Differential SLC6A4 methylation: a predictive epigenetic marker of adiposity from birth to adulthood.

BACKGROUND: The early life environment may influence susceptibility to obesity and metabolic disease in later life through epigenetic processes. SLC6A4 is an important mediator of serotonin bioavailability, and has a key role in energy balance. We tested the hypothesis that methylation of the SLC6A4 gene predicts adiposity across the life course. METHODS: DNA methylation at 5 CpGs within the SLC6A4 gene identified from a previous methyl binding domain array was measured by pyrosequencing. We measured DNA methylation in umbilical cord (UC) from children in the Southampton Women's Survey cohort (n = 680), in peripheral blood from adolescents in the Western Australian Pregnancy Cohort Study (n = 812), and in adipose tissue from lean and obese adults from the UK BIOCLAIMS cohort (n = 81). Real-time PCR was performed to assess whether there were corresponding alterations in gene expression in the adipose tissue. RESULTS: Lower UC methylation of CpG5 was associated with higher total fat mass at 4 years (p = 0.031), total fat mass at 6-7 years (p = 0.0001) and % fat mass at 6-7 years (p = 0.004). Lower UC methylation of CpG5 was also associated with higher triceps skinfold thickness at birth (p = 0.013), 6 months (p = 0.038), 12 months (p = 0.062), 2 years (p = 0.0003), 3 years (p = 0.00004) and 6-7 years (p = 0.013). Higher maternal pregnancy weight gain (p = 0.046) and lower parity (p = 0.029) were both associated with lower SLC6A4 CpG5 methylation. In adolescents, lower methylation of CpG5 in peripheral blood was associated with greater concurrent measures of adiposity including BMI (p ≤ 0.001), waist circumference (p = 0.011), subcutaneous fat (p ≤ 0.001) and subscapular, abdominal and suprailiac skinfold thicknesses (p = 0.002, p = 0.008, p = 0.004, respectively). In adipose tissue, methylation of both SLC6A4 CpG5 (p = 0.019) and expression of SLC6A4 (p = 0.008) was lower in obese compared with lean adults. CONCLUSIONS: These data suggest that altered methylation of CpG loci within SLC6A4 may provide a robust marker of adiposity across the life course.

Development, Epigenetics and Metabolic Programming.

It is now widely recognized that the environment in early life can have important effects on human growth and development, including the 'programming' of far-reaching effects on the risk of developing common metabolic and other noncommunicable diseases in later life. We have shown that greater childhood adiposity is associated with higher maternal adiposity, low maternal vitamin D status, excessive gestational weight gain and short duration of breast-feeding; maternal dietary patterns in pregnancy and vitamin D status have been linked with childhood bone mineral content and muscle function. Human studies have identified fetal liver blood flow adaptations and epigenetic changes as potential mechanisms that could link maternal influences with offspring body composition. In experimental studies, there is now substantial evidence that the environment during early life induces altered phenotypes through epigenetic mechanisms. Epigenetic processes, such as DNA methylation, covalent modifications of histones and non-coding RNAs, can induce changes in gene expression without a change in DNA base sequence. Such processes are involved in cell differentiation and genomic imprinting, as well as the phenomenon of developmental plasticity in response to environmental influences. Elucidation of such epigenetic processes may enable early intervention strategies to improve early development and growth.

Association between perinatal methylation of the neuronal differentiation regulator HES1 and later childhood neurocognitive function and behaviour.

BACKGROUND: Early life environments induce long-term changes in neurocognitive development and behaviour. In animal models, early environmental cues affect neuropsychological phenotypes via epigenetic processes but, as yet, there is little direct evidence for such mechanisms in humans. METHOD: We examined the relation between DNA methylation at birth and child neuropsychological outcomes in two culturally diverse populations using a genome-wide methylation analysis and validation by pyrosequencing. RESULTS: Within the UK Southampton Women's Survey (SWS) we first identified 41 differentially methylated regions of interest (DMROI) at birth associated with child's full-scale IQ at age 4 years. Associations between HES1 DMROI methylation and later cognitive function were confirmed by pyrosequencing in 175 SWS children. Consistent with these findings, higher HES1 methylation was associated with higher executive memory function in a second independent group of 200 SWS 7-year-olds. Finally, we examined a pathway for this relationship within a Singaporean cohort (n = 108). Here, HES1 DMROI methylation predicted differences in early infant behaviour, known to be associated with academic success. In vitro, methylation of HES1 inhibited ETS transcription factor binding, suggesting a functional role of this site. CONCLUSIONS: Thus, our findings suggest that perinatal epigenetic processes mark later neurocognitive function and behaviour, providing support for a role of epigenetic processes in mediating the long-term consequences of early life environment on cognitive development.

Role of DNA methyltransferase 1 on the altered eNOS expression in human umbilical endothelium from intrauterine growth restricted fetuses.

Reduced fetal growth associates with endothelial dysfunction and cardiovascular risk in both young and adult offspring and the nitric oxide (NO) system has been implicated in these effects. Epigenetic processes are likely to underlie such effects, but there is to date no evidence that endothelial dysfunction in early life results from epigenetic processes on key genes in the NO system, such as NOS3 (eNOS) and ARG2 (arginase-2). We determined basal DNA methylation status in NOS3 and ARG2 promoters, and DNA methyltransferase 1 (DNMT1) effect on eNOS and arginase-2 expression using human endothelial cells isolated from umbilical arteries (HUAEC) and veins (HUVEC) from control and intrauterine growth restricted (IUGR) fetuses. Compared with cells from control pregnancies, eNOS protein and mRNA levels were increased in HUAEC, but decreased in HUVEC, from IUGR, while arginase-2 levels were increased in IUGR-HUVEC. The NOS3 promoter showed a decrease in DNA methylation at CpG -352 in IUGR-HUAEC, and an increase in IUGR-HUVEC, when compared with control cells. Methylation in the hypoxia response element of the NOS3 promoter was increased in IUGR-HUAEC and decreased in HUVEC. Methylation in the AGR2 promoter in IUGR-HUVEC was decreased in a putative HRE, and without changes in IUGR-HUAEC. Silencing of DNMT1 expression normalized eNOS expression in IUGR endothelial cells, and restored the normal response to hypoxia in HUVEC, without effects on arginase-2. This data suggest that eNOS expression in IUGR-derived endothelial cells is programmed by altered DNA methylation, and can be reversed by transient silencing of the DNA methylation machinery.

Lower maternal body condition during pregnancy affects skeletal muscle structure and glut-4 protein levels but not glucose tolerance in mature adult sheep.

Suboptimal maternal nutrition and body composition are implicated in metabolic disease risk in adult offspring. We hypothesized that modest disruption of glucose homeostasis previously observed in young adult sheep offspring from ewes of a lower body condition score (BCS) would deteriorate with age, due to changes in skeletal muscle structure and insulin signaling mechanisms. Ewes were fed to achieve a lower (LBCS, n = 10) or higher (HBCS, n = 14) BCS before and during pregnancy. Baseline plasma glucose, glucose tolerance and basal glucose uptake into isolated muscle strips were similar in male offspring at 210 ± 4 weeks. Vastus total myofiber density (HBCS, 343 ± 15; LBCS, 294 ± 14 fibers/mm(2), P < .05) and fast myofiber density (HBCS, 226 ± 10; LBCS 194 ± 10 fibers/mm(2), P < .05), capillary to myofiber ratio (HBCS, 1.5 ± 0.1; LBCS 1.2 ± 0.1 capillary:myofiber, P < .05) were lower in LBCS offspring. Vastus protein levels of Akt1 were lower (83% ± 7% of HBCS, P < .05), and total glucose transporter 4 was increased (157% ± 6% of HBCS, P < .001) in LBCS offspring, Despite the reduction in total myofiber density in LBCS offspring, glucose tolerance was normal in mature adult life. However, such adaptations may lead to complications in metabolic control in an overabundant postnatal nutrient environment.

Associations between DNA methylation of a glucocorticoid receptor promoter and acute stress responses in a large healthy adult population are largely explained by lifestyle and educational differences.

BACKGROUND: Glucocorticoids are the key regulators of the biological stress response and act by binding to glucocorticoid receptors (GR). Expression of GR is altered by DNA methylation. Methylation patterns in GR promoters have been shown to be highly variable between individuals, but little is known about the functional consequences of this variation for the acute stress response. The present study investigated associations between methylation status of the GR 1-C promoter and cortisol, cardiovascular and perceived stress responses to a psychosocial stress protocol in a large healthy adult population. METHODS: A total of 725 overall healthy men and women, aged 55-60 years, participated in a standardized psychosocial stress protocol consisting of three different stressors. At different stages during the stress protocol, salivary cortisol levels, continuous blood pressure and heart rate (HR) levels as well as perceived stress were measured. Stress reactivity was calculated as the increase between basal and peak measurements. Methylation status of the GR 1-C promoter was assessed in DNA isolated from peripheral blood samples using a methylation sensitive PCR assay for 675 of the 725 participants. RESULTS: A decrease in methylation of the GR 1-C promoter was associated with a decrease in stress reactivity as indicated by lower cortisol and lower HR reactivity. A 1% decrease in GR 1-C methylation corresponded with a cortisol decrease by 0.14% (95% CI: 0.03-0.25, p=0.02) and an HR decrease by 0.10 bpm (0.03-0.16, p=0.003). Adjusting for sex, lifestyle and education largely abolished these associations. A decrease in methylation of the GR 1-C promoter was also associated with an increase in stress perception as indicated by higher perceived stress (0.03 points [0.00-0.06, p=0.05]), lower perceived performance (-0.03 points [-0.05 to -0.01], p=0.02), and lower perceived control (-0.03 points [-0.05 to 0.00], p=0.04). After adjusting for sex and educational level the associations were no longer statistically significant. GR 1-C methylation status was not associated with blood pressure responses to the stress protocol. DISCUSSION: Although effects were small, variation in methylation status in the GR 1-C promoter was associated with physical and perceived acute stress responses. Interestingly, these associations could largely be explained by differences in lifestyle and education.

Peri-implantation and late gestation maternal undernutrition differentially affect fetal sheep skeletal muscle development.

Poor prenatal nutrition is associated with a greater risk of adult glucose intolerance and insulin insensitivity in the offspring. Skeletal muscle is the primary tissue for glucose utilization, and insulin resistance in muscle is the earliest identifiable abnormality in the pre-diabetic patient. We investigated the effect of early and late gestation undernutrition on structure and markers of growth and glucose metabolism regulation in the fetal triceps brachii (TB, slow- and fast-twitch myofibres) and soleus (slow-twitch myofibres) muscles. Pregnant sheep were fed 100% nutrient requirements (C, n = 8) or a restricted diet peri-implantation (PI, n = 9; 40%, 1-31 days gestation (dGA) (term approximately 147)) or in late gestation (L, n = 6; 50%, 104-127 dGA). At 127 +/- 1 dGA we measured myofibre and capillary density in the fetal TB and soleus muscles, and mRNA levels in the TB of insulin receptor (InsR), glucose transporter-4 (GLUT-4) and type 1 insulin-like growth factor receptor (IGF-1R). Total myofibre and capillary densities were lower in the TB, but not the soleus, of PI and L fetuses. The predominant effect in the L group was on slow-twitch myofibres. In TB, InsR, GLUT-4 and IGF-1R mRNA levels were greater in L group fetuses. Our finding of reduced myofibre density is consistent with a redistribution of resources at the expense of specific peripheral tissues by early and late gestation undernutrition which may be mediated by a decrease in capillary density. The increase in key regulatory components of glucose uptake following late gestation undernutrition may constitute a short-term compensation to maintain glucose homeostasis in the face of fewer type I (insulin-sensitive) myofibres. However, together these adaptations may influence the risk of later metabolic disease and thus our findings have implications for future strategies aimed at improving maternal diet.