The Influences Of Glucose, Insulin, Advanced Glycation End Products, And Lipopolysaccharides On The Functions Of Monocyte-macrophage Cell Line THP-1Cells

Background and Objectives: Chronic low-grade inflammation is thecharacteristics of metabolic syndrome, such as obesity, type2diabetes, atherosclerosis,cardiovascular disease and chronic kidney disease. Macrophages play important roles inthe innate immune system in that activation and balancing of macrophage is the key inmodulating inflammation and its resolution. As a microenvironment of hyperglycemia，Hyperinsulinemia, and advanced glycation endproducts, the macrophages switch into aproinflammatory phenotype, producing Insulin-derived cytotoxic agents, such as TNF-α,IL-6and ROS, which might be related to delayed inflammation resolution and tissuedamage. Tight control of blood sugar is supposed to be very important in reversing theabnormal function of macrophage, but in studies that aimed to normalize blood glucosecontrol as in the NICE-SUGAR trial in critically ill patients and ACCORD trial inpatients around ten years of diabetes duration failed in reducing the all-cause mortalitiesassociated with diabetes. To the author’s knowlodge, until now there are still no studiesthat 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 onexperience of clinical practice. Therefore, we aim study the influence of different bloodglucose levels on functions of the macrophages’ pathogenic killing ability underdifferent microenvironment, i.e. different levels of insulin, AGE concentration, andinflammatory condition with lipopolysaccharide (mimicking “metabolic” endotoxemia).Methods:1. Basic cell cultureHuman monocytic cell line THP-1cells, obtained from Shanghai Cell TypeCulture Collection, were cultured in glucose free RPMI-1640medium supplied with10%heat-inactivated fetal calf serum and5.0mmol/L glucose in5%CO2atmosphere at 37℃. Cells were collected in confluence at the same condition.2. Experimental conditions(1) Cells were first incubated at the concentration of160nmol/L PMA after72hours and collected for further studies.(2) Cells were cultured in medium at different glucose concentrations (5.0,8.0,10.0,15.0,20.0,25.0mmol/L) for72hours and tested on production of reactive oxygenspecies and phagocytic ability.(3) Basal medium with different insulin concentrations (0mIU/L,20mIU/L,50mIU/L,200mIU/L and1000mIU/L) were used to mimic the three conditions met inclinical instances to study the effects of insulin. Cell cultured in medium with differentglucose concentrations were further cultured for another24h culture, collected untilanalysis.(4) Glycated albumin (AGE) was prepared by incubation of bovine serum albumin(BSA) with glucose at room temperature for90days. The cultured THP-1cells weredivided into two groups randomly. The first group, the cells were treated by BSA(100ug/ml) for48hours. The second group, the cells were treated by differentconcentrations of AGE (50,100,200,400ug/ml) for48hours.(5) To mimic the function of macrophages in condition of infection,lipopolysaccharide (LPS,1μg/ml) were added in the conditions of different glucose andinsulin concentrations, cells were incubated24hours and collected for further analysis.Results:1. Influence of glucose, AGE, insulin, and LPS on production of ROS ofTHP-1cell.(1) The production of ROS increased gradually from glucose concentration of5.0mM to25mM.(2) The production of ROS increased gradually from AGE concentration of50mg/L to400.0mg/L.(3) In the range of medium insulin concentrations, the production of ROS waslowest at the concentration of20mIU/L, then the production of ROS rose graduallyfrom50mIU/L until1000mIU/L.(4) Under stimulation of LPS (1μg/ml), LPS increased the production of ROS atevery level of glucose concentrations.(5) Under stimulation of LPS (1μg/ml), LPS increased the cell production of ROSat every level of insulin concentrations. (6) Under stimulation of LPS (1μg/ml), No different effects were observedbetween LPS free and LPS present conditions at every level of AGEs concentrations.2. Influence of glucose, AGE, insulin, and LPS on phagocytic ability of THP-1cell under different conditions.(1) At glucose concentration of5.0mM to10.0mM, The ability of phagocytosismaintained at a high level. Phagocytosis decreased significantly from glucoseconcentration of15.0mM.(2) Phagocytosis was significantly inhibited at every level of AGEsconcentrations.(3) From insulin concentration of20mIU/L to50mIU/L, phagocytosismaintained at a high level and was inhibited at concentration of200mIU/L and1000mIU/L.(4) Under stimulation of LPS (1μg/ml), LPS enhanced the cell phagocytosis atevery level of glucose concentrations. However, peak phagocytic ability were at glucoselevel of5mM, the ability declined from glucose level of8mM and up.(5) Under stimulation of LPS (1μg/ml), LPS enhanced phagocytosis at every levelof insulin concentrations.(6) Under stimulation of LPS (1μg/ml), no different effects were observedbetween LPS free and LPS present conditions at every level of AGEs concentrations.Conclusion: Both AGE and LPS can activate macrophages to produce ROS.Insulin, at concentration of physiological dose, acts as an anti-inflammatory, while atconcentration of hyperinsulinemia insulin promote inflammation. Glucose, atconcentration of5.0-10.0mM promote adequate level of ROS production andphagocytic ability.