DISCUSSION
The treatment with L. fermentum CECT5716 to HFD-fed mice improved
HFD-induced obesity, reducing body weight gain, which was associated
with an amelioration of glucose and lipid metabolism. The treatment did
not significantly modify energy intake, thus discarding any anorexigenic
effect, but it showed anti-inflammatory and immunomodulatory properties,
as seen before in different experimental models of intestinal
inflammation (Rodriguez-Nogales et al.,
2017; Rodríguez-Nogales et al., 2015).
Indeed, L. fermentum downregulated the expression of
pro-inflammatory mediators in HFD-fed mice as well as upregulated the
expression of key transcription factors that control adiponectin such as
PPARα (Kim et al., 2015). As in human
obesity, this was associated with an improvement in glucose and lipid
metabolism, most probably derived from the amelioration of the
obesity-associated insulin resistance, as shown by the impact of the
probiotic treatment on HOMA-IR values. Moreover, L. fermentumameliorated leptin resistance in liver and adipose tissue as well as
restored adiponectin expression in fat contents, which has been
previously reported for other probiotics, like different strains ofL. plantarum (Choi, Dong, Jeong,
Jung, Kim & Kim, 2019; Kwon et al.,
2020).
Obesity-associated insulin resistance is also characterized by
impairment of intracellular glucose uptake that is mediated by the
insulin-dependent receptor GLUT-4 (Vargas,
Podder & Carrillo Sepulveda, 2020), whose expression is reduced when
insulin resistance appears (Shepherd &
Kahn, 1999), similarly to that observed in the present study. L.
fermentum also improved insulin resistance, which was associated with
an increased expression of Glut-4 , ameliorating glycemic levels
and glucose utilization by target tissues, as previously reported for
other probiotics (Chen et al., 2018).
Besides, the obesity-associated inflammatory state is closely related to
the development of cardiovascular disease and endothelial dysfunction,
in part, mediated by leptin (Huby, Otvos
& Belin de Chantemele, 2016). Conversely, adiponectin has been shown
to correlate inversely with arterial hypertension and endothelial
dysfunction, since it facilitates the phosphorylation of endothelial
nitric oxide synthase (eNOS) enzyme and the subsequent production of
nitric oxide (NO), the most effective acetylcholine-induced
endothelium-derived relaxing factor in aorta
(Adya, Tan & Randeva, 2015).
Additionally, vascular endothelial and smooth muscle cells are also
targeting of the pro-inflammatory adipokines TNF-α and IL-6, which, as
well, increase expression of pro-inflammatory cytokines, including TNF-α
and IL-1β, as observed in the present study, and adhesion molecules.
This results in the activation of the renin-angiotensin system that
leads to inflammation of the vascular walls and development of
pre-atheromatous lesions, impairment of vasodilation in humans, most
probably through altering the expression and activity of eNOS and NADPH
oxidase (Walther et al., 2015). Thus, it
modifies the production of NO and superoxide anion
(O2-) facilitating vascular oxidative
stress (Didion, 2017). Interestingly,L. fermentum treatment reduced vascular expression of the
pro-inflammatory cytokines TNF-α and IL-1β in obese mice, as well as
inhibited the increased NADPH activity in the aortic tissue. This
suggests a reduction of ROS production and a higher NO bioavailability,
which could promote the restoration of the impaired
endothelium‐dependent relaxation to acetylcholine, similarly to that
reported previously for this probiotic in an experimental model of
systemic lupus erythematosus (Toral et
al., 2019).
Regarding the involvement of the gut in the pathogenesis of obesity,
there is a defect in the intestinal barrier function. This leads to
increased gut permeability (Teixeira et
al., 2012) that facilitates bacterial components translocation, like
LPS, that could reach systemic circulation and provoke metabolic
endotoxemia (Cani et al., 2007). LPS may
contribute to obesity-associated systemic inflammation upon binding to
its main receptor TLR-4, located in immune cells, liver, adipose tissue
and skeletal muscle. This promotes the activation of the transcription
factor NFκB and subsequent production and release of cytokines,
adipokines and ROS, thus altering glucose and lipid homeostasis
(Boutagy, McMillan, Frisard & Hulver,
2016). The present study confirms these observations, since obese mice
displayed reduced expression of the colonic markers of epithelial
integrity as well as increased LPS plasma levels and up-regulated
expression of Tlr-4 in liver, fat and aorta. Importantly,L. fermentum treatment significantly increased the colonic
expression of the different markers involved in gut integrity in obese
mice, thus restoring the intestinal barrier function and preventing
bacterial components translocation, since it reduced LPS plasma levels
and downregulated Tlr-4 expression. Other probiotics, likeL. sake OK67 (Lim, Jeong, Woo, Han
& Kim, 2016), L. gasseri (Kawano,
Miyoshi, Ogawa, Sakai & Kadooka, 2016)(and Bifidobacterium
adolescentis IM38 (Lim & Kim, 2017)
have also been reported to improve intestinal integrity and ameliorate
inflammation in obesity in mice. Closely related to the above, the
modulation of gut microbiota in obese mice exerted by L.
fermentum seems to play a key role. Changes in gut microbial
composition, mainly caused by external factors, can result in a dramatic
alteration of the symbiotic relationship between gut bacteria and the
host and promote the development of metabolic diseases, maybe by
facilitating a low-grade inflammation, as mentioned before
(Marchesi et al., 2016). In addition, it
is well established that gut microbiota composition is altered in
obesity (Ley, Backhed, Turnbaugh,
Lozupone, Knight & Gordon, 2005), consisting on an enrichment inFirmicutes (F) as well as a reduction in Bacteroidetes(B), both in humans (De Filippo et al.,
2010) and mice (Bagarolli et al., 2017).
Increased F/B ratio has been associated with a more efficient hydrolysis
of non-digestible polysaccharides in the intestinal lumen, so obese
individuals extract more calories and fat from food than lean ones
(Backhed et al., 2004). Our study agrees
with these observations since HFD-fed mice showed an increased F/B ratio
when compared with non-obese mice. However, L. fermentumtreatment was able to modulate gut microbiota composition, restoring the
main bacteria phyla to the normal values observed in control diet-fed
mice. The PCA analysis showed a clear separation between the clusters,
indicating a shift in the gut bacterial composition induced by the
probiotic. This amelioration of obesity-associated dysbiosis could be
associated with the reduction of energy assimilation and potentially
contribute to the beneficial effects observed. Additionally, in obese
patients different phyla have been reported to be increased, includingFusobacteria and Proteobacteri a, whereasVerrucobacteria is reduced (Crovesy,
Masterson & Rosado, 2020). Special attention has been paid to the
latter, which includes the bacteria Akkermansia muciniphila , a
mucin-degrading bacterium whose abundance is inversely related to body
weight and type 2 diabetes in mice
(Everard et al., 2013) and humans
(Karlsson, Onnerfalt, Xu, Molin, Ahrne &
Thorngren-Jerneck, 2012). Actually, it has been reported that A.
muciniphila treatment, or those that promote its abundance, could
reverse HFD-induced metabolic disorders
(Vezza et al., 2019). In the present
study, the proportion of the genus Akkermansia was reduced in
untreated obese mice, while increased after L. fermentumtreatment, maybe through an increment in colonic mucins production. This
probiotic could become a valid tool to acquire a properAkkermansia abundance that could have a therapeutic effect in
obesity, as demonstrated before (Chang et
al., 2019; El Hage, Hernandez-Sanabria &
Van de Wiele, 2017).
Furthermore, it has been reported that Erysipelotrichi ,
specifically, Clostridium spp , is associated with metabolic
syndrome and obesity in humans (Karlsson
et al., 2013) and mice (Chakraborti,
2015). Our results confirmed this since Erysipelotrichi class
and Clostridium spp abundance were increased in HFD-fed mice
while L. fermentum treatment reduced significantly their presence
in obese mice.
Regarding Bacteroidetes genus, a significant association between
HFD and Bacteroides abundance was found, being higher in lean
individuals than in obese ones (Castaner et
al., 2018). Thus, some strains of Bacteroides , such as B.
acidifaciens , have been reported to prevent obesity and improve insulin
sensitivity in mice (Yang et al., 2017).
Accordingly, in our study, untreated HFD-fed mice manifested a reducedBacteroides proportion in comparison with control diet-fed mice
whereas L. fermentum significantly increased it.
Gut metagenomic analysis, in both control diet and HFD mice, has
provided some insights into the mechanism of the microbiome to affect
weight gain and obesity. Therefore, within Bacteriodetes ,
lineages rich in genes involved in amino acid metabolism, translation,
and nucleotide metabolism, have been found less abundant in HFD mice,
while those genes for membrane transport and replication and repair were
increased. Similarly, HFD has been also described to modify gene
activation on Firmicutes phylum. Membrane transport (mostly ABC
transports), transcription and cell motility pathways were increased in
abundance, while those with more genes for carbohydrate and energy
metabolisms were decreased (Hildebrandt et
al., 2009). Since ABC transporters control the transport of a variety
of nutrients such as lipids, sugars, peptides and metals, an increase in
the number of these transporters could favor energy intake, as seen in
HFD-fed mice. Furthermore, a collection of genes involved in intake and
assimilation of sugars are also found to be more abundant in microbiome
samples from HFD-fed mice, as well as genes for phosphorus metabolism,
mainly phosphotransferase systems involved in the uptake and
assimilation of sugars (Greenblum,
Turnbaugh & Borenstein, 2012). These results were confirmed in our
study, where HFD-fed mice showed enrichment on genes responsible for
transport (including ABC transporter), bacterial secretion, motility and
sugars assimilation, among others, and were decreased after L.
fermentum administration, confirming the potential of this probiotic on
the modulation of gut microbiota.
In summary, HFD consumption promotes alterations in gut microbiota that
may increase intestinal permeability and LPS translocation, as well as
lead to insulin resistance, glucose homeostasis imbalance and systemic
low-grade inflammation. However, these obesity-related features were
reversed by changes in the gut microbiota profile induced by L.
fermentum CECT5716 administration, which suggests a potential use ofL. fermentum in clinical practice. In conclusion, the probiotic
treatment can be an important tool to prevent and treat patients with
obesity and metabolic syndrome.