-
Cells Dec 2019Changes in phosphoenolpyruvate (PEP) concentrations secondary to variations in glucose availability can regulate calcium signaling in T cells as this metabolite potently...
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 Ca/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/Ca axis, advancing a role for PEP as a second messenger communicating metabolism, calcium cell signaling, and tumor biology.
Topics: Calcium; Calcium Signaling; Cell Line, Tumor; Colonic Neoplasms; Cyclooxygenase 2; Cytosol; Gene Expression Regulation, Neoplastic; Glycolysis; HCT116 Cells; Humans; Interleukin-6; NFATC Transcription Factors; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxykinase (ATP); Proto-Oncogene Proteins c-myc
PubMed: 31861674
DOI: 10.3390/cells9010018 -
Molecules (Basel, Switzerland) Aug 2021Gluconeogenesis is a key interface between organic acid/amino acid/lipid and sugar metabolism. The aims of this article are four-fold. First, to provide a concise... (Review)
Review
Gluconeogenesis is a key interface between organic acid/amino acid/lipid and sugar metabolism. The aims of this article are four-fold. First, to provide a concise overview of plant gluconeogenesis. Second, to emphasise the widespread occurrence of gluconeogenesis and its utilisation in diverse processes. Third, to stress the importance of the vacuolar storage and release of Krebs cycle acids/nitrogenous compounds, and of the role of gluconeogenesis and malic enzyme in this process. Fourth, to outline the contribution of fine control of enzyme activity to the coordinate-regulation of gluconeogenesis and malate metabolism, and the importance of cytosolic pH in this.
Topics: Amino Acids; Citric Acid Cycle; Gluconeogenesis; Lipids; Malate Dehydrogenase; Nitrogen; Plants; Sugars
PubMed: 34500562
DOI: 10.3390/molecules26175129 -
Cancer & Metabolism Jan 2021Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is expressed in all cancer types examined and in neuroprogenitor cells. The gene is upregulated by amino...
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/Ca 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-C]glutamine and [U-C]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-C]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.
PubMed: 33413684
DOI: 10.1186/s40170-020-00236-3 -
IScience Apr 2023The Crabtree effect is defined as a rapid glucose-induced repression of mitochondrial oxidative metabolism and has been described in yeasts and tumor cells. Using...
The Crabtree effect is defined as a rapid glucose-induced repression of mitochondrial oxidative metabolism and has been described in yeasts and tumor cells. Using plate-based respirometry, we identified the Crabtree effect in normal (non-tumor) kidney proximal tubule epithelial cells (PTEC) but not in other kidney cells (podocytes or mesangial cells) or mammalian cells (C2C12 myoblasts). Glucose-induced repression of respiration was prevented by reducing glycolysis at the proximal step with 2-deoxyglucose and partially reversed by pyruvate. The late-stage glycolytic intermediates glyceraldehyde 3-phosphate, 3-phosphoglycerate, and phosphoenolpyruvate, but not the early-stage glycolytic intermediates or lactate, inhibited respiration in permeabilized PTEC and kidney cortex mitochondria, mimicking the Crabtree effect. Studies in diabetic mice indicated a pattern of increased late-stage glycolytic intermediates consistent with a similar pattern occurring . Our results show the unique presence of the Crabtree effect in kidney PTEC and identify the major mediators of this effect.
PubMed: 37091239
DOI: 10.1016/j.isci.2023.106462 -
Endocrinology and Metabolism (Seoul,... Feb 2024Sodium-dependent glucose cotransporter 2 (SGLT2) mediates glucose reabsorption in the renal proximal tubules, and SGLT2 inhibitors are used as therapeutic agents for...
BACKGRUOUND
Sodium-dependent glucose cotransporter 2 (SGLT2) mediates glucose reabsorption in the renal proximal tubules, and SGLT2 inhibitors are used as therapeutic agents for treating type 2 diabetes mellitus. This study aimed to elucidate the effects and mechanisms of SGLT2 inhibition on hepatic glucose metabolism in both serum deprivation and serum supplementation states.
METHODS
Huh7 cells were treated with the SGLT2 inhibitors empagliflozin and dapagliflozin to examine the effect of SGLT2 on hepatic glucose uptake. To examine the modulation of glucose metabolism by SGLT2 inhibition under serum deprivation and serum supplementation conditions, HepG2 cells were transfected with SGLT2 small interfering RNA (siRNA), cultured in serum-free Dulbecco's modified Eagle's medium for 16 hours, and then cultured in media supplemented with or without 10% fetal bovine serum for 8 hours.
RESULTS
SGLT2 inhibitors dose-dependently decreased hepatic glucose uptake. Serum deprivation increased the expression levels of the gluconeogenesis genes peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), glucose 6-phosphatase (G6pase), and phosphoenolpyruvate carboxykinase (PEPCK), and their expression levels during serum deprivation were further increased in cells transfected with SGLT2 siRNA. SGLT2 inhibition by siRNA during serum deprivation induces nuclear localization of the transcription factor forkhead box class O 1 (FOXO1), decreases nuclear phosphorylated-AKT (p-AKT), and p-FOXO1 protein expression, and increases phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK) protein expression. However, treatment with the AMPK inhibitor, compound C, reversed the reduction in the protein expression levels of nuclear p- AKT and p-FOXO1 and decreased the protein expression levels of p-AMPK and PEPCK in cells transfected with SGLT2 siRNA during serum deprivation.
CONCLUSION
These data show that SGLT2 mediates glucose uptake in hepatocytes and that SGLT2 inhibition during serum deprivation increases gluconeogenesis via the AMPK/AKT/FOXO1 signaling pathway.
Topics: Humans; AMP-Activated Protein Kinases; Diabetes Mellitus, Type 2; Gluconeogenesis; Glucose; Phosphoenolpyruvate Carboxykinase (ATP); Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Sodium; Sodium-Glucose Transporter 2; Sodium-Glucose Transporter 2 Inhibitors
PubMed: 38171209
DOI: 10.3803/EnM.2023.1786 -
Bioactive Materials Dec 2022Understanding mechanisms underlying the heterogeneity of multipotent stem cells offers invaluable insights into biogenesis and tissue development. Extracellular matrix...
Understanding mechanisms underlying the heterogeneity of multipotent stem cells offers invaluable insights into biogenesis and tissue development. Extracellular matrix (ECM) stiffness has been acknowledged as a crucial factor regulating stem cell fate. However, how cells sense stiffness cues and adapt their metabolism activity is still unknown. Here we report the novel role of mitochondrial phosphoenolpyruvate carboxykinase (PCK2) in enhancing osteogenesis in 3D ECM via glycolysis. We experimentally mimicked the physical characteristics of 3D trabeculae network of normal and osteoporotic bone with different microstructure and stiffness, observing that PCK2 promotes osteogenesis in 3D ECM with tunable stiffness in vitro and in vivo. Mechanistically, PCK2 enhances the rate-limiting metabolic enzyme pallet isoform phosphofructokinase (PFKP) in 3D ECM, and further activates AKT/extracellular signal-regulated kinase 1/2 (ERK1/2) cascades, which directly regulates osteogenic differentiation of MSCs. Collectively, our findings implicate an intricate crosstalk between cell mechanics and metabolism, and provide new perspectives for strategies of osteoporosis.
PubMed: 35415308
DOI: 10.1016/j.bioactmat.2022.03.036 -
Microbial Genomics Feb 2022Microbial phosphonate biosynthetic machinery has been identified in ~5 % of bacterial genomes and encodes natural products like fosfomycin as well as cell surface...
Microbial phosphonate biosynthetic machinery has been identified in ~5 % of bacterial genomes and encodes natural products like fosfomycin as well as cell surface decorations. Almost all biological phosphonates originate from the rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate (PnPy) catalysed by PEP mutase (Ppm), and PnPy is often converted to phosphonoacetaldehyde (PnAA) by PnPy decarboxylase (Ppd). Seven enzymes are known or likely to act on either PnPy or PnAA as early branch points en route to diverse biosynthetic outcomes, and these enzymes may be broadly classified into three reaction types: hydride transfer, aminotransfer, and carbon-carbon bond formation. However, the relative abundance of these branch points in microbial phosphonate biosynthesis is unknown. Also unknown is the proportion of -containing gene neighbourhoods encoding new branch point enzymes and potentially novel phosphonates. In this study we computationally sorted 434 -containing gene neighbourhoods based on these seven branch point enzymes. Unsurprisingly, the majority (56 %) of these pathways encode for production of the common naturally occurring compound 2-aminoethylphosphonate (AEP) or a hydroxylated derivative. The next most abundant genetically encoded intermediates were phosphonoalanine (PnAla, 9.2 %), 2-hydroxyethylphosphonate (HEP, 8.5 %), and phosphonoacetate (PnAc, 6 %). Significantly, about 13 % of the gene neighbourhoods could not be assigned to any of the seven branch points and may encode novel phosphonates. Sequence similarity network analysis revealed families of unusual gene neighbourhoods including possible production of phosphonoacrylate and phosphonofructose, the apparent biosynthetic use of the C-P lyase operon, and a virus-encoded phosphonate. Overall, these results highlight the utility of branch point inventories to identify novel gene neighbourhoods and guide future phosphonate discovery efforts.
Topics: Carbon; Genome, Bacterial; Organophosphonates
PubMed: 35188456
DOI: 10.1099/mgen.0.000781 -
Frontiers in Genetics 2022Cold tolerance is an important trait for sheep raised at high altitudes. Muscle tissue, comprising 30-40% of the total body mass, produces heat during cold exposure....
Cold tolerance is an important trait for sheep raised at high altitudes. Muscle tissue, comprising 30-40% of the total body mass, produces heat during cold exposure. However, little is known about the genetic mechanisms of this tissue and its role in thermogenesis in lambs. We examined genes in skeletal muscle tissue in a cold-adapted sheep breed, Altay, and a cold-intolerant sheep breed, Hu, when exposed to low air temperature. Three ewe-lambs of each breed were maintained at -5°C and three ewe-lambs of each breed were maintained at 20°C. After cold exposure for 25 days, the of each lamb was collected, and transcriptome profiles were sequenced and analyzed. The results of RNA-seq showed that the average reads among the four groups were 11.0 Gbase. The genome mapping rate averaged 88.1% and the gene mapping rate averaged 82.5%. The analysis of differentially expressed genes (DEGs) indicated that the peroxisome proliferator-activated receptors (PPAR), cAMP, and calcium signaling pathways and muscle contraction in muscle tissue were linked to thermogenesis in cold-exposed lambs. Furthermore, () increased glyceroneogenesis in cold-exposed Altay lambs, and (), (), and () were involved in the intake and transport of free fatty acids. In Hu sheep, cAMP biosynthesis from ATP hydrolysis was regulated by () and (). Skeletal muscle contraction was regulated by (). In conclusion, cold exposure altered the expression level of genes involved in heat production in muscle tissue. Some potential mechanisms were revealed, including calcium ion transport in the calcium signaling pathway, fatty acid metabolism in the PPAR signaling pathway, and cAMP biosynthesis in the cAMP signaling pathway. This study implied that skeletal muscle plays an important role in thermoregulation in lambs.
PubMed: 36338953
DOI: 10.3389/fgene.2022.1017458 -
Cellular and Molecular Life Sciences :... Jul 2022The citrate carrier (CIC) is an integral protein of the inner mitochondrial membrane which catalyzes the efflux of mitochondrial citrate (or other tricarboxylates) in... (Review)
Review
The citrate carrier (CIC) is an integral protein of the inner mitochondrial membrane which catalyzes the efflux of mitochondrial citrate (or other tricarboxylates) in exchange with a cytosolic anion represented by a tricarboxylate or a dicarboxylate or phosphoenolpyruvate. In this way, the CIC provides the cytosol with citrate which is involved in many metabolic reactions. Several studies have been carried out over the years on the structure, function and regulation of this metabolite carrier protein both in mammals and in many other organisms. A lot of data on the characteristics of this protein have therefore accumulated over time thereby leading to a complex framework of metabolic and physiological implications connected to the CIC function. In this review, we critically analyze these data starting from the multiple roles played by the mitochondrial CIC in many cellular processes and then examining the regulation of its activity in different nutritional and hormonal states. Finally, the metabolic significance of the citrate flux, mediated by the CIC, across distinct subcellular compartments is also discussed.
Topics: Animals; Carrier Proteins; Citrates; Cytosol; Mammals; Mitochondria
PubMed: 35842872
DOI: 10.1007/s00018-022-04466-0 -
Biochimica Et Biophysica Acta.... Nov 2020Mannose transporters constitute a superfamily (Man-PTS) of the Phosphoenolpyruvate Carbohydrate Phosphotransferase System (PTS). The membrane complexes are homotrimers... (Review)
Review
Mannose transporters constitute a superfamily (Man-PTS) of the Phosphoenolpyruvate Carbohydrate Phosphotransferase System (PTS). The membrane complexes are homotrimers of protomers consisting of two subunits, IIC and IID. The two subunits without recognizable sequence similarity assume the same fold, and in the protomer are structurally related by a two fold pseudosymmetry axis parallel to membrane-plane (Liu et al. (2019) Cell Research 29 680). Two reentrant loops and two transmembrane helices of each subunit together form the N-terminal transport domain. Two three-helix bundles, one of each subunit, form the scaffold domain. The protomer is stabilized by a helix swap between these bundles. The two C-terminal helices of IIC mediate the interprotomer contacts. PTS occur in bacteria and archaea but not in eukaryotes. Man-PTS are abundant in Gram-positive bacteria living on carbohydrate rich mucosal surfaces. A subgroup of IICIID complexes serve as receptors for class IIa bacteriocins and as channel for the penetration of bacteriophage lambda DNA across the inner membrane. Some Man-PTS are associated with host-pathogen and -symbiont processes.
Topics: Bacterial Proteins; Bacteriocins; Bacteriophages; Gram-Positive Bacteria; Mannose; Phosphotransferases; Protein Conformation, alpha-Helical; Protein Domains
PubMed: 32710850
DOI: 10.1016/j.bbamem.2020.183412