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Gut Microbiota and Its Possible Relationship With Obesity

      Obesity results from alterations in the body's regulation of energy intake, expenditure, and storage. Recent evidence, primarily from investigations in animal models, suggests that the gut microbiota affects nutrient acquisition and energy regulation. Its composition has also been shown to differ in lean vs obese animals and humans. In this article, we review the published evidence supporting the potential role of the gut microbiota in the development of obesity and explore the role that modifying the gut microbiota may play in its future treatment. Evidence suggests that the metabolic activities of the gut microbiota facilitate the extraction of calories from ingested dietary substances and help to store these calories in host adipose tissue for later use. Furthermore, the gut bacterial flora of obese mice and humans include fewer Bacteroidetes and correspondingly more Firmicutes than that of their lean counterparts, suggesting that differences in caloric extraction of ingested food substances may be due to the composition of the gut microbiota. Bacterial lipopolysaccharide derived from the intestinal microbiota may act as a triggering factor linking inflammation to high-fat diet-induced metabolic syndrome. Interactions among microorganisms in the gut appear to have an important role in host energy homeostasis, with hydrogen-oxidizing methanogens enhancing the metabolism of fermentative bacteria. Existing evidence warrants further investigation of the microbial ecology of the human gut and points to modification of the gut microbiota as one means to treat people who are overweight or obese.
      Fiaf (fasting-induced adipocyte factor), LPS (lipopolysaccharide), rRNA (ribosomal RNA)
      Obesity is a growing epidemic in many developed countries, including the United States, and is arousing increasing concern in developing countries, which have historically dealt with the burden of undernutrition.
      • Hensrud DD
      • Klein S
      Extreme obesity: a new medical crisis in the United States.
      The prevalence of obesity in adults has increased by more than 75% since 1980; currently, more than half of the US population is overweight, with nearly 1 in 3 adults being clinically obese.
      • Ogden CL
      • Yanovski SZ
      • Carroll MD
      • Flegal KM
      The epidemiology of obesity.
      Children are also increasingly overweight, suggesting that this epidemic will continue to worsen. Obesity is a major health problem because of its serious health consequences, including type 2 diabetes mellitus, cardiovascular diseases, pulmonary hypertension, obstructive sleep apnea, gastroesophageal reflux disease, musculoskeletal disorders, a variety of cancers, and a number of psychosocial concerns.
      • Hensrud DD
      • Klein S
      Extreme obesity: a new medical crisis in the United States.
      • Ogden CL
      • Yanovski SZ
      • Carroll MD
      • Flegal KM
      The epidemiology of obesity.
      Obesity has also been shown repeatedly to be associated with an increased risk of mortality.
      • Ogden CL
      • Yanovski SZ
      • Carroll MD
      • Flegal KM
      The epidemiology of obesity.
      The social and economic costs of obesity and its associated comorbidities are enormous and threaten to overwhelm an already overburdened health care system.
      • Ogden CL
      • Yanovski SZ
      • Carroll MD
      • Flegal KM
      The epidemiology of obesity.
      Obesity results from alterations in energy balance, ie, how the body regulates energy intake, expenditure, and storage. Because starvation poses a greater danger to an organism than overabundance, our biological systems are geared to better protect against weight loss than weight gain (ie, a thrifty genotype). Considerable effort has been made to improve the availability and stability of the food supply, resulting in an abundance of inexpensive, palatable, and energy-dense foods. Consequently, organisms adapted for a situation of insufficiency are now confronted with the easy availability of such foods.
      The physiologic processes that regulate weight and metabolism, including peripheral hunger and satiety signals, the central integration of this information, and the integrated gastrointestinal response to food intake, have received intense investigation, particularly during the past decade.
      • Korner J
      • Leibel RI
      To eat or not to eat—how the gut talks to the brain.
      • Huda MS
      • Wilding JP
      • Pinkney JH
      Gut peptides and the regulation of appetite.
      • Murphy KG
      • Dhillo WS
      • Bloom SR
      Gut peptides in the regulation of food intake and energy homeostasis.
      • Camilleri M
      Integrated upper gastrointestinal response to food intake.
      A person's weight and body composition are likely determined by interaction between his/her genetic makeup and social, cultural, behavioral, and environmental factors. Although energy intake has increased and physical activity has declined during the past few decades, these changes are difficult to quantify.
      • Hill JO
      Understanding and addressing the epidemic of obesity: an energy balance perspective.
      An increased intake of energy-dense foods, especially when combined with reduced physical activity, surely contributes to the high prevalence of obesity
      • Hill JO
      • Wyatt HR
      • Reed GW
      • Peters JC
      Obesity and the environment: where do we go from here?.
      ; however, the existence of complex systems that regulate energy balance requires that this paradigm be considered in a larger context.
      • Weigle DS
      Appetite and the regulation of body composition.
      • Morton GJ
      • Cummings DE
      • Baskin DG
      • Barsh GS
      • Schwartz MW
      Central nervous system control of food intake and body weight.
      Recent evidence suggests that the trillions of bacteria that normally reside within the human gastrointestinal tract, collectively referred to as the gut microbiota, affect nutrient acquisition and energy regulation; it further suggests that obese and lean people have different gut microbiota. These findings raise the possibility that the gut microbiota has an important role in regulating weight and may be partly responsible for the development of obesity in some people. This article examines the evidence supporting these claims and explores whether modifying the gut microbiota could one day be a treatment option for obesity.

      INDIGENOUS GUT MICROBIOTA

      Identification

      Until recently, our understanding of the human intestinal microbiota has been limited by reliance on conventional microbiological techniques (ie, selective culturing) and by our inability to culture many organisms in the gastrointestinal tract. With the development of methods for identifying gut microflora that do not require culturing (ie, molecular fingerprinting and ecological statistical approaches), a much more thorough and reliable assessment of the gut microbiota is now possible.
      • Gill SR
      • Pop M
      • DeBoy RT
      • et al.
      Metagenomic analysis of the human distal gut microbiome.
      • Palmer C
      • Bik EM
      • DiGiulio DB
      • Relman DA
      • Brown PO
      Development of the human infant intestinal microbiota [published online ahead of print June 26, 2007].
      • Eckburg PB
      • Bik EM
      • Bernstein CN
      • et al.
      Diversity of the human intestinal microbial flora.
      Specifically, the sequencing of 16S ribosomal RNA (rRNA) genes from amplified bacterial nucleic acid extracted from fecal material or mucosal samples has greatly facilitated the identification and classification of bacteria.
      • Macfarlane S
      • Macfarlane GT
      Bacterial diversity in the human gut.
      The study of entire microbial communities using metagenomic approaches based on these molecular methods has revealed a much greater diversity in the bacterial and archaeal domains than was previously thought to exist and has helped determine the community structure of several other previously unknown ecosystems.
      • Gill SR
      • Pop M
      • DeBoy RT
      • et al.
      Metagenomic analysis of the human distal gut microbiome.
      • Macfarlane S
      • Macfarlane GT
      Bacterial diversity in the human gut.
      • Frank DN
      • Pace NR
      Gastrointestinal microbiology enters the metagenomics era.
      • Hugenholtz P
      • Goebel BM
      • Pace NR
      Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity.
      • Amann RI
      • Ludwig W
      • Schleifer KH
      Phylogenetic identification and insitu detection of individual microbial cells without cultivation.
      For the purposes of this article, metagenomics refers to the study of all genes existing within the human genome and within the gut microbial genomes. Metagenomic approaches have tremendous potential to improve our understanding of the ways in which commensal and pathogenic microorganisms adapt themselves in humans.Figure 1 summarizes the steps involved in building a clone library, the most commonly used technique for molecular fingerprinting.
      Figure thumbnail gr1
      FIGURE 1Steps for building a clone library to fingerprint a complex microbial community. PCR = polymerase chain reaction; rRNA = ribosomal RNA.
      Using these techniques, investigators have estimated that the gastrointestinal tract in an adult human contains approximately 10
      • Palmer C
      • Bik EM
      • DiGiulio DB
      • Relman DA
      • Brown PO
      Development of the human infant intestinal microbiota [published online ahead of print June 26, 2007].
      microorganisms per milliliter of luminal content and harbors approximately 500 to 1000 distinctbacterial species.
      • Gill SR
      • Pop M
      • DeBoy RT
      • et al.
      Metagenomic analysis of the human distal gut microbiome.
      • Eckburg PB
      • Bik EM
      • Bernstein CN
      • et al.
      Diversity of the human intestinal microbial flora.
      • Ley RE
      • Bäckhed F
      • Turnbaugh PJ
      • Lozupone CA
      • Knight RD
      • Gordon JI
      Obesity alters gut microbial ecology.
      A very recent report suggests that this number is in fact much higher, with at least 1800 genera and between 15,000 and 36,000 species of bacteria.
      • Frank DN
      • St Amand AL
      • Feldman RA
      • Boedeker EC
      • Harpaz N
      • Pace NR
      Molecular-phlyogenetic characterization of microbial community imbalances in human inflammatory bowel disease.
      Life forms are divided into 3 domains: Eukaryota, the members of which contain a defined nuclear membrane separating the genome from cellular materials, and Bacteria and Archaea, which are prokaryotes lacking a DNA-containing nucleus. Prokaryotes are classified based on phylogeny (ie, 16S rRNA sequence similarities and differences). Although Archaea and Eukaryota domains are also represented in the gut, Bacteria clearly predominate.
      Sequencing the 16S rRNA gene from clone libraries has shown that uncultivated species and novel microorganisms constitute a substantial fraction of the gut microbiota (Table) . Using a cloning technique, Eckburg et al
      • Eckburg PB
      • Bik EM
      • Bernstein CN
      • et al.
      Diversity of the human intestinal microbial flora.
      recently conducted a comprehensive examination of the human colonic microbiota, finding that Bacteroidetes and Firmicutes account for more than 90% of all phylotypes of Bacteria and that Methanobrevibacter smithii, a hydrogen-consuming methanogen, dominates the Archaea domain.
      TABLEMajor Bacteria and Archaea Phyla and Genera Found in the Human Gut Microbiota
      Prokaryotic phyla were identified by using an alignment of the 18,348-sequence dataset from reference 18.
      PhylaRepresentative genera
      Bacteria
       FirmicutesRuminococcus
      Clostridium
      Peptostreptococcus
      Lactobacillus
      Enterococcus
       BacteroidetesBacteroides
       ProteobacteriaDesulfovibrio
      Escherichia
      Helicobacter
       Verrucomicrobia
      Not related to any known genera.
       ActinobacteriaBifidobacterium
       Cyanobacteria
      Not related to any known genera.
       Synergistes
      Not related to any known genera.
      Archaea
       EuryarchaeotaMethanobrevibacter
      a Prokaryotic phyla were identified by using an alignment of the 18,348-sequence dataset from reference
      • Ley RE
      • Bäckhed F
      • Turnbaugh PJ
      • Lozupone CA
      • Knight RD
      • Gordon JI
      Obesity alters gut microbial ecology.
      .
      b Not related to any known genera.
      As more microbial sequences become available, primers for real-time polymerase chain reaction will make it possible to quantify specific groups or species.
      • Ginzinger DG
      Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream.
      The growing database also allows design of molecular probes for quantitative real-time polymerase chain reaction, fluorescent in situ hybridization (FISH), and DNA microarray chips that identify specific bacterial species.

      Development

      Despite our limited understanding of the composition of the indigenous gut microbiota, evidence suggests that it is established within the first year of life
      • Palmer C
      • Bik EM
      • DiGiulio DB
      • Relman DA
      • Brown PO
      Development of the human infant intestinal microbiota [published online ahead of print June 26, 2007].
      • Berg RD
      The indigenous gastrointestinal microflora.
      and that the transformation to adult-type microbiota is likely triggered by multiple host and external factors,
      • Mackie RI
      • Sghir A
      • Gaskins HR
      Developmental microbial ecology of the neonatal gastrointestinal tract.
      • Gorbach SL
      Intestinal microflora.
      including the effects of the microbiota itself, developmental changes in the gut environment, and transition to an adult diet. The gut microbiota of the infant has long been thought to resemble that of the mother because most bacterial species are acquired during the birthing process.
      • Mackie RI
      • Sghir A
      • Gaskins HR
      Developmental microbial ecology of the neonatal gastrointestinal tract.
      However, this paradigm has been brought into question by recent evidence obtained using molecular techniques showing that children's stool samples do not resemble those of their parents more than those of other adults.
      • Palmer C
      • Bik EM
      • DiGiulio DB
      • Relman DA
      • Brown PO
      Development of the human infant intestinal microbiota [published online ahead of print June 26, 2007].
      The gut microbiota remains remarkably constant after transformation to adult-type microbiota; however, transient changes can occur, and, as recently demonstrated by Ley et al
      • Ley RE
      • Turnbaugh PJ
      • Klein S
      • Gordon JI
      Microbial ecology: human gut microbes associated with obesity.
      using culture-independent molecular methods, dietary factors can lead to long-term changes. This general stability is made possible by the recognition and tolerance of the infant-acquired microbiota by the gut immune system,
      • Ouwehand A
      • Isolauri E
      • Salminen S
      The role of the intestinal microflora for the development of the immune system in early childhood.
      which, by being exposed to and sampling microbial antigens, identifies them as normal. In contrast, the gut microbiota of one person can differ markedly from that of another; greater diversity is also seen in luminal (ie, stool) vs mucosal (ie, epithelial) compositions.
      • Eckburg PB
      • Bik EM
      • Bernstein CN
      • et al.
      Diversity of the human intestinal microbial flora.
      Comparative studies of adults with varying degrees of relatedness have shown that host genotype is more important than diet, age, and lifestyle in determining the composition of the gut microbiota.
      • Zoetendal EG
      • Akkermans ADL
      • Akkermans-van Vliet WM
      • de Visser JAGM
      • de Vos WM
      The host genotype affects the bacterial community in the human gastrointestinal tract.
      • Hopkins MJ
      • Sharp R
      • MacFarlane GT
      Age and disease related changes in intestinal bacterial populations assessed by cell culture, 16S rRNA abundance, and community cellular fatty acid profiles.
      The specific concentration and type of bacteria in the gastrointestinal tract are influenced by microhabitat variations throughout the gut, such as those in pH, oxygen, and nutrient availability.Figure 2 illustrates the key physiologic features of the human gut and the microbiological characteristics associated with them. Traditional culture-dependent microbiological studies have shown that the lower portion of the gastrointestinal tract has a higher bacterial count than the upper portion and that it is populated primarily by anaerobic bacteria, whereas the upper portion is populated largely by aerobic bacteria.
      • Berg RD
      The indigenous gastrointestinal microflora.
      • Rolf R
      Interactions among microorganisms of the indigenous intestinal flora and their influence on the host.
      Indeed, the terminal ileum is said to represent a transition zone between the aerobic microflora found in the proximal gut and the anaerobic organisms found in the colon.
      • Berg RD
      The indigenous gastrointestinal microflora.
      Once across the ileocecal valve, bacterial counts increase from 107 to 109/mL in the terminal ileum to approximately 1010 to 1012/mL in the colon.
      • Berg RD
      The indigenous gastrointestinal microflora.
      Recent molecular analyses indicate that the same bacteria phyla are present in the different anatomic regions of the gut and that only the relative abundance of the subgroups of the prevalent phyla varies.
      • Frank DN
      • St Amand AL
      • Feldman RA
      • Boedeker EC
      • Harpaz N
      • Pace NR
      Molecular-phlyogenetic characterization of microbial community imbalances in human inflammatory bowel disease.
      Figure thumbnail gr2
      FIGURE 2Key physiologic and microbiological features of the gut. Relative concentrations of bacteria and the pH at various locations within the adult gut are also noted. cfu = colony-forming unit.

      Metabolic Functions

      Studies using germ-free (ie, gnotobiotic) mice have shown that the gut microbiota is critical for maintaining normal gastrointestinal and immune function and normal digestion of nutrients.
      • Berg RD
      The indigenous gastrointestinal microflora.
      • Falk PG
      • Hooper LV
      • Midtvedt T
      • Gordon JI
      Creating and maintaining the gastrointestinal ecosystem: what we know and need to know from gnotobiology.
      • Neu J
      • Douglas-Escobar M
      • Lopez M
      Microbes and the developing gastrointestinal tract.
      • Macfarlane GT
      • Macfarlane S
      Human colonic microbiota: ecology, physiology and metabolic potential of intestinal bacteria.
      Although incompletely understood, thegut microbiota is implicated in a variety of host functions involving intestinal development and function, including epithelial turnover, immune modulation, gastrointestinal motility, and drug metabolism.
      • Ouwehand A
      • Isolauri E
      • Salminen S
      The role of the intestinal microflora for the development of the immune system in early childhood.
      • Rolf R
      Interactions among microorganisms of the indigenous intestinal flora and their influence on the host.
      • Abrams GD
      • Bishop JE
      Effect of the normal microbial flora on gastrointestinal motility.
      • Stappenbeck TS
      • Hooper LV
      • Gordon JI
      Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells.
      • Backhed F
      • Ley RE
      • Sonnenburg JL
      • Peterson DA
      • Gordon JI
      Hostbacterial mutualism in the human intestine.
      • Mathan VI
      • Wiederman J
      • Dobkin JF
      • Lindenbaum J
      Geographic differences in digoxin inactivation, a metabolic activity of the human anaerobic gut flora.
      The gut microbiota also has important metabolic functions, breaking down dietary toxins and carcinogens, synthesizing micronutrients, fermenting indigestible food substances, assisting in the absorption of certain electrolytes and trace minerals, and affecting the growth and differentiation of enterocytes and colonocytes through the production of short-chain fatty acids.
      • Macfarlane GT
      • Macfarlane S
      Human colonic microbiota: ecology, physiology and metabolic potential of intestinal bacteria.
      • Hooper LV
      • Midtvedt T
      • Gordon JI
      How host-microbial interactions shape the nutrient environment of the mammalian intestine.
      • Roberfroid MB
      • Bornet F
      • Bouley C
      • Cummings JH
      Colonic microflora: nutrition and health: summary and conclusions of an International Life Sciences Institute (ILSI) [Europe] workshop held in Barcelona, Spain.
      Finally, the normal gut microbiota helps prevent luminal colonization by pathogenic bacteria, such as Escherichia coli and Clostridia, Salmonella, and Shigella species.
      • DiBaise JK
      • Young RJ
      • Vanderhoof JA
      Enteric microbial flora, bacterial overgrowth and short bowel syndrome.
      • Gorbach SL
      Probiotics and gastrointestinal health.
      Findings from a recent study by Gill et al
      • Gill SR
      • Pop M
      • DeBoy RT
      • et al.
      Metagenomic analysis of the human distal gut microbiome.
      emphasize the important symbiotic contributions (in diversity and function) to the human metabolism made by the collection of microbial genomes known as the microbiome. After analyzing the fecal microbial community in healthy human participants, these investigators searched the DNA libraries for gene sequences that encode for enzymes known to participate in metabolism. They compared the translated enzyme sequences of the microbes to the ones in the human host and identified enzymes that affect host metabolism by maximizing the energy value of ingested food, promoting host homeostasis, and decontaminating the intestine.

      MICROBIAL CONTRIBUTIONS TO OBESITY

      The metabolic activities of the gut microbiota facilitate the extraction of calories from ingested dietary substances, help to store these calories in host adipose tissue for later use, and provide energy and nutrients for microbial growth and proliferation. Individual differences in energy recovery may provide a physiologic explanation for the observation that some obese patients do not seem to overeat. Indeed, it has been suggested that a person's gut microbiota has a specific metabolic efficiency and that certain characteristics of the microbiota composition might predispose to obesity.
      • Backhed F
      • Ley RE
      • Sonnenburg JL
      • Peterson DA
      • Gordon JI
      Hostbacterial mutualism in the human intestine.

      Dietary Energy Extraction

      In an elegant series of experiments, Backhed et al
      • Backhed F
      • Ding H
      • Wang T
      • et al.
      The gut microbiota as an environmental factor that regulates fat storage.
      found that young conventionally reared mice have a 40% higher body fat content and 47% higher gonadal fat content than germ-free mice even though they consumed less food than their germ-free counterparts. The distal gut microbiota from the normal mice was then transplanted into the gnotobiotic mice (a process known as conventionalization), resulting in a 60% increase in body fat within 2 weeks without any increase in food consumption or obvious differences in energy expenditure. This result supports the hypothesis that the composition of the gut microbiota affects the amount of energy extracted from the diet. The increase in body fat was accompanied by insulin resistance, adipocyte hypertrophy, and increased levels of circulating leptin and glucose.
      To elucidate potential underlying mechanisms, these investigators showed that the microbiota promoted absorption of monosaccharides from the gut and induced hepatic lipogenesis in the host, responses mediated by 2 signaling proteins, carbohydrate response element-binding protein (ChREBP) and liver sterol response element-binding protein type-1 (SREBP-1). Finally, using genetically modified (fasting-induced adipocyte factor [Fiaf]-knockout) mice, they showed that gut microbes suppress intestinal Fiaf, also known as angiopoietin-like protein 4. Fasting-induced adipocyte factor inhibits lipoprotein lipase activity, thereby catalyzing the release of fatty acids from lipoprotein-associated triacylglycerols, which are then taken up by muscle and adipose tissue. In the study, Fiaf suppression resulted in increased lipoprotein lipase activity in adipocytes and promoted storage of calories as fat, leading Backhed et al to postulate that energy regulation by the gut microbiota occurs through a number of interrelated microbial mechanisms. These mechanisms include fermentation of indigestible dietary polysaccharides to absorbable forms, intestinal absorption of monosaccharides and short-chain fatty acids with their subsequent conversion to fat within the liver, and regulation of host genes that promote deposition of fat in lipocytes.
      In a separate study designed to further explore the mechanism(s) underlying the resistance to obesity in germ-free mice, Backhed et al
      • Backhed F
      • Manchester JK
      • Semenkovich CF
      • Gordon JI
      Mechanisms underlying the resistance to diet-induced obesity in germ-free mice.
      studied germ-free mice consuming a Western-style, high-fat, sugar-rich diet. They determined that germ-free animals were protected from diet-induced obesity by 2 complementary but independent mechanisms that result in increased fatty acid metabolism: (1) elevated levels of Fiaf trigger the production of peroxisome proliferator-activated receptor γ coactivator, which is known to increase expression of genes encoding regulators of mitochondrial fatty acid oxidation; and (2) the activity of adenosine monophosphate-activated protein kinase, an enzyme that monitors cellular energy status, is increased. These findings suggest that the gut microbiota can affect both sides of the energy balance equation, influencing energy harvest from dietary substances (Fiaf) and affecting genes that regulate how energy is expended and stored.
      • Backhed F
      • Manchester JK
      • Semenkovich CF
      • Gordon JI
      Mechanisms underlying the resistance to diet-induced obesity in germ-free mice.
      In a proof-of-principle study, Turnbaugh et al
      • Turnbaugh PJ
      • Ley RE
      • Mahowald MA
      • Magrini V
      • Mardis ER
      • Gordon JI
      An obesity-associated gut microbiome with increased capacity for energy harvest.
      sought to understand how the gene content in the gut microbiota contributes to obesity. First, they characterized the distal gut microbiomes of genetically obese leptin-deficient (ob/ob) mice and their lean (ob/+ and +/+) littermates. Mice were used to avoid confounding variables such as diet, environment, and genotype that make such studies in humans difficult to interpret. In a series of experiments incorporating comparative metagenomics, these investigators showed that the microbiota in the ob/ob mice contained genes encoding enzymes that break down otherwise indigestible dietary polysaccharides. They also found more end products of fermentation (eg, acetate and butyrate) and fewer calories in the feces of the obese mice, leading them to speculate that the gut microbiota in these mice facilitate the extraction of additional calories from ingested food.
      To further show that the composition of the gut microbiota is important in determining weight, the investigators transferred the gut microbiota of either ob/ob mice or lean mice to lean gnotobiotic mice. After 2 weeks, the recipients of the microbiota from the ob/ob mice extracted more calories from food and also showed a significantly greater fat gain than did mice that received the microbiota from lean mice (mean percent of fat gain ± SD, 47%±8.3% vs 27%±3.6%; representing a difference of 4 kcal or 2% of total consumed calories based on the assumption that there are 9.3 kcal in a gram of fat).
      • Turnbaugh PJ
      • Ley RE
      • Mahowald MA
      • Magrini V
      • Mardis ER
      • Gordon JI
      An obesity-associated gut microbiome with increased capacity for energy harvest.
      These results suggest that differences in caloric extraction of ingested food substances may be determined by the composition of the gut microbiota, further supporting a microbial component in the pathogenesis of obesity. They also raise a number of questions. Do these small changes in energy extraction contribute to clinically meaningful differences in weight? How do conditions in the host (ie, genetic mutation in leptin in the ob/ob mouse) result in differences in the composition of the gut microbiota? Do these differences persist over time? Future studies are needed to clarify these issues.

      Chronic Systemic Inflammation

      On the basis of the recent demonstration that obesity and insulin resistance are associated with low-grade chronic systemic inflammation,
      • Wellen KE
      • Hotamisligil GS
      Inflammation, stress and diabetes.
      Cani et al
      • Cani PD
      • Amar J
      • Iglesias MA
      • et al.
      Metabolic endotoxemia initiates obesity and insulin resistance.
      postulated another mechanism linking the intestinal microbiota to the development of obesity. They hypothesized that bacterial lipopolysaccharide (LPS) derived from gram-negative bacteria residing in the gut microbiota acts as a triggering factor linking inflammation to high-fat diet-induced metabolic syndrome. In a series of experiments in mice fed a high-fat diet, they showed that (1) a high-fat diet increases endotoxemia and affects which bacterial populations are predominant in the intestinal microbiota (ie, it reduced both gram-negative [Bacteroides-related bacteria] and gram-positive bacteria [Eubacterium rectaleClostridium coccoides group and bifidobacteria], favoring an increase in the gram-negative to gram-positive ratio), and that (2) chronic metabolic endotoxemia induces obesity, insulin resistance, and diabetes. Using CD14 mutant mice fed a high-fat diet, they showed that metabolic endotoxemia triggers the expression of inflammatory cytokines (eg, tumor necrosis factor α, interleukin 1, interleukin 6, and plasminogenactivator inhibitor 1) via a CD14-dependent mechanism. A key molecule binding LPS at the surface of innate immune cells, CD14 triggers the secretion of proinflammatory cytokines.
      • Wright SD
      • Ramos RA
      • Tobias PS
      • Ulevitch RJ
      • Mathison JC
      CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein.
      It has been suggested that the LPS/CD14 system sets the tone of insulin sensitivity and regulates the onset of obesity and diabetes.
      • Cani PD
      • Amar J
      • Iglesias MA
      • et al.
      Metabolic endotoxemia initiates obesity and insulin resistance.
      Human studies have provided support for these findings. Treatment of humans with polymyxin B, an antibiotic that specifically targets gram-negative organisms, was shown to reduce LPS expression and hepatic steatosis.
      • Pappo I
      • Becovier H
      • Berry EM
      • Freund HR
      Polymyxin B reduces cecal flora, TNF production and hepatic steatosis during total parenteral nutrition in the rat.
      A more recent study reported that patients with type 2 diabetes had higher LPS levels than did a well-matched group of control participants without diabetes.
      • Creely SJ
      • McTernan PG
      • Kusminski CM
      • et al.
      Lipopolysaccharide activates an innate immune system response in human adipose tissue in obesity and type 2 diabetes.
      Figure 3 summarizes the possible mechanisms by which the gut microbial community can contribute to obesity.
      Figure thumbnail gr3
      FIGURE 3Mechanisms by which the intestinal microbiota may contribute to obesity. AMPK = adenosine monophosphate-activated protein kinase; ChREBP = carbohydrate response element-binding protein; Fiaf = fasting-induced adipocyte factor; LPL = lipoprotein lipase; LPS = lipopolysaccharide; PGC-1α = peroxisome proliferator-activated receptor γ coactivator 1α; SREBP-1 = sterol response element-binding protein type 1.

      Gut Microbiota Composition in Obese vs Lean Mice

      To assess the relative abundance of various types of gut bacteria in obese and lean mice, Ley et al
      • Ley RE
      • Bäckhed F
      • Turnbaugh PJ
      • Lozupone CA
      • Knight RD
      • Gordon JI
      Obesity alters gut microbial ecology.
      analyzed bacterial 16S rRNA gene sequences from the cecal microbiota of genetically obese (ob/ob) mice, their lean ob/+ and +/+ siblings, and their ob/+ mothers, all fed the same polysaccharide-rich diet. They found that the ob/ob mice had 50% fewer Bacteroidetes and correspondingly more Firmicutes than their lean littermates, a finding unrelated to differences in food consumption. These changes were seen throughout the division and were not due to an increase or decrease in the numbers of a few Bacteroidetes or Firmicutes members. The mechanisms responsible for this difference require further study. Ley et al also showed a strong association between kinship and distal gut microbial diversity, but the differences seen in obese mice occurred independently of kinship and sex. These results suggest that differences exist in the gut microbiota of obese vs lean mice, raising the possibility that the manipulation of gut microbiota could be a useful strategy for regulating energy balance in obese people.

      Gut Microbiota Composition in Obese vs Lean Humans

      To show the relevance of the animal experiments to humans, Ley et al
      • Ley RE
      • Turnbaugh PJ
      • Klein S
      • Gordon JI
      Microbial ecology: human gut microbes associated with obesity.
      serially monitored the fecal gut microbiota in 12 obese participants in a weight-loss program for a year, randomly assigning them to either a fat-restricted or carbohydrate-restricted low-calorie diet. As in the mice experiments, members of the Bacteroidetes and Firmicutes divisions dominated the microbiota, and bacterial flora showed remarkable intraindividual stability over time. Before diet therapy, obese participants had fewer Bacteroidetes and more Firmicutes than lean control participants. After weight loss, the relative proportion of Bacteroidetes increased, while Firmicutes decreased, a finding that correlated with the percentage of lost weight and not with changes in dietary caloric content. Bacteroidetes constituted approximately 3% of the gut bacteria before diet therapy and approximately 15% after successful weight loss. It is unknown why obese people have more Firmicutes. The host gut may have uncharacterized properties that select this bacterial phylum, which contains more than 250 genera and has diverse metabolic capabilities. For example, many of the Bacillus species are facultative aerobes, whereas the Clostridium species are obligate anaerobes. The vast diversity within Firmicutes may contribute to more efficient energy extraction from a variety of complex organic matter. Clearly, additional work is needed to better clarify the cause-and-effect relationship between obesity and the gut microbiota.

      Energy Homeostasis

      Although Bacteroidetes and Firmicutes are the dominant microbial organisms in the gut, methanogenic Archaea are also present. Archaeal methanogenesis improves the efficiency of polysaccharide fermentation by preventing the buildup of hydrogen and other reaction end products. In contrast, the formation of methane creates a large electron and energy sink; that energy is then unavailable for uptake by an animal. Cattle growers try to suppress methanogenesis in the cow's rumen for this reason. Unlike the rumen, which harbors acetate-utilizing methanogens such as Methanosarcina species,
      • Chen M
      • Wolin MJ
      Effect of monensin and lasalocid-sodium on the growth of methanogenic and rumen saccharolytic bacteria.
      the human gastrointestinal tract is dominated by hydrogen- and formate-oxidizing Methanobrevibacter species,
      • Eckburg PB
      • Bik EM
      • Bernstein CN
      • et al.
      Diversity of the human intestinal microbial flora.
      suggesting that acetate and butyrate produced by fermentative bacteria in the colon are not consumed by methanogens. By removing hydrogen and formate, Methanobrevibacter species may help the bacterial community produce more acetate and butyrate, which are important carbon sources for colon epithelium cells. As a result, this type of Bacteria-Archaea syntrophism in humans may lead to increased energy extraction from indigestible polysaccharide diets.
      To better understand the contributions of specific microbes, Samuel and Gordon
      • Samuel BS
      • Gordon JI
      A humanized gnotobiotic mouse model of hostarchaeal-bacterial mutualism.
      colonized the gut of germ-free mice with M smithii, Bacteroides thetaiotaomicron, or both. Bacteroides thetaiotaomicron is a common colonic bacteria that is highly efficient in glycan metabolism, allowing otherwise indigestible sugars to be metabolized and harvested as additional energy.
      • Comstock LE
      • Coyne MJ
      Bacteroides thetaiotamicron: a dynamic, niche-adapted human symbiont.
      Methanobrevibacter smithii is the most prominent Archaea in humans, constituting 10% of all anaerobes in the colons of healthy adults
      • Macfarlane GT
      • Macfarlane S
      Human colonic microbiota: ecology, physiology and metabolic potential of intestinal bacteria.
      ; however, the role of Archaea in human health remains uncertain. Samuel and Gordon
      • Samuel BS
      • Gordon JI
      A humanized gnotobiotic mouse model of hostarchaeal-bacterial mutualism.
      found that cocolonization with M smithii and B thetaiotaomicron increased the efficiency of energy extraction from dietary polysaccharides and the amount of host adiposity more than did colonization with either organism alone. Furthermore, Samuel et al
      • Samuel BS
      • Hansen EE
      • Manchester JK
      • et al.
      Genomic and metabolic adaptations of Methanobrevibacter smithii to the human gut.
      found that M smithii influenced the metabolism of B thetaiotaomicron, prompting it to consume mainly fructose-containing polysaccharides that break down into several substances, including formate, an important energy source of M smithii. These findings not only suggest a contribution of Archaea to digestive health but also show that interactions among microorganisms in the gut have a role in host energy homeostasis. These findings also raise the intriguing possibility of M smithii as a therapeutic target for reducing energy harvest in obese humans.

      MODIFYING THE ECOSYSTEM AS A THERAPEUTIC STRATEGY

      The best nonsurgical strategy for reversing obesity in the population may be to promote small but long-term changes in diet and physical activity that take advantage of our biological systems for regulating energy balance and preventing positive energy balance.
      • Korner J
      • Leibel RI
      To eat or not to eat—how the gut talks to the brain.
      Although the role of the gut microbiota in energy regulation remains undefined, the existence of systems that regulate energy balance suggests that microorganisms might have a substantial cumulative effect over time. Although clearly no substitute for proper diet and exercise, manipulation of the gut microbiota may represent a novel approach for treating obesity, one that has few adverse effects. Use of antibiotics, prebiotics, and probiotics may result in nonspecific modulation of the gut microbiota.

      Antibiotics

      Recently, Brugman et al
      • Brugman S
      • Klatter FA
      • Visser JT
      • et al.
      Antibiotic treatment partially protects against type 1 diabetes in the bio-breeding diabetes-prone rat: is the gut flora involved in the development of type 1 diabetes?.
      showed that antibiotic treatment decreased the incidence and delayed the onset of diabetes in a diabetes-prone rat model. Using FISH, they showed that the gut bacterial composition of rats that developed diabetes differed from that of those that did not. Specifically, rats that did not develop diabetes displayed a lower number of Bacteroides species. The investigators speculated that the antibiotic-induced alteration in the gut microbiota led to a reduction in the antigenic load and subsequent inflammation that usually leads to pancreatic β-cell destruction. Although the study by Brugman et al did not directly address obesity, it demonstrates the potential of modulating the intestinal microbiota as a therapeutic strategy.

      Prebiotics

      Prebiotic agents are nondigestible oligosaccharides that act as “fertilizers” of the colonic microbiota, enhancing the growth of beneficial commensal organisms (eg, Bifidobacterium and Lactobacillus species).
      • Roberfroid MB
      Functional foods: concepts and application to inulin and oligofructose.
      Fructo-oligosaccharides are prebiotic agents that are fermented by a number of colonic bacteria to modulate the growth of beneficial colonic bacteria. Inulin and oligofructose, naturally occurring fructo-oligosaccharides that are not digested in the upper gastrointestinal tract, have several functional and nutritional properties, including the ability to stimulate the growth of beneficial commensal organisms.
      • Roberfroid MB
      Functional foods: concepts and application to inulin and oligofructose.
      • Gibson GR
      • Beatty ER
      • Wang X
      • Cummings JH
      Selective stimulation of bifidobacteria in the human colon by oligofructose and inulin.
      In 2 recent studies in which rats were fed a standard
      • Cani PD
      • Dewever C
      • Delzenne NM
      Inulin-type fructans modulate gastrointestinal peptides involved in appetite regulation (glucagon-like peptide and ghrelin) in rats.
      or high-fat diet,
      • Cani PD
      • Neyrinck AM
      • Maton N
      • Delzenne NM
      Oligofructose promotes satiety in rats fed a high-fat diet: involvement of glucagon-like peptide-1.
      the addition of oligofructose to the diet reduced energy intake and consumption and protected against weight gain and fat-mass development, effects shown to be mediated by the modulation of endogenous gut peptides involved in appetite and weight regulation.
      • Delzenne NM
      • Cani PD
      • Daubioul C
      • Neyrinck AM
      Impact of inulin and oligofructose on gastrointestinal peptides.
      This result seems to contradict previously discussed findings suggesting that nondigestible polysaccharides may be responsible, at least in part, for increased weight in genetically obese mice because of increased energy extraction.
      • Turnbaugh PJ
      • Ley RE
      • Mahowald MA
      • Magrini V
      • Mardis ER
      • Gordon JI
      An obesity-associated gut microbiome with increased capacity for energy harvest.
      This discrepancy could result from a number of factors, including specific modulation of the gut microbiota (as yet poorly understood) and other physiologic effects of fibers, such as slowed gastric emptying and increased satiety.
      • Kleessen B
      • Hartmann L
      • Blaut M
      Oligofructose and long-chain inulin: influence on the gut microbial ecology of rats associated with a human faecal flora.
      Additional support for the role of prebiotics in reducing weight gain was provided by a study in which the addition of inulin and lupin-kernel fiber, also nondigestible starches, as fat replacers in a sausage patty was shown to reduce fat and energy intake in healthy humans.
      • Archer BJ
      • Johnson SK
      • Devereux HM
      • Baxter AL
      Effect of fat replacement by inulin or lupin-kernel fibre on sausage patty acceptability, post-meal perceptions of satiety and food intake in men.
      In a single-blind, crossover pilot study of 10 healthy people of normal weight, a 2-week treatment with oligofructose was shown to increase satiety after breakfast and dinner and to markedly reduce hunger and prospective food consumption after dinner, leading to a total energy intake per day that was 5% lower than that of the placebo group.
      • Cani PD
      • Joly E
      • Horsmans Y
      • Delzenne NM
      Oligofructose promotes satiety in healthy humans: a pilot study.
      In a recent study of the link between prebiotics and endotoxemia, Cani et al
      • Cani PD
      • Neyrinck AM
      • Fava F
      • et al.
      Selective increases of bifido-bacteria in gut microflora improve high-fat-diet-induced diabetes in mice through a mechanism associated with endotoxaemia.
      found that oligofructose increased the gut bifidobacterial content of high-fat diet-fed mice and that endotoxemia significantly and negatively correlated with Bifidobacterium species but with no other bacterial group. They also showed that, in high-fat oligofructose-treated-mice, a significant and positive correlation existed between Bifidobacterium species and improved glucose tolerance, glucose-induced insulin secretion, and normalized inflammatory tone. Although indirect, these lines of evidence present a rationale to warrant further investigation of the use of prebiotic supplementation to modify the gut microbiota in the management of food intake in people who are overweight and obese.

      Probiotics

      Probiotics are nonpathogenic live microorganisms that, when ingested, confer health benefits to the host.
      • Schrezenmeir J
      • de Vrese M
      Probiotics, prebiotics, and synbiotics—approaching a definition.
      Probiotics have generated considerable interest in recent years because studies investigating their use in a variety of clinical conditions, particularly diarrheal disorders, have yielded encouraging results.
      • Floch MH
      • Montrose DC
      Use of probiotics in humans: an analysis of the literature.
      A potential role for probiotics in the treatment of obesity has been suggested by 2 recent reports. Lee et al
      • Lee HY
      • Park JH
      • Seok SH
      • et al.
      Human originated bacteria, Lactobacillus rhamnosus PL60, produce conjugated linoleic acid and show anti-obesity effects in diet-induced obese mice.
      investigated the antiobesity effect of Lactobacillus rhamnosus PL60, a bacterium of human origin that produces conjugated linoleic acid, in diet-induced obese mice. Conjugated linoleic acid has been suggested to have a number of potential health effects in animal studies, including the ability to reduce body fat.
      • Park Y
      • Albright KJ
      • Liu W
      • Storkson JM
      • Cook ME
      • Pariza MW
      Effect of conjugated linoleic acid on body composition in mice.
      After 8 weeks of oral feeding with L rhamnosus PL60, mice lost weight without reducing energy intake. Further studies by these investigators suggested that the antiobesity effects were possibly related to apoptosis and messenger RNA expression in white adipose tissue. However, it should be noted that L rhamnosus PL60 did not reduce cell size in epididymal adipose tissue, and thus the decrease in the weight of white adipose tissues was due to reduction in cell number rather than in cell size. Because the number of adipose cells is constant in adult humans and only the cell size changes with obesity, the role of L rhamnosus PL60 in humans is unclear. Further supporting the inefficacy of this particular probiotic approach are the results of a recent randomized controlled study in which 122 obese humans were treated with 3.4 g of conjugated linoleic acid or placebo for 1 year.
      • Larsen TM
      • Toubro S
      • Gudmundsen O
      • Astrup A
      Conjugated linoleic acid supplementation for 1 y does not prevent weight or body fat regain.
      Sonnenburg et al
      • Sonnenburg JL
      • Chen CT
      • Gordon JI
      Genomic and metabolic studies of the impact of probiotics on a model gut symbiont and host.
      colonized germ-free mice with B thetaiotaomicron and Bifidobacterium longum, a commonly used probiotic. They found that, when B thetaiotaomicron encountered B longum, it expanded the range of polysaccharides targeted for degradation and did so independently of host genotype; however, it did not do so for all bifidobacteria (eg, Bifidobacterium animalis) with which it was cocultured. Similar metabolic effects were achieved with a probiotic from another division of Bacteria (Lactobacillus casei). In another demonstration that probiotics can exert metabolic effects on the host, Martin et al
      • Martin FPJ
      • Wang Y
      • Sprenger N
      • et al.
      Probiotic modulation of symbiotic gut microbial-host metabolic interaction in a humanized microbiome mouse model.
      administered probiotic beverages to germ-free mice that had been conventionalized with human baby flora. Using high-density data-generating spectroscopic techniques in combination with multivariate mathematical modeling, they showed that probiotic exposure resulted in distinct changes in the microbiome with associated metabolic alterations in a variety of tissues affecting energy, lipid, and amino acid metabolism. The importance of these findings to energy homeostasis and overall health in humans remains to be determined; however, they suggest that probiotics can alter the dynamics of the entire gut microbiota and show that these molecular approaches can be used to study the metabolic effects of probiotics on the host and the host's microbiome.

      FUTURE DIRECTIONS

      Studies are needed to clarify a number of issues related to the relationship between the gut microbiota and obesity. First, it remains to be determined whether small changes in caloric extraction, seen in several past studies, can result in clinically meaningful differences in weight in humans. In principle, small but persistent changes in energy homeostasis, in this case from increased energy extraction, should lead to changes in body composition and weight.
      • Hill JO
      Understanding and addressing the epidemic of obesity: an energy balance perspective.
      Second, the possible relationship between the gut microbiota—including previously cultured and uncultivated strains, as well as rare and abundant microbes—and the regulation of weight must be proved or disproved. In particular, it is essential to demonstrate unequivocally whether differences in gut microbiota in obese vs lean people are the cause or the result of obesity. In this regard, hormonal or other signals that potentially direct changes in the make-up of the gut microbiota need to be elucidated. Of immediate benefit will be metagenomic studies of the gut microbiota in mice with diet-induced obesity and microbiota transplant studies similar to those previously described in genetically induced obese mice. Third, the mechanisms responsible for the relative proportions of Bacteroidetes, Firmicutes, and Archaea in mice and humans must be explored. In particular, the environmental and genetic factors that determine the distinctive characteristics of each person's microbiota must be identified. Fourth, differences in surface-adherent mucosal microbial colonies (vs those found within the bowel lumen) must be defined for people who are obese and for those who have successfully lost weight. Similarly, the factors that affect the local microbial ecology, particularly of the adherent microbes, must be investigated and identified. Finally, a means to deliberately modify the gut microbiota must be proposed and then examined in well-controlled and closely monitored studies. Clinical trials assessing the efficacy of prebiotics and probiotics should evaluate participants' intestinal microbiota before and after therapy. Furthermore, given recent reports questioning the safety of probiotics,

      Besselink MG, van Santvoort HC, Buskens E, et al. Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial [published online ahead of print February 13, 2008]. Lancet, doi:10.1016/S0140-6736(08)60207-X.

      • Hoffman FA
      • Heimbach JT
      • Sanders ME
      • Hibberd PL
      Executive summary: scientific and regulatory challenges of development of probiotics as foods and drugs.
      further research is needed to establish the safety of this approach.

      CONCLUSION

      The worldwide obesity epidemic has intensified efforts to identify host and environmental factors that affect energy balance. One emerging finding is that the host and its microbiota have mutually beneficial and cooperative interactions. The evidence reviewed in this article, much of it obtained recently using powerful tools such as sequencing of 16S rRNA gene clone libraries, metagenomics, DNA microarrays, microbiota transplant, and gnotobiotic knock-out mice, suggests that the gut microbiota has a role in the regulation of energy balance and weight. It further suggests a role for a gut-derived factor, such as LPS, in the pathogenesis of obesity-related type 2 diabetes. Although these findings are promising, studies are needed both to better understand the causal relationship between gut microbiota of varied composition and the propensity to be obese or lean and to assess whether modulating the gut microbiota could help to reduce obesity.

      ADDENDUM

      After the manuscript had been accepted for publication, an article was published with relevance to our review. Kalliomäki et al
      • Kalliomäki M
      • Carmen Collado M
      • Salminen S
      • Isolauri E
      Early differences in fecal microbiota composition in children may predict overweight.
      prospectively followed children from birth to age 7 years. Fecal samples collected at ages 6 and 12 months were analyzed using a variety of molecular techniques. Higher numbers of bifidobacteria and lower numbers of Staphylococcus aureus were found in children who were normal weight at age 7 years than in those who were overweight-obese, suggesting that differences in the composition of the gut microbiota precede overweight-obesity.

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