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  1. 1. GLUTAMINE ATTENUATES THE TOXIC EFFECTS OF LEGUMES VIA STIMULATION OF HSP PRODUCTION Ramadass B , Dokladny K, Moseley P and Lin H C New Mexico Veterans Affairs Health systems and the University of New Mexico, Albuquerque, NM
  2. 2. Background <ul><li>Dietary components may be both beneficial and toxic. </li></ul><ul><li>An example of a dietary toxin is phytohemagglutinin (PHA), a lectin from red kidney beans (RKB). This lectin is toxic when consumed uncooked and may cause an acute noninfectious gastroenteritis (Biofactors 2004; 21(1-4) 399-401). </li></ul><ul><li>Brief, 24h exposure to a diet supplemented with raw red kidney beans (RRKB) leads to </li></ul><ul><ul><li>Reduced weight gain </li></ul></ul><ul><ul><li>Bacterial overgrowth </li></ul></ul><ul><ul><li>Increased intestinal permeability </li></ul></ul><ul><ul><li>Bacterial translocation </li></ul></ul>
  3. 3. Toxicity of Raw Red Kidney Beans <ul><li>This toxicity may be secondary to increased intestinal permeability as lectins have been shown to decrease trans-epithelial resistance in CaCO2 cells (Br J Pharmacol 2004 ;142(8):1219-26). </li></ul><ul><li>Previously, we found that feeding RRKB provides an experimental approach for inducing leaky gut and bacterial translocation which are seen in critical illness. </li></ul>
  4. 4. Glutamine in Critical Illness <ul><li>An example of a beneficial dietary component is glutamine. </li></ul><ul><li>Reduced plasma concentration of glutamine is seen in patients with critical illness. </li></ul><ul><li>Glutamine supplementation has been reported to have significant clinical benefits (J Nutr 2008 138(10):2040S-2044S). </li></ul><ul><li>In animals under experimental stress, glutamine protects by improving leaky gut and inhibiting bacterial translocation. </li></ul><ul><li>How glutamine exerts these benefits is poorly understood . </li></ul>
  5. 5. Reported Effects of Glutamine <ul><li>A major nutrient for intestinal epithelial cells. </li></ul><ul><li>Stimulates significant proliferation of intestinal epithelial cells (Gut 1999;44:608-614). </li></ul><ul><li>A precursor for glutathione (AJCN. 2009 Sep;90(3):814-821). </li></ul><ul><li>Decreases leaky gut (AJP 2009 Feb;296(2):G348-55). </li></ul><ul><li>Inhibits bacterial translocation (Am J Surg. 2010;199(1):35-42). </li></ul><ul><li>Increases host defense mechanisms including increased expression of heat shock proteins ( J Nutr 2008;138:740). </li></ul>
  6. 6. Heat Shock Proteins <ul><li>Heat shock proteins (HSP) are molecular chaperone proteins that are essential for proper protein folding. </li></ul><ul><li>HSPs play an important role in intestinal barrier function-HSP prevents heat stress-induced disruption of intestinal tight junction barrier, in part, via HSF-1 induced expression of occludin (AJP 2006 Feb;290(2):G204-12). </li></ul><ul><li>However, it is not known whether the protective effects of glutamine on intestinal barrier is explained by HSP? </li></ul>
  7. 7. Aim <ul><li>To examine this question, we tested the hypothesis that glutamine-induced HSP70 expression may decrease RRKB- induced leaky gut and bacterial translocation. </li></ul>
  8. 8. Methods <ul><ul><li>In-vivo </li></ul></ul><ul><ul><li>4 groups of rats were tested </li></ul></ul><ul><ul><ul><li>1. Control: Regular rat chow for 8 days </li></ul></ul></ul><ul><ul><ul><li>2. RRKB : Regular rat chow for 7 days switching to chow supplemented with 26% raw RKB on day 8 </li></ul></ul></ul><ul><ul><ul><li>3. Glutamine : 2% glutamine in drinking water + regular rat chow for 8 days . </li></ul></ul></ul><ul><ul><ul><li>4. Glutamine+RRKB : 2% glutamine in drinking water + regular rat chow for 7 days then switching to chow supplemented with 26% raw RKB on day 8. </li></ul></ul></ul><ul><li>At the end of day 8, animals were euthanized with liver and intestinal tissues collected as proximal (PI), mid (MI) or distal (DI) 1/3 of small intestine and colon. </li></ul>
  9. 9. Methods <ul><li>In-vitro </li></ul><ul><ul><li>1. CaCo-2 cells were treated with PHA (200µg/ml) and Trans-epithelial resistance was measured. </li></ul></ul><ul><ul><li>2. Caco-2 cells were co-transfected with constructs driving luciferase and doubly expressing HSP70 protein expression to create a cell line (HSP-70) that tested for gain of function. </li></ul></ul><ul><ul><li>3. After a 24h incubation for optimal luciferase expression, cells were subjected to PHA (200 µg/ml) for 48h followed by measuring luciferase activity as a marker for HSP70- mediated protection of protein folding. </li></ul></ul>
  10. 10. Experimental Outcomes <ul><ul><li>In-vivo </li></ul></ul><ul><ul><li>Intestinal Permeability : urinary recovery of lactulose and mannitol as represented by L/M ratio. </li></ul></ul><ul><ul><li>Bacterial Translocation: relative total bacterial load in liver tissue using q-PCR exploiting primers targeting 16s rRNA gene. </li></ul></ul><ul><ul><li>HSP expression: RT-PCR and HSP-70 protein availability by immunohistochemistry </li></ul></ul><ul><li>In-vitro </li></ul><ul><ul><li>Intestinal barrier function : transepithelial resistance (TER) </li></ul></ul><ul><ul><li>Protein folding : Luciferase activity since luminescence of this protein requires proper protein folding </li></ul></ul>
  11. 11. Results Glutamine Reduced RRKB-induced Leaky Gut <ul><ul><li>In-vivo </li></ul></ul>RRKB vs Glutamine+RRKB, P<0.001
  12. 12. Glutamine Reduced RRKB-induced Bacterial Translocation to the Liver. RRKB vs Glutamine+RRKB, P<0.001
  13. 13. Glutamine Increased HSP expression in Mid and Distal Small Intestine Glutamine vs Glutamine+RRKB, P<0.05
  14. 14. <ul><li>If HSP expression was inhibited by 24h of RRKB, how did glutamine have its protective effect on leaky gut and bacterial translocation on day 8? </li></ul><ul><li>Could the glutamine treatment have increased the availability of HSP70 protein even as HSP70 expression was suppressed on day 8? </li></ul>
  15. 15. HSP70 was still available on Day 8 <ul><li>Brown staining is positive for HSP-70. </li></ul><ul><li>Similar findings from Glutamine and Glutamine+ RRKB group. </li></ul><ul><li>Absence of significant staining for HSP-70. </li></ul><ul><li>Similar findings in Control and RRKB group. </li></ul>Control & RRKB groups Glutamine & Glutamine+RRKB groups
  16. 16. PHA Decreased TER in CaCO-2 cells In-vitro .
  17. 17. PHA Decreased Proteins Responsible for Intestinal Barrier Function
  18. 18. <ul><li>Since more than one barrier protein was affected by PHA, we tested the possibility of a more generalized disruption of protein folding and that the molecular chaperone protein HSP-70 may be involved. </li></ul>
  19. 19. Doubly-expressing HSP70 Cells are Protected from Lectin-induced Decrease in Protein Folding
  20. 20. Summary/Interpretation <ul><li>Glutamine decreased RRKB-induced increase in urinary L/M ratio suggesting that glutamine protects against leaky gut. </li></ul><ul><li>Glutamine decreased RRKB-induced increase in bacterial load in the liver suggesting that glutamine protects against bacterial translocation. </li></ul><ul><li>Glutamine alone increased HSP-70 expression on day 8, while adding RRKB treatment on day 8 during glutamine feeding eliminated this finding suggesting that a toxic effect of RRKB was the inhibition of HSP-70 expression. </li></ul>
  21. 21. <ul><li>However, when presence of HSP70 protein was examined, our data showed that Glutamine treatment provided enough HSP70 up-regulation to protect. </li></ul><ul><li>HSP70 gain of function showed that HSP70 was involved in the protection against RKB lectin-induced impairment of protein folding. </li></ul>
  22. 22. Conclusions <ul><li>Glutamine attenuates the toxic effects of legumes and legume lectin. </li></ul><ul><li>These toxic effects may depend on impairment of protein folding which may be overcome by heat shock protein. </li></ul>
  23. 23. Future Studies <ul><li>Testing the role of HSP70 using a “loss of function” approach. </li></ul><ul><li>Testing the inter-relationship between lectin, glutamine and HSP70 on protein folding. </li></ul>
  24. 24. Acknowledgment <ul><li>The authors gratefully acknowledge Tori Thomas for supporting Dr. Ramadass’ Postdoctoral fellowship. </li></ul><ul><li>Dr. Lin’s research is supported by the NIH, the VA Research Office and the Department of Defense. </li></ul>