Autophagy-Glycolysis Crosstalk Maintains Cellular Homeostasis [Apr. 22, 2025] DOJINDO LABORATORIES

Previous Science Note

Autophagy and glycolysis are fundamental to maintaining cellular energy homeostasis, yet their imbalance can lead to dysfunction and disease. Recent studies reveal how impaired autophagy disrupts glucose metabolism and how excessive glycolytic flux can suppress autophagy.This Science Note introduces new insights into the dynamic crosstalk between these pathways and its impact on cellular homeostasis.

Autophagy regulator ATG5 preserves cerebellar function by safeguarding its glycolytic activity (Nature Metabolism, 2025)
In cerebellar Purkinje cells involved in motor control, autophagy regulates glucose uptake by limiting the amount of GLUT2. Loss of autophagy leads to excessive glucose uptake and dysregulation of glycolysis, resulting in the accumulation of by-products such as lysophosphatidic acid and serine, which contribute to neuronal degeneration.

Highlighted technique: To assess changes in glucose uptake in autophagy-deficient Purkinje cells, the authors used cerebellar organotypic slice cultures from mice and evaluated glucose uptake capacity by fluorescent microscopy using a fluorescent analog of glucose.

Related technique Autophagic Flux Assay, Highly Sensitive Fluorescent Glucose Analog

Weak neuronal glycolysis sustains cognition and organismal fitness (Nature Metabolism, 2024)
Neurons maintain low glycolytic activity to maintain NAD⁺ levels, which are essential for sirtuin-mediated autophagy and mitochondrial homeostasis. Aberrant activation of glycolysis depletes NAD⁺, suppresses autophagy and leads to mitochondrial dysfunction and cognitive decline.

Highlighted technique: In this study, a mouse model was generated by overexpressing PFKFB3, a glycolysis-promoting enzyme that is normally degraded in neurons, to investigate the importance of weak neuronal glycolysis. Tissues and cells derived from these mice were used to examine NAD⁺ levels, mitochondrial activity, and autophagy.

Related technique NAD/NADH Assay, Glycolysis/OXPHOS Assay, Mitochondrial ROS Detection

Autophagy counters inflammation-driven glycolytic impairment in aging hematopoietic stem cells (Cell Stem Cell, 2024)
Age-related inflammation impairs glucose uptake in HSCs, causing metabolic stress and functional decline, while this stress triggers adaptive autophagy in a subset of HSCs that supports functional maintenance. Consistent with this mechanism, transient induction of autophagy by short-term fasting restores metabolism and revives the regenerative capacity of aged HSCs.

Highlighted technique: In this study, HSCs were isolated from aged mice subjected to a fasting/refeeding regimen and transplanted into recipient mice to assess their regenerative capacity. FCM was used to detect donor-specific markers and quantify the expansion of donor-derived cells, allowing evaluation of the functional restoration of HSCs by the fasting/refeeding treatment.

Related technique Autophagic Flux Assay, Extracellular OCR Assay, Glucose Uptake Assay

Application Note I
> NAD⁺ Depletion and Autophagy-Lysosomal Pathway Response

We determined how FK866-induced lysosomal deacidification affects the autophagy-lysosomal pathway. After staining with DAPGreen/DAPRed (for detecting autophagosome), or DALGreen (for detecting autolysosome), HeLa cells were starved in HBSS incubation and then treated with FK866 or Bafilomycin A1. Under the starvation condition, the fluorescent signals from all dyes increased, indicating the proceeding autophagy-lysosomal pathway. On the other hand, only DALGreen's signals were decreased in FK866 and Bafilomycin A1 treated cells with starvation conditions. These results clearly suggested that FK866 inhibits the autophagy-lysosomal pathway by NAD+ depletion-induced lysosomal deacidification.

Nampt inhibitor, FK866 inhibits the progress of autophagosome to autolysosome by lysosomal deacidification. A recent finding shows that the dysfunctional condition of nicotinamide adenine dinucleotide (NAD⁺) biosynthetic enzyme, Nampt induces lysosomal deacidification¹⁾. In this section, we tried to determine how NAD⁺ depletion-induced lysosomal deacidification affects the autophagy-lysosomal pathway. 1) Mikako Yagi, et. al., EMBO J., 40(8), e105268 (2021)

Application Note II
> Simultaneous Detection of Lysosomal and Mitochondrial Dysfunction

We tried the simultaneous detection of lysosomal and mitochondrial dysfunction in Hela cells treated with CCCP or Antimycin (AN). CCCP and AN are well-known inducers of mitochondrial ROS regarding loss of mitochondrial membrane potential. Recent research showed the result that CCCP induces not only mitochondrial ROS but also lysosomal neutralization. To detect mitochondrial ROS, HeLa cells were labeled by MitoBright ROS Deep Red - Mitochondrial Superoxide Detection, and the lysosomal mass and pH were detected separately with LysoPrime Green and pHLys Red. Co-staining with MitoBright ROS and Lysosomal dyes demonstrated that CCCP causes lysosomal neutralization and mitochondrial ROS induction at the same time.

Products in Use
- Lysosomal Acidic pH Detection Kit
(combination kit of LysoPrime Green / pHLys Red)
- MitoBright ROS Deep Red - Mitochondrial Superoxide Detection

Related Techniques

Target	Kit & Probes
First-time autophagy research	Autophagic Flux Assay Kit
Autophagy detection	DAPRed (Autophagosome detection), DALGreen (Autolysosome detection)
Mitophagy detection	Mitophagy Detection Kit
Highly Sensitive Fluorescent Glucose Analog	Glucose Uptake Assay Kit Blue/ Green / Red
Glycolysis/Oxidative phosphorylation Assay	Glycolysis/OXPHOS Assay Kit
Mitochondrial superoxide detection	MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
Oxygen consumption rate assay	Extracellular OCR Plate Assay Kit
Mitochondrial membrane potential detection	JC-1MitoMPDetection Kit, MT-1MitoMPDetection Kit
Apoptosis detection in multiple samples	Annexin V Apoptosis Plate Assay Kit
Cell proliferation/ cytotoxicity assay	Cell Counting Kit-8 and Cytotoxicity LDH Assay Kit-WST