- The long-term effect of ramipril on Gialpha2-protein and Protein Tyrosine Phosphatase 1B in an animal model of type 2 diabetes(OLETF rat).
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Jung Min Lee, Ok Ki Hong, Hyuk Sang Kwon, Sung Dae Moon, Sang Ah Chang, Hyun Shik Son, Kun Ho Yoon, Bong Yun Cha, Sung Koo Kang
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Korean Diabetes J. 2006;30(1):25-38. Published online January 1, 2006
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DOI: https://doi.org/10.4093/jkda.2006.30.1.25
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- BACKGROUND
The regulation of tyrosine phosphorylation/dephosphorylation is an important mechanism in various intracellular metabolism. Also impaired insulin signal transduction is important in pathogenesis of type 2 diabetes. It has been reported that PTP1B is a negative regulator of insulin action, and Gialpha2-protein is related to the regulation of PTP1B. Herein we investigated the long-term effects of ramipril on PTP1B/insulin signal protein interaction and the relation between Gialpha2 and PTP1B in animal model of type 2 diabetes (OLETF rat). METHODS: OLETF rats and age-matched LETO rats were divided into two groups. One group of rats received ramipril (10 mg/kg body weight) for 12 weeks, and another group did not. Finally, each group was divided into 2 subgroups, with or without insulin injection intravenously, before sacrifice. After sacrifice, tissues extracts of liver, hind limb muscle, and epididymal fat were obtained for quantification of PTP1B, Gialpha2, and several insulin signal proteins by western blotting. RESULTS: In liver and muscle, the levels of basal PTP1B and activated PTP1B of OLETF rats treated with ramipril and insulin were significantly decreased. The levels of Gialpha2, activated IRS-2, and activated p-85alpha were significantly increased in OLETF rats treated with ramipril and insulin. In adipose tissue, the levels of Gialpha2 and activated p-85alpha of OLETF rats treated with ramipril and insulin were slightly increased as in liver and muscle. But, the levels of basal PTP1B and activated PTP1B were significantly increased. And, the levels of activated IRS-1 and activated IRS-2 were decreased. CONCLUSION: These results suggest that the improvement of insulin sensitivity by treatment with ramipril was related to the decreased level of activated PTP1B. Also, we could suggest that the changes of activated PTP1B level was related with the changes of Gialpha2-protein. However, the results of adipose tissue were different from those of liver and muscle. So it seemed likely that there would be various major modulators for regulation of insulin signal pathway according to tissue.
- Effect of Gi-proteins on Insulin Binding, Internalization and Recycling of Insulin Receptor in Bovine Aorta Endothelial Cell.
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Hyuk Ho Kwon, Hyun Shik Son, Jung Min Lee, Seung Hyun Ko, Ok Ki Hong, Sung Dae Moon, Sang Ah Chang, Kun Ho Yoon, Bong Yun Cha, Kwang Woo Lee, Ho Young Son, Sung Koo Kang
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Korean Diabetes J. 2003;27(1):26-38. Published online February 1, 2003
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Abstract
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- BACKGROUND
Guanine nucleotide binding proteins (G-proteins) play important roles in the hormonal actions of many signal transduction systems. Possible roles for the Gi-protein in insulin action have been suggested. It is reported that Gi-protein is associated with insulin actions to a greater extent than Gs-protein. There are at least three different subtypes of Gi-proteins (Gi(alpha1), Gi(alpha2), and Gi(alpha3)), however, it is not certain which subtypes are associated with insulin receptors and their action. METHODS: To investigate the effects of Gi-proteins on insulin action, the Gi-proteins were overexpressed in cultured bovine aortic endothelial cells (BAEC), using the DNA-polylysine-adenovirus complex transfection method. After incubating for 24 hours, the BAEC were treated with 200 ng/mL insulin to evaluate the insulin binding, receptor internalization and recycling. RESULTS: The following results were found : 1) The binding of specific insulin bindings to the insulin receptors of endothelial cells were time-dependent, reaching their maximal levels in all cells after 30 minutes. The maximal specific bindings of the control, Gi(alpha1), Gi(alpha2), and Gi(alpha3) were 0.58+/-0.1, 0.54+/-0.08, 0.54+/-0.1, 0.53+/-0.09%, respectively. 2) The internalization of 125I-insulin, into endothelial cells, was assessed by the acid washing dissociation method, and occurred rapidly. There was a significant difference in the internalized radioactivity of the 125I-insulin in the overexpressed Gi(alpha2) protein group compared to the two groups. 3) The recycling of the insulin receptors in the three types of Gi-protein showed no significant difference between the three group. CONCLUSION: In conclusion, the Gi(alpha2) protein may be associated with internalization of the insulin-insulin receptor complex, and appears to be important in both the action of insulin and the intracellular processing of insulin receptors.
- 3-Dimensional Long Term Culture of Monolayer Cultured Dispersed Neonatal Porcine Pancreas Cells (NPCC).
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Sun Hee Suh, Kun Ho Yoon, Hyuk Sang Kwon, Ok Ki Hong, Jung Min Lee, Ki Ho Song, Soon Jib 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. 2002;26(5):383-395. Published online October 1, 2002
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Abstract
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We have reported porcine neonatal pancreas cell clusters (NPCCs) to be useful clinical alternative due to their growth potential and convenience. However, to apply the porcine NPCCs in human islet transplantation, there is a need to achieve in vitro maturation of porcine pancreas duct cells for the immediate cure of diabetes, and to escape hyperacute rejection. We have established a long-term 3D culture system of porcine pancreas duct cells for their in vitro induction in differentiated beta-cells. METHOD: For making NPCCs, pancreata from 1~3 days old pigs were minced, digested and cultured for 8 days. After 8 days, the cells were layered with Matrigel. After 50 days, the 3 dimensional cultures, the components of the reconstructed cell clusters were confirmed by three approaches: immunofluorescent staining, mea-surement of glucose stimulated insulin secretion and semiquantitative RT-PCR. RESULT: The monolayers of epithelial cells formed three-dimensional structures of cysts from which 50~200 micro meter diameter islet-like clusters of pancreas cells budded. The insulin and DNA contents, and the ratio of insulin/DNA, did not change significantly, even after 50 days of culturinge. The levels of insulin and galactosyl transferase mRNA showed a tendency to increase in the monolayer culture of the duct cells until day 8, after which the levels significantly decreased. However, the level of glucagon mRNA was maintained until day 50. Compared with their basal secretion at 5mM glucose, the cysts/cultivated porcine islet buds exposed to stimulatory 20mM glucose did not show difference in insulin secretion. CONCLUSION: We have shown the expansion of dispersed porcine neonatal pancreas cells in vitro, and the reconstruction of a three-dimensional structure, following Matrigel overlaying, but were unable to observe the transition of duct cells to beta cells, as observed in human duct cells. Further studies will be required to elucidate this difference.
- Effect of Overexpression of Gi Proteins on Insulin Actions in 3T3-L1 Adipocytes.
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Hyun Shik Son, Bong Yun Cha, Sung Dae Moon, Jung Min Lee, Ok Ki Hong, Sang Ah Chang, Yu Bae Ahn, Kun Ho Yoon, Kwang Woo Lee, Ho Young Son, Sung Koo Kang
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Korean Diabetes J. 2000;24(4):404-412. Published online January 1, 2001
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Abstract
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It has been reported that G proteins are involved in biological actions of insulin. Especially, Gi protein is more associated with insulin actions than Gs proteins. Gi protein has at least three different subtypes of Gi 1, Gi 2 and Gi 3 protein. However, it is not certain which subtypes of Gi proteins are associated with biological actions of insulin. METHODS: To investigate which subtypes of Gi proteins are associated with insulin action, we overexpressed three different kinds of Gi protein, Gi 1, Gi 2 and Gi 3 protein, in 3T3-L1 adipocytes using DNA-polylysine-adenovirus complex transfection method. After incubating for 2 hours, 3T3-L1 adipocytes were treated with 100 nM insulin for the evaluation of biological actions of insulin. Moreover, to elucidate insulin stimulated insulin receptor autophosphorylation and IRS-1 phosphorylation, 3T3-L1 adipocytes were stimulated with 100 nM insulin for 10 minutes, homogenized and immunoprecipitated with anti-phosphotyrosine antibody. RESULTS: Transfection with Gi 2 gene resulted in increment in insulin-stimulated [3H]2-deoxyglucose (DOG) uptake without affecting basal 2-DOG uptake, but not with Gi 1 and Gi 3 gene transfection. There was unchanged glycogen synthesis rate in all three Gialphasubtypes. Insulin-induced increments of insulin receptor autophos phorylation and IRS-1 phosphorylation were found in Gi 2 protein overexpressed group, only. CONCLUSION: These results suggest that Gi 2 protein may be associated with regulation of biological actions of insulin.
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