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PubMed2001

Butyrate and the cytokine-induced alpha1-proteinase inhibitor release in intestinal epithelial cells.

BACKGROUND: Alpha1-proteinase inhibitor (alpha1-PI), an anti-inflammatory protein thought to play a role in the intestinal inflammation, is synthesised by and released from the intestinal epithelial cells. IL-1beta is a key proinflammatory cytokine in the abnormal immune response that occurs in inflammatory bowel disease. Butyrate is a normal luminal constituent in the colon, known to be of benefit in preventing inflammatory bowel disease. Direct modes of action of butyrate in intestinal inflammation have been poorly studied so far. The aim of this study was to investigate the effects of butyrate on cytokine-mediated alpha1-PI release in intestinal epithelial cells. METHODS: Differentiated Caco-2 cells were incubated with IL-1beta in the presence or absence of 2 mM butyrate. Alpha1-PI expression in the cells was evaluated by Western blot analysis and alpha1-PI release by ELISA. RESULTS: Treatment with butyrate alone had no effect on alpha1-PI expression in differentiated Caco-2 cells. However, treatment of the cells with 2 mM butyrate significantly reduced the alpha1-PI level in IL-1beta-treated cells. In the cell culture medium, the presence of butyrate impaired the IL-1beta-induced alpha1-PI release to 17-35%. The treatment induced no change in the number of detached cells or the percentage of viable cells. CONCLUSION: Our data show that butyrate inhibits alpha1-PI release from Caco-2 colonocytes treated with IL-1beta. It is therefore likely that anti-inflammatory actions of butyrate occur via a mechanism that does not involve direct regulation of cytokine-induced anti-inflammatory protein expression in intestinal epithelial cells.

Butyrates↗
PubMed2002

Colonic epithelial atrophy induced by a fibre-free diet in rats is reversed by minimal amounts of luminal butyrate, but only in the short term.

BACKGROUND: Luminal butyrate may be trophic to the colonic epithelium, but this effect is poorly characterized. The aim of the present study was to define the dose-response, time-course, site-specificity and the dependence on background diet of the effects of butyrate on epithelial proliferation in normal distal colon, using an in vivo rat model of colonic substrate delivery. METHODS: Male Sprague-Dawley rats, maintained on a fibre-free diet, had butyrate infused twice daily into the colonic lumen via polyethylene tubes placed at laparotomy. Varying dose levels (0-80 micro mol/d; 4 d), site (caecal vs distal colonic), duration of infusions (1-5 weeks; 80 micro mol/d), or dietary fibre intake were investigated. Epithelial proliferative indices were assessed stathmokinetically. RESULTS: Four-day infusions of butyrate led to a progressive trophic effect (cells/crypt column increased from 37.9 +/- 1.6 at 0 micro mol/d to 44.7 +/- 1.2 at 80 micro mol/d) on fibre-deprived colonic mucosa, related linearly to the daily butyrate dose (P < 0.001, linear regression). This effect was mediated by increases in the number and proportion of mitoses, related to the square of the butyrate dose (P < 0.001 in each case, polynomial regression). Butyrate (80 micro mol/d) was associated with significantly higher cellularity (59.9 +/- 1.4) and mitotic activity (4.9 +/- 0.6) per crypt column compared to vehicle controls (50.3 +/- 1.6 and 0.9 +/- 0.2, respectively; P < 0.05, t-tests), at 1 and 3 weeks, but not at 5 weeks. Butyrate had similar effects on distal colonic crypt cellularity (62.0 +/- 1.5) when delivered caecally, but in rats fed a fibre-containing diet, colonic crypt cellularity (55.3 +/- 3.2) was similar to baseline (59.6 +/- 1.9). CONCLUSIONS: Trophic effects of butyrate are concentration-dependent and occur at low doses in the short term, but are not sustained over longer periods. They are seen only in a fibre-deprived state and appear to be independent of the site of administration.

Animals↗
PubMed2001

Resistance to butyrate-induced cell differentiation and apoptosis during spontaneous Caco-2 cell differentiation.

BACKGROUND & AIMS: The short-chain fatty acid butyrate induces cell cycle arrest, differentiation, and apoptosis in colon cancer cells, but often induces opposite effects in normal colonic epithelial cells. We determined whether response to butyrate is dependent on the basal differentiation status of colonic epithelial cells. METHODS: Caco-2 cells at progressive stages of differentiation were treated with butyrate, and endpoints were measured. RESULTS: Response of Caco-2 cells to butyrate was dependent on their differentiation status. Butyrate maximally stimulated cell cycle arrest, apoptosis, alkaline phosphatase activity, transepithelial resistance, cell migration, urokinase receptor expression, and interleukin 8 secretion in undifferentiated Caco-2 cells, whereas differentiated Caco-2 cells were essentially resistant to these effects. Consistently, butyrate selectively induced histone hyperacetylation in undifferentiated Caco-2 cells. This resistance was also observed during HT29cl.19A cell differentiation, but not in the nondifferentiating SW620 cell line. Finally, the rate of butyrate use significantly increased as Caco-2 cells underwent spontaneous differentiation. CONCLUSIONS: Colonic epithelial cells become progressively more refractory to the effects of butyrate during absorptive cell differentiation. We postulate that this resistance is caused by the rapid use of butyrate by differentiated Caco-2 cells, which likely results in low intracellular concentrations and subsequently in its inability to inhibit histone deacetylase.

Acetylation↗
PubMed2001

Butyrate suppression of colonocyte NF-kappa B activation and cellular proteasome activity.

Butyrate is derived from the microbial metabolism of dietary fiber in the colon where it plays an important role in linking colonocyte turnover and differentiation to luminal content. In addition, butyrate appears to have both anti-inflammatory and cancer chemopreventive activities. Using confocal microscopy and cell fractionation studies, butyrate pretreatment of a human colon cell line (HT-29 cells) inhibited the tumor necrosis factor-alpha (TNF-alpha)-induced nuclear translocation of the proinflammatory transcription factor NF-kappaB. Butyrate inhibited NF-kappaB DNA binding within 30 min of TNF-alpha stimulation, consistent with an inhibition of nuclear translocation. IkappaB.NF-kappaB complexes extracted from butyrate-treated cells were relatively resistant to in vitro dissociation by deoxycholate, suggesting a change in cellular IkappaB composition. Butyrate treatment increased p100 expression, an IkappaB that was not degraded upon TNF-alpha treatment. Butyrate also reduced the extent of TNF-alpha-induced IkappaB-alpha degradation and enhanced the presence of ubiquitin-conjugated IkappaB-alpha. The suppression of IkappaB-alpha degradation corresponded with a reduction in cellular proteasome activity as determined by in vitro proteasome assays and the increased presence of ubiquitin-conjugated proteins. The butyrate suppression of IkappaB-alpha degradation and proteasome activity may derive from its ability to inhibit histone deacetylases since the specific deacetylase inhibitor trichostatin A had similar effects. These results suggest a potential mechanism for the anti-inflammatory activity of butyrate and demonstrate the interplay between short chain fatty acids and cellular proteasome activity.

Active Transport, Cell Nucleus↗
PubMed2002

Luminal leptin enhances CD147/MCT-1-mediated uptake of butyrate in the human intestinal cell line Caco2-BBE.

In the intestine, butyrate constitutes the major energy fuel for colonocytes. However, little is known about the transport of butyrate and its regulation in the intestine. In this study we demonstrate that the monocarboxylate transporter (MCT-1) is apically polarized in model human intestinal epithelia and is involved in butyrate uptake by Caco2-BBE cell monolayers. The butyrate uptake by Caco2-BBE cell monolayers displayed conventional Michaelis-Menten kinetics and was found to be pH-dependent, Na(+)-independent, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-insensitive, and inhibited by the monocarboxylate transporter inhibitor alpha-cyano-4-hydroxycinnamate and by an excess of unlabeled butyrate. We show that MCT-1 associates with CD147 at the apical plasma membrane in Caco2-BBE cell monolayers. Using antisense CD147, we demonstrate that the association of CD147 with MCT-1 is critical for the butyrate transport activity. Interestingly, we show for the first time hormonal regulation of CD147/MCT-1 mediated butyrate uptake. Specifically, luminal leptin significantly up-regulates MCT-1-mediated butyrate uptake by increasing its maximal velocity (V(max)) without any modification in the apparent Michaelis-Menten constant (K(m)). Finally, we show that luminal leptin up-regulates butyrate uptake in Caco2-BBE monolayers by two distinct actions: (i) increase of the intracellular pool of MCT-1 protein without affecting CD147 expression and (ii) translocation of CD147/MCT-1 to the apical plasma membrane of Caco2-BBE cell monolayers.

Antigens, CD↗
PubMed2003

Anti-cancer effects of butyrate: use of micro-array technology to investigate mechanisms.

Epidemiological evidence suggests that a high intake of resistant starch and NSP protects against colo-rectal cancer. The mechanisms underlying this protection are thought to be mediated by the short-chain fatty acid butyrate, which is present in the colonic lumen in millimolar concentrations as a result of bacterial fermentation of carbohydrates that have resisted digestion in the small intestine. In vitro studies have shown that butyrate displays a host of chemo-preventative properties including increased apoptosis, reduced proliferation, down regulation of angiogenesis, enhanced immunosurveillance and anti-inflammatory effects in colo-rectal cancer cell lines. However, the molecular mechanisms underlying the apparent chemo-preventative actions of butyrate are largely unknown. The evidence supporting the role of butyrate as an anti-cancer agent is reviewed, with particular emphasis on those studies that have attempted to elucidate the mechanism of action of butyrate. Our understanding of the mechanistic action of butyrate and its role in cancer prevention is likely to advance considerably in this post-genomic era with the application of genomic and proteomic technologies. Studies are described that have used gene array and proteomic techniques to investigate the response of colo-rectal cancer cells to butyrate. These pioneering studies illustrate the potential of these technologies to help characterise the molecular responses of the cancer cell to butyrate, and to define the role of butyrate (and other nutrients) in the prevention of colo-rectal cancer.

Animals↗
PubMed2001

Butyrate induces glutathione S-transferase in human colon cells and protects from genetic damage by 4-hydroxy-2-nonenal.

Butyrate, one of the major products of gut fermentation, is known to inhibit proliferation, induce apoptosis and differentiation, and increase phase II enzyme activities in tumor cells, whereas little information is available on protective effects in less-transformed colon cells. The aim of this study was to investigate whether the chemoprotective mechanism of glutathione S-transferase (GST) induction by butyrate could also play a role in earlier stages of colon carcinogenesis and whether chemoresistance of cells toward the endogenous genotoxic risk factor 4-hydroxy-2-nonenal (HNE) could be a consequence of butyrate treatment. As cell models, we used the human tumor cell lines HT29 and HT29 clone 19A, a differentiated subclone with properties resembling primary colon cells. We determined the expression of GSTP1 protein (enzyme-linked immunosorbent assay), the major GST in HT29, GSTP1 mRNA (Northern blotting), GST activity, intracellular glutathione, and total protein. The genotoxic impact of HNE (100-200 microM) was compared in butyrate-treated and nontreated cells using single-cell microgel electrophoresis. Our results show that GSTP1 mRNA, GSTP1 protein, GST activity, and total protein were increased (1.2- to 2.5-fold) and glutathione levels were maintained after 24-72 h of incubation with 4 mM butyrate. Moreover, a marked reduction of HNE-induced genotoxicity was caused by preincubation with butyrate. Butyrate also induced the phosphorylation of extracellular signal-regulated kinases (ERK1/2, Western blotting) after 5-30 min, which indicates a regulation of GST expression by this signal pathway. Most effects were greater in HT29 parent cells than in clone cells. In conclusion, butyrate enhances expression of GST and other proteins in both cell lines, which leads to an enhanced chemoprotection, reducing the impact of HNE genotoxicity. Thus butyrate could play a role in early and later stages of cancer prevention by reducing exposure to relevant risk factors.

Aldehydes↗
PubMed2005

Intraruminal infusion of n-butyric acid induces an increase of ruminal papillae size independent of IGF-1 system in castrated bulls.

The objective of this study was to explore morphological alterations of rumen papillae induced by n-butyric acid in relation to the insulin-like growth factor (IGF) system in adult castrated bulls. Three animals fitted with rumen cannula were fed twice daily at a low and high nutritional level (LL and HL), i.e., at 1.1 x maintenance (M) and 1.6 x M, respectively. Diets contained artificial dried grass and concentrate (74:26 and 52:48). Bulls received no (B0) or daily intraruminal infusions of 500 g n-butyric acid (B500) over 14 d. The infusion started 1 h after the morning feeding (9:00) and lasted for 3.5 h. Thus, four treatments (BOLL, B500LL, BOHL, and B500HL) were compared. Blood and rumen mucosa samples from the atrium ruminis were taken at the last day of each period. Length, width and surface of rumen papillae were greater (p < 0.001) in BOHL than in BOLL. Treatment with n-butyric acid resulted in an increase of the papillae surface of 20-40% (p = 0.047) for both nutritional levels as compared to periods without n-butyric acid treatments. The higher nutritional level and intraruminal n-butyric acid infusion induced epithelial cell death. The percentage of proliferative cells was doubled by n-butyric acid treatment. The mRNA of IGF-1 and IGF type 1 receptor (IGF-1R), as well as IGF-1R binding capacity were unaffected by butyric acid treatments. The abundance of IGF-1 mRNA tended to be lower (p = 0.1) and IGF-1R abundance was lower (p = 0.03) in response to the HL. The plasma IGF-1 concentration was lower with butyric acid treatment (p < 0.01), but was unaffected by the nutritional level. In conclusion, under described experimental preconditions of daily short-time intraruminal n-butyric acid infusion alterations of rumen papillae morphology is not mediated by ruminal IGF type 1 receptor and by local IGF-1 expression in papillae in castrated bulls.

Animal Nutritional Physiological Phenomena↗
PubMed2001

Protective activity of butyrate on hydrogen peroxide-induced DNA damage in isolated human colonocytes and HT29 tumour cells.

Epidemiological studies support the involvement of short-chain fatty acids (SCFA) in colon physiology and the protective role of butyrate on colon carcinogenesis. Among the possible mechanisms by which butyrate may exert its anti-carcinogenicity an antioxidant activity has been recently suggested. We investigated the effects of butyrate and mixtures of SCFA (butyrate, propionate and acetate) on DNA damage induced by H(2)O(2) in isolated human colonocytes and in two human colon tumour cell lines (HT29 and HT29 19A). Human colonocytes were isolated from endoscopically obtained samples and the DNA damage was assessed by the comet assay. H(2)O(2) induced DNA damage in normal colonocytes in a dose-dependent manner which was statistically significant at concentrations over 10 microM. At 15 microM H(2)O(2) DNA damage in HT29 and HT29 19A cells was significantly lower than that observed in normal colonocytes (P < 0.01). Pre-incubation of the cells with physiological concentrations of butyrate (6.25 and 12.5 mM) reduced H(2)O(2) (15 microM) induced damage by 33 and 51% in human colonocytes, 45 and 75% in HT29 and 30 and 80% in HT29 19A, respectively. Treatment of cells with a mixture of 25 mM acetate + 10.4 mM propionate + 6.25 mM butyrate did not induce DNA damage, while a mixture of 50 mM acetate + 20.8 mM propionate + 12.5 mM butyrate was weakly genotoxic only towards normal colonocytes. However, both mixtures were able to reduce the H(2)O(2)-induced DNA damage by about 50% in all cell types. The reported protective effect of butyrate might be important in pathogenetic mechanisms mediated by reactive oxygen species, and aids understanding of the apparent protection toward colorectal cancer exerted by dietary fibres, which enhance the butyrate bioavailability in the colonic mucosa.

Biopsy↗
PubMed2005

Butyrate may enhance toxicological defence in primary, adenoma and tumor human colon cells by favourably modulating expression of glutathione S-transferases genes, an approach in nutrigenomics.

Butyrate, formed by bacterial fermentation of plant foods, has been suggested to reduce colon cancer risks by suppressing the proliferation of tumor cells. In addition, butyrate has been shown to induce glutathione S-transferases (GSTs) in tumor cell lines, which may contribute to the detoxification of dietary carcinogens. We hypothesize that butyrate also affects biotransformation in non-transformed colon cells. Thus, we have investigated the gene expression of drug metabolism genes in primary human colon tissue, premalignant LT97 adenoma and HT29 tumor cells cultured in an appropriate medium+/-butyrate. A total of 96 drug metabolism genes (including 12 GSTs) spotted on cDNA macroarrays (Superarray; n = 3) were hybridized with biotin-labeled cDNA probes. To validate the expression detected with Superarray, samples of LT97 cells were also analyzed with high density microarrays (Affymetrix U133A), which include biotransformation genes that overlap with the set of genes represented on the Superarray. Relative expression levels were compared across colon samples and for each colon sample+/-butyrate. Compared with fresh tissue, 13 genes were downregulated in primary cells cultivated ex vivo, whereas 8 genes were upregulated. Several genes were less expressed in LT97 (40 genes) or in HT29 (41 and 17 genes, grown for 72 and 48 h, respectively) compared with primary colon tissue. Butyrate induced GSTP1, GSTM2, and GSTA4 in HT29 as previously confirmed by other methods (northern blot/qPCR). We detected an upregulation of GSTs (GSTA2, GSTT2) that are known to be involved in the defence against oxidative stress in primary cells upon incubation with butyrate. The changes in expression detected in LT97 by Superarray and Affymetrix were similar, confirming the validity of the results. We conclude that low GST expression levels were favourably altered by butyrate. An induction of the toxicological defence system possibly contributes to reported chemopreventive properties of butyrate, a product of dietary fibre fermentation in the gut.

Adenoma↗
PubMed2003

Inhibition of histone deacetylase activity by butyrate.

This article reviews the effects of the short-chain fatty acid butyrate on histone deacetylase (HDAC) activity. Sodium butyrate has multiple effects on cultured mammalian cells that include inhibition of proliferation, induction of differentiation and induction or repression of gene expression. The observation that butyrate treatment of cells results in histone hyperacetylation initiated a flurry of activity that led to the discovery that butyrate inhibits HDAC activity. Butyrate has been an essential agent for determining the role of histone acetylation in chromatin structure and function. Interestingly, inhibition of HDAC activity affects the expression of only 2% of mammalian genes. Promoters of butyrate-responsive genes have butyrate response elements, and the action of butyrate is often mediated through Sp1/Sp3 binding sites (e.g., p21(Waf1/Cip1)). We demonstrated that Sp1 and Sp3 recruit HDAC1 and HDAC2, with the latter being phosphorylated by protein kinase CK2. A model is proposed in which inhibition of Sp1/Sp3-associated HDAC activity leads to histone hyperacetylation and transcriptional activation of the p21(Waf1/Cip1) gene; p21(Waf1/Cip1) inhibits cyclin-dependent kinase 2 activity and thereby arrests cell cycling. Pending the cell background, the nonproliferating cells may enter differentiation or apoptotic pathways. The potential of butyrate and HDAC inhibitors in the prevention and treatment of cancer is presented.

Acetyltransferases↗
PubMed2005

Prolonged butyrate treatment inhibits the migration and invasion potential of HT1080 tumor cells.

Butyrate, a normal constituent of the colonic luminal contents, is produced by the bacterial fermentation of dietary fibers and resistant starches. It has been hypothesized that butyrate may inhibit the invasion of tumor cells. The purpose of the present study was to investigate the effects of butyrate treatment on the growth, migration, and invasion characteristics of tumor HT1080 cells. HT1080 cells cultured in the presence of 0.5 and 1 mmol/L butyrate for 14 d exhibited an increase in the G(1) and G(2) fractions with a concomitant drop in the S-phase, thus showing slower cell growth. Interestingly, 0.5 and 1 mmol/L butyrate inhibited the migration and invasion rate of the tumor cells compared with the untreated (control) cells. The protein and mRNA levels of the tissue inhibitors of metalloproteinase-1 (TIMP-1) and TIMP-2 were significantly increased in HT1080 cells cultured with 0.5 and 1 mmol/L butyrate. Enzymatic activities and the mRNA level of the latent forms of matrix metalloproteinase (MMP), pro-MMP-2 and pro-MMP-9, were also increased in HT1080 cells cultured with 0.5 and 1 mmol/L butyrate. In contrast, the active form of MMP-2 was detectable by zymographic analysis in control but not butyrate-conditioned media. Collectively, these results demonstrate that prolonged and low-dose butyrate treatment increases both prometastasis MMP-2, -9 and antimetastasis TIMP-1, -2 expression, and the net effect of these increases is the inhibition of pro-MMP-2 activation and of tumor cell migration/invasion potential.

Butyrates↗
PubMed2001

Therapeutic effects of sodium butyrate on glioma cells in vitro and in the rat C6 glioma model.

OBJECTIVE: Preliminary in vitro studies have indicated that sodium butyrate inhibits the proliferation of cultured glioma cells and induces cellular differentiation, making it potentially useful as a therapeutic agent for patients with glioblastoma multiforme. The purpose of this study was to expand on the preliminary research by investigating the effects of sodium butyrate on multiple cell lines, explanted cells from glioblastoma tumor specimens, and in vivo in the rat C6 glioma brain tumor model. METHODS: Four malignant glioma cell lines (A-172, T98G, U118MG, and C6) and two primary cell cultures derived from human glioblastoma tumor specimens were treated with 2 mmol/L sodium butyrate for up to 72 hours. Sodium butyrate-induced effects on cell morphology, proliferation, cell cycle distribution, migration, glial fibrillary acidic protein staining, and S100beta protein content were determined. For in vivo studies, a total of 64 male Wistar-Furth rats underwent operations to implant C6 glioma cells stereotactically or were used as controls. The rats were treated with escalating doses of sodium butyrate by microinfusion with Alzet minipumps (Durect Corp., Cupertino, CA). RESULTS: Sodium butyrate treatment in vitro produced changes in morphology and glial fibrillary acidic protein expression indicative of cellular differentiation. In cell lines and explanted cells, sodium butyrate consistently inhibited glioblastoma cell proliferation (to 51 +/- 6% that of controls) and migration (to 46 +/- 17%). Intratumoral infusion of 40 mmol/L sodium butyrate prolonged the survival of Wistar-Furth rats with intracerebral C6 tumors (P = 0.013) without detectable toxicity. CONCLUSION: These data support further consideration of direct interstitial infusion of sodium butyrate in a Phase I clinical study for patients with recurrent glioblastoma multiforme.

Animals↗
PubMed2000

Phylogenetic relationships of butyrate-producing bacteria from the human gut.

Butyrate is a preferred energy source for colonic epithelial cells and is thought to play an important role in maintaining colonic health in humans. In order to investigate the diversity and stability of butyrate-producing organisms of the colonic flora, anaerobic butyrate-producing bacteria were isolated from freshly voided human fecal samples from three healthy individuals: an infant, an adult omnivore, and an adult vegetarian. A second isolation was performed on the same three individuals 1 year later. Of a total of 313 bacterial isolates, 74 produced more than 2 mM butyrate in vitro. Butyrate-producing isolates were grouped by 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism analysis. The results indicate very little overlap between the predominant ribotypes of the three subjects; furthermore, the flora of each individual changed significantly between the two isolations. Complete sequences of 16S rDNAs were determined for 24 representative strains and subjected to phylogenetic analysis. Eighty percent of the butyrate-producing isolates fell within the XIVa cluster of gram-positive bacteria as defined by M. D. Collins et al. (Int. J. Syst. Bacteriol. 44:812-826, 1994) and A. Willems et al. (Int. J. Syst. Bacteriol. 46:195-199, 1996), with the most abundant group (10 of 24 or 42%) clustering with Eubacterium rectale, Eubacterium ramulus, and Roseburia cecicola. Fifty percent of the butyrate-producing isolates were net acetate consumers during growth, suggesting that they employ the butyryl coenzyme A-acetyl coenzyme A transferase pathway for butyrate production. In contrast, only 1% of the 239 non-butyrate-producing isolates consumed acetate.

Adult↗
PubMed1997

Butyric acid-induced apoptosis of murine thymocytes, splenic T cells, and human Jurkat T cells.

The purpose of this study was to examine the effect of butyric acid, an extracellular metabolite from periodontopathic bacteria, on apoptosis induction in murine thymocytes, splenic T cells, and human Jurkat T cells. Butyric acid significantly suppressed T-cell viability in both a concentration- and time-dependent fashion. The results of DNA fragmentation assay indicated that butyric acid rapidly induced apoptosis in thymocytes (with 1.25 mM butyric acid and 6 h after treatment) and in splenic T cells and Jurkat cells (with 2.5 mM butyric acid and 16 h after treatment). Incubation of thymocytes or Jurkat cells with 5 mM butyric acid for 21 h resulted in the typical ladder pattern of DNA fragmentation. Furthermore, Jurkat cells treated with 5 mM butyric acid showed the characteristic pattern of apoptotic cells such as chromatin condensation and hypodiploid nuclei. Experiments with fractionated subpopulations of splenic T cells revealed that DNA fragmentation was predominantly observed in CD4+ T cells. Butyric acid-induced apoptosis of thymocytes was decreased by the protein kinase inhibitors H7 and staurosporine. These inhibitors were less effective with similarly treated splenic T cells and Jurkat cells. These data suggest that butyric acid, one of the volatile fatty acids produced by periodontopathic bacteria and one that easily penetrates the oral mucosa, can modulate the immunoregulatory cell population in periodontal tissue by inducing T-cell death through apoptosis.

Animals↗
PubMed1999

Lipopolysaccharide stimulates butyric acid-induced apoptosis in human peripheral blood mononuclear cells.

We previously reported that butyric acid, an extracellular metabolite from periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we examined the ability of butyric acid to induce apoptosis in peripheral blood mononuclear cells (PBMC) and the effect of bacterial lipopolysaccharide (LPS) on this apoptosis. Butyric acid significantly inhibited the anti-CD3 monoclonal antibody- and concanavalin A-induced proliferative responses in a dose-dependent fashion. This inhibition of PBMC growth by butyric acid depended on apoptosis in vitro. It was characterized by internucleosomal DNA digestion and revealed by gel electrophoresis followed by a colorimetric DNA fragmentation assay to occur in a concentration-dependent fashion. Butyric acid-induced PBMC apoptosis was accompanied by caspase-3 protease activity but not by caspase-1 protease activity. LPS potentiated butyric acid-induced PBMC apoptosis in a dose-dependent manner. Flow-cytometric analysis revealed that LPS increased the proportion of sub-G1 cells and the number of late-stage apoptotic cells induced by butyric acid. Annexin V binding experiments with fractionated subpopulations of PBMC in flow cytometory revealed that LPS accelerated the butyric acid-induced CD3(+)-T-cell apoptosis followed by similar levels of both CD4(+)- and CD8(+)-T-cell apoptosis. The addition of LPS to PBMC cultures did not cause DNA fragmentation, suggesting that LPS was unable to induce PBMC apoptosis directly. These data suggest that LPS, in combination with butyric acid, potentiates CD3(+) PBMC T-cell apoptosis and plays a role in the apoptotic depletion of CD4(+) and CD8(+) cells.

Adult↗
PubMed2003

Regulation of butyrate uptake in Caco-2 cells by phorbol 12-myristate 13-acetate.

Butyrate and the other short-chain fatty acids (SCFAs) are the most abundant anions in the colonic lumen. Also, butyrate is the preferred energy source for colonocytes and has been shown to regulate colonic electrolyte and fluid absorption. Previous studies from our group have demonstrated that the HCO(3)(-)/SCFA(-) anion exchange process is one of the major mechanisms of butyrate transport across the purified human colonic apical membrane vesicles and the apical membrane of human colonic adenocarcinoma cell line Caco-2 and have suggested that it is mainly mediated via monocarboxylate transporter-1 (MCT-1) isoform. However, little is known regarding the regulation of SCFA transport by various hormones and signal transduction pathways. Therefore, the present studies were undertaken to examine whether hydrocortisone and phorbol 12-myristate 13-acetate (PMA) are involved in a possible regulation of the butyrate/anion exchange process in Caco-2 cells. The butyrate/anion exchange process was assessed by measuring a pH-driven [(14)C]butyrate uptake in Caco-2 cells. Our results demonstrated that 24-h incubation with PMA (1 microM) significantly increased [(14)C]butyrate uptake compared with incubation with 4alphaPMA (inactive form). In contrast, incubation with hydrocortisone had no significant effect on butyrate uptake in Caco-2 cells compared with vehicle (ethanol) alone. Induction of butyrate uptake by PMA appeared to be via an increase in the maximum velocity (V(max)) of the transport process with no significant changes in the K(m) of the transporter for butyrate. Parallel to the increase in the V(max) of [(14)C]butyrate uptake, the MCT-1 protein level was also increased in response to PMA incubation. Our studies demonstrated that the butyrate/anion exchange was increased in response to PMA treatment along with the induction in the level of MCT-1 expression in Caco-2 cells.

Adenocarcinoma↗
PubMed2004

Butyrate specifically modulates MUC gene expression in intestinal epithelial goblet cells deprived of glucose.

The mucus layer covering the gastrointestinal mucosa is considered the first line of defense against aggressions arising from the luminal content. It is mainly composed of high molecular weight glycoproteins called mucins. Butyrate, a short-chain fatty acid produced during carbohydrate fermentation, has been shown to increase mucin secretion. The aim of this study was to test 1) whether butyrate regulates the expression of various MUC genes, which are coding for protein backbones of mucins, and 2) whether this effect depends on butyrate status as the major energy source of colonocytes. Butyrate was provided at the apical side of human polarized colonic goblet cell line HT29-Cl.16E in glucose-rich or glucose-deprived medium. In glucose-rich medium, butyrate significantly increased MUC3 and MUC5B expression (1.6-fold basal level for both genes), tended to decrease MUC5AC expression, and had no effect on MUC2 expression. In glucose-deprived medium, i.e., when butyrate was the only energy source available, MUC3 and MUC5B increase persisted, whereas MUC5AC expression was significantly enhanced (3.7-fold basal level) and MUC2 expression was strikingly increased (23-fold basal level). Together, our findings show that butyrate is able to upregulate colonic mucins at the transcriptional level and even better when it is the major energy source of the cells. Thus the metabolism of butyrate in colonocytes is closely linked to some of its gene-regulating effects. The distinct effects of butyrate according to the different MUC genes could influence the composition and properties of the mucus gel and thus its protective function.

Butyrates↗