Research into ‘the developmental origins of adult health and disease’ has made considerable progress over the past two decades, but a clear consensus on the exact nutrients involved and their mechanisms remains to be established. This problem relates, in part, to the long-term developmental time frame in which changes in metabolism and cardiovascular function occur. At the same time, the discipline has had to contend with the dual challenges of integrating findings from lifetime epidemiological studies that have largely focused on birth weight and its relationship (or otherwise) with adult disease and the ability to incorporate these results into appropriate nutritional intervention studies using animal models. It is now established that there are many potential influences on offspring outcome, including maternal body composition, age and parity, genetic constitution and macro- and micronutrient intake and handling, size, shape and number of offspring, their gender and type of lactational diet. Thus, it is not unexpected that as knowledge of the intricacies involved expands, a substantive number of publications have, in retrospect, been misleading because of inappropriate statistical analysis and/or mismatches in the different groups (of which some key examples will be summarised).
In the present brief review an attempt will be made to provide an overview of some of the major challenges for both human and animal studies, in conjunction with optimal experimental paradigms that may be utilised in future research aimed at elucidating the precise mechanisms by which changes in the maternal diet during reproduction can impact on the lifetime health of resulting offspring. Particular emphasis will be given to the applicability of the animal model that has been utilised and how similarities in the reproductive process may enable its best use in future examinations of the nutritional programming of adult health and disease.
Human models: historical and contemporary models of fetal programming
The majority of the early work conducted by David Barker and colleagues utilised data from historical cohorts born in the 1930s and benefitted from meticulous birth records, often kept by the same individual over long periods of time(Reference Barker, Bull and Osmond1). This approach has enabled clear relationships between not only the size and shape of an infant at birth but also its accompanying placental mass and later hypertension to be established(Reference Barker, Bull and Osmond1). Subsequently, the recruitment of long-term historical records from Finland has enabled longitudinal studies of infant growth to be related to adult insulin sensitivity(Reference Barker, Osmond and Forsén2). More recently, the use of nutritional interventions of preterm formula in randomised controlled studies has emphasised the impact of inappropriate growth in early infancy on later disease risk(Reference Singhal3). However, with more contemporary studies and the rise in both childhood and adult obesity it is now apparent that the translation of findings from such studies into current lifestyle interventions is very difficult. This situation results, at least in part, from the complexity of the biological processes involved and the challenge of effecting lifestyle changes in activity and dietary intake. At the same time, the causes of the ongoing epidemic of obesity, and the predicted increase in associated renal diseases and CVD, are multifactorial(Reference Keith, Redden and Katzmarzyk4) and may be either exacerbated, or reduced, by early dietary exposure(Reference Williams, Kurlak and Perkins5).
One consistent theme that is apparent from both historical and contemporary studies is that changes in nutrition at specific stages of pregnancy can have very different outcomes(Reference Symonds, Stephenson and Gardner6). This finding is not unexpected as different organs have critical and precise developmental windows that may be compromised, or enhanced, and thereafter permanently set for the rest of that individual's life. Importantly, adaptations of this type appear not only to be dependent on the period in which the mother's diet is altered but also the diet to which she is rehabilitated(Reference Reynolds, Godfrey and Barker7). One fundamental consideration is that the self-limitation in food intake between early-to-mid gestation that occurs commonly as a result of the nausea affecting approximately 90% of pregnant women in the UK may be directly linked to Western diets(Reference Pepper and Roberts8). The extent to which this factor relates to changes in placental function and/or fetal growth remains less clear, but there is a need to match global pre- and postnatal nutritional requirements in order to avoid accelerated growth and the concomitant increased risk of later obesity and metabolic complications(Reference Symonds9). Importantly, however, intergenerational acceleration mechanisms do not appear to make an important contribution to levels of raised childhood BMI within the population(Reference Davey Smith, Steer and Leary10).
The lactational environment and postnatal development
A further area requiring consideration is the relationship between the maternal diet in late pregnancy, its impact on mammary gland development and milk production and whether the infant is breast-fed or formula-fed(Reference Toschke, Martin and von Kries11). The higher macronutrient content of formula feed compared with breast milk, in conjunction with its fixed composition throughout a feed (unlike breast milk, the composition of which changes with time), will impact on the nutrient supply to the infant. It is therefore necessary to consider not only short-term but also long-term advantages of breast-feeding in relation to appetite regulation(Reference Sievers, Oldigs and Santer12). The type of lactational diet also impacts on other behavioural aspects, including sleep–awake activity cycles(Reference Lee13), so that extended breast-feeding may not only be beneficial in developing countries(Reference Heird14). Other confounding factors such as social class and smoking during pregnancy further determine postnatal diet(Reference Bogen, Hanusa and Whitaker15).
In summary, the relationship between nutrient supply during key stages of development from the time of conception to weaning is highly complex and requires careful in-depth consideration. It is necessary to conduct detailed animal experiments in a range of species in order to elucidate the mechanisms involved, be they epigenetic or related processes(Reference Symonds, Stephenson and Gardner16).
Animal models of nutritional programming
The main animal models that have been utilised to date to investigate the impact of maternal diet on long-term programming have been rats and sheep(Reference McMillen and Robinson17). These animals obviously have very different developmental patterns in the relationship between placental and fetal growth, maturity at birth and milk composition(Reference Prentice and Prentice18). The advantage of utilising rats is their very short gestational length. However, the type of diet they normally consume in the wild is very different from that fed to housed laboratory animals in which semi-purified diets are the norm. Such diets provide substantially greater differences in nutrients to pregnant rats than to controls and, therefore, should be considered ‘pharmacological’ as opposed to ‘physiological’ as they are outside the normal distribution of wild-type diets. For example, high-fat diets utilised to date in rat studies contain four times as much fat when compared with control diets and are at risk of being deficient in micronutrients. Not surprisingly, when fed a diet so rich in fat, maternal food intake is reduced(Reference Taylor, McConnell and Khan19). In addition, rats exhibit coprophagia, which has a substantial effect on nutrient flux and the ability to experimentally manipulate the intake of specific nutrients.
Appreciable placental growth necessarily continues up to term in the rat to meet the much higher protein demands for fetal growth compared with that of man or sheep(Reference Widdowson20). In large mammals, however, the maximal period of placental growth is early in pregnancy in order to meet the greatly increased nutrient requirements of the fetus in late gestation when its growth is exponential(Reference Symonds, Budge and Mostyn21). Furthermore, rats produce large litters whereas sheep and man only normally produce one (or two) offspring of comparable birth weight per pregnancy.
Comparative differences in organ development
Another substantive difference between rats and man (and rats and sheep) is that the development and maturation of the hypothalamic–pituitary system occurs after birth during the neonatal period in rats compared with during late gestation in sheep and man. Thus, any nutritional intervention that stops at birth in the rat will have substantially different outcomes compared with those extending into the neonatal period(Reference Symonds9). Other organs that continue to develop postnatally in the rat, compared with prenatally in sheep and man, include the kidney and brown adipose tissue(Reference Symonds, Stephenson and Gardner6) (Fig. 1). For example, in those species born with a mature hypothalamic–pituitary axis after a long gestation, such as in sheep and man, the majority of the appetite-controlling networks appear to be in place by mid- to late gestation(Reference Mühlhäusler, McMillen and Rouzaud22). This position is clearly in contrast to that in small mammals in which there is substantial development during the lactational period as the hypothalamic axis matures. It is thus not unexpected that leptin administration has such a marked effect on this process(Reference Bouret, Draper and Simerly23) and related processes(Reference Vickers, Gluckman and Coveny24, Reference Attig, Solomon and Ferezou25), which are not seen when given to the late-gestation fetus or neonatal sheep(Reference Mostyn, Bispham and Pearce26, Reference Yuen, Owens and Mühlhäusler27). Indeed, there is no postnatal leptin surge in either sheep(Reference Bispham, Budge and Mostyn28) or man(Reference Matsuda, Yokota and Iida29).
Nephron development occurs primarily after birth, as does brown fat, which in sheep and man was originally thought to be completely lost in the postnatal period(Reference Lean30), but is retained throughout the life cycle in the rodent. As such, brown adipose tissue has a fundamental impact on the energy balance of adult rats because it uniquely possesses uncoupling protein 1 and is thus able to rapidly generate large amounts of heat(Reference Cannon and Nedergaard31). Indeed, brown fat has a pivotal role in the regulation of thermogenesis, with a thermogenic potential of 300 W/g whilst all other tissues generate approximately 1 W/g(Reference Power32). Not unexpectedly, loss of uncoupling protein 1 is accompanied by an increase in fat mass, making it difficult to dissociate the dietary and related effects on changes in fat mass from adaptations in uncoupling protein 1 in this species(Reference Mostyn, Keisler and Webb33). Recently, indirect measurements in patients with cancer suggest that brown fat may be retained in small amounts in these individuals(Reference Nedergaard, Bengtsson and Cannon34). Its location is, however, primarily in the neck region(Reference Christensen, Clark and Morton35, Reference Goetze, Lavely and Ziessman36) and whether it has any functional importance in energy balance remains to be established.
Methodological considerations in the interpretation of metabolic programming
There have been two major problems with rat studies utilised in the assessment of long-term cardiovascular outcomes. First, in many studies blood pressure has only been measured using the tail-cuff technique in restrained and heated animals during the day when they are normally inactive, being nocturnal animals (for example, see Langley-Evans(Reference Langley-Evans37); Kwong et al.(Reference Kwong, Wild and Roberts38)). It is recognised that this approach can give very different results when compared with blood pressure measurements obtained using telemetry(Reference D'Angelo, Elmarakby and Pollock39); as such, it is only recommended for use in obviously hypertensive animals in which the technique was originally validated(Reference Bunag40). Thus, in the majority of studies in which it has been used in this area the tail-cuff technique is not informative in relation to the modest differences in blood pressure recorded in such normotensive rats. This factor may explain why in more recent studies the offspring born to dams fed a low-protein high-carbohydrate diet through pregnancy show either no difference or a reduction in blood pressure when measured using either telemetry or an indwelling arterial catheter(Reference Fernandez-Twinn, Ekizoglou and Wayman41, Reference Hoppe, Evans and Moritz42). Comparable findings are seen in offspring born to dams in which food intake is reduced by 50% through pregnancy compared with controls(Reference Brennan, Kaufman and Reynolds43). A further confounding factor in many of these early studies has been a failure to consider the dam as the experimental unit rather than her offspring(Reference Kwong, Wild and Roberts38), which is now recommended as standard practice(Reference Festing44). There also appears to be a marked divergence in the long-term outcomes between genders in rats that is primarily linked to the faster, as well as continued, growth of males compared with females(Reference Symonds and Gardner45).
Despite the fact that sheep are ruminants, they have proved invaluable in enabling the understanding of the nutritional and endocrine regulation of placento–fetal development. As in man, the primary metabolic substrate for fetal metabolism is glucose, for which GLUT 1 is the main placental regulator(Reference Dandrea, Wilson and Gopalakrishnan46). Glucose is thus transported across the placenta by active diffusion determined by its concentration in maternal blood(Reference Edwards, Symonds and Warnes47). In addition, not only does kidney development show a very similar ontogeny in sheep and man, but the distribution of total nephrons across the adult population is comparable(Reference Symonds, Budge, Mostyn and Watkins48). It is also feasible to obtain very consistent blood pressure recordings in the offspring using arterial cannulation whilst the animal is standing freely with continual access to its diet(Reference Gardner, Pearce and Dandrea49). At the same time, there is no discernable difference in blood pressure control or glucose regulation between genders when measured in intact adult sheep(Reference Symonds, Budge, Mostyn and Watkins48, Reference Gardner, Tingey and van Bon50).
Mismatched offspring and later outcomes?
Another confounding factor that has become apparent in a number of recent studies using sheep is a mismatch between the numbers of singletons and twins between nutritional groups. This mismatch has introduced erroneous conclusions, which is not unexpected given the pronounced differences in outcomes of human newborns that are also dependent on fetal number(Reference Hayes, Paul and Ness51). Appropriate statistical correction (as in the case of rat studies) for this factor is therefore necessary(Reference Festing44). Some of the more prominent examples of such mismatch between control and intervention groups are summarised in Table 1. Indeed, this mismatch is further confounded by using both twins from the same mother for whom the lactational environment will be further altered. In contrast, when one twin is reared alone by its mother, its growth rate matches that of a singleton, as does its later blood pressure control and glucose homeostasis(Reference Gardner, Tingey and van Bon50, Reference Gopalakrishnan, Gardner and Dandrea52).
It is now clear that future studies in the area will need to take into account not only changes in diet through pregnancy but also the relative contributions of gender, litter size, type and duration of lactation as well as weaning diets. These considerations will undoubtedly increase the time necessary to conduct such studies as well as their cost, but it is imperative that with the plethora of nutritional models already established these factors are optimised in order to elucidate the main pathways involved rather than pursuing models that may have little translational relevance(Reference Symonds, Stephenson and Budge53).
Conclusion
In summary, appropriate nutritional interventions are now available that can examine relevant short- and long-term outcomes and determine how contemporary diets impact on fat deposition, metabolic homeostasis and cardiovascular control in a consistent and validated manner. The completion of such studies may enable the determination of the optimum nutrition in terms of quantity and quality that may ultimately improve the health of future generations.
Acknowledgements
The authors declare no conflict of interest and acknowledge the support of the British Heart Foundation and the European Union Sixth Framework Programme for Research and Technical Development of the European Community – The Early Nutrition Programming Project (FOOD-CT-2005-007036) in their research.