Differentiation-Induced Insulin Secretion from Nonendocrine Cells with Engineered Human Proinsulin cDNA

Adenoviral vector-mediated insulin gene transfer in the mouse pancreas corrects streptozotocin-induced hyperglycemia

“…29 The most productive results have been achieved by using vector carrying furin-modified insulin. 18,20,[22][23][24][25][26][27] The studies reported here have demonstrated that injection of adenoviral vectors carrying furin-modified human insulin gene into the pancreatic parenchyma reverses hyperglycemia in streptozotocin-induced diabetic mice. The proinsulin translated from mRNA is processed, correctly localized in secretory vesicles, and prolongs survival.…”

“…[11][12][13][14][15][16][17] Several studies have shown that it is possible to process proinsulin to its mature form in non-␤-cells by engineering furin cleavage sites in the proinsulin mol- ecule. [18][19][20][21][22][23][24][25][26][27][28] When 293 cells are transfected with a human furin modified proinsulin cDNA, efficient processing and constitutive secretion of biologically active insulin was documented. 20 In THP-1 (monocyte-derived) and C2C12 (myoblast-derived) nonendocrine cell lines, transfection with a cDNA plasmid containing furin-modified human proinsulin gene produced mature human insulin.…”

“…20 In THP-1 (monocyte-derived) and C2C12 (myoblast-derived) nonendocrine cell lines, transfection with a cDNA plasmid containing furin-modified human proinsulin gene produced mature human insulin. 25 A hepatoma cell line was infected with adenoviral vector expressing human insulin with furin cleavage sites. Cells synthesized both proinsulin and mature insulin, which functionally was identical to that of authentically processed insulin.…”

Enhanced expression of a furin-cleavable proinsulin

“…To overcome this obstacle, various groups have used a variety of site-directed mutations to engineer proinsulin to be a substrate for furin (Yanagita et al 1992, Groskreutz et al 1994, Muzzin et al 1997, Short et al 1998, Yamasaki et al 1999, Shaw et al 2002. This enzyme (also known as PACE) is a Golgi-associated propeptide endoprotease that is present in the constitutive secretory pathway of virtually all cells (Van de Ven et al 1990).…”

Establishment of an artificial β-cell line expressing insulin under the control of doxycycline

“…To successfully mimic beta cell function, it is necessary to overcome three major obstacles: proinsulin synthesis, proinsulin processing, and mature insulin storage and regulated secretion. Our previous study (unpublished) and studies by other authors had demonstrated that correct proinsulin processing and insulin storage can be obtained in somatic cells transduced with the mutated proinsulin gene [10][11][12][13] , so the major problem to ectopic insulin expression is the difficulty of reconstructing in non-beta cells the highly regulated insulin secretion of the normal beta cells [14][15][16][17][18][19] .In 1992, Gossen and Bugard [20] developed the tetracycline (or doxycycline)-induced gene expression (Tet-on) system, which is useful for controlling the expression of targeted genes in quantitative manner and for determining the roles of the gene products in cellular functions [21][22][23][24][25][26] . In the present study, using this system, we generated the first non-beta cell line with deoxycycline-regulated insulin secretion.…”