Upregulation of MRNA TNF-α in Skeletal Muscle Tissue of Streptozotocin-Induced Diabetic RAT

  • Akbar Satria Fitriawan Universitas Respati Yogyakarta
  • Christin Wiyani Universitas Respati Yogyakarta
  • Endang Nurul Syafitri Universitas Respati Yogyakarta
  • Ririn Wahyu Widayati Universitas Respati Yogyakarta
Keywords: diabetes mellitus, TNF-α, inflammation, skeletal muscle


Inflammation is a molecular mechanism that linking obesity and ageing with insulin resistance in type 2 diabetes mellitus (DM). Although type 1 DM is primarily caused by insulin deficiency, but insulin resistance also prominent feature in this disease. It is not fully understood whether inflammation also contribute in insulin resistance phenotype in type 1 DM. This study aimed to assess mRNA TNF-α expression in the skeletal muscle tissue of type 1 diabetes mellitus rat model.This in-vivo study used 18 adults male wistar rats. The study conducted at Department of Anatomy, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada from July to October 2020. Male Wistar rats divided into control group (CDM, n=6), and diabetes mellitus group which is divided into 1-month DM group (DM1M, n=6), and 2-month DM group (DM2M, n=6). The DM model was conducted through single intraperitoneal injection of Streptozotocin 60 mg/kg Body Weight (BW). At the end of study, rats were sacrificed and the gastrocnemius muscle was harvested. The expression of mRNA TNF-α was measured by RT-PCR. Statistical analysis was conducted using One Way ANOVA test. Blood glucose level were significantly higher in DM groups compared to control group (p<0.05). The body weight of DM groups was significantly lower after 1 month and 2 months DM period compared to control group (p<0.05). DM groups demonstrated upregulation of mRNA TNF-α compared to control group (p<0.05). Type 1 diabetes mellitus model demonstrated upregulation of mRNA TNF-α in skeletal muscle tissue.


Akbarzadeh, A., Norouzian, D., Mehrabi, M. R., Jamshidi, S., Farhangi, A., Allah Verdi, A., Lame Rad, B. (2007). Induction of diabetes by Streptozotocin in rats. Indian Journal of Clinical Biochemistry, 22(2), 60–64. https://doi.org/10.1007/BF02913315
Al-Goblan, A. S., Al-Alfi, M. A., & Khan, M. Z. (2014). Mechanism linking diabetes mellitus and obesity. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 7, 587–591. https://doi.org/10.2147/DMSO.S67400
Association, A. D. (2014). Standards of medical care in diabetes-2014. Diabetes Care, 37(SUPPL.1), 14–80. https://doi.org/10.2337/dc14-S014
Chaturvedi, N., Sjoelie, A. K., Porta, M., Aldington, S. J., Fuller, J. H., Songini, M., & Kohner, E. M. (2001). Markers of insulin resistance are strong risk factors, for retinopathy incidence in type 1 diabetes: The EURODIAB prospective complications study. Diabetes Care, 24(2), 284–289. https://doi.org/10.2337/diacare.24.2.284
Chawla, A., Chawla, R., & Jaggi, S. (2016). Microvasular and macrovascular complications in diabetes mellitus : Distinct or continuum ? Indian Journal of Endocrinology and Metabolism, 20(4), 546–551. https://doi.org/10.4103/2230-8210.183480
Chen, E., Tsai, T. H., Lan, L., Saha, P., Chan, L., & Chang, B. H. (2017). PLIN2 is a Key Regulator of the Unfolded Protein Response and Endoplasmic Reticulum Stress Resolution in Pancreatic β Cells. Nature Publishing Group, (December 2016), 1–12. https://doi.org/10.1038/srep40855
Coll, T., Barroso, E., & Palomer, X. (2013). Oleate prevents saturated-fatty-acid-induced ER stress , inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism. Diabetologia, 56, 1372–1382. https://doi.org/10.1007/s00125-013-2867-3
Corrêa-Silva, S., Alencar, A. P., Moreli, J. B., Borbely, A. U., de S. Lima, L., Scavone, C., Calderon, I. M. P. (2018). Hyperglycemia induces inflammatory mediators in the human chorionic villous. Cytokine, 111(May), 41–48. https://doi.org/10.1016/j.cyto.2018.07.020
Damasceno, D. C., Netto, A. O., Iessi, I. L., Gallego, F. Q., Corvino, S. B., Dallaqua, B., … Rudge, M. V. C. (2014). Streptozotocin-induced diabetes models: Pathophysiological mechanisms and fetal outcomes. BioMed Research International, 2014. https://doi.org/10.1155/2014/819065
Donga, E., Dekkers, O. M., Corssmit, E. P. M., & Romijn, J. A. (2015). Insulin resistance in patients with type 1 diabetes assessed by glucose clamp studies: Systematic review and meta-analysis. European Journal of Endocrinology, 173(1), 101–109. https://doi.org/10.1530/EJE-14-0911
Elias, D., Prigozin, H., Polak, N., Rapoport, M., & Lohse, A. W. (1994). Autoimmune Diabetes Induced by the p-Cell Toxin STZ. Diabetes, 43(August), 992–998.
Finkel, T., & Holbrook, N. J. (2000). Oxidants, oxidative stress, and the biology of ageing. Nature, 408(November), 239–247.
Fitriawan, A. S., Widayati, R. W., Setyaningsih, W. A. W., Arfian, N., & Sari, D. C. R. (2019). Antidiabetic and hypolipidemic effect of centella asiatica extract in streptozotocin-induced diabetic rats. In Healthy and Active Aging (pp. 9–25).
Fresno, M., Alvarez, R., & Cuesta, N. (2011). Toll-like receptors, inflammation, metabolism and obesity. Archives of Physiology and Biochemistry, 117(February), 151–164. https://doi.org/10.3109/13813455.2011.562514
Garg, A. D., Kaczmarek, A., Krysko, O., Vandenabeele, P., Krysko, D. V, & Agostinis, P. (2012). ER stress-induced inflammation : does it aid or impede disease progression ? Trends in Molecular Medicine, 18(10), 589–598. https://doi.org/10.1016/j.molmed.2012.06.010
Gayathri, V., Lekshmi, P., & Padmanabhan, R. N. (2011). Anti-diabetes activity of ethanol extract of Centella asiatica (L.) urban (whole plant) in Streptozotocin-induced diabetic rats, isolation of an active fraction and toxicity evaluation of the extract. International Journal of Medicinal and Aromatic Plants, 1(3), 278–286.
Hackett, E., & Jacques, N. (2009). Type 2 diabetes pathophysiology and clinical features. Clinical Pharmacist, 475(December), 475–478. Retrieved from https://www.pharmaceutical-journal.com/files/rps-pjonline/pdf/cp200912_diabetes_features-475.pdf
Holman, N., Young, B., & Gadsby, R. (2015). Current prevalence of Type 1 and type 2 diabetes in adults and children in the UK. DIABETICMedicine DOI:, 32, 1119–1120. https://doi.org/10.1111/dme.12791
Hu, P., Han, Z., Couvillon, A. D., Kaufman, R. J., Exton, J. H. (2006). Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through ire1-mediated NFκB activation and down-regulation of TRAF2 expression. Molecular and Cellular Biology, 26(8), 3071–3084. https://doi.org/10.1128/MCB.26.8.3071
Ikebukuro, K., Adachi, Y., Yamada, Y., Fujimoto, S., Seino, Y., Oyaizu, H., Ikehara, S. (2002). Treatment of streptozotocin-induced diabetes mellitus by transplantation of islet cells plus bone marrow cells via portal vein in rats. Transplantation, 73(4), 512–518. https://doi.org/10.1097/00007890-200202270-00004
Ingerslev, B., Hansen, J. S., Hoffmann, C., Clemmesen, J. O., Secher, N. H., Scheler, M., Plomgaard, P. (2017). Angiopoietin-like protein 4 is an exercise-induced hepatokine in humans, regulated by glucagon and cAMP. Molecular Metabolism, 6(10), 1286–1295. https://doi.org/10.1016/j.molmet.2017.06.018
Kabir, A. U., Samad, M. Bin, Costa, N. M. D., Akhter, F., Ahmed, A., & Hannan, J. M. A. (2014). Anti-hyperglycemic activity of Centella asiatica is partly mediated by carbohydrase inhibition and glucose-fiber binding.
Kacerovsky, M., Brehm, A., Chmelik, M., Schmid, A. I., Szendroedi, J., Kacerovsky-Bielesz, G., Roden, M. (2011). Impaired insulin stimulation of muscular ATP production in patients with type 1 diabetes. Journal of Internal Medicine, 269(2), 189–199. https://doi.org/10.1111/j.1365-2796.2010.02298.x
Kahanovitz, L., Sluss, P. M., & Russell, S. J. (2017). Type 1 diabetes-a clinical perspective. Point of Care, 16(1), 37–40. https://doi.org/10.1097/POC.0000000000000125
Karpe, F., Dickmann, J. R., & Frayn, K. N. (2011). Fatty acids, obesity, and insulin resistance: time for a reevaluation. Perspectives in Diabetes, 60(October), 2441–2449. https://doi.org/10.2337/db11-0425
Lee, J. Y., & Hwang, D. H. (2006). The modulation of inflammatory gene expression by lipids: Mediation through toll-like receptors. Molecules and Cells, 21(2), 174–185.
Lenzen, S. (2008). The mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia, 51(2), 216–226. https://doi.org/10.1007/s00125-007-0886-7
Lin, Y., Berg, A. H., Iyengar, P., Lam, T. K. T., Giacca, A., Combs, T. P., Scherer, P. E. (2005). The hyperglycemia-induced inflammatory response in adipocytes: the role of reactive oxygen species. Journal of Biological Chemistry, 280(6), 4617–4626. https://doi.org/10.1074/jbc.M411863200
Liu, H., Sidiropoulos, P., Song, G., Lisa, J., Birrer, M. J., Stein, B., … Pope, R. M. (2002). TNF-α gene expression in macrophages: regulation by NFκB is independent of c-Jun or C/EBP β. Journal of Immunology, 164, 4277–4285. https://doi.org/10.4049/jimmunol.164.8.4277
Mali, K. K., Dias, R. J., Havaldar, V. D., & Yadav, S. J. (2017). Antidiabetic effect of garcinol on streptozotocin-induced diabetic rats. Indian Journal of Pharmaceutical Sciences, 79(3), 463–468. https://doi.org/10.4172/pharmaceutical-sciences.1000250
Mead, J. R., Irvine, S. A., & Ramji, D. P. (2002). Lipoprotein lipase: structure, function, regulation, and role in disease. Journal of Molecular Medicine, 80(12), 753–769. https://doi.org/10.1007/s00109-002-0384-9
Morey, M., O’Gaora, P., Pandit, A., & Hélary, C. (2019). Hyperglycemia acts in synergy with hypoxia to maintain the pro-inflammatory phenotype of macrophages. PLoS ONE, 14(8), 1–17. https://doi.org/10.1371/journal.pone.0220577
Morigny, P., Houssier, M., Mouisel, E., & Langin, D. (2016). Adipocyte lipolysis and insulin resistance. Biochimie, 125, 259–266. https://doi.org/10.1016/j.biochi.2015.10.024
Naidu, S. S. and M. D. (2019). Evaluation of protective effect of Centella asiatica leaves on pancreas function in diabetic rats. International Journal of Herbal Medicine, 7(1), 55–60.
Nguyen, M. T. A., Satoh, H., Favelyukis, S., Babendure, J. L., Imamura, T., Sbodio, J. I., Olefsky, J. M. (2005). JNK and tumor necrosis factor-α mediate free fatty acid-induced insulin resistance in 3T3-L1 adipocytes. Journal of Biological Chemistry, 280(42), 35361–35371. https://doi.org/10.1074/jbc.M504611200
Orchard, T., Olson, J., Erbey, J., & Williams, K. (2003). Insulin resistance–related factors, but not glycemia, predict coronary artery disease in type 1 diabetes. Diabetes, 26(5), 1374–1379. Retrieved from http://care.diabetesjournals.org/content/26/5/1374.short
Pahl, H. L. (1999). Activators and target genes of Rel/NF-κB transcription factors. Oncogene, 18(49), 6853–6866. https://doi.org/10.1038/sj.onc.1203239
Parameswaran, N., & Patial, S. (2010). Tumor necrosis factor-α signaling in macrophages. Critical Reviews in Eukaryotic Gene Expression, 20(2), 87–103. https://doi.org/10.1615/CritRevEukarGeneExpr.v20.i2.10
Portha, B., Levacher, C., Picon, L., & Rosselin, G. (1974). Diabetogenic effect of streptozotocin in the rat during the perinatal period. Diabetes, 23(11), 889–895. https://doi.org/10.2337/diab.23.11.889
Pournaghi, P., Sadrkhanlou, R.A., Hasanzadeh, S., & Foroughi, A. (2012). An investigation on body weights, blood glucose levels and pituitary-gonadal axis hormones in diabetic and metformin-treated diabetic female rats. Veterinary Research Forum : An International Quarterly Journal, 3(2), 79–84. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/25653751%0Ahttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC4312800
Rajamani, U., & Jialal, I. (2014). Hyperglycemia induces toll-like receptor-2 and -4 expression and activity in human microvascular retinal endothelial cells: Implications for diabetic retinopathy. Journal of Diabetes Research, 2014, 7–10. https://doi.org/10.1155/2014/790902
Rastellini, C., Shapiro, R., Corry, R., Fung, J. J., Starzl, T. E., & Rao, A. S. (1997). An attempt to reverse diabetes by delayed islet cell transplantation in humans. Transplant Proc. 1997, 29(4), 2238–2239.
Roden, M., Price, T. B., Perseghin, G., Petersen, K. F., Rothman, D. L., Cline, G. W., & Shulman, G. I. (1996). Mechanism of free fatty acid-induced insulin resistance in humans. Journal of Clinical Investigation, 97(12), 2859–2865. https://doi.org/10.1172/JCI118742
Saeedi, P., Petersohn, I., Salpea, P., Malanda, B., Karuranga, S., Unwin, N., Bright, D. (2019). Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045 : Results from the International Diabetes Federation Diabetes Atlas , 9 th edition. Diabetes Research and Clinical Practice, 157, 107843. https://doi.org/10.1016/j.diabres.2019.107843
Sasikala, S., Lakshminarasaiah, S., & Naidu, M. D. (2015). Antidiabetic activity of Centella asiatica on streptozotocin induced diabetic male albino rats. World Journal of Pharmaceutical Science, 3(8), 2321–3086.
Shi, H., Kokoeva, M. V, Inouye, K., Tzameli, I., Yin, H., & Flier, J. S. (2006). TLR4 links innate immunity and fatty acid – induced insulin resistance. The Journal of Clinical Investigation, 116(11), 3015–3025. https://doi.org/10.1172/JCI28898.TLRs
Steinberg, G. R., Michell, B. J., van Denderen, B. J. W., Watt, M. J., Carey, A. L., Fam, B. C., Kemp, B. E. (2006). Tumor necrosis factor α-induced skeletal muscle insulin resistance involves suppression of AMP-kinase signaling. Cell Metabolism, 4(6), 465–474. https://doi.org/10.1016/j.cmet.2006.11.005
Taniguchi, C. M., Emanuelli, B., & Kahn, C. R. (2006). Critical nodes in signalling pathways : insights into insulin action, 7(February), 85–96. https://doi.org/10.1038/nrm1837
Tanti, J. F., Ceppo, F., Jager, J., & Berthou, F. (2013). Implication of inflammatory signaling pathways in obesity-induced insulin resistance. Frontiers in Endocrinology, 3(JAN), 1–15. https://doi.org/10.3389/fendo.2012.00181
Weiss, R. B. (1982). Streptozocin: a review of its pharmacology, efficacy, and toxicity. Cancer Treat Rep, 66(3), 427–438.
Wu, J., & Yan, L. J. (2015). Streptozotocin-induced type 1 diabetes in rodents as a model for studying mitochondrial mechanisms of diabetic β cell glucotoxicity. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 8, 181–188. https://doi.org/10.2147/DMSO.S82272
Zhanga, Y., Li, X., Zhang, H., Zhao, Z., Peng, Z., Wang, Z., Li, X. (2018). Non-esterified fatty acids over-activate the tlr2 / 4-nf-κb signaling pathway to increase inflammatory cytokine synthesis in neutrophils from ketotic cows. Cellular Physiology and Biochemistry, 48, 827–837. https://doi.org/10.1159/000491913
Zheng, Y., Ley, S. H., & Hu, F. B. (2017). Global aetiology and epidemiology of type 2 diabetes mellitus and its complications. Nature Publishing Group, 14(2), 88–98. https://doi.org/10.1038/nrendo.2017.151