Abstract
Intrauterine or early postnatal high-fat diet (HFD) has substantial influences on adult offspring health; however, studies of HFD-induced maternal obesity on regulation of adult offspring bone formation are sparse. Here, we investigated the effects of HFD-induced maternal obesity on both fetal and adult offspring skeletal development. We found that HFD-induced maternal obesity significantly decreased fetal skeletal development, but enhanced fetal osteoblastic cell senescence signaling and significantly increased the expression of inflammatory factors of the senescence-associated secretory phenotype (SASP) in osteo-progenitors. It was found that p300/CBP activation led to H3K27 acetylation to increase the expression of senescence-related genes and PPARγ in embryonic mouse osteogenic calvarial cells from HFD obese dams. These results were recapitulated in human umbilical cord mesenchymal stem cells (UC MSCs) isolated from offspring of pregnant obese and lean mothers following delivery. Regardless of postnatal HFD challenge, adult offspring from HFD obese dams showed significantly suppressed bone formation. Such early involution of bone formation of adult offspring from HFD obese dams may at least in part due to histone acetylation, i.e., epigenetic regulation of genes involved in cell senescence signaling in pre-osteoblasts from prenatal development. These findings indicate fetal pre-osteoblastic cell senescence signaling is epigenetically regulated by maternal obesity to repress bone formation in adult offspring in rodents and suggest that at least some of these effects may also manifest in humans.
Introduction
Expression of certain genes in the genome can be regulated without altering the base sequence of DNA: e.g., through chromatin remodeling, DNA methylation, histone modifications (acetylation) and the expression of non-coding RNA (Vrtačnik et al. 2014). These mechanisms of epigenetics are important in regulating differentiation of different types of cells during both prenatal and postnatal development. Maternal nutrition appears to influence epigenetic alterations in the offspring and to program gene expression in key metabolic pathways, such as fatty acids and glucose metabolism (Levin & Govek 1998, King 2006, Shankar et al. 2008, Geraghty et al. 2016). Emerging evidence suggests that maternal obesity caused by excessive consumption of a high-calorie, high-fat diet (HFD) has a profound influence on the health of cardiovascular and skeletal system of the offspring during fetal development and infancy, as well as later on in childhood and into adulthood (Barker 2007, Samuelsson et al. 2008, McCurdy et al. 2009). The relevance of epigenetic control in bone-related gene expression, bone development and organ homeostasis, as well as in the onset and progression of musculoskeletal diseases, has also been increasingly shown (Lian 2015). We have recently reported the effects of maternal HFD on fetal osteogenic cell differentiation behaviors (Chen et al. 2012, 2016); however, the influence of HFD-induced maternal obesity on epigenetic regulation of offspring adult bone development has not been proven.
Bone mineralization within the skeletal envelope is impacted by variety of factors such as local paracrine molecules and hormones traveling through the bloodstream (Riggs et al. 2002), dietary factors and lifestyle, such as the amount of physical activity. Intrauterine programming of bone development leading to increased risk of osteoporosis in adulthood has been recently suggested (Dennison et al. 2013). Although there are no convincing data on determining the effect of obesity on early postnatal life bone quantity (Streeter et al. 2013, Kâ et al. 2013), feeding a HFD rich in saturated fat and cholesterol to experimental rodents has consistently been shown to inhibit bone formation (Parhami et al. 2001, Rosen & Bouxsein 2006, Zhao et al. 2008, Liang et al. 2009, Chen et al. 2010, 2013). Previous animal data indicated that inhibition of perinatal skeletal formation by HFD consumption during gestation (Liang et al. 2009, Chen et al. 2012) might be associated with DNA hypermethylation, suggesting intrauterine fetal skeletal developmental programming (Chen et al. 2012). We have recently provided evidence showing maternal HFD-induced obesity accelerates senescence signaling and therefore leads to altered glucose metabolism and mitochondrial function in osteo-progenitors derived from rats and humans (Chen et al. 2016), while these remain unrevealed in bone in postnatal adult offspring.
Cellular senescence is a general process, and it is thought to occur in most cells of organisms during aging and most notably within tumors (Campisi & d’Adda di Fagagna 2007, Collado et al. 2007). However, it was recently identified during mammalian embryonic development (Muñoz-Espín et al. 2013, Storer et al. 2013), leading to our interests in investigating links between gestational obesity and fetal cell senescence programming. In non-cancer cells, the (patho) physiologic role of cell senescence signaling is currently unknown. Although many senescence mediators have been characterized in rodent cells, induction of tumor suppressor genes p53, p21 and p16 are critical to the induction of senescence. Senescent cells increase the expression and secretion of numerous cytokines, chemokines, matrix metalloproteinases and other proteins that can alter local tissue environments. This feature was termed the senescence-associated secretory phenotype (SASP) (Coppé et al. 2008); this may be tracked by senescence-associated β-galactosidase (SAβG) activity, the most widely used marker to detect senescence both in vitro and in vivo (Dimri et al. 1995, Collado & Serrano 2006).
Relevant to the role of p300/CBP histone acetyltransferases (HATs), hyperacetylation of histone N-terminal tail lysines correlates strongly with active gene transcription (Bose et al. 2017). p300/CBP are together chiefly responsible for the global acetylation of histone H3 residues K18 and K27 and contribute to other locus-specific histone acetylation events. As in other tissues, intrauterine fetal skeletal programming is complex and may undergo postnatal reprogramming; however, histone modification by acetylation has been shown to be important for long-term regulation of cellular function (Kruhlak et al. 2001). This regulation occurs by inducing an open chromatin structure that correlates with gene activation. H3K-associated fetal tissue programming may affect several generations (Rando & Chang 2009) making it a difficult phenomenon to target for disease intervention. It remains unclear if an increase in H3K27 acetylation leads to epigenetic overexpression of target genes in embryonic osteoblastic cells and in bone of adult offspring. We hypothesize that HFD-induced maternal obesity increases H3K27 acetylation in osteogenic cells to change osteoblastic cell differentiation and activity.
Materials and methods
Animals and diets
Female 4-week-old C57BL6/J mice were purchased from Jackson Laboratory (https://www.jax.org/). Subsequently, these mice were divided into two groups: one group of mice received control semi-purified AIN-93G control diet (17% fat diet corn oil) and the other group of mice received a high-fat diet (by calories, 25% protein, 45% fat, and 30% carbohydrates). After 12 weeks of diet intervention (pre-pregnancy), mice were time-impregnated (n = 12 per group) by control male mice; this HFD-induced maternal obesity mouse model was similar to what we described previously in rats (Chen et al. 2012). Pregnant mice were individually housed in an Association for Assessment and Accreditation of Laboratory Animal Care-approved animal facility at the Arkansas Children’s Research Institute (Little Rock, AR) with constant humidity, lights on from 06:00 to 18:00 h, at 22°C. All animal procedures were approved by the Institutional Animal Care and Use Committee at University of Arkansas for Medical Sciences (UAMS, Little Rock, AR). One set of pregnant HFD and control diet mice were killed, and embryos were taken at gestational day 17.5 for analysis. The remainder of pregnant HFD and control diet mice were continued on diet regimens until pups were born. Male and female offspring from either HFD or control diet dams were weaned at 21 days of age onto either HFD or control diet, and diets were continued for 14 weeks.
Pregnant lean and obese women, and isolation of human UC MSCs
Twenty-four pregnant women were recruited for this study: twelve obese (pre-pregnancy BMI ≥30 kg/m2) and twelve non-obese (pre-pregnancy BMI between 19 and 25 kg/m2). After obtaining written informed consent during the first trimester of pregnancy, placentas and umbilical cords (UCs) were collected at UAMS following delivery. The protocol was approved by the Institutional Review Board at the UAMS. As described previously (Saben et al. 2014), mesenchymal stem cells (MSCs) from the UC matrix were isolated from the UCs, pooled and expanded. UC MSCs were counted and plated in growth media in a single well of a six-well plate. These UC MSCs were used for experiments described in the ‘Results’ section, and we utilized a subset of cells randomly from three obese mothers and from three lean mothers for the analysis in the current report.
Fetal and postnatal adult offspring tibial micro-computed tomography scan
We are not aware of any references of micro-computed tomography (μCT) for embryos; therefore, we used standard procedures for mouse tibia (see below), and bone volume was the only parameter analyzed. Adult offspring tibial μCT measurements of the trabecular and cortical compartments from the right tibial bone were evaluated using a Scanco μCT scanner (μCT-40; Scanco Medical AG, Bassersdorf, Switzerland) at 6 μm isotropic voxel size with X-ray source power of 55 kV and 145 μA and integration time of 300 ms. The gray-scale images were processed by using a low-pass Gaussian filter (σ = 0.8, support = 1) to remove noise, and a fixed threshold of 220 was used to extract the mineralized bone from the soft tissue and marrow phase. Cancellous bone was separated from the cortical regions by semi-automatically drawn contours. A total of 120 slices starting from about 0.1 mm distal to growth plate, constituting 0.70 mm of length, was evaluated for trabecular bone structure based on description by Bouxsein et al. (2010), and by using software provided by Scanco, as described in detail previously (Cao et al. 2009, Chen et al. 2009).
Fetal Alizarin red/alcian blue staining and isolation of embryonic calvarial cells
The gold standard Alizarin red/alcian blue staining for observation of fetal skeletal development and mineralization was used according to a method published previously (Chen et al. 2012). Pregnant female mice were killed on day 17 postcoitum (with 0 considered the day of the positive vaginal smear), and fetuses were collected. For the isolation of fetal calvarial cells, collagenase digestion of calvarial tissue was performed and cells from second and third digestion were collected and pooled. The detailed procedure for isolation of neonatal mouse and rat calvarial osteoblastic cells as previously published (Chen et al. 2012).
Cell cultures and real-time reverse transcription-polymerase chain reaction (real-time PCR)
Isolated fetal/embryonic mouse osteogenic calvarial cells (EOCCs) or human UC MSCs were cultured in α-MEM (# 41061-029, Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA), penicillin (100 units/mL), streptomycin (100 µg/mL) and glutamine (4 mM). α-MEM supplemented with 10% FBS, 1 mM ascorbyl-2-phosphate (Sigma-Aldrich) and 4 mM l-glutamine was used as osteoblast (OB) differentiation medium, while α-MEM supplemented with 10% FBS was used as control medium. Cell RNA from isolated in vitro-cultured cells and in vivo adult offspring bone tissue RNA were extracted using TRI Reagent (MRC Inc., Cincinnati, OH, USA) according to the manufacturer’s recommendation followed by DNase digestion and column cleanup using QIAGEN mini columns (Chen et al. 2016). Reverse transcription was carried out using an iScript cDNA synthesis kit from Bio-Rad. All primers for real-time PCR analysis used in this report were designed using Primer Express software 2.0.0 (Applied Biosystems).
Mouse and human cell SAβG activity assay, histology and immunostaining
Protein senescence-associated β-galactosidase (SAβG) activity assay was performed according to a method described previously (Thornburg et al. 2010). SAβG staining was performed on cultured cells either from HFD obese mice or control mice and on human UC MSCs from either obese or lean mothers using four-well chamber slides. The SAβG staining procedure was described previously (Chen et al. 2016). Standard antibody counterstaining for p53 and p21 (Cell Signaling) was performed after SAβG staining.
Western blot, inflammation antibody array and chromatin immunoprecipitation (ChIP)
Standard Western blots were performed using total protein isolated from cultured cells and bone tissue after aspiration of bone marrow. The following antibodies were used for Western blots: H3k27ac (#07-360, Millipore), p300/CBP (#P2859, Sigma), p300 (#05-257, Sigma), CBP (#SAB4500455, Sigma), β-actin (#A1978, Sigma), PPARγ (#ab191407, Abcam), and osteocalcin (#AB10911, Millipore). SuperSignal West Pico chemiluminescent substrate (Pierce) was used for developing blots. Mouse (#AAM-INF-1-8, RayBiotech, Inc) inflammation antibody array was performed according to the protocol provided by manufacturer. Procedure for standard ChIP assay using H3K27ac antibody (ChIP grade from Cell Signaling) was described previously (Chen et al. 2010), and information for all primers used for ChIP assay were listed in Supplementary Table 1 (see section on supplementary data given at the end of this article).
Measurements of bone formation markers and glucose and insulin levels in bone marrow plasma
Bone marrow plasma was prepared at the time of tissue harvest. Bone marrow was flushed out from the femur using 300 µL of PBS, vortexed and spun (1700 g ) for collecting supernatant as bone marrow plasma. The bone formation marker, bone-specific alkaline phophatase (ALP) was measured by Rat-MIDTM ALP ELISA (Nordic Biosciences Diagnostic, Herlev, Denmark). The bone marrow plasma total osteocalcin (Gla-OC) and undercarboxylated osteocalcin (Glu-OC) levels were measured by an ELISA-based kit from TAKARA (TAKARA Bio Inc.). The bone marrow plasma glucose and insulin levels were measured by Rat-LapsTM ELISA from Nordic Biosciences Diagnostic (Herlev, Denmark).
Statistical analyses
Data were expressed as means ± standard error. One-way and two-way ANOVA followed by Student–Newman–Keuls post hoc analysis was used to compare the treatment groups. Values were considered statistically significant at P < 0.05.
Results
HFD-induced maternal obesity impairs embryonic skeletal development and cell senescence signaling in mice
A maternal obese mouse model was developed by feeding female C57BL6/J mice with HFD for 12 weeks pre-pregnancy and during pregnancy (Supplementary Fig. 1, experimental design). At gestational day 17 (E17.5), embryos were taken, and fetal calvarial forming cells were isolated for experimental analysis. Dam body weight gains during the HFD period until offspring began weaning were presented (Supplementary Fig. 1). Fetuses from both HFD-obese and control diet dams were first μCT scanned. A total of 12 fetuses were analyzed: six fetuses (three males and three females) from six different HFD obese dams and six fetuses (three males and three females) from six different control diet dams. While fetal body weights were not found to be significantly different between HFD obese and control diet dams, μCT showed significantly reduced total bone volume (BV) in embryos from HFD-obese dams compared to those from control diet dams (Fig. 1A). There were no differences in BV between male and female fetuses within either diet (data not shown). After μCT scan, we performed Alizarin Red Staining on whole fetal skeleton for osteogenesis or bone mineralization. We observed less bone mineralization in both male and female embryos from HFD-obese dams compared to those male and female embryos from control diet dams (Fig. 1B).
Fetal calvarial osteo-progenitor cells were isolated by using collagenase type 1 digestion of very thin calvarias (one time digestion). These cells are embryonic osteo-progenitors because under appropriate stimulations, these cells can differentiate either toward adipocyte-like cells or osteoblastic cells (Chen et al. 2012). The embryonic calvarial osteo-progenitor cells from either HFD obese dams or control diet dams were cultured in chamber slides. Using methods established previously (Chen et al. 2016), exploratory senescence-associated β-galactosidase (SAβG) staining and counterstaining using p21, p53 antibody immunostaining were performed. Increased senescence signals were found in embryonic calvarial osteo-progenitor cells from HFD obese dams (Fig. 1C). To confirm these data, we performed SAβG activity measurements in proteins isolated from embryonic calvarial osteo-progenitor cells. The SAβG activity was significantly higher in embryonic calvarial osteo-progenitor cells from HFD-obese dams compared to those from control diet dams (Fig. 1D). Increased SAβG activity was accompanied by increased gene expression of p53 and p21 (Fig. 1E), two well-known mediators associated with cellular senescence, in embryonic calvarial osteo-progenitor cells from HFD obese dams. Senescent cells increase the expression and secretion of numerous cytokines, chemokines, MMPs and other proteins that can alter local tissue environments; this feature has been termed the SASP. We evaluated the SASP for embryonic calvarial osteo-progenitor cells from HFD-induced obese dams compared to embryonic calvarial osteo-progenitor cells from control dams, using an inflammatory antibody array (Fig. 1F). Strikingly, half of these SASP molecules were significantly upregulated in embryonic calvarial osteo-progenitor cells from HFD-induced obese dams compared to embryonic calvarial osteo-progenitor cells from control dams (Fig. 1F). These data suggest that embryonic skeletal development in HFD obese dams is characterized by cellular senescence promotion and inflammatory milieus.
Obesity-induced increases of senescence signaling in osteo-progenitors are associated with p300/CBP activation and H3K27 acetylation in both mice and humans
Proteins were isolated from calvarial cells of a subset of six male fetuses from six different dams, three from control dams and three from HFD dams. Western blots showed remarkable increases in acetylation of lysine 27 on histone H3 (H3K27ac), and expression of p300/CBP proteins (Fig. 2A). It has been shown that modification of histone tails plays an important role on regulating gene transcription and that hyperacetylation of histone N-terminal tail lysines (H3K27 as an example) correlates strongly with activation of hundreds of transcription factors including nuclear receptors such as PPARγ. We found that PPARγ expression was increased in embryonic calvarial osteo-progenitor cells from HFD-induced obese dams (Fig. 2A). To determine whether HFD maternal obesity enhances binding of H3K27ac to PPARγ and cellular senescence-associated target genes (p53 and p21), H3K27ac chromatin immunoprecipitation (ChIP) assays were carried out (Fig. 2B). We used antibody against H3K27ac and subsequent PCR amplification. We found that there were pronounced increases in the bindings of H3K27ac to the PPARγ gene and to p53 and p21 in embryonic calvarial osteo-progenitor cells from HFD obese dams compared to those cells from control diet dams (Fig. 2B).
Given the activations of H3K27 ac, p300/CBP, PPARγ and senescence signal changes in embryonic calvarial osteo-progenitor cells from HFD-induced obese mouse dams, we next considered if such events may occur in humans. Considering that embryonic calvarial osteo-progenitor cells are not feasible to study in humans, we studied senescence and p300/CBP/H3K27ac-associated epigenetic signals in isolated MSCs from human UCs. Experiments were conducted in human MSCs from a subset of obese (pre-pregnancy BMI ≥30 kg/m2) and control lean mothers (pre-pregnancy BMI between 19 and 25 kg/m2), random three per each group, following delivery. Information on the study subjects, the procedure and characterization of UC cells as MSCs have previously been described (Saben et al. 2014). UC MSCs were cultured in chamber slides. Using methods and similar approaches described earlier in cells from mice, exploratory SAβG staining and counterstaining using p21, p53 antibody immunostainings were performed. Clearly, when compared to non-obese mothers, (Fig. 3A) senescence protein markers in UC MSCs from obese mothers were substantially higher. Moreover, we collected proteins from cells cultured in six-well plates at confluency. Western blots showed obesity-associated increases in protein expression of p300, CBP and H3K27ac (Fig. 3B), similar to the data we showed above for fetuses from HFD mice. We also found increased expression of PPARγ in UC MSCs from obese mothers compared to those from lean mothers (Fig. 3C). The biological importance of senescent or pre-mature senescent non-cancer somatic cells is not fully understood, but it is reasonable to consider that increased senescence signaling in osteo-progenitors may persist to impair postnatal bone development. To determine whether maternal obesity enhances binding of H3K27ac to PPARγ and cellular senescence-associated target genes (p53 and p21) in human UC MSCs, H3K27ac chromatin immunoprecipitation (ChIP) assays were carried out (Fig. 4A). We used antibody against H3K27ac (recognize both human and mouse) and subsequent PCR amplification. We found that there were pronounced increases in the bindings of H3K27ac to the PPARγ gene and to p53 and p21 in UC MSCs from obese mothers compared to those cells from control lean mothers (Fig. 4B).
Adult offspring from HFD-induced maternal obese dams have decreased bone formation
Offspring from HFD obese and control diet dams were weaned, and a HFD challenge or control feeding began at 6 weeks of age. Therefore, we have total four groups for male (and female) offspring: control-control (control diet dam and control diet offspring); control-HFD (control diet dam and HFD offspring); HFD-control (HFD dam and control diet offspring) and HFD-HFD (HFD dams and HFD offspring). After 14 weeks of these post weaning diets, the HFD-HFD groups (male and female) gained significantly more weight than any other group (Supplementary Fig. 1).
Adult offspring left tibias were μCT scanned ex vivo for determining trabecular and cortical structural changes and BV. In males, compared to control-control as the standard group, the control-HFD group had significantly decreased bone volume (BV/TV), trabecular thickness (Tb.Th) and cortical thickness (Cort.Th); their increased structure model index (SMI) indicated mechanically inferior bone (Table 1). Strikingly, we found even more μCT parameters changed in the HFD-control group in both trabecular and cortical sites in male mice compared with control-control group animals (Table 1). Compared to control-control group, decreased bone volume (BV/TV), connective density (Conne-Dens), trabecular number (Tb.N) and trabecular thickness (Tb.Th), but increased trabecular spaces (Tb.Sp) were found in trabecular site of HFD-control group males (Table 1). At the cortical site, we found increased total CSA, periosteal perimeter, medullary area and endosteal perimeter in the HFD-control male group compared to the control-control group, indicating HFD-induced maternal obesity changes bone structure in offspring. More changes at the trabecular site were found in the male HFD-HFD group compared to the control-control group (Table 1). Overall, the impact of maternal or postnatal HFD was much more profound and numerous in male offspring compared to female offspring (Table 1).
Micro-CT parameters on trabecula and cortical of tibias of adult offspring from either HFD-induced obese or lean dams.
Male | Female | |||||||
---|---|---|---|---|---|---|---|---|
Control-control | Control-HFD | HFD-control | HFD-HFD | Control-control | Control-HFD | HFD-control | HFD-HFD | |
Trabecular | ||||||||
BV/TV | 0.28 ± 0.02c | 0.23 ± 0.04a | 0.24 ± 0.02b | 0.22 ± 0.03a | 0.16 ± 0.06 | 0.15 ± 0.02 | 0.14 ± 0.01 | 0.15 ± 0.02 |
Conn-dens. | 207.3 ± 47.8c,b | 163.6 ± 33.5c,b | 153.6 ± 13.2b | 138.5 ± 6.9a | 99.3 ± 31.6c | 57.9 ± 14.1a | 60.8 ± 13.4a,b | 68.1 ± 16.7b |
SMI | 1.38 ± 0.29a | 1.80 ± 0.19b | 1.55 ± 0.20a,b | 1.91 ± 0.20b,c | 2.0 ± 0.56 | 2.16 ± 0.21 | 2.31 ± 0.28 | 2.1 ± 0.2 |
Tb.N | 5.92 ± 0.51b,c | 5.56 ± 0.39b | 5.41 ± 0.12a,b | 5.31 ± 0.10a | 3.79 ± 0.43 | 3.47 ± 0.10 | 3.5 ± 0.15 | 3.69 ± 0.35 |
Tb.Th | 0.060 ± 0.003b,c | 0.056 ± 0.003a,b | 0.055 ± 0.002a | 0.057 ± 0.004b | 0.058 ± 0.006a | 0.065 ± 0.002b,c | 0.063 ± 0.002b | 0.062 ± 0.002a,b |
Tb.Sp | 0.152 ± 0.016a | 0.163 ± 0.016a,b | 0.167 ± 0.006b | 0.172 ± 0.006c | 0.26 ± 0.03 | 0.28 ± 0.007 | 0.28 ± 0.02 | 0.27 ± 0.03 |
vBMD | 891.2 ± 31.4 | 871.1 ± 3.5 | 885.2 ± 12.7 | 886.2 ± 13.1 | 907.1 ± 22.6 | 942.3 ± 34.6 | 922.3 ± 22.2 | 911.5 ± 15.3 |
Cortical | ||||||||
Cort CSA | 0.40 ± 0.03 | 0.38 ± 0.01 | 0.43 ± 0.04 | 0.42 ± 0.04 | 0.34 ± 0.03 | 0.35 ± 0.02 | 0.35 ± 0.01 | 0.33 ± 0.008 |
Cort.Th | 0.21 ± 0.01b | 0.20 ± 0.006a | 0.21 ± 0.01b | 0.21 ± 0.01b | 0.20 ± 0.01 | 0.21 ± 0.01 | 0.21 ± 0.007 | 0.20 ± 0.004 |
Total CSA | 0.73 ± 0.03a | 0.71 ± 0.02a | 0.80 ± 0.05b | 0.77 ± 0.09a | 0.62 ± 0.07 | 0.63 ± 0.04 | 0.60 ± 0.03 | 0.60 ± 0.02 |
Periosteal perim. | 2.34 ± 0.14a | 2.38 ± 0.07a | 2.53 ± 0.16b | 2.51 ± 0.32a,b | 2.13 ± 0.12 | 2.18 ± 0.07 | 2.11 ± 0.08 | 2.08 ± 0.07 |
Midshaft diam. | 0.61 ± 0.03 | 0.59 ± 0.008 | 0.62 ± 0.01 | 0.60 ± 0.02 | 0.57 ± 0.03 | 0.57 ± 0.02 | 0.56 ± 0.008 | 0.56 ± 0.004 |
Medullary area | 0.29 ± 0.01a | 0.28 ± 0.01a | 0.32 ± 0.02b | 0.31 ± 0.04a,b | 0.24 ± 0.04 | 0.24 ± 0.03 | 0.22 ± 0.02 | 0.22 ± 0.01 |
Endosteal perim. | 1.44 ± 0.05a | 1.49 ± 0.06a | 1.58 ± 0.1b | 1.54 ± 0.2a,b | 1.30 ± 0.14 | 1.32 ± 0.08 | 1.22 ± 0.08 | 1.27 ± 0.04 |
Medullary diam. | 0.39 ± 0.02 | 0.37 ± 0.006 | 0.40 ± 0.02 | 0.39 ± 0.007 | 0.36 ± 0.02b | 0.35 ± 0.02b | 0.35 ± 0.02b | 0.34 ± 0.009a |
Numbers are mean ± s.d., means with different letters differ significantly (P < 0.05, a < b < c) as determined by two-way ANOVA followed by Student-Newman-Keuls post hoc analysis for multiple pairwise comparisons. n = 7 per group in males and n = 5 per group in females.
BV/TV (%), bone volume/total volume; Conn-Dens (mm), connective tissue density; Cort CSA, cortical cross section area; Cort.Th, cortical thickness; SMI, structure model index; Tb.N (1/mm), trabecular number; Tb.Sp (mm); trabecular separation; Tb.Th (mm), trabecular thickness; vBMD, volumetric bone mineral density.
We performed ELISA measurements of glucose, insulin and bone formation markers (total osteocalcin, carboxylated osteocalcin and bone-specific ALP) in bone marrow plasma collected at the end of the post weaning feeding intervention. We found increased glucose and insulin levels and decreased levels of total osteocalcin and bone-specific ALP in the male HFD-HFD group compared to the control-control group (Table 2). We found decreased total osteocalcin level in the male HFD-control group compared to the control-control group. There was higher insulin level but decreased bone-specific ALP level in the male control-HFD group compared to the control-control group (Table 2). Compared to the significant changes of insulin and bone formation marker levels in male mice, there are differences in levels of those markers from the control-HFD, HFD-control groups compared to those in the control-control female mice, but not statistically different (Table 2). We only found significant changes of insulin and total osteocalcin levels in the female HFD-HFD group compared to the bone marrow plasma levels in the female control-control group (Table 2).
Bone marrow plasma concentrations of insulin, glucose and bone formation markers.
Male | Female | |||||||
---|---|---|---|---|---|---|---|---|
Control-control | Control-HFD | HFD-control | HFD-HFD | Control-control | Control-HFD | HFD-control | HFD-HFD | |
Glucose (mg/mL) | 0.61 ± 0.03a,b | 0.59 ± 0.025a | 0.63 ± 0.01b | 0.65 ± 0.015b,c | 0.63 ± 0.015 | 0.63 ± 0.015 | 0.62 ± 0.04 | 0.62 ± 0.025 |
Insulin (µg/L) | 0.19 ± 0.0045a | 0.21 ± 0.015b | 0.18 ± 0.015a | 0.22 ± 0.014b | 0.19 ± 0.021a | 0.21 ± 0.009b | 0.24 ± 0.041b,c | 0.22 ± 0.002b |
Total osteocalcin (ng/mL) | 38.4 ± 7.58c | 29.3 ± 8.13b | 18.7 ± 2.58a | 17.5 ± 5.23a | 29.4 ± 5.96b | 29.2 ± 7.65b | 24.4 ± 13.1a | 23.9 ± 4.79a |
Glu-osteocalcin (ng/mL) | 0.53 ± 0.15c | 0.45 ± 0.10b | 0.34 ± 0.14a | 0.42 ± 0.14b | 0.76 ± 0.21c | 0.61 ± 0.24b | 0.44 ± 0.15a | 0.53 ± 0.14b |
ALP (µUnits/min) | 0.45 ± 0.07b | 0.31 ± 0.07a | 0.45 ± 0.08b | 0.34 ± 0.05a | 0.41 ± 0.05 | 0.35 ± 0.08 | 0.34 ± 0.09 | 0.43 ± 0.03 |
Numbers are mean ± s.d., means with different letters differ significantly (P < 0.05, a < b < c) as determined by two-way ANOVA followed by Student-Newman-Keuls post hoc analysis for multiple pairwise comparisons. n = 7 per group in males and n = 5 per group in females.
ALP, alkaline phosphatase; Glu-osteocalcin, carboxylated osteocalcin.
Increased cell senescence signaling in embryonic osteo-progenitors from HFD dams persisted in bone of adult offspring
The results of these studies raise two additional questions at the molecular level. First, does the increased senescence signaling in osteo-progenitors from embryos derived from HFD dams persist into bone of adult offspring, i.e., through epigenetic regulation? Second, does increased senescence signaling in osteoblastic cells truly contribute to early bone degeneration or skeletal involution as mentioned previously? Obviously, it will be difficult and complicated to test second question. However, here we have begun to examine the first question. We isolated protein and RNA from mouse offspring bone. To assess if previously observed senescence signaling in fetal cells from HFD obese dams is also occurring in adult offspring bone, we measured SAβG activity in bone and bone marrow plasma. SAβG activity in bone was significantly higher in the male control-HFD, HFD-control and HFD-HFD groups compared to its activity in bone in the control-control group (Fig. 5A). Similar results were found in bone marrow plasma (Fig. 5B).
As an indirect measure of potential epigenetic events, mRNA expression in bone was evaluated. Transcript abundance of PPARγ in bone in the male control-HFD, HFD-control and HFD-HFD groups was significantly higher compared to its expression in bone from the control-control mice (Fig. 5C), while increased PPARγ mRNA expression was only found in the HFD-HFD group in female adult offspring (Fig. 5C). It was surprising, however, that we found osteocalcin, the mature osteoblast marker, mRNA expression was significantly lower in the control-HFD, HFD-control and HFD-HFD male and female groups compared to their respective control-control groups (Fig. 5D). Gene acetylation and cellular senescence signaling pathway involved in p300, CBP and PPARγ protein expression were also examined in bone from adult male mice. As shown in Fig. 6, p300, CBP, H3K27ac and PPARγ protein expression were clearly increased in bone from the control-HFD, HFD-control and HFD-HFD adult male offspring compared to their expression in bone from the control-control adult offspring (Fig. 6). Consistent with osteocalcin mRNA expression levels, osteocalcin protein expression was significantly lower in the control-HFD, HFD-control and HFD-HFD male groups compared to their respective control-control group (Fig. 6). These data indicated that increased senescence signaling in bone of adult offspring from HFD maternal obese mice, and offspring early life HFD are associated with protein acetylation, i.e., epigenetic regulation.
Discussion
The maternal environment, in particular, the nutritional status and obesity during pregnancy, has been shown to be important in the developmental trajectory of the fetus and to influence risk for chronic diseases such as diabetes, cardiovascular disease, osteoporosis and cancer in the offspring (Cooper et al. 2009, Godfrey et al. 2011). Maternal obesity, best known for predisposing offspring to obesity and metabolic diseases, has recently seen increased investigation in both animal models and humans on its effects on offspring tissue phenotype. The human skeleton is known as the largest endocrine organ; however, the fetal skeletal development and adult offspring bone formation associated with maternal obesity are still understudied. We previously hypothesized and provided new evidence (Chen et al. 2012, 2016) that the fetal skeleton represents another target for developmental programming and might lead to changes in ability to attain peak bone mass and thus alter the risk of osteoporosis in later life (Bedford et al. 2011). In the current study, we have, for the first time, using a mouse model combined with analysis of osteo-progenitors from human obese mothers, provided further evidence that maternal obesity regulates fetal osteoblastic cell senescence signaling and fetal and adult offspring skeletal development. We showed that regardless of postnatal HFD challenge, adult offspring from HFD obese dams have significantly suppressed bone formation. This phenomenon appeared to be much more robust in males compared to females. Such suppressed bone formation in adult offspring from HFD obese dams is a phenomenon of early bone involution/degeneration, and may be in part due to histone acetylation, i.e., epigenetic regulation of genes involved in cell senescence signaling in pre-osteoblasts. Although we have attempted to determine the links between epigenetic regulation of fetal pre-osteoblastic cell senescence signaling and altered embryonic and adult offspring skeletal development, under the condition of maternal obesity, such links may be universal for other tissue development in adult offspring in rodents and perhaps in humans.
The results revealed increased H3K27 acetylation in embryonic osteo-progenitors from HFD-induced obese mouse dams and in osteo-progenitors derived from umbilicus MSCs following delivery by pregnant obese mothers. Increased H3K27 acetylation is associated with increased p300/CBP activity. The mammalian p300/CBP is a pair of ubiquitously expressed, paralogous proteins that belong to a distinct family of histone acetyltransferases (HATs) (Bedford & Brindle 2012). Although increased p300/CBP activity enhances H3K27 acetylation and is consistent with previous evidence (Liu et al. 2014), activation of p300/CBP and increased H3K27 acetylation in embryonic osteo-progenitors associated with maternal obesity are new findings. p300/CBP are essential for animal development as deletion of either one in mice leads to early embryonic lethality (Kraus & Wong 2002). These two HATs have been shown to function as transcription co-activators for hundreds of transcription factors including nuclear receptors (NRs) (Chen et al. 2015), and here, we have shown PPARγ, peroxisome proliferator-activated receptor gamma, is a nuclear receptor. The role of PPARγ involving adipogenesis is well known; however, more functions of PPARγ on cellular signal transduction have been discovered recently. For example, it has been shown that overexpression or activation of PPARγ will in turn accelerate senescence pathways by inducing p16INK4α expression in a ligand-dependent manner (Tchkonia et al. 2010). In this regard, PPARγ was suggested as a molecule linking external or systemic factors (such as diet or obesity) and specific intra-cellular factors (such as the p16 gene) to control cellular senescence.
We have reported previously the effect of maternal obesity on fetal osteoblastic cell senescence pathways in a rat model (Chen et al. 2016), and the current results are consistent with those observations in general terms. Obesity may be considered to be associated with an increase in cellular senescence through systemic low-grade chronic inflammation (Kereliuk et al. 2017). From data presented in our current report, it supports that attenuation of physiological resolution of inflammation or low-grade inflammation occurred in fetal skeletal tissues from HFD-induced obese dams. Our results in mouse embryonic osteo-progenitors are consistent with previous evidence that cellular senescence promotes chronic inflammation through the SASP (Coppé et al. 2008). Moreover, we believe that increased cell senescence signaling in embryonic mouse calvarial cells and human MSCs and offspring in bone associated with maternal obesity are atypical. Specifically, in maternal obesity-associated embryonic osteogenic cells, increased PPARγ may interact with p53 and p21 to control senescence signaling in these cells. Supporting such a scenario, we have shown that HFD-induced postnatal obesity increases PPARγ expression to promote p53/p21-mediated cell senescence signaling in bone and osteoblastic cells (Chen et al. 2015). p300/CBP and increased H3K27 acetylation may be upstream of the team of PPARγ/p53/p21 in the involvement of embryonic osteogenic cell senescence signaling. Although our data suggest a novel phenomenon of maternal programming of pre-osteoblastic cell senescence signaling, obviously, signaling molecules we presented here are not bone cell type specific. Those cellular senescence-associated signaling molecules are expressed in variety of tissues; therefore, our data might implicate a universal and potential mechanistic explanation for poorly defined maternal pathophysiologic programming in other tissue systems.
While human studies, especially in postnatal offspring bone development, are important to the field, it is difficult to control for confounding factors over a lengthy and expensive study period, and genetic variability adds a high level of complexity. Therefore, rodent models with a common genetic background and carefully controlled dietary and activity conditions are useful for examining how altered HFD or obesity during gestation or early postnatal life influences the development of bone associated with obesity in the offspring. On the other hand, there are also significant limitations that need to be considered when using animals to model human pregnancy that can impact the generalizability of the findings. For example, the number of offspring per pregnancy, placentation, gestational length, parturition and different windows of fetal and neonatal cellular differentiation and organogenesis (Kereliuk et al. 2017). In addition, the normal macronutrient intake for rodents as a percent of total energy intake can be quite different from humans. Nonetheless, recent results from the Danish National Birth Cohort show that maternal poor-quality diet (i.e., a dietary pattern in which the most abundant food items are potatoes, french fries, white bread, pork, beef veal, mixed meat, cold meat and dressing sauce) is associated with significantly increased risk of childhood fractures in offspring (Petersen et al. 2015), supporting the idea that offspring skeletal health is influenced by the gestational environment and maternal diet (Bedford et al. 2011). Although human UC MSCs as we used in our study may not exactly represent fetal bone phenotype and bone progenitor functions, these cells are the most accessible fetal mesenchymal osteo-progenitor cells in infants during delivery. Accelerated senescence signaling pathway in those cells from obese mothers was very close finding to embryonic calvarial cells from HFD-induced obese mouse dams. It is acknowledged that despite clear outcomes, our findings in human cells are limited to a small sample size. Therefore, future clinical investigations of bone phenotype of offspring from obese mothers compared to those from lean mothers will be of interest.
Although mechanistic processes of normal development of bone and adipose tissue during early life are not well characterized, our current study elucidated the effect of maternal HFD on postnatal long bone development in mice. It is known, however, that maternal obesity enhances MSCs to form more committed pre-adipocytes and white adipocytes, which lead to adiposity in specific depots in offspring. This finding is consistent with our data in MSCs in our previous report from pregnant obese women and in a rat model (Chen et al. 2016). Previous evidence presented by us also suggested an epigenetic basis of such unbalanced differentiation potential between adipogenesis and osteoblastogenesis (Chen et al. 2016). This observation seems to be true from our current study; our data indicated that HFD in offspring from prenatal HFD dams significantly gain more weight than those from prenatal control diet dams. Moreover, significant changes in bone marrow plasma glucose and insulin levels and decreased bone formation marker osteocalcin and bone-specific ALP suggest HFD maternal obesity-related shifts in anabolic and catabolic responses that control bone homeostasis and explain postnatal bone phenotype characterized by µCT. However, it was surprising, without postnatal HFD challenge, weight gains in offspring from HFD obese mice had similar trends compared to those offspring from control diet dams (within postnatal study duration 20 weeks), yet, bone loss occurred. Such bone loss in offspring from HFD maternal obese mice is a phenomenon of early bone degeneration or early involution of bone formation. Indeed, HFD+control group had more effects on trabecular number than control+HFD group, while HFD+HFD had the greatest effect. We provided further data suggesting an association with accelerated bone senescence and epigenetic regulation. More in vivo studies may be needed to investigate that altered and epigenetically regulated senescence signaling in their precursors may interfere with adipogenic and osteogenic signals and therefore determine cell fate to either differentiate toward adipocytes or osteoblasts (Zhang et al. 2011, 2013). Moreover, our data showed that there may be difference in bone phenotype in offspring between male and female in response to either pre or postnatal HFD. Such gender differences in response to HFD have been shown in brown and white adipose tissue development (Strakovsky et al. 2014); sex chromosome or postnatal physiologic estrogen signaling may influence or dilute HFD-induced epigenetic regulation on tissue development. Nonetheless, it will be interesting for our future studies to determine in more detail of how sex chromosome or postnatal physiologic estrogen signaling has an effect on HFD-induced decreased postnatal skeletal development.
In summary, we have presented evidence suggesting epigenetic regulation of HFD-induced maternal obesity on both fetal and adult offspring skeletal development. We found CBP/p300 activation leads to H3K27 acetylation, which may increase cell senescence-related gene and PPARγ expression in EOCCs from HFD-obese dams, and in human UC MSCs isolated following delivery by obese and lean mothers. Adult offspring from HFD obese dams with standard control diet showed significantly suppressed bone formation. Such early involution of bone formation of adult offspring from HFD-obese dams is likely in part due to histone acetylation, i.e., epigenetic regulation of genes involved in cell senescence signaling in pre-osteoblasts from prenatal period.
Supplementary data
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-18-0244.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the United States Department of Agriculture (USDA)/Agricultural Research Service (ARS), project number # USDA-ARS Project 6026-51000-010-05S, to the Arkansas Children’s Nutrition Center.
Author contribution statement
J R C contributed to experiment design, performing experiments (senescence signaling determination, etc.), data analysis and writing paper. O P L contributed by performing experiments (Western blots, real-time PCR, ChIP, etc.) and data analysis. H J Z contributed by performing experiments (Western blots and real-time PCR, etc.). A W A contributed by performing experiments (micro-CT). K S contributed to experimental design.
Acknowledgements
The authors would like to thank the following people for their technical assistance: Matt Ferguson, Hoy Pittman and Bobby Fay. They thank Dr Aline Andres for providing human sample information. They thank John A Gregan for helpful editing of the manuscript.
References
Barker DJ 2007 The origins of the developmental origins theory. Journal of Internal Medicine 261 412–417. (https://doi.org/10.1111/j.1365-2796.2007.01809.x)
Bedford DC & Brindle PK 2012 Is histone acetylation the most important physiological function for CBP and p300? Aging 4 247–255. (https://doi.org/10.18632/aging.100453)
Bedford DC, Kasper LH, Wang R, Chang Y, Green DR & Brindle PK 2011 Disrupting the CH1 domain structure in the acetyltransferases CBP and p300 results in lean mice with increased metabolic control. Cell Metabolism 14 219–230. (https://doi.org/10.1016/j.cmet.2011.06.010)
Bose DA, Donahue G, Reinberg D, Shiekhattar R, Bonasio R & Berger SL 2017 RNA binding to CBP stimulates histone acetylation and transcription. Cell 168 135–149. (https://doi.org/10.1016/j.cell.2016.12.020)
Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ & Müller R 2010 Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. Journal of Bone and Mineral Research 25 1468–1486. (https://doi.org/10.1002/jbmr.141)
Campisi J & d’Adda di Fagagna F 2007 Cellular senescence: when bad things happen to good cells. Nature Reviews Molecular Cell Biology 8 729–740. (https://doi.org/10.1038/nrm2233)
Cao JJ, Gregoire BR & Gao H 2009 High-fat diet decreases cancellous bone mass but has no effect on cortical bone mass in the tibia in mice. Bone 44 1097–1104. (https://doi.org/10.1016/j.bone.2009.02.017)
Chen JR, Lazarenko OP, Haley RL, Blackburn ML, Badger TM & Ronis MJ 2009 Ethanol impairs estrogen receptor signaling resulting in accelerated activation of senescence pathways, whereas estradiol attenuates the effects of ethanol in osteoblasts. Journal of Bone and Mineral Research 24 221–230. (https://doi.org/10.1359/jbmr.081011)
Chen JR, Lazarenko OP, Wu X, Tong Y, Blackburn ML, Shankar K, Badger TM & Ronis MJ 2010 Obesity reduces bone density associated with activation of PPARγ and suppression of Wnt/β-catenin in rapidly growing male rats. PLoS ONE 5 e13704. (https://doi.org/10.1371/journal.pone.0013704)
Chen JR, Zhang J, Lazarenko OP, Kang P, Blackburn ML, Ronis MJ, Badger TM & Shankar K 2012 Inhibition of fetal bone development through epigenetic down-regulation of HoxA10 in obese rats fed high-fat diet. FASEB Journal 26 1131–1141. (https://doi.org/10.1096/fj.11-197822)
Chen JR, Zhang J, Lazarenko OP, Cao JJ, Blackburn ML, Badger TM & Ronis MJ 2013 Soy protein isolates prevent loss of bone quantity associated with obesity in rats through regulation of insulin signaling in osteoblasts. FASEB Journal 27 3514–3523. (https://doi.org/10.1096/fj.12-226464)
Chen JR, Lazarenko OP, Blackburn ML, Badger TM & Ronis MJ 2015 Soy protein isolate inhibits high-fat diet-induced senescence pathways in osteoblasts to maintain bone acquisition in male rats. Endocrinology 156 475–487. (https://doi.org/10.1210/en.2014-1427)
Chen JR, Lazarenko OP, Blackburn ML, Rose S, Frye RE, Badger TM, Andres A & Shankar K 2016 Maternal obesity programs senescence signaling and glucose metabolism in osteo-progenitors from rat and human. Endocrinology 157 4172–4183. (https://doi.org/10.1210/en.2016-1408)
Collado M & Serrano M 2006 The power and the promise of oncogene induced senescence markers. Nature Reviews Cancer 6 472–476. (https://doi.org/10.1038/nrc1884)
Collado M, Blasco MA & Serrano M 2007 Cellular senescence in cancer and aging. Cell 130 223–233. (https://doi.org/10.1016/j.cell.2007.07.003)
Cooper C, Harvey N, Cole Z, Hanson M & Dennison E 2009 Developmental origins of osteoporosis: the role of maternal nutrition. Advances in Experimental Medicine and Biology 646 31–39. (https://doi.org/10.1007/978-1-4020-9173-5_3)
Coppé JP, Patil CK, Rodier F, Sun Y, Muñoz DP, Goldstein J, Nelson PS, Desprez PY & Campisi J 2008 Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biology 6 2853–2868. (https://doi.org/10.1371/journal.pbio.0060301)
Dennison EM, Harvey NC & Cooper C 2013 Programming of osteoporosis and impact on osteoporosis risk. Clinical Obstetrics and Gynecology 56 549–555. (http://doi.org/10.1097/GRF.0b013e31829cb9b0)
Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, et al. 1995 A biomarker that identifies senescent human cells in culture and in aging skin in vivo. PNAS 92 9363–9367. (https://doi.org/10.1073/pnas.92.20.9363)
Geraghty AA, Lindsay KL, Alberdi G, McAuliffe FM & Gibney ER 2016 Nutrition during pregnancy impacts offspring's epigenetic status-evidence from human and animal studies. Nutrition and Metabolic Insights 16 41–47. (https://doi.org/10.4137/NMI.S29527)
Godfrey KM, Sheppard A, Gluckman PD, Lillycrop KA, Burdge GC, McLean C, Rodford J, Slater-Jefferies JL, Garratt E, Crozier SR, et al. 2011 Epigenetic gene promoter methylation at birth is associated with child's later adiposity. Diabetes 60 1528–1534. (https://doi.org/10.2337/db10-0979)
Kâ K, Rousseau MC, Lambert M, O'Loughlin J, Henderson M, Tremblay A, Alos N & Nicolau B 2013 Association between lean and fat mass and indicators of bone health in prepubertal caucasian children. Hormone Research in Paediatrics 80 154–162. (https://doi.org/10.1159/000354043)
Kereliuk SM, Brawerman GM & Dolinsky VW 2017 Maternal macronutrient consumption and the developmental origins of metabolic disease in the offspring. International Journal of Molecular Sciences 18 E1451. (https://doi.org/10.3390/ijms18071451)
King JC 2006 Maternal obesity, metabolism, and pregnancy outcomes. Annual Review of Nutrition 26 271–291. (https://doi.org/10.1146/annurev.nutr.24.012003.132249)
Kraus WL & Wong J 2002 Nuclear receptor-dependent transcription with chromatin. Is it all about enzymes? European Journal of Biochemistry 269 2275–2283. (https://doi.org/10.1046/j.1432-1033.2002.02889.x)
Kruhlak MJ, Hendzel MJ, Fischle W, Bertos NR, Hameed S, Yang XJ, Verdin E & Bazett-Jones DP 2001 Regulation of global acetylation in mitosis through loss of histone acetyltransferases and deacetylases from chromatin. Journal of Biological Chemistry 276 38307–38319. (https://doi.org/10.1074/jbc.M100290200)
Levin BE & Govek E 1998 Gestational obesity accentuates obesity in obesity-prone progeny. American Journal of Physiology 275 R1374–R1379. (https://doi.org/10.1152/ajpregu.1998.275.4.R1374)
Lian JB 2015 Epigenetic pathways regulating bone homeostasis. Bone 81 731–732. (https://doi.org/10.1016/j.bone.2015.05.036)
Liang C, Oest ME, Jones JC & Prater MR 2009 Gestational high saturated fat diet alters C57BL/6 mouse perinatal skeletal formation. Birth Defects Research: Part B, Developmental and Reproductive Toxicology 86 362–369. (https://doi.org/10.1002/bdrb.20204)
Liu Y, Wang L, Han R, Beier UH, Akimova T, Bhatti T, Xiao H, Cole PA, Brindle PK & Hancock WW 2014 Two histone/protein acetyltransferases, CBP and p300, are indispensable for Foxp3+ T-regulatory cell development and function. Molecular and Cellular Biology 34 3993–4007. (https://doi.org/10.1128/MCB.00919-14)
McCurdy CE, Bishop JM, Williams SM, Grayson BE, Smith MS, Friedman JE & Grove KL 2009 Maternal high-fat diet triggers lipotoxicity in the fetal livers of nonhuman primates. Journal of Clinical Investigation 119 323–335. (https://doi.org/10.1172/JCI32661)
Muñoz-Espín D, Cañamero M, Maraver A, Gómez-López G, Contreras J, Murillo-Cuesta S, Rodríguez-Baeza A, Varela-Nieto I, Ruberte J, Collado M, et al. 2013 Programmed cell senescence during mammalian embryonic development. Cell 155 1104–1118. (https://doi.org/10.1016/j.cell.2013.10.019)
Parhami F, Tintut Y, Beamer WG, Gharavi N, Goodman W & Demer LL 2001 Atherogenic high-fat diet reduces bone mineralization in mice. Journal of Bone and Mineral Research 16 182–188. (https://doi.org/10.1359/jbmr.2001.16.1.182)
Petersen SB, Rasmussen MA, Olsen SF, Vestergaard P, Mølgaard C, Halldorsson TI & Strøm M 2015 Maternal dietary patterns during pregnancy in relation to offspring forearm fractures: prospective study from the Danish National Birth Cohort. Nutrients 7 2382–2400. (https://doi.org/10.3390/nu7042382)
Rando OJ & Chang HY 2009 Genome-wide views of chromatin structure. Annual Review of Biochemistry 78 245–271. (https://doi.org/10.1146/annurev.biochem.78.071107.134639)
Riggs BL, Khosla S & Melton LJ 3rd 2002 Sex steroids and the construction and conservation of the adult skeleton. Endocrine Reviews 23 279–302. (https://doi.org/10.1210/edrv.23.3.0465)
Rosen CJ & Bouxsein ML 2006 Mechanisms of disease: is osteoporosis the obesity of bone? Nature Clinical Practice Rheumatology 2 35–43. (https://doi.org/10.1038/ncprheum0070)
Saben J, Thakali KM, Lindsey FE, Zhong Y, Badger TM, Andres A & Shankar K 2014 Distinct adipogenic differentiation phenotypes of human umbilical cord mesenchymal cells dependent on adipogenic conditions. Experimental Biology and Medicine 239 1340–1351. (https://doi.org/10.1177/1535370214539225)
Samuelsson AM, Matthews PA, Argenton M, Christie MR, McConnell JM, Jansen EH, Piersma AH, Ozanne SE, Twinn DF, Remacle C, et al. 2008 Diet-induced obesity in female mice leads to offspring hyperphagia, adiposity, hypertension, and insulin resistance: a novel murine model of developmental programming. Hypertension 51 383–392. (https://doi.org/10.1161/HYPERTENSIONAHA.107.101477)
Shankar K, Harrell A, Liu X, Gilchrist JM, Ronis MJ & Badger TM 2008 Maternal obesity at conception programs obesity in the offspring. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 294 R528–R538. (https://doi.org/10.1152/ajpregu.00316.2007)
Storer M, Mas A, Robert-Moreno A, Pecoraro M, Ortells MC, Di Giacomo V, Yosef R, Pilpel N, Krizhanovsky V, Sharpe J, et al.2013 Senescence is a developmental mechanism that contributes to embryonic growth and patterning. Cell 155 1119–1130. (https://doi.org/10.1016/j.cell.2013.10.041)
Strakovsky RS, Zhang X, Zhou D & Pan YX 2014 The regulation of hepatic Pon1 by a maternal high-fat diet is gender specific and may occur through promoter histone modifications in neonatal rats. Journal of Nutritional Biochemistry 25 170–176. (https://doi.org/10.1016/j.jnutbio.2013.09.016)
Streeter AJ, Hosking J, Metcalf BS, Jeffery AN, Voss LD & Wilkin TJ 2013 Body fat in children does not adversely influence bone development: a 7-year longitudinal study (EarlyBird 18). Pediatric Obesity 8 418–427. (https://doi.org/10.1111/j.2047-6310.2012.00126.x)
Tchkonia T, Morbeck DE, Von Zglinicki T, Van Deursen J, Lustgarten J, Scrable H, Khosla S, Jensen MD & Kirkland JL 2010 Fat tissue, aging, and cellular senescence. Aging Cell 9 667–684. (https://doi.org/10.1111/j.1474-9726.2010.00608.x)
Thornburg KL, Shannon J, Thuillier P & Turker MS 2010 In utero life and epigenetic predisposition for disease. Advances in Genetics 71 57–78. (https://doi.org/10.1016/B978-0-12-380864-6.00003-1)
Vrtačnik P, Marc J & Ostanek B 2014 Epigenetic mechanisms in bone. Clinical Chemistry and Laboratory Medicine 52 589–608. (https://doi.org/10.1515/cclm-2013-0770)
Zhang J, Lazarenko OP, Blackburn ML, Shankar K, Badger TM, Ronis MJ & Chen JR 2011 Feeding blueberry diets in early life prevent senescence of osteoblasts and bone loss in ovariectomized adult female rats. PLoS ONE 6 e24486. (https://doi.org/10.1371/journal.pone.0024486)
Zhang J, Lazarenko OP, Blackburn ML, Badger TM, Ronis MJ & Chen JR 2013 Blueberry consumption prevents loss of collagen in bone matrix and inhibits senescence pathways in osteoblastic cells. Age 35 807–820. (https://doi.org/10.1007/s11357-012-9412-z)
Zhao LJ, Jiang H, Papasian CJ, Maulik D, Drees B, Hamilton J & Deng HW 2008 Correlation of obesity and osteoporosis: effect of fat mass on the determination of osteoporosis. Journal of Bone and Mineral Research 23 17–29. (https://doi.org/10.1359/jbmr.070813)