The protein Poly was first discovered in Drosophila melanogaster, during a screening for third chromosome lethal mutations. Drosophila poly mutant larvae exhibit a slower rate of development. However, they reach the third instar larval stage and remain at that stage for 21 days before they die without reaching pupation. This phenotype is attributed to developmental impairment of the imaginal discs, therefore suggesting defects in cell growth and/or proliferation. During that stage, the mutant larvae develop melanotic masses. Poly is conserved and its homolog, Elp6, is one of the small subunits of the Elongator Complex, a complex involved in many cellular functions including transcription and translation. Drosophila larvae mutated at the Elp3 gene, the gene encoding the catalytic subunit of the Elongator complex, develop melanotic masses, a phenotype very similar to that of poly. The Heck laboratory published that Poly is a positive mediator of the Insulin Receptor/TOR (InR/TOR) pathway, which leads to protein, glycogen and fatty acid synthesis, regulates cell growth and apoptosis. It was shown that Poly interacts with InR, at least in some cases, therefore promoting cell growth and metabolism (Bolukbasi et al., 2012). The Drosophila genome shares 60% similarity to the human, with 77% of the genes attributed to a human disease having a Drosophila counterpart (Chien et al., 2002). In 2011, an intriguing study by Musselman et al. reported that feeding wild type Drosophila larvae with excessive amounts of sucrose led to the development of an insulin resistance phenotype similar to that of Type 2 Diabetes (T2D), thus rendering Drosophila an easily accessible T2D model. The phenotype included impaired metabolism, slower rate of development, and excessive accumulation of triglycerides (TAG) in the larval fat body. In my thesis research, I examined the involvement of Poly in insulin resistance - T2D using Drosophila as a model. The understanding of the connection between the protein and the disease came upon the discovery of a new form of Poly, Poly14. Poly14 is enriched in the Drosophila fat body, the equivalent of the human fat tissue and liver and its protein levels are significantly decreased when larvae are fed a high sucrose diet, compared to other types of diets – potentially linking the protein to the onset of T2D. Poly mRNA levels were also lower. To examine whether the overexpression of poly might be able to rescue the insulin resistance phenotype, two new Drosophila transgenes were generated with the ability to express the gene in a tissue of interest. In these two new transgenes, Poly is tagged with tRFP (Red Fluorescence Protein) at the N’- (UAS_N’RFPpoly) or the C’-terminus (UAS_polyC’RFP). Overexpression of Poly rescued the insulin resistance phenotype, therefore implicating Poly as a possible important regulator in the development of the insulin resistance phenotype. All of the above findings suggest a vital role of Poly in metabolism and the development of the insulin resistance/diabetic phenotype in Drosophila, providing us the opportunity for new tools in this very medically-relevant field of research.

Binge Eating Disorder (BED) is a deadly, psychiatric condition which affects about 10 million people in the USA. It is characterized by discrete and recurrent binge eating episodes consisting of rapid consumption of excessive amounts of highly palatable, energy-dense food (e.g. rich in sugars and fats) within discrete periods of time. Our laboratory has been focusing on the understanding of the behavioral, metabolic, and neurobiological factors underlying BED, through the development of an animal model of binge-like eating. This model is based on a limited access schedule in which rats are exposed 1-hour/day to a high-sucrose diet (HSD) in operant conditioning self-administration boxes. However, the consummatory and metabolic outcomes of exposing rats to a high-fat diet (HFD) in the same procedure are unknown. The aim of this thesis was to test the consummatory and metabolic effects of 1-hour limited access to either a HSD or a HFD in an operant rat model of binge-like eating. For this purpose, female rats were subjects of the binge-like eating procedure by limiting access to a HSD, a HFD, or a standard Chow diet. Our results show that limiting access to either a HSD or a HFD promoted binge-like eating as compared to control Chow diet. HSD binge-like eating was based on a true increase in the amount of food consumed, that is, an increased eating rate. Such suggests increase in palatability and a decrease in the home-cage standard chow intake, likely due to a negative contrast effect. Conversely, binge-like eating of the high-fat diet resulted from passive energy consumption due to the high energy-density of the food. Also, HFD binge-like eating was accompanied by neither increased eating rate nor rejection of the home-cage chow. Moreover, while HSD rats consumed less energy than HFD rats, the former were more energy efficient and gained more body weight than the latter. These results provide information on how the quality of food can deeply influence the behavioral and metabolic outcomes of binge-like eating. / 2022-06-07T00:00:00Z

Tesis por compendio / [ES] La obesidad representa un importante reto para la salud pública debido a su elevada prevalencia y a las comorbilidades asociadas. Las dietas hipercalóricas activan el sistema inmunitario intestinal y alteran la microbiota intestinal causando daños metabólicos en el organismo. De hecho, la pérdida de homeostasis inmunológica intestinal se considera un evento que precede a la aparición de la inflamación sistémica de bajo grado asociada a la obesidad. Dado que la microbiota intestinal es un factor modificable, su modulación puede convertirse en una oportunidad para reducir el impacto de la obesidad. Por ello la identificación de los factores que participan en la respuesta inflamatoria a las dietas obesogénicas y la búsqueda de alternativas terapéuticas basadas en la microbiota constituyen una vía de investigación prometedora para combatir la obesidad. Esta Tesis Doctoral evalúa el potencial de nuevos probióticos y estrategias dietéticas para combatir la obesidad basadas en sus propiedades inmunomoduladoras. En el Primer Capítulo investigamos el potencial anti-obesogénico del propil propano tiosulfinato (PTS), un compuesto organosulfurado derivado de la especie Allium, en dos dosis diferentes (0,1 o 1 mg/kg/día) utilizando un modelo murino de obesidad inducida por la dieta (DIO). Nuestros hallazgos demostraron los efectos protectores de PTS frente a la obesidad, ya que su administración redujo el peso corporal y mejoró la homeostasis de glucosa. En el tejido adiposo y el hígado, PTS redujo la inflamación y el metabolismo lipídico aberrante causado por la dieta obesogénica. Además, PTS incrementó la actividad termogénica en el tejido adiposo marrón y reforzó la función barrera intestinal. En vista de los modestos cambios en el ecosistema microbiano intestinal, concluimos que estos efectos no eran mediados por la microbiota. En el Segundo Capítulo evaluamos el potencial anti-obesogénico y el mecanismo de acción de una nueva bacteria llamada Phascolarctobacterium faecium DSM 32890. Para ello, realizamos diferentes experimentos in vitro e in vivo usando diferentes cultivos celulares (macrófagos derivados de médula ósea y células linfoides innatas intestinales del grupo 1 (LC1s)) y modelos murinos DIO (ratones C57BL/6J y Rag1-/-). El tratamiento de ratones alimentados con una dieta hipercalórica con P. faecium incrementó la proporción de los macrófagos M2 en el intestino, lo que contrarrestó el aumento de ILC1s y, en última instancia, mitigó la intolerancia a la glucosa y el aumento de peso corporal, independientemente de la viabilidad de la bacteria. Además, P. faecium reforzó la función barrera intestinal y evitó la inflamación sistémica causada por la dieta hipercalórica. Estos beneficios metabólicos se mantuvieron en ausencia de inmunidad adaptativa, pero se perdieron cuando la bacteria se coadministró con un inhibidor (GW2580) de la polarización de macrófagos M2. Por último, realizamos un amplio estudio con datos metagenómicos de 6.361 personas que mostró una relación inversa entre P. faecium y la obesidad, independientemente de la nacionalidad, el sexo o la edad, lo que sugiere la asociación de esta bacteria con la salud metabólica. En el Tercer Capítulo, investigamos la implicación de las ILC1s intestinales en la progresión de la obesidad y las alteraciones metabólicas asociadas. Para ello, evaluamos longitudinalmente la respuesta de las ILC1s y las consecuencias su depleción de ILC1s en un modelo murino DIO. En el intestino, el bloqueo de ILC1s evitó el aumento de macrófagos M1 e ILC2s y promovió la activación de la vía ILC3-IL22, aumentando la producción de mucina, la expresión de péptidos antimicrobianos y el número de células neuroendocrinas. Además, el bloqueo de ILC1s restableció el perfil microbiano y el metaboloma, acercándose al perfil asociado con la salud metabólica. En última instancia, estas mejoras se asociaron con una mayor secreción de hormonas intestinales, y una reducción de la insulinemia y la adiposidad. / [CA] L'obesitat és un repte per a la salut pública degut a la elevada prevalença i les comorbiditats. Les dietes hipercalòriques activen el sistema immunitari intestinal i alteren la microbiota intestinal causant danys metabòlics en l'organisme. De fet, la pèrdua d'homeòstasi immunològica a escala intestinal es considera un esdeveniment primerenc que precedeix l'aparició de la inflamació sistèmica de baix grau associada a l'obesitat. Atés que la microbiota intestinal és un factor modificable, la seua modulació pot convertir-se en una oportunitat per a reduir l'impacte de l'obesitat. Per això, la identificació dels factors que participen en la resposta inflamatòria a les dietes obesogèniques i la recerca d'alternatives terapèutiques basades en la microbiota son una via d'investigació prometedora per a combatre l'obesitat. Aquesta Tesi Doctoral avalua el potencial de nous probiòtics i estratègies dietètiques per a combatre l'obesitat basada en propietats immunomoduladores. En el Primer Capítol investiguem el potencial anti-obesogènic del propil propà tiosulfat (PTS), un compost organosulfurat derivat de l'espècie Allium, en dues dosis diferents (0,1 o 1 mg/kg/dia) utilitzant un model murí d'obesitat induïda per la dieta. Els resultats demostren els efectes protectors de PTS enfront a l'obesitat, ja que la seua administració va reduir el pes corporal i va millorar l'homeòstasi de glucosa. En el teixit adipós i el fetge, PTS va prevenir l'augment de la resposta inflamatòria i les alteracions del metabolisme lipídic causades per la dieta hipercalòrica. A més, PTS va incrementar l'activitat termogènica en el teixit adipós marró i millorà la funció barrera intestinal alterats per la dieta. Observàrem canvis modestos en la microbiota intestinal, concloent que els efectes no estan mediats de manera significativa per la microbiota. En el Segon Capítol avaluem el potencial anti-obesogènic i el mecanisme d'acció d'un nou bacteri Phascolarctobacterium faecium DSM 32890. Hem realitzat experiments in vitro i in vivo utilitzant diferents cultius cel·lulars (macròfags derivats de medul·la òssia i de cèl·lules limfoides innates intestinals del grup 1 (LC1s) i models murins d'obesitat induïda per la dieta (ratolins C57BL/6J, i Rag1-/- ). El tractament de ratolins alimentats amb una dieta hipercalòrica amb P. faecium incrementà la proporció dels macròfags M2 a l'intestí, contrarestant l'augment d'ILC1s i en última instància, mitigà la intolerància a la glucosa i l'augment del pes corporal, independentment de la viabilitat del bacteri. A més, P. faecium reforçà la funció bacterial intestinal i evità la inflamació sistèmica causada per la dieta hipercalòrica. Aquests beneficis metabòlics es mantenien en absència d'immunitat adaptativa, però es perderen quan el bacteri es coadministrà amb un inhibidor (GW2580) de la polarització de macròfags M2. Finalment, realitzàrem un ampli estudi amb dades metagenòmiques de 6.361 persones que mostrà una relació inversa entre P. faecium i l'obesitat, independentment de la nacionalitat, el sexe o l'edat, suggerint l'associació d'aquest bacteri amb la salut metabòlica. En el Tercer Capítol, investiguem la implicació de les ILC1s residents en l'intestí en la progressió de l'obesitat i les alteracions metabòliques associades. Evaluàrem longitudinalment la resposta de les ILC1s i les conseqüències de la depleció de ILC1s en un model murí d'obesitat. A l'intestí, el bloqueig de ILC1s va evitar l'augment de macròfags M1 i ILC2s, i va promoure l'activació de la via ILC3-IL22, augmentant la producció de mucina, l'expressió de pèptids antimicrobians i el nombre de cèl·lules neuroendocrines. El bloqueig de ILC1s va restablir la microbiota i el seu metaboloma, similar a l'estat saludable. Aquestes millores es van associar amb una major secreció d'hormones intestinals, i una reducció de la insulinèmia i l'adipositat. / [EN] Obesity is a major public health challenge due to its high prevalence, and association with metabolic comorbidities. Hypercaloric diets are known to overactivate the intestinal immune system and disrupt the microbiome, ultimately causing detrimental metabolic effects. The loss of intestinal immune homeostasis is considered an early step preceding the development of systemic low-grade inflammation associated with obesity and metabolic complications. In this regard, extensive evidence supports that the gut microbiome may be modified favorable and, thus, help to ameliorate these conditions. Hence, identifying factors triggering the low-grade inflammation and microbiome-base solutions to reduce the obesity burden represent promising avenues of research. This Doctoral Thesis aims to advance the knowledge and provide novel probiotics and dietary strategies to combat the burden of obesity based on their immunomodulatory properties to shape the metabolic response to the diet. In the First Chapter, we have investigated the anti-obesogenic potential of propyl propane thiosulfinate (PTS), an organo-sulfur compound derived from Allium species, at two different doses (0.1 or 1 mg/kg/day) using a murine model of diet-induced obesity (DIO). Our preclinical findings showed the protective effects of PTS against obesity, reducing body weight gain and maintaining glucose homeostasis, thus suggesting its potential to ameliorate the impact of the HFHSD. In the adipose tissue and the liver, PTS reduced inflammation and the aberrant lipid metabolism caused by the obesogenic diet. Additionally, PTS promoted thermogenic activity in the brown adipose tissue and enhance intestinal gut barrier defense. In view of the modest changes in the microbial ecosystem, we concluded that the effects of PTS were not mediated by the gut microbiota. In the Second Chapter, we have evaluated the anti-obesogenic potential and the mechanism of action of the new intestinal strain, Phascolarctobacterium faecium DSM 32890, isolated in our laboratory from a healthy volunteer. To that aim, we have performed different in vitro and in vivo experiments, including the use of different types of cell cultures (bone marrow-derived macrophages and group 1 of innate lymphoid cells (ILC1s)) and DIO murine models (wild-type C57BL/6J and Rag1-/- mice). Treatment of HFHSD-fed mice with P. faecium, regardless of its viability, shifted the macrophage phenotype towards an M2-type, which counteracted the obesity-induced increase in gut-resident ILC1s and ultimately mitigated glucose intolerance and body weight gain. Moreover, P. faecium treatment prevented systemic inflammation, boosted secretory immunoglobulin A production and induced antimicrobial peptide and interleukin 22 expression. These metabolic benefits were maintained in the absence of an adaptive immune system but were lost when the bacterium was co-administered with an inhibitor (GW2580) of M2 macrophage polarization. We confirmed that P. faecium was more prevalent in the gut metagenomes of non-obese adults regardless of nationality, sex or age, suggesting that it might contribute to safeguard metabolic health in humans. In the Third Chapter, we have investigated the involvement of gut-resident ILC1s in obesity progression and metabolic disruption. To address this goal, we evaluated longitudinally, in a DIO murine model, the ILC1s response to an obesogenic diet and the consequences of the ILC1s depletion. In the intestine, ILC1s depletion blunted the increases in M1 macrophages and ILC2s. Additionally, ILC1s depletion promoted the ILC3-IL22 pathway, increasing mucin production, the expression of antimicrobial gut peptides, and the number of neuroendocrine cells. Moreover, ILC1s depletion restored microbial and metabolomic profiles, resembling those associated with a healthy symbiotic state. The improvements in gut homeostasis were linked to a higher gut hormone secretion, and reduced insulinemia and adiposity. / Rebeca Liébana García has been beneficiary of an FPU contract (FPU 18/02026) and a mobility grant (EST22/00430) from Spanish Ministry of Universities. The experimental work conduced in this Doctoral Thesis has been funded by the Spanish Ministry of Science and Innovation (MICINN AGL2017-88801-P, PID2020-119536RB-I00), the Centre for the Development of Industrial Technology (CDTI, Ref 20170847), and the EU H2020 Marie Sklodowska Curie Actions (MSCA-IF “MicroILCs, GA: 8905454). / Liébana García, R. (2023). Effect and Mechanisms of Action of Intestinal Bacteria and Bioactive Compounds on the Immune System and Metabolism in Obesity Models [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/201910 / Compendio

Submitted by Maria Aparecida (cidazen@gmail.com) on 2017-04-26T12:29:41Z No. of bitstreams: 1 Bruno Araújo Serra Pinto.pdf: 5546725 bytes, checksum: 07839b14efcc510ac5ecfa62b980626e (MD5) / Made available in DSpace on 2017-04-26T12:29:41Z (GMT). No. of bitstreams: 1 Bruno Araújo Serra Pinto.pdf: 5546725 bytes, checksum: 07839b14efcc510ac5ecfa62b980626e (MD5) Previous issue date: 2017-04-07 / FAPEMA / Background: The epidemiological rise of metabolic syndrome (MS) is directly related to the exponential increase of added sugar consumption. Studies describes that MSmetabolic disorders, mainly insulin resistance and obesity, are related to development of oxidative stress, cognitive declines and dementias, and neuronal senescence acceleration. Even with several evidences correlating MS to neuronal damage, the molecular mechanisms involved are still unclear, and the endoplasmic reticulum stress (ER stress), in this context, could be placed like an intermediary condition that interconnects those morbidities. Objective: To investigate the deleterious effects of hippocampal ER stress about progression of cognitive, behavioral and motor declines in rats with metabolic syndrome-induced by sucrose-rich diet in different ages. Methods: Weaned Wistar rats were divided into 4 groups: two control groups (CTR, n = 7-9), fed a standard diet and followed up to 3 and 6-months-old, respectively; and two obese groups (HSD, n = 7), fed a sucrose-rich diet (25% sucrose) followed by the same periods. Was assessed in these groups: MS development; redox profile; Cognitive, behavioral and motor functions; And the hippocampal gene/protein expression of UPR sensors (Ire1α, Perk and Atf6), chaperones (Grp78, Grp94, Pdi, Calnexin and Calreticulin), neuronal plasticity (Bdnf), antioxidant defense (Nrf2), apoptosis (Bcl2, Chop and Parp-1) and senescence (p53 and p21). For aging control, rats at 20 months of age (OLD, n = 7) fed standard chow were included as aging control for gene/protein expression and neurological assessments. Results: The sucrose-rich diet was successful in establish the SM-phenotype. At 3 months, we observed central obesity even with lower energy intake, fasting and fed dysglycemia, hypertriglyceridemia, hapatic ectopic fat deposit, decreased lipolysis rates, glucose intolerance and hepatic insulin resistance. In unpublished data, we observed mild lipid peroxidation without exepressive antioxidant enzymes activity, and absence of peripheral insulin resistance. In animals with 6 months, we observed a deepening of metabolic dysfunctions encountered in 3-months-old. In addition we observed weight gain, free fatty acids, hyperinsulinemia, peripheral insulin resistance, increased lipid peroxidation, higher SOD, CAT and GPx reduction activity in 6-months-old rats. The lipolysis rate wasn't performed. Regarding the neurofunctional assessment at 3-months-old, the animals presented motor deficit and anxiogenic behavior, however without cognitive dysfunctions. In 6-months-animals, we observed anxiogenic behavior and important motor and cognitive impairments (learning and memory), similar to OLD group. Hippocampal molecular analysis revealed a different signaling between HSD groups of 3 and 6-months. In HSD at 3 months, we observed a switch-over from UPR-adaptive to pro-apoptotic signaling, marked by increased gene expression of Perk, Atf6 and Pdi A2 (adaptive), reduction of Grp78 and Bcl2, and increases of Chop and Caspase 3 (Apoptotic). In 6-months-HSD, we observed a complete failure of UPR adaptive signaling (UPR sensors and chaperones) and increased apoptotic signaling, featured by Bcl2 reduction and increased gene/protein expression of Chop. Additionally, we observed a reduction in the Bdnf gene expression and protein cleavage of Parp-1 compatible to calpain presence (necrosis/apoptosis marker). The expressions found in the 6-month-HSD were similar to OLD group, but the cell death markers (Chop and Calpain) were found only in HSD. As expected, senescence markers (p53 and p21) were increased in the OLD group and only p21 shown increased in HSD. Conclusions: Our data set supports that prolonged exposure to sucrose-rich diet promotes SM and oxidative stress, which disrupt hippocampal ER homeostasis, leading to senescence acceleration and cell death, and subsequently leads to severe cognitive, behavioral and motor impairments. / Introdução: O crescimento epidemiológico da síndrome metabólica (SM) está diretamente relacionado ao exponencial aumento do consumo de açucares de adição. Estudos descrevem que as desordens metabólicas que compõem a SM, principalmente a resistência insulínica e obesidade, estão relacionadas ao desenvolvimento de estresse oxidativo, declínios cognitivos, demências e aceleração da senescência neuronal. Mesmo com diversas evidências correlacionando a SM a danos neuronais, os mecanismos moleculares envolvidos ainda não são totalmente conhecidos. Neste contexto, o estresse do retículo endoplasmático (ERE) pode ser apontado como uma condição intermediária que interconecta estas morbidades. Objetivo: Investigar os efeitos deletérios do ERE hipocampal sobre a instalação de declínios cognitivos, comportamentais e motores em ratos com síndrome metabólica induzida por dieta rica em sacarose em diferentes faixas etárias. Métodos: Ratos Wistar com 21 dias de vida (desmame) foram divididos em 4 grupos: dois grupos controle (CTR, n = 7-9), alimentados com uma dieta padrão e acompanhados até os 3 e 6 meses de idade, respectivamente; e dois grupos obeso (HSD, n = 7), alimentados com dieta rica em sacarose (sacarose a 25%) acompanhados pelos mesmos períodos. Foi avaliado nos grupos: desenvolvimento de SM; perfil redox; funções cognitivas, comportamentais e motoras; e expressão gênica/proteica hipocampal de sensores da UPR (Ire1α, Perk e Atf6), chaperonas (Grp78, Grp94, Pdi, Calnexina e Calreticulina), plasticidade neuronal (Bdnf), defesa antioxidante (Nrf2), apoptose (Bcl2, Chop e Parp-1) e senescência (p53 e p21). Como um controle de envelhecimento, ratos com 20 meses de idade (OLD, n = 7) alimentados com dieta padrão foram incluídos aos experimentos de expressões gênica/proteica e avaliações neurológicas. Resultados: A dieta rica em sacarose teve sucesso em estabelecer o fenótipo de SM. Com 3 meses, o grupo HSD desenvolveu obesidade central mesmo com menor ingestão energética, disglicemia em estados de jejum e alimentado, hipertrigliceridemia, acúmulo de gordura ectópica hepática, diminuição da taxa de lipólise, intolerância à glicose e resistência hepática à insulina. Em dados não publicados, observamos discreta peroxidação lipídica sem expressiva atividade de enzimas antioxidantes e sem resistência insulínica periférica. Nos animais com 6 meses, observamos um aprofundamento das disfunções metabólicas dos animais de 3 meses. Adicionalmente, observamos ganho de peso, ácidos graxos livres, hiperinsulinemia, resistência insulínica periférica, maior peroxidação lipídica, maior atividade das enzimas SOD, CAT e redução da GPx. No que se refere à avaliação neurofuncional, aos 3 meses de idade, o grupo HSD apresentou déficit motor e comportamento ansiogênico, no entanto sem disfunções cognitivas. Contudo, nos animais de 6 meses observamos comportamento ansiogênico e importantes prejuízos motores e cognitivos (aprendizado e memória), semelhantes ao grupo OLD. A análise molecular hipocampal evidenciou uma sinalização diferente entre os grupos HSD de 3 e 6 meses. No HSD com 3 meses, observamos uma transição da sinalização adaptativa da UPR para a pró-apoptótica, marcada pelo aumento das expressões gênicas de Perk, Atf6 e Pdi A2 (adaptativa), redução da Grp78 e aumento da Chop e Caspase 3 (apoptótica). No HSD de 6 meses, observamos uma falência total da sinalização adaptativa da UPR (sensores da UPR e chaperonas), e aumento da sinalização apoptótica, caracterizada pela redução do Bcl2 e aumento da expressão gênica/proteica de Chop. Adicionalmente, observamos também redução da expressão gênica do Bdnf, redução da expressão proteica de Grp94 e clivagem proteica do Parp-1 compatível com a presença de Calpaína (marcador de necrose/apoptose). As expressões encontradas no HSD de 6 meses foram semelhante as alterações observadas no grupo OLD, mas os fatores de morte celular (Chop e Calpaína) foram encontrados apenas no HSD. Como esperado, os marcadores de senescência (p53 e p21) estavam aumentados no grupo OLD e apenas o p21 se mostrou aumentado no HSD. Conclusões: Nosso conjunto de dados apoia que a exposição prolongada à dieta rica em sacarose promove SM e estresse oxidativo, que perturba a homeostase do RE hipocampal, acarretando aceleração da senescência e morte celular, e subsequentes prejuízos cognitivos, comportamentais e motores.

La ménopause est un facteur de risques associé au développement des maladies métaboliques. Du fait de ce déficit hormonal, les femmes ménopausées pourraient présenter des risques particuliers une fois exposées à des agents chimiques capables de perturber le système endocrinien. Pour en savoir plus, nous avons mis au point un modèle de souris ovariectomisées supplémentées ou non en œstradiol (E2), et exposées de manière chronique à un mélange de polluants (TCDD, PCB153, DEHP et BPA) ajoutés à une alimentation obésogène. Chaque polluant est utilisé à une dose équivalente à la Dose Journalière Tolérable, et l’exposition recouvre différents stades de développement, incluant ou non la période maternelle (gestation + lactation). Nos résultats indiquent qu’en condition d’obésité induite par le régime, l’ovariectomie accentue la prise de poids, l’intolérance au glucose et l’insulino-résistance chez les souris femelles adultes. Ce phénotype est associé à une diminution de la signalisation œstrogénique dans le foie et une dérégulation de l’adipogenèse et de l’inflammation dans le tissu adipeux. De manière intéressante, le mélange de polluants réduit l’impact délétère de l’ovariectomie sur le phénotype métabolique, possiblement par une amélioration de la signalisation œstrogénique dans le foie et le tissu adipeux. Nos résultats suggèrent fortement que ces effets dépendent d’une exposition maternelle. En conclusion, nous montrons que notre mélange de polluants possède une activité œstrogéno-mimétique. Ceci pourrait avoir une implication dans la compréhension du rôle des polluants environnementaux dans le développement des maladies métaboliques chez la femme en transition ménopausique / Menopause is a risk factor associated with the development of metabolic diseases. Because of estrogen deficiency, postmenopausal women may be at particular risk when exposed to chemical agents that can disrupt the endocrine system. To get more insight, we have developed a model of ovariectomized mice supplemented or not with estradiol (E2), and chronically exposed to a mixture of pollutants (TCDD, PCB153, DEHP and BPA) added to an obesogenic diet. Each pollutant was used at a dose close to the Tolerable Daily Intake dose, and the exposure had covered different stages of development, including or not the maternal period (gestation + lactation). Our results indicate that in diet-induced obesity, ovariectomy triggered weight gain, increase glucose intolerance and insulin resistance. This phenotype was associated with decreased estrogenic signaling in liver and deregulation of adipogenesis and inflammation in adipose tissues. Interestingly, the mixture of pollutants reduced the deleterious impact of ovariectomy on the metabolic phenotype, possibly through an alleviation of estrogenic signaling in both liver and adipose tissues. These data were not reproduced in mice not exposed to pollutants during the maternal life. In conclusion, the mixture of pollutants displayed some estrogeno-mimetic activity. This could have implications in understanding the role of environmental pollutants in the development of metabolic diseases in women in menopausal transition.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A doença hepática gordurosa não alcoólica é uma desordem multifatorial causada principalmente por excesso nutricional e resistência à insulina, com prevalência estimada de 20-40% nos países ocidentais. A dieta hiperlipídica e/ou rica em sacarose pode influenciar no desenvolvimento da esteatose hepática associada à obesidade e a resistência à insulina. O fígado, por assumir papel central no controle metabólico, é um órgão alvo nos casos de excesso alimentar, ocasionando, principalmente, acúmulo de gotículas de gordura nos hepatócitos. Este trabalho teve como objetivo avaliar o início das alterações morfológicas e metabólicas no fígado e no tecido adiposo de camundongos suíços machos alimentados com dieta hiperlipídica e/ou rica em sacarose. Camundongos suíços machos aos três meses de idade foram divididos em quatro grupos nutricionais: dieta padrão (SC), dieta hiperlipídica (HF), dieta rica em sacarose (HSu) e dieta hiperlipídica rica em sacarose (HFHSu). Os animais receberam as respectivas dietas durante quatro semanas. A massa corporal, a ingestão alimentar e a tolerância oral à glicose foram avaliados. Ao sacrifício, o fígado e os depósitos de gordura corporal foram removidos e processados para análises histomorfométricas e moleculares. As amostras de sangue foram obtidas para análises bioquímicas plasmáticas. Os dados foram expressos como média e erro padrão da média e as diferenças foram testadas por one-way ANOVA com pós-teste de Holm-Sidak, e foi considerado o nível de significância de p<0,05. Os grupos HF e HFHSu apresentaram-se mais pesados quando comparados aos grupos SC e HSu. Os animais dos grupos HF, HSu e HFHSu apresentaram intolerância à glicose, esteatose hepática e aumento de triglicerídeos hepáticos quando comparados ao grupo SC (p<0,0005). Adicionalmente, houve elevação na expressão hepática das proteínas transportador de glicose 2 (GLUT-2), proteína de ligação ao elemento regulador do esterol 1-c (SREBP1-c), fosfoenolpiruvato carboxiquinase (PEPCK), glicose -6- fosfatase (G6PASE), substrato do receptor da insulinaI-1 (IRS-1) e proteína quinase B (AKt/ou PKB) e redução da expressão no fígado do receptor ativador de proliferação peroxissomal (PPAR-&#945;) nos grupos experimentais em comparação com o grupo SC (p<0,0005). A administração de dieta hiperlipídica e/ou rica em sacarose promoveu intolerância à glicose e danos hepáticos (hepatomegalia, esteatose, redução da beta-oxidação, aumento na lipogênese e na produção de glicose) em camundongos machos adultos. / The non-alcoholic fatty liver disease is a multifactorial disorder caused mainly by excess nutritional and insulin resistance, with an estimated prevalence of 20-40% in Western countries. The diet can to influence in the development of fatty liver associated with obesity and insulin resistance. The liver plays a central role in metabolic control, is a target organ, in case of excess of food, mainly causing accumulation of fat droplets in hepatocytes. This study aimed to evaluate the early morphological and metabolic changes in the liver and adipose tissue of male Swiss mice fed high-fat and/or high-sucrose diets. Twenty three-month-old male Swiss Webster mice were divided into 4 groups: standard chow (SC), high-fat diet (HF), high-sucrose diet (HSu) and high-fat-high-sucrose diet (HFHSu). Animals received the respective diets for 4 weeks; throughout the experiment, body mass, food intake and oral glucose tolerance were evaluated. After the mice were euthanized, the liver was removed and processed for histomorphometrical and molecular analysis. Blood samples were obtained for serum analysis. The data were tested by one-way ANOVA with a Holm-Sidak post-hoc test and were expressed as the mean standard error of the mean; the significance level was set at p < 0.05. The HF and HFHSu groups were heavier than the SC and HSu groups. Animals from the HF, HSu and HFHSu groups presented glucose intolerance, hepatomegaly, liver steatosis and augmented hepatic triglycerides when compared to the SC group (p<0.0005). Additionally, there was an elevation in glucose transporter 2 (GLUT-2), sterol regulatory element binding protein-1c (SREBP-1c), phosphoenolpyruvate carboxykinase (PEPCK), glucose 6 phosphatase (G6PASE), insulin receptor substrate 1 (IRS-1), protein kinase B (AKT/ or PKB) protein expression and a reduction in peroxisome proliferator-activated receptor alpha (PPAR-alpha) expression in liver from the experimental groups compared to those of the SC group (p<0.0005). Administration of high-fat and/or high-sucrose diets promoted glucose intolerance and liver damage (hepatomegaly, steatosis, reduced beta-oxidation, increased lipogenesis and glucose production) in adult male mice.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Ainda não está bem definido na literatura se uma dieta rica em sacarose, mesmo sendo isoenergética, provoca danos à saúde. Camundongos C57BL/6 foram alimentados com uma dieta controle (10% da energia proveniente de gordura, 8% da energia proveniente da sacarose - SC), uma dieta rica em sacarose (10% de energia proveniente da gordura, 32% da energia proveniente da sacarose - HSu), uma dieta hiperlipídica (42% da energia proveniente de gordura, 8% da energia proveniente da sacarose - HF) ou uma dieta combinada HF/HSu (42% da energia proveniente de gordura, 32% da energia proveniente da sacarose), durante oito semanas. Apesar da massa corporal e do índice de adiposidade não terem sofrido alteração, o grupo HSu apresentou hipertrofia dos adipócitos, o que também foi observado nos grupos HF e HF/HSu. Os grupos HF, HSu e HF/HSu foram intolerantes à glicose e apresentaram níveis séricos de insulina elevados. Os níveis séricos de leptina, resistina e proteína quimiotática de monócitos-1 (MCP-1) aumentaram, enquanto adiponectina sérica reduziu nos grupos HF, HSu e HF/HSu. No tecido adiposo, os animais HF, HSu e HF/HSu apresentaram maiores níveis de expressão protéica de leptina e níveis mais baixos de expressão protéica de adiponectina, em comparação ao grupo SC. Colesterol hepático foi maior nos grupos HF e HF/HSu, enquanto TG hepático foi maior nos grupos HSu e HF/HSu. Os animais dos grupos HF, HSu e HF/HSu apresentaram esteatose hepática, aumento da expressão protéica hepática de elemento regulador de esterol ligante da proteína 1 (SREBP-1c) e diminuição da expressão protéica do receptor ativador de proliferação peroxissomal alfa (PPAR-&#945;). Em conclusão, a dieta rica em sacarose não provoca obesidade nos animais, mas provoca alterações nos adipócitos (hipertrofia), intolerância à glicose, hiperinsulinemia, hiperlipidemia, esteatose hepática e aumento de citocinas inflamatórias. Os efeitos prejudiciais da dieta rica em sacarose, mesmo quando a sacarose substitui isocaloricamente o amido na alimentação, pode ter consequências para a saúde. / It is still unclear if an isoenergetic, sucrose-rich diet leads to health conse-quences. Mice were fed a control diet (10% energy from fat, 8% energy from sucrose - SC), a high-sucrose diet (10% energy from fat, 32% energy from sucrose - HSu), a high-fat diet (42% energy from fat, 8% energy from sucrose - HF) or a HF/HSu diet (42% energy from fat, 32% energy from sucrose) for eight weeks. Despite the un-changed body mass and adiposity indices, HSu presented adipocyte hypertrophy, which was also observed in the HF and HF/HSu. The HF, HSu and HF/HSu were glucose intolerant and had elevated serum insulin levels. The levels of leptin, resistin and Monocyte Chemotactic Protein-1 (MCP-1) increased, while the serum adiponectin decreased in the HF, HSu and HF/HSu. In the adipose tissue, the HF, HSu and HF/HSu showed higher levels of leptin protein expression and lower levels of adiponectin protein expression in comparison with the SC. Hepatic cholesterol was higher in the HF and HF/HSu, while hepatic TG was higher in the HSu and HF/HSu. Liver steatosis was higher, sterol regulatory element-binding protein-1c (SREBP-1c) hepatic expression was increased, and peroxisome proliferator-activated receptor-alpha (PPAR-&#945;) hepatic protein expression was decreased in the HF, HSu and HF/HSu in comparison with the SC. In conclusion, a sucrose-rich diet does not lead to a state of obesity but has the potential to cause changes in the adipocytes (hypertrophy) as well as glucose intolerance, hyperinsulinemia, hyperlipidemia, hepatic steatosis, and increases in the number of inflammatory cytokines. The deleterious effects of a sucrose-rich diet in an animal model, even when the sucrose replaces starch isocalorically in the feed, can have far-reaching consequences for health.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A doença hepática gordurosa não alcoólica é uma desordem multifatorial causada principalmente por excesso nutricional e resistência à insulina, com prevalência estimada de 20-40% nos países ocidentais. A dieta hiperlipídica e/ou rica em sacarose pode influenciar no desenvolvimento da esteatose hepática associada à obesidade e a resistência à insulina. O fígado, por assumir papel central no controle metabólico, é um órgão alvo nos casos de excesso alimentar, ocasionando, principalmente, acúmulo de gotículas de gordura nos hepatócitos. Este trabalho teve como objetivo avaliar o início das alterações morfológicas e metabólicas no fígado e no tecido adiposo de camundongos suíços machos alimentados com dieta hiperlipídica e/ou rica em sacarose. Camundongos suíços machos aos três meses de idade foram divididos em quatro grupos nutricionais: dieta padrão (SC), dieta hiperlipídica (HF), dieta rica em sacarose (HSu) e dieta hiperlipídica rica em sacarose (HFHSu). Os animais receberam as respectivas dietas durante quatro semanas. A massa corporal, a ingestão alimentar e a tolerância oral à glicose foram avaliados. Ao sacrifício, o fígado e os depósitos de gordura corporal foram removidos e processados para análises histomorfométricas e moleculares. As amostras de sangue foram obtidas para análises bioquímicas plasmáticas. Os dados foram expressos como média e erro padrão da média e as diferenças foram testadas por one-way ANOVA com pós-teste de Holm-Sidak, e foi considerado o nível de significância de p<0,05. Os grupos HF e HFHSu apresentaram-se mais pesados quando comparados aos grupos SC e HSu. Os animais dos grupos HF, HSu e HFHSu apresentaram intolerância à glicose, esteatose hepática e aumento de triglicerídeos hepáticos quando comparados ao grupo SC (p<0,0005). Adicionalmente, houve elevação na expressão hepática das proteínas transportador de glicose 2 (GLUT-2), proteína de ligação ao elemento regulador do esterol 1-c (SREBP1-c), fosfoenolpiruvato carboxiquinase (PEPCK), glicose -6- fosfatase (G6PASE), substrato do receptor da insulinaI-1 (IRS-1) e proteína quinase B (AKt/ou PKB) e redução da expressão no fígado do receptor ativador de proliferação peroxissomal (PPAR-&#945;) nos grupos experimentais em comparação com o grupo SC (p<0,0005). A administração de dieta hiperlipídica e/ou rica em sacarose promoveu intolerância à glicose e danos hepáticos (hepatomegalia, esteatose, redução da beta-oxidação, aumento na lipogênese e na produção de glicose) em camundongos machos adultos. / The non-alcoholic fatty liver disease is a multifactorial disorder caused mainly by excess nutritional and insulin resistance, with an estimated prevalence of 20-40% in Western countries. The diet can to influence in the development of fatty liver associated with obesity and insulin resistance. The liver plays a central role in metabolic control, is a target organ, in case of excess of food, mainly causing accumulation of fat droplets in hepatocytes. This study aimed to evaluate the early morphological and metabolic changes in the liver and adipose tissue of male Swiss mice fed high-fat and/or high-sucrose diets. Twenty three-month-old male Swiss Webster mice were divided into 4 groups: standard chow (SC), high-fat diet (HF), high-sucrose diet (HSu) and high-fat-high-sucrose diet (HFHSu). Animals received the respective diets for 4 weeks; throughout the experiment, body mass, food intake and oral glucose tolerance were evaluated. After the mice were euthanized, the liver was removed and processed for histomorphometrical and molecular analysis. Blood samples were obtained for serum analysis. The data were tested by one-way ANOVA with a Holm-Sidak post-hoc test and were expressed as the mean standard error of the mean; the significance level was set at p < 0.05. The HF and HFHSu groups were heavier than the SC and HSu groups. Animals from the HF, HSu and HFHSu groups presented glucose intolerance, hepatomegaly, liver steatosis and augmented hepatic triglycerides when compared to the SC group (p<0.0005). Additionally, there was an elevation in glucose transporter 2 (GLUT-2), sterol regulatory element binding protein-1c (SREBP-1c), phosphoenolpyruvate carboxykinase (PEPCK), glucose 6 phosphatase (G6PASE), insulin receptor substrate 1 (IRS-1), protein kinase B (AKT/ or PKB) protein expression and a reduction in peroxisome proliferator-activated receptor alpha (PPAR-alpha) expression in liver from the experimental groups compared to those of the SC group (p<0.0005). Administration of high-fat and/or high-sucrose diets promoted glucose intolerance and liver damage (hepatomegaly, steatosis, reduced beta-oxidation, increased lipogenesis and glucose production) in adult male mice.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Avaliar os efeitos benéficos do tratamento com óleo de peixe sobre mudanças metabólicas e morfológicas no pâncreas e tecido adiposo de camundongos C57BL/6 alimentados com dieta rica em lipídeos e sacarose (HLS).Camundongos machos da linhagem C57BL/6, foram alimentados com dieta padrão (P) ou dieta HLS. Aos 3 meses de idade, os camundongos do grupo HLS foram separados em grupo não-tratado (HLS) ou grupo tratado com óleo de peixe (HLS-Px, 1,5g/kg/dia). Aos 4 meses de idade os animais foram sacrificados. O grupo HLS apresentou aumento da massa corporal (MC) e no acúmulo do tecido adiposo total, porém o grupo HLS-Px apresentou menor MC e massa de tecido adiposo comparado ao grupo HLS. As concentrações de glicose plasmática e insulina não foram afetadas entre os grupos, no entanto os grupos HLS e HLS-Px apresentaram maior HOMA-IR. Os grupos HLS e HLS-Px apresentaram maiores concentrações plasmáticas do colesterol total e LDL-C, porém o grupo HLS-Px apresentou maior concentração plasmática do HDL-C e redução da concentração de triglicerídeos. Os adipócitos do grupo HLS apresentaram maior diâmetro quando comparado aos grupos controle e HLS-Px. A massa do pâncreas foi menor no grupo HLS-Px e as ilhotas pancreáticas apresentaram maior diâmetro no grupo HLS, quando comparado ao grupo controle. A expressão de insulina, glucagon e GLUT-2 mostrou-se forte em todas as ilhotas pancreáticas do grupo controle, mas o grupo HLS apresentou fraca expressão para o GLUT-2. Entretanto, HLS-Px apresentou maior expressão do GLUT-2. O tratamento com óleo de peixe foi capaz de reduzir o ganho de massa corporal e a concentração de triglicerídeos, assim como reduzir o acúmulo de tecido adiposo,hipertrofia dos adipócitos, das ilhotas pancreáticas, assim como prevenir a redução do GLUT-2 em camundongos C57BL/6. / To evaluate the fish oil treatment upon morphological and metabolic changes in the pancreas and adipose tissue of C57BL/6 mice fed high-fat-high-sucrose (HFHS) diet. Male C57BL/6 mice were fed HFHS chow or standard chow (SC). At 3 mo-old, HFHS mice were separated into untreated group (HFHS) or treated with fish oil (HFHS-Fo, 1.5 g/kg/day). At 4-mo-old animals were sacrificed. HFHS had increase in body mass (BM) and in total body fat, but HFHS-Fo had smaller BM and total body fat in relation to HFHS. Plasma glucose and insulin levels were not affected among the groups, but HFHS and HFHS-Fo had higher HOMA-IR ratio. HFHS and HFHS-FO had increased plasma total cholesterol and LDL-C, but HFHS-Fo increased plasma HDL-C and decreased triglycerides levels. The adipocytes size were greater in HFHS, when compared to SC and HFHS-Fo groups. HFHS-Fo had smaller pancreas mass and HFHS presented higher islet pancreatic diameter, when compared to SC group. SC group showed strong expression for insulin, glucagon and GLUT-2 in all pancreatic islets, and HFHS presented lesser expression for GLUT-2. However, HFHS-Fo presented increase of GLUT-2 expression. Fish oil treatment was able to reduce body mass gain and plasma TG, reduce fat pad adiposity as well as adipocyte and pancreatic islet hypertrophy, prevent decrease GLUT-2 in C57BL/6.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Avaliar os efeitos benéficos do tratamento com óleo de peixe sobre mudanças metabólicas e morfológicas no pâncreas e tecido adiposo de camundongos C57BL/6 alimentados com dieta rica em lipídeos e sacarose (HLS).Camundongos machos da linhagem C57BL/6, foram alimentados com dieta padrão (P) ou dieta HLS. Aos 3 meses de idade, os camundongos do grupo HLS foram separados em grupo não-tratado (HLS) ou grupo tratado com óleo de peixe (HLS-Px, 1,5g/kg/dia). Aos 4 meses de idade os animais foram sacrificados. O grupo HLS apresentou aumento da massa corporal (MC) e no acúmulo do tecido adiposo total, porém o grupo HLS-Px apresentou menor MC e massa de tecido adiposo comparado ao grupo HLS. As concentrações de glicose plasmática e insulina não foram afetadas entre os grupos, no entanto os grupos HLS e HLS-Px apresentaram maior HOMA-IR. Os grupos HLS e HLS-Px apresentaram maiores concentrações plasmáticas do colesterol total e LDL-C, porém o grupo HLS-Px apresentou maior concentração plasmática do HDL-C e redução da concentração de triglicerídeos. Os adipócitos do grupo HLS apresentaram maior diâmetro quando comparado aos grupos controle e HLS-Px. A massa do pâncreas foi menor no grupo HLS-Px e as ilhotas pancreáticas apresentaram maior diâmetro no grupo HLS, quando comparado ao grupo controle. A expressão de insulina, glucagon e GLUT-2 mostrou-se forte em todas as ilhotas pancreáticas do grupo controle, mas o grupo HLS apresentou fraca expressão para o GLUT-2. Entretanto, HLS-Px apresentou maior expressão do GLUT-2. O tratamento com óleo de peixe foi capaz de reduzir o ganho de massa corporal e a concentração de triglicerídeos, assim como reduzir o acúmulo de tecido adiposo,hipertrofia dos adipócitos, das ilhotas pancreáticas, assim como prevenir a redução do GLUT-2 em camundongos C57BL/6. / To evaluate the fish oil treatment upon morphological and metabolic changes in the pancreas and adipose tissue of C57BL/6 mice fed high-fat-high-sucrose (HFHS) diet. Male C57BL/6 mice were fed HFHS chow or standard chow (SC). At 3 mo-old, HFHS mice were separated into untreated group (HFHS) or treated with fish oil (HFHS-Fo, 1.5 g/kg/day). At 4-mo-old animals were sacrificed. HFHS had increase in body mass (BM) and in total body fat, but HFHS-Fo had smaller BM and total body fat in relation to HFHS. Plasma glucose and insulin levels were not affected among the groups, but HFHS and HFHS-Fo had higher HOMA-IR ratio. HFHS and HFHS-FO had increased plasma total cholesterol and LDL-C, but HFHS-Fo increased plasma HDL-C and decreased triglycerides levels. The adipocytes size were greater in HFHS, when compared to SC and HFHS-Fo groups. HFHS-Fo had smaller pancreas mass and HFHS presented higher islet pancreatic diameter, when compared to SC group. SC group showed strong expression for insulin, glucagon and GLUT-2 in all pancreatic islets, and HFHS presented lesser expression for GLUT-2. However, HFHS-Fo presented increase of GLUT-2 expression. Fish oil treatment was able to reduce body mass gain and plasma TG, reduce fat pad adiposity as well as adipocyte and pancreatic islet hypertrophy, prevent decrease GLUT-2 in C57BL/6.