Manon Dumont¹, Julia van Heck¹, Rick Meijer¹, Cees Tack¹, Rinke Stienstra^1,2

¹Department of Internal Medicine, Radboudumc, Nijmegen, ²Division of Human Nutrition and Health, Wageningen University, Wageningen

Manon.dumont@radboudumc.nl

Background: Glucose variability (GV) in diabetes is associated with an increased risk of developing vascular complications, in which inflammation is known to play a role. As macrophages are important contributors to vascular inflammation, this study aims to elucidate the molecular mechanisms and cellular pathways through which GV modulates macrophage inflammatory responses.

Methods: Monocyte-derived macrophages from healthy donors were exposed to glucose concentrations fluctuating between 0.5 and 29.5 mM every 24 hours for 4 days. Afterwards, glucose concentrations were stabilised to 15 mM. RNA sequencing was performed to uncover transcriptional changes in macrophages related to GV, in comparison to a condition kept constant at 15 mM glucose the entire time.

Results: We observed a total of 215 differentially expressed genes (DEGs) in macrophages exposed to glucose fluctuations as compared to macrophages exposed to constant glucose, which persisted after glucose levels were stabilised (FDR <0.05, n=3). Pathway-level enrichment analysis revealed that the highlighted DEGs are involved in upregulation of inflammatory signalling (Normalised enrichment score (NES) = 1.90, FDR <0.05), particularly via NFκB (NES = 1.78, FDR <0.05), in addition to increased expression of C-X-C chemokine receptor type 4 (FC = 1.34, FDR <0.05). Hypoxia signalling was also upregulated (NES = 2.19, FDR <0.05), including upregulation of VEGFA (Fold change (FC) = 1.42, FDR <0.05). Furthermore, genes related to glycolysis were upregulated after exposure to glucose fluctuations (NES = 1.74, FDR <0.05), including upregulation of SLC2A1/GLUT1 (Fold change = 1.63, FDR <0.05), whereas genes related to oxidative phosphorylation were downregulated (NES = -2.14, FDR <0.05).

Discussion/Conclusion: The results indicate that glucose fluctuations induce persistent transcriptional changes in macrophages that align with heightened inflammatory signalling, which suggests the involvement of cellular pathways specifically responsive to GV. Next, we aim to determine how these transcriptional changes shape macrophage functional responses in the context of vascular complications.