Low-, normal- and high-birth-weight pigs

There is overwhelming evidence from agriculture to demonstrate that small (or ‘runt’) piglets grow more slowly and become ‘fat’ adults⁽Reference Powell and Aberle^9, Reference Rehfeldt, Tuchscherer and Hartung²⁰⁾. In an investigation of a cohort of low-, middle- and heavy-birth-weight piglets up to 180 d postnatal age it was found that the low-birth-weight piglets grow more slowly but contain more perirenal adipose tissue and intramuscular fat than the middle-birth-weight piglets⁽Reference Poore and Fowden²¹⁾.

Several experimental studies have now compared the effects of physiological and biochemical outcomes in low- and normal-birth-weight piglets on adipose tissue in later life. Low-birth-weight piglets have been demonstrated to have significantly higher back fat at 12 months (P<0·05)⁽Reference Poore and Fowden²²⁾. However, despite the increase in fat depth, male pigs at 12 months of age have reduced plasma leptin and there are no associations between leptin and back fat⁽Reference Poore and Fowden²²⁾. Gender-specific effects of birth weight and other in utero and postnatal challenges are a common feature in DOHaD studies, with many experimental findings being gender, and also age, specific. The same cohort of low-birth-weight pigs have impaired glucose tolerance at 12 months of age⁽Reference Poore and Fowden²¹⁾.

Although the early epidemiological studies of DOHaD focused on nutrient restriction and/or socio-economic background linked to a low birth weight, offspring with high birth weight may also be at risk from obesity and other diseases in later life; the ‘U’-shaped curve⁽Reference Baker, Olsen and Sørensen²³⁾. On this basis, adipose tissue from piglets with low, normal or high birth weights were investigated on days 7 and 14 of age (neonatal period). It was found that uncoupling proteins (UCP) 2 and 3, which have a number of cellular roles including energy regulation and inflammation, are reduced in adipose tissue from low-birth-weight piglets on day 7, but not day 14⁽Reference Mostyn, Litten and Perkins²⁴⁾. In addition, UCP3 is positively related to plasma leptin in normal-sized pigs but negatively correlated in low- and high-birth-weight piglets on day 14, suggesting reduced activation⁽Reference Scarpace, Nicolson and Matheny²⁵⁾. Although the study only investigated the neonatal effects of low birth weight, a loss of the relationship between leptin and UCP3 may promote adipose tissue deposition. However, at this early stage of development there is no difference in adipose tissue TAG content. Low-birth-weight piglets do possess markedly more adipocytes⁽Reference Williams, Marten and Wilson²⁶⁾, in keeping with the earlier findings⁽Reference Spalding, Arner and Westermark¹⁵⁾ and suggesting an increased capacity for adipose tissue expansion and lipid accumulation. FABP3 and 4 (Fig. 1) and PPARγ are down regulated in low- and high-birth-weight piglets on day 7, but not day 14 in adipose tissue. Conversely, FABP3 and 4 are down-regulated in low- and high-birth-weight piglets in skeletal muscle on day 14. The reduced expression of PPARγ2 in adipose tissue of the low- and high-birth-weight piglets suggests delayed adipocyte development. This finding is partly confirmed by histological data, as the low-birth-weight piglets, but not the high-birth-weight piglets, have more adipocytes per visual field on day 7⁽Reference Williams, Marten and Wilson²⁶⁾. The reduction in FABP4, the ‘adipose tissue’-specific FABP, may be protective against the development of metabolic syndrome, as a number of recent studies suggest FABP4 is central to its onset through interaction with the insulin signalling and inflammatory pathways⁽Reference Boord, Maeda and Makowski^27–Reference Maeda, Cao and Kono²⁹⁾. A protective effect in this case seems unlikely, given the well-described ‘U’- or ‘J’-shaped relationship between birth weight and obesity and the metabolic syndrome in human studies⁽Reference Baker, Olsen and Sørensen^23, Reference Martorell, Stein and Schroeder³⁰⁾, but cannot be excluded because these animals have not been studied into adulthood.

Fig. 1. Adipose tissue gene expression on day 7 of postnatal age in low-body-weight (□), normal-body-weight (▪) and high-body-weight () piglets. Values are means with their standard errors represented by vertical bars for five animals per group. ^a,bMeans with unlike superscript letters were significantly different for each gene (P<0·05). BW, birth weight; FABP, fatty acid-binding protein. (Adapted from Williams et al.⁽Reference Williams, Marten and Wilson²⁶⁾.)

The Meishan breed

Early programming of adipose tissue in the Meishan breed of pig may provide protection against hypothermia and hypoglycaemia in this ancient oriental breed. The comparatively low birth weight of Meishan piglets compared with commercial lean breeds, associated with a high litter number, is not detrimental to offspring survival as the mortality rate is lower than that of commercial breeds⁽Reference Le Dividich, Mormede and Catheline³¹⁾. Adipose tissue PPARγ expression during the first week of life is reduced in Meishan piglets compared with that of piglets of a commercial breed⁽Reference Mostyn, Litten and Perkins³²⁾ and the expression of adiponectin and its receptor (adiponectin receptor 1) are below the level of detection at ⩽21 d of age⁽Reference Treece, Williams and McGivern³³⁾. A greater percentage of plurilocular adipocytes has been reported in Meishan piglets at ⩽30 d of age compared with a lean breed⁽Reference Hauser, Mourot and De Clercq³⁴⁾. Taken together these findings suggest that adipose tissue development is delayed in Meishan piglets, which may have a beneficial impact on nutrient partitioning and energy regulation during the neonatal period. Although older but prepubertal Meishan pigs (i.e. aged 80 d) have increased lipogenic potential compared with Large White pigs, the opposite has been found after puberty at 100 d, when several fat depots exhibit less lipogenic potential⁽Reference Mourot, Kouba and Bonneau³⁵⁾. These findings suggest that the mechanisms responsible for high fat accretion occur after weaning but before puberty⁽Reference Hauser, Mourot and De Clercq^34, Reference Treece, Williams and McGivern³⁶⁾.

Interestingly, milk leptin of Meishan sows is lower than that of commercial sows, suggesting that in the pig milk leptin does not reflect maternal fat stores or serum leptin⁽Reference Guay, Palin and Jacques Matte^37, Reference Mostyn, Sebert and Litten³⁸⁾. Milk leptin is, however, positively related to piglet growth rate, girth and body, gut, heart and spleen weight in Meishan piglets, suggesting a growth-promoting effect⁽Reference Mostyn, Sebert and Litten³⁸⁾. Despite milk leptin not reflecting adiposity, serum leptin is associated with backfat thickness in early pregnancy in Meishan–Landrace sows and increases significantly (P<0·05) up to day 25 of gestation (Fig. 2), when leptin receptor (total and long form) mRNA expression in homogenates prepared from whole fetuses is higher in Meishan–Landrace pigs than in Yorkshire–Landrace pigs⁽Reference Guay, Palin and Jacques Matte³⁷⁾.

Fig. 2. Serum leptin concentrations in two breeds of pregnant sow: Meishan–Landrace (□); Yorkshire–Landrace ().Values are means with their standard errors represented by vertical bars. ^a,bMeans with unlike superscript letters were significantly different between breeds (P<0·05). Mean value was significantly different from that at 25 d of gestation: *P<0·05. (Adapted from Guay et al.⁽Reference Guay, Palin and Jacques Matte³⁷⁾.)

The in utero factors that regulate Meishan adipose tissue are as yet unknown, but may reflect differences in glucocorticoid signalling⁽Reference Klemcke and Christenson^39, Reference Desautes, Sarrieau and Caritez⁴⁰⁾. Plasma cortisol concentrations of Meishan fetuses are 30% greater than those of age-matched white cross-bred (Yorkshire×Landrace×Large White×Chester White) fetuses, despite similar plasma adrenocorticotropin and cortisone⁽Reference Klemcke and Christenson³⁹⁾. At 6 weeks old Meishan pigs exhibit higher circulating cortisol concentrations under basal conditions yet demonstrate a reduced response to a stressful experience⁽Reference Desautes, Sarrieau and Caritez⁴⁰⁾. Furthermore, 7-week old Meishan piglets exhibit an enhanced response to adrenocorticotropin infusion compared with Large White piglets⁽Reference Hazard, Liaubet and SanCristobal⁴¹⁾. These increased basal and stimulated cortisol concentrations are likely to be a result of differential regulation in the Meishan adrenal gland, particularly via regulators of steroidogenesis⁽Reference Hazard, Liaubet and SanCristobal⁴¹⁾. The reported increased plasma cortisol levels, along with greater glucocorticoid receptor gene expression observed in adipose tissue from 4-d-old Meishan piglets, may represent a potential for greater lipid uptake⁽Reference Mostyn, Sebert and Litten³⁸⁾. Fig. 3 summarises the early-life programming of adipose tissue in the Meishan breed.

Fig. 3. Summary of early-life differences in adipose tissue of Meishan pigs in comparison with a typical ‘commercial’ lean porcine breed⁽Reference Hauser, Mourot and De Clercq^34, Reference Guay, Palin and Jacques Matte^37, Reference Mostyn, Sebert and Litten^38, Reference Mourot, Kouba and Bonneau⁶²⁾. ↑, Increased; ↓, decreased; GR, glucocorticoid receptor; adiporR1, adiponectin receptor 1.

Glucocorticoid administration

Glucocorticoid infusion has been utilised in many studies of the DOHaD hypothesis, in particular in small animals in which a suboptimal maternal diet is linked to overexposure of the fetus or neonate to glucocorticoids⁽Reference Budge, Stephenson and Symonds^42–Reference Langley-Evans⁴⁴⁾. However, the effect of a suboptimal maternal diet in a large-animal species has divergent responses in relation to maternal and offspring glucocorticoid concentrations, which are dependent on the type and timing of the nutritional challenge⁽Reference Budge, Stephenson and Symonds⁴²⁾. Studies of the effects of late-gestational glucocorticoid infusion on the growth of the fetus have demonstrated that the prepartum cortisol surge may be responsible for the normal decline in fetal growth rate observed towards term in the sheep⁽Reference Fowden, Szemere and Hughes⁴⁵⁾. Furthermore, glucocorticoid infusion has a pronounced impact on adipose tissue in the sheep when delivered near term. Maternal administration of the synthetic glucocorticoid dexamethasone on day 138 of gestation (term is approximately 147 d) enhances UCP1 abundance in perirenal adipose tissue (the most abundant adipose tissue depot in the fetal and newborn lamb) as well as thermogenic potential in prematurely-delivered sheep (day 140 of gestation)⁽Reference Clarke, Heasman and Symonds⁴⁶⁾. Infusing cortisol for 5 d to near-term (day 129 of gestation) fetal sheep produces an increase in UCP1 protein and UCP2 gene expression in perirenal adipose tissue. These early-life programming effects increase protein and mRNA for UCP1 to values comparable with those of a fetus at day 144 of gestation (Fig. 4), confirming another role for cortisol in the preparation of fetal adipose tissue for life after birth⁽Reference Mostyn, Pearce and Budge⁴⁷⁾.

Fig. 4. Uncoupling protein (UCP) 1 protein abundance in perirenal adipose tissue from fetal sheep that were infused for 5 d with cortisol (▪) or saline (9 g NaCl/l; □), adrenalectomised (Ax; ) or sham-operated (sham; ). Values are means with their standard errors represented by vertical bars. ^a,bMeans with unlike subscript letters were significantly different between groups (P<0·05). (Adapted from Mostyn et al.⁽Reference Mostyn, Pearce and Budge⁴⁷⁾).

In utero dietary challenges

Suboptimal nutrition during gestation has been demonstrated in several species to impact on offspring adiposity and can range from over- or undernutrition to placental restriction.

Studies of maternal nutritional restriction have played a substantial role in DOHaD research; many have demonstrated effects on adipose tissue⁽Reference Budge, Sebert and Sharkey¹⁹⁾. For example, offspring sampled at day 140 of gestation from ewes that have experienced nutrient restriction during early–mid gestation (day 28–day 80 of gestation), which is coincident with the period of maximal placental growth, exhibit increased perirenal adipose tissue at birth⁽Reference Bispham, Gopalakrishnan and Dandrea⁴⁸⁾. This adipose tissue also displays alterations in glucocorticoid signalling at day 140 of gestation and at 6 months postnatally⁽Reference Gnanalingham, Mostyn and Symonds⁴⁹⁾. Gene expression of the glucocorticoid receptor and 11β-hydroxysteroid dehydrogenase 1 are up regulated in perirenal adipose tissue from offspring of sheep that have experienced suboptimal nutrition during early–mid gestation; however, 11β-hydroxysteroid dehydrogenase 2 (which inactivates cortisol to cortisone) is reduced⁽Reference Gnanalingham, Mostyn and Symonds⁴⁹⁾. Cortisol has consistently been implicated in both obesity and the metabolic syndrome, which is predictable given the physiological traits observed in Cushing's syndrome⁽Reference Newell-Price, Bertagna and Grossman⁵⁰⁾. However, there is little evidence for raised plasma cortisol in obesity⁽Reference Rask, Olsson and Soderberg⁵¹⁾. Although plasma cortisol per se is not consistently elevated with increased fat mass in human subjects, tissue sensitivity to glucocorticoids is increased via alterations in the enzymes and receptors that regulate local levels⁽Reference Gnanalingham, Mostyn and Symonds⁴⁹⁾. Tissue sensitivity to cortisol is regulated by the enzyme 11β-hydroxysteroid dehydrogenase and the glucocorticoid receptor. Taken together these results suggest that these offspring, who have experienced suboptimal nutrition in utero early in gestation, may be at a higher risk of developing obesity and thus metabolic syndrome, which is in keeping with human findings from epidemiological studies⁽Reference Painter, Roseboom and Bleker⁶⁾. The findings of increased glucocorticoid sensitivity in these ovine offspring are similar to those observed in Meishan piglets, which are known to develop into obese adults.

In contrast, maternal nutrient restriction during late gestation (day 110–day147 of gestation), which is coincident with the period of maximal fetal growth and a parallel rise in fetal fat depots, results in reduced adiposity in the offspring. At days 1 and 30 postnatally offspring adipose tissue mRNA expression of glucocorticoid receptor and 11β-hydroxysteroid dehydrogenase 1 is lower and that of 11β-hydroxysteroid dehydrogenase 2 is higher in the nutrient-restricted group⁽Reference Gnanalingham, Mostyn and Symonds⁴⁹⁾. This adaptation may initially be protective against the development of visceral obesity or may simply represent a response to the reduced nutrient supply. However, the offspring have greater relative fat mass and impaired glucose tolerance at 1 year, which is associated with a reduction in GLUT4 protein abundance in adipose tissue⁽Reference Gardner, Tingey and Van Bon⁵²⁾. Again, this animal work complements the findings of human epidemiological studies. The Dutch Famine data highlighted that nutrient restriction during discrete periods of gestation leads to differential effects on body systems, with late-gestational nutrient restriction affecting intermediary metabolism (in particular glucose–insulin homeostasis) and increasing the risk of the offspring developing type 2 diabetes⁽Reference Painter, Roseboom and Bleker⁶⁾.

Postnatal dietary challenges

The influence of a secondary ‘insult’ on ovine offspring who have experienced maternal nutrient restriction during early–mid gestation has been investigated by restricting offspring physical activity and providing ad libitum feed. From weaning to 1 year of age offspring from control and nutrient-restricted sheep were raised in an obesogenic environment that produces a 60% reduction in physical activity compared with pasture-reared sheep⁽Reference Sebert, Hyatt and Chan^53, Reference Sharkey, Gardner and Fainberg⁵⁴⁾. At 1 year of age offspring that have experienced in utero nutrient restriction have a total body, subcutaneous and visceral fat weight similar to that of the animals born of control-fed mothers and reared in the obesogenic environment. The nutrient-restricted offspring do, however, exhibit a reduction in daily food intake when individually housed, an adaptation that is accompanied by increased plasma insulin, but not NEFA (Fig. 5). Indeed, raised plasma insulin may represent the primary adaptation promoting excess storage of nutrients in these offspring.

Fig. 5. Plasma insulin (ng/ml; □) and NEFA (mmol/l; ) from offspring of nutrient-restricted (NR) or control ewes that were reared in an obesogenic postnatal environment. Values are means with their standard errors represented by vertical bars. ^a,bMeans with unlike subscript letters were significantly different between groups (P<0·05). (Adapted from Sebert et al.⁽Reference Sebert, Hyatt and Chan⁵³⁾.)

There is evidence to suggest that metabolic syndrome and obesity disrupt endoplasmic reticulum function and cause protein misfolding, which can trigger the misfolded protein response, as documented by several reviews linking tissue stress responses to the metabolic syndrome⁽Reference Hotamisligil and Erbay^55, Reference Ozcan, Cao and Yilmaz⁵⁶⁾. In an attempt to alleviate stress within the endoplasmic reticulum, insulin signalling pathways are inhibited through activation of c-Jun N-terminal kinase 1⁽Reference Muoio and Newgard⁵⁷⁾, which impairs systemic glucose regulation⁽Reference Ozcan, Cao and Yilmaz⁵⁶⁾. Emerging evidence from the obese offspring born to nutrient-restricted mothers demonstrates an increase in phosphorylated c-Jun N-terminal kinase protein in perirenal adipose tissue, suggesting a down-regulation of insulin signalling⁽Reference Sharkey, Gardner and Fainberg⁵⁴⁾. Several other aspects of the unfolded protein response, inflammation and infiltration of pro-inflammatory macrophages are enhanced in obese offspring born to nutrient-restricted mothers⁽Reference Sharkey, Gardner and Fainberg⁵⁴⁾. This outcome may represent an in utero programming effect whereby total adipocyte number is reduced, thus increasing its susceptibility for cellular hypertrophy⁽Reference Heilbronn, Smith and Ravussin⁵⁸⁾.

Experimental placental restriction

Experimental restriction of placental growth results in reduced birth weight, increased early postnatal growth and increased adiposity in the offspring at 6 weeks of age⁽Reference De Blasio, Gatford and Robinson⁵⁹⁾ and impairs insulin sensitivity. Feeding behaviour is also reset at 2 weeks of age, at least in the 90 min after 1 h of fasting, when placental-restriction offspring exhibit a longer suckling time, which is predictive of catch-up growth and increased central adiposity, at least up to 6 weeks of age. The molecular adaptations that produce these physiological responses in response to placental restriction may be set in fetal life. A reduction in insulin-like growth factor 1 and leptin mRNA expression has been demonstrated in perirenal adipose tissue from placental-restriction fetuses at day 140–day 145 of gestation⁽Reference Duffield, Vuocolo and Tellam⁶⁰⁾, which could impair adipocyte proliferation and differentiation, thereby potentially increasing their susceptibility for hypertrophy in later life⁽Reference Heilbronn, Smith and Ravussin⁵⁸⁾. A reduction in leptin may simply reflect a reduction in lipid stores of the fetal perirenal adipose tissue⁽Reference Symonds, Phillips and Anthony⁶¹⁾. Together these findings suggest that offspring from an ovine model of intrauterine growth restriction display features that may predispose to obesity in later life, but to date have not demonstrated any long-term complications.

The various models of early-life programming of adipose tissue in the sheep are summarised in Fig. 6.

Fig. 6. Summary of adipose tissue programming effects in sheep subject to suboptimal in utero nutrition (either through dietary or placental manipulation) or a postnatal obesogenic environment in comparison with control animals⁽Reference Gnanalingham, Mostyn and Symonds^49, Reference Gardner, Tingey and Van Bon^52, Reference Sharkey, Gardner and Fainberg^54, Reference De Blasio, Gatford and Robinson^59, Reference Duffield, Vuocolo and Tellam⁶³⁾. ↑, Increased; ↓, decreased;=, similar; NR, nutrient-restricted; ER, endoplasmic reticulum; IGF1, insulin-like growth factor 1.