- Glucose-dependent Insulin Secretion from Genetically Engineered K-cells Using EBV-based Episomal Vector.
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Ju Hee Kim, Sung Dae Moon, Seung Hyun Ko, Yu Bai Ahn, Ki Ho Song, Hyang Sook Lim, Sook Kyung Lee, Soon Jip Yoo, Hyun Shik Son, Kun Ho Yoon, Bong Yun Cha, Ho Young Son, Sung Joo Kim, Je Ho Han
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Korean Diabetes J. 2007;31(1):9-21. Published online January 1, 2007
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DOI: https://doi.org/10.4093/jkda.2007.31.1.9
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Abstract
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- BACKGROUND
Type 1 diabetes mellitus is an autoimmune disease resulting in destruction of the pancreatic beta cells. Insulin gene therapy for these patients has been vigorously researched. The strategy for achieving glucose-dependent insulin secretion in gene therapy relies on glucose-responsive transcription of insulin mRNA and the constitutive secretory pathway of target non-beta cells. We observed that genetically engineered K-cells using Epstein-Barr virus (EBV)-based episomal vector can produce glucose-regulated insulin production. METHODS: Green fluorescent protein (GFP) or rat-preproinsulin (PPI) expression cassette transcriptionally controlled by the promoter of glucose dependent insulinotropic peptide (GIPP) is fused to pCEP4 containing the origin of replication (oriP) and Epstein-Barr virus nuclear antigen 1 (EBNA-1). CMV promoter was replaced by subcloning the GIPP into pCEP4 to generate pGIPP/CEP4. Two recombinant EBV-based episomal vectors, pGIPP/GFP/CEP4 and pGIPP/PPI/CEP4, were constructed. pGIPP/GFP/CEP4 and pGIPP/PPI/CEP4 containing K-cell specific GIPP were co-transfected into STC-1. K-cell was isolated from the clonal expansion of the fluorescent cells selected by hygromycin treatment in STC-1, and were analyzed for the expression of glucokinase (GK) or transcription factors involved in pancreas development. K-cells concurrently transfected with pGIPP/PPI/CEP4 and pGIPP/GFP/CEP4 were analyzed for the transcripts of PPI by RT-PCR, and for the glucose dependent insulin expression by immunocytochemistry or insulin assay using ultra-sensitive rat-specific insulin ELISA kit. RESULT: STC-1 was stably-transfected with pGIPP/GFP/CEP4 along with pGIPP/PPI/CEP4. Genetically selected fluorescent K-cells expressed GK and transcription factors involved in pancreas development. And K-cells transfected with pGIPP/PPI/CEP4 contained detectable levels of PPI transcripts and showed glucose-dependent immunoreactive insulin secretion. CONCLUSION: We identified genetically engineered K-cells which exert a glucose-dependent insulin expression using EBV-based episomal vector. The similarities between K-cells and pancreatic beta cells support that K-cells may make effective and ideal targeting cells for insulin gene therapy or alternative cell therapy.
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Citations
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- Relationship of traditional and nontraditional cardiovascular risk factors to coronary artery calcium in type 2 diabetes
Ju-Yeon Sim, Ju-Hee Kim, Yu-Bae Ahn, Ki-Ho Song, Je-Ho Han, Bong-Yun Cha, Sook-Kyung Lee, Sung-Dae Moon Korean Diabetes Journal.2009; 33(6): 466. CrossRef - Transdifferentiation of Enteroendocrine K-cells into Insulin-expressing Cells
Esder Lee, Jun Mo Yu, Min Kyung Lee, Gyeong Ryul Ryu, Seung-Hyun Ko, Yu-Bae Ahn, Sung-Dae Moon, Ki-Ho Song Korean Diabetes Journal.2009; 33(6): 475. CrossRef
- In Vitro Expansion and Differentiation of Islet Precursor Cells from Cultured Neonatal Porcine Pancreatic Tissue.
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Yu Bae Ahn, Kun Ho Yoon, Sun Hee Seo, Seung Hyun Ko, Ki Ho Song, Je Ho Han, Soon Jip Yoo, Hyun Sik Son, Moo Il Kang, Bong Yun Cha, Kwang Woo Lee, Ho Young Son, Sung Koo Kang
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Korean Diabetes J. 2000;24(3):310-322. Published online January 1, 2001
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- BACKGROUND
Neonatal porcine pancreas is an attractive alternative source for islet transplantation because of its growth potential and availability. Porcine neonatal pancreatic cell clusters (NPCCs) consist mainly of protodifferentiated cells expressing both the duct cell marker pancytokeratin and islet hormones. In this study, we investigated to expand and mature the pancreas duct cells contained in porcine NPCCs with extracellular matrix. METHODS: For NPCCs, pancreas obtained from neonatal pigs were minced, digested with collagenase and cultured overnight. Then NPCCs were further dispersed to small cell groups and cultured on HTB-9 extracellular matrix: the tissue attached and formed monolayer patches. At the 3rd and 8th days, tissue was fixed, immunostained for pancytokeratin (panCK), vimentin (VT) and islet hormones. RESULTS: During 5 days culture, the total cell numbers increased 3.2 fold on the matrix, and 1.6 fold on the sticky dish, respectively. Insulin positive cells (Ins+) were 6.0% of total cells at day 3 and increased 1.6 fold in numbers at day 8. There was significant increase in DNA content of NPCCs in monolayers on both sticky dishes and HTB-9 matrix. In contrast, insulin content of both groups decreased during culture periods. Until 8 days of culture after dispersion of porcine NPCC, most duct cells costained with panCK and VT. CONCLUSION: We observed NPCCs were composed of many of duct cells which were known to be endocrine precursor cells and monolayer culture of NPCC withextracellular matrix resulted in the proliferation and differentiation of pancreatic duct cells.
- Effect of Hyperglycemia on Internalization of Insulin-receptor Complexes in Human Umbilical Vein Endothelial Cells.
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Ki Ho Song, Yu Bae Ahn, Je Ho Han, Soon Jip Yoo, Kun Ho Yoon, Moo Il Kang, Bong Yun Cha, Kwang Woo Lee, Ho Young Son, Sung Koo Kang
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Korean Diabetes J. 1999;23(2):131-141. Published online January 1, 2001
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- BACKGROUND
It is well known that hyperglycemia activates protein kinase C (PKC) in vascular endothelial cells. However, the effect of hyperglycemia on internalization and recycling of insulin receptors by insulin in endothelial cells has not been examined thus far. METHODS: Human umbilical vein endothelial cells (HUVECs) were isolated from healthy, pregnant women. Confluent HUVECs were incubated in a culture media containing either 5 (NG group) or 25 mM glucose (HG group) for 4 days. Then, we measured the insulin binding, internalization and recycling of the insulin receptor and release of internalized insulin into the media. RESULTS: There was no difference in binding of 0.17 nM 125I-insulin between the two groups. However, the amount of internalized 125I-insulin, determined by the aeid washing method, was significantly greater in the HG group compared to the NG group. The addition of 10 pM 1-(5-isoquino-linesulfonyl)-2-methyl-piperazine (H7), a PKC inhibitor, to the HG group prevented the increase of internalization in 125I-insulin. In addition, preincubation with unlabeled insulin resulted in a decrease of 125I-insulin binding to a greater extent in the HG group compared with the NG group, indicating that high glucose levels increased internalizntion of insulin receptors. The high glucose-induced increase in internalization of insulin receptors was prevented by an addition of H7. Recycling of insulin receptors to the cell surface was not affected by high glucose. Internalized 125I-insulin released into media with time. The released amount of I-insulin in the HC group tended to be greater compared to the NG group. CONCLUSION: These results suggest that hyperglycemia may increase internalization of the insulin-receptor complexes in vascular endothelial cells through PKC activation.
- Prevalence of micro and macroalbuminuria in relation to hypertension and chronic diabetic vascular complications among type II diabeticpatients.
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Hyuk Ho Kwon, Je Ho Han, Jong Min Lee, Soon Jip Yoo, Hyun Sik Son, Kun Ho Yoon, Moo Il Kang, Jwan Soo Hong, Bong Yun Cha, Kwang Woo Lee, Ho Young Son, Sung Ku Kang
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Korean Diabetes J. 1992;16(4):317-324. Published online January 1, 2001
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- No abstract available.
- Relationship between magnesium and calcium to glucose stimulated insulin secretion in the perfused rat pancreas.
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Kun Ho Yoon, Soon Jip Yoo, Hyun Sik Son, Moo Il Kang, Kwan Soo Hong, Bong Youn Cha, Kwang Woo Lee, Ho Young Son, Sung Ku Kang
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Korean Diabetes J. 1991;15(1):63-71. Published online January 1, 2001
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- No abstract available.
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