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The Microbiome and Metabolome in Nonalcoholic Fatty Liver Disease
Chapter 27
The Microbiome and Metabolome in Nonalcoholic Fatty Liver Disease Silvia M. Ferolla1, Cla´udia A. Couto1, Maria de Lourdes A. Ferrari1, Luciana Costa Faria1, Murilo Pereira2 and Teresa C.A. Ferrari1 1
Department of Internal Medicine, Faculty of Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil; 2Post graduation in Functional
Clinical Nutrition, VP Institute, Cruzeiro do Sul University, São Paulo, Brazil
INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in the world [1]. It encompasses a spectrum of conditions that ranges from hepatic steatosis to steatosis associated with necroinflammatory lesions (nonalcoholic steatohepatitis/NASH), which may progress to fibrosis and cirrhosis. It is usually associated with metabolic syndrome (MS). The pathogenesis of NAFLD is related to insulin resistance (IR), as a key factor that initiates hepatic fat accumulation and, potentially, NASH [2,3]. The excessive deposition of triglyceride in the hepatocytes leads to a shift from carbohydrates to FFA mitochondrial beta-oxidation and may promote lipid peroxidation, and accumulation of reactive oxygen species (ROS). These compounds produce a variety of cellular stimuli with inflammatory response, hepatocellular injury, and fibrosis [2]. Nowadays, a growing interest has been devoted to guteliver axis (GLA) dysfunction, i.e., intestinal dysbiosis, bacterial overgrowth, and alteration of mucosa permeability, as relevant in NAFLD progression. It is also a possible alternative therapeutic target, in those patients unable to get benefits deriving from lifestyle modification, healthy diet, and physical activity promotion [4].
GuteLiver Axis The term “guteliver axis” is defined as the strong anatomical and functional interaction, between the gastrointestinal tract and the liver. GLA is characterized by bidirectional traffic. Nutrients and factors derived from gut lumen reach the liver, through the portal circulation. Bile acids produced by hepatocytes are released in the small intestine, through the biliary tract. However, this description is simplistic, as the GLA does not have only a nutritional role. is a complex structure, and the change of two of its components, namely gut barrier and intestinal microbiota, and seems to play an important role in liver lesions and NASH progression.
INTESTINAL ENVIRONMENT The gut microbiota composition is also influenced by diet, age, body weight, infections, medications, intestinal surgeries, and several liver diseases [5e7]. The gut microbiota is important to the host metabolism by secreting bioactive metabolites. It also participates in the development of the intestinal microvilli defense against pathogens, maintains gut immunity, performs the digestion of complex polysaccharides, synthesizes vitamins, and plays a role in fat storage [7e9]. The gut epithelium is a natural barrier that selects entry of useful substances present in the lumen, such as nutrients, and keeps at bay bacteria and their bioproducts. Tight junctions, specialized intercellular structures, assist this control. Derangement of the homeostasis between bacteria and the host, as occurs in small intestine bacterial overgrowth (SIBO),
Microbiome and Metabolome in Diagnosis, Therapy, and other Strategic Applications. https://doi.org/10.1016/B978-0-12-815249-2.00027-0 Copyright © 2019 Elsevier Inc. All rights reserved.
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may cause disruption of the intercellular tight junctions, and subsequent increase in intestinal permeability, leading to bacterial translocation (BT), i.e., transportation of bacteria and their products from the gut lumen into the blood [9]. The portal vein and the hepatic artery supply blood to the liver. The portal blood contains products of digestion and also microbial products derived from the gut microbiota. Therefore, the liver consists in the first site, of exposure and filtration of bacterial products from the intestine, such as lipopolysaccharides (LPS), lipopeptides, unmethylated DNA, and doublestranded RNA [9].
Endotoxin and Liver Inflammation The liver contains components of the immune system, such as macrophages, dendritic cells and natural killer T cells, which act as a first-line defense against endotoxin and microorganisms. Toll-like receptors (TLRs), present on the innate immune cells, consist of a family of type I transmembrane proteins, which recognize pathogen-associated molecular patterns (PAMPs), and damage associated molecular patterns (DAMPs) present on endogenous ligands. They initiate an adaptive immune response signaling cascade, leading to activation of proinflammatory genes, such as TNF-a IL-6, IL-8, and IL-12 genes. LPS, the active component of endotoxin, is the most studied PAMP. The liver is exposed to these PAMPs due to BT [40]. LPS has affinity to LPS-binding protein, which in turn, binds to CD14 and activates TLR4 in Kupffer cells, triggering an essential inflammatory cascade [7]. The synthesis of proinflammatory cytokines leads to prolonged inflammation and hepatocyte damage [10].
Intestinal Inflammasomes The TLRs, together with other sensors of PAMPs and DAMPs, are the inflammasomes, which are formed by a molecular macrocomplex that includes the enzyme caspase-1, whose activation causes the release of bioactive IL-1b and/or IL-18 [9]. Recent evidence suggests that the inflammasome is involved in NAFLD/NASH progression, via modulation of the gut microbiota [11]. Genetic inflammasome deficiency, associated with dysbiosis, determines increased concentration of bacterial products in the portal blood, which may exacerbate steatosis and increase tumor necrosis factor (TNF-a) expression [11]. Another mechanism that supports the role of the gut microbiota in the pathogenesis of NAFLD is the fact that certain types of bacteria inhibit gut epithelial expression of fasting-induced adipocyte factor (FIAF), a suppressor of lipoprotein lipase (LPL). FIAF is produced not only by the gut, but also by liver and adipose tissue, being an essential regulator of peripheral fat storage. By suppressing FIAF, the microbiota increases LPL activity in adipose tissue, enhancing the delivery of adipocyte-derived triglycerides, which determine storage of triacylglycerols in the liver [4]. Additionally, the microbiota is related to IR [12]. The transfer of gut microbiota from lean human donors to recipients with MS, via duodenal tube, resulted in increased insulin sensitivity within 6 weeks [13].
Bile Acids and Farnesoid X Receptor Bile acids are ligands of the farnesoid X receptor (FXR), which is expressed in the liver and gut [14]. The activation of FXR reduces circulating bile acids (feedback mechanism), and participates in the control of the gut-microbiota composition, and in the regulation of lipids and glucose homeostasis in the gut-liver axis. It is known to have a crucial role in hepatic de-novo lipogenesis, VLDL export, and plasma triglyceride turnover [4]. A recent human study showed low FXR protein expression in patients with NASH versus simple NAFLD, suggesting a protective role of FXR in liver disease progression [15].
Gut Microbiome in NAFLD Patients Obese individuals present predominance of Firmicutes and relative low proportion of Bacteroidetes [14]. In animal models, this has been associated with a propensity to NAFLD features like fasting hyperglycemia, insulin resistance, liver steatosis, and high expression of genes involved in de novo lipogenesis [16]. In humans with NASH, the microbiota composition also seems to have a lower proportion of Bacteroidetes, independently of hight fat diet and BMI. The low prevalence of Bacteroidetes might facilitate the growth of other phyla that are more efficient in harvesting energy from the diet [17]. Indeed, obese subjects present more H2-producing Prevotellaceae, and H2-utilizing methanogenic Archaea. The coexistence of H2-producing bacteria with H2-utilizing Archaea suggests that H2 transfer might increase energy uptake by the human large intestine in the obese person [18].
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Response to High-Fat Diet In two mice fed with high-fat diet (HFD) and with similar body weight gain, one mouse (called responder) developed hyperglycemia and presented high serum concentrations of proinflammatory cytokines, whereas the other (called nonresponder), did not. The gut microbiota was transplanted from both the responder, and the nonresponder, to germfree mice. These animals were fed with the same HFD, and also developed comparable obesity. The responder stoolreceiving mice presented fasting hyperglycemia, hyperinsulinemia, hepatic steatosis and high expression of genes involved in de novo lipogenesis. The nonresponder-receiver animals did not develop metabolic abnormalities. Gut microbiota associated with the NAFLD-prone and NAFLD-resistant phenotypes were, respectively, Firmicutes and Bacteroidetes [16]. Mouzaki et al. investigated the NASH human gut microbiota [19] and found a lower proportion of Bacteroides/ Prevotella ratio (herein referred to as Bacteroidetes), when compared to simple steatosis or healthy controls (living liver donors), independently of BMI and high fatty diet. NASH subjects also presented increased number of Clostridium coccoides in their stool, in comparison to the individuals with simple steatosis. In the experience of Zhu et al. [20] in NASH increased abundance of alcohol-producing Escherichia occurred, as well as high bloodeethanol levels, leading to increased oxidative stress, and liver inflammation due to alcohol metabolism. The gut microbiota also produced LPS, which contributes to liver damage, disrupts normal hepatocyte function, leads to mitochondrial oxidative stress, and reduces the clearance of toxins by the hepatocytes.
SIBO AND INCREASED INTESTINAL PERMEABILITY IN NAFLD PATIENTS Evidences suggest that NAFLD subjects present a high prevalence of small intestine bacterial overgrowth/SIBO [21e26] and increased gut permeability [24,27,28]. Most controlled trials demonstrated that the prevalence of SIBO in NAFLD, ranges from 50% to 77.8% [21,22,24,25]. SIBO in NASH individuals is also associated with the rise in hepatic TLR4 expression and release of interleukin (IL)-8 [25]. SIBO has been independently associated with the severity of liver steatosis in histology studies [23,24]. As liver biopsy is not always available, markers of hepatic damage, such as plasminogen activator inhibitor 1 (PAI-1), have been used [27,29]. Some studies have showed that high serum concentrations of PAI-1 are associated with hepatic steatosis, fibrosis [30,31], and elevated serum endotoxin concentrations [27,29]. SIBO may increase gut permeability, leading to endotoxemia, and oxidative stress in the liver. Miele et al. [24] showed high prevalence of SIBO, increased intestinal permeability (urinary excretion of 51Cr-ethylene diamine tetra acetate/ 51Cr-EDTA test), and disruption of the intercellular tight junctions of the gut (immunohistochemical analysis of zona occludens-1/ZO-1 expression in duodenal biopsy specimens), in subjects with NAFLD. Indeed, increased gut permeability was associated with severity of liver steatosis. Other authors, using the lactuloseemannitol test, also identified increased intestinal permeability in NAFLD patients [27,28]. Studies using plasma endotoxin levels and TLR4 expression (endotoxin receptor), as markers of intestinal permeability, found similar results [7,25,29,32]. According to them, NAFLD subjects present higher serum concentrations of the endotoxin core antibodies EndoCAb IgG (marker of endotoxin exposure) [32], increased endotoxin levels [27,29], and high expression of TLR4 in liver [29] and on CD14þ cells [25].
USE OF PROBIOTICS AND SYNBIOTICS IN NAFLD PATIENTS Several interventional studies [28,33e45] and meta-analyses [46e48], on the use of oral probiotics and synbiotics to modify gut microbiota in NAFLD subjects, have shown improvement of inflammatory markers, oxidative stress parameters and liver biochemistry. However, it is important to emphasize that the studies differ regarding probiotic doses, strains of bacteria and duration of treatment, which hamper the establishment of the best intervention.
CONCLUSIONS The evidences discussed here support the notion that gut microbiota changes induce an immune imbalance, leading to a state of metabolic alterations, hepatic fat accumulation, chronic inflammation, and, even NASH. More clinical studies are necessary, to better understand the cell-specific recognition and intracellular signaling events, involved in recognizing gut-derived microbes, and to set up an optimal balance in the GLA, in order to prevent and treat NAFLD.
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The role of small intestinal bacterial overgrowth, intestinal permeability, endotoxaemia, and tumour necrosis factor alpha in the pathogenesis of non-alcoholic steatohepatitis. Gut 2001;48:206e11. [22] Sajjad A, Mottershead M, Syn WK, Jones R, Smith S, Nwokolo CU. Ciprofloxacin suppresses bacterial overgrowth, increases fasting insulin but does not correct low acylated ghrelin concentration in non-alcoholic steatohepatitis. Aliment Pharmacol Ther 2005;22:291e9. [23] Sabaté JM, Jouët P, Harnois F, Mechler C, Msika S, Grossin M, Coffin B. High prevalence of small intestinal bacterial overgrowth in patients with morbid obesity: a contributor to severe hepatic steatosis. Obes Surg 2008;18:371e7. [24] Miele L, Valenza V, la Torre G, Montalto M, Cammarota G, Ricci R, Mascianà R, Forgione A, Gabrieli ML, Perotti G, Vecchio FM, Rapaccini G, Gasbarrini G, Day CP, Grieco A. Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease. Hepatology 2009;49:1877e87. [25] Shanab AA, Scully P, Crosbie O, Buckley M, O’Mahony L, Shanahan F, Gazareen S, Murphy E, Quigley EM. Small intestinal bacterial overgrowth in nonalcoholic steatohepatitis: association with toll-like receptor 4 expression and plasma levels of interleukin 8. Dig Dis Sci 2011;56:1524e34. [26] Kapil S, Duseja A, Sharma BK, Singla B, Chakraborti A, Das A, Ray P, Dhiman RK, Chawla Y. Small intestinal bacterial over growth and toll-like receptor signaling in patients with non-alcoholic fatty liver disease. J Gastroenterol Hepatol 2016;31:213e21. [27] Volynets V, Küper MA, Strahl S, Maier IB, Spruss A, Wagnerberger S, Königsrainer A, Bischoff SC, Bergheim I. Nutrition, intestinal permeability, and blood ethanol levels are altered in patients with nonalcoholic fatty liver disease (NAFLD). Dig Dis Sci 2012;57:1932e41. [28] Ferolla SM, Couto CA, Costa-Silva L, Armiliato GN, Pereira CA, Martins FS, Ferrari Mde L, Vilela EG, Torres HO, Cunha AS, Ferrari TC. Beneficial effect of synbiotic supplementation on hepatic steatosis and anthropometric parameters, but not on gut permeability in a population with nonalcoholic steatohepatitis. Nutrients 2016;28:E397. [29] Alessi MC, Bastelica D, Mavri A, Morange P, Berthet B, Grino M, Juhan-Vague I. Plasma PAI-1 levels are more strongly related to liver steatosis than to adipose tissue accumulation. Arterioscler Thromb Vasc Biol 2003;23:1262e8. [30] Bergheim I, Guo L, Davis MA, Lambert JC, Beier JI, Duveau I, Luyendyk JP, Roth RA, Arteel GE. Metformin prevents alcohol-induced liver injury in the mouse: critical role of plasminogen activator inhibitor-1. Gastroenterology 2006;130:2099e112. [31] Bergheim I, Guo L, Davis MA, Duveau I, Arteel GE. Critical role of plasminogen activator inhibitor-1 in cholestatic liver injury and fibrosis. J Pharmacol Exp Ther 2006;316:592e600.
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[32] Thuy S, Ladurner R, Volynets V, Wagner S, Strahl S, Königsrainer A, Maier KP, Bischoff SC, Bergheim I. Nonalcoholic fatty liver disease in humans is associated with increased plasma endotoxin and plasminogen activator inhibitor 1 concentrations and with fructose intake. J Nutr 2008;138:1452e5. [33] Loguercio C, de Simone T, Federico A, Terracciano F, Tuccillo C, di Chicco M, Cartenì M. Gut-liver axis: a new point of attack to treat chronic liver damage? Am J Gastroenterol 2002;97:2144e6. [34] Loguercio C, Federico A, Tuccillo C, Terracciano F, D’Auria MV, de Simone C, del Vecchio Blanco C. Beneficial effects of a probiotic VSL#3 on parameters of liver dysfunction in chronic liver diseases. J Clin Gastroenterol 2005;39:540e3. [35] Vajro P, Mandato C, Licenziati MR, Franzese A, Vitale DF, Lenta S, Caropreso M, Vallone G, Meli R. Effects of Lactobacillus rhamnosus strain GG in pediatric obesity-related liver disease. J Pediatr Gastroenterol Nutr 2011;52:740e3. [36] Aller R, de Luis DA, Izaola O, Conde R, Gonzalez Sagrado M, Primo D, de la Fuente B, Gonzalez J. Effect of a probiotic on liver aminotransferases in nonalcoholic fatty liver disease patients: a double blind randomized clinical trial. Eur Rev Med Pharmacol Sci 2011;15:1090e5. [37] Wong VW, Won GL, Chim AM, Chu WC, Yeung DK, Li KC, Chan HL. Treatment of nonalcoholic steatohepatitis with probiotics. A proof-ofconcept study. Ann Hepatol 2013;12:256e62. [38] Eslamparast T, Poustchi H, Zamani F, Sharafkhah M, Malekzadeh R, Hekmatdoost A. Synbiotic supplementation in nonalcoholic fatty liver disease: a randomized, double-blind, placebo-controlled pilot study. Am J ClinNutr 2014;99:535e42. [39] Alisi A, Bedogni G, Baviera G, Giorgio V, Porro E, Paris C, Giammaria P, Reali L, Anania F, Nobili V. Randomised clinical trial: the beneficial effects of VSL#3 in obese children with non-alcoholic steatohepatitis. Aliment Pharmacol Ther 2014;39:1276e85. [40] Famouri F, Shariat Z, Hashemipour M, Keikha M, Kelishadi R. Effects of probiotics on nonalcoholic fatty liver disease in obese children and adolescents. J Pediatr Gastroenterol Nutr 2017;64:413e7. [41] Mykhal’chyshyn HP, Bodnar PM, Kobyliak NM. Effect of probiotics on proinflammatory cytokines level in patients with type 2 diabetes and nonalcoholic fatty liver disease. Likars’ ka Sprava 2013;2:56e62. [42] Nabavi S, Rafraf M, Somi MH, Homayouni-Rad A, Asghari-Jafarabadi M. Effects of probiotic yogurt consumption on metabolic factors in individuals with nonalcoholic fatty liver disease. J Dairy Sci 2014;97:7386e93. [43] Sepideh A, Karim P, Hossein A, Leila R, Hamdollah M, Mohammad EG, Mojtaba S, Mohammad S, Ghader G, SeyedMoayed A. Effects of multistrain probiotic supplementation on glycemic and inflammatory indices in patients with nonalcoholic fatty liver disease: a double-blind randomized clinical trial. J Am Coll Nutr 2016;35:500e5. [44] Shavakhi A, Minakari M, Firouzian H, Assali R, Hekmatdoost A, Ferns G. Effect of a probiotic and metformin on liver aminotransferases in nonalcoholic steatohepatitis: a double blind randomized clinical trial. Int J Prev Med 2013;4:531e7. [45] Mofidi F, Poustchi H, Yari Z, Nourinayyer B, Merat S, Sharafkhah M, Malekzadeh R, Hekmatdoost A. Synbiotic supplementation in lean patients with non-alcoholic fatty liver disease: a pilot, randomised, double-blind, placebo-controlled, clinical trial. Br J Nutr 2017;117:662e8. [46] Ma YY, Li L, Yu CH, Shen Z, Chen LH, Li YM. Effects of probiotics on nonalcoholic fatty liver disease: a meta-analysis. World J Gastroenterol 2013;19:6911e8. [47] Khalesi S, Johnson DW, Campbell K, Williams S, Fenning A, Saluja S, Irwin C. Effect of probiotics and synbiotics consumption on serum concentrations of liver function test enzymes: a systematic review and meta-analysis. Eur J Nutr 2017. https://doi.org/10.1007/s00394-017-1568-y [Epub ahead of print]. [48] S Lavekar A, V Raje D, Manohar T, A Lavekar A. Role of probiotics in the treatment of nonalcoholic fatty liver disease: a meta-analysis. Euroasian J Hepato-Gastroenterol 2017;7:130e7.
The Microbiome and Metabolome in Nonalcoholic Fatty Liver Disease Silvia M. Ferolla1, Cla´udia A. Couto1, Maria de Lourdes A. Ferrari1, Luciana Costa Faria1, Murilo Pereira2 and Teresa C.A. Ferrari1 1
Department of Internal Medicine, Faculty of Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil; 2Post graduation in Functional
Clinical Nutrition, VP Institute, Cruzeiro do Sul University, São Paulo, Brazil
INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in the world [1]. It encompasses a spectrum of conditions that ranges from hepatic steatosis to steatosis associated with necroinflammatory lesions (nonalcoholic steatohepatitis/NASH), which may progress to fibrosis and cirrhosis. It is usually associated with metabolic syndrome (MS). The pathogenesis of NAFLD is related to insulin resistance (IR), as a key factor that initiates hepatic fat accumulation and, potentially, NASH [2,3]. The excessive deposition of triglyceride in the hepatocytes leads to a shift from carbohydrates to FFA mitochondrial beta-oxidation and may promote lipid peroxidation, and accumulation of reactive oxygen species (ROS). These compounds produce a variety of cellular stimuli with inflammatory response, hepatocellular injury, and fibrosis [2]. Nowadays, a growing interest has been devoted to guteliver axis (GLA) dysfunction, i.e., intestinal dysbiosis, bacterial overgrowth, and alteration of mucosa permeability, as relevant in NAFLD progression. It is also a possible alternative therapeutic target, in those patients unable to get benefits deriving from lifestyle modification, healthy diet, and physical activity promotion [4].
GuteLiver Axis The term “guteliver axis” is defined as the strong anatomical and functional interaction, between the gastrointestinal tract and the liver. GLA is characterized by bidirectional traffic. Nutrients and factors derived from gut lumen reach the liver, through the portal circulation. Bile acids produced by hepatocytes are released in the small intestine, through the biliary tract. However, this description is simplistic, as the GLA does not have only a nutritional role. is a complex structure, and the change of two of its components, namely gut barrier and intestinal microbiota, and seems to play an important role in liver lesions and NASH progression.
INTESTINAL ENVIRONMENT The gut microbiota composition is also influenced by diet, age, body weight, infections, medications, intestinal surgeries, and several liver diseases [5e7]. The gut microbiota is important to the host metabolism by secreting bioactive metabolites. It also participates in the development of the intestinal microvilli defense against pathogens, maintains gut immunity, performs the digestion of complex polysaccharides, synthesizes vitamins, and plays a role in fat storage [7e9]. The gut epithelium is a natural barrier that selects entry of useful substances present in the lumen, such as nutrients, and keeps at bay bacteria and their bioproducts. Tight junctions, specialized intercellular structures, assist this control. Derangement of the homeostasis between bacteria and the host, as occurs in small intestine bacterial overgrowth (SIBO),
Microbiome and Metabolome in Diagnosis, Therapy, and other Strategic Applications. https://doi.org/10.1016/B978-0-12-815249-2.00027-0 Copyright © 2019 Elsevier Inc. All rights reserved.
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may cause disruption of the intercellular tight junctions, and subsequent increase in intestinal permeability, leading to bacterial translocation (BT), i.e., transportation of bacteria and their products from the gut lumen into the blood [9]. The portal vein and the hepatic artery supply blood to the liver. The portal blood contains products of digestion and also microbial products derived from the gut microbiota. Therefore, the liver consists in the first site, of exposure and filtration of bacterial products from the intestine, such as lipopolysaccharides (LPS), lipopeptides, unmethylated DNA, and doublestranded RNA [9].
Endotoxin and Liver Inflammation The liver contains components of the immune system, such as macrophages, dendritic cells and natural killer T cells, which act as a first-line defense against endotoxin and microorganisms. Toll-like receptors (TLRs), present on the innate immune cells, consist of a family of type I transmembrane proteins, which recognize pathogen-associated molecular patterns (PAMPs), and damage associated molecular patterns (DAMPs) present on endogenous ligands. They initiate an adaptive immune response signaling cascade, leading to activation of proinflammatory genes, such as TNF-a IL-6, IL-8, and IL-12 genes. LPS, the active component of endotoxin, is the most studied PAMP. The liver is exposed to these PAMPs due to BT [40]. LPS has affinity to LPS-binding protein, which in turn, binds to CD14 and activates TLR4 in Kupffer cells, triggering an essential inflammatory cascade [7]. The synthesis of proinflammatory cytokines leads to prolonged inflammation and hepatocyte damage [10].
Intestinal Inflammasomes The TLRs, together with other sensors of PAMPs and DAMPs, are the inflammasomes, which are formed by a molecular macrocomplex that includes the enzyme caspase-1, whose activation causes the release of bioactive IL-1b and/or IL-18 [9]. Recent evidence suggests that the inflammasome is involved in NAFLD/NASH progression, via modulation of the gut microbiota [11]. Genetic inflammasome deficiency, associated with dysbiosis, determines increased concentration of bacterial products in the portal blood, which may exacerbate steatosis and increase tumor necrosis factor (TNF-a) expression [11]. Another mechanism that supports the role of the gut microbiota in the pathogenesis of NAFLD is the fact that certain types of bacteria inhibit gut epithelial expression of fasting-induced adipocyte factor (FIAF), a suppressor of lipoprotein lipase (LPL). FIAF is produced not only by the gut, but also by liver and adipose tissue, being an essential regulator of peripheral fat storage. By suppressing FIAF, the microbiota increases LPL activity in adipose tissue, enhancing the delivery of adipocyte-derived triglycerides, which determine storage of triacylglycerols in the liver [4]. Additionally, the microbiota is related to IR [12]. The transfer of gut microbiota from lean human donors to recipients with MS, via duodenal tube, resulted in increased insulin sensitivity within 6 weeks [13].
Bile Acids and Farnesoid X Receptor Bile acids are ligands of the farnesoid X receptor (FXR), which is expressed in the liver and gut [14]. The activation of FXR reduces circulating bile acids (feedback mechanism), and participates in the control of the gut-microbiota composition, and in the regulation of lipids and glucose homeostasis in the gut-liver axis. It is known to have a crucial role in hepatic de-novo lipogenesis, VLDL export, and plasma triglyceride turnover [4]. A recent human study showed low FXR protein expression in patients with NASH versus simple NAFLD, suggesting a protective role of FXR in liver disease progression [15].
Gut Microbiome in NAFLD Patients Obese individuals present predominance of Firmicutes and relative low proportion of Bacteroidetes [14]. In animal models, this has been associated with a propensity to NAFLD features like fasting hyperglycemia, insulin resistance, liver steatosis, and high expression of genes involved in de novo lipogenesis [16]. In humans with NASH, the microbiota composition also seems to have a lower proportion of Bacteroidetes, independently of hight fat diet and BMI. The low prevalence of Bacteroidetes might facilitate the growth of other phyla that are more efficient in harvesting energy from the diet [17]. Indeed, obese subjects present more H2-producing Prevotellaceae, and H2-utilizing methanogenic Archaea. The coexistence of H2-producing bacteria with H2-utilizing Archaea suggests that H2 transfer might increase energy uptake by the human large intestine in the obese person [18].
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Response to High-Fat Diet In two mice fed with high-fat diet (HFD) and with similar body weight gain, one mouse (called responder) developed hyperglycemia and presented high serum concentrations of proinflammatory cytokines, whereas the other (called nonresponder), did not. The gut microbiota was transplanted from both the responder, and the nonresponder, to germfree mice. These animals were fed with the same HFD, and also developed comparable obesity. The responder stoolreceiving mice presented fasting hyperglycemia, hyperinsulinemia, hepatic steatosis and high expression of genes involved in de novo lipogenesis. The nonresponder-receiver animals did not develop metabolic abnormalities. Gut microbiota associated with the NAFLD-prone and NAFLD-resistant phenotypes were, respectively, Firmicutes and Bacteroidetes [16]. Mouzaki et al. investigated the NASH human gut microbiota [19] and found a lower proportion of Bacteroides/ Prevotella ratio (herein referred to as Bacteroidetes), when compared to simple steatosis or healthy controls (living liver donors), independently of BMI and high fatty diet. NASH subjects also presented increased number of Clostridium coccoides in their stool, in comparison to the individuals with simple steatosis. In the experience of Zhu et al. [20] in NASH increased abundance of alcohol-producing Escherichia occurred, as well as high bloodeethanol levels, leading to increased oxidative stress, and liver inflammation due to alcohol metabolism. The gut microbiota also produced LPS, which contributes to liver damage, disrupts normal hepatocyte function, leads to mitochondrial oxidative stress, and reduces the clearance of toxins by the hepatocytes.
SIBO AND INCREASED INTESTINAL PERMEABILITY IN NAFLD PATIENTS Evidences suggest that NAFLD subjects present a high prevalence of small intestine bacterial overgrowth/SIBO [21e26] and increased gut permeability [24,27,28]. Most controlled trials demonstrated that the prevalence of SIBO in NAFLD, ranges from 50% to 77.8% [21,22,24,25]. SIBO in NASH individuals is also associated with the rise in hepatic TLR4 expression and release of interleukin (IL)-8 [25]. SIBO has been independently associated with the severity of liver steatosis in histology studies [23,24]. As liver biopsy is not always available, markers of hepatic damage, such as plasminogen activator inhibitor 1 (PAI-1), have been used [27,29]. Some studies have showed that high serum concentrations of PAI-1 are associated with hepatic steatosis, fibrosis [30,31], and elevated serum endotoxin concentrations [27,29]. SIBO may increase gut permeability, leading to endotoxemia, and oxidative stress in the liver. Miele et al. [24] showed high prevalence of SIBO, increased intestinal permeability (urinary excretion of 51Cr-ethylene diamine tetra acetate/ 51Cr-EDTA test), and disruption of the intercellular tight junctions of the gut (immunohistochemical analysis of zona occludens-1/ZO-1 expression in duodenal biopsy specimens), in subjects with NAFLD. Indeed, increased gut permeability was associated with severity of liver steatosis. Other authors, using the lactuloseemannitol test, also identified increased intestinal permeability in NAFLD patients [27,28]. Studies using plasma endotoxin levels and TLR4 expression (endotoxin receptor), as markers of intestinal permeability, found similar results [7,25,29,32]. According to them, NAFLD subjects present higher serum concentrations of the endotoxin core antibodies EndoCAb IgG (marker of endotoxin exposure) [32], increased endotoxin levels [27,29], and high expression of TLR4 in liver [29] and on CD14þ cells [25].
USE OF PROBIOTICS AND SYNBIOTICS IN NAFLD PATIENTS Several interventional studies [28,33e45] and meta-analyses [46e48], on the use of oral probiotics and synbiotics to modify gut microbiota in NAFLD subjects, have shown improvement of inflammatory markers, oxidative stress parameters and liver biochemistry. However, it is important to emphasize that the studies differ regarding probiotic doses, strains of bacteria and duration of treatment, which hamper the establishment of the best intervention.
CONCLUSIONS The evidences discussed here support the notion that gut microbiota changes induce an immune imbalance, leading to a state of metabolic alterations, hepatic fat accumulation, chronic inflammation, and, even NASH. More clinical studies are necessary, to better understand the cell-specific recognition and intracellular signaling events, involved in recognizing gut-derived microbes, and to set up an optimal balance in the GLA, in order to prevent and treat NAFLD.
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