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60 61 62 63 64 65 66 67 68 69 70 71 72 1,2 4,5 5 Max Nieuwdorp P. W. Gilijamse N. Pai Lee M. Kaplan 73 74 Departments of 1Vascular Medicine and 3Endocrinology and Metabolism, Academic Medical Center, University of Amsterdam, 75 Amsterdam, The Netherlands; 2Wallenberg Laboratory, Sahlgrenska Center for Cardiovascular and Metabolic Research, 76 University of Göteborg, Göteborg, Sweden; 4Department of Pediatric Gastroenterology & Nutrition, Boston Children’s Hospital, 77 Harvard Medical School, Boston, Massachusetts; and 5Obesity, Metabolism and Nutrition Institute and Gastrointestinal Unit, 78 Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 79 80 What other factors could contribute to insulin resistance 81 Intestinal microbes regulate metabolic function and energy balance; an altered microbial ecology is believed to in obese people? Based on findings from animal studies, the 82 contribute to the development of several metabolic dis- intestinal microbiota is involved in the development of in83 eases. Relative species abundance and metabolic charac- sulin resistance.5 Metabolic syndrome is a chronic inflam84 teristics of the intestinal microbiota change substantially matory disease, and translocation of intestinal bacteria has 85 in those who are obese or have other metabolic disorders been proposed to promote metabolic endotoxemia.6 Several 86 and in response to ingested nutrients or therapeutic studies have associated obesity with insulin resistance and agents. The mechanisms through which the intestinal increased levels of plasma lipopolysaccharide (LPS)-binding 87 microbiota and its metabolites affect host homeostasis are protein.7,8 Transplantation of fecal preparations from lean 88 just beginning to be understood. We review the relation- donors can temporarily improve insulin sensitivity.9 These Q789 90 ships between the intestinal microbiota and host metafindings indicate a relationship among the intestinal 91 bolism, including energy intake, use, and expenditure, in microbial, bacterial translocation, and host metabolism. 92 relation to glucose and lipid metabolism. These associaIt is a challenge to study the effects of the microbiota on 93 tions, along with interactions among the intestinal microhuman metabolism. The human intestinal microbiome in- 94 biota, mucus layer, bile acids, and mucosal immune (meta) 95 responses, reveal potential mechanisms by which the cludes 3000 to 5000 species that share a collective 10 High- 96 genome comprising nearly 5 million genes. microbiota affect metabolism. We discuss how controlled studies involving direct perturbations of microbial com- throughput sequencing has allowed for comprehensive an- 97 munities in human and animal models are required to alyses of the intestinal metagenome based on the genetic 98 material from fecal samples. When metagenomic analysis 99 identify effective therapeutic targets in the microbiota. data are compared with clinical symptoms or disease, the 100 analysis is called a metagenome-wide association study.11 101 Keywords: Obesity; Energy Expenditure; Bile Acids; Gut Such studies have shown significant differences in the 102 Microbiota. composition of the intestinal microbiota metagenome be- 103 tween metabolically healthy and unhealthy subjects.12,13 104 Studies of germ-free mice have implicated the intestinal 105 he obesity pandemic presents a considerable chal- microbiota in drug metabolism and macronutrient diges106 lenge for health care systems worldwide. More than tion,14 and antibiotic treatments aimed at reducing the 107 500 million people are obese, and the prevalence of obesity bacterial load in the intestine have profound effects on the 108 1 is increasing rapidly. Obese people are at risk for devel- production of metabolites by the gut microbiota.15 109 oping dyslipidemia, nonalcoholic fatty liver disease, and 110 2 insulin resistance. Notably, approximately one-half of all 111 obese people develop metabolic syndrome, which is 112 strongly associated with greater amounts of abdominal ad113 used in this paper: FMT, fecal microbial transplantation; ipose tissue, although little is understood about the mech- Abbreviations LPS, lipopolysaccharide; RYGB, Roux-en-Y gastric bypass; SCFA, short114 2 anisms of this association. However, obese insulin-resistant chain fatty acid; TMA, trimethylamine; TMAO, trimethylamine-N-oxide. 115 patients who undergo either subcutaneous adipose tissue 116 3 © 2014 by the AGA Institute removal or surgical removal of intra-abdominal adipose 0016-5085/$36.00 117 4 tissue do not have increased insulin sensitivity. http://dx.doi.org/10.1053/j.gastro.2014.02.008 118
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Obesity and insulin resistance are closely related to the presence of (visceral) adipose tissue inflammation. For example, adipose tissue produces numerous inflammatory cytokines,16 and there is evidence that visceral adipose tissue—and, to a lesser extent, subcutaneous adipose tissue—can promote insulin resistance.17 The presence of subcutaneous adipose tissue macrophages and crown-like structures (or accumulated CD68-positive macrophages) correlates with expression of genes that control inflammation,18 indicating a role for the innate immune system in insulin resistance.19 Changes in intestinal microbiota cause long-term changes in inflammation in obese subjects12; bacterial endotoxins such as LPS activate pattern recognition receptors such as Toll-like receptors, leading to an innate immune response and insulin resistance.20 The intestinal microbiota also produces many molecules that promote inflammation, such as peptidoglycans21 and flagellins,22 which activate inflammatory pathways, resulting in obesity and insulin resistance. Short-chain fatty acids (SFCAs) are produced by intestinal bacteria and may play important roles in dampening the adverse effects of intestinal pathogens on host metabolism. Diet-derived fibers are metabolized and fermented to SCFAs such as acetate, propionate, and butyrate by intestinal bacteria.23 Germ-free mice produce almost no SCFAs and therefore have altered responses to inflammatory stimuli.24 SCFAs may serve as an energy source for the intestinal epithelium and liver, given their transport predominantly via the portal vein after intestinal absorption. SCFAs might also modulate the immune response by reducing intestinal permeability.25 This hypothesis is supported by the observation that translocation of LPS from the gut to the portal vein produces obesity-associated low-grade inflammation and subsequent insulin resistance in mice, which can partly be reversed by administration of the propionate-producing bacteria Akkermansia muciniphila.26 Besides obesity, the intestinal microbiota might also be involved in atherogenesis. Specific dietary nutrients characterized by trimethylamine groups (eg, choline, phosphatidylcholine, and carnitine) are metabolized into the atherogenic compound trimethylamine-N-oxide (TMAO) by bacteria.27,28 Studies using germ-free mice or mice given broad-spectrum antibiotics showed that the intestinal microbiota is required for formation of trimethylamine (TMA) and TMAO.29 Further, bacterial colonization of germfree mice increases their plasma levels of TMAO, indicating that the intestinal microbiota is required for generation of TMA from sources of dietary choline or carnitine (such as eggs, milk, and red meat).30 For example, carnitine is an abundant nutrient in red meat, and the intestinal microbiota mediates production of TMAO from dietary L-carnitine.
Alteration of the Gut Microbiota in Obesity and Diabetes The intestinal microbiota is altered in humans and animal models of obesity.31 The intestinal (cecum-derived) microbiota of ob/ob mice has a 50% reduction in levels of
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Bacteroidetes and an increased proportion of Firmicutes compared with wild-type mice.32 The composition of the fecal microbiota of obese human subjects is similarly affected but changes with weight loss.33 Studies in germfree mice provide insights into the effects of the intestinal microbiota on host metabolism. Germ-free mice fed high-fat, high-sugar diets did not have the same metabolic disturbances as the littermates that were not germ free.34 Transfer of intestinal microbiota from obese mice resulted in significantly greater adiposity in recipients than transfer of microbiota from lean donors.35 One way in which intestinal microbes might affect host metabolism is by extracting calories from otherwise indigestible carbohydrates36; these carbohydrates are fermented by intestinal microbes to produce SCFAs.38 SCFAs may act as an energy substrate as they are absorbed by the intestinal epithelium and metabolized in the liver.37 Mouse models of obesity and human obese subjects have increased intestinal (cecal) levels of SCFA and decreased energy content in their feces.23 Studies have associated changes in proportions of Bacteroidetes and Firmicutes with obesity and metabolic syndrome. Metagenome-wide association studies by Qin et al13 (performed in China) and Karlsson et al12 (performed in Europe) reported metagenomic differences between a cohort of patients with type 2 diabetes mellitus and a group of healthy subjects. Clusters of genomic sequences were used as signatures for specific groups of bacteria, and each study found independently that the microbiota of subjects with type 2 diabetes mellitus had a lower proportion of butyrate-producing Clostridiales (Roseburia and Faecalibacterium prausnitzii), and greater proportions of Clostridiales that do not produce butyrate, as well as pathogens such as Clostridium clostridioforme. Other associations differed between the 2 studies. Karlsson et al12 detected an increased proportion of Lactobacillus gasseri and Streptococcus mutans (commensal bacteria in the mouth and upper intestinal tract) in their cohort with type 2 diabetes mellitus. Qin et al13 observed a greater proportion of Escherichia coli (which produce LPS to cause endotoxemia) in patients with type 2 diabetes mellitus. These studies raise interest in the association between type 2 diabetes mellitus and reduced production of butyrate because diets supplemented with butyrate were previously shown to prevent and reverse insulin resistance in mice that became obese on high-calorie diets and increase energy expenditure.38 Combined results from human and animal studies of obesity suggest that reduced butyrate production by the microbiota contributes to the development of insulin resistance.
Microbiota and Bile Acids Bile acids are secreted as glycine, taurine, or sulfate conjugates. Compounds excreted in bile reach the intestinal tract, where they can be deconjugated by gut microbiota, aided by populations of microorganisms with their own hydrolytic enzymes such as b-glucuronidase and sulfatases.39 Most liver-secreted bile acids (95%) are reabsorbed in the ileum to be taken up by the liver in the enterohepatic
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cycle. Only a small part of the bile acid pool escapes the enterohepatic cycle and travels toward the large intestine to be excreted in the feces. This excretion is accompanied by microbial deconjugation of glycine- and taurine-conjugated bile acid; germ-free mice were shown to have altered metabolism of bile acids.39–41 Follow-up studies showed that germ-free mice had increased levels of conjugated bile acids throughout the intestine, with no deconjugation, and strongly decreased fecal excretion.42 More recently, studies with antibiotics provided further support for the importance of intestinal microbiota in bile acid metabolism. Mice given ampicillin for 3 days increased biliary bile acid output 3-fold, whereas fecal output decreased 70%.43 We recently showed that vancomycin therapy (which eradicates gram-positive bacteria) of patients with insulin resistance also significantly altered the fecal bile acid pool, reducing the proportion of secondary bile acids, compared with primary bile acids, which is associated with reduced insulin sensitivity.44 Grampositive bacteria in the intestine therefore affect the bile acid pool, which alters regulation of lipid and glucose metabolism via the FXR45 and TGR546 signaling pathways.
Intestinal Microbiota and Host Physiology and Diseases Recent advances in sequencing technology and largescale information processing have increased our understanding of the diversity of the intestinal microbiota and its relationship to host physiology and diseases. Comprehensive characterizations of the microbiota have revealed rapid and profound effects of environmental and physiological changes on the composition and metabolic activities of the microbiota. Conversely, the microbial ecology has been correlated with specific diseases such as obesity and insulin resistance, and microbiota transfer studies in animals37 and humans6 have shown that alterations in the gut microbiota contribute to the pathogenesis of these disorders. Studies of development of the microbiota in children have provided important information about interactions between host tissues and the microbiota and how disruptions in the microbiota can contribute to disease development. Newborns have essentially no intestinal microbiota. However, low levels of bacterial translocation through the placental circulation likely provide a primitive bacterial community before birth.47 Nevertheless, the newborn intestine begins colonization by maternal and environmental bacteria during birth. The structure of this primary microbiota varies with mode of birth (cesarean section vs vaginal delivery), feeding (breastfeeding vs formula), and level of sanitation in the environment.48 After the first year of life, each infant develops a distinct microbial community; by approximately 3 years, microbial profiles resemble those of adults in patterns and diversity.49 The progressive maturation of the intestinal microbiota is likely to be important for the establishment of commensal and pathogen recognition by the immune system and determine later host-microbe interactions and susceptibility to infection, autoimmunity, and inflammatory diseases.
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The composition of the gut microbiota remains relatively 296 stable from late childhood to old age, when changes occur 297 again.50 These late changes often correspond with age- 298 related changes in diet and digestive physiology, but it is 299 not clear how these changes affect the gut microbiota. The 300 structure of the gut microbiota varies spatially along the 301 intestinal tract and cross-sectionally. The bacteria that 302 reside in close proximity to the mucosal surface are believed 303 to interact more closely with the immune system, whereas 304 bacteria that reside in the lumen may be more closely 305 associated with food and products of digestion, influencing 306 nutrient assimilation, signaling, and metabolic function. 307 Cani et al used the term “metabolic endotoxemia” to 308 describe the inflammatory state often associated with the 309 metabolic syndrome. This systemic inflammation is believed 310 to occur as bacteria translocates through the gastrointes- 311 tinal tract, increasing circulating levels of LPS. Obese sub- 312 jects with insulin resistance have increased circulating 313 levels of LPS compared with matched controls; endotoxemia 314 is independently associated with energy intake even in lean 315 men.6,51 Toll-like receptor 4–null mice are protected from 316 diet-induced obesity and associated insulin resistance.52 317 Antibiotics, which have been used against pathogenic 318 bacteria for nearly a century, also alter the commensal 319 bacterial community, affecting metabolic and immune 320 regulation. Short-term intake of antibiotics, via medication 321 or dietary exposure to antibiotic-treated animals, causes 322 shifts in the microbiota that have detrimental effects on 323 metabolism.44 Recent studies have associated cumulative Q8324 antibiotic exposure with rates of obesity and metabolic 325 disorders. Administration of subtherapeutic doses of anti- 326 biotics to young mice increases their adiposity and pro- 327 motes metabolic dysfunction.53 Similar observations have 328 been reported from epidemiological studies,54 highlighting 329 the importance of commensal intestinal bacteria for meta- 330 bolic homeostasis. 331
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Luminal Factors That Regulate Host Metabolism Commensal gut bacteria regulate immune and metabolic functions via several mechanisms.55 Bacteria release fatty acids, peroxidases, proteases, and bacteriocins that prevent pathogens from expanding in the intestinal community7; as previously mentioned, bacterial disaccharidases ferment unabsorbed dietary carbohydrates into SCFAs. Although increased generation of SCFAs could increase energy uptake from ingested plant carbohydrates, recent studies have shown that higher levels of SCFAs are generated by the microbiota in the small intestine of lean subjects.9 The signaling properties of the altered SCFAs might therefore be partly responsible for the metabolic and immunologic effects of the intestinal microbiota. The microbiota also regulates hepatic production of triglycerides by suppressing lipoprotein lipase inhibitors (angiopoietin-like 4, or fastinginduced adipose factor), resulting in continued expression of lipoprotein lipase and continued production of liver triglycerides.56 The microbiota has also been shown to regulate insulin sensitivity and the development of nonalcoholic
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fatty liver disease through its influence on the production of inflammatory cytokines such as tumor necrosis factor a.57 It is unclear whether these changes are mediated by gut luminal signaling events or the accumulation of bacterial products in the portal circulation. Diet is an important determinant of the composition of the gastrointestinal microbiota.58 Indigestible dietary carbohydrates are the main substrate available to intestinal bacteria, specifically resistant starches, dietary fibers, unabsorbed sugars, and alcohols. These are broken down via bacterial metabolism to complex monosaccharides and oligosaccharides and then to SCFAs. Other metabolites such as carbon dioxide and hydrogen are also produced, including branched-chain fatty acids, ammonia, choline, hydrogen sulfide, amines, phenols, indoles, and mercaptanes.59 These are important nutrients not only for the host but also for the commensal bacteria in the gut lumen. Low levels of choline in the diet have been associated with low levels of g-proteobacteria and high levels of Erysipelotrichi as well as hepatic steatosis in rodents and humans.60 Altered levels of choline may promote the development of nonalcoholic fatty liver disease through the effects of toxic methylamines and have been correlated with cardiovascular disease.27 SCFAs affect mediators of inflammation (such as tumor necrosis factor a and b, interleukin-6, interleukin-1b) and prevent activation of the transcription factor nuclear factor kB.61 Moreover, butyrate has been shown in animal and cell culture models to stimulate leptin production by adipocytes and GLP1 secretion by the L cells, whereas propionate is used for gluconeogenesis and lipogenesis in the liver.62 Butyrate may also have direct effects on gene expression by acting as a histone deacetylase inhibitor and has been shown to affect DNA methylation, proliferation, and differentiation in mammalian colonic epithelial cells.63 The prebiotic inulin caused a specific increase in Bifidobacterium species in mice with diet-induced obesity and a corresponding reduction in adiposity, most likely mediated via increased intestinal SCFAs.64 Identical findings were reported in obese mice on weight and glucose or lipid metabolism after dietary enrichment with pure butyrate.65 Likewise, dietary fats have distinct effects on the gut microbiota66; high-fat diets increase numbers of Firmicutes and Proteobacteria and subsequent inflammatory tone,67 potentially within 24 hours after the dietary composition is altered.58 The ability of the gut microbiota to affect host metabolism is therefore mediated by interactions between 4 key components: host nutrient intake, bile acids, luminal mucus layer, and the gut microbiota (and the SCFAs they produce). These interactions affect expression of hormones and signaling molecules, neuron activities, and immune function (Figure 1). The gastrointestinal mucus gel is divided into a secreted, loosely associated layer and a layer firmly attached to the mucosa.65 Mucus and degradation products of epithelial cells are powerful substrates for bacterial metabolism.68 Goblet cells produce a protective mucus layer that forms a barrier and provides lubricant for the gastrointestinal tract. Glycans, secreted by cells in the mucus layer, are a rich source of carbohydrates, comprising 50% to 80% of
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oligosaccharides by weight.69 Those with a deficiency in the production of fucose, a hexose sugar, are unable to glycosylate the terminal end of mucus glycans in the gut. Changes observed in the gut microbiota of (antibiotic-induced) fucose-deficient animals support a model in which the mucus layer interacts with and affects the quality of commensal mucosal bacteria.70 These data corroborate studies of vancomycin showing significant alterations in the commensal bacteria, with significant increases in bifidobacteria in mice and increased thickness of the luminal mucus layer.71 As previously described, proportions of A muciniphila, a mucin-degrading bacterium from the Verrucomicrobia, have been inversely correlated with body weight.26 Interestingly, diet-induced obese mice have decreased proportions of Akkermansia,72 whereas mice that have undergone gastric bypass surgery have increased proportions compared with before surgery. A prebiotic-enriched diet that contains oligofructose, a major constituent of the luminal mucin layer that activates intestinal goblet cells, increases concentrations of Akkermansia.26,73 The SCFAs can signal through several G protein–coupled receptors, including GPR41 and GPR43.74 GPR41 and GPR43 (encoded by FFAR2) are expressed by enteroendocrine cells and activated by SCFAs and other microbial metabolites.62 GPR41-knockout mice with conventional microbiota have reduced adiposity compared with wild-type mice with conventional microbiota, whereas germ-free wild-type and GPR41-knockout mice have similar levels of adiposity. These observations indicate a role for SCFA receptors in fat deposition.62 4-Cresol, a metabolite by-product of tyrosine metabolism, is believed to be produced by Clostridium, Bifidobacterium, and Bacteroides fragilis. Levels of 4-cresol metabolites in human urine correlate with diverse pathophysiologies, including obesity and inflammatory bowel disease, which are conditions that also have been associated with differences in the composition of the gut bacterial community.75
414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 Therapeutic Potential 454 of the Microbiota 455 The large amount of evidence that a healthy microbiota 456 is required for human health and protects against disease 457 has led to much interest in its therapeutic potential. Fecal 458 microbial transplantation (FMT) has been shown to be an 459 effective therapy for recurrent or refractory Clostridium 460 difficile–induced colitis; its success in early trials has led to 461 more widespread use. 462 The ability of the microbiota to transfer metabolic phe- 463 notypes among mice indicates the potential of FMT and 464 related approaches for treating metabolic disease in 465 patients. FMT from healthy, lean subjects to obese subjects 466 has been reported to improve peripheral insulin sensitivity.9 Q11467 These results provide an important proof of concept for 468 microbiota-directed treatment of metabolic disorders with a 469 distinct effect of the donor’s intestinal microbiota signature 470 (so-called superdonor). Microbial communities change with 471 body weight in rodents and humans. Proportions of A 472
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Figure 1. Mechanisms of bile acid and SCFA metabolism in the (A) physiological and (B) pathophysiological state. Altered SCFA production by intestinal microbiota leads to perturbations in bile acid, lipid, and glucose metabolism as well as increased intestinal permeability, resulting in aggravated metabolic endotoxemia and subsequent low-grade inflammation.
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muciniphila, a mucin-dependent butyrate-producing strain that resides in the mucus layer, are inversely correlated with body weight and metabolic profile.26 Oral gavage of A muciniphila led to a corresponding increase in gut colonization and reversed high-fat diet–induced metabolic changes such as gain in fat mass, metabolic endotoxemia, adipose tissue inflammation, and insulin resistance; these effects were not observed after transfer of heat-killed organisms. Akkermansia is only one of several bacterial genera in which the proportions are associated with metabolic function. Its ability to affect the metabolic phenotype, however, provides evidence that it might be possible to treat patients with defined intestinal bacterial populations (single or multiple organisms) rather than incompletely defined, patient-derived products. Increasing our understanding of the mechanisms by which the microbiota influence energy balance and metabolic function could lead to interventions (eg, foods or medications that target specific host factors or bacteria) that achieve the physiological effects of a healthy microbiota. The observation that bacterial expression of the cutC gene increased the risk of cardiovascular disease provides a case in point. The product of the cutC gene increases the conversion of choline to TMA, a potential promoter of cardiovascular pathology. Manipulating the microbiota to decrease the prevalence of cutC-expressing organisms is one therapeutic approach. Therapies aimed at decreasing cutC expression or those that counteract the yield of TMA or the effects of TMA itself could offer significant new alternatives for the treatment of these metabolic disorders.76 Investigating the mechanisms by which some microbiota induce satiety, increase insulin signaling, or provide other metabolic benefits could identify analogous approaches for treatment of obesity, diabetes, fatty liver disease, and other metabolic disorders associated with obesity (Figure 2).
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To date, the most effective sustainable treatment for weight reduction is Roux-en-Y gastric bypass (RYGB) surgery. Patients who undergo RYGB surgery lose w30% of their excess body weight and have significant improvements in glucose homeostasis within the first year after surgery.5 Although the mechanisms that produce these effects are not clear, the intestinal bacteria greatly change after the procedure, characterized by increased proportions of Gammaproteobacteria (Escherichia) and Verrucomicrobia (Akkermansia).5 To test the hypothesis that some of the effects of RYGB surgery are mediated by interactions between host cells and the microbiota, researchers created a mouse model of RYGB. When cecal contents were transferred from mice that underwent gastric bypass surgeries into germ-free mice, the recipients showed significant weight loss and decreased fat mass compared with the recipients of cecal contents from mice that underwent a sham procedure.72 On the other hand, the ability of the microbiota to transmit weight gain was recently shown using feces from weight-discordant pairs of human twins.77 Furthermore, transfer of fecal material from obese and lean littermates to germ-free mice produced a 15% to 17% greater increase in adiposity in recipients of microbiota from obese mice than lean mice. In a second set of experiments, recipients of microbiota from lean mice were cohoused with recipients of microbiota from obese mice and nonconventionalized germfree mice. The microbiota from lean recipients appeared to spread to all groups of mice affecting body weight.77 There are few data on the metabolic effects of FMT in humans. Recent studies have described alterations in the microbiota that induce changes in glucose9 and bile acid metabolism.78 In the first study, intestinal microbiota was transferred from lean human donors to recipients with metabolic syndrome via a postpyloric enteral feeding tube. A separate group of subjects underwent autologous
Figure 2. Three major pathways via which intestinal microbiota can affect human metabolism. Host metabolism and energy balance are influenced by an interplay of nutrients, bile acids, intestinal microbiota, and composition of the epithelial mucus layer. The processes in the intestinal lumen exert their effects systemically on metabolism via production of gut hormones (including glucose-dependent insulinotropic polypeptide and glucagon-like peptide), affecting (para)sympathetic tone and (dys)regulation of immune cell action. GI, gastrointestinal.
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transplant of intestinal microbiota (reinfusion of their own microbiota [controls]). Insulin sensitivity was measured in recipients of the allogeneic stool preparation at baseline and after 6 weeks using the hyperinsulinemic euglycemic clamp technique. Six weeks after allogeneic FMT, insulin sensitivity significantly increased, with higher median rates of glucose disappearance. Gut microbial diversity also increased after allogeneic FMT but was unchanged in the control group. Fecal SCFAs were also significantly altered after allogeneic FMT compared with controls. These experiments suggest that increased bacterial diversity is associated with reduced insulin resistance. However, this was a small study and FMT from only specific donors had beneficial effects (eg, superdonor),9 so further studies are needed and currently ongoing in our department. In conclusion, the intestinal microbiota is an important factor in the prevention and treatment of metabolic dysregulation. Studies that have examined the effects of transferring microbiota between conventional and germ-free rodents, humans and rodents, and humans and humans have shown its role in the development of obesity and subsequent insulin resistance. Whether and to what extent specific intestinal bacterial strains are relevant diagnostic and therapeutic targets remains to be proven.
References 1. Swinburn BA, et al. The global obesity pandemic: shaped by global drivers and local environments. Lancet 2011; 378:804–814. 2. Eckel RH, et al. The metabolic syndrome. Lancet 2010; 375:181–183. 3. Klein S, et al. Absence of an effect of liposuction on insulin action and risk factors for coronary heart disease. N Engl J Med 2004;350:2549–2557. 4. Fabbrini E, Tamboli RA, et al. Surgical removal of omental fat does not improve insulin sensitivity and cardiovascular risk factors in obese adults. Gastroenterology 2010;139. 448–445. 5. Kong LC, Tap J, Aron-Wisnewsky J, et al. Gut microbiota after gastric bypass in human obesity: increased richness and associations of bacterial genera with adipose tissue genes. Am J Clin Nutr 2013;98:16–24. 6. Amar J, Serino M, Lange C, et al; D.E.S.I.R. Study Group. Involvement of tissue bacteria in the onset of diabetes in humans: evidence for a concept. Diabetologia 2011; 54:3055–3061. 7. Cani PD, et al. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes 2007;56:1761–1772. 8. Moreno-Navarrete JM, et al. Circulating lipopolysaccharide-binding protein (LBP) as a marker of obesityrelated insulin resistance. Int J Obes (Lond) 2012;36: 1442–1449. 9. Vrieze A, et al. Transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome. Gastroenterology 2012;143: 913–916.e7. 10. Turnbaugh PJ, et al. The human microbiome project. Nature 2007;449:804–810.
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11. de Vos WM, Nieuwdorp M. Genomics: a gut prediction. Nature 2013;498:48–49. 12. Karlsson FH, et al. Gut metagenome in European women with normal, impaired and diabetic glucose control. Nature 2013;498:99–103. 13. Qin J, et al. A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature 2012;490:55–60. 14. Wikoff WR, et al. Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. Proc Natl Acad Sci U S A 2009;106:3698–3703. 15. Cho I, et al. Antibiotics in early life alter the murine colonic microbiome and adiposity. Nature 2012;488: 621–626. 16. Gesta S, et al. Evidence for a role of developmental genes in the origin of obesity and body fat distribution. Proc Natl Acad Sci U S A 2006;103:6676–6681. 17. Karastergiou K, et al. Distinct developmental signatures of human abdominal and gluteal subcutaneous adipose tissue depots. J Clin Endocrinol Metab 2013;98: 362–371. 18. Apovian CM, et al. Adipose macrophage infiltration is associated with insulin resistance and vascular endothelial dysfunction in obese subjects. Arterioscler Thromb Vasc Biol 2008;28:1654–1659. 19. Wentworth JM, et al. Pro-inflammatory CD11cþCD206þ adipose tissue macrophages are associated with insulin resistance in human obesity. Diabetes 2010;59: 1648–1656. 20. Cani PD, Delzenne NM. Gut microflora as a target for energy and metabolic homeostasis. Curr Opin Clin Nutr Metab Care 2007;10:729–734. 21. Clarke TB, et al. Recognition of peptidoglycan from the microbiota by Nod1 enhances systemic innate immunity. Nat Med 2010;16:228–231. 22. Vijay-Kumar M, et al. Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5. Science 2010;328:228–231. 23. Schwiertz A, et al. Microbiota and SCFA in lean and overweight healthy subjects. Obesity (Silver Spring) 2010; 18:190–195. 24. Maslowski KM, et al. Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature 2009;461:1282–1286. 25. Donohoe DR, et al. The microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon. Cell Metab 2011;13:517–526. 26. Everard A, et al. Cross-talk between Akkermansia muciniphila and intestinal epithelium controls dietinduced obesity. Proc Natl Acad Sci U S A 2013;110: 9066–9071. 27. Wang Z, et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature 2011; 472:57–63. 28. Bennett BJ, et al. Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation. Cell Metab 2013;17: 49–60. 29. Koeth RA, et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med 2013;19:576–585.
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30. Tang WH, et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med 2013;368:1575–1584. 31. de Vos WM, de Vos EA. Role of the intestinal microbiome in health and disease: from correlation to causation. Nutr Rev 2012;70(suppl 1):S45–S56. 32. Ley RE, et al. Obesity alters gut microbial ecology. Proc Natl Acad Sci U S A 2005;102:11070–11075. 33. Ley RE, et al. Microbial ecology: human gut microbes associated with obesity. Nature 2006;444:1022–1023. 34. Backhed F, et al. The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci U S A 2004;101:15718–15723. 35. Backhed F, et al. Mechanisms underlying the resistance to diet-induced obesity in germ-free mice. Proc Natl Acad Sci U S A 2007;104:979–984. 36. Tilg H. Obesity, metabolic syndrome, and microbiota: multiple interactions. J Clin Gastroenterol 2010;44(suppl 1): S16–S18. 37. Turnbaugh PJ, et al. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 2006;444:1027–1031. 38. Gao Z, et al. Butyrate improves insulin sensitivity and increases energy expenditure in mice. Diabetes 2009;58: 1509–1517. 39. Jones BV, et al. Functional and comparative metagenomic analysis of bile salt hydrolase activity in the human gut microbiome. Proc Natl Acad Sci U S A 2008; 105:13580–13585. 40. Wostmann BS. Intestinal bile acids and cholesterol absorption in the germfree rat. J Nutr 1973;103:982–990. 41. Abrams GD, Bishop JE. Effect of the normal microbial flora on gastrointestinal motility. Proc Soc Exp Biol Med 1967;126:301–304. 42. Madsen D, et al. Analysis of bile acids in conventional and germfree rats. J Lipid Res 1976;17:107–111. 43. Miyata M, et al. Administration of ampicillin elevates hepatic primary bile acid synthesis through suppression of ileal fibroblast growth factor 15 expression. J Pharmacol Exp Ther 2009;331:1079–1085. 44. Vrieze A, Out C, Fuentes S, et al. Vancomycin decreases insulin sensitivity and is associated with alterations in intestinal microbiota and bile acid composition in obese subjects with metabolic syndrome. J Hepatol 2013 Dec 6 [Epub ahead of print]. 45. Thomas C, et al. TGR5-mediated bile acid sensing controls glucose homeostasis. Cell Metab 2009;10:167–177. 46. Watanabe M, et al. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature 2006;439:484–489. 47. Jiménez E, Marín ML, et al. Is meconium from healthy newborns actually sterile? Res Microbiol 2008;159: 187–193. 48. Blustein J, Attina T, Liu M, et al. Association of caesarean delivery with child adiposity from age 6 weeks to 15 years. Int J Obes (Lond) 2013;37:900–906. 49. Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature 2012;486:207–214.
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50. Faith JJ, et al. The long-term stability of the human gut microbiota. Science 2013;341:1237439. 51. Amar J, Burcelin R, Ruidavets JB, et al. Energy intake is associated with endotoxemia in apparently healthy men. Am J Clin Nutr 2008;87:1219–1223. 52. Tsukumo DM, Carvalho-Filho MA, et al. Loss-of-function mutation in Toll-like receptor 4 prevents diet-induced obesity and insulin resistance. Diabetes 2007;56: 1986–1998. 53. Cho I, Yamanishi S, et al. Antibiotics in early life alter the murine colonic microbiome and adiposity. Nature 2012; 488:621–626. 54. Trasande L, Blustein J, et al. Infant antibiotic exposures and early-life body mass. Int J Obes (Lond) 2013;37:16–23. 55. Backhed F, et al. Host-bacterial mutualism in the human intestine. Science 2005;307:1915–1920. 56. Koliwad SK, Kuo T, Shipp LE, et al. Angiopoietin-like 4 (ANGPTL4, fasting-induced adipose factor) is a direct glucocorticoid receptor target and participates in glucocorticoid-regulated triglyceride metabolism. J Biol Chem 2009;284:25593–25601. 57. Henao-Mejia J, Elinav E, et al. Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature 2012;482:179–185. 58. Wu GD, Chen J, et al. Linking long-term dietary patterns with gut microbial enterotypes. Science 2011;334: 105–108. 59. Macfarlane GT, Macfarlane S. Bacteria, colonic fermentation, and gastrointestinal health. J AOAC Int 2012; 95:50–60. 60. Spencer MD, et al. Association between composition of the human gastrointestinal microbiome and development of fatty liver with choline deficiency. Gastroenterology 2011;140:976–986. 61. Luhrs H, et al. Butyrate inhibits NF-kappaB activation in lamina propria macrophages of patients with ulcerative colitis. Scand J Gastroenterol 2002;37:458–466. 62. Samuel BS, et al. Effects of the gut microbiota on host adiposity are modulated by the short-chain fatty-acid binding G protein-coupled receptor, Gpr41. Proc Natl Acad Sci U S A 2008;105:16767–16772. 63. Davie JR. Inhibition of histone deacetylase activity by butyrate. J Nutr 2003;133(suppl):2485S–2493S. 64. Cani PD, et al. Selective increases of bifidobacteria in gut microflora improve high-fat-diet-induced diabetes in mice through a mechanism associated with endotoxaemia. Diabetologia 2007;50:2374–2383. 65. Moran AP, Gupta A, Joshi L. Sweet-talk: role of host glycosylation in bacterial pathogenesis of the gastrointestinal tract. Gut 2011;60:1412–1425. 66. Le Chatelier E, Nielsen T, et al. Richness of human gut microbiome correlates with metabolic markers. Nature 2013;500:541–546. 67. Cotillard A, Kennedy SP, et al. Dietary intervention impact on gut microbial gene richness. Nature 2013; 500:585–588. 68. Blaut M, Clavel T. Metabolic diversity of the intestinal microbiota: implications for health and disease. J Nutr 2007;137(suppl 2):751S–755S.
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69. Corfield AP, et al. Mucins in the gastrointestinal tract in health and disease. Front Biosci 2001;6:D1321–D1357. 70. Pelissier MA, et al. Metronidazole effects on microbiota and mucus layer thickness in the rat gut. FEMS Microbiol Ecol 2010;73:601–610. 71. Hansen CH, et al. Early life treatment with vancomycin propagates Akkermansia muciniphila and reduces diabetes incidence in the NOD mouse. Diabetologia 2012; 55:2285–2294. 72. Liou AP, Paziuk M, Luevano JM Jr, et al. Conserved shifts in the gut microbiota due to gastric bypass reduce host weight and adiposity. Sci Transl Med 2013;5: 178ra41. 73. Kleessen B, Hartmann L, Blaut M. Fructans in the diet cause alterations of intestinal mucosal architecture, released mucins and mucosa-associated bifidobacteria in gnotobiotic rats. Br J Nutr 2003;89: 597–606. 74. Tazoe H, et al. Roles of short-chain fatty acids receptors, GPR41 and GPR43 on colonic functions. J Physiol Pharmacol 2008;59(suppl 2):251–262. 75. Ott SJ, et al. Reduction in diversity of the colonic mucosa associated bacterial microflora in patients with active inflammatory bowel disease. Gut 2004;53:685–693.
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76. Craciun S, Balskus EP. Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme. Proc Natl Acad Sci U S A 2012;109:21307–21312. 77. Ridaura VK, et al. Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science 2013; 341:1241214. 78. Weingarden AR, Chen C, et al. Microbiota transplantation restores normal fecal bile acid composition in recurrent Clostridium difficile infection. Am J Physiol Gastrointest Liver Physiol 2014;306:G310–G319.
1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 Received November 21, 2013. Accepted February 16, 2014. 1015 1016 Reprint requests 1017 Address requests for reprints to: Max Nieuwdorp, MD, PhD, Department of Internal and Vascular Medicine, AMC-UvA, Meibergdreef 9, Room F4-159.2, 1018 1091 EN Amsterdam, The Netherlands. e-mail: [email protected]; 1019 Lee M. Kaplan, MD, Obesity, Metabolism & Nutrition Institute, Massachusetts General Hospital, Boston, Massachusetts 02114; e-mail: 1020 [email protected]. Q5 1021 Conflicts of interest 1022 Q1 The authors disclose no conflicts. 1023 1024 Funding Supported by the 2012 EU RESOLVE Consortium (grant FP7-EU 305707 to Q21025 P.W.G.), National Institutes of Health grant DK088661 (to L.M.K.), and a Heart Q3 1026 and Stroke Foundation of Canada Junior Personnel Grant (HSFCR5 to N.P.). 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062
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60 61 62 63 64 65 66 67 68 69 70 71 72 1,2 4,5 5 Max Nieuwdorp P. W. Gilijamse N. Pai Lee M. Kaplan 73 74 Departments of 1Vascular Medicine and 3Endocrinology and Metabolism, Academic Medical Center, University of Amsterdam, 75 Amsterdam, The Netherlands; 2Wallenberg Laboratory, Sahlgrenska Center for Cardiovascular and Metabolic Research, 76 University of Göteborg, Göteborg, Sweden; 4Department of Pediatric Gastroenterology & Nutrition, Boston Children’s Hospital, 77 Harvard Medical School, Boston, Massachusetts; and 5Obesity, Metabolism and Nutrition Institute and Gastrointestinal Unit, 78 Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 79 80 What other factors could contribute to insulin resistance 81 Intestinal microbes regulate metabolic function and energy balance; an altered microbial ecology is believed to in obese people? Based on findings from animal studies, the 82 contribute to the development of several metabolic dis- intestinal microbiota is involved in the development of in83 eases. Relative species abundance and metabolic charac- sulin resistance.5 Metabolic syndrome is a chronic inflam84 teristics of the intestinal microbiota change substantially matory disease, and translocation of intestinal bacteria has 85 in those who are obese or have other metabolic disorders been proposed to promote metabolic endotoxemia.6 Several 86 and in response to ingested nutrients or therapeutic studies have associated obesity with insulin resistance and agents. The mechanisms through which the intestinal increased levels of plasma lipopolysaccharide (LPS)-binding 87 microbiota and its metabolites affect host homeostasis are protein.7,8 Transplantation of fecal preparations from lean 88 just beginning to be understood. We review the relation- donors can temporarily improve insulin sensitivity.9 These Q789 90 ships between the intestinal microbiota and host metafindings indicate a relationship among the intestinal 91 bolism, including energy intake, use, and expenditure, in microbial, bacterial translocation, and host metabolism. 92 relation to glucose and lipid metabolism. These associaIt is a challenge to study the effects of the microbiota on 93 tions, along with interactions among the intestinal microhuman metabolism. The human intestinal microbiome in- 94 biota, mucus layer, bile acids, and mucosal immune (meta) 95 responses, reveal potential mechanisms by which the cludes 3000 to 5000 species that share a collective 10 High- 96 genome comprising nearly 5 million genes. microbiota affect metabolism. We discuss how controlled studies involving direct perturbations of microbial com- throughput sequencing has allowed for comprehensive an- 97 munities in human and animal models are required to alyses of the intestinal metagenome based on the genetic 98 material from fecal samples. When metagenomic analysis 99 identify effective therapeutic targets in the microbiota. data are compared with clinical symptoms or disease, the 100 analysis is called a metagenome-wide association study.11 101 Keywords: Obesity; Energy Expenditure; Bile Acids; Gut Such studies have shown significant differences in the 102 Microbiota. composition of the intestinal microbiota metagenome be- 103 tween metabolically healthy and unhealthy subjects.12,13 104 Studies of germ-free mice have implicated the intestinal 105 he obesity pandemic presents a considerable chal- microbiota in drug metabolism and macronutrient diges106 lenge for health care systems worldwide. More than tion,14 and antibiotic treatments aimed at reducing the 107 500 million people are obese, and the prevalence of obesity bacterial load in the intestine have profound effects on the 108 1 is increasing rapidly. Obese people are at risk for devel- production of metabolites by the gut microbiota.15 109 oping dyslipidemia, nonalcoholic fatty liver disease, and 110 2 insulin resistance. Notably, approximately one-half of all 111 obese people develop metabolic syndrome, which is 112 strongly associated with greater amounts of abdominal ad113 used in this paper: FMT, fecal microbial transplantation; ipose tissue, although little is understood about the mech- Abbreviations LPS, lipopolysaccharide; RYGB, Roux-en-Y gastric bypass; SCFA, short114 2 anisms of this association. However, obese insulin-resistant chain fatty acid; TMA, trimethylamine; TMAO, trimethylamine-N-oxide. 115 patients who undergo either subcutaneous adipose tissue 116 3 © 2014 by the AGA Institute removal or surgical removal of intra-abdominal adipose 0016-5085/$36.00 117 4 tissue do not have increased insulin sensitivity. http://dx.doi.org/10.1053/j.gastro.2014.02.008 118
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Obesity and insulin resistance are closely related to the presence of (visceral) adipose tissue inflammation. For example, adipose tissue produces numerous inflammatory cytokines,16 and there is evidence that visceral adipose tissue—and, to a lesser extent, subcutaneous adipose tissue—can promote insulin resistance.17 The presence of subcutaneous adipose tissue macrophages and crown-like structures (or accumulated CD68-positive macrophages) correlates with expression of genes that control inflammation,18 indicating a role for the innate immune system in insulin resistance.19 Changes in intestinal microbiota cause long-term changes in inflammation in obese subjects12; bacterial endotoxins such as LPS activate pattern recognition receptors such as Toll-like receptors, leading to an innate immune response and insulin resistance.20 The intestinal microbiota also produces many molecules that promote inflammation, such as peptidoglycans21 and flagellins,22 which activate inflammatory pathways, resulting in obesity and insulin resistance. Short-chain fatty acids (SFCAs) are produced by intestinal bacteria and may play important roles in dampening the adverse effects of intestinal pathogens on host metabolism. Diet-derived fibers are metabolized and fermented to SCFAs such as acetate, propionate, and butyrate by intestinal bacteria.23 Germ-free mice produce almost no SCFAs and therefore have altered responses to inflammatory stimuli.24 SCFAs may serve as an energy source for the intestinal epithelium and liver, given their transport predominantly via the portal vein after intestinal absorption. SCFAs might also modulate the immune response by reducing intestinal permeability.25 This hypothesis is supported by the observation that translocation of LPS from the gut to the portal vein produces obesity-associated low-grade inflammation and subsequent insulin resistance in mice, which can partly be reversed by administration of the propionate-producing bacteria Akkermansia muciniphila.26 Besides obesity, the intestinal microbiota might also be involved in atherogenesis. Specific dietary nutrients characterized by trimethylamine groups (eg, choline, phosphatidylcholine, and carnitine) are metabolized into the atherogenic compound trimethylamine-N-oxide (TMAO) by bacteria.27,28 Studies using germ-free mice or mice given broad-spectrum antibiotics showed that the intestinal microbiota is required for formation of trimethylamine (TMA) and TMAO.29 Further, bacterial colonization of germfree mice increases their plasma levels of TMAO, indicating that the intestinal microbiota is required for generation of TMA from sources of dietary choline or carnitine (such as eggs, milk, and red meat).30 For example, carnitine is an abundant nutrient in red meat, and the intestinal microbiota mediates production of TMAO from dietary L-carnitine.
Alteration of the Gut Microbiota in Obesity and Diabetes The intestinal microbiota is altered in humans and animal models of obesity.31 The intestinal (cecum-derived) microbiota of ob/ob mice has a 50% reduction in levels of
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Bacteroidetes and an increased proportion of Firmicutes compared with wild-type mice.32 The composition of the fecal microbiota of obese human subjects is similarly affected but changes with weight loss.33 Studies in germfree mice provide insights into the effects of the intestinal microbiota on host metabolism. Germ-free mice fed high-fat, high-sugar diets did not have the same metabolic disturbances as the littermates that were not germ free.34 Transfer of intestinal microbiota from obese mice resulted in significantly greater adiposity in recipients than transfer of microbiota from lean donors.35 One way in which intestinal microbes might affect host metabolism is by extracting calories from otherwise indigestible carbohydrates36; these carbohydrates are fermented by intestinal microbes to produce SCFAs.38 SCFAs may act as an energy substrate as they are absorbed by the intestinal epithelium and metabolized in the liver.37 Mouse models of obesity and human obese subjects have increased intestinal (cecal) levels of SCFA and decreased energy content in their feces.23 Studies have associated changes in proportions of Bacteroidetes and Firmicutes with obesity and metabolic syndrome. Metagenome-wide association studies by Qin et al13 (performed in China) and Karlsson et al12 (performed in Europe) reported metagenomic differences between a cohort of patients with type 2 diabetes mellitus and a group of healthy subjects. Clusters of genomic sequences were used as signatures for specific groups of bacteria, and each study found independently that the microbiota of subjects with type 2 diabetes mellitus had a lower proportion of butyrate-producing Clostridiales (Roseburia and Faecalibacterium prausnitzii), and greater proportions of Clostridiales that do not produce butyrate, as well as pathogens such as Clostridium clostridioforme. Other associations differed between the 2 studies. Karlsson et al12 detected an increased proportion of Lactobacillus gasseri and Streptococcus mutans (commensal bacteria in the mouth and upper intestinal tract) in their cohort with type 2 diabetes mellitus. Qin et al13 observed a greater proportion of Escherichia coli (which produce LPS to cause endotoxemia) in patients with type 2 diabetes mellitus. These studies raise interest in the association between type 2 diabetes mellitus and reduced production of butyrate because diets supplemented with butyrate were previously shown to prevent and reverse insulin resistance in mice that became obese on high-calorie diets and increase energy expenditure.38 Combined results from human and animal studies of obesity suggest that reduced butyrate production by the microbiota contributes to the development of insulin resistance.
Microbiota and Bile Acids Bile acids are secreted as glycine, taurine, or sulfate conjugates. Compounds excreted in bile reach the intestinal tract, where they can be deconjugated by gut microbiota, aided by populations of microorganisms with their own hydrolytic enzymes such as b-glucuronidase and sulfatases.39 Most liver-secreted bile acids (95%) are reabsorbed in the ileum to be taken up by the liver in the enterohepatic
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cycle. Only a small part of the bile acid pool escapes the enterohepatic cycle and travels toward the large intestine to be excreted in the feces. This excretion is accompanied by microbial deconjugation of glycine- and taurine-conjugated bile acid; germ-free mice were shown to have altered metabolism of bile acids.39–41 Follow-up studies showed that germ-free mice had increased levels of conjugated bile acids throughout the intestine, with no deconjugation, and strongly decreased fecal excretion.42 More recently, studies with antibiotics provided further support for the importance of intestinal microbiota in bile acid metabolism. Mice given ampicillin for 3 days increased biliary bile acid output 3-fold, whereas fecal output decreased 70%.43 We recently showed that vancomycin therapy (which eradicates gram-positive bacteria) of patients with insulin resistance also significantly altered the fecal bile acid pool, reducing the proportion of secondary bile acids, compared with primary bile acids, which is associated with reduced insulin sensitivity.44 Grampositive bacteria in the intestine therefore affect the bile acid pool, which alters regulation of lipid and glucose metabolism via the FXR45 and TGR546 signaling pathways.
Intestinal Microbiota and Host Physiology and Diseases Recent advances in sequencing technology and largescale information processing have increased our understanding of the diversity of the intestinal microbiota and its relationship to host physiology and diseases. Comprehensive characterizations of the microbiota have revealed rapid and profound effects of environmental and physiological changes on the composition and metabolic activities of the microbiota. Conversely, the microbial ecology has been correlated with specific diseases such as obesity and insulin resistance, and microbiota transfer studies in animals37 and humans6 have shown that alterations in the gut microbiota contribute to the pathogenesis of these disorders. Studies of development of the microbiota in children have provided important information about interactions between host tissues and the microbiota and how disruptions in the microbiota can contribute to disease development. Newborns have essentially no intestinal microbiota. However, low levels of bacterial translocation through the placental circulation likely provide a primitive bacterial community before birth.47 Nevertheless, the newborn intestine begins colonization by maternal and environmental bacteria during birth. The structure of this primary microbiota varies with mode of birth (cesarean section vs vaginal delivery), feeding (breastfeeding vs formula), and level of sanitation in the environment.48 After the first year of life, each infant develops a distinct microbial community; by approximately 3 years, microbial profiles resemble those of adults in patterns and diversity.49 The progressive maturation of the intestinal microbiota is likely to be important for the establishment of commensal and pathogen recognition by the immune system and determine later host-microbe interactions and susceptibility to infection, autoimmunity, and inflammatory diseases.
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The composition of the gut microbiota remains relatively 296 stable from late childhood to old age, when changes occur 297 again.50 These late changes often correspond with age- 298 related changes in diet and digestive physiology, but it is 299 not clear how these changes affect the gut microbiota. The 300 structure of the gut microbiota varies spatially along the 301 intestinal tract and cross-sectionally. The bacteria that 302 reside in close proximity to the mucosal surface are believed 303 to interact more closely with the immune system, whereas 304 bacteria that reside in the lumen may be more closely 305 associated with food and products of digestion, influencing 306 nutrient assimilation, signaling, and metabolic function. 307 Cani et al used the term “metabolic endotoxemia” to 308 describe the inflammatory state often associated with the 309 metabolic syndrome. This systemic inflammation is believed 310 to occur as bacteria translocates through the gastrointes- 311 tinal tract, increasing circulating levels of LPS. Obese sub- 312 jects with insulin resistance have increased circulating 313 levels of LPS compared with matched controls; endotoxemia 314 is independently associated with energy intake even in lean 315 men.6,51 Toll-like receptor 4–null mice are protected from 316 diet-induced obesity and associated insulin resistance.52 317 Antibiotics, which have been used against pathogenic 318 bacteria for nearly a century, also alter the commensal 319 bacterial community, affecting metabolic and immune 320 regulation. Short-term intake of antibiotics, via medication 321 or dietary exposure to antibiotic-treated animals, causes 322 shifts in the microbiota that have detrimental effects on 323 metabolism.44 Recent studies have associated cumulative Q8324 antibiotic exposure with rates of obesity and metabolic 325 disorders. Administration of subtherapeutic doses of anti- 326 biotics to young mice increases their adiposity and pro- 327 motes metabolic dysfunction.53 Similar observations have 328 been reported from epidemiological studies,54 highlighting 329 the importance of commensal intestinal bacteria for meta- 330 bolic homeostasis. 331
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Luminal Factors That Regulate Host Metabolism Commensal gut bacteria regulate immune and metabolic functions via several mechanisms.55 Bacteria release fatty acids, peroxidases, proteases, and bacteriocins that prevent pathogens from expanding in the intestinal community7; as previously mentioned, bacterial disaccharidases ferment unabsorbed dietary carbohydrates into SCFAs. Although increased generation of SCFAs could increase energy uptake from ingested plant carbohydrates, recent studies have shown that higher levels of SCFAs are generated by the microbiota in the small intestine of lean subjects.9 The signaling properties of the altered SCFAs might therefore be partly responsible for the metabolic and immunologic effects of the intestinal microbiota. The microbiota also regulates hepatic production of triglycerides by suppressing lipoprotein lipase inhibitors (angiopoietin-like 4, or fastinginduced adipose factor), resulting in continued expression of lipoprotein lipase and continued production of liver triglycerides.56 The microbiota has also been shown to regulate insulin sensitivity and the development of nonalcoholic
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fatty liver disease through its influence on the production of inflammatory cytokines such as tumor necrosis factor a.57 It is unclear whether these changes are mediated by gut luminal signaling events or the accumulation of bacterial products in the portal circulation. Diet is an important determinant of the composition of the gastrointestinal microbiota.58 Indigestible dietary carbohydrates are the main substrate available to intestinal bacteria, specifically resistant starches, dietary fibers, unabsorbed sugars, and alcohols. These are broken down via bacterial metabolism to complex monosaccharides and oligosaccharides and then to SCFAs. Other metabolites such as carbon dioxide and hydrogen are also produced, including branched-chain fatty acids, ammonia, choline, hydrogen sulfide, amines, phenols, indoles, and mercaptanes.59 These are important nutrients not only for the host but also for the commensal bacteria in the gut lumen. Low levels of choline in the diet have been associated with low levels of g-proteobacteria and high levels of Erysipelotrichi as well as hepatic steatosis in rodents and humans.60 Altered levels of choline may promote the development of nonalcoholic fatty liver disease through the effects of toxic methylamines and have been correlated with cardiovascular disease.27 SCFAs affect mediators of inflammation (such as tumor necrosis factor a and b, interleukin-6, interleukin-1b) and prevent activation of the transcription factor nuclear factor kB.61 Moreover, butyrate has been shown in animal and cell culture models to stimulate leptin production by adipocytes and GLP1 secretion by the L cells, whereas propionate is used for gluconeogenesis and lipogenesis in the liver.62 Butyrate may also have direct effects on gene expression by acting as a histone deacetylase inhibitor and has been shown to affect DNA methylation, proliferation, and differentiation in mammalian colonic epithelial cells.63 The prebiotic inulin caused a specific increase in Bifidobacterium species in mice with diet-induced obesity and a corresponding reduction in adiposity, most likely mediated via increased intestinal SCFAs.64 Identical findings were reported in obese mice on weight and glucose or lipid metabolism after dietary enrichment with pure butyrate.65 Likewise, dietary fats have distinct effects on the gut microbiota66; high-fat diets increase numbers of Firmicutes and Proteobacteria and subsequent inflammatory tone,67 potentially within 24 hours after the dietary composition is altered.58 The ability of the gut microbiota to affect host metabolism is therefore mediated by interactions between 4 key components: host nutrient intake, bile acids, luminal mucus layer, and the gut microbiota (and the SCFAs they produce). These interactions affect expression of hormones and signaling molecules, neuron activities, and immune function (Figure 1). The gastrointestinal mucus gel is divided into a secreted, loosely associated layer and a layer firmly attached to the mucosa.65 Mucus and degradation products of epithelial cells are powerful substrates for bacterial metabolism.68 Goblet cells produce a protective mucus layer that forms a barrier and provides lubricant for the gastrointestinal tract. Glycans, secreted by cells in the mucus layer, are a rich source of carbohydrates, comprising 50% to 80% of
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oligosaccharides by weight.69 Those with a deficiency in the production of fucose, a hexose sugar, are unable to glycosylate the terminal end of mucus glycans in the gut. Changes observed in the gut microbiota of (antibiotic-induced) fucose-deficient animals support a model in which the mucus layer interacts with and affects the quality of commensal mucosal bacteria.70 These data corroborate studies of vancomycin showing significant alterations in the commensal bacteria, with significant increases in bifidobacteria in mice and increased thickness of the luminal mucus layer.71 As previously described, proportions of A muciniphila, a mucin-degrading bacterium from the Verrucomicrobia, have been inversely correlated with body weight.26 Interestingly, diet-induced obese mice have decreased proportions of Akkermansia,72 whereas mice that have undergone gastric bypass surgery have increased proportions compared with before surgery. A prebiotic-enriched diet that contains oligofructose, a major constituent of the luminal mucin layer that activates intestinal goblet cells, increases concentrations of Akkermansia.26,73 The SCFAs can signal through several G protein–coupled receptors, including GPR41 and GPR43.74 GPR41 and GPR43 (encoded by FFAR2) are expressed by enteroendocrine cells and activated by SCFAs and other microbial metabolites.62 GPR41-knockout mice with conventional microbiota have reduced adiposity compared with wild-type mice with conventional microbiota, whereas germ-free wild-type and GPR41-knockout mice have similar levels of adiposity. These observations indicate a role for SCFA receptors in fat deposition.62 4-Cresol, a metabolite by-product of tyrosine metabolism, is believed to be produced by Clostridium, Bifidobacterium, and Bacteroides fragilis. Levels of 4-cresol metabolites in human urine correlate with diverse pathophysiologies, including obesity and inflammatory bowel disease, which are conditions that also have been associated with differences in the composition of the gut bacterial community.75
414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 Therapeutic Potential 454 of the Microbiota 455 The large amount of evidence that a healthy microbiota 456 is required for human health and protects against disease 457 has led to much interest in its therapeutic potential. Fecal 458 microbial transplantation (FMT) has been shown to be an 459 effective therapy for recurrent or refractory Clostridium 460 difficile–induced colitis; its success in early trials has led to 461 more widespread use. 462 The ability of the microbiota to transfer metabolic phe- 463 notypes among mice indicates the potential of FMT and 464 related approaches for treating metabolic disease in 465 patients. FMT from healthy, lean subjects to obese subjects 466 has been reported to improve peripheral insulin sensitivity.9 Q11467 These results provide an important proof of concept for 468 microbiota-directed treatment of metabolic disorders with a 469 distinct effect of the donor’s intestinal microbiota signature 470 (so-called superdonor). Microbial communities change with 471 body weight in rodents and humans. Proportions of A 472
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Figure 1. Mechanisms of bile acid and SCFA metabolism in the (A) physiological and (B) pathophysiological state. Altered SCFA production by intestinal microbiota leads to perturbations in bile acid, lipid, and glucose metabolism as well as increased intestinal permeability, resulting in aggravated metabolic endotoxemia and subsequent low-grade inflammation.
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muciniphila, a mucin-dependent butyrate-producing strain that resides in the mucus layer, are inversely correlated with body weight and metabolic profile.26 Oral gavage of A muciniphila led to a corresponding increase in gut colonization and reversed high-fat diet–induced metabolic changes such as gain in fat mass, metabolic endotoxemia, adipose tissue inflammation, and insulin resistance; these effects were not observed after transfer of heat-killed organisms. Akkermansia is only one of several bacterial genera in which the proportions are associated with metabolic function. Its ability to affect the metabolic phenotype, however, provides evidence that it might be possible to treat patients with defined intestinal bacterial populations (single or multiple organisms) rather than incompletely defined, patient-derived products. Increasing our understanding of the mechanisms by which the microbiota influence energy balance and metabolic function could lead to interventions (eg, foods or medications that target specific host factors or bacteria) that achieve the physiological effects of a healthy microbiota. The observation that bacterial expression of the cutC gene increased the risk of cardiovascular disease provides a case in point. The product of the cutC gene increases the conversion of choline to TMA, a potential promoter of cardiovascular pathology. Manipulating the microbiota to decrease the prevalence of cutC-expressing organisms is one therapeutic approach. Therapies aimed at decreasing cutC expression or those that counteract the yield of TMA or the effects of TMA itself could offer significant new alternatives for the treatment of these metabolic disorders.76 Investigating the mechanisms by which some microbiota induce satiety, increase insulin signaling, or provide other metabolic benefits could identify analogous approaches for treatment of obesity, diabetes, fatty liver disease, and other metabolic disorders associated with obesity (Figure 2).
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To date, the most effective sustainable treatment for weight reduction is Roux-en-Y gastric bypass (RYGB) surgery. Patients who undergo RYGB surgery lose w30% of their excess body weight and have significant improvements in glucose homeostasis within the first year after surgery.5 Although the mechanisms that produce these effects are not clear, the intestinal bacteria greatly change after the procedure, characterized by increased proportions of Gammaproteobacteria (Escherichia) and Verrucomicrobia (Akkermansia).5 To test the hypothesis that some of the effects of RYGB surgery are mediated by interactions between host cells and the microbiota, researchers created a mouse model of RYGB. When cecal contents were transferred from mice that underwent gastric bypass surgeries into germ-free mice, the recipients showed significant weight loss and decreased fat mass compared with the recipients of cecal contents from mice that underwent a sham procedure.72 On the other hand, the ability of the microbiota to transmit weight gain was recently shown using feces from weight-discordant pairs of human twins.77 Furthermore, transfer of fecal material from obese and lean littermates to germ-free mice produced a 15% to 17% greater increase in adiposity in recipients of microbiota from obese mice than lean mice. In a second set of experiments, recipients of microbiota from lean mice were cohoused with recipients of microbiota from obese mice and nonconventionalized germfree mice. The microbiota from lean recipients appeared to spread to all groups of mice affecting body weight.77 There are few data on the metabolic effects of FMT in humans. Recent studies have described alterations in the microbiota that induce changes in glucose9 and bile acid metabolism.78 In the first study, intestinal microbiota was transferred from lean human donors to recipients with metabolic syndrome via a postpyloric enteral feeding tube. A separate group of subjects underwent autologous
Figure 2. Three major pathways via which intestinal microbiota can affect human metabolism. Host metabolism and energy balance are influenced by an interplay of nutrients, bile acids, intestinal microbiota, and composition of the epithelial mucus layer. The processes in the intestinal lumen exert their effects systemically on metabolism via production of gut hormones (including glucose-dependent insulinotropic polypeptide and glucagon-like peptide), affecting (para)sympathetic tone and (dys)regulation of immune cell action. GI, gastrointestinal.
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transplant of intestinal microbiota (reinfusion of their own microbiota [controls]). Insulin sensitivity was measured in recipients of the allogeneic stool preparation at baseline and after 6 weeks using the hyperinsulinemic euglycemic clamp technique. Six weeks after allogeneic FMT, insulin sensitivity significantly increased, with higher median rates of glucose disappearance. Gut microbial diversity also increased after allogeneic FMT but was unchanged in the control group. Fecal SCFAs were also significantly altered after allogeneic FMT compared with controls. These experiments suggest that increased bacterial diversity is associated with reduced insulin resistance. However, this was a small study and FMT from only specific donors had beneficial effects (eg, superdonor),9 so further studies are needed and currently ongoing in our department. In conclusion, the intestinal microbiota is an important factor in the prevention and treatment of metabolic dysregulation. Studies that have examined the effects of transferring microbiota between conventional and germ-free rodents, humans and rodents, and humans and humans have shown its role in the development of obesity and subsequent insulin resistance. Whether and to what extent specific intestinal bacterial strains are relevant diagnostic and therapeutic targets remains to be proven.
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1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 Received November 21, 2013. Accepted February 16, 2014. 1015 1016 Reprint requests 1017 Address requests for reprints to: Max Nieuwdorp, MD, PhD, Department of Internal and Vascular Medicine, AMC-UvA, Meibergdreef 9, Room F4-159.2, 1018 1091 EN Amsterdam, The Netherlands. e-mail: [email protected]; 1019 Lee M. Kaplan, MD, Obesity, Metabolism & Nutrition Institute, Massachusetts General Hospital, Boston, Massachusetts 02114; e-mail: 1020 [email protected]. Q5 1021 Conflicts of interest 1022 Q1 The authors disclose no conflicts. 1023 1024 Funding Supported by the 2012 EU RESOLVE Consortium (grant FP7-EU 305707 to Q21025 P.W.G.), National Institutes of Health grant DK088661 (to L.M.K.), and a Heart Q3 1026 and Stroke Foundation of Canada Junior Personnel Grant (HSFCR5 to N.P.). 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062
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