Diabetes & Metabolism Journal

Original Articles

Cell Cycle Progression of Vascular Smooth Muscle cell Through Modulation of p38 MAPK and GSK-3beta Activities Under High Glucose Condition.: Yang Ho Kang, In Ju Kim, Yong Ki Kim, Seok Man Son; Korean Diabetes J. 2005;29(5):418-431. Published online September 1, 2005

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Abstract PDF: BACKGOUND: Macroangiopathy, with atherosclerosis, is the leading cause of mortality and morbidity in diabetic patients. Vascular smooth muscle cells play a crucial role in atherosclerosis, as they proliferate, migrate and express genes that encode inducible growth factors. However, the mechanisms induced by hyperglycemia that accelerate the proliferative change of vascular smooth muscle cells in diabetes remain unclear. This study was aimed at clarifying the respective roles of hyperglycemia in the acceleration of vascular complications in diabetes, examine the effects of hyperglycemia on vascular smooth muscle cell proliferation and the possible underlying mechanisms, including cell cycle progression. METHODS: Primary cultured rat aortic RASMs were exposed to normal glucose(5 mmol/L D-glucose), high glucose(30 mmol/L D-glucose) or an osmotic control (5mmol/L D-glucose plus 24.5 mmol/L mannitol) for 72 hours. The effect of high glucose on cell proliferation was determined by assessing the cell count and BrdU incorporation. Proteins involved in the cell proliferation pathway (PDK1, Akt/PKB, p42/44 MAPK, p38 MAPK, GSK-3beta) and those in cell cycle progression (cdk4, cyclin D, cdk2, cyclin E and ppRb phosphorylation) were determined by Western blot analysis. cdk4 kinase and PKC activity assays were also performed. RESULTS: A high level of glucose increased both the cell count(P<0.01) and BrdU incorporation(P<0.01). The PDK1, Akt/PKB and p42/44 MAPK activities were not significantly increased. A high level of glucose significantly increased the activities of p38 MAPK (P<0.01) and GSK-3beta(P<0.05) and the expressions of cdk4, cyclin D and ppRb phosphorylation. The cdk4 (P<0.01) and PKC (P<0.05) activities were also significantly increased. The inhibition of protein kinase C with GF109203X markedly reduced the phosphorylations of p38 MAPK and GSK-3betaand the expressions of cdk4 and cyclin D. In addition, pretreatment with GF109203X decreased the cell number in response to a high glucose level. CONCLUSION: These findings suggest that a high level of glucose increases vascular smooth muscle cell proliferation, with the possible mechanism further increases the G1 to S phase cell cycle progression via the activation of PKC, p38 MAPK and GSK-3beta.

Increase in Fatty Acid Oxidation by AICAR: the Role of p38 MAPK.: Woo Je Lee, Jin Yob Kim, Sung Jin Bae, Eun Hee Koh, Sung Min Han, Hye Sun Park, Hyun Sik Kim, Min Seon Kim, Joong Yeol Park, Ki Up Lee; Korean Diabetes J. 2005;29(1):15-21. Published online January 1, 2005

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Abstract PDF: BACKGROUND
AMPK is an enzyme that increases glucose transport and fatty acid oxidation in skeletal muscle. The activation of AMPK stimulates fatty acid oxidation by decreasing the acetyl CoA carboxylase (ACC) activity and the concentration of malonyl-CoA. However, a recent study has reported a dissociation of AMPK activity and ACC phosphorylation in skeletal muscle during periods of prolonged exercise. This suggested that there is an additional mechanism for AMPK-induced fatty acid oxidation in skeletal muscle. METHODS: Plamitate oxidation was measured via the generation of [3H]-water generation from 9,10[3H]-palmitate after treating various concentrations of AICAR on the C2C12 mouse skeletal muscle cell line. Western analysis was used to test for the possible activation of p38 MAPK by AICAR. Involvement of p38 MAPK in the AICAR-induced increase in fatty acid oxidation was tested for by using SB203580, a p38 MAPK inhibitor. RESULTS: C2C12 cell treated with AICAR exhibited a dose-dependent increase in fatty acid oxidation compared to the cells that were not treated with AICAR. Western blot analysis revealed that phosphorylation of p38 MAPK was increased 2.5 folds after AICAR treatment. The increase of fatty acid oxidation with AICAR treatment was significantly inhibited by a treatment of SB203580; this indicated the involvement of p38 MAPK on the AICAR-induced increase in fatty acid oxidation. CONCLUSION: AICAR stimulated the fatty acid oxidation by activating p38 MAPK. This is a novel pathway by which AMPK activation in skeletal muscle increases the fatty acid oxidation

Mitogenic Effects and Signaling Pathway of Insulin-Like Growth Factor-I (IGF-I) in the Rat Beta Cell Line (INS-1).: In Kyung Jeong, Ja Young Kim, Hyung Joon Yoo, Myung Shik Lee, Moon Kyu Lee, Kwang Won Kim; Korean Diabetes J. 2004;28(6):478-489. Published online December 1, 2004

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Abstract PDF: BACKGROUND
Nutrients and growth factors are known to stimulate pancreatic beta cell mitogenesis. IGF-I acts as a survival factor by limiting apoptosis and stimulating proliferation in many cell types. However, the appropriate mitogenic signaling pathways have not been defined. The aim of this study is to elucidate the mitogenic effect and signaling pathways of IGF-I in the rat beta cell line (INS-I). METHODS: The studies were performed using the rat pancreatic beta cell line, INS-1. INS-1 cells were cultured in RPMI 1640 containing serum-free, 0.2% BSA and 11.1 mmol/L glucose media for 24 hours, and the cells were then treated with IGF-I and different concentrations of glucose or tyrosine phosphorylation inhibitors, or insulin. The cell proliferation was measured by the [3H]thymidine uptake and MTT assay. The cell cycle was analyzed by a flow cytometer by using propidium iodide staining. Western blot analyses were performed using antibodies against PY20 and phospho-MAPK. RESULTS: 1) MTT assay and the [3H]thymidine uptake showed that IGF-I stimulated the INS-1 cell proliferation in a dose dependent manner. Glucose was noted to independently increase the INS-1 cell proliferation. A combination of IGF-I and glucose has a synergistic effect on the proliferation of INS-I cells. Insulin did not influence on the mitogenic effect of IGF-I. 2) The S fraction of INS-1 cells treated with IGF-I was increased in a dose dependent manner. IGF-I stimulated the exit from G1 into the S phase of the cell cycle. 3) Investigation of the role of the PI3K and MAPK, by using of the inhibitors LY294002, wortmannin, and PD98059, demonstrated that the activation of MAPK, but not PI3K, required to stimulate the proliferation of INS-1 cells. 4) IGF-I stimulated the phosphorylation activation of pp60 and phospho-MAPK in the INS-1 cells. IGF-I induced the beta cell proliferation, and this was mediated via a signaling mechanism that was facilitated by MAPK. CONCLUSION: The proliferative effect of IGF-I on pancreatic beta cell seems to be mediated through MAPK signaling pathway.

The Role of Akt-1/PKBalpha on Insulin Action in 3T3-L1 Adipocyte.: Jung Min Lee, Hyun Shik Son, Hyuk Sang Kwon, Seung Ki Kwack, Seung Hyun Ko, Sang Ah Chang, Kun Ho Yoon, Bong Yun Cha, Kwang Woo Lee, Ho Young Son, Sung Koo Kang, Prem Sharma; Korean Diabetes J. 2002;26(4):274-285. Published online August 1, 2002

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Abstract PDF: BACKGROUND
S: Akt/PKB as a serine/threonine kinase is stimulated by insulin and other growth factors. And insulin stimulates glucose uptake by promoting the translocation of glucose transporter 4 (GLUT4) to the cell membrane. But, it is not clear that Akt/PKB, a downstream target of PI 3-kinase, is involved in glucose uptake pathway. In this study, we investigated the role of Akt/PKB, especially Akt-1, on insulin action in 3T3-L1 adipocyte. METHODS: We made recombinant Ad5.Akt-1 vector by the insertion of Akt-1 gene to adenoviral vector. And then, we overexpressed Akt-1 proteins(wild type and kinase inactive type) in 3T3-L1 adipocytes by using a adenoviral transfection method. We observed the changes of glucose uptake, glycogen synthesis, activities of mitogen-activated protein kinase (MAPK, also called extracellular signal-regulated kinase), p70 ribosomal s6 protein kinase (p70s6k), and glycogen synthase kinase 3 (GSK3) according to Akt-1 activity and insulin treatment. RESULTS: First, overexpression of Akt-1 did not affect to glucose uptake, whether insulin stimulates or not. Second, overexpression of Akt-1 did not affect the phosphorylation of p44/42-MAPK, either. Third, the glycogen synthesis was increased by overexpression of Akt-1. CONCLUSION: Akt-1 activation is necessary for glycogen synthesis, but is not essential for glucose transport in 3T3-L1 adipocytes.

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