Defining the ferroptotic phenotype of beta cells in type 1 diabetes and its inhibition as a potential antidiabetic strategy

Abstract

Introduction: Recently, the involvement of ferroptotic cell death in the reduction of β-cell mass in diabetes has been demonstrated. To elucidate the mechanisms of β-cell ferroptosis and potential antidiabetic effects of the ferroptosis inhibitor ferrostatin-1 (Fer-1) in vivo, a mouse model of type 1 diabetes (T1D) was used.

Methods: Animals were divided into three groups: control (vehicle-treated), diabetic (streptozotocin-treated, 40 mg/kg, from days 1-5), and diabetic treated with Fer-1 (1 mg/kg, from days 1-21). On day 22, glycemia and insulinemia were measured and pancreases were isolated for microscopic analyses.

Results: Diabetes disturbed general parameters of β-cell mass (islet size, β-cell abundance and distribution) and health (insulin and PDX-1 expression), increased lipid peroxidation in islet cells, and phagocytic removal of iron-containing material. It also downregulated the main players of the antiferroptotic pathway - Nrf2, GPX4, and xCT. In contrast, Fer-1 ameliorated the signs of deterioration of β-cell/islets, decreased lipid peroxidation, and reduced phagocytic activity, while upregulated expression of Nrf2 (and its nuclear translocation), GPX4, and xCT in β-cell/islets.

Discussion: Overall, our study confirms ferroptosis as an important mode of β-cell death in T1D and suggests antiferroptotic agents as a promising strategy for the prevention and treatment of diabetes.

Keywords: diabetes; ferroptosis; ferroptosis inhibitor; ferrostatin-1; β-cell death.

Figure 1

(A) Serum glucose levels and (B) serum insulin levels at day 22 of experiment; (C) islets surface area; (D) representative micrographs of islets, AZAN trichrome staining; (E) comparative immunohistochemical detection of insulin- and glucagon-positive cells at serial pancreatic sections; (F) immunohistochemical detection of PDX-1; (G) fibrosis score; (H) ratio of insulin-positive β (INS⁺) and (I) glucagon-positive α cells (GLK⁺); (J) average PDX-1 immunopositivity in islets of Langerhans from control (Ctrl), diabetic (DM), and Fer-1-treated diabetic animals (DM+Fer-1). Magnification and scale bar (D–F): ×40, 50 μm. Statistical significance (A–C, G–J): *p < 0.05, **p < 0.01, ***p < 0.001 – in comparison to Ctrl; ^#p < 0.05, ^##p < 0.01 – in comparison to DM.

Figure 2

Detection of (A) DNA fragmentation (green, TUNEL staining) combined with PI staining of nuclei (red) in islets of Langerhans (encircled), insert – a detail of a lymph node adjacent to pancreas; (B) quantification of DNA fragmentation inside islet nuclei (TUNEL fluorescence); (C, D) immunohistochemical detection and quantification of 4-HNE in pancreatic islets from control (Ctrl), diabetic (DM), and Fer-1-treated diabetic animals (DM +Fer-1); (E) Sudan III staining – lipofuscin detection inside islet cells (white arrows); (F) Pearl’s staining – phagocytic iron (Fe³⁺)-loaded cells in exocrine pancreas of diabetic animals (black arrows and insert) –iron-loaded cells;. Magnification and scale bar: (A) ×63, 25 μm; (D, F) ×40, 50 μm; (E) ×100, 20 μm.

Figure 3

Immunohistochemical detection and quantification of islet immunopositivity to (A, F) Nrf2 (insets on (A) – DAB channel of the original images, asterisk showing Nrf2 nuclear immunopositivity) (B, G) GPX4, (D, I) HO-1 and (E, J) PRDX-2. Immunofluorescence colocalization of (C) xCT (green) and insulin (red) in islets of Langerhans from control (Ctrl), diabetic (DM), and Fer-1-treated diabetic animals (DM+Fer-1) and (H) xCT immunopositivity of β-cells. Magnification and scale bar: (A, B, D, E) ×40, 50 μm; (C) ×63, 25 μm. Statistical significance (F–J): *p < 0.05, **p < 0.01, ***p < 0.001 – in comparison to Ctrl; ^##p < 0.01, ^###p < 0.001 – in comparison to DM.

Figure 4

Graphical summary of the results on the contribution of β-cell ferroptosis to the development and pathogenesis of T1D via modulation of Nrf2 and its downstream targets and its inhibition by ferrostatin-1 (Fer-1).