ATP Levels In Cells Research Articles

P15 Effects of coptisine on glucose metabolism and the mechanisms

Aim: Coptis chinensis (Chinese goldthread) has been widely used in Traditional Chinese Medicine for centuries and has a variety of pharmacological effects. In this study, we investigated the effect of coptisine on glucose related metabolic regulation in vitro and in vivo . Methods: Normal ICR mice, alloxan-induced type 1 diabetic mice and type 2 diabetic mice (KKay mice) were employed to explore the effect of coptisine on glucose metabolism in vivo . Human hepatoma cells (HepG2) and rat skeletal muscle myotube cells (L6) were used to investigate the effect of coptisine on the glucose metabolism in vitro . Results: In normal ICR mice, coptisine (10 mg/kg) decreased the fasting and fed blood glucose significantly without any significant effect on body weight. In alloxan-induced type 1 diabetic mice, coptisine decreased fasting and fed blood glucose. In type 2 diabetic Kkay mice, coptisine improved insulin tolerance and glucose tolerance but did not change LDL, TG, HDL or CHO level in serum. Our in vitro results showed that coptisine increased the glucose consumption and glucose uptake in HepG2 cells and L6 myotube cells. Coptisine significantly increased ATP level in HepG2 cells and decreased mitochondrial membrane potential in HepG2 cells. Furthermore, coptisine promoted hepatic mitochondrial F0F1-ATPase activity. Conclusion: In summary, coptisine has been shown to be effective in improving glucose metabolism in animal models. The effects of coptisine may be closely related with enhanced function of the mitochondria.

PKA activity exacerbates hypoxia-induced ROS formation and hypoxic injury in PC-12 cells

Hypoxia is a primary factor in many pathological conditions. Hypoxic cell death is commonly attributed to metabolic failure and oxidative injury. cAMP-dependent protein kinase A (PKA) is activated in hypoxia and regulates multiple enzymes of the mitochondrial electron transport chain, thus may be implicated in cellular energy depletion and hypoxia-induced cell death.Wild type (WT) PC-12 cells and PKA activity-deficient 123.7 PC-12 cells were exposed to 3, 6, 12 and 24h hypoxia (0.1% or 5% O2). Hypoxia, at 24h 0.1% O2, induced cell death and increased reactive oxygen species (ROS) in WT PC-12 cells. Despite lower ATP levels in normoxic 123.7 cells than in WT cells, hypoxia only decreased ATP levels in WT cells. However, menadione-induced oxidative stress similarly affected both cell types. While mitochondrial COX IV expression remained consistently higher in 123.7 cells, hypoxia decreased COX IV expression in both cell types.N-acetyl cysteine antioxidant treatment blocked hypoxia-induced WT cell death without preventing ATP depletion. Transient PKA catα expression in 123.7 cells partially restored hypoxia-induced ROS but did not alter ATP levels or COX IV expression.We conclude that PKA signaling contributes to hypoxic injury, by regulating oxidative stress rather than by depleting ATP levels. Therapeutic strategies targeting PKA signaling may improve cellular adaptation and recovery in hypoxic pathologies.

Sulforaphane Promotes Mitochondrial Protection in SH-SY5Y Cells Exposed to Hydrogen Peroxide by an Nrf2-Dependent Mechanism.

Sulforaphane (SFN; C6H11NOS2) is an isothiocyanate found in cruciferous vegetables, such as broccoli, kale, and radish. SFN exhibits antioxidant, anti-apoptotic, anti-tumor, and anti-inflammatory activities in different cell types. However, it was not previously demonstrated whether and how this natural compound would exert mitochondrial protection experimentally. Therefore, we investigated here the effects of a pretreatment (for 30min) with SFN at 5μM on mitochondria obtained from human neuroblastoma SH-SY5Y cells exposed to hydrogen peroxide (H2O2) at 300μM for 24h. We found that SFN prevented loss of viability in H2O2-treated SH-SY5Y cells. Furthermore, SFN decreased lipid peroxidation, protein carbonylation, and protein nitration in mitochondrial membranes of H2O2-exposed cells. Importantly, SFN enhanced the levels of both cellular and mitochondrial glutathione (GSH). SFN also suppressed the H2O2-mediated inhibition of mitochondrial components involved in the maintenance of the bioenergetics state, such as aconitase, α-ketoglutarate dehydrogenase, and succinate dehydrogenase, as well as complexes I and V. Consequently, SFN prevented the decline induced by H2O2 on the levels of ATP in SH-SY5Y cells. Silencing of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor by using small interfering RNA (siRNA) abolished the mitochondrial and cellular protection elicited by SFN. Therefore, SFN abrogated the H2O2-induced mitochondrial impairment by an Nrf2-dependent manner.

Abstract 448: Role of fructose in prostate cancer

Abstract The elevated level of glucose uptake and metabolism in cancers is the basis for the clinical localization of primary cancers and sites of metastasis by positron emission tomography (PET scanning), based on the enhanced cellular uptake of 2-deoxy-2-[18F]-fluoro-D-glucose (FDG). In prostate cancer (CaP), however, FDG-PET imaging has shown limited clinical applicability. This striking difference suggests that CaP cells utilize hexoses other than glucose, such as fructose, as the principal energy source. The purpose of this study was to determine whether or not fructose is a/the principal source of energy for CaP cells. mRNA and protein expression for the glucose transporter Glut-1 and fructose transporters Glut-2, Glut-5, Glut-7, Glut-9 and Glut-11 was analyzed in benign (PWR-1E, RWPE-1) and malignant (LNCaP, vCaP, LNCaP-C4-2, DU-145, and PC-3) human prostate cell lines using qRT-PCR and western blot, respectively. In addition, Glut(s) protein expression was analyzed on a tissue microarray containing 200 formalin-fixed paraffin-embedded benign and malignant human prostate tissues using immunohistochemistry. Fructose and glucose uptake was measured in vitro in benign and malignant human prostate cell lines using radiolabelled D-[U-14C]-fructose or 2-[1,2-3H]-deoxy-D-[3H]-glucose, respectively. Lastly, the effect of fructose or glucose on the levels of ATP, mitochondrial metabolism, and expression of the enzymes hexokinase-2 (HK2), type-C fructokinase (KHK-C), pyruvate kinase M2 (PKM2) and type-A lactate dehydrogenase (LDH-A) was analyzed in benign and malignant human prostate cell lines using chemiluminescence, seahorse, and qRT-PCR analyses, respectively. Our results indicated that expression of the fructose transporters, Glut-5 and Glut-9, was increased in CaP cell lines and in human CaP tissues compared to benign cell lines and benign prostate tissues, respectively. Glut-1 expression, however, did not differ between benign and malignant human prostate cells. Transport assays demonstrated that CaP cell lines have a higher capacity to transport fructose compared to benign cell lines. However, glucose uptake was not altered between benign and malignant human prostate cell lines. ATP levels in CaP cells were similar in the presence of fructose or glucose. Fructose, but not glucose, significantly altered mRNA expression of HK2, KHK-C, PKM2, and LDH-A in malignant human prostate cells. Taken together, our results suggest that fructose may represent an alternative energy source and may reprogram hexose metabolism in CaP cells. Citation Format: Daniela Carreño, Nestor Corro, Marcia Arredondo, Carmen Navarro, Verónica Torres, Viviana Montecinos, Paula Sotomayor, Francisco Nualart, Julio Cesar Cárdenas, Alejandro S. Godoy. Role of fructose in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 448. doi:10.1158/1538-7445.AM2017-448

Individual Amino Acid Supplementation Can Improve Energy Metabolism and Decrease ROS Production in Neuronal Cells Overexpressing Alpha-Synuclein.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein accumulation and loss of dopaminergic neurons in the substantia nigra (SN) region of the brain. Increased levels of alpha-synuclein have been shown to result in loss of mitochondrial electron transport chain complex I activity leading to increased reactive oxygen species (ROS) production. WT alpha-synuclein was stably overexpressed in human BE(2)-M17 neuroblastoma cells resulting in increased levels of an alpha-synuclein multimer, but no increase in alpha-synuclein monomer levels. Oxygen consumption was decreased by alpha-synuclein overexpression, but ATP levels did not decrease and ROS levels did not increase. Treatment with ferrous sulfate, a ROS generator, resulted in decreased oxygen consumption in both control and alpha-synuclein overexpressing cells. However, this treatment only decreased ATP levels and increased ROS production in the cells overexpressing alpha-synuclein. Similarly, paraquat, another ROS generator, decreased ATP levels in the alpha-synuclein overexpressing cells, but not in the control cells, further demonstrating how alpha-synuclein sensitized the cells to oxidative insult. Proteomic analysis yielded molecular insights into the cellular adaptations to alpha-synuclein overexpression, such as the increased abundance of many mitochondrial proteins. Many amino acids and citric acid cycle intermediates and their ester forms were individually supplemented to the cells with L-serine, L-proline, L-aspartate, or L-glutamine decreasing ROS production in oxidatively stressed alpha-synuclein overexpressing cells, while diethyl oxaloacetate or L-valine supplementation increased ATP levels. These results suggest that dietary supplementation with individual metabolites could yield bioenergetic improvements in PD patients to delay loss of dopaminergic neurons.

Protective role of α-lipoic acid in hyperuricemia-induced endothelial dysfunction.

The aim of the current study was to determine the effects of α-lipoic acid (LA) on hyperuricemia and endothelial dysfunction, and to uncover the underlying mechanism of its action. A hyperuricemic rat model was established by administration of uric acid (UA) and the rats were orally fed with 2 g/kg/day LA or phosphate-buffered saline. Primary rat aortic endothelial cells were subsequently isolated, and a cell viability assay, apoptosis assay, enzyme nitric oxide synthase (eNOS) activity assay and mitochondrial function assay were all performed. For the in vitro study, human umbilical vein endothelial cells were used and western blotting was performed to assess Akt signaling activity. The results of the current study indicated that LA inhibited apoptosis, enhanced eNOS activity and production of nitric oxide (NO), and rescued mitochondrial mass and function in uric acid (UA)-treated endothelial cells. LA activated Akt signaling and inhibition of Akt signaling abolished the effects of LA on cell viability, NO production, ROS production and ATP levels in UA-treated endothelial cells. Therefore, the current study demonstrated that LA attenuated oxidant stress and inhibited apoptosis in UA-treated endothelial cells by activating Akt signaling. The results indicate that LA may serve as a therapeutic approach to treat hyperuricemia-induced endothelial dysfunction.

The effect of G3 PAMAM dendrimer conjugated with B-group vitamins on cell morphology, motility and ATP level in normal and cancer cells

In a search for the safe vitamin carrier the PAMAM G3 dendrimer covalently substituted with 9 and 10 molecules of vitamin B7 (biotin) and B6 (pyridoxal), respectively (BC-PAMAM) was investigated. Dendrimer substitution with B-group vitamins significantly alters its biological properties as compared to native form. Observed effects on investigated cell parameters including morphology, adhesion, migration and ATP level were different for normal human fibroblasts (BJ) and squamous cell carcinoma (SCC-15) cell lines. BC-PAMAM revealed significantly less pronounced effects on investigated parameters, particularly at higher concentrations (5–50μM), which is relevant with its lower positive surface charge, as compared with native form. The bioconjugate, up to 50μM concentration, appeared to be a safe vitamin carrier to normal fibroblasts, without significant effect on their adhesion, shape and migration as well as on intracellular ATP level. In SCC-15 cells BC-PAMAM, at low concentrations (0.1–0.5μM), altered the cell shape and increase adhesion, whereas at higher concentrations opposite effects were seen. Measurements of cellular level of ATP showed that higher resistance of cancer cells to toxic effects of native PAMAM dendrimers may be due to higher energy supply of cancer cells.

Alterations of hepatocyte function with free radical generators and reparation or prevention with coffee polyphenols

Liver diseases are linked in the majority of cases to oxidative stress that antioxidants could neutralize with reducing liver injury. Chlorogenic acid, a coffee polyphenol, possesses antioxidant prosperities. The aim of this study was to evaluate in vitro preventive and corrective effects of cholorogenic acid in hepatocyte toxicity induced by free radicals. Hepatocytes were isolated from adult male Wistar rats. To determine corrective effects and reparation, cells were first exposed to two free radical generators (hydrogen peroxide/iron sulfate for hydroxyl radical formation, and phenazine methosulfate/nicotinamide adenine dinucleotide for superoxide anion formation) for 12H and thereafter treated by chlorogenic acid (1 and 10 μM final concentration) for another 12H. To show preventive effects, cells were pretreated by chlorogenic acid and thereafter exposed to free radical generators. Hepatocyte proliferation, glucose uptake, ATP contents, membrane fluidity and integrity, and intracellular redox status were investigated after 24H culture. The results showed that chlorogenic acid reversed the decrease in cell proliferation, glucose uptake and ATP levels, the increased LDH release and the reduced membrane fluidity and restored the oxidant/antioxidant status under oxidative stress. When pre-treated with chlorogenic acid, hepatocytes became very resistant to oxidative conditions and cellular homeostasis was maintained. In conclusion, chlorogenic acid displayed not only corrective but also preventive effects in hepatocytes exposed to oxidative stress and could be beneficial in patients with or at risk of liver diseases.

Abnormal epigenetic changes during differentiation of human skeletal muscle stem cells from obese subjects

BackgroundHuman skeletal muscle stem cells are important for muscle regeneration. However, the combined genome-wide DNA methylation and expression changes taking place during adult myogenesis have not been described in detail and novel myogenic factors may be discovered. Additionally, obesity is associated with low relative muscle mass and diminished metabolism. Epigenetic alterations taking place during myogenesis might contribute to these defects.MethodsWe used Infinium HumanMethylation450 BeadChip Kit (Illumina) and HumanHT-12 Expression BeadChip (Illumina) to analyze genome-wide DNA methylation and transcription before versus after differentiation of primary human myoblasts from 14 non-obese and 14 obese individuals. Functional follow-up experiments were performed using siRNA mediated gene silencing in primary human myoblasts and a transgenic mouse model.ResultsWe observed genome-wide changes in DNA methylation and expression patterns during differentiation of primary human muscle stem cells (myoblasts). We identified epigenetic and transcriptional changes of myogenic transcription factors (MYOD1, MYOG, MYF5, MYF6, PAX7, MEF2A, MEF2C, and MEF2D), cell cycle regulators, metabolic enzymes and genes previously not linked to myogenesis, including IL32, metallothioneins, and pregnancy-specific beta-1-glycoproteins. Functional studies demonstrated IL-32 as a novel target that regulates human myogenesis, insulin sensitivity and ATP levels in muscle cells. Furthermore, IL32 transgenic mice had reduced insulin response and muscle weight. Remarkably, approximately 3.7 times more methylation changes (147,161 versus 39,572) were observed during differentiation of myoblasts from obese versus non-obese subjects. In accordance, DNMT1 expression increased during myogenesis only in obese subjects. Interestingly, numerous genes implicated in metabolic diseases and epigenetic regulation showed differential methylation and expression during differentiation only in obese subjects.ConclusionsOur study identifies IL-32 as a novel myogenic regulator, provides a comprehensive map of the dynamic epigenome during differentiation of human muscle stem cells and reveals abnormal epigenetic changes in obesity.

Oridonin induces autophagy via inhibition of glucose metabolism in p53-mutated colorectal cancer cells

The Warburg effect is an important characteristic of tumor cells, making it an attractive therapeutic target. Current anticancer drug development strategies predominantly focus on inhibitors of the specific molecular effectors involved in tumor cell proliferation. These drugs or natural compounds, many of which target the Warburg effect and the underlying mechanisms, still need to be characterized. To elucidate the anticancer effects of a natural diterpenoid, oridonin, we first demonstrated the anticancer activity of oridonin both in vitro and in vivo in colorectal cancer (CRC) cells. Then miRNA profiling of SW480 cells revealed those intracellular signaling related to energy supply was affected by oridonin, suggesting that glucose metabolism is a potential target for CRC therapy. Moreover, our results indicated that oridonin induced metabolic imbalances by significantly inhibiting glucose uptake and reducing lactate export through significantly downregulating the protein levels of GLUT1 and MCT1 in vitro and vivo. However, the ATP level in oridonin-treated CRC cells was not decreased when oridonin blocked the glucose supply, indicating that oridonin induced autophagy process, an important ATP source in cancer cells. The observation was then supported by the results of LC3-II detection and transmission electron microscopy analysis, which confirmed the presence of autophagy. Furthermore, p-AMPK was rapidly deactivated following oridonin treatment, resulting in downregulation of GLUT1 and induction of autophagy in the cancer cells. Thus our finding helped to clarify the anticancer mechanisms of oridonin and suggested it could be applied as a glucose metabolism-targeting agent for cancer treatment.

Salvianolate protects H9c2 cells from hypoxia/reoxygenation injury-induced apoptosis by attenuating mitochondrial DNA oxidative damage

Objective: To investigate the possible mechanism related to the protective effects of salvianolate in H9c2 cells underwent hypoxia/reoxygenation (H/R)-injury. Methods: H9c2 cells were divided into four groups: control group, salvianolate group (S group), H/R group, and salvianolate+ H/R group(S+ H/R group), in which the H9c2 cells were pretreated with salvianolate before H/R-treatment.Apoptotic cells were detected by Tunel assays and AnnexinⅤ-FITC apoptosis detection kit.The intracellular ATP level, the change of mitochondrial membrane potential and the mitochondrial DNA oxidative damage were also determined in these groups. Results: (1) The apoptosis rate of H/R group(26.36±5.14)% was significantly higher compared to control group(2.71±1.66)%(P=0.000 4), which could be significantly reduced in S+ H/R group(17.28±4.75)%(P=0.012 8 vs. H/R group , P=0.003 9 vs. control group). The ratio of AnnexinⅤ and PI double positive cells in H/R group(28.23±6.73)% was significantly higher compared to control group(3.53±2.83)%(P=0.001 1), which was significantly reduced in S+ H/R group(18.10±4.56)%(P=0.037 2 vs. H/R group, P=0.038 3 vs. control group). (2)The ATP level of H9c2 cells in H/R group(49.05±10.12)% was significantly lower than in control group 100%(P=0.000 5), which was significantly increased in S+ H/R group(68.67±13.32)%(P=0.019 9 vs. H/R group). Confocal microscope showed that red fluorescence was dominant in the control group, red fluorescence was significantly reduced, while green fluorescence was significantly increased in H9c2 cells of H/R group and the fluorescence ratio of red to green in H/R group((37.13±8.47)%) was significantly decreased compared to control group (100%, P=0.000 1), fluorescence ratio of red to green was significantly increased in S+ H/R group((63.77±12.32)% vs. H/R group, P=0.007 3). (3)The mitochondrial DNA oxidative damage in different groups: there was only few 8-hydroxyguanine (8-OHdG) expression, which marked as green, in control group, and 8-OHdG expression was significantly upregulated in H/R group, moreover, the 8-OHdG was co-localized with mitochondria.The expression of 8-OHdG was significantly lower in S+ H/R group compared to H/R group. Conclusion: Salvianolate can reduce mitochondrial DNA oxidative damage, and protect mitochondrial function, thus inhibit myocardial cell apoptosis and eventually reduce the myocardial H/R-injury in H9c2 cells.

Induction of Apoptosis and Autophagy By Nampt Inhibition in Adult T-Cell Leukemia/Lymphoma and Leukemic Cell Lines

Introduction: Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with human T-cell leukemia virus type I (HTLV-1). Despite the recent advances in chemotherapy, allogeneic hematopoietic stem cell transplantation, and supportive care, the prognosis for patients with acute, lymphoma, or unfavorable chronic subtypes is one of the poorest among hematological malignancies. The identification of new molecular targets for ATL prevention and treatment is desired. SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone/protein deacetylase, plays crucial roles in various physiological processes, including aging and apoptosis. We previously reported that ATL patients had significantly higher SIRT1 protein levels and novel small-molecule SIRT1 inhibitors are highly effective against ATL cells.1,2 Nicotinamide phosphoribosyltransferase (Nampt) also known as pre-B-cell colony-enhancing factor 1 or visfatin is a rate-limiting enzyme in NAD+ biosynthesis, and it regulates intracellular ATP levels in mammalian cells. Most cancer cells (acute myeloid leukemia [AML], acute lymphoblastic leukemia [ALL], mantle cell lymphoma [MCL], chronic lymphocytic leukemia [CLL], and T-cell lymphoma) is sensitive to low concentrations of FK866, Nampt inhibitor, as measured in cytotoxicity and clonogenic assays.3Here, we assessed how Nampt is regulated in ATL cells and leukemic cell lines.Results: We observed that ATL patients had significantly higher SIRT1 and Nampt protein levels than healthy controls. FK866 induced significant growth inhibition and apoptosis (Annexin V+ cells and TUNEL) in leukemia/lymphoma cell lines (HTLV-1-related cell lines: S1T, MT-2; Jurkat and HL60). FK866 showed potent activities with GI50values of 0.63, 3.7, 1.0, and 3,4 nM for S1T, MT-2, Jurkat, and HL60 cells, respectively. FK866 also activated caspase activity (caspase-3, 8, and 9) with DNA fragmentation. However, a caspase inhibitor did not inhibit this caspase-dependent cell death. Interestingly, FK866 increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation as well as autophagy. Autophagy detection was also performed using the CytoID Autophagy detection kit. Autophagy levels are increased in the presence of STF-62247 pre-treated with bafilomycin A1, a specific inhibitor of vacuolar proton ATPase, whose inhibition is known to block the fusion of autophagosomes with lysosomes for 2 h. Thus, FK866 simultaneously caused apoptosis and autophagy.Conclusion:These results suggest that Nampt inhibitor is highly effective against ATL cells in caspase-dependent or -independent manners with autophagy, and that its clinical application might improve the prognosis of patients with this fatal disease.1. Kozako T, Aikawa A, Shoji T, et al. High expression of the longevity gene product SIRT1 and apoptosis induction by sirtinol in adult T-cell leukemia cells. Int J Cancer. 2012;131:2044-2055.2. Kozako T, Suzuki T, Yoshimitsu M, et al. Novel small-molecule SIRT1 inhibitors induce cell death in adult T-cell leukaemia cells. Sci Rep. 2015;5:11345.3. Nahimana A, Attinger A, Aubry D, et al. The NAD biosynthesis inhibitor APO866 has potent antitumor activity against hematologic malignancies. Blood. 2009;113:3276-3286. DisclosuresYoshimitsu:HUYA Bioscience International: Research Funding.

BTeam, a Novel BRET-based Biosensor for the Accurate Quantification of ATP Concentration within Living Cells.

ATP levels may represent fundamental health conditions of cells. However, precise measurement of intracellular ATP levels in living cells is hindered by the lack of suitable methodologies. Here, we developed a novel ATP biosensor termed “BTeam”. BTeam comprises a yellow fluorescent protein (YFP), the ATP binding domain of the ε subunit of the bacterial ATP synthase, and an ATP-nonconsuming luciferase (NLuc). To attain emission, BTeam simply required NLuc substrate. BTeam showed elevated bioluminescence resonance energy transfer efficiency upon ATP binding, resulted in the emission spectra changes correlating with ATP concentrations. By using values of YFP/NLuc emission ratio to represent ATP levels, BTeam achieved steady signal outputs even though emission intensities were altered. With this biosensor, we succeeded in the accurate quantification of intracellular ATP concentrations of a population of living cells, as demonstrated by detecting the slight distribution in the cytosol (3.7–4.1 mM) and mitochondrial matrix (2.4–2.7 mM) within some cultured cell lines. Furthermore, BTeam allowed continuous tracing of cytosolic ATP levels of the same cells, as well as bioluminescent imaging of cytosolic ATP dynamics within individual cells. This simple and accurate technique should be an effective method for quantitative measurement of intracellular ATP concentrations.

Cytotoxic Activity of Microalgal-derived Oxylipins against Human Cancer Cell lines and their Impact on ATP Levels

Oxylipins are metabolites derived from lipid peroxidation. The plant oxylipin methyl jasmonate (MJ) shows cytotoxic activity against cancer cell lines of various origins, with ATP-depletion being one of the mechanisms responsible for this effect. The cytotoxic activity of oxylipins (OXLs) isolated from the microalgae Chlamydomonas debaryana (13-HOTE) and Nannochloropsis gaditana (15-HEPE) was higher against UACC-62 (melanoma) than towards HT-29 (colon adenocarcinoma) cells. OXLs lowered the ATP levels of HT-29 and UACC-62 cells, but the effect was higher on the second cell line, which had higher basal ATP. This result proves a link between the cytotoxicity and the capability of these compounds to deplete ATP. In addition, the combination of 13-HOTE with the anticancer drug 5-fluorouracil (5-FU) induced a synergistic toxicity against HT-29 cells. These results highlight the therapeutic potential of oxylipins derived from microalgae.

StarD7 Protein Deficiency Adversely Affects the Phosphatidylcholine Composition, Respiratory Activity, and Cristae Structure of Mitochondria

Phosphatidylcholine (PC) is a major phospholipid of mitochondria, comprising 40-50% of both the outer and the inner membranes. However, PC must be imported from its production organelles because mitochondria lack the enzymes essential for PC biosynthesis. In a previous study, we found that StarD7 mediates the intracellular transfer of PC to mitochondria. Therefore, in this study, we analyzed the contribution of StarD7 to the maintenance of mitochondrial phospholipid content and function using siRNA-mediated knockdown and knock-out (KO) of the StarD7 gene in HEPA-1 cells. Real time analysis of respiratory activity demonstrated that the oxygen consumption rate and activity of mitochondrial complexes were impaired in StarD7-KD cells. To confirm these results, we established StarD7-KO HEPA-1 cells by double nicking using CRISPR/Cas9n. As expected, StarD7-KD and -KO cells showed a significant reduction in mitochondrial PC content. The ATP level and growth rate of KO cells were notably lower compared with wild-type cells when cultured in glucose-free galactose-containing medium to force cells to rely on mitochondrial ATP production. In KO cells, the level of the MTCO1 protein, a primary subunit of complex IV, was reduced without a concomitant decrease in its mRNA, but the level was restored when StarD7-I was overexpressed. StarD7-KO cells showed impaired formation of the mitochondrial supercomplexes and exhibited a disorganized cristae structure, with no changes in optic atrophy 1 protein. These findings indicate that StarD7 plays important roles in maintaining the proper composition of mitochondrial phospholipids as well as mitochondrial function and morphogenesis.

HDAC7 is overexpressed in human diabetic islets and impairs insulin secretion in rat islets and clonal beta cells

Aims/hypothesisPancreatic beta cell dysfunction is a prerequisite for the development of type 2 diabetes. Histone deacetylases (HDACs) may affect pancreatic endocrine function and glucose homeostasis through alterations in gene regulation. Our aim was to investigate the role of HDAC7 in human and rat pancreatic islets and clonal INS-1 beta cells (INS-1 832/13).MethodsTo explore the role of HDAC7 in pancreatic islets and clonal beta cells, we used RNA sequencing, mitochondrial functional analyses, microarray techniques, and HDAC inhibitors MC1568 and trichostatin A.ResultsUsing RNA sequencing, we found increased HDAC7 expression in human pancreatic islets from type 2 diabetic compared with non-diabetic donors. HDAC7 expression correlated negatively with insulin secretion in human islets. To mimic the situation in type 2 diabetic islets, we overexpressed Hdac7 in rat islets and clonal beta cells. In both, Hdac7 overexpression resulted in impaired glucose-stimulated insulin secretion. Furthermore, it reduced insulin content, mitochondrial respiration and cellular ATP levels in clonal beta cells. Overexpression of Hdac7 also led to changes in the genome-wide gene expression pattern, including increased expression of Tcf7l2 and decreased expression of gene sets regulating DNA replication and repair as well as nucleotide metabolism. In accordance, Hdac7 overexpression reduced the number of beta cells owing to enhanced apoptosis. Finally, we found that inhibiting HDAC7 activity with pharmacological inhibitors or small interfering RNA-mediated knockdown restored glucose-stimulated insulin secretion in beta cells that were overexpressing Hdac7.Conclusions/interpretationTaken together, these results indicate that increased HDAC7 levels caused beta cell dysfunction and may thereby contribute to defects seen in type 2 diabetic islets. Our study supports HDAC7 inhibitors as a therapeutic option for the treatment of type 2 diabetes.

Glucose-independent Acetate Metabolism Promotes Melanoma Cell Survival and Tumor Growth

Tumors rely on multiple nutrients to meet cellular bioenergetics and macromolecular synthesis demands of rapidly dividing cells. Although the role of glucose and glutamine in cancer metabolism is well understood, the relative contribution of acetate metabolism remains to be clarified. We show that glutamine supplementation is not sufficient to prevent loss of cell viability in a subset of glucose-deprived melanoma cells, but synergizes with acetate to support cell survival. Glucose-deprived melanoma cells depend on both oxidative phosphorylation and acetate metabolism for cell survival. Acetate supplementation significantly contributed to maintenance of ATP levels in glucose-starved cells. Unlike acetate, short chain fatty acids such as butyrate and propionate failed to prevent loss of cell viability from glucose deprivation. In vivo studies revealed that in addition to nucleo-cytoplasmic acetate assimilating enzyme ACSS2, mitochondrial ACSS1 was critical for melanoma tumor growth in mice. Our data indicate that acetate metabolism may be a potential therapeutic target for BRAF mutant melanoma.

Novel role of serine racemase in anti-apoptosis and metabolism

BackgroundSerine racemase (SR) catalyzes the production of d-serine, a co-agonist of the N-methyl-d-aspartate receptor (NMDAR). A previous report shows the contribution of SR in the NMDAR-mediated neuronal cell death process. Methods and resultsTo analyze the intrinsic role of SR in the cell death process, we established the epithelial human embryonic kidney 293T (HEK293T) cell lines expressing wild-type SR (SR-WT), catalytically inactive mutant SR (SR-K56G), and catalytically hyperactive mutant SR (SR-Q155D). To these cell lines, staurosporine (STS), which induces apoptosis, was introduced. The cells expressing SR-WT and SR-Q155D showed resistance to STS-induced apoptosis, compared with nontransfected HEK293T cells and cells expressing SR-K56G. The SR-WT cells also showed a significant higher viability than the SR-QD cells. Furthermore, we detected elevated phosphorylation levels of Bcl-2 at serine-70 and Akt at serine-473 and threonine-308, which are related to cell survival, in the cells expressing SR-WT and SR-Q155D. From the results of metabolite analysis, we found elevated levels of acetyl CoA and ATP in cells expressing SR-WT. ConclusionBecause SR has two enzymatic activities, namely, racemization and α, β-elimination, and SR-Q155D shows enhanced racemization and reduced α, β-elimination activities, we concluded that the racemization reaction catalyzed by SR may have a more protective role against apoptosis than the α, β-elimination reaction. Moreover, both of these activities are important for maximal survival and elevated levels of acetyl CoA and ATP. General significanceOur findings reveal the NMDAR-independent roles of SR in metabolism and cell survival.

ABCB1-overexpressing MDCK-II cells are hypersensitive to 3-bromopyruvic acid

AimsCancer cells, due to the Warburg effect, are more dependent on glycolysis than normal cells, so glycolytic inhibitor 3-bromopyruvic acid (3-BP) was proposed as a promising candidate for anticancer therapy. Overexpression of multidrug transporters is the main reason of resistance of cancer cells to chemotherapy. As the activity of multidrug transporters imposes an energetic burden on the cells, it can be expected that inhibition of ATP generation may exert a selective cytotoxicity to cells overexpressing multidrug transporters. The aim of this study was to compare the effect of 3-BP on the survival and ATP level in MDCK-II cells and MDCK-II cells overexpressing ABCB1 (Pgp) or ABCG2 (BCRP). Main methodsCell survival was measured with resazurin and with neutral red. ATP level was assayed with luciferin/luciferase kit. Luteolin transport was measured by an original method described in the paper. Key findings3-BP (10–200μM) induced a decrease of ATP level after 1-h incubation in all cell lines studied, more drastically in ABCB1-overexpressing cells. 50 and 200μM 3-BP significantly decreased cell viability; the effect was more pronounced for ABCB1-overexpressing cells. PSC833, inhibitor of ABCB1, ameliorated the toxic effect of 3-BP on MDCK-II ABCB1 cells and MDCK-II cells. 3-BP inhibited luteolin transport in MDCK-II ABCG2 cells. SignificanceThese results indicate that 3-BP shows selective toxicity against ABCB1- but not ABCG2-overexpressing cells, apparently due to enhanced ATP depletion but in a manner independent of the transport activity of Pgp, suggesting a novel mechanism of hypersensitivity of ABCB1-overexpressing cells to 3-BP.

GLUT1-mediated glucose uptake plays a crucial role duringPlasmodiumhepatic infection

SummaryIntracellular pathogens have evolved mechanisms to ensure their survival and development inside their host cells. Here, we show that glucose is a pivotal modulator of hepatic infection by the rodent malaria parasite Plasmodium berghei and that glucose uptake via the GLUT1 transporter is specifically enhanced in P. berghei‐infected cells. We further show that ATP levels of cells containing developing parasites are decreased, which is known to enhance membrane GLUT1 activity. In addition, GLUT1 molecules are translocated to the membrane of the hepatic cell, increasing glucose uptake at later stages of infection. Chemical inhibition of GLUT1 activity leads to a decrease in glucose uptake and the consequent impairment of hepatic infection, both in vitro and in vivo. Our results reveal that changes in GLUT1 conformation and cellular localization seem to be part of an adaptive host response to maintain adequate cellular nutrition and energy levels, ensuring host cell survival and supporting P. berghei hepatic development.