Interaction Of Glucose And Insulin On The Function And Bmi-1Gene Expression Of Monocyte Macrophage Cell Line THP-1Cells

Objective: Type2diabetes mellitus (DM) is characterized with chronic hyperglycemiaand alomost always, if not all, with hyperinsulinemia, indicating the association withinsulin resistance. Low-grade chronic inflammation is another accompanying feature inpatients with DM. Macrophage is recently shown to be a key inflammatory cell in thestarting fumy fire and important in the process of injury-repair and bacteria eridication.Hyperglycemia and hyperinsulinemia is sure in disturbing the adequate function ofmacrophage. No study is undertaken until now on threshold level of hyperglycemia andhyperinsulinemia and their interaction in which the function of macrophage iscomprised in balancing the ROS damage with effective clearing of injured tissue andinvading germs. Therefore, in this study we aim to study the different levels of glucose,and their interaction on the function and the expression of the proliferation associatedtranscriptor Bmi-1gene of macrophage.Methods:Human monocytic cell line THP-1cells were cultured in RPMI-1640medium(including the tendency for5.0mmol/L glucose) containing10%heat-inactived fetalbovine serum at37℃and5%CO2. Cells were collected at confluence. THP-1cellswere induced to differention with100nM phorbol-12-myristate-13-acetate (Sigma,USA) for72hours.THP-1cells were incubated under different concentrations containing finalconcentration of5.0,10.0and20.0mmol/L of glucose and different insulinconcentrations (0,20,50and200IU/L) for48hours respectively.For ROS production---Fluorescence Spectrophotometer analysis For phagocytic ability---Microplate System analysisFor Bmi-1expression---Real Time PCR and Western BlotResults:1. The effects of different levels of glucose and inuslin on the function of THP-1cell.Without insulin in medium, the ROS production of THP-1cells increased graduallyfrom5.0mmol/L to20.0mmol/L of glucose concentration. The phagocytotic ability didnot decrease at10.0mmol/L of glucose, but decreased significantly at20.0mmol/L ofglucose concentration.Under the condition of5mmol/L of glucose, insulin enhanced the ROS productionof THP-1after addition of insulin, and there was no significant difference in the ROSproduction through insulin concentration from20,50and to200IU/L. The phagocytoticability decreased from20,50, and to200IU/L of insulin levels.The lowest ROS production was observed at glucose level of10.0mmol/L withinsulin level of20IU/L, while the peak ROS production were at glucose level of20.0mmol/L with insulin level of200IU/L. The phagocytic ability maintained at a high levelwith glucose level of5and10.0mmol/L with insulin level of20IU/L.2. The effects of different levels of glucose and inuslin on the THP-1cell expresson ofBmi-1Increasing levels of glucose and insulin both decreased the expression of Bmi-1gene in THP-1cells. The suppression of Bmi-1expression both at mRNA level andprotein level was enhance with increase combination of glucose and insulin levels.Conclusions: A glucose level of less than10mmol/L with insulin level of20IU/L wasthe best environment for effective defence against invading germs. Although higher than10mmol/L of glucose and higher than20IU/L of insulin increase the killing ability（ROS production）, the ability of phagocytosis is decreased, therefore the efficiency ofbacteria killing will be compromised. Increasing levels of glucose or insulin, and theircombination reduced the expression of Bmi-1gene in THP-1macrophages. This effectneeds to be further studied.