Systemic availability and metabolism of colonic-derived short-chain fatty acids in healthy subjects: a stable isotope study

The short-chain fatty acids (SCFAs), acetate, propionate and butyrate, are bacterial metabolites that mediate the interaction between the diet, the microbiota and the host. In the present study, the systemic availability of SCFAs and their incorporation into biologically relevant molecules was quantified. Known amounts of 13C-labelled acetate, propionate and butyrate were introduced in the colon of 12 healthy subjects using colon delivery capsules and plasma levels of 13C-SCFAs 13C-glucose, 13C-cholesterol and 13C-fatty acids were measured. The butyrate-producing capacity of the intestinal microbiota was also quantified. Systemic availability of colonic-administered acetate, propionate and butyrate was 36%, 9% and 2%, respectively. Conversion of acetate into butyrate (24%) was the most prevalent interconversion by the colonic microbiota and was not related to the butyrate-producing capacity in the faecal samples. Less than 1% of administered acetate was incorporated into cholesterol and <15% in fatty acids. On average, 6% of colonic propionate was incorporated into glucose. The SCFAs were mainly excreted via the lungs after oxidation to 13CO2, whereas less than 0.05% of the SCFAs were excreted into urine. These results will allow future evaluation and quantification of SCFA production from 13C-labelled fibres in the human colon by measurement of 13C-labelled SCFA concentrations in blood

Butyrate protects Caco-2 cells from Campylobacter jejuni invasion and translocation

Invasion in and translocation across enterocytes are major events during Campylobacter jejuni-induced enteritis in humans. C. jejuni in vitro infection of cell monolayers typically results in loss of tight junction integrity, which could contribute to translocation. In the present study, we wanted to investigate whether butyrate is able to confer protection to Caco-2 cells against C. jejuni invasion, thus reducing paracellular permeability and limiting C. Jejuni translocation. Protection of Caco-2 cells against C. jejuni invasion was assessed using a gentamicin protection assay. Trans-well systems were used to investigate the impact of butyrate on translocation of C. jejuni across a Caco-2 monolayer and its effect on transepithelial resistance during infection. Butyrate protected Caco-2 cells against C. jejuni invasion in a concentration-dependent manner. Differentiated Caco-2 cells were less Susceptible to C. jejuni invasion than 3-d-old undifferentiated cells and higher concentrations of butyrate and longer incubation times were needed to become refractive for invasion. C. jejuni translocation over Caco-2 monolayers was reduced when monolayers were treated with butyrate and this was accompanied by an enhanced drop in transepithelial resistance. The present study showed that butyrate is able to protect Caco-2 cells front two major Virulence mechanisms of C. jejuni, namely invasion and translocation, but not from a decline in transepithelial resistance

Isolation and characterization of Faecalibacterium prausnitzii from calves and piglets.

The goal of our study was to isolate and characterize Faecalibacterium prausnitzii from fecal samples of healthy calves and piglets, in order to develop a novel probiotic for livestock animals. We identified 203 isolates of Faecalibacterium sp., which were clustered in 40 genetically distinct groups. One representative isolate from each cluster was selected for further characterization. The concentrations of the short chain fatty acids (SCFA) acetate, butyrate, propionate and isobutyrate in the culture media were measured by gas chromatography. We observed reduction in the concentration of acetate followed by concomitant increase in the concentration of butyrate, suggesting that the isolates were consuming acetate present in the media and producing butyrate. Butyrate production correlated positively with bacterial growth. Since butyrate has many benefits to the colonic epithelial cells, the selection of strains that produce higher amounts of butyrate is extremely important for the development of this potential probiotic. The effect of pH and concentration of bile salts on bacterial growth was also evaluated in order to mimic the conditions encountered by F. prausnitzii in vivo. The optimal pH for growth ranged between 5.5 and 6.7, while most isolates were inhibited by of the lowest concentration of bile salts tested (0.1%). Antimicrobial resistance profile showed that most isolates of Faecalibacterium sp. were resistant against ciprofloxacin and sulfamethoxazole-trimethoprim. More than 50% of the isolates were resistant to tetracycline, amikacin, cefepime and cefoxitin. A total of 19 different combinations of multidrug resistance were observed among the isolates. Our results provide new insights into the cultural and physiological characteristics of Faecalibacterium prausnitzii illustrating large variability in short chain fatty acid production, in vitro growth, sensitivity to bile salts, and antibiotic resistance and suggesting that future probiotic candidates should be carefully studied before elected for in vivo studies

Butyrate suppresses expression of neuropilin I in colorectal cell lines through inhibition of Sp1 transactivation

Background: Neuropilin is a transmembrane receptor for vascular endothelial growth factor (VEGF) and is expressed in normal endothelial cells and upregulated in cancer cells. Neuropilin-1 (NRP-1) has been shown to promote tumour cell migration and survival in colon cancer in response to VEGF binding. The expression profiles of neuropilins, associated co-receptors and known ligands have been mapped in three colorectal cell lines: Caco-2, HCT116 & HT29. We have previously shown that butyrate, a naturally occurring histone deacetylase inhibitor (HDACi) produced by fermentation of fibre in the colon, causes apoptosis of colon cancer cell lines. Results: Here we demonstrate that butyrate down-regulates NRP-1 and VEGF at the mRNA and protein level in colorectal cancer cell lines. NRP-1 is a known transcriptional target of Sp1, whose activity is regulated by acetylation. NRP-1 down-regulation by butyrate was associated with decreased binding affinity of Sp1 for canonical Sp-binding sites in the NRP-1 promoter. siRNA-mediated knock-down of Sp1 implied that Sp1 may have strong DNA binding activity but weak transactivation potential. Conclusion: The downregulation of the key apoptotic and angiogenesis regulator NRP-1 by butyrate suggests a novel contributory mechanism to the chemopreventive effect of dietary fibre

Experiments and simulations on short chain fatty acid production in a colonic bacterial community

Understanding how production of specific metabolites by gut microbes is modulated by interactions with surrounding species and by environmental nutrient availability is an important open challenge in microbiome research. As part of this endeavor, we explore interactions between F. prausnitzii, a major butyrate producer, and B. thetaiotaomicron, an acetate producer, under three different in vitro media conditions in monoculture and coculture. In silico Genome-scale dynamic flux balance analysis (dFBA) models of metabolism in the system using COMETS (Computation of Microbial Ecosystems in Time and Space) are also tested for explanatory, predictive and inferential power. Experimental findings indicate enhancement of butyrate production in coculture relative to F. prausnitzii monoculture but defy a simple model of monotonic increases in butyrate production as a function of acetate availability in the medium. Simulations recapitulate biomass production curves for monocultures and accurately predict the growth curve of coculture total biomass, using parameters learned from monocultures, suggesting that the model captures some aspects of how the two bacteria interact. However, a comparison of data and simulations for environmental acetate and butyrate changes suggest that the organisms adopt one of many possible metabolic strategies equivalent in terms of growth efficiency. Furthermore, the model seems not to capture subsequent shifts in metabolic activities observed experimentally under low-nutrient regimes. Some discrepancies can be explained by the multiplicity of possible fermentative states for F. prausnitzii. In general, these results provide valuable guidelines for design of future experiments aimed at better determining the mechanisms leading to enhanced butyrate in this ecosystem.https://www.biorxiv.org/content/10.1101/444760v1https://www.biorxiv.org/content/10.1101/444760v1Othe

Trap Response of Michigan Social Wasps (Hymenoptera: Vespidae) to the Feeding Attractants Acetic Acid, Isobutanol, and Heptyl Butyrate.

Nine species of social wasps were captured in traps baited with acetic acid, isobutanol, heptyl butyrate and combinations of acetic acid and either isobutanol or heptyl butyrate. Three yellowjacket species in the Vespula rufa species group were captured in traps (Vespula acadica (Sladen), Vespula consobrina (Saussure), Vespula vidua (Saussure)). They responded similarly, with attraction only to heptyl butyrate. Three yellowjacket species in the Vespula vulgaris species group were also captured in traps (Vespula vulgaris (L.), Vespula flavorpilosa Jacobson, Vespula maculifrons (Buyyson)). They responded similarly, with attraction primarily to the combination of acetic acid and isobutanol. The bald-faced hornet, Dolichovespula maculata (L.), was attracted to acetic acid and was more strongly attracted to the combination of acetic acid and isobutanol. The aerial yellowjacket, Dolichovespula arenaria (Fabr.), was attracted to isobutanol, and was more strongly attracted to the combination of acetic acid and isobutanol. These results add to our understanding of how to target various species of social wasps with chemical lures

Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line

Butyrate, a known histone deacetylase inhibitor (HDACi) and product of fibre fermentation, is postulated to mediate the protective effect of dietary fibre against colon cancer. The transcription factor Sp1 is a target of acetylation and is known to be associated with class I HDACs, including HDAC1. Sp1 is a ubiquitous transcription factor and Sp1-regulated genes include those involved in cell cycle regulation, apoptosis and lipogenesis: all major pathways in cancer development. The only known acetylated residue of Sp1 is lysine703 which resides in the DNA binding domain. Here we show that acetylated Sp1 loses p21- and bak-promoter -binding function in vitro. Furthermore treatment with a panel of HDAC inhibitors showed clustering of activities for a subset of inhibitors, causing G2 cell cycle arrest, Sp1 acetylation, p21 and Bak over-expression, all with very similar EC50 concentrations. These HDACi activities were not distributed according to the molecular class of compound. In order to mimic loss of binding, an siRNA strategy was used to reduce Sp1 expression. This resulted in altered expression of multiple elements of the p53/p21 pathway. Taken together our data suggest a mechanistic model for the chemopreventive actions of butyrate in colon epithelial cells, and provide new insight into the differential activities some classes of HDAC inhibitors

Comparing the effects of monensin and sodium butyrate on coccidia in post-weaned heifers

The emergence of antibiotic resistances has raised concerns in society about the widespread use of antibiotics, such as monensin, as growth enhancers in agriculture. Pressure to find alternatives has increased since the European Union banned the use of ionophorous antibiotics. Butyrate supplementation has been found to enhance growth in pre-weaned calves and it has been recently suggested to enhance growth in post-weaned heifers. In a recent study by Rice in 2017, there was a quadratic (P=0.03) response for coccidia counts as sodium butyrate was increased, with the lowest counts being at the intermediate doses. This suggested that butyrate has the potential to decrease coccidian in post-weaned heifers. Monensin is also used as a coccidiostat. While the objective of the study as a whole was to determine if sodium butyrate can replace monensin in the prevention of coccidiosis and in enhancing growth and feed efficiency, this report focuses only on the prevention of coccidiosis. At this time, only six of the forty Holstein heifers entered the study. The design was a 2 X 2 factorial arrangement of treatments in a randomized complete block design. Heifers were randomly assigned to 1 of 4 treatments: (1) carrier (control; C); (2) 0.75 g SB/kg of body weight + carrier (SB); (3) 1.0 mg M/kg of body weight + carrier (M); (4) (0.75 g SB/kg of body weight and 1.0 mg M/kg of body weight + carrier (SB/M). Heifers entered the study and were trained to Calan doors from week 12 to 13 age of life. Treatment began on week 14 of life and continued for 12 weeks. Fecal samples were taken from each calf weekly beginning in the covariate period for determination of coccidia. (Measurements were also taken to evaluate the growth of post weaned calves and apparent total tract nutrient digestibility). Oocyte counts were determined through the modified Wisconsin sugar floatation method. Due to the small sample size a statistical analysis was unable to be completed. However, the preliminary data suggested a negative trend in coccidian oocyte counts 2 as treatment progressed. This indicates that both sodium butyrate and monensin have the ability to decrease the shedding of coccidian oocytes, though the statistical significance is not known at this time

Effect of the Butyrate Prodrug Pivaloyloxymethyl Butyrate (AN9) on a Mouse Model for Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an early-onset motor neuron disease that leads to loss of muscle function. Butyrate (BA)-based compounds markedly improve the survival and motor phenotype of SMA mice. In this study, we examine the protective effects of the BA prodrug pivaloyloxymethyl butyrate (AN9) on the survival of SMNΔ7 SMA mice. Oral administration of AN9 beginning at PND04 almost doubled the average lifespan of SMNΔ7 SMA mice. AN9 treatment also increased the growth rate of SMNΔ7 SMA mice when compared to vehicle-treated SMNΔ7 SMA mice. In conclusion, BA prodrugs like AN9 have ameliorative effects on SMNΔ7 SMA mice