Neuronal Progenitor Maintenance Requires Lactate Metabolism and PEPCK-M-Directed Cataplerosis.

This study investigated the metabolic requirements for neuronal progenitor maintenance in vitro and in vivo by examining the metabolic adaptations that support neuronal progenitors and neural stem cells (NSCs) in their undifferentiated state. We demonstrate that neuronal progenitors are strictly dependent on lactate metabolism, while glucose induces their neuronal differentiation. Lactate signaling is not by itself capable of maintaining the progenitor phenotype. The consequences of lactate metabolism include increased mitochondrial and oxidative metabolism, with a strict reliance on cataplerosis through the mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) pathway to support anabolic functions, such as the production of extracellular matrix. In vivo, lactate maintains/induces populations of postnatal neuronal progenitors/NSCs in a PEPCK-M-dependent manner. Taken together, our data demonstrate that, lactate alone or together with other physical/biochemical cues maintain NSCs/progenitors with a metabolic signature that is classically found in tissues with high anabolic capacity.

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) is a pro-survival, endoplasmic reticulum (ER) stress response gene involved in tumor cell adaptation to nutrient availability.

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), encoded by the nuclear PCK2 gene, links TCA cycle intermediates and glycolytic pools through the conversion of mitochondrial oxaloacetate into phosphoenolpyruvate. In the liver PEPCK-M adjoins its profusely studied cytosolic isoform (PEPCK-C) potentiating gluconeogenesis and TCA flux. However, PEPCK-M is present in a variety of non-gluconeogenic tissues, including tumors of several origins. Despite its potential relevance to cancer metabolism, the mechanisms responsible for PCK2 gene regulation have not been elucidated. The present study demonstrates PEPCK-M overexpression in tumorigenic cells as well as the mechanism for the modulation of PCK2 abundance under several stress conditions. Amino acid limitation and ER stress inducers, conditions that activate the amino acid response (AAR) and the unfolded protein response (UPR), stimulate PCK2 gene transcription. Both the AAR and UPR lead to increased synthesis of ATF4, which mediates PCK2 transcriptional up-regulation through its binding to a putative ATF/CRE composite site within the PCK2 promoter functioning as an amino acid response element. In addition, activation of the GCN2-eIF2α-ATF4 and PERK-eIF2α-ATF4 signaling pathways are responsible for increased PEPCK-M levels. Finally, PEPCK-M knockdown using either siRNA or shRNA were sufficient to reduce MCF7 mammary carcinoma cell growth and increase cell death under glutamine deprivation or ER stress conditions. Our data demonstrate that this enzyme has a critical role in the survival program initiated upon stress and shed light on an unexpected and important role of mitochondrial PEPCK in cancer metabolism.

The mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) and glucose homeostasis: has it been overlooked?

BACKGROUND: Plasma glucose levels are tightly regulated within a narrow physiologic range. Insulin-mediated glucose uptake by tissues must be balanced by the appearance of glucose from nutritional sources, glycogen stores, or gluconeogenesis. In this regard, a common pathway regulating both glucose clearance and appearance has not been described. The metabolism of glucose to produce ATP is generally considered to be the primary stimulus for insulin release from beta-cells. Similarly, gluconeogenesis from phosphoenolpyruvate (PEP) is believed to be the primarily pathway via the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C). These models cannot adequately explain the regulation of insulin secretion or gluconeogenesis. SCOPE OF REVIEW: A metabolic sensing pathway involving mitochondrial GTP (mtGTP) and PEP synthesis by the mitochondrial isoform of PEPCK (PEPCK-M) is associated with glucose-stimulated insulin secretion from pancreatic beta-cells. Here we examine whether there is evidence for a similar mtGTP-dependent pathway involved in gluconeogenesis. In both islets and the liver, mtGTP is produced at the substrate level by the enzyme succinyl CoA synthetase (SCS-GTP) with a rate proportional to the TCA cycle. In the beta-cell PEPCK-M then hydrolyzes mtGTP in the production of PEP that, unlike mtGTP, can escape the mitochondria to generate a signal for insulin release. Similarly, PEPCK-M and mtGTP might also provide a significant source of PEP in gluconeogenic tissues for the production of glucose. This review will focus on the possibility that PEPCK-M, as a sensor for TCA cycle flux, is a key mechanism to regulate both insulin secretion and gluconeogenesis suggesting conservation of this biochemical mechanism in regulating multiple aspects of glucose homeostasis. Moreover, we propose that this mechanism may be important for regulating insulin secretion and gluconeogenesis compared to canonical nutrient sensing pathways. MAJOR CONCLUSIONS: PEPCK-M, initially believed to be absent in islets, carries a substantial metabolic flux in beta-cells. This flux is intimately involved with the coupling of glucose-stimulated insulin secretion. PEPCK-M activity may have been similarly underestimated in glucose producing tissues and could potentially be an unappreciated but important source of gluconeogenesis. GENERAL SIGNIFICANCE: The generation of PEP via PEPCK-M may occur via a metabolic sensing pathway important for regulating both insulin secretion and gluconeogenesis. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

Mitochondrial phosphoenolpyruvate carboxykinase promotes tumor growth in estrogen receptor-positive breast cancer via regulation of the mTOR pathway.

BACKGROUND: Tumor cells may aberrantly express metabolic enzymes to adapt to their environment for survival and growth. Targeting cancer-specific metabolic enzymes is a potential therapeutic strategy. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and links the tricarboxylic acid cycle and glycolysis/gluconeogenesis. Mitochondrial PEPCK (PEPCK-M), encoded by PCK2, is an isozyme of PEPCK and is distributed in mitochondria. Overexpression of PCK2 has been identified in many human cancers and demonstrated to be important for the survival program initiated upon metabolic stress in cancer cells. We evaluated the expression status of PEPCK-M and investigated the function of PEPCK-M in breast cancer. METHODS: We checked the expression status of PEPCK-M in breast cancer samples by immunohistochemical staining. We knocked down or overexpressed PCK2 in breast cancer cell lines to investigate the function of PEPCK-M in breast cancer. RESULTS: PEPCK-M was highly expressed in estrogen receptor-positive (ER+ ) breast cancers. Decreased cell proliferation and G0 /G1 arrest were induced in ER+ breast cancer cell lines by knockdown of PCK2. PEPCK-M promoted the activation of mTORC1 downstream signaling molecules and the E2F1 pathways in ER+ breast cancer. In addition, glucose uptake, intracellular glutamine levels, and mTORC1 pathways activation by glucose and glutamine in ER+ breast cancer were attenuated by PCK2 knockdown. CONCLUSION: PEPCK-M promotes proliferation and cell cycle progression in ER+ breast cancer via upregulation of the mTORC1 and E2F1 pathways. PCK2 also regulates nutrient status-dependent mTORC1 pathway activation in ER+ breast cancer. Further studies are warranted to understand whether PEPCK-M is a potential therapeutic target for ER+ breast cancer.

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.

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 carboxykinase is involved in antiviral immunity against Bombyx mori nucleopolyhedrovirus.

Phosphoenolpyruvate carboxykinase (PEPCK) has cytoplasmic isoform (PEPCK-C) and a mitochondrial isoform (PEPCK-M). PEPCK-C plays an important role in gluconeogenesis, but the function of PEPCK-M is largely unknown. In this study, we cloned two isoforms of PEPCK (BmPEPCK-1 and BmPEPCK-2; both of PEPCK-M) from the lepidopteran model Bombyx mori. BmPEPCK-1 and BmPEPCK-2 were adjacently located in the silkworm genome, and both contained 13 exons. The main difference in the sequences was the 13th exon and 3'UTR. The expression of BmPEPCK-1 was higher than that of BmPEPCK-2, the overexpression of which did not affect BmNPV proliferation. The expression levels of BmPEPCK-2 and ATG6/7/8/13 decreased after BmNPV infection. Overexpression of BmPEPCK-2 increased the expression of ATG6/7/8 and significantly decreased viral fluorescence and content, suggesting that BmPEPCK-2 suppressed the multiplication of BmNPV by increasing ATGs expression. These results revealed that PEPCK-M has an important function in antiviral immunity.

Computational evaluation of natural compounds as potential inhibitors of human PEPCK-M: an alternative for lung cancer therapy.

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide. Among its subtypes, non-small cell lung cancer (NSCLC) is the most common. Recently, the mitochondrial isoform of the enzyme phosphoenolpyruvate carboxykinase (HsPEPCK-M) was identified as responsible for the metabolic adaptation in the NSCLC allowing tumor growth even under conditions of glucose deficiency. This adaptation is possible due to the role of HsPEPCK-M in gluconeogenesis, converting the oxaloacetate to phosphoenolpyruvate in the presence of GTP, which plays an important role in the energetic support of these tumors. In this context, it was shown that the inhibition or knockdown of this enzyme was able to induce apoptosis in NSCLC under low glucose conditions. PURPOSE: In this study, novel putative inhibitors were proposed for the human PEPCK-M (HsPEPCK-M) based on a computer-aided approach. METHODS: Comparative modeling was used to generate 3D models for HsPEPCK-M. Subsequently, the set of natural compounds of the ZINC database was screened against HsPEPCK-M models using structure-based pharmacophore modeling and molecular docking approaches. The selected compounds were evaluated according to its chemical diversity and clustered based on chemical similarity. RESULTS: The pharmacophore hypotheses, generated based on known PEPCK inhibitors, were able to select 7,124 candidate compounds. These compounds were submitted to molecular docking studies using three conformations of HsPEPCK-M generated by comparative modeling. The aim was to select compounds with high predicted binding affinity for at least one of the conformations of HsPEPCK-M. After molecular docking, 612 molecules were selected as potential inhibitors of HsPEPCK-M. These compounds were clustered according to their structural similarity. Chemical profiling and binding mode analyses of these compounds allowed the proposal of four promising compounds: ZINC01656421, ZINC895296, ZINC00895535 and ZINC02571340. CONCLUSION: These compounds may be considered as potential candidates for HsPEPCK-M inhibitors and may also be used as lead compounds for the development of novel HsPEPCK-M inhibitors.

Mitochondrial GTP Links Nutrient Sensing to ß Cell Health, Mitochondrial Morphology, and Insulin Secretion Independent of OxPhos.

Mechanisms coordinating pancreatic ß cell metabolism with insulin secretion are essential for glucose homeostasis. One key mechanism of ß cell nutrient sensing uses the mitochondrial GTP (mtGTP) cycle. In this cycle, mtGTP synthesized by succinyl-CoA synthetase (SCS) is hydrolyzed via mitochondrial PEPCK (PEPCK-M) to make phosphoenolpyruvate, a high-energy metabolite that integrates TCA cycling and anaplerosis with glucose-stimulated insulin secretion (GSIS). Several strategies, including xenotopic overexpression of yeast mitochondrial GTP/GDP exchanger (GGC1) and human ATP and GTP-specific SCS isoforms, demonstrated the importance of the mtGTP cycle. These studies confirmed that mtGTP triggers and amplifies normal GSIS and rescues defects in GSIS both in vitro and in vivo. Increased mtGTP synthesis enhanced calcium oscillations during GSIS. mtGTP also augmented mitochondrial mass, increased insulin granule number, and membrane proximity without triggering de-differentiation or metabolic fragility. These data highlight the importance of the mtGTP signal in nutrient sensing, insulin secretion, mitochondrial maintenance, and ß cell health.

Changes in expression of serine biosynthesis and integrated stress response genes during myogenic differentiation of C2C12 cells.

Skeletal muscle is a highly metabolic and dynamic tissue that is formed through the complex and well-organised process of myogenesis. Although there is a good understanding about the role of the Muscle Regulatory Factors during myogenesis, little is known about the potential interplay of other metabolic proteins. The aim of this study was to determine the endogenous mRNA expression profile for a novel group of genes, recently associated with ß2-adrenergic agonist (BA) induced muscle hypertrophy in pigs [1], during myogenic differentiation in C2C12 cells and their response to dibutyryl cyclic-AMP (dbcAMP). These genes included mitochondrial phosphoenolpyruvate carboxykinase (PCK2/PEPCK-M), genes involved in serine biosynthesis (Phosphoglycerate dehydrogenase, PHGDH; Phosphoserine aminotransferase-1, PSAT1; Phosphoserine phosphatase, PSPH) and those involved in an integrated stress response (Asparagine synthetase, ASNS; Sestrin-2, SESN2; and Activating transcription factor-5, ATF5). A coordinated peak in endogenous PCK2, PHGDH, PSAT1, PSPH, ASNS, ATF5 and SESN2 mRNA expression was observed at day 2 of differentiation (P < 0.001) in C2C12 cells, which coincided with the peak in myogenin mRNA. Myotube hypertrophy was induced with dbcAMP (1 mM) treatment from day 0, thereby mimicking the in vivo BA response. Although dbcAMP treatment from day 0 induced larger myotubes and increased both myosin heavy chain-IIB (MyHC-IIB) and pyruvate carboxylase (PC) mRNA, the expression of PCK2, PHGDH, PSAT1 and ASNS mRNA were all unaffected. Treatment with dbcAMP from day 4 increased MyHC-IIB mRNA, however this was less dramatic compared to the response observed following treatment from day 0, but there was no effect on PC mRNA. There was also no effect of dbcAMP treatment from day 4 on PCK2, PHGDH, PSAT1 and ASNS mRNA. To conclude, the coordinated day 2 peak in endogenous expression of PCK2, PHGDH, PSAT1, PSPH, ASNS, ATF5 and SESN2 mRNA may relate to a shift in biosynthetic demand required to initiate myogenic differentiation. However, dbcAMP had no effect on the expression of these genes in vitro suggesting that the effects observed in BA-treated pigs might be via other signalling pathways from the activation of the ß2-adrenergic receptor, but independent of cAMP, or that there are species differences in the response.

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.

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) regulates the cell metabolism of pancreatic neuroendocrine tumors (pNET) and de-sensitizes pNET to mTOR inhibitors.

mTOR pathway activation and hypervascularity have been identified as important characteristics of pancreatic neuroendocrine tumors (pNETs). Agents targeting angiogenesis and mTOR, such as sunitinib and everolimus (RAD001), have been shown to result in progression-free survival of approximately 11 months in patients with advanced pNETs. Novel treatment is needed to extend survival. Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), which is encoded by PCK2, catalyzes the conversion of oxaloacetate to phosphoenolpyruvate. PEPCK-M has been demonstrated to potentiate cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK-C)-mediated gluconeogenesis and to play a critical role in the survival program initiated upon stress during metabolism in cancer cells. Elevated expression of PCK2 has been found in various tumors according to the results of The Cancer Genome Atlas project. However, the role of PEPCK-M aberration in cancers is not well understood. In the current study, we observed that 12 of 21 (57%) pNET patients had high expression of PEPCK-M in the tumors, whereas the normal islet cells had weak expression of PEPCK-M. Knockdown of PCK2 inhibited the proliferation of pNET cells and enhanced the sensitivity of pNET cells to mTOR inhibitors. Knockdown of PCK2 promoted glycolysis but reduced mitochondrial oxidative phosphorylation in pNET cells. The combination of mTOR inhibitors and an anti-glycolysis agent, 2-DG, synergistically or additively inhibited the proliferation of pNET cells, particularly for the cells with high expression of PEPCK-M. Therefore, targeting PEPCK-M or glycolysis combined with inhibiting mTOR is a potential therapeutic approach for the treatment of pNETs.