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.