O b e s i t y an d N A F L D The Role of Bacteria and Microbiota Ajay Duseja,

MBBS, MD, DM,

Yogesh Kumar Chawla,

MBBS, MD, DM*

KEYWORDS  Nonalcoholic fatty liver disease  Nonalcoholic steatohepatitis  Small intestinal bacterial overgrowth  Intestinal permeability  Endotoxemia  Toll-like receptors  Probiotics KEY POINTS  The term “gut microbiota” incorporates not just bacteria but also viruses and other microorganisms, such as protozoa, archaeal spp, yeasts, and parasites.  Gut–liver axis plays a central role in the pathogenesis of obesity and NAFLD, mainly through the crosstalk of the intestinal microbiota with the host immune system modulating inflammation, insulin resistance, and intestinal permeability.  Gut microbiota are linked to obesity through increased energy harvesting and storage and to NAFLD though the systemic inflammation, cytokines, and insulin resistance secondary to endotoxemia resulting from SIBO and increased gut permeability.

INTRODUCTION

The liver is a unique organ that receives blood supply from the portal vein and the hepatic artery. The blood in the portal vein, which drains from the mesenteric veins, contains not only products of digestion but also microbial products derived from microbes that colonize the gut. Therefore, the liver, which acts as the first site of filtration for such products, also becomes the first site of exposure to microbial products from the gut. Microbial products have been recognized to be responsible pathogenically for several disease situations of the liver and were earlier shown to be associated with hepatic encephalopathy and spontaneous bacterial peritonitis. What initially was termed as “gut flora” is now termed as “gut microbiota,” which is a preferred terminology because it incorporates not just bacteria but also viruses and other microorganisms, such as protozoa, archaeal spp, yeasts, and parasites. The human gut contains more than 1000 different bacterial species with 99% belonging to about 40 species.1 The bacterial number increases from 104 in the jejunum to 107 colony-forming units per gram of luminal content in the ileum

Disclosure and Conflict of Interest: The authors have nothing to disclose. Department of Hepatology, Post Graduate Institute of Medical Education and Research, Sector 12, Chandigarh 160012, India * Corresponding author. E-mail addresses: [email protected]; [email protected] Clin Liver Dis 18 (2014) 59–71 http://dx.doi.org/10.1016/j.cld.2013.09.002 1089-3261/14/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.

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and are mainly gram-negative aerobes and nonobligate anaerobes, whereas in the colon the number increases to 1012 colony-forming units per gram and are predominantly anaerobes. Genome of microbiota (microbiome) contains 100 times more genes than the human genome.2 The human gut is sterile in utero and the acquired microbiota at the time of birth depends on the mode of delivery (ie, vaginal or caesarean section). Establishment of further bacterial flora depends on whether the infant is on breast or bottle feed. Introduction of solid food in the diet establishes the adult-type of bacterial flora.3 The adult human gut bacterial flora consists predominantly of gram-positive Firmicutes (60%–80%) and gram-negative Bacteroidetes (20%–40%) that usually remains stable over time and is only subject to minor changes caused by age, diet, medication, infection, and intestinal surgery.4,5 Other bacteria in the human gut include, Bifidobacteria, Proteobacteria, Fusobacteria, Cyanobacteria, Verrcomicrobia, and Spirochaetes spp. Even though most of the adult gut flora consists of Firmicutes and Bacteroidetes there are differences at the species and strain level with less than 1% similarity in the bacterial species among individuals. The gut microbiota in addition to the energy harvesting by way of digestion of complex indigestible polysaccharides, synthesis of vitamins, and fat storage (discussed later) are an important source of energy to the colonocytes. Microorganisms also secrete several bioactive metabolites with diverse functions. Thus, the gut microbiota may be regarded as a microbial organ within the gut that contributes to multiple host processes including defense against pathogens by maintaining immunity at the level of gut, synthesis of several vitamins, and development of intestinal microvilli.1,6 This article reviews the literature on the association of gut microbiota with obesity and nonalcoholic fatty liver disease (NAFLD). GUT MICROBIOTA AND OBESITY

Estimated prevalence of overweight and obesity in US adults is close to 65% and 30%, respectively.7 The figures are likely to worsen in future because of the rising problem of overweight and obesity in children.8 Obesity is a lifestyle disease resulting from increased food intake and decreased physical activity especially in genetically predisposed individuals and has always been considered to be a state of “nutritional disequilibrium.” The gut microbiota are involved in energy harvesting and its storage in the adipose tissue. Microbiota help in extracting calories from otherwise indigestible polysaccharides in the diet with the help of glycoside hydrolases and polysaccharide lysases, enzymes that are absent in humans. Microbes convert these polysaccharides to monosaccharides and short-chain fatty acids in the colon, which after absorption cause the triglyceride synthesis in the liver.9 Microbes also suppress the intestinal epithelial expression of fasting-induced adipocyte protein (Fiaf), an inhibitor of lipoprotein lipase. Inhibition of the activity of Fiaf increases the lipoprotein lipase activity in the adipose tissue with increased storage of liver-synthesized triacyglycerols in the adipose tissue.1 Recent literature suggests the emerging role of gut microbiota in the development of obesity and its consequence including NAFLD. Most of the data are based on animal experiments but there is emerging human data that establishes the link between the gut microbiota, obesity, and NAFLD. One of the earlier animal studies evaluating the role of gut microbiota in obesity used germ-free (GF) mice and found that these animals despite consuming more food had 42% less body fat compared with normal animals.2 After transplantation of cecal microbiota from normal to GF mice there was 57% increase in the total body fat without any change in the energy expenditure and food intake. The results of the study suggested the role of gut microbiota in energy harvesting and fat storage.2 Similar

Obesity, NAFLD and Microbiota

results were shown in another study from the same group where despite high-calorie diet there was no gain in body weight in the mice in the absence of gut microbiota.10 Turnbaugh and colleagues11 transplanted the gut microbiota from obese ob/ob and lean (ob/1 or 1/1) mice into lean wild-type (WT) GF mice and found that animals that received the obese-type microbiota had higher fat gain (47%  8.3%) than the animals that received the lean-type microbiota (27%  3.6%) with no difference in energy expenditure and food intake between the two groups, thereby again suggesting the role of gut microbiota in the genesis of obesity. Studies have shown not only the quantitative difference in the gut microbiota between the obese and lean animals but also the differences in the bacterial species. A study comparing genetically obese leptin deficient (ob/ob) mice with lean (ob/1, 1/1) siblings and mothers, found that obese mice had less of Bacteroidetes (50% less) and more of Firmicutes in their gut compared with lean mice.12 Moreover the differences in the bacterial species between the two groups were not related to the difference in the food intake or total body mass. The same group also studied the microbial profiles in 12 obese human subjects and found that compared with lean subjects, obese subjects had lesser Bacteroidetes and more of Firmicutes.13 On assigning these subjects a fat-restricted or carbohydrate-restricted low-calorie diet for 1 year and sequencing 16 S rRNA genes from their stool samples, these subjects had an increase in Bacteroidetes spp and decrease in Firmicutes spp. The change in gut microbiota correlated with the weight loss of 6% in the fat-restricted group and 2% on carbohydrate-restricted diet.13 In another study, children were followed up until 7 years of age after baseline stool sample collection for gut microbiota at the age of 6 and 12 months. The study found that compared with lean, children who were overweight or obese at age 7 had less of Bacteroidetes spp and more of Staphylococcus aureus at the age 6 and 12 months.14 These studies support the concept that gut microbiota has a relationship with body weight and different gut microbiota composition has a bearing on determining the overweight and obesity in an individual. Pathogenesis of Microbiota-associated Obesity

Pathogenesis of microbiota-associated obesity is still unclear. Possible mechanism include increased energy harvest and fat storage, as described previously; induction of systemic inflammation; and its consequences and central effects on satiety (Box 1). The gut microbiota help in extracting calories from otherwise indigestible polysaccharides in the diet, converting these polysaccharides to monosaccharides and shortchain fatty acids in the colon, which after absorption cause triglyceride synthesis in the liver.11 Microbial inhibition of the activity of Fiaf increases the lipoprotein lipase

Box 1 Pathogenesis of microbiota-associated obesity  Increased energy harvest and fat storage  Conversion of polysaccharides to monosaccharides and short-chain fatty acids  Inhibition of the activity of Fiaf  Induction of systemic inflammation  Altering gut permeability and endotoxemia  Increased eCB system tone  Central effects on satiety

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activity in the adipose tissue with increased storage of triacyglycerols in the adipose tissue. This concept has been supported by animal studies showing the increased energy harvest and increase in body fat after transplantation of obese-type microbiota in GF mice.1 Stool examination of these animals showed more end products of fermentation and fewer calories suggesting increased energy extraction and storage by the obese-type microbiota.11 The decreased Bacteroidetes spp and increased Firmicutes spp in obese animals and humans could also suggest that Firmicutes spp possess more diverse enzymes capable of digesting and extracting calories from complex polysaccharides. By altering the gut permeability, the gut microbiota are also responsible for causing the endotoxemia and the systemic inflammation secondary to the downward signaling following the Toll-like receptor (TLR) stimulation (discussed later). Low-grade inflammation secondary to endotoxemia may be linked to obesity through increased endocannabinoid (eCB) system tone. Muccioli and colleagues15 showed in an animal model that gut microbiota modulate the intestinal eCB system tone, which in turn regulates gut permeability and plasma lipopolysaccharide (LPS) levels. By using CB(1) agonist and antagonist in lean and obese mouse models, they found that the eCB system controls gut permeability and adipose tissue physiology through LPS-eCB system regulatory loops and plays an important role in adipogenesis and obesity.15 GUT MICROBIOTA AND NAFLD

NAFLD is a constellation of conditions histologically characterized by mainly macrovesicular hepatic steatosis in individuals who do not consume alcohol in amounts generally considered to be harmful to the liver. It is a broad term consisting of patients with simple steatosis at one end of the spectrum, nonalcoholic steatohepatitis (NASH), NASH-related cirrhosis, and hepatocellular carcinoma at the other end. For some differences, NAFLD/NASH is an important cause of unexplained rise in hepatic transaminases, cryptogenic cirrhosis, and hepatocellular carcinoma worldwide.16,17 NAFLD is an extremely common liver disease with a prevalence as high as 46% in the general population.18 The figures increase with presence of risk factors, such as obesity and diabetes mellitus. In a systematic review including 1620 patients with severe obesity, prevalence of steatosis and NASH was 91% (range, 85%–98%) and 37% (range, 24%–98%), respectively, with cirrhosis in 1.7% (range, 1%–7%) of patients.19 Pathophysiologic Basis of NAFLD

In most of the subjects, NAFLD starts with an imbalance between energy intake and expenditure that leads to adipose tissue expansion. In addition, development of NAFLD is affected by genetic predisposition and environmental factors, such as diet and physical activity. Insulin resistance plays a major role in the pathogenesis of NAFLD. Originally, the pathogenesis of NAFLD was considered as two-hit process.20 Liver fat accumulation was the suggested first hit or the first step because of excessive triglyceride accumulation caused by a discrepancy between influx (from diet, increased hepatic uptake from subcutaneous and visceral adipose tissue, and de novo synthesis of hepatic fat) and decreased b-oxidation, decreased apolipoprotein B100 synthesis, and decreased very-low-density lipoprotein export from the liver. After the first hit (steatosis) a second hit, such as oxidative stress with accompanying inflammation, may cause progression to NASH in some patients.20 Recently, a modified two-hit, three-hit, and multiple-hit theories have been proposed as the possible pathophysiologic mechanisms in patients with NAFLD.21 According to the modified two-hit hypothesis, the accumulation of free

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fatty acids alone has been suggested to be sufficient to induce liver damage (concept of lipotoxicity), without recourse for a second hit, and triglyceride accumulation in the form of steatosis is considered protective by preventing free fatty acid–induced injury. According to the three-hit hypothesis, a third hit has been proposed as the inadequate hepatocyte regeneration and apoptosis. The multiple-hit hypotheses suggests that multiple events acting in parallel including lipotoxicity, increased oxidative stress, mitochondrial dysfunction, iron overload, and proinflammatory cytokines lead to NASH.21 Risk factors for NAFLD include increasing age, overweight or obesity, diabetes mellitus, hypertension, dyslipidemia, or metabolic syndrome usually associated with insulin resistance. The interethnic difference in the prevalence of NAFLD is believed to be related not only to different lifestyles but also to a strong genetic predisposition. Several genetic polymorphisms including hemochromatosis gene, apolipoprotein C3, MC4R, and Patatin-like phospholipase domain-containing protein 3 and TLR gene polymorphisms are associated with NAFLD.22,23 With respect to environmental factors influencing the risk of NAFLD and NASH, diet, exercise, and possibly gut microbiota are some of the candidates. The gut microbiota are involved in the pathogenesis of NAFLD by increasing gut permeability, causing low-grade inflammation, affecting dietary choline and bile acid metabolism, and by producing endogenous ethanol, ammonia, and acetaldehyde that are generally metabolized by the liver and are able to control Kupffer cell activity and cytokine production (Box 2). Small intestinal bacterial overgrowth and intestinal permeability

Tight junctions link the intestinal cells and play an important role in maintaining the integrity of the intestinal barrier. Occurrence of small intestinal bacterial overgrowth (SIBO) and increased intestinal permeability because of leaky tight junctions has been reported in patients with NAFLD. In an experimental study, Ga¨bele and colleagues24 induced NASH by feeding C57BL/6 mice a high-fat diet and then studied the effects of exposing the mice to 1% dextran sulfate sodium. By causing intestinal injury, combined administration of high-fat diet and dextran sulfate sodium not only worsened steatohepatitis, but also induced a profibrogenic response in the liver. The study suggested that the induction of intestinal inflammation by dextran sulfate sodium led to damage to intestinal barrier, LPS translocation, hepatic inflammation, and fibrogenesis in mice model of NASH.24 In one of the earlier human studies that assessed SIBO by a combined C-D-xylose and lactulose breath test and intestinal permeability by a dual lactulose rhamnose sugar test, it was found that 50% of patients with NASH had SIBO compared with 22% of SIBO in control subjects.25 In another study 18 patients with NASH were compared with ageand gender-matched control subjects and SIBO was assessed by the lactulose

Box 2 Pathogenesis of microbiota-associated NAFLD  Small intestinal overgrowth, increased gut permeability  Endotoxemia causing low-grade inflammation  Role of TLRs and inflammasomes  Altered dietary choline metabolism  Altered bile acid metabolism  Role of FXR receptors  Production of endogenous ethanol

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hydrogen breath test. SIBO was found to be more common in patients with NASH (77.78% vs 31.25%; P

Obesity and NAFLD: the role of bacteria and microbiota.

There are trillions of microorganisms in the human intestine collectively called gut microbiota. Obesity may be affected by the gut microbiota through...
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