Introduction
The insulin-like growth factor (IGF) system consists of two IGF ligands (IGF- I and IGF-II), two IGF receptors (IGF-1R and IGF-2R), and a group of at least six IGF-binding proteins (IGFBP-1 to 6) [
1]. These components are found in a variety of tissues or bodily fluids [
2] and act through endocrine, paracrine, and/or autocrine modes of action [
3]. As a result, the IGF system, with all components working together, regulates biological processes, including cell proliferation, differentiation, survival and apoptosis that are essential to life [
4,
5]. In particular, the IGF system plays an important role in the link between nutrition and postnatal growth and development [
6]. For example, fetal nutrient supply determines the fetal IGF axis [
7‐
9] and can continue shaping the IGF axis after birth, as revealed in rodents [
10] and humans [
11]. It has been shown that a maternal low-protein diet (LPD) is associated with a reduced fetal liver weight in rats [
12,
13]. The livers of progeny whose mothers were fed a high-protein diet during gestation were 8.3% heavier [
12,
13]. Accordingly, a maternal LPD causes a decrease in fetal hepatic
IGF-
I gene expression, but increases IGFBP levels in fetal plasma and liver, which may contribute to fetal growth retardation, as observed in rats [
14‐
18]. Previous studies have demonstrated that gene expression is associated with progesterone, IGF-I and IGFBP-5, which are upregulated in the fetus in response to maternal high-protein intake [
17,
19,
20]. In the case of large litters in pigs, the excess of dietary protein seems to be beneficial for preventing intrauterine growth retardation (IUGR) resulting from intrauterine crowding [
20]. Notably, recent studies have revealed that maternal protein supplementation during gestation affects human fetal body weight independent of IGF-I [
21]. In addition, hormones, especially those from the thyroid, are thought to be key players connecting the IGF growth axis and maternal factors/fetal nutrient supply. Thyroid hormones can regulate IGF-I production by activating the hepatic growth hormone receptor (GHR) [
22,
23], and they are sensitive to maternal nutritional reprogramming [
24,
25]. Nevertheless, the exact nature of these events is still unclear and warrants further exploration.
Interestingly, it has been well documented that epigenetic regulation modifies fetal gene expression during maternal nutrition programming. For instance, in cancer research, the IGFBP-3 gene is described to be vulnerable to epigenetic regulation, including DNA methylation and histone modification [
26]. It is worth mentioning that IGFBP-3 can block IGF-I and IGF-II from binding to their receptors and therefore plays an important role in cell proliferation stimulation. However, whether maternal dietary intervention affects gene expression in offspring via epigenetics has not been well explored. Nawathe et al. showed that DNA methylation is involved in changes of gene expression in the IGF axis in fetal growth disorders [
27], while maternal undernutrition alters hepatic metabolic gene transcription via DNA methylation and the modification of histones, including H3K4me3, H3K27me3 and H3K9me1 [
28,
29]. Moreover, modifications of H3K14ac and H3K9me3 are found to occur in fetal liver lipid metabolism when exposed to a high-fat diet in utero [
30].
Intriguingly, genes involved in the liver cell cycle and growth in newborn piglets are downregulated by histone modification when the dams are exposed to dietary supplementation with methyl donors [
31]. Building upon these studies, we hypothesized that epigenetic events during maternal nutrition programming may play a crucial role in modifying the liver IGF-I/IGFBP-3 growth axis in offspring.
Meishan (MS), a Chinese indigenous pig breed, pigs were used in the present study as an animal model to delineate the impact of a maternal high-protein diet on the hepatic IGF axis of offspring piglets at different life stages. Blood and liver samples were taken from sows at 70 days gestation, from piglets at birth, from weaned piglets at 35 days, and from fattened pigs at 180 days. We aimed to explore the possible epigenetic mechanisms on the regulation of specific genes involved in the IGF-I/IGFBP-3 pathway in the liver.
Discussion
In this study, we presented evidence demonstrating that a high-protein diet during pregnancy led to higher body and liver weights in newborns, as well as in weaning piglets. Endocrine factors such as insulin, sex hormones and thyroid hormones play important roles in fetal development and growth. One study demonstrated that a maternal high-protein/low-carbohydrate diet increases the insulin resistance of animals to maintain glucose homeostasis without affecting the basal insulin level [
35]. Accordingly, we reported that serum insulin levels were not changed in HP-exposed piglets. Sex hormones are considered an important factor and are thought to influence animal growth differently between males and females under maternal nutrition programming, but no sex disparity in body weight or hepatic growth axis was observed in our study regardless of treatment. Similarly, a human clinical trial revealed that postnatal diet affects sex steroid levels independent of infant size, differing from the prenatal nutrition intervention [
36]. This finding suggests that neonatal growth after birth may not be mediated directly by sex hormones. Rather, we postulate that the changed IGF axis may be more sensitive to thyroid hormones. It has been reported that hypothyroidism is closely linked to body weight and the growth rate of offspring in early life [
37‐
39]. Indeed, a recent study revealed that maternal high-protein diet consumption during pregnancy causes elevated body weight of offspring at birth without changing the IGF-I pathway [
21]. Similarly, we demonstrated that T3 and T4 levels in the serum were higher in the HP-exposed piglets with higher body weight. We found that the increased body weight tended to diminish toward adulthood. This finding might be explained by the adaptation response, i.e., slower growth rate in the growing stage/fattening period of pigs [
21]. Furthermore, we found that the IGF-1R protein content was increased, which was associated with elevated serum T3 levels at birth due to HP exposure. These results suggest that maternal high-protein intake promotes the activation of the IGF-I growth axis in the early life of offspring.
The IGF-IGFBP pathway is one of the most essential parts of growth axis regulation. Indeed, the regulation of IGF-dependent growth via maternal nutrition programming has been underlined, especially in IGUR models. In the current study, we demonstrated that liver IGF-1R protein content was increased at birth in response to high-protein exposure, indicating a stimulatory effect during the early life of offspring. This finding may explain the increased liver and body weights of neonatal and weaning piglets from the HP diet-fed sows compared to the weights of the control piglets. Interestingly, we found that at the weaning period (day 35), the IGFBP-3 levels in the serum and livers of the HP piglets were decreased. High-protein intake in pregnant women has been reported to be associated with reduced levels of IGF-II and IGFBP-3 in cord blood but to not affect IGF-I [
40]. In contrast, it is demonstrated that nutrition restriction during pregnancy in ewes tended to decrease IGFBP-3 expression in the fetus while lowering plasma IGF levels [
41,
42]. It is noteworthy that although IGFBP-3 was inclined to decrease, the IGFBP-2 level in fetal plasma was significantly increased due to maternal energy restriction [
12]. Importantly, it has been described that during fetal life, the predominant IGFBPs are IGFBP-1 and IGFBP-2, whereas IGFBP-3 becomes the most abundant protein later in life, accounting for 80% of all IGF binding. Thus, this may explain the different results of our study on sows and the previous studies on dams and women compared to the nutrition restriction study in ewes [
12].
On the other hand, it is widely accepted that poor fetal growth is associated with postnatal growth acceleration as a compensatory response and is linked to an increased risk of metabolic disorders in the long term [
43,
44]. The early growth retardation and later life compensatory growth have also been observed after weaning and in adulthood [
44,
45]. In this regard, the reduced IGFBP-3 expression reprogrammed by maternal high dietary protein may be an adaptation reaction to slow down growth from weaning to adult life. It is worth mentioning that the
IGFBP-
3 gene, but not the protein content, was still downregulated until adulthood in our study. This implies that post-transcriptional regulation may be involved. One possibility is that microRNAs target mRNA degradation and/or translational repression. Numerous studies have reported that maternal dietary supplementation regulates gene translation by modifying microRNA function [
31,
46]. However, whether the dissociation of the decreased
IGFBP3 mRNA and the unchanged IGFBP-3 protein expression in the liver of piglets is induced by a post-transcriptional mechanism is beyond the focus of the present study and may warrant future investigations.
The postnatal stage, especially the weaning period, is of critical importance for animal growth and for health in adult life [
47,
48]. Based on our data and data from other studies, maternal nutrition programming modulates liver growth and function in offspring predominantly via epigenetic regulation [
33,
49,
50]. Intriguingly, we demonstrated that the downregulated mRNA level of
IGFBP-
3 in the liver was associated with protein content. Cancer research has shown evidence that the
IGFBP-
3 gene is vulnerable to epigenetic regulation, including DNA methylation and histone modification [
26]. However, the understanding of the maternal–offspring
IGFBP-
3 gene expression regulation is limited. To support the notion that DNA methylation functions on the IGF axis in fetal growth [
27], our data revealed that CpG methylation was enhanced on the promoter of
IGFBP-
3 in HP piglets at weaning, thus blocking transcription to decrease
IGFBP-
3 mRNA expression.
Having described the altered DNA methylation status, we further studied the mechanisms by conducting ChIP analysis of the
IGFBP-
3 gene. We identified histone modification of the
IGFBP-
3 gene promoter in the offspring exposed to maternal high-protein intake in pigs for the first time. Total histone H3 status represents a whole genomic histone methylation; however, it was not changed in the liver of weaning HP piglets. It is known that maternal nutrition supplementation modifies liver metabolic genes via the modification of histones, including H3K4me3, H3K27me3 and H3k9me1, in the offspring [
29,
51]. For the specific gene
IGFBP-
3, enrichment changes in histone marks on the gene promoter region were in line with decreased
IGFBP-
3 transcription. Higher enrichment levels of H3K9me3 and H3K27me3 were associated with DNA hypermethylation in the H piglets at weaning. This suggests that high protein content in the maternal diet plays a dominant role in gene methylation to epigenetically modulate the hepatic growth axis in offspring. Furthermore, histone acetylation is a pivotal factor that regulates fetal gene expression [
52]. Although our data show that H3AC enrichment was not changed in this model, specific marks such as H3K27ac and H3K9ac should be measured in the future to confirm acetylation status in the offspring exposed to high-protein diet in pregnant dams.
In conclusion, our study reveals that a maternal high-protein diet given during gestation modulates the hepatic growth axis in weaning piglets. This may, at least partly, be attributed to the reprogramming of IGFBP-3 function by DNA methylation and histone modification. Our findings of epigenetic regulation of the IGFBP-3-mediated liver growth axis being involved in maternal nutrition programming provide an underlying mechanism of how dietary protein modulates fetal growth. Given that weaning is a critical period of life, a healthy growth pattern built from early life may reduce the risk of developing obesity in adulthood.