# The eIF5A hypusination inhibitor GC7 improves tolerance of pancreatic beta cells to ischemia/reperfusion

Hajar Ouahmi, Filippo Massa, Marc Cougnon, Isabelle Rubera, Gisèle Jarretou, Michel Tauc, Emmanuel van Obberghen, Antoine Sicard, Didier F Pisani

► To cite this version:

Hajar Ouahmi, Filippo Massa, Marc Cougnon, Isabelle Rubera, Gisèle Jarretou, et al.. The eIF5A hypusination inhibitor GC7 improves tolerance of pancreatic beta cells to ischemia/reperfusion. American Journal of Physiology - Cell Physiology, 2025, 330 (1), pp.C56-C69. 10.1152/ajpcell.00631.2025. hal-05417818

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Submitted on 19 Dec 2025

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<figure><img src="image_1.png" /></figure>
RESEARCH ARTICLE

# The eIF5A hypusination inhibitor GC7 improves tolerance of pancreatic beta cells to ischemia/reperfusion

Hajar Ouahmi,¹,² Ⓓ Filippo Massa,¹,² Marc Cougnon,¹ Ⓓ Isabelle Rubera,¹ Gisèle Jarretou,¹ Michel Tauc,¹
Emmanuel Van Obberghen,¹ Antoine Sicard,¹,²* and Didier F. Pisani¹*

¹LP2M, Université Côte d'Azur, CNRS, France and ²Service de Néphrologie Dialyse et Transplantation, Hôpital Pasteur 2, CHU Nice, Nice, France

Abstract

Transplantation of pancreatic islets, containing insulin-secreting beta cells, provides substantial benefits for individuals with type 1 diabetes. However, the low yield of the procedure limits its therapeutic potential, as many islets are lost during preparation and transplantation, primarily due to ischemia/reperfusion injuries and oxidative stress. N1-guanyl-1,7-diaminoheptane (GC7), an inhibitor of eIF5A hypusination, improves the resistance of various cells and organs to ischemia/reperfusion. Our study therefore explored whether GC7 treatment of beta cell models in vitro could serve as a strategy to enhance their resistance to ischemia/ reperfusion injuries. We treated rat INS-1 or mouse MIN6 cells with GC7 and analyzed insulin secretion, energetic metabolism, mitochondrial function, and both cell survival and oxidative stress under anoxia/reoxygenation conditions. In beta cells, eIF5A inhibition by GC7 treatment repressed transiently the mitochondrial activity, ATP production, and insulin secretion in response to glucose, which was linked to a metabolic shift from oxidative phosphorylation to anaerobic glycolysis. Following anoxia/reoxyge- nation to mimic ischemia/reperfusion, GC7 treatment significantly reduced oxidative stress while significantly improving cell sur- vival by >50%. Collectively, these findings are a proof of concept, demonstrating that GC7 treatment of beta cells enhances their resistance to ischemia-reperfusion injury. Hence, the use of GC7 appears as a promising strategy to improve pancreatic islet survival during transplantation.

**NEW & NOTEWORTHY** Treatment of rodent beta cell line with GC7, an inhibitor of eIF5A hypusination, reversibly slows down mitochondrial activity and shifts the cells' energy metabolism toward anaerobic glycolysis. As a result, GC7 treatment transiently inhibits ATP production and insulin secretion in response to glucose. These changes enhance the resistance of pancreatic beta cell line to ischemia/reperfusion by reducing oxidative stress and improving cell survival, highlighting the potential anti-ischemic benefits of GC7 for pancreatic islet transplantation.

anoxia; diabetes; metabolism; oxidative stress; transplantation

INTRODUCTION

Type 1 diabetes (T1D) is an autoimmune disease charac-
terized by a selective destruction of beta cells resulting in
insufficient insulin production and secretion. The current
treatment consists of multiple daily injections of exogenous
insulin (1, 2), which often fails to prevent long-term compli-
cations and can lead to life-threatening hypoglycemic epi-
sodes. Several new approaches are being developed to
improve the treatment of T1D and patients' quality of life,
including the increasing use of closed-loop insulin delivery,
immunosuppressive drugs, and mesenchymal stem cell
transplantation targeting autoimmunity (3, 4). Finally, beta
cell replacement using allogeneic primary islets combined
with immunosuppressive drugs (2) or stem cell-derived
islets has recently being tested with success (5). Among
these approaches, allogeneic transplantation of pancreatic

islets, which contain mainly α and beta cells (secreting glu-
cagon and insulin, respectively), has progressively emerged
as a promising alternative in patients with severe T1D or
associated with advanced kidney disease (2, 6–8). In this
approach, human pancreatic islets isolated from deceased
brain-dead donors are percutaneously injected into the
patient’s hepatic portal vein, from which they implant into
the liver, to restore the beta cell population and thus insulin
production and secretion (1). This procedure has been
shown in several clinical trials to avoid hypoglycemia and
to improve glycemic control and quality of life (6, 7, 9–11).
Consequently, islet transplantation is now used for patients
with T1D with severe hypoglycemia or after kidney trans-
plantation, in addition to islet autografts in patients with
benign pancreatic pathologies requiring a partial or total
pancreatectomy (12). Notwithstanding this progress, the
widespread use of pancreatic islet transplantation is still

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<footnote>*A. Sicard and D. F. Pisani contributed equally to this work.
Correspondence: D. F. Pisani (didier.pisani@univ-cotedazur.fr).
Submitted 8 August 2025 / Revised 25 August 2025 / Accepted 12 November 2025</footnote>
<figure><img src="image_4.png" /></figure>

hampered especially by islet loss resulting mainly from ischemia/reperfusion events during isolation from donor pancreas and in the first days posttransplantation. In addition, each patient requires multiple injections and consequently numerous donors (8). To tackle the issue of organ shortage, research efforts are being focused on means generating insulin-producing structures from sources with expandable properties, such as stem cells. In fact, considerable progress has been made leading to the first clinical trials in patients with T1D, prefiguring a ground-breaking advancement in the field (5). In addition to this promising strategy, there exists another one, which consists of limiting islet loss during isolation from donor pancreas to improve the yield of this procedure (8). Due to their high mitochondrial oxidative metabolism, which is essential to their function, beta cells, accounting for >50% of islet cells (13), are particularly sensitive to variations in oxygen availability (14) found during isolation procedure and transplantation in recipient patient. These ischemia (oxygen and nutrient deprivation) and reperfusion (sudden burst in oxygen and nutrient availability) conditions are both generating major oxidative stress leading to macromolecule damages and ultimately beta cell death (15). Thus, improving beta cell resistance to these oxygen variations is a critical factor and a promising strategy to enhance islet survival during the transplantation procedure (1, 9, 10).

Polyamines exert many essential cellular functions, including activation of the eukaryotic translation initiation factor eIF5A through the posttranslational addition of hypusine, which is generated from spermidine by the successive catalytic action of deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH) (16, 17). eIF5A is highly conserved across species from archaeobacteria to higher mammals including humans and is the only protein known to undergo hypusination. Using a specific competitive inhibitor of DHPS, N1-guanyl-1,7-diaminoheptane (GC7), we and others have shown that inhibition of eIF5A activation improves the resistance of mammalian tissues exposed to the noxious effects of ischemia/reperfusion (18-23). Indeed, in mouse kidney cells, we found that this protection against ischemia was secondary to a decrease in oxygen consumption due to a reversible shutdown of mitochondrial activity, with energy homeostasis maintained through anaerobic glycolysis, as well as to a limited oxidative stress and endoplasmic reticulum (ER) stress (18, 20, 22, 23). Notably, the effects of GC7 on metabolism and cellular phenotype were reversible, disappearing 24-48 h after its withdrawal (18-22, 24). An equivalent protection was found for mouse neurons when they are submitted to oxygen and glucose deprivation (21, 25). We extended these findings to rodent models of stroke (21) and renal ischemia/reperfusion (22, 23), and more importantly preclinical kidney transplantation models in pigs, where GC7 administration to the donor enhanced both early and long-term graft recovery (19, 20).

Given the critical role of oxygenation in islet survival during isolation and transplantation, we assessed the efficacy of GC7 in protecting beta cells against ischemia/reperfusion injury in vitro. Our findings showed that GC7 treatment protects INS-1 and MIN6 insulinoma cells from anoxia/reoxygenation by slowing down their metabolism and decreasing oxidative stress. Hence, the use of GC7 during the pancreatic islet transplantation procedure appears as a promising new way to improve the success of the transplantation.

# MATERIALS AND METHODS

## Reagents

When it is not specified, drugs, buffer solutions, fetal bovine serum (FBS), media, additives, and reagents were from Sigma-Aldrich Chimie (Saint-Quentin-Fallavier, France). N-guanyl-1,7-diaminoheptane (GC7) was synthesized by AtlanChim Pharma (Nantes, France) according to the previously described method (26).

## Cell Culture

Rat INS-1E insulinoma pancreatic stable beta cell line (RRID: CVCL_0351, INS-1 cells with high insulin secretion capacity, *Rattus norvegicus*, male) was maintained in RPMI 1640 containing 11 mM glucose supplemented with 10% (vol/vol) heat-inactivated FCS, 100 units/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 50 µM beta-mercaptoethanol.

Mouse MIN6 insulinoma stable pancreatic beta cell line (RRID: CVCL_0431, *Mus musculus*, sex unspecified) was maintained in DMEM containing 5.5 mM glucose supplemented with 10% (vol/vol) heat-inactivated FCS, 100 units/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 10 mM HEPES, and 100 µM beta-mercaptoethanol.

Cells were cultivated in humidified 5% (vol/vol) CO₂ and 95% (vol/vol) air at 37°C, and all experiments were performed the day after cell subconfluence for INS-1 and at 50% of confluence for MIN6 cells. The results presented correspond to at least three independent experiments (N) performed on cells from different passages and thaws, each experiment comprising "n" cell replicates from different plates.

For anoxia, cells were maintained for 3 h at 37°C in an airtight chamber containing a 100% N₂ atmosphere. Oxygen deprivation was controlled using an OXYBABY apparatus (WITT, Morsang sur Orge, France). Reoxygenation mimicking reperfusion was performed by placing cells for 2 h under 5% CO₂/95% air water-saturated atmosphere at 37°C. Anoxia/reoxygenation was validated by oxygen concentration follow-up and analysis of carbonic anhydrase (CaIX) and vascular endothelium growth factor (Vegf) mRNA levels, two HIF-1α target genes (Supplemental Fig. S1).

## Glucose-Stimulated Insulin Secretion

Cells were glucose-deprived for 1 h in Krebs-Ringer bicarbonate-HEPES buffer (KRBH) containing 1 mM glucose and 0.1% BSA (wt/vol). Then, the cells were incubated for 1 h 30 min in fresh KRBH containing 1- or 16-mM glucose, or 1 mM of glucose supplemented with KCl 30 mM. Centrifugated media and cell homogenates obtained by ethanol acid lysis were analyzed for insulin content using a mouse insulin ELISA kit (Cat. No. 10-1247-01; Mercodia AB, Uppsala, Sweden). To eliminate variations due to differences in cell number, insulin secretion was expressed as the percentage of the cellular insulin content, which is referred to as fractional insulin release.

## Cell Metabolism Analysis

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells were measured with a XF24 Extracellular Flux Analyzer (Seahorse Bioscience; Agilent Technologies, Courtaboeuf, France). Uncoupled and maximal
<figure><img src="image_5.png" /></figure>

OCR were determined using oligomycin (1.2 µM) and FCCP (1 µM), respectively. Rotenone and Antimycin-A (2 µM each) were used to inhibit mitochondrial respiration. All parameters were calculated and normalized following the approved procedures (27, 28). ATP production rates from OXPHOS or anaerobic glycolysis were calculated using the method described previously (29). Lactate was evaluated using the Lactate Glo-Assay (Cat. No. J5022; Promega, Charbonnières les Bains, France) and intracellular ATP using the ATPLite kit (Cat. No. 6016943; Revvity, Roissy-en-France, France) following the manufacturer's instructions.

## Protein Analysis

Whole proteins from cells were prepared using TNET lysis buffer [25 mM Tris-Cl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1% vol/vol Triton X-100, 0.5% vol/vol Nonidet P40, 1× protease, and phosphatase inhibitor cocktail (Sigma-Aldrich)]. Crude lysate was centrifuged at 10,000 g (30 min, 4°C). Supernatants containing soluble proteins were kept for analysis. Protein concentrations were evaluated using DC reagent assay (Bio-Rad, Marnes-la-Coquette, France), and 40 µg of proteins were blotted using SDS-PAGE basic protocol and Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad). Primary antibody incubation was performed overnight at 4°C. Primary antibodies were displayed in Table 1 and came from Abcam (Amsterdam, The Netherlands), Proteogenix (Schiltigheim, France), Thermo Fisher Scientific (Les Ulis, France), Sigma-Aldrich Chimie, and Cell Signaling Technology (Ozyme, Saint-Cyr-l'Ecole, France). They have been previously published (30) or validated as indicated by the manufacturers. Blots were incubated at room temperature (RT) for 30 min with an adequate horseradish peroxidase (HRP)-conjugated secondary antibody (bovine serum albumin 3% wt/vol, 1:5,000; Cat. No. 115-035-003, RRID: AB_10015289 and Cat. No. 111-035-003, RRID: AB_2313567; Jackson ImmunoResearch, Ely, UK). Detection was performed using chemiluminescent HRP substrate (Amersham, Freiburg, Germany) and Fusion FX imaging system (VILBER, Marne-la-Vallée, France). Band intensities were evaluated using Fiji Software (31).

## Immunofluorescence

Cells were fixed with 4% wt/vol paraformaldehyde for 10 min, permeabilized with 0.1% vol/vol Triton X-100/BSA 3% wt/vol for 10 min, and then sequentially incubated with anti-translocase of outer mitochondrial membrane 20 (TOMM20) primary antibody overnight at 4°C (Table 1) and with the relevant secondary antibody coupled to Alexa-594 (dilution

1:500; Cat. No. A-11005, RRID: AB_2534073; Thermo Fisher Scientific) for 30 min at RT. Finally, cells were washed with PBS containing DAPI (Sigma-Aldrich Chimie). Cells were mounted with Histomount Mounting Solution (Thermo Fisher Scientific) and visualized with an inverted epifluorescence Axiovert microscope (Carl Zeiss, Le Pecq, France) under oil immersion and using Hamatsu camera. Finally, pictures were taken and processed with Zen software (RRID: SCR_013672) (Carl Zeiss).

## Cell Survival Analysis

Viability of cells were evaluated using Calcein-AM 1 µM (Cat. No. C3099; Thermo Fisher Scientific) and Ethidium homodimer 4 µM (Cat. No. E1903; Sigma-Aldrich) to stain live and dead cells, respectively. Cells were incubated for 30 min at 37°C in the presence of both dyes and then washed two times with HBSS (w/o Phenol Red) before analysis. Fluorescent micrographs were recorded using an observer D1 microscope, captured with ZEN software (Carl Zeiss), and analyzed with Fiji software (RRID: SCR_002285) using homemade macro (31). Results are expressed as "survival rate" compared with control normoxia conditions.

## Oxidative Stress Analysis

Cells were grown in 96-wells black plate with clear bottom (PerkinElmer). For anoxia/reoxygenation experiments, cells were incubated directly before stress in HBSS containing 1% wt/vol FBS with at choice: 5 µM MitoSox Red (396/610 nm, Cat. No. M36008; Thermo Fisher Scientific) for the measurement of mitochondrial superoxide anion levels; 2 µM MitoTracker Green FM (490/520 nm, Cat. No. M7514; Thermo Fisher Scientific) for the determination of mitochondrial mass to normalized MitoSox signal; 5 µM CM-H2DCFDA (490/520 nm, Cat. No. C6827; Thermo Fisher Scientific), which is a probe mainly sensitive to hydroxyl radical and peroxynitrite; 2 µM Hoechst 33342 (360/490 nm, Cat. No. 62249; Thermo Fisher Scientific) to stain nuclei of live cells to normalize CM-H2DCFDA signal. Fluorescence intensity was determined after 3 h of anoxia followed by 2 h of reoxygenation at 37°C using a SP2000 Xenius plate reader (SAFAS, Monaco, Monaco).

## mRNA Expression Analysis

Our procedures follow the MIQE recommendations (32). Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction was performed using M-MLV-RT (Promega). SYBR qPCR premix Ex TaqII from Takara (Ozyme)

<table><caption>**Table 1.** *Details about antibodies*</caption><thead><tr><td>Name</td><td>Company</td><td>Reference</td><td>Origin</td><td>Target Size</td><td>Dilution Saturation</td><td>RRID</td></tr></thead><tbody><tr><td>elf5A</td><td>Abcam</td><td>ab137561</td><td>Rabbit polyclonal</td><td>17 kDa</td><td>1:1,000 milk</td><td>None</td></tr><tr><td>Hypusine</td><td>Proteogenix</td><td>PTX18841</td><td>Rabbit mAb</td><td>17 kDa</td><td>1:1,000 milk</td><td>None</td></tr><tr><td>HK2</td><td>Cell Signaling Technology</td><td>2867</td><td>Rabbit mAb</td><td>102 kDa</td><td>1:1,000 milk</td><td>AB_2232946</td></tr><tr><td>GLUT2</td><td>Abcam</td><td>ab54460</td><td>Rabbit polyclonal</td><td>50–70 kDa</td><td>1:750 milk</td><td>AB_880234</td></tr><tr><td>Beta-Tubulin</td><td>Invitrogen</td><td>MA5-16308</td><td>Mouse mAb</td><td>52 kDa</td><td>1:2,500 milk</td><td>AB_2537819</td></tr><tr><td>Beta-Actin</td><td>Sigma-Aldrich Chimie</td><td>A5441</td><td>Mouse mAb</td><td>42 kDa</td><td>1:2,500 milk</td><td>AB_476744</td></tr><tr><td>MitoProfile OXPHOS</td><td>Abcam</td><td>ab110413</td><td>Mouse mAbs</td><td>Multiple</td><td>1:250 BSA</td><td>AB_2629281</td></tr><tr><td>MitoProfile integrity</td><td>Abcam</td><td>ab110414</td><td>Mouse mAbs</td><td>Multiple</td><td>1:250 milk</td><td>AB_2687585</td></tr><tr><td>TOMM20</td><td>Sigma-Aldrich Chimie</td><td>WH0009804M1</td><td>Mouse mAb</td><td>14 kDa</td><td>1:100 BSA</td><td>AB_1843992</td></tr></tbody></table>

GLUT2, glucose transporter 2; HK2, hexokinase 2; TOMM20, translocase of outer mitochondrial membrane 20.
<figure><img src="image_6.png" /></figure>

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 glyceraldehyde-3-phosphate dehydrogenase (Gapdh) housekeeping genes and then quantified using the comparative ΔCt method. Primer sequences are available in Supplemental Fig. S2.

## Statistical Analysis

Data were analyzed by GraphPad Prism 7 software (RRID: SCR_002798) and using one or two-way ANOVA followed by

Tukey's post hoc test to assess statistical differences between multiple experimental groups. Differences were considered statistically significant with *P* < 0.05. Number of replicates are indicated in the legend of figures.

# RESULTS

## GC7 Exposure Induces a Time-Dependent Reduction in Insulin Secretion and ATP Levels in INS-1 Cells

We first verified the action of GC7 in INS-1 cells at various exposure times (1, 3, 6, and 16 h) by analyzing the ratio of

<figure><img src="image_7.png" /><figcaption>**Figure 1.** Time-dependent inhibition of eIF5A hypusination by GC7 and its cellular effect in INS-1 cells. Cells were treated with 30 µM of GC7 during 1 h, 3 h, 6 h or 16 h and then analyzed. A: time response of GC7 exposure on eIF5A hypusination evaluated by Western blot and quantification of the hypusinated/total eIF5A ratio (n = 4, N = 3). B and E: analysis of GSIS after various GC7 treatments. Insulin was measured in the presence of (G1) 1 mM glucose, (G16) 16 mM glucose, or (KCI) 1 mM glucose + 30 mM KCI and normalized to insulin cellular content. GSIS is expressed as a fold between basal and stimulated condition (n = 10–34, N = 3). C: insulin cellular contents normalized by protein quantity. D: time response of GC7 exposure on total intracellular ATP level (n = 11, N = 3). F: HK2 and GLUT2 protein levels assessed by Western blot (n = 4–5, N = 4). Scatter dot plots show individual values and means ± SD. Statistical significance of mean differences was tested by one-way ANOVA followed by Tukey's post hoc test. \*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*P < 0.0001. GC7, N1-guanyl-1,7-diaminoheptane; GLUT2, glucose transporter 2; GSIS, glucose-stimulated insulin secretion; HK2, hexokinase 2.</figcaption></figure>
<figure><img src="image_8.png" /></figure>

hypusinated eIF5A to total eIF5A (Fig. 1A and Supplemental Fig. S3). GC7 is a spermidine analog able to selectively inhibit DHPS by competition (30). It was used at the concentration of 30 µM allowing an optimal inhibition of hypusination in mammal cells and organs as previously demonstrated (20). As expected, treatment with GC7 resulted in up to a 50% reduction in eIF5A hypusination compared with untreated cells. The extent of inhibition was dependent on the duration of GC7 exposure. It was detectable after 1 h and reached a maximum after 16 h. Next, we analyzed the effect of GC7 on glucose-stimulated insulin secretion (GSIS). Cells were treated with 30 µM GC7 during 1–16 h and then washed and used to assess insulin secretion in response to 1 mM (G1) or 16 mM (G16) of glucose. Similar to its effect on eIF5A hypusination, the GC7-induced reduction in insulin secretion was observable within 1 h and reached its maximum in 16 h (Fig. 1B), without affecting significantly insulin content (Fig. 1C). Since increased intracellular ATP levels are a key trigger of insulin secretion, we measured the total cellular ATP level. We found that GC7-treated cells had decreased ATP levels compared with nontreated cells (Fig. 1D), which likely explains the inhibition of insulin secretion. To assess whether this could be attributed to impaired exocytosis, we measured insulin secretion at low glucose (1 mM) in the presence of depolarizing concentrations of KCl (30 mM). Six hours of GC7 treatment did not alter KCl-induced insulin secretion (Fig. 1E). Finally, we checked for an alteration of the first phase of glucose metabolism, uptake, and glycolysis, by analyzing key protein levels. Although we found no difference in hexokinase 2 (first rate-limiting enzyme of glycolysis) protein expression, we observed an increase in glucose transporter 2 (responsible of glucose cell entry) protein levels (Fig. 1F and Supplemental Fig. S3). These results supposed that the decrease in insulin secretion was not due to a defect in glucose uptake and its catabolism by glycolysis. Altogether, these results support the idea that GC7 interferes with GSIS by altering the metabolism of cells for secretion rather than by hindering the final stages of exocytosis.

For the following experiments, we selected 1 h and 16 h of GC7 treatment corresponding to its minimal and maximal effect, respectively.

## Inhibition of eIF5A Hypusination Slows Down Mitochondrial Metabolism of INS-1 Cells

eIF5A hypusination inhibition decreased intracellular ATP levels which can be linked to its well-known inhibitory effect on mitochondrial activity. Thus, we investigated mitochondrial metabolism of INS-1 cells treated or not with 30 µM GC7 for 1 h or 16 h (Fig. 2, A–D). As expected, in control cells, exposure to 16 mM glucose induced an increase in mitochondrial oxygen consumption rate (OCR) characteristic of a bioenergetic profile chiefly concerned with ATP synthesis through oxidative phosphorylation. In contrast, in GC7-treated cells, we observed a mitochondrial OCR inhibition of 20% after 1 h treatment and of 50% after 16 h (Fig. 2, A and B). It stands to reason that the OCR drop observed in GC7-treated cells was linked to a decrease in cellular ATP production (Fig. 2C).

In this context, it should be underlined that the maximal capacity of mitochondrial OCR was similar in control and GC7-treated cells (Fig. 2D), suggesting the absence of modification in the availability of mitochondria respiratory chains. This view was confirmed by evaluating

mitochondrial integrity and respiratory chain protein contents, using antibodies targeting subunits of the OXPHOS complexes and specific proteins of the mitochondrial compartments. The relative quantitative expression levels of complexes I to V were comparable in control and GC7-treated cells. Note that a similar lack of differences was observed for other proteins of the mitochondrial compartments (porins, cyclophilin D, and cytochrome C) (Fig. 2E and Supplemental Fig. S4). Finally, we searched for possible GC7 effects on mitochondria morphology by using TOMM20 immunodetection. Interestingly, although GC7 treatment (30 µM for 16 h) altered mitochondrial structure resulting in a loss of the tubular continuum observed in control cells and the appearance of rounded/swollen mitochondria (Fig. 2F), 1 h of GC7 treatment did not induce major changes but rather resulted in an intermediate phenotype characterized by a mixture of tubular and rounded mitochondria (Fig. 2F).

Altogether our results reveal that GC7 induced a slowdown in INS-1 cells mitochondrial activity associated with morphological changes, but without alterations in their intrinsic functional capacity.

## Inhibition of Hypusination Reroutes INS-1 Cell Metabolism to Glycolysis

To safeguard cell homeostasis, the natural response to a slowdown in oxidative phosphorylation is a shift toward anaerobic glycolysis. To explore this scenario, we performed an extracellular acidification rate evaluation by a Seahorse analysis. INS-1 cells treated or not with GC7 during 1 h or 16 h were first maintained in 1 mM glucose and thereafter stimulated with 16 mM of glucose. As shown in Fig. 3A, exposure to GC7 induced an increase in glucose consumption compared with untreated cells. To ascertain that the extracellular acidification was consecutive to a metabolic shift toward glycolysis anaerobia (use of pyruvate and NADH to form lactate plus NAD⁺), we quantified the expression of lactate dehydrogenase A and B (LDHA and LDHB) and, in the supernatant, the lactate levels 2 h after stimulation with 16 mM glucose. Interestingly, GC7 treatment increased dose dependently expression of *ldha* mRNA (subunit permitting lactate synthesis from pyruvate) without modifying *ldhb* mRNA levels (subunit permitting pyruvate synthesis from lactate), which is correlated with an increase in lactate excretion in GC7-treated cells (Fig. 3B). As expected, this fits together with an increase in ATP synthesis from anaerobic glycolysis (Fig. 3C) evaluated using ECAR (Fig. 3A) and OCR (Fig. 2A) values, as previously described (29). Despite this increase, the metabolic change due to GC7 treatment did not maintain ATP levels found in the basal situation, confirming results obtained for whole ATP cellular levels (Fig. 1D). Nevertheless, this lower ATP level was crucial to maintain the basal cellular activity essential for survival as cells cotreated for 16 h with GC7 and oxamate (10 mM), an inhibitor of LDH inhibiting lactate synthesis and efflux (Fig. 3C), displayed >90% of mortality (Fig. 3E). In contrast, cells treated with oxamate for 16 h, then with GC7 for 1 h, or left untreated, showed only 40% and <20% mortality, respectively (Fig. 3E). These results clearly confirmed
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 2.** Inhibition of hypusination in INS-1 cells slowed down their mitochondrial metabolism. INS-1 treated with GC7 30 µM during 1 h or 16 h was analyzed for mitochondrial metabolism and morphology. A: mitochondrial oxygen consumption traces and time of injection of the different compounds (n = 20, N = 3). Mitochondrial oxygen consumption rate (OCR) values (n = 16–20, N = 3) (B), ATP synthesis determined using oligomycin A values (n = 8–11, N = 3) (C), and maximal OCR using FCCP values (n = 8–11, N = 3) (D). E: evaluation of mitochondrial structural and respiratory chain protein contents by Western blot (n = 4–5, N = 3). F: mitochondrial network organization observed in control and GC7-treated INS-1 cells (30 µM for 16 h) using anti-TOMM20 antibody. Scatter dot plots show individual values and means ± SD. Curves show means ± SE. Statistical significance of mean differences was tested by one-way ANOVA followed by Tukey's post hoc test. \*P < 0.05; \*\*P < 0.001; \*\*\*P < 0.0001. au, arbitrary units; GC7, N1-guanyl-1,7-diamino-heptane; TOMM20, translocase of outer mitochondrial membrane 20.</figcaption></figure>
<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>**Figure 3.** Inhibition of hypusination shifted INS-1 cell metabolism to glycolysis. A–E: INS-1 cells were pretreated with GC7 30 µM during 1 h or 16 h, (C and E) in the presence or not of the LDH inhibitor oxamate (OXA, 10 mM, 16 h). At the end of treatment cells were (B and E) directly used for analysis or (A, C, and D) washed and maintained in 1 mM glucose basal then stimulated with glucose 16 mM. A: extracellular acidification rate (ECAR) expressed as % of 1 mM glucose or as mph/min/µg proteins (n = 16–20, N = 3). B: evaluation by qPCR of *Idha* and *Idhb* mRNA expression (N = 4). C: lactate efflux was evaluated in cell supernatant after addition of 16 mM glucose during 3 h and expressed as a rate of secretion per hour (n = 4–8, N = 3). D: assessment of ATP production rates by anaerobic glycolysis and oxidative phosphorylation (oxphos) expressed in pmol ATP/min/µg proteins and determined using ECAR and OCR values (n = 10–15, N = 3). E: survival rate evaluated using a live-dead fluorescence assay (n = 8–12, N = 3). Curves show means ± SE. Scatter dot plots show individual values and means ± SD. Stacked bars show means ± SD. Statistical significance of mean differences was tested by one-way ANOVA followed by Tukey's post hoc test. \*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.0001. GC7, N1-guanyl-1,7-diaminoheptane; OCR, oxygen consumption rate.</figcaption></figure>

that cells treated for 16 h with GC7 are dependent on ATP synthesis from anaerobic glycolysis.

## GC7-Induced Modulation of INS-1 Metabolism and GSIS Are Reversible

It is important to note that the GC7-induced inhibition of hypusination could be reversed by removing GC7 from the cell culture media (Fig. 4A and Supplemental Fig. S5). As a result, all previously described GC7 effects, including the metabolic shift (Fig. 4, B and C), were also reversed. In brief, intracellular ATP level (Fig. 4D) and mitochondrial morphology (Fig. 4E) were restored. To demonstrate the reversibility of the GC7-induced inhibition on the key INS-1 function, that is, GSIS, we used 3 and 6 h of GC7 treatment instead of 1 h which did not inhibit significantly GSIS. As shown in Fig. 4F, a 48-h withdrawal was not sufficient to undo the inhibition of GSIS after 16 h of GC7 treatment despite partial normalization of intracellular ATP levels (Fig. 4D). However, although 3 h and 6 h of GC7 exposure inhibited significantly GSIS, the 48-h removal restores a full GSIS (Fig. 4F) correlatively with ATP levels (Fig. 4D). Finally, the tendency of decreasing insulin content after 16 h of GC7 treatment was completely abolished 48 h after GC7 withdrawal (Fig. 4F).

## GC7-Mediated Inhibition of eIF5A Hypusination Prevents Oxidative Stress and Cell Death in INS-1 Cells Induced by Anoxia

INS-1 cells were treated with 30 µM GC7 for 1 h or 16 h and then washed before being exposed to stressful conditions that mimicked the ischemia/reperfusion situation (Fig. 5, A–D). To this end, cells were exposed to either normoxia (20% O₂ at 37°C for 5 h) or normothermic anoxia (≤0.1% O₂ at 37°C for 3 h) followed by a reoxygenation step with 20% O₂ at 37°C for 2 h. First, we measured the level of hypusination after a treatment of 1 h or 16 h, followed by normoxia (5 h) or anoxia/reoxygenation (A/R, 3 + 2 h). Under normoxic conditions, both durations of GC7 exposure resulted in inhibition of eIF5A hypusination (Fig. 5A and Supplemental Fig. S6). Although anoxia itself blocks hypusination due to the oxygen dependence of the DOHH activity, GC7 treatments exhibited an additive effect that was significant for a 16-h treatment (Fig. 1D and Supplemental Fig. S6). Interestingly, although untreated INS-1 cells exhibited a mortality rate of 70% after exposure to A/R, GC7 pretreatment for 1 h or 16 h reduced cell death following A/R by up to 20% and 50%, respectively (Fig. 5B). To investigate the protective mechanism against A/R injury, we investigated the effect on
<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 4.** Reversibility of the metabolic shift and mitochondrial remodeling due to GC7 treatment. INS-1 cells were treated for the indicated time (30 min to 16 h) with GC7, washed, and then analyzed 48 h later to determine recovery/reversion (rev groups) of eIF5A hypusination (n = 4, N = 3) (A), mitochondrial OCR (n = 10–11, N = 3) (B), basal ECAR (n = 9–11, N = 3) (C), ATP levels (n = 12, N = 3) (D), and mitochondrial network organization observed in control and GC7-treated INS-1 cells (30 µM for 1 or 16 h) using TOMM20 immunodetection (E). F: insulin secretion in response to glucose (n = 3–9, N = 3). GSIS is expressed as a fold between stimulated and basal condition. G: insulin content normalized by protein quantities (n = 6–9, N = 3). Scatter dot plots show individual values and means ± SD. Statistical significance of mean differences was tested by one-way ANOVA followed by Tukey's post hoc test. **P** < 0.01; ***P** < 0.001; ****P** < 0.0001. ECAR, extracellular acidification rate; GC7, N1-guanyl-1,7-diaminoheptane; GSIS, glucose-stimulated insulin secretion; OCR, oxygen consumption rate; TOMM20, translocase of outer mitochondrial membrane 20.</figcaption></figure>

oxidative stress which is a well-known deleterious process that occurs during anoxia and also reperfusion. Although A/R led to a sharp increase in oxidative stress in untreated cells, as assessed by the CM-H2DCFDA probe signal, GC7 treatments significantly reduced this rise (Fig. 5C). As insulin-producing cells are highly dependent on mitochondria metabolism, we assessed the involvement of mitochondrial reactive oxygen species (ROS) in oxidative stress due to A/R. Importantly, mitochondrial ROS production and oxidative stress level displayed the same profile, with an increase after A/R which was prevented by GC7 treatments (Fig. 5D).

To define if other sources of ROS were involved in oxidative stress found during anoxia/reoxygenation, we treated INS-1 cells with various inhibitors targeting key ROS-generating proteins and then submitted the cells to 4 h of anoxia and 2 h of reoxygenation. As displayed in Fig. 5E, neither XO/XDH inhibited with allopurinol and oxypurinol nor NOX and DUOX inhibited by apocynin and DPI seemed to be involved in ROS production leading to oxidative stress during anoxia/reoxygenation of INS-1 cells. Hence, this production appears to be restricted to a mitochondrial origin (Fig. 5E). Finally, as we have previously demonstrated that
<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>**Figure 5.** GC7 treatment protected INS-1 cells from deleterious effect of anoxia/reoxygenation. INS-1 cells were treated for 1 h or 16 h with GC7 and then exposed to either normoxia (Nx) or warm anoxia followed by a normothermic reoxygenation step (« A/R », ≤0.1% O₂ at 37°C for 3 h + 20% O₂ at 37°C for 2 h). A: hypusinated/total eIF5A ratio obtained from Western blot analysis in cells treated with GC7 30 µM during 1 h or 16 h plus 5 h corresponding to A/R time (n = 4, N = 3). B: survival rate evaluated using a live-dead fluorescence assay (n = 12–16, N = 3). Oxidative stress production evaluated with CM-H2DCFDA probe (n = 20–24, N = 3) (C), and mitochondrial ROS production with MitoSOX probe (n = 23, N = 3) (D). E: INS-1 cells were treated 1 h before anoxia with various inhibitors of ROS production as indicated and then analyzed for oxidative stress with CM-H2DCFDA. INS-1 cells maintained under normoxia the same time were used as control (n = 16–24, N = 3). F: INS-1 cells were pretreated with GC7 for 16 h or 1 h in the presence of pyocyanin (20 µM), a catalase inhibitor, during the last hour, then submitted to anoxia and analyzed for oxidative stress with CM-H2DCFDA (n = 16, N = 3). Hoechst (nuclei) and MitoGreen (mitochondria mass) probes were used to normalize corresponding fluorescent signal. Scatter dot plots show individual values and means ± SD. Statistical significance of mean differences was tested by one-way ANOVA followed by Tukey's post hoc test. \*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001. A/R, anoxia/reoxygenation; GC7, N1-guanyl-1,7-diaminoheptane; ROS, reactive oxygen species.</figcaption></figure>

GC7 increases antioxidant defense of cells (23), we checked for the involvement of catalase in the protection due to GC7. We exposed INS-1 cells, treated or not with GC7 1 h or 16 h, to the inhibitor of catalase, pyocyanin, and thereafter the cells were subjected to anoxia/reoxygenation 4 h/2 h. As expected, inhibition of catalase dramatically enhanced oxidative stress independently of GC7 treatment (Fig. 5F). Surprisingly, pyocyanin did not affect the antioxidant effect of GC7 as we retrieved protective effect against oxidative stress (Fig. 5F), excluding an effect of GC7 on catalase activity. Altogether these results implied that the GC7 antioxidant effect was only due to the restraint of mitochondrial ROS production.

GC7 Modulates the Metabolism and Protects Mouse Insulinoma Cells MIN6 from Anoxia

To avoid any cell-specific effects of GC7, we performed the same experiments in another beta cell line, the MIN6 insulinoma cells from mice. Similar to our observations in INS-1 cells, 16 h of treatment of MIN6 cells with 30 µM GC7

strongly inhibited oxidative phosphorylation in response to glucose (Fig. 6A) and simultaneously increased anaerobic glycolysis (Fig. 6B). This metabolic change was reversible 48 h after the removal of GC7 (Fig. 6, A and B). Also, this inhibition of mitochondrial activity by GC7 was linked to a strong decrease in the capacity of MIN6 cells to secrete insulin in response to glucose (Fig. 6C). To validate the ischemic potential of GC7 in MIN6 cells, we applied the same stress strategy as for INS-1 cells. In this situation, a 16-h treatment with GC7 protected the cells from anoxia-induced cell death (Fig. 6D) and simultaneously prevented the increase in oxidative stress (Fig. 6E) and mitochondrial ROS production (Fig. 6F) caused by this anoxia/reoxygenation sequence.

DISCUSSION

The management of type 1 diabetes (T1D) represent a major important public health challenge, due to the severe health consequences of the disease (1, 33) and the worrying
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>**Figure 6.** Metabolic modulation and anoxia protection due to GC7 treatment in MIN6 cells. A and B: MIN6 cells were treated for 16 h with GC7 (GC7 16 h) or not (CTRL) and then analyzed or washed and cultivated for additional 48 h (rev). Traces and individual values of mitochondrial oxygen consumption (OCR) (n = 11, N = 3) (A) and extracellular acidification (ECAR) (n = 10–11, N = 3) (B) rates in response to 1 or 16 mM glucose. Time of injection of the different compounds are indicated. C: MIN6 cells were treated for 8 h or 16 h and then submitted to 1 (G1) or 16 mM (G16) of glucose to determine glucose-stimulated insulin secretion (GSIS, secreted insulin/content) (n = 3, N = 3). D–F: MIN6 cells were treated for 8 h or 16 h with GC7 and then exposed to either normoxia (Nx) or warm anoxia followed by a normothermic reoxygenation step (A/R, ≤0.1% O₂ at 37°C for 3 h + 20% O₂ at 37°C for 2 h). D: survival rate evaluated using a live-dead fluorescence assay (n = 6–12, N = 3). Oxidative stress production evaluated with CM-H2DCFDA probe (n = 3–16, N = 3) (E), and mitochondrial ROS production with MitoSOX probe (n = 3–18, N = 3) (F). Hoechst (nuclei) and MitoGreen (mitochondria mass) probes were used to normalize corresponding fluorescent signal. Scatter dot plots show individual values and means ± SD. Curves show means ± SE. Statistical significance of mean differences was tested by one-way ANOVA followed by Tukey's post hoc test. \*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*P < 0.0001. GC7, N1-guanyl-1,7-diaminoheptane; ROS, reactive oxygen species.</figcaption></figure>

rise in incidence since COVID-19 pandemic, particularly among children (34–36). Despite significant advances in insulin therapy, current treatments still often fail to prevent severe hypoglycemic episodes and long-term complications, and hence to improve quality of life. Islet allograft transplantation has emerged in clinical trials as a promising alternative for patients with severe T1D, ameliorating metabolic outcomes, quality of life, and patient survival. However, its widespread clinical application is limited by two key factors, which are a shortage of organ donors and the low yield of the islet transplantation procedure, primarily due to ischemia/reperfusion-induced damage (1, 10).

Polyamines, which are essential for the hypusination of eIF5A, are present at elevated levels in both exocrine and endocrine pancreatic cells, with the highest concentrations found in insulin-producing beta cells, suggesting their critical role in pancreatic biology (37). The accumulation of polyamines in beta cells and eIF5A hypusination has been linked to T1D onset where they are associated with increased endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production (38, 39). Taken together, these findings make beta cells strong candidates for the protection by GC7, an inhibitor of eIF5A hypusination, which has been shown to alleviate oxidative stress, ER stress, and energetic stress in other cell types (20–23). Using insulinoma INS-1 and MIN6 cells, well-established models to study beta cell metabolism (40), we confirmed this hypothesis. Although it was already

demonstrated that GC7 protected INS-1 cells against ER stress (41, 42), we demonstrate here that GC7 effectively protects beta cells from anoxia/reoxygenation injury by reducing oxidative stress, mainly by limiting mitochondrial ROS production, the chief ischemia/reperfusion injury mechanism (15). However, this effect alone does not fully account for the robust protection observed in our survival assays. In fact, our data suggest that the metabolic actions of GC7 also play a pivotal role in enhancing the adaptive response to energetic stress during anoxic conditions. Specifically, we found that GC7 treatment slowed down mitochondrial activity, as evidenced by reduced oxygen consumption and diminished oxidative phosphorylation, while simultaneously increasing anaerobic glycolysis. This is particularly relevant because beta cells have fewer antioxidant enzymes (such as superoxide dismutase, glutathione peroxidase, and catalase) compared with other cell types, making them more susceptible to oxidative stress (43).

Regarding the functional impact of GC7 on beta cells, we observed a reduction in ATP levels and a reversible dose- and time-dependent inhibition of glucose-stimulated insulin secretion (GSIS) in relation to the extent of eIF5A hypusination inhibition. These findings align with the well-established relationship between ATP levels and GSIS. Furthermore, they substantiate previous studies showing that the depletion of polyamines impairs GSIS as well as pancreatic insulin content, and insulin gene transcription, the last two not being
<figure><img src="image_19.png" /></figure>

found in our conditions (44, 45). In vivo, mice lacking DHPS specifically in insulin-expressing cells develop beta cell dysfunction by 4 wk of age, resulting in reduced beta cell mass and plasma insulin levels (46). Furthermore, transgenic mice overexpressing SSAT, an enzyme involved in polyamine catabolism, exhibit spermidine depletion, which leads to decreased ATP levels in islets and impaired GSIS (47). Although eIF5A hypusination levels were not assessed, spermidine depletion suggests a concurrent reduction in DHPS activity (47). Interestingly, another study has shown that depleting polyamines and inhibiting hypusination in mouse models of T1D can have a protective effect, preserving beta cell function and mass, and hence delaying disease onset (48). Taken together, these findings suggested that sustained inhibition of eIF5A hypusination may impair beta cell function, particularly insulin production and secretion, a situation different from the transitory inhibition found in our work after acute GC7 treatment. Nevertheless, the effect of GC7 on INS-1 cells appears to be similar to that described previously (18, 20, 21). Mitochondrial silencing imposes a metabolic shift from oxidative phosphorylation to glycolysis, resulting in reduced ATP production and inhibition of GSIS, both of which are entirely reversible within 48 h. This cellular reprogramming limits energy consumption (for GSIS in beta cells) and, as a result, the low ATP level is sufficient to temporarily maintain the integrity, function, and survival of the cells, allowing them to survive in the event of oxygen and substrate deprivation, as is the case during ischemia, and waiting reperfusion. These results allow us to envisage that GC7-conditioned islets could be fully functional shortly after transplantation into a recipient.

Beta cells displayed few metabolic flexibility, compared, for example, with α cells (49), suggesting that GC7 must induce a pathway facilitating metabolic rewiring to anaerobic glycolysis not present in basal condition but which remains to be identified. It is well-described that hypusination of eIF5A is a crucial mechanism for mitochondrial protein translation (20, 50, 51) and import (52). Furthermore, its inhibition decreased strongly the level of proteins from the respiratory chain and mitochondrial activity. In our model, we did not observe this decrease in mitochondrial proteins despite a sharp decrease in mitochondrial activity. We can therefore assume that a key protein redirecting the flow of pyruvate from oxidative phosphorylation to lactate formation was involved. This could be due either to an inhibition of pyruvate transport and metabolism within mitochondria by inhibiting mitochondria pyruvate carrier or pyruvate dehydrogenase activity, or conversely to an increase in LDH activity (pyruvate -> lactate). The increased expression of *Ldha* mRNA (favoring pyruvate catabolism) without modification of *Ldhb* (favoring pyruvate anabolism) found in INS-1 cells treated with GC7 could augment this LDH activity, which is normally limited in beta cells (53). Moreover, the slight increase in insulin secretion without glucose stimulation found after sustained GC7 treatment could be related to this increase in LDHA, supporting in the same time the role of LDHB in the inhibition of basal insulin secretion (54). These crucial mechanisms linking eIF5A hypusination and metabolic choice need to be elucidated to better understand how inhibition of eIF5A function enables cells to become oxygen-independent. It should be noted that the mixed metabolism exhibited by INS-1 cells and their capacity

to anaerobic glycolysis, which does not replicate the higher oxidative phosphorylation metabolism of beta cells, resembled the metabolism of stem cell-derived islets (55). We can therefore assume that the GC7 strategy could also be useful for protecting insulin-producing cells derived from stem cells which already displayed metabolic flexibility.

Finally, it should be stressed that after their intraportal transplantation, islets lack a functional vascular system and have access only to the low oxygen level found in veins. Although islets acquire a functional capillary network within 1-2 wk, oxygenation remains diffusion-dependent in the early posttransplant period (56, 57). Various strategies, including islet encapsulation, have been developed to improve oxygenation (58-62), but these methods have not yet been successfully translated into clinical practice. GC7 treatment is clinically interesting to promote islet survival during transplantation as it reduced oxygen consumption in beta cells, enhancing their resistance to hypoxemia by decreasing cellular oxygen demand. This relative tolerance to hypoxia decreased oxidative stress and improved cell survival.

In our work, we demonstrate that the inhibition of hypusination by GC7 allows the survival of INS-1 and MIN6 cells during short anoxia and reoxygenation by silencing mitochondria and rewiring metabolism to anaerobic glycolysis. These insulinoma-derived cells are commonly used in vitro to study beta cell biology but do not reproduce the complex environment of pancreatic islets. Indeed, pancreatic islets are composed of numerous cell types with different sensibility to oxygen variation, beta cells being the more sensitive. Importantly, the islet cells are exposed differently to oxygen deprivation with a decreasing oxygen gradient going from the surface to the core of the islets. In addition, in vitro models do not allow to study the interaction between the different cells of the islets, especially for metabolites exchange, and the conclusions must be drawn cautiously considering these point of view. Another limitation of our findings was the anoxia/reoxygenation model mimicking ischemia/reperfusion in vivo, which has demonstrated its robustness in a previous study about organ transplantation but which cannot reproduce completely the complex situation found during islet transplantation procedure. Indeed, in that scenario, the islets are subjected to several cycles of ischemia/reperfusion of varying intensities. First, at the beginning of the process, when the donor pancreas is removed, warm ischemia occurs, followed by cold ischemia during organ storage. These episodes of ischemia are followed by partial reoxygenation when pancreas was digested to isolated the islets. Indeed, culture conditions do not reach optimal oxygen supply required normally for islet function and islets loose their intravascular network during these steps (63). Finally, when infused into the portal vein, which has low $pO_2$, the islets will eventually implant themselves in the liver parenchyma with limited blood supply for several days, generating additional ischemia (64) while awaiting effective neovascularization, which is mainly enabled by the recipient's endothelial cells (65). Thus, to clearly demonstrate the anti-ischemic action of GC7 suggested in the present work, we must transpose this study using primary islets prepared following standard procedure for transplantation and graft them into portal vein of type 1 diabetes models to faithfully reproduce the complex multicellular construction necessary for a successful islet transplantation.
<figure><img src="image_20.png" /></figure>

Conclusions

Our findings presented here suggest a potential clinical
application of GC7 as a preconditioning agent to improve
beta cells resistance to ischemia/reperfusion and thus islet
grafting outcomes. By improving the quality and quantity of
islets available for transplantation, this approach could facil-
itate access to pancreatic islet grafts for the many young
patients with T1D who are eagerly awaiting new treatments.

DATA AVAILABILITY

Data will be made available upon reasonable request.

SUPPLEMENTAL MATERIAL

Supplemental Figs. S1–S6: https://doi.org/10.6084/m9 figshare.
30234790.

ACKNOWLEDGMENTS

The authors greatly acknowledge the IRCAN Cytomed facilities that are supported by "le Cancéropôle PACA," "la Région Provence Alpes-Côte d'Azur," and "le Conseil Départemental 06."

GRANTS

This work was supported by a Grant from Société Francophone du Diabète (2022). H.O. has a PhD fellowship "Espoir de la Recherche 2022 from the Fondation pour la Recherche Médicale".

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by
the authors.

AUTHOR CONTRIBUTIONS

M.T., A.S., and D.F.P. conceived and designed research; H.O.,
F.M., M.C., I.R., G.J., and D.F.P. performed experiments; H.O., F.M.,
M.C., I.R., G.J., M.T., and D.F.P. analyzed data; H.O., F.M., I.R.,
E.V.O., A.S., and D.F.P. interpreted results of experiments; H.O.
and D.F.P. prepared figures; H.O. and D.F.P. drafted manuscript;
H.O., F.M., M.C., I.R., M.T., E.V.O., A.S., and D.F.P. edited and
revised manuscript; H.O., F.M., M.C., I.R., G.J., M.T., E.V.O., A.S.,
and D.F.P. approved final version of manuscript.

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