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Down-Regulation of the Monocarboxylate Transporter 1 Is Involved in Butyrate Deficiency During Intestinal Inflammation
Abstract
Butyrate oxidation is impaired in intestinal mucosa of patients with inflammatory bowel diseases (IBD). Butyrate uptake by colonocytes involves the monocarboxylate transporter (MCT) 1. We aimed to investigate the role of MCT1 in butyrate oxidation deficiency during colonic inflammation.
Colonic tissues were collected from patients with IBD or healthy controls and from rats with dextran sulfate sodium (DSS)-induced colitis. The intestinal epithelial cell line HT-29 was treated with interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). MCT1 expression was analyzed by real-time reverse-transcription polymerase chain reaction, Western blot, and immunofluorescence. Butyrate uptake and oxidation in HT-29 cells was assessed using [(14)C]-butyrate. The mechanism of MCT1 gene regulation was analyzed by nuclear run-on and reporter gene assays.
MCT1 messenger RNA (mRNA) and protein levels were markedly decreased in inflamed colonic mucosa of IBD patients and rats. In HT-29 cells, down-regulation of MCT1 mRNA and protein abundance by IFN-gamma and TNF-alpha correlated with a decrease in butyrate uptake and subsequent oxidation. IFN-gamma and TNF-alpha did not affect MCT1 mRNA stability but rather down-regulated gene transcription. We demonstrate that the cytokine response element is located in the proximal -111/+213 core region of the MCT1 promoter.
The data suggest that butyrate oxidation deficiency in intestinal inflammation is a consequence of reduction in MCT1-mediated butyrate uptake. This reinforces the proposition that butyrate oxidation deficiency in IBD is not a primary defect.
... Although precise mechanisms are not known, including the recycling mechanism and protein trafficking systems, calcineurin has been suggested to regulate cAMP degradation and affect cytoskeletal function [33,44,45]. In IBD patients, intestinal MCT1 protein expression is downregulated [46]. This is considered to be due to a decrease in butyrate-producing bacteria and inflammation itself. ...
... This is considered to be due to a decrease in butyrate-producing bacteria and inflammation itself. Interferon γ (IFN-γ) and TNF-α signaling downregulate the expression of MCT1 in HT-29 cells [46]. In pig colonic tissue and Caco-2 cells, TNF-α downregulated MCT1 expression [47]. ...
... Butyrate enhances mucosal barrier function; however, the barrier is disrupted during active inflammation [58]. The expression of MCT1 in the colon is reportedly decreased in patients with IBD and experimental colitis models [46,47,59]. In patients with active IBD, butyrate utilization and uptake are impaired (Figure 2). ...
The pathogenesis and refractory nature of inflammatory bowel disease (IBD) are related to multiple factors, including genetic factors, environmental factors, and abnormalities in gut microbial diversity, which lead to decreased levels of short-chain fatty acids (SCFAs). Among SCFAs, butyrate plays an important role in mucosal barrier maintenance, serves as an energy source in intestinal epithelial cells (IECs), and exhibits anti-inflammatory effects; therefore, it is a particularly important factor in gut homeostasis. Changes in gut microbiota and butyrate levels affect the outcomes of drug therapy for IBD. Butyrate is mainly absorbed in the large intestine and is transported by monocarboxylate transporter 1 (MCT1) and sodium-coupled monocarboxylate transporter 1 (SMCT1). During gut inflammation, butyrate utilization and uptake are impaired in IECs. Dysbiosis and low abundance of butyrate affect fecal microbiota transplantation and anticancer immunotherapy. Although butyrate administration has been reported as a treatment for IBD, its effects remain controversial. In this review, we discuss butyrate absorption and metabolism in patients with IBD and their relationship with drug therapy.
... The effect of CD inflammation on the expression of DMETs has not been extensively studied. In total, we found 13 publications that measured DMETs from CD patients in inflamed and [98,101,[105][106][107][108]. In addition, a case report study on a duodenum biopsy reported an increase in p-gp expression of greater than twofold in a CD patient, as compared with healthy specimens [109]. ...
... The gold standard for measuring absolute abundances of proteins in complex mixtures is now generally considered to be tandem mass spectrometry (LC/MS-MS) of protein digests, and in no case has such an analysis been reported. Six of the included publications had healthy volunteers as the control group [97,98,100,103,106,107], while the rest had non-IBD patients as the control group [99,105,108]. DMET abundances in the ileum, which is known to have a more dominant role in oral drugs than the colon, were reported in six studies. ...
... The expression of absorption, distribution, metabolism and excretion (ADME) proteins was reported in samples that were not differentiated for being in an active or inactive state of the disease [100,105], with an indication that the active state does not affect the expression of CYP3A4 and p-gp [100]. The degree of change of expression from normal was shown to be proportional to the tissue inflammation [103,105,106]. ...
Backgrond and Objective
Crohn’s disease (CD) is a chronic inflammatory bowel disease that affects a wide age range. Hence, CD patients receive a variety of drugs over their life beyond those used for CD itself. The changes to the integrity of the intestine and its drug metabolising enzymes and transporters (DMETs) can alter the oral bioavailability of drugs. However, there are other changes in systems parameters determining the fate of drugs in CD, and understanding these is essential for dose adjustment in patients with CD.Methods
The current analysis gathered all the available clinical data on the kinetics of drugs in CD (by March 2021), focusing on orally administered small molecule drugs. A meta-analysis of the systems parameters affecting oral drug pharmacokinetics was conducted. The systems information gathered on intestine, liver and blood proteins and other physiological parameters was incorporated into a physiologically based pharmacokinetic (PBPK) platform to create a virtual population of CD patients, with a view for guiding dose adjustment in the absence of clinical data in CD.ResultsThere were no uniform trends in the reported changes in reported oral bioavailability. The nature of the drug as well as the formulation affected the direction and magnitude of variation in kinetics in CD patients relative to healthy volunteers. Even for the same drug, the reported changes in exposure varied, possibly due to a lack of distinction between the activity states of CD. The highest alteration was seen with S-verapamil and midazolam, 8.7- and 5.3-fold greater exposure, respectively, in active CD patients relative to healthy volunteers. Only one report was available on liver DMETs in CD, and indicated reduced CYP3A4 activity. In a number of reports, mRNA expression of DMETs in the ileum and colon of CD patients was measured, focussing on P-glycoprotein (p-gp) transporter and CYP3A4 enzyme, and showed contradictory results. No data were available on protein expression in duodenum and jejunum despite their dominant role in oral drug absorption.Conclusion
There are currently inadequate dedicated clinical or quantitative proteomic studies in CD to enable predictive PBPK models with high confidence and adequate verification. The PBPK models for CD with the available systems parameters were able to capture the major physiological influencers and the gaps to be filled by future research. Quantification of DMETs in the intestine and the liver in CD is warranted, alongside well-defined clinical drug disposition studies with a number of index drugs as biomarkers of changes in DMETs in these patients, to avoid large-scale dedicated studies for every drug to determine the effects of disease on the drug’s metabolism and disposition and the consequential safety and therapeutic concerns.
... The available data on the protein expression of DTPs and DMEs in the colon of UC patients are limited. Most information is based on either mRNA data with limited correlation to actual protein expression, or semiquantitative Western Blot protein expression data (Blokzijl et al., 2007;Foley et al., 2021;Englund et al., 2007;Erdmann et al., 2019; Thibault et al., 2007). Moreover, most of these studies focus on a limited number of DTPs or DMEs comparing healthy subjects with UC patients in remission or with active inflammation. ...
... Another example is the decrease in butyrate uptake by the colonocytes due to the reduced MCT1 transporter expression. Butyrate is an important energy source for the colonocytes and promotes the production of anti-inflammatory cytokines, thereby protecting the cells against inflammation (Thibault et al., 2007;Salvi and Cowles, 2021). Lastly, MRP4 and P-gp are involved in the signaling pathways for cell survival and apoptosis (Johnstone et al., 2000;Wen et al., 2015). ...
The intestinal tract forms an important barrier against xenobiotics while allowing nutrients to pass. In ulcerative colitis (UC), a chronic inflammatory bowel disease, this barrier function is impaired leading to an abnormal immune response and inflammation of the colonic mucosa. Transporter proteins and metabolic enzymes are an integral part of the protective barrier in the gut and play an important role in the disposition of nutrients toxins and oral drugs. In this study, the protein expression of 13 transporters and 13 enzymes was determined in the sigmoid and rectum of UC patients in endoscopic remission and during active inflammation. In inflamed conditions (endoscopic Mayo sub-score 1, 2 or 3), a significant decrease (q<0.05) was observed in the median expression of the transporters P-gp (0.046 vs. 0.529 fmol/µg protein), MRP4 (0.003 vs. 0.023 fmol/µg protein) and MCT1 (0.287 vs. 1.090 fmol/µg protein), and the enzymes CYP3A5 (0.031 vs. 0.046 fmol/µg protein) and UGT2B7 (0.083 vs 0.176 fmol/µg protein). Moreover, during severe inflammation, the decrease was even more pronounced. Expression levels of other proteins were not altered during inflammation (e.g., OATP2B1, CYP3A4, CYP2B6 and UGT2B15). The results suggest a decreased transport and metabolism of xenobiotics in the colon of UC patients during active inflammation potentially altering local drug concentrations and thus treatment outcome.
... Increased dietary ber intake and SCFA are also associated with anti-in ammatory effects [3]. Of interest, BT utilization is impaired in the intestinal in amed mucosa of IBD patients [19] and one of the mechanisms involved consists in a defect in epithelial BT uptake [20], resulting in higher luminal concentrations of this SCFA. ...
... BT regulates in ammation by acting through two distinct mechanisms: (1) it interacts with cell membrane G protein-coupled receptors (GPR41, GPR43 and GPR109A) expressed in gut epithelium and immune cells, regulating downstream cell signal transduction mechanisms such as NF-κB pathway, MAPK and Ca 2+ , and (2) after entering colonic epithelial cells through transport proteins (such as MCT1 and SMCT1), it inhibits histone deacetylase [39][40][41]. Interestingly, a defect in BT epithelial uptake was described in IBD [19,20], resulting in higher luminal concentrations of this compound. So, we decided to evaluate if BT interferes with BA uptake, as a mechanism contributing to its anti-in ammatory effect. ...
Inflammatory bowel disease (IBD) is a group of chronic and life-threating inflammatory diseases of the gastrointestinal tract. The active intestinal absorption of bile salts is reduced in IBD, resulting in higher luminal concentrations of these agents that contribute to the pathophysiology of IBD-associated diarrhea. Butyrate is a short-chain fatty acid produced by colonic bacterial fermentation of dietary fibers. Butyrate utilization is impaired in the intestinal inflamed mucosa of IBD patients. The aim of this work is to investigate the link between IBD and bile acid absorption, by testing the effect of the pro-inflammatory cytokines TNF-a and IFN-γ and butyrate upon ³ H-taurocholate uptake by Caco-2 cells. The results obtained show that the proinflammatory cytokines TNF-a and IFN-γ inhibit Na ⁺ -independent, non-ASCT-mediated ³ H-TC uptake by Caco-2 cells. The inhibitory effect of these cytokines on ³ H-taurocholate uptake is PI3K- and JAK2-mediated. Moreover, butyrate in high but physiological concentrations was also found to inhibit ³ H-TC uptake and showed an additive effect with IFN-γ in reducing ³ H-taurocholate uptake. So, an interaction between fiber-derived butyrate and inflammatory cytokines appears to exist in relation to bile salt intestinal uptake in IBD, which may participate in the link between fiber intake and IBD.
... Indeed, a reduction in SCFA transportation has been noted in cases of active inflammatory bowel disease and several inflammatory cytokines reduce SCFA transporters in culture. [50][51][52][53] While we did not observe differences in RNA abundances of SLC16A1 (ie, MCT1) in rats with advanced diabetes, that does not preclude changes in protein levels and/ or other mechanisms associated with SCFA absorption and transportation. Overall, it is increasingly clear that hyperglycemia influences gastrointestinal physiology and diet-independent models should be considered to distinguish diabetes-specific mechanisms that influence SCFA uptake and metabolism. ...
Introduction
Colonocyte oxidation of bacterial-derived butyrate has been reported to maintain synergistic obligate anaerobe populations by reducing colonocyte oxygen levels; however, it is not known whether this process is disrupted during the progression of type 2 diabetes. Our aim was to determine whether diabetes influences colonocyte oxygen levels in the University of California Davis type 2 diabetes mellitus (UCD-T2DM) rat model.
Research design and methods
Age-matched male UCD-T2DM rats (174±4 days) prior to the onset of diabetes (PD, n=15), within 1 month post-onset (RD, n=12), and 3 months post-onset (D3M, n=12) were included in this study. Rats were administered an intraperitoneal injection of pimonidazole (60 mg/kg body weight) 1 hour prior to euthanasia and tissue collection to estimate colonic oxygen levels. Colon tissue was fixed in 10% formalin, embedded in paraffin, and processed for immunohistochemical detection of pimonidazole. The colonic microbiome was assessed by 16S gene rRNA amplicon sequencing and content of short-chain fatty acids was measured by liquid chromatography-mass spectrometry.
Results
HbA1c % increased linearly across the PD (5.9±0.1), RD (7.6±0.4), and D3M (11.5±0.6) groups, confirming the progression of diabetes in this cohort. D3M rats had a 2.5% increase in known facultative anaerobes, Escherichia–Shigella , and Streptococcus (false discovery rate <0.05) genera in colon contents. The intensity of pimonidazole staining of colonic epithelia did not differ across groups (p=0.37). Colon content concentrations of acetate and propionate also did not differ across UCD-T2DM groups; however, colonic butyric acid levels were higher in D3M rats relative to PD rats (p<0.01).
Conclusions
The advancement of diabetes in UCD-T2DM rats was associated with an increase in facultative anaerobes; however, this was not explained by changes in colonocyte oxygen levels. The mechanisms underlying shifts in gut microbe populations associated with the progression of diabetes in the UCD-T2DM rat model remain to be identified.
... SLC transporters have been reported to play crucial roles in maintaining the development, homeostasis, differentiation, and securing activation of immune cells, including T cells, NK cells, and macrophages 7 . Dysregulation of these transporters can trigger various immunologic diseases, including gout, asthma, inflammatory bowel disease, and Alzheimer's disease [8][9][10] . Of these transporters, monocarboxylate transporter 1 (MCT1, also known as SLC16A1) is responsible for the transportation of various monocarboxylates, including lactate and pyruvate, across the plasma membrane. ...
Monocarboxylate transporter 1 (MCT1) exhibits essential roles in cellular metabolism and energy supply. Although MCT1 is highly expressed in activated B cells, it is not clear how MCT1-governed monocarboxylates transportation is functionally coupled to antibody production during the glucose metabolism. Here, we report that B cell-lineage deficiency of MCT1 significantly influences the class-switch recombination (CSR), rendering impaired IgG antibody responses in Mct1f/fMb1Cre mice after immunization. Metabolic flux reveals that glucose metabolism is significantly reprogrammed from glycolysis to oxidative phosphorylation in Mct1-deficient B cells upon activation. Consistently, activation-induced cytidine deaminase (AID), is severely suppressed in Mct1-deficient B cells due to the decreased level of pyruvate metabolite. Mechanistically, MCT1 is required to maintain the optimal concentration of pyruvate to secure the sufficient acetylation of H3K27 for the elevated transcription of AID in activated B cells. Clinically, we found that MCT1 expression levels are significantly upregulated in systemic lupus erythematosus patients, and Mct1 deficiency can alleviate the symptoms of bm12-induced murine lupus model. Collectively, these results demonstrate that MCT1-mediated pyruvate metabolism is required for IgG antibody CSR through an epigenetic dependent AID transcription, revealing MCT1 as a potential target for vaccine development and SLE disease treatment.
... Interestingly, this was also previously reported by Thibault et al. 34 in a study on IBD patients, correlating the finding with the downregulation of butyrate transportation by MCT1 in IBD colonic mucosa, results in butyrate oxidation deficiency in intestinal inflammation. 34 Although this could be relevant, the study undertaken by Watanabe et al. 28 included mainly right-sided diverticula, where the concentration of butyrate has been reported to be lower at the distal end of the colon (sigmoid and rectum). 35 Clostridium cl. ...
Background and aims:
The role of the microbiota in diverticulosis and diverticular disease is underexplored. This systematic review aimed to assess all literature pertaining to the microbiota and metabolome associations in asymptomatic diverticulosis, symptomatic uncomplicated diverticular disease (SUDD), and diverticulitis pathophysiology.
Methods:
Seven databases were searched for relevant studies published up to September 28th , 2022. Data were screened in Covidence and extracted to Excel. Critical appraisal was undertaken using the Newcastle Ottawa Scale for case/control studies.
Results:
Of the 413 papers screened by title and abstract, 48 full-text papers were reviewed in detail with 12 studies meeting the inclusion criteria. Overall, alpha and beta diversity were unchanged in diverticulosis; however, significant changes in alpha diversity were evident in diverticulitis. A similar Bacteroidetes to Firmicutes ratio compared to controls was reported across studies. The genus-level comparisons showed no relationship with diverticular disease. Butyrate-producing microbial species were decreased in abundance suggesting a possible contribution to the pathogenesis of diverticular disease. Comamonas species was significantly increased in asymptomatic diverticulosis patients who later developed diverticulitis. Metabolome analysis reported significant differences in diverticulosis and SUDD, with upregulated uracil being the most consistent outcome in both. No significant differences were reported in the mycobiome.
Conclusion:
Overall, there is no convincing evidence of microbial dysbiosis in colonic diverticula to suggest that the microbiota contributes to the pathogenesis of asymptomatic diverticulosis, SUDD, or diverticular disease. Future research investigating microbiota involvement in colonic diverticula should consider an investigation of mucosa-associated microbial changes within the colonic diverticulum itself.
... SMCT1 and SMCT2 are expressed exclusively on the apical IEC membrane. A number of studies have demonstrated that inflammatory mediators such as TNFα and IFNγ repress MCT1 and SMCT1 expression and function in IEC [28,29] . A meta-analysis of 12 datasets comparing expression of MCT1, SMCT1 and SMCT2 in healthy and UC colonic tissue revealed a significant (range 17%-95%) decrease in MCT and SMCT expression in 10 of the 12 studies examined [27] . ...
Active episodes of inflammatory bowel disease (IBD), which include ulcerative colitis and Crohn's disease, coincide with profound shifts in the composition of the microbiota and host metabolic energy demand. Intestinal epithelial cells (IEC) that line the small intestine and colon serve as an initial point for contact for the microbiota and play a central role in innate immunity. In the 1980s, Roediger et al proposed the hypothesis that IBD represented a disease of diminished mucosal nutrition and energy deficiency ("starved gut") that strongly coincided with the degree of inflammation. These studies informed the scientific community about the important contribution of microbial-derived metabolites, particularly short-chain fatty acids (SCFA) such as butyrate, to overall energy homeostasis. Decades later, it is appreciated that disease-associated shifts in the microbiota, termed dysbiosis, places inordinate demands on energy acquisition within the mucosa, particularly during active inflammation. Here, we review the topic of tissue energetics in mucosal health and disease from the original perspective of that proposed by the starved gut hypothesis.
... It appears that increasing concentration of these fecal fatty acids is not only due to colonic mucosal damage and reduced mucosal integrity by inflammation, but also insufficient absorption of microbial metabolites into the intestine. NH 3 and other protein-derived metabolites in the lumen elicit inflammatory responses against the intestinal mucosa, which acts as a factor to reduce the expression of MCT1 [35]. In the colitis-induced group, there was a decrease in expression of transporters and an increase in fecal SCFA levels. ...
Background/objectives:
Chronic colitis is a risk factor for colorectal cancer (CRC) development in both animals and humans. Previously, we reported that a diet rich in protein (with casein as the protein source) significantly increased the risk of mouse CRC development in a dose-dependent manner. In this study, we investigated the effects of different protein sources on the risk of colitis development.
Materials/methods:
Balb/c mice were divided into 7 experimental groups: 20% casein (20C), 20C-dextran sulfate sodium (DSS), 40% casein-DSS (40CD), 40% whey protein-DSS (40WD), 40% soy protein-DSS (40SD), 40% white meat-DSS (40WMD), and 40% red meat-DSS (40RMD). Mice were fed an experimental diet for 4 wk and received 3% DSS in their drinking water for 6 days during the 4th wk of the experimental period.
Results:
Compared to other groups, the 40CD group showed the most aggravated colitis with increased disease activity and inflammatory markers. In the 40RMD group, interleukin (IL)-6 levels were the highest among all the groups. The 40SD group showed conflicting effects, for example, elevated mortality and disease activity but decreased nitric oxide (NO) levels. The 40WD group showed attenuated colitis with increased IL-10 levels and decreased NO levels. The 40WMD group showed conflicting effects, including decreased NO levels and elevated fecal lipocalin-2 and IL-6 levels.
Conclusions:
These results suggest that, at levels of 40% in the diet, casein and red meat exacerbate colitis, whereas whey protein mitigates it the most effectively.
... The high fecal butyrate concentrations in feces after DSS could be due to a decreased expression of MCT-1 and butyrate uptake, which has already been demonstrated in DSSinduced acute colitis [41]. If butyrate absorption was limited by DSS treatment, it would lead to altered metabolism by colonocytes [42]. ...
Ulcerative colitis (UC) patients often avoid foods containing fermentable fibers as some can promote symptoms during active disease. Pectin has been identified as a more protective fermentable fiber, but little has been done to determine the interaction between pectin and bioactive compounds present in foods containing that fiber type. Quercetin and chlorogenic acid, two bioactives in stone fruits, may have anti-cancer, anti-oxidant, and anti-inflammatory properties. We hypothesized that quercetin and chlorogenic acid, in the presence of the fermentable fiber pectin, may suppress the expression of pro-inflammatory molecules, alter the luminal environment, and alter colonocyte proliferation, thereby protecting against recurring bouts of UC. Rats (n = 63) received one of three purified diets (control, 0.45% quercetin, 0.05% chlorogenic acid) containing 6% pectin for 3 weeks before exposure to dextran sodium sulfate (DSS, 3% for 48 h, 3x, 2 wk separation, n = 11/diet) in drinking water to initiate UC, or control (no DSS, n = 10/diet) treatments prior to termination at 9 weeks. DSS increased the fecal moisture content (p < 0.05) and SCFA concentrations (acetate, p < 0.05; butyrate, p < 0.05). Quercetin and chlorogenic acid diets maintained SLC5A8 (SCFA transporter) mRNA levels in DSS-treated rats at levels similar to those not exposed to DSS. DSS increased injury (p < 0.0001) and inflammation (p < 0.01) scores, with no differences noted due to diet. Compared to the control diet, chlorogenic acid decreased NF-κB activity in DSS-treated rats (p < 0.05). Quercetin and chlorogenic acid may contribute to the healthy regulation of NF-κB activation (via mRNA expression of IκΒα, Tollip, and IL-1). Quercetin enhanced injury-repair molecule FGF-2 expression (p < 0.01), but neither diet nor DSS treatment altered proliferation. Although quercetin and chlorogenic acid did not protect against overt indicators of injury and inflammation, or fecal SCFA concentrations, compared to the control diet, their influence on the expression of injury repair molecules, pro-inflammatory cytokines, SCFA transport proteins, and NF-κB inhibitory molecules suggests beneficial influences on major pathways involved in DSS-induced UC. Therefore, in healthy individuals or during periods of remission, quercetin and chlorogenic acid may promote a healthier colon, and may suppress some of the signaling involved in inflammation promotion during active disease.
... 85 Interestingly, evidence suggests that inflammation may decrease butyrate-mediated uptake as well as the expression of both transporters. [95][96][97] Thus, one may speculate that the inflammatory state associated with obesity may downregulate transporter-mediated butyrate absorption. Limited literature is available on SCFA transport on the basolateral side of the membrane. ...
Evidence is increasing that disturbances in the gut microbiome may play a significant role in the etiology of obesity and type 2 diabetes. The short chain fatty acid butyrate, a major end product of the bacterial fermentation of indigestible carbohydrates, is reputed to have anti‐inflammatory properties and positive effects on body weight control and insulin sensitivity. However, whether butyrate has therapeutic potential for the treatment and prevention of obesity and obesity‐related complications remains to be elucidated. Overall, animal studies strongly indicate that butyrate administered via various routes (e.g., orally) positively affects adipose tissue metabolism and functioning, energy and substrate metabolism, systemic and tissue‐specific inflammation, and insulin sensitivity and body weight control. A limited number of human studies demonstrated interindividual differences in clinical effectiveness suggesting that outcomes may depend on the metabolic, microbial, and lifestyle‐related characteristics of the target population. Hence, despite abundant evidence from animal data, support of human data is urgently required for the implementation of evidence‐based oral and gut‐derived butyrate interventions. To increase the efficacy of butyrate‐focused interventions, future research should investigate which factors impact treatment outcomes including baseline gut microbial activity and functionality, thereby optimizing targeted‐interventions and identifying individuals that merit most from such interventions.
... Treatment with SB led to a concentration-and time-dependent upregulation of MCT1 mRNA in colonic epithelial cells (53). However, inflammation can lead to the dysregulation of MCT1 (54). In the present study, LPS decreased the expression of MCT1, while the mRNA expression of MCT1 increased in the LSB group, which indicated that inflammation caused the dysregulation of MCT1, but SB alleviates this effect. ...
This study aimed to evaluate whether sodium butyrate (SB) attenuates the ruminal response to LPS-stimulated inflammation by activating GPR41 in bovine rumen epithelial cells (BRECs). We examined the SB regulation of GPR41 and its impact on LPS-induced inflammation using GPR41 knockdown BRECs. The LPS-induced BRECs showed increases in the expression of genes related to pro-inflammation and decreases in the expression of genes related to tight junction proteins; these were attenuated by pretreatment with SB. Compared with that in LPS-stimulated BRECs, the ratio of phosphorylated NF-κB (p65 subunit) to NF-κB (p65 subunit) and the ratio of phosphorylated IκBα to IκBα were suppressed with SB pretreatment. The LSB group abated LPS-induced apoptosis and decreased the expression of Bax, Caspase 3, and Caspase 9 mRNA relative to the LPS group. In addition, the LSB group had a lower proportion of cells in the G0–G1 phase and a higher proportion of cells in the S phase than the LPS group. The mRNA expression of ACAT1 and BDH1 genes related to volatile fatty acid (VFA) metabolism were upregulated in the LSB group compared to those in LPS-induced BRECs. In addition, pretreatment with SB promoted the gene expression of GPR41 in the LPS-induced BRECs. Interestingly, SB pretreatment protected BRECs but not GPR41KD BRECs. Our results suggest that SB pretreatment protects against the changes in BRECs LPS-induced inflammatory response by activating GPR41.
... This reduced Mct1 expression is theorised to be related to AD-associated inflammation. This is supported by the knowledge that MCT1 expression is markedly reduced in humans with inflammatory bowel disease [57]. This study also identified a parallel reduction in the expression of 4F2hc and Lat2. ...
Alzheimer’s disease (AD) has traditionally been considered solely a neurological condition. Therefore, numerous studies have been conducted to identify the existence of pathophysiological changes affecting the brain and the blood-brain barrier in individuals with AD. Such studies have provided invaluable insight into possible changes to the central nervous system exposure of drugs prescribed to individuals with AD. However, there is now increasing recognition that extra-neurological systems may also be affected in AD, such as the small intestine, liver, and kidneys. Examination of these peripheral pathophysiological changes is now a burgeoning area of scientific research, owing to the potential impact of these changes on the absorption, distribution, metabolism, and excretion (ADME) of drugs used for both AD and other concomitant conditions in this population. The purpose of this review is to identify and summarise available literature reporting alterations to key organs influencing the pharmacokinetics of drugs, with any changes to the small intestine, liver, kidney, and circulatory system on the ADME of drugs described. By assessing studies in both rodent models of AD and samples from humans with AD, this review highlights possible dosage adjustment requirements for both AD and non-AD drugs so as to ensure the achievement of optimum pharmacotherapy in individuals with AD.
... Specifically, genes encoding enzymes responsible in butyrate oxidation/uptake are downregulated in inflamed mucosa of IBD patients [121][122][123]. Additionally, inflammatory cytokines inhibit butyrate uptake, oxidation (e.g., TNF-α), and MCT1/SLC16A1 expression [124][125][126]. These findings suggest that inflammation is directly related to SCFAs (specifically butyrate) synthesis, uptake, and metabolism, suggesting that SCFAs supplementation alone may not be sufficient to regain homeostasis. ...
Epithelial cells that line tissues such as the intestine serve as the primary barrier to the outside world. Epithelia provide selective permeability in the presence of a large constellation of microbes, termed the microbiota. Recent studies have revealed that the symbiotic relationship between the healthy host and the microbiota includes the regulation of cell–cell interactions at the level of epithelial tight junctions. The most recent findings have identified multiple microbial-derived metabolites that influence intracellular signaling pathways which elicit activities at the epithelial apical junction complex. Here, we review recent findings that place microbiota-derived metabolites as primary regulators of epithelial cell–cell interactions and ultimately mucosal permeability in health and disease.
... In UC and CD, expression of the SCFA transporter MCT1 was reduced, and butyrate uptake and oxidation were inhibited in UC. The change in SCFA transporter and receptor expression may be due to inflammation or alteration of gut microbiome composition [197,198]. Overall, alteration in the gut microbiota due to a HFD causes a reduction in SCFAs-producing bacteria-mainly butyrate-producing bacteria-and an increase in pathogenic bacteria, affecting the production and absorption of SCFAs. Overgrowth of pathobionts disrupts barrier function and causes inflammation, which results in the downregulation of SCFAs' receptor and transporter, as observed in IBD patients and in in vivo models of IBD. ...
The gut microbiota is a complex community of microorganisms that has become a new focus of attention due to its association with numerous human diseases. Research over the last few decades has shown that the gut microbiota plays a considerable role in regulating intestinal homeostasis, and disruption to the microbial community has been linked to chronic disease conditions such as inflammatory bowel disease (IBD), colorectal cancer (CRC), and obesity. Obesity has become a global pandemic, and its prevalence is increasing worldwide mostly in Western countries due to a sedentary lifestyle and consumption of high-fat/high-sugar diets. Obesity-mediated gut microbiota alterations have been associated with the development of IBD and IBD-induced CRC. This review highlights how obesity-associated dysbiosis can lead to the pathogenesis of IBD and CRC with a special focus on mechanisms of altered absorption of short-chain fatty acids (SCFAs)
... However, most SCFAs are absorbed in the proximal colon, which in humans is as yet unexamined in this context. Inflammatory bowel disorder (IBD) features a decreased absorption of butyrate due to a downregulation of MCT1 (Thibault et al., 2007). A similar downregulation has also been seen in ulcerative colitis (Fisel et al., 2018), which likely has a similar effect on SCFA absorption. ...
The role of the intestinal microbiota as a regulator of gut-brain axis signalling has risen to prominence in recent years. Understanding the relationship between the gut microbiota, the metabolites it produces, and the brain will be critical for the subsequent development of new therapeutic approaches, including the identification of novel psychobiotics. A key focus in this regard have been the short-chain fatty acids (SCFAs) produced by bacterial fermentation of dietary fibre, which include butyrate, acetate, and propionate. Ongoing research is focused on the entry of SCFAs into systemic circulation from the gut lumen, their migration to cerebral circulation and across the blood brain barrier, and their potential to exert acute and chronic effects on brain structure and function. This review aims to discuss our current mechanistic understanding of the direct and indirect influence that SCFAs have on brain function, behaviour and physiology, which will inform future microbiota-targeted interventions for brain disorders.
... In addition, the epithelial transporter-the cell-specific butyrate transporter (encoded by SLC16A1)-is downregulated in the inflamed colonic mucosa of patients with IBD. The reduction in butyrate and subsequent inhibition of β-oxidation may be particularly detrimental in the context of intestinal inflammation [96,97]. Administration of exogenous butyrate promotes resistance to experimental colitis [98]. ...
The unique biology of the intestinal epithelial barrier is linked to a low baseline oxygen pressure (pO2), characterised by a high rate of metabolites circulating through the intestinal blood and the presence of a steep oxygen gradient across the epithelial surface. These characteristics require tight regulation of oxygen homeostasis, achieved in part by hypoxia-inducible factor (HIF)-dependent signalling. Furthermore, intestinal epithelial cells (IEC) possess metabolic identities that are reflected in changes in mitochondrial function. In recent years, it has become widely accepted that oxygen metabolism is key to homeostasis at the mucosae. In addition, the gut has a vast and diverse microbial population, the microbiota. Microbiome–gut communication represents a dynamic exchange of mediators produced by bacterial and intestinal metabolism. The microbiome contributes to the maintenance of the hypoxic environment, which is critical for nutrient absorption, intestinal barrier function, and innate and/or adaptive immune responses in the gastrointestinal tract. In this review, we focus on oxygen homeostasis at the epithelial barrier site, how it is regulated by hypoxia and the microbiome, and how oxygen homeostasis at the epithelium is regulated in health and disease.
... Rather, butyrate deficiency appears to be a consequence of intestinal inflammation, instead of its cause. Furthermore, MCT1 is downregulated in inflamed intestinal tissue, thus decreasing intracellular butyrate availability and defeating the purpose of butyrate supplementation during active inflammation [68,69]. ...
The microbial metabolite butyrate serves as a link between the intestinal microbiome and epithelium. The monocarboxylate transporters MCT1 and SMCT1 are the predominant means of butyrate transport from the intestinal lumen to epithelial cytoplasm, where the molecule undergoes rapid β-oxidation to generate cellular fuel. However, not all epithelial cells metabolize butyrate equally. Undifferentiated colonocytes, including neoplastic cells and intestinal stem cells at the epithelial crypt base preferentially utilize glucose over butyrate for cellular fuel. This divergent metabolic conditioning is central to the phenomenon known as "butyrate paradox", in which butyrate induces contradictory effects on epithelial proliferation in undifferentiated and differentiated colonocytes. There is evidence that accumulation of butyrate in epithelial cells results in histone modification and altered transcriptional activation that halts cell cycle progression. This manifests in the apparent protective effect of butyrate against colonic neoplasia. A corollary to this process is butyrate-induced inhibition of intestinal stem cells. Yet, emerging research has illustrated that the evolution of the crypt, along with butyrate-producing bacteria in the intestine, serve to protect crypt base stem cells from butyrate's anti-proliferative effects. Butyrate also regulates epithelial inflammation and tolerance to antigens, through production of anti-inflammatory cytokines and induction of tolerogenic dendritic cells. The role of butyrate in the pathogenesis and treatment of intestinal neoplasia, inflammatory bowel disease and malabsorptive states is evolving, and holds promise for the potential translation of butyrate's cellular function into clinical therapies.
... In addition to microbial function, the utilization of SCFAs by host cells is also changed in patients with IBD. For example, proinflammatory cytokines TNF and IFN-γ decrease the expression of monocarboxylate transporter MCT1, a butyrate transporter, and further, MCT1 expression is markedly decreased in the inflamed colonic mucosa of IBD patients [72]. Several studies have demonstrated that butyrate oxidation and the expression of genes involved in butyrate oxidation are diminished in the intestinal mucosa of IBD patients [73][74][75]. ...
Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. Although the precise etiology of IBD is largely unknown, it is widely thought that diet contributes to the development of IBD. Diet shapes the composition of the gut microbiota, which plays critical roles in intestinal homeostasis. In contrast, intestinal inflammation induces gut dysbiosis and may affect the use of dietary nutrients by host cells and the gut microbiota. The interaction of diet and the gut microbiota is perturbed in patients with IBD. Herein, we review the current knowledge of diet and gut microbiota interaction in intestinal homeostasis. We also discuss alterations of diet and gut microbiota interaction that influence the outcome and the nutritional treatment of IBD. Understanding the complex relationships between diet and the gut microbiota provides crucial insight into the pathogenesis of IBD and advances the development of new therapeutic approaches.
... GPR41 and GPR43 have a role in the immune surveillance of the colonic mucosa towards microbial activity (67), whilst GPR109A inhibits NF-kB activation as a tumor suppressor (68). Active absorption of butyrate is reduced in IBD with decreased MCT1 expression in inflamed mucosa of UC patients (69), possibly due to high TNF-a levels (70) or reduced butyrate-producing bacteria, causing faulty oxidization (71,72), potentially indicating that butyrate reduces IL-8 expression mediated by GPR109A. ...
The diverse and dynamic microbial community of the human gastrointestinal tract plays a vital role in health, with gut microbiota supporting the development and function of the gut immune barrier. Crosstalk between microbiota-gut epithelium and the gut immune system determine the individual health status, and any crosstalk disturbance may lead to chronic intestinal conditions, such as inflammatory bowel diseases (IBD) and celiac disease. Microbiota-derived metabolites are crucial mediators of host-microbial interactions. Some beneficially affect host physiology such as short-chain fatty acids (SCFAs) and secondary bile acids. Also, tryptophan catabolites determine immune responses, such as through binding to the aryl hydrocarbon receptor (AhR). AhR is abundantly present at mucosal surfaces and when activated enhances intestinal epithelial barrier function as well as regulatory immune responses. Exogenous diet-derived indoles (tryptophan) are a major source of endogenous AhR ligand precursors and together with SCFAs and secondary bile acids regulate inflammation by lowering stress in epithelium and gut immunity, and in IBD, AhR expression is downregulated together with tryptophan metabolites. Here, we present an overview of host microbiota-epithelium-gut immunity crosstalk and review how microbial-derived metabolites contribute to host immune homeostasis. Also, we discuss the therapeutic potential of bacterial catabolites for IBD and celiac disease and how essential dietary components such as dietary fibers and bacterial tryptophan catabolites may contribute to intestinal and systemic homeostasis.
... For example, three flavonoids, quercetin, kaempferol, and delphinidin can bind to adenine and guanine (major groove), and thymine (minor groove) (Kanakis et al., 2005), and saffron derivate metabolites can interact with DNA guanine-quadruplexes (G4) (Hoshyar et al., 2012). These G4 motifs are stable structures related to gene promoter regulation and DNA methylation (Hardin et al., 1993;Mao et al., 2018); they can be dysregulated by aberrant DNA methylation due to folate deprivation Low levels of butyric acid in the gut Downregulation of SLC16A1 Decreased transport of butyric acid Thibault et al. (2007) mTORC complex Increased GLUT1 expression Downstream aerobic glycolysis in T cells to support their proliferation and effector function Song et al. (2020) Content courtesy of Springer Nature, terms of use apply. Rights reserved. ...
Background
In 1957, Francis Crick drew a linear diagram on a blackboard. This diagram is often called the “central dogma.” Subsequently, the relationships between different steps of the “central dogma” have been shown to be considerably complex, mostly because of the emerging world of small molecules. It is noteworthy that metabolites can be generated from the diet through gut microbiome metabolism, serve as substrates for epigenetic modifications, destabilize DNA quadruplexes, and follow Lamarckian inheritance. Small molecules were once considered the missing link in the “central dogma”; however, recently they have acquired a central role, and their general perception as downstream products has become reductionist. Metabolomics is a large-scale analysis of metabolites, and this emerging field has been shown to be the closest omics associated with the phenotype and concomitantly, the basis for all omics.Aim of reviewHerein, we propose a broad updated perspective for the flux of information diagram centered in metabolomics, including the influence of other factors, such as epigenomics, diet, nutrition, and the gut- microbiome.Key scientific concepts of reviewMetabolites are the beginning and the end of the flux of information.
... This is not due to a constitutive defect in the oxidative process but to a lower butyrate uptake by colonocytes caused by the downregulation of MCT1 expression in the inflamed mucosa. Such effect was confirmed in HT29 cells exposed to TNF-α and IFN-γ [42]. A low abundance of butyrate-producing bacteria and decreased butyrate availability possibly results in the same situation [17], reducing the butyrate oxidation by epithelial cells, decreasing ATP production, and finally contributing to mucosal lesion exacerbation. ...
This review describes current evidence supporting butyrate impact in the homeostatic regulation of the digestive ecosystem in health and inflammatory bowel diseases (IBDs). Butyrate is mainly produced by bacteria from the Firmicutes phylum. It stimulates mature colonocytes and inhibits undifferentiated malignant and stem cells. Butyrate oxidation in mature colonocytes (1) produces 70–80% of their energetic requirements, (2) prevents stem cell inhibition by limiting butyrate access to crypts, and (3) consumes oxygen, generating hypoxia and maintaining luminal anaerobiosis favorable to the microbiota. Butyrate stimulates the aryl hydrocarbon receptor (AhR), the GPR41 and GPR109A receptors, and inhibits HDAC in different cell types, thus stabilizing the gut barrier function and decreasing inflammatory processes. However, some studies indicate contrary effects according to butyrate concentrations. IBD patients exhibit a lower abundance of butyrate-producing bacteria and butyrate content. Additionally, colonocyte butyrate oxidation is depressed in these subjects, lowering luminal anaerobiosis and facilitating the expansion of Enterobacteriaceae that contribute to inflammation. Accordingly, gut dysbiosis and decreased barrier function in IBD seems to be secondary to the impaired mitochondrial disturbance in colonic epithelial cells.
... Gut epithelial cells express the monocarboxylate transporter MCT1, a primary ketone body transporter on the basolateral surface (83), and several papers suggest that gut epithelial cells are capable of utilizing ketone bodies from the vascular bed (84,85). As gastrointestinal inflammation and mucosal damage can impair butyrate uptake from the intestinal lumen (86,87), circulating ketones may provide a potential therapeutic option in certain patients with gastrointestinal disease. ...
There is a broad consensus in nutritional-microbiota research that high-fat (HF) diets are harmful to human health, at least in part through their modulation of the gut microbiota. However, various studies also support the inherent flexibility of the human gut and our microbiota's ability to adapt to a variety of food sources, suggesting a more nuanced picture. In this article, we first discuss some problems facing basic translational research and provide a different framework for thinking about diet and gut health in terms of metabolic flexibility. We then offer evidence that well-formulated HF diets, such as ketogenic diets, may provide healthful alternative fuel sources for the human gut. We place this in the context of cancer research, where this concern over HF diets is also expressed, and consider various potential objections concerning the effects of lipopolysaccharides, trimethylamine-N-oxide, and secondary bile acids on human gut health. We end by providing some general suggestions for how to improve research and clinical practice with respect to the gut microbiota when considering the framework of metabolic flexibility.
... MCT1 employs a symport with H + ions driven by a transmembrane gradient affecting the intracellular pH [44]. The presence of inflammation including inflammatory bowel disease or early stages of colorectal carcinogenesis is connected to the downregulation of MCT1 expression [45,46]. On the other hand, advanced stages of colorectal carcinoma (CRC) are associated with an upregulation of MCT1 expression [47] due to the intratumoral production and metabolism of lactate, another MCT1 substrate. ...
Postbiotics are health-promoting microbial metabolites delivered as a functional food or a food supplement. They either directly influence signaling pathways of the body or indirectly manipulate metabolism and the composition of intestinal microflora. Cancer is the second leading cause of death worldwide and even though the prognosis of patients is improving, it is still poor in the substantial part of the cases. The preventable nature of cancer and the importance of a complex multi-level approach in anticancer therapy motivate the search for novel avenues of establishing the anticancer environment in the human body. This review summarizes the principal findings demonstrating the usefulness of both natural and synthetic sources of postbotics in the prevention and therapy of cancer. Specifically, the effects of crude cell-free supernatants, the short-chain fatty acid butyrate, lactic acid, hydrogen sulfide, and β-glucans are described. Contradictory roles of postbiotics in healthy and tumor tissues are highlighted. In conclusion, the application of postbiotics is an efficient complementary strategy to combat cancer.
... The concentrations of compounds to be tested were chosen based on literature and our own previous works (e.g. Andrade et al., 2018;Araujo et al., 2013;Silva et al., 2017;Thibault et al., 2007;Wolczyk et al., 2016). ...
Obesity and type 2 diabetes mellitus (T2DM) associate with increased incidence and mortality from many cancers, including breast cancer. The mechanisms involved in this relation remain poorly understood. Our study aimed to investigate the in vitro effect of high levels of glucose, insulin, leptin, TNF-α, INF-γ and oxidative stress (induced with tert-butylhydroperoxide (TBH)), which are associated with T2DM, upon glucose uptake by breast cancer (MCF-7 and MDA-MB-231) and non-cancer (MCF-12A) cells and to correlate this effect with their effects upon cellular characteristics associated with cancer progression (cell proliferation, viability, migration, angiogenesis and apoptosis).
³H-DG uptake was markedly inhibited by a selective GLUT1 inhibitor (BAY-876) in all cell lines, proving that ³H-DG uptake is mainly GLUT1-mediated. TBH (2.5 μM), insulin (50 nM), leptin (500 ng/ml) and INF-y (100 ng/ml) stimulate GLUT1-mediated ³H-DG (1 mM) uptake by both ER-positive and triple-negative breast cancer cell lines. TBH and leptin, but not insulin and INF-γ, increase GLUT1 mRNA levels. Insulin and leptin (in both ER-positive and triple-negative breast cancer cell lines) and TBH (in the triple-negative cell line) have a proproliferative effect and leptin possesses a cytoprotective effect in both breast cancer cell lines that can contribute to cancer progression. The effects of TBH, insulin, leptin and INF-γ upon breast cancer cell proliferation and viability are GLUT1-dependent.
In conclusion, T2DM-associated characteristics induce changes in GLUT1-mediated glucose uptake that can contribute to cancer progression. Moreover, we conclude that BAY-876 can be a strong candidate for development of a new effective anticancer agent against breast cancer.
Various strategies in the microscale/nanoscale have been developed to improve oral absorption of therapeutics. Among them, gastrointestinal (GI)-transporter/receptor-mediated nanosized drug delivery systems (NDDSs) have drawn attention due to their many...
Butyrate, a metabolite produced by gut bacteria, has demonstrated beneficial effects in the colon and has been used to treat inflammatory bowel diseases. However, the mechanism by which butyrate operates remains incompletely understood. Given that oral butyrate can exert either a direct impact on the gut mucosa or an indirect influence through its interaction with the gut microbiome, this study aimed to investigate three key aspects: (1) whether oral intake of butyrate modulates the expression of genes encoding short-chain fatty acid (SCFA) transporters (Slc16a1, Slc16a3, Slc16a4, Slc5a8, Abcg2) and receptors (Hcar2, Ffar2, Ffar3, Olfr78, Olfr558) in the colon, (2) the potential involvement of gut microbiota in this modulation, and (3) the impact of oral butyrate on the expression of colonic SCFA transporters and receptors during colonic inflammation. Specific pathogen-free (SPF) and germ-free (GF) mice with or without DSS-induced inflammation were provided with either water or a 0.5% sodium butyrate solution. The findings revealed that butyrate decreased the expression of Slc16a1, Slc5a8, and Hcar2 in SPF but not in GF mice, while it increased the expression of Slc16a3 in GF and the efflux pump Abcg2 in both GF and SPF animals. Moreover, the presence of microbiota was associated with the upregulation of Hcar2, Ffar2, and Ffar3 expression and the downregulation of Slc16a3. Interestingly, the challenge with DSS did not alter the expression of SCFA transporters, regardless of the presence or absence of microbiota, and the effect of butyrate on the transporter expression in SPF mice remained unaffected by DSS. The expression of SCFA receptors was only partially affected by DSS. Our results indicate that (1) consuming a relatively low concentration of butyrate can influence the expression of colonic SCFA transporters and receptors, with their expression being modulated by the gut microbiota, (2) the effect of butyrate does not appear to result from direct substrate-induced regulation but rather reflects an indirect effect associated with the gut microbiome, and (3) acute colon inflammation does not lead to significant changes in the transcriptional regulation of most SCFA transporters and receptors, with the effect of butyrate in the inflamed colon remaining intact.
Studies in preclinical models support that the gut microbiota play a critical role in the development and progression of colorectal cancer (CRC). Specific microbial species and their corresponding virulence factors or associated small molecules can contribute to CRC development and progression either via direct effects on the neoplastic transformation of epithelial cells or through interactions with the host immune system. Induction of DNA damage, activation of Wnt/β-catenin and NF-κB proinflammatory pathways, and alteration of the nutrient's availability and the metabolic activity of cancer cells are the main mechanisms by which the microbiota contribute to CRC. Within the tumor microenvironment, the gut microbiota alter the recruitment, activation, and function of various immune cells, such as T cells, macrophages, and dendritic cells. Additionally, the microbiota shape the function and composition of cancer-associated fibroblasts and extracellular matrix components, fashioning an immunosuppressive and pro-tumorigenic niche for CRC. Understanding the complex interplay between gut microbiota and tumorigenesis can provide therapeutic opportunities for the prevention and treatment of CRC.
Gut bacteria provide benefits to the host and have been implicated in inflammatory bowel disease (IBD), where adherent-invasive E. coli (AIEC) pathobionts (e.g., strain LF82) are associated with Crohn’s disease. E. coli-LF82 causes fragmentation of the epithelial mitochondrial network, leading to increased epithelial permeability. We hypothesized that butyrate would limit the epithelial mitochondrial disruption caused by E. coli-LF82. Human colonic organoids and the T84 epithelial cell line infected with E. coli-LF82 (MOI = 100, 4 h) showed a significant increase in mitochondrial network fission that was reduced by butyrate (10 mM) co-treatment. Butyrate reduced the loss of mitochondrial membrane potential caused by E. coli-LF82 and increased expression of PGC-1$\alpha $α mRNA, the master regulator of mitochondrial biogenesis. Metabolomics revealed that butyrate significantly altered E. coli-LF82 central carbon metabolism leading to diminished glucose uptake and increased succinate secretion. Correlating with preservation of mitochondrial network form/function, butyrate reduced E. coli-LF82 transcytosis across T84-cell monolayers. The use of the G-protein inhibitor, pertussis toxin, implicated GPCR signaling as critical to the effect of butyrate, and the free fatty acid receptor three (FFAR3, GPR41) agonist, AR420626, reproduced butyrate’s effect in terms of ameliorating the loss of barrier function and reducing the mitochondrial fragmentation observed in E. coli-LF82 infected T84-cells and organoids. These data indicate that butyrate helps maintain epithelial mitochondrial form/function when challenged by E. coli-LF82 and that this occurs, at least in part, via FFAR3. Thus, loss of butyrate-producing bacteria in IBD in the context of pathobionts would contribute to loss of epithelial mitochondrial and barrier functions that could evoke disease and/or exaggerate a low-grade inflammation.
The gut is colonized by many commensal microorganisms, and the diversity and metabolic patterns of microorganisms profoundly influence the intestinal health. These microbial imbalances can lead to disorders such as inflammatory bowel disease (IBD). Microorganisms produce byproducts that act as signaling molecules, triggering the immune system in the gut mucosa and controlling inflammation. For example, metabolites like short-chain fatty acids (SCFA) and secondary bile acids can release inflammatory-mediated signals by binding to specific receptors. These metabolites indirectly affect host health and intestinal immunity by interacting with the intestinal epithelial and mucosal immune cells. Moreover, Tryptophan-derived metabolites also play a role in governing the immune response by binding to aromatic hydrocarbon receptors (AHR) located on the intestinal mucosa, enhancing the intestinal epithelial barrier. Dietary-derived indoles, which are synthetic precursors of AHR ligands, work together with SCFA and secondary bile acids to reduce stress on the intestinal epithelium and regulate inflammation. This review highlights the interaction between gut microbial metabolites and the intestinal immune system, as well as the crosstalk of dietary fiber intake in improving the host microbial metabolism and its beneficial effects on the organism.
Background and Purpose
Nonsteroidal anti‐inflammatory drugs (NSAIDs) can be associated with severe adverse digestive effects. This study examined the protective effects of the probiotic Saccharomyces boulardii CNCM I‐745 in a rat model of diclofenac‐induced enteropathy.
Experimental Approach
Enteropathy was induced in 40‐week‐old male rats by intragastric diclofenac (4 mg·kg⁻¹ BID for 14 days). S. boulardii CNCM I‐745 (3 g·kg⁻¹ BID by oral gavage) was administered starting 14 days before (preventive protocol) or along with (curative protocol) diclofenac administration. Ileal damage, inflammation, barrier integrity, gut microbiota composition and toll‐like receptors (TLRs)–nuclear factor κB (NF‐κB) pathway were evaluated.
Key Results
Diclofenac elicited intestinal damage, along with increments of myeloperoxidase, malondialdehyde, tumour necrosis factor and interleukin‐1β, overexpression of TLR2/4, myeloid differentiation primary response 88 (Myd88) and NF‐κB p65, increased faecal calprotectin and butyrate levels, and decreased blood haemoglobin levels, occludin and butyrate transporter monocarboxylate transporter 1 (MCT1) expression. In addition, diclofenac provoked a shift of bacterial taxa in both faecal and ileal samples. Treatment with S. boulardii CNCM I‐745, in both preventive and curative protocols, counteracted the majority of these deleterious changes. Only preventive administration of the probiotic counteracted NSAID‐induced decreased expression of MCT1 and increase in faecal butyrate levels. Occludin expression, after probiotic treatment, did not significantly change.
Conclusions and Implications
Treatment with S. boulardii CNCM I‐745 prevents diclofenac‐induced enteropathy through anti‐inflammatory and antioxidant activities. Such effects are likely to be related to increased tissue butyrate bioavailability, through an improvement of butyrate uptake by the enteric mucosa.
Inflammatory bowel disease (IBD) is a group of chronic and life-threating inflammatory diseases of the gastrointestinal tract. The active intestinal absorption of bile salts is reduced in IBD, resulting in higher luminal concentrations of these agents that contribute to the pathophysiology of IBD-associated diarrhea. Butyrate (BT) is a short-chain fatty acid produced by colonic bacterial fermentation of dietary fibers. BT utilization is impaired in the intestinal inflamed mucosa of IBD patients. Our aim was to investigate the link between IBD and bile acid absorption, by testing the effect of the pro-inflammatory cytokines TNF-α and IFN-γ and of BT upon 3H-TC uptake by Caco-2 cells. The proinflammatory cytokines TNF-α and IFN-γ inhibit Na+-independent, non-ASBT (sodium-dependent bile acid transporter)-mediated 3H-TC uptake by Caco-2 cells. The inhibitory effect of these cytokines on Na+-independent 3H-TC uptake is PI3K- and JAK/STAT1-mediated. These two compounds upregulate ASBT expression levels, but no corresponding increase in Na+-dependent component of 3H-TC is observed. Moreover, BT was also found to inhibit 3H-TC uptake and showed an additive effect with IFN-γ in reducing 3H-TC uptake. We conclude that an interaction between BT and bile acids appears to exist in IBD, which may participate in the link between diet, microbiota and IBD.
Background:
Mucosal healing is one of the principal therapeutic targets for ulcerative colitis (UC). Mitochondria are dynamic organelles that undergo constant fusion and fission; however, the process that is most conducive to mucosal healing remains unclear. This study investigated the role of mitochondrial fission in mucosal healing in UC patients.
Methods:
Quantitative polymerase chain reaction, Western blotting, and immunostaining were used to detect mitochondrial fission in UC patients and a dextran sulfate sodium-induced colitis model. Colonic organoids were used to investigate the role of mitochondrial fission in butyrate metabolism. Enzyme activity assays were performed to identify the key proteins involved in this mechanism.
Results:
It was found that inhibition of mitochondrial fission promoted mucosal healing in mice and that there was an increase in mitochondrial fission in colonic epithelial cells of UC patients. Excessive fission inhibits stem cell proliferation by impairing butyrate metabolism in colonic organoids. The mitochondrial fission antagonist P110 failed to promote mucosal healing in antibiotic-treated mice, and the addition of exogenous butyrate reversed this effect. Increased butyrate exposure in the colonic stem cell niche has also been observed in UC patients. Mechanistically, enzyme activity assays on colonic organoids revealed that excessive fission inhibits mitochondrial acetoacetyl-CoA thiolase activity via reactive oxygen species.
Conclusions:
Collectively, these data indicate that excessive mitochondrial fission suppresses mucosal repair by inhibiting butyrate metabolism and provides a potential target for mucosal healing in patients with ulcerative colitis.
The multiple beneficial effects of the short-chain fatty acid (SCFA) butyrate on human health, produced from fermentable carbohydrates by gut microbiota in the colon, are well known. At the intestinal level, butyrate regulates metabolism, helps in the transepithelial transport of fluids, inhibits inflammation, and induces the epithelial defense barrier. The liver receives a large amount of SCFAs along with the blood that travels from the gut via the portal vein. In many recent studies, butyrate has been reported to help prevent NAFLD, NASH, inflammation, cancer, and liver injuries. Butyrate ameliorates metabolic diseases, including insulin resistance and obesity, and has a direct relationship with preventing fatty liver diseases. Butyrate has different mechanisms of action, including strong regulatory effects on many genes expression by inhibiting the histone deacetylases (HDACs) and modulating cellular metabolism. The present review highlights the wide range of beneficial therapeutic and unfavorable side effects of butyrate, with a high potential for clinically important uses in several liver diseases.
Mucosal healing has emerged as a therapeutic goal to achieve lasting clinical remission in ulcerative colitis. Intestinal repair in response to inflammation presumably requires higher energy supplies for the restoration of intestinal barrier and physiological functions. However, epithelial energy metabolism during intestinal mucosal healing has been little studied, whereas inflammation-induced alterations have been reported in the main energy production site, the mitochondria. The aim of the present work was to assess the involvement of mitochondrial activity and the events influencing their function during spontaneous epithelial repair after colitis induction in mouse colonic crypts. The results obtained show adaptations of colonocyte metabolism during colitis to ensure maximal ATP production for supporting energetic demand by both oxidative phosphorylation and glycolysis in a context of decreased mitochondrial biogenesis and through mitochondrial function restoration during colon epithelial repair. In parallel, colitis-induced mitochondrial ROS production in colonic epithelial cells was rapidly associated with transient expression of GSH-related enzymes. Mitochondrial respiration in colonic crypts was markedly increased during both inflammatory and recovery phases despite decreased expression of several mitochondrial respiratory chain complex subunits after colitis induction. Rapid induction of mitochondrial fusion was associated with mitochondrial function restoration. Finally, in contrast with the kinetics expression of genes involved in mitochondrial oxidative metabolism and in glycolysis, the expression of glutaminase was markedly reduced in the colonic crypts both during colitis and repair phases. Overall, our data suggest that the epithelial repair after colitis induction is characterized by a rapid and transient increased capacity for mitochondrial ATP production in a context of apparent restoration of mitochondrial biogenesis and metabolic reorientation of energy production. The potential implication of energy production adaptations within colonic crypts to sustain mucosal healing in a context of altered fuel supply is discussed.
Butyrate (BT) is important in the prevention and inhibition of colorectal cancer (CRC). Inflammatory bowel disease, a risk factor for CRC, is associated with higher levels of proinflammatory cytokines and bile acids. The aim of this work was to investigate the interaction of these compounds in inhibiting BT uptake by Caco-2 cells, as a mechanism contributing to the link between IBD and CRC. TNF-α, IFN-γ, chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) markedly reduce 14C-BT uptake. All these compounds appear to inhibit MCT1-mediated BT cellular uptake at a posttranscriptional level, and, because their effect is not additive, they are most probably inhibiting MCT1 by a similar mechanism. Correspondingly, the antiproliferative effect of BT (MCT1-dependent) and of the proinflammatory cytokines and CDCA were not additive. In contrast, the cytotoxic effect of BT (MCT1-independent) and of the proinflammatory cytokines and CDCA were additive. In conclusion, proinflammatory cytokines (TNF-α and IFN-γ) and bile acids (DCA and CDCA) inhibit MCT1-mediated BT cellular uptake. These proinflammatory cytokines and CDCA were found to interfere with the antiproliferative effect of BT, mediated by an inhibitory effect upon MCT1-mediated cellular uptake of BT.
This is a thorough review of research on Autoimmune Diseases, with a particular focus on Inflammatory Bowel Diseases. This is a summary of what I learned through reading hundreds of research papers to develop a way of healing myself without the need of immunosuppressants normally used for treatment, and a long-term plan for maintaining health. Along the way I learned that most autoimmune diseases revolve around a similar underlying problem, the passage of bacteria through the intestinal wall. How our bodies respond to that depends on our genetics, the types of bacteria, as well as other factors, and ultimately determines how the resulting damage manifests itself. In my own case, my immune system responded hyperactively to the bacteria, causing severe localized damage as well as systemic effects-which gets labeled as ulcerative colitis. While the focus of the research began on that, much of it applies to other autoimmune diseases (Crohn's, multiple sclerosis, rheumatoid arthritis, etc.), and likely even some diseases that are not classified as autoimmune, such as Alzheimer's (as some research indicates that the accumulation of plaque in that disease is a consequence of bacteria from the intestines getting into the bloodstream and crossing the blood-brain barrier, triggering an inflammatory response in the brain). The paper presents the review of the research with a focus on building an understanding of what causes ulcerative colitis and other autoimmune diseases, and devising a "functional cure". I use that term to mean a treatment that leads to the elimination of any symptoms by fixing the underlying problems, but which does require some lasting lifestyle changes. A "protocol" is laid out at the end for others to follow to heal their intestinal lining and maintain health. 2
A disturbance in the symbiotic mutualism between the intestinal microbiome and the human host’s organism (syn. dysbiosis) accompanies the development of a variety of inflammatory and metabolic diseases that comprise the Metabolic Syndrome, chronic inflammatory gut diseases like Crohn’s disease, Non-alcoholic fatty liver disease (NAFLD) and cardiovascular diseases, among others. The changed uptake and effectiveness of short chain fatty acids (SCFAs) as well as an increase of the intestinal permeability are common, interdependent disease elements in this regard. Short chain fatty acids are end-products of intestinal bacterial fermentation and affect the mucosal barrier integrity via numerous molecular mechanisms. There is evidence to suggest, that SCFAs have a modulating influence on Signal transducer and activator of transcription 3 (STAT3) in intestinal epithelial cells. STAT3 is a central gene-transcription factor in signaling pathways of proliferation and inflammation. It can be activated by growth factors and other intercellular signaling molecules like the cytokine Oncostatin M (OSM). The mode of STAT3’s activation exhibits, finally, a decisive influence on the immunological balance at the intestinal mucosa. Therefore, the posttranslational modification of STAT3 under the influence of SCFAs is likely to be a very important factor within the development and -progression of dysbiosis-associated diseases. In this study, a clear positive in vitro-effect of the short chain fatty acid butyrate on the posttranslational serine727-phosphorylation of STAT3 and its total protein amount in the human adenocarcinoma cell line CACO2 is verified. Moreover, an increased gene expression of the OSM-receptor subunit OSMRβ can be observed after butyrate incubation. Histone deacetylase inhibition is shown to have a predominant role in these effects. Furthermore, a subsequent p38 MAPK-activation by Butyrate is found to be a key molecular mechanism regarding the STAT3-phosphorylation at serine727-residues. To consider the portion of butyrate receptor signaling in this context in future assays, a CACO-2 cell 3D-culture model is introduced in which an improvement of the GPR109A-receptor expression in CACO-2 cells is accomplished.
Multiple Sclerosis (MS) has been reported to be associated with intestinal inflammation and gut dysbiosis. To elucidate the underlying biology of MS-linked gut inflammation, we investigated gut infiltration of immune cells during the development of spontaneous experimental autoimmune encephalomyelitis (EAE) in humanized transgenic (Tg) mice expressing HLA-DR2a and human T cell receptor (TCR) specific for myelin basic protein peptide (MBP87-99)/HLA-DR2a complexes. Strikingly, we noted the simultaneous development of EAE and colitis, suggesting a link between autoimmune diseases of the central nervous system (CNS) and intestinal inflammation. Examination of the colon in these mice revealed the infiltration of MBP-specific Th17 cells as well as recruitment of neutrophils. Furthermore, we observed that fecal Lipocalin-2 (Lcn-2), a biomarker of intestinal inflammation, was significantly elevated and predominantly produced by the gut-infiltrating neutrophils. We then extended our findings to MS patients and demonstrate that their fecal Lcn-2 levels are significantly elevated compared to healthy donors (HDs). The elevation of fecal Lcn-2 levels correlated with reduced bacterial diversity and increased levels of other intestinal inflammation markers including neutrophil elastase and calprotectin. Of interest, bacteria thought to be beneficial for inflammatory bowel disease (IBD) such as Anaerobutyricum, Blautia, and Roseburia , were reduced in fecal Lcn-2-high MS patients. We also observed a decreasing trend in serum acetate (a short-chain fatty acid) levels in MS Lcn-2-high patients compared to HDs. Furthermore, a decrease in the relative abundance of Blautia massiliensis was significantly associated with a reduction of acetate in the serum of MS patients. This study suggests that gut infiltration of Th17 cells and recruitment of neutrophils are associated with the development of gut dysbiosis and intestinal inflammation, and that fecal Lcn-2 level is a sensitive biological indicator for gut dysbiosis in multiple sclerosis.
Background
Short-chain fatty acids (SCFAs) derived from gut bacteria are associated with protective roles in diseases ranging from obesity to colorectal cancers. Intake of microbially accessible dietary fibers (prebiotics) lead to varying effects on SCFA production in human studies, and gut microbial responses to nutritional interventions vary by individual. It is therefore possible that prebiotic therapies will require customizing to individuals.
Results
Here, we explored prebiotic personalization by conducting a three-way crossover study of three prebiotic treatments in healthy adults. We found that within individuals, metabolic responses were correlated across the three prebiotics. Individual identity, rather than prebiotic choice, was also the major determinant of SCFA response. Across individuals, prebiotic response was inversely related to basal fecal SCFA concentration, which, in turn, was associated with habitual fiber intake. Experimental measures of gut microbial SCFA production for each participant also negatively correlated with fiber consumption, supporting a model in which individuals’ gut microbiota are limited in their overall capacity to produce fecal SCFAs from fiber.
Conclusions
Our findings support developing personalized prebiotic regimens that focus on selecting individuals who stand to benefit, and that such individuals are likely to be deficient in fiber intake.
FLeuumVMoUypG_CgDJN_5gVideo Abstract
Crohn's disease affects the mucosal layer of the intestine, predominantly ileum and colon segments, with the potential to affect the expression of intestinal enzymes and transporters, and consequently, oral drug bioavailability. We carried out a quantitative proteomic analysis of inflamed and non-inflamed ileum and colon tissues from Crohn's disease patients and healthy donors. Homogenates from samples in each group were pooled and protein abundance determined by liquid chromatography-mass spectrometry (LC-MS). In inflamed Crohn's ileum, CYP3A4, CYP20A1, CYP51A1, ADH1B, ALPI, FOM1, SULT1A2, SULT1B1 and ABCB7 showed ≥10-fold reduction in abundance compared with healthy baseline. By contrast, only MGST1 showed ≥10 fold reduction in inflamed colon. Ileal UGT1A1, MGST1, MGST2, and MAOA levels increased by ≥2 fold in Crohn's patients, while only ALPI showed ≥2 fold increase in the colon. Counter-intuitively, non-inflamed ileum had a higher magnitude of fold change than inflamed tissue when compared with healthy tissue. Marked but non-uniform alterations were observed in the expression of various enzymes and transporters in ileum and colon compared with healthy samples. Modelling will allow improved understanding of the variable effects of Crohn's disease on bioavailability of orally administered drugs.
Intestinal environment disorder is a potential pathological mechanism of obesity. There is increasing evidence that disorders in the homeostasis of the intestinal environment can affect various metabolic organs, such as fat and liver, and lead to metabolic diseases. However, there are few therapeutic approaches for obesity targeting the intestinal environment. In this review, on the one hand, we discuss how intestinal microbial metabolites SCFA regulate intestinal function to improve obesity and the possible mechanisms and pathways related to obesity-related pathological processes (depending on SCFA-related receptors such as GPCRs, MCT and SMCT, and through epigenetic processes). On the other hand, we discuss dietary management strategies to enrich SCFA-producing bacteria and target specific SCFA-producing bacteria and whether fecal bacteria transplantation therapy to restore the composition of the gut microbiota to regulate SCFA can help prevent or improve obesity. Finally, we believe that it will be of great significance to establish a working model of gut– SCFA– metabolic disease development in the future for the improvement this human health concern.
Amino acids are crucial building blocks of living organisms. Together with their derivatives, they participate in many intracellular processes to act as hormones, neuromodulators, and neurotransmitters. For several decades amino acids have been studied for their potential as markers of various diseases, including inflammatory bowel diseases. Subsequent improvements in sample pretreatment, separation, and detection methods have enabled the specific and very sensitive determination of these molecules in multicomponent matrices—biological fluids and tissues. The information obtained from targeted amino acid analysis (biomarker-based analytical strategy) can be further used for early diagnostics, to monitor the course of the disease or compliance of the patients. This review will provide an insight into current knowledge about inflammatory bowel diseases, the role of proteinogenic amino acids in intestinal inflammation and modern analytical techniques used in its diagnosis and disease activity monitoring. Current advances in the analysis of amino acids focused on sample pretreatment, separation strategy, or detection methods are highlighted, and their potential in clinical laboratories is discussed. In addition, the latest clinical data obtained from the metabolomic profiling of patients suffering from inflammatory bowel diseases are summarized with a focus on proteinogenic amino acids.
1. The objectives of this study were to i) compare the effects of a commercial product providing encapsulated butyrate (EB) in combination with salinomycin in diets of broilers with impaired intestinal integrity and ii) to identify easy-to-measure biomarkers to evaluate intestinal integrity and health.
2. In total, 672 one-day-old male broilers (Ross 308) were randomly assigned to three experimental groups (eight replicates/group): no dietary supplement (control); EB (500 mg/kg, UltraGuard™-DUO, Devenish, Ireland); salinomycin (69 mg/kg feed, Sacox® 120). Impaired gut integrity was induced by a 10 times overdose of a commercially attenuated live vaccine against coccidiosis (Hipracox®, Hipra) on d 17 combined with a grower feed providing rye (50 g/kg diet).
3. Improved intestinal integrity and functionality were reflected by reduced fluorescein isothiocyanate-dextran (FITC-D) plasma levels, reduced bacterial translocation to the liver (on d 21) and increased plasma colouration level on d 21 after dietary supplementation of salinomycin, compared to a non-supplemented control diet. Both EB and salinomycin reduced plasma levels of D-lactate (P < 0.05).
4. An anti-inflammatory effect of salinomycin was indicated as the transient increase in circulating monocytes observed in the EB and control group from 20 to 28 d of age was slightly but not significantly reduced, in the salinomycin-fed group. Interestingly, greater expression of tumour necrosis factor α (TNF-α) and mucin 2 (MUC2) genes (P = 0.039 and P = 0.067, respectively) were detected in the group receiving salinomycin.
5. These effects may have collectively contributed to the significantly improved performance of broilers supplemented with salinomycin. The results indicated that EB at 500 mg/kg in feed, in contrast to salinomycin, neither supported gut health nor modulated intestinal integrity in broilers.
Type 2 diabetes mellitus (T2DM) is linked to an increased risk of breast cancer. We aimed to investigate how T2DM-associated characteristics (high levels of glucose, insulin, leptin, inflammatory mediators and oxidative stress) influence breast cancer carcinogenesis, in DMBA-treated (MCF-12ADMBA) and non-treated breast epithelial (MCF-12A) cell lines. Insulin (50 nM) promotes cell proliferation, ³H-DG uptake and lactic acid production in both cell lines. The stimulatory effects of insulin upon cell proliferation and ³H-DG uptake were hampered by rapamycin, LY294001 and BAY-876, in both cell lines. In conclusion, hyperinsulinemia, one important characteristic of T2DM, contributes to the initiation of breast cancer by a PI3K- and mTOR-dependent mechanism involving increased GLUT1-mediated glucose uptake.
Significance
The pro-proliferative effect of insulin in human breast epithelial DMBA-transformed and non-transformed cell lines is PI3K-, mTOR- and GLUT1-dependent.
Short chain fatty acids (SCFAs) produced by bacterial fermentation of dietary fiber exert myriad of beneficial effects including the amelioration of inflammation. SCFAs exist as anions at luminal pH, their entry into the cells depends on the expression and function of monocarboxylate transporters. In this regard, sodium-coupled monocarboxylate transporter-1 (SMCT-1) is one of the major proteins involved in the absorption of SCFA in the mammalian colon. However, very little is known about the mechanisms of regulation of SMCT-1 expression in health and disease. MicroRNAs (miRs) are known to play a key role in modulating gene expression. In silico analysis showed miR-29abc with highest context score and its binding region was conserved among mammals. The 3`-untranslated region (UTR) of human SMCT-1 gene was cloned into pmirGLO vector upstream of luciferase reporter and transiently transfected with miR-29a, b and c mimics into Caco-2 and/or T-84 cells. The presence of UTR of this gene significantly decreased luciferase activity compared to empty vector. Co-transfection with miR-29a, b or c resulted in further decrease in 3`UTR activity of SMCT-1 luciferase constructs. Mimic transfection significantly decreased SMCT-1 protein expression without altering mRNA expression. Further, the expression of miR-29a and c were significantly lower in mouse colon compared to small intestine, consistent with higher levels of SMCT-1 protein in the colon. Our studies demonstrated a novel finding in which miR-29a, b and c down-regulate SMCT-1 expression in colonic epithelial cells and may partly explain the differential expression of these transporters along the length of the GI tract.
Background
Short-chain fatty acids (SCFAs) derived from gut bacteria are associated with protective roles in diseases ranging from obesity to colorectal cancers. Intake of microbially accessible dietary fibers (prebiotics) lead to varying effects on SCFA production in human studies, and gut microbial responses to nutritional interventions vary by individual. It is therefore possible that prebiotic therapies will require customizing to individuals.
Results
Here, we explored prebiotic personalization by conducting a three-way crossover study of three prebiotic treatments in healthy adults. We found that within individuals, metabolic responses were correlated across the three prebiotics. Individual identity, rather than prebiotic choice, was also the major determinant of SCFA response. Across individuals, prebiotic response was inversely related to basal fecal SCFA concentration, which, in turn, was associated with habitual fiber intake. Experimental measures of gut microbial SCFA production for each participant also negatively correlated with fiber consumption, supporting a model in which individuals’ gut microbiota are limited in their overall capacity to produce fecal SCFAs from fiber.
Conclusions
Our findings support developing personalized prebiotic regimens that focus on selecting individuals who stand to benefit, and that such individuals are likely to be deficient in fiber intake.
The architecture of normal colonic mucosa suggest that terminally differentiated epithelial cells near the top of the crypt are extruded into the colonic lumen. Morphological studies have identified apoptotic cells among the differentiated phenotypes near the crypt-lumen interface, suggesting a link between pathways of differentiation, apoptosis, and cellular shedding. We studied these processes in HT29 and SW620 cells and found that compared to adherent cells, those cells which were shed during standard, uninduced culture conditions exhibited nonrandom DNA fragmentation characteristic of apoptosis. Moreover, these apoptotic cells, which accumulate in the media, exhibited a more differentiated phenotype. Because short-chain fatty acids (SCFAs) are natural effectors of colonic cell differentiation in vivo, we investigated the specificity of three 4-carbon atom SCFAs on potentiating differentiation and apoptosis, and thus accumulation of shed cells in the conditioned media, in these colonic carcinoma cell lines. Whereas the unbranched SCFA butyrate induced a more differentiated phenotype and enhanced apoptosis, two derivatives of butyrate, branched isobutyric acid and a nonmetabolizable fluorine-substituted analogue, heptafluorobutyric acid, were ineffective in inducing either differentiation or apoptosis. Thus, potentiated differentiation and apoptosis in colonic carcinoma cells were linked to SCFA structure and, most likely, utilization.
The short chain fatty acids, acetate, propionate, and butyrate are produced by colonic bacterial fermentation of non-starch polysaccharides. Butyrate is the major fuel source for the colonic epithelium and there is evidence to suggest that its oxidation is impaired in ulcerative colitis. Triplicate biopsy specimens were taken at colonoscopy from five regions of the large bowel in 15 sufferers of ulcerative colitis. These patients all had mild or quiescent colitis as assessed by clinical condition, mucosal endoscopic and histological appearance. The rate of oxidation of glucose, glutamine, and butyrate through to carbon dioxide was compared with that in biopsy specimens from 28 patients who had no mucosal abnormality. Butyrate (272 (199-368)) was the preferred fuel source for the colitic mucosa followed by glutamine (33 (24-62)) then glucose (7.2 (5.3-15)) pmol/micrograms/hour; medians and 95% confidence intervals, p < 0.01. There was no regional difference in the rate of utilisation of these metabolites. In the group with colitis the rate of butyrate oxidation to carbon dioxide was significantly impaired compared with that in normal mucosa decreasing from 472 (351-637) pmol/micrograms/hour to 272 (199-368) pmol/micrograms/hour; median and 95% confidence intervals, p = 0.016. The rate of glucose and glutamine utilisation were not significantly different between normal and colitic mucosa. These data confirm that in quiescent ulcerative colitis there is an impairment of butyrate oxidation.
Short chain fatty acids (SCFAs) are potentially valuable as a topical therapy for distal ulcerative colitis. The mechanism of action is unknown but may involve improved intracellular energy production as previous evidence indicates that colonocyte oxidation of butyrate is impaired in ulcerative colitis. No information is, however, available on human mucosal metabolism of acetate and propionate in either health or disease or the Vmax and Km values of butyrate oxidation. The aim of the study was to assess the kinetic parameters, Vmax and Km, of the complete oxidation of short chain fatty acids and glucose by human colonocytes and to explore whether a metabolic abnormality could be confirmed in patients with ulcerative colitis. Colonocytes were isolated from surgical specimens obtained from 14 patients with ulcerative colitis and eight control subjects. Incubations were performed in the presence of a concentration range of 14C-labelled acetate, propionate butyrate, and glucose. Oxidation rates were obtained by quantifying the production of 14CO2. Vmax and Km were calculated by computer fitting of the data to a Michaelis-Menten plot. No significant differences were shown in either Vmax or Km values of any of the SCFAs or glucose comparing controls and patients with ulcerative colitis. Comparing the results obtained regarding the individual SCFAs, the most striking difference was the considerably lower Km value of butyrate. The apparent Vmax of acetate tended to be higher than Vmax of propionate and butyrate. Vmax of glucose oxidation was significantly lower compared with the Vmax values of SCFA oxidation. The study shows the ability of isolated human colonocytes to utilise each of the three major SCFAs, but does not support a pathogenic role for defective metabolism of butyrate in ulcerative colitis. The considerably lower Km of butyrate oxidation supports a specific role of butyrate as an energy source for the colonic mucosa in both health and ulcerative colitis.
Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have antimitogenic, anticarcinogenic, antiinflammatory, and immunomodulatory properties. The molecular basis for these diverse properties is not known. Since the role of the nuclear factor NF-kappa B in these responses has been documented, we examined the effect of CAPE on this transcription factor. Our results show that the activation of NF-kappa B by tumor necrosis factor (TNF) is completely blocked by CAPE in a dose- and time-dependent manner. Besides TNF, CAPE also inhibited NF-kappa B activation induced by other inflammatory agents including phorbol ester, ceramide, hydrogen peroxide, and okadaic acid. Since the reducing agents reversed the inhibitory effect of CAPE, it suggests the role of critical sulfhydryl groups in NF-kappa B activation. CAPE prevented the translocation of the p65 subunit of NF-kappa B to the nucleus and had no significant effect on TNF-induced I kappa B alpha degradation, but did delay I kappa B alpha resynthesis. The effect of CAPE on inhibition of NF-kappa B binding to the DNA was specific, in as much as binding of other transcription factors including AP-1, Oct-1, and TFIID to their DNA were not affected. When various synthetic structural analogues of CAPE were examined, it was found that a bicyclic, rotationally constrained, 5,6-dihydroxy form was superactive, whereas 6,7-dihydroxy variant was least active. Thus, overall our results demonstrate that CAPE is a potent and a specific inhibitor of NF-kappa B activation and this may provide the molecular basis for its multiple immunomodulatory and antiinflammatory activities.
Increasing evidence points to a important role for inflammatory cytokines for the pathogenesis of Crohn's disease.
To compare the secretion rate of tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) by morphologically normal and inflamed intestinal mucosa from patients with Crohn's disease.
Organ cultures of intestinal biopsy specimens taken from areas of affected mucosa from patients with Crohn's disease spontaneously produced increased amounts of TNF-alpha, IL-1 beta, and IL-6 compared with controls but also biopsy specimens taken in macroscopically and microscopically unaffected areas in the same patients. Concentrations of IL-1 beta and IL-6 measured in the supernatant fluid of biopsy cultures were positively correlated with the degree of tissue involvement measured by both endoscopic and histological grading. By contrast, TNF-alpha concentrations were not correlated to endoscopic and histological grading.
These consistently raised TNF-alpha, IL-1 beta and IL-6 secretions by normal appearing mucosa from patients with Crohn's disease provide evidence for a sustained immune stimulation in Crohn's disease even in the absence of patent inflammation. The results shed a new light on the role of inflammatory cytokines in the onset of intestinal tissue damage in Crohn's disease and suggest that the range of intestinal lesions in Crohn's disease may be wider than suspected on the basis of regular endoscopic and histological examinations.
Colonic mucosal metabolism of butyrate may be impaired in ulcerative colitis. In this study we sought to confirm this observation, to determine if a similar change occurs in Crohn's colitis, and to establish whether a panenteric disorder of butyrate metabolism exists in either condition.
With use of a microculture technique, mucosal metabolic fluxes of 14[C]-labeled butyrate and 14[C]-labeled glutamine were measured as 14[C] carbon dioxide production in mucosal biopsy specimens from the colon and ileum in patients with ulcerative colitis, Crohn's colitis, and healthy bowel. Results were expressed as pmol/microg biopsy DNA/hour.
In the colon the mucosal metabolic fluxes of both butyrate and glutamine are reduced in both ulcerative colitis and Crohn's colitis compared with healthy controls. These changes were most marked in the presence of moderate to severe mucosal inflammation, there being no significant difference in mucosal metabolic flux between mildly inflamed mucosa and healthy controls. In the ileum the mucosal metabolic fluxes of butyrate and glutamine did not differ between healthy controls and those with either ulcerative colitis or Crohn's colitis.
Changes in colonic mucosal metabolism of butyrate and glutamine in inflammatory bowel disease occur as a consequence of the inflammatory process and are not peculiar to ulcerative colitis. Ileal mucosal metabolism is unchanged in ulcerative colitis and Crohn's colitis, indicating the absence of a panenteric abnormality of mucosal metabolism in these two conditions.
Sodium butyrate, a product of colonic bacterial fermentation, is able to inhibit cell proliferation and to stimulate cell differentiation of colonic epithelial cell lines. It has been proposed that these cellular effects could be linked to its ability to cause hyperacetylation of histone through the inhibition of histone deacetylase.
To analyse the molecular mechanisms of butyrate action on cell proliferation/differentiation and to compare them with those of trichostatin A, a well known inhibitor of histone deacetylase.
HT-29 cells were grown in the absence or presence of butyrate or trichostatin A. Cell proliferation and cell cycle distribution were studied after DNA staining by crystal violet and propidium iodide respectively. Cell cycle regulatory proteins were studied by western blot and reverse transcription-polymerase chain reaction. Cell differentiation was followed by measuring brush border enzyme activities. Histone acetylation was studied by acid/urea/Triton acrylamide gel electrophoresis.
Butyrate blocked cells mainly in the G(1) phase of the cell cycle, whereas trichostatin A was inhibitory in both G(1) and G(2) phases. Butyrate inhibited the mRNA expression of cyclin D1 without affecting its protein expression and stimulated the protein expression of cyclin D3 without affecting its mRNA expression. Trichostatin A showed similar effects on cyclin D1 and D3. Butyrate and trichostatin A stimulated p21 expression both at the mRNA and protein levels, whereas their effects on the expression of cyclin dependent kinases were slightly different. Moreover, butyrate strongly stimulated the activity of alkaline phosphatase and dipeptidyl peptidase IV, whereas trichostatin A had no effect. Finally, a six hour exposure to butyrate or trichostatin A induced histone H4 hyperacetylation. At 15 and 24 hours, histone H4 remained hyperacetylated in the presence of butyrate, whereas it returned to control levels in the presence of trichostatin A.
The data may explain how butyrate acts on cell proliferation/differentiation, and they show that trichostatin A does not reproduce every effect of butyrate, mainly because of its shorter half life.
Impaired colonocyte metabolism of butyrate has been implicated in the aetiopathogenesis of ulcerative colitis. Colonocyte butyrate metabolism was investigated in experimental colitis in mice.
Colitis was induced in Swiss outbred white mice by oral administration of 4% dextran sulphate sodium (DSS). Colonocytes isolated from colitic and normal control mice were incubated with [(14)C]butyrate or glucose, and production of (14)CO(2), as well as of intermediate metabolites (acetoacetate, beta-hydroxybutyrate and lactate), was measured. The effect of different substrate concentrations on oxidation was also examined.
Butyrate oxidation (micromol/h per mg protein; mean (SEM)) was significantly reduced in DSS colitis, values on day 7 of DSS administration being 0.177 (0.007) compared with 0.406 (0.035) for control animals (p<0.001). Glucose oxidation (micromol/h per mg protein; mean (SEM)) on day 7 of DSS administration was significantly higher than in controls (0.06 (0.006) v 0.027 (0.004), p<0.001). Production of beta-hydroxybutyrate was decreased and production of lactate increased in DSS colitis compared with controls. Increasing butyrate concentration from 10 to 80 mM enhanced oxidation in DSS colitis (0.036 (0.002) to 0.285 (0.040), p<0.001), although it continued to remain lower than in controls. Surface and crypt epithelial cells showed similar ratios of butyrate to glucose oxidation. When 1 mM DSS was added to normal colonocytes in vitro, it did not alter butyrate oxidation. The initial histological lesion of DSS administration was very patchy and involved crypt cells. Abnormal butyrate oxidation became apparent only after six days of DSS administration, at which time histological abnormalities were more widespread.
Colonocyte metabolism of butyrate, but not of glucose, is impaired in DSS colitis, and may be important in pathophysiology. Histological abnormalities preceded measurable defects in butyrate oxidation.
Proinflammatory cytokines are key factors in the pathogenesis of Crohn's disease (CD). Activation of nuclear factor kappa B (NFkappaB), which is involved in their gene transcription, is increased in the intestinal mucosa of CD patients. As butyrate enemas may be beneficial in treating colonic inflammation, we investigated if butyrate promotes this effect by acting on proinflammatory cytokine expression.
Intestinal biopsy specimens, isolated lamina propria cells (LPMC), and peripheral blood mononuclear cells (PBMC) were cultured with or without butyrate for assessment of secretion of tumour necrosis factor (TNF) and mRNA levels. NFkappaB p65 activation was determined by immunofluorescence and gene reporter experiments. Levels of NFkappaB inhibitory protein (IkappaBalpha) were analysed by western blotting. The in vivo efficacy of butyrate was assessed in rats with trinitrobenzene sulphonic acid (TNBS) induced colitis.
Butyrate decreased TNF production and proinflammatory cytokine mRNA expression by intestinal biopsies and LPMC from CD patients. Butyrate abolished lipopolysaccharide (LPS) induced expression of cytokines by PBMC and transmigration of NFkappaB from the cytoplasm to the nucleus. LPS induced NFkappaB transcriptional activity was decreased by butyrate while IkappaBalpha levels were stable. Butyrate treatment also improved TNBS induced colitis.
Butyrate decreases proinflammatory cytokine expression via inhibition of NFkappaB activation and IkappaBalpha degradation. These anti-inflammatory properties provide a rationale for assessing butyrate in the treatment of CD.
Regulation of NF-kappaB transactivation function is controlled at several levels, including interactions with coactivator proteins. Here we show that the transactivation function of NF-kappaB is also regulated through interaction of the p65 (RelA) subunit with histone deacetylase (HDAC) corepressor proteins. Our results show that inhibition of HDAC activity with trichostatin A (TSA) results in an increase in both basal and induced expression of an integrated NF-kappaB-dependent reporter gene. Chromatin immunoprecipitation (ChIP) assays show that TSA treatment causes hyperacetylation of the wild-type integrated NF-kappaB-dependent reporter but not of a mutant version in which the NF-kappaB binding sites were mutated. Expression of HDAC1 and HDAC2 repressed tumor necrosis factor (TNF)-induced NF-kappaB-dependent gene expression. Consistent with this, we show that HDAC1 and HDAC2 target NF-kappaB through a direct association of HDAC1 with the Rel homology domain of p65. HDAC2 does not interact with NF-kappaB directly but can regulate NF-kappaB activity through its association with HDAC1. Finally, we show that inhibition of HDAC activity with TSA causes an increase in both basal and TNF-induced expression of the NF-kappaB-regulated interleukin-8 (IL-8) gene. Similar to the wild-type integrated NF-kappaB-dependent reporter, ChIP assays showed that TSA treatment resulted in hyperacetylation of the IL-8 promoter. These data indicate that the transactivation function of NF-kappaB is regulated in part through its association with HDAC corepressor proteins. Moreover, it suggests that the association of NF-kappaB with the HDAC1 and HDAC2 corepressor proteins functions to repress expression of NF-kappaB-regulated genes as well as to control the induced level of expression of these genes.
Healthy colonocytes derive 60–70% of their energy supply from short-chain fatty acids, particularly butyrate. Butyrate has profound effects on differentiation, proliferation and apoptosis of colonic epithelial cells by regulating expression of various genes associated with these processes. We have previously shown that butyrate is transported across the luminal membrane of the colonic epithelium via a monocarboxylate transporter, MCT1. In this paper, using immunohistochemistry and in situ hybridisation histochemistry, we have determined the profile of MCT1 protein and mRNA expression along the crypt to surface axis of healthy human colonic tissue. There is a gradient of MCT1 protein expression in the apical membrane of the cells along the crypt-surface axis rising to a peak in the surface epithelial cells. MCT1 mRNA is expressed along the crypt-surface axis and is most abundant in cells lining the crypt. Analysis of healthy colonic tissues and carcinomas using immunohistochemistry and Western blotting revealed a significant decline in the expression of MCT1 protein during transition from normality to malignancy. This was reflected in a corresponding reduction in MCT1 mRNA expression, as measured by Northern analysis. Carcinoma samples displaying reduced levels of MCT1 were found to express the high affinity glucose transporter, GLUT1, suggesting that there is a switch from butyrate to glucose as an energy source in colonic epithelia during transition to malignancy. The expression levels of MCT1 in association with GLUT1 could potentially be used as determinants of the malignant state of colonic tissue.
British Journal of Cancer (2002) 86, 1262–1269. DOI: 10.1038/sj/bjc/6600264 www.bjcancer.com
© 2002 Cancer Research UK
and aims: Tumour necrosis factor alpha (TNF-alpha) induction of nuclear factor kappaB (NFkappaB) activation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). Trefoil factor family peptides TFF1, TFF2, and TFF3 exert protective, curative, and tumour suppressive functions in the gastrointestinal tract. In this study, we investigated effects of the TNF-alpha/NFkappaB regulatory pathway by TNF-alpha on expression of TFFs.
After TNF-alpha stimulation, expression of TFF genes was analysed by quantitative real time polymerase chain reaction and by reporter gene assays in the gastrointestinal tumour cell lines HT-29 and KATO III. Additionally, NFkappaB subunits and a constitutive repressive form of inhibitory factor kappaB (IkappaB) were transiently coexpressed. In vivo, morphological changes and expression of TFF3, mucins, and NFkappaB were monitored by immunohistochemistry in a rat model of 2,4,6-trinitrobenzene sulphonic acid induced colitis.
TNF-alpha stimulation evoked up to 10-fold reduction of TFF3 expression in the colon tumour cell line HT-29. Downregulation of reporter gene transcription of TFF3 was observed with both TNF-alpha and NFkappaB, and was reversible by IkappaB. In vivo, the increase in epithelial expression of NFkappaB coincided with reduced TFF3 expression during the acute phase of experimental colitis.
Downregulation of intestinal trefoil factor TFF3 is caused by repression of transcription through TNF-alpha and NFkappaB activation in vitro. In IBD, perpetual activation of NFkappaB activity may contribute to ulceration and decreased wound healing through reduced TFF3.
Impaired absorption of sodium (Na+) and water is a major factor in the pathogenesis of diarrhoea in ulcerative colitis (UC). Electrogenic Na+ absorption, present mainly in human distal colon and rectum, is defective in UC, but the molecular basis for this is unclear. The effect of UC on the expression of apical Na+ channels (ENaC) and basolateral Na+, K+-ATPase, the critical determinants of electrogenic Na+ transport, was therefore investigated in this study. Sigmoid colonic and/or proximal rectal mucosal biopsies were obtained from patients with mild to moderate UC, and patients with functional abdominal pain (controls). ENaC subunit expression was studied by immunohistochemistry, western blot analysis, and in situ hybridization, and Na+, K+-ATPase isoform expression was studied by immunohistochemistry, western blotting, and northern blot analysis. UC was associated with substantial decreases in the expression of the ENaC beta- and gamma-subunit proteins and mRNAs, whereas the decrease in ENaC alpha-subunit protein detected by immunolocalization was less marked. The levels of expression of Na+, K+-ATPase alpha1- and beta1-isoform proteins were also lower in UC patients than in controls, although there were no differences in Na+, K+-ATPase alpha1- and beta1-isoform mRNA levels between the two groups. Taken together, these results show that UC results mainly in decreased expression of the apical ENaC beta- and gamma-subunits, as well as the basolateral Na+, K+-ATPase alpha1- and beta1-isoforms. In conclusion, these changes provide a basis for the low/negligible levels of electrogenic Na+ absorption seen in the distal colon and rectum of UC patients, which contribute to the pathogenesis of diarrhoea in this disease.
Monocarboxylates such as lactate and pyruvate play a central role in cellular metabolism and metabolic communication between tissues. Essential to these roles is their rapid transport across the plasma membrane, which is catalysed by a recently identified family of proton-linked monocarboxylate transporters (MCTs). Nine MCT-related sequences have so far been identified in mammals, each having a different tissue distribution, whereas six related proteins can be recognized in Caenorhabditis elegans and 4 in Saccharomyces cerevisiae. Direct demonstration of proton-linked lactate and pyruvate transport has been demonstrated for mammalian MCT1-MCT4, but only for MCT1 and MCT2 have detailed analyses of substrate and inhibitor kinetics been described following heterologous expression in Xenopus oocytes. MCT1 is ubiquitously expressed, but is especially prominent in heart and red muscle, where it is up-regulated in response to increased work, suggesting a special role in lactic acid oxidation. By contrast, MCT4 is most evident in white muscle and other cells with a high glycolytic rate, such as tumour cells and white blood cells, suggesting it is expressed where lactic acid efflux predominates. MCT2 has a ten-fold higher affinity for substrates than MCT1 and MCT4 and is found in cells where rapid uptake at low substrate concentrations may be required, including the proximal kidney tubules, neurons and sperm tails. MCT3 is uniquely expressed in the retinal pigment epithelium. The mechanisms involved in regulating the expression of different MCT isoforms remain to be established. However, there is evidence for alternative splicing of the 5'- and 3'-untranslated regions and the use of alternative promoters for some isoforms. In addition, MCT1 and MCT4 have been shown to interact specifically with OX-47 (CD147), a member of the immunoglobulin superfamily with a single transmembrane helix. This interaction appears to assist MCT expression at the cell surface. There is still much work to be done to characterize the properties of the different isoforms and their regulation, which may have wide-ranging implications for health and disease. In the future it will be interesting to explore the linkage of genetic diseases to particular MCTs through their chromosomal location.
BACKGROUND
Sodium butyrate, a product of colonic bacterial fermentation, is able to inhibit cell proliferation and to stimulate cell differentiation of colonic epithelial cell lines. It has been proposed that these cellular effects could be linked to its ability to cause hyperacetylation of histone through the inhibition of histone deacetylase.
AIM
To analyse the molecular mechanisms of butyrate action on cell proliferation/differentiation and to compare them with those of trichostatin A, a well known inhibitor of histone deacetylase.
METHODS
HT-29 cells were grown in the absence or presence of butyrate or trichostatin A. Cell proliferation and cell cycle distribution were studied after DNA staining by crystal violet and propidium iodide respectively. Cell cycle regulatory proteins were studied by western blot and reverse transcription-polymerase chain reaction. Cell differentiation was followed by measuring brush border enzyme activities. Histone acetylation was studied by acid/urea/Triton acrylamide gel electrophoresis.
RESULTS
Butyrate blocked cells mainly in the G1 phase of the cell cycle, whereas trichostatin A was inhibitory in both G1 and G2 phases. Butyrate inhibited the mRNA expression of cyclin D1 without affecting its protein expression and stimulated the protein expression of cyclin D3 without affecting its mRNA expression. Trichostatin A showed similar effects on cyclin D1 and D3. Butyrate and trichostatin A stimulated p21 expression both at the mRNA and protein levels, whereas their effects on the expression of cyclin dependent kinases were slightly different. Moreover, butyrate strongly stimulated the activity of alkaline phosphatase and dipeptidyl peptidase IV, whereas trichostatin A had no effect. Finally, a six hour exposure to butyrate or trichostatin A induced histone H4 hyperacetylation. At 15 and 24 hours, histone H4 remained hyperacetylated in the presence of butyrate, whereas it returned to control levels in the presence of trichostatin A.
CONCLUSIONS
The data may explain how butyrate acts on cell proliferation/differentiation, and they show that trichostatin A does not reproduce every effect of butyrate, mainly because of its shorter half life.
BACKGROUND/AIMS Impaired colonocyte metabolism of butyrate has been implicated in the aetiopathogenesis of ulcerative colitis. Colonocyte butyrate metabolism was investigated in experimental colitis in mice. METHODS Colitis was induced in Swiss outbred white mice by oral administration of 4% dextran sulphate sodium (DSS). Colonocytes isolated from colitic and normal control mice were incubated with [14C]butyrate or glucose, and production of 14CO2, as well as of intermediate metabolites (acetoacetate, β-hydroxybutyrate and lactate), was measured. The effect of different substrate concentrations on oxidation was also examined. RESULTS Butyrate oxidation (μmol/h per mg protein; mean (SEM)) was significantly reduced in DSS colitis, values on day 7 of DSS administration being 0.177 (0.007) compared with 0.406 (0.035) for control animals (p<0.001). Glucose oxidation (μmol/h per mg protein; mean (SEM)) on day 7 of DSS administration was significantly higher than in controls (0.06 (0.006) v 0.027 (0.004), p<0.001). Production of β-hydroxybutyrate was decreased and production of lactate increased in DSS colitis compared with controls. Increasing butyrate concentration from 10 to 80 mM enhanced oxidation in DSS colitis (0.036 (0.002) to 0.285 (0.040), p<0.001), although it continued to remain lower than in controls. Surface and crypt epithelial cells showed similar ratios of butyrate to glucose oxidation. When 1 mM DSS was added to normal colonocytes in vitro, it did not alter butyrate oxidation. The initial histological lesion of DSS administration was very patchy and involved crypt cells. Abnormal butyrate oxidation became apparent only after six days of DSS administration, at which time histological abnormalities were more widespread. CONCLUSIONS Colonocyte metabolism of butyrate, but not of glucose, is impaired in DSS colitis, and may be important in pathophysiology. Histological abnormalities preceded measurable defects in butyrate oxidation.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
The transcription factor nuclear factor-kappaB (NF-kappaB) plays a central role in regulating immune and inflammatory responses. Because butyrate deficiency has been associated with inflammatory bowel disease, we examined the effect of butyrate on NF-kappaB activity in the human HT-29 colonic cell line.
The influence of butyrate (4 mmol/L) on NF-kappaB activity was determined using the gel mobility shift assay. The effect of butyrate on the expression of NF-kappaB subunits and inhibitory proteins was determined by immunoblotting. NF-kappaB-regulated gene expression was assayed by primer extension of intercellular adhesion molecule 1 and Mn superoxide dismutase messenger RNA, and by analysis of a transfected luciferase reporter.
Exposure of HT-29 cells to butyrate eliminated their constitutive NF-kappaB, p50 dimer activity. This inhibition corresponded with a reduction in p50 nuclear localization, without a reduction in expression. Butyrate also selectively modulated activation of NF-kappaB, suppressing its activation by tumor necrosis factor alpha and phorbol ester more than 10-fold, without affecting the activity induced by interleukin (IL)-1beta. Butyrate did, however, enhance formation of the stronger p65-p50 transcriptional activator in IL-1beta-stimulated cells. The changes in NF-kappaB activation did not correlate with changes in IkappaBalpha levels. Gene expression reflected DNA binding. The influence of butyrate on NF-kappaB may result in part from its ability to inhibit deacetylases because the specific deacetylase inhibitor trichostatin A has a similar effect.
These findings suggest that the influences of butyrate on colonic inflammatory responses may result in part from its influence on NF-kappaB activation. This activity of butyrate apparently involves its ability to inhibit deacetylases.
Oligonucleotide primers based on the human heart monocarboxylate transporter (MCT1) cDNA sequence were used to isolate a 544 bp cDNA product from human colonic RNA by reverse transcription‐polymerase chain reaction (RT‐PCR). The sequence of the RT‐PCR product was identical to that of human heart MCT1. Northern blot analysis using the RT‐PCR product indicated the presence of a single transcript of 3.3 kb in mRNA isolated from both human and pig colonic tissues. Western blot analysis using an antibody to human MCT1 identified a specific protein with an apparent molecular mass of 40 kDa in purified and well‐characterized human and pig colonic lumenal membrane vesicles (LMV).
Properties of the colonic lumenal membrane l ‐lactate transporter were studied by the uptake of L‐[U‐ ¹⁴ C]lactate into human and pig colonic LMV. l ‐lactate uptake was stimulated in the presence of an outward‐directed anion gradient at an extravesicular pH of 5.5. Transport of l ‐lactate into anion‐loaded colonic LMV appeared to be via a proton‐activated, anion exchange mechanism.
l ‐lactate uptake was inhibited by pyruvate, butyrate, propionate and acetate, but not by Cl ⁻ and SO 4 ²⁻ . The uptake of l ‐lactate was inhibited by phloretin, mercurials and α‐cyano‐4‐hydroxycinnamic acid (4‐CHC), but not by the stilbene anion exchange inhibitors, 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonic acid (DIDS) and 4‐acetamido‐4′‐isothiocyanostilbene‐2,2′‐disulphonic acid (SITS).
The results indicate the presence of a MCT1 protein on the lumenal membrane of the colon that is involved in the transport of l ‐lactate as well as butyrate across the colonic lumenal membrane. Western blot analysis showed that the abundance of this protein decreases in lumenal membrane fractions isolated from colonic carcinomas compared with that detected in the normal healthy colonic tissue.
Butyrate is a short chain fatty acid (SCFA) produced by bacterial fermentation of dietary fibers in the colon lumen which severely affects the proliferation of colon cancer cells in in vitro experiments. Although butyrate is able to interfere with numerous cellular targets including cell cycle regulator expression, little is known about butyrate metabolism and its possible involvement in its effect upon colon carcinoma cell growth. In this study, we found that HT-29 Glc−/+ cells strongly accumulated and oxidized sodium butyrate without producing ketone bodies, nor modifying oxygen consumption nor mitochondrial ATP synthesis. HT-29 cells accumulated and oxidized sodium acetate at a higher level than butyrate. However, sodium butyrate, but not sodium acetate, reduced cell growth and increased the expression of the cell cycle effector cyclin D3 and the inhibitor of the G1/S cdk-cyclin complexes p21/WAF1/Cip1, demonstrating that butyrate metabolism downstream of acetyl-CoA synthesis is not required for the growth-restraining effect of this SCFA. Furthermore, HT-29 cells modestly incorporated the 14C-labelled carbon from sodium butyrate into cellular triacylglycerols and phospholipids. This incorporation was greatly increased when d-glucose was present in the incubation medium, corresponding to the capacity of hexose to circulate in the pentose phosphate pathway allowing NADPH synthesis required for lipogenesis. Interestingly, when HT-29 cells were cultured in the presence of sodium butyrate, their capacity to incorporate 14C-labelled sodium butyrate into triacylglycerols and phospholipids was increased more than twofold. In such experimental conditions, HT-29 cells when observed under an electronic microscope, were found to be characterized by an accumulation of lipid droplets in the cytosol. Our data strongly suggest that butyrate acts upon colon carcinoma cells upstream of acetyl-CoA synthesis. In contrast, the metabolism downstream of acetyl-CoA [i.e. oxidation in the tricarboxylic acid (TCA) cycle and lipid synthesis] likely acts as a regulator of butyrate intracellular concentration.
The metabolism of the third component of complement (C3) has been investigated in four patients with ulcerative colitis, three patients with Crohn's disease and seven control subjects, using radioiodinated C3 prepared from fresh human plasma. Both the fractional catabolic rate and synthesis rate of C3 were increased in the patients with inflammatory bowel disease, although the serum-C3 levels were normal or raised. The results suggest that complement activation may play a role in the pathogenesis of mucosal inflammation in these diseases.
Suspensions of colonocytes (isolated colonic epithelial cells) were prepared from mucosa of the descending colon from 6 patients with quiescent ulcerative colitis (UC), 4 with acute UC, and 7 control subjects. In each group metabolic performance was investigated by assessing utilisation of n-butyrate, the main respiratory fuel of the colonic mucosa, as well as utilisation of glucose and glutamine. In both acute and quiescent UC oxidation of butyrate to CO2 and ketones was significantly lower than in the control tissues, and the decrease correlated with the state of the disease. Enhanced glucose and glutamine oxidation compensated for decreased butyrate oxidation in UC, indicating that colonocytes in colitis were not metabolically degenerate cells. Failure of butyrate oxidation reflects a variable yet definite metabolic defect in the mucosa in UC. Diminished oxidation of butyrate can explain the characteristic distribution of colitis along the colon, especially the frequency of UC in the distal colon. It is suggested that failure of fatty-acid (n-butyrate) oxidation in UC is an expression of an energy-deficiency disease of the colonic mucosa.
Isolated suspensions of colonocytes from the rat were used to assess utilization, interaction, and fate of metabolic substrates normally obtained from colonic bacteria (acetate, propionate, butyrate) or derived from the blood circulation to the colonic mucosa (D-glucose, acetoacetate, L-glutamine). The short-chain fatty acid n-butyrate (10 mM), on its own, accounted for 86% of the total oxygen consumption and suppressed oxidation of endogenous fuel by 82%. Ths value was not altered by the addition of acetoacetate (5 mM), of L-glutamine (5 mM), or of D-glucose (10 mM). Activation of short-chain fatty acids by colonocytes proceeded in the order of butyrate greater than acetate greater than propionate. D-Glucose on its own accounted for 30% of the oxygen consumption by colonocytes and hardly suppressed utilization of endogenous fuels. Colonocytes utilized ketone bodies (acetoacetate) and produced them (acetoacetate and beta-hydroxybutyrate) from short-chain fatty acids. Considering the interaction of substrates, isolated colonic epithelial cells utilized respiratory fuels in the preferential order of butyrate greater than acetoacetate greater than glutamine greater than glucose. The high rate of CO2 production from butyrate should be a worthwhile means of examining the functional activity of the colonic mucosa clinically and in vivo.
Previous studies have shown that butyrate is an important energy source for the distal colon, and that its metabolism may be defective in ulcerative colitis (UC). A similar metabolic defect in the ileum might account for the occurrence of 'pouchitis' in UC patients after colectomy. A method has been developed that allows the measurement of metabolism in ileocolonoscopic biopsy specimens, and this has been used to assess butyrate and glutamine metabolism in quiescent UC and controls. Preliminary experiments showed optimal metabolism of butyrate at 1 mmol/l. In controls glutamine metabolism was greater in the ascending (mean (SD)) (4.9 (3.2) nmol/h/micrograms protein) than in the descending colon (1.4 0.7)) (p < 0.05, Mann-Whitney U test), but butyrate metabolism was similar in the two regions (ascending 62.6 (44.2), descending 51.5 (32.0)). Consequently ratios of butyrate/glutamine metabolism were higher in the descending colon (20.6 (14.3)) than in the ascending colon (14.3 (9.6)) (p < 0.05). In UC, rates of butyrate metabolism were similar in the ascending (92.5 (58.3) nmol/h/micrograms protein) and descending (93.3 (115)) colon, and these were not significantly different from controls. In UC, glutamine metabolism was similar in the ascending (6.2 (7.7) nmol/h/micrograms protein) and descending colon (7.8 (7.9)); the metabolism in the descending colon was significantly greater than in controls (p < 0.01). Butyrate (135 (56) nmol/h/microgram protein) and glutamine (24.1 (16.2)) metabolism in the ileum in UC, were not significantly different from control values (butyrate 111 (57), glutamine 15.5 (15.6)). These results confirm that there is regional variation of nutrient utilisation throughout the colon, but they do not support the hypothesis that UC is caused by a deficiency of butyrate metabolism.
Although the interest in colonic mucosal metabolism of short-chain fatty acids is steadily increasing, the kinetic parameters Vmax (maximum velocity) and Km (Michaelis constant) of the complete oxidation of these acids into CO2 by colonic epithelial cells have not previously been determined.
Isolated rat colonocytes were incubated in the presence of a concentration range of 14C-labeled acetate, propionate, butyrate, and glucose. Oxidation rates were obtained by quantifying the production of 14CO2. Vmax and Km were calculated by computer-fitting of the data to a Michaelis-Menten plot.
The apparent Vmax values were similar comparing acetate, propionate, and butyrate (1.114 +/- 0.061, 0.991 +/- 0.072, and 1.007 +/- 0.070 mumol/min.g, respectively), but significantly lower for glucose (0.339 +/- 0.022 mumol/min.g). The corresponding Km values were all different and in the order of butyrate (0.184 +/- 0.017 mmol/L) < propionate (0.339 +/- 0.025 mmol/L) < acetate (0.487 +/- 0.019 mmol/L) < glucose (0.777 +/- 0.051 mmol/L). In substrate competition experiments, butyrate caused a strong noncompetitive inhibition of acetate oxidation and a mixed type of inhibition of propionate oxidation. Propionate inhibited the oxidation of acetate noncompetitively and that of butyrate competitively. Acetate only slightly inhibited the oxidation of propionate and butyrate.
Colonic epithelial cells seem to utilize short-chain fatty acids in a preferential order of butyrate > propionate > acetate. Oxidation of propionate or acetate, however, may provide the energy needed for cellular functions if the metabolism of butyrate is impaired or the luminal supply is limited.
Butyrate is an important energy source for the colon and its metabolism has been reported to be defective in ulcerative colitis. One mechanism for defective butyrate metabolism in patients with ulcerative colitis could be an enzyme deficiency in the beta-oxidation pathway of butyrate.
This study was undertaken to measure the activity of each enzyme involved in the beta-oxidation pathway of butyrate in colonic epithelium.
Patients with ulcerative colitis (n = 33), Crohn's colitis (n = 10), and control subjects with colorectal cancer or diverticular disease (n = 73) were studied.
Analysis was carried out using fluorometric and spectrophotometric techniques on homogenised epithelial biopsy specimens.
Significantly increased butyryl CoA dehydrogenase activity was found in mucosa from patients with ulcerative colitis (33.2 (28.3, 38.1) mumol/g wet weight/min:mean (95% CI)) compared with activity in mucosa from control patients (24.3 (20.9, 27.7) mumol/g wet weight/min:mean (95% CI)) p < 0.02. No significant increase in activity of the enzymes butyryl-CoA synthetase, crotonase or hydroxybutyryl-CoA dehydrogenase was found in patients with ulcerative colitis. In contrast the mucosal thiolase activity was significantly lower in those patients with quiescent colitis (3.21 (2.61, 3.81) mumol/g wet weight/min:mean (95% CI)) when compared with control mucosa (5.69 (5.09, 6.29) mumol/g wet weight/min:mean (95% CI)) p < 0.001. However, mucosal thiolase activity increases with the age of the donor patient and differences in the age range of the patient groups probably account for this finding.
This study shows no substantial deficiency of enzyme activity in the beta-oxidation pathway of butyrate in the mucosa of patients with ulcerative colitis in histological remission.
Impaired short-chain fatty acid metabolism by the colonocyte has been suggested as a pathogenic factor in ulcerative colitis (UC). The aim of this study was to measure in vivo butyrate metabolism in UC and to correlate butyrate oxidation with colonic permeability.
Butyrate oxidation was measured by means of a 14CO2-breath test after rectal instillation of 14C-butyrate. 51Cr-ethylenediaminetetraacetic acid (EDTA) was added to the enema, and the urinary % dose excretion of 51Cr-EDTA after 6 hours was a measure for permeability.
Patients with active extensive UC showed a significantly lower butyrate oxidation and increased colonic permeability in comparison to healthy controls. Butyrate oxidation correlated significantly negative with clinical activity. Oxidation of butyrate was not decreased in most patients with inactive extensive UC. In 3 patients with inactive disease and decreased oxidation, a relapse occurred within a few weeks after the test, whereas all patients with normal oxidation maintained their remission for at least 3 months. A significantly negative correlation existed between butyrate oxidation and colonic permeability.
Patients with active extensive UC have a decreased colonic butyrate oxidation. However, the fact that remission is associated with normal oxidation suggests that UC mucosa is not intrinsically altered in butyrate oxidation, making this unlikely to be a primary defect in UC.
GASTROENTEROLOGY 1999;117:745-746
Monocarboxylates such as lactate and pyruvate play a central role in cellular metabolism and metabolic communication between tissues. Essential to these roles is their rapid transport across the plasma membrane, which is catalysed by a recently identified family of proton-linked monocarboxylate transporters (MCTs). Nine MCT-related sequences have so far been identified in mammals, each having a different tissue distribution, whereas six related proteins can be recognized in Caenorhabditis elegans and 4 in Saccharomyces cerevisiae. Direct demonstration of proton-linked lactate and pyruvate transport has been demonstrated for mammalian MCT1-MCT4, but only for MCT1 and MCT2 have detailed analyses of substrate and inhibitor kinetics been described following heterologous expression in Xenopus oocytes. MCT1 is ubiquitously expressed, but is especially prominent in heart and red muscle, where it is up-regulated in response to increased work, suggesting a special role in lactic acid oxidation. By contrast, MCT4 is most evident in white muscle and other cells with a high glycolytic rate, such as tumour cells and white blood cells, suggesting it is expressed where lactic acid efflux predominates. MCT2 has a ten-fold higher affinity for substrates than MCT1 and MCT4 and is found in cells where rapid uptake at low substrate concentrations may be required, including the proximal kidney tubules, neurons and sperm tails. MCT3 is uniquely expressed in the retinal pigment epithelium. The mechanisms involved in regulating the expression of different MCT isoforms remain to be established. However, there is evidence for alternative splicing of the 5'- and 3'-untranslated regions and the use of alternative promoters for some isoforms. In addition, MCT1 and MCT4 have been shown to interact specifically with OX-47 (CD147), a member of the immunoglobulin superfamily with a single transmembrane helix. This interaction appears to assist MCT expression at the cell surface. There is still much work to be done to characterize the properties of the different isoforms and their regulation, which may have wide-ranging implications for health and disease. In the future it will be interesting to explore the linkage of genetic diseases to particular MCTs through their chromosomal location.
The short-chain fatty acid butyrate was readily taken up by Caco-2 cells. Transport exhibited saturation kinetics, was enhanced by low extracellular pH, and was Na(+) independent. Butyrate uptake was unaffected by DIDS; however, alpha-cyano-4-hydroxycinnamate and the butyrate analogs propionate and L-lactate significantly inhibited uptake. These results suggest that butyrate transport by Caco-2 cells is mediated by a transporter belonging to the monocarboxylate transporter family. We identified five isoforms of this transporter, MCT1, MCT3, MCT4, MCT5, and MCT6, in Caco-2 cells by PCR, and MCT1 was found to be the most abundant isoform by RNase protection assay. Transient transfection of MCT1, in the antisense orientation, resulted in significant inhibition of butyrate uptake. The cells fully recovered from this inhibition by 5 days after transfection. In conclusion, our data showed that the MCT1 transporter may play a major role in the transport of butyrate into Caco-2 cells.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
Butyrate is the principal source of energy for colonic epithelial cells, and has profound effects on their proliferation, differentiation and apoptosis. Transport of butyrate across the colonocyte luminal membrane is mediated by the monocarboxylate transporter 1 (MCT1). We have examined the regulation of expression of human colonic MCT1 by butyrate, in cultured colonic epithelial cells (AA/C1). Treatment with sodium butyrate (NaBut) resulted in a concentration- and time-dependent upregulation of both MCT1 mRNA and protein. At 2 mM butyrate, the magnitude of induction of mRNA (5.7-fold) entirely accounted for the 5.2-fold increase in protein abundance, and was mediated by both activation of transcription and enhanced mRNA stability. The other monocarboxylates found naturally in the colon, acetate and propionate, had no effect. The properties of butyrate uptake by AA/C1 cells were characteristic of MCT1. Induction of the MCT1 protein resulted in a corresponding increase in the maximal rate of butyrate transport. The V(max) for uptake of [U-(14)C]butyrate was increased 5-fold following pre-incubation with 2 mM NaBut, with no significant change in the apparent K(m). In conclusion, this study is the first to show substrate-induced regulation of human colonic MCT1. The basis of this regulation is a butyrate-induced increase in MCT1 mRNA abundance, resulting from the dual control of MCT1 gene transcription and stability of the MCT1 transcript. We suggest that butyrate-induced increases in the expression and resulting activity of MCT1 serve as a mechanism to maximise intracellular availability of butyrate, to act both as a source of energy and to influence processes maintaining cellular homeostasis in the colonic epithelium.
Uptake of butyrate across the colonocyte luminal membrane is mediated by the monocarboxylate transporter isoform 1 (MCT1). We have demonstrated previously that expression of human colonic MCT1 is responsive to butyrate, and that this involves the dual control of MCT1 gene transcription and stability of the MCT1 transcript. Here we describe the structural organization of the human MCT1 gene, and report the isolation and characterization of the MCT1 gene promoter. The MCT1 gene spans approximately 44 kb, and is organized as 5 exons intervened by 4 introns. The first of these introns is located in the 5'-UTR-encoding DNA, spans >26 kb, and thus accounts for approximately 60% of the entire transcription unit. Analysis of a 1.5 kb fragment of the MCT1 5'-flanking region, shows an absence of the classical TATA-Box motif. However, the region contains potential binding sites for a variety of transcription factors with known association with butyrate's action in the colon. In transient transfections the 5'-flanking region drives high-level expression of a luciferase reporter-gene in cells that endogenously express MCT1. Deletion analyses indicate that the cis-acting elements necessary for basal transcription of MCT1 are contained within the -70/+213 proximal sequence of the promoter.
The main limiting factor for sodium absorption in distal colon is the amiloride-sensitive epithelial sodium channel (ENaC). This study aimed to characterize mechanisms involved in the dysregulation of ENaC expression in ulcerative colitis (UC).
Epithelial preparations from surgically removed inflamed and control sigmoid colons were used. Active electrogenic Na(+) transport (J(Na)) was determined after 8-hour aldosterone stimulation in Ussing-chambers (corrected for the altered epithelial/subepithelial resistance ratio). Subsequently, ENaC alpha-, beta-, and gamma-subunits were analyzed immunohistochemically and in Western and Northern blots (corrected for the inflammatory increase in subepithelial protein content). To study gene regulation, the promoters of beta- and gamma-ENaC were analyzed in reporter gene assays.
In controls, aldosterone stimulated J(Na) and induced ENaC beta- and gamma-subunit expression, whereas this response was virtually abolished in UC. Preservation of surface epithelium in UC was indicated by unchanged ENaC alpha-subunit expression, which points also against a mere immaturity or epithelial cell loss. Inhibition of electrogenic sodium transport as well as beta- and gamma-ENaC mRNA expression could be mimicked in control colon by in vitro preexposure for 8 hours to tumor necrosis factor alpha and interferon gamma. Promoter analysis revealed that down-regulation of beta- and gamma-ENaC gene expression was primarily induced by tumor necrosis factor alpha.
We conclude that, in UC, elevated proinflammatory cytokines selectively impair beta- and gamma-ENaC expression, which contributes to diarrhea by reducing colonic sodium absorption.
We fractionated leukocytes from three donors into >90% pure samples of granulocytes, lymphocytes, and monocytes and tested them for transcriptional and translational expression of three physiologically-proven lactate transporters, monocarboxylate transporter 1(MCT1), MCT2, and MCT4, using RT-PCR and affinity-purified rabbit antibody (Ab) to the C-terminal segment of each human MCT. Transcripts of all three MCTs were identified in each leukocyte fraction by RT-PCR and proven by sequencing of fragments extracted after isolation on agarose gels. Transporter protein of the appropriate size was demonstrated for each of the monocarboxylate transporters MCTs in lymphocytes and monocytes by Western blot, while lower-molecular-weight bands were found in granulocytes and are presumed to be degraded forms, because they were blocked by antibody-antigen (Ab-Ag) preincubation. IHC demonstrated all three MCTs in methanol-fixed droplets of all three leukocyte fractions; stain was abolished on omission of the primary Ab. Plasmalemmal staining occurred with all MCTs in all leukocyte fractions. Because the K(m) for lactate increases approximately fivefold at each step, with MCT2<1<4, leukocytes must use the full range of lactate binding to survive in acidic and hypoxic environments. Except for MCT4 in lymphocytes, all the MCTs also stained leukocyte cytoplasm, often with distinct granularity. Nuclear membrane staining was also seen with MCT1 and MCT2, while platelet plasmalemma stained only with MCT2.
Butyrate serves as the major source of energy for colonic epithelial cells, and has profound effects on their proliferation, differentiation, and apoptosis. Transport of butyrate across the colonocyte luminal membrane is mediated by the monocarboxylate transporter, MCT1; the expression of which is down-regulated dramatically during colon carcinogenesis. We have proposed that the decline in MCT1 expression during colon carcinogenesis may reduce the intracellular availability of butyrate required to regulate expression of genes associated with the processes maintaining tissue homeostasis within the colonic mucosa.
To test this hypothesis we used the technique of RNA interference to inhibit MCT1 expression specifically, and determined the consequences of this inhibition on the ability of butyrate to exert its recognized effects in vitro using flow cytometry, immunofluorescence, Northern analysis, and Western analysis.
We show that inhibition of MCT1 expression, and hence butyrate uptake, has profound inhibitory effects on the ability of butyrate to regulate expression of key target genes: p21waf1/cip1 (p21), intestinal alkaline phosphatase (IAP), and cyclin D1, and their associated processes of proliferation and differentiation. In contrast, inhibition of MCT1 expression had no effect on the ability of butyrate to modulate expression of either bcl-XL or bak, and this was reflected in a corresponding lack of effect on butyrate induction of apoptosis.
Collectively, these results show the importance of MCT1 to the ability of butyrate to induce cell-cycle arrest and differentiation, and suggest fundamental differences in the mechanisms by which butyrate modulates specific aspects of cell function.
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