Neonatal Intramuscular Injection Of Plasmid DNA Encoding GLP-1 Affects Behavior And Hippocampal GR Expression In Adolescent Rats

In this study, neonatal (2-day-old) Wistar male rats were allocated to two groups and were intramuscularly injected with vacant plasmid (VP) or plasmid DNA encoding GLP-1 (GP), to investigate whether neonatal injection of GLP-1 over-expressing plasmid may cause persistent behavioral changes in adolescent rats. Elevated plus maze was applied to assess the anxiety-related behavior and EIA was employed to measure plasma corticosterone concentration. Real-time RT-PCR and Western blot analysis were used to quantitate hippocampal expression of GR and DNMT1 both at mRNA and protein levels. DMA methylation status of GR promoter exon 1₇ was determined by bisulfate sequencing PCR (BSP) and hippocampal expression of GLP-1R and NGFI-A was quantified to reveal the mechanisms underlying the action of GLP-1.1 Behavioral and Corticosterone DataThere were significant differences between the VP and GP rats in OE% (P = .024), OT% (P = .021) and (OE + CE) (P = .011) in the EPM test. Rats in GP group demonstrated significant higher number of entrances and time spent in the open arms, as compared to VP rats, indicating an anxiolytic effect. Nevertheless, the total number of entrances to both open and close arms was significantly lower in GP rats compared to VP counterparts, suggesting decreased mobility or activity in GP rats in general. GP rats showed slightly higher plasma corticosterone level compared to VP rats, but no statistical difference was detected between two groups (P = .42).2 Adrenal Weight IndexAdrenal gland of each rat was weighted and the index of adrenal weight was calculated by the formula: adrenal weight index = adrenal weight/body weight. The index of adrenal weights was: VP: 19.21±1.04, GP: 20.94±1.12. Statistical analysis revealed no difference between two groups (P = .172). 3 Expression of GR and DNMT1 in hippocampusA significant up-regulation of GR (P = .01) mRNA expression was determined in hippocampus of GP group, which was accompanied by significantly down-regulated DNMT1 expression (P = .024). Hippocampal DNMT1 protein levels were clearly reduced (P = .009), whereas GR showed a tendency of increase (P = .119) in GP rats as compared with VP rats. These results suggest that DNA methylation may be involved in GR transcriptional regulation.4 DNA Methylation of GR promoter exon 1₇Neither of the two CpG motifs within the NGFI-A binding site (CpG site 16 and 17) was methylated in the 36 subjects investigated. In contrast, all the remaining 15 CpG sites of the corresponding promoter exon 1₇ were hardly methylated other than some sporadic methylated sites. This implies that DNA methylation of promoter exon 1₇ may not underly the mechanism of GR up-regulation in GLP-1 -programmed rats.5 Hippocampal expression of GLP-1R and NGFI-A mRNAHippocampal GLP-1R (p = .009) and NGFI-A (p = .001) gene mRNA expression were significantly up-regulated in GP group as compared to VP group.Taken together, neonatal intramuscular injection of plasmid DNA encoding GLP-1 affects behavior and hippocampal GR expression in adolescent rats. Increased hippocampal NGFI-A mRNA expression induced by GLP-1 via GLP-1R signaling pathway, but not the methylation modification of GR promoter exon 1₇, may underlie the mechanism for the up-regulation of hippocampla GR expression and thus behavioral modifications.