Cell models for studying muscle insulin resistance
Keywords:
insulin resistance, cell model, muscle, cell line, treatment, concentration
Abstract
Type 2 diabetes is one of the most prevalent chronic diseases in the world today. Insulin resistance – a reduced responsiveness of tissues to insulin - is a hallmark of type 2 diabetes pathology. Skeletal muscle plays a pivotal role in glucose homeostasis - it is responsible for the majority of insulin-mediated glucose disposal and thus is one of the tissues most affected by insulin resistance.
To study the molecular mechanisms of a disease, researchers often turn to cell models – they are cheap, easy to use, and exist in a controlled environment with few unknown variables. Cell models for exploring muscle insulin resistance are constructed using primary cell cultures or immortalised cell lines and treating them with fatty acids, high insulin or high glucose. The choice of cell culture, concentration and duration of the treatment and the methods for measuring insulin sensitivity in order to confirm the model are rarely discussed. Choosing an appropriate and physiologically relevant model for a particular topic of interest is required in order for the results to be reproducible, relevant and translatable to more complex biological systems. Cell models enable research that would otherwise be inaccessible but, especially when studying human disease, they do not serve a purpose if they are not in line with the biological reality.
This review aims to summarise and critically evaluate the most commonly used cell models of muscle insulin resistance: the rationale for choosing these exact treatments and conditions, the protocols for constructing the models and the measurable outcomes used for confirming insulin resistance in the cells.
References
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6. Danoviz ME, Yablonka-Reuveni Z. Skeletal muscle satellite cells: background and methods for isolation and analysis in a primary culture system. In: Myogenesis. Totowa, NJ: Humana Press; 2012. p. 21–52.
7. Chavez JA, Summers SA. Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes. Arch Biochem Biophys. 2003 Nov 15;419(2):101–9.
8. Home Feed | ResearchGate [Internet]. Available from: https://www.researchgate.net/
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13. Roden M, Krssak M, Stingl H, Gruber S, Hofer A, Fürnsinn C, et al. Rapid impairment of skeletal muscle glucose transport/phosphorylation by free fatty acids in humans. Diabetes. 1999;48(2):358–64.
14. Yu C, Chen Y, Cline GW, Zhang D, Zong H, Wang Y, et al. Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem. 2002;277(52):50230–6.
15. Bonnard C, Durand A, Peyrol S, Chanseaume E, Chauvin M-A, Morio B, et al. Mitochondrial dysfunction results from oxidative stress in the skeletal muscle of diet-induced insulin-resistant mice. J Clin Invest. 2008;118(2):789–800.
16. Tamilarasan KP, Temmel H, Das SK, Al Zoughbi W, Schauer S, Vesely PW, et al. Skeletal muscle damage and impaired regeneration due to LPL-mediated lipotoxicity. Cell Death Dis. 2012;3(7):e354-8.
17. Krssak M, Petersen KF, Dresner A, Dipietro L, Vogel SM, Rothman DL, et al. Intramyocelular lipid concentrations are correlated with insulin sensitivity in humans: a 1H NMR spectroscopy study. Diabetologia. 1999;42(1):113–6.
18. Rifai N, Horvath AR, Wittwer CT E. Tietz fundamentals of clinical chemistry and molecular diagnostics. 8th ed. St. Louis, Missouri: Elsevier; 2019.
19. Richieri G V., Kleinfeld AM. Unbound free fatty acid levels in human serum. J Lipid Res. 1995;36(2):229–40.
20. Huang C, Somwar R, Patel N, Niu W, Török D, Klip A. Sustained exposure of L6 myotubes to high glucose and insulin decreases insulin-stimulated GLUT4 translocation but upregulates GLUT4 activity. Diabetes. 2002;51(7):2090–8.
21. C2C12 ATCC ® CRL-1772TM Mus musculus muscle [Internet]. Available from: https://www.lgcstandards-atcc.org/Products/All/CRL-1772.aspx?geo_country=rs#
22. Wiese RJ, Mastick CC, Lazar DF, Saltiel AR. Activation of mitogen-activated protein kinase and phosphatidylinositol 3’-kinase is not sufficient for the hormonal stimulation of glucose uptake, lipogenesis, or glycogen synthesis in 3T3-L1 adipocytes. Vol. 270, Journal of Biological Chemistry. 1995. p. 3442–6.
23. Yamamoto N, Ueda-Wakagi M, Sato T, Kawasaki K, Sawada K, Kawabata K, et al. Measurement of glucose uptake in cultured cells. Curr Protoc Pharmacol. 2015;71(1):12–4.
24. Hess SL, Suchin CR, Saltiel AR. The specific protein phosphatase inhibitor okadaic acid differentially modulates insulin action. J Cell Biochem. 1991;45(4):374–80.
25. Kumar N, Dey CS. Development of insulin resistance and reversal by thiazolidinediones in C2C12 skeletal muscle cells. Biochem Pharmacol. 2003;65(2):249–57.
26. Vlavcheski F, Den Hartogh DJ, Giacca A, Tsiani E. Amelioration of high-insulin-induced skeletal muscle cell insulin resistance by resveratrol is linked to activation of AMPK and restoration of GLUT4 translocation. Nutrients. 2020;12(4):914.
27. Wu W, Tang S, Shi J, Yin W, Cao S, Bu R, et al. Metformin attenuates palmitic acid-induced insulin resistance in L6 cells through the AMP-activated protein kinase/sterol regulatory element-binding protein-1c pathway. Int J Mol Med. 2015;35(6):1734–40.
28. Yang M, Wei D, Mo C, Zhang J, Wang X, Han X, et al. Saturated fatty acid palmitate-induced insulin resistance is accompanied with myotube loss and the impaired expression of health benefit myokine genes in C2C12 myotubes. Lipids Health Dis. 2013;12(1):104.
29. Meshkani R, Sadeghi A, Taheripak G, Zarghooni M, Gerayesh-Nejad S, Bakhtiyari S. Rosiglitazone, a PPARγ agonist, ameliorates palmitate-induced insulin resistance and apoptosis in skeletal muscle cells. Cell Biochem Funct. 2014;32(8):683–91.
30. Hirabara SM, Curi R, Maechler P. Saturated fatty acid-induced insulin resistance is associated with mitochondrial dysfunction in skeletal muscle cells. J Cell Physiol. 2010;222(1):187–94.
31. Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, Jung SY, et al. SIN1/MIP1 Maintains rictor-mTOR Complex Integrity and Regulates Akt Phosphorylation and Substrate Specificity. Cell. 2006 Oct 6;127(1):125–37.
32. Beg M, Abdullah N, Thowfeik FS, Altorki NK, McGraw TE. Distinct Akt phosphorylation states are required for insulin regulated Glut4 and Glut1-mediated glucose uptake. Vol. 6, eLife. 2017.
33. Sarbassov DD. Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR Complex. Science (80- ). 2005;307(5712):1098–101.
34. Frech M, Andjelkovic M, Ingley E, Reddy KK, Falck JR, Hemmings BA. High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity. J Biol Chem. 1997 Mar 28;272(13):8474–81.
35. Yang C, Aye CC, Li X, Diaz Ramos A, Zorzano A, Mora S. Mitochondrial dysfunction in insulin resistance: differential contributions of chronic insulin and saturated fatty acid exposure in muscle cells. Biosci Rep. 2012;32(5):465–78.
36. Coll T, Eyre E, Rodríguez-Calvo R, Palomer X, Sánchez RM, Merlos M, et al. Oleate reverses palmitate-induced insulin resistance and inflammation in skeletal muscle cells. J Biol Chem. 2008;283(17):11107–16.
37. Henique C, Mansouri A, Fumey G, Lenoir V, Girard J, Bouillaud F, et al. Increased mitochondrial fatty acid oxidation is sufficient to protect skeletal muscle cells from palmitate-induced apoptosis. J Biol Chem. 2010;285(47):36818–27.
38. Kwon B, Querfurth HW. Palmitate activates mTOR/p70S6K through AMPK inhibition and hypophosphorylation of raptor in skeletal muscle cells: Reversal by oleate is similar to metformin. Biochimie. 2015;118:141–50.
39. Yoneyama Y, Inamitsu T, Chida K, Iemura SI, Natsume T, Maeda T, et al. Serine Phosphorylation by mTORC1 Promotes IRS-1 Degradation through SCFβ-TRCP E3 Ubiquitin Ligase. iScience. 2018 Jul 27;5:1–18.
40. Gual P, Grémeaux T, Gonzalez T, Le Marchand-Brustel Y, Tanti JF. MAP kinases and mTOR mediate insulin-induced phosphorylation of Insulin Receptor Substrate-1 on serine residues 307, 612 and 632. Diabetologia. 2003 Nov;46(11):1532–42.
41. Zhang J, Gao Z, Yin J, Quon MJ, Ye J. S6K directly phosphorylates IRS-1 on Ser-270 to promote insulin resistance in response to TNF-α signaling through IKK2. J Biol Chem. 2008 Dec 19;283(51):35375–82.
42. MohammadTaghvaei N, Meshkani R, Taghikhani M, Larijani B, Adeli K. Palmitate enhances protein tyrosine phosphatase 1B (PTP1B) gene expression at transcriptional level in C2C12 skeletal muscle cells. Inflammation. 2011;34(1):43–8.
43. MohammadTaghvaei N, Taheripak G, Taghikhani M, Meshkani R. Palmitate-induced PTP1B expression is mediated by ceramide-JNK and nuclear factor κB (NF-κB) activation. Cell Signal. 2012;24(10):1964–70.
44. Warmington SA, Tolan R, McBennett S. Functional and histological characteristics of skeletal muscle and the effects of leptin in the genetically obese (ob/ob) mouse. Int J Obes. 2000;24(8):1040–50.
45. Tanner CJ, Barakat HA, Lynis Dohm G, Pories WJ, MacDonald KG, Cunningham PRG, et al. Muscle fiber type is associated with obesity and weight loss. Am J Physiol - Endocrinol Metab. 2002;282(6):E1191–6.
46. Tumova J, Malisova L, Andel M, Trnka J. Protective effect of unsaturated fatty acids on palmitic acid-induced toxicity in skeletal muscle cells is not mediated by PPARδ activation. Lipids. 2015;50(10):955–64.
47. Xiong CJ, Li PF, Song YL, Xue LX, Jia ZQ, Yao CX, et al. Insulin induces C2C12 cell proliferation and apoptosis through regulation of cyclin D1 and BAD expression. J Cell Biochem. 2013;114(12):2708–17.
48. Chen F, Qian LH, Deng B, Liu ZM, Zhao Y, Le YY. Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of nadph oxidase activation-driven oxidative stress. CNS Neurosci Ther. 2013;19(9):675–81.
49. Cai L, Li W, Wang G, Guo L, Jiang Y, Kang YJ. Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C–mediated caspase-3 activation pathway. Diabetes. 2002;51(6):1938–48.
2. Defronzo RA, Jacot E, Jequier E, Maeder E, Wahren J, Felber JP. The effect of insulin on the disposal of intravenous glucose: results from indirect calorimetry and hepatic and femoral venous catheterization. Diabetes. 1981;30(12):1000–7.
3. Partridge TA. Tissue culture of skeletal muscle. In: Basic Cell Culture Protocols. Totowa, NJ: Humana Press; 1997. p. 131–44.
4. Neville C, Rosenthal N, McGrew M, Bogdanova N, Hauschka S. Skeletal muscle cultures. In: Methods in Cell Biology. Cambridge, MA: Academic Press Inc.; 1997. p. 85–116.
5. Eberli D, Soker S, Atala A, Yoo JJ. Optimization of human skeletal muscle precursor cell culture and myofiber formation in vitro. Methods. 2009;47(2):98–103.
6. Danoviz ME, Yablonka-Reuveni Z. Skeletal muscle satellite cells: background and methods for isolation and analysis in a primary culture system. In: Myogenesis. Totowa, NJ: Humana Press; 2012. p. 21–52.
7. Chavez JA, Summers SA. Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes. Arch Biochem Biophys. 2003 Nov 15;419(2):101–9.
8. Home Feed | ResearchGate [Internet]. Available from: https://www.researchgate.net/
9. Shanik MH, Xu Y, Skrha J, Dankner R, Zick Y, Roth J. Insulin resistance and hyperinsulinemia: is hyperinsulinemia the cart or the horse? Diabetes Care. 2008;31(Supplement 2):S262-8.
10. Furnica RM, Istasse L, Maiter D. A severe but reversible reduction in insulin sensitivity is observed in patients with insulinoma. Ann Endocrinol (Paris). 2018;79(1):30–6.
11. Baldeweg SE, Golay A, Natali A, Balkau B, Del Prato S, Coppack SW. Insulin resistance, lipid and fatty acid concentrations in 867 healthy Europeans. Eur J Clin Invest. 2000;30(1):45–52.
12. Belfort R, Mandarino L, Kashyap S, Wirfel K, Pratipanawatr T, Berria R, et al. Dose-response effect of elevated plasma free fatty acid on insulin signaling. Diabetes. 2005;54(6):1640–8.
13. Roden M, Krssak M, Stingl H, Gruber S, Hofer A, Fürnsinn C, et al. Rapid impairment of skeletal muscle glucose transport/phosphorylation by free fatty acids in humans. Diabetes. 1999;48(2):358–64.
14. Yu C, Chen Y, Cline GW, Zhang D, Zong H, Wang Y, et al. Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem. 2002;277(52):50230–6.
15. Bonnard C, Durand A, Peyrol S, Chanseaume E, Chauvin M-A, Morio B, et al. Mitochondrial dysfunction results from oxidative stress in the skeletal muscle of diet-induced insulin-resistant mice. J Clin Invest. 2008;118(2):789–800.
16. Tamilarasan KP, Temmel H, Das SK, Al Zoughbi W, Schauer S, Vesely PW, et al. Skeletal muscle damage and impaired regeneration due to LPL-mediated lipotoxicity. Cell Death Dis. 2012;3(7):e354-8.
17. Krssak M, Petersen KF, Dresner A, Dipietro L, Vogel SM, Rothman DL, et al. Intramyocelular lipid concentrations are correlated with insulin sensitivity in humans: a 1H NMR spectroscopy study. Diabetologia. 1999;42(1):113–6.
18. Rifai N, Horvath AR, Wittwer CT E. Tietz fundamentals of clinical chemistry and molecular diagnostics. 8th ed. St. Louis, Missouri: Elsevier; 2019.
19. Richieri G V., Kleinfeld AM. Unbound free fatty acid levels in human serum. J Lipid Res. 1995;36(2):229–40.
20. Huang C, Somwar R, Patel N, Niu W, Török D, Klip A. Sustained exposure of L6 myotubes to high glucose and insulin decreases insulin-stimulated GLUT4 translocation but upregulates GLUT4 activity. Diabetes. 2002;51(7):2090–8.
21. C2C12 ATCC ® CRL-1772TM Mus musculus muscle [Internet]. Available from: https://www.lgcstandards-atcc.org/Products/All/CRL-1772.aspx?geo_country=rs#
22. Wiese RJ, Mastick CC, Lazar DF, Saltiel AR. Activation of mitogen-activated protein kinase and phosphatidylinositol 3’-kinase is not sufficient for the hormonal stimulation of glucose uptake, lipogenesis, or glycogen synthesis in 3T3-L1 adipocytes. Vol. 270, Journal of Biological Chemistry. 1995. p. 3442–6.
23. Yamamoto N, Ueda-Wakagi M, Sato T, Kawasaki K, Sawada K, Kawabata K, et al. Measurement of glucose uptake in cultured cells. Curr Protoc Pharmacol. 2015;71(1):12–4.
24. Hess SL, Suchin CR, Saltiel AR. The specific protein phosphatase inhibitor okadaic acid differentially modulates insulin action. J Cell Biochem. 1991;45(4):374–80.
25. Kumar N, Dey CS. Development of insulin resistance and reversal by thiazolidinediones in C2C12 skeletal muscle cells. Biochem Pharmacol. 2003;65(2):249–57.
26. Vlavcheski F, Den Hartogh DJ, Giacca A, Tsiani E. Amelioration of high-insulin-induced skeletal muscle cell insulin resistance by resveratrol is linked to activation of AMPK and restoration of GLUT4 translocation. Nutrients. 2020;12(4):914.
27. Wu W, Tang S, Shi J, Yin W, Cao S, Bu R, et al. Metformin attenuates palmitic acid-induced insulin resistance in L6 cells through the AMP-activated protein kinase/sterol regulatory element-binding protein-1c pathway. Int J Mol Med. 2015;35(6):1734–40.
28. Yang M, Wei D, Mo C, Zhang J, Wang X, Han X, et al. Saturated fatty acid palmitate-induced insulin resistance is accompanied with myotube loss and the impaired expression of health benefit myokine genes in C2C12 myotubes. Lipids Health Dis. 2013;12(1):104.
29. Meshkani R, Sadeghi A, Taheripak G, Zarghooni M, Gerayesh-Nejad S, Bakhtiyari S. Rosiglitazone, a PPARγ agonist, ameliorates palmitate-induced insulin resistance and apoptosis in skeletal muscle cells. Cell Biochem Funct. 2014;32(8):683–91.
30. Hirabara SM, Curi R, Maechler P. Saturated fatty acid-induced insulin resistance is associated with mitochondrial dysfunction in skeletal muscle cells. J Cell Physiol. 2010;222(1):187–94.
31. Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, Jung SY, et al. SIN1/MIP1 Maintains rictor-mTOR Complex Integrity and Regulates Akt Phosphorylation and Substrate Specificity. Cell. 2006 Oct 6;127(1):125–37.
32. Beg M, Abdullah N, Thowfeik FS, Altorki NK, McGraw TE. Distinct Akt phosphorylation states are required for insulin regulated Glut4 and Glut1-mediated glucose uptake. Vol. 6, eLife. 2017.
33. Sarbassov DD. Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR Complex. Science (80- ). 2005;307(5712):1098–101.
34. Frech M, Andjelkovic M, Ingley E, Reddy KK, Falck JR, Hemmings BA. High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity. J Biol Chem. 1997 Mar 28;272(13):8474–81.
35. Yang C, Aye CC, Li X, Diaz Ramos A, Zorzano A, Mora S. Mitochondrial dysfunction in insulin resistance: differential contributions of chronic insulin and saturated fatty acid exposure in muscle cells. Biosci Rep. 2012;32(5):465–78.
36. Coll T, Eyre E, Rodríguez-Calvo R, Palomer X, Sánchez RM, Merlos M, et al. Oleate reverses palmitate-induced insulin resistance and inflammation in skeletal muscle cells. J Biol Chem. 2008;283(17):11107–16.
37. Henique C, Mansouri A, Fumey G, Lenoir V, Girard J, Bouillaud F, et al. Increased mitochondrial fatty acid oxidation is sufficient to protect skeletal muscle cells from palmitate-induced apoptosis. J Biol Chem. 2010;285(47):36818–27.
38. Kwon B, Querfurth HW. Palmitate activates mTOR/p70S6K through AMPK inhibition and hypophosphorylation of raptor in skeletal muscle cells: Reversal by oleate is similar to metformin. Biochimie. 2015;118:141–50.
39. Yoneyama Y, Inamitsu T, Chida K, Iemura SI, Natsume T, Maeda T, et al. Serine Phosphorylation by mTORC1 Promotes IRS-1 Degradation through SCFβ-TRCP E3 Ubiquitin Ligase. iScience. 2018 Jul 27;5:1–18.
40. Gual P, Grémeaux T, Gonzalez T, Le Marchand-Brustel Y, Tanti JF. MAP kinases and mTOR mediate insulin-induced phosphorylation of Insulin Receptor Substrate-1 on serine residues 307, 612 and 632. Diabetologia. 2003 Nov;46(11):1532–42.
41. Zhang J, Gao Z, Yin J, Quon MJ, Ye J. S6K directly phosphorylates IRS-1 on Ser-270 to promote insulin resistance in response to TNF-α signaling through IKK2. J Biol Chem. 2008 Dec 19;283(51):35375–82.
42. MohammadTaghvaei N, Meshkani R, Taghikhani M, Larijani B, Adeli K. Palmitate enhances protein tyrosine phosphatase 1B (PTP1B) gene expression at transcriptional level in C2C12 skeletal muscle cells. Inflammation. 2011;34(1):43–8.
43. MohammadTaghvaei N, Taheripak G, Taghikhani M, Meshkani R. Palmitate-induced PTP1B expression is mediated by ceramide-JNK and nuclear factor κB (NF-κB) activation. Cell Signal. 2012;24(10):1964–70.
44. Warmington SA, Tolan R, McBennett S. Functional and histological characteristics of skeletal muscle and the effects of leptin in the genetically obese (ob/ob) mouse. Int J Obes. 2000;24(8):1040–50.
45. Tanner CJ, Barakat HA, Lynis Dohm G, Pories WJ, MacDonald KG, Cunningham PRG, et al. Muscle fiber type is associated with obesity and weight loss. Am J Physiol - Endocrinol Metab. 2002;282(6):E1191–6.
46. Tumova J, Malisova L, Andel M, Trnka J. Protective effect of unsaturated fatty acids on palmitic acid-induced toxicity in skeletal muscle cells is not mediated by PPARδ activation. Lipids. 2015;50(10):955–64.
47. Xiong CJ, Li PF, Song YL, Xue LX, Jia ZQ, Yao CX, et al. Insulin induces C2C12 cell proliferation and apoptosis through regulation of cyclin D1 and BAD expression. J Cell Biochem. 2013;114(12):2708–17.
48. Chen F, Qian LH, Deng B, Liu ZM, Zhao Y, Le YY. Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of nadph oxidase activation-driven oxidative stress. CNS Neurosci Ther. 2013;19(9):675–81.
49. Cai L, Li W, Wang G, Guo L, Jiang Y, Kang YJ. Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C–mediated caspase-3 activation pathway. Diabetes. 2002;51(6):1938–48.