Higher Butyrate Concentrations Research Articles

Ruminal fermentation kinetics in ewes offered a maize stover basal diet supplemented with cowpea hay, groundnut hay, cotton seed meal or maize meal.

The effect on ruminal digestive kinetics of supplementing maize stover (MS) with cowpea hay (CW), groundnut hay (GN), cotton seed meal (CSM) or maize meal (MM) was evaluated in a 5 x 5 Latin square using five Merino ewes with ruminal cannulae. CW and GN were offered at 30% w/w to MS, while CSM and MM were given at 15 g/kg0.75 per day. There were significant time (p < 0.001) and time x diet (p < 0.05) effects on pH but the effect of diet alone was not significant (p > 0.05). There was a significant (p < 0.05) increase in the concentration of ammonia with time on the supplemented diets. There were no significant (p > 0.05) differences in the concentrations of total volatile fatty acids (VFAs). Butyrate increased with supplementation (p < 0.05). Supplementation increased the concentrations of branched-chain and total VFAs with time (p < 0.05). The non-glucogenic ratio was not significantly (p > 0.05) lower on supplemented diets owing to the higher butyrate concentration. The degradation curve constants were not significantly different (p > 0.05), the average effective dry matter degradation being 27.80% +/- 0.618% at 0.05 h-1 flow rate. The chemical and physical characteristics of the cell wall of MS may have presented few sites for microbial colonization, so limiting degradation. These results showed that small quantities of forage supplements can improve the ruminal environment in animals subsisting on low-quality forages and form a basis for comparison with bought-in protein and energy sources.

Apoptosis cascade proteins are regulated in vivo by high intracolonic butyrate concentration: correlation with colon cancer inhibition.

The present study was aimed at evaluating the effect of high intracolonic butyrate concentrations, either through fermentation of a soluble fiber-enriched diet or via intracolonic butyrate instillation, on colon cancer in a chemically induced (dimethylhydrazine) rat model. The effects were tested in four groups of dimethylhydrazine-treated rats: (i) rats fed a standard diet, (ii) rats fed a diet enriched with 15% citrus pectin, a soluble fiber that ferments and produces a high concentration of intracolonic butyrate, (iii) rats fed a standard diet and intrarectally instilled with a sodium butyrate solution (50 mM), (iv) rats fed a standard diet and intrarectally instilled with sodium butyrate vehicle solution (100 mM NaCl). The apoptotic index in the distal colon of rats fed pectin was higher than in colonic tissue from rats fed a standard diet. The expression of caspase-1, a cysteine protease implicated in the regulation of programmed cell death, as detected by both Northern and Western analysis, showed the highest mRNA and protein levels in colonic tissue from rats intrarectally instilled with butyrate. Immunohistology confirmed the Western blot findings. Expression of the cleaved poly(ADP-ribose) polymerase product, a downstream nuclear substrate for caspase-3 in the apoptotic pathway, was elevated in both the pectin-fed and butyrate-instilled groups. Expression of the antiapoptotic protein Bcl-2 was significantly reduced following pectin feeding as well as butyrate instillation. The highest expression of Bcl-2 was observed in tumor tissue. A marked reduction in aberrant crypt number was observed in colonic tissue obtained from both the pectin-fed and butyrate-instilled groups relative to rats from the standard diet group. The average tumor volume per rat in both the pectin-fed and butyrate-instilled groups was significantly lower than in rats from the standard diet and the sodium butyrate vehicle-instilled groups. We conclude that high butyrate levels, either instilled or obtained following fermentation of soluble dietary fibers, inhibit early and late events in colon tumorigenesis by controlling the transcription expression and activity of key proteins involved in the apoptotic cascade.

Interaction between dexamethasone and butyrate in apoptosis induction: non-additive in thymocytes and synergistic in a T cell-derived leukemia cell line.

In thymocytes butyrate and trichostatin A are unable to augment dexamethasone-induced apoptosis. In cultured rat thymocytes the extent of apoptosis induced by dexamethasone alone did not increase by addition of 0.1 - 10 mM butyrate. Even more pronounced was the non-additive interrelationship between dexamethasone and trichostatin A, as trichostatin A-induced apoptosis was not only blocked by the presence of dexamethasone but dexamethasone-induced apoptosis was also partially inhibited in the presence of 0.1 - 0.5 microM trichostatin A. The fact that the non-additive relationship with dexamethasone for apoptosis induction was observed with both histone deacetylase inhibitors suggests that in thymocytes this phenomenon is related to histone acetylation. In contrast to this, in the human T cell-derived leukemia cell line CEM-C7H2, dexamethasone did not block butyrate- or trichostatin A-induced apoptosis; moreover, butyrate, in the concentration range of 0.1 - 1 mM, had a marked synergistic effect on dexamethasone-induced apoptosis. This synergism, however, was not mimicked by trichostatin A, indicating that the effect is not related to histone acetylation but rather due to a pleiotropic effect of butyrate. Furthermore, in CEM-C7H2 cells, at higher concentrations of butyrate (5 - 10 mM) or trichostatin A (0.4 - 0.8 microM), there was a minor but reproducible antagonistic effect of dexamethasone on apoptosis induced by each of the two histone deacetylase inhibitors, suggesting that this antagonistic effect too, is related to histone hyperacetylation.

Metabolism of short-chain fatty acids by rat colonic mucosa in vivo.

To determine the influence of substrate concentration and substrate interactions on short-chain fatty acid metabolism in vivo, a surgical procedure was established. Rats were surgically operated to cannulate a 5-cm segment of proximal colon, isolate the vasculature, and cannulate the right colic vein draining this segment. Thus metabolism was restricted to the defined colonic segment. The appearance of total (14)C and (14)CO(2) in the mesenteric blood stabilized after 30 min of perfusion. Increasing luminal concentrations of butyrate from 2 to 40 mmol/l resulted in linear increases in total (14)C, but (14)CO(2) production from [(14)C]butyrate increased as a function of concentration only up to 10 mmol/l and was stable at higher butyrate concentrations. In addition to CO(2), 3-hydroxybutyrate and lactate were major metabolites of acetate and butyrate in vivo. The presence of a mixture of alternative substrates in the lumen had no influence on the metabolism of butyrate to CO(2) but significantly reduced the metabolism of acetate to CO(2). When compared with young (4 mo old) animals, transport of butyrate was significantly lower for aged (48 mo old) animals, as evidenced by the rate of appearance in blood of total (14)C (P = 0.04) and (14)C in butyrate (P = 0.03), but metabolism was similar, since differences were not significant for (14)C in the major metabolites 3-hydroxybutyrate (P = 0.06) and CO(2) (P = 0.17). These results show that important aspects of short-chain fatty acid transport and metabolism are not predicted from data using isolated colonocytes but require study using an in vivo model.

Laboratory evaluation of previously fermented juice as a fermentation stimulant for lucerne silage

The potential use of forage juice anaerobically stored for 2 days (previously fermented juice, PFJ) as a silage additive was investigated. Lucerne (450 g) was ensiled in laboratory-scale silos (900 ml capacity) with or without sucrose (20 g kg−1) or PFJ (2.5 ml kg−1). Silages treated with both additives were also made and fermentation quality, in vitro dry matter digestibility and in vitro ruminal NH3 production were determined. The control silage showed very poor quality with high butyrate and NH3-N concentrations. Addition of sucrose significantly increased lactate but failed to inhibit the deterioration by clostridial fermentation. Use of PFJ further increased lactate and greatly reduced butyrate and NH3-N production during storage. The combination of sucrose and PFJ was more effective in improving fermentation than either additive on its own. In vitro digestibility appeared to be higher when lucerne was well preserved. Ruminal NH3 production in vitro was decreased in silages with sucrose and PFJ added at ensiling. This result indicates that use of PFJ can be a promising way to stimulate lactate production in some difficult direct cut silage. The potential of improving fermentation may be comparable to conventional sugar additives. © 1999 Society of Chemical Industry

Induction of human herpesvirus-8 DNA replication and transcription by butyrate and TPA in BCBL-1 cells.

Human herpesvirus-8 (HHV-8) is a gammaherpesvirus that is present primarily in a state of low level persistence in primary effusion lymphoma cell lines. Using BCBL-1 cells that harbour HHV-8 but lack Epstein-Barr virus, we demonstrate that sodium butyrate is much more effective than the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) at inducing high levels of class II and III virus transcription and viral DNA replication, but also initiates apoptosis. Apoptosis occurs prior to assembly of virions when high concentrations of butyrate (1-3 mM) are used, whereas reduction of butyrate concentration to 0.3 mM decreases the rate of apoptosis and results in production and secretion of enveloped virions that are visualized at high number by electron microscopy in approximately 20% of BCBL-1 cells. Butyrate induces much higher levels of multiple class II and class III transcripts than does TPA, including v-MIPI, v-IL-6, v-Bcl-2, vGPCR and ORF26. A decrease in concentration of butyrate from 3 to 0.3 mM delays the peak induction of these genes, but peak levels remain higher than peak levels in response to TPA. These studies indicate that the massive apoptosis induced by 3 mM butyrate could be diminished and delayed by reduction of butyrate concentration to 0.3 mM, thereby allowing expression of high levels of lytic-associated genes and production of high yields of HHV-8 virions.

Butyrate, aspirin and colorectal cancer.

In vitro, for animal cells generally, butyrate at millimolar concentrations is an inhibitor of growth. In vivo, however, colonocytes are able to grow in the environment of about 20 mM butyrate produced by bacterial fermentation on the luminal side of the colonic epithelium. An in vivo increase of the butyrate supply results in growth stimulation of cells in the colonic crypts. This discrepancy, namely, that in cell cultures butyrate is an inhibitor of growth, whereas in vivo it has a trophic effect, is the so called in vivo paradox of butyrate. In the present review it is pointed out that butyrate is an inhibitor of histone deacetylases and there is sufficient evidence for hyperacetylation being the mechanism of the in vitro growth-inhibiting effect of butyrate. As within animal cells hyperacetylation has to occur at a certain butyrate concentration (1-10 mM), it is postulated that the in vivo lack of inhibition and 'paradoxical' stimulation of growth is a result of a low intracellular steady state concentration of butyrate in the lower layers of the crypt in spite of the much higher butyrate concentration on the luminal side. As butyrate is the preferential source of energy for colonocytes, the in vivo trophic effect is not paradoxical, when in spite of an increase of the butyrate concentration in stool, the intracellular butyrate concentration of intestinal epithelial cells still remains below the inhibiting level. For mature non-dividing colonocytes which are programmed for apoptosis, there is no difference between the observations made in vitro or in vivo. Furthermore, recent developments are discussed which suggest that cyclo-oxygenase-2 may play an essential role in colonic carcinogenesis. Cyclo-oxygenase-2 is found to be expressed in most colorectal carcinomas, but not in normal non-transformed intestinal epithelial cells (DeWitt and Smith, 1995). Cyclo-oxygenase-2 overexpression makes intestinal epithelial cells resistant to butyrate-induced apoptosis (Tsujii and DuBois, 1995). This escape from butyrate-induced apoptosis appears to be an essential prerequisite for the development of colorectal cancer and suggests a functional role of butyrate in growth, differentiation and programmed cell death of colonic epithelial cells.

Net transfer of urea and ammonia across the ruminal wall of sheep

Texel wethers (68 +/- 2.5 kg BW) fitted with catheters in the ruminal veins and a mesenteric artery, blood flow probes on ruminal arteries, and a ruminal cannula were fed 500 g of orchardgrass hay every 12 h. During the last third of the feeding cycle, intraruminal injections were performed to evaluate the effect of urease activity, osmolality, and concentrations of NH3, butyrate, and CO2 in the rumen on urea and NH3 fluxes across the rumen wall. At pH 6.7, NH3 absorption increased with NH3 and butyrate concentrations in the rumen, and to a lesser extent with CO2 concentration. The increase in ruminal blood flow associated with CO2 and butyrate increase was always greater than the increase in NH3 absorption. Increasing ruminal osmolality slightly decreased NH3 absorption. Ruminal NH3 concentration and ruminal blood flow seemed to be the main determinant of NH3 absorption. Decreasing urease activity in the rumen decreased urea net transfer. The net transfer of urea to the rumen was stimulated by CO2. High concentrations of NH3 (330 mg of N/L) and butyrate (25 mM) in the rumen decreased urea net uptake, whereas osmolality (up to 420 mOsmol/L) did not affect it. Modifications in ruminal blood flow or water net movement across the ruminal wall did not seem to account for the effect of CO2, NH3, and butyrate on urea net uptake.

Relationship of Colonic Luminal Short-Chain Fatty Acids and pH to In Vivo Cell Proliferation in Rats

The mechanism by which fermentable fibers may stimulate colonic cell proliferation was tested using two types of fiber (highly fermentable pectin, and less fermentable wheat bran), measuring in vivo concentrations of short-chain fatty acids (SCFA) in the cecum and the proximal and distal colon of rats and correlating these concentrations with cell proliferation indices at the same locations within the intestine. Pectin supplementation resulted in higher concentrations of propionate in proximal and distal colon as compared with fiber-free controls, whereas wheat bran resulted in a higher concentration of butyrate at every site. In the cecum, pH had the strongest correlation to indices of cell proliferation. The lower the pH the greater the number of cells per crypt column (P < 0.05), cells per crypt circumference (P < 0.01), and total number of cells per crypt (P < 0.001). Butyrate had the strongest correlation between a specific SCFA and indices of cell proliferation. In the distal colon, butyrate concentration was positively correlated with number of cells per crypt column (P < 0.05) and total number of cells per crypt (P < 0.05). This study shows that different fibers are fermented to different SCFA in different amounts and that the in vivo concentration of certain of these SCFA is significantly correlated with cell proliferation indices.

Divergent effects of butyrate on the alkaline phosphatases of SW-620 cells

The human colon cancer cell line SW-620 produces two alkaline phosphatases (ALPs) which are not expressed by normal colon. They are the heat-stable, term-placental and the heat-labile, L-homoarginine-sensitive, liver/bone/kidney ALPs. Butyrate, an ALP inducer, has strikingly dissimilar effects on the activity of these enzymes: whereas high (2.0 mM) butyrate concentrations exclusively induce increased activity levels of liver/bone/kidney ALP, low (0.5 mM) concetrations increase the activity of both, albeit induction of term-placental ALP is less pronounced. These observations indicate that the effect of butyrate on the two ALPs is non-coordinate and suggest that their expression by SW-620 cells is independently modulated.

Resistance of Streptomyces cinnamonensis to butyrate and isobutyrate: production and properties of a new anti-isobutyrate (AIB) factor

Butyrate and isobutyrate (after isomerization to n-butyrate) are specific precursors for the biosynthesis of monensin A in Streptomyces cinnamonensis. High concentrations of both butyrate and isobutyrate (greater than 20 and 10 mM, respectively) were toxic to S. cinnamonensis plated on solid medium. Spontaneous mutants resistant to these substances were isolated. These new strains produced monensins at even higher concentrations of butyrate or isobutyrate, with an increased yield of monensin A. S. cinnamonensis produced an anti-isobutyrate (AIB) factor, which was originally found to be excreted by some isobutyrate-resistant stains growing on solid medium containing isobutyrate. On plates, the AIB factor efficiently counteracted toxic concentrations not only of isobutyrate, but also of acetate, propionate, butyrate, 2-methylbutyrate, valerate and isovalerate against S. cinnamonensis as well as other Streptomyces species. Although the AIB factor enabled normal growth, sporulation and monensin production on plates, it did not have positive effects on submerged cultures of S. cinnamonensis with isobutyrate. The partial purification of the AIB factor was achieved. The role of the AIB factor during spore germination on solid medium containing isobutyrate or its homologues is discussed.

Sodium butyrate selectively inhibits host cell glycoprotein synthesis in human fibroblasts infected with cytomegalovirus

Host cell as well as viral DNA synthesis in human fibroblasts infected with human cytomegalovirus was found to be largely resistant even to high concentrations of sodium butyrate. Likewise, production of viral progeny was reduced by 1-2 orders of magnitude but not abolished. On the other hand, the drug allowed (modified) glycosylation only of viral polypeptides whereas that of host proteins was suppressed. Immunofluorescence studies on living cells suggested that butyrate may interfere with processing and intracellular transport of virus-specific surface membrane antigens.

Production of Solvents by Clostridium acetobutylicum Cultures Maintained at Neutral pH

The formation of acetone and n-butanol by Clostridium acetobutylicum NCIB 8052 (ATCC 824) was monitored in batch culture at 35 degrees C in a glucose (2% [wt/vol]) minimal medium maintained throughout at either pH 5.0 or 7.0. At pH 5, good solvent production was obtained in the unsupplemented medium, although addition of acetate plus butyrate (10 mM each) caused solvent production to be initiated at a lower biomass concentration. At pH 7, although a purely acidogenic fermentation was maintained in the unsupplemented medium, low concentrations of acetone and n-butanol were produced when the glucose content of the medium was increased (to 4% [wt/vol]). Substantial solvent concentrations were, however, obtained at pH 7 in the 2% glucose medium supplemented with high concentrations of acetate plus butyrate (100 mM each, supplied as their potassium salts). Thus, C. acetobutylicum NCIB 8052, like C. beijerinckii VPI 13436, is able to produce solvents at neutral pH, although good yields are obtained only when adequately high concentrations of acetate and butyrate are supplied. Supplementation of the glucose minimal medium with propionate (20 mM) at pH 5 led to the production of some n-propanol as well as acetone and n-butanol; the final culture medium was virtually acid free. At pH 7, supplementation with propionate (150 mM) again led to the formation of n-propanol but also provoked production of some acetone and n-butanol, although in considerably smaller amounts than were obtained when the same basal medium had been fortified with acetate and butyrate at pH 7.

Stimulation of minor fetal hemoglobin synthesis in cord blood reticulocytes by butyrate

As it had been shown that sodium butyrate promoted hyperacetylation of nuclear histones the effect of 5, 10, and 50 mM sodium butyrate on the in vitro synthesis of Hb F Ic (an acetylated component of Hb F) and Hb F in cord blood reticulocytes was studied. The presence of 5 mM butyrate showed a significant increase in the production of Hb F Ic, whereas Hb F synthesis was unaffected. Higher concentrations of butyrate, however, had no stimulatory effect on Hb F Ic synthesis. Results of pulse-chase studies indicated that sodium butyrate inhibited the turnover of preformed Hb F Ic, probably by inhibiting deacetylase.

Effects of corticosteroids on the growth and metabolism of fibroblasts cultured from human skin.

Hydrocortisone, betamethasone 17-valerate, clobetasone butyrate and clobetasol propionate were tested in vitro for effects on the proliferation and metabolism of six strains of fibroblasts from normal human skin. The different cell strains gave similar results: hydrocortisone and betamethasone valerate slightly enhanced growth at all concentrations (0.0001-10 microgram/ml), but high concentrations of clobetasone butyrate and clobetasol propionate significantly reduced proliferation. Secretion of acid mucopolysaccharide was most inhibited by clobetasone butyrate and clobetasol propionate, then by betamethasone valerate, with hydrocortisone having the least effect. Clobetasol propionate and betamethasone valerate at 10 microgram/ml both reduced collagen synthesis by about 50%; other protein synthesis was less affected.