Abstract
DNA damage and DNA damage response (DDR) pathways in β-cells have received little attention especially in the context of type-2 diabetes. We postulate that p21 plays a key role in DDR by preventing apoptosis, associated through its overexpression triggered by DNA stand breaks (DSBs). Our results show that β-cells from chronic diabetic mice had a greater extent of DSBs as compared to their non-diabetic counterparts. Comet assays and nuclear presence of γH2AX and 53bp1 revealed increased DNA DSBs in 16 weeks old (wo) db/db β-cells as compared to age matched non-diabetic β-cells. Our study of gene expression changes in MIN6 cell line with doxorubicin (Dox) induced DNA damage, showed that the DDR was similar to primary β-cells from diabetic mice. There was significant overexpression of DDR genes, gadd45a and p21 after a 24-hr treatment. Western blot analysis revealed increased cleaved caspase3 over time, suggesting higher frequency of apoptosis due to Dox-induced DNA strand breaks. Inhibition of p21 by pharmacological inhibitor UC2288 under DNA damage conditions (both in Dox-induced MIN6 cells and older db/db islets) significantly increased the incidence of β-cell apoptosis. Our studies confirmed that while DNA damage, specifically DSBs, induced p21 overexpression in β-cells and triggered the p53/p21 cellular response, p21 inhibition exacerbated the frequency of apoptosis.
Highlights
DNA damage and DNA damage response (DDR) pathways in β-cells have received little attention especially in the context of type-2 diabetes
We were able to show, that were the comet tails significantly longer in 16 wo db/db islets as compared to agematched control islets, but they were much longer than 10 wo db/db islets (Fig. 1a). 10 wo db/db islets had lesser DNA damage compared to age-matched control islets which is not surprising given that at 10 weeks, db/db islets are expanding and secreting more insulin to compensate for peripheral insulin resistance
The percentage of DNA in comet tails of 16 wo db/db islet cells was 86% suggesting that a majority of islet cells during this time are subjected to some form of DNA damage (Fig. 1b)
Summary
DNA damage and DNA damage response (DDR) pathways in β-cells have received little attention especially in the context of type-2 diabetes. Our studies confirmed that while DNA damage, DSBs, induced p21 overexpression in β-cells and triggered the p53/p21 cellular response, p21 inhibition exacerbated the frequency of apoptosis. Glucolipotoxicity due to high fat diet in mice led to cellular senescence in β-cells[18] Another factor recently implicated in increased DNA damage was congenital hyperinsulinism in patients. In these rare cases, glucokinase mutations were seen to cause DNA double strand breaks (DSBs) in β-cells leading to dysfunction and apoptosis[7]. We further probed DDR gene expression in primary β-cells and in MIN6 cells exposed to Dox, to establish the β-cell response to DSBs. Our results show that DSBs are higher in older diabetic (db/db) islet cells compared to those from younger diabetic (db/db) mice. The evidence presented here points to increased DSBs in older db/db mice and that p21 plays an essential role in DDR and β-cell survival in diabetic β-cells with DSBs
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