Glycolytic Metabolites Research Articles

AMP-activated protein kinase mediates glycolysis in post-mortem breast muscle of broilers

The aim of this study was to confirm the role of AMP-activated protein kinase (AMPK) in the regulation of post-mortem breast muscle glycolysis in the broiler, we evaluated the effects of intraperitoneal injection of the specific AMPK activator (5-aminoimidazole-4-carboxamide ribonucleoside, AICAR) and inhibitor (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a] pyrimidine, Compound C) on AMPK phosphorylation (p-AMPK) level, meat quality, glycolytic metabolites and glycolytic enzymes activities in post-mortem breast muscle of broilers. Compared to the CON group, intraperitoneal injection of AIRCR increased p-AMPK level, glycolytic enzyme activities and lactate content in post-mortem breast muscle, increased lightness (L*) and drip loss, but decreased pH45min and pH24h. However, intraperitoneal injection of Compound C, decreased p-AMPK level, glycolytic enzyme activities, glucose-6-phosphate and lactate content in post-mortem breast muscle, and increased pH24h. Based on these results, we conclude that AMPK played a vital role in regulating post-mortem glycolysis in the breast muscle of broilers. HIGHLIGHTS Injection of AIRCR promoting muscle glycolysis. Injection of Compound C alleviating muscle glycolysis. AMPK played a vital role in regulating post-mortem muscle glycolysis.

Amino acid catabolism regulates hematopoietic stem cell proteostasis via a GCN2-eIF2α axis

Hematopoietic stem cells (HSCs) adapt their metabolism to maintenance and proliferation; however, the mechanism remains incompletely understood. Here, we demonstrated that homeostatic HSCs exhibited high amino acid (AA) catabolism to reduce cellular AA levels, which activated the GCN2-eIF2α axis, a protein synthesis inhibitory checkpoint to restrain protein synthesis for maintenance. Furthermore, upon proliferation conditions, HSCs enhanced mitochondrial oxidative phosphorylation (OXPHOS) for higher energy production but decreased AA catabolism to accumulate cellular AAs, which inactivated the GCN2-eIF2α axis to increase protein synthesis and coupled with proteotoxic stress. Importantly, GCN2 deletion impaired HSC function in repopulation and regeneration. Mechanistically, GCN2 maintained proteostasis and inhibited Src-mediated AKT activation to repress mitochondrial OXPHOS in HSCs. Moreover, the glycolytic metabolite, NAD+ precursor nicotinamide riboside (NR), accelerated AA catabolism to activate GCN2 and sustain the long-term function of HSCs. Overall, our study uncovered direct links between metabolic alterations and translation control in HSCs during homeostasis and proliferation.

Abstract 2327: Dysregulated TCA cycle is associated with endocrine therapy resistance in ER+ breast cancer cells

Abstract Estrogen receptor positive (ER+) breast cancer is the most common breast cancer subtype. Endocrine therapies, such as selective estrogen receptor modulators (SERM) and aromatase inhibitors (AI), along with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, are the current mainstays of treatment. Despite being initially effective, the majority of advanced ER+ breast cancers become resistant to the endocrine therapies. Therefore, it is important to understand the underlying molecular mechanisms that enable or promote resistance to endocrine therapies. Rapidly proliferating cells have a high energy demand and consequently consume greater quantities of glucose to fulfill their energy and anabolic demands to support cell growth. Using endocrine therapy sensitive and resistant breast cancer cell models we investigated the metabolic flux of glucose by growing the cells in the presence of the stable glucose isotope, UC13-Glucose and measured the C13-labelled metabolites of glycolysis and the TCA cycle. Glucose consumption was higher in endocrine therapy resistant MCF7/LCC9 (LCC9) cells compared with parental MCF7 (MCF7) and estrogen independent, endocrine therapy sensitive MCF7/LCC1 (LCC1) cells. The TCA cycle was noticeably impaired in LCC9 cells as the C-13 labelled citrate (m+4 and m+6) isotopomer was absent in these cells. In addition, C-13 labelled fumarate was very low in LCC9 cells with a concurrent increase in the succinate:fumarate ratio. These observations show that in LCC9 cells the TCA cycle is dysregulated at the succinate to fumarate conversion step, which is catalyzed by the succinate dehydrogenase (SDH) enzyme. SDH is a multimeric protein comprised of four different subunits and SDH assembly factor 2 (SDHAF2), which is a tumor suppressor gene. Further investigation revealed that SDHAF2 was under expressed in LCC9 cells compared with LCC1 cells, likely contributing to lower SDH activity. Intriguingly, SDH inhibition by dimethyl-malonate re-sensitized LCC9 cells to both Fulvestrant and 4-hydroxytamoxifen. Clinically, lower expression levels of SDHAF2 correlates with poor relapse free survival in ER+ breast cancer patients. In summary, our study suggests that accumulation of succinate driven by deficient SDH activity is functionally associated with endocrine therapy resistance in ER+ breast cancer cells. Citation Format: Surojeet Sengupta, Karla Andrade de Oliviera, Lu Jin, Robert Clarke. Dysregulated TCA cycle is associated with endocrine therapy resistance in ER+ breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2327.

Abstract LB081: GAS6-AXL inhibition by AVB-500 overcomes resistance to paclitaxel in endometrial cancer by decreasing tumor cell glycolysis

Abstract Objective/Background: Chemotherapy is often ineffective in advanced stage and aggressive histologic subtypes of endometrial cancer. Overexpression of the receptor tyrosine kinase AXL has been found to be associated with therapeutic resistance, metastasis, and poor prognosis in endometrial cancer. Our objective was to identify whether inhibition of AXL through a novel, selective targeted agent, AVB-500, would improve sensitivity to paclitaxel. Additionally, we sought to determine whether the mechanism for this improved sensitivity was through metabolic regulation. Methods: Immunohistochemistry (IHC) was performed on a tissue microarray containing specimens from patients with primary and metastatic uterine serous carcinoma. Blind scoring was performed by two reviewers. Kaplan-Meier methods were used to generate time-to-event curves. Cell viability was performed with high-grade endometrial, chemo-resistant cell lines, ARK1 and PUC198. Cells were treated with paclitaxel and with AVB-500+paclitaxel. Intraperitoneal (IP) ARK1 or PUC198 tumors were treated with vehicle, AVB-500, paclitaxel, or AVB-500+paclitaxel. A Seahorse Analyzer was used for glycolytic rate assays. Isotope tracing was used for in vivo metabolite abundance quantification. Results: We found that both GAS6 and AXL expression were higher by IHC in tumors with a poor response to chemotherapy compared to tumors with a good response to chemotherapy (GAS6: 40.9% vs. 30.4%, P=0.012; AXL: 60.7% vs. 42.7%, P=0.013). Median PFS and OS were longer in patients with low GAS6 expressing tumors compared to high GAS6 expressing tumors (PFS: 33.6 mo vs. 10.6 mo, P=0.003; OS: 39.5 mo vs. 27.7 mo, P=0.003). We showed that chemotherapy resistant endometrial cancer cell lines, ARK1 and PUC198 had improved sensitivity and synergy with paclitaxel when treated in combination with AVB-500. ARK1 and PUC198 in vivo IP models had significantly fewer tumors (ARK1: P=0.007; PUC198: P=0.029) and decreased tumor weight (ARK1: P<0.001; PUC198: P=0.006) when treated with AVB-500+paclitaxel vs. paclitaxel alone. Treatment with AVB-500 + paclitaxel decreased AKT signaling which resulted in a decrease in basal glycolysis. Finally, treatment with AVB-500 + paclitaxel decreased multiple glycolytic metabolites, including fructose-1,6-bisphosphate (P=0.002), 3-phosphoglyceric acid (P=0.014), phosphoenolpyruvate (P=0.013), and lactate (P=0.024) vs. treatment with paclitaxel alone. Conclusions: Our study provides strong pre-clinical rationale for combining AVB-500 with paclitaxel in aggressive endometrial cancer models. Additionally, continued identification of viable biomarkers is critical to the success of targeted therapies such as AVB-500. Given AXL’s role in glucose homeostasis, one or more glycolytic intermediates may prove to be a useful biomarker to help predict sensitivity in patients, thus tailoring who would benefit from this treatment combination. Citation Format: Shaina F. Bruce, Kevin Cho, Hollie Noia, Elena Lomonosova, Elizabeth Stock, Alyssa Oplt, Barbara Blachut, Mary M. Mullen, Lindsay M. Kuroki, Andrea R. Hagemann, Carolyn K. McCourt, Premal H. Thaker, Dineo Khabele, Matthew A. Powell, David G. Mutch, Leah P. Shriver, Gary J. Patti, Katherine C. Fuh. GAS6-AXL inhibition by AVB-500 overcomes resistance to paclitaxel in endometrial cancer by decreasing tumor cell glycolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB081.

Abstract 3133: Glucose is important for macrophage metabolism and response to immunotherapy in a pancreatic cancer model

Abstract Introduction: Pancreatic cancer (PC) is resistant to systemic therapies. This is at least in part due to the characteristically harsh tumor microenvironment which limits the delivery of systemic therapies and is not conducive to anti-tumor immune cell function. Unfortunately, development of novel therapeutics is arduous. Thus, identification of methods to augment the efficacy of existing therapeutics, such as immunotherapies, represents an attractive option. Herein, we attempt to sensitize PC to a colony stimulating factor 1 receptor (CSF1R) inhibitor by augmenting peripheral glucose levels. Methods Murine PC cells (KPC) and bone marrow derived macrophages were cultured in hypoglycemic (≤2.5mM) or hyperglycemic (25mM) conditions. Phenotypic (western blot, flow cytometry), metabolic (seahorse, LC-MS metabolomics), and cell survival (clonogenic assay) assays were performed. KPC cells were orthotopically injected into the pancreas of immunocompetent mice for survival studies. Mice were randomized to pexidartinib (a CSF1R inhibitor) or vehicle and received either normal water of 30% dextrose (D30). Results: In a co-culture experiment, tumor-supporting M2 macrophages increased KPC cell growth by 30%, whereas tumor-fighting M1 macrophages decreased KPC growth by 95%. M1 macrophages were poorly suited to survive in hypoglycemic conditions as compared to hyperglycemic conditions (relative survival: 38% vs 100%, p<0.01). M2 macrophage survival was equivalent (93% vs 100%, p=0.39). When cultured in hypoglycemic conditions, M1 macrophages adopted an M2-like metabolic profile (i.e., increased TCA cycle metabolites and decreased glycolytic metabolites) assessed by both seahorse and LC-MS metabolomics. As glucose concentrations decreased in culture media over time, protein levels of an M1 marker (inducible nitric oxide, iNOS) decreased and an M2 marker (arginase) appeared, suggesting a phenotypic switch. Pexidartinib augmented M1 iNOS protein levels and simultaneously decreased M2 arginase levels. On an in vitro flow cytometric analysis, the percentage of M2 macrophages (CD206+, CD301+, F480+, CD11b+) decreased as glucose concentrations were increased (33.7% vs 38.0%), and decreased further when pexidartinib was combined with hyperglycemia (20.5%). Mice treated with pexidartinib and D30 had improved median survival relative to mice receiving pexidartinib alone (median survival: 42 (IQR: 38, 52) vs 34 (IQR: 32, 38) days, p<0.05) in two independent experiments. Of note, pexidartinib alone did not improve survival as compared to vehicle or D30. Conclusion: Higher glucose conditions promote M1 macrophage survival and function, while preventing the switch to an M2 phenotype. Also, higher peripheral glucose levels appear to sensitize PC to a CSF1R inhibitor. We plan to validate these findings using other immunotherapeutics (e.g., checkpoint inhibitors). Citation Format: Jonathan J. Hue, Mehrdad Zarei, Hallie J. Graor, Erryk S. Katayama, Omid Hajihassani, Alexander W. Loftus, Luke D. Rothermel, Ali Vaziri-Gohar, Jordan M. Winter. Glucose is important for macrophage metabolism and response to immunotherapy in a pancreatic cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3133.

Celastrol alleviates high-fat diet-induced obesity via enhanced muscle glucose utilization and mitochondrial oxidative metabolism-mediated upregulation of pyruvate dehydrogenase complex

Celastrol, a natural triterpene from the Tripterygium wilfordii has been demonstrated to possess attributive properties to attenuate various animal models of obesity-associated conditions. The present study aimed to elucidate the putative targets of celastrol on intracellular glucose utilization and mitochondrial oxidative metabolism in the isolated quadriceps skeletal muscle of high-fat diet (HFD)-induced obese male C57BL6/J mice. Here we showed that celastrol remarkably attenuated obesity and insulin resistance through improvement of systemic glucose tolerance and insulin sensitivity. Enhanced mRNA transcription factors of key rate-limiting glycolytic and TCA cycle enzymes were observed following celastrol administration. The metabolic profiling revealed profound changes induced by celastrol administration on several key metabolites of glycolysis and tricarboxylic acid (TCA) cycle including glucose-1-phosphate, pyruvate, citrate, α-ketoglutarate, succinate and fumarate. Celastrol effectively increased mitochondrial oxidative functions via increased pyruvate dehydrogenase complex (PDC) activity and downregulated pyruvate dehydrogenase kinase 4 (PDK4) expressions. Enhanced succinate dehydrogenase (SDH) activity was noticed following celastrol co-supplementation, leading to a steady establishment of the electrochemical gradient across mitochondrial membrane for ATP production and mitochondrial biogenesis. In conclusion, the current findings accentuate the therapeutic potential of celastrol against HFD-induced obese mice via enhanced glucose utilization and mitochondrial oxidative metabolism-mediated upregulation of PDC activity in the skeletal muscle.

Lactate dehydrogenase as a surrogate marker for both PCR and survival in breast cancer patients who underwent neoadjuvant chemotherapy.

e12621 Background: Glucose metabolism in cancer cells produces various glycolytic metabolites, which can affect cancer progression and treatment outcome. Herein we evaluated the association of glucose transporters and enzymes involved in glucose metabolism with clinic-pathological factors and outcomes in breast cancer patients who underwent neoadjuvant chemotherapy(NAC). Methods: We selected 7 target transporters and enzymes(GLUT1, hexokinase 2, lactate dehydrogenase, MCT1, MCT4, IGF1RA, and IGF1RB) involved in glucose metabolism. Their tumoral expressions were scored both quantitively and qualitatively based on tissue microarrays and immunohistochemistry as previously described (Pinheiro et al, 2010) and then evaluated in relationships with pretreatment clinical/pathological characteristics and treatment outcomes including findings of 18F-FDG PET/CT,pathologic complete response(pCR), and relapse in breast cancer(stage IIA-IIIC) patients who underwent anthracycline or taxane-based NAC. Results: Of the 236 enrolled patients, 145(61.4%), 44(18.6%), and 47(19.9%) were luminal, HER2-enriched, and TNBC subtypes. Pathologic CR was determined 50(21.2%) and 57(24.2%) relapses, 42(17.8%) distant relapses, and 28 deaths were observed during median follow-up duration of 64.0 (32.3-114.9) months. The expression rates of the target molecules were 76.0, 31.1, 42.1, 54.9, 67.9, 78.4, and 49.8% for GLUT1, hexokinase 2 (HK2), lactate dehydrogenase(LD), MCT1, MCT4, IGF1RA, and IGF1RB, respectively. Among target molecules in glucose metabolism, LD was significantly associated with pCR(OR = 0.251; p- = 0.016) and distant disease-free survival (HR = 2.29; P = 0.019). Plus, LD expression shows a trend for a high SUVmax of primary tumor 18F-FDG PET/CT(OR = 1.797 P = 0.060). Conclusions: Tumoral expression of lactate dehydrogenase can be considered as a predictive marker for pCR to NAC and plus prognostic marker for survival in patients with breast cancer.

Mitochondrial respiration in B lymphocytes is essential for humoral immunity by controlling the flux of the TCA cycle.

To elucidate the function of oxidative phosphorylation (OxPhos) during B cell differentiation, we employ CD23Cre-driven expression of the dominant-negative K320E mutant of the mitochondrial helicase Twinkle (DNT). DNT-expression depletes mitochondrial DNA during B cell maturation, reduces the abundance of respiratory chain protein subunits encoded by mitochondrial DNA, and, consequently, respiratory chain super-complexes in activated B cells. Whereas B cell development in DNT mice is normal, B cell proliferation, germinal centers, class switch to IgG, plasma cell maturation, and Tcell-dependent as well as Tcell-independent humoral immunity are diminished. DNT expression dampens OxPhos but increases glycolysis in lipopolysaccharide and B cell receptor-activated cells. Lipopolysaccharide-activated DNT-B cells exhibit altered metabolites of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle and a lower amount of phosphatidic acid. Consequently, mTORC1 activity and BLIMP1 induction are curtailed, whereas HIF1α is stabilized. Hence, mitochondrial DNA controls the metabolism of activated B cells via OxPhos to foster humoral immunity.

1306-P: In Vivo Downregulation of Pancreatic Amylin in Diabetic Mice Improves Recognition Memory

Hypersecretion of amylin, a β-cell hormone that regulates satiation, is common in individuals with prediabetes and is associated with pancreatic amyloid deposition and type-2 diabetes. Evidence has emerged that increased circulating levels of amyloid-forming human amylin may potentially impair brain function. Because mouse amylin is non-amyloidogenic, we generated transgenic mice with conditional pancreatic expression of amyloid-forming human amylin to study how in vivo knockdown of human amylin expression influences brain function during the development of type-2 diabetes. Males and females were fed a high-fat diet starting at 3 months of age to induce amylin hypersecretion and glucose dysregulation. Males developed hyperglycemia at 5 months of age, whereas females showed glucose dysregulation 3-4 months later. At 5 months of age, human amylin-expressing male mice were randomly assigned to either amylin downregulation (by peritoneal tamoxifen injection) or control (maintained amylin expression) groups (n = 10/group) . Two months later, we assessed brain function with a novel object recognition test and performed comparative non-targeted metabolomics and global RNA-seq analyses of hippocampal tissue. Mice with downregulated human amylin showed enhanced recognition memory index (p < 0.001) and lower blood glucose levels (p < 0.001) compared to those that continued to express human amylin. This was associated with increased hippocampal levels of glycolysis metabolites, including lactic acid (p < 0.01) , glucose-6-phosphate (p = 0.06) , and fructose (p = 0.07) . Hippocampal gene-expression patterns between the two mouse groups revealed extensive compensatory changes in gene expression related to glucose metabolism. In conclusion, amylin downregulation in diabetic mice improves systemic glucose homeostasis and memory. Molecular processes associated with improved memory involve increased hippocampal glycolytic fluxes and compensatory gene expression. Disclosure V.Gopal viswanathan: None. M.Muehlbauer: None. J.R.Bain: Other Relationship; CardioEphEx, LLC. F.Despa: None. N.Verma: None. E.D.Winford: None. D.Kotiya: None. L.E.Radulescu: None. N.Leibold: None. K.C.Chen: None. S.Despa: None. D.A.Hill: None. Funding National Institutes of Health NS116058, AG057290, AG053999

Metabolic reprogramming by dual-targeting biomimetic nanoparticles for enhanced tumor chemo-immunotherapy

Cancer-associated fibroblasts (CAFs)-mediated metabolic support plays a vital role in tumorigenesis. The metabolic network between cancer cells and CAFs may serve as promising targets for cancer therapy. Here, aiming at targeted blockade of the metabolic support of CAFs to cancer cells, a biomimetic nanocarrier is designed by coating solid lipid nanoparticles containing chemotherapeutic paclitaxel (PTX) and glycolysis inhibitor PFK15 with hybrid membranes of cancer cells and activated fibroblasts. The nanoparticles possess outstanding dual-targeting ability which can simultaneously target cancer cells and CAFs. The encapsulated glycolysis inhibitor PFK15 can prevent the glycolysis of cancer cells and CAFs at the same time, thus increasing the chemosensitivity of cancer cells and blocking the metabolic support of CAFs to cancer cells. The results showed that the combination of PTX and PFK15 exhibited synergistic effects and inhibited tumor growth effectively. Moreover, the biomimetic nanoparticles obviously reduced the lactate production in the tumor microenvironment, leading to activated immune responses and enhanced tumor suppression. This work presents a facile strategy to destroy the metabolic network between cancer cells and CAFs, and proves the potential to elevate chemo-immunotherapy by glycolysis inhibition. Statement of significanceIn many solid tumors, most cancer cells produce energy and carry out biosynthesis through glycolysis, even in aerobic conditions. As the main tumor stromal cells, cancer-associated fibroblasts (CAFs) usually turn oxidative phosphorylation into aerobic glycolysis with metabolic reprogramming and provide high-energy glycolytic metabolites for cancer cells. The metabolic network between cancer cells and CAFs is regarded as the vulnerability among cancer cells. Moreover, lactate produced by cancer cells and CAFs through glycolysis often leads to the immunosuppressive tumor microenvironment. The present study provides an effective approach to destroy the metabolic network between cancer cells and CAFs and greatly improves the antitumor immune response by reducing lactate production, which serves as a promising strategy for combined chemo-immunotherapy mediated by glycolysis.

The CXCL12/CXCR4/ACKR3 Signaling Axis Regulates PKM2 and Glycolysis.

In response to CXCL12, CXCR4 and ACKR3 both recruit β-arrestin 2, regulating the assembly of interacting proteins that drive signaling and contribute to the functions of both receptors in cancer and multiple other diseases. A prior proteomics study revealed that β-arrestin 2 scaffolds pyruvate kinase M2 (PKM2), an enzyme implicated in shifting cells to glycolytic metabolism and poor prognosis in cancer. We hypothesized that CXCL12 signaling regulates PKM2 protein interactions, oligomerization, and glucose metabolism. We used luciferase complementation in cell-based assays and a tumor xenograft model of breast cancer in NSG mice to quantify how CXCR4 and ACKR3 change protein interactions in the β-arrestin-ERK-PKM2 pathway. We also used mass spectrometry to analyze the effects of CXCL12 on glucose metabolism. CXCL12 signaling through CXCR4 and ACKR3 stimulated protein interactions among β-arrestin 2, PKM2, ERK2, and each receptor, leading to the dissociation of PKM2 from β-arrestin 2. The activation of both receptors reduced the oligomerization of PKM2, reflecting a shift from tetramers to dimers or monomers with low enzymatic activity. Mass spectrometry with isotopically labeled glucose showed that CXCL12 signaling increased intermediate metabolites in glycolysis and the pentose phosphate pathway, with ACKR3 mediating greater effects. These data establish how CXCL12 signaling regulates PKM2 and reprograms cellular metabolism.

GAS6-AXL Inhibition by AVB-500 Overcomes Resistance to Paclitaxel in Endometrial Cancer by Decreasing Tumor Cell Glycolysis.

Chemotherapy is often ineffective in advanced-stage and aggressive histologic subtypes of endometrial cancer. Overexpression of the receptor tyrosine kinase AXL has been found to be associated with therapeutic resistance, metastasis, and poor prognosis. However, the mechanism of how inhibition of AXL improves response to chemotherapy is still largely unknown. Thus, we aimed to determine whether treatment with AVB-500, a selective inhibitor of GAS6-AXL, improves endometrial cancer cell sensitivity to chemotherapy particularly through metabolic changes. We found that both GAS6 and AXL expression were higher by immunohistochemistry in patient tumors with a poor response to chemotherapy compared with tumors with a good response to chemotherapy. We showed that chemotherapy-resistant endometrial cancer cells (ARK1, uterine serous carcinoma and PUC198, grade 3 endometrioid adenocarcinoma) had improved sensitivity and synergy with paclitaxel and carboplatin when treated in combination with AVB-500. We also found that in vivo intraperitoneal models with ARK1 and PUC198 cells had decreased tumor burden when treated with AVB-500 + paclitaxel compared with paclitaxel alone. Treatment with AVB-500 + paclitaxel decreased AKT signaling, which resulted in a decrease in basal glycolysis. Finally, multiple glycolytic metabolites were lower in the tumors treated with AVB-500 + paclitaxel than in tumors treated with paclitaxel alone. Our study provides strong preclinical rationale for combining AVB-500 with paclitaxel in aggressive endometrial cancer models.

Shikonin inhibited glycolysis and sensitized cisplatin treatment in non-small cell lung cancer cells via the exosomal pyruvate kinase M2 pathway

ABSTRACT The active ingredient of the traditional Chinese medicine comfrey is shikonin, a naphthoquinone compound. The focus of this study was to investigate the effect of shikonin on the proliferation, invasion, migration, and chemoresistance of non-small cell lung cancer (NSCLC) cells, and to explore its underlying molecular biological mechanisms. The results show that shikonin inhibited the viability, proliferation, invasion, and migration of NSCLC cells A549 and PC9, and induced apoptosis. As the inhibitor of pyruvate kinase M2 (PKM2), a key enzyme in glycolysis, shikonin inhibited glucose uptake and the production of lactate, the final metabolite of aerobic glycolysis. In vivo chemotherapeutic assay showed that shikonin reduced the tumor volume and weight in NSCLC mice model and increased the sensitivity to cisplatin chemotherapy. Histoimmunology experiments showed the combination of shikonin and cisplatin downregulated the expression of PKM2 and its transcriptionally regulated downstream gene glucose transporter 1 (Glut1) in tumor tissue. In an assessment of glucose metabolism, micro-PET/CT data showed a combination of shikonin and cisplatin inhibited the fluorodeoxy glucose (18F-FDG) uptake into tumor. Since exosomal PKM2 affected the sensitivity to cisplatin in NSCLC cells, we also demonstrated shikonin could inhibit exosome secretion and exosomal PKM2 through the administration of exosomal inhibitor GW4869. Furthermore, shikonin sensitized cisplatin treatment by reducing the extracellular secretion of exosomal PKM2. In conclusion, we suggest that shikonin not only inhibits PKM2 intracellularly but also reduces glycolytic flux and increases cisplatin sensitivity through the exosomal pathway.

A protein mutated in Parkinson's disease prevents damage of metabolites and proteins caused by a glycolytic metabolite

The accumulation of cellular damage is suspected to contribute to aging and neurodegenerative diseases. Oxidative stress and environmental factors likely play a role, but other damaging agents likely remain to be discoveredHere we report that an enzyme mutated in hereditary Parkinson’s disease achieves the seemingly impossible task to prevent damage caused by a glycolytic metabolite without affecting the metabolite itself. Inactivation of this enzyme in model systems ranging from flies to human cells leads to the accumulation of a wide range of metabolites and proteins damaged by a newly discovered covalent modification.Thus, this enzyme represents a highly conserved strategy to prevent damage in cells that perform glycolysis. Thereby, it interrupts a fundamental link between carbohydrate metabolism and a type of cellular damage that might contribute to the development of Parkinson’s disease.

The Integrated Stress Response and Eukaryotic Initiation Factor 2a Regulate Methylglyoxal-induced Pain Hypersensitivity

Diabetic neuropathic pain (DNP) is a debilitating condition. Current therapies are ineffective at treating DNP. We have previously demonstrated that methylglyoxal (MGO), a reactive metabolite of glycolysis that is elevated in the plasma of diabetic patients, pain hypersensitivity by engaging the integrated stress response (ISR) in mice. Induction of the ISR alters protein synthesis by inhibiting eukaryotic initiation factor 2α (eIF2α) and relying on eIF2A-mediated translation. We hypothesize that eIF2A regulates protein synthesis that contributes to pain hypersensitivity under conditions of ISR, especially after MGO application. We employed the recently characterized eIF2A-/- mouse to investigate how MGO treatment induces the ISR, facilitates eIF2A-mediated translation, and consequently causes pain hypersensitivity. We also subjected mice to tunicamycin (TUN), a well-known ISR inducer, and spared nerve injury (SNI). With donated human dorsal root ganglia (DRG) and spinal nerves (SpN), we aimed to translate our finding in rodents to humans. Intraplantar and intraperitoneal administrations of MGO induced pain hypersensitivity in a dose-dependent manner in male and female WT animals but not in eIF2A-/- mice. Following TUN and SNI, the eIF2A-/-¬¬ mice were protected against ISR-dependent pain (tunicamycin) but not against ISR-independent pain (SNI). MGO treatment of human DRG and SpN explants, and cultured human DRG neurons elevated p-eIF2α and eIF2A levels suggesting that MGO induces ISR not only in mice, but also in humans. Carboxyethyl lysine (CEL), a byproduct of reactive MGO and lysine residues, is increased in MGO treated cells and DRG and spinal nerve explants but not in tunicamycin treated explants, despite sharing elevated p-eIF2α and eIF2A levels. We demonstrate that eIF2A is necessary for the development of ISR-induced pain hypersensitivity. We further suggest that MGO and its byproduct, CEL, can be used as a biomarker to stratify DNP patients that would respond to ISR inhibitors like ISRIB. Diabetic neuropathic pain (DNP) is a debilitating condition. Current therapies are ineffective at treating DNP. We have previously demonstrated that methylglyoxal (MGO), a reactive metabolite of glycolysis that is elevated in the plasma of diabetic patients, pain hypersensitivity by engaging the integrated stress response (ISR) in mice. Induction of the ISR alters protein synthesis by inhibiting eukaryotic initiation factor 2α (eIF2α) and relying on eIF2A-mediated translation. We hypothesize that eIF2A regulates protein synthesis that contributes to pain hypersensitivity under conditions of ISR, especially after MGO application. We employed the recently characterized eIF2A-/- mouse to investigate how MGO treatment induces the ISR, facilitates eIF2A-mediated translation, and consequently causes pain hypersensitivity. We also subjected mice to tunicamycin (TUN), a well-known ISR inducer, and spared nerve injury (SNI). With donated human dorsal root ganglia (DRG) and spinal nerves (SpN), we aimed to translate our finding in rodents to humans. Intraplantar and intraperitoneal administrations of MGO induced pain hypersensitivity in a dose-dependent manner in male and female WT animals but not in eIF2A-/- mice. Following TUN and SNI, the eIF2A-/-¬¬ mice were protected against ISR-dependent pain (tunicamycin) but not against ISR-independent pain (SNI). MGO treatment of human DRG and SpN explants, and cultured human DRG neurons elevated p-eIF2α and eIF2A levels suggesting that MGO induces ISR not only in mice, but also in humans. Carboxyethyl lysine (CEL), a byproduct of reactive MGO and lysine residues, is increased in MGO treated cells and DRG and spinal nerve explants but not in tunicamycin treated explants, despite sharing elevated p-eIF2α and eIF2A levels. We demonstrate that eIF2A is necessary for the development of ISR-induced pain hypersensitivity. We further suggest that MGO and its byproduct, CEL, can be used as a biomarker to stratify DNP patients that would respond to ISR inhibitors like ISRIB.

Aminoaciduria and metabolic dysregulation during diabetic ketoacidosis: Results from the diabetic kidney alarm (DKA) study

ObjectiveWe examined changes in the excretion of various amino acids and in glycolysis and ketogenesis-related metabolites, during and after diabetic ketoacidosis (DKA) diagnosis, in youth with known or new onset type 1 diabetes (T1D). MethodsUrine samples were collected from 40 youth with DKA (52% boys, mean age 11 ± 4 years, venous pH 7.2 ± 0.1, blood glucose 451 ± 163 mg/dL) at 3 time points: 0–8 h and 12–24 h after starting an insulin infusion, and 3 months after hospital discharge. Mixed-effects models evaluated the changes in amino acids and other metabolites in the urine. ResultsConcentrations of urine histidine, threonine, tryptophan, and leucine per creatinine were highest at 0–8 h (148.8 ± 23.5, 59.5 ± 12.3, 15.4 ± 1.4, and 24.5 ± 2.4% of urine creatinine, respectively), and significantly decreased over 3 months (p = 0.028, p = 0.027, p = 0.019, and p < 0.0001, respectively). Urine histidine, threonine, tryptophan, and leucine per urine creatinine decreased by 10.6 ± 19.2, 0.7 ± 0.9, 1.3 ± 0.9, and 0.5 ± 0.3-fold, respectively, between 0 and 8 h and 3 months. ConclusionsIn our study, DKA was associated with profound aminoaciduria, suggestive of proximal tubular dysfunction analogous to Fanconi syndrome.

Adenylate Kinase Isozyme 3 Regulates Mitochondrial Energy Metabolism and Knockout Alters HeLa Cell Metabolism.

The balance between oxidative phosphorylation and glycolysis is important for cancer cell growth and survival, and changes in energy metabolism are an emerging therapeutic target. Adenylate kinase (AK) regulates adenine nucleotide metabolism, maintaining intracellular nucleotide metabolic homeostasis. In this study, we focused on AK3, the isozyme localized in the mitochondrial matrix that reversibly mediates the following reaction: Mg2+ GTP + AMP ⇌ Mg2+ GDP + ADP. Additionally, we analyzed AK3-knockout (KO) HeLa cells, which showed reduced proliferation and were detected at an increased number in the G1 phase. A metabolomic analysis showed decreased ATP; increased glycolytic metabolites such as glucose 6 phosphate (G6P), fructose 6 phosphate (F6P), and phosphoenolpyruvate (PEP); and decreased levels of tricarboxylic acid (TCA) cycle metabolites in AK3KO cells. An intracellular ATP evaluation of AK3KO HeLa cells transfected with ATeam plasmid, an ATP sensor, showed decreased whole cell levels. Levels of mitochondrial DNA (mtDNA), a complementary response to mitochondrial failure, were increased in AK3KO HeLa cells. Oxidative stress levels increased with changes in gene expression, evidenced as an increase in related enzymes such as superoxide dismutase 2 (SOD2) and SOD3. Phosphoenolpyruvate carboxykinase 2 (PCK2) expression and PEP levels increased, whereas PCK2 inhibition affected AK3KO HeLa cells more than wild-type (WT) cells. Therefore, we concluded that increased PCK2 expression may be complementary to increased GDP, which was found to be deficient through AK3KO. This study demonstrated the importance of AK3 in mitochondrial matrix energy metabolism.

OGA activated glycopeptide-based nano-activator to activate PKM2 tetramerization for switching catabolic pathways and sensitizing chemotherapy resistance

Tumor cells intensively engage in metabolic reprogramming for enhancing the availability of glycolytic metabolites and support cell proliferation. As the most important rate-limiting enzyme in aerobic glycolysis, activating the pyruvate kinase muscle isoform 2 (PKM2) from dimers to tetramers has become a key tumor chemotherapy method to control glucose metabolism. Herein, we developed a glycopeptide-based PKM2 nano-activator, which could induce the tetramerization of PKM2 based on serine bonding to Domain C of PKM2. The bound and trapped PKM2 tetramers significantly hindered glycolytic intermediates, prevented the nucleus translocation of dimeric PKM2, and ultimately inhibited the proliferation, chemoresistance and metastasis of tumor. The glycopeptide assembled into nanoparticles under aqueous conditions and in the circulation, which in situ transformed into PKM2 nano-activator with nanofibrillar structure after specifically activated by O-GlcNAcase recognition upregulated in a wide range of human tumors. Moreover, the glycopeptide-based PKM2 nano-activator successfully accumulated at the tumor sites and boosted the chemo-drug sensitivity against prostate and breast cancers. Attributed to these intriguing results, the newly developed glycopeptide-based PKM2 nano-activator can be envisioned a promising candidate for the treatment of tumors by switching catabolic pathways.

ADAPTOR PROTEIN Ruk/CIN85 PARTICIPATES IN THE METABOLIC CONTROL OF HUMAN BREAST ADENOCARCINOMA MCF-7 CELLS

Aim. To determine the role of Ruk/CIN85 in the control of breast adenocarcinoma cells metabolism, we performed systemic analysis of the activity levels/content of key enzymes/components of glycolysis and oxidative phosphorylation using as a model the weakly invasive human breast adenocarcinoma MCF-7 cell line (Mock); and its sublines with stable overexpression (G4 subline) and reverse down-regulation (G4vir subline) of the adaptor protein. Materials and methods. MCF-7 cells were cultured in the complete DMEM medium under standard conditions. Enzymes activity, content of metabolites and protein in cell extracts and the conditioned cell culture medium were estimated by spectrophotometric and fluorometric assays. Results. First of all, biochemical indexes of aerobic glycolysis, activity levels of some key glycolytic enzymes and metabolites were evaluated. A significant increase in the activity of these enzymes, aldolase A (ALDOA) and lactate dehydrogenase A (LDHA), was found in G4 cells compared to Mock by 1.3 and 1.6 times, respectively. In addition, in the conditioned medium of G4 cells, an increase in lactate content by 1.5 times compared with the control was found, which corresponded to a change in LDHA activity. Knockdown of Ruk/CIN85 expression level in G4 subline resulted in a significant decrease of these parameters compared to G4 cells, ALDOA – 4 times, LDHA - 1.4 times, and lactate production - 2.5 times. It should be noted that in G4vir cells, LDHA activity returned to level of control cells, while ALDOA activity and lactate content additionally decreased by 3 times and 1.6 times, respectively. Therefore, the observed changes in the intensity of glycolysis in MCF-7 sublines positively correlate with the expression level of adaptor protein studied. To assess the metabolic status of mitochondria, the level of activity of the Krebs cycle enzyme, NAD-dependent malate dehydrogenase (MDH2), the catalyst of last stage of the cycle, was determined. A 2-fold decrease in MDH2 activity was found in the MCF-7 G4 subline relative to control Mock cells, as well as an increase in this index by 2.4 times in G4vir cells to control values. Unlike glycolysis, we observed the opposite pattern with respect to the intensity of Krebs cycle reactions depending on the expression level of Ruk/CIN85. Conclusions. Use of limited proteolysis technique as the source of additional information for computer modeling allowed us to propose an improved model of 3D-structure of fibrinogen αC-regions. This model takes into account the behavior of αC-regions in the physiological condition and contributes to the general knowledge about fibrinogen structure.

OsCKX2 regulates phosphate deficiency tolerance by modulating cytokinin in rice

Cytokinin oxidase/dehydrogenases (CKXs) are key enzymes that degrade cytokinins (CTKs) and play an essential role in plant growth and development. The present study analyzed the phenotypic and physiological characteristics of OsCKX2 overexpressing (OE) and knockout (KO) rice plants after exposure to phosphate (Pi) deficiency and the transcriptome and metabolome to investigate the function of OsCKX2 in response to Pi deficiency. OsCKX2 KO plants demonstrated higher endogenous CTK levels than wild-type (WT) under Pi deficiency. Further analysis indicated more robust tolerance of OsCKX2 KO plants to Pi deficiency, which exhibited higher phosphorus concentration, larger shoot biomass, and lesser leaf yellowing under Pi deficiency; whereas the opposite was observed for OsCKX2 OE plants. Transcriptome and metabolome analyses revealed that overexpression of OsCKX2 downregulated the transcriptional levels of genes related to Pi transporters, membrane lipid metabolism, and glycolysis, and reduced the consumption of metabolites in membrane lipid metabolism and glycolysis. On the contrary, knockout of OsCKX2 upregulated the expression of Pi transporters, and increased the consumption of metabolites in membrane lipid metabolism and glycolysis. These results indicated that OsCKX2 impacted Pi uptake, recycling, and plant growth via Pi transporters, phospholipid hydrolysis, and glycolysis under Pi deficiency. Overall, OsCKX2 negatively regulated Pi deficiency tolerance by modulating CTKs in rice.