A suplementação dietética com AGCM induz resistência à insulina, redução de peso ponderal e aumento da adiposidade em ratos Wistar. Adipócitos isolados apresentam reduzidas captação de glicose estimulada por insulina e atividade/fosforilação da proteína AMPK. A expressão protéica do IR no tecido hepático está aumentada em animais tratados com AGCM com redução do grau de fosforilação, enquanto que o grau de fosforilação da proteína AKT permaneceu semelhante entre os grupos. Ilhotas pancreáticas isoladas apresentam redução na secreção de insulina quando incubadas com altas concentrações de glicose, diminuição do conteúdo total de insulina, hipersensibilidade a leucina e/ou arginina e aumento do percentual de morte celular com diminuída expressão da proteína AKT_1 . Desta forma, utilização em longo prazo dessa estratégia nutricional pode interferir no crescimento normal do indivíduo, na sensibilidade à insulina e possívelmente, desenvolvimento e instalação do diabetes. / The introduction of MCFA into diet induces insulin resistance, reduced body weight gain, and increased adiposity in Wistar rats. Isolated adipocytes have reduced insulin induced glucose uptake and phosphorylation/activation of AMPK protein. The insulin receptor protein expression is increased in liver of MCFA fed rats accompanied by reduced tyrosine phosphorylation, with similar AKT serine phosphorylation. Isolated pancreatic islets had reduced glucose stimulated insulin secretion due to high glucose exposure and reduced insulin content; higher insulin secretion induced by leucine and arginine, and increased apoptosis with reduced AKT protein level. In these regard, the chronic ingestion of MCFA may interfere with normal body growth, with the insulin sensitivity and may participate with the development of diabetes.

O polifenismo facultativo, observado entre rainhas e operárias em insetos altamente eussociais tem como estímulo inicial uma alimentação diferencial na fase larval que afeta tanto o desenvolvimento geral das larvas quanto a diferenciação de órgãos e sistemas, principalmente o sistema reprodutor das fêmeas. A via de sinalização por insulina (IIS) é uma das principais vias que integra o desenvolvimento geral de animais com as suas condições nutricionais. O objetivo desse trabalho foi verificar possíveis relações entre a via de sinalização por insulina e a diferenciação das castas em abelhas Apis mellifera. A partir de análises do genoma de Apis mellifera anotamos genes integrantes desta via e verificamos que há dois genes codificadores para receptores de insulina, InR1 e InR2. Os perfis de transcrição desses dois genes obtidos por RT-PCR quantitativa, em larvas de rainhas e operárias durante o período de troca de alimentação, demonstraram que há diferenças consideráveis nos padrões temporais e nos níveis dos transcritos para os receptores de insulina, InR1 (GB15492) e InR2 (GB18331), dentro de cada casta, como também entre as duas castas. Em rainhas verificamos uma interessante variação na transcrição de InR1, que no terceiro instar larval foi cerca de cinco vezes maior que a transcrição de InR2 e no quarto instar seguiu em níveis semelhantes ao de InR2. Essa variação de InR1 pode estar relacionada ao teor de proteínas da geléia real oferecida às larvas de rainhas no terceiro instar, que é maior do que teor de proteínas da geléia real oferecida a partir do quarto instar larval. Para as amostras de larvas de operárias observamos que os níveis dos transcritos dos dois receptores, InR1 e InR2, foram baixos no terceiro estágio larval e aumentaram, de maneira semelhante, até o início do quinto estágio larval, o que pode ter sido devido a algum composto existente na geléia de operária que estimule a transcrição dos genes para os receptores de insulina. Foram feitas análises complementares dos níveis de transcrição dos genes InR1 e InR2, em amostras de ovários, tanto de operárias quanto de rainhas, e em amostras de operárias adultas cultivadas em diferentes tipos de alimentações. Essas análises complementares evidenciaram que a transcrição dos genes para os receptores de insulina em Apis mellifera foi diferente nos ovários de ambas as castas, quando comparada às amostras de corpo inteiro, e que em operárias o transcrito do InR1 foi dominante ao longo de quase toda a vida adulta, sendo superado pelo transcrito InR2 apenas por volta de 13 e 15 dias.. Além disso, uma relação positiva entre o conteúdo de proteína e a transcrição de InR1 foi observada quando analisamos a sua transcrição em amostras de operárias adultas alimentadas com bee bread, uma dieta rica em proteína. Os resultados obtidos nesse trabalho, juntamente com os de Wheeler e colaboradores (2006), Seehus e colaboradores (2006), e Patel e colaboradores (2007), constituem as primeiras informações da via IIS em Apis mellifera, e servirão de base na busca da relação entre a dieta e os sinais downstream envolvidos na determinação de casta e diferenciação. / The initial stimulus that generates the facultative queen/worker polyphenism in highly social insects is a differential alimentation in the larval stages. It affects the general development of the larvae, as well as the differentiation of organs and systems, especially of the female reproductive system. The insulin signaling pathway (IIS) is one of the main pathways that integrates the general development of animals with their respective nutritional conditions. The aim of this work was to investigate the relationship between IIS and caste differentiation in the honey bee Apis mellifera. Using the available Apis mellifera genome information we annotated genes belonging to this pathway. We noted that there are two genes encoding putative insulin receptors, InR1 and InR2. The transcriptional profiles of these genes were obtained by quantitative RT-PCR of queen and workers larvae, giving special attention the period during which the larval diet changes. These results revealed considerable differences in the temporal patterns and levels of the transcripts of two the insulin receptor genes, InR1 (GB15492) and InR2 (GB18331) between the two castes and during their respective larval development. For queens we noted an interesting modulation in InR1 transcription: in the third larval instar it was about five fold higher than the transcription of InR2, but in the fourth instar both receptors were transcribed at similar levels. This variation in InR1 expression may be related to the protein content of royal jelly offered to the queen larvae in third instar, that is higher than the protein content of the royal jelly offered to fourth larvae instar. For the worker larvae samples we observed that transcripts levels of the two receptors, InR1 and InR2, were low in the third larval stage and increased in parallel until the onset of the fifth larval stage. This may have been due to some compound in the worker jelly which stimulates the transcription of both genes coding for insulin receptors. Complementary analysis of transcription levels of InR1 and InR2 were performed on ovaries of queen and worker larvae, and on adult workers maintained on different diets. These complementary analyses highlighted that transcription of the InR genes in the larval ovaries of Apis mellifera was differed from the whole body samples. In adult workers the expression of InR1 was dominant over InR2 during most of the adult life cycle, an inversion was only seen in 13 to 15 days old bees. Furthermore, a positive relationship between protein content and InR1 transcription was observed when analyzing its transcription in adult workers fed with bee bread, a protein-rich diet. The results of this work, in conjunction with those of Wheeler et al. (2007), Seehus et al. (2006) and Patel et al. (2007), are the first information on the IIS pathway in honey bees and they represent the basis for an in-depth pursuit on the relationship between diet and downstream signallng envolved in caste determination and differentiation.

Metabolic Syndrome Insulin Resistance Is Associated with Discordant Distribution of GLUT4 and the Insulin Receptor in Fast-Twitch and Slow-Twitch Muscle Fiber Types We have previously shown that We have previously shown that strength training alone improved insulin responsiveness in sedentary controls but not in metabolic syndrome subjects. Immunoblots of metabolic syndrome subjects[apos] muscle homogenates showed training-related increases in GLUT4 and mitochondrial enzymes was half that seen in the controls. To determine if this was due to changes primarily in fast-twitch fibers (strength fibers), we performed immunohistochemical (IHC) studies on muscle sections from these subjects to quantify fiber-specific changes in GLUT4, phospho-AMPK, phospho-mTOR, ATP synthase, and the insulin receptor. Signal intensity in confocal microscopic images was digitally quantified and the amount in each fiber type was adjusted by the fiber composition and the average size of each fiber type. Fiber type was classified using monoclonal antibodies against slow-twitch (type 1 fibers) and fast-twitch (type 2a and 2b fibers) myosin heavy chains. At baseline, both groups had slightly more insulin receptor in slow-twitch fibers, and most of the ATP synthase (mitochondrial marker) was in fast-twitch fibers. In controls, 55% of GLUT4 was in slow-twitch fibers, whereas metabolic syndrome subjects had only 33% of their GLUT4 in slow-twitch fibers. The IHC data showed modest increases in GLUT4 (9-25%), and substantial increases of ATP synthase (55-95%), and insulin receptors (44-104%) in both fiber types in both groups. Training-related increases were seen in phospho-AMPK (25% in slow-twitch, 15% in fast-twitch) only in the control subjects but no change in phospho-mTOR in either subject group. At baseline, metabolic syndrome subjects[apos] muscle had 56% of insulin receptors expressed in slow-twitch fibers, but only 33% of the GLUT4 was in these fibers. Thus, the untrained muscle composition of the metabolic syndrome subjects exhibited a mismatch between insulin receptors and GLUT4 in their fiber-specific distributions. This mismatch may contribute to the insulin resistance seen in the metabolic syndrome and may be involved in the diminished insulin sensitivity response to strength training in these subjects.

The overall objective of this thesis was to examine mechanisms involved in the acute regulation of sarcoplasmic reticulum (SR) Ca2+-handling properties by second messenger signaling pathways in skeletal and cardiac muscle. The aim of the first study (Chapter Two) was to characterize changes in the kinetic properties of sarco(endo)-plasmic reticulum Ca2+-ATPase (SERCA) proteins in cardiac and skeletal muscles in response to b-adrenergic, Ca2+-dependent calmodulin kinase II (CaMKII) and protein kinase C (PKC) signaling. The aim of the second study (Chapter Three) was to determine if insulin signaling could acutely regulate SERCA kinetic properties in cardiac and skeletal muscle. The aim of the final study (Chapter Four) was to determine if alterations in plasma glucose, epinephrine and insulin concentrations during exercise are able to influence SR Ca2+-handling properties in contracting human skeletal muscle. Data collected in Chapter Two and Chapter Three were obtained using tissue prepared from a group of 28 male Sprague-Dawley rats (9 weeks of age; mass = 280 ± 4 g: X ± S.E). Crude muscle homogenates (11:1 dilution) were prepared from selected hind limb muscles (soleus, SOL; extensor digitorum longus, EDL; the red portion of gastrocnemius, RG; and the white portion of gastrocnemius, WG) and the left ventricle (LV). Enriched SR membrane fractions, prepared from WG and LV, were also analyzed. A spectrophotometric assay was used to measure kinetic properties of SERCA, namely, maximal SERCA activity (Vmax), and Ca2+-sensitivity was characterized by both the Ca50, which is defined as the free Ca2+-concentration needed to elicit 50% Vmax, and the Hill coefficient (nH), which is defined as the relationship between SERCA activity and Ca2+f for 10 to 90% Vmax. The observations made in Chapter Two indicated that b-adrenergic signaling, activated by epinephrine, increased (P<0.05) Ca2+-sensitivity, as shown by a left-shift in Ca50 (i.e. reduced Ca50), without altering Vmax in LV and SOL but had no effect (P<0.05) on EDL, RG, or WG. Further analysis using a combination of cAMP, the PKA activator forskolin, and/or the PKA inhibitor KT5270 indicated that the reduced Ca50 in LV was activated by cAMP- and PKA-signaling mechanisms. However, although the reduced Ca50 in SOL was cAMP-dependent, it was not influenced by a PKA-dependent mechanism. In contrast to the effects of b-adrenergic signaling, CaMKII activation increased SERCA Ca2+-sensitivity, as shown by a left-shift in Ca50 and increased nh, without altering SERCA Vmax in LV but was without effect in any of the skeletal muscles examined. The PKC activator PMA significantly reduced SERCA Ca2+-sensitivity, by inducing a right-shift in Ca50 and decreased nH in the LV and all skeletal muscles examined. PKC activation also reduced Vmax in the fast-twitch skeletal muscles (i.e. EDL, RG and WG), but did not alter Vmax in LV or SOL. The results of Chapter Three indicated that insulin signaling increased SERCA Ca2+-sensitivity, as shown by a left-shift in Ca50 (i.e. reduced Ca50) and an increased nH, without altering SERCA Vmax in crude muscle homogenates prepared from LV, SOL, EDL, RG, and WG. An increase in SERCA Ca2+-sensitivity was also observed in enriched SERCA1a and SERCA2a vesicles when an activated form of the insulin receptor (A-INS-R) was included during biochemical analyses. Co-immunoprecipitation experiments were conducted and indicated that IRS-1 and IRS-2 proteins bind SERCA1a and SERCA2a in an insulin-dependent manner. However, the binding of IRS proteins with SERCA does not appear to alter the structural integrity of the SERCA Ca2+-binding site since no changes in NCD-4 fluorescence were observed in response to insulin or A-INS-R. Moreover, the increase in SERCA Ca2+-sensitivity due to insulin signaling was not associated with changes in the phosphorylation status of phospholamban (PLN) since Ser16 or Thr17 phosphorylation was not altered by insulin or A-INS-R in LV tissue. The data described in Chapter Four was collected from 15 untrained human participants (peak O2 consumption, VO2peak= 3.45 ± 0.17 L/min) who completed a standardized cycle test (~60% VO2peak) on two occasions during which they were provided either an artificially sweetened placebo (PLAC) or a 6% glucose (GLUC) beverage (~1.00 g CHO per kg body mass). Muscle biopsies were collected from the vastus lateralis at rest, after 30 min and 90 min of exercise and at fatigue in both conditions to allow assessment of metabolic and SR data. Glucose supplementation increased exercise ride time by ~19% (137 ± 7 min) compared to PLAC (115 ± 6 min). This performance increase was associated with elevated plasma glucose and insulin concentrations and reduced catecholamine concentrations during GLUC compared to PLAC. Prolonged exercise reduced (p<0.05) SR Ca2+-uptake, Vmax, Phase 1 and Phase 2 Ca2+-release rates during both PLAC and GLUC. However, no differences in SR Ca2+-handling properties were observed between conditions when direct comparisons were made at matched time points between PLAC and GLUC. In summary, the results of the first study (Chapter Two) indicate that b-adrenergic and CaMKII signaling increases SERCA Ca2+-sensitivity in the LV and SOL; while PKC signaling reduces SERCA Ca2+-sensitivity in all tissues. PKC activation also reduces Vmax in the fast-twitch skeletal muscles (i.e. EDL, RG, and WG) but has no effect on Vmax in the LV and SOL. The results of the second study (Chapter Three) indicate that insulin signaling acutely increases the Ca2+-sensitivity of SERCA1a and SERCA2a in all tissues examined, without altering the Vmax. Based on our observations, it appears that the increase in SERCA Ca2+-sensitivity may be regulated, in part, through the interaction of IRS proteins with SERCA1a and SERCA2a. The results of the final study (Chapter Four) indicate that alterations in plasma glucose, epinephrine and insulin concentrations associated with glucose supplementation during exercise, do not alter the time course or magnitude of reductions in SERCA or Ca2+-release channel (CRC) function in working human skeletal muscle. Although glucose supplementation did increase exercise ride time to fatigue in this study, our data does not reveal an association with SR Ca2+-cycling measured in vitro. It is possible that the strength of exercise signal overrides the hormonal influences observed in resting muscles. Additionally, these data do not rule out the possibility that glucose supplementation may influence E-C coupling processes or SR Ca2+-cycling properties in vivo.

Made available in DSpace on 2014-06-11T19:27:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-05Bitstream added on 2014-06-13T20:17:06Z : No. of bitstreams: 1 chiba_fy_me_araca.pdf: 754628 bytes, checksum: 34bc263d9874acda6daffee82f61b8b5 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Nos últimos anos, tem havido uma redução acentuada nos índices de cárie dentária em diversas regiões do mundo, fato que tem sido atribuído à exposição às substâncias fluoretadas, principalmente na forma de água de abastecimento público e dentifrício. Simultaneamente, nota-se a ocorrência do aumento da prevalência de fluorose dentária. Estudo realizado em 25 pacientes (15 a 30 anos de idade) com fluorose endêmica mostrou que 40% destes tinham a tolerância à glicose prejudicada, porém esta anomalia foi revertida com a remoção do excesso do flúor na água consumida. O NaF ocasiona inibição da glicólise, diminuição da secreção de insulina e hiperglicemia. Muitas destas respostas sugerem que o NaF pode ocasionar resistência à insulina. Se isto for confirmado, é recomendável diminuir a concentração de fluoreto nos dentifrícios utilizados principalmente por crianças diabéticas, pois a ingestão de pasta dental contendo flúor pode levar à piora na situação de saúde destas crianças. Sabendo-se que o fluoreto pode alterar o metabolismo de carboidratos, tornou-se fundamental caracterizar o efeito do NaF sobre: 1) a sensibilidade à insulina; 2) o grau de fosforilação em tirosina do substrato do receptor de insulina - pp185 (IRS-1/IRS-2); 3) a fluoremia, glicemia e insulinemia. Para tanto, foram utilizados ratos Wistar machos (1 mês de idade) castrados. Após 30 dias da castração, os animais foram divididos aleatoriamente em dois grupos: 1) grupo controle (CN), o qual foi submetido ao tratamento sem NaF, mas com uma solução de NaCl (9,54 mg/kg p.c.) que contém a mesma quantidade de sódio em relação à do grupo fluoreto de sódio; 2) grupo NaF (FN) que será submetido ao tratamento com NaF (4,0 mg de flúor/kg p.c.) na água de beber e na ração durante 42... / Over the last years, there has been a significant reduction in the incidence of dental caries in several regions of the world. This has been attributed to ingestion of fluoridated products, especially in the form of public water supplies and toothpaste. Simultaneously, there has been an increase in the prevalence of dental fluorosis. A study conducted in 25 patients (15 to 30 years of age) with endemic fluorosis showed that 40% of these had to impaired glucose tolerance, but this anomaly was reversed by removing the excess of fluoride from drinking water. NaF causes glycolysis inhibition, decrease on insulin secretion and hyperglycemia. These responses suggest that NaF can cause insulin resistance. If this is confirmed, the use of dentifrices with lower fluoride content is recommended, especially for diabetic children, for whom excessive F consumption may lead to worsening the condition of these children’s health. Knowing that F can interfere with carbohydrate metabolism, we felt it was important and fundamental to undertake a study to examine the chronic effect of NaF on: 1) insulin sensitivity; 2) pp185 (IRS-1/IRS-2) tyrosine phosphorylation in gastrocnemius muscle and liver of rats; 3) fluoremia, glycemia and insulinemia. For this study, castrated Wistar male rats (1 month of age) were used. Thirty days after castration, the animals were randomly divided in two groups: 1) control group (CN) which was subjected to treatment without NaF, but with a solution of NaCl (9.54 mg / kg bw) which contains the same amount of sodium in relation to the group NaF; 2) group NaF (FN) that was submitted to treatment with NaF administered in the drinking water and F contained in food pellets (F total inferred: 4.0 mg F / Kg bw / day in the form of NaF) during 42 days. After 6 weeks, the following experiments were conducted: 1) assessment... (Complete abstract, click electronic address below)

Orientador: Doris Hissako Sumida / Banca: Cléa Adas Saliba Garbin / Banca: Marília Afonso Rabelo Buzalaf / Resumo: Nos últimos anos, tem havido uma redução acentuada nos índices de cárie dentária em diversas regiões do mundo, fato que tem sido atribuído à exposição às substâncias fluoretadas, principalmente na forma de água de abastecimento público e dentifrício. Simultaneamente, nota-se a ocorrência do aumento da prevalência de fluorose dentária. Estudo realizado em 25 pacientes (15 a 30 anos de idade) com fluorose endêmica mostrou que 40% destes tinham a tolerância à glicose prejudicada, porém esta anomalia foi revertida com a remoção do excesso do flúor na água consumida. O NaF ocasiona inibição da glicólise, diminuição da secreção de insulina e hiperglicemia. Muitas destas respostas sugerem que o NaF pode ocasionar resistência à insulina. Se isto for confirmado, é recomendável diminuir a concentração de fluoreto nos dentifrícios utilizados principalmente por crianças diabéticas, pois a ingestão de pasta dental contendo flúor pode levar à piora na situação de saúde destas crianças. Sabendo-se que o fluoreto pode alterar o metabolismo de carboidratos, tornou-se fundamental caracterizar o efeito do NaF sobre: 1) a sensibilidade à insulina; 2) o grau de fosforilação em tirosina do substrato do receptor de insulina - pp185 (IRS-1/IRS-2); 3) a fluoremia, glicemia e insulinemia. Para tanto, foram utilizados ratos Wistar machos (1 mês de idade) castrados. Após 30 dias da castração, os animais foram divididos aleatoriamente em dois grupos: 1) grupo controle (CN), o qual foi submetido ao tratamento sem NaF, mas com uma solução de NaCl (9,54 mg/kg p.c.) que contém a mesma quantidade de sódio em relação à do grupo fluoreto de sódio; 2) grupo NaF (FN) que será submetido ao tratamento com NaF (4,0 mg de flúor/kg p.c.) na água de beber e na ração durante 42... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Over the last years, there has been a significant reduction in the incidence of dental caries in several regions of the world. This has been attributed to ingestion of fluoridated products, especially in the form of public water supplies and toothpaste. Simultaneously, there has been an increase in the prevalence of dental fluorosis. A study conducted in 25 patients (15 to 30 years of age) with endemic fluorosis showed that 40% of these had to impaired glucose tolerance, but this anomaly was reversed by removing the excess of fluoride from drinking water. NaF causes glycolysis inhibition, decrease on insulin secretion and hyperglycemia. These responses suggest that NaF can cause insulin resistance. If this is confirmed, the use of dentifrices with lower fluoride content is recommended, especially for diabetic children, for whom excessive F consumption may lead to worsening the condition of these children's health. Knowing that F can interfere with carbohydrate metabolism, we felt it was important and fundamental to undertake a study to examine the chronic effect of NaF on: 1) insulin sensitivity; 2) pp185 (IRS-1/IRS-2) tyrosine phosphorylation in gastrocnemius muscle and liver of rats; 3) fluoremia, glycemia and insulinemia. For this study, castrated Wistar male rats (1 month of age) were used. Thirty days after castration, the animals were randomly divided in two groups: 1) control group (CN) which was subjected to treatment without NaF, but with a solution of NaCl (9.54 mg / kg bw) which contains the same amount of sodium in relation to the group NaF; 2) group NaF (FN) that was submitted to treatment with NaF administered in the drinking water and F contained in food pellets (F total inferred: 4.0 mg F / Kg bw / day in the form of NaF) during 42 days. After 6 weeks, the following experiments were conducted: 1) assessment... (Complete abstract, click electronic address below) / Mestre

Le carcinome hépatocellulaire (CHC) est un cancer de mauvais pronostic. Les maladies chroniques du foie provoquent le développement d'une cirrhose qui évoluera éventuellement en CHC. Le récepteur de l'insuline (IR) est exprimé sous deux isoformes, IR-A et IR-B, qui résultent de l'épissage alternatif de l'ARNm. IR-B est l'isoforme majoritairement exprimé dans l'hépatocyte adulte et IR-A dans l'hépatocyte fœtal. Plusieurs travaux montrent une surexpression de IR-A dans différents cancers, associée à une signalisation oncogénique en réponse à l'IGF-II. Le rôle de la signalisation IGF-II/IR-A a peu été investigué dans le CHC. Notre étude porte sur le rôle biologique de la surexpression de IR-A dans les cellules de CHC au regard de la surexpression d'IGF-II. Une augmentation du ratio d'expression IR-A/IR-B est observée dans 70% des tumeurs de CHC par rapport au foie adjacent non tumoral, qui est significativement associée à l'expression de marqueurs clinicopathologiques de mauvais pronostic dont des marqueurs de cellules souches/progénitrices (CSP) et à une faible survie post-hépatectomie. Seules 9,4% des tumeurs surexpriment IGF-II. In vivo, la surexpression stable de IR-A, et non de IR-B, augmente la tumorigénicité de deux lignées de CHC disposant (Huh7) ou non (PLC/PRF5) d'une boucle autocrine d'IGF-II, et est associée à l'induction de différentes signatures pro-inflammatoires. Sans effet sur la prolifération, la surexpression de IR-A stimule la migration et l'invasion in vitro et augmente l'expression de marqueurs CSP. En conclusion, ces résultats identifient IR-A comme un nouvel acteur de la progression du CHC de façon indépendante d'une boucle autocrine d'IGF-II. / Hepatocellular carcinoma (HCC) is a poor prognosis cancer. Chronic liver diseases induce cirrhosis which will eventually evolve into HCC. The insulin receptor (IR) exists in two isoforms, IR-A and IR-B, resulting from mRNA alternative splicing. Adult hepatocytes predominantly express IR-B while fœtal hepatocytes mainly express IR-A. The overexpression of IR-A associated with IGF2 oncogenic signaling has been abundantly described in cancer cells. Little is known about IGF2/IR-A signaling in HCC. Our study aims to evaluate the biological functions associated to IR-A overexpression in HCC in relation to IGF2 overexpression. We observed that 70% of 85 HCC tumors showed upregulation of IR-A/IR-B ratio compared to adjacent nontumor tissue, which was significantly associated with clinicopathological markers of aggressive tumours such as stem/progenitor cell (SPC) markers and correlated with reduced patient survival after surgery. IGF2 upregulation was observed in only 9.4% of HCC. Stable overexpression of IR-A, but not IR-B, increased in vivo tumorigenicity in two HCC cell lines presenting (Huh7) or not (PLC/PRF5) an autocrine IGF2 secretion loop and was associated with the induction of different pro-inflammatory gene signatures. While IR-A overexpression did not promote cell proliferation in vitro, it stimulated migration and invasion and increased some SPC markers expression. Altogether these results highlight IR-A as a novel player in HCC progression irrespective of an IGF2 autocrine loop.

A administração de deidroepiandrosterona (DHEA) tem resultado em efeitos anti-diabetogênicos em animais de experimentação e no homem. Assim, o objetivo desse trabalho é avaliar o efeito do DHEA in vitro na expressão protéica do IR, do IRS-1, IRS-2, PI3-K, Akt, ERK-1/2; na expressão gênica do PDX-1, do PGC-1, da insulina, do GLUT-2 e da glicocinase; e avaliar a secreção estática de insulina de ilhotas pancreáticas de ratos. O cultivo das ilhotas por 24 horas com DHEA, não induziu nenhuma alteração tanto na expressão das proteínas quanto na secreção estática de insulina estimulada por glicose. Ocorreu aumento da fosforilação de ERK-1/2 e na expressão gênica do PGC-1. As células RINm5F, cultivadas por 72 horas com DHEA, apresentaram aumento da expressão total de IRS-1 e IRS-2. Concluímos, que 24 horas de cultura com ilhotas não é tempo suficiente para observar nenhuma alteração induzida pelo DHEA, na secreção de insulina, e na expressão das proteínas da via IRS/PI3-K/Akt. Células RINm5F podem ser um modelo alternativo para investigar os efeitos diretos do DHEA. / The dehydroepiandrosterone (DHEA) administration has resulted in reduction of abdominal fat and protection against insulin resistance from experimental animals and humans. So, the purpose of this project is measure the in vitro effects from DHEA: on protein expression of insulin receptor, the proteins IRS-1, IRS-2, PI3-K, Akt, and ERK-1/2; on gene expression of transcriptional factors PDX-1 and PGC-1, insulin, glucose transport GLUT-2 and glicocinase; and to measure the static insulin secretion, on cultured pancreatic islets of the rat. The culture of pancreatic islet for 24 hours with DHEA, did not induce nothing alteration on protein expression of the IR, IRS-1, IRS-2, PI3-K, Akt-1 and ERK-1/2, and static insulin secretion induced by glucose. However, happened increase ERK-1/2 phosphorylation and PGC-1 gene expression. The RINm5F cells, cultured by 72 hours, showed increase of the IRS-1 and IRS-2 expression. We conclude that 24 hours of the pancreatic islets culture are not sufficient time to look any alteration induced by DHEA, on insulin secretion, and on protein expression involved on IRS/PI3-K/Akt pathway. RINm5F cells can be an alternative model to research the direct effects from DHEA.

A suplementação dietética com AGCM induz resistência à insulina, redução de peso ponderal e aumento da adiposidade em ratos Wistar. Adipócitos isolados apresentam reduzidas captação de glicose estimulada por insulina e atividade/fosforilação da proteína AMPK. A expressão protéica do IR no tecido hepático está aumentada em animais tratados com AGCM com redução do grau de fosforilação, enquanto que o grau de fosforilação da proteína AKT permaneceu semelhante entre os grupos. Ilhotas pancreáticas isoladas apresentam redução na secreção de insulina quando incubadas com altas concentrações de glicose, diminuição do conteúdo total de insulina, hipersensibilidade a leucina e/ou arginina e aumento do percentual de morte celular com diminuída expressão da proteína AKT_1 . Desta forma, utilização em longo prazo dessa estratégia nutricional pode interferir no crescimento normal do indivíduo, na sensibilidade à insulina e possívelmente, desenvolvimento e instalação do diabetes. / The introduction of MCFA into diet induces insulin resistance, reduced body weight gain, and increased adiposity in Wistar rats. Isolated adipocytes have reduced insulin induced glucose uptake and phosphorylation/activation of AMPK protein. The insulin receptor protein expression is increased in liver of MCFA fed rats accompanied by reduced tyrosine phosphorylation, with similar AKT serine phosphorylation. Isolated pancreatic islets had reduced glucose stimulated insulin secretion due to high glucose exposure and reduced insulin content; higher insulin secretion induced by leucine and arginine, and increased apoptosis with reduced AKT protein level. In these regard, the chronic ingestion of MCFA may interfere with normal body growth, with the insulin sensitivity and may participate with the development of diabetes.

O polifenismo facultativo, observado entre rainhas e operárias em insetos altamente eussociais tem como estímulo inicial uma alimentação diferencial na fase larval que afeta tanto o desenvolvimento geral das larvas quanto a diferenciação de órgãos e sistemas, principalmente o sistema reprodutor das fêmeas. A via de sinalização por insulina (IIS) é uma das principais vias que integra o desenvolvimento geral de animais com as suas condições nutricionais. O objetivo desse trabalho foi verificar possíveis relações entre a via de sinalização por insulina e a diferenciação das castas em abelhas Apis mellifera. A partir de análises do genoma de Apis mellifera anotamos genes integrantes desta via e verificamos que há dois genes codificadores para receptores de insulina, InR1 e InR2. Os perfis de transcrição desses dois genes obtidos por RT-PCR quantitativa, em larvas de rainhas e operárias durante o período de troca de alimentação, demonstraram que há diferenças consideráveis nos padrões temporais e nos níveis dos transcritos para os receptores de insulina, InR1 (GB15492) e InR2 (GB18331), dentro de cada casta, como também entre as duas castas. Em rainhas verificamos uma interessante variação na transcrição de InR1, que no terceiro instar larval foi cerca de cinco vezes maior que a transcrição de InR2 e no quarto instar seguiu em níveis semelhantes ao de InR2. Essa variação de InR1 pode estar relacionada ao teor de proteínas da geléia real oferecida às larvas de rainhas no terceiro instar, que é maior do que teor de proteínas da geléia real oferecida a partir do quarto instar larval. Para as amostras de larvas de operárias observamos que os níveis dos transcritos dos dois receptores, InR1 e InR2, foram baixos no terceiro estágio larval e aumentaram, de maneira semelhante, até o início do quinto estágio larval, o que pode ter sido devido a algum composto existente na geléia de operária que estimule a transcrição dos genes para os receptores de insulina. Foram feitas análises complementares dos níveis de transcrição dos genes InR1 e InR2, em amostras de ovários, tanto de operárias quanto de rainhas, e em amostras de operárias adultas cultivadas em diferentes tipos de alimentações. Essas análises complementares evidenciaram que a transcrição dos genes para os receptores de insulina em Apis mellifera foi diferente nos ovários de ambas as castas, quando comparada às amostras de corpo inteiro, e que em operárias o transcrito do InR1 foi dominante ao longo de quase toda a vida adulta, sendo superado pelo transcrito InR2 apenas por volta de 13 e 15 dias.. Além disso, uma relação positiva entre o conteúdo de proteína e a transcrição de InR1 foi observada quando analisamos a sua transcrição em amostras de operárias adultas alimentadas com bee bread, uma dieta rica em proteína. Os resultados obtidos nesse trabalho, juntamente com os de Wheeler e colaboradores (2006), Seehus e colaboradores (2006), e Patel e colaboradores (2007), constituem as primeiras informações da via IIS em Apis mellifera, e servirão de base na busca da relação entre a dieta e os sinais downstream envolvidos na determinação de casta e diferenciação. / The initial stimulus that generates the facultative queen/worker polyphenism in highly social insects is a differential alimentation in the larval stages. It affects the general development of the larvae, as well as the differentiation of organs and systems, especially of the female reproductive system. The insulin signaling pathway (IIS) is one of the main pathways that integrates the general development of animals with their respective nutritional conditions. The aim of this work was to investigate the relationship between IIS and caste differentiation in the honey bee Apis mellifera. Using the available Apis mellifera genome information we annotated genes belonging to this pathway. We noted that there are two genes encoding putative insulin receptors, InR1 and InR2. The transcriptional profiles of these genes were obtained by quantitative RT-PCR of queen and workers larvae, giving special attention the period during which the larval diet changes. These results revealed considerable differences in the temporal patterns and levels of the transcripts of two the insulin receptor genes, InR1 (GB15492) and InR2 (GB18331) between the two castes and during their respective larval development. For queens we noted an interesting modulation in InR1 transcription: in the third larval instar it was about five fold higher than the transcription of InR2, but in the fourth instar both receptors were transcribed at similar levels. This variation in InR1 expression may be related to the protein content of royal jelly offered to the queen larvae in third instar, that is higher than the protein content of the royal jelly offered to fourth larvae instar. For the worker larvae samples we observed that transcripts levels of the two receptors, InR1 and InR2, were low in the third larval stage and increased in parallel until the onset of the fifth larval stage. This may have been due to some compound in the worker jelly which stimulates the transcription of both genes coding for insulin receptors. Complementary analysis of transcription levels of InR1 and InR2 were performed on ovaries of queen and worker larvae, and on adult workers maintained on different diets. These complementary analyses highlighted that transcription of the InR genes in the larval ovaries of Apis mellifera was differed from the whole body samples. In adult workers the expression of InR1 was dominant over InR2 during most of the adult life cycle, an inversion was only seen in 13 to 15 days old bees. Furthermore, a positive relationship between protein content and InR1 transcription was observed when analyzing its transcription in adult workers fed with bee bread, a protein-rich diet. The results of this work, in conjunction with those of Wheeler et al. (2007), Seehus et al. (2006) and Patel et al. (2007), are the first information on the IIS pathway in honey bees and they represent the basis for an in-depth pursuit on the relationship between diet and downstream signallng envolved in caste determination and differentiation.