Polyphenol effects on central leptin sensitivity in obesity

• Autores: Maria Ibars Serra
• Directores de la Tesis: Cinta Bladé (dir. tes.), Gerard Aragonès Bargalló (codir. tes.)
• Lectura: En la Universitat Rovira i Virgili ( España ) en 2017
• Idioma: español
• Tribunal Calificador de la Tesis: Catalina Picó Segura (presid.), Rosa María Lamuela Raventós (secret.), Chris Grill (voc.)
• Programa de doctorado: Programa de Doctorado en Nutrición y Metabolismo por la Universidad Rovira i Virgili
• Materias:
  • Química
    • Bioquímica
      • Hormonas
      • Procesos metabólicos
  • Ciencias médicas
    • Ciencias de la nutrición
      • Metabolismo energético
      • Enfermedades de la nutrición
• Enlaces
  • Tesis en acceso abierto en:  hdl.handle.net  TDX 
• Resumen

  • Objectives, results and main conclusions: Dietary polyphenols have been widely studied due to its beneficial effects on health. Furthermore, data regarding the effects of polyphenols on metabolism is promising for the prevention and treatment of cardiovascular disease, diabetes type II and obesity. It is of great interest to establish new approaches to decrease obesity since this condition is accompanied by increased health risk factors. In this sense, in vitro, in vivo and some human clinical trials tested the potential of polyphenols on obesity treatment as compounds with the ability to increase energy expenditure. Up to now, the vast majority of studies have focused on studying the effects of polyphenols improving obesity by its action in peripheral organs such as liver, adipose tissue and muscle. However, few studies investigated the effects of polyphenols on the regulation of energy homeostasis through their action in the central nervous system. Leptin is a key hormone on the regulation of energy homeostasis by signaling first order neurons in the arcuate nucleus of the hypothalamus to. Leptin activates POMC neurons and suppresses AgRP/NPY neurons activity producing satiety and increased energy expenditure. In obesity, leptin action is blunted which is reflected by increased circulating leptin levels that fail to counteract the increase in body weight, which is known as leptin resistance.. Therefore, the aim of this thesis was to evaluate the potential of polyphenols to improve leptin sensitivity in the hypothalamus in obesity and determine by which mechanisms these compounds modulate leptin signaling pathway. Previous research demonstrated that a high, pharmacologic, dose of grape seed proanthocyanidins (GSPE) is able to reduce body weight by inhibiting food intake and boosting energy expenditure. These are promising results for the use of GSPE as anti-obese compounds. However, further studies limiting the dose of GSPE to dietary doses are still necessary. Moreover, the satiating effects may be produced by additional mechanisms that regulate food intake and energy homeostasis such hypothalamic leptin signaling. Hence, we performed an experiment that consisted on first, the induction of obesity to animals using a cafeteria diet and after 10 weeks, when animals achieved at least a 10% increase on body weight, were supplemented with 25 mg/kg for a total of 3 weeks [Chapter 1]. This dose corresponds to a human intake of 284 mg GSPE/day by converting animal doses to human equivalent doses (HED) using the body surface area (BSA) normalization method and estimating the daily intake for a 60 kg adult. The human intake of proanthocyanidins ranges from 95 to 200 mg/day. Thus, GSPE doses administered in this study simulate the human proanthocyanidin intake. The effect of GSPE on central leptin signaling was evaluated using rat hypothalamus and determining the expression of genes that regulate leptin signaling pathway and downstream effectors. Firstly, hypothalamic pSTAT3 protein levels were determined as one of the gold standard to assess leptin cascade activation in vivo. The results show that obese animals supplemented with GSPE increased pSTAT3 levels compared to the cafeteria control group reaching similar values to the ones depicted by the lean group. Furthermore, Socs3 levels were also normalized as well as Ptp1b. According to these changes Pomc levels were remarkably enhanced by GSPE treatment. Therefore, the data obtained suggest that GSPE treatment increased leptin signaling in the cafeteria-diet fed rats. Importantly, POMC expression is linked to the induction of anorexigenic signals and animals supplemented with GSPE showed a significant reduction on food intake and a tendency to reduce adiposity after the treatment. Diet induced obesity and leptin resistance is characterized by increased hypothalamic inflammation and ER stress. Notably, in our experiment GSPE reduced hypothalamic inflammation, depicted by the decrease in inos mRNA levels, suggesting this effect could be one of the mechanisms involved in the improvement of leptin signaling induced by GSPE. Furthermore, it has been reported that Sirt1 activity is able to increase leptin sensitivity which lead us to evaluate Sirt1 expression in the hypothalamus. Interestingly, Sirt1 was overexpressed by GSPE treatment meaning that proanthocyanidins could also improve leptin sensitivity by this mechanism. Altogether these results indicate that GSPE increased central leptin sensitivity that was associated to a significant reduction of food intake. However, GSPE supplementation did not correct hyperleptinemia, body weight and adiposity at this dose and time length. For this reason we next focused on other phenolic compounds that could complement GSPE effects modulating leptin signaling and successfully reducing body weight [Chapter 2]. Many studies indicate that resveratrol and anthocyanins have the capacity to reduce body weight. Thus, we planned a first preliminary study to test the capacity of these types of polyphenols to modulate the leptin signaling pathway in the hypothalamus using healthy mice. The results showed that the daily treatment of mice with resveratrol, at a dose of 100 mg/kg body weight for 15 days, increased the energy expenditure in mice and overexpressed Obrb in the hypothalamus whereas an anthocyanin rich extract did not produce significant changes. According, some in vivo studies show that anthocyanins have anti-obesity properties by decreasing circulating leptin levels through the modulation of adipocytokine secretion and lipid metabolism in the adipose tissue.These results indicate a higher potential of resveratrol over the anthocyanins to improve leptin signaling in the hypothalamus and to reduce energy homeostasis.

    Due to the wide range of resveratrol doses used in animal studies studies, we decided to test the effect of a low, moderate and high dose of resveratrol using a diet-induced obesity model. Therefore, obese animals were supplemented with 50, 100 and 200 mg/kg of resveratrol which in HED correspond approximately to 486 mg, 970mg and 1.95 g resveratrol/day for an individual of 60 kg. These doses have previously been tested in human clinical trials and were generally well tolerated. The results demonstrated that the highest dose of resveratrol effectively corrects hyperleptinemia produced by the cafeteria diet and increased hypothalamic pSTAT3, suggesting that leptin sensitivity was improved. Interestingly, the highest dose of resveratrol produced a significant reduction of body weight, increased 24h energy expenditure and lipid oxidation without decreasing food consumption. Therefore, the high dose of resveratrol clearly exerted anti-obesity effects that were mediated, at least partly, by the restoration of leptin sensitivity in the hypothalamus of obese rats. The findings obtained in chapter 1 and 2 suggest that a mixture of 25 mg GSPE with 200 mg resveratrol by kg of body weight has the potential to successfully reverse obesity in obese animals since both types of polyphenols act as leptin sensitizer in the hypothalamus and GSPE modulate satiety whereas resveratrol induced energy expenditure, all of them key factors for body weight regulation. Fruits are an important polyphenol source in the human diet and their consumption is highly recommended to maintain health. Nowadays the consumption of seasonal and out-of season foods has generated some debate related to health effects and ecosystem sustainability. In this sense, the Xenohormesis Hypothesis states that plant molecules, such as polyphenols, are able to modulate mammalian physiology when consumed. These cues warn about environmental conditions where the plant developed allowing animals to adapt to the situation. Thus, we consider relevant to assess the effects produced by seasonal fruits rich in polyphenols when they are consumed out of season regarding their impact in the central leptin system and obesity [Chapter 3]. Leptin secretion is influenced by the light-cycle in seasonal animals which means the levels of this hormone fluctuate in accordance to a circannual rhythm. Seasonal animals increase their body stores and develop hyperleptinemia during summer and spring and gain less weight and decrease serum leptin during winter and autumn.

    For this study we used the Fisher-344 rat strain because its sensitivity to photoperiods (cita). Rats were placed in long-day (LD) (18:6h light:dark cycle) or short day (SD) (6:18h light:dark cycle) to simulate spring or autumn, respectively. Numerous studies report the metabolic protective effects of grape, grape by-products, cherries or their pure compounds, thus we have chosen grape and cherry as representative fruits of autumn and spring, respectively. In addition, this study was performed in both lean and dietary induced obesity models. Our results show that lean animals placed in SD photoperiod displayed decreased fat mass and serum leptin, despite no changes in body weight were observed. This data agrees with other studies using the same rat strain. Moreover, animals in SD showed a lower energy balance attributed to increased energy expenditure since no changes in cumulative food intake were detected. The gene expression of the components involved in leptin signaling pathway in the hypothalamus were not affected by photoperiod in lean animals.

    On the other hand, animals that consumed an obesogenic diet loose the photoperiodic regulation of fat mass but also showed a decreased energy balance in SD, in this case as a consequence of a reduction on the cumulative food intake. Contrarily to lean animals, in the obese model the photoperiod did not affect circulating leptin. However, central leptin system was affected, overexpressing Socs3 and Agrp during SD. In view of the previous results, we attempted to understand how the consumption of seasonal fruits in different photoperiod could affect central leptin system and energy balance in both lean and obese animals. In lean rats, grape and cherry consumption decreased the energy balance in a photoperiod independent manner. Interestingly, this effect was consequence of a reduced cumulative food intake in rats consuming grape whereas increased energy expenditure was observed in animals consuming cherry. Furthermore, both fruits increased Pomc mRNA levels in SD, which could explain the drop in energy balance observed in animals exposed to SD. Besides, cherry intake increased Obrb expression in SD suggesting that cherry consumption increases the central leptin sensitivity. However, Socs3 and Agrp, which are markers of attenuated leptin signaling, were overexpressed by cherry consumption in both LD and SD photoperiods. No effects of grape intake were observed in obese animals in either photoperiod. In contrast, cherries were very effective modulating the leptin system in obese rats in a photoperiod dependent mode. Specifically, cherry consumption produced an anorexigenic response in SD depicted by reduced cumulative food intake as well as downregulation of Agrp and Ptp1b meaning that leptin sensitivity was increased by this fruit in a photoperiod dependent manner. In addition, cherry intake effectively modulated receptors expressed in second order neurons, outside of the arcuate nucleus, Mc4r and Npy1r when was consumed at SD Therefore, cherry modulated leptin system when it was consumed out of season in both lean and obese animals. Importantly, in this study cherry modulated Agrp gene expression in photoperiod independent manner in both lean and obese animals, which could mean that AgRP neurons were more sensitive to cherry polyphenols compared to Pomc neurons. Interestingly, it has recently been demonstrated by researchers in Xu Laboratory that around 60-70% of AgRP neurons are located outside the blood brain barrier which makes them more susceptible to metabolic changes, since they are expose to blood borne substances. Then, we hypothesize that although polyphenols are able to cross the BBB, AgRP neurons are a good putative target for polyphenols since polyphenols circulating in systemic blood can directly interact with these neurons. This part of the thesis [Chapter 4] was performed in Professor Allison W. Xu laboratory, in the Diabetes Center at University of California, San Francisco. AgRP neurons are activated by fasting. However the specific mechanisms that modulate AgRP activity are not completely clarified. Recent data showed that AgRP neurons are activated by Gs protein-coupled receptors (GPCRs) which trigger a sustained increase in food intake. Adenosine signaling plays an important role in the CNS modulating neuronal activity by specifically binding to its receptors. Interestingly, adenosine receptor 2B (A2B receptor) is a Gs-coupled GPCR. Little is known about the role of this receptor in the CNS. Hence, we hypothesize that extracellular adenosine might bind the A2B receptor to sustain the firing in a positive feedback loop and to activate cellular responses in AgRP neurons. For this reason, we aimed to determine the role of A2B receptor in the nervous system and in AgRP neurons. The results obtained show that mice with a deletion of A2B in the nervous system reduced refeeding phenotype after 24-hour fasting, whereas deletion of A2B in AgRP neurons did not produce any change during refeeding. This could mean that A2B receptor exerts its effects through other pathways that also regulate feeding, such as neuropeptide Y (NPY) release or POMC neurons activity. Furthermore, mice with A2B deletion in AgRP neurons and challenged with 8-hour fasting at the beginning of the dark-phase, showed reduced cFos expression. Therefore, our results indicate that mutant mice presented a reduced neuronal activity in the ARC nucleus, where AgRP cell bodies are located. Altogether the results obtained in this study allows to suggest that during fasting AgRP neurons increase their firing rate and produce ATP in order to obtain energy. Adenosine is transported outside of the cell through the ENT1 transporter and, subsequently, extracellular adenosine levels rise. In this circumstance adenosine may bind A2B receptor and activate the transcription of AgRP gene which, in turn, leads to a positive feedback loop that keeps these neurons activated. This study brings novel insights about the modulation of AgRP neurons through A2B receptor which is a potential target to prevent or correct metabolic diseases. Future research on the field of bioactive compounds such as polyphenols may take advantage of these findings. In summary, different classes of polyphenols showed the ability to ameliorate energy homeostasis in obesity partly through the modulation of leptin signaling and improving leptin sensitivity in the hypothalamus. Therefore, these compounds are promising candidates for the design of functional foods that help to reduce obesity and the associated risk factors.

    Methodology:

    Rat and mouse handling: - Diet and treatment doses administration - Body weight measurements and body composition analysis - Metabolic analysis by indirect calorimetry - Food intake control - Euthanasia, organ dissection Experimental techniques: - Genotyping - RNA and protein isolation and quantification from tissues, mainly hypothalamus.

    - Determination of gene expression by qPCR and protein expression by Western Blot - Mouse brain cryosectioning for histological purposes and immunofluorescence.