PEPCK Activity Research Articles

Fibroblast growth factor 1 (FGF1) improves glucose homeostasis, modulates gut microbial composition, and reduces inflammatory responses in rainbow trout (Oncorhynchus mykiss) fed a high-fat diet

High-fat diets (HFDs) are widely used in aquaculture due to their lipid and protein-conserving effects, thereby reducing feed costs. However, prolonged feeding of HFD often leads to metabolic disorders in fish, such as disruption of hepatic lipid homeostasis, liver injury, and disruption of glucose homeostasis. Fibroblast growth factor 1 (FGF1) plays an essential role in controlling glucose levels in the body and dampening immune reactions. However, its impact on teleosts remains poorly researched. The therapeutic potential of recombinant FGF1 (rFGF1) was examined in a 6-week culture experiment involving rainbow trout (Oncorhynchus mykiss) that were fed an HFD. The results revealed that rFGF1 significantly reduced serum glucose levels and hepatic PEPCK and G6PC activities, but improved hepatic glycogen (P < 0.05), compared to the HFD + PBS group. Further experiments indicated that the inhibitory effect of rFGF1 on hepatic gluconeogenesis was mediated by the cAMP signaling pathway and was dependent on the high expression of PDE4D. In addition, rFGF1 increased hepatic glycogen content, which involves the AKT-GSK3β axis. Despite this increase, rFGF1 did not lead to glycogen storage disease, as shown by reduced hepatic inflammation as a result of decreased GOT (glutamic oxaloacetic transaminase), GPT (glutamic pyruvic transaminase), and elevated SOD (superoxide dismutase) in the rFGF1-treated group, accompanied by decreased il-1β, il-6, and xbp-1, and elevated nrf2 and number of hepatocyte autophagosomes. Alterations in gut microbes and short-chain fatty acids (SCFAs) were noted, indicating that rFGF1 caused a notable rise in intestinal Lactobacillus, acetic acid, and butyric acid levels. This study investigated the molecular mechanisms of rFGF1 on glucose metabolism and inflammatory responses in an HFD-fed rainbow trout model, providing new insights to improve the regulation of glucose metabolism in carnivorous fish.

Study on the Mechanism of GABA-Rich Adzuki Bean Regulating Blood Glucose Based on the IRS/PI3K/AKT Pathway.

The adzuki bean is a mature seed of the red bean leguminous plant, and people like to eat it because of its nutritious properties and moderate proportion of amino acids. Adzuki bean germination and the enrichment of GABA greatly improve the health effects of the adzuki bean. The effects of the GABA-rich adzuki bean on the expression of insulin-pathway-related genes and proteins in the liver of T2DM mice were studied via Western blotting and qPCR. The results showed that a GABA-rich adzuki bean diet could promote glycogen synthesis in the liver of T2DM mice, inhibit the activities of PEPCK and G-6-Pase, and significantly down-regulate the gene expression levels of PEPCK, G6PC and FOXO1 (p < 0.05) and the phosphorylation levels of FOXO1 and GSK3β. In addition, it can also up-regulate the expression of the AMPKα gene and down-regulate the expression of the SREBP1c gene to inhibit the synthesis of triglycerides and cholesterol in T2DM mice. Lipid accumulation in mice can alleviate glucose and lipid metabolism disorders and play an effective role in regulating blood glucose at liver tissue targets. This study suggested that the GABA-rich adzuki bean can improve hyperglycemia in type 2 diabetic mice by activating the IRS/PI3K/AKT signaling pathway in the liver.

Marine thermal fluctuation induced gluconeogenesis by the transcriptional regulation of CgCREBL2 in Pacific oysters

Marine thermal fluctuation profoundly influences energy metabolism, physiology, and survival of marine life. In the present study, short-term and long-term high-temperature stresses were found to affect gluconeogenesis by inhibiting PEPCK activity in the Pacific oyster (Crassostrea gigas), which is a globally distributed species that encounters significant marine thermal fluctuations in intertidal zones worldwide. CgCREBL2, a key molecule in the regulation of gluconeogenesis, plays a critical role in the transcriptional regulation of PEPCK in gluconeogenesis against high-temperature stress. CgCREBL2 was able to increase the transcription of CgPEPCK by either binding the promoter of CgPEPCK gene or activating CgPGC-1α and CgHNF-4α after short-term (6 h) high-temperature stress, while only by binding CgPEPCK after long-term (60 h) high-temperature stress. These findings will further our understanding of the effect of marine thermal fluctuation on energy metabolism on marine organisms.

Changes of glycolysis and gluconeogenesis key enzymes in the muscle of the shrimp Penaeus vannamei in response to hypoxia and reoxygenation

Although the shrimp Penaeus vannamei can tolerate oxygen-limited conditions, the organisms may suffer detrimental effects in several processes, including growth, reproduction, immune responses, and others. During hypoxia, the shrimp makes metabolic adjustments in energy use and glucose homeostasis. Glucose is hydrolyzed during glycolysis and synthesized from non-glycosidic metabolites in gluconeogenesis. Although these pathways share many enzymes, the processes are not exactly the reverse of each other. We studied the first and last irreversible steps of glycolysis and gluconeogenesis catalyzed by hexokinase/glucose-6-phosphatase (HK/G6Pase) and pyruvate kinase/pyruvate carboxylase/phosphoenolpyruvate carboxykinase (PK/PC/PEPCK), respectively, in muscle under hypoxia and hypoxia followed by reoxygenation to gain insights about the coordinated metabolic responses that maintain glucose balance during oxygen stress. For the first group, HK gene expression was induced in hypoxia while G6Pase was reduced in hypoxia and reoxygenation, whereas for enzymes activity, HK was maintained and G6Pase was only detected in reoxygenation. For the second group, gene expression increased in PK in hypoxia, in PC in reoxygenation and in both cases, the enzymatic activities were maintained. Mitochondrial PEPCK expression increased during hypoxia and cytosolic PEPCK decreased, while total PEPCK activity was maintained. Intracellular lactate decreased during hypoxia while glucose levels were maintained. Altogether the results show that in limited oxygen, the expression of glycolytic enzymes-genes are induced, the gluconeogenic counterparts are active, although with more varied responses to oxygen limited conditions, and intracellular glucose levels are maintained.

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

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

Fish oil minimises feed intake and improves insulin sensitivity in Zucker fa/fa rats.

Long-chain n-3 PUFA (LC n-3 PUFA) prevent, in rodents, insulin resistance (IR) induced by a high-fat and/or fructose diet but not IR induced by glucocorticoids. In humans, contrasting effects have also been reported. We investigated their effects on insulin sensitivity, feed intake (FI) and body weight gain in genetically insulin resistant male obese (fa/fa) Zucker (ZO) rats during the development of obesity. ZO rats were fed a diet supplemented with 7 % fish oil (FO) + 1 % corn oil (CO) (wt/wt) (ZOFO), while the control group was fed a diet containing 8 % fat from CO (wt/wt) (ZOCO). Male lean Zucker (ZL) rats fed either FO (ZLFO) or CO (ZLCO) diet were used as controls. FO was a marine-derived TAG oil containing EPA 90 mg/g + DHA 430 mg/g. During an oral glucose tolerance test, glucose tolerance remained unaltered by FO while insulin response was reduced in ZOFO only. Liver insulin sensitivity (euglycaemic-hyperinsulinaemic clamp + 2 deoxyglucose) was improved in ZOFO rats, linked to changes in phosphoenolpyruvate carboxykinase expression, activity and glucose-6-phosphatase activity. FI in response to intra-carotid insulin/glucose infusion was decreased similarly in ZOFO and ZOCO. Hypothalamic ceramides levels were lower in ZOFO than in ZOCO. Our study demonstrates that LC n-3 PUFA can minimise weight gain, possibly by alleviating hypothalamic lipotoxicity, and liver IR in genetically obese Zucker rats.

COVID-19-related hyperglycemia is associated with infection of hepatocytes and stimulation of gluconeogenesis

Occurrence of hyperglycemia upon infection is associated with worse clinical outcome in COVID-19 patients. However, it is still unknown whether SARS-CoV-2 directly triggers hyperglycemia. Herein, we interrogated whether and how SARS-CoV-2 causes hyperglycemia by infecting hepatocytes and increasing glucose production. We performed a retrospective cohort study including patients that were admitted at a hospital with suspicion of COVID-19. Clinical and laboratory data were collected from the chart records and daily blood glucose values were analyzed to test the hypothesis on whether COVID-19 was independently associated with hyperglycemia. Blood glucose was collected from a subgroup of nondiabetic patients to assess pancreatic hormones. Postmortem liver biopsies were collected to assess the presence of SARS-CoV-2 and its transporters in hepatocytes. In human hepatocytes, we studied the mechanistic bases of SARS-CoV-2 entrance and its gluconeogenic effect. SARS-CoV-2 infection was independently associated with hyperglycemia, regardless of diabetic history and beta cell function. We detected replicating viruses in human hepatocytes from postmortem liver biopsies and in primary hepatocytes. We found that SARS-CoV-2 variants infected human hepatocytesin vitrowith different susceptibility. SARS-CoV-2 infection in hepatocytes yields the release of new infectious viral particles, though not causing cell damage. We showed that infected hepatocytes increase glucose production and this is associated with induction of PEPCK activity. Furthermore, our results demonstrate that SARS-CoV-2 entry in hepatocytes occurs partially through ACE2- and GRP78-dependent mechanisms. SARS-CoV-2 infects and replicates in hepatocytes and exerts a PEPCK-dependent gluconeogenic effect in these cells that potentially is a key cause of hyperglycemia in infected patients.

FaGAPC2/FaPKc2.2 and FaPEPCK reveal differential citric acid metabolism regulation in late development of strawberry fruit.

Citric acid is the primary organic acid that affects the taste of strawberry fruit. Glycolysis supplies key substrates for the tricarboxylic acid cycle (TCA cycle). However, little is known about the regulatory mechanisms of glycolytic genes on citric acid metabolism in strawberry fruits. In this study, the citric acid content of strawberry fruit displayed a trend of rising and decreasing from the initial red stage to the full red stage and then dark red stage. Thus, a difference in citric acid metabolic regulation was suspected during strawberry fruit development. In addition, overexpression of either cytoplasm glyceraldehyde-3-phosphate dehydrogenase (FxaC_14g13400, namely FaGAPC2) or pyruvate kinase (FxaC_15g00080, namely FaPKc2.2) inhibited strawberry fruit ripening and the accumulation of citric acid, leading to a range of maturity stages from partial red to full red stage. The combined transcriptome and metabolome analysis revealed that overexpression of FaGAPC2 and FaPKc2.2 significantly suppressed the expression of phosphoenolpyruvate carboxykinase (FxaC_1g21491, namely FaPEPCK) but enhanced the content of glutamine and aspartic acid. Meanwhile, the activities of PEPCK and glutamate decarboxylase (GAD) were inhibited, but the activities of glutamine synthase (GS) were increased in FaGAPC2/FaPKc2.2-overexpressed fruit. Further, functional verification demonstrated that overexpression of FaPEPCK can promote strawberry fruit ripening, resulting in a range of maturity stage from full red to dark red stage, while the citric acid synthase (CS) activities and citric acid content were significantly decreased. Overall, this study revealed that FaGAPC2/FaPKc2.2 and FaPEPCK perform an important role in reducing citric acid content in strawberry fruit, and FaGAPC2/FaPKc2.2 mainly by promoting the GS degradation pathway and FaPEPCK mainly by inhibiting the CS synthesis pathway.

Responses to aluminum and cadmium of a RNAi sorghum line with decreased levels of phosphoenolpyruvate carboxylase 3 (PPC3)

Phosphoenolpyruvate carboxylase (PEPC) is an enzyme family with central roles in carbon and nitrogen metabolisms. In order to obtain knowledge about the function of specific PEPC isoenzymes, we have characterized RNAi sorghum lines with decreased level of PPC3 (Ppc3 lines). PPC3 is the main root PEPC, and participates in responses to salinity and ammonium stress. This prompted us to study the responses of Ppc3 lines to Al and Cd stress. Both Al and Cd treatments decreased germination rate, growth rate, and shoot and root biomass production. These detrimental effects were higher in Ppc3 lines than in Wt. Root PPC3 expression and PEPC activity was increased by Al and Cd in Wt, but not in Ppc3 lines. The treatments also increased PPC2 and PPCK gene expression, and PEPCk activity, but these changes did not substitute the lack of PPC3. The production and secretion of exudates by plant roots play an important role in plant tolerance to Al and Cd toxicity. Al (2 days of treatment) caused a 40-fold increase of the citrate content of root exudates, without differences between Wt and Ppc3 lines. On the contrary, Cd (7 days of treatment) caused a 400-fold increase of citrate in Wt exudates, but only a 30-fold increase in Ppc3 lines. These results indicate that although PPC3 is not necessary for early production of organic acids in root exudates, it is crucial for maintaining high level of synthesis and accumulation of citrate, and that PPC3 is the main PEPC isoenzyme responsible for this response to heavy metal stress.

Hepatic metabolite responses to 4-day complete fasting and subsequent refeeding in rats.

BackgroundFasting has been widely used to improve various metabolic diseases in humans. Adaptive fasting is necessary for metabolic adaptation during prolonged fasting, which could overcome the great advantages of short-term fasting. The liver is the main organ responsible for energy metabolism and metabolic homeostasis. To date, we lack literature that describes the physiologically relevant adaptations of the liver during prolonged fasting and refeeding. For that reason, this study aims to evaluate the response of the liver of Sprague-Dawley (SD) rats to prolonged fasting and refeeding.MethodsSixty-six male SD rats were divided into the fasting groups, which were fasted for 0, 4, 8, 12, 24, 48, 72, or 96 h, and the refeeding groups, which were refed for 1, 3, or 6 days after 96 h of fasting. Serum glucose, TG, FFA, β-hydroxybutyrate, insulin, glucagon, leptin, adiponectin and FGF21 levels were assessed. The glucose content, PEPCK activity, TG concentration and FFA content were measured in liver tissue, and the expression of genes involved in gluconeogenesis (PEPCK and G6Pase), ketogenesis (PPARα, CPT-1a and HMGCS2) and the protein expression of nutrient-sensing signaling molecules (AMPK, mTOR and SIRT1) were determined by RT-qPCR and western blotting, respectively.ResultsFasting significantly decreased the body weight, which was totally recovered to baseline after 3 days of refeeding. A 4-day fast triggered an energy metabolic substrate shift from glucose to ketones and caused serum hormone changes and changes in the protein expression levels of nutrient-sensing signaling molecules. Glycogenolysis served as the primary fuel source during the first 24 h of fasting, while gluconeogenesis supplied the most glucose thereafter. Serum FFA concentrations increased significantly with 48 h of fasting. Serum FFAs partly caused high serum β-hydroxybutyrate levels, which became an important energy source with the prolongation of the fasting duration. One day of refeeding quickly reversed the energy substrate switch. Nutrient-sensing signaling molecules (AMPK and SIRT1 but not mTOR signaling) were highly expressed at the beginning of fasting (in the first 4 h). Serum insulin and leptin decreased with fasting initiation, and serum glucagon increased, but adiponectin and FGF21 showed no significant changes. Herein, we depicted in detail the timing of the metabolic response and adaptation of the liver to a 4-day water-only fast and subsequent refeeding in rats, which provides helpful support for the design of safe prolonged and intermittent fasting regimens.

Irisin Regulates Hepatic Glucose Metabolism via AMPK and PI3K/Akt Activation

Irisin plays a crucial role in the glucose metabolism of mammals and fish. However, the mechanism by which irisin regulates hepatic glucose metabolism is not entirely known. To clarify irisin’s function and underlying mechanisms in common carp glucose metabolism, the FNDC5 expression patterns were detected after insulin/glucagon injection, long-term high-glucose, and high-fat diet feeding. In vivo, the activity of PFK and the synthesis of liver glycogen were significantly increased, while the enzyme activity of PEPCK was reduced after intraperitoneal injection with irisin. In contrast, irisin siRNA attenuated the brain and liver FNDC5a and FNDC5b expression, and the serum glucose was increased. Glut2, gs, hk, and pfk were considerably downregulated in the brain and liver, while pygl, pepck, and g6pase were remarkably upregulated after siRNA treatment. In vitro, irisin activates the phosphorylation level of AMPK and PI3K/Akt in primary hepatocytes. Compound C and wortmannin inhibit AMPK and PI3K/Akt, reversing irisin-induced expression of g6pase, fbpase, pepck, gk, gs, pfk, hk, and gsk3b. At the same time, compound C attenuated irisin-induced expression of pi3k and akt. Our research reveals the mechanisms of irisinA and irisinB as glucose metabolism regulators in teleost for the first time.

The Role of Palmitoleic Acid in Regulating Hepatic Gluconeogenesis through SIRT3 in Obese Mice

Hepatic gluconeogenesis is a crucial process to maintain glucose level during starvation. However, unabated glucose production in diabetic patients is a major contributor to hyperglycemia. Palmitoleic acid is a monounsaturated fatty acid (16:1n7) that is available from dietary sources. Palmitoleic acid exhibits health beneficial effects on diabetes, insulin resistance, inflammation, and metabolic syndrome. However, the mechanism by which palmitoleate reduces blood glucose is still unclear. SIRT3 is a key metabolism-regulating NAD+-dependent protein deacetylase. It is known that fasting elevates the expression of SIRT3 in the liver and it regulates many aspects of liver’s response to nutrient deprivation, such as fatty acid oxidation and ketone body formation. However, it is unknown whether SIRT3 also regulates gluconeogenesis. Our study revealed that palmitoleic acid reduced hepatic gluconeogenesis and the expression of SIRT3 under high-fat diet conditions. Overexpression of SIRT3 in the liver and hepatocytes enhanced gluconeogenesis. Further study revealed that SIRT3 played a role in enhancing the activities of gluconeogenic enzymes, such as PEPCK, PC, and MDH2. Therefore, our study indicated that under a high-fat diet, palmitoleic acid decreased gluconeogenesis by reducing enzymatic activities of PEPCK, PC, and MDH2 by down-regulating the expression of SIRT3.

Empagliflozin Inhibits Hepatic Gluconeogenesis and Increases Glycogen Synthesis by AMPK/CREB/GSK3β Signalling Pathway.

Increases in glucose production and decreases in hepatic glycogen storage induce glucose metabolic abnormalities in type 2 diabetes (T2DM). Empagliflozin, a sodium-dependent glucose transporter 2 (SGLT2) inhibitor, is an effective hypoglycemic drug; however, the effects of empagliflozin on hepatic gluconeogenesis and glycogenesis are still unclear. In this study, we investigated the effects and mechanisms of empagliflozin on hepatic gluconeogenesis and glycogenesis in vivo and in vitro. Empagliflozin was administered via gavage to db/db mice for 8 weeks, and human hepatocyte HL7702 cells were treated with empagliflozin after palmitic acid (PA) stimulation. Compared with the control db/db mice, empagliflozin-treated mice showed a significant reduction in urine glucose levels, blood glucose levels, body weight and intraperitoneal glucose tolerance test (IPGTT) blood glucose levels. Moreover, the expression levels and activities of key gluconeogenesis enzymes PEPCK and G6Pase were dramatically reduced in the empagliflozin-treated mice, and the protein expression levels of AMPK/CREB/GSK3β signalling pathway-related molecules were significantly changed. In HL7702 cells, empagliflozin ameliorated glucose production and PEPCK and G6Pase expression and activity. Empagliflozin could also prevent the decreases in glycogen content and regulate the protein expression levels of AMPK/CREB/GSK3β signalling pathway-related molecules. Then, we selected the AMPK agonist AICAR and inhibitor compound C to further verify the effects of the AMPK signalling pathway on hepatic gluconeogenesis and glycogen synthesis. The results of the 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AIACR) intervention in HL7702 cells were consistent with those of empagliflozin treatment, and the effects of empagliflozin were abolished by compound C. In summary, empagliflozin could maintain glucose homoeostasis by reducing gluconeogenesis and increasing glycogenesis through the AMPK/CREB/GSK3β signalling pathway.

The effect of coffee consumption on glucose homeostasis and redox-inflammatory responses in high-fat diet-induced obese rats

Coffee effects on glucose homeostasis in obesity remain controversial. We investigated whether coffee mitigates the negative effects on glucose metabolism induced by a high-fat diet and the interrelationships with redox-inflammatory responses. Rats were treated with: control (CT-); coffee (CT+) 3.9 g of freeze-dried coffee/kg of diet; high-fat (HF-); or high-fat + coffee 3.9 g of freeze-dried coffee/kg of diet (HF+) diet. The high-fat diet increased weight gain, feed efficiency, HOMA β, muscle and hepatic glycogen, intestinal CAT and SOD activity, hepatic protein (CARB) and lipid oxidation (MDA), muscle Prkaa1 mRNA and IL6 levels, and decreased food intake, hepatic GR, GPX and SOD activities, intestinal CARB, intestinal Slc2a2 and Slc5a1 and hepatic Prkaa1 and Prkaa2 mRNA levels, hepatic glucose-6-phosphatase and muscle hexokinase (HK) activities, compared to the control diet. The high-fat diet with coffee increased hepatic GST activity and TNF and decreased IL6 and intestinal glucosidase activity compared with the high-fat diet. The coffee diet increased muscle glycogen, hepatic CARB and PEPCK activity, and decreased hepatic GR and SOD activities and intestinal CARB, compared with the control diet. Coffee increased insulin levels, HOMA IR/β, FRAP, muscle Prkaa1 mRNA levels and hepatic and muscle phosphofructokinase-1, and it decreased intestinal CAT, hepatic Slc2a2 mRNA levels and muscle HK activity, regardless of the diet type. In conclusion, chronic coffee consumption improves antioxidant and anti-inflammatory responses, but does not ameliorate glucose homeostasis in a high-fat diet-induced obesity model. In addition, coffee consumption increases insulin secretion and promotes muscle glycogen synthesis in rats maintained on a control diet.

MiR-378a-3p Participates in Metformin's Mechanism of Action on C2C12 Cells under Hyperglycemia.

Metformin is the most used biguanide drug for the treatment of type 2 diabetes mellitus. Despite being mostly known for its hepatic anti-gluconeogenic effect, it is also known to modulate microRNAs (miRNAs, miRs) associated with metabolic diseases. The latter mechanism could be relevant for better understanding metformin’s mechanisms underlying its biological effects. In the current work, we found that metformin increases miR-378a-3p expression (p < 0.002) in C2C12 myoblasts previously exposed to hyperglycemic conditions. While the inhibition of miR-378a-3p was shown to impair metformin’s effect in ATP production, PEPCK activity and the expression of Tfam. Finally, mitophagy, an autophagic process responsible for the selective degradation of mitochondria, was found to be induced by miR-378a-3p (p < 0.04). miR-378a-3p stimulated mitophagy through a process independent of sestrin-2 (SESN2), a stress-responsible protein that has been recently demonstrated to positively modulate mitophagy. Our findings provide novel insights into an alternative mechanism of action of metformin involving miR-378a-3, which can be used in the future for the development of improved therapeutic strategies against metabolic diseases.

Effects of calcium treatment on malate metabolism and γ-aminobutyric acid (GABA) pathway in postharvest apple fruit

Calcium treatment effects on malate metabolism and the GABA pathway in ‘Cripps Pink’ apple fruit during storage were investigated. Postharvest apple fruit treated with 1% and 4% calcium chloride solutions were stored at 25 ± 1 °C. The 4% calcium treatment suppressed declines in titratable acidity and malate content and increased succinate and oxalate concentrations. Calcium treatment also reduced the respiration rate and decreased ethylene production peak during storage. Moreover, 4% calcium treatment significantly enhanced cyNAD-MDH and PEPC activities and upregulated MdMDH1, MdMDH2, MdPEPC1 and MdPEPC2 expression while inhibiting cyNADP-ME and PEPCK activities and downregulating MdME1, MdME4 and MdPEPCK2 expression. Surprisingly, calcium treatment changed the content of some free amino acids (GABA, proline, alanine, aspartic acid and glutamate), two of which (glutamate and GABA) are primary metabolites of the GABA pathway. Furthermore, calcium application enhanced GABA pathway activity by increasing MdGAD1, MdGAD2, MdGABA-T1/2 and MdSSADH transcript levels.

Kinetic and structural analysis of Escherichia coli phosphoenolpyruvate carboxykinase mutants

BackgroundPhosphoenolpyruvate carboxykinase (PEPCK) is a metabolic enzyme in the gluconeogenesis pathway, where it catalyzes the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and CO2. The substrates for Escherichia coli PEPCK are OAA and MgATP, with Mn2+ acting as a cofactor. Analysis of PEPCK structures have revealed amino acid residues involved in substrate/cofactor coordination during catalysis. MethodsKey residues involved in coordinating the different substrates and cofactor bound to E. coli PEPCK were mutated. Purified mutant enzymes were used for kinetic assays. The structure of some mutant enzymes were determined using X-ray crystallography. ResultsMutation of residues D269 and H232, which comprise part of the coordination sphere of Mn2+, reduced kcat by 14-fold, and significantly increased the Km values for Mn2+ and OAA. Mutation of K254 a key residue in the P-loop motif that interacts with MgATP, significantly elevated the Km value for MgATP and reduced kcat. R65 and R333 are key residues that interacts with OAA. The R65Q and R333Q mutations significantly increased the Km value for OAA and reduced kcat respectively. ConclusionsOur results show that mutation of residues involved in coordinating OAA, MgATP and Mn2+ significantly reduce PEPCK activity. K254 plays an important role in phosphoryl transfer, while R333 is involved in both OAA decarboxylation and phosphoryl transfer by E. coli PEPCK. General significanceIn higher organisms including humans, PEPCK helps to regulate blood glucose levels, hence PEPCK is a potential drug target for patients with non-insulin dependent diabetes mellitus.

Decarboxylation mechanisms of C4 photosynthesis in Saccharum spp.: increased PEPCK activity under water-limiting conditions

BackgroundC4 plants have been classified into three subtypes based on the enzymes used to decarboxylate C4 acids in the bundle sheath cells (NADP-ME, NAD-ME and PEPCK pathways). Evidences indicate that, depending on environmental factors, C4 plants may exhibit a certain degree of flexibility in the use of the decarboxylation mechanisms. In this context, the objective was to extend the knowledge on the degree of flexibility between the pathways of decarboxylation in sugarcane, a NADP-ME species, at different levels of water deficit.ResultsAn experiment was carried out with two cultivars - RB92579 (tolerant to water deficit) and SP80–3280 (susceptible to water deficit) subjected to moderate level (− 1.5 to − 1.8 MPa), severe level (below − 2.0 MPa) and recovery (48 h after rehydration) and changes in the activities of the enzymes involved in the three C4 mechanisms and in gene expression were investigated. Our results showed that sugarcane uses the PEPCK pathway as a decarboxylation mechanism in addition to the NADP-ME, which was more evident under water deficit conditions for both cultivars.ConclusionsThe results obtained here, show that sugarcane increases the use of the PEPCK pathway as a decarboxylation mechanism, in addition to the NADP-ME pathway, under conditions of water deficit, particularly in the tolerant cultivar.

The glycerol backbone of phospholipids derives from noncarbohydrate precursors in starved lung cancer cells.

Cancer cells are reprogrammed to consume large amounts of glucose to support anabolic biosynthetic pathways. However, blood perfusion and consequently the supply with glucose are frequently inadequate in solid cancers. PEPCK-M (PCK2), the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK), has been shown by us and others to be functionally expressed and to mediate gluconeogenesis, the reverse pathway of glycolysis, in different cancer cells. Serine and ribose synthesis have been identified as downstream pathways fed by PEPCK in cancer cells. Here, we report that PEPCK-M-dependent glycerol phosphate formation from noncarbohydrate precursors (glyceroneogenesis) occurs in starved lung cancer cells and supports de novo glycerophospholipid synthesis. Using stable isotope-labeled glutamine and lactate, we show that PEPCK-M generates phosphoenolpyruvate and 3-phosphoglycerate, which are at least partially converted to glycerol phosphate and incorporated into glycerophospholipids (GPL) under glucose and serum starvation. This pathway is required to maintain levels of GPL, especially phosphatidylethanolamine (PE), as shown by stable shRNA-mediated silencing of PEPCK-M in H23 lung cancer cells. PEPCK-M shRNA led to reduced colony formation after starvation, and the effect was partially reversed by the addition of dioleyl-PE. Furthermore, PEPCK-M silencing abrogated cancer growth in a lung cancer cell xenograft model. In conclusion, glycerol phosphate formation for de novo GPL synthesis via glyceroneogenesis is a newly characterized anabolic pathway in cancer cells mediated by PEPCK-M under conditions of severe nutrient deprivation.

Polyphenol-rich peach (Prunus persica L.) by-product exerts a greater beneficial effect than dietary fiber-rich by-product on insulin resistance and hepatic steatosis in obese rats

Peach juice by-product (PJBP) can prevent complications of obesity due to its high content of polyphenols and dietary fiber. This study aimed to evaluate the effect of a polyphenol-rich (PR-PJBP) and a dietary fiber-rich (DFR-PJBP) peach juice by-product on insulin resistance and hepatic steatosis in obese rats. Animals were fed with high fat and fructose diet (HFF) and supplemented with PR-PJBP and DFR-PJBP for eighteen weeks. PR-PJBP produced the greatest hypoglycemic effect by improving insulin resistance, increasing hepatic glycogen content, and decreasing hepatic PEPCK activity. Likewise, PR-PJBP decreased serum triglycerides levels, ameliorating hepatic steatosis, decreasing hepatic expression of Srebp1 and Fasn, and increasing hepatic Cpt1 expression. These results suggest that polyphenols from PJBP exert a greater beneficial effect on obesity-related complications than dietary fiber. These effects were associated with caffeoylquinic, p-coumaroylquinic and 4-feruloylquinic acids, and kaempferol derivatives. PR-PJBP could be used as an ingredient for the elaboration of functional foods.