Interrelation of adipose tissue macrophages and fibrosis in obesity.

Obesity is characterized by adipose tissue expansion, extracellular matrix remodelling and unresolved inflammation that contribute to insulin resistance and fibrosis. Adipose tissue macrophages represent the most abundant class of immune cells in adipose tissue inflammation and could be key mediators of adipocyte dysfunction and fibrosis in obesity. Although macrophage activation states are classically defined by the M1/M2 polarization nomenclature, novel studies have revealed a more complex range of macrophage phenotypes in response to external condition or the surrounding microenvironment. Here, we discuss the plasticity of adipose tissue macrophages (ATMs) in response to their microenvironment in obesity, with special focus on macrophage infiltration and polarization, and their contribution to adipose tissue fibrosis. A better understanding of the role of ATMs as regulators of adipose tissue remodelling may provide novel therapeutic strategies against obesity and associated metabolic diseases.

Chronic lymphocytic leukemia patient-derived xenografts recapitulate clonal evolution to Richter transformation.

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasm with a heterogeneous clinical behavior. In 5-10% of patients the disease transforms into a diffuse large-B cell lymphoma known as Richter transformation (RT), which is associated with dismal prognosis. Here, we aimed to establish patient-derived xenograft (PDX) models to study the molecular features and evolution of CLL and RT. We generated two PDXs by injecting CLL (PDX12) and RT (PDX19) cells into immunocompromised NSG mice. Both PDXs were morphologically and phenotypically similar to RT. Whole-genome sequencing analysis at different time points of the PDX evolution revealed a genomic landscape similar to RT tumors from both patients and uncovered an unprecedented RT subclonal heterogeneity and clonal evolution during PDX generation. In PDX12, the transformed cells expanded from a very small subclone already present at the CLL stage. Transcriptomic analysis of PDXs showed a high oxidative phosphorylation (OXPHOS) and low B-cell receptor (BCR) signaling similar to the RT in the patients. IACS-010759, an OXPHOS inhibitor, reduced proliferation, and circumvented resistance to venetoclax. In summary, we have generated new RT-PDX models, one of them from CLL cells that mimicked the evolution of CLL to RT uncovering intrinsic features of RT cells of therapeutical value.

The antioxidant l-Ergothioneine prevents cystine lithiasis in the Slc7a9-/- mouse model of cystinuria.

The high recurrence rate of cystine lithiasis observed in cystinuria patients highlights the need for new therapeutic options to address this chronic disease. There is growing evidence of an antioxidant defect in cystinuria, which has led to test antioxidant molecules as new therapeutic approaches. In this study, the antioxidant l-Ergothioneine was evaluated, at two different doses, as a preventive and long-term treatment for cystinuria in the Slc7a9-/- mouse model. l-Ergothioneine treatments decreased the rate of stone formation by more than 60% and delayed its onset in those mice that still developed calculi. Although there were no differences in metabolic parameters or urinary cystine concentration between control and treated mice, cystine solubility was increased by 50% in the urines of treated mice. We also demonstrate that l-Ergothioneine needs to be internalized by its transporter OCTN1 (Slc22a4) to be effective, as when administrated to the double mutant Slc7a9-/-Slc22a4-/- mouse model, no effect on the lithiasis phenotype was observed. In kidneys, we detected a decrease in GSH levels and an impairment of maximal mitochondrial respiratory capacity in cystinuric mice that l-Ergothioneine treatment was able to restore. Thus, l-Ergothioneine administration prevented cystine lithiasis in the Slc7a9-/- mouse model by increasing urinary cystine solubility and recovered renal GSH metabolism and mitochondrial function. These results support the need for clinical trials to test l-Ergothioneine as a new treatment for cystinuria.

Energy substrate metabolism, mitochondrial structure and oxidative stress after cardiac ischemia-reperfusion in mice lacking UCP3.

Myocardial ischemia-reperfusion (IR) injury may result in cardiomyocyte dysfunction. Mitochondria play a critical role in cardiomyocyte recovery after IR injury. The mitochondrial uncoupling protein 3 (UCP3) has been proposed to reduce mitochondrial reactive oxygen species (ROS) production and to facilitate fatty acid oxidation. As both mechanisms might be protective following IR injury, we investigated functional, mitochondrial structural, and metabolic cardiac remodeling in wild-type mice and in mice lacking UCP3 (UCP3-KO) after IR. Results showed that infarct size in isolated perfused hearts subjected to IR ex vivo was larger in adult and old UCP3-KO mice than in equivalent wild-type mice, and was accompanied by higher levels of creatine kinase in the effluent and by more pronounced mitochondrial structural changes. The greater myocardial damage in UCP3-KO hearts was confirmed in vivo after coronary artery occlusion followed by reperfusion. S1QEL, a suppressor of superoxide generation from site IQ in complex I, limited infarct size in UCP3-KO hearts, pointing to exacerbated superoxide production as a possible cause of the damage. Metabolomics analysis of isolated perfused hearts confirmed the reported accumulation of succinate, xanthine and hypoxanthine during ischemia, and a shift to anaerobic glucose utilization, which all recovered upon reoxygenation. The metabolic response to ischemia and IR was similar in UCP3-KO and wild-type hearts, being lipid and energy metabolism the most affected pathways. Fatty acid oxidation and complex I (but not complex II) activity were equally impaired after IR. Overall, our results indicate that UCP3 deficiency promotes enhanced superoxide generation and mitochondrial structural changes that increase the vulnerability of the myocardium to IR injury.

Impact of GLP-1 receptor agonist versus omega-3 fatty acids supplement on obesity-induced alterations of mitochondrial respiration.

Objective: To compare administration of the glucagon-like peptide-1 (GLP-1) analogue, exenatide, versus dietary supplementation with the omega-3 fatty acid-rich Calanus oil on obesity-induced alterations in mitochondrial respiration. Methods: Six-week-old female C57BL/6JOlaHSD mice were given high fat diet (HFD, 45% energy from fat) for 12 weeks to induce obesity. Thereafter, they were divided in three groups where one received exenatide (10 µg/kg/day) via subcutaneously implanted mini-osmotic pumps, a second group received 2% Calanus oil as dietary supplement, while the third group received HFD without any treatment. Animals were sacrificed after 8 weeks of treatment and tissues (skeletal muscle, liver, and white adipose tissue) were collected for measurement of mitochondrial respiratory activity by high-resolution respirometry, using an Oroboros Oxygraph-2k (Oroboros instruments, Innsbruck, Austria). Results: It was found that high-fat feeding led to a marked reduction of mitochondrial respiration in adipose tissue during all three states investigated - LEAK, OXPHOS and ETS. This response was to some extent attenuated by exenatide treatment, but not with Calanus oil treatment. High-fat feeding had no major effect on hepatic mitochondrial respiration, but exenatide treatment resulted in a significant increase in the various respiratory states in liver. Mitochondrial respiration in skeletal muscle was not significantly influenced by high-fat diet or any of the treatments. The precise evaluation of mitochondrial respiration considering absolute oxygen flux and ratios to assess flux control efficiency avoided misinterpretation of the results. Conclusions: Exenatide increased hepatic mitochondrial respiration in high-fat fed mice, but no clear beneficial effect was observed in skeletal muscle or fat tissue. Calanus oil did not negatively affect respiratory activity in these tissues, which maintains its potential as a dietary supplement, due to its previously reported benefits on cardiac function.

Glycosylation defects, offset by PEPCK-M, drive entosis in breast carcinoma cells.

On glucose restriction, epithelial cells can undergo entosis, a cell-in-cell cannibalistic process, to allow considerable withstanding to this metabolic stress. Thus, we hypothesized that reduced protein glycosylation might participate in the activation of this cell survival pathway. Glucose deprivation promoted entosis in an MCF7 breast carcinoma model, as evaluated by direct inspection under the microscope, or revealed by a shift to apoptosis + necrosis in cells undergoing entosis treated with a Rho-GTPase kinase inhibitor (ROCKi). In this context, curbing protein glycosylation defects with N-acetyl-glucosamine partially rescued entosis, whereas limiting glycosylation in the presence of glucose with tunicamycin or NGI-1, but not with other unrelated ER-stress inducers such as thapsigargin or amino-acid limitation, stimulated entosis. Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is upregulated by glucose deprivation, thereby enhancing cell survival. Therefore, we presumed that PEPCK-M could play a role in this process by offsetting key metabolites into glycosyl moieties using alternative substrates. PEPCK-M inhibition using iPEPCK-2 promoted entosis in the absence of glucose, whereas its overexpression inhibited entosis. PEPCK-M inhibition had a direct role on total protein glycosylation as determined by Concanavalin A binding, and the specific ratio of fully glycosylated LAMP1 or E-cadherin. The content of metabolites, and the fluxes from 13C-glutamine label into glycolytic intermediates up to glucose-6-phosphate, and ribose- and ribulose-5-phosphate, was dependent on PEPCK-M content as measured by GC/MS. All in all, we demonstrate for the first time that protein glycosylation defects precede and initiate the entosis process and implicates PEPCK-M in this survival program to dampen the consequences of glucose deprivation. These results have broad implications to our understanding of tumor metabolism and treatment strategies.

Orally administered Odoribacter laneus improves glucose control and inflammatory profile in obese mice by depleting circulating succinate.

BACKGROUND: Succinate is produced by both human cells and by gut bacteria and couples metabolism to inflammation as an extracellular signaling transducer. Circulating succinate is elevated in patients with obesity and type 2 diabetes and is linked to numerous complications, yet no studies have specifically addressed the contribution of gut microbiota to systemic succinate or explored the consequences of reducing intestinal succinate levels in this setting. RESULTS: Using germ-free and microbiota-depleted mouse models, we show that the gut microbiota is a significant source of circulating succinate, which is elevated in obesity. We also show in vivo that therapeutic treatments with selected bacteria diminish the levels of circulating succinate in obese mice. Specifically, we demonstrate that Odoribacter laneus is a promising probiotic based on its ability to deplete succinate and improve glucose tolerance and the inflammatory profile in two independent models of obesity (db/db mice and diet-induced obese mice). Mechanistically, this is partly mediated by the succinate receptor 1. Supporting these preclinical findings, we demonstrate an inverse correlation between plasma and fecal levels of succinate in a cohort of patients with severe obesity. We also show that plasma succinate, which is associated with several components of metabolic syndrome including waist circumference, triglycerides, and uric acid, among others, is a primary determinant of insulin sensitivity evaluated by the euglycemic-hyperinsulinemic clamp. CONCLUSIONS: Overall, our work uncovers O. laneus as a promising next-generation probiotic to deplete succinate and improve glucose tolerance and obesity-related inflammation. Video Abstract.

Detection of early seeding of Richter transformation in chronic lymphocytic leukemia.

Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS)high-B cell receptor (BCR)low-signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT.

Remission of obesity and insulin resistance is not sufficient to restore mitochondrial homeostasis in visceral adipose tissue.

Metabolic plasticity is the ability of a biological system to adapt its metabolic phenotype to different environmental stressors. We used a whole-body and tissue-specific phenotypic, functional, proteomic, metabolomic and transcriptomic approach to systematically assess metabolic plasticity in diet-induced obese mice after a combined nutritional and exercise intervention. Although most obesity and overnutrition-related pathological features were successfully reverted, we observed a high degree of metabolic dysfunction in visceral white adipose tissue, characterized by abnormal mitochondrial morphology and functionality. Despite two sequential therapeutic interventions and an apparent global healthy phenotype, obesity triggered a cascade of events in visceral adipose tissue progressing from mitochondrial metabolic and proteostatic alterations to widespread cellular stress, which compromises its biosynthetic and recycling capacity. In humans, weight loss after bariatric surgery showed a transcriptional signature in visceral adipose tissue similar to our mouse model of obesity reversion. Overall, our data indicate that obesity prompts a lasting metabolic fingerprint that leads to a progressive breakdown of metabolic plasticity in visceral adipose tissue.

Angiocrine polyamine production regulates adiposity.

Reciprocal interactions between endothelial cells (ECs) and adipocytes are fundamental to maintain white adipose tissue (WAT) homeostasis, as illustrated by the activation of angiogenesis upon WAT expansion, a process that is impaired in obesity. However, the molecular mechanisms underlying the crosstalk between ECs and adipocytes remain poorly understood. Here, we show that local production of polyamines in ECs stimulates adipocyte lipolysis and regulates WAT homeostasis in mice. We promote enhanced cell-autonomous angiogenesis by deleting Pten in the murine endothelium. Endothelial Pten loss leads to a WAT-selective phenotype, characterized by reduced body weight and adiposity in pathophysiological conditions. This phenotype stems from enhanced fatty acid ß-oxidation in ECs concomitant with a paracrine lipolytic action on adipocytes, accounting for reduced adiposity. Combined analysis of murine models, isolated ECs and human specimens reveals that WAT lipolysis is mediated by mTORC1-dependent production of polyamines by ECs. Our results indicate that angiocrine metabolic signals are important for WAT homeostasis and organismal metabolism.

Acute RyR1 Ca2+ leak enhances NADH-linked mitochondrial respiratory capacity.

Sustained ryanodine receptor (RyR) Ca2+ leak is associated with pathological conditions such as heart failure or skeletal muscle weakness. We report that a single session of sprint interval training (SIT), but not of moderate intensity continuous training (MICT), triggers RyR1 protein oxidation and nitrosylation leading to calstabin1 dissociation in healthy human muscle and in in vitro SIT models (simulated SIT or S-SIT). This is accompanied by decreased sarcoplasmic reticulum Ca2+ content, increased levels of mitochondrial oxidative phosphorylation proteins, supercomplex formation and enhanced NADH-linked mitochondrial respiratory capacity. Mechanistically, (S-)SIT increases mitochondrial Ca2+ uptake in mouse myotubes and muscle fibres, and decreases pyruvate dehydrogenase phosphorylation in human muscle and mouse myotubes. Countering Ca2+ leak or preventing mitochondrial Ca2+ uptake blunts S-SIT-induced adaptations, a result supported by proteomic analyses. Here we show that triggering acute transient Ca2+ leak through RyR1 in healthy muscle may contribute to the multiple health promoting benefits of exercise.

Mitochondrial cristae-remodeling protein OPA1 in POMC neurons couples Ca2+ homeostasis with adipose tissue lipolysis.

Appropriate cristae remodeling is a determinant of mitochondrial function and bioenergetics and thus represents a crucial process for cellular metabolic adaptations. Here, we show that mitochondrial cristae architecture and expression of the master cristae-remodeling protein OPA1 in proopiomelanocortin (POMC) neurons, which are key metabolic sensors implicated in energy balance control, is affected by fluctuations in nutrient availability. Genetic inactivation of OPA1 in POMC neurons causes dramatic alterations in cristae topology, mitochondrial Ca2+ handling, reduction in alpha-melanocyte stimulating hormone (α-MSH) in target areas, hyperphagia, and attenuated white adipose tissue (WAT) lipolysis resulting in obesity. Pharmacological blockade of mitochondrial Ca2+ influx restores α-MSH and the lipolytic program, while improving the metabolic defects of mutant mice. Chemogenetic manipulation of POMC neurons confirms a role in lipolysis control. Our results unveil a novel axis that connects OPA1 in POMC neurons with mitochondrial cristae, Ca2+ homeostasis, and WAT lipolysis in the regulation of energy balance.

Activation of the Integrated Stress Response and ER Stress Protect from Fluorizoline-Induced Apoptosis in HEK293T and U2OS Cell Lines.

The prohibitin (PHB)-binding compound fluorizoline as well as PHB-downregulation activate the integrated stress response (ISR) in HEK293T and U2OS human cell lines. This activation is denoted by phosphorylation of eIF2α and increases in ATF4, ATF3, and CHOP protein levels. The blockage of the activation of the ISR by overexpression of GRP78, as well as an increase in IRE1 activity, indicate the presence of ER stress after fluorizoline treatment. The inhibition of the ER stress response in HEK293T and U2OS led to increased sensitivity to fluorizoline-induced apoptosis, indicating a pro-survival role of this pathway after fluorizoline treatment in these cell lines. Fluorizoline induced an increase in calcium concentration in the cytosol and the mitochondria. Finally, two different calcium chelators reduced fluorizoline-induced apoptosis in U2OS cells. Thus, we have found that fluorizoline causes increased ER stress and activation of the integrated stress response, which in HEK293T and U2OS cells are protective against fluorizoline-induced apoptosis.

Effects of Lifestyle Intervention in Tissue-Specific Lipidomic Profile of Formerly Obese Mice.

Lipids are highly diverse in their composition, properties and distribution in different biological entities. We aim to establish the lipidomes of several insulin-sensitive tissues and to test their plasticity when divergent feeding regimens and lifestyles are imposed. Here, we report a proton nuclear magnetic resonance (1H-NMR) study of lipid abundance across 4 tissues of C57Bl6J male mice that includes the changes in the lipid profile after every lifestyle intervention. Every tissue analysed presented a specific lipid profile irrespective of interventions. Glycerolipids and fatty acids were most abundant in epididymal white adipose tissue (eWAT) followed by liver, whereas sterol lipids and phosphoglycerolipids were highly enriched in hypothalamus, and gastrocnemius had the lowest content in all lipid species compared to the other tissues. Both when subjected to a high-fat diet (HFD) and after a subsequent lifestyle intervention (INT), the lipidome of hypothalamus showed no changes. Gastrocnemius and liver revealed a pattern of increase in content in many lipid species after HFD followed by a regression to basal levels after INT, while eWAT lipidome was affected mainly by the fat composition of the administered diets and not their caloric density. Thus, the present study demonstrates a unique lipidome for each tissue modulated by caloric intake and dietary composition.

PEPCK-M recoups tumor cell anabolic potential in a PKC-ζ-dependent manner.

BACKGROUND: Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is expressed in all cancer types examined and in neuroprogenitor cells. The gene is upregulated by amino acid limitation and ER-stress in an ATF4-dependent manner, and its activity modulates the PEP/Ca2+ signaling axis, providing clear arguments for a functional relationship with metabolic adaptations for cell survival. Despite its potential relevance to cancer metabolism, the mechanisms responsible for its pro-survival activity have not been completely elucidated. METHODS: [U-13C]glutamine and [U-13C]glucose labeling of glycolytic and TCA cycle intermediates and their anabolic end-products was evaluated quantitatively using LC/MS and GC/MS in conditions of abundant glucose and glucose limitation in loss-of-function (shRNA) and gain-of-function (lentiviral constitutive overexpression) HeLa cervix carcinoma cell models. Cell viability was assessed in conjunction with various glucose concentrations and in xenografts in vivo. RESULTS: PEPCK-M levels linearly correlated with [U-13C]glutamine label abundance in most glycolytic and TCA cycle intermediate pools under nutritional stress. In particular, serine, glycine, and proline metabolism, and the anabolic potential of the cell, were sensitive to PEPCK-M activity. Therefore, cell viability defects could be rescued by supplementing with an excess of those amino acids. PEPCK-M silenced or inhibited cells in the presence of abundant glucose showed limited growth secondary to TCA cycle blockade and increased ROS. In limiting glucose conditions, downregulation of PKC-ζ tumor suppressor has been shown to enhance survival. Consistently, HeLa cells also sustained a survival advantage when PKC-ζ tumor suppressor was downregulated using shRNA, but this advantage was abolished in the absence of PEPCK-M, as its inhibition restores cell growth to control levels. The relationship between these two pathways is also highlighted by the anti-correlation observed between PEPCK-M and PKC-ζ protein levels in all clones tested, suggesting co-regulation in the absence of glucose. Finally, PEPCK-M loss negatively impacted on anchorage-independent colony formation and xenograft growth in vivo. CONCLUSIONS: All in all, our data suggest that PEPCK-M might participate in the mechanisms to regulate proteostasis in the anabolic and stalling phases of tumor growth. We provide molecular clues into the clinical relevance of PEPCK-M as a mechanism of evasion of cancer cells in conditions of nutrient stress.

NRF2 function in osteocytes is required for bone homeostasis and drives osteocytic gene expression.

Osteocytes, the most abundant bone cell type, are derived from osteoblasts through a process in which they are embedded in an osteoid. We previously showed that nutrient restriction promotes the osteocyte transcriptional program and is associated with increased mitochondrial biogenesis. Here, we show that increased mitochondrial biogenesis increase reactive oxygen species (ROS) levels and consequently, NRF2 activity during osteocytogenesis. NRF2 activity promotes osteocyte-specific expression of Dmp1, Mepe, and Sost in IDG-SW3 cells, primary osteocytes, and osteoblasts, and in murine models with Nfe2l2 deficiency in osteocytes or osteoblasts. Moreover, ablation of Nfe2l2 in osteocytes or osteoblasts generates osteopenia and increases osteoclast numbers with marked sexual dimorphism. Finally, treatment with dimethyl fumarate prevented the deleterious effects of ovariectomy in trabecular bone masses of mice and restored osteocytic gene expression. Altogether, we uncovered the role of NRF2 activity in osteocytes during the regulation of osteocyte gene expression and maintenance of bone homeostasis.

Pharmacology and preclinical validation of a novel anticancer compound targeting PEPCK-M.

BACKGROUND: Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate. The mitochondrial isozyme, PEPCK-M is highly expressed in cancer cells, where it plays a role in nutrient stress response. To date, pharmacological strategies to target this pathway have not been pursued. METHODS: A compound embodying a 3-alkyl-1,8-dibenzylxanthine nucleus (iPEPCK-2), was synthesized and successfully probed in silico on a PEPCK-M structural model. Potency and target engagement in vitro and in vivo were evaluated by kinetic and cellular thermal shift assays (CETSA). The compound and its target were validated in tumor growth models in vitro and in murine xenografts. RESULTS: Cross-inhibitory capacity and increased potency as compared to 3-MPA were confirmed in vitro and in vivo. Treatment with iPEPCK-2 inhibited cell growth and survival, especially in poor-nutrient environment, consistent with an impact on colony formation in soft agar. Finally, daily administration of the PEPCK-M inhibitor successfully inhibited tumor growth in two murine xenograft models as compared to vehicle, without weight loss, or any sign of apparent toxicity. CONCLUSION: We conclude that iPEPCK-2 is a compelling anticancer drug targeting PEPCK-M, a hallmark gene product involved in metabolic adaptations of the tumor.

Phosphoenolpyruvate from Glycolysis and PEPCK Regulate Cancer Cell Fate by Altering Cytosolic Ca2.

Changes in phosphoenolpyruvate (PEP) concentrations secondary to variations in glucose availability can regulate calcium signaling in T cells as this metabolite potently inhibits the sarcoplasmic reticulum Ca2+/ATPase pump (SERCA). This regulation is critical to assert immune activation in the tumor as T cells and cancer cells compete for available nutrients. We examined here whether cytosolic calcium and the activation of downstream effector pathways important for tumor biology are influenced by the presence of glucose and/or cataplerosis through the phosphoenolpyruvate carboxykinase (PEPCK) pathway, as both are hypothesized to feed the PEP pool. Our data demonstrate that cellular PEP parallels extracellular glucose in two human colon carcinoma cell lines, HCT-116 and SW480. PEP correlated with cytosolic calcium and NFAT activity, together with transcriptional up-regulation of canonical targets PTGS2 and IL6 that was fully prevented by CsA pre-treatment. Similarly, loading the metabolite directly into the cell increased cytosolic calcium and NFAT activity. PEP-stirred cytosolic calcium was also responsible for the calmodulin (CaM) dependent phosphorylation of c-Myc at Ser62, resulting in increased activity, probably through enhanced stabilization of the protein. Protein expression of several c-Myc targets also correlated with PEP levels. Finally, the participation of PEPCK in this axis was interrogated as it should directly contribute to PEP through cataplerosis from TCA cycle intermediates, especially in glucose starvation conditions. Inhibition of PEPCK activity showed the expected regulation of PEP and calcium levels and consequential downstream modulation of NFAT and c-Myc activities. Collectively, these results suggest that glucose and PEPCK can regulate NFAT and c-Myc activities through their influence on the PEP/Ca2+ axis, advancing a role for PEP as a second messenger communicating metabolism, calcium cell signaling, and tumor biology.