Synthesis of medium-chain-length polyhydroxyalkanoates in arabidopsis thaliana using intermediates of peroxisomal fatty acid beta-oxidation.

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the beta-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the beta-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.

Angiotensin II favors progression to heart failure in diabetic hearts: role of myocardial fatty acid oxidation downregulation

Purpose: Diabetic myocardium is particularly vulnerable to develop heart failure in response to chronic stress conditions including hypertension or myocardial infarction. We have recently observed that angiotensin II (Ang II)-mediated downregulation of the fatty acid oxidation pathway favors occurrence of heart failure by myocardial accumulation of lipids (lipotoxicity). Because diabetic heart is exposed to high levels of circulating fatty acid, we determined whether insulin resistance favors development of heart failure in mice with Ang II-mediated myocardial remodeling.Methods: To study the combined effect of diabetes and Ang II-induced heart remodeling, we generated leptin-deficient/insulin resistant (Lepob/ob) mice with cardiac targeted overexpression of angiotensinogen (TGAOGN). Left ventricular (LV) failure was indicated by pulmonary congestion (lung weight/tibial length>+2SD of wild-type mice). Myocardial metabolism and function were assessed during in vitro isolated working heart perfusion.Results: Forty-eight percent of TGAOGN mice without insulin resistance exhibited pulmonary congestion at the age of 6 months associated with increased myocardial BNP expression (+375% compared with WT) and reduced LV power (developed pressure x cardiac output; -15%). The proportion of mice presenting heart failure was markedly increased to 71% in TGAOGN mice with insulin resistance (TGAOGN/Lepob/ob). TGAOGN/Lepob/ob mice with heart failure exhibited further increase of BNP compared with failing non-diabetic TGAOGN mice (+146%) and further reduction of cardiac power (-59%). Mice with insulin resistance alone (Lepob/ob) did not exhibit signs of heart failure or LV dysfunction. Myocardial fatty acid oxidation measured during in vitro perfusion was markedly increased in non-failing hearts from Lepob/ob mice (+380% compared with WT) and glucose oxidation decreased (-72%). In contrast, fatty acid and glucose oxidation did not differ from Lepob/ob mice in hearts from TGAOGN/Lepob/ob mice without heart failure. However, both fatty acid and glucose oxidation were markedly decreased (-47% and -48%, respectively, compared with WT/Lepob/+) in failing hearts from TGAOGN/Lepob/ob mice. Reduction of fatty acid oxidation was associated with marked reduction of protein expression of a number of regulatory enzymes implied in fatty acid oxidation.Conclusions: Insulin resistance favors the progression to heart failure during chronic exposure of the myocardium to Ang II. Our results are compatible with a role of Ang II-mediated downregulation of fatty acid oxidation, potentially promoting lipotoxicity.

Fatty acids decrease IDX-1 expression in rat pancreatic islets and reduce GLUT2, glucokinase, insulin, and somatostatin levels.

IDX-1 (islet/duodenum homeobox-1) is a transcription factor expressed in the duodenum and pancreatic beta and delta cells. It is required for embryonic development of the pancreas and transactivates the Glut2, glucokinase, insulin, and somatostatin genes. Here we show that exposure of isolated rat pancreatic islets to palmitic acid induced a approximately 70% decrease in IDX-1 mRNA and protein expression as well as 40 and 65% decreases in the binding activity of IDX-1 for its cognate cis-regulatory elements of the Glut2 and insulin promoters, respectively. The inhibitory effect of palmitic acid required its mitochondrial oxidation since it was prevented by the carnitine palmitoyltransferase I inhibitor bromopalmitic acid. The palmitic acid effect on IDX-1 was correlated with decreases in GLUT2 and glucokinase expression of 40 and 25%, respectively, at both the mRNA and protein levels. Insulin and somatostatin mRNA expression was also decreased by 40 and 60%, whereas glucagon mRNA expression was not modified. After 48 h of exposure to fatty acids, total islet insulin, somatostatin, and glucagon contents were decreased by 85, 55, and 65%, respectively. At the same time, total hormone release was strongly stimulated (13-fold) for glucagon, whereas its was only marginally increased for insulin and somatostatin (1.5- and 1.7-fold, respectively). These results indicate that elevated fatty acid levels 1) negatively regulate Idx-1 expression; 2) decrease the expression of genes transactivated by IDX-1 such as those for GLUT2, glucokinase, insulin, and somatostatin; and 3) lead to an important increase in glucagon synthesis and secretion. Fatty acids thus have pleiotropic effects on pancreatic islet gene expression, and the negative control of Idx-1 expression may be an initial event in the development of these multiple defects.

Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors alpha and gamma.

Peroxisome proliferator-activated receptors (PPARs) alpha and gamma are key regulators of lipid homeostasis and are activated by a structurally diverse group of compounds including fatty acids, eicosanoids, and hypolipidemic drugs such as fibrates and thiazolidinediones. While thiazolidinediones and 15-deoxy-Delta12, 14-prostaglandin J2 have been shown to bind to PPARgamma, it has remained unclear whether other activators mediate their effects through direct interactions with the PPARs or via indirect mechanisms. Here, we describe a novel fibrate, designated GW2331, that is a high-affinity ligand for both PPARalpha and PPARgamma. Using GW2331 as a radioligand in competition binding assays, we show that certain mono- and polyunsaturated fatty acids bind directly to PPARalpha and PPARgamma at physiological concentrations, and that the eicosanoids 8(S)-hydroxyeicosatetraenoic acid and 15-deoxy-Delta12,14-prostaglandin J2 can function as subtype-selective ligands for PPARalpha and PPARgamma, respectively. These data provide evidence that PPARs serve as physiological sensors of lipid levels and suggest a molecular mechanism whereby dietary fatty acids can modulate lipid homeostasis.

Fatty acid signaling in Arabidopsis.

Many organisms use fatty acid derivatives as biological regulators. In plants, for example, fatty acid-derived signals have established roles in the regulation of developmental and defense gene expression. Growing numbers of these compounds, mostly derived from fatty acid hydroperoxides, are being characterized. The model plant Arabidopsis thaliana is serving a vital role in the discovery of fatty acid-derived signal molecules and the genetic analysis of their synthesis and action. The Arabidopsis genome sequencing project, the availability of large numbers of mutants in fatty acid biosynthesis and signal transduction, as well as excellent pathosystems, make this plant a tremendously useful model for research in fatty acid signaling. This review summarizes recent progress in understanding fatty acid signaling in A. thaliana and highlights areas of research where progress is rapid. Particular attention is paid to the growing literature on the jasmonate family of regulators and their role in defense against insects and microbial pathogens.

Differential involvement of peroxisome-proliferator-activated receptors alpha and delta in fibrate and fatty-acid-mediated inductions of the gene encoding liver fatty-acid-binding protein in the liver and the small intestine.

Liver fatty-acid-binding protein (L-FABP) is a cytoplasmic polypeptide that binds with strong affinity especially to long-chain fatty acids (LCFAs). It is highly expressed in both the liver and small intestine, where it is thought to have an essential role in the control of the cellular fatty acid (FA) flux. Because expression of the gene encoding L-FABP is increased by both fibrate hypolipidaemic drugs and LCFAs, it seems to be under the control of transcription factors, termed peroxisome-proliferator-activated receptors (PPARs), activated by fibrate or FAs. However, the precise molecular mechanism by which these regulations take place remain to be fully substantiated. Using transfection assays, we found that the different PPAR subtypes (alpha, gamma and delta) are able to mediate the up-regulation by FAs of the gene encoding L-FABP in vitro. Through analysis of LCFA- and fibrate-mediated effects on L-FABP mRNA levels in wild-type and PPARalpha-null mice, we have found that PPARalpha in the intestine does not constitute a dominant regulator of L-FABP gene expression, in contrast with what is known in the liver. Only the PPARdelta/alpha agonist GW2433 is able to up-regulate the gene encoding L-FABP in the intestine of PPARalpha-null mice. These findings demonstrate that PPARdelta can act as a fibrate/FA-activated receptor in tissues in which it is highly expressed and that L-FABP is a PPARdelta target gene in the small intestine. We propose that PPARdelta contributes to metabolic adaptation of the small intestine to changes in the lipid content of the diet.

Postinfarction heart failure in rats is associated with upregulation of GLUT-1 and downregulation of genes of fatty acid metabolism.

OBJECTIVES: Increasing evidence suggests that left ventricular remodeling is associated with a shift from fatty acid to glucose metabolism for energy production. The aim of this study was to determine whether left ventricular remodeling with and without late-onset heart failure after myocardial infarction is associated with regional changes in the expression of regulatory proteins of glucose or fatty acid metabolism. METHODS: Myocardial infarction was induced in rats by ligation of the left anterior descending coronary artery (LAD). In infarcted and sham-operated hearts the peri-infarction region (5-mm zone surrounding the region at risk), the interventricular septum and the right ventricular free wall were separated for analysis. RESULTS: At 8 and 20 weeks after LAD ligation, the peri-infarction region and the septum exhibited marked re-expression of atrial natriuretic factor [+252+/-37 and +1093+/-279%, respectively, in the septum (P<0.05)] and of alpha-smooth muscle actin [+34+/-10 and +43+/-14%, respectively, in the septum (P<0.05)]. At 8 weeks, when left ventricular hypertrophy was present without signs of heart failure, myocardial mRNA expression of glucose transporters (GLUT-1 and GLUT-4) was not altered, whereas mRNA expression of medium-chain acyl-CoA dehydrogenase (MCAD) was significantly reduced in the peri-infarction region (-25+/-7%; P<0.05). In hearts exhibiting heart failure 20 weeks after infarct-induction there was a change in all three ventricular regions of both mRNA and protein content of GLUT-1 [+72+/-28 and +121+/-15%, respectively, in the peri-infarction region (P<0.05)] and MCAD [-29+/-9 and -56+/-4%, respectively, in the peri-infarction region (P<0.05)]. CONCLUSION: In rats with large myocardial infarction, progression from compensated remodeling to overt heart failure is associated with upregulation of GLUT-1 and downregulation of MCAD in both the peri-infarction region and the septum.

Metabolic effects of parenteral nutrition enriched with n-3 polyunsaturated fatty acids in critically ill patients

BACKGROUND & AIMS: n-3 fatty acids are expected to downregulate the inflammatory responses, and hence may decrease insulin resistance. On the other hand, n-3 fatty acid supplementation has been reported to increase glycemia in type 2 diabetes. We therefore assessed the effect of n-3 fatty acids delivered with parenteral nutrition on glucose metabolism in surgical intensive care patients. METHODS: Twenty-four surgical intensive care patients were randomized to receive parenteral nutrition providing 1.25 times their fasting energy expenditure, with 0.25 g of either an n-3 fatty acid enriched-or a soy bean-lipid emulsion. Energy metabolism, glucose production, gluconeogenesis and hepatic de novo lipogenesis were evaluated after 4 days. RESULTS: Total energy expenditure was significantly lower in patients receiving n-3 fatty acids (0.015+/-0.001 vs. 0.019+/-0.001 kcal/kg/min with soy bean lipids (P<0.05)). Glucose oxidation, lipid oxidation, glucose production, gluconeogenesis, hepatic de novo lipogenesis, plasma glucose, insulin and glucagon concentrations did not differ (all P>0.05) in the 2 groups. CONCLUSIONS: n-3 fatty acids were well tolerated in this group of severely ill patients. They decreased total energy expenditure without adverse metabolic effects.

The peroxisomal Acyl-CoA thioesterase Pte1p from Saccharomyces cerevisiae is required for efficient degradation of short straight chain and branched chain fatty acids.

The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.

Increased flow of fatty acids toward beta-oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransferase activity or synthesizing medium-chain-length fatty acids.

Synthesis of polyhydroxyalkanoates (PHAs) from intermediates of fatty acid beta-oxidation was used as a tool to study fatty acid degradation in developing seeds of Arabidopsis. Transgenic plants expressing a peroxisomal PHA synthase under the control of a napin promoter accumulated PHA in developing seeds to a final level of 0. 06 mg g(-1) dry weight. In plants co-expressing a plastidial acyl-acyl carrier protein thioesterase from Cuphea lanceolata and a peroxisomal PHA synthase, approximately 18-fold more PHA accumulated in developing seeds. The proportion of 3-hydroxydecanoic acid monomer in the PHA was strongly increased, indicating a large flow of capric acid toward beta-oxidation. Furthermore, expression of the peroxisomal PHA synthase in an Arabidopsis mutant deficient in the enzyme diacylglycerol acyltransferase resulted in a 10-fold increase in PHA accumulation in developing seeds. These data indicate that plants can respond to the inadequate incorporation of fatty acids into triacylglycerides by recycling the fatty acids via beta-oxidation and that a considerable flow toward beta-oxidation can occur even in a plant tissue primarily devoted to the accumulation of storage lipids.

Decreased expression of peroxisome proliferator-activated receptor alpha and liver fatty acid binding protein after partial hepatectomy of rats and mice.

BACKGROUND AIMS: Marked changes in metabolism, including liver steatosis and hypoglycemia, occur after partial hepatectomy. Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a nuclear hormone receptor that is activated by fatty acids and involved in hepatic fatty acid metabolism and regeneration. Liver fatty acid binding protein (LFABP) is an abundant protein in liver cytosol whose expression is regulated by PPAR alpha. It is involved in fatty acid uptake and diffusion and in PPAR alpha signaling. The aim of this study was to investigate the expression of PPAR alpha and LFABP during liver regeneration. METHODS: Male Sprague-Dawley rats and male C57 Bl/6 mice were subjected to 2/3 hepatectomy and LFABP and PPAR alpha mRNA and protein levels were measured at different time points after surgery. The effect of partial hepatectomy was followed during 48 h in rats and 72 h in mice. RESULTS: PPAR alpha mRNA and protein levels were decreased 26 h after hepatectomy of rats. The LFABP mRNA and protein levels paralleled those of PPAR alpha and were also decreased 26 h after hepatectomy. In mice, the mRNA level was decreased after 36 and 72 h after hepatectomy. In this case, LFABP mRNA levels decreased more slowly after partial hepatectomy than in rats. CONCLUSIONS: A marked decrease in PPAR alpha expression may be important for changed gene expression, e.g. LFABP, and metabolic changes, such as hypoglycemia, during liver regeneration.

Role of the transcriptional factor C/EBPbeta in free fatty acid-elicited beta-cell failure.

Fatty acids can favour the development of Type 2 diabetes by reducing insulin secretion and inducing apoptosis of pancreatic beta-cells. Here, we show that sustained exposure of the beta-cell line MIN6 or of isolated pancreatic islets to the most abundant circulating fatty acid palmitate increases the level of C/EBPbeta, an insulin transcriptional repressor. In contrast, two unsaturated fatty acids, oleate and linoleate were without effect. The induction of C/EBPbeta elicited by palmitate was prevented by inhibiting the ERK1/2 MAP kinase pathway or by reducing mitochondrial fatty acid oxidation with an inhibitor of Carnitine Palmitoyl Transferase-1. Overexpression of C/EBPbeta mimicked the detrimental effects of palmitate and resulted in a drastic reduction in insulin promoter activity, impairment in the capacity to respond to secretory stimuli and an increase in apoptosis. Our data suggest a potential involvement of C/EBPbeta as mediator of the deleterious effects of unsaturated free fatty acids on beta-cell function.

Analysis of the beta-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway.

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Delta3,Delta2-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10-25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through beta-oxidation was more severely reduced in mutants devoid of Delta3,Delta2-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Delta3,Delta2-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.

The Peroxisomal Enzyme L-PBE Is Required to Prevent the Dietary Toxicity of Medium-Chain Fatty Acids.

Specific metabolic pathways are activated by different nutrients to adapt the organism to available resources. Although essential, these mechanisms are incompletely defined. Here, we report that medium-chain fatty acids contained in coconut oil, a major source of dietary fat, induce the liver ω-oxidation genes Cyp4a10 and Cyp4a14 to increase the production of dicarboxylic fatty acids. Furthermore, these activate all ω- and β-oxidation pathways through peroxisome proliferator activated receptor (PPAR) α and PPARγ, an activation loop normally kept under control by dicarboxylic fatty acid degradation by the peroxisomal enzyme L-PBE. Indeed, L-pbe(-/-) mice fed coconut oil overaccumulate dicarboxylic fatty acids, which activate all fatty acid oxidation pathways and lead to liver inflammation, fibrosis, and death. Thus, the correct homeostasis of dicarboxylic fatty acids is a means to regulate the efficient utilization of ingested medium-chain fatty acids, and its deregulation exemplifies the intricate relationship between impaired metabolism and inflammation.