Advertisement

Digestive Diseases and Sciences

, Volume 56, Issue 12, pp 3507–3516 | Cite as

l-Glutamine Supplementation Prevents Myenteric Neuron Loss and Has Gliatrophic Effects in the Ileum of Diabetic Rats

  • Renata Virginia Fernandes Pereira
  • Eleandro Aparecido Tronchini
  • Cristiano Massao Tashima
  • Eder Paulo Belato Alves
  • Mariana Machado Lima
  • Jacqueline Nelisis Zanoni
Original Article

Abstract

Background

Peripheral neuropathy caused chronically by diabetes mellitus is related to exacerbation of oxidative stress and a significant reduction in important endogenous antioxidants. l-Glutamine is an amino acid involved in defense mechanisms and is a substrate for the formation of glutathione, the major endogenous cellular antioxidant.

Aim

This study investigated the effects of 2% l-glutamine supplementation on peripheral diabetic neuropathy and enteric glia in the ileum in rats.

Methods

Male Wistar rats were divided into four groups: normoglycemics (N), normoglycemics supplemented with l-glutamine (NG), diabetics (D), and diabetics supplemented with l-glutamine (DG). After 120 days, the ileums were processed for HuC/D and S100 immunohistochemistry. Quantitative and morphometric analysis was performed.

Results

Diabetes significantly reduced the number of HuC/D-immunoreactive myenteric neurons per unit area and per ganglion in group D compared with normoglycemic animals (group N). l-Glutamine (2%) prevented neuronal death induced by diabetes (group DG) compared with group D. The glial density per unit area did not change with diabetes (group D) but was significantly reduced after l-glutamine supplementation (groups NG and DG). Ganglionic glial density was similar among the four groups. The neuronal area was not altered in groups D and DG. Glial size was reduced in group D; this was reversed by l-glutamine supplementation (group DG).

Conclusions

We concluded that 2% l-glutamine had neuroprotective effects directly on myenteric neurons and indirectly through glial cells, which had gliatrophic effects.

Keywords

Enteric nervous system Diabetic neuropathy Ileum Glial cells Glutamine 

Notes

Acknowledgments

We wish to thank Ana Paula de Santi Rampazzo, Maria Euride do Carmo Cancino, and Maria dos Anjos Fortunato for their excellent technical support.

References

  1. 1.
    Gabella G. Ultrastructure of the nerve plexuses of the mammalian intestine: The enteric glial cells. Neuroscience. 1981;6:425–436.PubMedCrossRefGoogle Scholar
  2. 2.
    Furness JB, Costa M. The Enteric Nervous System. New York, NY: Churchill Livingstone; 1987.Google Scholar
  3. 3.
    Liu W, Yue W, Wu R. Effects of diabetes on expression of glial fibrillary acidic protein and neurotrophins in rat colon. Auton Neurosci. 2010;154:79–83.PubMedCrossRefGoogle Scholar
  4. 4.
    Cabarrocas J, Savidge TC, Liblau RS. Role of enteric glial cells in inflammatory bowel disease. Glia. 2003;41:81–93.PubMedCrossRefGoogle Scholar
  5. 5.
    Ruhl A, Nasser Y, Sharkey KA. Enteric glia. Neurogastroenterol Motil. 2004;16:44–49.PubMedCrossRefGoogle Scholar
  6. 6.
    Ruhl A. Glial cells in the gut. Neurogastroenterol Motil. 2005;17:777–790.PubMedCrossRefGoogle Scholar
  7. 7.
    von Boyen G, Steinkamp M. The enteric glia and neurotrophic factors. Gastroenterol. 2006;44:985–990.CrossRefGoogle Scholar
  8. 8.
    von Boyen GB, Steinkamp M, Geerling I, et al. Proinflammatory cytokines induce neurotrophic factor expression in enteric glia: A key to the regulation of epithelial apoptosis in Crohn’s disease. Inflamm Bowel Dis. 2006;12:346–354.CrossRefGoogle Scholar
  9. 9.
    Cornet A, Savidge TC, Cabarrocas J, et al. Enterocolitis induced by autoimmune targeting of enteric glial cells: A possible mechanism in Crohn’s disease? Proc Natl Acad Sci USA. 2001;98:13306–13311.PubMedCrossRefGoogle Scholar
  10. 10.
    Bassotti G, Villanacci V, Fisogni S, et al. Enteric glial cells and their role in gastrointestinal motor abnormalities: introducing the neuro-gliopathies. World J Gastroenterol. 2007;13:4035–4041.PubMedGoogle Scholar
  11. 11.
    Bassotti G, de Roberto G, Castellani D, Sediari L, Morelli A. Normal aspects of colorectal motility and abnormalities in slow transit constipation. World J Gastroenterol. 2005;11:2691–2696.PubMedGoogle Scholar
  12. 12.
    Iantorno G, Bassotti G, Kogan Z, et al. The enteric nervous system in chagasic and idiopathic megacolon. Am J Surg Pathol. 2007;31:460–468.PubMedCrossRefGoogle Scholar
  13. 13.
    Bytzer P, Talley NJ, Leemon M, Young LJ, Jones MP, Horowitz M. Prevalence of gastrointestinal symptoms associated with diabetes mellitus: A population-based survey of 15,000 adults. Arch Intern Med. 2001;161:1989–1996.PubMedCrossRefGoogle Scholar
  14. 14.
    Zochodne DW. Diabetes mellitus and the peripheral nervous system: manifestations and mechanisms. Muscle Nerve. 2007;36:144–166.PubMedCrossRefGoogle Scholar
  15. 15.
    Du F, Wang L, Qian W, Liu S. Loss of enteric neurons accompanied by decreased expression of GDNF and PI3 K/Akt pathway in diabetic rats. Neurogastroenterol Motil. 2009;21:e114–e1229.CrossRefGoogle Scholar
  16. 16.
    Zanoni JN, de Miranda Neto MH, Bazotte RB, de Souza RR. Morphological and quantitative analysis of the neurons of the myenteric plexus of the cecum of streptozotocin-induced diabetic rats. Arq Neuropsiquiatr. 1997;55:696–702.PubMedCrossRefGoogle Scholar
  17. 17.
    Fregonesi CE, Miranda-Neto MH, Molinari SL, Zanoni JN. Quantitative study of the myenteric plexus of the stomach of rats with streptozotocin-induced diabetes. Arq Neuropsiquiatr. 2001;59:50–53.PubMedCrossRefGoogle Scholar
  18. 18.
    Zanoni JN, Buttow NC, Bazotte RB, Miranda Neto MH. Evaluation of the population of NADPH-diaphorase-stained and myosin-V myenteric neurons in the ileum of chronically streptozotocin-diabetic rats treated with ascorbic acid. Auton Neurosci. 2003;104:32–38.PubMedCrossRefGoogle Scholar
  19. 19.
    Tashima CM, Tronchini EA, Pereira RV, Bazotte RB, Zanoni JN. Diabetic rats supplemented with l-glutamine: A study of immunoreactive myosin-V myenteric neurons and the proximal colonic mucosa. Dig Dis Sci. 2007;52:1233–1241.PubMedCrossRefGoogle Scholar
  20. 20.
    De Freitas P, Natali MR, Pereira RV, Miranda Neto MH, Zanoni JN. Myenteric neurons and intestinal mucosa of diabetic rats after ascorbic acid supplementation. World J Gastroenterol. 2008;14:6518–6524.PubMedCrossRefGoogle Scholar
  21. 21.
    Pereira RV, de Miranda-Neto MH, da Silva Souza ID, Zanoni JN. Vitamin E supplementation in rats with experimental diabetes research: analysis of myosin-V and nNOS immunoreactive myenteric neurons from the terminal ileum. J Mol Histol. 2008;39:595–603.PubMedCrossRefGoogle Scholar
  22. 22.
    Wu G. Amino acids: Metabolism, functions, and nutrition. Amino Acids. 2009;37:1–17.PubMedCrossRefGoogle Scholar
  23. 23.
    Wu G, Fang YZ, Yang S, Lupton JR, Turner ND. Glutathione metabolism and its implications for health. J Nutr. 2004;134:489–492.PubMedGoogle Scholar
  24. 24.
    Loven D, Schedl H, Wilson H, et al. Effect of insulin and oral glutathione on glutathione levels and superoxide dismutase activities in organs of rats with streptozotocin-induced diabetes. Diabetes. 1986;35:503–507.PubMedCrossRefGoogle Scholar
  25. 25.
    Galli F. Amino acid and protein modification by oxygen and nitrogen species. Amino Acids. 2010.Google Scholar
  26. 26.
    Mannick JB. Regulation of apoptosis by protein S-nitrosylation. Amino Acids. 2007;32:523–526.PubMedCrossRefGoogle Scholar
  27. 27.
    Roth E, Oehler R, Manhart N, et al. Regulative potential of glutamine: relation to glutathione metabolism. Nutrition. 2002;18:217–221.PubMedCrossRefGoogle Scholar
  28. 28.
    Flaring UB, Rooyackers OE, Wernerman J, Hammarqvist F. Glutamine attenuates post-traumatic glutathione depletion in human muscle. Clin Sci (Lond). 2003;104:275–282.CrossRefGoogle Scholar
  29. 29.
    Wang J, Chen L, Li P, et al. Gene expression is altered in piglet small intestine by weaning and dietary glutamine supplementation. J Nutr. 2008;138:1025–1032.PubMedGoogle Scholar
  30. 30.
    Bergmeyer HU, Bernet E. d-Glucose determination with glucose oxidase and peroxidase. In: Methods of Enzymatic Analysis. 2nd ed. New York, NY: Verlag Chemie-Academic Press; 1974:1205–1215.Google Scholar
  31. 31.
    Koenig RJ, Peterson CM, Jones RL, Saudek C, Lehrman M, Cerami A. Correlation of glucose relation and hemoglobin AIc in diabetes mellitus. N Engl J Med. 1976;295:417–420.PubMedCrossRefGoogle Scholar
  32. 32.
    Stefanini M, De Martino C, Zamboni L. Fixation of ejaculated spermatozoa for electron microscopy. Nature. 1967;216:173–174.PubMedCrossRefGoogle Scholar
  33. 33.
    Lin Z, Gao N, Hu HZ, et al. Immunoreactivity of Hu proteins facilitates identification of myenteric neurons in guinea-pig small intestine. Neurogastroenterol Motil. 2002;14:197–204.PubMedCrossRefGoogle Scholar
  34. 34.
    Phillips RJ, Kieffer EJ, Powley TL. Loss of glia and neurons in the myenteric plexus of the aged Fischer 344 rat. Anat Embryol (Berl). 2004;209:19–30.CrossRefGoogle Scholar
  35. 35.
    Zanoni JN, De Freitas P, Pereira RV, Dos Santos Pereira MA, De Miranda Neto MH. Effects of supplementation with ascorbic acid for a period of 120 days on the myosin-V and NADPHd positive myenteric neurons of the ileum of rats. Anat Histol Embryol. 2005;34:149–153.PubMedCrossRefGoogle Scholar
  36. 36.
    Miranda Neto MH, Molinari SL, Natali MR, Sant’Ana DM. Regional differences in the number and type of myenteric neurons of the ileum of rats: A comparison of techniques of the neuronal evidentiation. Arq Neuropsiquiatr. 2001;59:54–59.PubMedCrossRefGoogle Scholar
  37. 37.
    Tsai PH, Liu JJ, Chiu WC, Pai MH, Yeh SL. Effects of dietary glutamine on adhesion molecule expression and oxidative stress in mice with streptozotocin-induced type 1 diabetes. Clin Nutr. 2011;30:124–129.PubMedCrossRefGoogle Scholar
  38. 38.
    Alves EP, Alves AM, Pereira RV, de Miranda Neto MH, Zanoni JN. Immunohistochemical study of vasoactive intestinal peptide (VIP) enteric neurons in diabetic rats supplemented with l-glutamine. Nutr Neurosci. 2010;13:43–51.PubMedCrossRefGoogle Scholar
  39. 39.
    Karaosmanoglu T, Aygun B, Wade PR, Gershon MD. Regional differences in the number of neurons in the myenteric plexus of the guinea pig small intestine and colon: an evaluation of markers used to count neurons. Anat Rec. 1996;244:470–480.PubMedCrossRefGoogle Scholar
  40. 40.
    Schneider LC, Perez GG, Banzi SR, Zanoni JN, Natali MR, Buttow NC. Evaluation of the effect of Ginkgo biloba extract (EGb 761) on the myenteric plexus of the small intestine of Wistar rats. J Gastroenterol. 2007;42:624–630.PubMedCrossRefGoogle Scholar
  41. 41.
    Zanoni JN, Hernandes L, Bazotte RB, Miranda Neto MH. Terminal ileum submucous plexus: study of VIP-ergic neurons of diabetic rats treated with ascorbic acid. Arq Neuropsiquiatr. 2002;60:32–37.PubMedCrossRefGoogle Scholar
  42. 42.
    Zanoni JN, Freitas P. Effects of ascorbic acid on the vasoactive intestinal peptide synthesis in the ileum submucous plexus of normal rats. Arq Gastroenterol. 2005;42:186–190.PubMedCrossRefGoogle Scholar
  43. 43.
    Pereira MA, Bagatin MC, Zanoni JN. Effects of the ascorbic acid supplementation on NADH-diaphorase myenteric neurons in the duodenum of diabetic rats. Biocell. 2006;30:295–300.PubMedGoogle Scholar
  44. 44.
    Roldi LP, Pereira RV, Tronchini EA, et al. Vitamin E (alpha-tocopherol) supplementation in diabetic rats: Effects on the proximal colon. BMC Gastroenterol. 2009;9:88.PubMedCrossRefGoogle Scholar
  45. 45.
    Shotton HR, Adams A, Lincoln J. Effect of aminoguanidine treatment on diabetes-induced changes in myenteric plexus of rat ileum. Auton Neurosci. 2007;132:16–26.PubMedCrossRefGoogle Scholar
  46. 46.
    Amores-Sánchez MI, Medina MA. Glutamine, as a precursor of glutathione, and oxidative stress. Mol Genet Metab. 1999;67:100–105.PubMedCrossRefGoogle Scholar
  47. 47.
    Matés JM, Pérez-Gómez C, Núñez de Castro I, Asenjo M, Márquez J. Glutamine and its relationship with intracellular redox status, oxidative stress and cell proliferation/death. Int J Biochem Cell Biol. 2002;34:439–458.PubMedCrossRefGoogle Scholar
  48. 48.
    Vincent AM, Russell JW, Low P, Feldman EL. Oxidative stress in the pathogenesis of diabetic neuropathy. Endocr Rev. 2004;25:612–628.PubMedCrossRefGoogle Scholar
  49. 49.
    Dutra F, Bechara EJ. Bioquímica e ação citotóxica de α-aminocetonas endógenas. Quimica Nova. 2005;28:483–491.CrossRefGoogle Scholar
  50. 50.
    Krammer HJ, Karahan ST, Sigge W, Kuhnel W. Immunohistochemistry of markers of the enteric nervous system in whole-mount preparations of the human colon. Eur J Pediatr Surg. 1994;4:274–278.PubMedCrossRefGoogle Scholar
  51. 51.
    Ferri GL, Probert L, Cocchia D, Michetti F, Marangos PJ, Polak JM. Evidence for the presence of S-100 protein in the glial component of the human enteric nervous system. Nature. 1982;297:409–410.PubMedCrossRefGoogle Scholar
  52. 52.
    Heizmann CW. The multifunctional S100 protein family. Methods Mol Biol. 2002;172:69–80.PubMedGoogle Scholar
  53. 53.
    Komuro T, Baluk P, Burnstock G. An ultrastructural study of neurons and non-neuronal cells in the myenteric plexus of the rabbit colon. Neuroscience. 1982;7:1797–1806.PubMedCrossRefGoogle Scholar
  54. 54.
    Iwata-Ichikawa E, Kondo Y, Miyazaki I, Asanuma M, Ogawa N. Glial cells protect neurons against oxidative stress via transcriptional up-regulation of the glutathione synthesis. J Neurochem. 1999;72:2334–2344.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Renata Virginia Fernandes Pereira
    • 1
  • Eleandro Aparecido Tronchini
    • 1
  • Cristiano Massao Tashima
    • 1
  • Eder Paulo Belato Alves
    • 1
  • Mariana Machado Lima
    • 1
  • Jacqueline Nelisis Zanoni
    • 1
  1. 1.Department of Morphological SciencesUniversidade Estadual de MaringáMaringáBrazil

Personalised recommendations