The operation of PEPCK increases light harvesting plasticity in C4 NAD-ME and NADP-ME photosynthetic subtypes: A theoretical study.

The repeated emergence of NADP-malic enzyme (ME), NAD-ME and phosphoenolpyruvate carboxykinase (PEPCK) subtypes of C4 photosynthesis are iconic examples of convergent evolution, which suggests that these biochemistries do not randomly assemble, but are instead specific adaptations resulting from unknown evolutionary drivers. Theoretical studies that are based on the classic biochemical understanding have repeatedly proposed light-use efficiency as a possible benefit of the PEPCK subtype. However, quantum yield measurements do not support this idea. We explore this inconsistency here via an analytical model that features explicit descriptions across a seamless gradient between C4 biochemistries to analyse light harvesting and dark photosynthetic metabolism. Our simulations show that the NADP-ME subtype, operated by the most productive crops, is the most efficient. The NAD-ME subtype has lower efficiency, but has greater light harvesting plasticity (the capacity to assimilate CO2 in the broadest combination of light intensity and spectral qualities). In both NADP-ME and NAD-ME backgrounds, increasing PEPCK activity corresponds to greater light harvesting plasticity but likely imposed a reduction in photosynthetic efficiency. We draw the first mechanistic links between light harvesting and C4 subtypes, providing the theoretical basis for future investigation.

Inhibition of Sodium-Glucose Cotransporter-2 during Serum Deprivation Increases Hepatic Gluconeogenesis via the AMPK/AKT/FOXO Signaling Pathway.

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.

Allosteric regulation of the lid domain of PCK2 as a novel strategy for modulating mitochondrial dynamics.

Aberrant PCK2 overexpression has been linked to an unfavorable prognosis and shorter survival, particularly in hepatocellular carcinoma (HCC). Thus, the inactivation of PCK2 provides a promising strategy for HCC treatment. In this study, we used a chemical genetic strategy to identify a natural-derived small-molecule cucurbitacin B (CuB) as a selective PCK2 inhibitor. CuB covalently bound to PCK2 at a unique Cys63 site, blocking the Ω-loop lid domain formation via a previously undisclosed allosteric mechanism. Additionally, targeted lipidomics analysis also revealed that CuB destroyed mitochondrial membrane integrity, leading to the disruption of mitochondrial fusion-fission dynamics. Taken together, this study highlights the discovery of a small-molecule CuB, which reprograms lipid metabolism for controlling mitochondrial dynamics via targeting PCK2 in cancer cells.

Sodium butyrate alleviates deoxynivalenol-induced porcine intestinal barrier disruption by promoting mitochondrial homeostasis via PCK2 signaling.

Deoxynivalenol (DON) is one of the most plentiful trichothecenes occurring in food and feed, which brings severe health hazards to both animals and humans. This study aims to investigate whether sodium butyrate (NaB) can protect the porcine intestinal barrier from DON exposure through promoting mitochondrial homeostasis. In a 4-week feeding experiment, 28 male piglets were allocated according to a 2 by 2 factorial arrangement of treatments with the main factors including supplementation of DON (< 0.8 vs. 4.0 mg/kg) and NaB (0.0 vs. 2 g/kg) in a corn/soybean-based diet. Dietary NaB supplementation mitigated the damaged mitochondrial morphology within the jejunal mucosa and the disrupted gut epithelial tight junctions irritated by DON. In IPEC-J2 cells, we found efficient recovery of the intestinal epithelial barrier occurred following NaB administration. This intestinal barrier reparation was facilitated by NaB-induced PCK2-mediated glyceroneogenesis and restoration of mitochondrial structure and function. In conclusion, we elucidated a mechanism of PCK2-mediated improvement of mitochondrial function by NaB to repair porcine intestinal barrier disruption during chronic DON exposure. Our findings highlight the promise of NaB for use in protecting against DON-induced gut epithelial tight junction disruption in piglets.

PEPCK and glucose metabolism homeostasis in arthropods.

The fat body is responsible for a variety of functions related to energy metabolism in arthropods, by controlling the processes of de novo glucose production (gluconeogenesis) and glycogen metabolism. The rate-limiting factor of gluconeogenesis is the enzyme phosphoenolpyruvate carboxykinase (PEPCK), generally considered to be the first committed step in this pathway. Although the study of PEPCK and gluconeogenesis has been for decades restricted to mammalian models, especially focusing on muscle and liver tissue, current research has demonstrated particularities about the regulation of this enzyme in arthropods, and described new functions. This review will focus on arthropod PEPCK, discuss different aspects to PEPCK regulation and function, its general role in the regulation of gluconeogenesis and other pathways. The text also presents our views on potentially important new directions for research involving this enzyme in a variety of metabolic adaptations (e.g. diapause), discussing enzyme isoforms, roles during arthropod embryogenesis, as well as involvement in vector-pathogen interactions, contributing to a better understanding of insect vectors of diseases and their control.

Biochemical, structural, and kinetic characterization of PPi -dependent phosphoenolpyruvate carboxykinase from Propionibacterium freudenreichii.

Phosphoenolpyruvate carboxykinases (PEPCK) are a well-studied family of enzymes responsible for the regulation of TCA cycle flux, where they catalyze the interconversion of oxaloacetic acid (OAA) and phosphoenolpyruvate (PEP) using a phosphoryl donor/acceptor. These enzymes have typically been divided into two nucleotide-dependent classes, those that use ATP and those that use GTP. In the 1960's and early 1970's, a group of papers detailed biochemical properties of an enzyme named phosphoenolpyruvate carboxytransphosphorylase (later identified as a third PEPCK) from Propionibacterium freudenreichii (PPi -PfPEPCK), which instead of using a nucleotide, utilized PPi to catalyze the same interconversion of OAA and PEP. The presented work expands upon the initial biochemical experiments for PPi -PfPEPCK and interprets these data considering both the current understanding of nucleotide-dependent PEPCKs and is supplemented with a new crystal structure of PPi -PfPEPCK in complex with malate at a putative allosteric site. Most interesting, the data are consistent with PPi -PfPEPCK being a Fe2+ activated enzyme in contrast with the Mn2+ activated nucleotide-dependent enzymes which in part results in some unique kinetic properties for the enzyme when compared to the more widely distributed GTP- and ATP-dependent enzymes.

The ancient CgPEPCK-1, not CgPECK-2, evolved into a multifunctional molecule as an intracellular enzyme and extracellular PRR.

Phosphoenolpyruvate carboxykinase (PEPCK) is a well-known lyase involved in gluconeogenesis, while their evolution and function differentiation have not been fully understood. In this study, by constructing a phylogenetic tree to examine PEPCKs throughout the evolution from poriferans to vertebrates, Mollusk was highlighted as the only phylum to exhibit two distinct lineages, Mollusca_PEPCK-1 and Mollusca_PEPCK-2. Further study of two representative members from Crassostrea gigas (CgPEPCK-1 and CgPEPCK-2) showed that they both shared conserved sequences and structural characteristics of the catalytic enzyme, while CgPEPCK-2 displayed a higher expression level than CgPEPCK-1 in all tested tissues, and CgPEPCK-1 was specifically implicated in the immune defense against LPS stimulation and Vibrio splendidus infection. Functional analysis revealed that CgPEPCK-2 had stronger enzymatic activity than CgPEPCK-1, while CgPEPCK-1 exhibited stronger binding activity with various PAMPs, and only the protein of CgPEPCK-1 increased significantly in hemolymph during immune stimulation. All results supported that distinct sequence and function differentiations of the PEPCK gene family should have occurred since Mollusk. The more advanced evolutionary branch Mollusca_PEPCK-2 should preserve its essential function as a catalytic enzyme to be more specialized and efficient, while the ancient branch Mollusca_PEPCK-1 probably contained some members, such as CgPEPCK-1, that should be integrated into the immune system as an extracellular immune recognition receptor.

Biallelic pathogenic variants in the mitochondrial form of phosphoenolpyruvate carboxykinase cause peripheral neuropathy.

Phosphoenolpyruvate carboxykinase (PCK) plays a critical role in cytosolic gluconeogenesis, and defects in PCK1 cause a fasting-aggravated metabolic disease with hypoglycemia and lactic acidosis. However, there are two genes encoding PCK, and the role of the mitochondrial resident PCK (encoded by PCK2) is unclear, since gluconeogenesis is cytosolic. We identified three patients in two families with biallelic variants in PCK2. One has compound heterozygous variants (p.Ser23Ter/p.Pro170Leu), and the other two (siblings) have homozygous p.Arg193Ter variation. All three patients have weakness and abnormal gait, an absence of PCK2 protein, and profound reduction in PCK2 activity in fibroblasts, but no obvious metabolic phenotype. Nerve conduction studies showed reduced conduction velocities with temporal dispersion and conduction block compatible with a demyelinating peripheral neuropathy. To validate the association between PCK2 variants and clinical disease, we generated a mouse knockout model of PCK2 deficiency. The animals present abnormal nerve conduction studies and peripheral nerve pathology, corroborating the human phenotype. In total, we conclude that biallelic variants in PCK2 cause a neurogenetic disorder featuring abnormal gait and peripheral neuropathy.

Two phosphoenolpyruvate carboxykinases with differing biochemical properties in Chlamydomonas reinhardtii.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible reaction of decarboxylation and phosphorylation of oxaloacetate (OAA) to generate phosphoenolpyruvate (PEP) and CO2 playing mainly a gluconeogenic role in green algae. We found two PEPCK isoforms in Chlamydomonas reinhardtii and we cloned, purified and characterised both enzymes. ChlrePEPCK1 is more active as decarboxylase than ChlrePEPCK2. ChlrePEPCK1 is hexameric and its activity is affected by citrate, phenylalanine and malate, while ChlrePEPCK2 is monomeric and it is regulated by citrate, phenylalanine and glutamine. We postulate that the two PEPCK isoforms found originate from alternative splicing of the gene or regulated proteolysis of the enzyme. The presence of these two isoforms would be part of a mechanism to finely regulate the biological activity of PEPCKs.

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.

The Mechanism Underlying the Hypoglycemic Effect of Epimedin C on Mice with Type 2 Diabetes Mellitus Based on Proteomic Analysis.

Type 2 diabetes mellitus (T2DM) has become a worldwide public health problem. Epimedin C is considered one of the most important flavonoids in Epimedium, a famous edible herb in China and Southeast Asia that is traditionally used in herbal medicine to treat diabetes. In the present study, the therapeutic potential of epimedin C against T2DM was ascertained using a mouse model, and the mechanism underlying the hypoglycemic activity of epimedin C was explored using a label-free proteomic technique for the first time. Levels of fasting blood glucose (FBG), homeostasis model assessment of insulin resistance (HOMA-IR), and oral glucose tolerance, as well as contents of malondialdehyde (MDA) and low-density lipoprotein cholesterol (LDL-C) in the 30 mg·kg-1 epimedin C group (EC30 group), were significantly lower than those in the model control group (MC group) (p < 0.05), while the contents of hepatic glycogen, insulin, and high-density lipoprotein cholesterol (HDL-C), as well as activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the EC30 group were notably higher than those in the MC group (p < 0.05). The structures of liver cells and tissues were greatly destroyed in the MC group, whereas the structures of cells and tissues were basically complete in the EC30 group, which were similar to those in the normal control group (NC group). A total of 92 differentially expressed proteins (DEPs) were enriched in the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In the EC30 vs. MC groups, the expression level of cytosolic phosphoenolpyruvate carboxykinase (Pck1) was down-regulated, while the expression levels of group XIIB secretory phospholipase A2-like protein (Pla2g12b), apolipoprotein B-100 (Apob), and cytochrome P450 4A14 (Cyp4a14) were up-regulated. According to the KEGG pathway assay, Pck1 participated in the gluconeogenesis and insulin signaling pathways, and Pla2g12b, Apob, and Cyp4a14 were the key proteins in the fat digestion and fatty acid degradation pathways. Pck1, Pla2g12b, Apob, and Cyp4a14 seemed to play important roles in the prevention and treatment of T2DM. In summary, epimedin C inhibited Pck1 expression to maintain FBG at a relatively stable level, promoted Pla2g12b, Apob, and Cyp4a14 expressions to alleviate liver lipotoxicity, and protected liver tissues and cells from oxidant stress possibly by its phenolic hydroxyl groups.

Directional regulation of cytosolic PEPCK catalysis is mediated by competitive binding of anions.

Phosphoenolpyruvate carboxykinase (PEPCK) is a well-characterized enzyme involved in primary glucose metabolism, responsible for catalyzing one of the key steps of gluconeogenesis. It is well demonstrated that PEPCK can efficiently catalyze the reversible interconversion of oxaloacetic acid (OAA) to phosphoenolpyruvate (PEP) in vitro, but the enzyme is typically ascribed a metabolic role that requires preferential catalysis in the direction of PEP synthesis in vivo. Here we present structural and functional data that demonstrate the preferential synthesis of PEP from OAA catalyzed by PEPCK in vivo is facilitated by anion-mediated enzyme inhibition that reduces enzyme activity more significantly in the direction of OAA synthesis than in the direction of PEP synthesis. From our studies we conclude that the specific binding of small, ubiquitous anions like chloride, present in millimolar concentrations under normal cellular conditions allows for metabolic control by restricting PEPCK to function in the direction of PEP synthesis.

Metformin can mitigate skeletal dysplasia caused by Pck2 deficiency.

As an important enzyme for gluconeogenesis, mitochondrial phosphoenolpyruvate carboxykinase (PCK2) has further complex functions beyond regulation of glucose metabolism. Here, we report that conditional knockout of Pck2 in osteoblasts results in a pathological phenotype manifested as craniofacial malformation, long bone loss, and marrow adipocyte accumulation. Ablation of Pck2 alters the metabolic pathways of developing bone, particularly fatty acid metabolism. However, metformin treatment can mitigate skeletal dysplasia of embryonic and postnatal heterozygous knockout mice, at least partly via the AMPK signaling pathway. Collectively, these data illustrate that PCK2 is pivotal for bone development and metabolic homeostasis, and suggest that regulation of metformin-mediated signaling could provide a novel and practical strategy for treating metabolic skeletal dysfunction.

Development of novel DNA marker for species discrimination of Fasciola flukes based on the fatty acid binding protein type I gene.

BACKGROUND: Multiplex polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) for nuclear phosphoenolpyruvate carboxykinase (pepck) and polymerase delta (pold), respectively, have been used to differentiate Fasciola hepatica, F. gigantica, and hybrid Fasciola flukes. However, discrimination errors have been reported in both methods. This study aimed to develop a multiplex PCR based on a novel nuclear marker, the fatty acid binding protein type I (FABP) type I gene. METHODS: Nucleotide sequence variations of FABP type I were analyzed using DNA samples of F. hepatica, F. gigantica, and hybrid Fasciola flukes obtained from 11 countries in Europe, Latin America, Africa, and Asia. A common forward primer for F. hepatica and F. gigantica and two specific reverse primers for F. hepatica and F. gigantica were designed for multiplex PCR. RESULTS: Specific fragments of F. hepatica (290 bp) and F. gigantica (190 bp) were successfully amplified using multiplex PCR. However, the hybrid flukes contained fragments of both species. The multiplex PCR for FABP type I could precisely discriminate the 1312 Fasciola samples used in this study. Notably, no discrimination errors were observed with this novel method. CONCLUSIONS: Multiplex PCR for FABP type I can be used as a species discrimination marker in place of pepck and pold. The robustness of the species-specific primer should be continuously examined using a larger number of Fasciola flukes worldwide in the future since nucleotide substitutions in the primer regions may cause amplification errors.

MiR-34a-5p promotes hepatic gluconeogenesis by suppressing SIRT1 expression.

Elevated hepatic gluconeogenesis is a major contributor of fasting hyperglycemia in diabetes. MicroRNAs (miRNAs) are tightly linked to glucose metabolism, but their role in hepatic gluconeogenesis remains largely unkown. In this current study, miR-34a-5p expression was significantly increased in liver tissues of db/db mice. Overexpression of miR-34a-5p promoted hepatic glucose production in mouse primary hepatocytes with increased expressions of gluconeogenic genes while miR-34a-5p inhibition displayed a contrary action. MiR-34a-5p overexpression in mouse primary hepatocytes repressed SIRT1 expression. SIRT1 inhibition by EX527 blocked phosphoenolpyruvate carboxykinase (PEPCK) protein degradation and enhanced hepatic gluconeogenesis. Treatment of A485 (a CBP/p300 inhibitor) decreased miR-34a-5p and PEPCK expressions in the livers of db/db mice, but elevated SIRT1 protein expression. In mouse primary hepatocytes, A485 exhibited a similar result. Overexpression of miR-34a-5p attenuated A485-inhibited gluconeogenic gene expressions and A485-induced SIRT1 protein expression. Finally, after miR-34a-5p was inhibited in the livers of db/db mice, hepatic glucose production and gluconeogenic gene expressions were markedly lowered. Our findings highlight a critical role of miR-34a-5p in the regulation of hepatic gluconeogenesis and miR-34a-5p may be a potential target in the treatment of type 2 diabetes.

FOXO1 is downregulated in obese mice subjected to short-term strength training.

Obesity is a worldwide health problem and is directly associated with insulin resistance and type 2 diabetes. The liver is an important organ for the control of healthy glycemic levels, since insulin resistance in this organ reduces phosphorylation of forkhead box protein 1 (FOXO1) protein, leading to higher hepatic glucose production (HGP) and fasting hyperglycemia. Aerobic physical training is known as an important strategy in increasing the insulin action in the liver by increasing FOXO1 phosphorylation and reducing gluconeogenesis. However, little is known about the effects of strength training in this context. This study aimed to investigate the effects of short-term strength training on hepatic insulin sensitivity and glycogen synthase kinase-3ß (GSK3ß) and FOXO1 phosphorylation in obese (OB) mice. To achieve this goal, OB Swiss mice performed the strength training protocol (one daily session for 15 days). Short-term strength training increased the phosphorylation of protein kinase B and GSK3ß in the liver after insulin stimulus and improved the control of HGP during the pyruvate tolerance test. On the other hand, sedentary OB animals reduced FOXO1 phosphorylation and increased the levels of nuclear FOXO1 in the liver, increasing the phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) content. The bioinformatics analysis also showed positive correlations between hepatic FOXO1 levels and gluconeogenic genes, reinforcing our findings. However, strength-trained animals reverted to this scenario, regardless of body adiposity changes. In conclusion, short-term strength training is an efficient strategy to enhance the insulin action in the liver of OB mice, contributing to glycemic control by reducing the activity of hepatic FOXO1 and lowering PEPCK and G6Pase contents.

Deacetylated nimbin analog N2 fortifies alloxan-induced pancreatic ß-cell damage in insulin-resistant zebrafish larvae by upregulating phosphoenolpyruvate carboxykinase (PEPCK) and insulin levels.

This study aims to evaluate the protective behaviour of N2, a semi-natural analog of nimbin, for its anti-diabetic efficacy against alloxan-induced oxidative damage and ß-cell dysfunction in in-vivo zebrafish larvae. A 500 µM of alloxan was exposed to zebrafish larvae for 24 h to induce oxidative stress in the pancreatic ß-cells and co-exposed with N2 to study the protection of N2 by inhibiting ROS by DCFH-DA, DHE and NDA staining along with Cellular damage, apoptosis and lipid peroxidation. The zebrafish was further exposed to 500 µM alloxan for 72 h to induce ß-cell destruction along with depleted glucose uptake and co-exposed to N2 to study the protective mechanism. Glucose levels were estimated, and PCR was used to verify the mRNA expression of phosphoenolpyruvate carboxykinase (PEPCK) and insulin. Alloxan induced (24 h) oxidative stress in the pancreatic ß-cells in which N2's co-exposure inhibited ROS by eliminating O-2 radicals and restoring the glutathione levels, thus preventing cellular damage and lipid peroxidation. The zebrafish exposed to 500 µM alloxan for 72 h was observed with ß-cell destruction along with depleted glucose uptake when stained with 2NBDG, wherein N2 was able to protect the pancreatic ß-cells from oxidative damage, promoted high glucose uptake and reduced glucose levels. N2 stimulated insulin production and downregulated PEPCK by inhibiting gluconeogenesis, attenuating post-prandial hyperglycemia. N2 may contribute to anti-oxidant protection against alloxan-induced ß-cell damage and anti-hyperglycemic activity, restoring insulin function and suppressing PEPCK expression.

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.

Role of PCK2 in the proliferation of vascular smooth muscle cells in neointimal hyperplasia.

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets (GSE28829, GSE43292, GSE100927, and GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation in vitro. Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 (PCK2) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in Pck2 knockout (KO) mice by wire injury and identified PCK2 expression in human femoral artery atheroma. Among six DEGs, only PCK2 and RGS1 showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with PCK2 siRNA. RNA sequencing of PCK2 siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of Pck2-KO mice and PCK2 was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.