The clearance of circulating glucose and long-chain fatty acids (FA) into skeletal muscle involves the translocation of glucose transporter GLUT4, fatty acid translocase (FAT/CD36), plasma membrane associated fatty acid binding protein (FABPpm) and fatty acid transport protein (FATP) 1 and 4 to the plasma membrane (PM). FAT/CD36 also appears to participate in the regulation of mitochondrial FA oxidation. Metabolic challenges are known to increase FA transport and/or oxidation, but whether this is solely attributable to the translocation of FAT/CD36 to the sarcolemma and/or mitochondria is unknown. Moreover, the signaling and trafficking pathways involved in the translocation of FA transporters are largely unexplored. In this thesis it was found that FA transport was markedly increased following insulin (+2.9-fold) or contraction (+1.7-fold) stimulation of skeletal muscle, along with the PM contents of FAT/CD36 (+78%, +55%,), FABPpm (+61%, +62%), FATP1 (+84%, +61%) and FATP4 (+60%, +66%) (p<0.05). Upon combining the two stimuli, only the translocation of FAT/CD36 (+179%) and FATP1 (+125%) to the PM was additive, suggesting that these transporters may reside in distinct insulin-sensitive and contraction-sensitive intracellular compartments. The translocation of FA transporters may involve the insulin-signaling protein Akt2. It was found that insulin-stimulated FA transport and PM translocation of FA transporters was essentially prevented in Akt2 knockout mice. Following contraction, FA transport was also markedly blunted, along with an impaired translocation of both FAT/CD36 and FATP1, but not FABPpm or FATP4. FA oxidation and mitochondrial FAT/CD36 appearance were also inhibited following muscle contraction in knockout mice (p<0.05). Whether the GLUT4 trafficking protein Munc18c is important for the vesicular re-distribution of FA transporters to the PM or mitochondria was also investigated. FA uptake was comparably increased 1.4 fold with insulin and contraction in both wildtype and heterozygous Munc18c-/+ mice, as were PM FA transporters FAT/CD36 (+82%, +84%), FABPpm (+39%, +43%), FATP1 (+40%, +38%) and FATP4 (+33%, +32%) (p<0.05). Contraction-stimulated mitochondrial FA oxidation was also increased similarly in wildtype (+39%) and Munc18c-/+ mice (+33%). These studies demonstrate that a number of FA transporters are involved in upregulating skeletal muscle FA transport, although their signaling and trafficking pathways may differ from that of GLUT4.

Glucagon-like peptide-1 (GLP-1) is an anti-diabetic intestinal L cell hormone. The monounsaturated fatty acid, oleic acid (OA), is an effective GLP-1 secretagogue that crosses the cell membrane by an unknown mechanism. Immunoblotting demonstrated the presence of fatty acid transport proteins (CD36 and FATP1, 3 and 4) in the murine GLUTag L cell model. The cells demonstrated specific 3H-OA uptake, which was dose-dependently inhibited by unlabeled-OA. Phloretin and SSO, inhibitors of carrier-mediated transport and CD36, respectively, also significantly decreased 3H-OA uptake, as did knocking down FATP4 by transfection of siRNA. OA dose-dependently increased GLP-1 secretion in GLUTag cells, while phloretin and FATP4 knockdown, but not SSO, decreased this response. OA injected directly into the ileum of wild-type mice increased plasma GLP-1 levels; in contrast, preliminary findings suggest decreased GLP-1 levels in FATP4 null mice at 60 min. Collectively, these findings indicate a role for FATP4 in OA-induced GLP-1 secretion.

Glucagon-like peptide-1 (GLP-1) is an anti-diabetic intestinal L cell hormone. The monounsaturated fatty acid, oleic acid (OA), is an effective GLP-1 secretagogue that crosses the cell membrane by an unknown mechanism. Immunoblotting demonstrated the presence of fatty acid transport proteins (CD36 and FATP1, 3 and 4) in the murine GLUTag L cell model. The cells demonstrated specific 3H-OA uptake, which was dose-dependently inhibited by unlabeled-OA. Phloretin and SSO, inhibitors of carrier-mediated transport and CD36, respectively, also significantly decreased 3H-OA uptake, as did knocking down FATP4 by transfection of siRNA. OA dose-dependently increased GLP-1 secretion in GLUTag cells, while phloretin and FATP4 knockdown, but not SSO, decreased this response. OA injected directly into the ileum of wild-type mice increased plasma GLP-1 levels; in contrast, preliminary findings suggest decreased GLP-1 levels in FATP4 null mice at 60 min. Collectively, these findings indicate a role for FATP4 in OA-induced GLP-1 secretion.

Fatty acid (FA) transport proteins are important regulators of FA uptake at the cell surface and the mitochondria where they are oxidized. Tight regulation of this process is necessary in order to meet metabolic requirements, while preventing excess lipid accumulation. In an obese state, there is an increase in FA uptake and increased storage of lipids in skeletal muscle, including diacylglycerol (DAG) and ceramides, which interfere with insulin-stimulated glucose uptake. Leptin administration has been shown to reduce muscle triacylglycerol accumulation and restore insulin response in obese rodents. However, it is not known whether this is mediated through a redistribution of the FA transport proteins to the cell surface and mitochondria. In addition to hyperglycemia, post-prandial lipidemia is also observed in the obese state, suggesting a resistance to insulin-stimulated FA uptake. The possibility that insulin-stimulated FA transporter translocation is impaired has received little attention. Lastly, while recent studies have demonstrated that the transverse (t)-tubules may be an important site for glucose uptake in muscle, this has not yet been examined with regards to the FA transporters. In the first study of this thesis, the recovery of insulin response with short-term (2 week) chronic leptin administration in high-fat fed rats was associated with a decrease in muscle reactive lipid species (DAG, ceramide) and an increase in markers of oxidative capacity. Contrary to our expectations, this was not mirrored by an alteration in the distribution of FA transport proteins (FAT/CD36 or FABPpm) at the sarcolemma or the two major mitochondrial populations. To gain further insight into FA transporters and their localization at the cell surface, the second study of this thesis analyzed both the sarcolemma and t-tubules (constitute 40 and 60% of the cell surface, respectively). The novel observation was made that the t-tubules contain FA transport proteins (FAT/CD36, FABPpm, FATP1 and FATP4), and that the distribution and response of these transporters to acute metabolic stimuli (insulin and muscle contraction) was unique from that of the sarcolemma. The third study of this thesis characterized the translocation of FA transport proteins in response to insulin in the obese, insulin resistant Zucker rat. FA transport proteins were chronically increased on both membrane fractions in muscle from the obese rats. Furthermore, a blunting of the insulin-induced translocation of FA transporters to both cell surface domains was observed, demonstrating that insulin resistance extends to the movement of FA as well as glucose transport proteins. The t-tubules appear to play an important role regarding substrate uptake. Together the data from this thesis suggests that a chronic elevation in FA transporters at both cell surface domains contributes to lipid accumulation in obese skeletal muscle, and that reduced sensitivity of both FA and glucose transport proteins to translocate in response to insulin may explain the lipidemia and hyperglycemia that often characterizes post-prandial situations in the obese condition. As the prevalence of obesity reaches epidemic proportions, research into the functional role of FA transport proteins in the progression of obesity related pathologies is warranted as we work to further our knowledge of this significant health issue. / Natural Sciences and Engineering Research Council, Canadian Institute of Health Research

No description available.

Die peroxisomalen ABC-Transporter Pxa1p und Pxa2p sind Halbtransporter. Genetische Studien ergaben Hinweise, dass sie zur Bildung aktiver Transporter heterodimerisieren und am Import von langkettigen Fettsäuren in die Peroxisomen von S. cerevisiae beteiligt sind. Es wurden epitopmarkierte Varianten der Proteine als Komplex isoliert. Damit wurde gezeigt, dass Pxa1p und Pxa2p ein stabiles Heterodimer bilden. Zur Charakterisierung der ATP Bindeeigenschaften wurden die Transporter mit 8-azido-[alpha-32P]-ATP inkubiert und kovalent verknüpft. Dabei konnte gezeigt werden, dass Pxa1p und Pxa2p eine unsymmetrische Bindung des ATP Analogons aufweisen. Pxa2p bindet deutlich mehr azido-ATP als Pxa1p, bei sehr ähnlichen Dissoziationskonstanten. Die reduzierte ATP Bindung von Pxa1p spiegelt sich durch degenerierte Sequenzmotive der an der ATP Bindung beteiligten Sequenzen wieder. Die isolierten ABC-Transporter wurden für ATPase Messungen eingesetzt. Sie zeigten eine basale ATPase Aktivität, die durch Zugabe langkettiger Coenzym A aktivierter Fettsäuren, wie Oleoyl-CoA und Palmitoyl-CoA stimulierbar war. Eine Lysin Mutation im Walker A Motiv von Pxa1p hatte keine Funktionalitätseinbuße zur Folge. Dieselbe Mutation bei Pxa2p führte im Wachstumstest auf Festmedium mit Ölsäure als Kohlenstoffquelle zu einem deutlich verlangsamten Wachstum. Diese Ergebnisse korrespondieren mit der beobachteten unsymmetrischen ATP Bindung von Pxa1p und Pxa2p, da bei dem schwächer bindenden Pxa1p die Mutation wirkungslos blieb. Keine Übereinstimmung war bei den ATPase Aktivitätsmessungen der aufgereinigten Mutanten zu verzeichnen. Beide Mutanten zeigten eine unbeeinträchtigte ATPase Aktivität. Die ABC-Transporter wurden in Proteoliposomen eingebaut und für Transportmessungen mit einem Spin-Label markierten Oleoyl-CoA verwendet. Die Transportmessungen zeigten einen ATP abhängigen Transport, woraus geschlossen wurde, dass Pxa1p-Pxa2p tatsächlich Coenzym A Ester langkettiger Fettsäuren transportiert. / The peroxisomal ABC-transporters Pxa1p and Pxa2p are half transporters. Previous genetic investigations have demonstrated that Pxa1p and Pxa2p have to dimerise in order to build a functional transporter, which is very likely involved in the import of long chain fatty acids into peroxisomes of S. cerevisiae. In this work, tagged versions of the proteins were purified as a complex. This proved for the building of a stable hetero dimer. For characterisation of the ATP binding properties, the transporters were incubated and cross linked with 8-azido-[alpha-32P]-ATP. This revealed an asymmetric binding of the ATP analogue. Pxa2p binds much more azido-ATP, than Pxa1p, while the dissociation constants are rather similar. The poorer ATP binding of Pxa1p is reflected by degenerated sequence motifs in the nucleotide binding fold. The purified ABC-transporters have been used for ATPase assays. They showed a basal ATPase activity, which could be stimulated by addition of long chain fatty acid CoAs, like oleoyl-CoA and palmitoyl-CoA. Mutants with a lysine mutation in the walker A motive of Pxa1p led to no functional impairment, while the corresponding lysine mutation in Pxa2p led to reduced growth on agar plates with oleic acid as sole carbon source. The result corresponds with the ATP binding properties of Pxa1p. Because of the poorer ATP binding, even in the wild type protein, the mutation was not supposed to have a big influence. No accordance was found in respect to the ATPase measurements of the isolated mutant proteins. Both mutants revealed unaffected ATPase activity. The purified ABC-transporters were reconstituted in proteoliposomes and used for translocation assays of a spin-labelled oleoyl-CoA derivative. The measurements revealed an ATP dependent transport of the oleoyl-CoA analogue. This led to the conclusion, that Pxa1p-Pxa2p is indeed the transporter of long chain acetyl CoA esters, which were transported in an ATP dependent manner.

No description available.

Coleoptera is an order of insects well known as beetles. Most coleopteran species are phytophagous insects and for this reason are essential to crop and storage pests such as the Tribolium castaneum. Lipid metabolism is vital for the biological functions of insects, playing a role in the generation of metabolic energy and other cellular processes. Fatty acid transport proteins (FATPs) play a crucial role in the transport of extracellular fatty acids to cells, have a conserved sequence between species and are involved in the synthesis of hormones and pheromones. Recent studies show that silencing the gene for FATPs through interfering RNAi techniques (RNAi) in insects affects fatty acid uptake and pheromone synthesis. This work aims to characterize proteins homologous to FATPs present in the genome of Tribolium castaneum, to evaluate the gene expression in tissues, developmental stages and insects treated with Orlistat and to evaluate the effect of FATP silencing on energy metabolism. Bioinformatics analyzes were performed with the amino acid sequences, and real-time PCR evaluated the gene expression. The effects of the drug Orlistat were evaluated through qPCR and analysis of nutritional index. The search for sequences in the T. castaneum genome revealed two sequences of proteins homologous to FATPs and bioinformatic analysis was performed. The study of the gene expression of FATPs by qPCR demonstrated more significant expression of the two genes in the fat body of larvae and many expressions in all stages of development of the insect, with higher expression in the pupa stage. The effects of Orlistat on the expression of FATPs evidenced the influence of diet composition on the regulation of the gene expression of these proteins. Gene silencing of TcasFATP was achieved, but no direct effects on the energetic dynamics of the larvae were observed since triacylglycerol levels, and β-oxidation rates were not affected. Thus, more detailed studies with the use of gene silencing will be necessary to characterize FATPs better and elucidate their role in insect energy metabolism. / FAPEAL - Fundação de Amparo à Pesquisa do Estado de Alagoas / Os coleópteros constituem uma ordem de insetos bastante conhecidos como besouros. A maioria das espécies de coleóptero são insetos fitófagos e por esta razão constituem importantes pragas de culturas e de armazenamento, como o Tribolium castaneum. O metabolismo de lipídeos é importante para as funções biológicas de insetos, exercendo papel na geração de energia metabólica e em outros processos celulares. As proteínas transportadoras de ácidos graxos (FATPs) exercem papel crucial no transporte de ácidos graxos extracelulares para as células, possuem sequência conservada entre as espécies e estão envolvidas na síntese de hormônios e feromônios. Estudos recentes mostram que o silenciamento do gene para FATPs através de técnicas de RNA de interferência (RNAi) em insetos afetam a absorção de ácidos graxos e a síntese de feromônio. Este trabalho tem como objetivo caracterizar proteínas homólogas à FATPs presentes no genoma de Tribolium castaneum, avaliar a expressão gênica nos tecidos, fases de desenvolvimento e em insetos tratados com Orlistate e avaliar o efeito do silenciamento de FATPs no metabolismo energético. Foram realizadas análises bioinformáticas com as sequências de aminoácidos e a avaliação da expressão gênica foi realizada por PCR em tempo real. Os efeitos do fármaco Orlistate foram avaliados através de qPCR e análise dos índices nutricionais. A busca de sequências no genoma do T. castaneum revelou duas sequências de proteínas homólogas à FATPs e a análise bioinformática foi realizada. O estudo da expressão gênica de FATPs por qPCR demonstrou maior expressão dos dois genes no corpo gorduroso de larvas e expressão considerável em todos os estágios de desenvolvimento do inseto, com maior expressão no estágio de pupa. Os efeitos do Orlistate na expressão das FATPs evidenciaram a influência da composição da dieta na regulação da expressão gênica dessas proteínas. O silenciamento gênico de TcasFATP foi alcançado, mas não foram observados efeitos diretos na dinâmica energética das larvas, pois os níveis de triacilglicerol e as taxas de β-oxidação não foram afetadas. Dessa forma, estudos mais detalhados com uso do silenciamento gênico serão necessários para melhor caracterização funcional das FATPs e elucidação do seu papel no metabolismo energético do inseto.