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The Human Gut Microbiome and Body Metabolism:Implications for
Obesity and DiabetesSridevi Devaraj,1,2 Peera Hemarajata,1,2 and
James Versalovic1,2*
BACKGROUND: Obesity,metabolic syndrome, and type 2diabetes are
major public health challenges. Recently,interest has surged
regarding the possible role of theintestinal microbiota as
potential novel contributors tothe increased prevalence of these 3
disorders.
CONTENT: Recent advances inmicrobial DNA sequenc-ing
technologies have resulted in the widespread appli-cation of
whole-genome sequencing technologies formetagenomic DNA analysis of
complex ecosystemssuch as the human gut. Current evidence suggests
thatthe gut microbiota affect nutrient acquisition, energyharvest,
and a myriad of host metabolic pathways.
CONCLUSION: Advances in the Human MicrobiomeProject and human
metagenomics research will leadthe way toward a greater
understanding of the impor-tance and role of the gut microbiome in
metabolic dis-orders such as obesity, metabolic syndrome,
anddiabetes. 2013 American Association for Clinical Chemistry
Obesity, metabolic syndrome, and type 2 diabetes aremajor public
health challenges, affecting approxi-mately 26 million children and
adults in the US. Morethan 8% of the US population has diabetes, of
which17.9 million people have the metabolic syndrome (1 ).During
the past 20 years, obesity has dramatically in-creased in
prevalence in the US. More than 1 in 3 USadults (36%) are obese,
and approximately 12.5 mil-lion (17%) of children and adolescents
(age 219 years)are obese (2 ). In theUS in 2010 (2 ), all of the
states hada prevalence of obesity of over 20%. The heterogeneityof
these disorders has been demonstrated through bothanthropometric
and genetic studies. These metabolicdisorders are believed to be
caused by a combination ofgenetic susceptibilities and lifestyle
changes. Recently,interest has surged in the possible role of the
intestinal
microbiome as a potential contributor to the rapidlyincreased
prevalence of obesity (35). This review fo-cuses on recent advances
in the understanding of thegut microbiome and techniques to assess
the micro-biome and its relationship to human body
metabolism,obesity,metabolic syndrome, and type 2 diabetes (Fig.
1).
The Human GutMicrobiome: The Toolkit behindthe Science
The widespread application of 16S rRNA gene se-quencing for
detection of bacterial pathogens and mi-crobial ecology has
provided a robust technical plat-form for the evaluation of the
bacterial composition ofthe human microbiome. Sequencing of 2
primary tar-gets within bacterial 16S rRNA genes yielded
valuablecompositional data pertaining to the human fecal
mi-crobiome of 242 healthy adults (6, 7 ). In the HumanMicrobiome
Project, 18 different body sites were sam-pled and sequenced. Stool
specimens were the singlespecimen type used to study the
intestinalmicrobiome.Previously published studies demonstrated the
varia-tion in composition of the gut microbiome among lo-cations
within the gastrointestinal tract in differentmammalian species.
For example, 16S rRNA gene se-quencing has been deployed to study
the maturationof murine cecal microbiota, and these studies
dem-onstrated the existence of a large number of yet-unidentified
bacteria that inhabit the mammalian in-testine (6 ). Such
sequencing strategies, which are cul-ture independent, are
essential for determiningbacterial composition of the microbiome
and its rela-tive stability and diversity over time. Thus, it is
essentialto develop robust experimental models of the
humanmicrobiome to delineate important mechanistic pro-cesses in
the development of human disease states.
Advances in sequencing technologies have resultedin the
widespread application of whole-genome (WG)3
sequencing technologies for metagenomic DNA anal-
1 Department of Pathology and Immunology, Baylor College of
Medicine and2 Department of Pathology, Texas Childrens Hospital,
Houston, TX.
* Address correspondence to this author at: Department of
Pathology, TexasChildrens Hospital, 1102 Bates Ave., Suite 830,
Houston, TX 77030. Fax832-825-1165; e-mail [email protected].
Received September 4, 2012; accepted January 16, 2013.Previously
published online at DOI: 10.1373/clinchem.2012.187617
3 Nonstandard abbreviations: WG, whole genome; NMR, nuclear
magnetic res-onance; GC-TOFMS, gas chromatography TOF mass
spectrometry; SCFA, short-chain fatty acid; CAZymes,
carbohydrate-active enzymes; CLA, conjugatedlinoleic acid; GLP-1,
glucagon-like peptide 1; RYGB, Roux-en-Y gastric bypass;HILIC-HPLC,
hydrophilic interaction liquid chromatographyHPLC; TNF,
tumornecrosis factor; GABA, -amino butyric acid; Fiaf,
fasting-induced adipocytefactor; LPS, lipopolysaccharide; TLR,
Toll-like receptor; BB, biobreeding.
Clinical Chemistry 59:4617628 (2013) Review
617
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ysis of complex ecosystems such as the human intestine(7 ). WG
sequencing strategies provide microbial com-positional as well as
functional information. WG datacan be used to infer bacterial
composition, and thesedata yield information similar to that
generated by 16SrRNA gene sequencing. The genome sequences ofhighly
abundant species are well represented in a set ofrandom shotgun
reads, whereas less abundant speciesare represented by fewer
sequences generated in a next-generation sequencing run. This
relative richness per-mits the comprehensive measurement of the
composi-
tional responses of an ecosystem to dietary changes,drug
therapy, epigenetic alterations, and environmen-tal perturbations.
Alternatively, most genes (usuallyapproximately 2000 genes per
bacterium) in themicro-biome are sequenced so that metabolic and
other func-tional pathways can be evaluated in each
individualsmetagenome. FunctionalWG data provide opportuni-ties to
find out which metabolic pathways are affectedand how the
microbiome may contribute mechanisti-cally to health and disease
states. This technology cre-ates the formidable challenge of
managing vast data
Fig. 1. Hyperglycemia (HG) and increased free fatty acids (FFA),
which are hallmarks of obesity, metabolicsyndrome, and diabetes,
combined with a high-fat, highglycemic load diet, could result in
increased activation ofthe inflammasome complex as well as increase
the activation of macrophages via increased TLR activation
andnuclear factor B (NF-B) activation.
Increased metabolic endotoxemia may occur and activate the TLR4
pathway via the adapter protein, MyD88, leading to immunecell
activation and inflammation. Also, macrophages could infiltrate the
adipose tissue and activate mitogen-activated proteinkinases, such
as c-Jun aminoterminal kinase (JNK) and NF-B, resulting in
increased cross-talk and adipose-tissuederivedadipokines. A
hyperglycemic and high fat diet could also result in changes to the
gut microbiome by altering the content ofhistidine, glutamate,
SCFAs, and other factors and promote gut-barrier dysfunction and
conditions prevalent in obesity,metabolic syndrome, and diabetes by
altering the host response. All of these metabolic alterations that
result in increasedsystemic inflammation, macrophage activity, and
TLR activation contribute to the increased cardiometabolic burden
in obesity,diabetes, and metabolic syndrome.
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618 Clinical Chemistry 59:4 (2013)
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sets. Advances in next-generation DNA sequencingyielded 576.7 Gb
of microbial DNA sequence data,which were generated with an
Illumina genome an-alyzer (Illumina) from total DNA from the stool
sam-ples of 124 European adults (8 ). The relationship be-tween the
commensalmicrobiota that comprise the gutmicrobiota and those that
are in the intestinal barrier iscomplex and differs spatially
throughout different ar-eas of the gastrointestinal tract. Fecal
metagenomicsmeasures ecosystem changes in stool or the distal
intes-tine, but it does not compare the microbiomes in dif-ferent
regions of the intestine. It is also important tonote that
metagenomic analysis of fecal samples doesnot include all important
molecular interactionswithin the gastrointestinal tract. Turnbaugh
et al. haveproposed the idea of a core set of functions within
themicrobiome, and the tools of proteomics and metabo-lomics may be
required for more in-depth functionalanalyses (7, 9 ). From a
systems perspective, meta-genomic analyses may provide further
details on spe-cific intraindividual changes and thus have major
im-plications for personalized medicine strategies.
Metatranscriptomics, metaproteomics, and meta-bonomics will be
useful to explore the functional as-pects of the gut microbiome
from the top down. Real-time analysis of the intestinal microbiome
is a usefultool in the development of personalized approaches
totargeted therapies. Metabonomics can be described asthe study of
metabolic responses to chemicals, the en-vironment, and diseases
and involves the computa-tional analysis of spectral metabolic data
that provideinformation on temporal changes to specific
metabo-lites. In addition, metabonomics provides global met-abolic
profiling of an individual in real time. It is pos-sible, with such
approaches, to elucidate complexpathways and networks that are
altered in specific dis-ease states. The combination ofmetabolic
profiling andmetagenomic studies of gut microbiota permits thestudy
of host andmicrobial metabolism in great detail.Such analysis of
functional components of the micro-biome that affect metabolism and
human health is re-ferred to as functional metagenomics.
Metagenomics and the science of the human mi-crobiome have
arrived at the forefront of biology pri-marily because of major
technical and conceptual de-velopments. The major technical
development was thedeployment in many centers of next-generation
DNAsequencing technologies with greatly enhanced capa-bilities for
sequencing collections of microbial ge-nomes in the metagenome.
Technological advanceshave created newopportunities for the pursuit
of large-scale sequencing projects that were difficult to imaginea
decade ago. The key conceptual development was theemerging paradigm
of the essential nature of complexmicrobial communities and their
importance to mam-
malian biology andhumanhealth anddisease. TheHu-manMicrobiome
Project was approved inMay 2007 as1 of 2 major components (in
addition to the humanepigenomics program) of NIH RoadMap version
1.5(now known as the Common Fund). Recently, 2 sem-inal reports
from theHumanMicrobiome Project con-sortium (10, 11) described
investigations in which apopulation of 242 healthy adults were
sampled at 15 or18 body sites up to 3 times, 5177 microbial
taxonomicprofiles were generated from 16S rRNA genes, andmore than
3.5 T bases of metagenomic sequences weregenerated. In addition, in
parallel, the Human Micro-biome Project consortium has sequenced
approxi-mately 800 human-associated reference genomes. Thisresource
will provide a framework for future studies ofdisease states and a
reference collection of healthy humanmicrobiome data. The data set
will enable future investi-gations into the epidemiology and
ecology of the humanmicrobiome invariousdisease states, and
treatment strat-egies will evolve from these studies. Using
compositionaland functional approaches, the relationships
betweenpathological variations in the gutmicrobiome and
severaldisease states have been delineated.
Urine metabolomics provides an opportunity forstudies of the
microbiomes impact on whole-bodymetabolism. The advantages of using
urinary samplesinclude relatively large sample volumes and the
conve-nience of noninvasive collection. In addition, urinesamples
can be used for the investigation of the chro-nology of metabolic
changes and thus are a valuabletool for investigations related to
the pathogenesis orprogression of disease and for screening and
diagnosisas well as prognostic evaluation. The methods com-monly
used for metabolic profiling of urine includeprocedures such as
nuclear magnetic resonance(NMR) spectroscopy, LC-MS, GC-MS, and gas
chro-matography TOFmass spectrometry (GC-TOFMS). Ina recent seminal
report, theNicholson group describeda method for urine collection
and storage that empha-sizes the importance ofmidstreamurine
collection andthe addition of urease before the freezing of urine
sam-ples. This method will eventually be used for
metabolicprofiling. Before analyses by GC-MSbased tech-niques,
urease activity is terminated with ethanol ormethanol and then
derivatized by subjecting the sam-ple to oximation followed by
trimethylsilyl derivatiza-tion (12). Because of the various sample
preparationsteps, it is important to use biological QC samples
andcheck the validity of the data that are obtained fromGC-MSbased
techniques that use principal compo-nent analysis. GC-MSbased
metabolomic studies in-clude several steps such as baseline
correction, noisereduction, deconvolution, peak area calculation,
andretention time alignment, and these steps help to gen-erate
consistent data. Several different commercially
The Human Gut Microbiome and Body Metabolism Review
Clinical Chemistry 59:4 (2013) 619
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available software programs can assist in such correc-tion
strategies before data analyses. Also, in urinarymetabolic
profiling, it is important to normalize data(on the basis of
volume, creatinine content, and othervariables) to obtain
meaningful information and min-imize the influence of dilution.
Using this protocol,investigators were able to undertake
high-throughputmetabolic profiling of approximately 400600
metab-olites in 120 urine samples per week. High-throughputanalyses
by NMR spectroscopy or MS are metabolic-profiling strategies that
are widely used to provideglobal metabolic overviews of
humanmetabolism (1316). Coupled with computational multivariate
analy-sis, these methods provide a deeper understanding ofdisease
states and can lead to biomarker discovery. Thisapproach
facilitates the quantification of environmen-tal influences on the
host genome and human health.As part of large-scale clinical
studies, this analyticalstrategy has been successfully applied to
disease statessuch as hypertension (17), ischemic heart disease
(18),diabetes (19), and obesity (20).
Metabonomics can be quite challenging becausethe chemical space
associatedwith the endogenousme-tabolites is highly diversified,
and thus complete meta-bolic information for any sample is hard to
deciphercompletely. Common analytical technologies used
inmetabolomics and metabonomics include NMR spec-troscopy, LC-MS,
and GC-MS, as well as GC-TOFMS.These different analytical
techniques have their ownstrengths and weaknesses and are usually
used in anintegrated fashion such that each of these
analyticalplatforms can provide complementary data; the selec-tion
of particular analytical techniques depends on thestudy questions
that are being posed. NMR has the ad-vantage of being rapid,
nondestructive to samples, andapplicable to intact biomaterials
rich in chemical struc-tural information. NMR requires minimal
samplepreparation and can be used to investigate a mixture ofor
several different metabolites in a single sample.However, MS-based
strategies have the advantages ofincreased sensitivity, accuracy,
precision, and repro-ducibility compared to NMR. Furthermore, the
cou-pling of GC to TOFMS offers several additional advan-tages such
as reduced analysis time and greateraccuracy with respect to peak
deconvolution.
The GutMicrobiome: Beyond Composition toFunction
andMetabolism
The gut microbial community includes approximately1014 bacteria
that normally reside in the gastrointesti-nal tract, reaching a
microbial cell number that greatlyexceeds the number of human cells
of the body. Thecollective genomeof thesemicroorganisms
(themicro-biome) containsmillions of genes (a rapidly expanding
number) compared to roughly 20 00025 000 genes inthe human
genome. This microbial factory contrib-utes to a broad range of
biochemical and metabolicfunctions that the human body could not
otherwiseperform (21). Although diet-induced changes in
gutmicrobiota occur within a short time frame (134days after a diet
switch), the changes are readily revers-ible (22, 23). In animal
models, the ratio of the mostprominent intestinal bacterial phyla,
the Bacteroidetesand Firmicutes, is altered in response to
dietarychanges (22, 23). Disruption of the energy equilibriumleads
to weight gain. Mouse model studies have dem-onstrated the
relationship between energy equilibrium,diet, and the composition
of the gut microbiome.Transplantation of the gut microbiota from
obese do-nors resulted in increased adiposity in recipients
com-pared to a similar transfer from lean donors.
Recent evidence suggests that the gut microbiotaaffect nutrient
acquisition, energy harvest, and a myr-iad of host metabolic
pathways (24). Recent findingsraise the possibility that the gut
microbiota has an im-portant role in regulating weight and may be
partlyresponsible for the development of obesity. Initial evi-dence
of the relationship between obesity and gut mi-crobial composition
was reported 3 decades ago, whensurgically induced weight loss
through gastric bypasssurgery and weight gain through lesions of
the ventro-medial hypothalamic nucleus were found to be associ-ated
with changes in gut microbial ecology (25, 26).These earlier
studies used culture-dependent methods,which detect a minority of
microbes harbored in thegut. In recent years, the ability to obtain
a thoroughpicture of gut microbial communities has improved bythe
introduction of molecular, culture-independenttechniques based on
ribosomal 16S rRNA gene se-quencing. Jumpertz et al. (27) performed
an inpatientenergy balance study in 12 lean and 9 obese
individualsas they consumed 2 calorically distinct diets for
briefperiods of time, and these investigators
simultaneouslymonitored the gut microbiota by performing
pyrose-quencing studies of bacterial 16S rRNA genes presentin feces
and bymeasuring ingested and stool calories bybomb calorimetry.
This study showed that altered nu-trient load (i.e., high calories
vs low calories) inducedrapid changes in the bacterial composition
of the hu-man gut microbiota, and these changes correlated wellwith
stool energy loss in lean individuals. Increasedproportions of
Firmicutes and corresponding reduc-tions in Bacteroidetes taxa were
associated with an in-creased energy harvest of approximately 150
kcal.These data point to a strong link between gut micro-biome
composition and nutrient absorption in hu-mans, and such studies
need to be confirmed withlarger numbers of study participants.
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620 Clinical Chemistry 59:4 (2013)
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The gut microbiome is very important in main-taining both
gastrointestinal and immune function aswell as being crucial for
the digestion of nutrients, andthis notion has been confirmed by
studies of germ-freemice (2830). Important metabolic functions of
thegut microbiome include the catabolism of dietary tox-ins and
carcinogens, synthesis of micronutrients, fer-mentation of
indigestible food substances, and assist-ing in the absorption of
electrolytes and minerals. Inaddition, the production of
short-chain fatty acids(SCFAs) by the gutmicrobiome affects growth
and dif-ferentiation of enterocytes and colonocytes. Differ-ences
in the metabolic activities of the gut microbiomemay contribute to
variation in caloric extraction fromingested dietary substances,
storage of calories in adi-pose tissue, and energy availability for
microbial pro-liferation. Such differences in the gut microbiome
arealso responsible for the variation in the ability of
anindividuals capacity to harvest energy, which may ex-plain
aspects of obesity. Differences in gut microbialcomposition and its
metabolic efficiency may be re-sponsible for the predisposition of
an individual tometabolic disorders such as obesity and diabetes
(31).
The gut microbiome can affect whole-body me-tabolism and alter
physiological parameters inmultiplebody compartments (32). In one
study (33), gnotobi-otic mice had increased quantities of
phosphocholineand glycine in the liver and increased quantities of
bileacids in the intestine. The gut microbiome also influ-ences
kidney homeostasis by modulating quantities ofkey cell-volume
regulators such as betaine and choline(33). A more recent study
showed specific differencesin the patterns of bile acids present
and reduced overallbile acid diversity in germ-free vs conventional
rats(34). Compared to conventional rats, germ-free ratshave
increased concentrations of conjugated bile acidsthat can
accumulate in the liver and the heart.
The GutMicrobiome and CarbohydrateMetabolism
Carbohydrates are an important nutritional compo-nent for
mammals and the mammalian microbiome,including the gut microbiota.
Mammals absorb simplesugars, including galactose and glucose, in
the proxi-mal jejunum via specific sugar transporters. Mamma-lian
enzymes hydrolyze disaccharides (sucrose, lactose,maltose) and
starches to constituentmonosaccharides,but have limited abilities
to hydrolyze other polysac-charides. As a consequence, every day a
bulk of undi-gested plant polysaccharides (cellulose, xylan, and
pec-tin) and partially digested starch reaches microbialcommunities
in the distal gut. By hostingmetabolicallyactive microbiota capable
of hydrolyzing complex car-bohydrates, mammals avoid the need to
evolve com-
plex enzymes that are required to break down the vari-ety of
polysaccharides in the diet. Microbes, bycontrast, contain many
genes encoding a variety ofcarbohydrate-active enzymes (CAZymes) in
the hu-man microbiome (35). Microbial CAzymes that con-stitute the
mammalian host repertoire include glyco-side hydrolases,
carbohydrate esterases, glycosyltransferases, and polysaccharide
lyases (35). Microbesgain access to abundant readily fermentable
carbonsources that would otherwise be wasted by the host andmay use
these complex carbohydrate substrates to sus-tain viable,
functionally robustmicrobial communitiesand generate bioactive
signals that affect mammalianmetabolism.
Intestinal bacterial taxa differ with respect to theirabilities
to utilize dietary and host-derived carbohy-drates (e.g., mucus
components) (23, 36). Bacterio-detes (23, 36) also have been
demonstrated to easilyassimilate dietary carbohydrates, because
members ofthis bacterial phylum possess several carbohydrate
uti-lization pathways. However, in situations of
dietarycarbohydrate starvation, gut bacteria catabolize mu-cins in
the gastrointestinal tract as a carbohydratesource, thereby
potentially compromising the mucuslayer adjacent to the epithelium.
In addition to Bacte-roides, strains of the genus Bifidobacterium
containgenes encoding glycan-foraging enzymes that enablethese gut
bacteria to acquire nutrients from host-derived glycans (37).
Besides their capacity to hydro-lyze starch, gut microbes have
developed the ability todegrade numerous plant and host-derived
glycoconju-gates (glycans) and glycosaminoglycans including
cel-lulose, chondroitin sulfate, hyaluronic acid, mucins,and
heparin. Microbial catabolic enzymes such as en-doglycosidases may
act on dietary substrates to releasecomplex N-glycans from human
milk and other dairysources (38). Fluctuations in diet may have
functionalconsequences for bacteria and the host so that the
can-nibalization of indigenousmammalian carbohydratesmay result in
augmentation of beneficial features, pre-vention of diseases, or
predisposition to different dis-ease states. For example,
bifidobacteria grown on hu-man milk oligosaccharides stabilize
tight junctionformation in the epithelium and promote the
secretionof the antiinflammatory cytokine, interleukin-10 (39).The
biogeography of the microbiome may be relevantbecause specific
genes/pathways such as simple carbo-hydrate transport
phosphotransferase systems aremore prominent in the small intestine
than in the colon(40). Probing into pathways which are affected by
al-terations in the gut microbiome (e.g., carbohydratestorage and
utilization) will yield new knowledge onthe role of
human-associated microbes in the develop-ment of several metabolic
disorders in humans.
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The GutMicrobiome and Fatty AcidMetabolism
Intestinal bacteria including probiotics produce a di-verse
array of fatty acids that may have health-promoting effects.
Intestinal bifidobacteria produceconjugated linoleic acid (CLA),
and CLA appears tomodulate the fatty acid composition in the liver
andadipose tissue in murine models (41). In addition toconjugated
and free fatty acids, intestinal bacteria gen-erate SCFAs (i.e.,
acetate, butyrate, propionate) by fer-menting dietary carbohydrates
(fiber) that humanscannot digest themselves. A recent study showed
thatgerm-freemice are devoid of SCFAs, indicating the im-portance
of the gutmicrobiota for SCFA production inthe intestine (42).
Acetate is the dominant SCFA typein humans, and this SCFA appears
to play an intriguingrole in the modulation of 5AMP-activated
protein ki-nase activity and macrophage infiltration in
adiposetissue (43). SCFAs such as propionate can be used forde novo
glucose or lipid synthesis and serve as an en-ergy source for the
host.
SCFAs may function as microbe-derived signalsthat influence
carbohydrate metabolism and gut phys-iology by stimulating
mammalian peptide secretionand serving as energy sources for gut
epithelial cells.SCFAs can stimulate glucagon-like peptide 1
(GLP-1)secretion via the G-proteincoupled receptor FFAR2(free fatty
acid receptor 2) in the colonic mucosa (44).By stimulating GLP-1
secretion, bacterial SCFAs pro-vide signals that suppress glucagon
secretion, induceglucose-dependent insulin secretion, and promote
glu-cose homeostasis. An enteroendocrinological pathwayis proposed
in which SCFAs stimulate the secretion ofpeptide YY, a hormone that
is released by ileal andcolonic epithelial cells in response to
feeding and seemsto suppress the appetite (45). High-fat diets
supple-mented with butyrate prevented and reversed
insulinresistance in dietary-obese mice. At the same
time,butyrate-producing bacteria and fecal butyrate con-centrations
decline with diets containing reducedamounts of specific
carbohydrates (46). The SCFApropionate modulates energy homeostasis
by promot-ing GPR41 (G protein-coupled receptor
41)-mediatedactivation of sympathetic neurons, in contrast to
ke-tone bodies (47). The ability to modulate sympatheticoutflow
provides another mechanism linking the gutmicrobiome to the enteric
nervous system, energy ex-penditure, and metabolic homeostasis.
Roux-en-Y gastric bypass (RYGB) surgery is ama-jor bariatric
intervention to treat morbid obesity. Be-fore surgery, increased
quantities of Bacteroidetes wereobserved, but reductions in
Bacteroidetes and en-hanced quantities of Proteobacteria were
detected fol-lowing surgery (48). These microbial population
shiftslikely change the metabolite profiles and relative pre-
ponderance of different fatty acids, including SCFAs.These
results are supported by a recent animal study.Nonobese rats with
RYGB had decreased amounts ofFirmicutes and Bacteroidetes and
significantly in-creased amounts (52-fold higher concentrations)
ofProteobacteria compared with sham-operated rats(49). Obesity is a
proinflammatory state. It wasshown that the abundance of
butyrate-producingFaecalibacterium prausnitzii species is
negatively as-sociated with biomarkers of inflammation beforeand
after RYGB, indicating that this bacterial speciesmay contribute to
maintaining a healthy gut (48 ).Thus, surgical interventions in the
gastrointestinaltract may have profound effects on gut
microbialcomposition, SCFA production, and the mamma-lian immune
system.
Interestingly, a recent study (50) has demon-strated that
subtherapeutic administration of antibiot-ics alters the population
structure of the gut micro-biome as well as itsmetabolic
capabilities. In this study,investigators administered
subtherapeutic doses of an-tibiotics to youngmice, resulting in
increased adiposityin young mice and increased levels of the
incretinGIP-1. In addition, these investigators observed
sub-stantial taxonomic changes in the microbiome (in-creased
Lachnospiraceae and Firmicutes and decreasedBacteroidetes), changes
in key genes involved in themetabolism of carbohydrates to SCFAs
(increased lev-els of acetate, propionate, and butyrate), increases
incolonic SCFA levels, and alterations in the regulation ofhepatic
metabolism of lipids and cholesterol. Thus,modulation of murine
metabolic homeostasis can beachieved by altering the gut microbiota
through anti-biotic manipulation.
The GutMicrobiome and Amino AcidMetabolism
Beneficial microbes such as bifidobacteria and lactoba-cilli
produce biologically active compounds derivedfrom amino acids,
including a variety of biogenicamines.Dietary components include
proteins and pep-tides thatmay be hydrolyzed to amino acids by
luminalproteinases and peptidases. Amino acids derived fromdietary
protein sources may serve as substrates for lu-minal bioconversion
by the gut microbiome. Diversemicrobial enzymes may contribute to
mammalianamino acidmetabolism by generating bioactivemetab-olites
in the intestine. One such class of enzymes,amino acid
decarboxylases, is widely prevalent in gutmicrobes, and these
microbial enzymes, when com-bined with amino acid transport
systems, link dietarycompounds with microbial metabolism and
signalingwith the gut mucosa (Fig. 2).
Combinations of metabolomics strategies, includ-ing MS, HPLC,
and NMR, are leading to discoveries of
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622 Clinical Chemistry 59:4 (2013)
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metabolites and small compounds derived from
thehumanmicrobiome.With the use of hydrophilic inter-action liquid
chromatographyHPLC (HILIC-HPLC),bioactive molecules derived from
gut microbes wereisolated as antiinflammatory, tumor necrosis
factor(TNF)-inhibitory compounds and HILIC-HPLC frac-tions were
analyzed by MS and NMR. One such mi-crobial signal and biogenic
amine, histamine, wasidentified and quantified in TNF-inhibitory
HILIC-HPLC fractions derived from Lactobacillus reuterifound in
breast milk and the gut (51 ). Histamine isproduced from
L-histidine via histidine decarboxyl-ase, which is present in some
fermentative bacteriaincluding probiotic lactobacilli. One
constituent ofthe gut microbiome, L. reuteri, is able to convert
adietary component, L-histidine, into an immuno-regulatory signal,
histamine, which suppresses pro-inflammatory TNF production via
histamine type 2receptors in the intestinal epithelium. Other
exam-ples of microbe-facilitated amino acid metabolisminclude the
generation of -amino butyric acid(GABA) from glutamate via
glutamate decarboxyl-ase (52 ) and the production of putrescine
fromornithine. The identification of these bacterial bio-active
metabolites and their corresponding mecha-nisms of action with
respect to immunomodulationmay lead to improved antiinflammatory
strategiesfor chronic immune-mediated diseases. Such
antiin-flammatory amino acid metabolites may amelioratepathologic
processes in obesity and diabetes.
The GutMicrobiome and BodyMetabolism:Obesity and
Inflammation
The incidence of overweight and obesity has reachedepidemic
proportions. Data reported by the CDC andthe National Health and
Nutrition Examination Sur-vey indicated that, in 2008, an estimated
1.5 billionadults were overweight, and more than 200 millionmen and
almost 300 million women were obese bythese criteria. Worldwide
obesity has more than dou-bled in the last 2 decades. Obesity is
associated with acluster of metabolic and systemic disorders such
as in-sulin resistance, type 2 diabetes, fatty liver disease,
ath-erosclerosis, and hypertension. The major cause ofobesity is a
positive energetic balance resulting from anincreased energy intake
from the diet and a decreasedenergy output associated with low
physical activity. Inaddition to alterations in diet and physical
activity re-sulting in obesity, genetic differences contribute
toobesity and cause differences in energy storage and ex-penditure.
Furthermore, growing evidence suggeststhat the gut microbiota
represents an important factorcontributing to the host response to
nutrients. A land-mark study byTurnbaugh et al. (53)was one of the
firststudies to show how the gene content in the gut micro-biota
contributes to obesity. The microbiomes ob-tained from the distal
gut of genetically obese leptin-deficientmice (ob/ob) and their
lean littermates (ob/and /) were compared. In this study,
investigatorsreported that the microbiota in the ob/ob mice
con-
Fig. 2. Intestinal microbes may play an important role in
host-microbiota interactions via luminal conversion.
Nutrients consumed by the host may be converted by intestinal
microbes into several bioactive compounds that could affectthe
health and disease states of the host and the intestinal
microbiota. SCFAs short-chain fatty acids. Reproduced
withpermission from Hemarajata et al. (78 ).
The Human Gut Microbiome and Body Metabolism Review
Clinical Chemistry 59:4 (2013) 623
-
tained genes encoding enzymes that hydrolyze indi-gestible
dietary polysaccharides. Increased amounts offermentation end
products (such as acetate and bu-tyrate) and decreased calories
were found in the feces ofobese mice. These data suggest that the
gut microbiotain this mouse model promoted the extraction of
addi-tional calories from the diet.
The composition of the gut microbiome seems tobe important in
regulating body weight (54). To dem-onstrate this point,
investigators conducted experi-ments in which they transplanted the
gutmicrobiota ofeither ob/ob mice or lean mice to lean
gnotobioticmice. After 2 weeks, mice that received microbiotafrom
the ob/ob mice were able to extract more caloriesfrom food and also
showed a significantly greater fatgain than mice that received the
microbiota from leanmice. Thus, differences in caloric extraction
of ingestedfood substances may be largely a result of the
compo-sition of the gut microbiota. These data support a piv-otal
role for the gut microbiome in the pathogenesis ofobesity and
obesity-related disorders.
Manipulation of the gutmicrobiotamay be an im-portant
therapeutic strategy to regulate energy balancein individuals who
are obese, diabetic, or have a diag-nosis of metabolic syndrome. In
genetically obese,ob/ob mice and their lean counterparts fed the
samepolysaccharide-rich diet, Ley et al. analyzed bacterial16S rRNA
gene sequences from the cecal microbiotaand reported that ob/ob
mice had 50% fewer Bacte-roidetes and correspondingly more
Firmicutes thantheir lean littermates and this difference was
unrelatedto differences in food consumption.
Backhed et al. (55) confirmed these findings andfound that
young, conventionally reared mice had a40% higher body fat content
and 47% higher gonadalfat content than germ-free mice, although
their foodconsumption was less than their germ-free counter-parts.
When the distal gut microbiota from young,conventionally-reared
mice were transplanted into thegnotobiotic mice, a 60% increase in
body fat within 2weeks was observed, without any increase in food
con-sumption or energy expenditure. This increase in bodyfat was
accompanied by increased insulin resistance,adipocyte hypertrophy,
and increased concentrationsof circulating leptin and glucose.
Mechanistic studiesdemonstrated that the microbiota promoted
absorp-tion of monosaccharides from the gut and induced he-patic
lipogenesis in the host. These responses werelargely mediated via
upregulation of 2 signaling pro-teins, ChREBP (carbohydrate
response element-binding protein) and liver SREBP-1 (sterol
responseelement-binding protein type-1). In addition, with
ge-netically modified fasting-induced adipocyte
factor(Fiaf)-knockoutmice, gutmicrobeswere also shown tosuppress
intestinal Fiaf (56).
Several studies have highlighted the pivotal role ofinflammation
in the metabolic processes leading to themetabolic syndrome,
obesity, and diabetes. Cani et al.(5759) postulated another
mechanism linking the in-testinal microbiota to the development of
obesity. Theauthors hypothesized that bacterial
lipopolysaccharide(LPS) derived from gram-negative bacteria
residing inthe gut microbiota may be the trigger for increased
in-flammation observed in high-fat dietinducedmetabolicsyndrome.
Ina seriesof experiments inmice fedahigh-fatdiet, the investigators
showed evidence of pronouncedendotoxemia, associated with
reductions in both gram-negative (Bacteroides-related bacteria) and
gram-positivebacteria (Eubacterium rectaleClostridium
coccoidesgroup and bifidobacteria), and an increased ratio
ofgram-negative to gram-positive bacteria. The authorsof this
report suggested that chronic metabolic endo-toxemia induces
obesity, insulin resistance, anddiabetes.
In human experiments, Ley et al. (60) and Ravus-sin et al. (61)
serially monitored the fecal gut microbi-ota in 12 obese
individuals who participated in aweight-loss program for a year by
following either afat-restricted or carbohydrate-restricted
low-caloriediet. Similar to experiments in mice, in humans a
rela-tive abundance ofmicrobiota that belonged to the
Bac-teroidetes and Firmicutes phyla was found, and themi-crobiota
showed remarkable intraindividual stabilityover time. Before the
initiation of the low-calorie diet, arelative abundance of
Firmicutes and decreasedamounts of Bacteroidetes were detected in
the obeseparticipants compared with the nonobese controls. Af-ter
weight loss, increased amounts of Bacteroidetes(3% to 15%) and a
decreased abundance of Firmicuteswere observed, and these changes
correlated with thepercentage of weight loss and not with changes
in di-etary caloric content. These human studies confirmedanimal
data suggesting that alterations in gutmicrobialcomposition are
associated with obesity. Cause and ef-fect relationships between
obesity and changes in thegut microbiota remain unclear. Kalliomki
et al., in aprospective study of children from birth to age 7
years(62), collected fecal specimens at 6 and 12 months ofage. This
report documented an abundance of Bifido-bacterium taxa and
decreased proportions of Staphylo-coccus aureus in children who
were whose weight waswithin reference intervals at age 7 years than
in thosewho were overweight or obese. Although they did notexamine
factors such as diet and physical activity, thesedata suggest that
differences in the composition of thegut microbiota precede
overweight and obesity status.Antibiotics have also been shown to
pervasively affectgut microbial composition. A 5-day course of
orallyadministered ciprofloxacin decreased substantially
thediversity of the fecalmicrobial community (63). In this
Review
624 Clinical Chemistry 59:4 (2013)
-
study, although most of the microbial community re-vived within
4 weeks after administration of cipro-floxacin, some other genera
failed to reappear even af-ter treatment with the antibiotic for 6
months (63).
The GutMicrobiome andMetabolism: Diabetes andtheMetabolic
Syndrome
Toll like receptors (TLRs) are pattern recognition re-ceptors
that are important in mediating inflammationand immunity. Increased
amounts of TLRs are presenton cell surfaces in patients with
obesity, diabetes, andmetabolic syndrome (63). Recently,
investigators haveexplored the role of the gut microbiome in
regulatingTLR-mediated insulin resistance. Mice deficient in
themicrobial pattern-recognition receptor TLR5 dis-played
hyperphagia, became obese, and developed fea-tures of the metabolic
syndrome, including hyperten-sion, hypercholesterolemia, and
insulin resistancesecondary to dysregulation of interleukin-1
signaling(43). When gut microbiomes from these mice
weretransplanted into germ-free mice with an intact toll-like
receptor 5 (TLR5) gene, recipient mice developedsimilar features of
themetabolic syndrome, which sug-gests that the intestinal
microbiome was the key deter-minant of this disease phenotype. In
another study,TLR2-deficient mice developed obesity, insulin
resis-tance, and glucose intolerance, and the gut micro-biomes of
TLR2-deficient mice had a greater abun-dance of Firmicutes and
fewer Actinobacteria of thegenus Bifidobacterium (64).
Administration of an anti-biotic cocktail eliminated many of the
Firmicutes andresulted in improved insulin activity and glucose
toler-ance. In addition to improved insulin activity and glu-cose
tolerance, because lower levels of Bifidobacteriumspp. contribute
to increased gut permeability, thischange in the gut microbiomemay
result in a leaky gutand yield increased concentrations of
endotoxins suchas LPS in the circulation. The immune system
recog-nizes LPS as a microbial pattern triggering TLR signal-ing
and causing inflammation. Both obesity and in-flammation tend to
cause diabetes, so the loss of TLR2in these mice leads to changes
in their gut bacteria,which result in a greater risk of diabetes
mellitus. In-deed, increased amounts of TLR2 have been observedon
monocytes of patients with metabolic syndrome,type 1 and type 2
diabetes compared with matchedcontrols (6572). TLR2 deficiency in
diabetic mice re-sults in decreased development of complications of
di-abetes such as diabetic nephropathy (68). Possibly, thegut
microbiome plays a crucial role in regulating dia-betic
vasculopathies, and this area will be an importantarea of future
investigation.
With regard to the role of the microbiome in met-abolic syndrome
and associated abnormalities, studies
in germ-free mice demonstrate that they are
protectedfromobesity, insulin resistance, dyslipidemia, and
fattyliver disease/nonalcoholic steatohepatitis when fed ahigh-fat
Western diet (69). In contrast, following thecolonization with
microbiota from conventionallyraisedmice, the body fat content in
the originally germ-free mice increased up to 60% in 14 days. This
wasassociated with increased insulin resistance, althoughthe food
intake was reduced. The metabolic syndromeaffects 1 in 3 US adults
and leads to an increased pro-pensity of diabetes and
cardiovascular disease (70). In asingle human study in patients
with the metabolic syn-drome, Zupancic et al. (71) studied Amish
men andwomen with varying body mass indices. In 310
studyparticipants, gut microbiota were characterized bydeep
pyrosequencing of bar-coded PCR ampliconsfrom the V1V3 region of
the 16S rRNA gene. Theywere able to identify 3 communities of
interacting bac-teria in the gut microbiota, analogous to
previouslyidentified gut enterotypes. Network analysis identified22
bacterial species and 4 operational taxonomic unitsthat were either
positively or inversely correlated withmetabolic syndrome traits,
suggesting that certainmembers of the gut microbiota can contribute
to themetabolic syndrome. It is important that future studiesfocus
on delineation of specific components of the gutmicrobiome that
contribute to visceral obesity, dysgly-cemia, dyslipidemia,
hypertension, and insulin resis-tance associated with this
population (71). Nonalco-holic fatty liver disease is the hepatic
manifestation ofmetabolic syndrome and the leading cause of
chronicliver disease in the Western world. Using differentmouse
models of inflammasome deficiency, such asmice deficient in Asc
(apoptosis-associated speck-likeprotein containing a caspase
recruitment domain),NLRP3 (nucleotide-binding domain, leucine rich
fam-ily, pyrin containing 3), caspase, or interleukin 18,
theauthors showed significant alterations in gut microbi-ota as
evidenced by increased members of Bacterio-detes and decreased
members of Firmicutes in thesemouse models. More severe hepatic
steatosis and in-flammation were found, as evidenced by
increasedTLRs (mainly TLR 4 and 9) and secretion of hepaticTNF-.
Importantly, the authors speculated that in-creased hepatic
steatosis was due to intestinal bacterialproducts acting as
agonists for TLR4 and 9 and enter-ing the liver via the portal
circulation (72). Further-more, these pathologic changes result in
exacerbationof hepatic steatosis and obesity (72). Thus, altered
in-teractions between the gut microbiota and the host,produced by
defective inflammasome sensing, maygovern the rate of progression
of multiple abnormali-ties associated with metabolic syndrome.
Intestinal microbiomes have also been studied inrelation to
insulin resistance in patients with type 2
The Human Gut Microbiome and Body Metabolism Review
Clinical Chemistry 59:4 (2013) 625
-
diabetes. Larsen et al. reported that there was a signifi-cant
reduction in the relative abundance of Firmicutesand Clostridia in
adults with type 2 diabetes when theyused the technique of deep
tag-encoded sequencing (73).Additionally, the ratios
ofBacteroidetes toFirmicutes andBacteroidesPrevotella toC.
coccoidesEubacterium rectalegroups were correlated with increased
fasting glucoselevels in these patients. In this study, Larsen et
al.showed that in addition to the decreased abundance ofFirmicutes,
the Betaproteobacteria levels were signifi-cantly increased in
diabetic patients compared to non-diabetic controls and their
abundance significantlycorrelated with their plasma glucose
concentrations(r 0.46, P 0.05) (73). Such findings are
intriguingand prompt questions regarding how microbial com-position
and correspondingmetabolites may influencewhole-body metabolism in
humans and contribute toinsulin resistance and diabetes.
Interestingly, the gut microbiome also regulatestype 1 diabetes.
Type 1 diabetes is an autoimmune dis-ease, which is due to the
specific destruction of theendocrine insulin-secreting pancreatic
cells resultingin an insulinopenic state. Type 1 diabetes also
predis-poses to microvascular and macrovascular complica-tions.
Data have emerged on the critical role of thegastrointestinal
microbiota in the protection or thetriggering of type 1 diabetes
(74). In 2 models of dia-betes, the NOD (nonobese diabetic) mouse
and thebiobreeding (BB) rat, the incidence of spontaneoustype 1
diabetesmellitus can be affected by themicrobialenvironment in the
animal-housing facility or by expo-sure tomicrobial stimuli (75).
Furthermore, the recog-nition of bacterial determinants from
intestinal micro-biota may trigger type 1 diabetes. TLRs are
innatepattern-recognition receptors involved in host defenseand
maintenance of tissue integrity. TLR signaling ismediated through
the adapter proteinMyD88, and thedeletion ofMyD88 protects from
atherosclerosis. Micelacking MyD88 were protected against insulitis
(74),and this phenomenon depends on commensal mi-crobes because
germ-free MyD88 knockout mice de-velop robust diabetes.
In BB rats (a model of type 1 diabetes), Lactobacil-lus species
present in feces (L. johnsonii and L. reuteri)were negatively
correlated with type 1 diabetes devel-opment (76), possibly via
modulation of the intestinalmucosal protein and oxidative stress
response leadingto lower quantities of proinflammatory cytokines
suchas interferon . Thus, therapeuticmodulation aimed ataltering
the gut microbiome may be beneficial in re-tarding the development
of diabetes.
Alterations in the gutmicrobiota contribute to thedevelopment of
autoimmune disorders such as type 1diabetes. Fecal samples were
obtained from 4 pairs ofmatched participants in a case control
study (77).
More than 30 billion nucleotide bases of Illumina
shot-gunmetagenomic data were analyzed, and the findingsrevealed
significantly increased proportions of path-ways and modules
involved in carbohydrate metabo-lism and stress responses in cases
compared to con-trols. Other differences included the relative
quantitiesof genes involved in adhesion, motility, and sulfur
me-tabolism, which were more abundant in cases, whereasgenes with
roles in DNA and protein metabolism,amino acid synthesis, and
aerobic respiration weremore abundant in controls. The 16S rRNA
data werealso mined for indications of changes in
microbialcomposition. At the phylum level, numbers of
Actino-bacteria, Bacteroidetes, and Proteobacteria were
signif-icantly increased in cases, whereas Firmicutes,
Fuso-bacteria, Tenericutes, and Verrucomicrobia werehigher in
controls (P 0.001). At the genus level, theabundance of Bacteroides
was much greater in cases,whereas Prevotella was much more abundant
in con-trols. Furthermore, the total number of bacteria thatproduce
lactic acid and butyrate was greater in controlsthan in cases.
Thus, these data suggest that such lactate-and butyrate-producing
bacteriamay be beneficial andmaintain a healthy gut and that
dysregulation of thesebacteria can lead to reduction in optimal
mucin syn-thesis, as identified in individuals with
autoimmunediseases, and contribute to development of type 1
dia-betes. These exciting findings need to be confirmed inlarger
study populations.
In conclusion, randomized clinical studies shouldhelp define the
features of the gut microbiome thatcontribute to obesity and
diabetes epidemics in definedpopulations. In addition, mechanistic
studies of thehuman microbiome will be instructive and have
ther-apeutic implications. Furthermore, advances in tech-nology,
such as 16S rRNA sequencing, WG metag-enomics, and metabolomics in
metabolic diseases willenable scientists to mine large data sets
for disease-contributing features. Large databases and
bioinfor-matics resources such as those derived from the Hu-man
Microbiome Project will lead the way toward agreater understanding
of the importance and role ofthe gut microbiome in metabolic
disorders such asobesity, metabolic syndrome, and diabetes, and
thesestudies may provide therapeutic strategies to reducethe
aggregate cardiometabolic burden in humanpopulations.
AuthorContributions:All authors confirmed they have contributed
tothe intellectual content of this paper and have met the following
3 re-quirements: (a) significant contributions to the conception
and design,acquisition of data, or analysis and interpretation of
data; (b) draftingor revising the article for intellectual content;
and (c) final approval ofthe published article.
Review
626 Clinical Chemistry 59:4 (2013)
-
Authors Disclosures or Potential Conflicts of
Interest:Uponman-uscript submission, all authors completed the
author disclosure form.Disclosures and/or potential conflicts of
interest:
Employment or Leadership:None declared.Consultant or Advisory
Role:None declared.Stock Ownership:None declared.
Honoraria:None declared.
Research Funding: NIH P30 DK56338-06A2; J. Versalovic, NIHUH3
DK083990, NIH R01 AT004326-01A1, and NIH R01DK065075-01.
Expert Testimony:None declared.Patents:None declared.
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Review
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