Decrease In Glucose Consumption Research Articles

Triple negative breast cancer migration is modified by mitochondrial metabolism alteration induced by natural extracts of C. spinosa and P. alliacea

Tumor metabolism is a crucial aspect of cancer development, and mitochondria plays a significant role in the aggressiveness and metastasis of tumors. As a result, mitochondria have become a promising therapeutic target in cancer treatment, leading to the development of compounds known as mitocans. In our group, we have consolidated the search of anticancer therapies based on natural products derived from plants, obtaining extracts such as P2Et from Caesalpinia spinosa and Anamu-SC from Petiveria alliacea, which have been shown to have antitumor activities in different cancer models. These extracts, due to their complex molecular composition, can interfere with multiple functions during tumor progression. To better understand how these natural products operate (P2Et and Anamu-SC), we constructed a model using 4T1 murine breast cancer cells with reduced expression of genes associated with glycolysis (Hexokinase-2) and mitochondrial function (Cqbp). The results indicate that the cells were more sensitive to the Anamu-SC extract, showing significant decreases in glucose consumption, ATP production, and oxygen consumption rate. Additionally, we observed changes in mitochondrial function, which reduced the cells’ ability to migrate, particularly when C1qbp was silenced. This triple-negative breast cancer model allows us to identify potential natural products that can modulate tumor cell metabolism.

Tensin 4 facilitates aerobic glycolysis, migration and invasion of colorectal cancer cells through the β‑catenin/c‑Myc signaling pathway.

Tensin 4 (TNS4) is overexpressed in multiple cancers, including colorectal cancer (CRC), and is associated with a poor prognosis of patients with CRC. However, the role and underlying mechanisms of TNS4 in CRC have yet to be elucidated. The expression of TNS4 in CRC tissues were analyzed by immunohistochemistry. Cell migration and invasion were assessed in vitro using Transwell assay. Western blot and reverse transcription (RT)-quantitative (q)PCR were used to investigate the molecular mechanisms by which TNS4 regulates aerobic glycolysis, migration and invasion of CRC cells. The present study demonstrated that TNS4 was highly expressed in the cancer tissues of patients with CRC and significantly associated with the tumor-node-metastasis stages. TNS4 silencing led to a significant decrease in glucose consumption and lactate production in CRC cells, and knockdown of TNS4 suppressed the migration and invasion of CRC cells via aerobic glycolysis through the β-catenin/c-Myc pathway. Notably, treatment with DASA-58, an activator of glycolysis, or SKL2001, an activator of β-catenin/c-Myc signaling, significantly reversed the effect of TNS4 knockdown on aerobic glycolysis, migration and invasion of CRC cells. Collectively, these results suggest that TNS4 may act as a novel regulator of aerobic glycolysis, migration and invasion of CRC cells by modulating β-catenin/c-Myc signaling, providing a new potential biomarker and therapeutic target in CRC.

Abstract A105: Pharmacological inhibition of nicotinamide adenine dinucleotide (NAD+) production enzyme nicotinamide phosphoribosyltransferase (NAMPT) impairs cellular survival, energy metabolism, and tumor growth in neuroblastoma (NB) models

Abstract Background: Neuroblastoma is the most common extracranial solid tumor in children. High-risk patients have a poor prognosis with an overall survival rate of <50%, thus novel therapeutic strategies are critically needed. To address this need, we performed a high-throughput drug screen and identified nicotinamide phosphoribosyltransferase inhibitors (NAMPTis) as highly active against neuroblastoma cells compared to other cancer cell types. NAMPT is the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway, which cancer cells preferentially utilize to generate NAD+, a key co-factor that plays essential roles in energy metabolism. In this study, we investigated the translational potential and mechanistic effects of NAMPT inhibition in preclinical neuroblastoma models. Methods/Results: Using 3 NAMPTis on a panel of 10 neuroblastoma cell lines, we confirmed that a majority of these models were highly responsive to NAMPTis by Incucyte Live-Cell proliferation analysis. Notably, we observed complete growth inhibition in most models at doses <3.2nM of OT-82, a clinically relevant NAMPTi with a more favorable toxicity profile. On-target activity was confirmed by dose-dependent depletion of intracellular NAD+ in cells treated with OT-82. As NAD+ is necessary for energy metabolism, particularly ATP production, we performed a Cell-Titer Glo assay to indirectly assess ATP levels and observed decreases in all models with OT-82 treatment. Glycolysis and oxidative phosphorylation (OXPHOS) are two processes involved in ATP production that utilize NAD+, thus we measured activity of both processes via biochemical and extracellular flux analyses. With OT-82 treatment, we observed decreases in glucose consumption and lactate production in most models, and decreases in OXPHOS in some cell lines. Finally, an in vivo pilot study testing OT-82 in an orthotopic neuroblastoma PDX model demonstrated antitumor activity. Conclusion: Together, these data show that neuroblastoma is susceptible to disruption of the NAMPT pathway and suggest that NAMPTis have translational potential as a novel therapy for neuroblastoma patients. Citation Format: Sophia Varriano, Unsun Lee, Victor Collins, Rosa Nguyen, Sameer Issaq, Ming Sun, Ariana Nelson, Choh Yeung, Craig Thomas, Arnulfo Mendoza, Carol Thiele, Christine Heske. Pharmacological inhibition of nicotinamide adenine dinucleotide (NAD+) production enzyme nicotinamide phosphoribosyltransferase (NAMPT) impairs cellular survival, energy metabolism, and tumor growth in neuroblastoma (NB) models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A105.

NSCLC presents metabolic heterogeneity, and there is still some leeway for EGF stimuli in EGFR-mutated NSCLC

BackgroundMetabolic remodeling is crucial in carcinogenesis and cancer progression. Oncogenic mutations may promote metabolic reprogramming in cancer cells to support their energy and biomass requirements. EGFR mutations are commonly found in non-small cell lung cancer (NSCLC) and may induce NSCLC metabolic rewiring. Whether EGFR-driven metabolic reprogramming triggers cell vulnerabilities with therapeutic potential remains unknown. MethodsThe role of EGFR signaling activation by EGF was investigated using NSCLC cell lines with different EGFR and KRAS status: A549 (EGFR WT and KRAS c.34G > A), H292 (EGFR WT and KRAS WT) and PC-9 (EGFR exon 19 E746-A750 deletion and KRAS WT). The effect of EGF on NSCLC cell death and cell cycle was evaluated using flow cytometry, and cell migration was assessed through wound healing. EGFR, HER2, MCT1, and MCT4 expression was analyzed through immunofluorescence or western blotting. We explored the impact of glucose and lactate bioavailability on NSCLC cells' metabolic profile using nuclear magnetic resonance (NMR) spectroscopy. Moreover, the expression of several relevant metabolic genes in NSCLC cells or patient samples was determined by RT-qPCR. ResultsWe showed that cell lines presented different metabolic profiles, and PC-9 cells were the most responsive to EGF stimulus, as they showed higher rates of cell proliferation and migration, together with altered metabolic behavior. By inhibiting EGFR with gefitinib, a decrease in glucose consumption was observed, which may be related to the fact that despite PC-9 harbor EGFR mutation, they still express the EGFR WT allele. The analysis of NSCLC patients' RNA showed a correlation between MCT1/MCT4 and GLUT1 expression in most cases, indicating that the metabolic information can serve as a reference in patients' follow-up. ConclusionTogether, this study shows that NSCLC cell lines have heterogeneous metabolic profiles, which may be underlaid by different genetic profiles, revealing an opportunity to identify and stratify patients who can benefit from metabolism-targeted therapies.

Baicalein suppresses rectal cancer progression through inhibition of cellular glycolysis and intracellular adenosine triphosphate level

This study explored the mechanism of baicalein on rectal cancer. Human rectal adenocarcinoma cells HR8348 were exposed to low, moderate and high concentration of baicalein (5, 10, and 20 μmol/L), respectively, for 48 h with untreated control group, followed by analysis of glucose consumption, lactic acid and denosine triphosphate (ATP) content, cell proliferation and expression of phosphatidylinositol-3-kinase (PI3K), protein kinase B(AKT), and Hypoxia-Inducible Factor 1-Alpha (HIF-1α). Cell viability in groups of baicalein decreased at 24, 48, and 72 h after treatment, with lowest cell viability in high-dose group (P <0.05), especially at 48 and 72 h in both medium and high dose groups (P <0.05). Of note, baicalein dose-dependently induced cell apoptosis and reduced levels of PI3K, AKT, and HIF-1α. Moreover, glucose consumption and lactic acid content gradually decreased in the absence of baicalein, and the increased concentration of baicalein was associated with more dramatical decrease in glucose consumption. The lowest glucose consumption [(6.49±2.23) μmol/105 cells] and lactic acid content [(16.90±3.77) μmol/105 cells] was noticed in the high-dose group. Baicalein decreased ATP content, facilitating energy metabolism in rectal cancer cells (P <0.05). Baicalein inhibits PI3K/Akt pathway to down-regulate HIF-1α, thereby suppressing cell viability and proliferation of rectal cancer HR8348 cells. This is due to inhibition of glycolysis and intracellular energy metabolism, which provides a new insight into new treatment method for rectal cancer.

Advanced in Vitro Safety Assessment of Herbal Medicines for the Treatment of Non-Psychotic Mental Disorders in Pregnancy.

When confronted with non-psychotic mental disorders, pregnant women often refrain from using synthetic drugs and resort to herbal medicines such as St. John’s wort, California poppy, valerian, lavender, and hops. Nevertheless, these herbal medicines have not yet been officially approved in pregnancy due to lack of safety data. Using a variety of in vitro methods (determination of cytotoxicity, apoptosis induction, genotoxicity, effects on metabolic properties, and inhibition/induction of differentiation) in a commonly used placental cell line (BeWo b30), we were previously able to show that extracts from these plants are likely to be safe at the usual clinical doses. In the present work, we wanted to extend our safety assessment of these herbal medicines by 1) looking for possible effects on gene expression and 2) using the same in vitro methods to characterize effects of selected phytochemicals that might conceivably lead to safety issues. Proteomics results were promising, as none of the five extracts significantly affected protein expression by up- or down-regulation. Protopine (contained in California poppy), valerenic acid (in valerian), and linalool (in lavender) were inconspicuous in all experiments and showed no adverse effects. Hyperforin and hypericin (two constituents of St. John’s wort) and valtrate (typical for valerian) were the most obvious phytochemicals with respect to cytotoxic and apoptotic effects. A decrease in cell viability was evident with hypericin (≥1 µM) and valtrate (≥10 µM), whereas hyperforin (≥3 µM), hypericin (30 µM) and valtrate (≥10 µM) induced cell apoptosis. None of the tested phytochemicals resulted in genotoxic effects at concentrations of 0.1 and 1 µM and thus are not DNA damaging. No decrease in glucose consumption or lactate production was observed under the influence of the phytochemicals, except for valtrate (at all concentrations). No compound affected cell differentiation, except for hyperforin (≥1 µM), which had an inhibitory effect. This study suggests that extracts from St. John’s wort, California poppy, valerian, lavender, and hops are likely to be safe during pregnancy. High plasma concentrations of some relevant compounds—hyperforin and hypericin from St. John’s wort and valtrate from valerian—deserve special attention, however.

Monitoring the mechanism of anti-cancer agents to inhibit colorectal cancer cell proliferation: Enzymatic biosensing of glucose combined with molecular docking

Glucose, a major energy source in cellular metabolism, has a significant role in cell growth. The increase in glucose uptake is a distinguishing hallmark in cancer cells. A key step in glucose utilization is the transport of glucose to the cancer cells for supplying their additional energy. The glucose transporter (or GLUT) family is a membrane protein which facilitates the uptake of glucose in most cancer cell types. Given the increased glucose level in cancer cells and the regulatory role of GLUTs in glucose uptake, it is required to combine both experimental and theoretical studies to develop new methods to monitor cell proliferation. Herein, for the first time, a new strategy was proposed to evaluate the cell proliferation of HT-29 based on glucose consumption in the presence of resveratrol (RSV) as an anticancer agent. A hybrid nanocomposite of carbon nanofibers and nitrogen-doped graphene quantum dots was used to design an enzymatic sensor for the selective and sensitive determination of glucose in cancer cells. The results obtained from the voltammetric technique were compared with the conventional colorimetric assay. A good correlation was observed between the proliferation rate and glucose utilization by cancer cells. As it was observed, RSV induces a decrease in glucose consumption, indicating lower glucose uptake efficiency for HT-29 cells. Molecular docking studies reveal that RSV can block the interaction of glucose with the GLUT family. This is one of the possible mechanisms for the decrease of glucose level followed by the reduction of cell proliferation in the presence of RSV. Compared with traditional methods, in vitro electrochemical techniques benefit from simple, nontoxic, sensitive and low-cost detection assays and hence serve as a novel tool to pursue the growth inhibition of cancer cell in response to anti-cancer agents.

Removal of citrate from PAS‐III additive solution improves functional and biochemical characteristics of buffy‐coat platelet concentrates stored for 7 days, with or without INTERCEPT pathogen reduction

Deterioration in quality of platelet concentrates (PCs) during storage results from the appearance of storage lesions affecting the hemostatic functions and posttransfusion survival of platelets. These lesions depend on the preparation and pathogen inactivation methods used, duration of storage, and platelet additive solutions (PASs) present in storage bags. We investigated the effects of citrate contained in third-generation PAS (PAS-III) on storage lesions in buffy-coat PCs with or without photochemical (amotosalen-ultraviolet A) treatment over 7 days. Platelet counts were conserved in all groups during storage, as was platelet swirling without appearance of macroscopic aggregates. Glycoprotein (GP) IIbIIIa and GPVI expression remained stable, whereas GPIbα declined similarly in all groups during storage. Removal of citrate from PAS-III, resulting in global reduction of citrate from 11 to 5 mM, led to a significant decrease in glucose consumption, which largely countered a modest deleterious effect of photochemical treatment. Citrate reduction also resulted in decreased lactate generation and better maintenance of pH during storage, while photochemical treatment had no impact on these parameters. Moreover, citrate-free storage significantly reduced exposure of P-selectin and the apoptosis signal phosphatidylserine, thereby abolishing the activating effect of photochemical treatment on both parameters. Citrate reduction benefited platelet aggregation to various agonists up to Day 7, whereas PCT had no impact on these responses. Removal of citrate from PAS-III has a beneficial impact on platelet metabolism, spontaneous activation, and apoptosis, and improves platelet aggregation, irrespective of photochemical treatment, which should allow transfusion of platelets with better and longer-lasting functional properties.

Oxidative Pentose Phosphate Pathway Enzyme 6-Phosphogluconate Dehydrogenase Plays a Key Role in Breast Cancer Metabolism.

Simple SummaryCancer cells alter their metabolism to maintain their high need for energy, produce enough macromolecules for biosynthesis, and preserve their redox status. The investigation of cancer cell-specific metabolic alterations has vital importance to identify targets to be exploited for therapeutic development. The pentose phosphate pathway (PPP) is often highly activated in tumor cells to maintain redox level, as this pathway takes an important role in reactive oxygen species detoxification. PPP also yields ribose-5-phosphate, a five-carbon sugar essential for synthesizing nucleotides necessary for DNA replication and cell proliferation. In this study, we inhibited one of the key enzymes of this biochemical pathway and observed the main functions of this enzyme in breast cancer cells. We have demonstrated that inhibition of this enzyme reduces cell proliferation and leads to cell cycle arrest and apoptosis. Besides that, we showed that the inhibition of this enzyme causes an alteration in cellular metabolism. All these findings indicate that targeting this enzyme with specific pharmacological inhibitors is an effective strategy in fighting cancer.The pentose phosphate pathway (PPP) plays an essential role in the metabolism of breast cancer cells for the management of oxidative stress and the synthesis of nucleotides. 6-phosphogluconate dehydrogenase (6PGD) is one of the key enzymes of the oxidative branch of PPP and is involved in nucleotide biosynthesis and redox maintenance status. Here, we aimed to analyze the functional importance of 6PGD in a breast cancer cell model. Inhibition of 6PGD in MCF7 reduced cell proliferation and showed a significant decrease in glucose consumption and an increase in glutamine consumption, resulting in an important alteration in the metabolism of these cells. No difference in reactive oxygen species (ROS) production levels was observed after 6PGD inhibition, indicating that 6PGD, in contrast to glucose 6-phosphate dehydrogenase, is not involved in redox balance. We found that 6PGD inhibition also altered the stem cell characteristics and mammosphere formation capabilities of MCF7 cells, opening new avenues to prevent cancer recurrance after surgery or chemotherapy. Moreover, inhibition of 6PGD via chemical inhibitor S3 resulted in an induction of senescence, which, together with the cell cycle arrest and apoptosis induction, might be orchestrated by p53 activation. Therefore, we postulate 6PGD as a novel therapeutic target to treat breast cancer.

Short-Chain Hydroxyl CoA Dehydrogenase Probably is the Central Player in DNA Replication and Glucose Consumption in Human Non-Small Lung Carcinoma Cell Line

: Hydroxyl CoA Dehydrogenase (HADH) is one of the key enzymes in fatty acid β-oxidation. Recently, Hydroxyl CoA Dehydrogenase gene mutation and knockdown were found to be correlated with hyperinsulinemia and central nervous system diseases. As the HADH is one of the critical enzymes in the β-oxidation pathway, the interconnection between HADH and tumorigenicity still is unclear. So, we used Short hairpin RNA (ShRNA) to knock down short-chain hydroxyl CoA dehydrogenase (HADHSC) in human non-small lung carcinoma cell line, H1299, followed by checking cell proliferation, DNA replication, and mRNA level of some the most essential enzymes in glycolysis cycle and Krebs. Cell proliferation was checked by comparing the cell numbers in knockdown and control cells. DNA replication in the H1299 cell line was studied after applying 5-ethynyl 2’-deoxyuridine (EDU) and 4’-6 diamidino-2-phenylindol (DAPI) DNA synthesis Assay. The data revealed a significant decrease in cell proliferation and DNA replication in the cells that the HADHSC was knocked down compared to the control cells. Besides, mRNA levels of the enzymes that needed adenosine triphosphate (ATP) for their activity were decreased abruptly. Furthermore, lactate dehydrogenase (LDHA) mRNA level decreased, and glucose uptake assay showed a tremendous decrease in glucose consumption by H1299 cells with HADHSC knockdown.

Penfluridol triggers mitochondrial-mediated apoptosis and suppresses glycolysis in colorectal cancer cells through down-regulating hexokinase-2.

Penfluridol, a commonly used antipsychotic agent in a clinical setting, exhibits potential anticancer properties against various human malignancies. Here, we investigated the effect of penfluridol on the biological behavior of colorectal cancer (CRC) cells. Cell viability and clonogenic potential were detected by the cell counting kit-8 and colony formation assay. The cell apoptosis and cell cycle distribution were quantified through flow cytometry. Caspase-3 activity, glucose consumption, lactate production, and intracellular ATP levels were evaluated using the corresponding commercial detection kits. The protein levels of related genes were detected through western blotting. Mitochondrial membrane potential was detected using JC-1 staining. A CRC xenograft tumor model was used to validate the antitumor activity of penfluridol in vivo. Penfluridol reduced cell survival and promoted apoptotic cell death effectively through the mitochondria-mediated intrinsic pathway in a dose-dependent manner. Furthermore, the process of glycolysis in HCT-116 and HT-29 cells was inhibited upon penfluridol treatment, as evidenced by the decrease in glucose consumption, lactate production, and intracellular ATP levels. Further mechanistic studies revealed that penfluridol influenced cell apoptosis and glycolysis in CRC cells by downregulating hexokinase-2 (HK-2). The proapoptotic effect and glycolytic inhibition-induced by penfluridol were effectively reversed by HK-2 overexpression. Consistent with in vitro results, penfluridol could also suppress tumor growth and trigger apoptosis in vivo. Penfluridol triggers mitochondrial-mediated apoptosis and induces glycolysis inhibition via modulating HK-2 in CRC and provides a theoretical basis to support penfluridol as a repurposed drug for CRC patients.

Identification of Gliotoxin isolated from marine fungus as a new pyruvate kinase M2 inhibitor

Pyruvate kinase M2 (PKM2) functions as an important rate-limiting enzyme of aerobic glycolysis that is involved in tumor initiation and progression. However, there are few studies on effective PKM2 inhibitors. Gliotoxin is a marine-derived fungal secondary metabolite with multiple biological activities, including immunosuppression, cytotoxicity, and et al. In this study, we found that Gliotoxin directly bound to PKM2 and inhibited its glycolytic activity in a dose-dependent manner accompanied by the decreases in glucose consumption and lactate production in the human glioma cell line U87. Moreover, Gliotoxin suppressed tyrosine kinase activity of PKM2, leading to a dramatic reduction in Stat3 phosphorylation in U87 cells. Furthermore, Gliotoxin suppressed cell viability in U87 cells, and cytotoxicity of Gliotoxin on U87 cells was obviously augmented under hypoxia condition compared to normal condition. Finally, Gliotoxin was demonstrated to induce cell apoptosis of U87 cells and synergize with temozolomide. Our findings identify Gliotoxin as a new PKM2 inhibitor with anti-tumor activity, which lays the foundation for the development of Gliotoxin as a promising anti-tumor drug in the future.

Ginsenoside 20(S)-Rg3 upregulates HIF-1α-targeting miR-519a-5p to inhibit the Warburg effect in ovarian cancer cells.

The Warburg effect, one of the metabolic hallmarks of cancer, is responsible for rapid energy production through a high rate of aerobic glycolysis. Ginsenoside 20(S)-Rg3 antagonizes the Warburg effect in ovarian cancer cells by upregulating some microRNAs, including miR-519a-5p, that target key enzymes involved in aerobic glycolysis. How 20(S)-Rg3-upregulated miR-519a-5p influences the Warburg effect in ovarian cancer cells remains poorly defined, however. Here we report that while overexpression of miR-519a-5p in ovarian cancer cells inhibited the Warburg effect, inhibition of miR-519a-5p negated the suppressive action of 20(S)-Rg3 against the Warburg effect as evidenced by a decrease in glucose consumption, lactate production and HK2 expression. We identified HIF-1α as a direct target of miR-519a-5p via luciferase reporter assays and demonstrated the counteraction by overexpressed HIF-1α of 20(S)-Rg3-suppressed Warburg effect. Further, 20(S)-Rg3 decreased DNMT3A-mediated DNA methylation in the promoter region of its precursor gene, leading to an increase in the level of miR-519a-5p. In conclusion, 20(S)-Rg3 upregulates miR-519a-5p via reducing DNMT3A-mediated DNA methylation to inhibit HIF-1α-stimulated Warburg effect in ovarian cancer.

Tetraconazole alters the methionine and ergosterol biosynthesis pathways in Saccharomyces yeasts promoting changes on volatile derived compounds.

The influence of antifungal tetraconazole residues (either as an active substance or as a commercial formulation product) on the fermentative activity of Saccharomyces cerevisiae yeast was evaluated in pasteurized Garnacha red must by using laboratory-scale fermentation assays. The presence of this fungicide promoted a slight decrease in glucose consumption. Volatile fermentative-derived compounds were evaluated in deep. Statistically significant changes were found in methionol (with a mean decrease of around 24%), fatty acids (with increments ranged from 23% to 66%), and ethyl esters (with increases ranged from 23% to 145%) contents when grape musts were enriched with the commercial formulation at both contamination levels assayed. Based on protein mass fingerprinting analysis, it was possible to relate these variations on volatiles content with changes in the activity of several enzymes (Met3p, Met14p, Adh2p, Hmg1p, Erg5p, Erg6p, Erg11p, and Erg20p) involved in the secondary metabolism of yeasts.

Ginsenoside Rg1 ameliorates palmitic acid-induced insulin resistance in HepG2 cells in association with modulating Akt and JNK activity.

Hepatic insulin resistance can be induced by excess dietary intake of saturated fat. Ginsenoside Rg1 (GRg1), the major active ginsenoside enriched in tonic food ginseng, was reported to help alleviate liver diseases. In the present study, GRg1 was evaluated for its impact on palmitic acid (PA)-induced hepatic insulin resistance model in vitro. Insulin resistance in HepG2 cells was induced by 0.5 mM PA exposure for 24 h and then the effect of GRg1 on cellular glucose consumption was measured. Expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphate (G6Pase) were analyzed by Western blot and quantitative real-time polymerase chain reaction. Activation of protein kinases and transcript factor was analyzed by measuring protein phosphorylation. The influence of GRg1 on reactive oxygen species (ROS) production in HepG2 was also examined. GRg1 reversed PA-induced decrease in glucose consumption of HepG2 cells by downregulating gluconeogenesis genes G6pase and PEPCK. GRg1 increased Akt activation but inhibited JNK activation in PA-challenged HepG2 cells. Cellular ROS level was elevated in insulin-resistant HepG2 cells but was reduced by GRg1. Together these findings indicate that GRg1 protects against hepatic insulin resistance via preserving insulin signaling sensitivity and is a promising alternative medicine.

Changes of Metabolic Phenotype of Cardiac Progenitor Cells During Differentiation: Neutral Effect of Stimulation of AMP-Activated Protein Kinase.

Cardiac progenitor cells (CPCs) in the adult mammalian heart, as well as exogenous CPCs injected at the border zone of infarcted tissue, display very low differentiation rate into cardiac myocytes and marginal repair capacity in the injured heart. Emerging evidence supports an obligatory metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) during stem cells differentiation, suggesting that pharmacological modulation of metabolism may improve CPC differentiation and, potentially, healing properties. In this study, we investigated the metabolic transition underlying CPC differentiation toward cardiac myocytes. In addition, we tested whether activators of adenosine monophosphate-activated protein kinase (AMPK), known to promote mitochondrial biogenesis in other cell types would also improve CPC differentiation. Stem cell antigen 1 (Sca1+) CPCs were isolated from adult mouse hearts and their phenotype compared with more mature neonatal rat cardiac myocytes (NRCMs). Under normoxia, glucose consumption and lactate release were significantly higher in CPCs than in NRCMs. Both parameters were increased in hypoxia together with increased abundance of Glut1 (glucose transporter), of the monocarboxylic transporter Mct4 (lactate efflux mediator) and of Pfkfb3 (key regulator of glycolytic rate). CPC proliferation was critically dependent on glucose and glutamine availability in the media. Oxygen consumption analysis indicates that, compared with NRCMs, CPCs exhibited lower basal and maximal respirations with lower Tomm20 protein expression and mitochondrial DNA content. This CPC metabolic phenotype profoundly changed upon in vitro differentiation, with a decrease of glucose consumption and lactate release together with increased abundance of Tnnt2, Pgc-1α, Tomm20, and mitochondrial DNA content. Proliferative CPCs express both alpha1 and -2 catalytic subunits of AMPK that is activated by A769662. However, A769662 or resveratrol (an activator of Pgc-1α and AMPK) did not promote either mitochondrial biogenesis or CPC maturation during their differentiation. We conclude that although CPC differentiation is accompanied with an increase of mitochondrial oxidative metabolism, this is not potentiated by activation of AMPK in these cells.

Panax ginseng Total Protein Facilitates Recovery from Dexamethasone-Induced Muscle Atrophy through the Activation of Glucose Consumption in C2C12 Myotubes.

Background The clinical anti-inflammatory drug dexamethasone (DEX) can cause many side effects such as muscle atrophy for long-term use. Muscle atrophy induced by DEX may be caused by decrease of glucose consumption. Panax ginseng C.A. Meyer was previously considered to be an antiatrophic agent for glucocorticoid- (GC-) treated therapies. As one of the main components, it remains unclear whether ginseng total protein (GP) facilitates recovery from muscle atrophy induced by DEX. Methods In this study, GP was extracted and purified with Sephadex-G50. C2C12 myoblasts was induced with 2% horse serum to differentiate into C2C12 myotubes. Cell viability was analyzed by the MTT assay, and Ca2+ concentration was analyzed by a flow cytometer. The release of lactic dehydrogenase (LDH) and the glucose consumption were analyzed by spectrophotometry. The phosphorylation of AMP-activated protein kinase (AMPK), phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt) and the expression of glucose transporter 4 (GLUT4) were analyzed by Western blotting. The phosphorylation of AS160 was quantified by Immunofluorescence staining. Results We found that GP increased cell viability and increased myotube diameter in high-dose DEX-treated C2C12 myotubes for 24 h, but this activity was not found in the enzymatic hydrolyzed GP group. GP reduced muscle atrophy by decreasing the expression of key proteins such as muscle RING-finger protein-1 and muscle atrophy F-box, reducing the Ca2+ concentration, and decreasing the release of LDH in DEX-injured C2C12 myotubes. Moreover, GP improved glucose consumption and increased the phosphorylation of AMPK, PI3K, Akt, and AS160 and the expression of GLUT4 in DEX-treated C2C12 myotubes. Conclusion The results of this study suggest that GP has effects on recovering DEX-induced muscle atrophy and cell injury, which may improve glucose consumption via the AMPK and PI3K/Akt pathways in high-dose DEX-treated C2C12 myotubes. This study provides in vitro mechanistic insights into the recovery of muscle atrophy with GP treatment.

MiR-603 targeted hexokinase-2 to inhibit the malignancy of ovarian cancer cells

The Warburg effect, characterized by energy production through a high rate of aerobic glycolysis, is a metabolic hallmark of cancer cells. We previously found that ginsenoside 20(S)-Rg3 upregulated miR-603 and impaired the malignancy of ovarian cancer cells by inhibiting the Warburg effect. However, the precise functional role of miR-603 in ovarian cancer progression remains poorly defined. Here, we report that the level of miR-603 in ovarian cancer tissues is significantly lower than that in para-tumor tissues. Overexpression of miR-603 in ovarian cancer cells inhibits the Warburg effect as evidenced by a decrease in glucose consumption, lactate production and hexokinase-2 (HK2) expression, reduces cell proliferation in vitro, and weakens their migration and invasion. Further, miR-603 directly targets HK2 as indicated in a luciferase reporter assay. In contrast to agomiR-NC, agomiR-603 treatment significantly inhibits tumor growth in vivo and the Warburg effect, which is illustrated by a decreased uptake of 18F-FDG in subcutaneous xenografts and HK2 downregulation. Finally, miR-603 is negatively regulated by DNMT3A-mediated DNA methylation in the promoter region of its precursor gene, suggesting that 20(S)-Rg3 antagonizes DNMT3A-mediated DNA methylation to impair growth, migration and invasion of ovarian cancer cells. In conclusion, miR-603 is a tumor suppressor targeting HK2 in ovarian cancer and its low level may result from DNMT3A-mediated methylation.

By blocking hexokinase-2 phosphorylation, limonin suppresses tumor glycolysis and induces cell apoptosis in hepatocellular carcinoma.

IntroductionThe purpose of present study was to investigate the effect of limonin on tumor glycolysis and the underlying mechanisms in hepatocellular carcinoma (HCC).MethodsCell proliferation and colony formation assays were performed to evaluate the potency of limonin against HCC cells in vitro. The glucose consumption and lactate production after limonin treatment was determined. The effect of limonin on hexokinase-2 (HK-2) activity was assessed and the mitochondrial location of HK-2 was studied by immunoprecipitation. Cell apoptosis and protein expression were detected by flow cytometry and western blotting respectively. Protein overexpression by plasmid transfection was adopted to investigate the molecular mechanisms.ResultsHCC proliferation and colony formation were inhibited by limonin in vitro. With the suppression of HK-2 activity, the glycolytic level in HCC cells was substantially reduced, which was evidenced by the decrease of glucose consumption and lactate production. The phosphorylation of HK-2 was substantially inhibited by limonin, which resulted in the disassociation of HK-2 from mitochondria. Due to the reduction of HK-2 in mitochondria, increasing Bax were shifted to the mitochondria and gave rise to the release of cytochrome C, which induced HCC cells to subject to mitochondria-mediated apoptosis. Mechanism investigations revealed that the decrease of HK-2 phosphorylation was mainly due to the inhibition of Akt activity. In Akt exogenously overexpressed HCC cells, limonin-mediated cell proliferation inhibition, glycolysis suppression and apoptosis induction were significantly impaired.ConclusionLimonin inhibited the tumor glycolysis in hepatocellular carcinoma by suppressing HK-2 activity, and the suppression of HK-2 was closely related to the decrease of Akt activity.

Ghrelin acts as energy status sensor of male reproduction by modulating Sertoli cells glycolytic metabolism and mitochondrial bioenergetics

Ghrelin is a growth hormone-releasing peptide that has been suggested to interfere with spermatogenesis, though the underling mechanisms remain unknown. We studied the effect of ghrelin in human Sertoli cells (hSCs) metabolic phenotype. For that, hSCs were exposed to increasing concentrations of ghrelin (20, 100 and 500 pM) mimicking the levels reported in obese, normal weight, and severely undernourished individuals. The metabolite production/consumption was determined. The protein levels of key glycolysis-related transporters and enzymes were assessed. The lactate dehydrogenase (LDH) activity was measured. Mitochondrial complexes protein levels and mitochondria membrane potential were also measured. We showed that hSCs express the growth hormone secretagogue receptor. At the concentration present in the plasma of normal weight men, ghrelin caused a decrease of glucose consumption and mitochondrial membrane potential in hSCs, though LDH activity and lactate production remained unchanged, illustrating an alteration of glycolytic flux efficiency. Exposure of hSCs to levels of ghrelin found in the plasma of severely undernourished individuals decreased pyruvate consumption and mitochondrial complex III protein expression. All concentrations of ghrelin decreased alanine and acetate production by hSCs. Notably, the effects of ghrelin levels found in severely undernourished individuals were more pronounced in hSCs metabolic phenotype highlighting the importance of a proper eating behavior to maintain male reproductive potential. In conclusion, ghrelin acts as an energy status sensor for hSCs in a dose-dependent manner, showing an inverse association with the production of lactate, thus controlling the nutritional support of spermatogenesis.