Abstract
We have established insulin-secreting cell line, L1-INS/fur cells, by engineering 3T3-L1 murine preadipocytes with human preproinsulin cDNA. Analysis with HPLC, mass spectrometry and immunological assay identified human insulin in the culture medium. Notably, secretion of insulin from L1-INS/fur cell was increased 8.2 times higher after induction of cellular differentiation. The increment of insulin secretion during differentiation was further enhanced by additive treatment with thiazolidinedione, a promoting agent of adipocyte differentiation. This observation strongly suggests that the enhancement of insulin secretion is tightly associated with cellular differentiation process itself. Expression rate of the insulin transgene was not changed after the additional treatment with thiazolidinedione. On the other hand, furin gene expression by Northern analysis showed an increase, and Western analysis revealed even more reduction in cellular content of proinsulin. These results indicate that mechanism of the observed enhancement in insulin secretion during the differentiation is mainly due to increased capacity of the proinsulin processing by induction of furin. Results of our present study will provide important information on cell-based therapy using undifferentiated progenitors and tissue stem cells. (Mol Cell Biochem 268: 1–8, 2005)
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Kolodka TM, Finegold M, Moss L, Woo SL: Gene therapy for diabetes mellitus in rats by hepatic expression of insulin. Proc Natl Acad Sci USA 92: 3293–3297, 1995
Simpson AM, Tuch BE, Swan MA, Tu J, Marshall GM: Functional expression of the human insulin gene in a human hepatoma cell line (HEP G2). Gene Ther 2: 223–231, 1995
Yamasaki K, Sasaki T, Nemoto M, Eto Y, Tajima N: Differentiation-induced insulin secretion from nonendocrine cells with engineered human proinsulin cDNA. Biochem Biophys Res Commun 26: 5361–5365, 1999
Groskreutz DJ, Sliwkowski MX, Gorman CM: Genetically engineered proinsulin constitutively processed and secreted as mature, active insulin. J Biol Chem 269: 6241–6245, 1994
Simonson GD, Groskreutz DJ, Gorman CM, MacDonald MJ: Synthesis and processing of genetically modified human proinsulin by rat myoblast primary cultures. Hum Gene Ther 7: 71–78, 1996
Auricchio A, Gao GP, Yu QC, Raper S, Rivera VM, Clackson T, Wilson JM: Constitutive and regulated expression of processed insulin following in vivo hepatic gene transfer. Gene Ther 9: 963–971, 2002
Weissman IL: Stem cells: Units of development, units of regeneration, and units in evolution. Cell 100: 157–168, 2000
Sasaki T, Fujimoto K, Sakai K, Nemoto M, Nakai N, Tajima N: Gene and cell-based therapy for diabetes mellitus: Endocrine gene therapeutics. Endocr Pathol 14: 141–144, 2003
Mann R, Mulligan RC, Baltimore D: Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33: 153–159, 1983
Sasaki T, Hansen RS, Gartler SM: Hemimethylation and hypersensitivity are early events in transcriptional reactivation of human inactive X-linked genes in a hamster × human somatic cell hybrid. Mol Cell Biol 9: 3819–3826, 1992
Fujimoto K, Shibasaki T, Yokoi N, Kashima Y, Matsumoto M, Sasaki T, Tajima N, Iwanaga T, Seino S: Piccolo, a Ca2 + sensor in pancreatic beta-cells. Involvement of cAMP-GEFII/Rim2/Piccolo complex in cAMP-dependent exocytosis. J Biol Chem 277: 50497–50502, 2002
Green H, Kehinde O: An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion. Cell 5:19–27, 1975
Tafuri SR: Troglitazone enhances differentiation, basal glucose uptake, and Glut1 protein levels in 3T3-L1 adipocytes. Endocrinology 137: 4706–4712, 1996
Halvorsen YD, Bond A, Sen A, Franklin DM, Lea-Currie YR, Sujkowski D, Ellis PN, Wilkison WO, Gimble JM: Thiazolidinediones and glucocorticoids synergistically induce differentiation of human adipose tissue stromal cells: Biochemical, cellular, and molecular analysis. Metabolism 50: 407–413, 2001
Rajagopal J, Anderson WJ, Kume S, Martinez OI, Melton DA: Insulin staining of ES cell progeny from insulin uptake. Science 299: 363, 2003
Croissandeau G, Basak A, Seidah NG, Chretien M, Mbikay M: Proprotein convertases are important mediators of the adipocyte differentiation of mouse 3T3-L1 cells. J Cell Sci 115: 1203–1211, 2002
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Fujimoto, K., Sasaki, T., Nemoto, M. et al. Enhanced insulin secretion from engineered 3T3-L1 preadipocytes by induction of cellular differentiation. Mol Cell Biochem 268, 1–8 (2005). https://doi.org/10.1007/s11010-005-0608-8
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DOI: https://doi.org/10.1007/s11010-005-0608-8