Enzyme Phosphoenolpyruvate Carboxykinase Research Articles

Carotid body denervation improves hyperglycemia in obese mice.

The carotid bodies (CBs) have been implicated in glucose abnormalities in obesity via elevation of activity of the sympathetic nervous system. Obesity-induced hypertension is mediated by insulin receptor (INSR) signaling and by leptin, which binds to the leptin receptor (LEPRb) in CB and activates transient receptor potential channel subfamily M member 7 (TRPM7). We hypothesize that in mice with diet-induced obesity, hyperglycemia, glucose intolerance, and insulin resistance will be attenuated by the CB denervation (carotid sinus nerve dissection, CSND) and by knockdown of Leprb, Trpm7, and Insr gene expression in CB. In series of experiments in 75 male diet-induced obese (DIO) mice, we performed either CSND (vs. sham) surgeries or shRNA-induced suppression of Leprb, Trpm7, or Insr gene expression in CB, followed by blood pressure telemetry, intraperitoneal glucose tolerance and insulin tolerance tests, and measurements of fasting plasma insulin, leptin, corticosterone, glucagon and free fatty acids (FFAs) levels, hepatic expression of gluconeogenesis enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G-6-Pase) mRNA and liver glycogen levels. CSND decreased blood pressure, fasting blood glucose levels and improved glucose tolerance without any effect on insulin resistance. CSND did not affect any hormone levels and gluconeogenesis enzymes, but increased liver glycogen level. Genetic knockdown of CB Leprb, Trpm7, and Insr had no effect on glucose metabolism. We conclude that CB contributes to hyperglycemia of obesity, probably by modulation of the glycogen-glucose equilibrium. Diabetogenic effects of obesity on CB in mice do not occur via activation of CB Leprb, Trpm7, and Insr.NEW & NOTEWORTHY This paper provides first evidence that carotid body denervation abolishes hypertension and improves fasting blood glucose levels and glucose tolerance in mice with diet-induced obesity. Furthermore, we have shown that this phenomenon is associated with increased liver glycogen content, whereas insulin sensitivity and enzymes of gluconeogenesis were not affected.

Enhanced glucose homeostasis via Clostridium symbiosum-mediated glucagon-like peptide 1 inhibition of hepatic gluconeogenesis in mid-intestinal bypass surgery.

The small intestine is known to play a crucial role in the development and remission of diabetes mellitus (DM). However, the exact mechanism by which mid-small intestinal bypass improves glucose metabolism in diabetic rats is not fully understood. To elucidate the mechanisms by which mid-small intestinal bypass improves glucose metabolism. Streptozotocin (STZ) was used to induce DM in Sprague-Dawley (SD) rats at a dose of 60 mg/kg. The rats were then randomly divided into two groups: The mid-small intestine bypass (MSIB) group and the sham group (underwent switch laparotomy). Following a 6-wk recovery period post-surgery, the rats underwent various assessments, including metabolic parameter testing, analysis of liver glycogen levels, measurement of key gluconeogenic enzyme activity, characterization of the gut microbiota composition, evaluation of hormone levels, determination of bile acid concentrations, and assessment of the expression of the intestinal receptors Takeda G protein-coupled receptor 5 and farnesoid X receptor. The MSIB group of rats demonstrated improved glucose metabolism and lipid metabolism, along with increased hepatic glycogen content. Furthermore, there was a decrease in the expression of the key gluconeogenic enzymes phosphoenolpyruvate carboxykinase 1 and glucose-6-phosphatase. Importantly, the MSIB group exhibited a substantial increase in the abundances of intestinal Lactobacillus, Clostridium symbiosum, Ruminococcus gnavus, and Bilophila. Moreover, higher levels of secondary bile acids, such as intestinal lithocholic acid, were observed in this group. Remarkably, the changes in the gut microbiota showed a significant correlation with the expression of key gluconeogenic enzymes and glucagon-like peptide 1 (GLP-1) at 6 wk postoperatively, highlighting their potential role in glucose regulation. These findings highlight the beneficial effects of mid-small intestine bypass on glucose metabolism and the associated modulation of the gut microbiota. The findings of this study demonstrate that the introduction of postoperative intestinal Clostridium symbiosum in the mid-small intestine contributes to the enhancement of glucose metabolism in nonobese diabetic rats. This improvement is attributed to the increased inhibition of hepatic gluconeogenesis mediated by GLP-1, resulting in a favorable modulation of glucose homeostasis.

In vitro, in vivo, and in silico evaluation of the glucocorticoid receptor antagonist activity of 3,6-dibromocarbazole.

3,6-Dibromocarbazole is a novel environmental contaminant which is currently detected in several environmental media worldwide. This work aims to investigate the anti-glucocorticoid potency and endocrine disrupting effects of 3,6-dibromocarbazole. In vitro experiments indicated that 3,6-dibromocarbazole possessed glucocorticoid receptor (GR) antagonistic activity and inhibited dexamethasone-induced GR nuclear translocation. 3,6-Dibromocarbazole reduced the expression levels of glucocorticoid responsive genes including glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), fatty acid synthase (FAS), and tyrosine aminotransferase (TAT), and further disrupted the protein expression of two key enzymes PEPCK and FAS in gluconeogenesis. In vivo experiments showed that 3,6-dibromocarbazole induced abnormal development of zebrafish embryos and disrupted the major neurohormones involved in activation of hypothalamic-pituitary-adrenocortical (HPA) axis in zebrafish larvae. The results of molecular docking and molecular dynamics simulation contributed to explain the antagonistic effect of 3,6-dibromocarbazole. Taken together, this work identified 3,6-dibromocarbazole as a GR antagonist, which might exert endocrine disrupting effects by interfering the pathway of gluconeogenesis.

Protein-Protein Interactions and Quantitative Phosphoproteomic Analysis Reveal Potential Mitochondrial Substrates of Protein Phosphatase 2A-B'ζ Holoenzyme.

Protein phosphatase 2A (PP2A) is a heterotrimeric conserved serine/threonine phosphatase complex that includes catalytic, scaffolding, and regulatory subunits. The 3 A subunits, 17 B subunits, and 5 C subunits that are encoded by the Arabidopsis genome allow 255 possible PP2A holoenzyme combinations. The regulatory subunits are crucial for substrate specificity and PP2A complex localization and are classified into the B, B', and B" non-related families in land plants. In Arabidopsis, the close homologs B'η, B'θ, B'γ, and B'ζ are further classified into a subfamily of B' called B'η. Previous studies have suggested that mitochondrial targeted PP2A subunits (B'ζ) play a role in energy metabolism and plant innate immunity. Potentially, the PP2A-B'ζ holoenzyme is involved in the regulation of the mitochondrial succinate/fumarate translocator, and it may affect the enzymes involved in energy metabolism. To investigate this hypothesis, the interactions between PP2A-B'ζ and the enzymes involved in the mitochondrial energy flow were investigated using bimolecular fluorescence complementation in tobacco and onion cells. Interactions were confirmed between the B'ζ subunit and the Krebs cycle proteins succinate/fumarate translocator (mSFC1), malate dehydrogenase (mMDH2), and aconitase (ACO3). Additional putative interacting candidates were deduced by comparing the enriched phosphoproteomes of wild type and B'ζ mutants: the mitochondrial regulator Arabidopsis pentatricopeptide repeat 6 (PPR6) and the two metabolic enzymes phosphoenolpyruvate carboxylase (PPC3) and phosphoenolpyruvate carboxykinase (PCK1). Overall, this study identifies potential PP2A substrates and highlights the role of PP2A in regulating energy metabolism in mitochondria.

Effects of Starvation and Refeeding on Glucose Metabolism and Immune Responses in Macrobrachium rosenbergii.

Starvation is a common challenge for aquatic animals in both natural and cultured environments. To investigate the effects of starvation and refeeding on glucose metabolism and immunity in Macrobrachium rosenbergii, prawns were starved for 14days and then refed for 7days. Results showed that both glucose and trehalose levels decreased significantly at the beginning of starvation, followed by a significant decrease in glycogen content in the hepatopancreas and muscle. Triglyceride and total protein reserves were also mobilized under starvation, with a slightly quicker response from triglycerides. The mRNA levels of glycolysis (glucokinase) and anabolism-related enzymes (glycogen branching enzyme, diacylglycerol acyltransferase, and transpeptidase) decreased during starvation, while gluconeogenic potential was induced, as indicated by up-regulated transcriptional levels of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase) and catabolism-related enzymes (glycogen debranching enzyme, adipose triglyceride lipase, and cathepsin B). Starvation also stimulated the expression of the crustacean hyperglycemic hormone and inhibited insulin-like peptide expression, indicating their potential role in glucose metabolism regulation. In addition, starvation increased the mRNA levels of superoxide dismutase and prophenoloxidase, indicating an influence on the immune system. After bacterial infection, starved prawns showed enhanced activity of non-specific immunological parameters and reduced mortality. Refeeding for 7days led to a recovery of physiological and biochemical indices and transcriptional levels of metabolism/immune-related genes. Our findings provide a better understanding of the mechanisms underlying energy utilization, metabolic adaptation, and immune response to starvation in M. rosenbergii.

C4 Equivalent Decarboxylation Competence in Tropical Orchids

Tropical xero-epiphytic orchids undergo a photosynthetic mechanism called ‘Crassulacean Acid Metabolism’, a modification of Calvin–Benson cycle. This anabolic process aids in the long-term retention of organically fixed CO2, imprinting them as active sequestrators and fixers of gaseous CO2. The malate decarboxylation competence of enzymes—phosphoenolpyruvate carboxykinase (PEPCK), Nicotinamide Adenine Dinucleotide malic enzyme (NAD-ME) and Nicotinamide Adenine Dinucleotide Phosphate malic enzyme (NADP-ME) in xero-epiphytic orchids that concentrate CO2 by employing RuBisCO to embark on a carboxylation route is defined in this paper. Physiological parameters like photosynthetic rates, and quantum yield, accompanied by histomorphometry were analysed. Our study indicated PEPCK as the dominant decarboxylating enzyme in tropical orchids. The efficiency of PEPCK was reinforced by two additional malic enzymes, which are dependent on cofactors, namely Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate. These results indicated the need to appraise the potential of tropical orchids as strategic plant contenders for CO2 triggered greenhouse effect mitigator.

Pyruvate Dehydrogenase Inhibition Leads to Decreased Glycolysis, Increased Reliance on Gluconeogenesis and Alternative Sources of Acetyl-CoA in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive disease characterized by poor outcomes and therapy resistance. Devimistat is a novel agent that inhibits pyruvate dehydrogenase complex (PDH). A phase III clinical trial in AML patients combining devimistat and chemotherapy was terminated for futility, suggesting AML cells were able to circumvent the metabolic inhibition of devimistat. The means by which AML cells resist PDH inhibition is unknown. AML cell lines treated with devimistat or deleted for the essential PDH subunit, PDHA, showed a decrease in glycolysis and decreased glucose uptake due to a reduction of the glucose transporter GLUT1 and hexokinase II. Both devimistat-treated and PDHA knockout cells displayed increased sensitivity to 2-deoxyglucose, demonstrating reliance on residual glycolysis. The rate limiting gluconeogenic enzyme phosphoenolpyruvate carboxykinase 2 (PCK2) was significantly upregulated in devimistat-treated cells, and its inhibition increased sensitivity to devimistat. The gluconeogenic amino acids glutamine and asparagine protected AML cells from devimistat. Non-glycolytic sources of acetyl-CoA were also important with fatty acid oxidation, ATP citrate lyase (ACLY) and acyl-CoA synthetase short chain family member 2 (ACSS2) contributing to resistance. Finally, devimistat reduced fatty acid synthase (FASN) activity. Taken together, this suggests that AML cells compensate for PDH and glycolysis inhibition by gluconeogenesis for maintenance of essential glycolytic intermediates and fatty acid oxidation, ACLY and ACSS2 for non-glycolytic production of acetyl-CoA. Strategies to target these escape pathways should be explored in AML.

Secoisolariciresinol Diglucoside (SDG) from flaxseed in the prevention and treatment of diabetes mellitus

This review focuses on the role of reactive oxygen species (ROS) on the development of type 1 and type 2 diabetes and its treatment with secoisolariciresinol diglucoside (SDG) isolated from flaxseed which is an antioxidant and suppresses phosphoenolpyruvate carboxykinase (PEPCK) gene expression, a ratelimiting enzyme in the gluconeogenesis in the liver. Role of ROS in the development of type 1 diabetes [diabetic prone Bio Breeding (BBdp) rats and streptozotocin-induced diabetic (STZ) rats and type 2 diabetes (Zucker diabetic fatty female rats, ZDF rats)] has been discussed. Oxidative stress has been assessed by measuring serum and pancreatic malondialdehyde (MDA), pancreatic chemiluminescence (pancreatic-CL) and oxygen radical producing activity of white blood cells (WBCCL). Diagnosis of diabetes was made by hyperglycaemia and glucosuria. Incidence of diabetes was 100 % in SDZ rats, 72 % in BBdp rats and 100 % in ZDF rats by the age of 72 days. Development of diabetes was associated with increases in the serum and pancreatic MDA, WBC-CL and pancreatic-CL and glycated haemoglobin (HbA1 c). SDG prevented the development of diabetes by 75 % in STZ rats, by 71 % in BBdp rats and by 20 % at 72 days of age in ZDF rats. However, 80 % of the rats which did not develop diabetes by 72 days of age, developed diabetes later on, suggesting that SDG treatment delays the development of diabetes in ZDF rats. Treatment with SDG decreased the levels of serum and pancreatic MDA, WBC-CL and pancreatic-CL. In conclusion, development of type 1 and type 2 diabetes is mediated through oxidative stress and the prevention or delay in the development of diabetes with SDG could be due to its antioxidant activity and its suppressant effect on PEPCK enzyme. Lignan complex which contains 34 % to 38 % of SDG is effective in lowering serum glucose and HbA1 c in type 2 diabetes in humans.

Exercise postconditioning reduces ischemic injury via suppression of cerebral gluconeogenesis in rats.

Pre-stroke exercise conditioning reduces neurovascular injury and improves functional outcomes after stroke. The goal of this study was to explore if post-stroke exercise conditioning (PostE) reduced brain injury and whether it was associated with the regulation of gluconeogenesis. Adult rats received 2h of middle cerebral artery (MCA) occlusion, followed by 24h of reperfusion. Treadmill activity was then initiated 24h after reperfusion for PostE. The severity of the brain damage was determined by infarct volume, apoptotic cell death, and neurological deficit at one and three days after reperfusion. We measured gluconeogenesis including oxaloacetate (OAA), phosphoenolpyruvate (PEP), pyruvic acid, lactate, ROS, and glucose via ELISA, as well as the location and expression of the key enzyme phosphoenolpyruvate carboxykinase (PCK)-1/2 via immunofluorescence. We also determined upstream pathways including forkhead transcription factor (FoxO1), p-FoxO1, 3-kinase (PI3K)/Akt, and p-PI3K/Akt via Western blot. Additionally, the cytoplasmic expression of p-FoxO1 was detected by immunofluorescence. Compared to non-exercise control, PostE (*p<.05) decreased brain infarct volumes, neurological deficits, and cell death at one and three days. PostE groups (*p<.05) saw increases in OAA and decreases in PEP, pyruvic acid, lactate, ROS, glucose levels, and tissue PCKs expression on both days. PCK-1/2 expressions were also significantly (*p<.05) suppressed by the exercise setting. Additionally, phosphorylated PI3K, AKT, and FoxO1 protein expression were significantly induced by PostE at one and three days (*p<.05). In this study, PostE reduced brain injury after stroke, in association with activated PI3K/AKT/FoxO1 signaling, and inhibited gluconeogenesis. These results suggest the involvement of FoxO1 regulation of gluconeogenesis underlying post-stroke neuroprotection.

PEPCK Gene for Enhanced Photosynthesis and Salinity Stress Tolerance in Rice: A Review

Phosphoenolpyruvate carboxykinase (PEPCK) is an enzyme of the lyase family utilized in the gluconeogenesis pathway in plants. It converts oxaloacetate into Phosphoenolpyruvate (PEP) and carbon dioxide. PEPCK acts as a prime decarboxylase cytosolic enzyme and may have exhibited a positive response against salinity (main abiotic stress) stress in certain plants. Transgenic plants (C3) with high-level expression of C4 enzymes PEPC or PEPCK can serve as a perfect example for increasing the crop photosynthetic capacity through genetic engineering. In this review, we have focused on the recent advances in the utilization of the PEPCK gene and its role in increasing photosynthesis as well as in abiotic stress tolerance.

The impact of vitamin A supplementation on thyroid function and insulin sensitivity: implication of deiodinases and phosphoenolpyruvate carboxykinase in male Wistar rats

PurposeVitamin A is an essential nutrient with vital biological functions. The present study investigated the effect of different doses of vitamin A palmitate at different time intervals on thyroid hormones and glycemic markers.MethodsMale rats were administrated vitamin A palmitate at different doses (0, 0.7, 1.5, 3, 6, and 12 mg/kg, oral) and samples were collected at different time intervals of 2, 4, and 6 weeks. The levels of vitamin A, thyroid hormones (T3, T4, and TSH), deiodinases (Dio1 and Dio3), glycemic markers (blood insulin and fasting glucose levels, HOMA IR and HOMA β), retinol-binding protein 4 (RBP4) and the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) were measured.ResultsThe findings demonstrated that long-term supplementation with high doses of vitamin A palmitate resulted in hypothyroidism (lower T3 and T4 levels and elevated TSH levels) as well as upregulation of Dio1 and Dio3 expression levels. This effect was associated with elevated glucose and insulin levels, enhanced HOMA IR, and decreased HOMA B index. In addition, prolonged vitamin A supplementation significantly increased RBP4 levels that upregulated the expression of PEPCK.ConclusionHigh doses of vitamin A supplementation increased the risk of hypothyroidism, modulated insulin sensitivity, and over a long period, increased the incidence of type 2 diabetes mellitus associated with oxidative stress and hepatitis.

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.

Carpachromene Ameliorates Insulin Resistance in HepG2 Cells via Modulating IR/IRS1/PI3k/Akt/GSK3/FoxO1 Pathway.

Insulin resistance contributes to several disorders including type 2 diabetes and cardiovascular diseases. Carpachromene is a natural active compound that inhibits α-glucosidase enzyme. The aim of the present study is to investigate the potential activity of carpachromene on glucose consumption, metabolism and insulin signalling in a HepG2 cells insulin resistant model. A HepG2 insulin resistant cell model (HepG2/IRM) was established. Cell viability assay of HepG2/IRM cells was performed after carpachromene/metformin treatment. Glucose concentration and glycogen content were determined. Western blot analysis of insulin receptor, IRS1, IRS2, PI3k, Akt, GSK3, FoxO1 proteins after carpachromene treatment was performed. Phosphoenolpyruvate carboxykinase (PEPCK) and hexokinase (HK) enzymes activity was also estimated. Viability of HepG2/IRM cells was over 90% after carpachromene treatment at concentrations 6.3, 10, and 20 µg/mL. Treatment of HepG2/IRM cells with carpachromene decreased glucose concentration in a concentration- and time-dependant manner. In addition, carpachromene increased glycogen content of HepG2/IRM cells. Moreover, carpachromene treatment of HepG2/IRM cells significantly increased the expression of phosphorylated/total ratios of IR, IRS1, PI3K, Akt, GSK3, and FoxO1 proteins. Furthermore, PEPCK enzyme activity was significantly decreased, and HK enzyme activity was significantly increased after carpachromene treatment. The present study examined, for the first time, the potential antidiabetic activity of carpachromene on a biochemical and molecular basis. It increased the expression ratio of insulin receptor and IRS1 which further phosphorylated/activated PI3K/Akt pathway and phosphorylated/inhibited GSK3 and FoxO1 proteins. Our findings revealed that carpachromene showed central molecular regulation of glucose metabolism and insulin signalling via IR/IRS1/ PI3K/Akt/GSK3/FoxO1 pathway.

Growth hormone promotes hepatic gluconeogenesis by enhancing BTG2\u2013YY1 signaling pathway

Growth hormone (GH) is one of the critical factors in maintaining glucose metabolism. B-cell translocation gene 2 (BTG2) and yin yang 1 (YY1) are key regulators of diverse metabolic processes. In this study, we investigated the link between GH and BTG2–YY1 signaling pathway in glucose metabolism. GH treatment elevated the expression of hepatic Btg2 and Yy1 in primary mouse hepatocytes and mouse livers. Glucose production in primary mouse hepatocytes and serum blood glucose levels were increased during GH exposure. Overexpression of hepatic Btg2 and Yy1 induced key gluconeogenic enzymes phosphoenolpyruvate carboxykinase 1 (PCK1) and glucose-6 phosphatase (G6PC) as well as glucose production in primary mouse hepatocytes, whereas this phenomenon was markedly diminished by knockdown of Btg2 and Yy1. Here, we identified the YY1-binding site on the Pck1 and G6pc gene promoters using reporter assays and point mutation analysis. The regulation of hepatic gluconeogenic genes induced by GH treatment was clearly linked with YY1 recruitment on gluconeogenic gene promoters. Overall, this study demonstrates that BTG2 and YY1 are novel regulators of GH-dependent regulation of hepatic gluconeogenic genes and glucose production. BTG2 and YY1 may be crucial therapeutic targets to intervene in metabolic dysfunction in response to the GH-dependent signaling pathway.

Effects of crustacean hyperglycaemic hormone RNA interference on regulation of glucose metabolism in Litopenaeus vannamei after ammonia-nitrogen exposure.

To unveil the adaptation of Litopenaeus vannamei to elevated ambient ammonia-N, crustacean hyperglycaemic hormone (CHH) was knocked down to investigate its function in glucose metabolism pathway under ammonia-N exposure. When CHH was silenced, haemolymph glucose increased significantly during 3-6 h, decreased significantly during 12-48 h and recovered to the control groups' level at 72 h. After CHH knock-down, dopamine (DA) contents reduced significantly during 3-24 h, which recovered after 48 h. Besides, the expressions of guanylyl cyclase (GC) and DA1R in the hepatopancreas decreased significantly, while DA4R increased significantly. Correspondingly, the contents of cyclic AMP (cAMP), cyclic GMP (cGMP) and diacylglycerol (DAG) and the expressions of protein kinase A (PKA), protein kinase G (PKG), AMP active protein kinase α (AMPKα) and AMPKγ were significantly down-regulated, while the levels of protein kinase C (PKC) and AMPKβ were significantly up-regulated. The expressions of cyclic AMP response element-binding protein (CREB) and GLUT2 decreased significantly, while GLUT1 increased significantly. Moreover, glycogen content, glycogen synthase and glycogen phosphorylase activities in hepatopancreas and muscle were significantly increased. Furthermore, the levels of key enzymes hexokinase, pyruvate kinase and phosphofructokinase in glycolysis (GLY), rate-limiting enzymes citrate synthase in tricarboxylic acid and critical enzymes phosphoenolpyruvate carboxykinase, fructose diphosphate and glucose-6-phosphatase in gluconeogenesis (GNG) were significantly decreased in hepatopancreas. These results suggest that CHH affects DA and then they affect their receptors to transmit glucose metabolism signals into the hepatopancreas of L. vannamei under ammonia-N stress. CHH acts on the cGMP-PKG-AMPKα-CREB pathway through GC, and CHH affects DA to influence cAMP-PKA-AMPKγ-CREB and DAG-PKC-AMPKβ-CREB pathways, thereby regulating GLUT, inhibiting glycogen metabolism and promoting GLY and GNG. This study contributes to further understand glucose metabolism mechanism of crustacean in response to environmental stress.

Gluconeogenic enzyme PCK1 deficiency promotes CHK2 O-GlcNAcylation and hepatocellular carcinoma growth upon glucose deprivation.

Although cancer cells are frequently faced with a nutrient- and oxygen-poor microenvironment, elevated hexosamine-biosynthesis pathway (HBP) activity and protein O-GlcNAcylation (a nutrient sensor) contribute to rapid growth of tumor and are emerging hallmarks of cancer. Inhibiting O-GlcNAcylation could be a promising anticancer strategy. The gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 (PCK1) is downregulated in hepatocellular carcinoma (HCC). However, little is known about the potential role of PCK1 in enhanced HBP activity and HCC carcinogenesis under glucose-limited conditions. In this study, PCK1 knockout markedly enhanced the global O-GlcNAcylation levels under low-glucose conditions. Mechanistically, metabolic reprogramming in PCK1-loss hepatoma cells led to oxaloacetate accumulation and increased de novo uridine triphosphate synthesis contributing to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. Meanwhile, deletion of PCK1 also resulted in AMPK-GFAT1 axis inactivation, promoting UDP-GlcNAc synthesis for elevated O-GlcNAcylation. Notably, lower expression of PCK1 promoted CHK2 threonine 378 O-GlcNAcylation, counteracting its stability and dimer formation, increasing CHK2-dependent Rb phosphorylation and HCC cell proliferation. Moreover, aminooxyacetic acid hemihydrochloride and 6-diazo-5-oxo-L-norleucine blocked HBP-mediated O-GlcNAcylation and suppressed tumor progression in liver-specific Pck1-knockout mice. We reveal a link between PCK1 depletion and hyper-O-GlcNAcylation that underlies HCC oncogenesis and suggest therapeutic targets for HCC that act by inhibiting O-GlcNAcylation.

Characterization of Anoectochilus roxburghii polysaccharide and its therapeutic effect on type 2 diabetic mice

Anoectochilus roxburghii is a traditional herb in China that can be potentially used to treat diabetes. A novel polysaccharide ARLP-W was isolated from Anoectochilus roxburghii by chromatography on DEAE-52 cellulose. Chemical analysis indicated that ARLP-W (8.1 × 104 Da) was mainly composed of mannose and glucose. The main linkages of glycosidic bonds of ARLP-W were β-1, 4-Manp and α-1, 4-Glcp. The terminal Glcp was connected to Manp-via O-3. RT-qPCR and western blotting analysis showed that ARLP-W caused a significant reduction in the levels of the key gluconeogenesis enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) in the liver. The results of the insulin resistance tests indicated that ARLP-W increased glucose absorption. These results indicate that ARLP-W has a good therapeutic effect on type 2 diabetes and can assist with further development and application treatment of diabetes.

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.

Lipocalin-2: a role in hepatic gluconeogenesis via AMP-activated protein kinase (AMPK).

Evidence is accumulating that lipocalin2 (LCN2) is implicated in insulin resistance and glucose homeostasis, but the underlying possible mechanisms remain unclear. This study is to investigate the possible linkage between LCN2 and AMP-activated protein kinase (AMPK) or forkhead transcription factor O1 (FoxO1), which influences insulin sensitivity and gluconeogenesis in liver. LCN2 knockout (LCN2KO) mice and wild-type littermates were used to evaluate the effect of LCN2 on insulin sensitivity and hepatic gluconeogenesis through pyruvate tolerance test (PTT), glucose tolerance test (ipGTT), insulin tolerance test (ITT), and hyperinsulinemic-euglycemic clamps, respectively. LCN2KO mice and WT mice in vivo, and in vitro HepG2 cells were co-transfected with adenoviral FoxO1-siRNA (Ad-FoxO1-siRNA) or adenovirus expressing constitutively active form of AMPK (Ad-CA-AMPK), or dominant negative adenovirus AMPK (Ad-DN-AMPK), the relative mRNA and protein levels of two key gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6P) were measured. Improved insulin sensitivity and inhibited gluconeogenesis in the LCN2KO mice were confirmed by pyruvate tolerance tests and hyperinsulinemic-euglycemic clamps. Nuclear FoxO1 and its downstream genes PEECK and G6P were decreased in the livers of the LCN2KO mice, and AMPK activity was stimulated and directly phosphorylated FoxO1. In vitro, AMPK activity was inhibited in HepG2 cells overexpressing LCN2 leading to a decrease in phosphorylated FoxO1 and an increase in nuclear FoxO1. The present study demonstrates that LCN2 regulates insulin sensitivity and glucose metabolism through inhibiting AMPK activity, and regulating FoxO1 and its downstream genes PEPCK/G6P, which regulate hepatic gluconeogenesis.

Phosphoenolpyruvate Carboxykinase (PCK) in the Brain Gluconeogenic Pathway Contributes to Oxidative and Lactic Injury After Stroke.

To demonstrate the role of the rate-limiting and ATP-dependent gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK) in oxidative and lactic stress and the effect of phenothiazine on PCK after stroke, a total of 168 adult male Sprague Dawley rats (3months old, 280-300g) underwent 2-h intraluminal middle cerebral artery occlusion (MCAO) and reperfusion for 6, 24, 48h, or 7days. Phenothiazine (chlorpromazine and promethazine (C+P)) (8mg/kg) and 3-mercaptopicolinic acid (3-MPA, a PCK inhibitor, 100μM) were administered at reperfusion onset. The effects of phosphoenolpyruvate, 3-MPA, or PCK knockdown were studied in neuronal cultures subjected to oxygen/glucose deprivation. Reactive oxygen species, lactate, phosphoenolpyruvate (PEP; a gluconeogenic product), mRNA, and protein of total PCK, PCK-1, and PCK-2 increased after MCAO and oxygen-glucose deprivation (OGD). Oxaloacetate (a gluconeogenic substrate) decreased, while PEP and glucose were increased, suggesting reactive gluconeogenesis. These changes were attenuated by phenothiazine, 3-MPA, or PCK shRNA. PCK-1 and -2 existed primarily in neurons, while the effects of ischemic stroke on the PCK expression were seen predominately in astrocytes. Thus, phenothiazine reduced infarction and oxidative/lactic stress by inhibiting PCKs, leading to functional recovery.