Type‐2 Diabetic/Obese Patients with Associated Vascular Disease Have Increased Circulating Acetylated HMGB1

Abstract

IntroductionType‐2 Diabetes (T2D) is highly associated with peripheral artery disease (PAD), with both PAD and T2D highly associated with chronic inflammation. High mobility group box 1 (HMGB1) is key in regulating both inflammation and diabetes. Acetylated HMGB1 is the extracellularly released form of HMGB1 from post translational modifications (PTM) that occur after nuclear HMGB1 turnover. Acetylated HMGB1 has significant cytokine/chemokine activity. Plasma HMGB1 high molecular weight (HMW) complexes have been identified in the literature as PTM in chronic inflammation. Glycoprotein cytokine Osteoprotegerin (OPG) is a known marker for vascular disease, with research showing that high levels of OPG correlate tightly with increased levels of HMGB1 in T2D patients with PAD. We hypothesize that obese, T2D patients will display elevated levels of circulating acetylated HMGB1 in HMW complexes and that we will find associated high circulating OPG in same patients.MethodsPlasma was collected from 25 male/female adult patients diagnosed with chronic T2D sorted by sex, obese or lean, w/ and w/o vascular disease. Healthy and trauma patients plasma was used as experimental controls. Diluted plasma (1:25) were denatured in SDS page gels and ran for immunoblots with luminescence using the primary antibodies HMGB1 (abcam ab18256) and acetyl‐HMGB1 (Aviva OASG03545). OPG quantification was measured in same plasma samples as above by ELISA (Thermofisher TNFRSF11B) following the manufacturer protocol. Data is represented as mean±SEM. A total of 5‐10 samples per experimental group were analyzed, representative images of immunoblots are shown. Statistical analysis was performed using t‐test and ANOVA with Tukey’s correction.ResultsImmunoblot analysis showed that there is an increase in plasma HMGB1 in plasma samples from T2D, obese patients with vascular disease; predominantly acetylated HMGB1 in HMW complexes (>100kDa) were present within obese‐T2D patients compared to both healthy patients and trauma patients. Since we wanted to understand if vascular disease was associated in plasma samples from these patients, we performed OPG quantification. When we analyzed our cohort, results showed increased OPG in obsese‐T2D patients compared to lean T2D patients with a clear difference by sex (males>females). Obese T2D males with arterial injury have significantly higher circulating OPG compared to lean T2D males with arterial injury.ConclusionsThe drastically higher HMW complexes found uniquely in obese/T2D patients identifies acetylated HMGB1 as the predominant circulating form of HMGB1 in obese/T2D patients. Findings show there might be some association of elevated circulating OPG and acetylated HMGB1 as potential biomarkers. Future studies will focus on understanding the role of acetyl‐HMGB1 in the disease progression of T2D/obese patients with vascular disease.