The pancreatic cell can respond in the long term to hyperglycemia both with an increased capacity for insulin production and, in susceptible individuals, with apoptosis. 30 min after the treatment of STZ). The mice in the second group (= 23) were injected i.p. with STZ (50 mg?kg?1) followed in 30 min by glucose (8.76C10.0 mg?g?1). The mice in the third group (= 25) were injected i.p. with STZ (50 mg/kg) followed in 30 min by glucosamine (4.98 mg?g?1). Additionally, 50 mice were divided into five groups and injected i.p. with STZ at doses of 55, 60, 65, 75, and 80 mg?kg?1. The blood glucose was monitored frequently before sacrifice 26 hr later. Hybridization. The paraffin and frozen sections of mouse pancreas were prepared and hybridized as described (21). The antisense insulin 35S-cRNA riboprobe was synthesized from the 350-bp rat insulin I cDNA in pBlueScript KS (Stratagene), using T3 polymerase. The sense 35S-cRNA riboprobe, to detect the transgenic antisense mouse glutamine:fructose-6-phosphate amidotransferase (GFAT) mRNA, was synthesized from the 2 2.1-kb mouse GFAT cDNA in pT7T3 (Amersham Pharmacia), using T7 RNA polymerase. After hybridization, the slides were exposed to x-ray film for 3C10 days. The slides also were dipped in photographic emulsion, exposed, developed, and counterstained with hematoxylin and eosin, then subjected to microscopic examination. Terminal Deoxynucleotide Transferase-Mediated dUTP Nicked-End Labeling (TUNEL) Assay. The TUNEL assay was performed as described (22) in paraffin-embedded sections of the pancreases by using an cell death detection kit (Boehringer Mannheim). The endogenous perioxidase activity was blocked by immersing the sections in 0.3% H2O2 in methanol for 30 min before cell permeablization. Nonspecific binding of the peroxidase-coupled antifluorescein antibody was blocked with PBS containing 3% BSA for 20 min. Positive cells were visualized by using peroxidase substrate enhancer and 3,3-diaminobenzidine tetrahydrochloride substrate (Boehringer Mannheim), and sections were counterstained with hematoxylin. For a pancreas to be scored as apoptotic, all islets had to display TUNEL positivity. Generation of Transgenic Mice with Cell-Specific Expression of Mouse GFAT Antisense Gene. The 2 2.2-kb mouse GFAT cDNA (23), consisting of 150 bp 5 untranslated region and complete coding sequence, was inserted in the antisense direction between TP-434 cost the TP-434 cost rat insulin II promoter (RIP) (24) as well as the simian virus 40 (SV40) little T-antigen intron and polyadenylation sequences. The complete 4.4-kb fragment, containing the RIP-mGFAT (antisense)-SV40 construct, was excised through the cloning vector, purified, and microinjected into fertilized eggs from SJLXB6 mice. From the 29 living births, seven creator mice had been determined by PCR evaluation of tail suggestion DNA using oligonucleotide primers hybridizing towards the RIP and mGFAT series. The ensuing 1.28-kb PCR product spans the RIP-mGFAT junction. Gene dosage was dependant on slot-blot analysis, as well as the transgenic mouse range, termed 3C4, with the best gene dosage was used in most of research. For uniformity, 3- to 4-month-old man mice out of this range TP-434 cost had been used for the next studies, although identical results had been obtained using the additional lines, females, and pets over 5 weeks old. hybridization having a sense-oriented mouse GFAT [35S]cRNA probe was performed to look for the cell-specific expression from the antisense transgene in islets. Immunohistochemical staining TP-434 cost with RL2 mAbs was performed as before in the transgenic mice and their wild-type littermates 1.5 hr when i.p. injection with STZ (50 mg?kg?1) and glucose (10.0 mg?g?1, 30 min after TP-434 cost the treatment of STZ). Treatment of RIP-mGFAT (AS) Mice with Multiple Low Doses of STZ. Multiple low doses of STZ treatment (5 Rabbit polyclonal to AK3L1 consecutive days, 40 mg?kg?1) was performed as described (25) in 14 transgenic mice and 16 littermates (from three different lines). The diabetes was assessed by blood glucose measurements every 3 days and histological analysis 14 and 28 days after the last injection of STZ. Treatment of RIP-mGFAT (AS) Mice with STZ in Combination with Glucose or Glucosamine. The trangenic mice and their littermates were injected with either STZ (50 mg?kg?1) and glucose (8.76C10 mg?g?1, = 41, 21 transgenic mice and 20 wild-type littermates), or.