The capacity to effectively adapt metabolism to environmental demands is crucial for cell viability, proliferation, and function. Recent discoveries in Cell Research have highlighted the role of the lysosomal pool of AMPK in promoting glutaminolysis during glucose shortage through the activation of a PDZD8-GLS1 axis.

To manage fluctuations in nutrient availability and energy requirement, organisms and cells employ a variety of biological mechanisms to sense, transport, store and utilize substrates. The implementation of both short- and long-term responses to nutrient stress and elevated energy requirements is essential for maintaining cellular function and survival.1 In eukaryotes, the highly conserved 5′-AMP-activated protein kinase (AMPK) is a key regulator of cellular energy status, acting as a sensor of adenine nucleotides. In the context of low cellular energy, activation of AMPK controls cell metabolism by shifting from an ATP-consuming anabolic mode to an ATP-generating catabolic mode in order to restore cellular energy balance.2 While AMPK is activated by the canonical pathway following energy deficit (i.e., increases in cellular ADP:ATP and/or AMP:ATP ratios), recent evidence indicates that AMPK is also activated by glucose deprivation through a non-canonical pathway in the absence of any detectable changes in cellular energy status.2 This AMP/ADP-independent glucose sensing mechanism may represent one of the ancestral roles of AMPK, as it is evolutionarily conserved in the AMPK yeast ortholog SNF1.2 Glucose deprivation selectively activates a pool of AMPK localized at the surface of lysosome by a mechanism involving detection of reduced fructose-1,6-bisphosphate (FBP) levels by the glycolytic enzyme aldolase and formation of a v-ATPase–AXIN–LKB1–AMPK complex (Fig. 1).3 However, how lysosomal AMPK activation orchestrates metabolic responses and rewires cellular metabolism in low glucose conditions remains incompletely understood.

Glucose deprivation reduces intracellular FBP levels which are sensed by aldolase, leading to the formation of a lysosomal complex containing v-ATPase, Ragulator, AXIN, and LKB1, and activation of an organelle-specific pool of AMPK. ER-localized PDZD8 is next phosphorylated at Thr527 by AMPK, promoting interaction with and activation of the mitochondrial GLS1 at the ER–mitochondria contact site and increasing glutaminolysis. CR-enhanced glutaminolysis may contribute to lifespan and healthspan extension in C. elegans and mice. Figure was created with BioRender.com.

To address this knowledge gap, Li et al.4 investigated the lysosomal AMPK-dependent mechanisms that regulate metabolic flexibility during glucose deprivation. In glucose-starved mouse embryonic fibroblasts (MEFs), a sequential utilization of alternative carbon sources was observed, with glutamine being metabolized first (within 2 h) and fatty acids being utilized at a later stage (after 12 h). Similar kinetics were observed in vivo in the livers and skeletal muscles of starved mice, with enhanced glutaminolysis occurring after 8 h followed by increased fatty acid oxidation (FAO) after 16 h. This effect was associated with a rapid increase in oxygen consumption rate (OCR), indicating that glutamine is catabolized before fatty acids in the mitochondria, and is suppressed by knockdown of either AMPK or glutaminase 1 (GLS1), the rate-limiting enzyme in glutaminolysis. To identify the AMPK target(s) involved, a pull-down assay with pan-phospho-AMPK substrate antibodies was performed using the subcellular fractions of glucose-starved MEFs enriched in mitochondria-associated endoplasmic reticulum (ER) membranes. By mass spectrometry, they identified the PDZ domain containing 8 (PDZD8), an intrinsic ER membrane protein,5 as a new AMPK substrate. Interestingly, mutation of the AMPK phosphorylation site in PDZD8 (Thr527) blocked the increase in glutaminolysis but not FAO following glucose deprivation. Furthermore, the PDZD8-Thr527 phosphorylation was abolished in MEFs lacking the components of the lysosomal AMPK glucose-sensing pathway, demonstrating the specific involvement of the lysosomal AMPK pool in low glucose-induced glutaminolysis. At the molecular level, the authors demonstrated that AMPK-mediated phosphorylation of PDZD8 alleviates the autoinhibitory effect exerted by its N-terminal region on its C-terminal region, thereby facilitating its interaction with GLS1 at the ER–mitochondria contact sites and GLS1 activation (Fig. 1).

To investigate the physiological implications of the AMPK-PDZD8-GLS1 axis, mice with a specific deletion of PDZD8 and expressing an AMPK-unphosphorylatable PDZD8-T527A mutant in either skeletal muscle or macrophages were generated.4 In skeletal muscle, the fasting-induced increase in glutaminolysis and mitochondrial OCR was abolished in mice with muscular PDZD8 replaced with PDZD8-T527A mutant, confirming a role for the AMPK-PDZD8 axis in the regulation of skeletal muscle metabolism during acute glucose starvation. Moreover, bone marrow-derived macrophages from PDZD8 mutant mice showed impaired glutaminolysis and pro-inflammatory cytokine secretion in response to lipopolysaccharide (LPS). This defect in response to LPS was also observed in vivo in both macrophage-specific PDZD8 mutant mice and mice treated with pharmacological inhibitors of GLS1, suggesting a role for the AMPK-PDZD8-GLS1 axis in metabolic reprogramming and pro-inflammatory functions of immune cells. However, it is difficult to reconcile these results with previous studies showing that glutaminolysis-derived α-ketoglutarate orchestrates macrophage polarization by promoting alternative (M2) activation and restricting classical (M1) pro-inflammatory activation through epigenetic remodeling and inhibition of the NF-κB pathway, respectively.6,7 Further studies are therefore required to clarify this point and elucidate the exact role of lysosomal AMPK in immune cell metabolic and functional plasticity.

In a second study, Li et al.8 examined the role of AMPK-dependent PDZD8 phosphorylation and enhanced glutaminolysis in calorie restriction (CR)-induced lifespan extension. Remarkably, the AMPK-PDZD8-GLS1 signaling axis was shown to be conserved in Caenorhabditis elegans and mutation of the AMPK phosphorylation site on pdzd-8 (the PDZD8 homolog in C. elegans) reduced glutamine catabolism and inhibited lifespan extension in response to low glucose-mimicking conditions or CR. Conversely, the phospho-mimetic pdzd-8 mutant promoted glutaminolysis and extended lifespan, even in nematodes lacking the AMPK homolog aak-2. Mechanistically, the AMPK-PDZD8-GLS1 axis triggers a transient and mild mitochondrial oxidative stress during CR leading to the expression of reactive oxygen species (ROS)-scavenging enzymes, a feature reminiscent of mitohormesis.9 Similarly, CR-induced phosphorylation of PDZD8-Thr527 and transient mitochondrial ROS in skeletal muscles from 8-month-old mice, independently of the AMPK canonical pathway.8 Interestingly, CR improved muscle fitness, an anti-aging effect that was not observed in aged mice expressing the AMPK-unphosphorylatable PDZD8-T527A mutant (Fig. 1). Nevertheless, it remains unclear the contribution of glutaminolysis to improving healthspan in mammals.

In summary, these insightful studies from Li and colleagues have elucidated the role of the AMPK-PDZD8-GLS1 axis in intrinsic cellular metabolism, highlighting that the regulation of glutaminolysis is intricately linked to glucose availability sensed at the lysosome. While these studies provide new insights into lysosomal AMPK function, particularly in the context of lifespan and healthspan extension, several key questions remain unanswered regarding the potential beneficial or detrimental effects of targeting this specific pathway. For instance, glutamine-dependent metabolic reprogramming is supported by AMPK activation for the long-term survival of cancer cells following matrix detachment.10 Overall, further research is needed to clarify the broader implications of lysosomal AMPK in regulating metabolic plasticity and treating diseases.