PhD Marion De Toledo
email: marion.detoledo@igmm.cnrs.fr

Isabel C Lopez-Mejia

Patricia Cavelier

Marine Pratlong

Célia Barrachina

Xavier Gromada

Jean-Sébastien Annicotte

Jamal Tazi
email: jamal.tazi@igmm.cnrs.fr

PhD Carine Chavey
email: carine.chavey@igmm.cnrs.fr

Lamin C counteracts glucose intolerance in aging, obesity and diabetes through β-cell adaptation

Aging-dependent changes in tissue function are associated with the development of metabolic diseases. However, the molecular connections linking aging, obesity and diabetes remain unclear. Lamin A, lamin C and progerin, products of the Lmna gene, have antagonistic functions on energy metabolism and lifespan. Lamin C, albeit promoting obesity, increases lifespan suggesting that this isoform is crucial for maintaining healthy conditions under metabolic stresses. Since β-cell loss during obesity or aging leads to diabetes, we investigated the contribution of lamin C to β-cell function in physiopathological conditions. We demonstrate that aged lamin C-only expressing mice (Lmna LCS/LCS ) become obese but remain glucose tolerant, due to adaptive mechanisms including increased β-cell mass and insulin secretion. Triggering diabetes in young mice revealed that Lmna LCS/LCS animals normalize their fasting glycemia by both increasing insulin secretion and regenerating -cells. Genome-wide analyses combined to functional analyses revealed an increase of mitochondrial biogenesis and global translational rate in Lmna LCS/LCS islets, two major processes involved in insulin secretion. Altogether, our results demonstrate for the first time, that the sole expression of lamin C protects from glucose intolerance through a -cell adaptive transcriptional program during metabolic stresses highlighting Lmna gene processing as a new therapeutic target for diabetes treatment.

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Diabetes

Although recent reports highlighted that Lmna isoforms are involved in the structure and function of the exocrine pancreas (9), their contribution to the endocrine pancreas and, more particularly, in insulin-producing -cells remain to be investigated. We hypothesized that lamin C might contribute to maintain adequate glucose homeostasis during aging through a role on pancreatic β-cell plasticity or regeneration. Using a comprehensive analysis of the metabolic phenotype of Lmna LCS/LCS mice using different models related to β-cell failure (i.e. aging, STZinduced diabetes and obesity-induced diabetes), we demonstrate that exclusive lamin C expression favors β-cell mass, insulin production and insulin secretion in conditions where dual expression of lamin A and lamin C from the Lmna gene are accompanied by abnormal glucose homeostasis. This adaptive effect on β-cell mass and function is associated with an increase in mitochondrial activity and of global translation rates, providing a molecular link between lamin C and β-cell fitness during aging. Using a model of T2D (diet-induced obesity, DIO), we report that Lmna LCS/LCS mice enhance insulin secretion to adapt to DIO and to maintain normoglycemia.

Finally, in streptozotocin-induced diabetes mice -cell failure was rescued in Lmna LCS/LCS through increased regeneration of β-cells. Altogether, our findings demonstrate that the exclusive expression of lamin C improves β-cell function and mass during metabolic stress and suggest that the manipulation of Lmna isoforms can be targeted to counteract diabetes.

Introduction

Lamin A, lamin C and progerin proteins, produced by alternative RNA processing of the Lmna gene, are intermediate filaments that bridge peripheral heterochromatin to the nuclear envelope (1), thus participating in the maintenance of chromatin architecture (2,3) and in transcriptional regulation (4). Mutations in the Lmna gene result in laminopathies, including progeria, which lead to metabolic abnormalities such as lipodystrophy, hypertriglyceridemia, insulin resistance and metabolic syndrome, thus suggesting an important role for A-type lamins in the regulation of glucose and lipid metabolism (5). Comparing Knock-in mice expressing all three lamin isoforms (lamin A, lamin C and progerin) or exclusively lamin C isoform (Lmna LCS/LCS ), we previously reported antagonistic functions of Lmna isoforms in energy expenditure and lifespan, pointing out a key function for this gene in the control of energy homeostasis (6).

Despite showing increased body weight, Lmna LCS/LCS have an unexpectedly increased lifespan suggesting a role for lamin C in improving metabolic health during aging.

Pancreatic  cells play a critical role in maintaining glucose homeostasis through the secretion of insulin, and their dysfunction results in diabetes. Whereas type 1 diabetes (T1D) is an autoimmune disease that results from the destruction of -cells, type 2 diabetes (T2D) is due to energy imbalance and is characterized by impaired peripheral insulin sensitivity followed by defects in insulin secretion. The common hallmark of T1D and T2D is an inadequate supply of functional insulin-producing -cells, leading to the dysregulation of glucose metabolism and associated metabolic complications (7). The restoration of normal β-cell mass and function has therefore become of great interest for the generation of novel anti-diabetic drugs. In this context, β-cell regeneration has been extensively explored in mice and humans. However, the mechanisms involved in the functional plasticity required for the regeneration of β-cell mass remain poorly understood (7,8).

Research Design and Methods

Animal experiments and ethics statement

Lmna LCS/LCS Knock-In mice were bred onto C57BL/6J background as described (10). All animal procedures were conducted in accordance with the French national animal care guidelines. Mice were maintained in pathogen-free conditions in our animal facility (E34-172-16). Mice were housed under a 12-h light/dark cycle and given an ad libitum regular chow diet (A04; Safe). All experiments were conducted by authorized personnel (agreements MDT34-433; CC-I-34UnivMontp-f1-12) and approved by the Ethics Committee of the Languedoc-Roussillon region Montpellier (CEEA-LR-12003). Male mice were used for in vivo experiments and isolated islets were prepared from male or female mice.

Histology and immunohistochemistry

Pancreas sections (4µm) were deparaffinized, rehydrated, stained with hematoxylin and eosin (HE) and visualized with a Nanozoomer. Islets diameter were measured using NDP View software.

For immunohistochemistry, rehydrated sections were incubated in citrate buffer at 95°C for 30min. After blocking, sections were incubated in primary antibodies (see table 2) overnight at 4°C and were detected using appropriate secondary antibodies. Nuclei were counterstained with Hoechst® 33342.

Metabolic analysis and generation of diabetic mouse models

For IPGTT, 18 h-fasted mice were injected intraperitoneally with glucose (1.7g/kg), and glycemia was measured using a blood glucometer (Roche). Insulin levels were quantified using Mouse Ultrasensitive Insulin ELISAs (ALPCO). To induce T2D, 12-week-old mice had unrestricted acces to a HFD (ResearchDiet) for 25 weeks. To induce acute β-cell loss, 13 weeks old mice were injected i.p. with a single high dose (200mg/kg) of streptozotocin (Sigma-Aldrich) dissolved in 0.1M sodium citrate buffer (pH4.4).

Pancreatic islet Isolation and Glucose-stimulated insulin secretion

Mouse islets were isolated by type V collagenase digestion (Sigma) of pancreata. After separation in a density gradient medium (Histopaque®-1119, Sigma), islets were handpicked.

They were then cultured for 18-20h at 37°C in a 95% air-5% CO 2 atmosphere in RPMI-1640 (ThermoFisher) containing 10% FCS and 100 µg/ml penicillin-streptomycin.

To evaluate insulin secretion, 3 replicates of 5 islets were preincubated for 1h at 37°C in KRB buffer. After low (3mM) or high (20mM) glucose stimulation, secreted insulin and total insulin cell content were measured. Data are expressed as a ratio of secreted insulin/total insulin content.

Total pancreatic insulin and glucagon content

Pancreata were lysed in 2ml Acid/Ethanol lysis buffer and incubated O/N at 4°C. After neutralization with 10% of 1M Na2CO3, total insulin and glucagon content were measured by ELISA (Mouse Ultrasensitive Insulin elisa-ALPCO, Mercodia Glucagon ELISA -10 μL).

RT-qPCR analysis and mitochondrial DNA quantification

Total RNA was extracted from isolated islets using TRIzol (Life Technologies, USA).. Samples were quantified with a NanoDrop ND-1000. 1.5 μg RNA were then converted to cDNA with the Maxima First Strand cDNA Synthesis Kit (#K1642, Thermo Scientific). Gene expression was normalized to cyclophillin and TBP.

For mitochondrial DNA quantification, islets were digested using proteinaseK. qPCR analysis was performed with primers specific for mitochondrial genes (mtND5, mt16s) and normalized to nuclear genes (LN11, HK2).

qPCR was performed with a LightCycler® 480 and LightCycler® 480 SYBR GreenI Master (Roche). Primer sequences are provided in Supplemental Table 1.

Ultrastructural transmission electron microscopy

After isolation, 150 islets were immersed in a solution of 2.5% glutaraldehyde. After dehydration, samples were embedded in EmBed812. 70nm sections were counterstained with uranyl acetate and observed on a Hitachi-7100 microscope.

Seahorse experiments and metabolic flux analysis

Oxygen consumption rate (OCR) was measured in 96-well plates using a XFe96-Seahorse (Agilent). Briefly, 25 islets/well were plated in poly-D-lysine-coated wells in XF-assay medium containing 3mM glucose, 1mM sodium pyruvate and 1mM glutamine (pH7.4) for 1 h at 37°C without CO 2 . OCRs were monitored in response to glucose (20mM), oligomycin (2μM), FCCP (1μM) and rotenone (0.5μM). OCRs measurements were normalized by total DNA level using a Qubit dsDNA HS assay kit (LifeTechnologies).

Incorporation of 35 S-Met/Cys into isolated islets

Seventy isolated islets were incubated in Met/Cys-depleted RPMI medium (Sigma) for 30min and then supplemented with 50μCi 35 S-Met/Cys EasyTag Express (PerkenElmer) for For Peer Review Only Diabetes 30min at 37°C. Islets were lysed in RIPA buffer. 10µg of total protein were size-fractionated on 4-12% gradient SDS-PAGE. The gel was dried and quantified using a PhosphoImager (GE-Healthcare).

Flow cytometry

Islets dissociated with 0.05% Trypsine-0.5 mM EDTA were fixed with 1% PFA and permeabilised with 0.05% Triton X-100. After blocking, cells were incubated anti-insulin antibody, washed, reacted with a FITC-conjugated antibody and analyzed by FACSCANTO II (Becton Dickinson).

RNA isolation, RNA-seq, RRBS, ATAC-seq and data analysis

Total RNA was extracted using TRIzol and quantified on Qubit (Life Technologies). RNA quality was verified using RNA pico-chips on a 2100-Bioanalyzer (Agilent). Total DNA was purified using MasterPure TM Complete DNA (Illumina). Libraries for RNA-seq and RRBS were prepared and sequenced by the MGX sequencing platform in Montpellier (France).

For ATAC-seq analyses, islets were cryopreserved in 50%FCS, 40%RPMI, 10%DMSO and processed by ActiveMotif.

All data are deposited in GEO-NCBI under accession number GSE122361.

Statistical analysis

Results were expressed as the mean ± SEM. Statistical comparison were made using student's t-test. Statistically significant diffrencies are indicated as *p<0.05, **p 0.01, ***p 0.001, ****p 0.0001

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A detailed description of the all procedures and bioinformatic analyses can be found in Supplementary Methods.

Data and Resource Availability

The resources generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request. The genome wide datas generated and/or analyzed during the current study are deposited in GEO-NCBI under accession number GSE122361.

Results

Increased obesity in aged Lmna LCS/LCS mice is associated to improved glucose tolerance

To evaluate whether lamin C regulates β-cell function, we evaluated glucose homeostasis in Lmna LCS/LCS mice (6). Initially designed to generate a mouse model of progeria, the c.1827C>T;p.G609G point mutation was introduced into a floxed neo cassette (Supplemental data 1A). This cassette was inserted in intron-10 of the Lmna gene, downstream of the polyadenylation site of lamin C, thus preventing the formation of prelamin A transcripts by blocking lamin A-specific splicing (10). The conditional mutant allele (Lmna LCS ) resulted in the exclusive expression of lamin C. This was confirmed by comparing its levels in brain, liver, pancreas and muscle from Lmna +/+ (wild type, WT) and Lmna LCS/LCS mice (Supplemental data 1B).

We observed increased expression of lamin C in Lmna LCS/LCS mice compared to WT in most tissues except in the brain, where the lamin C/lamin A ratio is already high even in WT mice, as previously shown (11).

With the experimental Lmna LCS/LCS model established, we then investigated the metabolic phenotype of these mice. The Lmna LCS/LCS mice gained more weight than control mice as they aged (Fig 1A). This increase in boby weight is mainly due to an increase in white adipose tissue without any further change in body composition as shown in our previous study (6). Body weight and fasting glucose levels in Lmna +/+ and Lmna LCS/LCS mice were similar until 25 weeks of age (Fig 1A -B). Interestingly, the significant increase in body weight was observed from 30 weeks of age in Lmna LCS/LCS mice, but was even more pronounced at 75 weeks of age. The following aging experiments are thus conducted by comparing "young" (25 weeks old) to "old" animals (75 weeks old). We observed a significant increase in fasting glycemia in control mice as they aged (Fig 1B

Lmna LCS/LCS mice have increased β-cell mass and insulin secretion during aging

Our observations in aged Lmna LCS/LCS mice prompted us to study whether lamin C could contribute to pancreatic islet function by regulating pancreatic mass and/or insulin secretion.

Old Lmna LCS/LCS mice had heavier pancreata, whereas no differences in mass were observed between genotypes in young animals (Fig 2A). Histological analyses revealed that while young Lmna +/+ and Lmna LCS/LCS pancreata were microscopically similar, pancreata from old Lmna LCS/LCS mice displayed significantly larger islets of Langerhans compared to Lmna +/+ mice (Fig 2B). This effect was further amplified in the pancreata of "very old" (141 weeks old) Lmna LCS/LCS mice, as evidenced by hyperplasic islets (Supplemental data 1C). In young animals, measurements of whereas no changes were observed on glucagon-positive -cells (Supplemental data 2A). In addition, when compared to young mice, old Lmna LCS/LCS mice exhibited an increased number of islets per pancreas (Fig 2F). Furthermore, total pancreatic insulin level was increased in LCS mice compared to control mice (Fig 2G) whereas global glucagon level was not significantly different (supplemental data 2B). These data demonstrate that the expression of lamin C protects from aging-induced β-cell mass loss by increasing the size and the number of islets of Langerhans and the number of insulin-positive cells.

We then investigated the functionality of these abundant and large Lmna LCS/LCS islets through glucose-stimulated insulin secretion (GSIS) experiments. Although no difference in GSIS between genotypes was observed in pancreatic islets isolated from young animals, islets derived from old Lmna LCS/LCS mice displayed a 29.5% higher GSIS than age-matched control islets after 20mM glucose stimulation (Fig 2H). Interestingly, no difference of insulin secretion in basal, 2.8mM glucose stimulation was observed, suggesting that the increased insulinemia observed in fasted Lmna LCS/LCS mice (Fig 1D) could be due to a cumulative effect of the increased number of islets and their improved insulin secretion capacity.

To confirm the direct effect of lamin C on β-cells, we next generated MIN6 pancreatic βcell lines in which lamin A expression was abolished using CRISPR/Cas9-mediated genome editing (MIN6 LCO ). Targeting exon 11 of the Lmna gene resulted in a complete depletion of lamin A expression. In the MIN6 LCO cells expressing only lamin C (Supplemental data 3A) insulin secretion in response to a 20mM glucose stimulation was increased compared to the control MIN6 WT cells (Supplemental data 3B), indicating that lamin C can enhance insulin secretion and improve β-cell function.

Taken together, these results showed that old Lmna LCS/LCS mice have increased β-cell number and improved insulin secretion in response to glucose both in vitro and in vivo. These adaptive mechanisms may account for the normoglycemia observed in old Lmna LCS/LCS mice despite their obesity.

Increased insulin secretion in Lmna LCS/LCS mice can counteract obesity-induced type 2 diabetes

To counteract obesity, β-cells need to increase their secretory capacity by progressive expansion of their mass (12). To evaluate how Lmna LCS/LCS β-cell function adapts to metabolic challenges, we investigated the capacity of Lmna LCS/LCS -derived islets to counteract hyperglycemia observed during obesity-induced T2D in young mice. Challenging 12 weeks old Lmna +/+ and Lmna LCS/LCS mice with a 60%kcal high-fat diet (HFD) for 25 weeks resulted in increased body weight (Fig 3A). This body weight gain was significantly higher in Lmna LCS/LCS mice and was associated to increased food intake when compared to the control mice (Supplemental data 4). No differences were observed between genotypes in the consumption of regular chow diet (CD). Monitoring of fasting glycemia over the course of HFD revealed that during the first 10 weeks, Lmna LCS/LCS mice were hyperglycemic compared to control mice, probably due to their higher body weight (Fig 3B). However, at week 15 under HFD, a significant improvement in fasting glycemia was observed. At week 25, Lmna LCS/LCS mice demonstrated a further improvement in glucose tolerance (Fig 3C ) and showed increased insulin secretion in response to in vivo glucose stimulation (Fig 3D). GSIS experiments using islets isolated from HFD-fed mice for 25 weeks demonstrated that while Lmna +/+ -derived islets failed to secrete insulin in response to glucose, islets from HFD-fed Lmna LCS/LCS mice efficiently secreted insulin in response to glucose (Fig 3E).

These results demonstrate that Lmna LCS/LCS mice efficiently counteract HFD-induced hyperglycemia by increasing insulin secretion. Taken together with our aging studies, these For Peer Review Only Diabetes results suggest that a lamin C-specific adaptive mechanism is engaged by insulin-producing cells to maintain glucose responsiveness in order to counteract the effects of aging and obesityinduced T2D.

Increased -cell regeneration in streptozotocin-injected Lmna LCS/LCS mice

To further explore the role of lamin C on β-cell number and function, we evaluated the capacity of Lmna LCS/LCS mice to regenerate β-cells in an acute β-cell loss context. 13 weeks old Lmna +/+ and Lmna LCS/LCS animals were treated with a single dose of streptozotocin (STZ, 200 mg/kg) to mimic diabetes, and the regeneration of β-cells was measured 18 days after STZ injection. 3 days after STZ treatment, Lmna +/+ and Lmna LCS/LCS mice became diabetic with no significant difference in fasting glucose level (Fig 4A). From day 4, while Lmna +/+ mice remained hyperglycemic, blood glucose levels in Lmna LCS/LCS mice rapidly decreased with a complete normalization of glycemia 18 days after STZ injection. Moreover, we observed approximately 15% of body weight loss during the first 4 days following STZ treatment for both genotypes. 18 days after STZ injection Lmna +/+ mice remained as leaner as they were before the treatment, while Lmna LCS/LCS mice recovered their initial body weight (Supplemental data 5A). Consistent with our previous observations, Lmna LCS The increased number of islets in Lmna LCS/LCS mice led us to hypothesize that Lmna LCS/LCS mice are either protected from STZ-induced apoptosis and/or are able to efficiently regenerate β-cells after STZ-induced damage. 72h after STZ treatment, β-cells were strongly depleted equally in Lmna +/+ and Lmna LCS/LCS mice as shown by representative immunostaining as well as β-cells mass quantification (Fig 4D). The susceptibility of β-cells to STZ-induced apoptosis was confirmed by immunostaining (Supplemental data 5B, left panel), and quantitative assessments 48h after STZ treatment using TUNEL assays showed no significant difference in the number of apoptotic cells between Lmna +/+ and Lmna LCS/LCS mice (Supplemental data 5B, right panel). This finding was further confirmed in the MIN6 β-cell line treated with STZ, in which no differences in the number of apoptotic cells were observed between MIN6 WT and MIN6 LCO cells (Supplemental data 5C).

We next studied β-cell regeneration after STZ treatment in Lmna LCS/LCS mice. The presence of proliferating cells after STZ treatment was quantified using phospho-histone H3 (phosho-H3) antibody staining on pancreatic sections at day 9 post-STZ injection. Pancreata from Lmna LCS/LCS mice had a significantly higher number of phospho-H3 positive cells compared to control mice (Fig 4E ), suggesting an increased regenerative capacity in Lmna LCS/LCS mice. To identify the functional profile of these newly generated cells, we next performed coimmunostaining for phosho-H3 (red) and different pancreatic markers, including insulin (βcells), lectin (ductal cells), glucagon (-cells) and somatostatin (-cells). Co-immunostaining was observed for phosho-H3 and insulin, as well as lectin but not with glucagon or somatostatin, suggesting that proliferating cells observed in Lmna LCS/LCS mice may arise from duplication of pre-existing β-cells and from ductal cells (Fig 4F) two well-known processes for -cell neogenesis (8).

Altogether, these results suggest that Lmna LCS/LCS mice are able to counteract STZinduced diabetes by regenerating β-cells.

Lmna LCS/LCS mice display a transcriptional reprogramming of genes involved in mitochondria and translation

Given the important role of lamins in chromatin organization and in the control of gene expression (4), we next evaluated the transcriptional status, DNA methylation and chromatin accessibility of islets isolated from 75 weeks old Lmna +/+ and Lmna LCS/LCS mice. RNA-sequencing based analysis (RNA-seq) revealed that 1507 genes (9.3% of the sequenced genes) were significantly differentially regulated between genotypes (p-value0.05), with 1024 genes upregulated and 483 genes downregulated in Lmna LCS/LCS mice relative to controls (Fig 5A). Gene ontology enrichment analysis showed an enrichment of genes associated with mitochondria (15.92%) and global translation (15.59%). Most genes associated with mitochondrial function (205/240 genes) and translation pathways (169/235 genes) were significantly upregulated in Lmna LCS/LCS islets compared to Lmna +/+ islets. Reduced representation bisulfite sequencing (RRBS)-based methylome analysis revealed that the observed variations in gene expression were not correlated to changes in DNA methylation (Fig 5B). Indeed, independent of the region of the genome that was analyzed (Supplemental data 6), no differentially methylated cytosines (DMC) were found in 1383868 cytosines tested. Moreover, no differentially methylated regions (DMR) were found in 872305 tested regions (GEO accession number: GSE122361). In order to evaluate whether the transcriptional dysregulation was due to a global remodelling of the chromatin compaction and/or accessibility, we performed Assay for Transposable-Accessible For Peer Review Only Diabetes Chromatin with high-throughput sequencing (ATAC-seq) on pancreatic islets isolated from old Lmna +/+ vs Lmna LCS/LCS mice. ATAC-seq results revealed that no difference in chromatin accessibility profiles were observed between Lmna +/+ and Lmna LCS/LCS isolated islets (Fig 5C ), suggesting that the transcriptional dysregulations observed in RNA-seq analysis were not due to changes in global chromatin accessibility upon the exclusive expression of lamin C.

To go further into the molecular mechanisms by which lamins could modulate transcription programs, we analyzed the chromatin architecture by investigating the spatial nuclear localization of selected histone marks that are known to modulate gene expression (13).

Immunofluorescence analyses of pancreatic sections showed an enrichment of the active enhancer mark H3K27Ac at the nuclear periphery in Lmna LCS/LCS β-cells compared to control (Fig RNA-seq data also highlighted a role for lamin C in the regulation of protein translationrelated genes in islets from old Lmna LCS/LCS mice. In particular, genes involved in ribosome synthesis (Rpl and Mrpl protein family) and in the initiation phase of eukaryotic translation (eIFs, eukaryotic initiation factor) were overexpressed in Lmna LCS/LCS islets. qPCR experiments confirmed that the expression of genes directly implicated in different steps of translation was significantly increased in Lmna LCS/LCS islets compared to Lmna +/+ islets (Fig 6E). Because ribosome synthesis is tightly correlated with ribosomal RNA (rRNA) synthesis, we also measured 18S and 28S rRNA levels in isolated islets and found that their expression levels were 1. Altogether, these data suggest that the increased insulin secretion observed in Lmna LCS/LCS mice is likely due to an increase in global translation rates, which enhances insulin production. The secretion is supported by an increased number of functional mitochondria that play a crucial role in insulin exocytosis upon glucose stimulation.The observed lamin C-mediated increase in insulin production and secretion may explain the improved metabolic phenotype observed in obese, aged Lmna LCS/LCS mice. It has been shown that β-cell turnover decreases with aging due to the onset of senescence [START_REF] Aguayo-Mazzucato | β Cell Aging Markers Have Heterogeneous Distribution and Are Induced by Insulin Resistance[END_REF]. Indeed, the expression of p16 Ink4a , a key marker of senescence, limits islet proliferation and regeneration in an age-dependent manner [START_REF] Krishnamurthy | p16INK4a induces an age-dependent decline in islet regenerative potential[END_REF]. Conversely, the downregulation of p16 Ink4a in a Pten-null background reduces the age-induced decline of β-cell proliferation and enhances islet mass in aged mice [START_REF] Zeng | PTEN controls β-cell regeneration in aged mice by regulating cell cycle inhibitor p16ink4a[END_REF], as observed in our aged Lmna LCS/LCS mice. While one may speculate that a decrease in cellular senescence in our Lmna LCS/LCS mice may underlie the observed maintenance of β-cell mass during aging, no difference in the expression of the senescence marker p16 Ink4a was observed in our Lmna LCS/LCS mice versus controls (data not shown), suggesting that delayed senescence does not contribute to the maintenance of functional islets in our model. This hypothesis is further strengthened by the observation that β-cells which exclusively express lamin C adapt to pathological conditions unrelated to aging, such as diabetes induced in young mice.

Discussion

The specific functions of lamin A versus lamin C in β-cell maintenance and function during aging and/or metabolic stress is a central question. A limitation of the Lmna LCS/LCS model is that we cannot rule out potential contributing effects of lamin C in other organs, as well as an effect of lamin A deficiency in cells expressing lamin C only. Since the gene targeting strategy employed here to generate the Lmna LCS/LCS model precludes to specifically express lamin C only in β cells, and subsequently perform lineage-tracing experiments, this question remains to be addressed. Interestingly, mice expressing lamin A only (Lmna LAO ) are healthy with no detectable phenotypical or histopathological abnormalities, despite a complete loss of lamin C expression [START_REF] Coffinier | Direct Synthesis of Lamin A, Bypassing Prelamin A Processing, Causes Misshapen Nuclei in Fibroblasts but No Detectable Pathology in Mice[END_REF], even in 24-month-old mice. Challenging these Lmna LAO mice with HFD or STZ treatment may help to accurately characterize their specific functions in glucose homeostasis.

To decipher the molecular mechanism by which lamin C regulates pancreatic islet functions, we analyzed the impact of lamin C exclusive expression using genome-wide DNA methylation, ATAC-seq and high-throughput RNA sequencing.

For Peer Review Only Diabetes RNA-seq analysis revealed that the expression of genes involved in mitochondrial function and protein translation was altered in aged Lmna LCS/LCS islets compared to controls.

Unlike the decline of mitochondrial function observed in aging-associated diseases such as diabetes [START_REF] Sebastián | Mitochondrial Dynamics: Coupling Mitochondrial Fitness with Healthy Aging[END_REF], we found increased mitochondrial activity in aged Lmna LCS/LCS islets that likely contributes to the observed improvement in insulin secretion. It is interesting to note that decreased mitochondrial biogenesis was previously observed in adipose tissue from Lmna LCS/LCS mice aged 45 weeks (6). Contrary to white adipose tissue where mitochondria are relatively less abundant and crucial for adipocyte homeostasis in normal diet and temperature conditions, mitochondrial function in the β-cell is directly coupled to insulin biosynthesis and insulin exocytosis. Since both studies were not conducted in animals at the same ages (45 weeks (6) vs 75 weeks old in the present study), we cannot exclude an age-dependent effect in these tissues.

We can however propose that the differential expression of lamin isoforms may have a tissuespecific impact on mitochondrial gene transcription.

RNA-seq analysis and translation rate analysis revealed an increase of global protein synthesis in the islets of aged-Lmna LCS/LCS associated with an increased lifespan as described in our previous study (6). It is widely accepted that reduced protein synthesis promotes longevity [START_REF] Gonskikh | Alterations of the translation apparatus during aging and stress response[END_REF]. Indeed, protein biosynthesis in aging is accompanied by more frequent production of damaged proteins due to transcriptional or translational errors and co-translational protein misfolding. The increased lifespan associated to an upregulation of global protein synthesis in our aged-Lmna LCS/LCS mice suggests that proteins synthetized within the pancreatic islets are well-folded and biologically active, possibly due to the maintenance of an efficient protein quality control system. This hypothesis is supported by our demonstration that insulin, the major protein synthesized by β-cells, is properly secreted and efficient during aging, since aged-Lmna LCS/LCS mice remain glucose tolerant and normoglycemic. This functional insulin Page 21 of 60 For Peer Review Only Diabetes overproduction may contribute to the maintenance of a positive feedback loop of insulinregulated protein synthesis via the mTOR pathway [START_REF] Proud | Regulation of protein synthesis by insulin[END_REF].

How Lmna isoforms are able to modulate gene transcription in β-cells remains to be elucidated. Previous studies have described epigenetic dysregulation in pancreatic islets during T2D, showing differential methylation pattern associated with islet dysfunctions [START_REF] Volkov | Whole-genome Bisulfite Sequencing of Human Pancreatic Islets Reveals Novel Differentially Methylated Regions in Type 2 Diabetes Pathogenesis[END_REF] and changes in the open chromatin landscape of human islets [START_REF] Bysani | ATAC-seq reveals alterations in open chromatin in pancreatic islets from subjects with type 2 diabetes[END_REF]. Moreover, DNA methylation is a dynamic process in aging β-cells. De novo methylation of proliferation-related genes and demethylation of genes related to β-cell function increases insulin secretion during aging [START_REF] Avrahami | Aging-Dependent Demethylation of Regulatory Elements Correlates with Chromatin State and Improved β Cell Function[END_REF].

Nevertheless, we did not observe any difference in DNA methylation in Lmna LCS/LCS versus wild type pancreatic islets. This finding shows that the selective expression of lamin C does not influence overall DNA methylation in pancreatic islets and points to another regulatory mechanism.

In metazoan nuclei, specific lamina-associated domains (LADs) contribute to the overall spatial organization of the genome and are associated with gene repression [START_REF] Van Steensel | Lamina-Associated Domains: Links with Chromosome Architecture, Heterochromatin, and Gene Repression[END_REF]. Cellular defects in cells from progeria patients include widespread alterations in chromatin organization, such as the loss of heterochromatin domains and changes in epigenetic markers [START_REF] Mccord | Correlated alterations in genome organization, histone methylation, and DNA-lamin A/C interactions in Hutchinson-Gilford progeria syndrome[END_REF]. Furthermore, it has been shown that lamin A/C can modulate transcription through the regulation of epigenetic factors [START_REF] Cesarini | Lamin A/C sustains PcG protein architecture, maintaining transcriptional repression at target genes[END_REF]. Therefore, the expression of only one Lmna isoform may have a crucial impact on the spatial compartmentalization of inactive or active chromosome domains, which in turn may have consequences on transcriptional dysregulation. Surprisingly, we did not observe any global changes in chromatin accessibility through ATAC-seq experiments in our mouse models. Nevertheless, Lmna LCS/LCS pancreas show an active enhancer mark (H3K27Ac) relocalization to peripheral heterochromatin. The localization of an active histone mark in an heterochromatin environment has already been described showing that acetylation of histone in silent domains drive their mislocalization and derepression [START_REF] Cabianca | Active chromatin marks drive spatial sequestration of heterochromatin in C. elegans nuclei[END_REF]. Furthermore, there is increasing evidence for active and regulated transcription in the heterochromatin at the nuclear periphery [START_REF] Saksouk | Constitutive heterochromatin formation and transcription in mammals[END_REF]. Altogether, these observations lead us to speculate that the phenotype observed in Lmna LCS/LCS mice could be due to transcriptional reactivation of a subset of mitochondrial and translational genes through histone marks relocalization rather than by reorganizing global chromatin accessibility or modifying DNA methylation. The role of lamin C in the nuclear localization of specific loci and their respective histone marks requires further investigations.

In conclusion, Lmna LCS/LCS mice present an enhanced adaptive response to the increased metabolic demands during the course of diabetes and aging. This is probably due to efficient glucose-sensing and insulin production by their neogenerated islets [START_REF] Wortham | Mechanisms of β-cell functional adaptation to changes in workload[END_REF]. Our study reveals an important role for lamin C in pancreatic -cells, namely a protective role against metabolic defects arising from -cell dysfunction. This effect may promote the healthy aging of the Lmna LCS/LCS mice, and sustain their extended lifespan (6). The discovery of new drugs targeting Lmna alternative splicing may represent an innovative therapeutic strategy to counteract aging -cell decline and improve diabetes treatment. 80°C for later analysis of insulin content by ELISA (Mouse Ultrasensitive Insulin elisa-ALPCO, USA). Insulin content is collected after islets lysis in an Acid/Ethanol lysis buffer. Data are expressed as a ratio of insulin secreted/total insulin content.

For MIN6 GSIS assays, prior to glucose stimulation, cells were first starved in KRB buffer without glucose for 1h.

Total pancreatic insulin and glucagon content

Mouse pancreata were snap-frozen in liquid nitrogen immediately after collection. Samples were reduced into powder, resuspended in 2ml Acid/Ethanol lysis buffer and incubated O/N at 4°C. After sonication, samples were neutralized with 10% of 1M Na2CO3 and centrifuged.

Total Insulin and Glucagon content were measured by ELISA (Mouse Ultrasensitive Insulin elisa-ALPCO, Mercodia Glucagon ELISA -10 μL).

Flow cytometry

The islets were first dissociated into a free cell suspension with 0.05%Trypsine-0.5 mM EDTA. Cells were fixed with 1% PFA in PBS for 10 minutes, washed with PBS and permeabilised with 0.05% Triton X-100 for 10 minutes at 4°C. After blocking with PBS containing 1% BSA, 2% donkey serum, 50 mM glycine, and 0.5% gelatin, cell suspension was incubated with a guinea pig anti-insulin antibody (ab7842; Abcam) for 30 minutes at 4°C, washed and then reacted with a FITC-conjugated donkey anti-guinea pig antibody (F(ab')2 fragment; Jackson ImmunoResearch Labs). The cell suspension was then washed and analyzed by FACSCANTO II (Becton Dickinson).

MIN6 cell culture and apoptosis

The mouse insulinoma MIN6 pancreatic β-cell line (AddexBio) was maintained at 37°C in DMEM High Glucose, Glutamax, and sodium pyruvate (#31966-021, Thermo Fisher Scientific, MA, USA) supplemented with 15% fetal calf serum, 0.05 mM betamercaptoethanol and 100 µg/ml penicillin-streptomycin in an atmosphere of 95% air/5% CO2.

To evaluate the MIN6-induced apoptosis, cells were treated with STZ (12 mM) before staining with Annexin-V-FITC (Invitrogen) and propidium iodide and then analyzed by flow cytometry (FACSCalibur, Becton Dickinson).

CRISPR-Cas9-mediated LCO MIN6 cell line

To generate the MIN LCO cell line, we targeted exon 11 of the Lmna gene to enhance the use of the polyA site of lamin C located in intron 10. Gene-specific CRISPR sgRNA sequences were selected to generate a non-homologous end joining in exon 11 of the Lmna gene (gene ID: 4000) (Exon11-2119: 5'-ACTCGAAGCTTCCGCAGTGTGG-3').

The generated sgRNAs were inserted into the SIGMA pU6-gRNA. Both pU6-gRNA and pCMV-Cas9wt-2A-GFP or pCMV-Cas9 D10A-2A-GFP (SIGMA) were cotransfected into MIN6 cells using Lipofectamine 3000 Reagent (Thermo Fisher Scientific). GFP-positive cells were sorted by flow cytometry (FACSCalibur, Becton Dickinson), diluted, reseeded in 100 mm petri dishes and cultured for 2 weeks to obtain isolated clones. After handpicking clones for isolation, cells were collected and analyzed by western blot.

Histological studies

For paraffin treatment, pancreases were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned (4µm), and stained with hematoxylin and eosin (HE) following standard procedures for morphological evaluation. HE staining of pancreatic sections was visualized with a Nanozoomer slide scanner (Hamamatsu). Scanned images were analyzed with NDP View software. The mean diameter of the Langerhans islets was determined by measuring the diameter of at least 150 islets per mouse (n=5) using NDP View software.

Immunohistochemistry and image acquisition

For immunohistochemistry, pancreas sections were deparaffinized, rehydrated, and incubated for antigen retrieval in citrate buffer at 95°C for 30 min. Nonspecific antibody binding was blocked by incubation with PBS containing 1%BSA, 0.05% Tween20 for 30 min at room temperature. Sections were then incubated in primary antibodies (see table 2) overnight at 4°C. After being washed, primary antibodies were detected using appropriate secondary antibodies. Nuclei were counterstained with Hoechst® 33342 (2µg/ml, ThermoFischer Scientific).

β-cell apoptosis after STZ treatment was determined using a Click-iT™ TUNEL Colorimetric IHC Detection Kit (#C10625, Thermofischer scientific).

Widefield images were acquired using a Metamorph-driven (Molecular Devices, Sunnyvale, CA) Leica DM6000 fluorescence microscope with a 100X HCX Plan 0.

RT-qPCR analysis

Total RNA was extracted from isolated islets using TRIzol (Life Technologies, USA).

After precipitation in isopropanol, RNA pellets were washed in cold 75% ethanol and resuspended in nuclease-free water. Samples were quantified and evaluated for purity (260nm/280-nm and 260-nm/230-nm ratio) with a NanoDrop ND-1000 spectrophotometer. Total RNA (1.5 μg) was then converted to cDNA with the Maxima First Strand cDNA Synthesis Kit (#K1642, Thermo Scientific, US). Gene expression was quantified by SYBR green real-time PCR on the LightCycler® 480 System (Roche Applied Science). Reactions were run in triplicate. The mean expression value for the housekeeping genes cyclophillin and TBP was used as an internal control to normalize variability in expression. Primer sequences are provided in Supplemental Table 1.

Mitochondrial DNA quantification

Isolated islets were digested 1h at 55 °C in a lysis buffer containing 100 mM Tris (pH 8), 5 mM EDTA, 0.2% SDS, 200 mM NaCl, and 0.1mg/ml proteinase K for DNA extraction.

Quantitative PCR (qPCR) analysis of DNA samples diluted at 1 ng/μl was performed with primers specific for mitochondrial genes (mtND5, mt16s) and normalized to the mean of two nuclear genes (LN11, HK2). qPCR was performed with a LightCycler® 480 and LightCycler® 480 SYBR Green I Master (Roche).

RNA-Seq, RRBS, ATAC-Seq and Data analysis

RNA-Seq

RNA-Seq libraries construction

RNA-Seq libraries were constructed with the Truseq stranded mRNA sample preparation (Low throughput protocol) kit from Illumina. 400ng of total RNA were used for the construction of the libraries. Using poly-T oligo attached magnetic beads, total RNA was purified in order to obtain only poly-A mRNA. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using SuperScript II reverse transcriptase, Actinomycin D and random hexamer primers. The Second strand cDNA was synthesized by replacing dTTP with dUTP. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with 15 cycles of PCR. The final cDNA libraries were validated with a Fragment Analyzer (Advanced Analytical, Ankeny, IA) and quantified using the Kapa Library quantification kit (Kapabiosystems). On 1 sequencing lane of a flowcell V4, and 1 sequencing lane of a flowcell V2, the 6 libraries were pooled in equal proportions, denatured with NaOH were identified using Fisher's exact test with the weight method that is implemented in the topGO package. As confidence threshold we used a P-value of 1%. To perform this analysis the differentially expressed genes were compared with those of all known genes present in the annotation. The GO categories were found in the Org.Mm.eg.db package based on the gene reporter EntrezGeneID.

Reduced Representation Bisulfite Sequencing

Libraries construction

Library construction was performed using the Premium RRBS kit (Diagenode). Briefly, for each sample, 100ng of intact genomic DNA were digested using MspI for 12 hours at 37°C. DNA ends were repaired and Diagenode indexed adaptors were ligated to each end of the repaired DNA. Each ligated DNA was quantify by qPCR using the Kapa Library quantification kit (Kapabiosystems) on a LightCycler 480 (Roche Life Science) prior to pooling (8 samples per pool). Each pool was submited to bisulfite conversion and desalted. Optimal PCR cycle number was determined by qPCR using the Kapa Library quantification kit (Kapabiosystems) before the final enrichment PCR. Once purified using magnetic beads (AMPure XP, Beckman Coulter), library pools were verified on Fragment Analyzer (AATI) and precisely quantified by qPCR using the Kapa Library quantification kit (Kapabiosystems). Each pool was denatured, diluted to a 18 pM concentration and spiked with a 10 % phiX Illumina's library before clustering. Clustering and sequencing were performed in paired end 2*50nt, 2 lanes per pool , according to the manufacturer's instructions on a Hiseq2500 using Rapid V2

clustering and SBS reagents.

Analysis of RRBS data

to Benjamini-Hochberg multiple testing correction) was less than 0.05 and the difference of methylation percent above 25%.

Assay for Transposase-Accessible Chromatin (ATAC-Seq)

Cells were harvested and frozen in culture media containing 50% FCS, 40% RPMI and 10% DMSO. Cryopreserved cells were sent to Active Motif to perform the ATAC-seq assay. The cells were then thawed in a 37 o C water bath, pelleted, washed with cold PBS, and tagmented as previously described (12), with some modifications based on (13). Briefly, cell pellets were resuspended in lysis buffer, pelleted, and tagmented using the enzyme and buffer provided in the Nextera Library Prep Kit (Illumina). Tagmented DNA was then purified using the MinElute PCR purification kit (Qiagen), amplified with 10 cycles of PCR, and purified using Agencourt AMPure SPRI beads (Beckman Coulter). Resulting material was quantified using the KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems), and sequenced with PE42 sequencing on the NextSeq 500 sequencer (Illumina).

Analysis of ATAC-seq data was very similar to the analysis of ChIP-Seq data. Reads were aligned using the BWA algorithm (mem mode; default settings). Duplicate reads were removed, only reads mapping as matched pairs and only uniquely mapped reads (mapping quality >= 1) were used for further analysis. Alignments were extended in silico at their 3'ends to a length of 200 bp and assigned to 32-nt bins along the genome. The resulting histograms (genomic "signal maps") were stored in bigWig files. Peaks were identified using the MACS 2. For differential analysis, reads were counted in all merged peak regions (using Subread, v1.5.2), and the replicate groups for each condition were compared using DESeq2 (v1.24.0).

Page 57 of 60

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  islet size (Fig 2C, upper panel) and staining of insulin and glucagon-positive cells within the pancreatic islets (Fig 2D, upper panel) revealed no differences, whereas old Lmna LCS/LCS animals displayed enlarged islets with an increase in insulin-positive cells, but no differences in the number of glucagon-positive cells (Fig 2C and 2D, lower panel). Quantification of the -/-cell ratio confirmed an increased number of insulin-positive -cells in aged Lmna LCS/LCS mice (Fig 2E)

  /LCS showed improved glucose tolerance 18 days after STZ injection (Fig 4B), suggesting functional insulin-producing cells in STZ-treated Lmna LCS/LCS mice. Morphological analysis of the pancreata 18 days post-STZ injection revealed that the number of insulin-positive cells was dramatically reduced in Lmna +/+ islets (Fig 4C, left panel). Conversely, Lmna LCS/LCS insulin-positive β-cells recovered efficiently, with comparable staining of insulin-and glucagon-positive cells before and after STZ treatment. Overall, 18 days after STZ injection, a 2.5-fold increase in insulin-positive cells was observed in the pancreas of Lmna LCS/LCS mice compared to control mice (Fig 4C, right panel).

  5D, left panel) whereas no difference was observed for the localization of the repressive mark H3K9Me3 (Fig 5D, right panel). Interestingly, the distribution of the H3K27Ac epigenetic mark to the nuclear periphery is associated with a modification of the nuclear architecture. Indeed, we observed that the chromatin is relocated to the periphery of the nucleus in Lmna LCS/LCS mice as indicated by Hoechst staining (fig 5D) without changing the global chromatin accessibility as shown by our ATAC-seq analysis. Taken together, our transcriptomic and epigenomic wide studies demonstrate that the transcriptional reprogramming of genes involved in mitochondrial function and global translation cannot be assigned to either changes in DNA methylation or chromatin accessibility signatures (RRBS and ATAC-seq) but more likely to the relocalisation of epigenomic marks and/or a remodeling of the nuclear architecture. Lmna LCS/LCS pancreatic islets display increased mitochondrial biogenesis and increased global translation Since mitochondrial homeostasis is critical for both the maintenance of β-cell function and lifespan promotion (14), we next focused on the regulation of mitochondrial gene expression and function. First, we validated our RNA-seq data by quantitative RT-PCR analysis and confirmed that Lmna LCS/LCS -derived islets expressed increased levels of key genes involved in the control of mitochondrial function, including the genes encoding electron transport chain proteins (Nduf, Atp5 and Cox protein family, Fig 6A). In addition, the amount of mitochondrial DNA relative to nuclear DNA was significantly higher in Lmna LCS/LCS islets (Fig 6B). Ultrastructural analysis of β-cells by transmission electron microscopy (Fig 6C) together with the oxygen consumption rate (OCR) of islets (Fig 6D) further supported a higher number of functional mitochondria in Lmna LCS/LCS islets. Taken together, these results suggest that both mitochondrial number and activity are increased in pancreatic islets isolated from Lmna LCS/LCS mice.

  5-fold increased in Lmna LCS/LCS islets (Fig 6F). To confirm that translation rates are increased in Lmna LCS/LCS islets, we measured 35 S-Met/Cys incorporation and found that global protein synthesis levels were significantly increased in Lmna LCS/LCS islets (Fig 6G). Since insulin is the major protein synthetized in β-cells, we measured insulin content in Lmna +/+ and Lmna LCS/LCS isolated islets. Since Lmna LCS/LCS islets are larger than control islets, classical insulin content measurement may not quantitatively reflect the abundance of insulin per cell. Therefore, we performed FACS analysis to measure intracellular insulin levels in dissociated β-cells. As shown in Fig 6H, quantification of FACS experiments revealed that insulin production was increased by approximately 25% in Lmna LCS/LCS β-cells.

  Loss of β-cell function and mass is involved in the development of T1D and T2D, and may contribute to the metabolic complications observed during aging. Our results demonstrate for the first time that the exclusive expression of lamin C acts as a key regulator of β-cell function and regeneration, thus protecting against hyperglycemia observed in several forms of metabolic stresses, such as aging, diet-induced obesity and STZ-induced diabetes. By combining in vitro, ex vivo and in vivo studies, we report that lamin C expression increases β-cell number during aging, counteracts β-cell failure during diet-induced obesity and promotes β-cell regeneration after STZ treatment. Most importantly we demonstrate that lamin C modulates a specific transcriptional program that contributes to enhance the translation of insulin and promotes insulin secretion through a mitochondria-coupled mechanism.

Figure 1 :

 1 Abbreviations:

Figure 2 :

 2 Figure 2: Lmna LCS/LCS mice show increased β-cell mass and insulin secretion during aging

Figure 4 :

 4 Figure 4: Streptozotocin-injected Lmna LCS/LCS mice display increased -cell regeneration

Figure 5 :

 5 Figure 5: Lmna LCS/LCS mice display a transcriptional reprogramming of genes involved in

Figure 6 :

 6 Figure 6: Lmna LCS/LCS pancreatic islets display increased mitochondrial biogenesis and

de

  Toledo et al. Supplemental data 6 Distribution of the analyzed CpG sites in various regions of the genome.

  1.0 algorithm at a cutoff of p-value 1e-7, without control file, and with thenomodel option. Peaks that were on the ENCODE blacklist of known false ChIP-Seq peaks were removed. Signal maps and peak locations were used as input data to Active Motifs Page 56 of 60 For Peer Review Only Diabetes proprietary analysis program, which creates Excel tables containing detailed information on sample comparison, peak metrics, peak locations and gene annotations.

  ). Interestingly Lmna LCS/LCS mice remained normoglycemic, although an increased body weight is typically associated with hyperglycemia. These findings prompted us to analyze glucose homeostasis in aged Lmna LCS/LCS mice. Glucose clearance was similar in young animals of both genotypes (Fig 1C, left panel), whereas old Lmna LCS/LCS mice showed improved glucose tolerance compared to age-matched Lmna +/+ mice (Fig 1C, right panel). As expected, no differences in basal and glucose-stimulated insulin secretion were observed in young animals from either genotype (Fig 1D, left panel). Basal and stimulated insulin secretion was, however, increased in old Lmna LCS/LCS mice compared to old WT mice (Fig 1D, right panel). Altogether, these data suggest that although Lmna LCS/LCS mice become obese as they age, their glucose clearance and insulin secretion are improved, suggesting that lamin C may modulate the function of pancreatic islets of Langerhans during aging.

  Mean food intake of CD or HFD in 30-week-old Lmna +/+ and Lmna LCS/LCS mice (n=8 per group).

							Diabetes Diabetes Diabetes							Page 42 of 60 Page 44 of 60
	B A	Food intake(g/mouse/day)	number of α-cell/islet section 0 5 10 15 20 A A	0 20 40 60 80 Young CD 70 55 35 Lmna LCS/LCS ns ns Lmna +/+ M I N 6 w t kDa M I N 6 L C O	HFD Lamin C Lamin A Actin ****	number of α-cell/islet section	0 20 40 60 80 Old	ns	Lmna +/+	Lmna LCS/LCS	Lmna +/+	Lmna LCS/LCS	Lmna +/+	Lmna LCS/LCS
			B	Lmna +/+	Lmna LCS/LCS		Lmna +/+	Lmna LCS/LCS
	Lmna +/+ Lmna LCS/LCS		B	A) Lamin A and lamin C expression in CRISPR/Cas9-generated Lmna +/+ Lmna LCS/LCS 0 2 4 6 8 10 ns Low Glucose High Glucose Total pancreatic Glucagon (ng) 0.0 0.5 1.0 1.5 MIN6 wt Insulin secretion (%content) MIN6 LCO ****
				MIN6 wt , MIN6 LCO and MIN6 LAO cells as assessed by western
				blot.
			A) Quantification of number of a-cells per islet section in young vs old B) GSIS in MIN6 wt and MIN6 LCO cells. The results were
		Lmna +/+ and Lmna LCS/LCS mice (n=4 per group) normalized to insulin content. A) Body weight curve over time in Lmna +/+ and Lmna LCS/LCS mice during STZ treatment
		(n=5 per group).
			B) Glucagon content from total pancreata of old Lmna +/+ and All values are expressed as the mean ± SEM. ****p< 0.0001
		Lmna LCS/LCS mice (n=6 per group) and ns not significant by Student's unpaired t-test. B) Representatives images (left panel) of TUNEL staining (green) on pancreatic sections
		from Lmna +/+ and Lmna LCS/LCS mice obtained 48 h after STZ treatment (n=3 per group).
		All values are expressed as the mean ± SEM. ns not significant by Insulin is stained in red, and nuclei are stained with Hoechst (scale bar 50 µm).
		Student's unpaired t-test. Quantification (right panel) of the number of TUNEL positive cells per islets.
		C) FACS quantification of mean percentages of apoptotic MIN6 wt and MIN6 LCO cells
		treated with or without STZ (12 mM) for 5 h.
		All values are expressed as the mean ± SEM. *p<0.05 and ns not significant by Student's
		unpaired t-test.
							For Peer Review Only For Peer Review Only For Peer Review Only
									de Toledo et al. Supplemental data 2 de Toledo et al. Supplemental data 3 de Toledo et al. Supplemental data 5

ns For Peer Review Only Diabetes de Toledo et al. Supplemental data 4

  Leica 63×/1.4 oil HCX PL APO CS objective controlled using the Leica LAS AF software.

	7 -1.4 NA oil objective (NA 1.32, Leica, Melville, NY) and a Coolsnap HQ2 camera (Photometrics, Woburn, MA). Confocal images were acquired using a Confocal Leica SP5-SMDmicroscope After isolation, 150 islets of Langerhans per mice and per genotype were immersed in a solution of 2.5% glutaraldehyde in Sorensen's buffer (0.1 M, pH 7.4) overnight at 4 °C. They were rinsed twice in Sorensen's buffer and post-fixed in a 0.5% osmic acid for 2 hours at room temperature in the dark. Tissues were dehydrated in a graded series of ethanol solutions (30-100%) and embedded in EmBed 812 with an Automated Microwave Tissue Processor for Electronic Microscopy, Leica EM AMW. Thin sections (70 nm; Leica-Reichert Ultracut E) of each block were collected at different levels. These sections were counterstained with uranyl acetate and observed on a Hitachi 7100 transmission electron microscope (TEM) in the Centre de Ressources en Imagerie Cellulaire de Montpellier and a Ultrastructural transmission electron microscopy (TEM) (France).

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Acknowledgements

This work was supported by grants from OSEO-ISI CaReNA (I 13 03 008W to JT), from E.G.I.D (ANR-10-LABX-46 to J-S. A.), ANR (BETAPLASTICITY, ANR-17-CE14-0034 to J-S. A.), EFSD (J-S.A.), I-SITE ULNE (EpiRNA-Diab to J-S. A.), Société Francophone du Diabetes (J-S.A.), INSERM, CNRS, Lille University, Association pour la Recherche sur le Diabète (to J-S.A.), Conseil Régional Hauts de France and Métropole Européenne de Lille (to X.G. and J-S.A.) and from the Swiss National Science Foundation (Ambizione PZ00P3-168077 to ICLM). We thank David Lleres and Cyril Esnault for discussions and scientific critique. The authors are grateful to MRI and RHEM for assistance in cytometry and experimental histology, as well as the animal facility platforms RAM and ZEFI. We especially thank Sylvain de Rossi, Myriam Boyer and Chantal Cazevieille for their technical assistance. We thank Magali A. Ravier for advice for the islet isolation from mice and Amal Makrini for the bioinformamatics support. Marion de Toledo and Carine Chavey are the guarantors of this work.

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Authors' contribution:

CC and MdT designed, performed and interpreted the majority of the experiments. PC performed histological analysis. MP and CB contributed to the RNA-seq and RRBS analysis as well as the bioinformatics analysis. XG gave advice for islet isolation from mice. ICLM contributed to the aging experiments. CC and MdT wrote the manuscript. ICLM, JT and J-SA reviewed and approved the manuscript.

Conflict of interest:

The authors have declared that no conflict of interest exists. E) Quantification of / cells ratio per pancreatic section in young and old Lmna +/+ and Lmna LCS/LCS mice (n=4) F) Quantification of isolated islets number after collagenase digestion in young and old Lmna +/+ and Lmna LCS/LCS mice. G) Global insulin content from total pancreata of old Lmna +/+ and Lmna LCS/LCS mice (n=6 per group) H) Glucose-stimulated insulin secretion (GSIS) under low (3mM) or high (20mM) glucose concentration from isolated islets from young and old Lmna +/+ and Lmna LCS/LCS mice (n=4 per group). The results were normalized to total insulin content.

All values are expressed as the mean ± SEM. **p 0.01, ***p 0.001, n.s not significant by Student's unpaired t-test.

Supplementary material Western Blot

Mouse brain, liver, pancreas and muscle tissue samples were snap-frozen in liquid nitrogen immediately after collection. Samples were then reduced into powder form with a tissue grinder, homogenized and dissolved at 150 mg/ml in a buffer containing 63mM Tris-HCl (pH7.5), 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol and 8M urea. Samples were stored at -80°C. Protein extracts were size-fractionated on 4-12% gradient SDS-polyacrylamide Bis-Tris gels (ThermoFischer Scientific) and transferred to nitrocellulose membranes for Western blotting. Western blots were probed with a 1:1000 diluton of anti-lamin A/C (sc-20681-Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1:4000 dilution of anti-Actin, clone C4 (#MAB1501 -Millipore, MA, USA).

Food intake

Food intake was measured three times a week over a period of one month with ten animals per genotype in individual cages.

Glucose-stimulated insulin secretion

For insulin secretion, 5 islets of 150-200μm diameter kept in 1,5ml microtubes were pre-incubated for 1 h at 37°C in Krebs-Ringer bicarbonate (KRB) buffer containing: 115mM NaCl, 4.7mM KCl, 6.2mM CaCl2, 20mM NaHCO3, 1.2mM MgSO4, 1.2mM KH2PO4, 16mM HEPES, 0.5% Free Faty Acid BSA, 10mM glucose. The pre-incubation medium was then replaced with KRB buffer supplemented with different glucose concentrations (Low: 3mM; High: 20mM). After incubation for 1 h at 37°C, the supernatant was collected and stored at -For Peer Review Only Diabetes and diluted to 16 pM before clustering. Cluster formation, primer hybridization and singleend read, 50 cycles sequencing were performed on cBot and HiSeq2500 (Illumina, San Diego, CA) respectively. Image analyses and base calling were performed using the Illumina HiSeq Control Software and the Real-Time Analysis component. Demultiplexing was performed using Illumina's conversion software (bcl2fastq 2.18). The quality of the raw data was assessed using FastQC from the Babraham Institute and the Illumina software SAV (Sequencing Analysis Viewer). Potential contaminants were monitored with the FastQ Screen software from the Babraham Institute.

RNA-Seq data analysis

We aligned RNA-seq reads to the mouse genome (UCSC mm9) with the splice junction mapper TopHat 2.1.1 (1), which used Bowtie 2.2.9 (2). We downloaded gene model annotations from the UCSC database (genes.gtf 31 August 2017) and GeneIDs from the NCBI (gene2refseq.gz, 31 August 2017). Final read alignments having more than 3 mismatches were discarded. We performed gene counting using the union mode of HTSeq-count 0.9.0 (3). Before statistical analysis, genes with less than 15 reads (cumulating all the analyzed samples) were filtered out. Counts were normalized using the Relative Log Expression (RLE) method as implemented in the Bioconductor (4) package edgeR 3.16.5 (5). Differentially expressed (DE) genes were identified using 3 different statistical methods: DESeq 1.26.0 (6), EdgeR, and DESeq2 1.14.1 (7). We corrected P-values for multiple testing using the Benjamini-Hochberg FDR method (8). Genes with adjusted P-value <0.05 were declared 'differentially expressed'. The final lists of DE genes were the genes common to the 3 tests.

To perform the functional analysis of the resulting list of genes with the Gene Ontology (GO) annotations, the topGO(9) package from Bioconductor was used. Overrepresented GO terms

The sequencing quality control of RRBS data was identical to the one performed for RNA-Seq data. The un-methylated cytosine added at the end-repair step during library preparation was used as a built-in bisulfite conversion efficiency control. Sequences that contained the adapter on their 3' end were trimmed with cutadapt (https://cutadapt.readthedocs.org/en/stable/; minimaloverlap length between the read and the adapter of 5; discard trimmed reads shorter than 15). The option "--trimmed-only" was used to discard reads that did not contain the adapter. The percentage of non-bisulfite converted cytosines was then calculated as the number of times the filled in position at the 2nd base from the 3' end was not converted to T. Mean ± sd bisulfite conversion was 99.55 ± 0.03% for the read 1 and 99.63 ± 0.02% for the read 2 for the 24 samples.

Trim Galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) was used to perform adapter trimming and quality control in two subsequent steps in order to remove the 2 additional bases that contained a cytosine which were artificially introduced at the end-repair step during the library preparation (--rrbs option). The mm9 mouse reference genome was prepared for Bismark 0.17.0 using bismark_genome_preparation script.

Bismark software (version 0.17.0) from the Babraham Institute (10) was used to align trimmed RRBS reads to the mouse genome. Then, bismark_methylation_extractor extracted the methylation call for every single C analyzed. Differential methylation analysis was performed using the methylSig (v0.4.4) Bioconductor R package (11). A site specific analysis was done for each CpG site covered by min 10 and max 500 reads using local information to improve variance estimation. A tiled analysis was also conducted by using the total number of cytosines in a windows of 25 bp. A cytosine or a region was declared differentially methylated if the adjusted p-value (according


Charlotte Depeux
email: charlotte.depeux@gmail.com

Ascel Samba-Louaka

Thomas Becking

Christine Braquart-Varnier

Jérôme Moreau

Jean-François Lemaître

Tiffany Lemaître

Hélène Laverre

François-Xavier Paulhac

Jean-Michel Dechaume-Moncharmont

Gaillard

Tiffany Laverre

Hélène Paulhac

François-Xavier Dechaume-Moncharmont

Jean-Michel Gaillard

Sophie Beltran-Bech

The crustacean Armadillidium vulgare (Latreille, 1804) (Isopoda: Oniscoidea), a new promising model for the study of cellular senescence

Keywords: immunosenescence, telomerase reverse transcriptase (TERT), β-galactosidase activity

HAL is

INTRODUCTION

Many hypotheses have tried to explain why senescence is a quasi-ubiquitous phenomenon in the living organisms. For instance, the disposable soma theory proposed the senescence process as a result of damages accumulation over time [START_REF] Kirkwood | Evolution of ageing[END_REF]. These damages are strongly influenced by the environment, leading to trade-offs between the different functions (e.g., between reproduction and somatic maintenance) and shaping a high diversity of senescence patterns across species and populations, among individuals, and between sexes.

One current challenge is to understand the selective forces and mechanisms driving this diversity of senescence patterns.

At the cellular level, senescence corresponds to the cellular deterioration leading to stop the cellular cycle [START_REF] Campisi | Cellular senescence: when bad things happen to good cells[END_REF]. As ageing is associated with cellular senescence [START_REF] Herbig | Cellular senescence in aging primates[END_REF][START_REF] Wang | DNA damage response and cellular senescence in tissues of aging mice[END_REF][START_REF] Lawless | Quantitative assessment of markers for cell senescence[END_REF], many biomolecular parameters potentially inform about senescence and can therefore be valuable tools for studying this process (Bernardez de [START_REF] Bernardez De | Assessing cell and organ senescence biomarkers[END_REF]. For example, the evolution of the integrity and efficiency of immune cells is particularly relevant to study cellular senescence because a diminution of the number of effective immune cells with increasing age takes place in both vertebrates (e.g., [START_REF] Cheynel | Immunosenescence patterns differ between populations but not between sexes in a long-lived mammal[END_REF] and invertebrates (e.g., [START_REF] Park | Cellular immunosenescence in adult male crickets, Gryllus assimilis[END_REF]. Another marker used to study cellular senescence is the enzymatic activity of the b-galactosidase. This enzyme is a hydrolase that transforms polysaccharides into monosaccharides. The lysosomal activity of this enzyme is increased when the cell enters in senescence [START_REF] Dimri | A biomarker that identifies senescent human cells in culture and in aging skin in vivo[END_REF][START_REF] Itahana | Methods to detect biomarkers of cellular senescence: the senescence-associated β-galactosidase assay[END_REF]. This phenomenon occurs in senescent cells of many organisms ranging from humans [START_REF] Gary | Quantitative assay of senescence-associated βgalactosidase activity in mammalian cell extracts[END_REF] to honeybees [START_REF] Hsieh | Honeybee trophocytes and fat cells as target cells for cellular senescence studies[END_REF]. Another protein linked to the cellular senescence process is the telomerase, a ribonucleoprotein complex composed by two essential components, the telomerase reverse transcriptase (TERT) and the telomerase RNA (TR) and other accessorial proteins [START_REF] Podlevsky | The telomerase database[END_REF]. Telomerase lengthens the ends of telomeres (i.e., DNA sequences located at the end of chromosomes that protect chromosome integrity and shorten after each cell division).

Cell senescence arises when the telomere length becomes critically short [START_REF] Chiu | Replicative senescence and cell immortality: The role of telomeres and telomerase[END_REF][START_REF] Shay | Senescence and immortalization: role of telomeres and telomerase[END_REF]. Telomerase activity depends on the species, age, as well as the type of tissue (e.g., [START_REF] Gomes | Telomere biology in Metazoa[END_REF]. For instance, telomerase is active during the development before birth and after only in stem and germ cells in humans [START_REF] Liu | Telomere lengthening early in development[END_REF][START_REF] Morgan | Telomerase regulation and the intimate relationship with aging[END_REF] whereas in Daphnia pulicaria Forbes, 1893 the telomerase activity in all tissues of the body decreases with increasing age [START_REF] Schumpert | Telomerase activity and telomere length in Daphnia[END_REF]. The TERT is essential in the telomerase protein complex and has been shown to be related to cell survival in humans [START_REF] Cao | TERT regulates cell survival independent of telomerase enzymatic activity[END_REF]. The TERT has also been detected in numerous species including vertebrates, fungi, ciliates, and insects [START_REF] Robertson | Canonical TTAGG-repeat telomeres and telomerase in the honey bee, Apis mellifera[END_REF][START_REF] Podlevsky | The telomerase database[END_REF].

Because the patterns of senescence are strongly diversified within the living world, it seems essential to study organisms displaying markedly different life histories strategies to understand the causes and mechanisms underlying this diversity. Thus, invertebrates are increasingly used in experimental studies of senescence [START_REF] Stanley | Aging and immunosenescence in invertebrates[END_REF][START_REF] Ram | Invertebrates as model organisms for research on aging biology[END_REF].

In addition to share similarities with vertebrates in terms of senescence, they can be manipulated experimentally and they are easier to monitor throughout their entire lifetime [START_REF] Ram | Invertebrates as model organisms for research on aging biology[END_REF]. These advantages make them models of choice for studying senescence. Here, we propose the common woodlouse Armadillidium vulgare [START_REF] Latreille | Tableau méthodique des Insectes[END_REF] as a promising new model for studying senescence. Woodlouse can live beyond three years and display sex-specific senescence patterns in natural populations [START_REF] Paris | Population characteristics of the terrestrial isopod Armadillidium vulgare in California grassland[END_REF]. In addition, one study has already reported evidence of immune-senescence in this species [START_REF] Sicard | Variations of immune parameters in terrestrial isopods: a matter of gender, aging and Wolbachia[END_REF].

In this context, we tested the suitability of β-galactosidase activity, immune cell parameters, and the TERT gene expression to cause age-specific responses in A. vulgare.

According to reports in the literature, we expected an increase in β-galactosidase activity, and a decrease of both TERT gene expression and immune cell viability and density in A. vulgare.

As males have higher adult survival than females [START_REF] Paris | Population characteristics of the terrestrial isopod Armadillidium vulgare in California grassland[END_REF], cellular senescence patterns are also expected to be sex-specific in this species.

MATERIALS AND METHODS

Biological model

Individuals of A. vulgare used in the experiments were obtained from a wild population collected in Denmark in 1982. These individuals have been maintained on moistened soil under the natural photoperiod of Poitiers, France (46.58°N, 0.34°E) at 20 °C, and fed ad libitum with dried linden leaves and carrots. Crosses were monitored to control and promote genetic diversity. For each clutch obtained, individuals were sexed, and brothers and sisters were separated to ensure virginity. Woodlice are promiscuous and only breed when they are one-year old during their first breeding season in the spring. Females carry their offspring in a ventral pouch (marsupium) and can produce up to three clutches per season. In the common woodlouse, individuals molt throughout their lifetimes, with approximate one molt per month.

During this process all the cells of the concerned tissues are renewed at 20 °C [START_REF] Steel | Mechanisms of coordination between moulting and reproduction in terrestrial isopod crustacea[END_REF].

The brain, the nerve cord, and gonads, however, are not renewed during molting and are therefore relevant candidates for tissue-specific study of senescence in this species. Woodlice were classified in three different age categories: juvenile (before first reproduction, birth to one-year old), young (one-year old, first year of reproduction), and old (up to two-year old), which are very rare in natural populations [START_REF] Paris | Population characteristics of the terrestrial isopod Armadillidium vulgare in California grassland[END_REF]. As the woodlouse is continuous grower, juveniles and small individuals could not be used for certain experiments, especially when they required protein extraction or hemolymph collection. Thus, we were only able to test in them the telomerase expression. Old individuals were sampled according to the number of individuals available in our breeding; sometimes we had two-year-old individuals and sometimes three-year-old ones. Moreover, males and females were tested separately to assess the impact of sex.

Measurement of β-galactosidase activity

To test the impact of age on β-galactosidase activity, 180 individuals were used: 90 young (i.e., six-month old, 45 males, and 45 females) and 90 old (two-year old, 45 males, and 45 females).

Individuals were dissected separately in Ringer solution (sodium chloride (NaCl) 394 mM, potassium chloride (KCl) 2 mM, calcium chloride (CaCl2) 2 mM, sodium bicarbonate (NaHCO3) 2 mM) and nerve cord was removed. To obtain a sufficient amount of protein, we made pools of five nerve cords from five different individuals of the same age. The five nerve cords were filed in 500 µl of Lyse buffer 1X (Chaps detergent ((3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate) 5 mM, citric acid (C6H807) 40 mM, sodium phosphate (Na3PO4) 40 mM, benzamidine (C7H8N2) 0.5 mM, and PMSF (phenymethylsulfonyle (C7H7FO2S) 0.25 mM, pH 6) [START_REF] Gary | Quantitative assay of senescence-associated βgalactosidase activity in mammalian cell extracts[END_REF], and then centrifuged at 15,000g at 4 °C for 30 min. The supernatant was taken and kept at -80°C until its utilization. The protein concentration was determined by the BCA (bicinchoninic acid assay) assay (Thermo Fisher Scientific, Waltham, MA, USA) and homogenized at 0.1 mg ml -1 . The β-galactosidase activity was measured as described by [START_REF] Gary | Quantitative assay of senescence-associated βgalactosidase activity in mammalian cell extracts[END_REF]. Briefly, 100 µl of extracted protein at the concentration of 0.1 mg ml -1 were added to 100 µl of reactive 4-methylumbelliferyl-D-galactopyranoside (MUG) solution in a 96-wellmicroplate.

The MUG reactive, in contact to β-galactosidase, leads by hydrolysis to the synthesis of 4methylumbelliferone (4-MU), which is detectable using fluorescent measurements. Measures were performed by the multimode Mithrax microplate reader (LB940 HTS III, excitation filter: 120 nm, emission filter 460 nm; Berthold Technologies, Bad Wildbad, Germany) for 120 min. Two technical replicates were measured for each nerve pool.

Measurement of immune cell parameters

To test the impact of age on the immune cell parameters (i.e., density, viability, and size), we were able to undertake individual tests because of our previous experience. It was therefore not necessary to carry out pools of hemolymph unlike proteins or DNA to make the measurements. Sixty mature individuals were used: 30 young (i.e., one-year old, 15 males, and 15 females) and 30 old (three-year old, 15 males, and 15 females) individuals.

To study the impact of age on the immune parameters, a hole was bored in the middle of the sixth segment and 3 µl of haemolymph were collected per individual with an eyedropper and deposited promptly in 15 µl of anticoagulant solution (MAS-EDTA (EDTA (ethylenediaminetetraacetic (C10H16N208)) 9 mM, trisodium citrate (Na3C6H5O7) 27 mM, sodium chloride (NaCl) 336 mM, glucose (C6H12O6) 115 mM, pH 7 [START_REF] Rodriguez | Characterisation of shrimp haemocytes and plasma components by monoclonal antibodies[END_REF]).

individuals were used for this experiment. For each group we tested the expression level of the TERT gene in two different tissues: the nerve cord (somatic line) and gonads (germinal line).

Animals were washed by immersion for 30 sec in a 30% sodium hypochlorite solution (NaClO) followed by two 30-sec immersions in distilled water. Tissues were dissected in Ringer solution (sodium chloride 394 mM, potassium chloride (KCl) 2 mM, calcium chloride (CaCl2) 2 mM, sodium bicarbonate (NaHCO3) 2 mM) and deposited by pools of five units for each tissues on TRIzol reagent (Invitrogen) to extract RNA according to the manufacturer's protocol after a cell disintegration using a Vibra Cell 75,185 sonicator (amplitude of 30%).

Total RNA was quantified by NanoDrop technology and stored at -80 °C until use. Reverse transcriptions (RT) were made from 500 ng of RNA previously extracted and using the SuperScript TM IV Reverse Transcriptase kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Primers were designed using the identified gene: primer TERT_F: 5'-AGGGAAAACGATGCACAACC-3' and primer TERT_R: 5'-GTTCGCCAAATGTTCGCAAC-3' (see Supplementary material S1). Quantitative RT-PCR was performed using 0.6 µl of each primer (10 µM), 2.4 µl of nuclease-free water, and 1.5 µl of cDNA template and the LightCycler LC480 system (Roche, Pleasanton, CA, USA ) as follows: 10 min at 95 °C, 45 cycles of 10 s at 95 °C, 10 s at 60 °C, and 20 s at 72 °C.

Expression levels of target genes were normalized based on the expression level of two reference genes previously established: the ribosomal protein L8 (RbL8) and the Elongation Factor 2 (EF2) [START_REF] Chevalier | The immune cellular effectors of terrestrial isopod Armadillidium vulgare: Meeting with their invaders, Wolbachia[END_REF].

Statistical analyses.

All statistical analyses were performed using the R software v.3.5.2 (R. Core [START_REF] Team | R: A language and environment for statistical computing[END_REF].

The β-galactosidase activity was analyzed with linear mixed effect models using the package lme4 [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF]. As two technical replicates were measured for each pool, the model included the pools fitted as a random effect, age and sex and their two-way interaction as fixed factors.

Concerning the immune parameters, linear models with Gaussian distribution were fitted to analyze variation in the cell size and viability. A linear model of the cell number (logtransformed; [START_REF] Ives | For testing the significance of regression coefficients, go ahead and logtransform count data[END_REF] was fitted for the cell density.

The level of TERT expression according to age in the two different tissues were compared by a Kruskal-Wallis rank sum test in combination with Nemenyi's post hoc multiple comparison test with the Tuckey correction using R package PMCMR [START_REF] Pohlert | The pairwise multiple comparison of mean ranks package (PMCMR). R package[END_REF].

RESULTS

β-galactosidase activity

The β-galactosidase activity was higher in old (i.e., two-year old) than in young (i.e., sixmonth old) individuals (c 2 1 = 6.15, P = 0.013; Fig. 1). We also detected a higher βgalactosidase activity in females than in males (c 2 1 = 7.26, P = 0.007; Fig. 1).

<Fig. 1> Immune cells parameters

Cell size was higher in three-year-old than in one-year-old individuals (F1,58 = 8.54, P = 0.005; Fig. 2A). Conversely, the cell density was higher in one-year-old than in three-year-old individuals (F1,58 = 4.33, P = 0.01; Fig. 2B). Concerning the immune cell viability, a statistically significant interaction occurred between age and sex, with a relatively lower immune cell viability in three-year-old females (F3,56 = 6.85, P = 0.01; Fig. 2C). No sex effect was detected on cell size (F2,57 = 0.76, P = 0.38; Fig. 2A) or cell density (F2,57 = 0.32, P = 0.57, Fig. 2B).

<Fig. 2> TERT gene expression

The TERT gene expression decreased with increasing age in nerve cords (c 2 3 = 23.30, P < 0.001; Fig. 3A). TERT expression was higher in four-month-old individuals compared to twoyear-old and three-year-old individuals (P = 0.001 in both cases) and in one-year-old individuals compared to three-years-old individuals (P = 0.038), without any detectable sex effect (c 2 1 = 0.14, P = 0.70; Fig. 3A). In gonads, the TERT gene expression was much higher in females (c 2 1 = 17.81, P < 0.001; Fig. 3B) and tended to decrease with increasing age (c 2 3 = 7.5, P = 0.057; Fig. ,3B) as the TERT gene expression tended to be higher in four-month-old females compared to three-year-old females (P = 0.054). A general tendency was also observed in males (c 2 1 = 7.34, P = 0.061; Fig. 3B), the TERT gene expression tending to be higher in two-year-old individuals compared to one-year-old and three-year-old individuals (P = 0.14 and P = 0.12, respectively; Fig. 3B).

<Fig. 3>

DISCUSSION

We tested several effective physiological biomarkers of vertebrate senescence to assess whether they could also be used in invertebrates such as the common woodlouse. Immune cells showed an increase in their size and a decrease in their density and viability with increasing age. The activity of the β-galactosidase enzyme in nerve cords increased. The TERT gene expression decreased with increasing age in nerve cords of males and females and in the gonads of females. In contrast, the TERT gene expression was very low in the male gonads to suggest a role on the cellular senescence status in this tissue. The difference regarding the expression of TERT in the nerve cords and gonads underlies the importance of organ choice to perform such an analysis.

Our study is in line with previous studies that had previously revealed the possibility of using vertebrate biomarkers in invertebrates [START_REF] Hsieh | Honeybee trophocytes and fat cells as target cells for cellular senescence studies[END_REF][START_REF] Park | Cellular immunosenescence in adult male crickets, Gryllus assimilis[END_REF][START_REF] Schumpert | Telomerase activity and telomere length in Daphnia[END_REF]. By testing a set of different physiological biomarkers of vertebrate senescence, often studied independently, our study supports both ideas that routinely used biomarkers in vertebrates can be adapted in invertebrates and that the senescence process is quasi-ubiquitous in the living world and can be expressed in a similar way in very different organisms.

Previous studies have shown that the probabilities to survive decrease with increasing age in A. vulgare [START_REF] Paris | Population characteristics of the terrestrial isopod Armadillidium vulgare in California grassland[END_REF]. The cellular damages accumulated during the life of the isopod could be the cause of cell senescence and therefore the driving force behind actuarial senescence [START_REF] Harman | Aging: a theory based on free radical and radiation chemistry[END_REF][START_REF] Barja | The flux of free radical attack through mitochondrial DNA is related to aging rate[END_REF][START_REF] Barja | Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals[END_REF][START_REF] Finkel | Oxidants, oxidative stress and the biology of ageing[END_REF]. In A. vulgare, the 2-and 3-year-old individuals could have therefore accumulated more cellular damages during their lifetime, leading to the cellular senescence we report.

Our study also revealed a strong difference between sexes on the response of biomarkers to age changes. At a given age, females display higher β-galactosidase activity and lower immune cell viability than males, as if they age faster than males. Between-sex differences in lifespan have been reported in A. vulgare with a longer lifespan in males than in females [START_REF] Geiser | Further observations on the sex-ratios of terrestrial isopods[END_REF][START_REF] Paris | Population characteristics of the terrestrial isopod Armadillidium vulgare in California grassland[END_REF]. Exact differences in actuarial senescence patterns (i.e., age-specific changes in survival probabilities) remain to be quantified in A.

vulgare but such differences are quite common both in vertebrates and invertebrates [START_REF] Tidière | Does sexual selection shape sex differences in longevity and senescence patterns across vertebrates? A review and new insights from captive ruminants[END_REF][START_REF] Marais | Sex gap in aging and longevity: can sex chromosomes play a role?[END_REF]. One of the main hypotheses proposed to explain sex differences in longevity or senescence patterns relies on different resource allocation strategies between sexes [START_REF] Vinogradov | Male reproductive strategy and decreased longevity[END_REF][START_REF] Bonduriansky | Sexual selection, sexual conflict and the evolution of ageing and life span[END_REF]. Females carry their offspring in their marsupium for one month, giving nutrients and protection, thus, they allocate more energy to reproduction than males that do not provide any parental care. The fact that the lifespan of A. vulgare is shorter in females [START_REF] Paris | Population characteristics of the terrestrial isopod Armadillidium vulgare in California grassland[END_REF] thus supports a role of differential sex allocation in this species. Moreover, the difference between males in TERT expression in gonads also suggest a difference in sex allocation according both to age and sex. The increased in TERT expression in male gonad in two-years-old individuals should be the result of a terminal investment in reproduction for males: by increasing their TERT expression and thus potentially their telomerase activity they should provide a better fitness to their offspring. This allocation may be lost when males are too old (three-year old).

Sex differences in resource allocation strategies could also be driven by environmental conditions [START_REF] Shertzer | State-dependent energy allocation in variable environments: life history evolution of a rotifer[END_REF]. Our physiological biomarkers of vertebrate senescence revealed sex differences, and as supported in [START_REF] Depeux | Impact of temperature and photoperiod impact on survival and biomarkers of senescence in common woodlouse[END_REF], they could constitute useful tools to identify other factors involved in variations in senescence patterns, such as environmental stressors. Moreover, if these biomarkers seem to predict better the physiological age than chronological age, notably in terms of survival and reproduction, they could correspond to biomarkers of senescence in the woodlouse [START_REF] Baker | Biomarkers of aging[END_REF][START_REF] Simm | Potential biomarkers of ageing[END_REF][START_REF] Sprott | Biomarkers of aging and disease: Introduction and definitions[END_REF].

The physiological biomarkers of vertebrate senescence thus respond to age changes in A. vulgare, a species that represents a new invertebrate model of aging. The parameters that predict the chronological age of A. vulgare individuals might offer reliable biomarkers, especially if their measurements are related to both reproductive and survival prospects more than to the chronological age of individuals. The availability of its genome, the ease of its breeding, its particularity to continue growing during its lifespan, and its adaptations to

Figure captions Figure 1 .

 captions1 Figure captions

Figure 1 .

 1 Figure 1. Immune cell size (A), density (B), and viability (C) according to age and sex in

Figure 2 .

 2 Figure 2. Relative expression level of TERT expression in nerve cords (A) and in gonads (B)

ACKNOWLEDGEMENTS

We would like to thank Sylvine Durand, Richard Cordaux, Isabelle Giraud, and Bouziane Moumen for our constructive discussions as well as Marius Bredon, Carine Delaunay, Maryline Raimond, and Alexandra Lafitte for technical assistance. The preprint of this work has been deposited in BioRxiv (https://doi.org/10.1101/583914). This work was supported by the 2015-2020 State-Region Planning Contract and European Regional Development Fund and intramural funds from the Centre National de la Recherche Scientifique (CNRS) and the University of Poitiers. JFL and JMG were supported by a grant from the Agence Nationale de la Recherche (ANR-15-CE32-0002-01 to JFL). Our research also received funding from the Appel à projets de recherche collaborative inter-équipes 2016-2017 by the laboratory Ecologie et Biologie des Interactions.

Then, 6 µl of trypan blue at 0.4% (Invitrogen, Carlsbad, CA, USA) were added to color the dead cells. Thereafter, 10 µl of this solution were deposed in a counting slide (Invitrogen Coutness®; Thermo Fisher Scientific). The immune cell density, the immune cell viability, and the immune cell size were evaluated using an automated cell counter (Invitrogen Countess®).

Measurement of TERT gene expression

The identification of the telomerase reverse transcriptase (TERT) gene was first performed from the A. vulgare genome [START_REF] Chebbi | The genome of Armadillidium vulgare (Crustacea, Isopoda) provides insights into sex chromosome evolution in the context of cytoplasmic sex determination[END_REF]. In order to test whether this gene was present and preserved in crustaceans, phylogenetic analyses were undertaken upstream (see Supplementary material S1-S4). This gene has been found in crustacean transcriptomes and the topology of the TERT-gene tree follows the phylogenetic relationships between the involved species (Supplementary S3), suggesting a conserved role of the TERT gene.

Gene expression

We tested the effect of age on the expression of TERT gene within four different age groups: 1) four-month old, 2) one-year old, 3) two-year old, and 4) three-year old. Females and males were tested separately by pools of five individuals in one-, two-, and three-year-old groups and by pools of seven individuals in a four-month-old group as smaller individuals provide less biological material. All conditions required four replicates for each sex. A total of 176 terrestrial life as well as the presence of cellular senescence on our senescence biomarkers make it a promising candidate to study senescence.

SUPPLEMENTARY MATERIAL

Supplementary material is available at Journal of Crustacean Biology online. S1. Phylogenetic analysis protocol of the TERT gene in crustaceans. S2. TERT-gene alignment used for the phylogenetic analysis. S3. TERT-gene tree following the phylogenetic relationships between the species involved. S4. TERT-gene tree following the phylogenetic relationships between the species involved (Newick format).