cAMP activates the generation of reactive oxygen species and inhibits the secretion of IL-6 in peripheral blood mononuclear cells from type 2 diabetic patients

Abstract

Peripheral blood mononuclear cells (PBMNC) from patients with type 2 diabetes (DM2) have generated higher levels of reactive oxygen species (ROS) that were higher than those in cells from healthy individuals. In the presence of a cAMP-elevating agent, ROS production was significantly activated in PBMNC from DM2 patients but it was inhibited in cells from healthy subjects. Higher levels of IL-6 has been detected in the supernatant of PBMNC cultures from DM2 patients in comparison with healthy controls. When cells were cultured in the presence of a cAMP-elevating agent, the level of IL-6 decreased has by 46% in the supernatant of PBMNC from DM2 patients but it remained unaltered in controls. No correlations between ROS and IL-6 levels in PBMNC from DM2 patients or controls have been observed. Secretions of IL-4 or IFNgamma by PBMNC from patients or controls have not been affected by the elevation of cAMP. cAMP elevating agents have activated the production of harmful reactive oxidant down modulated IL-6 secretion by these cells from DM2 patients, suggesting an alteration in the metabolic response possibly due to hyperglicemia. The results suggest that cAMP may play an important role in the pathogenesis of diabetes.

Figure 1

Reactive Oxygen species (ROS) generation by peripheral blood mononuclear cells from type 2 diabetic patients—PBMNC = peripheral blood mononuclear cells; the values were compared by Student “t” test; p < 0.05 were considered as significant; RLU/min = Relative Light Units per minute; PBS = phosphate buffered saline. ROS production was greater in DM2 than in healthy control. DM2 = type 2 diabetic patients; PBMNC = peripheral blood mononuclear cells.

Figure 2

Effect of cyclic AMP (cAMP) on ROS production in PBMNC from type 2 diabetic patients in comparison to healthy subjects. RLU/min = Relative Light Units per minutes; PBMNC= Peripheral blood mononuclear cells; The average were compared by Student “t” test and p < 0.05 was considered as a significant difference.

Figure 3

The effect of cAMP on ROS production in PBMNC from DM2 patients and from healthy subjects. The results are expressed in the form of the ratio E/C for individual subjects, where E refers to cells cultured in the presence of cAMP (experiment) and C refers to cells cultured in the absence of the additive (Control). E/C = [RLU/min produced by PBMNC in the presence of cAMP]/[RLU/min produced by PBMNC in the absence of cAMP]. RLU/min = Relative Light Units per minutes; PBMNC= peripheral blood mononuclear cells.

Figure 4

Quantification of IL-6 in supernatant of cultured PBMNC. PBMNC (peripheral blood mononuclear cells) were cultured in RPMI-1640 and the culture supernatant, cell-free, was assayed for interleukin 6 (IL-6). Comparison of IL-6 in supernatant of PBMNC from DM2 and Healthy control. Peripheral blood mononuclear cells (PBMNC) were cultured in RPMI-16-40 for 48 h at 37°C. The supernatant cell-free was used for quantification of inteleukin 6 (IL-6) in an ELISA commercial kit. The values were compared by Student “t” test and the difference between IL-6 from healthy control and from type diabetic patients (DM2) was significant.

Figure 5

(A and B) represent typical curves of kinetics studies on reactivie oxygen species (ROS) generation by peripheral blood mononuclear cells (PBMNC) either from healthy control (A) or from type 2 diabetic patients (B) in the presence or in the absence of cyclic AMP (cAMP).