http://informahealthcare.com/arp ISSN: 1381-3455 (print), 1744-4160 (electronic) Arch Physiol Biochem, 2014; 120(3): 99–111 ! 2014 Informa UK Ltd. DOI: 10.3109/13813455.2014.940352

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ORIGINAL ARTICLE

Dietary enrichment with alpha-linolenic acid during pregnancy attenuates insulin resistance in adult offspring in mice K. S. Hollander1, C. Tempel Brami2, F. M. Konikoff1, M. Fainaru3, and A. Leikin-Frenkel1,3 1

Minerva Center for Cholesterol, Gallstones Research and Lipid Metabolism in the Liver, 2G.S.W. Faculty of Life Sciences, and 3Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Abstract

Keywords

Objective: Our objective was to test the contribution of dietary enrichment in essential or saturated fatty acids, in normocaloric diets, on the lipid accumulation and insulin resistance in the adult offspring in a C57Bl6/J mice model. Methods: Pregnant mothers were fed normocaloric diets containing 6% fat enriched in essential fatty acids (EFA): alpha-linolenic (ALA-18:3, n-3), linoleic (LA-18:2, n-6), or saturated fatty acids (SFA). After a washing-out period with regular diet, the offspring received a high-fat diet before euthanization. Results: Adult mice fed maternal ALA showed lower body weight gain and lower liver fat accumulation, lower HOMA index and lower stearoyl-CoA desaturase (SCD1) activity than those fed maternal SFA. Conclusion: The results observed using this novel model suggest that ALA in maternal diet may have the potential to inhibit insulin resistance in adult offspring.

alpha-linolenic acid, dietary enrichment, insulin resistance, pregnancy

Introduction Obesity is currently one of the most threatening global epidemics, posing a major risk for insulin resistance, dyslipidemia, cardiovascular diseases, hypertension and non-alcoholic fatty liver disease (NAFLD) (Bruce & Hanson, 2010; James et al., 2004). The foetal origins hypothesis proposed by Barker was inspired by evidence that adult type 2 diabetes and cardiovascular disease are initiated during early development in response to undernutrition (Barker et al., 1989). Thus, developmental nutrition may impact health through the early activation of gene expression and/or the metabolic pathways involved in developing obesity and insulin resistance in adult offspring (Bouret, 2009; Demmelmair et al., 2006; Samuelsson et al., 2008; Symonds et al., 2009). During the course of mammalian development, fatty acids are transferred to the foetus through the placenta and their composition depends, to a great extent, on the maternal diet (Hornstra, 2000; Innis, 2005). The potential of nutritional fatty acids to improve long-term health outcomes critically depends, therefore, on their appropriate supply at key stages of foetal growth, such as tissue differentiation and/or consolidation of the starting point of the nutrition-related metabolic or endocrine pathways.

Correspondence: Alicia Leikin-Frenkel, Sackler School of Medicine and the Bert W. Strassburger Lipid Center, Sheba Medical Center, Tel-Hashomer, Israel. Tel: 972-3-5302939. Mobile: 972-545301475. Fax: 972-305304431. E-mail: [email protected]; [email protected]

History Received 15 December 2013 Revised 17 June 2014 Accepted 25 June 2014 Published online 17 July 2014

It is known that dietary saturated fatty acids (SFA) increase the risk of obesity, insulin resistance and NAFLD by regulating the hepatic gene expression involved in lipogenesis (Vallim & Salter, 2010). In turn, essential fatty acids (EFA), alpha-linolenic (ALA) and linoleic (LA) (Burr & Burr, 1930) as well as their metabolic products, polyunsaturated fatty acids (PUFA), decrease the risk of obesity and insulin resistance (Carpentier et al., 2006), regulate cell function, growth and the expression of genes (Georgiadi et al., 2012; Guri et al., 2006; Nakamura et al., 2004), as well as controlling energy storage and oxidation (Lo´pez et al., 2008; Rossmeisl et al., 2012). Fatty acids are metabolized by fatty acid desaturases, which are important targets of nutritional fatty acid regulation in metabolic diseases (Das, 2005; Sartore et al., 2008). Among these desaturases, Stearoyl-CoA desaturase (SCD1) plays a prominent role in obesity, energy and fat metabolism (Merino et al., 2010; Zhou et al., 2009) and its tissue levels are directly correlated with insulin resistance (Bouret, 2010; Sampath et al., 2007; Waters & Ntambi, 1996). Although it has been recognized that nutrition influences the early onset of diseases (Innis, 2011; Newnham et al., 2002; Widdowson & McCance, 1963), the role of fat in developmental nutrition is less understood. Most research has focused mainly on the high-fat content in the maternal diet or the consequent obese or diabetic condition of the mother (Buckley et al., 2005; Chechi & Cheema, 2006). Few studies have looked at the quality of fatty acids during development (Brown et al., 2010; Hussain et al., 2013; Ibrahim et al., 2009) in which specific fatty acids were provided as part of a

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Arch Physiol Biochem, 2014; 120(3): 99–111

high-calorie diet and/or also provided after weaning. Such protocols make it difficult to interpret the influence of fatty acids per se during foetal development. Based on our previous work showing that maternal dietary fatty acids enhance the expression levels of desaturases in embryos (Vaisman & Leikin-Frenkel, 2003), we hypothesized that the quality of the dietary fatty acids in development, beyond the amount of fat, calories or maternal status, may modulate fat accumulation in the offspring. With the aim of understanding the role of ALA and LA vs SFA enrichment in the maternal diet in the long-term prevention of fat accumulation and insulin resistance in the offspring, we have developed a novel dietary murine model with maternal diets containing the same amounts of calories and total fat as regular chow but differentially enriched in ALA, LA or SFA. The results showing the dietary impact of those fatty acids on the long-term modulation of fat accumulation and insulin resistance in the adult offspring are presented in this paper.

Materials and methods Animals and diets C57Bl6/J mice, 4 to 5 weeks old, were obtained from the animal facility of Tel-Aviv University. The studies were approved by the Institutional Committee for Animal Experiments at Tel Aviv University, Tel Aviv, Israel. Female mice were fed four isocaloric diets prepared with fat-free chow diet (Koffolk, Israel) to which commercially available oils were added at a ratio of 6%: soybean (RD), safflower (LA), flaxseed (ALA) or coconut oil (SFA). Soybean was chosen since it is the oil in the regular chow diet employed in the animal’s facilities (Figure 1). Three to five females/group received the experimental diet two weeks pre-conception and during pregnancy and lactation. Males received regular chow diet except during the mating period

RD

ALA

during which they shared their female mates’ diets (one male for every two females). Pups were exposed to their mother’s diet during lactation and were fed, after weaning, regular chow for two months, called the washing-out period. Each test group was then divided into two subgroups receiving either regular chow or high-fat diet (HFD: 60% ± 3% calories from lard) for another two months, until euthanization by an overdose of anaesthetic (Xylazine-Ketanal),after an overnight (12 h) fasting. Tissue samples were obtained under liquid nitrogen and were kept frozen (70  C) until further use. Plasma was separated from blood and was kept frozen (20  C) until use. At all stages, the animals had free access to food and water and were kept in ventilated rooms under a light/dark cycle of 12 h/12 h. Guidelines for the use and care of the animals at Tel Aviv University’s Animal House were followed. Animals’ weight and fat parameters Food consumption and body weight were monitored weekly. Body weight gain (BWG) was calculated by referring a particular day’s weight (n) to that of day one of that particular period: pregnancy, lactation, washing out or challenge: ([Body weight day n  Body weight day 1]/[Body weight day 1] %). Body fat was detected and quantified by MRI and percent lean body mass was calculated as (LBM ¼ Body weight in grams  Amount of body fat in grams)/Body weight % where (Amount of body fat in grams ¼ body mass in grams  [% body fat by MRI/100]). Biochemical determinations Oral glucose tolerance test (OGTT) was performed following 8-hour fast and oral administration of 2 g/kbw glucose. Blood was taken from the retro-orbital vein and the basal fasting glucose level was measured (Time 0). Consecutive blood samples were taken every 30 min up to two hours.

LA

SFA

Offspring Post-weaning Diet (2 moths)

RD

RD

RD

RD

Offspring Challenge Diet (2 Months)

RD

HFD

RD

HFD

RD

HFD

RD

HFD

Figure 1. Animal model. The upper row arrows indicate the four different diets provided to female groups before and during pregnancy and lactation (n ¼ 3–5 females/group), eight weeks altogether. The middle row arrows indicate the regular diet provided after weaning, for two months, to all the offspring born to dams fed each different diet. This was called the washing-out period. The lower row arrows indicate the two different diets, either RD or HFD provided for two months, after the washing-out period, to the offspring born from dams fed each different diet. This was called the RD or HFD challenge period and lasted two months until euthanization.

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DOI: 10.3109/13813455.2014.940352

Dietary enrichment with alpha-linolenic acid during pregnancy

Plasma glucose levels were measured by using a Precision QID sensor MediSense (Abbott Laboratories Co., MediSense Inc., Bedford, MA). The area under the curve was calculated by the trapezoidal rule. Plasma insulin levels were determined after 8-hour fast using an insulin immuno-assay kit (MRC Mouse Insulin, Elisa 96T, Mercodia AB, Sweden). The HOMA index was calculated as Plasma glucose [mM]  Plasma insulin [mU/ml]/22.5. Plasma lipids: NEFA, triacylglycerides, LDL, HDL and total cholesterol were determined by commercial kits (Wako, USA and Boehringer, Germany) and quantified with an Advia 1650 autoanalyser (Bayer, Leverkusen, Germany). Fatty acid analysis Fatty acids were analysed as methyl ester derivatives (FAME) by gas chromatography (GC) in a Varian, 3800 Series (Walnut Creek, CA) chromatograph (FID) with a fused silica SGE capillary column 30  0.025 and Varian Star Workstation Advance Application software, version 6x. Mouse plasma samples were processed for the analysis of fatty acids as follows: 100 ml plasma, kept frozen at 20  C before use, were taken into a screw-capped tube (teflon-lined) containing 5 mg heptadecanoic acid as Internal Standard. 1 ml 5% SO4H2 in methanol was added. The tubes were gassed with nitrogen, closed tightly and heated at 85  C for 1.5 h with occasional shaking, several times. After cooling, 1 ml of hexane was added, the tubes’ content was mixed and, after a short centrifugation, the hexane layer was extracted into a new tube. Before GLC analysis, the hexane extracts were concentrated by evaporation under nitrogen. One-twentieth of the final resuspension was applied in 1 ul hexane into the gas chromatograph. The fatty acids’ profiles were compared to that of a known mixture of fatty acids of animal source, PUFA2 (Supelco, USA) for identification (Glaser et al., 2010). SCD1 activity and mRNA expression SCD1 activity was measured as the index calculated by determining the ratio between the products and substrates of the surrogate markers 16:1/16:0 and18:1/18:0 taken from the plasma fatty acid profile (Warensjo et al., 2008). Total RNA was extracted utilizing TriReagent (SigmaAlderich, Jerusalem, Israel) according to the manufacturer’s protocol. Extracted RNA underwent reverse transcription (RT) to form cDNA by means of the ABgeneÔ or VersoÔ RT-PCR Systems. The TaqManÕ Gene Expression AssayPre-Made for SCD-1 as well as Beta Actin were obtained from Applied Biosystems, Israel (Agentek Ltd.), catalogue number: 4331182. The Assay ID for SCD-1 is Mm01197142_m1 and it has an amplicon length of 71 bp. Reaction products were normalised according to expression of the Beta Actin gene (Assay ID of Mm00607939_s1 and amplicon length of 115 bp) and presented as fold change compared to the RD data in each group. Western blot analysis Livers were homogenized and lysed in RIPA buffer containing 50 mM Tris-HCl, pH ¼ 8.0, 150 mM sodium chloride,

101

1% NP-40, 0.5% sodium deoxycholate, 0.5% SDS, added freshly with protease and phosphatase inhibitors, followed by a 30 min incubation on ice and centrifugation for 20 min at 4  C at 13 000 g. Cleared supernatants were taken, and protein concentrations were determined by BCA assay (Pierce). Cell lysates were added with 4X Laemmli sample buffer, boiled and separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes. After non-specific blocking with skim milk or BSA for 1 h, the membranes were incubated with anti-SCD(CD.E10):sc-5820 (Santa Cruz), or anti- -actin (Santa Cruz) overnight at 4  C or 1 h at room temperature. Following, the membranes were washed three times with Trisbuffered saline added with 0.1% Tween 20 (TBST) and incubated with an appropriate HRP-conjugated secondary antibody. Membranes were washed three times with TBST, incubated with an ECL solution (Pierce) and exposed to X-ray films. The film was analyzed by densitometry and areas, measured by OptiQuant program, corresponding to SCD1 protein were normalized according to the area of actin protein in the same gel for each sample. Results are presented as fold change compared to the RD treatment in the male or female group. Fat determination Body fat (BF) was determined by MRI experiments performed on a 7T/30 system (Bruker, Rheinstetten, Germany) using a 7.5-cm volume coil. The mice were anaesthetized with isoflurane (1–3%) in 1 l/min oxygen and the respiration rate was monitored and maintained 30–40 breaths min1. Coronal T1w images were acquired with Spin echo TR 600 ms, TE 12 ms, ns 2, 256  128 with zero filling to 256  256, slice thickness 0.8 mm, 24 contiguous slices, FOV 8.5 cm, no fat suppression and with in-plane resolution 332 um. Total fat volume (subdermal and intraperitoneal) was measured by summing up the fat pixels by automatic segmentation of the fat using image J 1.4 and multiplying it by the voxel volume. Total animal volume was also measured, allowing us to extract the percentage of fat for each animal. Tissue lipids: The liver samples were weighed and homogenized with saline at a ratio of 1:5 (w:v) in plastic tubes on ice. Lipids were extracted from an aliquot of the liver homogenate according to the procedure of Folch et al. (1957). The total amount (total liver lipids, TLL) was calculated after aliquot evaporation to constant weight (Leikin-Frenkel et al., 2008). Epididymal fat was excised and weighted. Liver neutral lipids were detected with the lysochrome ORO (C26H24N4O) and visualized by light microscopy with a 40 magnification (Mehlem et al., 2013). Statistical analyses For comparing the means of treatment amongst the maternal diet groups, one-way ANOVA analysis was implemented followed by an appropriate post hoc test: Bonferroni, LSD, Tukey HSD and Scheffe. ANOVA was performed on the Citrix database from Tel Aviv University using SSPS versions 15, 17 and 18. Statistically significant results were reported according to a p value equal to or less than 0.05. All data are shown ± SEM unless stated otherwise.

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Results

Dams’ well-being was not affected by maternal dietary fatty acid composition

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Animal model The animal model consisted of feeding four groups of mothers’ four diets differing only in their fatty acid composition, with the same amount of fat and total calories as regular chow. After weaning, the offspring received RD during a washing-out period and were then exposed to an RD or HFD challenge diet (Figure 1). The dietary composition is shown in Table 1(A). Total fat given to the offspring in the HFD challenge period was of animal origin and six times higher than in RD, providing 58.3% of the total calories. The dietary fatty acid composition shows that the proportions of myristic (14:0), palmitic (16:0) and stearic acids were highest in SFA and stearic acids were highest (18:0) in RD (Table 1B). As expected, linoleic acid (18:2 n-6) was highest in LA and also in HFD, whereas alpha-linolenic acid (18:3 n-3) was highest in ALA. The n-3/n-6 ratio was 1.2 in ALA, 10 times higher than in the other fatty acid-enriched diets.

Table 1. Diet composition. (A)

Dietary composition (g%)

Protein Carbohydrate Fat* Otherz Total Calories from fat (%) (B)

Maternal diet

Offspring HFD

20.1 53.8 6.0 20.1 100.0 12.6

13.2 35.4 38.6 12.8 100.0 58.3

Dietary fatty acid composition (mole %)

Maternal diet

RD

ALA

LA

SFA

Offspring HFD

Fatty acids 14:0 16:0 16:1n-7 18:0 18:1n-9 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:5n-3 22:6n-3 n3/n6

0.2 10.2 0.3 3.5 42.0 35.1 6.7 0.3 0.3 0.4 0.0 0.2

0.0 10.3 0.3 4.8 23.7 28.0 31.6 0.1 0.3 0.2 0.3 1.2

0.2 11.6 0.3 5.0 28.4 47.9 4.7 0.5 0.3 0.3 0.2 0.1

15.2 24.9 0.1 8.5 20.3 28.1 2.1 0.2 0.2 0.4 0.0 0.1

0.9 23.6 0.8 15.1 39.3 17.6 1.0 0.2 0.2 0.0 0.4 0.1

*Fat in maternal diet was oil of different vegetable origin: Soybean for RD, flaxseed for ALA, safflower for LA and coconut for SFA. Fat in offspring high fat diet (HFD) was lard. zFat free chow diet contained fibre, ash, amino acids, minerals and vitamins added. (A) All diets fed to dams during pre-conception, pregnancy and lactation were prepared by mixing 94 g% fat-free chow diet with 6 g% different fats/oils: RD-soybean, ALA-flaxseed, LA-safflower, SFA-coconut. HFD diet fed adult offspring during the challenge period was prepared by mixing 61.18 g% fat-free chow diet with 38 g% lard. Fat-free chow diet contained fibre, ash, amino acids, minerals and vitamins. (B) Dietary fatty acid composition. Fatty acid composition of the four diets provided to the dams in the present model RD, ALA, LA, SFA and high-fat diet (HFD) provided to the offspring during the challenge period. The lower row presents the ratio between n-3 and n-6 fatty acids in each diet. The predominant fatty acids or n-3/n-6 ratio in each diet are indicated in bold.

The number of pregnant mothers was similar in all four test diets as was also the food intake, about 10.4 ± 1.2 g/day (not shown). Maternal body weight was similar during pregnancy independently of the diet as were also OGTT with no significant differences between the diets, and plasma lipids (Figure 2A–C). Pups’ weight The numbers of pups born per mother and their weight at birth and after weaning were similar for all dietary treatments (Table 2A and B). Food consumption was similar for all the groups after weaning (Table 2C). Offspring biochemical parameters were affected differently by maternal dietary fatty acid composition Lipids: After HFD challenge plasma lipids were significantly higher for SFA fed males than for RD, except for LDL cholesterol, which showed no significant changes. A similar trend was observed in females, although the differences were significant only for LDL and total cholesterol (Table 3A). Insulin: After HFD challenge SFA males had insulin plasma levels higher than ALA and LA and similar to RD (Table 3B, left). SFA females also had higher insulin levels (n.s.) than other diets, 14.7 ± 2.1 mU/ml whereas ALA had lower values (n.s.). After RD challenge, insulin plasma levels in ALA males were lower than in other diets. In females insulin values were similar for all the maternal dietary treatments (Table 3B, right). NEFA: After HFD challenge SFA males had NEFA significantly higher values than RD, whereas ALA and LA had lower values than RD. ALA females had significantly lower values than all other diets (Table 3B, right) and those of LA had similar values than RD. After RD challenge, NEFA plasma levels in males and females were lower for ALA than in other diets (Table 3B, right). ALA-enriched maternal diet lowered whereas SFA-enriched maternal diet increased glucose and insulin in the offspring OGTT is represented as plasma glucose levels as a function of time and also as Area under the Curve in Figure 3(A). After HFD challenge males OGTT, values were significantly lower in ALA than in SFA animals after 60 min. SFA glucose levels, 14 mmol/l, remained higher at 120 min, indicating, possibly, insulin resistance (Figure 3A, upper left). SFA females had basal glucose levels of 10 mmol/L, almost twice that in the other dietary treatments. It increased to 16 mmol/L after 30 min and remained high towards the end of the test, which is an indication of insulin resistance. The plasma glucose values were significantly higher than those of the RD animals all throughout the OGTT (Figure 3A, lower right).The area under the curve shows higher values for SFA males and females than for the other diets (Figure 3A, HFD lower left). After RD challenge there were no differences in the OGTT in males and females fed different maternal diets (Figure 3A, RD higher and lower).

Dietary enrichment with alpha-linolenic acid during pregnancy

DOI: 10.3109/13813455.2014.940352

Table 2. Dams and offspring parameters.

DAMS BODY WEIGHT 40

RD n-3 n-6 SFA

35

(A) Number of mothers/diet Pups born/mother and pups/litter

g

30

parturition

25

103

RD

ALA

LA

SFA

3±1 6.3 ± 1.8

3±1 5.1 ± 1.6

3±1 5.1 ± 1.4

3±1 5.2 ± 1.8

(B)

Offspring body weight (g)

20

Birth Lactation Washing Out Males Females

15 13

16

17

20

21

OGTT

1.3 ± 0.2 8.8 ± 1.8

1.2 ± 0.1 9.3 ± 1.1

1.3 ± 0.1 8.6 ± 1.3

23.4 ± 2.2 24.9 ± 1.4 24.2 ± 0.7 23.6 ± 1.9 20.0 ± 1.2 19.4 ± 1.5 19.7 ± 1.1 19.2 ± 0.7

(C)

Food consumption (g/day)

Plasma Glucose(mmol/l)

20

3.7 ± 0.5 15

4.5 ± 0.6

3.8 ± 1.0

4.4 ± 0.6

(A) Number of mothers per diet, pups per mother in each diet and pups per mother per litter. (B) Offspring body weight at birth, after weaning and after 2 month washing-out period with RD. (C) Offspring food consumption (n ¼ 6).

10 5 0 0

30

60

90

Table 3. Plasma biochemical parameters in the offspring.

120

Time (min) Area under the Curve

mmol/l*min

2000

(A)

Total cholesterol

TG

HDL

LDL

mg/dl

1500 1000 500 0 RD

ALA

LA

SFA

Maternal Diet PLASMA LIPIDS

200

Total Cholesterol

HDL

LDL

TG

Male RD-HFD 77.3 ± 3.5a ALA-HFD 88.3 ± 22.3 LA-HFD 94.5 ± 6.4 SFA-HFD 106.0 ± 12.7b

44.0 ± 3.0a 66.7 ± 11.5 64.5 ± 9.2 78.0 ± 21.2b

33.9 ± 1.8a 35.7 ± 19.3 40.8 ± 2.8 48.2 ± 0.9b

34.6 ± 2.1 39.3 ± 12.9 41.0 ± 1.4 42.5 ± 9.2

Female RD-HFD ALA-HFD LA-HFD SFA-HFD

60.0 ± 1.4 61.0 ± 4.5 63.3 ± 10.2 73.0 ± 8.9

24.8 ± 7.5 22.7 ± 9.8 30.4 ± 2.2 36.1 ± 5.5

21.0 ± 5.6a 29.3 ± 3.5a 34.3 ± 1.5a 43.3 ± 4.0b

59.0 ± 12.7a 68.3 ± 3.8a 77.3 ± 1.5b 93.7 ± 7.4c

HFD challenge

150 mg/dl

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Pregnancy (days)

1.2 ± 0.3 8.6 ± 1.2

100

(B)

50 0 RD

ALA

LA

SFA

Maternal Diet

Figure 2. Mother’s parameters. (A) Body weight of dams during and after pregnancy. (B) Oral glucose tolerance test (OGTT) representative of the last week of pregnancy (upper panel) and area under the curve (lower panel). (C) Plasma lipids measured in blood received during the last week of pregnancy (n ¼ 3).

After the HFD challenge, the HOMA index in SFA males and females was significantly higher, more than twice the values of those in other diets. ALA males and females had significantly lower HOMA indices, 2.9 and 3.2 respectively, than those on other diets (Figure 3B, left). After the RD challenge, the HOMA index was similar for males and females fed different maternal diets (Figure 3B, right). Maternal dietary fatty acid composition differentially affected offspring body weight gain and adiposity Weight: During the HFD challenge, the body weight gain was significantly higher from the third week, for SFD males than

Male RD ALA LA SFA Female RD ALA LA SFA

Insulin mU/ml

NEFA mEq/l

11.61 ± 1.56a,b 1481.31 ± 165.5a 10.54 ± 0.19b 950.10 ± 108.8b a 12.15 ± 1.09 969.03 ± 173.4b 13.76 ± 0.95a 1564.08 ± 158.2a 12.4 ± 1.5 11.6 ± 1.9 13.1 ± 2.01 14.7 ± 2.1

1235.16 ± 196a 727.87 ± 63.6b 931.93 ± 154.60a 1337.19 ± 120.50a

RD challenge Insulin mU/ml

NEFA mEq/l

11.1 ± 1.9a 631.5 ± 10.0a 10.0 ± 0.6a 510.8 ± 51.2a 10.7 ± 1.3a 827.3 ± 5.1b 12.41 ± 1.0b 950.31 ± 102.6b 10.50 ± 0.87 907.2 ± 127.8a 11.29 ± 1.76 509.4 ± 58.2b 11.24 ± 1.42 875.2 ± 100.9a 11.48 ± 1.31 1284.9 ± 119.3a

(A) Plasma lipids in males (upper) and females (lower) after the HFD Challenge period. (B) Plasma insulin and non-esterified fatty acids (NEFA) after the HFD (left) or the RD challenge period (right) in male (upper) and female (lower) offspring. Means (n ¼ 6) without a common letter difference p50.05 between the maternal dietary treatments.

ALA and LA. In females, differences in body weight were observed from the second week of the HFD challenge (Figure 4A, upper left). The body weight gain was, at the end of the HFD feeding, significantly higher for males and females fed maternal SFA (20%), than for those fed ALA or LA maternal diets (Figure 4B, left). During the RD challenge the body weight gain was about 12%, lower for ALA and LA

(A)

Plasma Glucose (mmol/l)

(B)

(mmol/l)*min

60

90

120

RD

ALA

LA

SFA

1500

2000

1500

2000

0

4

8

12

ALA

RD-HFD b ALA-HFD LA-HFD SFA-HFD

RD

a

ALA

LA

a

SFA

c

0

4

8

12

HFD

RD

a

RD

ALA

b

LA

a

SFA

c

0

4

8

12

HOMA index

RD

ALA

LA

SFA

0

4

8

12

0

3.89 2.91 4.56 8.77

30

0

0

0

Area under the Curve

SFA

b

120

0 LA

a

90

0 ALA

a

60

0

a

30

500

0

5

10

15

5

10

15

500

1500

2000

0

b

b

a

500

SFA

b

120

b

a,b

a

500

LA

a

90

5

10

15

1000

a

60

b

b

a

20

1000

RD

a

30

b

a,b

a

20

Oral Glucose Tolerance Test 20

MALES

1000

0

RD ALA LA SFA

FEMALES

RD Challenge

1000

1500

2000

0

5

10

15

20

MALES

HFD Challenge

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0

RD

RD

30

ALA

ALA

LA

LA

120

SFA

SFA

Time (min)

90

Maternal Diet

60

FEMALES

K. S. Hollander et al.

Figure 3. Insulin resistance parameters in the offspring. (A) Oral glucose tolerance test (OGTT) and (B) HOMA index in males and females after the after the HFD (left) or the RD challenge period (right). Means (n ¼ 6) without a common letter difference p50.05 between the maternal dietary treatments indicated in the x axis, as described in Methods.

HOMA -IR

104 Arch Physiol Biochem, 2014; 120(3): 99–111

(A)

(B)

Body weight (g)

5

b

6

7

SFA

a

8

25

30

0

LA

0

ALA

5

10

5

10

RD

b

4 1

15

3

30 29 28 27 26 25 24 23 22 21 20 19 18

15

2

b b

a a

20 FD H

a

1

RD ALA LA SFA

MALES

20

25

30

30 29 28 27 26 25 24 23 22 21 20 19 18

HFD Challenge

RD

a

2

3

ALA

b

4

5

FEMALES

LA

b

6

7

a

0

5

10

15

20

25

30

Body Weight Gain

SFA

8

a a a,b b

30 29 28 27 26 25 24 23 22 21 20 19 18

Body weight

a

RD

1

2

b

4

ALA

3

5

MALES

LA

b

6

7

SFA

a

8

0

5

10

15

20

25

30

30 29 28 27 26 25 24 23 22 21 20 19 18

RD Challenge

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Figure 4. Offspring body weight after the HFD challenge period. (A) Weekly body weight in the offspring, indicated in the y axis in grams, during the 8 weeks of challenge period in males and females. (B) Body weight gain at the end of HFD (left) or the RD challenge period (right). Means (n ¼ 6) without a common letter difference p50.05 between the maternal dietary treatments indicated in the x axis, as described in Methods.

BWG (%)

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than for RD and SFA, in males and about 10% higher for SFA than other diets in females. At the end of HFD challenge, body fat in ALA males was lower than those on other diets (Figure 5A). SFA males had

body fat significantly higher than males on any other diet, 24.3%, and ALA had significantly lower body fat, 16.9%, than the others. Mild changes were observed in the lean body mass of males. ALA females had significantly lower body fat,

Offspring Body Fat MALES body fat %

100 90 80 70 60 50 40 30 20 10 0

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% 100 90 80 70 60 50 40 30 20 10 0

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(A)

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SFA

Maternal Diet Figure 5. Body and tissue lipids. (A) Body fat and lean body mass expressed as percent of the total body mass during the last week of HFD challenge period. (B) Epididymal fat (EF) representing white adipose tissue, in males. The numbers express the ratio between EF/body weight % at the end of HFD challenge period. (C) Liver fat expressed as mg fat/g (upper) and observed by microscopy after ORO staining (lower) in males after HFD challenge diet. Means (n ¼ 6) without a common letter difference p50.05. The x axis indicates that the animals received different maternal diets, as described in Methods.

Dietary enrichment with alpha-linolenic acid during pregnancy

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22.7%, than those on any other diet. Epididymal fat, measured in relation to the body mass, was significantly lower in ALA males, 1%, than in RD, 2.3% or SFA, 3% (Figure 5B). Total liver weight was not affected by maternal fatty acid diets, neither in males nor in females (not shown). However, liver fat was significant lower in ALA and LA males, 94 and 105 mg/g liver respectively, than in the other groups. RD and SFA males had higher fat levels, about 180 mg/g liver indicative of NAFLD (Figure 5C, upper).The steatosis developed in those animals can be observed in the ORO stain of liver slices (Figure 5C, lower). LA and SFA females had liver fat levels significantly higher, 65 and 75 mg/g liver respectively, than RD, 40 mg/g liver and ALA (not shown). However, none of the females developed NAFLD. Maternal dietary ALA or SFA differentially affected plasma fatty acids composition and SCD1 in the offspring Plasma fatty acid analysis showed main changes in ALA and LA males, which had significantly higher levels of 18:0 and lower levels of 18:1 than those other diets (Table 4). Minor fatty acids like 18:3 n-6 were higher in LA-HFD and SFA-HFD than in other diets. Females’ plasma fatty acids showed a similar trend, without significant changes. SCD1 index, calculated from the surrogate markers in the plasma fatty acids profile, was lowest for ALA and LA males, and half that in RD and SFA animals for both SCD1 substrates: 16:0 and 18:0 (Figure 6A, left upper and middle panels). SFA females displayed the highest SCD1 index amongst all diets (Figure 6A, right upper and middle panels). SCD1 index in epididymal fat was the lowest in ALA males (Figure 6A, left lower panel). Liver SCD1 mRNA expression levels after HFD challenge, in animals fed ALA, LA and SFA diets are shown as fold change compared to those fed maternal RD diet. SFA males had significantly higher values than other diets (Figure 6B, left). ALA females had significantly lower values than those fed other diets and SFA had 20% higher values than RD

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(Figure 6B, right). Liver SCD1 protein immunoblot is compared to that of actin and presented as fold change compared to the maternal RD treatment for males (Figure 6C, left) and females (Figure 6C, right) showing that ALA animals have lower levels than other maternal diets.

Discussion This study shows that early foetal exposure to ALA-enriched maternal normo-caloric diets attenuates, whereas SFA conversely enhances insulin resistance and adiposity in adult offspring. To the best of our knowledge, this is the first report on the effects of maternal fatty acids in normo-caloric diets on the adult offspring exposed to a lipogenic diet. It is worth noting that the dams were healthy during pregnancy (Figure 2), which suggests that the long-term effects of the maternal diets in the adult offspring were associated with the differential fatty acids’ quality and not with their mothers’ health condition (Chechi & Cheema, 2006). To determine that a new model was developed, characterize by a normal amount of calories and fat in the maternal diets, with different fatty acid composition (Table 1). A post-weaning washing-out period was designed to eliminate the presence of the dietary maternal fatty acids and an HFD challenge period was intended to induce fat accumulation and insulin resistance (Figure 1) in the adult offspring. The present results also confirm our previous findings showing the impact of maternal dietary fatty acids in early development on the expression of desaturases (Vaisman & Leikin-Frenkel, 2003). Maternal dietary ALA attenuates whereas SFA differentially promotes increased plasma lipids, NEFA and insulin. In agreement with other dietary models using maternal diets enriched in saturated fatty acids (Buckley et al., 2005), SFA-fed offspring had elevated plasma lipids after the HFD challenge (Table 3A). Male and female offspring fed maternal SFA showed increased insulin and NEFA levels after the HFD period (Table 3B, left) in agreement with other works showing a direct correlation between insulin and NEFA plasma levels

Table 4. Offspring plasma fatty acid composition. Plasma fatty acid composition mole % Males

Fatty acid 14:0 16:0 16:1n-7 18:0 18:1n-9 18:2n-6 18:3n-6 18:3n-3 20:3n-6 20:4n-6 20:5n-3 22:4n-6 22:6n-3

Females

RD

ALA

LA

SFA

RD

ALA

LA

SFA

0.2 ± 0.1a 31.8 ± 2.1a 0.5 ± 0.1 16.7 ± 1.9 18.2 ± 1.4b 16.1 ± 1.9a 0±0 0.1 ± 0.1 0.7 ± 0b 10.0 ± 1.2 0.1 ± 0b 0.2 ± 0.1 5.4 ± 0.5

0.4 ± 0.1 40.7 ± 4.7b 0.3 ± 0.2 21.2 ± 2.9a 14.1 ± 1.5a 10.3 ± 2.4 0.2 ± 0 0.2 ± 0 0.0 ± 0a 8.0 ± 2.2 0.0 ± 0a 0.6 ± 0.6 4.0 ± 1.1

0.4 ± 0 39.7 ± 2.7b 0.3 ± 0.1 21.6 ± 1.5a 12.3 ± 2.3a 10.5 ± 2.2 2.9 ± 1.7a 0.2 ± 0 0.0 ± 0a 8.7 ± 0.8 0.0 ± 0a 0.2 ± 0 4.6 ± 0.6

0.5 ± 0 36.7 ± 3.4a,b 0.6 ± 0.2 15.1 ± 0.9 20.0 ± 2.7b 11.2 ± 0.7 0.7 ± 0.3 0.2 ± 0.1 0.3 ± 0.1c 6.9 ± 2.9 0.4 ± 0.1c 0.4 ± 0.3 3.3 ± 0.2

0.5 ± 0.1 38.4 ± 3.8 0.4 ± 0.2 20.3 ± 3.5 18.9 ± 2.4 11.2 ± 3.5 0.2 ± 0.1 0.2 ± 0.1 0.5 ± 0.2 6.3 ± 1.1 0.2 ± 0.2 0.4 ± 0.1 2.8 ± 1.1

0.3 ± 0 35.2 ± 3.9 0.5 ± 0.1 20.4 ± 2.9 18.9 ± 1.0 13.0 ± 1.6 0.3 ± 0.1 0.1 ± 0 0.8 ± 0.1 7.6 ± 1.2 0.1 ± 0.0 0.3 ± 0.1 3.1 ± 1.9

0.3 ± 0.1 38.4 ± 1.1 0.5 ± 0.2 18.9 ± 2.0 17.6 ± 0.7 11.9 ± 1.7 0.2 ± 0.1 0.2 ± 0.1 0.5 ± 0.1 7.2 ± 0.2 0.1 ± 0.1 0.5 ± 0.2 3.7 ± 0.3

0.3 ± 0.0 35.8 ± 3.8 0.7 ± 0.3 18.9 ± 0.3 21.4 ± 0.5 11.5 ± 0.3 0.2 ± 0.1 0.1 ± 0.0 0.5 ± 0.3 6.4 ± 0.4 0.0 ± 0.0 0.4 ± 0.1 3.7 ± 0.1

Fatty acids are expressed as mole % in males (left) and females (right) after the HFD challenge diet. Means (n ¼ 6) without a common letter difference p50.05 between the maternal dietary treatments indicated in the x axis, as described in Methods.

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MALES

FEMALES SCD1 activity

(A)

16:1/16:0

Liver 0.025 0.02 0.015 0.01 0.005 0

a

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b

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LA

SFA

b a

RD

a

a

ALA

LA

SFA

LA

SFA

1.5

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1.0 b

b 0.5 0.0

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18:1/18:0

Fatty Acid Ratio

b

a

1.60 1.20 0.80 0.40 0.00

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Epidydimal Fat 0.4

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0.3 b

0.2 0.1 0.0 RD

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1.5 Foldchange

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a

a

b

1.5

1 HFD

b

1

0.5

0.5

0

0

a,b

SCD1 immunoblotting

(C) SCD1 Actin 2.0 Foldchange

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0.025 0.02 0.015 0.01 0.005 0

a

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c a b

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2.0 a

a

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1.0

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0.5 0.0

0.0 RD

ALA

LA

SFA

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Maternal Diet

Figure 6. SCD1 index, mRNA and protein levels. (A) Represents the total body SCD1 index calculated from the plasma fatty acids profile for palmitic (16:0) and stearic acids (18:0) and their mono-unsaturated products, palmitoleic (16:1) and oleic (18:1). (B) Represents the SCD1 mRNA expression levels in male (left) and female (right) offspring after the HFD challenge diet in liver, calculated as fold change compared to the RD in the same group. Means (n ¼ 6) without a common letter difference p50.05. The x axis indicates that the different maternal diets received by animals, as described in Methods and the determinations were performed after the HFD challenge diet. (C) Represents the SCD1 immunoblot images in male (left) and female (right) offspring after the HFD challenge diet in liver, compared with the actin levels in the same samples. The graphs represent SCD1 protein levels normalized by actin levels and calculated as fold change compared to the RD in the same group. The picture is representative of triplicates from the same treatments. The x axis indicates the different maternal diets received by animals, as described in Methods, and the determinations were performed after the HFD challenge diet.

(Salgin et al., 2012). Maternal ALA, conversely, led to lower insulin and NEFA values and also to lower HOMA index values in the male offspring. This, together with the higher OGTT response for SFA animals and a lower one for ALA,

indicate divergent long-term effects of both diets on the physiological and metabolic pathways involved in insulin and glucose homeostasis in the adult offspring. ALA reduction and SFA increase of insulin levels were more systematically

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detected in males than in females (Figure 3B, Table 3), in agreement with the gender-related long-term effects of the perinatal dietary fatty acids shown previously by Korotkova et al. (2005). Moreover, recent studies have shown differences by sex in the protective effects of estrogen on diet-induced insulin resistance in mice (Riant et al., 2009). ALA attenuated whereas SFA promoted body weight and adiposity. The differential effect of maternal dietary fatty acids on body weight could be assessed after the HFD challenge period. Male and female offspring fed maternal SFA showed a higher weight gain during this period (Figure 4A and B) than animals fed ALA and LA. Male offspring fed maternal RD and SFA showed a 12% increase weight gain higher than animals fed ALA and LA while females fed SFA increased only 5% of their body weight, but still higher than other diets. The inhibitory effects of ALA were also observed in adiposity. As expected, the offspring fed SFA were significant higher whereas ALA had lower BF and epididymal fat after HFD, among other groups (Figure 5A and B). Moreover, fatty liver (NAFLD) development was prevented in males fed maternal ALA and LA but not in those fed RD and SFA (Figure 5C). Interestingly, and contrary to the findings of Spruss et al. (2012), females did not develop NAFLD. It may be assumed that the beneficial effects of maternal dietary ALA and the detrimental ones of SFA develop through their differential modulation of metabolic pathways leading to adipogenesis. Those effects were only observed clearly after HFD but not RD in adult life, thus indicating that lipid synthesis and/or accumulation are modified due to the early exposure to maternal fatty acids. Numerous enzymes like acyl CoA carboxylase, fatty acid synthase, desaturases and elongases as well as nuclear factors/transcription factors are involved in lipid biosynthesis and oxidation. Some key hepatic targets of fatty acid control include PPAR (, /,

), SREBP-1, ChREBP, MLX, NFB, and FoxO1 or factors controlling transcription factor function (e.g., IB, PGC1) (Jump, 2011). Maternal fatty acids could be involved in the regulation of their expression early in life, contributing to the diverging long-term metabolic design observed in the present work. Maternal ALA and SFA differently modified offspring SCD1 after HFD. Plasma fatty acid profiles in the offspring after the HFD challenge period showed differences in monounsaturated fatty acids correlated with higher SCD1 activity. As shown by the indices relating to both fatty acids substrate, palmitic and stearic (16:0 and 18:0) and their products palmitoleic and oleic (16:1 and 18:1) respectively ((Table 4), SFA increased, whereas ALA and LA decreased the enzyme activity. The critical role of SCD1 in the control of lipid metabolism and associated insulin resistance in adult rodents has been described previously (Dobrzyn et al., 2008; Ntambi et al., 2002). It has also been shown that EFA decreased whereas SFA induced SCD1 expression (Kahn et al., 2006; Lee et al., 2006). As shown here, maternal ALA also attenuated the increase in SCD1 expression induced by SFA in the adult offspring (Figure 6B). A differential effect of maternal diets on males and females was maybe explained by the differential induction of SCD1 expression by estrogen described by

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Mark-Kappeler et al. (2011). In addition, and similar to the enzyme activity SCD1 protein levels were reduced by maternal ALA and increased by SFA in males. In females, ALA decreased SCD1 expression and protein levels (Figure 6C).The different profiles of SCD1 activity, mRNA expression and protein levels, beyond the ascribed hormonal differences among males and females, may be found in a number of other factors possibly affected also by the diets like post-transcriptional modifications, enzyme half life, membrane configuration affecting, other components of the electron transport chain involved in desaturation and substrate availability etc. Importantly, SCD1 deficiency has been shown to activate metabolic pathways that promote -oxidation and decrease lipogenesis in liver and skeletal muscles (Martins et al., 2012; Poudyal et al., 2012; Rossmeisl et al., 2012). Therefore, our results point to the potential of ALA as a dietary factor in pregnancy able to have long-term inhibitory effects on the HFD-induced SCD1 activity and mRNA expression and associated pathways, leading to higher adiposity (Merino et al., 2010; Zhou et al., 2009). Altogether, we have described here the inhibitory effect of maternal dietary ALA and, conversely the stimulatory effect of SFA on insulin resistance, adiposity, NAFLD and the expression and activity of SCD1 in the adult offspring after an HFD challenge. LA showed similarity to ALA for most IR parameters, although ALA, from both EFA, was the most consistent and efficient maternal fatty acid able to produce all the observed effects (Deckelbaum & Torrejon, 2012; Leikin & Shinitzky, 1995; Pouteau et al., 2010). In addition, a further explanation for the maternal dietary fatty acids’ long-term effects on the offspring, it may invoke cellular membrane structural changes that could account for different fatty acid desaturase activities and higher activities of the receptors (Manco et al., 2004; Sampath & Ntambi, 2006) and transporters involved in glucose uptake (Pouteau et al., 2010). In fact, a positive direct effect of dietary ALA, such as the decrease of SCD1 activity (Kahn et al., 2006) in adult animals, has been previously described. Likewise, palmitic acid, saturated, has been shown (Martins et al., 2012) to reduce insulin receptor expression and activity as well as its phosphorylation. However, in the present model the exposure to maternal dietary fatty acids occurred at the very earliest stages of life. It is noteworthy that the long-term effects were observed four months later in the adult offspring in particular after the exposure to HFD, not RD, which contained new and different fatty acids. Even though this is a relatively unexplored area, two facts are already clear: first, that maternal fatty acids did affect the adult offspring phenotype; and second, that ALA and SFA had divergent effects that clearly manifested after an HFD challenge leading to adiposity. The particular fatty acids enriching maternal diets in the present model may be looked upon as the cause of early metabolic or genetic modifications, with long-term opposite effects on glucose and lipid metabolic pathways. Thus, permanent changes may be induced in desaturases and elongases as well as nuclear factors/transcription factors involved in lipid biosynthesis and oxidation including PPAR (, /, ), SREBP-1, ChREBP, MLX, NFB, and FoxO1 or co-activators like IB and PGC1) (Jump, 2011). We propose, moreover, that ALA or SFA may differentially

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trigger several mechanisms such as epigenetic modifications in SCD1 or related regulatory factors (Aagaard-Tillery et al., 2008; Heerwagen et al., 2010; Mauvoisin & Mounier, 2011; Rhee et al., 2012), membrane modification with longterm effects on receptor and transporters and others. Schwenk et al. (2013) have recently reported a differential effect of lipids or carbohydrates on the hepatic expression of SCD1 associated with altered promoter methylation, indicating that diets affect lipid metabolism in the liver via epigenetic mechanisms. The results presented in this work may be attributed to the specific effects of dietary maternal ALA during development (Ntambi et al., 2002; Rossmeisl et al., 2012) but they may also be attributed to its long-chain metabolic products (Leikin & Shinitzky, 1995; Ntambi et al., 2002) or to the proportion of n-3/n-6 (ALA/LA) in the diets or the ALA deficiency in the SFA maternal diet (Heerwagen et al., 2013). SCD1 is an important component in the regulation of bodily metabolism (Liu et al., 2011) and a highly promising target in combating obesity and its related complications. There are four known stearoyl-CoA desaturase (SCD) enzyme isoforms in mouse and two in humans required for the biosynthesis of monounsaturated fatty acids, mainly oleate (Miyazaki et al., 2005) and their role has not been fully unveiled. If our findings in this murine model are relevant to human nutrition, the manipulation of dietary maternal fatty acid in gestation may be considered a tool to achieve long-term metabolic and/or permanent genetic changes in several pathways affecting enzymes involved in adipogenesis, SCD1 or its regulatory elements or other. ALA in gestation may lead to a healthier phenotype in the adult offspring.

Conclusions The murine nutritional model presented here proved useful for assessing the role of ALA and SFA in maternal normocaloric diets, in inhibiting or stimulating, respectively, adiposity and insulin resistance in the adult offspring exposed to HFD.

Acknowledgements This study was supported by the Moshe Ishai Center for the Investigation of Natural Food on the Quality of Human Health Grant, the Research Authority of Tel Aviv University and partial funding from the Minerva Foundation, Germany. The authors are grateful to Prof. Amiram Raz, Faculty of Life Sciences, Tel Aviv University, for allowing KSH the use of the Gas Chromatograph; to Drs Aviv Shaish, Ayelet Harari, Yehuda Kamari, Hana Levkovitz and Tal Almog at the Bert Strassburger Lipid Center, Sheba, Tel Hashomer, for helpful comments on the manuscript (AS, AH, YK) and technical assistance with ORO and immunoblotting (HL, TA).

Declaration of interest Moshe Ishai Center for the Investigation of Natural Food on the Quality of Human Health Grant, the Research Authority of Tel Aviv University Grant and partial funding from the Minerva Foundation, Germany.

Arch Physiol Biochem, 2014; 120(3): 99–111

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