Bindu Chandrasekharan

Diabetes and the enteric nervous system

Abstract  Diabetes is associated with several changes in gastrointestinal (GI) motility and associated symptoms such as nausea, bloating, abdominal pain, diarrhoea and constipation. The pathogenesis of altered GI functions in diabetes is multifactorial and the role of the enteric nervous system (ENS) in this respect has gained significant importance. In this review, we summarize the research carried out on diabetes‐related changes in the ENS. Changes in the inhibitory and excitatory enteric neurons are described highlighting the role of loss of inhibitory neurons in early diabetic enteric neuropathy. The functional consequences of these neuronal changes result in altered gastric emptying, diarrhoea or constipation. Diabetes can also affect GI motility through changes in intestinal smooth muscle or alterations in extrinsic neuronal control. Hyperglycaemia and oxidative stress play an important role in the pathophysiology of these ENS changes. Antioxidants to prevent or treat diabetic GI motility problems have therapeutic potential. Recent research on the nerve–immune interactions demonstrates inflammation‐associated neurodegeneration which can lead to motility related problems in diabetes.

Colonic motor dysfunction in human diabetes is associated with enteric neuronal loss and increased oxidative stress

Background  Gastrointestinal dysfunction is very common in diabetic patients. We assessed the changes in the colonic enteric nervous system using colectomy specimens and intestinal biopsies from diabetic subjects and age‐matched controls.

Targeted Deletion of Neuropeptide Y (NPY) Modulates Experimental Colitis

Background Neurogenic inflammation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). We examined the role of neuropeptide Y (NPY) and neuronal nitric oxide synthase (nNOS) in modulating colitis. Methods Colitis was induced by administration of dextran sodium sulphate (3% DSS) or streptomycin pre-treated Salmonella typhimurium (S.T.) in wild type (WT) and NPY (NPY−/−) knockout mice. Colitis was assessed by clinical score, histological score and myeloperoxidase activity. NPY and nNOS expression was assessed by immunostaining. Oxidative stress was assessed by measuring catalase activity, glutathione and nitrite levels. Colonic motility was assessed by isometric muscle recording in WT and DSS-treated mice. Results DSS/S.T. induced an increase in enteric neuronal NPY and nNOS expression in WT mice. WT mice were more susceptible to inflammation compared to NPY−/− as indicated by higher clinical & histological scores, and myeloperoxidase (MPO) activity (p<0.01). DSS-WT mice had increased nitrite, decreased glutathione (GSH) levels and increased catalase activity indicating more oxidative stress. The lower histological scores, MPO and chemokine KC in S.T.-treated nNOS−/− and NPY−/−/nNOS−/− mice supported the finding that loss of NPY-induced nNOS attenuated inflammation. The inflammation resulted in chronic impairment of colonic motility in DSS-WT mice. NPY –treated rat enteric neurons in vitro exhibited increased nitrite and TNF-α production. Conclusions NPY mediated increase in nNOS is a determinant of oxidative stress and subsequent inflammation. Our study highlights the role of neuronal NPY and nNOS as mediators of inflammatory processes in IBD.

Adenosine 2B receptors (A2BAR) on enteric neurons regulate murine distal colonic motility

Delayed colonic emptying leading to constipation is a significant health concern. We investigated the role of adenosine 2B receptor (A2BAR) in modulating distal colonic motility using wild‐type and A2BAR‐knockout (A2BAR–/–) mice. Colon motility was assessed using stool characteristics and colonic transit. Distal colonic ganglia, isolated by laser capture microdissection, were tested for A2BAR expression by RT‐ PCR. The distal colon contraction and relaxation responses were assessed by electrical field stimulation (EFS) in presence of A2BAR agonists, antagonists or inhibitors of nitric oxide (NO) and guanylate cyclase. Nitrite levels were measured in enteric neuronal cultures exposed to A2BAR agonists/antagonists. A2BAR–/– mice display increased stool retention, decreased stool frequency, delayed colonic emptying, and decreased circular muscle relaxation. RT‐PCR identified A2BAR expression in distal colonic ganglia. EFS studies revealed that enteric neuronal A2BAR is essential for distal colonic relaxation, and A2BAR antagonists can inhibit relaxation. Enteric neurons stimulated with A2BAR agonists produced more nitrite than cultures treated with antagonists. We demonstrate an essential role of A2BAR in regulating distal colon relaxation, as A2BAR activation is linked to NO signaling. Hence targeting the colonic A2BAR could represent a novel therapeutic strategy to treat constipation.— Chandrasekharan, B. P., Kolachala, V. L., Dalmasso, G., Merlin, D., Ravid, K., Sitaraman, S. V., Srinivasan, S. Adenosine 2B receptors (A2BAR) on enteric neurons regulate murine distal colonic motility. FASEB J. 23, 2727–2734 (2009)

Tumor necrosis factor-neuropeptide y cross talk regulates inflammation, epithelial barrier functions, and colonic motility

Background:Neuro-immune interactions play a significant role in regulating the severity of inflammation. Our previous work demonstrated that neuropeptide Y (NPY) is upregulated in the enteric nervous system during murine colitis and that NPY knockout mice exhibit reduced inflammation. Here, we investigated if NPY expression during inflammation is induced by tumor necrosis factor (TNF), the main proinflammatory cytokine. Methods:Using primary enteric neurons and colon explant cultures from wild type and NPY knockout (NPY−/−) mice, we determined if NPY knockdown modulates TNF release and epithelial permeability. Further, we assessed if NPY expression is inducible by TNF in enteric neuronal cells and mouse model of experimental colitis, using the TNF inhibitors-etanercept (blocks transmembrane and soluble TNF) and XPro1595 (blocks soluble TNF only). Results:We found that enteric neurons express TNF receptors (TNFR1 and R2). Primary enteric neurons from NPY−/− mice produced less TNF compared with wild type. Further, TNF activated NPY promoter in enteric neurons through phospho-c-Jun. NPY−/− mice had decreased intestinal permeability. In vitro, NPY increased epithelial permeability through phosphatidyl inositol-3-kinase (PI3-K)-induced pore-forming claudin-2. TNF inhibitors attenuated NPY expression in vitro and in vivo. TNF inhibitor–treated colitic mice exhibited reduced NPY expression and inflammation, reduced oxidative stress, enhanced neuronal survival, and improved colonic motility. XPro1595 had more protective effects on neuronal survival and motility compared with etanercept. Conclusions:We demonstrate a novel TNF–NPY cross talk that modulates inflammation, barrier functions, and colonic motility during inflammation. It is also suggested that selective blocking of soluble TNF may be a better therapeutic option than using anti-TNF antibodies.

Glial cell line-derived neurotrophic factor enhances neurogenin3 gene expression and β-cell proliferation in the developing mouse pancreas

Glial cell line-derived neurotrophic factor (GDNF) is a factor produced by glial cells that is required for the development of the enteric nervous system. In transgenic mice that overexpress GDNF in the pancreas, GDNF has been shown to enhance beta-cell mass and improve glucose control, but the transcriptional and cellular processes involved are not known. In this study we examined the influence of GDNF on the expression of neurogenin3 (Ngn3) and other transcription factors implicated in early beta-cell development, as well as on beta-cell proliferation during embryonic and early postnatal mouse pancreas development. Embryonic day 15.5 (E15.5) mouse pancreatic tissue when exposed to GDNF for 24 h showed higher Ngn3, pancreatic and duodenal homeobox gene 1 (Pdx1), neuroD1/beta(2), paired homeobox gene 4 (Pax4), and insulin mRNA expression than tissue exposed to vehicle only. Transgenic expression of GDNF in mouse pancreata was associated with increased numbers of Ngn3-expressing pancreatic cells and higher beta-cell mass at embryonic day 18 (E18), as well as higher beta-cell proliferation and Pdx1 expression in beta-cells at E18 and postnatal day 1. In the HIT-T15 beta-cell line, GDNF enhanced the expression of Pax6. This response was, however, blocked in the presence of Pdx1 small interfering RNA (siRNA). Chromatin immunoprecipitation studies using the HIT-T15 beta-cell line demonstrated that GDNF can influence Pdx1 gene expression by enhancing the binding of Sox9 and neuroD1/beta(2) to the Pdx1 promoter. Our data provide evidence of a mechanism by which GDNF influences beta-cell development. GDNF could be a potential therapeutic target for the treatment and prevention of diabetes.

Neuropeptide Y (NPY) promotes inflammation-induced tumorigenesis by enhancing epithelial cell proliferation.

We have demonstrated that neuropeptide Y (NPY), abundantly produced by enteric neurons, is an important regulator of intestinal inflammation. However, the role of NPY in the progression of chronic inflammation to tumorigenesis is unknown. We investigated whether NPY could modulate epithelial cell proliferation and apoptosis, and thus regulate tumorigenesis. Repeated cycles of dextran sodium sulfate (DSS) were used to model inflammation-induced tumorigenesis in wild-type (WT) and NPY knockout (NPY-/-) mice. Intestinal epithelial cell lines (T84) were used to assess the effects of NPY (0.1 µM) on epithelial proliferation and apoptosis in vitro. DSS-WT mice exhibited enhanced intestinal inflammation, polyp size, and polyp number (7.5 ± 0.8) compared with DSS-NPY-/- mice (4 ± 0.5, P < 0.01). Accordingly, DSS-WT mice also showed increased colonic epithelial proliferation (PCNA, Ki67) and reduced apoptosis (TUNEL) compared with DSS-NPY-/- mice. The apoptosis regulating microRNA, miR-375, was significantly downregulated in the colon of DSS-WT (2-fold, P < 0.01) compared with DSS-NPY-/--mice. In vitro studies indicated that NPY promotes cell proliferation (increase in PCNA and β-catenin, P < 0.05) via phosphatidyl-inositol-3-kinase (PI3-K)-β-catenin signaling, suppressed miR-375 expression, and reduced apoptosis (increase in phospho-Bad). NPY-treated cells also displayed increased c-Myc and cyclin D1, and reduction in p21 (P < 0.05). Addition of miR-375 inhibitor to cells already treated with NPY did not further enhance the effects induced by NPY alone. Our findings demonstrate a novel regulation of inflammation-induced tumorigenesis by NPY-epithelial cross talk as mediated by activation of PI3-K signaling and downregulation of miR-375. NEW & NOTEWORTHY Our work exemplifies a novel role of neuropeptide Y (NPY) in regulating inflammation-induced tumorigenesis via two modalities: first by enhanced proliferation (PI3-K/pAkt), and second by downregulation of microRNA-375 (miR-375)-dependent apoptosis in intestinal epithelial cells. Our data establish the existence of a microRNA-mediated cross talk between enteric neurons producing NPY and intestinal epithelial cells, and the potential of neuropeptide-regulated miRNAs as potential therapeutic molecules for the management of inflammation-associated tumors in the gut.

199 TNF-α Induced Expression of Enteric Neuronal Neuropeptide Y (NPY) Influences Intestinal Epithelial Barrier Functions

Introduction: The integrity of the intestinal epithelium is crucial to maintain boundaries between luminal contents and mucosal immune system. Increased epithelial permeability has been noted in 10-20% of pre-symptomatic Crohns disease patients. We investigated if enteric neurons producing neuropeptide Y (NPY) could influence epithelial permeability and modulate TNF-α signaling. Methods: Caco2BBE epithelial cells were grown on transwell plates and NPY was added to the basolateral medium (0.1 μM). The epithelial permeability was measured by transepithelial resistance (TER) and flux of FITC-Dextran (4 Kd) at 2 and 24 h. The changes in the expression of tight junction proteins were compared in control and NPY-treated cells by real-time polymerase chain reaction (qRT-PCR) and Western blotting. The enteric neurons transfected with NPY promoter constructs (-1078 (full length), -952, -836, -769, -728, -597, -448, -278) were treated with TNF-α, and NPY promoter activity was assessed by luciferase assay. NPY expression in enteric neurons treated with TNF-α was also determined by qRT-PCR. Results: The addition of NPY increased the TER and FITC-Dextran flux across the CaCo2-BBE monolayer (p<0.05), thus increasing colonic epithelial permeability. NPY also induced a two-fold increase in the claudin-2 expression at mRNA (p< 0.01) and protein (p< 0.05) levels. NPY mRNA was up regulated by two-fold in TNF-α treated enteric neurons (p< 0.05) as seen by qRT-PCR. Luciferase assay demonstrated a significant increase in the NPY promoter activity, specifically in the regions between -728 and -836 of the NPY promoter (p< 0.05. Transcription factor (TRANSFAC) analysis showed Activator Protein-1 (AP-1) binding sites in this region of the NPY promoter, supporting the responsiveness to TNF-α. Conclusions: TNF-α upregulates NPY expression in enteric neurons; NPY in turn alters barrier functions of the colonic epithelium via modulation of leaky tight junction proteins like Claudin-2. Taken together, enteric neurons producing NPY aggravate pro-inflammatory signaling and increases epithelial permeability. Our studies demonstrate a role of NPY regulation of epithelial permeability and participation of enteric neuronal signaling in propagating inflammation during inflammatory bowel disease.

S1269 TLR4 Signaling Plays An Important Role in Hyperglycemia-Induced Enteric Neuronal Apoptosis and Colonic Dysmotility

G A A b st ra ct s more stable. Methods: Abdominal radiographs performed for SitzMark localization and transit during a validation trial in chronic constipation visualized the SmartPill in 17 films from healthy subjects and 49 from constipated patients. Capsule location was classified as in the right, left, or rectosigmoid colon based on accepted bony landmarks. Colon contractions >25 mmHg and motility indices (MI) from the capsule were quantified from 1 hr before to 1 hr after the time each radiograph was taken. Mean pH and pH variability (standard deviation of all pH values within the 2 hr time period for each individual—this is postulated to be a quantitative measure of fecal mixing) were obtained from the same times. Results: Contraction numbers, MI, and pH values progressively increased from right to rectosigmoid colon while pH variability decreased in proximal to distal manner (Table). For 90% of recordings with >70 contractions, the capsule localized to the left or rectosigmoid. For 89% of recordings with MI >70, the capsule localized to the left or rectosigmoid. For 88% of recordings with pH >7.5, the capsule localized to the left or rectosigmoid colon.Conclusions: The right colon shows relatively less contractile activity and more acidic pH suggesting fermentation, fecal mixing and absorption while more distal regions exhibit more intense contractility and less acidity suggesting propagation. Site-specific differences in motor activity and pH of the unprepared human colon provide insight into colon physiology.