Maternal Fat Supplementation during Late Pregnancy and Lactation

Article pubs.acs.org/JAFC

Maternal Fat Supplementation during Late Pregnancy and Lactation Influences the Development of Hepatic Steatosis in Offspring Depending on the Fat Source Marina Llopis,† Juana Sánchez,† Teresa Priego, Andreu Palou,* and Catalina Picó Molecular Biology, Nutrition and Biotechnology (Nutrigenomics), University of the Balearic Islands (UIB) and Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Carretera de Valldemossa Km 7.5, Palma de Mallorca 07122, Spain ABSTRACT: In this study we investigate the effects of maternal supplementation with different fat sources (margarine, olive oil, or butter) during pregnancy and lactation on offspring metabolic health in adulthood and under obesogenic conditions. In adulthood and under a high-fat (HF) diet, the margarine group showed lower body fat content than the butter group and was also protected against the increase in hepatic lipid content occurring in the other groups, whereas the butter group showed signs of more advanced hepatic steatosis. Under an HF diet, all fat-supplemented animals showed greater hepatic expression levels of fatty acid oxidation-related genes compared to their normal-fat diet counterparts, with higher levels in the margarine group. Under these conditions, the margarine group also showed higher white adipose tissue mRNA levels of adipogenic genes than the other fat-supplemented groups. Thus, compared to other fat sources, offspring from margarine-supplemented dams seem to be more protected from metabolic alterations related to the HF diet, particularly concerning hepatic fat accumulation. KEYWORDS: metabolic programming, fatty liver, maternal nutrition, dietary fat

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INTRODUCTION According to Barker’s fetal origin of adult disease hypothesis, perturbations in the gestational milieu influence the development of disease later in life.1 The Dutch famine study represents an emblematic example of fetal programming of obesity and its related disorders due to maternal undernutrition during the gestational period.2 Studies in animal models have also evidenced that maternal undernutrition during gestation has long-term consequences on offspring metabolic energy systems, increasing the predisposition to develop obesity,3−5 with the outcomes depending on the severity and period of undernutrition, as well as on the gender.6 However, nowadays in Western societies, maternal undernutrition only concerns a small percentage of the population because of the abundant food supply.7 More and more women are therefore obese and consuming a calorific or fat-rich diet when pregnant.8 Thus, the characterization of the effects of overconsumption during pregnancy and lactation on offspring metabolic health becomes of great interest. Maternal high-fat (HF) diet feeding during pregnancy and lactation in rodents has been shown to result in a phenotype of the offspring that closely resembles the human metabolic syndrome.8 These animals present greater adiposity and body mass,7,9−11 abnormal glucose homeostasis10,12 and serum lipid profiles,10,13,14 and increased blood pressure.14,15 Moreover, these animals are more prone to accumulate an excessive amount of triglycerides (TGs) in the liver,9,16,17 the hallmark of nonalcoholic fatty liver disease (NAFLD), which is well recognized as being part of the metabolic syndrome.18 This ranges from simple fatty liver (hepatic steatosis) to a potentially progressive form, nonalcoholic steatohepatitis (NASH), which may lead to liver fibrosis and cirrhosis, resulting in increased morbidity and mortality.18 Alterations in different metabolic © 2014 American Chemical Society

pathways, including enhanced fatty acid release from adipose tissue, increased de novo fatty acid synthesis, and decreased βoxidation, can lead to the development of hepatic steatosis;18 hence, white adipose tissue (WAT) and liver, which are the main tissues involved in lipid metabolism, are relevant in the development of this disease. Therefore, it is accepted that early exposure to excess fat, especially during fetal and early postnatal life, which are periods showing the highest adaptability and vulnerability to external factors, may have negative effects on the later metabolic health of the offspring. However, in addition to the amount of fat, the type of fat eaten during this period may also be determinant. In this sense, we previously found that margarine oversupply, compared to other fat sources consumed in Western diets, such as olive oil and butter, during late pregnancy and lactation, programs the offspring for increased leptin sensitivity and for a lower preference for fat food and hence provides relative protection against body mass gain in adulthood, particularly under an obesogenic environment. 19 We now report specific effects of maternal supplementation with olive oil, margarine, or butter during late pregnancy and lactation on the risk of obesity-related metabolic diseases in offspring, particularly the development of hepatic steatosis in adulthood, which could be attributed to different programming effects on liver and/or WAT metabolism. Received: Revised: Accepted: Published: 1590

November 27, 2013 January 13, 2014 January 22, 2014 January 22, 2014 dx.doi.org/10.1021/jf405161e | J. Agric. Food Chem. 2014, 62, 1590−1601

Journal of Agricultural and Food Chemistry

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with 8% calories from fat), and the other group was exposed to a highfat (HF) diet (containing 4.7 kcal/g, with 45% calories from fat, Research Diets, Inc., New Brunswick, NJ). The HF diet contained 5.5% calories from soybean oil and 39.5% from lard. Body mass and food intake were followed until the age of 6 months. At this age, the body fat content (by EchoMRI-700, Echo Medical Systems, LLC, Houston, TX) was recorded and blood samples were collected (in heparinized containers) from the saphenous vein, under fasting animals deprived of food for 12 h. All groups include at least seven animals (n = 7−12). At the age of 6 months, the animals were killed by decapitation under fed conditions, during the first 2 h of the beginning of the light cycle. The liver and various WAT depotsgonadal, retroperitoneal, mesenteric, and inguinalwere rapidly removed. All samples were weighed, immediately frozen in liquid nitrogen, and stored at −70 °C. The retroperitoneal was the depot selected as representative of the WAT to perform mRNA expression. Blood was also collected and centrifuged at 1000g for 10 min to collect the plasma, which was stored at −20 °C until analysis. Quantification of Insulin and Glucose Concentration and Calculation of the Homeostatic Model Assessment for Insulin Resistance. The blood glucose concentration was measured with an Accu-Chek glucometer (Roche Diagnostics, Barcelona, Spain). The plasma insulin concentration was measured using a rat insulin enzymelinked immunosorbent assay (ELISA) kit (Mercodia AB, Uppsala, Sweden) following standard procedures. The homeostatic model assessment for insulin resistance (HOMAIR) was used to assess insulin resistance. It was calculated from the fasting insulin and glucose concentrations using the formula of Matthews et al.:21 HOMA-IR = [fasting glucose concentration (mmol/ L) × fasting insulin concentration (mU/L)]/22.5. Quantification of Plasma Levels of Triglycerides, Nonesterified (or Free) Fatty Acids, and β-Hydroxybutyrate and Hepatic Lipid Content. Commercial enzymatic colorimetric kits were used for the determination of plasma triglyceride (Sigma, Madrid, Spain), nonesterified (or free) fatty acids (NEFAs) (Wako Chemicals GmbH, Neuss, Germany), and 3-hydroxybutyrate (BEN S.r.l.Biochemical enterprise) levels. Total lipids were extracted from about 600 mg of hepatic tissue and quantified by the method of Folch et al.22 Histological Studies. A histological study was performed on the liver. A piece of fresh dissected liver (n = 6) was fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4 °C. The samples were then washed in phosphate buffer and dehydrated in a graded series of ethanol, cleared in xylene, and embedded in paraffin blocks. Sections of the tissues 5 μm thick were stained with hematoxylin and eosin. Liver sections were classified into four grades depending on fat accumulation following the Burnt et al. classification.23 Grade 0 was assigned when no fat accumulation was found in the liver, grade 1 when fat vacuoles were seen in less than 33% of hepatocytes, grade 2 when 33−66% of hepatocytes were affected by fat vacuoles, and grade 3 when fat vacuoles were found in more than 66% of hepatocytes. Liver images from light microscopy were digitalized and analyzed using Axio Vision software (Carl Zeiss Imaging Solutions). RNA Extraction. Total RNA was extracted from the liver and retroperitoneal WAT by an E.Z.N.A. RNA purification system (Omega Biotek, Inc., Norcross, GA) according to the manufacturer’s instructions. Isolated RNA was quantified using the NanoDrop ND1000 spectrophotometer (NadroDrop Technologies Wilmington, DE) and its integrity confirmed using agarose gel electrophoresis. RT-qPCR Analysis. Real-time polymerase chain reaction was used to measure mRNA expression levels of selected genes in the liver and retroperitoneal WAT in the offspring of the different groups of animals at the age of 6 months: specifically, CD36, sterol regulatory element binding protein 1c (SREBP1c), fatty acid synthase (FAS), stearoyl coenzyme A desaturase 1 (SCD1), peroxisome proliferator activated receptor α (PPARα), fibroblast growth factor 21 (FGF21), liver carnitine palmitoyltransferase 1a (CPT1L), pyruvate dehydrogenase kinase 4 (PDK4), glucokinase (GK), pyruvate kinase 4 (PK), insulin

MATERIALS AND METHODS

Ethical Approval. The animal protocol followed in this study was reviewed and approved by the Bioethical Committee of our University, and guidelines for the use and care of laboratory animals of the University were followed. Animals and Experimental Design. Virgin female Wistar rats (Charles River Laboratories Spain, SA, Barcelona, Spain), housed at 22 °C with a period of light/dark of 12 h each (lights on from 08:00 to 20:00) and with free access to food and water, were mated with male rats. The day of conception (day 0 of pregnancy) was determined by examination of vaginal smears for the presence of sperm, and then female rats were single caged. Pregnant rats were supplemented with 30% of the normal caloric intake of a group of dams fed ad libitum in the form of different fat sources (refined olive oil, margarine, or butter) during late pregnancy and lactation (from day 14 of pregnancy to day 21 of lactation), as previously described.19 The fatty acid profile of the different fat sources has been previously described20 and is shown in Table 1. Supplementation of the different fat sources was carried out

Table 1. Fatty Acid Composition of Fat Sources Used in the Experimental Design19,a fatty acid

olive oil

margarine

butter

SFAs

nd nd 0.63 12.7 0.91 nd nd 14.3

nd 4.56 1.54 13.9 1.28 nd nd 21.2

2.06 6.44 15.7 36.9 10.7 0.77 0.86 73.5

C16:1n7 C18:1n7 C18:1n9c C24:1 MUFAs

1.05 1.87 70.6 nd 74.0

nd 1.04 20.9 1.72 23.8

1.53 0.42 16.5 0.61 19.8

C18:2n6 C18:3n6 C18:3n3 CLA 9,11 C20:3n6 C22:5n3

7.70 nd 1.02 nd nd nd 8.72 7.70 1.02

41.9 nd 5.52 nd nd 1.48 49.0 42.0 7.01

1.17 0.64 0.75 0.79 0.12 0.52 3.99 1.93 1.27

C10:0 C12:0 C14:0 C16:0 C18:0 C20:0 C23:0

PUFAs n-6 n-3 a

Data are the means of three different measurements expressed as a percentage of total fat. SFAs = saturated fatty acids, MUFAs = monounsaturated fatty acids, PUFAs = polyunsaturated fatty acids, and nd = nondetectable levels. The bold numbers indicate the total of each category of fatty acid in each fat source. by gavage once a day. As described,19 no differences were observed in the total amount of kilocalories ingested by dams during the supplementation period among fat-supplemented groups. Thus, different outcomes observed in the offspring may be attributed to the different fat sources of maternal supplementation and not to the intake of different amount of calories. The offspring of these animals are referred to as olive oil, margarine, and butter groups, respectively. The offspring of dams fed ad libitum (control group) were also followed. At day 1 after delivery, excess pups in each litter were removed to keep 10 pups per dam. At weaning animals were fed a standard diet until the age of 4 months; then they were divided into two groups: one group continued under a normal-fat (NF) diet (standard diet containing 3.2 kcal/g, 1591

dx.doi.org/10.1021/jf405161e | J. Agric. Food Chem. 2014, 62, 1590−1601


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Table 2. Body Mass, Fat Content, and Mass of Different WAT Depots and Liver of 6 Month Old Male Offspring of Dams Supplemented with 30% of the Normal Caloric Intake with Different Fat Sources (Olive Oil, Margarine, or Butter) during Late Pregnancy and Lactationa

a

Animals were fed a standard NF diet until the age of 4 months and then an NF or HF diet until the age of 6 months. The double vertical line indicates that the ANOVA analysis was assessed without the control group. Data are means ± SEM (n = 7−12). Statistics: D, effect of diet (NF or HF); T, effect of maternal supplementation (p < 0.05, two-way ANOVA). a ≠ b, LSD post hoc analysis. Key: *, different from their respective NFdiet-fed group (p < 0.05, Student’s t test); ↑, different versus the control (p < 0.05, Student’s t test). receptor (INSR), and insulin receptor substrate 1 (IRS1) in the liver and lipoprotein lipase (LPL), CD36, muscle CPT1, adipose triglyceride lipase (ATGL), glucose transporter 4 (GLUT4), hexokinase II (HK), PPARγ2, SREBP1c, FAS, acetyl coenzyme A carboxylase (ACC), glycerol-3-phosphate acyltransferase (GPAT), and InsR in WAT. 18S (in liver) and β-actin (in WAT) were used as reference genes. A 0.25 μg of total RNA (in a final volume of 5 μL) was denatured at 65 °C for 10 min and then reverse transcribed to cDNA using MuLV reverse transcriptase (Applied Biosystems, Madrid, Spain) at 20 °C for 15 min and 42 °C for 30 min with a final step of 5 min at 95 °C in an Applied Biosystems 2720 thermal cycler (Madrid, Spain). Each polymerase chain reaction (PCR) was performed from diluted (1/ 20) cDNA template, forward and reverse primers (1 μM each), and Power SYBER Green PCR Master Mix (Applied Biosystems, Foster City, CA). Primers, obtained from Sigma (Madrid, Spain), for the different genes are described in ref 24. Real-time PCR was performed using the Applied Biosystems StepOnePlus real-time PCR system with the following profile: 10 min at 95 °C, followed by a total of 40 twotemperature cycles (15 s at 95 °C and 1 min at 60 °C). To verify the purity of the products, a melting curve was produced after each run according to the manufacturer’s instructions. The threshold cycle (Ct) was calculated by the instrument’s software (StepOne Software v2.0), and the relative expression of each mRNA was calculated as previously described.25 Western Blot Analysis of ATGL in Retroperitoneal WAT. The amount of ATGL in retroperitoneal WAT was determined by Western blot. This protein was chosen because it is considered to be the main triacylglycerol lipase in adipose tissue and catalyzes the first step of lipid hydrolysis.26 Briefly, tissue was homogenized at 4 °C in 1:5 (w/v) in RIPA buffer containing Halt protease and phosphatase inhibitor cocktail (Thermo Fisher, Rockford, IL). Western blot was performed

in a 4−15% Criterion TGX precast gel (BioRad, Madrid, Spain), transferred to a nitrocellulose membrane. ATGL antibody was obtained from Cayman Chemical (Ann Arbor, MI). Membranes were also incubated with anti-β-actin antibody (Cell Signaling, Inc., Hercules, CA) to ensure the equal loading. Specific infrared (IR)-dyed secondary anti-IgG antibodies (LI-COR Biosciences, Lincoln, NE) were used. For IR detection, membranes were scanned in an Odyssey imager (LI-COR); the bands were quantified using the software Odyssey v3.0 (LI-COR). Statistical Analysis. All data are expressed as the mean ± SEM (n = 7−12). Multiple comparisons were assessed by repeated measures ANOVA and two-way ANOVA to determine the effects of different factors (maternal fatty acid source supplementation and type of diet). Differences between groups were assessed by one-way ANOVA followed by least significant difference (LSD) post hoc comparison to assess statistical differences between the groups. Single comparisons were assessed by Student’s t test or pair t test analysis. Principal component analysis (PCA) was performed to assess relationships between the adiposity of the animals, the lipid content in the liver, and the expression levels of the genes measured in the liver and WAT (a total of 27 variables). Two PCA models were constructed, one for all samples receiving the NF diet and another for all those receiving the HF diet. The score plot of PC1 versus PC2 was used to examine the potential contribution of these components to discriminate the four groups of animals (control, olive oil, margarine, and butter). The analyses were performed with SPSS for Windows (SPSS, Chicago, IL). The threshold of significance was defined at p < 0.05.

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RESULTS Body Mass, Body Fat, and Hepatic Lipid Content. As previously described for the same cohort of animals,19 no significant differences were found regarding the body mass of 1592



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Figure 1. (A) Hepatic lipid content, (B) numerical grading of NASH in liver biopsies, and (C) representative hematoxylin and eosin staining of histological liver samples from 6 month old male offspring of dams supplemented with 30% of the normal caloric intake with different fat sources (olive oil, margarine, or butter) during late pregnancy and lactation. Animals were fed a standard NF diet until the age of 4 months and then an NF or HF diet until the age of 6 months. Scale bar = 20 μm. V = terminal hepatic venule. The dotted line indicates that the ANOVA analysis was assessed without the control group. Data are the mean ± SEM (n = 7−12). Statistics: D, effect of diet (NF or HF); T, effect of maternal supplementation (p < 0.05, two-way ANOVA). a ≠ b, LSD post hoc analysis. Key: *, different from their respective NF-diet-fed group (p < 0.05, Student’s t test); ↓, different versus the control (p < 0.05, Student’s t test).

Table 3. Circulating Glucose, Insulin, Nonesterified Fatty Acid (NEFA), Triglyceride, and β-Hydroxybutyrate Levels and HOMA-IR in Male Offspring of Dams Supplemented with 30% of the Normal Caloric Intake with Different Fat Sources (Olive Oil, Margarine, or Butter) during Late Pregnancy and Lactation, at 6 months of Age under ad Libitum Feeding Conditions, and after 12 h of Fastinga

a

Animals were fed a standard NF diet until the age of 4 months and then an NF or HF diet until the age of 6 months. The double vertical line indicates the ANOVA analysis was assessed without the control group. Data are means ± SEM (n = 7−12). Statistics: within normal-fat conditions, a ≠ b, LSD post hoc analysis; within high-fat conditions, y ≠ x, LSD post hoc analysis. Key: #, different from their respective ad-libitum-fed group (p < 0.05. paired t test); *, different from their respective NF-diet-fed group (p < 0.05. Student’s t test); ↑/↓, different versus the control (p < 0.05, Student’s t test). 1593



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Figure 2. Expression of genes related to energy metabolism (FA uptake and oxidation, lipogenesis, glucose uptake and metabolism, and insulin signaling) in liver of 6 month old male offspring of dams supplemented with 30% of the normal caloric intake with different fat sources (olive oil, margarine, or butter) during late pregnancy and lactation. C = control, O = olive oil, M = margarine, and B = butter. Animals were fed a standard NF diet until the age of 4 months and then an NF or HF diet until the age of 6 months. The dotted line indicates that the ANOVA analysis was assessed without the control group. Data are means ± SEM (n = 7−12). Genes determined were CD36, sterol regulatory element binding protein 1c (SREBP1c), fatty acid synthase (FAS), stearoyl coenzyme A desaturase 1 (SCD1), peroxisome proliferator activated receptor α (PPARα), fibroblast growth factor 21 (FGF21), liver carnitine palmitoyltransferase 1a (CPT1L), pyruvate dehydrogenase kinase 4 (PDK4), glucokinase (GK), pyruvate kinase 4 (PK), insulin receptor (INSR), and insulin receptor substrate 1 (IRS1). Statistics: D, effect of diet (NF or HF); T, effect of maternal supplementation; D×T, interaction between diet and maternal supplementation (p < 0.05, two-way ANOVA). a ≠ b, LSD post hoc analysis. Key: *, different from their respective NF-diet-fed group (p < 0.05, Student’s t test); ↑, different versus the control (p < 0.05, Student’s t test).

were found under the HF diet. No differences between groups were found in liver mass, under either the NF or HF diet. Figure 1A shows the hepatic lipid content in the different groups of animals. Animals exposed to the HF diet presented higher hepatic lipid content compared to animals maintained under the NF diet (p < 0.05, Student’s t test), with the exception of animals in the margarine group. Notably, within animals exposed to the HF diet, animals in the margarine group presented lower hepatic lipid content compared to the controls (p < 0.05, Student’s t test). Moreover, the margarine groups (NF and HF analyzed together) showed lower hepatic lipid content compared to the butter groups (p < 0.05, LSD post hoc). Histological analysis of the liver (Figure 1B,C) revealed evidence of NAFLD in animals exposed to the HF diet, especially in the olive oil and butter groups. The control and margarine groups showed a lower incidence of steatosis. In addition, although the control and margarine groups did not show evidence of hepatic steatosis under NF diet conditions,

the different groups of animals at the age of 6 months, depending on maternal fat supplementation, under either an NF or an HF diet (Table 2). As expected, the body mass of animals under an HF diet was higher than that of animals under an NF diet (p < 0.05, two-way ANOVA), the difference being higher and significant by Student’s t test in the olive oil group. Animals exposed to an HF diet presented higher body fat content, higher adiposity index (calculated as the percentage of the sum of the four fat depots measuredinguinal, mesenteric, retroperitoneal, and epididymaldivided by the mass of the animal), and greater mass of the individual fat depots (p < 0.05, two-way ANOVA). Nevertheless, the body fat content and mass of the inguinal WAT in the margarine group under an HF diet were not different compared to their NF-diet-fed counterparts (by Student’s t test), and the values were significantly lower than those of the butter group. The offspring of all fat-supplemented groups displayed greater mass of the inguinal WAT under NF diet conditions compared to the control group (p < 0.05, Student’s t test), but no differences 1594



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Figure 3. Expression of genes related to energy metabolism (FA uptake and oxidation, lipolysis, glucose uptake and metabolism, lipogenesis, and insulin signaling) in the retoperitoneal white adipose tissue (rpWAT) of 6 month old male offspring of dams supplemented with 30% of the normal caloric intake with different fat sources (olive oil, margarine, or butter) during late pregnancy and lactation. C = control, O = olive oil, M = margarine, and B = butter. Animals were fed a standard NF diet until the age of 4 months and then an NF or HF diet until the age of 6 months. The dotted line indicates that the ANOVA analysis was assessed without the control group. Data are means ± SEM (n = 7−12). Genes determined were lipoprotein lipase (LPL), CD36, muscle carnitine palmitoyltransferase 1a (CPT1m), adipose triglyceride lipase (ATGL), glucose transporter 4 (GLUT4), hexoquinase II (HK), peroxisome proliferator activated receptor γ 2 (PPARγ2), sterol regulatory element binding protein 1c (SREBP1c), fatty acid synthase (FAS), acetyl coenzyme A carboxylase (ACC), glycerol-3-phosphate acyltransferase (GPAT), and insulin receptor (INSR). Statistics: D, effect of diet (NF or HF); T, effect of maternal supplementation; D×T, interaction between diet and maternal supplementation (p < 0.05, two-way ANOVA). a ≠ b, x ≠ y, LSD post hoc analysis. Key: *, different from their respective NF-diet-fed group (p < 0.05, Student’s t test); ↑ /↓, different versus control (p < 0.05, Student’s t test).

were higher under the HF diet conditions compared to those under the NF diet conditions (p < 0.05, Student’s t test). In addition, in animals exposed to the NF diet, an increase in NEFA levels was observed as an effect of fasting (p < 0.05, pair t test), except in the margarine group, where differences did not reach statistical significance; however, under the HF diet, NEFA levels remained unchanged with fasting, with the exception of the animals in the margarine group, which showed lower levels under fasting conditions compared to the feeding state (p < 0.05, pair t test). Concerning TGs, all groups showed increased triglyceridemia under the HF diet compared to the NF diet (p < 0.05, Student’s t test), with the exception of the margarine group, where differences did not reach statistical significance. Fasting produced a decrease in TG levels in the margarine group, similar to the controls, under both NF and HF diet conditions (p < 0.05, pair t test); however, this decrease was not observed in the olive oil and butter groups fed the NF diet. Animals in

some evidence of steatosis was already seen at this age in the olive oil group and particularly in the butter group. Glucose, Insulin, NEFA, TG, and β-Hydroxybutyrate Circulating Levels. Table 3 shows circulating parameters in the different groups of animals fed an NF or HF diet and under ad libitum and 12 h fasting conditions. As expected, the glucose and insulin levels decreased with fasting in all groups (p < 0.05, repeated measures analysis and pair t test). No differences were observed in the glucose and insulin levels as an effect of maternal fat supplementation, under either the NF or HF diet. Notably, animals in the butter group exposed to an HF diet presented higher glucose levels under ad libitum feeding conditions compared to their NF-diet-fed counterparts (p < 0.05, Student’s t test). No significant differences were found in HOMA-IR. Regarding circulating NEFA, no significant differences were found under ad libitum feeding conditions between the different groups of animals. In all of them, circulating levels 1595



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of WAT because it is a major internal fat pad present in rats and is involved in the development of insulin resistance and type 2 diabetes.27 Results showing the expression of selected genes involved in nutrient handling and metabolism in this fat depot are summarized in Figure 3. Animals in the olive oil group showed higher LPL mRNA levels compared to the other fat-supplemented groups, under both NF and HF diets (p < 0.05, one-way ANOVA). Concerning CD36, their mRNA levels were higher in the fatsupplemented groups under the HF diet compared to their NF diet counterparts (p < 0.05; two-way ANOVA), with higher significant differences by Student’s t test in the margarine group. Concerning lipolysis and fatty acid oxidation genes, under HF diet conditions, animals in the margarine group showed higher ATGL mRNA expression levels than the olive oil and butter groups, whereas, under the NF diet, they presented lower CPT1M mRNA levels compared to animals in the olive oil group (p < 0.05, one-way ANOVA). Regarding ATGL, differences similar to those of the mRNA levels were also found in their protein levels, although in this case differences did not reach statistical significance (Figure 4).

the margarine group, under either the NF or HF diet, displayed lower fasting TG levels compared to the olive oil and butter groups (p < 0.05, one-way ANOVA). Regarding ketone bodies, NF-diet-fed animals in the margarine and butter groups showed higher β-hydroxybutyrate (BHB) levels than the olive oil group (one-way ANOVA), under ad-libitum-fed conditions; the margarine group also presented higher levels than the controls, under both fed and fasting conditions (p < 0.05, Student’s t test). Under fed conditions, the HF-diet-fed olive oil and butter groups showed increased BHB levels compared to their NF-diet-fed counterparts (p < 0.05, Student’s t test). Fasting produced a significant increase in BHB levels in HF-diet-fed animals in the olive oil group and in the margarine group, under both NF and HF diet conditions (p < 0.05, pair t test), but no changes were found in the other groups. Expression of Genes Related to Energy Metabolism in the Liver. Results showing the expression of selected genes involved in nutrient handling and metabolism in the liver are summarized in Figure 2. HF diet feeding differently affected CD36 expression levels in the distinct fat-supplemented groups (interactive effect between diet and maternal treatment, p < 0.05, two-way ANOVA). Whereas levels tended to increase in the olive oil and margarine groups as an effect of the HF diet (with levels in the latter being higher compared to those of the control and butter groups), they tended to decrease in the butter group. Concerning lipogenesis-related genes, HF-dietfed animals in the margarine group showed higher mRNA levels of SREBP1c than the olive oil and butter groups, and both the olive oil and margarine groups also showed higher levels than the controls. FAS and SCD1 mRNA expression levels were decreased in control animals under HF diet conditions, but only SCD1 showed a significant decrease in the fat-supplemented groups, and the decrease was not as marked as in the control group. Compared to the controls, HF-diet-fed animals in the olive oil group and in the butter group showed higher mRNA levels of FAS and SCD1, respectively. The expression levels of genes related to fatty acid oxidation (PPARα, FGF21, CPT1L, and PDK4) increased in the fatsupplemented groups as an effect of the HF diet but not in the control animals (p < 0.05, two-way ANOVA). Therefore, under the HF diet, all fat-supplemented groups revealed a greater expression of FGF21, CPT1L, and PDK4 compared to the controls (with the exception of the butter group for the latter gene). The increase in gene expression as an effect of HF diet feeding was particularly remarkable for CPT1L, and especially in the margarine group, which showed a 2.45-fold increase. Under the HF diet, animals in the margarine group also showed higher mRNA levels of PPARα compared to the butter group (p < 0.05, one-way ANOVA). Under the NF diet, animals in the butter group displayed lower expression levels of PPARα compared to the controls (p = 0.06, Student’s t test). GK and PK mRNA expression levels followed a similar pattern in the different groups of animals. Compared to the controls, the HF-diet-fed olive oil and margarine groups presented higher GK and PK mRNA levels (p < 0.05, Student’s t test). These animals, together with the HF-diet-fed animals of the butter group, also showed higher INSR mRNA levels than the controls (p < 0.05, Student’s t test). No significant changes were found concerning IRS1 between the different groups of animals Expression of Genes Related to Energy Metabolism in WAT. The retroperitoneal fat pad was chosen as representative

Figure 4. ATGL protein levels (measured by Western blotting) in retroperitoneal white adipose tissue (rpWAT) of 6 month old male offspring of dams supplemented with 30% of the normal caloric intake with different fat sources (olive oil, margarine, or butter) during late pregnancy and lactation. Animals were fed a standard NF diet until the age of 4 months and then an NF or HF diet until the age of 6 months. The dotted line indicates that the ANOVA analysis was assessed without the control group. Representative results are shown in the top panels. In the bottom panels, results represent the mean ± SEM (n = 7−12) expressed in arbitrary units (AU).

Animals of the fat-supplemented groups showed lower GLUT4 and HK mRNA levels under the HF diet compared to their NF-diet-fed counterparts (p < 0.05, two-way ANOVA), with a higher significant difference by Student’s t test in the olive oil group (for both genes) and in the margarine group (for the latter gene). No significant changes were found in the control group as an effect of HF diet feeding, although a 1596



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Figure 5. (A) Principal component loadings for the first two principal components (PCs). Data were spread by diet to assess possible relationships between gene expression in the tissues studied (liver and rpWAT) and the adiposity index and the hepatic lipid content under both dietary conditions (NF and HF diets). The most significant loadings are indicated with an asterisk. (B) Score plot representation.

tendency to lower levels was found concerning GLUT4. Notably, animals in the olive oil group displayed the highest mRNA levels of these genes under NF diet conditions, and these levels were significantly higher compared to those of the butter group (for GLUT4) and those of both the margarine and butter groups (for HK). Under an HF diet, animals of the fat-supplemented groups showed lower mRNA expression levels of the adipogenic factor SREBP1c and of genes related to the synthesis of fatty acids (ACC1 and FAS) and TG (GPAT) with respect to those found under an NF diet (p < 0.05, two-way ANOVA), with some differences between groups. Specifically, HF-diet-fed animals in the margarine group did not show significant differences (by Student’s t test) compared to their NF-diet-fed counterparts concerning SREBP1c, ACC, and GPAT mRNA levels, as also occurred for the latter in the animals in the butter group. A pattern similar to that of the SREBP1c gene was found for the other adipogenic factor, PPARγ. HF-diet-fed animals in the olive oil and butter groups showed lower mRNA levels compared to their NF-diet-fed counterparts (p < 0.05, Student’s t test), while no changes were found in the margarine group as an effect of the HF diet. Control animals also showed a significant decrease in the expression levels of SREBP1c, ACC, and FAS under the HF diet, but no changes were observed concerning PPARγ and GPAT (p < 0.05, Student’s t test), compared to their NF-diet-fed counterparts. Animals in the olive oil group fed an NF diet showed higher mRNA levels of

GPAT with respect to animals in the margarine and butter groups (p < 0.05, one-way ANOVA). Concerning the insulin receptor, animals in the butter group presented higher mRNA levels than animals in the margarine group under the NF diet (p < 0.05, one-way ANOVA), but their expression levels were decreased when these animals were fed the HF diet (p < 0.05, Student’s t test). No significant changes were found in the other groups of animals as an effect of HF diet feeding. Principal Component Analyses. The results of principal component analysis (PCA) of 27 variables are shown in Figure 5A. The data were spread by diet to assess possible relationships between gene expression in the studied tissues (liver and WAT) and the adiposity index and the hepatic lipid content under both dietary conditions (NF and HF diets). The first and second components were responsible for over 40% of the total variance in both conditions (43% in samples under the NF diet and 47% in those under the HF diet). Under NF diet conditions, PC1 was mainly characterized by liver transcripts related to glycolysis and lipogenesis, such as PK (0.895), SREBP1c (0.801), SCD1 (0.788), and FAS (0.767). Under the HF diet, the same transcripts mentioned above were important in the PC1, but other liver transcripts related to fat oxidation such as FGF21 (0.765), CPT1L (0.627), and PDK4 (0.626) were also of great importance. Concerning PC2, under the NF diet, the variables with higher loadings were liver transcripts related to fat oxidation [FGF21 (0.809), CPT1L (0.714), PDK4 (0.653), PPARα (0.635)] and insulin signaling [INSR 1597



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affect offspring susceptibility to develop this disease, with different degrees of severity depending on the type of diet they are exposed to in adulthood. HF diet conditions in adulthood have been previously described to lead to increased hepatic content of lipids.28 However, perinatal conditions have also been proved to be determinant. In fact, Bruce et al. showed that HF diet exposure during both development and postweaning periods was worse than HF diet exposure in the postweaning period alone,16 and a maternal HF diet led to the development of NAFLD later in life, even if a control diet was given in the postweaning period.16,17 Thus, maternal HF diet during early periods of development may increase susceptibility to hepatic steatosis and inflammation in adult offspring, which may result in a more severe form when animals are also exposed to adult nutritional insults.16 Interestingly, we show here that this effect depends not only on the ingestion of fat per se but also on the maternal source of fat during the early stage of life. To ascertain which metabolic pathway could be responsible for the observed effects of maternal supplementation with different fat sources, we analyzed the expression levels of selected genes involved in energy metabolism in liver and WAT of adult animals, under both NF and HF diet conditions. The results revealed that differences between the groups were made particularly evident when animals were exposed to HF diet conditions in adulthood. Under an HF diet, we observed increased hepatic expression levels of β-oxidation-related genes, such as PPARα, FGF21, CPT1L, and PDK4, in the offspring of all fat-supplemented groups, and the increase for most of them was more marked in the margarine group. HF-diet-fed animals in the margarine group also presented higher expression levels of CD36 compared to animals in the butter group and the controls, which suggests increased hepatic uptake of fatty acids. An increase in the fatty acid influx to the liver has been related to the development of hepatic steatosis;28 however, this does not appear to be the case in these conditions, suggesting that fatty acids taken up by the liver may be efficiently oxidized, in accordance with the increased levels in ketone bodies found in the margarine group compared to the other groups, particularly animals in the olive oil and control groups. This assumed increase in the uptake and oxidation of fatty acids by the liver in animals in the margarine group could be a mechanism to avoid increased circulating levels of NEFA, which has been related to insulin resistance progression.29 In fact, animals in the margarine group seemed to be protected against the increased triglyceridemia observed in the other groups under an HF diet and showed lower circulating levels of NEFA than the other groups under these conditions. This could be indicative of lower WAT lipolytic capacity, but contrasts with the presence in this tissue of higher mRNA levels (and to some extent also of the protein) of the key rate-limiting enzyme for lipolysis, ATGL, compared to those of the other fat-supplemented groups. Therefore, it is plausible that although WAT in these animals appears to have a greater capacity to release NEFA under fasting conditions, other tissues, as seen for the liver, could be more efficient in removing them from circulation. All in all, these results suggest that animals in the margarine group are programmed to be more efficient in the processing and handling of lipids under HF diet conditions, in relation to a better lipid clearance from circulation, and hence are protected against the ectopic accumulation of fat in tissues other than adipose tissue.

(0.627)] and WAT transcripts related to fat uptake [CD36 (0.745) and LPL (0.734)]. Under the HF diet, PC2 was mainly characterized by hepatic lipid content (−0.631), adiposity index (−0.532), and WAT transcripts, such as ATGL (0.898), PPARγ (0.857), INSR (0.730), and SREBP (0.613). The score plots of PC1 versus PC2 of all the samples in both dietary conditions are shown in Figure 5B. As can be seen, no discriminant model was found for animals of the different groups under an NF diet. In contrast, under an HF diet, control animals were separated from the animals of the fatsupplemented groups and were located in quadrant C, with the lowest scores for PC1. Animals in the margarine group were located in quadrant B, completely separate from the control animals. The olive oil and butter groups were mainly located in quadrant A, and no discrimination between these two groups was achieved.

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DISCUSSION Maternal intake of excess fat during pregnancy and lactation in rodents has been reported to be associated with negative outcomes on metabolic health in offspring, increasing the susceptibility to develop obesity and other related metabolic diseases.8 Studies performed in this respect are generally based on saturated fat sources. However, we previously described that maternal supplementation with excess fat during late pregnancy and lactation may have different outcomes in offspring depending on the source of fat. In particular, margarine, compared to other fats such as olive oil and butter, was shown to program the offspring for increased leptin sensitivity and a lower preference for fat food in adulthood.19 Here, by using the same cohort of animals, we also aimed to assess whether maternal lipid oversupply (with margarine, olive oil, or butter) during late pregnancy and lactation may also differently affect the metabolic health of offspring and hence the risk of obesityrelated metabolic diseases, particularly the development of hepatic steatosis. We show here that although maternal supplementation with the different fat sources did not result in significant differences regarding body mass in the adult offspring, margarine supplementation conferred partial protection against the increase in body fat content, particularly subcutaneous fat, observed in the other groups when exposed to an HF diet. This protection was not seen under NF diet conditions, where the three fat-supplemented groups showed higher subcutaneous fat content than the control group. Concerning hepatic lipid content, HF-diet-fed animals from the different groups showed increased lipid accumulation compared to their NF-diet-fed counterparts with the exception of animals in the margarine group, which seemed to present a certain protection against lipid accumulation in the liver. Histological analyses of liver samples essentially confirmed the results obtained from lipid quantification, but showed little incidence of hepatic steatosis signs in HF-diet-fed control animals, which was similar to that of animals in the margarine group under the HF diet. On the other hand, although animals in the olive oil and butter groups under the HF diet presented hepatic lipid levels similar to those of the control animals, histological analysis unveiled that these animals presented a more advanced form of the disease, particularly the animals in the butter group. Indeed, even under an NF diet, some animals in the olive oil group and more acutely in the butter group presented histological signs of NAFLD, suggesting that maternal olive oil and particularly butter oversupply during critical periods of development may 1598



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According to previous results,28,30 exposure of control animals to HF diet conditions brings about a decrease in the expression of lipogenic genes (FAS and SCD1) in the liver; however, this decrease was diminished in the offspring of all fatsupplemented groups. This lower downregulation could be related to the increased mRNA levels of INSR, suggesting higher insulin signaling, and to higher mRNA levels of the transcription factor SREBP1c, also occurring in these animals with the exception of those in the butter group. SREBP1c mediates the effects of insulin on the expression of different lipogenic genes, such as GK, ACC, FAS, ELOVL6, SCD1, and GPAT.18 In fact, HF-diet-fed animals in the olive oil and margarine groups showed increased expression levels of hepatic GK and PK compared to the controls. This may suggest a higher rate of fatty acid synthesis from glucose. Hence, lower downregulation of de novo lipogenesis under HF diet conditions may lead to higher hepatic accumulation of TG, as found in animals in the olive oil and butter groups. Animals in the margarine group, despite having increased lipogenesis capacity compared to the controls, appear to be more efficient in burning this excess fat supply and would better respond against hepatic steatosis under an HF diet. Therefore, increased β-oxidation capacity in spite of the higher capacity of de novo fatty acid synthesis appears to be responsible for the protection against hepatic steatosis in animals in the margarine group under HF diet conditions. This response seems to be different from that occurring in control animals under these obesogenic conditions, because they did not show increased lipogenic capacity in the liver, but in turn fatty acid oxidation capacity was not activated. Differences in the expression of lipogenic genes, depending on maternal fat supplementation, were also observed in WAT. PPARγ is a master transcriptional regulator of adipogenesis. Animals in the margarine group did not show the downregulation in the expression levels of PPARγ and SREBP occurring in the other groups of animals under HF diet conditions. This indicate that these animals may have a greater lipogenesis capacity in the WAT than the other fatsupplemented groups, but this could be counterbalanced with the increased ATGL mRNA expression levels in WAT of these animals compared to the other fat-supplemented groups under HF diet conditions. Hepatic steatosis has been associated with insulin resistance, and several rodent models have shown that a decrease in the hepatic TG pool correlates with improved insulin sensitivity.31,32 Here we did not find clear evidence of insulin resistance (in terms of serum parameters and HOMA-IR) in any group of animals, not even in those presenting signs of hepatic steatosis. Nevertheless, it should be mentioned that animals in the butter group fed an HF diet presented decreased expression levels of INSR in WAT compared to their NF-diet-fed counterparts, suggesting that these animals, which also presented the more advanced form of NAFLD, may be more prone to developing insulin resistance. Conversely, animals in the olive oil group, particularly under NF diet conditions, appear to be more efficient in the glucose clearance from circulation, since they showed the highest mRNA levels of GLUT4 and HK in WAT. This might indicate that animals in the olive oil group are metabolically better programmed for a correct glucose metabolism when under an NF diet. However, with the challenge of an HF diet, these animals do not seem to be that metabolically flexible and hence displayed a higher increase in body fat content than animals in the margarine group and

presented signs of liver steatosis. The predictive adaptative response hypothesis proposes that the degree of mismatch between the pre- and postnatal environments is an important determinant of subsequent diseases.8 Notably, we previously described that the intake of excess olive oil by dams during late pregnancy and lactation was associated with decreased adipogenesis and increased thermogenic capacity in weaned animals, affecting body mass gain and body fat during the suckling period.20 According to the mentioned hypothesis, later effects of this type of fat during the perinatal period appear to be dependent on postweaning diet and conditions. To figure out what changes in gene expression patterns (in the liver and WAT) were more related with two parameters related to metabolic health, such as adiposity and hepatic lipid content, a PCA was performed. This analysis showed that, under an HF diet, adiposity and lipid content in the liver were highly associated (in a negative way) with transcript levels in WAT related to lipolysis (ATGL) and to insulin sensitivity and adipogenesis (INSR, PPARγ, and SERBP). Thus, it can be interpreted that increased expression levels of these genes under an HF diet may be relevant to avoid excessive adiposity and TG accumulation in tissues other than WAT, particularly the liver. Interestingly, these variables were characteristic of principal component 2, and the margarine group was the one with the highest values of this component. In addition, PCA also showed the significance of the liver transcripts related to energy uptake and adipogenesis (GH, PK, and FAS) and also to fat oxidation (CPT1L, PDK4, and FGF21), all these transcripts with high scores in PC1 (in both dietary conditions). Curiously, control animals under an HF diet presented low values of this component compared to all the other groups. Therefore, although the distribution of animals under the NF diet was disperse, among HF-diet-fed animals, the control and the margarine groups were clearly distinguished between each other and from the olive oil and butter groups. The specific factors underlying the effects of maternal supplementation with different fat sources are not known. Differences described in milk fatty acid composition depending on the type of maternal fat during lactation 20 could influence the different outcomes. In addition, the putative involvement of the leptin protein in the beneficial effects of margarine oversupply compared to other fat sources may be considered, taking into account that margarine-supplemented dams (in the same cohort of dams) were described to present higher milk leptin concentration compared to olive oil dams at day 12 of lactation.19 Leptin is present in maternal milk,33 and its intake during the suckling period has been described to exert protecting effects against the development of later obesity and related metabolic pathologies, such as hepatic steatosis, under obesogenic conditions.25,34 It could be speculated that the greater supply of milk-borne leptin to animals in the margarine group could account for some of the beneficial effects found in this group of animals compared to the other fat-supplemented groups. However, further studies need to be done to confirm the involvement of milk-borne leptin and to ascertain whether other factors may be involved. In conclusion, maternal HF diet feeding during pregnancy and lactation affects offspring susceptibility to metabolic diseases, but the consequences depend not only on the intake of fat per se but also on the type of fat supplied. That is, maternal supplementation with excess fat from margarine during late gestation and lactation, in comparison with other types of fat, appears to program the offspring for healthier lipid 1599



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(5) Palou, M.; Priego, T.; Sanchez, J.; Palou, A.; Pico, C. Sexual dimorphism in the lasting effects of moderate caloric restriction during gestation on energy homeostasis in rats is related with fetal programming of insulin and leptin resistance. Nutr. Metab. 2010, 7, 69. (6) Pico, C.; Palou, M.; Priego, T.; Sanchez, J.; Palou, A. Metabolic programming of obesity by energy restriction during the perinatal period: different outcomes depending on gender and period, type and severity of restriction. Front. Physiol. 2012, 3, 436. (7) Bayol, S. A.; Simbi, B. H.; Stickland, N. C. A maternal cafeteria diet during gestation and lactation promotes adiposity and impairs skeletal muscle development and metabolism in rat offspring at weaning. J. Physiol. 2005, 567, 951−61. (8) Armitage, J. A.; Taylor, P. D.; Poston, L. Experimental models of developmental programming: consequences of exposure to an energy rich diet during development. J. Physiol. 2005, 565, 3−8. (9) Bayol, S. A.; Simbi, B. H.; Fowkes, R. C.; Stickland, N. C. A maternal “junk food” diet in pregnancy and lactation promotes nonalcoholic fatty liver disease in rat offspring. Endocrinology 2010, 151, 1451−61. (10) Guo, F.; Jen, K. L. High-fat feeding during pregnancy and lactation affects offspring metabolism in rats. Physiol. Behav. 1995, 57, 681−6. (11) Buckley, A. J.; Keseru, B.; Briody, J.; Thompson, M.; Ozanne, S. E.; Thompson, C. H. Altered body composition and metabolism in the male offspring of high fat-fed rats. Metabolism 2005, 54, 500−7. (12) Taylor, P. D.; McConnell, J.; Khan, I. Y.; Holemans, K.; Lawrence, K. M.; Asare-Anane, H.; Persaud, S. J.; Jones, P. M.; Petrie, L.; Hanson, M. A.; Poston, L. Impaired glucose homeostasis and mitochondrial abnormalities in offspring of rats fed a fat-rich diet in pregnancy. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2005, 288, R134−9. (13) Karnik, H. B.; Sonawane, B. R.; Adkins, J. S.; Mohla, S. High dietary fat feeding during perinatal development of rats alters hepatic drug metabolism of progeny. Dev. Pharmacol. Ther. 1989, 14, 135−40. (14) Khan, I. Y.; Taylor, P. D.; Dekou, V.; Seed, P. T.; Lakasing, L.; Graham, D.; Dominiczak, A. F.; Hanson, M. A.; Poston, L. Genderlinked hypertension in offspring of lard-fed pregnant rats. Hypertension 2003, 41, 168−75. (15) Langley-Evans, S. C. Intrauterine programming of hypertension in the rat: nutrient interactions. Comp. Biochem. Physiol., A: Physiol. 1996, 114, 327−33. (16) Bruce, K. D.; Cagampang, F. R.; Argenton, M.; Zhang, J.; Ethirajan, P. L.; Burdge, G. C.; Bateman, A. C.; Clough, G. F.; Poston, L.; Hanson, M. A.; McConnell, J. M.; Byrne, C. D. Maternal high-fat feeding primes steatohepatitis in adult mice offspring, involving mitochondrial dysfunction and altered lipogenesis gene expression. Hepatology 2009, 50, 1796−808. (17) Gregorio, B. M.; Souza-Mello, V.; Carvalho, J. J.; Mandarim-deLacerda, C. A.; Aguila, M. B. Maternal high-fat intake predisposes nonalcoholic fatty liver disease in C57BL/6 offspring. Am. J. Obstet. Gynecol. 2010, 203, 495 e1−8. (18) Postic, C.; Girard, J. Contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: lessons from genetically engineered mice. J. Clin. Invest. 2008, 118, 829−38. (19) Sanchez, J.; Priego, T.; Garcia, A. P.; Llopis, M.; Palou, M.; Pico, C.; Palou, A. Maternal supplementation with an excess of different fat sources during pregnancy and lactation differentially affects feeding behavior in offspring: putative role of the leptin system. Mol. Nutr. Food Res. 2012, 56, 1715−28. (20) Priego, T.; Sanchez, J.; Garcia, A. P.; Palou, A.; Pico, C. Maternal dietary fat affects milk Fatty Acid profile and impacts on weight gain and thermogenic capacity of suckling rats. Lipids 2013, 48, 481−95. (21) Matthews, D. R.; Hosker, J. P.; Rudenski, A. S.; Naylor, B. A.; Treacher, D. F.; Turner, R. C. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985, 28, 412−9.

partitioning and handling and in turn prevents, to some extent, hepatic steatosis under obesogenic conditions. This effect seems to be associated with an activation of the metabolic pathways related to adipogenesis and lipolysis in WAT, as well as with increased fatty acid oxidation in the liver. Conversely, butter oversupply during this critical period of development may promote hepatic steatosis in the offspring, even when animals are fed a balanced diet after weaning.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +34 971173170. Fax: +34 971173426. E-mail: andreu. [email protected]. Author Contributions †

M.L. and J.S. contributed equally to this work.

Funding

This work was supported by the Spanish Government (Grants AGL2009-11277 and AGL2012-33692), the European Union (BIOCLAIMS, Grant FP7-244995), and the Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red Fisiopatologiá de la Obesidad y Nutrición, CIBERobn. Our Laboratory is a member of the European Research Network of Excellence NuGO (The European Nutrigenomics Organization; EU Contract FP6-506360). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank E. Ceresi for his work in the liver histological analysis. ABBREVIATIONS USED ACC, actetyl coenzyme A carboxylase; ATGL, adipose triglyceride lipase; BHB, β-hydroxybutyrate; CPT1, carnitine palmitoyltransferase; FAS, fatty acid synthase; FGF21, fibroblast growth factor 21; GPAT, glycerol-3-phosphate acyltransferase; GK, glucokinase; GLUT4, glucose transporter 4; HK, hexokinase II; HF, high fat; INSR, insulin receptor; IRS1, insulin receptor substrate 1; LPL, lipoprotein lipase; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; NEFA, nonesterified (or free) fatty acid; NF, normal fat; PPAR, peroxisome proliferator activated receptor; PCA, principal component analysis; PDK4, pyruvate dehydrogenase kinase 4; PK, pyruvate kinase 4; SCD1, stearoyl coenzyme A desaturase 1; SREBP1c, sterol regulatory element binding protein 1c; TG, triglyceride; WAT, white adipose tissue

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REFERENCES

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Maternal Fat Supplementation during Late Pregnancy and Lactation

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