Marie Michèle Boulet

Catherine Calzada

Magali Pettazzoni

Taïssia Lelekov-Boissard

Charline Buisson

Mathilde Di Filippo

Annie Durand

Stéphanie Lambert-Porcheron

Julie-Anne Nazare

Philippe Moulin

Marie Michèle Boulet

Marie-Caroline Michalski

Ph.D David Cheillan
email: david.cheillan@chu-lyon.fr

A meal rich in palm oil or butter modifies the sphingolipid profile of postprandial

Keywords: cholesteryl esters, Cer, ceramides, ceramides, chylomicrons, dietary lipids, tandem mass spectrometry, type 2 diabetes. Highlights

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Introduction

It is now well established that the plasma lipidome is modified in metabolic diseases, such as type 2 diabetes (T2D). Besides changes in major lipid classes (triglycerides (TG), cholesteryl esters (CE), nonesterified cholesterol and phospholipids (PL)), plasma concentrations of some sphingolipid (SP) classes, such as ceramides (Cer), sphingomyelins (SM), sphingosine-1-phosphate (S1P) and GM3 ganglioside (GM3), among others, are altered in the plasma of T2D patients compared to healthy control subjects [START_REF] Haus | Plasma ceramides are elevated in obese subjects with type 2 diabetes and correlate with the severity of insulin resistance[END_REF][START_REF] Gorska | Concentrations of sphingosine and sphinganine in plasma of patients with type 2 diabetes[END_REF][START_REF] Carlsson | Changes in Serum Sphingomyelin After Roux-en-Y Gastric Bypass Surgery Are Related to Diabetes Status[END_REF][START_REF] Sato | Circulating levels of ganglioside GM3 in metabolic syndrome: A pilot study[END_REF] and recent studies validated plasma SP as candidate biomarkers of cardiovascular disease [START_REF] Poss | Machine learning revels serum sphingolipids as cholesterol-independent biomarkers of coronary artery disease[END_REF].

There is also growing interest in the lipidomic analysis of plasma lipoproteins, since the distribution and the concentrations of lipids differ across lipoprotein classes [START_REF] Wiesner | Lipid profiling of FPLCseparated lipoprotein fractions by electrospray ionization tandem mass spectrometry[END_REF]. Dyslipidemia in T2D results in significant alterations of lipoprotein lipids which have a contributing impact on the functional properties of lipoproteins leading to increased cardiovascular risk in metabolic diseases. However, most studies have investigated modifications of major core lipids in cholesterol-rich lipoproteins (LDL and HDL) compared to studies focusing on minor surface lipids such as SP. Regarding T2D, a few studies reported that minor lipid composition of HDL and LDL is modified [START_REF] Boon | Ceramides contained in LDL are elevated in type 2 diabetes and promote inflammation and skeletal muscle insulin resistance[END_REF][START_REF] Ståhlman | Dyslipidemia, but not hyperglycemia and insulin resistance, is associated with marked alterations in the HDL lipidome in type 2 diabetic subjects in the DIWA cohort: Impact on small HDL particles[END_REF], causing alteration in their functionality [START_REF] Kontush | Unraveling the complexities of the HDL lipidome[END_REF]. For example, compared to particles from control subjects, HDL from diabetic patients are depleted in S1P [START_REF] Brinck | Diabetes Mellitus Is Associated With Reduced High-Density Lipoprotein Sphingosine-1-Phosphate Content and Impaired High-Density Lipoprotein Cardiac Cell Protection[END_REF][START_REF] Denimal | Significant abnormalities of the HDL phosphosphingolipidome in type 1 diabetes despite normal HDL cholesterol concentration[END_REF], and this could alter their cardioprotective function [START_REF] Brinck | Diabetes Mellitus Is Associated With Reduced High-Density Lipoprotein Sphingosine-1-Phosphate Content and Impaired High-Density Lipoprotein Cardiac Cell Protection[END_REF]. It was also shown that modification in phosphatidylcholine (PC) and SM contents of HDL2 and HDL3 could affect their capacity to manage cholesterol trafficking in cells expressing SR-B1 [START_REF] Yancey | High density lipoprotein phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor BI[END_REF]. LDL from dyslipidemic T2D patients contained more diacylglycerol and less CE in their lipid core as well as a reduction in SM, Cer and lysophosphatidylcholine [START_REF] Ståhlman | Clinical dyslipidaemia is associated with changes in the lipid composition and inflammatory properties of apolipoprotein-B-containing lipoproteins from women with type 2 diabetes[END_REF].

Besides LDL and HDL, elevated concentrations of triglyceride-rich lipoproteins (TGRL) are recognized as a risk factor for ischaemic cardiovascular diseases [START_REF] Zilversmit | Atherogenesis: A postprandial phenomenon[END_REF]. TGRL are constituted of chylomicrons (CM), VLDL as well as their remnants, whose composition varies according to the amount and type of dietary fats [START_REF] Boulet | Postprandial Triglyceride-Rich Lipoproteins from Type 2 Diabetic Women Stimulate Platelet Activation Regardless of the Fat Source in the Meal[END_REF]. Many epidemiologic studies have well established that an increase of postprandial TG is an independent risk factor for cardiovascular events incidence, especially in diabetic women compared to men [START_REF] Borén | Postprandial hypertriglyceridemia as a coronary risk factor[END_REF][START_REF] Bansal | Fasting compared with nonfasting triglycerides and risk of cardiovascular events in women[END_REF][START_REF] Nordestgaard | Nonfasting triglycerides and risk of myocardial infarction, ischemic heart disease, and death in men and women[END_REF]. Among postprandial lipids, polar lipids such as SP are present in various foods, particularly dairy products mainly in the form of SM [START_REF] Vesper | Sphingolipids in food and the emerging importance of sphingolipids to nutrition[END_REF]. Recent studies from our laboratory showed that, compared to soybean polar lipids, the ingestion of milk polar lipids in mice led to an early increase in postprandial lipemia, a faster clearance of CM from plasma as well as increased concentrations of SM and C24:1 Cer in CM [START_REF] Lecomte | Milk Polar Lipids Affect In Vitro Digestive Lipolysis and Postprandial Lipid Metabolism in Mice[END_REF]. In post-menopausal overweight women, a 4-week milk polar lipid supplementation lowered total-, LDL-cholesterol and TG at fasting and postprandially [START_REF] Vors | Milk polar lipids reduce lipid cardiovascular risk factors in overweight postmenopausal women: towards a gut sphingomyelin-cholesterol interplay[END_REF] and decreased some SM and Cer species both in plasma and CM [START_REF] Barz | Milk polar lipids favorably alter circulating and intestinal ceramide and sphingomyelin species in postmenopausal women[END_REF]. Specific nutritional interventions, such as the PREDIMED trial, showed positive associations between specific Cer species (C16:0, C22:0, C24:1 and C24:0) and the incidence of cardiovascular events [START_REF] Wang | Plasma ceramides, Mediterranean diet, and incident cardiovascular disease in the PREDIMED Trial (Prevención con Dieta Mediterránea)[END_REF]. Thus, there is growing interest towards the modulation of the SP composition of the diet to prevent metabolic disorders and/or improve/restore metabolic status [START_REF] Barz | Alterations of endogenous sphingolipid metabolism in cardiometabolic diseases: Towards novel therapeutic approaches[END_REF].

Our objectives were to characterize the SP profile of plasma and TGRL from T2D patients and more specifically, to study the SP composition of plasma and TGRL isolated before and after a mixed breakfast meal including a realistic amount of lipids (20g) from a vegetable palm oil source or from butter.

Material and Methods

Study participants

Thirty women with T2D were recruited for a parallel-group clinical trial described in a previous publication (COMPLETE, clinical trial registration number at ClinicalTrials.gov: NCT03561571) [START_REF] Boulet | Postprandial Triglyceride-Rich Lipoproteins from Type 2 Diabetic Women Stimulate Platelet Activation Regardless of the Fat Source in the Meal[END_REF]. The primary outcome of the COMPLETE study was to determine the effect of fasting and postprandial TGRL on thromboxane B2, a marker of platelet activation [START_REF] Boulet | Postprandial Triglyceride-Rich Lipoproteins from Type 2 Diabetic Women Stimulate Platelet Activation Regardless of the Fat Source in the Meal[END_REF]. Analysis of the sphingolipidome of plasma and TGRL was one of the predefined secondary outcomes. After a 12 hour overnight fast, blood was drawn and the patients ingested a breakfast containing 20g lipids from either hazelnut-cocoa spread (Palm Nut) rich in palm oil (n=15) or Butter (n=15). A second blood sample was collected 4 hours (T240) after the ingestion of the breakfast.

In order to validate the fasting SP phenotyping of COMPLETE T2D volunteers with that of healthy subjects, fasting plasma from six healthy women (control, CTL) who were part of a previous trial (FiTaBlé, clinical trial registration number at ClinicalTrials.gov: NCT03717311) were used. CTL women had normal fasting glycemia, triglyceridemia and cholesterol concentrations. Both trials were approved by a national Scientific Ethics Committee (COMPLETE ID-RCB: 2018-A01414-51; FiTaBlé ID-RCB : 2018-A00949-46) and conducted in accordance with the principles of the Helsinki declaration. Informed written consent was obtained from all subjects.

Blood was collected into EDTA tubes and plasma was immediately isolated by centrifugation (1500 × g, 10 min, at 4 °C) and stored in aliquots at -20°C. The clinical and metabolic characteristics of the participants are shown in Table 1. T2D patients were obese, had a long history of diabetes, mild dyslipidemia with mild hypertriglyceridemia, normal HDL cholesterol and moderately high LDL cholesterol. Regarding the anti-diabetic and lipid lowering medication, 28 of 30 patients had metformin, 18 had sulfonylurea, 11 had glucagon-like peptide 1 analog, 8 had gliptin, and 1 had acarbose. In addition, 17 of 30 had statins and 1 had a combined therapy of statins and ezetimibe. None was treated with fibrates or insulin. Healthy CTL subjects had all the parameters mentioned above in the expected normal range.

Test Breakfasts Composition

Both experimental breakfasts contained 20g lipids and were similar in carbohydrates, proteins, and caloric contents (supplemental Table S1). The Palm Nut breakfast provided 439 calories with 51.1%, 40.9%, and 6.5% of energy as carbohydrates, lipids including a commercial hazelnut-cocoa refined palm oil spread (60 g), and proteins, respectively. The Butter breakfast was composed of 51.7% carbohydrates, 40.9% lipids including commercial butter (22.5 g, 82% fat) and 4.6% proteins for a total of 440 calories. Regarding the total fatty acid (FA) composition of both fats, Palm Nut mostly contained palmitic (C16:0, 33.2%) and oleic (C18:1 n-9, 45.2%) acids. Butter contained 78.8% saturated fatty acids, including 42.2% palmitic acid [START_REF] Boulet | Postprandial Triglyceride-Rich Lipoproteins from Type 2 Diabetic Women Stimulate Platelet Activation Regardless of the Fat Source in the Meal[END_REF]. Regarding SP, Palm Nut in the breakfast contained 0.03 mg Cer, 1.03 mg SM and 0.03 mg GM3 while 22.5g butter contained 0.38 mg Cer, 6.70 mg SM and 0.28 mg GM3.

Isolation of TGRL fractions and determination of protein concentration

TGRL fractions were isolated from the plasma by ultracentrifugation at 9777 × g for 1 hour at 12°C using a Beckman Coulter Optima TLX ultracentrifuge and a Beckman TLA 100.2 rotor. Top 100 µL of the floating layer corresponding to TGRL with density <1.000 g/ml was collected and immediately stored at -20°C. Protein concentration of the fractions was measured using a modified Lowry method [START_REF] Lowry | J Protein measurement with the Folin phenol reagent[END_REF] prior to storage. The hydrodynamic diameter of TGRL was measured by dynamic light scattering at 25 °C with a Zeta-Sizer NanoS (Malvern, UK) [START_REF] Boulet | Postprandial Triglyceride-Rich Lipoproteins from Type 2 Diabetic Women Stimulate Platelet Activation Regardless of the Fat Source in the Meal[END_REF].

Determination of plasma and TGRL sphingolipid compositions 2.4.1. Lipid extraction

Lipids from plasma (10 µL) and TGRL fractions (100 µL) were extracted in a single phase chloroform:methanol (1:2) procedure after addition of a mix of internal standards in methanol (details in supplemental Table S2). The lipid containing phase was evaporated with nitrogen and dried lipid extracts were stored at -20°C.

Saponification and purification

Dried lipid extracts were resuspended in 1.5 ml of chloroform:methanol (1:2) and saponified with 150µl of 1M KOH solution. Samples were incubated for two hours at 37°C and pH was adjusted to neutrality with acetic acid. Purification was performed on a solid phase extraction (SPE) C18 column by a double extraction using methanol:H2O (1:1). Elution was successively done with methanol:H2O (12:1) for GM3 and chloroform:methanol (1:2) for Cer and SM. Samples were evaporated with nitrogen and homogenized with chloroform:methanol (2:1) before direct injection in the mass spectrometer.

Tandem mass spectrometry analysis

Samples were homogenised in chloroform:methanol (1:2 v/v) and analysed by direct flow injection on a triple-quadrupole mass spectrometer (API 4500 QTRAP MS/MS; Sciex Applied Biosystems, Toronto, Canada) in the positive ionization mode for Cer and SM and in the negative ionization mode for GM3 (supplemental Table S2). These three classes of SP were quantified separately with the Multi Reaction Monitoring (MRM) method with a flow rate of 200 μl/min (analysis time of 3 minutes). The concentration of each molecular species (12 species for each class) was calculated from the ratio of its signal to that of the corresponding internal standard. Total Cer, SM and GM3, corresponded to the sum of the various species. Data were expressed either as µmol/L plasma or µmol/g TGRL protein. Results are presented based on the assumption of sphingosine d18:1 as the major sphingoid base for determined GM3, SM and Cer species.

Determination of plasma and TGRL sphingoid bases (sphingosine-1-phosphate, sphingosine, sphinganine) and sphingosylphosphorylcholine compositions 2.5.1. Lipid extraction

Extraction of sphingoid bases and sphingosylphosphorylcholine (SPC) was based on the method of Pettazzoni et al. [START_REF] Pettazzoni | LC-MS/MS multiplex analysis of lysosphingolipids in plasma and amniotic fluid: A novel tool for the screening of sphingolipidoses and Niemann-Pick type C disease[END_REF]. Plasma and TGRL fractions (200 µL) were precipitated with 500 µL of a mix of internal standards in methanol and 500 µL of 2% H3PO4 (details in supplemental Table S2). Samples were centrifuged and the supernatant was directly transferred in SPE columns Oasis cartridge (MCX, Waters Corp) previously conditioned with 2% H3PO4 and methanol. Columns were washed with 2% formic acid and elution was performed successively with 0.2% formic acid in methanol and 2% NH4OH in methanol. Lipid extracts were evaporated with nitrogen and redissolved with 500 µL of 0.2 % formic acid in 37.5% acetonitrile.

Liquid chromatography and tandem mass spectrometry analysis

Liquid chromatography (LC) -tandem mass spectrometry (MS/MS) analysis was performed using two LC pumps (LC20AD) and an autosampler (SIL20AC) (Prominence Liquid Chromatograph, Shimadzu©, Kyoto, Japan) coupled to an API 4500 QTrap (Sciex Applied Biosystems, Toronto, Canada) operated in the MRM mode. MS/MS parameters and retention time (RT) for each molecule are described in supplemental Table S2. Positive ionization mode was used for sphinganine (Spha), sphingosine (Spho) and SPC; negative ionization mode for sphingosine-1-phosphate (S1P). Reverse phase LC was performed on a C8 column (Uptisphere® 120 Å, 3 µm, 2.1 mm x 50 mm, Interchim©) maintained at 25°C (phase A: formic acid 0.2% in H2O, phase B: formic acid 0.2% in acetonitrile). The following gradient was run at a flow rate of 0.4 mL/min. The gradient was initiated at 37.5% of phase A, 0 to 4 min : 37.5 to 50 % B, 4 to 8 min : 50% B, 8 to 8.5 min : 50 to 75% B, 8.5 to 9.5 min : 75% B, 9.5 to 10 min : 75 to 37.5% B, 10 to 13 min : 37.5 % B). Fifty microliters were injected and the total run time was 13 minutes. Quantification was performed with the Analyst software version 1.6.2, using IS and a nine point external calibration curve (linear regression). Data were expressed in nmol/L or nmol/g TGRL protein.

Statistical analysis

Data were expressed as means ± SEM. Normality of data distribution was assessed using d'Agostino-Pearson normality test. Differences between T240 and T0 were assessed using paired Student's t-test or Wilcoxon test. Differences were considered significant at P<0.05 (2-sided test). All statistical analyses were performed using GraphPad Prism 8.0 (Graphpad Software, San Diego, CA).

Results

The sphingolipid concentrations of plasma and TGRL are modified in fasting type 2 diabetic patients compared to fasting control subjects

Plasma sphingolipids

Characterization of T2D volunteers of COMPLETE study at fasting shows that total Cer and GM3 concentrations were higher by 34% and 28% respectively in plasma from these T2D women compared to plasma from CTL subjects (T2D Cer: 15.5 ± 0.6 µM vs CTL Cer: 11.0 ± 0.7 µM; T2D GM3: 12.6 ± 0.7 µM vs CTL GM3: 10.2 ± 0.3 µM) (Fig. 1A). No differences in plasma SM concentrations were observed between T2D patients and CTL subjects. Regarding sphingoid bases and lysosphingolipids, S1P, Spho and Spha concentrations were similar between T2D patients and CTL subjects whereas decreased concentrations of SPC were found in T2D patients (T2D SPC: 3.5 ± 0.5 nmol/L vs CTL SPC: 10.2 ± 2.1 nmol/L) (Fig. 1B). Quantitative analyses of 12 molecular species within each class of SP showed differences between T2D patients and CTL subjects (Fig. 2). In Cer, most species were higher in plasma from T2D patients, especially the most abundant very long chain ones C22:0 (+55%), C24:1 (+34%) and C24:0 Cer (+65%) (Fig. 2A). Most molecular species of SM were similar between T2D and CTL, excepted for a slight increase in minor C16:1 and C18:1 SM species (Fig. 2B). In GM3, increased concentrations of GM3 containing saturated fatty acids, i.e. C18:0 (+54%), C20:0 (+75%), C22:0 (+78%) and C24:0 (+31%), as well as C22:1 (+22%) were found in plasma from T2D patients (Fig. 2C).

Total plasma T2D Cer concentrations were positively correlated with plasma TG (r=0.42, P=0.021), total cholesterol (r=0.61, P=0.0004), LDL-cholesterol (r=0.43, P=0.018) and apoB concentrations (r=0.46, P=0.014). Amongst molecular species of Cer, C16:0 Cer was also positively correlated with plasma TG (r=0.46, P=0.021), total cholesterol (r=0.52, P=0.021), LDL-cholesterol (r=0.40, P=0.021) and apoB levels (r=0.38, P=0.021). Plasma most abundant very long chain C22:0 Cer and C24:0 Cer species were correlated with TG (r=0.48, P=0.007 and r=0.47, P=0.008, respectively), and C22:0+C24:0+C24:1 Cer species were correlated with total cholesterol (r=0.61, P=0.0004, r=0.56, P=0.001 and r=0.49, P=0.006, respectively). No correlation was found between SP and glycemic parameters (either glycemia, HbA1C, HOMA-IR).

TGRL sphingolipids

No significant differences were observed between total Cer, SM and GM3 concentrations in T2D patients vs CTL subjects when concentrations were expressed per mg of protein (Fig. 1C). Regarding sphingoid bases and SPC, only S1P concentrations, expressed per mg protein, was increased in T2D TGRL (Fig. 1D). Of note, when results were expressed per L of plasma, SM, GM3, S1P and SPC concentrations were higher in TGRL from T2D patients compared to those from CTL subjects, whereas no change in Cer concentrations was observed (data not shown).

The sphingolipid concentrations of TGRL are modified in T2D patients after the ingestion of a single meal

COMPLETE study in T2D women shows that the ingestion of 20 g lipids in a single meal led to modifications of SP concentrations in postprandial diabetic TGRL compared to fasting T2D TGRL (Fig. 3) without any change in plasma SP (supplemental Table S3). In particular, total TGRL Cer concentrations increased by 45% at T240 after the intake of the Palm Nut meal and by 40% following the Butter breakfast compared to fasting TGRL Cer concentrations (0.51 ± 0.01 vs 0.35 ± 0.04 µmol/g protein in the Palm Nut group, 0.83 ± 0.09 vs 0.60 ± 0.09 µmol/g protein) (Fig. 3A). SM concentrations tended to increase in postprandial TGRL from Palm Nut group (7.14 ± 0.53 vs 6.56 ± 1.47 µmol/g protein, P=0.09) but no change was noted in postprandial TGRL SM from the Butter group. Neither meal modified the concentrations of postprandial GM3, sphingoid bases and SPC (Fig. 3B). Amongst the molecular species of TGRL Cer, C16:0, C22:0, C24:1 and C24:0 increased postprandially after the Palm Nut or Butter breakfast (P=0.07 for C22:0 Cer in the Butter group) (Fig. 4A). Accordingly, as an overall marker of atherogenic species, the sum of C16:0+C22:0+C24:1+C24:0 Cer was different between fasting TGRL and postprandial TGRL both in the Palm Nut group (0.17 ± 0.03 µmol/g protein vs 0.30 ± 0.04 µmol/g protein, P=0.0006, +78%) and in the Butter group (0.35 ± 0.07 µmol/g protein vs 0.53 ± 0.07 µmol/g protein, P=0.014, +52%). There was no effect of either meal on the molecular species concentrations of TGRL SM and GM3 (Fig. 4B andC).

Postprandial sphingolipids are correlated with triglycerides in TGRL from T2D patients

A positive correlation was observed in the Palm Nut group between postprandial changes ( T240-T0) of TGRL C22:0 Cer, C24:0 Cer, summed C16:0+C22:0+C24:1+C24:0 Cer concentrations and postprandial changes in plasma TG, TGRL TG as well as  TGRL total C16:0 concentrations (Figure 5). No significant correlation in the Butter group was noted regarding these changes. Total  TGRL Cer concentrations (0.16 ± 0.05 µmol/g protein) tended to be correlated with  TGRL total C16:0 concentrations (118.3 ± 35.6 nmol/mg protein i.e. C16:0 concentration in postprandial TGRL total lipids: 231.5 ± 42.3 nmol/mg protein minus C16:0 concentration in fasting TGRL lipids: 113.2 ± 19 nmol/mg protein) in the Palm Nut group (r=0.49, P=0.06) while no significant correlations were observed in the Butter group. In the Butter group, total  TGRL Cer concentrations tended to be associated with  TGRL mean diameters (r=0.46, P=0.08).

Discussion

In the present study, targeted lipidomic analyses of SP by MS/MS reveal detailed compositions of SP classes of TGRL from fasting T2D patients, expanding data on altered SP concentrations in T2D plasma.

First, our data show that plasma Cer concentrations were increased (+34%) in fasting obese women with T2D from COMPLETE study compared to healthy women, with the greatest increase being observed for the most abundant molecular species, C24:0 Cer (+65%) followed by C22:0 Cer and C24:1 Cer. Our data are consistent with reported elevated plasma Cer (+30%) in fasting obese T2D patients [START_REF] Haus | Plasma ceramides are elevated in obese subjects with type 2 diabetes and correlate with the severity of insulin resistance[END_REF], with increased concentrations of C18:0, C20:0, C24:1 Cer quantified subspecies and C24:0 Cer being the most abundant plasma ceramide subspecies. We also observed increased concentrations of plasma total GM3 and more precisely of saturated fatty acids subspecies in T2D patients, in accordance with higher serum levels of GM3 in hypertriglyceridemic patients or T2D patients with severe obesity [START_REF] Sato | Circulating levels of ganglioside GM3 in metabolic syndrome: A pilot study[END_REF]. The positive correlations that we observed between plasma Cer, specific C16:0 Cer concentrations and total cholesterol, LDL-cholesterol as well as TG confirm that Cer and its transport into lipoproteins are altered by the dysregulated metabolism of lipids in diabetes [START_REF] Laaksonen | Plasma ceramides predict cardiovascular death in patients with stable coronary artery disease and acute coronary syndromes beyond LDLcholesterol[END_REF][START_REF] Havulinna | Circulating ceramides predict cardiovascular outcomes in the population-based FINRISK 2002 Cohort[END_REF]. Regarding SM, the most abundant SP class, no change of its concentration was evidenced in obese T2D patients, while plasma SM levels have been reported to be lower in diabetic patients than in non-diabetic patients before bariatric surgery [START_REF] Carlsson | Changes in Serum Sphingomyelin After Roux-en-Y Gastric Bypass Surgery Are Related to Diabetes Status[END_REF]. By contrast, an increase of total SM levels was observed in TGRL from T2D patients. Such an increase could reflect higher cholesterol absorbers as shown in VLDL [START_REF] Agren | Postprandial lipemic response and lipoprotein composition in subjects with low or high cholesterol absorption efficiency[END_REF]. A novel observation of the present study is the detection of S1P in TGRL and their increased concentrations in T2D patients vs CTL subjects.

It is well established that in the postprandial state, CM and their remnants are the main carriers of dietary lipids, including TG, CE and polar lipids, and that the transient accumulation of TG in CM generally constitutes the first and main change in the circulation [START_REF] Dubois | Effects of graded amounts (0-50 g) of dietary fat on postprandial lipemia and lipoproteins in normolipidemic adults[END_REF]. We previously showed in our COMPLETE randomized study that a 20g fat meal led to increased concentrations of TG in postprandial TGRL isolated from T2D obese women [START_REF] Boulet | Postprandial Triglyceride-Rich Lipoproteins from Type 2 Diabetic Women Stimulate Platelet Activation Regardless of the Fat Source in the Meal[END_REF]. Besides TG, TGRL are constituted of a polar surface including SP that possesses biological functions of specific interest in T2D and cardiovascular risk. In this ancillary part of the COMPLETE study, we show that the intake of this single meal enriched in saturated fats increased total Cer concentrations in postprandial TGRL compared to fasting TGRL. Detailed analyses of TGRL molecular species revealed specific increase of C16:0 and very-long chain molecular species (C22:0, C24:0 and C24:1). It is known that following a meal, absorbed dietary lipids are taken up by enterocytes which incorporate most of them into CM for secretion. Amongst fatty acids, palmitic acid (C16:0) is the precursor of de novo Cer synthesis whom first step involves the condensation of palmitoyl-CoA and serine via serine palmitoyl transferase (SPT) [START_REF] Mandon | Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver[END_REF]. In the breakfast given to our T2D patients, palmitic acid represented at least 33% of total lipids in Butter and Palm Nut. Therefore, the observed increased Cer concentrations in postprandial TGRL are likely to result from a de novo synthesis of Cer in the intestine through the supply of dietary palmitic acid. These results support studies performed in mice showing that a palm oil bolus increased plasma total Cer 4 hours after the gavage and that pre-treatment of rodents by myriocin, an inhibitor of SPT, abolished the increase of total Cer, suggesting a de novo ceramide synthesis in the small intestine [START_REF] Tran | Short term palmitate supply impairs intestinal insulin signaling via ceramide production[END_REF]. In human studies, lipid infusion increased plasma Cer levels [START_REF] Watt | Regulation of plasma ceramide levels with fatty acid oversupply: evidence that the liver detects and secretes de novo synthesised ceramide[END_REF] and saturated fatty acid overfeeding of overweight people for several weeks increased plasma Cer [START_REF] Luukkonen | Saturated fat is more metabolically harmful for the human liver than unsaturated fat or simple sugars[END_REF] including very long chain species, C24:0 [START_REF] Rosqvist | Overeating Saturated Fat Promotes Fatty Liver and Ceramides Compared With Polyunsaturated Fat: A Randomized Trial[END_REF] and C24:1 [START_REF] Luukkonen | Saturated fat is more metabolically harmful for the human liver than unsaturated fat or simple sugars[END_REF].

Regarding SM, we found here that total SM concentrations tended to increase in postprandial TGRL from the Palm Nut group compared to fasting concentrations. FA composition of plasma SM has been described to be rather stable [START_REF] Nilsson | Absorption and lipoprotein transport of sphingomyelin[END_REF] and no effect of a 5-week supplementation on SM molecular species distribution amongst lipoproteins classes has been evidenced in young adults [START_REF] Myher | Effect of saturated and unsaturated fat diets on molecular species of phosphatidylcholine and sphingomyelin of human plasma lipoproteins[END_REF]. Although SP are present in dairy-derived foods, it is very unlikely that the increased content of SP in TGRL is reflective of a dietary contribution of pre-formed SP rather than de novo synthesis, due to the very limited amount of SP in the Palm Nut (most likely traces via its skim milk powder content) and Butter breakfasts. We did not detect any difference of changes ( T240 -T0) of total TGRL Cer concentrations between the Palm Nut and Butter groups, despite differences in FA compositions of the meals (33% C16:0 and 45% C18:1 n-9 in the Palm Nut or 79% saturated fatty acids including 42% C16:0 in the butter). Therefore, our results indicate that the single intake of 20g lipids containing at least 33% C16:0 is sufficient to modify the SP profile of intestine-derived TGRL and the FA composition of Cer. The effects of other dietary fatty acids, including monounsaturated fatty acids and polyunsaturated fatty acids, on the sphingolipid profile of TGRL certainly deserve further investigation.

Many experimental in vitro studies showed the role of distinct ceramides as bioactive lipid mediators and biomarkers of insulin resistance, T2D, and coronary artery disease [START_REF] Choi | Ceramides and other sphingolipids as drivers of cardiovascular disease[END_REF]. A recent meta-analysis highlighted the importance of studying the molecular species of Cer that are significantly associated with the risk of adverse cardiovascular events in patients [START_REF] Mantovani | Ceramides and risk of major adverse cardiovascular events: A metaanalysis of longitudinal studies[END_REF]. Our results in type diabetic patients having ingested the palm oil-based breakfast showed positive correlations between C22:0 Cer, C24:0 Cer, summed C16:0+C22:0+C24:1+C24:0 Cer and TG in TGRL, suggesting a contributing (direct or indirect) effect of TGRL Cer in potential deleterious effects of increased postprandial TG levels in metabolic diseases. Such a correlation was not found after butter consumption. In this regard, functional pro-aggregant effects of postprandial TGRL on platelets were evidenced in our COMPLETE study [START_REF] Boulet | Postprandial Triglyceride-Rich Lipoproteins from Type 2 Diabetic Women Stimulate Platelet Activation Regardless of the Fat Source in the Meal[END_REF].

In the prospective case-cohort PREDIMED study enrolling participants with high cardiovascular risk [START_REF] Wang | Plasma ceramides, Mediterranean diet, and incident cardiovascular disease in the PREDIMED Trial (Prevención con Dieta Mediterránea)[END_REF], a positive association was documented between plasma C16:0+C22:0+C24:1+C24:0 Cer and the incidence of cardiovascular events. However, further studies are needed to test the potential biological effects of Cer with different acyl-chain lengths.

Although the small sample size is a limitation of our study, it is the first time to our knowledge that the distribution of SP classes and molecular species in fasting and postprandial TGRL from T2D patients is described. Our study only included women with T2D, because of higher risk for cardiovascular morbidity and mortality compared to men with diabetes, so our results would need to be confirmed in T2D men and patients with obesity. In conclusion, the ingestion of a single 20g fat-rich meal from a vegetable or dairy source by T2D patients led to increased concentrations of deleterious molecular species of Cer in postprandial TGRL to a similar extent than the diabetes status vs healthy status in the fasting state. Ceramides in TGRL, although minor components of the lipidome, can be modulated by dietary lipids and could contribute to the increased atherogenicity of postprandial TGRL in metabolic diseases beyond the traditional risk factors for atherothrombosis. editing; M-C.M. : conceptualization, formal analysis, writing -review and editing, visualization, supervision, funding acquisitions; D.C. : conceptualization, methodology, validation, formal analysis, investigation, data curation, writing -review and editing, resources, supervision. All co-authors read, commented, and approved the manuscript.

Fig. 1 .

 1 Fig.1. Total sphingolipid concentrations in plasma and TGRL from fasting type 2 diabetic patients

Fig. 2 .

 2 Fig. 2. Plasma sphingolipid molecular species in fasting type 2 diabetic patients of COMPLETE

Fig. 3 .Fig. 4 .Fig. 5 .

 345 Fig. 3. Sphingolipid concentrations in triglyceride-rich lipoproteins from type 2 diabetic patients

  Figure 1

  Figure 5

Table 1 .

 1 Clinical and metabolic characteristics of women with type 2 diabetes in the COMPLETE study and comparison with healthy control women.

	COMPLETE study

Acknowledgements

We thank the nurses and the medical team of the department of Endocrinology, Metabolic Disease, Diabetes and Nutrition at Louis Pradel hospital and the technical team of the sphingolipid analysis platform at Laboratoire de Biochimie et de Biologie Moléculaire.

Funding

This work was supported by Inserm and promoted by Hospices Civils de Lyon. MMB received a doctoral contract from the Ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation and a doctoral grant from the Nouvelle Société Francophone d'Athérosclérose. CC thanks the CNRS.

Other authors have no conflict of interest to declare. The trial had no funding by any industrial company.