Milk polar lipids favorably alter circulating and intestinal ceramide and sphingomyelin species in postmenopausal women

Mélanie Le Barz, Cécile Vors, Emmanuel Combe, Laurie Joumard-Cubizolles, Manon Lecomte, Florent Joffre, Michèle Trauchessec, Sandra Pesenti, Emmanuelle Loizon, Anne-Esther Breyton, Emmanuelle Meugnier, Karène Bertrand, Jocelyne Drai, Chloé Robert, Annie Durand, Charlotte Cuerq, Patrice Gaborit, Nadine Leconte, Annick Bernalier-Donadille, Eddy Cotte, Martine Laville, Stéphanie Lambert-Porcheron, Lemlih Ouchchane, Hubert Vidal, Corinne Malpuech-Brugère, David Cheillan, Marie-Caroline Michalski, Mélanie Le Barz, Cécile Vors, Emmanuel Combe, Laurie Joumard-Cubizolles, Manon Lecomte, Florent Joffre, Michèle Trauchessec, Sandra Pesenti, Emmanuelle Loizon, Anne-Esther Breyton, Emmanuelle Meugnier, Karène Bertrand, Jocelyne Drai, Chloé Robert, Annie Durand, Charlotte Cuerq, Patrice Gaborit, Nadine Leconte, Annick Bernalier-Donadille, Eddy Cotte, Martine Laville, Stéphanie Lambert-Porcheron, Lemlih Ouchchane, Hubert Vidal, Corinne Malpuech-Brugère, David Cheillan, Marie-Caroline Michalski

Abstract

BACKGROUNDHigh circulating levels of ceramides (Cer) and sphingomyelins (SM) are associated with cardiometabolic diseases. The consumption of whole fat dairy products, naturally containing such polar lipids (PL), is associated with health benefits, but the impact on sphingolipidome remains unknown.METHODSIn a 4-week randomized controlled trial, 58 postmenopausal women daily consumed milk PL-enriched cream cheese (0, 3, or 5 g of milk PL). Postprandial metabolic explorations were performed before and after supplementation. Analyses included SM and Cer species in serum, chylomicrons, and feces. The ileal contents of 4 ileostomy patients were also explored after acute milk PL intake.RESULTSMilk PL decreased serum atherogenic C24:1 Cer, C16:1 SM, and C18:1 SM species (Pgroup < 0.05). Changes in serum C16+18 SM species were positively correlated with the reduction of cholesterol (r = 0.706), LDL-C (r = 0.666), and ApoB (r = 0.705) (P < 0.001). Milk PL decreased chylomicron content in total SM and C24:1 Cer (Pgroup < 0.001), parallel to a marked increase in total Cer in feces (Pgroup < 0.001). Milk PL modulated some specific SM and Cer species in both ileal efflux and feces, suggesting differential absorption and metabolization processes in the gut.CONCLUSIONMilk PL supplementation decreased atherogenic SM and Cer species associated with the improvement of cardiovascular risk markers. Our findings bring insights on sphingolipid metabolism in the gut, especially Cer, as signaling molecules potentially participating in the beneficial effects of milk PL.TRIAL REGISTRATIONClinicalTrials.gov, NCT02099032, NCT02146339.FUNDINGANR-11-ALID-007-01; PHRCI-2014: VALOBAB, no. 14-007; CNIEL; GLN 2018-11-07; HCL (sponsor).

Keywords: Cardiovascular disease; Cholesterol; Clinical Trials; Lipid rafts; Metabolism.

Conflict of interest statement

Conflict of interest: This work was supported in part by a grant from the French Dairy Interbranch Organisation (CNIEL). MCM has received research funding for other research projects from CNIEL, Danone-Nutricia Research, and Sodiaal-Candia R&D. MCM has consultancy activities for food, fats and oils, and dairy companies. MCM is a member of the scientific advisory board of ITERG, the Industrial Technical Centre for the oils and fats business sector. These activities had no link with the present study. FJ was, and KB is, an employee of ITERG. PG is an employee of ACTALIA Produits Laitiers, an Agri-Food Technical Institute, with a strong specialization in dairy research and development, and food safety. ML had research collaborations with Mondelez and Bridor without link with the present study. HV has research collaborations with PiLeJe and Roquette without link with the present study.

Figures

Figure 1. Design of VALOBAB-C and VALOBAB-D…
Figure 1. Design of VALOBAB-C and VALOBAB-D clinical trials and graphical summary of analyses performed on predefined secondary outcomes.
In the VALOBAB-C clinical trial, 58 postmenopausal women were supplemented with test cream cheese containing either 0, 3, or 5 g of milk PL during 4 weeks. In the VALOBAB-D trial, 4 ileostomy patients were subjected to the acute consumption of the 3 test cheeses following a crossover study design. In both trials, during the exploratory visit, overnight fasted volunteers received a standardized breakfast rich in fat and sugars at time 0 and a meal containing the test cream cheese at time 240 minutes of the postprandial period. Tables and figures reporting specific results are listed. Cer, ceramides; CMRF, chylomicron-rich fraction; MFGM, milk fat globule membrane; PL, polar lipids; SP, sphingolipids; SM, sphingomyelins; TAG, triacylglycerols. Molecular structures were drawn using the LIPIDMAPS tool.
Figure 2. Major correlations between the impacts…
Figure 2. Major correlations between the impacts of milk PL supplementation on blood lipids and on serum SM and Cer (VALOBAB-C).
(A–C, and G) Spearman’s correlations between blood lipids and serum SM and Cer species. All data are expressed as ΔV2–V1 at fasting. Yellow, all groups were considered for the analysis (A and G) (n = 30); blue, the 2 groups supplemented with either 3 or 5 g–milk PL only (n = 20); and orange, the control group only (n = 10). For panels A–C, and G, asterisks in bold represent correlations that remain significant after adjustment for clinical center, quartiles of volunteer age, and waist circumference. (D–F and H–K) Graphs illustrating specific Spearman’s correlations between the intervention impact on C16+18 SM species and on LDL-C (D), total C (E), and ApoB48 (F); between C20+22 Cer species proportions (%) and LDL-C (H), and total C (I); between C24-26 Cer species proportions (%) and LDL-C (J), and total C (K). Apo, apolipoprotein; C, cholesterol; Cer, ceramides; CTL, control; HDL, high density lipoprotein; LDL, low density lipoprotein; NEFA, nonesterified fatty acids; PL, polar lipids; SM, sphingomyelin; TAG, triacylglycerols.
Figure 3. Milk PL supplementation during 4…
Figure 3. Milk PL supplementation during 4 weeks modulate SM and Cer molecular composition of plasma CMRF.
(A and B) Kinetics of ΔV2–V1 CMRF SM and Cer normalized by CMRF TAG content. (C–H) Molecular composition analysis of specific SM and Cer species in CMRF after normalization by CMRF TAG content: C16:1 SM (C), C20:1 SM (D), C24:1 SM (E), C16:1 Cer (F), C22:0 Cer (G), and C24:1 Cer (H). Data are presented as mean ± SEM (n = 6 / group). The vertical dotted line represents the intake of the meal including the control or milk PL–rich dairy (according to group). The Pgroup and Pposthoc are shown for the postprandial period from 120 to 480 minutes. Statistical analysis was done using a linear mixed model followed by Tukey-Kramer’s post hoc test. Pposthoc corresponds altogether to PCTL versus 3g-PL; PCTL versus 5g-PL and P3g versus 5g-PL. Results are presented based on the assumption of sphingosine d18:1 as the major sphingoid base for determined SM and Cer species. Cer, ceramides; CMRF, chylomicron-rich fraction; CTL, control; PL, polar lipids; SM, sphingomyelins; TAG, triacylglycerols. See Supplemental Table 3 and Supplemental Figure 2, VALOBAB-C trial.
Figure 4. Milk PL ingestion modulates SM…
Figure 4. Milk PL ingestion modulates SM and Cer species in ileal efflux in ileostomy patients.
(A) Cumulated enrichment over 0–480 minutes of total SM and Cer. (B and C) Molecular composition of ileal content efflux after 8 h of accumulation in SM (B) and Cer (C) species. Data are expressed in μmol and presented as mean ± SEM (n = 4/group), and empty circles represent individual values. Statistical analysis was done using 1-way ANOVA followed by Tukey’s post hoc test (normal data) or Friedman’s test followed by Dunn’s post hoc test (nonnormal data). Letters “a” and “b” indicate statistically different (P < 0.05) intervention effects between groups as calculated by post hoc analysis. Results are presented based on the assumption of sphingosine d18:1 as the major sphingoid base for determined SM and Cer species. Cer, ceramides; CTL, control; PL, polar lipids; SP, sphingolipids; SM, sphingomyelins. See Supplemental Figure 3, VALOBAB-D trial.
Figure 5. Effect of milk PL supplementation…
Figure 5. Effect of milk PL supplementation during 4 weeks on SM and Cer species excreted in feces.
(A and B) Molecular composition of SM (A) and Cer (B) in fecal samples (ΔV2–V1). Data are presented as mean ± SEM (control, n = 9; 3 g–PL, n = 7; 5 g–PL, n = 8) and expressed in μmol/g of lyophilized feces. Empty circles represent individual values. (C–M) Variations of specific SP species present in fecal samples were also determined and expressed as percentage of total SM and Cer: C16:0 SM (C), C22:0 SM (D), C24:0 SM (E), C16:1 SM (F), C22:1 SM (G), C24:1 SM (H), C16:0 Cer (I), C22:0 Cer (J), C24:0 Cer (K), C16:1 Cer (L), C22:1 Cer (M), and C24:1 Cer species (N) (ΔV2–V1). Statistical analysis was done using nonparametric analysis (nonnormal data). Letters “a” and “b” indicate statistically different (P < 0.05) intervention effects between groups as calculated by post hoc analysis. Results are presented based on the assumption of sphingosine d18:1 as the major sphingoid base for determined SM and Cer species. Cer, ceramides; CTL, control; PL, polar lipids; SM, sphingomyelins. See Supplemental Table 4, VALOBAB-C trial.

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