Enteric Neuropathy Can Be Induced by High Fat Diet In Vivo and Palmitic Acid Exposure In Vitro

Objective

Obese and/or diabetic patients have elevated levels of free fatty acids and increased susceptibility to gastrointestinal symptoms. Since the enteric nervous system is pivotal in regulating gastrointestinal functions alterations or neuropathy in the enteric neurons are suspected to occur in these conditions. Lipid induced intestinal changes, in particular on enteric neurons, were investigated in vitro and in vivo using primary cell culture and a high fat diet (HFD) mouse model.

Design

Mice were fed normal or HFD for 6 months. Intestines were analyzed for neuronal numbers, remodeling and lipid accumulation. Co-cultures of myenteric neurons, glia and muscle cells from rat small intestine, were treated with palmitic acid (PA) (0 – 10⁻³ M) and / or oleic acid (OA) (0 – 10⁻³ M), with or without modulators of intracellular lipid metabolism. Analyses were by immunocyto- and histochemistry.

Results

HFD caused substantial loss of myenteric neurons, leaving submucous neurons unaffected, and intramuscular lipid accumulation in ileum and colon. PA exposure in vitro resulted in neuronal shrinkage, chromatin condensation and a significant and concentration-dependent decrease in neuronal survival; OA exposure was neuroprotective. Carnitine palmitoyltransferase 1 inhibition, L-carnitine- or alpha lipoic acid supplementation all counteracted PA-induced neuronal loss. PA or OA alone both caused a significant and concentration-dependent loss of muscle cells in vitro. Simultaneous exposure of PA and OA promoted survival of muscle cells and increased intramuscular lipid droplet accumulation. PA exposure transformed glia from a stellate to a rounded phenotype but had no effect on their survival.

Conclusions

HFD and PA exposure are detrimental to myenteric neurons. Present results indicate excessive palmitoylcarnitine formation and exhausted L-carnitine stores leading to energy depletion, attenuated acetylcholine synthesis and oxidative stress to be main mechanisms behind PA-induced neuronal loss.High PA exposure is suggested to be a factor in causing diabetic neuropathy and gastrointestinal dysregulation.     

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA2�� (VIA)-deficient mice

Ca²⁺-independent phospholipase A₂β (iPLA₂β) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA₂β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M_1,3,5 receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA₂β^−/−, iPLA₂β^+/−, and iPLA₂β^+/+ mice, and [1-¹⁴C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J_in, representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA₂β^−/− or iPLA₂β^+/− compared with iPLA₂β^+/+ mice showed widespread and significant baseline reductions in k* and J_in for DHA. Arecoline increased both parameters in brain regions of iPLA₂β^+/+ mice but quantitatively less so in iPLA₂β^−/− and iPLA₂β^+/− mice. Consistent with iPLA₂β’s reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA₂β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M_1,3,5 receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.     

Protective Role of P2Y2 Receptor against Lung Infection Induced by Pneumonia Virus of Mice

ATP released in the early inflammatory processes acts as a danger signal by binding to purinergic receptors expressed on immune cells. A major contribution of the P2Y₂ receptor of ATP/UTP to dendritic cell function and Th2 lymphocyte recruitment during asthmatic airway inflammation was previously reported. We investigated here the involvement of P2Y₂ receptor in lung inflammation initiated by pneumonia virus of mice infection. We demonstrated that P2Y₂ ^−/− mice display a severe increase in morbidity and mortality rate in response to the virus. Lower survival of P2Y₂ ^−/− mice was not significantly correlated with excessive inflammation despite the higher level of neutrophil recruiters in their bronchoalveolar fluids. Interestingly, we observed reduced ATP level and lower numbers of dendritic cells, CD4⁺ T cells and CD8⁺ T cells in P2Y₂ ^−/− compared to P2Y₂ ^+/+ infected lungs. Lower level of IL-12 and higher level of IL-6 in bronchoalveolar fluid support an inhibition of Th1 response in P2Y₂ ^−/− infected mice. Quantification of DC recruiter expression revealed comparable IP-10 and MIP-3α levels but a reduced BRAK level in P2Y₂ ^−/− compared to P2Y₂ ^+/+ bronchoalveolar fluids. The increased morbidity and mortality of P2Y₂ ^−/− mice could be the consequence of a lower viral clearance leading to a more persistent viral load correlated with the observed higher viral titer. The decreased viral clearance could result from the defective Th1 response to PVM with a lack of DC and T cell infiltration. In conclusion, P2Y₂ receptor, previously described as a target in cystic fibrosis therapy and as a mediator of Th2 response in asthma, may also regulate Th1 response protecting mice against lung viral infection.     

Distribution of P2Y<Subscript>2</Subscript> receptors in the guinea pig enteric nervous system and its coexistence with P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> receptors,neuropeptide Y,nitric oxide synthase and calretinin

The distribution of P2Y₂ receptor-immunoreactive (ir) neurons and fibers and coexistence of P2Y₂ with P2X₂ and P2X₃ receptors, neuropeptide Y (NPY), calretinin (CR), calbindin (CB) and nitric oxide synthase (NOS) was investigated with immunostaining methods. The results showed that P2Y₂-ir neurons and fibers were distributed widely in myenteric and submucous plexuses of the guinea pig stomach corpus, jejunum, ileum and colon. The typical morphology of P2Y₂-ir neurons was a long process with strong positive staining on the same side of the cell body. The P2Y₂-ir neurons could be Dogiel type 1. About 40–60% P2X₃-ir neurons were immunoreactive for P2Y₂ in the myenteric plexus and all the P2X₃-ir neurons expressed the P2Y₂ receptor in the submucosal plexus; almost all the NPY-ir neurons and the majority of CR-ir neurons were also immunoreactive for P2Y₂, especially in the myenteric plexus of the small intestine; no P2Y₂-ir neurons were immunoreactive for P2X₂ receptors, CB and NOS. It is shown for the first time that S type/Dogiel type 1 neurons with fast P2X and slow P2Y receptor-mediated depolarizations could be those neurons expressing both P2Y₂-ir and P2X₃-ir and that they are widely distributed in myenteric and submucosal plexuses of guinea pig gut.     

Distribution of P2Y6 and P2Y12 receptor: their colocalization with calbindin, calretinin and nitric oxide synthase in the guinea pig enteric nervous system

The distribution of P2Y₆ and P2Y₁₂ receptor-immunoreactive (ir) neurons and fibers and their coexistence with calbindin, calretinin and nitric oxide synthase (NOS) has been investigated with single and double labeling immunostaining methods. The results showed that 30–36% of the ganglion cells in the myenteric plexus are strongly P2Y₆ receptor-ir neurons; they are distributed widely in the myenteric plexus of stomach, jejunum, ileum and colon, but not in the submucosal plexus, with a typical morphology of multipolar neurons with a long axon-like process. About 42–46% of ganglion cells in both the myenteric and submucosal plexuses show P2Y₁₂ receptor-ir. About 28–35% of P2Y₆ receptor-ir neurons were found to coexist with NOS and 41–47% of them coexist with calretinin, but there was no coexistence of P2Y₆ receptor-ir with calbindin. In contrast, all P2Y₁₂ receptor-ir neurons were immunopositive for calbindin, although occasionally P2Y₁₂ receptor-ir neurons without calbindin immunoreactivity were found, while none of the P2Y₁₂ receptor-ir neurons were found to coexist with calretinin or NOS in the gastrointestinal system of guinea pig. The P2Y₁₂ receptor-ir neurons coexpressing calbindin-ir in the small intestine are Dogiel type II/AH, intrinsic primary afferent neurons.     

Lipopolysaccharide-Induced Loss of Cultured Rat Myenteric Neurons - Role of AMP-Activated Protein Kinase

Objective

Intestinal barrier function is vital for homeostasis. Conditions where the mucosal barrier is compromised lead to increased plasma content of lipopolysaccharide (LPS). LPS acts on Toll-like receptor 4 (TLR4) and initiates cellular inflammatory responses. TLR4 receptors have been identified on enteric neurons and LPS exposure causes neuronal loss, counteracted by vasoactive intestinal peptide (VIP), by unknown mechanisms. In addition AMP activated protein kinase (AMPK) stimulation causes loss of enteric neurons. This study investigated a possible role of AMPK activation in LPS-induced neuronal loss.

Design

Primary cultures of myenteric neurons isolated from rat small intestine were used. Cultures were treated with LPS (0.2–20 µg/mL) with and without TAK1-inhibitor (5Z)-7-Oxozeaenol (10⁻⁶ M) or AMPK inhibitor compound C (10⁻⁵ M). AMPK-induced neuronal loss was verified treating cultures with three different AMPK activators, AICAR (10⁻⁴−3×10⁻³ M), metformin (0.2–20 µg/mL) and A-769662 (10⁻⁵−3×10⁻⁴ M) with or without the presence of compound C (10⁻⁵ M). Upstream activation of AMPK-induced neuronal loss was tested by treating cultures with AICAR (10⁻³ M) in the presence of TAK1 inhibitor (5Z)-7-Oxozeaenol (10⁻⁶ M). Neuronal survival and relative numbers of neurons immunoreactive (IR) for VIP were evaluated using immunocytochemistry.

Results

LPS caused a concentration dependent loss of neurons. All AMPK activators induced loss of myenteric neurons in a concentration dependent manner. LPS-, AICAR- and metformin-,but not A-769662-, induced neuronal losses were inhibited by presence of compound C. LPS, AICAR or metformin exposure increased the relative number of VIP-IR neurons; co-treatment with (5Z)-7-Oxozeaenol or compound C reversed the relative increase in VIP-IR neurons induced by LPS. (5Z)-7-Oxozeaenol, compound C or A-769662 did not per se change neuronal survival or relative numbers of VIP-IR neurons.

Conclusion

AMPK activation mimics LPS-induced loss of cultured myenteric neurons and LPS-induced neuronal loss is counteracted by TAK1 and AMPK inhibition. This suggests enteric neuroimmune interactions involving AMPK regulation.     

P2Y2R Deficiency Attenuates Experimental Autoimmune Uveitis Development

We aimed to study the role of the nucleotide receptor P2Y₂R in the development of experimental autoimmune uveitis (EAU). EAU was induced in P2Y₂^+/+ and P2Y₂^-/- mice by immunization with IRBP peptide or by adoptive transfer of in vitro restimulated semi-purified IRBP-specific enriched T lymphocytes from spleens and lymph nodes isolated from native C57Bl/6 or P2Y₂^+/+ and P2Y₂^-/- immunized mice. Clinical and histological scores were used to grade disease severity. Splenocytes and lymph node cell phenotypes were analyzed using flow cytometry. Semi-purified lymphocytes and MACS-purified CD4⁺ T lymphocytes from P2Y₂^+/+ and P2Y₂^-/- immunized mice were tested for proliferation and cytokine secretion. Our data show that clinical and histological scores were significantly decreased in IRBP-immunized P2Y₂^-/- mice as in P2Y₂^-/- mice adoptively transfered with enriched T lymphocytes from C57Bl/6 IRBP-immunized mice. In parallel, naïve C57Bl/6 mice adoptively transferred with T lymphocytes from P2Y₂^-/- IRBP-immunized mice also showed significantly less disease. No differences in term of spleen and lymph node cell recruitment or phenotype appeared between P2Y₂^-/- and P2Y₂^+/+ immunized mice. However, once restimulated in vitro with IRBP, P2Y₂^-/- T cells proliferate less and secrete less cytokines than the P2Y₂^+/+ one. We further found that antigen-presenting cells of P2Y₂^-/- immunized mice were responsible for this proliferation defect. Together our data show that P2Y₂^-/- mice are less susceptible to mount an autoimmune response against IRBP. Those results are in accordance with the danger model, which makes a link between autoreactive lymphocyte activation, cell migration and the release of danger signals such as extracellular nucleotides.     

The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

Various radioligands have been used to characterize and quantify the platelet P2Y₁₂ receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y₁ and P2Y₁₂. We used the [³H]PSB-0413 selective P2Y₁₂ receptor antagonist radioligand to reevaluate the number of P2Y₁₂ receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [³H]PSB-0413 bound to 425 ± 50 sites/platelet (K_D = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y₁₂, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y₁ ligand MRS2179 and the P2X₁ ligand α,β-Met-ATP did not displace [³H]PSB-0413 binding. Patients with severe P2Y₁₂ deficiency displayed virtually no binding of [³H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y₁₂ receptor had normal binding. Studies in mice showed that: (1) [³H]PSB-0413 bound to 634 ± 87 sites/platelet (K_D = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [³H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y₁₂ receptors, to identify patients with P2Y₁₂ deficiencies or quantify the effect of P2Y₁₂ targeting drugs.     

Resistance to Diet-Induced Obesity and Associated Metabolic Perturbations in Haploinsufficient Monocarboxylate Transporter 1 Mice

The monocarboxylate transporter 1 (MCT1 or SLC16A1) is a carrier of short-chain fatty acids, ketone bodies, and lactate in several tissues. Genetically modified C57BL/6J mice were produced by targeted disruption of the mct1 gene in order to understand the role of this transporter in energy homeostasis. Null mutation was embryonically lethal, but MCT1 ^+/− mice developed normally. However, when fed high fat diet (HFD), MCT1 ^+/− mice displayed resistance to development of diet-induced obesity (24.8% lower body weight after 16 weeks of HFD), as well as less insulin resistance and no hepatic steatosis as compared to littermate MCT1 ^+/+ mice used as controls. Body composition analysis revealed that reduced weight gain in MCT1 ^+/− mice was due to decreased fat accumulation (50.0% less after 9 months of HFD) notably in liver and white adipose tissue. This phenotype was associated with reduced food intake under HFD (12.3% less over 10 weeks) and decreased intestinal energy absorption (9.6% higher stool energy content). Indirect calorimetry measurements showed ∼ 15% increase in O₂ consumption and CO₂ production during the resting phase, without any changes in physical activity. Determination of plasma concentrations for various metabolites and hormones did not reveal significant changes in lactate and ketone bodies levels between the two genotypes, but both insulin and leptin levels, which were elevated in MCT1 ^+/+ mice when fed HFD, were reduced in MCT1 ^+/− mice under HFD. Interestingly, the enhancement in expression of several genes involved in lipid metabolism in the liver of MCT1 ^+/+ mice under high fat diet was prevented in the liver of MCT1 ^+/− mice under the same diet, thus likely contributing to the observed phenotype. These findings uncover the critical role of MCT1 in the regulation of energy balance when animals are exposed to an obesogenic diet.     

P2Y2 receptor agonist with enhanced stability protects the heart from ischemic damage in vitro and in vivo

Extracellular nucleotides acting via P2 receptors play important roles in cardiovascular physiology/pathophysiology. Pyrimidine nucleotides activate four G protein-coupled P2Y receptors (P2YRs): P2Y₂ and P2Y₄ (UTP-activated), P2Y₆, and P2Y₁₄. Previously, we showed that uridine 5′-triphosphate (UTP) activating P2Y₂R reduced infarct size and improved mouse heart function after myocardial infarct (MI). Here, we examined the cardioprotective role of P2Y₂R in vitro and in vivo following MI using uridine-5′-tetraphosphate δ-phenyl ester tetrasodium salt (MRS2768), a selective and more stable P2Y₂R agonist. Cultured rat cardiomyocytes pretreated with MRS2768 displayed protection from hypoxia [as revealed by lactate dehydrogenase (LDH) release and propidium iodide (PI) binding], which was reduced by P2Y₂R antagonist, AR-C118925 (5-((5-(2,8-dimethyl-5H-dibenzo[a,d][7]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide). In vivo, echocardiography and infarct size staining of triphenyltetrazolium chloride (TTC) in 3 groups of mice 24 h post-MI: sham, MI, and MI+MRS2768 indicated protection. Fractional shortening (FS) was higher in MRS2768-treated mice than in MI alone (40.0 ± 3.1 % vs. 33.4 ± 2.7 %, p < 0.001). Troponin T and tumor necrosis factor-α (TNF-α) measurements demonstrated that MRS2768 pretreatment reduced myocardial damage (p < 0.05) and c-Jun phosphorylation increased. Thus, P2Y₂R activation protects cardiomyocytes from hypoxia in vitro and reduces post-ischemic myocardial damage in vivo.     

Aquaporin-1 Tunes Pain Perception by Interaction with Nav1.8 Na+ Channels in Dorsal Root Ganglion Neurons

Aquaporin-1 (AQP1) water channels are expressed in the plasma membrane of dorsal root ganglion (DRG) neurons. We found reduced osmotic water permeability in freshly isolated DRG neurons from AQP1^−/− versus AQP1^+/+ mice. Behavioral studies showed greatly reduced thermal inflammatory pain perception in AQP1^−/− mice evoked by bradykinin, prostaglandin E₂, and capsaicin as well as reduced cold pain perception. Patch clamp of freshly isolated DRG neurons showed reduced action potential firing in response to current injections. Single action potentials after pulse current injections showed reduced maximum inward current, suggesting impaired Na_v1.8 Na⁺ function. Whole-cell Na_v1.8 Na⁺ currents in Na_v1.8-expressing ND7-23 cells showed slowed frequency-dependent inactivation after AQP1 transfection. Immunoprecipitation studies showed AQP1- Na_v1.8 Na⁺ interaction, which was verified in live cells by single-particle tracking of quantum dot-labeled AQP1. Our results implicate the involvement of AQP1 in DRG neurons for the perception of inflammatory thermal pain and cold pain, whose molecular basis is accounted for, in part, by reduced Na_v1.8-dependent membrane Na⁺ current. AQP1 is, thus, a novel target for pain management.     

Group 1B phospholipase A₂ deficiency protects against diet-induced hyperlipidemia in mice

Excessive absorption of products of dietary fat digestion leads to type 2 diabetes and other obesity-related disorders. Mice deficient in the group 1B phospholipase A₂ (Pla2g1b), a gut digestive enzyme, are protected against diet-induced obesity and type 2 diabetes without displaying dietary lipid malabsorption. This study tested the hypothesis that inhibition of Pla2g1b protects against diet-induced hyperlipidemia. Results showed that the Pla2g1b^−/− mice had decreased plasma triglyceride and cholesterol levels compared with Pla2g1b^+/+ mice subsequent to feeding a high-fat, high-carbohydrate (hypercaloric) diet. These differences were evident before differences in body weight gains were observed. Injection of Poloxamer 407 to inhibit lipolysis revealed decreased VLDL production in Pla2g1b^−/− mice. Supplementation with lysophosphatidylcholine, the product of Pla2g1b hydrolysis, restored VLDL production rates in Pla2g1b^−/− mice and further elevated VLDL production in Pla2g1b^+/+ mice. The Pla2g1b^−/− mice also displayed decreased postprandial lipidemia compared with Pla2g1b^+/+ mice. These results show that, in addition to dietary fatty acids, gut-derived lysophospholipids derived from Pla2g1b hydrolysis of dietary and biliary phospholipids also promote hepatic VLDL production. Thus, the inhibition of lysophospholipid absorption via Pla2g1b inactivation may prove beneficial against diet-induced hyperlipidemia in addition to the protection against obesity and diabetes.     

P2Y1 Receptor Activation of the TRPV4 Ion Channel Enhances Purinergic Signaling in Satellite Glial Cells

Transient receptor potential (TRP) ion channels of peripheral sensory pathways are important mediators of pain, itch, and neurogenic inflammation. They are expressed by primary sensory neurons and by glial cells in the central nervous system, but their expression and function in satellite glial cells (SGCs) of sensory ganglia have not been explored. SGCs tightly ensheath neurons of sensory ganglia and can regulate neuronal excitability in pain and inflammatory states. Using a modified dissociation protocol, we isolated neurons with attached SGCs from dorsal root ganglia of mice. SGCs, which were identified by expression of immunoreactive Kir4.1 and glutamine synthetase, were closely associated with neurons, identified using the pan-neuronal marker NeuN. A subpopulation of SGCs expressed immunoreactive TRP vanilloid 4 (TRPV4) and responded to the TRPV4-selective agonist GSK1016790A by an influx of Ca²⁺ ions. SGCs did not express functional TRPV1, TRPV3, or TRP ankyrin 1 channels. Responses to GSK1016790A were abolished by the TRPV4 antagonist HC067047 and were absent in SGCs from Trpv4^−/− mice. The P2Y₁-selective agonist 2-methylthio-ADP increased [Ca²⁺]_i in SGCs, and responses were prevented by the P2Y1-selective antagonist MRS2500. P2Y₁ receptor-mediated responses were enhanced in TRPV4-expressing SGCs and HEK293 cells, suggesting that P2Y₁ couples to and activates TRPV4. PKC inhibitors prevented P2Y₁ receptor activation of TRPV4. Our results provide the first evidence for expression of TRPV4 in SGCs and demonstrate that TRPV4 is a purinergic receptor-operated channel in SGCs of sensory ganglia.     

Regional expression of P2Y4 receptors in the rat central nervous system

P2Y receptors are G protein-coupled receptors composed of eight known subunits (P2Y_{1, 2, 4, 6, 11, 12, 13, 14}), which are involved in different functions in neural tissue. The present study investigates the expression pattern of P2Y₄ receptors in the rat central nervous system (CNS) using immunohistochemistry and in situ hybridization. The specificity of the immunostaining has been verified by preabsorption, Western blot, and combined use of immunohistochemistry and in situ hybridization. Neurons expressing P2Y₄ receptors were distributed widely in the rat CNS. Heavy P2Y₄ receptor immunostaining was observed in the magnocellular neuroendocrine neurons of the hypothalamus, red nucleus, pontine nuclei, mesencephalic trigeminal nucleus, motor trigeminal nucleus, ambiguous nucleus, inferior olive, hypoglossal nucleus, and dorsal motor vagus nucleus. Both neurons and astrocytes express P2Y₄ receptors. P2Y₄ receptor immunostaining signals were mainly confined to cell bodies and dendrites of neurons, suggesting that P2Y₄ receptors are mainly involved in regulating postsynaptic events. In the hypothalamus, all the vasopressin (VP) and oxytocin (OT) neurons and all the orexin A neurons were immunoreactive for P2Y₄ receptors. All the neurons expressing P2Y₄ receptors were found to express N-methyl-d-aspartate receptor 1 (NR1). These data suggest that purines and pyrimidines might be involved in regulation of the release of the neuropeptides VP, OT, and orexin in the rat hypothalamus via P2Y₄ receptors. Further, the physiological and pathophysiological functions of the neurons may operate through coupling between P2Y₄ receptors and NR1.     

Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation

The hypothesis tested in this study was that cholesterol esterification by ACAT2 would increase cholesterol absorption efficiency by providing cholesteryl ester (CE) for incorporation into chylomicrons. The assumption was that absorption would be proportional to Acat2 gene dosage. Male ACAT2^+/+, ACAT2^+/−, and ACAT2^−/− mice were fed a diet containing 20% of energy as palm oil with 0.2% (w/w) cholesterol. Cholesterol absorption efficiency was measured by fecal dual-isotope and thoracic lymph duct cannulation (TLDC) methods using [³H]sitosterol and [¹⁴C]cholesterol tracers. Excellent agreement among individual mice was found for cholesterol absorption measured by both techniques. Cholesterol absorption efficiency in ACAT2^−/− mice was 16% compared with 46–47% in ACAT2^+/+ and ACAT2^+/− mice. Chylomicrons from ACAT2^+/+ and ACAT2^+/− mice carried ∼80% of total sterol mass as CE, whereas ACAT2^−/− chylomicrons carried >90% of sterol mass in the unesterified form. The total percentage of chylomicron mass as CE was reduced from 12% in the presence of ACAT2 to ∼1% in ACAT2^−/− mice. Altogether, the data demonstrate that ACAT2 increases cholesterol absorption efficiency by providing CE for chylomicron transport, but one copy of the Acat2 gene, providing ∼50% of ACAT2 mRNA and enzyme activity, was as effective as two copies in promoting cholesterol absorption.     

Migration and differentiation of transplanted enteric neural crest-derived cells in murine model of Hirschsprung’s disease

Stem cell therapy offers the potential of rebuilding the enteric nervous system (ENS) in the aganglionic bowel of patients with Hirschsprung’s disease. P0-Cre/Floxed-EGFP mice in which neural crest-derived cells express EGFP were used to obtain ENS stem/progenitor cells. ENS stem/progenitor cells were transplanted into the bowel of Ret^−/− mouse, an animal model of Hirschsprung’s disease. Immunohistochemical analysis was performed to determine whether grafted cells gave rise to neurons in the recipient bowel. EGFP expressing neural crest-derived cells accounted for 7.01 ± 2.52 % of total cells of gastrointestinal tract. ENS stem/progenitor cells were isolated using flow cytometry and expanded as neurosphere-like bodies (NLBs) in a serum-free culture condition. Some cells in NLBs expressed neural crest markers, p75 and Sox10 and neural stem/progenitor cells markers, Nestin and Musashi1. Multipotency of isolated ENS stem/progenitor cells was determined as they differentiated into neurons, glial cells, and myofibloblasts in culture. When co-cultured with explants of hindgut of Ret^−/− mice, ENS stem/progenitor cells migrated into the aganglionic bowel and gave rise to neurons. ENS stem/progenitor cells used in this study appear to be clinically relevant donor cells in cell therapy to treat Hirschsprung’s disease capable of colonizing the affected bowel and giving rise to neurons.     

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.     

Colonic motor dysfunctions in a mouse model of high-fat diet-induced obesity: an involvement of A<Subscript>2B</Subscript> adenosine receptors

Adenosine A_2B receptors (A_2BR) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of A_2BR in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic A_2BR localization was examined by immunofluorescence. The role of A_2BR in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, A_2BR were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the A_2BR antagonist MRS1754 enhanced electrically induced NK₁-mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A_2B receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both A_2BR ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of A_2BR expression. A_2BR, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity.     

Characterization of eicosanoid synthesis in a genetic ablation model of ceramide kinase

Multiple reports have demonstrated a role for ceramide kinase (CERK) in the production of eicosanoids. To examine the effects of the genetic ablation of CERK on eicosanoid synthesis, primary mouse embryonic fibroblasts (MEFs) and macrophages were isolated from CERK^−/− and CERK^+/+ mice, and the ceramide-1-phosphate (C1P) and eicosanoid profiles were investigated. Significant decreases were observed in multiple C1P subspecies in CERK−/− cells as compared to CERK^+/+ cells with overall 24% and 48% decreases in total C1P. In baseline experiments, the levels of multiple eicosanoids were significantly lower in the CERK^−/− cells compared with wild-type cells. Importantly, induction of eicosanoid synthesis by calcium ionophore was significantly reduced in the CERK^−/− MEFs. Our studies also demonstrate that the CERK^−/− mouse has adapted to loss of CERK in regards to airway hyper-responsiveness as compared with CERK siRNA treatment. Overall, we demonstrate that there are significant differences in eicosanoid levels in ex vivo CERK^−/− cells compared with wild-type counterparts, but the effect of the genetic ablation of CERK on eicosanoid synthesis and the serum levels of C1P was not apparent in vivo.     

Targeted Deletion of Kcne2 Impairs HCN Channel Function in Mouse Thalamocortical Circuits

Background

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the pacemaking current, I_h, which regulates neuronal excitability, burst firing activity, rhythmogenesis, and synaptic integration. The physiological consequence of HCN activation depends on regulation of channel gating by endogenous modulators and stabilization of the channel complex formed by principal and ancillary subunits. KCNE2 is a voltage-gated potassium channel ancillary subunit that also regulates heterologously expressed HCN channels; whether KCNE2 regulates neuronal HCN channel function is unknown.

Methodology/Principal Findings

We investigated the effects of Kcne2 gene deletion on I_h properties and excitability in ventrobasal (VB) and cortical layer 6 pyramidal neurons using brain slices prepared from Kcne2 ^+/+ and Kcne2 ^−/− mice. Kcne2 deletion shifted the voltage-dependence of I_h activation to more hyperpolarized potentials, slowed gating kinetics, and decreased I_h density. Kcne2 deletion was associated with a reduction in whole-brain expression of both HCN1 and HCN2 (but not HCN4), although co-immunoprecipitation from whole-brain lysates failed to detect interaction of KCNE2 with HCN1 or 2. Kcne2 deletion also increased input resistance and temporal summation of subthreshold voltage responses; this increased intrinsic excitability enhanced burst firing in response to 4-aminopyridine. Burst duration increased in corticothalamic, but not thalamocortical, neurons, suggesting enhanced cortical excitatory input to the thalamus; such augmented excitability did not result from changes in glutamate release machinery since miniature EPSC frequency was unaltered in Kcne2 ^−/− neurons.

Conclusions/Significance

Loss of KCNE2 leads to downregulation of HCN channel function associated with increased excitability in neurons in the cortico-thalamo-cortical loop. Such findings further our understanding of the normal physiology of brain circuitry critically involved in cognition and have implications for our understanding of various disorders of consciousness.