MicroRNAs Regulate Hypothalamic AT2R Expression in Hypercaloric Diet‐Induced Epigenetic Programming of Male Mice Offspring

Abstract

One in ten adults are living with diabetes worldwide and it is expected to be one in eight adults by the year 2045. Maternal epigenetic programming is one of the determinants of disease susceptibility in offspring. The renin‐angiotensin system contributes to metabolic homeostasis through the balance between Angiotensin II (Ang‐II) binding to Angiotensin II Type 1 receptor (AT1R) and Angiotensin II Type 2 receptor (AT2R). We have shown that maternal hypercaloric (HCD) diet leads to a pre‐diabetic state exclusively in adult male offspring. Additionally, we showed a paradoxical reversal of hyperglycemia in programmed males challenged with Ang‐II. Here, we hypothesize that this hypoglycemic response in programmed males is due to a shift in Ang‐II binding from AT1R towards the protective AT2R. To test this hypothesis, C57BL6/J dams were fed HCD or regular diet (RD) for 1 month and mated with RD‐fed males. After weaning, offspring were maintained on RD for 3 months resulting in two groups: HCD‐RD (programmed) and RD‐RD (control). Half of each group was implanted with a subcutaneous Ang‐II osmotic pump (200 ng/kg/min/2 weeks). Another osmotic pump filled with AT2R blocker, PD123319 (10 mg/kg/day) was implanted in a subgroup of Ang‐II programmed mice resulting in 5 groups: HCD‐RD, RD‐RD, HCD‐RD+Ang‐II, RD‐RD+Ang‐II and HCD‐RD+Ang‐II+PD123319 (n=7‐10/group). A glucose tolerance test was performed before and after drugs infusion. Following euthanasia, half of each hypothalamus was harvested for protein quantification using simple western while the other halves were processed for microRNA (miRNA) sequencing. Results show that HCD‐RD+Ang‐II+PD123319 had elevated glucose levels compared to HCD‐RD+Ang‐II mice at 15 min (355.7 ± 32 vs. 279.8 ± 19.66 mg/dL, p=0.04), 30 min (405 ± 18.54 vs. 263.5 ± 22.68mg/dL, p=0.0013) and 60 min (298.3 ± 19.75 vs.189.8 ± 21.35 mg/dL, p=0.0036). On the other hand, there was no difference in the glycemic levels of HCD‐RD+Ang‐II+PD123319 compared to RD‐RD+Ang‐II groups at 15 min (355.7 ± 32.97 vs. 349.0 ± 32.06 mg/dL, p=0.8), 30 min (405.0 ± 18.54 vs. 405.8 ± 44.28 mg/dL, p=0.9) and 60 min (298.3 ± 19.75 vs. 230.0 ± 35.52 mg/dL, p=0.1). AT2R protein expression was higher in the HCD‐RD compared to RD‐RD (p=0.04) and in HCD‐RD+Ang‐II compared to RD‐RD+Ang‐II (p=0.03). For miRNA sequencing, miRNA 124‐3p, targeting AT2R was differentially expressed in the HCD‐RD compared to RD‐RD (FC=1.4, p=0.04) as well as HCD‐RD compared to HCD‐RD+Ang‐II (FC=0.74, p=0.0001). Whereas, no change in the miRNA expression was depicted in the non‐programmed groups; RD‐RD vs RD‐RD+Ang‐II (FC=0.97, p=0.85). In conclusion, maternal hypercaloric diet epigenetic programming takes place probably through regulating the gene expression of AT2R via changing miRNA ‐124 levels that works on the post‐transcriptional modification level.