Effect of Insulin on the Cell Proliferation and Cell Cycle Progression in Fibroblasts

Yoon, Jeong Bin; Sim, Woo Young; Haw, Choong Rim; Kim, Sung -Soo

Abstract

BACKGROUND: Insulin exerts an effect on cell proliferation and inhibition of apoptosis. However, the actions of insulin on cell cycle progression and signal transduction pathway are not well understood and insulin shows diverse effects on cell proliferation depending on cell types.
OBJECTIVE
We attempted to understand the underlying mechanism by which insulin exerts this proliferative effect on 3T3 L1 fibroblasts by various markers of cell proliferation. METHOD: We investigated the effect of insulin on cell proliferation by [3H]thymidine incorporation, analyzing the cell cycle stages by flow cytometric measurement of DNA content per cell, cell counting, analysing cell division as well as the signal transduction pathway of insulin by measuring of phosphatidylinositol 3-kinase(PI3-kinase) and p44/42 mitogen-activated protein kinase (p44/42 MAPK/ERK) activity.
RESULTS
The results were as follows: 1. Total accumulated [3H]thymidine incorporation increased approximately two-fold with insulin over the 0.5% serum control at 48 h incubation, and the maximal rate of DNA synthesis was observed during 8-12 h incubation and continuously declined until 48 h without a second increase in DNA synthnesis.. 2. The flow cytometric analysis of cell population distribution showed that insulin increased the cell population in S phase. 3. After insulin treatment for 48 h, cell number was increased approximately 45% in comparison with 0.5% serum control. 4. The cell division analysed after staining 3T3 L1 fibroblasts with carboxyfluorescein diacetate succinimidyl ester (CFSE). Cell division occured only once in 24h after insulin treatment.. 5. Insulin stimulated PI3-kinase and p44/42 MAPK/ERK activity about three- and two-folds, respectively.
CONCLUSION
Taken together, this data indicates that insulin stimulated the transit from G0/G1 to S phase, progressed cell cycle through G2/M phase, increased the cell number and PI3-kinase, p44/42 MAPK/ERK stimulate cell proliferation. However, under our experimental conditions, insulin has a limited efficacy for late cell cycle events required for completion of miosis and cell cycle progression into the second round and the increase of the cell number by insulin was much less than the increase of the PI3-kinase and p44/42 MAPK/ERK activity. Therefore, the authors think that another pathways other than PI3-kinase or p44/42 MAPK/ERK might be involved in the effect of insulin on cell proliferation.