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Su1886 Molecular Identification and Functional Characterization of the Human Colonic Thiamine Pyrophosphate Transporter
the cytosolic heteroquatramer protein complex, enabling it to bind the ER, which can generate PCTV to transport the chylomicron from ER to Golgi. The chylomicron output into lymph is correlated to intestinal luminal phosphatidylcholine (PC). Luminal PC is absorbed as lyso-phosphatidylcholine (lyso-PC). We previously showed that the dietary lipids are absorbed from the apical membrane by Caveolin-1 containing Cytosolic Endocytic Vesicles (CEV). We tested the hypothesis that lyso-PC activates the PKCζ; detach it from CEV, enabling PKCζ to phosphorylate Sar1b. Methods: Cytosol was isolated from rats whose intestinal PC was altered by (A) bile diversion, no PC (B) saline infusion, low PC (C) chow fed, normal PC (D) fat fed, high PC and (E) PC infusion, very high PC. PKCζ activity was measured by phosphorylation of PKCζ pseudo substrate. Lyso-PC was measured by HPLC. Results: PKCζ was activated by lyso-PC, non-linear regression curve for PKCζ vs. lyso-PC, calculated as Km=1.49 ± 0.244 nM and Vmax = 1.12 ± 1.058 nM. The amount of cytosolic lyso-PC in A to E ranges from 0 to 0.45 nM, suggesting that the amount of cytosolic lysoPC is always within a range to control PKCζ activation. PKCζ activity ranges from 0 to 0.7 Arbitrary Unit in A to E. Post absorptive CEV contain PKCζ by western blot but PKCζ detachment from the CEV is proportional to cytosolic lyso-PC as estimated by western blot of CEV. Biotinylation of r-PKCζ showed a conformational change on activation by lyso-PC. We conclude that PKCζ on CEV was activated by lyso-PC, changes its conformation and eluted from vesicles, enter into the cytosol and phosphorylate Sar1b, split the heteroquatramer protein complex to release FABP1. Now free FABP1 can bind to ER membrane for PCTV formation, which transports chylomicrons from ER to Golgi. Fig. 1 illustrated the scheme for control of dietary lipid transport.
Su1886
Background: Colonic microbiota synthesizes considerable amount of vitamin B1 in the form of thiamine pyrophosphate (TPP). Recent functional studies from our laboratory have shown the existence of a specific, high-affinity, and regulated carrier-mediated uptake system for TPP in human colonocytes (Am. J. Physiol., 303:G389-95,2012). Nothing, however, is known about the molecular identity of this system. Here, we show that the system involved is a product of the SLC44A4 gene, a previously uncharacterized membrane protein with an unknown function. Methods: BLASTP algorithm and the SOSUI and TMHMM trans-membrane helixes prediction programs were used in searches for mammalian homologs of the TPP ABC transporter permease of Treponema denticola. The human SLC44A4 was identified as a potential candidate and its cDNA was cloned from human colonic NCM460 cells. 3 H-TPP uptake studies were performed on confluent ARPE19 cells, transiently expressing SLC44A4. Expression of SLC44A4 was assessed by Western blot analysis and qRT-PCR. Metabolism of TPP was determined by mean of thin-layer chromatography. Results: Expression of the SLC44A4 cDNA in human retinal pigment epithelial ARPE19 cells showed a significant (> 5 folds) induction in uptake of 3H-TPP. The induced uptake was found to be: 1) temperature- and energy-dependent, but Na+- and pH-independent, 2) saturable as a function of 3H-TPP concentration with an apparent Km of 0.17 ± 0.064 μM, and 3) highly specific for TPP and is not affected by free thiamine, thiamine monophosphate (TMP), or a host of other compounds including choline. Expression of the human TPP transporter (hTPPT) at the protein and mRNA levels was found to be high in the colon but negligible in the small intestine. Cell surface biotinylation assay and live-cell confocal imaging of epithelia showed apical membrane domain expression of the hTPPT-YFP protein. Studies with mouse colonocytes (mice also express the TPPT) have shown that the transported TPP is subjected to considerable and time-dependent metabolism to TMP and free thiamine. Conclusion: These results report, for the first time, the molecular identification and characterization of a specific and high affinity TPP uptake system in human colonocytes. The findings also lend further support that the microbiota-generated TPP is nutritionally available to the host, and especially to the local colonocytes. [Supported by NIH grant R37 DK 56061 to HMS]. Su1887 Mechanism of Inhibition of Na-Glutamine Co-Transport by Prostaglandin E2 in Intestinal Epithelial Cells Soudamani Singh, Subha Arthur, Uma Sundaram Background: Assimilation of the most important nutrient for the enterocytes, namely glutamine, is mediated by Na-dependent glutamine co-transport (NGcT) which is present on the brush border membrane (BBM) of the enterocytes in the mammalian small intestine. NGcT is known to be inhibited in the chronically inflamed rabbit small intestine secondary to a reduction in the number of the co-transporters. Prostaglandin E2 (PGE2) is markedlyincreased in enterocytes during chronic enteritis. Whether the inhibition of NGcT during chronic enteritis may be mediated by PGE2 is unknown. Hypothesis: PGE2 mediates the inhibition of NGcT during chronic intestinal inflammation. Aim: Determine the effect of PGE2 on NGcT inenterocytesand determine its mechanism of action. Methods: Rat small intestinal epithelial cells (IEC-18) grown to confluenceon 24 well plates wereutilized. Nadependent glutamine uptake was performed using tritiated glutamine in intact cells. Na/KATPase activity was determined by measuring the inorganic phosphate released. Western blots were performed using rat-specific antibodies.Results: Na-dependent glutamine uptake in IEC-18 cells was significantlyinhibited by PGE2 (NGcT was 2169±188 nmol/mg protein/ 2 min in control cells and 832±44 in PGE2 treated cells, n=4, p<0.05). Since Na/K-ATPase provides the favorable NGcT, it was measured and found diminished (18.5±1.4 nmol/mg protein/min in control and 11±1.3 nmol/mg protein/min in PGE2 treated cells, n=5, p<0.05). PGE2 is known to mediate its action via protein kinase A (PKA). Thus, when cells were pretreated with PKA inhibitor, Rp-cAMP, it reversed the inhibition of NGcTby PGE2 (2386±183 nmol/mg protein/2 min in Rp-cAMPtreated cells p<0.05, n=4). Rp-cAMP also reversed the inhibition of Na/K-ATPase (20.1±1.4 nmol/mg protein/min in Rp-cAMPtreated cells,n=5, p<0.05). Preliminary kinetic studies revealed that the mechanismof inhibition of NGcT by PGE2 in IEC-18 cells was secondary to a decrease in the maximal rate of uptake without a significant alteration in the affinity for glutamine. Western blot studies demonstrated that the immune reactive levels of B0AT1 decreased in IEC-18 cells treated with PGE2 and they were reversed by Rp-cAMP pre-treatment. Conclusions: These studies demonstratethat PGE2 through PKA inhibits Na-dependent glutamine co-transport in intestinal epithelial cells. At the transporter level, thisinhibition is not secondary to an alteration in the Na extruding capacity of the cells. Themechanism of inhibition issecondary to a decrease in co-transporter numbers without a significant change in affinityof the co-transporter. This mechanism of inhibition of Na-glutamine co-transport in-vitro isidentical to that seen in the rabbit model of inflammatory bowel disease.
Su1885 Prolonged High-Fat Feeding Decreases SGLT1 and GLUT2 Activity and Blunts Their Upregulation by Glucose-Dependent Insulinotropic Polypeptide (GIP) in Obese Mice Steven D. Coon, Vazhaikkurichi M. Rajendran, Satish K. Singh Background: Na-glucose cotransporter-1 (SGLT1) mediates jejunal glucose absorption in the normal mouse. However, in a short-term, high-fat-fed mouse model of obesity, both glucose tranporter-2 (GLUT2) and SGLT1 participate in glucose absorption. The incretin hormone, GIP, augments SGLT1 activity in normal mice, while in obese mice GIP augments SGLT1 only slightly while enhancing apical GLUT2 expression and activity. Aim: To determine the effects of GIP on jejunal SGLT1 and GLUT2 activity in a long term high-fat-fed mouse model of obesity. Methods: Mice were fed a high-fat vs. normal diet for up to 9 months to generate obese and control animals respectively. The mechanisms of glucose absorption and the effects of GIP were assessed in jejunum mounted under voltage-clamp conditions in animals that were fed their respective diets for 5, 7 and 9 months. Mucosal to serosal (m-s) 3H-3-O-Methyl glucose fluxes were measured to determine total glucose absorption (i.e., both SGLT1 and GLUT2), while short circuit current (Isc) was measured to determine SGLT1-mediated electrogenic glucose absorption. Western blot analyses were performed to determine the expression level of SGLT1 and GLUT2 protein in biotinylated apical membranes isolated from normal and obese mouse jejunum. Results: At 5 months of feeding, GIP enhanced glucose absorption in obese animals primarily via GLUT2 (untreated vs GIP treated: 0.7±0.1 vs 2.3±0.3 μEq/h.cm2; p < 0.03) , and actually decreased SGLT1 activity (untreated vs GIP treated: 110 vs 49 μA/cm2; p < 0.05). The observed GIP-dependent enhancement of GLUT2 activity in obese mice at 5 mo (2.3 μEq/h.cm2) subsequently and significantly fell to 1.0±0.4 and 0.8±0.2 μEq/h.cm2 at 7 and 9 mo respectively. In control mice fed a normal diet, SGLT1 remained the predominant glucose uptake mechanism and continued to be augmentable by GIP throughout the 9 mo observation period. Westernblot analyses of biotinylated brush border membranes revealed that, in parallel with the decrease in glucose absorption, both GLUT2 and SGLT1 expression were reduced at 7 and 9 mos in obese but not control mice. Conclusions: High-fat diet feeding enhances GIPaugmented glucose absorption by activation and enhanced apical expression of GLUT2 at 5 mo. However, with continued high-fat feeding (7 & 9 months) glucose absorption is decreased by reducing apical expression of SGLT1 and GLUT2 and by blunting GIP-dependent augmentation of SGLT1 and GLUT2 activity.
Su1888 Mucosal Sucrase Activity Is Diminished Following Bariatric Roux-en-Y Surgery Antone R. Opekun, Katie Fracolli, Mary L. Brandt, Vadim Sherman, Karol Hernandez, Bruno P. Chumpitazi Obesity, defined as a body mass index of >30 kg/m2 and is a pandemic currently affecting approximately 35% of the United States population. For morbid obesity, there are very few weight loss therapies that have proven efficacious in the long-term, with the exception of the Roux-en-Y gastric bypass (RYGB) surgery. Several studies have been conducted to elucidate the mechanisms of weight loss after RYGB, but our understanding remains incomplete. Minimal research has been performed to investigate the role of carbohydrate malabsorption after RYGB surgery. Sucrose is a major component of ingested dietary carbohydrates
S-493
AGA Abstracts
AGA Abstracts
Molecular Identification and Functional Characterization of the Human Colonic Thiamine Pyrophosphate Transporter Svetlana Nabokina, Katsuhisa Inoue, Veedamali S. Subramanian, Judith E. Valle, Hiroaki Yuasa, Hamid M. Said
Su1886
Background: Colonic microbiota synthesizes considerable amount of vitamin B1 in the form of thiamine pyrophosphate (TPP). Recent functional studies from our laboratory have shown the existence of a specific, high-affinity, and regulated carrier-mediated uptake system for TPP in human colonocytes (Am. J. Physiol., 303:G389-95,2012). Nothing, however, is known about the molecular identity of this system. Here, we show that the system involved is a product of the SLC44A4 gene, a previously uncharacterized membrane protein with an unknown function. Methods: BLASTP algorithm and the SOSUI and TMHMM trans-membrane helixes prediction programs were used in searches for mammalian homologs of the TPP ABC transporter permease of Treponema denticola. The human SLC44A4 was identified as a potential candidate and its cDNA was cloned from human colonic NCM460 cells. 3 H-TPP uptake studies were performed on confluent ARPE19 cells, transiently expressing SLC44A4. Expression of SLC44A4 was assessed by Western blot analysis and qRT-PCR. Metabolism of TPP was determined by mean of thin-layer chromatography. Results: Expression of the SLC44A4 cDNA in human retinal pigment epithelial ARPE19 cells showed a significant (> 5 folds) induction in uptake of 3H-TPP. The induced uptake was found to be: 1) temperature- and energy-dependent, but Na+- and pH-independent, 2) saturable as a function of 3H-TPP concentration with an apparent Km of 0.17 ± 0.064 μM, and 3) highly specific for TPP and is not affected by free thiamine, thiamine monophosphate (TMP), or a host of other compounds including choline. Expression of the human TPP transporter (hTPPT) at the protein and mRNA levels was found to be high in the colon but negligible in the small intestine. Cell surface biotinylation assay and live-cell confocal imaging of epithelia showed apical membrane domain expression of the hTPPT-YFP protein. Studies with mouse colonocytes (mice also express the TPPT) have shown that the transported TPP is subjected to considerable and time-dependent metabolism to TMP and free thiamine. Conclusion: These results report, for the first time, the molecular identification and characterization of a specific and high affinity TPP uptake system in human colonocytes. The findings also lend further support that the microbiota-generated TPP is nutritionally available to the host, and especially to the local colonocytes. [Supported by NIH grant R37 DK 56061 to HMS]. Su1887 Mechanism of Inhibition of Na-Glutamine Co-Transport by Prostaglandin E2 in Intestinal Epithelial Cells Soudamani Singh, Subha Arthur, Uma Sundaram Background: Assimilation of the most important nutrient for the enterocytes, namely glutamine, is mediated by Na-dependent glutamine co-transport (NGcT) which is present on the brush border membrane (BBM) of the enterocytes in the mammalian small intestine. NGcT is known to be inhibited in the chronically inflamed rabbit small intestine secondary to a reduction in the number of the co-transporters. Prostaglandin E2 (PGE2) is markedlyincreased in enterocytes during chronic enteritis. Whether the inhibition of NGcT during chronic enteritis may be mediated by PGE2 is unknown. Hypothesis: PGE2 mediates the inhibition of NGcT during chronic intestinal inflammation. Aim: Determine the effect of PGE2 on NGcT inenterocytesand determine its mechanism of action. Methods: Rat small intestinal epithelial cells (IEC-18) grown to confluenceon 24 well plates wereutilized. Nadependent glutamine uptake was performed using tritiated glutamine in intact cells. Na/KATPase activity was determined by measuring the inorganic phosphate released. Western blots were performed using rat-specific antibodies.Results: Na-dependent glutamine uptake in IEC-18 cells was significantlyinhibited by PGE2 (NGcT was 2169±188 nmol/mg protein/ 2 min in control cells and 832±44 in PGE2 treated cells, n=4, p<0.05). Since Na/K-ATPase provides the favorable NGcT, it was measured and found diminished (18.5±1.4 nmol/mg protein/min in control and 11±1.3 nmol/mg protein/min in PGE2 treated cells, n=5, p<0.05). PGE2 is known to mediate its action via protein kinase A (PKA). Thus, when cells were pretreated with PKA inhibitor, Rp-cAMP, it reversed the inhibition of NGcTby PGE2 (2386±183 nmol/mg protein/2 min in Rp-cAMPtreated cells p<0.05, n=4). Rp-cAMP also reversed the inhibition of Na/K-ATPase (20.1±1.4 nmol/mg protein/min in Rp-cAMPtreated cells,n=5, p<0.05). Preliminary kinetic studies revealed that the mechanismof inhibition of NGcT by PGE2 in IEC-18 cells was secondary to a decrease in the maximal rate of uptake without a significant alteration in the affinity for glutamine. Western blot studies demonstrated that the immune reactive levels of B0AT1 decreased in IEC-18 cells treated with PGE2 and they were reversed by Rp-cAMP pre-treatment. Conclusions: These studies demonstratethat PGE2 through PKA inhibits Na-dependent glutamine co-transport in intestinal epithelial cells. At the transporter level, thisinhibition is not secondary to an alteration in the Na extruding capacity of the cells. Themechanism of inhibition issecondary to a decrease in co-transporter numbers without a significant change in affinityof the co-transporter. This mechanism of inhibition of Na-glutamine co-transport in-vitro isidentical to that seen in the rabbit model of inflammatory bowel disease.
Su1885 Prolonged High-Fat Feeding Decreases SGLT1 and GLUT2 Activity and Blunts Their Upregulation by Glucose-Dependent Insulinotropic Polypeptide (GIP) in Obese Mice Steven D. Coon, Vazhaikkurichi M. Rajendran, Satish K. Singh Background: Na-glucose cotransporter-1 (SGLT1) mediates jejunal glucose absorption in the normal mouse. However, in a short-term, high-fat-fed mouse model of obesity, both glucose tranporter-2 (GLUT2) and SGLT1 participate in glucose absorption. The incretin hormone, GIP, augments SGLT1 activity in normal mice, while in obese mice GIP augments SGLT1 only slightly while enhancing apical GLUT2 expression and activity. Aim: To determine the effects of GIP on jejunal SGLT1 and GLUT2 activity in a long term high-fat-fed mouse model of obesity. Methods: Mice were fed a high-fat vs. normal diet for up to 9 months to generate obese and control animals respectively. The mechanisms of glucose absorption and the effects of GIP were assessed in jejunum mounted under voltage-clamp conditions in animals that were fed their respective diets for 5, 7 and 9 months. Mucosal to serosal (m-s) 3H-3-O-Methyl glucose fluxes were measured to determine total glucose absorption (i.e., both SGLT1 and GLUT2), while short circuit current (Isc) was measured to determine SGLT1-mediated electrogenic glucose absorption. Western blot analyses were performed to determine the expression level of SGLT1 and GLUT2 protein in biotinylated apical membranes isolated from normal and obese mouse jejunum. Results: At 5 months of feeding, GIP enhanced glucose absorption in obese animals primarily via GLUT2 (untreated vs GIP treated: 0.7±0.1 vs 2.3±0.3 μEq/h.cm2; p < 0.03) , and actually decreased SGLT1 activity (untreated vs GIP treated: 110 vs 49 μA/cm2; p < 0.05). The observed GIP-dependent enhancement of GLUT2 activity in obese mice at 5 mo (2.3 μEq/h.cm2) subsequently and significantly fell to 1.0±0.4 and 0.8±0.2 μEq/h.cm2 at 7 and 9 mo respectively. In control mice fed a normal diet, SGLT1 remained the predominant glucose uptake mechanism and continued to be augmentable by GIP throughout the 9 mo observation period. Westernblot analyses of biotinylated brush border membranes revealed that, in parallel with the decrease in glucose absorption, both GLUT2 and SGLT1 expression were reduced at 7 and 9 mos in obese but not control mice. Conclusions: High-fat diet feeding enhances GIPaugmented glucose absorption by activation and enhanced apical expression of GLUT2 at 5 mo. However, with continued high-fat feeding (7 & 9 months) glucose absorption is decreased by reducing apical expression of SGLT1 and GLUT2 and by blunting GIP-dependent augmentation of SGLT1 and GLUT2 activity.
Su1888 Mucosal Sucrase Activity Is Diminished Following Bariatric Roux-en-Y Surgery Antone R. Opekun, Katie Fracolli, Mary L. Brandt, Vadim Sherman, Karol Hernandez, Bruno P. Chumpitazi Obesity, defined as a body mass index of >30 kg/m2 and is a pandemic currently affecting approximately 35% of the United States population. For morbid obesity, there are very few weight loss therapies that have proven efficacious in the long-term, with the exception of the Roux-en-Y gastric bypass (RYGB) surgery. Several studies have been conducted to elucidate the mechanisms of weight loss after RYGB, but our understanding remains incomplete. Minimal research has been performed to investigate the role of carbohydrate malabsorption after RYGB surgery. Sucrose is a major component of ingested dietary carbohydrates
S-493
AGA Abstracts
AGA Abstracts
Molecular Identification and Functional Characterization of the Human Colonic Thiamine Pyrophosphate Transporter Svetlana Nabokina, Katsuhisa Inoue, Veedamali S. Subramanian, Judith E. Valle, Hiroaki Yuasa, Hamid M. Said