Global transcriptomic analysis of the arcuate nucleus following chronic glucocorticoid treatment

Abstract

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.

Stress and the Brain: An Emerging Role for Selenium

“…Our knowledge of the ability of glucocorticoids to regulate the selenoproteins was recently expanded to the brain by Wray et al (2019). In this study, chronic corticosterone administration increased gene expression of SelenoP and Dio2, while decreasing expression of the selenium recycling enzyme selenocysteine lyase (Scly), in the arcuate nucleus (Arc) of the hypothalamus, a brain region with high GCR expression.…”

Selenocysteine β-Lyase: Biochemistry, Regulation and Physiological Role of the Selenocysteine Decomposition Enzyme

“…A study that performed RNA-sequencing analysis of the hypothalamic arcuate nuclei of mice chronically treated with corticosterone revealed a remarkable downregulation of the Scly gene in this region. Strikingly, in the first two days of glucocorticoid treatment, the mRNA for two selenoproteins, SelenoP, and iodothyronine deiodinase 2 ( Dio2 ), were upregulated [74]. The enzyme DIO2 a key regulator of thyroid hormone activation in cells, particularly in the hypothalamic-pituitary axis [75,76].…”

“…The enzyme DIO2 a key regulator of thyroid hormone activation in cells, particularly in the hypothalamic-pituitary axis [75,76]. Upregulation of the Dio2 gene in mice was maintained upon chronic treatment with corticosterone [74], implying that, expression or activity of this selenoprotein could be dependent on the absence of SCLY.…”

Selenium and hydrogen selenide: essential micronutrient and the fourth gasotransmitter?

“…This enzyme, first isolated in 1982 [15], catalyses decomposition of selenocysteine to hydrogen selenide and alanine, utilising pyridoxal 5'-phosphate as a cofactor [15]. SCL has been found in the human liver, kidney, heart and adrenal and muscle tissue in decreasing order of specific activity [17], and can be regulated by hypoxia, oxidative stress, pro-inflammatory cytokines and glucocorticoids [18][19][20]. Intracellularly, the dietary oxidation product selenate is first reduced to selenite [21] and then to hydrogen selenide, either by thioredoxin reductase [22] or through a series of redox reactions coupled to reduced glutathione (GSH) [23].…”