Nutrient Condition in the Microenvironment Determines Essential Metabolisms of CD8(+) T Cells for Enhanced IFN gamma Production by Metformin

Metformin (Met), a first-line drug for type 2 diabetes, lowers blood glucose levels by suppressing gluconeogenesis in the liver, presumably through the liver kinase B1-dependent activation of AMP-activated protein kinase (AMPK) after inhibiting respiratory chain complex I. Met is also implicated as a drug to be repurposed for cancers; its mechanism is believed identical to that of gluconeogenesis inhibition. However, AMPK activation requires high Met concentrations at more than 1 mM, which are unachievable in vivo. The immune-mediated antitumor response might be the case in a low dose Met. Thus, we proposed activating or expanding tumor-infiltrating CD8(+) T cells (CD8TILs) in a mouse model by orally administering Met in free drinking water. Here we showed that Met, at around 10 mu M and a physiologically relevant concentration, enhanced production of IFN gamma,TNF alpha and expression of CD25 of CD8(+) T cells upon TCR stimulation. Under a glucose-rich condition, glycolysis was exclusively involved in enhancing IFN gamma production. Under a low-glucose condition, fatty acid oxidation or autophagy-dependent glutaminolysis, or both, was also involved. Moreover, phosphoenolpyruvate carboxykinase 1 (PCK1), converting oxaloacetate to phosphoenolpyruvate, became essential. Importantly, the enhanced IFN gamma production was blocked by a mitochondrial ROS scavenger and not by an inhibitor of AMPK. In addition, IFN gamma production by CD8TILs relied on pyruvate translocation to the mitochondria and PCK1. Our results revealed a direct effect of Met on IFN gamma production of CD8(+) T cells that was dependent on differential metabolic pathways and determined by nutrient conditions in the microenvironment

Metformin-ROS-Nrf2 connection in the host defense mechanism against oxidative stress, apoptosis, cancers, and ageing

Reactive oxygen species (ROS) acts as a second messenger to trigger biological responses in low concentrations, while it is implicated to be toxic to biomolecules in high concentrations. Mild inhibition of respiratory chain Complex I by metformin at physiologically relevant concentrations stimulates production of low-level mitochondrial ROS. The ROS seems to induce anti-oxidative stress response via activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione peroxidase (GPx), which results in not only elimination of ROS but also activation of cellular responses including resistance to apoptosis, metabolic changes, cell proliferation, senescence prevention, lifespan extension, and immune T cell activation against cancers, regardless of its effect controlling blood glucose level and T2DM. Although metformin's effect against T2DM, cancers, and ageing, are believed mostly attributed to the activation of AMP-activated protein kinase (AMPK), the cellular responses involving metformin-ROS-Nrf2 axis might be another natural asset to improve healthspan and lifespan

Evidence for Hypoxia-Induced Shift in ATP Production from Glycolysis to Mitochondrial Respiration in Pulmonary Artery Smooth Muscle Cells in Pulmonary Arterial Hypertension

Background: The metabolic state of pulmonary artery smooth muscle cells (PASMCs) from patients with pulmonary arterial hypertension (PAH) is not well understood. In this study, we examined the balance between glycolysis and mitochondrial respiration in non-PAH-PASMCs and PAH-PASMCs under normoxia and hypoxia. Methods: We investigated the enzymes involved in glycolysis and mitochondrial respiration, and studied the two major energy-yielding pathways (glycolysis and mitochondrial respiration) by measuring extracellular acidification rate (ECAR) and cellular oxygen consumption rate (OCR) using the Seahorse extracellular flux technology. Results: Under both normoxia and hypoxia, the mRNA and protein levels of pyruvate dehydrogenase kinase 1 and pyruvate dehydrogenase were increased in PAH-PASMCs compared with non-PAH-PASMCs. The mRNA and protein levels of lactate dehydrogenase, as well as the intracellular lactate concentration, were also increased in PAH-PASMCs compared with non-PAH-PASMCs under normoxia. However, these were not significantly increased in PAH-PASMCs compared with non-PAH-PASMCs under hypoxia. Under normoxia, ATP production was significantly lower in PAH-PASMCs (59 ± 5 pmol/min) than in non-PAH-PASMCs (70 ± 10 pmol/min). On the other hand, ATP production was significantly higher in PAH-PASMCs (31 ± 5 pmol/min) than in non-PAH-PASMCs (14 ± 3 pmol/min) under hypoxia. Conclusions: There is an underlying change in the metabolic strategy to generate ATP production under the challenge of hypoxia

Blocking EP4 downregulates tumor metabolism and synergizes with anti-PD-1 therapy to activate natural killer cells in a lung adenocarcinoma model

Prostaglandin E2 (PGE2), a product of the cyclooxygenase (COX) pathway, is produced by tumors and surrounding stromal cells. It stimulates tumor progression, promotes angiogenesis, and suppresses the antitumor response. Pharmacological inhibition of PGE2 synthesis has been shown to suppress tumor initiation and growth in vivo. In the current study, we demonstrated that the growth of the Ptgs2-deficient the 3LL lung adenocarcinoma cell line was downregulated in vivo through natural killer (NK) cell activation and a reduction in the population of polymorphonuclear leukocyte-myeloid-derived suppressor cells (PMN-MDSCs) and tumor associated macrophages (TAMs). Based on these results, the therapeutic effect of ONO-AE3–208 (EP4i), an inhibitor of EP4 (a PGE2 receptor), combined with anti-PD-1Ab was evaluated. EP4i, but not anti-PD-1 Ab, decreased tumor metabolism including glycolysis, fatty acid oxidation, and oxidative phosphorylation. EP4i induced IFNγ production from only NK cells (not from T cells) and a shift from M2- to M1-like macrophages in TAMs. These effects were further enhanced by anti-PD-1 Ab treatment. Although CD8T cell infiltration was increased, IFNγ production was not significantly altered, even with combination therapy. Tumor hypoxia was ameliorated by either EP4i or anti-PD-1 Ab treatment, which was further affected by the combination. Normalization of tumor vessels was significant only for the combination therapy. The results indicate a novel effect of EP4i for the metabolic reprogramming of tumors, revealed unique features of EP4i that can synergize with anti-PD-1Ab to promote IFNγ production of NK cells, polarize TAMs into the M1-phenotype, and reduce hypoxia through normalization of the tumor vasculature

Blocking EP4 downregulates tumor metabolism and synergizes with anti-PD-1 therapy to activate natural killer cells in a lung adenocarcinoma model

Prostaglandin E2 (PGE2), a product of the cyclooxygenase (COX) pathway, is produced by tumors and surrounding stromal cells. It stimulates tumor progression, promotes angiogenesis, and suppresses the antitumor response. Pharmacological inhibition of PGE2 synthesis has been shown to suppress tumor initiation and growth in vivo. In the current study, we demonstrated that the growth of the Ptgs2-deficient the 3LL lung adenocarcinoma cell line was downregulated in vivo through natural killer (NK) cell activation and a reduction in the population of polymorphonuclear leukocyte-myeloid-derived suppressor cells (PMN-MDSCs) and tumor associated macrophages (TAMs). Based on these results, the therapeutic effect of ONO-AE3–208 (EP4i), an inhibitor of EP4 (a PGE2 receptor), combined with anti-PD-1Ab was evaluated. EP4i, but not anti-PD-1 Ab, decreased tumor metabolism including glycolysis, fatty acid oxidation, and oxidative phosphorylation. EP4i induced IFNγ production from only NK cells (not from T cells) and a shift from M2- to M1-like macrophages in TAMs. These effects were further enhanced by anti-PD-1 Ab treatment. Although CD8T cell infiltration was increased, IFNγ production was not significantly altered, even with combination therapy. Tumor hypoxia was ameliorated by either EP4i or anti-PD-1 Ab treatment, which was further affected by the combination. Normalization of tumor vessels was significant only for the combination therapy. The results indicate a novel effect of EP4i for the metabolic reprogramming of tumors, revealed unique features of EP4i that can synergize with anti-PD-1Ab to promote IFNγ production of NK cells, polarize TAMs into the M1-phenotype, and reduce hypoxia through normalization of the tumor vasculature