Commensal microbe-derived acetate suppresses NAFLD/NASH development via hepatic FFAR2 signalling in mice.

BACKGROUND: Non-alcoholic liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome, and it can progress to non-alcoholic steatohepatitis (NASH). Alterations in the gut microbiome have been implicated in the development of NAFLD/NASH, although the underlying mechanisms remain unclear. RESULTS: We found that the consumption of the prebiotic inulin markedly ameliorated the phenotype of NAFLD/NASH, including hepatic steatosis and fibrosis, in mice. Inulin consumption resulted in global changes in the gut microbiome, including concomitant enrichment of the genera Bacteroides and Blautia, and increased concentrations of short-chain fatty acids, particularly acetate, in the gut lumen and portal blood. The consumption of acetate-releasing resistant starch protected against NAFLD development. Colonisation by Bacteroides acidifaciens and Blautia producta in germ-free mice resulted in synergetic effects on acetate production from inulin. Furthermore, the absence of free fatty acid receptor 2 (FFAR2), an acetate receptor, abolished the protective effect of inulin, as indicated by the more severe liver hypertrophy, hypercholesterolaemia and inflammation. These effects can be attributed to an exacerbation of insulin resistance in the liver, but not in muscle or adipose tissue. CONCLUSION: These findings demonstrated that the commensal microbiome-acetate-FFAR2 molecular circuit improves insulin sensitivity in the liver and prevents the development of NAFLD/NASH. Video abstract.

An acetate-yielding diet imprints an immune and anti-microbial programme against enteric infection.

OBJECTIVES: During gastrointestinal infection, dysbiosis can result in decreased production of microbially derived short-chain fatty acids (SCFAs). In response to the presence of intestinal pathogens, we examined whether an engineered acetate- or butyrate-releasing diet can rectify the deficiency of SCFAs and lead to the resolution of enteric infection. METHODS: We tested whether a high acetate- or butyrate-producing diet (HAMSA or HAMSB, respectively) condition Citrobacter rodentium infection in mice and assess its impact on host-microbiota interactions. We analysed the adaptive and innate immune responses, changes in gut microbiome function, epithelial barrier function and the molecular mechanism via metabolite sensing G protein-coupled receptor 43 (GPR43) and IL-22 expression. RESULTS: HAMSA diet rectified the deficiency in acetate production and protected against enteric infection. Increased SCFAs affect the expression of pathogen virulence genes. HAMSA diet promoted compositional and functional changes in the gut microbiota during infection similar to healthy microbiota from non-infected mice. Bacterial changes were evidenced by the production of proteins involved in acetate utilisation, starch and sugar degradation, amino acid biosynthesis, carbohydrate transport and metabolism. HAMSA diet also induced changes in host proteins critical in glycolysis, wound healing such as GPX1 and epithelial architecture such as EZR1 and PFN1. Dietary acetate assisted in rapid epithelial repair, as shown by increased colonic Muc-2, Il-22, and anti-microbial peptides. We found that acetate increased numbers of colonic IL-22 producing TCRαß+CD8αß+ and TCRγδ+CD8αα+ intraepithelial lymphocytes expressing GPR43. CONCLUSION: HAMSA diet may be an effective therapeutic approach for fighting inflammation and enteric infections and offer a safe alternative that may impact on human health.

Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disorder with a high prevalence, especially in industrialized countries. Dysbiosis of the intestinal microbiota has been observed in RA patients. For instance, new-onset untreated RA (NORA) is associated with the underrepresentation of the Clostridium cluster XIVa, including Lachnospiraceae, which are major butyrate producers, although the pathological relevance has remained obscure. Follicular regulatory T (TFR) cells play critical regulatory roles in the pathogenesis of autoimmune diseases, including RA. Reduced number of circulating TFR cells has been associated with the elevation of autoantibodies and disease severity in RA. However, the contribution of commensal microbe-derived butyrate in controlling TFR cell differentiation remains unknown. METHODS: We examined the contribution of microbe-derived butyrate in controlling autoimmune arthritis using collagen-induced arthritis (CIA) and SKG arthritis models. We phenotyped autoimmune responses in the gut-associated lymphoid tissues (GALT) in the colon and joint-draining lymph nodes in the CIA model. We developed an in vitro CXCR5+Bcl-6+Foxp3+ TFR (iTFR) cell culture system and examined whether butyrate promotes the differentiation of iTFR cells. FINDINGS: Microbe-derived butyrate suppressed the development of autoimmune arthritis. The immunization of type II collagen (CII) caused hypertrophy of the GALT in the colon by amplifying the GC reaction prior to the onset of the CIA. Butyrate mitigated these pathological events by promoting TFR cell differentiation. Butyrate directly induced the differentiation of functional TFR cells in vitro by enhancing histone acetylation in TFR cell marker genes. This effect was attributed to histone deacetylase (HDAC) inhibition by butyrate, leading to histone hyperacetylation in the promoter region of the TFR-cell marker genes. The adoptive transfer of the butyrate-treated iTFR cells reduced CII-specific autoantibody production and thus ameliorated the symptoms of arthritis. INTERPRETATION: Accordingly, microbiota-derived butyrate serves as an environmental cue to enhance TFR cells, which suppress autoantibody production in the systemic lymphoid tissue, eventually ameliorating RA. Our findings provide mechanistic insights into the link between the gut environment and RA risk. FUNDING: This work was supported by AMED-Crest (16gm1010004h0101, 17gm1010004h0102, 18gm1010004h0103, and 19gm1010004s0104 to KH), the Japan Society for the Promotion of Science (JP17KT0055, JP16H01369, and JP18H04680 to KH; JP17K15734 to DT), Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects (KH), Keio Gijuku Fukuzawa Memorial Fund for the Advancement of Education and Research (DT), the SECOM Science and Technology Foundation (KH), the Cell Science Research Foundation (KH), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (DT), the Suzuken Memorial Foundation (KH and DT), the Takeda Science Foundation (KH and DT), The Science Research Promotion Fund, and The Promotion and Mutual Aid Corporation for Private Schools of Japan (KH).

Erratum: Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

This corrects the article DOI: 10.1038/ni.3713.

Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

Gut dysbiosis might underlie the pathogenesis of type 1 diabetes. In mice of the non-obese diabetic (NOD) strain, we found that key features of disease correlated inversely with blood and fecal concentrations of the microbial metabolites acetate and butyrate. We therefore fed NOD mice specialized diets designed to release large amounts of acetate or butyrate after bacterial fermentation in the colon. Each diet provided a high degree of protection from diabetes, even when administered after breakdown of immunotolerance. Feeding mice a combined acetate- and butyrate-yielding diet provided complete protection, which suggested that acetate and butyrate might operate through distinct mechanisms. Acetate markedly decreased the frequency of autoreactive T cells in lymphoid tissues, through effects on B cells and their ability to expand populations of autoreactive T cells. A diet containing butyrate boosted the number and function of regulatory T cells, whereas acetate- and butyrate-yielding diets enhanced gut integrity and decreased serum concentration of diabetogenic cytokines such as IL-21. Medicinal foods or metabolites might represent an effective and natural approach for countering the numerous immunological defects that contribute to T cell-dependent autoimmune diseases.

Dietary butyrylated high-amylose starch reduces azoxymethane-induced colonic O(6)-methylguanine adducts in rats as measured by immunohistochemistry and high-pressure liquid chromatography.

O(6)-methyl guanine (O(6)MeG) adducts are major toxic, promutagenic, and procarcinogenic adducts involved in colorectal carcinogenesis. Resistant starch and its colonic metabolite butyrate are known to protect against oncogenesis in the colon. In this study, we hypothesized that a dietary intervention that specifically delivers butyrate to the large bowel (notably butyrylated high-amylose maize starch [HAMSB]) would reduce colonic levels of O(6)MeG in rats shortly after exposure to the deoxyribonucleic acid (DNA) alkylating agent azoxymethane (AOM) when compared with a low-amylose maize starch (LAMS). A further objective was to validate an immunohistochemistry (IHC) method for quantifying O(6)MeG against a high-performance liquid chromatography method using fluorescence and diode array detection. Rats were fed either LAMS or HAMSB diets for 4 weeks followed by a single injection of AOM or saline and killed 6 hours later. After AOM exposure, both IHC and high-performance liquid chromatography method using fluorescence and diode array detection measured a substantially increased quantity of DNA adducts in the colon (P<.001). Both techniques demonstrated equally that consumption of HAMSB provided a protective effect by reducing colonic adduct load compared with the LAMS diet (P<.05). In addition, IHC allowed visualization of the O(6)MeG distribution, where adduct load was reduced in the lower third of the crypt compartment in HAMSB-fed rats (P=.036). The apoptotic response to AOM was higher in the HAMSB-fed rats (P=.002). In conclusion, the reduction in O(6)MeG levels and enhancement of the apoptotic response to DNA damage in the colonic epithelium through consumption of HAMSB provide mechanistic insights into how HAMSB protects against colorectal tumorigenesis.

Housing experimental rats in solid-based cages with digestible bedding may confound outcomes of nutritional studies.

BACKGROUND: Rats used in nutritional studies are often kept in wire-based cages to prevent ingestion of bedding and minimise ingestion of faeces. However, wire-based cages are criticised because of potential negative animal welfare implications. This study investigated the effects of wire and solid-based cages with corncob bedding on large bowel fermentation and microbiota. Rats were group housed in wire or solid-based cages and fed either a low-fibre (LF) diet or a high-fibre (HF) diet composed of resistant starch for 4 weeks. RESULTS: Bedding material was observed in faeces of rats housed in solid-based cages. Caging type and diet altered large bowel fermentation variables and bacterial populations. Caecal digesta weight was lower in rats fed HF diet and maintained on bedding than in HF-fed rats maintained on wire. Bacteria abundance associated with fibre fermentation was higher in LF-diet fed rats maintained on bedding compared with LF-fed rats housed on wire. CONCLUSION: Maintaining rats in solid-based cages with corncob bedding alters large bowel fermentation and bacterial communities owing to ingestion of bedding. These changes may confound outcomes of nutritional studies, particularly those investigating the health effects of fibres. The use of wire-based caging may be justified in research of this type.

Butyrylated starch affects colorectal cancer markers beneficially and dose-dependently in genotoxin-treated rats.

Population studies suggest that greater dietary fiber intake may lower colorectal cancer (CRC) risk, possibly through the colonic bacterial fermentative production of butyrate. Butyrylated starch delivers butyrate to the colon of humans with potential to reduce CRC risk but high doses may exacerbate risk through promoting epithelial proliferation. Here we report the effects of increasing dietary butyrylated high amylose maize starch (HAMSB) on azoxymethane (AOM) induced distal colonic DNA damage, cell proliferation, mucus layer thickness and apoptosis in rats. Five groups of 15 rats were fed AIN-93G based diets containing 0-40% HAMSB for 4 weeks then injected with (AOM) and killed 6 hours later. Large bowel total SCFA, acetate and butyrate pools and hepatic portal venous plasma total SCFA, acetate and butyrate concentrations were higher with greater HAMSB intake. Distal colonic epithelial apoptotic index and colonic mucus thickness increased, while DNA single strand breaks decreased dose-dependently with greater HAMSB intake. Colonocyte proliferation rates were unaffected by diet. These data suggest that increasing large bowel butyrate may reduce the risk of CRC in a dose dependent manner by enhancing apoptotic surveillance in the colonic epithelium for damaged cells without promoting the risk of tumorigenesis through increased cell proliferation.

Dietary supplementation of propionylated starch to domestic cats provides propionic acid as gluconeogenic substrate potentially sparing the amino acid valine.

In strict carnivorous domestic cats, a metabolic competition arises between the need to use amino acids for gluconeogenesis and for protein synthesis both in health and disease. The present study investigated the amino acid-sparing potential of propionic acid in cats using dietary propionylated starch (HAMSP) supplementation. A total of thirty cats were fed a homemade diet, supplemented with either HAMSP, acetylated starch (HAMSA) or celite (Control) for three adaptation weeks. Propionylated starch was hypothesised to provide propionic acid as an alternative gluconeogenic substrate to amino acids, whereas acetic acid from HAMSA would not provide any gluconeogenic benefit. Post-adaptation, a 5-d total faecal collection was carried out to calculate apparent protein digestibility coefficients. Fresh faecal and blood samples were collected to analyse fermentation endproducts and metabolites. The apparent protein digestibility coefficients did not differ between supplements (P = 0·372) and were not affected by the protein intake level (P = 0·808). Faecal propionic acid concentrations were higher in HAMSP than in HAMSA (P = 0·018) and Control (P = 0·003) groups, whereas concentrations of ammonia (P = 0·007) were higher in HAMSA than in HAMSP cats. Tendencies for or higher propionylcarnitine concentrations were observed in HAMSP compared with HAMSA (P = 0·090) and Control (P = 0·037) groups, and for tiglyl- + 3-methylcrotonylcarnitine concentrations in HAMSP as compared with Control (P = 0·028) cats. Methylmalonylcarnitine concentrations did not differ between groups (P = 0·740), but were negatively correlated with the protein intake level (r -0·459, P = 0·016). These results suggest that HAMSP cats showed more saccharolytic fermentation patterns than those supplemented with HAMSA, as well as signs of sparing of valine in cats with a sufficient protein intake.

Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells.

Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (Treg) cells, which have a central role in the suppression of inflammatory and allergic responses. However, the molecular mechanisms by which commensal microbes induce colonic Treg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic Treg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of Treg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of Treg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4(+) CD45RB(hi) T cells in Rag1(-/-) mice. Treatment of naive T cells under the Treg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of Treg cells. Our findings provide new insight into the mechanisms by which host-microbe interactions establish immunological homeostasis in the gut.

Butyrylated starch increases colonic butyrate concentration but has limited effects on immunity in healthy physically active individuals.

BACKGROUND: Butyrate delivery to the large bowel may positively modulate commensal microbiota and enhance immunity. OBJECTIVE: To determine the effects of increasing large bowel butyrate concentration through ingestion of butyrylated high amylose maize starch (HAMSB) on faecal biochemistry and microbiota, and markers of immunity in healthy active individuals. DESIGN: Male and female volunteers were assigned randomly to consume either two doses of 20 g HAMSB (n = 23; age 37.9 +/- 7.8 y; mean +/- SD) or a low amylose maize starch (LAMS) (n = 18; age 36.9 = 9.5 y) twice daily for 28 days. Samples were collected on days 0, 10 and 28 for assessment of faecal bacterial groups, faecal biochemistry, serum cytokines and salivary antimicrobial proteins. RESULTS: HAMSB led to relative increases in faecal free (45%; 12-86%; mean; 90% confidence interval; P = 0.02), bound (950%; 563-1564%; P < 0.01) and total butyrate (260%; 174-373%; P < 0.01) and faecal propionate (41%; 12-77%; P = 0.02) from day 0 to day 28 compared to LAMS. HAMSB was also associated with a relative 1.6-fold (1.2- to 2.0-fold; P < 0.01) and 2.5-fold (1.4- to 4.4-fold; P = 0.01) increase in plasma IL-10 and TNF-alpha but did not alter other indices of immunity. There were relative greater increases in faecal P. distasonis (81-fold (28- to 237-fold; P < 0.01) and F. prausnitzii (5.1-fold (2.1- to 12-fold; P < 0.01) in the HAMSB group. CONCLUSIONS: HAMSB supplementation in healthy active individuals promotes the growth of bacteria that may improve bowel health and has only limited effects on plasma cytokines.

Resistant starches protect against colonic DNA damage and alter microbiota and gene expression in rats fed a Western diet.

Resistant starch (RS), fed as high amylose maize starch (HAMS) or butyrylated HAMS (HAMSB), opposes dietary protein-induced colonocyte DNA damage in rats. In this study, rats were fed Western-type diets moderate in fat (19%) and protein (20%) containing digestible starches [low amylose maize starch (LAMS) or low amylose whole wheat (LAW)] or RS [HAMS, HAMSB, or a whole high amylose wheat (HAW) generated by RNA interference] for 11 wk (n = 10/group). A control diet included 7% fat, 13% protein, and LAMS. Colonocyte DNA single-strand breaks (SSB) were significantly higher (by 70%) in rats fed the Western diet containing LAMS relative to controls. Dietary HAW, HAMS, and HAMSB opposed this effect while raising digesta levels of SCFA and lowering ammonia and phenol levels. SSB correlated inversely with total large bowel SCFA, including colonic butyrate concentration (R(2) = 0.40; P = 0.009), and positively with colonic ammonia concentration (R(2) = 0.40; P = 0.014). Analysis of gut microbiota populations using a phylogenetic microarray revealed profiles that fell into 3 distinct groups: control and LAMS; HAMS and HAMSB; and LAW and HAW. The expression of colonic genes associated with the maintenance of genomic integrity (notably Mdm2, Top1, Msh3, Ung, Rere, Cebpa, Gmnn, and Parg) was altered and varied with RS source. HAW is as effective as HAMS and HAMSB in opposing diet-induced colonic DNA damage in rats, but their effects on the large bowel microbiota and colonocyte gene expression differ, possibly due to the presence of other fiber components in HAW.

Butyrate delivered by butyrylated starch increases distal colonic epithelial apoptosis in carcinogen-treated rats.

Animal studies show that increasing large bowel butyrate concentration through ingestion of butyrylated or resistant starches opposes carcinogen-induced tumorigenesis, which is consistent with population data linking greater fiber consumption with lowered colorectal cancer (CRC) risk. Butyrate has been shown to regulate the apoptotic response to DNA damage. This study examined the impact of increasing large bowel butyrate concentration by dietary butyrylated starch on the colonic epithelium of rats treated with the genotoxic carcinogen azoxymethane (AOM). Four groups of 10 male rats were fed AIN-93G based-diets containing either low amylose maize starch (LAMS), LAMS with 3% tributyrin, 10% high amylose maize starch (HAMS) or 10% butyrylated HAMS (HAMSB). HAMS and HAMSB starches were cooked by heating in water. After 4 weeks, rats were injected once with AOM and killed 6 h later. Rates of apoptosis and proliferation were measured in colonic epithelium. Short-chain fatty acid concentrations in large bowel digesta and hepatic portal venous plasma were higher in HAMSB than all other groups. Apoptotic rates in the distal colon were increased by HAMSB and correlated with luminal butyrate concentrations but cellular proliferation rates were unaffected by diet. The increase in apoptosis was most marked in the base and proliferative zone of the crypt. Regulation of luminal butyrate using HAMSB increases the rates of apoptotic deletion of DNA-damaged colonocytes. We propose this pro-apoptotic function of butyrate plays a major role reducing tumour formation in the AOM-treated rat and that these data support a potential protective role of butyrate in CRC.

Butyrate esterified to starch is released in the human gastrointestinal tract.

BACKGROUND: Short-chain fatty acids (SCFAs) maintain human colonic function and may help prevent colonic disease. A study with ileostomists showed that starches acylated with specific SCFAs largely survive passage through the small intestine, but the percentage released in the colon has not been established. OBJECTIVE: The objective was to determine the percentage of ingested esterified butyrate released in the human gastrointestinal tract. DESIGN: The study was a randomized, crossover, controlled trial consisting of baseline and four 2-wk periods during which 16 volunteers consumed diets low in resistant starch plus 20 and 40 g cooked high-amylose maize starch (HAMS: HAMS20 or HAMS40) or butyrylated HAMS (HAMSB20 or HAMSB40) daily. HAMSB20 contained 31.8 mmol esterified butyrate. Complete 48-h fecal collections were made on days 2-3 and 12-13 of each period. RESULTS: Free fecal butyrate concentrations were higher after HAMSB40 than after HAMSB20 (P < 0.005) and HAMS (P < 0.0001) and higher than baseline data (P < 0.0001). Fecal esterified butyrate concentrations were highest in the HAMSB40 (days 12-13; P < 0.0001) group, and concentrations in the HAMSB40 (days 2-3) and HAMSB20 groups were higher than those in the HAMS groups and those at baseline (P < 0.0001). Ingestion of HAMSB20 and HAMSB40 resulted in the release of 26.8 ± 1.0 and 50.2 ± 2.4 mmol butyrate/d (days 12-13) (84.2 ± 3.0% and 79.0 ± 3.1% of total ingested esterified butyrate), respectively, in the gastrointestinal tract. By calculation, ∼57.2% of ingested esterified butyrate was released in the colon. Microbial analysis showed that this release was probably facilitated mainly by Parabacteroides distasonis, which increased in abundance with HAMSB40 (days 12-13) (P < 0.001). CONCLUSIONS: This study shows that cooked butyrylated starch delivers esterified butyrate to the human colon effectively and has the potential to improve human bowel health. This trial is registered in the Australian Clinical Trials Registry as ACTRN012606000398505.

Is the tissue persistence of O(6)-methyl-2'-deoxyguanosine an indicator of tumour formation in the gastrointestinal tract?

Azoxymethane (AOM) is a methylating agent capable of inducing mutations in DNA by forming adducts with DNA bases. It has been used to understand the mechanisms involved in colon carcinogenesis. Of the adducts formed in response to AOM, O(6)-methyl-2'-deoxy-guanosine (O(6)-mdGua) is the most mutagenic. Based on studies in rodents of the abundance and persistence of DNA adducts in various tissues after treatment with alkylating agents, previous results suggest, as a generalization, that the longer O(6)-mdGua adducts remain unrepaired in the cells of a tissue, the greater the risk for tumorigenesis. To test this hypothesis, we have built on these studies, expanding the number of tissues in which O(6)-mdGua abundance and persistence were examined and correlating these data with tumour distribution and abundance in rats maintained for 26 weeks after the treatment with AOM. Our study revealed firstly the existence of groups of tissues that developed relatively large amounts (proximal and distal colon, proximal small intestine (SI), liver and kidney) and relatively low levels (stomach, distal SI, bladder, spleen, blood and lung) of O(6)-mdGua after AOM exposure. Secondly, while all tissues showed an increase in adduct levels at 6h after mutagen treatment and most showed a significant drop in adduct levels between 6h and 48h (stomach, proximal and distal SI, liver, spleen, blood and lung), one group of tissues displayed O(6)-mdGua levels that did not decrease at 48h (proximal and distal colon, kidney and bladder). Predictably, the colon displayed tumours 26 weeks after treatment. Interestingly, however, the proximal SI also displayed significant tumour formation at that time. Our findings demonstrate (1) a direct association between exposure to O(6)-mdGua and tumours of the distal colon and (2) a dissociation of the relationship between adduct clearance and tumorigenesis in the SI. This diversity of response in the gastrointestinal tract warrants further analysis.

Bifidobacteria can protect from enteropathogenic infection through production of acetate.

The human gut is colonized with a wide variety of microorganisms, including species, such as those belonging to the bacterial genus Bifidobacterium, that have beneficial effects on human physiology and pathology. Among the most distinctive benefits of bifidobacteria are modulation of host defence responses and protection against infectious diseases. Nevertheless, the molecular mechanisms underlying these effects have barely been elucidated. To investigate these mechanisms, we used mice associated with certain bifidobacterial strains and a simplified model of lethal infection with enterohaemorrhagic Escherichia coli O157:H7, together with an integrated 'omics' approach. Here we show that genes encoding an ATP-binding-cassette-type carbohydrate transporter present in certain bifidobacteria contribute to protecting mice against death induced by E. coli O157:H7. We found that this effect can be attributed, at least in part, to increased production of acetate and that translocation of the E. coli O157:H7 Shiga toxin from the gut lumen to the blood was inhibited. We propose that acetate produced by protective bifidobacteria improves intestinal defence mediated by epithelial cells and thereby protects the host against lethal infection.

Dietary resistant and butyrylated starches have different effects on the faecal bacterial flora of azoxymethane-treated rats.

Epidemiological studies have suggested that dietary fibre lowers the risk of colorectal cancer, which may be due to increased butyrate production from colonic fermentation of a type of fibre, resistant starch (RS). The present study investigated the effects of dietary RS and butyrylated RS on the faecal microbiota of rats treated with azoxymethane. A total of four groups of nine rats were fed diets containing either standard maize starch (low-amylose maize starch (LAMS), low RS), LAMS with 3 % tributyrin (LAMST), cooked 10 % high-amylose maize starch (HAMS, high RS) or cooked 10 % butyrylated HAMS (HAMSB). Faecal samples were examined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. Multivariate analysis demonstrated no differences between faecal microbiota before treatment but revealed differences in DGGE patterns between diet groups, with the exception of the two low-RS groups (LAMS and LAMST). Subsequent analysis identified eleven DGGE bands contributing significantly to the differentiation between diets. These phylotypes belonged to Clostridiales (five), Lactobacillus (one) and Bacteroidetes (five) lineages. Rats fed HAMS had increased concentration of propionate in their distal colonic digesta and developed faecal populations containing Ruminococcus bromii-like bacteria. HAMSB increased propionate and butyrate concentrations in distal colonic digesta and was associated with the appearance of two non-butyrate-producing bacteria, Lactobacillus gasseri and Parabacteroides distasonis. In conclusion, supplementation with specific dietary RS leads to changes in faecal microbiota profiles that may be associated with improved bowel health.

Butyrylated starch increases large bowel butyrate levels and lowers colonic smooth muscle contractility in rats.

The short-chain fatty acids acetate, propionate, and butyrate are produced by colonic bacterial fermentation of carbohydrates. Butyrate is important in the regulation of the colonocyte cell cycle and gut motility and may also reduce the risk of large bowel cancer. We have shown that dietary butyrylated starch can deliver butyrate to the large bowel in a sustained manner. We hypothesized that ingestion of butyrylated starch increases large bowel butyrate levels and decreases colonic contractility. Groups of male Sprague-Dawley rats (n = 8) were fed AIN-93G-based diet containing a highly digestible low-amylose maize starch (LAMS) control or 5% or 10% butyrylated LAMS (LAMSB) for 10 days. We found that cecal but not colonic tissue weight as well as cecal and distal colonic digesta weights and fecal output were higher in LAMSB fed rats. Butyrylated LAMS lowered digesta pH throughout the large bowel. Cecal, proximal, and distal colonic butyrate pools and portal venous butyrate concentrations were higher in rats fed LAMSB. Electrically stimulated and receptor-dependent carbachol and prostaglandin E(2)-induced isotonic contractions were lower in isolated intact sections of proximal colon (P < .05) but not the terminal ileum after 10% LAMSB ingestion. These results demonstrated that elevation of butyrate levels in the large bowel of the rat correlated with reduction of contractile activity of the colonic musculature, which may assist in the reabsorption of water and minerals.