Orientadora: Profa. Dra. Maria Camila Almeida / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2018. / Mentol é um agonista do canal TRPM8 que mimetiza a sensação de frio e recruta mecanismos termorregulatórios de defesa ao frio em mamíferos, promovendo hipertermia e aumentando gasto energético. Por esse motivo, mentol tem sido sugerido como um medicamento para o tratamento da obesidade. Neste estudo, buscamos avaliar a influência do tratamento diário com mentol sobre a massa corporal e sobre os efetores termorregulatórios em ratos Wistar e em camundongos deficientes em leptina (ob/ob). Os animais foram tratados topicamente com mentol 5% por 1, 3 ou 9 dias consecutivos, enquanto a massa corporal, ingestão alimentar, concentração de leptina sérica, temperatura abdominal, consumo de oxigênio, vasoconstricção cutânea, comportamento de preferência térmica e morfologia de adipócitos brancos e marrons foram analisados. Além disso, a expressão de c-Fos foi quantificada no núcleo parabraquial lateral (LPB) e na área pré-óptica mediana (MPA). Em ratos, mentol promoveu hipertermia em todos os dias de tratamento, devido ao aumento do metabolismo e vasoconstrição cutânea, sem afetar a ingestão de alimento ou a concentração sérica de leptina, resultando em menor ganho de tecido adiposo branco e, consequentemente, menor ganho de massa corporal. À medida que o tratamento progrediu, o aumento do metabolismo e a hipertermia induzidos por mentol foram atenuados, mas não abolidos. Além disso, a vasoconstrição cutânea foi potencializada e um aumento no comportamento de busca por calor foi induzido. No entanto, nenhum efeito sobre o ganho de massa corporal foi observado em camundongos ob/ob tratados com mentol, embora em camundongos C57Bl (controles), observamos diminuição no ganho de massa corporal após tratamento com mentol. Além disso, a expressão de c-Fos induzida por mentol no LPB e no MPA não foi alterada ao longo do tratamento. Em conjunto, esses resultados sugerem que, embora mudanças ocorram no recrutamento dos termoefetores durante o tratamento de curto prazo, mentol aumenta o metabolismo sem afetar o consumo de alimento em roedores não obesos em crescimento, o que resulta na inibição do ganho normal de massa corporal ao reduzir o ganho de tecido adiposo branco. Além disso, corroborando com estudos prévios, a estimulação periférica dos canais TRPM8 recruta termoefetores de defesa ao frio via LPB-POA, uma vez que mentol ativa neurônio nestas áreas encefálicas. Contudo, as alterações no recrutamento dos termoefetores em resposta ao tratamento de curto prazo com mentol não é devido a uma alteração na ativação neuronal no LPB ou na MPA. / Menthol is a TRPM8 channel agonist that mimics cold sensation and activates thermoregulatory cold-defense mechanisms in mammals, promoting hyperthermia and increasing energy expenditure. For this reason, menthol has been suggested as an anti-obesity drug. In this study, we aimed at evaluating the influence of daily repeated menthol treatments on body mass and thermoregulatory effectors in Wistar rats and leptin-deficient ob/ob mice. Animals were topically treated with 5% menthol for 1, 3 or 9 consecutive days, while body mass, food intake, serum leptin, abdominal temperature, oxygen consumption, cutaneous vasoconstriction, thermal preference and brown and white adipocyte morphology were analysed. Also, c-Fos expression was quantified in the lateral parabrachial nucleus (LPB) and in the medial preoptic area (MPA). In rats, menthol promoted hyperthermia on all days of treatment, due to an increase in metabolism and cutaneous vasoconstriction, without affecting food intake or serum leptin concentration, resulting in less white adipose tissue gain and, consequently, reduced body mass gain in menthol-hyperthermic rats. As the treatment progressed, the menthol-induced increases in metabolism and hyperthermia were attenuated, but not abolished. Moreover, cutaneous vasoconstriction was potentiated and an increase in the warmth-seeking behavior was induced. However, no effect on body mass gain was observed in menthol-treated ob/ob mice, although C57Bl mice (controls) also presented reduced body mass gain after menthol treatment. Also, menthol-induced c-Fos expression in the MPA and in the LPB was not altered along the treatment. Taken together, the results suggest that, although changes occur in thermoeffector recruitment during the course of shortterm treatment, menthol increases metabolism without affecting food consumption in growing, non-obese rodents, which results in inhibition of normal body mass gain by reducing white adipose tissue gain. Also, in agreement with the previous studies, the peripheral stimulation of the TRPM8 channels recruits cold defense thermoeffectors via LPB-POA, since menthol activates neuron in these encephalic areas. However, changes in the thermoeffectors recruitment in response to short-term menthol treatment are not due to a change in neuronal activity in the LPB or in the MPA.

Les décès attribués à un choc septique à la suite d’une infection sévère augmentent chez les diabétiques et surviennent assez fréquemment dans les unités de soins intensifs. Le diabète sucré et le choc septique augmentent la production d’espèces réactives oxygénées et de cytokines pro-inflammatoires, lesquelles activent le facteur de transcription nucléaire Kappa B conduisant à l’induction du récepteur B1 (RB1) des kinines. Le diabète induit par la streptozotocine (STZ) augmente l’expression du RB1 dans divers tissus périphériques, le cerveau et la moelle épinière. Les lipopolysaccharides bactériens (LPS), souvent utilisés pour induire le choc septique, induisent aussi le RB1. L’objectif de ce travail vise à démontrer la contribution du RB1 des kinines dans l’exacerbation du choc septique pendant le diabète. Des rats Sprague-Dawley (225-250 gr) traités à la STZ (65 mg/kg, i.p.) ou le véhicule ont reçu quatre jours plus tard les LPS (2 mg/kg, i.v.) ou le véhicule en présence ou pas d’un antagoniste du RB1 (SSR240612, 10 mg/kg) administré par gavage. La température corporelle a été mesurée pendant 24h après le traitement. Le SSR240612 a aussi été administré à 9h AM et 9h PM et les rats sacrifiés à 9h AM le jour suivant après un jeûne de 16 h. Les effets de ces traitements ont été mesurés sur les taux plasmatiques d’insuline et de glucose, l’œdème et la perméabilité vasculaire (dans divers tissus avec la technique du Bleu d’Evans) ainsi que sur l’expression du RB1 (PCR en temps réel) dans le cœur et le rein. L’augmentation de la température corporelle après traitement au LPS chez les rats traités ou pas à la STZ a été bloquée par le SSR240612. L’antagoniste a normalisé l’hyperglycémie et amélioré la déficience en insuline chez les rats STZ. Le SSR240612 a inhibé l’œdème et réduit la perméabilité vasculaire dans les tissus des rats diabétiques traités ou pas avec les LPS. La surexpression du RB1 chez les rats traités au STZ et/ou LPS était renversée par le SSR240612. Cet antagoniste a prévenu la mortalité causée par les LPS et LPS plus STZ. Les effets anti-pyrétique, anti-inflammatoire et anti-diabétique du SSR240612 suggèrent que le RB1 puisse représenter une cible thérapeutique valable pour le traitement de la co-morbidité associée au choc septique dans le diabète. / Death attributed to septic shock following severe infection increases in diabetic patients and occurs quite frequently in intensive care units. Diabetes mellitus and septic shock increase the production of reactive oxygen species and pro-inflammatory cytokines leading to higher kinin B1 receptor (RB1) expression that is mediated by the activation of the transcriptional nuclear factor Kappa B. Streptozotocin (STZ)-induced diabetes increased the expression of RB1 in rat peripheral tissues, brain and spinal cord. Bacterial lipopolysaccharides (LPS) commonly used to induce septic shock in animal models, also induce RB1. Our objective is to study the contribution of kinin RB1 in the increased morbidity and mortality associated with the combination of these two diseases. Sprague-Dawley rats (225-250g) treated with STZ (65 mg/kg, ip) or vehicle received four days later LPS (2 mg/kg, iv) or vehicle in the presence or absence of the RB1 antagonist, SSR240612 (10 mg/kg), administered by gavage. Body temperature was monitored for 24h after treatment. In addition, SSR240612 was administered twice (9h AM and 9h PM) and rats were sacrificed the following morning at 9h AM after 16 h of fasting to measure the impact on plasma insulin and glucose, oedema and vascular permeability in various tissues (with the technique of Evans Blue) and on the expression of RB1 (real-time PCR) in heart and kidney. The increase in body temperature caused by treatment with LPS both in STZ-diabetic and non-diabetic rats was blocked by SSR240612. The antagonist normalized hyperglycaemia and improved insulin deficiency in STZ rats. SSR240612 inhibited oedema and reduced vascular permeability in all tissues from diabetic rats treated or not with LPS. The overexpression of RB1 induced by LPS and STZ was blocked by SSR240612. Pharmacological blockade of B1R with SSR240612 prevented the mortality induced by LPS and STZ plus LPS. Thus the anti-pyretic, anti-inflammatory and anti-diabetic effects of SSR240612 suggest that kinin RB1 is a promising therapeutic target for the treatment of co-morbidity associated with septic shock in diabetes.

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