early low-fat diet enriched with linolenic acid reduces liver … · 2016. 6. 14. · (dyasis...

Laurent Demizieux,1 Fabiana Piscitelli,2 Stephanie Troy-Fioramonti,1

Fabio Arturo Iannotti,2 Simona Borrino,2 Joseph Gresti,1 Tania Muller,1

Jerome Bellenger,3 Cristoforo Silvestri,2 Vincenzo Di Marzo,2 and Pascal Degrace1

Early Low-Fat Diet Enriched WithLinolenic Acid Reduces LiverEndocannabinoid Tone and ImprovesLate Glycemic Control After a High-FatDiet Challenge in MiceDiabetes 2016;65:1824–1837 | DOI: 10.2337/db15-1279

Evidence suggests that alterations of glucose and lipidhomeostasis induced by obesity are associated with theelevation of endocannabinoid tone. The biosynthesis of thetwo main endocannabinoids, N-arachidonoylethanolamineand 2-arachidonoyl-glycerol, which derive from arachi-donic acid, is influenced by dietary fatty acids (FAs). Weinvestigated whether exposure to n-3 FA at a young agemay decrease tissue endocannabinoid levels and preventmetabolic disorders induced by a later high-fat diet (HFD)challenge. Three-week-old mice received a 5% lipid dietcontaining lard, lard plus safflower oil, or lard plus linseedoil for 10 weeks. Then, mice were challenged with a 30%lard diet for 10 additional weeks. A low n-6/n-3 FA ratio inthe early diet induces a marked decrease in liver endo-cannabinoid levels. A similar reduction was observed intransgenic Fat-1 mice, which exhibit high tissue levels ofn-3 FA compared with wild-type mice. Hepatic expres-sion of key enzymes involved in carbohydrate and lipidmetabolism was concomitantly changed. Interestingly,some gene modifications persisted after HFD challengeand were associated with improved glycemic control.These findings indicate that early dietary interventionsbased on n-3 FA may represent an alternative strategyto drugs for reducing endocannabinoid tone and improv-ing metabolic parameters in the metabolic syndrome.

The endocannabinoid (EC) system (ECS) is known to playa crucial role in energy homeostasis. Regulation by this

system takes place at the central level by changing foodintake (1), and at the peripheral level by the modificationof energy metabolism (2). An overactive ECS plays a cru-cial role in obesity by increasing food intake (3) and lipo-genesis (4), by downregulating catabolic reactions (5,6),and by promoting fat accumulation and alteration ofglucose homeostasis. As a consequence, treating obesityby decreasing ECS activity has been considered. A phar-macological approach was developed, leading to the com-mercialization of an inverse agonist of the cannabinoidreceptor type-1 (CB1R) rimonabant. However, this drugwas withdrawn from the market because of its undesiredcentral nervous system side effects (7). Meanwhile, thedownregulation of ECS tone in peripheral tissues involvedin energy homeostasis, either with non–brain-penetrantCB1R blockers, or inhibitors of the biosynthesis of endog-enous CB1R agonists, is still considered to be a potentialapproach to counter the adverse events observed in obe-sity (8).

The ECS is defined as a set of endogenous ligands (ECs),synthesized and degraded by specific enzymes and receptorsthat are able to bind these molecules. It includes two mem-brane receptors, CB1R and CB2R, and two main endogenousagonists, N-arachidonoyl-ethanolamine (AEA [or ananda-mide]) and 2-arachidonoyl-glycerol (2-AG). AEA is typicallysynthesized by the enzyme N-acylphosphatidylethanolaminephospholipase D (NAPE-PLD), although alternative pathways

1Team Pathophysiology of Dyslipidemia, Faculty of Sciences Gabriel, INSERM UMR866“Lipides, Nutrition, Cancer,” Université de Bourgogne Franche-Comté, Dijon, France2Endocannabinoid Research Group, Institute of Biomolecular Chemistry, ConsiglioNazionale delle Ricerche, Pozzuoli, Naples, Italy3Team Lipid Transfer Proteins and Lipoprotein Metabolism, Faculty of SciencesGabriel, INSERM UMR866 “Lipides, Nutrition, Cancer,” Université de BourgogneFranche-Comté, Dijon, France

Corresponding authors: Vincenzo Di Marzo, [email protected], and PascalDegrace, [email protected].

Received 11 September 2015 and accepted 28 March 2016.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1279/-/DC1.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

1824 Diabetes Volume 65, July 2016

METABOLISM

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exist, whereas 2-AG is formed by the action of diacylglycerollipase a or b upon diacylglycerols. After release, ECs aresubjected to rapid breakdown by degrading enzymessuch as fatty acid (FA) amide hydrolase (FAAH) forN-acylethanolamines and monoacylglycerol lipase (MAGL)for 2-AG (9) (Fig. 1A).

AEA and 2-AG are lipids, and both ultimately derivefrom arachidonic acid (AA [or C20:4n-6]) via AA-containingphospholipids. AA is endogenously biosynthesized fromthe essential FA (10) linoleic acid (LA [or C18:2n-6]). As aconsequence, a dietary supplementation in LA (11,12) orAA 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 docosahex-aenoic acid (DHA [or C22:6n-3]), cause a decrease in 2-AGand AEA levels because of the replacement of AA in phos-pholipids with such FAs (13,14).

It is well known that dietary n-3 FAs exert beneficialeffects on obesity (15). For instance, n-3 FAs have the abil-ity to reduce ectopic fat and inflammation in Zucker ratsand to reduce glucose intolerance that is generally as-sociated with obesity (16). Thus, they were shown toimprove insulin sensitivity and gluconeogenesis in rodents(17,18). Evidence, involving transcription factors such ascAMP-responsive element–binding protein H (CREBH) (19)

and sterol regulatory element–binding protein (20), for theexistence of a direct causal link between EC tone and theability of n-3 FAs to improve some of these features of obe-sity has recently been shown (16). This emphasizes the po-tential benefits of nutritional interventions in the treatmentof pathologies related to obesity, particularly using n-3 FAs.

Accumulating evidence (21) shows that dietary factors,including lipids, in developmental periods such as fetallife, infancy, and early childhood, are associated with obe-sity risk later in life. In line with this, it has been shownthat early overnutrition leads to persistent dysregulationin leptin and insulin sensitivity (22), along with enhancedinflammatory response (23). In addition to excess calories,the FA composition of the perinatal diet is also an impor-tant factor in the nutritional programming of the meta-bolic phenotype in adulthood. Recent studies (24) haveshown that perinatal exposure to a rich n-6 FA diet is ableto induce obesity and to affect body fat mass across gen-erations. On the other hand, the beneficial role of dietaryn-3 FA during early life has been emphasized because n-3FA deficiency increases adiposity in guinea pigs (25). Thisidea was reinforced by the study of Massiera et al. (26),showing that the addition of a-linolenic acid (a-LNA [orC18:3n-3]) to an LA-rich high-fat diet (HFD) under iso-lipidic and isocaloric conditions reduces the deleterious

Figure 1—Schematic illustration of the pathways for biosynthesis and degradation of ECs (A) and the experimental study design (B). AEA,N-arachidonoyl-ethanolamine; 2-AG, 2-arachidonoyl-glycerol; DAGL, diacylglycerol lipase; ECs, endocannabinoids; FAAH, fatty acid am-ide hydrolase; MAGL, monoacylglycerol lipase; NAPE-PLD, N-acylphosphatidylethanolamine phospholipase D.

diabetes.diabetesjournals.org Demizieux and Associates 1825

effect of n-6 FAs when given to mouse pups. The beneficialpreventive effect of n-3 FA is not restricted to a HFD. Evenin a normolipidic diet, they are able to limit HFD-inducedinsulin resistance (IR) and hepatic steatosis, two main fea-tures of obesity (27). In keeping with this, the improve-ment of glucose and lipid homeostasis was also observedin transgenic Fat-1 mice, which can synthesize n-3 FA fromn-6 FA without the need for dietary supplementation (28).

In this study, we aimed to investigate the role of theFA composition of a normolipidic diet (n-6 vs. n-3 FA) inthe early prevention of HFD-induced obesity and itsrelationship with EC tone. Our starting hypothesis wasthat a n-3 FA–enriched diet given to weaning mice candecrease ECS activity, which in turn prevents HFD-induced metabolic disorders. To support our data, we alsoexamined liver EC tone in Fat-1 mice presenting consis-tently high levels of n-3 FA in their tissues.

RESEARCH DESIGN AND METHODS

Animals and DietsOfficial French regulations (#87848) for the use and careof laboratory animals were followed throughout the ex-perimental period. The experimental protocol was approvedby the local ethics committee for animal experimentation(#BX0622). Three-week-old C57BL/6J male mice werepurchased from Charles River Laboratories (Saint-Germain-Nuelles, France). Mice surviving the stress of delivery wererandomly separated in three series of 14–16 animals re-ceiving different low-fat diets (LFDs; 5% w/w total lipids)for 10 weeks as schematized in Fig. 1B. Animals werehoused individually on a 12-h/12-h light/dark scheduleat 22–23°C with ad libitum access to water and food.Lipids in lard series came exclusively from pork fat, whilelard was partially substituted with safflower oil (SAF) orlinseed oil (LIN) in SAF and LIN series, respectively.The FA composition of the custom diets manufacturedby SSNIF (Soest, Germany) is presented in Table 1. After

10 weeks, half of the population of each series was usedfor analyses, and the rest was challenged with an HFD(30% w/w total lipids from lard) for an additional periodof 10 weeks. C57BL/6J transgenic fat-1 mice were gener-ated and housed as described previously (29).

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

Glucose Tolerance and Insulin Tolerance TestsFor an oral glucose tolerance test (OGTT) and an insulintolerance test (ITT), mice respectively received an oralload (2 g/kg) of a D-glucose solution (20% w/v) or an in-traperitoneal injection of insulin (0.5 UI/kg; Actrapid;Novo Nordisk, Paris, France) after a 6-h fast. OGTTs andITTs were performed in the same mice within a 3-day in-terval. Glycemia was measured at 0, 15, 30, 45, 60, 90,and 120 min directly in blood sampled from the tail veinwith a My Life Pura Glucose Meter (Ypsomed, Paris,France). During an OGTT, larger blood samples (25 ml)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 weredetermined using an Ultrasensitive Mouse ELISA Kit(eurobio, Les Ulis, France).

Lipid and EC AnalysesFor the determination of total FA composition in tissueand diets, lipids were extracted according to the methodof Folch et al. (30). Concentrations were determined using

Table 1—Diet and liver total FA composition

FA

Diet (% total FA) Liver after early LF diet Liver after HF diet challenge

Lard SAF LIN Lard SAF LIN Lard SAF LIN

C14:0 2.1 1.2 1.7

C16:0 27.7 17.6 24.0 24.40 6 0.56 22.49 6 0.24 25.36 6 0.22 25.19* 6 0.25 25.34* 6 0.36 26.23* 6 0.35

C16:1n-7 3.1 1.7 2.6 7.69a 6 1.14 4.00b 6 0.21 7.29a 6 0.28 4.38* 6 0.14 4.14 6 0.12 4.26* 6 0.16

C18:0 16.7 9.1 14.4 3.71a 6 0.19 7.97b 6 0.54 4.83a 6 0.17 3.91 6 0.44 3.82* 6 0.43 3.92* 6 0.43

C18:1n-9 40.5 28.1 37.4 44.47a 6 0.19 22.75b 6 0.81 35.63c 6 0.67 47.75* 6 1.63 46.49* 6 1.65 45.58* 6 1.73

C18:2n-6 8.6 40.9 10.0 5.11a 6 0.09 18.0b 6 0.51 7.34c 6 0.18 5.92* 6 0.33 6.33* 6 0.44 6.68 6 0.37

C18:3n-3 0.4 0.8 9.2

C20:4n-6 5.49a 6 0.36 14.0b 6 0.80 3.94c 6 0.09 5.23* 6 0.82 5.09* 6 0.83 5.05* 6 0.79

C22:6n-3 1.14a 6 0.17 2.34b 6 0.09 7.25c 6 0.13 0.27a* 6 0.22 1.37b* 6 0.22 1.99b* 6 0.40

n-6/n-3 22.3 52.7 1.1 8.72a 6 0.10 13.46b 6 0.73 1.03c 6 0.02 8.73a 6 0.63 9.34a* 6 0.59 6.36b* 6 0.50

Liver FAs are expressed as the mean 6 SEM (n = 7–9). Different superscript letters (a, b, c) indicate significant statistical differencesbetween groups after early LF diet or after HF diet challenge at P , 0.05. Values are not indicated when FA content did not reach1 g/100 g in at least one series. *Significant statistical differences between HF diet challenge vs. corresponding early LF dietmeans (P , 0.05).

1826 Dietary Fatty Acids and Endocannabinoid Tone Diabetes Volume 65, July 2016

C17:0 as the internal standard, after methylation accordingto the procedure of Christie (31) and separation by gaschromatography, as previously described (32). ECs and con-geners were purified from lipid extracts and determined byisotope dilution liquid chromatography–atmospheric pres-sure chemical ionization–mass spectrometry using deuter-ated standards as described in the study by Bartelt et al. (33).

Western Blot AnalysisThe isolation and quantification of total proteins fromtissues was performed as previously described (34). Briefly,from each animal that was previously anesthetized, theliver was isolated and immediately frozen in liquid nitro-gen. Each tissue was subsequently washed in cold PBS(without Ca2+ and Mg2+, pH 7.4) and homogenized in alysis solution containing the following: 150 mmol/L NaCl,1 mmol/L EDTA, 1% (v/v) Triton X-100, 2.5 mmol/Lsodium pyrophosphate, 1 mmol/L b-glycerophosphate,1 mmol/L Na3VO4, 20 mmol/L Tris-HCl, pH 8, and 1%SDS, plus protease inhibitors, at pH 7.4. Lysates were in-cubated for 30 min at 4°C on a shaker and then were centri-fuged for 15 min at 13,000g at 4°C. Supernatants weretransferred into clear tubes and quantified by DC ProteinAssay (Bio-Rad). Subsequently, the samples (60–80 mg oftotal protein) were boiled for 5 min in Laemmli SDS load-ing buffer and loaded on 8–10% SDS-PAGE and then trans-ferred to a polyvinylidene fluoride membrane. Filters wereincubated 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) wasused to check for equal protein loading. Reactive bandswere detected by chemiluminescence by the use of ClarityWestern ECL substrate (Bio-Rad). Images were analyzed ona Chemi-Doc station with Quantity One Software (Bio-Rad).

Enzyme AssaysFAAH and MAGL activity was measured as previouslydescribed (34). In particular, 2-AG hydrolysis was mea-sured by incubating the 10,000g liver cytosolic fraction(100 mg/sample) in Tris-HCl 50 mmol/L, at pH 7.0 at37°C for 20 min, with synthetic 2-arachidonoyl-[3H]-glycerol(40 Ci/mmol; ARC, St. Louis, MO) properly diluted with2-AG (Cayman Chemicals) to the final concentration of10 mmol/L. The amount of [3H]-glycerol produced wasmeasured by scintillation counting of the aqueous phaseafter extraction of the incubation mixture with 2 volumesof CHCl3/CH3OH (1/1; v/v). AEA hydrolysis was mea-sured by incubating the 10,000g liver membrane fraction(70–100 mg/sample) in Tris-HCl l50 mmol/L, at pH 9–10at 37°C for 30 min, with synthetic N-arachidonoyl-[14C]-ethanolamine (55 mCi/mmol; ARC, St. Louis, MO) prop-erly diluted with AEA (Tocris Bioscience, Avonmouth,Bristol, U.K.) to the final concentration of 2 mmol/L.The amount of [14C]-ethanolamine produced was mea-sured by scintillation counting of the aqueous phase.

Activities were calculated in picomoles of substrate hydro-lyzed 3 minutes 3 milligrams of protein.

RT-PCRTotal mRNAs from tissues were extracted with Tri-Reagent(Euromedex, Souffelweyersheim, France), and 1 mg ofRNA was reverse transcribed using the Iscript cDNA Kit(Bio-Rad). Real-time PCR was performed as described pre-viously (6) using a StepOnePlus Real-Time PCR System(Life Technologies, Saint-Aubin, France). Primer sequencesused for amplification are indicated in SupplementaryTable 1. For each gene, a standard curve was establishedfrom four cDNA dilutions (1:10 to 1:10,000) and used todetermine the relative gene expression variation after nor-malization with the geometric mean of three housekeep-ing genes (TATA box binding protein, L38, and 18S).

Statistical AnalysisResults are expressed as the means 6 SEM. Data wereanalyzed statistically using two-way ANOVA followed bythe Tukey post hoc test, or using the Student t test. Dif-ferences were considered significant at P , 0.05.

RESULTS

Liver Total FA CompositionLard used for the preparation of the LFD was partiallyreplaced by SAF or LIN to modify the proportions of LA anda-LNA, while limiting background variations. In this way, thefinal 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 offeeding with the different LFDs was estimated through theanalysis of total liver FA composition (Table 1). As expected,when compared with lard, the SAF diet increased the propor-tions of LA and AA in liver lipids at the expense of C18:1n-9,while LIN induced an increase in DHA. This resulted instrong alterations of tissue n-6/n-3 FA ratios. After challeng-ing the animals with a 30% lard oil diet for 10 weeks, theliver SAF and LIN FA profiles became very close to that of thelard diet. Nevertheless, the n-6/n-3 FA ratio in liver LINremained slightly lower than those for lard and SAF.

Tissue EC LevelsSwitching 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 levelsin the liver (Fig. 2). By comparing the SAF group with thelard group, we aimed to reflect the impact of a specificelevation of LA levels on tissue EC contents. Elevating di-etary LA levels increased only 2-AG levels in the liver. Like-wise, the specific impact of the elevation of n-3 FA levels inthe diet on EC levels was estimated by comparing the LINgroup with lard group. In these conditions, AEA levels weresignificantly lower in the liver of LIN mice compared withlard mice, while the same trend was observed for 2-AG. Inother tested tissues (Supplementary Fig. 1), a huge nutri-tional alteration in the n-6/n-3 FA ratio in the diet alwayssignificantly reduced AEA levels, except for the visceral ad-ipose tissue (VAT). The levels of 2-AG were concomitantlyreduced in brain and muscle but was not modified in sub-cutaneous adipose tissue (SCAT) or in VAT.





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

Transcriptomic and Proteomic Analysis of ECSTranscriptomic and proteomic analysis of the ECS arepresented in Figure. 3A–C. Data concerning FAAH andMAGL, which mainly hydrolyze AEA and 2-AG, respec-tively, indicated that the liver protein levels and activityof MAGL were more influenced by the composition of theearly diet than those of FAAH. In particular, both the pro-tein levels and activity of MAGL were reduced by the SAFdiet compared with other diets, which is in accordance withthe higher levels of 2-AG in this group. Protein levels ofNAPE-PLD, which is involved in the formation of AEA,were also reduced by the SAF and LIN diets comparedwith the lard diet. Regarding CB1R, liver mRNA levelswere unchanged, whatever the diet.

Challenging mice with a 30% lard diet induced a morepronounced alteration of liver enzymes linked to ECS. Datarelated to FAAH indicated a strong downregulation, whileNAPE-PLD levels were increased, suggesting an inverseregulation of EC degradation and biosynthetic pathways infavor of an elevation of AEA levels. The HFD concomitantlyinduced the mRNA expression of CB1R in the liver for allgroups. Interestingly, the impact of the HFD sometimesappeared to depend on the nutritional history. In partic-ular, CB1R mRNA expression was lower in the LIN groupcompared with the SAF group, and FAAH protein levelswere higher in the LIN group than in the lard group.

Body Composition, Plasma Parameters, and GlycemicControlBody weight and fat pad relative mass measured at the endof the early LFD did not differ among the three groups

(Table 2). Nevertheless, LIN induced a significant decreasein total fat mass, as determined by EchoMRI. The particu-lar sensitivity of the liver to the different early diets wasindicated by the decrease in relative mass and lipid contentinduced by SAF and LIN compared with lard. Similarly, mus-cle lipids were also the highest for the lard group (data notshown). As expected, total body fat, fat pad weight, andtissue lipid levels were increased after the HFD challenge,but no differences were observed among groups.

Early feeding with SAF and LIN experimental dietsimproved some important plasma parameters related toglucose and lipid homeostasis compared with lard (Table2). Notably, fasting triglyceridemia and cholesterolemiawere reduced after the SAF and LIN diets. Although thesediets did not affect glycemia, insulin levels in these groupswere higher than those in lard group, suggesting a possi-ble impairment of b-cell function by early and prolongedexposure to a lard diet. Interestingly, levels of plasmaadiponectin were significantly higher in animals eating aLIN diet than in those eating a lard diet, pointing out aputative impact of n-3 FA on adipose tissue metabolism.Data relative to glycemic control (Fig. 4A and B) showedthat only the LIN diet exerted slight changes on glucoseclearance after oral glucose load or insulin administration.

As expected, HFD challenge induced marked variations inmost of the metabolic parameters measured (Table 2).Glycemia, cholesterolemia, insulinemia, and adiponectin andleptin levels increased in all groups. The slight hyperglycemiaassociated with the compensatory hyperinsulinemiareflected the onset of an insulin-resistant state, no mat-ter which early diet was consumed. However, calcula-tions derived from OGTT results and insulin response(DI0–15/DG0–15 [ratio of insulin production to glucoseload] and DI0 [oral disposition index]) suggested an alter-ation in b-cell function for mice fed with the lard dietonly (Table 2). Data also revealed that animals fed withthe diet enriched in n-3 FA over the 10 weeks after wean-ing had higher plasma adiponectin levels and better gly-cemic control after HFD challenge. Notably, OGTT, ITT,

Figure 2—Effect of early diets and later HFD challenge on liver AEA and 2-AG contents. Three-week-old mice were fed a 5% lipid dietcontaining lard, lard plus SAF, or lard plus LIN for 10 weeks (early LF diet). Then all mice were challenged with a 30% lard diet for 10additional weeks (HF diet challenge). Results are expressed as the mean 6 SEM (n = 4–6). Different superscript letters (a, b, c) indicatesignificant statistical differences between groups after early LF diet or after HF diet challenge at P < 0.05. *Significant statistical differencesbetween HF diet challenge and corresponding early LF diet means (P < 0.05).



Figure 3—Effect of early diets and later HFD challenge on the regulation of proteins involved in liver ECS activity. Three-week-old micewere fed a 5% lipid diet containing lard, lard plus SAF, or lard plus LIN for 10 weeks (early LF diet). Then all mice were challenged with a30% lard diet for 10 additional weeks (HF diet challenge). A: Gene expression analysis of CB1R, NAPE-PLD (denoted as NAPE), and FAAH.B: Representative immunoblots of liver protein analysis of FAAH, NAPE-PLD (denoted as NAPE), and MAGL with graphic densitometryquantification. C: FAAH and MAGL activity measured in four separate samples, as described in RESEARCH DESIGN AND METHODS. Results areexpressed as the mean6 SEM (n = 4–6). Different superscript letters (a, b, c) indicate significant statistical differences between groups afterearly LF diet or after HF diet challenge at P< 0.05. *Significant statistical differences between HF diet challenge and corresponding early LFdiet means (P < 0.05). a-tub, a-tubulin; CON, control.


and HOMA-IR results were improved in the LIN groupcompared with the other groups (Fig. 4C and D and Table2). In addition, t0–30min insulin production in response toglucose load was also significantly higher in the LIN groupthan in the lard group (Fig. 4E).

Expression of Genes Related to Carbohydrate andLipid Metabolism in the LiverThe fact that early feeding with LIN induced long-lastingpositive effects on glycemic control prompted us to studythe impact of the different diets on the expression ofgenes related to carbohydrate and lipid metabolism in theliver (Fig. 5). Thus, early feeding with the LIN diet signif-icantly decreased mRNA levels of the key gluconeogenicenzyme genes PEPCK and glucose-6-phosphate (G6P), aswell as that of glucokinase gene (GCK), FA synthase (FAS),stearoyl-CoA desaturase 1 (SCD1), and the transcriptionfactor CREBH, compared with the lard group. Data alsorevealed that young mice fed with the diet exclusivelycontaining lard as a lipid source showed the highestmRNA expression of FAT/CD36 (FA translocase) and LPLgenes related to FA uptake. This observation also applied totumor necrosis factor-a (TNF-a) mRNA levels, suggestingan elevated liver inflammatory status in the livers of miceeating the lard diet, which was decreased by the SAF andLIN diets.

Whatever the early diet, feeding mice with HFD-inducedchanges 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 theLIN diet showed lower expression of several genes afterHFD challenge compared with other diets, suggesting thatthe addition of n-3 FA in the early diet may induce biologicalimprinting mechanisms controlling gene expression. In thisway, PEPCK, CREBH, LPL, and TNF-a mRNA levels werelower in the LIN group than in the SAF and lard groups.

EC Levels and Gene Expression in Fat-1 Mice LiverFor further insight into the impact of n-3 FA tissueenrichment on ECS tone, we measured EC content in thelivers of Fat-1 mice (Fig. 6). These transgenic animals areable to endogenously synthesize n-3 FA from n-6 FA andconsequently exhibit high levels of n-3 FA in their tissues.Interestingly, EC levels were strongly reduced in Fat-1mouse livers (Fig. 6A). We also found that the expressionof FAAH, PEPCK, G6P, and FAS genes was lower in Fat-1than in wild-type mice (Fig. 6B).

DISCUSSION

The objective of this study was twofold. First, we exploredwhether early exposure to a diet enriched in n-3 or n-6 FAcould concomitantly influence EC tone in several mousetissues and metabolic parameters, with particular atten-tion to the liver, which is considered to be the mostvulnerable organ after nutritional programming duringthe perinatal period. Second, we examined the long-termeffects of these postnatal nutritional manipulations byexamining whether they were associated with alterations

Table 2—Body composition and plasma parameters

After early LF diet After HF diet challenge

Lard(n = 7)

SAF(n = 7)

LIN(n = 7)

Lard(n = 7)

SAF(n = 9)

LIN(n = 7)

BW (g) 22.7 6 0.74 22.8 6 0.47 23.5 6 0.74 35.4* 6 1.60 36.3* 6 2.13 36.4* 6 1.63

Liver (% of BW) 4.43a 6 0.07 3.61b 6 0.10 3.61b 6 0.07 4.00 6 0.19 4.17 6 0.25 3.78 6 0.22

pVAT (% of BW) 2.19 6 0.17 2.23 6 0.08 2.34 6 0.17 6.19 6 0.34 5.91 6 0.42 6.33 6 0.29

iSCAT (% of BW) 1.33a 6 0.07 1.02b 6 0.04 1.16c 6 0.06 2.74 6 0.28 2.84 6 0.31 2.82 6 0.18

Fat mass (% of BW) 13.9a 6 0.46 14.8 a 6 0.95 12.9 b 6 0.27 33.3* 6 1.73 33.4* 6 2.43 34.3* 6 1.88

TGs (mg/mL) 0.43a 6 0.04 0.27b 6 0.03 0.29b 6 0.04 0.27* 6 0.02 0.32 6 0.03 0.28 6 0.02

Cholesterol (mg/mL) 1.30a 6 0.06 1.17b 6 0.09 1.07b 6 0.12 1.68* 6 0.12 1.71* 6 0.12 1.69* 6 0.05

Adiponectin (mg/mL) 15.5a 6 2.17 20.9b 6 3.07 22.3b 6 2.59 28.66a* 6 1.69 31.75ab* 6 1.61 35.54b* 6 2.16

Leptin (pg/mL) 40.1 6 26.6 16.1 6 10.5 21.0 6 10.1 526.4* 6 129.1 890.5* 6 156.1 622.6* 6 141.3

Insulin (pg/mL) 25.4a 6 15.4 108.3b 6 36.0 131.5b 6 62.7 715.5* 6 168.3 837.0* 6 151.5 755.9* 6 119.2

Glucose (g/L) 1.76 6 0.06 1.70 6 0.02 1.63 6 0.10 2.07* 6 0.14 2.09* 6 0.11 1.97* 6 0.08

HOMA-IR 20.0ab 6 2.63 22.7a 6 3.40 15.4bc 6 1.98

DI0–15/DG0–15 2176a 6 220 2,344b 6 646 1,993b 6 504

DI0 20.002a 6 0.005 0.056b 6 0.014 0.060b 6 0.013

All parameters except glucose levels were measured from blood and tissue samples collected on the day of sacrifice from overnight-fasted animals. Glucose levels and calculations were determined from blood samples collected during OGTT experiments initiated withanimals fasted for 6 h. BW, body weight; DI0 –15/DG0–15, insulin production to glucose load (mIU/mmol); DI0, oral disposition index(mmol21) = (DI0–15/DG0–15)*1/fasting insulin; HOMA-IR, fasting glucose (mmol/L)*fasting insulin (mIU/L)/22.5; iSCAT, inguinal subcuta-neous adipose tissue; pVAT, periepididymal adipose tissue. Results are expressed as the mean 6 SEM (n = 7–9). Different superscriptletters (a, b, c) indicate significant statistical differences between groups after early LF diet or after HF diet challenge at P , 0.05.*Significant statistical differences between HF diet challenge and corresponding early LF diet means (P , 0.05).


of metabolic parameters in response to a later HFDchallenge. Our data indicate in particular that exposureto an n-3 FA–enriched diet at an early age induces amarked reduction in liver ECS activity associated with

an alteration of key enzymes involved in liver carbohy-drate and lipid metabolism. In addition, we observedthat some of the liver gene expression modificationsinduced by n-3 FA feeding in the first weeks of life

Figure 4—Effect of early diets and later HFD challenge on glycemic control. Three-week-old mice were fed a 5% lipid diet containing lard,lard plus SAF, or lard plus LIN for 10 weeks (early LF diet) and were subjected to an OGTT (2 g/kg) (A) and an ITT (0.5 IU/kg) (B). Then allmice were challenged with a 30% lard diet for 10 additional weeks (HF diet challenge) and subjected to an OGTT (C) and an ITT (D). E:Plasma insulin appearance after oral glucose load (2 g/kg) was determined after HF diet challenge. Results are expressed as the mean6 SEM(n = 7–9). *P < 0.05, LIN vs. lard and SAF. †P < 0.05, LIN vs. SAF. $P < 0.05, LIN vs. lard. AU, arbitrary units; AUC, area under the curve.


persisted after HFD challenge and were associated withan improved glycemic control.

A recent series of studies carried out by Alvheim et al.(11,12,14) highlighted the importance of the dietary LAon EC tone. These works demonstrated that excessive LA

consumption elevates tissue EC levels and is associatedwith metabolic alterations. Here, some important issuesconcerning the impact of dietary FA, and particularly n-3FA, on ECS activity were also emphasized. First, we ob-served that the substitution of 0.5% lard for LIN in the

Figure 5—Expression of genes related to carbohydrate and lipid metabolism in the liver. Three-week-old mice were fed a 5% lipid dietcontaining lard, lard plus SAF, or lard plus LIN for 10 weeks (early LF diet). Then all mice were challenged with a 30% lard diet for10 additional weeks (HF diet challenge). The results are expressed as the mean 6 SEM (n = 6–9). Different superscript letters (a, b) indicatesignificant statistical differences between groups after early LF diet or after HF diet challenge at P < 0.05. *Significant statistical differencesbetween HF diet challenge vs. corresponding early LF diet means (P < 0.05).


early diet was sufficient to decrease EC levels in brain,liver, and muscle. Because EC tissue levels were generallydecreased when the diet was enriched in a-LNA, regard-less of changes in LA levels, it might be suggested that

EC synthesis is more influenced by n-3 than n-6 dietaryFA. The fact that increasing the amount of LA in the dietwhile a-LNA levels remained constant did not induce amarked variation in EC levels also supports this assumption.

Figure 6—AEA and 2-AG contents and expression of genes related to the ECS and the carbohydrate and lipid metabolism in the livers ofFat-1 mice. A: Liver AEA and 2-AG contents. B: Gene expression analysis. Results are expressed as the mean 6 SEM (n = 5). *Significantstatistical differences between groups at P < 0.05. WT, wild type.


In line with this, the decrease in liver AEA level inducedby long-term administration of LIN compared with lardcould not be due to a net decrease in the supply of n-6biosynthetic precursors of EC because LA levels were inthe same range in the two diets. Instead, competitionbetween n-6 and n-3 FA for elongation and desaturationsteps may be crucial for EC synthesis in these conditions.We also found here that early dietary n-3 FA reduces EClevels in the brain and the skeletal muscle. Decreases inboth AEA and 2-AG, with the former persisting afterHFD challenge, were observed in the LIN group com-pared 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 interventionsbased on n-3 FA, might constitute an alternative strategyto “global” CB1R blockers to reduce ECS overactivity and,subsequently, various parameters of the metabolic syn-drome, while possibly limiting the consequences on brainfunction.

Animals from the lard series were fed with a 5% lipiddiet, consisting mainly of long chain–saturated and mono-unsaturated FA (MUFA), and were initially prone to the in-duction of metabolic disorders related to IR. So it was notsurprising to observe that replacing part of the lard withSAF or LIN in the diet was able to limit the alterationsof some metabolic parameters, such as triglyceridemia,cholesterolemia, and liver and muscle lipid content,which is in agreement with previous studies (37). Thelower insulin levels observed in lard compared with SAFand LIN mice may also represent an impairment of b-cellfunction induced by prolonged exposure to saturated FA(38). However, a notable finding from this work is thatonly the diet enriched with a-LNA induced specificchanges in the liver expression of genes involved in glu-coneogenesis and de novo lipogenesis. Thus, the LIN dietdecreased PEPCK, G6P, and GCK mRNA levels, suggestinga slowing down of liver glucose production. Moleculardata also indicated that dietary n-3 FA reduces the liverexpression of SCD1 and FAS, suggesting a decrease in FAde novo synthesis, which was further illustrated by thelower liver lipid content observed in this group. Thesefindings concur with those of other studies (39,40) show-ing an inhibitory effect of n-3 FA on gluconeogenesis andde novo lipogenesis, two key actors in IR setup. Althoughthe changes were not statistically significant, the LINdiet also tended to improve glucose and insulin tolerancecompared with the lard diet, suggesting that strongerpositive effects of the diet might have occurred with longertreatment.

Our findings are reminiscent of those observed inFat-1 transgenic mice, which can endogenously synthe-size n-3 FA from n-6 FA, and consequently show elevatedlevels of DHA in the liver compared with wild-type mice.They also appear to be protected from HFD-inducedglucose intolerance, dyslipidemia, and liver steatosis(10,41). A recent study (28) indicated that Fat-1 micedisplay reduced capacity for gluconeogenesis and lipid

synthesis, as suggested by the low hepatic protein ex-pression of PEPCK, G6P, acetyl-CoA carboxylase (ACC),and FAS in the liver. Interestingly, in addition to confirminglower mRNA levels of these enzymes, we demonstrated thatAEA and 2-AG levels are also strongly reduced in the livers ofFat-1 mice.

The observation that tissue enrichment with n-3 FA(by dietary or transgenic manipulation) induces a signif-icant decrease in ECS tone in the liver and muscle supportsthe possible existence of a direct causal link between thedecrease in EC tone induced by LIN diet and the improve-ment of metabolic parameters observed in our study. Therole of the ECS in regulating glucose homeostasis is wellknown (19,42–44). In particular, studies using liver-specificCB1R knock-out mice demonstrated that hepatic CB1Ractivation is both necessary and sufficient to account fordiet-induced hepatic IR (42). In primary hepatocytes, directCB1R activation was found to induce glucose production byincreasing the expression of CREBH and gluconeogenicgenes (19). CREBH is a liver-specific transcription factorrecently described as a crucial actor in the regulationof hepatic glucose metabolism in mammals. CREBH hasbeen shown to be induced by fasting or insulin-resistantstates in rodents and to activate the transcription ofPEPCK or G6Pase gene. Consistent with this, we observedthat mice fed with the LIN diet show low levels of PEPCK,G6P, and CREBH, which are associated with a reducedactivity of the ECS in the liver. Therefore, it is conceivablethat the low gluconeogenic gene expression observed inthe livers of these mice is mediated by reduced CREBHexpression observed in response to the decrease in ECtone induced by n-3 FA exposure.

Liu et al. (45) have recently proposed a functional linkbetween the ECS and de novo lipogenesis pathways thatcould also apply to our findings. The authors identifiedhepatic MUFAs generated via SCD1 as endogenous in-hibitors of FAAH in the liver, and thereby as being re-sponsible for elevated hepatic levels of AEA. Here, theaddition of LIN to the diet increased n-3 FA levels in liverlipids without depressing n-6 FA levels, but decreased theC18:1/C18:0 ratio, suggesting that part of the effects mightbe due to desaturase activity modification. Thus, the lowAEA levels observed in the liver of LIN mice could resultfrom the downregulation of FAS and SCD1 gene expression,and by low liver MUFA production, which in turn would in-crease the degradation of AEA by FAAH. The increase in ECSactivity induced by HFD challenge is also in agreement withthe potential impact of MUFA on EC biosynthesis. Thus,long-term administration of the lipogenic diet might haveincreased NAPE-PLD levels and decreased FAAH activity,thus leading to higher AEA levels.

Altogether, our results suggest that decreasing ECSactivity by introducing n-3 FA into the early diet inducesliver gene expression changes that may contribute tocarbohydrate and lipid metabolism improvements. Thereduction in fat mass expansion observed in LIN mice alsosuggests a general amelioration of energy homeostasis.


It is widely accepted that the nutritional environmentand weight gain in the first years of life are associatedwith the risk of developing metabolic disorders. It hasbeen shown that during the postnatal period, metabolicimprinting may occur and create a predisposition to anearly onset and aggravation of metabolic disorders in-duced by exposure to HFD later in life (46). In the currentstudy, the impact of early diets on the susceptibility todevelop metabolic disorders induced by a subsequent HFDwas tested by challenging mice with a 30% lard-based dietfor 10 weeks. Evidence has accumulated indicating a tonicoveractivation of ECS after HFD-induced obesity (14,35,47).Nevertheless, data from the literature (12,14,48) also sug-gest that the effect of HFD feeding on peripheral EC levelsmay depend on the FA composition of the diet. In thecurrent study, we consistently observed that tissue ECcontents were generally higher after long-term adminis-tration of an HFD, whatever the early LFD. Liver tran-scriptomic and proteomic analyses related to the ECSalso supported a stimulatory effect of HFD on EC tone,as indicated by the marked increase in NAPE-PLD andCB1R expression along with the concomitant decrease inFAAH expression and activity. However, an importantfinding concerns EC levels in the inguinal AT, whichwere lowered in all groups after HFD challenge, whichis in agreement with previous works (47,49) concerningthe impact of HFD on adipose depots of both rodentsand humans. Because leptin and insulin were stronglyincreased after HFD challenge, it might be suggestedthat the decrease in SCAT EC levels is due to the actionof these hormones on EC production, as previouslyshown (50,51). This possibility implies that our dietaryconditions did not yet alter SCAT metabolism to such adegree that it became resistant to hormonal control. Thefact that adiponectin levels were increased by eating anHFD suggests that fat depots still had the capacity toexpand by recruiting new adipocytes. Indeed, adiponec-tin production is suppressed as adipocytes become hy-pertrophic and macrophages infiltrate the tissue (52).So, it would be informative to determine whether ECSactivity is still decreased in other, more severe, models ofobesity in which the SCAT shows excessive hypertrophyand metabolic stress.

Feeding mice with 30% lard caused metabolic disordersthat are typically attributed to diet-induced obesity in-dependent of the postnatal nutritional history. However,the metabolic consequences were somehow limited, likelybecause of the total lipid content and the duration of thediet, which were not elevated. Thus, mice became fatter,and showed liver TG accumulation, hyperinsulinemia,hyperleptinemia, slight hyperglycemia, and hypercholes-terolemia. While the differences concerning liver EC toneand lipid composition induced by the LIN early diet didnot persist after HFD challenge, interestingly, we ob-served better glucose tolerance in these animals comparedwith the lard and SAF groups. Data further suggest thatthe better glycemic control observed in mice fed the early

LIN diet was not dependent on b-cell function but ratherwas associated with an improvement in insulin sensitiv-ity. This may be closely related to the reduced expressionof some key genes involved in lipid and carbohydratehomeostasis in the liver of LIN-fed mice. Although dif-ferences were not always quite statistically significant forgenes taken individually, the concomitant decrease in CB1R,PEPCK, G6P, CREBH, FAS, SCD1, SREBP1c, FAT/CD36,LPL, and TNF-a mRNA levels collectively suggests theexistence of a metabolic imprinting set up by the earlyn-3 FA exposure period.

In conclusion, our results strongly support the possibil-ity that early dietary n-3 FA induces a decrease in liver ECtone, giving rise to modifications persisting later in life andpromoting resistance toward metabolic complications in-duced by an obesogenic diet. These findings support theemerging notion that dietary n-3 FA could be an alternativestrategy to drug use to reduce the overactivity of peripheralECS, and consequently to improve or prevent metabolicdisorders related to obesity.

Acknowledgments. The authors thank Serge Monier (Plateforme deCytométrie) from INSERM UMR866, Lipides, Nutrition, Cancer, for excellenttechnical assistance.Funding. This work was supported by funds from the Regional Council ofBurgundy and Groupe Lipides et Nutrition.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. L.D. and P.D. designed and analyzed theexperiments, wrote the manuscript, and performed experiments related to FAcompositions and metabolic and molecular parameters. F.P., S.B., and C.S.determined the tissue endocannabinoid contents. S.T.-F. performed experimentsrelated to FA compositions and metabolic and molecular parameters andreviewed the written draft of the manuscript. F.A.I. performed Western blottingand enzyme activity experiments. J.G. and T.M. performed experiments related toFA compositions and metabolic and molecular parameters. J.B. produced Fat-1mouse tissue samples. V.D. designed and analyzed the experiments and wrotethe manuscript. V.D. and P.D. are the guarantors of this work and, as such, hadfull access to all the data in the study and take responsibility for the integrity ofthe data and the accuracy of the data analysis.

References1. Di Marzo V, Goparaju SK, Wang L, et al. Leptin-regulated endocannabinoidsare involved in maintaining food intake. Nature 2001;410:822–8252. Quarta C, Mazza R, Obici S, Pasquali R, Pagotto U. Energy balance regu-lation by endocannabinoids at central and peripheral levels. Trends Mol Med2011;17:518–5263. Williams CM, Kirkham TC. Anandamide induces overeating: mediation by centralcannabinoid (CB1) receptors. Psychopharmacology (Berl) 1999;143:315–3174. Mallat A, Lotersztajn S. Endocannabinoids and their role in fatty liver dis-ease. Dig Dis 2010;28:261–2665. Jeong WI, Osei-Hyiaman D, Park O, et al. Paracrine activation of hepaticCB1 receptors by stellate cell-derived endocannabinoids mediates alcoholic fattyliver. Cell Metab 2008;7:227–2356. Jourdan T, Demizieux L, Gresti J, et al. Antagonism of peripheral hepaticcannabinoid receptor-1 improves liver lipid metabolism in mice: evidence fromcultured explants. Hepatology 2012;55:790–7997. Buggy Y, Cornelius V, Wilton L, Shakir SA. Risk of depressive episodes withrimonabant: a before and after modified prescription event monitoring studyconducted in England. Drug Saf 2011;34:501–509


8. Di Marzo V, Després JP. CB1 antagonists for obesity–what lessons have welearned from rimonabant? Nat Rev Endocrinol 2009;5:633–6389. Di Marzo V. Endocannabinoids: synthesis and degradation. Rev PhysiolBiochem Pharmacol 2008;160:1–2410. Li J, Li FR, Wei D, et al. Endogenous v-3 polyunsaturated fatty acid pro-duction confers resistance to obesity, dyslipidemia, and diabetes in mice. MolEndocrinol 2014;28:1316–132811. Alvheim AR, Torstensen BE, Lin YH, et al. Dietary linoleic acid elevates theendocannabinoids 2-AG and anandamide and promotes weight gain in mice fed alow fat diet. Lipids 2014;49:59–6912. Alvheim AR, Torstensen BE, Lin YH, et al. Dietary linoleic acid elevatesendogenous 2-arachidonoylglycerol and anandamide in Atlantic salmon (Salmosalar L.) and mice, and induces weight gain and inflammation in mice. Br J Nutr2013;109:1508–151713. Berger A, Crozier G, Bisogno T, Cavaliere P, Innis S, Di Marzo V. Ananda-mide and diet: inclusion of dietary arachidonate and docosahexaenoate leads toincreased brain levels of the corresponding N-acylethanolamines in piglets. ProcNatl Acad Sci U S A 2001;98:6402–640614. Alvheim AR, Malde MK, Osei-Hyiaman D, et al. Dietary linoleic acid elevatesendogenous 2-AG and anandamide and induces obesity. Obesity (Silver Spring)2012;20:1984–199415. Sato A, Kawano H, Notsu T, et al. Antiobesity effect of eicosapentaenoic acidin high-fat/high-sucrose diet-induced obesity: importance of hepatic lipogenesis.Diabetes 2010;59:2495–250416. Batetta B, Griinari M, Carta G, et al. Endocannabinoids may mediate theability of (n-3) fatty acids to reduce ectopic fat and inflammatory mediators inobese Zucker rats. J Nutr 2009;139:1495–150117. Kalupahana NS, Claycombe K, Newman SJ, et al. Eicosapentaenoicacid prevents and reverses insulin resistance in high-fat diet-induced obesemice via modulation of adipose tissue inflammation. J Nutr 2010;140:1915–192218. Hein GJ, Chicco A, Lombardo YB. Fish oil normalizes plasma glucose levelsand improves liver carbohydrate metabolism in rats fed a sucrose-rich diet.Lipids 2012;47:141–15019. Chanda D, Kim DK, Li T, et al. Cannabinoid receptor type 1 (CB1R) signalingregulates hepatic gluconeogenesis via induction of endoplasmic reticulum-boundtranscription factor cAMP-responsive element-binding protein H (CREBH) in pri-mary hepatocytes. J Biol Chem 2011;286:27971–2797920. Furuya DT, Poletto AC, Freitas HS, Machado UF. Inhibition of cannabinoidCB1 receptor upregulates Slc2a4 expression via nuclear factor-kB and sterolregulatory element-binding protein-1 in adipocytes. J Mol Endocrinol 2012;49:97–10621. Zalewski BM, Patro B, Veldhorst M, et al. Nutrition of infants and youngchildren (1-3 years) and its effect on later health: a systematic review of currentrecommendations (EarlyNutrition Project). Crit Rev Food Sci Nutr. 9 March 2015[Epub ahead of print]22. Glavas MM, Kirigiti MA, Xiao XQ, et al. Early overnutrition results in early-onset arcuate leptin resistance and increased sensitivity to high-fat diet. Endo-crinology 2010;151:1598–161023. Liu Z, Lim CY, Su MY, et al. Neonatal overnutrition in mice exacerbateshigh-fat diet-induced metabolic perturbations. J Endocrinol 2013;219:131–14324. Massiera F, Barbry P, Guesnet P, et al. A Western-like fat diet is sufficient toinduce a gradual enhancement in fat mass over generations. J Lipid Res 2010;51:2352–236125. Pouteau E, Aprikian O, Grenot C, et al. A low alpha-linolenic intake duringearly life increases adiposity in the adult guinea pig. Nutr Metab (Lond) 2010;7:826. Massiera F, Saint-Marc P, Seydoux J, et al. Arachidonic acid and prosta-cyclin signaling promote adipose tissue development: a human health concern?J Lipid Res 2003;44:271–27927. González-Périz A, Horrillo R, Ferré N, et al. Obesity-induced insulin re-sistance and hepatic steatosis are alleviated by omega-3 fatty acids: a role forresolvins and protectins. FASEB J 2009;23:1946–1957

28. Romanatto T, Fiamoncini J, Wang B, Curi R, Kang JX. Elevated tissueomega-3 fatty acid status prevents age-related glucose intolerance in fat-1transgenic mice. Biochim Biophys Acta 2014;1842:186–19129. Bellenger J, Bellenger S, Bataille A, et al. High pancreatic n-3 fatty acidsprevent STZ-induced diabetes in fat-1 mice: inflammatory pathway inhibition.Diabetes 2011;60:1090–109930. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation andpurification of total lipides from animal tissues. J Biol Chem 1957;226:497–50931. Christie WW. A simple procedure for rapid transmethylation of glycerolipidsand cholesteryl esters. J Lipid Res 1982;23:1072–107532. Niot I, Gresti J, Boichot J, et al. Effect of dietary n-3 and n-6 poly-unsaturated fatty acids on lipid-metabolizing enzymes in obese rat liver. Lipids1994;29:481–48933. Bartelt A, Orlando P, Mele C, et al. Altered endocannabinoid signalling aftera high-fat diet in Apoe(-/-) mice: relevance to adipose tissue inflammation, he-patic steatosis and insulin resistance. Diabetologia 2011;54:2900–291034. Iannotti FA, Piscitelli F, Martella A, et al. Analysis of the “endocannabinoidome”in peripheral tissues of obese Zucker rats. Prostaglandins Leukot Essent FattyAcids 2013;89:127–13535. Matias I, Petrosino S, Racioppi A, Capasso R, Izzo AA, Di Marzo V.Dysregulation of peripheral endocannabinoid levels in hyperglycemia andobesity: effect of high fat diets. Mol Cell Endocrinol 2008;286(Suppl. 1):S66–S7836. Di Marzo V, Griinari M, Carta G, et al. Dietary krill oil increases docosa-hexaenoic acid and reduces 2-arachidonoylglycerol but not N-acylethanolaminelevels in the brain of obese Zucker rats. Int Dairy J 2010;20:231–23537. Sealls W, Gonzalez M, Brosnan MJ, Black PN, DiRusso CC. Dietary poly-unsaturated fatty acids (C18:2 omega6 and C18:3 omega3) do not suppresshepatic lipogenesis. Biochim Biophys Acta 2008;1781:406–41438. Elks ML. Chronic perifusion of rat islets with palmitate suppresses glucose-stimulated insulin release. Endocrinology 1993;133:208–21439. Delarue J, LeFoll C, Corporeau C, Lucas D. N-3 long chain polyunsaturatedfatty acids: a nutritional tool to prevent insulin resistance associated to type 2diabetes and obesity? Reprod Nutr Dev 2004;44:289–29940. Rossi AS, Lombardo YB, Chicco AG. Lipogenic enzyme activities and glu-cose uptake in fat tissue of dyslipemic, insulin-resistant rats: effects of fish oil.Nutrition 2010;26:209–21741. White PJ, Arita M, Taguchi R, Kang JX, Marette A. Transgenic restoration oflong-chain n-3 fatty acids in insulin target tissues improves resolution capacityand alleviates obesity-linked inflammation and insulin resistance in high-fat-fedmice. Diabetes 2010;59:3066–307342. Liu J, Zhou L, Xiong K, et al. Hepatic cannabinoid receptor-1 mediates diet-induced insulin resistance via inhibition of insulin signaling and clearance inmice. Gastroenterology 2012;142:1218–1228.e143. Silvestri C, Di Marzo V. The endocannabinoid system in energy homeostasisand the etiopathology of metabolic disorders. Cell Metab 2013;17:475–49044. Troy-Fioramonti S, Demizieux L, Gresti J, Muller T, Vergès B, Degrace P.Acute activation of cannabinoid receptors by anandamide reduces gastrointes-tinal motility and improves postprandial glycemia in mice. Diabetes 2015;64:808–81845. Liu J, Cinar R, Xiong K, et al. Monounsaturated fatty acids generated viastearoyl CoA desaturase-1 are endogenous inhibitors of fatty acid amide hy-drolase. Proc Natl Acad Sci USA 2013;110:18832–1883746. Sullivan EL, Grove KL. Metabolic imprinting in obesity. Forum Nutr 2010;63:186–19447. Starowicz KM, Cristino L, Matias I, et al. Endocannabinoid dysregulation inthe pancreas and adipose tissue of mice fed with a high-fat diet. Obesity (SilverSpring) 2008;16:553–56548. Piscitelli F, Carta G, Bisogno T, et al. Effect of dietary krill oil supplemen-tation on the endocannabinoidome of metabolically relevant tissues from high-fat-fed mice. Nutr Metab (Lond) 2011;8:51


49. Annuzzi G, Piscitelli F, Di Marino L, et al. Differential alterations of theconcentrations of endocannabinoids and related lipids in the subcutaneous ad-ipose tissue of obese diabetic patients. Lipids Health Dis 2010;9:4350. Matias I, Gonthier MP, Orlando P, et al. Regulation, function, and dysreg-ulation of endocannabinoids in models of adipose and beta-pancreatic cells andin obesity and hyperglycemia. J Clin Endocrinol Metab 2006;91:3171–3180

51. D’Eon TM, Pierce KA, Roix JJ, Tyler A, Chen H, Teixeira SR. The role ofadipocyte insulin resistance in the pathogenesis of obesity-related elevations inendocannabinoids. Diabetes 2008;57:1262–126852. Skurk T, Alberti-Huber C, Herder C, Hauner H. Relationship between adi-pocyte size and adipokine expression and secretion. J Clin Endocrinol Metab2007;92:1023–1033


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