Improving feed intake and rumen fermentation in lambs using mixed-dimensional attapulgite clay to adsorb naturally occurring mycotoxins.

This experiment was conducted to evaluate the effects of including a mixed-dimensional attapulgite clay (MDA) into a naturally moldly diet for Hu lambs. Fifty male Hu lambs with similar initial body weight (28.24 ±â€…1.80 kg) were randomly allocated into five dietary treatments: a basal diet containing naturally occurring mycotoxins with 0, 0.5, 1.0, and 2.0 kg/t MDA, and basal diet with a commercial mycotoxin adsorbent Solis with montmorillonite as the major component at 1 kg/t. Both MDA and Solis increased average daily gain (ADG) and dry matter intake (DMI; P ≤ 0.004), and there was no difference in growth performance between MDA and Solis (P ≥ 0.26). The final body weight, DMI, and ADG were linearly increased with increasing MDA supplementation (P < 0.01). Lambs treated with both MDA and Solis demonstrated greater apparent digestibility of dry matter (DM), organic matter (OM), and energy compared with the control group (P ≤ 0.03), and there were no differences in nutrient digestibilities between MDA and Solis (P ≥ 0.38). Digestibility of CP was linearly increased with the increasing MDA supplementation (P = 0.01). Neither MDA nor Solis affected rumen total volatile fatty acid (TVFA) concentration (P ≥ 0.39), but decreased the acetate-to-propionate ratio and molar proportion of n-butyrate (P ≤ 0.01), and MDA also increased the concentration of ammonia (P = 0.003). Besides, increasing MDA supplementation linearly reduced the acetate-to-propionate ratio and molar proportion of n-butyrate (P = 0.01), but linearly and quadratically increased the concentration of ammonia (P ≥ 0.003). These results showed that the incorporation of MDA into a naturally moldy diet of Hu lambs yielded comparable results to the Solis product, with higher growth performance and nutrient digestibility but lower acetate-to-propionate ratio observed. In conclusion, including ≥ 1 kg/t of MDA in high mycotoxin risk diets for growing lambs improves feed intake and rumen fermentation.

The issue of mycotoxin-contaminated animal feed has consistently presented a significant challenge in relation to animal health and production. The mixed-dimensional attapulgite clay (MDA) has been proven effective in binding polar mycotoxins such as aflatoxin, while also effectively adsorbing hydrophobic or weakly polar mycotoxins such as zearalenone (ZEN) and ochratoxin. Therefore, this study was undertaken to assess the impact of MDA inclusion in mycotoxin-contaminated diets on performance and rumen fermentation variables in lambs. The results indicated that MDA not only significantly improved the growth performance and nutrient digestibility of Hu lambs but also enhanced the molar proportion of propionate and ammonia concentration, and reduced the acetate to propionate ratio and the molar proportion of n-butyrate.

Gut microbiota modulate CD8+ T cell immunity in gastric cancer through Butyrate/GPR109A/HOPX.

The gut microbiota and Short-chain fatty acids (SCFAs) can influence the progression of diseases, yet the role of these factors on gastric cancer (GC) remains uncertain. In this work, the analysis of the gut microbiota composition and SCFA content in the blood and feces of both healthy individuals and GC patients indicated that significant reductions in the abundance of intestinal bacteria involved in SCFA production were observed in GC patients compared with the controls. ABX mice transplanted with fecal microbiota from GC patients developed more tumors during the induction of GC and had lower levels of butyric acid. Supplementation of butyrate during the induction of gastric cancer along with H. pylori and N-methyl-N-nitrosourea (MNU) in WT in GPR109A-/-mice resulted in fewer tumors and more IFN-γ+ CD8+ T cells, but this effect was significantly weakened after knockout of GPR109A. Furthermore, In vitro GC cells and co-cultured CD8+ T cells or CAR-Claudin 18.2+ CD8+ T cells, as well as in vivo tumor-bearing studies, have indicated that butyrate enhanced the killing function of CD8+ T cells or CAR-Claudin 18.2+ CD8+ T cells against GC cells through G protein-coupled receptor 109A (GPR109A) and homologous domain protein homologous box (HOPX). Together, these data highlighted that the restoration of gut microbial butyrate enhanced CD8+ T cell cytotoxicity via GPR109A/HOPX, thus inhibiting GC carcinogenesis, which suggests a novel theoretical foundation for GC management against GC.

Anti-obesity effect of butyrate links to modulation of gut microbiome and epigenetic regulation of muscular circadian clock.

The role of the muscle circadian clock in regulating oxidative metabolism exerts a significant influence on whole-body energy metabolism; however, research on the connection between the muscle circadian clock and obesity is limited. Moreover, there is a lack of studies demonstrating the regulatory effects of dietary butyrate on muscle circadian clock and the resulting antiobesity effects. This study aimed to investigate the impacts of dietary butyrate on metabolic and microbiome alterations and muscle circadian clock in a diet-induced obesity model. Male Sprague-Dawley rats were fed a high-fat diet with or without butyrate. Gut microbiota and serum metabolome were analyzed, and molecular changes were examined using tissues and a cell line. Further correlation analysis was performed on butyrate-induced results. Butyrate supplementation reduced weight gain, even with increased food intake. Gut microbiome analysis revealed an increased abundance of Firmicutes in butyrate group. Serum metabolite profile in butyrate group exhibited reduced amino acid and increased fatty acid content. Muscle circadian clock genes were upregulated, resulting in increased transcription of fatty acid oxidation-related genes. In myoblast cells, butyrate also enhanced pan-histone acetylation via histone deacetylase inhibition, particularly modulating acetylation at the promoter of circadian clock genes. Correlation analysis revealed potential links between Firmicutes phylum, including certain genera within it, and butyrate-induced molecular changes in muscle as well as phenotypic alterations. The butyrate-driven effects on diet-induced obesity were associated with alterations in gut microbiota and a muscle-specific increase in histone acetylation, leading to the transcriptional activation of circadian clock genes and their controlled genes.

Butyrate Prevents the Pathogenic Anemia-Inflammation Circuit by Facilitating Macrophage Iron Export.

Most patients with inflammatory bowel disease (IBD) develop anemia, which is attributed to the dysregulation of iron metabolism. Reciprocally, impaired iron homeostasis also aggravates inflammation. How this iron-mediated, pathogenic anemia-inflammation crosstalk is regulated in the gut remains elusive. Herein, it is for the first time revealed that anemic IBD patients exhibit impaired production of short-chain fatty acids (SCFAs), particularly butyrate. Butyrate supplementation restores iron metabolism in multiple anemia models. Mechanistically, butyrate upregulates ferroportin (FPN) expression in macrophages by reducing the enrichment of histone deacetylase (HDAC) at the Slc40a1 promoter, thereby facilitating iron export. By preventing iron sequestration, butyrate not only mitigates colitis-induced anemia but also reduces TNF-α production in macrophages. Consistently, macrophage-conditional FPN knockout mice exhibit more severe anemia and inflammation. Finally, it is revealed that macrophage iron overload impairs the therapeutic effectiveness of anti-TNF-α antibodies in colitis, which can be reversed by butyrate supplementation. Hence, this study uncovers the pivotal role of butyrate in preventing the pathogenic circuit between anemia and inflammation.

ASTRAGALOSIDE â £ MODULATES GUT MACROPHAGES M1/M2 POLARIZATION BY RESHAPING GUT MICROBIOTA AND SHORT CHAIN FATTY ACIDS IN SEPSIS.

ABSTRACT: M1 macrophage-mediated inflammation is critical in sepsis. We previously found the protective role of astragaloside intravenous (AS-IV) in sepsis-associated gut impairment, whose specific mechanism remains unknown. Gut microbiota modulates gut homeostatic balance to avoid excessive inflammation. Here, we aimed to investigate effects of AS-IV on gut macrophages polarization and potential roles of gut microbiota and short chain fatty acids (SCFAs) in septic gut damage. Mice were pretreated by AS-IV gavage for 7 days before cecal ligation and puncture. M1 polarization of gut lamina propria macrophages (LpMs) was promoted by cecal ligation and puncture, accompanied by abnormal cytokines release and intestinal barrier dysfunction. NLRP3 inflammasome was activated in M1 LpMs. 16S rRNA sequencing demonstrated gut microbiota imbalance. The levels of acetate, propionate, and butyrate in fecal samples decreased. Notably, AS-IV reversed LpMs M1/M2 polarization, lightened gut inflammation and barrier injury, reduced NLRP3 inflammasome expression in LpMs, restored the diversity of gut microbiome, and increased butyrate levels. Similarly, these benefits were mimicked by fecal microbiota transplantation or exogenous butyrate supplementation. In Caco-2 and THP-1 cocultured model, LPS and interferon γ caused THP-1 M1 polarization, Caco-2 barrier impairment, abnormal cytokines release, and high NLRP3 inflammasome expression in THP-1 cells, all of which were mitigated by butyrate administration. However, these protective effects of butyrate were abrogated by NLRP3 gene overexpression in THP-1. In conclusion, AS-IV can ameliorate sepsis-induced gut inflammation and barrier dysfunction by modulating M1/M2 polarization of gut macrophages, whose underlying mechanism may be restoring gut microbiome and SCFA to restrain NLRP3 inflammasome activation.

Bacillus subtilis and Macleaya cordata extract regulate the rumen microbiota associated with enteric methane emission in dairy cows.

BACKGROUND: Ruminant livestock production is a considerable source of enteric methane (CH4) emissions. In a previous study, we found that dietary inclusions of Bacillus subtilis (BS) and Macleaya cordata extract (MCE) increased dry matter intake and milk production, while reduced enteric CH4 emission in dairy cows. The objective of this study was to further elucidate the impact of feeding BS and MCE on rumen methanogenesis in dairy cows using rumen metagenomics techniques. RESULTS: Sixty dairy cows were blocked in 20 groups of 3 cows accordingly to their live weight, milk yield, and days in milk, and within each group, the 3 cows were randomly allocated to 1 of 3 treatments: control diet (CON), control diet plus BS (BS), and control diet plus MCE (MCE). After 75 days of feeding experimental diets, 12 cows were selected from each treatment for collection of rumen samples for the metagenomic sequencing. Results showed that BS decreased ruminal acetate and butyrate, while increased propionate concentrations, resulting in decreased acetate:propionate ratio. The metagenomics analysis revealed that MCE reduced relative abundances of Methanobrevibacter wolinii, Methanobrevibacter sp. AbM4, Candidatus Methanomassiliicoccus intestinalis, Methanobrevibacter cuticularis, Methanomicrobium mobile, Methanobacterium formicicum, and Methanobacterium congolense. Both BS and MCE reduced relative abundances of Methanosphaera sp. WGK6 and Methanosphaera stadtmanae. The co-occurrence network analysis of rumen bacteria and archaea revealed that dietary treatments influenced microbial interaction patterns, with BS and MCE cows having more and stronger associations than CON cows. The random forest and heatmaps analysis demonstrated that the Halopenitus persicus was positively correlated with fat- and protein-corrected milk yield; Clostridium sp. CAG 269, Clostridium sp. 27 14, Haloarcula rubripromontorii, and Methanobrevibacter curvatus were negatively correlated with rumen acetate and butyrate concentrations, and acetate:propionate ratio, whereas Selenomonas rumiantium was positively correlated with those variables. CONCLUSIONS: The present results provided new information for mitigation of enteric methane emissions of dairy cows by feeding BS and MCE to influence rumen microbial activities. This fundamental knowledge is essential for developing enteric CH4 reduction strategies to mitigate climate change and reduce dietary energy waste. Video Abstract.

Blocked conversion of Lactobacillus johnsonii derived acetate to butyrate mediates copper-induced epithelial barrier damage in a pig model.

BACKGROUND: High-copper diets have been widely used to promote growth performance of pigs, but excess copper supplementation can also produce negative effects on ecosystem stability and organism health. High-copper supplementation can damage the intestinal barrier and disturb the gut microbiome community. However, the specific relationship between high-copper-induced intestinal damage and gut microbiota or its metabolites is unclear. OBJECTIVE: Using fecal microbiota transplantation and metagenomic sequencing, responses of colonic microbiota to a high-copper diet was profiled. In addition, via comparison of specific bacteria and its metabolites rescue, we investigated a network of bacteria-metabolite interactions involving conversion of specific metabolites as a key mechanism linked to copper-induced damage of the colon. RESULTS: High copper induced colonic damage, Lactobacillus extinction, and reduction of SCFA (acetate and butyrate) concentrations in pigs. LefSe analysis and q-PCR results confirmed the extinction of L. johnsonii. In addition, transplanting copper-rich fecal microbiota to ABX mice reproduced the gut characteristics of the pig donors. Then, L. johnsonii rescue could restore decreased SCFAs (mainly acetate and butyrate) and colonic barrier damage including thinner mucus layer, reduced colon length, and tight junction protein dysfunction. Given that acetate and butyrate concentrations exhibited a positive correlation with L. johnsonii abundance, we investigated how L. johnsonii exerted its effects by supplementing acetate and butyrate. L. johnsonii and butyrate administration but not acetate could correct the damaged colonic barrier. Acetate administration had no effects on butyrate concentration, indicating blocked conversion from acetate to butyrate. Furthermore, L. johnsonii rescue enriched a series of genera with butyrate-producing ability, mainly Lachnospiraceae NK4A136 group. CONCLUSIONS: For the first time, we reveal the microbiota-mediated mechanism of high-copper-induced colonic damage in piglets. A high-copper diet can induce extinction of L. johnsonii which leads to colonic barrier damage and loss of SCFA production. Re-establishment of L. johnsonii normalizes the SCFA-producing pathway and restores colonic barrier function. Mechanistically, Lachnospiraceae NK4A136 group mediated conversion of acetate produced by L. johnsonii to butyrate is indispensable in the protection of colonic barrier function. Collectively, these findings provide a feasible mitigation strategy for gut damage caused by high-copper diets. Video Abstract.

Gut Microbial Metabolite Butyrate and Its Therapeutic Role in Inflammatory Bowel Disease: A Literature Review.

Background and objective: Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is a chronic inflammatory disorder characterized by aberrant immune responses and compromised barrier function in the gastrointestinal tract. IBD is associated with altered gut microbiota and their metabolites in the colon. Butyrate, a gut microbial metabolite, plays a crucial role in regulating immune function, epithelial barrier function, and intestinal homeostasis. In this review, we aim to present an overview of butyrate synthesis and metabolism and the mechanism of action of butyrate in maintaining intestinal homeostasis and to discuss the therapeutic implications of butyrate in IBD. Methods: We searched the literature up to March 2023 through PubMed, Web of Science, and other sources using search terms such as butyrate, inflammation, IBD, Crohn's disease, and ulcerative colitis. Clinical studies in patients and preclinical studies in rodent models of IBD were included in the summary of the therapeutic implications of butyrate. Results: Research in the last two decades has shown the beneficial effects of butyrate on gut immune function and epithelial barrier function. Most of the preclinical and clinical studies have shown the positive effect of butyrate oral supplements in reducing inflammation and maintaining remission in colitis animal models and IBD patients. However, butyrate enema showed mixed effects. Butyrogenic diets, including germinated barley foodstuff and oat bran, are found to increase fecal butyrate concentrations and reduce the disease activity index in both animal models and IBD patients. Conclusions: The current literature suggests that butyrate is a potential add-on therapy to reduce inflammation and maintain IBD remission. Further clinical studies are needed to determine if butyrate administration alone is an effective therapeutic treatment for IBD.

Matrix-entrapped fibers create ecological niches for gut bacterial growth.

Insoluble plant cell walls are a main source of dietary fiber. Both chemical and physical fiber structures create distinct niches for gut bacterial utilization. Here, we have taken key fermentable solubilized polysaccharides of plant cell walls and fabricated them back into cell wall-like film forms to understand how fiber physical structure directs gut bacterial fermentation outcomes. Solubilized corn bran arabinoxylan (Cax), extracted to retain some ferulate residues, was covalently linked using laccase to form an insoluble cell wall-like film (Cax-F) that was further embedded with pectin (CaxP-F). In vitro fecal fermentation using gut microbiota from three donors was performed on the films and soluble fibers. Depending on the donor, CaxP-F led to higher relative abundance of recognized beneficial bacteria and/or butyrate producers-Akkermansia, Bifidobacterium, Eubacterium halii, unassigned Lachnospiraceae, Blautia, and Anaerostipes-than free pectin and Cax, and Cax-F. Thus, physical form and location of fibers within cell walls form niches for some health-related gut bacteria. This work brings a new understanding of the importance of insoluble cell wall-associated fibers and shows that targeted fiber materials can be fabricated to support important gut microbiota taxa and metabolites of health significance.

Dietary Resveratrol Butyrate Monoester Supplement Improves Hypertension and Kidney Dysfunction in a Young Rat Chronic Kidney Disease Model.

Chronic kidney disease (CKD) remains a public health problem. Certain dietary supplements can assist in the prevention of CKD progression. In this regard, resveratrol is a polyphenol and has a potential therapeutic role in alleviating CKD. We previously utilized butyrate in order to improve the bioavailability of resveratrol via esterification and generated a resveratrol butyrate monoester (RBM). In this study, the hypothesis that RBM supplementation is able to protect against kidney dysfunction and hypertension was tested by using an adenine-induced CKD model. For this purpose, three-week-old male Sprague Dawley rats (n = 40) were equally categorized into: group 1-CN (sham control); group 2-CKD (adenine-fed rats); group 3-REV (CKD rats treated with 50 mg/L resveratrol); group 4-MEL (CKD rats treated with 25 mg/L RBM); and group 5-MEH (CKD rats treated with 50 mg/L RBM). At the end of a 12-week period, the rats were then euthanized. The adenine-fed rats displayed hypertension and kidney dysfunction, which were attenuated by dietary supplementation with RBM. The CKD-induced hypertension coincided with: decreased nitric oxide (NO) bioavailability; augmented renal protein expression of a (pro)renin receptor and angiotensin II type 1 receptor; and increased oxidative stress damage. Additionally, RBM and resveratrol supplementation shaped distinct gut microbiota profiles in the adenine-treated CKD rats. The positive effect of high-dose RBM was shown together with an increased abundance of the genera Duncaniella, Ligilactobacillus, and Monoglobus, as well as a decrease in Eubacterium and Schaedierella. Importantly, the mechanism of action of the RBM supplementation may be related to the restoration of NO, rebalancing of the RAS, a reduction in oxidative stress, and alterations to the gut microbiota. Moreover, RBM supplementation shows promise for the purposes of improving CKD outcomes and hypertension. As such, further translation to human studies is warranted.

A comparison of post-ruminal provision of Ca-gluconate and Ca-butyrate on growth performance, gastrointestinal barrier function, short-chain fatty acid absorption, intestinal histology, and brush-border enzyme activity in beef heifers.

The objective of this study was to compare the effects of post-ruminal provision of Ca-butyrate (CaB) when delivered via abomasal dosing, and Ca-gluconate (CaG) when provided ruminally using a rumen protected form or using an unprotected form via abomasal dosing on short-chain fatty acid (SCFA) concentration throughout the GIT, nutrient digestibility, GIT barrier function, ruminal SCFA absorption, ruminal morphometrics, intestinal brush border enzyme activity, and blood parameters for beef heifers. Thirty-two beef heifers fitted with ruminal cannulas were used in a randomized complete block design and assigned to one of four treatments: 1) negative control (ruminal infusion of double-distilled water; CON); 2) abomasal infusion of CaB (AB; 0.0029% of BW); 3) abomasal infusion of CaG (AG; 0.0077% of BW); and 4) ruminal infusion of a hydrogenated fat-embedded CaG (RG; 0.0192% of BW) to provide ruminal protection. Excluding CON, treatments were designed to deliver the same amount of butyrate in the small intestine. Heifers were housed in individual pens and DMI was limited to 95% of voluntary intake to minimize a potential confounding effect of DMI on treatment responses. Total GIT barrier function was assessed on day 17 and SCFA disappearance was evaluated on day 21 using the temporarily isolated and washed reticulo-rumen technique. On day 28, heifers were slaughtered, and ruminal and colonic digesta were collected to assess SCFA concentration. Additionally, ruminal, jejunal, and colonic tissues were collected to assess SCFA fluxes and regional barrier function ex vivo using the Ussing chamber technique. For colonic digesta, both AB and CaG treatments reduced the proportion of acetate (P < 0.05) and increased the proportion on propionate (P < 0.05) compared to CON. Relative to CON, AB but not CaG treatments increased in vivo ruminal disappearance of total SCFA (P = 0.01), acetate (P = 0.03), propionate (P = 0.01), and butyrate (P > 0.01). Treatments did not affect (P ≥ 0.10) acetate and butyrate fluxes in the ruminal and colonic tissues when measured ex vivo; however, when compared with CON, AB tended to decrease (P = 0.09) mannitol flux across ruminal tissue. In addition, mannitol flux was affected (P < 0.01) by region, with greater mannitol flux across the jejunum than rumen and colon. We conclude that while both abomasal infusion of CaB and CaG affect the molar proportion of acetate and propionate in the colon, only abomasal CaB stimulated ruminal SCFA absorption for growing beef heifers.

Butyrate, a short-chain fatty acid (SCFA), has received attention due to its ability to promote gastrointestinal (GIT) health and development. However, butyrate in its free form presents a strong odor, limiting its use in diet formulation. Supplementation of butyrate precursors, such as gluconate, have been studied to enhance butyrate production in the GIT. This study evaluated the effects of post-ruminal infusion of Ca-butyrate (AB; 0.0029% of BW) and Ca-gluconate (AG; 0.0077% of BW) and ruminal infusion of a hydrogenated fat-embedded Ca-gluconate (RG; 0.0192% of BW) relative to control (CON; ruminal infusion of double-distilled water). Thirty-two beef heifers fitted with ruminal cannulas were fed for 28 d and GIT barrier function and ruminal SCFA absorption were assessed. At slaughter, the rumen, jejunum, and colon tissues were collected and barrier function and SCFA fluxes were assessed ex vivo. Relative to CON, AB but not AG and RG increased in vivo ruminal SCFA absorption and tended to increase ex vivo barrier function. Thus, the data presented in this study shows that butyrate and gluconate do not function through the same mode of action in the GIT of beef heifers.

Butyrate Attenuates Hepatic Steatosis Induced by a High-Fat and Fiber-Deficient Diet via the Hepatic GPR41/43-CaMKII/HDAC1-CREB Pathway.

SCOPE: Hepatic steatosis is a major health issue that can be attenuated by a healthy diet. This study investigates the effects and molecular mechanisms of butyrate, a dietary fiber metabolite of gut microbiota, on lipid metabolism in hepatocytes. METHODS AND RESULTS: This study examines the effects of butyrate (0-8 mM) on lipid metabolism in primary hepatocytes. The results show that butyrate (2 mM) consistently inhibits lipogenic genes and activates lipid oxidation-related gene expression in hepatocytes. Furthermore, butyrate modulates lipid metabolism genes, reduces fat droplet accumulation, and activates the calcium/calmodulin-dependent protein kinase II (CaMKII)/histone deacetylase 1 (HDAC1)-cyclic adenosine monophosphate response element binding protein (CREB) signaling pathway in the primary hepatocytes and liver of wild-type (WT) mice, but not in G-protein-coupled receptor 41 (GPR41) knockout and 43 (GPR43) knockout mice. This suggests that butyrate regulated hepatic lipid metabolism requires GPR41 and GPR43. Finally, the study finds that dietary butyrate supplementation (5%) ameliorates hepatic steatosis and abnormal lipid metabolism in the liver of mice fed a high-fat and fiber-deficient diet for 15 weeks. CONCLUSION: This work reveals that butyrate improves hepatic lipid metabolism through the GPR41/43-CaMKII/HDAC1-CREB pathway, providing support for consideration of butyrate as a dietary supplement to prevent the progression of NAFLD induced by the Western-style diet.

Short-chain fatty acids ameliorate necrotizing enterocolitis-like intestinal injury through enhancing Notch1-mediated single immunoglobulin interleukin-1-related receptor, toll-interacting protein, and A20 induction.

Single immunoglobulin interleukin-1-related receptor (SIGIRR), toll-interacting protein (TOLLIP), and A20 are major inhibitors of toll-like receptor (TLR) signaling induced postnatally in the neonatal intestine. Short-chain fatty acids (SCFAs), fermentation products of indigestible carbohydrates produced by symbiotic bacteria, inhibit intestinal inflammation. Herein, we investigated the mechanisms by which SCFAs regulate SIGIRR, A20, and TOLLIP expression and mitigate experimental necrotizing enterocolitis (NEC). Butyrate induced NOTCH activation by repressing sirtuin 1 (SIRT1)-mediated deacetylation of the Notch intracellular domain (NICD) in human intestinal epithelial cells (HIECs). Overexpression of NICD induced SIGIRR, A20, and TOLLIP expression. Chromatin immunoprecipitation revealed that butyrate-induced NICD binds to the SIGIRR, A20, and TOLLIP gene promoters. Notch1-shRNA suppressed butyrate-induced SIGIRR/A20 upregulation in mouse enteroids and HIEC. Flagellin (TLR5 agonist)-induced inflammation in HIEC was inhibited by butyrate in a SIGIRR-dependent manner. Neonatal mice fed butyrate had increased NICD, A20, SIGIRR, and TOLLIP expression in the ileal epithelium. Butyrate inhibited experimental NEC-induced intestinal apoptosis, cytokine expression, and histological injury. Our data suggest that SCFAs can regulate the expression of the major negative regulators of TLR signaling in the neonatal intestine through Notch1 and ameliorate experimental NEC. Enteral SCFAs supplementation in preterm infants provides a promising bacteria-free, therapeutic option for NEC.NEW & NOTEWORTHY Short-chain fatty acids (SCFAs), such as propionate and butyrate, metabolites produced by symbiotic gut bacteria are known to be anti-inflammatory, but the mechanisms by which they protect against NEC are not fully understood. In this study, we reveal that SCFAs regulate intestinal inflammation by inducing the key TLR and IL1R inhibitors, SIGIRR and A20, through activation of the pluripotent transcriptional factor NOTCH1. Butyrate-mediated SIGIRR and A20 induction represses experimental NEC in the neonatal intestine.

Chlorogenic Acid and Quercetin in a Diet with Fermentable Fiber Influence Multiple Processes Involved in DSS-Induced Ulcerative Colitis but Do Not Reduce Injury.

Ulcerative colitis (UC) patients often avoid foods containing fermentable fibers as some can promote symptoms during active disease. Pectin has been identified as a more protective fermentable fiber, but little has been done to determine the interaction between pectin and bioactive compounds present in foods containing that fiber type. Quercetin and chlorogenic acid, two bioactives in stone fruits, may have anti-cancer, anti-oxidant, and anti-inflammatory properties. We hypothesized that quercetin and chlorogenic acid, in the presence of the fermentable fiber pectin, may suppress the expression of pro-inflammatory molecules, alter the luminal environment, and alter colonocyte proliferation, thereby protecting against recurring bouts of UC. Rats (n = 63) received one of three purified diets (control, 0.45% quercetin, 0.05% chlorogenic acid) containing 6% pectin for 3 weeks before exposure to dextran sodium sulfate (DSS, 3% for 48 h, 3x, 2 wk separation, n = 11/diet) in drinking water to initiate UC, or control (no DSS, n = 10/diet) treatments prior to termination at 9 weeks. DSS increased the fecal moisture content (p < 0.05) and SCFA concentrations (acetate, p < 0.05; butyrate, p < 0.05). Quercetin and chlorogenic acid diets maintained SLC5A8 (SCFA transporter) mRNA levels in DSS-treated rats at levels similar to those not exposed to DSS. DSS increased injury (p < 0.0001) and inflammation (p < 0.01) scores, with no differences noted due to diet. Compared to the control diet, chlorogenic acid decreased NF-κB activity in DSS-treated rats (p < 0.05). Quercetin and chlorogenic acid may contribute to the healthy regulation of NF-κB activation (via mRNA expression of IκΒα, Tollip, and IL-1). Quercetin enhanced injury-repair molecule FGF-2 expression (p < 0.01), but neither diet nor DSS treatment altered proliferation. Although quercetin and chlorogenic acid did not protect against overt indicators of injury and inflammation, or fecal SCFA concentrations, compared to the control diet, their influence on the expression of injury repair molecules, pro-inflammatory cytokines, SCFA transport proteins, and NF-κB inhibitory molecules suggests beneficial influences on major pathways involved in DSS-induced UC. Therefore, in healthy individuals or during periods of remission, quercetin and chlorogenic acid may promote a healthier colon, and may suppress some of the signaling involved in inflammation promotion during active disease.

The short-chain fatty acid butyrate accelerates vascular calcification via regulation of histone deacetylases and NF-κB signaling.

Recent studies have shown that short-chain fatty acids (SCFAs), primarily acetate, propionate and butyrate, play a crucial role in the pathogenesis of cardiovascular disease. Whether SCFAs regulate vascular calcification, a common pathological change in cardiovascular tissues, remains unclear. This study aimed to investigate the potential role of SCFAs in vascular calcification. Using cellular and animal models of vascular calcification, we showed that butyrate significantly enhanced high phosphate (Pi)-induced calcification and osteogenic transition of vascular smooth muscle cells (VSMC) in vitro, whereas acetate and propionate had no effects. Subsequent studies confirmed that butyrate significantly promoted high Pi-induced aortic ring calcification ex vivo and high dose vitamin D3 (vD3)-induced mouse vascular calcification in vivo. Mechanistically, butyrate significantly inhibited histone deacetylase (HDAC) expression in VSMCs, and a pan HDAC inhibitor Trichostatin A showed similar inductive effects on calcification and osteogenic transition of VSMCs to butyrate. In addition, the SCFA sensing receptors Gpr41 and Gpr109a were primarily expressed by VSMCs, and butyrate induced the rapid activation of NF-κB, Wnt and Akt signaling in VSMCs. Intriguingly, the NF-κB inhibitor SC75741 significantly attenuated butyrate-induced calcification and the osteogenic gene Msx2 expression in VSMCs. We showed that knockdown of Gpr41 but not Gpr109a attenuated butyrate-induced VSMC calcification. This study reveals that butyrate accelerates vascular calcification via its dual effects on HDAC inhibition and NF-κB activation. Our data provide novel insights into the role of microbe-host interaction in vascular calcification, and may have implications for the development of potential therapy for vascular calcification.

Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on milk fat, rumen environment and biohydrogenation, and rumen protozoa in lactating cows fed diets with increased risk for milk fat depression.

Biohydrogenation-induced milk fat depression (MFD) is a reduction in milk fat synthesis caused by bioactive fatty acids (FA) produced during altered ruminal microbial metabolism of unsaturated FA. The methionine analog 2-hydroxy-4-(methylthio)butanoate (HMTBa) has been shown to reduce the shift to the alternate biohydrogenation pathway and maintain higher milk fat yield in high-producing cows fed diets lower in fiber and higher in unsaturated FA. The objective of this experiment was to verify the effect of HMTBa on biohydrogenation-induced MFD and investigate associated changes in rumen environment and fermentation. Twenty-two rumen cannulated high-producing Holstein cows [168 ± 66 d in milk; 42 ± 7 kg of milk/d (mean ± standard deviation)] were used in a randomized design performed in 2 blocks (1 = 14 cows, 2 = 8 cows). Treatments were control (corn carrier) and HMTBa (0.1% of diet dry matter). The experiment included a 7-d covariate period followed by 3 phases that fed diets with increasing risk of MFD. The diet during the covariate and low-risk phase (7 d) was 32% neutral detergent fiber with no additional oil. The diet during the moderate-risk phase (17 d) was 29% neutral detergent fiber with 0.75% soybean oil. Soybean oil was increased to 1.5% for the last 4 d. The statistical model included the random effect of block and time course data were analyzed with repeated measures including the random effect of cow and tested the interaction of treatment and time. There was no effect of block or interaction of block and treatment or time. There was no overall effect of treatment or treatment by time interaction for dry matter intake, milk yield, and milk protein concentration and yield. Overall, HMTBa increased milk fat percent (3.2 vs. 3.6%) and yield (1,342 vs. 1,543 g/d) and there was no interaction of treatment and dietary phase. Additionally, HMTBa decreased the concentration of trans-10 18:1 in milk fat and rumen digesta. Average total ruminal concentration of volatile FA across the day and total-tract dry matter and fiber digestibility were not affected by HMTBa, but HMTBa increased average rumen butyrate and decreased propionate concentration and increased total protozoa abundance. Additionally, HMTBa increased the fractional rate of α-linoleic acid clearance from the rumen following a bolus predominantly driven by a difference in the first 30 min. Plasma insulin was decreased by HMTBa. In conclusion, HMTBa prevented the increase in trans FA in milk fat associated with MFD through a mechanism that is independent of total volatile FA concentration, but involves modification of rumen biohydrogenation. Decreased propionate and increased butyrate and ruminal protozoa may also have functional roles in the mechanism.

Screening of the heterotrophic microalgae strain for the reclamation of acid producing wastewater.

For the sustainable development of the environment, to reduce the high cost and low productivity of microalgae biofuel, nine microalgae strains were screened to study the growh and nutrient removal properties under heterotrophic culture by using the waste carbon source of volatile fatty acids (VFAs). Chlorella sorokiniana (C.sorokiniana) was selected as the best strain with the highest biomass concentration of 0.77 g L-1, specific growth rate of 0.25 d-1, biomass productivity of 91.43 mg L-1 d-1, total nitrogen removal efficiency of 95.96% and total phosphorus removal efficiency of 93.42%. To study the utilization potential of acid-producing wastewater by heterotrophic microalgae, actual acid-producing wastewater was recycled three times for the utilization of C.sorokiniana. After the three utilization cultivation, the removal rates of COD, total nitrogen, ammonia nitrogen, and total phosphorus were 74.44%, 88.05%, 79.08%, and 82.69%, respectively. The total utilization rates of acetic acid, propionic acid, and butyric acid were 58.99%, 70.54%, and 81.52%, respectively. In addition, the highest lipid content of 39.15% and protein content of 42.43% achieved at the third cultivation. After the first cultivation, the composition and diversity of the microbial community structure changed dramatically, with Protebacteria, Bacteroidota, Hydrogenophaga, and Algoriphagus becoming enriched. These results showed a promising way of coupling wastewater treatment with biomass production for long-term sustainability of microalgae lipid production.

Short Communication: Supplementation with calcium butyrate causes an increase in the percentage of oxidative fibers in equine gluteus medius muscle.

Optimal athletic performance requires meeting the energetic demands of the muscle fibers, which are a function of myosin ATPase enzymatic activity. Skeletal muscle with a predominant oxidative metabolism underlies equine athletic success. Sodium butyrate, a short-chain fatty acid, can affect muscle fiber composition in pigs. To determine if a similar scenario exists in horses, 12 adult Thoroughbred geldings (7.4 ± 0.6 yr of age; mean ± SEM) were fed 16 g of calcium butyrate (CB) or an equivalent amount of carrier (CON) daily for 30 d in a crossover design. Middle gluteal muscle biopsies were collected before and after the feeding trial for immunohistochemical determination of fiber type, and RNA and protein isolation. After 30 d, CB increased (P < 0.05) the percentage of type IIA fibers and tended (P = 0.13) to reduce the numbers of type IIX fibers in comparison to control (CON). No changes (P > 0.05) in type I, IIA, or IIX fiber size were observed in response to CB. No differences (P > 0.05) were noted in the abundance of succinate dehydrogenase (SDH) protein or activity between horses receiving CB or CON. Myogenin mRNA abundance was unaffected (P > 0.05) by 30 d of CB supplementation. The increase in type IIA fibers in the absence of altered mitochondrial SDH enzymatic activity suggests that CB affects myosin ATPase expression independent of altered metabolism.

The largest tissue in the body, skeletal muscle, is a heterogeneous mix of fibers that are categorized based on their primary source of energy production and speed of contraction. Evidence suggests that Thoroughbred horses with a greater percentage of type IIA, fast-twitch, oxidative fibers are more successful than those with fewer. Pigs fed a diet supplemented with butyrate contained a greater percentage of oxidative muscle fibers. This study examined the ability of calcium butyrate (CB), a short-chain fatty acid, to alter muscle fiber composition in horses. Adult Thoroughbred geldings were supplemented with a placebo or CB for 30 d, and gluteus medius muscle biopsies were retrieved and analyzed for fiber type, myogenin expression, and succinate dehydrogenase (SDH) activity. Results demonstrate a small increase in the percentage of type IIA fibers without a change in SDH activity, a marker of oxidative metabolism. Myogenin expression remained unaffected by CB supplementation. These efforts underscore the need for further research to validate improved exercise performance in response to CB supplementation and identify a mechanism of action for the fatty acid in the equine skeletal muscle.

Effect of feeding calcium gluconate embedded in a hydrogenated fat matrix on feed intake, gastrointestinal fermentation and morphology, intestinal brush border enzyme activity and blood metabolites in growing lambs.

Gluconate salts have been identified as a butyrate precursor when fed to non-ruminant species and may increase the butyrate concentration in the large intestine supporting gastrointestinal health and development. The objective of this study was to evaluate the dose response of hydrogenated fat-embedded calcium gluconate (HFCG) on performance and gastrointestinal tract (GIT) development in growing lambs. Thirty-two wether lambs were used in a randomized complete block design and assigned to 1 of 4 treatments differing in the inclusion of HFCG: 0.0% (CON), 0.075% (LOW), 0.30% (MED), and 0.60% of the diet (HIGH). Lambs were allocated into individual pens and fed ad libitum with feed delivered twice daily. Feed intake was recorded daily, and body weight (BW) was assessed at the beginning and the end of the 29-d period. Blood was sampled on day 21, prior to feeding and 6 h post-feeding to evaluate changes in ß-hydroxybutyrate, glucose, and insulin concentrations. Total fecal collection was conducted during days 25 to 28 to assess apparent total tract digestibility. On day 29, lambs were slaughtered, and the entire GIT was separated by region to enable sampling of tissue and digesta. Data were analyzed to assess linear, quadratic, and cubic effects of HFCG dose. Final BW, average daily gain, and dry matter intake decreased linearly (P ≤ 0.02) with increasing HFCG. Increasing inclusion of HFCG linearly decreased (P = 0.01) the thickness of the stratum corneum in ruminal papillae but did not affect other strata (P ≥ 0.34). Omasal digesta weight linearly decreased (P = 0.01) as the concentration of HFCG increased and abomasal digesta weight was cubically affected (P = 0.03) the increasing dose of HFCG. Short-chain fatty acid concentration in the cecum was cubically affected (P < 0.01) with increasing dose of HFCG where low dose had the greatest concentration. Moreover, increasing the dietary supply of HFCG linearly increased the proportion of acetate (P = 0.04) in the cecum and linearly decreased the proportion of propionate in the digesta of both the cecum (P < 0.01) and colon (P = 0.01). Colon crypt depth was quadratically (P = 0.03) affected with the increasing dose of HFCG, where lambs fed MED had greatest crypt depth. We conclude that feeding HFCG to growing lambs did not increase butyrate concentration in the large intestine and consequently does not increase the absorptive surface area of the whole tract, the size of the GIT, or the functionality of the intestine.

Gluconate salts have been reported to be metabolized by microbes in the gastrointestinal tract to yield butyrate. Butyrate has shown potential to enhance functionality of the gastrointestinal tract by increasing the absorptive surface area, enzyme activity, and the barrier function. This study evaluated the inclusion of four levels of hydrogenated fat-embedded Ca-gluconate (HFCG; 0.0%, 0.075%, 0.30%, and 0.60% of the diet) designed to increase the production of butyrate in the large intestine. Thirty-two wether lambs were fed for 28 d, slaughtered, and eviscerated to allow complete evaluation of the gastrointestinal tract and its contents. Growth and dry matter intake decreased linearly with increasing dose of HFCG. Dose of HFCG cubically affected short-chain fatty acid concentration in the cecum with increased concentrations at the 0.075% dose. Moreover, increasing dose of HFCG linearly increased the proportion of acetate and linearly decreased the proportion of propionate in the cecum without altering the proportion of butyrate. Thus, the supplementation of HFCG did not increase butyrate concentration in the large intestine and did not enhance gastrointestinal tract function.

Effects of dietary butyrate supplementation and oral nonsteroidal anti-inflammatory drug administration on serum inflammatory markers and productivity of dairy cows during the calving transition.

Dairy cattle experience inflammation during the calving transition period, and butyrate and nonsteroidal anti-inflammatory drugs (NSAID) are expected to reduce the inflammation. Our objective was to evaluate the effects of dietary butyrate supplementation and oral NSAID administration on feed intake, serum inflammatory markers, plasma metabolites, and milk production of dairy cows during the calving transition period. Eighty-three Holstein cows were used in the experiment with a 2 × 2 factorial arrangement of treatments. The cows were blocked by parity and calving date, and randomly assigned to a dietary butyrate or control supplement, and NSAID or a placebo oral administration. Experimental diets were iso-energetic containing calcium butyrate at 1.42% of diet dry matter (DM) or the control supplement (1.04% commercial fat supplement and 0.38% calcium carbonate of diet DM). The close-up diets contained 13.3% starch and 42.4% neutral detergent fiber on a DM basis, and were fed from 28 d before expected calving date until calving. The postpartum diets contained 22.1% starch and 34.1% neutral detergent fiber on a DM basis and were fed from calving to 24 d after calving. Oral NSAID (1 mg of meloxicam/kg of body weight) or placebo (food dye) was administered 12 to 24 h after calving. Dietary butyrate supplementation and oral NSAID administration did not affect milk yield or postpartum serum concentrations of amyloid A and haptoglobin. However, butyrate-fed cows increased plasma fatty acid concentration on d -4 relative to calving (501 vs. 340 µEq/L) and tended to increase serum haptoglobin concentration (0.23 vs. 0.10 mg/mL). There was a supplement by drug interaction effect on plasma glucose concentration on d 4; in cows administered the placebo drug, butyrate supplementation decreased plasma glucose concentration compared with control-fed cows (62.8 vs. 70.1 mg/dL). Butyrate-fed cows tended to have lower milk crude protein yield compared with cows fed the control diet (1.21 vs. 1.27 kg/d). Dietary butyrate supplementation and oral NSAID administration did not have overall positive effects on production performance of dairy cows during the calving transition period.