Early Low-Fat Diet Enriched With Linolenic Acid Reduces Liver Endocannabinoid Tone and Improves Late Glycemic Control After a High-Fat Diet Challenge in Mice Free

The endocannabinoid (EC) system (ECS) is known to play a crucial role in energy homeostasis. Regulation by this system takes place at the central level by changing food intake (1), and at the peripheral level by the modification of energy metabolism (2). An overactive ECS plays a crucial role in obesity by increasing food intake (3) and lipogenesis (4), by downregulating catabolic reactions (5,6), and by promoting fat accumulation and alteration of glucose homeostasis. As a consequence, treating obesity by decreasing ECS activity has been considered. A pharmacological approach was developed, leading to the commercialization of an inverse agonist of the cannabinoid receptor type-1 (CB1R) rimonabant. However, this drug was withdrawn from the market because of its undesired central nervous system side effects (7). Meanwhile, the downregulation of ECS tone in peripheral tissues involved in energy homeostasis, either with non–brain-penetrant CB1R blockers, or inhibitors of the biosynthesis of endogenous CB1R agonists, is still considered to be a potential approach to counter the adverse events observed in obesity (8).

The ECS is defined as a set of endogenous ligands (ECs), synthesized and degraded by specific enzymes and receptors that are able to bind these molecules. It includes two membrane receptors, CB1R and CB2R, and two main endogenous agonists, N-arachidonoyl-ethanolamine (AEA [or anandamide]) and 2-arachidonoyl-glycerol (2-AG). AEA is typically synthesized by the enzyme N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD), although alternative pathways exist, whereas 2-AG is formed by the action of diacylglycerol lipase α or β upon diacylglycerols. After release, ECs are subjected to rapid breakdown by degrading enzymes such as fatty acid (FA) amide hydrolase (FAAH) for N-acylethanolamines and monoacylglycerol lipase (MAGL) for 2-AG (9) (Fig. 1A).

AEA and 2-AG are lipids, and both ultimately derive from arachidonic acid (AA [or C20:4n-6]) via AA-containing phospholipids. AA is endogenously biosynthesized from the essential FA (10) linoleic acid (LA [or C18:2n-6]). As a consequence, a dietary supplementation in LA (11,12) or AA itself (13) is able to elevate the tissue contents of EC. Conversely, diets enriched with n-3 polyunsaturated FAs, such as eicosapentaenoic acid (C20:5n-3) and docosahexaenoic acid (DHA [or C22:6n-3]), cause a decrease in 2-AG and AEA levels because of the replacement of AA in phospholipids with such FAs (13,14).

It is well known that dietary n-3 FAs exert beneficial effects on obesity (15). For instance, n-3 FAs have the ability to reduce ectopic fat and inflammation in Zucker rats and to reduce glucose intolerance that is generally associated with obesity (16). Thus, they were shown to improve insulin sensitivity and gluconeogenesis in rodents (17,18). Evidence, involving transcription factors such as cAMP-responsive element–binding protein H (CREBH) (19) and sterol regulatory element–binding protein (20), for the existence of a direct causal link between EC tone and the ability of n-3 FAs to improve some of these features of obesity has recently been shown (16). This emphasizes the potential benefits of nutritional interventions in the treatment of pathologies related to obesity, particularly using n-3 FAs.

Accumulating evidence (21) shows that dietary factors, including lipids, in developmental periods such as fetal life, infancy, and early childhood, are associated with obesity risk later in life. In line with this, it has been shown that early overnutrition leads to persistent dysregulation in leptin and insulin sensitivity (22), along with enhanced inflammatory response (23). In addition to excess calories, the FA composition of the perinatal diet is also an important factor in the nutritional programming of the metabolic phenotype in adulthood. Recent studies (24) have shown that perinatal exposure to a rich n-6 FA diet is able to induce obesity and to affect body fat mass across generations. On the other hand, the beneficial role of dietary n-3 FA during early life has been emphasized because n-3 FA deficiency increases adiposity in guinea pigs (25). This idea was reinforced by the study of Massiera et al. (26), showing that the addition of α-linolenic acid (α-LNA [or C18:3n-3]) to an LA-rich high-fat diet (HFD) under isolipidic and isocaloric conditions reduces the deleterious effect of n-6 FAs when given to mouse pups. The beneficial preventive effect of n-3 FA is not restricted to a HFD. Even in a normolipidic diet, they are able to limit HFD-induced insulin resistance (IR) and hepatic steatosis, two main features of obesity (27). In keeping with this, the improvement of glucose and lipid homeostasis was also observed in transgenic Fat-1 mice, which can synthesize n-3 FA from n-6 FA without the need for dietary supplementation (28).

In this study, we aimed to investigate the role of the FA composition of a normolipidic diet (n-6 vs. n-3 FA) in the early prevention of HFD-induced obesity and its relationship with EC tone. Our starting hypothesis was that a n-3 FA–enriched diet given to weaning mice can decrease ECS activity, which in turn prevents HFD-induced metabolic disorders. To support our data, we also examined liver EC tone in Fat-1 mice presenting consistently high levels of n-3 FA in their tissues.

Official French regulations (#87848) for the use and care of laboratory animals were followed throughout the experimental period. The experimental protocol was approved by the local ethics committee for animal experimentation (#BX0622). Three-week-old C57BL/6J male mice were purchased from Charles River Laboratories (Saint-Germain-Nuelles, France). Mice surviving the stress of delivery were randomly separated in three series of 14–16 animals receiving different low-fat diets (LFDs; 5% w/w total lipids) for 10 weeks as schematized in Fig. 1B. Animals were housed individually on a 12-h/12-h light/dark schedule at 22–23°C with ad libitum access to water and food. Lipids in lard series came exclusively from pork fat, while lard was partially substituted with safflower oil (SAF) or linseed oil (LIN) in SAF and LIN series, respectively. The FA composition of the custom diets manufactured by SSNIF (Soest, Germany) is presented in Table 1. After 10 weeks, half of the population of each series was used for analyses, and the rest was challenged with an HFD (30% w/w total lipids from lard) for an additional period of 10 weeks. C57BL/6J transgenic fat-1 mice were generated and housed as described previously (29).

Body composition (fat mass, lean mass, and total body water) was measured by EchoMRI (Echo Medical Systems, Houston, TX). Plasma triglyceride (TG) and cholesterol concentrations were determined using commercial kits (DyaSis Diagnostics, Grabels, France). Adiponectin levels were measured by ELISA from Merck Millipore (Darmstadt, Germany), while leptin and insulin plasma content was determined using a Luminex-based Bio-Plex Pro mouse assay (Bio-Rad, Marnes-La-Coquette, France).

For an oral glucose tolerance test (OGTT) and an insulin tolerance test (ITT), mice respectively received an oral load (2 g/kg) of a d-glucose solution (20% w/v) or an intraperitoneal injection of insulin (0.5 UI/kg; Actrapid; Novo Nordisk, Paris, France) after a 6-h fast. OGTTs and ITTs were performed in the same mice within a 3-day interval. Glycemia was measured at 0, 15, 30, 45, 60, 90, and 120 min directly in blood sampled from the tail vein with a My Life Pura Glucose Meter (Ypsomed, Paris, France). During an OGTT, larger blood samples (25 µl) were collected from tail in tubes containing EDTA (Sarstedt, Nümbrecht, Germany) to measure insulinemia at time 0, 15, 30, and 60 min after glucose load. Insulin levels were determined using an Ultrasensitive Mouse ELISA Kit (eurobio, Les Ulis, France).

For the determination of total FA composition in tissue and diets, lipids were extracted according to the method of Folch et al. (30). Concentrations were determined using C17:0 as the internal standard, after methylation according to the procedure of Christie (31) and separation by gas chromatography, as previously described (32). ECs and congeners were purified from lipid extracts and determined by isotope dilution liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry using deuterated standards as described in the study by Bartelt et al. (33).

The isolation and quantification of total proteins from tissues was performed as previously described (34). Briefly, from each animal that was previously anesthetized, the liver was isolated and immediately frozen in liquid nitrogen. Each tissue was subsequently washed in cold PBS (without Ca²⁺ and Mg²⁺, pH 7.4) and homogenized in a lysis solution containing the following: 150 mmol/L NaCl, 1 mmol/L EDTA, 1% (v/v) Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na₃VO₄, 20 mmol/L Tris-HCl, pH 8, and 1% SDS, plus protease inhibitors, at pH 7.4. Lysates were incubated for 30 min at 4°C on a shaker and then were centrifuged for 15 min at 13,000g at 4°C. Supernatants were transferred into clear tubes and quantified by DC Protein Assay (Bio-Rad). Subsequently, the samples (60–80 µg of total protein) were boiled for 5 min in Laemmli SDS loading buffer and loaded on 8–10% SDS-PAGE and then transferred to a polyvinylidene fluoride membrane. Filters were incubated overnight at 4°C with the following antibodies: 1) mouse anti-FAAH clone 4H8 (dilution 1:1,000; Sigma-Aldrich); 2) rabbit anti–NAPE-PLD (dilution 1:2,500; Abnova, Taipei, Taiwan); 3) rabbit anti-MAGL (dilution 1:200; Cayman Chemicals, Ann Arbor, MI). The monoclonal anti-tubulin clone B-5–1-2 (dilution 1:5,000; Sigma-Aldrich) was used to check for equal protein loading. Reactive bands were detected by chemiluminescence by the use of Clarity Western ECL substrate (Bio-Rad). Images were analyzed on a Chemi-Doc station with Quantity One Software (Bio-Rad).

FAAH and MAGL activity was measured as previously described (34). In particular, 2-AG hydrolysis was measured by incubating the 10,000g liver cytosolic fraction (100 µg/sample) in Tris-HCl 50 mmol/L, at pH 7.0 at 37°C for 20 min, with synthetic 2-arachidonoyl-[³H]-glycerol (40 Ci/mmol; ARC, St. Louis, MO) properly diluted with 2-AG (Cayman Chemicals) to the final concentration of 10 μmol/L. The amount of [³H]-glycerol produced was measured by scintillation counting of the aqueous phase after extraction of the incubation mixture with 2 volumes of CHCl₃/CH₃OH (1/1; v/v). AEA hydrolysis was measured by incubating the 10,000g liver membrane fraction (70–100 µg/sample) in Tris-HCl l50 mmol/L, at pH 9–10 at 37°C for 30 min, with synthetic N-arachidonoyl-[¹⁴C]-ethanolamine (55 mCi/mmol; ARC, St. Louis, MO) properly diluted with AEA (Tocris Bioscience, Avonmouth, Bristol, U.K.) to the final concentration of 2 μmol/L. The amount of [¹⁴C]-ethanolamine produced was measured by scintillation counting of the aqueous phase. Activities were calculated in picomoles of substrate hydrolyzed × minutes × milligrams of protein.

Total mRNAs from tissues were extracted with Tri-Reagent (Euromedex, Souffelweyersheim, France), and 1 µg of RNA was reverse transcribed using the Iscript cDNA Kit (Bio-Rad). Real-time PCR was performed as described previously (6) using a StepOnePlus Real-Time PCR System (Life Technologies, Saint-Aubin, France). Primer sequences used for amplification are indicated in Supplementary Table 1. For each gene, a standard curve was established from four cDNA dilutions (1:10 to 1:10,000) and used to determine the relative gene expression variation after normalization with the geometric mean of three housekeeping genes (TATA box binding protein, L38, and 18S).

Results are expressed as the means ± SEM. Data were analyzed statistically using two-way ANOVA followed by the Tukey post hoc test, or using the Student t test. Differences were considered significant at P < 0.05.

Lard used for the preparation of the LFD was partially replaced by SAF or LIN to modify the proportions of LA and α-LNA, while limiting background variations. In this way, the final n-6/n-3 FA ratios in lard, SAF, and LIN diets were 22, 53 and 1, respectively (Table 1). The impact of 10 weeks of feeding with the different LFDs was estimated through the analysis of total liver FA composition (Table 1). As expected, when compared with lard, the SAF diet increased the proportions of LA and AA in liver lipids at the expense of C18:1n-9, while LIN induced an increase in DHA. This resulted in strong alterations of tissue n-6/n-3 FA ratios. After challenging the animals with a 30% lard oil diet for 10 weeks, the liver SAF and LIN FA profiles became very close to that of the lard diet. Nevertheless, the n-6/n-3 FA ratio in liver LIN remained slightly lower than those for lard and SAF.

Switching the n-6/n-3 FA ratio from 53 to 1 in the diet (SAF vs. LIN diet) affected both AEA and 2-AG tissue levels in the liver (Fig. 2). By comparing the SAF group with the lard group, we aimed to reflect the impact of a specific elevation of LA levels on tissue EC contents. Elevating dietary LA levels increased only 2-AG levels in the liver. Likewise, the specific impact of the elevation of n-3 FA levels in the diet on EC levels was estimated by comparing the LIN group with lard group. In these conditions, AEA levels were significantly lower in the liver of LIN mice compared with lard mice, while the same trend was observed for 2-AG. In other tested tissues (Supplementary Fig. 1), a huge nutritional alteration in the n-6/n-3 FA ratio in the diet always significantly reduced AEA levels, except for the visceral adipose tissue (VAT). The levels of 2-AG were concomitantly reduced in brain and muscle but was not modified in subcutaneous adipose tissue (SCAT) or in VAT.

Whatever the early diet that was administered, feeding the HFD raised liver AEA and 2-AG levels (Fig. 2). However, the increase was not significant for 2-AG in SAF series because levels were already elevated at the end of the LFD. EC levels were also generally increased after the HFD in brain, VAT, and muscle. Otherwise, SCAT appeared to be differently influenced by the HFD regardless of the early diet, as suggested by the decrease in EC levels (Supplementary Fig. 1).

Transcriptomic and proteomic analysis of the ECS are presented in Figure. 3A–C. Data concerning FAAH and MAGL, which mainly hydrolyze AEA and 2-AG, respectively, indicated that the liver protein levels and activity of MAGL were more influenced by the composition of the early diet than those of FAAH. In particular, both the protein levels and activity of MAGL were reduced by the SAF diet compared with other diets, which is in accordance with the higher levels of 2-AG in this group. Protein levels of NAPE-PLD, which is involved in the formation of AEA, were also reduced by the SAF and LIN diets compared with the lard diet. Regarding CB1R, liver mRNA levels were unchanged, whatever the diet.

Challenging mice with a 30% lard diet induced a more pronounced alteration of liver enzymes linked to ECS. Data related to FAAH indicated a strong downregulation, while NAPE-PLD levels were increased, suggesting an inverse regulation of EC degradation and biosynthetic pathways in favor of an elevation of AEA levels. The HFD concomitantly induced the mRNA expression of CB1R in the liver for all groups. Interestingly, the impact of the HFD sometimes appeared to depend on the nutritional history. In particular, CB1R mRNA expression was lower in the LIN group compared with the SAF group, and FAAH protein levels were higher in the LIN group than in the lard group.

Body weight and fat pad relative mass measured at the end of the early LFD did not differ among the three groups (Table 2). Nevertheless, LIN induced a significant decrease in total fat mass, as determined by EchoMRI. The particular sensitivity of the liver to the different early diets was indicated by the decrease in relative mass and lipid content induced by SAF and LIN compared with lard. Similarly, muscle lipids were also the highest for the lard group (data not shown). As expected, total body fat, fat pad weight, and tissue lipid levels were increased after the HFD challenge, but no differences were observed among groups.

Early feeding with SAF and LIN experimental diets improved some important plasma parameters related to glucose and lipid homeostasis compared with lard (Table 2). Notably, fasting triglyceridemia and cholesterolemia were reduced after the SAF and LIN diets. Although these diets did not affect glycemia, insulin levels in these groups were higher than those in lard group, suggesting a possible impairment of β-cell function by early and prolonged exposure to a lard diet. Interestingly, levels of plasma adiponectin were significantly higher in animals eating a LIN diet than in those eating a lard diet, pointing out a putative impact of n-3 FA on adipose tissue metabolism. Data relative to glycemic control (Fig. 4A and B) showed that only the LIN diet exerted slight changes on glucose clearance after oral glucose load or insulin administration.

As expected, HFD challenge induced marked variations in most of the metabolic parameters measured (Table 2). Glycemia, cholesterolemia, insulinemia, and adiponectin and leptin levels increased in all groups. The slight hyperglycemia associated with the compensatory hyperinsulinemia reflected the onset of an insulin-resistant state, no matter which early diet was consumed. However, calculations derived from OGTT results and insulin response (DI_0–15/DG_0–15 [ratio of insulin production to glucose load] and DI₀ [oral disposition index]) suggested an alteration in β-cell function for mice fed with the lard diet only (Table 2). Data also revealed that animals fed with the diet enriched in n-3 FA over the 10 weeks after weaning had higher plasma adiponectin levels and better glycemic control after HFD challenge. Notably, OGTT, ITT, and HOMA-IR results were improved in the LIN group compared with the other groups (Fig. 4C and D and Table 2). In addition, t_0–30min insulin production in response to glucose load was also significantly higher in the LIN group than in the lard group (Fig. 4E).

The fact that early feeding with LIN induced long-lasting positive effects on glycemic control prompted us to study the impact of the different diets on the expression of genes related to carbohydrate and lipid metabolism in the liver (Fig. 5). Thus, early feeding with the LIN diet significantly decreased mRNA levels of the key gluconeogenic enzyme genes PEPCK and glucose-6-phosphate (G6P), as well as that of glucokinase gene (GCK), FA synthase (FAS), stearoyl-CoA desaturase 1 (SCD1), and the transcription factor CREBH, compared with the lard group. Data also revealed that young mice fed with the diet exclusively containing lard as a lipid source showed the highest mRNA expression of FAT/CD36 (FA translocase) and LPL genes related to FA uptake. This observation also applied to tumor necrosis factor-α (TNF-α) mRNA levels, suggesting an elevated liver inflammatory status in the livers of mice eating the lard diet, which was decreased by the SAF and LIN diets.

Whatever the early diet, feeding mice with HFD-induced changes in the expressions of genes responsive to insulin, such as PEPCK and GCK, which were, respectively, decreased and increased. We further noticed that mice prefed with the LIN diet showed lower expression of several genes after HFD challenge compared with other diets, suggesting that the addition of n-3 FA in the early diet may induce biological imprinting mechanisms controlling gene expression. In this way, PEPCK, CREBH, LPL, and TNF-α mRNA levels were lower in the LIN group than in the SAF and lard groups.

For further insight into the impact of n-3 FA tissue enrichment on ECS tone, we measured EC content in the livers of Fat-1 mice (Fig. 6). These transgenic animals are able to endogenously synthesize n-3 FA from n-6 FA and consequently exhibit high levels of n-3 FA in their tissues. Interestingly, EC levels were strongly reduced in Fat-1 mouse livers (Fig. 6A). We also found that the expression of FAAH, PEPCK, G6P, and FAS genes was lower in Fat-1 than in wild-type mice (Fig. 6B).

The objective of this study was twofold. First, we explored whether early exposure to a diet enriched in n-3 or n-6 FA could concomitantly influence EC tone in several mouse tissues and metabolic parameters, with particular attention to the liver, which is considered to be the most vulnerable organ after nutritional programming during the perinatal period. Second, we examined the long-term effects of these postnatal nutritional manipulations by examining whether they were associated with alterations of metabolic parameters in response to a later HFD challenge. Our data indicate in particular that exposure to an n-3 FA–enriched diet at an early age induces a marked reduction in liver ECS activity associated with an alteration of key enzymes involved in liver carbohydrate and lipid metabolism. In addition, we observed that some of the liver gene expression modifications induced by n-3 FA feeding in the first weeks of life persisted after HFD challenge and were associated with an improved glycemic control.

A recent series of studies carried out by Alvheim et al. (11,12,14) highlighted the importance of the dietary LA on EC tone. These works demonstrated that excessive LA consumption elevates tissue EC levels and is associated with metabolic alterations. Here, some important issues concerning the impact of dietary FA, and particularly n-3 FA, on ECS activity were also emphasized. First, we observed that the substitution of 0.5% lard for LIN in the early diet was sufficient to decrease EC levels in brain, liver, and muscle. Because EC tissue levels were generally decreased when the diet was enriched in α-LNA, regardless of changes in LA levels, it might be suggested that EC synthesis is more influenced by n-3 than n-6 dietary FA. The fact that increasing the amount of LA in the diet while α-LNA levels remained constant did not induce a marked variation in EC levels also supports this assumption. In line with this, the decrease in liver AEA level induced by long-term administration of LIN compared with lard could not be due to a net decrease in the supply of n-6 biosynthetic precursors of EC because LA levels were in the same range in the two diets. Instead, competition between n-6 and n-3 FA for elongation and desaturation steps may be crucial for EC synthesis in these conditions. We also found here that early dietary n-3 FA reduces EC levels in the brain and the skeletal muscle. Decreases in both AEA and 2-AG, with the former persisting after HFD challenge, were observed in the LIN group compared with the SAF group. On the whole, these findings, in agreement with previous data in adult mice (35) and rats (34,36), confirm that early dietary interventions based on n-3 FA, might constitute an alternative strategy to “global” CB1R blockers to reduce ECS overactivity and, subsequently, various parameters of the metabolic syndrome, while possibly limiting the consequences on brain function.

Animals from the lard series were fed with a 5% lipid diet, consisting mainly of long chain–saturated and monounsaturated FA (MUFA), and were initially prone to the induction of metabolic disorders related to IR. So it was not surprising to observe that replacing part of the lard with SAF or LIN in the diet was able to limit the alterations of some metabolic parameters, such as triglyceridemia, cholesterolemia, and liver and muscle lipid content, which is in agreement with previous studies (37). The lower insulin levels observed in lard compared with SAF and LIN mice may also represent an impairment of β-cell function induced by prolonged exposure to saturated FA (38). However, a notable finding from this work is that only the diet enriched with α-LNA induced specific changes in the liver expression of genes involved in gluconeogenesis and de novo lipogenesis. Thus, the LIN diet decreased PEPCK, G6P, and GCK mRNA levels, suggesting a slowing down of liver glucose production. Molecular data also indicated that dietary n-3 FA reduces the liver expression of SCD1 and FAS, suggesting a decrease in FA de novo synthesis, which was further illustrated by the lower liver lipid content observed in this group. These findings concur with those of other studies (39,40) showing an inhibitory effect of n-3 FA on gluconeogenesis and de novo lipogenesis, two key actors in IR setup. Although the changes were not statistically significant, the LIN diet also tended to improve glucose and insulin tolerance compared with the lard diet, suggesting that stronger positive effects of the diet might have occurred with longer treatment.

Our findings are reminiscent of those observed in Fat-1 transgenic mice, which can endogenously synthesize n-3 FA from n-6 FA, and consequently show elevated levels of DHA in the liver compared with wild-type mice. They also appear to be protected from HFD-induced glucose intolerance, dyslipidemia, and liver steatosis (10,41). A recent study (28) indicated that Fat-1 mice display reduced capacity for gluconeogenesis and lipid synthesis, as suggested by the low hepatic protein expression of PEPCK, G6P, acetyl-CoA carboxylase (ACC), and FAS in the liver. Interestingly, in addition to confirming lower mRNA levels of these enzymes, we demonstrated that AEA and 2-AG levels are also strongly reduced in the livers of Fat-1 mice.

The observation that tissue enrichment with n-3 FA (by dietary or transgenic manipulation) induces a significant decrease in ECS tone in the liver and muscle supports the possible existence of a direct causal link between the decrease in EC tone induced by LIN diet and the improvement of metabolic parameters observed in our study. The role of the ECS in regulating glucose homeostasis is well known (19,42–44). In particular, studies using liver-specific CB1R knock-out mice demonstrated that hepatic CB1R activation is both necessary and sufficient to account for diet-induced hepatic IR (42). In primary hepatocytes, direct CB1R activation was found to induce glucose production by increasing the expression of CREBH and gluconeogenic genes (19). CREBH is a liver-specific transcription factor recently described as a crucial actor in the regulation of hepatic glucose metabolism in mammals. CREBH has been shown to be induced by fasting or insulin-resistant states in rodents and to activate the transcription of PEPCK or G6Pase gene. Consistent with this, we observed that mice fed with the LIN diet show low levels of PEPCK, G6P, and CREBH, which are associated with a reduced activity of the ECS in the liver. Therefore, it is conceivable that the low gluconeogenic gene expression observed in the livers of these mice is mediated by reduced CREBH expression observed in response to the decrease in EC tone induced by n-3 FA exposure.

Liu et al. (45) have recently proposed a functional link between the ECS and de novo lipogenesis pathways that could also apply to our findings. The authors identified hepatic MUFAs generated via SCD1 as endogenous inhibitors of FAAH in the liver, and thereby as being responsible for elevated hepatic levels of AEA. Here, the addition of LIN to the diet increased n-3 FA levels in liver lipids without depressing n-6 FA levels, but decreased the C18:1/C18:0 ratio, suggesting that part of the effects might be due to desaturase activity modification. Thus, the low AEA levels observed in the liver of LIN mice could result from the downregulation of FAS and SCD1 gene expression, and by low liver MUFA production, which in turn would increase the degradation of AEA by FAAH. The increase in ECS activity induced by HFD challenge is also in agreement with the potential impact of MUFA on EC biosynthesis. Thus, long-term administration of the lipogenic diet might have increased NAPE-PLD levels and decreased FAAH activity, thus leading to higher AEA levels.

Altogether, our results suggest that decreasing ECS activity by introducing n-3 FA into the early diet induces liver gene expression changes that may contribute to carbohydrate and lipid metabolism improvements. The reduction in fat mass expansion observed in LIN mice also suggests a general amelioration of energy homeostasis.

It is widely accepted that the nutritional environment and weight gain in the first years of life are associated with the risk of developing metabolic disorders. It has been shown that during the postnatal period, metabolic imprinting may occur and create a predisposition to an early onset and aggravation of metabolic disorders induced by exposure to HFD later in life (46). In the current study, the impact of early diets on the susceptibility to develop metabolic disorders induced by a subsequent HFD was tested by challenging mice with a 30% lard-based diet for 10 weeks. Evidence has accumulated indicating a tonic overactivation of ECS after HFD-induced obesity (14,35,47). Nevertheless, data from the literature (12,14,48) also suggest that the effect of HFD feeding on peripheral EC levels may depend on the FA composition of the diet. In the current study, we consistently observed that tissue EC contents were generally higher after long-term administration of an HFD, whatever the early LFD. Liver transcriptomic and proteomic analyses related to the ECS also supported a stimulatory effect of HFD on EC tone, as indicated by the marked increase in NAPE-PLD and CB1R expression along with the concomitant decrease in FAAH expression and activity. However, an important finding concerns EC levels in the inguinal AT, which were lowered in all groups after HFD challenge, which is in agreement with previous works (47,49) concerning the impact of HFD on adipose depots of both rodents and humans. Because leptin and insulin were strongly increased after HFD challenge, it might be suggested that the decrease in SCAT EC levels is due to the action of these hormones on EC production, as previously shown (50,51). This possibility implies that our dietary conditions did not yet alter SCAT metabolism to such a degree that it became resistant to hormonal control. The fact that adiponectin levels were increased by eating an HFD suggests that fat depots still had the capacity to expand by recruiting new adipocytes. Indeed, adiponectin production is suppressed as adipocytes become hypertrophic and macrophages infiltrate the tissue (52). So, it would be informative to determine whether ECS activity is still decreased in other, more severe, models of obesity in which the SCAT shows excessive hypertrophy and metabolic stress.

Feeding mice with 30% lard caused metabolic disorders that are typically attributed to diet-induced obesity independent of the postnatal nutritional history. However, the metabolic consequences were somehow limited, likely because of the total lipid content and the duration of the diet, which were not elevated. Thus, mice became fatter, and showed liver TG accumulation, hyperinsulinemia, hyperleptinemia, slight hyperglycemia, and hypercholesterolemia. While the differences concerning liver EC tone and lipid composition induced by the LIN early diet did not persist after HFD challenge, interestingly, we observed better glucose tolerance in these animals compared with the lard and SAF groups. Data further suggest that the better glycemic control observed in mice fed the early LIN diet was not dependent on β-cell function but rather was associated with an improvement in insulin sensitivity. This may be closely related to the reduced expression of some key genes involved in lipid and carbohydrate homeostasis in the liver of LIN-fed mice. Although differences were not always quite statistically significant for genes taken individually, the concomitant decrease in CB1R, PEPCK, G6P, CREBH, FAS, SCD1, SREBP1c, FAT/CD36, LPL, and TNF-α mRNA levels collectively suggests the existence of a metabolic imprinting set up by the early n-3 FA exposure period.

In conclusion, our results strongly support the possibility that early dietary n-3 FA induces a decrease in liver EC tone, giving rise to modifications persisting later in life and promoting resistance toward metabolic complications induced by an obesogenic diet. These findings support the emerging notion that dietary n-3 FA could be an alternative strategy to drug use to reduce the overactivity of peripheral ECS, and consequently to improve or prevent metabolic disorders related to obesity.

Acknowledgments. The authors thank Serge Monier (Plateforme de Cytométrie) from INSERM UMR866, Lipides, Nutrition, Cancer, for excellent technical assistance.

Funding. This work was supported by funds from the Regional Council of Burgundy and Groupe Lipides et Nutrition.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. L.D. and P.D. designed and analyzed the experiments, wrote the manuscript, and performed experiments related to FA compositions and metabolic and molecular parameters. F.P., S.B., and C.S. determined the tissue endocannabinoid contents. S.T.-F. performed experiments related to FA compositions and metabolic and molecular parameters and reviewed the written draft of the manuscript. F.A.I. performed Western blotting and enzyme activity experiments. J.G. and T.M. performed experiments related to FA compositions and metabolic and molecular parameters. J.B. produced Fat-1 mouse tissue samples. V.D. designed and analyzed the experiments and wrote the manuscript. V.D. and P.D. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.