Stable isotope tracing to probe dietary fibre-associated gut microbes and metabolites in the prevention of NASH

The stable isotope labeling strategy provide direct footprints of inulin in the gut metagenome and metabolome, thereby shedding the limelight on the key gut microbes and metabolites involved in NASH prevention.
Published in Microbiology
Stable isotope tracing to probe dietary fibre-associated gut microbes and metabolites in the prevention of NASH
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Not all dietary fibres are equal for NASH prevention

In recent decades, overnutrition and obesity have led to the dramatic rise in the incidence of non-alcoholic fatty liver disease (NAFLD), which has become a global health burden. A proportion of patients with NAFLD develops non-alcoholic steatohepatitis (NASH), with inflammation and hepatocyte damage. As no FDA-approved drugs is available for targeting NASH, an area of our interest is to study whether dietary modification can be an alternative approach for NASH prevention or treatment. In this study, we first questioned if the types of fibre matter in NASH prevention. We chose one soluble fibre, inulin and one insoluble fibre, cellulose, and investigated their preventive effects in two mouse models of NASH. We discovered that inulin is more effective than cellulose in preventing NASH in mice. So, the type of fibre is critical for NASH prevention.

How do we trace bugs and metabolites associated with fibre consumption?

A distinct feature of soluble fibre vs insoluble fibre is the ability of gut microbes to utilize the former as an energy source. We therefore hypothesized that soluble fibre-associated gut microbes and metabolites might play a critical role in NASH prevention. Indeed, shot-gun metagenome sequencing and non-targeted metabolomics demonstrated that inulin, but not cellulose, significantly modified gut microbial and metabolic compositions, implying that gut microbiota and metabolites might contribute to the beneficial effect of soluble fibre.

Nevertheless, the key question remains which are the key bacteria and metabolites involved in alleviation of NASH? To answer this question, we employed 13C-stable isotope labeling using uniformly labeled 13C-inulin added to the mouse diet. The basic principle is that 13C-inulin will be assimilated by gut microbes and incorporated into their genomic DNA, which make their genome DNA “heavier”. “Heavy” DNA fraction from inulin-utilizing microbes are then isolated from non-labelled microbial DNA by gradient density ultra-centrifugation, followed by metagenome profiling to identify enriched bacterial species. Simultaneously, we performed LC-MS to uncover 13C-labelled metabolites that are associated with inulin fermentation by gut microbes.

What are the key findings?

By tracking the carbon flow from dietary inulin into microbial genome DNA or metabolites, we identified a series of 13C-labelled bacteria including Bacteroides uniformis, Bacteroides acidifaciens, Parabacteroides distasonis, and 13C-labelled metabolites such as long chain fatty acids, adenosine, vitamins, etc, that are enriched as a consequence of fermentation by gut microbes. On the contrary, cellulose was minimally utilized by gut microbes. In vivo validation work showed that P. distasonis was the most effective bacteria in suppressing NASH in mice. Concordantly, P. distasonis is regarded as a gut commensal bacterium that is highly enriched in healthy subjects compared to patients with diabetes. Among the 13C labelled metabolites, pentadecanoic acid was consistently enriched in mice stool and portal vein serum. More importantly, it was a metabolic product of P. distasonis in vitro and also enriched in P. distasonis mono-colonized germ-free mice. Functional validation in mice revealed that pentadecanoic acid is protective in NASH by suppressing liver damage (ALT and AST) and proinflammatory cytokines (TNF-α and IL-6). Collectively, we identified an inulin P. distasonis-pentadecanoic acid axis that suppresses NASH development.

P. distasonis and its metabolite works by fixing the “leaky” gut in NASH

An intact gut barrier function as an important defensive line for the host against pathogens and their products. We found that mice with diet-induced NASH developed a “leaky gut”, which exacerbated hepatic inflammation via increased translocation of lipopolysaccharides (LPS), major bacterial toxins that potently activate the immune system. On the other hand, either the administration of inulin, P. distasonis, or pentadecanoic acid could fix “leaky gut” by restoring tight junction integrity. Consequently, upregulation of NASH related pathways including lipogenesis and chemokine signaling pathway in our NASH mouse models were consistently suppressed by the administration of inulin, P. distasonis, or pentadecanoic acid.

Future implications

Till now, gut fermentation of dietary fibre is largely a “black box” with little knowledge of the key bacteria and metabolites in association with its beneficial effects. Our stable isotope labeling strategy provide direct footprints of inulin in the gut metagenome and metabolome, thereby shedding the limelight on the key gut microbes and metabolites involved in NASH prevention. Our approach is generalizable for tracing the interplay of fibre as well as other nutrients with gut microbes and their collateral effect on human health and disease.

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