Distinct Shift in Beta-Cell Glutaredoxin 5 Expression Is Mediated by Hypoxia and Lipotoxicity Both In Vivo and In Vitro

Abstract

Histomorphological and functional alterations in pancreatic islet composition directly correlate with hyperglycemia severity. Progressive deterioration of metabolic control in subjects suffering from type 2 diabetes is predominantly caused by impaired beta-cell functionality. The glutaredoxin system is supposed to wield protective properties for beta-cells. Therefore, we sought to identify a correlation between the structural changes observed in diabetic pancreatic islets with altered glutaredoxin 5 expression, in order to determine an underlying mechanism of beta-cell impairment. Islets of db/db mice presenting with uncontrolled diabetes were assessed in terms of morphological structure and insulin, glucagon, and glutaredoxin 5 expression. MIN6 cell function and glutaredoxin 5 expression were analyzed after exposure to oleic acid and hypoxia. Islets of diabese mice were marked by typical remodeling and distinct reduction of, and shifts, in localization of glutaredoxin 5-positive cells. These islets featured decreased glutaredoxin 5 as well as insulin and glucagon content. In beta-cell culture, glutaredoxin 5 protein and mRNA expression were decreased by hypoxia and oleic acid but not by leptin treatment. Our study demonstrates that glutaredoxin 5 expression patterns are distinctively altered in islets of rodents presenting with uncontrolled diabesity. In vitro, reduction of islet-cell glutaredoxin 5 expression was mediated by hypoxia and oleic acid. Thus, glutaredoxin 5-deficiency in islets during diabetes may be caused by lipotoxicity and hypoxia.

Keywords: MIN6; db mouse; diabetes mellitus type 2; glutaredoxin; hypoxia; islet remodeling; lipotoxicity; rodent diabesity.

Figure 1

Blood glucose level and body weight of db/db and C57BL/6 mice. (A) Blood glucose level of db/db and C57BL/6 mice. (B) Body weight of db/db and C57BL/6 mice. Values are mean ± SEM (n = 12–15 mice), black bars represent db/db mice, white bars represent C57BL/6 mice, *** denotes p < 0.0001.

Figure 2

Morphology and quantification of db/db and C57BL/6 islet extent. (A–C) Representative images taken of db/db and C57BL/6 islets using light microscopy in comparison: (A) typical small db islet with lost demarcation to exocrine issue, (B) example of extensive db islets, and (C) generic C57 islet (fuchsine red: insulin, bars indicate 50 µm). (D) Quantification of mean islet area as measured with ImageJ corresponding to islet extent. Black bars represent db/db mice, white bars represent C57BL/6 mice, n = 6 mice, * denotes p < 0.05.

Figure 3

Islet composition and insulin/glucagon content of db/db and C57BL/6 islets. (A–F) Representative images taken of immunostained db/db and C57BL/6 islets stained for insulin and glucagon (green: insulin, red: glucagon, bars indicate 50 µm). (G,H) Quantification of relative islet insulin and absolute glucagon area. (I) Quantification of islet glucagon/insulin area ratio. (J,K) Quantification of insulin and glucagon fluorescent intensity. Black bars represent db/db mice, white bars represent C57BL/6 mice, n = 6 mice, ** denotes p < 0.005, * denotes p < 0.05.

Figure 4

Grx5 expression patterns in db/db and C57BL/6 mice and quantification of Grx5 area, fluorescent intensity, Grx5 to insulin ratio and nuclei staining. (A–C,E–G) Representative images taken of immunostained db/db and C57BL/6 islets stained for insulin and Grx5 (green: insulin, red: Grx5, bars indicate 50 µm). (D,H) Schematics of Grx5 staining in db/db and C57BL/6 islets (black: high intensity staining to white: no signal detected). (I) Quantification of islet Grx5 area normalized to islet extent. (J) Quantification of Grx5 fluorescent intensity. (K) Quantification of Grx5 to insulin ratio. (L) Quantification of nuclei stained for Grx5. Black bars represent db/db mice, white bars represent C57BL/6 mice, n = 6 mice, *** denotes p < 0.0001, ** denotes p < 0.005, * denotes p < 0.05.

Figure 5

Insulin and Grx5 protein expression and Grx5 mRNA expression in MIN6 cells upon exposure to recombinant leptin for 2 and 48 h. (A) MIN6 lysate insulin content. (B) MIN6 supernatant insulin content. (C) MIN6 lysate Grx5 content. (D) MIN6 Grx5 mRNA expression. Black bars represent 2 h, white bars represent 48 h. n = 9 runs, *** denotes p < 0.001.

Figure 6

Insulin and Grx5 protein and mRNA expression in MIN6 cells upon exposure to hypoxia (2% O₂) and oleic acid for 24 h. (A) MIN6 supernatant insulin content. (B) MIN6 INS2 mRNA expression. (C) MIN6 lysate Grx5 content. (D) MIN6 MTT assay after 24 h of normoxic oleic acid treatment. Black bars represent normoxic atmosphere, white bars hypoxic atmosphere. n = minimum of 3 runs. *** denotes p < 0.001, ** denotes p < 0.01, * denotes p < 0.05.