Endocrine Abstracts

Background: Sepsis is the primary cause of mortality in intensive care units. It is characterized by compromised function of several vital organs, including the adrenal glands. However, little is known about the mechanisms involved in this process. Recent experimental data demonstrated a potential role of iron overload and related iron-dependent, lipid peroxide driven form of regulated necrosis known as ferroptosis in sepsis-induced multiorgan damage. In a previous study, we demonstrated that human adrenocortical carcinoma cells NCI-H295R are sensitive to ferroptosis resulting from glutathione peroxidase 4 (GPX4) inhibition. However, whether sepsis also triggers ferroptosis in the adrenal gland remains unexplored until now.

Objectives: The main objective of this study was to investigate whether sepsis-associated conditions such as enhanced steroidogenesis and inflammation can induce ferroptosis in adrenocortical cells.

Methods: In order to mimic septic conditions in vitro, NCI-H295R cells were either exposed to forskolin, a well-known activator of steroidogenesis, or to inflammatory cytokine interleukin 6 (IL6). Necrosis induction (positive annexin V and PI staining) as well as lipid peroxidation (C11-Bodipy staining) were analyzed 24–48 h thereafter. Furthermore, an expression of ferroptosis-relevant molecules was subsequently analyzed on gene and protein level by qPCR and western blot respectively. Finally, the expression those regulatory molecules was additionally assessed in the adrenals of mice treated with LPS.

Results: Our study demonstrated robust expression of two major modulators of ferroptosis, GPX4 and long-chain-fatty-acid-CoA ligase 4 (ACSL4), in the human and mouse adrenal cortex. Stimulation of NCI-H295R cells with either forskolin or IL6, as well as administration of bacterial LPS to mice, enhanced ACSL4 expression in adrenocortical cells. Furthermore in vitroresults demonstrated that stimulation with forskolin and IL6 moderately increased lipid peroxidation and number of necrotic NCI-H295R cells, which indirectly suggested ferroptosis induction. However, while addition of the ferroptosis inhibitor ferrostatin-1 mitigated forskolin-mediated induction of lipid peroxidation, it rescued only a fraction of NCI-H295R adrenal cells from necrosis, suggesting potential involvement of a different form of cell death. Indeed, in vitro co-treatment of those cells with an apoptosis inhibitor fmk-zVad demonstrated a similar effect. In addition, we have found that IL-6 can decrease RSL3-mediated ferroptosis.

Conclusions: In summary, our findings suggest that septic conditions may promote ferroptosis and/or apoptosis in adrenocortical cells. However, further experiments, including the evaluation of GPX4 activity and caspase activation, as well as the validation of results using primary adrenal cells, are essential to gain a more thorough understanding of this process.