Link between gut-microbiome derived metabolite and shared gene-effects with hepatic steatosis and fibrosis in NAFLD

Abstract

Previous studies have shown that gut-microbiome is associated with nonalcoholic fatty liver disease (NAFLD). We aimed to examine if serum metabolites, especially those derived from the gut-microbiome, have a shared gene-effect with hepatic steatosis and fibrosis. This is a cross-sectional analysis of a prospective discovery cohort including 156 well-characterized twins and families with untargeted metabolome profiling assessment. Hepatic steatosis was assessed using magnetic-resonance-imaging proton-density-fat-fraction (MRI-PDFF) and fibrosis using MR-elastography (MRE). A twin additive genetics and unique environment effects (AE) model was used to estimate the shared gene-effect between metabolites and hepatic steatosis and fibrosis. The findings were validated in an independent prospective validation cohort of 156 participants with biopsy-proven NAFLD including shotgun metagenomics sequencing assessment in a subgroup of the cohort. In the discovery cohort, 56 metabolites including 6 microbial metabolites had a significant shared gene-effect with both hepatic steatosis and fibrosis after adjustment for age, sex and ethnicity. In the validation cohort, 6 metabolites were associated with advanced fibrosis. Among them, only one microbial metabolite, 3-(4-hydroxyphenyl)lactate, remained consistent and statistically significantly associated with liver fibrosis in the discovery and validation cohort (fold-change of higher-MRE versus lower-MRE: 1.78, P < 0.001 and of advanced versus no advanced fibrosis: 1.26, P = 0.037, respectively). The share genetic determination of 3-(4-hydroxyphenyl)lactate with hepatic steatosis was R_G :0.57,95%CI:0.27-0.80, P < 0.001 and with fibrosis was R_G :0.54,95%CI:0.036-1, P = 0.036. Pathway reconstruction linked 3-(4-hydroxyphenyl)lactate to several human gut-microbiome species. In the validation cohort, 3-(4-hydroxyphenyl)lactate was significantly correlated with the abundance of several gut-microbiome species, belonging only to Firmicutes, Bacteroidetes and Proteobacteria phyla, previously reported as associated with advanced fibrosis. Conclusion: This proof of concept study provides evidence of a link between the gut-microbiome and 3-(4-hydroxyphenyl)lactate that shares gene-effect with hepatic steatosis and fibrosis. (Hepatology 2018).

Figure 1. Shared gene-effect between serum metabolites and hepatic steatosis and fibrosis

Shared gene-effect of the serum metabolites is presented in a polar plot as genetic determination estimates (R_G) adjusted for age, sex and ethnicity colored based on eight metabolomics superpathways (in the central inset).

Figure 2. 56 serum metabolites shared gene-effect with hepatic steatosis and fibrosis

The variation of the 56 serum metabolites, colored based on eight superpathways, with shared gene-effect with hepatic steatosis and fibrosis is depicts as log₂ fold change in participant with NAFLD versus non NAFLD as defined by MRI-PDFF≥5% (left panel) and in participant with higher measurement of MRE (>4.17 kPa) versus lower measurement of MRE (<1.67kPa) (right panel). The 6 serum metabolites derived from the gut microbiome are underlined in red based upon the Human Metabolome Database http://www.hmdb.ca/.

Figure 3. 3-(4-hydroxyphenyl)lactate gut microbiome-linked metabolites is associated with liver fibrosis

Only one serum metabolites linked to the gut microbiome was significantly associated with liver fibrosis in both the discovery and validation cohort.

Figure 4. 3-(4-hydroxyphenyl)lactate is associated with liver steatosis and fibrosis

Median scaled intensity and 95% confidence interval of 3-(4-hydroxyphenyl)lactate in A. Biopsy-proven NAFLD cohort in individuals with versus without advanced fibrosis. B. In the Twins and Family cohort in individuals with higher (>4.61 kPa) versus lower MRE measurement (<1.61 kPa) and in individuals with NAFLD (MRI-PDFF≥5%) versus non-NAFLD (MRI-PDFF<5%). Scaled intensity was obtained by re-scaling raw data to have median equal to 1. C. 3-(4-hydroxyphenyl)lactate had significant shared gene-effect with both hepatic steatosis and fibrosis in AE twins model.

Figure 5. 3-(4-hydroxyphenyl)lactate derived from human gut microbiome

A. Association network of bacterial species from human gut microbiome (identified using the Human microbiome project database (http://www.hmpdacc.org/resources/data_browser.php.) that encodes for the three bacterial enzymes (oranges boxes) catalyzing 3-(4-hydroxyphenyl)lactate and phenyllactate depicted as circles colored by phylum: Proteobacteria in purple, Firmicutes in yellow and Actinobacteria in pink and numbers in italic next to each species name represent unique Taxonomy Database identifiers. Abbreviations: HPPR: hydroxyphenylpyruvate reductase, FldA: cinnamoyl-CoA:phenyllactate CoA-transferase. B. Spearman correlation between 3-(4-hydroxyphenyl)lactate and phenyllactate and the abundance of bacterial species in the human microbiome, only significant correlation are represented, *p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001 after adjustment for age and sex. Framed bacterial species have been previously reported to be significantly associated with the presence of advanced fibrosis (19).

Figure 6. Gut-microbiome derived metabolites and development of NAFLD: a potential cross talk