Ontogeny of Diurnal Variation of Adrenal Clock Genes and the Expression of Star and Mc2r and Its Relationship with Plasma Corticosterone Rhythm in Rats

Presentation Number: MON-0387
Date of Presentation: June 23rd, 2014

Silvia Ruiz Roa*1, Rogério Lenotti Zuliani2, Paula C. L. Elias2, Margaret De Castro2, Lucila Elias2, Jose Antunes-Rodrigues2 and Ayrton C. Moreira2
1Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil, 2Ribeirao Preto Medical School - University of Sao Paulo, Ribeirao Preto, Brazil

Abstract

Introduction: The circadian rhythm of hypothalamic-pituitary-adrenal (HPA) axis depends on clock genes expressed in the hypothalamic suprachiasmatic nucleus (SCN) and also in peripheral oscillators. The circadian rhythm of plasma corticosterone (B) levels is established at post natal day 16 (P16) in rats. However, the ontogeny of diurnal variation of adrenal clock genes and steroidogenesis related genes is still unknown. Objectives: The aim of this study was to compare the ontogeny of diurnal variation of adrenal clock genes and Star and Mc2r expression and the rhythm of B secretion. Material and Methods: We studied neonate male Wistar rats born to mothers kept under a 12h light; 12h dark cycle (lights on at 0700h, zeitgeber time-ZT0). Litters were decapitated every 4h over a 24h period on days P6, P16 and P24. Trunk blood was collected for B measurement by RIA. Right adrenals were stored until RNA extraction. The expression levels of the mRNA of Clock, Bmal1, Per1, Per2, Per3, Cry1, Cry2, Rev-Erbα, Rorα, Star and Mc2r were measured by RT-PCR and analyzed by Mann-Whitney test. The significance level was set at α=0.05. All procedures were approved by the Animal Bioethics Committee. Results: The ZT0- ZT12 mean plasma B levels (µg/dL) were 0.57±0.0- 0.57±0.0, (P=1.0); 1.24±0.71- 3.74±0.94, (P<0.01); 0.67±0.14- 10.2±3.42, (P<0.01) at P6, P16 and P24 respectively. Plasma B levels at ZT12 were higher than ZT0 from 16 days of age. There were no changes in the adrenal gene expression of Clock and Rorα between ZT0 and ZT12 in all groups. At ZT12 in P6, Bmal1 and Mc2r gene expression were higher (P<0.01) and Per2 and Per3 (P<0.01) were lower than ZT0. In P16 at ZT12 there was a lower expression of Mc2r (P<0.01). At ZT12 in P16 and P24, Per1, Per2, Per3, Cry1 and Star gene expression were higher than ZT0 (P<0.05). In contrast, Bmal1 had a lower expression at ZT12 in P16 and P24 (P<0.01). In P24 at ZT12 there was a higher expression of Cry2 and Rev-Erbα (P<0.01) than at ZT0. Conclusion: These results indicate that diurnal variation of Bmal1, Per1, Per2, Per3, Cry1, Cry2, Rev-Erbα, Star and Mc2r genes is detectable in the adrenal gland of neonatal rats. In addition, daily expression of Per1,2,3, Cry1 and Star in the adrenal is parallel to plasma B rhythm from P16 on. The mechanisms underlying the progressive maturation of the HPA axis and its predominant vespertine activity in rats involve besides a neurohumoral circuitry also an intrinsic adrenal molecular components.

 

Nothing to Disclose: SRR, RLZ, PCLE, MD, LE, JA, ACM