Abstract:

Chemicals have been shown to induce epigenetic changes that alter glucose metabolism genes, potentially leading to insulin resistance and increasing the risk of metabolic disorders like type 2 diabetes. This study was aimed to assess histopathological and epigenetic changes in insulin signalling molecules in STZ-induced type-2 diabetic rats and the possible therapeutic role of hesperidin. Hesperidin (100mg/kg b.wt) was administered to STZ-induced rats and assessed for its protective role and epigenetic mechanisms in the gastrocnemius muscle. Diabetic rats exhibited significant increase (p<0.05) in renal function markers such as urea (60, 140, 80, 70, and 79 mg/dL) and creatinine (0.9, 2, 1.2, 1.1, and 1.0 mg/dL), oxidative stress markers, while antioxidant enzymes such as superoxide dismutase (0.9, 0.5,0.8, 0.87 and U/mg protein) and catalase (1, 0.4,0.86, 0.92 and 1.13 U/mg protein) were markedly lower (p<0.05). Histopathological analysis revealed a decrease and disruption in muscle fibres. The mRNA expression of insulin signalling molecules PI3K (1, 0.6, 0.8, 1.1, and 1 fold) and Munc18 (1, 0.6, 0.8, 1, and 0.9) was significantly (p<0.01) reduced in diabetic groups. Epigenetic studies showed CpG island methylation in the promoter regions of GLUT4, Akt, and IR genes in diabetic rats. However, hesperidin treatment restored the detrimental changes caused by diabetogenic agent, streptozotocin. The present study concludes that hesperidin plays a central role in regulating epigenetic mechanisms of insulin signalling molecules and GLUT4 translocation in skeletal muscle and thereby protects the muscle cells.

References:

[1].  Herman, W. H., 2017, The Global Burden of Diabetes: An Overview. 10.1007/978-3-319-41559-8_1.

[2].  Pyrzynska, K., 2022, Hesperidin: A Review on Extraction Methods, Stability and Biological Activities. Nutrients, 14(12), 2387. doi: 10.3390/nu14122387

[3].  Jayaraman, S., Natarajan, S. R., Ponnusamy, B., Veeraraghavan, V. P. and Jasmine, S., 2023. Unlocking the potential of beta sitosterol: Augmenting the suppression of oral cancer cells through extrinsic and intrinsic signalling mechanisms. The Saudi Dental Journal, 35(8), pp.1007-1013.

[4].  Jayaraman, R., Subramani, S., Sheik Abdullah S. H., & Udaiyar M., 2018, Antihyperglycemic effect of hesperidin, a citrus flavonoid, extenuates hyperglycemia and exploring the potential role in antioxidant and antihyperlipidemic in streptozotocin-induced diabetic rats. Biomedicine and Pharmacotherapy, 97, 98-106. doi: 10.1016/j.biopha.2017.10.102.

[5].  Al-Ishaq, R. K., Abotaleb M., Kubatka P., Kajo K., & Büsselberg D., 2019, Flavonoids and Their Anti-Diabetic Effects: Cellular Mechanisms and Effects to Improve Blood Sugar Levels. Biomolecules, 9(9), 430. doi:10.3390/biom9090430.

[6].  Sundaram, R., Nandhakumar E., & Haseena B. H., 2019, Hesperidin, a citrus flavonoid ameliorates hyperglycemia by regulating key enzymes of carbohydrate metabolism in streptozotocin-induced diabetic rats, Toxicology Mechanisms and Methods, 29:9, 644-653.

[7].  de Souza P, da Silva R. C. V., Mariano, L. N. B., Dick, S. L., & Ventura GC, Cechinel-Filho V, 2022, Diuretic and Natriuretic Effects of Hesperidin, a Flavanone Glycoside, in Female and Male Hypertensive Rats. Plants (Basel), 21, 12(1):25.

[8].  Sruthi, M. A., Mani, G., Ramakrishnan, M. and Selvaraj, J., 2023, Dental caries as a source of Helicobacter pylori infection in children: An RT‐PCR study. International Journal of Paediatric Dentistry, 33(1), pp.82-88.

[9].  Peng, P., Jin J., Zou G., Sui Y., Han Y, Zhao D., & Liu L., 2021. Hesperidin prevents hyperglycemia in diabetic rats by activating the insulin receptor pathway. Experimental and Therapeutic Medicine, 21(1):53.

[10]. Mahmoud, A. M., Ahmed, O. M., Ashour, M. B., et al., 2015, In vivo and in vitro antidiabetic effects of citrus flavonoids; a study on the mechanism of action. International Journal of Diabetes in Developing Countries, 35(3), 250-263.

[11]. Krishnan, R. P., Pandiar, D., Ramani, P. and Jayaraman, S., 2025, Molecular profiling of oral epithelial dysplasia and oral squamous cell carcinoma using next generation sequencing. Journal of Stomatology, Oral and Maxillofacial Surgery, 126(4), p.102120.

[12]. Mahmoud, A. M., 2016, Hesperidin as a Promising Anti-Diabetic Flavonoid: the Underlying Molecular Mechanism. Int Journal of Food and Nutritional Science, 3, 313-314.

[13]. Holliday, R., 2006, Epigenetics: a historical overview. Epigenetics, 1: 76– 80.

[14]. Weinhold. B., 2006, Epigenetics: the science of change. Environ Health Perspect, 114(3), A160-7.

[15]. Berger, S. L., Kouzarides, T., Shiekhattar, R., Shilatifard, A., 2009, An operational definition of epigenetics. Genes & Development, 23(7), 781-3. doi: 10.1101/gad.1787609.

[16]. Jayaraman, S., Natarajan, S. R., Veeraraghavan, V. P. and Jasmine, S., 2023, Unveiling the anti-cancer mechanisms of calotropin: Insights into cell growth inhibition, cell cycle arrest, and metabolic regulation in human oral squamous carcinoma cells (HSC-3). Journal of Oral Biology and Craniofacial Research, 13(6), pp.704-713.

[17]. Moosavi, A., Motevalizadeh A. A., 2016, Role of Epigenetics in Biology and Human Diseases. Iranian Biomedical Journal, 20(5), 246-58. doi: 10.22045/ibj.2016.01.

[18]. Kawada, J., 1992, New hypotheses for the mechanisms of streptozotocin and alloxan inducing Diabetes mellitus. Yakugaku Zasshi, 112(11), 773-91. Japanese. doi: 10.1248/ yakushi1947. 112.11_773

[19]. Szkudelski, T., 2001, The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiological Research, 50(6):537-46.

[20]. Chen, S., Gan, D., Lin, S., Zhong, Y., Chen M., Zou X., Shao Z., & Xiao, G., 2022, Metformin in aging and aging-related diseases: clinical applications and relevant mechanisms. Theranostics, 12(6), 2722-2740. doi: 10.7150/thno.71360.

[21]. Gurina, T. S., Simms, L., Histology, Staining. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books /NBK557663/

[22]. Tahir, R. A., Zheng, D. A., Nazir, A., & Qing, H., 2019, A review of computational algorithms for CpG islands detection. Journal of Biosciences, 44(6), 143.

[23]. Patrick, P. L., Lam., Mitsuyo O., Subhankar D., Yu H., Tairan Q., Tao L., Dan Z., Youhou, K., Yunfeng, L., Maria, K., Li X., Wilson C. Y., et al., 2013, Munc18b Is a Major Mediator of Insulin Exocytosis in Rat Pancreatic β-Cells. Diabetes, 62(7), 2416–2428. https://doi.org/10.2337/db12-1380

[24]. Świderska E., Strycharz, J., Wróblewski, A., Szemraj J., Drzewoski J., & Śliwińska A., 2020, Role of PI3K/AKT Pathway in Insulin-Mediated Glucose Uptake [Internet]. Blood Glucose Levels. IntechOpen; 2020. http://dx.doi.org/10.5772/intechopen.80402

[25].Fathima, J. S., Jayaraman, S., Sekar, R. and Syed, N. H., 2024, The role of MicroRNAs in the diagnosis and treatment of oral premalignant disorders. Odontology, pp.1-10.

[26]. Ivorra, M. D., Paya, M., Villar, A., 1989, A review of natural products and plants as potential antidiabetic drugs. Journal of Ethnopharmacology, 27(3), 243–275.

[27]. Anil, B., 2010, Gaikwad, Jeena Gupta, Kulbhushan Tikoo; Epigenetic changes and alteration of Fbn1 and Col3A1 gene expression under hyperglycaemic and hyperinsulinaemic conditions. Biochemistry Journal, 432 (2), 333–341. doi: https://doi.org/10.1042/BJ20100414

[28]. Yasothkumar, D., Ramani, P., Jayaraman, S., Ramalingam, K. and Tilakaratne, W. M., 2024, Expression Profile of Circulating Exosomal microRNAs in Leukoplakia, Oral Submucous Fibrosis, and Combined Lesions of Leukoplakia and Oral Submucous Fibrosis. Head and Neck Pathology, 18(1), p.28.

[29]. Bansal, A., & Pinney, S. E., 2017, DNA methylation and its role in the pathogenesis of diabetes. Pediatr Diabetes, 18(3),167-177. doi: 10.1111/pedi.12521.

[30]. Sagar, S., Ramani, P., Moses, S., Gheena, S. and Selvaraj, J., 2024, Correlation of salivary cytokine IL-17A and 1, 25 dihydroxycholecalciferol in patients undergoing orthodontic treatment. Odontology, pp.1-10.

[31]. Thomas, D. D., Corkey, B. E., Istfan N.W., & Apovian C. M., 2019, Hyperinsulinemia: An Early Indicator of Metabolic Dysfunction. Journal of the Endocrine Society, 3(9),1727-1747. doi: 10.1210/js.2019-00065.

[32]. Wilcox. G., 2005, Insulin and insulin resistance. Clinical Biochemist Reviews, 2005 26(2), 19-39.

[33]. Zhao, C., Yang C., Wai, S. T. C., Zhang Y. P., Portillo M, Paoli P., Wu Y., San Cheang W., Liu B., Carpéné C., Xiao J., & Cao H., 2019, Regulation of glucose metabolism by bioactive phytochemicals for the management of type 2 diabetes mellitus. Critical Reviews in Food Science and Nutrition; 59(6):830-847. doi: 10.1080/10408398.2018.1501658.

[34]. Stanley M. P. P., & Kamalakkannan N., 2006, Rutin improves glucose homeostasis in streptozotocin diabetic tissues by altering glycolytic and gluconeogenic enzymes. Journal of Biochemical and Molecular Toxicology, 20(2), 96-102. doi: 10.1002/jbt.20117.

[35]. Latha M, & Pari L. 2003, Antihyperglycaemic effect of Cassia auriculata in experimental diabetes and its effects on key metabolic enzymes involved in carbohydrate metabolism. Clinical and Experimental Pharmacology and Physiology, 30(1‐2):38-43.

[36]. Jayachandran, M., Zhang, T., Ganesan, K., Xu, B., Chung, & S.S.M. .2018, Isoquercetin ameliorates hyperglycemia and regulates key enzymes of glucose metabolism via insulin signaling pathway in streptozotocin-induced diabetic rats. European Journal of Pharmacology, 82:112–120.

[37]. Pari, L, & Rajarajeswari, N., 2009. Efficacy of coumarin on hepatic key enzymes of glucose metabolism in chemical induced type 2 diabetic rats. Chemico-Biological Interactions, 181:292–296.

[38]. Gothandam, K., Ganesan, V. S., Ayyasamy, T., & Ramalingam, S., 2019, Antioxidant potential of theaflavin ameliorates the activities of key enzymes of glucose metabolism in high fat diet and streptozotocin–induced diabetic rats. Redox Report, 24:41–50.

[39]. Tian, M., Han, Y. B., Zhao, C. C., et al. 2021, Hesperidin alleviates insulin resistance by improving HG-induced oxidative stress and mitochondrial dysfunction by restoring miR-149. Diabetol Metab Syndr 13:50. https://doi.org/10.1186/s13098-021-00664-1.

[40]. Chen C, Peng S, Chen F, Liu L, Li Z, Zeng G, Huang Q. 2017, Protective effects of pioglitazone on vascular endothelial cell dysfunction induced by high glucose via inhibition of IKKα/β-NFκB signaling mediated by PPARγ in vitro. Canadian Journal of Physiology and Pharmacology, 95(12), 1480-1487. doi: 10.1139/cjpp-2016-0574.

[41]. Copps, K.D.;,White, M.F. 2012, Regulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1 and IRS2. Diabetologia, 55:2565–2582.

[42]. Chuang, W.T., Yen, C.C., Huang, C.S., Chen, H.W., Lii, C. K., 2020, 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, 15228–15238.

[43]. Boucher, J., Kleinridders, A., Kahn, C. R., 2014, Insulin receptor signaling in normal and insulin-resistant states. Cold Spring Harbor Perspectives in Biology, 2014, 6, a009191.

[44]. Zhang, Y. Xu, W., Huang, X., Zhao, Y., Ren, Q., Hong, Z., Huang, M., Xing, X., 2018, Fucoxanthin ameliorates hyperglycemia, hyperlipidemia and insulin resistance in diabetic rats partially through IRS-1/PI3K/Akt and AMPK pathways. Journal of Functional Foods, 48, 515–524.

[45]. Cai, S., Sun, W., Fan, Y., Guo, X., Xu, G., Xu, T., Hou, Y., Zhao, B., Feng, & X., Liu, 2016. T. Effect of mulberry leaf (Folium Mori) on insulin resistance via IRS-1/PI3K/Glut-4 signalling pathway in type 2 diabetes mellitus rats. Pharmaceutical Biology, 54, 2685–2691.

[46]. Hsu, W. H., Hsiao, P. J., Lin, P. C., Chen, S. C., Lee, M. Y., Shin, S. J., 2017. Effect of metformin on kidney function in patients with type 2 diabetes mellitus and moderate chronic kidney disease. Oncotarget, 9(4), 5416-5423. doi: 10.18632/oncotarget.23387.

[47]. Bandeira Sde, M, Guedes Gda, S, da Fonseca, L. J., Pires, A. S., Gelain, D. P., Moreira J. C., Rabelo, L. A., Vasconcelos, S. M., Goulart, M. O., 2012, Characterization of blood oxidative stress in type 2 diabetes mellitus patients: increase in lipid peroxidation and SOD activity. Oxid Med Cell Longev, 819310. doi: 10.1155/2012/819310.