Gut Microbiota and Modulation of Liver Damage in NAFLD
Gut Microbiota and Modulation of Liver Damage in NAFLD
Several experimental data suggest that gut-derived endotoxin and GM composition can act as a
 "second hit" or insult to convert hepatic SS to NASH and cause both local hepatic and
 systemic inflammation.This study's aim is to analyze microbiota diversity, providing
 information both on intestinal microbial composition and on the metabolic processes linked to
 them. In addition, we will correlate, for the first time, GM composition to hepatic and white
 adipose tissue gene expression patterns of interest and serum and fecal markers possibly
 related to impaired fat storage and inflammation. We aim to provide preliminary data to
 design future intervention studies with pre- or probiotics or bile acid derivatives to
 prevent/treat inflammation and fibrosis in NAFLD patients.
Non-alcoholic fatty liver disease (NAFLD) includes benign hepatic simple steatosis (SS) and
 the more severe steatohepatitis (NASH), which is characterized by inflammation and fibrosis
 potentially leading to cirrhosis and hepatocellular carcinoma. Current research is focused on
 what are the risk factors and determinants of NASH. NAFLD is associated with the metabolic
 syndrome, and the prevalence has been described to be up to 98% in morbidly obese individuals
 undergoing bariatric surgery. NAFLD in non-obese subjects have a different clinical profile
 than in obese individuals, being associated with insulin resistance (IR), differential
 distribution of visceral adipose tissue, recent increase in body weight and/or presence of
 overweight, intake of high cholesterol diet and genetic background.

 Gut microbiota (GM) regulates fat metabolism in mice. In humans its alterations have been
 linked to diabetes, obesity, IR, atherosclerosis and inflammation, SS and NAFLD.

 Several experimental data suggest that gut-derived endotoxin and GM composition can act as a
 "second hit" or insult to convert hepatic SS to NASH and cause both local hepatic and
 systemic inflammation. With regard to human studies, Muozaki et al. have recently showed, by
 using a polymerase chain reaction (PCR) TaqMan system approach, that obese patients with
 NASH, have a lower percentage of fecal Bacteroidetes (Bacteroidetes to total bacteria counts)
 compared to both SS and healthy controls and a higher percentage of C. coccoides compared to
 those with SS . In addition, Zhu et al. showed in pediatric subjects, by using a 16S
 ribosomal RNA detection method, an unique pattern of enterotypes in patients with NASH, in
 obese individuals with no sign of liver damage and in lean healthy controls. Finally, Wai-Sun
 Wong et al. showed, also using a 16S ribosomal RNA detection method, that a small group of
 Chinese NASH patients demonstrated fecal dysbiosis but not significant changes in
 biodiversity compared to healthy subjects. Finally, inflammation in patients with symptomatic
 atherosclerosis has been shown to be associated with lower levels of butyrate producing gut
 bacteria such as Roseburia.

 Among the possible factors involved in determining NAFLD severity, serum bile acid (BA)
 concentration and its post-prandial variations have been recently linked to the regulation of
 body weight, liver fat and inflammation and glucose and lipid metabolism. These BA regulatory
 functions are mediated by their interaction with the farnesoid X receptor (FXR)and the G
 Protein-Coupled BA Receptor 1 (GPBAR1 or TGR5) at both hepatic and subcutaneous adipose
 tissue levels. No human study has been directed to investigating the mechanisms through which
 GM composition influences inflammation and fibrosis in both obese and non-obese patients with
 NAFLD.

 Liver biopsy is clinically advisable during bariatric surgery, due to the high prevalence of
 NAFLD and NASH in morbidly obese patients. It has been previously suggested that the high
 prevalence of histologically proven NAFLD in patients with gallstones may also justify
 routine liver biopsy during cholecystectomy, even in non-obese subjects, to establish the
 diagnosis, stage, and possible therapy. The latter suggestion has been very recently
 reinforced by the evidence that, in humans, cholecystectomy may represent an independent risk
 factor for NAFLD detected at ultrasounds and by the experimental demonstration that
 cholecystectomy increases hepatic triglycerides content.

 In the present research project we will study patients with histologically proven SS or with
 NASH. Liver biopsy will be performed during bariatric surgery (sleeve gastrectomy) or
 cholecystectomy in patients with preoperative evidence of NAFLD at ultrasounds. We will
 compare GM composition using, for the first time, the most accurate method available, that is
 metagenomic shotgun. This method allows to analyze microbiota diversity, providing
 information both on intestinal microbial composition and on the metabolic processes linked to
 them. In addition, we will correlate, for the first time, GM composition to hepatic and, only
 in the obese patients, also to white adipose tissue gene expression patterns of interest and
 serum and fecal markers possibly related to impaired fat storage and inflammation. We aim to
 provide preliminary data to design future intervention studies with pre- or probiotics or
 bile acid derivatives to prevent/treat inflammation and fibrosis in NAFLD patients.
Inclusion Criteria:

 1. Male or female, equal or over 18 years old

 2. Eligible for Sleeve Gastrectomy for obesity with BMI 35-50 kg/m2

 3. Eligible for Cholecystectomy for symptomatic gallstones and bright liver at
 ultrasounds

 4. Alcohol consumption is less than 20 g/d

 Exclusion Criteria:

 1. Having liver disease of other etiology

 2. Having advanced liver disease

 3. Having abnormal coagulation or other reason contraindicating a Liver Biopsy

 4. On regular intake of medications known to cause or exacerbate steatohepatitis or
 antibiotic, pre- or probiotics in the previous 3 months

 5. Use of vitamin E or fish oil supplements in the previous 2 months

 6. Alcohol consumption of more than 20 g/dl

 7. Inflammatory bowel diseases

 8. previous gastrointestinal surgery modifying the anatomy (prior to bariatric surgery)

 9. Pregnancy or lactating state