bs_bs_banner

doi:10.1111/cga.12062

Congenital Anomalies 2014; 54, 195–219

195

ORIGINAL ARTICLE

Seeking genes responsible for developmental origins of health and disease from the fetal mouse liver following maternal food restriction Tetsuo Ogawa1*, Junko Shibato1, Randeep Rakwal1,2,3, Tomomi Saito1, Gaku Tamura1, Makiko Kuwagata1 and Seiji Shioda1 Department of Anatomy, Showa University School of Medicine, Tokyo, 2Faculty of Life and Environmental Sciences, and 3Organization for Educational Initiatives, University of Tsukuba, Ibaraki, Japan 1

ABSTRACT

Low birthweight resulting from a nonoptimal fetal environment is correlated epidemiologically to a higher risk of adult diseases, and which has also been demonstrated using animal models for maternal undernutrition. In this study, we subjected pregnant mice to 50% food restriction (FR), and profiled gene expression and promoter DNA methylation genome-wide using the fetal livers. The fact that effect of food restriction is opposite between before and after birth encouraged us to hunt for genes that are expressed oppositely to adult calorie restriction (CR) using the maternal livers. Among oppositely regulated genes, we identified trib1 (tribbles homolog 1). Using genetically modified mice, trib1 has been shown to have a demonstrable contribution to a risk of hypertriglyceridaemia and insulin resistance. Our data showed that the trib1 expression and its promoter DNA methylation could be affected physiologically (by maternal nutrition), and therefore might be a strong candidate gene for developmental origins of adult diseases. Furthermore, lepr (leptin receptor) gene was downregulated by maternal FR, indicating its potential role in induction of obesity and diabetes. Gene expression as well as promoter DNA methylation profiling revealed that glucocorticoid receptor target genes were regulated by maternal FR. This supports previous studies that suggest an important role of fetal glucocorticoid exposure in the mechanism of developmental origins of diseases. Our transcriptomics profiling data also suggested that maternal FR impaired development of the immune system. An inventory of candidate genes responsible for developmental origins of health and disease is presented and discussed in this study. Key Words: developmental origins of health and disease, lepr, maternal undernutrition, promoter methylation microarray, trib1

INTRODUCTION Looking at people around you in daily life reveals interesting variations in body shape and food habits. Some people eat a lot but keep a slim body, whereas some people claim to eat less but are fat. Similarly, there are people who live longer while others die early, even under a similar environment. In Japan, for example, people tend to live long lives. However, living longer is not always an indicator of good health. Some very senior citizens remain healthy while others at the same age are bed-ridden. How can we explain Correspondence: Tetsuo Ogawa, PhD, Department of Anatomy, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan. Email: [email protected] Received January 30, 2014; revised and accepted April 3, 2014. *Present address: Department of Physiology, Saitama Medical University, 38 Morohongo Moroyama-machi, Iruma-gun, Saitama 350-0495, Japan.

these differences? Developmental origins of health and disease (DOHaD) concept holds the key to understanding why some people are at a higher or lower risk. Non-optimal fetal environments resulting in low birthweight have epidemiologically been associated with increased risks for adult diseases such as hypertension, cardiovascular disease, and type 2 diabetes (Barker and Osmond 1986; Barker et al. 1993, 2002a, 2002b). Studies on some populations in South Africa, Finland, and India demonstrated that children born with a low birthweight and who underwent rapid postnatal weight gain (catch up growth) were most likely to develop type 2 diabetes in adulthood (Crowther et al. 1998; Forsen et al. 2000; Yajnik 2000). This pattern of fetal and childhood growth was also reported to make them susceptible to the development of hypertension and coronary heart disease later in life (Eriksson et al. 1999; Cheung et al. 2000; Singhal et al. 2007). These epidemiological studies established the DOHaD concept (Barker 2007). The theory considers that nutritional and/or other environmental factors during critical periods of gestation have the potential to permanently “program” the structure and/or function of emerging organ systems. Complete understanding of the mechanisms underlying DOHaD is a distant goal but some early progress has been reported. Further increase in unraveling the mechanisms will definitely contribute to the prevention of adult diseases, with potential positive affects to healthy human aging. As a mechanism for their epidemiological findings, Hales and Barker have proposed the “thrifty phenotype hypothesis” (Hales and Barker 2001). In this hypothesis those authors considered that fetal undernutrition induces adaptations to maximize chances of survival in prospected postnatal environments of poor nutrition. During these adaptations, undernutrition would permanently impact on wholebody metabolism by modifying the programming of the genes and their expression profiles. These adaptations would be beneficial only if they were born into their prospected poor environment. However, those would be side-effects if they were to be exposed to the opposite environment (rich nutrition). We considered that understanding how gene expression in fetal tissues is affected during these adaptations holds the key to unveil the mechanisms for developmental origins. Animal studies are important to investigate the DOHaD theory. A study on mice successfully demonstrated that intrauterine undernutrition produced by 50% food restriction (FR) induced low birthweight and catch-up growth during the first postnatal week, and thereafter the offspring developed impaired glucose tolerance and obesity in adulthood (Jimenez-Chillaron et al. 2006). Previously, phenotypes of metabolic syndrome, such as hyperinsulinemia, hyperleptinemia, hyperphagia, hypertension, and obesity were also induced in the rat offspring from mother exposed to 70% FR or low protein diet (Vickers et al. 2000, 2005; Woods et al. 2001). In addition, “shortened life span” was also demonstrated in the mouse offspring following maternal undernutrition (Ozanne and Hales 2004). © 2014 Japanese Teratology Society

196

T. Ogawa et al.

To facilitate understanding of the DOHaD mechanism/s, gene expression and DNA methylation analyses have been reported. Gluckman and co-workers demonstrated that intrauterine undernutrition (70% FR) induced decreases in the expression of nr3c1 (glucocorticoid receptor) and ppara (peroxisome proliferator activated receptor alpha) genes as well as hypermethylation of their promoters in the rat offspring (Gluckman et al. 2007). Two other subsequent studies by Lillycrop and co-workers reported upregulation of these genes with hypomethylation of their promoter DNA following maternal low protein diet in rats (Lillycrop et al. 2007, 2008). Global gene expression profiling on the visceral fat pads following maternal low protein diet was also reported focussing on the adipose tissue (Guan et al. 2005). In their analysis, a number of genes involved in pre-adipocyte proliferation, adipocyte differentiation, and lipogenesis, as well as angiogenesis, and extracellular matrix remodeling were found to be upregulated (Guan et al. 2005). Those analytical results simply reflected adiposity, providing few clues to the mechanisms involved therein. To get an insight into the underlying mechanisms, analysis on the developing tissues was required. A more recent study in mice has examined DNA methylation in the fetal liver following maternal protein restriction (9% vs. 18%; from gestation day 0) by using the CpG island microarray chip (van Straten et al. 2010). Those authors reported that prenatal protein restriction induced hypermethylation in lxr (liver X-receptor) promoter, which is a nuclear receptor critically involved in the control of cholesterol and fatty acid metabolism. Calorie restriction (CR), a dietary intervention that is low in calories but maintains proper nutrition, is well known to prevent metabolic syndrome (Redman and Ravussin 2011). In animal experiments including rodents, CR is the most effective nutritional intervention for preventing chronic disease, slowing ageing, and extending lifespan (Masoro 2005; Mair and Dillin 2008; Omodei and Fontana 2011). We planned our research focusing on the fact that CR in adults leads to a healthy life, while prenatal CR induces a non-healthy life with an increased risk for diseases (the effect is completely opposite!). In the present study, the focus was toward transcriptome (genome-wide) profiling of genes in the fetal mouse liver following maternal 50% FR. Considering the opposite effects of nutrition between the fetus and adult, we further analyzed our fetal array data by comparing with data from their mothers. In addition, we performed global DNA methylation analysis using a mouse promoter DNA microarray chip, and compared the results with data of the gene expression analysis. Finally, we presented a list of candidate genes responsible for DOHaD. Considering the demonstration of shortened life span in mouse offspring exposed to maternal undernutrition, those genes may include potential longevity genes.

METHODS Animals and tissue collection The mice (inbred strain C57BL/6J) used in this study were purchased from Japan SLC (Hamamatsu, Japan), and housed at the Animal Institution facility in Showa University. Regular cages with bedding materials were used to accommodate the mice, which were kept in a ventilated animal room with controlled temperature and relative humidity with a 12:12 h light : dark schedule (lights turned on at 08.00 hours). Animals had access to chow (CE-2, CLEA, Tokyo Japan) and tap water ad libitum, and all animal experiments were perfomed exactly as previously reported (Ogawa et al. 2011a). The day the vaginal plug was observed was designated embryonic © 2014 Japanese Teratology Society

day 0 (ED0) and gestation day 0 (GD0). Pregnant mice were exposed to 50% food restriction (FR) from GD10 to 18 and caesarean section was performed between 10.00–12.00 hours on GD18. Amount of CE-2 chow supplied to the FR group was calculated as 50% of CE-2 consumed by the control group each gestation day. The control group was supplied with chow ad libitum. Food consumption of each animal is shown in Supporting Information Table S1 (2.0–3.1 g was supplied in the FR group based on the comsumption of the control group). Considering the potential stress induced by removal of animals from the home cage, pregnant mice were killed by cervical dislocation within 5 min of leaving the animal room. The fetuses were taken out and anesthetized on ice-cold phosphate buffered saline (PBS). The fetuses were dissected under a dissection microscope, and fetal tissues were carefully removed avoiding any other tissue contamination. The liver was collected from six mothers in each group. Fetal liver was collected from two male fetuses from each mother. The liver was also collected from mothers to compare with fetal liver in gene expression analysis. The harvested livers were immediately immersed in liquid nitrogen (N2) and stored at −80°C in a deep freezer. Some livers from littermate male fetuses were immersed in 4% paraformaldehyde, and then processed as sections (4 μm) for classical hematoxylin-eosin staining to check for induction of cell death and/or any changes in the cellular population following FR. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Showa University.

Global gene expression (transcriptomics) analysis For maternal and fetal livers, each replicate tissue was individually ground to a very fine powder with liquid N2, and stored at −80°C until used. Extraction of total RNA and quality determination, followed by cDNA synthesis and its subsequent check using the expression of house-keeping gene (GAPDH) by reverse transcription-polymerase chain reaction (RT-PCR) were performed exactly as described previously (Ogawa et al. 2011a). The primers are listed in Table 1. Total RNA extracted from mother and fetal livers for each control and FR was pooled in each group, prior to DNA microarray analysis (Agilent mouse whole genome 4 × 44 K; G4122F). We designed three pools in each group (Control and FR). One pool consisted of one fetus from each mother (n = 6), which contained fetuses from all mothers. The second pool consisted of three fetuses (litter mates of the 1st pool) from three mothers (one fetus from each mother). The third pool collected fetuses from the other three mothers. We performed DNA microarray analysis on these three pools. We searched for genes that were altered commonly among those three pools. Microarray experiment (labelling, hybridization, and dye-swap approach) was performed exactly as described previously (Ogawa et al. 2011a; Hori et al. 2012a). Briefly, to select differentially expressed genes by the dye-swap approach, we considered genes that were upregulated in chip 1 (Cy3/Cy5 label for control and FR, respectively) but downregulated in chip 2 (Cy3/Cy5 label for FR and control, respectively), for the first pool of fetal liver. Similarly the same selection criteria was applied for chip 3 and 4 (mother liver tissue), for chip 5 and 6 (the second pool of fetal liver), and for chip 7 and 8 (the third pool of fetal liver). For the detection of significantly differentially expressed genes between control and treated samples each slide image was processed by Agilent Feature Extraction software (version 9.5.3.1) (for details see [Ogawa et al. 2011a]). The list of genes searched in the fetal liver was further

Gene symbol

GAPDH Trib1 Gadd45b Ubd Ctgf Insig1 Gimap5 Ptx3 Lepr Lepr 1 Rb Lepr 2 Rc Lepr 3 Ra Rag1 Trib3 Fabp4 Fst Il7r (alpha) Gpr88 Fasn Sphk1 Hmgcs1 Il7 Cd40 Fgf1 Pdk1 Mbd1 Ddit4 Ddit4l Rps6 Vldlr Pck1

NM_008084 NM_144549 NM_008655 NM_023137 NM_010217 NM_153526 NM_175035 NM_008987 NM_146146 NM_146146 NM_010704 NM_001122899 NM_009019 NM_175093 NM_024406 NM_008046 NM_008372 NM_022427 NM_007988 NM_025367 AK078743 NM_008371 NM_011611 NM_010197 NM_172665 NM_013594 NM_029083 NM_030143 NM_009096 NM_013703 NM_011044

Glyceraldehyde-3-phosphate dehydrogenase Tribbles homolog 1 (Drosophila) Growth arrest and DNA-damage-inducible 45 beta Ubiquitin D Connective tissue growth factor Insulin induced gene 1 GTPase, IMAP family member 5 Pentraxin related gene Leptin receptor, transcript variant 1 Leptin receptor, transcript variant 1 Leptin receptor, transcript variant 2 Leptin receptor, transcript variant 3 Recombination activating gene 1 Tribbles homolog 3 (Drosophila) Fatty acid binding protein 4, adipocyte Follistatin Interleukin 7 receptor G-protein coupled receptor 88 Fatty acid synthase Sphingosine kinase 1 Similar to hydroxymethylglutaryl-CoA synthase Interleukin 7 CD40 antigen, transcript variant 1 Fibroblast growth factor 1 Pyruvate dehydrogenase kinase, isoenzyme 1 Methyl-CpG binding domain protein 1 DNA-damage-inducible transcript 4 DNA-damage-inducible transcript 4-like Ribosomal protein S6 Very low density lipoprotein receptor, transcript variant 1 Phosphoenolpyruvate carboxykinase 1, cytosolic

Description gctacactgaggaccaggttgt ttcttcgagaaggactttggag tcttgggttcgtatctggactt aagagtggcagtttgctctttc agcctgtcaagtttgagctttc tgtccagctgtccttgacttta cctacccgaggggatatagtct catcctgttttcttatggcaca tagtgtgaggaggtacgtggtg atgaagagcaagggtttatcca tgaagatgatggaatgaagtgg aatttccaaaagagaacggaca cagtttcaactcacagcgtttc gtctctcctccacagtctgacc tcctcctcgaaggtttacaaaa tcttgaagtgaagcattctgga ctttcctctaggtcccctgact ctctacacgtggaggaacgag aatccatcatcaacatcatcca gagctgatggtatgtgaagctg ccaaacgctcctctaatttttg ctggtgaactgcacaagtaagg aaaaggtggtcaagaaaccaaa aaatgcccactcagaaatcact ccacattccgaagctcttaaac cctgaagaggatggagaagaga aagatccaggggctgttaagtt aaagctggataggatcgtgtgt agaagatgatgtccgccagtat ggaggaattggcaacataaaaa gtggaggagatcgacaggtatc

Nucleotide sequence (5′-3′): left

Primer design for reverse transcription-polymerase chain reaction (RT-PCR) validation experiment

Accession (gene)

Table 1

ctcctgttattatgggggtctg ttatctgacagcgcatcatctt actggatcagggtgaagtgaat atcattcattgatccaccttcc tgtcccttacttcctggcttta tacatcctgcttccactctgaa tcttgtacacagtgggcttctg cttttcttggccaatctgtagg agccacttcattccatcatctt cagtcaaaagcacaccactctc tggcatctaaactgcaacctta caaccgtcacaccattatcatt tcatggtaaacccagagctttt cttggccagcaaggtatatttc gccatctagggttatgatgctc ctgggagagagcttaggacaaa agagcaagcattccagactttc acaaaagggtaaacgtcaatgg ccactgactcttcacagaccag atagccccaccttctagctttc tcacattatgacactgccttcc tgtagatttctcagctgcttgg tcttggctcatctcaaacttca ttatcctctgtggctgattgtg ctagccagctactccacgttct ctgaattccaggttcagctttt ctaacacaggggacagtccttc ctaaatctgggatttcctggtg gtgaggacagcctacgtctctt agatgggaggaagaaaggagac cattggtgtcaaatgcaaactt

Nucleotide sequence (5′-3′): right 306 251 315 324 279 313 291 277 336 288 393 329 323 336 288 328 302 342 310 263 261 251 321 301 344 291 254 337 263 266 332

Product size (bp)

Seeking genes responsible for DOHaD 197

© 2014 Japanese Teratology Society

198 analyzed following our previous studies (Hori et al. 2012a, 2012b). In this analysis, each gene was examined for its functional category, and subcategory. To re-check the microarray data, RT-PCR, as detailed above, was performed on candidate genes using 3′-UTR gene-specific primers (Table 1). Global DNA methylation analysis Preparation of genomic DNA Genomic DNA was extracted from 10 mg of liver tissue powder (see above) using the Blood and Cell Culture DNA Kit Mini (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. The quantity and quality of genomic DNA were determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm. (A260/A280 ratio of 1.8–2.0, A260/230 ratio of >2.0). The extracted genomic DNA was electrophoresed on a 1.6% agarose gel in 1 × TAE buffer to check its quality, and importantly, to confirm that there was no contamination with RNA. Sonication of genomic DNA The purified genomic DNA was sonicated using a water-bath type sonicator as follows: 5 μg genomic DNA was diluted in 250 μL TE buffer pH 8.0 in a 1.5 mL sterile microfuge tube; sonicated for 10 s followed by incubation for 10 s in ice-cold water; repeated 25 times; and 10 μL was loaded into the well of a 1.6% agarose gel to verify fragment size of DNA (mean size should be 100–800 bp; average 350 bp). Immunoprecipitation of methylated DNA (MeDIP) Methylated DNA was immunoprecipitated (IP) using MethylMiner Methylated (Invitrogen, CA, USA) following the manufacturer’s protocol. The aqueous supernatant (methylated DNA fraction) was transferred to a new microfuge tube, followed by addition of 1 μL glycogen (20 mg/mL stock), 1/10 volume of 3 M sodium acetate (pH 5.2), two volmues of 100% ethanol, and mixed well by tapping. The mixture was precipitated at −80°C for 30 min or overnight. Next, the pellet was obtained by centrifugation at 11,430 g for 15 min (4°C) after carefully removing the supernatant. The pellets were washed by adding 1000 μL of 70% ethanol. To do so, the pellet was resuspended by mixing well and spinned down at 11,430 g for 5 min (4°C), followed by air-drying for 5 min. Finally, the dried DNA pellet was resuspended in 60 μL of DNase-free water. Analysis of methylated DNA expression by microarray Immunoprecipitated DNA (methylated DNA) and total genomic DNA (reference DNA) were labeled with Cy3 and Cy5 respectively, using Agilent Genomic Labeling kit PLUS (Agilent Technologies, Santa Clara, CA, USA) and hybridized to the SurePrint G3 Mouse Promoter 2 × 400 K G4876A DNA chip (Agilent Technologies). Following hybridization (Agilent Oligo aCGH Hybridization Kit) and washing (Agilent Oligo aCGH/ChIP-on-chip Wash Buffer), the arrays were scanned using a Agilent G2505C microarray scanner and raw data were obtained using Feature Extraction Ver.9.5.3. The differential DNA methylation expression levels for each probe by fold changes (>1.5/

Seeking genes responsible for developmental origins of health and disease from the fetal mouse liver following maternal food restriction.

Low birthweight resulting from a non-optimal fetal environment is correlated epidemiologically to a higher risk of adult diseases, and which has also ...
1MB Sizes 5 Downloads 3 Views