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Oh, Woo, Kim, Kim, and Kim: Glucose Regulated Production of Human Insulin in Genetically Modified Myoblast Cell Line (C2C12)
Original Article
Journal of Korean Endocrine Society

Journal of Korean Endocrine Society 2006; 21(6): 526-535.

Published online: 20 December 2006

DOI: https://doi.org/10.3803/jkes.2006.21.6.526

Glucose Regulated Production of Human Insulin in Genetically Modified Myoblast Cell Line (C2C12)

Seungjoon Oh, Jeong-Taek Woo, Sung Woon Kim, Jin-Woo Kim, Young Seol Kim

Department of Internal Medicine, College of Medicine, Kyung Hee University, Korea.

Received 1 February 2006       Accepted 21 December 2006

Copyright © 2006 Korean Endocrine Society

Abstract

Background

To develop somatic gene therapy model for diabetes mellitus, it is the most important to control it by glucose concentration. In order to develop the myoblasts that produce insulin by glucose concentration, the transfection of genes of human insulin, rat glucokinase and rat GLUT2 was conducted using C2C12, the murine myoblast cell line.

Methods

pMLC-hINSmut plasmid vector to which human insulin cDNA was inserted in C2C12 cell line, pCB7/GLUT2 and pCB7/GK to which GLUT2 and glucokinase were inserted. Based on the inserted gene, C2C12/INS-GLUT2, C2C12/INS-GK and C2C12/INS-GK-GLUT2 were prepared. In each cell line, its mRNA and protein expression were measured. Also, the capability of producing insulin in low glucose (2.7 mM) and high glucose (25 mM) were compared.

Results

1. It was observed that C2C12/INS-GLUT2, C2C12/INS-GK, C2C12/INS-GK-GLUT2 cell line expressed mRNA and protein of transfected genes, respectively.
2. As for the insulin production depending on the glucose concentration in C2C12/INS, it slightly increased from 0.049 ± 0.003 µU/106 cells/hr to 0.197 ± 0.022 µU/106 cells/hr. However, in C2C12/GK-GLUT2-INS, it showed the most evident increase: from 0.251 ± 0.074 µU/106 cells/hr to 1.325 ± 0.221 µU/106 cells/hr.
3. The expression of insulin gene decreased in proportion to the insulin production capability, reaching the minimum point at the 8th week.

Conclusion

Genetically engineered murine myoblast secreted insulin depending on the glucose concentration in vitro and was able to cause its decrement when transplanted. However, it should be continued to study the method to maintain the consistent genetic expression in somatic cell therapy.

Keywords: Glucokinase, GLUT2, Insulin, Myocyte

Figures and Tables

Fig. 1
Schematic of expression plasmids. A. generalized map of plasmid used for expression of human insulin; B. generalized map of plasmids used for expression of rat glucokinase, or rat GLUT2.
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Fig. 2
RT-PCR analysis of insulin, glucokinase, GLUT2 mRNA in C2C12 derived cell lines at 8th week after transfection. In the C2C12/GK-GLUT2-INS cell line in which glucokinase, GLUT2 and insulin gene were all transfected, all of three genes were expressed and other cells expressed mRNA depending on inserted genes.
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Fig. 3
Western blot analysis of glucokinase or GLUT2 protein in C2C12-derived cell line at the 8th week after transfection. The arrow in the right side of the figure indicates glucokinase in Fig. 3A and GLUT2 in Fig. 3B, respectively. C, C2C12; 1 and 2, C2C12/GK; 3 and 4, C2C12/INS-GK; 5, C2C12/INS-GLUT2; 6, C2C12/INS-GK-GLUT2; 7 and 8, C2C12/GLUT2; 9 and 10, C2C12/INS-GLUT2; 11, C2C12/INS-GK-GLUT2; 12, C2C12/INS-GK.
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Fig. 4
Effect of glucose concentration on insulin secretion in C2C12 cell lines overexpressing insulin gene and GLUT2 or glucokinase gene or both at 8th week after transfection (n = 4). *P < 0.05.
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Fig. 5
RT-PCR analysis of insulin mRNA and daily insulin production in C2C12 derived cell line.
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Table 1
Sequences of PCR primer sets used to amplify each cDNA in this study
jkes-21-526-i001

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