Journal of Gastroenterology

, Volume 45, Issue 6, pp 646–655 | Cite as

The formation of intracellular glyceraldehyde-derived advanced glycation end-products and cytotoxicity

  • Jun-ichi Takino
  • Yuka Kobayashi
  • Masayoshi TakeuchiEmail author
Original Article—Liver, Pancreas, and Biliary Tract



Nonalcoholic steatohepatitis (NASH) is a feature of metabolic syndrome. Advanced glycation end-products (AGEs) are formed by the Maillard reaction, which contributes to aging and to certain pathological complications of diabetes. A recent study has suggested that glyceraldehyde-derived AGEs (Glycer-AGEs) are elevated in the sera of patients with NASH. Furthermore, immunohistochemistry of Glycer-AGEs showed intense staining in the livers of patients with NASH. The present study aimed to examine the effect of intracellular Glycer-AGEs on hepatocellular carcinoma (Hep3B) cells.


Cell viability was determined by the WST-1 assay. The slot blot and Western blot were used to detect intracellular Glycer-AGEs, and their localization was analyzed by confocal microscopy. Real-time reverse transcription-polymerase chain reaction was used to quantify the mRNA for the acute phase reactant C-reactive protein (CRP).


Glyceraldehyde (GA), which is the precursor of Glycer-AGEs, induced a concentration- and time-dependent increase in cell death, which was associated with an increase in intracellular Glycer-AGEs formation. Aminoguanidine (AG), which prevents AGEs formation, inhibited the formation of intracellular Glycer-AGEs and prevented cell death. Among the intracellular Glycer-AGEs that were formed, heat shock cognate 70 (Hsc70) was identified as a GA-modified protein, and its modification reduced the activity of Hsc70. Furthermore, intracellular Glycer-AGEs increased the CRP mRNA concentration.


These results suggest that intracellular Glycer-AGEs play important roles in promoting inflammation and hepatocellular death.


Advanced glycation end-products Glyceraldehyde Heat shock cognate 70 Inflammation Nonalcoholic steatohepatitis 



This work was supported in part by grants from the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research (B), #19300254).


  1. 1.
    Ludwig J, Viggiano TR, McGill DB, Oh BJ. Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clin Proc. 1980;55:434–8.PubMedGoogle Scholar
  2. 2.
    Angulo P. Nonalcoholic fatty liver disease. N Engl J Med. 2002;346:1221–31.CrossRefPubMedGoogle Scholar
  3. 3.
    Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology. 1999;116:1413–9.CrossRefPubMedGoogle Scholar
  4. 4.
    Dam-Larsen S, Franzmann M, Andersen IB, Christoffersen P, Jensen LB, Sørensen TI, et al. Long term prognosis of fatty liver: risk of chronic liver disease and death. Gut. 2004;53:750–5.CrossRefPubMedGoogle Scholar
  5. 5.
    Marchesini G, Brizi M, Bianchi G, Tomassetti S, Bugianesi E, Lenzi M, et al. Nonalcoholic fatty liver disease: a feature of the metabolic syndrome. Diabetes. 2001;50:1844–50.CrossRefPubMedGoogle Scholar
  6. 6.
    Chitturi S, Abeygunasekera S, Farrell GC, Holmes-Walker J, Hui JM, Fung C, et al. NASH and insulin resistance: insulin hypersecretion and specific association with the insulin resistance syndrome. Hepatology. 2002;35:373–9.CrossRefPubMedGoogle Scholar
  7. 7.
    Marchesini G, Bugianesi E, Forlani G, Cerrelli F, Lenzi M, Manini R, et al. Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology. 2003;37:917–23.CrossRefPubMedGoogle Scholar
  8. 8.
    Day CP, James OF. Steatohepatitis: a tale of two “hits”? Gastroenterology. 1998;114:842–5.CrossRefPubMedGoogle Scholar
  9. 9.
    Al-Abed Y, Kapurniotu A, Bucala R. Advanced glycation end products: detection and reversal. Methods Enzymol. 1999;309:152–72.CrossRefPubMedGoogle Scholar
  10. 10.
    Vlassara H, Palace MR. Diabetes and advanced glycation endproducts. J Intern Med. 2002;251:87–101.CrossRefPubMedGoogle Scholar
  11. 11.
    Glomb MA, Monnier VM. Mechanism of protein modification by glyoxal and glycolaldehyde, reactive intermediates of the Maillard reaction. J Biol Chem. 1995;270:10017–26.CrossRefPubMedGoogle Scholar
  12. 12.
    Thornalley PJ, Langborg A, Minhas HS. Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose. Biochem J. 1999;344:109–16.CrossRefPubMedGoogle Scholar
  13. 13.
    Takeuchi M, Bucala R, Suzuki T, Ohkubo T, Yamazaki M, Koike T, et al. Neurotoxicity of advanced glycation end-products for cultured cortical neurons. J Neuropathol Exp Neurol. 2000;59:1094–105.PubMedGoogle Scholar
  14. 14.
    Yamagishi S, Amano S, Inagaki Y, Okamoto T, Koga K, Sasaki N, et al. Advanced glycation end products-induced apoptosis and overexpression of vascular endothelial growth factor in bovine retinal pericytes. Biochem Biophys Res Commun. 2002;290:973–8.CrossRefPubMedGoogle Scholar
  15. 15.
    Yamagishi S, Inagaki Y, Okamoto T, Amano S, Koga K, Takeuchi M, et al. Advanced glycation end product-induced apoptosis and overexpression of vascular endothelial growth factor and monocyte chemoattractant protein-1 in human-cultured mesangial cells. J Biol Chem. 2002;277:20309–15.CrossRefPubMedGoogle Scholar
  16. 16.
    Hyogo H, Yamagishi S, Iwamoto K, Arihiro K, Takeuchi M, Sato T, et al. Elevated levels of serum advanced glycation end products in patients with non-alcoholic steatohepatitis. J Gastroenterol Hepatol. 2007;22:1112–9.CrossRefPubMedGoogle Scholar
  17. 17.
    Yoshida T, Yamagishi S, Nakamura K, Matsui T, Imaizumi T, Takeuchi M, et al. Pigment epithelium-derived factor (PEDF) inhibits advanced glycation end product (AGE)-induced C-reactive protein expression in hepatoma cells by suppressing Rac-1 activation. FEBS Lett. 2006;580:2788–96.CrossRefPubMedGoogle Scholar
  18. 18.
    Iwamoto K, Kanno K, Hyogo H, Yamagishi S, Takeuchi M, Tazuma S, et al. Advanced glycation end products enhance the proliferation and activation of hepatic stellate cells. J Gastroenterol. 2008;43:298–304.CrossRefPubMedGoogle Scholar
  19. 19.
    Takeuchi M, Makita Z, Bucala R, Suzuki T, Koike T, Kameda Y. Immunological evidence that non-carboxymethyllysine advanced glycation end-products are produced from short chain sugars and dicarbonyl compounds in vivo. Mol Med. 2000;6:114–25.PubMedGoogle Scholar
  20. 20.
    Brownlee M, Vlassara H, Kooney A, Ulrich P, Cerami A. Aminoguanidine prevents diabetes-induced arterial wall protein cross-linking. Science. 1986;232:1629–32.CrossRefPubMedGoogle Scholar
  21. 21.
    Nicholls K, Mandel TE. Advanced glycosylation end-products in experimental murine diabetic nephropathy: effect of islet isografting and of aminoguanidine. Lab Invest. 1989;60:486–91.PubMedGoogle Scholar
  22. 22.
    Khalifah RG, Baynes JW, Hudson BG. Amadorins: novel post-Amadori inhibitors of advanced glycation reactions. Biochem Biophys Res Commun. 1999;257:251–8.CrossRefPubMedGoogle Scholar
  23. 23.
    Price DL, Rhett PM, Thorpe SR, Baynes JW. Chelating activity of advanced glycation end-product inhibitors. J Biol Chem. 2001;276:48967–72.CrossRefPubMedGoogle Scholar
  24. 24.
    Carbone DL, Doorn JA, Kiebler Z, Sampey BP, Petersen DR. Inhibition of Hsp72-mediated protein refolding by 4-hydroxy-2-nonenal. Chem Res Toxicol. 2004;17:1459–67.CrossRefPubMedGoogle Scholar
  25. 25.
    Rodríguez-Ariza A, López-Sánchez LM, González R, Corrales FJ, López P, Bernardos A, et al. Altered protein expression and protein nitration pattern during d-galactosamine-induced cell death in human hepatocytes: a proteomic analysis. Liver Int. 2005;25:1259–69.CrossRefPubMedGoogle Scholar
  26. 26.
    Daugaard M, Rohde M, Jäättelä M. The heat shock protein 70 family: highly homologous proteins with overlapping and distinct functions. FEBS Lett. 2007;581:3702–10.CrossRefPubMedGoogle Scholar
  27. 27.
    Canbakan B, Senturk H, Tahan V, Hatemi H, Balci H, Toptas T, et al. Clinical, biochemical and histological correlations in a group of non-drinker subjects with non-alcoholic fatty liver disease. Acta Gastroenterol Belg. 2007;70:277–84.PubMedGoogle Scholar
  28. 28.
    Bell DS, Allbright E. The multifaceted associations of hepatobiliary disease and diabetes. Endocr Pract. 2007;13:300–12.PubMedGoogle Scholar
  29. 29.
    Feldstein AE, Canbay A, Angulo P, Taniai M, Burgart LJ, Lindor KD, et al. Hepatocyte apoptosis and FAS expression are prominent features of human nonalcoholic steatohepatitis. Gastroenterology. 2003;125:437–43.CrossRefPubMedGoogle Scholar
  30. 30.
    Ribeiro PS, Cortez-Pinto H, Solá S, Castro RE, Ramalho RM, Baptista A, et al. Hepatocyte apoptosis, expression of death receptors, and activation of NF-kappaB in the liver of nonalcoholic and alcoholic steatohepatitis patients. Am J Gastroenterol. 2004;99:1708–17.CrossRefPubMedGoogle Scholar
  31. 31.
    Takeuchi M, Yamagishi S. Alternative routes for the formation of glyceraldehyde-derived AGEs (TAGE) in vivo. Med Hypotheses. 2004;63:453–5.CrossRefPubMedGoogle Scholar
  32. 32.
    Taniguchi S, Okinaka M, Tanigawa K, Miwa I. Difference in mechanism between glyceraldehyde- and glucose-induced insulin secretion from isolated rat pancreatic islets. J Biochem. 2000;127:289–95.PubMedGoogle Scholar
  33. 33.
    Takahashi H, Tran PO, LeRoy E, Harmon JS, Tanaka Y, Robertson RP. d-Glyceraldehyde causes production of intracellular peroxide in pancreatic islets, oxidative stress, and defective beta cell function via non-mitochondrial pathways. J Biol Chem. 2004;279:37316–23.CrossRefPubMedGoogle Scholar
  34. 34.
    Sakai K, Matsumoto K, Nishikawa T, Suefuji M, Nakamaru K, Hirashima Y, et al. Mitochondrial reactive oxygen species reduce insulin secretion by pancreatic beta-cells. Biochem Biophys Res Commun. 2003;300:216–22.CrossRefPubMedGoogle Scholar
  35. 35.
    Hamelin M, Mary J, Vostry M, Friguet B, Bakala H. Glycation damage targets glutamate dehydrogenase in the rat liver mitochondrial matrix during aging. FEBS J. 2007;274:5949–61.CrossRefPubMedGoogle Scholar
  36. 36.
    Kumar PA, Kumar MS, Reddy GB. Effect of glycation on alpha-crystallin structure and chaperone-like function. Biochem J. 2007;408:251–8.CrossRefPubMedGoogle Scholar
  37. 37.
    Schalkwijk CG, van Bezu J, van der Schors RC, Uchida K, Stehouwer CD, van Hinsbergh VW. Heat-shock protein 27 is a major methylglyoxal-modified protein in endothelial cells. FEBS Lett. 2006;580:1565–70.CrossRefPubMedGoogle Scholar
  38. 38.
    Gomes RA, Miranda HV, Silva MS, Graça G, Coelho AV, Ferreira AE, et al. Yeast protein glycation in vivo by methylglyoxal. Molecular modification of glycolytic enzymes and heat shock proteins. FEBS J. 2006;273:5273–87.CrossRefPubMedGoogle Scholar
  39. 39.
    Yoneda M, Mawatari H, Fujita K, Iida H, Yonemitsu K, Kato S, et al. High-sensitivity C-reactive protein is an independent clinical feature of nonalcoholic steatohepatitis (NASH) and also of the severity of fibrosis in NASH. J Gastroenterol. 2007;42:573–82.CrossRefPubMedGoogle Scholar
  40. 40.
    Targher G, Bertolini L, Rodella S, Lippi G, Franchini M, Zoppini G, et al. NASH predicts plasma inflammatory biomarkers independently of visceral fat in men. Obesity. 2008;16:1394–9.CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2010

Authors and Affiliations

  • Jun-ichi Takino
    • 1
  • Yuka Kobayashi
    • 1
  • Masayoshi Takeuchi
    • 1
    Email author
  1. 1.Department of Pathophysiological Science, Faculty of Pharmaceutical SciencesHokuriku UniversityKanazawaJapan

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