Functional variants in the sucrase-isomaltase gene associate with increased risk of irritable bowel syndrome.

OBJECTIVE: IBS is a common gut disorder of uncertain pathogenesis. Among other factors, genetics and certain foods are proposed to contribute. Congenital sucrase-isomaltase deficiency (CSID) is a rare genetic form of disaccharide malabsorption characterised by diarrhoea, abdominal pain and bloating, which are features common to IBS. We tested sucrase-isomaltase (SI) gene variants for their potential relevance in IBS. DESIGN: We sequenced SI exons in seven familial cases, and screened four CSID mutations (p.Val557Gly, p.Gly1073Asp, p.Arg1124Ter and p.Phe1745Cys) and a common SI coding polymorphism (p.Val15Phe) in a multicentre cohort of 1887 cases and controls. We studied the effect of the 15Val to 15Phe substitution on SI function in vitro. We analysed p.Val15Phe genotype in relation to IBS status, stool frequency and faecal microbiota composition in 250 individuals from the general population. RESULTS: CSID mutations were more common in patients than asymptomatic controls (p=0.074; OR=1.84) and Exome Aggregation Consortium reference sequenced individuals (p=0.020; OR=1.57). 15Phe was detected in 6/7 sequenced familial cases, and increased IBS risk in case-control and population-based cohorts, with best evidence for diarrhoea phenotypes (combined p=0.00012; OR=1.36). In the population-based sample, 15Phe allele dosage correlated with stool frequency (p=0.026) and Parabacteroides faecal microbiota abundance (p=0.0024). The SI protein with 15Phe exhibited 35% reduced enzymatic activity in vitro compared with 15Val (p<0.05). CONCLUSIONS: SI gene variants coding for disaccharidases with defective or reduced enzymatic activity predispose to IBS. This may help the identification of individuals at risk, and contribute to personalising treatment options in a subset of patients.

Dense genotyping of immune-related loci identifies HLA variants associated with increased risk of collagenous colitis.

OBJECTIVE: Collagenous colitis (CC) is a major cause of chronic non-bloody diarrhoea, particularly in the elderly female population. The aetiology of CC is unknown, and still poor is the understanding of its pathogenesis. This possibly involves dysregulated inflammation and immune-mediated reactions in genetically predisposed individuals, but the contribution of genetic factors to CC is underinvestigated. We systematically tested immune-related genes known to impact the risk of several autoimmune diseases for their potential CC-predisposing role. DESIGN: Three independent cohorts of histologically confirmed CC cases (N=314) and controls (N=4299) from Sweden and Germany were included in a 2-step association analysis. Immunochip and targeted single nucleotide polymorphism (SNP) genotype data were produced, respectively, for discovery and replication purposes. Classical human leucocyte antigen (HLA) variants at 2-digit and 4-digit resolution were obtained via imputation from single marker genotypes. SNPs and HLA variants passing quality control filters were tested for association with CC with logistic regression adjusting for age, sex and country of origin. RESULTS: Forty-two markers gave rise to genome-wide significant association signals, all contained within the HLA region on chromosome 6 (best p=4.2×10-10 for SNP rs4143332). Among the HLA variants, most pronounced risk effects were observed for 8.1 haplotype alleles including DQ2.5, which was targeted and confirmed in the replication data set (p=2.3×10-11; OR=2.06; 95% CI (1.67 to 2.55) in the combined analysis). CONCLUSIONS: HLA genotype associates with CC, thus implicating HLA-related immune mechanisms in its pathogenesis.

Bidirectional communication between the Aryl hydrocarbon Receptor (AhR) and the microbiome tunes host metabolism.

The ligand-induced transcription factor, aryl hydrocarbon receptor (AhR) is known for its capacity to tune adaptive immunity and xenobiotic metabolism-biological properties subject to regulation by the indigenous microbiome. The objective of this study was to probe the postulated microbiome-AhR crosstalk and whether such an axis could influence metabolic homeostasis of the host. Utilising a systems-biology approach combining in-depth 1H-NMR-based metabonomics (plasma, liver and skeletal muscle) with microbiome profiling (small intestine, colon and faeces) of AhR knockout (AhR-/-) and wild-type (AhR+/+) mice, we assessed AhR function in host metabolism. Microbiome metabolites such as short-chain fatty acids were found to regulate AhR and its target genes in liver and intestine. The AhR signalling pathway, in turn, was able to influence microbiome composition in the small intestine as evident from microbiota profiling of the AhR+/+ and AhR-/- mice fed with diet enriched with a specific AhR ligand or diet depleted of any known AhR ligands. The AhR-/- mice also displayed increased levels of corticosterol and alanine in serum. In addition, activation of gluconeogenic genes in the AhR-/- mice was indicative of on-going metabolic stress. Reduced levels of ketone bodies and reduced expression of genes involved in fatty acid metabolism in the liver further underscored this observation. Interestingly, exposing AhR-/- mice to a high-fat diet showed resilience to glucose intolerance. Our data suggest the existence of a bidirectional AhR-microbiome axis, which influences host metabolic pathways.

Exploring the genetics of irritable bowel syndrome: a GWA study in the general population and replication in multinational case-control cohorts.

OBJECTIVE: IBS shows genetic predisposition, but adequately powered gene-hunting efforts have been scarce so far. We sought to identify true IBS genetic risk factors by means of genome-wide association (GWA) and independent replication studies. DESIGN: We conducted a GWA study (GWAS) of IBS in a general population sample of 11,326 Swedish twins. IBS cases (N=534) and asymptomatic controls (N=4932) were identified based on questionnaire data. Suggestive association signals were followed-up in 3511 individuals from six case-control cohorts. We sought genotype-gene expression correlations through single nucleotide polymorphism (SNP)-expression quantitative trait loci interactions testing, and performed in silico prediction of gene function. We compared candidate gene expression by real-time qPCR in rectal mucosal biopsies of patients with IBS and controls. RESULTS: One locus at 7p22.1, which includes the genes KDELR2 (KDEL endoplasmic reticulum protein retention receptor 2) and GRID2IP (glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein), showed consistent IBS risk effects in the index GWAS and all replication cohorts and reached p=9.31×10(-6) in a meta-analysis of all datasets. Several SNPs in this region are associated with cis effects on KDELR2 expression, and a trend for increased mucosal KDLER2 mRNA expression was observed in IBS cases compared with controls. CONCLUSIONS: Our results demonstrate that general population-based studies combined with analyses of patient cohorts provide good opportunities for gene discovery in IBS. The 7p22.1 and other risk signals detected in this study constitute a good starting platform for hypothesis testing in future functional investigations.

Gut microbial communities modulating brain development and function.

Mammalian brain development is initiated in utero and internal and external environmental signals can affect this process all the way until adulthood. Recent observations suggest that one such external cue is the indigenous microbiota which has been shown to affect developmental programming of the brain. This may have consequences for brain maturation and function that impact on cognitive functions later in life. This review discusses these recent findings from a developmental perspective.

Evaluation of the cyclooxygenase inhibiting effects of six major cannabinoids isolated from Cannabis sativa.

Cyclooxygenase enzymes (COX-1 and COX-2) catalyse the production of prostaglandins from arachidonic acid. Prostaglandins are important mediators in the inflammatory process and their production can be reduced by COX-inhibitors. Endocannabinoids, endogenous analogues of the plant derived cannabinoids, occur normally in the human body. The Endocannabinoids are structurally similar to arachidonic acid and have been suggested to interfere with the inflammatory process. They have also been shown to inhibit cancer cell proliferation. Anti-inflammatory effects of cannabinoids and endocannabinoids have been observed, however the mode of action is not yet clarified. Anti-inflammatory activity (i.e., inhibition of COX-2) is proposed to play an important role in the development of colon cancer, which makes this subject interesting to study further. In the present work, the six cannabinoids tetrahydrocannabinol (Δ9-THC), tetrahydrocannabinolic acid (Δ9-THC-A), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabigerol (CBG) and cannabigerolic acid (CBGA), isolated from Cannabis sativa, were evaluated for their effects on prostaglandin production. For this purpose an in vitro enzyme based COX-1/COX-2 inhibition assay and a cell based prostaglandin production radioimmunoassay were used. Cannabinoids inhibited cyclooxygenase enzyme activity with IC50 values ranging from 1.7·10⁻³ to 2.0·10⁻4 M.

Decreased fat storage by Lactobacillus paracasei is associated with increased levels of angiopoietin-like 4 protein (ANGPTL4).

BACKGROUND: Intervention strategies for obesity are global issues that require immediate attention. One approach is to exploit the growing consensus that beneficial gut microbiota could be of use in intervention regimes. Our objective was to determine the mechanism by which the probiotic bacteria Lactobacillus paracasei ssp paracasei F19 (F19) could alter fat storage. Angiopoietin-like 4 (ANGPTL4) is a circulating lipoprotein lipase (LPL) inhibitor that controls triglyceride deposition into adipocytes and has been reported to be regulated by gut microbes. METHODOLOGY/PRINCIPAL FINDINGS: A diet intervention study of mice fed high-fat chow supplemented with F19 was carried out to study potential mechanistic effects on fat storage. Mice given F19 displayed significantly less body fat, as assessed by magnetic resonance imaging, and a changed lipoprotein profile. Given that previous studies on fat storage have identified ANGPTL4 as an effector, we also investigated circulating levels of ANGPTL4, which proved to be higher in the F19-treated group. This increase, together with total body fat and triglyceride levels told a story of inhibited LPL action through ANGPTL4 leading to decreased fat storage. Co-culture experiments of colonic cell lines and F19 were set up in order to monitor any ensuing alterations in ANGPTL4 expression by qPCR. We observed that potentially secreted factors from F19 can induce ANGPTL4 gene expression, acting in part through the peroxisome proliferator activated receptors alpha and gamma. To prove validity of in vitro findings, germ-free mice were monocolonized with F19. Here we again found changes in serum triglycerides as well as ANGPTL4 in response to F19. CONCLUSIONS/SIGNIFICANCE: Our results provide an interesting mechanism whereby modifying ANGPTL4, a central player in fat storage regulation, through manipulating gut flora could be an important gateway upon which intervention trials of weight management can be based.

Guidance for substantiating the evidence for beneficial effects of probiotics: current status and recommendations for future research.

Probiotic bacteria are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. There is a growing interest in probiotics within the scientific community, with consumers, and in the food industry. The interactions between the gut and intestinal microbiota and between resident and transient microbiota define a new arena in physiology, an understanding of which would shed light on the "cross-talk" between humans and microbes. The different beneficial effects of specific probiotic strains may be translated into different health claims. However, there is a need for comprehensive and harmonized guidelines on the assessment of the characteristics and efficacy of probiotics and of foods containing them. An international expert group of ILSI has evaluated the published evidence of the functionality of different probiotics in 4 areas of (human) application: 1) metabolism, 2) chronic intestinal inflammatory and functional disorders, 3) infections, and 4) allergy. Based on the existing evidence, concrete examples of demonstration of benefits and gaps are listed, and guidelines and recommendations are defined that should help design the next generation of probiotic studies.

Guidance for substantiating the evidence for beneficial effects of probiotics: impact of probiotics on digestive system metabolism.

Probiotic bacteria have been studied for their potential impact on the metabolism of dietary components in the small intestine lumen including lactose digestion, metabolism of lipids such as cholesterol, and oxalate metabolism. In the large intestine, they contribute to the metabolism of otherwise indigestible dietary carbohydrates (e.g., prebiotics) and have a favorable effect on colonic protein and ammonia metabolism, although their effect on the digestive fate of phytochemicals and xenobiotics is still uncertain. Probiotics also influence metabolism in the host tissues, in particular the gastrointestinal mucosa and the liver. Underlying mechanisms include supply of additional enzymatic activities in the gut lumen and alterations of the composition or metabolic pattern of the gut resident microbiota. For future studies, selection of probiotic strains should include assessment of their metabolic activities, and the outcome of the intervention studies should also take into account the composition of the probiotic matrix and the background diet of the target population. New technologies such as metabolomics hold great promise for assessment of probiotics functionality.

N-Nitrosopiperidine and N-Nitrosodibutylamine induce apoptosis in HepG2 cells via the caspase dependent pathway.

The human hepatoma cell line (HepG2) exhibited a dose and time-dependent apoptotic response following treatment with N-Nitrosopiperidine (NPIP) and N-Nitrosodibutylamine (NDBA), two recognized human carcinogens. Our results showed a significant apoptotic cell death (95%) after 24h treatment with NDBA (3.5 mM), whereas it was necessary to use high doses of NPIP (45 mM) to obtain a similar percentage of apoptotic cells (86%). In addition, both extrinsic (caspase-8) and intrinsic pathway (caspase-9) could be implicated in the N-Nitrosamines-induced apoptosis. This study also addresses the role of reactive oxygen species (ROS) as intermediates for apoptosis signaling. A significant increase in ROS levels was observed after NPIP treatment, whereas NDBA did not induce ROS. However, N-acetylcysteine (NAC) did not block NPIP-induced apoptosis. All these findings suggest that NPIP and NDBA induce apoptosis in HepG2 cells via a pathway that involves caspases but not ROS.

Intestinal microbiota regulate xenobiotic metabolism in the liver.

BACKGROUND: The liver is the central organ for xenobiotic metabolism (XM) and is regulated by nuclear receptors such as CAR and PXR, which control the metabolism of drugs. Here we report that gut microbiota influences liver gene expression and alters xenobiotic metabolism in animals exposed to barbiturates. PRINCIPAL FINDINGS: By comparing hepatic gene expression on microarrays from germfree (GF) and conventionally-raised mice (SPF), we identified a cluster of 112 differentially expressed target genes predominantly connected to xenobiotic metabolism and pathways inhibiting RXR function. These findings were functionally validated by exposing GF and SPF mice to pentobarbital which confirmed that xenobiotic metabolism in GF mice is significantly more efficient (shorter time of anesthesia) when compared to the SPF group. CONCLUSION: Our data demonstrate that gut microbiota modulates hepatic gene expression and function by altering its xenobiotic response to drugs without direct contact with the liver.

Genetic mapping of Mom5, a novel modifier of Apc(Min)-induced intestinal tumorigenesis.

The initial purpose of this study was to assess the role of estrogen receptor beta (ERbeta) in intestinal tumorigenesis by examining the effects of an ERbeta knockout (ERbeta(-/-)) on Apc(Min) mice. In order to accomplish this goal on a uniform genetic background, we were required to backcross the ERbeta knockout from the 129P2 genetic background to the B6 genetic background for 10 generations. Midway through this process, we performed a test cross in which mice from the N(5) backcross generation of the ERbeta knockout strain were intercrossed with Apc(Min/+) mice to obtain Apc(Min/+) ERbeta(+/+), Apc(Min/+) ERbeta(+/-) and Apc(Min/+) ERbeta(-/-) mice. Intestinal tumorigenesis in the N(5)F(2) mice was evaluated at 14 weeks of age. The analysis of the impact of ERbeta in the N(5) cross was complicated by segregating 129P2-derived alleles that affected tumor number and were unlinked to ERbeta. Genetic linkage analysis of this cross permitted the localization of a single genetic modifier of tumor number in Apc(Min/+) mice. This locus, Modifier of Min 5 (Mom5), maps to proximal mouse chromosome 5; the 129P2 allele of this locus is associated with a 50% reduction in mean intestinal tumor number. Through in silico analysis and confirmatory sequencing, we have identified the Rad50-interacting protein-1 gene as a strong candidate for Mom5.

Disruption of estrogen receptor signaling enhances intestinal neoplasia in Apc(Min/+) mice.

Estrogen receptors (ERs) [ERalpha (Esr1) and ERbeta (Esr2)] are expressed in the human colon, but during the multistep process of colorectal carcinogenesis, expression of both ERalpha and ERbeta is lost, suggesting that loss of ER function might promote colorectal carcinogenesis. Through crosses between an ERalpha knockout and Apc(Min) mouse strains, we demonstrate that ERalpha deficiency is associated with a significant increase in intestinal tumor multiplicity, size and burden in Apc(Min/+) mice. Within the normal intestinal epithelium of Apc(Min/+) mice, ERalpha deficiency is associated with an accumulation of nuclear beta-catenin, an indicator of activation of the Wnt-beta-catenin-signaling pathway, which is known to play a critical role in intestinal cancers. Consistent with the hypothesis that ERalpha deficiency is associated with activation of Wnt-beta-catenin signaling, ERalpha deficiency in the intestinal epithelium of Apc(Min/+) mice also correlated with increased expression of Wnt-beta-catenin target genes. Through crosses between an ERbeta knockout and Apc(Min) mouse strains, we observed some evidence that ERbeta deficiency is associated with an increased incidence of colon tumors in Apc(Min/+) mice. This effect of ERbeta deficiency does not involve modulation of Wnt-beta-catenin signaling. Our studies suggest that ERalpha and ERbeta signaling modulate colorectal carcinogenesis, and ERalpha does so, at least in part, by regulating the activity of the Wnt-beta-catenin pathway.

Antiapoptotic effects of dietary antioxidants towards N-nitrosopiperidine and N-nitrosodibutylamine-induced apoptosis in HL-60 and HepG2 cells.

The aim of this work was to determine the effect of vitamin C, diallyl disulfide (DADS) and dipropyl disulfide (DPDS) towards N-nitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA)-induced apoptosis in human leukemia (HL-60) and hepatoma (HepG2) cell lines using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. None of the vitamin C (5-50 microm), DADS and DPDS (1-5 microm) concentrations selected induced a significant percentage of apoptosis. In simultaneous treatments, vitamin C, DADS and DPDS reduced the apoptosis induced by NPIP and NDBA in HL-60 and HepG2 cells (around 70% of reduction). We also investigated its scavenging activities towards reactive oxygen species (ROS) produced by NPIP and NDBA using 2',7'-dichlorodihydrofluorescein diacetate in both cell lines. ROS production induced by both N-nitrosamine was reduced to control levels by vitamin C (5-50 microm) in a dose-dependent manner. However, DADS (5 microm) increased ROS levels induced by NPIP and NDBA in HL-60 (40 and 20% increase, respectively) and HepG2 cells (18% increase), whereas DPDS was more efficient scavenger of ROS at the lowest concentration (1 microm) in both HL-60 (52 and 25% reduction, respectively) and HepG2 cells (24% reduction). The data demonstrated that the scavenging ability of vitamin C and DPDS could contribute to inhibition of the NPIP- and NDBA-induced apoptosis. However, more than one mechanism, such as inhibition of phase I and/or induction of phase II enzymes, could be implicated in the protective effect of dietary antioxidants towards NPIP- and NDBA-induced apoptosis in HL-60 and HepG2 cells.

NMR metabolomic analysis of fecal water from subjects on a vegetarian diet.

A vegetarian diet rich in phytochemicals may prevent colon carcinogenesis by affecting biochemical processes in the colonic mucosa. Compounds passing the digestive system reaching the colon could potentially be detected in fecal water. We previously reported that intact fecal water samples from human volunteers significantly decreased prostaglandin production and COX-2 protein expression in colonic cells. The aim with the present study was to further study the composition of the fecal waters, using NMR spectroscopy and multivariate data analysis, and to trace the COX-2 inhibiting activity. Intact fecal water samples and fractions thereof were analyzed for their ability to inhibit prostaglandin E2 production in the human colon cell line HT-29. The majority of the tested aqueous phases derived from intact fecal water showed ability to inhibit prostaglandin production in cells (13.8+/-1.34% inhibition, p=0.01). NMR analysis indicated the presence of significant quantities of amino acids and fatty acids. Major metabolites included; acetic acid, butanoic acid, propanoic acid, glutamic acid and alanine. Smaller amounts of glycine and fumaric acid, which are known to have anti-inflammatory and anti-tumorigenic properties, were also detected. This study describes for the first time NMR metabolomic analysis of fecal water from subjects on a vegetarian diet.

Inhibition by vitamin C of apoptosis induced by N-nitrosamines in HepG2 and HL-60 cells.

The aim of this study was to evaluate the effect of vitamin C towards N-nitrosopyrrolidine (NPYR)- and N-nitrosodimethylamine (NDMA)-induced apoptosis in human hepatoma (HepG2) and leukemia (HL-60) cell lines using flow cytometry analysis and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay (TUNEL). None of the vitamin C concentrations tested (1-100 microM) caused cytotoxicity in HepG2 cells. However, there were significant losses of HL-60 cells viability, measured by MTT assay, 72 h after treatment with 50 and 100 microM vitamin C (29 and 46%, respectively). Moreover, an increase of lactate dehydrogenase release was significant with 50 microM at 72 h (28%) and with 100 microM of vitamin C at 48 and 72 h (27 and 36%, respectively). Also, the percentage of apoptotic HL-60 cells found in TUNEL assay increased to 21% when they were treated with 100 microM vitamin C for 72 h. Thus, in subsequent simultaneous treatments with NPYR (30 and 50 mM) or NDMA (27 and 68 mM) and vitamin C, concentrations of 5-50 microM vitamin C were used. Our results revealed that vitamin C, at all concentrations and times tested, reduced the apoptosis induced by NPYR and NDMA in both cell lines, showing a similar effect in HepG2 and HL-60 cells towards NPYR (50 mM)--65 and 63% of reduction, respectively--whereas towards NDMA (27 mM) the inhibition was higher in HL-60 than in HepG2 cells--75 and 57%, respectively. Therefore, our findings suggest that inhibition of apoptosis may be one of the mechanisms by which vitamin C exerts its protective effect.