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Diabetes & Metabolism 35 (2009) 262–272
Review
Intestinal microflora and metabolic diseases
M. Serino a,b,∗ , E. Luche a,b , C. Chabo a,b , J. Amar c , R. Burcelin a,b,∗
b
a U858, Institut national de la santé et de la recherche médicale (Inserm), Toulouse, France
IFR31, institut de médecine moléculaire de Rangueil (I2MR), université de Toulouse Paul-Sabatier, 1, avenue Jean-Poulhes,
31432 Toulouse cedex 4, France
c Rangueil Hospital, Department of Therapeutics, Toulouse, France
Received 12 March 2009; accepted 15 March 2009
Available online 5 May 2009
Abstract
Recent advances in molecular sequencing technology have allowed researchers to answer major questions regarding the relationship between a
vast genomic diversity—such as found in the intestinal microflora—and host physiology. Over the past few years, it has been established that, in
obesity, type 1 diabetes and Crohn’s disease—to cite but a few—the intestinal microflora play a pathophysiological role and can induce, transfer
or prevent the outcome of such conditions. A few of the molecular vectors responsible for this regulatory role have been determined. Some are
related to control of the immune, vascular, endocrine and nervous systems located in the intestines. However, more important is the fact that the
intestinal microflora-to-host relationship is bidirectional, with evidence of an impact of the host genome on the intestinal microbiome. This means
that the ecology shared by the host and gut microflora should now be considered a new player that can be manipulated, using pharmacological
and nutritional approaches, to control physiological functions and pathological outcomes. What now remains is to demonstrate the molecular
connection between the intestinal microflora and metabolic diseases. We propose here that the proinflammatory lipopolysaccharides play a causal
role in the onset of metabolic disorders.
© 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Diabetes; Obesity; Metagenome; Inflammation; Lipopolysaccharides; Review
Résumé
Flore intestinale et maladies métaboliques.
Les progrès récents des méthodes de séquençage à ultrahaut débit ont permis de répondre à des questions fondamentales concernant la relation
entre la flore intestinale, vecteur de la plus grande diversité génétique de notre organisme, et la physiologie de l’hôte. Il est maintenant établi
qu’au cours de l’obésité, du diabète de type 1 et de la maladie de Crohn, pour n’en citer que quelques unes, la flore intestinale joue un rôle
important. Ce microbiote peut induire, transférer ou prévenir le développement de ces maladies. Certains acteurs moléculaires responsables de
ces effets ont été identifiés et seraient relatifs au contrôle du système immunitaire, du développement vasculaire ou de fonctions endocrines et
nerveuses principalement et initialement localisées dans l’intestin. Il faut également considérer que la relation entre l’hôte et la flore intestinale
est bidirectionnelle. En effet, certaines données démontrent l’impact de l’hôte sur la flore intestinale. Désormais, une écologie mutualisée entre
la flore intestinale et l’hôte doit être considérée comme un nouvel acteur de la physiologie et physiopathologie. Cet acteur peut être manipulé
par des approches pharmacologiques et nutritionnelles afin de contrôler la physiologie humaine et ses dysfonctionnements. Les mécanismes
moléculaires doivent encore être démontrés. Nous proposons dans cette revue que les lipopolysaccharides bactériens, hautement inflammatogènes,
sont responsables de l’initiation du développement des maladies métaboliques.
© 2009 Elsevier Masson SAS. Tous droits réservés.
Mots clés : Diabète ; Obésité ; Métagénome ; Inflammation ; Lipopolysaccharides ; Revue
1. Introduction
∗
Corresponding authors.
E-mail addresses: [email protected] (M. Serino),
[email protected] (R. Burcelin).
1262-3636/$ – see front matter © 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.diabet.2009.03.003
Our human microflora are an inherited feature resulting from
our evolution. With the celebration of 200 years since the birth of
Charles Darwin, it makes sense to consider that the development
M. Serino et al. / Diabetes & Metabolism 35 (2009) 262–272
of human physiology could be the consequence of environmental factors such as microflora, which might even be thought of
as a selection factor among humans. Indeed, the selection of the
species comprising our microflora today could be considered to
be the result of evolution, with some strains selected on the basis
of our evolving genome. This means that the host–microflora
relationship could be the result of a bidirectional selection process that has now established a shared ecology among the
different members of the microflora and the host.
However, the molecular bases of this ecology and its interaction with the host need deciphering if we are to have access to the
potentially vast new spectrum of strategies for the management
of human physiology and treatment of diseases. Cancer and the
inflammatory and metabolic diseases are among the first in line
for the use of microflora-based therapeutic strategies.
Given the wide diversity of our entire body’s microflora, this
review focuses on the intestinal microflora, and its relationship with metabolic diseases and other related disorders. The
choice of this narrow field of discussion is driven by the intuitive
concept that feeding our body is also feeding our intestinal flora.
Consequently, both the genome and metagenome contribute to
the subtle balance of mechanisms involved in metabolism. We
discuss here some of the most recent findings on the molecular
mechanisms through which the intestinal microflora influence
metabolic diseases.
2. Intestinal microflora: a mutual ecology
The intestine is inhabited by trillions of microorganisms,
including bacteria, fungi and the Archaea. Two sets of microorganisms have been described. In one, the tasks performed by
some members are partially known and are most likely beneficial to the host. However, the vast majority of species are neither
beneficial nor harmful to human physiology, and their function
remains completely unknown. These species are termed “normal
flora”, or “microbiota”. It is estimated that 500 to 1000 different
species of bacteria live in the human body [1].
The precise composition of the intestinal microflora varies
among individuals and animals, with evidence to suggest that
its establishment occurs during the first year of life [2], and that
host and external environmental factors, as well as changes in
the gut environment and in the microbiota itself, can trigger
changes from childhood to adulthood [3,4]. The intestinal colonization that follows birth represents the host’s earliest contact
with microbes [3]. Indeed, microbes from the mother and surrounding environment colonize the gastrointestinal tract of the
infant until the dense, complex microbiota have developed. The
succession of microbes colonizing the intestinal tract is most
marked during early development, when the feeding mode shifts
from breast- and formula-feeding to weaning and the introduction of solid food. In infants, the microbiota develops rapidly
and remains unstable during the first year of life, but becomes
more stable after that. This initial stage of microbiota establishment could be the key modulating moment in the establishment
of a healthy microbiota in the individual. It is thought that food
is one of the major determinants of the intestinal microbiota
and, therefore, has important consequences for the development
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of adult physiology and, hence, pathology. The indigenous gut
microbiotas are considered to be among the first stimuli to trigger
mechanisms leading to the generation of immunophysiological
regulation [3,5,6]. They provide crucial signals for the development of the immune system in infancy and set the immunological
“tone” in adulthood.
This finding has also been observed in germ-free mice,
where oral administration of ovalbumin antigen in neonate mice
blocked the inflammatory Th1-mediated response, which usually triggers the production of interferon (IFN)-gamma and
IgG2a, in favour of a Th2 tolerogenic response, characterized
by IgE, IgG1 and interleukin (IL)-4 in the adult mice [7]. Part of
the intestinal mucosal barrier function is formed by a common
mucosal immune system that communicates with the various
mucosal surfaces of the body. Local T-cell immunity is an important aspect of the specific intestinal immune system [8]. T-cell
reactivity is programmed during its initial stages of activation
by mucosal dendritic cells, which are thought to play key roles
in regulating immune responses in the antigen-rich gastrointestinal environment [8,9]. Defects in this regulatory system
are believed to lead to the two main forms of inflammatory
bowel disease (IBD)—Crohn’s disease (CD) and ulcerative colitis (UC) [9,10]. This means that the programming of dendritic
cell function by intestinal microflora might be involved in the
occurrence of inflammatory diseases [8].
A detailed description of the role played by the intestinal
microflora in the immune system is beyond the scope of this
review. In brief, the first intuitive function attributed to the
intestinal microflora is their capacity to break down certain
nutrients, such as plant carbohydrates, that the host would otherwise be unable to digest [11], thus allowing the host to recover
more energy from ingested food and, hence, better development.
Another function is the synthesis of bioactive molecules, such as
phytoestrogens [12], that are able to deeply interact with human
physiology [13] (Fig. 1).
Similarly, the microflora could benefit from the intestinal
environmental conditions of a healthy host, ensuring longerlasting dissemination. This idea has been described for many
years and is now well established [14]. Some bacteria carry
out fermentation of the complex, indigestible carbohydrates of
plants, and synthesize vitamins such as folic acid, vitamin K
and biotin [15,16]. Other beneficial bacteria in the normal flora
include the Lactobacillus species, which convert milk protein to
lactic acid in the gut [17]. The presence of such bacterial colonies
also inhibits the growth of potentially pathogenic bacteria (usually through competitive exclusion), which is why some of these
“good” bacteria are sold as probiotic dietary supplements [18].
This shows that a mutually beneficial ecology is being shared
among different members of the microflora and the host. Furthermore, some bacterial functions are involved in host functions
such as intestinal epithelial turnover, immune modulation, drug
metabolism, gastrointestinal tract motility regulation, mucosal
barrier fortification, angiogenesis, intestinal homoeostasis and
postnatal intestinal maturation [19–25].
The vast majority of these commensal bacteria are anaerobic,
able to survive and grow in an environment lacking oxygen, and
some normal flora bacteria can act as opportunistic pathogens in
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Fig. 1. Intestinal microflora ecology and its functions. The human intestine harbours roughly 1014 microorganisms, comprising bacteria, fungi and protozoa. Bacteria
represent the majority of the gut flora, accounting for 60% of the total. The gut microflora has multiple functions, as represented here.
cases of lowered immunity. Escherichia coli, for example, lives
in the colon and, as an extensively studied model organism, is
probably the best understood bacterial species [26]. Mutated
strains of this gut commensal, such as E. coli O157:H7, can
cause disease. However, it remains “dormant” in a healthy host.
Microflora composition varies along the gastrointestinal tract
(from the stomach to the small and large intestines), according
to factors such as nutrient availability, pH and oxygen concentrations. The bacterial count increases from the upper to
the lower parts of the gastrointestinal tract, ranging from 102
colony-forming units (cfu)/mL in the stomach to 1012 cfu/mL
in the colon, with species–specific metabolic characteristics that
vary according to their localization. The upper portion of the
gastrointestinal tract is mostly colonized by aerobic bacteria,
while the lower portion is colonized by anaerobic species; this is
because oxygen concentration decreases from the duodenum to
the colon. The pH also influences the numbers of bacteria found
in the different parts of the gastrointestinal tract: the stomach
has the smallest number due to its low pH of 1–2, whereas the
pH increases within the small and large intestines from 5 (colon)
to 7.
Our understanding of the complex gut microflora ecology (Fig. 1) has been improved by molecular biology tools
that extend the study of microorganisms to those that are
not cultivable [27–29]. Sequencing of the gene encoding for
16S ribosomal RNA in nucleic acids extracted from faeces
or other luminal samples is a useful tool for the phylogenetic identification of bacteria [30]. Using this molecular
approach, it is now possible to study the complexity of all
genes of the gut microflora, an analysis termed “metagenomics”. This approach provides a better understanding of the
entire genome of the intestinal microflora, including the relationship between the commensal and pathogenic strains. The
human colonic microbiota is composed of Bacteroidetes (Bacteroides species) and Firmicutes (Clostridium, Lactobacillus,
Enterococcus), which together account for more than 90% of
the gut bacterial phyla [29]. Molecular technologies such as
fluorescent in situ hybridization (FISH) and DNA microarray chips allow the identification of specific bacterial species.
However, besides a qualitative analysis, there is also the possibility of designing strain-specific real-time polymerase chain
reaction (PCR) primers for performing quantitative analyses
[31].
The advent of these molecular methods has generated enough
data to demonstrate that, in a given individual, the composition
of the intestinal microflora remains virtually the same throughout life, with no dramatic changes [32]. It has been suggested
that this stability is conferred by the intestinal immune system long-term exposure to microbial antigens from childhood
to adulthood, allowing it to identify bacterial antigens as normal
[19].
3. Intestinal microflora and inflammatory diseases
The role of the intestinal microflora has been largely
described in inflammatory diseases such as IBD, CD and UC.
Although beyond the scope of this review, some concepts are
helpful for understanding the origin of metabolic disorders.
Indeed, it is now thought that obesity and diabetes are associated
with a poor inflammatory status, leading to impaired insulin
action and adipose tissue plasticity (Figs. 2 and 3) [33,34].
This means that the origin of metabolic inflammation could
have some traits in common with inflammatory diseases, albeit
of lesser intensity. We briefly review here some of the main
concepts.
3.1. Intestinal microflora, CD and UC
IBDs such as CD and UC represent abnormal immune
responses of the gastrointestinal tract leading to adverse clini-
M. Serino et al. / Diabetes & Metabolism 35 (2009) 262–272
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Fig. 2. Intestinal microflora changes and metabolic implications. Ingested components such as high-fat foods, antibiotics, dietary fibre (prebiotics) and bacterial
additives (probiotics) can change the intestinal microflora ecology, leading to an unbalanced Firmicutes–Bacteroidetes ratio. This imbalance can affect metabolism,
leading to a pathological state (obesity and diabetes).
cal outcomes. CD is associated with excessive IL-12, IL-23 and
IFN-gamma production that affects the wall of the small intestine, leading to inflammation and, often, granulomas. Patients
report gastrointestinal symptoms such as abdominal pain, diar-
rhoea and rectal bleeding, as well as systemic symptoms of
weight loss, fever and fatigue; they can also develop fistulae
[35]. UC is associated with excess IL-13 production that primarily affects the colon, with chronic inflammation of the mucosa
Fig. 3. Nutrition-induced intestinal microflora changes and their metabolic consequences. A change in nutrition induces changes in the intestinal microflora.
In particular, a fat-enriched diet decreases the Gram-positive–Gram-negative ratio, which is associated with increased lipopolysaccharide (LPS) absorption via
mechanisms involving lipoproteins and paracellular translocation. LPS then activates cells from the immune system in the liver, adipose tissues and muscles. The
increased inflammation eventually triggers insulin resistance and adipose tissue plasticity.
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[36]. More important, an atypical Th2 response mediated by
non-classical natural-killer T (NKT) cells producing the IL-13 is
characterized by an exacerbated cytotoxic potential in epithelial
cells, altering the integrity of the intestinal wall and, thus, leading to ulceration. Certainly, metabolic diseases are not associated
with this degree of inflammation. However, an impaired innate
immune system, in which hypersensitive macrophages or lymphocytes can generate an over immune response, could lead to
excess cytokine production, thereby increasing systemic inflammation. An increased inflammatory status is now accepted to be
at least partly responsible for impaired insulin action [33,34].
Recent data have shown that, when differential gene expression
was determined by microarray along the longitudinal axis of
the small intestine of C57BL/6J mice fed a diabetogenic highfat diet, after 2, 4 and 8 weeks of the dietary intervention, the
most pronounced effects were observed in the middle section
of the small intestine [37]. Numerous genes coding for cell
cycles, lipid metabolism and inflammation were identified. From
this, the authors concluded that high-fat-feeding modulates biological processes, especially those related to lipid metabolism
and inflammation. In addition, they found that the differential
expression of potential signaling molecules could provoke systemic effects in peripheral organs by influencing their metabolic
homoeostasis.
Another mechanism common to intestinal inflammatory and
metabolic diseases is related to changes in the intestinal mucosa.
Using conventional culture techniques, Swidsinski et al. [38]
demonstrated greater amounts of mucosa-associated bacteria
(those in the mucous layer and at the epithelial surface) in tissue
biopsies obtained from IBD patients vs controls. Indeed, concentrations of mucosa bacteria increased progressively with the
severity of disease. The authors suggested that changes in the
mucosal flora in IBD are not secondary to inflammation, but
the result of a specific host response. They further hypothesized
that a healthy mucosa is capable of blocking faecal bacteria
and that this function is profoundly disturbed in patients with
IBD. This was demonstrated by the fact that most germ-free
(sterile) rodents have no intestinal inflammation or immune
activation, but rapidly develop disease and pathogenic immune
responses after colonization by specific pathogen-free enteric
bacteria [39]. Although the processes of intestinal inflammatory diseases and diabetes are different, the mucosa hypothesis
offers a mechanism that increases contact between the intestinal microflora and cells of the innate immune system, thereby
increasing inflammation. The same reasoning could apply as
regards an impaired anti-inflammatory mechanism. Sellon et al.
[40] showed that mice with targeted deletion of the IL-10 gene
spontaneously developed enterocolitis when maintained under
conventional conditions, but developed only colitis when kept
in specific pathogen-free environments. This study confirmed
the hypothesis that enteric bacteria are necessary for the development of spontaneous colitis and immune system activation in
IL-10-deficient mice.
As for hypotheses based on what is known of the role of
intestinal microflora in the development of intestinal inflammatory diseases, there are indeed mechanisms in common between
metabolic diseases and IBDs, albeit with considerably less
inflammation in metabolic diseases. Taken together, the innate
and acquired immune systems are good candidates, as both
are involved in the interplay between intestinal microflora and
the inflammatory/anti-inflammatory cytokine balance. A good
example of this idea is the role of the intestinal microflora in the
control of type 1 diabetes (T1D).
4. Intestinal microflora and T1D
T1D is a T-cell-mediated autoimmune disease that results in
destruction of the insulin-producing beta cells of the pancreas.
T1D is known as “childhood”, “juvenile” or “insulin-dependent”
diabetes, although it is not exclusively seen in childhood,
as demonstrated by its growing incidence in adults. So far,
there are no preventative measures against T1D, and the initiating cause of the immune-system damage is still not fully
understood, although diet and exercise can help. However, a
provocative and growing body of evidences show that the intestinal microflora is involved in the development of T1D. Previous
hints showed that the spontaneous incidence of T1D in an animal model—non-obese diabetic (NOD) mice—could be due
to the animal microbial environment [41] or microbial stimuli
[42,43].
Recently, the interaction between the gut microflora and
innate immune system showed that the microflora could be considered a T1D-predisposing factor [44]. In their first series of
experiments, the authors found that specific pathogen-free NOD
mice lacking the MyD88 protein (a key component for recognition of microbial stimuli by innate immune system receptors)
were able to resist T1D, suggesting that the lack of MyD88 protein affected the autoimmune T cells systemically, preventing
the destruction of the pancreatic beta cells. Excess activation by
microbial antigen could lie at the origin of the autoimmunity.
However, a surprising conclusion came with the finding that
MyD88 knockout mice without intestinal microflora still developed T1D. This paradoxically suggested that immunization by
microflora prevented the overt activity of the immune system,
leading to autoimmunity.
In fact, colonization of the NOD germ-free mice with a
specific microbial combination, comprising bacterial phyla normally present in the human intestine, attenuated T1D. This
meant that, first, a given flora combination was responsible for
the occurrence of T1D by a mechanism requiring the MyD88
inflammatory signaling molecule and that, second, a different
intestinal flora group could protect against T1D by a mechanism that prevented overt activation of the immune system.
In addition to these surprising data, a new finding was that,
in addition to the capacity of intestinal microflora to control
host functions such as the innate immune system or nutrient
absorption (Fig. 1) [45], the host itself was able to control
the metagenome. This was again demonstrated in mice lacking
the MyD88 protein with different intestinal microflora, characterized by a reduced Firmicutes–Bacteroidetes ratio, and an
increase in Lactobacillaceae, Rikenellaceae and Porphyromonadaceae families of bacteria in the caecal contents. This change
in flora suggested that the host could select a particular intestinal flora to enable regulation of various new bodily functions.
M. Serino et al. / Diabetes & Metabolism 35 (2009) 262–272
For example, T1D is associated with increased intestinal permeability [46], lymphocytic infiltration in the mucosa [47] and
morphological changes [48] in the BioBreeding (BB) rat, a
model of spontaneous T1D. In humans, Bosi et al. [49] showed
that, following the oral administration of two probes—lactulose
and mannitol—urinary excretion over the following 5 h was
dramatically increased, suggesting augmentation of intestinal
uptake of the probes. All subjects with islet autoimmunity
analyzed by the investigators showed increased intestinal permeability to the disaccharide lactulose, indicative of intestinal
barrier damage. This suggests that numerous other proinflammatory substances might be able to cross the epithelial barrier and
reach the immune system to generate and increase an inflammatory response. Substances such as the highly proinflammatory
lipopolysaccharides (LPS) and peptidoglycans (PGN) are excellent candidates.
5. Intestinal microflora, obesity and type 2 diabetes
(T2D)
In the USA, roughly one million diabetic patients are newly
diagnosed each year. Given this growing number of cases, as
well as the numbers of undiagnosed cases (one-third of patients
are not aware of their disease), the treatment of diabetes remains
a major therapeutic challenge that the entire population needs
to address. Obesity has now been classified as the newest epidemic, as its occurrence is on the increase in Western countries.
The World Health Organization (WHO) has estimated that 600
million people will be obese by 2025 [50]. This corresponds to
a doubling of the current obese population. The rise in excessive weight gain in the developed countries is associated with
metabolic and cardiovascular diseases due to combined genetic
and environmental factors.
Although numerous hypotheses have been proposed to link
these factors with obesity, no clear unifying concept has yet
emerged. In terms of environmental factors, a noteworthy point
is that reducing dietary fibre induces a cascade of metabolic
impairments, leading to excess body weight, and cardiovascular and diabetic complications. Recent evidence also shows
that the gut microflora is determinant of body weight and the
amount (size) of adipose tissue, and is involved in intestinal
permeability (Fig. 2). This suggests that the microflora play
an important role in the development of T2D and obesity, two
linked pathologies that highlight an epidemiological progression
[45,51,52].
A novel hypothesis was recently proposed wherein the intestinal microflora could be considered a causative factor of obesity
[52–54]. Briefly, it proposes that a specific intestinal microflora
combination is responsible for increased energy storage. Two
mechanisms are then involved: first, the microflora increase the
bioavailability of energy intake by transforming non-digestible
fibre into absorbable nutrients; and second, the microflora regulate intestinal gene expression. Specifically, gut bacteria could
reduce the intestinal expression of fasting-induced adipocyte
factor (FIAF), a protein that inhibits lipoprotein lipase activity.
Its inhibition would favour the release of free fatty acids (FFA)
from lipoprotein particles, making FFA more readily available
267
to the liver and other tissues, which would then store them more
efficiently [55].
Similarly, a change in the intestinal microflora could favour
the production of short-chain fatty acids by means of their own
metabolism. Such molecules are activators of triglyceride synthesis.
These two arguments could explain why axenic (or germfree) mice are leaner than their conventional counterparts [53]. In
contrast, colonization of axenic mice by conventional microflora
rapidly increased their body weight [53]. This was associated
with decreased expression of FIAF and modulation of other
genes involved in glucose and lipid metabolism [54], thus supporting the involvement of intestinal microflora in the control of
energy metabolism.
Intestinal microflora composition itself is changed in
metabolic diseases (Fig. 2) [45,51,56–60]. Genetically ob/ob
mice are characterized by a 50% reduction in the number
of Bacteroidetes, which is balanced by an increased number of Firmicutes, compared with lean mice. This was not
due to a change in the quality of food, but may have been
induced by the large amount of food consumed by the obese
mice [61]. However, independent of food quantity, the quality of food is certainly a regulating factor in metagenomic
profiles (the intestinal microflora). Recently, we found that
feeding mice a high-fat diet induces a dramatic change of
the metagenomic profile of the intestinal microflora. This was
associated with a general reduction in the total amount of intestinal bacteria and a reduction in Gram-positive bacteria, while
the proportion of Gram-negative bacteria increased (Fig. 3)
[57–60].
Changes in the intestinal microflora have also been reported
in human studies (Fig. 2). Obese patients are characterized
by a greater disturbance of the Firmicutes–Bacteriodetes ratio
[45,51,56,61], which was subsequently reduced and almost normalized when the patients underwent several weeks of body
weight loss [51]. In addition, this showed that the intestinal
microflora can be adapted by nutritional therapy, which means
that metagenomic imprinting is reversible.
6. Intestinal microflora and increased metabolic
endotoxaemia
A raised hepatic low-density lipoprotein (LDL)–high-density
lipoprotein (HDL) ratio defines dyslipidaemia, an important
feature of metabolic diseases. The LPS, the principal component of the outer membrane of Gram-negative bacteria and
the most powerful of the proinflammatory molecules, is also
a component of lipoprotein particles [62,63]. The latter have
been proposed to buffer LPS in response to septic shock
[63], and to prevent an overt inflammatory response and fatality. As for the proinflammatory characteristics of LPS and
its association with a fat-enriched diet and dyslipidaemia, we
have already demonstrated that this bacterial component is a
triggering factor in metabolic diseases induced by a high-fat
diet [57]. When mice were fed a high-fat diet for 4 weeks,
their levels of LPS in plasma increased two- to three fold.
It further increased throughout the duration of the treatment
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before reaching a plateau. The increased endotoxaemia—called
“metabolic endotoxaemia”—is a pathological condition associated with quantitative and qualitative changes in the intestinal
microflora. A high-fat diet increased the Gram-negative–Grampositive ratio in the caecum by reducing the Gram-positive count
(Fig. 3). The mechanisms for this remain unknown, but might
be related to the reduced dietary fibre in the fat-enriched diet.
Indeed, dietary fibre supplementation can reverse changes in the
intestinal microflora induced by a fat-enriched diet [60]. Furthermore, by implanting a subcutaneous osmotic minipump to
deliver a continuous subcutaneous infusion of LPS, we causally
linked LPS to metabolic disease. All features characteristic of
the metabolic syndrome—fasting hyperinsulinaemia, glucose
intolerance, visceral weight gain, hepatic lipid overload and
insulin resistance—were induced by continuous subcutaneous
LPS infusion alone.
The link between high-fat-feeding in mice and metabolic
endotoxaemia has also been demonstrated in humans. Amar et
al. [64] showed that fat intake is associated with endotoxaemia
in apparently healthy people. We conducted a dietary survey
that also assessed other metabolic parameters such as total and
HDL cholesterol, and plasma triglyceride levels, in 1015 randomly recruited subjects. Plasma was collected in the morning
after overnight fasting. Plasma LPS levels were also measured
in a subsample of 201 men. We found that energy intake was
associated with increased LPS plasma levels. In contrast, carbohydrate and protein intakes were not associated with increased
endotoxaemia. In mice, the proportion of fat in the diet controlled the extent of metabolic endotoxaemia independent of
the amount of food ingested [64]. Thus, the authors demonstrated that fat is a highly efficient transporter of LPS from the
intestinal lumen to the bloodstream. A molecular demonstration of the role of LPS in the onset of metabolic diseases was
made by studying CD14 (LPS receptor)-deficient mice, in which
the development of obesity, insulin resistance, hepatic steatosis
and most of the metabolic features associated with metabolic
diseases were delayed [57]. Either LPS or a high-fat diet can
induce adipose tissue infiltration and inflammation, which was
totally blunted in the mutant mice. In addition, antibiotherapy
can reverse the metabolic phenotype [58]. For this reason, it is
now thought that metabolic endotoxaemia is a triggering factor
in the onset of metabolic diseases induced by a high-fat diet.
However, many questions regarding the role of LPS still need
to be addressed. The tissues targeted by LPS, for instance are
as yet unknown, and the cells of the innate immune system
involved in the inflammatory process also need to be determined.
6.1. A linear concept and other hypotheses
So far, this review has found that increased fat ingestion or changes in the intestinal microflora can modulate host
gene expression (Fig. 1) and corresponding metabolic functions (Fig. 2). The nutritional and microbiota signal for this
could be LPS. The role of lipoprotein synthesis and release
by the intestines, and the role of paracellular ingestion of
microbiota particles, could be the mechanisms involved in
LPS absorption. A change in the structure of the gut barrier, altering the architecture of the modulating tight junctions,
would affect intestinal paracellular permeability, resulting in
metabolic endotoxaemia. Other processes by which the intestinal microflora could control tight junctions—and, hence, LPS
absorption—might involve molecular factors in the control of
metabolic diseases. The subsequent local inflammation could
also induce abnormal stimulation of the intestinal immune
system, leading to localized inflammatory reactions in the
intestinal barrier that, in turn, could trigger a systemic inflammatory reaction against white adipose tissue, muscle and
the liver, finally resulting in insulin resistance and T2D
(Fig. 3).
7. Changing the intestinal microflora
7.1. Antibiotic treatments
As the intestinal microflora could be a causal triggering factor
of metabolic diseases, Cani et al. [58] treated control, highfat-fed and ob/ob mice with broad-spectrum antibiotics. They
used ampicillin and neomycin as examples of antibiotics that
are poorly absorbed (or unabsorbed in the case of neomycin) by
the organism to reduce the occurrence of systemic effects [65].
The results showed that changes in the gut microbiota induced
by antibiotic treatment reduced metabolic endotoxaemia and the
caecal content of LPS in both the high-fat-fed and ob/ob mice.
The reduced endotoxaemia correlated with improved glucose
tolerance and decreases in body weight gain, fat-mass development, inflammation, oxidative stress and macrophage infiltration
marker mRNA expression in visceral adipose tissue. In addition,
the use of ob/obxCD14–/– mutant mice showed that the lack
of CD14 reduced inflammatory markers and metabolic impairments [58].
Likewise, Membrez et al. [66] tested a similar antibiotherapy
against the gut microbiota in two different mouse models with
insulin resistance. In this study, the investigators used a combination of norfloxacin and ampicillin, as this treatment is known
to result in maximum suppression of the numbers of caecal aerobic and anaerobic bacteria in ob/ob mice [67]. After 2 weeks
of intervention with the antibiotic combination, both the ob/ob
and the diet-induced obese and insulin-resistant mice showed
significant improvements in fasting glycaemia and oral glucose
tolerance. The authors found that this effect was independent
of food intake or adiposity because pair-fed ob/ob mice were
as glucose intolerant as the control ob/ob mice. Reduced liver
triglycerides and increased liver glycogen concentrations were
also associated with the improved glucose tolerance. In addition, the antibiotic treatment in ob/ob mice showed antidiabetic
effects by reducing endotoxaemia and increasing adiponectin
levels.
In this study, the investigators discovered that modulation of
the gut microbiota improved glucose tolerance in mice by altering the expression of the hepatic and intestinal genes involved
in inflammation and metabolism, and changing the hormonal,
inflammatory and metabolic status of the host.
M. Serino et al. / Diabetes & Metabolism 35 (2009) 262–272
7.2. Prebiotic treatment
One key question still remains: why is the intestinal
microflora in obese patients and mice associated with an
increased Firmicutes bacterial count? The answer may be
related to changes in the amount of dietary fibre consumed
nowadays. The data show the importance of the intestinal
microflora in controlling body weight, confirmed by the observation that dietary fibre—which can also change the gut
microflora [60]—is associated with metabolic changes. Dietary
fibre is a strong inducer of fermenting bacteria [68–71]. The
molecular relationship between dietary fibre, microflora and
an improved metabolic status is still unknown. However, our
recent work has shown that the intestinal microflora can
regulate inflammation, which is closely linked to metabolic
diseases [58]. The latter are characterized by a low inflammatory status, which contributes to the impaired insulin action
[72].
Recently, it has been shown that high-fat-feeding contributes
to the induction of moderate inflammation, characterized by
cytokine secretion (TNF-␣, IL-6, IL-1␤) that strongly impairs
insulin action and signaling [33,73]. The result is that the glucose transported by muscle or produced in the liver is no longer
controlled by insulin, thereby resulting in hyperglycaemia. Consequently, hyperinsulinaemia develops to compensate for the
hyperglycaemia, and glucose is then stored in adipose tissue,
leading to obesity.
Other recent findings propose that dietary fibre can improve
the metabolic status of diabetic mice by reducing inflammation [71]. This effect could be due to improved secretion
of glucagon-like peptide-1 (GLP-1), a gut hormone which
increases glucose-stimulated insulin secretion [74,75]. This idea
is physiologically relevant as GLP-1 is secreted into the hepatoportal vein and controls glucose metabolism (insulin secretion,
peripheral glucose utilization and vascular blood flow) [76–81].
Increasing the secretion of this hormone at its physiological site
of production may be a therapeutic advantage in light of other
strategies that aim to increase GLP-1 secretion in the systemic
blood by pharmacological means. Intestinal endocrine function may also be improved when the deleterious effects of a
high-fat diet that triggers inflammation is blunted by dietary
fibre.
The control of GLP-1 secretion by inflammation could
have a major impact on human health as this hormone is
the basis of three new antidiabetic drugs that are already
on the market [82,83]. However, the molecular mechanisms
linking high-fat-feeding, inflammation and dietary fibre are
still unknown. Prebiotics are non-digestible food oligosaccharides that beneficially affect the host by selectively stimulating
the growth and/or activity of one or a limited number
of bacteria in the colon, thereby improving host health
[84].
At present, only two dietary non-digestible oligosaccharides
fulfill all the criteria for prebiotic classification: fructooligosaccharides (FOS); and galactooligosaccharides (GOS).
Classification as a prebiotic food ingredient is based on the
following criteria:
269
• it is neither hydrolyzed nor absorbed in the upper part of the
gastrointestinal tract;
• it is selectively fermented by one or a limited number of
potentially beneficial bacteria in the colon;
• it alters the composition of the colonic microbiota toward a
healthier composition;
• it preferably induces effects that are beneficial to host health
[85].
In fact, not all oligosaccharides—such as mannanoligosaccharides (MOS)—fit this definition. They may confer other
positive benefits, but are minimally utilized by commensal
bacteria (Bifidobacterium and Lactobacillus species). Also, prebiotics are usually carbohydrates (such as oligosaccharides),
but other non-carbohydrate molecules can be used. The most
common form of prebiotic is soluble fibre. We reported that
high-fat-feeding was associated with more endotoxaemia and
fewer Bifidobacterium species in the caecal contents in mice
[58]. Cani et al. [71] found that oligofructose can restore
the bifidobacteria count in high-fat-fed mice, and this finding
positively correlated with improved glucose tolerance, glucoseinduced insulin secretion, decreased endotoxaemia, and plasma
and adipose tissue proinflammatory cytokines. Indeed, Watanabe et al. [86] have now confirmed the beneficial effect of
FOS supplementation in female BALB/c mice fed a synthetic
diet for 3 weeks compared with control mice that did not
receive FOS. The mice were then epicutaneously immunized
with 2,4-dinitrofluorobenzene, after which the mice continued
to receive their respective diets. Five days after immunization,
the mice were ear-challenged with hapten, but the subsequent
ear-swelling was significantly reduced in the FOS-supplemented
mice compared with the control-diet-fed mice. Using denaturing
gradient gel electrophoresis (DGGE), the investigators assessed
changes in the intestinal microbiota and found that the numbers of bifidobacteria (Bifidobacterium pseudolongum, assessed
by sequence analysis), but not lactobacilli, were significantly
higher in the FOS-supplemented mice compared with the mice
fed the control diet. Ear-swelling was also negatively correlated
with the numbers of bifidobacteria found in the faeces. The
authors concluded that supplementing the diet with prebiotics
leads to changes in the intestinal microflora that improve health.
This means that strategies involving prebiotics should further
improve patients’ well-being and health.
7.3. Probiotic treatment
Probiotics are dietary supplements that contain potentially
beneficial bacteria or yeasts. According to the current definition of the United Nations’ Food and Agriculture Organization
(FAO)/WHO, probiotics are “live microorganisms which, when
administered in adequate amounts, confer a health benefit on
the host”. Interest in probiotics has recently increased, with
studies investigating their beneficial roles in the treatments
of various diseases. Rautava et al. [87] showed the beneficial
effects of Lactobacillus rhamnosus GG and B. lactis Bb-12 in
reducing the risks of early acute otitis media and antibiotic
use, and recurrent respiratory infections, in the first year of
270
M. Serino et al. / Diabetes & Metabolism 35 (2009) 262–272
life. Probiotics can also improve the effects of certain drugs
such as gliclazide, a sulphonylurea with beneficial extrapancreatic effects in diabetics. Al-Salami et al. [88] showed a
threefold probiotic-induced reduction in gliclazide bioavailability in healthy rats whereas it increased uptake (by twofold) in
diabetic rats, leading to a reduction in blood glucose levels by
insulin-independent mechanisms. In addition, probiotics can be
administered as a food supplement in yogurt, for example [89].
In their study, Bajaj et al. found that probiotic-complemented
yogurt was able to reverse minimal hepatic encephalopathy
(MHE), the preclinical stage of overt hepatic encephalopathy
(OHE), a significant condition that affects up to 60% of cirrhosis
patients.
Yadav et al. [90] found that supplementing with the probiotics L. acidophilus and L. casei increased the efficacy of
dahi (Greek yogurt) in suppressing streptozotocin-induced diabetes in rats by inhibiting insulin depletion, preserving diabetic
dyslipidaemia, and inhibiting lipid peroxidation and nitrite
formation. In ob/ob mice, a genetic mouse model of obesityrelated non-alcoholic fatty liver disease and T2D, Li et al.
[91] found that 4 weeks of treatment with VSL#3, a probiotic
combination of bifidobacteria, lactobacilli, and Streptococcus
thermophilus, decreased both hepatic total fatty-acid content
and serum alanine aminotransferase. These benefits were associated with normalization of hepatic fatty-acid ␤-oxidation and
reduction of hepatic NF-␬B activity and uncoupling protein
(UCP)-2 expression, suggesting that VSL#3 therapy improves
hepatic insulin resistance and lipid metabolism. The benefits
of VSL#3 probiotics were also demonstrated by Ma et al.
[92] in another mouse model of high-fat-diet-induced obesity, steatosis and insulin resistance. Oral probiotic treatment
significantly improved the depleted numbers of hepatic NKT
cells, insulin resistance and hepatic steatosis. The effect was
NKT-dependent, resulting from attenuation of inflammatory signalling.
8. Microflora as a friend
Mazmanian et al. [93] showed that a specific component of
the intestinal microflora can directly interact with the innate
immune system of the host, resulting in a beneficial effect.
In their elegantly written report, the investigators showed
that Bacteroides fragilis can protect animals against experimental colitis induced by Helicobacter hepaticus, a potential
pathogenic commensal of the intestinal microflora. The benefit depends on the production of a single specific molecule
produced by B. fragilis called ‘polysaccharide A’ (PSA). In
fact, animals with a mutant variant of B. fragilis lacking PSA
cannot resist H. hepaticus colonization, leading to disease and
proinflammatory cytokine production in the colon. In addition, the purified molecule of PSA alone is enough to protect
against H. hepaticus colonization and even against chemically
induced (trinitrobenzene sulphonic acid, TNBS) experimental
colitis. PSA protection is mediated by IL-10-producing CD4+
T cells.
In conclusion, the present review shows, for the first time,
that specific intestinal microflora components can produce
molecules that are contributory to host health. Indeed, the intestinal microflora can act, via the production of specific molecules,
not only as an enemy of the host, but also—as described
above—as a new friend to improve the defense mechanisms
of the host (Figs. 1–3).
9. Conflicts of interest
The authors have no conflicts of interest to declare in relation
to this report.
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