Date on Master's Thesis/Doctoral Dissertation

8-2004

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Committee Chair

Epstein, Paul N.

Author's Keywords

Antioxidants; Islet transplantation; Diabetes; Pancreatic beta-cells

Subject

Pancreatic beta cells; Islands of Langerhans

Abstract

Pancreatic beta cells are extremely vulnerable to destruction by Reactive Oxygen Species (ROS). In type 1 diabetes and islet transplantation ROS are thought to be involved in the loss of beta cells. To test the role of antioxidant in islet transplantation. In our lab we have determined that transgenic overexpression of the antioxidant protein metallothionein (MT) in pancreatic beta cells provides broad resistance to oxidative stress by scavenging most kinds of ROS. A direct test of hypoxia/reperfusion sensitivity was shown that MT markedly reduced ROS production and improved islet cell survival. Furthermore, in both syngeneic transplantation and allotransplantation, MT islets preserved high insulin content and extended the duration of euglycemia two-fold longer than nontransgenic islets. The time course of protection suggested that the major mode of MT action may have been protection from hypoxia or hypoxia/reperfusion. To test the role of antioxidants in type 1 diabetes, three lines of antioxidant transgenic NOD mice were produced with beta-cell specific overexpression of MT, catalase (Cat) or MnSOD. Unexpectedly, the two cytosolic antioxidants, MT and Cat, but not mitochondrial MnSOD, dramatically hastened both spontaneous onset diabetes and cyclophosphamide (CYP) induced diabetes in NOD mice. MT and Cat transgenic beta-cells died by apoptosis more rapidly than control beta-cells. These data indicate that cytoplasmic ROS may have some protective role in beta-cells against type 1 diabetes, which is a role that has been recognized in some other cell types. To elucidate this protective mechanism, we assessed the status of the P13K/Akt/Foxo-1/PDX-1 pathway, one of the most important survival pathways in the beta-cells. Western blots of islets from transgenic and control NOD mice showed that both "in vivo" after CYP injection and "in vitro" after cytokine treatment phosphorylation of Akt and Foxo-1, and PDX-1 expression were significantly reduced in transgenic islets. "In vitro" MT sensitized NOD islets to cytokine induced cell death even though MT efficiently scavenged cytokine induced ROS production. Orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor rescued the sensitizing effect of MT to cytokine toxicity. Our data imply that elevated cytosolic antioxidants may result in higher PTP activity in beta-cells by protecting PTPs from ROS, thereby cause decreased beta-cell survival and accelerating type 1 diabetes in NOD mice. The data from this project demonstrated that overexpression of antioxidants protects islets from ROS damage produced during early phase islet transplantation but sensitizes beta-cells to diabetes in NOD mice.

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