The intestinal microbiota plays a critical role in maintaining human health; however, the mechanisms governing the normal homeostatic number and composition of these microbes are largely unknown. ...Previously it was shown that intestinal alkaline phosphatase (IAP), a small intestinal brush border enzyme, functions as a gut mucosal defence factor limiting the translocation of gut bacteria to mesenteric lymph nodes. In this study the role of IAP in the preservation of the normal homeostasis of the gut microbiota was investigated.
Bacterial culture was performed in aerobic and anaerobic conditions to quantify the number of bacteria in the stools of wild-type (WT) and IAP knockout (IAP-KO) C57BL/6 mice. Terminal restriction fragment length polymorphism, phylogenetic analyses and quantitative real-time PCR of subphylum-specific bacterial 16S rRNA genes were used to determine the compositional profiles of microbiotas. Oral supplementation of calf IAP (cIAP) was used to determine its effects on the recovery of commensal gut microbiota after antibiotic treatment and also on the colonisation of pathogenic bacteria.
IAP-KO mice had dramatically fewer and also different types of aerobic and anaerobic microbes in their stools compared with WT mice. Oral supplementation of IAP favoured the growth of commensal bacteria, enhanced restoration of gut microbiota lost due to antibiotic treatment and inhibited the growth of a pathogenic bacterium (Salmonella typhimurium).
IAP is involved in the maintenance of normal gut microbial homeostasis and may have therapeutic potential against dysbiosis and pathogenic infections.
Background
Patients with type 1 diabetes have shown an increase in circulating cytokines, altered lipoprotein metabolism and signs of vascular dysfunction in response to high‐fat meals. Intestinal ...alkaline phosphatase (IAP) regulates lipid transport and inflammatory responses in the gastrointestinal tract. We therefore hypothesized that changes in IAP activity could have profound effects on gut metabolic homeostasis in patients with type 1 diabetes.
Methods
Faecal samples of 41 nondiabetic controls and 46 patients with type 1 diabetes were analysed for IAP activity, calprotectin, immunoglobulins and short‐chain fatty acids (SCFAs). The impact of oral IAP supplementation on intestinal immunoglobulin levels was evaluated in C57BL/6 mice exposed to high‐fat diet for 11 weeks.
Results
Patients with type 1 diabetes exhibited signs of intestinal inflammation. Compared to controls, patients with diabetes had higher faecal calprotectin levels, lower faecal IAP activities accompanied by lower propionate and butyrate concentrations. Moreover, the amount of faecal IgA and the level of antibodies binding to oxidized LDL were decreased in patients with type 1 diabetes. In mice, oral IAP supplementation increased intestinal IgA levels markedly.
Conclusion
Deprivation of protective intestinal factors may increase the risk of inflammation in the gut – a phenomenon that seems to be present already in patients with uncomplicated type 1 diabetes. Low levels of intestinal IgA and antibodies to oxidized lipid epitopes may predispose such patients to inflammation‐driven complications such as cardiovascular disease and diabetic nephropathy. Importantly, oral IAP supplementation could have beneficial therapeutic effects on gut metabolic homeostasis, possibly through stimulation of intestinal IgA secretion.
Diet soda consumption has not been associated with tangible weight loss. Aspartame (ASP) commonly substitutes sugar and one of its breakdown products is phenylalanine (PHE), a known inhibitor of ...intestinal alkaline phosphatase (IAP), a gut enzyme shown to prevent metabolic syndrome in mice. We hypothesized that ASP consumption might contribute to the development of metabolic syndrome based on PHE’s inhibition of endogenous IAP. The design of the study was such that for the in vitro model, IAP was added to diet and regular soda, and IAP activity was measured. For the acute model, a closed bowel loop was created in mice. ASP or water was instilled into it and IAP activity was measured. For the chronic model, mice were fed chow or high-fat diet (HFD) with/without ASP in the drinking water for 18 weeks. The results were that for the in vitro study, IAP activity was lower (p < 0.05) in solutions containing ASP compared with controls. For the acute model, endogenous IAP activity was reduced by 50% in the ASP group compared with controls (0.2 ± 0.03 vs 0.4 ± 0.24) (p = 0.02). For the chronic model, mice in the HFD + ASP group gained more weight compared with the HFD + water group (48.1 ± 1.6 vs 42.4 ± 3.1, p = 0.0001). Significant difference in glucose intolerance between the HFD ± ASP groups (53 913 ± 4000.58 (mg·min)/dL vs 42 003.75 ± 5331.61 (mg·min)/dL, respectively, p = 0.02). Fasting glucose and serum tumor necrosis factor-alpha levels were significantly higher in the HFD + ASP group (1.23- and 0.87-fold increases, respectively, p = 0.006 and p = 0.01). In conclusion, endogenous IAP’s protective effects in regard to the metabolic syndrome may be inhibited by PHE, a metabolite of ASP, perhaps explaining the lack of expected weight loss and metabolic improvements associated with diet drinks.
A diet high in fiber is associated with a decreased incidence and growth of colon cancers. Butyrate, a four-carbon short-chain fatty acid product of fiber fermentation within the colon, appears to ...mediate these salutary effects. We sought to determine the molecular mechanism by which butyrate mediates growth inhibition of colonic cancer cells and thereby to elucidate the molecular link between a high-fiber diet and the arrest of colon carcinogenesis. We show that concomitant with growth arrest, butyrate induces p21 mRNA expression in an immediate-early fashion, through transactivation of a promoter cis-element(s) located within 1.4 kb of the transcriptional start site, independent of p53 binding. Studies using the specific histone hyperacetylating agent, trichostatin A, and histone deacetylase 1 indicate that growth arrest and p21 induction occur through a mechanism involving histone hyperacetylation. We show the critical importance of p21 in butyrate-mediated growth arrest by first confirming that stable overexpression of the p21 gene is able to cause growth arrest in the human colon carcinoma cell line, HT-29. Furthermore, using p21-deleted HCT116 human colon carcinoma cells, we provide convincing evidence that p21 is required for growth arrest to occur in response to histone hyperacetylation, but not for serum starvation nor postconfluent growth. Thus, p21 appears to be a critical effector of butyrate-induced growth arrest in colonic cancer cells, and may be an important molecular link between a high-fiber diet and the prevention of colon carcinogenesis.
BACKGROUND & AIMS: The permeability of intestinal epithelial monolayers increases after exposure to nitric oxide. The aim of this study was to investigate the role of excessive NO production on ...intestinal barrier function in rats injected with lipopolysaccharide (LPS).
METHODS: Rats were injected with saline or LPS (5 mg/ kg). Bacterial translocation to mesenteric lymph nodes, liver, and spleen was assessed 24 hours after LPS injection. Mucosal permeability was determined by loading fluorescein-labeled dextran (mol wt, 4000 daltons) into an intestinal segment and measuring its appearance in plasma. Intestinal mucosal mitochondrial respiration was assessed using 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide.
RESULTS: Intestinal tissue from LPS-challenged rats showed upregulation of inducible NO synthase (iNOS) messenger RNA expression and subsequent up-regulation of iNOS enzymatic activity. Plasma concentrations of nitrite plus nitrate (NO2-/NO3-) were increased for at least 24 hours after injection of LPS. Treatment with the selective iNOS inhibitor, aminoguanidine, inhibited iNOS enzymatic activity and overproduction of NO2-/NO3-. LPS-induced bacterial translocation was reduced by aminoguanidine. LPS-induced intestinal hyperpermeability was ameliorated by both aminoguanidine and another selective iNOS inhibitor, S-methylisothiourea. LPS depressed intestinal mucosal mitochondrial function, and this effect was ameliorated by aminoguanidine.
CONCLUSIONS: Overproduction of NO may contribute to intestinal barrier dysfunction in LPS challenged rats, possibly by interfering with mitochondrial oxidative metabolism.
(Gastroenterology 1997 Oct;113(4):1246-57)
Aims
To examine whether co‐administration of intestinal alkaline phosphatase (IAP) with antibiotics early in life may have a preventive role against metabolic syndrome (MetS) in mice.
Methods
A total ...of 50 mice were allocated to four treatment groups after weaning. Mice were treated with azithromycin (AZT) ± IAP, or with no AZT ± IAP, for three intermittent 7‐day cycles. After the last treatment course, the mice were administered a regular chow diet for 5 weeks and subsequently a high‐fat diet for 5 weeks. Body weight, food intake, water intake, serum lipids, glucose levels and liver lipids were compared. 16S rRNA gene pyrosequencing was used to determine the differences in microbiome composition.
Results
Exposure to AZT early in life rendered mice susceptible to MetS in adulthood. Co‐administration of IAP with AZT completely prevented this susceptibility by decreasing total body weight, serum lipids, glucose levels and liver lipids to the levels of control mice. These effects of IAP probably occur as a result of changes in the composition of specific bacterial taxa at the genus and species levels (e.g. members of Anaeroplasma and Parabacteroides).
Conclusions
Co‐administration of IAP with AZT early in life prevents mice from susceptibility to the later development of MetS. This effect is associated with alterations in the composition of the gut microbiota. IAP may represent a novel treatment against MetS in humans.
Histone acetylation and cancer Archer, Sonia Y; Hodint, Richard A
Current opinion in genetics & development,
04/1999, Letnik:
9, Številka:
2
Journal Article
Recenzirano
In the past year, several papers have been published which implicate a link between alterations in chromatin structure and the development of cancer. Both histone hyperacetylation and hypoacetylation ...appear to be important in the neoplastic process, depending on the target gene involved. In the case of colon cancer, induction of the p21 gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis.
Rescue with tissue-engineered small intestine (TESI) after massive small bowel resection (MSBR).
Short bowel syndrome is a morbid product of massive small bowel resection. We report the first ...replacement of a vital organ by tissue engineering with TESI after MSBR.
Ten male Lewis rats underwent TESI implantation with green fluorescent protein (GFP)-marked cells (TESI+, n = 5) or sham laparotomy (TESI-, n = 5) followed by MSBR. Side-to-side anastomosis of TESI to proximal small intestine was performed or omitted. TESIO animals underwent implantation of engineered intestine with no further surgery. Weights were measured QOD until day 40. Transit times were measured. DNA assay was performed with computer morphometry. Northern blots of RNA were probed for intestinal alkaline phosphatase (IAP) and villin. Hematoxylin and eosin, S100, and smooth muscle actin immunohistochemistry were performed. Blood was collected at sacrifice.
All 10 rats initially lost then regained weight. The initial rate of weight loss was higher in TESI+ versus TESI-, but the nadir was reached a week earlier with more rapid weight gain subsequently to 98% preoperative weight on day 40 in animals with engineered intestine versus 76% (P < 0.03). Serum B12 was higher at 439 pg/mL versus 195.4 pg/mL. IAP mRNA appeared greater in TESI+ than TESIO, with constant villin levels. Histology revealed appropriate architecture including nerve. GFP labeling persisted.
Anastomosis of TESI significantly improved postoperative weight and B12 absorption after MSBR. IAP, a marker of differentiation in intestinal epithelium, is present in TESI, and GFP labeling was accomplished.
Incubation of enterocytic monolayers with interferon (IFN)-gamma increases nitric oxide (NO) production and permeability, but NO synthesis inhibitors ameliorate the development of IFN-gamma-induced ...hyperpermeability. Induction of inducible nitric oxide synthase (iNOS), an isoform of the enzyme responsible for NO biosynthesis, is often enhanced by the synergistic effects of multiple cytokines. Moreover, many of the cytopathic effects of NO are mediated by peroxynitrite, which is produced by the reaction of NO with superoxide radical anion. In the present study, we sought to determine whether combinations of several proinflammatory cytokines, including IFN-gamma, interleukin-1beta, and tumor necrosis factor-alpha, have synergistic effects on the induction of iNOS expression and/or hyperpermeability to hydrophilic solutes in cultured enterocytic monolayers. We also assessed the effects of aminoguanidine (a relatively selective iNOS inhibitor), L-N(G)-monomethyl arginine (an isoform-nonselective NO synthase inhibitor), and Tiron (a superoxide radical anion scavenger) on the development of cytokine-induced hyperpermeability.
Caco-2 monolayers were incubated under control conditions or with IFN-gamma, interleukin-1beta, or tumor necrosis factor-alpha alone, pairwise combinations of these cytokines, or all three cytokines together (cytomix; CM). iNOS messenger RNA (mRNA) expression was assessed using Northern blot analysis. The permeability of Caco-2 monolayers growing on permeable supports in bicameral chambers was assessed by measuring the apical-to-basolateral flux of fluorescein disulfonic acid. The concentration of nitrate plus nitrite in culture supernatants, an indirect measure of NO production, was determined using the Griess reaction.
After 24 hrs of incubation, up-regulation of iNOS mRNA expression was evident only in cells exposed to IFN-gamma-containing formulations. Expression of iNOS mRNA was far greater in cells incubated with CM than in cells treated with IFN-gamma alone or either of the two-component IFN-gamma-containing cytokine combinations. Compared with IFN-gamma, CM resulted in a higher rate of NO production over 48 hrs of incubation. The permeability of Caco-2 monolayers increased by approximately six-fold and approximately 20-fold after incubation for 48 hrs with IFN-gamma alone and CM, respectively. The increase in permeability induced by incubation with CM was significantly ameliorated by the addition of aminoguanidine, L-N(G)-monomethyl arginine, or Tiron.
IFN-gamma-containing combinations of cytokines are potent inducers of iNOS in cultured enterocytic monolayers. Peroxynitrite may be an important mediator of cytokine-induced gut epithelial hyperpermeability.
The short-chain fatty acid (SCFA) butyrate is produced via anaerobic bacterial fermentation within the colon and is thought to be protective in regard to colon carcinogenesis. Although butyrate (C4) ...is considered the most potent of the SCFA, a variety of other SCFA also exist in the colonic lumen. Butyrate is thought to exert its cellular effects through the induction of histone hyperacetylation. We sought to determine the effects of a variety of the SCFA on colon carcinoma cell growth, differentiation and apoptosis. HT-29 or HCT-116 (wild-type and p21-deleted) cells were treated with physiologically relevant concentrations of various SCFA, and histone acetylation state was assayed by acid-urea-triton-X gel electrophoresis and immunoblotting. Growth and apoptotic effects were studied by flow cytometry, and differentiation effects were assessed using transient transfections and Northern blotting. Propionate (C3) and valerate (C5) caused growth arrest and differentiation in human colon carcinoma cells. The magnitude of their effects was associated with a lesser degree of histone hyperacetylation compared with butyrate. Acetate (C2) and caproate (C6), in contrast, did not cause histone hyperacetylation and also had no appreciable effects on cell growth or differentiation. SCFA-induced transactivation of the differentiation marker gene, intestinal alkaline phosphatase (IAP), was blocked by histone deacetylase (HDAC), further supporting the critical link between SCFA and histones. Butyrate also significantly increased apoptosis, whereas the other SCFA studied did not. The growth arrest induced by the SCFA was characterized by an increase in the expression of the p21 cell-cycle inhibitor and down-regulation of cyclin B1 (CB1). In p21-deleted HCT-116 colon cancer cells, the SCFA did not alter the rate of proliferation. These data suggest that the antiproliferative, apoptotic and differentiating properties of the various SCFA are linked to the degree of induced histone hyperacetylation. Furthermore, SCFA-mediated growth arrest in colon carcinoma cells requires the p21 gene.
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