Poly(ADP-ribose) polymerase-deficient mice are protected from streptozotocin-induced diabetes

Abstract

Streptozotocin (STZ) selectively destroys insulin-producing beta islet cells of the pancreas providing a model of type I diabetes. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme whose overactivation by DNA strand breaks depletes its substrate NAD+ and then ATP, leading to cellular death from energy depletion. We demonstrate DNA damage and a major activation of PARP in pancreatic islets of STZ-treated mice. These mice display a 500% increase in blood glucose and major pancreatic islet damage. In mice with homozygous targeted deletion of PARP (PARP -/-), blood glucose and pancreatic islet structure are normal, indicating virtually total protection from STZ diabetes. Partial protection occurs in PARP +/- animals. Thus, PARP activation may participate in the pathophysiology of type I diabetes, for which PARP inhibitors might afford therapeutic benefit.

Figure 1

PARP −/− mice are protected from STZ-induced hyperglycemia. Within 1 week of i.p. injection of 200 mg/kg STZ, wild-type mice show markedly elevated blood glucose that peaks at 2 weeks and remains elevated at 6 weeks. PARP −/− mice do not become hyperglycemic after injection but rather show a blood glucose profile that parallels that of control wild-type mice injected with vehicle only. PARP +/− mice show no increase in blood glucose at 1 week, but at 2 weeks they become as hyperglycemic as treated wild-type mice and remain hyperglycemic 6 weeks after injection. Values are means ± SEM for three trials with at least 5 mice per group.

Figure 2

Western blot analysis of pancreatic proteins with anti-PARP antibody shows that PARP protein is absent from pancreas of PARP −/− mice and abundant in PARP +/+ pancreas. PARP +/− pancreas shows approximately half of the PARP +/+ level of PARP protein. The blot shown is typical of four independent replications.

Figure 3

Pancreatic islets are protected from STZ-induced B cell loss in PARP −/− mice. In wild-type and PARP +/− mice, pancreatic islets were small and distorted 6 weeks after a single i.p. injection of 200 mg/kg of STZ, as judged by staining with hematoxylin/eosin staining. Immunohistochemistry for insulin demonstrated that the majority of islet atrophy was caused by a marked loss of B cells. Glucagon immunoreactive cells appeared relatively spared but were no longer arranged peripherally in the islet. In contrast, islets from PARP −/− mice showed no evidence of atrophy (as judged by hematoxylin/eosin staining), B cell loss (as judged by insulin staining), or altered cellular arrangement (as judged by normal glucagon staining), and were histologically similar to vehicle treated PARP +/+ mice. The number and distribution of insulin and glucagon immunoreactive cells were also similar to those from islets from untreated controls. Results are representative of two trials with at least 5 mice per group.

Figure 4

STZ treatment damages DNA in PARP +/+, PARP +/−, and PARP −/− mice. PUNT+ labeling of DNA strand breaks in mouse pancreatic islet cells shows that i.p. injection of 200 mg/kg STZ elicits comparable DNA damage 5 days later in PARP +/+, PARP +/−, and PARP −/− mice. Baseline pancreatic islet cell DNA damage is higher in PARP +/− and PARP −/− mice than in PARP +/+ mice. These results are samples of at least five independent replications.

Figure 5

PARP activation in mouse pancreas after STZ treatment. Wild-type pancreatic PARP activity is increased 5 days after treatment with 200 mg/kg of STZ. PARP −/− mice, however, show negligible basal PARP activity both before and 5 days after STZ treatment. PARP +/− mice have intermediate levels of basal PARP activity and PARP activation after STZ treatment. Values are means ± SEM for 3 trials of at least 5 mice per group. ∗, PARP +/+ STZ, significantly different from basal PARP +/+ pancreatic PARP activity, P < 0.01 (Student’s t test). ∗ PARP +/− STZ, significantly different from STZ-treated PARP +/+ pancreatic PARP activity, P < 0.01 (Student’s t test). ∗, PARP −/− STZ, significantly different from STZ-treated PARP +/− pancreatic PARP activity, P < 0.01 (Student’s t test).

Figure 6

PARP activation after STZ treatment is localized to islets in PARP +/+ pancreas. Emulsion was used to visualize microscopically in situ PARP activation using the PARIS detection technique. Pancreatic sections in this figure were adjacent to those used in Figs. 4 and 5. All PARIS sections shown were verified for the presence of islets through additional hematoxylin/eosin staining of adjacent pancreatic sections (data not shown). PARP +/+ pancreas shows some degree of basal PARP activity diffusely spread throughout the pancreas, which is greater than that seen in PARP +/− mice. There is a virtual absence of basal PARP activity in PARP −/− mice. Five days after i.p. injection of 200 mg/kg STZ, PARP +/+ mice show dramatic activation of PARP localized specifically to islet cells. This PARP activation corresponds to the induction of DNA damage shown in Fig. 4. After STZ treatment of PARP +/− mice, however, there is much less distinct and robust PARP activation in islet cells than in PARP +/+ mice. PARP −/− pancreas shows no PARIS signal after STZ treatment. Incorporation of the PARP inhibitor benzamide into the PARIS assay in pancreatic sections from STZ-treated PARP +/+ and PARP +/− mice abolishes PARIS signal, as this assay is specific for in situ PARP activity. PARIS signal in STZ-treated PARP −/− mice is unchanged with incorporation of benzamide into the PARIS assay.