Frédéric Fumeron
email: frederic.fumeron@inserm.fr

Anthony Nicolas

Jean-Philippe Bastard

Soraya Fellahi

Léonore Wigger

Mark Ibberson

Céline Cruciani-Guglielmacci

Hervé Le Stunff

Gilberto Velho

Christophe Magnan

D E S I R Study

Michel Marre

Beverley Balkau

Ronan Roussel

Dairy consumption is associated with lower

Keywords: dairy product intake, dihydroceramides, ceramides, general population, longitudinal study

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Introduction

Sphingolipids, in particular ceramides and dihydroceramides, have been recognized as playing a large role in lipotoxicity, that is known to be associated with impaired glucose metabolism and type 2 diabetes [START_REF] Chaurasia | Ceramides -Lipotoxic inducers of metabolic disorders[END_REF]. In the French prospective D.E.S.I.R. (Data from the Epidemiological Study on the Insulin-Resistance syndrome) cohort, we have shown that high levels of plasma ceramides and dihydroceramides are associated with type 2 diabetes, and that dihydroceramides are higher in people who progressed to type 2 diabetes, even dihydroceramides measured 9 years before the onset of the disease [START_REF] Wigger | Plasma dihydroceramides are diabetes susceptibility biomarker candidates in mice and humans[END_REF]. We replicated these results in another cohort, the CoLaus study [START_REF] Wigger | Plasma dihydroceramides are diabetes susceptibility biomarker candidates in mice and humans[END_REF]. Other recent cohort studies yielded similar results with ceramides, but did not report plasma dihydroceramide concentrations [START_REF] Lemaitre | Circulating sphingolipids, insulin, HOMA-IR, and HOMA-B: The Strong Heart Family Study[END_REF][START_REF] Neeland | Relation of plasma ceramides to visceral adiposity, insulin resistance and the development of type 2 diabetes mellitus: the Dallas Heart Study[END_REF].

Type 2 diabetes is a multifactorial disease, resulting from interactions between genetic susceptibility and environmental factors, including diet. The influence of dairy product intake in type 2 diabetes risk is controversial, but meta-analyses including large cohorts seem to show a beneficial effect of dairy intake [START_REF] Gijsbers | Consumption of dairy foods and diabetes incidence: a dose-response meta-analysis of observational studies[END_REF][START_REF] Gao | Dairy products consumption and risk of type 2 diabetes: systematic review and dose-response meta-analysis[END_REF][START_REF] Aune | Dairy products and the risk of type 2 diabetes: a systematic review and dose-response meta-analysis of cohort studies[END_REF][START_REF] Schwingshackl | Food groups and risk of type 2 diabetes mellitus: a systematic review and meta-analysis of prospective studies[END_REF]. In D.E.S.I.R., we have shown an inverse association between dairy consumption at baseline and onset of impaired fasting glycaemia/type 2 diabetes and of the metabolic syndrome, over a 9-year follow-up [START_REF] Fumeron | Dairy consumption and the incidence of hyperglycemia and the metabolic syndrome: results from a french prospective study, Data from the Epidemiological Study on the Insulin Resistance Syndrome (DESIR)[END_REF][START_REF] Fumeron | Dairy products and the metabolic syndrome in a prospective study, DESIR[END_REF].

Although ceramides are considered to be an important cardiometabolic risk factor [11],

there are few published studies reporting their relation with diet in humans, and to our knowledge, none concerned dairy products. Our aim in this report is to study relationships between dairy consumption and concentrations of dihydroceramides and ceramides in the D.E.S.I.R. cohort.

Subjects and Methods

Population

The D.E.S.I.R. study is a prospective cohort of men and women, aged 30-65 years, recruited from volunteers who were offered periodic health examinations free of charge by the French Social Security system, in 10 health examination centers from the western part of France.

They were clinically and biologically evaluated at visits every 3 years, and the final examination was 9 years after inclusion [START_REF] Balkau | Predicting diabetes: clinical, biological, and genetic approaches: data from the Epidemiological Study on the Insulin Resistance Syndrome (DESIR)[END_REF]. For our previous studies on ceramides, all incident cases of type 2 diabetes and a random sample from the entire pool of people as controls were selected (n=295). For the present study, we include only the 105 controls without diabetes because disease per se might be a confounding factor. Supporting this concept, we observed no association between dairy products and dihydroceramides and ceramides in people with diabetes (data not shown).

The D.E.S.I.R. study was approved by the ethics committee of the Kremlin Bicêtre Hospital, and all participants signed an informed consent.

Measurements

Weight, height, and waist circumferences were measured by trained personnel. Venous blood samples were collected in the morning after participants had fasted for 12 h. Systolic and diastolic arterial pressures were measured after 5 min of rest in a supine position, with a mercury sphygmomanometer adapted for arm size. Two measures of blood pressure were taken, and means were used for the analysis. A detailed description of laboratory measurements including fasting triglycerides, and glucose is provided elsewhere [START_REF] Balkau | Predicting diabetes: clinical, biological, and genetic approaches: data from the Epidemiological Study on the Insulin Resistance Syndrome (DESIR)[END_REF]. Total adiponectin was assayed in fasting plasma EDTA samples from 3249 people from the D.E.S.I.R. cohort selected at random, collected at baseline and kept frozen at -80°C. An ELISA kit from Alpco®, Eurobio, Courtaboeuf, France was used. According to the manufacturer, intra-and inter-assay coefficients of variation for adiponectin assay were less than 6.0%.At baseline, then at years 3, 6 and 9, dihydroceramides and ceramides were determined by mass spectrometry, on thawed samples kept at -80°C, in a subset of the cohort [START_REF] Wigger | Plasma dihydroceramides are diabetes susceptibility biomarker candidates in mice and humans[END_REF]. Lipid extraction, mass spectrometry (MS) based lipid detection and data processing for human samples was performed by Zora Biosciences Oy (Espoo, Finland) [START_REF] Jung | High throughput quantitative molecular lipidomics[END_REF] as described previously [START_REF] Wigger | Plasma dihydroceramides are diabetes susceptibility biomarker candidates in mice and humans[END_REF]. In the present study, we analyzed the 105 people who did not progress to diabetes, because disease per se might be a confounding factor.. Characteristics of participants are shown in table 1.

Type 2 diabetes was defined as fasting plasma glucose ≥ 7 mmol/l or treatment by glucose lowering agents.

A 23-item questionnaire was completed by each participant, to determine the frequency and level of consumption of different foods. This questionnaire has been validated by comparison with the dietary history method, with 30 minute interviews by trained dieticians [START_REF] Lasfargues | Validity of a short self-administered dietary questionnaire compared with a dietetic interview[END_REF]. This validation study enabled the determination of multivariable linear regression equations to estimate the main nutrient intakes as well as total calories, from the questions on the consumption of different foods. Two items concerned dairy products: cheese, milk and other dairy products (except cheese). One portion was defined as 30 g for cheese, and 125 mL for milk or dairy products. There were three possible responses for cheese intake (0-1 portion/day; 2-3 /day; > 3/day), and four for the intake of dairy products (except cheese) (never; < 1 portion/day; 1-2 /day; > 2 /day) [START_REF] Fumeron | Dairy consumption and the incidence of hyperglycemia and the metabolic syndrome: results from a french prospective study, Data from the Epidemiological Study on the Insulin Resistance Syndrome (DESIR)[END_REF][START_REF] Fumeron | Dairy products and the metabolic syndrome in a prospective study, DESIR[END_REF]. In the present study, these responses were dichotomized as low/high: <2 portions/day / ≥2 portions/day for cheese and < 1 portion/day /  1 portion/day for dairy products (except cheese), in order to optimize group sizes for comparisons.

Statistics

General characteristics of the studied sample are described by means ± SD, medians (interquartile range) or %. The associations between plasma dihydroceramide and ceramide concentrations (natural log values) at four follow-up times with baseline dairy intake, sex and their interactions, were tested by analysis of covariance (ANCOVA) for repeated measures, adjusted for age, BMI, alcohol intake, total energy intake, physical activity, and fasting plasma cholesterol, triglycerides and glucose. ANCOVA tested the average effects of dairy intake and the interactions with sex and time. Results are shown as least squares adjusted geometric means with 95% confidence intervals.

All statistical analyses used SYSTAT 13®software for Windows.

Results

Total dihydroceramide and ceramide concentrations were similar in men and women (Table 1), but men had higher triglyceride concentrations, as well as higher alcohol and energy intakes; adiponectin concentrations were higher in women. Dairy intake (except cheese) was similar in men and women, but women ate less cheese.

A higher consumption of dairy products (except cheese) was associated with total plasma dihydroceramides during follow-up, in interaction with sex (P=0.004): dihydroceramide concentrations at follow-up were lower in women with high consumption as compared to the low consumers (P=0.03) but in men, no significant association was found (Table 2). In women, plasma dihydroceramides significantly increased during the follow-up in low consumers (P=0.01) but not in high consumers. No differences in total plasma ceramides according to dairy (except cheese) consumption was found in the whole population (Table 3).

Nevertheless, there was a trend for lower total plasma ceramides in women with a high intake of dairy (except cheese) (P=0.08) (Table 3).

When looking at the different dihydroceramide species, on average, all were higher at followup in women with low dairy (except cheese) consumption (Figure 1). Two specific ceramide species (out of nine) were also significantly associated with dairy consumption in women: Cer(d18:1/26:0) and Cer(d18:1/26:1) (Figure 1).

Low and high cheese intake was associated with different changes in plasma dihydroceramides and ceramides during follow-up (P=0.04 for time X cheese consumption interactions for both dihydroceramides and ceramides) (Table 4), however, when looking at changes in high and low consumers separately, no significant change over the follow-up was observed in any group.

Discussion

In this study, we have shown that plasma dihydroceramides, predictive markers of type 2 diabetes, were lower in women from the D.E.S.I.R. study with a higher consumption of dairy products except cheese, i.e. fresh dairy products. This result is consistent with our previous data indicating that dairy intake is associated with a lower risk of impaired fasting glucose/type 2 diabetes [START_REF] Fumeron | Dairy consumption and the incidence of hyperglycemia and the metabolic syndrome: results from a french prospective study, Data from the Epidemiological Study on the Insulin Resistance Syndrome (DESIR)[END_REF].

A high-fat diet in mice increased ceramide concentrations concomitantly with insulin resistance [START_REF] Wigger | Plasma dihydroceramides are diabetes susceptibility biomarker candidates in mice and humans[END_REF], but despite the fact that ceramides and related molecules are now recognized as important cardiometabolic risk factors, few dietary influences on plasma ceramides and dihydroceramides have been described in humans [START_REF] Summers | Could ceramides become the new cholesterol?[END_REF]. Diet induced weight loss has been shown to be associated with a decrease in intramyocellular dihydroceramide levels, but not ceramide levels [START_REF] Dube | Effects of weight loss and exercise on insulin resistance, and intramyocellular triacylglycerol, diacylglycerol and ceramide[END_REF]. A healthy Nordic diet lowered some plasma ceramide species in people with the metabolic syndrome [START_REF] Lankinen | Healthy nordic diet alters the plasma lipidomic profile in adults with features of metabolic syndrome in a multicenter randomized dietary intervention[END_REF]. A diet rich in saturated fat increased ceramides and dihydroceramides when compared to diets rich in unsaturated fat or carbohydrates [START_REF] Luukkonen | Saturated fat is more metabolically harmful for the human liver than unsaturated fat or simple sugars[END_REF]. In PREDIMED, a score summing concentrations of four ceramides was associated with cardiovascular risk and modified the effects of a Mediterranean diet on cardiovascular outcomes, nevertheless this score was not modified by a Mediterranean diet [START_REF] Wang | Plasma ceramides, mediterranean diet, and incident cardiovascular disease in the PREDIMED trial (Prevencion con Dieta Mediterranea)[END_REF].

The relationship between dairy and (dihydro)ceramides might explain at least in part, the negative association of dairy with impaired fasting glucose/ type 2 diabetes in the D.E.S.I.R. study [START_REF] Fumeron | Dairy consumption and the incidence of hyperglycemia and the metabolic syndrome: results from a french prospective study, Data from the Epidemiological Study on the Insulin Resistance Syndrome (DESIR)[END_REF]. Actually, it has been shown that ceramides play a deleterious role in insulin resistance [START_REF] Chaurasia | Ceramides -Lipotoxic inducers of metabolic disorders[END_REF], but this is less clear for dihydroceramides, which are ceramide precursors.

Dihydroceramides were previously considered as inert sphingolipid precursors but they have now been proposed as biomarkers of metabolic dysfunction [START_REF] Siddique | Dihydroceramides: From bit players to lead actors[END_REF]. Dihydroceramides are markers of the ceramide de novo synthesis pathway. This pathway might be especially at risk for metabolic disease when compared to the other pathways. In patients with NAFLD, liver dihydroceramides and ceramides were increased in those with high as compared to low insulin resistance, while other markers of ceramide synthesis, by sphingomyelin hydrolysis or salvage pathway, were unchanged [START_REF] Luukkonen | Hepatic ceramides dissociate steatosis and insulin resistance in patients with non-alcoholic fatty liver disease[END_REF]. In the same study, serum adiponectin was lower in the high insulin resistance group. Although there is no clear mechanism for the results found in the present study, adiponectin might play a role in the observed association between (dihydro)ceramides and dairy intake. Adiponectin activates ceramidase by its two receptors, which can contribute to lower concentrations of ceramides via increased degradation [START_REF] Holland | Receptor-mediated activation of ceramidase activity initiates the pleiotropic actions of adiponectin[END_REF].

Low fat dairy product intake [START_REF] Niu | Low-fat dairy, but not whole-/high-fat dairy, consumption is related with higher serum adiponectin levels in apparently healthy adults[END_REF], and dietary patterns characterized by low fat dairy and whole grain [START_REF] Yannakoulia | A dietary pattern characterized by high consumption of whole-grain cereals and low-fat dairy products and low consumption of refined cereals is positively associated with plasma adiponectin levels in healthy women[END_REF] or by dairy and bread [24] have been positively associated with adiponectin concentrations. As mentioned in our previous papers, it can be assumed that in France, the majority of cheese consumed is high-fat, while other dairy products such as milk and yogurts are low-fat [START_REF] Fumeron | Dairy consumption and the incidence of hyperglycemia and the metabolic syndrome: results from a french prospective study, Data from the Epidemiological Study on the Insulin Resistance Syndrome (DESIR)[END_REF][START_REF] Fumeron | Dairy products and the metabolic syndrome in a prospective study, DESIR[END_REF]. In women in the D.E.S.I.R. cohort, a higher dairy (but not cheese) consumption was associated with higher adiponectin concentrations (high consumption: 6.84mg/L (adjusted mean) ± 1.02 (SEM) vs. low consumption: 6.35mg/L ± 1.03; P=0.04 by ANCOVA). We also observed negative but non-significant correlations of adiponectin with dihydroceramides and ceramides in women, likely because of the small sample size of people with both measurements. This is consistent with the hypothesis that it is mainly the low fat fresh dairy intake that is associated with high adiponectin, which is associated with lower ceramides, leading to a lower risk of type 2 diabetes. In the D.E.S.I.R. study, cheese consumption (high fat) was not significantly associated with (dihydro)ceramides nor with impaired fasting glycemia/type 2 diabetes [START_REF] Fumeron | Dairy consumption and the incidence of hyperglycemia and the metabolic syndrome: results from a french prospective study, Data from the Epidemiological Study on the Insulin Resistance Syndrome (DESIR)[END_REF][START_REF] Fumeron | Dairy products and the metabolic syndrome in a prospective study, DESIR[END_REF].

Nevertheless, the association between dihydroceramides and dairy intake was statistically significant only in women. This indicates that it can only be a partial explanation for the lower hyperglycemia risk associated with dairy consumption, since there was no evidence of interaction between sex and dairy consumption on the risk of hyperglycemia in D.E.S.I.R. [START_REF] Fumeron | Dairy consumption and the incidence of hyperglycemia and the metabolic syndrome: results from a french prospective study, Data from the Epidemiological Study on the Insulin Resistance Syndrome (DESIR)[END_REF][START_REF] Fumeron | Dairy products and the metabolic syndrome in a prospective study, DESIR[END_REF]. This work has some strengths and several limitations. Among the strengths are the prospective nature of the data. The size of our study may seem limited, however repeated measurements of ceramides were performed, which is not the case in larger cohort studies with only one single measurement [START_REF] Lemaitre | Circulating sphingolipids, insulin, HOMA-IR, and HOMA-B: The Strong Heart Family Study[END_REF][START_REF] Neeland | Relation of plasma ceramides to visceral adiposity, insulin resistance and the development of type 2 diabetes mellitus: the Dallas Heart Study[END_REF]. We were also able to adjust for confounders such as lifestyle factors. The relationships we observed are modest, but they were obtained after adjustment for lifestyle variables, including physical activity, smoking, alcohol consumption, and energy intake. One of the main limitations is that our questionnaire does not allow the separation of low-fat and high-fat products, or to estimate the effects of different dairy products. However, we could separate cheese from other dairy products. As already stated, it can be assumed that in France, cheese is high-fat, but that the milk included in other dairy products is low fat.

In conclusion, our data show that in women from the general population, higher fresh dairy product consumption is associated with lower concentrations of plasma dihydroceramides, a protective marker for the onset of type 2 diabetes. Further studies will be needed to clear up the potential mechanism of this association. a by t-test on continuous variables (on Ln values for those given as medians) or chi square on grouped variables (given as %).

a by two-way (sex, intake) ANCOVA (on Ln values) for repeated measures, adjusted for age, BMI, alcohol intake, total energy intake, physical activity, and plasma cholesterol, triglycerides, and glucose. No statistically significant interaction effect forintake X time was observed.

b by one-way (intake) ANCOVA for repeated measures, same adjustments. No statistically significant interaction effect for intake X time was observed.

Figure caption

Figure 1 Plasma

 1 Figure 1Plasma specific dihydroceramide and ceramide concentrations in women without diabetes at baseline or at follow-up, according to their intake of dairy products (except cheese). The D.E.S.I.R. Study. P-values are for the average intake effects during the follow-up by ANCOVA for repeated measures, adjusted for age, BMI, alcohol intake, total energy intake, physical activity, and fasting plasma cholesterol, triglycerides, and glucose. No statistically significant interaction between intake and time was observed. Data are adjusted geometric means with 95% CI. (A) Dihydroceramide (d18:0/16:0) (B) Dihydroceramide (d18:0/22:0) (C) Dihydroceramide (d18:0/23:0) (D) Dihydroceramide (d18:0/24:0) (E) Dihydroceramide (d18:0/24:1) (F) Ceramide (d18:1/26:0) (G) Ceramide (d18:1/26:1)

Table 1 . Baseline characteristics of the people without diabetes selected for ceramide analysis. The D.E.S.I.R. Study

 1

	Men	Women	P a

b By two-way ANCOVA (on Ln values) for repeated measures, same adjustments as above. One way ANCOVA, in low intake: P time=0.27, in high intake: P time= 0.63

Acknowledgements

The D.E.S.I.R. Study Group. INSERM U1018: B. Balkau, P. Ducimetière, E. Eschwège; INSERM U367: F. Alhenc-Gelas; CHU D'Angers: Y Gallois, A. Girault; Centre de Recherche des Cordeliers, INSERM U1138, Bichat Hospital: F. Fumeron, M. Marre, R. Roussel; CHU de Rennes: F. Bonnet; CNRS UMR8090, Lille: A. Bonnefond, S. Cauchi, P. Froguel; Univ Paris Descartes, UMR1153, Paris: F .Rancière; Centres d'Examens de Santé: Alençon, Angers, Blois, Caen, Chateauroux, Chartres, Cholet, Le Mans, Orléans, Tours; Institut de Recherche Médecine Générale: J. Cogneau; General practitioners of the region; Institut inter-régional pour la Santé: C. Born, E. Caces, M. Cailleau, O Lantieri, J.G. Moreau.

Funding

This research was supported by grants from CNIEL (National Interprofessional Center for the Dairy Economy) and IMIDIA (Innovative Medicines Initiative Joint Undertaking) (grant agreement no. 155005). The D.E.S.I.R. study has been funded by INSERM contracts with Caisse nationale de l'assurance Maladie des Travailleurs Salariés (CNAMTS), Lilly, Novartis Pharma, and Sanofi-Aventis; INSERM (Réseaux en Santé Publique, Interactions entre les déterminants de la santé, Cohortes Santé TGIR 2008); the Association Diabète Risque Vasculaire; the Fédération Française de Cardiologie; La Fondation de France; Association de Langue Française pour l'Etude du Diabète et des Maladies Métaboliques (ALFEDIAM)/Société Francophone de Diabétologie (SFD); l'Office National Interprofessionnel des Vins 11 (ONIVINS); Ardix Medical; Bayer Diagnostics; Becton Dickinson; Cardionics; Merck Santé; Novo Nordisk; Pierre Fabre; Roche; Topcon.

The analysis and interpretation of the data has been done without the participation of these organizations and companies.

Disclosure of interest

Frédéric Fumeron has received research support and honoraria from CNIEL (National Interprofessional Center for the Dairy Economy). The other authors declare that they have no competing interest. a by two-way (sex, intake) ANCOVA (on Ln values) for repeated measures, adjusted for age, BMI, alcohol intake, total energy intake, physical activity, and plasma cholesterol, triglycerides and glucose. No statistically significant interaction effect for intake X time was observed.

b by one-way (intake) ANCOVA for repeated measures, same adjustments. No statistically significant interaction effect for intake X time was observed, but in women, in low intake group: P(time)=0.01, in high intake group: P(time)= 0.95. Data are adjusted geometric means [95% CI]. No significant interaction effect intake X sex was observed.

a By two-way ANCOVA (on Ln values) for repeated measures, adjusted for age, BMI, alcohol intake, total energy intake, physical activity, and plasma cholesterol, triglycerides, and glucose. One way ANCOVA, in low intake group: P (time)=0.14, in high intake group: P (time)= 0.11.