Microglial Function during Glucose Deprivation: Inflammatory and Neuropsychiatric Implications

Abstract

Inflammation is increasingly recognized as a contributor to the pathophysiology of neuropsychiatric disorders, including depression, anxiety disorders and autism, though the factors leading to contextually inappropriate or sustained inflammation in pathological conditions are yet to be elucidated. Microglia, as the key mediators of inflammation in the CNS, serve as likely candidates in initiating pathological inflammation and as an ideal point of therapeutic intervention. Glucose deprivation, as a component of the pathophysiology of ischemia or occurring transiently in diabetes, may serve to modify microglial function contributing to inflammatory injury. To this end, primary microglia were cultured from postnatal rat brain and subject to glucose deprivation in vitro. Microglia were characterized for their proliferation, phagocytic function and secretion of inflammatory factors, and tested for their capacity to respond to a potent inflammatory stimulus. In the absence of glucose, microglia remained capable of proliferation, phagocytosis and inflammatory activation and showed increased release of inflammatory factors after presentation of an inflammatory stimulus. Glucose-deprived microglia demonstrated increased phagocytic activity and decreased accumulation of lipids in lipid droplets over a 48-h timecourse, suggesting they may use scavenged lipids as a key alternate energy source during metabolic stress. In the present manuscript, we present novel findings that glucose deprivation may sensitize microglial release of inflammatory mediators and prime microglial functions for both survival and inflammatory roles, which may contribute to psychiatric comorbidities of ischemia, diabetes and/or metabolic disorder.

Keywords: Depression; Diabetes; Hypoglycemia; Inflammation; Ischemia; Microglia.

Fig. 1

Glucose deprivation is insufficient to induce inflammatory release from microglia. a Schematic timeline of treatment paradigm. For each condition, GD began 1 or 24 h (for 24 or 48 h total time points, respectively) prior to 24 h 100 ng/ml LPS treatment. b–d Release of NO (b), IL1β (c) and TNF (d) is significantly increased by treatment with LPS. GD increases release of NO and IL1β significantly above the LPS treated, normal glucose condition at 24 and 48 h, respectively (N = 4). MTT assay (e), a measure of oxidative phosphorylation, was significantly decreased in microglia subject to glucose deprivation relative to normal glucose at 24 h, and in the presence of LPS at 24 or 48 h (N = 8). Numbers indicate mean cell density for each condition as determined from a parallel assay plate. N.D. not determinable. Asterisk indicates significant groupwise differences by two-way ANOVA between control and LPS and Double asterisk indicates pairwise significance by Bonferroni’s post hoc

Fig. 2

Glucose deprivation alters the function, but not the viability and proliferation, of microglia. Experiments were performed as in Fig. 1a, 24 h total time course. Immunofluorescence microscopy did not reveal any morphological differences between microglia cultured in normal glucose (a) or glucose deprived (b) media. Microglia were labelled with (i) Hoechst 33342, (ii) Bodipy 493/503, and (iii) Iba1, arrowheads in the overlay (iv) indicate microglia showing detectable lipid droplets as indicated by Bodipy 493/503 staining. c Phagocytosis as a portion of cells taking up fluorescent beads was not significantly affected by glucose deprivation (N = 6). d Phagocytic uptake, in terms of mean uptake of fluorescent beads per cell, was significantly increased with glucose deprivation (N = 6). e Proliferation, as measured by incorporation of the nucleotide analogue EdU, was not significantly affected by glucose deprivation (N = 3). Asterisk indicates groupwise differences between control and LPS treatment by two-way ANOVA, Dagger indicates pairwise difference between normal glucose and GD by Mann-Whitney U test

Fig. 3

Glucose deprivation alters the accumulation of lipid droplets. Accumulation of lipid droplets was quantified by staining with BODIPY 493/503 and integration of green fluorescence within the soma of microglia identified by Hoechst 33342 and CD68 labelling, and is indicative of an increased size and/or number of lipid droplets. Fluorescence was significantly increased over the course of 48 h in the normal glucose condition, but not during glucose deprivation. Both normal and glucose-deprived cells showed significant increases in BODIPY 493/503 staining after LPS stimulation at either 24 or 48 h. Asterisk indicates significant groupwise differences by two-way ANOVA between control and LPS and Double asterisk indicates pairwise significance by Bonferroni’s post hoc (N = 4)