Search

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.

Protein turnover through de novo synthesis is critical for sustainable cellular functions. We previously found that glucose consumption rate in Corynebacterium glutamicum under anaerobic conditions increased at temperature higher than the upper limit of growth temperature. Here, we showed that production of lactic and succinic acids increased at higher temperature for long-term (48 h) anaerobic reaction in metabolically engineered strains. At 42 °C, beyond the upper limit of growth temperature range, biomass-specific lactic acid production rate was 8% higher than that at 30 °C, the optimal growth temperature. In contrast, biomass-specific succinic acid production rate was highest at 36 °C, 28% higher than that at 30 °C, although the production at 42 °C was still 23% higher than that at 30 °C. As enzymes are usually unstable at high temperatures, we investigated whether protein turnover of metabolic enzymes is required for the production of lactic and succinic acids under these conditions. Interestingly, when de novo protein synthesis was inhibited by addition of chloramphenicol, after 6 h, only succinic acid production was inhibited. Because glycolytic enzymes are involved in both lactic and succinic acids synthesis, enzymes in the anaplerotic pathway and the tricarboxylic acid (TCA) cycle leading to succinic acid synthesis were likely to be responsible for its decreased production. Among the five enzymes examined, the specific activity of only pyruvate carboxylase was drastically decreased after 48 h at 42 °C. Thus, the de novo synthesis of pyruvate carboxylase is required for long-term production of succinic acid. Graphical abstract KEY POINTS: • Long-term reaction for organic acids can be improved at temperature beyond ideal growth conditions. • De novo synthesis of pyruvate carboxylase is required for long-term succinic acid production.

Recombinant yeast pyruvate carboxylase (PYC2) expression was previously shown to be an effective metabolic engineering strategy for reducing lactate formation in a number of relevant mammalian cell lines, but, in the case of CHO cells, did not consistently lead to significant improvement in terms of cell growth, product titer and energy metabolism efficiency. In the present study, we report on the establishment of a PYC2-expressing CHO cell line producing a monoclonal antibody and displaying a significantly altered lactate metabolism compared to its parental line. All clones exhibiting strong PYC2 expression were shown to experience a significant and systematic metabolic shift toward lactate consumption, as well as a prolonged exponential growth phase leading to an increased maximum cell concentration and volumetric product titer. Of salient interest, PYC2-expressing CHO cells were shown to maintain a highly efficient metabolism in fed-batch cultures, even when exposed to high glucose levels, thereby alleviating the need of controlling nutrient at low levels and the potential negative impact of such strategy on product glycosylation. In bioreactor operated in fed-batch mode, the higher maximum cell density achieved with the PYC2 clone led to a net gain (20%) in final volumetric productivity., (Copyright © 2015. Published by Elsevier B.V.)

As C4-dicarboxylic acids could replace C4-petrochemicals, the reductive tricarboxylic acid (TCA) pathway was overexpressed in Pichia pastoris for production of the C4-dicarboxylic acids. Three expression cassettes which carried the pyruvate carboxylase gene (pc), the cytoplasmic malate dehydrogenase gene (mdh1) and the retargeted fumarase gene (Tfum) were integrated into the chromosomal DNA of P. pastoris GS115 alone or jointly. Multicopy integrations were screened using quantitative PCR for C4-dicarboxylic acid overaccumulation. The results showed that the highest titer in 96 h of fumaric, malic and succinic acid (0.76, 42.28 and 9.42 g l(-1)) was obtained by co-expression of pc and mdh1 in P. pastoris. This is the first report about multiple genes engineered in P. pastoris for C4-dicarboxylic acid production. The strain Pp-PC-MDH1, moreover, has a significant potential to produce malic acid in aerobic conditions., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Anabolic biosynthesis requires precursors supplied by the Krebs cycle, which in turn requires anaplerosis to replenish precursor intermediates. The major anaplerotic sources are pyruvate and glutamine, which require the activity of pyruvate carboxylase (PC) and glutaminase 1 (GLS1), respectively. Due to their rapid proliferation, cancer cells have increased anabolic and energy demands; however, different cancer cell types exhibit differential requirements for PC- and GLS-mediated pathways for anaplerosis and cell proliferation. Here, we infused patients with early-stage non-small-cell lung cancer (NSCLC) with uniformly 13C-labeled glucose before tissue resection and determined that the cancerous tissues in these patients had enhanced PC activity. Freshly resected paired lung tissue slices cultured in 13C6-glucose or 13C5,15N2-glutamine tracers confirmed selective activation of PC over GLS in NSCLC. Compared with noncancerous tissues, PC expression was greatly enhanced in cancerous tissues, whereas GLS1 expression showed no trend. Moreover, immunohistochemical analysis of paired lung tissues showed PC overexpression in cancer cells rather than in stromal cells of tumor tissues. PC knockdown induced multinucleation, decreased cell proliferation and colony formation in human NSCLC cells, and reduced tumor growth in a mouse xenograft model. Growth inhibition was accompanied by perturbed Krebs cycle activity, inhibition of lipid and nucleotide biosynthesis, and altered glutathione homeostasis. These findings indicate that PC-mediated anaplerosis in early-stage NSCLC is required for tumor survival and proliferation.

Cancer cells exhibit altered metabolism compared with that of the surrounding tissue. There is hope that these reprogrammed metabolic pathways in tumors hold the key to advances for both cancer imaging and therapy. Translation of observations in cultured cancer cells to live tumors, however, has proven to be highly complex, and robust methods to analyze metabolic activity in primary human tumors are sorely needed. In this issue of the JCI, Sellers et al. use perioperative administration of isotope-labeled glucose to lung cancer patients to differentiate metabolic pathways between tumors and benign lung. They identify pyruvate carboxylation, a reaction that enables glucose-derived carbon to replenish TCA cycle intermediates, as a key component of anabolic metabolism in tumor cells.

Unlike short interfering RNAs (siRNAs), which are commonly designed to repress a single messenger RNA (mRNA) target through perfect base pairing, microRNAs (miRNAs) are endogenous small RNAs that have evolved to concurrently repress multiple mRNA targets through imperfect complementarity. MicroRNA target recognition is primarily determined by pairing of the miRNA seed sequence (nucleotides 2-8) to complementary match sites in each mRNA target. Whereas siRNA technology is well established for single target knockdown, the design of artificial miRNAs for multi-target repression is largely unexplored. We designed and functionally analysed over 200 artificial miRNAs for simultaneous repression of pyruvate carboxylase and glutaminase by selecting all seed matches shared by their 3' untranslated regions. Although we identified multiple miRNAs that repressed endogenous protein expression of both genes, seed-based artificial miRNA design was highly inefficient, as the majority of miRNAs with even perfect seed matches did not repress either target. Moreover, commonly used target prediction programs did not substantially discriminate effective artificial miRNAs from ineffective ones, indicating that current algorithms do not fully capture the features important for artificial miRNA targeting and are not yet sufficient for designing artificial miRNAs. Our analysis suggests that additional factors are strong determinants of the efficacy of miRNA-mediated target repression and remain to be discovered., (© The Author(s) 2014. Published by Oxford University Press.)

Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.

Succinic acid is a specialty chemical having numerous applications in industrial, pharmaceutical and food uses. One of the major challenges in the succinate fermentation process is eliminating the formation of byproducts. In this study, we describe eliminating byproduct formate and improving succinate productivity by reengineering a high succinate producing E. coli strain SBS550MG-Cms243(pHL413Km). The NAD(+)-dependent formate dehydrogenase gene (fdh1) of Candida boidinii was coexpressed with Lactococcus lactis pyruvate carboxylase (pycA) under the control of Ptrc and PpycA promoters in plasmid pHL413KF1. The newly introduced fdh1 converts 1 mol of formate into 1 mol of NADH and CO2. The reengineered strain SBS550MG-Cms243(pHL413KF1) retains the reducing power of formate through an increase in NADH availability. In anaerobic shake flask fermentations, the parent strain SBS550MG-Cms243(pHL413Km) consumed 99.86 mM glucose and produced 172.38 mM succinate, 16.16 mM formate and 4.42 mM acetate. The FDH bearing strain, SBS550MG-Cms243(pHL413KF1) consumed 98.43 mM glucose and produced 171.80 mM succinate, 1mM formate and 5.78 mM acetate. Furthermore, external formate supplementation to SBS550MG(pHL413KF1) fermentations resulted in about 6% increase in succinate yields as compared to SBS550MG(pHL413Km). In an anaerobic fed-batch bioreactor process, the average glucose consumption rate, succinate productivity, and byproduct formate concentration of SBS550MG(pHL413Km) was 1.40 g/L/h, 1g/L/h, and 17 mM, respectively. Whereas, the average glucose consumption rate, succinate productivity and byproduct formate concentration of SBS550MG(pHL413KF1) was 2 g/L/h, 2 g/L/h, 0-3 mM respectively. A high cell density culture of SBS550MG(pHL413KF1) showed further improvement in succinate productivity with a higher glucose consumption rate. Reduced levels of byproduct formate in succinate fermentation broth would provide an opportunity for reducing the cost associated with downstream processing, purification, and waste disposal., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Anorexia-cachexia is a common and severe cancer-related complication but the underlying mechanisms are largely unknown. Here, using a mouse model for tumour-induced anorexia-cachexia, we screened for proteins that are differentially expressed in the hypothalamus, the brain's metabolic control centre., Methods: The hypothalamus of tumour-bearing mice with implanted methylcholanthrene-induced sarcoma (MCG 101) displaying anorexia and their sham-implanted pair-fed or free-fed littermates was examined using two-dimensional electrophoresis (2-DE)-based comparative proteomics. Differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry., Results: The 2-DE data showed an increased expression of dynamin 1, hexokinase, pyruvate carboxylase, oxoglutarate dehydrogenase, and N-ethylmaleimide-sensitive factor in tumour-bearing mice, whereas heat-shock 70 kDa cognate protein, selenium-binding protein 1, and guanine nucleotide-binding protein Gα0 were downregulated. The expression of several of the identified proteins was similarly altered also in the caloric-restricted pair-fed mice, suggesting an involvement of these proteins in brain metabolic adaptation to restricted nutrient availability. However, the expression of dynamin 1, which is required for receptor internalisation, and of hexokinase, and pyruvate carboxylase were specifically changed in tumour-bearing mice with anorexia., Conclusion: The identified differentially expressed proteins may be new candidate molecules involved in the pathophysiology of tumour-induced anorexia-cachexia.

The yeast Torulopsis glabrata CCTCC M202019, which is used for industrial pyruvate production, was chosen to explore the suitability of engineering this multi-vitamin auxotrophic yeast for increased malate production. Various metabolic engineering strategies were used to manipulate carbon flux from pyruvate to malate: (i) overexpression of pyruvate carboxylase and malate dehydrogenase; (ii) identification of the bottleneck in malate production by model iNX804; (iii) simultaneous overexpression of genes RoPYC, RoMDH and SpMAE1. Using these strategies, 8.5gL(-1) malate was accumulated in the engineered strain T.G-PMS, which was about 10-fold greater than that of the control strain T.G-26. The results presented here suggest that T. glabrata CCTCC M202019 is a promising candidate for industrial malate production., (© 2013 Elsevier Inc. All rights reserved.)

There is an imperative need for expression systems allowing the efficient and robust manufacturing of high quality glycoproteins. In the present work, HEK-293 cells stably expressing interferon-α2b were further engineered with the insertion of the yeast pyruvate carboxylase 2 gene. In batch cultures, marked reductions in lactate and ammonia production were observed compared to the parental cell clone. Although the maximum specific growth rate remained unchanged, the altered metabolism led to a 2-fold increase in maximum cell density and 33% increase in the integral of viable cell concentration and interferon production yield. The underlying metabolic changes were further investigated using various (13)C-labeled substrates and measuring the resulting lactate mass isotopomer distributions. Simultaneous metabolite and isotopomer balancing allowed the accurate determination of key intracellular fluxes. Such detailed and quantitative knowledge about the central carbon metabolism of the cells is instrumental to further support the development of high-yield fed-batch processes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pyruvate carboxylase (PC) is a mitochondrial enzyme that catalyses the carboxylation of pyruvate to oxaloacetate thereby allowing supplementation of citric acid cycle intermediates. The presence of PC in skeletal muscle is controversial. We report here, that PC protein is easily detectable by streptavidin blot and describe the presence of considerable amounts of PC in cultured human myotubes and in human muscle tissue., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Escherichia coli strain DC1515, deficient in glucose phosphotransferase (ptsG), lactate dehydrogenase (ldhA) and pyruvate:formate lyase (pflA), is a promising candidate for the fermentative production of succinate. To further improve the succinate producing capability of DC1515, we constructed plasmid pTrchisA-pyc with heterogenous pyruvate carboxylase (pyc) from Bacillus subtilis 168 under the Trc promoter and introduced it into DC1515. We used lactose as a substitute of IPTG to induce pyc. We optimized the culture conditions such as the lactose addition time, the lactose concentration and the culture temperature after induction for succinate production. We also explored the effect of lactose supplement during the fermentation. The results showed that pyc can be expressed under lactose induction in the fermentative medium with 15 g/L glucose due to the deficient of ptsG in DC1515. Under optimized conditions, the final succinate concentration reached to 15.17 g/L, which was 1.78-fold higher than that of control strain. If complementing lactose twice to the concentration of 1 g/L during the fermentation, the final succinate concentration could further reach to 17.54 g/L. This work might provide valuable information for gene expression in E. coli strains using lactose as inducer for succinate production in a glucose-medium. Due to the reduced cost, E. coli is becoming a more promising strain for succinate production through fermentation.

The mitochondrial enzyme, pyruvate carboxylase (PC; EC 6.4.1.1) is considered to play a significant role in the intermediary metabolism of neural tissue. PC-catalyzed carboxylation of pyruvate to oxaloacetate is a major anaplerotic reaction in brain. Anaplerosis is essential for homeostasis of the members of the tricarboxylic acid (TCA) cycle. Several biochemical pathways rely on withdrawing TCA cycle members. Prominent among these are biosynthesis of fatty acids and of non-essential amino acids such as aspartate, asparagine, glutamate and glutamine, gluconeogenesis, glycogen synthesis, and regeneration of NADPH. The expression of PC in brain has already been described and assigned to astrocytes. Since pyruvate carboxylase deficiency is associated with malformations of the brain, e.g., inadequate development of the corpus callosum and the lack of myelination, one can hypothesize that PC may be expressed also in glial cells other than astrocytes. Therefore, the expression of PC was investigated in cultured oligodendroglial, microglial, and ependymal cells. As assessed by RT-PCR, all these cultures contain PC mRNA. This mRNA is generated in a transcription process that is regulated by the "distal class" of promoters of the PC gene. The expression of PC among cultured glial cells was studied with a rabbit antiserum by immunoblotting and immunocytochemistry. The results indicate that PC is not only expressed in cultured astroglial cells but also in cultured oligodendrocytes, microglial cells, and ependymocytes. It appears that the intermediary metabolism of these cells includes the anaplerotic action of PC as well as possibly also functions of the enzyme in biosynthetic pathways and the provision of NADPH for defense against reactive oxygen species.

Colostrum feeding and glucocorticoid administration affect glucose metabolism and insulin release in calves. We have tested the hypothesis that dexamethasone as well as colostrum feeding influence insulin-dependent glucose metabolism in neonatal calves using the euglycemic-hyperinsulinemic clamp technique. Newborn calves were fed either colostrum or a milk-based formula (n=14 per group) and in each feeding group, half of the calves were treated with dexamethasone (30 microg/[kg body weight per day]). Preprandial blood samples were taken on days 1, 2, and 4. On day 5, insulin was infused for 3h and plasma glucose concentrations were kept at 5 mmol/L+/-10%. Clamps were combined with [(13)C]-bicarbonate and [6,6-(2)H]-glucose infusions for 5.5h (i.e., from -150 to 180 min, relative to insulin infusion) to determine glucose turnover, glucose appearance rate (Ra), endogenous glucose production (eGP), and gluconeogenesis before and at the end of the clamp. After the clamp liver biopsies were taken to measure mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC). Dexamethasone increased plasma glucose, insulin, and glucagon concentrations in the pre-clamp period thus necessitating a reduction in the rate of glucose infusion to maintain euglycemia during the clamp. Glucose turnover and Ra increased during the clamp and were lower at the end of the clamp in dexamethasone-treated calves. Dexamethasone treatment did not affect basal gluconeogenesis or eGP. At the end of the clamp, dexamethasone reduced eGP and PC mRNA levels, whereas mitochondrial PEPCK mRNA levels increased. In conclusion, insulin increased glucose turnover and dexamethasone impaired insulin-dependent glucose metabolism, and this was independent of different feeding.

The ability of dairy cattle to adapt to changes in nutrient intake requires appropriately responsive expression of several key genes in liver. Holstein cows were used in 2 experiments to determine the effect of short-term feed restriction on expression of mRNA for gluconeogenic and ureagenic enzymes in liver. In experiment 1, cows were fed a total mixed diet for ad libitum intake for a 5-d period followed by 5 d of 50% of their previous 5-d ad libitum intake followed by 10 d of ad libitum feeding. Liver biopsies and blood samples were obtained on d 5, 10, and 20 of the experiment, the last day of each feeding period. Pyruvate carboxylase (PC) mRNA increased with feed restriction, but phosphoenolpyruvate carboxykinase (PEPCK) was unchanged. Expression of carbamoyl phosphate synthetase (CPS-I), argininosuccinate synthetase, and ornithine transcarbamylase mRNA were not altered by feed restriction; however, CPS-I mRNA expression tended to increase during realimentation. In experiment 2, cows were fed for ad libitum intake for 5 d and then fed 50% of previous intake for 5 d. Liver biopsy samples collected on d 5 and 10 were used for PC mRNA, PEPCK mRNA, and in vitro measure of gluconeogenesis from radiolabelled propionate and lactate. The data indicate expression of genes for key metabolic processes in liver of lactating cows is responsive to feeding level. Expression of PC mRNA is part of the adaptive response to feed intake restriction and is matched by increased capacity for gluconeogenesis from lactate.

The insect cell baculovirus expression vector system (BEVS) is one of the most commonly used expression systems for recombinant protein production. This system is also widely used for the production of recombinant virus and virus-like particles. Although several published reports exist on recombinant protein expression using insect cells, information dealing with their metabolism in vitro is relatively scarce. In this work we have analyzed the metabolism of glucose and glutamine, the main carbon and/or energy compounds, of the two most commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5). Radiolabeled substrates have been used to determine the flux of glucose carbon entering the tricarboxylic acid cycle (TCA) and the pentose phosphate (PP) pathway by direct measurement of 14CO2 produced. The percentage of total glucose metabolized to CO2 via the TCA cycle was higher in the case of the Sf-9 (2.7%) compared to Tn-5 (0.6%) cells, while the percentage of glucose that is metabolized via the PP pathway was comparable at 14% and 16% for the two cell lines, respectively. For both cell lines, the remaining 83% of glucose is metabolized through other pathways generating, for example, lactate, alanine, etc. The percentage of glutamine oxidized in the TCA cycle was approximately 5-fold higher in the case of the Tn-5 (26.1%) as compared to the Sf-9 cells (4.6%). Furthermore, the changes in the metabolic fluxes of glucose and glutamine in Tn-5-PYC cells, which have been engineered to express a cytosolic pyruvate carboxylase, have been studied and compared to the unmodified cells Tn-5. As a result of this metabolic engineering, significant increase in the percentage of glucose oxidized in the TCA cycle (3.2%) as well as in the flux through the PP pathway (34%) of the Tn-5-PYC were observed.

Metabolic engineering has been defined as a directed improvement of product formation or cellular properties by modification of specific biochemical pathways or introduction of new enzymatic reactions by recombinant DNA technology. The use of metabolic flux analysis (MFA) has helped in the understanding of the key limitation in the metabolic pathways of cultured animal cells. The MFA of the major nutrients glucose and glutamine showed that the flux of glucose to the TCA cycle and its subsequent utilization is limited as a result of the lack of certain key enzymes in the pathway. One of the key enzymes controlling this flux is pyruvate carboxylase. Introduction of this enzyme into mammalian cells has been shown to improve the utilization of glucose and limit the production of lactate and ammonia, which are deleterious to cell growth. In the present work a yeast pyruvate carboxylase gene has been introduced into mammalian (HEK 293) and insect (Trichoplusia ni High-Five) cells, resulting in the cytosolic expression of the enzyme. In both cases the resulting transfected cells were able to utilize glucose and glutamine more efficiently and produce lower amounts of lactate and ammonia. Differences in the amino acid utilization pattern were also observed, indicating changes in the basic metabolism of the cells. The performance of the transfected cells as expression systems for adenovirus and baculovirus vectors, respectively, has also been examined. The results obtained and their impact on the process development for protein and viral vector production are discussed.

Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology.

Anaplerotic enzyme reactions are those which replenish tricarboxylic acid intermediates that are withdrawn for the synthesis of biomass. In this study, we examined recombinant protein production in Escherichia coli containing activity in an additional anaplerotic enzyme, pyruvate carboxylase. In batch fermentations, the presence of pyruvate carboxylase resulted in 68% greater production of the model protein, beta-galactosidase, 41% greater cell yield, and 57% lower acetate concentration. We discuss why these results indicate that acetate concentration does not limit cell growth and protein synthesis, as predicted by other researchers, and suggest instead that the rate of acetate formation represents an inefficient consumption of glucose carbon, which is reduced by the presence of pyruvate carboxylase.

Recently, a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to reconstitute the missing link between glycolysis and TCA, thus increasing the flux of glucose into the TCA and resulting in a higher intracellular ATP content. Now, these metabolically engineered cells have been additionally transfected with a plasmid bearing the gene for human erythropoietin. EPO yield and substrate-specific productivity of the recombinant BHK-21 cells have been compared to control cells without the PYC2-gene but transfected with the plasmid coding for the expression of the selection genes and EPO. PYC2-expressing clones showed a 2-fold higher glucose-specific productivity and a 2-fold higher product concentration in a continuously perfused bioreactor. Moreover, the PYC2 expression enabled the cells to become more resistant to low glucose concentrations in the culture medium. They could produce at nearly maximum productivity under glucose-limiting conditions of 0.05-1 gl(-1) that guaranteed a reduced accumulation of lactate in fed-batch production systems. Due to the fact that PYC2-expressing cells are characterized by reduced glucose consumption, a prolonged production phase in bioreactors can be maintained. Based on the demand not to fall short of 80% cell viability for the production, EPO could be produced for 2 days (30%) longer compared to the control due to a more economic exploitation of glucose, and the prolonged viability period of the cells using a batch cultivation driven by glutamine limitation.

The objective of this study was to profile phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC) mRNA expression in the liver of dairy cattle during the peripartum transition and determine changes in abundance of these mRNA in response to protein fed during the prepartum period. Thirty-eight multiparous Holstein cows were fed diets containing either 12% crude protein (CP) and 26% rumen undegradable protein (RUP), 16% CP and 26% RUP, 16% CP and 33% RUP, or 16% CP and 40% RUP on a dry-matter basis beginning 28 d before expected calving. After calving, all cows were fed a common diet through 56 d in milk (DIM). Northern analysis of RNA from liver biopsy samples obtained on days -28, -14, +1, +28, and +56 relative to calving indicated that PC and PEPCK mRNA expression were responsive to onset of lactation but not to prepartum protein or RUP concentration. Abundance of PEPCK mRNA was similar at -28, -14, and +1 DIM but was elevated by +28 and +56 DIM relative to precalving levels. Liver PC mRNA abundance was elevated on +1 DIM, remained elevated through 28 DIM, and declined to precalving levels by 56 DIM. The activity of PC enzyme was correlated (r2 = 0.89) with PC mRNA abundance. The data demonstrate increased abundance of PC mRNA during the early transition period followed by increased abundance of PEPCK mRNA during the postpartum period and suggest increased potential metabolism of lactate, pyruvate, and amino acids that contribute to the liver pyruvate pool.

A previous study on the gene structure of rat pyruvate carboxylase revealed that two tissue-specific promoters are responsible for the production of multiple transcripts with 5'-end heterogeneity (Jitrapakdee, S., Booker, G. W., Cassady, A. I., and Wallace, J. C. (1997) J. Biol. Chem. 272, 20522-20530). Here we report transcription and translation regulation of pyruvate carboxylase (PC) expression during development and in genetically obese rats. The abundance of PC mRNAs was low in fetal liver but increased by 2-4-fold within 7 days after birth, concomitant with an 8-fold increase in the amount of immunoreactive PC and its activity and then decreased during the weaning period. Reverse transcriptase polymerase chain reaction analysis indicated that the proximal promoter was activated during the suckling period and reduced in activity at weaning. In genetically obese Zucker rats, adipose PC was 4-5-fold increased, concomitant with a 5-6-fold increase in mRNA level. Reverse transcriptase-polymerase chain reaction analysis also showed that the proximal promoter was activated in the hyperlipogenic condition. Conversely, transcription of the proximal promoter was not detectable in various liver cell lines, suggesting that this promoter was not functional under cell culture conditions. In rat pancreatic islets and insulinoma cells, only transcripts D and E, generated from the distal promoter of the PC gene, were expressed. Glucose increased PC transcripts from the distal promoter when the insulinoma cells were maintained in 10 mM glucose. We conclude that the proximal promoter of the rat PC gene plays a major role in gluconeogenesis and lipogenesis, whereas the distal promoter is necessary for anaplerosis. In vitro translation and in vivo polysome profile analysis indicated that transcripts C and E were translated with similar translational efficiencies that are substantially greater than that of transcript D, suggesting that 5'-untranslated regions play a role in translational control.

We have developed a new vector for the stable expression of recombinant protein in mammalian cells. In this vector, designated pEFIRES-P, both the recombinant cDNA and the puromycin resistant gene (pac) are transcribed as a single message driven by the strong human polypeptide chain elongation factor 1alpha promoter. The presence of an internal ribosome entry site ensures any clones that are resistant to puromycin also express high levels of recombinant protein encoded by cDNA cloned upstream from the pac gene. We report the use of this vector to generate stable cell lines expressing human pyruvate carboxylase and show that following transfection and clonal selection, all twenty colonies that conferred high dose resistance to puromycin also expressed very high levels of functional pyruvate carboxylase. Use of pEFIRES-P should permit the rapid and efficient generation of stable cell lines for the characterisation of recombinant proteins., (Copyright 1998 Academic Press.)

We have identified regions in the promoters of the PYC1 and PYC2 genes from Saccharomyces cerevisiae involved in their regulation in different culture conditions. In the case of PYC1, a UAS in the region between -330/-297 and three repressing sequences with the common central core CCGCC at positions -457, -432 and -399 were identified. Specific binding of nuclear proteins to the -330/-214 DNA fragment was abolished in rtg mutants suggesting a role for the RTG genes in the control of PYC1 expression. In the case of the PYC2 promoter, elimination of a fragment from -417 to -291 brings about a two-fold decrease in the expression in repressed conditions and a similar increase in derepression.

Pyruvate carboxylase (PYC) is a biotin-dependent enzyme catalyzing the anaplerotic conversion of pyruvate to oxaloacetate in Rhizobium etli strain CE3. A pyc::Tn5 mutant had severely reduced growth, or failed to grow on sugars, three-carbon organic acids or glycerol, consistent with these substrates being metabolized via pyruvate. Transconjugants expressing a pyc::beta-glucuronidase gene fusion had slightly increased apparent pyc transcription during growth on pyruvate as compared to succinate, similar to the modest carbon source dependent changes in PYC activity reported previously. Biotin supplementation of cultures growing on pyruvate dramatically increased PYC activity but not apparent pyc transcription. Bacteroids isolated from bean nodules did not contain detectable PYC activity while apparent pyc transcription occurred at a moderate level.

The synthesis of pyruvate carboxylase (PC) was studied by using quantitative immunoblot analysis with an antibody raised against PC purified from Rhodobacter capsulatus and was found to vary 20-fold depending on the growth conditions. The PC content was high in cells grown on pyruvate or on carbon substrates metabolized via pyruvate (lactate, D-malate, glucose, or fructose) and low in cells grown on tricarboxylic acid (TCA) cycle intermediates or substrates metabolized without intermediate formation of pyruvate (acetate or glutamate). Under dark aerobic growth conditions with lactate as a carbon source, the PC content was approximately twofold higher than that found under light anaerobic growth conditions. The results of incubation experiments demonstrate that PC synthesis is induced by pyruvate and repressed by TCA cycle intermediates, with negative control dominating over positive control. The content of PC in R. capsulatus cells was also directly related to the growth rate in continuous cultures. The analysis of intracellular levels of pyruvate and TCA cycle intermediates in cells grown under different conditions demonstrated that the content of PC is directly proportional to the ratio between pyruvate and C4 dicarboxylates. These results suggest that the regulation of PC synthesis by oxygen and its direct correlation with growth rate may reflect effects on the balance of intracellular pyruvate and C4 dicarboxylates. Thus, this important enzyme is potentially regulated both allosterically and at the level of synthesis.

We have identified and characterized multiple mRNA transcripts of rat and human pyruvate carboxylases [EC 6.4.1.1] using rapid amplification of cDNA 5' ends-polymerase chain reaction (RACE-PCR). Five alternative forms of rat pyruvate carboxylase cDNAs have been identified in liver, kidney, brain, and adipose tissue and these are expressed in a tissue-specific manner. Two alternative forms of human pyruvate carboxylase cDNA have also been identified in liver. These pyruvate carboxylase cDNAs have a common coding region but differ in their 5' untranslated regions (5'UTRs), suggesting that they are generated by alternative splicing of the primary transcript. Southern blot analysis of restriction enzyme digested rat genomic DNA revealed that pyruvate carboxylase is encoded by a single copy gene.

The activity and level of hepatic pyruvate carboxylase (PC) has been reported to be altered by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment in mice. If alteration in PC level/activity by TCDD influences TCDD toxicity, one would expect to observe an early post-exposure reduction in PC mRNA. To examine the molecular events responsible for the alteration of PC activity in livers of TCDD-treated mice, we designed a synthetic DNA oligonucleotide probe specific for PC mRNA. Northern blot analysis on RNA extracts from hepatic tissue at various times and doses post-TCDD exposure were done. Furthermore, to elucidate the role of the dioxin Ah locus on alterations of PC activity by TCDD, we utilized C57BL/6J (Ahb/b, Ah high TCDD affinity) mice and a congenic (Ahd/d, AH low TCDD affinity) mouse strain. At 8 days post TCDD treatment, a dose-dependent reduction of hepatic PC mRNA levels was observed in Ahb/b mice. The response, reduction in PC mRNA levels, in the Ahb/b strain was about 10-fold greater than that of comparably exposed congenic Ahd/d mice. These results indicate that previously reported reductions in PC activity/level by TCDD treatment of mice is a consequence of a reduction in PC mRNA levels and that the effect requires a competent Ah receptor.

Pancreatic islets were cultured for 1 day in the presence of 1 to 20 mM glucose and islet proteins were separated on polyacrylamide gels and transferred to nitrocellulose. Pyruvate carboxylase and an unidentified biotin-containing protein were visualized with [125I]streptavidin followed by autoradiography. The amount of pyruvate carboxylase was proportional to the concentration of glucose. Estimates of the amount of the enzyme in islets were made by comparing the density of the islet pyruvate carboxylase band with a standard curve of various amounts of authentic pyruvate carboxylase. This indicated that the enzyme comprised 0.4% of total islet protein. Net synthesis of the enzyme was increased by cAMP and methyl succinate. A nuclear run-on assay showed that glucose caused increases in pyruvate carboxylase and pyruvate dehydrogenase E1 alpha subunit transcripts and decreases in branched chain ketoacid dehydrogenase E1 alpha transcripts in rat insulinoma (RINm5F) cells. Pancreatic islets cultured in the presence of 1 mM glucose for 1 day cannot respond to glucose with insulin release. Previous studies demonstrated that carbon flux into the citric acid cycle intermediates via both carboxylation and decarboxylation is decreased in glucose-incapacitated islets (M. J. MacDonald, 1993, Arch. Biochem. Biophys. 300, 205-214), 1993). The current results support the idea that carboxylation of glucose-derived pyruvate, as well as decarboxylation of pyruvate, is important for glucose-induced insulin secretion.

The arylhydrocarbon receptor (AhR) plays a central role in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity in animals. The investigations described here provide evidence that support a role for the AhR in TCDD-mediated pyruvate carboxylase (PC) level/activity reductions in mice. Pyruvate carboxylase plays a pivotal role in gluconeogenesis and in supplying carbon units for the citric acid cycle. Delivered ip in a corn oil carrier, TCDD suppresses PC activity/amount at doses as low as 1 microgram/kg in responsive C57BL/6J(Ahb/b) mice. Corn oil alone injected ip into mice at 4 mL/kg appears to be an inducer that increases the amount and activity of PC. However, TCDD suppresses this induction. In the Ahb/b mouse, PC levels and activity are reduced to 10% of control values at a dose of 75 micrograms/kg. A time-course experiment shows that the PC reductions are apparent within 16 hours post-TCDD exposure. Here we report investigations on the PC/TCDD response using a congenic C57BL/6J(Ahd/d) mouse strain having an AhR with a low affinity for TCDD. If the PC/TCDD response is AhR mediated, the congenic mouse strain (Ahd/d) would require much higher doses of TCDD to suppress PC. In the Ahd/d mice, we observe that an approximately 60-fold increase in TCDD dose is necessary to produce a PC/TCDD effect. We also find that in Ahd/d mice, corn oil does not induce an increase in PC activity/amounts, as reported for Ahb/b mice.

Peroxidase-conjugated avidin was used to detect the endogenous avidin-binding proteins in rat tissues. By a transblot method, avidin-peroxidase interacted with proteins of mitochondrial fractions of rat liver with estimated molecular weights of 120,000 and 74,000. The proteins were identified as pyruvate carboxylase (120 kDa, pI 6.4) and methylcrotonyl-CoA carboxylase (74 kDa, pI 7.2) by two-dimensional gel electrophoresis and transblot method. The estimated molecular weight of an additional band (220,000) detected in the cytosolic fraction of rat liver was consistent with acetyl-CoA carboxylase. Intense staining also occurred with kidney, heart, ovary and adipose tissue, moderate with large and small intestine, cerebrum and cerebellum, and very faint with testis, lung and spleen. The sections of rat liver embedded in LR white were incubated with avidin-colloidal gold conjugate and examined under an electron microscope. The glutaraldehyde-perfused rat liver blocks were also incubated with streptavidin-ferritin conjugate and the ultrathin sections were cut and examined. The majority of gold and ferritin particles were found in the mitochondria of liver cells. No other cellular compartment was labeled except the cytosol which accounted for approximately 20% of the total labeling of the hepatocytes. The present procedure is a simple, rapid and inexpensive method for detecting the intracellular localization of endogenous avidin-binding proteins in the cells.

The level of pyruvate carboxylase in Pseudmonas citronellolis is controlled by the carbon source of the growth medium. The activity of the enzyme is highest in cells grown on lactate or glucose and virtually absent in cells grown on malate or aspartate. Double labeling studies with 3H- and 14C-labeled leucine confirm that pyruvate carboxylase is synthesized in the presence of lactate but not in the presence of aspartate. The studies also show that coordinated regulation occurs at the level of the synthesis of the two polypeptides which make up pyruvate carboxylase in P. citronellolis, rather than at the stages of their assembly into protomers or the biotinylation of the apoenzyme. There is no evidence for control of the catalytic acitivity of the holoenzyme via effectors. In all other varieties of pyruvate carboxylase examined thus far, the enzyme appears to be constitutive with regulation accomplished either through effector modulation of holoenzyme activity (pyruvate carbox-lase from animal sources, yeast, several species of bacteria) or through control of the biotinylation of the apoenzyme by holocarboxylase synthetase (Bacillus stearothermophilus, yeast).

1. The effect of aeration on the key enzymes of gluconeogenesis was studied in baker's yeast (Saccharomyces cerevisiae) and in a nonrespiratory variant of S. cerevisiae grown under glucose limitation. 2. In baker's yeast phosphoenolpyruvate carboxykinase, hexosediphophatase and isocitrate lyase were completely repressed under anaerobic conditions. Their repression could be partially reversed by using intense aeration. 3. In the nonrespiratory variant these enzymes were absent independently of aeration. 4. Pyruvate carboxylase of baker's yeast showed maximal activity under anaerobic conditions. In the nonrespiratory variant pyruvate carboxylase had low activity under both anaerobic and aerobic conditions.

Eight cases of isolated human pyruvate carboxylase deficiency were examined from seven families. Although all patients presented with a chronic lacticacidemia, two particular patients presented with the added features of hyperammonemia, citrullinemia, and hyperlysinemia. When cultured skin fibroblasts from these patients were examined for their ability to synthesize [3H]biotin-containing proteins, it was found that the two patients who presented with hyperammonemia, citrullinemia, and hyperlysinemia did not synthesise a protein of the correct subunit molecular weight (Mr = 125 K daltons) corresponding to pyruvate carboxylase. In addition, when skin fibroblast proteins were labeled with [35S]methionine, cross-reacting material (CRM) corresponding to pyruvate carboxylase was immunoprecipitated by antipyruvate carboxylase antiserum in most patients, but again the two patients with the atypical presentation showed no CRM. We propose that the different clinical presentation of human pyruvate carboxylase deficiency is a manifestation of two different mutations in the pyruvate carboxylase gene, one that results in the synthesis of a relatively inactive pyruvate carboxylase protein CRM(+ve) and one that results in the lack of expression of the gene in the form of a recognizable protein CRM(-ve).

Cell extracts of Pseudomonas aeruginosa strain PAO were found to contain pyruvate carboxylase activity. Specific activities were minimal when cells were grown on Casamino Acids, acetate, or succinate, but were three- to fourfold higher when cells were grown in glucose, gluconate, glycerol, lactate, or pyruvate minimal media. The reaction in crude cell extracts and in partially purified preparations was dependent on pyruvate, adenosine 5'-triphosphate, and Mg(2+), but was not affected by either the presence or absence of acetyl coenzyme A. Activity was nearly totally inhibited by avidin and this inhibition was substantially blocked by free biotin in incubation mixtures. Cell extracts were shown to fix (14)CO(2) in a reaction that had these same characteristics. Eight pleiotropic, carbohydrate-negative mutant strains of the organism were isolated after nitrosoguanidine mutagenesis. Each mutant strain grew normally in acetate, succinate, and citrate minimal media but failed to utilize glucose, gluconate, 2-ketogluconate, mannitol, glycerol, lactate, and pyruvate as sole sources of carbon and energy. These strains were found by quantitative transductional analysis with phage F116 to form a single linkage group. Cell extracts of each mutant strain were either lacking or severely deficient in pyruvate carboxylase activity. Spontaneous revertants of five of the eight strains were isolated and found to recover simultaneously both pyruvate carboxylase activity and the ability to utilize each of the C(6) and C(3) compounds. A second linkage group of similar mutant strains that grew on the C(3) compounds was found to contain normal levels of pyruvate carboxylase activity, but each strain was deficient in an enzyme of the Entner-Doudoroff pathway.

The differentiation of mouse 3T3-L1 cells is characterized by an accumulation of cytosolic triglyceride and marked increase in many enzymatic activities involved in triglyceride biosynthesis. The specific activity of one such enzyme, pyruvate carboxylase, increases at least 20-fold and is due to a parallel increase in the intracellular concentration of the protein. Pulse-labeling experiments demonstrated that the increase in the specific activity of pyruvate carboxylase was due to an increase in the rate of enzyme synthesis. In the differentiated cell, pyruvate carboxylase represented 1.9% of the total cellular protein and 1% of the protein radiolabeled during a 1-h pulse. This was 35-and 28-fold higher than in the undifferentiated cell, respectively. The turnover of pyruvate carboxylase in the differentiated cell was similar to that in the undifferentiated cell with the enzyme having a half-life of 28-35 h. The half-life of apopyruvate carboxylase in avidin-treated 3T3-L1 cells was 24 h, indicating that the turnover of the apoenzyme was not significantly different than that of the holoenzyme. Radiolabeling pyruvate carboxylase with [14C]biotin and [3H]leucine demonstrated that the turnover of biotin associated with the enzyme was identical to the turnover of the enzymatic protein.

The specific activity of pyruvate carboxylase [pyruvate:carbon-dioxide ligase (ADP-forming); EC 6.4.1.1] in 3T3-L1 cells increases approximately 20-fold when these cells differentiate to an adipocyte-like form [Mackall, J. C. & Lane, M. D. (1977) Biochem. Biophys. Res. Commun. 79, 720-725]. A specific antibody to the purified rat liver enzyme quantitatively precipitated pyruvate carboxylase from 3T3-L1 crude homogenates. Use of this immunological technique permitted us to demonstrate that the increase in pyruvate carboxylase activity is due to an increase in the intracellular concentration of the enzyme. The content of pyruvate carboxylase in differentiated 3T3-L1 cells is sufficiently high (1-2% of total protein) that the increase in this large protein (subunit M(r) = 130,000) can be visualized when 3T3-L1 crude extracts are subjected to electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. When 3T3-L1 cells differentiated in the presence of avidin, they contained less than 5% of the pyruvate carboxylase activity of cells that differentiated in the absence of avidin. However, the immunoprecipitable pyruvate carboxylase content of the avidin-treated cells was essentially the same as that of cells that differentiated without avidin. Full activity of the enzyme was rapidly restored in the avidin-treated cells upon the addition of excess biotin. The recovery of activity was closely correlated with the incorporation of [(14)C]biotin into immunoprecipitable pyruvate carboxylase. The rapidity with which the activity was restored and the insensitivity of the process to inhibitors of protein synthesis strongly suggest that the apoenzyme of pyruvate carboxylase accumulates during differentiation in the presence of avidin.