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Keywords:

  • developmental programming;
  • epigenetics;
  • inflammation

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest
  7. Funding
  8. References
  9. Supporting Information

Aim

Metabolic programming via components of the maternal diet during gestation may play a role in the development of different aspects of the metabolic syndrome. Using a mouse model, we aimed to characterize the role of maternal western-type diet in the development of non-alcoholic fatty liver disease (NAFLD) in the offspring.

Methods

Female mice were fed either a western (W) or low-fat control (L) semisynthetic diet before and during gestation and lactation. At weaning, male offspring were assigned either the W or the L diet, generating four experimental groups: WW, WL, LW and LL offspring. Biochemical, histological and epigenetic indicators were investigated at 29 weeks of age.

Results

Male offspring exposed to prenatal and post-weaning western-style diet (WW) showed hepatomegaly combined with accumulation of hepatic cholesterol and triglycerides. This accumulation was associated with up-regulation of de novo lipid synthesis, inflammation and dysregulation of lipid storage. Elevated hepatic transaminases and increased expression of Tnfa, Cd11, Mcp1 and Tgfb underpin the severity of liver injury. Histopathological analysis revealed the presence of advanced steatohepatitis in WW offspring. In addition, alterations in DNA methylation in key metabolic genes (Ppara, Insig, and Fasn) were detected.

Conclusion

Maternal dietary fat intake during early development programmes susceptibility to liver disease in male offspring, mediated by disturbances in lipid metabolism and inflammatory response. Long-lasting epigenetic changes may underlie this dysregulation.

Growth and development in utero is a complex and dynamic process, requiring interacting components from the mother and foetus to ensure optimal growth and survival throughout pregnancy (Belkacemi et al. 2010). Depending on the interactions between the mother, the placenta and the foetus, the supply of macro- and micro-nutrients and endocrine signals is critical in the early phase of life.

Epidemiological studies have been the first to link this early phase of life to developing obesity, cardiovascular disease, type-2 diabetes and increased central adiposity in adults (Forsdahl 1977, Barker & Osmond 1986). This link has been coined the Developmental Origins of Health and Disease (DOHaD) hypothesis. Originally, the focus of these studies was on the increased disease risk observed in individuals with a low birthweight, and hence the role of undernutrition in utero (Ravelli et al. 1998, 1999, Roseboom et al. 1999).

Undernutrition, however, has been nowadays replaced by over-nutrition in western society, affecting mothers and unborn children before or during gestation. Currently, in the USA, 18–35% of pregnant woman are clinically obese (Ehrenberg et al. 2004). Similarly, estimates from the UK suggest that 20% of pregnant woman registering for primary care are obese (Kanagalingam et al. 2005). Obesity during pregnancy is associated with increased risk of complications such as gestational hypertension, pre-eclampsia, gestational diabetes mellitus and delivery of a large-for-gestational-age infant. Longitudinal studies have shown strong associations between infants born large for gestational age, an independent risk factor for childhood obesity, and high gestational weight gain of the mother (Nohr et al. 2008). In addition, maternal pre-pregnancy BMI is a better predictor of childhood obesity than high newborn birthweight (Rising & Lifshitz 2008, Catalano et al. 2009, 2012, Laitinen et al. 2012). Childhood obesity, as a major risk factor for adult obesity, early type-2 diabetes and cardiovascular disease (Dietz 1998, Waters et al. 2011), indicates a possible rise in the prevalence of metabolic diseases.

The mechanistic link between maternal obesity and offspring health, by shared environment, is not yet completely understood. Animal studies support the concept that maternal over-nutrition during critical periods can result in long-term phenotypic changes in offspring, for example hyperphagia, increased adiposity, reduced locomotor activity, hypertension and insulin resistance (Gniuli et al. 2008, Samuelsson et al. 2008, Dunn & Bale 2009).

A number of studies have shown that these phenotypic changes, induced by prenatal nutrition, involve altered epigenetic regulation of specific genes (Jimenez-Chillaron et al. 2012), leading to metabolic dysregulation in the offspring. Nevertheless, there is limited information about the effect of maternal dietary fat intake on epigenetic processes and health outcome in offspring.

Very recently, several groups have described that gestational energy-rich diets can programme hepatic steatosis or even non-alcoholic steatohepatitis (NASH) in the offspring in rodent models (Bruce et al. 2009, Elahi et al. 2009, Gregorio et al. 2010, Oben et al. 2010, Ashino et al. 2012, Li et al. 2012, Kjaergaard et al. 2013, Mouralidarane et al. 2013). Often, these studies compare rodent chow with different diets based on lard as the main fat source (35, 45 or 60%), sometimes combined with high fructose intake or condensed milk. The combination of these studies clearly shows the close interrelationship between maternal diet and foetal liver physiology.

However, because of the complex composition of the diets used in these studies, it is impossible to identify the relevant ingredients which underlie this metabolic programming. Hence, the aim of this study was to characterize the long-term consequences of gestational over-nutrition in a mouse model and to pinpoint the molecular mechanisms involved using semi-synthetic diets only. The study focusses on the interactions between the prenatal and post-natal environment that together modulate the susceptibility to obesity and related diseases. We report that prenatal exposure to western diet (6 weeks before as well as during pregnancy and lactation) followed by post-weaning western diet feeding leads to increased weight gain, lipid accumulation and inflammation in the liver, the hallmarks NASH. We further correlate these phenotypic changes with epigenetic modifications in a selection of genes.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest
  7. Funding
  8. References
  9. Supporting Information

Experimental animals

All mice were housed in a light- and temperature-controlled facility (lights on 7:00–19:00 hours, 21 °C) with free access to drinking water and food. Experimental procedures were approved by the Ethics Committees for Animal Experiments of the University of Groningen. To study the effects of exposure to a western-style diet during development, 5-week-old female C57BL/6 mice were purchased from Harlan (Horst, the Netherlands) and randomly assigned to either a semi-synthetic energy-rich-western diet (W) (4.73 kcal g−1; 45% kcal fat, 20% kcal protein, 35% kcal carbohydrate; D12451, Research Diets, New Brunswick) or a semi-synthetic low-fat-control diet (L) (3.85 kcal g−1; 10% kcal fat, 20% kcal protein, 70% carbohydrate; D12450B, Research Diets). After 6 weeks on their respective diets, mice were mated with males. Dams failing to become pregnant were allowed to remate. Dams were maintained on their diets throughout pregnancy and lactation. Weight gain and food intake were measured weekly during this period. Dams were allowed to deliver spontaneously and left undisturbed with their litters for 24 h. This resulted in six litters in the control diet group and six litters in the maternal western diet group. Litter sizes were standardized to 5–6 pups, to ensure no litter was nutritionally biased. At weaning, male offspring were assigned to western or low-fat feeding, generating four experimental groups: LL (n = 7), WL (n = 7), LW (n = 10) and WW (n = 8), which represents prenatal and lactation vs. post-weaning diet. Offspring body weight and food consumption were recorded at weekly intervals. Offspring were terminated by heart puncture under isoflurane anaesthesia. Tissues were collected, weighed and snap frozen in liquid nitrogen and stored at −80 °C until biochemical analysis and RNA isolation.

Insulin tolerance test

Intraperitoneal insulin tolerance test (IPITT) was performed after 24 weeks on the W diet. Insulin tolerance was assessed after a four-hour fast with a single bolus of insulin (0.5 U kg−1, Actapid; Novo Nordisk, Bagsvaerd, Denmark) intraperitoneally at time 0, and tail venous blood glucose concentration was monitored at -10, 10, 20, 30, 40, 50, 60, 75 and 90 min with a glucometer in conscious animals.

Plasma and liver measurements

Blood glucose levels were measured with a Lifescan EuroFlash glucose meter (Lifescan Benelux, Beerse, Belgium). Plasma insulin concentrations were determined using an Ultrasensitive Mouse Insulin ELISA kit (Alpco Diagnostics, Salem, NH, USA). Livers were homogenized in ice-cold phosphate-buffered saline. Hepatic lipids were extracted according to Bligh and Dyer (Bligh & Dyer 1959). Commercially available kits (total and free cholesterol (DiaSys Diagnostic Systems, Holzheim, Germany), non-esterified-free fatty acids (Wako, Neuss, Germany) and triglycerides (Roche Diagnostics, Mannheim, Germany)) were used to determine the lipid profiles in plasma and liver. Fatty acid composition was determined using gas chromatography after methylation as described previously (Lepage & Roy 1986). Levels of the ketone body metabolite, 3-hydroxybutyric acid, in the plasma were determined using a commercial kit (BioAssay Systems, Heyward, CA, USA). Immunoreactive hormones were determined on a Luminex 100 instrument (luminex, Oosterhout, the Netherlands) by multiplex immunoassay (Biorad, Hercules, CA, USA) according to manufacturer's protocol.

Histological analysis

Liver sections were fixed in 4% formalin and paraffin embedded prior to sectioning. Fixed liver sections were stained with haematoxylin–eosin for visual assessment of steatosis and cellular infiltrate. The severity of liver condition was quantified by a certified veterinary pathologist using the Kleiner scoring system (Kleiner et al. 2005). The average score for each histological characteristic in each group was used. In situ immunohistochemistry was performed on 4-μm-thick sections of paraffin-embedded liver samples with a three-step avidin–biotin technique according to the manufacturer's instructions (LSAB2, Dako, Copenhagen, Denmark). The primary antibody was anti-CD3 (Dako) rabbit polyclonal antibody recognizing lymphocytes and primary antibodies against F4/80 (1 : 200; BioLegend, San Diego, CA, USA) for macrophages. The number of lymphocytes/macrophages within the liver section was evaluated by counting positive cells per 400× field in ten random fields.

RNA extraction and expression microarray

Total RNA was extracted from liver and white adipose tissue (WAT) samples using the TRI Reagent method (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer's protocol. Integrity and concentration of RNA were determined with Experion (Biorad). Purified total RNA from 6 liver samples per group was used for global gene expression analysis (mouseWG6 v2.0, Illumina, San Diego, CA, USA). One experimental and 1 biological outlier were excluded, so final n = 6, 5, 5, 6 (LL, WL, LW, WW). Mouse WG6 arrays were analysed using the MADMAX pipeline (Lin et al. 2011; Wageningen, the Netherlands). After quantile and ComBat normalization, data were prefiltered (FC1.1) and statistically analysed (IBMT); genes passing the FDR of 5% (LL vs. WL, 1 probe; LW vs. WW, 269 probes) were added to our list. An FDR of 10% would result in 6 and 716 probes respectively (data not shown here). Metacore™ enrichment analysis was performed on the extracted list. Comparison was between LW vs. WW, because LL vs. WL analysis yielded 1 probe. Array Data are deposited at the GEO database (GSE44901).

Quantitative RT-PCR

Complementary DNA was obtained using the reverse transcription procedure with Moloney Murine Leukaemia Virus-reverse transcriptase (M-MLV-RT) (Sigma-Aldrich) with random primers according to the protocol of the manufacturer. cDNA levels were measured by TAQMAN RT-PCR using an ABI PRISM 7900 sequence detector (Applied Biosystems, Nieuwekerk a/d IJssel, the Netherlands) against a calibration curve of pooled cDNA solutions. PCR was performed on all samples available [n = 7, 7, 10, 8 (LL, WL, LW, WW)]. Hepatic expression levels were normalized to 36b4 and adipocyte expression levels to Eef2. The sequences of primers and probes published earlier are deposited at RTPrimerDB (www.rtprimerdb.org), unpublished primers and probe sequences are available on request.

DNA isolation, bisulphite conversion and gene-specific DNA methylation analysis

Genomic DNA was isolated from liver using the Allprep DNA/RNA isolation kit (Qiagen, Venlo, the Netherlands) following the manufacturer's protocol. Bisulphite modification of 500 ng genomic DNA was performed utilizing the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA) according to manufacturer's instructions. The bisulphite-converted DNA (20–25 ng μL−1) was stored at −20 °C until use. DNA amplifications were performed on bisulphite-treated DNA using commercially available assays for Fasn (PM00238448, Qiagen), Insig (PM00203847, Qiagen) and Lpl (PM00405118, Qiagen). PCR primers were designed for Lxra, Ppara and Pgc1a (Table S1). Modified DNA (20–25 ng) was amplified in a total volume of 25 μL. Two microlitre of the PCR product was analysed on agarose gel. The methylation status was examined by pyrosequencing using a Qiagen pyromark Q24 system with 10 μl of the PCR product. Pyrosequencing reactions were set up using PyroGold Reagent kit (Qiagen) according to the manufacturer's instructions. The methylation levels at the target CpGs were evaluated by converting the resulting pyrograms to numerical values for peak heights and expressed either as the percentage of methylation of individual CpG sites or as the mean of all CpGs analysed at a given sequence.

Data analysis and statistics

Data were tested for normal distribution, then reported as Tukey's Box and Wiskers plot using median with 25th to 75th percentile intervals, unless stated otherwise. Plots were created using the Graphpad Prism 5.0 software package. Statistical analysis between prenatal exposure was performed using the Mann–Whitney U-test, and the level of significance was set at P < 0.05. Statistical analyses were performed using SPSS 20.0 for Windows (SPSS Inc, Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest
  7. Funding
  8. References
  9. Supporting Information

Characteristics of the dams

Female mice received a western-style diet (W) before and throughout pregnancy and lactation. The different dietary treatments did not affect body weight, fasting plasma glucose, insulin, cholesterol and non-esterified fatty acid levels among dams at weaning (Table 1). Importantly, food intake was 6.00 g per mouse per day in the western diet group and 7.29 g per mouse per day in the control group, leading to similar daily caloric intake. Plasma triglyceride levels were elevated at time of weaning in western diet-fed dams (Table 1). Neither litter size (low-fat; n = 5–8 vs. western; n = 5–10) nor birthweight [low-fat; 1.31 (1.10–1.53) vs. western 1.11 (0.90–1.40)] was influenced by prenatal western diet.

Table 1. Physiological characteristics of dams at weaning
 LW
  1. L, low-fat control; W, western-style diet.

  2. Values are median (min-max).

  3. L (n = 6); W (n = 6).

  4. a

    P < 0.05.

Body weight (g) 27.05 (25.18–27.42) 28.71 (26.06–32.07)
Plasma
Glucose (mmol L−1)6.45 (5.70–7.20)7.20 (5.60–8.80)
Insulin (ng mL−1)0.43 (0.18–3.78)0.28 (0.17–1.08)
Triglycerides (mmol L−1)0.26 (0.18–0.31) 0.50 (0.31–0.83) *
Cholesterol (mmol L−1)2.49 (1.80–2.76)3.35 (0.86–4.07)
Non-esterified fatty acids (mmol L−1)0.38 (0.30–0.51)0.44 (0.38–0.59)

Maternal western diet had limited effects on plasma parameters in the offspring at adult age

To elucidate the long-term effects of maternal diet, all metabolic parameters of offspring were compared based on the prenatal treatment; LL vs. WL and LW vs. WW.

Despite the small effects seen in western-fed dams, WW offspring reached a greater body weight compared with LW offspring (P < 0.05, Fig. 1a). This difference in body weight did not relate to food intake (Fig. S1).

image

Figure 1. Effects of maternal western-style diet feeding on hepatic lipids. Phenotypic characterization of 29-week-old male offspring. LL (n = 7), WL (n = 7), LW (n = 10), WW (n = 8). (a) Body weight at 29 weeks of age. (b) Livers from LL, WL, LW and WW offspring show differences in size and gross morphology. (c) Percentage of liver weight vs. total body weight. (d) Liver triglycerides and total cholesterol. (e) saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA) composition. *P < 0.05; **P < 0.01. Data expressed as median with percentiles (25–75).

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Analysis of fasted plasma lipids revealed similar levels of β-hydroxybutyrate, non-esterified fatty acids (NEFA), triglycerides (TG) and total cholesterol (CHOL) between prenatal treatment groups (Table 2). No difference was detected on fasting plasma glucose and insulin over the course of the experiment (Table S2). In addition, no decrease in insulin sensitivity was detected between offspring during an insulin tolerance test (Fig. S2).

Table 2. Plasma parameters of male offspring at 29 weeks of age
 LLWLLWWW
  1. Values are median (min-max).

  2. a

    P < 0.05, WL vs. LL.

  3. b

    P < 0.01, WW vs. LW.

n 7 7108
Triglycerides (mmol L−1)0.42 (0.26–0.50)0.59 (0.33–0.71)0.52 (0.34–0.73)0.49 (0.18–0.83)
Cholesterol (mmol L−1)3.66 (2.72–3.91)3.42 (1.98–3.92)4.65 (2.80–5.89)4.65 (2.56–6.67)
Free cholesterol (mmol L−1)2.34 (1.99–2.61)2.31 (1.97–2.53)2.49 (1.64–2.83)2.76 (2.22–3.32)
Esterified cholesterol (mmol L−1)1.08 (0.38–1.83)1.12 (0.01–1.54)1.94 (0.31–4.25)2.06 (0.31–3.65)
Non-esterified fatty acids (mmol L−1)0.53 (0.40–0.69)0.50 (0.38–0.90)0.53 (0.38–0.69)0.42 (0.31–0.58)
Β-hydroxybutyrate (mmol L−1)0.75 (0.58–1.97)0.60 (0.33–1.01)0.32 (0.22–1.38)0.22 (0.17–0.50)
Leptin (ng mL−1)8.14 (2.89–22.18)11.53 (0.58–29.35)31.00 (22.53–99.37)40.27 (27.62–54.19)
Resistin (ng mL−1)45.69 (23.57–109.23)66.79 (13.25–86.45)136.71 (79.94–254.32)132.41 (54.54–184.80)
GLP−1 (pg mL−1)43.71 (31.75–61.23)27.84 (9.30–72.31)#32.76 (4.72–78.22)31.97 (22.01–72.31)
PAI−1 (ng mL−1)1.23 (0.96–1.55)1.34 (0.97–2.71)1.66 (1.27–4.30)1.55 (1.14–4.04)
Glucagon (pg mL−1)322.26 (243.22–393.81)270.74 (100.08–924–98)273.98 (218.49–655.42)285.81 (147.50–969.98)
Ghrelin (ng mL−1)14.24 (5.02–20.24)13.83 (5.66–34.81)10.41 (6.69– 14.24)6.82 (5.66–8.83)**
GIP (pg mL−1)229.48 (189.01–273.83)224.31 (161.94–286.45)264.96 (195.60–396.46)231.20 (161.32–247.38)**

Plasma adipokines and adipose tissue-derived hormones were analysed (Table 2). Gastric inhibitory polypeptide (GIP) (P < 0.01, WW vs. LW) and plasma GLP-1, glucagon-like peptide (P < 0.05, WL vs. LL) were decreased in offspring prenatally exposed to the western diet.

Maternal western diet induces fat accumulation in the liver of offspring on western diet

While, livers of LL and WL offspring were regarded as very similar in size [LL 1.20(0.80–1.80) vs. WL 1.40(0.90–1.80)], WW offspring displayed alterations in liver size (LW 1.55(1.20–2.70) vs. WW 2.20(1.80–3.70), P < 0.01] and gross morphology (Fig. 1b). Moreover, this increase in size was still seen when comparing relative liver mass (to body weight) of WW with LW offspring (Fig. 1c, P < 0.01).

This increased liver mass parallels with hepatic lipid analysis. Accumulation of triglycerides (P < 0.01, vs. LW) and cholesterol (P < 0.05, vs. LW) was measured in the livers of WW offspring (Fig. 1d). The relative amount of saturated fatty acids (SFA) was not affected by prenatal western diet feeding. However, the relative abundance of monounsaturated fatty acids was elevated (MUFA, P < 0.01, vs. LW), while a marked reduction in polyunsaturated fatty acids (PUFA, P < 0.05, vs. LW) was observed in WW offspring (Fig. 1e). The relative amounts of stearate (18 : 0), arachidonate (20 : 4) and docosanoic acid (22 : 0) were markedly decreased in WW offspring (P < 0.05, vs. LW), whereas these were increased in WL offspring (P < 0.05, vs. LL). In contrast, the relative amounts of palmitoleate (16 : 1), oleate (18 : 1), cis-vaccenate (18 : 1) and eicosenoate (20 : 1) were markedly increased in WW offspring (P < 0.05 vs. LW) (Fig. S3).

Maternal fat intake primes the progression towards development of steatohepatitis

Plasma alanine aminotransferase (ALT), a marker for hepatic damage, was normal in LL and WL offspring, but increased in the WW offspring compared with LW offspring (Fig. 2a, P < 0.05, WW vs. LW). Analysis of the aspartate aminotransferase (AST) showed similar results (Fig. 2b, P < 0.01, WW vs. LW), indicating that maternal western diet feeding led to increased liver damage in the offspring exposed to post-weaning western diet.

image

Figure 2. Exposure to western-style diet during early developmental phases and adult life induces hepatic steatosis. (a) Plasma ALT levels. (b) Plasma AST levels. (c) NAS activity score. (d) H&E staining, (e) CD3 and (f) F4/80 stains of representative liver sections. *P < 0.05; **P < 0.01. Data expressed as median with percentiles (25–75). LL (n = 7), WL (n = 7), LW (n = 10), WW (n = 8).

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In parallel, histological analysis revealed clear evidence of liver injury in WW offspring, as confirmed by the Kleiner NAFLD Activity Score (Fig. 2c). Offspring fed the low-fat diet post-weaning showed relatively normal hepatic architecture, without ballooning and with slight inflammation. As described above, post-weaning western diet led to a hepatic fatty liver in adult LW offspring. In addition, maternal western diet strengthened the disease progression to acute steatohepatitis (NASH) by greater fat infiltration in the peri-portal zone and increased steatosis in distinct panacinar zones compared with LW offspring (Fig. 2d). Increased presence of lymphocytes (CD3 staining, Fig. 2e) and macrophages (F4/80 staining, Fig. 2f) confirmed the boosted inflammation in the liver of WW offspring.

Increased hepatic inflammation and fibrogenesis in WW offspring

To characterize the molecular consequences of maternal western diet feeding, we assessed the hepatic gene expression profile by Illumina WG6 microarrays. Metacore enrichment analysis indicated alterations in regulatory pathways, processes and process networks between LW and WW offspring groups. Retinol metabolism and arachidonic acid metabolism were affected, which can lead to disturbed circadian rhythm and drive to the metabolic syndrome through altered glycolysis and fatty acid metabolism in post-weaning western diet groups. The gene expression profile of WW offspring has uniquely changed in respect to inflammatory pathways, which is in concordance with our histological data (Table 3).

Table 3. Summary of Microarray-generated Metacore analyses in LW vs. WW offspring
 P-valueFDR
Differentially regulated pathways
Protein folding and mutation_Angiotensin system maturation0.000000.00001
Linoleic acid/Rodent version0.000080.00928
Differentially regulated processes
Response to organic substance0.000000.00000
Response to stress0.000000.00000
Response to endogenous stimulus0.000000.00000
Response to hormone stimulus0.000000.00000
Response to wounding0.000000.00000
Wound healing0.000000.00000
Regulation of biological quality0.000000.00000
Response to chemical stimulus0.000000.00000
Cellular response to hormone stimulus0.000000.00000
Cellular response to chemical stimulus0.000000.00000
Differentially regulated networks
Inflammation_IL-6 signalling0.000070.00952
Development_Skeletal muscle development0.000330.02318
Development_Regulation of angiogenesis0.000870.03423
Cell adhesion_Integrin priming0.000970.03423
Blood coagulation0.001760.03709
Cell adhesion_Cell-matrix interaction0.001840.03709

The interaction between disrupted lipid homeostasis and immune response is implicated in the pathogenesis of NASH. Among key inflammatory genes, Tnfa was up-regulated in prenatally exposed WL (vs. LL) and WW offspring (P < 0.05, vs. LW). In addition, Mcp1 was highly induced in WW offspring (P < 0.05, vs. LW), as well as Cd11 (P < 0.05, vs. LW) and Cd68 (P < 0.01, vs. LW). The hepatic fibrosis marker, Tgfb, showed elevated mRNA level only in the WW offspring with NASH phenotype (P < 0.05, vs. LW) (Fig. 3a).

image

Figure 3. Effect of prenatal diet on mRNA expression in the liver. (a) mRNA levels of genes involved in de novo lipogenesis in liver. (b) mRNA levels of regulatory genes in liver. (c) mRNA level of inflammatory markers in liver. *P < 0.05; **P < 0.01; ***P < 0.001. Data expressed as median with percentiles (25–75), LL (n = 7), WL (n = 7), LW (n = 10), WW (n = 8).

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Maternal fat intake induces hepatic lipogenic gene expression, in parallel to lipid accumulation

To further explore the hepatic lipid accumulation in WW offspring, we assessed the expression of genes involved in fatty acid synthesis and oxidation (Fig. 3b,c). Expression of several key genes involved in hepatic lipogenesis was found to be increased in WW offspring relative to LW, such as the master lipogenic transcription factor Srebp1c (P < 0.05) and target genes Fasn (P < 0.05), Scd1 (P < 0.001), Acc1 (P < 0.05) and Elovl6 (P < 0.05). The expression of Lxra was not affected by prenatal western diet feeding. However, the expression of Rxra, encoding for the heterodimer partner of LXR and the PPARs, was reduced in WW offspring compared with LW offspring (P < 0.05). In addition, a reduced mRNA level of Ppara was detected (P < 0.01, WW vs. LW), without changes in expression of target genes. The mRNA level of Cidea and Cidec, involved in lipid droplet formation, was increased in offspring exposed to the western-style diet prenatally (WW vs. LW).

Maternal western diet increases transcription of genes involved in lipid synthesis and inflammation in adipose tissue

As metabolic disorders are associated with adipose tissue, we examined this tissue in more detail. Alterations in the transcript level of the fatty acid synthesis regulating genes were observed in white adipose tissue (WAT). Scd1 and Elovl6 were down-regulated, while Dgat2 (P < 0.05), Fatp4 (Slc27a4, P < 0.05), Lpl (P < 0.05) and Fabp4 (P < 0.05) were up-regulated in WW offspring compared with LW offspring. Furthermore, offspring exposed to the western-style diet during developmental periods showed higher expression level of Srebp1a (P < 0.05, LL vs. WL and P < 0.05, WW vs. LW), Srebp1c (P < 0.01, LL vs. WL and not significant, WW vs. LW) and Srebp2 (P < 0.09, LL vs. WL and P < 0.01, WW vs. LW) (Fig. S4a).

In parallel, the inflammatory status of WAT was characterized because low-level chronic inflammation of white adipose tissue is a well-documented phenomenon in animal and human obesity. Gene expression level of the macrophage-related markers such as F4/80 (P < 0.001, LL and WL vs. LW and WW), Cd11 (P < 0.001, LL and WL vs. LW and WW) and Tnfa (P < 0.01, LL and WL vs. LW and WW) was all markedly increased in the LW and WW offspring compared with LL and WL offspring. Moreover, WW offspring had a significantly higher expression of the profibrotic Timp1 (P < 0.01, WW vs. LW) and higher transcript level of Il6 although it was not significant, relative to LW offspring (Fig. S4b). The expression of these genes was not only affected by post-weaning western feeding alone, demonstrating significant positive interaction between prenatal exposure to western diet in inducing adipose tissue inflammation.

Maternal western diet modifies the hepatic DNA methylation pattern of Lpl, Fasn, Insig2, Lxra, Pgc1a and the Ppara imprinted region

DNA methylation changes have been shown to be important for long-term regulation of gene expression. We selected several key metabolic genes for methylation analyses. Our focus was on genes previously identified to be involved in epigenetic programming (see Jimenez-Chillaron et al. 2012, and Pruis et al. 2013, for background information). Genes that have a regulatory function in metabolism and a relatively CpG rich promoter were analysed: Lxra, Insig2, Ppara, Pgc1a, Fasn and Lpl. The results of the DNA methylation analysis in liver tissues are shown in Figure 4. Lxra, Insig2, Ppara, Fasn and Lpl genes showed higher methylation levels of at least one individual CpG position related to the maternal diet. Pgc1a, on the other hand, showed reduced methylation at the first CpG position (LW vs WW). Only for Fasn was the difference in methylation so high that also the average of all methylated sites was increased (WW vs. LW).

image

Figure 4. Levels of DNA methylation in key metabolic genes in the liver. Methylation levels of consecutive CpG positions and their average of (a) Lxra, (b) Insig2, (c) Ppara, (d) Pgc1a, (e) Fasn and (f) Lpl. *P < 0.05. Data expressed as median with interquartile range (25–75), LL (n = 7), WL (n = 7), LW (n = 10), WW (n = 8).

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest
  7. Funding
  8. References
  9. Supporting Information

This study investigated the role of a maternal western diet (rich in energy, fat and cholesterol) on the development of adult metabolic disease susceptibility of the offspring. Using a mouse model of maternal western-style diet feeding, we further strengthen the concept that early developmental phases are critical to the development and progression of the metabolic syndrome. In our study, WW offspring – the combination of maternal and offspring western diet feeding – presented increased weight gain, hepatic hyperlipidaemia as well as increased liver injury, confirmed by histopathological characterization and verification by hepatic expression of inflammatory markers. Whether these physiological changes, seen in offspring prenatally exposed to western diet, are induced by elevated dietary fat content per se or are instead due to a change in dietary fatty acid composition was not addressed in this experimental design.

Murine models have consistently described lipid abnormalities in offspring exposed to a high-fat diet during early phases of development (Bruce et al. 2009, Elahi et al. 2009, Gregorio et al. 2010, Oben et al. 2010, Ashino et al. 2012, Li et al. 2012, Kjaergaard et al. 2013, Kruse et al. 2013, Mouralidarane et al. 2013). We here compared western-type diet (moderate fat and cholesterol) to matched semisynthetic control diet. In contrast to these studies, where obesogenic diets have been used, our feeding regimen did not lead to maternal obesity or maternal insulin resistance, because dams on western diet had a comparable caloric intake as control dams by eating less. We have started feeding the different diets several weeks before mating, to guarantee a steady-state situation in the dams and to avoid abrupt metabolic changes during pregnancy. Otherwise, direct stress effects of a dietary change on the embryo and foetus could have occurred. Still, our data clearly indicate that maternal western diet intake, even without maternal obesity and/or insulin resistance, has an effect on the metabolism of offspring, predisposing them to development of NAFLD later in life. Hence, our study extends the data from our colleagues with the novel finding that maternal obesity is not required for programming of NAFLD, but that an isocaloric shift from carbohydrate to fat in the diet is already sufficient.

The pathogenesis of NAFLD is considered a two-hit model. The ‘first hit’ (fat accumulation) sensitizes the liver to injuries caused by one or more additional factors, while the ‘second hit’ leads to the development of fibrosis (Day & James 1998). Following this suggested model, the ‘first hit’ in this study could be the dysregulated fat metabolism in WW offspring, derived from an increased de novo lipogenesis, increased lipid storage and decreased oxidation.

Increased de novo lipogenesis in this model could be mediated through increased expression of Srebp1c and downstream target genes. The hepatic SREBP1c-mediated pathways are known to be activated by insulin and Lxra (Fon Tacer & Rozman 2011). In our model, however, the WW offspring did neither show an alteration in the transcript level of Lxra, nor in the expression of corresponding Lxra target genes. It has been shown that PUFA suppress the activity of Srebp1c, independent of Lxra, by enhanced degradation of Srebp1c mRNA and stimulated proteasomal degradation of SREBP proteins (Deng et al. 2002). Therefore, we suggest that the increased level of Srebp1c is induced by the decreased level of hepatic PUFA.

In addition to decreased levels of hepatic PUFA in WW offspring, there was an increase in MUFA. This tilt of the balance towards MUFA formation may be caused by the up-regulation of Scd1, shown to play a key role in adequate storage and hence the development of hepatic steatosis (Li et al. 2009). Adequate storage of lipids is further promoted by the CIDE family proteins (Yonezawa et al. 2011, Zhou et al. 2012). Increased hepatic mRNA expression of Cidea and Cidec was present in WW offspring indicative of abnormal elevation of lipid droplet formation. Cidea mRNA expression has been shown to be highly correlated with the development of hepatic steatosis in humans (Zhou et al. 2012). CIDEC also mediated the development of hepatic steatosis (Matsusue et al. 2008). In the development of NAFLD, CIDE family members could be a promising sign of altered lipid accumulation.

The ‘second hit’ in the development of NASH encompasses a variety of factors. These factors include hepatocyte organelle (particularly mitochondria) malfunction, peroxisome proliferator-activated receptor dysfunction and inflammation (Zhan & An 2010). Firstly, mitochondrial dysfunction has been proposed as an important mechanism in the development of NASH in offspring from high-fat diet-fed dams (Bruce et al. 2009). While we did not address electron transport activity, WW offspring show signs of altered mitochondrial function, for example microvesicular fat accumulation, decreased plasma β-hydroxybutyrate and down-regulation of Ppara mRNA. We could assume that peroxisomal and mitochondrial β-oxidation systems are not appropriately up-regulated to match the increased production of fatty acids.

Secondly, inflammation could be a key process to initiate the development of NASH. In our model, pre- and post-weaning western diet feeding led to increased Tnfa levels in the liver, and this increase has been shown to be involved in the recruitment of circulating macrophages, activation of Kupffer cells and hepatic stellate cells. Likewise, the higher expression of Tgfb in WW offspring indicates the initiation of these intracellular signalling cascades through the activation of hepatic stellate cells. This recruitment in WW offspring contributed to the more severe liver injury seen.

Moreover, in WAT, there was an increased expression of inflammatory markers such as Cd38 and Timp1. Interestingly, under obese conditions, low-level chronic inflammation and macrophage infiltration into white adipose tissue are well-documented phenomenon in NAFLD (Rius et al. 2012).

Among the different mechanisms that could lead to interindividual differences, the epigenetic regulation of gene expression has emerged as a potentially important contributor. One such contributor, which has been established as playing a role in the programming of offspring through maternal nutrition, is the methylation of CpG islands (discussed in Jimenez-Chillaron et al. 2012). We have previously described, in a programming model of protein restriction, that the CpG methylation of Lxra is altered (van Straten et al. 2010). In contrast to the protein restriction model, we now found no differences in Lxra methylation. However, another important regulator, Ppara, showed significant methylation differences in several CpG positions; this might explain the loss of up-regulation of this gene. This finding has been described earlier in a model of maternal protein restriction (Lillycrop et al. 2008). The regulatory region of Ppara belongs to the most sensitive metabolic programming target, in a nutritional perspective. Our results further support the theory that maternal over-nutrition leads to altered DNA methylation pattern of the offspring during their development and influencing their later health. However, we could not establish this relationship for all genes studied, indicating that other regulatory factors may be active.

In summary, hepatic triglyceride accumulation in WW offspring was attributed to up-regulation of de novo fatty acid synthesis via activation of Srebp1c, failure to up-regulate mitochondrial B-oxidation and fatty acid export. We propose that the major force in the disease progression from NAFLD to NASH of WW offspring is inflammation due to programmed effects of maternal western diet. First epigenetic changes have been described here, but a genome-wide approach will be required to unravel the complete underlying network of regulation.

We are very grateful to Angelika Jurdzinski for animal care and excellent technical assistance.

Funding

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest
  7. Funding
  8. References
  9. Supporting Information

This research was performed within the framework of CTMM, the Center for Translational Molecular Medicine (www.ctmm.nl), project PREDICCt (Grant 01C-104) and supported by the Dutch Heart Foundation, Dutch Diabetes Research Foundation and Dutch Kidney Foundation.

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  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest
  7. Funding
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest
  7. Funding
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
apha12197-sup-0001-fig1.tifTIFF image403KFigure S1. Average food intake from weaning until 28 weeks of age.
apha12197-sup-0002-fig2.tifTIFF image232KFigure S2. Insulin tolerance test.
apha12197-sup-0003-fig3.tifTIFF image571KFigure S3. Hepatic fatty acid composition.
apha12197-sup-0004-fig4.tifTIFF image1096KFigure S4. White adipose tissue gene expression.
apha12197-sup-0005-Supplementary-Tables.docxWord document21KTable S1. List of primers used for pyrosequencing of bisulfite converted genomic DNA. Table S2. Plasma glucose and insulin levels at different time-points.

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