Hyperglycemia Suppress Diacylglycerol Kinase-α Expression in Human Neutrophils

Inflammation and oxidative stress are important factors in the pathogenesis of diabetic complications. We have previously shown that poor glycemic control results in significantly increased oxidative burst and protein kinase C (PKC) activity in diabetic neutrophils. The physiologic activator of PKC in neutrophils is diacylglycerol (DAG), which acts as a second messenger in signaling events and is catabolyzed by diacylglycerol kinase (DGK)α. DAG and PKC activity are increased in diabetic neutrophils.

Aim: The aim of this study was to investigate DGK-α expression and activity in neutrophils from diabetic subjects.

Methods: 41 Type 2 diabetic subjects classified according to the ADA criteria and 41 systemically and periodontally healthy subjects were recruited. Peripheral blood neutrophils were isolated and superoxide generation was measured. DAG content was analyzed and DGK-α message and protein were measured by quantitative real-time PCR and Western blotting, respectively. To simulate hyperglycemia in vitro, differentiated HL-60 cells were cultured with normal glucose (5mM), high glucose (25mM), and the RAGE ligand S100b (5ug/ml).

Results: In diabetic neutrophils, poor glycemic control (HbA1c >8.0%) was associated with significantly higher superoxide release. DAG was increased in diabetic neutrophils. The mRNA and protein expression of DGK-α in neutrophils from the poorly controlled group were significantly lower (30%; p<0.05). In cell culture experiments with healthy human neutrophils and differentiated HL-60 cells, hyperglycemia and/or S100b significantly reduced DGK-α expression.

Conclusion: In uncontrolled diabetes, neutrophils are primed to produce excess oxidative stress. The molecular mechanism includes a down regulation of DGK-α and an accumulation of cytoplasmic DAG resulting in chronic activation of PKC and increased inflammation.