Glucose modulation of the inflammatory phenotype of SIM-A9 microglial cells

Introduction: Microglia play a central role in neuroinflammation. Their immune function is highly sensitive to changes in the brain’s microenvironment, particularly in nutrient availability. However, most current research in this area relies on glucose concentrations in culture that are not physiologically relevant. This study seeks to establish a more relevant model for investigating microglial immunometabolism, focusing on how glucose availability affects the metabolic and inflammatory responses of SIM-A9 microglial cells. Additionally, the role of oleic acid (OA) in modulating glucose induced changes was examined.

Methods: To define the culture model, SIM-A9 mouse microglial cells were cultured in DMEM/F-12 or 1 mM glucose Neurobasal media, and cellular confluency, viability, and expression and release of pro-inflammatory markers were tested. SIM A9 cells were then cultured in varying glucose concentrations (1, 2, 5, and 17.5 mM), and treated with or without 100 µM oleic acid (OA). Inflammatory responses were assessed by measuring mRNA expression and release of pro-inflammatory cytokines (TNFα and IL1β) by RT-qPCR and ELISA. Lipopolysaccharide (LPS, 2.5 ng/ml) was used as a pro-inflammatory inducer. Reliance on glucose metabolism was analysed by monitoring glucose uptake, the expression of the glycolysis marker (Pfkfb3), and oxidative phosphorylation (OxPhos) complex levels by western blotting. GLUT1 and GLUT3 expression were measured by RT-qPCR and western blotting to assess glucose transporter regulation.

Results: SIM-A9 cells maintained confluency in 1 mM glucose without an elevated pro inflammatory response compared to culture in DMEM/F-12. In the absence of 4 OA or LPS, culture in 5 mM glucose resulted in the most prominent pro-inflammatory response (57% increase of TNFα release and 80-fold increase of expression, compared to 1 mM glucose NB). OA significantly reduced TNFα release and expression in low glucose, with the highest reduction at 5 mM. In contrast, culture in 17.5 mM glucose produced inconsistent results, suggesting this concentration may not be relevant for modelling the brain’s glucose environment. OA treatment also induced a shift away from glycolysis, where reduced pro inflammatory activation was observed, while increasing OxPhos activity.

Conclusion: This study highlights the need for a more physiologically relevant model in microglial immunometabolism research and suggest that both glucose and lipid availability and metabolism can modulate SIM-A9 microglial function through both glucose and lipid metabolism. This could provide important insights into neurodegenerative diseases, such as Alzheimer’s disease, where metabolic dysregulation and chronic inflammation are key pathological factors.