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The Effect and Possible Mechanism of High Insulin on CE Accumulation in RAW264.7Cells

Author: Tian
Tutor: LiaoDuanFang; XieZhiZhong
School: Nanhua University
Course: Pharmacology
Keywords: SREBP-1 HSL ACAT-1 HI Macrophage Cholesterol
CLC: R96
Type: Master's thesis
Year: 2012
Downloads: 40
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Abstract


Objective: To explore the effects of high level of insulin on cholesterol esteraccumulation in macrophages and its possible mechanism.Methods: Mouse-derived RAW264.7macrophage cells were cultured in high-glucoseDMEM medium, and interfered with10μg/ml chol:MβCD and differentconcentrations of insulin. Intracellular cholesterol ester content was detected by oilred O staining and enzymic fluorescence method. RT-PCR and Western Blot methodwere used to measure mRNA and protein levels of sterol regulatory element bindingprotein-1(SREBP-1), hormone-sensitive lipase (HSL), and acyl coenzyme Acholesterol acyl transferase-1(ACAT-1), respectively. Moreover, we used smallinterfering RNA to knockdown the endogenous SREBP-1. Within the SREBP-1silenced RAW264.7cells, we measured the level of intracellular cholesterol esters anddetected mRNA and protein expression of HSL and ACAT-1.Results: Treated with10μg/ml chol:MβCD and different concentrations ofinsulin(10-6mol/L or10-9mol/L) for48hours, we found high-dose glucose treatedgroup(25mmol) didn’t increase intracellular cholesteryl ester contents compare tolow-dose group treated group(5mmol) in RAW264.7cells. Treated with10μg/mlchol:MβCD for48hours, intracellular cholesteryl ester contents in RAW264.7cells ofhigh-dose insulin treated group (10-6mol/L, HI group) were obviously higher thanthat of normal/physiological-dose insulin treated group (10-9mol/L, NI group).High-dose insulin treatment also increased SREBP-1but decreased HSL mRNA andprotein expression, while it had no effects on ACAT-1. Furthermore, it was found thatSREBP-1knockdown ameliorated high-dose insulin induced macrophage-derivedfoam cell formation, and up-regulated both mRNA and protein expression of HSLsignificantly.Conclusion: The abnormally high level insulin will up-regulate macrophagesSREBP-1expression, thus inhibiting the expression of HSL, lower intracellular cholesterol ester decomposition, and accelerated foam cell formation.

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