etection of Gene Expression Associated in The Regulation of Blood Glucose on Insulin Resistance Mice Model Using qPCR Method

• Main Author: Fitriana, Fathin
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/44977
• Institution: Institut Teknologi Bandung

Type 2 diabetes mellitus is characterized by the resistance of target tissues to insulin. Insulin is a hormone secreted by pancreatic ?cells and the secretion is triggered by the presence of glucose and free fatty acids with high concentration in the blood. The incidence of type 2 diabetes mellitus increased due to diet and unhealthy lifestyle. In type 2 diabetes mellitus, there are several genes that are consistently associated with an increased risk of type 2 diabetes and insulin resistance. Gene expression level that play roles in blood glucose regulation can be determined quantitatively using quantitative realtime PCR (qPCR). The basic principle of qPCR is the lower Ct value means there were more target genes in the initial sample. This study aimed to detect the expression of genes involved in the regulation of blood glucose including Peroxisome Proliferator-Activated Receptor gamma (PPAR?), leptin and resistin in normal and insulin resistance induced mice using qPCR method. In this study, insulin resistance was induced in male Swiss Webster mice by administrating high fat and sugar emulsion. The mice were divided into two groups, normal and insulin resistance group which consisted of 3 mice in a group. Upon induction for 4 weeks, confirmation of the successful insulin resistance induction was conducted using an oral glucose tolerance test and the results were calculated as rate constant of insulin tolerance test (KITT). All mice were sacrificed, then the liver and perirenal fat tissue were isolated. Total RNA which was isolated from the liver and perirenal fat tissue was reverse transcribed into cDNA and then quantified by qPCR. The qPCR results were calculated by the relative quantification method Livak which compared the expression of target genes relative to the normalisator or housekeeping genes. Two pairs of primer for each target gene and ?-actin the housekeeping gene were designed in silico and the selection was done both in silico and in vitro by qPCR. Specificity was determined from the melting curve of amplification products by primer of the target genes. The detection of the target gene amplification products was done by using the fluorescence dye SYBR Green. Based on the KITT results, administration of high fat and sugar emulsion with a volume of 0.01 ml/g body weight caused insulin resistance on induced group after 4 weeks induction. The result of quantification showed that the expression of PPAR?in perirenal fat tissue in insulin resistance model group was lower than the normal group. Meanwhile, leptin and resistin expression in insulin resistance model group was significantly higher compared to the normal group.