2024 年 71 巻 3 号 p. 209-222
The observational findings of Barker’s original epidemiological studies were generalized as the Barker hypothesis and extended as the Developmental Origins of Health and Disease (DOHaD) theory. Barker et al. proposed that low birthweight (LBW) was associated with the occurrence of various noncommunicable diseases (NCDs) later in life. In other words, LBW itself is associated with the development of NCDs. This led to the DOHaD theory which proposed that an organism may have a specific period of developmental plasticity that is highly sensitive to the factors in its environment, and that combinations of acquired constitution and environmental factors may adversely affect health and risk the formation of NCDs. Due to undernutrition during the fetal period, the fetus acquires an energy-saving constitution called a thrifty phenotype due to adaptations of the metabolic and endocrine systems. It has been suggested that stimuli experienced early in development can persist throughout life and induce permanent physiological changes that predispose to NCDs. It has since become clear that the adverse environmental effects during the prenatal period are also intergenerationally and transgenerationally inherited, affecting the next generation. It has been shown that nutritional interventions such as methyl-donner and epigenome editing can restore some of the impaired functions and reduce the risk of developing some diseases in the next generation. This review thus outlines the mechanisms underlying various disease risk formations and their genetic programs for the next generation, which are being elucidated through studies based on our fetal undernutrition rat models.
WHO defines noncommunicable diseases (NCDs) as those conditions that “tend to be of long duration and are the result of a combination of genetic, physiological, environmental and behavioural factors,” and that these chronic diseases “disproportionately affect people in low- and middle-income countries, where more than three quarters of global NCD deaths (31.4 million) occur” [1]. Risk factors include modifiable behaviors, such as tobacco use, physical inactivity, unhealthy diet, and alcohol abuse, while metabolic risk factors include elevated blood pressure, overweight/obesity, hyperglycemia, and dyslipidemia. It is now known that not only the postnatal but also the prenatal environment has long-term effects on the development of disease risk. Clinical epidemiologists Barker et al. conducted surveys in some parts of England and found that both ischemic heart disease and its underlying metabolic syndrome were more common in low birthweight (LBW) populations [2-4]. Based on the assumption that this phenomenon was the result of the fetus adapting to an unfavorable environment, they proposed the so-called thrifty phenotype hypothesis [5]. In other words, those who have adapted to a poor nutritional environment during the fetal period are more likely to develop diabetes and death from ischemic heart disease than those who lived in a rich one. This idea has been supported by various subsequent studies and developed into the current Developmental Origins of Health and Disease (DOHaD) [6]. We review here how environment induces epigenomic alterations that shape post-developmental risk of disease and how they affect the next generation.
During the relatively short period of the Dutch famine from December 1944 to April 1945, a pregnant mother’s calorie intake decreased to about 400–800 calories per day. Children born from women who were pregnant (early, middle, or late pregnancy) during the famine were associated with differences in birthweight and subsequent incidence of adult disease [7]. Infants who were calorie-restricted during late pregnancy had lower birthweights, but those whose mothers were exposed to famine during early and middle pregnancy had normal birthweights [8, 9]. In addition, infants whose mothers were exposed to famine during middle and late pregnancy showed decreased glucose tolerance after growth [10], whereas infants whose mothers were exposed to famine during early pregnancy had a higher risk of atherosclerosis [11] and higher body mass index (BMI) [12]. The data derived from the Dutch famine studies have thus shown that the timing of maternal exposure to famine has a critical effect on the programming of the later phenotype of their fetus.
On the other hand, the Leningrad famine lasted for more than 800 days (from September 1941 to January 1943). The Dutch epidemiological study mentioned above found that severe undernutrition during early infancy, pregnancy, and critical periods of infant development is associated with a variety of NCDs later in life, including not only diabetes and cardiovascular disease but also depression. However, unlike the Dutch famine, the Leningrad famine did not show an increased incidence of insulin resistance, dyslipidemia, hypertension, or coronary artery disease [13].
The contrasts between the Dutch famine and the Leningrad famine helped inspire the so-called mismatch hypothesis. In the case of the Leningrad famine, fetal survivors acquired the so-called thrifty phenotype, that is, the ability to adapt to the poor postnatal nutritional environment that occurred during the Leningrad famine. By contrast, the Dutch famine was of a relatively short period. Infants exposed to the Dutch famine were exposed to a rich nutritional environment after birth and showed compensatory or catch-up growth (CG) in early childhood, when the intrauterine adaptation that supported short-term survival became incompatible with the subsequent environment. Therefore, it is thought that the Dutch famine fetus has to pay for the intrauterine adaptation since the rapid improvement of the nutritional environment after birth induces a mismatch between the newborn’s constitution (thrifty phenotype) and its favorable environment after birth. These hypotheses are now called predictive adaptive responses (PAR)/mismatch hypotheses.
Barker et al. conducted epidemiological studies of the geographical distribution of diseases in England and Wales and found that there was a positive geographic correlation between infant mortality from 1921 to 1925 and ischemic heart disease mortality from 1968 to 1978. These observations led to the hypothesis that poor nutritional status in infancy associated with a specific cause of death (ischemic heart disease) later in life. The scientific papers written by Barker were the most influential early publications in the field and led to the Barker hypothesis or DOHaD [2-4, 6]. The Barker hypothesis stimulated global interest in the field of developmental plasticity. Since then, Gillman et al. extended DOHaD into a theory that “recognizes the broader scope of developmental cues, extending from the oocyte to the infant and beyond, and the concept that the early life environment has widespread consequences for later health” [14]. At present, undesirable environmental factors at the developmental stage, such as fertilization, embryonic period, fetal period, and infant period, are associated with the health of adults and old age and various NCDs through epigenetic alterations, that has come to be considered as risk factors. Prenatal malnutrition is thus thought to cause the fetus to acquire the thrifty phenotype. However, there are many unclear points about the difference of phenotype in the overall picture of specific changes in the endocrine metabolism system. Further research is needed to pin down the identity of the thrifty phenotype more exactly in order to help prevent postnatal growth PAR/mismatch and the onset of ever-increasing metabolic disorders.
In Japan, an increase in the rate of LBW infants has been reported to be remarkably elevated for over a decade, and it is an urgent task to control the future development of NCDs in these children [15-17]. Among various causes of LBW, low BMI before pregnancy and poor weight gain during pregnancy are the major causes. Most Japanese young women regard being lean as beautiful and want to avoid being obese. In the past, some pregnant women were subjected to weight restrictions. Since some pregnant women are likely to restrict their diet to control their weight, it is possible that the newborn baby may be underweight at birth due to malnutrition. Based on DOHaD, these infants can be considered to be at high risk of future NCDs. However, verification of the mechanism underlying NCD onset caused by LBW in humans is time-consuming and would encounter many ethical problems. Therefore, to elucidate the thrifty phenotype and PAR/mismatch hypothesis, we need to create a rodent model of a calorie-restricted diet during pregnancy.
DNA-based signaling is higher fidelity than other mechanisms which are much less robust, resulting in differences in the timescales of reliable signaling. Two distinction notions that are often confused are genetic (that is, DNA-based) versus epigenetic inheritance mechanisms. Epigenetics is one of the mechanisms that can be influenced by the environment during the prenatal period in ways that persist and appear during subsequent development. Epigenetics is a transcriptional control mechanism that occurs via acquired modifications to chromatin formed by genomic DNA wrapped around histones [17]. A large piece of genomic DNA is wrapped around a histone core to form a nucleosome structure, which is then neatly folded and housed in the nucleus. The histone core is an octamer composed of two each of four histone proteins H2A, H2B, H3, and H4, and the C-terminus of the histone proteins undergoes various chemical modifications. In particular, the C-terminus of H3 and H4 is highly conserved from yeasts to humans, and modifications of methylation and acetylation have a strong effect on gene expression [18]. DNA is also methylated in the same way as histone proteins. DNA methylation occurs predominantly at cytosines in CpG dinucleotides, with approximately 70–80% of all CpG sites methylated, with the exception of CpG islands and other gene regulatory sequences. Mammals have maintenance DNA methyltransferase (DNMT1) and de novo DNA methyltransferase (DNMT3A and DNMT3B) [19], and DNA methylation of the gene promoter region is associated with the silencing of gene expression by blocking the binding of transcription factors [20]. In contrast, noncoding RNAs do not encode proteins, especially microRNAs (miRNAs), which are other factors that control the expression of genomic information and regulate epigenetic regulation [21-23]. miRNAs are also regulated by exogenous factors, altering gene activity by inhibiting translation or degrading messenger RNA (mRNA). There is little evidence that miRNA expression patterns induced by the environment can be inherited. However, since miRNAs are part of the genetic code, it is possible that DNA influences miRNA activity and promotes inheritance. Although little is known about the expression of miRNA transcription, the best-characterized miRNAs originate from introns or exons of protein-coding or nonprotein-coding genes [24-26]. It was once thought that the sperm genome was transcriptionally quiescent and contributed only to the restoration of polyploidy in the fertilized egg. However, it has become clear that a set of functional RNAs exists and has been characterized in mature sperm that are delivered to the oocyte at fertilization and contribute to early embryonic development, thus influencing the phenotypic outcome of the offspring [27]. Small noncoding RNAs (sncRNAs) are potential carriers of non-genetic information in sperm [28, 29], and tRNA-derived small RNAs (tsRNAs) and miRNAs are most abundant in mature sperm. Sperm tsRNAs have been identified as molecular mediators of paternal experiences such as high-fat diets [30, 31], low-protein diets [32], and stress [33]. Moreover, miRNAs are possible mediators of the epigenetic inheritance of transgenerational changes [34-36]. Noncoding RNA-mediated epigenetic regulation is thus thought to be strongly related to paternal inheritance. In this way, the mechanism of information transmission to the next generation via the epigenome is being rapidly elucidated.
The epigenome is partially inherited through mitosis and is essential for controlling tissue differentiation and cellular responsiveness. The epigenome of a cell or tissue is determined by both the DNA sequence and exposure of the cell or organism to its environment. Epigenetic information that is partially stabilized during mitosis establishes memory of past exposures, especially during developmental transitions. The epigenome thus integrates genomic influences with developmental and environmental exposures and is increasingly recognized to play an important role in the pathophysiology of disease. Furthermore, epigenetic changes and their phenotypes due to environmental stimuli are inherited not only by F1 generation offspring but also F2 and F3 ones, the mechanism of which is presently being elucidated. When adults are exposed to a stimulus, their germline, as well as the germline of the fetus in pregnant females, is exposed to the same stimuli [37, 38]. The terms “intergenerational” and “transgenerational” are often used to describe such effects, but they need to be clarified. Transgenerational effects refer only to phenomena that cannot be attributed to the direct effects of a specific trigger on the affected organism. For example, environmental stimuli can directly affect gestational embryos (and oocytes that have already formed within mammalian female embryos), so that true transgenerational inheritance is only possible to define after F3 generations [38, 39]. Only phenotypic changes that occur in the F2 (for male inheritance) or the F3 (for female inheritance) generations, and the effects over shorter timescales should be described as parental or intergenerational effects. Epigenome changes caused by malnutrition in the fetus and infancy persist for a long time until adulthood, even after recovery from starvation and resuming normal growth, and result in loss of gene function [40]. In DOHaD, disturbing the gene expression mechanisms via those epigenetic changes becomes a risk of disease onset. Studies using rodent models have clearly demonstrated that the diet of paternal, maternal, or grandmother generations influences gene expression in offspring via the epigenome [41].
While evidence for germline non-genetic inheritance of phenotypes and diseases continues to grow in animal models, there are few reports of this phenomenon in humans. Evidence of germline-based non-genetic inheritance in humans links ancestral lifetime exposures with changes in DNA methylation or small RNA expression in germ cells, and between ancestral experience down to great-grandchildren. Several studies have reported small changes in DNA methylation. It is unclear whether small differences in DNA methylation affect target gene expression and phenotype, even if the changes are statistically significant. Also, many of the reported methylation differences are localized near genes, and it is unclear whether these differences actually lead to changes in gene expression. Senaldi and Smith-Raska have reviewed transgenerational inheritance diseases with several epigenomic alterations [42]. One of the most compelling examples of germline-based non-genetic inheritance is fetal exposure to elevated thyroid hormone levels. Residents of the Azorean island of São Miguel had a high frequency of autosomal dominant mutations in the thyroid hormone (TH) receptor beta (THRβ) [43]. Wild-type fetuses that did not inherit the mutation are exposed to high levels of thyroid hormone in utero when the mother carries a heterozygous THRβ mutation. A wild-type child of a THRβ heterozygous mother exhibited a LBW, consistent with her high levels of TH in utero exposure. These wild-type individuals had impaired thyroid-stimulating hormone (TSH) repression, revealing reduced genetic sensitivity to thyroid hormone. The same pattern was observed in the F3 generation, whose great-grandmother had a THRβ mutation and whose grandfather had been exposed to high levels of TH in utero. Unfortunately, the details of the epigenetic changes are unknown, although the authors speculated the possibility of the changes occurring. A study using the Swedish Uppsala Birth Cohort Multigenerational Study has demonstrated that the paternal grandfather’s prepubertal food abundance was associated with increased grandchild mortality. When paternal grandfathers had sufficient access to food before puberty, male grandchildren were at higher risk of dying from cancer [44]. A similar study in China has found that exposure to starvation in utero increases the risk of type 2 diabetes. The increased risk of diabetes was strongest when both parents were starved in utero. A Dutch famine study also has demonstrated that children born during the famine were smaller than those born the year before the famine and were at higher risk of metabolic and cardiovascular diseases in adulthood. The study showed hypomethylation of insulin-like growth factor (IGF)-2, an important gene for intrauterine growth and development, in individuals exposed to starvation in utero compared with unexposed same-sex siblings [45]. In addition, children of prenatally undernourished fathers had an increased incidence of obesity and higher BMI than those of unexposed ones [46]. Whether it truly reflects epigenetic intergenerational inheritance remains to be seen, although research in this area of human disease makes it very likely that there are predisposing factors for serious diseases that have yet to be elucidated. In fact, there are many diseases for which there is no known causative DNA mutation, despite evidence of a strong genetic component. Germ cells undergo extensive epigenetic reprogramming from early embryonic to mature germ cells. Well-known reprogramming stages occur from early embryonic development through puberty. Germ cells are thought to become more susceptible to environmental influences during these reprogramming stages. Information transfer is hypothesized to occur through epigenetic changes in the sperm, oocyte, or both sets of gametes. Maternal separations early in life and chronic fluctuating stress in late adulthood of mice alter the levels of several miRNAs in sperm [47, 48]. Surprisingly, an injection of stress-regulated miRNAs into normally fertilized eggs recapitulates the effects of transgenerational transmission of behavioral, hormonal, and gene expression defects in the offspring [49]. However, the molecular mechanisms underlying transgenerational inheritance remain unclear (Fig. 1). A carefully designed study would be required to show that epigenomic changes induced by environmental influences are inherited across generations of human subjects, and that the underlying epigenetic mechanisms have been determined. We urgently need to establish national and international collaborative multigenerational cohort studies to address the question of whether intergenerational and transgenerational inheritance contributes to the risk of NCDs in a prospective study with a well-defined time-order parameter.
Mechanisms of transfer of information about the parental environment over generations, and methods and effects of intervention. Many mechanisms of transfer of information about the environmental experience can underlie inheritance over generations in both genome-associated (e.g., covalent modifications of histones, miRNA, tsRNA, and DNA methylation, among others) and genome-independent (e.g., microbiome) transfer. Paternal effects are not always mediated by gametes but may possibly act via the mother indirectly. Intervention methods reportedly include the effects of diet, exercise, postnatal care, and supplementations of methyl modulators (e.g., folate, vitamin B12, betaine, etc.). Furthermore, the role of sperm sncRNA in transmitting acquired traits has been mainly investigated in mouse models. Sperm tsRNA contributes to the intergenerational transmission of maternal environment-induced predisposition to NCDs in their offspring.
The raison d’être of the genome is that although the base sequence does not change, the epigenome changes depending on the situation, and various cells can be produced from a single fertilized egg. Moreover, by maintaining the epigenomic changes, they also ensure post-differentiation homeostasis. Individuals can flexibly adapt to various environments by changing the epigenome without changing the genome sequence. However, if epigenomic alterations formed by some environmental factors remain uncorrected for a long period of time, they can become risk factors for disease development. Moreover, when rat pups were separated from their mothers immediately after birth, the glucocorticoid receptor (GR) gene in the central nervous system methylated, its expression was suppressed, and the action of stress hormones was attenuated [50]. It was thus shown that gene expression regulation by epigenetic changes takes place not only in the embryonic and fetal period but also in the postnatal environment, and is involved in health and disease development (Fig. 1). It is becoming clear that the epigenome that has been altered in the environment can be restored by subsequent artificial intervention in the environment. Although the exact molecular mechanism is presently unknown, improvements in obesity [51], exercise therapy [52, 53], and epigenetic changes caused by metformin administration [54] have also been reported, and safe and efficient methods are being explored (Fig. 2).
Schematic representation of inter- (F2) and trans- (F3) generational inheritance and methyl modulator intervention. Schematic representation of methyl modulator intervention: Our fetal undernourished model rats were offspring born to mothers who were fed a low-carbohydrate (LC, blue arrow) diet throughout pregnancy period. For methyl modulator intervention, a methyl modulator (Methyl, green arrow) diet enriched with folic acid, vitamin B12, choline, zinc, and betaine was fed to lactating maternal rats for 1 week immediately after birth or fed to offspring for 1 week after weaning. However, restoration of impaired glucocorticoid negative feedback regulation was effective only on lactating maternal rats for 1 week immediately after birth. Next-generation offspring (F2 and F3) were fed a standard diet during all the periods. Surprisingly, methyl modulator intervention during the lactating period also restored impaired glucocorticoid negative feedback regulation in the F3 generation. Future studies are expected to determine whether these methyl modulators are effective in intervention at the timing after the F2 generation. SC, standard chow; LC, low-carbohydrate diet, Methyl, methyl modulator diet; NBW, normal birthweight; LBW, low birthweight.
The main body of epigenetic gene expression control is the attachment and detachment of methyl to the cytosine bases in DNA, hence epigenetics is reversible. Even if it is once methylated, it can be demethylated to restore the function of the gene. In fact, some nutrients have been shown to restore epigenetic changes. Folic acid is an essential water-soluble vitamin for vertebrates. It is incorporated as an essential cofactor for nucleotide synthesis and the generation of S-adenosylmethionine (SAM), which functions as a methyl group donor for DNA, RNA, and proteins [55]. Maternal folate deficiency causes severe neural tube defects [56] and craniofacial abnormalities [57] in offspring. Although the prevalence of these defects is significantly reduced by folic acid supplementation before and during pregnancy, these birth defects remain prevalent worldwide [58, 59]. Maternal and newborn vitamin B12 concentrations are associated with DNA methylation at multiple CpGs in offspring blood [60]. By contrast, acute vitamin B12 supplementation can improve behavioral measures that are associated with depression-like behavior in mice [61]. The effects of folate and vitamin B12 are thus related to epigenetic changes in the offspring (and probably in the next generation as well) owing to the accessibility of the DNA during early development. Supplementation with choline and betaine has been studied not only as a potential hepatoprotective substrate but also as an epigenetic controller [62]. Although the details of the restoration mechanism remain unknown, it has been hypothesized that SAM, a metabolite of the folate-methionine cycle, affects epigenomic modification because it is known to be a methyl donor for DNA and proteins.
The effects of folic acid/methionine cycle metabolite supplementation on sperm are also being elucidated. The folic acid supplementation group recorded a statistically significant improvement in survival pregnancy rate compared to the control group [63, 64]. No substantial changes in oocyte fertility or embryo quality were detected between the supplementation and placebo groups. No changes in general health, sperm counts, or imprinted gene methylation were detected at doses of 10x or more the recommended daily dose of folic acid. However, only very high doses (20-fold) of folate for 12 months showed hypomethylation of sperm [65]. Folic acid supplementation has thus been reported to improve sperm function. In addition, the effects of folate supplementation on the epigenome, as well as on sperm morphology and function, have been demonstrated. Hoek et al. conducted a systematic review and meta-analysis to present evidence of an association between paternal folate status and sperm quality, fertility, congenital malformations, and placental weight [66]. Paternal folate deficiency is associated with increased birth defects in children such as craniofacial and musculoskeletal malformations. DNA methylation analysis and subsequent functional analysis using mouse models have identified differential methylation in sperm of genes involved in chronic diseases such as development, cancer, diabetes, autism, and schizophrenia [67]. However, we believe that long-term administration of excessive folic acid aimed at causing hypomethylation is potentially dangerous. Actually, these safety concerns are contrary to the 2015 WHO statement that “high folic acid intake has not reliably been shown to be associated with negative healing effects” [68]. A high-dose folic acid administration to humans should still be carried out with caution, and sufficient appropriate evidence must be accumulated. On the other hand, the effect of supplementation with other methyl donors is limited. The supplementation of betaine may be a useful agent for increasing semen quality, fertility, and welfare, and to improve the breeding strategy of breeder males in hot climates [69]. It is likely that betaine supplements improved semen parameters in sperm motility and viability, and influenced DNA fragmentation during heat stress with reduction in serum homocysteine concentrations [70]. Unfortunately, the effects of these supplements on the next generation via the epigenome still need to be verified.
In addition to nutritional intervention, exercise has been reported to reduce the risk of disease development in the next generation through epigenomic changes (Fig. 2). In fact, it is clear that exercise itself modifies the epigenome. Voisin et al. conducted a retrospective review on physical activity and DNA methylation and showed that an individual’s methylation profile changes in a dose-dependent, gene-specific, and tissue-specific manner in response to physical activity; that long-term (chronic) exercise primarily affects methylation levels of metabolic genes in skeletal muscle; and that a threshold of exercise intensity is reached, leading to active demethylation of metabolic genes [71]. Barrez and Zierath reviewed the current understanding of mechanisms by which lifestyle factors affect the epigenetic landscape in type 2 diabetes mellitus and obesity, and evidence from the past few years on the potential mechanisms by which diet and exercise affect the epigenome over several generations [72]. Thus, evidence is emerging that exercise interventions have transgenerational effects.
The postnatal environment has also been shown to affect the epigenome of offspring. Szyf et al. proposed a mechanism linking maternal behavior and epigenetic programming, and opened up the prospect that similar epigenetic mutations generated early in life may play a role in producing interindividual differences in human behavior [73]. Epigenetic factors also serve as mediators to coordinate gut microbiota within the host. Aiming to dissect this interplay mechanism, Wu et al. reviewed the research profile of gut microbiota and epigenetics in detail, and further interpreted the biofunctions of this interplay (i.e., the improvement of metabolic disturbances) [74]. Moreover, it has been shown that feeding a choline diet alters the epigenome and metabolism through the intestinal microbiota, which also affects the next generation. Thus, prebiotics and probiotics are also expected to become effective intervention methods to reduce the incidence of diseases in LBW infants [75]. We hope that evidence of these intervention methods will accumulate in the future and that more secure and safer preemptive medical care will be realized.
Offspring born from maternal rats, which were fed with diets that restricted 40% of their carbohydrate-derived calories throughout pregnancy, were born with a LBW of approximately 20–25% [76]. There was no difference in gestational age, number of offspring, and sex ratio. When we gave the mother rats a standard diet ad libitum after birth, they were able to provide sufficient milk. By weaning day, many rat offspring showed CG within the average of –2SD of the weight of control rats. Some of them showed non-CG (NCG), however, and had short body length and LBW (LBW-NCG).
We investigated the growth hormone (GH)–IGF-1 axis to clarify the endocrine mechanism of CG, and reported the findings [76]. Although there was no difference in the blood GH concentration, the blood IGF-1 concentration in the LBW-NCG rat was lower than that in LBW-CG and control rat. Most of the IGF-1 in the blood was produced in the liver. When comparing tissue content and mRNA expression level in the liver, those of LBW-NCG were also lower than those of the control and LBW-CG rats. Considering the possibility that the GH signal was impaired in LBW-NCG rats, the expression level of GH receptor in the liver was examined. The expression levels of mRNA and protein in LBW-NCG rats were lower than those in controls and LBW-CG.
In order to clarify the mechanism by which the expression level of GH receptor in the liver of LBW-NCG rats was decreased, we focused on miRNA. An miRNA is a factor that binds mainly to the 3'-UTR of mRNA, regulates post-transcriptional expression by lacking mRNA stability and inhibiting translation. By searching the database (targetscan.org), we found five miRNAs which have a nucleotide sequence that binds to the GH receptor. The expression of miR-322 in the liver of LBW-NCG rats was significantly increased compared to the control and LBW-CG. The expression level of miR-322 showed significant negative correlation with the body length and the expression level of GH receptor mRNA in the liver. Although the mechanism underlying the increase in miR-322 expression in the liver of LBW-NCG rats has yet to be clarified, increased expression of miR-322 most likely suppressed GH receptor expression and decreased GH signal transduction in the liver of LBW-NCG rats. It has been shown that undernutrition during the embryonic period is adapted to the postnatal poor nutrition environment by changing the gene expression in peripheral organs and thus reducing the body size. These changes are thus considered to be due to the thrifty phenotype. Such LBW is caused not only by prenatal undernutrition but also by nicotine exposure. Prenatal nicotine exposure caused a decrease in blood IGF-1 levels, similar to prenatal undernutrition, but it is thought that the mechanism is not mediated by miR-322 and GHR, but by some other mechanism. However, LBW model rats exposed to nicotine during the prenatal period also showed elevated blood insulin levels when exposed to a high-fat diet [77]. In other words, LBW, albeit from different causes, has been proven to predispose to the risk of NCDs.
LWB or preterm birth is associated with an increased risk of hypertension, proteinuria, and kidney disease, which develop later in life. The mechanisms that mediate fetal programming of hypertension have been extensively studied in three major organ systems: the kidney (decreased nephron number, activation of the renin-angiotensin system, or increased renal sympathetic nerve activity); the vasculature system (alterations in structure or impaired vasodilation); and the neuroendocrine system (hypothalamic-pituitary-adrenal upregulation or altered adaptation to stress) [78-80]. In our analysis of the fetal malnutrition rat model, no abnormalities were found in the kidneys or blood vessels (including renin-angiotensin-aldosterone system). However, elevated blood corticosterone levels were observed in LBW rats exposed to a high-fat diet. Excess glucocorticoids often survive inactivation by 11β-hydroxysteroid dehydrogenase-2 in the kidney and bind to mineralocorticoid receptors. As a result, blood pressure increases due to sodium reabsorption and increased fluid volume. Excessive and prolonged glucocorticoid exposure is associated with a high cardiovascular and metabolic burden. We thus found that LBW rats had elevated blood pressure by high-fat diet exposure after growth [81]. We have previously reported a negative feedback mechanism of glucocorticoids after restraint stress and abnormalities of the mechanism in LBW rat. When rats were exposed to restraint stress, the blood corticosterone concentration rose to at a peak value of 30–60 minutes, and the value decreases with time even if the stress exposure was continued [82]. In our study, prolonged elevation of blood corticosterone levels was observed after restraint stress in LBW rats. We showed that the expression of miR-449a, an miRNA involved in the down-regulation of pituitary corticotropin releasing factor (CRF) receptors, impaired induction in the pituitary of restraint-exposed LBW rats [83, 84]. In the pituitary gland of LBW rats, the expression of growth arrest-specific 5 (Gas5), a long noncoding RNA, was increased. Gas5 has a sequence homologous to a glucocorticoid responsive element (GRE) which is a DNA binding sequence of the GR [85]. It is thus conjectured that the increase of Gas5 competitively inhibited the binding between the GR and GRE, and reduced the glucocorticoid action in the pituitary of LBW. Since the expression of miR-449a is induced by glucocorticoids, the impaired induction of miR-449a expression in LBW may be due to increased expression of Gas5. As described above, the blood corticosterone concentration was significantly higher in high-fat diet exposed LBW than high-fat diet exposed control rats, and the blood pressure was increased by high-fat diet exposure. The expression of pituitary miR-449a was induced in the control rats exposed to the high-fat diet compared with the standard chow-fed control rats, whereas the expression of miR-449a failed to be induced in the pituitary of the high-fat diet-exposed LBW rats. It can thus be conjectured that the impairment of glucocorticoid action and miR-449a induction in the pituitary of high-fat diet-exposed LBW rats may be responsible for the increase in blood corticosterone concentration and the increase in blood pressure. CRF receptors are widely expressed in the brain [86]. Its expression is seen not only in neurons but also in microglia [87]. Microglia are known to modulate synapses [88]. In other words, if CRF elevates, it is expected to change various neural activities including the autonomic nervous system. The relationship between elevated blood pressure and the autonomic nervous system is of interest, and further studies on it are needed as a future research topic.
On the other hand, glucocorticoids affect not only blood pressure maintenance but also metabolism. Recently, Nguyen et al. performed a detailed analysis of metabolism and gene expression in GR-administered diet-induced obese rats [89]. They revealed that prolonged treatment of GR modulator reversed body weight gain and adiposity in animals fed a high-fat diet. Glucocorticoid action is reduced by elevation of Gas5 expression in the pituitary of our LBW rats. It is still unclear whether elevated Gas5 expression occurs throughout the body, and not just in the pituitary, but changes in the metabolic system due to loss of the glucocorticoid action of LBW may be different from those in normal rats. It is quite possible that one of the causes of the thrifty phenotype formation is hyperglucocorticoidemia. In addition, glucocorticoids affect growth. Magiakou reviewed growth disturbance by adrenal hypersecretion of cortisol, and has reported that height is usually compromised by advanced skeletal maturation or by suppressed growth, particularly in the first years of life, due to excess glucocorticoid treatment in congenital adrenal hyperplasia, and that final height is reduced with congenital adrenal hyperplasia [90]. Growth disorders are also characteristic of the etiology of Cushing’s syndrome when they occur during childhood and adolescent growth, and their final height remains reduced even after surgical treatment. This is apparently due to direct or indirect growth impairment by hypercortisolism during the disease, followed by inadequate CG. One possible cause of the small body size is due to the trade-off of the thrifty phenotype which is most likely caused by an increase in blood glucocorticoids due to the impairment of negative feedback. Glucocorticoids affect the differentiation of various cells. Prenatal cortisol exposure was shown to have a significant effect on adipose differentiation in the fetus. Richards et al. have reported the effects of cortisol on epicardial adipose tissue, a visceral fat pad associated with adverse cardiovascular conditions in adults, using ewe model [91], and discuss the effects of maternal cortisol. They explained that maternal cortisol differs substantially from maternal nutritional and placental-restricted cortisol, which influences changes in the adipocytokines in parallel with feeding the fetus. In other words, it is not only the metabolic effects of glucocorticoids, but also the systemic effects of cortisol that may affect cellular programming, perhaps via the epigenome. Therefore, it can be said that the high glucocorticoid state caused by the abnormal negative feedback of glucocorticoids is a factor that increases the risk of developing various NCDs.
LBW affects the negative feedback dysregulation of glucocorticoids, and other hormone secretion patterns. The leptin surge in the mouse model is accelerated postnatally in the mouse LBW model [92]. Leptin, a hormone produced in white adipose tissue, acts on the hypothalamus and regulates appetite, energy metabolism, cardiovascular function, and glucose metabolism [93]. However, leptin administration to neonatal ob/ob mice exacerbates diet-induced obesity [94]. Therefore, leptin may have other mechanisms besides stimulating the hypothalamus directly to regulate food intake. Alternatively, high concentrations of leptin immediately after birth may reduce leptin sensitivity. Since leptin also activates the sympathetic nerve, it is conjectured that the change in leptin sensitivity formed by leptin surge in infancy strongly influences the autonomic nerve function after growth. Intracerebroventricular injection of leptin has been reported to cause efferent sympathetic excitation of rat kidney, adrenal gland, lumbar spine, liver, and brown and white adipose tissue [95]. Increased leptin after food intake acts on the brain to suppress appetite, resulting in activation of the sympathetic nervous system and changes in gastric function. Postnatal leptin surge is considered to determine leptin sensitivity [96], and elimination of early leptin surge is likely to lead to overeating and obesity [97]. On the other hand, administration of leptin to neonatal rats causes early-onset hypertension and cardiac dysfunction during adult life [98]. Leptin administration to newborn mice reduces glucose tolerance in adulthood [99]. Wu et al. have demonstrated that daily injection of ob/ob mice with leptin between P8 and P16, mimicking the postnatal leptin surge, largely rescued the ability of these mice to acquire the developmentally induced beige adipocytes at P20, which was associated with enhanced sympathetic nerve innervation. They explained that the postnatal leptin surge is essential for the developmentally induced beige adipocyte formation in mice, possibly through increasing sympathetic nerve innervation [100]. Changes in postnatal leptin secretion thus affect not only food intake but also the metabolic system via the autonomic nervous system.
In an epidemiological study of the Dutch famine, the offspring of women exposed to the famine during early and middle pregnancy did not show any difference in birthweight, but changes in body composition after growth were observed, suggesting that there was a risk of disease onset in the future [101]. On the other hand, an epidemiological study in Spain has reported an association between next-generation birthweight and the risk of developing placental-mediated diseases [102]. In addition, epidemiological studies in the United States have reported that black infants born to LBW mothers had lighter birthweights, suggesting the possible existence of racial differences [103]. These results suggest that various factors are involved in the impact on the birthweight of the next generation, and that the quality and duration of malnutrition are among the most important factors. Therefore, LBW male and female rats who failed to show CG by the weaning period were mated to obtain F2 offspring. The mated F1 generation dams were fed a standard chow diet ad libitum during gestation period, but the birthweight of the F2 generation offspring was significantly lower than that of the control rats, and the growth of their body length was shorter until the weaning period. Furthermore, when the males and females of the F2 generation that failed to show CG were mated to obtain the F3 generation and the F4 generation as well, the birthweight of the offspring was significantly lower than those of control rats [76]. We also demonstrated that impaired corticosterone negative feedback regulation after exposure to restraint stress is also transgenerationally inherited across generations, at least to the F3 generation [104] (Fig. 2). Furthermore, in order to clarify whether LBW is influenced by paternal or maternal effects, we used small body size rats that failed to achieve CG in either the mother or the father role to mate with rats. When the dams were fed a standard chow diet ad libitum during the gestation period, the birthweight was low in all combinations, and there was no difference between males and females due to the influence of the birthweight of the father or mother. Small body size rats with LBW and NCG showed increased miR-322 expression and decreased GH receptor expression in the liver, and reduced blood IGF-1 levels. It was shown that the gene was transgenerationally inherited to at least the F4 generation [77]. However, the DNA methylation of miR-322 seen in the F1 generation was not recapitulated in the F2 and later generations, and the mechanism of miR-322 upregulation remains unclear.
We also studied the intervention effect of methyl modulators using our model rats. We attempted intervention with a diet supplemented with folic acid and vitamin B12 to restore the glucocorticoid response in LBW rats. The methyl modulator diet, prepared according to the previous report, was fed to lactating or third-trimester mother rats or post-weaning offspring [105]. Contents of the methyl modulator diet are shown in Fig 2. As a result, when comparing blood corticosterone levels after exposure to restraint stress, the corticosterone levels were restored to the control level when intervened during the first week after delivery or during the third trimester of pregnancy. However, the intervention was ineffective for 3 days after delivery or after weaning (Fig. 2). Therefore, we found that there is a small window which can be effective in restoring the abnormalities in glucocorticoid negative feedback regulation by methyl modulator supplementation. However, it is a fact that methyl modulator intervention method has an appropriate timing and span in which the intervention effect can be exerted [87]. The effects of proper timing of intervention are inherited by the next generation, suggesting that methyl modulator intervention affects the epigenomic modification of a specific gene region and the mechanisms underlying the regulation and maintenance of epigenomes. According to the Dutch Hunger Winter studies, individuals whose mothers were pregnant during the famine had higher methylation of some genes and lower methylation of others as compared with those who were not prenatally exposed to famine [44, 88, 89]. These methylation differences may explain the likelihood of these individuals developing certain diseases later in life [90-92]. To further examine these changes, we will be conducting more wide-ranging and comprehensive epigenomic analyses in order to elucidate the mechanisms by which the effects of fetal undernutrition are inherited intergenerationally and transgenerationally (Fig. 2).
DOHaD research in humans takes a long period of several decades to obtain results, and once the results are available, it may be too late to respond to the ongoing declining birthrate and aging population. In order to overcome this time constraint, it is necessary to search for the risk factors and biomarkers for DOHaD-related NCDs using model animals that mimic human pathologies such as obesity and diabetes. In addition, various intervention studies that cannot be performed on humans due to ethical restrictions can be performed in animal experiments. By combining the findings obtained from animal experiments with the results of human clinical studies and cohort studies and applying them to humans, we believe that it will be possible to help promote human health and prevent diseases. Regarding “intergenerational transmission of disease onset risk,” which is one of the important issues to be solved in this field, Heard and Martienssen have stated that animal experiments using well-controlled mammals and cohort studies with appropriately adjusted backgrounds are necessary for its elucidation [39]. In other words, in order to apply the results obtained from DOHaD research to clinical practice, it is necessary to organically combine the knowledge obtained from animal experiments and clinical studies using human cohorts.
CG, catch-up growth; CRF, corticotropin-releasing factor; CRF-R, CRF receptor; DNMT, DNA methyltransferase; DOHaD, Developmental Origins of Health and Disease; Gas, growth arrest-specific; GH, growth hormone; GR, glucocorticoid receptor; GRE, glucocorticoid responsive element; IGF, insulin-like growth factor; LBW, low birthweight; LOF, loss of function; miRNA, microRNA; NCD, noncommunicable disease; NCG, noncatch-up growth; PAR, predictive adaptive responses; POMC, proopiomelanocortin; PVN, paraventricular nucleus of the hypothalamus; SAM, S-adenosylmethionine; sncRNA, small noncoding RNA; TH, thyroid hormone; THR, thyroid hormone receptor; tRNA, transfer RNA; TSH, thyroid-stimulating hormone; tsRNA, tRNA-derived small RNA; UTR, untranslated region
The authors (TN and NS) have nothing to disclose. Norimasa Sagawa is a member of Endocrine Journal’s Editorial Board.
