BACKGROUND:Influence of dietary protein and its interaction with dietary fiber (DF) on gut microbiota, fermentation, and host immunity, particularly immunoglobulin A (IgA) responses, is not fully understood. OBJECTIVES:This study examined how gut microbiota adapt to dietary protein, subsequently influencing fermentation profiles and IgA responses, and how these effects are modulated by coingestion of vegetable fiber (VF). METHODS:Male Wistar rats (5 wk old) were fed one of 6 diets varying in protein (casein, soy, and egg white) and DF (cellulose, VF) source for 14 d (n = 6/group). Cecal microbial composition, organic acids, ammonia, IgA, and immune-related gene expression as well as fecal IgA were analyzed. Data were analyzed using 2-way or aligned rank transform analysis of variance (ANOVA). Microbiota composition was assessed using permutational multivariate ANOVA and ANOVA-like differential expression tool version 2, and Spearman's rank correlation was applied for microbial co-occurrence network construction and correlation analysis. RESULTS:Soy protein-VF diet yielded the highest alpha-diversity, whereas egg white protein-VF diet yielded the lowest across multiple indices (P < 0.05). Beta-diversity analysis confirmed distinct clustering among dietary groups (P < 0.001), whereas network analysis showed that protein source affected community structure. Soy protein-VF diet showed an increase in n-butyrate production relative to soy protein-cellulose diet (49.09 compared with 17.28 μmol/cecum, P < 0.05). Egg white protein-cellulose diet showed the highest ammonia production that was suppressed by VF coingestion (155.52 compared with 61.66 μmol/cecum, P < 0.05). Notably, cecal IgA showed a positive correlation with ammonia (ρ = 0.67, P-adj. < 0.01). CONCLUSIONS:In rats, dietary protein and its interaction with VF are associated with compositionally distinct microbial signatures that influence fermentation profiles and IgA responses in the cecum. These findings highlight the importance of considering protein-fiber combinations when designing dietary interventions to optimize gut health.

High-amylose maize starch (HAMS) can lead to succinate accumulation in the rat colon depending on the colonic microbiota. Since succinate is primarily produced via the vitamin B12 (VB12)-dependent succinate pathway, limited VB12 availability in the colon may impair fermentation. While a portion of dietary VB12 may reach the colon, most of it is absorbed in the upper gastrointestinal tract, potentially resulting in an insufficient supply for colonic bacteria. This study aimed to determine the minimum caecal VB12 concentration required to prevent succinate accumulation and to assess whether dietary cobalt (Co), a structural component of VB12 and its analogues, promotes microbial VB12 analogue synthesis. Sprague-Dawley male rats were used in three experiments. In Experiment 1, HAMS-fed rats were given diets with increasing VB12 doses. Caecal succinate concentrations decreased dose-dependently, with a predicted threshold of 74 pmol/g VB12 required to prevent accumulation. In Experiment 2, rats were fed HAMS diets with varying Co levels. Co supplementation significantly increased VB12-equivalent concentrations, measured by microbiological assay, from 27 to 915 pmol/g without altering cobalamin concentrations, suggesting enhanced microbial synthesis of VB12 analogues. Caecal succinate levels decreased with increasing Co intake, mimicking the effects of dietary VB12. In Experiment 3, rats were fed HAMS diets with or without high-dose Co to confirm these effects and assess microbiota changes. Co supplementation restored the abundance of Akkermansia, which utilises VB12 and its analogues. These findings suggest that maintaining sufficient colonic VB12 - through direct supplementation or Co-stimulated microbial production - may help mitigate HAMS-induced succinate accumulation and support balanced colonic fermentation.

Small intestinal villous atrophy, a frequent complication of cancer chemotherapy, impairs nutrient absorption and worsens participant quality of life. Although the glucagon-like peptide-2 (GLP-2) analog teduglutide can promote villus regeneration, its clinical application is limited. We hypothesized that dietary low-digestible glucans (LDGs) would enhance GLP-2 secretion and accelerate mucosal recovery. This study evaluated whether LDGs stimulate GLP-2 secretion and facilitate recovery from 5-fluorouracil (5-FU)-induced villous atrophy in rats. Male Sprague-Dawley rats were treated with 5-FU to induce villous atrophy, followed by diets containing resistant maltodextrin (RMD) or isomaltodextrin as LDGs. Portal GLP-2 concentrations, small intestinal villus height, maltase activity, and cecal short-chain fatty acid concentrations were assessed at 3 and 6 days after LDG supplementation. LDG supplementation significantly increased portal GLP-2 concentrations and accelerated recovery of villus height in the small intestine, especially in the ileum, compared with controls. Notably, villus height in the RMD group recovered within 3 days, whereas 6 days were required in controls. Both RMD and isomaltodextrin increased cecal tissue weight, and LDGs induced only a transient reduction in cecal acetate concentration. However, when 5-FU was administered concurrently, LDGs did not promote villus recovery, suggesting that their effect requires preserved epithelial proliferative capacity. In summary, LDGs promote rapid recovery of small intestinal villi after chemotherapy-induced injury, possibly through enhanced GLP-2 secretion. Dietary LDGs may offer a novel nutritional intervention to support mucosal recovery in participants undergoing cancer chemotherapy.

Since the core protein of mucin in the digesta of the stomach and small intestine, which is less affected by bacteria, remains intact, mucin content can be measured by enzyme-linked immunosorbent assay (ELISA). However, the mucin core protein in bacteria-rich colon digesta and feces is partially hydrolyzed by bacterial enzymes and not fully recognized by mucin antibodies, so mucin cannot be accurately quantified by ELISA. This method quantifies the glycan content linked to the mucin core protein and expresses mucin content in the colon digesta and feces as the equivalent of O-linked oligosaccharide chain. Although mucin glycans are also hydrolyzed by colonic bacteria, this method is a more accurate and simple way to measure mucin content in the digesta of the large intestine and feces than the ELISA method.

Mucinase consists of some proteases, glycosidases, sulfatases, and sialidases. It is not practical to measure individual enzyme activities when measuring mucinase activity. In this method, mucinase activity is measured using porcine gastric mucin as a substrate and feces as an enzyme source. This description includes fecal pellet preparation, reaction procedure of mucinase, measurement of reducing sugars liberated during the procedure, and determination of nitrogen content in the fecal preparations.

Public attention to the correlation between excessive sucrose consumption and metabolic syndrome has facilitated research on its underlying mechanisms. Recent studies have newly identified the intestine, rather than the liver, as the predominant site of dietary fructose metabolism, which corrects the previous concepts and links the promotion of fatty liver and hyperlipidemia to gut microbiota dysbiosis, although the specific mode of action remains unclear. Here, we used microbiota suppression to identify the causative bacteria. Male Wistar rats were fed a starch or high-sucrose diet and orally administered two independent antibiotic treatments for four weeks. Metronidazole, neomycin, ampicillin, vancomycin, and their mixture were used to explore highly sucrose-responsive bacteria. Metronidazole and the mixture decreased or showed a tendency to decrease high-sucrose diet-induced increases in liver weight, plasma triglyceride levels, and fatty acid synthesis-related gene expression (ATP citrate lyase, fatty acid synthase, and malic enzyme). To further investigate the ability of metronidazole to ameliorate high-sucrose diet-induced abnormalities in lipid metabolism, three nitroimidazole antibiotics (metronidazole, tinidazole, and ornidazole) were used. Only metronidazole reduced the liver weight and decreased plasma triglyceride levels on a high-sucrose diet. Among the high-sucrose diet induction effects, five bacteria suppressed by metronidazole were screened, belonging to the family Rikenellaceae, genera Bacteroides, Blautia, and Dorea, and species Eubacterium dolichum. Overall, suppression of the gut microbiota by metronidazole-specific action, rather than the nitroreductase activity of tinidazole and ornidazole, was responsible for the amelioration of high-sucrose diet-induced fatty liver and hypertriglyceridemia. Five causative bacteria were identified in this study.

Previous attempts to limit fat intake failed to reduce metabolic syndrome incidence, spotlighting excessive sucrose consumption phenomenon. Recent studies challenge the traditional belief, actually fructose is predominantly metabolized in the small intestine, not just the liver. Evidence supports sucrose overconsumption leads to hepatic lipid accumulation via gut microbiome modulation, but the mechanisms and prevention strategies remain unclear. This study investigates the impact of osmolytes inositol and taurine, which are abundant in citrus fruits and seafood respectively, on lipid metabolism in rats consuming excessive sucrose. Five-week-old male Wistar rats fed with control diet, high sucrose diet, and high sucrose diet with inositol or taurine for 28 days. High sucrose diet-induced accumulation of hepatic triglyceride level was reduced only by inositol. Both inositol and taurine decreased hepatic SREBP1, ChREBP, lipogenesis enzymes mRNA levels increased by a high sucrose diet. Taurine also significantly increased the fatty acid uptake major player CD36 mRNA levels in liver. Expression of genes related to fatty acid degradation and transport remained unchanged. In cecal microbiome, high sucrose diet groups showed a decrease in alpha diversity and diverged in beta diversity from the starch diet group, while inositol and taurine did not significantly affect these aspects. Ten bacterial features including genera such as Bacteroides, Parabacteroides, Odoribacter, Streptococcus, etc., were filtered out related to the ameliorative effects of inositol and taurine on lipid metabolism. Overall, both inositol and taurine modulated hepatic lipogenesis and microbiome. However, due to taurine’s enhancement of fatty acid uptake, only inositol improved steatosis in high sucrose diet rats.

Background & aims Excess sucrose intake induces metabolic syndrome. In human, abnormal lipids metabolism like obesity, hyperlipidemia and fatty liver are induced. However, excess sucrose causes different phenotypes in different species. Based on our previous study, excess sucrose induced fatty liver and hyperlipidemia in rats. The phenotypes and mechanism of abnormal lipid metabolism in mice is unclear. We investigated the different phenotypes in 5 strains of mice and the relationship between gut microbiome and abnormal lipid metabolism in C57BL/6N mice. Methods We examined the effect of a high sucrose diet in 5 different strains of mice. Besides, to find out the relationship between gut microbiome and metabolic disorder induced by excess sucrose, C57BL/6N mice were fed with a high sucrose diet with or without antibiotics cocktail. Results A high sucrose diet induced obesity and fatty liver in inbred mice, whereas did not induce hyperlipidemia in all strains of mice. Moreover, a high sucrose diet changed the composition of gut microbiota in C57BL/6N mice. Antibiotics treatment alleviated the abnormal lipid metabolism induced by high sucrose diet by changing the composition of gut short chain fatty acids. Conclusions These results indicates that the phenotypes of metabolic syndrome are influenced by genetic factors. Furthermore, the dysbiosis of gut microbiome caused by excess sucrose may contribute to the development of abnormal lipid metabolism via its metabolites.

Since propionate exerts several physiological effects, maintenance of its normal colonic fermentation is essential. To investigate whether vitamin B-12 (VB12) is essential for normal propionate fermentation by colonic bacteria, via the succinate pathway, we examined if high-amylose cornstarch (HACS) feeding activated such a pathway, if high HACS feeding impaired propionate fermentation, and if oral VB12 supplementation normalized propionate fermentation. Male rats were given control, 20% HACS or 3% fucose diets (Expt. 1); a VB12-free control diet or one supplemented with 5-30% HACS (Expt. 2); and the 20% HACS diet supplemented with 0.025-25 mg/kg of VB12 (Expt. 3), for 14 d. HACS feeding significantly increased cecal succinate concentration, activating the succinate pathway (Expt. 1). Cecal cobalamin concentration in 20% and 30% HACS groups was about 75% of that in the control group (Expt. 2). Cecal succinate and propionate concentrations significantly increased and decreased in 30% HACS groups, respectively, compared with the control group. Although HACS group supplemented with 0.025 mg/kg of VB12 had a low concentration of cecal propionate, adding high amounts of VB12 to HACS diets provided sufficient amounts of VB12 to rat ceca and increased cecal propionate concentration (Expt. 3). Compared with the non-HACS group, the relative abundance of Akkermansia muciniphila, but not Bacteroides/Phocaeicola, was lower in the HACS counterpart and showed improvement with increased VB12 doses. To summarize, feeding high HACS decreased and increased cecal VB12 and succinate concentrations, respectively. Furthermore, colonic delivery of sufficient amounts of VB12 to rats likely reduced accumulation of succinate and normalized propionate fermentation.

To investigate whether the oral intake of slowly digestible α-glucan (SDG) could have a trophic (i.e., thickening) effect on their ileal mucosae, for 10 d, rats were given control (non-SDG), 10% isomaltodextrin (IMD) or 10% resistant maltodextrin (RMD) diets. In addition, experimental rat groups were further divided into two groups each and their diets either had or had not 1% sodium carboxymethylcellulose (CMC) added as a thickening agent. In the jejuna and the ilea, compared with control rats, the villus length and the mucosal thickness, but not the crypt depth, were significantly greater in the RMD-fed rats, with the trophic effect being weaker in the IMD-fed rats than in the RMD-fed rats. The colonic crypt depth was significantly greater in SDG groups than in the control group. The concentration of plasma glucagon-like peptide (GLP)-2 in the portal veins of the RMD group but not the IMD group was significantly higher than in the control group, with no effect of CMC supplementation on its concentration. The concentrations of cecal short-chain fatty acids did not significantly increase with SDG supplementation except for propionate concentration of the IMD-supplemented rats, compared with those in the control rats. We concluded that SDGs, especially RMD, thickened the mucosae of the rat distal small intestines. In particular, this effect of RMD but not IMD could have resulted from increased glucose available as a secretagogue of the trophic hormone GLP-2, in the ileum.

Dietary factors, affect Akkermansia muciniphila (AM) abundance in the colon, have attracted attention, driven by the inverse correlation between AM abundance and metabolic disorders. We prepared skate-skin mucin (SM), porcine stomach mucin (PM), and rat gastrointestinal mucin (RM). SM contained more sulfated sugars and threonine than PM or RM. Rats were fed a control diet or diets including SM, PM, or RM (15 g/kg), or SM (12 g/kg) from 5 different threonine contents for 14 d. Cecal total bacteria and AM were less and more numerous, respectively, in SM-fed rats than the others, but SM did not affect microbial species richness. Low-threonine SM did not induce AM proliferation. The in vitro fermentation with human feces showed that the rate of AM increase was greater with SM than PM. Collectively, heavy SM sulfation facilitates a priority supply of SM-derived amino sugars and threonine that promotes AM proliferation in rats and human feces.

Excess sucrose intake has been found to be a major factor in the development of metabolic syndrome, especially in promoting nonalcoholic fatty liver disease. The excess fructose is believed to targets the liver to promote de novo lipogenesis, as described in major biochemistry textbooks. On the contrary, in this study, we explored the possible involvement of gut microbiota in excess sucrose-induced lipid metabolic disorders, to validate a novel mechanism by which excess sucrose causes hepatic lipid metabolic disorders via alterations to the gut microbial community structure. Wistar male rats were fed either a control starch diet or a high-sucrose diet for 4 weeks. Half of the rats in each group were treated with an antibiotic cocktail delivered via drinking water for the entire experimental period. After 4 weeks, rats fed with the high-sucrose diet showed symptoms of fatty liver and hyperlipidemia. The architecture of cecal microbiota was altered in rats fed with high-sucrose diet as compared to the control group, with traits including increased ratios of the phyla Bacteroidetes/Firmicutes, reduced α-diversity, and diurnal oscillations changes. Antibiotic administration rescued high-sucrose diet-induced lipid accumulation in the both blood and liver. Levels of two microbial metabolites, formate and butyrate, were reduced in rats fed with the high-sucrose diet. These volatile short-chain fatty acids might be responsible for the sucrose-induced fatty liver and hyperlipidemia. Our results indicate that changes in the gut microbiota induced by a high-sucrose diet would promote the development of nonalcoholic fatty liver disease via its metabolites, such as short-chain fatty acids.

The brain is rich in long-chain PUFA, which play an essential role in its development and functions. Here, we examined the impact of maternal n-3 PUFA intake deficiency during gestation and lactation on the development of glial cells in the pup's developing cerebral cortex. In addition, using myelination as indicator and the anti-myelin basic protein as measurement to establish the relationship between the number of glial fibrillary acidic protein (GFAP)-positive cells and the development of oligodendrocytes, we determined the myelination state of the somatosensory cortex at postnatal day 14. Rat dams were fed either a control (Cont) or an n-3 PUFA-deficient (Def) diet for 60 d (acclimatisation: 14 d; gestation: 21 d; and lactation: 21 d). Pups lactated from dams throughout the experiment. The distribution pattern of astrocytes in pups on postnatal day 7 was immunohistochemically analysed using GFAP and brain lipid binding protein (BLBP) as markers for mature astrocytes and astrocyte-specific radial glial cells, respectively. It was observed that, when compared with Cont pups, GFAP-positive cells decreased, BLBP-positive cells increased and myelinated structures were sparser in the somatosensory cortices of Def pups. In the open field test on postnatal day 21, behavioural parameters did not differ between groups. Our results indicated that inhibited maturation of astrocytes caused by maternal n-3 PUFA deficiency hindered the development of brain glial cells of neonatal rats; hence, maternal n-3 PUFA intake during the gestation and lactation periods may have been crucial for the brain cell composition of pups.

Pectin enhances mucin secretion in the rat small intestine. However, what structural features of pectin to stimulate mucin secretion remain unclear. The study aimed to clarify active constituents of pectin using a human goblet cell line, HT29-MTX. Various pectins at 100 mg/L commonly stimulated MUC5AC secretion, irrespective of their differences in molecular size, plant origin and degree of methoxylation, whereas other dietary fiber materials at 100 mg/L did not show any effects, except fucoidan. Hairy region concentrate (HRC) and its further fractions (F1-F3) were prepared by polygalacturonase treatment of citrus pectin and successive anion exchange chromatography. Neutral sugars, such as galactose and arabinose were enriched in these fractions. HRC and F1-F3 at 30 mg/L significantly increased MUC5AC secretion, which were 3 times more potent compared with a starting material (citrus pectin). Further, a dose-dependent study showed that F1 significantly increased MUC5AC secretion from at 0.3 mg/L, much stronger than that of mucin-secretagogue lipopolysaccharides. Rats consumed 5% apple pectin diet showed significant increases of luminal mucin contents and Muc2 expression in the small intestine, while the luminal mucin contents in rats consumed 1.5% HRC diet were increased by 24% compared to those in rats consumed control diet, but the difference did not reach significant. Thus, HRC is supposed to be active constituents of mucin-secretory effect of pectin in vitro. At present, however, the effect of HRC has not been verified in vivo.

Abstract Fecal microbiota in seven different monogastric animal species, elephant, horse, human, marmoset, mouse, pig and, rat were compared using the same analytical protocol of 16S rRNA metagenome. Fecal microbiota in herbivores showed higher alpha diversity than omnivores except for pigs. Additionally, principal coordinate analysis based on weighted UniFrac distance demonstrated that herbivores and pigs clustered together, whereas other animal species were separately aggregated. In view of butyrate‐ and lactate‐producing bacteria, predominant genera were different depending on animal species. For example, the abundance of Faecalibacterium, a known butyrate producer, was 8.02% ± 3.22% in human while it was less than 1% in other animal species. Additionally, Bifidobacterium was a predominant lactate producer in human and marmoset, while it was rarely detected in other omnivores. The abundance of lactate‐producing bacteria in herbivores was notably lower than omnivores. On the other hand, herbivores as well as pig possess Fibrobacter, a cellulolytic bacterium. This study demonstrated that fecal microbiota in herbivorous animals is similar, sharing some common features such as higher alpha diversity and higher abundance of cellulolytic bacterium. On the other hand, omnivorous animals seem to possess unique fecal microbiota. It is of interest that pigs, although omnivore, have fecal microbiota showing some common features with herbivores.

BACKGROUND Intestinal mucins escape digestion and enter the large bowel where they are degraded by the microbiota. To what extent and how mucins impact large-bowel physiology remain unclear. OBJECTIVE This study examined the large-bowel fermentation characteristics of mucins and mucin-derived O-glycan sugars and whether they affect gut immunity. METHODS Mucin secretion from the terminal ileum was determined from feces of ileorectostomized male Wistar rats (age 6 wk) fed an AIN76-based control diet (CD) for 15 d (experiment 1). Normal male Wistar rats (age 6 wk; 4 wk for experiment 4) were fed CD ± porcine stomach mucin (PM) at 6 or 12 g/kg diet, equivalent to 1.5 and 3 times the daily mucin secretion, for 14 d (experiment 2); CD ± N-acetylglucosamine (GlcNAc), fucose, or N-acetylneuraminic acid at 10 g/kg diet for 14 d (experiment 3); or CD ± PM (15 g/kg diet) or GlcNAc (10 g/kg diet) for 29 d (experiment 4). SCFAs, microbial composition, and cecal O-glycan content were assessed. IgA+ plasma cells and regulatory T cells and inflammatory cytokine expression in the cecum were evaluated (experiment 4). RESULTS Daily mucin secretion corresponded to 43.2 μmol of O-glycans. Cecal O-glycan contents were comparable between CD- and PM-fed rats. PM-fed rats harbored more mucin-degrading bacteria. Cecal concentrations of acetate (+37%) and n-butyrate (+73%) were higher in 12-g/kg PM diet-fed rats versus CD (P < 0.05). Among O-glycan sugars, only GlcNAc produced higher n-butyrate concentrations (+68%) versus CD (P < 0.05), with increased numbers of butyrate-producing bacteria. GlcNAc increased the abundance of IgA+ plasma cells (+29%) and regulatory T cells (+33%) versus CD, whereas PM increased IgA+ plasma cells (+25%) (all P < 0.05). GlcNAc and PM decreased expression of Tnfa (-30%, -40%) and Ifng (-30%, -70%) versus CD (all P < 0.05). CONCLUSIONS Mucin-derived O-glycans act as endogenous fiber and maintain mucosal immune homeostasis via large-bowel SCFA production in rats.

β-glucan is a generic term for insoluble dietary fibers exerting various effects on the immune system. As a group, β-glucans are non-cellulose polysaccharides composed of a glucopyranose as the main constituent sugar with β configuration, having a β- (1,3)-linked glucopyranose main chain as a common feature. β-glucans are absorbed through the intestine. Since the 1980s, there have been many studies reporting various effects of β-glucans on the immune system, including reports on receptors, that have slowly clarified their recognition system and action mechanisms. However, these studies focused mostly on treatments of infectious diseases and tumors; thus, the effects of β-glucans ingested in food as dietary fiber and their mechanisms of action remain largely unknown. The uptake of β-glucan into the body may be resemble that of proteins, which are soluble polymers, and insoluble material such as dietary fiber. Dietary fibers have varied structures, with wide-ranging solubility and physiological effects. Understanding whether these substances are actually taken up, how they exert their effects, and their metabolism after being taken up are important issues when considering the functionality and safety of dietary fibers.

AbstractTo investigate whether oral intake of highly branched α-glucan isomaltodextrin (IMD) could stimulate ileal glucagon-like peptide-1 (GLP-1) secretion, we examined (1) the digestibility of IMD, (2) the digestion and absorption rates of IMD, in rat small intestine and (3) portal GLP-1 concentration in rats given IMD. In Expt 1, ileorectostomised rats were given a 3 % IMD diet for 10 d. Separately, a 16-h in vitro digestion of IMD, using porcine pancreatic α-amylase and brush-border membrane vesicles from rat small intestine, was conducted. In Expt 2, upon 24-h fasting, rats were given any of glucose, IMD and high-amylose maize starch (HAMS) (1 g/kg of body weight). In Expt 3, caecectomised rats were given 0·2 % neomycin sulphate and a 5 % IMD diet for 10 d. The in vivo and in vitro digestibility of IMD was 70–80 %. The fraction of IMD digested in vitro for the first 120 min was 67 % of that in maize starch. The AUC for 0–120 min of plasma glucose concentration was significantly lower in HAMS group and tended to be lower in IMD group than in the glucose group. Finally, we also observed that, when compared with control rats, glucose of IMD significantly stimulated and improved the concentration of portal active GLP-1 in antibiotic-administered, caecectomised rats. We concluded that IMD was slowly digested and the resulting glucose stimulated GLP-1 secretion in rat small intestine. Oral delivery of slowly released IMD glucose to the small intestine probably exerts important, yet unknown, physiological effects on the recipient.