Use of Embryo Transfer to Determine the Effects of a Maternal Diet Enriched in Methyl Components on Body Weight and Serum Glucose Concentrations in Viable Yellow Offspring.

Abstract

Epigenetics might account for various disease states, including obesity and diabetes, which are not explained by classic genetics. The best characterized animal model in which an epigenetic change in a gene is inherited and leads to well defined pathological outcomes is the viable yellow (Avy) mouse, which possess coat colors ranging from fully yellow to mottled, to completely brown, depending on degree of methylation of an intracisternal A particle (IAP) inserted in the promoter of their agouti gene. Yellow Avy mice with a demethylated IAP develop “yellow obese syndrome”, typified by obesity and diabetes; whereas, brown Avy, siblings, with a methylated IAP site, and black (a/a) siblings remain healthy. Addition of nutrients, likely to enrich the pool of methyl donors and vitamin co-factors required for methylation, to the diet of dams from the time of conception to term has been reported to increase DNA methylation of the IAP promoter site, such that more offspring are brown and less prone to “yellow-obese syndrome”. However, no previous study has determined when offspring are most sensitive to components in maternal diet. Herein, we used an embryo transfer approach to determine when during this time period, offspring are sensitive to methyl components. C57Bl6J females, maintained on either AIN93G control (C) or methyl-enriched (M) diet, were bred to Avy/a males. Zygotes from these matings were transferred into CD1 recipient females, also on either C or M diet. Thus, there were four groups (C to C, 20 litters, 84 pups; C to M, 22 litters, 108 pups; M to C, 14 litters, 79 pups; M to M, 9 litters, 50 pups). Coat color of the pups was assessed at 21 days of age, and all pups were maintained on the C diet. Each week, a subset of each group was weighed and saphenous vein blood serum collected and assayed for glucose by using a Bayer Contour blood glucose monitoring system. Our initial statistical assessment of coat color differences was by Chi-Squared analysis, and, based on this analysis, it appeared that there was a significant shift in offspring coat color based on donor and recipient diet combinations with M to M unexpectantly yielding more yellow coat color offspring (P values ranging from 0.001 to 0.04). However, when data were analyzed by GLIMMOX, which treats the litter rather than individual pups as a unit and is the more appropriate statistical test, there was no difference in coat color between the four groups (P = 0.81). At 40 days of age, offspring born to C to C and M to C groups weighed less than C to M and M to M offspring (C to C = 23.3 ± 1.4 g, M to C = 23.3 ± 1.1 g, C to M = 27.4 ± 1.4 g, M to M = 29.2 ± 1.5 g, P < 0.05). At this age, offspring in C to C group demonstrated lower serum glucose concentrations than mice in the other three groups (C to C = 126.6 ± 11.6 mg/dl, C to M = 160.0 ± 12.0 mg/dl, M to C = 155.6 ± 9.3 mg/dl, and M to M = 177.1 ± 12.1 mg/dl, P < 0.05). These studies demonstrated that when litter is treated as the experimental unit, there were no differences in coat color brought about by increased methyl donors in maternal diet. However, offspring exposed to M diet during gestation were heavier and exhibited higher serum glucose concentrations than those exposed to a control diet throughout gestation. These results suggest that methyl supplements or other components in the methyl-enriched diet influence the embryonic developmental outcome from periconception to term and possibly even increase the risk for adult-onset diseases, including obesity and diabetes. (poster)