Hormonal effects on glucose and ketone metabolism in a perfused liver of an elasmobranch, the North Pacific spiny dogfish, Squalus suckleyi.

Hormonal influence on hepatic function is a critical aspect of whole-body energy balance in vertebrates. Catecholamines and corticosteroids both influence hepatic energy balance via metabolite mobilization through glycogenolysis and gluconeogenesis. Elasmobranchs have a metabolic organization that appears to prioritize the mobilization of hepatic lipid as ketone bodies (e.g. 3-hydroxybutyrate [3-HB]), which adds complexity in determining the hormonal impact on hepatic energy balance in this taxon. Here, a liver perfusion was used to investigate catecholamine (epinephrine [E]) and corticosteroid (corticosterone [B] and 11-deoxycorticosterone [DOC]) effects on the regulation of hepatic glucose and 3-HB balance in the North Pacific Spiny dogfish, Squalus suckleyi. Further, hepatic enzyme activity involved in ketogenesis (3-hydroxybutyrate dehydrogenase), glycogenolysis (glycogen phosphorylase), and gluconeogenesis (phosphoenolpyruvate carboxykinase) were assessed in perfused liver tissue following hormonal application to discern effects on hepatic energy flux. mRNA transcript abundance key transporters of glucose (glut1 and glut4) and ketones (mct1 and mct2) and glucocorticoid function (gr, pepck, fkbp5, and 11ßhsd2) were also measured to investigate putative cellular components involved in hepatic responses. There were no changes in the arterial-venous difference of either metabolite in all hormone perfusions. However, perfusion with DOC increased gr transcript abundance and decreased flow rate of perfusions, suggesting a regulatory role for this corticosteroid. Phosphoenolpyruvate carboxykinase activity increased following all hormone treatments, which may suggest gluconeogenic function; E also increased 3-hydroxybutyrate dehydrogenase activity, suggesting a function in ketogenesis, and decreased pepck and fkbp5 transcript abundance, potentially showing some metabolic regulation. Overall, we demonstrate hormonal control of hepatic energy balance using liver perfusions at various levels of biological organization in an elasmobranch.

Elucidation of the Mechanisms of Inter-domain Coupling in the Monomeric State of Enzyme I by High-pressure NMR.

The catalytic cycle of Enzyme I (EI), a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate, is characterized by a series of local and global conformational rearrangements. This multistep process includes a monomer-to-dimer transition, followed by an open-to-closed rearrangement of the dimeric complex upon PEP binding. In the present study, we investigate the thermodynamics of EI dimerization using a range of high-pressure solution NMR techniques complemented by SAXS experiments. 1H-15N TROSY and 1H-13C methyl TROSY NMR spectra combined with 15N relaxation measurements revealed that a native-like engineered variant of full-length EI fully dissociates into stable monomeric state above 1.5 kbar. Conformational ensembles of EI monomeric state were generated via a recently developed protocol combining coarse-grained molecular simulations with experimental backbone residual dipolar coupling measurements. Analysis of the structural ensembles provided detailed insights into the molecular mechanisms driving formation of the catalytically competent dimeric state, and reveals that each step of EI catalytical cycle is associated with a significant reduction in either inter- or intra-domain conformational entropy. Altogether, this study completes a large body work conducted by our group on EI and establishes a comprehensive structural and dynamical description of the catalytic cycle of this prototypical multidomain, oligomeric enzyme.

The shikimate pathway: gateway to metabolic diversity.

Covering: 1997 to 2023The shikimate pathway is the metabolic process responsible for the biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Seven metabolic steps convert phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) into shikimate and ultimately chorismate, which serves as the branch point for dedicated aromatic amino acid biosynthesis. Bacteria, fungi, algae, and plants (yet not animals) biosynthesize chorismate and exploit its intermediates in their specialized metabolism. This review highlights the metabolic diversity derived from intermediates of the shikimate pathway along the seven steps from PEP and E4P to chorismate, as well as additional sections on compounds derived from prephenate, anthranilate and the synonymous aminoshikimate pathway. We discuss the genomic basis and biochemical support leading to shikimate-derived antibiotics, lipids, pigments, cofactors, and other metabolites across the tree of life.

Transcriptional and translational flux optimization at the key regulatory node for enhanced production of naringenin using acetate in engineered Escherichia coli.

As a key molecular scaffold for various flavonoids, naringenin is a value-added chemical with broad pharmaceutical applicability. For efficient production of naringenin from acetate, it is crucial to precisely regulate the carbon flux of the oxaloacetate-phosphoenolpyruvate (OAA-PEP) regulatory node through appropriate pckA expression control, as excessive overexpression of pckA can cause extensive loss of OAA and metabolic imbalance. However, considering the critical impact of pckA on naringenin biosynthesis, the conventional strategy of transcriptional regulation of gene expression is limited in its ability to cover the large and balanced solution space. To overcome this hurdle, in this study, pckA expression was fine-tuned at both the transcriptional and translational levels in a combinatorial expression library for the precise exploration of optimal naringenin production from acetate. Additionally, we identified the effects of regulating pckA expression by validating the correlation between phosphoenolpyruvate kinase (PCK) activity and naringenin production. As a result, the flux-optimized strain exhibited a 49.8-fold increase compared with the unoptimized strain, producing 122.12 mg/L of naringenin. Collectively, this study demonstrated the significance of transcriptional and translational flux rebalancing at the key regulatory node, proposing a pivotal metabolic engineering strategy for the biosynthesis of various flavonoids derived from naringenin using acetate. ONE-SENTENCE SUMMARY: In this study, transcriptional and translational regulation of pckA expression at the crucial regulatory node was conducted to optimize naringenin biosynthesis using acetate in E. coli.

Wheat germ peptide improves glucose metabolism and insulin resistance in HepG2 hepatocytes via regulating SOCS3/IRS1/Akt pathway.

Evidence has demonstrated that oxidative stress plays a crucial role in regulating cellular glucose metabolism. In previous studies, wheat germ peptide (WGP) was found to effectively mitigate oxidative stress induced by high glucose. Based on the information provided, we hypothesized that WGP could exhibit antihyperglycemic and anti-insulin-resistant effects in cells. The insulin-resistant cell model was established by insulin stimulation. The glucose consumption, glycogen content, and the activities of hexokinase and pyruvate kinase following WGP treatment were measured. The protein expression of SOCS3, phosphorylated insulin receptor substrate-1 (p-IRS1), IRS1, phosphorylated protein kinase B (p-Akt), Akt, glucose transporter 2 (GLUT2), phosphorylated GSK 3ß, GSK 3ß, FOXO1, G6P, and phosphoenolpyruvate carboxykinase were assessed by western blot analysis. Our results demonstrated that WGP treatment increased cellular glucose consumption and glycogen synthesis and enhanced hexokinase and pyruvate kinase activities. Additionally, WGP treatment was observed to cause a significant reduction in the expression of SOCS3, FOXO1, G6P, and phosphoenolpyruvate carboxykinase, as well as in the ratio of p-IRS1/IRS1. Conversely, the expression of GLUT2 and the ratios of p-Akt/Akt and p-GSK3ß/GSK3ß were upregulated by WGP. These findings suggested that WGP can activate the SOCS3/IRS1/Akt signaling pathway, thus promoting the phosphorylation of GSK-3ß and increasing the expression of FOXO1 and GLUT2, which contribute to enhancing glycogen synthesis, inhibiting gluconeogenesis, and promoting glucose transport in insulin-resistant HepG2 cells.

KATP channel activity and slow oscillations in pancreatic beta cells are regulated by mitochondrial ATP production.

Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP-sensitive potassium channel (KATP ) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that pyruvate kinase (PK) in close proximity to beta cell KATP locally produces the ATP that inhibits KATP activity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside-out patches inhibited KATP . To test the relative contributions of local vs. mitochondrial ATP production, we recorded KATP activity using mouse beta cells and INS-1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of KATP activity by PEP + ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well-known action of protons to inhibit KATP . In cell-attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on KATP channel closure by glucose, further suggesting that PK is not an important regulator of KATP . In contrast, addition of mitochondrial inhibitors robustly increased KATP activity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that oxidative phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial oxidative phosphorylation is the primary controller of KATP in pancreatic beta cells. KEY POINTS: Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit KATP activity in excised patches. PEP solutions only inhibit KATP activity if the pH is unbalanced. Modulating pyruvate kinase has minimal effects on KATP activity. Mitochondrial inhibition, in contrast, robustly potentiates KATP activity in cell-attached patches. Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via oxidative phosphorylation to close KATP . Mitochondrial oxidative phosphorylation is therefore the main source of the ATP that inhibits the KATP activity of pancreatic beta cells.

Neddylation of phosphoenolpyruvate carboxykinase 1 controls glucose metabolism.

Neddylation is a post-translational mechanism that adds a ubiquitin-like protein, namely neural precursor cell expressed developmentally downregulated protein 8 (NEDD8). Here, we show that neddylation in mouse liver is modulated by nutrient availability. Inhibition of neddylation in mouse liver reduces gluconeogenic capacity and the hyperglycemic actions of counter-regulatory hormones. Furthermore, people with type 2 diabetes display elevated hepatic neddylation levels. Mechanistically, fasting or caloric restriction of mice leads to neddylation of phosphoenolpyruvate carboxykinase 1 (PCK1) at three lysine residues-K278, K342, and K387. We find that mutating the three PCK1 lysines that are neddylated reduces their gluconeogenic activity rate. Molecular dynamics simulations show that neddylation of PCK1 could re-position two loops surrounding the catalytic center into an open configuration, rendering the catalytic center more accessible. Our study reveals that neddylation of PCK1 provides a finely tuned mechanism of controlling glucose metabolism by linking whole nutrient availability to metabolic homeostasis.

Functional and structural characterization of Streptococcus pneumoniae pyruvate kinase involved in fosfomycin resistance.

Glycolysis is the primary metabolic pathway in the strictly fermentative Streptococcus pneumoniae, which is a major human pathogen associated with antibiotic resistance. Pyruvate kinase (PYK) is the last enzyme in this pathway that catalyzes the production of pyruvate from phosphoenolpyruvate (PEP) and plays a crucial role in controlling carbon flux; however, while S. pneumoniae PYK (SpPYK) is indispensable for growth, surprisingly little is known about its functional properties. Here, we report that compromising mutations in SpPYK confers resistance to the antibiotic fosfomycin, which inhibits the peptidoglycan synthesis enzyme MurA, implying a direct link between PYK and cell wall biogenesis. The crystal structures of SpPYK in the apo and ligand-bound states reveal key interactions that contribute to its conformational change as well as residues responsible for the recognition of PEP and the allosteric activator fructose 1,6-bisphosphate (FBP). Strikingly, FBP binding was observed at a location distinct from previously reported PYK effector binding sites. Furthermore, we show that SpPYK could be engineered to become more responsive to glucose 6-phosphate instead of FBP by sequence and structure-guided mutagenesis of the effector binding site. Together, our work sheds light on the regulatory mechanism of SpPYK and lays the groundwork for antibiotic development that targets this essential enzyme.

PCK1 is a key regulator of metabolic and mitochondrial functions in renal tubular cells.

Phosphoenolpyruvate carboxykinase 1 (PCK1 or PEPCK-C) is a cytosolic enzyme converting oxaloacetate to phosphoenolpyruvate, with a potential role in gluconeogenesis, ammoniagenesis, and cataplerosis in the liver. Kidney proximal tubule cells display high expression of this enzyme, whose importance is currently not well defined. We generated PCK1 kidney-specific knockout and knockin mice under the tubular cell-specific PAX8 promoter. We studied the effect of PCK1 deletion and overexpression at the renal level on tubular physiology under normal conditions and during metabolic acidosis and proteinuric renal disease. PCK1 deletion led to hyperchloremic metabolic acidosis characterized by reduced but not abolished ammoniagenesis. PCK1 deletion also resulted in glycosuria, lactaturia, and altered systemic glucose and lactate metabolism at baseline and during metabolic acidosis. Metabolic acidosis resulted in kidney injury in PCK1-deficient animals with decreased creatinine clearance and albuminuria. PCK1 further regulated energy production by the proximal tubule, and PCK1 deletion decreased ATP generation. In proteinuric chronic kidney disease, mitigation of PCK1 downregulation led to better renal function preservation. PCK1 is essential for kidney tubular cell acid-base control, mitochondrial function, and glucose/lactate homeostasis. Loss of PCK1 increases tubular injury during acidosis. Mitigating kidney tubular PCK1 downregulation during proteinuric renal disease improves renal function.NEW & NOTEWORTHY Phosphoenolpyruvate carboxykinase 1 (PCK1) is highly expressed in the proximal tubule. We show here that this enzyme is crucial for the maintenance of normal tubular physiology, lactate, and glucose homeostasis. PCK1 is a regulator of acid-base balance and ammoniagenesis. Preventing PCK1 downregulation during renal injury improves renal function, rendering it an important target during renal disease.

Augmenting TCR signal strength and ICOS costimulation results in metabolically fit and therapeutically potent human CAR Th17 cells.

IL-17-producing antigen-specific human T cells elicit potent antitumor activity in mice. Yet, refinement of this approach is needed to position it for clinical use. While activation signal strength regulates IL-17 production by CD4+ T cells, the degree to which T cell antigen receptor (TCR) and costimulation signal strength influences Th17 immunity remains unknown. We discovered that decreasing TCR/costimulation signal strength by incremental reduction of αCD3/costimulation beads progressively altered Th17 phenotype. Moreover, Th17 cells stimulated with αCD3/inducible costimulator (ICOS) beads produced more IL-17A, IFNγ, IL-2, and IL-22 than those stimulated with αCD3/CD28 beads. Compared with Th17 cells stimulated with the standard, strong signal strength (three beads per T cell), Th17 cells propagated with 30-fold fewer αCD3/ICOS beads were less reliant on glucose and favored the central carbon pathway for bioenergetics, marked by abundant intracellular phosphoenolpyruvate (PEP). Importantly, Th17 cells stimulated with weak αCD3/ICOS beads and redirected with a chimeric antigen receptor that recognizes mesothelin were more effective at clearing human mesothelioma. Less effective CAR Th17 cells generated with high αCD3/ICOS beads were rescued by overexpressing phosphoenolpyruvate carboxykinase 1 (PCK1), a PEP regulator. Thus, Th17 therapy can be improved by using fewer activation beads during manufacturing, a finding that is cost effective and directly translatable to patients.

Phosphoenolpyruvate induces endothelial dysfunction and cell senescence through stimulation of metabolic reprogramming.

Endothelial dysfunction is a key early link in the pathogenesis of atherosclerosis, and the accumulation of senescent vascular endothelial cells causes endothelial dysfunction. Phosphoenolpyruvate (PEP), which is a high-energy glycolytic intermediate, protects against ischemia-reperfusion injury in isolated rat lung, heart, and liver tissue by quickly providing ATP. However, it was reported that serum PEP concentrations are 13-fold higher in healthy elderly compare to the young. Unlike that of other cell types, the energy required for the physiological function of endothelial cells is mainly derived from glycolysis. Recently, it is unclear whether circulating accumulation of PEP affects endothelial cell function. In this study, we found for the first time that 50-250 µM of PEP significantly promoted THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs) through increased expression of vascular endothelial adhesion factor 1 (VCAM1) and intercellular adhesion factor 1 (ICAM1) in HUVECs. Meanwhile, 50-250 µM of PEP decreased the expression of endothelial nitric oxide synthase (eNOS) and cellular level of nitric oxide (NO) in HUVECs. Moreover, PEP increased levels of ROS, enhanced the numbers of SA-ß-Gal-positive cells and upregulated the expression of cell cycle inhibitors such as p21, p16 and the phosphorylation level of p53 on Ser15, and the expression of proinflammatory factors including TNF-α, IL-1ß, IL-6, IL-8, IL-18 and MCP-1 in HUVECs. Furthermore, PEP increased both oxygen consumption rate (OCR) and glycolysis rate, and was accompanied by reduced NAD+/NADH ratios and enhanced phosphorylation levels of AMPKα (Thr172), p38 MAPK (T180/Y182) and NF-κB p65 (Ser536) in HUVECs. Notably, PEP had no significant effect on hepG2 cells. In conclusion, these results demonstrated that PEP induced dysfunction and senescence in vascular endothelial cells through stimulation of metabolic reprogramming.

Phosphoenolpyruvate regulates the Th17 transcriptional program and inhibits autoimmunity.

Aerobic glycolysis, a metabolic pathway essential for effector T cell survival and proliferation, regulates differentiation of autoimmune T helper (Th) 17 cells, but the mechanism underlying this regulation is largely unknown. Here, we identify a glycolytic intermediate metabolite, phosphoenolpyruvate (PEP), as a negative regulator of Th17 differentiation. PEP supplementation or inhibition of downstream glycolytic enzymes in differentiating Th17 cells increases intracellular PEP levels and inhibits interleukin (IL)-17A expression. PEP supplementation inhibits expression of signature molecules for Th17 and Th2 cells but does not significantly affect glycolysis, cell proliferation, or survival of T helper cells. Mechanistically, PEP binds to JunB and inhibits DNA binding of the JunB/basic leucine zipper transcription factor ATF-like (BATF)/interferon regulatory factor 4 (IRF4) complex, thereby modulating the Th17 transcriptional program. Furthermore, daily administration of PEP to mice inhibits generation of Th17 cells and ameliorates Th17-dependent autoimmune encephalomyelitis. These data demonstrate that PEP links aerobic glycolysis to the Th17 transcriptional program, suggesting the therapeutic potential of PEP for autoimmune diseases.

An obesogenic diet impairs uncoupled substrate oxidation and promotes whitening of the brown adipose tissue in rats.

Brown adipose tissue (BAT) is rich in mitochondria containing uncoupling protein 1 (UCP1), and dissipates energy through thermogenesis. However, even though BAT mass and its UCP1 content increase in rodents chronically fed a high-fat sucrose-enriched (HFS) diet, marked expansion of adiposity still occurs in these animals, suggesting insufficient BAT-mediated HFS diet-induced thermogenesis. Thus, the objective of this study was to investigate the metabolic and molecular mechanisms that regulate BAT thermogenesis in HFS-induced obesity. To accomplish this, rats were fed either a standard chow or HFS diet for 8 weeks. Subsequently, glucose and fatty acid metabolism and the molecular mechanisms underlying these processes were assessed in freshly isolated primary BAT adipocytes. Despite increasing BAT mass and its UCP1 content, the HFS diet reduced uncoupled glucose and palmitate oxidation in BAT adipocytes. It also markedly diminished tyrosine hydroxylase content and lipolysis in these cells. Conversely, glucose uptake, lactate production, glycerol incorporation into lipids, palmitate incorporation into triacylglycerol (TAG), phosphoenolpyruvate carboxykinase and glycerol kinase levels, and lipoprotein lipase and cluster of differentiation 36 gene expression were increased. In summary, a HFS diet enhanced glyceroneogenesis and shifted BAT metabolism toward TAG synthesis by impairing UCP1-mediated substrate oxidation and by enhancing fatty acid esterification in intact brown adipocytes. These adaptive metabolic responses to chronic HFS feeding attenuated BAT thermogenic capacity and favoured the development of obesity. KEY POINTS: Despite increasing brown adipose tissue (BAT) mass and levels of thermogenic proteins such as peroxisome proliferator-activated receptor γ coactivator 1α, carnitine palmitoyltransferase 1B and uncoupling protein 1 (UCP1), an obesogenic high-fat sucrose-enriched (HFS) diet attenuated uncoupled glucose and fatty acid oxidation in brown adipocytes. Brown adipocytes diverted glycerol and fatty acids toward triacylglycerol (TAG) synthesis by elevating the cellular machinery that promotes fatty acid uptake along with phosphoenolpyruvate carboxykinase and glycerol kinase levels. The HFS diet increased glucose uptake that supported lactate production and provided substrate for glyceroneogenesis and TAG synthesis in brown adipocytes. Impaired UCP-1-mediated thermogenic capacity and enhanced TAG storage in BAT adipocytes were consistent with reduced adipose triglyceride lipase and tyrosine hydroxylase levels in HFS diet-fed animals.

Loss of hepatic phosphoenolpyruvate carboxykinase 1 dysregulates metabolic responses to acute exercise but enhances adaptations to exercise training in mice.

Acute exercise increases liver gluconeogenesis to supply glucose to working muscles. Concurrently, elevated liver lipid breakdown fuels the high energetic cost of gluconeogenesis. This functional coupling between liver gluconeogenesis and lipid oxidation has been proposed to underlie the ability of regular exercise to enhance liver mitochondrial oxidative metabolism and decrease liver steatosis in individuals with nonalcoholic fatty liver disease. Herein we tested whether repeated bouts of increased hepatic gluconeogenesis are necessary for exercise training to lower liver lipids. Experiments used diet-induced obese mice lacking hepatic phosphoenolpyruvate carboxykinase 1 (KO) to inhibit gluconeogenesis and wild-type (WT) littermates. 2H/13C metabolic flux analysis quantified glucose and mitochondrial oxidative fluxes in untrained mice at rest and during acute exercise. Circulating and tissue metabolite levels were determined during sedentary conditions, acute exercise, and refeeding postexercise. Mice also underwent 6 wk of treadmill running protocols to define hepatic and extrahepatic adaptations to exercise training. Untrained KO mice were unable to maintain euglycemia during acute exercise resulting from an inability to increase gluconeogenesis. Liver triacylglycerides were elevated after acute exercise and circulating ß-hydroxybutyrate was higher during postexercise refeeding in untrained KO mice. In contrast, exercise training prevented liver triacylglyceride accumulation in KO mice. This was accompanied by pronounced increases in indices of skeletal muscle mitochondrial oxidative metabolism in KO mice. Together, these results show that hepatic gluconeogenesis is dispensable for exercise training to reduce liver lipids. This may be due to responses in ketone body metabolism and/or metabolic adaptations in skeletal muscle to exercise.NEW & NOTEWORTHY Exercise training reduces hepatic steatosis partly through enhanced hepatic terminal oxidation. During acute exercise, hepatic gluconeogenesis is elevated to match the heightened rate of muscle glucose uptake and maintain glucose homeostasis. It has been postulated that the hepatic energetic stress induced by elevating gluconeogenesis during acute exercise is a key stimulus underlying the beneficial metabolic responses to exercise training. This study shows that hepatic gluconeogenesis is not necessary for exercise training to lower liver lipids.

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.

Phosphoenolpyruvate reallocation links nitrogen fixation rates to root nodule energy state.

Legume-rhizobium symbiosis in root nodules fixes nitrogen to satisfy the plant's nitrogen demands. The nodules' demand for energy is thought to determine nitrogen fixation rates. How this energy state is sensed to modulate nitrogen fixation is unknown. Here, we identified two soybean (Glycine max) cystathionine ß-synthase domain-containing proteins, nodule AMP sensor 1 (GmNAS1) and NAS1-associated protein 1 (GmNAP1). In the high-nodule energy state, GmNAS1 and GmNAP1 form homodimers that interact with the nuclear factor-Y C (NF-YC) subunit (GmNFYC10a) on mitochondria and reduce its nuclear accumulation. Less nuclear GmNFYC10a leads to lower expression of glycolytic genes involved in pyruvate production, which modulates phosphoenolpyruvate allocation to favor nitrogen fixation. Insight into these pathways may help in the design of leguminous crops that have improved carbon use, nitrogen fixation, and growth.

Metabolic adaptation and ATP homeostasis in Pseudomonas fluorescens exposed to phosphate stress.

Phosphate (Pi) is essential for life as it is an integral part of the universal chemical energy adenosine triphosphate (ATP), and macromolecules such as, DNA, RNA proteins and lipids. Despite the core roles and the need of this nutrient in living cells, some bacteria can grow in environments that are poor in Pi. The metabolic mechanisms that enable bacteria to proliferate in a low phosphate environment are not fully understood. In this study, the soil microbe Pseudomonas (P.) fluorescens was cultured in a control and a low Pi (stress) medium in order to delineate how energy homeostasis is maintained. Although there was no significant variation in biomass yield in these cultures, metabolites like isocitrate, oxaloacetate, pyruvate and phosphoenolpyruvate (PEP) were markedly increased in the phosphate-starved condition. Components of the glycolytic, glyoxylate and tricarboxylic acid cycles operated in tandem to generate ATP by substrate level phosphorylation (SLP) as NADH-producing enzymes were impeded. The α-ketoglutarate (KG) produced when glutamine, the sole carbon nutrient was transformed into phosphoenol pyruvate (PEP) and succinyl-CoA (SC), two high energy moieties. The metabolic reprogramming orchestrated by isocitrate lyase (ICL), phosphoenolpyruvate synthase (PEPS), pyruvate phosphate dikinase (PPDK), and succinyl-CoA synthetase fulfilled the ATP budget. Cell free extract experiments confirmed ATP synthesis in the presence of such substrates as PEP, oxaloacetate and isocitrate respectively. Gene expression profiling revealed elevated transcripts associated with numerous enzymes including ICL, PEPS, and succinyl-CoA synthetase (SCS). This microbial adaptation will be critical in promoting biological activity in Pi-poor ecosystems.

Engineering of global transcription factor FruR to redirect the carbon flow in Escherichia coli for enhancing L-phenylalanine biosynthesis.

BACKGROUND: The catabolite repressor/activator protein (FruR) is a global regulatory protein known to control the expression of several genes concerned with carbon utilization and energy metabolism. This study aimed to illustrate effects of the FruR mutant on the L-phenylalanine (L-PHE) producing strain PHE01. RESULTS: Random mutagenesis libraries of fruR generated in vitro were first integrated into the chromosome of PHE01 by CRISPR/Cas9 technique, and then the best mutant PHE07 (FruRE173K) was obtained. With this mutant, a final L-PHE concentration of 70.50 ± 1.02 g/L was achieved, which was 23.34% higher than that of PHE01. To better understand the mechanism, both transcriptomes and metabolomes of PHE07 were carried out and compared to that of PHE01. Specifically, the transcript levels of genes involved in gluconeogenesis pathway, pentose phosphate pathway, Krebs cycle, and glyoxylate shunt were up-regulated in the FruRE173K mutant, whereas genes aceEF, acnB, and icd were down-regulated. From the metabolite level, the FruRE173K mutation led to an accumulation of pentose phosphate pathway and Krebs cycle products, whereas the products of pyruvate metabolism pathway: acetyl-CoA and cis-aconic acid, were down-regulated. As a result of the altered metabolic flows, the utilization of carbon sources was improved and the supply of precursors (phosphoenolpyruvate and erythrose 4-phosphate) for L-PHE biosynthesis was increased, which together led to the enhanced production of L-PHE. CONCLUSION: A novel strategy for L-PHE overproduction by modification of the global transcription factor FruR in E. coli was reported. Especially, these findings expand the scope of pathways affected by the fruR regulon and illustrate its importance as a global regulator in L-PHE production.

Structural analysis of the pseudaminic acid synthase PseI from Campylobacter jejuni.

Campylobacter jejuni PseI is a pseudaminic acid synthase that condenses the 2,4-diacetamido-2,4,6-trideoxy-l-altrose sugar (6-deoxy AltdiNAc) and phosphoenolpyruvate to generate pseudaminic acid, a sialic acid-like 9-carbon backbone α-keto sugar. Pseudaminic acid is conjugated to cell surface proteins and lipids and plays a key role in the mobility and virulence of C. jejuni and other pathogenic bacteria. To provide insights into the catalytic mechanism of PseI, we performed a structural study on PseI. PseI forms a two-domain structure and assembles into a domain-swapped homodimer. The PseI dimer has two cavities, each of which accommodates a metal ion using conserved histidine residues. A comparative analysis of structures and sequences suggests that the cavity of PseI functions as an active site that binds the 6-deoxy AltdiNAc and phosphoenolpyruvate substrates and mediates their condensation. Furthermore, we propose the substrate binding-induced structural rearrangement of PseI and predict 6-deoxy AltdiNAc recognition residues that are specific to PseI.

The antidiabetic effects of Bifidobacterium longum subsp. longum BL21 through regulating gut microbiota structure in type 2 diabetic mice.

Bifidobacterium longum subsp. longum BL21 (BL21) possesses hypoglycemic activity, but its anti-diabetic mechanism has rarely been illustrated. In the present work, the effects of BL21 on type 2 diabetes mellitus (T2DM) were investigated in diabetic mice induced via a high-fat diet combined with streptozotocin (STZ). Our data indicated that BL21 at a dose of 109 CFU per day significantly lowered the levels of fasting blood glucose and alleviated insulin resistance in diabetic mice. Meanwhile, BL21 enhanced the anti-oxidative capacity, increased the hepatic glycogen content, and significantly decreased the gene expression levels of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in the livers of diabetic mice. Endotoxemia-related inflammation and impaired intestinal barrier function in diabetic mice were also improved using BL21. More importantly, the disturbance of intestinal flora was regulated by BL21, including increased levels of the genera Akkermansia, Alloprevotella, Bacteroides, and Alistipes and decreased levels of Lachnospiraceae_NK4A136_group, Mucispirillum, and Odoribacter. Collectively, the amelioration of T2DM via BL21 supplementation might be partially attributed to regulation of the parameters related to glucose metabolism and the modulation of gut microbiota. Therefore, BL21 could be a potential functional food for ameliorating T2DM.