of lethal amounts of lipid peroxides in cell membranes leads to a form of cell death known as ferroptosis (Dixon et al.,2012;Stockwell et al.,2017;Stockwell and Jiang,2020).Conse-quently,cells are equipped with strong antioxidant defense systems that constantly dissipate toxic lipid peroxides gen-erated in cellular membranes,thereby maintaining cell via-bility and homeostasis (Zheng and Conrad,2020).The most powerful anti-ferroptosis defense system is believed to be mediated by glutathione peroxidase 4 (GPX4),a glutathione peroxidase that uses glutathione as its cofactor to reduce lipid hydroperoxides to non-toxic lipid alcohols (Fig.1)(Zheng and Conrad,2020).A variety of ferroptosis inducers(FINs) act to inactivate GPX4 or deplete glutathione,causing an imbalance between the production and detoxification of lipid peroxides that subsequently induces ferroptotic cell death (Yang et al.,2014).Genetic ablation of GPX4 can have the same effect (Friedmann Angeli et al.,2014).     

Fatty Acid Activation in Cyanobacteria Mediated by Acyl-Acyl Carrier Protein Synthetase Enables Fatty Acid Recycling

In cyanobacteria fatty acids destined for lipid synthesis can be synthesized de novo, but also exogenous free fatty acids from the culture medium can be directly incorporated into lipids. Activation of exogenous fatty acids is likely required prior to their utilization. To identify the enzymatic activity responsible for activation we cloned candidate genes from Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 and identified the encoded proteins as acyl-acyl carrier protein synthetases (Aas). The enzymes catalyze the ATP-dependent esterification of fatty acids to the thiol of acyl carrier protein. The two protein sequences are only distantly related to known prokaryotic Aas proteins but they display strong similarity to sequences that can be found in almost all organisms that perform oxygenic photosynthesis. To investigate the biological role of Aas activity in cyanobacteria, aas knockout mutants were generated in the background of Synechocystis sp. PCC 6803 and S. elongatus PCC 7942. The mutant strains showed two phenotypes characterized by the inability to utilize exogenous fatty acids and by the secretion of endogenous fatty acids into the culture medium. The analyses of extracellular and intracellular fatty acid profiles of aas mutant strains as well as labeling experiments indicated that the detected free fatty acids are released from membrane lipids. The data suggest a considerable turnover of lipid molecules and a role for Aas activity in recycling the released fatty acids. In this model, lipid degradation represents a third supply of fatty acids for lipid synthesis in cyanobacteria.Cyanobacteria present a diverse group of Gram-negative bacteria capable of oxygenic photosynthesis (Margulis, 1975). Their two photosystems, as well as other genetic and morphological similarities, identified them as putative predecessors of chloroplasts of eukaryotic plants (Wallace, 1982; Pakrasi, 1995). The structural similarities of cyanobacteria and chloroplasts are reflected in part by equivalence of biochemical pathways and their components. For instance, cyanobacterial fatty acid and glycerolipid compositions closely resemble those of the inner envelope and thylakoid membranes of chloroplasts (Roughan et al., 1980; Heinz and Roughan, 1983). In cyanobacteria, as well as in chloroplasts, fatty acids are synthesized by a type II fatty acid synthase (FAS) complex utilizing a freely dissociable acyl carrier protein (ACP; Froehlich et al., 1990). The products of FAS are released as acyl ACPs and may serve directly as substrates for acyltransferases, incorporating the fatty acids into membrane lipids (Frentzen et al., 1983). The substrate specificity of the acyltransferases establishes in cyanobacteria as well as in plastids the typical prokaryotic fatty acid pattern characterized by C16 fatty acids esterified to the sn-2 position. The correspondence of metabolic pathways between cyanobacteria and chloroplasts is reflected by the shared presence of closely related enzymes that catalyze key reactions. Besides the many similarities, however, there are also clear discrepancies that in part account for the fact that cyanobacteria are unicellular organisms, whereas chloroplasts are embedded in the metabolism of a eukaryotic cell. In terms of lipid metabolism, such differences become obvious if one considers the fact that the plastidial FAS also supplies the extraplastidic compartment with fatty acids (Browse et al., 1986). Fatty acid export from the chloroplast necessitates the release of synthesized acyl chains from ACP to allow transport across both envelope membranes. The release is achieved by the action of acyl-ACP thioesterases that hydrolyze the acyl-ACP thioester to liberate the fatty acid (Voelker et al., 1997). In cyanobacteria such export would obviously result in an unfavorable loss of fatty acids, and consequently homologous proteins to acyl-ACP thioesterases cannot be found here. Whereas cyanobacteria seem to be unable to release fatty acids enzymatically from their activated state, all cyanobacterial genomes available to date encode an activity most likely responsible for the activation of free fatty acids. The respective sequences are annotated as acyl-CoA synthetases. Conserved motifs in the amino acid sequence identify these proteins as members of the well-established superfamily of AMP-binding proteins. This protein family comprises several hundred amino acid sequences spreading across all organisms analyzed so far. The family members are annotated in the PROSITE database under entry number PS00455. Although these predicted fatty acid-activating enzymes of cyanobacteria are annotated as acyl-CoA synthetases due to their sequence similarity to proteins with such enzymatic activity, there is a much higher degree of similarity to certain AMP-binding proteins of plant origin with less-well-established function. These plant proteins are predicted to reside in chloroplasts and one member of this subgroup from Arabidopsis (Arabidopsis thaliana) designated as AAE15 was recently described as acyl-ACP synthetase. The conclusions were based on the comparison of enzymatic activity between plant extracts of wild-type and knockout mutant lines (Koo et al., 2005). Whereas the biological role of this activity remained largely elusive, it was shown that the capacity of plant extracts to elongate supplied medium fatty acids depended on AAE15 activity. Since the elongation of medium chain fatty acids in the plastid depends on the FAS requiring acyl ACPs, it was concluded that the fatty acids must have been activated by ACP. The elongated fatty acids ultimately appeared in membrane lipids. Together these findings suggested that AAE15 is an acyl-ACP synthetase.Besides encoding a protein homologous to AAE15 from Arabidopsis, cyanobacteria are also able to utilize exogenous fatty acids like it was shown for isolated chloroplasts. It is well established that feeding different cyanobacteria with free fatty acids results in the incorporation of these fatty acids into membrane lipids. For this process the activation of the fatty acids is believed to be essential. This causal relationship was clearly shown at least for other unicellular organisms like Escherichia coli and yeast (Saccharomyces cerevisiae) where the deletion of acyl-CoA synthetase activity resulted in the inability to utilize exogenous fatty acids (Overath et al., 1969; Knoll et al., 1995). It is not easy to assess how regularly cyanobacterial cells are exposed to exogenous free fatty acids in nature but at least for marine strains this is most likely a rather artificial situation. Therefore, it can be speculated that the capacity to activate free fatty acids might be of different relevance in the lipid metabolism of cyanobacteria in vivo.In this article, we investigated the fatty acid metabolism of cyanobacteria. We isolated candidate genes potentially encoding enzymes involved in fatty acid activation from the strains Synechocystis sp. PCC 6803 (hereafter Synechocystis) and Synechococcus elongatus PCC 7942 (hereafter Synechococcus) and performed heterologous expression in E. coli. The recombinant proteins were shown to possess acyl-ACP synthetase activity with broad substrate specificity. Knockout mutant strains deficient in acyl-ACP synthetase activity were characterized by secretion of endogenous free fatty acids into the culture medium. Combined with labeling experiments, the results suggest an essential role for acyl-ACP synthetase in fatty acid recycling in cyanobacteria.     

Specific identification of free and esterified fatty acids in tissue sections

Synopsis The histochemical method of Adamset al. (1966) for demonstrating triglycerides in tissue sections was applied to kidneys exhibiting a wide variety of disease states. It became apparent, as would be expected, that the existing method demonstrates not only triglycerides but also free fatty acids in the same section. Even though the presence of free fatty acids could be detected in the control sections, their existence made it impossible to identify triglycerides with certainty.A modification is described which employs a potassium hydroxide-dioxan mixture to saponify and extract selectively free fatty acids from tissue sections. Fatty acids in free form can be demonstrated separately, in parallel sections, from those esterified as triglyceride. This modified technique was applied to frozen sections of formalin-fixed human and rat tissues, revealing distinct and highly characteristic distribution patterns for these two forms of fatty acid.     

Saturated Fatty Acid Requirer of Neurospora crassa

Dietary saturated fatty acids containing 12- to 18-carbon atoms satisfy growth requirements of Neurospora crassa mutant cel (previously named ol; Perkins et al., reference 11); unsaturated fatty acids are synthesized by direct desaturation when an appropriate saturate is available. Odd-chain saturates, 15 carbons and 17 carbons long, satisfy the requirement, and elaidic acid (18:1 Delta(9)trans) results in slow growth. Oleic acid and other cis-unsaturated fatty acids do not satisfy growth requirements; however, oleic acid plus elaidic acid result in growth at a faster rate than elaidate alone. The use of a spin-label fatty acid reveals that hyphae produced by cel during a slow basal level of growth have lipids that reflect a relatively rigid state of viscosity compared to wild type. cel Supplemented with fatty acids and wild type supplemented in the same way have lipids of the same viscosities as reflected by electron spin resonance.     

Effects of genetic background on thermoregulation and fatty acid-induced uncoupling of mitochondria in UCP1-deficient mice

An interaction between free fatty acids and UCP1 (uncoupling protein-1) leading to de-energization of mitochondria was assumed to be a key event for triggering heat production in brown fat. Recently, Matthias et al., finding indistinguishable de-energization of isolated brown fat mitochondria by fatty acids in UCP1-deficient mice and control mice, challenged this assumption (Matthias, A., Jacobsson, A., Cannon, B., and Nedergaard, J. (1999) J. Biol. Chem. 274, 28150-28160). Since their results were obtained using UCP1-deficient and control mice on an undefined genetic background, we wanted to determine unambiguously the phenotype of UCP1 deficiency with the targeted Ucp1 allele on congenic C57BL/6J and 129/SvImJ backgrounds. UCP1-deficient congenic mice have a very pronounced cold-sensitive phenotype; however, deficient mice on the F1 hybrid background were resistant to cold. We propose that heterosis provides a mechanism to compensate for UCP1 deficiency. Contrary to the results of Matthias et al., we found a significant loss of fatty acid-induced de-energization, as reflected by membrane potential and oxygen consumption, in brown fat mitochondria from UCP1-deficient mice. Unlike cold sensitivity, fatty acid-induced uncoupling of mitochondria was independent of the genetic background of UCP1-deficient mice. We propose that intracellular free fatty acids directly regulate uncoupling activity of UCP1 in a manner consistent with models described in the literature.     

Transformation of fatty acids catalyzed by cytochrome P450 monooxygenase enzymes of Candida tropicalis

Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in beta-oxidation convert these substrates to long-chain alpha,omega-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is omega-hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C(18:1)), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to omega-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C(14:0)) much more effectively. Both enzymes, in particular CYP52A17, also oxidized omega-hydroxy fatty acids, ultimately generating the alpha,omega-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for beta-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.     

Peroxisomal ATP Import Is Essential for Seedling Development in Arabidopsis thaliana

Several recent proteomic studies of plant peroxisomes indicate that the peroxisomal matrix harbors multiple ATP-dependent enzymes and chaperones. However, it is unknown whether plant peroxisomes are able to produce ATP by substrate-level phosphorylation or whether external ATP fuels the energy-dependent reactions within peroxisomes. The existence of transport proteins that supply plant peroxisomes with energy for fatty acid oxidation and other ATP-dependent processes has not previously been demonstrated. Here, we describe two Arabidopsis thaliana genes that encode peroxisomal adenine nucleotide carriers, PNC1 and PNC2. Both proteins, when fused to enhanced yellow fluorescent protein, are targeted to peroxisomes. Complementation of a yeast mutant deficient in peroxisomal ATP import and in vitro transport assays using recombinant transporter proteins revealed that PNC1 and PNC2 catalyze the counterexchange of ATP with ADP or AMP. Transgenic Arabidopsis lines repressing both PNC genes were generated using ethanol-inducible RNA interference. A detailed analysis of these plants showed that an impaired peroxisomal ATP import inhibits fatty acid breakdown during early seedling growth and other β-oxidation reactions, such as auxin biosynthesis. We show conclusively that PNC1 and PNC2 are essential for supplying peroxisomes with ATP, indicating that no other ATP generating systems exist inside plant peroxisomes.The β-oxidation of fatty acids, a process that exclusively occurs within peroxisomes in plants and yeast, plays an important role in storage oil mobilization to support seedling establishment of oilseed plants, such as Arabidopsis thaliana (Graham and Eastmond, 2002; Baker et al., 2006; Graham, 2008). Upon germination, fatty acids are released from storage oil triacylglycerol (TAG) by lipolysis, degraded via β-oxidation in specialized peroxisomes, termed glyoxysomes, and subsequently converted to sucrose, which drives growth and development until seedlings become photoautotrophic (Graham and Eastmond, 2002; Baker et al., 2006; Graham, 2008). Before the fatty acids can enter β-oxidation, they are imported into peroxisomes by a peroxisomal ATP binding cassette (ABC) transporter, variously known as CTS (COMATOSE), At PXA1 (Arabidopsis peroxisomal ABC transporter), or PED3 (peroxisomal defective 3) and hereafter referred to as CTS (Zolman et al., 2001; Footitt et al., 2002; Hayashi et al., 2002). Subsequently, the imported fatty acids are activated by esterification to CoA. This ATP-dependent reaction within peroxisomes is catalyzed by long-chain acyl-CoA synthetases 6 and 7 (LACS6 and LACS7, respectively), which are named according to their substrate specificity for long-chain fatty acids, which are significant components of seed storage oil in Arabidopsis (Fulda et al., 2002, 2004).In Saccharomyces cerevisiae, two mechanisms exist for import and activation of fatty acids, depending on chain length (Hettema et al., 1996). Long-chain fatty acids (C16 and C18) are converted to acyl-CoA esters in the cytosol prior to transport by the heterodimeric peroxisomal ABC transporter, Pxa1p/Pxa2 (Hettema et al., 1996). By contrast, short- and medium-chain fatty acids (≤C14) that enter the peroxisomes by passive diffusion or by an unknown transport protein are activated within peroxisomes (Hettema et al., 1996). The possibility cannot be excluded, though, that CTS imports the corresponding CoA derivatives, as is the case for the yeast Pxa1p/Pxa2p heterodimer (Hettema et al., 1996; Verleur et al., 1997), implicating a cytosolic activation of the fatty acids, catalyzed by a hitherto unknown enzyme. The actual substrates transported by CTS in Arabidopsis have not yet been experimentally determined (Theodoulou et al., 2006). However, the sucrose-dependent seedling growth phenotype of the lacs6 lacs7 double knockout mutant demonstrated that peroxisomal activation is essential for lipid mobilization to provide energy for early seedling growth (Fulda et al., 2004). The lacs6 lacs7 mutant is impaired in the degradation of fatty acids, leading to growth arrest shortly after germination (Fulda et al., 2004).Besides fatty acid mobilization, β-oxidation is also involved in generation of signaling molecules, such as the phytohormones auxin and fatty acid–derived jasmonic acid (JA) (Zolman et al., 2000; Schaller et al., 2004; Delker et al., 2007). By analogy to fatty acids released from storage oil, the precursors of these signaling molecules require CoA esterification before they can enter β-oxidation (Baker et al., 2006; Goepfert and Poirier, 2007). While the enzymes responsible for ATP-dependent activation of natural auxin (indole butyric acid [IBA]) and proherbicide 2,4-dichlorophenoxybutyric acid (2,4-DB) are currently unknown, several enzymes belonging to the acyl-activating enzyme (AAE) family have been implicated in jasmonate biosynthesis (Schneider et al., 2005; Koo et al., 2006; Kienow et al., 2008). Moreover, several as yet uncharacterized members of the large AAE family carry a putative peroxisome targeting signal (PTS) and thus might be good candidates to activate the additional β-oxidation substrates within peroxisomes (Shockey et al., 2002, 2003).In the case where activation of fatty acids or other substrates takes place within peroxisomes, the question arises as to how these ATP-dependent reactions are supplied with ATP. It is currently unknown whether plant peroxisomes are able to produce ATP by substrate-level phosphorylation or whether they depend on external ATP to supply energy-dependent reactions within peroxisomes. So far, transport proteins that supply plant peroxisomes with energy for fatty acid oxidation have not been characterized. However, in bakers'' yeast, a peroxisomal adenine nucleotide transporter, ANT1, that is required for the ATP-dependent activation of medium-chain fatty acids inside peroxisomes has been characterized (Palmieri et al., 2001).ATP transport proteins play an important role in the distribution of the primary agent coupling endergonic and exergonic reactions in every cellular compartment (Winkler and Neuhaus, 1999). In Arabidopsis and other plants, various adenine nucleotide carriers have been identified at the molecular level. The mitochondrial ADP/ATP carrier mediates the export of ATP that is synthesized in the mitochondrion to provide energy for cellular metabolism (Heimpel et al., 2001; Haferkamp et al., 2002). The plastidial ATP/ADP transporter (nucleotide transporter) is involved in ATP uptake by both chloroplasts and heterotrophic plastids, to enable the nocturnal ATP supply required for chlorophyll biosynthesis (Reiser et al., 2004; Reinhold et al., 2007), as well as by heterotrophic plastids to drive starch biosynthesis (Batz et al., 1992; Tjaden et al., 1998). Yet another ATP/ADP antiporter located in the endoplasmic reticulum (ER) membrane provides energy by importing ATP into the ER for the accumulation of ER-related storage lipids and proteins (Leroch et al., 2008).In this study, we identified two novel peroxisomal adenine nucleotide carrier proteins (PNC1 and PNC2) from Arabidopsis. Colocalization studies demonstrated that these proteins are targeted to peroxisomes. Yeast complementation and in vitro ATP uptake assays showed that both PNC1 and PNC2 catalyze the counterexchange of ATP with AMP. Using an inducible RNA interference (RNAi) repression strategy, we further established several transgenic Arabidopsis lines with reduced expression levels of both PNC1 and PNC2. Our results showed that import of ATP into peroxisomes that is catalyzed by PNC1 and PNC2 is essential for activation of fatty acids during seedling germination and plays a role in other β-oxidation reactions in peroxisomes, such as auxin metabolism. Analysis of PNC1 and PNC2 repression lines further indicates that no other ATP generating systems exist inside plant peroxisomes and that ATP import is the only way to supply the peroxisomal matrix with ATP.     

The Path to Insulin Resistance: Paved with Ceramides?

Obesity-associated, system-wide elevations in free fatty acids, tumor necrosis factor alpha, and glucocorticoids increase intracellular lipid metabolites and promote insulin resistance. In this issue, Holland et al. (2007) provide pharmacological and genetic evidence that ceramide plays a key role in the development of insulin resistance induced by these factors.     

In vivo effects of octanoate and oleate on glucose-6-phosphate dehydrogenase during germination of Pinus pinea seeds.

During our investigation on the effect of some fatty acids on the germination of Pinus pinea seeds (Vincenzini et al., 1973), we noted a marked decrease of G6PDH activity when octanoate or oleate were added to the culture medium. In an attempt to provide biochemical information on the effect of free fatty acids on G6PDH activity, in vivo, during Pinus pinea seeds germination, we tested different concentrations of octanoate and oleate added to the culture medium. Moreover other enzymes of fundamental metabolic pathways were also considered, particularly 6PGDH, the second enzyme of HMS.     

A monotopic membrane protein goes solo

Carnitine palmitoyltransferases (CPTs) are part of the enzymatic system that imports fatty acids into mitochondria. The crystal structure of rat CPT-2 by Rufer et al. (2006) (this issue of Structure) reveals a Y-shaped tunnel for binding the CoA and acyl-carnitine substrates and a hydrophobic insert mediating membrane association.     

CYP704B1 Is a Long-Chain Fatty Acid ω-Hydroxylase Essential for Sporopollenin Synthesis in Pollen of Arabidopsis

Sporopollenin is the major component of the outer pollen wall (exine). Fatty acid derivatives and phenolics are thought to be its monomeric building blocks, but the precise structure, biosynthetic route, and genetics of sporopollenin are poorly understood. Based on a phenotypic mutant screen in Arabidopsis (Arabidopsis thaliana), we identified a cytochrome P450, designated CYP704B1, as being essential for exine development. CYP704B1 is expressed in the developing anthers. Mutations in CYP704B1 result in impaired pollen walls that lack a normal exine layer and exhibit a characteristic striped surface, termed zebra phenotype. Heterologous expression of CYP704B1 in yeast cells demonstrated that it catalyzes ω-hydroxylation of long-chain fatty acids, implicating these molecules in sporopollenin synthesis. Recently, an anther-specific cytochrome P450, denoted CYP703A2, that catalyzes in-chain hydroxylation of lauric acid was also shown to be involved in sporopollenin synthesis. This shows that different classes of hydroxylated fatty acids serve as essential compounds for sporopollenin formation. The genetic relationships between CYP704B1, CYP703A2, and another exine gene, MALE STERILITY2, which encodes a fatty acyl reductase, were explored. Mutations in all three genes resulted in pollen with remarkably similar zebra phenotypes, distinct from those of other known exine mutants. The double and triple mutant combinations did not result in the appearance of novel phenotypes or enhancement of single mutant phenotypes. This implies that each of the three genes is required to provide an indispensable subset of fatty acid-derived components within the sporopollenin biosynthesis framework.The biopolymer sporopollenin is the major component of the outer walls in pollen and spores (exines). It is highly resistant to nonoxidative physical, chemical, and biological treatments and is insoluble in both aqueous and organic solvents. While the stability and resistance of sporopollenin account for the preservation of ancient pollen grains for millions of years with nearly full retention of morphology (Doyle and Hickey, 1976; Friis et al., 2001), these same qualities make it extremely difficult to study the chemical structure of sporopollenin. Thus, although the first studies on the composition of sporopollenin were reported in 1928 (Zetzsche and Huggler, 1928), the exact structure of sporopollenin remains unresolved. At present, it is thought that sporopollenin is a complex polymer primarily made of a mixture of fatty acids and phenolic compounds (Guilford et al., 1988; Wiermann et al., 2001).Fatty acids were first implicated as sporopollenin components when ozonolysis of Lycopodium clavatum and Pinus sylvestris exine yielded significant amounts of straight- and branched-chain monocarboxylic acids, characteristic fatty acid breakdown products (Shaw and Yeadon, 1966). More recently, improved purification and degradation techniques coupled with analytical methods, such as solid-state ¹³C-NMR spectroscopy, Fourier transform infrared spectroscopy, and ¹H-NMR, have shown that sporopollenin is made up of polyhydroxylated unbranched aliphatic units and also contains small amounts of oxygenated aromatic rings and phenylpropanoids (Guilford et al., 1988; Ahlers et al., 1999; Domínguez et al., 1999; Bubert et al., 2002). Biochemical studies using thiocarbamate herbicide inhibition of the chain-elongating steps in the synthesis of long-chain fatty acids and radioactive tracer experiments provided further evidence that lipid metabolism is involved in the biosynthesis of sporopollenin (Wilwesmeier and Wiermann, 1995; Meuter-Gerhards et al., 1999).Relatively little is known about the genetic network that determines sporopollenin synthesis. However, several Arabidopsis (Arabidopsis thaliana) genes implicated in exine biosynthesis encode proteins with sequence homology to enzymes that are involved in fatty acid metabolism. Mutations in MALE STERILITY2 (MS2) eliminate exine and affect a protein with sequence similarity to fatty acyl reductases; the predicted inability of ms2 plants to reduce pollen wall fatty acids to the corresponding alcohols suggests that this reaction is a key step in sporopollenin synthesis (Aarts et al., 1997). The FACELESS POLLEN1 (FLP1) gene, whose loss causes the flp1 exine defect, encodes a protein similar to those involved in wax synthesis (Ariizumi et al., 2003). The no exine formation1 (nef1) mutant accumulates reduced levels of lipids, and the NEF1 protein was suggested to be involved in either lipid transport or the maintenance of plastid membrane integrity, including those plastids in the secretory tapetum of anthers, where many of the sporopollenin components are synthesized (Ariizumi et al., 2004). The dex2 mutant has mutations in the evolutionarily conserved anther-specific cytochrome P450, CYP703A2 (Morant et al., 2007), which catalyzes in-chain hydroxylation of saturated medium-chain fatty acids, with lauric acid (C12:0) as a preferred substrate (Morant et al., 2007). A recently described gene, ACOS5, encodes a fatty acyl-CoA synthetase that has in vitro preference for medium-chain fatty acids (de Azevedo Souza et al., 2009). Mutations in all of these genes compromise exine formation.Here, we describe an evolutionarily conserved cytochrome P450, CYP704B1, and demonstrate that this gene is essential for exine biosynthesis and plays a role different from that of CYP703A2. Heterologously expressed CYP704B1 catalyzed ω-hydroxylation of several saturated and unsaturated C14-C18 fatty acids. These results suggest the possibility that ω-hydroxylated fatty acids produced by CYP704B1, together with in-chain hydroxylated lauric acids provided by the action of CYP703A2, may serve as key monomeric aliphatic building blocks in sporopollenin formation. Analyses of the genetic relationships between CYP704B1, MS2, and CYP703A2 suggest that all three genes are involved in the same pathway within the sporopollenin biosynthesis framework.     

IL-1β activation as a response to metabolic disturbances

IL-1β is a major regulator of islet inflammation in type 2 diabetes. Several factors contribute to the induction of islet-derived IL-1β, including glucose, free fatty acids, and leptin. A recent report in Nature Immunology (Masters et?al., 2010) identifies amyloid polypeptide as an additional enhancer of IL-1β production.     

Long, saturated chains: tasty domains for kinases of insulin resistance

The mechanistic basis of how cells respond to increased fatty acids (FAs) is murky but potentially involves receptor-mediated activation or inhibition by different FA classes. Holzer et?al. (2011) recently propose in Cell that expansion of intracellular membrane microdomains induced by saturated FA recruit and activate c-Src for JNK activation.     

Power surge: supporting cells "fuel" cancer cell mitochondria

An emerging paradigm in tumor metabolism is that catabolism in host cells "fuels" the anabolic growth of cancer cells via energy transfer. A study in Nature Medicine (Nieman et al., 2011) supports this; they show that triglyceride catabolism in adipocytes drives ovarian cancer metastasis by providing fatty acids as mitochondrial fuels.