Phosphoenolpyruvate Carboxykinase Activity Research Articles

Dietary thiamine modulates carbohydrate metabolism, antioxidant status, and alleviates hypoxia stress in oriental river prawn Macrobrachium nipponense (de Haan)

Hypoxia is one of the challenges in prawns aquaculture. However, the role of thiamine, which is a coenzyme in carbohydrate metabolism with antioxidant properties, in reducing hypoxia in prawns aquaculture is currently unknown. We investigated the effects of thiamine on antioxidant status, carbohydrate metabolism and acute hypoxia in oriental river prawn, Macrobrachium nipponense. One thousand eight hundred prawns (0.123 ± 0.003 g) were fed five diets (60 prawns each tank, six replicates per diet) supplemented with graded thiamine levels (5.69, 70.70, 133.67, 268.33 and 532.00 mg/kg dry mater) for eight weeks and then exposed to hypoxia stress for 12 h followed by reoxyegnation for 12 h. The results showed that, under normoxia, prawns fed the 133.67 or 268.33 mg/kg thiamine diet had significantly lower glucose 6-phosphatedehydrogenase, succinate dehydrogenase and phosphoenolpyruvate carboxykinase activities than those fed the other diets. Moreover, total antioxidant capacity (T-AOC) increased significantly when prawns were fed the 133.67 mg/kg thiamine diet. Superoxide dismutase (SOD) activity and malonaldehyde (MDA) content also increased significantly when prawns were fed the 268.33 or 532.00 mg/kg thiamine diet under hypoxia. And the significantly increased SOD activity and MDA level also observed in prawns fed 532.00 mg/kg thiamine under reoxygenation. Under normoxia, prawns fed the 70.70 or 133.67 mg/kg thiamine diet decreased the mRNA expressions of AMP-activated protein kinase-alpha (AMPK-α), pyruvate dehydrogenase-E1-α subunit (PDH-E1-α) and hypoxia-inducible factor-1s (HIF-1α, HIF-1β), but increased the mRNA expressions of phosphofructokinase (PFK) significantly. After 12 h of hypoxia, the energy metabolism related genes (AMPK-β, AMPK-γ, PFK, PDH-E1-α), hypoxia-inducible factor related genes (HIF-1α, HIF-1β) and thiamine transporter gene (SLC19A2) were up-regulated significantly in prawns fed the 133.67 or 268.33 mg/kg thiamine diets. After 12 h of reoxygenation, prawns fed the 133.67 or 268.33 mg/kg diet significantly decreased the SOD activity, MDA level and SLC19A2 mRNA expression compared with other diets. The optimum thiamine was 161.20 mg/kg for minimum MDA content and 143.17 mg/kg for maximum T-AOC activity based on cubic regression analysis. In summary, supplementing 143.17 to 161.20 mg/kg thiamine in the diets for M. nipponense improves the antioxidant capacity under normoxia and reduces the oxidative damage under hypoxia stress.

Simulation of the crosstalk between glucose and acetaminophen metabolism in a liver zonation model.

The liver metabolizes a variety of substances that sometimes interact and regulate each other. The modeling of a single cell or a single metabolic pathway does not represent the complexity of the organ, including metabolic zonation (heterogeneity of functions) along with liver sinusoids. Here, we integrated multiple metabolic pathways into a single numerical liver zonation model, including drug and glucose metabolism. The model simulated the time-course of metabolite concentrations by the combination of dynamic simulation and metabolic flux analysis and successfully reproduced metabolic zonation and localized hepatotoxicity induced by acetaminophen (APAP). Drug metabolism was affected by nutritional status as the glucuronidation reaction rate changed. Moreover, sensitivity analysis suggested that the reported metabolic characteristics of obese adults and healthy infants in glucose metabolism could be associated with the metabolic features of those in drug metabolism. High activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphate phosphatase in obese adults led to increased APAP oxidation by cytochrome P450 2E1. In contrast, the high activity of glycogen synthase and low activities of PEPCK and glycogen phosphorylase in healthy infants led to low glucuronidation and high sulfation rates of APAP. In summary, this model showed the effects of glucose metabolism on drug metabolism by integrating multiple pathways into a single liver metabolic zonation model.

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

Obesity is a worldwide health problem and is directly associated with insulin resistance and type 2 diabetes. The liver is an important organ for the control of healthy glycemic levels, since insulin resistance in this organ reduces phosphorylation of forkhead box protein 1(FOXO1) protein, leading to higher hepatic glucose production (HGP) and fasting hyperglycemia. Aerobic physical training is known as an important strategy in increasing the insulin action in the liver by increasing FOXO1 phosphorylation and reducing gluconeogenesis. However, little is known about the effects of strength training in this context. This study aimed to investigate the effects of short-term strength training on hepatic insulin sensitivity and glycogen synthase kinase-3β(GSK3β) and FOXO1 phosphorylation in obese (OB) mice. To achieve this goal, OB Swiss mice performed the strength training protocol (one daily session for 15 days). Short-term strength training increased the phosphorylation of protein kinase B and GSK3β in the liver after insulin stimulus and improved the control of HGP during the pyruvate tolerance test. On the other hand, sedentary OB animals reduced FOXO1 phosphorylation and increased the levels of nuclear FOXO1 in the liver, increasing the phosphoenolpyruvate carboxykinase(PEPCK) and glucose-6-phosphatase (G6Pase) content. The bioinformatics analysis also showed positive correlations between hepatic FOXO1 levels and gluconeogenic genes, reinforcing our findings. However, strength-trained animals reverted to this scenario, regardless of body adiposity changes. In conclusion, short-term strength training is an efficient strategy to enhance the insulin action in the liver of OB mice, contributing to glycemic control by reducing the activity of hepatic FOXO1 and lowering PEPCK and G6Pase contents.

Glucocorticoids decrease thermogenic capacity and increase triacylglycerol synthesis by glycerokinase activation in the brown adipose tissue of rats.

Although it is well established that glucocorticoids inactivate thermogenesis and promote lipid accumulation in interscapular brown adipose tissue (IBAT), the underlying mechanisms remain unknown. We found that dexamethasone treatment (1 mg/kg) for 7 days in rats decreased the IBAT thermogenic activity, evidenced by its lower responsiveness to noradrenaline injection associated with reduced content of mitochondrial proteins, respiratory chain protein complexes, noradrenaline, and the β3 -adrenergic receptor. In parallel, to understand better how dexamethasone increases IBAT lipid content, we also investigated the activity of the ATP citrate lyase (ACL), a key enzyme of de novo fatty acid synthesis, glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway, and the three glycerol-3-P generating pathways: (1) glycolysis, estimated by 2-deoxyglucose uptake, (2) glyceroneogenesis, evaluated by phosphoenolpyruvate carboxykinase activity and pyruvate incorporation into triacylglycerol-glycerol, and (3) direct phosphorylation of glycerol, investigated by the content and activity of glycerokinase. Dexamethasone increased the mass and the lipid content of IBAT as well as plasma levels of glucose, insulin, non-esterified fatty acid, and glycerol. Furthermore, dexamethasone increased ACL and G6PD activities (79% and 48%, respectively). Despite promoting a decrease in the incorporation of U-[14 C]-glycerol into triacylglycerol (~54%), dexamethasone increased the content (~55%) and activity (~41%) of glycerokinase without affecting glucose uptake or glyceroneogenesis. Our data suggest that glucocorticoid administration reduces IBAT thermogenesis through sympathetic inactivation and stimulates glycerokinase activity and content, contributing to increased generation of glycerol-3-P, which is mostly used to esterify fatty acid and increase triacylglycerol content promoting IBAT whitening.

Biosynthesis of gold nanoparticles using leaf extract of Dittrichia viscosa and in vivo assessment of its anti-diabetic efficacy

Several studies have reported the anti-diabetic effect of biologically synthesized gold nanoparticles (AuNPs). This study was designed to investigate the in vivo anti-diabetic activity of AuNPs synthesized using the leaf extract of Dittrichia viscosa in a high-fat diet (HFD)/streptozotocin (STZ)-induced diabetes in rats. AuNPs were synthesized using the leaf extract of D. viscosa, and the synthesized AuNPs were characterized by UV–visible spectrophotometer, dynamic light scattering (DLS), zeta potential, and transmission electron microscopy (TEM). To study the anti-hyperglycemic effect of the AuNPs formed using D. viscosa extract, adult male Sprague–Dawley rats were divided into three groups (6–8 rats/group) as follows: control group, a diabetic group without treatment, and a diabetic group treated intraperitoneally with a daily injection of AuNPs at a dose of 2.5 mg/kg for 21 days. Diabetes was induced by maintaining the rats on HFD for 2 weeks, followed by a single intraperitoneal injection of 45 mg/kg of STZ. Serum and liver samples were collected at the end of the treatment period and used to measure glucose levels and hepatic gene expression and activity of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in the liver gluconeogenic pathway. The AuNPs formed using D. viscosa extract were mainly spherical with a size range between 20 and 50 nm with good stability and dispersity, as indicated by the zeta potential and DLS measurements. Treatment with AuNP significantly lowered the blood glucose level, the gene expression, and the activity of hepatic PEPCK in comparison to the diabetic untreated group (P < 0.05). This study suggests that AuNPs synthesized using D. viscosa leaf extract can alleviate hyperglycemia in HFD/STZ-induced diabetes in rats, which could be through the reduction of hepatic gluconeogenesis by inhibiting the expression and activity of the hepatic PEPCK gene.Graphical abstractSchematic illustration of the biosynthesis of AuNPs showing their distinctive morphology under the EM. The generated particles were injected into animals and serum glucose levels were reported in addition to the PEPCK expression and activity

Scaling with body mass and age in glycolytic enzymes of domestic dogs.

Animals produce ATP through oxidative phosphorylation using oxygen, but cellular energy can also be obtained through glycolysis when oxygen is not present at sufficient levels. Although most mammals of larger body mass have longer life spans, small dog breeds tend to outlive large breeds. Primary fibroblast cells from larger breeds of dogs have previously been shown to have increased dependency on glycolytic phenotypes across their lifespan. Different levels of activity of the glycolytic enzymes pyruvate kinase (PK), lactate dehydrogenase (LDH), and phosphoenolpyruvate carboxykinase (PEPCK) may provide insight to a mechanism that leads to the different metabolic phenotype observed in different sized breeds as they age. In this study, 1) we measured the activities of PK, LDH, and PEPCK in primary fibroblasts from dogs of different breed sizes and age classes and 2) measured the activities of PK and LDH inplasmafrom dogs of different breed sizes and age classes. We found that there was no significant relationship between body mass and PK, LDH and PEPCK activity in primary fibroblasts. Further, there were not significant differences with activity in these enzymes for old dogs compared to young dogs. In plasma, we found a negative correlation between PK activity and body mass and no relationship between LDH activity and body mass. There was a negative relationship between LDH activity and age in dogs. Further, while a negative correlational relationship between PK activity and age was only marginal, a best subsets regression model demonstrated a significant marginal effect of age on PK activity. PK and LDH may provide intermediates for other metabolic pathways in small breeds. However, large breed dogs may demonstrate a deficiency in metabolism at the PK level, a cellular metabolic pathway that may potentially aid in tumor progression.

Activating Effects of the Bioactive Compounds From Coffee By-Products on FGF21 Signaling Modulate Hepatic Mitochondrial Bioenergetics and Energy Metabolism in vitro.

Coffee by-products contain bioactive compounds that have been shown to have the capacity to modulate human metabolism. The goal of this study was to investigate the effects of the main bioactive compounds in coffee by-products and two aqueous extracts from the coffee husk and silverskin on the activation of fibroblast growth factor 21 (FGF21) signaling and the subsequent regulation of mitochondrial bioenergetics and lipid and glucose metabolism. HepG2 cells treated with palmitic acid (PA) were used in a non-alcoholic fatty liver disease (NAFLD) cell model. The bioactive compounds from coffee by-products (50 μmol L−1) and the aqueous extracts from the coffee silverskin and coffee husk (100 μg mL−1) increased ERK1/2 phosphorylation and the secretion of FGF21 (1.3 to 1.9-fold). Coffee by-products' bioactive compounds counteracted inflammation and PA-triggered lipotoxicity. Oxidative stress markers (ROS, mitochondrial superoxide, and NADPH oxidase) and the activity of antioxidant enzymes (superoxide dismutase and catalase) were modulated through the activation of Nrf2 signaling. Mitochondrial bioenergetics were regulated by enhancing respiration and ATP production via PGC-1α, and the expression of oxidative phosphorylation complexes increased. Coffee by-products' bioactive compounds decreased lipid accumulation (23–41%) and fatty acid synthase activity (32–65%) and triggered carnitine palmitoyltransferase-1 activity (1.3 to 1.7-fold) by activating AMPK and SREBP-1c pathways. The GLUT2 expression and glucose uptake were increased (58–111%), followed by a promoted glucokinase activity (55–122%), while glucose production and phosphoenolpyruvate carboxykinase activity were reduced due to IRS-1/Akt1 regulation. The bioactive compounds from coffee by-products, primarily chlorogenic and protocatechuic acids, could regulate hepatic mitochondrial function and lipid and glucose metabolism by activating FGF21 and related signaling cascades.

Phytochemicals from the Cocoa Shell Modulate Mitochondrial Function, Lipid and Glucose Metabolism in Hepatocytes via Activation of FGF21/ERK, AKT, and mTOR Pathways.

The cocoa shell is a by-product that may be revalorized as a source of bioactive compounds to prevent chronic cardiometabolic diseases. This study aimed to investigate the phytochemicals from the cocoa shell as targeted compounds for activating fibroblast growth factor 21 (FGF21) signaling and regulating non-alcoholic fatty liver disease (NAFLD)-related biomarkers linked to oxidative stress, mitochondrial function, and metabolism in hepatocytes. HepG2 cells treated with palmitic acid (PA, 500 µmol L−1) were used in an NAFLD cell model. Phytochemicals from the cocoa shell (50 µmol L−1) and an aqueous extract (CAE, 100 µg mL−1) enhanced ERK1/2 phosphorylation (1.7- to 3.3-fold) and FGF21 release (1.4- to 3.4-fold) via PPARα activation. Oxidative stress markers were reduced though Nrf-2 regulation. Mitochondrial function (mitochondrial respiration and ATP production) was protected by the PGC-1α pathway modulation. Cocoa shell phytochemicals reduced lipid accumulation (53–115%) and fatty acid synthase activity (59–93%) and prompted CPT-1 activity. Glucose uptake and glucokinase activity were enhanced, whereas glucose production and phosphoenolpyruvate carboxykinase activity were diminished. The increase in the phosphorylation of the insulin receptor, AKT, AMPKα, mTOR, and ERK1/2 conduced to the regulation of hepatic mitochondrial function and energy metabolism. For the first time, the cocoa shell phytochemicals are proved to modulate FGF21 signaling. Results demonstrate the in vitro preventive effect of the phytochemicals from the cocoa shell on NAFLD.

Effects of different chromium sources on growth performance, serum biochemical, hepatopancreas glycometabolism enzymes activities, IR, GLUT2 and SGLT1 gene expression of common carp ( Cyprinus carpio )

A 60-day feeding trial was conducted to evaluate the effects of different chromium sources on the common carp. Four diets (32.2% crude protein and 6% lipids of dry matter) were formulated to add chromium oxide (Cr2O3), chromium picolinate (CrPic) and chromium methionine (CrMet) with 2 mg/kg Cr3+, respectively, and one basal diet. Each diet was randomly assigned to triplicate groups of 60 juvenile common carp (approximately 40.95 ± 4.80 g). The results indicated that fish fed CrMet diets had significantly higher weight gain, specific growth rate and feed efficiency than other groups (p < 0.05). No significant differences in the whole body compositions except the lipid content significantly increased in CrMet diets (p < 0.05). Fish that were fed chromium diets had significantly higher lactic dehydrogenase and creatine kinase in serum, succinic acid dehydrogenase and lactic acid in muscle, pyruvate kinase and glycogen synthase in hepatopancreas, lower glucose concentration and phosphoenolpyruvate carboxykinase activities in hepatopancreas than those given the basal diet (p < 0.05). Insulin receptor (IR) in serum, IR gene expression in hepatopancreas and sodium-dependent glucose transporter 1 (SGLT1) in intestinal was increased (p < 0.05), while the cortisol decreased significantly in the dietary of CrMet or CrPic (p < 0.05). CrMet supplementation also increased HK activities, up-regulated glucose transporter 2 (GLUT2) in hepatopancreas (p < 0.05). In conclusion, dietary addition of CrMet improved growth performance, adjusted glycometabolism enzyme activities, IR, GLUT2 and SGLT1 gene expression and had a significant impact on the carbohydrate utilization of juvenile common carp fed high corn diets compared with CrPic and Cr2O3.

Differential expression of gluconeogenic enzymes in early- and late-stage diabetes: the effect of Citrullus colocynthis (L.) Schrad. Seed extract on hyperglycemia and hyperlipidemia in Wistar-Albino rats model

BackgroundThe medicinal plant Citrullus colocynthis (L.) Schrad. (C. colocynthis) may benefit patients at different phases of diabetes by attuning to contrasting situations. Our primary objective was to find the mechanism(s) behind the antidiabetic/anti-hyperlipidemic effects of C.colocynthis seed aqueous extract (CCAE) in two different stages of type 2 diabetes (T2D) in rats.MethodsFasting blood sugar (FBS) levels, body weights, and the degree of impaired glucose tolerance (IGT) were measured in healthy nondiabetic control rats (Con), as well as rats with early and late stages of T2D, denoted as ET2D and LT2D, respectively. CCAE was intraperitoneally (IP) injected for 28 days. In the end, the hepatic mRNA expression levels of the following genes were determined by RT-PCR: glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), insulin-dependent sterol regulatory element-binding protein-1c (SREBP-1c), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), peroxisome proliferator-activated receptor alpha (PPARα), and carnitine palmitoyltransferase I (CPT1). The liver was examined by hematoxylin and eosin (H&E) and Oil-Red O staining. CCAE was partially analyzed by HPLC-DAD.ResultsET2D and LT2D were characterized by differentially elevated FBS, deteriorated bodyweight, and significant IGT compared to Con. Hepatosteatoses of varying morphologies and higher hepatic expression of G6Pase than PRPCK in ET2D versus the opposite in LT2D further confirmed the divergent nature of metabolic aberrations. At the end of 28 days, the high levels of FBS, alkaline phosphatase (ALP), triglyceride (TG), urea, hepatic protein carbonyl content (PCC), and alanine and aspartate aminotransferases (AST and ALT, respectively) persisted in untreated LT2D. CCAE ameliorated oxidative stress and upregulated PPARα expression in diabetic groups and Con; it downregulated CPT1 expression in the LT2D group. CCAE’s ability to lower FBS and serum and hepatic TG in both ET2D and LT2D indicated its ability to act via different mechanisms. Ferulic acid (Fer A) and rutin hydrate (RH) were detected in CCAE.ConclusionCCAE lowered the FBS in ET2D via inhibiting the hepatic G6Pase expression (glycogenolysis). In LT2D, CCAE abated sugar levels by diverting PEPCK activity, preferably towards glyceroneogenesis than gluconeogenesis. The preserved triglyceride/fatty acid (TG/FA) cycle, the upregulated PPARα, and the downregulated CPT1 gene expressions reduced serum and hepatic TG.

Changes in gluconeogenesis pathways and key genes associated with mass mortality in the clam Meretrix petechialis upon Vibrio infection

Mass mortality events frequently occur in marine bivalves growing in natural habitats and artificial culture environments due to pathogen infection. The clam Meretrix petechialis is an important commercial bivalve species, and the clam culture industry has suffered frequent mass mortality events during summer. However, the pathogenesis of the host is still poorly understood. In this study, M. petechialis was challenged with Vibrio parahaemolyticus, and the clams were sampled at different infection phases and physiological statuses. The transcriptome analysis of the clam hepatopancreas in the onset phase of Vibrio challenge showed the significant enrichment of multiple metabolism-related signaling pathways, including peroxisome proliferator-activated receptors (PPARs), adenosine 5′-monophosphate activated protein kinase (AMPK), phosphatidylinositol 3′-kinase (PI3K)-Akt signaling pathways and glycolysis/gluconeogenesis, which revealed the involvement of metabolism-related signaling pathways in host immune defense. Weighted correlation network analysis (WGCNA) revealed that key enzymes involved in gluconeogenesis were enriched in this phase, suggesting that gluconeogenesis may be associated with infection-induced mortality in clams. Examinations of the glucose content, phosphoenolpyruvate carboxykinase (PEPCK) activity and the expression of key genes involved in gluconeogenesis showed that gluconeogenesis was repressed during Vibrio infection. An analysis of the expression of eight key genes in the PI3K-Akt signaling pathway between moribund and surviving individuals implied that gluconeogenesis was related to clam mass mortality. The results might help us to understand the molecular mechanism of death outbreak events and provide potential markers to detect the physiological status of this clam species.

Dietary chromium modulates glucose homeostasis and induces oxidative stress in Pacific white shrimp (Litopenaeus vannamei).

While chromium (Cr) has been recognized as an essential nutrient for all animals, and dietary supplementation can be beneficial, it can also be toxic. The present study aimed to investigate the contrasting effects of dietary chromium in Pacific white shrimp Litopenaeus vannamei. Five experimental diets were formulated to contain Cr at levels of 0.82 (Cr0.82, unsupplemented diet), 1.01 (Cr1.01), 1.22 (Cu1.22), 1.43 (Cr1.43) and 1.63 (Cr1.63) mg/kg and were fed to shrimp for 8 weeks. Highest weight gain was recorded in shrimp fed the diet containing 1.22 mg/kg Cr. Shrimp fed the diet containing the highest level of Cr (1.63 mg/kg) showed the lowest weight gain and clear signs of oxidative stress and apoptosis as evidenced by higher levels of H2O2, malondialdehyde and 8-hydroxydeoxyguanosine, and expression of caspase 2, 3, 5, and lower contents of total and oxidized glutathione, and expression of Cu/Zn sod, cat, gpx, mt, bcl2. Chromium supplementation promoted glycolysis and inhibited gluconeogenesis as shown by increased activities of hexokinase, phosphofructokinase and pyruvate kinase, and reduced activity of phosphoenolpyruvate carboxykinase in shrimp fed the diet containing 1.43 mg/kg Cr. Shrimp fed the diet with 1.63 mg/kg Cr had lowest contents of crustacean hyperglycemic hormone and insulin like peptide in hemolymph. Expression of genes involved in insulin signaling pathway and glycose metabolism including insr, irs1, pik3ca, pdpk1, akt, acc1, gys, glut1, pk, hk were up-regulated, and foxO1, gsk-3β, g6pc, pepck were down-regulated in shrimp fed the diets supplemented with Cr. This study demonstrated that optimum dietary supplementation of Cr had beneficial effects on glucose homeostasis and growth, whereas excess caused oxidative damage and impaired growth. The results contribute to our understanding of the biological functions of chromium in shrimp.

Differential expression of PEPCK isoforms is correlated to Aedes aegypti oogenesis and embryogenesis.

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.

Tyr‐Asp inhibition of glyceraldehyde 3‐phosphate dehydrogenase affects plant redox metabolism

How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr‐Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr‐Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3‐phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr‐Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched‐chain amino acid‐containing dipeptides, but not by Tyr‐Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small‐molecule regulators at the nexus of stress, protein degradation, and metabolism.

Synthesis and Biological Activity of Acinetobacter calcoaceticus IMV B-7241 Surfactants Depending on Monovalent Cations Content in Cultivation Medium

Microbial surfactants (biosurfactants) are multifunctional preparations due to a combination of physicochemical (reduction of surface and interfacial tension, emulsifying activity) and biological (antimicrobial and antiadhesive activity, the ability to destroy biofilms) properties. However, the disadvantage of biosurfactants synthesized as a complex of compounds is the possibility of changing the biological activity depending on the conditions of producer cultivation. Aim. To study the effect of potassium and sodium cations on the NADP+-dependent glutamate dehydrogenase activity of cell-free extract of Acinetobacter calcoaceticus ІМV B-7241 with subsequent appropriate modification of the nutrient medium composition and determination of antimicrobial and anti-adhesive activity of surfactant synthesized. Methods. A. calcoaceticus ІМV B-7241 strain was grown in media containing 2% of sunflower oil waste as a carbon source, as well as various concentrations of potassium and sodium chloride (basal – 1.0 g/l NaCl, medium #1 that did not contains NaCl, medium #2 in which the concentration of NaCl was 2.0 g/l, medium #3 in which the concentration of NaCl and KCl was 1.0 g/l each). The surfactants were extracted from the supernatant liquid culture with a modified Folch mixture. Antiadhesive activity and the degree of biofilms degradation were determined by spectrophotometric method, antimicrobial activity − by the indicator of the minimum inhibitory concentration (MIC). Activity of enzymes of surface-active aminolipids biosynthesis (NADP+-dependent glutamate dehydrogenase) and glycolipids (phosphoenolpyruvate (PEP) carboxylase, PEP-synthetase, PEP-carboxykinase, trehalose phosphate synthase) were analyzed in cell-free extracts obtained after the destruction of cells by ultrasound. Results. It was found that potassium and sodium cations in concentrations of 50 and 100 mM are inhibitors of NADP+-dependent glutamate dehydrogenase, and in lower concentrations (5–20 mM) – activators of this enzyme, as well as PEP-carboxykinase and PEP-synthetase. The increase in the biosurfactant concentration to 6.1−7.7 g/l during cultivation of A. calcoaceticus ІМV B-7241 in medium #1 and #3 was due to the predominant synthesis of glycolipids under such conditions, which was evidenced by the increase in 1.8−6.5 times in the activity of PEP-carboxylase, PEP-carboxykinase, PEP-synthetase and trehalose phosphate synthetase compared to the indicators on the basal medium. The concentration of surfactants synthesized in the basal medium was 3.6 g/l, but such surfactants were characterized by the highest antimicrobial and anti-adhesive activity. Their MIC against the test-cultures of studied bacteria (Pseudomonas sp. MI-2, Bacillus subtilis BT-2, Escherichia coli IEM-1, Staphylococcus aureus BMS-1, Enterobacter cloaceae C-8) and fungi (Candida albicans D-6, Rhizopus nigricans P1, Aspergillus niger P-3, Fusarium culmorum T-7) were 0.88−56 μg/ml and were by 2−3 orders of magnitude lower compared to established for surfactants synthesized in modified media #1–3. In the case of treatment of abiotic materials with surfactant solutions obtained on the basal medium, the adhesion of bacteria and fungi was on average 10–20% lower than after surface treatment by the surfactant synthesized in modified media. In the presence of 148−296 μg/ml of surfactants obtained in the basal medium, destruction of S. aureus BMS-1 and B. subtilis BT-2 biofilms was 45−66%, and C. albicans D-6 yeast – 39−44%. Under the action of similar concentrations of surfactants synthesized in modified media, the destruction of bacterial and yeast biofilms was lower: 6-52 and 20–46%, respectively. Conclusions. The obtained results are consistent with the data of our previous studies on the possibility of regulating the antimicrobial and antiadhesive activity of surfactants in the process of producer cultivation by changing the content of cations in the medium, which are inhibitors/activators of enzymes responsible for the synthesis of components of the surfactants complex, which have certain biological properties.

Insulin-mediated glycemic responses and glucose homeostasis in black sea bream (Acanthopagrus schlegelii) fed different carbohydrate sources

The present study was conducted to investigate the effects of dietary carbohydrate sources on insulin mediated glycemic responses and glucose homeostasis in black seabream. Four isonitrogenous (48%) and isolipidic (12%) diets were formulated to contain 20% glucose (GLU), sucrose (SUC), dextrin (DEX) and wheat starch (WS), respectively. The black seabream was randomly distributed into 12 tanks (n = 3) at a stocking density of 12 fish per tank. After 2 weeks of feeding, fishes were starved for 48 h then re-fed and sampled at 0, 1, 3, 6, 12, and 24 h. The results showed that the concentration of serum glucose increased at 1 h, peaked at 3 h, decreased at 12 h, and returned to basal level at 24 h in all dietary groups (p < 0.05). In contrast, there were significant differences in postprandial insulin levels among all treatments, especially in the DEX group, which showed higher insulin level at 3 h after feeding (p < 0.05). Compared with 0 h, liver glycogen in the WS and DEX groups increase significantly at 6 h and was depleted at 24 h (p < 0.05). Hepatic enzyme activities such as glucokinase (GCK) and pyruvate kinase (PK) were influenced by sampling time and dietary carbohydrate sources (p < 0.05). Fish fed with the DEX diet showed the highest hepatic GCK and PK activities and the lowest phosphoenol pyruvate carboxykinase (PEPCK) activity at 3 h among all the treatments. Slightly but not significantly higher phosphofructo kinase (PFK) activity was observed in fish fed with the SUC diet than those fed with the GLU diet at 6 h post prandial (p > 0.05). The relative expression of insulin signaling pathways such as insulin receptor substrate1 (irs1) and protein kinase B (akt1) were significantly up-regulated at 3 h in the DEX group (p < 0.05). Meanwhile, the mRNA expression levels of glucokinase (gk) and pyruvate kinase (pk) were sensitive to hepatic glycolysis, and their expressions were up-regulated at 1, 3, 6 h, especially at 3 h in the DEX group. Genes such as glucose 6 phosphatase (g6pc) and phosphoenol pyruvate carboxykinase (pepck) played an important role in hepatic gluconeogenesis, and their expressions were inhibited at 1, 3 h. Overall, the results of present study indicated that dietary carbohydrate sources resulted in a hyperglycaemic situation 3 h after feeding, most likely as a response to glucose loading. Moreover, glucose metabolic responses were regulated by different carbohydrate sources at different time points, and plays a important role in clearing glucose through increased activity of insulin, glycolysis, and glycogenesis, along with gluconeogenesis suppression.

Comprehensive Analysis of C. glutamicum Anaplerotic Deletion Mutants Under Defined d-Glucose Conditions.

Wild-type C. glutamicum ATCC 13032 is known to possess two enzymes with anaplerotic (C4-directed) carboxylation activity, namely phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx). On the other hand, C3-directed decarboxylation can be catalyzed by the three enzymes phosphoenolpyruvate carboxykinase (PEPCk), oxaloacetate decarboxylase (ODx), and malic enzyme (ME). The resulting high metabolic flexibility at the anaplerotic node compromises the unambigous determination of its carbon and energy flux in C. glutamicum wild type. To circumvent this problem we performed a comprehensive analysis of selected single or double deletion mutants in the anaplerosis of wild-type C. glutamicum under defined d-glucose conditions. By applying well-controlled lab-scale bioreactor experiments in combination with untargeted proteomics, quantitative metabolomics and whole-genome sequencing hitherto unknown, and sometimes counter-intuitive, genotype-phenotype relationships in these mutants could be unraveled. In comparison to the wild type the four mutants C. glutamiucm Δpyc, C. glutamiucm Δpyc Δodx, C. glutamiucm Δppc Δpyc, and C. glutamiucm Δpck showed lowered specific growth rates and d-glucose uptake rates, underlining the importance of PCx and PEPCk activity for a balanced carbon and energy flux at the anaplerotic node. Most interestingly, the strain C. glutamiucm Δppc Δpyc could be evolved to grow on d-glucose as the only source of carbon and energy, whereas this combination was previously considered lethal. The prevented anaplerotic carboxylation activity of PEPCx and PCx was found in the evolved strain to be compensated by an up-regulation of the glyoxylate shunt, potentially in combination with the 2-methylcitrate cycle.

Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity

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.

Changes to hepatic nutrient dynamics and energetics in rainbow trout (Oncorhynchus mykiss) following exposure to and recovery from hydraulic fracturing flowback and produced water

Hydraulic fracturing flowback and produced water (FPW) is a highly complex and heterogenous wastewater by-product of hydraulic fracturing practices. To date, no research has examined how FPW exposure to freshwater biota may affect energetic homeostasis following subsequent induction of detoxification processes. Rainbow trout (Oncorhynchus mykiss) were acutely exposed for 48 h to either 2.5% or 7.5% FPW, and hepatic metabolism was assessed either immediately or following a 3-week recovery period. Induction of xenobiotic metabolism was observed with an 8.8-fold increase in ethoxyresorufin-O-deethylase (EROD) activity after 48 h exposure to 7.5% FPW, alongside a 10.3-fold increase in the mRNA abundance of cyp1a, both of which returned to basal level after three weeks. Glucose uptake capacity was elevated by 6.8- and 12.9-fold following 2.5% and 7.5% FPW exposure, respectively, while alanine uptake was variable. Activity measurements and mRNA abundance of key enzymes involved in hepatic metabolism indicated that aerobic metabolism was maintained with exposure, as was glycolysis. Gluconeogenesis, as measured by phosphoenolpyruvate carboxykinase (PEPCK) activity, decreased by ~30% 48 h following 2.5% FPW exposure and ~20% 3 weeks after 7.5% FPW exposure. The abundance of pepck mRNA activity followed similar, yet non-significant, trends. Finally, a delayed increase in amino acid catabolism was observed, as glutamate dehydrogenase (GDH) activity was increased 2-fold in 7.5% FPW exposed fish when compared to saline control fish at the 3-week time point. We provide evidence to suggest that although hepatic metabolism is altered following acute FPW exposure, metabolic homeostasis generally returns 3-weeks post-exposure.

Effects of dietary carbohydrate types on growth performance, innate immunity, antioxidant ability and glucose metabolism of brook troutSalvelinus fontinalis

The study evaluated the effects of different types of dietary carbohydrates on growth, immunity, antioxidant capacity and glucose metabolism enzyme activities in the juvenile brook trout. Fish (13.65 ± 0.06 g) were fed five isonitrogenous and isolipidic diets that contained 150g kg-1 glucose, sucrose, dextrin, pregelatinized corn starch (PS) or raw corn starch (RS) respectively. Weight gain, specific growth rate and protein efficiency ratio were significantly higher in fish fed dextrin than the other treatments. The serum alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (AKP) and glucose were higher in fish fed glucose than those fed dextrin, PS or RS. Hepatic superoxide dismutase (SOD), catalase (CAT) and lysozyme activity, as well as total antioxidant capacity, were lower in fish fed glucose or sucrose than the other treatments. Moreover, the highest malondialdehyde (MDA) level was reported in the glucose group. Furthermore, fish fed glucose had significantly lower hepatic glucokinase (GK), phosphofructokinase (PFK) and phosphoenolpyruvate carboxykinase (PEPCK) activities than those fed dextrin, PS or RS. The results suggest that dextrin is the optimal carbohydrate source for brook trout, while incorporating glucose into the diet suppresses innate immunity, increases oxidative stress and interferes with glucose metabolism, resulting into a reduction in growth.