Dominique Hermier
email: dominique.hermier@agroparistech.fr

Annaïg Lan

Frédérique Tellier

Anne Blais

Marta Grauso Culetto

Véronique Mathé

Yannick Bellec

Lionel Gissot

Philippe Schmidely

Jean-Denis Faure

and metabolic effects of dietary camelina sphingolipids during the metabolic syndrome onset in mice

Keywords: sphingolipid, Camelina sativa, metabolic syndrome, digestibility, acyl chain

Sphingolipids appear as a promising class of components susceptible to prevent the onset of the metabolic syndrome (MetS). Gut availability and effects of Camelina sativa sphingolipids were investigated in a mouse model of dietary-induced metabolic syndrome. Seed meals from two Camelina sativa lines enriched respectively in C24-and C16-NH 2-glycosyl-inositolphosphoryl-ceramides (NH 2 GIPC) were used in hypercaloric diets. After 5 weeks on these two hypercaloric diets, two markers of the MetS were alleviated (adiposity and insulin resistance) as well as inflammation markers and colon barrier dysfunction. A more pronounced effect was observed with the C16-NH 2 GIPC-enriched HC diet, in particular for colon barrier function. Despite a lower digestibility, C16-NH 2 GIPC were more prevalent in the intestine wall. Sphingolipids provided as camelina meal can therefore counteract some deleterious effects of a hypercaloric diet in mice at the intestinal and systemic levels.

Interestingly, these beneficial effects seem partly dependent on sphingolipid acyl chain length.

INTRODUCTION

The metabolic syndrome (MetS) is a cluster of abnormalities including visceral obesity, dyslipidemia, hyperglycemia, and increased blood pressure, which results in a higher risk of cardiovascular diseases and type-2 diabetes [START_REF] Alberti | Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity[END_REF] .

Its prevalence is increasing worldwide and makes it a major public health challenge. This increase is associated with several features of lifestyle such as physical inactivity, psychosocial stress, environmental contaminants, and, as a major determinant, dietary habits [START_REF] Cornier | The metabolic syndrome[END_REF] . High caloric intake, typical of Western-type diet rich in saturated fatty acids (SFA) and sugars, is a major causative factor of the onset of the MetS, which is characterized by early events such insulin resistance (IR) and a low-grade inflammation (LGI) [START_REF] Araujo | Impact of high-fat diet on the intestinal microbiota and small intestinal physiology before and after the onset of obesity[END_REF][START_REF] Dandona | Macronutrient intake induces oxidative and inflammatory stress: potential relevance to atherosclerosis and insulin resistance[END_REF] . These alterations are known to originate, at least partly, from an allostatic stress due to the Western-type diet, which is characterized by microbial dysbiosis [START_REF] Tremaroli | Functional interactions between the gut microbiota and host metabolism[END_REF] , together with local inflammation [START_REF] Ji | Nutrient-induced inflammation in the intestine[END_REF] and physiological dysfunction of the intestinal wall [START_REF] Araujo | Impact of high-fat diet on the intestinal microbiota and small intestinal physiology before and after the onset of obesity[END_REF] . The resulting endotoxemia enhances the low-grade inflammation (LGI) at the systemic level, which, in turn, amplifies the initiation of the MetS [START_REF] Cani | Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat dietinduced obesity and diabetes in mice[END_REF][START_REF] Minihane | Low-grade inflammation, diet composition and health: current research evidence and its translation[END_REF] In addition to the rodent studies, both observation and intervention human studies confirmed the link between nutrients, gut dysfunction, LGI and initiation of the MetS, and in particular IR [START_REF] Minihane | Low-grade inflammation, diet composition and health: current research evidence and its translation[END_REF][START_REF] Nagpal | Gut microbiota in health and disease: an overview focused on metabolic inflammation[END_REF] . In consequence, new preventive and therapeutic strategies for MetS, including lifestylebased changes involving nutrition, must be established, tested and improved in specifically susceptible and vulnerable people, such as the at-risk, obese subjects. In these patients, the challenge is to prevent or delay the onset of IR and low-grade inflammation, and of cardiometabolic complications. Among the different possible solutions are diet changes and/or diet complementation.

As part of dietary candidates susceptible to prevent or alleviate the onset of the MetS, and particularly of IR, sphingolipids (SL) appear as a new and promising class of components. Indeed, body SL originate from endogenous synthesis, but may also be provided by the diet from animal or vegetal products. SL are composed of a sphingoid base (or long-chain base, LCB) acylated by a long or very long-chain FA and bearing a more or less complex polar head group [START_REF] Berkey | Sphingolipids and plant defense/disease: the "death" connection and beyond[END_REF] . They are essential components of cell membranes and are involved in cell signalling in both plants and animals. They are associated, together with animal cholesterol or plant phytosterols, into membrane micro-domains (caveolae and lipid rafts) [START_REF] Lipina | Sphingolipids: agents provocateurs in the pathogenesis of insulin resistance[END_REF] . These micro-domains are more ordered and less fluid than the rest of the plasma membrane, which favours docking, clustering and functionality of insulin receptor and other membrane receptors [START_REF] Lipina | Sphingolipids: agents provocateurs in the pathogenesis of insulin resistance[END_REF][START_REF] Langeveld | Glycosphingolipids and insulin resistance[END_REF] . Interestingly, the length of the SL acyl chain is an important structural feature modulating the properties of these micro-domains and subsequent translocation and downstream signalling of insulin receptor [START_REF] Park | Ablation of very long acyl chain sphingolipids causes hepatic insulin resistance in mice due to altered detergent-resistant membranes[END_REF] . A number of disparate, but convincing data, suggest that some plant SL (phytoSL) would improve some features of the MetS, such as dyslipidemia [START_REF] Duivenvoorden | Dietary sphingolipids lower plasma cholesterol and triacylglycerol and prevent liver steatosis in APOE*3Leiden mice[END_REF][START_REF] Snel | The effect of dietary phytosphingosine on cholesterol levels and insulin sensitivity in subjects with the metabolic syndrome[END_REF] , IR/glucose intolerance [START_REF] Snel | The effect of dietary phytosphingosine on cholesterol levels and insulin sensitivity in subjects with the metabolic syndrome[END_REF][START_REF] Murakami | Improved high-fat diet-induced glucose intolerance by an oral administration of phytosphingosine[END_REF][START_REF] Yunoki | Dietary sphingolipids ameliorate disorders of lipid metabolism in Zucker fatty rats[END_REF][START_REF] Zigmond | Beta-glycosphingolipids improve glucose intolerance and hepatic steatosis of the Cohen diabetic rat[END_REF] and non-alcoholic steatohepatitis [START_REF] Duivenvoorden | Dietary sphingolipids lower plasma cholesterol and triacylglycerol and prevent liver steatosis in APOE*3Leiden mice[END_REF][START_REF] Zigmond | Beta-glycosphingolipids improve glucose intolerance and hepatic steatosis of the Cohen diabetic rat[END_REF][START_REF] Lalazar | Oral Administration of beta-Glucosylceramide for the Treatment of Insulin Resistance and Nonalcoholic Steatohepatitis: Results of a Double-Blind, Placebo-Controlled Trial[END_REF][START_REF] Zigmond | beta-glycosphingolipids ameliorated non-alcoholic steatohepatitis in the Psammomys obesus model[END_REF] .

There is also some evidence that dietary SL from both animal and plant origin may exert anti-inflammatory effects at the extra-digestive level, including in the postprandial state [START_REF] Norris | Dietary sphingolipids: potential for management of dyslipidemia and nonalcoholic fatty liver disease[END_REF][START_REF] Zhang | Betaglucosylceramide ameliorates liver inflammation in murine autoimmune cholangitis[END_REF] . Other beneficial effects of phytoSL have been described at the intestine level, in mice models of chemically-induced colorectal cancer or inflammatory bowel diseases [START_REF] Arai | Effects of Dietary Plant-Origin Glucosylceramide on Bowel Inflammation in DSS-Treated Mice[END_REF][START_REF] Bryan | Sphingolipids as Mediators in the Crosstalk between Microbiota and Intestinal Cells: Implications for Inflammatory Bowel Disease[END_REF][START_REF] Camp | Therapeutic implications of bioactive sphingolipids: A focus on colorectal cancer[END_REF][START_REF] Duan | Oral glucosylceramide reduces 2,4dinitrofluorobenzene induced inflammatory response in mice by reducing TNF-alpha levels and leukocyte infiltration[END_REF][START_REF] Mazzei | Suppression of intestinal inflammation and inflammation-driven colon cancer in mice by dietary sphingomyelin: importance of peroxisome proliferator-activated receptor gamma expression[END_REF][START_REF] Yamashita | Effects of Dietary Plant-origin Glucosylceramide on Colon Cytokine Contents in DMH-treated Mice[END_REF] . In contrast, when it comes to nutritionally-induced rather than chemically-induced, allostatic intestinal stress, it remains unknown if dietary phytoSL may alleviate microbial dysbiosis and/of gut barrier function alterations, which could explain some beneficial systemic consequences.

The mechanisms by which phytoSL exert their beneficial effects are still not elucidated and since it is a large and diversified lipid family, the role of the different structural components (polar head, acyl chains, long chain base) remains to be deciphered. Indeed, plants have a large variety of SL with more than 300 molecular species, some of which being tissue specific (29) . They are characterized by specific polar heads like glycosyl-inositol-phosphorylceramides (GIPC) and LCB like phytosphingosine (PHS, t18:1). Seeds also accumulate specific amino-derivate GIPC (NH 2 GIPC) [START_REF] Tellier | Comparative plant sphingolipidomic reveals specific lipids in seeds and oil[END_REF] .

Taken together, available data suggest that plant-derived SL can have beneficial effects on the MetS onset. However, all previous studies were performed by using purified SL and, most often, plant SL were administered orally or intra-peritoneally, and not as diet ingredient. The objective of the present study was therefore to assess (i) whether plant SL provided as part of the diet (i.e. as a food ingredient in a food matrix) could counteract MetS onset and (ii) whether SL acyl chain length is important in modulating the physiological and metabolic impact of dietary phytoSL. Because bioavailability is an essential prerequisite to extra-digestive effects, digestibility of phytoSL with different acyl chain length had also to be determined. Plant SL originating from two isogenic camelina cultivars in which SL were differing only by their fatty acid (FA) chain length were provided as seed meal to mice fed for 5 weeks with high-fat/high sucrose diets. The first outcome was to assess the bioavailability of dietary phytoSL at the digestive level, by using plant sphingolipidomic profiling, and their possible impact on intestinal function. The second one was to evaluate if dietary phytoSL could slower the onset of the MetS, especially as regards dyslipidemia and IR. Finally, the hypothesis that acyl chain length may modulate digestibility and bioactivity of phytoSL was also tested for both the outcomes.

MATERIALS AND METHODS

Selection of C16-SL accumulating camelina line. Camelina sativa cultivar "Céline" was grown in glasshouse conditions as described previously [START_REF] Bellec | Evaluation of the potential for interspecific hybridization between Camelina sativa and related wild Brassicaceae in anticipation of field trials of GM camelina[END_REF] . To generate plants accumulating SL rich in C16 FA (C16-SL) , the coding sequence of the Arabidopsis ceramide synthase AtLOH2 (At3g19260) was PCR amplified using LOH2EcoRI-F and LOH2XhoI-R primers modified with EcoRI and XhoI restriction enzymes (LOH2EcoRI-F, GGAATTCATGGAATCGGTATCATCACG; LOH2XhoI-R, CCGCTCGAGCTAATCATCATCATCCTCTG) and cloned behind the soybean glycinin promoter into the pBinGlyRed2 vectors after removal of the Luciferase coding sequence using the EcoRI/XhoI enzymes. Plants were transformed by flower dip infiltration with Agrobacterium tumefasciens carrying pBinGlyRed2::AtLOH2 and T1 seeds were selected based on their red fluorescence [START_REF] Morineau | Selective gene dosage by CRISPR-Cas9 genome editing in hexaploid Camelina sativa[END_REF] . A total of 107 T2 progeny were produced and screened for their respective seed SL content by LC-MS/MS. Eight T3 progeny were eventually selected and amplified and rescreened for their seed SL content. A homozygous line named LOH2 Y with the highest C16-SL content was finally selected and amplified as T4 and T5 generations. Complete SL profile was carried out on T5 seeds, as described below.

A total of 1476 wild type camelina and 1593 LOH2 Y T4 plants were cultivated side by side in the glasshouse, resulting in the production of 6.1 kg of Céline seeds (of which SL-FA are mostly C24) and 5.4 kg of T5 LOH2 Y seeds (rich in C16-SL). Camelina seeds were crushed with an Oilprinz KK8 F universal (Kern Kraft, Reut, Germany) at 60 rpm and with opening of 8 mm to produce CamC24 and CamC16 meal and oil from Céline and LOH2 Y seeds respectively. Macronutriment composition of the camelina meal was determined as previously described [START_REF] De Andrade | Effect of duodenal infusion of trans10,cis12-CLA on milk performance and milk fatty acid profile in dairy goats fed high or low concentrate diet in combination with rolled canola seed[END_REF] and was (in weight %): 36.5/39.4 protein, 19.7/19.8 lipid, 29.4/28.8 fibre, 5.79/5.83 ashes and 5.70/5.81 humidity for the CamC24 and CamC16, respectively. Fibre typology was the same in both meals (data not shown).

Animals and diets. Thirty-three 7-week old male C57BL/6JOlaHSD mice (Envigo, Gannat, France) were housed in individual cages with wired floor, at 22 ± 1°C under 12-h light/dark cycles (light on at 07:00 am) and acclimated to local conditions for 1 week. During the last 3 days of the week, 27 mice were progressively switched from the standard pelleted chow diet (SAFE A04, Augy, France) to the control hypercaloric (HC-Con) diet (see below). At 8 weeks of age, mice were divided into 4 groups and fed during 5 weeks with either the same chow diet as before (normocaloric diet, NC group, n=6) or one of the three hypercaloric (HC) diets, two providing one of the two camelina meals (HC-CamC24 and HC-CamC16, n=9) and the third one a control HC diet (HC-Con n=9) (Supplemental Table 1). Mice had free access to food and tap water. Individual body weight was recorded weekly. The study was performed according to the European directive for the use and care of laboratory animals (2010/63/UE), and received the agreement of the local animal ethics committee and of the ministerial committee for animal experimentation (Ref 20151I0317422745.v4-APAFIS#2701). HC diets were prepared freshly twice a week, stored at 4°C, and provided daily to mice as soft pellets. CamC24 and CamC16 meals from the commercial Céline cultivar and LOH2 Y line were incorporated into the HC diets (respectively HC-CamC24 and HC-CamC16). Camelina oil obtained from Céline seed crushing was kept at 4°C and incorporated into the HC-Con diet at the same level as in the two HC-CamC24 and HC-CamC16 diets. It contains as previously described mostly polyunsaturated FA (53%) and monounsaturated FA (29%), as previously described [START_REF] Morineau | Selective gene dosage by CRISPR-Cas9 genome editing in hexaploid Camelina sativa[END_REF] . SL analysis of this oil showed the absence of NH 2 GIPC, the major SL in both camelina meals. Sphingolipid were extracted from camelina meals, oils and diets, as described previously (29) . CamC24 and CamC16 meals contained the same levels (in nmol/g) of total phytoSL and NH 2 GIPC, which was the major SL in both meals (92%) (Supplemental Table 2). In contrast, C24-NH 2 GIPC accounted for 82% of total NH 2 GIPC in CamC24, whereas C16-NH 2 GIPC accounted for 90% of total NH 2 GIPC in CamC16. The amount of camelina meals to be incorporated into the HC-CamC24 and HC-CamC16 diets (107g/kg) was calculated according to the SL content of the camelina meals, the mice food intake (about 4 g/d for a HC diet) and the amount of purified plant SL given to rodents in the literature (4-8 mg/d in mice, without indication of their body weight) [START_REF] Duivenvoorden | Dietary sphingolipids lower plasma cholesterol and triacylglycerol and prevent liver steatosis in APOE*3Leiden mice[END_REF][START_REF] Murakami | Improved high-fat diet-induced glucose intolerance by an oral administration of phytosphingosine[END_REF] . SL profile of the HC-CamC24 and HC-CamC16 diets reflected that of the respective meals, with NH 2 GIPC being the major SL with mostly by 24 carbons FA in CamC24 meal, and by 16 carbons FA in CamC16 meal (Table 1). As expected, HC-Con diet contained very little NH 2 GIPC, whereas the NC diet was surprisingly rich in SL, and especially in NH 2 GIPC, probably as a consequence of the high level of plant-based materials (e.g. soya bean) in the commercial chow.

In vivo measurements and sample collection. Food intake during 48 h was monitored after 6 d and 4 wk on the experimental diets. Feces were collected and weighed for further lipidomics at the end of the 48 h. Part of the feces was freeze-dried before assaying the dry matter content [START_REF] De Andrade | Effect of duodenal infusion of trans10,cis12-CLA on milk performance and milk fatty acid profile in dairy goats fed high or low concentrate diet in combination with rolled canola seed[END_REF] . Apparent digestibility of the dry matter was calculated as follows:

(intake-feces content)/intake*100. SL apparent digestibility was calculated by applying the same formula to SL content measured in food and feces.

The body composition (fat mass and lean mass) was measured in vivo after 3 weeks on the experimental diets by dual energy X-ray absorptiometry (DEXA), using a Lunar PIXImus densitometer (GE Medical Systems, Buc, France). The stability of the device was controlled by a measurement of a phantom before each session. The mice were anesthetized by isoflurane inhalation during the measurement. Analysis of the images was performed with the software provided with the device (Lunar PIXImus v2.10, GE Medical Systems, Buc, France), using auto-thresholding.

An oral glucose tolerance test (OGTT) was performed after 3 weeks on the experimental diets. Six hours fasted mice were weighed and a blood sample (~25 μL) was taken from the tail vein ~15 min before (t0) gavage, then after 15, 30, 60, and 120 min following orally administration of a glucose solution (2 g/kg). Blood glucose concentration was immediately assayed using a standard glucometer (Onetouch, LifeScan, Boulogne-Billancourt, France).

Blood was centrifuged (10 min, 1600 g, 4°C) and plasma stored at -20°C for subsequent assay of insulin.

After 5 weeks on the experimental diets, mice were fasted for 6 h. After a blood sample has been taken from the tail vein for glucose determination, mice were deeply anesthetized by isoflurane inhalation (IsoFlo, Axience, Pantin, France). Blood was taken by cardiac puncture before mice were euthanized by exsanguination. The abdominal cavity was then opened and the following parts were removed and weighed: liver, epididymal adipose tissue (EpAT), kidneys, spleen, small intestine, caecum and colon. The luminal contents of the caecum and the colon were removed and weighed. Jejunum, colon and EpAT samples were snap-frozen in liquid nitrogen and stored at -80°C. Blood samples were centrifuged as described above for plasma extraction and storage.

Ex vivo intestinal measurements.

To assess potential changes in intestinal barrier function, electrophysiological measurements were performed as previously described [START_REF] Beaumont | Epithelial response to a high-protein diet in rat colon[END_REF] with the following modifications: each mouse proximal colon was opened along the mesenteric line and mounted on an EasyMount Ussing chamber insert (Physiologic Instrument Inc, San Diego, CA, USA) with an exposed area of 0.2 cm 2 . Mounted tissues were left to equilibrate their trans-epithelial potential (V t , mV) 45 min before clamping them to 0 mV to obtain the short-circuit current (I sc , μA/cm 2 ). The trans-epithelial electrical conductance (G t , mS/cm 2 ) was obtained using the Ohm law by the voltage deviation when the tissue was current pulsed with ± 5 μA.

Paracellular permeability to fluorescein isothiocyanate (FITC)-dextran 4000 (FD4, Sigma) was determined during a 100-min period in Ussing chambers. Soon after tissue mounting (t 0 ), FD4 was added to the chamber mucosal side at the final concentration of 0.250 mg/mL. Serosal samples were collected at t 0 and after 100 min. To test for tissue viability at the end of the experiment, carbachol (CCh, 10 -4 M) was applied at the serosal side verifying the activation of the calcium-dependent chloride secretion by the I sc increase.

Biochemical analyses and physiological measurements. Blood glucose concentration was

determined with an Accu-Chek® glucometer (Roche Diagnostics, Meylan, France). Plasma triglyceride concentrations were determined by colorimetric enzymatic methods using a commercial kit adapted for use in a 96-well microplate reader (Molecular Devices, Saint-Grégoire, France), and obtained from Randox (Roissy, France) [START_REF] Fossati | Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide[END_REF][START_REF] Takayama | A new enzymatic method for determination of serum choline-containing phospholipids[END_REF] . Plasma insulin was determined using enzyme-linked immuno-assay (Mouse insulin ELISA, Mercodia, Paris, France). Homeostasis model assessement of insulin resistance (HOMA-IR) index was calculated using the formula: [fasting insulin (pmol/L) x fasting glucose (mmol/L)]/22.5.

Sphingolipidomics. SL were extracted from feces, jejunum and colon as described previously (36) . A volume of 500 µL isopropanol:hexane:water (55:20:25, v/v) and 10 µL standard mix [0.01 nmol of C12-Cer (d18:1-h12:0), 0.1 nmol of C12-GluCer (d18:1-h12:0) and 0.2 nmol of ganglioside (GM1)] were added to material and ground using a Polytron homogenizer. The plunger was rinsed with 500 µL of extraction solvent and the sample incubated at 60°C for 15 min. After centrifugation at 4000 rpm for 5 min at room temperature, the supernatant was recovered and the pellet extracted once more with 1 mL of solvent. Supernatants were combined and dried with a speed Vac. Then the samples were incubated at 50°C for 1 h with 500 µL of 33% methylamine solution in ethanol-water (7:3) and dried under nitrogen and resuspended by sonication in 100 µL of tetrahydrofuran-methanol-water (2:1:2) containing 0.1% formic acid and filtrated prior to analysis by liquid chromatography coupled to mass spectrometry (UPLC-MS/MS). UPLC-MS/MS analyses were carried out on a Waters Acquity UPLC system coupled to a Waters Xevo tandem quadrupole mass spectrometer equipped with an ESI source (Waters, Milford, MA, USA). Chromatographic conditions, mass spectrometric parameters, and multiple reaction monitoring (MRM) methods were defined as described previously [START_REF] Tellier | Comparative plant sphingolipidomic reveals specific lipids in seeds and oil[END_REF] .

Gene expression.

Total RNA was extracted from EAT and colon samples using Trizol reagent (Invitrogen, Carlsbad, CA, USA). qRT-PCR was performed with mouse-specific primers (Eurogentec, Angers, France) to quantify mRNA level of the following genes : interleukin-6 (Il-6), interleukin-1β (Il-1β) interleukin-10 (Il-10) and tumor necrosis factor-α (Tnf-α) in both EAT and colon, plasminogen activator inhibitor-1 (Pai-1) and monocyte chemoattractant protein-1 (Mcp-1) in EAT, and claudin 1 (Cldn1) and zonula occludens (Tjp1) in the colon.

After cDNA synthesis from mRNA using High Capacity cDNA Reverse Transcription Kit (ThermoFisher-Life Technologies, Les Ulis, France), Real-Time PCR was performed subsequently on cDNA using the power SYBR Green PCR master mix and StepOne Real-Time PCR system (ThermoFisher-Life Technologies, Les Ulis, France). cDNA samples were assayed in triplicate and gene expression levels for each sample were normalized relative to hypoxanthine-guanine phosphoribosyltransferase gene (Hprt) with 2 -∆∆Ct calculation.

Statistical analyses. Data are reported as means ± standard deviations. They were analysed using the XLSTAT application (Addinsoft, Paris, France). Significance level was set at P<0.05.

The effects of energy level (HC vs NC) and of camelina meal (HC-Con vs HC-CamC16/C24) were tested using a 2-way ANOVA. When diet effect was significant, differences between means were tested for significance using the post-hoc Tukey procedure.

RESULTS.

Production of Camelina seeds with different sphingolipid contents. The high expression of the C16 specific Ceramide synthase LOH2 from Arabidopsis in Camelina sativa led to seeds with opposite sphingolipid profile (Supplemental Figure 1). Camelina seeds and meals (cake fraction from seeds) from commercial cultivar Céline (CamC24) presented characteristic sphingolipid profile with high levels of NH 2 GIPC almost exclusively with very long chain FA (C24). Conversely, the LOH2 expressing line LOH2 Y (CamC16) accumulated almost exclusively C16 NH 2 GIPC albeit the levels of total sphingolipids remained similar to CamC24. C16 GluCer was also increased and C24 GluCer strongly decreased in CamC16 (Supplemental Table 2). A similar trend was also observed for Cer and hCer but at a much lower scale. The most abundant LCB in seeds were t18:0 and t18:1 in both lines even if d18:1 and d18:0 were increased in LOH2 Y due to the accumulation of C16 NH 2 GIPC and GluCer .The accumulation of C16 SL had a weak impact on plant fitness with a slight delay of germination and slower early growth rate as well as a lower seed yield and weight for LOH2 Y (data not shown).

Body weight and associated parameters. During the 5 wk of MetS induction, all mice gained

weight, but to a different extent (Figure 1). After 5 wk on the experimental diets, mice on the HC-Con diet exhibited a higher weight gain (8.8±1.2g) than mice of the NC group, which gained 3.5±1.1 g only (Supplemental Table 3). In both HC-Cam groups, body weight gain, as well as body weight after 2, 3 and 5 weeks on their respective diets, did not differ significantly from the NC group, but were significantly lower than in the HC-Con group (Figure 1 and Supplemental Table 3). Food intake (as kcal/d) tended to parallel body weight, and was the highest in the HC-Con group after 6 d and 4 wk, whereas there was no difference between groups at both times when energy intake was adjusted for body weight (Supplemental Table 3). Apparent digestibility (% intake of dry matter that was not found in feces) was equivalent after 6 d or 4 wk on the same experimental diets. After 6 d, it was the lowest in the NC group, intermediary in the HC-Con group and the highest in the HC-CamC24 and HC-CamC16 groups, whereas after 4 wk, it remained the highest in the HC-CamC24 group only (Supplemental Table 3). Body composition. After 3 wk on the experimental diets, the lean mass was the same in all groups, whereas the fat mass (absolute and relative to body weight) was as expected higher in the three HC groups than in the NC group, without significant effect of the camelina meals (Table 2). After 5 wk, mice of the HC-Con group were significantly heavier than those of the NC group whereas mice of both HC-Cam groups showed intermediate body weight (Figure 1 and Table 2). Similarly, when compared to the NC group, adiposity (absolute and relative weight of the EAT) was globally increased in the three HC groups, but to a lower extent in the HC-Cam groups. While liver and small intestine weights were similar in all groups, the weights of the caecum and the colon, as well as of the caecum content, were significantly lower in the three HC groups.

Blood and tissue markers associated with the MetS. During an OGTT after 3 weeks on the experimental diets, glucose and insulin plasma concentration increased sharply during 15 min, then returned to fasting values after 120 min in all groups (Figure 2). When compared to the NC group, the HC-Con group exhibited higher fasting glucose and insulin concentration (t0) and AUC over 120 min, as well as higher HOMA-IR index (Table 3 and Figure 2). Besides, the increase in glucose concentration between t0 and t30 min, which takes into account the difference in fasting glycemia between groups ( glucose), was twofold higher in the HC-Con group than in the NC group. In both HC-Cam groups, fasting glucose concentration and AUC over 120 min were similar to those of the HC-Con group, and higher than in the NC-Con group, whereas insulin AUC and HOMA did not differ from the NC-Con group and were therefore lower than in the HC-group. Interestingly, insulin fasting plasma concentration in HC-CamC16 mice did not differ from the NC mice and was significantly lower than in the HC-Con mice, which was not the case in mice HC-CamC24 mice. In the two HC-Cam groups,  glucose between t30 and t0 of the OGTT was intermediary between NC-and HC-Con groups.

At the end of the experiment, after 5 weeks on the experimental diets, fasting glucose concentrations did not differ from those after 3 weeks, remaining higher in the three HC groups than in the NC group (Table 3). Fasting triglyceridemia was identical in NC-and HC-Con groups, but was surprisingly 2-fold lower in the 2 HC-Cam groups. Compared to the NC-Con group, fasting cholesterolemia was higher in all the HC groups but only significantly in both HC-Cam groups.

Transcript levels of a few inflammatory and vascular markers were higher in mice fed the HC diets: Mcp-1 and Pai-1 in the adipose tissue (P = 0.011 and 0.002, respectively) and Il-1 in the colon (P = 0.039). However, according to the post-hoc Tukey test, and as compared to the NC group, Mcp-1 and Il-1  s were significantly higher in the HC-Con group only, and Pai-1 in the HC-Cam groups only (Table 4). Other mRNA levels of inflammatory markers (Il-6, Il-1β, Il-10 and Tnf-α in the adipose tissue and Il-6, Il-10, Tnf-α in the colon) did not differ between groups, neither did those of the tight junction protein encoding genes Tjp1 and Cldn1 in the colon (data not shown).

Colon epithelial barrier and permeability. Measurement of electrical parameters in Ussing

chambers was performed on proximal colon (Table 5). There was no difference in the FD4 permeability within the diet groups, which indicated that paracellular pathway of the gut barrier function in the colon was not altered (data not shown). The colon transepithelial voltage (V t ), which is usually negative at the colonocyte luminal side, was more negative in the 3 HC groups than in the control NC group, this effect being significantly less pronounced in the HC-CamC16 group. Conversely, the short-circuit current (I sc ) was higher in the three HC groups, with no difference between them. Trans-epithelial conductance (G t ) in the three HC groups did not differ from that in the NC group, however the HC-CamC16 and the HC-Cam24 groups showed the highest and lowest values respectively.

SL intake, excretion and tissue profile. Apparent NH 2 GIPC digestibility (Table 6) was calculated after 4 wk of experimental diets, using the in vivo measurement of food intake and fecal excretion and the analysis of complete plant sphingolipidomic profiles of diets (Table 1) and feces (data not shown). Since NH 2 GIPC accounted for nearly 80% of total SL in HC-CamC16 and HC-Cam24 diets, intake and excretion were calculated in this fraction only.

Mean individual NH 2 GIPC dietary intake, expressed as absolute amounts, grossly reflected their dietary profile. It was very low in the HC-Con group, much higher in the NC group (over 40 fold increase), and even further increased in both HC-Cam groups by over 80 fold, with no significant difference between the two groups. In contrast, NH 2 GIPC excretion differed markedly between the HC-Cam groups. Excretion rate of NH 2 GIPC in the HC-CamC16 group was 2.5-fold higher than that in the HC-CamC24 group. Consequentely, the apparent digestibility of dietary NH 2 GIPC was significantly lower (80%) in the HC-CamC16 than in the three other groups (87-94%).

Concentration of the most abundant plant SL (NH 2 GIPC and GluCer with C16 and C24 FA)

was determined in the jejunum and colon walls (Table 7). Jejunum and colon of the NC group contained similar amount of C24-NH 2 GIPC and C24-GluCer, while the latter was a minor component of the diet. Jejunum and colon of the HC-Con mice were nearly devoid of phytoSL, as expected from their low level in the HC-Con diet. On the contrary, plant NH 2 GIPC and GluCer were found in the intestine on mice fed with the camelina meals. As expected C24-SL were the most prominent SL in the HC-CamC24 group, while C16-SL was the most abundant in the HC-CamC16 group, with NH 2 GIPC being the most abundant SL in both groups. Tissue content of the various SL species was lower in the colon than in the jejunum, however small levels of plant NH 2 GIPC were still present in the colon. Finally, the total concentration of SL (GluCer + NH 2 GIPC) in the intestine of HC-CamC16 mice was approximately 2-fold higher in the jejunum (12.5 nmol/g) and in 5-fold higher in the colon (2.14 nmol/g) than in HC-CamC24 mice (6.40 and 0.40 nmol/g in the jejunum and colon, respectively). Interestingly, while C24-GluCer ratio between colon and jejunum was similar in the two HC-Cam groups (0.38 vs 0.41), this ratio was much lower in the HC-Cam24 group than in the HC-Cam16 group for C24-NH 2 GIPC (0.05 vs 0.26) and even lower in the HC-Cam16 group for C16-NH 2 GIPC (0.05 vs 0.16).

DISCUSSION.

Digestive bioavailability of camelina SL. In most in vivo studies aiming to highlight the mechanisms involved in potentially beneficial effects of plant SL at the systemic level, a dramatic gap remains concerning the bioavailability of plant SL when provided in the diet.

Both animal and plant SL are hydrolysed into LCB, FA, and the polar head group in the small intestine, then eventually absorbed, reesterified, and secreted into the lymph [START_REF] Fujii | Selective Absorption of Dietary Sphingoid Bases from the Intestine via Efflux by P-Glycoprotein in Rats[END_REF][START_REF] Ishikawa | Dietary glucosylceramide is absorbed into the lymph and increases levels of epidermal sphingolipids[END_REF][START_REF] Sugawara | Digestion of maize sphingolipids in rats and uptake of sphingadienine by Caco-2 cells[END_REF][START_REF] Sugawara | Intestinal absorption of dietary maize glucosylceramide in lymphatic duct cannulated rats[END_REF] . As regards phytoSL, it is needed to document to what extent they are digested then absorbed, in which form they are transported and made available to various tissues, and which molecular species are stored in various body compartments.

To our knowledge, the present study quantifies, for the first time, intestinal digestibility of a major phytoSL (NH 2 GIPC, accounting for nearly 80% of total SL in the HC-Cam diets)

when provided as a dietary component. Camelina NH 2 GIPC in the two HC-Cam diets exhibited the same LCB profile, and only differed in their FA length (mainly C24 in the CamC24 meal and C16 in the CamC16 meal). Although apparent digestibility of dietary NH 2 GIPC was high, it was lower in the HC-CamC16 group, which could be accounted for by a higher fecal excretion. However, the higher NH 2 GIPC content in the gut wall of the HC-Camc16 fed group, suggests some metabolic specificity during reesterification and excretion into the lymph(Table 7). Besides, beyond this demonstration of the NH 2 GIPC availability at the digestive level, NH 2 GIPC-containing SL, or their metabolites, have to be sought in other body compartments, this lack of information being a limitation of the present study.

Effects of the camelina meal on the early markers of the MetS. Plant SL have been shown

to exert beneficial effects on some risk factors associated with the MetS, such as altered glucose homeostasis, abnormal lipid profile, and low-grade chronic inflammation. In the present study, we used our mouse model of the early steps of the MetS to explore the effects of dietary plant SL provided by camelina meal in a hypercaloric diet [START_REF] Hermier | NO synthesis from arginine is favored by alpha-linolenic acid in mice fed a high-fat diet[END_REF] . As expected from this model, after 5 wk on a hypercaloric diet rich in saturated FA and sucrose (HC-Con group), mice exhibited (as compared to those on a normocaloric diet, NC-group) an increase in body weight and adiposity (Table 2 and Figure 1), and in fasting glycemia (Table 4). After only 3 wk, these alterations were already detectable and were accompanied by higher values of insulinemia and HOMA-IR index (Table 3), and of glucose and insulin postprandial AUC (Figure 2). These findings are consistent with data obtained in mice fed a HC diet rich in saturated FA [START_REF] Lam | Effects of dietary fat profile on gut permeability and microbiota and their relationships with metabolic changes in mice[END_REF] .

When SL was provided as camelina meal in the HC diet (HC-CamC24 and -C16 groups), body weight gain and adiposity were reduced, but not the lean mass (Figure 1 and Table 2).

This specific effect of camelina meals on adiposity was not accounted for by a lower food intake, since it was the same in all groups after 6 d and 4 wk when normalized for body weight. Whether this effect is due to the presence of SL in the diet is questionable, since no significant effect of phytoSL was reported for body weight, fat mass or body mass index in previous studies (14-17, 43, 44) . In our study, the presence of other components of the camelina meal, such as fibers, may be responsible for a lower nutrient absorption, and subsequent bioavailability, lowering body storage of lipids, either exogenous (dietary) or endogenous (de novo synthesised). However, a specific effect of phytoSL may not be precluded, since a very recent study showed that mice fed a high-fat diet supplemented with milk polar lipids containing 25% SL gained less weight than mice on the high-fat diet alone [START_REF] Milard | Milk Polar Lipids in a High-Fat Diet Can Prevent Body Weight Gain: Modulated Abundance of Gut Bacteria in Relation with Fecal Loss of Specific Fatty Acids[END_REF] .

In parallel, in mice fed by either camelina meal, fasting insulinemia and HOMA-IR index were normalized, as well as insulinemia AUC during an OGTT, and triglyceridemia decreased (Table 3 and Figure 2). These findings are grossly consistent with previous studies using purified SL or LCB in rodent mice models exhibiting features of the MetS or in human subjects with a MetS. Indeed, various SL or LCB decreased triglyceridemia in mice or sand rats on a hypercaloric diet [START_REF] Duivenvoorden | Dietary sphingolipids lower plasma cholesterol and triacylglycerol and prevent liver steatosis in APOE*3Leiden mice[END_REF][START_REF] Zigmond | beta-glycosphingolipids ameliorated non-alcoholic steatohepatitis in the Psammomys obesus model[END_REF] whereas phytosphingosine decreased glycemia in subjects with a MetS [START_REF] Snel | The effect of dietary phytosphingosine on cholesterol levels and insulin sensitivity in subjects with the metabolic syndrome[END_REF] and improved glucose homeostasis in human subjects with a MetS and various rodent models, [START_REF] Snel | The effect of dietary phytosphingosine on cholesterol levels and insulin sensitivity in subjects with the metabolic syndrome[END_REF][START_REF] Murakami | Improved high-fat diet-induced glucose intolerance by an oral administration of phytosphingosine[END_REF][START_REF] Yunoki | Dietary sphingolipids ameliorate disorders of lipid metabolism in Zucker fatty rats[END_REF][START_REF] Zigmond | Beta-glycosphingolipids improve glucose intolerance and hepatic steatosis of the Cohen diabetic rat[END_REF][START_REF] Lalazar | Oral Administration of beta-Glucosylceramide for the Treatment of Insulin Resistance and Nonalcoholic Steatohepatitis: Results of a Double-Blind, Placebo-Controlled Trial[END_REF] . In contrast, the present study showed no effect on cholesterolemia, which is inconsistent with previous studies that repeatedly reported a decrease in plasma total, LDL, or non-HDL cholesterol in various rodent models and in subjects with a MetS [START_REF] Duivenvoorden | Dietary sphingolipids lower plasma cholesterol and triacylglycerol and prevent liver steatosis in APOE*3Leiden mice[END_REF][START_REF] Snel | The effect of dietary phytosphingosine on cholesterol levels and insulin sensitivity in subjects with the metabolic syndrome[END_REF][START_REF] Yunoki | Dietary sphingolipids ameliorate disorders of lipid metabolism in Zucker fatty rats[END_REF][START_REF] Zigmond | beta-glycosphingolipids ameliorated non-alcoholic steatohepatitis in the Psammomys obesus model[END_REF] . One originality of our short-term study is the demonstration that, when focusing on the onset of the MetS, an early alteration of glucose homeostasis, as demonstrated by insulin resistance occurrence both at the fasting state and during an OGTT, may be counteracted by a phytoSL-rich matrix after only 3 weeks on the experimental diets.

The mechanisms by which dietary SL may exert systemic effects remain largely speculative. First, in response to various SL or LCB, a decrease in the postprandial absorption of dietary triglycerides and cholesterol has been suggested, and would explain the downstream effects on lipid metabolism at the hepatic or plasma level [START_REF] Duivenvoorden | Dietary sphingolipids lower plasma cholesterol and triacylglycerol and prevent liver steatosis in APOE*3Leiden mice[END_REF][START_REF] Chung | Dietary sphingomyelin lowers hepatic lipid levels and inhibits intestinal cholesterol absorption in high-fat-fed mice[END_REF] . Second, gene regulation by SL or LCB, including activation of various PPARs, and resulting in beneficial effects on insulin sensitivity, glucose tolerance and lipid metabolism have been also demonstrated [START_REF] Yunoki | Dietary sphingolipids ameliorate disorders of lipid metabolism in Zucker fatty rats[END_REF][START_REF] Zigmond | beta-glycosphingolipids ameliorated non-alcoholic steatohepatitis in the Psammomys obesus model[END_REF][START_REF] Chung | Dietary sphingomyelin lowers hepatic lipid levels and inhibits intestinal cholesterol absorption in high-fat-fed mice[END_REF] . Finally, it has been recently shown that animal SL may affect gut microbiota in mice in a way that would limit some detrimental effects of HF diets [START_REF] Milard | Milk Polar Lipids in a High-Fat Diet Can Prevent Body Weight Gain: Modulated Abundance of Gut Bacteria in Relation with Fecal Loss of Specific Fatty Acids[END_REF][START_REF] Norris | Milk sphingomyelin improves lipid metabolism and alters gut microbiota in high fat diet-fed mice[END_REF] . All these mechanisms, which remain to be investigated in our model, would contribute to lower adiposity and slower insulin resistance in mice fed the HC-Cam diets (Table 2).

In response to a diet rich in saturated FA and sucrose, the increased adipose tissue often features an inflammatory phenotype [START_REF] Dandona | Macronutrient intake induces oxidative and inflammatory stress: potential relevance to atherosclerosis and insulin resistance[END_REF] . In the present study that targeted the initial steps of the MetS, there was no evidence that gene expression of pro-inflammatory cytokines was enhanced in the adipose tissue, despite an increased adiposity, and providing SL-rich camelina meal had no significant effect. Gene expression of two others inflammatory markers, Mcp-1 and Pai-1, was enhanced in response to the HC diet (HC-Con group).

Attenuation of

Mcp-1 mRNA level may be related to the lower adiposity of mice on both HC-Cam diets, whereas the reason why the HC-Cam diets aggravated the mRNA level of Pai-1, remains to be investigated.

To our knowledge, the present study, provides the first evidence that a mixture of natural plant SL, essentially in the form of NH 2 GIPC, and provided in their original matrix, exerted some beneficial effects during the MetS onset. However, the role of other camelina meal components, such as phospholipids, polyphenols or specific fibers cannot be excluded [START_REF] Chaturvedi | Camelina sativa: An Emerging Biofuel Crop[END_REF][START_REF] Smit | Effects of feeding camelina cake to weaned pigs on safety, growth performance, and fatty acid composition of pork[END_REF] .

Effects of the camelina meal on the intestine wall. When compared to the NC group, the HC-Con diet enhanced a few inflammation markers in the colon and the adipose tissue (Table 4). In contrast, in mice fed the camelina meals, mRNA levels of Mcp-1 in the adipose tissue and of Il-1β in the colon did not differ from those in the NC group (Table 4). The role of SL in inflammatory processes is still in debate, but a few studies support the hypothesis that dietary phytoSL (as maize GluCer) or animal SL (as sphingomyelin) alleviate gut inflammation in rodent models [START_REF] Arai | Effects of Dietary Plant-Origin Glucosylceramide on Bowel Inflammation in DSS-Treated Mice[END_REF][START_REF] Bryan | Sphingolipids as Mediators in the Crosstalk between Microbiota and Intestinal Cells: Implications for Inflammatory Bowel Disease[END_REF][START_REF] Camp | Therapeutic implications of bioactive sphingolipids: A focus on colorectal cancer[END_REF][START_REF] Duan | Oral glucosylceramide reduces 2,4dinitrofluorobenzene induced inflammatory response in mice by reducing TNF-alpha levels and leukocyte infiltration[END_REF][START_REF] Mazzei | Suppression of intestinal inflammation and inflammation-driven colon cancer in mice by dietary sphingomyelin: importance of peroxisome proliferator-activated receptor gamma expression[END_REF][START_REF] Yamashita | Effects of Dietary Plant-origin Glucosylceramide on Colon Cytokine Contents in DMH-treated Mice[END_REF] . How plant SL may improve intestinal inflammation remains nonetheless highly speculative. A study using Caco-2 cells suggested that SL may interact with epithelial cells without being absorbed. In these in vitro conditions, plant GluCer remained at the cell surface, but decreased LPS-induced production of inflammatory cytokines, maybe by affecting the interactions between LPS and its TLR at the cell surface [START_REF] Yamashita | Effects of Dietary Plant-origin Glucosylceramide on Colon Cytokine Contents in DMH-treated Mice[END_REF] .

Similar interactions with non-digested plant SL or their metabolites may contribute in vivo to modulate the inflammatory status of the intestine wall. The intestinal microbiota is likely to contribute to these interactions, since it is able to metabolize animal SL, which would contribute to regulate cytokine production [START_REF] Bryan | Sphingolipids as Mediators in the Crosstalk between Microbiota and Intestinal Cells: Implications for Inflammatory Bowel Disease[END_REF][START_REF] Norris | Dietary and Endogenous Sphingolipid Metabolism in Chronic Inflammation[END_REF] .

Interestingly, epithelial integrity of the colon was specifically modified by the HC-CamC16 diet, as demonstrated by its effects on transepithelial voltage (V t ) and epithelial conductance (G t ) (Table 5). V t describes the unidirectional transport of sodium from the luminal to the serosal side of the epithelium. This potential difference is usually negative at the colonocyte luminal side, and was even more negative with HC diets. This effect of HC-diets reflects an increase in the driving force of the colonocytes to allow the sodium, chloride and water absorption from the colon lumen, and therefore a loss of permeability. This decrease in V t may be related to the high fat content of the diet, known to alter electrical parameters of transcellular barrier function [START_REF] Lam | Effects of dietary fat profile on gut permeability and microbiota and their relationships with metabolic changes in mice[END_REF][START_REF] Muller | Gut barrier impairment by high-fat diet in mice depends on housing conditions[END_REF][START_REF] Tomas | High-fat diet modifies the PPAR-gamma pathway leading to disruption of microbial and physiological ecosystem in murine small intestine[END_REF] . This damage was attenuated in mice given the diet containing camelina SL rich in C16 FA (HC-CamC16). V t increase in HC-Camc16 mice was also associated with higher epithelial conductance (G t ) which was not due to a loss of epithelial permeability, at least via the paracellular pathway, because it was not accompanied by an increase in the apical-to-basal flux of the paracellular marker FD4 in any group. The absence of dietary effects on the gene expression of two tight junction proteins, claudin 1 (Cldn1) and zonula occludens (Tjp1), confirms that the paracellular permeability was not affected by any of the HC diets. Rather, we suggest that, in the colon of the HC-CamC16 mice, this increased G t may reflect a compensatory mechanism that counteracts the increase of actively transported ions (as evidenced by the higher short-circuit current -I c -in all HC groups) by increasing passive movement of ions. This would explain the lower value of V t in the HC- In conclusion, phytoSL provided in a plant matrix delays the onset of the MetS, by reducing adiposity and insulin resistance. Involved mechanisms at the intestinal and systemic levels remain mostly speculative at this initial stage, but it may be hypothesized that some of the effects observed, including on the adipose tissue and blood parameters, results from an impact of phytoSL or their metabolites at the intestinal level. Moreover, some of these effects are modulated by the FA length of the SL, since they are sometimes more pronounced in presence of C16-NH 2 GIPC. More researchs are required to uncover the importance of FA length in the interactions between phytoSL, the intestinal wall, and systemic effects. In this context, interactions of phytoSL and their metabolites with the microbiota may be a promising avenue of investigation. Body weight (g) was measured longitudinally in the fed state for 37d. At d0, after they were acclimated to local conditions for 1 week, mice that have been given progressively the HC-Con diet during this 1 st week, were switched to the HC-Con, HC-CamC24 and HC-CamC16 according to their experimental group.

Values are means ± standard deviations for 6 and 9 mice in the NC and the three HC-groups, respectively.

NC, normo-caloric; HC, hyper-caloric; Con, control; CamC24, camelina meal C24 (from the commercial Céline cultivar rich in C24 fatty acids); CamC16, camelina meal C16 (from the experimental LOH2 cultivar rich in C16 fatty acids).

  CamC16 group. Nevertheless, further investigations would be needed to determine the precise mechanisms involved in the action of camelina SL (native or digested products) on activity and/or expression of colon transporters and exchangers that ensured colon barrier integrity and flux. Yet, it can be concluded from the present study than differences in the FA profile of dietary plant SL, and particularly the enrichment of C16 FA in the experimental LOH2 Y line (to the detriment of C24 FA in the commercial Céline cultivar) modulate the deleterious effects of a high-fat diet on colon epithelial barrier function, and more specifically on transepithelial permeability.

Figure 1 .Figure 2 .

 12 Figure 1. Body weight curve and body weight gain upon 5 week feeding with the

Figure 1 .

 1 Figure 1. Body weight curve.

Figure 2 .

 2 Figure 2. Plasma kinetics of glucose and insulin during an OGTT after 3 weeks on the

Table 1 . SL composition of the experimental diets (nmol/g).

 1 dissection and sampling. They thank Ophélie Dhumez (UMR Modélisation systémique Appliquée aux Ruminants) for the determination of nutrient composition of the camelina meals.

	NC, normo-caloric; HC, hyper-caloric; Con, control; CamC24, camelina meal C24 (from the
	commercial Céline cultivar rich in C24 fatty acids); CamC16, camelina meal C16 (from the
	experimental LOH2 cultivar rich in C16 fatty acids); hCer, hydroxyceramide; GluCer, glucosyl-
	ceramide; NH 2 GIPC, aminoglycosyl-inositol-phosphoryl-ceramide.

Table 2 . Body composition after 3 and 5 weeks on the experimental diets

 2

	Values are means ± standard deviations for 6 and 9 mice in the NC and the three HC-groups,
	respectively. Mean values within a row, not sharing a same superscript letter, were
	significantly different at P<0.05.
	NC, normo-caloric; HC, hyper-caloric; Con, control; CamC24, camelina meal C24 (from the
	commercial Céline cultivar rich in C24 fatty acids); CamC16, camelina meal C16 (from the
	experimental LOH2 cultivar rich in C16 fatty acids); EAT, epididymal adipose tissue.

Table 3 . Blood markers associated with a MetS after 3 weeks (during an OGTT) and 5 weeks on the experimental diets.

 3

	Values are means ± standard deviations for 6 and 9 mice in the NC and the three HC-groups,
	respectively. Mean values within a row, not sharing a same superscript letter, were
	significantly different at P<0.05.
	NC, normo-caloric; HC, hyper-caloric; Con, control; CamC24, camelina meal C24 (from the
	commercial Céline cultivar rich in C24 fatty acids); CamC16, camelina meal C16 (from the
	experimental LOH2 cultivar rich in C16 fatty acids).
	HOMA-IR: homeostasis model assessment of insulin resistance

Table 4 . Relative gene expression level of some markers of low-grade inflammation.

 4 Values are means ± standard deviations for 6 and 8-9 mice in the NC and the three HCgroups, respectively. They were calculated according to the 2 -ΔΔCt , where ΔCt = (Ct target gene -Ct HPRT) normalized to the NC group. Mean values within a row, not sharing a same superscript letter, were significantly different at P<0.05. NC, normo-caloric; HC, hyper-caloric; Con, control; CamC24, camelina meal C24 (from the commercial Céline cultivar rich in C24 fatty acids); CamC16, camelina meal C16 (from the experimental LOH2 cultivar rich in C16 fatty acids).

		NC	HC-Con	HC-CamC24 HC-CamC16	P ANOVA
	EAT
	Mcp-1	1.19±0.72 a 3.55±2.07 b 2.79±1.10 ab	2.08±0.77 ab	0.0168
	Pai-1	1.24±0.81 a 2.67±1.02 ab 4.12±1.64 b	3.17±1.28 b	0.0021
	Colon
	Il-1β	1.03±0.25 a 2.03±1.02 b 1.60±0.39 ab	1.58±0.44 ab	0.0707

Table 5 . Electrical measurement of colon epithelium parameters in Ussing chambers.

 5 Measures were acquired after 45-minutes epithelium equilibration in recording buffer. V t , trans-epithelial voltage; I sc , short-circuit current; G t , trans-epithelial electrical conductance.Values are means ± standard deviations for 3-4 mice in each group.Mean values within a row, not sharing a same superscript letter, were significantly different at P<0.05. NC, normo-caloric; HC, hyper-caloric; Con, control; CamC24, camelina meal C24 (from the commercial Céline cultivar rich in C24 fatty acids); CamC16, camelina meal C16 (from the experimental LOH2 cultivar rich in C16 fatty acids).

		NC	HC-Con	HC-CamC24 HC-CamC16 P ANOVA
	V t (mV)	-2.78±0.15 a -8.45±0.83 b -9.08±1.32 b -5.90±1.30 c <0.0001
	I sc (μA/cm 2 )	59±10 a	166±17 b	149±14 b	142±3 b	<0.0001
	G t (mS/cm 2 )	16.8±2.2 ab	16.5±1.8 ab	14.0±2.4 a	20.1± 3.3 b	0.0396

Table 6 . Intake, excretion, and apparent digestibility of NH 2 GIPC after 4 weeks on the experimental diets.

 6 Values are means ± standard deviations for 6 and 9 mice in the NC and the three HC-groups, respectively. Mean values within a row, not sharing a same superscript letter, were significantly different at P<0.05.

	NC, normo-caloric; HC, hyper-caloric; Con, control; CamC24, camelina meal C24 (from the
	commercial Céline cultivar rich in C24 fatty acids); CamC16, camelina meal C16 (from the
	experimental LOH2 cultivar rich in C16 fatty acids).

Acknowledgements

The authors acknowledge the valuable contribution of Célia Carbonne, MSc student in UMR Physiologie de la Nutrition et du Comportement Alimentaire (PNCA), who contributed to the running of the study, and of Morgane Dufay (UMR PNCA) for animal care and help with

Funding: This study was funded by Scientific direction and valorisation of AgroParisTech (2015 incitative call, NUTRICAM project). It has benefited from a French State grant (LabEx Saclay Plant Sciences-SPS, ref. ANR-10-LABX-0040-SPS), managed by the French National Research Agency under an "Investments for the Future" program (ref. ANR-11-IDEX-0003-02).

Supporting information.

Supplemental Table 1. Composition and nutritional values of the experimental diets. Supplemental Table 2. SL composition of the camelina meals (nmol/g) Supplemental Table 3. Body weight gain and food intake Supplemental Figure 1. NH 2 GIPCs and GlCers profiles in Camelina sativa seeds from the commercial Céline cultivar (CamC24) and from the LOH2 Y line (CamC16).


Christophe Carrincazeaux

Michel Grossetti

Damien Talbot
email: damien.talbot@u-bordeaux4.fr

Clusters, Proximities and Networks

In the Marshallian tradition of industrial districts, alongside theories of regional systems [START_REF] Cooke | Regional innovation systems, clusters, and the knowledge economy[END_REF], national innovation systems [START_REF] Lundvall | National Systems of Innovation: Towards a Theory of Innovation and Interactive Learning[END_REF], local productive systems [START_REF] Courlet | Industries, Territoires et Politiques Publiques[END_REF] or innovative environments (Camagni & Maillat, 2006), the cluster concept is usually treated in its spatial dimension, considering quite correctly that geographic proximity between firms constitutes a real factor in the diffusion of information and knowledge [START_REF] Porter | Clusters and the new economics of competition[END_REF]. The current international fascination with the concept of clusters, both amongst politicians and economic researchers, is rooted in the hypothesis that proximity displays characteristics that are conducive to the preservation and even reinforcement of relationships, irrespective of the area of activity in question. Policies encouraging the polarization of innovation activities are based on the following reasoning [START_REF] Torre | Clusters et syste `mes locaux d'innovation, Retour critique sur les hypothe `ses naturalistes de la transmission des connaissances a `l'aide des cate ´gories de l'economie de la proximite[END_REF]: (1) for innovation to occur, knowledge must be produced or appropriated; (2) by its very nature, knowledge cannot be appropriated in its entirety, and this induces effects wherein the knowledge spills over to other companies; (3) inasmuch as distance constitutes an obstacle to the diffusion of tacit knowledge-which can only be transferred via imitation and practice-geographic proximity is conducive to diffusion.

A proximity-based approach offers a complementary analysis of the concept of clusters, transcending a geographic proximity approach per se and incorporating a broader notion of coordination. Since agents are present both "here and elsewhere" [START_REF] Rallet | L'e ´conomie de proximite ´s[END_REF]-here since they are located in a geographic space within which they entertain relationships with neighbours, elsewhere because actors clearly entertain remote relationships with other agents-a person can be "near" someone from whom s/he is geographically distant. The idea here is that alongside spatial proximity, there is also such a thing as a relational type of non-spatial proximity. Geographic proximity is a representation that individuals have of the distance that separates them in space from objects and/or other individuals [START_REF] Torre | Proximity and localization[END_REF]. Since this assessment is made from a distance, it is relative in two different ways: because the distance is weighted by transportation time and costs, as a result of the material structuring of space; and because the distance that separates individuals, organizations or cities is also a representation, a judgement that causes people to see themselves, in a binary fashion, as being situated "close" or "far away". This conception of geographic proximity is based on an enriched conception of space, apprehended as an active construction of relationships and not as a simple functional context. According to the authors, non-spatial proximity is related, albeit not exhaustively, to organized proximity [START_REF] Torre | Proximity and localization[END_REF], socio-economic proximity [START_REF] Bouba-Olga | Socio-e ´conomie de proximite ´[END_REF] and institutional and organizational proximities (Talbot & Kirat, 2005;Talbot, 2008) (cf. Carrincazeaux et al., in this issue, on the scientific trajectory of the French Proximity School). Everyone agrees that proximity's relational aspect should be construed (much like geographic proximity) both as a potential that can facilitate coordination and also as a constraint (cultural limitations to cooperation, negative externalities, etc.) (Pecqueur & Zimmermann, 2004). The idea here is that the conditions for coordination, apprehended as constructs, need to be updated. All in all, interlinking these two types of proximity provides an analytical vision of the way actors coordinate themselves in a geographic space. Here clusters are viewed as a paradigmatic example covering the fundamental categories of proximity.

This special issue 1 adheres to the latter vision by offering a series of articles that study how a cluster's actors link proximity's spatial and non-spatial dimensions in their relationships with other agents. It is in this linkage between local and global connections, between the here and the elsewhere, and in the recombination of knowledge that this enables, that we will be able to see clusters as open systems more than as closed ones.

Where innovation processes are involved, it is important to show that although diffusion within the knowledge cluster is crucial for innovation, it is also important to transfer knowledge about the environment towards the cluster. Here we will need a network concept alongside our proximity construct to emphasize the importance of the relational dimension of the innovation process, specifically because it enables a linkage of the local with the non-local.

We start with a contribution by C. Carrincazeaux, Y. Lung and J. Vicente, who offer a historical analysis of the scientific trajectory of a group of French researchers, called "Dynamics of Proximity". After revisiting the group's leading publications, the authors outline the main issues characterizing this school of thought, presented as research agenda items. Two main questions are how should proximity be measured and what role does proximity play between interactions and institutions. The former theme may not be explicitly discussed in this issue, but the latter does play a major role. In actual fact, this was the title of the Fifth Annual Proximity Conference held in June 2006 at Bordeaux, which gave birth to the articles featured here.

M. Grossetti's article belongs to this latter school of thought and focuses on different levels of action. He demonstrates the need to distinguish relationships between individuals and relationships between organizations (firms, research laboratories, public organizations for economic development). The effects of spatial proximity are easier to understand when we analyse the interactions between these levels of action. Relationships between individuals help to establish relationships between organizations, but once launched, these can become autonomous and pursue their own logic. The existence of a local inter-individual network encourages local exchanges between organizations, without turning them into prisoners of said networks. Above all, it highlights the importance of the "mediation resources" that help to liberate people from interpersonal networks. Here, the effects of proximity derive from the geography of networks and from mediation resources. They are not explained by the need to transfer tacit knowledge.

ITFs (International Trade Fairs) are one of the mediation resources that companies use to access non-local partners. M. Ramı ´rez-Pasillas, in a study of the Lammhult cluster in Sweden, has analysed how firms mobilize spatial and relational proximities to accentuate the transfer of ITFs' knowledge towards clusters. The author shows that firms located in the cluster acquire knowledge due to their participation in these ITFs, knowledge that is then recombined and diffused within the cluster via local networks. The combination of networks and mediations (ITFs) produces a cluster that is localized yet also connected to international exchange flows.

Along these same lines, F. Belussi, A. Sammarra and S. R. Sedita detect, within a "life science cluster" located in Emilia-Romagnia (Italy) and comprised of firms and public research organizations, the presence of an "open innovation model". They note that, within a cluster, firms' innovation benefits as much from geographic proximity as from social embeddedness, interactions with local institutions, knowledge spillovers and globalization processes that multiply the sources of innovation, thanks for example to cooperation efforts with international actors. They also show that a positive relationship exists between "external R&D networking, in-house R&D investment, and innovative performance", for both firms and public research organizations. In short, this is a promising opening.

J. Wickham and A. Vecchi then underline the importance of global connections in creating local knowledge and sparking innovation. In a study of the "Irish Software industry in Dublin", they highlight links between business travel and cluster dynamics, studying the way in which global links are constructed and maintained by different "relational arrangements". Lastly, J. M. Zuliani adheres to a vision of clusters construed as open systems in a study on the growing porosity of certain sectors of activity. He notes that product-based or branch of activity-based analysis is becoming less and less effective at characterizing industrial clusters' productive organization. One example is provided by embedded systems produced in Toulouse, which developed initially as per a sectorial logic before progressively shifting due to the convergence of competencies and knowledge held by the aeronautical, spatial and automobile electronic industries. What we have here are local prime contractors sharing subcontracted information technology (IT) services; examples of cooperation between industrialists and engineering academies; and staff movements within a local labour market. This has led to the emergence of a "local system of competencies", one whose specificities are situated upstream of the products that firms sell.

The convergence of the articles compiled here illustrates the fertility of approaches based on a detailed analysis of coordination and networks at different levels. These approaches show that the existence of local configurations like clusters is not contrary to the growth of non-local exchanges, since it results from a complex architecture of individual networks, inter-firm relationships and different mediation systems.

Note

1. The present issue is partially derived from communications made at the Fifth Annual Proximity Conference, entitled "Proximity, between interactions and institutions", held in Bordeaux in June 2006 and funded by the Aquitaine Regional Authority. Some of the texts presented here were translated as part of the "Trajectories of innovation" programme, also funded by the Aquitaine Region.

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