4. Discussion
Dysregulation of circadian rhythm homeostasis has been associated with various disorders of lipid metabolism, including obesity(Li et al., 2020). In this sense, melatonin has been explored as a treatment for obesity and other metabolic disorders. Since conflicting results have been reported(Genario, Cipolla-Neto, Bueno, & Santos, 2021; Loloei et al., 2019), a more in-depth investigation is required to develop innovative therapeutic strategies. With this aim, we evaluated the effect of agomelatine, a melatonergic agonist, in experimental obesity in mice, and compared it to melatonin and metformin, the most prescribed antidiabetic drug.
Our results show that agomelatine treatment lowered body weight gain and fat accumulation in a similar manner to previously observed for melatonin(de Farias et al., 2019) and metformin(Ji, Wang, & Li, 2019), as well as improved obesity-associated glucose intolerance. This confirms previous experimental observations that associate weight decrease and enhanced insulin sensitivity with the regulation of metabolic clock and/or the increase of the energy expenditure/intake ratio(Cherngwelling et al., 2021; Farias et al., 2019). Although some preclinical studies have indicated that melatonin lowers body weight and visceral fat accumulation(de Farias et al., 2019; Farias et al., 2019), this has not been confirmed in humans(Mantele et al., 2012). However, it has been observed that agomelatine administration in patients with night eating syndrome reduces body weight, related to restoration of sleep patterns and sleep-related eating disorders(Milano et al., 2013), and its anti-obesogenic has been recently reported in HFD-fed rats(Cherngwelling et al., 2021), in agreement with our observations.
Regarding the lipid profile, whereas melatonin only reduced HDL-cholesterol, agomelatine supplementation showed a significant improvement in the cholesterol profile. This effect could be related to the direct effect of the circadian rhythms regulating dietary lipid absorption in intestinal enterocytes(Hussain & Pan, 2012). Metformin treatment also ameliorated the hypercholesterolemic status induced by HFD, an effect widely described in humans and mice(Gonzalez & Jiang, 2017), lightening the severity of high fat induced hepatic steatosis.
The accumulation of fat tissue in obesity is linked to a chronic low-grade inflammation, with elevated circulating proinflammatory mediators, such as TNF-α and IL-6 secreted by the liver and fat tissue(Ellulu, Patimah, Khaza’ai, Rahmat, & Abed, 2017). This drives immune cell recruitment and activation of inflammatory signalling pathways, such as the c-Jun N-terminal kinase (JNK)-related signalling, which interferes with insulin signalling, and therefore, with the glucose metabolism(Lee, Giraud, Davis, & White, 2003). Consistently, HFD led to augmented expression of different pro-inflammatory mediators, including Tnf-α , Il-1β ,Il-6 and Mcp-1 , in adipose tissue and liver. While all the treatments reduced their expression in the liver, only agomelatine reduced them in fat tissue. Likewise, all treatments significantly lowered HFD-induced Jnk-1 up-regulation, in line with previous results(Farias et al., 2019). Nevertheless, this is the first report of such anti-inflammatory activity for agomelatine treatment in obese mice, which could participate in the improvement of insulin signalling and glucose metabolism.
The excessive accumulation of adipose tissue can also produce an alteration in adipokine levels, such as leptin and adiponectin. Leptin, apart from suppressing appetite, is considered a pro-inflammatory mediator associated with insulin resistance(Yadav et al., 2013). In obesity, decreased expression of the leptin receptor in liver and fat lead to leptin resistance and excessive leptin release(Yadav et al., 2013). Conversely, adiponectin is an anti-inflammatory and insulin-sensitizing mediator that suppresses hepatic glucose production(Sharma, McClung, & Abraham, 2016). Altered leptin and adiponectin expression profiles were observed in this study. Lower fat expression of adiponectin in obese mice, which agrees with previous studies(Wu et al., 2021), was only significantly upregulated by agomelatine and metformin. Whilst metformin and melatonin have been widely assessed for their impact on adipokine production in experimental and human studies of obesity or diabetes(Ferreira-Hermosillo et al., 2020; Su et al., 2016), this is the first evidence of beneficial impact of agomelatine on adiponectin and leptin expression.
Besides, obesity-related insulin resistance implies intracellular glucose uptake impairment due to reduced GLUT-4 expression. Such effect was observed in HFD-fed mice and counteracted by agomelatine and metformin, but not melatonin, which neither has previously shown a significant effect on pinealectomized animals(Nogueira et al., 2011). Also related with insulin resistance in obesity is the role of AMPK, involved in the translocation of GLUT-4 transporters to the membrane and the inhibition of liver gluconeogenesis and inflammatory pathways(Ruderman, Carling, Prentki, & Cacicedo, 2013). Antidiabetic drugs, including metformin, act as insulin sensitizers through AMPK activation(Lu et al., 2019). Hence,Ampk expression in liver and fat was partially restored by metformin, but also agomelatine. These results, together with the decreased glycaemia, confirm the capacity of the agomelatine treatment to improve insulin sensitivity and glucose homeostasis facilitated by central and peripheral target tissues. In addition to energy metabolism, AMPK is also recognized as a regulatory node for immune responses(O’Neill & Hardie, 2013). AMPK activation inhibits two major immune signalling pathways, nuclear factor-κB (NF-κB) and signal transducer and activator of transcription (STAT), reducing proinflammatory cytokines expression(Salminen, Hyttinen, & Kaarniranta, 2011). This anti-inflammatory effect was also evidenced mainly in agomelatine-treated mice, reducing cytokine expression and immune cell infiltration.
Under pathological conditions, like obesity, the pro-inflammatory milieu stimulates the proliferation immature myeloid cells (IMCs) and block the differentiation into mature myeloid populations, causing the accumulation of myeloid-derived suppressor cells (MDSCs) (Ly6C+CD11b+). The liver is the major organ where IMCs accumulate(Budhwar, Verma, Verma, Rai, & Singh, 2018), and, in agreement with previous studies(Sundara Rajan & Longhi, 2016), we observed an increase of MDSCs in obese mice. HFD could lead to immune activation and recruitment, as shown above by increased IL-6 liver expression, and reported for NAFLD patients, explaining the impaired myeloid differentiation(Braunersreuther, Viviani, Mach, & Montecucco, 2012). Interestingly, all treatments restored its accumulation as well as Il-6 expression levels in this experimental model, which has also previously been observed for metformin in vivo (Hayashi et al., 2019) and in vitro (Xu et al., 2019).
Macrophages are key regulators of the inflammatory process, reacting to a wide variety of stimuli, including metabolic signals. It is well known that the accumulation of inflammatory macrophages in the liver and in the fat contributes to the deregulation of glucose homeostasis, obesity-induced inflammation, and hepatic fibrosis. The hepatic macrophage population (CD45+CD11bint) was increased in obese mice and, both melatonin and metformin reduced it, as expected from other studies(de Farias et al., 2019; Woo et al., 2014). Interestingly, agomelatine showed an even stronger effect at the highest dose, and it also restored macrophage population in the adipose tissue, confirming the improvement of the inflammatory response in association with the metabolic status.
Obesity has also been associated with an increased gut permeability, which positively correlates with HOMA-IR index and is aggravated by liver injury(Teixeira, Souza, et al., 2012). In line with the loss of mucosal integrity, we observed a down-regulation of intestinal epithelial markers in obese mice, which could enable the access of bacterial components, such as LPS, into the circulation, contributing to the underlaying inflammation(Luther et al., 2015). Agomelatine and melatonin increased the expression of these markers and, as a consequence, counteracted the upregulation of liver Tlr-4expression, which correlates with LPS plasma levels(Diez-Echave et al., 2020). This impact in TLR4, which promotes NFκB signalling and the subsequent release of cytokines, adipokines and ROS, could also explain the beneficial effect of agomelatine, connecting intestinal permeability with improved inflammatory response and glucose and lipid homeostasis.
Regarding microbiota composition, as commented before, obesity-associated dysbiosis may contribute to metabolic endotoxemia and thus low-grade systemic inflammation. Interestingly, it has been previously shown that melatonin and metformin treatments can also reverse gut dysbiosis associated with metabolic endotoxemia in animal models of obesity(Ren et al., 2018; Zhang & Hu, 2020). Thus, we studied microbial composition and observed a decrease in microbial richness, evenness and diversity associated with HFD intake. Agomelatine has shown for the first time to produce marked shifts in the obese gut microbiome and restore the balance between Firmicutes andBacteroidetes, of interest for the management of the metabolic syndrome and obesity(Shen et al., 2013). The increase in Firmicutes /Bacteroidetes (F/B) ratio has been associated with a more efficient hydrolysis of non-digestible polysaccharides and an increased caloric use in obese individuals(Shen et al., 2013). Other alterations described in obese patients, such as reducedVerrucomicrobia phylum(Crovesy, Masterson, & Rosado, 2020), were also observed in our model and restored by agomelatine treatment. At lower taxonomic levels, agomelatine also normalized the composition of microbiota whilst metformin only had a partial effect. It is particularly interesting the increase in Verrumicrobiales containing Akkermansia muciniphila , a mucin-degrading bacterium whose abundance is inversely associated with body weight in obese mice and type 2 diabetes(Abuqwider, Mauriello, & Altamimi, 2021). Treatments that stimulate its growth have shown to alleviate HFD-induced metabolic disorders(Abuqwider, Mauriello, & Altamimi, 2021), which points this as an interesting mechanism that could underlie agomelatine’s beneficial effects.
The ”dialogue” between the intestinal microbiota and the host primarily relies on their biochemical pathways and metabolites produced, finding an altered functional profile with HFD, which was evidenced in our study. Imputed gene expression and pathway analysis showed that agomelatine treatment correlates with an increase in glycolysis, gluconeogenesis and lipid metabolism, and underrepresentation of genes involved in the transport (including ABC transporter), bacterial secretion, PPAR signalling, fatty acid biosynthesis, motility and sugars assimilation(Greenblum, Turnbaugh, & Borenstein, 2012).Of note, agomelatine, together with metformin, increased the abundance of butyrate-producing bacteria, which have been describe to protects animals from HFD-induced obesity, attenuating fat gain and insulin resistance(Henagan et al., 2015). These results could be associated with the modification of the Bacteroidetes and Lactobacillus abundance, which participate in butyrate generation via lactate production(Le Chatelier et al., 2013), whilst the increase in propionate-producing bacteria could relate toA. muciniphila, a propionate producer bacteria(Louis & Flint, 2017). Moreover, propionate plays a key role in counteracting cholesterol synthesis, being the ratio acetate/propionate crucial for cholesterol and lipid metabolism regulation(Wong, de Souza, Kendall, Emam, & Jenkins, 2006). These results support the beneficial effect observed with agomelatine and highlight its therapeutic potential for the modulation of the gut microbiota in obesity.
All the alterations observed in obesity, and in this experimental model, such as insulin signalling impairment, intestinal dysbiosis and systemic inflammation, can affect endothelial function. An increased NADPH enzyme activity and the subsequent production of reactive oxygen species inactivates NO and impair vessel dilation, which contributes to the pathogenesis of the metabolic syndrome(Rovella et al., 2021). The decreased enzyme activity observed with agomelatine treatment could explain the enhanced endothelial relaxation, as well as the general improvement of the underlying condition.
In conclusion, the melatonergic agonist agomelatine improves glucose intolerance, insulin resistance, lipid metabolism and inflammatory status associated with HFD-induced obesity. Moreover, it has shown the ability to ameliorate the gut dysbiosis that characterizes this condition. These properties may support the use of agomelatine as a novel therapeutic tool to manage human obesity, which displays a better pharmacokinetic profile than melatonin and more global effects than this and metformin, the most used drug nowadays.