Intestinal microbiota and the control of glucose homeostasis and lipid metabolism
in the host
The human intestinal microbiota is dominated by five phyla: Firmicutes, Bacteroidetes,
Actinobacteria, Proteobacteria, and Verrucomicrobia. In adults, more than 80% of the
species belong to just two phyla, Firmicutes and Bacteroidetes. Short chain fatty
acids (SCFA) are catabolic end-products from intestinal microbial fermentation. Acetate,
propionate and butyrate are the most abundant (Ríos-Covián et al., 2016a) whilst branched
chain fatty acids (BCFA; isobutyrate, valerate, and isovalerate), the organic acids
lactate, succinate, formate, and gases, can be also formed.
In humans, the main fermentable sources of SCFA are undigested dietary polysaccharides;
amino acids and proteins may constitute additional substrates for colonic fermentation,
whereas host-derived glycoproteins contribute to a limited extent. BCFA can be formed
at considerably lower proportions than SCFA from branched-chain amino acids (BCAA;
valine, leucine, and isoleucine). Threonine renders propionate and butyrate, whereas
glutamate, histidine, lysine, arginine, and alanine give rise to acetate and butyrate
formation; additionally, the intestinal microbiota contributes to the production of
amino acids available to the host through de novo biosynthesis (Neis et al., 2015).
Moreover, metabolic cross-feeding, that is the utilization of end products from the
carbohydrate catabolism of a given microorganism by another one, strongly influences
the final balance of intestinal SCFA. It occurs mainly for the formation of butyrate
from acetate or lactate, is considerably lower for butyrate conversion to propionate,
and very scarce between propionate and acetate (Den Besten et al., 2013).
Intestinal SCFA can incorporate into the enterohepatic circulation, being metabolized
in the liver and reaching other extra-intestinal locations (Den Besten et al., 2014).
Increasing evidence supports a regulatory role for SCFA in glucose homeostasis and
lipid metabolism, in which intestinal SCFA ligands FFAR2 and FFAR3 and the glucagon-like
peptide are involved. In the liver propionate is gluconeogenic whereas acetate and
butyrate are lipogenic. Recent studies evidence that propionate and butyrate activate
the intestinal gluconeogenesis (De Vadder et al., 2014), the glucose synthesized serving
as a homeostatic signal in the portal system, to control hepatic gluconeogenesis (causal
factor of insulin resistance and type 2 diabetes) and improving whole-body glucose
homeostasis. Moreover, propionate flux through the liver reduces visceral and liver
fat by decreasing intrahepatic triglycerides (Chambers et al., 2015). Propionate inhibits
hepatic lipogenesis and cholesterogenesis promoted by acetate (Demigne et al., 1995)
whereas propionate and butyrate inhibit lipolysis and lipogenesis and increase the
incorporation of glucose mediated by insulin into the adipose tissue (Heimann et al.,
2015). These observations prompt to the acetate/propionate ratio as an indicator for
the potential contribution of intestinal SCFA to body lipid metabolism. Additionally,
the improvement of glucose homeostasis promoted by dietary fiber seems to be associated
with elevated fluxes of SCFA from the intestinal lumen to other organs rather than
with the fecal SCFA concentrations (Den Besten et al., 2014).
Several metabolic disorders as obesity, insulin resistance, and metabolic syndrome
are associated with impairment of the metabolism of carbohydrates and lipids by the
host, and are accompanied by changes in the gut microbiota (Turnbaugh et al., 2009;
Bervoets et al., 2013). Higher levels and altered patterns of SCFA (Fernandes et al.,
2014; Salazar et al., 2015) and changes in the Firmicutes to Bacteroidetes ratio,
have been repeatedly reported in obese individuals (Ley et al., 2006; Turnbaugh et
al., 2006). Nonetheless, contradictory results published so far on the relative abundance
of both phyla exclude its use as a broadly applicable marker.
Increases in plasma circulating BCAA and aromatic amino acids (phenylalanine and tyrosine)
were related with higher risk of type 2 diabetes and insulin resistance (Utzschneider
et al., 2016), having been suggested that the altered functionality of the intestinal
microbiota (also affecting de novo biosynthesis of amino acids) determines these differential
profiles of circulating amino acids (Neis et al., 2015).
Increasing protein intake (Pillot et al., 2009) and gastric surgery (Liou et al.,
2013) have demonstrated efficacy for weight control and improvement of glucose homeostasis,
partly related to the increase of propionate (De Vadder et al., 2014), and enrichment
of intestinal Bacteroidetes/Bacteroides (Furet et al., 2010; Jumpertz et al., 2011;
Liou et al., 2013). In contrast, a significant reduction in butyrate and certain butyrate-producing
Firmicutes have been associated with diets containing low amounts of fiber and carbohydrates
(Duncan et al., 2007, 2008; Walker et al., 2011). These suggest a microbiota unbalance
in obese subjects, or under inadequate diets, which is partly restored following gastric
surgery or by introducing weight-loss diets. However, some microbiome alterations
seem to persist after dietary interventions, facilitating post-dieting weight regain
(Thaiss et al., 2016). This stresses the importance of achieving a full restoration
of the intestinal microbiota after dietary treatments, including proper balanced microbial
metabolic products, to ensure durable effects.
A focus on the genus Bacteroides and the production of propionate in the intestinal
microbial ecosystem
The order Bacteroidales is the most abundant Gram-negative bacteria, colonizing the
human gut at densities up to 5–8 × 1010 CFU per gram of feces (Zitomersky et al.,
2011). Among the predominant genera are Bacteroides and Prevotella. These microorganisms
can use a wide range of dietary soluble polysaccharides that are firstly released
from vegetable fiber in the intestine by microbial primary degraders (Martens et al.,
2011). The genus Bacteroides displays a high flexibility to adapt to the nutritional
conditions of the intestinal environment (Comstock and Coyne, 2003), being able to
use dietary or host-derived glycans according to the nutrient availability (Sonnenburg
et al., 2005). Bacteroides can also incorporate amino acids from outside (Smith and
MacFarlane, 1998) which could be used to maintain cell structures and as an energy
source.
Three different biochemical pathways have been identified in colonic bacteria for
propionate formation (Reichardt et al., 2014). The succinate pathway is the only one
for propionate production from hexoses by Bacteroidetes, although some Negativicutes
(family Veillonellaceae, phylum Firmicutes) can form propionate by utilizing succinate.
The acrylate pathway is used for the conversion of lactate into propionate by very
few bacterial genera within the phylum Firmicutes, whereas deoxy-sugars (fucose and
rhamnose) are converted through the propanediol pathway by some Proteobacteria and
members of the Lachnospiraceae family (phylum Firmicutes). Akkermansia muciniphila
(phylum Verrucomicrobia) has been identified as a key propionate producing mucin-degrading
species (Derrien et al., 2004).
Notably, several studies point to Bacteroidetes as the largest propionate producers
in the human gut (Salonen et al., 2014; Aguirre et al., 2016). Interestingly, by modifying
microbiota composition with antibiotic treatment in mice, Zhao et al. (2013) found
a strong correlation between fecal levels of SCFA and the abundance of Bacteroides
and other members of the phylum Bacteroidetes.
The type of carbohydrates and availability of organic nitrogen sources modify In vitro
the metabolism of Bacteroides
SCFA and organic acids formed in cultures of Bacteroides (acetate, succinate, lactate,
and propionate) depend on the type of fermentable substrates, generation time and
incubation period (Kotarski and Salyers, 1981; Rios-Covian et al., 2013, 2015, 2016b).
Propionate is generally favored at long generation times, with complex carbohydrates,
and under carbon source limitation.
We have studied the metabolism of Bacteroides fragilis growing in media containing
different carbohydrates and nitrogen sources. Catabolic end-products formed in the
presence of carbohydrates in non-defined peptone and yeast extract containing medium
(BM; Rios-Covian et al., 2015) with respect to a minimal medium without no organic
nitrogen source (MM; Rios-Covian et al., 2016b) evidenced higher SCFA and organic
acids production in the former medium, when it was supplemented with bacterial exopolysaccharides
(EPS), which are complex structures synthesized by some bacteria (Figure 1A). Acetate
accounted for 30–54% of the total products formed in any condition, constituting a
fundamental way for obtaining energy by this bacterium. An inverse correlation was
found between the production of propionate plus succinate and that of lactate (Rios-Covian
et al., 2015, 2016b; Figure 1A), this last being favored in the absence of organic
nitrogen sources and with rapid fermentable carbohydrates, as occurs in MM added with
glucose. Conversely, a shift toward propionate formation appears to occur in the presence
of organic nitrogen when EPS are present. This probably reflects a preferential use
of the glucolytic pathway and acetate formation for obtaining energy and keeping redox
balance by B. fragilis in the presence of rapidly fermentable carbohydrates; in contrast,
when complex/slowly fermentable carbohydrates are available and amino acids are present,
carbon skeletons from amino acids could be incorporated at the level of pyruvate;
in such conditions the propionate-succinate pathway seems to be potentiated as a way
for energy obtaining whilst serving to restore cell redox balance (Rios-Covian et
al., 2015; Figure 1B). Proteomics and gene expression analyses reinforced the hypothesis
of the activation of amino acids catabolism and the succinate pathway in B. fragilis
grown in BM with EPS (Rios-Covian et al., 2015). Therefore, the preferential metabolic
route for energy production and redox maintaining, and the final metabolic products
formed by B. fragilis, may be largely dependent on carbohydrates and nitrogen sources
available. These results suggest the possibility of regulating the metabolism of Bacteroides
by controlling dietary carbohydrate/protein balance.
Figure 1
The metabolic versatility of
Bacteroides
and the modulation of its metabolism through diet may impact human health. (A) The
relative proportions of the different organic acids and SCFA produced by cultures
of Bacteroides fragilis at 24 h of incubation in non-defined peptone and yeast extract
containing medium (BM; Rios-Covian et al., 2015) and in minimal medium without no
organic nitrogen source (MM; Rios-Covian et al., 2016b) and supplemented with glucose,
or with exopolysaccharides produced by Bifidobacterium strains (EPS E44 and EPS R1),
are represented in shaded circles. The table at the top right side indicates total
concentration (mM) of SCFA plus organic acids produced by B. fragilis in the different
culture conditions. (B) Schematic representation of catabolic routes for the formation
of SCFA and organic acids by B. fragilis. Thick bold arrows indicate pathways probably
favored in MM supplemented with glucose (left side) or in BM supplemented with bacterial
EPS (right side). (C) Schematic representation of the general hypothesis on how re-shaping
the intestinal Bacteroides metabolism through the adequate balance of dietary proteins
and carbohydrates could influence human health. On the one hand, changes occurring
in the profile of SCFA and organic acids produced by this bacterium could act on the
host carbohydrates and lipids metabolism directly or through cross-feeding or other
microbial interaction mechanisms. On the other hand, the metabolism of intestinal
Bacteroides may modify blood circulating amino acids in the host, which have been
related with some metabolic disorders. OAA, oxaloacetate; SCFA, short chain fatty
acidis; BCAA, branched chain amino acids; PEP, phosphoenolpyruvate.
Moreover, when analyzing the amino acids in cultures of B. fragilis added with different
carbohydrates, we found a decrease in the concentration of leucine, isoleucine and
phenylalanine after incubation in any condition, whereas valine and tyrosine showed
much less increases or slight decreases in EPS as compared to glucose (Supplementary
Material Table 2 in Rios-Covian et al., 2015). This points to a potential capacity
of B. fragilis (as may probably occur with other Bacteroidetes) for regulating BCAA
and aromatic amino acids levels in its growth environment.
Modulation of the intestinal Bacteroides by dietary carbon/nitrogen sources: a tool
for restoring the intestinal microbiota metabolic balance
Under sufficient organic nitrogen, the mildly acidic pH (5.5) stimulates butyrate
producing species in the human colon curtailing the growth of Bacteroides that was
however maximized at pH 6.5 (Walker et al., 2005). The pH in the caecum is about 5.7
but gradually increases to 6.7 in the rectum. Dietary fiber fermentation promotes
a slight decrease of the luminal pH whereas high protein/amino acids fermentation,
favored by low carbohydrate availability, causes slightly pH increases (Smith and
MacFarlane, 1998). Interestingly, a recent study with mice demonstrated that diet-microbiome
interactions are driven by the pattern of protein and carbohydrate intake (Holmes
et al., 2017). Moreover, some experiments with gnotobiotic mice support shifts in
Bacteroides metabolic functions as a response to dietary changes (McNulty et al.,
2013; Wu et al., 2015).
The studies just commented support the interdependence between diet, gut microbiome
and host metabolism, and allow to hypothesize that the combination of dietary organic
nitrogen sources with appropriate carbohydrates may be used to modify the metabolic
activity of colonic Bacteroides populations by modifying the profile of organic acids
formed and enhancing propionate formation in some parts of the large intestine while
promoting shifts toward healthier profiles of serum amino acids (Figure 1C).
Within this “scenario,” the potential role that the functional control and metabolic
reprogramming of Bacteroides through diet may play in the regulation of the host metabolism
deserves more attention. It is essential to decipher to what extent organic nitrogen
sources and carbohydrates could affect the different species of prominent intestinal
bacteria, such as the genus Bacteroides. An important question raised is whether changes
in SCFA and organic acids profile induced by remodeling the metabolic activity of
Bacteroides through adequate dietary interventions would influence other less nutritionally
versatile gut beneficial microbes through the enhancement of cross-feeding or other
microbial interaction mechanisms. Omics, including metabolomics/metabonomics, applied
to the analysis of microbial cultures, animal models, and human samples are necessary
for understanding host and microbiota metabolic remodeling as a response to dietary
combinations of organic nitrogen/carbohydrates.
The potential to re-shape the metabolism of Bacteroides with specific combinations
of dietary carbohydrates-proteins based on their composition, structure and availability
in the gut, merits further study. The final aim should be designing diets based on
nutrient components targeted at modulating the metabolism of Bacteroides, which may
interact with other intestinal beneficial microbes, in order to restore the metabolic
balance of the microbiota to promote durable host's health effects.
Author contributions
DR, NS, MG, and Cd conceived the idea and designed the structure of the manuscript.
All authors contributed significantly to the experimental data compared in the Figure
1A. Cd and DR drafted the manuscript and Figure. All authors have critically red,
corrected, and approved the final version of the manuscript and agree with the opinions
expressed here.
Funding
The work of the research group in the matter of this article is being currently financed
by projects AGL2013-43770-R from Plan Estatal de I+D+I (Spanish Ministry of Economy
and Competitiveness, MINECO) and by Grant GRUPIN14-043 from Plan Regional de Investigación
del Principado de Asturias, Spain. Both national and regional grants received cofounding
from European Union FEDER funds. DR-C was the recipient of a predoctoral FPI fellowship
and NS benefits from a Juan de la Cierva post-doctoral contract, both granted by MINECO.
Conflict of interest statement
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.