Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Nov 8;1(1):3.
doi: 10.1186/1740-2557-1-3.

Insulin expressing hepatocytes not destroyed in transgenic NOD mice

Muhammad T Tabiin  1 , Christopher P White, Grant Morahan, Bernard E Tuch
Affiliations

Insulin expressing hepatocytes not destroyed in transgenic NOD mice

Muhammad T Tabiin et al. J Autoimmune Dis. .

Abstract

BACKGROUND: The liver has been suggested as a suitable target organ for gene therapy of Type 1 diabetes. However, the fundamental issue whether insulin-secreting hepatocytes in vivo will be destroyed by the autoimmune processes that kill pancreatic beta cells has not been fully addressed. It is possible that the insulin secreting liver cells will be destroyed by the immune system because hepatocytes express major histocompatibility complex (MHC) class I molecules and exhibit constitutive Fas expression; moreover the liver has antigen presenting activity. Together with previous reports that proinsulin is a possible autoantigen in the development of Type 1 diabetes, the autoimmune destruction of insulin producing liver cells is a distinct possibility. METHODS: To address this question, transgenic Non-Obese Diabetic (NOD) mice which express insulin in the liver were made using the Phosphoenolpyruvate Carboxykinase (PEPCK) promoter to drive the mouse insulin I gene (Ins). RESULTS: The liver cells were found to possess preproinsulin mRNA, translate (pro)insulin in vivo and release it when exposed to 100 nmol/l glucagon in vitro. The amount of insulin produced was however significantly lower than that produced by the pancreas. The transgenic PEPCK-Ins NOD mice became diabetic at 20-25 weeks of age, with blood glucose levels of 24.1 +/- 1.7 mmol/l. Haematoxylin and eosin staining of liver sections from these transgenic NOD PEPCK-Ins mice revealed the absence of an infiltrate of immune cells, a feature that characterised the pancreatic islets of these mice. CONCLUSIONS: These data show that hepatocytes induced to produce (pro)insulin in NOD mice are not destroyed by an ongoing autoimmune response; furthermore the expression of (pro)insulin in hepatocytes is insufficient to prevent development of diabetes in NOD mice. These results support the use of liver cells as a potential therapy for type 1 diabetes. However it is possible that a certain threshold level of (pro)insulin production might have to be reached to trigger the autoimmune response.

PubMed Disclaimer

Figures

Figure 1
Figure 1
A-C. The PEPCK-Ins transgene. Schematic representation (A). Example of PCR screening of PEPCK-Ins transgenic mice (B). Lanes 1–5 and lanes 10–14 are PCR reactions with genomic DNA extracted from mice that developed from microinjected mouse eggs. Primers specific for the PEPCK-Ins transgene were used for lanes 1–9 (30 cycles) while primers specific for mouse insulin 1 were used for lanes 10–18 (30 cycles). Lanes 6 and 15 are PCR negative controls (no DNA added). Lanes 7 and 16 are PCR reactions to demonstrate the specificity of the primers used (wild type NOD mouse DNA added). Lanes 8, 9, 17 and 18 are half copy spiked and plasmid controls respectively (positive controls). Example of Southern Blotting for the PEPCK-Ins transgene (C). Lane 1 One copy spiked sample (100 ng Balb/c genomic DNA + 102fg PEPCK-Ins plasmid), lane 2 genomic DNA from tail tip of F2 PEPCK-Ins mouse, lanes 3 and 4 are DNA from pups which died before weaning and lanes 5–7 are DNA from still born pups. Each lane was loaded with 15μg of genomic DNA that was digested with Xba I and Pst I to release the transgene.
Figure 2
Figure 2
A-B. Mouse insulin 1 mRNA. RT-PCR (A). Lanes 1A-6A are RT-PCR reactions using primers specific for mouse insulin 1 mRNA (30 cycles). Lanes 1B-6B are RT-PCR reactions using primers specific for mouse GAPDH mRNA (30 cycles). Lanes 1–4 are total RNA from the liver of progeny from founder T645-18, lane 5 is total RNA from the liver of progeny from founder T647-2 and lane 6 total RNA from the pancreas of progeny from founder T645-18. RPA for mouse insulin 1 mRNA (B). Lane 1 pancreatic total RNA, lane 2 liver total RNA from T647-2 progeny and lanes 3–7 liver total RNA from T645-18 progeny.
Figure 3
Figure 3
A-F. In-situ hybridisation. Pancreatic islets from a diabetic transgenic mouse illustrating insulitis, (A) antisense and (B) sense. Transgenic liver from a diabetic transgenic mouse (C) antisense and (D) sense. Wild type liver from a diabetic NOD mouse (E) antisense and (F) sense.
Figure 4
Figure 4
Incidence of diabetes in F1 and F2 PEPCK-Ins transgenic NOD mice versus wild type F1 and F2 NOD mice. PEPCK-Ins NOD males total n = 14, PEPCK-Ins NOD females n = 25, wild type NOD males n = 45 and wild type NOD females n = 51.
Figure 5
Figure 5
Staining of the liver of diabetic transgenic PEPCK-Ins mice with haematoxylin and eosin. (Black Bar = 20μm).
Figure 6
Figure 6
A-D. Haematoxylin and eosin, and insulin staining of pancreatic sections from transgenic PEPCK-Ins NOD mice. H & E staining of a pancreatic islet from a diabetic transgenic mouse illustrating insulitis (A), and from a normoglycaemic transgenic mouse (B). Insulin staining of a pancreatic islet from a diabetic transgenic mouse showing few remaining β cells (C) and from a normoglycaemic transgenic mice (D). Black Bar = 20μm for A-C, and 40μm for D.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Simpson AM, Marshall GM, Tuch BE, Maxwell L, Szymanska B, Tu J, Beynon S, Swan MA, Camacho M. Gene therapy of diabetes: glucose-stimulated insulin secretion in a human hepatoma cell line (HEPG2ins/g) Gene Ther. 1997;4:1202–1215. doi: 10.1038/sj.gt.3300527. - DOI - PubMed
    1. Tuch BE, Szymanska B, Yao M, Tabiin MT, Gross DJ, Holman S, Swan MA, Humphrey RK, Marshall GM, Simpson AM. Function of a genetically modified human liver cell line that stores, processes and secretes insulin. Gene Ther. 2003;10:490–503. doi: 10.1038/sj.gt.3301911. - DOI - PubMed
    1. Vollenweider F, Irminger JC, Gross DJ, Villa-Korniaroff L, Halban PA. Processing of proinsulin by transfected hepatoma (FAO) cells. J Biol Chem. 1992;267:14629–14636. - PubMed
    1. Gros L, Montoliu L, Riu E, Lebrigand L, Bosch F. Regulated production of mature insulin by non-β-cells. Hum Gene Ther. 1997;8:2249–2259. - PubMed
    1. Lu D, Tamemoto H, Shibata H, Saito I, Takeuchi T. Regulatable production of insulin from primary cultured hepatocytes–insulin production is upregulated by glucagon and cAMP and down regulated by insulin. Gene Ther. 1998;5:888–895. doi: 10.1038/sj.gt.3300677. - DOI - PubMed