Author(s):
Rita Rakhmawati, Mae Sri Hartati Wahyuningsi, Arko Jatmiko Wicaksono, Mustofa Mustofa, Ahmad Hamim Sadewa
Email(s):
maeshw@ugm.ac.id
DOI:
10.52711/0974-360X.2025.00726 
Address:
Rita Rakhmawati1,5, Mae Sri Hartati Wahyuningsih2,4*, Arko Jatmiko Wicaksono2,4, Mustofa Mustofa2, Ahmad Hamim Sadewa3
1Department of Medicine and Health Science Doctorate Program, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia.
2Department of Pharmacology and Therapy, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia.
3Department of Biochemistry, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia.
4Center for Herbal Medicine, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia.
5Department of Pharmacy, Faculty of Mathematics and Natural Sciences,Universitas Sebelas Maret, Surakarta, Indonesia.
*Corresponding Author
Published In:
Volume - 18,
Issue - 10,
Year - 2025
ABSTRACT:
Insulin resistance is one of the main risk factors for the development of diabetes mellitus. The search for natural ingredient-based drugs for antidiabetic treatment is widely carried out through experimental evidence in the laboratory. An in vitro model has advantages in easily maintaining and replicating. It is an appropriate choice for studying insulin resistance. The critical point in conducting insulin experiments in the early stages is to make the skeletal muscle C2C12 myotube form into insulin-resistant cells. For this reason, suitable types of insulin resistance inducers, effective concentrations, and administered inducer methods need to be discussed. This systematic review provides an overview of types of insulin resistance inducers in the C2C12 in vitro method, the effective concentrations in the molecular study of insulin resistance, and the technique administered by the inducer. Methods: The PubMed and Scopus databases were searched from 2012–2022. Results: Palmitic acid is one of the popular insulin resistance inducers. Concentrations of 0.20-0.75 mM palmitic acid are sufficient to cause insulin resistance. Inducing methods were administered simultaneously or in phases, together with the compound. The difference in the addition of inducers in forming the C2C12 cell model of insulin resistance is related to the research objectives for preventive/protective or curative purposes. The methods were successfully reported in establishing an insulin-resistant C2C12 cell model.
Cite this article:
Rita Rakhmawati, Mae Sri Hartati Wahyuningsi, Arko Jatmiko Wicaksono, Mustofa Mustofa, Ahmad Hamim Sadewa. Establishing an Insulin-Resistance C2C12 Cell Model: A Systematic Review. Research Journal of Pharmacy and Technology. 2025;18(10):5027-4. doi: 10.52711/0974-360X.2025.00726
Cite(Electronic):
Rita Rakhmawati, Mae Sri Hartati Wahyuningsi, Arko Jatmiko Wicaksono, Mustofa Mustofa, Ahmad Hamim Sadewa. Establishing an Insulin-Resistance C2C12 Cell Model: A Systematic Review. Research Journal of Pharmacy and Technology. 2025;18(10):5027-4. doi: 10.52711/0974-360X.2025.00726 Available on: https://rjptonline.org/AbstractView.aspx?PID=2025-18-10-65
REFERENCES:
1. Abdul-Ghani MA. DeFronzo RA. Pathogenesis of Insulin Resistance in Skeletal Muscle. Journal Biomed Biotechnology. 2010; 2010: 476279. doi: 10.1155/2010/476279.
2. Saeedi P. Petersohn I. Salpea P. Malanda B. Karuranga S. Unwin N. Colagiuri S. Guariguata L. Motala AA. Ogurtsova K. Shaw J. E. Bright D. Williams R et al. Global and Regional Diabetes Prevalence Estimates for 2019 And Projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas. 9th edition. Diabetes Research and Clinical Practice. 2019; 157: 107843. https://doi.org/10.1016/j.diabres.2019.107843.
3. Luo J. Hou Y. Xie M. Ma W. Shi D. Jiang. B et al. CYC31. A Natural Bromophenol PTP1B Inhibitor Activates Insulin Signaling and Improves Long Chain Fatty Acid Oxidation in C2C12 Myotubes. Marine Drugs. 2020; 18(267): https://doi.org/10.3390/md18050267
4. Dahlén, A. D., Dashi, G., Maslov, I., Attwood, M. M., Jonsson, J., Trukhan, V., and Schiöth, H. B. Trends in Antidiabetic Drug Discovery: FDA Approved Drugs, New Drugs in Clinical Trials and Global Sales. Frontiers in Pharmacology. 2022; 12, 1–16. https://doi.org/10.3389/fphar.2021.807548
5. Sujatha Kumari, M., Kiran Babu, M., Sulthana, M. R., Srinivas, M., and Prasanthi, C. Diabetes mellitus: Present status and drug therapy updates. Research Journal of Pharmacy and Technology. 2014; 7(1), 84–94.
6. Pandeya, S. N., Kumar, R., Kumar, A., and Pathak, A. K. Antidiabetics Review on Natural Products. Research Journal of Pharmacy and Technology. 2010; 3(2), 300–318.
7. Keerthana, K., and Jothi, G. Determination of In vitro antidiabetic potential of Aqueous and Ethanol extracts and isolated fraction of Zanthoxylum armatum DC stem bark. Research Journal of Pharmacy and Technology. 2020; 13(8), 3681. https://doi.org/10.5958/0974-360x.2020.00651.4
8. Preethi, P. J. Review Article Herbal medicine for diabetes mellitus : A Review. Asian J. Pharm. Res. 2013' 3(2), 57–70.
9. Devaliya, R., and Shirsat, M. A review on indigenous medicinal plants for diabetes mellitus. Research Journal of Pharmacy and Technology. 2017; 10(8), 2828–2836. https://doi.org/10.5958/0974-360X.2017.00499.1
10. Keerthana, K., and Jothi, G. Determination of in vitro antidiabetic potential of Aqueous and Ethanol extracts and isolated fraction of Zanthoxylum armatum DC stem bark. Research Journal of Pharmacy and Technology. 2020; 13(8), 3681. https://doi.org/10.5958/0974-360x.2020.00651.4
11. Doke SK. Dhawale SC. Alternatives to Animal Testing: A Review. Saudi Pharmaceutical Journal. 2015; 23(3): 223–229. https://doi.org/10.1016/j.jsps.2013.11.002
12. Antony, J., Satadal, D., Manisha, C., Peet Thomas, Victoria Jeyarani, and Choephel, T. In-vitro cell line models and assay methods to study the anti-diabetic activity. Research Journal of Pharmacy and Technology. 2019; 12(5); 2200–2206. https://doi.org/10.5958/0974-360X.2019.00367.6
13. Yudhani R D. Sari Y. Nugrahaningsih DAA. Sholikhah EN. Rochmanti M. Purba AKR. Khotimah H. Nugrahenny D. Mustofa M et al. In Vitro Insulin Resistance Model: A Recent Update. Journal of Obesity. 2023. https://doi.org/10.1155/2023/1964732
14. Jakovljevic NK. Pavlovic K. Zujovic T. Kravic-Stevovic T. Jotic A. Markovic I Lalic NM et al. In Vitro Models of Insulin Resistance: Mitochondrial Coupling is Differently Affected in Liver and Muscle Cells. Mitochondrion. 2021; 61: 165–173. https://doi.org/10.1016/j.mito.2021.10.001
15. Wong, C. Y., Al-Salami, H., and Dass, C. R. C2C12 cell model: its role in understanding of insulin resistance at the molecular level and pharmaceutical development at the preclinical stage. Journal of Pharmacy and Pharmacology, 2020; 72(12), 1667–1693. https://doi.org/10.1111/jphp.13359
16. Batra, S., Batra, N., Pareek, A., and Nagori, B. P. In vitro study of Cayratia trifolia (L.) domin on isolated rat hemidiaphragm by glucose uptake method. Research Journal of Pharmacy and Technology. 2012; 5(5), 691–693.
17. Yang C. Aye CC. Li X. Diaz Ramos A. Zorzano A. Mora S et al. Mitochondrial Dysfunction in Insulin Resistance: Differential Contributions of Chronic Insulin and Saturated Fatty Acid Exposure in Muscle Cells. Bioscience Reports. 2012; 32(5): 465–478. https://doi.org/10.1042/BSR20120034
18. Thomson MJ. Williams MG. Frost SC. Development of Insulin Resistance in 3T3-L1 Adipocytes. Journal of Biological Chemistry. 1997; 272(12): 7759–7764. https://doi.org/10.1074/jbc.272.12.7759
19. Henriksen JE. Alford F. Ward GM. Beck-Nielsen H et al. Risk and Mechanism of Dexamethasone-Induced Deterioration of Glucose Tolerance in Non-Diabetic First-Degree Relatives of NIDDM Patients. Diabetologia. 1997; 40(12): 1439–1448. https://doi.org/10.1007/s001250050847
20. Sakoda H. Ogihara T. Anai M. Funaki M. Inukai K. Katagiri H. Fukushima Y. Onishi Y. Ono H. Fujishiro M. Kikuchi M. Oka Y. Asano T et al. Dexamethasone-Induced Insulin Resistance in 3T3-L1 Adipocytes is Due to Inhibition of Glucose Transport Rather than Insulin Signal Transduction. Diabetes. 2000; 49(10): 1700–1708. https://doi.org/10.2337/diabetes.49.10.1700
21. Biradar, K. V, Patil, C. B., Chivde, B. V, Malipatil, M. H., and Sanjivkumar, D. Effect of Superoxide Dismutase Mimetic Tempol on Dexamethasone Induced Insulin Resistance- Role of Oxidative Stress. ABSTRACT : Research J. Pharmacology and Pharmacodynamics. 2011; 3(3), 134–137.
22. Mariappan MM. Feliers D. Mummidi S. Choudhury GG. Kasinath BS et al. High Glucose, High Insulin, and Their Combination Rapidly Induce Laminin-β1 Synthesis by Regulation of mRNA Translation in Renal Epithelial Cells. Diabetes. 2007; 56(2): 476–485. https://doi.org/10.2337/db05-1334
23. Moller DE. Potential Role of TNF-α in the Pathogenesis of Insulin Resistance and Type 2 Diabetes. Trends in Endocrinology and Metabolism. 2000; 11(6): 212–217. https://doi.org/10.1016/S1043-2760(00)00272-1
24. Miles PDG. Romeo OM. Higo K. Cohen A. Rafaat K. Olefsky JM et al. TNF-α-Induced Insulin Resistance In Vivo And Its Prevention by Troglitazone. Diabetes. 1997; 46(11): 1678–1683. https://doi.org/10.2337/diab.46.11.1678
25. Yang Q. Zhu Z. Wang L. Xia H. Mao J. Wu J. Kato K. Li H. Zhang J. Yamanaka K. An Y. The Protective Effect of Silk Fibroin on High Glucose-Induced Insulin Resistance in HepG2 Cells. Environmental Toxicology and Pharmacology. 2019; 69: 66-71. doi: 10.1016/j.etap.2019.04.001.
26. de Luca C. Olefsky JM. Inflammation and Insulin Resistance. FEBS Letters. 2008; 582(1): 97–105. https://doi.org/10.1016/j.febslet.2007.11.057
27. Pittas AG. Joseph NA. Greenberg AS. Adipocytokines and Insulin Resistance. Journal of Clinical Endocrinology and Metabolism. 2004; 89(2): 447–452. https://doi.org/10.1210/jc.2003-031005
28. Rabe K. Lehrke M. Parhofer KG. Broedl UC et al. Adipokines and Insulin Resistance. Molecular Medicine. 2008; 14(11–12): 741–751. https://doi.org/10.2119/2008-00058.Rabe
29. Sharma P. Sri Swetha Victoria V. Praneeth Kumar P. Karmakar S. Swetha M. Reddy A et al. Cross-Talk Between Insulin Resistance and Nitrogen Species in Hypoxia Leads to Deterioration of Tissue and Homeostasis. International Immunopharmacology. 2023; 122(12): 110472. https://doi.org/10.1016/j.intimp.2023.110472
30. Lee BH. Hsu WH. Liao. TH. Pan TM et al. The Monascus Metabolite Monascin Against TNF-α-Induced Insulin Resistance via Suppressing PPAR-γ Phosphorylation in C2C12 Myotubes. Food and Chemical Toxicology. 2011; 49(10): 2609–2617. https://doi.org/10.1016/j.fct.2011.07.005
31. Nursucihta S. Thai’in HA. Putri MD Utami DN. Ghani AP et al. Antianemia Activity of Parkia speciosa hassk Seed Ethanolic Extract. Traditional Medicine Journal. 2014; 19(05); 49–54.
32. Gong L. Zou Z. Huang L. Guo S. Xing D et al. Photobiomodulation Therapy Decreases Free Fatty Acid Generation and Release in Adipocytes to Ameliorate Insulin Resistance in Type 2 Diabetes. Cellular Signalling. 2020; 67: 109491. https://doi.org/10.1016/j.cellsig.2019.109491
33. Li HB. Yang YRY. Mo ZJ. Ding Y. Jiang WJ et al. Silibinin Improves Palmitate-Induced Insulin Resistance in C2C12 Myotubes by Attenuating IRS-1/PI3K/Akt Pathway Inhibition. Brazilian Journal of Medical and Biological Research. 2015; 48(5): 440–446. https://doi.org/10.1590/1414-431X20144238
34. Schütze S. Berkovic D. Tomsing O. Unger C. Krönke M. Tumor necrosis Factor Induces Rapid Production of 1′2′Diacylglycerol by a Phosphatidylcholine-Specific Phospholipase C. Journal of Experimental Medicine. 1991; 174(5): 975–988. https://doi.org/10.1084/jem.174.5.975
35. Adams JM. Pratipanawatr T. Berria R. Wang E. DeFronzo RA. Sullards MC. Mandarino LJ. Ceramide Content Is Increased in Skeletal Muscle from Obese Insulin-Resistant Humans. Diabetes. 2004; 53(1): 25–31. https://doi.org/10.2337/diabetes.53.1.25
36. Malik SA. Acharya JD. Mehendale NK. Kamat SS. Ghaskadbi SS et al. Pterostilbene Reverses Palmitic Acid-Mediated Insulin Resistance in HepG2 Cells by Reducing Oxidative Stress and Triglyceride Accumulation. Free Radical Research. 2019; 53(7): 815–827. https://doi.org/10.1080/10715762.2019.1635252
37. Park JM. Lee JS. Song JE. Sim YC. Ha SJ. Hong EK et al. Cytoprotective Effect of Hispidin Against Palmitic Acid-Induced Lipotoxicity in C2C12 Myotubes. Molecules. 2015; 20(4): 5456–5467. https://doi.org/10.3390/molecules20045456
38. Ayeleso TB. Ramachela K. Mukwevho E. Aqueous-Methanol Extracts of Orange-Fleshed Sweet Potato (Ipomoea batatas) Ameliorate Oxidative Stress and Modulate Type 2 Diabetes-Associated Genes in Insulin-Resistant C2C12 Cells. Molecules. 2018; 23(2058). https://doi.org/10.3390/molecules23082058
39. Bakar MHA. Tan JS. Improvement of Mitochondrial Function by Celastrol in Palmitic Acid-Treated C2C12 Myotubes via Activation of the PI3K-Akt Signaling Pathway. Biomedicine and Pharmacotherapy. 2017; 93: 903–912. https://doi.org/10.1016/j.biopha.2017.07.021
40. Lin CH. Kuo YH. Shih CC. Eburicoic Acid: A Triterpenoid Compound from Antrodia camphorata Displays Antidiabetic and Antihyperlipidemic Effects in Palmitic Acid-Treated C2C12 Myotubes and High-Fat Diet-Fed Mice. International Journal of Molecular Sciences. 2017; 18(2314). https://doi.org/10.3390/ijms18112314
41. Xu Q. Luo J. Wu N. Zhang R. Shi D et al. BPN. A Marine-Derived PTP1B Inhibitor Activates Insulin Signaling and Improves Insulin Resistance in C2C12 Myotubes. International Journal of Biological Macromolecules. 2018; 106: 379–386. https://doi.org/10.1016/j.ijbiomac.2017.08.042
42. Yoon HJ. Bang MH. Kim H. Imm JY et al. Improvement of Palmitic Acid-Induced Insulin Resistance in C2C12 Skeletal Muscle Cells Using Platycodon grandiflorum Seed Extracts. Food Bioscience. 2018; 25(07): 61–67. https://doi.org/10.1016/j.fbio.2018.08.002
43. Zhu R. Zheng J. Chen L. Gu B. Huang S et al. Astragaloside IV Facilitates Glucose Transport in C2C12 Myotubes through the IRS1/AKT Pathway and Suppresses the Palmitic Acid-Induced Activation of the IKK/IB Pathway. International Journal of Molecular Medicine. 2016; 37(6): 1697–1705. https://doi.org/10.3892/ijmm.2016.2555
44. Sánchez-Alegría K. Bastián-Eugenio CE. Vaca L. Arias C. Palmitic Acid Induces Insulin Resistance by a Mechanism Associated with Energy Metabolism and Calcium Entry In Neuronal Cells. Federation of American Societies for Experimental Biology Journal. 2021; 35(7). https://doi.org/10.1096/fj.202100243R
45. Zhao M. Zhang ZF. Ding Y. Wang JB. Li Y et al. Astragalus Polysaccharide Improves Palmitic Acid-Induced Insulin Resistance by Inhibiting PTP1B and NF-κB in C2C12 Myotubes. Molecules. 2012; 17(6): 7083–7092. https://doi.org/10.3390/molecules17067083
46. Feng XT. Wang TZ. Leng J. Chen Y. Liu JB. Liu Y. Wang WJ et al. Palmitic Acid Contributes to Insulin Resistance Through Downregulation of the Src-Mediated Phosphorylation of Akt in C2C12 Myotubes. Bioscience. Biotechnology and Biochemistry. 2012; 76(7): 1356–1361. https://doi.org/10.1271/bbb.120107
47. Liu L. Zhang X. Chen F. Hu J. Zeng B et al. The Establishment of the Insulin Resistance Model. BIO Web of Conferences. 2017; 8: 03005. https://doi.org/10.1051/bioconf/20170803005
48. Alkhateeb H. Chabowski A. Glatz JFC. Luiken JFP. Bonen A et al. Two Phases of Palmitic Acid-Induced Insulin Resistance in Skeletal Muscle: Impaired GLUT4 Translocation is Followed by a Reduced GLUT4 Intrinsic Activity. American Journal of Physiology - Endocrinology and Metabolism. 2007; 293(3): 783–793. https://doi.org/10.1152/ajpendo.00685.2006
49. Nguyen MT. Min KH. Lee W. MiR-183-5p Induced by Saturated Fatty Acids Hinders Insulin Signaling by Downregulating Irs-1 In Hepatocytes. International Journal Molecular Science. 2022; 23(6): 2979. doi: 10.3390/ijms23062979.
50. Kim JS. Lee H. Jung CH. Lee SJ. Ha. TY. Ahn J et al. 2018. Chicoric Acid Mitigates Impaired Insulin Sensitivity by Improving Mitochondrial Function. Bioscience. Biotechnology and Biochemistry. 82(7). 1197–1206. https://doi.org/10.1080/09168451.2018.1451742
51. Dai X. Ding Y. Zhang Z. Cai X. Bao L. Li Y et al. Quercetin But Not Quercitrin Ameliorates Tumor Necrosis Factor-Alpha-Induced Insulin Resistance in C2C12 Skeletal Muscle Cells. In Biological and Pharmaceutical Bulletin 2013; 36(5).
52. Shyur LF. Varga V. Chen CM. Mu SC. Chang YC. Li SC et al. Extract of white Sweet Potato Tuber Against TNF-α-induced Insulin Resistance by Activating the PI3K/Akt pathway in C2C12 Myotubes. Botanical Studies. 2021; 62(7). https://doi.org/10.1186/s40529-021-00315-8
53. Jia XK. Huang JF. Huang XQ. Li XY. Huang MQ. Zhu HC. Li GP. Lan ML. Yu ZW. Xu W. Wu SS. et al. Alismatis Rhizoma Triterpenes Alleviate High-Fat Diet-Induced Insulin Resistance in the Skeletal Muscle of Mice. Evidence-Based Complementary and Alternative Medicine. 2021. https://doi.org/10.1155/2021/8857687
54. Li M. Zhang Y. Cao Y. Zhang D. Liu L. Guo Y et al. Wang C. Icariin ameliorates Palmitic Acid-Induced Insulin Resistance by Reducing Thioredoxin-Interacting Protein (TXNIP) and Suppressing ER Stress in C2C12 Myotubes. Frontiers in Pharmacology. 2018; 9(OCT). https://doi.org/10.3389/fphar.2018.01180
55. Chen J. Zhu J. Meng X. Aronia melanocarpa Anthocyanin Extracts are an Effective Regulator Of Suppressor Of Cytokine Signaling 3-Dependent Insulin Resistance in HepG2 and C2C12 Cells. Journal of Functional Foods. 2020; 75(September): 104258. https://doi.org/10.1016/j.jff.2020.104258
56. Vandanmagsar B. Yu Y. Simmler C. Dang TN. Kuhn P. Poulev A. Ribnicky DM. Pauli GF. Floyd ZE et al. Bioactive Compounds from Artemisia dracunculus L. Activate AMPK Signaling in Skeletal Muscle. Biomedicine and Pharmacotherapy. 2021; 143(06). https://doi.org/10.1016/j.biopha.2021.112188
57. Chuang WT. Yen CC. Huang CS. Chen HW. Lii CK et al. Benzyl Isothiocyanate Ameliorates High-Fat Diet-Induced Hyperglycemia by Enhancing Nrf2-Dependent Antioxidant Defense-Mediated IRS-1/AKT/TBC1D1 Signaling and GLUT4 Expression in Skeletal Muscle. Journal of Agricultural and Food Chemistry. 2020; 68(51): 15228–15238. https://doi.org/10.1021/acs.jafc.0c06269
58. Fujiwara Y. Tsukahara C. Ikeda N. Sone Y. Ishikawa T. Ichi I. Koike T. Aoki Y et al. Oleuropein Improves Insulin Resistance in Skeletal Muscle by Promoting The Translocation of GLUT4. Journal of Clinical Biochemistry and Nutrition. 2017; 61(3): 196–202. https://doi.org/10.3164/jcbn.166120
59. Park JM. Lee JS. Song JE. Sim YC. Ha SJ. Hong EK. Cytoprotective Effect of Hispidin Against Palmitic Acid-Induced Lipotoxicity in C2C12 Myotubes. Molecules. 2015; 20(4): 5456–5467. https://doi.org/10.3390/molecules20045456
60. Feng XT. Wang TZ. Chen Y. Liu JB. Liu Y. Wang WJ et al. Pollen typhae Total Flavone Improves Insulin-Induced Glucose Uptake Through the Β-Arrestin-2-Mediated Signaling In C2C12 Myotubes. International Journal of Molecular Medicine. 2012; 30(4): 914–922. https://doi.org/10.3892/ijmm.2012.1061
61. Li S. Li H. Yang D. Yu X. Irwin DM. Niu G. Tan H. Excessive Autophagy Activation and Increased Apoptosis Are Associated with Palmitic Acid-Induced Cardiomyocyte Insulin Resistance. Journal of Diabetes Research. 2017. https://doi.org/10.1155/2017/2376893