β-hydroxybutyrate improves the redox status, cytokine production, and phagocytic potency of glucose-deprived HMC3 human microglia-like cells

Neurology Biochemistry Life Sciences Immunology Medicine Cell Biology
β-hydroxybutyrate HMC3 cytokines glucose deprivation human microglia phagocytosis

Research Report: β-Hydroxybutyrate Improves Redox State, Cytokine Secretion, and Phagocytic Ability of Glucose-Deprived HMC3 Human Microglia

Introduction

Microglia are resident neuroimmune cells in the brain, accounting for 5-12% of total neuroglial cells, with strong migration, proliferation, and phagocytic abilities. For example, microglia maintain a healthy brain microenvironment by phagocytosing apoptotic cellular debris in the brain parenchyma. In cases of central nervous system infection or injury, microglia migrate to the site of injury, phagocytose invading pathogens or debris from dead cells, and help damaged tissue recover by releasing inflammatory cytokines, chemokines, and growth factors. Although microglia exhibit complex functional diversity, it is unclear how impairment of glucose metabolism affects their function. Glucose metabolic disorders are common in almost all neurodegenerative and psychiatric diseases, such as stroke, Parkinson’s disease, Alzheimer’s disease, substance use disorders, anxiety, and depression. Diabetic patients may also experience temporary hypoglycemia when receiving insulin treatment. Understanding the effects of glucose deprivation on microglial function is of great significance for the mechanistic study and treatment of these diseases.

Paper Source

This paper was written by Anil Kumar Rana, Babita Bhatt, and Mohit Kumar et al., from the National Agri-Food Biotechnology Institute and Regional Centre for Biotechnology. It was published in the Journal of Neuroimmune Pharmacology in 2024. The DOI is 10.1007/s11481-024-10139-5.

Research Workflow

This study investigated changes in phenotypic state, redox state, cytokine secretion, and phagocytic ability of the human microglial cell line HMC3 cultured in vitro under glucose deprivation conditions. Additionally, the study explored the restorative effects of β-hydroxybutyrate (BHB) as a metabolic supplement on these characteristics.

Experimental Design and Methods

  1. Cell Culture and Treatment:

    • HMC3 cells were cultured in high glucose (25mM) DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin and streptomycin antibiotics.
    • At 80–90% confluence, cells were replaced with fresh high glucose DMEM for 24 hours. Glucose deprivation and BHB supplementation experiments used glucose-free DMEM and glucose-free DMEM supplemented with 5mM BHB to culture cells for 24 hours.

  2. MTT Assay:

    • MTT reduction depends on NAD(P)H-dependent oxidoreductases and is a direct indicator of oxidative phosphorylation and pentose phosphate pathway activity. The experiment verified the effect of glucose deprivation on cellular metabolic activity by detecting cellular oxidative phosphorylation levels.

  3. Nitrite Level Determination:

    • Griess reagent was used to detect nitrite levels in cells under different conditions to assess the redox state of the cells.

  4. Cell Death Assay:

    • Trypan blue staining method was used to study cell death. Trypan blue dye cannot pass through intact cell membranes and is repelled by negatively charged dye, but can enter damaged dead cells.

  5. Superoxide Levels:

    • Dihydroethidium (DHE) dye was used to detect superoxide levels in cells exposed to different conditions, evaluating the cell’s antioxidant stress state.

  6. Mitochondrial Staining:

    • MitoTracker™ Red dye was used to assess the mitochondrial health of cells.

  7. Quantitative PCR:

    • Quantitative reverse transcription PCR (qPCR) was used to analyze mRNA expression of genes.

  8. Enzyme-Linked Immunosorbent Assay (ELISA):

    • Used for quantitative detection of cytokine levels such as TNF, IL-1β, IL-6, and IL-10 secreted by HMC3 cells under normal and glucose-deprived conditions.

  9. Phagocytosis Assay:

    • Fluorescently labeled latex beads were used as phagocytic substrates to evaluate the phagocytic ability of cells under different treatment conditions.

  10. Western Blot:

    • Used to detect the expression levels of proteins such as Nox2, BDH1, and SCOT in HMC3 cells under normal and glucose-deprived conditions.

Research Results

  1. Effects of Glucose Deprivation on Energy Metabolism and Redox State:

    • MTT assay results showed that glucose deprivation significantly reduced the metabolic activity of HMC3 cells (p<0.0001), and mitochondrial health was also impaired (decreased Mitotracker staining). Although cells did not show death (trypan blue staining), overall energy metabolism and redox state were impaired.
    • Cells showed increased Nox2 mRNA expression (p=0.007) under glucose deprivation conditions, but protein levels decreased. Additionally, superoxide and nitrite levels were significantly reduced, indicating impaired antioxidant stress capacity of cells under glucose deprivation conditions.

  2. Effects of Glucose Deprivation on Cytokine Secretion:

    • qPCR analysis showed increased IL-1β mRNA expression (p=0.0009) and significantly decreased TNF mRNA expression (p=0.0009) under glucose deprivation conditions. IL-6 mRNA expression showed no significant change.
    • ELISA results further confirmed decreased secretion of IL-1β and TNF under glucose deprivation conditions, while IL-6 and IL-10 secretion showed no significant changes.

  3. Effects of Glucose Deprivation on Phagocytic Ability:

    • qPCR analysis found significantly increased mRNA expression of TREM2 (p=0.016) and CD68 (p=0.0006) under glucose deprivation conditions. However, functional phagocytosis experiments (latex beads) showed that glucose deprivation significantly reduced the phagocytic activity of cells.

  4. Restorative Effects of BHB Supplementation:

    • BHB supplementation significantly restored IL-1β secretion (p=0.0328) and phagocytic activity (p=0.0261) in glucose-deprived HMC3 cells.
    • Mechanistically, BHB supplementation increased BDH1 protein expression (p=0.0423), but SCOT expression showed no significant change. Additionally, BHB supplementation improved mitochondrial health (significantly increased MTT reduction levels, p<0.0001).

Conclusion

This study demonstrates that glucose metabolic disorders may affect the phagocytic and neuroimmune functions of microglia, potentially exacerbating pathological changes in neurological diseases. Supplementation with ketone bodies (such as BHB) can serve as an alternative energy source, restoring energy metabolism, redox state, cytokine secretion, and phagocytic ability of microglia under glucose deprivation conditions. Therefore, BHB may serve as a potential metabolic therapy to improve these pathological conditions. In the future, further research is needed to validate these findings in more complex in vivo environments.

Research Highlights

  • First in-depth exploration of the effects of glucose deprivation on energy metabolism, redox state, cytokine secretion, and phagocytic ability of human HMC3 microglia.
  • Found that glucose deprivation significantly reduced the metabolic activity and antioxidant stress capacity of microglia, and weakened their cytokine secretion and phagocytic function.
  • Proposed BHB supplementation as an alternative energy source that can effectively restore microglial function under glucose deprivation conditions.