The microbiome in non-alcoholic fatty liver disease: associations and implications
Eugenia A. Lina, Gillian M. Barlowa, Ruchi Mathurb
Cedars-Sinai Medical Center, Los Angeles, CA, USA
Title: Intestinal microbiota in patients with nonalcoholic fatty liver disease
Authors: Mouzaki M, Comelli EM, Arendt BM, Bonengel J, Fung SK, Fischer SE, McGilvray ID, Allard JP
Journal: Hepatology 2013;58:120-127
Title: Fecal microbiome and volatile organic compound metabolome in obese humans with nonalcoholic fatty liver disease
Authors: Raman M, Ahmed I, Gillevet PM, Probert CS, Ratcliffe NM, Smith S, Greenwood R, Sikaroodi M, Lam V, Crotty P, Bailey J, Myers RP, Rioux KP
Journal: Clin Gastroenterol Hepatol 2013;11:868-875, e1-e3
aGI Motility Program, Department of Medicine (Eugenia A. Lin, Gillian M. Barlow); bDivision of Endocrine Diabetes and Metabolism (Ruchi Mathur), Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA
Conflict of Interest: None
Correspondence to: Ruchi Mathur, MD, FRCP(C), Director of Diabetes, Division of Endocrine Diabetes and Metabolism, Department of Medicine, Cedars-Sinai Medical Center 131 Becker Building, 8700 Beverly Blvd, Los Angeles, CA 90048, Tel.: +310 423 4774, Fax: +310 423 0440, e-mail: Ruchi.Mathur@cshs.org
Received 11 December 2013; accepted 12 December 2013
Summary
Nonalcoholic fatty liver disease (NAFLD) is an increasingly significant public health problem as the risk factors for NAFLD such as obesity, insulin resistance and type II diabetes have also become more prevalent [1]. The composition of the intestinal microbiota (IM) has been previously implicated in the pathogenesis of obesity in clinical and animal studies, suggesting that obese humans exhibit a reduction in microbial diversity coupled with an increase in the ratios of Firmicutes to Bacteroidetes compared to lean humans [2,3]. Colonic bacteria may also have effects on the human host through intestinal absorption and delivery to the liver via the portal vein [4]. Therefore two recent papers examining the potential role of IM in NAFLD, by Mouzaki et al [5] and Raman et al [6] respectively, are both timely and relevant.
In the first of these, Mouzaki et al performed a prospective, cross-sectional study to determine whether differences in IM composition could contribute to the development of NAFLD [5]. Fifty total subjects constituted three groups of varying NAFLD disease progression: 11 simple steatosis (SS), 22 nonalcoholic steatoheaptitis (NASH), and 17 living liver donors as healthy controls (HC). Subjects in the SS group represent the mildest form of NAFLD, while the NASH group represents the most severe form and can lead to cirrhosis. Liver biopsy confirmed NASH, SS, or healthy liver in controls, and subjects in the NAFLD spectrum underwent extensive assessment to rule out other liver diseases. All subjects completed 7-day food and activity records, and provided stool samples for analysis. Quantitative real-time polymerase chain reaction measured microbial levels in stool samples from each subject. Levels of Bacteroides/Precotella (Bacteriodetes), Clostridium leptum, C. coccoides, bifidobacteria, Escherichia coli, total bacteria and Archaea were determined. Subjects in the NASH group had a lower ratio of Bacteriodetes to total bacterial counts in comparison to the SS or HC groups (P=0.006) and higher C. coccoides compared to the SS group (P=0.04). Interestingly, the authors were only able to detect Archaea in five HC, two SS and two NASH subjects, which limited the statistical power for any comparisons. After accounting for BMI and fat intake, no statistical difference remained in C. coccoides levels between groups, but there was still an independent significant association between subjects with NASH and a lower relative abundance of Bacteriodetes.
The authors concluded that these data provide evidence that the composition of the IM may play a role in the development of NAFLD. As microbes have established inflammatory roles, the authors also proposed that future studies may further elucidate the effects of the IM on the liver and the mechanisms underlying the development and progression of SS and NASH [5].
In the second study, Raman et al performed an observational case-control study that investigated the colonic microbiome and other volatile organic compounds (VOC) in obese NAFLD patients and healthy controls [6]. Thirty obese NAFLD subjects and thirty healthy controls participated in the study. Neither group was subject to dietary restrictions, and the obese NAFLD participants had not undertaken any lifestyle modifications [6]. All provided stool samples for characterization of IM by multitag pyrosequencing and of VOC profiles by gas chromatography-mass spectrometry. In contrast to the results of Mouzaki et al [5], no discernable differences were found between the relative abundance of Bacteroidetes in obese NAFLD subjects vs. healthy controls. The researchers also did not find a correlation between BMI and the proportion of Firmicutes to Bacteroidetes as previously described by Ley et al [7], although the authors did note that the subjects in their study mainly had class I obesity, whereas the subjects in Ley et al had class III and class IV obesity. The authors did find small but significant differences between obese NAFLD patients and controls at the family and genus levels, including an over-representation of Lactobacillus and selected members of phylum Firmicutes in NAFLD subjects, while members of Ruminococcaceae (genus: Oscillibacter) were significantly under-represented. However, it is possible that these differences were due to obesity rather than NAFLD, as the controls were not obese. The authors concluded that there is a shift in the fecal microbiome and a significantly altered fecal VOC profile in obese NAFLD subjects, and propose that, as hepatotropic or hepatotoxic factors, these microbial VOC metabolites may be related to NAFLD [6].
Opinion
Taken together, the results of these two studies support that the gut microbiome is altered in subjects with NAFLD, although further studies such as microbiota transplantation experiments would be required to conclusively determine which of the identified differences are causal as opposed to resulting from the effects of the disease state on the IM. There are several important differences between the two studies. First, Raman et al performed multitag pyrosequencing to characterize the fecal microbiome (within the limits of classification achievable with currently available databases, as discussed by the authors) [6], whereas the study by Mouzaki et al employed Q-PCR to detect levels of specific microbes [5]. Although the mean BMI in the NASH [5] and NAFLD [6] groups in the two studies was similar, the range of BMIs in the NASH group studied by Mouzaki et al (24.2-49.5) was greater than that of the NAFLD group studied by Raman et al (29-35). Additionally, the study by Mouzaki et al was limited to Caucasians, and also compared dietary intake and physical activity levels between groups and correlated these with microbial levels, which was not undertaken by Raman et al, who also enrolled subjects of all races [6]. Lastly, the diagnosis of NAFLD was made based on laboratory measurements coupled with sonographic findings in the study by Raman et al [6], whereas Mouzaki et al used laboratory measurements and histologic analyses of liver biopsies, and further stratified subjects as having SS or NASH [5]. These differences in study design make it difficult to directly compare the results of the two studies. Nonetheless, there is evidence for a BMI-independent association between decreased Bacteriodetes and NASH [5], and some of the differences at the family and genus levels identified by Raman et al may also be associated with NAFLD. It is interesting that in the study by Mouzaki et al, the authors were unable to detect Archaea in the majority of study subjects [5]. In humans, the predominant Archaea are members of the order Methanobacteriales (methanogens), with Methanobrevibacter smithii being the most prevalent [8-10], and these can typically be detected in 70% of ‘normal’ human subjects [8]. That Archaea were not detected in more subjects by Mouzaki et al may result from differences between study populations, and/or may also be reflective of differences in the isolation techniques used. There are particular difficulties to working with M. smithii and other Archaea – as anerobes, stool analysis may be difficult if specific conditions are not met, and the primers used can also be critical. In addition, we note that both of these studies used stool samples for their analyses. Although this is typical of the majority of studies of the gut microbiota, the small intestine is a far more important site for digestion and absorption. Small intestinal microbes influence digestion and absorption directly, producing enzymes that assist the host in utilizing non-digestible carbohydrates and host-derived glycoconjugates; in deconjugating; dehydroxylating bile acids, and in cholesterol reduction and the biosynthesis of vitamins [11-15]. Our group has recently demonstrated that there are significant differences in the microbial populations in the small bowel and colon (personal communication with Dr Mark Pimentel), findings which are also supported by other studies [16]. These findings illustrate the importance of studying the microbiome in the small bowel directly (by obtaining duodenal aspirates) rather than relying on stool. Nonetheless, these pilot studies provide evidence that alterations in the microbiome may contribute to the development of NAFLD, and larger studies that include obese controls followed by animal studies are warranted to further elucidate the potential contributions of gut microbes and their metabolites to the pathogenesis of NAFLD. With recent advances in sequencing technologies, it should be possible to perform next-generation sequencing analyses of larger numbers of NAFLD subjects, including both obese and non-obese controls, in order to delineate the role of altered microbial populations in the development and progression of this disease.
References
1. Abu-Shanab A, Quigley EM. The role of the gut microbiota in nonalcoholic fatty liver disease. Nat Rev Gastroenterol Hepatol 2010;7:691-701.
2. Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes associated with obesity. Nature 2006;444:1022-1023.
3. Clemente JC, Ursell LK, Parfrey LW, Knight R. The impact of the gut microbiota on human health: an integrative view. Cell 2012;148:1258-1270.
4. Iacono A, Raso GM, Canani RB, Calignano A, Meli R. Probiotics as an emerging therapeutic strategy to treat NAFLD: focus on molecular and biochemical mechanisms. J Nutr Biochem 2011;22:699-711.
5. Mouzaki M, Comelli EM, Arendt BM, et al. Intestinal microbiota in patients with nonalcoholic fatty liver disease. Hepatology 2013;58:120-127.
6. Raman M, Ahmed I, Gillevet PM, et al. Fecal microbiome and volatile organic compound metabolome in obese humans with nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol 2013;11:868-875 e1-3.
7. Ley RE, Backhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI. Obesity alters gut microbial ecology. Proc Natl Acad Sci USA 2005;102:11070-11075.
8. Weaver GA, Krause JA, Miller TL, Wolin MJ. Incidence of methanogenic bacteria in a sigmoidoscopy population: an association of methanogenic bacteria and diverticulosis. Gut 1986;27:698-704.
9. Pochart P, Lemann F, Flourie B, Pellier P, Goderel I, Rambaud JC. Pyxigraphic sampling to enumerate methanogens and anaerobes in the right colon of healthy humans. Gastroenterology 1993;105:1281-1285.
10. Miller TL, Wolin MJ. Enumeration of Methanobrevibacter smithii in human feces. Arch Microbiol 1982;131:14-18.
11. Gill SR, Pop M, Deboy RT, et al. Metagenomic analysis of the human distal gut microbiome. Science 2006;312:1355-1359.
12. Hooper LV, Midtvedt T, Gordon JI. How host-microbial interactions shape the nutrient environment of the mammalian intestine. Annu Rev Nutr 2002;22:283-307.
13. Backhed F, Ding H, Wang T, et al. The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA 2004;101:15718-15723.
14. Ridlon JM, Kang DJ, Hylemon PB. Bile salt biotransformations by human intestinal bacteria. J Lipid Res 2006;47:241-259.
15. Conly JM, Stein K, Worobetz L, Rutledge-Harding S. The contribution of vitamin K2 (menaquinones) produced by the intestinal microflora to human nutritional requirements for vitamin K. Am J Gastroenterol 1994;89:915-923.
16. Gu S, Chen D, Zhang JN, et al. Bacterial community mapping of the mouse gastrointestinal tract. PLoS ONE 2013;8:e74957