Biotin concentration affects anaplerotic reactions functioning in glutamic acid production in Corynebacterium glutamicum.

The study investigates the effect of biotin concentration on the role of anaplerotic reactions catalysed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) in glutamic acid production by Corynebacterium glutamicum. C. glutamicum requires biotin for its growth, and its glutamic acid production can be induced by the addition of Tween 40 or penicillin or by biotin limitation. The biotin enzyme PC and the non-biotin enzyme PEPC catalyse two anaplerotic reactions to supply oxaloacetic acid to the TCA cycle in C. glutamicum. Therefore, they are crucial for glutamic acid production in this bacterium. In this study, we investigated the contribution of each anaplerotic reaction to Tween 40- and penicillin-induced glutamic acid production using disruptants of PEPC and PC. In the presence of 20 µg l-1 biotin, which is sufficient for growth, the PEPC-catalysed anaplerotic reaction mainly contributed to Tween 40- and penicillin-induced glutamic acid production. However, when increasing biotin concentration 10-fold (i.e. 200 µg l-1), both PC- and PEPC-catalysed reactions could function in glutamic acid production. Western blotting revealed that the amount of biotin-bound PC was reduced by the addition of Tween 40 and penicillin in the presence of 20 µg l-1. However, these induction treatments did not change the amount of biotin-bound PC in the presence of 200 µg l-1 biotin. These results indicate that both anaplerotic reactions are functional during glutamic acid production in C. glutamicum and that biotin concentration mainly affects which anaplerotic reactions function during glutamic acid production.

Glucagon promotes increased hepatic mitochondrial oxidation and pyruvate carboxylase flux in humans with fatty liver disease.

We assessed in vivo rates of hepatic mitochondrial oxidation, gluconeogenesis, and ß-hydroxybutyrate (ß-OHB) turnover by positional isotopomer NMR tracer analysis (PINTA) in individuals with metabolic-dysfunction-associated steatotic liver (MASL) (fatty liver) and MASL disease (MASLD) (steatohepatitis) compared with BMI-matched control participants with no hepatic steatosis. Hepatic fat content was quantified by localized 1H magnetic resonance spectroscopy (MRS). We found that in vivo rates of hepatic mitochondrial oxidation were unaltered in the MASL and MASLD groups compared with the control group. A physiological increase in plasma glucagon concentrations increased in vivo rates of hepatic mitochondrial oxidation by 50%-75% in individuals with and without MASL and increased rates of glucose production by ∼50% in the MASL group, which could be attributed in part to an ∼30% increase in rates of mitochondrial pyruvate carboxylase flux. These results demonstrate that (1) rates of hepatic mitochondrial oxidation are not substantially altered in individuals with MASL and MASLD and (2) glucagon increases rates of hepatic mitochondrial oxidation.

Pyruvate Carboxylase Attenuates Myocardial Ischemia-Reperfusion Injury in Heart Transplantation via Wnt/ß-Catenin-Mediated Glutamine Metabolism.

The ischemia-reperfusion process of a donor heart during heart transplantation leads to severe mitochondrial dysfunction, which may be the main cause of donor heart dysfunction after heart transplantation. Pyruvate carboxylase (PC), an enzyme found in mitochondria, is said to play a role in the control of oxidative stress and the function of mitochondria. This research examined the function of PC and discovered the signaling pathways controlled by PC in myocardial IRI. We induced IRI using a murine heterotopic heart transplantation model in vivo and a hypoxia-reoxygenation cell model in vitro and evaluated inflammatory responses, oxidative stress levels, mitochondrial function, and cardiomyocyte apoptosis. In both in vivo and in vitro settings, we observed a significant decrease in PC expression during myocardial IRI. PC knockdown aggravated IRI by increasing MDA content, LDH activity, TUNEL-positive cells, serum cTnI level, Bax protein expression, and the level of inflammatory cytokines and decreasing SOD activity, GPX activity, and Bcl-2 protein expression. PC overexpression yielded the opposite findings. Additional research indicated that reducing PC levels could block the Wnt/ß-catenin pathway and glutamine metabolism by hindering the movement of ß-catenin to the nucleus and reducing the activity of complex I and complex II, as well as ATP levels, while elevating the ratios of NADP+/NADPH and GSSG/GSH. Overall, the findings indicated that PC therapy can shield the heart from IRI during heart transplantation by regulating glutamine metabolism through the Wnt/ß-catenin pathway.

Acetate enables metabolic fitness and cognitive performance during sleep disruption.

Sleep is essential for overall health, and its disruption is linked to increased risks of metabolic, cognitive, and cardiovascular dysfunctions; however, the molecular mechanisms remain poorly understood. This study investigated how sleep disturbances contribute to metabolic imbalance and cognition impairment using a chronic sleep fragmentation (SF) mouse model. SF mice exhibited impaired cognition, glucose metabolism, and insulin sensitivity compared with controls. We identified increased acetate levels in hypothalamic astrocytes as a defensive response in SF mice. Through acetate infusion or astrocyte-specific Acss1 deletion to elevate acetate levels, we observed mitigated metabolic and cognitive impairments in SF mice. Mechanistically, acetate binds and activates pyruvate carboxylase, thereby restoring glycolysis and the tricarboxylic acid cycle. Among individuals most commonly affected by SF, patients with obstructive sleep apnea exhibited elevated acetate levels when coupled with type 2 diabetes. Our study uncovers the protective effect of acetate against sleep-induced metabolic and cognitive impairments.

Hyperpolarized [2-13C, 3-2H3]Pyruvate Detects Hepatic Gluconeogenesis In Vivo.

The feasibility of hyperpolarized [2-13C, 3-2H3]pyruvate for probing gluconeogenesis in vivo was investigated in this study. Whereas hyperpolarized [1-13C]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 13C. To prolong the T1 relaxation time, [2-13C, 3-2H3]pyruvate was synthesized and dissolved with D2O after dynamic nuclear polarization. The T1 of [2-13C, 3-2H3]pyruvate in D2O could be improved by 76.9% (79.6 s at 1 T and 74.5 s at 3 T) as compared to [2-13C]pyruvate in water. Hyperpolarized [2-13C, 3-2H3]pyruvate with D2O dissolution was applied to rat livers in vivo under normal feeding and fasting conditions. A gluconeogenic product, [2-13C]phosphoenolpyruvate, was observed at 149.9 ppm from fasted rats only, highlighting the utility of [2-13C, 3-2H3]pyruvate in detecting key gluconeogenic enzyme activities such as pyruvate carboxylase and phosphoenolpyruvate carboxykinase in vivo.

Clinical, biochemical, and molecular profiles of three Sri Lankan neonates with pyruvate carboxylase deficiency.

Objectives: Pyruvate carboxylase, a mitochondrial enzyme, catalyses the conversion of glycolytic end-product pyruvate to tricarboxylic acid cycle intermediate, oxaloacetate. Rare pyruvate carboxylase deficiency manifests in three clinical and biochemical phenotypes: neonatal onset type A, infantile onset type B and a benign C type. The objective of this case series is to expand the knowledge of overlapping clinical and biochemical phenotypes of pyruvate carboxylase deficiency. Case presentation: We report three Sri Lankan neonates including two siblings, of two unrelated families with pyruvate carboxylase deficiency. All three developed respiratory distress within the first few hours of birth. Two siblings displayed typical biochemical findings reported in type B. The other proband with normal citrulline, lysine, moderate lactate, paraventricular cystic lesions, bony deformities, and a novel missense, homozygous variant c.2746G>C [p.(Asp916His)] in the PC gene, biochemically favoured type A. Conclusions: Our findings indicate the necessity of prompt laboratory investigations in a tachypneic neonate with coexisting metabolic acidosis, as early recognition is essential for patient management and family counselling. Further case studies are required to identify overlapping symptoms and biochemical findings in different types of pyruvate carboxylase deficiency phenotypes.

Contributions of the anaplerotic reaction enzymes pyruvate carboxylase and phosphoenolpyruvate carboxylase to l-lysine production in Corynebacterium glutamicum.

Anaplerotic reactions catalyzed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) have important roles in the production of l-lysine to replenish oxaloacetic acid (OAA) in Corynebacterium glutamicum. However, the relative contributions of these enzymes to l-lysine production in C. glutamicum are not fully understood. In this study, using a parent strain (P) carrying a feedback inhibition-resistant aspartokinase with the T311I mutation, we constructed a PC gene-deleted mutant strain (PΔPC) and a PEPC gene-deleted mutant strain (PΔPEPC). Although the growth of both mutant strains was comparable to the growth of strain P, the maximum l-lysine production in strains PΔPC and PΔPEPC decreased by 14% and 49%, respectively, indicating that PEPC strongly contributed to OAA supply. l-Lysine production in strain PΔPC slightly decreased during the logarithmic phase, while production during the early stationary phase was comparable to production in strain P. By contrast, strain PΔPEPC produced l-lysine in an amount comparable to the production of strain P during the logarithmic phase; l-lysine production after the early stationary phase was completely stopped in strain PΔPEPC. These results indicate that OAA is supplied by both PC and PEPC during the logarithmic phase, while only PEPC can continuously supply OAA after the logarithmic phase.

Engineering the reductive tricarboxylic acid pathway in Aureobasidium pullulans for enhanced biosynthesis of poly-L-malic acid.

Aureobasidium pullulans produced poly-L-malic acid (PMA) as the main metabolite in fermentation but with relatively low productivity and yield limiting its industrial application. In this study, A. pullulans ZX-10 was engineered to overexpress cytosolic malate dehydrogenase (MDH) and pyruvate carboxylase (PYC) and PMA synthetase (PMS) using a high-copy yeast episomal plasmid with the gpdA promoter from Aspergillus nidulans. Overexpressing endogenous PMS and heterologous MDH and PYC from Aspergillus oryzae respectively increased PMA production by 19 % - 37 % (0.64 - 0.74 g/g vs. 0.54 g/g for wild type) in shake-flask fermentations, demonstrating the importance of the reductive tricarboxylic acid (rTCA) pathway in PMA biosynthesis. A. pullulans co-expressing MDH and PYC produced 96.7 g/L PMA at 0.90 g/L∙h and 0.68 g/g glucose in fed-batch fermentation, which were among the highest yield and productivity reported. The engineered A. pullulans with enhanced rTCA pathway is advantageous and promising for PMA production.

Zonated iron deposition in the periportal zone of the liver is associated with selectively enhanced lipid synthesis.

BACKGROUND AND AIMS: Disorders in liver lipid metabolism have been implicated in a range of metabolic conditions, including fatty liver and liver cancer. Altered lipid distribution within the liver, shifting from the pericentral to the periportal zone under pathological circumstances, has been observed; however, the underlying mechanism remains elusive. Iron, an essential metal, exhibits a zonal distribution in the liver similar to that of lipids. Nevertheless, the precise relationship between iron and lipid distribution, especially in the pericentral and periportal zones, remains poorly understood. METHODS: We conducted comprehensive in vitro and in vivo experiments, combining with in situ analysis and RNA sequencing, aiming for a detailed exploration of the causal relationship between iron accumulation and lipid metabolism. RESULTS: Our research suggests that iron overload can disrupt the normal distribution of lipids within the liver, particularly in the periportal zone. Through meticulous gene expression profiling in both the pericentral and periportal zones, we identified pyruvate carboxylase (PC) as a pivotal regulator in iron overload-induced lipid accumulation. Additionally, we revealed that the activation of cyclic adenosine monophosphate response element binding protein (CREB) was indispensable for Pc gene expression when in response to iron overload. CONCLUSIONS: In summary, our investigation unveils the crucial involvement of iron overload in fostering hepatic lipid accumulation in the periportal zone, at least partly mediated by the modulation of Pc expression. These insights offer new perspectives for understanding the pathogenesis of fatty liver diseases and their progression.

Anemoside B4, a new pyruvate carboxylase inhibitor, alleviates colitis by reprogramming macrophage function.

OBJECTIVES: Colitis is a global disease usually accompanied by intestinal epithelial damage and intestinal inflammation, and an increasing number of studies have found natural products to be highly effective in treating colitis. Anemoside B4 (AB4), an abundant saponin isolated from Pulsatilla chinensis (Bunge), which was found to have strong anti-inflammatory activity. However, the exact molecular mechanisms and direct targets of AB4 in the treatment of colitis remain to be discovered. METHODS: The anti-inflammatory activities of AB4 were verified in LPS-induced cell models and 2, 4, 6-trinitrobenzene sulfonic (TNBS) or dextran sulfate sodium (DSS)-induced colitis mice and rat models. The molecular target of AB4 was identified by affinity chromatography analysis using chemical probes derived from AB4. Experiments including proteomics, molecular docking, biotin pull-down, surface plasmon resonance (SPR), and cellular thermal shift assay (CETSA) were used to confirm the binding of AB4 to its molecular target. Overexpression of pyruvate carboxylase (PC) and PC agonist were used to study the effects of PC on the anti-inflammatory and metabolic regulation of AB4 in vitro and in vivo. RESULTS: AB4 not only significantly inhibited LPS-induced NF-κB activation and increased ROS levels in THP-1 cells, but also suppressed TNBS/DSS-induced colonic inflammation in mice and rats. The molecular target of AB4 was identified as PC, a key enzyme related to fatty acid, amino acid and tricarboxylic acid (TCA) cycle. We next demonstrated that AB4 specifically bound to the His879 site of PC and altered the protein's spatial conformation, thereby affecting the enzymatic activity of PC. LPS activated NF-κB pathway and increased PC activity, which caused metabolic reprogramming, while AB4 reversed this phenomenon by inhibiting the PC activity. In vivo studies showed that diisopropylamine dichloroacetate (DADA), a PC agonist, eliminated the therapeutic effects of AB4 by changing the metabolic rearrangement of intestinal tissues in colitis mice. CONCLUSION: We identified PC as a direct cellular target of AB4 in the modulation of inflammation, especially colitis. Moreover, PC/pyruvate metabolism/NF-κB is crucial for LPS-driven inflammation and oxidative stress. These findings shed more light on the possibilities of PC as a potential new target for treating colitis.

In silico Analysis of Two Novel Variants in the Pyruvate Carboxylase (PC) Gene Associated with the Severe Form of PC Deficiency.

Background: Inborne errors of metabolism are a common cause of neonatal death. This study evaluated the acute early-onset metabolic derangement and death in two unrelated neonates. Methods: Whole-exome sequencing (WES), Sanger sequencing, homology modeling, and in silico bioinformatics analysis were employed to assess the effects of variants on protein structure and function. Results: WES revealed a novel homozygous variant, p.G303Afs*40 and p.R156P, in the pyruvate carboxylase (PC) gene of each neonate, which both were confirmed by Sanger sequencing. Based on the American College of Medical Genetics and Genomics guidelines, the p.G303Afs*40 was likely pathogenic, and the p.R156P was a variant of uncertain significance (VUS). Nevertheless, a known variant at position 156, the p.R156Q, was also a VUS. Protein secondary structure prediction showed changes in p.R156P and p.R156Q variants compared to the wild-type protein. However, p.G303Afs*40 depicted significant changes at C-terminal. Furthermore, comparing the interaction of wild-type and variant proteins with the ATP ligand during simulations, revealed a decreased affinity to the ATP in all the variants. Moreover, analysis of Single nucleotide polymorphism impacts on PC protein using Polyphen-2, SNAP2, FATHMM, and SNPs&GO servers predicted both R156P and R156Q as damaging variants. Likewise, free energy calculations demonstrated the destabilizing effect of both variants on PC. Conclusion: This study confirmed the pathogenicity of both variants and suggested them as a cause of type B Pyruvate carboxylase deficiency. The results of this study would provide the family with prenatal diagnosis and expand the variant spectrum in the PC gene,which is beneficial for geneticists and endocrinologists.

Comprehensive quantification of metabolic flux during acute cold stress in mice.

Cold-induced thermogenesis (CIT) is widely studied as a potential avenue to treat obesity, but a thorough understanding of the metabolic changes driving CIT is lacking. Here, we present a comprehensive and quantitative analysis of the metabolic response to acute cold exposure, leveraging metabolomic profiling and minimally perturbative isotope tracing studies in unanesthetized mice. During cold exposure, brown adipose tissue (BAT) primarily fueled the tricarboxylic acid (TCA) cycle with fat in fasted mice and glucose in fed mice, underscoring BAT's metabolic flexibility. BAT minimally used branched-chain amino acids or ketones, which were instead avidly consumed by muscle during cold exposure. Surprisingly, isotopic labeling analyses revealed that BAT uses glucose largely for TCA anaplerosis via pyruvate carboxylation. Finally, we find that cold-induced hepatic gluconeogenesis is critical for CIT during fasting, demonstrating a key functional role for glucose metabolism. Together, these findings provide a detailed map of the metabolic rewiring driving acute CIT.

Comprehensive isotopomer analysis of glutamate and aspartate in small tissue samples.

Stable isotopes are powerful tools to assess metabolism. 13C labeling is detected using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS). MS has excellent sensitivity but generally cannot discriminate among different 13C positions (isotopomers), whereas NMR is less sensitive but reports some isotopomers. Here, we develop an MS method that reports all 16 aspartate and 32 glutamate isotopomers while requiring less than 1% of the sample used for NMR. This method discriminates between pathways that result in the same number of 13C labels in aspartate and glutamate, providing enhanced specificity over conventional MS. We demonstrate regional metabolic heterogeneity within human tumors, document the impact of fumarate hydratase (FH) deficiency in human renal cancers, and investigate the contributions of tricarboxylic acid (TCA) cycle turnover and CO2 recycling to isotope labeling in vivo. This method can accompany NMR or standard MS to provide outstanding sensitivity in isotope-labeling experiments, particularly in vivo.

Case Report: Prenatal neurological injury in a neonate with pyruvate carboxylase deficiency type B.

Background: Pyruvate carboxylase (PC) is a key enzyme for gluconeogenesis. PC deficiency (PCD) is an extremely rare autosomal recessive metabolic disease and is divided into three types. Type B PCD is clinically featured by lactic acidosis, hyperammonemia, hypercitrullinemia, hypotonia, abnormal movement, and seizures. Case presentation: Here, we report the first case of type B PCD in China, presenting with intractable lactic acidosis shortly after birth. A compound heterozygous mutation in the PC gene was identified by whole-exome sequencing, NM_001040716.2: c.1154_1155del and c.152G>A, which were inherited from her asymptomatic parents, respectively. Furthermore, prenatal neuroradiological presentations including widened posterior horns of lateral ventricles, huge subependymal cysts, and increased biparietal diameter and head circumference were concerned. Symptomatic treatment was taken and the infant died at 26 days. Conclusion: To our knowledge, this is the minimum gestational age (22w5d) that's when the prenatal onset of the neuroradiologic phenotype of PCD was observed. PCD has a poor prognosis and lacks an effective treatment, so this paper is shared to highlight the importance of PCD prenatal diagnosis in the absence of family history.

The adipose-derived stem cell peptide ADSCP2 alleviates hypertrophic scar fibrosis via binding with pyruvate carboxylase and remodeling the metabolic landscape.

AIM: The purpose of this study was to investigate the function and mechanism of a novel peptide derived from adipose-derived stem cell-conditioned medium (ADSC-CM). METHODS: Mass spectrometry was applied to identify expressed peptides in ADSC-CM obtained at different time points. The cell counting kit-8 assay and quantitative reverse transcription polymerase chain reactions were performed to screen the functional peptides contained within ADSC-CM. RNA-seq, western blot, a back skin excisional model of BALB/c mice, the peptide pull-down assay, rescue experiments, untargeted metabolomics, and mixOmics analysis were performed to thoroughly understand the functional mechanism of selected peptide. RESULTS: A total of 93, 827, 1108, and 631 peptides were identified in ADSC-CM at 0, 24, 48, and 72 h of conditioning, respectively. A peptide named ADSCP2 (DENREKVNDQAKL) derived from ADSC-CM inhibited collagen and ACTA2 mRNAs in hypertrophic scar fibroblasts. Moreover, ADSCP2 facilitated wound healing and attenuated collagen deposition in a mouse model. ADSCP2 bound with the pyruvate carboxylase (PC) protein and inhibited PC protein expression. Overexpressing PC rescued the reduction in collagen and ACTA2 mRNAs caused by ADSCP2. Untargeted metabolomics identified 258 and 447 differential metabolites in the negative and positive mode, respectively, in the ADSCP2-treated group. The mixOmics analysis, which integrated RNA-seq and untargeted metabolomics data, provided a more holistic view of the functions of ADSCP2. CONCLUSION: Overall, a novel peptide derived from ADSC-CM, named ADSCP2, attenuated hypertrophic scar fibrosis in vitro and in vivo, and the novel peptide ADSCP2 might be a promising drug candidate for clinical scar therapy.

Immunodetection of Pyruvate Carboxylase Expression in Human Astrocytomas, Glioblastomas, Oligodendrogliomas, and Meningiomas.

Pyruvate carboxylase (PC) is an enzyme catalyzing the carboxylation of pyruvate to oxaloacetate. The enzymatic generation of oxaloacetate, an intermediate of the Krebs cycle, could provide the cancer cells with the additional anaplerotic capacity and promote their anabolic metabolism. Recent studies revealed that several types of cancer cells express PC. The gained anaplerotic capability of cells mediated by PC correlates with their expedited growth, higher aggressiveness, and increased metastatic potential. By immunohistochemical staining and immunoblotting analysis, we investigated PC expression among samples of different types of human brain tumors. Our results show that PC is expressed by the cells in glioblastoma, astrocytoma, oligodendroglioma, and meningioma tumors. The presence of PC in these tumors suppose that PC could support the anabolic metabolism of their cellular constituents by its anaplerotic capability.

Stepwise metabolic engineering of Corynebacterium glutamicum for the production of phenylalanine.

Corynebacterium glutamicum was metabolically engineered to produce phenylalanine, a valuable aromatic amino acid that can be used as a raw material in the food and pharmaceutical industries. First, a starting phenylalanine-producer was constructed by overexpressing tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and phenylalanine- and tyrosine-insensitive bifunctional enzyme chorismate mutase prephenate dehydratase from Escherichia coli, followed by the inactivation of enzymes responsible for the formation of dihydroxyacetone and the consumption of shikimate pathway-related compounds. Second, redirection of the carbon flow from tyrosine to phenylalanine was attempted by deleting of the tyrA gene encoding prephenate dehydrogenase, which catalyzes the committed step for tyrosine biosynthesis from prephenate. However, suppressor mutants were generated, and two mutants were isolated and examined for phenylalanine production and genome sequencing. The suppressor mutant harboring an amino acid exchange (L180R) on RNase J, which was experimentally proven to lead to a loss of function of the enzyme, showed significantly enhanced production of phenylalanine. Finally, modifications of phosphoenolpyruvate-pyruvate metabolism were investigated, revealing that the inactivation of either phosphoenolpyruvate carboxylase or pyruvate carboxylase, which are enzymes of the anaplerotic pathway, is an effective means for improving phenylalanine production. The resultant strain, harboring a phosphoenolpyruvate carboxylase deficiency, synthesized 50.7 mM phenylalanine from 444 mM glucose. These results not only provided new insights into the practical mutations in constructing a phenylalanine-producing C. glutamicum but also demonstrated the creation of a potential strain for the biosynthesis of phenylalanine-derived compounds represented by plant secondary metabolites.

Overexpression of Setaria italica phosphoenolpyruvate carboxylase gene in rice positively impacts photosynthesis and agronomic traits.

C4 plants have the inherent capacity to concentrate atmospheric CO2 in the vicinity of RuBisCo, thereby increasing carboxylation, and inhibiting photorespiration. Carbonic anhydrase (CA), the first enzyme of C4 photosynthesis, converts atmospheric CO2 to HCO3-, which is utilized by PEPC to produce C4 acids. Bioengineering of C4 traits into C3 crops is an attractive strategy to increase photosynthesis and water use efficiency. In the present study, we isolated the PEPC gene from the C4 plant Setaria italica and transferred it to C3 rice. Overexpression of SiPEPC resulted in a 2-6-fold increment in PEPC enzyme activity in transgenic lines with respect to non-transformed control. Photosynthetic efficiency was enhanced in transformed plants, which was associated with increased ФPSII, ETR, lower NPQ, and higher chlorophyll accumulation. Water use efficiency was increased by 16-22% in PEPC transgenic rice lines. Increased PEPC activity enhanced quantum yield and carboxylation efficiency of PEPC transgenic lines. Transgenic plants exhibited higher light saturation photosynthesis rate and lower CO2 compensation point, as compared to non-transformed control. An increase in net photosynthesis increased the yield by (23-28.9%) and biomass by (24.1-29%) in transgenic PEPC lines. Altogether, our findings indicate that overexpression of C4-specific SiPEPC enzyme is able to enhance photosynthesis and related parameters in transgenic rice.

CRISPR Cas9-mediated ablation of pyruvate carboxylase gene in colon cancer cell line HT-29 inhibits growth and migration, induces apoptosis and increases sensitivity to 5-fluorouracil and glutaminase inhibitor.

Pyruvate carboxylase (PC) is an important anaplerotic enzyme that replenishes the tricarboxylic acid cycle (TCA) intermediates. It prevents the collapse of the TCA cycle upon its intermediates are removed during high anabolic demand. We have recently shown that overexpression of PC protein was associated with staging, metastasis and poor survival of colorectal cancer patients. Herein, we generated the PC knockout (PC KO) colon cancer cell lines, HT-29, by CRISPR-Cas9 technique, as a model to understand the role of this enzyme in colorectal cancer. The PC KO HT-29 cell lines had no detectable PC protein and did not show abnormal cellular or nuclear structures. However, PC KO HT-29 cells showed a 50-60% reduction in their growth rate and a 60-70% reduction in migration. The deficient growth phenotype of PC KO HT-29 cells was associated with apoptotic induction with no apparent cell cycle disruption following five days of growth. Down-regulation of key lipogenic enzymes, including acetyl-CoA carboxylase-1 and fatty acid synthase, was also associated with growth inhibition, suggesting that the de novo lipogenesis is impaired. Furthermore, PC KO HT-29 cells were 50% and 60% more sensitive to 5-fluorouracil and glutaminase inhibitor, CB-839, at their IC50 concentrations, respectively, following 48 h exposure. The increased cytotoxicity of CB-839 to PC KO HT-29 cells was associated with increased poly (ADP-ribose) polymerase cleavage. However, this was not observed with PC KO cells exposed to 5-fluorouracil, suggesting that PC KO HT-29 cells were prone to CB-839-induced apoptosis. Collectively, these findings indicate that ablation of PC expression in HT-29 cells disrupts the metabolic homeostasis of cells and inhibits proliferation and migration, accompanied by apoptotic induction. This study highlights the crucial role of PC in supporting the survival of HT-29 cells during exposure to chemotherapeutic drugs.

Requirement of hepatic pyruvate carboxylase during fasting, high fat, and ketogenic diet.

Pyruvate has two major fates upon entry into mitochondria, the oxidative decarboxylation to acetyl-CoA via the pyruvate decarboxylase complex or the biotin-dependent carboxylation to oxaloacetate via pyruvate carboxylase (Pcx). Here, we have generated mice with a liver-specific KO of pyruvate carboxylase (PcxL-/-) to understand the role of Pcx in hepatic mitochondrial metabolism under disparate physiological states. PcxL-/- mice exhibited a deficit in hepatic gluconeogenesis and enhanced ketogenesis as expected but were able to maintain systemic euglycemia following a 24 h fast. Feeding a high-fat diet to PcxL-/- mice resulted in animals that were resistant to glucose intolerance without affecting body weight. However, we found that PcxL-/- mice fed a ketogenic diet for 1 week became severely hypoglycemic, demonstrating a requirement for hepatic Pcx for long-term glycemia under carbohydrate-limited diets. Additionally, we determined that loss of Pcx was associated with an induction in the abundance of lysine-acetylated proteins in PcxL-/- mice regardless of physiologic state. Furthermore, liver acetyl-proteomics revealed a biased induction in mitochondrial lysine-acetylated proteins. These data show that Pcx is important for maintaining the proper balance of pyruvate metabolism between oxidative and anaplerotic pathways.