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

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

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

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

Gene expression of <i>INS1</i>, <i>Grx1</i>, and <i>Grx5</i> in db/db and db/+ islets.

<p>Gene expression was evaluated by qRT-PCR. (a) <i>INS1</i> expression declined in both groups of mice in relation to their age, but controls exhibited significantly higher expression levels at all time points. (b) <i>Grx1</i> 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) <i>Grx5</i> 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 <i>p</i> < 0.0001, ** denotes <i>p</i> < 0.005.</p

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

<p>(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 <i>μ</i>m). (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 <i>p</i> < 0.0001.</p

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

<p>(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 <i>p</i> < 0.0001.</p

Summary.

<p>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.</p

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

<p>(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 <i>μ</i>m²). (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 <i>p</i> < 0.0001, ** denotes <i>p</i> < 0.005, * denotes <i>p</i> < 0.05.</p

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

<p>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 <i>μ</i>m².</p

Paracrine regulation and improvement of β-cell function by thioredoxin

The failure of insulin-producingβ-cells is the underlying cause of hyperglycemia in diabetes mellitus.β-cell decay has been linked to hypoxia, chronic inflammation,and oxidative stress. Thioredoxin (Trx) proteins are major actors in redox signaling and essential for signal transduction and the cellular stress response. We haveanalyzed the cytosolic, mitochondrial, and extracellular Trx system proteins in hypoxic and cytokine-induced stress usingβ-cell culture, isolated pancreatic islets, andpancreatic islet transplantation modelling low oxygen supply.Protein levels of cytosolic Trx1 and Trx reductase (TrxR) 1 significantly decreased, while mitochondrial Trx2 and TrxR2 increased upon hypoxia and reox-ygenation. Interestingly, Trx1 was secreted byβ-cells during hypoxia. Moreover, murine and human pancreatic islet grafts released Trx1 upon glucose stimulation.Survival of transplanted islets was substantially impaired by the TrxR inhibitor auranofin.Since a release was prominent upon hypoxia, putative paracrine effects of Trx1 onβ-cells were examined. In fact, exogenously added recombinant hTrx1 mitigatedapoptosis and preserved glucose sensitivity in pancreatic islets subjected to hypoxia and inflammatory stimuli, dependent on its redox activity. Human subjects werestudied, demonstrating a transient increase in extracellular Trx1 in serum after glucose challenge. This increase correlated with better pancreatic islet function.Moreover, hTrx1 inhibited the migration of primary murine macrophages.In conclusion, our study offers evidence for paracrine functions of extracellular Trx1 that improve the survival and function of pancreaticβ-cells