Korean Diabetes J.  2009 Dec;33(6):475-484. 10.4093/kdj.2009.33.6.475.

Transdifferentiation of Enteroendocrine K-cells into Insulin-expressing Cells

• Lee E
• Yu JM
• Lee MK
• Ryu GR
• Ko SH
• Ahn YB
• Moon SD
• Song KH

• KMID: 2298053
• DOI: http://doi.org/10.4093/kdj.2009.33.6.475

Abstract

BACKGROUND
Despite a recent breakthough in human islet transplantation for treating type 1 diabetes mellitus, the limited availability of donor pancreases remains a major obstacle. Endocrine cells within the gut epithelium (enteroendocrine cells) and pancreatic beta cells share similar pathways of differentiation during embryonic development. In particular, K-cells that secrete glucose-dependent insulinotropic polypeptide (GIP) have been shown to express many of the key proteins found in beta cells. Therefore, we hypothesize that K-cells can be transdifferentiated into beta cells because both cells have remarkable similarities in their embryonic development and cellular phenotypes. METHODS: K-cells were purified from heterogeneous STC-1 cells originating from an endocrine tumor of a mouse intestine. In addition, a K-cell subclone expressing stable Nkx6.1, called "Kn4-cells," was successfully obtained. In vitro differentiation of K-cells or Kn4-cells into beta cells was completed after exendin-4 treatment and serum deprivation. The expressions of insulin mRNA and protein were examined by RT-PCR and immunocytochemistry. The interacellular insulin content was also measured. RESULTS: K-cells were found to express glucokinase and GIP as assessed by RT-PCR and Western blot analysis. RT-PCR showed that K-cells also expressed Pdx-1, NeuroD1/Beta2, and MafA, but not Nkx6.1. After exendin-4 treatment and serum deprivation, insulin mRNA and insulin or C-peptide were clearly detected in Kn4-cells. The intracellular insulin content was also increased significantly in these cells. CONCLUSION: K-cells are an attractive potential source of insulin-producing cells for treatment of type 1 diabetes mellitus. However, more experiments are necessary to optimize a strategy for converting K-cells into beta cells.

Keyword

Differentiation; Enteroendocrine cells; K-cell; Nkx6.1 protein; Pancreatic beta-cell

MeSH Terms

Animals
Blotting, Western
C-Peptide
Diabetes Mellitus, Type 1
Embryonic Development
Endocrine Cells
Enteroendocrine Cells
Epithelium
Female
Glucokinase
Humans
Immunohistochemistry
Insulin
Insulin-Secreting Cells
Intestines
Islets of Langerhans Transplantation
Mice
Pancreas
Peptides
Phenotype
Pregnancy
Proteins
RNA, Messenger
Tissue Donors
Venoms
C-Peptide
Glucokinase
Insulin
Peptides
Proteins
RNA, Messenger
Venoms

Figure

• 

  Fig. 1 Design of Nkx6.1-expressing vector. The Nkx6.1-expressing vector (pcDNA3.1 + Nkx6.1) was made as shown in this cartoon.

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  Fig. 2 RT-PCR of transcription factors mRNA. K-cells were found to express transcription factors which were all present in mouse islets except Nkx6.1.

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  Fig. 3 RT-PCR of insulin 1 mRNA. Insulin1 band was weakly detected in K-cells after 7 day-treatment with 10 mM nicotinamide and 10 pM exendin-4 in serum-free medium. 1, Mouse islets; 1-1, K-cells; 2, K-cells, 7 days in serum-free medium; 3, K-cells, 7 days in serum-free medium with 10 mM nicotinamide; 4, K-cells, 7 days in serum-free medium with 10 mM nicotinamide and 10 pM exendin-4.

• 

  Fig. 4 RT-PCR of insulin 1 and Nkx6.1 mRNA. Insulin1 band was clearly detected in Kn4-cells after 7 day-treatment with 10 mM nicotinamide and 10 pM exendin-4 in serum-free medium. 1, Rat islets; 1-1, Mouse islets; 2, K-cells, 7 days in serum-free medium; 3, K_pcDNA-cells, 7 days in serum-free medium; 4, Kn4-cells, 7 days in serum-free medium; 5, K-cells, 7 days in serum-free medium with 10 mM nicotinamide and 10 pM exendin-4; 6, K_pcDNA-cells, 7 days in serum-free medium with 10 mM nicotinamide and 10 pM exendin-4; 7, Kn-4 cells, 7 days in serum-free medium with 10 mM nicotinamide and 10 pM exendin-4.

• 

  Fig. 5 Fluorescent microscopy for insulin immunocytochemistry in K-cells and transdifferentiated Kn4-cells. Some of differentiated Kn4-cells were found to be insulin-positive (A, Insulin staining in Ins-1 cells, ×200; B, Insulin staining in K-cells, ×200; C, Insulin staining in differentiated Kn4-cells, ×400; D, GFP expression, ×400).

• 

  Fig. 6 Confocal microscopy for C-peptide immunocytochemistry in transdifferentiated Kn4-cells. Some of differentiated Kn4-cells were found to be C-peptide-positive (A, Nuclei staining with DAPI; B, GFP expression; C, C-peptide staining D, Merged image, ×400).

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  Fig. 7 Intracellular content of insulin. Insulin content was significantly higher in transdifferentiated Kn4-cells as compared with K-cells. *P < 0.05.

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  Fig. 8 Cell morphology of Kn4-cells before and after 7 day-treatment with 10 mM nicotinamide and 10 pM exendin-4 in serum-free medium (×400).

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