Korean Diabetes J.  2009 Dec;33(6):466-474. 10.4093/kdj.2009.33.6.466.

Treatment of Type 1 Diabetes through Genetically Engineered K-cell Transplantation in a Mouse Model

• Sim JY
• Kim JH
• Ahn YB
• Song KH
• Han JH
• Cha BY
• Lee SK
• Moon SD

• KMID: 2298052
• DOI: http://doi.org/10.4093/kdj.2009.33.6.466

Abstract

BACKGROUND
K-cells function as targets for insulin gene therapy. In a previous study, we constructed EBV-based plasmids expressing rat preproinsulin controlled by glucose-dependent insulinotropic polypeptide promoters. In the present study, we attempted to correct hyperglycemia in vivo using genetically engineered K-cells in a mouse model of type 1 diabetes. METHODS: K-cells expressing insulin were transplanted under the kidney capsules of STZ-induced diabetic mice. The blood glucose levels and body weights of the experimental animals were measured daily. After four weeks, the mice were injected intra-peritoneally with 2 g/kg glucose following a 6 hr fast. Blood glucose levels were measured immediately following glucose injections. All animals were sacrificed at the end of the glucose tolerance study, and pancreas and graft-bearing kidney tissue samples were stained with antibodies against insulin, glucagon, and C-peptide. RESULTS: The body weights of K-cell-transplanted diabetic mice increased after transplantation, whereas those of untreated diabetic control mice continued to decline. The blood glucose levels of K-cell-transplanted diabetic mice decreased gradually during the two weeks following transplantation. After intra-peritoneal injection of glucose into K-cell-transplanted diabetic mice, blood glucose levels increased at 30 minutes, and were restored to the normal range between 60 and 90 minutes, while untreated control diabetic mice continued to experience hyperglycemia. Kidney capsules containing transplanted K-cells were removed, and sections were stained with anti-insulin antibodies. We detected insulin-positive cells in the kidney capsules of K-cell-transplanted diabetic mice, but not in untreated control mice. CONCLUSION: We detected glucose-dependent insulin secretion in genetically engineered K-cells in a mouse model of type 1 diabetes. Our results suggest that genetically modified insulin producing K-cells may act as surrogate beta-cells to effectively treat type 1 diabetes.

Keyword

Epstein-Barr virus; Gastric inhibitory polypeptide; Gene therapy; K-cell; Plasmid

MeSH Terms

Animals
Antibodies
Blood Glucose
Body Weight
C-Peptide
Capsules
Gastric Inhibitory Polypeptide
Genetic Therapy
Glucagon
Glucose
Herpesvirus 4, Human
Hyperglycemia
Insulin
Kidney
Mice
Pancreas
Plasmids
Protein Precursors
Rats
Reference Values
Transplants
Antibodies
Blood Glucose
C-Peptide
Capsules
Gastric Inhibitory Polypeptide
Glucagon
Glucose
Insulin
Protein Precursors

Figure

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  Fig. 1 Schematic presentation of the construct which is EBV-based plasmid expressing PPI or GFP transcriptionally controlled by GIP promoter (GIPP). A. Plasmid expressing GFP as control. B. Plasmid expressing PPI. GFP, green fluorescent protein; GIP, glucose dependent insulinotropic peptide; PPI, preproinsulin.

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  Fig. 2 Average of body weight variation. Experiments on all nude mice were measured body weight every day. Body weight changes of normal control mice and K-cell transplanted diabetic mice were similarly increased, but only untreated STZ-induced diabetic mice maintain low body weight (16~19 g). Values are means. K-cells were transplanted at day 0. *P < 0.05 vs. normal control mice or K-cell transplanted diabetic mice group.

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  Fig. 3 Blood glucose level after K-cell transplantation. Glucose levels of normal control mice show within normal limits (less then 180 mg/dL). Those of untreated STZ-induced diabetic mice were irregular and constant high level. Similarly, those of K-cell transplanted diabetes mice were high, but it was getting lower gradually during the experimental period. Values are means. K-cells were transplanted at day 0. *P < 0.05 vs. untreated STZ-induced diabetic mice group.

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  Fig. 4 Glucose tolerance tests of normal control mice and K-cell treated diabetic mice or untreated STZ-induced diabetic mice. Values are means of each group (n = 3). *P < 0.05 vs. normal control mice or K-cell transplanted diabetic mice group.

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  Fig. 5 Immunohistochemistry of insulin expression in graft-bearing kidney and pancreas of the normal control mice, untreated STZ-induced diabetic mice or K-cell transplanted diabetic mice (×400).

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  Fig. 6 Immunofluorescence study for insulin expression in pancreas and graft-bearing kidney of normal control mice, untreated STZ-induced diabetic mice or K-cell transplanted diabetic mice (×400).

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