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GLUTAMINE ATTENUATES THE TOXIC EFFECTS OF LEGUMES VIA

STIMULATION OF HSP PRODUCTION

Ramadass B, Dokladny K, Moseley P and Lin H CNew Mexico Veterans Affairs Health systems and the University of New

Mexico, Albuquerque, NM

Background• Dietary components may be both beneficial and

toxic.

• 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).

• Brief, 24h exposure to a diet supplemented with raw red kidney beans (RRKB) leads to– Reduced weight gain– Bacterial overgrowth– Increased intestinal permeability– Bacterial translocation

Toxicity of Raw Red Kidney Beans

• 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).

• Previously, we found that feeding RRKB provides an experimental approach for inducing leaky gut and bacterial translocation which are seen in critical illness.

Glutamine in Critical Illness• An example of a beneficial dietary component is

glutamine.

• Reduced plasma concentration of glutamine is seen in patients with critical illness.

• Glutamine supplementation has been reported to have significant clinical benefits (J Nutr 2008 138(10):2040S-2044S).

• In animals under experimental stress, glutamine protects by improving leaky gut and inhibiting bacterial translocation.

• How glutamine exerts these benefits is poorly understood.

Reported Effects of Glutamine• A major nutrient for intestinal epithelial cells.

• Stimulates significant proliferation of intestinal epithelial cells (Gut 1999;44:608-614).

• A precursor for glutathione (AJCN. 2009 Sep;90(3):814-821).

• Decreases leaky gut (AJP 2009 Feb;296(2):G348-55).

• Inhibits bacterial translocation (Am J Surg. 2010;199(1):35-42).

• Increases host defense mechanisms including increased expression of heat shock proteins ( J Nutr

2008;138:740).

Heat Shock Proteins• Heat shock proteins (HSP) are molecular chaperone

proteins that are essential for proper protein folding.

• 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).

• However, it is not known whether the protective effects of glutamine on intestinal barrier is explained by HSP?

Aim

• To examine this question, we tested the hypothesis that glutamine-induced HSP70 expression may decrease RRKB- induced leaky gut and bacterial translocation.

MethodsIn-vivo4 groups of rats were tested

1. Control: Regular rat chow for 8 days2. RRKB: Regular rat chow for 7 days switching to chow

supplemented with 26% raw RKB on day 83. Glutamine: 2% glutamine in drinking water + regular

rat chow for 8 days .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.

• 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.

Methods• In-vitro

1. CaCo-2 cells were treated with PHA (200µg/ml) and Trans-epithelial resistance was measured.

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.

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.

Experimental Outcomes• In-vivo• Intestinal Permeability: urinary recovery of lactulose and

mannitol as represented by L/M ratio.

• Bacterial Translocation: relative total bacterial load in liver tissue using q-PCR exploiting primers targeting 16s rRNA gene.

• HSP expression: RT-PCR and HSP-70 protein availability by immunohistochemistry

• In-vitro• Intestinal barrier function: transepithelial resistance (TER)

• Protein folding: Luciferase activity since luminescence of this protein requires proper protein folding

ResultsGlutamine Reduced RRKB-induced Leaky Gut

In-vivo

RRKB vs Glutamine+RRKB, P<0.001

Glutamine Reduced RRKB-induced Bacterial Translocation to the Liver.

RRKB vs Glutamine+RRKB, P<0.001

Glutamine Increased HSP expression in Mid and Distal Small Intestine

Glutamine vs Glutamine+RRKB, P<0.05

• 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?

• Could the glutamine treatment have increased the availability of HSP70 protein even as HSP70 expression was suppressed on day 8?

HSP70 was still available on Day 8

•Brown staining is positive for HSP-70.

•Similar findings from Glutamine and Glutamine+ RRKB group.

Glutamine & Glutamine+RRKB groups

•Absence of significant staining for HSP-70.

•Similar findings in Control and RRKB group.

Control & RRKB groups

In-vitro

PHA Decreased TER in CaCO-2 cells

.

PHA Decreased Proteins Responsible for Intestinal Barrier Function

• 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.

Doubly-expressing HSP70 Cells are Protected from Lectin-induced Decrease in Protein Folding

Summary/Interpretation

• Glutamine decreased RRKB-induced increase in urinary L/M ratio suggesting that glutamine protects against leaky gut.

• Glutamine decreased RRKB-induced increase in bacterial load in the liver suggesting that glutamine protects against bacterial translocation.

• 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.

• However, when presence of HSP70 protein was examined, our data showed that Glutamine treatment provided enough HSP70 up-regulation to protect.

• HSP70 gain of function showed that HSP70 was involved in the protection against RKB lectin-induced impairment of protein folding.

Conclusions

• Glutamine attenuates the toxic effects of legumes and legume lectin.

• These toxic effects may depend on impairment of protein folding which may be overcome by heat shock protein.

Future Studies• Testing the role of HSP70 using a “loss of

function” approach.

• Testing the inter-relationship between lectin, glutamine and HSP70 on protein folding.

Acknowledgment

• The authors gratefully acknowledge Tori Thomas for supporting Dr. Ramadass’ Postdoctoral fellowship.

• Dr. Lin’s research is supported by the NIH, the VA Research Office and the Department of Defense.