Hypoxia-Inducible Factor 1α Stabilization Restores Epigenetic Control of Nitric Oxide Synthase 1 Expression and Reverses Gastroparesis in Female Diabetic Mice.

BACKGROUND & AIMS: Although depletion of neuronal nitric oxide synthase (NOS1)-expressing neurons contributes to gastroparesis, stimulating nitrergic signaling is not an effective therapy. We investigated whether hypoxia-inducible factor 1α (HIF1A), which is activated by high O2 consumption in central neurons, is a Nos1 transcription factor in enteric neurons and whether stabilizing HIF1A reverses gastroparesis. METHODS: Mice with streptozotocin-induced diabetes, human and mouse tissues, NOS1+ mouse neuroblastoma cells, and isolated nitrergic neurons were studied. Gastric emptying of solids and volumes were determined by breath test and single-photon emission computed tomography, respectively. Gene expression was analyzed by RNA-sequencing, microarrays, immunoblotting, and immunofluorescence. Epigenetic assays included chromatin immunoprecipitation sequencing (13 targets), chromosome conformation capture sequencing, and reporter assays. Mechanistic studies used Cre-mediated recombination, RNA interference, and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated epigenome editing. RESULTS: HIF1A signaling from physiological intracellular hypoxia was active in mouse and human NOS1+ myenteric neurons but reduced in diabetes. Deleting Hif1a in Nos1-expressing neurons reduced NOS1 protein by 50% to 92% and delayed gastric emptying of solids in female but not male mice. Stabilizing HIF1A with roxadustat (FG-4592), which is approved for human use, restored NOS1 and reversed gastroparesis in female diabetic mice. In nitrergic neurons, HIF1A up-regulated Nos1 transcription by binding and activating proximal and distal cis-regulatory elements, including newly discovered super-enhancers, facilitating RNA polymerase loading and pause-release, and by recruiting cohesin to loop anchors to alter chromosome topology. CONCLUSIONS: Pharmacologic HIF1A stabilization is a novel, translatable approach to restoring nitrergic signaling and treating diabetic gastroparesis. The newly recognized effects of HIF1A on chromosome topology may provide insights into physioxia- and ischemia-related organ function.

[Electroacupuncture promotes gastrointestinal motility by activating autophagy of Cajal interstitial cells via downregulating PI3K/Akt/mTOR signaling pathway in stomach of diabetic gastro-paresis rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Zusanli" (ST36), "Sanyinjiao" (SP6) and "Liangmen" (ST21) on gastrointestinal motility, blood glucose content and expression of autophagy-related proteins 1 light chain 3 (LC3), p62, phosphatidyli-nositol-3 kinase (PI3K), protein kinase B (Akt), p-Akt and mammalian target protein of rapamycin (mTOR) of interstitial cells of Cajal (ICCs) in the cultured gastric antrum cells in diabetic gastroparesis (DGP) rats, so as to reveal its mechanisms underlying improvement of DGP. METHODS: A total of 45 Sprague Dawley (SD) rats were randomly divided into blank control, model, EA, medication (3-methyladenine, 3-MA) and EA+3-MA groups, with 9 rats in each group. The DGP model was established by intraperitoneal injection of 2% streptozotocin (STZ) combined with high-fat and high sugar diet for 8 weeks. The gastric emptying rate was measured by using gavage of phenol red (to measure the propelling length of the phenol red/total length of small intestine ×100%). The symptom score (mental state, coat color and luster, behavior and activity, stool traits) of rats was observed every week and the blood glucose content was measured by using a glucometer. EA (20 Hz/100 Hz, 2 mA) was applied to unilateral ST36, SP6 and ST21 alternatively for 15 min, once daily, 5 days a week for 3 weeks. Rats of the 3-MA and 3-MA+EA groups received intraperitoneal injection of 3-MA (30 mg·kg-1·d-1, 10 mg/mL), once daily, 5 days a week for 3 weeks. After 15 days' intervention, the rats were operated for gastric emptying rate test, specimen collection, isolation, and culture of primary ICCs. The expression levels of microtubule associated protein LC3, p62, PI3K, Akt, p-Akt and mTOR of ICCs of cultured gastric antrum cells were detected using Western blot, and the number of autophagosomes in ICC of gastric antrum was observed under transmission electron microscope. RESULTS: Compared with the blank control group, the symptom score, blood glucose, and the expression levels of p62, class Ⅰ PI3K, Akt, p-Akt and mTOR proteins were increased significantly (P<0.01), while the gastric emptying rate and ratio of LC3Ⅱ/LC3Ⅰ and the expression level of class Ⅲ PI3K protein were significantly decreased (P<0.05, P<0.01) in the model group. In comparison with the model group, the increase of symptom score, blood glucose, and expression levels of p62, class Ⅰ PI3K, Akt, p-Akt and mTOR proteins and the decrease of gastric empty rate and LC3Ⅱ/LC3Ⅰ ratio and the expression level of class Ⅲ PI3K protein were all reversed in both EA and EA+3-MA groups (P<0.05, P<0.01), rather than in the 3-MA group. In addition, 3-MA also reversed modeling-induced increase of class Ⅰ PI3K, Akt, p-Akt and mTOR proteins expression (P<0.01). No significant differences were found between the EA and EA+3-MA in downregulating the levels of symptom score and blood glucose content, and in upregulating gastric empty rate(P>0.05). The effect of EA was notably superior to that of EA+3-MA in upregulating the ratio of LC3Ⅱ/LC3Ⅰ and the expression level of class Ⅲ PI3K protein, and in downregulating the expression of p62, class Ⅰ PI3K, Akt, p-Akt and mTOR proteins (P<0.05, P<0.01). The findings of transmission electron microscopy showed obvious swelling, breakage of some mitochondrial cristae in the ICC cells of antrum and no autophagosomes in the model group and 3-MA group, which was milder in the damage of mitochondrial cristae and marked increase in the autophagosomes in both EA and EA+3-MA groups. CONCLUSION: EA can improve the gastrointestinal motility and symptoms in DGP rats, which may be related to its functions in downregulating PI3K/Akt/mTOR signaling to promote autophagy level of ICC.

[Effect of electroacupuncture combined with Zhuang-medicine-thread moxibustion on expression of apoptosis-related factors in gastric antrum of diabetic gastroparesis rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) combined with Zhuang-medicine-thread moxibustion on expression of apoptosis-related factors in gastric antrum of diabetic gastroparesis (DGP) rats, so as to explore its mechanism underlying improvement of DGP. METHODS: Male SD rats were randomly divided into normal, model, medication, EA, Zhuang-medicine-thread moxibustion (moxibustion) and EA+moxibustion (combination) groups (12 rats in each group). The DGP model was established by intraperitoneal injection of streptozotocin (STZ). Rats of the medication group were treated by gavage of 0.15 mg/mL mosapride citrate suspension （10 mL/kg）. EA (10 Hz/50 Hz, 2 mA, 20 min) or Zhuang-medicine-thread moxibustion (3 cones) was applied to "Zhongwan" (CV12), bilateral "Neiguan" (PC6) and bilateral "Sanyinjiao" (SP6) of the related groups, once a day for 3 weeks. The blood glucose, gastric emptying rate and intestinal propulsion rate of rats were measured. The apoptosis index of gastric antrum cells were observed by TUNEL staining. The protein and mRNA expressions of Caspase-3, B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in gastric antrum were detected by Wes-tern blot and real-time quantitative PCR, respectively. RESULTS: Compared with the normal group, the blood glucose, the apoptosis index, the protein and gene expressions of Caspase-3 and Bax were significantly increased (P<0.01), and the gastric emptying rate, intestinal propulsive rate, the protein and gene expressions of Bcl-2 were considerably decreased (P<0.01) in the model group. In contrast to the model group, the blood glucose in the EA, moxibustion and combination groups, the apoptosis index in the 4 treatment groups, as well as Caspase-3 protein, Bax protein and mRNA expressions in the medication, EA and combination groups, Caspase-3 protein and mRNA, Bax mRNA expressions in the moxibustion group were significantly decreased (P<0.01, P<0.05); while the gastric emptying rate and intestinal propulsive rate in the 4 treatment groups, and Bcl-2 protein and mRNA expressions in the medication and combination groups, Bcl-2 mRNA expressions in the EA and moxibustion groups were obviously increased (P<0.01). The effects of EA+moxibustion were significantly superior to those of simple EA, moxibustion or medication in increasing gastric emptying rate and intestinal propulsive rate, and in lowering blood glucose (P<0.05, P<0.01). And the effects of the combination treatment were better than those of EA in lowering Caspase-3 protein and Bax mRNA expressions (P<0.01), and in increasing Bcl-2 protein and mRNA expressions (P<0.05, P<0.01). Also the effects of the combination treatment were better than those of moxibustion in lowering the apoptosis index, Caspase-3 protein, and Bax protein and mRNA expressions (P<0.01, P<0.05), and in increasing Bcl-2 protein expression (P<0.05). CONCLUSION: EA combined with Zhuang-medicine-thread moxibustion can reduce blood glucose and improve gastrointestinal motility in DGP rats, which may be related to its effect in regulating of Caspase-3, Bax and Bcl-2 expression.

[Effect of electroacupuncture combined with Zhuang-medicine-thread moxibustion on silent information regulator-1/nuclear factor κB signaling pathway in gastric antrum of diabetic gastroparesis rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) combined with Zhuang-medicine-thread moxibustion on silent information regulator-1 (SIRT1)/nuclear factor kappa B (NF-κB) signal pathway and inflammatory factor expression in gastric antrum tissue of diabetic gastroparesis (DGP) rats, so as to explore its mechanism underlying improvement of DGP. METHODS: Male SD rats were randomly divided into normal, model, medication, EA, Zhuang-medicine-thread moxibustion (moxibustion) and EA+moxibustion groups (n=12 per group). The DGP model was established by intraperitoneal injection of streptozotocin (STZ). Rats of the medication group were treated by gavage of 0.15 mg/mL mosapride citrate suspension. EA (10 Hz /50 Hz, 2 mA) or moxibustion (3 cones) or EA+moxibustion was applied to "Zhongwan"(CV12), bilateral "Neiguan"(PC6) and bilateral "Sanyinjiao"(SP6) of the related group for 20 min, once a day for 3 weeks. Blood glucose, gastric emptying rate and intestinal propulsion rate were measured. The levels of serum interleukin (IL)-6, IL-8, IL-10 and tumor necrosis factor-α(TNF-α) were detected by ELISA; the phosphorylation level of the phosphorylated inhibitor of nuclear factor κBα inhibitor (pIκ-Bα), the protein and mRNA expression of NF-κB p65 and SIRT1 in the gastric antrum tissue were detected by Western blot and real-time quantifitative PCR, respectively. RESULTS: (1) Compared with the normal group, the levels of blood glucose, serum IL-6, IL-8, TNF-α, and gastric pIκ-Bα and NF-κB p65 protein and mRNA expressions were significantly increased (P<0.01), and the gastric emptying rate, intestinal propulsion rate, serum IL-10 level, and SIRT1 protein and mRNA expressions were considerably decreased in the model group (P<0.01). (2) In contrast to the model group, the blood glucose in the EA, moxibustion and EA+moxibustion groups, serum IL-6, IL-8 and TNF-α levels in the 4 treatment groups, as well as NF-κB p65 protein expression in the medication and EA+moxibustion groups, and NF-κB p65 mRNA expression and pIκ-Bα protein and mRNA expression in the 4 treatment groups were significantly decreased (P<0.01, P<0.05); while the gastric emptying rate and intestinal propulsive rate and IL-10 content in the 4 treatment groups, and SIRT1 protein and mRNA expression in the medication and EA+moxibustion groups were obviously increased (P<0.05, P<0.01). (3) The effects of EA+moxibustion were significantly superior to those of simple EA and moxibustion in increasing gastric emptying rate, IL-10, SIRT1 protein expression (P<0.05, P<0.01), and in lowering IL-8 and TNF-α contents, pIκ-Bα protein and mRNA expression and NF-κB p65 mRNA expression (P<0.05, P<0.01). No significant differences were found among the 4 intervention groups in promoting the intestinal propulsive rate and among the EA, moxibustion and EA+moxibustion groups in lowering blood glucose (P>0.05). CONCLUSION: EA combined with Zhuang-medicine-thread moxibustion can effectively reduce the level of serum inflammatory factors and regulate SIRT1/NF-κB signal pathway in DGP rats, which may contribute to its function in improving gastrointestinal movement.

miR-10b-5p Rescues Diabetes and Gastrointestinal Dysmotility.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICCs) and pancreatic ß cells require receptor tyrosine kinase (KIT) to develop and function properly. Degeneration of ICCs is linked to diabetic gastroparesis. The mechanisms linking diabetes and gastroparesis are unclear, but may involve microRNA (miRNA)-mediated post-transcriptional gene silencing in KIT+ cells. METHODS: We performed miRNA-sequencing analysis from isolated ICCs in diabetic mice and plasma from patients with idiopathic and diabetic gastroparesis. miR-10b-5p target genes were identified and validated in mouse and human cell lines. For loss-of-function studies, we used KIT+ cell-restricted mir-10b knockout mice and KIT+ cell depletion mice. For gain-of-function studies, a synthetic miR-10b-5p mimic was injected in multiple diabetic mouse models. We compared the efficacy of miR-10b-5p mimic treatment vs antidiabetic and prokinetic medicines. RESULTS: miR-10b-5p is highly expressed in ICCs from healthy mice, but drastically depleted in ICCs from diabetic mice. A conditional knockout of mir-10b in KIT+ cells or depletion of KIT+ cells in mice leads to degeneration of ß cells and ICCs, resulting in diabetes and gastroparesis. miR-10b-5p targets the transcription factor Krüppel-like factor 11 (KLF11), which negatively regulates KIT expression. The miR-10b-5p mimic or Klf11 small interfering RNAs injected into mir-10b knockout mice, diet-induced diabetic mice, and TALLYHO polygenic diabetic mice rescue the diabetes and gastroparesis phenotype for an extended period of time. Furthermore, the miR-10b-5p mimic is more effective in improving glucose homoeostasis and gastrointestinal motility compared with common antidiabetic and prokinetic medications. CONCLUSIONS: miR-10b-5p is a key regulator in diabetes and gastrointestinal dysmotility via the KLF11-KIT pathway. Restoration of miR-10b-5p may provide therapeutic benefits for these disorders.

Protein-elongating mutations in MYH11 are implicated in a dominantly inherited smooth muscle dysmotility syndrome with severe esophageal, gastric, and intestinal disease.

Gastrointestinal motility disorders include a spectrum of mild to severe clinical phenotypes that are caused by smooth muscle dysfunction. We investigated the genetic etiology of severe esophageal, gastric, and colonic dysmotility in two unrelated families with autosomal dominant disease presentation. Using exome sequencing, we identified a 2 base pair insertion at the end of the myosin heavy chain 11 (MYH11) gene in all affected members of Family 1 [NM_001040113:c.5819_5820insCA(p.Gln1941Asnfs*91)] and a 1 base pair deletion at the same genetic locus in Proband 2 [NM_001040113:c.5819del(p.Pro1940Hisfs*91)]. Both variants are predicted to result in a similarly elongated protein product. Heterozygous dominant negative MYH11 pathogenic variants have been associated with thoracic aortic aneurysm and dissection while biallelic null alleles have been associated with megacystis microcolon intestinal hypoperistalsis syndrome. This report highlights heterozygous protein-elongating MYH11 variants affecting the SM2 isoforms of MYH11 as a cause for severe gastrointestinal dysmotility, and we hypothesize that the mechanistic pathogenesis of this disease, dominant hypercontractile loss-of-function, is distinct from those implicated in other diseases involving MYH11 dysfunction.

Proteomics in gastroparesis: unique and overlapping protein signatures in diabetic and idiopathic gastroparesis.

Macrophage-based immune dysregulation plays a critical role in development of delayed gastric emptying in diabetic mice. Loss of anti-inflammatory macrophages and increased expression of genes associated with pro-inflammatory macrophages has been reported in full-thickness gastric biopsies from gastroparesis patients. We aimed to determine broader protein expression (proteomics) and protein-based signaling pathways in gastric biopsies of diabetic (DG) and idiopathic gastroparesis (IG) patients. Additionally, we determined correlations between protein expressions, gastric emptying, and symptoms. Full-thickness gastric antrum biopsies were obtained from nine DG patients, seven IG patients, and five nondiabetic controls. Aptamer-based SomaLogic tissue scan that quantitatively identifies 1,305 human proteins was used. Protein fold changes were computed, and differential expressions were calculated using Limma. Ingenuity pathway analysis and correlations were carried out. Multiple-testing corrected P < 0.05 was considered statistically significant. Seventy-three proteins were differentially expressed in DG, 132 proteins were differentially expressed in IG, and 40 proteins were common to DG and IG. In both DG and IG, "Role of Macrophages, Fibroblasts and Endothelial Cells" was the most statistically significant altered pathway [DG false discovery rate (FDR) = 7.9 × 10-9; IG FDR = 6.3 × 10-12]. In DG, properdin expression correlated with GCSI bloating (r = -0.99, FDR = 0.02) and expressions of prostaglandin G/H synthase 2, protein kinase C-ζ type, and complement C2 correlated with 4 h gastric retention (r = -0.97, FDR = 0.03 for all). No correlations were found between proteins and symptoms or gastric emptying in IG. Protein expression changes suggest a central role of macrophage-driven immune dysregulation in gastroparesis, specifically, complement activation in diabetic gastroparesis.NEW & NOTEWORTHY This study uses SOMAscan, a novel proteomics assay for determination of altered proteins and associated molecular pathways in human gastroparesis. Seventy-three proteins were changed in diabetic gastroparesis, 132 in idiopathic gastroparesis compared with controls. Forty proteins were common in both. Macrophage-based immune dysregulation pathway was most significantly affected in both diabetic and idiopathic gastroparesis. Proteins involved in the complement and prostaglandin synthesis pathway were associated with symptoms and gastric emptying delay in diabetic gastroparesis.

Activation of Nrf2 attenuates delayed gastric emptying in obesity induced diabetic (T2DM) female mice.

Diabetic gastroparesis (GP) is a clinical syndrome characterized by delayed gastric emptying (DGE). Loss of Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) led to reduced nNOSα mediated gastric motility and DGE. The molecular signaling of cinnamaldehyde (CNM) mediated Nrf2 activation and its mechanistic role on DGE were further investigated in obese/T2D female mice. Adult female homozygous Nfe2l2-/- (C57BL/6J) and their wild-type (WT) littermates (Nfe2l2+/+) mice were fed with high fat diet (HFD; Obese/T2D model), or normal diet (ND) with or without CNM (50 mg/kg b.w; i.p). Supplementation of CNM attenuated (p < 0.05) DGE in WT female but not in Nrf2 KO Obese/T2D mice. CNM (1) normalized serum estradiol-17ß levels, (2) induced gastric Nrf2 and phase II antioxidant enzymes through extracellular signal-regulated kinase, (ERK)/c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK), (3) reduced glucose synthase kinase 3 beta (GSK3ß) and aryl hydrocarbon receptor (AhR) and this was associated with (4) increased estrogen receptor expression, BH4 (Cofactor of nNOS) biosynthesis enzyme GCH-1 and nNOSα dimerization in WT Obese/T2 diabetic female mice. In addition, CNM restored impaired nitrergic relaxation in hyperglycemic conditions. These findings emphasize the importance of Nrf2 in maintaining nNOSα mediated GE and may have a translational relevance to treat obese/diabetic gastroparesis in women.

Gastroparesis is associated with decreased FOXF1 and FOXF2 in humans, and loss of FOXF1 and FOXF2 results in gastroparesis in mice.

BACKGROUND AND AIMS: The transcription factors FOXF1 and FOXF2 have been implicated in the development of the gastrointestinal tract but their role in adults or in gastrointestinal diseases is poorly understood. We have recently shown that expression of serum response factor (SRF), a transcription factor whose activity is modulated by FOXF proteins, is decreased in the stomach muscularis of patients with gastroparesis. The aim of the current study was to determine whether FOXF expression is decreased in gastroparesis patients and whether loss of FOXF1 and/or FOXF2 from adult smooth muscle is sufficient to impair gastric emptying in mice. METHODS: Full-thickness stomach biopsy samples were collected from control subjects and from patients with gastroparesis. mRNA was isolated from the muscularis externa, and FOXF mRNA expression levels were determined by quantitative reverse transcriptase (RT)-PCR. Foxf1 and Foxf2 were knocked out together and separately from smooth muscle cells in adult mice, and the subsequent effect on liquid gastric emptying and contractile protein expression was determined. KEY RESULTS: Expression of FOXF1 and FOXF2 is decreased in smooth muscle tissue from gastroparesis patients. Knockout of Foxf1 and Foxf2 together, but not alone, from mouse smooth muscle resulted in delayed liquid gastric emptying. Foxf1/2 double knockout mice had decreased expression of smooth muscle contractile proteins, SRF, and myocardin in stomach muscularis. CONCLUSIONS AND INFERENCES: Our findings suggest that decreased expression of FOXF1 and FOXF2 may be contributing to the impaired gastric emptying seen in gastroparesis patients.

Delayed gastric emptying in diabetic rats caused by decreased expression of cystathionine gamma lyase and H2 S synthesis: in vitro and in vivo studies.

BACKGROUND: This study aimed to evaluate the role of H2 S on gastric emptying rate (GER) and also to determine the effect of gastric distention on mRNA and protein expression of cystathionine ß-lyase (CBS) and cystathionine γ-synthase (CSE) in diabetic-gastroparetic and normal rats. METHODS: Adult normal rats intraperitoneally received either propargylglycine (PAG), L-cysteine or NaHS 30 min prior to GER marker (acetaminophen) to investigate H2 S involvement in GER and the same protocols were performed in diabetes-induced gastroparesis rats. The role of calcitonin gene related peptide (CGRP) neurons in the prokinetic effect of endogenous H2 S on GER was determined. The level of CBS and CSE expressions in response to gastric distention were also determined. The effect of H2 S on frequency and tension of spontaneous contractions of gastric smooth muscle strips was investigated. KEY RESULTS: Our results showed that: (i) H2 S and L-cysteine increased GER in gastroparetic and normal rats. (ii) The increased levels of CSE expression in response to gastric distention in diabetic rats were lower than in normal rats. (iii) PAG inhibited the excitatory effect of capsaicin on GER and on tension of spontaneous contractions of strips. (iv) Hydrogen sulphide increased the frequency and tension of spontaneous contractions of gastric strip muscles in normal and diabetic rats. CONCLUSIONS & INFERENCES: The results showed that delayed GER in diabetic rats can be due to down-regulation of H2 S biosynthesis enzyme, CSE and suggested that a potential prokinetic role for H2 S to treat the delayed gastric emptying in diabetic patients.

DNA microarray SNP associations with clinical efficacy and side effects of domperidone treatment for gastroparesis.

BACKGROUND: Domperidone treatment for gastroparesis is associated with variable efficacy as well as the potential for side effects. DNA microarray single nucleotide polymorphism (SNP) analysis may help to elucidate the role of genetic variability on the therapeutic effectiveness and toxicity of domperidone. AIM: The aim of this study was to identify SNPs that are associated with clinical efficacy and side effects of domperidone treatment for gastroparesis from DNA microarray experiments. This will help develop a strategy for rational selection of patients for domperidone therapy. METHODS: DNA samples extracted from the saliva of 46 patients treated with domperidone were analyzed using Affymetrix 6.0 SNP microarrays. Then least angle regression (LARS) was used to select SNPs that are related to domperidone efficacy and side effects. Decision tree based prediction models were constructed with the most correlated features selected by LARS. RESULTS: Using the most stable SNP selected by LARS a prediction model for side effects of domperidone achieved (95 ± 0)% true negative rate (TN) and (78 ± 11)% true positive rate (TP) in nested leave-one-out tests. For domperidone efficacy, the prediction based on five most stable SNPs achieved (85 ± 7)% TP and (61±4)% TN. Five identified SNPs are related to ubiquitin mediated proteolysis, epithelial cell signaling, leukocyte, cell adhesion, and tight junction signaling pathways. Genetic polymorphisms in three genes that are related to cancer and hedgehog signaling were found to significantly correlate with efficacy of domperidone. CONCLUSION: LARS was found to be a useful tool for statistical analysis of domperidone-related DNA microarray data generated from a small number of patients.

Loss of NRF2 impairs gastric nitrergic stimulation and function.

Emerging research suggests that antioxidant gene expression has the potential to suppress the development of gastroparesis. However, direct genetic evidence that definitively supports this concept is lacking. We used mice carrying a targeted disruption of Nfe2l2, the gene that encodes the transcription factor NRF2 and directs antioxidant Phase II gene expression, as well as mice with a targeted disruption of Gclm, the modifier subunit for glutamate-cysteine ligase, to test the hypothesis that defective antioxidant gene expression contributes to development of gastroparesis. Although expression of heme oxygenase-1 remained unchanged, expression of GCLC, GCLM, SOD1, and CAT was down-regulated in gastric tissue from Nrf2(-/-) mice compared to wild-type animals. Tetrahydrobiopterin oxidation was significantly elevated and nitrergic relaxation was impaired in Nrf2(-/-) mouse gastric tissue. In vitro studies showed a significant decrease in NO release in Nrf2(-/-) mouse gastric tissue. Nrf2(-/-) mice displayed delayed gastric emptying. The use of Gclm(-/-) mice demonstrated that the loss of glutamate-cysteine ligase function enhanced tetrahydrobiopterin oxidation while impairing nitrergic relaxation. These results provide genetic evidence that loss of antioxidant gene expression can contribute to the development of gastroparesis and suggest that NRF2 represents a potential therapeutic target.

Altered expression of Ano1 variants in human diabetic gastroparesis.

Diabetes affects many organs including the stomach. Altered number and function of interstitial cells of Cajal (ICC), the gastrointestinal pacemaker cells, underlie a number of gastrointestinal motility disorders, including diabetic gastroparesis. In the muscle layers, ICC selectively express Ano1, thought to underlie classical Ca(2+)-activated Cl(-) currents. Mice homozygous for Ano1 knock-out exhibit abnormal ICC function and motility. Several transcripts for Ano1 are generated by alternative splicing of four exons. Here, we report expression levels of transcripts encoded by alternative splicing of Ano1 gene in gastric muscles of patients with diabetic gastroparesis and nondiabetic control tissues. Expression of mRNA from two alternatively transcribed exons are significantly different between patients and controls. Furthermore, patients with diabetic gastroparesis express mRNA for a previously unknown variant of Ano1. The 5' end of this novel variant lacks exons 1 and 2 and part of exon 3. Expression of this variant in HEK cells produces a decreased density of Ca(2+)-activated Cl(-) currents that exhibit slower kinetics compared with the full-length Ano1. These results identify important changes in expression and splicing of Ano1 in patients with diabetic gastroparesis that alter the electrophysiological properties of the channel. Changes in Ano1 expression in ICC may directly contribute to diabetic gastroparesis.

Domperidone treatment for gastroparesis: demographic and pharmacogenetic characterization of clinical efficacy and side-effects.

BACKGROUND: Domperidone is a useful alternative to metoclopramide for treatment of gastroparesis due to better tolerability. Effectiveness and side-effects from domperidone may be influenced by patient-related factors including polymorphisms in genes encoding drug-metabolizing enzymes, drug transporters, and domperidone targets. AIMS: The aim of this study was to determine if demographic and pharmacogenetic parameters of patients receiving domperidone are associated with response to treatment or side-effects. METHODS: Patients treated with domperidone for gastroparesis provided saliva samples from which DNA was extracted. Fourteen single-nucleotide polymorphisms (SNPs) in seven candidate genes (ABCB1, CYP2D6, DRD2, KCNE1, KCNE2, KCNH2, KCNQ1) were used for genotyping. SNP microarrays were used to assess single-nucleotide polymorphisms in the ADRA1A, ADRA1B, and ADRA1D loci. RESULTS: Forty-eight patients treated with domperidone participated in the study. DNA was successfully obtained from each patient. Age was associated with effectiveness of domperidone (p=0.0088). Genetic polymorphism in KCNH2 was associated with effectiveness of domperidone (p=0.041). The efficacious dose was associated with polymorphism in ABCB1 gene (p=0.0277). The side-effects of domperidone were significantly associated with the SNPs in the promoter region of ADRA1D gene. CONCLUSIONS: Genetic characteristics associated with response to domperidone therapy included polymorphisms in the drug transporter gene ABCB1, the potassium channel KCNH2 gene, and α1D--adrenoceptor ADRA1D gene. Age was associated with a beneficial response to domperidone. If verified in a larger population, this information might be used to help determine which patients with gastroparesis might respond to domperidone and avoid treatment in those who might develop side-effects.

Serotonergic signalling in the stomach and duodenum of patients with gastroparesis.

Serotonin (5-HT) is involved in the regulation of motoric and sensory functions of the upper gastrointestinal tract. The aim of the current study was to determine whether serotonergic signalling is altered in patients with idiopathic gastroparesis. Mucosal biopsy specimens were collected from the duodenum, antrum and fundus of 11 patients with idiopathic gastroparesis and 11 healthy controls. Neuroendocrine cells, specifically 5-HT producing cells, were counted after immunohistochemistry, and non-neuronal mRNA expression levels of tryptophan hydroxylase (TPH)-1, 5-HT transport protein (SERT), 5-HT3 and 5-HT4 receptor were quantified by real time RT-PCR. The number of 5-HT producing cells was comparable between patients and controls. No difference in expression of TPH-1 (rate limiting enzyme in 5-HT biosynthetic pathway) and SERT (responsible for 5-HT uptake) was found between patients and controls (P > 0.05). In the duodenum, the expression of the 5-HT3 receptor subunits and the 5-HT4 receptor was comparable between both groups. However, the 5-HT4(c) splice variant was expressed more abundantly in healthy controls compared to patients (P = 0.015). This study suggests that the delayed gastric emptying and upper abdominal symptoms in idiopathic gastroparesis do not result from altered mucosal 5-HT biosynthetic and uptake capacity.