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Metformin is a biguanide widely prescribed to treat Type II diabetes that has gained interest as an antineoplastic agent. Recent work suggests that metformin directly antagonizes cancer cell growth through its actions on complex I of the mitochondrial electron transport chain (ETC). However, the mechanisms by which metformin arrests cancer cell proliferation remain poorly defined. Here we demonstrate that the metabolic checkpoint kinases AMP-activated protein kinase (AMPK) and LKB1 are not required for the antiproliferative effects of metformin. Rather, metformin inhibits cancer cell proliferation by suppressing mitochondrial-dependent biosynthetic activity. We show that in vitro metformin decreases the flow of glucose- and glutamine-derived metabolic intermediates into the Tricarboxylic Acid (TCA) cycle, leading to reduced citrate production and de novo lipid biosynthesis. Tumor cells lacking functional mitochondria maintain lipid biosynthesis in the presence of metformin via glutamine-dependent reductive carboxylation, and display reduced sensitivity to metformin-induced proliferative arrest. Our data indicate that metformin inhibits cancer cell proliferation by suppressing the production of mitochondrial-dependent metabolic intermediates required for cell growth, and that metabolic adaptations that bypass mitochondrial-dependent biosynthesis may provide a mechanism of tumor cell resistance to biguanide activity.

Dendritic cells (DCs) are first responders of the innate immune system that integrate signals from external stimuli to direct context-specific immune responses. Current models suggest that an active switch from mitochondrial metabolism to glycolysis accompanies DC activation to support the anabolic requirements of DC function. We show that early glycolytic activation is a common program for both strong and weak stimuli, but that weakly activated DCs lack long-term HIF-1α-dependent glycolytic reprogramming and retain mitochondrial oxidative metabolism. Early induction of glycolysis is associated with activation of AKT, TBK, and mTOR, and sustained activation of these pathways is associated with long-term glycolytic reprogramming. We show that inhibition of glycolysis impaired maintenance of elongated cell shape, DC motility, CCR7 oligomerization, and DC migration to draining lymph nodes. Together, our results indicate that early induction of glycolysis occurs independent of pro-inflammatory phenotype, and that glycolysis supports DC migratory ability regardless of mitochondrial bioenergetics. The activation of dendritic cells (DC) is associated with a metabolic switch from oxidative to glycolytic metabolism. Here, the authors show that both strong and weak stimuli cause an immediate increase in glycolysis, but only strong stimuli induce long-term glycolytic reprogramming.

One of the major metabolic changes associated with cellular transformation is enhanced nutrient utilization, which supports tumor progression by fueling both energy production and providing biosynthetic intermediates for growth. The liver kinase B1 (LKB1) is a serine/threonine kinase and tumor suppressor that couples bioenergetics to cell-growth control through regulation of mammalian target of rapamycin (mTOR) activity; however, the influence of LKB1 on tumor metabolism is not well defined. Here, we show that loss of LKB1 induces a progrowth metabolic program in proliferating cells. Cells lacking LKB1 display increased glucose and glutamine uptake and utilization, which support both cellular ATP levels and increased macromolecular biosynthesis. This LKB1-dependent reprogramming of cell metabolism is dependent on the hypoxia-inducible factor-1α (HIF-1α), which accumulates under normoxia in LKB1-deficient cells and is antagonized by inhibition of mTOR complex I signaling. Silencing HIF-1α reverses the metabolic advantages conferred by reduced LKB1 signaling and impairs the growth and survival of LKB1-deficient tumor cells under low-nutrient conditions. Together, our data implicate the tumor suppressor LKB1 as a central regulator of tumor metabolism and growth control through the regulation of HIF-1α–dependent metabolic reprogramming.

A characteristic of memory T (T M) cells is their ability to mount faster and stronger responses to reinfection than naïve T (T N) cells do in response to an initial infection. However, the mechanisms that allow this rapid recall are not completely understood. We found that CD8 T M cells have more mitochondrial mass than CD8 T N cells and, that upon activation, the resulting secondary effector T (T E) cells proliferate more quickly, produce more cytokines, and maintain greater ATP levels than primary effector T cells. We also found that after activation, T M cells increase oxidative phosphorylation and aerobic glycolysis and sustain this increase to a greater extent than T N cells, suggesting that greater mitochondrial mass in T M cells not only promotes oxidative capacity, but also glycolytic capacity. We show that mitochondrial ATP is essential for the rapid induction of glycolysis in response to activation and the initiation of proliferation of both T N and T M cells. We also found that fatty acid oxidation is needed for T M cells to rapidly respond upon restimulation. Finally, we show that dissociation of the glycolysis enzyme hexokinase from mitochondria impairs proliferation and blocks the rapid induction of glycolysis upon T-cell receptor stimulation in T M cells. Our results demonstrate that greater mitochondrial mass endows T M cells with a bioenergetic advantage that underlies their ability to rapidly recall in response to reinfection.

The identification of cancer-associated mutations in the tricarboxylic acid (TCA) cycle enzymes isocitrate dehydrogenases 1 and 2 (IDH1/2) highlights the prevailing notion that aberrant metabolic function can contribute to carcinogenesis. IDH1/2 normally catalyse the oxidative decarboxylation of isocitrate into α-ketoglutarate (αKG). In gliomas and acute myeloid leukaemias, IDH1/2 mutations confer gain-of-function leading to production of the oncometabolite R -2-hydroxyglutarate (2HG) from αKG. Here we show that generation of 2HG by mutated IDH1/2 leads to the activation of mTOR by inhibiting KDM4A, an αKG-dependent enzyme of the Jumonji family of lysine demethylases. Furthermore, KDM4A associates with the DEP domain-containing mTOR-interacting protein (DEPTOR), a negative regulator of mTORC1/2. Depletion of KDM4A decreases DEPTOR protein stability. Our results provide an additional molecular mechanism for the oncogenic activity of mutant IDH1/2 by revealing an unprecedented link between TCA cycle defects and positive modulation of mTOR function downstream of the canonical PI3K/AKT/TSC1-2 pathway. Oncogenic mutations of isocitrate dehydrogenases 1 and 2 result in the production of the oncometabolite R-2-hydroxyglutarate. Here the authors show that the oncometabolite promotes mTOR activation in a PTEN/PI3K-independent manner by regulating DEPTOR stability via inhibition of KDM4A activity.

The mammalian adenosine monophosphate-activated protein kinase (AMPK) is a serine-threonine kinase protein complex that is a central regulator of cellular energy homeostasis. However, the mechanisms by which AMPK mediates cellular responses to metabolic stress remain unclear. We found that AMPK activates transcription through direct association with chromatin and phosphorylation of histone H2B at serine 36. AMPK recruitment and H2B Ser³⁶ phosphorylation colocalized within genes activated by AMPK-dependent pathways, both in promoters and in transcribed regions. Ectopic expression of H2B in which Ser³⁶ was substituted by alanine reduced transcription and RNA polymerase II association to AMPK-dependent genes, and lowered cell survival in response to stress. Our results place AMPK-dependent H2B Ser³⁶ phosphorylation in a direct transcriptional and chromatin regulatory pathway leading to cellular adaptation to stress.

Pulmonary macrophages have two distinct ontogenies: long-lived embryonically-seeded alveolar macrophages (AM) and bone marrow-derived macrophages (BMDM). Here, we show that after infection with a virulent strain of Mycobacterium tuberculosis (H37Rv), primary murine AM exhibit a unique transcriptomic signature characterized by metabolic reprogramming distinct from conventional BMDM. In contrast to BMDM, AM failed to shift from oxidative phosphorylation (OXPHOS) to glycolysis and consequently were unable to control infection with an avirulent strain (H37Ra). Importantly, healthy human AM infected with H37Ra equally demonstrated diminished energetics, recapitulating our observation in the murine model system. However, the results from seahorse showed that the shift towards glycolysis in both AM and BMDM was inhibited by H37Rv. We further demonstrated that pharmacological (e.g. metformin or the iron chelator desferrioxamine) reprogramming of AM towards glycolysis reduced necrosis and enhanced AM capacity to control H37Rv growth. Together, our results indicate that the unique bioenergetics of AM renders these cells a perfect target for Mtb survival and that metabolic reprogramming may be a viable host targeted therapy against TB.

Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor FLCN, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally occurring FLCN mutations predisposing to BHD are predicted to produce truncated proteins unable to bind AMPK, pointing to the critical role of this interaction in the tumor suppression mechanism. Here, we demonstrate that FLCN is an evolutionarily conserved negative regulator of AMPK. Using Caenorhabditis elegans and mammalian cells, we show that loss of FLCN results in constitutive activation of AMPK which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function.

Whether screening the metabolic activity of immune cells facilitates discovery of molecular pathology remains unknown. Here we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome sequencing identified germline mutations in SDHA , which encodes succinate dehydrogenase subunit A, in all three patients with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1–Nrf2 system to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency. Hess and colleagues perform metabolic screens in B cells from patients with primary antibody deficiency and find that germline mutations in the succinate dehydrogenase subunit SDHA drive the expression of the cytokine IL-6 in patients with persistent polyclonal B cell lymphocytosis.

Activation of AMPK has been associated with pro‐atrophic signaling in muscle. However, AMPK also has anti‐inflammatory effects, suggesting that in cachexia, a syndrome of inflammatory‐driven muscle wasting, AMPK activation could be beneficial. Here we show that the AMPK agonist AICAR suppresses IFNγ/TNFα‐induced atrophy, while the mitochondrial inhibitor metformin does not. IFNγ/TNFα impair mitochondrial oxidative respiration in myotubes and promote a metabolic shift to aerobic glycolysis, similarly to metformin. In contrast, AICAR partially restored metabolic function. The effects of AICAR were prevented by the AMPK inhibitor Compound C and were reproduced with A‐769662, a specific AMPK activator. AICAR and A‐769662 co‐treatment was found to be synergistic, suggesting that the anti‐cachectic effects of these drugs are mediated through AMPK activation. AICAR spared muscle mass in mouse models of cancer and LPS induced atrophy. Together, our findings suggest a dual function for AMPK during inflammation‐driven atrophy, wherein it can play a protective role when activated exogenously early in disease progression, but may contribute to anabolic suppression and atrophy when activated later through mitochondrial dysfunction and subsequent metabolic stress. Synopsis Cachexia is a co‐morbidity characterized by the loss of skeletal muscle that arises in patients with pro‐inflammatory diseases, like cancer. Activators of AMPK were found to protect against inflammation‐induced muscle atrophy, demonstrating the potential of targeting AMPK for therapy in cachexia. The AMPK activator AICAR, but not metformin, protected C2C12 myotubes from IFNγ/TNFα‐driven atrophy. The differential effects of AICAR and metformin were associated with the ability to restore or inhibit mitochondrial function during inflammation, suggesting that the mechanism of AMPK activation affects the outcome of treatment. The anti‐cachectic properties of AICAR were impaired by treatment with the AMPK inhibitor Compound C and were synergistic with the AMPK activator A‐769662, suggesting that the effects of AICAR were mediated by AMPK activation. AICAR, but not metformin, was effective at preventing muscle mass loss in mice in both the C26 model of cancer cachexia and an endotoxin model of sepsis. Collectively, this study suggests that treatment with AMPK activators during the early stages of cachexia could be a novel avenue for the development of therapies. Graphical Abstract Cachexia is a co‐morbidity characterized by the loss of skeletal muscle that arises in patients with pro‐inflammatory diseases, like cancer. Activators of AMPK were found to protect against inflammation‐induced muscle atrophy, demonstrating the potential of targeting AMPK for therapy in cachexia.