SRA

Cushing's syndrome is one of the most common endocrinopathies in adult dogs. It is a consequence of chronic hypercortisolemia (HC), that may present a pathological or iatrogenic origin. Among other alterations, Cushing's syndrome involves the differential modulation of lipid metabolism, i.e., while enhanced lipolysis is observed in subcutaneous adipose tissue, an increased accumulation is observed in visceral adipose tissue (VAT). VAT acts in a variety of mechanisms besides energy storage, playing an important role as an endocrine organ. Strikingly, the effects of HC in VAT were never investigated in the dog. Thus, in the present project the transcriptome of visceral adipose tissue (VAT) of dogs sampled before and after long-term HC was investigated. HC was induced by subcutaneously implanted ACTH-releasing pumps and canine VAT was collected before and after treatment, followed by deep RNA sequencing (RNA-Seq) analysis. A total of 1190 differentially expressed genes (DEG) were found in response to ACTH treatment (P and FDR < 0.01). Genes upregulated post-ACTH were mostly associated with translation; in contrast, terms enriched in downregulated DEGs were related to cell adhesion and migration, intracellular signaling, immune response, extracellular matrix and angiogenesis. Transcript counts of HSD11B2, enzyme responsible for the conversion of active cortisol into cortisone, and of the glucocorticoid receptor, were decreased after ACTH treatment, while HSD11B1, activator of cortisol, were increased. Furthermore, treatment further appeared to modulate insulin signaling, hormonal sensitivity, cell proliferation, extracellular matrix organization, lipolysis, vasculogenesis and immune signaling. These results allowed the identification of factors that might play an important role in HC-related expansion of VAT, e.g., TIMP4, FGF1, CCR2, CXCR4 and HSD11B1/2. Similarities in the effects of chronic hypercortisolemia on VAT of dogs and humans were further highlighted. Overall design: Hypercortisolemia (HC) was induced in five adult Beagle dogs by the permanent administration of tetracosactide, a synthetic ACTH hormone, which stimulates endogenous cortisol secretion, using osmotic pumps for 25 weeks. The pumps were implanted subcutaneously in the dorsolateral region of the neck and replaced every 4 weeks. Tetracosactide dosage started at 1.3-1.95 µg/kg/day, increasing to a final dosage of 6-10 µg/kg/day. After 25 weeks of treatment, HC was confirmed with a positive ACTH stimulation and a low-dose dexamethasone suppression test. Visceral adipose tissue was collected by laparotomy prior to treatment (Pre-ACTH samples) and after the 25 weeks of tetracosactide administration (Post-ACTH samples). RNA-seq was then performed with Next Generation Sequencing methodology and the effects of prolonged HC were evaluated by pairwise comparison between Pre-ACTH and Post-ACTH groups.