Adrenergic signalling is part of the sympathetic nervous system and is activated upon stimulation by the catecholamines epinephrine and norepinephrine. This regulates heart rate, energy mobilization, digestion and helps to divert blood flow to important organs. Insulin is released to regulate metabolism of carbohydrates, fats and proteins, mainly by taking up glucose from the blood. The insulin and the catecholamine hormone systems are normally working as opposing metabolic regulators and are therefore thought to antagonize each other. One of the major regulators involved in insulin signalling is the mechanistic target of rapamycin (mTOR). There are two different complexes of mTOR; mTORC1 and mTORC2, and they are essential in the control of cell growth, metabolism and energy homeostasis. Since mTOR is one of the major signalling nodes for anabolic actions of insulin it was thought that catecholamines might oppose this action by inhibiting the complexes. However, lately there are studies demonstrating that this may not be the case. mTOR is for instance part of the adrenergic signalling pathway resulting in hypertrophy of cardiac and skeletal muscle cells and inhibition of smooth muscle relaxation and helps to regulate browning in white adipose tissue and thermogenesis in brown adipose tissue (BAT). In this thesis I show that β-adrenergic signalling leading to glucose uptake occurs independently of insulin in skeletal muscle and BAT, and does not activate either Akt or mTORC1, but that the master regulator of this pathway is mTORC2. Further, my co-workers and I demonstrates that β-adrenergic stimulation in skeletal muscle and BAT utilizes different glucose transporters. In skeletal muscle, GLUT4 is translocated to the plasma membrane upon stimulation. However, in BAT, β-adrenergic stimulation results in glucose uptake through translocation of GLUT1. Importantly, in both skeletal muscle and BAT, the role of mTORC2 in β-adrenergic stimulated glucose uptake is to regulate GLUT-translocation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

A digestão nos insetos ocorre no intestino médio de forma compartimentada. A digestão inicial dos polímeros ocorre no interior da membrana peritrófica. Os oligômeros resultantes difundem-se para o espaço luminal exterior à membrana peritrófica onde são atacados por outras enzimas. Na digestão final os dímeros resultantes são hidrolisados por enzimas imobilizadas na superfície do epitélio do intestino médio. Após o processo de digestão final os monômeros são absorvidos pelas células do epitélio intestinal. Os Díptera ditos superiores, incluindo a mosca doméstica, apresentam peculiaridades digestivas que aparentemente resultam de adaptações para digerir uma dieta que consiste principalmente de bactérias. No ventrículo anterior ocorre uma diminuição no conteúdo de amido do bolo alimentar. Na porção seguinte, o bolo alimentar passa para o ventrículo médio onde as bactérias são mortas pela ação combinada de baixo pH, uma lisozima digestiva e uma proteinase tipo catepsina D. O material liberado das bactérias é digerido no ventrículo posterior, como ocorre no ventrículo inteiro da maioria dos insetos de outros grupos taxonômicos. Com o objetivo de compreender a peculiar digestão em Musca domestica, foram utilizadas suas larvas para identificar funcionalmente as regiões absortivas de nutrientes, identificar as moléculas envolvidas na absorção de nutrientes, identificar as moléculas envolvidas com tamponamento e fluxos de fluidos intestinais, sequenciar as enzimas digestivas principais e identificar os seus sítios de secreção. Experimentos fisiológicos de absorção de glicose e análises de atividade enzimática permitiram acessar de maneira direta os aspectos da digestão. Contudo, experimentos de sequenciamento de bibliotecas de cDNA, análise de sequências transcritas e verificação de expressão de genes em diferentes tecidos foram abordagens fundamentais na identificação das moléculas subjacentes aos processos fisiológicos intestinas de Musca domestica. Os indícios de que absorção de glicose no intestino de Musca domestica se dê por transportadores do tipo SGLT, com a possível participação de facilitadores do tipo GLUT, permitem estabelecer um foco para futuros estudos. A descrição de sequências relacionadas ao tamponamento intestinal permitiu ampliar a discussão sobre tal processo. Ao detalhar os sítios de expressão da subunidade a da V-ATPase, do canal de cloreto e do transportador de amônia foi possível testar o modelo de tamponamento proposto anteriormente e propor a participação de outras moléculas no processo. Sequências correspondentes as atividades de carboxipeptidase, maltase e aminopeptidase descritas na literatura foram pesquisadas, gerando sequências candidatas a codificarem as referidas enzimas. Com isso, é possível descrever a digestão de oligômeros e dímeros com base nos genes transcritos e nas sequências de aminoácidos que formam as enzimas digestivas. A descoberta da sequência que transcreve uma metaloproteinase, por sua vez, abre caminhos para a descrição e caracterização de sua atividade proteolítica nos tecidos digestivos da larva de Musca domestica. Essa análise permitiu também elucidar a localização dos sítios de expressão e, portanto, as zonas de secreção de enzimas. De maneira geral, este estudo contribuiu para a compreensão de diversos aspectos da digestão de Musca domestica, elucidando questões da particular fisiologia digestiva desse inseto. / Digestion in insects occurs in the midgut in a compartmentalized way. Initial digestion takes place inside the peritrophic membrane. The resulting oligomers diffuse into the luminal space outside the peritrophic membrane where they are hydrolyzed by other enzymes. In the final digestion, the resulting dimers are hydrolyzed by enzymes immobilized on the midgut epithelium. After the final digestion, the monomers are absorbed by intestinal epithelial cells. The so-called higher Diptera, including the house fly, have digestive peculiarities apparently resulting of adaptations to digest a diet consisting mainly of bacteria. In the anterior midgut there is a decrease in the starch content of the food bolus. The bolus now passes into the middle midgut, where bacteria are killed by the combined action of low pH, a special lysozyme and a cathepsin D-like proteinase. Finally, the material released by bacteria is digested in the posterior midgut, as is observed in the whole midgut of insects of other taxa. In order to understand the peculiar digestion in Musca domestica, the larvae were used to identify (a) the functionally the nutrient absorptive regions, (b) the molecules involved in the absorption of nutrients, (c) the molecules involved in buffering and fluid flows, (d) the cDNA sequences corresponding to intestinal digestive enzymes, (e) the main sites of secretion. Physiological experiments of glucose absorption and enzyme activity analysis allowed a direct access to aspects of digestion. Otherwise, cDNA library sequencing followed by sequence annotation and tissue-specific expression analysis were fundamental approaches in the understanding of intestinal physiology of Musca domestica. Evidence that glucose absorption in the gut of Musca domestica occurs through SGLT-like transporters, with the possible participation of facilitators GLUT-like, allowed us to establish a focus for future studies. The description of cDNA sequences corresponding to proteins putatively responsible for intestinal buffering widened the discussion of this process. The finding of the expression sites of V-ATPase subunits, chloride channel, and ammonia transporter led to revising the present buffering model and the inclusion of other molecules in the process. The cDNA sequences corresponding to the activities of carboxypeptidase, aminopeptidase and maltase described in the literature were searched for as candidate sequences to encode those enzymes. This made it possible to describe the digestion of oligomers and dimers based on transcribed genes and enzyme amino acid sequences. The discovery of the metalloproteinase transcribing sequence opened a new research line: the description and characterization of its proteolytic activity in the midgut of the Musca domestica larvae. This study also allowed elucidating the location of digestive enzyme expression sites and, therefore, the putative zones of enzyme secretion. Overall, this study contributed to understanding many aspects of digestion of Musca domestica, clarifying aspects of the peculiar digestive physiology of this insect.

Morphologic and metabolic change is a known property of the adult brain. A number of behavioural tasks alter local cerebral blood flow and glucose utilisation. The expression of the glucose transporter 1 (GLUT1), which allows the entry of glucose to the brain, also has been shown to change in response to long-lasting neuronal activation. However, little is known about the effect of acute neuronal activation on GLUT1 expression. Using immunohistochemistry and Western blot, we investigated cerebral GLUT1 expression and vasculature density in mice undergoing a 48-hour voluntary wheel running period. The results showed that the striatum was the main region where GLUT1 protein was up-regulated: There was a trend for GLUT1 expression and blood vessels density to be associated with the distance run during the experiment. These results indicate that short-term increased neuronal activation is associated with rapid changes in glucose transport and possibly vascular remodelling.

Morphologic and metabolic change is a known property of the adult brain. A number of behavioural tasks alter local cerebral blood flow and glucose utilisation. The expression of the glucose transporter 1 (GLUT1), which allows the entry of glucose to the brain, also has been shown to change in response to long-lasting neuronal activation. However, little is known about the effect of acute neuronal activation on GLUT1 expression. Using immunohistochemistry and Western blot, we investigated cerebral GLUT1 expression and vasculature density in mice undergoing a 48-hour voluntary wheel running period. The results showed that the striatum was the main region where GLUT1 protein was up-regulated: There was a trend for GLUT1 expression and blood vessels density to be associated with the distance run during the experiment. These results indicate that short-term increased neuronal activation is associated with rapid changes in glucose transport and possibly vascular remodelling.

Morphologic and metabolic change is a known property of the adult brain. A number of behavioural tasks alter local cerebral blood flow and glucose utilisation. The expression of the glucose transporter 1 (GLUT1), which allows the entry of glucose to the brain, also has been shown to change in response to long-lasting neuronal activation. However, little is known about the effect of acute neuronal activation on GLUT1 expression. Using immunohistochemistry and Western blot, we investigated cerebral GLUT1 expression and vasculature density in mice undergoing a 48-hour voluntary wheel running period. The results showed that the striatum was the main region where GLUT1 protein was up-regulated: There was a trend for GLUT1 expression and blood vessels density to be associated with the distance run during the experiment. These results indicate that short-term increased neuronal activation is associated with rapid changes in glucose transport and possibly vascular remodelling.

The structure-function relationship explains how the human erythrocyte glucose transport protein (GLUT1) catalyzes sugar transport across the plasma membrane. This work investigates the glucose transport mechanism, the structural arrangement and dynamics of GLUT1 membrane-spanning α-helices, the molecular basis for glucose transport regulation by ATP, and how cysteine accessibility contributes to GLUT1 structure. A rapid kinetics approach was applied to examine the conformational changes GLUT1 undergoes during the transport cycle. To transition from a global to molecular focus, a novel mass spectrometry technique was developed to resolve GLUT1 sequence that is associated either with membrane embedded GLUT1 subdomains or with water exposed domains. By studying accessibility changes of specific amino acids to covalent modification by a Sulfo-NHS-LC-Biotin probe, specific protein regions associated with glucose transport modulation by ATP were identified. Finally, mass spectrometry was applied to examine cysteine residue accessibility under native and reducing conditions. This thesis presents data supporting the isolation of an intermediate, occluded GLUT1 conformational state that temporally bridges import and export configurations during glucose translocation. Our results confirm that amphipathic α-helices line the translocation pathway and promote interactions with the aqueous environment and substrate. In addition, we show that GLUT1 is conformationally dynamic, undergoes reorganization in the cytoplasmic region in response to ATP modulation, and that GLUT1 contains differentially exposed cysteine residues that affect its folding.

Morphologic and metabolic change is a known property of the adult brain. A number of behavioural tasks alter local cerebral blood flow and glucose utilisation. The expression of the glucose transporter 1 (GLUT1), which allows the entry of glucose to the brain, also has been shown to change in response to long-lasting neuronal activation. However, little is known about the effect of acute neuronal activation on GLUT1 expression. Using immunohistochemistry and Western blot, we investigated cerebral GLUT1 expression and vasculature density in mice undergoing a 48-hour voluntary wheel running period. The results showed that the striatum was the main region where GLUT1 protein was up-regulated: There was a trend for GLUT1 expression and blood vessels density to be associated with the distance run during the experiment. These results indicate that short-term increased neuronal activation is associated with rapid changes in glucose transport and possibly vascular remodelling.

A digestão nos insetos ocorre no intestino médio de forma compartimentada. A digestão inicial dos polímeros ocorre no interior da membrana peritrófica. Os oligômeros resultantes difundem-se para o espaço luminal exterior à membrana peritrófica onde são atacados por outras enzimas. Na digestão final os dímeros resultantes são hidrolisados por enzimas imobilizadas na superfície do epitélio do intestino médio. Após o processo de digestão final os monômeros são absorvidos pelas células do epitélio intestinal. Os Díptera ditos superiores, incluindo a mosca doméstica, apresentam peculiaridades digestivas que aparentemente resultam de adaptações para digerir uma dieta que consiste principalmente de bactérias. No ventrículo anterior ocorre uma diminuição no conteúdo de amido do bolo alimentar. Na porção seguinte, o bolo alimentar passa para o ventrículo médio onde as bactérias são mortas pela ação combinada de baixo pH, uma lisozima digestiva e uma proteinase tipo catepsina D. O material liberado das bactérias é digerido no ventrículo posterior, como ocorre no ventrículo inteiro da maioria dos insetos de outros grupos taxonômicos. Com o objetivo de compreender a peculiar digestão em Musca domestica, foram utilizadas suas larvas para identificar funcionalmente as regiões absortivas de nutrientes, identificar as moléculas envolvidas na absorção de nutrientes, identificar as moléculas envolvidas com tamponamento e fluxos de fluidos intestinais, sequenciar as enzimas digestivas principais e identificar os seus sítios de secreção. Experimentos fisiológicos de absorção de glicose e análises de atividade enzimática permitiram acessar de maneira direta os aspectos da digestão. Contudo, experimentos de sequenciamento de bibliotecas de cDNA, análise de sequências transcritas e verificação de expressão de genes em diferentes tecidos foram abordagens fundamentais na identificação das moléculas subjacentes aos processos fisiológicos intestinas de Musca domestica. Os indícios de que absorção de glicose no intestino de Musca domestica se dê por transportadores do tipo SGLT, com a possível participação de facilitadores do tipo GLUT, permitem estabelecer um foco para futuros estudos. A descrição de sequências relacionadas ao tamponamento intestinal permitiu ampliar a discussão sobre tal processo. Ao detalhar os sítios de expressão da subunidade a da V-ATPase, do canal de cloreto e do transportador de amônia foi possível testar o modelo de tamponamento proposto anteriormente e propor a participação de outras moléculas no processo. Sequências correspondentes as atividades de carboxipeptidase, maltase e aminopeptidase descritas na literatura foram pesquisadas, gerando sequências candidatas a codificarem as referidas enzimas. Com isso, é possível descrever a digestão de oligômeros e dímeros com base nos genes transcritos e nas sequências de aminoácidos que formam as enzimas digestivas. A descoberta da sequência que transcreve uma metaloproteinase, por sua vez, abre caminhos para a descrição e caracterização de sua atividade proteolítica nos tecidos digestivos da larva de Musca domestica. Essa análise permitiu também elucidar a localização dos sítios de expressão e, portanto, as zonas de secreção de enzimas. De maneira geral, este estudo contribuiu para a compreensão de diversos aspectos da digestão de Musca domestica, elucidando questões da particular fisiologia digestiva desse inseto. / Digestion in insects occurs in the midgut in a compartmentalized way. Initial digestion takes place inside the peritrophic membrane. The resulting oligomers diffuse into the luminal space outside the peritrophic membrane where they are hydrolyzed by other enzymes. In the final digestion, the resulting dimers are hydrolyzed by enzymes immobilized on the midgut epithelium. After the final digestion, the monomers are absorbed by intestinal epithelial cells. The so-called higher Diptera, including the house fly, have digestive peculiarities apparently resulting of adaptations to digest a diet consisting mainly of bacteria. In the anterior midgut there is a decrease in the starch content of the food bolus. The bolus now passes into the middle midgut, where bacteria are killed by the combined action of low pH, a special lysozyme and a cathepsin D-like proteinase. Finally, the material released by bacteria is digested in the posterior midgut, as is observed in the whole midgut of insects of other taxa. In order to understand the peculiar digestion in Musca domestica, the larvae were used to identify (a) the functionally the nutrient absorptive regions, (b) the molecules involved in the absorption of nutrients, (c) the molecules involved in buffering and fluid flows, (d) the cDNA sequences corresponding to intestinal digestive enzymes, (e) the main sites of secretion. Physiological experiments of glucose absorption and enzyme activity analysis allowed a direct access to aspects of digestion. Otherwise, cDNA library sequencing followed by sequence annotation and tissue-specific expression analysis were fundamental approaches in the understanding of intestinal physiology of Musca domestica. Evidence that glucose absorption in the gut of Musca domestica occurs through SGLT-like transporters, with the possible participation of facilitators GLUT-like, allowed us to establish a focus for future studies. The description of cDNA sequences corresponding to proteins putatively responsible for intestinal buffering widened the discussion of this process. The finding of the expression sites of V-ATPase subunits, chloride channel, and ammonia transporter led to revising the present buffering model and the inclusion of other molecules in the process. The cDNA sequences corresponding to the activities of carboxypeptidase, aminopeptidase and maltase described in the literature were searched for as candidate sequences to encode those enzymes. This made it possible to describe the digestion of oligomers and dimers based on transcribed genes and enzyme amino acid sequences. The discovery of the metalloproteinase transcribing sequence opened a new research line: the description and characterization of its proteolytic activity in the midgut of the Musca domestica larvae. This study also allowed elucidating the location of digestive enzyme expression sites and, therefore, the putative zones of enzyme secretion. Overall, this study contributed to understanding many aspects of digestion of Musca domestica, clarifying aspects of the peculiar digestive physiology of this insect.

Protein-mediated sugar transport is nominally absent in normoxic (adequately oxygenated) pigeon erythrocytes. Following exposure to metabolic inhibitors (cyanide or carbonylcyanide-p-trifluoromethoxyphenylhydrazone), pigeon red cells transport sugars by a saturable, stereoselective pathway that is inhibited by cytochalasin B or forskolin. The sugar transport capacity of fully poisoned cells is consistent with a transporter density of approximately 30 carriers per erythrocyte. Immunoblot analyses and competition ELISA indicate that pigeon red cells contain approximately 200 copies of an integral plasma membrane protein immunologically related to the glucose transporter isoform GLUT1. GLUT1 is quantitatively restricted to the plasma membrane at all times. Pigeon red cells and brain lack proteins immunologically related to the sugar transporter isoforms GLUT3 and GLUT4. Specific immunodepletion of red cell GLUT1 content results in the subsequent loss of reconstitutable protein-mediated sugar transport. These findings demonstrate that avian erythrocyte sugar transport is mediated by a GLUT1-like sugar transport protein and that sugar transport stimulation by metabolic inhibitors results from derepression of cell surface sugar transport proteins. Lysis-resealing experiments suggest that derepression is a glutathione (OSH) dependent phenomenon. This mechanism of transport regulation contrasts with insulin stimulation of sugar transport in muscle and adipose tissue which is believed to result from recruitment of intracellular sugar transporters to the plasma membrane.

Exercise has been considered a cornerstone of diabetes prevention and treatment for decades, but the benefits of resistance training are less clear. Nineteen non-diabetic subjects (10 metabolic syndrome, 9 sedentary controls) underwent 8 weeks of supervised resistance training. After training, strength and V̇ O2max increased by 10% in both groups. Percent body fat decreased in subjects with the metabolic syndrome. Additionally, lean body mass increased in both groups (p<0.05). Expression of glucose transporter protein-4 (GLUT4), the principle insulin-responsive glucose transporter, increased significantly in both groups. 5-adenosine monophosphateactivated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) expression increased in both groups, indicating increased protein synthesis and mitochondrial biogenesis. Markers of insulin resistance measured by a euglycemic hyperinsulinemic clamp did not improve in subjects with the metabolic syndrome but increased significantly in control subjects (13%). Resistance training upregulates intracellular signaling pathways that may be beneficial for ameliorating the metabolic syndrome.