Measurement of autophagic flux from human peripheral blood mononuclear cells in health and cardiometabolic disease

Introduction: Autophagy is a conserved intracellular degradation pathway responsible for the turnover of damaged organelles and proteins. Dysregulation of autophagy has been implicated in cardiometabolic diseases, including Type 2 Diabetes Mellitus (T2DM). However, most prior human studies rely on static autophagy markers rather than dynamic assessments of autophagic flux. Whether autophagic flux is altered in T2DM remains unclear. We hypothesized that autophagic flux (LC3-II accumulation following lysosomal inhibition) and Transcription Factor EB (TFEB) expression would be reduced while sequestome 1 (p62/SQSTM1) expression would be increased in peripheral blood mononuclear cells (PBMCs) from individuals with T2DM compared with healthy controls. Methods: Peripheral whole blood was sampled from healthy participants (n = 5) and patients with T2DM (n = 4). Whole blood was treated with the autophagy inhibitor, bafilomycin A1 (100 nM, 60 minutes), or equal volume DMSO, and peripheral blood mononuclear cells (PBMCs) were isolated via density centrifugation. Markers of autophagy such as LC3BI and II, TFEB, and p62/SQSTM1 were determined via Western blot. Autophagic flux (ΔLC3-II) was calculated as the difference in LC3-II expression between bafilomycin A1 and vehicle conditions. Proteins of interest were normalized to β-actin or GAPDH. Using mean gray value, data are presented as fold change from healthy control. Data are expressed as mean±SD, and significance was defined as P< 0.05. Results: TFEB expression was not different between healthy and T2DM participants at baseline (Fold Change: 3.0±3.8 vs. 1.0±1.5 healthy controls; P = 0.27). Neither expression of p62 (Fold Change: 0.57±0.46 vs. 1.0±1.3; P = 0.62) or autophagic flux (ΔLC3II Fold Change: 8.1±11.6 vs. 1.0±2.0; P = 0.27) was different between T2DM and healthy controls at baseline. Conclusion: In this pilot sample, PBMC autophagic flux and TFEB expression were not significantly altered in individuals with T2DM. The substantial variability observed highlights the need for larger cohorts and complementary approaches. Ongoing work will assess TFEB subcellular localization and autophagy dynamics using nuclear fractionation, qPCR, and confocal imaging, as well as mechanistic perturbations including high-glucose exposure, viral gene delivery, and siRNA-based modulation. Funding for this project is supported by NHLBI 5R00HL161491-04 (WEH) This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.