Proceedings of The Physiological Society

University of Manchester (2010) Proc Physiol Soc 19, C144

Oral Communications

The effects of chronic ACTH treatment on adrenal cell hypertrophy, proliferation and apoptosis in mice

R. Menzies1, D. R. Dunbar1, L. J. Mullins1, J. J. Mullins1, M. A. Bailey1, C. J. Kenyon1

1. Centre for Cardiovascular Science, The University of Edinburgh, Edinburgh, United Kingdom.

Conditions of ACTH excess cause increased glucocorticoid production and adrenal hypertrophy. Infusion of ACTH into mice causes a three-fold increase in adrenal mass and a five-fold increase in plasma corticosterone (Dunbar et al, 2010). We have used a combination of microarray, histology and immunohistochemistry to identify gene pathways and cell types involved in ACTH-induced adrenal hypertrophy. Under isofluorane anaesthesia (2% in oxygen), osmotic minipumps containing either ACTH (2.5μg/d, n=5) or saline (n=5) were implanted into male C57BL/6J mice. After 2 weeks, mice were killed by decapitation and the adrenal glands weighed and either fixed in formalin for immunohistochemistry and morphometric analysis or processed for microarray analysis of gene expression using Affymetrix Mouse Genome 430 2.0 GeneChips (Dunbar et al, 2010). Data are mean±SE and comparisons were made by ANOVA. Adrenal mass was higher in ACTH-treated mice than controls (0.28±0.03 vs 0.11±0.01 mg/g body weight; P<0.01). After normalisation and correction for multiple testing, the expression of 1217 genes was found to be affected (P <0.001; ≥ 2-fold) by ACTH treatment: 513 were upregulated and 704 were downregulated. Several upregulated genes were involved in cell division, exemplified by increased expression of Ki67 (Entrez Gene ID 17345). Immunohistochemistry located Ki67-positive cells predominantly at the zona glomerulosa/fasciculata interface. The number of Ki67-positive cells was increased 3-fold (P < 0.01) by ACTH treatment. Several genes known to be activated in hypertrophied cells were differentially expressed. These included Cdkn1a/b/c (cyclin dependent kinase inhibitors, Entrez Gene IDs 12575, 12576 and 12577), associated with growth arrest, and tRNA synthetase genes, required for amino acid incorporation into proteins. ACTH treatment caused a two fold increase in zona fasciculata cell area (314±16 vs 153±8µm2, P < 0.001). This region of hypertrophy is distinct from the that of cell proliferation and is consistent with enhanced glucocorticoid synthesis. Several genes associated with apoptosis were decreased by ACTH treatment. The effect of ACTH on two of these genes was confirmed by quantitative PCR: clusterin (Entrez Gene ID 12759) and caspase 12 (Entrez Gene ID 12364) were decrease by 2.6 and 1.8 fold respectively, P<0.05). In sections from vehicle treated mice, Tunel staining located apoptosing cells predominantly at the inner margin of the zona reticularis. In ACTH-treated mice, apoptotic cells were fewer in number (P<0.05) and more widely distributed. In conclusion, ACTH increases adrenal mass by causing cell hypertrophy and cell hyperplasia and reducing apoptosis. These are distinct processes linked to different cell types within the adrenal cortex and are regulated by separate gene changes.

Where applicable, experiments conform with Society ethical requirements