Ewa Augustin
email: augustin@chem.pg.gda.pl

Anna Moś-Rompa

Dorota Nowak-Ziatyk
email: dorota-nowak@o2.pl

Jerzy Konopa
email: konopa@chem.pg.gda.pl

Antitumor 1-nitroacridine derivative C-1748, induces apoptosis, necrosis or senescence in human colon carcinoma HCT8 and HT29 cells

Keywords: C-1748, apoptosis, necrosis, senescence, reactive oxygen species (ROS)

C-1748 is a DNA binding agent with potent antitumor activity, especially towards prostate and colon carcinoma xenografts in mice. Here, we elucidated the nature of cellular response of human colon carcinoma HCT8 and HT29 cells to C-1748 treatment, at biological relevant concentrations (EC 90 and their multiplicity). Cell cycle analysis showed gradual increase of HCT8 cells with sub-G1 DNA content (25% after 72 h) considered as apoptotic. Hypodiploid cell population increased up to 60% upon treatment with 4xEC 90 concentration of the drug. Compared with HCT8 cells, the fraction of sub-G1 HT29 cells did not exceed 14%, even following 4-fold dose escalation. Morphological changes and biochemical markers such as: phosphatydylserine externalization, apoptotic DNA breaks, mitochondrial dysfunction, caspase activation, confirmed the presence of considerable amount of apoptotic HCT8 cells but only a low amount of apoptotic HT29 cells. Next, we demonstrated that HCT8 cells surviving after exposure to C-1748 were in state of senescence, based on altered cell morphology and expression of a pH 6-dependent galactosidase. On the contrary, no -galactosidase staining was observed in HT29 cells after C-1748 treatment. Moreover, prolonged drug incubation (up to 168 h) resulted in massive detachment of cells from culture plates, what together with Annexin V/PI results, indicated that necrosis was the main response of HT29 cells to C-1748 treatment. We also determined the ability of C-1748 to induce reactive oxygen species (ROS) in colon cancer cells and demonstrated, that generation of ROS was not essential for C-1748-induced apoptosis and cytotoxic activity of this drug.

The 1-nitroacridines are DNA-binding and potent antitumor agents synthesized at Gdańsk University of Technology [START_REF] Ledochowski | Ledakrinanticancerous medicine 1-nitro-9(3dimethylamino)-acridine x 2HCl x H 2 O[END_REF]. Among them, the most active compound Ledakrin (Nitracrine) was in 1970's registered as an antitumor drug in Poland [START_REF] Gniazdowski | Nitracrine and its congenersan overview[END_REF].

Ledakrin was recommended especially for treatment of ovary carcinoma, but also effectively used against colon, breast and lung cancers [START_REF] Bratkowska -Seniów | Morphologic blood pathern in patients treated with Ledakrin[END_REF][START_REF] Kwaśniewska -Rokocińska | Preliminary observations on the Ledakrin effects in treatment of patients with lung carcinoma[END_REF]. However, due to severe side effects and high general toxicity, Ledakrin was desisted from further clinical application. Modification of chemical properties of 1-nitroacridines, especially decreasing the ability of nitro group in position 1 to undergo reduction, by introducing to position 4, para to the nitro group, en electron donating-methyl group, resulted in development of 4-methyl-1-nitroacridines with high antitumor activity towards broader spectrum of tumors and lower toxicity than parental 1-nitroacridines [START_REF] Chen | Preclinical studies of 1-nitroacridine derivatives: Effective anti-tumor agents for prostate cancer[END_REF][START_REF] Tadi | Pre-clinical evaluation of 1-nitroacridine derived chemotherapeutic agent that has preferential cytotoxic activity towards prostate cancer[END_REF]. The most active 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748, Fig. 1), was found to have significant cytotoxic activity in vitro towards cancer cell lines and high antitumor activity against several prostate (LnCaP, JCA, PC3, TSU) and colon carcinoma (HCT8) xenografts in nude mice [START_REF] Tadi | Pre-clinical evaluation of 1-nitroacridine derived chemotherapeutic agent that has preferential cytotoxic activity towards prostate cancer[END_REF]. Pre-clinical toxicology studies in rodent and dogs showed C-1748 to have very low systemic toxicity along with a lowered mutagenic potential [START_REF] Ashok | Preclinical toxicological evaluation of a putative prostate cancer specific 1nitroacridine derivative in rodent[END_REF][START_REF] Ashok | Preclinical toxicology and pathology of 9-(2'-hydroxyethylamino)-4-methyl-1nitroacridine (C-1748), a novel anti-cancer agent in male Beagle dogs[END_REF][START_REF] Narayanan | Comparative analysis of mutagenic potency of 1-nitroacridine derivatives[END_REF]. C-1748, as well as Ledakrin and other 1nitroacridines, after metabolic activation bind covalently to DNA [START_REF] Konopa | Potent antitumor 4substituted-1-nitroacridines DNA crosslinking agents[END_REF] and induced DNA crosslinking in tumor cells, which was crucial for biological activity of these compounds [START_REF] Konopa | The mode of action of cytotoxic and antitumor 1-nitroacridines. III. In vivo interstrand crosslinking of DNA of mammalian or bacterial cells by 1-nitroacridines[END_REF][START_REF] Pawlak | Cytotoxic and antitumor activity of 1nitroacridines as an aftereffect of their interstrand DNA cross-linking[END_REF][START_REF] Szostek | Interstrand crosslinking of DNA in tumor cells by a new group od antitumor 1-nitroacridines[END_REF].

Recently, it has been shown that C-1748 significantly induced DNA double strand breaks in prostate cancer LnCaP cells and, to smaller extend, in leukemia HL60 cells, less sensitive to the action of this compound [START_REF] Tadi | Pre-clinical evaluation of 1-nitroacridine derived chemotherapeutic agent that has preferential cytotoxic activity towards prostate cancer[END_REF]. Moreover, in LnCaP cells, C-1748 treatment markedly downregulated androgen receptor but upregulated The molecular mechanisms by which chemotherapeutic agents kill tumor cells are numerous and depend on a variety of parameters including the drug and tumor types. It is widely accepted that tumor cells exposed to cytotoxic drugs die mainly from apoptosis. Moreover, in many types of tumor cells accelerated or premature senescence, a form of growth arrest after exposure to ionizing radiation or drugs that induce DNA damage, has been observed [START_REF] Schmitt | Cellular senescence and cancer treatment[END_REF][START_REF] Gewirtz | Accelarated senescence: An emerging role in tumor cell response to chemotherapy and radiation[END_REF]. It is still not clear what determines if a cell undergoes senescence or apoptosis. Although most cells are capable of both, these processes seem to be exclusive [START_REF] Campisi | Cellular senescence: when bad things happen to good cells[END_REF]. Moreover, in many types of cells the influence of p53 function can promote senescence arrest and apoptotic cell death [START_REF] Hattangadi | Influence of p53 and caspase 3 activity on cell death and senescence in response to methotrexate in the breast tumor cell[END_REF]. However, there is also evidence for accelerated senescence that is independent of p53 [START_REF] Roberson | Escape from therapy-induced accelerated cellular senescence in p53-null lung cancer cells in human lung cancers[END_REF].

The current studies were designed to elucidate the nature of the response of human colon cancer HCT8 and HT29 cells to C-1748 in terms of growth arrest and/ or cell death. Additionally, we determined the ability of C-1748 to induce reactive oxygen species (ROS) production, and investigated the correlation between ROS generation and cell death and cytotoxic activity of the drug. In a view of recent finding that a form of senescence arrest may occur in response to antitumor drugs, we also examined the ability of C-1748 to induce senescence. Cell growth inhibition was assessed by cell counting using Coulter Counter, model ZB1 according to the method describe by Bhuyan et al. [START_REF] Bhuyan | Lethality, DNA alkylation and cell cycle effects of adozelesin (U-73975) on rodent and human cells[END_REF]. Briefly, cells (1x10 5 ) were seeded in 24-well microculture plates and treated with various C-1748 concentrations for 72 h. A dose response curve was plotted and used to calculate drug concentration that yielded 90% of inhibition of cells growth (EC 90 ).

Cell culture and growth inhibition assay

Cell cycle distribution by flow cytometry

Asynchronous HCT8 and HT29 cells were exposed to C-1748 at concentrations corresponding to EC 90 values and their multiplicity for 3 -72 h. After drug treatment, adherent cells were trypsinized, combined with nonadherent cells, collected by centrifugation for 5 min at 4 o C, at 1,000 rpm, washed twice with ice-cold phosphate-buffered saline (PBS) and then fixed in ice-cold 80% ethanol overnight at -20˚C. Next, cells were centrifuged for 5 min at 4˚C, at 1,000 rpm, washed twice with PBS and resuspended in 1ml of DNA staining solution (20 μg/ml of propidium iodide PI and 100 μg/ml of RNase A in PBS), for 30 min in the dark. Flow cytometric analyses were performed using a FACScan cytometer (Becton Dickinson, San Jose, CA, USA). At least 1x10 4 cells were collected and the data analysis was performed using the WinMDI software (J. Trotter, Scripps Research Institute, San Diego, CA, USA). All experiments were repeated at least three times.

Cell morphology assessment

Nuclear morphology was examined under fluorescence microscope (OLYMPUS BX60) after staining with 4',6-diaminidino-2-phenylindole (DAPI).

TUNEL assay

Apoptotic DNA degradation following by C-1748 treatment was studied by the TUNEL assay. The commercially accessible Apoptosis Detection kit (Roche Diagnostics, Manheim, Germany) was used and prepared according to the supplier's recommended protocol. Briefly, following treatment, cells (1x10 6 ) were centrifuged, washed twice in PBS and gently resuspended in 1% formaldehyde in PBS and kept on ice for 15 min. Fixed cells were washed with PBS and resuspended in 70% ethanol. Dehydrated cells were incubated overnight at -20˚C. After rehydration in PBS for 5 min cells were resuspended in 50 μl of reaction buffer (5U terminal transferase, 2.5 mM cobalt chloride, 0.5 mM biotin-dUTP and TdT buffer containing 0.2 M potassium cocadylate, 25 mM TRIS-HCl, 0.25 mg/ml BSA, pH 6.6) and incubated at 37˚C for 30 min. Then, cells were washed with rinsing buffer (PBS containing 0.1% Triton X-100, 0.5% BSA), resuspended in 100 μl of staining buffer containing 2.5 μg/ml FITC-avidin, 4xSSC buffer, 0.1% Triton X-100 and 5% (w/v) non-fat dry milk, and incubated for 30 min at room temperature in the dark. The pellets were then washed twice with rinsing buffer, resuspended in PBS containing 5 μg/ml PI and 100 μg/ml RNase A, and incubated at room temperature for 30 min. The red (PI) and green (FITC) fluorescence were measured with FACScan flow cytometer.

A c c e p t e d M a n u s c r i p t 9 2.6. Phosphatidylserine externalization Annexin V binding assay was performed using Annexin-V-Fluos Staining kit (Roche Diagnostics, Manheim, Germany) according to the manufacturer's specification. Briefly, after drug incubation, cells (1x10 6 ) were trypsinized, rinsed with HEPES/NaOH buffer (10 mM Hepes pH 7.4, 140 mM NaCl, 5 mM CaCl 2 ), pelleted and resuspended in 50 μl of Annexin V-FITC diluted in binding buffer containing PI.

Cells were incubated for 15 min at room temperature in the dark and diluted with 400 l of HEPES/NaOH buffer and analyzed by flow cytometry within 1 h. Bivariate flow cytometry and data analysis were performed as described for TUNEL assay.

Measurement of mitochondrial transmembrane potential (ΔΨm)

Changes in mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry using JC-1 dye (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide, Molecular Probes, Eugene, OR, USA). After treatment, cells (5x10 5 ) were trypsinized and washed twice with PBS and incubated with medium containing JC-1 (10 μg/ml) for 15 min at room temperature in the dark. Finally, cells were washed and resuspended in 1 ml of PBS for flow cytometry analysis. The loss of ΔΨm was monitored based on a decrease in JC-1 red fluorescence concurrent with an increase in green fluorescence.

Caspase-3 activity measurements

Caspase-3 activation was determined using the Active Caspase-3 Apoptosis kit (BD Pharmingen, San Diego, CA, USA) according to the manufacturer's instruction. Cells (1x10 6 ) stained with FITC-conjugated anti-active caspase-3 antibody, were analyzed by flow cytometry.

A c c e p t e d M a n u s c r i p t 10 2.9. Determination of cellular senescence pH 6.0-dependent β-galactosidase expression was used as a marker for senescence along with senescent related morphology. Following C-1748-treatment, cells (1x10 5 ) were washed twice with PBS and fixed in 2% formaldehyde and 0.2% glutaraldehyde in PBS for 5 min. The cells were then washed again with PBS and stained with X-gal ( 5-bromo-4-chloro-3-indolyl-β-D-galactosidase) staining solution (1 mg/ml X-gal, 40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl 2 ). Following overnight incubation at 37 ○ C, the cells were washed twice with PBS, and photographed under a light microscope.

Western blot analysis

Western blot analysis of whole cell lysates was performed as described previously [START_REF] Augustin | Induction of G2/M phase agrest and apoptosis of human leukemia Wells by potent antitumor triazoloacridinone C-1305[END_REF]. Membrane was probed with primary mouse monoclonal anti-p53 antibody (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:1000 and anti-actin (Sigma-Aldrich, St. Louis, MO, USA) diluted in 1:1000 and then incubated with horseradish peroxidase-conjugated secondary antibody (anti-mouse 1:2000 for p53, anti-mouse for actin 1:10 000) and visualized by enhanced chemiluminescence kit (Pierce Biotechnology, Rockford, IL, USA). fluorescence was examined by flow cytometry on a FACScan cytometer. Samples incubated with hydrogen peroxide (0.01%) were used as a positive control. All experiments were repeated at least three times.

Measurement of ROS generation

Statistical analysis

Results are expressed as means  S.D. A paired t-test was used to compare two groups, and one-way ANOVA and Dunnett's t-test was used for multiple comparisons. The criterion for statistical significance was set at p  0.05. The effect of the 1-nitroacridine derivative C-1748 on cell cycle progression of colon cancer cells, was determined by flow cytometry, following treatment with the drug (EC 90 concentration) for various incubation times (3-72 h). Flow cytometry analyses showed that starting from 3 h of C-1748 treatment, HCT8 cells underwent a transient arrest in G1 and G2/M phases of the cell cycle, however the number of cells in these phases was gradually decreased during prolonged drug incubation (Fig. 2 A). These cell cycle changes were accompanied by a time-dependent appearance of typical for late stage of apoptosis, sub-G1 peak (25% after 72 h of treatment), as well as by the appearance of polyploid cell population (about 30% after 72 h of drug treatment, Tab. 1). The incubation of HCT8 cells with higher drug concentration (4xEC 90 ), significantly increased the percentage of sub-G1 cells (up to 60% after 72 h, Fig. 2 C, Tab. 2). Treatment of HT29 cells with C-1748 at EC 90 concentration resulted in less evident perturbations of the cell cycle. Starting from 3 h of drug treatment, a slight increase in G1 phase was observed. Accumulation of cells at G2/M compartment was detectable after 24 h of drug treatment and following longer incubation, the number of cells in this phase significantly decreased (Fig. 2 B).

RESULTS

Effect of C-1748 on HCT8 and HT29 cell growth and cell cycle distribution

Apoptotic cell morphology

Flow cytometric analysis of cells in sub-G1 region indicated that colon cancer cells treated with C-1748 underwent apoptosis. To confirm these data, morphological changes of cells treated with C-1748 were assessed using fluorescence microscopy following DAPI staining. As shown in Fig. 3 A and B, untreated cells exhibited unchanged morphology, characteristic for colon cancer cells. After 12 h of C-1748 treatment at EC 90 concentration, only a few apoptotic HCT8 cells were visible.

Prolonged drug incubation (48 and 72 h), as well as two-fold increased drug concentration, resulted in appearance of numerous cells exhibiting morphological changes typical for apoptosis, with cell shrinkage, chromatin condensation and fragmentation into discrete bodies. By contrast, only a few apoptotic HT29 cells were observed following C-1748 treatment at EC 90 concentration and its multiplicity. At 72 h of C-1748 exposure some of HT29 cells were shrunken and formed the structure of apoptotic bodies.

DNA degradation

TUNEL assay was used to investigate whether C-1748 induces apoptotic DNA fragmentation in human colon carcinoma cells. This method, based on the labeling

Annexin V/PI dual staining

To further confirm that C-1748 induces apoptosis, HCT8 and HT29 cells treated with the drug at EC 90 concentrations were stained with Annexin V-FITC and propidium iodide, and analyzed by flow cytometry. This assay is based on the translocation of phosphatidylserine (PS) from the inner leaflet of the plasma membrane to the cell surface in the early stage of apoptosis. Positive staining with fluorescently labelled Annexin V correlates with loss of plasma membrane polarity but precedes the complete loss of membrane integrity that accompanies later stages of cell death resulting from either apoptosis and necrosis. In contrast, PI can only enter cells after loss of their membrane integrity. Thus, dual staining with Annexin V and PI allows clear to discriminate between unaffected cells (Annexin V-/PI-), early apoptotic cells (Annexin V+/PI-), late apoptotic cells (Annexin V+/PI+) and primary necrotic cells (Annexin V-/PI+). As indicated in the Fig. 5 A Populations of early and late apoptotic HT29 cells reached only about 10% after 72 h of C-1748 exposure. Interestingly, starting from 48 h, the population of necrotic cells (Annexin V-/PI+) significantly increased and at 72 h reached  30%. Prolonged incubation with the drug (up to 144 h) resulted in intensive Annexin V-/PI+ (38%) as well as Annexin V+/PI+ (32%) staining without preceeding Annexin V+/PI-staining, and was accompanied by massive detachment of cells from culture plates (data not shown).

Changes in mitochondrial transmembrane potential

Mitochondrial transmembrane potential  m , an electrical potential required for several mitochondrial functions including ATP generation, is dramatically reduced early during apoptosis. The disruption of the  m generally defines not only an early, but already irreversible stage of apoptosis [START_REF] Ly | The mitochondrial membrane potential (deltapsi(m)) in apoptosis[END_REF]. The loss of  m is routinely used as an indicator of mitochondria damage during apoptosis. Therefore, next we examined the effect of C-1748 on  m using mitochondria specific membrane potential probe JC-1. As compared with untreated cells, a time-dependent increase in the proportion of HCT8 and HT29 cells with a reduced  m was noted following C-1748 treatment at EC 90 concentration (Fig. 6 A). Starting from 6 h, the population of HCT8 cells with low  m steadily increased, and reached approximately 53% after

Caspase-3 activity

It is well established that in most cases, apoptosis requires the participation of endogenous cellular enzymescaspases. Among the identified caspases, activation of caspase-3 is a crucial in numerous types of cells leading to the execution of apoptosis [START_REF] Janicke | Emerging role of caspase-3 in apoptosis[END_REF][START_REF] Yang | Caspase-3 status is a determinant of differential responses to genistein between MD-MB-231 and MCF-7 breast cancer cells[END_REF]. Therefore, we examined, whether caspase-3 was involved in apoptosis induced by C-1748 in colon cancer cells. After 3, 6 and 12 h of treatment with C-1748 at EC 90 concentration, we observed only a small amount of cells with active caspase-3 in both colon cancer cell lines (Fig. 6 B). However, in the case of HCT8 cells, 24 h of C-1748 treatment resulted in significant increase in number of cells with active caspase-3 (21%), and after 48 and 72 h of incubation, the percentage of cells with active caspase-3 reached about 40% and 60%, respectively.

Compared to HCT8 cells, the percentage of HT29 cells expressing active caspase-3 after C-1748 treatment, was markedly lower (17% after 72 h).

Induction of cellular senescence

DNA damage by a variety of compounds has been shown to induce cellular senescence in various cancer cells. Senescent cells are generally characterized by a reduction in proliferating capacity, adoption of a flattened and enlarged cell shape, and the appearance of senescence-associated -galactosidase (SA--gal) activity [START_REF] Dimri | A biomarker that identifies senescent human cells in culture and aging skin in vivo[END_REF]. Because C-1748 induced apoptosis only in part of HCT8 and HT29 cells, we investigated whether these cells underwent senescence following C-1748 treatment.

A c c e p t e d M a n u s c r i p t

In HCT8 cells increased -galactosidase staining relative to control was observed 72 h after treatment with C-1748. The number of SA--gal-positive cells continued to increase and after 168 h of C-1748 exposure, the whole population of HCT8 cells presented -galactosidase staining. Together with increased -galactosidase staining, all HCT8 cells had an enlarged and flattened morphology typical for senescent cells (Fig. 7 A). In contrast, we did not observe any -galactosidase staining, as well as senescent morphology, in HT29 cells after C-1748 treatment (Fig.

B).

Several recent studies have shown that drug-induced senescence depends on p53 status [START_REF] Hattangadi | Influence of p53 and caspase 3 activity on cell death and senescence in response to methotrexate in the breast tumor cell[END_REF]. However, there are also evidences, that senescence has been observed in cells both with and without functional p53 [START_REF] Roberson | Escape from therapy-induced accelerated cellular senescence in p53-null lung cancer cells in human lung cancers[END_REF]. Therefore we conducted experiments to examine the influence of p53 protein on the senescence process induced by C-1748 in HCT8 cells. Western blot analysis revealed that the total p53 level gradually decreased following C-1748 treatment (Fig. 7 C). Simultaneously, the number of SA--gal-positive cells increased after prolonged drug incubation up to 168 h (Fig. 7A). The obtained results suggested that senescence process induced by C-1748 in HCT8 cells was independent on p53 status.

Generation of ROS by C-1748 and effects of antioxidants on cell viability and C-1748-induced cell death

A numerous of evidence has accumulated indicating involvement of oxidative stress in the mode of action of various bioactive substances, including anti-cancer agents [START_REF] Kovacic | Mechanisms of anti-cancer agents: emphasis on oxidative stress and electron transfer[END_REF]. Reactive oxygen species (ROS) are frequently generated by redox cycling via electron transfer groups, including nitro group in aromatic compounds like C-1748. Stimulation of formation of ROS can lead to oxidative stress, involved in the induction of necrotic or apoptotic cell death. To determine whether this event occurs in C-1748-induced apoptosis, we examined the generation of ROS by flow cytometric analysis following staining with 2', 7'dichlorofluorescin diacetate (DCFH-DA), which is converted to highly fluorescent 2', 7'dichlorofluorescin (DCF) with the presence of intracellular ROS. HCT8 and HT29 cells were incubated with C-1748 at EC 90 concentration for 0, 1, 2, 3, 4, 5 and 24 h prior to collection, and analyzed by flow cytometry for DCF fluorescence. Cells with a high level of DCF fluorescence, correlated with ROS generation, were shifted right on the x axis. As shown in Fig. 8 A, an approximately 2-fold increase in generation of DCF-sensitive ROS in HT29 cells was evident as early as 1 h after C-1748 treatment and then showed a steady level up to 24 h. In HCT8 cells, a small increase in ROS generation was detected after 1 h of C-1748 exposure and reached its maximum levels within 4 h. For comparison, in positive controls, in which HCT8 or HT29 cells were treated with 0.01% H 2 O 2 for 30 min prior to analysis, an approximately 2-fold increase in DCF fluorescence intensity was observed (data not shown). To further elucidate whether the ability to generate of ROS is essential for C-1748-induced apoptosis, we investigated the effects of antioxidants on C-1748-induced cell death as well as their influence on cell viability following C-1748 exposure. The colon cancer cells were pretreated with increasing concentrations of N-acetyl-L-cysteine (NAC) or melatonin for 1 h and then were incubated for further 72 h with the drug (HCT80.04 M; HT290.21 M). After treatment, cell viability and the phosphatidylserine (PS) externalization as a marker of apoptotic cells were determined. As shown in Fig. 8 B and 8 C, two studied antioxidants: NAC and melatonin had no effect on cell viability. Moreover, NAC did not protect HCT8 cells against C-1748-induced apoptosis because the level of Annexin V positive/PI negative cells did not change following A c c e p t e d M a n u s c r i p t pretreatment with antioxidant (Fig. 8 D with reference to results presented on Fig. 5 A).

DISCUSSION

The aim of the current study was to investigate and characterize the cellular response of human colon cancer HCT8 and HT29 cells to C-1748 treatment.

It was previously shown that C-1748 binds to DNA non-covalently (by intercalation) and covalently, and after metabolic activation forms interstrand

A c c e p t e d M a n u s c r i p t 20 crosslinks in cellular DNA [START_REF] Szostek | Interstrand crosslinking of DNA in tumor cells by a new group od antitumor 1-nitroacridines[END_REF]. Covalent binding and ability to form interstrand DNA crosslinks plays a crucial role for the biological activity of this drug [START_REF] Konopa | The mode of action of cytotoxic and antitumor 1-nitroacridines. III. In vivo interstrand crosslinking of DNA of mammalian or bacterial cells by 1-nitroacridines[END_REF][START_REF] Pawlak | Cytotoxic and antitumor activity of 1nitroacridines as an aftereffect of their interstrand DNA cross-linking[END_REF][START_REF] Szostek | Interstrand crosslinking of DNA in tumor cells by a new group od antitumor 1-nitroacridines[END_REF]. It is worth to point out that on the molecular level, C-1748 produced rather the same mechanism of action (interstrand DNA crosslinking) [START_REF] Konopa | The mode of action of cytotoxic and antitumor 1-nitroacridines. III. In vivo interstrand crosslinking of DNA of mammalian or bacterial cells by 1-nitroacridines[END_REF][START_REF] Pawlak | Cytotoxic and antitumor activity of 1nitroacridines as an aftereffect of their interstrand DNA cross-linking[END_REF][START_REF] Szostek | Interstrand crosslinking of DNA in tumor cells by a new group od antitumor 1-nitroacridines[END_REF]. The question arises whether such common mechanism of action will trigger the same cellular response in different cancer cell lines.

The inhibition of tumor cells proliferation seems to be the first effect of action of anticancer drugs, including C-1748. The other cellular effects, such apoptosis, necrosis or senescence occur with some delay in relation to inhibition of cell proliferation. Therefore, it was very important to performed all experiments at biologically relevant concentration of the drug. From the therapeutics point of view the most advantageous would be the concentration of the drug corresponding to its EC 100 value. Because this concentration is impossible to estimate, in our studies we applied concentration of the drug corresponding to its EC 90 values in all our experiments (concentration which caused 90% of inhibition of cells proliferation).

Two colon cancer cell lines used in this work were selected due to their high sensitivity to C-1748. In addition, C-1748 exhibited potent antitumor activity against HCT8 xenografts in mice [START_REF] Tadi | Pre-clinical evaluation of 1-nitroacridine derived chemotherapeutic agent that has preferential cytotoxic activity towards prostate cancer[END_REF].

Cell cycle analysis showed time and dose-dependent appearance of sub-G1 population of HCT8 cells, considered as apoptotic. In contrary to HCT8 cells, the fraction of sub-G1 population of HT29 cells following C-1748 treatment was considerable lower and did not exceed 14%, even following 4-fold increase of drug concentration (Fig. 2 C), which suggested that apoptotic response of HT29 cells to C-1748 treatment was somewhat weaker.

A c c e p t e d M a n u s c r i p t 21

Apoptosis is characterized by several biochemical criteria, including different kinetics of phosphatydilserine (PS) exposure on the leaflet of the plasma membrane, changes in mitochondrial membrane permeability, caspase activation, internucleosomal DNA cleavage, release of intermembrane space mitochondrial proteins [START_REF] Krysko | Apoptosis and necrosis: detection, discrimination and phagocytosis[END_REF] as well as by a series of morphological changes such as formation of apoptotic bodies, nuclear and cytoplasmic condensation, chromatin fragmentation, shrinkage of cells and bleb formation [START_REF] Rello | Morphological criteria to distinguish cell death induced by apoptotic and necrotic treatments[END_REF]. However, necrosis is characterized mostly in negative terms by absence of caspase activation, cytochrome c release, DNA oligonucleosomal fragmentation, presence of plasma membrane permeabilization [START_REF] Galluzzi | Cell death modalities: classification and pathophysiological implications[END_REF]. Therefore, identification of characteristic morphological and biochemicals markers of apoptosis makes it possible to distinguish apoptosis from other forms of cell death.

In our study the induction of apoptosis by C-1748 in human colon cancer cells was confirmed by several biochemical markers, such as: DNA degradation (TUNEL assay), loss of mitochondrial membrane potential  m , caspase-3 activity and phosphatidylserine externalization, as well as by morphological examination of the cells. Cellular morphology of both tested human colon cancer cells progressively changed with increasing duration of C-1748 exposure. Prolonged drug incubation resulted in the appearance of disintegrated cells, as evidenced by apoptotic bodies, and cells with condensed nuclear chromatin (Fig. 3). These morphological observations together with the appearance of sub-G1 populations confirmed the presence of considerable amount of apoptotic HCT8 cells and only small amount of HT29 cells, following treatment with C-1748.

To corroborate cell morphology-based observations, the apoptotic process induced by C-1748 was additionally monitored by TUNEL assay. Exposure of colon 35% in HCT8 cells and was smaller (12%) in HT29 cells (Fig. 4). These data correlated with those obtained from cell cycle analysis, where 25% of the HCT8 and 14% of the HT29 all population represented cells with sub-G1 DNA content.

Moreover, cells with apoptotic DNA fragmentation were derived from each phase of the cell cycle which was consistent with the lack of cell cycle arrest in the specific phase.

Most apoptotic pathways converge on the mitochondria, inducing the disruption of the mitochondrial transmembrane potential  m . The loss of mitochondrial membrane potential  m is early and already irreversible stage of apoptosis [START_REF] Ly | The mitochondrial membrane potential (deltapsi(m)) in apoptosis[END_REF]. Moreover, mitochondrial dysfunction during apoptosis is very often associated with the activation of caspases [START_REF] Slee | Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -8, and -10 in a caspase-9-dependent manner[END_REF]. Exposure to C-1748 resulted in timedependent decrease of mitochondrial membrane potential  m in HCT8 and HT29 cells (Fig. 6 A). However, the significant increase in the number of cells with active caspase-3 was observed in HCT8 cells. After 72 h of incubation with the drug, 60% of HCT8 cells had active caspase-3, but in the case of HT29 cells caspase-3 was activated only in 10% of cell population (Fig. 6 necrosis [START_REF] Kim | Mitochondrial permeability transition: a common pathway to necrosis and apoptosis[END_REF].

The mode of cell death induced by C-1748 was further confirmed by Annexin V/PI dual staining. In HCT8 cells the percentage of Annexin V+/PI+ cells was increased by 48% at 72 h of C-1748 treatment, whereas in HT29 cells, the fraction of early and late apoptotic cells reached only 10% after 72 h of drug treatment (Fig. 5 A and B). Simultaneously, 72 h incubation resulted in dramatic increase of necrotic (Annexin V-/PI+) HT29 cells (30%). This effect was further augmented upon 144 h of drug exposure. Compared with HT29 cells, only small population of necrotic cells (9% after 72 h) was observed in HCT8 cells treated with C-1748. Collectively, these findings confirmed that apoptosis plays a major role in death of HCT8 cells in response to C-1748, whereas HT29 cells die mainly be necrosis.

Next, we investigated whether C-1748-treated cells, which did not die by apoptosis or necrosis, might undergo accelerated senescence. We demonstrated that the HCT8 cells, surviving after exposure to C-1748, were in state of senescence, based on altered cell morphology and increased -galactosidase staining relative to control (Fig. 7 A). In contrast to HCT8 cells, no -galactosidase staining could be observed in HT29 cells after C-1748 treatment (Fig. 7 B). Moreover, the prolonged C-1748 and other 1-nitroacridines require metabolic activation before they exhibit any cytotoxic and antitumor activity [START_REF] Szostek | Interstrand crosslinking of DNA in tumor cells by a new group od antitumor 1-nitroacridines[END_REF][START_REF] Gorlewska | Products of metabolic activation of the antitumor drug Ledakrin (Nitracrine) in vitro[END_REF]. It was shown that metabolic transformations of these compounds occurred in several steps including the reduction of nitro group. One electron reduction of nitro group is usually the first step of 1-nitroacridine's metabolism [START_REF] Gorlewska | Products of metabolic activation of the antitumor drug Ledakrin (Nitracrine) in vitro[END_REF]. Nitroradical anion formed in this step is able to transform the molecular oxygen into ROS. Therefore, we determined the ability of C-1748 to induce ROS and we established the relevance of this effect for the induction of apoptosis and cytotoxic activity of this drug. In our study 2-fold increase in generation of DCF-sensitive ROS in HT29 cells was observed after 1 h of C-1748 treatment, whereas in HCT8 cells maximum level of ROS generation was reached after 4 h of incubation with the drug (Fig. 8 A). Moreover, two antioxidants: N-acety-Lcysteine (NAC) and melatonin, that neutralize reactive oxygen species, had no effect on cell death induced by C-1748, as well as on cell viability. Taken together, these results strongly suggest that oxidative stress may not be a casual factor in the antitumor agents, such as adriamycin [START_REF] Di | Apoptosis, autophagy, accelerated senescence and reactive oxygen in response of human breast tumor cells to Adriamycin[END_REF]. Therefore, the divergent cellular response triggered by the same chemotherapeutic agent should be especially considered in cancer therapy of heterogenous population of tumor cells in patients, e.g. in designing of multidrug therapy.

ACKNOWLEDGMENTS

This work was support by State Committee for Scientific Research (KBN), Poland, grant no. 3PO5A11824. The authors thank Dr. Anna Skwarska from our Department for critical reading of the article and for valuable comments. We also thank Prof.

A c c e p t e d M a n u s c r i p t 5 estrogen

 5 receptor what was a further validation of the cytotoxicity of C-1748 in this cells[START_REF] Tadi | Pre-clinical evaluation of 1-nitroacridine derived chemotherapeutic agent that has preferential cytotoxic activity towards prostate cancer[END_REF].

1 .

 1 Chemicals and antibodies 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine C-1748 was synthesized as monochloride by Dr Barbara Wysocka-Skrzela in the Department of Pharmaceutical Technology and Biochemistry at Gdańsk University of Technology, Poland. Stock solution of the drug in 50% ethanol was freshly prepared just before use. Proteinase K and N-acetyl-L-cysteine (NAC) were purchased from Merck (Darmstadt, Germany). RNase A, DAPI, PI, SA -gal, melatonin, mouse monoclonal anti-p53 antibody, mouse monoclonal anti--actin antibody were obtained from Sigma-Aldrich (St. Louis, MO, USA). JC-1 was purchased from Molecular Probes (Eugene, OR, USA). The following chemicals were obtained form Roche Diagnostics (Manheim, Germany): Annexin V-Fluos Staining Kit, terminal transferase, cobalt chloride, biotin-dUTP and TdT buffer. FITC-conjugated Monoclonal Active Caspase-3 Antibody Apoptosis Kit was purchased from BD Pharmingen (San Diego, CA, USA).

  HCT8 human ileocecal adenocarcinoma cell line was kindly provided by R.K. Tiwari from Department of Microbiology and Immunology, New York Medical College, Valhalla, USA and HT29 human adenocarcinoma colon cell line was obtained from D. Banerjee from Department of Medicine and Pharmacology, Cancer Institute of New Jersey, New Jersey, USA. HCT8 and HT29 cells were grown in RPMI 1640 medium (GIBCO BRL Life Technologies, Paisley, United Kingdom) and McCoy's 5A medium (Sigma-Aldrich, St Louis, MO, USA) respectively, supplemented with 10% heat-inactivated fetal bovine serum and antibiotics (100 g/ml streptomycin, 100 U/ml penicillin), in a 5% CO 2 atmosphere at 37 o C. Under these growth conditions the cell-A c c e p t e d M a n u s c r i p t 7 doubling time was 24 h for both cell lines. All experiments were performed with cells in the exponential phase of growth.

A c c e p t e d M a n u s c r i p t 8

 8 Briefly, after drug incubation, cells (5x10 5 ) were spun onto microscopic slides, fixed in methanol : acetic acid solution (3:1) for 15 min and stained with DAPI (1 g/ml) for 5 min. Cells were regarded as apoptotic based on the presence of condensed, fragmented chromatin.

Intracellular

  ROS generation was monitored by flow cytometry, using fluorescent product of DCFH-DA oxidation, 2', 7'dichlorofluorescin, DCF (SIGMA, St Louis, MO, USA). HCT8 and HT29 cells were treated with C-1748 at concentration corresponding to EC 90 value for the indicated times. Cells (1x10 6 ) were then harvested by tripsinization, washed twice with PBS, and loaded with 20 M DCFH-A c c e p t e d M a n u s c r i p t 11 DA. Cells were incubated at 37C for 30 min, washed twice in PBS, and green (DCF)

A

  c c e p t e d M a n u s c r i p t The growth of the human colon cancer HCT8 and HT29 cells in the presence of various concentrations of C-1748, ranging from 0.001 to 10 M, was determined by cell counting using Coulter Counter. After a continuous 72 h exposure, C-1748 significantly inhibited growth of colon cancer cells in a concentration-dependent manner (data not shown). The concentration of the drug required to inhibit cell growth by 90% (EC 90 ) demonstrated that HCT8 cells were more sensitive to C-1748 treatment than HT29 cells (EC 90 0.04 M for HCT8 cells and 0.21 M for HT29 cells, respectively). EC 90 concentration was used in all the subsequent experiments regarding the cellular response of colon cancer cells to C-1748.

A c c e p t e d M a n u s c r i p t 13

 13 Starting from 3 h of C-1748 exposure the population of polyploid cells increased and reached the highest level after 24 h (20%) and then began to decrease. Between 48 and 72 h, the reduction in G2/M arrested and polyploid cells was accompanied by a concomitant increase in sub-G1 population (about 14% after 72 h, Tab. 1). In contrary to HCT8 cells, increased concentration of C-1748 (4xEC 90 ) did not result in considerable increase of the number of HT29 cells undergoing apoptosis in terms of sub-G1 DNA content (Fig.2C, Tab. 2).

  A c c e p t e d M a n u s c r i p t 14 the apoptotic DNA fragments by exogenous terminal deoxytransferase (TdT) and biotin-dUTP/FITC-avidin together with propidium iodide staining, allows to determine whether the DNA fragmentation observed in the treated cells occurred in particular phase of the cell cycle. As indicated in the Fig. 4 (right panel), the population of HCT8 cells with apoptotic DNA breaks remained small after 3 h and 12 h of C-1748 exposure at EC 90 concentration and increased to approximately 35% after 72 h. On the contrary, the population of HT29 cells with apoptotic DNA breaks was markedly smaller and reached the level of 12% after 72 h of C-1748 treatment. Biparametric flow cytometry analysis also revealed that cells with apoptotic DNA breaks derived from all phases of the cell cycle.

  , HCT8 cells remained largely unaffected for the first 6 h of C-1748 treatment. Prolonged drug incubation resulted in A c c e p t e d M a n u s c r i p t 15 progressive appearance of early and late apoptotic cells, and after 72 h of drug exposure, these populations together reached  48%. Moreover, upon drug treatment progression from early (Annexin V+/PI-) to late stages (Annexin V-/PI+) of apoptosis was visible. The fraction of Annexin V-/PI+ (primary necrosis) cells reached 8.8% after 72 h of incubation with C-1748. Treatment of HT29 cells with C-1748 resulted in significantly lower apoptotic cell population compared to HCT8 cells (Fig. 5 B).

  exposure to C-1748. In the case of HT29 cells, the population with low  m started to increase after 12 h of C-1748 treatment and reached 47% after prolonged incubation with the drug (72 h).

A c c e p t e d M a n u s c r i p t 22 cancer

 22 HCT8 and HT29 cells to C-1748 at EC 90 concentration resulted in the appearance of TUNEL-positive cells. The fraction of cells with fragmented DNA was

  B). The activation of caspase-3 concomitantly with the dissipation of  m confirmed that apoptosis was the main response of HCT8 cells to C-1748 treatment. In contrast, the disruption of mitochondrial membrane potential  m , which also can result from membrane permeabilization, together with the presence of only small population of HT29 cells with active caspase-3 indicated that necrosis was responsible for the death of colon cancer HT29 cells after C-1748 treatment. The increased permeabilization of cellular membranes, including mitochondrial membrane which provides to decrease of A c c e p t e d M a n u s c r i p t 23 mitochondrial transmembrane potential  m has been very often observed during

  drug incubation (up to 168 h) resulted in massive detachment of cells from culture plates. These observations, together with Annexin V/PI results, clearly indicate that necrosis plays the main role in the response of HT29 cells to C-1748 exposure.It is worth to point out, that on the cellular level C-1748, at biologically relevant concentrations corresponding to its EC 90 values, induces different cellular response (apoptosis, necrosis or senescence) in human colon carcinoma cell lines. A possible explanation of such diverse cellular response may be the status of p53 protein. HCT8A c c e p t e d M a n u s c r i p t 24 cells with wild p53 underwent mainly apoptosis and senescence. On the contrary, HT29 cells with mutated, non functional p53 underwent mainly necrosis (apoptotic response was very weak), and did not exhibit features of senescence. Western blot experiments revealed that the total p53 level gradually decreased in HCT8 cells upon C-1748 treatment and after 168 h there was no p53 protein signal from Western blot analysis (Fig.7 C). On the other hand, the number of SA--gal-positive HCT8 cells continued to increase and after 168 h of C-1748 exposure the whole population presented -galactosidase staining (Fig.7 A). These results suggested that senescence process induced by C-1748 was independent on p53 protein. Thus, further studies are needed to indicate other factors responsible for the diverse cellular response of colon cancer cells to C-1748 treatment.

A c c e p t e d M a n u s c r i p t 25 pathway

 25 of C-1748-mediated apoptosis and has no influence on cytotoxic activity of this drug. In summary, our results indicated that C-1748 at biological relevant concentration corresponding to EC 90 values, induced different cellular response in human colon carcinoma cells. HCT8 cells underwent transient G1 and G2/M phase arrest followed by apoptosis. Moreover, prolonged drug treatment led to the induction of senescence of HCT8 cells. In contrast, the apoptotic response of HT29 cells to C-1748 exposure was significantly weaker and was preceded by rather stable G1 phase arrest. However, HT29 cells did not undergo senescence and died mainly by necrosis. Additionally, we demonstrated that C-1748 at EC 90 concentration induced reactive oxygen species (ROS), but generation of ROS was not essential for C-1748-induced apoptosis and cytotoxic activity of this drug. Importantly, we demonstrated that one chemotherapeutic agent, C-1748, induces different cellular effects (apoptosis, necrosis, senescence) in two colon cancer cell lines. Moreover, such diverse cellular response was observed even within one colon cancer cell population. Similar observations have been made for other

Figure 1 .

 1 Figure 1. Chemical structure of 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine derivative, C-1748.

A c c e p t e d M a n u s c r i p t

Figure 2 .

 2 Figure 2. Changes in DNA content following treatment with C-1748 at EC 90 concentration for

Figure 3 .

 3 Figure 3.Changes in nuclear morphology of HCT8 (A) and HT29 (B) cells following treatment

Figure 4 .

 4 Figure 4. TUNEL assay for HCT8 and HT29 cells exposed to C-1748 at EC 90 concentration for

Figure 5 .

 5 Figure 5.

Figure 6 .

 6 Figure 6.

A c c e p t e d M a n u s c r i p t 33 Figure 7 .

 337 Figure 7.

Figure 8 .

 8 Figure 8.

Figure

  Figure

Table 1 .

 1 Percentage of sub-G1 and cell cycle distribution of HCT8 and HT29 cells treated with 0.04 M (1xEC 90 ) or 0,21 M (1xEC 90 ) C-1748 respectively, for the time indicated. Data are presented as mean ± SD of triplicate experiments.

	Table
	Cell	Time of
	line	treatment	Sub-G1	G1	S	G2/M	> 4n
		[h]
		0	1.5 ± 0.10	35.6 ± 0.30	23.5 ± 0.50	39.4 ± 1.20	4.3 ± 0.50
		3	1.4 ± 0.25	41.4 ± 2.28	8.0 ± 2.10	40.0 ± 1.25	9.7 ± 0.25
	HCT8	6	1.7 ± 0.35	27.4 ± 1.39	12.2 ± 0.80	47.3 ± 4.08	12.1 ± 1.32
		24	7.6 ± 0.61	20.3 ± 1.24	15.9 ± 1.34	33.2 ± 4.45	24.0 ± 0.13
		48	17.9 ± 1.21	18.5 ± 0.98	12.1 ± 3.40	27.5 ± 1.36	24.5 ± 0.74
		72	25.0 ± 1.79	14.0 ± 0.40	11.2 ± 3.40	19.7 ± 0.54	30.8 ± 3.78
		0	2.7 ± 0.30	45.0 ± 0.80	13.9 ± 1.80	28.2 ± 0.20	9.5 ± 0.15
		3	1.6 ± 0.15	50.5 ± 4.09	15.3 ± 0.21	25.1 ± 1.56	8.1 ± 0.10
	HT29	6	1.8 ± 0.37	45.4 ± 0.66	14.6 ± 1.15	27.8 ± 2.60	10.5 ± 1.10
		24	4.1 ± 0.12	30.6 ± 1.46	11.8 ± 0.94	32.9 ± 2.84	20.8 ± 1.25
		48	9.6 ± 0.55	43.9 ± 2.91	13.2 ± 2.16	24.6 ± 2.51	9.5 ± 0.13
		72	14.0 ± 0.29	46.5 ± 5.64	17.0 ± 1.61	16.0 ± 0.35	6.9 ± 0.50

Table 2 .

 2 Percentage of sub-G1 and cell cycle distribution of HCT8 and HT29 cells treated with 0.16 M (4xEC 90 ) or 0,84 M (4xEC 90 ) C-1748 respectively, for the time indicated. Data are presented as mean ± SD of triplicate experiments.

	Cell	Time of
	line	treatment	Sub-G1	G1	S	G2/M	> 4n
		[h]
		0	1.5 ± 0.10	35.6 ± 0.30	23.5 ± 0.50	39.4 ± 1.2	4.3 ± 0.50
		3	2.0 ± 0.04	31.3 ± 0.50	12.5 ± 2.86	31.7 ± 5.69	21.2 ± 2.8
	HCT8	6	2.0 ± 0,45	23.7 ± 0.37	13.2 ± 2.93	35.8 ± 2.31	24.0 ± 0.90
		24	48.8 ± 0.10	15.2 ± 2.16	8.0 ± 1.34	15.0 ± 0.47	13.2 ± 2.4
		48	60.4 ± 4.55	15.8 ± 0.15	10.5 ± 0.30	8.9 ± 0.35	5.0 ± 0.85
		72	57.8 ± 0.03	15.3 ± 0.73	11.7 ± 0.58	9.3 ± 0.54	6.3 ± 0.47
		0	2.7 ± 0.30	45.0 ± 0.80	13.9 ± 1.80	28.2 ± 0.20	9.5 ± 0.15
		3	2.4 ± 0.30	38.1 ± 2.09	31.0 ± 3.54	12.4 ± 0.23	17.1 ± 2.43
	HT29	6	2.3 ± 0.12	33.9 ± 1.97	28.9 ± 0.68	16.1 ± 1.80	19.7 ± 3.50
		24	9.9 ± 0.18	30.5 ± 0.86	25.4 ± 1.35	13.1 ± 2.30	14.7 ± 0.47
		48	14.5 ± 0.23	37.5 ± 2.54	23.0 ± 0.80	12.9 ± 0.34	13.2 ± 0.59
		72	18.9 ± 0.23	32.2 ± 1.56	19.0 ± 0.95	11.9 ± 0.12	17.9 ± 1.12

Table A c c e p t e d M a n u s c r i p 1

Jacek Witkowski, Department of Pathophysiology, Medical University of Gdańsk, for help in flow cytometry studies.

ROS generation

Graphical Abstract


Laurène Martinez
email: laurene.martinez@unice.fr

Marion Berenguer
email: marion.berenguer@unice.fr

M Christine Bruce
email: bruce@unice.fr

Yannick Le Marchand-Brustel

Roland Govers
email: govers@unice.fr

Rosiglitazone increases cell surface GLUT4 levels in 3T3-L1 adipocytes through an enhancement of endosomal recycling

Keywords: GLUT4, thiazolidinedione, rosiglitazone, diabetes type 2, insulin resistance, adipocyte

Insulin induces a translocation of the glucose transporter GLUT4 from intracellular storage compartments towards the cell surface in adipocytes and skeletal muscle cells, allowing the cells to take up glucose. In type 2 diabetes-associated insulin resistance, the efficiency of this process is reduced. The thiazolidinediones, widely prescribed as anti-diabetic therapy, are generally regarded as insulin-sensitizers. The aim of this study was to evaluate the effect of the thiazolidinedione rosiglitazone (BRL 49653) on GLUT4 in adipocytes. When applied during differentiation, rosiglitazone dose dependently augmented GLUT4 expression along with the formation of lipid droplets. Intriguingly, its presence during differentiation led to increases in both cell surface GLUT4 levels and insulin-sensitivity of GLUT4 translocation in mature adipocytes. Treatment of fully differentiated adipocytes with rosiglitazone also led to increases in GLUT4 at the plasma membrane. Rosiglitazone similarly affected cell surface levels of the endosomal transferrin receptor, but did not alter the GLUT4 internalization rate.

The augmentation in cell surface GLUT4 levels was maintained in adipocytes that were rendered insulin resistant in vitro by a 24 hour insulin treatment and moreover in these cells rosiglitazone also fully restored insulin-induced GLUT4 translocation. We conclude that in adipocytes, rosiglitazone increases cell surface GLUT4 levels by increasing its endosomal recycling and restores insulin-induced GLUT4 translocation in insulin resistance. These results implicate novel modes of action on GLUT4 that are all likely to contribute to the insulin-sensitizing effect of rosiglitazone in type 2 diabetes.

A c c e p t e d M a n u s c r i p t

Introduction

Since their initial availability 15 years ago, the thiazolidinediones (TZDs) have been widely prescribed as medication for type 2 diabetes (T2D) [START_REF] Martens | Metabolic and additional vascular effects of thiazolidinediones[END_REF][START_REF] Wilding | Thiazolidinediones, insulin resistance and obesity: Finding a balance[END_REF]. In addition to a direct effect on insulin-regulated glucose homeostasis in vivo (i.e. reducing insulin resistance), TZD administration has been associated with an amelioration of plasma lipid profiles, fat distribution, blood pressure, inflammation, endothelial dysfunction, and atherosclerosis [START_REF] Wilding | Thiazolidinediones, insulin resistance and obesity: Finding a balance[END_REF][START_REF] Ceriello | Thiazolidinediones as anti-inflammatory and anti-atherogenic agents[END_REF].

TZDs are agonists for the transcription factor peroxisome proliferator-activated receptorgamma (PPARγ). This nuclear receptor regulates the synthesis of proteins involved in glucose and lipid homeostasis and is mostly present in adipose tissue [START_REF] Spiegelman | PPAR-gamma: adipogenic regulator and thiazolidinedione receptor[END_REF]. PPARγ, together with C/EBP, is regarded as the master regulator of fat cell differentiation. TZDs increase efficacy and kinetics of adipocyte differentiation [START_REF] Hallakou | Pioglitazone induces in vivo adipocyte differentiation in the obese Zucker fa/fa rat[END_REF][START_REF] Hallakou | Pioglitazone-induced increase of insulin sensitivity in the muscles of the obese Zucker fa/fa rat cannot be explained by local adipocyte differentiation[END_REF] and suppress TNFα-mediated inhibition of adipocyte differentiation [START_REF] Ohsumi | Troglitazone prevents the inhibitory effects of inflammatory cytokines on insulininduced adipocyte differentiation in 3T3-L1 cells[END_REF][START_REF] Szalkowski | Antidiabetic thiazolidinediones block the inhibitory effect of tumor necrosis factor-alpha on differentiation, insulin-stimulated glucose uptake, and gene expression in 3T3-L1 cells[END_REF]. In vivo, PPARγ ablation is accompanied by an absence of all types of adipose tissue [START_REF] Barak | PPAR gamma is required for placental, cardiac, and adipose tissue development[END_REF], while in vitro its inactivation results in a downregulation of the expression of genes that confer the characteristics of mature adipocytes [START_REF] Tamori | Role of peroxisome proliferator-activated receptor-gamma in maintenance of the characteristics of mature 3T3-L1 adipocytes[END_REF]. While the primary (adipogenic) action of TZDs occurs in adipose tissue, their insulin-sensitizing effects mostly take place in skeletal muscle [START_REF] Hammarstedt | Improved insulin sensitivity and adipose tissue dysregulation after short-term treatment with pioglitazone in non-diabetic, insulin-resistant subjects[END_REF]. While these two actions may seem relatively unrelated, the effects in muscle could be indirect and possibly due to a reversal of fatty acid accumulation in muscle resulting from an increased fatty acid metabolism in adipose tissue [START_REF] Martin | PPARgamma activators improve glucose homeostasis by stimulating fatty acid uptake in the adipocytes[END_REF]. In addition, TZD-mediated PPARγ activation alters the secretion of adipokines that are involved in the regulation of insulin sensitivity and glucose metabolism (e.g. TNFα and leptin), leading to improvements in insulin action in multiple insulin-sensitive tissues [START_REF] Wilding | Thiazolidinediones, insulin resistance and obesity: Finding a balance[END_REF][START_REF] Hallakou | Pioglitazone-induced increase of insulin sensitivity in the muscles of the obese Zucker fa/fa rat cannot be explained by local adipocyte differentiation[END_REF].

In any case, it is unlikely that the low PPARγ levels in muscle and liver times lower than in adipose tissue) are directly responsible for some of the insulin-sensitizing effects as A c c e p t e d M a n u s c r i p t 4 TZDs do not display insulin-sensitizing effects in mice lacking adipose tissue [START_REF] Chao | Adipose tissue is required for the antidiabetic, but not for the hypolipidemic, effect of thiazolidinediones[END_REF] and TZDs hardly activate PPARγ-responsive genes in muscle and liver [START_REF] Albrektsen | Novel genes regulated by the insulin sensitizer rosiglitazone during adipocyte differentiation[END_REF].

In insulin resistance in vivo, TZD troglitazone restores decreased GLUT4 expression levels in adipose tissue of the T2D animal model OLETF (Otsuka Long-Evans Tokushima Fatty rat) [START_REF] Furuta | Troglitazone improves GLUT4 expression in adipose tissue in an animal model of obese type 2 diabetes mellitus[END_REF]. An increase in GLUT4 expression in adipocytes was also described for rosiglitazone in db/db mice [START_REF] Young | Repeat treatment of obese mice with BRL 49653, a new potent insulin sensitizer, enhances insulin action in white adipocytes. Association with increased insulin binding and cell-surface GLUT4 as measured by photoaffinity labeling[END_REF]. However, suppression of insulin resistance in the absence of alterations in GLUT4 expression has been reported for englitazone in adipocytes of high fat diet rats [START_REF] Stevenson | The antihyperglycemic agent englitazone prevents the defect in glucose transport in rats fed a high-fat diet[END_REF] and for englitazone and pioglitazone in muscle of glucocorticoid-treated rats [START_REF] Weinstein | Effects of thiazolidinediones on glucocorticoid-induced insulin resistance and GLUT4 glucose transporter expression in rat skeletal muscle[END_REF]. In insulin resistance in vitro, rosiglitazone has been suggested to rescue impaired glucose transport by restoring baseline levels of reactive oxygen species (ROS) [START_REF] Houstis | Reactive oxygen species have a causal role in multiple forms of insulin resistance[END_REF]. However, it is not known whether and how GLUT4 is involved in this rescue. In any case, there is no report yet that describes the effects of rosiglitazone on GLUT4 in vitro.

Here, we investigated the effects of rosiglitazone on GLUT4 in (pre)adipocytes, notably its expression and cell surface levels. In addition, we examined whether the effect on cell surface amounts was specific for GLUT4 and maintained under conditions of insulin resistance.

A c c e p t e d M a n u s c r i p t 5

Materials and methods

Materials

3T3-L1 murine preadipocytes were obtained from the American Type Culture Collection ATCC/LGC Standards (Teddington, UK) and Plat-E cells, used for the generation of retrovirus, were kindly provided by Dr Toshio Kitamura (University of Tokyo, Tokyo, Japan).

Sera were from PAA (Pasching, Austria), media and HEPES from Invitrogen (Carlsbad, CA, USA), and insulin from Lilly (Suresnes, France). Monoclonal anti-HA antibody was obtained from Covance (Emeryville, CA, USA), fluorescent antibodies and TOPRO3 from Molecular Probes/Invitrogen (Carlsbad, CA, USA), anti-transferrin receptor from Zymed/Invitrogen (Carlsbad, CA, USA), and antibodies against GLUT4 and insulin receptor and HRPconjugated secondary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Antibody against syntaxin-13 was generously provided by Dr Rytis Prekeris (University of Colorado, Denver, CO, USA). Anti-tubulin antibody, mouse IgG antibody and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rosiglitazone, troglitazone, and pioglitazone were from Alexis/Axxora (Lausen, Switzerland). Unless indicated otherwise, rosiglitazone was used at a concentration of 10 μM. pBABE vector was kindly provided by Dr Hartmut Land (University of Rochester, Rochester, NY, USA).

Molecular biology

The cDNA encoding HA-GLUT4 (GLUT4 with an HA epitope tag in its first luminal domain) inserted in pBABE-puro vector is described elsewhere [START_REF] Govers | High-throughput analysis of the dynamics of recycling cell surface proteins[END_REF]. A pBABE-puro vector containing the cDNA encoding the human transferrin receptor with an HA-tag at its C-

Cell culture

As the 3T3-L1 preadipocytes originally obtained from ATCC appeared to be very heterologous and differentiated very poorly, an individual clone of cells was isolated from these preadipocytes and tested for growth, differentiation capacity, and insulin-induced GLUT4 translocation. Cultures of this clone contained at least 95% adipocytes upon differentiation. 3T3-L1 preadipocytes were cultured, infected with retrovirus, and differentiated as described before [START_REF] Govers | High-throughput analysis of the dynamics of recycling cell surface proteins[END_REF]. For differentiation, preadipocytes were cultured for three days in DMEM/FBS, containing 700 nM insulin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 250 nM dexamethasone, and 0.1 g/l biotin. Unless stated otherwise, preadipocytes were differentiated in the absence of rosiglitazone. Adipocytes were used for experiments 8 to 12 days after onset of differentiation, with medium changed two days prior to the experiment.

For the induction of insulin resistance, 3T3-L1 adipocytes were cultured for 24 hours in DMEM supplemented with 2 g/l BSA and 100 nM insulin [START_REF] Maier | Long-term insulin treatment of 3T3-L1 adipocytes results in mis-targeting of GLUT4: implications for insulin-stimulated glucose transport[END_REF], followed by an extensive washing procedure, consisting of washing the wells with DMEM, supplemented with 20 mM HEPES and 2 g/l BSA, leaving the cells for 20 minutes in DMEM/HEPES/BSA, and repeating these two steps for an additional five times.

For all other experiments, cells were serum-starved for 2 hours in DMEM containing 2 g/l BSA.

A c c e p t e d M a n u s c r i p t 7

Fluorescence-based techniques

The fluorescence-based assay for the detection of relative amounts of GLUT4 at the cell surface has been described in detail elsewhere [START_REF] Govers | High-throughput analysis of the dynamics of recycling cell surface proteins[END_REF]. Briefly, HA-GLUT4-expressing 3T3-L1 adipocytes grown in 96 well plates were fixed and immunolabeled using anti-HA antibody after the cells had been incubated in the absence or presence of the permeabilizing agent saponin to label GLUT4 at the cell surface or total cellular GLUT4, respectively. For microscopy, cell were incubated, fixed, and labeled as for the fluorescence-based assay.

Nuclei were stained using TOPRO3. For the quantitative detection of endogenous GLUT4 and fat droplets in differentiating cells, non-infected (pre)adipocytes were cultured in black clear-bottom well plates and labeled with anti-GLUT4 1F8 antibody and 0.1 mg/l of the lipid stain nile red (within the same wells). Both anti-GLUT4 antibody and nile red displayed negligible background levels (evident from preadipocyte cultures). For quantitative analysis, the fluorescence signal related to HA(-GLUT4), GLUT4 and nile red was measured using the bottom-reading mode in a fluorescence microtiter plate reader (FLUOstar Galaxy, BMG Labtechnologies; Offenburg, Germany). Background fluorescence (preadipocytes) was subtracted from GLUT4 and nile red measurements. For microscopy, fluorescent label was visualized using a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss, Göttingen, Germany) in the C3M MicorBio Cell Imaging Facility.

To measure GLUT4 internalization, a method adapted from [START_REF] Williams | Golgin-160 is required for the Golgi membrane sorting of the insulin-responsive glucose transporter GLUT4 in adipocytes[END_REF] was used. Adipocytes were cultured on coverslips, differentiated, serum-starved, and incubated for 20 minutes with 100 nM insulin, upon which anti-HA antibody was added to the medium. After 1 to 4 minutes, the cells were washed extensively with ice-cold PBS on ice, fixed with paraformaldehyde, permeabilized with saponin and incubated with Alexa488-conjugated goat-anti-mouse The analysis was performed by quantifying all consecutive views along a horizontal line across the coverslip. The number of cells with intracellular label or label exclusively at the cell surface was determined for each view. The percentage of HA-positive cells with internalized label was determined for 5 consecutive views and averaged for at least 10 x 5 views. In total, these views represented more than 200 HA-positive cells.

Glucose uptake

Adipocytes, grown in 12 well plates, were incubated for 5 minutes in KRP/BSA ( HRP-conjugated secondary antibodies were visualized using chemiluminescence reagent (PerkinElmer) and a CCD camera-based imager (LAS-3000, Fujifilm; Tokyo, Japan).

Relative intensities were quantitated using MultiGauge software (Fujifilm).

Statistics

All data are presented as means ± SD. Experiments were repeated at least three times.

Representative experiments are shown. For 96 well plate and glucose uptake experiments, 4

wells and 3 wells were used for each data point, respectively. Comparisons between individual data sets were evaluated using two-tailed Student's t-tests and comparisons between dose-response curves were evaluated using F-tests (GraphPad Prism software). Doseresponse data were fitted based on a nonlinear four-parameter (variable slope) sigmoidal dose

Rosiglitazone augments GLUT4 expression along adipocyte differentiation

TZDs are known for their positive effect on the differentiation of preadipocytes into adipocytes. Remarkably, a detailed analysis of the enhancement of differentiation is lacking.

To test TZD rosiglitazone in our cell system, we evaluated its effect on GLUT4 expression and lipid accumulation in 3T3-L1 (pre)adipocytes, differentiated with standard or suboptimal differentiation protocols (Fig. 1). Using a standard 3 day differentiation protocol (700 nM insulin, 500 μM IBMX, and 250 nM dexamethasone), virtually all preadipocytes differentiated well into adipocytes, as evidenced by the appearance of GLUT4 and nile redpositive fat droplets. Rosiglitazone did not seem to have any effect under these conditions.

Serial dilution of the differentiation cocktail revealed that when insulin, IBMX, and dexamethasone concentrations were reduced to one eighth of the original concentrations, the cells stopped to differentiate (i.e. absence of GLUT4 and nile red signal). However, in this case, rosiglitazone restored differentiation. Rosiglitazone did not induce GLUT4 expression or lipid accumulation in the absence of insulin, IBMX, and dexamethasone. Quantitative analysis of GLUT4 expression and fat accumulation in cells treated with various concentrations of insulin, dexamethasone and IBMX revealed that rosiglitazone did not act on the differentiation process by enhancing insulin action, but that it enhanced the effects of IBMX and/or dexamethasone (Fig. 1B). Examining cells that had been exposed to one eighth of the differentiation cocktail and various concentrations of rosiglitazone demonstrated that fat accumulation was slightly more sensitive to rosiglitazone than GLUT4 expression (Fig. 2A). Half-maximal concentrations of rosiglitazone for fat accumulation and GLUT4 expression were 15 nM and 27 nM, respectively. A small discrepancy between GLUT4

A c c e p t e d M a n u s c r i p t 11 expression and fat accumulation was also observed when the cells were treated for various time periods with differentiation cocktail in the absence or presence of rosiglitazone (Fig. 2B).

Exposure of differentiating preadipocytes to rosiglitazone increases cell surface GLUT4 levels in fully differentiated adipocytes

We evaluated the effect of early exposure of the cells to rosiglitazone (during differentiation) on cell surface GLUT4 levels in fully differentiated adipocytes (Fig. 3). Preadipocytes expressing HA-GLUT4 were incubated with a standard differentiation cocktail in the absence or presence of rosiglitazone for 2 to 5 days and cultured further in the absence of insulin, IBMX, dexamethasone, and rosiglitazone for a total of 11 days. Adipocytes were then treated or not for 20 minutes with 100 nM insulin and cell surface GLUT4 levels were determined.

The use of rosiglitazone during the differentiation process increased cell surface GLUT4 levels in the absence of insulin for all time periods tested and also for the three day differentiation condition with insulin stimulation (Fig. 3A). This led to a reduction in the relative effect of insulin on cell surface GLUT4 levels (Fig. 3B).

Exposure of differentiated adipocytes to rosiglitazone increases basal cell surface GLUT4 levels and insulin sensitivity

To determine whether the effect of rosiglitazone on GLUT4 in adipocytes is restricted to its presence during the differentiation process, we differentiated 3T3-L1 preadipocytes in the absence of rosiglitazone and treated the cells with rosiglitazone after differentiation (Fig. 4).

This approach demonstrated that rosiglitazone gradually but largely augmented cell surface GLUT4 levels after up to five days of treatment (Fig. 4A). In contrast, cell surface GLUT4

A c c e p t e d M a n u s c r i p t 12 levels after acute insulin stimulation were comparable between control and rosiglitazonetreated adipocytes (Fig. 4A). Fluorescence confocal microscopy of control and rosiglitazonetreated adipocytes confirmed these results (Fig. 4B). Moreover, this increase in cell surface GLUT4 levels in basal but not insulin-stimulated cells was paralleled by increases in basal glucose uptake and an absence of increased glucose uptake under conditions where the cells were stimulated with high concentrations of insulin (Fig. 4C). Cells that were stimulated with submaximal concentrations of insulin (1 nM) still displayed a rosiglitazone-induced augmentation of glucose uptake. Western blot analysis of rosiglitazone-treated cells revealed that the increase in glucose uptake could not be accounted for by an increase in GLUT4 expression. However, in accordance with previous studies [START_REF] Hernandez | Rosiglitazone produces insulin sensitisation by increasing expression of the insulin receptor and its tyrosine kinase activity in brown adipocytes[END_REF], rosiglitazone increased the expression of the insulin receptor (Fig. 4D,E). The increase in cell surface GLUT4 levels was not specific to rosiglitazone as other TZD family members, pioglitazone and troglitazone, also displayed similar effects on GLUT4 (Fig. 4F).

Treatment of differentiated adipocytes for five days with various concentrations of rosiglitazone resulted in a dose response curve for cell surface GLUT4 expression with an EC50 value of 22 nM and maximum effects at 0.5 μM (Fig. 5A). This response was highly similar to that of preadipocytes regarding their rosiglitazone-enhanced differentiation (Fig. 2A). Acutely stimulating control-and rosiglitazone-treated adipocytes with various concentrations of insulin revealed that rosiglitazone not only augmented cell surface GLUT4 levels but also sensitized the 3T3-L1 adipocytes to insulin. Half-maximal insulin concentrations dropped from 0.90 nM to 0.38 nM (P<0.0001) in adipocytes treated with rosiglitazone during the early phase of their differentiation (Fig. 5B) and from 0.76 nM to 0.07 nM (P<0.05) in adipocytes treated with rosiglitazone after differentiation (Fig. 5C).

A c c e p t e d M a n u s c r i p t 13

The effect of rosiglitazone on basal cell surface GLUT4 levels is long-lasting

We next addressed the question whether rosiglitazone treatment of adipocytes induced a temporary effect on GLUT4 or whether this was a long-term (lasting) effect. Adipocytes were treated with rosiglitazone at various time periods before the analysis of cell surface GLUT4 levels, as indicated in Fig. 6A. Unfortunately, culturing the adipocytes for extended periods increased basal cell surface levels, even in the absence of rosiglitazone. Nevertheless, our study revealed that a 5 day rosiglitazone treatment followed by a rosiglitazone-free incubation period of 8 days was not different from a rosiglitazone treatment during the final 5 days (Fig. 6B). This indicates that the effect of rosiglitazone on GLUT4 is long-lasting (irreversible during the time course of the experiment), likely due to a change in cell properties.

Rosiglitazone also increases cell surface levels of the transferrin receptor

To test whether the effect of rosiglitazone treatment is specific for GLUT4, we studied its effect on the transferrin receptor (TfR) (Fig. 7). While only ~50% of the intracellular GLUT4 pool is present in endosomes, cytoplasmic TfR is present exclusively in endosomes and therefore marks a subpopulation of the intracellular GLUT4-containing compartments [START_REF] Livingstone | Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes[END_REF].

To compare the TfR with GLUT4 using similar methodologies, we studied a TfR that was extracellularly tagged with an HA epitope. Immunofluorescence microscopy demonstrated that the localization of the tagged TfR was similar to the localization of endogenous TfR (Suppl. fig. S1). A quantitative analysis demonstrated that the TfR displayed quite a remarkable insulin-responsiveness (Fig. 7A). Though less pronounced than for GLUT4, the TfR translocated towards the cell surface in response to insulin. Similarly to GLUT4, the amount of TfR at the cell surface augmented with rosiglitazone (Fig. 7B), while the effects of A c c e p t e d M a n u s c r i p t 14 rosiglitazone and insulin were not additive, indicating that the effect of rosiglitazone is not specific to GLUT4 and that rosiglitazone may augment endosomal recycling kinetics.

Rosiglitazone does not affect GLUT4 internalization

As rosiglitazone affects both GLUT4 and TfR, it is tempting to speculate that rosiglitazone alters cell surface GLUT4 levels by acting on its internalization rate. To test this possibility, adipocytes were incubated for 5 days in the presence or absence of 10 μM rosiglitazone and internalization was measured (Fig. 8). Cells were incubated with anti-HA antibody for very short time periods to ensure that anti-HA-bound GLUT4 did not yet return to the cell surface after its internalization. Next, the percentage of cells with internalized label was determined.

This revealed that rosiglitazone did not inhibit GLUT4 internalization, indicating that another step in the intracellular trafficking of GLUT4 (and of TfR) is altered by rosiglitazone.

Rosiglitazone restores insulin-induced GLUT4 translocation in adipocytes that are rendered insulin-resistant in vitro

TZDs have been studied for their potency in reversing insulin resistance in 3T3-L1 adipocytes. However, these studies have focused on the reversal of the reduction in insulininduced glucose uptake. Hence, it remains uncertain whether the TZD effects on insulin resistance can be accounted for by a change in the cellular expression of GLUT4, by an increase in cell surface GLUT4 levels, or by another mechanism. Therefore, we addressed the question whether rosiglitazone pretreatment could prevent the relative decrease in cell surface GLUT4 levels induced by a 24 hour insulin treatment (Fig. 9). This treatment clearly induced insulin resistance as a subsequent 20 minute stimulation of the cells with 1, 10, and 100 nM A c c e p t e d M a n u s c r i p t 15 insulin resulted in reduced cell surface GLUT4 levels compared to control cells (Fig. 9A).

However, in rosiglitazone-treated adipocytes, acute insulin-induced GLUT4 translocation was not impaired by a 24 hour insulin pretreatment. As the insulin effects in rosiglitazone-treated cells are smaller compared to non-treated cells, we also examined the effects of shorter rosiglitazone treatments on reduced cell surface GLUT4 levels (Fig. 9B). This demonstrated that after two days of treatment, rosiglitazone completely restored acute insulin responsiveness of GLUT4 translocation in cells subjected to a 24 hour insulin incubation, without largely enhancing basal cell surface levels. These data indicate that rosiglitazone augments basal cell surface GLUT4 levels and independently restores the response of GLUT4 to insulin stimulation under conditions that induce insulin resistance. To evaluate whether rosiglitazone rescues the decrease in GLUT4 expression that has been associated with insulin resistance in vivo and in vitro [START_REF] Maier | Long-term insulin treatment of 3T3-L1 adipocytes results in mis-targeting of GLUT4: implications for insulin-stimulated glucose transport[END_REF][START_REF] Shepherd | Glucose transporters and insulin action--implications for insulin resistance and diabetes mellitus[END_REF], we treated adipocytes for three days with or without insulin and rosiglitazone. This demonstrated that the insulin-induced downregulation of GLUT4 expression (~50% after three days) is not rescued by rosiglitazone (Fig. 9C).

Furthermore, our studies demonstrated that reductions in GLUT4 expression due to TNF-α treatment are not rescued by rosiglitazone either (data not shown).

A c c e p t e d M a n u s c r i p t 16

Discussion

While adipose tissue is not the main storage site for glucose in the postprandial state, glucose metabolism in this organ is essential for whole body glucose homeostasis, via a direct regulation of the synthesis and secretion of adipokines that have important effects in other parts of the body. Adipocyte-specific knock-out of GLUT4 results in insulin resistance in muscle and liver and an enhanced secretion of adipokines, amongst which serum retinol binding protein 4 (RBP4) [START_REF] Abel | Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver[END_REF][START_REF] Yang | Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes[END_REF]. RBP4 levels have been demonstrated to be elevated in insulin resistance and T2D. Moreover, its overexpression causes insulin resistance while its genetic deletion enhances insulin sensitivity. Interestingly, rosiglitazone has been shown to restore serum RBP4 levels in insulin resistant mice [START_REF] Yang | Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes[END_REF]. Given that both GLUT4 and rosiglitazone regulate adipokine secretion, adipocytic GLUT4 could very well be implicated in the rosiglitazone-induced adipokine-dependent amelioration of whole body glucose homeostasis in T2D.

Our results support this hypothesis as they show that rosiglitazone increases basal cell surface GLUT4 levels and glucose uptake by adipocytes. In addition to its enhancement of GLUT4 expression throughout adipocyte differentiation, this TZD augmented the amount of GLUT4 at the plasma membrane of adipocytes when administered either during (pre)adipocyte differentiation or afterwards. So, even while the time-scale of the effect of insulin and rosiglitazone on GLUT4 traffic are distinct, rosiglitazone clearly partially mimics insulin action. Moreover, even when administered after the cells had been fully differentiated, its effect on GLUT4 was irreversible, suggesting that rosiglitazone changes adipocyte properties.

Possibly, rosiglitazone, via its interaction with transcription factor PPARγ, may change the expression of one or several proteins that play a regulatory role in endosomal recycling such A c c e p t e d M a n u s c r i p t as rab, SNARE, or sorting nexin proteins [START_REF] Cai | Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle[END_REF][START_REF] Van Kerkhof | Sorting nexin 17 facilitates LRP recycling in the early endosome[END_REF]. In support of this, the rosiglitazoneinduced effects on GLUT4 required several days of treatment. In contrast, troglitazone has been shown to largely alter cell surface GLUT4 levels in muscle and fat cells within 15 and 24 hours of treatment, respectively [START_REF] Yonemitsu | Troglitazone induces GLUT4 translocation in L6 myotubes[END_REF][START_REF] Shintani | Troglitazone not only increases GLUT4 but also induces its translocation in rat adipocytes[END_REF]. Troglitazone concentrations that were found to increase cell surface GLUT4 levels in these studies were much higher than the concentrations of rosiglitazone we found to be effective, but this correlates well with the lower affinity of troglitazone for PPARγ [START_REF] Adams | Activators of peroxisome proliferator-activated receptor gamma have depot-specific effects on human preadipocyte differentiation[END_REF][START_REF] Camp | Differential activation of peroxisome proliferator-activated receptor-gamma by troglitazone and rosiglitazone[END_REF]. Neither in muscle cells [START_REF] Yonemitsu | Troglitazone induces GLUT4 translocation in L6 myotubes[END_REF] nor in adipocytes [START_REF] Shintani | Troglitazone not only increases GLUT4 but also induces its translocation in rat adipocytes[END_REF][START_REF] Huang | Annexin II is a thiazolidinedioneresponsive gene involved in insulin-induced glucose transporter isoform 4 translocation in 3T3-L1 adipocytes[END_REF] (Fig. 3A,C) were the effects of glitazones and high concentrations of insulin additive. However, rosiglitazone treatment of ob/ob mice has been reported to increase glucose uptake and cell surface GLUT4 levels in primary adipocytes stimulated with insulin ex vivo, but this was most probably due to an increase in GLUT4 expression [START_REF] Young | Repeat treatment of obese mice with BRL 49653, a new potent insulin sensitizer, enhances insulin action in white adipocytes. Association with increased insulin binding and cell-surface GLUT4 as measured by photoaffinity labeling[END_REF]. Basal cell surface GLUT4 levels were found to be increased by troglitazone in L6 muscle cells [START_REF] Yonemitsu | Troglitazone induces GLUT4 translocation in L6 myotubes[END_REF] and primary adipocytes [START_REF] Shintani | Troglitazone not only increases GLUT4 but also induces its translocation in rat adipocytes[END_REF],

and by rosiglitazone and troglitazone in 3T3-L1 adipocytes (this study). Furthermore, troglitazone was found to induce a significant (50%) increase in GLUT4 expression levels in adipocytes [START_REF] Shintani | Troglitazone not only increases GLUT4 but also induces its translocation in rat adipocytes[END_REF], while this was not the case for rosiglitazone in our studies.

Our studies have suggested that the effect of rosiglitazone is not specific for GLUT4, as rosiglitazone also increased basal but not insulin-enhanced cell surface levels of the transferrin receptor (TfR). The predominantly endosome-localized TfR is constitutively recycling towards the plasma membrane, while its recycling kinetics are increased even further upon acute insulin stimulation [START_REF] Foran | Protein kinase B stimulates the translocation of GLUT4 but not GLUT1 or transferrin receptors in 3T3-L1 adipocytes by a pathway involving SNAP-23, synaptobrevin-2, and/or cellubrevin[END_REF]. As both GLUT4 and TfR surface levels are increased upon rosiglitazone treatment, we propose that rosiglitazone increases endosomal recycling kinetics. For GLUT4 this increase is quite dramatic, as GLUT4 recycles extremely slowly in non-stimulated cells [START_REF] Govers | Insulin increases cell surface GLUT4 levels by dose dependently discharging GLUT4 into a cell surface recycling pathway[END_REF][START_REF] Karylowski | GLUT4 is retained by an intracellular cycle of vesicle formation and fusion with endosomes[END_REF]. As the rosiglitazone effects appear not to be further enhanced by insulin, similar molecular systems involved in endosomal recycling may be implicated in the long-term rosiglitazone effects and in acute insulin action. The rosiglitazoneinduced increase in the expression of the insulin receptor (Fig. 3D) could imply an activation of the insulin signaling cascade under basal conditions. However, neither the insulin receptor nor its downstream effector Akt were phosphorylated under basal conditions in rosiglitazonetreated adipocytes (data not shown). Our data demonstrate that the increase in cell surface GLUT4 levels is not due to changes in its internalization rate. In contrast, metformin has been demonstrated to inhibit GLUT4 endocytosis and thereby to augment insulin-induced increases in the amount of GLUT4 at the plasma membrane [START_REF] Yang | Long-term metformin treatment stimulates cardiomyocyte glucose transport through an AMP-activated protein kinase-dependent reduction in GLUT4 endocytosis[END_REF], which is clearly different from our findings. Since rosiglitazone, as well as other TZDs, affect the synthesis and secretion of adipokine/cytokines, such as adiponectin [START_REF] Maeda | PPARgamma ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein[END_REF], RBP4 [START_REF] Yang | Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes[END_REF], leptin [START_REF] Nolan | Effect of troglitazone on leptin production. Studies in vitro and in human subjects[END_REF], Il-6 [START_REF] Lagathu | Chronic interleukin-6 (IL-6) treatment increased IL-6 secretion and induced insulin resistance in adipocyte: prevention by rosiglitazone[END_REF], and TNF-α [START_REF] Okuno | Troglitazone increases the number of small adipocytes without the change of white adipose tissue mass in obese Zucker rats[END_REF], it cannot be excluded that the effect on endosomal recycling is mediated in part via an alteration in adipokine secretion (autocrine effect). Alternatively, this effect on endocytic recycling could be mediated (in part) via annexin II, which is involved in insulin-induced GLUT4 translocation, and whose expression levels are elevated upon troglitazone treatment [START_REF] Huang | Annexin II is a thiazolidinedioneresponsive gene involved in insulin-induced glucose transporter isoform 4 translocation in 3T3-L1 adipocytes[END_REF]. Finally, TZDs might enhance endosomal recycling via altering intracellular ROS (reactive oxygen species) levels, as TZDs have been shown to display antioxidant properties [START_REF] Houstis | Reactive oxygen species have a causal role in multiple forms of insulin resistance[END_REF].

These antioxidant properties may also be implicated in the effects of rosiglitazone on GLUT4 in insulin resistance. Previously, the reduction in glucose uptake in insulin-resistant 3T3-L1 adipocytes has been shown to be suppressed by ROS scavenger MnTBAP as well as by rosiglitazone, while rosiglitazone decreased intracellular ROS as effectively as MnTBAP [START_REF] Szalkowski | Antidiabetic thiazolidinediones block the inhibitory effect of tumor necrosis factor-alpha on differentiation, insulin-stimulated glucose uptake, and gene expression in 3T3-L1 cells[END_REF][START_REF] Houstis | Reactive oxygen species have a causal role in multiple forms of insulin resistance[END_REF]. However, it is unclear whether the TZD-mediated restoration of glucose uptake was mediated via a change in glucose transporter expression level or via restoration of the rate of insulin-induced GLUT4 translocation. Our studies indicate that rosiglitazone completely A c c e p t e d M a n u s c r i p t 19 restores the response of GLUT4 to insulin stimulation under conditions that normally induce insulin resistance. This was already evident after two days of rosiglitazone treatment, before major changes in basal cell surface GLUT4 levels occurred. This effect is likely to be mediated via a restoration of insulin signaling [START_REF] Peraldi | Thiazolidinediones block tumor necrosis factoralpha-induced inhibition of insulin signaling[END_REF] and possibly due to an increase in the expression of the insulin receptor (Fig. 3). It has been reported that in primary adipocytes from rosiglitazone-treated ob/ob mice GLUT4 expression is increased, concurrent with an increase in cell surface GLUT4 levels under conditions of insulin stimulation [START_REF] Young | Repeat treatment of obese mice with BRL 49653, a new potent insulin sensitizer, enhances insulin action in white adipocytes. Association with increased insulin binding and cell-surface GLUT4 as measured by photoaffinity labeling[END_REF]. However, our studies have demonstrated that the reduction in GLUT4 expression that is associated with insulin resistance is not reversed by rosiglitazone, suggesting that at least in adipocytes rendered insulin-resistant in vitro, rosiglitazone acts mainly on GLUT4 translocation and not on GLUT4 expression.

In conclusion, using the 3T3-L1 adipocyte cell model, we have investigated for the first time the effects of rosiglitazone on GLUT4 in vitro and have demonstrated that treatment of adipocytes for several days with rosiglitazone results in an irreversible effect on endosomal protein traffic, in an augmentation of GLUT4 at the PM, and in an increased glucose uptake.

A second, independent effect of rosiglitazone was seen in insulin resistant cells, where it fully restored insulin-induced GLUT4 translocation. While it remains to be determined by which molecular mechanisms rosiglitazone affects endosomal GLUT4 recycling and GLUT4 insulin-sensitivity in insulin resistance, these effects are likely to contribute to the rosiglitazone-mediated enhancement of glucose homeostasis in vivo. Adipocytes cultured in 96 well plates were treated and labeled as described for (A), but fluorescence was quantified using a fluorescence microtiter plate reader. Each well was used for both GLUT4 and lipid measurements. The amount of GLUT4 and lipid in adipocytes differentiated using the standard differentiation protocol (700 nM insulin, 500 μM IBMX, and 250 nM dexamethasone) was set to 100%. Note that rosiglitazone increased GLUT4 expression and lipid accumulation by enhancing the effects of IBMX and/or dexamethasone.

The absence of a dominant role for insulin in the differentiation process (in the absence and presence of rosiglitazone) cannot be explained by the presence of insulin in the sera, as the concentration of insulin in the culture medium was as low as 3 pM.

  sequence of the PCR part was verified by sequence analysis (Cogenics/Beckman Coulter).

A c c e p t e d M a n u s c r i p t 8 antibody.

 8 Adipocytes were analyzed by epifluorescence microscopy using a 63X objective.

A c c e p t e d M a n u s c r i p t 9 transferred

 9 to PVDF membrane. Membranes were incubated with the indicated antibodies.

(

  log) -response curve according to the equation Y = bottom+(Top-Bottom)/(1+10^((LogEC50-X)*HillSlope)). Differences between data sets were considered statistically different when P<0.05.

Fig. 1 .

 1 Figure legends

Fig. 2 .

 2 Fig. 2. Effect of varying the concentration and length of rosiglitazone treatment on GLUT4

A c c e p t e d M a n u s c r i p t 25 periods

 25 to either one fourth (175 nM insulin, 125 μM IBMX, and 62 nM dexamethasone) or half of the standard differentiation cocktail (350 nM insulin, 250 μM IBMX, and 125 nM dexamethasone) in the absence or presence of 10 μM rosiglitazone. Cells were cultured further to a total of 8 days and GLUT4 and lipid was quantitated. Note the subtle differences in the kinetics of the increases in GLUT4 and lipid, which is most evident at 36 and 72 hours of treatment (dashed lines).

Fig. 3 .Fig. 4 .

 34 Fig. 3. Changes in cell surface GLUT4 levels in 3T3-L1 adipocytes differentiated in the

Fig. 5 .M a n u s c r i p t 27 (Fig. 6 .

 5276 Fig. 5. Dose response curves of rosiglitazone and insulin on cell surface GLUT4 levels in

Fig. 7 .

 7 Fig. 7. The intracellular traffic of the transferrin receptor is affected by rosiglitazone in

Fig. 8 .

 8 Fig. 8. Rosiglitazone does not impair GLUT4 internalization. Insulin-stimulated HA-GLUT4-

Fig. 9 .

 9 Fig. 9. Rosiglitazone prevents reduced cell surface GLUT4 levels in insulin resistance. (A)

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Acknowledgments

We thank Rytis Prekeris for anti-syntaxin13 antibody, Hartmut Land for pBABE vector, Toshio Kitamura for Plat-E cells, and Alain Ktorza (Institut de Recherches Servier, Suresnes, France) for helpful suggestions, and acknowledge the C3M MicorBio Cell Imaging Facility. This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and the University of Nice-Sophia-Antipolis, the Conseil Régional Provence-Alpes-Côte d'Azur, and the Conseil Général des Alpes Maritimes. RG is a recipient of an INSERM Avenir grant and LM is supported by an INSERM/Région Provence-Alpes-Côte d'Azur/Servier grant.