Gut Microbiota and Modulation of Liver Damage in NAFLD

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

• Male or female, equal or over 18 years old
• Eligible for Sleeve Gastrectomy for obesity with BMI 35-50 kg/m2
• Eligible for Cholecystectomy for symptomatic gallstones and bright liver at ultrasounds
• Alcohol consumption is less than 20 g/d

Exclusion criteria

• Having liver disease of other etiology
• Having advanced liver disease
• Having abnormal coagulation or other reason contraindicating a Liver Biopsy
• On regular intake of medications known to cause or exacerbate steatohepatitis or antibiotic, pre- or probiotics in the previous 3 months
• Use of vitamin E or fish oil supplements in the previous 2 months
• Alcohol consumption of more than 20 g/dl
• Inflammatory bowel diseases
• previous gastrointestinal surgery modifying the anatomy (prior to bariatric surgery)
• Pregnancy or lactating state