Obesity is a growing public health threat, potentially affecting emotional, and physical health with a relevant mortality rate and a high burden of disease for Western societies. Core symptoms of this disease are disturbance of eating habits and inability to control body weight, thus leading to an imbalance between energy intake and expenditure. In recent years several approaches have been used to treat obesity, yet the efficacy of the few medications proven useful remains a matter of debate. Thus, effective and safe treatments are urgently needed (Patel and Stanford, 2018). Efforts should be made to disclose the causes that make individuals more vulnerable to the development of obesity, in order to gain useful information for alleviating or even preventing occurrence of this condition. Genetic predisposition has been implicated in disease susceptibility (Stice, 2002; Striegel-Moore and Bulik, 2007), however, differences in phenotype heterogeneity point also to the relevant role of the environment and lifestyle (Hill et al., 2003). Molecular research has formed the basis to understand how environmental factors (i.e., dieting) may facilitate disease progression by engaging epigenetic mechanisms (van Dijk et al., 2015), helping also to understand the increasing epidemic of the disease in the last years within the same genome. It is in fact well known that the latter mechanisms can evoke transient changes (although how long they actually last is unclear) in gene expression, involving chemical modifications of DNA that do not affect the actual DNA sequence of the organism. The understanding of how transcriptional regulation might affect individual risk of developing obesity represents a major challenge in research and may provide invaluable help for the development of preventive strategies, or of more effective therapeutics. Central regulation of food intake is rather complex, and thus different endogenous key players should be considered. For instance, it is clear that rewarding properties of food, possibly leading to drug-like food addiction (D’Addario et al., 2014; Mancino et al., 2015), are responsible for disease development and share neuronal pathways that seem to overlap with drug addiction (Volkow and Wise, 2005). These neural circuitries, driving eating behaviors in the brain to ensure food-intake and to regulate caloric balance, are thus controlled not only by homeostatic mechanisms but also by reward systems to promote motivational, hedonically driven feeding (Coccurello and Maccarrone, 2018). Among the endogenous systems involved in both rewarding and homeostatic mechanisms, a relevant role is played by both the endocannabinoid (ECS) and the opioid (EOS) systems (Cota et al., 2003a; Tanda and Goldberg, 2003), which functionally interact with each other in mediating neurological functions (Manzanares et al., 1999; Tanda and Goldberg, 2003). In feeding regulation, both ECS and EOS seem to contribute to the reward aspect of eating (Saper et al., 2002; Yeomans and Gray, 2002; Cota et al., 2003b). Of note, several preclinical studies reported that both opioid and cannabinoid receptor agonists stimulate food intake (Reid, 1985; Foltin et al., 1988; Williams et al., 1998; Glass et al., 1999; Williams and Kirkham, 1999), that is instead reduced by antagonists (Recant et al., 1980; Marks-Kaufman et al., 1984; Colombo et al., 1998; Di Marzo et al., 2001; Glass et al., 2002; Hildebrandt et al., 2003; Ravinet Trillou et al., 2003; Statnick et al., 2003; Vickers et al., 2003) with suppression of body weight gain in rodents. It has been also observed in human trials that opioid antagonists (naloxone, naltrexone, or nalmefene) may be helpful in the short term to suppress appetite, even though caution should be taken for possible long term use because of side effects and limited weight loss (Nathan and Bullmore, 2009). In this context, it should be recalled that rimonabant, a selective type 1 cannabinoid receptor (CB₁) antagonist able to reduce body weight (Rinaldi-Carmona et al., 1995; Di Marzo and Despres, 2009), was an approved drug on the European market, but was withdrawn because of an increased risk of psychiatric disorders (Johansson et al., 2009).

Moreover, combination therapies [i.e., Contrave (Apovian et al., 2013) and Qysmia (Gadde et al., 2011)] also held promise for obesity treatment, and preclinical studies performed using a combination of opioid and cannabinoid antagonists (naloxone and nor-BNI with rimonabant) demonstrated enhanced feeding reduction when compared to that evoked by rimonabant alone (Lockie et al., 2011, 2015).

Against this background, the aim of this study was to advance our knowledge about the molecular mechanisms engaging ECS and EOS in the development of obesity, in order to identify disease biomarkers and disclose new molecular clues to be targeted by innovative strategies of pharmacological intervention. By using a well-established rat model of Diet-Induced Obesity (DIO), we first investigated regulation of ECS and EOS genes expression during disease progression in the hypothalamus, a brain region involved in appetite regulation (Bazhan and Zelena, 2013), where the role of ECS (Berthoud, 2012; Zeltser et al., 2012; Ramírez-López et al., 2015) and EOC (Dum et al., 1983; Nogueiras et al., 2012; Romero-Picó et al., 2018) in feeding response modulation has been deeply investigated and already reported. We followed gene expression regulation at the beginning of obesity development (i.e., after 5 weeks on high fat diet), as well as when the phenotype was well-established (i.e., after 21 weeks on high fat diet). Among the epigenetic mechanisms possibly responsible for altered gene expression, we focused on DNA methylation, that consists of a methyl group transfer to the position 5 of the cytosine pyrimidine ring in a cytosine guanine dinucleotide (CpG), which ultimately blocks the binding of transcription factors thus causing chromatin compaction and gene silencing (Zhu et al., 2016). Of note, by using the same DIO animal model considered here, we already showed the epigenetic regulation of other obesity genes in the progression of the disorder (Cifani et al., 2015).

In addition, in this work, we also analyzed the DNA methylation status of key ECS and EOS genes in peripheral blood mononuclear cells (PBMCs) from a subset of humans with obesity. It is important to recall that PBMCs contain the complete epigenetic machinery present in neurons (Arosio et al., 2014), and are considered a convenient substitute for cerebral markers (Woelk et al., 2011) that are readily accessible and reflects the molecular processes occurring in the central nervous system (Gladkevich et al., 2004). PBMCs have been already proposed in obesity studies as a valuable tool to monitor metabolic recovery in weight loss strategies (Reynés et al., 2015), and thus their study in this disorder is of clear relevance to detect altered pathways (metabolic or signaling) that could be transferred to the brain. Moreover, it has been well-documented since many years the activation of the ECS in human obese subjects and the regulation of EOS components in obesity and related eating disorders in the periphery (Jimerson and Wolfe, 2004).

The first outcome of this study is the selective up-regulation of the expression of Cnr1, the gene coding for CB₁, and of Oprm1, the gene coding for MOP, in the hypothalamus of rats exposed to high fat diet for 5 and 21 weeks. These alterations were present at both time-points analyzed for CB₁, that appeared to be engaged in long-lasting effects, and only at the beginning of obesity development for MOP, that appeared to be engaged in obesity onset only. Of note, we failed to observe any alteration in any other component of the ECS and of the EOS, speaking in favor of a distinct role of the two receptors in obesity.

Our data are thus consistent with many studies that already showed the hyperphagic role of CB₁ (Williams and Kirkham, 1999; Jamshidi and Taylor, 2001; Verty et al., 2005; Koch et al., 2015) and MOP (Smith et al., 2002) in this brain region. Hypothalamic administration of the endocannabinoid anandamide (Jamshidi and Taylor, 2001) or of the cannabis extract tetrahydrocannabinol (THC) (Verty et al., 2005), both primarily acting though CB₁ in the brain, rapidly increases food intake in rats. Studies focused on the hypothalamus documented also that high-fat feeding increases MOP protein levels in Wistar rats (Smith et al., 2002), as well as in rats susceptible to obesity (Barnes et al., 2006).

The link between these two receptors in the promotion of feeding has been proposed by Kock and colleagues, who showed that this effect can be due to CB₁ activation on POMC neurons leading to the release of β-endorphin, an opioid neuropeptide acting on MOP (Koch et al., 2015).

Despite the great amount of research showing CB₁ levels dysregulation centrally in animal models of obesity (South and Huang, 2008; Massa et al., 2010), there is less information about altered hypothalamic Cnr1 mRNA levels, which anyhow have been reported by some (Kempf et al., 2007; Gamelin et al., 2016; Ramirez-Lopez et al., 2016). Consistently with our present findings, Cnr1 mRNA levels increased in female offspring hypothalami from rat dams fed a high caloric diet (Ramirez-Lopez et al., 2016). Moreover, receptor knockdown in mice hypothalamus induced a reduction in body weight while increasing energy expenditure (Cardinal et al., 2012). On the other hand, receptor gene expression was down-regulated in the hypothalamus of rats fed with a palatable high-energy diet (Timofeeva et al., 2009). It seems noteworthy that in a recent analysis of the transcriptional regulation of ECS components in an animal model of binge eating behavior, we observed a selective epigenetic regulation of fatty acid amide hydrolase, the key enzyme for the degradation of anandamide, instead, Cnr1 and all other ECS components were not affected (Pucci et al., 2018). This seems of particular relevance if one aims at finding specific biomarkers for different eating disorders and related disturbances (such as obesity).

The involvement of MOP gene regulation in obesity has also been investigated. MOP KO mice fed a high-fat diet were resistant to obesity (Tabarin et al., 2005), even though others also showed that MOP KO mice on a standard diet increased body weight in adulthood, when compared to wild-type littermates (Han et al., 2006). Analysis of gene expression showed increased MOP mRNA levels in hypohalami of the offspring of obese pregnant mice (Vucetic et al., 2010; Grissom et al., 2014), whereas no changes were observed in mice fed from weaning with high-fat diet for around 15 weeks (Vucetic et al., 2011).

In order to characterize MOP and CB₁ transcriptional regulation, we sought to compare hypothalamic genes DNA methylation. We observed a consistent reduction of the epigenetic hallmark at Cnr1 promoter, in four of the eight CpG sites under study as well as in their methylation average, but only at the earliest time-point (5 weeks). However, no changes were observed in MOP DNA methylation. These findings suggest that the temporary hypomethylation at Cnr1 gene at the beginning of obesity development might be responsible of the early changes observed in gene expression; yet, it does not seem necessary in the long term, when chromatin might be in a poised state with mRNA levels resulting still high. At any rate, this is the first report showing DNA methylation of Cnr1 in obesity, whereas others have already addressed MOP epigenetic regulation in mice reward-related brain regions of the offspring of pregnant dams exposed to chronic high-fat diet (Vucetic et al., 2011). In partial agreement with our data, also the latter studies failed to show alterations of methyl CpG-binding protein, as well as of histone acetylation, at MOP gene promoter in the hypothalamus (Vucetic et al., 2011). Very recently the hypothalamic increase in histone acetylation was instead reported at Cnr1 gene promoter, and was linked to increased hypothalamic receptor expression (Almeida et al., 2019).

With the aim to extend the study of CB₁ and MOP receptors to human obesity, we assessed whether PBMCs might mirror central nervous system defects, and we then evaluated DNA methylation at CNR1 and OPRM1 gene promoters in PBMCs of humans with obesity. We failed to observe any difference between controls and humans with obesity in the overall population, yet age-based stratification of the data clearly showed a significant reduction of the epigenetic hallmark at both CNR1 and OPRM1 promoters in younger (<30 years old) humans with obesity. As for CNR1, an age-dependent modulation was observed at one of the five CpG sites analyzed, as well as at their average, possibly driving an up-regulation of gene expression compared with normal weight subjects. Instead, in the same population DNA methylation was reduced in four of the five CpG sites analyzed. These differences for both receptors did not occur in subjects older than 30 years. Our findings appear of particular relevance if one attempts to identify early disease biomarkers. In agreement with this, a lower DNA methylation was also observed at genes promoters of humans with obesity with a shorter time length from disorder onset, and in those with lower BMI.

At the clinical level, it seems noteworthy that an increased CNR1 expression in obesity has been shown at the peripheral level (Pagano et al., 2007; Sarzani et al., 2009), but never in blood samples from human subjects. Moreover, to the best of our knowledge, OPRM1 expression in humans with obesity has not been studied yet, with the only available PET clinical study in different brain regions reporting lower MOP availability in obese subjects compared to controls (Karlsson et al., 2015).

Overall, our findings in human peripheral samples confirm the data observed centrally in the animal model, showing that regulation of CNR1 and OPRM1 genes is altered mainly at the early stage of phenotype development, at least in terms of the epigenetic hallmarks analyzed.

In conclusion, epigenetic switching appears dynamic and temporary in the preclinical and clinical models used here to investigate CNR1 and OPRM1. Alterations of DNA methylation in these two genes, observed earlier in the obese phenotype as a result of early exposure (i.e., to high-fat diet or, for instance, to stressful conditions), disappear at a later stage in life. Thus, here we clearly show the relevant role of CNR1 and OPRM1 transcriptional regulation as a possible biomarker for obesity and, due to the reversible nature of the epigenetic hallmark, our data open new avenue for environmental strategies of intervention. As yet, conventional environmental strategies for the treatment of obesity include lifestyle modifications of diet and physical activity, and are often insufficient (Blevins, 2010). However, this might be also due to the lack of an early diagnosis. In line with this, our data support the relevance of a preventive therapeutic approach based on environmental factors.

Of relevance, a very recent study has reported altered DNA methylation on obesity-related genes in healthy adolescents, and these alterations were suggested as early life changes possibly associated with elevated disease risk in adulthood (He et al., 2019).

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

This study was carried out in accordance with the recommendations of “name of guidelines, name of committee” with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Ethic Committee of IRCCS – Istituto Auxologico Italiano (RBFR12DELS_004/05C302_2013).

This study was carried out in accordance with the recommendations of Institutional Guidelines and complied with the Italian Ministry of Health and associated guidelines from European Communities Council Directive. The protocol was approved by Italian Ministry of Health no. 1610/2013.

MP, CD, CC, and MM conceived and designed the experiments. CD, MP, MM, VV, SM, EZ, and MMas performed the experiments. CD, MS, LP, MP, and CC analyzed the data. CD, CC, and AS contributed reagents, materials, and analysis tools. CD, MP, and MMac wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.