Expression Of Citrate Synthase Research Articles

Aberrant Expression of Citrate Synthase is Linked to Disease Progression and Clinical Outcome in Prostate Cancer.

PurposeCitrate synthase (CS) is a rate-limiting enzyme in the citrate cycle and is capable of catalyzing oxaloacetate and acetyl-CoA to citrate. CS has been uncovered to be upregulated in a variety of cancers, and its expression and clinical significance in prostate cancer (PCa) remain unknown.MethodsIn this study, we examined the association between CS expression level and clinicopathological features of prostate cancer patients in a TMA cohort and the public cancer database (The Cancer Genome Atlas-Prostate Adenocarcinoma, TCGA-PRAD). The CS knockdown cell lines were constructed to study the effects of CS downregulation on proliferation, colony formation, migration, invasion, and cell cycle of prostate cancer cells in vitro. And the effect of CS downregulation on tumor growth in mice was studied in vivo. In addition, the metabolomics and mitochondrial function were detected in the CS knockdown cell lines.ResultsCS expression level in PCa tissues was higher than that in normal tissues (P < 0.05). CS upregulation was significantly associated with high Gleason score (P < 0.05), advanced pathological stage (P < 0.001), and biochemical recurrence (P < 0.001). Functionally, decreased expression of CS inhibited PCa cell proliferation, colony formation, migration, invasion and cell cycle in vitro, and inhibited tumor growth in vivo. In addition, CS downregulation exerted potential inhibitory effects on the lipid metabolism and mitochondrial function of PCa cells.ConclusionIn conclusion, these findings suggested that CS upregulation may contribute to the aggressive progression and poor prognosis of PCa patients, which might be partially associated with its influences on the cell lipid metabolism and mitochondrial function.

Role of Glutamate Synthase in Biofilm Formation by Bacillus subtilis.

Bacillus subtilis forms robust biofilms in the presence of large amounts of carbon sources, such as glycerol. However, little is known about the importance of the metabolic systems, or the relationship between metabolic systems and regulatory systems, involved in biofilm formation. Glutamate synthase, encoded by gltAB, is an enzyme that converts 2-ketoglutarate (a tricarboxylic acid [TCA] cycle intermediate) and glutamine into glutamate, which is a general amino group donor in metabolism. Here, we show that a ΔgltA mutant exhibited early arrest of biofilm formation in complex medium containing glycerol. This phenotype was not due to glutamate auxotrophy. Consistent with its biofilm formation phenotype, the ΔgltA mutant exhibited an early decrease in expression of the epsA and tapA operons, which are responsible for production of biofilm matrix polymers. This resulted from decreased activity of their regulator, Spo0A, as evidenced by reduced expression of other Spo0A-regulated genes in the ΔgltA mutant. The ΔgltA mutation prevented biofilm formation only in the presence of large amounts of glycerol. Moreover, limited expression of citrate synthase (but not other TCA enzymes) restored biofilm-forming ability to the ΔgltA mutant. These results indicate that the ΔgltA mutant accumulates an inhibitory intermediate (citrate) in the TCA cycle in the presence of large amounts of glycerol. The ΔgltA mutant formed biofilms when excess iron was added to the medium. Taken together, the data suggest that accumulation of citrate ions by the ΔgltA mutant causes iron shortage due to chelation, which prevents activation of Spo0A and causes defective biofilm formation.IMPORTANCEBacillus subtilis, a model organism for bacterial biofilm formation, forms robust biofilms in a medium-dependent manner. Although the regulatory network that controls biofilm formation has been well studied, the importance of the underlying metabolic systems remains to be elucidated. The present study demonstrates that a metabolic disorder in a well-conserved metabolic system causes accumulation of an inhibitory metabolic intermediate that prevents activation of the system that regulates biofilm formation. These findings increase our understanding of the coordination between cellular metabolic status and the regulatory networks governing biofilm formation.

ALA protects against ERS-mediated apoptosis in a cochlear cell model with low citrate synthase expression

A/J mouse is a model of age-related hearing loss (AHL). Mutation in the citrate synthase (Cs) gene of the mouse plays an important role in the hearing loss and degeneration of cochlear cells. To investigate the pathogenesis of cochlear cell damage in A/J mice resulted from Cs mutation, we downregulated the expression level of CS in HEI-OC1, a cell line of mouse cochlea, by shRNA. The results showed that low CS expression led to low ability of cell proliferation. Further study revealed an increase level of reactive oxygen species (ROS), activation of ATF6 mediated endoplasmic reticulum stress (ERS) and high expression levels of caspase12 and Bax in the cells. Moreover, the AEBSF, an ATF6 inhibitor, could reduce the expression levels of caspase-12 and Bax by inhibiting the hydrolysis of ATF6 in the cells. Finally, antioxidant alpha-lipoic acid (ALA) reduced the ROS levels and the apoptotic signals in the cell model with low CS expression. We therefore conclude that the ERS mediated apoptosis, which is triggered by ROS, may be involved in the cell degeneration in the cochleae of A/J mice.

Influence of bariatric surgery induced weight loss on oxidative DNA damage

Obesity is associated with elevated cancer risk, which may be represented by elevated genomic damage. Oxidative stress plays a key role in obesity related detrimental health consequences including DNA oxidation damage. The elevated cancer risk in obesity may be a consequence. Weight loss has been shown to reduce genomic damage, but the role of oxidative stress in that has not been clarified.The aim of this study is therefore to investigate the influence of bariatric surgery induced weight loss on DNA oxidation damage in morbidly obese subjects. For this aim, we used cryopreserved peripheral blood mononuclear cells in the FPG comet assay. Advanced protein oxidation products and 3-nitrotyrosine were measured as oxidative and nitrative protein stress markers. Furthermore, expression of oxidative stress related proteins HSP70 and Nrf2 as well as mitochondrial enzyme citrate synthase and NADPH oxidase subunit p22 phox were analysed.Our findings revealed significantly reduced DNA strand breaks, but DNA base oxidation was not reduced. We observed significant reduction in plasma AOPPs and 3-nitrotyrosine, which indicated an improvement in oxidative/nitrative stress. However, expression of HSP70 and Nrf2 were not altered after weight loss. In addition, expression of citrate synthase and p22 phox were also unaltered.Overall, bariatric surgery induced significant reduction in excess body weight and improved the patients’ health status, including reduced DNA strand breaks and slightly improved antioxidant status in some of the investigated endpoints, while cellular ROS formation and DNA oxidation damage stayed unaltered. This complex situation may be due to combined beneficial effects of weight loss and burdening of the body with fat breakdown products. In the future, collecting samples two years after surgery, when patients have been in a weight plateau for some time, might be a promising approach.

Four‐week Ketogenic Diet Improves Aerobic Metabolic Capacity in Extensor Digitrum Longus Muscle of Sedentary Male Rats

Ketogenic diet is an extremely high‐fat diet with low or absence of carbohydrate in the total nutrient. Thus, this diet causes ketone body production from the liver and shifts energy sources from carbohydrate to fat. Whereas previous studies have suggested that ketogenic diet ameliorates brain disorder such as drug‐resistant epilepsy, its influence on peripheral tissue is unclear. Skeletal muscle is an important organ for locomotion and metabolism. Recent studies have suggested that the ketogenic diet changes the energy metabolic properties in the skeletal muscle as well; however, the results are contradictory. Moreover, it remains unknown whether the ketogenic diet affects the muscle contractile function. Therefore, this study aimed to clarify the effect of ketogenic diet on muscle performance and biochemical adaptation in rat skeletal muscle. This study was approved by Institute for Animal Experimentation at St. Marianna University School of Medicine. Male Wistar rats (8 weeks of age) were assigned to either control group (CON) or ketogenic diet group (KD). CON and KD were fed for 4 weeks either control diet (10% fat, 10% protein, 80% carbohydrate) or ketogenic diet (90% fat, 10% protein, 0% carbohydrate), respectively. Body mass was recorded once a week. After the 4‐week feeding period, the animals were anesthetized, and the blood sample was collected to analyze blood ketone concentration. The right extensor digitrum longus (EDL) muscle was used to evaluate the twitch force, tetanic force, and fatigue ex vivo. The left EDL was homogenized and used to analyze the myosin heavy chain composition, the protein expression of metabolic enzymes, and the citrate synthase activity. Body mass was lower in KD than CON at third and fourth week of the feeding period. Blood ketone concentration in KD was significantly higher than CON. There was no statistical difference in the twitch and tetanic forces as well as muscle fatigue between CON and KD. However, the citrate synthase activity in KD was significantly higher than CON. The protein expression of citrate synthase, succinate dehydrogenase, cytochrome c oxidase subunit 4, and 3‐hydroxyacyl‐CoA dehydrogenase in KD was significantly higher than CON. Moreover, there was a shift of myosin heavy chain from type IIb to IIx in KD. Therefore, we conclude that 4‐week ketogenic diet improves skeletal muscle aerobic capacity without obstructing muscle contractile function in the sedentary male rats.Support or Funding InformationThis work was supported by MEXT/JSPS KAKENHI Grant Numbers JP24650334, JP18K17767.

Transcription Factor TaDof1 Improves Nitrogen and Carbon Assimilation Under Low-Nitrogen Conditions in Wheat

A single transcription factor is known to coordinate expression of a set of metabolites in a biochemical pathway; its use therefore can be a functional strategy in generating plants with desired traits. Triticum aestivum Dof1 (TaDof1) transcription factor is mainly associated with improved nitrogen assimilation in plants. In the current research, the transgenic wheat overexpressing TaDof1 transcription factor, under a constitutive promoter, was developed by Agrobacterium-mediated transformation. The two elite wheat cultivars (Galaxy and Faisalabad-2008) were selected for transformation study. The T0 plants were subjected to screening using selection medium containing herbicide BASTA. PCR results confirmed that only 8 out of 31 plants possessed the complete TaDof1 cassette. A transformation efficiency of 0.46% for Galaxy and 0.08% for Faisalaad-2008 was obtained. The quantitative RT-PCR was performed on T1 plants grown under nitrogen-limiting conditions. A substantial rise in the expression of citrate synthase (CS), isocitrate dehydrogenase (ICDH), phosphoenolpyruvate carboxylase (PEPC), and pyruvate kinase (PK) genes regulated by TaDof1 was observed after 4 weeks of nitrogen stress in T1 plants. The maximum fold increase of 464 was recorded for ICDH. Our findings indicate a cooperative modification of nitrogen and carbon metabolisms since they are intimately linked together. Overexpression of TaDof1 in wheat resulted in a significant increase in various agronomic traits. Furthermore, various physiological and biochemical markers (chlorophyll, protein, and soluble sugar contents) exhibited a profound change in TaDof1 transgenic plants in comparison with wild type plants. The results clearly depict the merits of employing transcription factors in engineering plant metabolisms.

Changes in biomarker levels and myofiber constitution in rat soleus muscle at different exercise intensities.

Although exercise affects the function and structure of skeletal muscle, our knowledge regarding the biomedical alterations induced by different intensities of exercise is incomplete. Here we report on the changes in biomarker levels and myofiber constitution in the rat soleus muscle induced by exercise intensity. Male adult rats at 7weeks of age were divided into 3 groups by exercise intensity, which was set based on the accumulated lactate levels in the blood using a treadmill: stationary control (0m/min), aerobic exercise (15m/min), and anaerobic exercise (25m/min). The rats underwent 30min/day treadmill training at different exercise intensities for 14days. Immediately after the last training session, the soleus muscle was dissected out in order to measure the muscle biomarker levels and evaluate the changes in the myofibers. The mRNA expression of citrate synthase, glucose-6-phosphate dehydrogenase, and Myo D increased with aerobic exercise, while the mRNA expression of myosin heavy-chain I and Myo D increased in anaerobic exercise. These results suggest that muscle biomarkers can be used as parameters for the muscle adaptation process in aerobic/anaerobic exercise. Interestingly, by 14days after the anaerobic exercise, the number of type II (fast-twitch) myofibers had decreased by about 20%. Furthermore, many macrophages and regenerated fibers were observed in addition to the injured fibers 14days after the anaerobic exercise. Constitutional changes in myofibers due to damage incurred during anaerobic exercise are necessary for at least about 2weeks. These results indicate that the changes in the biomarker levels and myofiber constitution by exercise intensity are extremely important for understanding the metabolic adaptations of skeletal muscle during physical exercise.

Amplification of Mitochondrial Activity in the Healing Response Following Rotator Cuff Tendon Injury

Mitochondrial function following rotator cuff tendon injury (RCI) influences the tendon healing. We examined the mitochondrial morphology and function under hypoxia in the shoulder tendon tissue from surgically-induced tenotomy-RCI rat model and cultured swine tenocytes. The tendon tissue was collected post-injury on 3–5 (Group-A), 10–12 (Group-B), and 22–24 (Group-C), days and the corresponding contralateral tendons were used as control for each group. There was higher protein expression of citrate synthase (P < 0.0001) [10.22 MFI (mean fluorescent intensity)] and complex-1 (P = 0.0008) (7.86 MFI) in Group-A and Group-B that decreased in Group-C [(P = 0.0201) (5.78 MFI and (P = 0.7915) (2.32 MFI), respectively] compared to control tendons. The ratio of BAX:Bcl2 (Bcl2 associated x protein:B cell lymphoma 2) in RCI tendons increased by 50.5% (Group-A) and 68.4% (Group-B) and decreased by 25.8% (Group-C) compared to normoxic controls. Hypoxia increased β-tubulin expression (P = 0067) and reduced PGC1-α (P = 0412) expression in the isolated swine tenocytes with no effect on the protein expression of Complex-1 (P = 7409) and citrate synthase (P = 0.3290). Also, the hypoxic tenocytes exhibited about 4-fold increase in mitochondrial superoxide (P < 0.0001), altered morphology and mitochondrial pore integrity, and increase in mitochondrial density compared to normoxic controls. These findings suggest the critical role of mitochondria in the RCI healing response.

Regulation of expression of the mitochondrial and peroxisomal forms of citrate synthase in maize during germination and in response to light

Expression of genes encoding the mitochondrial and peroxisomal forms of citrate synthase (EC 2.3.3.1) was studied in maize (Zea mays L.) in scutella during germination and in leaves depending on light regime. During germination, citrate synthase activity increased in scutella both in mitochondria and in fatty-acid metabolizing peroxisomes (glyoxysomes) by day 6 and then declined. This was preceded by the peak of expression of the genes encoding the mitochondrial (Csy1) and peroxisomal (Csy2) forms of citrate synthase occurring on the day 3 of germination, after which the expression of Csy1 gradually and of Csy2 sharply declined. The decrease of expression of both genes was followed by the increase of promoter methylation which was more intensive for the gene encoding the mitochondrial form. In leaves, the activity of the mitochondrial form was much higher than that of the peroxisomal form and increased in darkness, while the peroxisomal form was almost undetectable in darkness and increased in the light. The mitochondrial form was inhibited by white and red light while the peroxisomal form was induced by white, red and blue light indicating the involvement of phytochrome and cryptochrome. The mechanism of light regulation of citrate synthase involved promoter methylation leading to the inhibition of corresponding genes and exhibiting opposite patterns for Csy1 and Csy2. Citrate synthase was purified from mitochondria and glyoxysomes of maize scutellum. The mitochondrial form had higher optimum pH as compared to the glyoxysomal form and possessed higher affinity to oxaloacetate and acetyl-CoA. It is concluded that expression of citrate synthase during germination and in response to light is regulated by methylation of promoters of corresponding genes.

Schooling improves critical swimming performance in zebrafish (Danio rerio)

The critical swimming performance (Ucrit) of fish has typically been measured on individuals given a step-based test that ends in fatigue. Many of the fish given this test naturally travel in schools; a concern is that the Ucrittest underestimates the natural performance of schooling fish, as there are hydrodynamic benefits to schooling. We addressed whether Ucritwas improved by schooling by giving zebrafish (Danio rerio) Ucrittests individually or in groups of three, five, or ten. We found that fish swam faster in schools and that schools grew more cohesive as flow speed increased. The increased Ucritowed to a greater number of fish swimming longer at lower speeds. In support of this, fish with the greatest Ucrittended to have lower lactate and greater expression of citrate synthase. Our results indicate that Ucritmay increase when fish are tested in groups and that this may relate to energy savings, as well as differences in individual physiology.

The effect of very-low-calorie diet on mitochondrial dysfunction in subcutaneous adipose tissue and peripheral monocytes of obese subjects with type 2 diabetes mellitus.

Mitochondrial dysfunction is a potentially important player in the development of insulin resistance and type 2 diabetes mellitus (T2DM). We investigated the changes of mRNA expression of genes encoding main enzymatic complexes of mitochondrial respiratory chain in subcutaneous adipose tissue (SCAT) and peripheral monocytes (PM) of 11 subjects with simple obesity (OB), 16 obese patients with T2DM and 17 healthy lean subjects (C) before and after very low-calorie diet (VLCD) using quantitative real time PCR. At baseline in SCAT, both T2DM and OB group had decreased mRNA expression of all investigated mitochondrial genes with the exception of 2 complex I (NDUFA 12) and complex IV (COX 4/1) enzymes in OB subjects. In contrast, in PM only the expression of complex I enzymes NDUFA 12 and MT-ND5 was reduced in both T2DM and OB subjects along with decreased expression of citrate synthase (CS) in T2DM group. Additionally, T2DM subjects showed reduced activity of pyruvate dehydrogenase and complex IV in peripheral blood elements. VLCD further decreased mRNA expression of CS and complex I (NT-ND5) and II (SDHA) enzymes in SCAT and complex IV (COX4/1) and ATP synthase in PM of T2DM group, while increasing the activity of complex IV in their peripheral blood elements. We conclude that impaired mitochondrial biogenesis and decreased activity of respiratory chain enzymatic complexes was present in SCAT and PM of obese and diabetic patients. VLCD improved metabolic parameters and ameliorated mitochondrial oxidative function in peripheral blood elements of T2DM subjects but had only minor and inconsistent effect on mitochondrial gene mRNA expression in SCAT and PM.

STAT3 Regulation of Citrate Synthase Is Essential during the Initiation of Lymphocyte Cell Growth

Citrate is a required carbon precursor for de novo fatty acid and membrane lipid synthesis. However, the pathways regulating intracellular citrate, particularly during the initial transition from a resting state to cell growth, remain unclear. Here, we show that STAT3 is among the first signaling events activated in resting lymphocytes following growth factor stimulation. During this period, the inhibition of STAT3 blocks the expression of citrate synthase (CS) and reduces the levels of intracellular citrate. As a consequence of CS loss and the reduction in citrate, cells are unable to grow or proliferate in response to extracellular growth factors. These effects were due to STAT3-dependent transcriptional regulation of CS, as exogenous addition of citrate could restore fatty acid synthesis, cell growth, and proliferation. Taken together, our studies reveal that transcription-dependent control of CS is essential for regulating the initiation of cell growth.

Repeated maximal-intensity hypoxic exercise superimposed to hypoxic residence boosts skeletal muscle transcriptional responses in elite team-sport athletes.

To determine whether repeated maximal-intensity hypoxic exercise induces larger beneficial adaptations on the hypoxia-inducible factor-1α pathway and its target genes than similar normoxic exercise, when combined with chronic hypoxic exposure. Lowland elite male team-sport athletes underwent 14days of passive normobaric hypoxic exposure [≥14h·day-1 at inspired oxygen fraction (Fi O2 ) 14.5-14.2%] with the addition of six maximal-intensity exercise sessions either in normobaric hypoxia (Fi O2 ~14.2%; LHTLH; n=9) or in normoxia (Fi O2 20.9%; LHTL; n=11). A group living in normoxia with no additional maximal-intensity exercise (LLTL; n=10) served as control. Before (Pre), immediately after (Post-1) and 3weeks after (Post-2) the intervention, muscle biopsies were obtained from the vastus lateralis. Hypoxia-inducible factor-1α subunit, vascular endothelial growth factor, myoglobin, peroxisome proliferator-activated receptor-gamma coactivator 1-α and mitochondrial transcription factor A mRNA levels increased at Post-1 (all P≤0.05) in LHTLH, but not in LHTL or LLTL, and returned near baseline levels at Post-2. The protein expression of citrate synthase increased in LHTLH (P<0.001 and P<0.01 at Post-1 and Post-2, respectively) and LLTL (P<0.01 and P<0.05 at Post-1 and Post-2, respectively), whereas it decreased in LHTL at Post-1 and Post-2 (both P<0.001). Combined with residence in normobaric hypoxia, repeated maximal-intensity hypoxic exercise induces short-term post-intervention beneficial changes in muscle transcriptional factors that are of larger magnitude (or not observed) than with similar normoxic exercise. The decay of molecular adaptations was relatively fast, with most of benefits already absent 3weeks post-intervention.

Reduced expression of citrate synthase leads to excessive superoxide formation and cell apoptosis

A/J mice are a mouse model of age-related hearing loss. It has been demonstrated that a mutation in gene of citrate synthase (CS) contributes to the early onset of hearing loss occurring at about one month of age. To understand the effects of a decreased CS activity that results from the mutation in Cs gene on hearing loss in A/J mice, human kidney cell line (293T) was transiently transfected with short hairpin RNA for Cs (shRNA-Cs) to reduce expression of CS. In comparison with those of cells transfected with a scrambled sequence (shRNA-NC), the oxygen consumption rate and adenosine trisphosphate (ATP) production level were decreased in 293T cells transfected with shRNA-Cs. Meanwhile, excessive superoxide production was induced as determined by mitochondrial superoxide formation assay (MitoSOX) and superoxide dismutase 2 (SOD2) detection. Moreover, the expression levels of BIP (binding immunoglobulin protein) and CHOP (CCAAT/enhancer-binding protein-homologous protein), markers of endoplasmic reticulum stress, were upregulated. Furthermore, apoptosis related molecule caspase-3 and the mitochondrial membrane potential were reduced. It is therefore concluded that downregulation of Cs expression in 293T cells leads to low level of ATP production, excessive superoxide formation and cell apoptosis, which implies a possible mechanism for hearing loss in A/J mice.

Genetic switches and related tools for controlling gene expression and electrical outputs of Geobacter sulfurreducens.

Physiological studies and biotechnology applications of Geobacter species have been limited by a lack of genetic tools. Therefore, potential additional molecular strategies for controlling metabolism were explored. When the gene for citrate synthase, or acetyl-CoA transferase, was placed under the control of a LacI/IPTG regulator/inducer system, cells grew on acetate only in the presence of IPTG. The TetR/AT system could also be used to control citrate synthase gene expression and acetate metabolism. A strain that required IPTG for growth on D-lactate was constructed by placing the gene for D-lactate dehydrogenase under the control of the LacI/IPTG system. D-Lactate served as an inducer in a strain in which a D-lactate responsive promoter and transcription repressor were used to control citrate synthase expression. Iron- and potassium-responsive systems were successfully incorporated to regulate citrate synthase expression and growth on acetate. Linking the appropriate degradation tags on the citrate synthase protein made it possible to control acetate metabolism with either the endogenous ClpXP or exogenous Lon protease and tag system. The ability to control current output from Geobacter biofilms and the construction of an AND logic gate for acetate metabolism suggested that the tools developed may be applicable for biosensor and biocomputing applications.

Controlling Citrate Synthase Expression by CRISPR/Cas9 Genome Editing for n-Butanol Production in Escherichia coli.

Genome editing using CRISPR/Cas9 was successfully demonstrated in Esherichia coli to effectively produce n-butanol in a defined medium under microaerobic condition. The butanol synthetic pathway genes including those encoding oxygen-tolerant alcohol dehydrogenase were overexpressed in metabolically engineered E.coli, resulting in 0.82 g/L butanol production. To increase butanol production, carbon flux from acetyl-CoA to citric acid cycle should be redirected to acetoacetyl-CoA. For this purpose, the 5'-untranslated region sequence of gltA encoding citrate synthase was designed using an expression prediction program, UTR designer, and modified using the CRISPR/Cas9 genome editing method to reduce its expression level. E.coli strains with decreased citrate synthase expression produced more butanol and the citrate synthase activity was correlated with butanol production. These results demonstrate that redistributing carbon flux using genome editing is an efficient engineering tool for metabolite overproduction.

N-acetylcysteine inhibits kinase phosphorylation during 3T3-L1 adipocyte differentiation

ABSTRACTObjectives: Reports investigating the effects of antioxidants on obesity have provided contradictory results. We have previously demonstrated that treatment with the antioxidant N-acetylcysteine (NAC) inhibits cellular triglyceride (Tg) accumulation as well as total cellular monoamine oxidase A (MAOA) expression in 3T3-L1 mature adipocytes (Calzadilla et al., Redox Rep. 2013;210–218). Here we analyzed the role of NAC on adipogenic differentiation pathway.Methods: Assays were conducted using 3T3-L1 preadipocytes (undifferentiated cells: CC), which are capable of differentiating into mature adipocytes (differentiated cells: DC). We studied the effects of different doses of NAC (0.01 or 1 mM) on DC, to evaluate cellular expression of phospho-JNK½ (pJNK½), phospho-ERK½ (pERK½) and, mitochondrial expression of citrate synthase, fumarate hydratase and MAOA.Results: Following the differentiation of preadipocytes, an increase in the expression levels of pJNK½ and pERK½ was observed, together with mitotic clonal expansion (MCE). We found that both doses of NAC decreased the expression of pJNK½ and pERK½. Consistent with these results, NAC significantly inhibited MCE and modified the expression of different mitochondrial proteins.Discussion: Our results suggested that NAC could inhibit Tg and mitochondrial protein expression by preventing both MCE and kinase phosphorylation.

The novel choline kinase inhibitor ICL-CCIC-0019 reprograms cellular metabolism and inhibits cancer cell growth.

The glycerophospholipid phosphatidylcholine is the most abundant phospholipid species of eukaryotic membranes and essential for structural integrity and signaling function of cell membranes required for cancer cell growth. Inhibition of choline kinase alpha (CHKA), the first committed step to phosphatidylcholine synthesis, by the selective small-molecule ICL-CCIC-0019, potently suppressed growth of a panel of 60 cancer cell lines with median GI50 of 1.12 μM and inhibited tumor xenograft growth in mice. ICL-CCIC-0019 decreased phosphocholine levels and the fraction of labeled choline in lipids, and induced G1 arrest, endoplasmic reticulum stress and apoptosis. Changes in phosphocholine cellular levels following treatment could be detected non-invasively in tumor xenografts by [18F]-fluoromethyl-[1,2–2H4]-choline positron emission tomography. Herein, we reveal a previously unappreciated effect of choline metabolism on mitochondria function. Comparative metabolomics demonstrated that phosphatidylcholine pathway inhibition leads to a metabolically stressed phenotype analogous to mitochondria toxin treatment but without reactive oxygen species activation. Drug treatment decreased mitochondria function with associated reduction of citrate synthase expression and AMPK activation. Glucose and acetate uptake were increased in an attempt to overcome the metabolic stress. This study indicates that choline pathway pharmacological inhibition critically affects the metabolic function of the cell beyond reduced synthesis of phospholipids.

Oral Corticosterone Administration Reduces Insulitis but Promotes Insulin Resistance and Hyperglycemia in Male Non‐obese Diabetic Mice

Steroid‐induced diabetes via oral delivery of glucocorticoids is the most common form of drug‐induced hyperglycemia in adults and children. Because a variety of pathological situations require a chronic glucocorticoid regimen in humans, appropriate experimental models are required to examine and understand the metabolic alterations associated with such an approach. Therefore, the purpose of this study was to investigate how chronic oral corticosterone administration impacted specific metabolic parameters in male non‐obese diabetic mice. These mice are genetically susceptible to spontaneous development of diabetes due to destruction of pancreatic islet β‐cells, which includes T‐lymphocyte infiltration into the pancreatic tissue. Therefore, these mice are a model system of a genetically at risk population in which to test both the immunosuppressive and diabetogenic properties of oral steroid delivery. The experimental design was six weeks of corticosterone delivery via drinking water (100μg/mL), followed by a four week washout phase. Control mice received 1% ethanol vehicle in the drinking water. All mice were 12 weeks of age when the study began. Three weeks after corticosterone delivery, there was reduced sensitivity to insulin measured by insulin tolerance test (ITT) over a 2h period, which culminated with a 22% increase in area under the curve AUC in the corticosterone group when compared with the vehicle control (p &lt; 0.05). Overt hyperglycemia (&gt;250mg/dL) and a 12% increase in body weight (31.3g) in the corticosterone group relative to vehicle control (28g; p &lt; 0.01) were apparent six weeks after steroid delivery. While there was no significant difference in the absolute number of islets per pancreas between the groups, mice receiving corticosterone had a 2.5‐fold increase in insulin‐positive area (p &lt; 0.05) relative to the vehicle control group. This increase in insulin‐positive area is most likely a compensatory expansion of the islet β‐cell population in response to insulin resistance (as measured by ITT) in the periphery. The mice in the corticosterone group also displayed a 60% decrease in insulitis (islet immune cell invasion) compared with vehicle control group six weeks after oral corticosterone delivery. In addition, there were marked increases in expression of citrate synthase (7415‐fold up vs. vehicle) and glycogen synthase (6972‐fold up vs. vehicle) genes in skeletal muscle of mice in the corticosterone group relative to vehicle control. Corticosterone‐induced hyperglycemia and changes in muscle gene expression were all completely reversed by the end of the washout phase, indicating that the metabolic parameters measured were reversible. These results are similar to those reported with human subjects receiving chronic oral delivery of glucocorticoids, indicating that male NOD mice may be an excellent translational model to mimic steroid‐induced diabetes in a genetically susceptible population.Support or Funding InformationNIH P20 GM103528

Abstract C118: Choline kinase inhibition with the novel pharmacological inhibitor ICL-CCIC-0019 reprograms cellular metabolism and inhibits cancer cell growth

Abstract The glycerophospholipid phosphatidylcholine (PC) is the most abundant phospholipid species of eukaryotic membranes and essential for structural integrity and signaling function of cell membranes required for cancer cell growth. PC is synthesized via the CDP-choline pathway, whereby choline kinase alpha (CHKA) denotes the first committed step in this sequence of enzymatic reactions. CHKA phosphorylates choline to form phosphocholine and its overexpression in many solid tumors is linked to progression of normal cells to malignancy. We developed the highly selective, choline-competitive small-molecule ICL-CCIC-0019 (IC50 of 0.27±0.06 μM). Across a panel of 131 human kinases, the inhibitor showed minimal off-target effects (only 5 kinases were inhibited more than 20% at a concentration of 10 μM). ICL-CCIC-0019 potently inhibited growth of a panel of 60 cancer cell lines with median GI50 of 1.12 μM. Importantly, proliferation of normal cells was only minimally affected (MCF-10A and ST-T1b: GI50 30-120 μM). ICL-CCIC-0019 decreased phosphocholine levels and the fraction of labeled choline in lipids, and induced G1 arrest, endoplasmic reticulum stress and apoptosis. Changes in phosphocholine cellular levels following treatment could be detected non-invasively in tumor xenografts by 18F-fluoromethyl-[1,2-2H4]-choline positron emission tomography. Pharmacokinetic modeling revealed that the macro parameter Ki denoting the net irreversible uptake rate was significantly decreased in tumor after 48 hours (Ki (1/min): control, 0.0054±0.00060; ICL-CCIC-0019, 0.0032±0.00064), confirming in vivo target inhibition. This resulted in potent antitumor activity in HCT116 xenografts. We further reveal a previously unappreciated effect of choline metabolism on mitochondria function. Comparative metabolomics demonstrated that phosphatidylcholine pathway inhibition leads to a metabolically stressed phenotype analogous to mitochondria toxin treatment but without reactive oxygen species activation. Drug treatment decreased TCA cycle activity, oxygen consumption rate and elevated extracellular acidification rate. This was associated with a reduction of citrate synthase expression and AMP kinase activation. Glucose and acetate uptake were increased in an attempt to overcome the metabolic stress. This study indicates that choline pathway pharmacological inhibition is a valid therapeutic strategy and critically affects the metabolic function of the cell beyond reduced synthesis of phospholipids. This work was supported in part by Cancer Research UK-Engineering and Physical Sciences Research Council Grant C2536/A10337. Citation Format: Sebastian Trousil, Maciej Kalisczcak, Zachary Schug, Quang-De Nguyen, Giampaolo Tomasi, Rosy Favicchio, Diana Brickute, Robin Fortt, Frazer J. Twyman, Laurence Carroll, Andrew Kalusa, Naveenan Navaratnam, Thomas Adejumo, David Carling, Eyal Gottlieb, Eric O. Aboagye. Choline kinase inhibition with the novel pharmacological inhibitor ICL-CCIC-0019 reprograms cellular metabolism and inhibits cancer cell growth. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C118.