Hypothalamic mechanisms mediating glucocorticoid-induced metabolic effects

Abstract

Glucocorticoids (GCs) are widely prescribed for inflammatory and autoimmune conditions. However, patients often develop 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. Therefore, this thesis aimed to identify genes and pathways in the hypothalamic arcuate nucleus (ARC) that are altered with GC treatment, and evaluate their contribution to GC-induced metabolic abnormalities. To better characterize the metabolic phenotype prior to RNA-seq, corticosterone (Cort; 75μg/ml) was administered in the drinking water to male C57Bl/6J mice for 2 days or 4 weeks. Cort treatment produced an early and sustained increase in food intake, with a delayed increase in body weight and adipose tissue mass. Insulin was elevated at 2 days, which progressed to fasting hyperinsulinemia and insulin resistance by 4 weeks. Indirect calorimetry revealed a persistent increase in respiratory exchange ratio, but no change in energy expenditure with 2 weeks GC treatment. However, UCP1 in brown adipose tissue (BAT) was decreased at 4 weeks, suggesting a delayed decrease in energy expenditure. To identify genes and pathways altered in the ARC, RNA-seq was performed on isolated arcuate nuclei from 2 day and 4 week Cort treated mice. RNA-seq revealed a multitude of genes altered at both timepoints, however the wealth of secondary effects at 4 weeks made interpretation of this dataset difficult. Subsequent work focused on genes altered at 2 days that are predicted to contribute to GC-induced hyperphagia and obesity. RNA-seq identified 131 upregulated and 100 downregulated genes after 2 days of Cort treatment. Among the genes altered were known GC-regulated genes, including Cdkn1a, Fkbp5, Mt1, Mt2, as well as some involved in the control of energy balance, such as Agrp, Ghsr, and Nmb. Results were confirmed by qRT-PCR, with a strong correlation between techniques. Thus, RNA-seq has identified candidate genes for investigation. One gene of interest was type-II iodothyronine deiodinase (Dio2), which increases the local availability of triiodothyronine (T3), and in turn increases food intake. With 2 day Cort treatment Dio2 increased (2-fold) in the ARC, which was confirmed by in situ hybridization. To determine the contribution of Dio2 in the development of GC-induced hyperphagia and obesity, AAV-directed CRISPR-Cas9 injections were used to knock down Dio2 in the MBH. Lack of a specific anti-DIO2 antibody meant that it was not possible to quantify DIO2 knockdown. However, the efficiency of CRISPR guide RNAs was pre-validated in vitro and correct targeting of the MBH was determined by immunofluorescence, and by quantification of Cas9 mRNA in MBH micro-punches. Dio2 knockdown mice were then challenged with Cort for 4 weeks, and showed a mild attenuation in BAT weight gain, as well as a 50% reduction in the GC-induced increase in Agrp. However, Dio2 knockdown conferred no protection from GC-induced hyperphagia, obesity, or hyperglycemia. This study has provided further insight into the development of GC side effects, and has identified several hypothalamic genes that may contribute to these effects. Future studies will further investigate the hypothalamic mechanisms driving GC-induced metabolic side effects, thus informing the development of targeted therapies to prevent the GC-induced metabolic sequelae.

Date of Award	31 Dec 2018
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Anne White (Supervisor) & Erika Harno (Supervisor)