A comparison of gene expression of redox sensitive genes and the corresponding proteins encoded in type 2 diabetes mellitus patients with those in healthy subjects

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A comparison of gene expression of redox sensitive genes and the corresponding proteins encoded in type 2 diabetes mellitus patients with those in healthy subjects

 

Author: Chui, Tat Kong
Title: A comparison of gene expression of redox sensitive genes and the corresponding proteins encoded in type 2 diabetes mellitus patients with those in healthy subjects
Degree: M.Sc.
Year: 2012
Subject: Diabetes -- Molecular aspects.
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Health Technology and Informatics
Pages: xviii, 173 p. : ill. ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2520217
URI: http://theses.lib.polyu.edu.hk/handle/200/6560
Abstract: Diabetes Mellitus (DM) is a common metabolic syndrome, especially type 2 (T2DM). Oxidative stress has been reported to cause insulin resistance and endothelial dysfunction, both of which are commonly found in DM. Elevated level of glucose can result in increased formation of reactive oxygen species (ROS) that causes oxidative stress leading to endothelial cells and pancreatic β cells damage. Redox sensitive genes and proteins include HO-1, Nrf2, KEAP-1 and Bach 1. These are either upregulated or downregulated in response to oxidative stress. HO-1, with potent anti-oxidant, anti-inflammatory and anti-proliferative effects, is considered a critical molecule against oxidative stress. Nrf2 activates various oxidative defensive genes, including HO-1, and provides an anti-inflammatory and cytoprotective functions. Keap-1 binds, ubiquitinates and degrades Nrf2 until oxidants modify Keap-1 leading to dissociation of Nrf2 from the Keap-1-Nrf2 complex, thus freeing Nrf2 to activate various cytoprotective genes. Bach-1 acts as a transcriptional repressor of HO-1 activation until haem binds to Bach-1 and displaces it from enhancers of HO-1, making the enhancers available to Nrf2, a transcriptional activator of HO-1. These types of complex but well coordinated controls not only maintain haem homeostasis but also defend against oxidative stress. In this exploratory molecular study, the expression of the genes of interest (HO-1, Nrf2, Keap-1 and Bach-1) and the corresponding proteins level of HO-1 was measured and compared in white cells from the buffy coat of normal subjects (n=8) and the lymphocytes of Type 2 DM patients (n=8) using the techniques of real time PCR and Western blotting. In addition, relationships between HO-1 protein content and factors that could be expected to associate with this, such as glycaemic control, antioxidant status, and polymorphisms in HO-1 were explored.
Results showed no statistically significant differences in expression of HO-1, Nrf2 and Keap-1 between the two groups of samples, though the variation of expressions in both normal subjects and T2DM patients was very high, but the expression of Bach-1 in the T2DM subjects was statistically significant lower than that in the normal subjects. Significant correlation was found between HO-1 gene expression and protein expression, Nrf2 expression and HO-1 expression as well as Nrf2 expression and Keap-1 expression in T2DM samples. In addition, the protein expression of HO-1 of normal subjects was not statistically different from that of T2DM patients and the variation of expression in both normal subjects and T2DM patients was also very high. Besides, the HO-1 protein expression of T2DM patients had no significant correlation with the level of fasting blood glucose, HbA1c, total bilirubin and FRAP. Moreover, the allele type of HO-1 (GT)(n) microsatellite DNA repeats polymorphism had no significant correlation with HO-1 expression in T2DM patients. This study is the first to explore such redox gene expression and protein content in samples from normal and T2DM subjects and to investigate factors that may influence HO-1 gene and protein expression in T2DM. No clear differences or associations were found except statistical difference of Bach-1 expression existed between the two groups of samples and significant correlation between HO-1 gene expression and protein expression, Nrf2 expression and HO-1 expression as well as Nrf2 expression and Keap-1 expression in T2DM samples were seen. Nonetheless, useful pilot data have been gathered to support future studies looking at the influence of oxidative stress and glycaemic control on redox sensitive cytoprotection in T2DM.

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