Global transcriptomic analysis of the arcuate nucleus following chronic glucocorticoid treatment

Jonathan R. Wray, Alison Davies, Charlotte Sefton, Tiffany-Jayne Allen, Antony Adamson, Philip Chapman, Brian Y.H. Lam, Giles S.H. Yeo, Anthony P. Coll, Erika Harno, Anne White

Glucocorticoids (GCs) are extensively prescribed medicines due to their potent immunosuppressive and anti-inflammatory properties. Prolonged high-dose GC use is associated with a range of metabolic side effects, including hyperphagia, obesity, and abnormal glucose homeostasis. A potential region involved in driving these GC-induced side effects is the hypothalamic arcuate nucleus. Wray et al. used a global transcriptomic approach to investigate the effects of GCs in the hypothalamus. This revealed a variety of genes involved in energy regulation. The range of both known and novel candidate genes indicates that multiple mechanisms may contribute to the metabolic abnormalities observed with GC treatment.

Objective: Glucocorticoids (GCs) are widely prescribed medications that are well recognized to cause adverse metabolic effects including hyperphagia, obesity, and hyperglycemia. These effects have been recapitulated in a murine model of GC excess, and we hypothesize that they are mediated, in part, through central mechanisms. This study aimed to identify genes in the hypothalamic arcuate nucleus (ARC) that are altered with GC treatment and evaluate their contribution to GC-induced metabolic abnormalities.

Methods: Corticosterone (Cort; 75 μg/ml) was administered in the drinking water to male C57Bl/6J mice for 2 days or 4 weeks. Phenotypic analysis of each group was undertaken and central and peripheral tissues were collected for biochemical and mRNA analyses. Arcuate nuclei were isolated by laser capture microdissection and tissue analyzed by RNA-seq.

Results: RNA-seq analysis of ARC tissue from 4 week Cort treated mice revealed 21 upregulated and 22 downregulated genes at a time when mice had increased food intake, expansion of adipose tissue mass, and insulin resistance. In comparison, after 2 days Cort treatment, when the main phenotypic change was increased food intake, RNA-seq identified 30 upregulated and 16 downregulated genes. Within the genes altered at 2 days were a range of novel genes but also those known to be regulated by GCs, including Fkbp5, Mt2, Fam107a, as well as some involved in the control of energy balance, such as Agrp, Sepp1, Dio2, and Nmb. Of the candidate genes identified by RNA-seq, type-II iodothyronine deiodinase (Dio2) was chosen for further investigation as it was increased (2-fold) with Cort, and has been implicated in the control of energy balance via the modulation of hypothalamic thyroid hormone availability. Targeted knockdown of Dio2 in the MBH using AAV-mediated CRISPR-Cas9 produced a mild attenuation in GC-induced brown adipose tissue weight gain, as well as a 56% reduction in the GC-induced increase in Agrp. However, this conferred no protection from GC-induced hyperphagia, obesity, or hyperglycemia.

Conclusions: This study identified a comprehensive set of genes altered by GCs in the ARC and enabled the selection of key candidate genes. Targeted knockdown of hypothalamic Dio2 revealed that it did not mediate the chronic GC effects on hyperphagia and hyperglycemia.