The influence of newly synthesized hybrid Thiazolidinedione (TZD)-methoxy on the expression and acetylation of diabetes related genes

Abstract

Type 2 diabetes mellitus is a metabolic illness that is categorized by fasting plasma glucose levels above 7 mmol/1. It is preceded by insulin resistance which is triggered by increased dietary fats and sedentary lifestyle. Type 2 diabetes is often associated with the down regulation of critical genes that confer protection against the disease, especially genes involved in glucose transport and lipid oxidation (NRF-1 , GLUT4, MEF2A). Furthermore, diabetes is also associated with decreased expression and activity of various histone acetyl transferases (pCAF/p300, GCN5) involved in acetylation of these gene promoters. Therefore, finding the means to up-regulate genes that confer protection against diabetes can be a positive step towards the discovery of new and effective therapeutic modalities in the treatment of type 2 diabetes. Though metformin has been widely used as the most potent drug in treating and managing type 2 diabetes, it still does not cure the disease and is associated with some side effects. Therefore, this study was aimed at investigating whether the newly synthesized Thiosemicarbazone-triazole hybrid compound can be a better therapeutic modality in the treatment of type 2 diabetes than metformin. This study was conducted in C2C 12 myotubes, which are mouse embryonic stem cells. Palmitate was introduced in this cell line to induce insulin resistance . A cell viability test was done using MTT assay kit to establish an optimal Thiazolidinedione (TZD) concentration to be used in these cells. There were five treatment groups in this study, namely, control (normal), palmitate (to induce insulin resistance) , palmitate + metformin, palmitate + TZD and TZD only. Complementary Deoxyribonucleic acid (cDNA) was synthesized from total ribonucleic acid (RNA) extracted from myotubes and amplified iv using Quantitative Polymerase Chain Reaction (qPCR). To study the influence of TZD on transcriptional factor binding to their respective cis-element DNA binding domains, Chromatin lmmunoprecipitation (ChlP) was used to investigate the binding extent of MEF2A to the GLUT4 promoter. Furthermore, the effect of this TZD on antioxidant properties was also assessed. In this respect, Trolox Equivalent Antioxidant Capacity (TEAC) was used to measure radical scavenging activity while Ferric Reducing Antioxidant Power (FRAP) assay was used to measure the ferric reducing antioxidant power of the hybrid compound. To study the breakdown of glucose in the cel ls, glucose oxidase assay was conducted according to the manufacturer's instructions. Adenosine triphosphate (ATP) synthesis was also examined using a standard ATP assay kit as per the manufacturer's protocol. The results in this study showed that most of the genes involved in glucose transport and lipid metabolism assessed (NRF-1, GLUT4, MEF2A) and those involved in acetylation (pCAF/p300, GCN5) were upregulated by the hybrid compound 2d' higher than metformin. Furthermore, regarding protein-DNA complex interactions, the TZD increased MEF2A-GLUT4 binding. The TZD also showed high antioxidant activity at different degrees. As a result, TZD has potential in deterring the commencement and development of diseases caused by free radical etiology such as type 2 diabetes mellitus. The TZD treatments led to increased glucose oxidase activity while the ATP content was found to be even higher. This study demonstrated that our newly synthesized TZD upregulated and increased gene expression and acetylation of diabetes related genes. These findings are a crucial step towards the development of new drugs that may be critical in the better management and treatment of diabetes.