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Obesity and high-fat diet (HFD) consumption result in hypothalamic inflammation and metabolic dysfunction. While the TLR4 activation by dietary fats is a well-characterized pathway involved in the neuronal and glial inflammation, the role of its accessory proteins in diet-induced hypothalamic inflammation remains unknown. Here, we demonstrate that the knockdown of TLR4-interactor with leucine-rich repeats (Tril), a functional component of TLR4, resulted in reduced hypothalamic inflammation, increased whole-body energy expenditure, improved the systemic glucose tolerance and protection from diet-induced obesity. The POMC-specific knockdown of Tril resulted in decreased body fat, decreased white adipose tissue inflammation and a trend toward increased leptin signaling in POMC neurons. Thus, Tril was identified as a new component of the complex mechanisms that promote hypothalamic dysfunction in experimental obesity and its inhibition in the hypothalamus may represent a novel target for obesity treatment.

Aims: To evaluate the effects of hyperlipidic diet on the mouse prostate and also investigate if physical exercise is able to restore such effects. Methodology: Adult male Swiss mice were fed with a balanced (ND) or hyperlipidic diet (45% saturated fat, HD) for 16 weeks. Half were submitted to a sedentary (NDS and HDS) or exercise routine (swimming- NDE and HDE) for 8 weeks. Then, the prostate was analyzed by immunoreactions (proliferating cell nuclear antigen- PCNA, androgen receptor- AR, and estrogen receptor-ERβ), western blotting (ERK 1/2), and caspase-3 activity. Results: We found that saturated fat uptake promoted 16% weight gain, increased fat-mass and hyperglycaemia, as well as reduced testosterone levels. In addition, HD atrophied prostate secretory epithelium and stimulated cell proliferation through higher expression of AR and activation of ERK signaling. Additionally, saturated fat reduced prostatic ERβ content. Physical exercise per se promoted an anabolic effect by increasing testosterone and stimulating cell proliferation in the prostate of sedentary animals. Finally, exercise was able to restore the proliferative signals caused by the hyperlipidic diet on prostate. Conclusion: We suggest that the combination of hyperlipidic diet and sedentary lifestyle could negatively affected some prostate stimulating pathways that could trigger proliferative diseases in mice and physical exercise may be an interesting strategy to reverse such effects.

Swiss mice belong to an outbred strain of mice largely used as a model for experimental obesity induced by high fat diet (HFD). We have previously demonstrated that a given cohort of age-matched Swiss mice is hallmarked by heterogeneous changes in body weight when exposed to HFD. The reasons underlying such variability, however, are not completely understood. Therefore we aimed to clarify the mechanisms underlying the variability in spontaneous weight gain in age-matched male swiss mice. To achieve that, individuals in a cohort of age-matched male Swiss mice were categorized as prone to body mass gain (PBMG) and resistant to body mass gain (RBMG). PBMG animals had higher caloric intake and body mass gain. RBMG and PBMG mice had a similar reduction in food intake when challenged with leptin but only RBMG exhibited a drop in ghrelin concentrations after refeeding. PBMG also showed increased midbrain levels of ghrelin receptor (Ghsr) and Dopamine receptor d2 (Drd2) mRNAs upon refeeding. Pharmacological blockade of GHSR with JMV3002 failed to reduce food intake in PMBG mice as it did in RBMG. On the other hand, the response to JMV3002 seen in PBMG was hallmarked by singular transcriptional response in the midbrain characterized by a simultaneous increase in both tyrosine hydroxylase (Th) and Proopiomelanocortin (Pomc) expressions. In conclusion, our data show that differences in the expression of genes related to the reward system in the midbrain as well as in ghrelin concentrations in serum correlate with spontaneous variability in body mass and food intake seen in age-matched male Swiss mice.

In a public dataset of transcripts differentially expressed in selected neuronal subpopulations of the arcuate nucleus, we identified TLR4-interactor with leucine-rich repeats (Tril) as a potential candidate for mediating the harmful effects of a high-fat diet in proopiomelanocortin (POMC) neurons. The non-cell-specific inhibition of Tril in the arcuate nucleus resulted in reduced hypothalamic inflammation, protection against diet-induced obesity associated with increased whole-body energy expenditure and increased systemic glucose tolerance. The inhibition of Tril, specifically in POMC neurons, resulted in a trend for protection against diet-induced obesity, increased energy expenditure and increased hypothalamic sensitivity to leptin. Thus, Tril emerges as a new component of the complex mechanisms that promote hypothalamic dysfunction in experimental diet-induced obesity.

Chronic HFD intake induces low-grade inflammation in the hypothalamus, which is characterized by proinflammatory cytokine overproduction. This process occurs, at least in part, via TLR4 activation by circulant saturated fatty acids. The TLR4 signaling in the brain is mediated by the TLR4 interactor with leucine-rich repeats (TRIL), an upstream accessory protein. Our main purpose was to determine the putative involvement of TRIL in diet-induced hypothalamic inflammation. We employed male, 8-12 week-old C57BL and POMCCRE mice fed on chow or HFD. The mRNA levels in the hypothalamus were measured by RT-PCR, western blot analyses was used for determination of protein content and immunofluorescence microscopy for evaluate the distribution of TRIL in arcuate nucleus (ARC) of hypothalamus and leptin signaling in POMC-expressing neurons. Finally, bilateral viral injections were performed to non-specific knockdown of TRIL in ARC using shRNA-based lentivirus and Cre-dependent AAV to knockdown TRIL specifically in POMC neurons. The non-specific knockdown of TRIL in the ARC of mice fed on HFD attenuated body weight gain despite no alterations in food intake, improved glucose homeostasis and increased energy expenditure. Moreover, nlrp3, il1β and hspa5 mRNA expression were decreased upon knockdown of TRIL. POMC-specific knockdown of TRIL reduced body fat with no changes in food intake or body weight gain in mice fed on HFD for 8 weeks. The inhibition of TRIL in POMC neurons resulted in a trend of improved leptin signaling in those neurons. We also found increased expression of thermogenic genes in the iBAT and improved fasting glucose. POMC-specific inhibition of TRIL in mice fed on HFD for long-term (24 weeks) did not prevent POMC loss or diminishes cleaved-caspase 3 in ARC. Collectively our results suggest that TRIL is involved in the TLR4-mediated early response to dietary fats and TRIL targeting counteract hypothalamic inflammation and partially restores metabolic homeostasis. Disclosure A. Moura-Assis: None. J. Junior: None. J.M. Gaspar: None. L. Velloso: None. P.S. Nogueira: None. Funding São Paulo Research Foundation (2016/01245-5)

Excessive fat consumption increases the level of fatty acids (FAs) in the blood, which reach the hypothalamus and damage the circuit related to energy balance. In the present study, we used palmitate in a primary culture of purified astrocytes to mimic the fat-rich environment found in obesity. Our results showed increased glial fibrillary acidic protein (GFAP) reactivity in hypothalamic astrocytes compared to cortical astrocytes. In addition, palmitate-treated astrocytes showed no significant changes in cytokine expression and an upregulation of glutathione in the culture medium that may serve as an intrinsic neuroprotective property against excess FA. Additionally, purified hypothalamic neurons were incubated with palmitate-treated astrocyte-conditioned medium (MPAL). MPAL treated-neurons exhibited a reduction in excitatory synapses and enhanced neuritogenesis. Our results suggest that hypothalamic astrocytes react to palmitate differently than cortical astrocytes and influence the behavior of the neural network related to energy balance. Our work brings a better understanding of the interactions among hypothalamic neurons in a high FA environment, similarly to obesity induced by a high-fat diet.

This study examined physiological variables of animals fed with a high-fat diet (HFD) or with a normal diet (ND) subjected to swimming at low and moderate level. Over 16 weeks, a group of animals was fed with HFD or ND, and at the 8 weeks, they started swimming with 50% or 80% of the maximum load achieved in the progressive work test. Weekly, body weight and the amount of ingested food were registered. The glycemic level was measured at the beginning, middle and at the end of the experiment. Adipose tissue, gastrocnemius muscles and hearts were collected for morphometry. The results showed that the animals fed an HFD had a minor caloric intake; however, the HFD increased body weight and adiposity, likely causing cardiac hypertrophy and an increase in the glycemic level. In this context, swimming with an 80% load contributed positively to weight control, adiposity, glycemic level, to control cardiac hypertrophy and induce hypertrophy in the gastrocnemius muscle. All parameters assessed showed better results for the ND animals. Therefore, the importance of fat consumption was emphasized in relation to obesity onset. The practice of swimming with an 80% load produced greater benefits than swimming with a 50% load for overweight treatment.

The arcuate and the paraventricular and lateral hypothalamic nuclei, related to hunger and satiety control, are generally compromised by excess fatty acids. In this situation, fatty acids cause inflammation via TLR4 (toll like receptor 4) and the nuclei become less responsive to the hormones leptin and insulin, contributing to the development of obesity. In this work, these nuclei were analyzed in animals fed with high-fat diet and submitted to swimming without and with load for two months. For this, frontal sections of the hypothalamus were immunolabelled with GFAP (glial fibrillary acidic protein), synaptophysin, IL-6 (interleukin 6) and TLR4. Also, proteins extracted from the hypothalamus were analyzed using Western blotting (GFAP and synaptophysin), fluorometric analysis for caspases 3 and 7, and CBA (cytometric bead array) for Th1, Th2, and Th17 profiles. The high-fat diet significantly caused overweight and, in the hypothalamus, decreased synapses and increased astrocytic reactivity. The swimming with load, especially 80 % of the maximum load, reduced those consequences. The high-fat diet increased TLR4 in the arcuate nucleus and the swimming exercise with 80 % of the maximum load showed a tendency of reducing this expression. Swimming did not significantly influence the inflammatory or anti-inflammatory cytokines in the hypothalamus or in plasma. The high-fat diet in sedentary animals increased the expression of caspases 3 and 7 and swimming practice reduced this increment to levels compatible with animals fed on a normal diet. The set of results conclude that the impact of swimming on the damage caused in the hypothalamus by a high-fat diet is positive. The different aspects analyzed in here point to better cellular viability and conservation of the synapses in the hypothalamic nuclei of overweight animals that practiced swimming with a load.

Obesity is characterized by the excess of body fat and, therefore, may cause musculoskeletal alterations that can negatively influence the tendons. Such overweight-influenced alterations are exercise sensitive though. Morphological and biochemical alterations were reported in the calcaneal tendon of mice submitted to a lipid-rich diets along with practicing exercises, with the following groups: normal diet without exercise (ND), normal diet with exercise (NDex), lipid-rich diet without exercise (LD), lipid-rich diet without exercise (LDex). The calcaneal tendons were removed and subjected to histological and biochemical analysis. Layers of the tissue were stained with Hematoxylin and Eosin, Picrosirius Red and Von Kossa while a protein dosage was conduce by the Bradford method. The morphologicals analysis there was no statistical difference concerning the number of fibroblasts among the groups. Groups submitted to exercises showed higher amount of collagen and non-collagenous protein deposition. The lipid-rich diet without exercse group had a more disorganized collagen matrix with intense basophilia. The same group had areas of calcification confirmed by Von Kossa technique. Practicing physical activity, such as swimming, can improve the changes caused in the calcaneal tendon in mice submitted to a lipid-rich diets, having a better collagen organization and the synthesis.

Obesity-associated hypothalamic inflammation plays an important role in the development of defective neuronal control of whole body energy balance. Because dietary fats are the main triggers of hypothalamic inflammation, we hypothesized that CD1, a lipid-presenting protein, may be involved in the hypothalamic inflammatory response in obesity. Here, we show that early after the introduction of a high-fat diet, CD1 expressing cells gradually appear in the mediobasal hypothalamus. The inhibition of hypothalamic CD1 reduces diet-induced hypothalamic inflammation and rescues the obese and glucose-intolerance phenotype of mice fed a high-fat diet. Conversely, the chemical activation of hypothalamic CD1 further increases diet-induced obesity and hypothalamic inflammation. A bioinformatics analysis revealed that hypothalamic CD1 correlates with transcripts encoding for proteins known to be involved in diet-induced hypothalamic abnormalities in obesity. Thus, CD1 is involved in at least part of the hypothalamic inflammatory response in diet-induced obesity and its modulation affects the body mass phenotype of mice.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico O consumo de dietas hiperlipídicas, ricas em ácidos graxos saturados, induz o ganho de peso corporal e a expressão de citocinas pro inflamatórias que podem ativar vias de apoptose em neurônios que controlam fome e saciedade no hipotálamo. A atividade física e a mudança de hábito alimentar representam alguns tratamentos para a obesidade. Assim, o foco do trabalho foi estudar a resposta corporal de animais submetidos à dieta hiperlipídica e treinamento físico, com exercício de natação, e analisar a reatividade glial, densidade sinaptica, morte celular e perfil de expressão de citocinas no hipotálamo. Após 16 semanas de tratamento com dietas, sendo as 8 últimas com atividade física, os animais tiveram o hipotálamo extraído para análise dos núcleos arqueado, paraventricular e lateral. Os grupos que ingeriram dieta hiperlipídica (HFD) mostraram aumento de peso corporal, hipofagia, maior adiposidade, maior taxa glicêmica, juntamente, com maior reatividade astroglial, menor densidade sináptica, maior TNF e IL-6, e, maior expressão das proteínas caspases 3 e 7 clivadas, em relação aos grupos que foram alimentados com dieta padrão (ND). O grupo HFD que praticou natação com carga de 80% da carga máxima atingida no teste progressivo de esforço foi o que mostrou melhores resultados para os dados fisiológicos e para as dosagens de citocinas e caspases, bem como, mostraram menor reatividade astroglial e maior densidade sináptica, indicando um papel protetor do exercício físico praticado nesse trabalho. The consumption of diets rich in saturated fatty acids induces a gain in body weight and the expression of cytokines pro-inflammatory that can enable the process of apoptosis in neurons that control food intake and satiety in the hypothalamus. Physical activity and the change of dietary habits are the treatments for obesity. Thus, the focus of the study was to evaluate the response of animals submitted to fat diet and physical training, with the exercise of swimming, and analyze the glial reactivity, synapse density, cell death and profile of expression of cytokines in the hypothalamus. After 16 weeks of treatment with diets, being the 8 latest with physical activity, the animals had the hypothalamus extracted for analysis of arcuate, paraventricular and lateral nuclei. The groups that ingested the high fat diet (HFD) showed increased body weight, hypofagia, greater adiposity, increased glucose, along with greater astroglial reactivity, lower synaptic density, greater TNF and IL-6, and, greater expression of s 3 and 7 caspases in relation to the groups that were fed with standard diet (ND). The HFD group that practiced swimming with a load of 80% of the maximum load reached in the progressive effort test showed better results for physiological data and for the determination of cytokine and caspases levels, as well as, showed lower astroglial reactivity and higher synaptic density, indicating a protective role of physical exercise practiced in this work. Dissertação (Mestrado)