Growth Factor‐Dependent Proliferation of the Pancreatic β‐cell Line βTC‐tet: An Assay for β‐cell Mitogenic Factors

Abstract

The ability to expand normal pancreatic islet β cells in culture would significantly advance the prospects of cell therapy for diabetes. A number of growth factors can stimulate limited islet cell replication, however other factors may exist which are more effective β-cell-specific mitogens. The search for novel β-cell growth factors has been hampered by the lack of a β-cell-specific proliferation assay. We developed a simple and sensitive assay for β-cell growth factors based on a conditionally-transformed mouse β-cell line (βTC-tet). These cells express the SV40 T antigen (Tag) oncoprotein under control of the tetracycline (Tc) operon regulatory system. In the presence of Tc, Tag expression is tightly shut off and the cells undergo complete growth arrest. Here we show that the growth-arrested cells can proliferate in response to growth factors in the absence of Tag. Using this assay, a number of growth factors previously shown to be mitogenic to a mixed islet cell population were found to induce proliferation of pure β cells. We conclude that growth-arrested βTC-tet cells can be employed in a survey of factors from various sources for identifying novel factors with β-cell mitogenic activity.Key words: β-cell lines, cell proliferation, growth factors, mitogenicity, tetracycline-regulated gene expression INTRODUCTIONBeta-cell transplantation represents an attractive approach for replacement of the ____________________ *Corresponding author: tel: 972-3-640 7701; fax. 972-3-640 9950; e-mail: sefrat@post.tau.ac.il Int. Jnl. Experimental Diab. Res., 3:69-74, 2002 © 2002 Taylor and Francis 1560 damaged β cells in type I diabetes. One major obstacle to β-cell therapy is the lack of an abundant cell supply. A number of rodent β-cell lines have been developed by oncogenic transformation, however this approach has proven difficult to adapt to human β cells. Development of efficient ways for in vitro expansion of normal islets would significantly advance the prospects of diabetes cell therapy. Adult islet cells are difficult to propagate in culture, although they maintain a proliferative capacity, as evidenced by a limited mitogenic response to a number of growth factors [1,2]. Members of the growth hormone family, in particular placental lactogen (PL) and prolactin (PRL), induce replication in neonatal rat islet cells [3]. These activities may be responsible for islet mass expansion in pregnancy. Significant mitogenic effects of hepatocyte growth factor (HGF) have been observed on human fetal and adult islets [4] and mouse islets [5]. Insulin-like growth factors (IGF) I and II, and platelet-derived growth factor, affect fetal rat islet cell growth [6,7]. IGF I has also been shown to stimulate replication of cultured rat insulinoma cells [8], and IGF II activates the growth of both normal [9] and transformed [10] mouse β cells in vivo. The Reg protein, produced by pancreatic acinar cells and regenerating islets, is another candidate for a β-cell growth factor [11]. These findings suggest that islet cells possess cell su...

Using Mass Spectrometry to Detect, Differentiate, and Semiquantitate Closely Related Peptide Hormones in Complex Milieu: Measurement of IGF-II and Vesiculin

“…The MIN6 cell line secreted approximately 3 times more insulin but much lower amounts of IGF-II and vesiculin than the BTC6-F7 cell line. Both vesiculin and IGF-II were secreted in much lower quantities than total insulin; these concentrations of IGF-II are close to those previously shown to stimulate ␤-cell proliferation (4,24).…”

“…Two 10-L injections were performed per sample. To prepare a 5-point standard curve, 1 mL of nonconditioned medium (serum-free, phenol red-free with 0.1% BSA [w/v]) was constituted with peptide (2,4,8,16, and 40 ng/mL of vesiculin; 80, 40, 20, 10, and 5 ng/mL of IGF-II) and processed through SPE columns as above. A second 5-point standard curve, which encompassed the lower limit of detection for vesiculin, was analyzed; this consisted of 1 mL of nonconditioned media constituted with vesiculin (0.1, 0.5, 1, 2, and 4 ng/mL vesiculin).…”

“…Placental lactogen, prolactin, hepatocyte growth factor, and PTHrP are among several factors that have been shown to induce islet ␤-cell survival if not replication (3,4). IGF-II is a peptide hormone/growth factor in the same superfamily as insulin and has been highlighted as an excellent candidate for a ␤-cell-specific growth factor.…”

A gene expression network model of type 2 diabetes links cell cycle regulation in islets with diabetes susceptibility

“…Widespread (Petrik et al 1999) or local (Devedjian et al 2000;Okamoto et al 2006) overexpression of Igf2 has been shown to promote islet cell hyperplasia, and application of Igf2 to betacells in culture induces proliferation (Milo-Landesman and Efrat 2002). Recent genome-wide association studies have identified genetic variants in IGF2BP2 that are associated with type 2 diabetes in human patients (Saxena et al 2007;Scott et al 2007;Zeggini et al 2007).…”

IGF2: an endocrine hormone to improve islet transplant survival

“…IGF2 has been shown to promote pancreatic b-cell survival against apoptotic stimuli in vitro and in vivo (Rabinovitch et al 1982, Swenne et al 1987, Hogg et al 1993, Ilieva et al 1999, Robitaille et al 2003, Jourdan et al 2011 and induce proliferation in a growth-arrested mouse b-cell line (Milo-Landesman & Efrat 2002). IGF2 protects pre-diabetic NOD mouse islets from the cytotoxic actions of IL1b by the mechanisms that include a reduction in apoptosis (Hill et al 1999b).…”