Tau expression and phosphorylation in enteroendocrine cells.

Background and objective: There is mounting evidence to suggest that the gut-brain axis is involved in the development of Parkinson's disease (PD). In this regard, the enteroendocrine cells (EEC), which faces the gut lumen and are connected with both enteric neurons and glial cells have received growing attention. The recent observation showing that these cells express alpha-synuclein, a presynaptic neuronal protein genetically and neuropathologically linked to PD came to reinforce the assumption that EEC might be a key component of the neural circuit between the gut lumen and the brain for the bottom-up propagation of PD pathology. Besides alpha-synuclein, tau is another key protein involved in neurodegeneration and converging evidences indicate that there is an interplay between these two proteins at both molecular and pathological levels. There are no existing studies on tau in EEC and therefore we set out to examine the isoform profile and phosphorylation state of tau in these cells. Methods: Surgical specimens of human colon from control subjects were analyzed by immunohistochemistry using a panel of anti-tau antibodies together with chromogranin A and Glucagon-like peptide-1 (two EEC markers) antibodies. To investigate tau expression further, two EEC lines, namely GLUTag and NCI-H716 were analyzed by Western blot with pan-tau and tau isoform specific antibodies and by RT-PCR. Lambda phosphatase treatment was used to study tau phosphorylation in both cell lines. Eventually, GLUTag were treated with propionate and butyrate, two short chain fatty acids known to sense EEC, and analyzed at different time points by Western blot with an antibody specific for tau phosphorylated at Thr205. Results: We found that tau is expressed and phosphorylated in EEC in adult human colon and that both EEC lines mainly express two tau isoforms that are phosphorylated under basal condition. Both propionate and butyrate regulated tau phosphorylation state by decreasing its phosphorylation at Thr205. Conclusion and inference: Our study is the first to characterize tau in human EEC and in EEC lines. As a whole, our findings provide a basis to unravel the functions of tau in EEC and to further investigate the possibility of pathological changes in tauopathies and synucleinopathies.

Perinatal supplementation of 4-phenylbutyrate and glutamine attenuates endoplasmic reticulum stress and improves colonic epithelial barrier function in rats born with intrauterine growth restriction.

Intrauterine growth restriction (IUGR) can affect the structure and function of the intestinal barrier and increase digestive disease risk in adulthood. Using the rat model of maternal dietary protein restriction (8% vs. 20%), we found that the colon of IUGR offspring displayed decreased mRNA expression of epithelial barrier proteins MUC2 and occludin during development. This was associated with increased mRNA expression of endoplasmic reticulum (ER) stress marker XBP1s and increased colonic permeability measured in Ussing chambers. We hypothesized that ER stress contributes to colonic barrier alterations and that perinatal supplementation of dams with ER stress modulators, phenylbutyrate and glutamine (PG) could prevent these defects in IUGR offspring. We first demonstrated that ER stress induction by tunicamycin or thapsigargin increased the permeability of rat colonic tissues mounted in Ussing chamber and that PG treatment prevented this effect. Therefore, we supplemented the diet of control and IUGR dams with PG during gestation and lactation. Real-time polymerase chain reaction and histological analysis of colons from 120-day-old offspring revealed that perinatal PG treatment partially prevented the increased expression of ER stress markers but reversed the reduction of crypt depth and goblet cell number in IUGR rats. In dextran sodium sulfate-induced injury and recovery experiments, the colon of IUGR rats without perinatal PG treatment showed higher XBP1s mRNA levels and histological scores of inflammation than IUGR rats with perinatal PG treatment. In conclusion, these data suggest that perinatal supplementation with PG could alleviate ER stress and prevent epithelial barrier dysfunction in IUGR offspring.

Perinatal undernutrition alters intestinal alkaline phosphatase and its main transcription factors KLF4 and Cdx1 in adult offspring fed a high-fat diet.

Nutrient restriction during gestation and/or suckling is associated with an increased risk of developing inflammation, obesity and metabolic diseases in adulthood. However, the underlying mechanisms, including the role of the small intestine, are unclear. We hypothesized that intestinal adaptation to the diet in adulthood is modulated by perinatal nutrition. This hypothesis was tested using a split-plot design experiment with 20 controls and 20 intrauterine growth-retarded (IUGR) rats aged 240 days and randomly assigned to be fed a standard chow or a high-fat (HF) diet for 10 days. Jejunal tissue was collected at necropsy and analyzed for anatomy, digestive enzymes, goblet cells and mRNA levels. Cecal contents and blood serum were analyzed for alkaline phosphatase (AP). IUGR rats failed to adapt to HF by increasing AP activity in jejunal tissue and cecal content as observed in controls. mRNA levels of transcription factors KLF4 and Cdx1 were blunted in jejunal epithelial cell of IUGR rats fed HF. mRNA levels of TNF-α were lower in IUGR rats. They also displayed exacerbated aminopeptidase N response and reduced jejunal goblet cell density. Villus and crypt architecture and epithelial cell proliferation increased with HF in both control and IUGR rats. Serum AP tended to be lower, and serum levamisole inhibition-resistant AP fraction was lower, in IUGR than controls with HF. Serum fatty acids and triglycerides were higher in IUGR rats and higher with HF. In conclusion, the adult intestine adapts to an HF diet differentially depending on early nutrition, jejunal AP and transcription factors being blunted in IUGR individuals fed HF.

CpG-ODN and MPLA prevent mortality in a murine model of post-hemorrhage-Staphyloccocus aureus pneumonia.

Infections are the most frequent cause of complications in trauma patients. Post-traumatic immune suppression (IS) exposes patients to pneumonia (PN). The main pathogen involved in PN is Methicillin Susceptible Staphylococcus aureus (MSSA). Dendritic cells () may be centrally involved in the IS. We assessed the consequences of hemorrhage on pneumonia outcomes and investigated its consequences on DCs functions. A murine model of hemorrhagic shock with a subsequent MSSA pneumonia was used. Hemorrhage decreased the survival rate of infected mice, increased systemic dissemination of sepsis and worsened inflammatory lung lesions. The mRNA expression of Tumor Necrosis Factor-alpha (TNF-α), Interferon-beta (IFN-ß) and Interleukin (IL)-12p40 were mitigated for hemorrhaged-mice. The effects of hemorrhage on subsequent PN were apparent on the pDCs phenotype (reduced MHC class II, CD80, and CD86 molecule membrane expression). In addition, hemorrhage dramatically decreased CD8(+) cDCs- and CD8(-) cDCs-induced allogeneic T-cell proliferation during PN compared with mice that did not undergo hemorrhage. In conclusion, hemorrhage increased morbidity and mortality associated with PN; induced severe phenotypic disturbances of the pDCs subset and functional alterations of the cDCs subset. After hemorrhage, a preventive treatment with CpG-ODN or Monophosphoryl Lipid A increased transcriptional activity in DCs (TNF-α, IFN-ß and IL-12p40) and decreased mortality of post-hemorrhage MSSA pneumonia.

Butyrate utilization by the colonic mucosa in inflammatory bowel diseases: a transport deficiency.

The short-chain fatty acid butyrate, which is mainly produced in the lumen of the large intestine by the fermentation of dietary fibers, plays a major role in the physiology of the colonic mucosa. It is also the major energy source for the colonocyte. Numerous studies have reported that butyrate metabolism is impaired in intestinal inflamed mucosa of patients with inflammatory bowel disease (IBD). The data of butyrate oxidation in normal and inflamed colonic tissues depend on several factors, such as the methodology or the models used or the intensity of inflammation. The putative mechanisms involved in butyrate oxidation impairment may include a defect in beta oxidation, luminal compounds interfering with butyrate metabolism, changes in luminal butyrate concentrations or pH, and a defect in butyrate transport. Recent data show that butyrate deficiency results from the reduction of butyrate uptake by the inflamed mucosa through downregulation of the monocarboxylate transporter MCT1. The concomitant induction of the glucose transporter GLUT1 suggests that inflammation could induce a metabolic switch from butyrate to glucose oxidation. Butyrate transport deficiency is expected to have clinical consequences. Particularly, the reduction of the intracellular availability of butyrate in colonocytes may decrease its protective effects toward cancer in IBD patients.

Down-regulation of the monocarboxylate transporter 1 is involved in butyrate deficiency during intestinal inflammation.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Neuropeptide AF and FF modulation of adipocyte metabolism. Primary insights from functional genomics and effects on beta-adrenergic responsiveness.

The presence of a neuropeptide AF and FF receptor (NPFF-R2) mRNA in human adipose tissue (Elshourbagy, N. A., Ames, R. S., Fitzgerald, L. R., Foley, J. J., Chambers, J. K., Szekeres, P. G., Evans, N. A., Schmidt, D. B., Buckley, P. T., Dytko, G. M., Murdock, P. R., Tan, K. B., Shabon, U., Nuthulaganti, P., Wang, D. Y., Wilson, S., Bergsma, D. J., and Sarau, H. M. (2000) J. Biol. Chem. 275, 25965-25971) suggested these peptides, principally recognized for their pain modulating effects, may also impact on adipocyte metabolism, an aspect that has not been explored previously. Our aim was thus to obtain more insights into the actions of these peptides on adipocytes, an approach initially undertaken with a functional genomic assay. First we showed that 3T3-L1 adipocytes express both NPFF-R1 and NPFF-R2 transcripts, and that NPAF binds adipocyte membranes with a nanomolar affinity as assessed by surface plasmon resonance technology. Then, and following a 24-h treatment with NPFF or NPAF (1 microm), we have measured using real-time quantitative reverse transcriptase-PCR the mRNA steady state levels of already well characterized genes involved in key pathways of adipose metabolism. Among the 45 genes tested, few were modulated by NPFF ( approximately 10%) and a larger number by NPAF ( approximately 27%). Interestingly, NPAF increased the mRNA levels of beta2- and beta3-adrenergic receptors (AR), and to a lesser extent those of beta1-ARs. These variations in catecholamine receptor mRNAs correlated with a clear induction in the density of beta2- and beta3-AR proteins, and in the potency of beta-AR subtype-selective agonists to stimulate adenylyl cyclase activity. Altogether, these data show that NPFF-R1 and NPFF-R2 are functionally present in adipocytes and suggest that besides their well described pain modulation effects, NPAF and to a lesser extent NPFF, may have a global impact on body energy storage and utilization.