The tricarboxylic acid cycle activity in cultured primary astrocytes is strongly accelerated by the protein tyrosine kinase inhibitor tyrphostin 23

Accepted Manuscript The tricarboxylic acid cycle activity in cultured primary astrocytes is strongly accelerated by the protein tyrosine kinase inhibitor tyrphostin 23 Michaela C. Hohnholt, Eva-Maria Blumrich, Helle S. Waagepetersen, Ralf Dringen PII:

S0197-0186(16)30282-0

DOI:

10.1016/j.neuint.2016.11.008

Reference:

NCI 3951

To appear in:

Neurochemistry International

Received Date: 3 September 2016 Revised Date:

9 November 2016

Accepted Date: 14 November 2016

Please cite this article as: Hohnholt, M.C., Blumrich, E.-M., Waagepetersen, H.S., Dringen, R., The tricarboxylic acid cycle activity in cultured primary astrocytes is strongly accelerated by the protein tyrosine kinase inhibitor tyrphostin 23, Neurochemistry International (2016), doi: 10.1016/ j.neuint.2016.11.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

NCI-2016-174-R1, second revised version

The tricarboxylic acid cycle activity in cultured primary astrocytes is strongly accelerated by the protein tyrosine

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kinase inhibitor tyrphostin 23

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Dringen2,3

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Michaela C. Hohnholt1, Eva-Maria Blumrich2,3 , Helle S. Waagepetersen1 and Ralf

Department of Drug Design and Pharmacology, Faculty of Health and Medical Science,

University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark 2

Centre for Biomolecular Interactions Bremen, Faculty 2 (Biology/Chemistry), University of

Bremen, PO. Box 330440, D-28334 Bremen, Germany.

Centre for Environmental Research and Sustainable Technology, Leobener Strasse,

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D-28359 Bremen, Germany

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Address correspondence to: Michaela C. Hohnholt Department of Drug Design and Pharmacology Faculty of Health and Medical Science University of Copenhagen Universitetsparken 2 2100 Copenhagen Denmark Email: [email protected] Abbreviations: DMF: N,N-dimethylformamide; GCMS: gas chromatography mass spectrometry; I: isotopomer; IB: incubation buffer; LDH: lactate dehydrogenase; M: mass of the unlabeled molecule; MCL: molecular carbon labeling; MTBSTFA: N-methyl-N-(tertbutyldimethylsilyl) trifluoroacetamide; PDH: pyruvate dehydrogenase; PKM2: pyruvate kinase isoform M2; PTKs: protein tyrosine kinases; T23: tyrphostin 23; TCA: tricarboxylic acid; X: number of labeled carbon atoms; α-KG: α-ketoglutarate

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ACCEPTED MANUSCRIPT Abstract Tyrphostin 23 (T23) is a well-known inhibitor of protein tyrosine kinases and has been considered as potential anti-cancer drug. T23 was recently reported to acutely stimulate the glycolytic flux in primary cultured astrocytes. To investigate whether T23 also affects the

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tricarboxylic acid (TCA) cycle, we incubated primary rat astrocyte cultures with [U13

C]glucose in the absence or the presence of 100 µM T23 for 2 h and analyzed the

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metabolite pattern. These incubation conditions did not compromise cell viability and

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confirmed that the presence of T23 doubled glycolytic lactate production. In addition, T23treatment strongly increased the molecular carbon labeling of the TCA cycle intermediates

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citrate, succinate, fumarate and malate, and significantly increased the incorporation of

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labelling into the amino acids glutamate, glutamine and aspartate. These results clearly demonstrate that, in addition to glycolysis, also the mitochondrial TCA cycle is strongly accelerated after exposure of astrocytes to T23, suggesting that a protein tyrosine kinase may

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be involved in the regulation of the TCA cycle in astrocytes.

Highlights:

T23 stimulates glycolytic flux in cultured astrocytes.

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Molecular carbon labeling from [U-13C]glucose is enhanced in tricarboxylic acid cycle

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intermediates.

Mitochondrial metabolism of brain astrocytes is accelerated by T23 exposure.

Key words: Astrocytes; lactate; metabolism; tyrphostins; tricarboxylic acid cycle

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ACCEPTED MANUSCRIPT 1. Introduction Tyrphostins are structurally similar to tyrosine (Banbury et al., 2003) and were developed as selective synthetic inhibitors of protein tyrosine kinases (PTKs) (Levitzki, 1992). As such, tyrphostins showed promissing results in cancer therapy (Bojko et al., 2015; Levitzki and

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Gazit, 1995). Tyrphostin 23 (T23) was identified as an inhibitor of epidermal growth factor receptor (EGFR) induced signaling (Ligęza et al., 2011). However, concerning brain cells T23 has also been used as a tool to study the role of tyrosine kinases in regulation of volume

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dependent chloride channels in cultured astrocytes (Crépel et al., 1998; Mongin et al., 1999), to block the uptake of nanoparticles in cultured astrocytes (Pickard et al., 2011) and neurons

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(Petters and Dringen, 2015), to interfere with dopamine uptake in rat neurons (Hoover et al., 2007) and to modulate the hypo-osmotic release of taurine and aspartate from cultured astrocytes (Franco et al., 2001; Mongin et al., 1999). Very recently T23 was reported to also affect the basal metabolism of brain cells as it substantially accelerates glycolytic flux in

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cultured astrocytes (Blumrich et al., 2016).

In brain, astrocytes are considered to have important metabolic functions and to be essential

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partners of neurons in energy metabolism and neurotransmission (Contreras, 2015; Hirrlinger and Dringen, 2010; Schousboe et al., 2014; Winkler and Hirrlinger, 2015). In astrocytes,

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glycolysis is considered an important ATP generating pathway (Bolaños, 2016; Hall et al., 2012). Although, it has been debated, cultured astrocytes are in particular glycolytic (Allaman et al., 2015; Blumrich et al., 2016; Carpenter et al., 2015; Halim et al., 2010)(Bouzier-Sore and Pellerin, 2013; San Martin et al., 2013). A variety of compounds, including T23, has been shown in the past years to stimulate glycolytic flux and lactate release from cultured astrocytes (Blumrich et al., 2016) although for several of these compounds the mechanism involved in glycolysis stimulation remains to be elucidated.

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ACCEPTED MANUSCRIPT Despite being considered as mainly glycolytic cells, cultured astrocytes have also an active mitochondrial metabolism. Upon inhibition of mitochondrial respiration (Bolanos et al., 1994; Scheiber and Dringen, 2011), astrocytes increase their glycolytic rate, which indicates that these cells can compensate at least partly a loss of mitochondrial ATP production via

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glycolysis (Bolaños, 2016). The tricarboxylic acid (TCA) cycle localized in the mitochondrial matrix oxidizes the carbon backbone of acetyl residues derived from glycolytically generated pyruvate. The oxidation steps are associated with the generation of reducing equivalents in the

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form of NADH and FADH2 which are subsequently used in the synthesis of ATP by oxidative phosphorylation. The TCA cycle is metabolically connected to the amino acid glutamate

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(Nissen et al., 2015; Olsen and Sonnewald, 2015). The neurotransmitter glutamate that is taken up by astrocytes can also become converted to glutamine by glutamine synthetase, which can subsequently be released by astrocytes and afterwards taken up by neurons (glutamate-glutamine cycle) for neurotransmitter recycling (Parpura et al., 2016; Schousboe et

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al., 2014). The expression and activity of glutamate dehydrogenase in astrocytes (Rothe et al., 1990) provide the machinery for the oxidation of glutamate to α-ketoglutarate and therefore enables glutamate to serve as substrate for ATP production (McKenna, 2013). A decrease in

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glutamate dehydrogenase activity leads to an increase in glucose utilization by astrocytes,

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highlighting the role of glutamate as an energy substrate (Pajecka et al., 2015) and the connection between glycolytic and mitochondrial metabolism.

We have very recently reported that T23 substantially accelerates glycolytic flux in cultured astrocytes (Blumrich et al., 2016). In order to investigate whether also mitochondrial pathways of astrocytes may be affected by T23, we applied T23 to rat cultured primary astrocytes in the presence of [U-13C]glucose and investigate the isotopomer pattern in TCA cycle intermediates and TCA cycle derived amino acids. We clearly demonstrate that T23, in addition to the known stimulation of glycolytic flux, also strongly accelerates the 13C labeling 4

ACCEPTED MANUSCRIPT of TCA cycle intermediates and the labeling of the amino acids glutamate, glutamine and aspartate.

2. Materials and Methods

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2.1. Materials

Dulbecco’s modified Eagles’s medium (DMEM) was obtained from Invitrogen-Gibco (Darmstadt, Germany) and fetal calf serum (FCS) and penicillin G/streptomycin sulfate

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solution were purchased from Biochrom (Berlin, Germany). T23, N-methyl-N-(tertbutyldimethylsilyl) trifluoroacetamide (MTBSTFA) and N-N-dimethylformamide (DMF)

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were obtained from Sigma-Aldrich (Steinheim, Germany or St. Louis, MO, USA). DGlucose-13C6 (99% enrichment) was from Cambridge Isotope Laboratories (Andover, USA). Gas chromatography mass spectrometry (GCMS) chemicals and columns were purchased from Agilent Technologies (Santa Clara, CA, USA). The enzymes glucose-6-phosphate

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dehydrogenase, hexokinase, lactate dehydrogenase (LDH) and glutamate pyruvate transaminase were from Roche Diagnostics (Mannheim, Germany). Other chemicals and buffer ingredients were from Sigma (Steinheim, Germany), Applichem (Darmstadt, Germany)

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or Merck (Darmstadt, Germany). Sterile Nunclon-5-cm cell culture dishes and unsterile 96-

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well plates were obtained from Sarstedt (Nümbrecht, Germany).

2.2. Astrocyte cultures

Preparation of astrocyte-rich primary cultures was performed as described previously (Hamprecht and Löffler, 1985; Tulpule et al., 2014). The cells harvested from the whole brain of newborn Wistar rats were seeded at a density of 600.000 cell/mL in 5 cm-cell culture dishes in 5 mL culture medium (90% DMEM containing 25 mM glucose, 10% FCS, 1 mM pyruvate, 18 U/mL penicillin G and 18 µg/mL streptomycin sulfate). The cultures were grown in the humidified atmosphere of a Sanyo (Osaka, Japan) incubator with 10% CO2. Every 7th 5

ACCEPTED MANUSCRIPT day as well as one day before the experimental incubation the culture medium was renewed. Confluent cultures of an age between 18 and 30 days were used for experiments. The cultures prepared and grown in this way are characterized by a strong enrichment in astrocytes, but

2014; Tulpule et al., 2014).

2.3. Experimental incubations 13

C labeling experiments. For

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Astrocyte cultures in 5 cm dishes were used for metabolite

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contain minor contaminations of oligodendrocytes and microglial cells (Petters and Dringen,

incubations, the culture medium was removed, the cells were washed once with 3 mL pre-

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warmed (37°C) incubation buffer (IB; 145 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 0.8 mM Na2HPO4, 20 mM HEPES adjusted at 37°C to pH 7.4 with NaOH) and incubated for 2 h at 37°C in 2.5 mL IB containing 5 mM [U-13C]glucose without T23 (controls) or with 100 µM T23. These conditions were chosen as they have been shown to

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cause a maximal stimulating effect of T23 on astrocytic glycolysis (Blumrich et al., 2016). To stop the incubations, the incubation medium was harvested and the cells were washed with 1.5 mL ice-cold phosphate-buffered saline (PBS; 10 mM potassium phosphate buffer pH 7.4

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containing 150 mM NaCl). The incubation media were transferred into reaction tubes and used later to assess extracellular LDH activity, lactate content and glucose consumption as

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well the carbon labeling of selected extracellular metabolites. For analysis of 13C labeled TCA cycle intermediates and amino acids cell extracts were prepared with ethanol as previously described in detail (Walls et al., 2014).

2.4. Determination of glycolytic flux and cell viability The glycolytic flux of cultured astrocytes was determined by measuring the lactate production and glucose consumption by the cells as previously described in detail (Tulpule et al., 2014). Extracellular lactate was meassured using a enzymatic assay with LDH and glutamate 6

ACCEPTED MANUSCRIPT pyruvate transaminase in 10 µL samples of the media harvested after the incubation. The extracellular glucose concentration was determined in 10 µL media samples by a coupled enzymatic assay with hexokinase and glucose-6-phosphate dehydrogenase. The difference in extracellular glucose concentration before and after the incubation of the cells was calculated

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as glucose consumption. The release of the cytosolic enzyme LDH was determined as previously described (Dringen et al., 1998; Tulpule et al., 2014) using 10 µL samples of the harvested incubation media to investigate a potential loss in cell membrane integrity of

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cultured astrocytes by the treatment.

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2.5. Extraction of cell cultures for GCMS measurement and determination of protein content

The washed cells were extracted as previously described (Walls et al., 2014). 1.5 mL ice-cold 70% ethanol was applied to each dish of washed cells, the cells were scraped off using a

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rubber policeman and the lysate was transferred into a 2 mL reaction tube. Additional 500 µL 70 % ethanol were used to wash the dish and the ethanol fractions of one dish were combined. After centrifugation (20 min at 4°C at 12.045g; Eppendorf MINI-Spin, Hamburg, Germany)

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the supernatant was transferred into a new reaction tube and dried in a speed-vac (Eppendorf, Hamburg, Germany) at room temperature, while the centrifugation pellets were dried

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overnight on air and used for protein determination. For protein determination, the protein pellets were lysed for 1 h in 0.5 M NaOH under continuous shaking and the protein content was determined by the Lowry method (Lowry et al., 1951) using bovine serum albumin as standard protein.

2.6. Metabolic mapping of metabolites of interest by GCMS Dried cell extracts from one dish were resuspended in 250 µL pure H2O and 125 µL of the resuspended cell extracts were used for GCMS analysis. Extraction and derivatization of 7

ACCEPTED MANUSCRIPT samples was performed by a modification of a previously described method (Mawhinney et al., 1986; Walls et al., 2014). Briefly, the pH of the GCMS aliquot was adjusted to 1-2 (2 M HCl), dried under nitrogen flow and extracted with 96 % ethanol/benzene. After drying under nitrogen flow, the organic extraction was performed a second time, followed by derivatization

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of metabolites with 14 % DMF/ 86 % MTBSTFA. Analysis of unlabeled standards and cell extracts was performed on a gas chromatograph (Agilent Technologies 7820A chromatograph, J&W GC column HP-5MS, parts no. 19091S-433; Agilent, Glostrup,

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Denmark) coupled to a mass spectrometer (Agilent Technologies, 5977E). To calculate the isotope enrichment of the metabolites of interest a published method (Biemann, 1962) was 13

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used and the values were corrected for natural abundance of

C by using the unlabeled

standards. For each isotopomer M+X (M = mass of the unlabeled molecule, X = number of labeled carbon atoms) the data are presented as percent of the total pool of the metabolite.

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2.7. Calculation of molecular carbon labeling (MCL)

The average percent of labeled carbon atoms for each metabolite was calculated as described previously (Bak et al., 2006a). Labeled alanine for example will contain between one and 13

C atoms (M+1, M+2 or M+3). The percentage of the individual isotopomers (that

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three

means: M+1, M+2 or M+3) were multiplied by the number of carbons labeled (that means: 1

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for M+1, 2 for M+2 or 3 for M+3), summed up and expressed as percent of the total number of carbon atoms.

2.8. Calculation of the cycling ratio The cycling ratio is an indicator of TCA cycle activity (Bak et al., 2012). Glycolytic metabolism of [U-13C]glucose produces [1,2-13C]acetyl-Coenzyme A (CoA), which enters the TCA cycle continuously. Thus, the number of

13

C atoms increases over time for each TCA

cycle intermediate. Initially in the first turn of the TCA cycle, [1,2-13C]acetyl-CoA forms 8

ACCEPTED MANUSCRIPT double labeled (M+2) citrate by condensing with unlabeled oxaloacetate and subsequently other (M+2) TCA cycle intermediates and TCA cycle-derived amino acids. Glutamate is formed either by transamination or oxidative deamination of α-ketoglutarate (α-KG). In the second and further turns of the TCA cycle [1,2-13C]acetyl-CoA condenses with already

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double-labeled oxaloacetate and the number of labeled carbon atoms in TCA cycle intermediates and derived amino acids increases. The cycling ratio is calculated by dividing the labeling arising from second and later turns with that from the first turn using the

for aspartate):

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Cycling ratio (aspartate) = (I(M+1)+I(M+3)+I(M+4))/I(M+2)

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following formula from the % labeled isotopomer (I) of each metabolite (given is the example

A higher cycling ratio means a higher TCA cycle activity within a given time frame.

2.9. Presentation of data

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The values shown are means ± SD of data obtained in experiments performed on three independently prepared astrocyte cultures. Analysis for statistical significance was performed by the two-tailed paired t-test (comparison of two data sets; indicated by asterisks). p>0.05

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was considered as not significant.

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ACCEPTED MANUSCRIPT 3. Results 3.1. Effects of T23 on cell viability and glycolytic flux in primary astrocytes To investigate whether T23 may affect mitochondrial metabolism of astrocytes, we incubated primary cultures of astrocytes for 2 h with [U-13C]glucose in the absence or the presence of

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100 µM T23. The media of the cultures were collected to determine extracellular LDH activity (Fig. 1A), extracellular lactate content (Fig. 1B) as well as cellular glucose consumed (Fig. 1C), while the cells were extracted for metabolite analysis by GCMS. For all incubation

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conditions, the extracellular LDH activity was below 10% of the initial total cellular LDH activity (Fig. 1A), demonstrating that the incubation conditions used did not severely

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compromised cell viability. After incubation in the presence of T23, the extracellular lactate content was found increased almost 3-fold (6.6 ± 0.5 µmol/mg) as compared to incubations in the absence of T23 (2.3 ± 0.1 µmol/mg) (Fig. 1B). A similar stimulation by T23 was found for the glucose consumption, which was also significantly elevated from 1.6 ± 0.2 µmol/mg

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(absence of T23) to 4.1 ± 0.7 µmol/mg for incubations with T23 (Fig. 1C). These data demonstrate that T23 stimulates glycolysis also in astrocytes grown in 5 cm dishes, with a different medium volume to cell number ratio, compared to that of cultures in wells of 24-

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well dishes previously used for similar experiments (Blumrich et al., 2016).

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3.2. Effects of T23 on the 13C labeling of lactate and alanine After the 2 h incubation of astrocytes with [U-13C]glucose substantial amounts of 13C labeling were found in extracellular and cellular lactate and alanine (Fig. 2). Compared to control cells (treated in the absence of T23), the MCL of intracellular lactate was not significantly altered in T23-treated astrocytes (Fig. 2A), while that of alanine was decreased from 34.5 ± 7.4 % (control cells) to 19.5 ± 5.2 % in astrocytes that had been exposed to T23 (Fig. 2B). In the medium, the MCL of lactate was significantly increased from 74.7 ± 1.8 % (control astrocytes) to 87.8 ± 1.2 % (T23-treated astrocytes) (Fig. 2C), while the MCL of alanine was 10

ACCEPTED MANUSCRIPT slightly but significantly decreased from 53.1 ± 1.7 % (control) to 48.7 ± 1.8 % in T23 treated astrocytes (Fig. 2D). The majority of the labeling of intracellular lactate and alanine represented lactate M+3 and alanine M+3 both in control astrocytes and T23-treated astrocytes (data not shown) which is a consequence of the direct metabolism of [U13

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C]glucose to pyruvate and subsequent reduction and transamination to lactate and alanine,

respectively. However, also substantial amounts of unlabelled lactate and alanine were determined which are likely to be derived from metabolites (i.e., glycogen, intermediates of

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onset of the incubation with [U-13C]glucose.

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glycolysis and pentose phosphate pathway) that had been present in the cells already at the

3.3. Labeling of cellular TCA cycle intermediates

To investigate the influence of T23 on TCA cycle metabolism, the MCL of TCA cycle

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intermediates after exposure to [U-13C]glucose was determined. Compared to control astrocytes (incubation without T23), the MCL values of the three TCA cycle intermediates citrate, succinate and malate were found strongly and significantly increased after T23

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treatment to values of 190 ± 35 % (citrate), 229 ± 85 % (succinate) and 246 ± 24 % (malate) (Fig. 3A,B,D), while the MCL of fumarate was in T23-treated cells 151 ± 47 % of the values

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determined for control cells (Fig. 3C) and this difference did not reach the level of significance.

These high enrichments may arise from increased glycolysis as demonstrated by the accelerated glucose consumption and lactate production (see 3.1), but the TCA cycling ratios are needed in order to evaluate changes in the TCA cycle metabolism. Thus, the TCA cycling ratios were calculated to investigate whether the TCA cycling was affected by the incubation with T23. For all the four investigated TCA cycle intermediates citrate, succinate, fumarate

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ACCEPTED MANUSCRIPT and malate the cycling ratios were significantly and strongly increased in astrocytes that had been incubated with T23 compared to cells incubated without T23 (Fig. 3E-H). For a more detailed analysis of the influence of T23 on the TCA cycle, the labeling of the individual isotopomers of the four TCA cycle intermediates was analyzed. Direct labeling of

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TCA cycle intermediates from [U-13C]glucose gives rise to TCA cycle intermediates labeled in two carbon atoms (M+2). The labeling in M+2 isotopomers of citrate (Fig. 4A), succinate (Fig. 4B) and fumarate (Fig. 4C) did not differ for cells incubated in the absence (control) or

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in the presence of T23. In contrast, the labeling in malate M+2 was significantly increased

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from 13.8 ± 2.0 % (control) to 21.4 ± 1.6 % after incubation in the presence of T23 (Fig. 4D). These differences in the labeling of the various TCA cycle intermediates are a consequence of compartmentation of the metabolites in various pool sizes, which are not all in equilibrium with each other. The isotopomers M+1, M+3, M+4 and M+5 of citrate (Fig. 4A) showed a significantly higher labeling in cells that had been exposed to T23 compared to control cells

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(Fig. 4A). For both succinate and malate, the isotopomers M+1, M+3 and M+4 were significantly stronger labeled in T23-exposed cells compared to control cells (Fig. 4B,D), while for fumarate only the M+4 isotopomer was significantly stronger labeled in T23-treated

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cells compared to control cells (Fig. 4C). The significant increase in malate M+2 suggests that

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the entrance of glucose-derived carbon into the TCA cycle is increased. In addition, the higher labeling of the isotopes M+1, M+3, M+4 and M+5 in the different TCA cycle intermediates suggest that the TCA cycling and potential other pathways such as pyruvate recycling are accelerated by T23 in astrocytes.

3.4. 13C Labeling of cellular amino acids

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ACCEPTED MANUSCRIPT In T23-treated astrocytes, the MCL of the cellular amino acids aspartate, glutamate and glutamine were increased from 11.3 ± 1.9 % (control, aspartate), 10.8 ± 2.5 % (control, glutamate) and 8.0 ± 1.9 % (control, glutamine) to 35.4 ± 5.5 % (aspartate), 26.1 ± 6.9 % (glutamate) and 21.8 ± 5.0 % (glutamine) (Fig. 5A-C). As the three amino acids glutamate,

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glutamine and aspartate are directly linked to the TCA cycle intermediates α-ketoglutarate and oxaloacetate, their labelling patterns correspond closely to that of the TCA cycle intermediates and reflect TCA cycling activity. Therefore, in analogy to the cycling ratio of

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TCA intermediates, we determined a corresponding TCA cycling ratio for the amino acids. Also the TCA cycling ratios of the TCA cycle intermediate-derived amino acids were

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increased from 1.2 ± 0.1 (aspartate), 0.8 ± 0.1 (glutamate) and 0.9 ± 0.1 (glutamine) in the absence of T23 to 2.0 ± 0.2 (aspartate), 2.0 ± 0.3 (glutamate) and 2.0 ± 0.4 (glutamine) in the presence of T23 (Fig. 5D-F).

Analysis of the labeling in the individual isotopomers of the TCA cycle intermediate-derived

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amino acids revealed that, in contrast to the TCA cycle intermediates, the labeling in M+2 of aspartate, glutamate and glutamine was significantly increased (Fig. 6). Also the labeling of

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the other isotopomers M+1, M+3 and M+4 of aspartate (Fig. 6A) and the isotopomers M+1, M+3, M+4 and M+5 of glutamate (Fig. 6B) was strongly increased, while in glutamine also

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the labeling of the isotopomers M+3, M+4 and M+5 was significantly increased (Fig. 6C).

3.5. Labeling of extracellular citrate and glutamine Cultured astrocytes are known to release citrate (Sonnewald et al., 1991; Waagepetersen et al., 2001) and glutamine (Bak et al., 2006a; Waagepetersen et al., 2001) into the medium. Thus, the labeling in these two extracellular metabolites was investigated after the 2 h incubation with [U-13C]glucose in the absence or the presence of T23. Compared to control cells, the 13

ACCEPTED MANUSCRIPT MCL of extracellular citrate was strongly elevated from 10.0 ± 1.1 % to 28.3 ± 1.7 % after incubation of the cells with T23 (Fig. 7A). In addition, the labeling of the individual isotopomers M+2, M+4, M+5 and M+6 was significantly higher in T23-treated cells compared with control cells, whereas the labeling of the citrate isotopomers M+1 and M+3

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did not differ to that of control cells (Fig. 7B). The MCL of extracellular glutamine was significantly increased from 5.1 ± 0.8 % in the absence of T23 to 21.1 ± 1.6 % in the presence of T23 (Fig. 7C) and the labeling of all isotopomers of extracellular glutamine from M+1 to

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M+5 was significantly increased (Fig. 7D).

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ACCEPTED MANUSCRIPT 4. Discussion In the current study we have investigated the acute metabolic consequences of an exposure of cultured astrocytes to the protein tyrosine kinase inhibitor T23. In addition to a strong acceleration of the glycolytic flux, T23 also accelerated the 13C labeling from [U-13C]glucose

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of TCA cycle intermediates and the amino acids glutamate, glutamine and aspartate which are generated from TCA cycle intermediates in viable cultured astrocytes. The observed strong acceleration of glucose consumption and lactate release confirms results published very

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recently (Blumrich et al., 2016) and includes T23 to a group of structurally diverse

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compounds which are able to acutely stimulate astrocytic lactate production (Blumrich et al., 2016). The molecular mechanisms of the observed stimulation of glycolytic flux by T23 appear not to involve AMP kinase but rather other phosphorylation/dephosphorylation events which regulate glycolytic flux in astrocytes (Blumrich et al., 2016).

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To test whether TCA cycle metabolism is influenced by the presence of T23, cultured astrocytes were incubated in the presence of [U-13C]glucose to track the carbon atoms as described before (Walls et al., 2014). After T23 exposure the labeling of cellular TCA cycle

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intermediates, of the TCA-cycle derived amino acids glutamate, aspartate and glutamine as

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well as of extracellular lactate, citrate and glutamine was strongly enhanced while the labeling of cellular and extracellular alanine, a product of the transamination of pyruvate was found significantly decreased. This strongly suggests that T23 induces an alteration of metabolism which directs carbon atoms derived from glycolysis more strongly towards mitochondrial TCA cycle metabolism.

As reported before for cultured mouse astrocytes from cortex (Skytt et al., 2010; Sonnewald et al., 1993; Sonnewald et al., 1991) or from cerebellum (Waagepetersen et al., 2001), also in 15

ACCEPTED MANUSCRIPT rat astrocyte cultures a strong labeling from [U-13C]glucose of the TCA cycle intermediates citrate, fumarate and malate and of the TCA cycle intermediate-derived amino acids aspartate, glutamate and glutamine was observed. Small differences in the percent labeling observed between the different studies are likely to be a consequence of differences in the preparation

After incubation with

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and culturing of the cells, species differences and variations in the experimental incubations.

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C glucose, cultured astrocytes have been shown to release labeled

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lactate (Skytt et al., 2010), alanine (Skytt et al., 2010), citrate (Sonnewald et al., 1991; Waagepetersen et al., 2001; Westergaard et al., 1994) and glutamine (Skytt et al., 2010;

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Waagepetersen et al., 2001). The labeling of extracellular lactate or alanine in rat astrocyte cultures was higher than that in the cellular lactate or alanine pool, consistent with a previous report using mouse astrocytes (Skytt et al., 2010). Also the cellular labeling pattern of citrate is different from that of extracellular citrate, as in cells the M+2 isotopomer is the

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predominant one, whereas in extracellular citrate the M+3 isotopomer is also abundant. These differences in the cellular and extracellular labeling patterns of metabolites are consistent with the concept of a compartmentalized metabolism in astrocytes (Sonnewald et al., 1993;

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Waagepetersen et al., 2001). Differences in the cellular and extracellular labeling pattern of one metabolite can only occur if two pools of one metabolite exist and these two pools are not

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in direct exchange as discussed in detail before (Waagepetersen et al., 2001). Compartmentation of metabolites likely directs metabolites in specific metabolic pathways supporting a particular purpose. The relevance of compartmentalized metabolism of astrocytes for neighboring neurons may depend on the metabolic circumstances and on signals exchanged between astrocytes and neurons. Compartmentation may help astrocytes to direct part of their own metabolism towards a product that could support neurons during a given demanding situation.

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ACCEPTED MANUSCRIPT Exposure to T23 did strongly increase the MCL and the TCA cycling of most TCA cycle intermediates and of the TCA cycle intermediate-derived amino acids. Analysis of the individual isotopomers revealed that especially the higher labeled isotopomers (M+3 and M+4) were found increased after T23 application compared to the direct labeling derived

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from [U-13C]glucose. Higher M+3 labeling compared to M+4 suggests a high contribution of pyruvate carboxylation compared to repeated cycling (Waagepetersen et al., 2001). In addition, also the citrate M+5 is strongly increased, which may be formed by condensation of

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pyruvate carboxylation-derived oxaloacetate with [1,2-13C]acetyl-CoA. However, in rat astrocytes T23 increases both M+3 and M+4 to around the same extent, suggesting that T23

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may strongly increase TCA cycling and pyruvate carboxylation.

Activation of pyruvate dehydrogenase (PDH) by the PDH kinase inhibitor dichloroacetate has been shown to increase the entrance of pyruvate into the TCA cycle by 13C labeled substrates

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in intact hearts (Lewandowski, 1992; Lloyd et al., 2003), liver (Large et al., 1997) and brain (Park et al., 2013) and to decrease the pyruvate carboxylase flux in the liver (Large et al., 1997). As also T23 caused an increase in the label entering the TCA cycle via PDH, a direct

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or indirect activation of PDH may be involved in the observed stimulation of TCA cycling in T23-treated astrocytes, which is consistent with the observed strong increase in the labeling of

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M+4 of citrate, fumarate and aspartate. Thus, the alterations in the isotopomer patterns after T23 exposure are consistent with an increase in PDH dependent catabolism and TCA cycle cycling. Interestingly, the increase in isotopomer labeling in the TCA cycle derived amino acids was more extensive compared to that observed for the respective TCA cycle intermediates. The amino acids are usually higher labeled than the TCA cycle intermediates (Skytt et al., 2010; Waagepetersen et al., 2001) and thus, the amino acid pool may be a sink for the labeling.

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ACCEPTED MANUSCRIPT In addition to an action of T23 on the TCA cycle, T23 may also affect the respiratory chain which in turn would indirectly modulate TCA cycle activity. Some tyrosine kinase inhibitors have been shown to uncouple or to inhibit the respiratory chain depending on the experimental conditions (Soltoff, 2004; Young et al., 1993) or to lower the mitochondrial

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membrane potential (Liang and Ullyatt, 1998). However, preliminary experiments revealed that T23 did not acutely affect the oxygen consumption of mitochondria isolated from cultured astrocytes (data not shown), suggesting that T23 is unlikely to uncouple or inhibit

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mitochondrial respiration in astrocytes. The changes observed in labeling of metabolites are

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only compatible with an uncoupling of the mitochondrial respiration.

The known potential of tyrphostins as inhibitor of PTKs suggests that T23 affects astrocytic metabolism via an inhibition of a PTK. However, the responsible enzyme has so far not been identified for astrocytes. Likely cellular targets of a T23-sensitive PTK are PDH, enzymes of

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the TCA cycle or complexes of the respiratory chain. In the murine hematopoietic Ba/F3 cell line, a group of metabolic enzymes have been identified, including lactate dehydrogenase A, glucose-6-phosphate dehydrogenase and mitochondrial malate dehydrogenase 2, that are

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tyrosine-phosphorylated (Hitosugi et al., 2009), suggesting that reversible phosphorylation by PTK and a phosphatase can modulate the activity of these enzymes and subsequently the

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metabolic flux. A dimeric pyruvate kinase isoform M2 (PKM2) that is less active has been suggested to shuttle glucose derived carbon more towards syntheses of lipids and amino acids than the higher active tetrameric isoform which favours glycolysis and lactate production (Dang, 2009). PKM2 has been shown to be expressed in astrocytes, which carries an inducible nuclear localisation signal allowing the cells to regulate their glycolytic flux according to the overall energy conditions (Zhang et al., 2014). This hints towards a more active TCA cycle, which might be a prerequisit in order to handle the potential increase in lipid and amino acid synthesis. However, induction of protein expression would have required more time and 18

ACCEPTED MANUSCRIPT appears unlikely to cause the dramatic change in astrocytic metabolism observed within 2 h after exposure to T23. It is more likely that the rapid activation of glycolysis and TCA cycling in the presence of T23 is a consequence of a rapid modulation by a T23-sensitive kinase of one or more enzymes, in glycolysis, PDH or the TCA cycle. Currently, we do not have

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experimental evidence which allows to conclude whether the T23-induced acceleration of TCA cycling is a consequence of an accelerated glycolytic flux or whether accelerated TCA

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cycling causes stimulation of glycolytic flux.

Mitochondrial metabolism of brain cells is increasingly discussed as a potential key factor in

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neurodegenerative diseases like Alzheimer’s or Parkinson’s disease (Swerdlow, 2016). As in neurodegenerative diseases, a reduced bioenergetic capacity is observed and it has been suggested that increasing the bioenergetic capacity may be beneficial (Swerdlow, 2016). Thus, the stimulation of both glycolysis and TCA cycle metabolism by T23 in brain cells may

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indeed boost ATP production and subsequently be beneficial for conditions characterized by impaired astrocytic energy metabolism. In addition to the cellular TCA cycle intermediates and amino acids, also the MCL labeling of extracellular lactate, citrate and glutamine was

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strongly increased after T23 treatment. In brain, these metabolites are considered to have important extracellular functions, lactate as energy substrate (Carpenter et al., 2015; Pellerin

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and Magistretti, 2012), citrate to complex cations such as iron and regulate the extracellular homeostasis of Ca2+ and Mg2+ (Grootveld et al., 1989; Gutteridge, 1992; Westergaard et al., 1994) and glutamine as precursor for neuronal glutamate (Bak et al., 2006b). Thus, an improved generation and astrocytic release of lactate, citrate and glutamine after treatment with T23 may also be beneficial for other cells of the brain.

Tyrphostins showed promissing results in cancer therapy (Bojko et al., 2015; Levitzki and Gazit, 1995). The results of the present study may help to understand a component of the 19

ACCEPTED MANUSCRIPT potential of T23 as anti-cancer drug. Cancer cells are known to have a higly glycolytic phenotype (Warburg, 1956; Wolf et al., 2011) which is a potential target for anti-cancer drugs (Tennant et al., 2010). Therefore an additional T23-mediated increase in glycolysis and TCA cycle may render tumor cells more prone to other anti-cancer treatments. However, studies are

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requested to clarify whether T23 has similar stimulating effects on the metabolism of tumor cells as those reported here in cultured astrocytes and how such metabolic changes mediated

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by T23 may affect tumor growth and progression.

In summary, the protein tyrosine kinase inhibitor T23 efficiently stimulates glycolysis and

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TCA cycle in cultured astrocytes, suggesting that these processes are regulated by phosphorylation/dephosphorylation events which involve at least one protein tyrosine kinase. This process involves T23-induced alteration of metabolism which more strongly directs carbon atoms derived from glycolysis towards mitochondrial TCA cycle metabolism. Further

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studies are now required to identify the mechanisms involved in the alteration of astrocytic metabolism by T23 and to test for the potential of T23 to also affect glucose metabolism of others types of brain cells. In addition, it would be interesting to study whether T23 may also

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alter metabolism of astrocytes in vivo.

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Acknowledgement

Dr. Stefan Stolte (UFT, Bremen) kindly provided the Speed-Vac to dry the ethanol extracts. Michaela C. Hohnholt thanks the German “Deutsche Forschungsgemeinschaft” for financial support (HO 5204/2-1).

Conflict of interest The authors have no conflict of interest to declare. References: 20

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ACCEPTED MANUSCRIPT Figure legends Figure 1: Effects of T23 on the glycolytic flux and the viability of cultured astrocytes. The cells were incubated with [U-13C]glucose in the absence (control) or the presence of 100 µM T23 for 2 h. The extracellular LDH activity (A), the extracellular lactate content (B) and the

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glucose consumed (C) were determined. The data shown represent mean ± SD of values obtained from 3 independently prepared cultures. Significance of difference between the values obtained for cells treated in the absence or the presence of T23 are indicated (*p<0.05,

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**p<0.01).

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Figure 2: Effects of T23 on the molecular carbon labeling (MCL) of cellular (A,B) and extracellular (C,D) lactate (A,C) and alanine (B,D). The cells were incubated with [U13

C]glucose in the absence (control) or the presence of 100 µM T23 for 2 h. The MCL (%)

was obtained by analyzing the cell extracts or the incubation media by GCMS for lactate and

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alanine isotopomers. The data shown are mean ± SD of values obtained from 3 independently prepared cultures. Significance of difference between the values obtained for cells treated in

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the absence or the presence of T23 is indicated (***p<0.001).

Figure 3: Effects of T23 on the molecular carbon labeling (MCL) (A-D) and cycling ratios

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(E-H) of the TCA cycle intermediates citrate (A,E), succinate (B,F), fumarate (C,G) and malate (D,H) in cultured astrocytes. The cells were incubated for 2 h with [U-13C]glucose in the absence or the presence of 100 µM T23. To obtain the MCL (%) of the indicated metabolites, the cell extracts were analyzed by GCMS. The data shown are mean ± SD of values obtained from 3 independently prepared cultures. Significance of difference between the values obtained for cells treated in the absence or the presence of T23 is indicated (**p<0.01, ***p<0.001).

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ACCEPTED MANUSCRIPT Figure 4: Effects of T23 on the distribution of 13C-labeled isotopomers of cellular citrate (A), succinate (B), fumarate (C) and malate (D). The cells were incubated for 2 h with [U13

C]glucose in the absence (control) or presence of 100 µM T23. To obtain the distribution of

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C-labeling isotopomers of the indicated metabolites, the cell extracts were analyzed by

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GCMS. The data shown are mean ± SD of values obtained from 3 independently prepared cultures. Significance of difference between the values obtained for cells treated in the

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absence or the presence of T23 is indicated (*p<0.05, **p<0.01 and ***p<0.001).

Figure 5: Effects of T23 on the molecular carbon labeling (MCL) (A-C) and cycling ratios

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(D-F) of the amino acids aspartate (A,D), glutamate (B,E) and glutamine (C,F) in cultured astrocytes. The cells were incubated for 2 h with [U-13C]glucose in the absence (control) or the presence of 100 µM T23. To obtain the MCL (%) of the indicated metabolites, the cell extracts were analyzed by GCMS. The data shown are mean ± SD of values obtained from 3

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independently prepared cultures. Significance of difference between the values obtained for cells treated in the absence or the presence of T23 is indicated (***p<0.001).

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Figure 6: Effects of T23 on the percentage distribution of

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aspartate (A), glutamate (B) and glutamine (C). The cells were incubated for 2 h with [U13

of

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C]glucose in the absence (control) or the presence of 100 µM T23. To obtain the distribution C-labeling isotopomers of the indicated metabolites, the cell extracts were analyzed by

GCMS. The data shown are mean ± SD of values obtained from 3 independently prepared cultures. Significance of difference between the values obtained for cells treated in the absence or the presence of T23 is indicated (**p<0.01 and ***p<0.001).

Figure 7: Effects of T23 on the MCL (A,C) and distribution of 13C-labeled isotopomers (B,D) of extracellular citrate (A,B) and extracellular glutamine (C,D). The cells were incubated for 2 26

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cells treated in the absence or the presence of T23 is indicated (***p<0.001).

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