Gene therapy of diabetes: glucose-stimulated insulin secretion in a human hepatoma cell line (HEP G2ins/g)

Abstract

In order to design a feasible somatic cell gene delivery sysgogues. While glucose responsiveness commenced at a tem for the treatment of type I diabetes, a suitable cell type lower concentration than normal islets, a secretion curve needs to be determined. We have previously shown that approaching normal physiological conditions was generthe stable transfection of the full-length insulin cDNA into ated. Immunoelectron microscopy revealed the presence the human liver cell line, (HEP G2ins) resulted in synthesis, of insulin-containing granules, similar in size and appearstorage and acute regulated release of insulin to analogues ance to those of the normal beta cell. These results demof cAMP, but not to the physiological stimulus glucose. In onstrate that while it is most likely that the HEP G2ins/g attempting to explain the lack of glucose responsiveness cell line predominantly secretes insulin via the constitutive of the HEP G2ins cells we have stably transfected these pathway, significant acute regulated release was seen in cells with the human islet glucose transporter GLUT 2 response to glucose, and thus represents significant pro-(HEP G2ins/g cells). The HEP G2ins/g cell clones exhibit gress in the creation of a genetically engineered 'artificial glucose-stimulated insulin secretion and glucose potentibeta cell' from a human hepatocyte cell line. ation of the secretory response to nonglucose secreta-

Function of a genetically modified human liver cell line that stores, processes and secretes insulin

“…Our experience with the HUH7-ins cell line extends our results with another human liver cell line, HEP G2ins/g, which also synthesized, stored and secreted insulin in a regulated manner when exposed to glucose in tissue culture. 6 The ability of the transplanted HEP G2ins/g cells to secrete (pro)insulin in response to glucose was impaired, with the cells having no immediate effect on the blood glucose levels of the diabetic recipient mice. 24 This was different from the results we obtained with transplanted HUH7-ins cells ( Figure 10).…”

“…Large vacuoles were also present in another human insulin-producing cell we have created, HEP G2ins/g cells. 6 No secretory vesicles containing granules were observed in HUH7 cells, but there were a small number of vacuoles. The secretory vesicles present in HUH7-ins cells, at 170-230 nm diameter, were smaller than the structures termed membrane sacs 300 nm in diameter, found in normal b cells.…”

“…NIT 1 insulinoma cells previously gave similar results with this immunogold technique. 6 There was no non-specific labeling in the nucleus or elsewhere in the cell. Control HUH7 cells were negative as were HUH7-ins and MIN6 cells in the absence of the primary antibody.…”

“…Proinsulin was not detectable in the cell extracts, as determined by an RIA specific for proinsulin. 6 Clones 2, 9 and 13, which were used for the acute stimulation experiments described previously, contained the largest amount of insulin, 6.970.8, 5.970.3 and 4.771.0 pmol/10 6 cells (n¼5), respectively. The percent of insulin in each cell of these three clones secreted each day was 3.6, 5.1 and 4.3, respectively.…”

“…[5][6][7][8][9][10][11][12][13] Results of a previous study in our laboratory have shown that insertion of human insulin cDNA (pC 2 ) and the glucose transporter GLUT2 into the neoplastic human hepatoma cell line HEP G2 resulted in synthesis and storage of (pro)insulin in structures resembling the secretory granules of normal pancreatic b bcells. 6 These HEP G2ins/g cells exhibited regulated, near-physiological secretion of (pro)insulin in response to glucose. In contrast, insertion of insulin into two rat liver cell lines, FAO 7 and H4-II-E 8 , resulted in synthesis and release of insulin, but not its storage or regulated secretion.…”