Alterations in secondary gut metabolites derived from the microbial fermentation of food in the gut have significant effects on various aspects of host physiology. Our recent studies on obese mice treated with Orlistat, an antiobesity treatment, revealed a significantly altered gut microbial profile marked by an over-abundance of Proteobacteria and alterations in secondary gut metabolites. In this study, we determined effect of fecal metabolites from high-fat diet fed mice treated with Orlistat (HFDOrl) on colonic epithelial cells in relation to inflammation, barrier function, mitochondrial activity, reactive oxygen species (ROS) levels, and oxidative stress. Quantitative PCR was used to measure intestinal mRNA expression of oxidative stress, inflammation, apoptosis, and gut barrier function genes in mice on a high fat diet with and without Orlistat treatment versus those fed a low-fat diet (HFDOrl, HFD, Normal diet-fed [ND] respectively). Alterations to antioxidant function in HCT-116-ARE-luciferase stable cell line and mitochondrial function in Caco-2 cells was analyzed under oxidative stress with exposure to aqueous fecal extracts from HFDOrl, HFD, and ND groups. The results of this study indicate that a significant increase in anti-oxidative response was observed based on the luciferase activity of HCT-116-ARE-luciferase stable cells. Increased maximal respiration and mitochondrial ROS under oxidative stress was also detected in confluent Caco-2 cells resulting from exposure to fecal extracts from the HFDOrl group compared with the HFD group and pure Orlistat. Furthermore, mice from the HFDOrl group exhibited a significant increase in colonic epithelial expression of oxidative markers (Nrf-2 and SOD-2), inflammation-related markers (IL-6 and TNF-α), and gut barrier function markers (Muc-2 and Occludin). Taken together, the results suggest that Orlistat treatment in the HFD group causes changes in secondary gut metabolites which affect the colonic redox state and may eventually lead to the development of inflammatory, oxidative, and mitochondrial dysfunction at the cellular level.

Curtailing the absorption of triglycerides (TGs) is a preferred pathway for treating obesity. Our previous study demonstrated that the water-soluble fraction from potato could inhibit the lipase activity of patatin, one of the major proteins in potato. This aqueous fraction was purified and concentrated by deproteination and reversed-phase chromatography to investigate the effectiveness against obesity. Biochemical analyses indicated that the fraction non-competitively inhibited pancreatic lipase (PLase) with a half-maximal inhibitory concentration of 10.17 µg/mL, and was named as potato-derived lipase inhibitory fraction (PI). Animal studies on C57BL/6 mice showed that in mice fed a high-fat diet (HFD), PI treatment resulted in reductions in body weight gain, adipose fat deposition, and liver TGs, and ameliorated the gut microbiome dysbiosis caused by HFD feeding; meanwhile, orlistat, a well-known lipase inhibitor, diverged the gut microbiome profile in mice fed a HFD. High resolution electronspray ionization-Orbitrap tandem mass spectrometry identified gallic acid, 4-hydroxybenzoic acid, and protocatechuic acid, which are known to have lipase inhibitory activities, in PI. However, these compounds could not reconstitute comparable specific inhibitory activity of PI inferring the existence of another inhibitory compound(s) to be identified in PI.

A maternal diet rich in dietary fiber, such as β-glucan, plays a crucial role in the offspring’s acquisition of gut microbiota and the subsequent shaping of its microbiome profile and metabolome. This in turn has been shown to aid in neurodevelopmental processes, including early microglial maturation and immunomodulation via metabolites like short chain fatty acids (SCFAs). This study aimed to investigate the effects of oat β-glucan supplementation, solubilized by citric acid hydrolysis, from gestation to adulthood. Female C57BL/6J mice were orally supplemented with soluble oat β-glucan (ObG) or carboxymethyl cellulose (CMC) via drinking water at 200 mg/kg body weight during breeding while the control group received 50 mg/kg body weight of carboxymethyl cellulose. ObG supplementation increased butyrate production in the guts of both dams and 4-week-old pups, attributing to alterations in the gut microbiota profile. One-week-old pups from the ObG group showed increased neurodevelopmental markers similar to four-week-old pups that also exhibited alterations in serum markers of metabolism and anti-inflammatory cytokines. Notably, at 8 weeks, ObG-supplemented pups exhibited the highest levels of spatial memory and cognition compared to the control and CMC groups. These findings suggest a potential enhancement of neonatal neurodevelopment via shaping of early-life gut microbiome profile, and the subsequent increased later-life cognitive function.

Background: Although the food quality of soy protein is known to be as good as that of animal proteins, some soybean proteins are not susceptible to digestion and remain undigested in the intestine. We hypothesized that digestion-resistant soy proteins might interact with the intestinal membrane, microbes, and metabolites, and change the intestinal physiology or the profile of the gut microbiome. Objective: To identify the Protease-Resistant Soy Proteins (PRSPs) and their interaction with intestinal membrane proteins by MS, and to assess the functions of PRSPs in the small intestine. Methods: Soy proteins were sequentially digested with pepsin and pancreatin, and the PRSPs were identified by SDS-PAGE and MS. Intestinal cell membrane proteins interacting with PRSPs were isolated by affinity purification and photo-affinity crosslinking, and identified using MS/MS. Inhibition of cholesterol absorption to lipoprotein-depleted intestinal cells, CaCo-2, and hepatic cells, HepG2, was measured in the presence and absence of PRSPs. FITC-conjugated Gram-positive, Lactobacillus plantarum, and Gram-negative bacteria, Escherichia coli, were incubated with Ca- Co-2 cells in the presence of PRSPs to investigate the regulation of bacterial cell binding to intestinal epithelial cells by PRSPs. Results: MS/MS of PRSPs identified glycinin, β-conglycinin, trypsin inhibitors, lipoxygenase, and sucrose-binding protein. MS analysis also identified the intestinal membrane proteins bound to PRSPs. The functions of the identified interacting proteins included ion transportation, carbohydrate- binding, cytoskeleton formation, hydrolysis, cell-cell junction formation, and cholesterol/steroid- binding. In particular, apolipoprotein E, aminopeptidase N, and Niemann-Pick C1-like protein 1 are known to be involved in cholesterol absorption in the small intestine. The inhibition of cholesterol absorption by CaCo-2 and HepG2 cells by PRSPs confirmed the MS results. Binding of L. plantarum and E. coli to CaCo-2 cells was efficiently inhibited by PRSPs. Conclusion: PRSPs can interact with intestinal membrane proteins, and regulate cholesterol absorption by intestinal epithelial cell and interactions of the gut microbiome. Soy protein in the intestine acts as a nutrient, and triggers changes in intestinal functions by interacting with intestinal cells, microorganisms, and nutrients. These findings will provide valuable new functional information about the effects of soy proteins on human health.