Pyruvate Carboxylase Activity Research Articles

Abstract 2327: Pyruvate carboxylase rescues MCF10A-ras cell viability in extracellular matrix detached conditions

Abstract Breast cancer is the most diagnosed cancer in women, and metastasis accounts for 90% of cancer related deaths. In order to metastasize, cancer cells must survive in conditions where they are detached from the extracellular matrix (ECM). Untransformed cells decrease energy metabolism when detached from ECM, but oncogenic transformation results in rescued energy metabolism of detached cells. Expression of the anaplerotic enzyme pyruvate carboxylase (PC) is required for breast to lung metastasis, and may play a role in promoting ECM detached cell survival by supporting metabolic plasticity. PC activity results in the carboxylation of pyruvate to synthesize the TCA cycle intermediate oxaloacetate (OAA). We hypothesize that PC expression is required for cell survival in ECM detached conditions. The Harvey-ras transformed MCF10A human breast epithelial cell line (MCF10A-ras) were utilized for these studies. ECM detachment was modeled using Poly-HEMA coated plates in combination with MTT assays to measure cell viability. qPCR was used to quantify mRNA abundance of metabolic genes. Protein expression was quantified using western blot. PC activity was determined using [13C6]-glucose flux analysis by comparing 3 carbon labeled (M+3) citrate, malate, and aspartate. Results show a 94% increase in PC mRNA expression and a 90% increase in PC protein expression, in detached cells compared to attached cells. In addition PC knockdown decreases viability in ECM detached conditions significantly by 15%, while PC overexpression increases viability by 17%. Further, replenishing the TCA cycle with OAA, the product of the PC reaction, rescues PC knockdown cells by 14%. Increased PC activity in detached cells was determined by a 23% increase in M+3 malate and a 61% increase in M+3 aspartate. However, an 11% decrease in M+3 citrate was observed, suggesting that detached cells do not utilize increased PC expression to replenish the TCA cycle intermediate, citrate. Given the larger relative increase in PC activity demonstrated by M+3 aspartate, we hypothesized that PC activity is required for detached cell survival to provide substrate for aspartate synthesis. Providing aspartate (2.5 mM), a downstream product of PC, in detached cells with PC knockdown rescued cell viability with no benefit of aspartate supplementation in detached PC expressing cells. Further, the aspartate synthesizing enzyme, glutamate OAA transferase 2 (GOT2) mRNA expression was unchanged in detached cells. In summary PC expression and activity is increased in detached cells compared to attached cells, and OAA produced by the PC reaction is utilized by cells to synthesize aspartate and maintain cell viability in detached conditions. PC may be a useful target for future treatments aimed at preventing metastasis. Citation Format: Madeline P. Sheeley, Dorothy Teegarden. Pyruvate carboxylase rescues MCF10A-ras cell viability in extracellular matrix detached conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2327.

Glutamate Dehydrogenase Is Important for Ammonia Fixation and Amino Acid Homeostasis in Brain During Hyperammonemia.

Impaired liver function may lead to hyperammonemia and risk for hepatic encephalopathy. In brain, detoxification of ammonia is mediated mainly by glutamine synthetase (GS) in astrocytes. This requires a continuous de novo synthesis of glutamate, likely involving the action of both pyruvate carboxylase (PC) and glutamate dehydrogenase (GDH). An increased PC activity upon ammonia exposure and the importance of PC activity for glutamine synthesis has previously been demonstrated while the importance of GDH for generation of glutamate as precursor for glutamine synthesis has received little attention. We therefore investigated the functional importance of GDH for brain metabolism during hyperammonemia. To this end, brain slices were acutely isolated from transgenic CNS-specific GDH null or litter mate control mice and incubated in aCSF containing [U-13C]glucose in the absence or presence of 1 or 5 mM ammonia. In another set of experiments, brain slices were incubated in aCSF containing 1 or 5 mM 15N-labeled NH4Cl and 5 mM unlabeled glucose. Tissue extracts were analyzed for isotopic labeling in metabolites and for total amounts of amino acids. As a novel finding, we reveal a central importance of GDH function for cerebral ammonia fixation and as a prerequisite for de novo synthesis of glutamate and glutamine during hyperammonemia. Moreover, we demonstrated an important role of the concerted action of GDH and alanine aminotransferase in hyperammonemia; the products alanine and α-ketoglutarate serve as an ammonia sink and as a substrate for ammonia fixation via GDH, respectively. The role of this mechanism in human hyperammonemic states remains to be studied.

Characterizing the Molecular Basis of the Allosteric Activation of Pyruvate Carboxylase by Acetyl CoA

Pyruvate carboxylase (PC) is a central metabolic enzyme that produces oxaloacetate from pyruvate and bicarbonate in an ATP-dependent manner. The catalytic activity of PC contributes to glucose homeostasis and has been implicated in enhanced tumorigenesis and metastasis in certain cancers. PC activity is allosterically activated by acetyl CoA, and allosterically inhibited by L-aspartate. The binding of these effector molecules is mutually exclusive. The binding site for acetyl-CoA has largely been identified through previous structural studies, but the exact binding site for the acetyl moiety is unknown, partly as a consequence of the enzyme-catalyzed hydrolysis of acetyl CoA. The acetyl moiety is a particularly important feature of acetyl CoA activation, contributing to the significantly enhanced allosteric activation of acetyl CoA relative to CoA. Here, we have employed non-hydrolysable and truncated analogs of acetyl-CoA to more precisely probe the acetyl moiety binding site in PC. We have determined that the acetyl moiety contributes greatly to the binding of the molecule but that it does not alter the degree of activation. Additionally, kinetic studies strongly suggest that L-aspartate competes for the acetyl moiety binding site with acetyl CoA. A definition of the acetyl moiety binding site further clarifies the mechanism of acetyl CoA binding and offers a more detailed molecular description of allosteric regulation in this central metabolic enzyme.

Fat Induces Glucose Metabolism in Nontransformed Liver Cells and Promotes Liver Tumorigenesis.

Hepatic fat accumulation is associated with diabetes and hepatocellular carcinoma (HCC). Here, we characterize the metabolic response that high-fat availability elicits in livers before disease development. After a short term on a high-fat diet (HFD), otherwise healthy mice showed elevated hepatic glucose uptake and increased glucose contribution to serine and pyruvate carboxylase activity compared with control diet (CD) mice. This glucose phenotype occurred independently from transcriptional or proteomic programming, which identifies increased peroxisomal and lipid metabolism pathways. HFD-fed mice exhibited increased lactate production when challenged with glucose. Consistently, administration of an oral glucose bolus to healthy individuals revealed a correlation between waist circumference and lactate secretion in a human cohort. In vitro, palmitate exposure stimulated production of reactive oxygen species and subsequent glucose uptake and lactate secretion in hepatocytes and liver cancer cells. Furthermore, HFD enhanced the formation of HCC compared with CD in mice exposed to a hepatic carcinogen. Regardless of the dietary background, all murine tumors showed similar alterations in glucose metabolism to those identified in fat exposed nontransformed mouse livers, however, particular lipid species were elevated in HFD tumor and nontumor-bearing HFD liver tissue. These findings suggest that fat can induce glucose-mediated metabolic changes in nontransformed liver cells similar to those found in HCC. SIGNIFICANCE: With obesity-induced hepatocellular carcinoma on a rising trend, this study shows in normal, nontransformed livers that fat induces glucose metabolism similar to an oncogenic transformation.

Isotope tracing reveals glycolysis and oxidative metabolism in childhood tumors of multiple histologies

Survival among children with high-risk solid tumors remains poor. Reprogrammed metabolism promotes tumor growth and may contain therapeutic liabilities. Tumor metabolism has been assessed in adults using intra-operative 13C-glucose infusions. Pediatric tumors differ from adult cancers in their low mutational burden and derivation from embryonic tissues. Here we used 13C infusions to examine tumor metabolism in children, comparing phenotypes among tumor types and between childhood and adult cancers. Patients recruited to study NCT03686566 received an intra-operative infusion of [U-13C]glucose during tumor resection to evaluate central carbon pathways in the tumor, with concurrent metabolomics to provide a broad overview of metabolism. Differential characteristics were determined using multiple comparison tests and mixed effect analyses. We studied 23 tumors from 22 patients. All tumors analyzed by [U-13C]glucose contained labeling in glycolytic and tricarboxylic acid (TCA) cycle intermediates. Labeling in the TCA cycle indicated activity of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), with PDH predominating. Neuroblastomas had high lactate labeling relative to other childhood cancers and lung cancer, and were distinguished by abundant tyrosine catabolites consistent with catecholamine synthesis. Intra-operative [U13C]glucose infusions are safe and informative in pediatric cancer. Tumors of various histologies use glycolysis and oxidative metabolism, with subtype-selective differences evident from this small cohort. Expanding this cohort may uncover predictive biomarkers and therapeutic targets from tumor metabolism. N.C.I grants to P.L. (R21CA220090-01A1) and R.J.D. (R35CA22044901); H.H.M.I. funding to R.J.D.; Children's Clinical Research Advisory Committee funding to K.J.

Tumor Cells Dictate Anti-Tumor Immune Responses by Altering Pyruvate Utilization and Succinate Signaling in Cd8 <sup>+</sup> T Cells

The tumor microenvironment (TME) is a unique metabolic niche that can inhibit CD8+ T cell metabolism and cytotoxic activity. It has been a challenge to dissect the metabolic interplay between tumors and T cells. Consequently, the mechanisms whereby the TME regulates CD8+ T cell function are not completely understood. Here we establish an in vitro system that recapitulates the metabolic niche of the TME and allows us to define cell-specific metabolism. We identify tumor-derived lactate as a direct inhibitor of CD8+ T cell cytotoxicity, revealing an unexpected metabolic shunt in the tricarboxylic acid (TCA) cycle in CD8+ T cells. Metabolically fit cytotoxic T cells shunt succinate out of the TCA cycle to promote autocrine signaling via the succinate receptor (SUCNR1), a G-protein coupled cell surface receptor. This leaves CD8+ T cells reliant on pyruvate carboxylase (PC) to replenish TCA cycle intermediates (anaplerosis). In contrast, lactate reduces PC-mediated anaplerosis, thus depleting the TCA cycle and reducing SUCNR1 signaling. Notably, inhibition of pyruvate dehydrogenase (PDH) is sufficient to override lactate-mediated reprogramming, and to restore PC activity, succinate secretion, and autocrine activation of SUCNR1 both in vitro and in vivo using syngeneic tumor models. Thus, dissecting the CD8+ T cell-specific metabolic adaptation to the TME unexpectedly revealed that a TCA cycle succinate shunt and plasticity in pyruvate flux is a crucial determinant of CD8+ T cell anti-tumor activity. Furthermore, we identify PDH as a potential drug target to allow CD8+ T cells to retain cytotoxicity and overcome a lactate-rich TME. These studies demonstrate that the TME remodels CD8+ T cell pyruvate metabolism, leading to a reduction in CD8+ T cell cytotoxicity and anti-tumor immunity.

Assessment of hepatic pyruvate carboxylase activity using hyperpolarized [1-13 C]-l-lactate.

To evaluate the utility of hyperpolarized [1-13 C]-l-lactate to detect hepatic pyruvate carboxylase activity in vivo under fed and fasted conditions. [1-13 C]-labeled sodium L-lactate was polarized using a dynamic nuclear polarizer. Polarization level and the T1 were measured in vitro in a 3 Telsa MR scanner. Two groups of healthy rats (fasted vs. fed) were prepared for in vivo studies. Each rat was anesthetized and intravenously injected with 60-mM hyperpolarized [1-13 C]-l-lactate, immediately followed by dynamic acquisition of 13 C (carbon-13) MR spectra from the liver at 3 Tesla. The dosage-dependence of the 13 C-products was also investigated by performing another injection of an equal volume of 30-mM hyperpolarized [1-13 C]-l-lactate. T1 and liquid polarization level of [1-13 C]-l-lactate were estimated as 67.8 s and 40.0%, respectively. [1-13 C]pyruvate and [1-13 C]alanine, [13 C]bicarbonate ( ) and [1-13 C]aspartate were produced from hyperpolarized [1-13 C]-l-lactate in rat liver. Smaller and larger aspartate were measured in the fed group compared to the fasted group. Pyruvate and alanine production were increased in proportion to the lactate concentration, whereas the amount of and aspartate production was consistent between 30-mM and 60-mM lactate injections. This study demonstrates that a unique biomarker of pyruvate carboxylase flux, the appearance of [1-13 C]aspartate from [1-13 C]-l-lactate, is sensitive to nutritional state and may be monitored in vivo at 3 Tesla. Because [13 C] is largely produced by pyruvate dehydrogenase flux, these results suggest that the ratio of [1-13 C]aspartate and [13 C] (aspartate/ ) reflects the saturable pyruvate carboxylase/pyruvate dehydrogenase enzyme activities.

Glucose-dependent partitioning of arginine to the urea cycle protects β-cells from inflammation.

Chronic inflammation is linked to diverse disease processes, but the intrinsic mechanisms that determine cellular sensitivity to inflammation are incompletely understood. Here, we show the contribution of glucose metabolism to inflammation-induced changes in the survival of pancreatic islet β-cells. Using metabolomics, biochemical and functional analyses, we investigate the protective versus non-protective effects of glucose in the presence of pro-inflammatory cytokines. When protective, glucose metabolism augments anaplerotic input into the TCA cycle via pyruvate carboxylase (PC) activity, leading to increased aspartate levels. This metabolic mechanism supports the argininosuccinate shunt, which fuels ureagenesis from arginine and conversely diminishes arginine utilization for production of nitric oxide (NO), a chief mediator of inflammatory cytotoxicity. Activation of the PC-urea cycle axis is sufficient to suppress NO synthesis and shield cells from death in the context of inflammation and other stress paradigms. Overall, these studies uncover a previously unappreciated link between glucose metabolism and arginine-utilizing pathways via PC-directed ureagenesis as a protective mechanism.

Autosomal dominant transmission of transient neonatal lactic acidosis: a case report

BackgroundLactic acidosis is a common finding in neonates, in whom mitochondrial dysfunction is often secondary to tissue hypoperfusion, respiratory failure, and/or sepsis. Primary (non-physiological) lactic acidosis is comparatively rare, and suggests the presence of an inborn error of mitochondrial energy metabolism. Optimal medical management and accurate prognostication requires the correct determination of the etiology of lactic acidosis in a given patient. Unfortunately, genetic diagnoses are rare and highly variable for neonates presenting with primary lactic acidosis; individual case reports may offer the most promise for treatment considerations.The mitochondrion is a complex molecular machine incorporating the products of > 1000 distinct nuclear genes. Primary lactic acidoses are therefore characterized by high genetic heterogeneity and a specific genetic diagnosis currently remains out of reach in most cases. Most mitochondriopathies with neonatal onset follow autosomal recessive inheritance and carry a poor prognosis. Here we detail the case of a father and daughter with dominantly-inherited, resolving (i.e. transient) neonatal hyperlactatemia due to complex IV deficiency. We found no other published descriptions of benign transient complex IV deficiency with autosomal dominant inheritance.Case presentationBoth individuals presented as neonates with unexplained, marked lactic acidosis suggesting a primary mitochondrial disorder. Within the first weeks of life, elevated blood lactate levels normalized. Their clinical and developmental outcomes were normal. Biochemical studies in the proband showed multiple abnormalities consistent with a complex IV respiratory chain defect. Cultured skin fibroblasts showed an elevated lactate-to-pyruvate ratio, deficient complex IV activity, and normal pyruvate dehydrogenase and pyruvate carboxylase activities. Whole-exome sequencing of the proband and both parents did not identify a causative mutation.ConclusionWe conclude that the proband and her father appear to have a dominant form of transient neonatal hyperlactatemia due to heterozygous changes in an as-yet unidentified gene. This transient neonatal complex IV deficiency should be considered in the differential diagnosis of primary neonatal hyperlactatemia; notable clinical features include autosomal-dominant inheritance and an apparently benign postnatal course. This report exemplifies the growing differential diagnosis for neonatal lactic acidosis and highlights the importance of both physician counselling and the use of family history in communicating with parents.

Cold acclimation of mesophyll conductance, bundle-sheath conductance and leakiness in Miscanthus×giganteus.

The cold acclimations of mesophyll conductance (gm ), bundle-sheath conductance (gbs ) and the CO2 concentrating mechanism (CCM) of C4 plants have not been well studied. Here, we estimated the temperature response of gm , gbs and leakiness (ϕ), the amount of concentrated CO2 that escapes the bundle-sheath cells, for the chilling-tolerant C4 plant Miscanthus×giganteus grown at 14 and 25°C. To estimate these parameters, we combined the C4 -enzyme-limited photosynthesis model and the Δ13 C discrimination model. These combined models were parameterised using in vitro activities of carbonic anhydrase (CA), pyruvate, phosphate dikinase (PPDK), ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and phosphoenolpyruvate carboxylase (PEPc). Cold-grown Miscanthus plants increased in vitro activities of RuBisCO and PPDK but decreased PEPc activity compared with warm-grown plants. Mesophyll conductance and gbs responded strongly to measurement temperatures but did not differ between plants from the two growth temperatures. Furthermore, modelling showed that ϕ increased with measurement temperatures for both cold-grown and warm-grown plants, but was only marginally larger in cold-grown compared with warm-grown plants. Our results in Miscanthus support that gm and gbs are unresponsive to growth temperature and that the CCM is able to acclimate to cold through increased activity of PPDK and RuBisCO.

AMP-activated protein kinase (AMPK) regulates astrocyte oxidative metabolism by balancing TCA cycle dynamics.

AMP-activated protein kinase (AMPK) is an important energy sensor located in cells throughout the human body. From the periphery, AMPK is known to be a metabolic master switch controlling the use of energy fuels. The energy sensor is activated when the energy status of the cell is low, initiating energy-producing pathways and deactivating energy-consuming pathways. All brain cells are crucially dependent on energy production for survival, and the availability of energy substrates must be closely regulated. Intriguingly, the role of AMPK in the regulation of brain cell metabolism has been sparsely investigated, particularly in astrocytes. By investigating metabolism of 13 C-labeled energy substrates in acutely isolated hippocampal slices and cultured astrocytes, with subsequent mass spectrometry analysis, we here show that activation of AMPK increases glycolysis as well as the capacity of the TCA cycle, that is, anaplerosis, through the activity of pyruvate carboxylase (PC) in astrocytes. In addition, we demonstrate that AMPK activation leads to augmented astrocytic glutamate oxidation via pyruvate recycling (i.e., cataplerosis). This regulatory mechanism induced by AMPK activation is mediated via glutamate dehydrogenase (GDH) shown in a CNS-specific GDH knockout mouse. Collectively, these findings demonstrate that AMPK regulates TCA cycle dynamics in astrocytes via PC and GDH activity. AMPK functionality has been shown to be hampered in Alzheimer's and Parkinson's disease and our findings may therefore add to the toolbox for discovery of new metabolic drug targets.

Inhibition of Anaplerosis Attenuated Vascular Proliferation in Pulmonary Arterial Hypertension

Vascular remodeling is considered a key event in the pathogenesis of pulmonary arterial hypertension (PAH). However, mechanisms of gaining the proliferative phenotype by pulmonary vascular cells are still unresolved. Due to well-established pyruvate dehydrogenase (PDH) deficiency in PAH pathogenesis, we hypothesized that the activation of another branch of pyruvate metabolism, anaplerosis, via pyruvate carboxylase (PC) could be a key contributor to the metabolic reprogramming of the vasculature. In sugen/hypoxic PAH rats, vascular proliferation was found to be accompanied by increased activation of Akt signaling, which upregulated membrane Glut4 translocation and caused upregulation of hexokinase and pyruvate kinase-2, and an overall increase in the glycolytic flux. Decreased PDH activity and upregulation of PC shuttled more pyruvate to oxaloacetate. This results in the anaplerotic reprogramming of lung vascular cells and their subsequent proliferation. Treatment of sugen/hypoxia rats with the PC inhibitor, phenylacetic acid 20 mg/kg, starting after one week from disease induction, significantly attenuated right ventricular systolic pressure, Fulton index, and pulmonary vascular cell proliferation. PC inhibition reduced the glycolytic shift by attenuating Akt-signaling, glycolysis, and restored mitochondrial pyruvate oxidation. Our findings suggest that targeting PC mediated anaplerosis is a potential therapeutic intervention for the resolution of vascular remodeling in PAH.

Antioxidant-Conjugated Peptide Attenuated Metabolic Reprogramming in Pulmonary Hypertension

Pulmonary arterial hypertension (PAH) is a chronic cardiopulmonary disorder instigated by pulmonary vascular cell proliferation. Activation of Akt was previously reported to promote vascular remodeling. Also, the irreversible nitration of Y350 residue in Akt results in its activation. NitroAkt was increased in PAH patients and the SU5416/Hypoxia (SU/Hx) PAH model. This study investigated whether the prevention of Akt nitration in PAH by Akt targeted nitroxide-conjugated peptide (NP) could reverse vascular remodeling and metabolic reprogramming. Treatment of the SU/Hx model with NP significantly decreased nitration of Akt in lungs, attenuated right ventricle (RV) hypertrophy, and reduced RV systolic pressure. In the PAH model, Akt-nitration induces glycolysis by activation of the glucose transporter Glut4 and lactate dehydrogenase-A (LDHA). Decreased G6PD and increased GSK3β in SU/Hx additionally shunted intracellular glucose via glycolysis. The increased glycolytic rate upregulated anaplerosis due to activation of pyruvate carboxylase in a nitroAkt-dependent manner. NP treatment resolved glycolytic switch and activated collateral pentose phosphate and glycogenesis pathways. Prevention of Akt-nitration significantly controlled pyruvate in oxidative phosphorylation by decreasing lactate and increasing pyruvate dehydrogenases activities. Histopathological studies showed significantly reduced pulmonary vascular proliferation. Based on our current observation, preventing Akt-nitration by using an Akt-targeted nitroxide-conjugated peptide could be a useful treatment option for controlling vascular proliferation in PAH.

A Non-canonical Function of BMAL1 Metabolically Limits Obesity-Promoted Triple-Negative Breast Cancer.

SummaryThe epidemiological association between disrupted circadian rhythms and metabolic diseases is implicated in increased risk of human breast cancer and poor therapeutic outcomes. To define a metabolic phenotype and the underlying molecular mechanism, we applied chronic insulin treatment (CIT) to an in vitro model of triple-negative breast cancer to directly address how BMAL1, a key circadian transcription factor, regulates cancer cell respiration and governs tumor progression. At the cellular level, BMAL1 suppresses the flexibility of mitochondrial substrate usage and the pyruvate-dependent mitochondrial respiration induced by CIT. We established an animal model of diet-induced obesity/hyperinsulinemia and observed that BMAL1 functions as a tumor suppressor in obese, but not lean, mice. Downregulation of BMAL1 is associated with higher risk of metastasis in human breast tumors. In summary, loss of BMAL1 in tumors confers advantages to cancer cells in both intrinsic mitochondrial metabolism and extrinsic inflammatory tumor microenvironment during pre-diabetic obesity/hyperinsulinemia.

Real-time hyperpolarized 13C magnetic resonance detects increased pyruvate oxidation in pyruvate dehydrogenase kinase 2/4\u2013double knockout mouse livers

The pyruvate dehydrogenase complex (PDH) critically regulates carbohydrate metabolism. Phosphorylation of PDH by one of the pyruvate dehydrogenase kinases 1–4 (PDK1–4) decreases the flux of carbohydrates into the TCA cycle. Inhibition of PDKs increases oxidative metabolism of carbohydrates, so targeting PDKs has emerged as an important therapeutic approach to manage various metabolic diseases. Therefore, it is highly desirable to begin to establish imaging tools for noninvasive measurements of PDH flux in rodent models. In this study, we used hyperpolarized (HP) 13C-magnetic resonance spectroscopy to study the impact of a PDK2/PDK4 double knockout (DKO) on pyruvate metabolism in perfused livers from lean and diet-induced obese (DIO) mice and validated the HP observations with high-resolution 13C-nuclear magnetic resonance (NMR) spectroscopy of tissue extracts and steady-state isotopomer analyses. We observed that PDK-deficient livers produce more HP-bicarbonate from HP-[1-13C]pyruvate than age-matched control livers. A steady-state 13C-NMR isotopomer analysis of tissue extracts confirmed that flux rates through PDH, as well as pyruvate carboxylase and pyruvate cycling activities, are significantly higher in PDK-deficient livers. Immunoblotting experiments confirmed that HP-bicarbonate production from HP-[1-13C]pyruvate parallels decreased phosphorylation of the PDH E1α subunit (pE1α) in liver tissue. Our findings indicate that combining real-time hyperpolarized 13C NMR spectroscopy and 13C isotopomer analysis provides quantitative insights into intermediary metabolism in PDK-knockout mice. We propose that this method will be useful in assessing metabolic disease states and developing therapies to improve PDH flux.

Active pyruvate dehydrogenase and impaired gluconeogenesis in orthotopic hepatomas of rats

BackgroundTherapies targeting altered activity of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) have been proposed for hepatomas. However, the activities of these pathways in hepatomas in vivo have not been distinguished. Here we examined pyruvate entry into the tricarboxylic acid (TCA) cycle through PDH versus PC in vivo using hepatoma-bearing rats. MethodsHepatoma-bearing rats were generated by intrahepatic injection of H4IIE cells. Metabolism of 13C-labeled glycerol, a physiological substrate for both gluconeogenesis and energy production, was measured with 13C NMR analysis. The concentration of key metabolites and the expression of relevant enzymes were measured in hepatoma, surrounding liver, and normal liver. ResultsIn orthotopic hepatomas, pyruvate entry into the TCA cycle occurred exclusively through PDH and the excess PDH activity compared to normal liver was attributed to downregulated pyruvate dehydrogenase kinase (PDK) 2/4. However, pyruvate carboxylation via PC and gluconeogenesis were minimal, which was linked to downregulated forkhead box O1 (FoxO1) by Akt activity. In contrast to many studies of cancer metabolism, lactate production in hepatomas was not increased which corresponded to reduced expression of lactate dehydrogenase. The production of serine and glycine in hepatomas was enhanced, but glycine decarboxylase was downregulated. ConclusionsThe combination of [U-13C3]glycerol and NMR analysis enabled investigation of multiple biochemical processes in hepatomas and surrounding liver. We demonstrated active PDH and other related metabolic alterations in orthotopic hepatomas that differed substantially not only from the host organ but also from many earlier studies with cancer cells.

Inhibition of Anaplerotic Glutaminolysis Underlies Selenite Toxicity in Human Lung Cancer.

Large clinical trials and model systems studies suggest that the chemical form of selenium dictates chemopreventive and chemotherapeutic efficacy. Selenite induces excess ROS production, which mediates autophagy and eventual cell death in non-small cell lung cancer adenocarcinoma A549 cells. As the mechanisms underlying these phenotypic effects are unclear, the clinical relevance of selenite for cancer therapy remains to be determined. The authors' previous stable isotope-resolved metabolomics and gene expression analysis showed that selenite disrupts glycolysis, the Krebs cycle, and polyamine metabolism in A549 cells, potentially through perturbed glutaminolysis, a vital anaplerotic process for proliferation of many cancer cells. Herein, the role of the glutaminolytic enzyme glutaminase 1 (GLS1) in selenite's toxicity in A549 cells and in patient-derived lung cancer tissues is investigated. Using [13 C6 ]-glucose and [13 C5 ,15 N2 ]-glutamine tracers, selenite's action on metabolic networks is determined. Selenite inhibits glutaminolysis and glutathione synthesis by suppressing GLS1 expression, and blocks the Krebs cycle, but transiently activates pyruvate carboxylase activity. Glutamate supplementation partially rescues these anti-proliferative and oxidative stress activities. Similar metabolic perturbations and necrosis are observed in selenite-treated human patients' cancerous lung tissues ex vivo. The results support the hypothesis that GLS1 suppression mediates part of the anti-cancer activity of selenite both in vitro and ex vivo.

Cardiolipin-induced activation of pyruvate dehydrogenase links mitochondrial lipid biosynthesis to TCA cycle function

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial bioenergetics, recent evidence in the yeast model indicates that CL is also essential for intermediary metabolism. To gain insight into the function of CL in energy metabolism in mammalian cells, here we analyzed the metabolic flux of [U-13C]glucose in a mouse C2C12 myoblast cell line, TAZ-KO, which is CL-deficient because of CRISPR/Cas9-mediated knockout of the CL-remodeling enzyme tafazzin (TAZ). TAZ-KO cells exhibited decreased flux of [U-13C]glucose to [13C]acetyl-CoA and M2 and M4 isotopomers of tricarboxylic acid (TCA) cycle intermediates. The activity of pyruvate carboxylase, the predominant enzyme for anaplerotic replenishing of the TCA cycle, was elevated in TAZ-KO cells, which also exhibited increased sensitivity to the pyruvate carboxylase inhibitor phenylacetate. We attributed a decreased carbon flux from glucose to acetyl-CoA in the TAZ-KO cells to a ∼50% decrease in pyruvate dehydrogenase (PDH) activity, which was observed in both TAZ-KO cells and cardiac tissue from TAZ-KO mice. Protein-lipid overlay experiments revealed that PDH binds to CL, and supplementing digitonin-solubilized TAZ-KO mitochondria with CL restored PDH activity to WT levels. Mitochondria from TAZ-KO cells exhibited an increase in phosphorylated PDH, levels of which were reduced in the presence of supplemented CL. These findings indicate that CL is required for optimal PDH activation, generation of acetyl-CoA, and TCA cycle function, findings that link the key mitochondrial lipid CL to TCA cycle function and energy metabolism.

Loss of the Retinoblastoma Protein Modulates Glucose Metabolism in Lung Cancer

Dysregulated metabolism is a hallmark of cancer cells and is driven in part by specific genetic alterations in various oncogenes or tumor suppressors. The retinoblastoma protein (Rb) is a tumor suppressor that was initially characterized in regulating cell cycle progression but recent studies have highlighted a functional role for Rb in controlling cellular metabolism. As such, we aim to characterize the metabolic phenotype caused by genetic loss of Rb in a mouse model of lung cancer. We have employed the Kras+/LSLG12D/Rb1lox/lox and Kras+/LSLG12D/Rb1+/+ transgenic model to study the metabolic effects of Rb1 loss in lung cancer. Cre‐exposed Kras+/LSLG12D/Rb1lox/lox mice exhibit increased tumor burden as well as decreased overall survival compared to Kras+/LSLG12D/Rb1+/+ mice. Our tracer studies using bolus dosing of 13C‐glucose revealed an increase in glucose carbon incorporation into select glycolytic intermediates. Consistent with this result, Rb1 depleted tumors expressed higher hexokinase 2 and glucose transporter 1. Additionally, compared to Rb1‐proficient tumors, Rb1 null tumors exhibit decreased one and three carbon labeled aspartate, which is indicative of pyruvate carboxylase (PC) activity. However, Rb1−/− tumors were found to express higher PC compared to Rb1+/+ tumors, suggesting these tumors may utilize an alternative nutrient source for TCA anaplerosis other than glucose. Taken together, these data have identified several metabolic genes under Rb control, and suggest Rb may regulate differential nutrient utilization in lung tumors.Support or Funding InformationNCI RO1CA166327, American Cancer Society RSG 13‐139‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Central Role of Glucokinase in Glucose Homeostasis: A Perspective 50 Years After Demonstrating the Presence of the Enzyme in Islets of Langerhans.

It is hypothesized that glucokinase (GCK) is the glucose sensor not only for regulation of insulin release by pancreatic β-cells, but also for the rest of the cells that contribute to glucose homeostasis in mammals. This includes other cells in endocrine pancreas (α- and δ-cells), adrenal gland, glucose sensitive neurons, entero-endocrine cells, and cells in the anterior pituitary. Glucose transport is by facilitated diffusion and is not rate limiting. Once inside, glucose is phosphorylated to glucose-6-phosphate by GCK in a reaction that is dependent on glucose throughout the physiological range of concentrations, is irreversible, and not product inhibited. High glycerol phosphate shuttle, pyruvate dehydrogenase, and pyruvate carboxylase activities, combined with low pentose-P shunt, lactate dehydrogenase, plasma membrane monocarboxylate transport, and glycogen synthase activities constrain glucose-6-phosphate to being metabolized through glycolysis. Under these conditions, glycolysis produces mostly pyruvate and little lactate. Pyruvate either enters the citric acid cycle through pyruvate dehydrogenase or is carboxylated by pyruvate carboxylase. Reducing equivalents from glycolysis enter oxidative phosphorylation through both the glycerol phosphate shuttle and citric acid cycle. Raising glucose concentration increases intramitochondrial [NADH]/[NAD+] and thereby the energy state ([ATP]/[ADP][Pi]), decreasing [Mg2+ADP] and [AMP]. [Mg2+ADP] acts through control of KATP channel conductance, whereas [AMP] acts through regulation of AMP-dependent protein kinase. Specific roles of different cell types are determined by the diverse molecular mechanisms used to couple energy state to cell specific responses. Having a common glucose sensor couples complementary regulatory mechanisms into a tightly regulated and stable glucose homeostatic network.