Butyrate Treatment Research Articles

Butyrate influences intracellular levels of adenine and adenine derivatives in the fungus Penicillium restrictum

Butyrate, a small fatty acid, has an important role in the colon of ruminants and mammalians including the inhibition of inflammation and the regulation of cell proliferation. There is also growing evidence that butyrate is influencing the histone structure in mammalian cells by inhibition of histone deacetylation. Butyrate shows furthermore an antimicrobial activity against fungi, yeast and bacteria, which is linked to its toxicity at a high concentration. In fungi there are indications that butyrate induces the production of secondary metabolites potentially via inhibition of histone deacetylases. However, information about the influence of butyrate on growth, primary metabolite production and metabolism, besides lipid catabolism, in fungi is scarce. We have identified the filamentous fungus Penicillium (P.) restrictum as a susceptible target for butyrate treatment in an antimicrobial activity screen. The antimicrobial activity was detected only in the mycelium of the butyrate treated culture. We investigated the effect of butyrate ranging from low (0.001mM) to high (30mM), potentially toxic, concentrations on biomass and antimicrobial activity. Butyrate at high concentrations (3 and 30mM) significantly reduced the fungal biomass. In contrast P. restrictum treated with 0.03mM of butyrate showed the highest antimicrobial activity. We isolated three antimicrobial active compounds, active against Staphylococcus aureus, from P. restrictum cellular extracts treated with butyrate: adenine, its derivate hypoxanthine and the nucleoside derivate adenosine. Production of all three compounds was increased at low butyrate concentrations. Furthermore we found that butyrate influences the intracellular level of the adenine nucleoside derivate cAMP, an important signalling molecule in fungi and various organisms.In conclusion butyrate treatment increases the intracellular levels of adenine and its respective derivatives.

Effect of abomasal butyrate infusion on gene expression in the duodenum of lambs

A previous study infusing butyrate into the abomasum of sheep produced increased oxygen, glucose, glutamate, and glutamine uptake by the portal-drained viscera. These changes were thought to be partially due to increases in glycolysis and cell proliferation. The purpose of this study was to evaluate the duodenum transcriptome of control and butyrate-treated lambs to determine whether genes involved in these pathways were altered. Polled Dorset lambs (n = 9) received a pulse dose of either butyrate (10 mg/kg BW) or an equal volume of a buffered saline solution (1 mL/kg BW) daily at the time of feeding. Lambs were euthanized approximately 4 h after treatment/feeding on d 21, and a sample of duodenal mucosa was obtained from which total RNA was isolated for microarray analysis. A total of 230 genes were differentially expressed (P < 0.05). Pathway analyses performed with the differentially expressed genes revealed glycolysis, fatty acid activation/biosynthesis, UDP-N-acetyl-ᴅ-galactosamine biosynthesis, γ-Linolenate biosynthesis, and mitochondrial ʟ-carnitine shuttle pathways up-regulated by the butyrate treatment. Additionally, expression of functional gene clusters related to mitochondrial function was found to be enriched (P < 0.05) with the butyrate treatment. These data could partially explain the metabolite flux changes that were observed with the butyrate treatment; specifically the increase in glucose uptake and glycolysis pathway upregulation and the increased oxygen uptake and upregulation of mitochondria function-related genes.

Butyrate Inhibits Cancerous HCT116 Colon Cell Proliferation but to a Lesser Extent in Noncancerous NCM460 Colon Cells.

Butyrate, an intestinal microbiota metabolite of dietary fiber, exhibits chemoprevention effects on colon cancer development. However, the mechanistic action of butyrate remains to be determined. We hypothesize that butyrate inhibits cancerous cell proliferation but to a lesser extent in noncancerous cells through regulating apoptosis and cellular-signaling pathways. We tested this hypothesis by exposing cancerous HCT116 or non-cancerous NCM460 colon cells to physiologically relevant doses of butyrate. Cellular responses to butyrate were characterized by Western analysis, fluorescent microscopy, acetylation, and DNA fragmentation analyses. Butyrate inhibited cell proliferation, and led to an induction of apoptosis, genomic DNA fragmentation in HCT116 cells, but to a lesser extent in NCM460 cells. Although butyrate increased H3 histone deacetylation and p21 tumor suppressor expression in both cell types, p21 protein level was greater with intense expression around the nuclei in HCT116 cells when compared with that in NCM460 cells. Furthermore, butyrate treatment increased the phosphorylation of extracellular-regulated kinase 1/2 (p-ERK1/2), a survival signal, in NCM460 cells while it decreased p-ERK1/2 in HCT116 cells. Taken together, the activation of survival signaling in NCM460 cells and apoptotic potential in HCT116 cells may confer the increased sensitivity of cancerous colon cells to butyrate in comparison with noncancerous colon cells.

Effect of Butyrate on Collagen Expression, Cell Viability, Cell Cycle Progression and Related Proteins Expression of MG-63 Osteoblastic Cells.

AimsButyric acid is one major metabolic product generated by anaerobic Gram-negative bacteria of periodontal and root canal infection. Butyric acid affects the activity of periodontal cells such as osteoblasts. The purposes of this study were to investigate the effects of butyrate on MG-63 osteoblasts.MethodsMG-63 cells were exposed to butyrate and cell viability was estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mRNA and protein expression of type I collagen and cell cycle-related proteins were measured by reverse-transcriptase polymerase chain reaction (RT-PCR), western blotting or immunofluorescent staining. Cellular production of reactive oxygen species (ROS) was analyzed by 2',7'-dichlorofluorescein (DCF) fluorescence flow cytometry.ResultsExposure to butyrate suppressed cell proliferation, and induced G2/M (8 and 16 mM) cell cycle arrest of MG-63 cells. Some cell apoptosis was noted. The mRNA expression of cdc2 and cyclin-B1 decreased after exposure to butyrate. The protein expression of type I collagen, cdc2 and cyclin B1 were decreased, whereas the expression of p21, p27 and p57 was stimulated. Under the treatment of butyrate, ROS production in MG-63 cells markedly increased.ConclusionsThe secretion of butyric acid by periodontal and root canal microorganisms may inhibit bone cell growth and matrix turnover. This is possibly due to induction of cell cycle arrest and ROS generation and inhibition of collagen expression. These results suggest the involvement of butyric acid in the pathogenesis of periodontal and periapical tissue destruction by impairing bone healing responses.

Effects of early intervention with sodium butyrate on lipid metabolism-related gene expression and liver metabolite profiles in neonatal piglets

This study was conducted to investigate the effects of early intervention with sodium butyrate on the lipid metabolism and liver metabolite profiles in neonatal piglets. Ten litters of newborn crossbred Duroc×Landrace×Large White piglets were randomly assigned to the control or sodium butyrate treatment with 5 litters per treatment. Piglets in the sodium butyrate treatment were orally administered with 7–13mL sodium butyrate solution (150mmol/L) per day from d 0–6, whereas the piglets in the control treatment were treated with the same dose of physiological saline. At the age of 7 and 21 d, 1 piglet from each litter was sacrificed. The liver, backfat, and loin muscle of piglets were collected for gene expression or metabolome analysis. The results showed that sodium butyrate treatment had no effect on the growth performance of piglets. Sodium butyrate decreased the concentration of cholesterol (P<0.05) and tended to decrease high density lipoprotein-cholesterol (P=0.079) in the serum on d 7, while there was no difference in serum metabolites between 2 treatments on d 21. Real-time PCR assay showed that sodium butyrate down-regulated the mRNA expression of lipogentic genes sterol regulatory element binding protein 1c and fatty acid synthetize in the liver on d 7 (P<0.05). Adipocyte markers leptin, fatty acid binding protein 4, and peroxisome proliferatoractivated receptor γ expression in backfat were up-regulated (P<0.05) by sodium butyrate treatment on d 7. Gas chromatography–mass spectrometry analysis showed that sodium butyrate mainly affected liver glycolysis metabolism and gluconeogenesis metabolism of piglets on d 7 and 21. These results indicate that early oral administration of sodium butyrate could change the lipid metabolism by decreasing fatty acid synthesis, and modulate hepatic metabolite profiles.

Single-dose infusion of sodium butyrate, but not lactose, increases plasma β-hydroxybutyrate and insulin in lactating dairy cows

Several studies have identified beneficial effects of butyrate on rumen development and intestinal health in preruminants. These encouraging findings led to further investigations related to butyrate supplementation in the mature ruminant. However, the effects of elevated butyrate concentrations on rumen metabolism have not been investigated, and consequently the maximum tolerable dosage rate of butyrate has not been established. Therefore, the first objective of this work was to evaluate the effect of a short-term increase in rumen butyrate concentration on key metabolic indicators. The second objective was to evaluate the source of butyrate, either directly dosed in the rumen or indirectly supplied via lactose fermentation in the rumen. Jugular catheters were inserted into 4 ruminally fistulated Holstein cows in a 4×4 Latin square with 3-d periods. On d 1 of each period, 1h after feeding, cows were ruminally dosed with 1 of 4 treatments: (1) 2L of water (CON), (2) 3.5g/kg of body weight (BW) of lactose (LAC), (3) 1g/kg of BW of butyrate (1GB), or (4) 2g/kg of BW of butyrate (2GB). Sodium butyrate was the source of butyrate, and NaCl was added to CON (1.34g/kg of BW), LAC (1.34g/kg of BW), and 1GB (0.67g/kg of BW) to provide equal amounts of sodium as the 2GB treatment. Serial plasma and rumen fluid samples were collected during d 1 of each period. Rumen fluid pH was greater in cows given the 1GB and 2GB treatments compared with the cows given the LAC treatment. Cows administered the 1GB and 2GB treatments had greater rumen butyrate concentrations compared with LAC. Those cows also had greater plasma butyrate concentrations compared with cows given the LAC treatment. Plasma β-hydroxybutyrate was greater and insulin tended to be greater for butyrate treatments compared with LAC. No difference in insulin was found between the 1GB and 2GB treatments. Based on plasma and rumen metabolites, singly infusing 3.5g/kg of BW of lactose into the rumen is not as effective at providing a source of butyrate as compared with singly infusing 1 or 2g/kg of BW of butyrate into the rumen. Additionally, rumen pH, rumen butyrate, plasma β-hydroxybutyrate, glucose, and plasma butyrate were less affected in cows administered the 1GB treatment than in cows given the 2GB treatment. This finding suggests that singly dosing 1g/kg of BW of butyrate could serve as the maximum tolerable concentration for future research.

Abstract P162: Butyrate, a Microbial Metabolite, Attenuates Angiotensin II-induced Hypertension and Gut Dysbiosis

Objectives: We have previously established that hypertension (HTN) is associated with gut dysbiosis in rat models and patients with high blood pressure. Gut dysbiosis was associated with an increase in Firmicutes/Bacteriodetes ratio and a decrease in butyrate-producing microbial populations. This led us to hypothesize that infusion of butyrate would overcome gut microbial dysbiosis induced butyrate deficiency and reverse angiotensin II (Ang II)-induced HTN. Design and Method: Four groups of C57BL6 mice were infused with either saline, Ang II (1000ng/kg/min), Ang II + sodium butyrate (1mg/kg), or sodium butyrate alone for 4 weeks. Fecal samples were analyzed by 16S bacterial rDNA sequencing for gut microbiome identification. Intestinal leukocytes were analyzed using FACS. Results: Ang II induced increase in MAP was significantly attenuated in mice co-administrated with butyrate (Control, 102.1±5.7 mmHg; Ang II, 148.3±8.1 mmHg; Ang II+Butyrate, 120.5±11.2 mmHg). Microbiota analysis demonstrated a significant increase of gut dysbiosis in Ang II-HTN that was normalized by butyrate treatment (F/B ratio: Control, 2.6; Ang II, 5.4; Ang II+Butyrate, 2.0). Principal Coordinates Analysis indicated each group in the input phylogenetic tree had significantly different microbial communities. LEfSe analysis showed that there were decreases of beneficial bacteria such as Lactobacillus and Bifidobacterium , and increases of Corynebacterium and Staphylococcus at the genus level of Ang II-HTN mice. Furthermore, inflammatory status of the gut as evidenced by the level of mucosal T cells in lamina propria from these groups showed that there was an increase of CCR2 + Th17 cells in Ang II-HTN mice, but not in butyrate co-treated mice (Control, 15.7%; Ang II, 28.4%; Ang II+Butyrate, 15%). Conclusions: These observations show that gut dysbiosis and the decrease of butyrate producing bacteria are associated with Ang II-HTN. Thus, supplementing butyrate in Ang II treated mice attenuated HTN and reversed gut dysbiosis, as well as normalizing the intestinal Th17. These data suggest butyrate producing bacteria could be considered as a novel probiotic therapy for hypertension.

Effects of Sodium Butyrate Treatment on Histone Modifications and the Expression of Genes Related to Epigenetic Regulatory Mechanisms and Immune Response in European Sea Bass (Dicentrarchus Labrax) Fed a Plant-Based Diet.

Bacteria that inhabit the epithelium of the animals’ digestive tract provide the essential biochemical pathways for fermenting otherwise indigestible dietary fibers, leading to the production of short-chain fatty acids (SCFAs). Of the major SCFAs, butyrate has received particular attention due to its numerous positive effects on the health of the intestinal tract and peripheral tissues. The mechanisms of action of this four-carbon chain organic acid are different; many of these are related to its potent regulatory effect on gene expression since butyrate is a histone deacetylase inhibitor that play a predominant role in the epigenetic regulation of gene expression and cell function. In the present work, we investigated in the European sea bass (Dicentrarchus labrax) the effects of butyrate used as a feed additive on fish epigenetics as well as its regulatory role in mucosal protection and immune homeostasis through impact on gene expression. Seven target genes related to inflammatory response and reinforcement of the epithelial defense barrier [tnfα (tumor necrosis factor alpha) il1β, (interleukin 1beta), il-6, il-8, il-10, and muc2 (mucin 2)] and five target genes related to epigenetic modifications [dicer1(double-stranded RNA-specific endoribonuclease), ehmt2 (euchromatic histone-lysine-N-methyltransferase 2), pcgf2 (polycomb group ring finger 2), hdac11 (histone deacetylase-11), and jarid2a (jumonji)] were analyzed in fish intestine and liver. We also investigated the effect of dietary butyrate supplementation on histone acetylation, by performing an immunoblotting analysis on liver core histone extracts. Results of the eight-week-long feeding trial showed no significant differences in weight gain or SGR (specific growth rate) of sea bass that received 0.2% sodium butyrate supplementation in the diet in comparison to control fish that received a diet without Na-butyrate. Dietary butyrate led to a twofold increase in the acetylation level of histone H4 at lysine 8, but showed no effect on the histone H3 at Lys9. Moreover, two different isoforms of histone H3 that might correspond to the H3.1 and H3.2 isoforms previously found in terrestrial animals were separated on the immunoblots. The expression of four (il1 β, il8, irf1, and tnfα) out of seven analyzed genes related to mucosal protection and inflammatory response was significantly different between the two analyzed tissues but only il10 showed differences in expression due to the interaction between tissue and butyrate treatment. In addition, butyrate caused significant changes in vivo in the expression of genes related to epigenetic regulatory mechanisms such as hdac11, ehmt2, and dicer1. Statistical analysis by two-way ANOVA for these genes showed not only significant differences due to the butyrate treatment, but also due to the interaction between tissue and treatment.

Effect of feeding sodium butyrate in the late finishing period on Salmonella carriage, seroprevalence, and growth of finishing pigs

Pork is an important source of human salmonellosis and low-cost on-farm control measures may provide a useful element in reducing the prevalence of this pathogen in food. This study investigated the effectiveness of dietary supplementation with sodium butyrate administered to finisher pigs for ∼4-weeks prior to slaughter to control Salmonella shedding on highly contaminated farms.Two trials (A and B) were conducted on two commercial pig farms, which had a history of high Salmonella seroprevalence. In both trials, pens (14 pens of 12 pigs/pen in Trial A and 12 pens of 12–17 pigs/pen in Trial B) were randomly assigned to a control (finisher feed without additive) or a treatment group (the same feed with 3kg sodium butyrate/t) for 24–28days, depending on the trial. Faeces were collected from each pig on days 0, 12 and 24/28, and blood, caecal digesta and ileocaecal/mesenteric lymph nodes were collected from the slaughterhouse. Pigs were weighed at the start and end of the trials, feed intake was recorded, and carcass quality parameters were recorded at slaughter.In Trial A, Salmonella shedding was reduced in the treatment compared to the control group at the end of the trial (30% versus 57% probability of detecting Salmonella in faeces, respectively; p<0.001). This reflected the serology results, with detection of a lower seroprevalence in the treatment compared to the control group using the 20% optical density cut-off (69.5% versus 89%; p=0.001). However, no effect on faecal shedding or seroprevalance was observed in Trial B, which may be explained by the detection of a concomitant infection with Lawsonia intracellularis. No significant differences in Salmonella recovery rates were observed in the caecal digesta or lymph nodes in either trial. Furthermore, feed intake, weight gain, and feed conversion efficiency (FCE) did not differ between groups (p>0.05) in either trial. Numerical improvements in weight gain and FCE were found with sodium butyrate treatment, which gave a cost benefit of €0.04/kg of live-weight gain.Overall, results suggest that strategic feeding of sodium butyrate, at 3kg/t of feed, to finishing pigs for 24–28days prior to slaughter was effective in reducing Salmonella shedding and seroprevalance but perhaps only in the absence of co-infection with other pathogens. However, sodium butyrate supplementation at this rate did not influence intestinal carriage, nor did it reduce seroprevalence to below the cut-off used for the high Salmonella risk category in Ireland (50%), or significantly improve growth performance.

Rescue of Isolated GH Deficiency Type II (IGHD II) via Pharmacologic Modulation of GH-1 Splicing.

Isolated GH deficiency (IGHD) type II, the autosomal dominant form of GHD, is mainly caused by mutations that affect splicing of GH-1. When misspliced RNA is translated, it produces a toxic 17.5-kDa GH isoform that reduces the accumulation and secretion of wild-type-human GH (wt-hGH). Usually, isolated GHD type II patients are treated with daily injections of recombinant human GH in order to maintain normal growth. However, this type of replacement therapy does not prevent toxic effects of the 17.5-kDa GH isoform on the pituitary gland, which can eventually lead to other hormonal deficiencies. Here, we tested the possibility to restore the constitutive splicing pattern of GH-1 by using butyrate, a drug that mainly acts as histone deacetylase inhibitor. To this aim, wt-hGH and/or different hGH-splice site mutants (GH-IVS3+2, GH-IVS3+6, and GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GHRHR) (GC-GHRHR). Upon butyrate treatment, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed increased GH transcript level, intracellular GH content, and GH secretion when compared with the corresponding untreated condition. The effect of butyrate was most likely mediated by the alternative splicing factor/splicing factor 2. Overexpression of alternative ASF/SF2 in the same experimental setting, indeed, promoted the amount of full-length transcripts thus increasing synthesis and secretion of the 22-kDa GH isoform. In conclusion, our results support the hypothesis that modulation of GH-1 splicing pattern to increase the 22-kDa GH isoform levels can be clinically beneficial and hence a crucial challenge in GHD research.

Butyrate upregulates endogenous host defense peptides to enhance disease resistance in piglets via histone deacetylase inhibition.

Butyrate has been used to treat different inflammatory disease with positive outcomes, the mechanisms by which butyrate exerts its anti-inflammatory effects remain largely undefined. Here we proposed a new mechanism that butyrate manipulate endogenous host defense peptides (HDPs) which contributes to the elimination of Escherichia coli O157:H7, and thus affects the alleviation of inflammation. An experiment in piglets treated with butyrate (0.2% of diets) 2 days before E. coli O157:H7 challenge was designed to investigate porcine HDP expression, inflammation and E. coli O157:H7 load in feces. The mechanisms underlying butyrate-induced HDP gene expression and the antibacterial activity and bacterial clearance of macrophage 3D4/2 cells in vitro were examined. Butyrate treatment (i) alleviated the clinical symptoms of E. coli O157:H7-induced hemolytic uremic syndrome (HUS) and the severity of intestinal inflammation; (ii) reduced the E. coli O157:H7 load in feces; (iii) significantly upregulated multiple, but not all, HDPs in vitro and in vivo via histone deacetylase (HDAC) inhibition; and (iv) enhanced the antibacterial activity and bacterial clearance of 3D4/2 cells. Our findings indicate that butyrate enhances disease resistance, promotes the clearance of E. coli O157:H7, and alleviates the clinical symptoms of HUS and inflammation, partially, by affecting HDP expression via HDAC inhibition.

HDAC inhibitors cause site-specific chromatin remodeling at PU.1-bound enhancers in K562 cells.

BackgroundSmall molecule inhibitors of histone deacetylases (HDACi) hold promise as anticancer agents for particular malignancies. However, clinical use is often confounded by toxicity, perhaps due to indiscriminate hyperacetylation of cellular proteins. Therefore, elucidating the mechanisms by which HDACi trigger differentiation, cell cycle arrest, or apoptosis of cancer cells could inform development of more targeted therapies. We used the myelogenous leukemia line K562 as a model of HDACi-induced differentiation to investigate chromatin accessibility (DNase-seq) and expression (RNA-seq) changes associated with this process.ResultsWe identified several thousand specific regulatory elements [~10 % of total DNase I-hypersensitive (DHS) sites] that become significantly more or less accessible with sodium butyrate or suberanilohydroxamic acid treatment. Most of the differential DHS sites display hallmarks of enhancers, including being enriched for non-promoter regions, associating with nearby gene expression changes, and increasing luciferase reporter expression in K562 cells. Differential DHS sites were enriched for key hematopoietic lineage transcription factor motifs, including SPI1 (PU.1), a known pioneer factor. We found PU.1 increases binding at opened DHS sites with HDACi treatment by ChIP-seq, but PU.1 knockdown by shRNA fails to block the chromatin accessibility and expression changes. A machine-learning approach indicates H3K27me3 initially marks PU.1-bound sites that open with HDACi treatment, suggesting these sites are epigenetically poised.ConclusionsWe find HDACi treatment of K562 cells results in site-specific chromatin remodeling at epigenetically poised regulatory elements. PU.1 shows evidence of a pioneer role in this process by marking poised enhancers but is not required for transcriptional activation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0065-5) contains supplementary material, which is available to authorized users.

Interspecies nuclear transfer using fibroblasts from leopard, tiger, and lion ear piece collected postmortem as donor cells and rabbit oocytes as recipients.

Skin fibroblast cells were obtained from a small piece of an ear of leopard, lion, and tiger collected postmortem and attempts were made to synchronize the skin fibroblasts at G0/G1 of cell cycle using three different approaches. Efficiency of the approaches was tested following interspecies nuclear transfer with rabbit oocytes as recipient cytoplasm. Fluorescence-activated cell sorting revealed that the proportion of G0/G1 cells increased significantly (P < 0.05) when cells subjected to serum starvation, contact inhibition, and 3mM sodium butyrate (NaBu) treatment when compared with cycling cells. However, 3mM NaBu treatment caused alterations in cell morphology and increase in dead cells. Thus, interspecies nuclear transfer was carried out using fibroblast cells subjected to contact inhibition for 72h, serum starvation for 48h, and cells treated with 1.0mM NaBu for 48h. The fusion rates, the proportion of fused couplets that cleaved to two-cell and developed to blastocyst, were highest in all three species when the donor cells were treated with 1.0mM NaBu for 48h. But, the blastocyst percentage of interspecies nuclear embryos (5-6%) was significantly lower when compared with rabbit-rabbit nuclear transfer embryos (22.9%). In conclusion, fibroblast cells of leopard, lion, and tiger were successfully synchronized and used for the development of blastocysts using rabbit oocytes as recipient cytoplasm.

In Vivo Evaluation of Butyrate, a Histone Deacetylase Inhibitor Effect on Lipid Profile of Apo E−/− Mice Fed on Atherogenic Diet

A large body of evidence indicates diets high in fiber are correlated with lower incidences of adverse cardiovascular events including atherosclerotic vascular disease by cholesterol lowering effects. Although, butyrate, a natural product of dietary fiber fermentation produced in the colon appears to have a role in the cardio protective effects of fiber, its influence on hyperlipidemic conditions is not clear. In the present study, ApoE−/− knockout atherogenic mouse model fed on non‐atherogenic or atherogenic diets were treated with or without butyrate (50mg/kg, ip, six days/week; N=8 animals/group) for 18 weeks to determine the effect of butyrate on atherogenic diet related hyperlipidemic state. All animals were handled and treated by strictly following the Institutional guidelines approved by the Institutional Animal Care and Use Committee, there were no mortalities and animals were not under any stress. At the end of treatment, blood samples were collected for lipid analysis. The results indicate that chronic butyrate treatment causes no significant changes in overall lipid profiles both in groups fed on non‐atherogenic and atherogenic diet compared to their respective no butyrate treatment controls. However, butyrate treatment significantly reduced body weights of animals that were fed on atherogenic diet compared to the weights of animals fed on non‐atherogenic diet. The results appears to show that butyrate exerts a potential protective role in modulating vascular health by promoting lower body weight even with no change in atherogenic diet induced hyperlipidemic state. It is possible other mechanism(s) may be playing a role in butyrate‐induced lower body weights of animals fed with atherogenic diet.Support or Funding InformationThis study was made possible, in part, by research infrastructure support from grant number 2G12MD007605 from the NIMHD/NIH

Butyrate Plays Differential Roles in Cellular Signaling in Cancerous HCT116 and Noncancerous NCM460 Colon Cells

Butyrate, an intestinal microbiota metabolite of dietary fiber, exhibits chemoprevention effects in colon. However, the mechanistic action of butyrate at the cellular level remains to be determined. We hypothesize that butyrate plays differential roles in cancerous and non‐cancerous cells through signaling pathways regulating histone deacetylation, apoptosis, and cellular survival. We tested this hypothesis by exposing cancerous HCT116 or non‐cancerous NCM460 colon cells to physiological relevant doses of butyrate (0.25–2 mmol/L). Cellular responses were characterized by protein expression (Western blotting), fluorescent microscopy, acetylation, and DNA fragmentation analyses. Upon exposure to butyrate (2 mmol/L) for 72 hours, cell proliferation was reduced by 93% in HCT116 cells, compared to 57% in NCM460 cells. In addition, butyrate treatment led to an induction of apoptosis, histone deacetylation, genomic DNA fragmentation and p21 tumor suppressor expression in HCT116 cells, but to a smaller extent in NCM460 cells. In contrast, butyrate increased the phosphorylation of extracellular‐regulated kinase 1/2 (ERK1/2) in NCM460 cells but not in HCT116 cells. Taken together, the activation of survival signaling in NCM460 cells and apoptotic signaling in HCT116 cells may confer the increased sensitivity of cancerous colon cells to butyrate in comparison with noncancerous colon cells.Support or Funding InformationThis work was supported by USDA‐ARS, CRIS project (5450‐51000‐050‐00D).

Acetylation of p53 Protein at Lysine 120 Up-regulates Apaf-1 Protein and Sensitizes the Mitochondrial Apoptotic Pathway

The p53 tumor suppressor controls cell growth, metabolism, and death by regulating the transcription of various target genes. The target-specific transcriptional activity of p53 is highly regulated. Here we demonstrate that acetylation of p53 at Lys-120 up-regulates its transcriptional activity toward Apaf-1, a core component in the mitochondrial apoptotic pathway, and thus sensitizes caspase activation and apoptosis. We found that histone deacetylase (HDAC) inhibitors, including butyrate, augment Lys-120 acetylation of p53 and thus Apaf-1 expression by inhibiting HDAC1. In p53-null cells, transfection of wild-type but not K120R mutant p53 can restore the p53-dependent sensitivity to butyrate. Strikingly, transfection of acetylation-mimicking K120Q mutant p53 is sufficient to up-regulates Apaf-1 in a manner independent of butyrate treatment. Therefore, HDAC inhibitors can induce p53 acetylation at lysine 120, which in turn enhances mitochondrion-mediated apoptosis through transcriptional up-regulation of Apaf-1.

Butyrate enhances antibacterial effects while suppressing other features of alternative activation in IL-4-induced macrophages.

The short-chain fatty acid butyrate is produced by fermentation of dietary fiber by the intestinal microbiota; butyrate is the primary energy source of colonocytes and has immunomodulatory effects. Having shown that macrophages differentiated with IL-4 [M(IL-4)s] can suppress colitis, we hypothesized that butyrate would reinforce an M(IL-4) phenotype. Here, we show that in the presence of butyrate M(IL-4)s display reduced expression of their hallmark markers Arg1 and Ym1 and significantly suppressed LPS-induced nitric oxide, IL-12p40, and IL-10 production. Butyrate treatment likely altered the M(IL-4) phenotype via inhibition of histone deacetylation. Functionally, M(IL-4)s treated with butyrate showed increased phagocytosis and killing of bacteria, compared with M(IL-4) and this was not accompanied by enhanced proinflammatory cytokine production. Culture of regulatory T cells with M(IL-4)s and M(IL-4 + butyrate)s revealed that both macrophage subsets suppressed expression of the regulatory T-cell marker Foxp3. However, Tregs cocultured with M(IL-4 + butyrate) produced less IL-17A than Tregs cocultured with M(IL-4). These data illustrate the importance of butyrate, a microbial-derived metabolite, in the regulation of gut immunity: the demonstration that butyrate promotes phagocytosis in M(IL-4)s that can limit T-cell production of IL-17A reveals novel aspects of bacterial-host interaction in the regulation of intestinal homeostasis.

Suppressing activity of tributyrin on hepatocarcinogenesis is associated with inhibiting the p53-CRM1 interaction and changing the cellular compartmentalization of p53 protein.

Hepatocellular carcinoma (HCC), an aggressive and the fastest growing life-threatening cancer worldwide, is often diagnosed at intermediate or advanced stages of the disease, which substantially limits therapeutic approaches for its successful treatment. This indicates that the prevention of hepatocarcinogenesis is probably the most promising approach to reduce both the HCC incidence and cancer-related mortality. In previous studies, we demonstrated a potent chemopreventive effect of tributyrin, a butyric acid prodrug, on experimental hepatocarcinogenesis. The cancer-inhibitory effect of tributyrin was linked to the suppression of sustained cell proliferation and induction of apoptotic cell death driven by an activation of the p53 apoptotic signaling pathway. The goal of the present study was to investigate the underlying molecular mechanisms linked to tributyrin-mediated p53 activation. Using in vivo and in vitro models of liver cancer, we demonstrate that an increase in the level of p53 protein in nuclei, a decrease in the level of cytoplasmic p53, and, consequently, an increase in the ratio of nuclear/cytoplasmic p53 in rat preneoplastic livers and in rat and human HCC cell lines caused by tributyrin or sodium butyrate treatments was associated with a marked increase in the level of nuclear chromosome region maintenance 1 (CRM1) protein. Mechanistically, the increase in the level of nuclear p53 protein was associated with a substantially reduced binding interaction between CRM1 and p53. The results demonstrate that the cancer-inhibitory activity of sodium butyrate and its derivatives on liver carcinogenesis may be attributed to retention of p53 and CRM1 proteins in the nucleus, an event that may trigger activation of p53-mediated apoptotic cell death in neoplastic cells.

Site-Specific Identification of Lysine Acetylation Stoichiometries in Mammalian Cells.

Functional characterization of the lysine acetylation pathway requires quantitative measurement of the modification abundance at the stoichiometry level. Here, we developed a systematic workflow for global untargeted identification of site-specific Lys acetylation stoichiometries in mammalian cells. Our strategy includes an optimized protocol for in vitro chemical labeling of unmodified lysine with stable isotope-encoded acetyl-NHS ester, deep proteomic profiling with a high resolution mass spectrometer, and a new software tool for quantitative analysis and stoichiometry determination. The workflow was validated using in vitro chemically labeled BSA and synthetic peptides with multiple Lys acetylations at various positions. In the proof-of-concept study, we applied the strategy to analyze the proteome of HeLa cells and determined the stoichiometries of over 600 acetylation sites with good reproducibility. Sodium butyrate treatment induced a significant increase of acetylation stoichiometries in HeLa cells. Analysis of site-specific stoichiometry dynamics revealed the coregulation of closely positioned acetylation sites on histones H3 and H4 upon treatment.

Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis.

PurposeButyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells.MethodsHuman colorectal cancer cell lines (HCT-116 and HT-29) were treated with sodium butyrate at concentrations ranging from 0.5–5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining), and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot.ResultsSodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II), beclin-1, and autophagocytosis-associated protein (Atg)3. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin) and genetic (siRNA targeting BIP and CHOP) methods, the induction of BIP, PDI, IRE1a, and LC3-II was blocked, but PARP cleavage was markedly enhanced.DiscussionTaken together, these results suggested that sodium butyrate-induced autophagy was mediated by endoplasmic reticulum stress, and that preventing autophagy by blocking the endoplasmic reticulum stress response enhanced sodium butyrate-induced apoptosis. These results provide novel insights into the anti-tumor mechanisms of butyric acid.