Diabetes Metab J.  2021 Jan;45(1):86-96. 10.4093/dmj.2019.0160.

Notch1 Has an Important Role in β-Cell Mass Determination and Development of Diabetes

• Eom YS
• Gwon AR
• Kwak KM
• Youn JY
• Park H
• Kim KW
• Kim BJ

• KMID: 2514077
• DOI: http://doi.org/10.4093/dmj.2019.0160

Abstract

Notch signaling pathway plays an important role in regulating pancreatic endocrine and exocrine cell fate during pancreas development. Notch signaling is also expressed in adult pancreas. There are few studies on the effect of Notch on adult pancreas. Here, we investigated the role of Notch in islet mass and glucose homeostasis in adult pancreas using Notch1 antisense transgenic (NAS).

Western blot analysis was performed for the liver of 8-week-old male NAS mice. We also conducted an intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test in 8-week-old male NAS mice and male C57BL/6 mice (control). Morphologic observation of pancreatic islet and β-cell was conducted in two groups. Insulin secretion capacity in islets was measured by glucose-stimulated insulin secretion (GSIS) and perifusion.

NAS mice showed higher glucose levels and lower insulin secretion in IPGTT than the control mice. There was no significant difference in insulin resistance. Total islet and β-cell masses were decreased in NAS mice. The number of large islets (≥250 µm) decreased while that of small islets (<250 µm) increased. Reduced insulin secretion was observed in GSIS and perifusion. Neurogenin3, neurogenic differentiation, and MAF bZIP transcription factor A levels increased in NAS mice.

Our study provides that Notch1 inhibition decreased insulin secretion and decreased islet and β-cell masses. It is thought that Notch1 inhibition suppresses islet proliferation and induces differentiation of small islets. In conclusion, Notch signaling pathway may play an important role in β-cell mass determination and diabetes.

Keyword

Diabetes mellitus; Glucose; Insulin-secreting cells; Insulin secretion; Receptors, Notch

Figure

• 

  Fig. 1 Notch1 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. 2 Protein 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. 3 Notch1 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. 4 Notch1 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. 5 Notch1 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. 6 Notch1 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. 7 Neurogenin3 (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).

• 

Reference

1. American Diabetes Association. 2. Classification and diagnosis of diabetes: standards of medical care in diabetes-2018. Diabetes Care. 2018; 41:S13–S27.

2. Cernea S, Dobreanu M. Diabetes and beta cell function: from mechanisms to evaluation and clinical implications. Biochem Med (Zagreb). 2013; 23:266–280.

3. Chen C, Cohrs CM, Stertmann J, Bozsak R, Speier S. Human beta cell mass and function in diabetes: recent advances in knowledge and technologies to understand disease pathogenesis. Mol Metab. 2017; 6:943–957.

4. Karadimos MJ, Kapoor A, El Khattabi I, Sharma A. β-Cell preservation and regeneration for diabetes treatment: where are we now? Diabetes Manag (Lond). 2012; 2:213–222.

5. Naujok O, Burns C, Jones PM, Lenzen S. Insulin-producing surrogate β-cells from embryonic stem cells: are we there yet? Mol Ther. 2011; 19:1759–1768.

6. Apelqvist A, Li H, Sommer L, Beatus P, Anderson DJ, Honjo T, et al. Notch signalling controls pancreatic cell differentiation. Nature. 1999; 400:877–881.

7. Murtaugh LC, Stanger BZ, Kwan KM, Melton DA. Notch signaling controls multiple steps of pancreatic differentiation. Proc Natl Acad Sci U S A. 2003; 100:14920–14925.

8. Kim W, Shin YK, Kim BJ, Egan JM. Notch signaling in pancreatic endocrine cell and diabetes. Biochem Biophys Res Commun. 2010; 392:247–251.

9. Gridley T. Notch signaling and inherited disease syndromes. Hum Mol Genet. 2003; 12 Spec No 1:R9–R13.

10. Krebs LT, Xue Y, Norton CR, Shutter JR, Maguire M, Sundberg JP, et al. Notch signaling is essential for vascular morphogenesis in mice. Genes Dev. 2000; 14:1343–1352.

11. Xue Y, Gao X, Lindsell CE, Norton CR, Chang B, Hicks C, et al. Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum Mol Genet. 1999; 8:723–730.

12. Cheng P, Zlobin A, Volgina V, Gottipati S, Osborne B, Simel EJ, et al. Notch-1 regulates NF-kappaB activity in hemopoietic progenitor cells. J Immunol. 2001; 167:4458–4467.

13. Shelly LL, Fuchs C, Miele L. Notch-1 inhibits apoptosis in murine erythroleukemia cells and is necessary for differentiation induced by hybrid polar compounds. J Cell Biochem. 1999; 73:164–175.

14. Kopinke D, Brailsford M, Pan FC, Magnuson MA, Wright CV, Murtaugh LC. Ongoing Notch signaling maintains phenotypic fidelity in the adult exocrine pancreas. Dev Biol. 2012; 362:57–64.

15. Bartolome A, Zhu C, Sussel L, Pajvani UB. Notch signaling dynamically regulates adult β cell proliferation and maturity. J Clin Invest. 2019; 129:268–280.

16. Suzuki K, Bonner-Weir S, Hollister J, Weir GC. A method for estimating number and mass of islets transplanted within a membrane device. Cell Transplant. 1996; 5:613–625.

17. Wang RN, Kloppel G, Bouwens L. Duct- to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats. Diabetologia. 1995; 38:1405–1411.

18. Rosenberg L, Brown RA, Duguid WP. A new approach to the induction of duct epithelial hyperplasia and nesidioblastosis by cellophane wrapping of the hamster pancreas. J Surg Res. 1983; 35:63–72.

19. Rosenberg L, Duguid WP, Healy M, Clas D, Vinik AI. Reversal of diabetes by the induction of islet cell neogenesis. Transplant Proc. 1992; 24:1027–1028.

20. Finegood DT, Scaglia L, Bonner-Weir S. Dynamics of beta-cell mass in the growing rat pancreas. Estimation with a simple mathematical model. Diabetes. 1995; 44:249–256.

21. Fujimoto WY, Metz SA. Phasic glucose-stimulated insulin secretion by neonatal rat pancreatic islet cells. Enhancement by sodium salicylate. Diabetes. 1984; 33:872–878.

22. Bergstrom RW, Fujimoto WY, Teller DC, de Haen C. Oscillatory insulin secretion in perifused isolated rat islets. Am J Physiol. 1989; 257:E479–E485.

23. Aguayo-Mazzucato C, Bonner-Weir S. Stem cell therapy for type 1 diabetes mellitus. Nat Rev Endocrinol. 2010; 6:139–148.

24. Juhl K, Bonner-Weir S, Sharma A. Regenerating pancreatic beta-cells: plasticity of adult pancreatic cells and the feasibility of in-vivo neogenesis. Curr Opin Organ Transplant. 2010; 15:79–85.

25. Hanley NA, Hanley KP, Miettinen PJ, Otonkoski T. Weighing up beta-cell mass in mice and humans: self-renewal, progenitors or stem cells? Mol Cell Endocrinol. 2008; 288:79–85.

26. Talchai C, Xuan S, Lin HV, Sussel L, Accili D. Pancreatic β cell dedifferentiation as a mechanism of diabetic β cell failure. Cell. 2012; 150:1223–1234.

27. Accili D, Talchai SC, Kim-Muller JY, Cinti F, Ishida E, Ordelheide AM, et al. When β-cells fail: lessons from dedifferentiation. Diabetes Obes Metab. 2016; 18 Suppl 1:117–122.

28. Pajvani UB, Shawber CJ, Samuel VT, Birkenfeld AL, Shulman GI, Kitajewski J, et al. Inhibition of Notch signaling ameliorates insulin resistance in a FoxO1-dependent manner. Nat Med. 2011; 17:961–967.

29. Edlund H. Pancreatic organogenesis: developmental mechanisms and implications for therapy. Nat Rev Genet. 2002; 3:524–532.

30. Wang H, Brun T, Kataoka K, Sharma AJ, Wollheim CB. MAFA controls genes implicated in insulin biosynthesis and secretion. Diabetologia. 2007; 50:348–358.

31. Aramata S, Han SI, Kataoka K. Roles and regulation of transcription factor MafA in islet beta-cells. Endocr J. 2007; 54:659–666.

32. Yang YP, Thorel F, Boyer DF, Herrera PL, Wright CV. Context-specific α- to-β-cell reprogramming by forced Pdx1 expression. Genes Dev. 2011; 25:1680–1685.

33. Ahnfelt-Ronne J, Hald J, Bodker A, Yassin H, Serup P, Hecksher-Sorensen J. Preservation of proliferating pancreatic progenitor cells by delta-Notch signaling in the embryonic chicken pancreas. BMC Dev Biol. 2007; 7:63.

34. Hart A, Papadopoulou S, Edlund H. Fgf10 maintains notch activation, stimulates proliferation, and blocks differentiation of pancreatic epithelial cells. Dev Dyn. 2003; 228:185–193.