STZ-induced diabetes mouse model

Insulin-dependent diabetes mellitus was experimentally induced by administering the β-cell toxin Streptozotocin (STZ) to 8-week-old, male wild-type mice (C57BL/6J) according to the multiple low-dose injection protocol of the Diabetic Complications Consortium (50 mg/kg BW; i.p for 5 days versus vehicle (Na-Citrate Buffer)). One group received daily injections of polyethylene-glycosylated (PEGylated)-insulin (25 nmol/kg/day in 5 μl/g) to achieve normoglycemia. PEGylated insulin was synthesized by N-terminal amine reductive amination with 20K methoxy PEG propionaldehyde. In brief, human insulin was dissolved in 50 mM sodium acetate buffer (pH 5.0) and 50% acetonitrile. A 30-fold excess of sodium cyanoborohydride and a 1.5-fold excess of methoxy PEG propionaldehyde (M-ALD-20K, JenKem Technology USA, Plano, TX) was added to the insulin-containing buffer for 3 h at room temperature with stirring. Purification by reverse phase chromatography on a C-8 column in 0.1% TFA acetonitrile solvents yielded PEGylated insulin at greater than 95% purity.