Single-Cell Analysis for Glycogen Localization and Metabolism in Cultured Astrocytes

Abstract

Cerebral glycogen is principally localized in astrocytes rather than in neurons. Glycogen metabolism has been implicated in higher brain functions, including learning and memory, yet the distribution patterns of glycogen in different types of astrocytes have not been fully described. Here, we applied a method based on the incorporation of 2-NBDG, a d-glucose fluorescent derivative that can trace glycogen, to investigate glycogen's distribution in the brain. We identified two types of astrocytes, namely, 2-NBDG I (glycogen-deficient) and 2-NBDG II (glycogen-rich) cells. Whole-cell patch-clamp and fluorescence-activated cell sorting (FACS) were used to separate 2-NBDG II astrocytes from 2-NBDG I astrocytes. The expression levels of glycogen metabolic enzymes were analyzed in 2-NBDG I and 2-NBDG II astrocytes. We found unique glycogen metabolic patterns between 2-NBDG I and 2-NBDG II astrocytes. We also observed that 2-NBDG II astrocytes were mainly identified as fibrous astrocytes but not protoplasmic astrocytes. Our data reveal cell type-dependent glycogen distribution and metabolism patterns, suggesting diverse functions of these different astrocytes.

Visualizing Subcellular Enrichment of Glycogen in Live Cancer Cells by Stimulated Raman Scattering

“…After validating the high imaging sensitivity and specificity together with metabolic phenotyping using this SRS strategy of d 7 -glucose labeling, we compared our results to those from fluorescence imaging of glycogen using 2-NBDG in live cells. 2-NBDG has been reported to be capable of being incorporated into glycogen through glycogenesis. , We coincubated HeLa cells with 100 μM 2-NBDG and 25 mM d 7 -glucose and performed correlative fluorescence and SRS imaging. Surprisingly, almost no fluorescence signal from 2-NBDG colocalized with signals from the CD G channel (Figure A, fluorescence (blue) vs CD G (red), white arrowed).…”

“…2-NBDG has been reported to be capable of being incorporated into glycogen through glycogenesis. 17,18 We co-incubated HeLa cells with 100 μM of 2-NBDG and 25 mM d 7 -glucose and performed correlative fluorescence and SRS imaging. Surprisingly, almost no fluorescence signal from 2-NBDG colocalized with signals from the CD G channel (Fig.…”

“…Mass spectrometry-based methods such as nano secondary ion mass spectrometry (nano SIMS) or transmission electron microscopy (TEM) are sample-destructive and not compatible for live-cell studies. Fluorescence imaging of 2-NBDG, a popular fluorescent glucose analogue, offers subcellular and live-cell visualization , but has a few limitations from the fluorophore labeling. First, 2-NBDG is significantly more hydrophobic compared to regular glucose, which can yield an affinity to lipids that decreases the specificity of 2-NBDG labeling of glycogen. , Second, 2-NBDG is nonmetabolizable after phosphorylation and may not provide downstream metabolic information …”

Brain-Type Glycogen Phosphorylase Is Crucial for Astrocytic Glycogen Accumulation in Chronic Social Defeat Stress-Induced Depression in Mice

“…Primary astrocytes were prepared as previously described ( Zhu et al, 2020 ). Astrocytes were collected from newborn C57BL/6J mice.…”

Network Analysis of miRNA and mRNA Changes in the Prelimbic Cortex of Rats With Chronic Neuropathic Pain: Pointing to Inflammation

“…The total RNA from PL tissues was isolated using a tissue total RNA extraction kit (Tiangen, DP431) according to the manufacturer's instructions. The specific experimental steps are as described in the previous article (Zhu et al, 2020). The mRNA was retrotranscribed into cDAN a total of 1 µg using a PrimeScriptTM RT reagent Kit (TaKaRa).…”