Supplementary MaterialsSupplementary information 41598_2017_3873_MOESM1_ESM

Supplementary MaterialsSupplementary information 41598_2017_3873_MOESM1_ESM. initial YM90K hydrochloride phases of T2D, beta-cells can compensate for the increased insulin demand by expansion and/or by producing more insulin. However, as the disease progresses, the capacity of beta-cells to compensate for the increased insulin demand is reduced due to an increase in cellular stress and beta-cell loss2. Among the YM90K hydrochloride factors that contribute to beta-cell dysfunction in diabetes, oxidative stress is thought to play an important role3. Notably, beta-cells express very low levels of antioxidant enzymes, such as catalase and superoxide-dismutase, which make them susceptible to oxidative damage4, 5. Indeed, oxidative stress is frequently observed in islets of both human diabetes patients and mouse models of insulin resistance6C8. Additionally, treatment of beta-cells with hydrogen peroxide (H2O2) leads to cytoplasmic translocation of essential transcription factors for beta-cell maturation and function, YM90K hydrochloride including MafA. A similar pattern of loss of transcription factors involved in maturation occurs in islets from T2D donors4. In line with these findings, antioxidant treatments and the overexpression of antioxidant YM90K hydrochloride enzymes in diabetic mouse models can delay diabetes development and/or enhances beta-cell function9C11. In contrast to the damaging effect of oxidative stress in the context of insulin resistance, H2O2 plays a role in beta-cell function under normal physiological conditions. For instance, glucose-stimulated insulin secretion is impaired by the removal of H2O2 12, 13. Additionally, treatment of pregnant mice with the antioxidant N-Acetyl cysteine (NAC) results in defects in normal beta-cell development in the offspring14, 15. Thus, one can hypothesize that the effect of ROS on beta-cells is context dependent. Whereas in diabetes, chronic oxidative stress could contribute to beta-cell loss and dysfunction, under physiological conditions, moderate levels of ROS maintain beta-cell function. However, whether ROS are important for additional aspects of beta-cell biology, for instance their ability to undergo expansion remains to be addressed. In the present study we used zebrafish as an model to investigate the role of H2O2 in the control of beta-cell proliferation. By applying genetic and pharmacological manipulations of H2O2 levels, we show that H2O2 is required for beta-cell proliferation in response to nutrients. To support our findings, we also used cultured rat beta-cells as an model. Using a genetically-encoded reporter of H2O2, we show that glucose stimulates a rapid H2O2-increase in beta-cells, we performed whole mount hybridization for and at 5 dpf. While transcripts are not detected in the beta-cells of WT larvae, hybridization (WISH) for in Rabbit polyclonal to PMVK WT and expression was not detected in the beta-cells of WT larvae whereas transcripts. (B) Double WISH for (brown) and (purple) showing overlap between the and transcripts in and might seem un-physiological if directly applied to cells, however, the YM90K hydrochloride actual concentration that reaches the internal organs of the zebrafish larvae is likely much lower20, 21. To directly test if lower levels of H2O2 levels can promote proliferation when applied directly to mammalian beta-cells, we treated INS-1 cells for five days with low, nontoxic levels of exogenously added H2O2 or with the reducing agent N-acetyl-cysteine (NAC) as a negative control. Because H2O2 is metabolized within an hour in cell culture medium22, we supplied H2O2 every 24?hours, while NAC was only added in the beginning of the experiment. Treatment of INS-1 cells with 0.25?M H2O2 produced a significant increase in cell numbers compared to controls (Fig.?4E). Only addition of low levels of H2O2, ranging from 0.01 to 1 1?M, promoted cell proliferation in a dose-dependent manner, whereas higher levels of H2O2 (10?M) did not show a stimulatory effect. In line with the positive effect of H2O2 on proliferation, reducing ROS levels with 2?mM NAC decreased proliferation (Fig.?4E). These results show a conserved role of H2O2 in stimulating beta-cell proliferation with much lower concentrations eliciting a proliferative response as compared to promoter drives simultaneous expression of H2B GFP and dsRED. Due to the significantly faster maturation of GFP compared to dsRED, those beta-cells that exhibit GFP but no dsRED expression are newly-formed while older beta-cells co-express both GFP and dsRED16. Thereby, we incubated larvae with.