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Inhibition of Intestinal Biotin Absorption by Chronic Alcohol Feeding:Cellular and Molecular Mechanisms
Su2050 Effect of Antidiabetic Drugs on the Intestinal Regulation of PepT1 Patrick Hindlet, Claire Barraud, Laura Boschat, Robert Farinotti, Andre Bado, Marion Buyse Background and aims: PepT1 plays a major role in nitrogen supply by mediating the intestinal absorption in a di- and tripeptides form of 70% of total nitrogen. We previously showed that PepT1 is regulated in a murin model of type 2 diabetes (T2D)/obesity. We therefore examined the effect of two major drugs commonly used in the T2D treatment, namely metformin and the PPARγ activator rosiglitazone (RSG) In Vitro in a first step and then in our model of T2D/obesity. Methods: For In Vitro studies, Caco2 cells were treated for 3-7 days with metformin (10 mM) and/or RSG (10 μM), the PPARγ inhibitor SR202 (300 μM), the AMPK inhibitor Compound C (10 μM), the mTOR inhibitor rapamycin (0.2 nM). PepT1 activity was studied following the Papp of 3H-Gly-Sar on Caco2 cells grown on Transwell®. Protein and mRNA expression were studied by western-blotting and qPCR respectively. The 4E binding protein 1 (4EBP-1) phosphorylation was studied by ELISA. For In Vivo studies, C57Bl6 male mice were fed a high fat (HF) diet for 6 weeks and received metformin (250 mg/kg/d) and/or RSG (8mg/kg/d) per os for 7 days and PepT1 activity was evaluated following Gly-Sar transport by the jejunal loop method. Results: In Vitro, a 7-day treatment by RSG induced a 1.3 and 1.6-fold increase in PepT1 activity and protein expression without any change in PepT1 mRNA expression. This regulation of the mRNA translation was mediated by the activation of the PPARγ receptor and through the mTOR pathway thereafter. Exploring the downstream signaling of mTOR activation, we showed that neither the S6 ribosomal protein, nor 4EBP-1 were implicated in the effect of RSG on PepT1. Metformin induced a 2-fold AMPK-dependant decrease in PepT1 transcriptionnal expression that totally reversed the effect of RSG and which conducted in a similar down regulation in the protein expression and the activity. Interestingly, metformin also reduced the phosphorylation of 4EBP-1, leading us to postulate that the drug could also modulate PepT1 expression by inhibiting its mRNA translation. In Vivo, the HF diet induced a drastic decrease of Gly-Sar transport mediated by PepT1. As expected, a 7-days RSG treatment increased PepT1 activity, whereas metformin alone had no effect but still reversed the increase in PepT1 activity induced by RSG. Conclusions: Taken together, these data indicate that PepT1 is regulated by the PPARγ agonist rosiglitazone In Vitro by enhancing its mRNA translation. On the other hand, metformin produces a strong down regulation of the transporter and acts at 2 crucial steps of PepT1 processing by inhibiting its mRNA expression and inhibiting the translation. The discrepancy between In Vivo and In Vitro experiments concerning the effect of metformin may be mediated by modification of hormonal sensitivity.
A. Cytosol from enterocytes absorbing 3H-oleate was placed under an OptiPred gradient and 3H-dpm determined. B. Light fractions (DRM) were immunoblotted for the indicated proteins.
Su2051 Inhibition of Intestinal Biotin Absorption by Chronic Alcohol Feeding:Cellular and Molecular Mechanisms Sandeep B. Subramanya, Veedamali S. Subramanian, Jeyan S. Kumar, Robert Hoiness, Hamid M. Said Background: The water-soluble vitamin biotin is essential for normal cellular functions and its deficiency leads to a variety of clinical abnormalities. Mammals obtain biotin from exogenous sources via intestinal absorption, a process mediated by the sodium-dependent multivitamin transporter (SMVT). Chronic alcohol use in humans is associated with a significant reduction in plasma biotin levels. Little, however, is known about the cellular and molecular mechanisms involved in this inhibition, and thus, were investigated in this study. Methods: Adult male albino Wistar rats and transgenic mice carrying the human SLC5A6 gene 5'-regulatory regions were used as an In Vivo model of alcohol delivery and Caco-2 cells as an In Vitro model systems were used in this study. The rats were fed (for 4 weeks) a Lieber-DeCarli ethanol liquid-diet (36% calorie); control rats were pair-fed the same diet but without ethanol. Carrier-mediated entry and exit of 3H-biotin into and out of enterocytes was examined using purified jejunal brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV), respectively. Carrier-mediated colonic uptake of 3H-biotin was determined using colonic sheets. Changes in expression of SMVT protein, mRNA, and heterogenous mRNA (hnRNA) levels were determined by Western blot, realtime PCR, and by semi quantitative PCR. In chronic alcohol-fed (25% calorie) transgenic mice, changes in carrier mediated biotin uptake in jejunal enterocytes, mRNA, and SLC5A6 promoter activity was determined. Biotin uptake and SLC5A6 P1 and P2 promoter activity was determined in Caco-2 cells exposed to chronic alcohol. Results: Carrier-mediated biotin
A. Immunodepletion of Caveolin-1, -2, and -3 from cytosol derived from enterocytes absorbing 3H-oleate. The % 3H-oleate remaining in the cytosol is shown. B. Immunoblots of the cytosol as in A. were obtained after each round of immunodepletion of Caveolin-1, -2, and -3, the band density measured and compared to native cytosol. Su2049 Localization, Structure and Regulation of AMP-Activated Protein Kinase in the Small Intestine Elodie Harmel, Benoit Viollet, Carole Garofalo, Lea Emonnot, Martine Laville, Emile Levy INTRODUCTION: Mammalian 5' AMP-activated protein kinase (AMPK) acts as a sensor of cellular energy homeostasis. It stimulates multiple metabolic pathways that increase energy production and switches off processes that consume energy. Dysfunction of this fascinating enzyme affects lipid metabolism and vital organ functions. Intriguingly, although AMPK's importance in controlling lipid metabolism has been well established in skeletal muscle and the liver, its role in regulating energy storage and depletion in enterocytes remains unexplored.
S-543
AGA Abstracts
AGA Abstracts
In fact, little attention has been paid to the small intestine, the first organ to face nutrients and an organ closely associated with diabetic postprandial dyslipidemia. OBJECTIVES: To assess whether (i) AMPK is highly expressed in particular regions of the intestine and displays a tissue-specific hetrotrimer composition; (ii) AMPK acts to monitor and restore energy in this localization, regulates lipid homeostasis and responds to diverse pharmacological signals. METHODS: Studies were performed with control and AMPKα1-/- mice as well as with the human intestinal Caco-2 cell line. RESULTS: Our data have first shown the presence of all the AMPK subunits (α1/α2/β1/β2/γ1/γ2/γ3) in Caco-2 cells and intestinal epithelial cells of control mice. Analysis of gene and protein expression by RT-PCR and Western blot, respectively, revealed a marked difference in abundance of AMPK subunits with the predominance of AMPKα1. The prevalence of α1/β2/γ1 heterotrimer was confirmed by co-immunoprecipitation. Further experiments indicated that AMPKα1 is present mainly in the jejunum and ileum and to a lesser extent in the duodenum and colon. AMPK was found activated by 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) (p<0.001), which led in increased phosphorylation of the downstream target acetyl-CoA carboxylase (ACC), thereby resulting in the inhibition of fatty acid synthesis. Interestingly, no influence was noted in 3-hydroxy-3 methylglutaryl-CoA reductase (HMG-CoAR), the key regulatory enzyme of cholesterol synthesis. Reciprocally, inhibition of AMPK phosphorylation by compound C raised ACC phosphorylation and activity (p<0,001) without affecting HMGCoAR. Finally, depletion of glucose significantly activated AMPK and inhibited ACC whereas high glucose concentration down-regulated AMPK phosphorylation while activating ACC. CONCLUSIONS: Our findings uncover the localization, structure, function and regulation of AMPK in the small intestine. Ongoing studies will clarify AMPK role in pathophysiological conditions.
AGA Abstracts
uptake by jejunal BBMV and BLMV from rats chronically fed the alcohol liquid diet was significantly (p<0.05) lower than pair-fed control rats. This decrease in biotin uptake was associated with a significant decrease in the level of expression of SMVT protein, mRNA, and hnRNA, levels. Carrier-mediated biotin uptake by colonic sheets was also significantly (p<0.05) inhibited by chronic alcohol feeding. In chronic alcohol-fed transgenic mice, jejunal enterocytes showed a significant inhibition in carrier mediated biotin uptake (p<0.01), which was associated with marked decrease in SMVT mRNA level and activity of the firefly luciferase reporter gene compared to pair-fed control transgenic mice. Chronic alcohol exposed Caco2 cells showed a significant decrease in carrier mediated biotin uptake and in the activity of the individual promoters P1 and P2 of human SLC5A6 gene. Conclusion: These studies show that chronic alcohol feeding/exposure inhibits cellular and molecular parameters of intestinal biotin uptake process, and that the effect is (at least in part) being exerted at the transcriptional level(supported by grants from NIH and VA).
Su2054 Cytotoxicity of Fecal Water is Associated to Increased Concentrations of Alcohols Karen Windey, Vicky De Preter, Jean Herman, Thierry Louat, Greet Vandermeulen, Kristin Verbeke Introduction: Colonic fermentation of carbohydrates and proteins leads to the production of short chain fatty acids, whereas protein fermentation also results in branched chain fatty acids, phenols, sulphides and amines. Some of these metabolites are potentially toxic. In the present study, we modified the degree of protein fermentation by changing protein intake and investigated whether fecal metabolites associated with cytotoxicity originate from protein fermentation. Materials and methods: After a 1-week run-in period with normal protein (NP) intake, 20 healthy volunteers followed an isocaloric high protein (HP, 30%) and a low protein (LP, 9%) diet for 2 weeks in a randomized cross-over study. Fibre and fat intake were kept constant. During the run-in period and during the second week of each diet period the volunteers completed a dietary journal and collected urine for 48h and feces for 72h. Dietary composition was analyzed. Colonic protein fermentation was estimated from the urinary concentration of p-cresol. Profiles of volatile organic compounds (VOC) were analysed in fecal samples using GC-MS. Metabolite profiles were compared using cluster analysis. Fecal water cytotoxicity was determined using the WST-1 assay and expressed as fold dilution at which 50% of the cells survived (fold dilution, FD50). Cytotoxicity was related to the metabolite profiles. Results are expressed as average and standard deviation. Results: Protein intake accounted for 27 ± 4,15% of energy intake during the HP diet, 15 ± 2,19% during the NP and 12 ± 1,76% during the LP diet. Urinary p-cresol excretion was significantly correlated with protein intake (r=0,314; p=0,015). Fecal water cytotoxicity was not correlated with protein intake (p>0.05). The average cytotoxic dilution of the NP samples was 52,3 ± 24,9 compared to 39,3 ± 22,2 of the HP samples and 45,5 ± 25,8 of the LP samples. Cluster analysis of metabolite patterns in fecal samples according to cytotoxicity revealed a separation between the high toxicity (FD50 > 75) samples and the low toxicity (FD50 < 50) samples. This separation was mainly due to the presence of alcohols and the sulfur-containing compounds, allylisocyanate and 2,4-dithiapentane, in the high toxicity samples and the presence of cycloalkanes and cycloalkenes in the low toxicity samples. SCFA did not contribute to the clustering of the samples.
Su2052 High-Fat Diet-Induced Neuropathy of the Enteric Nervous System and the Effect of Alpha-7 Nicotinic Acetylcholine Receptor Agonist, DMAB-Anabaseine Dihydrochloride Onesmo B. Balemba, Chloe M. Stenkamp-Strahm, Joshua Cady, Ailene MacPherson Excessive calorie and high-fat diet ingestion cause obesity and type two diabetes mellitus (T2DM). Disruption of gastrointestinal (GI) microbial community structure and subsequent inflammation are key pathobiological events that occur before noticeable weight gain and insulin resistance. The enteric nervous system (ENS) plays a critical role in homeostatic adaptation to hypercaloric feeding. An altered expression of neurotransmitters and neuronal cell death has been seen in the ENS of T2DM patients, but the impact of a high-fat diet on the ENS is not fully understood. We are studying how a high-fat diet affects the ENS and whether the partial alpha 7-nicotinic acetylcholine receptor (α7-nAChR) agonist DMABanabaseine dihydrochloride (DMAB) reverses obesity and ENS neuropathy in a mouse model. C57/BL6 mice were fed either a standard chow or 72% fat diet for two and eight weeks, monitoring weight gain every other day. They were tested for glucose tolerance prior to euthanasia by measuring blood glucose after 6-hour fasting, and then 30 min. and 1-hour after 10 mg/kg intraperitoneal glucose injections. Immunohistochemistry was used to study calbindin, calcitonin gene-related peptide (CGRP), substance P and S-100 protein immunoreactivities in the ENS using the longitudinal smooth muscle-myenteric plexus (LMMP) and submucosal (SMP) wholemount preparations and 10 μm full-thickness cryostat sections from duodenum. A Nikon Eclipse fluorescent microscope and Metamorph 2 software were used for morphometric analysis of tissue innervation indexes. We observed that two weeks was enough time to cause a significant weight gain and glucose intolerance, suggesting an onset of insulin resistance. ENS changes observed after two weeks include a reduction in S100P density and the innervation indices of CGRP, calbindin and substance P. After eight weeks, S100P and substance P continue to be reduced while the CGRP innervation index showed no change and that of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), became increased. Treating fat-fed mice for four weeks with DMAB (10 mg Kg-1 subcutaneously) normalized weekly weight, although it did not lessen glucose intolerance. DMAB treatment increased ChAT mRNA levels by 3-14 fold compared to control, suggesting that an up-regulation of ChAT could be occurring. Collectively, our findings suggest that a high-fat diet and subsequent lipotoxicity disrupts enteric glial cells responsible for the upkeep and maintenance of nerve cells and the regulation of mucosal barrier function. The diet alters neurochemical expression in the ENS suggesting altered neurotransmission. DMAB and possibly other α7-nAChR agonists are promising therapies for the prevention and treatment of obesity, T2DM and other metabolic disorders that merit further study.
Su2055 Increased Copper Intake During Iron Deficiency Partly Ameliorates the IronDeficient Phenotype: A Possible Adaptive Mechanism in Iron Homeostasis Perungavur Ranganathan, Yan Lu, Lingli Jiang, Changae Kim, James F. Collins We sought to test the hypothesis that increased copper intake in the setting of iron deficiency would enhance serum ferroxidase (i.e. ceruloplasmin [Cp]) activity and positively alter iron homeostasis. The combined effect of alterations in dietary iron and copper was thus investigated in rats by tracking biological and biochemical parameters of iron and copper homeostasis. Copper deprivation increased hepatic iron levels over controls (54%), and also increased serum iron as compared to iron deprived rats (~5-fold). Increasing copper to ~5X the normal level had no effect on iron levels or hepatic copper levels in controls, but serum copper increased by ~145% in iron deficient rats. Hemoglobin and hematocrit levels increased ~2fold in iron deficient rats fed a high copper diet. Real-time PCR experiments revealed increased expression of Cp in copper deficient rats and decreased expression of hepcidin in liver under low Fe conditions. Western analyses of sera showed increased Cp protein with low Fe diet and low Fe diet with extra copper, and decreased Cp with low Cu. This pattern was also reflected by (in-gel) enzyme activity assay for ferroxidase, by two different methods (pPD and ferrozine). Similar activity-assays, performed spectrophotometrically, clearly demonstrated that serum ferroxidase activity was enhanced by iron deprivation and by intake of higher copper levels (in the control and iron deficient groups). This investigation has thus demonstrated that increased copper intake, reflected by increased liver and serum copper levels, can enhance serum ferroxidase activity in rats, which in turn may play a key role in the partial amelioration of the iron deficient phenotype.
Su2053 Disorders in AMPK and Insulin Signalling Pathways in the Intestine of Insulin-Resistant and Diabetic Psammomys Obesus Elodie Harmel, Ali Bendjoudi, Mounib Elchebly, Benoit Viollet, Ehud Ziv, Edgard Delvin, Martine Laville, Emile Levy Emerging evidence supports the link between insulin resistance (IR), type 2 diabetes (T2D) and aberrant intestinal lipid and lipoprotein metabolism. However, the abnormalities in intestinal signalling pathways were not systematically investigated in these metabolic disorders. RESEARCH DESIGN AND METHODS: The aims of the present study were to determine whether aberrant AMP-activated protein kinase (AMPK) and insulin signalling pathways characterize the jejunum and ileum of IR and T2D Psammomys obesus sand rat, an animal model of IR and T2D, and is associated with overproduction of triglyceride (TG)rich lipoproteins. RESULTS: Concomitant with their enhanced lipogenesis as well as their higher efficiency in de novo TG synthesis, apolipoprotein B-48 biogenesis and TG-rich lipoprotein assembly in conditions of IR and T2D, intestinal tissues exhibited :(a) a downregulation of AMPK signalling pathway essentially typified by a decrease in AMPKα-Thr172 phosphorylation, AMPKα protein mass and gene expression of the isoforms AMPKα1 and AMPKα2; (b) a reduction in acetyl CoA carboxylase (ACC)-ser79 phosphorylation without alterations in total ACC expression, suggesting a rise in activity of both ACC1 (responsible for fatty acids (FA) synthesis) and ACC2 (responsible for the inhibition of FA oxidation); (c) a decline in the gene and protein expression of carnitine palmitoyl transferase, supporting an inhibition of β-oxidation; (d) a trend of lessening of a second signalling pathway portrayed by the diminution of the Akt and glycogen synthetase kinase-3β phosphorylation; and (e) an activation of the stress-responsive p38-mitogen-activated protein kinase exemplified by its augmented phosphorylation. CONCLUSIONS: These data suggest that a highly coordinated network between AMPK and insulin signaling pathways are fully required for normal lipid synthesis and lipoprotein production in the small intestine of Psammomys obesus.
AGA Abstracts
Su2056 Electroacupuncture (EA) via Chronically Implanted Electrodes Improves Glucose Tolerance in Obese and Diabetic Rats Jieyun Yin, Xiaohong Xu, Shiying Li, Jiande Chen Hypoglycemic effect of EA has been indicated recently in anesthetized rats. It is however, unknown whether there is a similar effect in conscious rats. The other issue for EA to treat diabetes is the feasibility of chronic application of EA. We propose a novel method of EA using chronically implanted electrodes. The aims of the study were to test the feasibility of EA using chronic electrodes and to assess effects of EA on postprandial glycemia in various rodent models. Methods: Regular (N=8), diet-induced obese (DIO, N=12), and Zucker diabetic fatty (type-2 diabetes, ZDF, N=8) rats were chronically implanted with a pair of 3mm stainless steel electrodes at acupoints CV12 and CV4 with the connecting wires subcutaneously tunneled to and externalized at the back of the neck. After recovery, oral glucose tolerance tests (OGTT) were performed with EA and sham-EA on different days. Two sets of EA parameters were tested in regular rats: A: train-on of 2s, train-off of 3s, 0.5ms, 25Hz, 6mA; Parameter B: same as A except 10mA; in DIO and ZDF rats, only parameter B was used. The blood glucose level was measured at baseline and every 15 min for 90 min after glucose ingestion. The percent postprandial increase in blood glucose was calculated at each time point and the average increase during the 90-min period was also assessed and compared. Results: 1) In regular rats, in comparison with sham-EA, the blood glucose increase after oral glucose was not altered with EA of parameter A, but significantly attenuated with EA of parameter B (60% vs. 44%, P<0.02). 2) EA of parameter B also significantly attenuated the postprandial glucose increase in both DIO rats (47% vs. 35%, P=0.005) and type-2 ZDF rats (152% vs.132%, P=0.02). The postprandial glucose increase was higher in ZDF rats than that in DIO or regular rats (P<0.01). 4) Chronically implanted electrodes at the acupuncture points were not dislodged; no adverse events were noted,
S-544
A. Cytosol from enterocytes absorbing 3H-oleate was placed under an OptiPred gradient and 3H-dpm determined. B. Light fractions (DRM) were immunoblotted for the indicated proteins.
Su2051 Inhibition of Intestinal Biotin Absorption by Chronic Alcohol Feeding:Cellular and Molecular Mechanisms Sandeep B. Subramanya, Veedamali S. Subramanian, Jeyan S. Kumar, Robert Hoiness, Hamid M. Said Background: The water-soluble vitamin biotin is essential for normal cellular functions and its deficiency leads to a variety of clinical abnormalities. Mammals obtain biotin from exogenous sources via intestinal absorption, a process mediated by the sodium-dependent multivitamin transporter (SMVT). Chronic alcohol use in humans is associated with a significant reduction in plasma biotin levels. Little, however, is known about the cellular and molecular mechanisms involved in this inhibition, and thus, were investigated in this study. Methods: Adult male albino Wistar rats and transgenic mice carrying the human SLC5A6 gene 5'-regulatory regions were used as an In Vivo model of alcohol delivery and Caco-2 cells as an In Vitro model systems were used in this study. The rats were fed (for 4 weeks) a Lieber-DeCarli ethanol liquid-diet (36% calorie); control rats were pair-fed the same diet but without ethanol. Carrier-mediated entry and exit of 3H-biotin into and out of enterocytes was examined using purified jejunal brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV), respectively. Carrier-mediated colonic uptake of 3H-biotin was determined using colonic sheets. Changes in expression of SMVT protein, mRNA, and heterogenous mRNA (hnRNA) levels were determined by Western blot, realtime PCR, and by semi quantitative PCR. In chronic alcohol-fed (25% calorie) transgenic mice, changes in carrier mediated biotin uptake in jejunal enterocytes, mRNA, and SLC5A6 promoter activity was determined. Biotin uptake and SLC5A6 P1 and P2 promoter activity was determined in Caco-2 cells exposed to chronic alcohol. Results: Carrier-mediated biotin
A. Immunodepletion of Caveolin-1, -2, and -3 from cytosol derived from enterocytes absorbing 3H-oleate. The % 3H-oleate remaining in the cytosol is shown. B. Immunoblots of the cytosol as in A. were obtained after each round of immunodepletion of Caveolin-1, -2, and -3, the band density measured and compared to native cytosol. Su2049 Localization, Structure and Regulation of AMP-Activated Protein Kinase in the Small Intestine Elodie Harmel, Benoit Viollet, Carole Garofalo, Lea Emonnot, Martine Laville, Emile Levy INTRODUCTION: Mammalian 5' AMP-activated protein kinase (AMPK) acts as a sensor of cellular energy homeostasis. It stimulates multiple metabolic pathways that increase energy production and switches off processes that consume energy. Dysfunction of this fascinating enzyme affects lipid metabolism and vital organ functions. Intriguingly, although AMPK's importance in controlling lipid metabolism has been well established in skeletal muscle and the liver, its role in regulating energy storage and depletion in enterocytes remains unexplored.
S-543
AGA Abstracts
AGA Abstracts
In fact, little attention has been paid to the small intestine, the first organ to face nutrients and an organ closely associated with diabetic postprandial dyslipidemia. OBJECTIVES: To assess whether (i) AMPK is highly expressed in particular regions of the intestine and displays a tissue-specific hetrotrimer composition; (ii) AMPK acts to monitor and restore energy in this localization, regulates lipid homeostasis and responds to diverse pharmacological signals. METHODS: Studies were performed with control and AMPKα1-/- mice as well as with the human intestinal Caco-2 cell line. RESULTS: Our data have first shown the presence of all the AMPK subunits (α1/α2/β1/β2/γ1/γ2/γ3) in Caco-2 cells and intestinal epithelial cells of control mice. Analysis of gene and protein expression by RT-PCR and Western blot, respectively, revealed a marked difference in abundance of AMPK subunits with the predominance of AMPKα1. The prevalence of α1/β2/γ1 heterotrimer was confirmed by co-immunoprecipitation. Further experiments indicated that AMPKα1 is present mainly in the jejunum and ileum and to a lesser extent in the duodenum and colon. AMPK was found activated by 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) (p<0.001), which led in increased phosphorylation of the downstream target acetyl-CoA carboxylase (ACC), thereby resulting in the inhibition of fatty acid synthesis. Interestingly, no influence was noted in 3-hydroxy-3 methylglutaryl-CoA reductase (HMG-CoAR), the key regulatory enzyme of cholesterol synthesis. Reciprocally, inhibition of AMPK phosphorylation by compound C raised ACC phosphorylation and activity (p<0,001) without affecting HMGCoAR. Finally, depletion of glucose significantly activated AMPK and inhibited ACC whereas high glucose concentration down-regulated AMPK phosphorylation while activating ACC. CONCLUSIONS: Our findings uncover the localization, structure, function and regulation of AMPK in the small intestine. Ongoing studies will clarify AMPK role in pathophysiological conditions.
AGA Abstracts
uptake by jejunal BBMV and BLMV from rats chronically fed the alcohol liquid diet was significantly (p<0.05) lower than pair-fed control rats. This decrease in biotin uptake was associated with a significant decrease in the level of expression of SMVT protein, mRNA, and hnRNA, levels. Carrier-mediated biotin uptake by colonic sheets was also significantly (p<0.05) inhibited by chronic alcohol feeding. In chronic alcohol-fed transgenic mice, jejunal enterocytes showed a significant inhibition in carrier mediated biotin uptake (p<0.01), which was associated with marked decrease in SMVT mRNA level and activity of the firefly luciferase reporter gene compared to pair-fed control transgenic mice. Chronic alcohol exposed Caco2 cells showed a significant decrease in carrier mediated biotin uptake and in the activity of the individual promoters P1 and P2 of human SLC5A6 gene. Conclusion: These studies show that chronic alcohol feeding/exposure inhibits cellular and molecular parameters of intestinal biotin uptake process, and that the effect is (at least in part) being exerted at the transcriptional level(supported by grants from NIH and VA).
Su2054 Cytotoxicity of Fecal Water is Associated to Increased Concentrations of Alcohols Karen Windey, Vicky De Preter, Jean Herman, Thierry Louat, Greet Vandermeulen, Kristin Verbeke Introduction: Colonic fermentation of carbohydrates and proteins leads to the production of short chain fatty acids, whereas protein fermentation also results in branched chain fatty acids, phenols, sulphides and amines. Some of these metabolites are potentially toxic. In the present study, we modified the degree of protein fermentation by changing protein intake and investigated whether fecal metabolites associated with cytotoxicity originate from protein fermentation. Materials and methods: After a 1-week run-in period with normal protein (NP) intake, 20 healthy volunteers followed an isocaloric high protein (HP, 30%) and a low protein (LP, 9%) diet for 2 weeks in a randomized cross-over study. Fibre and fat intake were kept constant. During the run-in period and during the second week of each diet period the volunteers completed a dietary journal and collected urine for 48h and feces for 72h. Dietary composition was analyzed. Colonic protein fermentation was estimated from the urinary concentration of p-cresol. Profiles of volatile organic compounds (VOC) were analysed in fecal samples using GC-MS. Metabolite profiles were compared using cluster analysis. Fecal water cytotoxicity was determined using the WST-1 assay and expressed as fold dilution at which 50% of the cells survived (fold dilution, FD50). Cytotoxicity was related to the metabolite profiles. Results are expressed as average and standard deviation. Results: Protein intake accounted for 27 ± 4,15% of energy intake during the HP diet, 15 ± 2,19% during the NP and 12 ± 1,76% during the LP diet. Urinary p-cresol excretion was significantly correlated with protein intake (r=0,314; p=0,015). Fecal water cytotoxicity was not correlated with protein intake (p>0.05). The average cytotoxic dilution of the NP samples was 52,3 ± 24,9 compared to 39,3 ± 22,2 of the HP samples and 45,5 ± 25,8 of the LP samples. Cluster analysis of metabolite patterns in fecal samples according to cytotoxicity revealed a separation between the high toxicity (FD50 > 75) samples and the low toxicity (FD50 < 50) samples. This separation was mainly due to the presence of alcohols and the sulfur-containing compounds, allylisocyanate and 2,4-dithiapentane, in the high toxicity samples and the presence of cycloalkanes and cycloalkenes in the low toxicity samples. SCFA did not contribute to the clustering of the samples.
Su2052 High-Fat Diet-Induced Neuropathy of the Enteric Nervous System and the Effect of Alpha-7 Nicotinic Acetylcholine Receptor Agonist, DMAB-Anabaseine Dihydrochloride Onesmo B. Balemba, Chloe M. Stenkamp-Strahm, Joshua Cady, Ailene MacPherson Excessive calorie and high-fat diet ingestion cause obesity and type two diabetes mellitus (T2DM). Disruption of gastrointestinal (GI) microbial community structure and subsequent inflammation are key pathobiological events that occur before noticeable weight gain and insulin resistance. The enteric nervous system (ENS) plays a critical role in homeostatic adaptation to hypercaloric feeding. An altered expression of neurotransmitters and neuronal cell death has been seen in the ENS of T2DM patients, but the impact of a high-fat diet on the ENS is not fully understood. We are studying how a high-fat diet affects the ENS and whether the partial alpha 7-nicotinic acetylcholine receptor (α7-nAChR) agonist DMABanabaseine dihydrochloride (DMAB) reverses obesity and ENS neuropathy in a mouse model. C57/BL6 mice were fed either a standard chow or 72% fat diet for two and eight weeks, monitoring weight gain every other day. They were tested for glucose tolerance prior to euthanasia by measuring blood glucose after 6-hour fasting, and then 30 min. and 1-hour after 10 mg/kg intraperitoneal glucose injections. Immunohistochemistry was used to study calbindin, calcitonin gene-related peptide (CGRP), substance P and S-100 protein immunoreactivities in the ENS using the longitudinal smooth muscle-myenteric plexus (LMMP) and submucosal (SMP) wholemount preparations and 10 μm full-thickness cryostat sections from duodenum. A Nikon Eclipse fluorescent microscope and Metamorph 2 software were used for morphometric analysis of tissue innervation indexes. We observed that two weeks was enough time to cause a significant weight gain and glucose intolerance, suggesting an onset of insulin resistance. ENS changes observed after two weeks include a reduction in S100P density and the innervation indices of CGRP, calbindin and substance P. After eight weeks, S100P and substance P continue to be reduced while the CGRP innervation index showed no change and that of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), became increased. Treating fat-fed mice for four weeks with DMAB (10 mg Kg-1 subcutaneously) normalized weekly weight, although it did not lessen glucose intolerance. DMAB treatment increased ChAT mRNA levels by 3-14 fold compared to control, suggesting that an up-regulation of ChAT could be occurring. Collectively, our findings suggest that a high-fat diet and subsequent lipotoxicity disrupts enteric glial cells responsible for the upkeep and maintenance of nerve cells and the regulation of mucosal barrier function. The diet alters neurochemical expression in the ENS suggesting altered neurotransmission. DMAB and possibly other α7-nAChR agonists are promising therapies for the prevention and treatment of obesity, T2DM and other metabolic disorders that merit further study.
Su2055 Increased Copper Intake During Iron Deficiency Partly Ameliorates the IronDeficient Phenotype: A Possible Adaptive Mechanism in Iron Homeostasis Perungavur Ranganathan, Yan Lu, Lingli Jiang, Changae Kim, James F. Collins We sought to test the hypothesis that increased copper intake in the setting of iron deficiency would enhance serum ferroxidase (i.e. ceruloplasmin [Cp]) activity and positively alter iron homeostasis. The combined effect of alterations in dietary iron and copper was thus investigated in rats by tracking biological and biochemical parameters of iron and copper homeostasis. Copper deprivation increased hepatic iron levels over controls (54%), and also increased serum iron as compared to iron deprived rats (~5-fold). Increasing copper to ~5X the normal level had no effect on iron levels or hepatic copper levels in controls, but serum copper increased by ~145% in iron deficient rats. Hemoglobin and hematocrit levels increased ~2fold in iron deficient rats fed a high copper diet. Real-time PCR experiments revealed increased expression of Cp in copper deficient rats and decreased expression of hepcidin in liver under low Fe conditions. Western analyses of sera showed increased Cp protein with low Fe diet and low Fe diet with extra copper, and decreased Cp with low Cu. This pattern was also reflected by (in-gel) enzyme activity assay for ferroxidase, by two different methods (pPD and ferrozine). Similar activity-assays, performed spectrophotometrically, clearly demonstrated that serum ferroxidase activity was enhanced by iron deprivation and by intake of higher copper levels (in the control and iron deficient groups). This investigation has thus demonstrated that increased copper intake, reflected by increased liver and serum copper levels, can enhance serum ferroxidase activity in rats, which in turn may play a key role in the partial amelioration of the iron deficient phenotype.
Su2053 Disorders in AMPK and Insulin Signalling Pathways in the Intestine of Insulin-Resistant and Diabetic Psammomys Obesus Elodie Harmel, Ali Bendjoudi, Mounib Elchebly, Benoit Viollet, Ehud Ziv, Edgard Delvin, Martine Laville, Emile Levy Emerging evidence supports the link between insulin resistance (IR), type 2 diabetes (T2D) and aberrant intestinal lipid and lipoprotein metabolism. However, the abnormalities in intestinal signalling pathways were not systematically investigated in these metabolic disorders. RESEARCH DESIGN AND METHODS: The aims of the present study were to determine whether aberrant AMP-activated protein kinase (AMPK) and insulin signalling pathways characterize the jejunum and ileum of IR and T2D Psammomys obesus sand rat, an animal model of IR and T2D, and is associated with overproduction of triglyceride (TG)rich lipoproteins. RESULTS: Concomitant with their enhanced lipogenesis as well as their higher efficiency in de novo TG synthesis, apolipoprotein B-48 biogenesis and TG-rich lipoprotein assembly in conditions of IR and T2D, intestinal tissues exhibited :(a) a downregulation of AMPK signalling pathway essentially typified by a decrease in AMPKα-Thr172 phosphorylation, AMPKα protein mass and gene expression of the isoforms AMPKα1 and AMPKα2; (b) a reduction in acetyl CoA carboxylase (ACC)-ser79 phosphorylation without alterations in total ACC expression, suggesting a rise in activity of both ACC1 (responsible for fatty acids (FA) synthesis) and ACC2 (responsible for the inhibition of FA oxidation); (c) a decline in the gene and protein expression of carnitine palmitoyl transferase, supporting an inhibition of β-oxidation; (d) a trend of lessening of a second signalling pathway portrayed by the diminution of the Akt and glycogen synthetase kinase-3β phosphorylation; and (e) an activation of the stress-responsive p38-mitogen-activated protein kinase exemplified by its augmented phosphorylation. CONCLUSIONS: These data suggest that a highly coordinated network between AMPK and insulin signaling pathways are fully required for normal lipid synthesis and lipoprotein production in the small intestine of Psammomys obesus.
AGA Abstracts
Su2056 Electroacupuncture (EA) via Chronically Implanted Electrodes Improves Glucose Tolerance in Obese and Diabetic Rats Jieyun Yin, Xiaohong Xu, Shiying Li, Jiande Chen Hypoglycemic effect of EA has been indicated recently in anesthetized rats. It is however, unknown whether there is a similar effect in conscious rats. The other issue for EA to treat diabetes is the feasibility of chronic application of EA. We propose a novel method of EA using chronically implanted electrodes. The aims of the study were to test the feasibility of EA using chronic electrodes and to assess effects of EA on postprandial glycemia in various rodent models. Methods: Regular (N=8), diet-induced obese (DIO, N=12), and Zucker diabetic fatty (type-2 diabetes, ZDF, N=8) rats were chronically implanted with a pair of 3mm stainless steel electrodes at acupoints CV12 and CV4 with the connecting wires subcutaneously tunneled to and externalized at the back of the neck. After recovery, oral glucose tolerance tests (OGTT) were performed with EA and sham-EA on different days. Two sets of EA parameters were tested in regular rats: A: train-on of 2s, train-off of 3s, 0.5ms, 25Hz, 6mA; Parameter B: same as A except 10mA; in DIO and ZDF rats, only parameter B was used. The blood glucose level was measured at baseline and every 15 min for 90 min after glucose ingestion. The percent postprandial increase in blood glucose was calculated at each time point and the average increase during the 90-min period was also assessed and compared. Results: 1) In regular rats, in comparison with sham-EA, the blood glucose increase after oral glucose was not altered with EA of parameter A, but significantly attenuated with EA of parameter B (60% vs. 44%, P<0.02). 2) EA of parameter B also significantly attenuated the postprandial glucose increase in both DIO rats (47% vs. 35%, P=0.005) and type-2 ZDF rats (152% vs.132%, P=0.02). The postprandial glucose increase was higher in ZDF rats than that in DIO or regular rats (P<0.01). 4) Chronically implanted electrodes at the acupuncture points were not dislodged; no adverse events were noted,
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