Effect of sub-toxic chlorpyrifos on redox sensitive kinases and insulin signaling in rat L6 myotubes

  • Shrijana Shrestha
  • Vijay Kumar Singh
  • Sajib Kumar Sarkar
  • Balasubramanian Shanmugasundaram
  • Kadirvelu Jeevaratnam
  • Bidhan Chandra Koner
Research Article



Sub-chronic exposures to chlorpyrifos, an organophosphorus pesticide is associated with incidence of diabetes mellitus. Biochemical basis of chlorpyrifos-induced diabetes mellitus is not known. Hence, effect of its sub-toxic exposure on redox sensitive kinases, insulin signaling and insulin-induced glucose uptake were assessed in rat muscle cell line.


In an in vitro study, rat myoblasts (L6) cell line were differentiated to myotubes and then were exposed to sub-toxic concentrations (6 mg/L and 12 mg/L) of chlorpyrifos for 18 h. Then total anti-oxidant level in myotubes was measured and insulin-stimulated glucose uptake was assayed. Assessment of activation of NFκB & p38MAPK and insulin signaling following insulin stimulation from tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and serine phosphorylation of Akt were done in myotubes after chlorpyrifos exposure by western blot (WB) and compared with those in vehicle-treated controls.


The glucose uptake and total antioxidant level in L6-derived myotubes after sub-toxic exposure to chlorpyrifos were decreased in a dose-dependent manner. As measured from band density of WB, phosphorylation levels increased for redo-sensitive kinases (p38MAPK and IκBα component of NFκB) and decreased for IRS-1 (at tyrosine 1222) and Akt (at serine 473) on insulin stimulation following chlorpyrifos exposure as compared to those in controls.


We conclude that sub-toxic chlorpyrifos exposure induces oxidative stress in muscle cells activating redox sensitive kinases that impairs insulin signaling and thereby insulin-stimulated glucose uptake in muscle cells. This probably explains the biochemical basis of chlorpyrifos-induced insulin resistance state and diabetes mellitus.


Chlorpyrifos Type 2 diabetes mellitus Insulin signaling Insulin resistance NFκB p38MAPK 



Type 2 diabetes mellitus




Insulin Receptor Substrate-1


Redox sensitive kinases


Nuclear factor kappa B

p38 MAPK

p38 Mitogen-Activated Protein Kinase


3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide


Dulbecco'’s modified Eagle'’s medium


Fetal bovine serum



We acknowledge Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India (F.No. SB/SO/HS/0024/2013) for providing extramural funding to meet the expenditure of the study.

Compliance with ethical standards

Conflict of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.


  1. 1.
    Lee DH, Lee IK, Porta M, Steffes M, Jacobs DR. Relationship between serum concentrations of persistent organic pollutants and the prevalence of metabolic syndrome among non-diabetic adults: results from the National Health and Nutrition examination survey 1999-2002. Diabetologia. 2007;50:1841–51.CrossRefGoogle Scholar
  2. 2.
    Everett CJ, Matheson EM. Biomarkers of pesticide exposure and diabetes in the 1999 – 2004 National Health and Nutrition examination survey. Environ Int. 2010;36:398–401.CrossRefGoogle Scholar
  3. 3.
    World Health Organization. WHO Specifications and evaluations for public health pesticides (Chlorpyrifos). 2009 p32. Available from: Accessed 13 04 2018.
  4. 4.
    Acker CI, Nogueira CW. Chlorpyrifos acute exposure induces hyperglycemia and hyperlipidemia in rats. Chemosphere. 2012;89:602–8.CrossRefGoogle Scholar
  5. 5.
    Mohamed WR, Mehany ABM, Hussein RM. Alpha lipoic acid protects against chlorpyrifos-induced toxicity in Wistar rats via modulating the apoptotic pathway. Environ Toxicol Pharmacol. 2018;59:17–23.CrossRefGoogle Scholar
  6. 6.
    Ojha A, Gupta YK. Evaluation of genotoxic potential of commonly used organophosphate pesticides in peripheral blood lymphocytes of rats. Hum Exp Toxicol. 2015;34:390–400.CrossRefGoogle Scholar
  7. 7.
    Tang G, Yao J, Zhang X, Lu N, Zhu KY. Comparison of gene expression profiles in the aquatic midge (Chironomus tentans) larvae exposed to two major agricultural pesticides. Chemosphere. 2018;194:745–54.CrossRefGoogle Scholar
  8. 8.
    Pallotta MM, Barbato V, Pinton A, Acloque H, Gualtieri R, Talevi R, et al. In vitro exposure to CPF affects bovine sperm epigenetic gene methylation pattern and the ability of sperm to support fertilization and embryo development. Environ Mol Mutagen. 2018.Google Scholar
  9. 9.
    Shin H-S, Seo J-H, Jeong S-H, Park S-W, Park Y, Son S-W, et al. Exposure of pregnant mice to chlorpyrifos-methyl alters embryonic H19 gene methylation patterns. Environ Toxicol. 2014;29:926–35.CrossRefGoogle Scholar
  10. 10.
    Montgomery MP, Kamel F, Saldana TM, Alavanja MCR, Sandler DP. Incident diabetes and pesticide exposure among licensed pesticide applicators: agricultural health study, 1993–2003. Am J Epidemiol. 2008;167:1235–46.CrossRefGoogle Scholar
  11. 11.
    Elsharkawy EE, Yahia D, El-Nisr NA. Sub-chronic exposure to chlorpyrifos induces hematological, metabolic disorders and oxidative stress in rat: attenuation by glutathione. Environ Toxicol Pharmacol. 2013;35:218–27.CrossRefGoogle Scholar
  12. 12.
    Lee YH, Giraud J, Davis RJ, White MF. c-Jun N-terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade. J Biol Chem. 2003;278:2896–902.CrossRefGoogle Scholar
  13. 13.
    Peris-Sampedro F, Cabré M, Basaure P, Reverte I, Domingo JL, Teresa Colomina M. Adulthood dietary exposure to a common pesticide leads to an obese-like phenotype and a diabetic profile in apoE3 mice. Environ Res. 2015;142:169–76.CrossRefGoogle Scholar
  14. 14.
    Petersen KF, Shulman GI. Etiology of insulin resistance. Am J Med. 2006;119:S10–6.CrossRefGoogle Scholar
  15. 15.
    DeFronzo RA, Tripathy D. Skeletal muscle insulin resistance is the primary defect in type 2 diabetes. Diabetes Care. 2009;32(Suppl 2):S157–63.CrossRefGoogle Scholar
  16. 16.
    Ziel FH, Venkatesen N, Davidson MB. Glucose transport is rate limiting for skeletal muscle glucose metabolism in normal and STZ-induced diabetic rats. Diabetes. 1988;37:885–90.CrossRefGoogle Scholar
  17. 17.
    Ren JM, Marshall BA, Gulve EA, Gao J, Johnson DW, Holloszy JO, et al. Evidence from transgenic mice that glucose transport is rate-limiting for glycogen deposition and glycolysis in skeletal muscle. J Biol Chem. 1993;268:16113–5.PubMedGoogle Scholar
  18. 18.
    Zierath JR, Krook A, Wallberg-Henriksson H. Insulin action and insulin resistance in human skeletal muscle. Diabetologia. 2000;43:821–35.CrossRefGoogle Scholar
  19. 19.
    Maegawa H, Shigeta Y, Egawa K, Kobayashi M. Impaired autophosphorylation of insulin receptors from abdominal skeletal muscles in nonobese subjects with NIDDM. Diabetes. 1991;40:815–9.CrossRefGoogle Scholar
  20. 20.
    Krook A, Björnholm M, Galuska D, Jiang XJ, Fahlman R, Myers MG, et al. Characterization of signal transduction and glucose transport in skeletal muscle from type 2 diabetic patients. Diabetes. 2000;49:284–92.CrossRefGoogle Scholar
  21. 21.
    Saulsbury MD, Heyliger SO, Wang K, Johnson DJ. Chlorpyrifos induces oxidative stress in oligodendrocyte progenitor cells. Toxicology. 2009;259:1–9.CrossRefGoogle Scholar
  22. 22.
    Chiapella G, Flores-Martín J, Ridano ME, Reyna L, Magnarelli de Potas G, Panzetta-Dutari GM, et al. The organophosphate chlorpyrifos disturbs redox balance and triggers antioxidant defense mechanisms in JEG-3 cells. Placenta. 2013;34:792–8.CrossRefGoogle Scholar
  23. 23.
    Ambali SF, Akanbi DO, Shittu M, Giwa A, Oladipo OO, Ayo JO. Chlorpyrifos-induced clinical, hematological and biochemical changes in Swiss albino mice- mitigating effect by co-administration of vitamins C and E. Life Sci J. 2010;7:37–44.Google Scholar
  24. 24.
    Uchendu C, Ambali SF, Ayo JO, Esievo KAN, Umosen AJ. Erythrocyte osmotic fragility and lipid peroxidation following chronic co-exposure of rats to chlorpyrifos and deltamethrin, and the beneficial effect of alpha-lipoic acid. Toxicol Rep. 2014;1:373–8.CrossRefGoogle Scholar
  25. 25.
    Morgan MJ, Liu Z. Crosstalk of reactive oxygen species and NF-κB signaling. Cell Res. 2011;21:103–15.CrossRefGoogle Scholar
  26. 26.
    Son Y, Cheong Y-K, Kim N-H, Chung H-T, Kang DG, Pae H-O. Mitogen-activated protein kinases and reactive oxygen species: how can ROS activate MAPK pathways? J Signal Transduct. 2011;2011:1–6.CrossRefGoogle Scholar
  27. 27.
    Maddux BA, See W, Lawrence JCJ, Goldfine AL, Goldfine ID, Evans JL. Protection against oxidative stress-induced insulin resistance in rat L6 muslce cells by micromolar concentrations of alpha-lipoic acid. Diabetes. 2001;50:404–10.CrossRefGoogle Scholar
  28. 28.
    Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes Dev. 2004;18:2195–224.CrossRefGoogle Scholar
  29. 29.
    Archuleta TL, Lemieux AM, Saengsirisuwan V, Teachey MK, Lindborg KA, Kim JS, et al. Oxidant stress-induced loss of IRS-1 and IRS-2 proteins in rat skeletal muscle: role of p38 MAPK. Free Radic Biol Med. 2009;47:1486–93.CrossRefGoogle Scholar
  30. 30.
    Evans JL, Goldfine IRAD, Maddux BA, Grodsky GM, Francisco S. Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocr Rev. 2002;23:599–622.CrossRefGoogle Scholar
  31. 31.
    Karin M, Gallagher E. From JNK to pay dirt: Jun kinases, their biochemistry, physiology and clinical importance. IUBMB Life (International Union Biochem Mol Biol Life). 2005;57:283–95.CrossRefGoogle Scholar
  32. 32.
    Hotamisligil GS. The role of TNFalpha and TNF receptors in obesity and insulin resistance. J Intern Med. 1999;245:621–5.CrossRefGoogle Scholar
  33. 33.
    Slotkin TA, Brown KK, Seidler FJ. Developmental exposure of rats to chlorpyrifos elicits sex-selective hyperlipidemia and hyperinsulinemia in adulthood. Environ Health Perspect. 2005;113:1291–4.CrossRefGoogle Scholar
  34. 34.
    Abdollahi M, Donyavi M, Pournourmohammadi S, Saadat M. Hyperglycemia associated with increased hepatic glycogen phosphorylase and phosphoenolpyruvate carboxykinase in rats following subchronic exposure to malathion. Comp Biochem Physiol C Toxicol Pharmacol. 2004;137:343–7.CrossRefGoogle Scholar
  35. 35.
    Pournourmohammadi S, Farzami B, Ostad SN, Azizi E, Abdollahi M. Effects of malathion subchronic exposure on rat skeletal muscle glucose metabolism. Environ Toxicol Pharmacol. 2005;19:191–6.CrossRefGoogle Scholar
  36. 36.
    Shrestha S, Singh VK, Shanmugasundaram B, Sarkar SK, Jeevaratnam K, Koner BC. The Effect of Chlorpyrifos, an Organophosphorus Pesticide, on Glucose Uptake in Whole Blood. J Drug Metab Toxicol. 2016;7.Google Scholar
  37. 37.
    Gangemi S, Gofita E, Costa C, Teodoro M, Briguglio G, Nikitovic D, et al. Occupational and environmental exposure to pesticides and cytokine pathways in chronic diseases (Review). Int J Mol Med. 2016;1012–20.CrossRefGoogle Scholar
  38. 38.
    Rösen P, Nawroth PP, King G, Möller W, Tritschler HJ, Packer L. The role of oxidative stress in the onset and progression of diabetes and its complications: a summary of a Congress Series sponsored by UNESCO-MCBN, the American Diabetes Association and the German Diabetes Society. Diabetes Metab Res Rev. 2001;17:189–212.CrossRefGoogle Scholar
  39. 39.
    Batista JES, Sousa LR, Martins IK, Rodrigues NR, Posser T, Franco JL. Data on the phosphorylation of p38MAPK and JNK induced by chlorpyrifos in Drosophila melanogaster. Data Br. 2016;9:32–4.CrossRefGoogle Scholar
  40. 40.
    Park JH, Ko J, Park YS, Park J, Hwang J, Koh HC. Clearance of Damaged Mitochondria Through PINK1 Stabilization by JNK and ERK MAPK Signaling in Chlorpyrifos-Treated Neuroblastoma Cells. Mol Neurobiol. 2017;54:1844–57.CrossRefGoogle Scholar
  41. 41.
    Hommelberg PPH, Plat J, Langen RCJ, Schols AMWJ, Mensink RP. Fatty acid-induced NF-kappaB activation and insulin resistance in skeletal muscle are chain length dependent. Am J Physiol Endocrinol Metab. 2009;296:E114–20.CrossRefGoogle Scholar
  42. 42.
    Moxham C, Tabrizchi A, Davis R, Malbon C. Jun N-terminal kinase mediates activation of skeletal muscle glycogen synthase by insulin in vivo. J Biol Chem. 1996;271:30765–73.CrossRefGoogle Scholar
  43. 43.
    Guo JH, Wang H, Malbon CC. Conditional, tissue-specific expression of Q205L Gai2in vivo mimics insulin activation of c-Jun N-terminal kinase and p38 kinase. J Biol Chem. 1998;273:16487–93.CrossRefGoogle Scholar
  44. 44.
    Aguirre V, Uchida T, Yenush L, Davis R, White MF. The c-Jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307). J Biol Chem. 2000;275:9047–54.CrossRefGoogle Scholar
  45. 45.
    Yuan M. Reversal of Obesity- and Diet-Induced Insulin Resistance with Salicylates or Targeted Disruption of Ikkbeta. Science. 2001;293:1673–7.CrossRefGoogle Scholar
  46. 46.
    Hirosumi J, Tuncman G, Chang L, Görgün CZ, Uysal KT, Maeda K, et al. A central, role for JNK in obesity and insulin resistance. Nature. 2002;420:333–6.CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Department of BiochemistryMaulana Azad Medical CollegeNew DelhiIndia
  2. 2.Department of Biochemistry and Molecular BiologyPondicherry UniversityPuducherryIndia

Personalised recommendations