Mickael Couty

Mohamed Amiche
email: mohamed.amiche@upmc.fr

Antitumor Activity and Mechanism of Action of the Hormonotoxin, an LHRH Analog Conjugated to Dermaseptin-B2, a Multifunctional Antimicrobial Peptide

Keywords: anticancer drug, antimicrobial peptide (AMP), dermaseptin, frog skin peptides, LHRH, prostate cancer, Phyllomedusa bicolor, therapeutic peptides

Prostate cancer represents the most common cancer in men. For patients with advanced or metastatic form, treatments will be able to slow down the progression but cannot cure it even with the used of new targeted therapies. In this context, the development of innovative drugs resulting from the exploration of biodiversity could open new therapeutic alternatives. Dermaseptin-B2 (DRS-B2), a natural multifunctional antimicrobial peptide isolated from the Amazonian frog skin, has been reported to possess antitumor and antiangiogenic activities. To improve DRS-B2 pharmacological properties and target prostate tumor cells more speci cally, we have developed a chimeric molecule, called Hormonotoxin (H-B2) which is composed of a DRS-B2 combined with a hormonal analog, d-Lys 6 -LHRH, to target LHRH-Receptor which is overexpressed in more than 85% of prostate cancers. In vitro H-B2 has a signi cant antiproliferative effect on the PC3 tumor cell line, with an IC 50 value close to that of DRS-B2. The antitumor activity of H-B2 was con rmed in vivo in mouse model xenografted with PC3 tumors and appears to be better tolerated than DRS-B2. Biophysical experiments showed that the addition of the hormonal analog to DRS-B2 did not alter either its secondary structure or its biological activity. Combination of different experimental approaches indicated that H-B2 induces cell death by an apoptotic mechanism whereas DRS-B2 was shown to induce it by necrosis. These results could explain the H-B2 less toxicity compared to DRS-B2. H-B2 represents a promising targeting approach for cancer therapy.

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Introduction

Prostate cancer (PCa) is the second most frequent cancer diagnosis made in men and the fth leading cause of death worldwide in 2018 1 . For most men with PCa, their disease will follow an indolent course.

The 5-year survival rates are encouraging: 98% and 83% in the USA and Europe, respectively 2 . Localized PCa may be cured with surgery or radionuclide therapy, however, the disease recurs in approximately 20 to 30% of men treated for localized PCa and advanced disease is associated with poor outcomes.

Concerning chemotherapy, the current standard treatment for hormone sensitive metastatic PCa is androgen deprivation by luteinizing hormone-releasing hormone (LHRH) agonists or antagonists that induce castration by blocking the gonadotropic axis. Although most men with metastatic PCa initially respond to this Androgen-Deprivation Therapy (ADT), inevitably their cancer progresses on this treatment to a disease state known as castration-resistant prostate cancer (CRPC). Despite the appearance of a panel of new therapies in this indication since 2010, such as second-generation hormone therapy (abiraterone, enzalutamide), taxane-based chemotherapy (docetaxel, cabazitaxel), immunotherapy (sipuleucel-T), targeted therapy (ipilimumab, an anti-CTLA-4 antibody) and metabolic radiotherapy (Ra-223) for men with bone metastasis, the median survival for men with metastatic castration-resistant prostate cancer (mCRPC) is less than 2 years 2 . In this context, the development of innovative therapies therefore represents a major challenge in the hope of offering to patients a more effective treatment combining low toxicity, reduced side effects and resistance associated with conventional therapies.

Among these new molecules, those from biodiversity could represent a particular interest. A group of interesting peptides from natural sources are antimicrobial peptides (AMPs) [START_REF] Tornesello | Antimicrobial Peptides as Anticancer Agents: Functional Properties and Biological Activities[END_REF] . Indeed, in recent years, an increasing number of articles show that these AMPs are in fact multi-functional peptides such as anticancer agents, immuno-modulators, chemokines, vaccine adjuvants, or regulators of innate defense [4][START_REF] Santos | Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans[END_REF][6] .

From this group of AMPs, the cationic antimicrobial peptides (CAPs) offer a concrete development path derived from biodiversity. Characterized for nearly thirty years, they were initially studied for their antimicrobial virtues. Indeed, a growing number of studies have shown that some of these peptides, which are toxic to bacteria but not to normal mammalian cells, have a broad spectrum of cytotoxic activity against cancer cells. Electrostatic interactions between the positive charges carried by CAPs and the anionic components of cell membranes are considered to be the major elements involved in the selective destruction of cancer cells [START_REF] Hoskin | Studies on anticancer activities of antimicrobial peptides[END_REF] . Among CAPs, dermaseptins B2 and B3 (DRS-B2 and DRS-B3) are 2 natural antimicrobial peptides isolated from the skin of an Amazonian tree frog of the genus Phyllomedusa bicolor 8,[START_REF] Amiche | A consistent nomenclature of antimicrobial peptides isolated from frogs of the subfamily Phyllomedusinae[END_REF] . Our team has initially reported signi cant antitumor activity of DRS-B2 and DRS-B3 on various human cell lines including prostate cancer while they have no effect on normal cells [START_REF] Santos | Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans[END_REF][START_REF] Van Zoggel | Antitumor and angiostatic peptides from frog skin secretions[END_REF] . Furthermore, the anti-tumor activity of DRS-B2 was con rmed in vivo in PC3 prostatic tumor cell line xenografted in athymic mice model. Treatment with DRS-B2 at 2.5mg/kg body weight six times a week in peritumoral reduced the tumor growth of 50% after 35 days. Concerning the mechanism of action of DRS-B2, our previous studies suggested a rapid mechanism of cell death, with aggregation at the plasma membrane of cancer cells and penetration into the cytoplasm and the nucleus [START_REF] Santos | Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans[END_REF][START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF] .

These polycationic peptides, that appear to interact and speci cally cross the membrane of the tumor cells, and with no effect on normal cells represent an innovative technological platform for the development and design of original molecules that can be used in the targeted treatment of cancers resistant to current therapies. However, its in-vivo use could question the problem of signi cant toxicity regarding the doses used. To reduce the toxicity of pharmacological molecule, the targeted therapy could represent a promising way. The concept of hormonotoxin (H-B2) was born from this problem, based on tumor targeting by associating DRS-B2 with a hormone (H) whose receptor is over-expressed on the tumor surface. This concept is related to the immunotoxin approach, which combines a toxin with a monoclonal antibody [START_REF] Pastan | Immunotoxin therapy of cancer[END_REF] . The speci c interaction of the ligand with its receptor would ideally allow targeting tumor cells while optimizing the interaction of the peptide with the membrane. The advantage of peptides is the simplicity of their production by chemical synthesis. Numerous studies have shown that many membrane receptors are over-expressed on the surface of cancer cells and that there are natural or synthetic peptide ligands (agonists or antagonists) that bind to them with very good a nity and selectivity [START_REF] Schally | Use of analogs of peptide hormones conjugated to cytotoxic radicals for chemotherapy targeted to receptors on tumors[END_REF] . This is the case of the luteinizing hormone-releasing hormone receptor (LHRH-R), whose expression rate is greater than 80% in endometrial, ovarian and prostate cancer cells [START_REF] Halmos | High incidence of receptors for luteinizing hormone-releasing hormone (LHRH) and LHRH receptor gene expression in human prostate cancers[END_REF] . Available data strongly suggest that about 91% of prostate cancers express LHRH-R high-a nity binding sites [START_REF] Engel | Drug Insight: clinical use of agonists and antagonists of luteinizinghormone-releasing hormone[END_REF] . In these cancers, in vitro proliferation may be inhibited by the analogues of LHRH in a dose-and timedependent manner [START_REF] Engel | Drug Insight: clinical use of agonists and antagonists of luteinizinghormone-releasing hormone[END_REF][16][START_REF] Völker | Expression of receptors for luteinizing hormone-releasing hormone in human ovarian and endometrial cancers: frequency, autoregulation, and correlation with direct antiproliferative activity of luteinizing hormone-releasing hormone analogues[END_REF] . As a result, LHRH-R appears to be an ideal target for the development of personalized therapeutic treatment of various human cancers. An example is the fusion of LHRH or its analogues with various bacterial and plant toxins that have been used to target and kill cancer cells expressing LHRH receptors [18][START_REF] Curtis | Novel LHRH-receptor-targeted cytolytic peptide, EP-100: rst-in-human phase I study in patients with advanced LHRH-receptor-expressing solid tumors[END_REF][START_REF] Engel | Targeted chemotherapy of endometrial, ovarian and breast cancers with cytotoxic analogs of luteinizing hormone-releasing hormone (LHRH)[END_REF][START_REF] Leuschner | Human prostate cancer cells and xenografts are targeted and destroyed through luteinizing hormone releasing hormone receptors[END_REF][START_REF] Zhu | A conjugate of methotrexate and an analog of luteinizing hormone releasing hormone shows increased e cacy against prostate cancer[END_REF] .

In this paper we reported the design and synthesis of the chimeric peptide H-B2, a molecule composed of dermaseptin-B2 coupled to an LHRH analogue, and studies of its structure and biological activities in vitro and in vivo, as well as the deciphering of its antitumor mechanism of action.

Results

The Hormonotoxin H-B2 induces a signi cant antiproliferative effect on the hormone-resistant prostate tumor cell line PC3 and has a low hemolytic activity.

Preliminary experiments were performed to investigate the existence of a correlation between LHRH receptor expression and the effect of H-B2. For this purpose, we used four different prostate cell lines (PC3, DU145, 22RV1 and BPH-1) which are presented in Table 1. The PC3, DU145, and 22Rv1 cell lines were used as models for androgen-independent prostate cancer. PC3 and DU145 were derived from bone and brain metastasis, respectively. 22Rv1 cells were originally derived from a primary site of an advanced PCa that was serially transplanted in nude mice and BPH-1 is a prostatic hyperplasic cell line. Western blot analysis indicated that LHRH-R is strongly expressed by metastatic PC3 and DU145 cells, but its expression is very low in 22Rv1 and absent in BPH-1 cells (Fig. 2A). These results are in agreement with studies indicating the overexpression of LHRH-R in human prostatic cancer cells and then could be used to evaluate the anti-proliferative effect of H-B2 [START_REF] Völker | Expression of receptors for luteinizing hormone-releasing hormone in human ovarian and endometrial cancers: frequency, autoregulation, and correlation with direct antiproliferative activity of luteinizing hormone-releasing hormone analogues[END_REF] . Dose-response of H-B2 and DRS-B2 after 48 h of treatment was evaluated on the different prostate cell lines expressing or not LHRH-R as indicated in Fig. 2A.

Table 1 List of cell lines and their characteristics

Cell line

Characteristics

PC3

Adenocarcinoma derived from bone metastasis DU145 Adenocarcinoma derived from brain metastasis 22Rv1 Adenocarcinoma derived from a PCa primary tumor that was serially transplanted in nude mice.

BPH-1 Benign Prostatic Hyperplasia Cell Line

Results show that the viability of all cell lines was reduced dose-dependently by DRS-B2 and H-B2 (Fig. 2B). The concentrations of peptides required to inhibit 50% (IC 50 ) of cell proliferation are reported in Fig. 2C. Moreover, comparison of the IC 50 shows that H-B2 was slightly more potent than DRS-B2 against cancer cells that express LHRH-R compared to BPH-1 cells that do not express LHRH receptor (Fig. 2B,2C). Since PC3 cells present a relatively good sensitivity to H-B2 and DRS-B2, we decide to use this cell line for further investigation concerning the in vivo study of H-B2 and its antiproliferative mechanism of action.

To assess the functional role of LHRH-R on the H-B2 antiproliferative activities, LHRH-R was knocked down in PC3 cells. The expression level of LHRH-R was validated by WB analysis (Fig. 3A). The viability test showed that knockdown of LHRH-R in PC3 cells slightly decreased the sensitivity of PC3 cells to H-B2 as compared to si-control or parental PC3 cells who express LHRH-R (Fig. 3B). The calculated IC 50 was 3.73 µM for siLHRH-R PC3 compared to 2.95 µM for si-control PC3 and 3.05 µM for PC3 parental cells.

Collectively, these results indicated that the antiproliferative activity of H-B2 is higher in cells which express LHRH receptor, such as prostate cancer cells.

Since CAPs could present hemolytic activity, H-B2 and DRS-B2 were tested on human red blood cells. The hemolytic activity was determined by incubation of DRS-B2 or H-B2 at different doses with human erythrocytes. In parallel, the erythrocytes were incubated in the presence of 0.2% (v/v) Triton representing the positive control (100% hemolysis). Results presented in Fig. 4 show that DRS-B2 and H-B2 have a low hemolytic activity. Indeed, while some cationic antimicrobial peptides show hemolytic activity, DRS-B2 and H-B2 do not cause more than 10% hemolysis at the highest doses tested (50 µM) and could be considered as low cytotoxicity peptides. This nding is encouraging and essential for in vivo testing and for potential therapeutic use.

Hormonotoxin H-B2 signi cantly inhibits tumor growth in xenografted mice without measurable side effects.

To investigate the antitumor e cacy of the H-B2, we performed in vivo experiments using xenografting PC3 tumor cells in nude mice. Fourteen days after injection of PC3 cells, tumors of around 100 mm 3 were developed. The mice were then randomized into four groups (n = 6 mice/group) and treated by IP injection of 2.5 and 5 mg/kg of H-B2 or 2.5 mg/kg of DRS-B2 or vehicle, 3 times per week for ve weeks.

The results showed that H-B2 has inhibited PC3 tumor growth in a dose-dependent manner (Fig. 5). The inhibition of the tumor growth was about 35% and 54% when mice were treated with H-B2 at 2.5 mg/kg and 5 mg/kg, respectively (Fig. 5A). At the dose of 2.5 mg/kg, H-B2 has a better antitumor effect (35%) than DRS-B2 (26%) (Fig. 5A). Similar results were obtained by analyzing the weights of tumors harvested from tumor-bearing mice (Fig. 5B).

To further de ne the effect of H-B2 on tumor proliferation in vivo, Ki67 labeling was performed on tumor sections from each group. As shown in Fig. 5C, the proliferation index was signi cantly inhibited in H-B2treated mice (35% inhibition at 2.5 mg/kg and 68% at 5 mg/kg) as compared to the control group.

Taken together, these results indicate that HB2 has a better antitumor effect than DRS-B2 and less toxicity in mice (data not shown).

Mechanism of antitumor action of the Hormonotoxin H-B2 in comparison with DRS-B2

The mechanism of antitumor action of these two peptides was addressed by studying, on the one hand, their bioactive structure by three spectroscopic approaches, CD, uorescence, and 2D-NMR in the presence of micelles mimicking the plasma membranes of the target cells and, on the other hand, by measuring the cell viability after double annexin V-FITC and PI staining, the cytotoxic activity by measuring the cytoplasmic LDH released, and the DNA fragmentation induced by these peptides.

Addition of the hormonal analogue (d-Lys 6 -LHRH) to the DRS-B2 did not alter the secondary structure of the hormonotoxin H-B2.

Preliminary indications of the peptides secondary structures obtained by CD measurements in PBS and in zwitterionic detergent (DPC) at different concentrations are shown in Fig. 6. The CD spectrum of DRS-B2 and H-B2 in PBS buffer showed that these peptides have very little ordered structure. However, there is a clear change in the spectra of these two peptides as soon as the critical micellar concentration (CMC) of DPC is reached (Fig. 6A.1 for DRS-B2 and 6B.1 for H-B2) that indicates an enhanced helical content, with minima at 208 and 222 nm. Deconvolution of the spectra allows us to quantify the relative proportions of the secondary structures of our peptides. Thus, in the absence of a micelle in the medium, the observed αhelix ratio is about 11% for DRS-B2 and 10% for HB2 as shown in Fig. 6A.2 and 6B.2, respectively. When the concentration of DPC is increased to 5 mM, the α-helicity rates then reach 25% and 55% for H-B2 and DRS-B2 respectively.

The presence of a W residue in the sequence of DRS-B2 and H-B2 allows us to study the in uence of the microenvironment on the structure of these two peptides. Indeed, the uorescence emission maximum of W is between 320 and 355 nm when excited at 290 nm, and the maximum emission wavelength re ects the exposure of W to the solvent. This uorescence is measured in aqueous solution (PBS 1x) for observation in a non-structural environment (the peptides does not form an α-helix in water) and in micellar solution to study the effect of a lipid-like microenvironment (Fig. 6A.3 and 6B.3). We observe that beyond 1 mM, which is the CMC of DPC, the uorescence emission maxima of DRS-B2 and H-B2 shift to shorter wavelengths ("blue shift") and show a strong increase in uorescence intensity (hyperchromic shift). These spectral changes re ect a change from hydrophilic to hydrophobic environment that can be explained either by the burial of the W residue within the hydrophobic layers of DPC micelles, or by burying the W after folding of the peptide following its conformational changes.

Finally, the structure of H-B2 was investigated using NMR in micellar DPC solution. This technique allows a direct analysis of peptide conformation and exibility at the residue level. For each residue of the peptide chain, the chemical shift deviation (CSD) of the Hα protons were calculated (Fig. 7A). This corresponds to the difference between the observed chemical shift of the Hα protons and the "random coil" chemical shift (obtained in the absence of structuring and corresponding to values taken from a library of short unstructured peptides). CSD values which deviate from 0 ppm (< - 0.1 or > + 0.1 ppm) are typically used to detect the presence of secondary structures. In the DRS-B2 portion of hormonotoxin H-B2, most residues exhibit signi cantly negative Hα CSD values (average - 0.23 ppm), indicating the presence of stable helical conformations, as evidenced by CD. H-B2 forms an α-helix along virtually the entire length of the DRS-B2 part (residues 2-31), with greater stability in the rst half of the helix. The CSD values are very close to those observed for DRS-B2 alone, proving that the C-terminal attachment of the LHRH segment does not affect the helical structure of the DRS-B2 segment. On the other hand, the LHRH segment appears to be less structured, with possibly the formation of turn structures.

The linewidth of HN protons was also analyzed by measuring the intensity of [START_REF] Engel | Drug Insight: clinical use of agonists and antagonists of luteinizinghormone-releasing hormone[END_REF] N-1 H crosspeaks on natural abundance [START_REF] Engel | Drug Insight: clinical use of agonists and antagonists of luteinizinghormone-releasing hormone[END_REF] N-1 H 2D HSQC spectrum (Fig. 7B). The proton linewidth is in uenced by peptide dynamics and interaction with the larger molecular weight DPC micelle. The LHRH segment shows a different behavior from the DRS-B2 segment in H-B2, with stronger [START_REF] Engel | Drug Insight: clinical use of agonists and antagonists of luteinizinghormone-releasing hormone[END_REF] N-1 H crosspeaks intensities. This shows that the LHRH segment is more dynamic and does not appear to strongly interact with the micelle.

The cell death induced by H-B2 and DRS-B2 uses two different mechanisms that are related to apoptosis and necrosis, respectively.

To obtain more information about how H-B2 acts on PC3 tumor cells, the anti-proliferative mechanism of action of this peptide was studied in comparison with that of DRS-B2. Since the known anti-proliferative effect of DRS-B2 on tumor cells resembled more to a membrane killing-like effect, experiments concerning the cytotoxic effect, the induction of apoptosis or necrosis were investigated with H-B2 in comparison with DRS-B2.

A key feature of cells undergoing apoptosis, necrosis, and other forms of cellular damage could be analyzed by measuring the activity of cytoplasmic lactate dehydrogenase (LDH) released by damaged cells. Previously, we have shown that DRS-2 increased this release [START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF] . Thus, PC3 cells were treated with different concentrations (1, 5, 7.5 and 10 µM) of H-B2 or DRS B2 for 24 h and the amount of released cytoplasmic LDH into the medium was measured (Fig. 8). Cells treated with Triton X100 0.9% (v/v) were used as an internal positive control. The results show a low release of cytoplasmic LDH when the PC3 cells are treated with 1 µM of H-B2 or DRS-B2 with 15 and 10% release respectively. The LDH release is maximal upon addition of 5 µM or higher doses of these peptides. However, this maximum release was limited to 65-70%. These data suggested that the cytotoxic effect of H-B2 on PC3 cells is comparable to that of DRS-B2.

To further characterize the effect of H-B2 and DRS-B2 on cell death in PC3 cells, we rst examined the viability of cells treated with these peptides by ow cytometry using annexin V-FITC and PI double staining of the cells. As shown in Fig. 9, ow cytometry analysis showed that H-B2 promoted cell apoptosis in a dose-dependent manner. After being treated with H-B2 for 24 h, 4%, 15%, and 63% of double staining A-V + /PI -which corresponds to early apoptotic cells were found in PC3 cells treated with 1 µM, 2.5 µM, and 5 µM, respectively. These values were signi cantly higher than that of untreated cells (1%) and similar (63.5%) to cells treated with H 2 O 2 used as a positive control. DRS-B2 presented a lower effect with only 5.5% of double staining A-V+/PI-when PC3 cells were treated with DRS-B2 at 2.5 µM, which agrees with results described by Van Zoggel et al [START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF] .

DNA fragmentation represents the nal stage of apoptosis. To examine apoptosis through DNA fragmentation induced by H-B2 or DRS-B2, we used the terminal deoxynucleotidyl transferase (Tdt) dUTP Nick-End Labeling (TUNEL) assay, in which DNA strand breaks are detected by enzymatic labeling of free 3'-OH ends with modi ed nucleotides.

The analysis of DNA fragmentation in PC3 cells by the TUNEL technique shows that the percentage of fragmented DNA increases with H-B2 treatments. Treatment of PC3 cells with 5 µM H-B2 induces a DNA fragmentation level of 69% compared to untreated cells (1.5%), whereas it reaches only 9% when treated with the same concentration of DRS-B2. (Fig. 10). PC3 cells treated with 10 nM Taxotere or 1 µM of Staurosporin as positive controls show 96% and 86% DNA fragmentation, respectively (Fig. 10). These data con rm that the mechanism of PC3 cell death induced by H-B2 is different from that induced by DRS-B2, which is necrotic, and could rather correspond to an apoptotic mechanism, in agreement with the ow cytometry results described previously.

Discussion

With 1.1 million prostate cancers diagnosed worldwide in 2012, of which approximately 6% are metastatic from the start, the development of treatments for mCPRC is a global necessity with a promising market. The development of treatments for the management of castration-resistant metastatic patients is necessary because currently available treatments only allow a few months of survival gain mainly due to problems of drug resistance of cancer cells. In this context, the use of natural or synthetic peptides could offer interesting therapeutic approaches. To limit peripheral toxicity and increase local concentration, numerous cytotoxic molecules conjugated with peptide hormones such as LHRH or somatostatin whose receptors are widely overexpressed on the surface of tumor membranes have been developed [START_REF] Schally | Cancer chemotherapy based on targeting of cytotoxic peptide conjugates to their receptors on tumors[END_REF][START_REF] Schally | Peptide analogs in the therapy of prostate cancer[END_REF][START_REF] Nagy | Targeting of cytotoxic luteinizing hormone-releasing hormone analogs to breast, ovarian, endometrial, and prostate cancers[END_REF][26][START_REF] Seitz | Preclinical evaluation of properties of a new targeted cytotoxic somatostatin analog, AN-162 (AEZS-124), and its effects on tumor growth inhibition[END_REF] . Currently, only one cytotoxic peptide is in clinical development (Phase 2 completed), AEZS-108 (zoptarelin doxorubicin) 28 .

Starting from our previous studies on the anticancer activity of the CAP dermaseptin-B2 (DRS-B2), a natural peptide issued from the biodiversity, we have developed a synthetic chimeric H-B2 peptide combining DRS-B2 and LHRH peptides [START_REF] Santos | Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans[END_REF][START_REF] Van Zoggel | Antitumor and angiostatic peptides from frog skin secretions[END_REF][START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF] . The use of LHRH was justi ed by the fact that 86% of prostate cancers express the LHRH receptor. A peptide targeting this receptor therefore should increase the speci city for cancer cells and decrease the in vivo toxicity of DRS-B2.

At the structural level, the coupling of the peptide hormone (H) LHRH to the C-terminus of DRS-B2 does not modify the structure of DRS-B2 nor its conformational behavior in a membrane environment. The characteristics essential to the cytotoxic mode of action of these polycationic peptides and to the targeted molecular interaction are therefore preserved. CAPs are supposed to act on cancer cells in which the outer layer of the plasma membrane is highly negatively charged as for bacteria plasma membranes.

In the case of cancer cells, the negatively charged membrane is due to the presence of phosphatidylserine, negatively charged mucin proteins or highly sulfated GAGs (5, 25). After binding dermaseptin peptides accumulate in a carpet like manner on the outside of a lipid bilayer until a threshold concentration is reached, causing them to form pores in which the peptides are inserted with the phospholipid headgroups of the membrane. We have shown that H-B2 was structured as a continuous helix in its "toxin" portion (DRS-B2) and that the "hormone" ligand portion remains free to interact with LHRH-R.

In vitro, H-B2 was slightly more effective than DRS-B2 on the proliferation of the various prostate cancer cell lines tested. Even if the speci city of interaction with LHRH-R could not be demonstrated physically, the knock down expression of the LRHR receptor by RNA interference on PC3 cells slightly decreases the e ciency of H-B2, thus pointing out that addition of the peptide hormone to the DRS-B2 was bene t. We could also mention that cells expressing high level of LHRH receptor like PC3 and DU145 presented a modest but signi cative gain in IC 50 when compared with those of DRS-B2 alone. Targeting cancer cells with an overexpressed receptor like LHRH is a strategy that could permit to improve the e ciency of a peptide like DRS-B2 to gain in its ED 50 . However, in the case of H-B2, this gain is low. We could imagine that even if the LHRH receptor is overexpressed on PC3 cells, its concentration at the surface of the plasma membrane is too low to guide enough DRS-B2 to membrane to complete its carpet like structure to further form pores in the plasma membrane. Since saturation of the LHRH receptor by H-B2 could not be su cient to kill cells, therefore, additional part of DRS-B2 in H-B2 structure could be necessary to do it.

In vivo H-B2 inhibited tumor growth by more than 50% and signi cantly decreased proliferation without any major side effects. Since the addition of the hormone peptide to the DRS-B2 permits a better tolerance when injected in mice, we could conclude that despite signi cant improvement in IC 50 on the cell line, the hormone peptide improved the in vivo activity of DRS-B2 since we could treat mice with 5 mg/kg H-B2 without side effects instead of 2.5 mg/kg with DRS-B2. It is interesting to note that the treatment of prostate tumor cells with H-B2 is globally well tolerated. We did not observe any major abnormalities in the blood workup after H-B2 injection. Importantly, at a dose of 10 mg/kg, DRS-B2 is lethal while H-B2 at the same dose was well tolerated in toxicity tests, with no behavioral changes in the mice or weight loss. The design of the H-B2 chimeric peptide thus made it possible to circumvent the toxicity of DRS-B2 while maintaining its anti-tumor e cacy.

Concerning the mechanism of action, the combination of different experimental approaches used in this study such as cell viability by ow cytometry, cytotoxicity by cytoplasmic LDH release, and DNA fragmentation by TUNEL essay allowed us to show that the mechanism of cell death induced by H-B2 is probably different from that of DRS-B2 and might be similar to apoptosis. Indeed, we showed that both peptides have the same effect on cytoplasmic LDH release but a distinctly different cell labeling when cell viability is analyzed by ow cytometry and DNA fragmentation by TUNEL assays. The differences between the effect of the two peptides observed in the TUNEL assay show that H-B2 induces DNA fragmentation up to 70% in contrast to DRS-B2 which is less than 10%. These results are also in agreement with those obtained in 2012 by Van Zoggel et al who showed a double Annexin V+/PI+ labeling of PC3 cells treated with DRS-B2, suggesting a necrotic cell death mechanism [START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF] .

Apart from DRS-B2 previously studied in the laboratory, two new members of the dermaseptin family have been recently reported to exhibit antitumor activities: Dermaseptin-PP from Phyllomedusa palliata and Dermaseptin-PT9 from Phyllomedusa tarsius [START_REF] Dong | Novel Frog Skin-Derived Peptide Dermaseptin-PP for Lung Cancer Treatment: In vitro/vivo Evaluation and Anti-tumor Mechanisms Study[END_REF][START_REF] Li | A Novel Dermaseptin Isolated from the Skin Secretion of Phyllomedusa tarsius and Its Cationicity-Enhanced Analogue Exhibiting Effective Antimicrobial and Anti-Proliferative Activities[END_REF] . Both exhibited antiproliferative activity against various human tumor cells, rapid LDH release activity, and an apoptosis-like cell death mechanism. Among these dermaseptins, the DRS-B2 presented the higher ED 50 on various tumor cells.

Finally, the study of the hemolytic effect of the different peptides allowed us to observe that both DRS-B2 and H-B2 have a low hemolytic activity, which is encouraging in the perspective of a therapeutic approach. Further in vivo studies on mice could be necessary to obtain more pharmacokinetic and toxicology information to complete the study.

Experimental Procedures Peptide synthesis.

The LHRH analogue containing a dK residue (d for D con guration of the Lys residue) in position 6

(peptide A: pEHWSY(dK)LRG-amide) and DRS-B2 (peptide B:

GLWSKIKEVGKEAAKAAAKAAGKAALGACSEAV-acid) were synthesized by X'PROCHEM Company (Lille, France). The chimeric peptide H-B2 (Fig. 1A) composed of the peptide A grafted by its epsilon NH 2 of the dK 6 residue with the COOH-terminal of the peptide B is determined to be >95% pure by RP-HPLC (Fig. 1B) and its molecular weight determined by ESI-mass spectrometry is [M+4H]/4=1105.2 (Fig. 1C). The peptide is dissolved in sterile water for experimental use.

Circular dichroism (CD) spectroscopy of peptides.

The helical structure of HB2 and DRS-B2 were analyzed by CD spectroscopy using a Jobin Yvon CD6 dichrograph linked to a PC micro-processor as described [START_REF] Galanth | Mechanism of antibacterial action of dermaseptin B2: interplay between helixhinge-helix structure and membrane curvature strain[END_REF] . Brie y, measurements were calibrated with (+)-10-camphorsulfonic acid and performed with 10 mM HB2 or DRS-B2 diluted in PBS alone or with increasing concentrations of dodecylphosphocholin (DPC) (10, 30, 100, 1000 and 5000 μM) at 25 C using a quartz cuvette (Hellma) with a path length of 0.1 cm. Spectra, recorded in 1 nm steps, were averaged over ve scans, and corrected for the base line. The CD spectra were deconvoluted using CDNN Software [START_REF] Böhm | Quantitative analysis of protein far UV circular dichroism spectra by neural networks[END_REF] . Circular dichroism measurements are reported as Δε/n, where Δε is the dichroic increment (M -1 cm -1 )

and n is the number of residues in the peptide. The α-helix content of peptides was obtained using the relation: Pα = -[Δε 222nm x10] (Pα: percentage of α-helix; Δε 222nm : dichroic increment per residue at 222 nm) [START_REF] Zhong | Environment affects amino acid preference for secondary structure[END_REF] .

Fluorescence of tryptophan-containing peptides performed as previously described by Dos Santos et al. [START_REF] Santos | Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans[END_REF] Emission spectra were recorded on a Jobin-Yvon Fluoromax II instrument (HORIBA Jobin-Yvon, France) equipped with an Ozone-free 150 W xenon lamp. The excitation wavelength was 290 nm, and the emission spectra were acquired at 300-360 nm. At least ve measurements for each titration point were recorded with an integration time of 1 s. The HB2 and DRS-B2 concentration in PBS was 2 μM, and the DPC concentration varied from 0 to 1000 μM. Tryptophan uorescence was determined by subtracting spectra without peptide.

Nuclear Magnetic Resonance (NMR) Spectroscopy. Cell culture.

Human PCa cell lines PC3, DU145 and 22Rv1 were purchased from ATCC (American Type Culture Collection and were cultured in RPMI supplemented with 10% of fetal bovine serum (FBS). Human hyperplasic cell line BPH1 was purchased from the German Collection of Cell Cultures (DSMZ, Braunschweig, Germany). BPH1 were maintained in RPMI 1640 supplemented by 10% FBS, 20 ng/mL testosterone and 1% insulin-transferrin-selenium. Cell cultures were maintained at 37°C and 5% CO 2 in humidi ed atmosphere. All culture reagents were purchased from Life Technologies (Cergy-Pontoise, France). All experiments were performed as previously described by Dos Santos et al. [START_REF] Santos | Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans[END_REF] Cell-viability assays.

Cells were seeded at a density of 5×10 3 cells/well in 96-multiwell plates in complete medium and incubated for 24 h at 37˚C in a controlled humidi ed 7% CO 2 environment. Cells were then treated with DRS-B2 or HB2 as indicated, for 48 h. Cell viability was measured using the 3-(4,5-dimethylthiazol2-yl)diphenyltetrazolium bromide (MTT) dye method (Sigma, Saint Quentin Fallavier, France) according to the manufacturer's instructions. Each experiment was performed in triplicate and at least three independent experiments as previously described by Dos santos et al. [START_REF] Santos | Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans[END_REF] . IC 50 values were determined by GraphPad Prism 5.0 (GraphPad Software, USA).

Small RNA (siRNA) Assay.

PC3 cell silencing of LHRH-R were performed using Lipofectamine RNaiMax (Invitrogen) according to the manufacturer's protocol as previously described by Elahouel et al. [START_REF] Elahouel | Pleiotrophin exerts its migration and invasion effect through the neuropilin-1 pathway[END_REF] . Predesigned siRNAs of either nontargeting siRNA (GGCUACGUCCAGGAG CGCACCTT) as a control or target-speci c siRNAs (AAGCAUGGAUUGGAUCAGUAATT) to knock-down LHRH-R were obtained from Euro ns Genomics.

Brie y, cells were plated overnight at 50-60% con uence, then transfected with 10 nM of either nontargeting siRNA or LHRH-R target-speci c siRNAs, in serum-and antibiotic-free Opti-MEM medium (Invitrogen). After 24 h, transfected cells were treated with HB2 as indicated for 24 h. In addition, transfection e ciency was evaluated by Western blot analysis 24-48 h after transfection.

Western blotting was performed as previously described by Elahouel et al. [START_REF] Elahouel | Pleiotrophin exerts its migration and invasion effect through the neuropilin-1 pathway[END_REF] Cell lysates were prepared with RIPA extraction buffer containing anti-protease inhibitors (Roche). Equal amounts of total proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, then transferred onto polyvinylidene di uoride membrane (Millipore) using standard protocols. Nonspeci c binding was prevented by incubating the membranes in 5% (w/v) nonfat milk in PBS containing 0.2% (v/v) Tween-20 (blocking buffer (BB) for 1 h and then incubated with primary antibodies in diluent (5% nonfat milk in TBS-Tween) for 1 h. Membranes were subsequently incubated overnight at 4°C with antibodies, rabbit anti-GnRH-R (Abcam,183079 ; 1 : 2000) or mouse anti-β-actin (SantaCruz Biotechnology; 1 : 2000) and then with HRP-conjugated secondary antibody for 1h at RT (Cell Signaling Technology ; 1:2000). Immunocomplexes were revealed using a chemiluminescence detection system kit "SuperSignal™ West Pico PLUS" (Thermoscienti c) and visualized using the GBox system (Syngene).

Hemolysis assessment.

The hemolytic activity of H-B2 and DRS-B2 was determined using fresh human erythrocytes from a healthy donor that was prepared as follows: 4 mL of whole blood was collected and centrifuged at 900 g for 10 min at 4°C to separate plasma from erythrocytes. The pellet was then rinsed with PBS pH 7.4 and centrifuged at 900 g for 10 min at 4°C. After counting and making a red cell solution at 4 × 10 8 erythrocytes/mL (diluted in PBS pH 7.4), 50 µL of a diluted peptide solution was added in cascade to which 50 µL of the erythrocyte solution was added (made in triplicate). After 1 hour of incubation at 37°C, the tubes were centrifuged at 12 000 rpm for 15 seconds at 4°C. The supernatant was then recovered. The hemoglobin present in the erythrocytes was determined in the supernatants via a plate reader at 450 nm. A parallel incubation in the presence of 0.2% (v/v) Triton was carried out to determine the absorbance associated with 100% hemolysis. Immunohistochemical analysis of the tumors were performed as previously described by Van Zoggel et al. [START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF] . The xed tumors were embedded in para n and then 6 µm sections were prepared. The tumors sections were depara nized, antigen unmasking was performed, and endogenous peroxidase activity was inactivated with a 2% hydrogen peroxide solution for 10 minutes. Unspeci c staining was blocked using Power Block Universal reagent (Biogenex Laboratories/Microm Microtech, Francheville, France) for 10 min at 37°C. Tissues were then incubated 2 hours at room temperature with anti-human Ki67 antibody (Mouse monoclonal, M7240, Dako, 1:50) for 2 hours. Immuno-complexes were revealed using HRP conjugated secondary antibodies and the DAB substrate. Tissues were then counterstained with hematoxylin and cover slipped with Mowiol mounting medium. Quanti cation of Ki67 positive stained cells was quanti ed by image J software analysis on the whole tumor section.

Lactate dehydrogenase (LDH) release assay.

The cytoplasm LDH release assay was performed as previously described [START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF] . Brie y, PC3 cells were grown in a 96 well plate (1,500 cells/well/100 μL) in complete medium and treated with various concentrations of H-B2 or DRS-B2. Cell membrane integrity was evaluated by measuring the LDH activity released into the culture media 24 hours after peptide exposure. The CytoTox96 non-radioactive cytotoxicity assay (Promega; Charbonnières-les-Bains, France) was performed according to the manufacturer's instructions and quanti ed by measuring the absorbance at 490 nm. The 100% cytotoxicity corresponds to the LDH released with treatment of the cells with Triton X100 at 0.9% (v/v).

Apoptosis analysis by ow cytometry.

For the apoptosis assay, an FITC-Annexin-V (A-V) and Propidium Iodide (PI) double staining method was used. PC3 cells were grown in a 12-well plate and treated or not with H-B2, DRS-B2 or hydrogen peroxide as described previously [START_REF] Santos | Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans[END_REF] . 24 hours after exposure, cell viability was evaluated by ow cytometry analysis with FITC-Annexin-V (A-V) and propidium iodide staining. Medium and trypsinized cells were collected and washed with PBS. After centrifugation, cells were suspended in PBS to obtain a cell density of 0.5 × 106 cells per mL. One mL of this cell extract was centrifuged, suspended in 200 μL PBS, transferred to a microtiterplate with round bottom and centrifuged again. Resulted cell pellet was resuspended in 200 μL of Binding Buffer 1x (BD PharmingenTM), containing 5 μL of FITC-Annexin-V (BD PharmingenTM) and incubated during 10 minutes in dark at room temperature. The cells were washed with PBS and incubated with 200 μL of Binding Buffer 1x containing PI ( nal concentration 1 μg/mL) (BD Pharmin-genTM)) for 5 minutes in dark at room temperature. Flow cytometry analysis was performed with LSR Fortessa X20 analyzer (BD Biosciences) and FlowJo V10 software.

Terminal transferase-mediated dUTP Nick End-Labelling (TUNEL) assay.

PC3 cells were cultured in 6-well plates at 150,000 cells per well in complete medium and treated or not

The

  NMR sample was prepared in 550 μL of H 2 O/D 2 O (90:10 v/v) in the presence of 85 mM DPC-d 38 (d 38-dodecylphosphocholin, MAPCHO â -12-d 38 , Avanti Polar Lipids) and using a peptide concentration of 1 mM. The pH was set to 4.1 using microliter amounts of NaOH 0.1 M. DSS was added at a concentration of 0.11 mM for chemical shift calibration. The NMR spectra were acquired at 45°C on a 500 MHz (11.7 T) Avance III Bruker â spectrometer equipped with a TCI cryoprobe. The following experiments were recorded: 2D 1 H-1 H TOCSY (66 ms mixing time), 2D 1 H-1 H NOESY (150 ms mixing time), 2D natural abundance[START_REF] Engel | Drug Insight: clinical use of agonists and antagonists of luteinizinghormone-releasing hormone[END_REF] N-1 H and 2D[START_REF] Schally | Use of analogs of peptide hormones conjugated to cytotoxic radicals for chemotherapy targeted to receptors on tumors[END_REF] C-1 H HSQC experiments, as described[START_REF] Galanth | Mechanism of antibacterial action of dermaseptin B2: interplay between helixhinge-helix structure and membrane curvature strain[END_REF] .

with 1 or 2 . 5 Figure 1

 251 Figures

Figure 2 of

 2 Figure 2

Figure 5

 5 Figure 5

Figure 7 Structure

 7 Figure 7

Figure 8

 8 Figure 8

Figure 9 PC3

 9 Figure 9

  Tumor xenograft Studieswere performed as previously described by Van Zoggel et al.[START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF] . PC3 (2 × 10 6 ) cells were injected subcutaneously into the right ank of 4-week-old male NMRI nude mice (Janvier, Le Genest-Saint-Isle,

	France). When the tumor volume reached approximately 100 mm 3 , the mice were randomly divided into
	four groups (n=6): control (PBS), DRS-B2 (2.5 mg/kg), H-B2 (2.5 mg/kg), H-B2 (5 mg/kg) by
	intraperitoneal injection twice a week. Tumor size was measured two times per week with a caliper and
	tumor volume was calculated with the formula: V= 4/3π*R1 2 *R2 whereby radius 1 (R1), radius 2 (R2). At
	the end of the experiment, the mice were sacri ced, and body weight was measured. The tumors were
	isolated, weighted and then xed in formalin.
	Ethics statement.
	mouse experiments were performed according to ARRIVE guidelines on animal care and approved by
	Charles Darwin Animal Experimentation Ethics Committee (CEEACD/N°5) and conducted in compliance
	with European Community.

ACKNOWLEDGMENTS

We thank Aurelie Guguin and Adeline Henry from the Flow Cytometry facility of IMRB (institute Mondor de recherche biomedicale).

a second labeling with DAPI, intercalating DNA was performed by incubating for 15 minutes at room temperature with a 1 µg/mL solution. The different labelling steps were performed avoiding any exposure to light.

Imaging was performed with an Axiolmager M2 epi uorescence microscope. The images obtained by microscopy were recorded with the Zen 2012 software and analyzed with the ImageJ© software. Each image underwent a thresholding of uorescence calibrated on the positive control, and which was preserved for all other images. The software then analyzed the number of DAPI and uorescein uorescence events to calculate the ratio between the total number of DAPI-labeled cell nuclei and the number of uorescein-labeled nuclei with fragmented DNA. The experiment was performed in triplicate.

Statistical analysis.

The statistical analyses were performed using the GraphPad PrismTM version 4.00 software from GraphPad Software Inc. (San Diego, CA, USA). The results are expressed as the means ± standard deviation (SD) or standard error mean (SEM) of at least three determinations for each test from three independent experiments. Statistical analyses were carried out using the unpaired t-test. The statistical signi cance of the differences is given as *p < 0.05; ** p < 0.01; ***p < 0.001; ns: not signi cant.

COMPETING INTERESTS:

The authors have declared that no competing interests exist.


Marc Cougnon

Romain Carcy

Nicolas Melis

Isabelle Rubera

Christophe Duranton

Karine Dumas

Jean-François Tanti

Catherine Pons

Nicolas Soubeiran

Marina Shkreli

Thierry Hauet

Luc Pellerin

Sébastien Giraud

Nicolas Blondeau

Michel Tauc
email: michel.tauc@univ-cotedazur.fr

Didier F Pisani

Inhibition of eIF5A hypusination reprogrammes metabolism and glucose handling in mouse kidney

Keywords: metabolic switch, GC7, GLUT, glycolysis, glucose reabsorption, kidney

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The eukaryotic initiation factor eIF5A is the only protein known to be activated by the post translational transformation of a specific lysine residue to hypusine through the so-called hypusination process [START_REF] Park | Identification of hypusine, an unusual amino acid, in a protein from human lymphocytes and of spermidine as its biosynthetic precursor[END_REF] which is mediated by the successive catalytic action of deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) [START_REF] Park | Hypusine, a polyamine-derived amino acid critical for eukaryotic translation[END_REF] . A unique feature of eIF5A is that it seems necessary for translation of a limited number of mRNA through its ability to bind RNA in a sequence specific manner [START_REF] Xu | Identification of mRNA that binds to eukaryotic initiation factor 5A by affinity co-purification and differential display[END_REF] . eIF5A was also shown to be involved in the synthesis of only 5% of the total protein content in mammalian cells [START_REF] Li | eIF5A Promotes Translation Elongation, Polysome Disassembly and Stress Granule Assembly[END_REF] and particularly those containing polyproline sequences [START_REF] Gutierrez | eIF5A promotes translation of polyproline motifs[END_REF] . eIF5A is present as two isoforms: eIF5A1 which is ubiquitously expressed and eIF5A2 which is restricted to few tissues and is a hallmark of numerous types of cancer [START_REF] Nakanishi | Targeting the polyamine-hypusine circuit for the prevention and treatment of cancer[END_REF] . eIF5A and its hypusination step has been also described as involved in HIV-1 replication [START_REF] Schroeder | In silico design, synthesis, and screening of novel deoxyhypusine synthase inhibitors targeting HIV-1 replication[END_REF] , malaria disease [START_REF] Kaiser | Translational control of eIF5A in various diseases[END_REF] or diabetes [START_REF] Maier | The unique hypusine modification of eIF5A promotes islet beta cell inflammation and dysfunction in mice[END_REF][START_REF] Mastracci | Hypusinated eIF5A is expressed in the pancreas and spleen of individuals with type 1 and type 2 diabetes[END_REF] . Interestingly, a strong relationship between hypoxia tolerance and the hypusination of eIF5A has been highlighted in drosophila model [START_REF] Vigne | The role of polyamines in protein-dependent hypoxic tolerance of Drosophila[END_REF] . Considering this new concept we have recently shown in mammals that the specific inhibition of eIF5A hypusination by the spermidine analogue N1-guanyl-1,7-diamine-heptane (GC7) [START_REF] Lee | Branched-chain and unsaturated 1,7-diaminoheptane derivatives as deoxyhypusine synthase inhibitors[END_REF] is able to enhance the ischemic tolerance both at the cellular and tissue level in a rat kidney model of ischemia/reperfusion injury [START_REF] Melis | Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome[END_REF] .

Based on these results, we successfully used GC7 both in preclinical model of kidney transplantation in pig [START_REF] Giraud | The inhibition of eIF5A hypusination by GC7, a preconditioning protocol to prevent brain death-induced renal injuries in a preclinical porcine kidney transplantation model[END_REF] and in a transient focal cerebral ischemia (tFCI) model in mice [START_REF] Bourourou | Inhibition of eIF5A hypusination pathway as a new pharmacological target for stroke therapy[END_REF] . Indeed, acute systemic administration of GC7 in the donor allowed better functional recovery of the kidney graft in the first case [START_REF] Melis | Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome[END_REF][START_REF] Giraud | The inhibition of eIF5A hypusination by GC7, a preconditioning protocol to prevent brain death-induced renal injuries in a preclinical porcine kidney transplantation model[END_REF] and reduced the infarct volume, and motor and cognitive post-stroke deficits in the second [START_REF] Bourourou | Inhibition of eIF5A hypusination pathway as a new pharmacological target for stroke therapy[END_REF] . At the cellular level one of the first observations reported on cultured mouse kidney cells treated with GC7 was a metabolic shift from aerobic oxidative phosphorylation toward anaerobic glycolysis decreasing consequently oxygen consumption. In parallel, GC7 treated kidney cells displayed a mitochondrial remodeling characterized by a downregulation of respiratory chain complexes expression and activity while the ATP content remained high [START_REF] Melis | Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome[END_REF] . These observations have been recently corroborated in macrophages [START_REF] Puleston | Polyamines and eIF5A Hypusination Modulate Mitochondrial Respiration and Macrophage Activation[END_REF] .

In vertebrates the ischemia tolerance depends in part upon their ability to urgently use anaerobic glycolysis to ensure their energy supply. This switch is time dependent and not adapted to an acute episode of ischemia. Thus, the ischemic tolerance reported through GC7 treatment suggests a pharmacological conditioning metabolic effect. Nonetheless, the molecular mechanism that support this process remains to be unraveled. Proximal cells manage glucose reabsorption while they do not use it as a source of energy for their own [START_REF] Uchida | Substrate specificity to maintain cellular ATP along the mouse nephron[END_REF] . The transepithelial transport of glucose occurs in two steps [START_REF] Ghezzi | Physiology of renal glucose handling via SGLT1, SGLT2 and GLUT2[END_REF] : 1) an apical membrane entry via SGLT1 and SGLT2, two sodium-linked cotransporters 2) a basolateral export mediated by the facilitated glucose transporters GLUT1 and GLUT2. In this study, we aimed to understand how kidney cells conciliate glucose reabsorption function and anaerobic glycolysis under GC7 treatment. We finally propose that the pharmacological inhibition of eIF5A hypusination by GC7 reversibly reprograms the use of glucose by the proximal cells toward glycolysis parallelly to an impairment of renal glucose reabsorption due to GLUT1 misexpression. This leads to a deep and reversible cell metabolism remodeling allowing survival of kidney cells to oxygen deprivation. These results highlight a therapeutic opportunity in conditions of glucose homeostasis unbalance.

Material & Methods.

Reagents N-guanyl-1,7-diaminoheptane (GC7) was synthesized by AtlanChim Pharma (Saint-Herblain, France) according to the methods described by Jasiulionis et al. [START_REF] Jasiulionis | Inhibition of eukaryotic translation initiation factor 5A (eIF5A) hypusination impairs melanoma growth[END_REF] Canagliflozin was purchased from InVitrogen (Cergy Pontoise, France). Culture media, buffer solutions, fetal bovine serum (FBS) and other culture reagents were from Sigma-Aldrich (Saint-Quentin Fallavier, France).

Animals

The experiments were conducted in accordance with the French and European regulations (2010/63/EU directive) for the care and use of research animals and were approved by national experimentation committees (MESR N°: APAFIS#22670-2019101811258232). 10-week-old C57BL/6 male mice from Janvier Laboratory (France) were maintained at housing temperature (22°C) and 12:12-hour light-dark cycles, with ad libitum access to food and water.

Mice were daily treated with GC7 (3 mg/kg in saline solution, intraperitoneal injection, n=12, "GC7" group) or with vehicle only (saline solution NaCl 0.9% w/v, intraperitoneal injection, n=12, "ctrl" group) for three days. After the first injection, mice were individually housed and acclimated alone in calorimetric cages (Oxylet-Physiocage Panlab, Bioseb, Vitrolles, France) for 24 hours before experimental measurements.

Cell Culture

Renal proximal convoluted tubule cells (PCT) were obtained from primary cultures of murine proximal tubule segments, immortalized with pSV3neo vector and were cultured as previously described [START_REF] Barrière | Swelling-activated Chloride and Potassium Conductance in Primary Cultures of Mouse Proximal Tubules. Implication of KCNE1 Protein[END_REF][START_REF] Barrière | CFTR null mutation altered cAMP-sensitive and swelling-activated Cl-currents in primary cultures of mouse nephron[END_REF] . Cultures were classically maintained in a 5% CO2/95% air water-saturated atmosphere in M1 medium (DMEM/F12, Glutamine, SVF, EGF, T3, dexamethasone, ITS, G418).

All experiments were performed the day after cell confluence.

Cell metabolism analysis

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of PCT cells were determined using an XF24 Extracellular Flux Analysedr (Seahorse Bioscience). Uncoupled and maximal OCR were determined using oligomycin (1.2 µM) and FCCP (1 µM). Rotenone and Antimycin-A (2 µM each) were used to inhibit mitochondrial respiration. All parameters were calculated as described previously [START_REF] Brand | Assessing mitochondrial dysfunction in cells[END_REF] .

Cell survival analysis

The Fluorescent LIVE/DEAD® Cell Viability/Cytotoxicity Assay Kit (Invitrogen, France) was used on PCT cells (24-well plates), according to the manufacturer's protocol. Fluorescent micrographs were recorded using an observer D1 microscope (Zeiss, Germany) and analysed using imageJ software.

Protein analysis

Whole proteins from cells and tissues were prepared using TNET lysis buffer (25 mM Tris-Cl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% Nonidet P40, 1x protease inhibitor cocktail and 1x Phosphostop mix (Roche Diagnostics, Meylan, France)). In addition, tissues were solubilized using Precellys tissue homogenizer in ice-cold buffer and using CK14 beads (Bertin Technologies, Ozyme). For membrane protein enriched lysates, cells were washed twice with cold PBS, once with cold water and finally incubated in hypotonic buffer (Tris 10 mM, EDTA 1 mM, pH7.5, 1x protease inhibitor cocktail) 10 min on ice. Cells were scratched and disrupted using a 25G needle. Crude lysate was centrifuged first at 2000 g (10 min, 4°C) to eliminate undisrupted cells and nuclei, and then at 10000 g (30 min, 4°C). Pellet containing membranes was resuspended in hypotonic buffer containing protease inhibitors.

Protein concentration was evaluated by BCA assay (PIERCE, Thermo Scientific, France) and blotted using SDS-PAGE basic protocol. Primary antibody incubation was performed overnight at 4°C (eIF5A, Abcam #32443; GLUT1, Abcam #ab54460; GLUT2, Cell Signaling #54460; hypusine-eIF5A, [START_REF] Melis | Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome[END_REF] ; β-actin, sigma # A5441) and then with adequate HRP-conjugated secondary antibodies (Jackson ImmunoResearch, Interchim, France) (30 minutes, 1:10000, RT). Detection was performed using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Molsheim, France) and Fuji apparatus. Band intensities were evaluated using PCBas Software.

Lactate dehydrogenase (#K2228, APExBIO, Clinisciences) and glyceraldehyde 3-phosphate dehydrogenase (#K680, BioVision, Clinisciences) cells content were evaluated using activity assay and following manufacturer's instructions.

Biochemical parameter analysis

Urinary creatinine was assayed by colorimetric reaction (NaOH 0.75N/Picric acid 0.04 M V/V) detected at 520 nm. Glucose was evaluated directly in plasma, urine and cell supernatant using Glucose-Glo Assay (Promega) and following manufacturer's instructions. Cell glucose efflux was directly measured in glucose free media at indicated time. Cell glucose consumption was evaluated by assay of glucose decrease in full media containing 4.5 mg/L glucose at indicated time and normalized by glucose efflux measured at the same time.

Glucose uptake was measured using 2-deoxy-d-[ 3 H]-glucose (2-DG). After two washes with PBS, cells were incubated 10 min with 100 µM 2-DG and 1 µCi 2-deoxy-D-[ 3 H] glucose. Culture plates were put on ice and rinsed with ice-cold PBS. Cells were scraped in 0.5N NaOH, neutralized with 0.5N HCl and 2-deoxy-d-[ 3 H]-glucose uptake was measured by liquid scintillation counting of cell lysate with a beta-counter.

Ion concentrations (Cl -, lactate, Na + , K + ) were evaluated by ion chromatography analysis. All biological samples (plasma, urine and cell supernatant) as well as ion standard solutions were previously deproteinized by addition of acetonitrile (dilution 1:1 volume). Samples were strongly mixed and centrifuged at 12.000g (10min at 4°C). Ion concentrations of the supernatant were determined using an ion chromatography Dionex ICS-5000 plus system (Thermo Scientific). The system included an autosampler, pumps, eluent generator and conductivity detectors. The system was equipped with 2 eluent generator cartridges (Dionex EGC500KOH; Dionex EGC500MSA), an anion column (IonPac CS17, 2 mm) and a cation column (IonPac AS-11 HC, 2mm). Ion concentrations were determined using Chromeleon software (Thermo Scientific) by measuring surface area of the peaks and were compared to the corresponding ion standard profiles.

mRNA analysis

Procedures follow MIQE recommendations [START_REF] Bustin | The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments[END_REF] . Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. In addition, tissues were solubilized using Precellys tissue homogenizer in TRIzol reagent and using CK14 beads. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using M-MLV-RT (Promega). SYBR qPCR premix Ex TaqII from Takara (Ozyme, France) was used for quantitative PCR (qPCR), and assays were run on a StepOne Plus ABI real-time PCR machine (PerkinElmer Life and Analytical Sciences, Boston). The expression of selected genes was normalized to that of the TATA-box binding protein (TBP) and β-actin housekeeping genes, and then quantified using the comparative-ΔCt method. Primer sequences are available upon request.

Histology and immunohistochemistry

Freshly sampled tissues were fixed in 10% buffered formalin overnight at RT and then paraffinembedded. Embedded tissues were cut in 5 μm sections and dried 30 min at 55°C. For immunohistochemistry, sections were then deparaffinized in xylene, rehydrated through alcohol, and washed in phosphate-buffered saline (PBS). Antigen retrieval was performed using Vector unmasking reagent (Vector Laboratories). Sections were blocked with MOM blocking solution (MOM kit, Vector Laboratories), then incubated with mouse monoclonal primary antibody anti-GLUT1 (Abcam, #ab40084) diluted in MOM diluent for overnight at 4°C. Detection was performed with Alexa647-conjugated donkey anti-mouse secondary antibody (ThermoFisher Scientific).

GLUT1 stained kidney sections were sequentially scanned for far red signal allowing imaging of a large area of the sections. GLUT1 signal analysis was then performed using ImageJ software (Supplemental Fig. 4).

Statistical analysis

Data were analysed using GraphPad Prism 6 software. Calorimetry data were analysed using two-way ANOVA multiple comparison and activity using multiple t-test. Other data were analysed by Mann-Whitney (2 groups) or Kruskal-Wallis (more than 2 groups) test to assess statistical differences between experimental groups. Differences were considered statistically significant with p<0.05. Data were displayed as scatter plot of independent values and group mean values ± SD.

Results

GC7 promotes anaerobic glycolysis.

We firstly show that GC7, which induced a decrease of eIF5A hypusination in the kidney in vivo [START_REF] Melis | Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome[END_REF] , had the same effect in our in vitro model of PCT cells. Fig. 1a shows that GC7 treatment (24 h, 30 µM) induced a decrease of eIF5A hypusination in these cells issued from proximal tubules [START_REF] L'hoste | CFTR mediates cadmium-induced apoptosis through modulation of ROS level in mouse proximal tubule cells[END_REF] . The dose of 30 µM we used was previously shown to be the lowest dose with a maximal protective effect on the anoxia induced cell death [START_REF] Melis | Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome[END_REF] . Since the metabolic switch observed involves a mitochondrial down-regulation [START_REF] Melis | Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome[END_REF] we investigated how GC7-induced switching from oxidative phosphorylation to glycolysis affects glucose and the overall metabolism of PCT cells treated 24 hours with GC7. Mitochondrial oxygen consumption rate (OCR) of GC7 treated cells maintained in glucose free medium was very low and not modulated by addition of 10 mM glucose. By contrast, PCT control cells exhibited a higher rate of basal O2 consumption but remained also insensitive to the addition of glucose (Fig. 1b). Using a substrate free medium, we demonstrated that PCT control cells increased their mitochondrial O2 consumption upon addition of 2 mM glutamine instead of glucose, and that GC7 treated cells were unable to increase their O2 consumption whatever the substrate used (Fig. 1c). The pH analysis of both types of cells revealed an increase in extracellular acidification upon glucose addition (Fig. 1d,e) and not with that of glutamine (Fig. 1e). However, this increase was three-fold higher in GC7 treated cells (Fig. 1d,e). This metabolic analysis demonstrated that GC7 treatment switched PCT cells from amino-acids dependent oxidative phosphorylation and used glucose instead of glutamine as energetic substrate through the anaerobic glycolysis pathway.

GC7 enhances glucose consumption

We then analysed the kinetic of lactate efflux and glucose consumption in PCT cells after 24 hours of GC7 treatment (Fig. 2a-b). Correlatively to media acidification analysis, the lactate efflux was increased (three-fold more at 8 hours) in GC7 treated cells (Fig. 2a). This was associated to a significant increase in glucose consumed by the cells (Fig. 2b). Looking for a modification in glucose flux, we analysed the glucose uptake using 2-deoxy-D-[ 3 H] glucose. Such analysis revealed a significant decrease of glucose uptake in GC7 treated cells (Fig. 2c). This modification was not linked to a decrease in Na + dependent glucose transporters SGLT1 and SGLT2 (responsible of glucose uptake from nephron lumen by proximal cells in vivo) mRNA expression (Fig. 2d).

SGLT2 glucose entry is essential to GC7 treated cell metabolism and survival

As glucose enters proximal cells mainly by the Na + -glucose transporters SGLT2, and in a lower extent by SGLT1 [START_REF] Ghezzi | Physiology of renal glucose handling via SGLT1, SGLT2 and GLUT2[END_REF] in proximal tubule cells, we used the SGLT2 inhibitor canagliflozin to disrupt this crucial step in control and GC7 treated cells (30 µM, 24h). Pretreatment (4 h) as well as acute treatment with 10 µM canagliflozin blunted medium acidification of PCT cells upon addition of glucose and consequently anaerobic glycolysis (Fig. 3 a-b). Moreover live/dead analysis after 24 hours of treatment with 10 µM canagliflozin revealed a cytotoxic effect of the SGLT2 inhibition leading to a mortality of approximately 75% in GC7 treated cells, but not in control canaglifozin treated PCT cells and untreated cells that both remained alive (Fig. 3c). Thus, glucose entry via SGLT2 transporter is required for survival of GC7 treated PCT cells contrary to control cells.

Glycolysis is the exclusive energy source in GC7 treated cells

To evaluate the importance of glucose as energy source for GC7 treated PCT cells, we treated or not PCT cells with GC7 (30 µM, 24h) and analysed their metabolism by sequential addition of glucose and 2-deoxy-glucose (2-DG) (Fig. 4a-b). As previously shown, the respiratory capacity of PCT cells were not modified after glucose addition and were insensitive to 2-DG (Fig. 4a).

Differently, glucose addition increased slightly extracellular acidification rate (ECAR) in control cells and strongly in GC7 treated ones whereas 2-DG inhibited it close to the basal level (Fig. 4b). This is confirmed by the measurement of lactate efflux that showed that 2-DG treatment inhibited the release of lactate in control and GC7 treated PCT cells (Fig. 4c). Thus, the weak glycolytic activity displayed by control cells was notably increased by GC7. Survival of PCT cells, which displayed a basal glycolysis, was not affected by addition of 2-DG (Fig. 4d). Indeed, these cells use glutamine-fueled oxidative phosphorylation as energy source (Fig. 1). By contrast, GC7 treated cells displayed more than 90 % of mortality under 2-DG treatment (Fig. 4d), demonstrating clearly that glucose dependent glycolysis is the unique energy source of the GC7 treated cells.

GC7 inhibits glucose efflux in proximal cells and represses GLUT1 expression

As proximal cells mediate glucose reabsorption into the interstitial milieu in vivo, we looked for their in vitro glucose efflux capacity. As shown in Fig. 5a, the glucose efflux was decreased in GC7 treated PCT cells and reaches a more than two-fold difference after 8 hours of treatment. In proximal cells the glucose efflux corresponds to a passive transport through both the GLUT1 and GLUT2 facilitated transporters expressed at the basolateral membrane, so we analysed the effect of GC7 on their expression. The GLUT1 protein expression was impaired in GC7 treated PCT cells whereas that of GLUT2 was not significantly modified (Fig. 5b).

LDH and PDH activities are not involved in the GC7-induced metabolic shift.

The lack of oxidative phosphorylation could be due to a high activity of lactate dehydrogenase (LDH) reducing pyruvate availability to enter mitochondria. We did not find any difference between untreated and GC7 treated cells in LDH activity in whole cell lysates (Supplemental Fig. 1a).

Moreover, we used oxamate, a noncompetitive inhibitor of LDH. Oxamate treatment (100 mM) of control and GC7 treated cells inhibited strongly the lactate efflux (Supplemental Fig. 1b) and in a lesser extent media acidification (Supplemental Fig. 1c). Analysis of oxygen consumption did not reveal any effect of oxamate in control and GC7 treated cells (Supplemental Fig. 1d). Thus, increasing pyruvate availability in PCT cells did not favor its use by mitochondria. Moreover, oxamate treatment did not lead to cell death neither in control nor in GC7 treated cells (Supplemental Fig. 1e) suggesting that cell survival was not affected under oxamate treatment contrarily to canaglifozin or 2DG. These results demonstrate that GC7-induced metabolic shift was not linked to LDH activity.

Pyruvate dehydrogenase (PDH), a key enzyme of OXPHOS, can be inactivated by multiple phosphorylations of serine residues (Ser232, Ser239 and Ser300) catalyzed by pyruvate dehydrogenase kinases (PDK). In control PCT cells PDH was highly phosphorylated and these phosphorylations were decreased in the presence of 5 mM dichloroacetate (DCA) (Supplemental Fig. 2a) an inhibitor of PDK, which maintained PDH in an active form. Surprisingly, in GC7 treated cells all residues were found dephosphorylated close to the values found after DCA treatment (Supplemental Fig. 2a). As DCA promotes pyruvate entry into the Krebs cycle, we checked for oxygen consumption in GC7 treated and untreated cells and submitted to DCA (5 mM, 24h). As expected, DCA increased OCR in control cells allowing activation of PDH. In contrast no change was observed in GC7 treated cells (Supplemental Fig. 2b) demonstrating that pyruvate cannot be used as substrate for oxidative phosphorylation and is preferentially reduced in lactate. In the same way no change by DCA treatment was found in lactate efflux (Supplemental Fig. 2c) as well as the cell survival rate (Supplemental Fig. 2d) of GC7 treated cells.

The GC7 induced metabolic switch is fully reversible.

We next analysed the recovery of the OXPHOS pathway following GC7 removal. Confluent PCT cells were treated for 24 h with 30 µM GC7. Afterward GC7 was removed and cells were maintained in culture medium for an additional period of 72 h. Seahorse experiments revealed that GC7 pre-treated PCT cells recovered a basal OCR close to the one measured in control condition (Fig. 6a) in the presence of 10 mM glucose. In the same way the ECAR of these cells returned to the values observed in control cells (Fig. 6b). This indicates that PCT cells are able to recover an OXPHOS confirming the reversion of the GC7 effect. From a functional point of view, Fig. 6c shows that 72 h after GC7 removal the glucose efflux of PCT cells, which is fully impaired by GC7, is hugely recovered. Parallelly the glucose consumption tends to return toward the value observed in control cells (Fig. 6d). These data demonstrate that the mitochondrial "silencing" induced by GC7 is reversible as far as both the metabolic and functional features of PCT cells are concerned. This conclusion was reinforced by the fact that GLUT1 expression which is fully repressed by GC7 is back following the recovery period (Fig. 6e).

Impact of GC7 treatment on mice physiological parameters

We looked in vivo for the metabolic and main physiological effects associated to the overall modifications observed in vitro. Mice were treated daily with GC7 (IP, 3mg/kg/day; "GC7" group) or with vehicle only (NaCl 0.9% w/v; "ctrl" group) for 72 hours, according to the protocol demonstrated to have beneficial effect in rodent and pig models [START_REF] Melis | Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome[END_REF][START_REF] Giraud | The inhibition of eIF5A hypusination by GC7, a preconditioning protocol to prevent brain death-induced renal injuries in a preclinical porcine kidney transplantation model[END_REF][START_REF] Bourourou | Inhibition of eIF5A hypusination pathway as a new pharmacological target for stroke therapy[END_REF] . No difference was found between control and GC7 treated mice for sodium, potassium and chloride ion contents in plasma and urine (Supplemental Fig. 3a). Interestingly, while glucose and lactate plasma levels were unchanged upon GC7 treatment (Supplemental Fig. 3b), urinary glucose and lactate were significantly higher in GC7 compared to control mice when reported to urinary creatinine (Fig. 7a).

Moreover, urinary creatinine was not significantly different between the two groups suggesting an unaltered renal function (Fig. 7b). To verify that these phenotypes were linked to GC7 effect, we confirmed that eiF5A hypusination was decreased in the kidney after 3 days of GC7 treatment (Fig. 7c).

GLUT1 expression is impaired in kidneys from GC7 treated mice.

As the increase in glucosuria and lactaturia found in mice could be due to the altered expression of glucose transporters shown in vitro (Fig. 6e), we studied the effect of GC7 treatment on the expression of glucose transporters in mice kidneys. As observed in PCT cells we retrieved a decrease in the protein level of GLUT1, but not of GLUT2, in whole kidney protein extract from GC7 treated mice (Fig. 7c). To confirm the GLUT1 misexpression in kidney, we evaluated its expression in renal cortical region by immunohistochemistry using sagittal kidney sections from control and GC7-treated mice. As displayed in Fig. 7d, the expression of GLUT1 is clearly noticeable in control kidneys at the basolateral membrane of the tubules while its expression is impaired in term of labeling intensity (Fig. 7d) and number of positive tubules in kidneys from GC7-treated mice as evaluated by quantifying the labelled areas of each sections (Fig. 7e, Supplemental Fig. 4 and5).

Discussion

We have previously demonstrated that inhibition of eIF5A hypusination, through conditioning treatment with the spermidine analogue GC7, led to anoxic tolerance in kidney cells [START_REF] Melis | Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome[END_REF][START_REF] Giraud | The inhibition of eIF5A hypusination by GC7, a preconditioning protocol to prevent brain death-induced renal injuries in a preclinical porcine kidney transplantation model[END_REF] and neurons [START_REF] Bourourou | Inhibition of eIF5A hypusination pathway as a new pharmacological target for stroke therapy[END_REF] both in vitro and in vivo. Tolerance to anoxia due to GC7 treatment has been shown to be linked to an inhibition of mitochondrial activity together with an increase in anaerobic glycolysis that allowed cell survival by maintaining basal ATP synthesis and membrane integrity. Here we are advancing in understanding this mechanism by demonstrating a phenotypical switch of the proximal tubule cells treated with GC7, from a full functional state to a survival state based on the fate of glucose. In in vivo control condition proximal convoluted tubular cells uptake glucose via SGLT2 transporters family in the S1 segment and drive it out to the interstitial space in vivo using the high affinity GLUT1 and low affinity GLUT2 facilitated glucose transporters [START_REF] Hediger | Molecular physiology of sodium-glucose cotransporters[END_REF] . Moreover, these cells do not use this glucose as an energy source for their own and rather generate ATP from oxidative phosphorylation of glutamine [START_REF] Uchida | Substrate specificity to maintain cellular ATP along the mouse nephron[END_REF][START_REF] Sekine | Chapter 6 -Solute Transport, Energy Consumption, and Production in the Kidney[END_REF] . Herein, we demonstrate that GC7 treatment inhibits the efflux of glucose (i.e. glucose reabsorption) and mitochondrial oxidative phosphorylation, which leads to the establishment of anaerobic glycolysis to ensure the energy demand necessary for cell survival (Fig. 8). Interestingly, while under GC7 treatment the cells were unable to use another source of energy than glucose, this capacity was not lost as the cells retrieve normal metabolism and function after GC7 removal [START_REF] Melis | Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome[END_REF][START_REF] Giraud | The inhibition of eIF5A hypusination by GC7, a preconditioning protocol to prevent brain death-induced renal injuries in a preclinical porcine kidney transplantation model[END_REF] . This functional and metabolic shift allows cells to become transiently independent of the environmental oxygen and thus to resist to the deep decrease in oxygen concentration resulting either from anoxia in vitro or ischemia in vivo [START_REF] Melis | Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome[END_REF] . With regards to kidney transplant where the GC7conditioning lasts a couple of days after treatment, cells resist to the high deleterious reoxygenation step characterizing the reperfusion step in vivo [START_REF] Giraud | The inhibition of eIF5A hypusination by GC7, a preconditioning protocol to prevent brain death-induced renal injuries in a preclinical porcine kidney transplantation model[END_REF] . Anoxia tolerance of proximal tubular cells is a crucial challenge as they are highly dependent on oxidative metabolism and they do not use glucose as energy source [START_REF] Uchida | Substrate specificity to maintain cellular ATP along the mouse nephron[END_REF] . Thus, opportunistically the GC7 takes advantage of this high availability of glucose to make these cells independent of oxygen by imposing the implementation of an anaerobic glycolytic metabolism.

The functional and metabolic shift observed in vitro in proximal tubule cells treated by GC7 leads to a decrease in glucose reabsorption by the kidney in vivo. Indeed, the increase in glucosuria highlights a deficit in glucose reabsorption and the increase in lactaturia is linked to the anaerobic glycolysis setting up along the nephron. This is reinforced by the fact that lactate plasma level is not modified by GC7 treatment. Surprisingly the decrease in glucose uptake found in vitro was not linked to sglt expression alteration, while sglt glucose transport is required for PCT cells survival. SGLT co-transporters are electrogenic due to their coupling with Na + and their activities depend both on the plasma membrane potential and the inward Na + gradient, thus we suspect that GC7 treatment could modify the membrane potential and/or the inward directed sodium gradient resulting in an apparent independence between their expressions and transport capacities. We cannot exclude a more general effect of GC7 on whole body metabolism which could be compensated by other physiological mechanisms. Hepatic clearance is the main mechanism regulating hyperlactatemia, in addition to other tissues as skeletal muscles, heart and kidney proximal tubule which clear lactate by converting it to pyruvate [START_REF] Phypers | Lactate physiology in health and disease[END_REF] . In vitro we demonstrate that proximal cells are unable to use lactate as substrate, suggesting that kidney cells are not involved in lactate clearance. We know that GC7 can perform this metabolic switch in other organs and cells such as the brain and neurons [START_REF] Bourourou | Inhibition of eIF5A hypusination pathway as a new pharmacological target for stroke therapy[END_REF] . Therefore, considering our findings in kidney cells, it is tempting to assume that an epithelium with equivalent glucose metabolism may also be targeted and thereby protected by GC7.

In vitro we demonstrated that inhibition of eIF5A hypusination leads to a shift from mitochondrial oxidative phosphorylation to anaerobic glycolysis. Nevertheless, the pathway governing GC7 effect still needs to be clarified. Indeed, inhibition of mitochondrial activity could promote metabolic shift and favor anaerobic glycolysis leading to inhibition of glucose efflux to preserve cell integrity. The low level of PDH phosphorylation found in GC7 treated cells, demonstrating the inability of mitochondria to perform oxidative phosphorylation even in the presence of pyruvate, could suggest that the mitochondria "silencing" occurred before the onset of glycolysis. Another way would be that the increase in glucose availability, due to GLUT1 misexpression, enhances glucose metabolism concomitantly to inhibition of mitochondria activity, which ultimately leads to an anaerobic metabolism. In the context of an increased glycolysis a parallel point has also to be studied in the future: it is the effect of GC7 on the pentose pathway involving glucose 6 phosphate that is produced early in glycolysis and that provides carbon skeleton for the synthesis of nucleic acids. So many questions that deserve to be studied in order to fill in the gaps of the molecular mechanisms linking eIF5A hypusination inhibition to metabolic shift. Puleston and collaborators demonstrated that GC7 treatment of macrophages leads to an inhibition of several mitochondrial proteins explaining a shift to anaerobic glycolysis [START_REF] Puleston | Polyamines and eIF5A Hypusination Modulate Mitochondrial Respiration and Macrophage Activation[END_REF] . These authors explained the decrease in a subset of mitochondrial proteins by a specific defect of their translation due to a specific motif hypersensitive to eIF5A hypusination [START_REF] Puleston | Polyamines and eIF5A Hypusination Modulate Mitochondrial Respiration and Macrophage Activation[END_REF] . These interesting results obtained in vitro involves that all cells displaying mitochondrial metabolism must be sensitive to GC7 treatment, which may not be the case especially in vivo were GC7 treatment did not imply a general shift toward anaerobic glycolysis in our model and others [START_REF] Maier | The unique hypusine modification of eIF5A promotes islet beta cell inflammation and dysfunction in mice[END_REF][START_REF] Melis | Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome[END_REF][START_REF] Giraud | The inhibition of eIF5A hypusination by GC7, a preconditioning protocol to prevent brain death-induced renal injuries in a preclinical porcine kidney transplantation model[END_REF][START_REF] Bourourou | Inhibition of eIF5A hypusination pathway as a new pharmacological target for stroke therapy[END_REF][START_REF] Robbins | Inhibition of deoxyhypusine synthase enhances islet {beta} cell function and survival in the setting of endoplasmic reticulum stress and type 2 diabetes[END_REF] .

It is known that dhps deficiency (the gene encoding DHS) or chronic treatment with DHS inhibitors, as GC7, ameliorate glucose tolerance and glycemia in various mouse models of diabetes (HFD [START_REF] Levasseur | Hypusine biosynthesis in β cells links polyamine metabolism to facultative cellular proliferation to maintain glucose homeostasis[END_REF][START_REF] Turpaev | The protein synthesis inhibitor brusatol normalizes high-fat diet-induced glucose intolerance in male C57BL/6 mice: role of translation factor eIF5A hypusination[END_REF] , STZ [START_REF] Maier | The unique hypusine modification of eIF5A promotes islet beta cell inflammation and dysfunction in mice[END_REF][START_REF] Tersey | Protective Effects of Polyamine Depletion in Mouse Models of Type 1 Diabetes: Implications for Therapy[END_REF] , humanized mouse model of T1D [START_REF] Imam | eIF5A inhibition influences T cell dynamics in the pancreatic microenvironment of the humanized mouse model of Type 1 Diabetes[END_REF] , db/db 33 ; NOD [START_REF] Colvin | Deoxyhypusine synthase promotes differentiation and proliferation of T helper type 1 (Th1) cells in autoimmune diabetes[END_REF] ). All these exciting studies relied hypusination inhibition effect to beta cell mass protection and decreased pancreas inflammation. Unfortunately, none of these works studied peripheral effects of GC7 and especially its impact on intestine and renal functions, as we demonstrated in this work. Thus, it will be very interesting to further analysed GC7 effect on glucose reabsorption functions in mouse models of diabetes in order to determine if eIF5A hypusination inhibition in this context could participate to glucose tolerance improvement. One of the more striking result of this study is the drastic inhibition of GLUT1 expression consecutive to GC7 treatment, an effect that is fully reversible in a couple of days and devoid of side effects in our time frame. Indeed, the physiological consequence of this misexpression leads to an inhibition of glucose reabsorption and an enhancement of glucosuria.

This could be of importance in clinic since it has been shown that an antisense GLUT1 transgene could protect mesangial cells from glucose induction of GLUT1 and fibronectin expression that could be beneficial in the setting of diabetes [START_REF] Heilig | Antisense GLUT-1 protects mesangial cells from glucose induction of GLUT-1 and fibronectin expression[END_REF] . The figure 8 summarizes the molecular effects we evidenced and that are driven by GC7 treatment regarding glucose handling. The remaining question is the link between eIF5A hypusination and GLUT1 expression. Indeed, GLUT1 amino acid sequence does not include proline rich regions and its encoding mRNA sequence does not contain consensus sequences described up to now as having a link with eIF5A [START_REF] Xu | Identification of mRNA that binds to eukaryotic initiation factor 5A by affinity co-purification and differential display[END_REF][START_REF] Mandal | Genome-wide analyses and functional classification of proline repeat-rich proteins: potential role of eIF5A in eukaryotic evolution[END_REF] . Thus, future investigations are warranted to establish additional target motifs and recognition sites of hypusinated eIF5A.

An important point is the complete reversion of the GC7 effect that demonstrates a nonharmful effect on the mitochondrial network but rather a transitory "silencing" effect. In conclusion such a reversible pharmacological reprogramming of glucose handling and oxygen dependence by GC7 represents a pharmacological opportunity in ischemic as well as hyperglycemia associated pathologies from renal origin.

Authors contributions

Michel Tauc, Didier Pisani: Equal contribution, conceptualization, project administration, interpretation, planning administration, writing, funding acquisition, supervision. Confluent PCT cells were treated or not with 30 µM GC7 for 24 hours and then GC7 was removed and cells were maintained for an additional period of 72 h and analysed. a, b/ Cells were analysed using Seahorse technology to evaluate oxygen consumption (a) and extracellular acidification (b) in the presence of 10 mM glucose. Addition of other compounds are indicated. Dots displayed mean ± SD of 3 independent experiments. * p<0.05 evaluated using Mann-Whitney. c/ At the end of treatment cells were deprived in glucose and the efflux of glucose was measured in the next 8 hours. d/ Measurement of cell glucose consumption corresponding to the difference between measurement of the media glucose content decrease (in the presence of glucose in media) and media glucose content increase (after glucose deprivation). Dots displayed mean ± SD of 3 independent experiments. * p<0.05 evaluated using Mann-Whitney tests. 'Ctrl rev' are control cells at 72 h without GC7 treatment and 'GC7 rev' correspond to cells 72 h after GC7 removal. 'Ctrl' and 'GC7' are cells analysed after 24 h. (e) Western blot analysis of GLUT1 in membrane enriched protein lysate from PCT cells following a 24h GC7 treatment (30µM) and after 72h of GC7 washout (72h rev). β-Actin was used as loading control. MW: molecular weight. * p<0.05 evaluated using Mann-Whitney test. Mice were treated 3 days with GC7 (3 mg/kg, i.p., "GC7" group) or vehicle only (NaCl, "ctrl" group) and metabolic parameters were analysed at the end of the treatment. a/ Glucosuria (glucose/creatinine) and lactaturia (lactate/creatinine) measured in bladder urine. b/ urinary creatinine (mg.dL -1 ). c/ Hypusination of eIF5A and GLUT1 and GLUT2 protein levels evaluated by western blot using four kidney protein extract for each group. MW: molecular weight. Graphic

Figure legends

Fig. 1 .

 1 Fig. 1. GC7 induces a switch in cell metabolism and substrate preference.

Fig. 2 GC7

 2 Fig. 2 GC7 increases glucose consumption and lactate efflux.

Fig. 3 .

 3 Fig. 3. SGLT2 glucose entry is essential to GC7 treated cell metabolism and survival.

Fig. 4 .

 4 Fig. 4. Glycolysis is essential to GC7 treated cell metabolism and survival.

Fig. 5 .

 5 Fig. 5. GC7 modulates glucose flux in PCT cells.

Fig. 6 .

 6 Fig. 6. The effect of GC7 is fully reversible.

Fig. 7 .

 7 Fig. 7. Impact of a 3-days GC7 treatment on kidney glucose metabolism.

  displayed hypusine/total eIF5A ratio and GLUT/β-Actin ratio using optical density band values. d/ Mosaic reconstruction of whole kidney sections from untreated and GC7 treated mice displaying GLUT1 signals in cortical region. Magnification of a representative cortical regions from each group. Scale bars are indicated. e/ quantification of GLUT1 immunofluorescence. Graphics displayed scatter plot of 4 (c), 6 (a, b) or 10 (e) independent values and mean ± SD. * p<0.05 evaluated using Mann-Whitney test.

Fig. 8 .

 8 Fig. 8. Schema of GC7 effects on glucose handling in proximal cells.

Acknowledgments

This work was supported by a grant DPM 20121125559 from the Fondation pour la Recherche Médicale (FRM), a grant from the Société de Réanimation de Langue Française (SRLF) and a grant (project KIRI) from the Agence Nationale pour la Recherche (ANR). The authors greatly acknowledge the C3M Animal core facility as well as the IRCAN Cytomed, Histology and Molecular and Cellular Imaging (PICMI) facilities that are supported by "le Cancéropôle PACA", "la Région Provence Alpes-Côte d'Azur" and "le Conseil Départemental 06".

Competing interests

The authors declare no competing financial interests