Differential expression of islet glutaredoxin 1 and 5 with high reactive oxygen species production in a mouse model of diabesity

Abstract

The onset and progression of diabetes mellitus type 2 is highly contingent on the amount of functional beta-cell mass. An underlying cause of beta-cell decay in diabetes is oxidative stress, which markedly affects the insulin producing pancreatic cells due to their poor antioxidant defence capacity. Consequently, disturbances of cellular redox signaling have been implicated to play a major role in beta-cell loss in diabetes mellitus type 2. There is evidence suggesting that the glutaredoxin (Grx) system exerts a protective role for pancreatic islets, but the exact mechanisms have not yet been elucidated. In this study, a mouse model for diabetes mellitus type 2 was used to gain further insight into the significance of Grx for the islets of Langerhans in the diabetic metabolism. We have observed distinct differences in the expression levels of Grx in pancreatic islets between obese, diabetic db mice and lean, non-diabetic controls. This finding is the first report about a decrease of Grx expression levels in pancreatic islets of diabetic mice which was accompanied by declining insulin secretion, increase of reactive oxygen species (ROS) production level, and cell cycle alterations. These data demonstrate the essential role of the Grx system for the beta-cell during metabolic stress which may provide a new target for diabetes mellitus type 2 treatment.

Fig 1. Body weight and fasting blood glucose level of db/db and db/+ mice.

(a) Body weight of db/db and db/+ mice. (b) Fasting blood glucose levels of db/db and db/+ mice. Data depicted from 6, 12, and 18 weeks of age, corresponding to pancreatectomy. Values are mean ± SEM (n = 11-40 mice), black bars represent db/db mice, white bars represent db/+ mice, *** denotes p < 0.0001.

Fig 2. Morphology and quantification of area, proliferation rate, and fluorescent intensity of db/db and db/+ islets.

(a, b, f, g, k, l) Representative images taken of immunostained islets at 6, 12 and 18 weeks are shown in comparison (green: insulin, blue: nuclei; bars indicate 100 μm²). (c, h, m) Quantification of mean islet area as measured with ImageJ corresponding to insulin staining area. (d, i, n) Proliferation of islet cells analyzed by Ki-67 positive cells per islet. (e, j, o) Semiquantitative analysis of insulin staining. Black bars represent db/db mice, white bars represent db/+ mice, n = 3 mice, *** denotes p < 0.001, ** denotes p < 0.005.

Fig 3. Gene expression of INS1, Grx1, and Grx5 in db/db and db/+ islets.

Gene expression was evaluated by qRT-PCR. (a) INS1 expression declined in both groups of mice in relation to their age, but controls exhibited significantly higher expression levels at all time points. (b) Grx1 expression was higher in db/+ mice at all time points. A slight decrease in controls was observed, while db/db animals featured a gap at 12 weeks of age. (c) Grx5 expression decreased in both groups with age with higher levels in db/+ islets. Values are mean ± SEM (n = 4—6 mice) and normalized with beta-actin, black bars represent db/db mice, white bars represent db/+ mice, *** denotes p < 0.0001, ** denotes p < 0.005.

Fig 4. Qualitative comparison of the Grx system in db/db and db/+ islets.

Representative monochrome pictures of Grx staining pattern captured of islets of 12 weeks old db/db and db/+ mice. (a, e) Grx1, (b, f) Grx2, (c, g) Grx3, (d, h) Grx5. Staining patterns suggested higher expression in db/+ mice. The difference was most pronounced for Grx1 and 5. 200x, yellow circles indicate islets, bars indicate 100 μm².

Fig 5. Representative images of Grx1 and 5 staining, quantification of Grx to insulin ratio, and fluorescent intensity of db/db and db/+ islets.

(a, b, e, f, i, j, m, n, q, r, u, v) Representative images taken of immunostained islets at 6, 12 and 18 weeks are shown in comparison (green: Grx1 / 5, red: insulin, bars indicate 50 μm²). (c, g, k, o, s, w) Semiquantitative analysis of Grx1 / 5 staining. (d, h, l, p, t, x) Quantification of Grx 1 / 5 to insulin staining ratio. Black bars represent db/db mice, white bars represent db/+ mice, n = 3 mice, *** denotes p < 0.0001, ** denotes p < 0.005, * denotes p < 0.05.

Fig 6. Representative images of ROS measurements and quantification in islets of db/db and db/+ mice.

(a, b, d, e, g, h) Representative images show DCF stained pancreatic islets without any treatment as well as upon treatment with either glucose or TNF-alpha (bars represent 75 μm). (c, f, i) Quantification of DCT fluorescence intensity revealed significantly higher ROS production in db/db islets with a more pronounced rise after exposure to high glucose and TNF-alpha treatment in comparison to db/+ islets. Values are mean ± SEM (n = 54—139 islets), black bars represent db/db islets, white bars represent db/+ islets, *** denotes p < 0.0001.

Fig 7. Summary.

Both gluco- and lipotoxicity are extracellular promoters of ROS generation. ROS are harmful to cellular elements as they catalyze their glutathionylation. When the cell’s antioxidant capacity is depleted, cell death occurs. Regarding the beta-cell, ROS impair insulin secretion. Grx1 and 5 wield protective properties. Grx1 is a major actor in de-glutathionylation, thereby reversing the harmful effects of ROS on its targets, exerting anti-apoptotic and pro-proliferative effects, and preserving insulin secretion. Grx5 has impact on the respiratory chain and cellular iron homeostasis by transferring iron-sulfur clusters to respective apoproteins. Hence, it supports cell viability and function, allows proliferation and counteracts iron accumulation which would promote ROS formation.