All ETDs from UAB

Advisory Committee Chair

Timothy W Garvey

Advisory Committee Members

Pi-Ling Chang

Yuchang Fu

Document Type

Thesis

Date of Award

2019

Degree Name by School

Master of Science (MS) School of Health Professions

Abstract

Our previous data indicates that glucose-toxicity is dependent upon upregulation of TRIB3 expression in muscle. TRIB3 is a glucose-responsive gene and, as a pseudokinase, it blocks phosphorylation of AKT resulting in insulin resistance. The purpose of the current study was to identify the role of the differentiation process and the minimal functional sequence of TRIB3 promoter region in modulating glucose-induced TRIB3 gene expression in C2C12 myocytes. First, C2C12 myocytes were collected every 48 hours during the whole differentiation, totally 4 intervals. Before collection, cells were treated with either 5mmol glucose or 25mmol glucose for 24 hours. Under 5mmol glucose, the statistically significant increase of TRIB3 mRNA expression began at Day 5 with a 1.4-fold change and continued to reach at 4.2-fold increase at Day 7 compared with Day 1 (p<0.05). Additionally, at each time point, high glucose treatment (25mmol) significantly increased TRIB3 mRNA and protein expression compared with the corresponding controls (p<0.05). Besides, as the differentiation was ongoing, TRIB3 mRNA and protein levels under high glucose dramatically amplified achieving an almost 4 times elevation at the fully differentiated status compared with Day 1 (p<0.05). These results suggest that cell differentiation will also induce TRIB3 gene expression and plays a critical role in glucose-induced TRIB3 expression. In the second portion, a 2.4 Kb DNA fragment of the TRIB3 gene (-1700 to +737) will be subcloned into a pMetLuc-Reporter vector and transfected into C2C12 myoblasts treated with normal or high glucose. The longest promoter/reporter constructs (-1700/ +737) displayed the most activities under glucose stimulation with 1.5-fold elevation (p<0.05) compared with the basal promoter activity. Moreover, the intermediated constructs (-400/+737) showed similar promoter activity as the longer constructs (-980/+737), which significantly responded to high glucose with a 1.44-fold increase (p<0.05). Nonetheless, a further deletion construct (-290/+737), showed a significantly lower basal promoter activity (p<0.05) as well as no response to glucose stimulation. In conclusion, 1) TRIB3 expression and its response to glucose rise during cell differentiation in muscle cells. 2) Glucose induction depends on the regulation of promoter activity and especially an element residing at -400 to +290 of the promoter. 3) This work provides insight into relevant targets to prevent or reduce glucose-induced insulin resistance in muscle in diabetes.

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