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

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.