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Postoperative fatigue (POF) is the most common and long-lasting complication after surgery, which brings heavy burden to individuals and society. Recently, hastening postoperative recovery receives increasing attention, but unfortunately, the mechanisms underlying POF remain unclear. Propofol is a wildly used general anesthetic in clinic, and inspired by the rapid antidepressant effects induced by ketamine at non-anesthetic dose, the present study was undertaken to investigate the anti-fatigue effects and underlying mechanisms of propofol at a non-anesthetic dose in 70% hepatectomy induced POF model in rats. We first showed here that single administration of propofol at 0.1 mg/kg ameliorated acute POF in hepatectomy induced POF rats. Based on metabonomics analysis, we hypothesized that propofol exerted anti-fatigue activity in POF rats by facilitating free fatty acid (FFA) oxidation and gluconeogenesis. We further confirmed that propofol restored the deficit in FFA oxidation and gluconeogenesis in POF rats, as evidenced by the elevated FFA utilization, acetyl coenzyme A content, pyruvic acid content, phosphoenolpyruvic acid content, hepatic glucose output and glycogen storage. Moreover, propofol stimulated glucagon secretion and up-regulated expression of cAMP-response element binding protein (CREB), phosphorylated CREB, peroxlsome prolifeator-activated receptor-γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinade1 and carnitine palmitoltransferase 1A. In summary, our study suggests for the first time that propofol ameliorates acute POF by promoting glucagon-regulated gluconeogenesis via CREB/PGC-1α signaling and accelerating FFA beta-oxidation.

Finding reliable cheap sources for producing chemicals and materials is always challenging. During recent decades, photosynthetic organisms such as cyanobacteria, which used CO2 as a carbon source for making products, have attracted a great deal of attention. Among cyanobacteria, Synechocystis sp. PCC 6803 has been considered as a model strain and has some desirable features that make it suitable for use as an industrial strain. Pyruvate kinase (PK) catalyzes the transformation of phosphoenolpyruvate (PEP) to pyruvate in the last step of glycolysis that is an essential enzyme to produce adenosine triphosphate (ATP) in all organisms. Therefore, it plays a critical role in regulating cell metabolism. However, active and allosteric sites of PK and allosteric mechanisms governing PK activity are poorly understood in many bacteria. This study was aimed to provide more insight into PKs of Synechocystis sp. PCC 6803, using in silico methods. The results indicated that predicted structures of PKs from Synechocystis sp. PCC 6803 are reliable and can be considered for further studies. Molecular docking studies suggested that for predicted structures of sll0587 and sll1275, respectively, there are three and two possible active or allosteric sites. Furthermore, molecular interaction analysis of modeled structures proposes that sll0587 is strongly inhibited by ATP and when ATP concentration is low, this isoenzyme is active.

Background/Aim: Cancer is the most fatal disease worldwide whose most lethal characteristics are invasion and metastasis. Hepatocellular carcinoma (HCC) is one of the most fatal cancers worldwide. HCC often shows encapsulation, which is related to better prognosis. In this study, proteomic analysis of HCC tissues with and without encapsulation was performed, in order to elucidate the factors which play important roles in encapsulation., Materials and Methods: Five HCC tissues surrounded by a capsule and five HCC tissues which broke the capsule were obtained from patients diagnosed with HCC who underwent surgical liver resection. Protein samples from these tissues were separated by two-dimensional gel electrophoresis (2-DE), and the protein spots whose expression was different between encapsulated and non-encapsulated HCC tissues were identified through gel imaging analysis software. The selected protein spots were analyzed and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS)., Results: Two-DE analysis showed 14 spots whose expression was different between encapsulated and non-encapsulated HCC tissues. Of these, 9 were up-regulated and 5 were downregulated in HCC tissues without encapsulation. The validation by Western blot confirmed that leucine aminopeptidase 3 (LAP3) and phosphoenolpyruvate carboxykinase mitochondrial (PCK2) were up-regulated significantly in HCC tissues with a capsule, compared to HCC tissues that broke the capsule., Conclusion: These findings suggest that LAP3 and PCK2 could be factors responsible for the maintenance of encapsulation in HCC tissues., peer-reviewed

Larvae of the goldenrod gall fly, Eurosta solidaginis, rely on a freeze tolerance strategy to survive the sub-zero temperatures of Canadian winter. Critical to their survival is the accumulation of polyol cryoprotectants and global metabolic rate depression, both of which require the regulation of glycolysis and reorganization of carbohydrate metabolism. This study explored the role that pyruvate kinase (PK) regulation plays in this metabolic reorganization. PK was purified from control (5 °C-acclimated) and frozen (-15 °C-acclimated) larvae and enzyme kinetic properties, structural stability, and post-translational modifications were examined in both enzyme forms. The Km phosphoenolpyruvate (PEP) of frozen PK was 20% higher than that of control PK, whereas the Vmax of frozen PK was up to 50% lower than that of control PK at the lowest assay temperature, suggesting inhibition of the enzyme during the winter. Additionally, the activity and substrate affinity of both forms of PK decreased significantly at low assay temperatures, and both forms were regulated allosterically by a number of metabolites. Pro-Q™ Diamond phosphoprotein staining and immunoblotting experiments demonstrated significantly higher threonine phosphorylation of PK from frozen animals while acetylation and methylation levels remained constant. Together, these results indicate that PK exists in two structurally distinct forms in E. solidaginis. In response to conditions mimicking the transition to winter, PK appears to be regulated to support metabolic rate depression, the accumulation of polyol cryoprotectants, and the need for extended periods of anaerobic carbohydrate metabolism to allow the animal to survive whole-body freezing.

6-Phosphofructokinase-1-kinase (PFK) tetramers catalyse the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate (F16BP). Vertebrates have three PFK isoforms (PFK-M, PFK-L, and PFK-P). This study is the first to compare the kinetics, structures, and transcript levels of recombinant human PFK isoforms. Under the conditions tested PFK-M has the highest affinities for F6P and ATP (K0.5ATP 152 µM; K0.5F6P 147 µM), PFK-P the lowest affinities (K0.5ATP 276 µM; K0.5F6P 1333 µM), and PFK-L demonstrates a mixed picture of high ATP affinity and low F6P affinity (K0.5ATP 160 µM; K0.5F6P 1360 µM). PFK-M is more resistant to ATP inhibition compared with PFK-L and PFK-P (respectively, 23%, 31%, 50% decreases in specificity constants). GTP is an alternate phospho donor. Interface 2, which regulates the inactive dimer to active tetramer equilibrium, differs between isoforms, resulting in varying tetrameric stability. Under the conditions tested PFK-M is less sensitive to fructose 2,6-bisphosphate (F26BP) allosteric modulation than PFK-L or PFK-P (allosteric constants [K0.5ATP+F26BP/K0.5ATP] 1.10, 0.92, 0.54, respectively). Structural analysis of two allosteric sites reveals one may be specialised for AMP/ADP and the other for smaller/flexible regulators (citrate or phosphoenolpyruvate). Correlations between PFK-L and PFK-P transcript levels indicate that simultaneous expression may expand metabolic capacity for F16BP production whilst preserving regulatory capabilities. Analysis of cancer samples reveals intriguing parallels between PFK-P and PKM2 (pyruvate kinase M2), and simultaneous increases in PFK-P and PFKFB3 (responsible for F26BP production) transcript levels, suggesting prioritisation of metabolic flexibility in cancers. Our results describe the kinetic and transcript level differences between the three PFK isoforms, explaining how each isoform may be optimised for distinct roles.

Translating ribosomes require elongation factor P (EF-P) to incorporate consecutive prolines (XPPX) into nascent peptide chains. The proteome of Corynebacterium glutamicum ATCC 13032 contains a total of 1,468 XPPX motifs, many of which are found in proteins involved in primary and secondary metabolism. We show here that synthesis of EIIGlc , the glucose-specific permease of the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) encoded by ptsG, is strongly dependent on EF-P, as an efp deletion mutant grows poorly on glucose as sole carbon source. The amount of EIIGlc is strongly reduced in this mutant, which consequently results in a lower rate of glucose uptake. Strikingly, the XPPX motif is essential for the activity of EIIGlc , and substitution of the prolines leads to inactivation of the protein. Finally, translation of GntR2, a transcriptional activator of ptsG, is also dependent on EF-P. However, its reduced amount in the efp mutant can be compensated for by other regulators. These results reveal for the first time a translational bottleneck involving production of the major glucose transporter EIIGlc , which has implications for future strain engineering strategies.

The micropeptide adropin encoded by the clock-controlled energy homeostasis–associated gene is implicated in the regulation of glucose metabolism. However, its links to rhythms of nutrient intake, energy balance, and metabolic control remain poorly defined. Using surveys of Gene Expression Omnibus data sets, we confirm that fasting suppresses liver adropin expression in lean C57BL/6J (B6) mice. However, circadian rhythm data are inconsistent. In lean mice, caloric restriction (CR) induces bouts of compulsive binge feeding separated by prolonged fasting intervals, increasing NAD-dependent deacetylase sirtuin-1 signaling important for glucose and lipid metabolism regulation. CR up-regulates adropin expression and induces rhythms correlating with cellular stress-response pathways. Furthermore, adropin expression correlates positively with phosphoenolpyruvate carboxokinase-1 (Pck1) expression, suggesting a link with gluconeogenesis. Our previous data suggest that adropin suppresses gluconeogenesis in hepatocytes. Liver-specific adropin knockout (LAdrKO) mice exhibit increased glucose excursions following pyruvate injections, indicating increased gluconeogenesis. Gluconeogenesis is also increased in primary cultured hepatocytes derived from LAdrKO mice. Analysis of circulating insulin levels and liver expression of fasting-responsive cAMP-dependent protein kinase A (PKA) signaling pathways also suggests enhanced responses in LAdrKO mice during a glucagon tolerance test (250 µg/kg intraperitoneally). Fasting-associated changes in PKA signaling are attenuated in transgenic mice constitutively expressing adropin and in fasting mice treated acutely with adropin peptide. In summary, hepatic adropin expression is regulated by nutrient- and clock-dependent extrahepatic signals. CR induces pronounced postprandial peaks in hepatic adropin expression. Rhythms of hepatic adropin expression appear to link energy balance and cellular stress to the intracellular signal transduction pathways that drive the liver fasting response.

Red cell pyruvate kinase deficiency is the most common glycolytic defect causing congenital nonspherocytic hemolytic anemia. Pyruvate kinase is the enzyme involved in the last step of glycolysis – the transfer of a phosphate group from phosphoenolpyruvate producing the enolate of pyruvate and ATP (50 % of total energy ATP of erythrocytes). ATP deficiency directly shortened red cell lifespan. Affected red blood cells are destroyed in the splenic capillaries, leading to the development of chronic hemolytic anemia. It is an autosomal recessive disease, caused by homozygous and compound heterozygous mutations in the PKLR gene. There are no exact data on the incidence of pyruvate kinase deficiency, but the estimated frequency varies from 3: 1,000,000 to 1:20,000. The clinical features of the disease and the severity are highly variable. Diagnosis of pyruvate kinase deficiency is based on the determination of pyruvate kinase activity and molecular genetic study of the PKLR gene. The variety of clinical manifestations, possible complications, as well as the inaccessibility of diagnostic methods complicate the diagnosis.

Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation—in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)—was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239—which participates in a web of interactions of the protein with G6P—was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.

Objective To study the effect of lncRNA HAND2-AS1 on gastric adenocarcinoma (GA) cell property and explore its specific mechanism. Methods Data on stomach adenocarcinoma (STAD) were analyzed to screen differentially expressed lncRNA HAND2-AS1. RNA22-HAS database and dual luciferase reporter assay were applied to confirm the target relationship between HAND2-AS1/HIF3A and miR-184. The HAND2-AS1 and miR-184 expressions in tissue or cells were determined by qRT-PCR and Western blot. Besides, after GA cells (AGS) cultured in normoxic and hypoxic condition, phosphoenolpyruvate (PEP) and lactic acid were quantified by Phosphoenolpyruvate Fluorometric Assay Kit and Lactic Acid Detection kit, respectively. Additionally, colony formation assay, transwell invasion and migration assays were used to evaluate the abilities of cell invasion, migration, and proliferation in distinct conditions. Results The HAND2-AS1 and HIF3A expressions were down-regulated and miR-184 expression was up-regulated in GA tissues and cells. Dual luciferase reporter assay confirmed HAND2-AS1 and HIF3A were targeted by miR-184. AGS cell proliferation abilities were restrained by HAND2-AS1 and HIF3A overexpression and enhanced by miR-184, as well as migration and invasion abilities. In addition, HAND2-AS1 rescued enhanced AGS cell proliferation, cell migration, cell invasion abilities and glycolytic process caused by hypoxia via miR-184/HIF3A. Conclusion LncRNA HAND2-AS1 could inhibit GA cell proliferation, migration and invasion abilities and glycolytic process induced by hypoxia through miR-184/HIF3A signaling.

Hepatic glucose metabolism signaling downstream of insulin can diverge to multiple pathways including AKT. Genetic studies suggest that AKT is necessary for insulin to suppress gluconeogenesis. To specifically address the role of AKT2, the dominant liver isoform of AKT in the regulation of gluconeogenesis genes, we generated hepatocytes lacking AKT2 (Akt2−/−). We found that, in the absence of insulin signal, AKT2 is required for maintaining the basal level expression of phosphoenolpyruvate carboxyl kinase (PEPCK) and to a lesser extent G6Pase, two key rate-limiting enzymes for gluconeogenesis that support glucose excursion due to pyruvate loading. We further showed that this function of AKT2 is mediated by the phosphorylation of cyclic AMP response element binding (CREB). Phosphorylation of CREB by AKT2 is needed for CREB to induce the expression of PEPCK and likely represents a priming event for unstimulated cells to poise to receive glucagon and other signals. The inhibition of gluconeogenesis by insulin is also dependent on the reduced FOXO1 transcriptional activity at the promoter of PEPCK. When insulin signal is absent, this activity appears to be inhibited by AKT2 in manner that is independent of its phosphorylation by AKT. Together, this action of AKT2 on FOXO1 and CREB to maintain basal gluconeogenesis activity may provide fine-tuning for insulin and glucocorticoid/glucagon to regulate gluconeogenesis in a timely manner to meet metabolic needs.

Phosphoenolpyruvate carboxylase (PEPC) is a carbon-fixing enzyme with critical roles in seed development. Previously we observed a positive correlation between PEPC activity and protein content in mature seeds among soybean cultivars and varietal differences of PEPC activity in immature seeds, which is concordant with seed protein accumulation. Here, we report a PEPC isoform (Gmppc2) which is preferentially expressed in immature soybean seeds at the late maturation stage. Gmppc2 was co-expressed with enzyme genes involved in starch degradation: α-amylase, hexokinase, and α-glucan phosphorylase. Gmppc2 was developmentally induced in the external seed coats, internal seed coats, hypocotyls, and cotyledons at the late maturation stage. The expression of Gmppc2 protein was negatively regulated by the application of a nitrogen fertilizer, which suppressed nodule formation. These results imply that Gmppc2 is involved in the metabolism of nitrogen originated from nodules into seeds, and Gmppc2 might be applicable as a biomarker of seed protein content. Abbreviations: PEP: phosphoenolpyruvate; PEPC: phosphoenolpyruvate carboxylase; RNA-Seq: RNA sequencing; PCA: principal component analysis; SE: standard error

The protein and oil contents in soybean seeds are major factors in seed quality. Seed proteins and oils are synthesized from sucrose and nitrogenous compounds transported into maturing seeds. In this study, we compared changes in the activity of phosphoenolpyruvate carboxylase (PEPC) and the accumulation profiles of protein and oil in maturing seeds of two soybean cultivars, which exhibit different protein and oil contents in seeds, to determine the interrelationships of them. A principal component analysis indicated a concordance of seed PEPC activity with the protein content, but did not with the oil content. PEPC activity per seed was highest in the late maturation stage, when the physiological status of the vegetative organs drastically changed. The high-protein cultivar had higher PEPC activity compared to the low-protein cultivar. These results highlight the biological role of PEPC in the synthesis of protein, therefore it was implied that PEPC could be a biomarker in soybean breeding. Abbreviations: ANOVA: analysis of variance; DS: developmental stage; DW: dry weight; FW: fresh weight; NIR: near infrared; PEP(C): phosphoenolpyruvate (carboxylase); PC(A): principal component (analysis); S.E.: standard error; WC: water content.

Background Phosphoenolpyruvate carboxykinase (PCK) has been almost exclusively recognized as a critical enzyme in gluconeogenesis, especially in the liver and kidney. Accumulating evidence has shown that the enhanced activity of PCK leads to increased glucose output and exacerbation of diabetes, whereas the defects of PCK result in lethal hypoglycemia. Genetic mutations or polymorphisms are reported to be related to the onset and progression of diabetes in humans. Scope of review Recent studies revealed that the PCK pathway is more complex than just gluconeogenesis, depending on the health or disease condition. Dysregulation of PCK may contribute to the development of obesity, cardiac hypertrophy, stroke, and cancer. Moreover, a regulatory network with multiple layers, from epigenetic regulation, transcription regulation, to posttranscription regulation, precisely tunes the expression of PCK. Deciphering the molecular basis that regulates PCK may pave the way for developing practical strategies to treat metabolic dysfunction. Major conclusions In this review, we summarize the metabolic and non-metabolic roles of the PCK enzyme in cells, especially beyond gluconeogenesis. We highlight the distinct functions of PCK isoforms (PCK1 and PCK2), depict a detailed network regulating PCK's expression, and discuss its clinical relevance. We also discuss the therapeutic potential targeting PCK and the future direction that is highly in need to better understand PCK-mediated signaling under diverse conditions.

The industrial yeast Pichia pastoris can utilize amino acids as the sole source of carbon. It possesses a post-transcriptional regulatory circuit that governs the synthesis of cytosolic glutamate dehydrogenase 2 (GDH2) and phosphoenolpyruvate carboxykinase (PEPCK), key enzymes of amino acid catabolism. Here, we demonstrate that the post-transcriptional regulatory circuit is activated during carbon starvation resulting in the translation of GDH2 and PEPCK mRNAs. GDH2 and PEPCK synthesis is abrogated in Δatg1 indicating a key role for autophagy or an autophagy-related process. Finally, carbon-starved Δgdh2 and Δpepck exhibit poor survival. This study demonstrates a key role for amino acid catabolism during carbon starvation, a phenomenon hitherto unreported in other yeast species.

Chronic heat stress (CHS) induces metabolic changes in skeletal muscle from growth to maintenance that jeopardizes growth performance, carcass traits, and meat quality of pigs. We investigated the protective effect of dietary organic selenium (hydroxy-4-methylselenobutanoic acid, OH-SeMet) on CHS-induced skeletal muscle damages of growing pigs, and the corresponding responses of selenoproteins. A total of 40 ((Landrace ×Yorkshire) × Duroc) pigs with an average live weight of 49.64 ± 2.48 kg were used in this 4-week trial. Pigs were randomly allotted to 5 groups: The control group was raised on a basal diet in a thermoneutral environment (22 ± 2 °C), and four CHS groups were raised on a basal diet and supplemented with Se 0.0, 0.2, 0.4, and 0.6 mg/kg as OH-SeMet, respectively, in hyperthermal condition (33 ± 2 °C). CHS resulted in significant decrease of growth performance, carcass traits, and meat quality, which were associated with reduced (p &lt, 0.05) serum alkaline phosphatase (ALP) and total superoxide dismutase (T-SOD) and increased (p &lt, 0.05) serum creatine (CK), sarcous heat shock protein 70 (HSP70), glucokinase (GCK), phosphoenolpyruvate carboxykinase (PEPCK), and malondialdehyde (MDA) contents. Meanwhile, four metabolism-related genes and seven selenoprotein encoding genes were abnormally expressed in skeletal muscle. Dietary OH-SeMet addition partially alleviated the negative impact of CHS on carcass traits and improved meat quality. These improvements were accompanied by the increase in Se deposition, the anti-oxidative capacity of serum and muscle, and protein abundance of GPX1, GPX3, GPX4, and SELENOP. Supplementation with 0.6 mg Se/kg (OH-SeMet) restored the sarcous PEPCK, and 0.4 and 0.6 mg Se/kg (OH-SeMet) restored all abnormally expressed metabolism-related and selenoprotein encoding genes. In summary, dietary supplementation with OH-SeMet beyond Se requirement mitigated CHS-induced depression of carcass traits and meat quality of pigs associated with optimal skeletal metabolism, enhanced antioxidant capacity, and regulation of selenoproteins in skeletal muscle of pigs.

In order to characterize the molecular mechanisms by which leucine regulates carbohydrate metabolism and energy homeostasis, juvenile crucian carps (Carassius auratus gibelio var. CAS III) fed with a high carbohydrate diet were supplemented with different levels of dietary leucine: 0% (Leu0), 0.4% (Leu4), 0.8% (Leu8), 1.2% (Leu12), 1.6% (Leu16), 2.0% (Leu20), and 5.0% (Leu50). After 8 weeks, RNA sequencing was performed on samples collected from the Leu0, Leu8, Leu12 and Leu50 groups. Differentially expressed genes were then detected and analyzed. The results showed a total of 91.6 Gb of clean bases were generated. Moreover, a total of 1131, 5254, and 1539 DEGs were detected in Leu8, Leu12, and Leu50 compared with Leu0, respectively, encompassing 161 common DEGs. STEM analysis elucidated four significant clusters of DEGs that were associated with “glycerophospholipid metabolism,” “glycerolipid metabolism,” “PPAR signaling pathway,” and “adipocytokine signaling pathway.” Moreover, the mRNA expression levels of acyl-CoA synthetase long chain family member 5 (ACSL5), choline kinase beta (CHKB), cryptochrome-1 (CRY1), lon protease homolog 2, peroxisomal isoform X2 (LONP2), lipin 1 (LPIN1), membrane bound O-acyltransferase domain containing 2 (MBOAT2), phosphoenolpyruvate carboxykinase 1 (PEPCK), and uridine-cytidine kinase 2b (UCK2b) were then further investigated in all leucine treatment groups at starvation times of 0 h, 24 h, and 48 h. The results revealed that the expression levels of UCK2b and MBOAT2 were negatively correlated with the addition of leucine, whereas CHKB, LONP2, CRY1, PEPCK, and LPIN1 were positively correlated. In conclusion, dietary leucine supplementation below 1.2% enhanced carbohydrate metabolism in juvenile crucian carp fed with a high-carbohydrate diet, whereas concentrations above 2.0% is a better choice for energy homeostasis under starvation.

Clinical applications of antiangiogenic agents profoundly affect tumor cell behaviors via the resultant hypoxia. To date, how the hypoxia regulates tumor cells remains unclear. Here, we show that hypoxia promotes the growth of human breast tumorigenic cells that repopulate tumors [tumor-repopulating cells (TRCs)] in vitro and in vivo. This stimulating effect is ascribed to hypoxia-induced reactive oxygen species (ROS) that activates Akt and NF-κB, dependent on the attenuated tricarboxylic acid (TCA) cycle. We find that fumarate is accumulated in the TCA cycle of hypoxic TRCs, leading to glutathione succination, NADPH/NADP+ decrease, and an increase in ROS levels. Mechanistically, hypoxia-increased HIF-1α transcriptionally downregulates the expression of mitochondrial phosphoenolpyruvate carboxykinase (PCK2), leading to TCA cycle attenuation and fumarate accumulation. These findings reveal that hypoxia-reprogrammed TCA cycle promotes human breast TRCs growth via a HIF-1α-downregulated PCK2 pathway, implying a need for a combination of an antiangiogenic therapy with an antioxidant modulator.

In the last step of glycolysis Pyruvate kinase catalyzes the irreversible conversion of ADP and phosphoenolpyruvate to ATP and pyruvic acid, both crucial for cellular metabolism. Thus pyruvate kinase plays a key role in controlling the metabolic flux and ATP production. The hallmark of the activity of different pyruvate kinases is their tight modulation by a variety of mechanisms including the use of a large number of physiological allosteric effectors in addition to their homotropic regulation by phosphoenolpyruvate. Binding of effectors signals precise and orchestrated movements in selected areas of the protein structure that alter the catalytic action of these evolutionarily conserved enzymes with remarkably conserved architecture and sequences. While the diverse nature of the allosteric effectors has been discussed in the literature, the structural basis of their regulatory effects is still not well understood because of the lack of data representing conformations in various activation states. Results of recent studies on pyruvate kinases of different families suggest that members of evolutionarily related families follow somewhat conserved allosteric strategies but evolutionarily distant members adopt different strategies. Here we review the structure and allosteric properties of pyruvate kinases of different families for which structural data are available.

During the peripartum period, dairy cows experience both an increase in circulating fatty acid (FA) profile and a change in circulating FA profile, which have been shown to alter regulation of gluconeogenic genes. The objective was to quantify gene expression of key enzymes involved in gluconeogenesis and FA transport into the mitochondria in primary hepatocytes in response to exposure to an FA mixture mimicking what is circulating in a transition dairy cow with or without enrichment of C16:0, C18:0, and C18:1. Primary hepatocytes were isolated from 4 Holstein bull calves 3 d of age (± standard deviation 2 d) and cultured. Twenty-four hours after plating, treatments were applied to the cells for 24-h incubation. Treatments consisted of (1) control (1% BSA), (2) 0.75 mM FA cocktail (3% C14:0, 27% C16:0, 23% C18:0, 31% C18:1, 8% C18:2, and 8% C18:3 to mimic the FA profile of dairy cattle at calving), (3) 0.90 mM FA cocktail, (4) 0.75 mM FA cocktail + 0.15 mM C16:0, (5) 0.75 mM FA cocktail + 0.15 mM C18:0, and (6) 0.75 mM FA cocktail + 0.15 mM C18:1. After harvest in Trizol (Life Technologies, Carlsbad, CA), samples were stored at -80°C until RNA extraction, purification, and reverse transcription. Abundance of mRNA was measured using quantitative real-time PCR. Expression of genes of interest [carnitine palmitoyltransferase 1A, pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase (PCK1), mitochondrial phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase] was calculated relative to the average abundance of 2 reference genes (ribosomal protein L32 and glyceraldehyde 3-phosphate dehydrogenase), which were the most stable out of 3 tested. Data were analyzed using PROC MIXED (SAS version 9.4; SAS Institute, Cary, NC) with the fixed effect of treatment and calf in the random statement. The addition of FA compared with the 1% BSA treatment increased the expression of carnitine palmitoyltransferase 1A and cytosolic PCK1. Enrichment with individual FA did not further regulate pyruvate carboxylase or PCK1 beyond that achieved by the basal profile. These results suggest that shifts in circulating FA profile within a biological range, without a difference in the total FA concentration, have minimal effects on transcriptional regulation of hepatic gluconeogenic genes in primary bovine hepatocytes.

Background:The phosphoenolpyruvate carboxykinase (PEPCK) isoforms are considered as rate-limiting enzymes for gluconeogenesis and glyceroneogenesis pathways. PEPCK exhibits several interesting features such as a) organelle-specific isoforms (cytosolic and a mitochondrial) in vertebrate clade, b) tissue-specific expression of isoforms and c) organism-specific requirement of ATP or GTP as a cofactor. In higher organisms, PEPCK isoforms are intricately regulated and activated through several physiological and pathological stimuli such as corticoids, hormones, nutrient starvation and hypoxia. Isoform-specific transcriptional/translational regulation and their interplay in maintaining glucose homeostasis remain to be fully understood. Mounting evidence indicates the significant involvement of PEPCK isoforms in physiological processes (development and longevity) and in the progression of a variety of diseases (metabolic disorders, cancer, Smith–Magenis syndrome).Objective:The present systematic review aimed to assimilate existing knowledge of transcriptional and translational regulation of PEPCK isoforms derived from cell, animal and clinical models.Conclusion:Based on current knowledge and extensive bioinformatics analysis, in this review we have provided a comparative (epi)genetic understanding of PCK1 and PCK2 genes encompassing regulatory elements, disease-associated polymorphisms, copy number variations, regulatory miRNAs and CpG densities. We have also discussed various exogenous and endogenous modulators of PEPCK isoforms and their signaling mechanisms. A comprehensive review of existing knowledge of PEPCK regulation and function may enable identification of the underlying gaps to design new pharmacological strategies and interventions for the diseases associated with gluconeogenesis.

Objective The liver regulates the availability of insulin to other tissues and is the first line insulin response organ physiologically exposed to higher insulin concentrations than the periphery. Basal insulin during fasting inhibits hepatic gluconeogenesis and glycogenolysis, whereas postprandial insulin peaks stimulate glycogen synthesis. The molecular consequences of chronic insulin deficiency for the liver have not been studied systematically. Methods We analyzed liver samples of a genetically diabetic pig model (MIDY) and of wild-type (WT) littermate controls by RNA sequencing, proteomics, and targeted metabolomics/lipidomics. Results Cross-omics analyses revealed increased activities in amino acid metabolism, oxidation of fatty acids, ketogenesis, and gluconeogenesis in the MIDY samples. In particular, the concentrations of the ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) and of retinol dehydrogenase 16 (RDH16), which catalyzes the first step in retinoic acid biogenesis, were highly increased. Accordingly, elevated levels of retinoic acid, which stimulates the expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK1), were measured in the MIDY samples. In contrast, pathways related to extracellular matrix and inflammation/pathogen defense response were less active than in the WT samples. Conclusions The first multi-omics study of a clinically relevant diabetic large animal model revealed molecular signatures and key drivers of functional alterations of the liver in insulin-deficient diabetes mellitus. The multi-omics data set provides a valuable resource for comparative analyses with other experimental or clinical data sets., Highlights • MIDY pigs were used to study consequences of insulin-deficient diabetes for the liver. • RDH16 and HMGCS2 were drivers of stimulated gluconeogenesis and ketogenesis in MIDY pigs. • Hepatic immune functions and extracellular matrix were reduced in MIDY pigs. • This multi-omics data resource is valuable for analyses of other liver omics data sets.

Tribbles related homolog 1 is the mammalian ortholog of Tribbles, which controls cell division and migration during development in Drosophila. TRB1 is a pseudokinase and functions as a scaffold protein. Recent findings suggest that TRB1 plays important roles in hepatic lipid metabolism and participates in insulin resistance. However, the underlying mechanisms have not yet been elucidated. Here, we demonstrate that TRB1 suppresses FOXO1 transcriptional activity to downregulate the expression of G6Pase and PEPCK, which encode gluconeogenic rate-limiting enzymes. TRB1 knockdown enhances FOXO1 binding to the gluconeogenic gene promoters. It also increases FOXO1 acetylation and recruits CBP to the binding sequence of FOXO1. These results suggest that TRB1 suppresses the expression of G6Pase and PEPCK by attenuating FOXO1 transcriptional activity and negatively regulates gluconeogenesis.

Objective: Recent studies demonstrate circulating serum spexin levels are reduced in obesity or type 2 diabetes mellitus (T2DM) patients and may play a role in glucose metabolism. The mechanism underlying is not known. In this study, we explore whether spexin has a role in insulin resistance and hepatic glucose metabolism. Methods: The correlation between serum spexin levels and the homeostasis model assessment of insulin resistance (HOMA-IR) was studied in newly diagnosed T2DM patients. After intraperitoneal injection of exogenous spexin for 8 weeks, the effect of spexin on exogenous glucose infusion rates (GIR), and hepatic glucose production (HGP) were assessed by extended hyperinsulinemic-euglycemic clamp in high-fat-diet (HFD)-induced rats. Glucose concentration with CRISPR/Cas9-mediated disruption of spexin expression in HepG2 cells culture was observed. Expression of transcription factors (Forkhead box O1, FoxO1 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha, PGC-1α) and key enzymes (G-6-Pase and PEPCK) of gluconeogenesis pathway were observed in vitro and in vivo. Results: The serum spexin level was significantly low in newly diagnosed T2DM patients as compared with healthy patients and significantly negatively correlated with the HOMA-IR values. Exogenous spexin treatment resulted in weight loss and decrease of HOMA-IR value in high-fat-diet (HFD)-induced rats. The exogenous glucose infusion rates (GIR) were higher in the HFD + spexin group than that in the HFD group (358 ± 32 vs. 285 ± 24 μmol/kg/min, P < 0.05). Steady-state hepatic glucose production (HGP) was also suppressed by ~50% in the HFD + spexin group as compared with that in the HFD group. Furthermore, spexin inhibited gluconeogenesis in dose-dependent and time-dependent manner in the insulin-resistant cell model. CRISPR/Cas9-mediated knockdown of spexin in HepG2 cells activated gluconeogenesis. Moreover, spexin was shown regulating gluconeogenesis by inhibiting FoxO1/PGC-1α pathway, and key gluconeogenic enzymes, (PEPCK and G-6-Pase) in both HFD-induced rats and insulin-resistant cells. Conclusions: Spexin plays an important role in insulin resistance in HFD-induced rats and insulin-resistant cells. Regulation of the effects of spexin on insulin resistance may hold therapeutic value for metabolic diseases.

Tropical theileriosis caused by Theileria annulata obligate parasite that infect ruminant animals, including Bos taurus. The disease results massive economic losses in livestock production worldwide. Here we describe cloning, expression and both biochemical and structural characterization of beta enolase from Bos taurus in vitro and in silico. The interconversion of 2‑phosphoglycerate to phosphoenolpyruvate was catalyzed by enolase is a metalloenzyme in glycolytic pathway and gluconeogenesis. Enolase from Bos taurus was cloned, expressed and the protein was purified at 95% purity using cobalt column by affinity chromatography. The optimum enzymatic activity was calculated at pH 6.5. For the first time in the literature, the kinetic parameters of the enzyme, Vmax and Km, were measured as 0.1141 mM/min and 0.514 mM, respectively. Besides, Bos taurus enolase 3-dimensional structure was built by homology modelling to be used in silico analyses. The interactions of the enzyme-substrate complex were elucidated by molecular dynamics simulations for 100 ns. These interactions were found to be the same as experimentally determined interactions in yeast. These results would enable further structure based drug design studies with the biochemical characterization of the host organism Bos taurus enolase enzyme in vitro and the elucidation of behavior of enzyme-substrate complex in silico.

The Gram-negative bacterium Vibrio cholerae adapts to changes in the environment by selectively producing the necessary machinery to take up and metabolize available carbohydrates. The import of fructose by the fructose-specific phosphoenolpyruvate (PEP) phosphotransferase system (PTS) is of particular interest because of its putative connection to cholera pathogenesis and persistence. Here, we describe the expression and regulation of fruB, which encodes an EIIA-FPr fusion protein as part of the fructose-specific PTS in V. cholerae. Using a series of transcriptional reporter fusions and additional biochemical and genetic assays, we identified Cra (catabolite repressor/activator) and cAMP receptor protein (CRP) as regulators of fruB expression and determined that this regulation is dependent upon the presence or absence of PTS sugars. Cra functions as a repressor, downregulating fruB expression in the absence of fructose when components of PTSFru are not needed. CRP functions as an activator of fruB expression. We also report that Cra and CRP can affect fruB expression independently; however, CRP can modulate cra expression in the presence of fructose and glucose. Evidence from this work provides the foundation for continued investigations into PTSFru and its relationship to the V. cholerae life cycle. IMPORTANCEVibrio cholerae is the causative agent of cholera disease. While current treatments of care are accessible, we still lack an understanding of the molecular mechanisms that allow V. cholerae to survive in both aquatic reservoirs and the human small intestine, where pathogenesis occurs. Central to V. cholerae’s survival is its ability to use available carbon sources. Here, we investigate the regulation of fruB, which encodes a protein central to the import and metabolism of fructose. We show that fruB expression is controlled by the transcriptional regulators Cra and CRP. This work contributes toward a clearer understanding of how carbon source availability impacts the physiology and, potentially, the persistence of the pathogen.

Our previous research demonstrated that compound licochalcone E can reduce glucose tolerance and lipid metabolism in diabetic rats, although its mechanism remains unknown. Here, we used palmitic acid (PA) to establish a PA-treated HepG2 model, and then examined glucose uptake, glucose consumption, and blood lipids to evaluate the effects of licochalcone E within the safe dose range in the model. Polymerase chain reaction (PCR) was used to detect the expression levels of key genes associated with liver gluconeogenesis; enzyme-linked immunosorbent assay (ELISA) was deployed to evaluate the concentration of inflammatory factors; and laser confocal microscopy and western blot were used to determine the levels of reactive oxygen species (ROS) and NLRP3 inflammasome signaling pathway-related proteins, respectively. Finally, molecular simulations were exploited to validate the interaction between licochalcone E and the NLRP3 inflammasome. The results demonstrated that licochalcone E showed no toxicity in the dose range of 2.5–40 μM. In this dose range, licochalcone E substantially increased the uptake and consumption of glucose in the insulin resistance model and dose-dependently reduced the concentration of total cholesterol. The PCR results indicated that licochalcone E dose-dependently reduced the expression of Glucose-6-phosphatase (G6Pase) and Phosphoenolpyruvate carboxykinase (PEPCK) genes and increased the expression of Glucose Transporter 4 (Glut4) in PA-treated HepG2. Moreover, the ELISA results revealed that licochalcone E significantly reduced the expression of TNF-α, IL-1β, and IL-18. Confocal microscopy results showed that licochalcone E dramatically reduced the generation of ROS and the expressions of NLRP3 and its downstream caspase-1 in PA-treated HepG2 model. Western blot results further indicated that licochalcone E significantly reduced the expression of NLRP3, caspase-1 and IL-1β in the model. Additionally, molecular simulations demonstrated that licochalcone E has good binding affinity for the NLPR3 inflammasome. We concluded that licochalcone E has the potential to be used as an insulin sensitizer by reducing the release of ROS and inflammatory factors following inhibition of the NLPR3 signaling pathway.

Alport Syndrome (AS) is a genetic disorder characterized by impaired kidney function. The development of a noninvasive tool for early diagnosis and monitoring of renal function during disease progression is of clinical importance. Hyperpolarized 13C MRI is an emerging technique that enables non-invasive, real-time measurement of in vivo metabolism. This study aimed to investigate the feasibility of using this technique for assessing changes in renal metabolism in the mouse model of AS. Mice with AS demonstrated a significant reduction in the level of lactate from 4- to 7-week-old, while the levels of lactate were unchanged in the control mice over time. This reduction in lactate production in the AS group accompanied a significant increase of PEPCK expression levels, indicating that the disease progression in AS triggered the gluconeogenic pathway and might have resulted in a decreased lactate pool size and a subsequent reduction in pyruvate-to-lactate conversion. Additional metabolic imaging parameters, including the level of lactate and pyruvate, were found to be different between the AS and control groups. These preliminary results suggest that hyperpolarized 13C MRI might provide a potential noninvasive tool for the characterization of disease progression in AS.

Introduction of C4 photosynthetic traits into C3 crops is an important strategy for improving photosynthetic capacity and productivity. Here, we report the research results of a variant line of sorghum–rice (SR) plant with big panicle and high spikelet density by introducing sorghum genome DNA into rice by spike-stalk injection. The whole-genome resequencing showed that a few sorghum genes could be integrated into the rice genome. Gene expression was confirmed for two C4 photosynthetic enzymes containing pyruvate, orthophosphate dikinase and phosphoenolpyruvate carboxykinase. Exogenous sorghum DNA integration induced a series of key traits associated with the C4 pathway called “proto-Kranz” anatomy, including leaf thickness, bundle sheath number and size, and chloroplast size in bundle sheath cells. Significantly, transgenic plants exhibited enhanced photosynthetic capacity resulting from both photosynthetic CO2-concentrating effect and improved energy balance, which led to an increase in carbohydrate levels and productivity. Furthermore, such rice plant exhibited delayed leaf senescence. In summary, this study provides a proof for the feasibility of inducing the transition from C3 leaf anatomy to proto-Kranz by spike-stalk injection to achieve efficient photosynthesis and increase productivity.

Muscle glycogen depletion has been proposed as one of the main causes of fatigue during exercise. However, few studies have addressed the contribution of liver glycogen to exercise performance. Using a low-intensity running protocol, here, we analyzed exercise capacity in mice overexpressing protein targeting to glycogen (PTG) specifically in the liver (PTGOE mice), which show a high concentration of glycogen in this organ. PTGOE mice showed improved exercise capacity, as determined by the distance covered and time ran in an extenuating endurance exercise, compared with control mice. Moreover, fasting decreased exercise capacity in control mice but not in PTGOE mice. After exercise, liver glycogen stores were totally depleted in control mice, but PTGOE mice maintained significant glycogen levels even in fasting conditions. In addition, PTGOE mice displayed an increased hepatic energy state after exercise compared with control mice. Exercise caused a reduction in the blood glucose concentration in control mice that was less pronounced in PTGOE mice. No changes were found in the levels of blood lactate, plasma free fatty acids, or β-hydroxybutyrate. Plasma glucagon was elevated after exercise in control mice, but not in PTGOE mice. Exercise-induced changes in skeletal muscle were similar in both genotypes. These results identify hepatic glycogen as a key regulator of endurance capacity in mice, an effect that may be exerted through the maintenance of blood glucose levels.

Valproate (VPA) is a widely used mood stabilizer, but its therapeutic mechanism of action is not understood. This knowledge gap hinders the development of more effective drugs with fewer side effects. Using the yeast model to elucidate the effects of VPA on cellular metabolism, we determined that the drug upregulated expression of genes normally repressed during logarithmic growth on glucose medium and increased levels of activated (phosphorylated) Snf1 kinase, the major metabolic regulator of these genes. VPA also decreased the cytosolic pH (pHc) and reduced glycolytic production of 2/3-phosphoglycerate. ATP levels and mitochondrial membrane potential were increased, and glucose-mediated extracellular acidification decreased in the presence of the drug, as indicated by a smaller glucose-induced shift in pH, suggesting that the major P-type proton pump Pma1 was inhibited. Interestingly, decreasing the pHc by omeprazole-mediated inhibition of Pma1 led to Snf1 activation. We propose a model whereby VPA lowers the pHc causing a decrease in glycolytic flux. In response, Pma1 is inhibited and Snf1 is activated, resulting in increased expression of normally repressed metabolic genes. These findings suggest a central role for pHc in regulating the metabolic program of yeast cells.

Portable and quantitative detection of Escherichia coli (E. coli) has the potential to reform clinical diagnostics, food safety, and environmental monitoring. At present, most commercial devices used for pathogen detection have disadvantages such as expensive, highly complex operations, or limited detection specificity. Using the common luminometer and the properties of pyruvate kinase utilizing phosphoenolpyruvate to generate adenosine triphosphate (ATP), we have developed a method that could specifically quantify E. coli. The system is based on a sandwich hybridization procedure wherein both oligonucleotide probes recognize each end of the target of pathogenic 16S rRNAs segment. The detection probe DNA-conjugated pyruvate kinase can link ATP production to the detection of pathogenic nucleic acid in the samples. The luminometer-based system is capable of detecting E. coli with single bacteria resolution. The platform should be easily used to the detection of many other toxic analytes through the application of suitable functional-DNA recognition elements.

Background Emergence of Candida glabrata, which causes potential life-threatening invasive candidiasis, has been widely associated with high morbidity and mortality. In order to cause disease in vivo, a robust and highly efficient metabolic adaptation is crucial for the survival of this fungal pathogen in human host. In fact, reprogramming of the carbon metabolism is believed to be indispensable for phagocytosed C. glabrata within glucose deprivation condition during infection. Methods In this study, the metabolic responses of C. glabrata under acetate growth condition was explored using high-throughput transcriptomic and proteomic approaches. Results Collectively, a total of 1482 transcripts (26.96%) and 242 proteins (24.69%) were significantly up- or down-regulated. Both transcriptome and proteome data revealed that the regulation of alternative carbon metabolism in C. glabrata resembled other fungal pathogens such as Candida albicans and Cryptococcus neoformans, with up-regulation of many proteins and transcripts from the glyoxylate cycle and gluconeogenesis, namely isocitrate lyase (ICL1), malate synthase (MLS1), phosphoenolpyruvate carboxykinase (PCK1) and fructose 1,6-biphosphatase (FBP1). In the absence of glucose, C. glabrata shifted its metabolism from glucose catabolism to anabolism of glucose intermediates from the available carbon source. This observation essentially suggests that the glyoxylate cycle and gluconeogenesis are potentially critical for the survival of phagocytosed C. glabrata within the glucose-deficient macrophages. Conclusion Here, we presented the first global metabolic responses of C. glabrata to alternative carbon source using transcriptomic and proteomic approaches. These findings implicated that reprogramming of the alternative carbon metabolism during glucose deprivation could enhance the survival and persistence of C. glabrata within the host.

Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry, saturation-transfer difference nuclear magnetic resonance and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. In addition, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemical reaction with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress., P. L. -M. was funded by a PhD fellowship from the ‘‘la Caixa’’ Foundation and received financial support from the Universidad de Zaragoza, Fundación Bancaria Ibercaja, and Fundación CAI (CM 1/16) during his stay at the UW–Madison. We acknowledge Ministerio de Economía, Industria y Competitividad for grant AGL2015-66177 (J. A. C.), the University of Zaragoza for grant UZ-2015-BIO-01 (J. A. C.), and OTRI (University of Zaragoza), Ministerio de Ciencia, Innovación y Universidades for grants CTQ2013-44367-C2-2-P and BFU2016-75633-P (R. H. -G.) and BFU2016-78232-P (A. V. -C.), Gobierno de Aragón for grants E34_R17 and LMP58_18 (R. H. -G.) and E45_17R (J. A. C and A. V. -C.) and FEDER (2014–2020) funds for “Building Europe from Aragón”. R. H. -G. and A. V. -C. thank the ARAID foundation for support. The research leading to these results also received funding from the FP7 (2007–2013) under BioStruct-X (grant agreement N283570 and BIOSTRUCTX_5186). We also thank the ALBA synchrotron radiation source, in particular, the beamline XALOC facility. J. A. and I. D. acknowledge access to UEA Faculty of Science Research Facilities. J. A. acknowledges funding support from the Biotechnology and biological Sciences Research Council (BBSRC; BB/P010660/1) and the Spanish Ministry of Science, Innovation and Universities through grant PID2019-109395GB-100. T. H. acknowledges a BBSRC DTP studentship. Financial support from the Universidad de Sevilla (Acciones Especiales del VI Plan Propio de Investigación y Transferencia) is also acknowledged. This work was also supported by National Institutes of Health (NIH) Grant GM065386 (J. M. D.), NIH National Research Service Award T32 GM007215 (J. B.) and the National Science Foundation Graduate Research Fellowship Program (NSF-GRFP) DGE-1256259 (J. B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Liver is the central metabolic hub that coordinates carbohydrate and lipid metabolism. The bioactive derivative of vitamin A, retinoic acid (RA), was shown to regulate major metabolic genes including phosphoenolpyruvate carboxykinase, fatty acid synthase, carnitine palmitoyltransferase 1, and glucokinase among others. Expression levels of these genes undergo profound changes during adaptation to fasting or in metabolic diseases such as type 1 diabetes (T1D). However, it is unknown whether the levels of hepatic RA change during metabolic remodeling. This study investigated the dynamics of hepatic retinoid metabolism and signaling in the fed state, in fasting, and in T1D. Our results show that fed-to-fasted transition is associated with significant decrease in hepatic retinol dehydrogenase (RDH) activity, the rate-limiting step in RA biosynthesis, and downregulation of RA signaling. The decrease in RDH activity correlates with the decreased abundance and altered subcellular distribution of RDH10 while Rdh10 transcript levels remain unchanged. In contrast to fasting, untreated T1D is associated with upregulation of RA signaling and an increase in hepatic RDH activity, which correlates with the increased abundance of RDH10 in microsomal membranes. The dynamic changes in RDH10 protein levels in the absence of changes in its transcript levels imply the existence of posttranscriptional regulation of RDH10 protein. Together, these data suggest that the downregulation of hepatic RA biosynthesis, in part via the decrease in RDH10, is an integral component of adaptation to fasting. In contrast, the upregulation of hepatic RA biosynthesis and signaling in T1D might contribute to metabolic inflexibility associated with this disease.

Melatonin priming is an effective strategy to improve the germination of aged oat (Avena sativa L.) seeds, but the mechanism involved in its time-course responses has remained largely unknown. In the present study, the phenotypic differences, ultrastructural changes, physiological characteristics, and proteomic profiles were examined in aged and melatonin-primed seed (with 10 &mu, M melatonin treatment for 12, 24, and 36 h). Thus, 36 h priming (T36) had a better remediation effect on aged seeds, reflecting in the improved germinability and seedlings, relatively intact cell ultrastructures, and enhanced antioxidant capacity. Proteomic analysis revealed 201 differentially abundant proteins between aged and T36 seeds, of which 96 were up-accumulated. In melatonin-primed seeds, the restoration of membrane integrity by improved antioxidant capacity, which was affected by the stimulation of jasmonic acid synthesis via up-accumulation of 12-oxo-phytodienoic acid reductase, might be a candidate mechanism. Moreover, the relatively intact ultrastructures enabled amino acid metabolism and phenylpropanoid biosynthesis, which were closely associated with energy generation through intermediates of pyruvate, phosphoenolpyruvate, fumarate, and &alpha, ketoglutarate, thus providing energy, active amino acids, and secondary metabolites necessary for germination improvement of aged seeds. These findings clarify the time-course related pathways associated with melatonin priming on promoting the germination of aged oat seeds.

Phosphoenolpyruvate carboxykinase (PEPCK) is a gluconeogenic enzyme with a cytosolic (Pck1/PEPCK-C) and mitochondrial (Pck2/PEPCK-M) isoform. Here we investigate the effect of 3-mercaptopicolinic acid (3-MPA), a PEPCK inhibitor, on C2C12 muscle cells. We report that Pck2 mRNA is 50–5000-fold higher than Pck1 during C2C12 myogenesis, indicating Pck2 is the predominant PEPCK isoform. C2C12 cell proliferation was inhibited in a dose-dependent manner following 48 h 3-MPA treatment (0.01–1 mM). C2C12 myogenic differentiation was significantly induced following 3-MPA treatment (0.25, 0.5, 1 mM) from day 0 of differentiation, demonstrated by increased creatine kinase activity, fusion index and myotube diameter; likewise, the myosin heavy chain (MyHC)-IIB isoform (encoded by Myh4) is an indicator of hypertrophy, and both porcine MYH4-promoter activity and endogenous Myh4 mRNA were also significantly induced. High doses (0.5 and/or 1 mM) of 3-MPA reduced mRNA expression of Pck2 and genes associated with serine biosynthesis (Phosphoglycerate dehydrogenase, Phgdh; phosphoserine aminotransferase-1, Psat1) following treatment from days 0 and 4. To conclude, as Pck2/PEPCK-M is the predominant isoform in C2C12 cells, we postulate that 3-MPA promoted myogenic differentiation through the inhibition of PEPCK-M. However, we were unable to confirm that 3-MPA inhibited PEPCK-M enzyme activity as 3-MPA interfered with the PEPCK enzyme assay, particularly at 0.5 and 1 mM.

Although corticosteroid-mediated hepatic gluconeogenic activity in response to stress has been extensively studied in fishes and other vertebrates, there is little information on the stress response in basal vertebrates. In sea lamprey (Petromyzon marinus), a representative member of the most basal extant vertebrate group Agnatha, 11-deoxycortisol and deoxycorticosterone are the major circulating corticosteroids. The present study examined changes in circulating glucose and 11-deoxycortisol concentrations in response to a physical stressor. Furthermore, the gluconeogenic actions of 11-deoxycortisol and deoxycorticosterone were examined. Within 6 h of exposure of larval and juvenile sea lamprey to an acute handling stress, plasma 11-deoxycortisol levels increased 15- and 6-fold, respectively, and plasma glucose increased 3- and 4-fold, respectively. Radiometric receptor binding studies revealed that a corticosteroid receptor (CR) is present in the liver at lower abundance than in other tissues (gill and anterior intestine) and that the binding affinity of the liver CR was similar for 11-deoxycortisol and deoxycorticosterone. Transcriptional tissue profiles indicate a wide distribution of cr transcription, kidney-specific transcription of steroidogenic acute regulatory protein (star) and liver-specific transcription of phosphoenolpyruvate carboxykinase (pepck). Ex vivo incubation of liver tissue with 11-deoxycortisol resulted in dose-dependent increases in pepck mRNA levels. Finally, intraperitoneal administration of 11-deoxycortisol and deoxycorticosterone demonstrated that only 11-deoxycortisol resulted in an increase in plasma glucose. Together, these results provide the first direct evidence for the gluconeogenic activity of 11-deoxycortisol in an agnathan, indicating that corticosteroid regulation of plasma glucose is a basal trait among vertebrates.

Glucokinase (GK), a key regulator of hepatic glucose metabolism in the liver and glucose sensor and mediator in the secretion of insulin in the pancreas, is not studied in detail for its therapeutic application in diabetes. Herein, we study the alteration in GK activity during hyperinsulinemia-induced insulin resistance in HepG2 cells. We also investigated the link between GK and Bcl-2-associated death receptor (BAD) during hyperinsulinemia. There are emerging demands for GK activators from natural resources, and we selected vanillic acid (VA) to evaluate its potential as GK activators during hyperinsulinemia in HepG2 cells. VA is a phenolic compound and a commonly used food additive in many food industries. We found that VA safeguarded GK inhibition during hyperinsulinemia significantly in HepG2 cells. VA also prevented the depletion of glycogen synthesis during hyperinsulinemia, which is evident from protein expression studies of phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, glycogen synthase, and glycogen synthase kinase-3β. This was associated with activation of BAD activity, which was also confirmed by Western blotting. Molecular docking revealed strong binding between GK active site and VA, supporting their strong interaction. These are the first in vitro data to indicate the beneficial properties of VA with respect to insulin resistance induced by hyperinsulinemia by GK activation. Since it is activated via BAD, the hypoglycemia associated with general GK activation is not expected here and therefore has significant implications for future therapies against diabetes.

The mosquito Aedes aegypti undertakes a shift in carbohydrate metabolism during embryogenesis, including an increase in the activity of phosphoenolpyruvate carboxykinase (PEPCK), a key gluconeogenic enzyme, at critical steps of embryo development. All eukaryotes studied to date present two PEPCK isoforms, namely PEPCK-M (mitochondrial) and PEPCK-C (cytosolic). In A. aegypti, however, these proteins are so far uncharacterized. In the present work we describe two A. aegypti PEPCK isoforms by sequence alignment, protein modeling, and transcription analysis in different tissues, as well as PEPCK enzymatic activity assays in mitochondrial and cytoplasmic compartments during oogenesis and embryogenesis. First, we characterized the protein sequences compared to other organisms, and identified conserved sites and key amino acids. We also performed structure modeling for AePEPCK(M) and AePEPCK(C), identifying highly conserved structural sites, as well as a signal peptide in AePEPCK(M) localized in a very hydrophobic region. Moreover, after blood meal and during mosquito oogenesis and embryogenesis, both PEPCKs isoforms showed different transcriptional profiles, suggesting that mRNA for the cytosolic form is transmitted maternally, whereas the mitochondrial form is synthesized by the zygote. Collectively, these results improve our understanding of mosquito physiology and may yield putative targets for developing new methods for A. aegypti control.

Pancreatic β-cells couple nutrient metabolism with appropriate insulin secretion. Here, we show that pyruvate kinase (PK), which converts ADP and phosphoenolpyruvate (PEP) into ATP and pyruvate, underlies β-cell sensing of both glycolytic and mitochondrial fuels. Plasma membrane-localized PK is sufficient to close K(ATP) channels and initiate calcium influx. Small-molecule PK activators increase the frequency of ATP/ADP and calcium oscillations and potently amplify insulin secretion. PK restricts respiration by cyclically depriving mitochondria of ADP, which accelerates PEP cycling until membrane depolarization restores ADP and oxidative phosphorylation. Our findings support a compartmentalized model of β-cell metabolism in which PK locally generates the ATP/ADP required for insulin secretion. Oscillatory PK activity allows mitochondria to perform synthetic and oxidative functions without any net impact on glucose oxidation. These findings suggest a potential therapeutic route for diabetes based on PK activation that would not be predicted by the current consensus single-state model of β-cell function.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal gastrointestinal malignancies due to its characteristics of local invasion and distant metastasis. Purine element binding protein &alpha, (PUR&alpha, ) is a DNA and RNA binding protein, and recent studies have showed that abnormal expression of PUR&alpha, is associated with the progression of some tumors, but its oncogenic function, especially in ESCC progression, has not been determined. Based on the bioinformatic analysis of RNA-seq and ChIP-seq data, we found that PUR&alpha, affected metabolic pathways, including oxidative phosphorylation and fatty acid metabolism, and we observed that it has binding peaks in the promoter of mitochondrial phosphoenolpyruvate carboxykinase (PCK2). Meanwhile, PUR&alpha, significantly increased the activity of the PCK2 gene promoter by binding to the GGGAGGCGGA motif, as determined though luciferase assay and ChIP-PCR/qPCR. The results of Western blotting and qRT-PCR analysis showed that PUR&alpha, overexpression enhances the protein and mRNA levels of PCK2 in KYSE510 cells, whereas PUR&alpha, knockdown inhibits the protein and mRNA levels of PCK2 in KYSE170 cells. In addition, measurements of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) indicated that PUR&alpha, promoted the metabolism of ESCC cells. Taken together, our results help to elucidate the molecular mechanism by which PUR&alpha, activates the transcription and expression of PCK2, which contributes to the development of a new therapeutic target for ESCC.

© 2020 Gil-Gil et al., Fosfomycin is a bactericidal antibiotic, analogous to phosphoenolpyruvate, that exerts its activity by inhibiting the activity of MurA. This enzyme catalyzes the first step of peptidoglycan biosynthesis, the transfer of enolpyruvate from phosphoenolpyruvate to uridine-diphosphate-N-acetylglucosamine. Fosfomycin is increasingly being used, mainly for treating infections caused by Gram-negative multidrug-resistant bacteria. The mechanisms of mutational resistance to fosfomycin in Stenotrophomonas maltophilia, an opportunistic pathogen characterized by its low susceptibility to commonly used antibiotics, were studied in the current work. None of the mechanisms reported so far for other organisms, which include the production of fosfomycin-inactivating enzymes, target modification, induction of an alternative peptidoglycan biosynthesis pathway, and the impaired entry of the antibiotic, are involved in the acquisition of such resistance by this bacterial species. Instead, the unique cause of resistance in the mutants studied is the mutational inactivation of different enzymes belonging to the Embden-Meyerhof-Parnas central metabolism pathway. The amount of intracellular fosfomycin accumulation did not change in any of these mutants, showing that neither inactivation nor transport of the antibiotic is involved. Transcriptomic analysis also showed that the mutants did not present changes in the expression level of putative alternative peptidoglycan biosynthesis pathway genes or any related enzyme. Finally, the mutants did not present an increased phosphoenolpyruvate concentration that might compete with fosfomycin for its binding to MurA. On the basis of these results, we describe a completely novel mechanism of antibiotic resistance based on mutations of genes encoding metabolic enzymes., Work in the laboratory is supported by Instituto de Salud Carlos III (grant RD16/0016/0011) and cofinanced by the European Development Regional Fund “A Way to Achieve Europe,” by grant S2017/BMD-3691 InGEMICS-CM, funded by Comunidad de Madrid (Spain) and European Structural and Investment Funds, and by the Spanish Ministry of Economy and Competitivity (BIO2017-83128-R). T.G.-G. is the recipient of an FPI fellowship from MINECO.

Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is a key enzyme for gluconeogenesis that is positively regulated by propionate in bovines at the transcription level. The specific elements that determine propionate responsiveness within the bovine PCK1 promoter are unknown. In silico promoter analysis of the bovine PCK1 gene revealed several clusters of transcription factor binding sites. In the present study, we determined the essentiality of the putative cyclic AMP response element (CRE) at -94 through -87 bp and the 2 putative hepatic nuclear factor 4α (HNF4α) binding elements at +68 through +72 and -1,078 through -1,074, respectively, in mediating bovine PCK1 promoter responses to propionate and other regulators, including butyrate, cyclic AMP (cAMP), and glucocorticoids. The wild-type bovine PCK1 promoter [PCK1(WT)] was ligated to a luciferase reporter gene and transfected into rat hepatoma (H4IIE) cells. Activities of PCK1(WT) were induced by approximately 2-, 2-, 4-, 8-, 9-, 18-, and 16-fold respectively when exposed to cAMP (as 1.0 mM 8-Br-cAMP), 5.0 μM dexamethasone, cAMP + dexamethasone, 2.5 mM propionate, cAMP + propionate, cAMP + dexamethasone + propionate, and 2.5 mM butyrate. Seven mutants lacking either one single site, 2 of the 3 sites, or all 3 sites, generated by site-directed mutagenesis, were tested. Responses to propionate and all other treatments were completely abolished when CRE at -94 through -87 bp and HNF4α at +68 through +72 bp were both deleted. Our data indicate that these 2 regulatory elements act synergistically to mediate the bovine PCK1 promoter responses to propionate as well as butyrate, cAMP, and dexamethasone. The activation of PCK1 through these regulatory elements serves to activate the metabolic potential of bovine toward gluconeogenesis when the primary substrate for gluconeogenesis, propionate, is also present.

Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F420., F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance. IMPORTANCE Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F420.

To test the hypothesis that myricitrin (MYR) improves type 2 diabetes, we examined the effect of MYR on hyperglycemia, glucose intolerance, hepatic steatosis, and inflammation in high-fat diet (HFD) and streptozotocin (STZ)-induced type 2 diabetic mice. Male C57BL/6J mice were randomly divided into three groups: non-diabetic, diabetic control, and MYR (0.005%, w/w)-supplemented diabetic groups. Diabetes was induced by HFD and STZ, and MYR was administered orally for 5 weeks. Myricitrin exerted no significant effects on food intake, body weight, fat weight, or plasma lipids levels. However, MYR significantly decreased fasting blood glucose levels, improved glucose intolerance, and increased pancreatic &beta, cell mass compared to the diabetic control group. Myricitrin administration also markedly increased glucokinase mRNA expression and activity as well as lowered glucose-6-phosphatase and phosphoenolpyruvate carboxykinase mRNA expression and activity in the liver. In addition, liver weight, hepatic triglyceride content, and lipid droplet accumulation were markedly decreased following MYR administration. These changes were seemingly attributable to the suppression of the hepatic lipogenic enzymes&mdash, fatty acid synthase and phosphatidate phosphohydrolase. Myricitrin also significantly lowered plasma MCP-1 and TNF-&alpha, levels and the mRNA expression of hepatic pro-inflammatory genes. These results suggest that MYR has anti-diabetic potential.

The pathophysiology of type 2 diabetes mellitus (T2DM) is characterized by not only insulin resistance, but also the abnormal regulation of glucagon secretion, suggesting that antagonizing the glucagon-induced signaling pathway has therapeutic potential in the treatment of T2DM. Although various Kampo medicines (traditional herbal medicines) are often utilized to ameliorate the symptoms of T2DM, their effects on glucagon signaling have not yet been clarified. In the present study, we examined the effects of nine types of representative Kampo formulations prescribed for T2DM on glucagon-induced CREB activation in HEK293T cells stably expressing glucagon receptor (Gcgr) and a hepatic cell line HepG2. Among these Kampo medicines, Rokumigan, Hachimijiogan, and Goshajinkigan significantly suppressed the glucagon-induced transactivation of the cAMP-responsive element (CRE)-binding protein (CREB) by inhibiting its interaction with CREB-binding protein (CBP), which led to a reduction in the expression of phosphoenolpyruvate carboxykinase (PEPCK) mRNA. Furthermore, among the crude drugs commonly contained in these three Kampo medicines, Rehmannia Root (Jio), Moutan Bark (Botampi), and Cornus Fruit (Shanzhuyu) exerted inhibitory effects on glucagon-induced CREB activation. Collectively, the present results provide a novel mechanism, the inhibition of glucagon signaling, by which Rokumigan, Hachimijiogan, and Goshajinkigan improve the symptoms of T2DM., Cell biology; Pharmaceutical science; Biochemistry; Molecular biology; Health sciences; Pharmacology; Type 2 diabetes mellitus; Kampo medicines; Glucagon; CREB; PEPCK

Clonorchiasis, caused by chronic infection with Clonorchis sinensis (C. sinensis), is an important food-borne parasitic disease that seriously afflicts more than 35 million people globally, resulting in a socioeconomic burden in endemic regions. C. sinensis adults long-term inhabit the microaerobic and limited-glucose environment of the bile ducts. Energy metabolism plays a key role in facilitating the adaptation of adult flukes to crowded habitat and hostile environment. To understand energy source for adult flukes, we compared the component and content of free amino acids between C. sinensis-infected and uninfected bile. The results showed that the concentrations of free amino acids, including aspartic acid, serine, glycine, alanine, histidine, asparagine, threonine, lysine, hydroxylysine, and urea, were significantly higher in C. sinensis-infected bile than those in uninfected bile. Furthermore, exogenous amino acids could be utilized by adult flukes via the gluconeogenesis pathway regardless of the absence or presence of exogenous glucose, and the rate-limiting enzymes, such as C. sinensis glucose-6-phosphatase, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and pyruvate carboxylase, exhibited high expression levels by quantitative real-time PCR analysis. Interestingly, no matter whether exogenous glucose was present, inhibition of gluconeogenesis reduced the glucose and glycogen levels as well as the viability and survival time of adult flukes. These results suggest that gluconeogenesis might play a vital role in energy metabolism of C. sinensis and exogenous amino acids probably serve as an important energy source that benefits the continued survival of adult flukes in the host. Our study will be a cornerstone for illuminating the biological characteristics of C. sinensis and the host-parasite interactions., Author summary Clonorchiasis, closely related to cholangiocarcinoma and hepatocellular carcinoma, has led to a negative socioeconomic impact in global areas especially some Asian endemic regions. Owing to the emergence of drug resistance and hypersensitivity reactions after the massive and repeated use of praziquantel as well as the lack of effective vaccines, searching for new strategies that prevent and treat clonorchiasis has become an urgent matter. Clonorchis sinensis, the causative agent of clonorchiasis, long-term inhabits the microaerobic and limited-glucose environment of the bile ducts. Adequate nutrients are essential for adult flukes to resist the adverse condition and survive in the crowed habitat. Studies on energy metabolism of adult flukes are beneficial for further exploring host-parasite interactions and developing novel anti-parasitic drugs. Our results suggest that gluconeogenesis probably plays a vital role in energy metabolism of Clonorchis sinensis and exogenous amino acids might be an essential energy source for adult flukes to successfully survive in the host. Our foundational study opens a new avenue for explaining energy metabolism of Clonorchis sinensis and provides a valuable strategy that the gluconeogenesis pathway will be a potential and novel target for the prevention and treatment of clonorchiasis.

The phenolic profiles, hypoglycemic activity, and molecular mechanism of the effect on type 2 diabetes mellitus (T2DM) of four highland barley varieties were investigated in the present study. The fundamental phenolics in highland barley were ferulic acid, naringin, and catechin, which mainly existed in bound form. These varieties showed favorable hypoglycemic activity via inhibition of &alpha, glucosidase and &alpha, amylase activities, enhancement of glucose consumption, glycogen accumulation and glycogen synthase 2 (GYS2) activity, and down-regulation of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) activities. Specifically, ZQ320 variety exhibited the strongest hypoglycemic activity compared to the other varieties. Highland barley phenolics could inhibit gluconeogenesis and motivate glycogen synthesis via down-regulating the gene expression of G6Pase, PEPCK, and glycogen synthase kinase 3&beta, (GSK3&beta, ), while activating the expression of insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3 kinase (PI3K), serine/threonine kinase (Akt), GYS2, and glucose transporter type 4 (GLUT4). Therefore, phenolics from highland barley could be served as suitable candidates for therapeutic agent in T2DM to improve human health.

Hepatic nuclear factor 4 alpha (HNF4α) drives the expression of apolipoprotein B (ApoB), microsomal triglyceride transfer protein (MTP) and phospholipase A2 G12B (PLA2G12B), governing hepatic very-low-density lipoprotein (VLDL) production and secretion. Andrographolide (AP) is a major constituent isolated from Andrographis paniculata. We found that AP can disrupt the interaction between HNF4α and its coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α). Virtual docking and mutational analysis indicated that arginine 235 of HNF4α is essential for binding to AP. As a consequence of antagonizing the activity of HNF4α, AP suppresses the expression of ApoB, MTP and PLA2G12B and reduces the rate of hepatic VLDL secretion in vivo. AP additionally reduced gluconeogenesis via down-regulating the expression of HNF4α target genes phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6pc). Collectively, our results suggest that AP affects liver function via modulating the transcriptional activity of HNF4α.