The Influences Of Blood Glucose Levels On The Function Of Neutrophil-moncyte Cell Line HL-60 Cells

Background and Objectives: Patients with diabetes are prone to suffer from bacterial infection, diabetic chronic complications. Neutrophils play critical roles in host defense against various bacterial infections. Treatment of hyperglycemia decrease infection rate and enhances the patient recover from infection. But in studies that aimed to normalize blood glucose control as in the NICE-SUGAR trial in critically ill patients and ACCORD trial in patients around ten years of duration failed in reducing total mortality associated with diabetes. Until now there are still no studies that aimed to explore the optimal range of glucose levels and excursion in diabetes. Most of the current consensus on target levels of blood glucose was based on experience of clinical practice. Therefore, we aim study the influence of different blood glucose level on functions of the pathogen kill neutrophils in a fashion of serial increase in modulating glucose concentration in step increment, and in states that mimic the existence of hyperinsulinemia and bacteria (endotoxemia).Methods:1. Basic cell cultureA human myeloid leukemia cell line HL-60 cells, maintained in the Department of Hematology, Dalian Medical university College of Laboratory Medicine, were cultured in glucose free RPMI-1640 medium supplied with 10% heat-inactivated fetal calf serum and 5.0 mmol/L glucose in 5%CO2 atmosphere at 37℃. Cell were collected in exponential growth stage at the same condition, and treated with different factors.2. Experimental conditions(1) Different glucose levels (3.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 15.0, 20.0 mmol/L, and 20 mmol/L mannitol as an osmotic control) were to be tested on the functions of reactive oxygen species production and phagocytosis. Cells were cultured for 72 hours and collected for further analysis.(2) Cell culture medium with different insulin concentrations (0 mIU/L, 20 mIU/L as physiological dose, 50 mIU/L as stress dose, 1000 mIU/L as extreme hyperinsulinemia) were used to mimic the three conditions met in clinical instances to study the effects of insulin. Cell cultured in medium with different glucose concentrations were further cultured for another 24h culture, collected until analysis.(3) Cells were first incubated in the concentration of 160 nmol/L PMA after 24 hours and collected further studies. To mimic the function of neutrophils in condition of infection, lipopolysaccharide (LPS, 10μg/ml) were added in the conditions of different glucose and insulin concentrations, cells were incubated 8 hours and collected for further analysis.Results:1. Influence on the phagocytic ability of HL-60 cell under different conditions of glucose, LPS and insulin.(1) The cell phagocytic ability increased gradually from glucose concentration of 5.0 mmol/L, reached maximum at 8.0 mmol/L (P<0.05), and returned baseline at 10.0mmol/L.(2) Under stimulation of LPS (10μg/ml), peak phagocytic ability were at glucose level of 3.5 mmol/L (P<0.05), the ability declined to similar levels as in LPS free medium at the same equivalent glucose level of 6.0 mmol/L, the picture went as in conditions of increase in glucose conditions.(3) On culture at the existence of insulin, the phagocytic increments at 5.0 mmol/L and 10.0 mmol/L of glucose concentration were inhibited, there was no peak effect observed on different insulin conditions and LPS free condition. No different effects were observed between LPS free and LPS present conditions.2. Influence on production of ROS of HL-60 cell under different conditions of glucose, LPS and insulin.(1) There was no observed effect on ROS production of HL-60 cells from glucose concentration of 5.0 mmol/L until 20.0 mmol/L.(2) LPS increased the cell production of ROS at every level of glucose concentrations (P<0.05). No peak was observed.(3) At the presence of LPS, insulin at concentration of 20 mIU/L or 50 mIU/L did not increase or inhibit the production of ROS, and the levels of ROS produced were similar to the effects at the presence of LPS, but when the medium contained 1000 mIU/L insulin, the production of ROS was significantly inhibited at every level of glucose concentrations.Conclusions:Glucose could increase the functions of phagocytosis at concentration of 5.0 ~ 10.0 mmol/L and does not affect on production of ROS in condition of insulin deficiency, Insulin has no effects on the phagocytotic ability and ROS production at pathogen free condition. Pathogen product, such as LPS increases the production of ROS, while it does affect the phagocytotic ability. Insulin at concentrations of physiological and stress-associated moderate hyperinsulinemia has no effect on ROS production at the presence of LPS, but extreme hyperinsulinemia inhibits the ROS production.