M1739 Luminal Propionate Sensing via G-Protein in Lipid Rafts in Colonic Secretory Response

insulin resistance by activating PPARγ in adipose tissue and promoting adipocytes differentiation. Methods. In Vivo experiments in lean (fa/+) and obese (fa/fa) Zucker rats were carried out. Animals were housed on a 12-h light-dark cycle and fed standard laboratory diet ad libitum. Drugs administration was started at 15 weeks of age. Animals were randomized into 4 groups (12 per group) and administered with the semi-synthetic FXR ligand 6ethyl chenodeoxycholic acid (6E-CDCA) (10 mg/kg), or rosiglitazone (10 mg/kg) or the combination of the two (5 mg/kg each) once daily for 7 weeks. Results. The administration of 6E-CDCA reduced hyperinsulinemia, hyperglycemia and hyperlipidemia, decreased the circulating levels of FFAs, cholesterol , HDL, ALT, AST, γGT and ameliorated the insulin sensitivity as demonstrated by positive effects on OGGT and ITT. In the liver, 6E-CDCA administration ameliorated insulin resistance by increasing the levels of p-AKT at ser473 and by decreasing the liver level of p-IRS at ser312. Moreover, it decreased triglyceride, FFA and cholesterol content. In the muscle, 6E-CDCA decreased tryglicerides, FFAs and cholesterol levels. The histological analysis (oil-red oil staining) demonstrated a reduction in fat content in response to the FXR ligand, but not the PPARγ ligand. In addition, 6ECDCA reduced body weight gain and fat mass when compared with rosiglitazone. 6E-CDCA upregulated the liver expression of SHP, PPARα, APOC2, PGC1α and downregulated TNFα and Cyp7α1 as evaluated with RT-PCR. 6E-CDCA administration reduced mean arterial pressure, heart weight and troponin plasma level. Conclusions. This is the first report of the beneficial effects of a FXR ligand in a rodent model of NASH. FXR administration to fa/fa rats ameliorates lipid homeostasis and glusose homeostasis and attenuates insulin resistance. These data ground the use of FXR ligands in NASH patients.

M1738

Caveolae and their main membrane proteins (caveolin-1, -2 and -3) concentrate receptors, G proteins, effector enzymes, and associated signaling molecules so as to facilitate coordinated generation and degradation of second messengers such as cAMP and cGMP. In gastrointestinal smooth muscle cGMP levels are regulated via feedback mechanisms involving cGMP-dependent kinase (PKG)-mediated phosphorylation of soluble guanylyl cyclase (sGC) and cGMPspecific phsophodiesterase 5 (PDE5) resulting in decreased synthesis and increased degradation of cGMP. We have previously shown that caveolin-1 and -3 are expressed in gastrointestinal smooth muscle, and that caveolin-1 is associated with PDE5, but not with sGC in the basal state. We hypothesize that binding of PDE5 to caveolin-1 inhibits PDE5 activity, thereby reducing cGMP degradation and enhancing cGMP/PKG signaling. AIM. To examine the role of caveolin-1 in nitric oxide (NO)-induced muscle relaxation and colonic propulsion. METHODS. Colonic segments were isolated from wild type and caveolin-1 knockout (caveolin-1-/-) mice. Relaxation, i.e., decrease in carbachol-induced tension, in response to NOdonor (sodium nitroprusside, SNP) was measured isometrically with a force-displacement transducer. Descending relaxation in response to mucosal stroking was measured in threecompartment, flat-sheet preparation. The velocity of propulsion of artificial fecal pellets that mimic native pellets in size and shape was measured in isolated whole segments of the colon. Cyclic GMP levels were measured by RIA and PDE5 activity was measured by ionexchange chromatography using [3H]cGMP as substrate. RESULTS. SNP stimulated PDE5 activity, cGMP formation and induced relaxation in a concentration-dependent manner in colonic muscle strips. SNP-stimulated PDE5 activity was augmented, whereas cGMP formation and muscle relaxation were attenuated in colonic muscle strips from caveolin-1-/- mice. In the peristalsis preparation, mucosal stroking elicited stimulus-dependent descending relaxation caudad to the stimulus, which was significantly inhibited (58%-73% inhibition) in colonic segments from caveolin-1-/- mice compared to wild type mice. Consistent with the reduced descending relaxation, the velocity of pellet propulsion was significantly slower in colonic segments from caveolin-1-/- mice compared to wild type mice (0.72 mm/s in colonic segments from wild type mice vs. 0.38 mm/s in colonic segments from caveolin-1 -/mice) suggesting that caveolin-1 promotes colonic propulsion. CONCLUSION. Caveolin1 enhances cGMP-mediated muscle relaxation and colonic propulsion by binding PDE5 and inhibiting cGMP degradation.

M1736 Blockade of Angiotensin II Type 1 Receptor Reduces Hepatocellular Lipid Accumulation in An In Vitro Model of Steatosis : A Potential Implication for the Treatment of Non-Alcoholic Fatty Liver Disease Keishi Kanno, Takuya Ikuta, Sosuke Matsuda, Nobusuke Kishikawa, Susumu Tazuma Background/Aims: A distinct subgroup of angiotensin II type 1 receptor (AT1R) blockers (ARBs) have been reported to suppress the development of hepatic steatosis. These effects were generally explained by selective peroxisome proliferator-activated receptor (PPAR)γ modulating properties of ARBs, independent of their AT1R blocking actions. However, in our previous experiments mice lacking AT1R exhibited resistance against methionine-choline deficient (MCD) diet-induced steatohepatitis when compared to wild type mice (Nabeshima Y, et al. Gastroenterology 2007;132(4):A819.). This observation suggests the possibility that the blockade of AT1R itself might contribute to hepatocellular lipid metabolism. To explore the direct role for AT1R in hepatocellular steatosis, we utilized In Vitro model of cellular steatosis in which human hepatoma cell line HepG2 was overloaded with free fatty acids (FFA). Methods: AT1R expression in HepG2 was confirmed by Western blot analysis and real-time PCR. Cellular steatosis was induced by overload of 200 or 400 μM FFA mixture (2:1 oleate: palmitate). The role for AT1R in cellular steatosis was investigated by pharmacological blockade using telmisartan (10 μM) as well as by gene silencing by RNA interference targeting AT1R. Cellular lipid content was assessed by Nile Red assay. The expressions of PPARα, its target genes, and the other PPAR isoforms were quantified by real-time PCR. Results: Western blot analysis revealed AT1R protein expression in HepG2. In addition, real-time PCR demonstrated similar level of AT1R mRNA in HepG2 as compared to human kidney tissue. Following the treatment of FFA mixture for 24 h, intracellular lipid was increased by 4-5-fold as quantified by Nile Red assay. These changes were markedly attenuated by the presence of telmisartan by 60-70 %. AT1R depletion by siRNA also resulted in significant decrease (-15-30 %) in cellular lipid deposition following FFA exposure. This was also associated with significant up-regulation of PPARα (+41%) and its target genes, acyl-CoA synthetase 1 and apolipoprotein B 100 (+27 %, +21 %, respectively). In contrast, gene expressions of the other PPAR isoforms, PPARγ and PPARβ(δ) were not altered by AT1R depletion Conclusion: Our data indicate that AT1R plays a key biological role in the regulation of hepatic lipid metabolism presumably through induction of PPARα. Up-regulation of PPARα would likely increase the sensitivity to agonist, and thus clinical usage of ARBs together with PPARα ligand such as fibrates might have synergistic effects on hepatic lipid metabolism as a therapeutic strategy for non-alcoholic fatty liver disease.

M1739 Luminal Propionate Sensing via G-Protein in Lipid Rafts in Colonic Secretory Response Takaji Yajima Background: G-protein coupled signal transduction plays a significant role in nutrient sensing in the gut. Addition of luminal propionate and other short-chain fatty acids resulted in transient chloride secretion and increased short-circuit current (Isc) and conductance in rat colon In Vitro. The Isc responses to propionate were remarkably inhibited by luminal procaine, and serosal atropine and TTX, indicating via an enteric reflex involving a mucosal sensory mechanism and cholinergic motor nerves. Objective: The present study aimed to examine an involvement of G-protein signal transduction in Isc response to luminal propionate sensing in rat distal colon. Methods: The mucosa-submucosa preparations from the distal colon of SD rats (230-270g) were used in all experiments. The tissue mounted in an Ussing chamber was voltage clumped at zero potential to measure Isc. Cholesterol rich membrane domain including lipid rafts was fractionated by a sucrose-gradient ultra-centrifugation after a detergent free extraction from the isolated colonic crypt. Results: The increase in Isc induced by luminal propionate was significantly inhibited by prior addition of luminal mastoparan (MP) and methyl-β-cyclodextrin (MβCD), 66% and 81%, respectively. On the other hand, the increases in Isc evoked by serosal ACh (0.05mM) and electrical field stimulation (10Hz, 90sec) were not inhibited by MP and MβCD. The inhibitory effects of MP and MβCD indicate the involvement of a G-protein and a cholesterol rich membrane domain, lipid rafts, in the luminal propionate sensing. As the Isc response to propionate was desensitized against the following stimulation, we analyzed an uncoupling of G-protein and flotillin-1 in lipid rafts by Western blotting. A 10-min stimulation with luminal propionate (5mM) caused uncoupling of Gαq/11 and flotillin-1 in the crypt isolated from the distal colon. Conclusion: The present results suggest that the secretory response to luminal propionate sensing in the rat distal colon is mediated probably via Gαq/11 in lipid rafts.

M1737 Acetaldehyde May Induce Hepatic Fibrosis via the Expression of Cannabinoid Receptor Eleonora Patsenker, Gunda Millonig, Sebastian Mueller, Felix Stickel, Helmut-Karl Seitz Background: The endocannabinoid system is involved in the regulation of hepatic fibrosis, steatosis and inflammation. CB1 and CB2 receptors (CBR1 and CBR2) are substantially upregulated during liver injury and mediate pro- and anti-fibrogenic effects. Recent data strongly support the hypothesis that cannabinoids mediate hepatic fibrosis since daily cannabis smoking was found to be an independent risk factor for the progression of fibrosis in chronic hepatitis C. Aims and Methods: To investigate the role of CBRs in alcoholic liver disease (ALD), 56 paraffin-embedded and 24 frozen liver tissues from patients with different severities of ALD were analyzed. CBR1 and CBR2 immunohistochemistry was performed with polyclonal anti-CBR1 antibodies. CBR1 and CBR2, procollagen alpha1(I) (PCI) and alpha-smooth muscle actin (aSMA) transcription was quantified by TaqMan PCR. For In Vitro experiments, human hepatic stellate cells (HSC) were treated with acetaldehyde, a toxic and profibrogenic metabolite of alcohol, at 50-200μM for 6 and 24h in serum-free conditions. Results: CBR1 immunostainings in ALD revealed expression of CBR1 predominantly in fibrotic septa of liver sections showing advanced fibrosis, whereas in tissues with no or low fibrosis stages CBR1 immunoreactivity was less pronounced or absent. At mRNA level, CBR1 correlated positively with fibrosis stage, while CBR2 expression showed an inverse correlation. CBR1 induction did not correlate with PCI expression in ALD. In Vitro, acetaldehyde induced CBR1 mRNA about 2-fold after 24h of incubation at 200μM, whereas CBR2 mRNA was unaffected. Conclusions: Our results show that CBR1 signaling may be of relevance in hepatic fibrogenesis which may be enhanced by acetaldehyde, the first metabolite of ethanol oxidation. Thus, cannabis smoking may potentially aggravate the development of fibrosis in ALD.

M1740 Natriuretic Peptides Cause Relaxation of Lower Esophageal Sphincter Through a Novel Brain Natriuretic Peptide-Preferring Natriuretic Peptide Receptor Shih-Che Huang Atrial natriuretic peptide (ANP) produces relaxation in the lower esophageal sphincter. The subtype of the natriuretic peptide receptor mediating this relaxation is unclear. To characterize natriuretic peptide receptor in the lower esophageal sphincter, we measured relaxation of transverse strips from the guinea pig lower esophageal sphincter caused by ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and des[Gln18, Ser19, Gly20, Leu21, Gly22]ANP(4-23) amide (cANP(4-23)), a selective natriuretic peptide receptor 3 (NPR3) agonist. In carbachol or sarafotoxin S6c-contracted lower esophageal sphincter strips, BNP caused moderate, sustained and concentration-dependent relaxation. ANP and CNP caused mild relaxation whereas cANP(4-23) did not cause any relaxation. The relative potencies for natriuretic peptide to cause relaxation were BNP > ANP = CNP. BNP was more potent than ANP and CNP in causing relaxation of the lower esophageal sphincter. This could not be accounted for by NPR1, 2 or 3 and suggests the existence of a novel natriuretic peptide receptor. Furthermore, the BNP-induced relaxation was not affected by tetrodotoxin or atropine, indicating a direct effect. The relaxant response of BNP was abolished by charybdotoxin and attenuated by the cGMP inhibitor Rp-8CPT-cGMPS but not affected

A-421

AGA Abstracts

AGA Abstracts

Caveolin-1 Regulates Colonic Propulsion By Inhibiting cGMP-Specific PDE5 Activity and Enhancing cGMP/PKG-Mediated Muscle Relaxation John R. Grider, Sunila Mahavadi, Gracious R. Ross, Hamid I. Akbarali, Karnam S. Murthy