- 11β-HSD
-
11β-hydroxysteroid dehydrogenase
- GC
-
glucocorticoid
- GR
-
glucocorticoid receptor
Maternal nutrition and programming of adult disease
It is now well established that a sub-optimal environment in utero can have pronounced effects on the development of the fetus and thus confer greater risk of disease in later life. The process whereby a stimulus or insult at a sensitive or critical period of development has long-term effects is termed programming. This was first investigated by the retrospective cohort studies of Barker et al. during the late 1980s who established that individuals with low birth weight, who were short or thin at birth, or who were small in relation to the placental size, were at increased risk of metabolic disease such as hypertension and impaired glucose tolerance in adulthood( Reference Barker, Bull and Osmund 1 , Reference Hales, Barker and Clark 2 ).
It is presumed that low birth weight and disproportionate body size are indicative of a lack of nutrients and/or oxygen during gestation, reflecting adaptations that the fetus has made to sustain its normal development( Reference Barker 3 ). The highest prevalence of impaired glucose tolerance and diabetes was seen in individuals who were lean at birth and became obese as adults suggesting that it is actually the mismatch between the pre- and postnatal environment that causes these effects( Reference Hales, Barker and Clark 2 ). This is further supported by later studies using the sheep as a model for maternal undernutrition, which have shown similar detrimental outcomes on the metabolism of the offspring who are adequately nourished, with or without changes in birth weight( Reference Serbert, Dellschaft and Chan 4 – Reference Gardner, Tingey and Van Bom 6 ).
Animal models for nutritional programming
Animal models are required to examine the underlying physiological, biochemical and molecular mechanisms behind the nutritional programming of offspring disease in a manner that would be unethical in human subjects. One advantage of such models is that the effect of nutrition can be assessed independently of confounding factors such as genetics, other environmental factors and social status under precisely controlled conditions.
Various species have been used as models to study the effects of nutrition on fetal programming, the most common being the rat and the sheep( Reference McMullen and Mostyn 7 ). Although rodents are small, inexpensive and are ideal in multivariate experiments, there are numerous differences between rodents and human subjects. One of the major limitations is that rodents are altricial animals, born with an undeveloped brain and endocrine system, with significant maturation of organs during the weaning period.
Due to the differences in the digestive system of sheep and human subjects (sheep are ruminants), sheep have been used primarily to investigate the effects of global undernutrition during pregnancy rather than specific macronutrient manipulations such as low protein or high fat. In contrast to the rat, sheep have a similar rate of pre- and postnatal growth to human subjects, and only produce one or two offspring, weighing between 3 and 6 kg, not unlike human subjects( Reference McMullen and Mostyn 7 ).
Primates are the ideal animal models due to their similarities to human subjects, but long lifespan, expensive housing and ethical considerations limit their use( Reference Weatherall 8 ).
In recent years, the pig has been more widely used as an animal model for human disease and is particularly useful for nutritional studies because of the similarities to human subjects in terms of the physiology and anatomy of the digestive system( Reference Litten-Brown, Corson and Clarke 9 – Reference Pond, Miller, Ullrey and Lewis 11 ). The digestive functions of each segment of the gastrointestinal tract are similar with comparable enzyme activities and organ secretions( Reference Pond, Miller, Ullrey and Lewis 11 ). In addition, the endocrine and paracrine control of gastrointestinal tract growth, motility and overall function, appear to be similar. Finally, both pigs and human subjects are able to utilise some fibre as a source of energy due to the fermentation that occurs in the large intestine( Reference Pond, Miller, Ullrey and Lewis 11 ).
Nutrient requirements during infancy, growth, reproduction and lactation are similar between man and pig( Reference Miller and Ullrey 10 – Reference Burrin, Pond and Mersmann 12 ). In addition, the neonatal pig is comparable to the human infant with respect to the stage of development and function of several organ systems. The large litter size, which allows for multiple comparisons based on birth weight, and the high postnatal growth rate also makes the pig an attractive candidate for nutritional intervention studies.
Low-protein maternal diets
Following the findings by Barker et al.( Reference Barker, Bull and Osmund 1 – Reference Barker 3 ), proof of principle was required in order to establish the cellular and molecular mechanisms behind programming effects. Due to the importance of protein in growth and development, many studies, particularly in rats, focused on the effects of maternal protein restriction on the offspring.
Protein restriction during pregnancy in rats has been shown in some studies to produce low-birth-weight offspring with higher blood pressure in early adulthood( Reference Langley-Evans, Philips and Benediktsson 13 – Reference Bertram, Trowern and Copin 15 ). In addition, in the longer term, these offspring exhibit reduced insulin sensitivity and perturbed TAG metabolism, as indicated by raised plasma cholesterol and TAG, although birth weight was unaffected( Reference Ehruma, Salter and Sculley 16 ). Interestingly, these changes have been demonstrated to take place despite no alterations in overall energy intake of the mothers( Reference Langley-Evans, Welham and Jackson 14 , Reference Ehruma, Salter and Sculley 16 ), or differences in body weight or fat mass of the offspring( Reference Ehruma, Salter and Sculley 16 ).
The role of glucocorticoids
The precise mechanisms linking maternal malnutrition, particularly low-protein diets, to metabolic disease in the offspring are still unknown. However, studies in both sheep and rats have strongly suggested that glucocorticoids (GC) play a key role( Reference Langley-Evans, Philips and Benediktsson 13 , Reference Bertram, Trowern and Copin 15 , Reference Whorwood, Firth and Budge 17 ). It has been proposed that fetal overexposure to maternal GC may trigger programming events in utero that establish persistent increases in GC hormone action in the offspring throughout life, although this is yet to be consistently established( Reference Bertram, Trowern and Copin 15 , Reference Whorwood, Firth and Budge 17 ).
GC excess has been linked to the clinical observations associated with the metabolic syndrome( Reference Arnaldi, Angeli and Atkinson 18 , Reference Covar, Leung and McCormick 19 ). For example, patients with Cushing's disease who have increased secretion of cortisol, normally due to a pituitary tumour, can develop abdominal obesity, hypertension, hyperlipidaemia and insulin resistance( Reference Arnaldi, Angeli and Atkinson 18 ). Also, clinical administration of GC to treat acute and chronic inflammatory diseases has been associated with similar adverse metabolic effects( Reference Covar, Leung and McCormick 19 ). It is therefore possible that programmed alterations in GC sensitivity may play a role linking maternal nutrient availability, fetal growth and metabolic disease risk.
Cortisol, the principal GC in human subjects, sheep and pigs, but not rodents, is regulated by the activity of the hypothalamic–pituitary–adrenal axis, a neuro-endocrine feedback loop, and is secreted in response to stress or low levels of circulating cortisol (Fig. 1)( Reference Bamberger, Schulte and Chrousos 20 ). Individual sensitivity to GC are highly regulated at a tissue level by intracellular expression of the GC receptor (GR), and the enzymes 11β-hydroxysteroid dehydrogenase (11β-HSD) types 1 and 2 at the level of gene transcription (Fig. 1)( Reference Bamberger, Schulte and Chrousos 20 ). The two isoforms of 11β-HSD are located in the endoplasmic reticulum and are responsible for the tissue-specific inter-conversion of the less biologically active cortisone to cortisol. 11β-HSD1 behaves as an 11-oxo-reductase, catalysing the conversion of cortisone to bioactive cortisol( Reference Stewart and Krozowski 21 ). Conversely 11β-HSD2 acts as an 11-oxo-dehydrogenase and catalyses the opposite reaction( Reference Stewart and Krozowski 21 ). The two isozymes are products of two different genes and have distinct tissue distributions, with 11β-HSD1 expressed primarily in the liver, adipose tissue, kidney and brain, and 11β-HSD2 mainly in the kidney and salivary glands( Reference Wang 22 , Reference Walker and Stewart 23 ).
Fig. 1. Diagrammatic representation of cortisol secretion via the hypothalamic–pituitary–adrenal (HPA) axis and its regulation at a tissue-specific level. CRH, corticotrophin releasing hormone; ACTH, adrenal corticotrophic hormone; 11β-HSD, 11β-hydroxysteroid dehydrogenase; GR, glucocorticoid receptor.
11β-HSD2 is highly expressed in feto-placental tissues and is thought to play a key role in protecting the fetus from overexposure to maternal cortisol( Reference Langley-Evans, Philips and Benediktsson 13 , Reference Whorwood, Firth and Budge 17 , Reference Edwards, Benediktsson and Lindsay 24 – Reference Condon, Gosden and Gardener 26 ). In the rat, the effects of the maternal low-protein diet on reducing offspring birth weight and programming of hypertension and dysregulation of glucose metabolism are potentially mediated by the inhibition of placental 11β-HSD2( Reference Langley-Evans, Philips and Benediktsson 13 , Reference Bertram, Trowern and Copin 15 , Reference Ehruma, Salter and Sculley 16 , Reference Langley-Evans and Nwagwu 27 ). This is also observed in human studies where a positive relationship between placental 11β-HSD2 activity and fetal weight has been identified( Reference Stewart, Rogerson and Mason 25 ).
Studies in both rats and sheep have shown that maternal diet programmes increased GC sensitivity at a tissue-specific level in both the fetal, neonatal and adult offspring, probably due to the reduction in placental 11β-HSD2( Reference Bertram, Trowern and Copin 15 , Reference Whorwood, Firth and Budge 17 ). In sheep, GR expression is increased in the adrenals, liver, lungs, perirenal adipose tissue and kidney of neonatal offspring born to ewes which were nutrient restricted during early-mid-gestation (Table 1)( Reference Whorwood, Firth and Budge 17 ). In addition, there was a 50% reduction in 11β-HSD2 expression in all tissues in which this key enzyme was found to be abundant, such as in the kidneys and adrenals( Reference Whorwood, Firth and Budge 17 ). 11β-HSD1 expression was unaffected by maternal diet, except in perirenal adipose tissue, where there was a 2-fold increase in mRNA abundance( Reference Whorwood, Firth and Budge 17 ). Importantly, these effects were observed without any significant alterations in the fetal metabolic or endocrine environment( Reference Clarke, Heasman and Juniper 28 , Reference Brameld, Mostyn and Dandrea 29 ). Similar findings were shown in a study on rats in which dams were protein restricted throughout gestation (Table 1)( Reference Bertram, Trowern and Copin 15 ). These offspring were shown to have decreased expression of 11β-HSD2 in the kidney from birth until adulthood (5 months of age), with no effect on 11β-HSD1 expression( Reference Bertram, Trowern and Copin 15 ). In addition, GR mRNA and protein expression were increased in peripheral tissues, such as the kidneys and lungs, from both late fetal (day 20) and neonatal offspring up to 12 weeks of age( Reference Bertram, Trowern and Copin 15 ). Therefore, this suggests that an increase in GC sensitivity in the offspring due to sub-optimal maternal nutrition is associated with an increased risk of metabolic disease( Reference Bertram, Trowern and Copin 15 , Reference Whorwood, Firth and Budge 17 ).
Table 1. Summary of effects of a maternal low-protein diet or nutrient restriction during pregnancy on offspring development
C, control; LP, low-protein; ↓, decreased in low-protein or nutrient-restricted gestational groups; GR, glucocorticoid receptor; 11β-HSD, 11β-hydroxysteroid dehydrogenase; ↑, increased in low-protein or nutrient-restricted gestational groups; BW, body weight; NR, nutrient restricted.
High-protein diets
Despite the tendency for dietary protein intake to exceed recommended values in the western world, particularly in younger women( Reference Rolland-Cachera, Bellisle and Deheeger 30 ), investigations into the effects of a high-protein diet during pregnancy on offspring development are limited( Reference Kramer and Kakuma 31 – Reference Zimanyi, Bertram and Black 35 ). At present, there is insufficient evidence in human subjects that a high protein intake or protein supplementation during pregnancy affects offspring birth weight or postnatal growth due to conflicting outcomes of epidemiological studies( Reference Kramer and Kakuma 31 , Reference Mathews, Yudkin and Neil 32 ).
In rats, a reduction in offspring birth weight, similar to that observed with protein restriction, has been demonstrated when dams were fed an isoenergetic, high-protein diet (40%) throughout gestation( Reference Daenzer, Ortmann and Klaus 33 ). These offspring showed accelerated postnatal growth and by the age of 9 weeks had increased fat mass in comparison with those born to mothers fed an adequate amount of protein (20%) during gestation( Reference Daenzer, Ortmann and Klaus 33 ). In contrast, other rat studies with similar levels of protein supplementation, have not demonstrated any effects on offspring birth weight( Reference Thone-Reineke, Kalk and Dorn 34 , Reference Zimanyi, Bertram and Black 35 ), but have shown an increase in blood pressure of the male pups by 4 weeks of age( Reference Thone-Reineke, Kalk and Dorn 34 ).
We have previously reported that maternal protein supplementation throughout pregnancy in the sow, increases offspring pre-weaning mortality with no effects on litter size or piglet birth weight( Reference Almond, Bikker and Lomax 36 ). The reasons for this increase in mortality are unclear, but it could be linked to constipation in sows because of the reduced fibre content of the protein supplemented diets, through the removal of sugarbeet pulp, enabling the diets to be balanced for energy. Constipation in sows is a common problem in the pig industry and has been associated with a number of adverse outcomes including farrowing problems, mastitis and failure of milk let down, all of which could increase piglet mortality( Reference Muirhead and Alexander 37 ).
Despite some similarities between maternal protein restriction and supplementation investigations in rats( Reference Daenzer, Ortmann and Klaus 33 , Reference Thone-Reineke, Kalk and Dorn 34 ), our recent study was the first to investigate the effects of protein supplementation (16·3% v. 12·3% control) on GC sensitivity. The study demonstrated that offspring born to sows fed a protein-supplemented diet throughout pregnancy exhibited an increase in gene expression of both GR and 11β-HSD1 in the liver at 1 week( Reference Almond, Bikker and Lomax 38 ) and 6 months of age (K Almond, P Bikker, M Lomax, M E Symonds and A Mostyn, unpublished results), suggesting that these animals were more sensitive to GC. These results are similar to the effects observed in offspring born to mothers who were protein restricted or under-nourished during pregnancy( Reference Bertram, Trowern and Copin 15 , Reference Whorwood, Firth and Budge 17 ). It is possible that this is due to enhanced transfer of maternal GC to the fetus, resulting in an increase in GC sensitivity in the peripheral tissues of the offspring( Reference Bertram, Trowern and Copin 15 , Reference Whorwood, Firth and Budge 17 ). In addition to the increase in both GR and 11β-HSD1 gene expression with maternal protein supplementation, the liver weight (taken as a percentage of body weight) of these animals were increased at 6 months of age (K Almond, P Bikker, M Lomax, M E Symonds and A Mostyn, unpublished results). The reasons for this are as yet unclear, and investigations are still ongoing, as both lipid and glycogen content were unaffected by maternal diet (K Almond, P Bikker, M Lomax, M E Symonds and A Mostyn, unpublished results).
Despite these differences in liver weight and gene expression, at present, no other differences or adverse health effects have been demonstrated in the variables measured in these offspring at 6 months of age.
Conclusions
The consequences of sub-optimal nutrition through alterations in the macronutrient content of the maternal diet will not simply be reflected in altered neonatal body composition and increased mortality, but are likely to continue into adulthood and confer greater risk of metabolic disease. The mechanisms behind this nutritional programming are beginning to be elucidated, with particular focus on GC action. The liver is of key importance in these studies due to its primary role in metabolism and maintaining whole body energy balance.
This study investigated the programming effects of protein supplementation in pigs throughout pregnancy. We have demonstrated an up-regulation of genes involved in GC sensitivity, such as GR and 11β-HSD1, in the liver, but have yet to detect any other significant changes in these piglets, with no differences observed in body weight or composition. This increase in GC sensitivity was similar to the programming effects observed following maternal protein restriction or global undernutrition during pregnancy( Reference Bertram, Trowern and Copin 15 , Reference Whorwood, Firth and Budge 17 ). Taken together, these findings suggest that the type of nutritional insult pregnancy is not important and that maternal under- and over-nutrition may cause similar programming effects. Therefore, these findings could have important implications in determining the programming effects of maternal diet on human disease risk.
The importance of these findings for the pig industry are not clear, as no phenotypic differences were observed between offspring of protein supplemented, compared to control fed mothers; however, protein supplementation significantly increased offspring mortality. It is currently unknown whether meat quality would be affected by the alterations in maternal dietary protein, although further work is currently being carried out to investigate the effects on muscle quality to validate this. However, this study may have important implications if these offspring were to become breeding stock and were allowed to grow and develop past 6 months of age.
Acknowledgements
This work was funded by The University of Nottingham and Schothorst Feed Research in the Netherlands. Thanks also to EARNEST, which co-funded the work at Schothorst, and, in addition, a small grant from the Endocrinology Society which supported the laboratory work. The authors have no conflicts of interest to declare with this paper. K.A. wrote the manuscript and carried out the laboratory work and literature review all as part of a PhD. P.B., M.L., M.S. and A.M. supervised the PhD, as well as securing the funding for this project, helped with various aspects of the pig management and laboratory work and also gave advice and ideas.
