Circulating microbiota-derived metabolites: a "liquid biopsy?

Abstract

Background/objectives: Non-alcoholic fatty liver disease (NAFLD) causes a wide spectrum of liver damage, from simple steatosis (SS) to cirrhosis. SS and non-alcoholic steatohepatitis (NASH) cannot be distinguished by clinical or laboratory features. Dysregulation of the gut microbiota is involved in NASH pathogenesis. The aim of this study was to assess the relationship between microbiota-derived metabolites and the degrees of NAFLD; also, to investigate whether these metabolites could be included in a panel of NASH biomarkers.

Subjects/methods: We used liquid chromatography coupled to triple-quadrupole-mass spectrometry (LC-QqQ) analysis to quantify choline and its derivatives, betaine, endogenous ethanol, bile acids, short-chain fatty acids and soluble TLR4 in serum from women with normal weight (n = 29) and women with morbid obesity (MO) (n = 82) with or without NAFLD. We used real-time polymerase chain reaction (RT-PCR) analysis to evaluate the hepatic and intestinal expression level of all genes studied (TLR2, TLR4, TLR9, LXRα, SREBP1C, ACC1, FAS, PPARα, CPT1α, CROT, SREBP2, ABCA1, ABCG1 and FXR in the liver; TLR2, TLR4, TLR5, TLR9, GLP-1R, DPP-4, FXR and PPARɣ in the jejunum) in 82 women with MO with normal liver histology (NL, n = 29), SS (n = 32), and NASH (n = 21).

Results: Hepatic FAS, TLR2, and TLR4 expression were overexpressed in NAFLD patients. TLR2 was overexpressed in NASH patients. In women with MO with NAFLD, we found upregulation of intestinal TLR9 expression and downregulation of intestinal FXR expression in women with NASH. Circulating TMAO, glycocholic acid and deoxycholic acid levels were significantly increased in NAFLD patients. Endogenous circulating ethanol levels were increased in NASH patients in comparison to those in SS patients.

Conclusions: These findings suggest that the intestine participates in the progression of NAFLD. Moreover, levels of certain circulating microbiota-related metabolites are associated with NAFLD severity and could be used as a "liquid biopsy" in the noninvasive diagnosis of NASH.

Fig. 1

Histologic features, grading, and staging of NAFLD (own images). Histological evaluation of liver sections stained with Hematoxylin-eosin, 200×: a SS group: Normal architecture amb macrovesicular steatosis. b NASH group: Macrovesicular steatosis, ballooning degeneration, and lobular inflammation

Fig. 2

Hepatic expression of genes related to lipid metabolism, FXR and Toll-like receptors in women with morbid obesity (n = 82) classified according to liver histopathology: normal liver (NL), simple steatosis (SS), and non-alcoholic steatohepatitis (NASH). The Mann-Whitney U test or Kruskal-Wallis test was used to determine differences between groups. SREBP2, sterol regulatory element-binding protein 2; ABCA1, ATP-binding cassette A1; ABCG, ATP-binding cassette G; CPT1α, carnitine palmitoyl transferase 1 alpha; CROT, carnitine O-Octanoyltransferase; SREBP1C, sterol regulatory element-binding protein 1c; PPARα, peroxisome proliferator-activated receptor alpha; LXRα, liver X receptor alpha; ACC1, acetyl-CoA carboxylase 1; FAS, fatty acid synthase; FXR, farnesoid X receptor; TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; and TLR9, Toll-like receptor 9. p < 0.05 was considered statistically significant

Fig. 3

Intestinal mRNA expression of Toll-like receptors, DPP-4, FXR and PPARɣ in women with morbid obesity (n = 82) classified according to liver histopathology: normal liver (NL), simple steatosis (SS), and non-alcoholic steatohepatitis (NASH). Mann-Whitney’s U test or Kruskal-Wallis test was used to determine differences between groups. TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; TLR5, Toll-like receptor 5; TLR9, Toll-like receptor 9; DDP-4, dipeptidyl peptidase-4; FXR, farnesoid X receptor; PPARγ, peroxisome proliferator-activated receptor gamma. p < 0.05 was considered statistically significant