Can probiotics & prebiotics reduce the impact of high protein diets on gut barrier function by modulating the microbiota in sex-dependent manners?

Gut walls provide physical barriers preventing harmful products of bacteria and digestion from entering the blood. Loss of barrier function (leaky-gut) leads to passage of these molecules into the blood, chronic low-grade inflammation and cardiovascular disease [1, 2].

Some dietary protein reaches the colon where it could drive the expansion of proteolytic bacteria, resulting in higher concentrations of metabolic end-products, including phenol and ammonia, which have been shown to reduce barrier function in vitro [3, 4] but not in vivo.

Some in vitro studies demonstrate that specific probiotics and prebiotics can reduce the effects of high-protein availability on the production of bacterial metabolites associated with leaky gut [5, 6]. However, the results have been inconsistent and often use hydrolysed proteins which do not reach the colon. In addition, protein from single sources is often used. However, we have preliminary data showing highly significant differences in proteolytic bacteria-derived metabolite production depending on the source of the protein (Figure 1a).

We have identified significant sex-based differences in microbiota composition and metabolite production in vitro in response to high levels of protein (Figure 1b-d). We have also independently demonstrated that the effect of both prebiotics and probiotics on immunity are highly sex-dependent in vivo [7] (Figure 1e).

Pigs are valuable, tractable, preclinical models for human nutrition studies since they share many characteristics of gastrointestinal physiology, immunology and gut microbiology. Thus, results from this pig study will be highly translatable for human healthcare. This PhD will determine whether specific prebiotics and probiotics can reduce the negative impacts of high protein diets on intestinal barrier function in a sex-dependent manner.
Objectives

Objective 1: Determine if production of bacterial-derived metabolites linked with increased gut permeability (eg phenol, ammonia) are increased in line with increased non-hydrolysed mixed-source (eg whey, milk, soya, pea, fish) protein availability using verified continuous culture in vitro gut model systems inoculated with human faeces (Figure 1f).

Objective 2: Identify prebiotics and probiotics (eg GOS, FOS, inulin, Bifidobacteria lactis, Lactobacillus casei) which have the most significant impact on reducing these bacterial metabolites associated with reduced barrier function using the in vitro system above.

Objective 3: Explore if supernatants from the models above have a direct impact on the expression of tight cell junction (TCJ)-associated protein expression (eg ZO-1, E-cadherin) using a combination of human colonic cell lines and quantitative fluorescence immunohistology.

Objective 4: Determine if the benefits of the prebiotic and/or probiotic identified in objective 2 can be observed in vivo using a pig model for humans (Figure 2a). Bacterial population dynamics, metabolite production and TCJ-associated protein expression (and immunity) will be assessed using 16S sequencing, gas-chromatography/mass spectroscopy and quantitative fluorescence immunohistology respectively. Quantification of the expression of immune-associated proteins will be achieved using fluorescence histology.

Objective 5: Determine if there are sexually dimorphic effects in all the above using sex-balanced treatment and control groups throughout and by using 'sex' as a factor during statistical analyses.

Objective 6: Statistical approaches will be used to generate a global overview of intestinal barrier function, the gut microbiota, immunity, host metabolism and host-microbe co-metabolism. This will generate mechanistic understanding of the biological processes associated with increased dietary protein, microbiota modification and determine whether sexual disparity occurs.