L-lysine protects C2C12 myotubes and 3T3-L1 adipocytes against high glucose damages and stresses

Autoři: S. Mehdi Ebrahimi aff001;  S. Zahra Bathaie aff001;  Nassim Faridi aff001;  Mohammad Taghikhani aff001;  Manouchehr Nakhjavani aff002;  Soghrat Faghihzadeh aff003
Působiště autorů: Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran aff001;  Endocrinology and Metabolism Research Center (EMRC), Vali-Asr Hospital, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran aff002;  Department of Statistics, Zanjan University of Medical Sciences, Zanjan, Iran aff003
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225912


Hyperglycemia is a hallmark of diabetes, which is associated with protein glycation and misfolding, impaired cell metabolism and altered signaling pathways result in endoplasmic reticulum stress (ERS). We previously showed that L-lysine (Lys) inhibits the nonenzymatic glycation of proteins, and protects diabetic rats and type 2 diabetic patients against diabetic complications. Here, we studied some molecular aspects of the Lys protective role in high glucose (HG)-induced toxicity in C2C12 myotubes and 3T3-L1 adipocytes. C2C12 and 3T3-L1 cell lines were differentiated into myotubes and adipocytes, respectively. Then, they were incubated with normal or high glucose (HG) concentrations in the absence/presence of Lys (1 mM). To investigate the role of HG and/or Lys on cell apoptosis, oxidative status, unfolded protein response (UPR) and autophagy, we used the MTT assay and flow cytometry, spectrophotometry and fluorometry, RT-PCR and Western blotting, respectively. In both cell lines, HG significantly reduced cell viability and induced apoptosis, accompanying with the significant increase in reactive oxygen species (ROS) and nitric oxide (NO). Furthermore, the spliced form of X-box binding protein 1 (XBP1), at both mRNA and protein levels, the phosphorylated eukaryotic translation initiation factor 2α (p-eIf2α), and the Light chain 3 (LC3)II/LC3I ratio was also significantly increased. Lys alone had no significant effects on most of these parameters; but, treatment with HG plus Lys returned them all to, or close to, the normal values. The results indicated the protective role of Lys against glucotoxicity induced by HG in C2C12 myotubes and 3T3-L1 adipocytes.

Klíčová slova:

Adipocytes – Apoptosis – Autophagic cell death – Cell differentiation – Endoplasmic reticulum – Nitric oxide – Toxicity


1. Del Prato S. Role of glucotoxicity and lipotoxicity in the pathophysiology of Type 2 diabetes mellitus and emerging treatment strategies. Diabetic Medicine. 2009;26(12):1185–92. doi: 10.1111/j.1464-5491.2009.02847.x 20002468

2. Li J, Niu X-L, Madamanchi NR. Leukocyte antigen-related protein tyrosine phosphatase negatively regulates hydrogen peroxide-induced vascular smooth muscle cell apoptosis. Journal of Biological Chemistry. 2008;283(49):34260–72. doi: 10.1074/jbc.M806087200 18854310

3. Lavrentyev EN, Malik KU. High glucose-induced Nox1-derived superoxides downregulate PKC-βII, which subsequently decreases ACE2 expression and ANG (1–7) formation in rat VSMCs. American Journal of Physiology-Heart and Circulatory Physiology. 2009;296(1):H106–H18. doi: 10.1152/ajpheart.00239.2008 18978194

4. Reddy AB, Ramana KV, Srivastava S, Bhatnagar A, Srivastava SK. Aldose reductase regulates high glucose-induced ectodomain shedding of tumor necrosis factor (TNF)-α via protein kinase C-δ and TNF-α converting enzyme in vascular smooth muscle cells. Endocrinology. 2008;150(1):63–74. doi: 10.1210/en.2008-0677 18772236

5. Kim J-a, Montagnani M, Koh KK, Quon MJ. Reciprocal relationships between insulin resistance and endothelial dysfunction. Circulation. 2006;113(15):1888–904. doi: 10.1161/CIRCULATIONAHA.105.563213 16618833

6. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature. 2001;414(6865):813–20. doi: 10.1038/414813a 11742414

7. Stitt AW, Jenkins AJ, Cooper ME. Advanced glycation end products and diabetic complications. Expert opinion on investigational drugs. 2002;11(9):1205–23. doi: 10.1517/13543784.11.9.1205 12225243

8. Tan KC, Chow W-S, Ai VH, Metz C, Bucala R, Lam KS. Advanced glycation end products and endothelial dysfunction in type 2 diabetes. Diabetes care. 2002;25(6):1055–9. doi: 10.2337/diacare.25.6.1055 12032114

9. Wu J, Kaufman R. From acute ER stress to physiological roles of the unfolded protein response. Cell Death & Differentiation. 2006;13(3):374–84.

10. Lin JH, Walter P, Yen TB. Endoplasmic reticulum stress in disease pathogenesis. Annu Rev pathmechdis Mech Dis. 2008;3:399–425.

11. Zhao L, Ackerman SL. Endoplasmic reticulum stress in health and disease. Current opinion in cell biology. 2006;18(4):444–52. doi: 10.1016/j.ceb.2006.06.005 16781856

12. Adastra KL, Chi MM, Riley JK, Moley KH. A differential autophagic response to hyperglycemia in the developing murine embryo. Reproduction. 2011;141(5):607–15. doi: 10.1530/REP-10-0265 21367963

13. Yao J, Tao Z-F, Li C-P, Li X-M, Cao G-F, Jiang Q, et al. Regulation of autophagy by high glucose in human retinal pigment epithelium. Cellular Physiology and Biochemistry. 2014;33(1):107–16. doi: 10.1159/000356654 24481000

14. Momoi T. Caspases involved in ER stress-mediated cell death. Journal of chemical neuroanatomy. 2004;28(1–2):101–5. doi: 10.1016/j.jchemneu.2004.05.008 15363495

15. Frederick KK, Michaelis VK, Corzilius B, Ong TC, Jacavone AC, Griffin RG, et al. Sensitivity-enhanced NMR reveals alterations in protein structure by cellular milieus. Cell. 2015;163(3):620–8. doi: 10.1016/j.cell.2015.09.024 26456111; PubMed Central PMCID: PMC4621972.

16. Singh P, Mahadi F, Roy A, Sharma P. Reactive oxygen species, reactive nitrogen species and antioxidants in etiopathogenesis of diabetes mellitus type-2. Indian journal of clinical Biochemistry. 2009;24(4):324–42. doi: 10.1007/s12291-009-0062-6 23105858

17. Pacher P, Obrosova IG, Mabley JG, Szabó C. Role of nitrosative stress and peroxynitrite in the pathogenesis of diabetic complications. Emerging new therapeutical strategies. Current medicinal chemistry. 2005;12(3):267–75. doi: 10.2174/0929867053363207 15723618

18. Engin F, Hotamisligil GS. Restoring endoplasmic reticulum function by chemical chaperones: an emerging therapeutic approach for metabolic diseases. Diabetes, obesity & metabolism. 2010;12 Suppl 2:108–15. doi: 10.1111/j.1463-1326.2010.01282.x 21029307.

19. Jafarnejad A, Bathaie S, Nakhjavani M, Hassan M, Banasadegh S. The improvement effect of L‐Lys as a chemical chaperone on STZ‐induced diabetic rats, protein structure and function. Diabetes/metabolism research and reviews. 2008;24(1):64–73. doi: 10.1002/dmrr.769 17879961

20. Perlmutter DH. Chemical chaperones: a pharmacological strategy for disorders of protein folding and trafficking. Pediatric research. 2002;52(6):832–6. doi: 10.1203/00006450-200212000-00004 12438657

21. Welch WJ, Brown CR. Influence of molecular and chemical chaperones on protein folding. Cell stress & chaperones. 1996;1(2):109.

22. Tsubuku S, Mochizuki M, Mawatari K, Smriga M, Kimura T. Thirteen-week oral toxicity study of L-lysine hydrochloride in rats. International journal of toxicology. 2004;23(2):113–8. doi: 10.1080/10915810490444415 15204731.

23. Bathaie SZ, Nobakht BF, Mirmiranpour H, Jafarnejad A, Moosavi-Nejad SZ. Effect of chemical chaperones on glucose-induced lysozyme modifications. The protein journal. 2011;30(7):480–9. doi: 10.1007/s10930-011-9353-x 21882049

24. Mirmiranpour H, Bathaie SZ, Khaghani S, Nakhjavani M, Kebriaeezadeh A. Investigation of the mechanism (s) involved in decreasing increased fibrinogen activity in hyperglycemic conditions using L-lysine supplementation. Thrombosis research. 2012;130(3):e13–e9. doi: 10.1016/j.thromres.2012.04.010 22575419

25. Mirmiranpour H, Khaghani S, Bathaie SZ, Nakhjavani M, Kebriaeezadeh A, Ebadi M, et al. The Preventive Effect of L-Lysine on Lysozyme Glycation in Type 2 Diabetes. Acta Medica Iranica. 2016;54(1):24–31. 26853287

26. Bahmani F, Bathaie SZ, Aldavood SJ, Ghahghaei A. Prevention of alpha-crystallin glycation and aggregation using l-lysine results in the inhibition of in vitro catalase heat-induced-aggregation and suppression of cataract formation in the diabetic rat. International journal of biological macromolecules. 2019;132:1200–7. doi: 10.1016/j.ijbiomac.2019.04.037 30965074.

27. Yaffe D, Saxel O. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature. 1977;270(5639):725. doi: 10.1038/270725a0 563524

28. Kirchberger M. Excitation and contraction of skeletal muscle. Physiological Basis of Medical Practice. 1991:62–102.

29. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry. 1976;72(1–2):248–54.

30. Ramirez-Zacarias J, Castro-Munozledo F, Kuri-Harcuch W. Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochemistry and Cell Biology. 1992;97(6):493–7.

31. Green LM, Reade JL, Ware CF. Rapid colormetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines. Journal of immunological methods. 1984;70(2):257–68. doi: 10.1016/0022-1759(84)90190-x 6609997

32. Winterbourn CC. The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells. Biochimica et Biophysica Acta (BBA)-General Subjects. 2014;1840(2):730–8.

33. Wojtala A, Bonora M, Malinska D, Pinton P, Duszynski J, Wieckowski MR. Methods to monitor ROS production by fluorescence microscopy and fluorometry. Methods in enzymology. 542: Elsevier; 2014. p. 243–62. doi: 10.1016/B978-0-12-416618-9.00013-3 24862270

34. Schmidt J, Rinaldi S, Scalbert A, Ferrari F, Achaintre D, Gunter M, et al. Plasma concentrations and intakes of amino acids in male meat-eaters, fish-eaters, vegetarians and vegans: a cross-sectional analysis in the EPIC-Oxford cohort. European Journal of Clinical Nutrition. 2016 70 306–12. doi: 10.1038/ejcn.2015.144 26395436

35. Boyvin L, Séri KL, Aké JA, Djaman JA. Lysine and threonine plasma concentrations in Ivorian patients living with human immunodeficiency virus. Journal of AIDS and HIV Research. 2017;9(9):194–201. doi: 10.5897/JAHR2017.0438

36. Verzola D, Fama A, Villaggio B, Di Rocco M, Simonato A, D'Amato E, et al. Lysine triggers apoptosis through a NADPH oxidase-dependent mechanism in human renal tubular cells. Journal of inherited metabolic disease. 2012;35(6):1011–9. doi: 10.1007/s10545-012-9468-z 22403019.

37. Baynes JW, Thorpe SR. The role of oxidative stress in diabetic complications. Current Opinion in Endocrinology, Diabetes and Obesity. 1996;3(4):277–84.

38. Ihara Y, Toyokuni S, Ichida K, Odaka H. Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes. Diabetes. 1999;48(4):927. doi: 10.2337/diabetes.48.4.927 10102716

39. Berkels R, Bertsch A, Zuther T, Dhein S, Stockklauser K, Rösen P, et al. Evidence for a NO synthase in porcine platelets which is stimulated during activation/aggregation. European journal of haematology. 1997;58(5):307–13. doi: 10.1111/j.1600-0609.1997.tb01676.x 9222285

40. Wever RM, Lüscher TF, Cosentino F, Rabelink TJ. Atherosclerosis and the two faces of endothelial nitric oxide synthase. Circulation. 1998;97(1):108–12. doi: 10.1161/01.cir.97.1.108 9443438

41. Madar Z, Kalet-Litman S, Stark AH. Inducible nitric oxide synthase activity and expression in liver and hepatocytes of diabetic rats. Pharmacology. 2005;73(2):106–12. doi: 10.1159/000081952 15528954

42. Stadler K, Jenei V, von Bölcsházy G, Somogyi A, Jakus J. Increased nitric oxide levels as an early sign of premature aging in diabetes. Free Radical Biology and Medicine. 2003;35(10):1240–51. doi: 10.1016/s0891-5849(03)00499-4 14607523

43. Llorens S, Nava E. Cardiovascular diseases and the nitric oxide pathway. Current vascular pharmacology. 2003;1(3):335–46. doi: 10.2174/1570161033476637 15320480

44. Liaudet L, Gnaegi A, Rosselet A, Markert M, Boulat O, Perret C, et al. Effect of L-lysine on nitric oxide overproduction in endotoxic shock. British journal of pharmacology. 1997;122(4):742–8. doi: 10.1038/sj.bjp.0701419 9375972; PubMed Central PMCID: PMC1564977.

45. Al-Malki AL. Suppression of acute pancreatitis by L-lysine in mice. BMC complementary and alternative medicine. 2015;15:193. doi: 10.1186/s12906-015-0729-x 26100532; PubMed Central PMCID: PMC4476087.

46. Rahmanpour R, Bathaie SZ. Histone H1 structural changes and its interaction with DNA in the presence of high glucose concentration in vivo and in vitro. Journal of Biomolecular Structure and Dynamics. 2011;28(4):575–86. doi: 10.1080/07391102.2011.10508596 21142225

47. Jiang M, Jia L, Jiang W, Hu X, Zhou H, Gao X, et al. Protein disregulation in red blood cell membranes of type 2 diabetic patients. Biochem Biophys Res Commun. 2003;309(1):196–200. doi: 10.1016/s0006-291x(03)01559-6 12943682.

48. Krantz S, Lober M, Thiele M, Teuscher E. Diminished adhesion of endothelial aortic cells on fibronectin and collagen layers after nonenzymatic glycation. Experimental and Clinical Endocrinology & Diabetes. 1988;91(02):155–60.

49. Pandey VK, Mathur A, Kakkar P. Emerging role of Unfolded Protein Response (UPR) mediated proteotoxic apoptosis in diabetes. Life sciences. 2019;216:246–58. doi: 10.1016/j.lfs.2018.11.041 30471281.

50. Rabhi N, Salas E, Froguel P, Annicotte JS. Role of the unfolded protein response in beta cell compensation and failure during diabetes. Journal of diabetes research. 2014;2014:795171. doi: 10.1155/2014/795171 24812634; PubMed Central PMCID: PMC4000654.

51. Mahdavifard S. Investigation of the effect of "poly-antiglycating agents" on early, intermediates and end products of glycation in the diabetic atherosclerotic rat model and in vitro. Tehran: Tarbiat Modares University; 2014.

52. He C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy. Annual review of genetics. 2009;43:67–93. doi: 10.1146/annurev-genet-102808-114910 19653858

53. Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nature reviews Molecular cell biology. 2012;13(2):89–102. doi: 10.1038/nrm3270 22251901.

54. Lin H, Liu XB, Yu JJ, Hua F, Hu ZW. Antioxidant N-acetylcysteine attenuates hepatocarcinogenesis by inhibiting ROS/ER stress in TLR2 deficient mouse. PLoS One. 2013;8(10):e74130. doi: 10.1371/journal.pone.0074130 24098333; PubMed Central PMCID: PMC3788783.

55. Ding W, Zhang X, Huang H, Ding N, Zhang S, Hutchinson SZ, et al. Adiponectin protects rat myocardium against chronic intermittent hypoxia-induced injury via inhibition of endoplasmic reticulum stress. PLoS One. 2014;9(4):e94545. doi: 10.1371/journal.pone.0094545 24718591; PubMed Central PMCID: PMC3981809.

56. Mehrpour M, Esclatine A, Beau I, Codogno P. Autophagy in health and disease. 1. Regulation and significance of autophagy: an overview. American journal of physiology Cell physiology. 2010;298(4):C776–85. doi: 10.1152/ajpcell.00507.2009 20089931.

57. Codogno P, Mehrpour M, Proikas-Cezanne T. Canonical and non-canonical autophagy: variations on a common theme of self-eating? Nature reviews Molecular cell biology. 2011;13(1):7–12. doi: 10.1038/nrm3249 22166994.

58. Rubinsztein DC, Cuervo AM, Ravikumar B, Sarkar S, Korolchuk V, Kaushik S, et al. In search of an "autophagomometer". Autophagy. 2009;5(5):585–9. doi: 10.4161/auto.5.5.8823 19411822.

59. Penas C, Font-Nieves M, Fores J, Petegnief V, Planas A, Navarro X, et al. Autophagy, and BiP level decrease are early key events in retrograde degeneration of motoneurons. Cell death and differentiation. 2011;18(10):1617–27. doi: 10.1038/cdd.2011.24 21436843; PubMed Central PMCID: PMC3172115.

60. Clarke R, Cook KL, Hu R, Facey CO, Tavassoly I, Schwartz JL, et al. Endoplasmic reticulum stress, the unfolded protein response, autophagy, and the integrated regulation of breast cancer cell fate. Cancer research. 2012;72(6):1321–31. doi: 10.1158/0008-5472.CAN-11-3213 22422988; PubMed Central PMCID: PMC3313080.

61. Verfaillie T, Salazar M, Velasco G, Agostinis P. Linking ER Stress to Autophagy: Potential Implications for Cancer Therapy. International journal of cell biology. 2010;2010:930509. doi: 10.1155/2010/930509 20145727; PubMed Central PMCID: PMC2817393.

62. Sato T, Ito Y, Nedachi T, Nagasawa T. Lysine suppresses protein degradation through autophagic–lysosomal system in C2C12 myotubes. Mol Cell Biochem 2014;391:37–46. doi: 10.1007/s11010-014-1984-8 24532005

63. Sato T, Ito Y, Nagasawa T. Regulatory effects of the L-lysine metabolites, L-2-aminoadipic acid and L-pipecolic acid, on protein turnover in C2C12 myotubes. Bioscience, biotechnology, and biochemistry. 2016:1–8. doi: 10.1080/09168451.2015.1056512 27427787.

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