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Cutaneous leishmaniasis (CL) is a neglected public health problem in developing countries including Iran. CL is caused by different species of Leishmania parasites and results in morbidity and complications in patients with active lesions. Since protozoa are intracellular parasites, cellular immunity plays an essential role in controlling the infection. Cellular immunity is induced immediately after the infection and persists for many years after recovery. Studies over the past decades have identified the important role of CD4+ T cells and their T-helper subtypes in the immune response to CL. Finding antigens capable of producing Th1-dominant immune responses can play an effective role in preventing CL. This study examines the protective role of phosphoenolpyruvate carboxykinase (PEPCK), a critical enzyme for protozoan survival and reproduction. The potential of targeting PEPCK for the development of novel prevention strategies against Chagas disease is explored. Detailed findings presented herein underscore the critical role of PEPCK as an immunogenic antigen in parasite metabolism, gluconeogenesis, and energy homeostasis. Notably, the elimination of PEPCK alters parasite metabolic activity and attenuates Leishmania pathogenicity. These results position PEPCK as a promising vaccine candidate and therapeutic target for CL, pending further validation. [ABSTRACT FROM AUTHOR]

Hyperglycemia affects approximately 16.7% of pregnancies worldwide, with gestational diabetes mellitus (GDM) accounting for nearly 84% of these cases. GDM, characterized by glucose intolerance during pregnancy, presents significant health risks. Rosmarinus officinalis (RO) and Centella asiatica (CA) are known for their antidiabetic and antioxidant properties, including the ability to enhance insulin secretion and inhibit phosphoenolpyruvate carboxykinase (PEPCK) expression in the gluconeogenesis pathway. This study aims to assess the impact of a nanoemulsion of RO and CA combination (ROCA) on heart rate, superoxide dismutase (SOD), nuclear factor erythroid 2-related factor 2a (Nrf2a), sirtuin 1 (SIRT-1), and tyrosine hydroxylase (TH) expression in zebrafish larvae model of GDM. GDM was induced by exposure of zebrafish embryo to a 3% glucose solution in an embryonic medium. The GDM phenotype was confirmed by elevated PEPCK expression as a hyperglycemia marker. GDM zebrafish was administered RO-CA nanoemulsion at concentrations of 2.5, 5, and 10 μg/mL.from 2 hpf to 72 hpf. Heart rate was monitored using stereoscopic imaging connected to a camera, while expression levels of PEPCK, Nrf2a, SOD, SIRT-1, and TH were quantified using reverse transcriptase polymerase chain reaction (RT-PCR). Results revealed a significant decrease in PEPCK expression in the treatment groups compared to the glucose untreated group. Notably, the nanoemulsion maintained heart rate frequency and upregulated Nrf2a, SOD, SIRT-1, and TH expression, particularly at a concentration of 2.5 μg/mL. Overall, these findings suggest that the RO-CA nanoemulsion exhibits enhanced antioxidant activity and holds promise as a potential cardioprotective agent in GDM. [ABSTRACT FROM AUTHOR]

Recently, the increase in marine temperatures has become an important global marine environmental issue. The ability of energy supply in marine animals plays a crucial role in avoiding the stress of elevated temperatures. The investigation into anaerobic metabolism, an essential mechanism for regulating energy provision under heat stress, is limited in mollusks. In this study, key enzymes of four anaerobic metabolic pathways were identified in the genome of scallop Chlamys farreri, respectively including five opine dehydrogenases (CfOpDHs), two aspartate aminotransferases (CfASTs) divided into cytoplasmic (CfAST1) and mitochondrial subtype (CfAST2), and two phosphoenolpyruvate carboxykinases (CfPEPCKs) divided into a primitive type (CfPEPCK2) and a cytoplasmic subtype (CfPEPCK1). It was surprising that lactate dehydrogenase (LDH), a key enzyme in the anaerobic metabolism of the glucose–lactate pathway in vertebrates, was absent in the genome of scallops. Phylogenetic analysis verified that CfOpDHs clustered according to the phylogenetic relationships of the organisms rather than substrate specificity. Furthermore, CfOpDHs, CfASTs, and CfPEPCKs displayed distinct expression patterns throughout the developmental process and showed a prominent expression in muscle, foot, kidney, male gonad, and ganglia tissues. Notably, CfASTs displayed the highest level of expression among these genes during the developmental process and in adult tissues. Under heat stress, the expression of CfASTs exhibited a general downregulation trend in the six tissues examined. The expression of CfOpDHs also displayed a downregulation trend in most tissues, except CfOpDH1/3 in striated muscle showing significant up-regulation at some time points. Remarkably, CfPEPCK1 was significantly upregulated in all six tested tissues at almost all time points. Therefore, we speculated that the glucose–succinate pathway, catalyzed by CfPEPCK1, serves as the primary anaerobic metabolic pathway in mollusks experiencing heat stress, with CfOpDH3 catalyzing the glucose–opine pathway in striated muscle as supplementary. Additionally, the high and stable expression level of CfASTs is crucial for the maintenance of the essential functions of aspartate aminotransferase (AST). This study provides a comprehensive and systematic analysis of the key enzymes involved in anaerobic metabolism pathways, which holds significant importance in understanding the mechanism of energy supply in mollusks. [ABSTRACT FROM AUTHOR]

Abstract Background In metabolic stress, the cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) enzyme is involved in energy production through the gluconeogenesis pathway. PEPCK-C deficiency is a rare childhood-onset autosomal recessive metabolic disease caused by PCK1 genetic defects. Previous studies showed a broad clinical spectrum ranging from asymptomatic to recurrent hypoglycemia with/without lactic acidosis, encephalopathy, seizures, and liver failure. Results In this article, we discuss the occurrence of PEPCK-C deficiency in four families from the United Arab Emirates and Oman. All patients presented with unexplained hypoglycemia as a common feature. Two out of the seven patients presented with episodes of encephalopathy that resulted in seizures and neuroregression leading to global developmental delay and one patient had a neonatal presentation. Observed biochemical abnormalities include elevated lactate, transaminases, and tricarboxylic acid cycle metabolites in most patients. Elevated creatine kinase was documented in two patients. Whole exome sequencing revealed two novel (c.574T > C, and c.1268 C > T) and a previously reported splice site (c.961 + 1G > A) PCK1 variant in the affected families. Conclusion Patients become vulnerable during intercurrent illness; thus, prevention and prompt reversal of a catabolic state are crucial to avoid irreversible brain damage. This report will help to expand the clinical understanding of this rare disease and recommends screening for PEPCK-C deficiency in unexplained hypoglycemia.

Background: In metabolic stress, the cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) enzyme is involved in energy production through the gluconeogenesis pathway. PEPCK-C deficiency is a rare childhood-onset autosomal recessive metabolic disease caused by PCK1 genetic defects. Previous studies showed a broad clinical spectrum ranging from asymptomatic to recurrent hypoglycemia with/without lactic acidosis, encephalopathy, seizures, and liver failure. Results: In this article, we discuss the occurrence of PEPCK-C deficiency in four families from the United Arab Emirates and Oman. All patients presented with unexplained hypoglycemia as a common feature. Two out of the seven patients presented with episodes of encephalopathy that resulted in seizures and neuroregression leading to global developmental delay and one patient had a neonatal presentation. Observed biochemical abnormalities include elevated lactate, transaminases, and tricarboxylic acid cycle metabolites in most patients. Elevated creatine kinase was documented in two patients. Whole exome sequencing revealed two novel (c.574T > C, and c.1268 C > T) and a previously reported splice site (c.961 + 1G > A) PCK1 variant in the affected families. Conclusion: Patients become vulnerable during intercurrent illness; thus, prevention and prompt reversal of a catabolic state are crucial to avoid irreversible brain damage. This report will help to expand the clinical understanding of this rare disease and recommends screening for PEPCK-C deficiency in unexplained hypoglycemia. [ABSTRACT FROM AUTHOR]

Hepatic dysfunction is commonly observed in subjects with hyperthyroidism. Hepassocin is a hepatokine playing an important role in metabolic diseases and exhibiting a hepatic protective effect. Nevertheless, the relationship between hepassocin and hyperthyroidism was still unknown. In the present study, a total of 36 subjects with Graves' disease were enrolled, and we found that the alanine aminotransferase (ALT) levels were significantly decreased in parallel with the decrement in serum hepassocin concentrations at 6 months after standard treatment for hyperthyroidism. In addition, HepG2 cell line was used to investigate the role of hepassocin in hyperthyroidism-induced hepatic dysfunction. Treatment of hepassocin recombinant protein in HepG2 cells dose-dependently decreased triiodothyronine (T3)-induced ALT and aspartate aminotransferase (AST) elevation. Moreover, hepassocin significantly increased the expression of phosphoenolpyruvate carboxykinase (PEPCK) in a dose-dependent manner. Deletion of hepassocin in HepG2 cells reversed the effects of T3 on PEPCK expressions. Furthermore, we found that T3 increased the expression of hepassocin through a hepatocyte nuclear factor 1α-dependent pathway. Taken together, these results indicated a compensatory increase in serum hepassocin might have a protective role in hyperthyroidism-induced hepatic dysfunction. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is a major cause of human death. Mortality is primarily due to metastatic organ colonization, with the liver being the main organ affected. We modeled metastatic CRC (mCRC) liver colonization using patient-derived primary and metastatic tumor xenografts (PDX). Such PDX modeling predicted patient survival outcomes. In vivo selection of multiple PDXs for enhanced metastatic colonization capacity upregulated the gluconeogenic enzyme PCK1, which enhanced liver metastatic growth by driving pyrimidine nucleotide biosynthesis under hypoxia. Consistently, highly metastatic tumors upregulated multiple pyrimidine biosynthesis intermediary metabolites. Therapeutic inhibition of the pyrimidine biosynthetic enzyme DHODH with leflunomide substantially impaired CRC liver metastatic colonization and hypoxic growth. Our findings provide a potential mechanistic basis for the epidemiologic association of anti-gluconeogenic drugs with improved CRC metastasis outcomes, reveal the exploitation of a gluconeogenesis enzyme for pyrimidine biosynthesis under hypoxia, and implicate DHODH and PCK1 as metabolic therapeutic targets in CRC metastatic progression.

Abstract Background Microbial production of naringenin has received much attention owing to its pharmaceutical applicability and potential as a key molecular scaffold for various flavonoids. In the microbial fermentation, a cheap and abundant feedstock is required to achieve an economically feasible bioprocess. From this perspective, utilizing acetate for naringenin production could be an effective strategy, with the advantages of both low-cost and abundant feedstock. For the efficient production of naringenin using acetate, identification of the appropriate regulatory node of carbon flux in the biosynthesis of naringenin from acetate would be important. While acetyl-CoA is a key precursor for naringenin production, carbon flux between the TCA cycle and anaplerosis is effectively regulated at the isocitrate node through glyoxylate shunt in acetate metabolism. Accordingly, appropriate rerouting of TCA cycle intermediates from anaplerosis into naringenin biosynthesis via acetyl-CoA replenishment would be required. Results This study identified the isocitrate and oxaloacetate (OAA) nodes as key regulatory nodes for the naringenin production using acetate. Precise rerouting at the OAA node for enhanced acetyl-CoA was conducted, avoiding extensive loss of OAA by fine-tuning the expression of pckA (encoding phosphoenolpyruvate carboxykinase) with flux redistribution between naringenin biosynthesis and cell growth at the isocitrate node. Consequently, the flux-optimized strain exhibited a significant increase in naringenin production, a 27.2-fold increase (with a 38.3-fold increase of naringenin yield on acetate) over that by the unoptimized strain, producing 97.02 mg/L naringenin with 21.02 mg naringenin/g acetate, which is a competitive result against those in previous studies on conventional substrates, such as glucose. Conclusions Collectively, we demonstrated efficient flux rerouting for maximum naringenin production from acetate in E. coli. This study was the first attempt of naringenin production from acetate and suggested the potential of biosynthesis of various flavonoids derived from naringenin using acetate.

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. [ABSTRACT FROM AUTHOR]

Metabolic reprogramming is commonly accompanied by alterations in the expression of metabolic enzymes. These metabolic enzymes not only catalyze the intracellular metabolic reaction, but also participate in a series of molecular events to regulate tumor initiation and development. Thus, these enzymes may act as promising therapeutic targets for tumor management. Phosphoenolpyruvate carboxykinases (PCKs) are the key enzymes involved in gluconeogenesis, which mediates the conversion of oxaloacetate into phosphoenolpyruvate. Two isoforms of PCK, namely cytosolic PCK1 and mitochondrial PCK2, has been found. PCK not only participates in the metabolic adaptation, but also regulates immune response and signaling pathways for tumor progression. In this review, we discussed the regulatory mechanisms of PCKs expression including transcription and post-translational modification. We also summarized the function of PCKs in tumor progression in different cellular contexts and explores its role in developing promising therapeutic opportunities.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible reaction of decarboxylation and phosphorylation of oxaloacetate (OAA) to generate phosphoenolpyruvate (PEP) and CO2 playing mainly a gluconeogenic role in green algae. We found two PEPCK isoforms in Chlamydomonas reinhardtii and we cloned, purified and characterised both enzymes. ChlrePEPCK1 is more active as decarboxylase than ChlrePEPCK2. ChlrePEPCK1 is hexameric and its activity is affected by citrate, phenylalanine and malate, while ChlrePEPCK2 is monomeric and it is regulated by citrate, phenylalanine and glutamine. We postulate that the two PEPCK isoforms found originate from alternative splicing of the gene or regulated proteolysis of the enzyme. The presence of these two isoforms would be part of a mechanism to finely regulate the biological activity of PEPCKs. [ABSTRACT FROM AUTHOR]

Phosphoenolpyruvate carboxykinase (PEPCK or PCK) catalyzes the first rate-limiting step in hepatic gluconeogenesis pathway to maintain blood glucose levels. Mammalian cells express two PCK genes, encoding for a cytoplasmic (PCPEK-C or PCK1) and a mitochondrial (PEPCK-M or PCK2) isoforms, respectively. Increased expressions of both PCK genes are found in cancer of several organs, including colon, lung, and skin, and linked to increased anabolic metabolism and cell proliferation. Here, we report that the expressions of both PCK1 and PCK2 genes are downregulated in primary hepatocellular carcinoma (HCC) and low PCK expression was associated with poor prognosis in patients with HCC. Forced expression of either PCK1 or PCK2 in liver cancer cell lines results in severe apoptosis under the condition of glucose deprivation and suppressed liver tumorigenesis in mice. Mechanistically, we show that the pro-apoptotic effect of PCK1 requires its catalytic activity. We demonstrate that forced PCK1 expression in glucose-starved liver cancer cells induced TCA cataplerosis, leading to energy crisis and oxidative stress. Replenishing TCA intermediate α-ketoglutarate or inhibition of reactive oxygen species production blocked the cell death caused by PCK expression. Taken together, our data reveal that PCK1 is detrimental to malignant hepatocytes and suggest activating PCK1 expression as a potential treatment strategy for patients with HCC.

Hepatic dysfunction is commonly observed in subjects with hyperthyroidism. Hepassocin is a hepatokine playing an important role in metabolic diseases and exhibiting a hepatic protective effect. Nevertheless, the relationship between hepassocin and hyperthyroidism was still unknown. In the present study, a total of 36 subjects with Graves’ disease were enrolled, and we found that the alanine aminotransferase (ALT) levels were significantly decreased in parallel with the decrement in serum hepassocin concentrations at 6 months after standard treatment for hyperthyroidism. In addition, HepG2 cell line was used to investigate the role of hepassocin in hyperthyroidism-induced hepatic dysfunction. Treatment of hepassocin recombinant protein in HepG2 cells dose-dependently decreased triiodothyronine (T3)-induced ALT and aspartate aminotransferase (AST) elevation. Moreover, hepassocin significantly increased the expression of phosphoenolpyruvate carboxykinase (PEPCK) in a dose-dependent manner. Deletion of hepassocin in HepG2 cells reversed the effects of T3 on PEPCK expressions. Furthermore, we found that T3 increased the expression of hepassocin through a hepatocyte nuclear factor 1α-dependent pathway. Taken together, these results indicated a compensatory increase in serum hepassocin might have a protective role in hyperthyroidism-induced hepatic dysfunction.

The yeast Komagataella phaffii (a.k.a. Pichia pastoris) harbours a unique glutamate utilization pathway in which the cytosolic enzymes glutamate dehydrogenase 2 (GDH2), aspartate aminotransferase 2 (AAT2) and phosphoenolpyruvate carboxykinase (PEPCK) catalyze the sequential conversion of glutamate to α‐ketoglutarate, oxaloacetate and phosphoenolpyruvate respectively. GDH2 and PEPCK are essential for glutamate catabolism. Their synthesis is induced by autophagy during carbon starvation and are essential for cell survival. Here, we demonstrate that GDH2 and PEPCK reciprocally regulate each other's protein levels during glutamate catabolism such that GDH2 is downregulated in Δpepck and PEPCK is downregulated in Δgdh2. We further demonstrate that sequential conversion of glutamate to α‐ketoglutarate and oxaloacetate by GDH2 and AAT2, respectively, is essential for PEPCK synthesis in cells metabolizing glutamate. Our studies indicate that translation of GDH2 mRNA is induced by glutamate while oxaloacetate derived from glutamate is likely to be the inducer of PEPCK mRNA translation during glutamate catabolism. Thus, GDH2‐ and PEPCK‐catalyzed reactions are essential for ATP generation and gluconeogenesis respectively during carbon starvation and glutamate catabolism in K. phaffii. We conclude that K. phaffii harbours a unique translational regulatory circuit in which substrates of GDH2 and PEPCK act as inducers of their synthesis, a phenomenon not reported in any yeast species. Take Away: GDH2 and PEPCK reciprocally regulate each other's protein levels in Komagataella phaffiiGlutamate regulates the translation of GDH2 mRNA during glutamate metabolismOxaloacetate regulates the translation of PEPCK mRNA during glutamate metabolismThus, GDH2 and PEPCK synthesis is induced by their own substrates in K. phaffii [ABSTRACT FROM AUTHOR]

D-Pantothenic acid (DPA), also known as vitamin B5 is associated with several biological functions and its deficiency causes metabolic and energetic disorders in humans. Fortification of foods with DPA is the viable option to address this risk. DPA biological production route employs pantoate-β-alanine ligase (PBL) as the key enzyme, which avoids the tedious and time-consuming optical resolution process. The selection of an efficient PBL enzyme is vital for the biological production of DPA. In this study, the panC gene encoding PBL from Escherichia coli, Bacillus megaterium, Corynebacterium glutamicum and Bacillus subtilis was expressed in B. megaterium. B. subtilis derived panC exhibited high PBL activity 61.62 ± 2.15 U/mL. Co-expression of phosphoenolpyruvate carboxykinase (pckA) did not improve the DPA production in B. megaterium. Biocatalytic fed-batch fermentation with externally supplemented precursor substrates (D-pantoic acid and β-alanine) improved DPA titer to 45.56 ± 0.53 g/L. Daily dietary requirements of DPA for different age groups (including babies, small children, athletes and elderly people) is steadily increasing and the improved DPA production addressed in this study offers advantage for its application in fortification of food products meeting the emerging nutritional demand. [ABSTRACT FROM AUTHOR]

Abstract Background Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is expressed in all cancer types examined and in neuroprogenitor cells. The gene is upregulated by amino acid limitation and ER-stress in an ATF4-dependent manner, and its activity modulates the PEP/Ca2+ signaling axis, providing clear arguments for a functional relationship with metabolic adaptations for cell survival. Despite its potential relevance to cancer metabolism, the mechanisms responsible for its pro-survival activity have not been completely elucidated. Methods [U-13C]glutamine and [U-13C]glucose labeling of glycolytic and TCA cycle intermediates and their anabolic end-products was evaluated quantitatively using LC/MS and GC/MS in conditions of abundant glucose and glucose limitation in loss-of-function (shRNA) and gain-of-function (lentiviral constitutive overexpression) HeLa cervix carcinoma cell models. Cell viability was assessed in conjunction with various glucose concentrations and in xenografts in vivo. Results PEPCK-M levels linearly correlated with [U-13C]glutamine label abundance in most glycolytic and TCA cycle intermediate pools under nutritional stress. In particular, serine, glycine, and proline metabolism, and the anabolic potential of the cell, were sensitive to PEPCK-M activity. Therefore, cell viability defects could be rescued by supplementing with an excess of those amino acids. PEPCK-M silenced or inhibited cells in the presence of abundant glucose showed limited growth secondary to TCA cycle blockade and increased ROS. In limiting glucose conditions, downregulation of PKC-ζ tumor suppressor has been shown to enhance survival. Consistently, HeLa cells also sustained a survival advantage when PKC-ζ tumor suppressor was downregulated using shRNA, but this advantage was abolished in the absence of PEPCK-M, as its inhibition restores cell growth to control levels. The relationship between these two pathways is also highlighted by the anti-correlation observed between PEPCK-M and PKC-ζ protein levels in all clones tested, suggesting co-regulation in the absence of glucose. Finally, PEPCK-M loss negatively impacted on anchorage-independent colony formation and xenograft growth in vivo. Conclusions All in all, our data suggest that PEPCK-M might participate in the mechanisms to regulate proteostasis in the anabolic and stalling phases of tumor growth. We provide molecular clues into the clinical relevance of PEPCK-M as a mechanism of evasion of cancer cells in conditions of nutrient stress.

Objective To investigate the role of mammalian sterile line 20-like kinas 1(MST1) and its regulatory mechanism in hepatic gluconeogenesis of mouse. Methods The mRNA expression of MST1 in the liver of db/db mice and db/m mice was detected by RT-qPCR. MST1 was overexpressed with adenovirus to detect glucose output capacity in mouse primary hepatocytes. Luciferase reporter assay was adopted to detect the effect of MST1 on phosphoenolpyruvate carboxykinase (PEPCK) promoter activity in HepG2 cells. Forkhead transcription factor O1 (FOXO1) was knocked down by interfering adenovirus in primary mouse liver cells to detect the effect of MST1 overexpression on the mRNA levels of PEPCK. Results The mRNA level of MST1 was significantly upregulated in the liver of db/db mice (P＜0.001). MST1 overexpression enhanced glucose output in mouse primary hepatocytes (P＜0.05). In HepG2 cells, MST1 overexpression increased the mRNA of PEPCK (P＜0.001) and the activity of PEPCK promoter (P＜0.01). Furthermore, MST1-induced increase in PEPCK mRNA was abolished when FOXO1 was knocked down by interfering adenovirus. Conclusions MST1 promotes hepatic gluconeogenesis by regulating the mRNA expression of PEPCK dependent on FOXO1.

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

The feasibility of hyperpolarized [2- 13 C, 3- 2 H 3 ]pyruvate for probing gluconeogenesis in vivo was investigated in this study. Whereas hyperpolarized [1- 13 C]pyruvate has clear access to metabolic pathways that convert pyruvate to lactate, alanine, and bicarbonate, its utility for assessing pyruvate carboxylation and gluconeogenesis has been limited by technical challenges, including spectral overlap and an obscure enzymatic step that decarboxylates the labeled carbon. To achieve unambiguous detection of gluconeogenic products, the carbonyl carbon in pyruvate was labeled with 13 C. To prolong the T 1 relaxation time, [2- 13 C, 3- 2 H 3 ]pyruvate was synthesized and dissolved with D 2 O after dynamic nuclear polarization. The T 1 of [2- 13 C, 3- 2 H 3 ]pyruvate in D 2 O could be improved by 76.9% (79.6 s at 1 T and 74.5 s at 3 T) as compared to [2- 13 C]pyruvate in water. Hyperpolarized [2- 13 C, 3- 2 H 3 ]pyruvate with D 2 O dissolution was applied to rat livers in vivo under normal feeding and fasting conditions. A gluconeogenic product, [2- 13 C]phosphoenolpyruvate, was observed at 149.9 ppm from fasted rats only, highlighting the utility of [2- 13 ]pyruvate in detecting key gluconeogenic enzyme activities such as pyruvate carboxylase and phosphoenolpyruvate carboxykinase 2 H 3 ]pyruvate in detecting key gluconeogenic enzyme activities such as pyruvate carboxylase and phosphoenolpyruvate carboxykinase in vivo .

Citrate is one of the most important metabolites determining the flavour of citrus fruit. It has been reported that nitrogen supply may have an impact on acid level of fruit. Here, the relationship between nitrogen metabolism and citrate catabolism was studied in pumelo juice sacs. Differences in metabolites, gene expression and flux distributions were analyzed in juice sacs incubated in medium with and without NH 4 +. Compared with those incubated with NH 4 +, juice sacs under nitrogen deficiency exhibited enhanced flux through phosphoenolpyruvate carboxykinase (PEPCK) and accelerated consumption of citrate, while the other two TCA cycle efflux points, through malic enzyme (ME) and glutamate dehydrogenase (GDH), were both repressed. Consistent with the estimated fluxes, the expression of PEPCK1 was upregulated under nitrogen deficiency, while that of GDH1 , GDH2 , NAD-ME1 and NADP-ME2 were all repressed. Thus, we propose that PEPCK1 contributes to citrate degradation under nitrogen limitation. • Metabolism in pomelo juice sacs was analyzed systematically and quantitatively with stoichiometric metabolic flux analysis. • Nitrogen deficiency accelerated decrease in citrate content of juice sacs. • Nitrogen deficiency enhanced reaction of PEPCK and suppressed other TCA cycle exporting reactions. • CitPEPCK1 gene expression was activated under different conditions where inorganic nitrogen is absent. [ABSTRACT FROM AUTHOR]

In order to better study the structure and function of phosphoenolpyruvate carboxylase, the phosphoenolpyruvate carboxylase gene is cloned and expressed by PCR (polymerase chain reaction), double enzyme digestion and cell transformation. The results show that the new phosphoenolpyruvate carboxylase gene of Escherichia coli is successfully amplified and cloned into the prokaryotic expression vector pET-30a, and then introduced into Escherichia coli BL21 expression system. The phosphoenolpyruvate carboxylase of Escherichia coli is successfully induced by IPTG. The phosphoenolpyruvate carboxylase can be efficiently expressed by using the proposed method, which provides preparation for further scale expression, purification and application.

The aim of this study was to explore the possible mechanisms of Ficus carica leaf (FCL) extract in suppressing hepatic gluconeogenesis in diabetic mice. Diabetic mice (streptozotocin-induced) received 1 g/kg of FCL extract twice a day for 6 weeks. Fasting blood glucose levels were measured and a 2-h oral glucose tolerance test was conducted. AMP-activated protein kinase (AMPK), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), and peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) expression was examined. HepG2 hepatocytes were treated with FCL extract and an AMPK inhibitor (compound C) or agonist (AICAR), and PEPCK, G6pase, PGC-1α, AMPK, forkhead transcription factor O1 (FOXO1), and hepatic nuclear factor 4α (HNF4α) expression was determined. The results showed that FCL extract inhibited the expression of PEPCK and G6Pase in the liver of diabetic mice and HepG2 hepatocytes. FCL extract activated AMPK and decreased PGC-1α, HNF4α, and FOXO1 expression. The AMPK inhibitor attenuated those effects through inhibiting gluconeogenesis, while the AMPK agonist partially enhanced gluconeogenesis. In conclusion, FCL extract inhibits hepatic gluconeogenesis via activation of AMPK and down-regulation of gluconeogenic enzymes.

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.

Melatonin plays physiological roles in various critical processes, including circadian rhythms, oxidative stress defenses, anti‐inflammation responses, and immunity; however, the current understanding of the role of melatonin in hepatic glucose metabolism is limited. In this study, we examined whether melatonin affects gene expression of the key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK). We found that melatonin treatment increased PEPCK mRNA levels in rat highly differentiated hepatoma (H4IIE) cells and primary cultured hepatocytes. In addition, we found that melatonin induction was synergistically enhanced by dexamethasone, whereas it was dominantly inhibited by insulin. We also report that the effect of melatonin was blocked by inhibitors of mitogen‐activated protein kinase/extracellular signal‐regulated protein kinase (MAPK/ERK), RNA polymerase II, and protein synthesis. Furthermore, the phosphorylated (active) forms of ERK1 and ERK2 (ERK1/2) increased 15 min after melatonin treatment. We performed luciferase reporter assays to show that melatonin specifically stimulated promoter activity of the PEPCK gene. Additional reporter analysis using 5′‐deleted constructs revealed that the regulatory regions responsive to melatonin mapped to two nucleotide regions, one between −467 and −398 nucleotides and the other between −128 and +69 nucleotides, of the rat PEPCK gene. Thus, we conclude that melatonin induces PEPCK gene expression via the ERK1/2 pathway at the transcriptional level, and that induction requires de novo protein synthesis. [ABSTRACT FROM AUTHOR]

Ficus deltoidea var. deltoidea Jack (FD) is a well-known plant used in Malay folklore medicine to lower blood glucose in diabetic patients. For further research of the antihyperglycemic mechanisms, the protein tyrosine phosphatase 1B (PTP1B)-inhibitory effect of FD was analysed both in vitro and in vivo. To optimise a method for FD extraction, water, 50, 70, 80, 90 and 95 % ethanol extracts were prepared and determined for their total phenolic and triterpene contents, and PTP1B-inhibition capacity. Among the tested extracts, 70 % ethanol FD extract showed a significant PTP1B inhibition (92·0 % inhibition at 200 µg/ml) and high phenolic and triterpene contents. A bioassay-guided fractionation of the 70 % ethanol extract led to the isolation of a new triterpene (3β,11β-dihydroxyolean-12-en-23-oic acid; F3) along with six known compounds. In vivo, 4 weeks’ administration of 70 % ethanol FD extract (125, 250 and 500 mg/kg/d) to streptozotocin–nicotinamide-induced type 2 diabetic rats reversed the abnormal changes of blood glucose, insulin, total Hb, GLUT2, lipid profile, and oxidative stress in liver and pancreas. Moreover, FD reduced the mRNA expression of the key gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and glucose 6-phosphatase) and restored insulin receptor and GLUT2 encoding gene (Slc2a2) expression. In addition, FD significantly down-regulated the hepatic PTP1B gene expression. These results revealed that FD could potentially improve insulin sensitivity, suppress hepatic glucose output and enhance glucose uptake in type 2 diabetes mellitus through down-regulation of PTP1B. Together, our findings give scientific evidence for the traditional use of FD as an antidiabetic agent.

Abstract Objective Global climate change has resulted in the decline of health and condition of various coral reefs worldwide. Here, we describe expression profiles of Acropora muricata collected during opposing seasons in Otsuki, Kochi, Japan to define the capacity of corals to cope with changing environmental conditions. Coral communities in Otsuki experience large temperature fluctuations between the winter (~ 16 °C) and summer (~ 27 °C). Results Coral nubbins that were collected in the summer showed no change in photochemical efficiency when exposed to thermal or cold stress, while winter samples showed a decrease in photochemical health when subjected to thermal stress. Under cold stress, corals that were collected in the summer showed an up-regulation of actin-related protein and serine/threonine protein kinase, while corals collected during the winter did not show any cellular stress. On the other hand, under thermal stress, the most notable change was the up-regulation of phosphoenolpyruvate carboxykinase in corals that were collected during the winter season. Our observations in the differential genes expressed under temperature-derived stress suggest that A. muricata from Kochi may maintain physiological resilience due to the frequently encountered environmental stress, and this may play a role in the coral’s thermal tolerance.

Copyright of China Tropical Medicine is the property of China Tropical Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

On the basis of our previous studies of microbial L-valine production under oxygen deprivation, we developed isobutanol-producing Corynebacterium glutamicum strains. The artificial isobutanol synthesis pathway was composed of the first three steps of the L-valine synthesis pathway; and the subsequent Ehrlich Pathway: pyruvate was converted to 2-ketoisovalerate in the former reactions; and the 2-keto acid was decarboxylated into isobutyraldehyde, and subsequently reduced into isobutanol in the latter reactions. Although there exists redox cofactor imbalance in the overall reactions, i.e., NADH is generated via glycolysis whereas NADPH is required to synthesize isobutanol, it was resolved by taking advantage of the NAD-preferring mutant acetohydroxy acid isomeroreductase encoded by ilvC TM and the NAD-specific alcohol dehydrogenase encoded by adhA. Each enzyme activity to synthesize isobutanol was finely tuned by using two kinds of lac promoter derivatives. Efficient suppression of succinate by-production and improvement of isobutanol yield resulted from inactivation of pckA , which encodes phosphoenolpyruvate carboxykinase, whereas glucose consumption and isobutanol production rates decreased because of the elevated intracellular NADH/NAD+ ratio. On the other hand, introduction of the exogenous Entner–Doudoroff pathway effectively enhanced glucose consumption and productivity. Overexpression of phosphoenolpyruvate:carbohydrate phosphotransferase system specific to glucose and deletion of ilvE , which encodes branched-chain amino acid transaminase, further suppressed by-products and improved isobutanol productivity. Finally, the produced isobutanol concentration reached 280 mM at a yield of 84% (mol/mol glucose) in 24 h. • Isobutanol producing strains of Corynebacterium glutamicum were developed. • Each expression level of enzymes to synthesize isobutanol was optimized. • Succinate by-production was suppressed by disrupting pckA in addition to ppc. • Disruption of pckA also increased the NADH/NAD+ ratio, and decreased productivity. • Introduction of the exogenous Entner-Doudoroff pathway increased productivity. [ABSTRACT FROM AUTHOR]

Aim: The study was aimed at assessing the effect of β-sitosterol on carbohydrate metabolic enzymes in high-fat diet and sucrose-induced type-2 diabetic rats. Experimental design: Adult male Wistar rats were used and divided into four groups: Group I: Control; Group II: Type-2 diabetic rats; Group III: Type-2 diabetic rats treated with β-sitosterol (20 mg/kg b.wt/day, orally for 30 days); and Group IV: Type-2 diabetic rats treated with metformin (50 mg/kg b.wt/day, orally for 30 days). After 30 days of treatment, animals were anesthetized; liver tissues were dissected out and used for the assessment of glucokinase, glucose-6-phosphatase, glycogen synthase, glycogen phosphorylase, and phosphoenolpyruvate carboxykinase (PEPCK) enzyme activities which were studied by spectrophotometric analysis. Results: The activity of glucose-6-phosphatase, glycogen phosphorylase, and PEPCK was significantly raised (P < 0.05) in type-2 diabetic rats. Conversely, glucokinase and glycogen synthase activities were significantly reduced (P < 0.05) in type-2 diabetic rats. Oral administration of the plant sterol (β-sitosterol) could effectively normalize the detrimental changes caused by high diet induction to that of the control and standard drug metformin. Conclusion: The present study shows that β-sitosterol regulates carbohydrate metabolic enzymes in the liver, thereby β-sitosterol may have therapeutic potential candidate for the management of type-2 diabetes. [ABSTRACT FROM AUTHOR]

Ficus deltoidea var. deltoidea Jack (FD) is a well-known plant used in Malay folklore medicine to lower blood glucose in diabetic patients. For further research of the antihyperglycemic mechanisms, the protein tyrosine phosphatase 1B (PTP1B)-inhibitory effect of FD was analysed both in vitro and in vivo. To optimise a method for FD extraction, water, 50, 70, 80, 90 and 95 % ethanol extracts were prepared and determined for their total phenolic and triterpene contents, and PTP1B-inhibition capacity. Among the tested extracts, 70 % ethanol FD extract showed a significant PTP1B inhibition (92·0 % inhibition at 200 µg/ml) and high phenolic and triterpene contents. A bioassay-guided fractionation of the 70 % ethanol extract led to the isolation of a new triterpene (3β,11β-dihydroxyolean-12-en-23-oic acid; F3) along with six known compounds. In vivo, 4 weeks' administration of 70 % ethanol FD extract (125, 250 and 500 mg/kg/d) to streptozotocin–nicotinamide-induced type 2 diabetic rats reversed the abnormal changes of blood glucose, insulin, total Hb, GLUT2, lipid profile, and oxidative stress in liver and pancreas. Moreover, FD reduced the mRNA expression of the key gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and glucose 6-phosphatase) and restored insulin receptor and GLUT2 encoding gene (Slc2a2) expression. In addition, FD significantly down-regulated the hepatic PTP1B gene expression. These results revealed that FD could potentially improve insulin sensitivity, suppress hepatic glucose output and enhance glucose uptake in type 2 diabetes mellitus through down-regulation of PTP1B. Together, our findings give scientific evidence for the traditional use of FD as an antidiabetic agent. [ABSTRACT FROM AUTHOR]

Glyceroneogenesis is important for the maintenance of fat content in white adipose tissue (WAT). An increase in WAT, and especially the pattern of fat distribution, specifically in visceral depots, potentially contributes to cardiovascular and metabolic diseases, such as type 2 diabetes mellitus, myocardial infarction and hypertension. Recent studies have shown important differences in glyceroneogenesis of different fat sites under the administration of glucocorticoids (GCs). Such differences need to be analysed with criteria evidencing the parameter studied, the type of corticoid, the form of administration and also the tissue studied. PubMed, Scopus and Virtual Health Library were used to search for articles that analysed the effect of GCs on glyceroneogenesis in different sites of adipose tissue in mammals and primary cultures. GCs decrease the glyceroneogenesis in epididymal WAT (EWAT) and also decrease the expression of the mRNA, content and activity of phosphoenolpyruvate carboxykinase (PEPCK-C), key enzyme of glyceroneogenesis. However, in retroperitoneal WAT (RWAT), although there is no consensus about the effect of GCs on PEPCK mRNA, GCs increase PEPCK-C activity and glyceroneogenesis flux. In inguinal WAT (IWAT) an in vitro study showed an increase in the PEPCK mRNA induced by dexamethasone. However, prednisolone does not change glyceroneogenesis flux. In interscapular brown adipose tissue (IBAT) prednisolone or dexamethasone does not change PEPCK-C activity in control diet-fed rats but led to a decrease in PEPCK-C activity in fasted- or high-fat/low-carbohydrate diet-fed rats, as well as in suckling rats. Despite that fact that GCs have different potencies, the same dose of dexamethasone reduces PEPCK-C activity in EWAT, but not in RWAT and IBAT from control-diet fed rats. In summary, the data presented in this article show that GCs differentially regulate glyceroneogenesis in different sites of adipose tissue. Further experiments are needed to firmly establish our hypothesis and clarify the mechanisms involved. • Glucocorticoids (GCs) decrease glyceroneogenesis in epididymal adipose tissue (WAT). • Dexamethasone increases glyceroneogenesis and PEPCK activity in retroperitoneal WAT. • Dexamethasone increases PEPCK mRNA levels in inguinal WAT. • Dexamethasone does not affect PEPCK-C activity it in interscapular brown adipose tissue. • GCs effect on PEPCK does not determinate the classical effect of Cushing's syndrome. [ABSTRACT FROM AUTHOR]

Changes in phosphoenolpyruvate (PEP) concentrations secondary to variations in glucose availability can regulate calcium signaling in T cells as this metabolite potently inhibits the sarcoplasmic reticulum Ca2+/ATPase pump (SERCA). This regulation is critical to assert immune activation in the tumor as T cells and cancer cells compete for available nutrients. We examined here whether cytosolic calcium and the activation of downstream effector pathways important for tumor biology are influenced by the presence of glucose and/or cataplerosis through the phosphoenolpyruvate carboxykinase (PEPCK) pathway, as both are hypothesized to feed the PEP pool. Our data demonstrate that cellular PEP parallels extracellular glucose in two human colon carcinoma cell lines, HCT-116 and SW480. PEP correlated with cytosolic calcium and NFAT activity, together with transcriptional up-regulation of canonical targets PTGS2 and IL6 that was fully prevented by CsA pre-treatment. Similarly, loading the metabolite directly into the cell increased cytosolic calcium and NFAT activity. PEP-stirred cytosolic calcium was also responsible for the calmodulin (CaM) dependent phosphorylation of c-Myc at Ser62, resulting in increased activity, probably through enhanced stabilization of the protein. Protein expression of several c-Myc targets also correlated with PEP levels. Finally, the participation of PEPCK in this axis was interrogated as it should directly contribute to PEP through cataplerosis from TCA cycle intermediates, especially in glucose starvation conditions. Inhibition of PEPCK activity showed the expected regulation of PEP and calcium levels and consequential downstream modulation of NFAT and c-Myc activities. Collectively, these results suggest that glucose and PEPCK can regulate NFAT and c-Myc activities through their influence on the PEP/Ca2+ axis, advancing a role for PEP as a second messenger communicating metabolism, calcium cell signaling, and tumor biology. [ABSTRACT FROM AUTHOR]

Changes in phosphoenolpyruvate (PEP) concentrations secondary to variations in glucose availability can regulate calcium signaling in T cells as this metabolite potently inhibits the sarcoplasmic reticulum Ca2+/ATPase pump (SERCA). This regulation is critical to assert immune activation in the tumor as T cells and cancer cells compete for available nutrients. We examined here whether cytosolic calcium and the activation of downstream effector pathways important for tumor biology are influenced by the presence of glucose and/or cataplerosis through the phosphoenolpyruvate carboxykinase (PEPCK) pathway, as both are hypothesized to feed the PEP pool. Our data demonstrate that cellular PEP parallels extracellular glucose in two human colon carcinoma cell lines, HCT-116 and SW480. PEP correlated with cytosolic calcium and NFAT activity, together with transcriptional up-regulation of canonical targets PTGS2 and IL6 that was fully prevented by CsA pre-treatment. Similarly, loading the metabolite directly into the cell increased cytosolic calcium and NFAT activity. PEP-stirred cytosolic calcium was also responsible for the calmodulin (CaM) dependent phosphorylation of c-Myc at Ser62, resulting in increased activity, probably through enhanced stabilization of the protein. Protein expression of several c-Myc targets also correlated with PEP levels. Finally, the participation of PEPCK in this axis was interrogated as it should directly contribute to PEP through cataplerosis from TCA cycle intermediates, especially in glucose starvation conditions. Inhibition of PEPCK activity showed the expected regulation of PEP and calcium levels and consequential downstream modulation of NFAT and c-Myc activities. Collectively, these results suggest that glucose and PEPCK can regulate NFAT and c-Myc activities through their influence on the PEP/Ca2+ axis, advancing a role for PEP as a second messenger communicating metabolism, calcium cell signaling, and tumor biology. [ABSTRACT FROM AUTHOR]

The purpose of this study was to examine the effects of chronic exercise training (running 30 m/min, 10% grade, 90 min/d for 8-10 weeks) on specific renal enzyme activities involved with the gluconeogenic pathway in the fed and 24-hr fasted state in rats. A portion of the kidney (containing the cortex and medulla) was homogenized from which cytosolic (c) and mitochondrial (m) fractions were separated. Maximal gluconeogenic enzyme activities were assessed for: phosphoenolpyruvate carboxykinase (cPEPCK), fructose 1,6-bisphosphatase (cFBP), pyruvate carboxylase (mPC), aspartate aminotransferase (cAspAT), alanine aminotransferase (cAlaAT), and lactate dehydrogenase (cLDH). In the fed state, there was no significant difference between groups in any of the enzymes examined (nmoles/min x mg protein--1): cPEPCK (25.8 ± 1.7), cFBP (106.8 ± 7.1), mPC (20.7 ± 1.8), cAspAT (1047.1 ± 38.6), cAlaAT (52.3 ± 4.3), and cLDH (1728.6 ± 163.2). After the 24-hr fast, there was a significant increase in cPEPCK (52.4 ± 2.9 and 52.0 ± 2.1) and mPC (44.6 ± 4.3 and 47.6 ± 4.9), control and trained, respectively. These results suggest that the maximal enzyme activities for cPEPCK and mPC can be augmented as a result of fasting that was independent of the training status. [ABSTRACT FROM AUTHOR]

Gluconeogenesis is a key interface between organic acid/amino acid/lipid and sugar metabolism. The aims of this article are four-fold. First, to provide a concise overview of plant gluconeogenesis. Second, to emphasise the widespread occurrence of gluconeogenesis and its utilisation in diverse processes. Third, to stress the importance of the vacuolar storage and release of Krebs cycle acids/nitrogenous compounds, and of the role of gluconeogenesis and malic enzyme in this process. Fourth, to outline the contribution of fine control of enzyme activity to the coordinate-regulation of gluconeogenesis and malate metabolism, and the importance of cytosolic pH in this.

C4 plants show higher photosynthetic capacity and resource use efficiency than C3 plants. However, the genetic variations of these traits and their regulatory factors in C4 plants still remain to be resolved. We investigated physiological, biochemical, and structural traits involved in photosynthesis and photosynthetic water and nitrogen use efficiencies (PWUE and PNUE) in 22 maize lines and four teosinte lines from various regions of the world. Net photosynthetic rate (PN) ranged from 32.1 to 46.5 μmol m−2 s−1. PN was positively correlated with stomatal conductance, transpiration rate, and chlorophyll, nitrogen and soluble protein contents of leaves, but not with specific leaf weight. PN was positively correlated with the activities of ribulose-1,5-bisphosphate carboxylase/oxygenase and the C4-acid decarboxylases, NADP-malic enzyme and phosphoenolpyruvate carboxykinase, but not with the activity of phosphoenolpyruvate carboxylase. Leaf structural traits (stomatal parameters, leaf thickness, and interveinal distance) were not correlated with PN. These data suggest that physiological and biochemical traits are involved in the genetic variation of PN, but structural traits are not directly involved. PWUE is in the lower class of values reported for C4 plants, whereas PNUE is in the highest class of values reported for C4 plants. PNUE was negatively correlated with leaf nitrogen content but not significantly correlated with PN. PWUE was not correlated with δ13C values of leaves, indicating difficulty in using δ13C values as an indicator of PWUE of maize. In general, teosinte lines showed lower PN but higher PWUE than maize lines.

As a key molecular scaffold for various flavonoids, naringenin is a value-added chemical with broad pharmaceutical applicability. For efficient production of naringenin from acetate, it is crucial to precisely regulate the carbon flux of the oxaloacetate-phosphoenolpyruvate (OAA-PEP) regulatory node through appropriate pckA expression control, as excessive overexpression of pckA can cause extensive loss of OAA and metabolic imbalance. However, considering the critical impact of pckA on naringenin biosynthesis, the conventional strategy of transcriptional regulation of gene expression is limited in its ability to cover the large and balanced solution space. To overcome this hurdle, in this study, pckA expression was fine-tuned at both the transcriptional and translational levels in a combinatorial expression library for the precise exploration of optimal naringenin production from acetate. Additionally, we identified the effects of regulating pckA expression by validating the correlation between phosphoenolpyruvate kinase (PCK) activity and naringenin production. As a result, the flux-optimized strain exhibited a 49.8-fold increase compared with the unoptimized strain, producing 122.12 mg/L of naringenin. Collectively, this study demonstrated the significance of transcriptional and translational flux rebalancing at the key regulatory node, proposing a pivotal metabolic engineering strategy for the biosynthesis of various flavonoids derived from naringenin using acetate., One-Sentence Summary: In this study, transcriptional and translational regulation of pckA expression at the crucial regulatory node was conducted to optimize naringenin biosynthesis using acetate in E. coli., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Pancreatic β-cells express the gluconeogenic enzymes glucose 6-phosphatase (G6Pase), fructose 1,6-bisphosphatase (FBP), and phosphoenolpyruvate (PEP) carboxykinase (PCK), which modulate glucose-stimulated insulin secretion (GSIS) through their ability to reverse otherwise irreversible glycolytic steps. Here, we review current knowledge about the expression and regulation of these enzymes in the context of manipulating them to improve insulin secretion in diabetics. Because the regulation of gluconeogenic enzymes in β-cells is so poorly understood, we propose novel research avenues to study these enzymes as modulators of insulin secretion and β-cell dysfunction, with especial attention to FBP, which constitutes an attractive target with an inhibitor under clinical evaluation at present. Glucose must be metabolized into pyruvate through the glycolytic pathway in β-cells for proper oscillatory and biphasic insulin secretion. Chronic impairment of glycolysis negatively impacts β-cell function, leading to hyperglycemia and diabetes. Functional gluconeogenic enzymes that reverse the glycolytic pathway are expressed in β-cells and modulate glucose-stimulated insulin secretion (GSIS). Dysregulation of gluconeogenic enzymes in β-cells is associated with impaired GSIS, so they constitute interesting pharmacological targets for intervention in insulin secretion. Deeper understanding of the regulation of gluconeogenic enzymes in β-cells is critical to unravel their physiological and pathological role and to discover new points of intervention for antidiabetic therapeutics. [ABSTRACT FROM AUTHOR]

Despite being a carnivorous fish species, cobia (Rachycentron canadum) can utilize high levels of dietary carbohydrate (up to 360 g kg−1). By contrast, rainbow trout (also carnivorous) cannot, due to the absence of molecular induction of glycolytic enzyme and inhibition of gluconeogenic enzyme gene expressions such as pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK). We hypothesized that this phenomenon is species-specific and will not be observed in cobia. Our results show that, at the molecular level, the mRNA abundance of the important glycolytic (PK) and gluconeogenic (PEPCK) enzymes in cobia liver are regulated by dietary carbohydrate-to-lipid (CHO:L) ratios and nutritional status (fed, unfed, and refed). Significantly upregulated hepatic PK and depressed PEPCK gene expressions were observed when the fish were fed with an increasing CHO/L-ratio diet or were refed. However, in contrast to gene expression, there was no significant effect of dietary CHO/L ratios on PK enzyme activity. The decrease in PEPCK activity was significantly found between low CHO/L ratio and high CHO/L ratio diets, whereas the moderate CHO/L ratio group showed intermediate values. But PEPCK activity appeared to be independent of nutritional status. These results suggest that nutritional regulation is obvious, at least at the molecular level, in the key hepatic enzymes (PK and PEPCK) of the glucose metabolism pathway, in response to different dietary CHO/L ratios and to the transition from being starved to fed. Determining whether other key enzymes involved in hepatic glucose metabolism contribute to glucose tolerance in cobia is necessary for further investigation of this phenomenon at the enzymatic and molecular levels. [ABSTRACT FROM AUTHOR]

Dysregulated hepatic gluconeogenesis is a hallmark of insulin resistance and type 2 diabetes mellitus (T2DM). Although existing drugs have been proven to improve gluconeogenesis, achieving this objective with functional food is of interest, especially using conjugated linoleic acid (CLA) found in dairy products. Both cis‐9, trans‐11 (c9,t11) and trans‐10, cis‐12 (t10,c12) isomers of CLA were tested in human (HepG2) and rat (H4IIE) hepatocytes for their potential effects on gluconeogenesis. The hepatocytes exposed for 24 h with 20 μM of c9,t11‐CLA had attenuated the gluconeogenesis in both HepG2 and H4IIE by 62.5% and 80.1%, respectively. In contrast, t10,c12‐CLA had no effect. Of note, in HepG2 cells, the exposure of c9,t11‐CLA decreased the transcription of gluconeogenic enzymes, cytosolic phosphoenolpyruvate carboxykinase (PCK1) by 87.7%, and glucose‐6‐phosphatase catalytic subunit (G6PC) by 38.0%, while t10,c12‐CLA increased the expression of G6PC, suggesting the isomer‐specific effects of CLA on hepatic glucose production. In HepG2, the peroxisome proliferator‐activated receptor (PPAR) agonist, rosiglitazone, reduced the glucose production by 72.9%. However, co‐administration of c9,t11‐CLA and rosiglitazone neither exacerbated nor attenuated the efficacy of rosiglitazone to inhibit glucose production; meanwhile, t10,c12‐CLA abrogated the efficacy of rosiglitazone. Paradoxically, PPARγ antagonist GW 9662 also led to 70.2% reduction of glucose production and near undetectable PCK1 expression by abrogating CLA actions. Together, while the precise mechanisms by which CLA isomers modulate hepatic gluconeogenesis directly or via PPAR warrant further investigation, our findings establish that c9,t11‐CLA suppresses gluconeogenesis by decreasing PEPCK on hepatocytes. [ABSTRACT FROM AUTHOR]

Abstract Rainbow trout have, as salmonid fish species, undergone sequential genome duplication events in their evolutionary history. In addition to a teleost-specific whole genome duplication approximately 320–350 million years ago, rainbow trout and salmonids in general underwent an additional salmonid lineage-specific genome duplication event approximately 80 million years ago. Through the recent sequencing of salmonid genome sequences, including the rainbow trout, the identification and study of duplicated genes has become available. A particular focus of interest has been the evolution and regulation of rainbow trout gluconeogenic genes, as recent molecular and gene expression evidence points to a possible contribution of previously uncharacterized gluconeogenic gene paralogues to the rainbow trout long-studied glucose intolerant phenotype. Since the publication of the initial rainbow trout genome draft, resequencing and annotation have further improved genome coverage. Taking advantage of these recent improvements, we here identify a salmonid-specific genome duplication of ancestral mitochondrial phosphoenolpyruvate carboxykinase 2 isoenzyme, we termed pck2a and pck2b. Cytosolic phosphoenolpyruvate carboxykinase (Pck1) and, more recently mitochondrial Pck2 , are considered to be the rate-limiting enzymes in de novo gluconeogenesis. Following in silico confirmation of salmonid pck2a and pck2b evolutionary history, we simultaneously profiled cytosolic pck1 and mitochondrial pck2a and pck2b expression in rainbow trout liver under several experimental conditions known to regulate hepatic gluconeogenesis. Cytosolic pck1 abundance was increased by nutritional (diets with a high protein to carbohydrate ratio compared to diets with a low carbohydrate to protein ratio) and glucoregulatory endocrine factors (glucagon and cortisol), revealing that the well-described transcriptional regulation of pck1 in mammals is present in rainbow trout. Conversely, and in contrast to mammals, we here describe endocrine regulation of pck2a (decrease in abundance in response to glucagon infusion), and nutritional, social-status-dependent and hypoxia-dependent regulation of pck2b. Specifically, pck2b transcript abundance increased in trout fed a diet with a low protein to carbohydrate ratio compared to a diet with a high protein to carbohydrate ratio, in dominant fish compared to subordinate fish as well as hypoxia. This specific and differential expression of rainbow trout pck2 ohnologues is indicative of functional diversification, and possible functional consequences are discussed in light of the recently highlighted gluconeogenic roles of mitochondrial pck2 in mammalian models. Highlights • The salmonid lineage experienced pck2 genome duplication, yielding pck2a and pck2b • pck1 is largely regulated by nutritional and endocrine stimuli, as mammals • Transcript abundance of pck2a and pck2b is differentially regulated suggesting possible sub- or neo-funtionalization • Transcript abundance pck2b a isinduced by carbohydrate-rich diets and hypoxia, but decreased in socially subordinate fish [ABSTRACT FROM AUTHOR]

Copyright of Journal of Hebei University of Science & Technology is the property of Hebei University of Science & Technology, Journal of Hebei University of Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Phosphoenolpyruvate carboxykinase (PEPCK) is known to exist as mitochondrial (PEPCK‐M) and cytosolic isoforms (PEPCK‐C). They are involved in glucose production and play vital roles in regulating glucose homeostasis. This study aimed to (1) clone and characterize PEPCK‐C cDNA from golden pompano fish (Trachinotus ovatus), which are known to have poor utilization of dietary carbohydrates, and (2) analyze the regulation of its expression by varying dietary carbohydrate‐to‐lipid ratios and nutritional status. A full length golden pompano PEPCK‐C cDNA fragment of 2,652 bp was cloned, which contains an open reading frame of 1,875 bp encoding 624 amino acids and shows a high homology to cobia (Rachycentron canadum) PEPCK‐C sequence (92% similarity). The analysis of tissue distributions of PEPCK‐C mRNA showed that a high abundance of PEPCK‐C was expressed in golden pompano liver, followed by kidney; while lower expression levels were found in the heart and intestine. In liver, PEPCK is under the regulation of dietary composition and nutritional status at the enzymatic and molecular levels. Our results indicate that PEPCK‐C plays a modulating role in the adaptation of hepatic gluconeogenesis to different nutrient conditions. Thus, the absence of molecular inhibition of gluconeogenic PEPCK gene expression in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), which can at least partially account for their poor utilization of dietary carbohydrate, is specific to these species and is not observed in golden pompano. Further studies that examine, at the enzymatic and molecular levels, other key enzymes involved in hepatic glucose metabolism are necessary to understand the poor utilization of carbohydrates in golden pompano. [ABSTRACT FROM AUTHOR]

Hepatitis B virus X protein (HBx) can stimulate the transcription of phosphoenolpyruvate carboxykinase (PEPCK), a rate‐determining enzyme in gluconeogenic pathway. Two isoforms of PEPCK exist, a cytoplasmic form (PCK1) and a mitochondrial isoform (PCK2). The current study investigated the direct effect of HBx‐stimulated PEPCK on hepatitis B virus (HBV) replication. We showed that PCK1 rather than PCK2 was upregulated by HBx. We also demonstrated that overexpression of PCK1 decreased HBV replication, whereas inhibition of PCK1‐enhanced HBV replication. Furthermore, we found overexpression of PCK1 led to reduced expression of peroxisome proliferator‐activated receptor‐coactivator 1α (PGC‐1α) and peroxisome proliferator‐activated receptor γ (PPAR‐γ), whereas knocking down PCK1 resulted in an increased expression of PGC‐1α and PPAR‐γ. When PPAR‐γ was inhibited, knocking down PCK1 could not induce the apparent enhanced HBV replication. Our data suggested that PCK1 induced by HBx led to decreased HBV replication through the downregulation of PGC‐1α and PPAR‐γ. Thus, our study demonstrates a negative‐feedback loop involving PCK1 and HBV may provide a balanced cell environment for HBV persistent infection. [ABSTRACT FROM AUTHOR]

Graphical abstract AMP-activated protein kinase (AMPK) plays pivotal roles in insulin sensitivity and glucose homeostasis. PEPCK, G6pase, PGC-1α, FOXO1, HNF4α are the key enzymes and transcription factors in gluconeogenesis. PGC-1α, FOXO1, HNF4α can regulate G6Pase and PEPCK enzymes gene expression. Highlights • Ficus carica leaf (FCL) extract possesses hypoglycemic effects. • FCL extract inhibited PEPCK and G6Pase expression in diabetic mice and cells. • FCL extract activated AMPK and decreased the expressions of PGC-1α, HNF4α, and FOXO1. • FCL extract inhibits hepatic gluconeogenesis via the activation of AMPK. Abstract The aim of this study was to explore the possible mechanisms of Ficus carica leaf (FCL) extract in suppressing hepatic gluconeogenesis in diabetic mice. Diabetic mice (streptozotocin-induced) received 1 g/kg of FCL extract twice a day for 6 weeks. Fasting blood glucose levels were measured and a 2-h oral glucose tolerance test was conducted. AMP-activated protein kinase (AMPK), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), and peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) expression was examined. HepG2 hepatocytes were treated with FCL extract and an AMPK inhibitor (compound C) or agonist (AICAR), and PEPCK, G6pase, PGC-1α, AMPK, forkhead transcription factor O1 (FOXO1), and hepatic nuclear factor 4α (HNF4α) expression was determined. The results showed that FCL extract inhibited the expression of PEPCK and G6Pase in the liver of diabetic mice and HepG2 hepatocytes. FCL extract activated AMPK and decreased PGC-1α, HNF4α, and FOXO1 expression. The AMPK inhibitor attenuated those effects through inhibiting gluconeogenesis, while the AMPK agonist partially enhanced gluconeogenesis. In conclusion, FCL extract inhibits hepatic gluconeogenesis via activation of AMPK and down-regulation of gluconeogenic enzymes. [ABSTRACT FROM AUTHOR]

The fat body is responsible for a variety of functions related to energy metabolism in arthropods, by controlling the processes of de novo glucose production (gluconeogenesis) and glycogen metabolism. The rate-limiting factor of gluconeogenesis is the enzyme phosphoenolpyruvate carboxykinase (PEPCK), generally considered to be the first committed step in this pathway. Although the study of PEPCK and gluconeogenesis has been for decades restricted to mammalian models, especially focusing on muscle and liver tissue, current research has demonstrated particularities about the regulation of this enzyme in arthropods, and described new functions. This review will focus on arthropod PEPCK, discuss different aspects to PEPCK regulation and function, its general role in the regulation of gluconeogenesis and other pathways. The text also presents our views on potentially important new directions for research involving this enzyme in a variety of metabolic adaptations (e.g. diapause), discussing enzyme isoforms, roles during arthropod embryogenesis, as well as involvement in vector-pathogen interactions, contributing to a better understanding of insect vectors of diseases and their control. [Display omitted] • The activity of PEPCK is critical for embryo development in arthropods. • PEPCK-M and PEPCK-C have different contributions to energy metabolism regulation and development of arthropods. • PEPCK activity increases to maintain glucose synthesis in diapause or quiescence in arthropods. • PEPCK isoforms respond differently to blood feeding in hematophagous arthropods. • PEPCK activity is associated with gluconeogenesis as a mechanism to cope with hypo- or hyperosmotic stress in arthropods. • PEPCK plays an important role in the vector-pathogen interaction. [ABSTRACT FROM AUTHOR]