Pre-clinical evaluation of novel pharmacological targets for the treatment of obesity and eating disorders: focus on Oleoylethanolamide and the oxytocinergic system

Overweight, obesity and eating disorders, such as anorexia nervosa (AN), are complex conditions, influenced by a variety of factors, including genetic, environmental, psychological, and societal elements, and are becoming a serious medical and public health problem. Obesity has been highly associated with a cluster of metabolic dysfunctions leading to insulin resistance and then a higher chance of developing T2DM. In this scenario, OEA, a naturally occurring bioactive lipid belonging to the family of NAEs, has received great attention for its biological properties. It has been demonstrated, over the last two decades from my laboratory and other researchers, that OEA exerts a plethora of protective effects including anti-obesity, anti-inflammatory, and antioxidant properties thus supporting its potential use for the treatment of obesity and eating disorders. Therefore, by using a rat model of early-onset obesity, the aim of the II chapter of this thesis was to evaluate first whether rats with an early-onset obese phenotype showed cardiac and skeletal muscle alteration related to obesity, and then whether chronic peripheral administration of OEA might revert such alterations; moreover, also the effect of OEA on food intake and body weight was evaluated. To this aim, young male Wistar-Han rats were exposed to a HFD (60% of the Kcal from fats) or LFD (10% of the Kcal from fats) for 7 weeks. After the induction of the pre-obese phenotype, both groups were daily treated subchronically for 2 weeks either with vehicle or with OEA at the dose of 10 mg/kg, intraperitoneally. Food intake and body weight were monitored during the entire experiment. At the end of the treatment rats were sacrificed and the heart and the gastrocnemius were collected and processed for different analyses to assess morphological changes, lipid content, molecular pathways related to energy signalling, mitochondrial dynamics and ATP production. At the end of the 7 weeks of the diet exposure, we observed only a slight difference in body weight between HFD and LFD animals of about 7%. Rats exposed to the HFD exhibited increased triacylglycerol levels in skeletal muscle paralleled with a reduction of the carnitine-palmitoyl transferase 1 (CPT-1, an enzyme that regulates the entry of long chain fatty acids into mitochondria, where then undergo β-oxidation) but not in the cardiac muscle. We then analysed the activation of Akt (also known as protein-kinase B, a promoter of energy production) and PDK1 (3-phosphoinositide-dependent protein kinase-1), implicated in Akt signalling pathway. We found an increased activation of Akt and PDK1 in the heart of HV group, well correlating with AMPK decrease in HV group, since these two parameters are oppositely regulated. AMPK is an important sensor of energy balance being allosterically activated by a rise in the AMP/ATP ratio. In the skeletal muscle instead, we did not detect any significant changes in pAkt/Akt and pPDK1/PDK ratios in HV animals, and contrary to what we observed in the heart, in the skeletal muscle of HV rats was observed an increased AMPK activation that OEA was able to lower back to levels comparable to control animals. In the heart, the variations in Akt, PDK1 and AMPK signalling were not paralleled with differences in expression and activities of mitochondrial, as well as ATP production and mitochondrial biogenesis. Instead in the skeletal muscle, the augmented activation of AMPK signalling is paralleled to an increased expression of mitochondrial complexes I, II and IV and activity of complexes I and II and a decreased production of ATP. In this scenario, mitochondrial biogenesis is known to be a compensatory response to mitochondrial damage and insufficient ATP production, with the production of new, healthy mitochondria by mtDNA replication and the recruitment of PGC-1 and Nrf2 which are known to stimulate mitochondrial biogenesis. Additionally, histological changes indicating fibrosis in both muscle tissues of rats exposed to HFD. In all parameters measured, OEA treatment was able to restore the alterations due to the exposure to an HFD. These findings provide important novel information on the antiobesity effect of OEA, shedding new light on its therapeutic potential effect in the treatment of obesity and obesity-related alterations. Regarding OEA mechanism of action, it is now well recognized that the pro-satiety effect of OEA is strictly dependent on the involvement of key brain hypothalamic and hindbrain areas. Indeed, OEA’s satiety action is strictly connected to the recruitment of the oxytocinergic system. Oxytocin (OXY) is an anorexigenic neuropeptide well known for its involvement in delivery and lactation. Recently, OXY raised interest for its action spanning from the homeostatic to hedonic modulation of feeding behaviour. Moreover, OXY promotes social interactions and recognition of conspecifics that rely on olfaction in most species. Indeed, beyond food enjoyment, as part of quality of life, smell has the ability to transfer and regulate emotional conditions, and thus impacts social relationships, at various stages across life. In this context, anorectic patients revealed alterations in the activation of both homeostatic and non-homeostatic (hedonic)-related brain areas, as well as deficits of the olfactory system. Therefore, through this III chapter, we aimed to evaluate a possible alteration of the central oxytocinergic system in a genetic animal model of anorexia, the anx/anx model. This model exhibits poor appetite and a lean appearance, making it a suitable model for studying AN. To reach our aim, in collaboration with Prof. Ida Nilsson at Karolinska Institutet (Stockholm, Sweden), we mapped the oxytocinergic system in brain areas involved in both homeostatic and hedonic eating behaviour, as well as brain areas belonging to the olfactory system. Moreover, neurohypophysial and plasma levels of OXY has been investigated. Furthermore, to deeper understand the degree of involvement of the oxytocinergic system in AN, we profiled the whole transcriptome from both PVN and SON neurons, the two main brain nuclei involved in OXY synthesis. To our surprise, we did not find a difference in OXY-receptor (OXY-R) immunoreactivity in hypothalamic brain areas related to homeostatic regulation of food intake. We instead found a decreased OXY-R immunoreactivity within brainstem areas, where OXY action is interconnected with signals raising from the gut aimed at regulating energy need-based aspects of food intake. Interestingly, we found a very coherent expression pattern of OXY-R within brain areas related to olfaction, in particular an increased immunoreactivity in most of the areas analysed. Briefly, these brain areas analyzed all receive direct inputs from the main olfactory bulb, and each of these has a different role in shaping coming olfactory cues, at levels of social memory, association between odours and social cues, reward related to both social and food stimuli and driving rapid attention to olfactory cues. Our results obtained from the transcriptomic analyses of the PVN and SON highlighted the alteration of genes associated with activation of microglia related to persistent inflammation, response of glial cells, regulation of neuronal plasticity and neurogenesis; interestingly, most of the genes are associated with other neuropsychiatric diseases. Therefore, the results reported in this work of thesis have the potential to unravel new targets for the development of pharmacological treatments for obesity and eating disorders, two major health problems worldwide.