A novel dipeptidyl peptidase IV inhibitor DA-1229 ameliorates streptozotocin-induced diabetes by increasing β-cell replication and neogenesis  Jae Min Cho, Hye Won Jang, Hwanju Cheon, Yeon Taek Jeong, Do-Hoon Kim, Yu-Mi Lim, Song-hyen Choi, Eun-kyoung Yang, Chang- Yell Shin, Moon Ho Son, Soon Hoe Kim, Heung-Jae Kim, Myung-Shik Lee  Diabetes Research and Clinical Practice  Volume 91, Issue 1, Pages 72-79 (January 2011) DOI: 10.1016/j.diabres.2010.10.012 Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 The effects of DA-1229 on glycemic profile in diabetic mice. (A) Male C57BL/6 mice rendered diabetic after administration of 100mg/kg STZ were fed chow containing 0.3% DA-1229 (STZ-DA) or control chow (STZ-NC), and non-fasting glucose levels were monitored weekly. Control C57BL/6 mice were also fed 0.3% DA-1229 (CON-DA) or control chow (CON-NC). (B) After feeding with chow containing 0.3% DA-1229 or control chow for 10 weeks, IPGTT was done as described in Section 2 (*P<0.05; **P<0.01; ***P<0.001, relative to STZ-NC mice). Diabetes Research and Clinical Practice 2011 91, 72-79DOI: (10.1016/j.diabres.2010.10.012) Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 The effects of DA-1229 on DPP IV activity, GLP-1 and insulin level. (A) Ten weeks after feeding 0.3% DA-1229, fasting plasma DPP IV enzyme activity was measured as described in Section 2. (B) In the same mice, fasting GLP-1 level was determined by ELISA. (C) Ten weeks after feeding 0.3% DA-1229 or control chow, serum was obtained before (0) and 15min after intraperitoneal glucose injection to overnight fasted mice for measurement of insulin level by ELISA (*P<0.05; **P<0.01; ***P<0.001). Diabetes Research and Clinical Practice 2011 91, 72-79DOI: (10.1016/j.diabres.2010.10.012) Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 The effects of DA-1229 on β-cell mass. (A) After 13 weeks of feeding 0.3% DA-1229, pancreatic sections were obtained for insulin IHC using a specific antibody (100×). (B) After insulin IHC, the mean volume density of β-cells (Vvβ) was assessed by point counting method as described in Section 2 (**P<0.01). Diabetes Research and Clinical Practice 2011 91, 72-79DOI: (10.1016/j.diabres.2010.10.012) Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 The effects of DA-1229 on β-cells replication and neogenesis. (A) Sixteen hours after intraperitoneal injection of 100mg/kg BrdU, double IHC for insulin and BrdU was done to evaluate BrdU incorporation into β-cells as a measure of β-cell replication. (B) Arrow heads indicate representative BrdU-incorporating β-cells in STZ-DA mice (left, 100×; right, 200×). (C) The number of small β-cell unit comprising three insulin+ cells at a maximum was determined after insulin IHC as a measure of β-cell neogenesis. (D) Arrows indicate representative small β-cell units in STZ-DA mice (100×). (E) Insulin IHC was conducted using pancreatic sections from STZ-DA and STZ-NC mice, and ductal area was examined to search for possible presence of ductal cells expressing insulin (black arrow, ductal cells expressing insulin; white arrow, islet). Insulin+ cells were not found in ductal cells of STZ-NC mice (data not shown) (200×) (*P<0.05; **P<0.01). Diabetes Research and Clinical Practice 2011 91, 72-79DOI: (10.1016/j.diabres.2010.10.012) Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Expression of PDX-1 after administration of DPP IV inhibitor. (A) Double immunofluorescent staining for PDX-1 and insulin was done to study the expression of PDX-1, a downstream mediator of GLP-1, in islet cells after administration of DA-1229 to mice rendered diabetic by STZ treatment by confocal microscopy (400×). (B) Relative fluorescence intensity of PDX-1 staining was measured and normalized to islet area (n=2) (P<0.05). (C) After double immunofluorescent staining for PDX-1 and insulin, ductal area and small β-cell units representing β-cell neogenesis were examined to study possible expression of PDX-1 in them (400×) (P<0.05). Diabetes Research and Clinical Practice 2011 91, 72-79DOI: (10.1016/j.diabres.2010.10.012) Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions