Cardiac HMGB1/TLR4 Activation in Pre‐Type 2 Diabetes: A Potential Mechanism Triggering Diabetic Cardiovascular Complications

Abstract

The prevalence of type 2 diabetes mellitus (T2DM) continues to increase at an alarming rate, to the point of epidemic standards, within the United States. One of the major, potentially lethal, complications of T2DM is the development of cardiovascular disease. Toll‐like receptor 4 (TLR4), a key component of innate immunity, has been implicated in diabetic complications due to its resultant increase in inflammation and oxidative stress. While previous findings from our lab have identified that vascular TLR4 plays a major role in DM vascular complications, the activation of TLR4 in the heart from pre‐T2DM has yet to be explored. We hypothesize that TLR4 signaling is activated in the heart of pre‐diabetic rats and is linked to an early inflammatory state. Male Goto‐Kakizaki (GK) rats, a non‐obese model of T2DM, were utilized in this study, with eight and sixteen week old animals representing the pre‐T2DM and T2DM groups, respectively. Wistar rats served as the non‐T2DM (control) group. As expected, blood glucose levels (mg/dL) were elevated in the T2DM group (162.75± 11.32 vs. 85.83 ± 8.25 control group, p<0.001, n=6). A slight increase in blood glucose levels were observed in the pre‐T2DM group (121 ± 7.75 vs. 85.83 ±8.25 control group, p<0.05). T2DM rats exhibited increased systolic blood pressure (SBP) (149± 12mmHg), an expected cardiovascular complication in T2DM, while pre‐T2DM rats displayed normal SBP (112 ± 7 mmHg). To determine whether pre‐T2DM conditions modulate TLR4 signaling in cardiac tissue, western blot analysis was performed. Cardiac TLR4 protein expression was markedly elevated in the pre‐T2DM group (3.1±0.1 fold increase vs. T2DM, p<0.01, n=4), paralleled by increased NFKβ expression (2.4 ± 0.2 fold increase vs. T2DM, p<0.05, n=4), an inflammatory marker and downstream target of TLR4 signaling. MyD88, an adaptor protein of TLR4 signaling, remained unaltered in cardiac tissue of pre‐T2DM subjects, suggesting that cardiac TLR4 activation in pre‐T2DM may occur via an MyD88‐independent mechanism. Of note, we found that high‐mobility group box 1 protein (HMGB1) expression was markedly increased in pre‐T2DM hearts (1.3 fold increase vs. control, p<0.01, n=4). HMGB1 is a damage‐associated molecular pattern (DAMP) molecule that has been suggested to be an endogenous TLR4 ligand. This finding implies HMGB1 may be a sterile ligand candidate in the activation of TLR4‐mediated inflammation in pre‐diabetic milieu. In conclusion, our results suggest that HMGB1/TLR4/NFKβ signaling pathway is activated in pre‐T2DM hearts. Therefore, strategies to target either HMGB1 or TLR4 pose a potential alternative therapy in the prevention of diabetic cardiovascular complications.Support or Funding InformationNIH