Review

Microbes and Metabolism

Understanding the role of gut

microbiomehost metabolic signal
disruption in health and disease
Elaine Holmes, Jia V. Li, Thanos Athanasiou, Hutan Ashrafian and
Jeremy K. Nicholson
Department of Surgery and Cancer, Imperial College London, London, UK

There is growing awareness of the importance of the gut

microbiome in health and disease, and recognition that
the microbe to host metabolic signalling is crucial to
understanding the mechanistic basis of their interaction.
This opens new avenues of research for advancing
knowledge on the aetiopathologic consequences of dysbiosis with potential for identifying novel microbiallyrelated drug targets. Advances in both sequencing technologies and metabolic profiling platforms, coupled
with mathematical integration approaches, herald a
new era in characterizing the role of the microbiome
in metabolic signalling within the host and have far
reaching implications in promoting health in both the
developed and developing world.
Implications of hostmicrobiome metabolic interactions
in health and disease
The symbiosis between the human host and gut microbiota
can trigger specific biological responses both locally and
systemically. Of the 55 bacterial divisions, only two are
prominent in mammalian gut microbiota (Bacteroidetes
and Firmicutes), and it is, therefore, likely that host eukaryotic organisms have co-evolved with gut microbiota in
the context of this symbiosis to achieve a physiological
homeostasis [1]. Individual bacterial species present
unique pathological effects and, similarly, shifts in gut
bacterial colonies can also prompt specific disease-inducing
activity (dysbiosis) or disease-protective activity (probiosis). This balance between the bacterial host defence reinforcement and proinflammatory pathology was first
proposed by the 1908 Nobel laureate Elie Metchnikoff
who noted the therapeutic benefits of acid-producing lactic
bacilli [2]. The beneficial effects of gut microbiota include:
(i) immune-cell development and homeostasis (Th1 vs. Th2
and Th17), (ii) food digestion, (iii) supporting fat metabolism, (iv) epithelial homeostasis, (v) enteric nerve regulation and (vi) promoting angiogenesis. Conversely,
maladapted microbial ecology can impair many of these
homeostatic and physiological signals so that they result in
a number of disease states that include allergy, inflammatory bowel disease (IBD), obesity, cancer and diabetes. We
Corresponding author: Nicholson, J.K. (j.nicholson@imperial.ac.uk)

describe the relationship of the microbiome with specific

physiological and pathological states and explore the metabolome as a window for monitoring the activity of the gut
microbiome via microbialmammalian cometabolism.
Moreover, we identify potential avenues for exploitation
of this hostmicrobial metabolic axis with respect to improving human health.
Metabolic profiling studies, mainly adopting mass spectrometric (MS) and NMR spectroscopic platforms (Box 1) to
measure the metabolic composition of biological samples,
have been instrumental in characterizing a wide variety of
diseases and have been used for biomarker screening,
elucidating mechanistic information relating to disease
aetiology and in monitoring responses to therapeutic interventions [3,4]. Subtle changes in metabolic profiles in
response to physiological perturbations or environmental
stimuli have also been described [5,6]. In many such
studies, the panel of diagnostic biomarkers has included
several metabolites of gut microbial or microbialmammalian cometabolic origin. To probe the exact nature of the
metabolic handshake between the mammalian host and
the resident gut microbiota, several animal models of
simplified microbiota have been developed including
germ-free animals, antibiotic-treated rodent models
(resulting in a temporary knockout effect of selected bacterial groups) and animals with transplanted microbiota,
such as the Schaedler microbiota (consisting of eight bacteria including Escherichia coli var. mutabilis, Streptococcus faecalis, Lactobacillus acidophilus, Lactobacillus
salivarius, group N Streptococcus, Bacteroides distasonis,
a Clostridium sp. and an extremely oxygen-sensitive fusiform bacterium) [7] or human infant microbiota. The consequences of these microbial modifications on the host
metabolism are summarized in Box 2 with a brief discussion of their advantages and limitations as a model for
understanding the hostmicrobiome functional relationship.
Given the proximity of the gut microbiota with the
intestine, it is unsurprising that the microbiome is implicated in intestinal diseases. However, the reach of the gut
microbiota has been shown to extend far beyond local
effects to remote organ systems such as the brain and
encompasses more processes than inflammation. Dysbiosis

0966-842X/$ see front matter ! 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2011.05.006 Trends in Microbiology, July 2011, Vol. 19, No. 7

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Box 1. Common spectroscopic techniques for metabolic profiling

Spectroscopic technologies commonly used for the metabolic profiling
of biological fluids and tissues can be used in either an untargeted
mode allowing for screening of the global metabolic composition of
metabolites without the necessity of preselecting a set of analytes, or
alternatively, can be tailored to measure selected classes of metabolite
such as lipids or bile acids. No single analytical method can measure
the total set of metabolites present in a biological sample owing to
analytical limitations and, therefore, the choice of analytical platform is
driven by prior knowledge of the nature of metabolic disruption,
platform availability, cost, robustness and biological matrix.
Mass spectrometry (MS): metabolites in complex mixtures can be
measured using MS-based methods, which discriminate molecules
according to their mass to charge (m/z) ratio. For the purpose of
metabolic profiling of biofluids, MS methods are generally prefaced
with a separation technology such as gas chromatography (GC-MS),
high performance or ultra performance liquid chromatography
(HPLC-MS or UPLC-MS) or capillary electrophoresis (CE-MS). These
platforms tend to exhibit high sensitivity and have been widely
applied to targeted metabolite classes (e.g. lipids, bile acids and
SCFAs) in the context of characterizing disease. Metabolite identification is achieved by use of existing databases of chromatographic
retention times and m/z values and by ion fragmentation patterns.
NMR spectroscopy: exploits the property of spin that nuclei
possess. These nuclei can exist in discrete orientations that relate to
discrete energy states when placed in a magnetic field. Spectra are
measured following perturbation of the system with radiofrequency
pulses and are expressed in the frequency domain, which carries
information on the molecular structure of chemical constituents
based on their chemical shift with respect to a reference standard,
signal intensities and splitting patterns. 1H is typically the choice of
nucleus for high throughput metabolic screening. NMR is an
extremely robust technology and requires little or no sample
preparation. For a comprehensive review of the strengths and
limitations of these profiling methods, see [91].

of the gut microbiota has been implicated in several modern epidemics in the Western world, the most notable being
IBD, certain cancers, heart disease and metabolic syndrome and associated risk factors such as obesity and
hypertension (Table 1). We summarize the role of the
microbiome in the aetiology and development of several
diseases and examine concurrent changes in the metabolome (Table 2). Furthermore, we explore the potential for
exploiting this hostmicrobial relationship with respect to
developing new therapeutic interventions.

Mathematical modelling and data integration: spectra obtained

from biological samples are highly multivariate and complex with
various degrees of signal overlap. Interpretation of spectra with
respect to identifying patterns of metabolites relating to a given
physiological or pathological condition is typically achieved using
data reduction and multivariate statistical analysis methods. The
general aim is to classify or predict objects by identifying inherent
patterns in a set of indirect measurements and to relate these
classifications to the metabolites or signals that strongly weigh the
classification in order to identify candidate biomarkers. These
methods operate by compressing the variation in a data set into a
smaller number of components based on latent variables and can
operate either with or without the incorporation of classification
information in the model. As with the profiling methods themselves,
each pattern recognition technique has strengths and limitations, and
the choice of method is made based on considerations such as the
priority of classification over biomarker identification, sensitivity of
method, input data and number of missing values. It is generally
appropriate to use more than one method for the purpose of validation
and maximizing the extraction of information. Common methods
include principal components analysis (PCA), partial least squares
(PLS), PLS-discriminant analysis (PLS-DA), clustering algorithms, selforganizing maps, neural networks and genetic algorithms. Detailed
descriptions of multivariate methods can be found in [92].
Typically, for ascertaining the covariation of mammalian metabolites with microbial composition and identifying potential biological
associations between specific bacterial species or families and
metabolic profiles of biofluids or tissues, relatively simple correlation
methods have been adopted. Outputs of microbial composition of
faeces including DGGE, FISH and 454 sequencing data have variously
been used to generate correlation matrices with urinary, faecal, serum
or intestinal tissue profiles [46,93] although bidimensional partial
least squares algorithms and other more complex methods can be
used to integrate two disparate -omic datasets [94].

IBD
IBD represents two disorders of chronic intestinal inflammation, ulcerative colitis (UC) and Crohns disease (CD),
both of which are associated with a complex genetic susceptibility together with clear evidence for the involvement
of environmental triggers. Evidence implicating the role of
microbiota in IBD was initially derived from observations
that some antibiotics improved the disease course of
patients with this disorder whereas several animal models
of IBD required bacterial colonisation for inflammation to

Box 2. Metabolic signatures of microbially-modulated animal models

The metabolic phenotypes of several classic models of microbial
modulation have been characterized using NMR and MS methods:
Germ-free: several strains of rat and mice have been profiled. NMR
studies have addressed differences in urinary metabolic signatures
between conventional mice (C3H/HeJ) and their germ-free counterparts (e.g. 3-hydroxylcinnamic acid, 4-hydroxypropionic acid, hippurate and phenylacetylglucine), liver (glutathione, glycine, hypotaurine
and trimethylamine N-oxide), intestine (alanine, aspartate, glutamate,
lactate, taurine-conjugated bile acids, tyrosine and glycine) and
kidney (betaine, choline, ethanolamine, inosine and myo-inositol)
[95]. Another study on Sprague Dawley germ-free rats has shown the
different urinary levels of 2-oxoglutarate, formate, trimethylamine Noxide, hippurate, 4-hydroxypropionic acid, 3-hydroxypropionic acid
and plasma levels of betaine, glucose and lactate compared with
conventional rats [96]. A targeted UPLC-MS method for profiling bile
acids has shown that the relative proportion of taurine-conjugated
bile acids in the liver, kidney, heart and plasma of germ-free rats is
markedly higher than in conventional animals [97].
Germ-free conventionalization: to understand the metabolic trajectory in acclimatized germ-free rats, male Fischer 344 germ-free rats

350

were examined and found to have increased urinary levels of

phenylacetylglycine after 6 h following introduction to a standard
laboratory environment. 3-Hydroxypropionic acid was observed at
day 12 and increased up to day 17. The urinary metabolic profile was
within the control range by day 21, suggesting the establishment of a
stable gut microbiota [98].
Germ-free colonized with simplified microbiota: NMR and
UPLC-MS profiling have also been applied to investigate the
global metabolic signatures of plasma, urine, faecal extracts, liver
tissues and ileal flushes from humanized microbiome mice treated
with probiotics (e.g. Lactobacillus paracasei or Lactobacillus
rhamnosus) [99] and prebiotics (e.g. galactosyl-oligosaccharide)
[100].
Antibiotic treatment: NMR urine profiles of antibiotic treated
rodents show a similarity with germ-free rat models with lower
concentrations of phenolics and other microbial metabolites including hippurate, phenylacetylglycine, trimethylamine, dimethylamine
and higher oligosaccharides and choline. Particularly, hippurate level
shifted back to control level within 19 days post a single vancomycin
dose in female NMRI mice [101].

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Table 1. Examples of disease models with modulated gut microbiota

Disease
Obesity
Obesity

Animal model
Mice
Mice

Experimental design
Conventionally reared vs. germ-free
Conventionally reared vs. germ-free

Obesity

Mice

FIAF genetic knockouts

Obesity

Obese ob/ob vs. lean ob/+ and ob+/+

Obesity

Mice (genetic obese

leptin-deficient ob/ob)
Mice (genetic obese
leptin-deficient ob/ob)
Mice

Obesity

Mice

Obese ob/ob vs. lean ob/+, ob+/+ and

ob/+ mothers
Colonization of germ-free mice

Obesity and
diabetes

Mice

Non-mutant vs. CD14 mutant mice

Diabetes

Mice

Diabetes

Rats

NOD (non-obese diabetic)

MyD88 knockout mice
vs. non-mutant mice
Biobreeding diabetes resistant
vs. biobreeding diabetes prone

IBD

Humans

IBD patients

IBD

Mice

IL10-deficient mutants
vs. non-mutants

Cancer

Mice

Tgfb1-null mice vs. non-mutants

Cancer

Mice

Cancer
Cancer

Mice
Mice

Germ-free IL10-deficient mice

vs. germ-free non-mutants
ApcMin vs. non-mutants
Helicobacter-free C3H/HeN
mice vs. C57BL/6 FL-N/35 mice

Cardiovascular
dyslipidaemia

Mice

Cardiovascular
dyslipidaemia

Hamsters

Obesity

Obese ob/ob vs. lean ob/+ and ob+/+

Normal chow-fed mice

vs. high fat-fed mice
vs. high fat-oligofructose-fed mice
Grain sorghum lipid
extract-fed hamsters

occur [8]. When compared with controls, patients with IBD

demonstrate a reduced diversity of Firmicutes and Bacteroidetes, and mucous-invading bacteria such as E. coli have
been associated with disease-specific activity [9]. Genetic
studies confirm a role for the underlying microbialhost
interaction in IBD pathogenesis. These include the genomic regions of nucleotide-binding oligomerisation domain
containing 2 (NOD2), which is an intracellular receptor
that recognizes proteins found in bacterial cell wall species.
Patients with CD demonstrate an association with the
NOD2 gene including three polymorphisms that weaken
the host peptidoglycan response. Carriers of NOD2 have a
1.754-fold increased risk of CD and are clinically more
likely to undergo surgical gastrointestinal (GI) resection.
The mechanisms linking NOD2 require further investigation; however, the microbialhost interaction is likely to be
modulated by an underlying cytokine environment, and
disease development occurs only when these animals
are colonized by normal gut bacteria [10]. In a study of

Mechanism
Increased food consumption
Increased gut monosaccharide absorption
and induction of hepatic lipogenesis
Fat storage (increased lipoprotein
lipase activity)
Genes encoding enzymes that breakdown
otherwise indigestible polysaccharides
Increased fermentation

Refs
[69]
[69]

Decreased Bacteroidetes, increased

Firmicutes
Increased energy extraction by bacterial
interactions
Bacterial LPS from Gram-negative bacteria
triggers inflammation in response to
a high fat diet, which results in metabolic
syndrome
A dysfunction in the microbial responsive
immune system can lead to autoimmunity
and diabetes
Administration of Lactobacillus johnsonii
isolated from biobreeding diabetes
resistant delays the onset of type
1 diabetes in biobreeding diabetes prone
Reduced diversity of Firmicutes and
Bacteroidetes
Gut inflammation associated with
IL10 deficiency occurs in the presence
of normal gut bacteria
GI cancer develops in the context of normal
gut bacteria with a lack of TGFB1
Colitis-associated colorectal cancer can be
reversed by colonisation with normal gut microbiota
GI polyps only occur in a germ-free environment
Enteric microbiota define hepatocellular
carcinoma risk in mice exposed to carcinogenic
chemicals or hepatitis virus transgenes
Endotoxaemia significantly and negatively
correlates with Bifidobacterium spp.

[50,51]

Bifidobacteria increased in grain sorghum

lipid extract-fed hamsters and positively
associated with HDL plasma cholesterol

[69]
[70]
[70]

[71]
[61]

[41]

[72]

[9]
[10]

[20]
[21]
[22]
[73]

[59]

[74]

interleukin-10 (IL10)-deficient mice, which spontaneously

develop colitis, gas chromatography (GC)-MS analysis
identified a shift towards a higher plasma low-density
lipoprotein (LDL) to very low-density lipoprotein (VLDL)
ratio and higher plasma levels of lactate, pyruvate and
citrate with lower levels of glucose consistent with increased fatty acid oxidation and glycolysis [11]. Chemically-induced murine models of colitis have also shown
systematic differentiation in plasma and tissue profiles
from matched control animals consisting of altered succinate, indole-3-acetate, glutamate and glutamine, which
tracked the developmental phases of colitis and its severity
[12]. Metabolic profiling studies of IBD in humans have
shown systematic differentiation of CD and UC based on
urinary and faecal metabolite profiles. Although reduced
concentrations of butyrate, acetate, methylamine and trimethylamine and increased excretion of amino acids suggestive of malabsorption were characteristic of both
conditions, the extent of metabolic perturbation was greater
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in the CD group and was accompanied by increased faecal

glycerol, which was not seen in the UC group [13]. The
differential urinary signature of IBD from CD and UC
includes alteration of hippurate, 4-cresyl sulfate and formate, all potential metabolites of gut microbial activity [14].

It has recently been shown via genome-wide association

studies (GWAS) that there are overlapped genetic signatures of CD and UC, and furthermore, that IBD is genetically linked to mutations associated with a number of
non-GI diseases [15]. Given that these GI conditions are

Table 2. Examples of metabolites associated with microbial metabolism or microbialhost cometabolism and illustrations of
associations with disease
Metabolite class

Sample type

Metabolites

SCFAs

Plasma,
faeces,
tissues

Acetate, butyrate,
propionate

Polyamines

Urine,
plasma,
faeces,
tissues

Putrescine,
cadaverine

Methylamines
and products of
choline degradation

Urine,
plasma,
faeces,
tissues

Benzoates

Urine,
plasma

Methylamine,
dimethylamine,
dimethylglycine,
trimethylamine,
trimethylamine
N-oxide
Benzoic acid (plasma),
hippurate (urine),
2-hydroxyhippurate

Chlorogenic acids

Urine,
plasma

Protein putrefaction products

Urine,
Tyrosine
plasma,
faeces

Tryptophan

Urine,
plasma,
faeces

Phenylalanine

Urine,
plasma,
faeces
Urine,
plasma,
faeces

Bile acids

352

Dihydroferulic acid,
dihydroferulic acid-3-Osulfate, ferulic acid-4-Osulfate, dihydroferulic
acid glucuronide,
feruloylglycine
4-cresyl sulfate
4-cresyl
glucuronide

Origin of metabolites and examples of association with

disease
Microbes ferment substances presented in the large
intestine, including indigestible oligosaccharides, dietary
plant polysaccharides or fibre, non-digested proteins and
intestinal mucin, and produce SCFAs.
Protein putrefaction by a range of anaerobic
microorganisms can result in amines such as putrescine
and cadaverine, can exert adverse impact including
genotoxicity on the host. These polyamines have been
previously found to increase resistance of Neisseria
gonorrhoeae to mediators of the innate human host
defence. Putrescine has also been proposed as a urinary
marker for diabetes in a diet-induced mouse model of
diabetes.
Dietary choline can be metabolized into methylamines by
the gut microbes and has been shown to be modulated in
dietary-induced models of obesity, diabetes and
cardiovascular disease.

Refs
[75]

[76,77]

[77,78]

Decreased concentrations of urinary hippurate have been

consistently reported as characteristic of obesity in both
animal models and humans. 2-Hydroxyhippurate was
shown to be positively correlated with colorectal cancer in
a clinical study whereas high urinary levels of hippurate
and dimethylamine were characteristic of diabetes in a rat
model.
Chlorogenic acids are known antioxidants and have been
beneficially linked to health, and have been shown to
lower blood pressure in a human cohort with essential
hypertension.

[7981]

Decreased plasma concentrations of indoxylsulfate and

indoleacetate were key discriminators in a rat model of
depression. Indoxyl sulfate and phenylacetylglutamine
have been found in higher concentrations in the plasma of
diabetic individuals compared to non-diabetics. Abnormal
urinary excretion of phenylacetylglutamine, hippurate and
hydroxyhippurates has been reported in autistic children.

[63,8385]

Conjugated bile acids are cholesterol derivatives

synthesized in the liver and contain a steroid ring
conjugated with glycine or taurine. Bile acids are well
known to facilitate lipid absorption and signal systemic
endocrine functions to regulate triglyceride, cholesterol,
glucose and energy homeostasis. Gut microbiota have
been shown to modify the bile acid profiles through a
broad range of reactions such as deconjugation
(hydrolysis of bile salt conjugates to form free bile acids),
dehydroxylation, oxidation (dehydrogenation) and
sulfation, resulting in the formation of secondary and
tertiary bile acids. Deoxycholate has been reported to be
higher in the plasma of diabetics.

[84,86]

[82]

Indoleactylacetate,
indoleactylglycine,
indolelactate,
3-hydroxyindole,
indoxyl sulfate
Phenylacetlyglycine,
phenylacetylglutamine
Cholic acid, hyocholic
acid, deoxycholic acid,
chenodeoxycholic acid,
hyodeoxycholic acid,
ursodeoxycholic acid,
glycocholic acid,
glycodeoxylcholic acid,
glycochenodeoxycholic
acid, taurocholic acid,
taurohyocholic acid,
taurodeoxylcholic acid,
taurochenoxycholic acid

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Table 2 (Continued )
Metabolite class

Sample type

Metabolites

Lipids

Plasma,
faeces

Acylglycerols,
sphingomyelin,
cholesterol,
phosphatidylcholines,
phosphoethanolamines,
triglycerides

Organic acids

Urine,
plasma,
faeces

Lactate, formate

Origin of metabolites and examples of association with

disease
Gut microbiota regulate lipid synthesis and metabolism
directly through various microbial activities. Undigested
and non-absorbed glycerides in colon may be hydrolysed
into free fatty acids and glycerol by bacterial lipases.
Glycerol can be subsequently converted into
3-hydroxypropanal (3-HPA) and then 1,3-propanediol
(1,3-PDO) by a NAD+-dependent oxidoreductase by a wide
range of colonic bacteria including the genera Klebsiella,
Enterobacter, Citrobacter, Clostridum and Lactobacillus
members. Lactobacilli, particularly Lactobacillus reuteri,
are most efficient in accumulating 3-HPA.
High plasma levels of lactate and formate have been found
to be characteristic of several parasitic infections and urinary
formate has also been shown to inversely correlate with
high blood pressure.

also associated with gut microbial abnormalities, this begs

the question as to whether a significant transgenomic network process is in operation linking both microbial and
human genes to disease aetiopathogenesis. These studies
collectively support a key role for the microbiota in IBDs
with contribution to the immunological component of the
disease as well as exerting direct metabolic effects. However, the mechanisms by which these microbial alterations
contribute to IBD pathogenesis remain unknown. Thus,
specific studies probing key functionally-active microbiota
are of importance.
Cancer
The association of the gut microbiota with cancer is most
commonly observed with GI tumours, as expected, although there are examples of these microbiota modifying
the cancer risk to other systems such as in breast tumours
[16]. The bacterium Helicobacter pylori has been proposed
to have an aetiological relationship with gastric cancer [17]
and has been applied to track the early migration of Homo
sapiens suggesting that humans were already infected by
H. pylori before their migrations from Africa [18] and have
demonstrated a direct gut microbehost relationship ever
since [19]. Colonic cancer is the third most common cancer
in industrialized countries and is the second leading cause
of cancer deaths. Several knockout mice models have
elucidated a growing understanding of colonic cancers
and gut microbiota. Transforming growth factor beta 1
(Tgfb1)-null mice develop colonic cancer in the presence
of conventional gut microbiota [20], whereas the germ-free
Il10-deficient mice are resistant to colitis-associated colorectal cancer and resistance is lost upon colonization with
normal gut microbiota [21]. Conversely, tumour-prone
ApcMin mice demonstrate only a modest reduction in GI
polyps when raised in a germ-free environment [22]. These
effects of the gut microbiota on cancer development reflect
a complex interplay between the gut genome and the
physiological environment of the host and the microbiota.
Other examples include colonic cancer models with disordered transforming growth factor beta (TGFB) signalling,
such as Tgfb1-null, Smad3-null and Rag2-null mice, and
predispose the development of cancer with pro-inflammatory gut bacterial species such as H. pylori and Helicobacter
hepaticus [23,24].

Refs
[8789]

[4,90]

Metabolic profiling studies in colon and other cancers

have also drawn attention to the role of the gut microbiota in
aetiogenesis showing profound modulation of lipid and sterol pathways, and changes in faecal amino acids, short-chain
fatty acids (SCFAs) and amines [2527]. The underlying
mechanisms of shifts in microbial ecology contributing to
colonic tumourigenesis include dietary changes and subsequent shifts in metabolic expression. Specifically, there is an
epidemiological association between colorectal cancer and
raised sulfide production by sulfur-reducing bacteria (SRB)
such as Desulfovibrio vulgaris that is typically seen in meatrich Western diets [28]. Here, SRBs compete with methanogenic bacteria for hydrogen to produce hydrogen sulfide.
This is supported by evidence that demonstrates that sulfide
acts as an oxidation inhibitor of the SCFA n-butyrate in the
colonic epithelium. Butyrate is a potent histone deacetylase
inhibitor that has epigenic activity in colonocytes and microRNA-dependent p21 gene expression activity leading to
colonic cancer prevention [29] while also offering important
epithelial regulatory activities such as ion absorption, membrane lipid control, cellular detoxification and mucus formation. An association between 4-cresol and colon cancer
[30] has been reported. The production of 4-cresol is dependent upon intestinal environmental factors such as the
composition of the microbiota, food intake and pH of the
intestinal tract [31]. 4-Cresol is synthesized from tyrosine
and phenylalanine via 4-hydroxylphenylacetate by gut
microbiota. Clostridium difficile and certain Lactobacillus
strains are known to produce p-cresol by decarboxylation of
4-hydroxyphenylacetate [32,33]. Subsequently, 4-cresol is
excreted in the form of 4-cresyl glucuronide and 4-cresyl
sulfate in urine [34] through glucuronidation and sulfation.
Marked changes in the urinary composition of gut microbial metabolites have been found to be characteristic of
several other cancers. For example, quinolinate, 4-hydroxybenzoate and gentisate concentrations were higher in
kidney cancer patients whereas 3-hydroxyphenylacetate
and 4-hydroxybenzoate were anticorrelated with renal cancer [35]. Urinary hippurate, 4-hydroxyphenylacetate and
formate, among other metabolites, have been reported to
differentiate ovarian cancer from breast cancer patients and
healthy controls [36], and urinary hippurate was found to be
one of the strongest discriminators of lung cancer [37] and
has also been found in relatively high concentrations in
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OBESITY
Steatosis

Metabolic syndrome
risk factors

Adipokines

Sex steroids

Inflammation
Free fatty acids

Gut
microbiome

Insulin resistance

Oxidative
stress

Lipid
peroxidation

Inflammatory
cytokines

Metabolic
surgery
(BRAVE
Effects)

Sex steroid
receptor

Bile acid
metabolites

Insulin

Sex hormone
binding globulin

Insulin & IGF-1

receptor

Diabetes, Metabolic Syndrome,Cardiovascular Disease & Cancer

TRENDS in Microbiology

Figure 1. Schematic of the gut microbial contribution to obesity and related diseases.

tumour tissue itself [38]. A GC-MS analysis of urine from

patients with osteosarcoma also identified that gut microbial metabolites, in particular putrescine, hippurate and 4hydroxyphenylpropionate, comprised part of the metabolic
signature of the disease [37]. Further evidence of the contribution of the microbiota to cancer is the finding that one of
the most discriminatory metabolites in a study of 50 breast
cancer patients and matched controls was 4-hydroxyphenylacetate [39], a microbial product of tyrosine degradation.
Adding to the challenge of identifying biomarkers of
specific cancers and elucidating potential aetiological roles
of the gut microbiota is the impact of diet on both the
microbiome composition and the disease. For example, the
Western diet alters the colonic proportions of bile acids to
increase the proportion of primary bile acids transformed
to secondary bile acids by intestinal bacteria. Deoxycholic
acid (one of the secondary bile acids) is associated with
several models of carcinogenesis and correlated with the
enzymatic activity of 7a-dehydroxylating bacteria [40].
Several species of Clostridium demonstrate high and low
7a-dehydroxylase activity, which could represent a novel
target for modifying GI cancer risk. The central role of the
gut microbiota in cancer and obesity is illustrated in
Figure 1. Therefore, in addition to the direct relationship
of particular microorganisms with specific cancers, such as
H. pylori with gastric cancer, the gut microbiota also
contribute indirectly via cholesterol and lipid metabolism
with growing evidence that obesity is associated with
cancer risk and poorer prognosis.
Diabetes
The global epidemic of obesity is associated with a dramatic increase in the prevalence of type 2 diabetes mellitus
(T2DM). T2DM is characterized by insulin resistance
354

whereas type 1 diabetes mellitus is characterized by a loss

of insulin-producing beta cells in the pancreatic islets of
Langerhans, which results in insulin deficiency. The notion
that gut microbiota are key players in the onset and
development of diabetes is becoming more widely accepted
as the evidence base grows. Experimental evidence has
shown that the non-obese diabetic (NOD) mouse lacking
the innate microbial-recognition immune system receptor
MyD88 is resistant to type 1 diabetes [41]. If MyD88
knockout mice were depleted of normal intestinal flora,
type 1 diabetes would ensue, whereas if NOD mice were
colonized with the altered Schaedler flora (ASF), the diabetes would be attenuated [41]. This alluded to the role of
gut microbiota and the innate immune receptor MyD88 in
priming the immune system in the context of type 1
diabetes autoimmunity. As type 2 diabetes is strongly
associated with obesity, these two conditions could be
linked by an underlying physiological process including
the disordered regulation of gut bacterial profiles.
From the metabolic profiling literature, several studies
have reported modulated choline degradation products
and bile acids as characteristic of insulin resistance and
diabetes in animal models. Studies involving high fat dietinduced initiation of insulin resistance in animal models
found alterations in the plasma lipids and in the concentrations of urinary metabolites such as phenylacetate,
hydroxyphenylacetylglycine and hydroxyindoleacetic acid,
although host species and strain influenced the exact
metabolic composition of the biofluids in these animal
models [4245]. Low urinary concentrations of hippurate
and higher concentrations of dimethylamine have been
found in Zucker obese and Goto-kakizaki rats in comparison with the respective control strains [43,46]. A streptozotocin-induced rat model of diabetes reported acetate,

Review
ethanol and lactate among the key discriminatory metabolites in addition to a modulated plasma lipid profile [44].
NMR spectroscopy was applied to characterize the plasma
profiles of congenic strains of rats derived from crossing a
diabetic and control animal. Quantitative trait loci were
used to generate a correlation matrix with the plasma
metabolite profiles and linkage to benzoate was found to
be a consequence of deletion of a uridine diphosphate
glucuronosyltransferase [47]. The ability of the microbiota
to influence the expression of diabetes in animal models
and the clear impact upon the choline degradation pathway in diabetic animals and man emphasizes the potential
of the microbes to contribute to disease aetiology and
expression.
Obesity
Obesity has traditionally been considered to be a disorder
of energetic and nutritional surplus, which in some cases is
associated with a genetic predisposition. Recently, however, the evidence for the role of the gut microbiome has
offered new insight into aspects of our understanding of
obesity pathogenesis. These include the association of gut
microbiota with the following: intestinal permeability,
systemic quantity of adipose tissue and body weight.
The initial association between gut bacterial species and
weight gain was derived from studies where decreasing
dietary fibre intake resulted in excess body weight and
diabetes, and was hypothesized to result from a change in
gut microbiota as a consequence of altered nutrient supply
and digestion. Subsequently, it has been demonstrated
that consumption of a high fat diet results in a decrease
of total gut bacterial levels and an increase in Gramnegative bacteria. Four bacterial mechanisms have been
identified to result in excess bodily energy gain: (i) microbiota increase energy bioavailability by transforming increased proportions of non-digestible food into
biochemically absorbable nutrients; (ii) the influence of
intrinsic bacterial metabolism to generate and raise systemic levels of SCFAs to activate triglyceride synthesis;
(iii) high fat diets can result in a responsive bacterial
metabolism resulting in pathology (such as microbial conversion of choline to methylamines leading to a choline
deficient state, which induces liver disease); and (iv) the
ability of the microbiome in regulating gut gene expression
to favour an obese state. This could occur through the
reduction of lipoprotein lipase activity through the inhibition of angiopoietin-like 4 (Angptl4) and fasting-induced
adipocyte factor (FIAF) to increase free fatty acids and
adipose levels [48,49].
Experimental evidence has revealed that germ-free mice
are less obese than normal controls but gain weight and
have decreased Angptl4 expression following colonization
by conventional gut bacteria [1]. The ob/ob leptin-deficient
obese mouse has a microbiome with a 50% reduction in
Bacteriodetes and a concurrent increase in Firmicutes,
which might be associated with increased food consumption
in these animals. Obese humans also demonstrate an alteration of the Firmicutes to Bacteroidetes ratio that can be
altered by weight loss [50,51]. Furthermore, bacterial modulation of obesity is also suggested by the transmission of
obesogenic bacterial profiles in ex-germ-free mice [52]. The

Trends in Microbiology July 2011, Vol. 19, No. 7

fact that consistent differences have been noted in the

metabolic profiles of animals and humans following caloric
restriction also concurs with the notion that body weight and
weight change is associated with alteration in microbial
composition. Among these microbial signature changes include increased urinary excretion of hippurate, 4-hydroxyphenylacetic acid, phenylacetylglycine and decreased
acetate and lactate [53,54].
Bariatric surgery offers the most consistent method of
weight loss in morbidly obese patients, and also achieves
the resolution of metabolic dysfunction (including the resolution of T2DM). Bariatric surgery procedures modify the
gut microbiome to achieve a low inflammatory and weight
loss profile that might reverse the effects of the metabolic
syndrome [55]. Work on a bariatric animal model has
revealed that these operations work through the BRAVE
effects: (i) bile flow alteration, (ii) reduction of gastric size,
(iii) anatomical gut rearrangement and altered flow of
nutrients, (iv) vagal manipulation and (v) enteric gut
hormone modulation [56]. Furthermore, substantial shifts
of the main gut phyla following metabolic surgery (in a
rodent animal model) towards higher levels of Proteobacteria and lower levels of both Firmicutes and Bacteroidetes
have been demonstrated as compared to controls [57]. The
microbial effects of successful weight loss and anti-diabetic
operations could lead to novel mechanisms and therapies
for obesity and T2DM. In a recent study by Mutch et al.
[58], where weight loss was achieved in obese subjects
following Roux-en-Y gastric bypass surgery, marked
alterations in the plasma profile were found with many
classes of metabolites changing post surgery. In addition to
lipids, indoles, fatty acids and amino acids, 4-cresyl sulfate
was characteristic of the obese profiles [58] and might
indicate an altered clostridial profile. To date, investigating microbial functions in obesity have been approached
via both weight gain (obese patients and murine models)
and weight loss (bariatric surgery in human and murine
models) with good consensus that body weight influences
or is influenced by the host microbiota. The exact nature of
this hostmicrobial interplay requires further mechanistic
investigation.
Cardiovascular dyslipidaemia and metabolic
endotoxaemia
Dyslipidaemia is a disorder of lipoprotein metabolism,
which results in a systemic increase of disease-inducing
blood lipids such as cholesterol, triglycerides and LDL
particles and a decrease in the levels of protective lipids
such as high-density lipoprotein (HDL) particles. A sustained high fat diet can induce dyslipidaemia that results
in a proinflammatory response including an increase of
Gram-negative bacterial outer membrane lipopolysaccharide (LPS) levels to result in a metabolic endotoxaemia
demonstrable in both rodents [59] and humans [60]. An
infusion of LPS into rodents caused clinical features of
metabolic syndrome such as weight gain, insulin resistance and hepatic lipid overload. Fat has been demonstrated to be a highly efficient transporter of LPS from the gut
lumen to the bloodstream and its effects can be delayed in
mice lacking the LPS-CD14 receptor [61]. Non-fatty diets
are associated with decreased levels of LPS and lipoprotein
355

Review
particles can buffer its proinflammatory effects. High fat
diets demonstrate a shift in gut bacterial ecology by increasing the Gram-negative:Gram-positive ratio whereas
an increased fibre intake has been demonstrated to reverse
these changes [59] and heralds a possible avenue to promote dietary therapy for the prevention of metabolic endotoxaemia and atherogenic dislipidaemia.
Neuropathology
The composition of the intestinal microbiota is extremely
relevant in neurogastroenterology, which deals with the
interactions of the central nervous system and the gut
(gutbrain axis). Several neuropathological diseases are
thought to be associated with the gut microbiota. Autism is
a disorder of neural development with impaired social
behaviour and often involves GI symptoms. Previous studies examined the faecal microbial profiles of autistic children, which indicated 10-fold higher numbers of
Clostridium spp. compared with healthy subjects [62].
Many species of Clostridium are known to produce neurotoxins, which could contribute to the autism spectrum.
Metabolic alterations in gut hostmicrobial cometabolism
including higher urinary levels of hippurate and phenylacetylglutamine and tryptophan/nicotinic acid metabolism
have been observed in autistic children [63]. The bile acids
have been shown to differ across various neurological
conditions. For example, glycocholate (GCA), glycodeoxycholate (GDCA) and glycochenodeoxycholate have been
shown to be altered in plasma profiles in Alzheimers
disease (AD) [64], whereas tauroursodeoxycholic has been
shown to modulate p53-mediated apoptosis in AD and to be
neuroprotective in Huntingtons disease [64]. Clearly, gut
microbiota not only exert a local effect on the GI tract but
also impact remote organs such as the brain through
chemical signalling. Further investigation of the gutbrain
axis may provide valuable mechanistic insight into a range
of neuropathological and developmental diseases.
Concluding remarks
Advances in technology in both metabolic profiling and
microbial phenotyping methods have improved our ability
to derive correlations between the microbial and metabolic
phenotypes, and mathematical modelling tools have been
developed to accommodate high density data such as those
generated by metabonomic and metagenomic methods.
New methods of integrating these -omics datasets have
been developed to extract correlations between specific
microbes and metabolites. Several methods ranging from
simple correlations to bidirectional partial least squares
approaches have been explored (Box 1), but more work is
needed both on the preprocessing and data modelling
components of this integration process. One limitation of
the technology at present is that most of the microbial
phenotyping tools, such as fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis
(DGGE) and 454 sequencing, map the content of the
microbes present without giving any indication of activity.
A much needed breakthrough is to further profile the
microbiotal metabolism by establishing which microbial
products are formed directly from specific substrates
(originating from the human diet or faecal mucins) using
356

Trends in Microbiology July 2011, Vol. 19, No. 7

stable-isotope labelling (U-13C glucose) approaches [65]. In

the disease areas summarized in this review and beyond,
there is scope for further elucidation of the role of the gut
microbiota and development of potential therapeutic targets based on the underpinning metabolic linkages or even
on the microbes themselves.
Although the fact that there is a strong relationship
between the mammalian host and its enteric microbiota,
which impacts upon health, cannot be disputed, much of
the literature is confusing and consensus on the exact
mechanisms by which microbes modulate disease processes has not yet been reached. For example the ratio of
Firmicutes:Bacteroidetes has been reported to differ in
obesity in several studies, whereas in other studies, no
modulation of this ratio has been found with weight gain or
loss. This may be in part due to the fact that the Grampositive and Gram-negative bacteria in the gut are independent and thus ratios of particular bacterial groups or
families may be uninformative. Sequencing analysis at a
more refined level may provide a better understanding of
the structure and function of the microbiota. A recent
international study has identified three robust clusters
of microbiota (known as enterotypes) that are not nation
or continent specific. Metagenomic reads from populations
at different geographical locations were mapped using
DNA sequence homology to 1511 reference genomes to
reveal that intestinal microbiota variation is generally
stratified, not continuous. This confirmed the existence
of a select series of hostmicrobial symbiotic relationships,
where certain genes are significantly correlated with age
and physiological characteristics such as body mass index.
These enterotypes can be applied to identify novel biomarkers or targets of disease [66].
To date, most of the research into the effect of microbial
metabolism on the host has been focused on diseases that
predominate in the Western world. One potential avenue
for exploiting the hostmicobiome metabolic crosstalk is to
use the knowledge of the mechanisms involved in weight
gain to promote thrifty bacteria in populations in developing countries where malnutrition is one of the biggest
clinical problems. Studies in humans and in animal models
of parasitic infection have indicated that a three-way
relationship exists between the host and its enteric microbiota and parasites, with each parasite causing specific
changes in the gut microbial contribution to the urine,
plasma and faecal metabolomes. Therefore, there is clear
potential for adapting the analytical strategies and knowledge gained from other disease areas and applying them to
promote health in developing countries.
Exploration of the gutbrain axis and the role of the
microbiota in modifying behaviour is also an exciting but
underdeveloped area of research. The possibility of understanding the metabolic basis of the link between the gut
and brain and, moreover, the ability to influence this
connection is tantalizing. For example, germ-free mice
have been shown to have lower neurotrophic factor expression levels and to mount a more severe elevation in corticosterone levels than specific pathogen free (SPF) mice.
Moreover, it was shown that this response could be reversed by colonization with Bifidobacterium infantis or
reconstitution with SPF faeces but potentiated by E. coli

Review
[67]. The potential for uncovering new bacterial targets or
dietary strategies for treating neurological aspects of such
diseases is enormous. Some specific probiotics such as
Lactobacillus farciminis have been demonstrated to have
an impact on spinal neuronal activation [68].
As the drive towards research consortia strengthens,
multidisciplinary teams with the capacity for combining
microbial phenotyping, metabolic profiling and clinical
expertise become a reality and should serve to develop
the current understanding of the metabolic language of
mammalianmicrobial communication. The benefits of
attaining this knowledge are clear. The gut microbiome
functions as a virtual organ and significantly extends the
metabolic capacity of the host. This transgenomic metabolism offers a new paradigm for developing novel therapies
for many diseases and has potential to beneficially impact
upon a range of acute and chronic pathologies.
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