Impact of Gut Microbiota and Diet on the Development of Atherosclerosis in Apoe-/- Mice.

Objective- To investigate the effect of gut microbiota and diet on atherogenesis. Approach and Results- Here, we investigated the interaction between the gut microbiota and diet on atherosclerosis by feeding germ-free or conventionally raised Apoe-/- mice chow or Western diet alone or supplemented with choline (which is metabolized by the gut microbiota and host enzymes to trimethylamine N-oxide) for 12 weeks. We observed smaller aortic lesions and lower plasma cholesterol levels in conventionally raised mice compared with germ-free mice on a chow diet; these differences were not observed in mice on a Western diet. Choline supplementation increased plasma trimethylamine N-oxide levels in conventionally raised mice but not in germ-free mice. However, this treatment did not affect the size of aortic lesions or plasma cholesterol levels. Gut microbiota composition was analyzed by sequencing of 16S rRNA genes. As expected, the global community structure and relative abundance of many taxa differed between mice fed chow or a Western diet. Choline supplementation had minor effects on the community structure although the relative abundance of some taxa belonging to Clostridiales was altered. Conclusions- In conclusion, the impact of the gut microbiota on atherosclerosis is dietary dependent and is associated with plasma cholesterol levels. Furthermore, the microbiota was required for trimethylamine N-oxide production from dietary choline, but this process could not be linked to increased atherosclerosis in this model.

Monovalerin and trivalerin increase brain acetic acid, decrease liver succinic acid, and alter gut microbiota in rats fed high-fat diets.

PURPOSE: Short-chain fatty acids (SCFA) are known for their anti-inflammatory properties and may also prevent against the development of metabolic diseases. This study investigated possible effects of two valeric acid esters, monovalerin (MV) and trivalerin (TV) in rats fed high-fat diets. METHODS: Four groups of rats were given a low-fat diet (LF) or a high-fat control diet (HFC) with or without supplementation of MV or TV (5 g/kg) for 3 weeks (n = 7/group). SCFA (caecum, blood, liver and brain), succinic acid (liver), microbiota (caecum), lipid profile (liver and blood) and the inflammatory biomarker, lipopolysaccharide-binding protein (blood) were analysed at the end of the experiment. RESULTS: Supplementation of MV and TV to a high-fat diet increased 1.5-fold the amounts of acetic acid in the brain and 1.7-fold serum concentration of valeric acid, whereas liver succinic acid was reduced by 1.5-fold. Although liver triglyceride levels were higher in both MV and TV groups compared with the LF group, liver LDL/HDL ratio was lower in the MV group (P < 0.05). The caecal microbiota composition was altered, with threefold higher abundance of Bacteroidetes and higher ratio of Bacteroidetes/Firmicutes in the MV group compared with the HFC and LF groups. Acetic acid in the brain was negatively correlated with TM7, family S24-7 and rc4-4, and positively associated to Tenericutes and Anaeroplasma. CONCLUSIONS: The present study shows that MV and TV in the specified dose can affect caecal microbiota composition and, therefore, bacterial metabolites in the liver, serum and brain as well as the lipid profile in the liver.

Effects of Proteases from Pineapple and Papaya on Protein Digestive Capacity and Gut Microbiota in Healthy C57BL/6 Mice and Dose-Manner Response on Mucosal Permeability in Human Reconstructed Intestinal 3D Tissue Model.

Cysteine proteases obtained from the stem of pineapple or papaya latex, bromelain and papain, respectively, exhibit a broad spectrum of beneficial effects on human health. However, their effects on gut microbiota composition or dose-manner effects on the intestinal integrity of healthy tissue have not been evaluated. In this study, C57BL/6 young, healthy mice were fed bromelain or papain in a dose of 1 mg per animal/day for three consecutive days, followed by the assessment of digestive protein capacity, intestinal morphology and gut microbiota composition. Furthermore, a human reconstructed 3D tissue model EpiIntestinal (SMI-100) was used to study the effects of 1, 0.1 and 10 mg/mL doses of each enzyme on tissue integrity and mucosal permeability using TEER measurements and passage of Lucifer Yellow marker from the apical to the basolateral side of the mucosa. The results indicated that fruit proteases have the potential to modulate gut microbiota with decreasing abundance of Proteobacteria and increasing beneficial Akkermansia muciniphila. The enhancement of pancreatic trypsin was observed in bromelain and papain supplementation, while bromelain also increased the thickness of the ileal mucosa. Furthermore, an in vitro study showed a dose-dependent interruption in epithelial integrity, which resulted in increased paracellular permeability by the highest doses of enzymes. These findings define bromelain and papain as promising enzymatic supplementation for controlled enhancement of paracellular uptake when needed, together with beneficial effects on the gut microbiota.

Influence of Leptin and Adiponectin Supplementation on Intraepithelial Lymphocyte and Microbiota Composition in Suckling Rats.

Dietary components in early life play a role in both microbiota and intestinal immune system maturation in mammalian species. Adipokines, as endogenously produced hormones from breast milk, may have an impact on this process. The aim of the present study was to establish the influence of leptin and adiponectin supplementation during suckling on the intraepithelial lymphocyte composition, intestinal barrier function, intestinal gene expression, and gut microbiota in rat. For this purpose, newborn Wistar rats were supplemented daily with leptin, adiponectin, or whey protein concentrate during the first 21 days of life. Lymphocyte composition was established by immunofluorescence staining and flow cytometry analysis; intestinal gene expression by real-time PCR and cecal microbiota were analyzed through 16S rRNA gene sequencing. Although leptin and adiponectin were able to increase the Tc TCRαß+ and NKT cell proportion, they decreased the NK cell percentage in IEL. Moreover, adipokine supplementation differentially modified CD8+ IEL. While the supplementation of leptin increased the proportion of CD8αα+ IEL (associated to a more intestinal phenotype), adiponectin enhanced that of CD8αß+ (related to a peripheral phenotype). Furthermore, both adipokines enhanced the gene expression of TNF-α, MUC-2, and MUC-3, and decreased that of FcRn. In addition, the adipokine supplementations decreased the abundance of the Proteobacteria phylum and the presence of Blautia. Moreover, leptin-supplemented animals had lower relative abundance of Sutterella and a higher proportion of Clostridium genus, among others. However, supplementation with adiponectin resulted in lower abundance of the Roseburia genus and a higher proportion of the Enterococcus genus. In conclusion, the supplementation with leptin and adiponectin throughout the suckling period had an impact on both the IEL composition and the gut microbiota pattern, suggesting a modulatory role of these adipokines on the development of intestinal functionality.

Effects of barley variety, dietary fiber and ß-glucan content on bile acid composition in cecum of rats fed low- and high-fat diets.

Diet-induced obesity and insulin resistance have been linked to changes in bile acid (BA) profiles, which in turn are highly dependent on the dietary composition and activity of the gut microbiota. The objective of the present study was to investigate whether the type and level of fiber had an effect on cecal BA composition when included in low- and high-fat diets. Groups of rats were fed two barley varieties, which resulted in three test diets containing three levels of ß-glucans and two levels of dietary fiber. BAs were preconcentrated using hollow fiber liquid-phase microextraction and quantified by gas chromatography. The amount of the secondary BAs, lithocholic-, deoxycholic- and hyodexycholic acids was generally higher in groups fed high-fat diets compared with corresponding acids in groups fed low-fat diets (P<.05). In contrast, most of the primary and the secondary BAs, ursodeoxycholic acid and ß- and ω-muricholic acids, were two to five times higher (P<.05) in groups fed low-fat diets than in groups fed high-fat diets. This was particularly true for groups fed the highest level of ß-glucans and in some cases also the medium level. The BA profile in the gut was strongly dependent on the amount and type of dietary fiber in the diet, which may be useful in the prevention/treatment of diseases associated with changes in BA profiles.

Impact of Rye Kernel-Based Evening Meal on Microbiota Composition of Young Healthy Lean Volunteers With an Emphasis on Their Hormonal and Appetite Regulations, and Blood Levels of Brain-Derived Neurotrophic Factor.

Rye kernel bread (RKB) evening meals improve glucose tolerance, enhance appetite regulation and increase satiety in healthy volunteers. These beneficial effects on metabolic responses have been shown to be associated with increased gut fermentation. The present study aimed to elucidate if RKB evening meals may cause rapid alterations in microbiota composition that might be linked to metabolic-, immune-, and appetite- parameters. Gut-brain axis interaction was also studied by relating microbiota composition to amount of brain-derived neurotrophic factor (BDNF) in blood plasma. Nineteen healthy volunteers, ten women and nine men aged 22-29 years, BMI < 25 (NCT02093481) participated in the study performed in a crossover design. Each person was assigned to either white wheat bread (WWB) or RKB intake as a single evening meal or three consecutive evenings. Stool and blood samples as well as subjective appetite ratings were obtained the subsequent morning after each test occasion, resulting in four independent collections per participant (n = 76). DNA was extracted from the fecal samples and V4 hypervariable region of the bacterial 16S rRNA genes was sequenced using next generation sequencing technology. Higher abundance of Prevotella and Faecalibacterium with simultaneous reduction of Bacteroides spp. were observed after RKB meals compared to WWB. The associations between metabolic test variables and microbiota composition showed a positive correlation between Bacteroides and adiponectin levels, whereas only Prevotella genus was found to have positive association with plasma levels of BDNF. These novel findings in gut-brain interactions might be of importance, since decreased levels of BDNF, that plays an essential role in brain function, contribute to the pathogenesis of several major neurodisorders, including Alzheimer's. Thus, daily consumption of Faecalibacterium- and/or Prevotella-favoring meals should be investigated further for their potential to prevent neurodegenerative processes in the brain.

Barley Products of Different Fiber Composition Selectively Change Microbiota Composition in Rats.

SCOPE: Several dietary fiber properties are suggested to be important for the profiling of the microbiota composition, but those characteristics are rather unclear. Whether different physico-chemical properties of barley dietary fiber influence the gut microbiota composition is investigated. METHODS AND RESULTS: Seven diets containing equal amounts of dietary fiber from barley malts, brewer's spent grain (BSG), and barley extracts, resulting in varying amounts of ß-glucan, soluble arabinoxylan, and insoluble arabinoxylan in the diets were given to conventional rats. Malts increased microbiota alpha diversity more than BSG and the extracts. The intake of soluble arabinoxylan was related to Akkermansia and propionic acid formation in the cecum of rats, whereas ß-glucan and/or insoluble arabinoxylan were attributed to some potentially butyrate-producing bacteria (e.g., Lactobacillus, Blautia, and Allobaculum). CONCLUSION: This study demonstrates that there is a potential to stimulate butyrate- and propionate-producing bacteria in the cecum of rats with malt products of specific fiber properties. Moreover, BSG, a by product from beer production, added to malt can possibly be used to further modulate the microbiota composition, toward a higher butyric acid formation. A complex mixture of fiber as in the malts is of greater importance for microbiota diversity than purer fiber extracts.

Effects of monobutyrin and tributyrin on liver lipid profile, caecal microbiota composition and SCFA in high-fat diet-fed rats.

Butyric acid has been shown to have suppressive effects on inflammation and diseases related to the intestinal tract. The aim of the present study was to investigate whether supplementation of two glycerol esters, monobutyrin (MB) and tributyrin (TB), would reach the hindgut of rats, thus having an effect on the caecal profile of SCFA, microbiota composition and some risk markers associated with chronic inflammation. For this purpose, rats were fed high-fat diets after adding MB (1 and 5 g/kg) and TB (5 g/kg) to a diet without any supplementation (high-fat control; HFC). A low-fat (LF) diet was also included. In the liver, total cholesterol concentrations, LDL-cholesterol concentrations, LDL:HDL ratio, and succinic acid concentrations were reduced in rats given the MB and TB (5 g/kg) diets, compared with the group fed the HFC diet. These effects were more pronounced in MB than TB groups as also expressed by down-regulation of the gene Cyp8b1. The composition of the caecal microbiota in rats fed MB and TB was separated from the group fed the HFC diet, and also the LF diet, as evidenced by the absence of the phylum TM7 and reduced abundance of the genera Dorea (similar to LF-fed rats) and rc4-4. Notably, the caecal abundance of Mucispirillum was markedly increased in the MB group compared with the HFC group. The results suggest that dietary supplementation of MB and TB can be used to counteract disturbances associated with a HFC diet, by altering the gut microbiota, and decreasing liver lipids and succinic acid concentrations.