NOTES
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CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article was reported.
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AUTHOR CONTRIBUTIONS
Conception or design: K.W.K., B.J.K.
Performing the experiments: Y.S.E., A.R.G., K.M.K., J.Y.Y., H.P.
Acquisition, analysis, or interpretation of data: Y.S.E., A.R.G., K.W.K., B.J.K.
Drafting the work or revising: Y.S.E., A.R.G., B.J.K.
Final approval of the manuscript: Y.S.E, B.J.K.
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FUNDING
This study was supported by a grant from the Korea Health21 R&D project, Ministry for Health, Welfare, and Family Affairs, Republic of Korea (No. HI14C2539) and grants from the Gachon University Gil Medical Center, Republic of Korea (No. RD 2013-03 and No. RD 2013-50).
Fig. 1Notch1 antisense construct (NAS-C) is expresssed in 4-week-old NAS mice. Mouse tail genotyping of NAS-C was performed using polymerase chain reaction (PCR) in 4-week-old male NAS and control mice.
Fig. 2Protein expression levels of Notch1 are reduced in 8-week-old male Notch1 antisense transgenic (NAS) mice compared to control mice. (A) Protein expression levels of Notch1, 2, 3, and 4 in the liver by Western blotting and (B) The relative ratio of protein expression level of Notch1, 2, 3, and 4 in two groups. Equal protein loading was confirmed by β-actin (control, n=1; NAS, n=2).
Fig. 3Notch1 inhibition worsens glucose profile and decreases insulin secretion without changing insulin resistance in 8-week-old male Notch1 antisense transgenic (NAS) mice. (A) Body weight and casual glucose level. (B) Glucose profile in intraperitoneal insulin tolerance test (IPGTT) (glucose 2 g/kg). (C) Area under the curve (AUC) of glucose during IPGTT. (D) Serum insulin level at 0, 15, and 30 minutes in IPGTT. (E) Glucose profile in IPITT. (F) AUC of glucose during IPITT. All data are represented as the mean±standard deviation. aP<0.01, bP<0.001 compared to the control mice (control, n=16; NAS, n=16).
Fig. 4Notch1 inhibition decreases islet mass and increases the numbers of small islets compared to large islets. (A) H&E staining and immunohistochemistry (IHC) of pancreatic section. (B) Islet diameter of 8-week-old male Notch1 antisense transgenic (NAS) and control mice. (C) Islet area of 8-week-old male NAS and control mice. (D) Islet number/4 mm2 of 8-week-old male NAS and control mice. (E) Islet number/4 mm2 according to the diameter (<250 µm vs. ≥250 µm). All data are represented as the mean±standard deviation. aP<0.05, bP<0.01, and cP<0.001 compared to the control mice (control, n=6; NAS, n=6).
Fig. 5Notch1 inhibition decreases β-cell mass and does not affect the α/β-cell ratio. (A) Immunohistochemistry (IHC) of islet. (B) Representative images of islets stained with immunofluorescence in 8-week-old male Notch1 antisense transgenic (NAS) and control mice pancreas section. (C) α/β-cell diameter in 8-week-old male NAS and control mice. (D) α/β-cell area of 8-week-old male NAS and control mice. (E) α/β-cell ratio in three groups divided by size (<0.003, 0.003 to 0.03, and >0.03 mm2). All data are represented as the mean±standard deviation. aP<0.05 compared to the control mice (control, n=6; NAS, n=6).
Fig. 6Notch1 inhibition decreases insulin secretion in islet of 8-week-old male Notch1 antisense transgenic (NAS) compared to control mice. (A) Insulin secretion induced by glucose stimulation from 3.3 to 16.7 mmol/L. (B) Insulin release from perifused pancreas of control and NAS mice. All data are represented as the mean±standard deviation. aP<0.001 compared to the control mice (control, n=4; NAS, n=4).
Fig. 7Neurogenin3 (Ngn3), neurogenic differentiation (NeuroD), and MAF bZIP transcription factor A (Maf-A) are increased in 8-week-old male Notch1 antisense transgenic (NAS) mice compared to control mice. Real-time polymerase chain reaction was performed on the transcription factors including Ngn3, NeuroD, Maf-A, pancreatic and duodenal homeobox 1 (Pdx1), and insulin 1 (Ins1). Total mRNA was isolated from mice islet. All data represented as mean±standard deviation. aP<0.05 compared with control mice (control, n=3; NAS, n=4).