Zéphyra Appriou

Kévin Nay

Nicolas Pierre

Dany Saligaut

Luz Lefeuvre-Orfila

Brice Martin

Thibault Cavey

Martine Ropert

Olivier Loréal

Françoise Rannou-Bekono

Frédéric Derbré
email: frederic.derbre@univ-rennes2.fr

Skeletal muscle ceramides do not contribute to physical inactivity-induced insulin resistance

Keywords: NF-ΚB, HOMA-IR, AMP kinase, Akt, triglycerides

Physical inactivity increases the risk to develop type 2 diabetes, a disease characterized by a state of insulin resistance. By promoting inflammatory state, ceramides are especially recognized to alter insulin sensitivity in skeletal muscle. The present study was designed to analyze, in mice, whether muscle ceramides contribute to physical inactivity-induced insulin resistance. For this purpose, we used the wheel lock model to induce a sudden reduction of physical activity, in combination with myriocin treatment, an inhibitor of de novo ceramide synthesis. Mice were assigned to 3 experimental groups: voluntary wheel access group (Active), a wheel lock group (Inactive) and wheel lock group treated with myriocin (Inactive-Myr). We observed that 10 days of physical inactivity induces hyperinsulinemia and increases HOMA-IR. The muscle ceramide content was not modified by physical inactivity and myriocin. Thus, muscle ceramides do not play a role in physical inactivityinduced insulin resistance. In skeletal muscle, insulin-stimulated Akt phosphorylation and inflammatory pathway were not affected by physical inactivity whereas a reduction of GLUT4 content was observed. Based on these results, physical inactivity-induced insulin resistance seems related to a reduction in GLUT4 content rather than defects in insulin signaling. We observed in inactive mice that myriocin treatment improves glucose tolerance, insulin-stimulated Akt, AMPK activation and GLUT4 content in skeletal muscle. Such effects occur regardless of changes in muscle ceramide content. These findings open promising research perspectives to identify new mechanisms of action for myriocin on insulin sensitivity and glucose metabolism.

INTRODUCTION

Physical inactivity is now recognized as a global pandemic promoting the development of numerous chronic diseases including coronary heart diseases, type 2 diabetes, cancer or dementia [START_REF] Booth | Role of Inactivity in Chronic Diseases: Evolutionary Insight and Pathophysiological Mechanisms[END_REF][START_REF] Pedersen | The diseasome of physical inactivity--and the role of myokines in muscle--fat cross talk[END_REF]. Each year, non-communicable chronic diseases kill 36 million people worldwide, including 17.3 million of deaths due to cardiovascular diseases and 1.3 million due to diabetes [START_REF] Lee | Effect of physical inactivity on major noncommunicable diseases worldwide: an analysis of burden of disease and life expectancy[END_REF]. Nine million of these deaths occur before 60 years old, while over 5.3 million deaths could be averted every year if all inactive people performed only 15 to 30 min/day of moderate physical exercise [START_REF] Lee | Effect of physical inactivity on major noncommunicable diseases worldwide: an analysis of burden of disease and life expectancy[END_REF]. In addition to morbidity and premature mortality, the economic burden of physical inactivity for national governments is estimated worldwide to 53.8 billion US dollars for health-care systems, and 13.7 billion US dollars for productivity losses [START_REF] Ding | The economic burden of physical inactivity: a global analysis of major non-communicable diseases[END_REF].

For the World Health Organization, a person is considered as physically inactive if he doesn't meet any of these 3 criteria: 30 min of moderate-intensity physical activity on at least 5 days every week, 20 min of vigorous-intensity physical activity on at least 3 days every week, or an equivalent combination achieving 600 metabolic equivalent (MET)-min per week [START_REF] Hallal | Global physical activity levels: surveillance progress, pitfalls, and prospects[END_REF]. Two main experimental approaches are used to understand the metabolic disorders related to this deleterious behavior [START_REF] Pierre | From physical inactivity to immobilization: Dissecting the role of oxidative stress in skeletal muscle insulin resistance and atrophy[END_REF]: 1) physical inactivity, induced in humans by the reduction of the daily number of steps performed (from 10000 to less than 5000) [START_REF] Knudsen | Changes in insulin sensitivity precede changes in body composition during 14 days of step reduction combined with overfeeding in healthy young men[END_REF][START_REF] Krogh-Madsen | A 2-wk reduction of ambulatory activity attenuates peripheral insulin sensitivity[END_REF][START_REF] Reynolds | Acute inactivity impairs glycemic control but not blood flow to glucose ingestion[END_REF] and the wheel locked model in rodents [START_REF] Roberts | Potential clinical translation of juvenile rodent inactivity models to study the onset of childhood obesity[END_REF]; 2) immobilization, induced by hindlimb unloading in rodents and bed rest in humans. Even if the latter represents an interesting approach to explore metabolic changes occurring with dramatic reduction of skeletal muscle activity [START_REF] Bergouignan | Physical inactivity as the culprit of metabolic inflexibility: evidence from bed-rest studies[END_REF], it can be considered as too extreme compared to what physically inactive people really experience [START_REF] Pierre | From physical inactivity to immobilization: Dissecting the role of oxidative stress in skeletal muscle insulin resistance and atrophy[END_REF][START_REF] Roberts | Potential clinical translation of juvenile rodent inactivity models to study the onset of childhood obesity[END_REF]. In the present study, we thus choose the wheel lock model to study physical inactivity in mice.

The physiological mechanisms responsible for the development of chronic diseases related to physical inactivity has been deeply explored during the last decade [START_REF] Booth | Role of Inactivity in Chronic Diseases: Evolutionary Insight and Pathophysiological Mechanisms[END_REF][START_REF] Gratas-Delamarche | Physical inactivity, insulin resistance, and the oxidative-inflammatory loop[END_REF][START_REF] Pedersen | The diseasome of physical inactivity--and the role of myokines in muscle--fat cross talk[END_REF][START_REF] Pierre | From physical inactivity to immobilization: Dissecting the role of oxidative stress in skeletal muscle insulin resistance and atrophy[END_REF]. Among the proposed mechanisms, insulin resistance associated to chronic hyperinsulinemia is considered as a key triggering event promoting lipid storage and obesity [START_REF] Softic | Insulin concentration modulates hepatic lipid accumulation in mice in part via transcriptional regulation of fatty acid transport proteins[END_REF][START_REF] Stumvoll | Suppression of systemic, intramuscular, and subcutaneous adipose tissue lipolysis by insulin in humans[END_REF], but also tumor growth and cancer [START_REF] Tsujimoto | Association between hyperinsulinemia and increased risk of cancer death in nonobese and obese people: A population-based observational study[END_REF]. Insulin resistance is clinically defined as the inability of a known quantity of exogenous or endogenous insulin to increase glucose uptake and utilization. In humans, several studies reported that a sudden reduction of physical activity causes whole-body insulin resistance after only few days [START_REF] Knudsen | Changes in insulin sensitivity precede changes in body composition during 14 days of step reduction combined with overfeeding in healthy young men[END_REF][START_REF] Krogh-Madsen | A 2-wk reduction of ambulatory activity attenuates peripheral insulin sensitivity[END_REF][START_REF] Reynolds | Acute inactivity impairs glycemic control but not blood flow to glucose ingestion[END_REF]. In this context, the drastic reduction of energy expenditure rapidly occurring in skeletal muscle is currently considered as the primary event promoting insulin resistance [START_REF] Booth | Role of Inactivity in Chronic Diseases: Evolutionary Insight and Pathophysiological Mechanisms[END_REF]. The related gain of fat mass and the chronic low grade inflammatory state observed in inactive people are generally proposed as responsible for insulin resistance development [START_REF] Booth | Role of Inactivity in Chronic Diseases: Evolutionary Insight and Pathophysiological Mechanisms[END_REF][START_REF] Pedersen | The diseasome of physical inactivity--and the role of myokines in muscle--fat cross talk[END_REF]. However, insulin resistance occurs after few days of physical inactivity whereas systemic inflammatory markers (e.g. TNF-α, IL-6) or visceral fat mass generally increase after several weeks in both humans and rodent experiments [START_REF] Hamburg | Physical inactivity rapidly induces insulin resistance and microvascular dysfunction in healthy volunteers[END_REF][START_REF] Krogh-Madsen | A 2-wk reduction of ambulatory activity attenuates peripheral insulin sensitivity[END_REF][START_REF] Olsen | Metabolic responses to reduced daily steps in healthy nonexercising men[END_REF][START_REF] Rector | Cessation of daily exercise dramatically alters precursors of hepatic steatosis in Otsuka Long-Evans Tokushima Fatty (OLETF) rats[END_REF]. Thus, adipose tissue and inflammation processes do not appear to be the culprits of early insulin resistance.

Physical inactivity, whatever the experimental model, induces a shift in fuel metabolism in favor of carbohydrate oxidation and in detriment of lipid oxidation, resulting in an accumulation of intramuscular lipids [START_REF] Bergouignan | Effect of physical inactivity on the oxidation of saturated and monounsaturated dietary Fatty acids: results of a randomized trial[END_REF][START_REF] Laye | Cessation of daily wheel running differentially alters fat oxidation capacity in liver, muscle, and adipose tissue[END_REF][START_REF] Momken | Resveratrol prevents the wasting disorders of mechanical unloading by acting as a physical exercise mimetic in the rat[END_REF]. Lipid accumulation in skeletal muscle is well known to be related with insulin resistance, especially due to their conversion into ceramides [START_REF] Samuel | Mechanisms for insulin resistance: common threads and missing links[END_REF]. Ceramides are bioactive mediators involved in cell responses to stress, and their increase in skeletal muscle is known to induce insulin resistance through the inhibitory phosphorylation of proteins of the insulin pathway including insulin receptor substrate-1/2 (IRS1/2) and phosphatidylinositol-3-kinase, (PI3-kinase) [START_REF] Chavez | Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes[END_REF]. Ceramides are synthesized through both stimulation of sphingomyelinase-mediated hydrolysis of membrane sphingomyelin, and de novo synthesis pathway consisting of the condensation of palmitoyl-CoA with serine [START_REF] Hannun | Ceramide in the eukaryotic stress response[END_REF]. Whereas accumulation of intramuscular ceramides has been extensively explored in the context of obesity [START_REF] Schmitz-Peiffer | Targeting Ceramide Synthesis to Reverse Insulin Resistance[END_REF][START_REF] Ussher | Inhibition of De Novo Ceramide Synthesis Reverses Diet-Induced Insulin Resistance and Enhances Whole-Body Oxygen Consumption[END_REF], their role in the onset of insulin resistance related to physical inactivity remains poorly understood.

We hypothesized that physical inactivity rapidly increases ceramide synthesis in skeletal muscle, which in turn would induce insulin resistance. Therefore, the present study was designed to analyze, in mice, whether inhibition of ceramide synthesis prevents insulin resistance observed after a short period of physical inactivity. For this purpose, we used: 1) the wheel lock model to study the effect of physical inactivity; 2) myriocin, an inhibitor of de novo synthesis of ceramides [START_REF] Salaun | Myriocin prevents muscle ceramide accumulation but not muscle fiber atrophy during short-term mechanical unloading[END_REF].

MATERIAL AND METHODS

All procedures described below were approved by the French Ministry of Higher Education and Research in accordance with the local committee on Ethics in Research of Rennes (veterinary service of health and animal protection, authorization 01259.03).

Animal care and protocol. To mimic the effects of physical inactivity in mice, we used the wheel lock model first developed by Frank Booth's research group [START_REF] Roberts | Potential clinical translation of juvenile rodent inactivity models to study the onset of childhood obesity[END_REF]. Twenty-two male C57BL/6J mice were obtained at weaning (3 weeks) and allowed to acclimatize for 1 week. The animals were housed in temperature controlled room (21 ± 2°C) with a 12-h:12-h light/dark cycle and received standard rodent chow and water ad libitum. After 1 week, mice were separated into individual cages all equipped with a voluntary running wheel outfitted with a lap counter (IntelliBio, Nancy, France). Food intake and covered distance were daily noticed whereas body weight was recorded every week. The distance daily covered increased the first two weeks and remained unchanged during the next 4 weeks. At the end of this period, mice were assigned to 3 experimental groups with equal mean and standard deviation for daily covered distance: voluntary wheel access group (Active, n=7), a wheel lock group (Inactive, n=7) and wheel lock group treated with myriocin (Inactive-Myr, n=8).

Myriocin was daily injected (0.3 mg/kg, i.p) as previously described [START_REF] Hojjati | Effect of myriocin on plasma sphingolipid metabolism and atherosclerosis in apoE-deficient mice[END_REF][START_REF] Lee | Effects of sphingolipid synthesis inhibition on cholesterol gallstone formation in C57BL/6J mice[END_REF]. In the same schedule, Active and Inactive groups received saline vehicle. After 10 days of physical inactivity, mice were sacrificed in an overnight fasting state. Mice were anesthetized with a ketamine-xylazine-butorphanol cocktail. Adipose tissue, rectus femoris (RF) and right tibialis anterior (TA) muscles were removed, and then immediately frozen in liquid nitrogen. Left tibialis anterior muscles were removed, and then submitted to an ex vivo insulin sensitivity test. Immediately following tissue harvest, mice were euthanized via exsanguination of the heart. Intracardiac blood was collected into dry tubes and centrifuged (1,500 g, 10 min) for serum sampling.

Glucose tolerance assessment. Oral glucose tolerance test (OGTT) was performed on the morning one day before sacrifice. Mice were fasted for 6h before the test. Then, glucose was administrated by oral gavage (1 g/kg body weight). Glucose values were obtained at rest and 15, 30, 45, 60, 90, 120 min after glucose gavage from tail blood samples. Glucose level was determined using a glucose meter (Freestyle Papillon Vision). Wheels were locked during the whole time of fasting and OGTT.

Ex vivo muscle insulin sensitivity test. As previously described [START_REF] Tardif | Oleate-enriched diet improves insulin sensitivity and restores muscle protein synthesis in old rats[END_REF], left tibialis anterior muscles were longitudinally divided in two strips (20-25 mg) and each strip was preincubated for 30 min in 3 mL of modified Krebs Ringer buffer (120 mM NaCl, 4.8 mM KCl, 25 mM NaHCO 3 , 2.5 mM CaCl 2 , 1.24 mM NaH 2 PO 4 , 1.25 mM MgSO 4 , 8 mM D-Glucose, 2 mM sodium pyruvate, 2 mM HEPES, pH 7.4) saturated with a 95% O 2 and 5% CO 2 mix at 37°C under stirring.

One of the two strips was stimulated with 20 nM insulin for 30 minutes, then muscle samples were frozen in liquid nitrogen until analysis. Muscle insulin sensitivity was assessed by measuring the phosphorylation state of Akt on serine 473, an intermediate of the insulin pathway.

Serum parameters. Glucose concentrations were performed using Automated Beckman Coulter (Beckman Coulter, Brea, CA). Serum insulin concentrations were measured by enzyme-linked immunosorbent assay (ELISA) according to manufacturer's instructions (Millipore, St Louis, MO, USA). Insulin sensitivity was determined by calculating HOMA-IR according to the following formula [START_REF] Matthews | Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man[END_REF]: HOMA-IR = [fasting glucose (mmol/l)] × [fasting insulin (μU/ml)] /22.5.

Quantification of muscle triglycerides. Muscle triglycerides were determined by using DiaSys kit (Diagnostic System, Grabels, France) following a preliminary organic phase extraction according to Bligh & Dyer's method [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF]. Briefly, 30 mg of right tibialis anterior samples were crushed with 300 μL of 150 mM sodium chloride. Then 150 μL of muscle homogenates were extracted with 600 μL of a methanol-chloroform mixture (1:1, v/v). The organic layers were collected after centrifugation (10,000g for 10 min) and dried under nitrogen. Dry samples were reconstituted in 37.5 μL of isopropanol/acetonitrile/water mixture (2:1:1, v/v/v) and 10 μL were analyzed according to the manufacturer recommendations.

Quantification of muscle sphingolipids and ceramides. Sphingolipids were extracted from ≈ 30 mg of homogenized tibialis anterior muscles using acidified cyclohexane/isopropanol mixture (60:40, v/v, 0.1% formic acid) and purified on NH 2 SPE cartridges (silica gel cartridges, 100 mg) to obtain distinct fractions of ceramides and sphingomyelins [START_REF] Bodennec | A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges[END_REF]. The sphingolipid fractions were then quantified by UHPLC-ESI-MS/MS using an Acquity H-Class UHPLC system (Waters, Milford, MA) combined with a Waters Xevo TQD triple quadrupole mass spectrometer. Lipid extracts were injected onto a C18 BEH column (2.1 mm x 50.0 mm, 1.7-µm particles; Waters) held to 43°C to separate all species of ceramides and sphingomyelins with two different LC elution gradients. For ceramide species separation, the gradient started at 95% of eluent B (mobile phase of water and of methanol with 1% formic acid and 5 mM ammonium formate), up to 98% in 4 min, then rapidly decreased to 95% for 0.1 min, and was maintained for 2 min. For sphingomyelin species separation, the gradient started at 95% of eluent B, up to 99% for 6 min, then rapidly decreased to 95% for 0.1 min, and was maintained for 1.4 min. Multiple reaction-monitoring mode in positive electrospray ionization was used to quantify each species of ceramides and sphingomyelins. The source heater temperature hold at 150°C, and the capillary voltage was set at 3.2 kV. The flow rate of desolvation gas was of 650 l/h at 350°C, and the cone voltage varied from 26-58 V. Argon was used as the collision gas, and collision energies varied from 12-40 eV. Data analyses were performed by Mass Lynx software version 4.1 (Waters, Manchester, UK). Sphingolipid quantification was possible with calibration curves constructed by plotting the peak area ratios of analyses to the respective internal standard against concentration using a linear regression model. The quantification measurements were performed using the TargetLinks software (Waters). CAATGCCAACTCTCGTCAACAG and R: CATCCAGGTCACCTTCTCGG). GLUT4 was normalized using three reference genes (PPPIA, HPRT, RPL19) according to geNorm analysis [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF].

Western Blotting. Cytosolic protein extraction was performed from rectus femoris muscle in cold lysis buffer containing 10 mM Tris-HCl, pH 7.4, 0.5 M sucrose, 50 mM NaCl, 5 mM EDTA, 30 mM Na 4 P 2 O 7 , 1% NP-40, 0.25% sodium deoxycholate, 50 mM NaF, 100 µM sodium orthovanadate and proteases inhibitors cocktail (Sigma P8340, 5 µl/ml). The samples were homogenized using a Polytron homogenizer at 4°C. Each sample was then incubated on ice for 30 min followed by 3 x 10 s of sonication. The homogenates were then centrifugated at 12,000g for 12 min at 4°C. The protein concentration of the supernatant was determined by a Lowry assay using bovine serum albumin (BSA) as standard. Samples were then diluted in SDS-PAGE sample buffer [50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.1% bromophenol blue], and heated 5 min at 95°C until analyses. Fifty micrograms of proteins were resolved on 12.5% SDS-PAGE. The proteins were transferred at 240 mA for 90 min onto a 0.2-µm nitrocellulose membrane. Membranes were blocked with 5% BSA or nonfat dry milk in TBST (Tris-buffered saline -0.05% Tween-20) for 1 h at room temperature. Membranes were incubated overnight at 4°C with appropriate primary antibodies: AKT

(1:1000, Cell Signaling), p-AKT Ser473 (1:1000, Cell Signaling), AMPK (1:1000, Cell Signaling), p-AMPK Thr172 (1:1000, Cell Signaling), p65 (1:1000, Cell Signaling), p-p65 Ser536 (1:1000, Cell Signaling), IRS-1 (1:1000, Cell Signaling), pIRS-1 Ser302 (1:1000, Cell Signaling), STAT3 (1:1000, Cell Signaling), p-STAT3 Ser727 , IκBα (1:1000, Cell Signaling), GLUT4 (1:1000, Abcam), and α-actin

(1:700, Sigma Aldrich). Thereafter, membranes were washed with TBST and incubated for 1 h at room temperature with infrared dye-conjugated secondary antibodies (LI-COR, Lincoln, NE, USA).

After washing, blots were captured using the Odyssey Imaging System (LI-COR). All blots were scanned, densitometric analysis of the bands was conducted using GS-800 Imaging densitometer and QuantityOne software. Phosphospecific signal was normalized to the total signal to estimate the ratio of activated markers.

Statistical analysis. Data are presented as mean ± SEM. Normality and equality of variances were checked using a Kolmogorov-Smirnov and Fischer test, respectively. A one-way analysis of variance (ANOVA) was performed to compare each parameter between the 3 experimental groups. When appropriate, the Fisher LSD test was used as a post-hoc analysis. If normality and/or equal variance tests failed, we checked the significance using one-way ANOVA on ranks (Kruskal-Wallis). When appropriate, the Dunn's test was used as a post-hoc analysis. For all statistical analyses, the significance level was set at 0.05. Data were analyzed using the statistical package GraphPad Prism version 6.02 for Windows (GraphPad Software, La Jolla, California).

RESULTS

Physical activity levels, body weight, food intake and visceral fat mass. During the 10 days of wheel lock, Active mice exhibited a mean daily physical activity levels of 3.90 ± 0.78 km/day. Daily food intake was lower in both Inactive and Inactive-Myr mice compared to Active mice (-7.9% and -7.6%, p=0.007 and 0.01, respectively, Table 1). After 10 days of wheel lock, body weight significantly increased only in Active mice. No significant difference of body weight was observed between the 3 experimental groups before and after the 10 days of physical inactivity. Visceral fat mass did not differ between the 3 experimental groups at the end of the protocol (Table 1).

Muscle triglycerides, ceramides and sphingomyelin content. We first observed that muscle triglyceride (TG) content tends to increase in Inactive mice (p=0.09, Fig. 1A) whereas it increases in the Inactive-Myr group (p=0.027, Fig. 1A). Contrary to our hypothesis, physical inactivity did not modify total, saturated and unsaturated ceramides content in muscle (Fig. 1B). Individual ceramides species ranging from C16:0 to C24:1 remained also unchanged after 10 days of physical inactivity (Fig. 1C and1D).

Surprisingly, muscle ceramides content was not affected by 10 days of myriocin treatment (Fig. 1B, 1C and 1D). Total, saturated and unsaturated sphingomyelin content also remained unchanged with physical inactivity and after myriocin treatment (Fig. 1E).

Whole-body insulin sensitivity and glucose tolerance. The effect physical inactivity and myriocin treatment on whole-body insulin sensitivity was assessed by measuring the circulating level of glucose and insulin. Fasting glycemia remained unaffected with physical inactivity and myriocin treatment (Fig. 2A). Serum insulin levels were higher in both Inactive and Inactive-Myr groups compared to Active group (p=0.025 and 0.015, respectively, Fig. 2B). Such results were associated with a higher HOMA-IR index in Inactive compared to Active mice (p=0.045, Fig. 2C), whereas this index did not differ between Inactive-Myr and Active mice (p=0.17, Fig. 2C). Thus, myriocin seems to prevent physical inactivity-induced whole-body insulin resistance. An OGTT was also performed at the end of the protocol to observe the effects of physical inactivity and myriocin on glucose tolerance. Here, we did not observe significant difference in glucose concentrations (at all time points) and area under the curve (AUC) between Active and Inactive mice (Fig. 2D andE). However, we reported that Inactive-Myr mice exhibited an AUC significantly lower compared to both Active (p=0.019, Fig. 2D) and

Inactive mice (p<0.001, Fig. 2D). This effect was due to a lower glucose levels both at the beginning and the end of OGTT (15 min, 30 min, 90 and 120 min, p<0.05, Fig. 2E). Taken together, our results

indicate that myriocin improves glucose tolerance.

Proximal insulin signaling and GLUT4 content in skeletal muscle. We observed a deleterious effect of physical inactivity on whole-body insulin sensitivity and a beneficial effect of myriocin on glucose tolerance. To investigate the mechanism regulating such effects, we first evaluated the integrity of insulin signaling in skeletal muscle by measuring, ex vivo, basal and insulin-stimulated Akt activation.

We observed that insulin stimulation increased phospho-Akt levels in Active (p=0.019), Inactive (p=0.029) and Inactive-Myr (p<0.001, Fig. 3A). Whereas the magnitude of these responses did not differ between Active and Inactive mice, Inactive-Myr mice exhibited a higher muscle activation of Akt in response to insulin when compared to both Active and Inactive mice (p=0.046 and p=0.036, respectively, Fig. 3A). Glucose uptake is recognized to be regulated by insulin signaling and GLUT4 pool in skeletal muscle [START_REF] Pierre | From physical inactivity to immobilization: Dissecting the role of oxidative stress in skeletal muscle insulin resistance and atrophy[END_REF]. In the present study, we observed that physical inactivity reduced GLUT4 protein (p=0.043, Fig. 3B and3E), but not GLUT4 mRNA (Fig. 3C). Interestingly, we observed that myriocin treatment prevent the reduction of muscle GLUT4 protein induced by physical inactivity (p=0.005, Fig. 3B). At the mRNA level, GLUT4 was reduced by myriocin in Inactive-Myr compared to Inactive mice (p=0.031, Fig. 3B). As AMP-activated protein kinase (AMPK) is recognized to regulate GLUT4 expression and it translocation to the membrane [START_REF] Mcgee | AMP-activated protein kinase regulates GLUT4 transcription by phosphorylating histone deacetylase 5[END_REF], we decided to measure the levels of AMPK activation in skeletal muscle. We reported that AMPK activation remained unchanged in Inactive compared to Active mice (Fig. 3D andE).

Interestingly, we showed that AMPK activation was higher in myriocin-treated mice compared to both Active and Inactive mice (p=0.031 and p=0.003, respectively, Fig. 3D and3E). In insulin-resistant rodent models, chronic hyperphosphorylation of IRS1 on Ser 302 has been identified as playing a key role in muscle insulin resistance [START_REF] Morino | Muscle-Specific IRS-1 Ser→Ala Transgenic Mice Are Protected From Fat-Induced Insulin Resistance in Skeletal Muscle[END_REF]. In our experiments, no significant change was observed in the phosphorylation state of IRS1 on Ser 302 between the 3 experimental groups (Fig. 4A and4E). Muscle insulin resistance is also related to activation of NF-ΚB and IL6/STAT3 inflammatory signaling pathways [START_REF] Gratas-Delamarche | Physical inactivity, insulin resistance, and the oxidative-inflammatory loop[END_REF]. Here, we reported that physical inactivity combined or not with myriocin treatment did not modulate phospho-p65 and IKBα (Fig. 4C and 4E), two well-recognized markers of NF-KB activation [START_REF] Christian | The Regulation of NF-κB Subunits by Phosphorylation[END_REF]. Similarly, phosphorylation of STAT3 on Ser 727 remained unchanged in the three experimental groups (Fig. 4B and4E).

DISCUSSION

Muscle ceramides are known to play a key role in the development of insulin resistance during high fat nutritional intake, but their role in physical inactivity-induced insulin resistance are poorly understood. Our data support that early insulin resistance observed with physical inactivity is not due to ceramide accumulation and disrupted insulin signaling in skeletal muscle, but could be caused by a reduction in muscle GLUT4 content. We also observed that 10 days of myriocin treatment in inactive mice improve glucose tolerance, but surprisingly through a mechanism that appears independent of changes in muscle ceramide content.

Immobilization rapidly causes both in humans and rodents a reduction in fatty acid (FA) transport to mitochondria and in mitochondrial FA β-oxidation, resulting in an accumulation of intramuscular lipids [START_REF] Bergouignan | Effect of physical inactivity on the oxidation of saturated and monounsaturated dietary Fatty acids: results of a randomized trial[END_REF][START_REF] Kwon | Intramyocellular ceramides and skeletal muscle mitochondrial respiration are partially regulated by Toll-like receptor 4 during hindlimb unloading[END_REF][START_REF] Laye | Cessation of daily wheel running differentially alters fat oxidation capacity in liver, muscle, and adipose tissue[END_REF][START_REF] Momken | Resveratrol prevents the wasting disorders of mechanical unloading by acting as a physical exercise mimetic in the rat[END_REF][START_REF] Salaun | Myriocin prevents muscle ceramide accumulation but not muscle fiber atrophy during short-term mechanical unloading[END_REF]. Saturated FA oxidation, including palmitate, are particularly reduced in skeletal muscle during immobilization [START_REF] Bergouignan | Physical inactivity as the culprit of metabolic inflexibility: evidence from bed-rest studies[END_REF]. Interestingly, palmitate is well identified to increase the expression of serine palmitoyltransferase 2 (SPT2), a key enzyme in ceramide biosynthesis [START_REF] Erickson | AICAR inhibits ceramide biosynthesis in skeletal muscle[END_REF]. Ceramides have been identified as key bioactive sphingolipids in the development of muscle insulin resistance, especially the C:16 and C:18 moieties, which are the most abundant in skeletal muscle [START_REF] Chung | Intramyocellular Ceramides: Subcellular Concentrations and Fractional De Novo Synthesis in Postabsorptive Humans[END_REF][START_REF] Perreault | Intracellular localization of diacylglycerols and sphingolipids influences insulin sensitivity and mitochondrial function in human skeletal muscle[END_REF]. Based on previous data from our laboratory and others showing in rodents that C:16 and C:18 ceramides increase in skeletal muscle after 1 or 2 weeks of immobilization [START_REF] Kwon | Intramyocellular ceramides and skeletal muscle mitochondrial respiration are partially regulated by Toll-like receptor 4 during hindlimb unloading[END_REF][START_REF] Salaun | Myriocin prevents muscle ceramide accumulation but not muscle fiber atrophy during short-term mechanical unloading[END_REF], we hypothesized that 10 days of physical inactivity induced by wheel lock will also cause muscle ceramide accumulation.

Contrary to immobilization, we observed that total and saturated ceramide levels in muscle remained unchanged after 10 days of physical inactivity. This was also the case for muscle TG which are increase after immobilization [START_REF] Kwon | Intramyocellular ceramides and skeletal muscle mitochondrial respiration are partially regulated by Toll-like receptor 4 during hindlimb unloading[END_REF][START_REF] Salaun | Myriocin prevents muscle ceramide accumulation but not muscle fiber atrophy during short-term mechanical unloading[END_REF], while we reported only a trend with physical inactivity. In those studies, ceramides content has been measured in rodent soleus muscle that mainly contain type 1 fibers. As in the present study ceramides content has been measured in tibialis anterior that contain type 2 fibers, it could partly explain why immobilization and physical inactivity induce a distinct effect on muscle ceramides content. Alternatively, immobilization is a more drastic model than physical inactivity, this could also explain why muscle ceramides content increase in one case but not the other, and this independently of muscle fiber type [START_REF] Salaun | Myriocin prevents muscle ceramide accumulation but not muscle fiber atrophy during short-term mechanical unloading[END_REF].

Despite these limitations, the present study supports that muscle ceramides are not responsible of insulin resistance induced by physical inactivity. Nevertheless we cannot exclude that other lipid intermediates (e.g diacylglycerol and long chain acyl-CoA) could play a role in physical inactivityinduced muscle insulin resistance.

In rodents, the wheel lock model is currently the closest model to mimic human physical inactivity [START_REF] Roberts | Potential clinical translation of juvenile rodent inactivity models to study the onset of childhood obesity[END_REF]. Using this model in mice, we observed that 10 days of physical inactivity are sufficient to induce hyperinsulinemia and to increase the HOMA-IR, both considered as hallmarks of insulin resistance [START_REF] Singh | Surrogate markers of insulin resistance: A review[END_REF]. These results are in accordance with previous studies conducted in Sprague-Dawley or OLETF rats after 7 days of wheel lock [START_REF] Rector | Changes in skeletal muscle mitochondria in response to the development of type 2 diabetes or prevention by daily wheel running in hyperphagic OLETF rats[END_REF][START_REF] Rector | Cessation of daily exercise dramatically alters precursors of hepatic steatosis in Otsuka Long-Evans Tokushima Fatty (OLETF) rats[END_REF][START_REF] Teich | Curcumin limits weight gain, adipose tissue growth, and glucose intolerance following the cessation of exercise and caloric restriction in rats[END_REF]. Few data are available about the effects of the wheel lock model on glucose tolerance. Teich and colleagues recently observed that 7 days of wheel lock were sufficient to affect glucose tolerance in young Sprague-Dawley male rats [START_REF] Teich | Curcumin limits weight gain, adipose tissue growth, and glucose intolerance following the cessation of exercise and caloric restriction in rats[END_REF]. In the present study, we did not report an effect on glucose tolerance after 10 days of wheel lock. Our results are in accordance with data obtained in humans exposed to a reduction in daily number of steps during 5 or 14 days, where no change in glucose tolerance was reported [START_REF] Knudsen | Changes in insulin sensitivity precede changes in body composition during 14 days of step reduction combined with overfeeding in healthy young men[END_REF][START_REF] Reynolds | Acute inactivity impairs glycemic control but not blood flow to glucose ingestion[END_REF]. All together, these results sustain that the wheel lock model is a realistic experimental set-up to study, in mice, human physical inactivity and its role in chronic diseases development.

Skeletal muscle being responsible of 80% of whole-body glucose uptake under insulin stimulation [START_REF] Defronzo | Effects of insulin on peripheral and splanchnic glucose metabolism in noninsulin-dependent (type II) diabetes mellitus[END_REF], early insulin resistance occurring with physical inactivity is mainly attributed to alterations in skeletal muscle glucose uptake [START_REF] Knudsen | Changes in insulin sensitivity precede changes in body composition during 14 days of step reduction combined with overfeeding in healthy young men[END_REF][START_REF] Krogh-Madsen | A 2-wk reduction of ambulatory activity attenuates peripheral insulin sensitivity[END_REF].

Interestingly, using the wheel lock model during only 2 days, Kump and Booth observed a reduction of insulin-stimulated 2-deoxyglucose uptake in skeletal muscle [START_REF] Kump | Alterations in insulin receptor signalling in the rat epitrochlearis muscle upon cessation of voluntary exercise[END_REF]. Similar results were also reported in rats submitted to muscle unloading after 24h [START_REF] Kawamoto | Immobilization rapidly induces muscle insulin resistance together with the activation of MAPKs (JNK and p38) and impairment of AS160 phosphorylation[END_REF][START_REF] O'keefe | Development of whole-body and skeletal muscle insulin resistance after one day of hindlimb suspension[END_REF]. However, the mechanisms by which physical inactivity and muscle unloading rapidly induce muscle insulin resistance remain unclear. Krogh-Madsen and colleagues (2010) have reported that a reduction of muscle Akt phosphorylation during hyperinsulinemic euglycemic clamp occurred after 2 weeks of reduction of ambulatory activity, thus supporting the idea that insulin signaling was early affected by physical inactivity. However, if the clamp is the gold standard to assess peripheral insulin sensitivity, it appears less appropriate to assess insulin signaling. Indeed, presence of circulating hormones or cytokines (e.g. leptin, adiponectin, IL-6) and muscle contractility are all factors affecting muscle insulin signaling during clamp, independently of direct action of insulin. For these reasons, we decided to explore insulin-stimulated muscle Akt phosphorylation in exvivo conditions to determine whether insulin signaling was affected with a short period of physical inactivity. In this condition, we observed that Akt phosphorylation in inactive mice did not differ from active animals, suggesting that insulin signaling until Akt remains unaffected after 10 days of physical inactivity. To support such results, we explored the effects of physical inactivity on inflammatory pathways recognized to affect insulin signaling. Here, neither NF-κB nor IL-6/STAT3 signaling pathways were affected in skeletal muscle of inactive mice. Further, in response to pro-inflammatory state IRS1 is inhibited by a phosphorylation on its Ser 302 [START_REF] Gratas-Delamarche | Physical inactivity, insulin resistance, and the oxidative-inflammatory loop[END_REF][START_REF] Hage Hassan | Sustained Action of Ceramide on the Insulin Signaling Pathway in Muscle Cells: IMPLICATION OF THE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN KINASE[END_REF][START_REF] Morino | Muscle-Specific IRS-1 Ser→Ala Transgenic Mice Are Protected From Fat-Induced Insulin Resistance in Skeletal Muscle[END_REF]. In the present study, we found that physical inactivity did not modify the phosphorylation state IRS1 Ser302 . Taken together, these results indicate that, in the context of physical inactivity, the onset of insulin resistance is not due an inflammatory process affecting the skeletal muscle.

Our data support that muscle insulin signaling does not appear affected by a short period of physical inactivity. In accordance with [START_REF] Kump | Alterations in insulin receptor signalling in the rat epitrochlearis muscle upon cessation of voluntary exercise[END_REF], we reported that 10 days of wheel lock caused a significant reduction of GLUT4 pool in skeletal muscle. These results support the idea that inactivity-induced muscle insulin resistance would be rather related to a decrease of muscle GLUT4 pool than a defect in insulin signaling. Interestingly, we observed that mRNA coding for GLUT4 was not affected suggesting that the decrease of GLUT4 pool would be due to an increase of its degradation. It is well established that chronic insulin stimulation causes a decrease of GLUT4 content in adipocytes, mainly due to an accelerated GLUT4 degradation in the lysosomes [START_REF] Liu | The SUMO conjugating enzyme Ubc9 is a regulator of GLUT4 turnover and targeting to the insulin-responsive storage compartment in 3T3-L1 adipocytes[END_REF]Ma et al., 2014, p. 4;[START_REF] Sargeant | Effect of insulin on the rates of synthesis and degradation of GLUT1 and GLUT4 glucose transporters in 3T3-L1 adipocytes[END_REF]. Similar cellular events could occur in skeletal muscle of inactive. Such mechanisms need to be explored in further experiments.

To explore the role of ceramides in physical inactivity-induced insulin resistance, we treated inactive mice with myriocin, an inhibitor of de novo synthesis of ceramides. As previously reported in others experimental models including Zucker diabetic rats or high fat diet-fed mice [START_REF] Holland | Inhibition of Ceramide Synthesis Ameliorates Glucocorticoid-, Saturated-Fat-, and Obesity-Induced Insulin Resistance[END_REF][START_REF] Ussher | Inhibition of De Novo Ceramide Synthesis Reverses Diet-Induced Insulin Resistance and Enhances Whole-Body Oxygen Consumption[END_REF], we observed that myriocin treatment improved glucose tolerance in inactive mice.

Interestingly, we concomitantly observed in skeletal muscle that myriocin improves muscle insulin signaling and AMK activation, and prevents the decrease of GLUT4 pool induced by physical inactivity. This result is in accordance with the data of Liu and colleagues (2013) demonstrating that myriocin prolonged yeast lifespan by activating key signaling pathways controlling stress resistance and energy metabolic homeostasis including AMPK signaling pathway. Thus, our results support that the beneficial effect of myriocin on glucose tolerance may be mediated by an improvement of both insulin-and AMPK-dependent muscle glucose uptake [START_REF] Jensen | Contraction-stimulated glucose transport in muscle is controlled by AMPK and mechanical stress but not sarcoplasmatic reticulum Ca2+ release[END_REF]. Contrary to our hypothesis, the benefits of myriocin treatment on glucose tolerance are not associated to modulation in muscle ceramide concentrations. The previous studies reporting in mice a beneficial effect of myriocin on glucose tolerance and insulin sensitivity were systematically associated to a prevention of muscle ceramide accumulation induced by high fat diet or pathological genetic background [START_REF] Holland | Inhibition of Ceramide Synthesis Ameliorates Glucocorticoid-, Saturated-Fat-, and Obesity-Induced Insulin Resistance[END_REF][START_REF] Ussher | Inhibition of De Novo Ceramide Synthesis Reverses Diet-Induced Insulin Resistance and Enhances Whole-Body Oxygen Consumption[END_REF][START_REF] Yang | Central role of ceramide biosynthesis in body weight regulation, energy metabolism, and the metabolic syndrome[END_REF]. Herein, we found that myriocin could also act on insulin sensitivity independently from ceramides, thus highlighting the need to explore new mechanisms linking myriocin and insulin sensitivity.

In summary, our results support that muscle ceramide accumulation and inflammatory pathways do not play a role in physical inactivity-induced insulin resistance. Our data support that insulin resistance observed after a short period of physical inactivity appears related to a reduction in muscle GLUT4 content rather than to defects of muscle insulin signaling.

DISCLOSURES

No conflict of interest, financial or otherwise, are declared by the author(s). 25.2 ± 0.5 25.8± 0.2 24.8 ± 0.2 10 days post-locking 25.8 ± 0.6 $ 25.9 ± 0.2 24.5 ± 0.3 Food intake (g/day)

4.16 ± 0.05 3.83 ± 0.09 * 3.84 ± 0.08 * Physical activity (km/day) 3.90 ± 0.78 --------------------------Visceral fat mass (g/100 g BW)

2.12 ± 0.19 2.30 ± 0.25 2.08 ± 0.15 Data are presented as mean ± SEM (n=7-8/group). Significant differences (p< 0.05) are indicated as follows: comparison vs. values before wheel locking ($), comparison vs. Active group (*)

  RNA extraction and quantitative real-time PCR. Total RNA extraction from frozen rectus femoris was performed with Trizol® (Invitrogen, France) according to the manufacturer's instructions. The RNA quality and quantity were assessed by FlashGel DNA System® and Nanodrop® spectrophotometry, respectively. Reverse transcription was performed on a T100 Thermal Cycler (Bio-Rad) with iScript TM cDNA synthesis kit (Bio-Rad) from 1 µg total RNA. Real time PCR experiments were done on a CFX96 Real Time System (Bio Rad). Samples were analyzed in duplicate in 10 µl reaction volume containing 4.8 µl IQ TM Sybr ® GreenSuperMix (Bio-Rad), 0.1 µl of each primer (100 nM final) and 5 µl of diluted cDNA. The following primer sequences were used: GLUT4: (F: GCCTGCCCGAAAGAGTCTAA and R: CATTGATGCCTGAGAGCTGTTG); PPIA (F: and R: CAGGACTCCTCGTATTTGCAG); RPL19 (F:

Figure 1 .Figure 2 .Figure 3 .Figure 4 .

 1234 Figure 1. Effects of physical inactivity and myriocin treatment on lipids profile. Data are

Table 1 . Effects of physical inactivity and myriocin treatment on body weight, food intake and visceral fat mass.

 1

	Active	Inactive	Inactive-Myr

ACKNOWLEDGMENTS

The authors thank Véronique Ferchaud-Roucher and Mickael Croyal (Corsaire platform, CRNH, UMR1280, Nantes) for technical help in sphingolipid analyses.

GRANTS

This study was supported by grants from the Brittany Research Council (SAD program -MusFer n°8802) and ID2Santé-Bretagne (PICAM project).


Professor Jean-François Viel
email: jean-francois.viel@univ-rennes1.fr

Yoann Mallet

Christina Raghoumandan

Philippe Quenel

Philippe Kadhel

Florence Rouget

Luc Multigner

Impact of Saharan dust episodes on preterm births in Guadeloupe (French West Indies)

Keywords: Preterm birth, particulate matter, desert dust, Guadeloupe archipelago, French West Indies CI Confidence interval OR Odds ratio PM 10 Particulate matter ≤ 10 µm

Objectives Large amounts of mineral dust are transported from their African sources in the Saharan-Sahel region to the Caribbean Sea, generating peak exposures to particulate matter ≤10 µm (PM 10 ).

This study aimed to investigate the impact of Saharan dust episodes on preterm births in the Guadeloupe archipelago.

Methods

The study population consisted of 909 pregnant women who were enrolled in the TIMOUN mother-child cohort between 2004 and 2007. Desert dust episodes were assessed from PM 10 concentrations recorded at the unique background air quality monitoring station located in Pointe-à-Pitre. For each woman, the daily PM 10 concentrations were averaged over the entire pregnancy, and the proportion of days with intense dust episodes (≥ 55 µg PM 10 /m 3 ) during pregnancy was calculated. Weighted logistic regression models adjusting for known individual socio-medical risk factors were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for preterm birth.

Results

During pregnancy, the mean PM 10 concentrations ranged from 13.17 to 34.92 µg/m 3 , whereas the proportion of intense dust events ranged from 0.00 to 19.41%. Increased adjusted ORs were found for both the mean PM 10 concentrations and the proportion of intense dust events (OR 1.40, 95% CI 1.08 to 1.81, and OR 1.54, 95% CI 1.21 to 1.98 per standard deviation change, respectively). Restriction to spontaneous preterm births produced similar ORs but with wider 95% CIs.

Conclusion

Considering the personal and social burden of this adverse pregnancy outcome, this finding is of importance for both health care workers and policy makers to provide necessary preventive measures.

INTRODUCTION

Desert dust is a natural contributor to atmospheric particulate matter (PM) worldwide. Large amounts of mineral dust are transported from their African sources in the Saharan-Sahel region to the Caribbean Sea. [START_REF] Prospero | Understanding the transport and impact of African dust on the Caribbean basin[END_REF] The archipelago of Guadeloupe (French West Indies) is periodically exposed to desert dust between April and October, generating peak exposures that can last several days and exceed the PM ≤ 10 µm (PM 10 ) health-based standards (WHO: 20 µg/m 3 , European Union: 40 µg/m 3 , as an annual average).

The available epidemiological evidence of the impact of desert dust episodes on human health is inconsistent. In Europe, although some studies have linked sandy dust storms to a range of adverse health outcomes including respiratory and cardiovascular conditions and mortality, others have found no harmful effect. [START_REF] Karanasiou | Health effects from Sahara dust episodes in Europe: literature review and research gaps[END_REF] The same inconsistencies emerge from studies published more recently. [START_REF] Neophytou | Particulate matter concentrations during desert dust outbreaks and daily mortality in Nicosia, Cyprus[END_REF][START_REF] Alessandrini | Saharan dust and the association between particulate matter and daily hospitalisations in Rome, Italy[END_REF][START_REF] Reyes | Impact of Saharan dust particles on hospital admissions in Madrid (Spain)[END_REF][START_REF] Stafoggia | Desert dust outbreaks in Southern Europe: contribution to daily PM₁₀ concentrations and short-term associations with mortality and hospital admissions[END_REF] In the Caribbean region, a handful of studies is available. [START_REF] Monteil | Saharan dust clouds and human health in the English-speaking Caribbean: what we know and don't know[END_REF] On the island of Barbados, no relationship was found between desert dust concentrations and asthmatic attendances at the Queen Elizabeth Hospital for the period of 1996-1997, [START_REF] Prospero | Relationship between African dust carried in the Atlantic trade winds and surges in pediatric asthma attendances in the Caribbean[END_REF] whereas on the island of Trinidad, Saharan dust clouds were associated with increased acute pediatric asthma admissions. [START_REF] Gyan | African dust clouds are associated with increased paedriatric asthma accident and emergency admissions on the Caribbean island of Trinidad[END_REF] On the island of Martinique, PM 10 concentrations were associated with increased hospital admissions for cardiovascular or pulmonary conditions over the period of 2001-2006. [START_REF] Blateau | Health effects from desert dust episodes in Martinique, 2001-2006[END_REF] A recent study conducted in the Guadeloupe archipelago found a significant association between the PM 10 and PM 2.5-10 pollutants contained in the Saharan dust and visits to the emergency department for children with asthma in 2011. [START_REF] Cadelis | Short-term effects of the particulate pollutants contained in Saharan dust on the visits of children to the emergency department due to asthmatic conditions in Guadeloupe (French Archipelago of the Caribbean)[END_REF] It is worth noting that these studies used an ecological design with time-series analyses, associating a number of health events on a particular day with air pollution levels on the same or recent days.

Regarding maternal exposure to anthropogenic particulate pollution (originating from the burning of fossil fuels in vehicles, power plants or various industrial processes), evidence from available studies is indicative of an association with preterm birth. [START_REF] Stieb | Ambient air pollution, birthweight and preterm birth: a systematic review and meta-analysis[END_REF] Therefore, the influence of desert dust particulate pollution on pregnancy outcomes appears plausible and could be of public health importance because Sahara dust transport may greatly increase the ambient levels of PM (particularly in Southern Europe and in some Atlantic islands). [START_REF] Karanasiou | Health effects from Sahara dust episodes in Europe: literature review and research gaps[END_REF] Surprisingly, to the best of our knowledge, only one study has reported on the influence of Saharan dust episodes on pregnancy (in Spain). [START_REF] Dadvand | Saharan dust episodes and pregnancy[END_REF] The Guadeloupe archipelago represents a unique opportunity to assess the potential impact of Saharan dust episodes on preterm birth for two main reasons. First, because of the absence of heavy industries and the year-round presence of trade winds (dispersing gaseous and chemical pollutants), anthropogenic particle pollution is low in this area (approximately 20 μg/m 3 ). [START_REF] Euphrasie-Clotilde | The relationship between coastal West African dust level and Caribbean island dust[END_REF] As a result, desert dust natural pollution is the main factor affecting PM 10 concentrations. Second, a high rate of preterm birth (15.8%) is observed in Guadeloupe. [START_REF] Rouget | Medical and sociodemographic risk factors for preterm birth in a French Caribbean population of African descent[END_REF] This high preterm birth rate is not fully understood, but it could be partly due to the African origin of its population, which as such presents a refractory high rate, common to populations of African descent, irrespective of their current geographic location and independent of socioeconomic status and obstetrical care. [START_REF] Muglia | The enigma of spontaneous preterm birth[END_REF] However, other than African ancestry, there is room for other unknown or unstudied risk factors (including environmental exposures) to explain this high rate of preterm births.

Therefore, this study aimed to investigate the impact of Saharan dust episodes on preterm birth among a population of pregnant women in the Guadeloupe archipelago using prospectively collected individual data.

METHODS

Setting and study design

Guadeloupe (part of the French West Indies) is an archipelago situated in the Caribbean Sea. It covers an area of 1,628 km 2 and has a population of 450,000 inhabitants. This study relies on the TIMOUN mother-child cohort fully described elsewhere. [START_REF] Rouget | Medical and sociodemographic risk factors for preterm birth in a French Caribbean population of African descent[END_REF] Briefly, between 2004 and 2007, 1068 women attending check-up visits at public hospitals or dispensaries during their third trimester of pregnancy and having resided in Guadeloupe for more than 3 years were enrolled in the cohort. At inception, the participants completed a standardized questionnaire during a face-to-face interview with midwives. The questionnaire covered sociodemographic characteristics, medical and obstetrical history, and various lifestyle factors. After delivery, information was collected from midwives, pediatricians and hospital medical records about the medical history of the pregnancy, delivery, perinatal conditions, and measurements and health status of the newborn at birth. Following Rouget et al., [START_REF] Rouget | Medical and sociodemographic risk factors for preterm birth in a French Caribbean population of African descent[END_REF] we excluded women not born in the Caribbean (n = 110), cases involving multiple births (n = 25), severe birth defects (n = 8) and induced pregnancies after fertility treatment (n = 15), resulting in a study sample of 911 women (one case involved both fertility treatment and multiple births).

Preterm births

Gestational age in weeks was estimated by the obstetricians in charge of follow-up. It was based on the first day of the last menstrual period and was confirmed or corrected by ultrasound. Preterm birth was defined as a birth before 37 completed weeks of gestational age. We distinguished between spontaneous and medically induced preterm birth. The latter results from the induction of delivery due to maternal or fetal factors (either by medication or by caesarean section) before the onset of spontaneous labor. Elective induced labor was not practiced at the delivery centers involved in this study. Desert dust episodes were assessed in two ways. First, the mean daily PM 10 concentrations (µg /m 3 ) were considered. However, as we could not distinguish between natural (desert dust) and anthropogenic concentrations, we also used a dust episode index, designed by Gwad'Air for daily transmission to local authorities and assumed to be more specific. It mainly relies on ATMO subindexes, daily indicators of air quality widely used in France. [START_REF] Van Den Elshout | Comparing urban air quality in Europe in real time. A review of existing air quality indices and the proposal of a common alternative[END_REF] These sub-indexes, ranging from 1 (very good) to 10 (very poor), are calculated for each of four pollutants (sulphur dioxide, nitrogen dioxide, ozone and PM 10 ) on the basis of its concentration compared to a specific reference scale.

Desert dust exposure assessment

Drawing upon the ATMO PM 10 sub-index category, the contrasts observed with the other subindexes, and the usual local meteorological conditions, Gwad'Air empirically defines a dust episode index as following: absent (0 to 27 µg PM 10 /m 3 ), light (28 to 38 µg PM 10 /m 3 ), moderate (39 to 54 µg PM 10 /m 3 ), and intense (≥ 55 µg PM 10 /m 3 ). As a categorical exposure estimate, we used the presence of an intense episode. Because of routine maintenance or machine failure independent of any desert dust episodes, there were a few missing days of data (14, 12, 21, and 13 in 2005, 2006, 2007, and 2008, respectively).

For each woman, daily measures of PM 10 concentrations were averaged over the entire pregnancy, and the proportion of days with intense dust episodes during pregnancy was calculated. However, we had to account for the lack of exposure data in the entire year of 2004, which included two full pregnancies (and 234 partial pregnancies). Therefore, we were forced to exclude those two pregnancies from the study sample. For the 909 remaining women, the number of days with available exposure measurements was used as the denominator of the intense dust episode proportion.

Covariates

In-depth statistical analyses have previously resulted in a list of individual risk factors for preterm birth in the TIMOUN cohort. [START_REF] Rouget | Medical and sociodemographic risk factors for preterm birth in a French Caribbean population of African descent[END_REF] The corresponding risk factors (all referring to the index pregnancy) were considered in this study as covariates and introduced into the statistical models: place of inclusion (i.e., the healthcare facility where pregnant women were enrolled: University hospital, local hospital, local antenatal care clinic), maternal age (< 20, 20-34, > 34 years), marital status (single, living as a couple, single and living with family), years of education (< 5, 5-12, > 12), body mass index (< 18.5, 18.5-25.0, > 25.0 kg/m 2 ), parity (0, 1, ≥ 2), prior preterm birth (yes/no), prior miscarriage (yes/no), prior induced abortions (yes/no), lupus (yes/no), history of asthma (yes/no), chronic hypertension (yes/no), gestational weight gain (< 275, 275-675, > 675 g/week), gestational hypertension (yes/no), gestational diabetes (yes/no), urinary tract infection (yes/no), and sex of the newborn (male/female).

Data analysis

Multivariate logistic regression models were used to produce odds ratios (ORs) and their 95% confidence intervals (CIs) for associations between desert dust exposure and preterm birth while adjusting for all individual risk factors previously described. As exposure estimates, averaged PM 10 concentrations and the proportion of intense dust episodes during pregnancy were considered in turn. To compare the impact of both exposure estimates, we calculated the ORs per standard deviation change (3.08 µg/m 3 and 4.06 % for the mean PM 10 concentration and the proportion of intense dust episodes, respectively).

To compensate for differences in missing daily exposure measurements, each case in the input data set was weighted by the proportion of gestation length with available exposure measurements.

Therefore, the more complete the exposure assessment, the more important the observation. We repeated analyses considering spontaneous and medically induced preterm births separately.

Statistical analyses were performed using R software (R Foundation for Statistical Computing, Vienna, Austria, 2018).

RESULTS

Characteristics of the study population

The women's pregnancies in this study occurred during the time period from 30 March 2004 to 30 July 2007. The sociodemographic and lifestyle factors of the study population are reported in Table 1.

Most women were enrolled in the study at the University Hospital, were aged between 20 and 34 years old, were living with a partner and had completed between 5 and 12 years of education.

Approximately two-fifths were overweight or obese before pregnancy. Regarding the outcome, 142 women (15.6%) delivered preterm among the 909 total women: 67 of them (47.2%) were spontaneous preterm births, and 74 (52.1%) were medically induced preterm births (for one woman, the type of preterm birth could not be identified).

Characteristics of exposure

The average PM 10 concentration observed during the time period of 2005-2008 was 26.78 µg/m 3 .

Table 2 presents the distribution of desert dust exposure estimates during pregnancy for the entire cohort and according to the outcome (term / preterm birth). Mean PM 10 concentrations ranged from 13.17 to 34.92 µg/m 3 , whereas the proportion of intense dust events ranged from 0.00 to 19.41%.

Figure 1 describes the cumulative distribution of both exposure estimates according to term/preterm births. Distributions are shifted to the right for preterm births, above 28 µg PM 10 /m 3 (Figure 1a) and 7% of intense dust episodes (Figure 1b).

Associations between preterm birth and desert dust exposure

Table 3 presents the crude and adjusted associations between desert exposure estimates and preterm births. Except for the association between spontaneous preterm births and mean PM 10 concentrations, the crude and adjusted ORs were very similar.

When considering all preterm births, increased adjusted ORs were found for both the mean PM 10 concentrations and the proportion of intense dust events (OR 1.40, 95% CI 1.08 to 1.81, p=0.01 and OR 1.54, 95% CI 1.21 to 1.98, p=0.0006 respectively). To determine whether these associations vary with distance to the monitoring station (potentially reflecting some exposure misclassification), we introduced an interaction term between distance (split at the median) and exposure estimates into the regression models. No significant interaction effects were observed (p=0.75 and p=0.52, for the mean PM 10 concentration and the proportion of intense dust episodes, respectively).

Restriction to spontaneous preterm births produced similar adjusted ORs but with wider 95% CIs (OR 1.39, 95% CI 0.93 to 2.06, p=0.10 and OR 1.63, 95% CI 1.13 to 2.35, p=0.009 for mean PM 10 concentrations and the proportion of intense dust events, respectively). Regarding induced preterm births, lower ORs were found, but they were still greater than one (with CIs including unity). * For one woman, the type of preterm birth was unknown. † The covariates for which we adjusted were place of inclusion, maternal age, marital status, years of education, body mass index, parity, prior preterm birth, prior miscarriage, prior induced abortions, lupus, asthma, chronic hypertension, gestational weight gain, gestational hypertension, gestational diabetes, urinary tract infection, sex of the newborn. ‡ The odds ratios were calculated per standard deviation change.

DISCUSSION

This research adds new evidence to the health effects of Saharan dust episodes by showing an association between maternal pregnancy exposure (as assessed by the mean PM 10 concentration and the proportion of intense dust events) and preterm birth.

The present study has many strengths, including the study setting, the longitudinal design, and extensive information on covariates. Guadeloupe can be considered a natural laboratory for disentangling the influence of desert dust natural pollution and anthropogenic pollution because according to Euphrasie-Clotilde et al., [START_REF] Euphrasie-Clotilde | The relationship between coastal West African dust level and Caribbean island dust[END_REF] on average, the concentration of anthropogenic pollution does not exceed 20 µg PM 10 /m 3 (despite some possible traffic-related pollution hot-spots). This study took advantage of the TIMOUN mother-child cohort, an archipelago-wide cohort, that collected information on potential maternal socioeconomic, behavioral, and medical confounders at an early stage of pregnancy and therefore before outcomes occurred. To minimize residual confounding, we deliberately adjusted for numerous risk factors highlighted previously by Rouget et al. [START_REF] Rouget | Medical and sociodemographic risk factors for preterm birth in a French Caribbean population of African descent[END_REF] However, this study has some limitations. Exposure to Saharan dust was assessed through one single background air quality monitoring station (situated in the center of Guadeloupe) regardless of the subject's residential location. These monitor-based estimates may have introduced some nondifferential measurement error for pregnant women living far from the monitoring station due to the presumed spatial heterogeneity of air pollutants relating to topography and local micro sources, although the absence of interaction with distance to the monitoring station is reassuring. Alternative exposure estimates could be derived from desert dust modeling (such as the Navy Aerosol Analysis and Prediction System -NAAPS -model) or from satellite imagery (such as the Moderate Resolution

Imaging Spectroradiometer -MODIS -aerosol optical thickness product). However, considering the lower accuracy of these proxies and the need for retrospective exposure assessment (time period of 2004-2008), we preferred to rely on a well-established ground-based instrument. The population size and the number of events may appear modest but do not limit the statistical power of the analysis to identify significant findings.

In the Guadeloupe archipelago, the average PM 10 concentration observed during the time period the crude ORs, the potential for confounding is small. In other words, these results suggest that desert dust episodes influence preterm birth above and beyond the wide range of established risk factors. That higher ORs were found for spontaneous preterm births than medically induced preterm births is not unexpected. Although some underlying disorders are common to both outcomes (e.g., preeclampsia), other specific conditions that cause induced preterm births are not assumed to be influenced by prenatal air pollution exposure, although a recent case-crossover study suggested that a short-term increase in PM 2.5 concentrations is a potential trigger of placental abruption. [START_REF] Ananth | Exposures to air pollution and risk of acuteonset placental abruption: a case-crossover study[END_REF] The underlying biological pathways of the effect of particulate pollution on pregnancy are complex and multiple, given the chemical and physical heterogeneity of air pollutants and the various stages of fetal vulnerability during pregnancy. Researchers have, however, suggested the alteration of maternal-placental exchanges, endocrine disruption, oxidative pathways and alteration of maternal host-defense mechanisms as possible mechanisms. [START_REF] Slama | Meeting report: atmospheric pollution and human reproduction[END_REF] However, compared to anthropogenic particle pollution, coarse particles coming from desert sources could bring their own toxicity because desert dust clouds carry heavy metals and biological components (such as bacteria, viruses, fungi and endotoxins) to the downwind countries. [START_REF] Morman | The role of airborne mineral dusts in human disease[END_REF] At variance with our results, Dadvand et al. [START_REF] Dadvand | Saharan dust episodes and pregnancy[END_REF] observed a small but statistically significant increase in gestational age at delivery in association with the number of episodic days during the third trimester and entire pregnancy. However, their study was conducted in another geographical region, with differences in climate pattern, distance from dust sources and morphology or chemical composition of the particles.

In conclusion, the current study suggests that exposure to desert dust episodes is associated with preterm birth, while adjusting for sociodemographic and medical risk factors. Considering the personal and social burden of this adverse pregnancy outcome, we believe that this finding is of importance for both health care workers and policy makers to provide necessary preventive measures (such as communicating personalized information to pregnant women about a desert dust cloud moving to the coastline).

  Exposure data recorded at the urban background station located in Pointe-à-Pitre (central part of Guadeloupe, latitude 16°14'55" N, longitude 61°32'36" W) were provided by Gwad'Air (the official air quality monitoring agency for Guadeloupe), from 2005 to 2008. No other air quality monitoring station was operating in the Guadeloupe archipelago during this period of time. The median bird fly distance between the place of residence and the air quality monitoring station siting was 13.03 km (minimum 0.11 km, maximum 38.89 km).

  of 2005-2008 (26.78 µg/m 3 ) ranks high compared to those observed in the seventeenth largest French cities (mainland France) during the time period of 2007-2010 (range: 19.3 to 31.8 µg PM 10 /m 3 ). 18 Associations are consistent across exposure estimates when calculating ORs per standard deviation change suggesting that PM 10 concentrations recorded at the urban background monitoring station correctly reflect desert dust concentrations. Because the adjusted ORs do not contrast with

Figure 1

 1 Figure 1 Cumulative distribution of the mean PM 10 concentration (µg/m 3 ) (A) and the proportion of intense dust episodes (B) during pregnancy according to term (thick points) / preterm (thin points) births (TIMOUN cohort, Guadeloupe archipelago, French West Indies, 2005-2007).

Table 1

 1 Distribution of sociodemographic and lifestyle factors

	in the study population (n=909, TIMOUN cohort, Guadeloupe
	archipelago, French West Indies, 2005-2008)
		N (%)
	Maternal place of birth
	Guadeloupe or Martinique	795 (87.4)
	Other Caribbean islands	114 (12.6)
	Place of inclusion
	University hospital	605 (66.6)
	Local hospital	222 (24.4)
	Local antenatal care clinic	82 (9.0)
	Maternal age (years)
	<20	74 (8.1)
	20-34	535 (58.9)
	>34	300 (33.0)
	Marital status
	Single	229 (25.2)
	Living with partner	466 (51.4)
	Single living with family	189 (20.8)
	Missing	25 (2.6)
	Education (years)
	<5	62 (6.8)
	5-12	663 (73.0)
	>12	184 (20.2)
	Body mass index (kg/m²)
	<18.5	55 (6.1)
	18.5-25.0	455 (50.0)
	>25	379 (41.7)
	Missing	20 (2.2)
	Mother's employment during pregnancy
	Yes	378 (41.6)
	No	530 (58.3)
	Missing	1 (0.01)

Table 2

 2 Descriptive statistics of desert dust exposure estimates during pregnancy on the entire study population and according to the mode of onset of labor (TIMOUN cohort, Guadeloupe archipelago, French West Indies,2005-2008)

	N	Mean ± SD	Min	Max

Table 3

 3 Crude and adjusted Odds Ratios for the risk of preterm birth according to desert dust exposure estimates and mode of onset of labor (TIMOUN cohort, Guadeloupe archipelago, French West Indies,2005-2008)

		No. of	No. of			Crude		Adjusted	†
	Desert dust exposure estimates	preterm	term
		births *	births	OR	‡	95% CI	OR	‡	95% CI
	All births	142	767
	PM 10 concentrations (3.08 µg/m 3 )			1.36	1.07, 1.72	1.40	1.08, 1.81
	Proportion of intense desert dust episodes (4.06%)			1.38	1.11, 1.72	1.54	1.21, 1.98
	Spontaneous preterm and term births	67	767
	PM 10 concentrations (3.08 µg/m 3 )			1.44	1.01, 2.05	1.39	0.93, 2.06
	Proportion of intense desert dust episodes (4.06%)			1.53	1.11, 2.10	1.63	1.13, 2.35
	Induced preterm and term births	74	767
	PM 10 concentrations (3.08 µg/m 3 )			1.28	0.93, 1.76	1.30	0.91, 1.86
	Proportion of intense desert dust episodes (4.06%)			1.25	0.93, 1.68	1.35	0.96, 1.90

Abbreviations: CI, confidence interval; OR, odds ratio; PTB, preterm birth.

Acknowledgements

We thank Sylvaine Cordier for implementing the TIMOUN cohort. We are indebted to Léah Michineau, Christine Monfort, and Céline Garbin for valuable technical assistance. We thank our partners in the BrumiSaTerre research team (Stéphanie Guyomard, Antoine Talarmin, Séverine Ferdinand, Erika Upegui, Fabien Mercier, and Gülen Ayhan) for fruitful discussions.

Funding

This work was supported by the French National Research Program for Environmental and Occupational Health of ANSES (EST-2016/1/015).

What is already known on this subject

• Large amounts of mineral dust are transported from the Saharan-Sahel region to the Caribbean Sea, generating peak exposures to particulate matter ≤10 µm (PM 10 ).

• The influence of desert dust particulate pollution on pregnancy outcomes appears plausible.

What are the new findings

• This research adds new evidence to the health effects of Saharan dust episodes by showing an association between maternal pregnancy exposure (as assessed by the mean PM 10 concentration and the proportion of intense dust events) and preterm birth while adjusting for sociodemographic and medical risk factors.

How might this impact on policy or clinical practice in the foreseeable future?

• Considering the personal and social burden of this adverse pregnancy outcome, this finding is of importance for both health care workers and policy makers to provide necessary preventive measures (such as communicating personalized information to pregnant women about a desert dust cloud moving to the coastline).

Contributors JFV generated the idea for the paper and prepared an analytical plan. YM and JFV conducted all data analyses. JFV prepared the first draft on which all co-authors commented.

Competing interests None declared

Patient consent Written informed consent was obtained from all pregnant women included in the study.

Ethics approval

This study was approved by the Guadeloupean Ethics Committee for studies involving human subjects.