TIMP-1 Transgenic Mice Recover From Diabetes Induced By Multiple Low-dose Streptozotocin

Background and objective: Type 1 diabetes results from autoimmune destruction of the insulin-producingβcells of pancreatic islets, and the capacity for self-replication ofβcells in the adult is too limited to result in a significant regeneration following extensive tissue injury. Tissue inhibitor of metalloproteinase-1 (TIMP-1) is one representative of the natural matrix metalloproteinase (MMP) inhibitor family, which is known to possess a broad range of biological activities, including inhibition of metalloproteinase activity, regulation of proliferation, and apoptosis of a variety of cell types, and, depending on the context, differential regulation of angiogenic and inflammatory responses. Recent study suggested that TIMP-1 could prevent pancreaticβ-cell from cytokine-mediated apoptosis in vitro, and the facts that MMP was involved in high fat-induced diabetes in ZDF rats, and that deficiency of TIMP-3 was involved in the pathogenesis of diabetes and vascular inflammation of insulin receptor haploinsufficient mice, have been reported recently^9,10. These observations let us hypothesize and explore whether overexpression of TIMP-1 in pancreatic cells could counteract the cytotoxicity or even reverse the diabetes induced by multiple low-doses of streptozotocin (MLDS) in vivo.Methods: We firstly characterized the high expression of human TIMP-1 mRNA in pancreas of transgenic mice by Northern blot and RT-PCR, and then evaluated physiological influence of overexpression of human TIMP-1 on metabolic parameters of transgenic mice by glucosemeters, RIA, immunohistochemistry. Secondly, to induce diabetic hyperglycemia, streptozotocin was administered at 40mg/kg body weight to 6-week-old both TIMP-1 transgenic mice and nontransgenic mice for consecutive 5 days, with tail vein glucose level monitored for up to 40weeks. Furthermore, histological and immunohistochemical analysis were performed to investigate the effects of TIMP-1 on insulitis, apoptosis, proliferation of islets cell andβcell mass before and after MLDS treatment. Finally, NIT-1 cell were transfected with the hTIMP- 1-pcDNA3.1, and stable transfectants were selected to demonstrate the survival and mitogenic effect of TIMP-1 in an independent system; To explore the mechanism of the effect of TIMP-1, MMP inhibitor GM6001 and doxycycline, PI3K inhibitor LY294002 and MEK inhibitor U0126 were applied, western blot analysis of phosphorylation of Akt and ERK1/2 were carried out.Results: (1) High level of hTIMP-1 mRNA was dected in transgenic mice. There were no significant differences between transgenic mice and nontransgenic mice in metabolic parameters including body weight, fasting blood glucose, serum insulin level, percent ofαcell, intraperitoneal glucose tolerance test, insulin tolerance test. 2 weeks after MLDS administration, nontransgenic mice developed severe hyperglycemia, with polydipsia, polyphagia and body weight loss and hypoinsuliminia, while transgenic mice showed mild hyperglycemia (TG: 13.0±1.3 mmol/L vs. Con: 24.5±1.6mmol/L, P < 0.0001) and hypoinsulinemia. Blood glucose of transgenic mice slowly increased to 24.4±1.6 mmol/L 12 weeks after MLDS, but 75% of them returned to normal levels 40 weeks after MLDS, with restored normal insulin level and increase of body weight. (2)Historical analysis showed that about 40% of islets of nontransgenic mice presented variouse degrees of insulitis 2 weeks after MLDS, while infiltrated lymphocytes were hardly seen in islets of transgenic mice, indicating that overexpression of TIMP-1 in transgenic mice almost completely prevented insulitis induced by MLDS. TUNEL analysis suggested the percent of apoptotic islets cells in transgenic mice is less than that of nontransgenic mice both after MLDS and a single high-dose of streptozotocin. BrdU uptake assay showed that proliferatingβcells were significantly increased in transgenic mice compared with nontransgenic littermates. In consist with above finding,βcell mass of transgenic mice was greater than that of nontransgenic mice, and restored to normal level 40 weeks post MLDS, although both of them decreased significantly after MLDS administration. (3)Survival and mitogenic effects of TIMP-1 was demonstrated in insulinoma NIT-1 cells, and was neutralized by PI3K inhibitor and MEK inhibitor. Western blot analysis showed that overexpression of TIMP-1 stimulates the phosphorylation of Akt and ERK. While above finding was not seen in MMP inhibitor treated NIT-1 cell.Conclusions: The present study demonstated, for the first time, that overexpression of TIMP-1 can counteract the cytotoxicity and insulitis induced by MLDS, enhance the proliferation ofβcells in vivo, normalize the blood glucose, and prolong the survival in the transgenic mice, indicating that TIMP-1 may be a potential target for prevention and treatment of type 1 diabetes. Akt and ERK pathway may be involved in the beneficial effects of TIMP-1.