Developmental programming, epigenetics and obesity

During critical periods of embryonic, fetal and early postnatal development, environmental influences can affect mammalian development, leading to permanent changes in the regulation of energy balance. There is increasing acceptance of the idea that such developmental programming of energy balance regulation is an important factor in the current worldwide obesity epidemic⁽ Reference Atkinson, Pietrobelli and Uauy ^{1 ,} Reference Gluckman, Hanson and Zimmet ^{2 )}. To guide the exploration of underlying biologic mechanisms, 15 years ago Waterland & Garza⁽ Reference Waterland and Garza ^{3 )} proposed the mechanistic construct of metabolic imprinting to describe adaptive responses to early nutrition that are characterised by a limited period of sensitivity during development (a ‘critical window’), a persistent effect lasting through adulthood, a specific and measurable outcome and a dose–response or threshold relation between exposure and outcome. Of the five potential mechanisms they elaborated⁽ Reference Waterland and Garza ^{3 )}, the potential for metabolic imprinting to occur via induced alterations in epigenetic gene regulation has received the most attention.

Epigenetics is the study of mitotically heritable alterations in gene expression potential that are not caused by changes in DNA sequence⁽ Reference Jaenisch and Bird ^{4 )}. First recognised for their importance in genomic imprinting, X-inactivation and silencing of retrotransposons⁽ Reference Bestor, Edwards and Boulard ^{5 )}, epigenetic mechanisms are now recognised to play a key role in stabilising gene expression potential in differentiated cells⁽ Reference Kundaje, Meuleman and Ersnt ^{6 )}. Various molecular mechanisms are known to participate in epigenetic regulation, including DNA methylation, covalent modification of histone proteins, autoregulatory DNA-binding proteins and non-coding RNA. Our research focuses on DNA methylation, which in mammalian somatic tissues occurs predominantly at cytosines in CpG dinucleotides. CpG methylation is recognised as the most stable epigenetic mark⁽ Reference Cedar and Bergman ^{7 )}, and is hence an excellent candidate mechanism of metabolic imprinting. Indeed, during critical periods of early development, nutrition and other environmental exposures can induce alterations in DNA methylation that persist to adulthood, in both rodents⁽ Reference Dolinoy, Huang and Jirtle ^{8 –} Reference Weaver, Cervoni and Champagne ^{11 )} and human subjects⁽ Reference Dominguez-Salas, Moore and Baker ^{12 ,} Reference Waterland, Kellermayer and Laritsky ^{13 )}.

Human and animal model data show that epigenetic dysregulation can cause obesity. As recently reviewed⁽ Reference Waterland ^{14 )}, adult-onset obesity in cloned mice⁽ Reference Tamashiro, Wakayama and Akutsu ^{15 )} suggests epigenetic causality in obesity, and individually variable hyperphagic obesity of isogenic heterozygous agouti viable yellow (A^vy ) mice, which correlates with A^vy methylation⁽ Reference Waterland and Jirtle ^{9 ,} Reference Morgan, Sutherland and Martin ^{16 )}, provides a clear example of epigenetic dysregulation causing obesity. In human subjects, sporadic cases of Prader–Willi syndrome (a neurodevelopmental syndrome characterised by hyperphagic obesity, among other symptoms)⁽ Reference Goldstone ^{17 )} are caused by aberrant hypermethylation and epigenetic silencing of a region of chromosome 15.

Despite these compelling examples, it remains unclear whether epigenetic dysregulation plays a major role in the current obesity epidemic. This is due to multiple obstacles to the study of epigenetic aetiology in human subjects, including the inherent cell-type specificity of epigenetic regulation, the influence of genetic variation on epigenetic variation, the potential for reverse causality, and the overall complexity of epigenetic regulation⁽ Reference Waterland ^{14 ,} Reference Waterland and Michels ^{18 )}. For these reasons, controlled studies in isogenic mouse models now offer the best opportunities to understand how interindividual epigenetic variation influences energy balance regulation. Our recent investigations into two such models have led to surprising insights.

Two mouse models of developmental programming of energy balance show alterations in physical activity but not food intake

The first of these is a mouse model of transgenerational effects of maternal obesity on the offspring. Fifteen years ago Levin proposed that in pregnancies complicated by maternal obesity the intrauterine environment may present an unfavourable milieu to the developing fetus, leading to aberrant development of central mechanisms for regulation of energy balance⁽ Reference Levin ^{19 )}. If, in this manner, maternal obesity during pregnancy promotes positive energy balance (i.e. energy storage in the form of adipose tissue) in her offspring, over successive generations this could lead to transgenerational amplification of obesity. A^vy/a mice provide an attractive model in which to test this because they are spontaneously hyperphagic and become severely obese as adults, but unlike most genetic mouse models of obesity, the females remain fertile. We passed the obesity-promoting A^vy allele through the female germline for four generations⁽ Reference Waterland, Travisano and Tahiliani ^{20 )}. Consistent with the feed-forward hypothesis, even among this isogenic population, the offspring of fatter mothers were themselves fatter as adults. Consequently, average adult body weight and adiposity increased over successive generations. In this experiment, we also studied another four-generation lineage that was treated identically as the first, but provided a methyl-supplemented diet known to promote DNA methylation during development⁽ Reference Waterland and Jirtle ^{9 ,} Reference Wolff, Kodell and Moore ^{21 )}. In the methyl-supplemented group, transgenerational amplification of obesity was completely prevented, suggesting a potential role for DNA methylation in this phenomenon⁽ Reference Waterland, Travisano and Tahiliani ^{20 )}. It is important to clarify that although epigenetic mechanisms affecting body-weight regulation may be involved within each generation, we believe it unlikely that the transgenerational effect in this model reflects transgenerational epigenetic inheritance. As in many examples of transgenerational effects, recapitulation is a more likely mechanism than transgenerational epigenetic inheritance⁽ Reference Waterland ^{14 )}.

We conducted a follow-up study in the A^vy model to answer two questions: (1) When is the critical period for the effect of maternal obesity (prenatal or postnatal)?; (2) Is positive energy balance in this model due to increased food intake or reduced energy expenditure? We cross-fostered newborn mice at birth between lean a/a and obese A^vy /a dams to examine the effect of being exposed to an obese A^vy /a mother either during fetal development or during the suckling period⁽ Reference Baker, Li and Kohorst ^{22 )}. These studies provided several surprising findings. First, we found that offspring of A^vy /a dams are growth restricted at birth (despite normal placental weight), suggesting placental insufficiency in A^vy /a dams. When offspring of obese A^vy /a dams were fostered to lean a/a dams (OL offspring) they caught up to normal body weight by weaning. Most interestingly, fetal growth restriction and subsequent catch-up growth led to increased body weight and adiposity in adulthood, but only in female offspring. Overall, although these data indicate that growth restriction during prenatal development is an essential component of the programming mechanism⁽ Reference Baker, Li and Kohorst ^{22 )}, they do not rule out a potential critical role for catch-up growth during the suckling period.

To answer the second question, metabolic cage studies were performed to measure home cage activity, energy expenditure and food intake. The results of these studies were completely consistent with the growth and body composition data. Whereas food intake of female OL offspring was not different from that of controls, their levels of physical activity (home cage activity) and energy expenditure were reduced directly after weaning, and remained so in adulthood. In fact, the persistently blunted spontaneous physical activity (SPA) of OL females appeared to completely explain their increased adiposity as adults⁽ Reference Baker, Li and Kohorst ^{22 )}. Notably, recent studies in three different human populations showed that perinatal undernutrition leads to increased obesity in adulthood, specifically in females⁽ Reference Robinson ^{23 )}. Our data suggest that this may be occurring by permanent blunting of physical activity.

Remarkably, in a completely different mouse model of developmental programming of obesity, we found a similar female-specific effect on physical activity. Fostering newborn rodents to small litters (SL) is a simple method to achieve overnutrition during the suckling period. When rats or mice are fostered to SL during the suckling period, by the age of weaning they are consistently heavier than control pups fostered in normal-sized litters⁽ Reference Aubert, Suquet and Lemonnier ^{24 ,} Reference Kennedy ^{25 )}. Suckling period overnutrition permanently alters body-weight regulation and glucose metabolism of SL rodents. Although all pups are provided the same diet (ad lib) after weaning, SL offspring remain heavier and fatter in adulthood⁽ Reference Kennedy ^{25 ,} Reference Cryer and Jones ^{26 )}, and exhibit impaired glucose tolerance⁽ Reference Aubert, Suquet and Lemonnier ^{24 ,} Reference Waterland and Garza ^{27 )} and dysregulated endocrine pancreas function⁽ Reference Waterland and Garza ^{27 )}. We performed metabolic cage studies to determine if the persistent change in energy balance regulation of SL mice is caused by altered food intake or energy expenditure⁽ Reference Li, Kohorst and Zhang ^{28 )}. At postnatal day 1 (P1), mice were fostered to SL (four pups/litter) or control litters of nine pups. As previously reported, SL males and females were heavier and fatter than control mice at weaning (P21) and remained so as adults (P180). Our metabolic studies showed no group differences at P25, but by adulthood (P180) we observed blunted energy expenditure correlated with reduced SPA in SL mice. Although similar trends were observed in both sexes, the effects were significant (P<0·001) only in females⁽ Reference Li, Kohorst and Zhang ^{28 )}. Hence, together with our data in the A^vy model of fetal growth restriction and catch-up growth⁽ Reference Baker, Li and Kohorst ^{22 )}, these findings suggest that females are particularly sensitive to developmental programming of energy balance.

Biological determinants and developmental programming of physical activity

Most importantly, integrating these two very different models of early nutritional influences on the development of energy balance regulation, in which both food intake and physical activity were extensively characterised by state-of-the-art methods, our results suggest that developmental programming of energy balance may be mediated principally at the level of physical activity. This insight is particularly timely, in that modernisation is leading to a worldwide epidemic of physical inactivity⁽ Reference Kohl, Craig and Lambert ^{29 )}. Physical activity clearly has a genetic component⁽ Reference Bauman, Reis and Sallis ^{30 ,} Reference de Vilhena e Santos, Katzmarzyk and Seabra ^{31 )} and plays an important role in protecting some individuals from obesity⁽ Reference Garland, Schutz and Chappell ^{32 –} Reference Kotz, Teske and Billington ^{34 )}. Based on a scholarly review of the literature on the biological basis of physical activity, Rowland proposed that each individual is endowed with a specific set point (the activity-stat) that determines his or her propensity for physical activity⁽ Reference Rowland ^{35 )}. Although most have considered the activity-stat to be genetically determined, it could also be subject to developmental programming. Indeed, in addition to our recent mouse studies, previous studies have found evidence of developmental programming of the activity stat. Before considering those studies, it is helpful to point out that physical activity is typically classified as either voluntary exercise or SPA. Voluntary exercise is physical activity that is not directly needed for survival or influenced by any outside factors, whereas SPA is all physical activity that is not voluntary exercise, including activities of daily-living and pacing and fidgeting⁽ Reference Garland, Schutz and Chappell ^{32 )}. In rodents, running wheel activity is the universal model for voluntary exercise⁽ Reference Garland, Schutz and Chappell ^{32 )}, and home cage activity is indicative of SPA⁽ Reference Kotz, Teske and Billington ^{34 )}.

In what we believe was the first reported animal data suggesting developmental programming of physical activity, Vickers et al.⁽ Reference Vickers, Breier and McCarthy ^{36 )} studied a rat model of maternal undernutrition (30 % of ad libitum intake) throughout pregnancy, and examined effects on the offspring. They performed brief (15 min) measurements of home cage activity (SPA) when the offspring were both juveniles (P35) and adults (P145), and found reduced SPA in offspring of undernourished dams. Importantly, other than our data described above, this is the only study we are aware of that measured effects on physical activity both shortly after weaning and in adulthood. In this manner, the study demonstrated that maternal undernutrition during pregnancy caused reduced physical activity in the offspring shortly after weaning, and this persisted to adulthood. Two subsequent studies examined variants of another rat model of gestational undernutrition, assigning pregnant dams to a normal or low-protein diet during gestation⁽ Reference Sutton, Centanni and Butler ^{37 )} or gestation through weaning⁽ Reference Ohishi, Wang and Akane ^{38 )}. In the first study, offspring of control and undernourished dams were cross-fostered at birth to control dams to limit the exposure (undernutrition) to prenatal development. Voluntary exercise (wheel running) was measured over a 2-week period in adolescent (P56) offspring of undernourished dams. Both male and female offspring of undernourished dams engaged in much less voluntary exercise than control offspring⁽ Reference Sutton, Centanni and Butler ^{37 )}. The other study of maternal low-protein diet⁽ Reference Ohishi, Wang and Akane ^{38 )} differed from the first in that the exposure started at day 10 of gestation and continued through the end of the suckling period. Rather than reduced physical activity, this study reported increased motor activity in adult (P72) offspring of undernourished dams. Interestingly, as in our studies, this effect was observed only in female offspring⁽ Reference Ohishi, Wang and Akane ^{38 )}. Combining our data (discussed above) with these earlier studies, the results of these four diverse models of developmental programming suggest that, indeed, the setting of one's activity stat can be affected by nutritional exposures during prenatal and early postnatal development. Now, the challenge is to understand how this occurs, and determine whether similar processes occur in man.