letter nature genetics • volume 31 • may 2002 111 β-cell–specific deletion of the Igf1 receptor leads to hyperinsulinemia and glucose intolerance but does not alter β-cell mass Rohit N. Kulkarni 1 , Martin Holzenberger 2 , David Q. Shih 3 , Umut Ozcan 1 , Markus Stoffel 3 , Mark A. Magnuson 4 & C. Ronald Kahn 1 1 Research Division, Joslin Diabetes Center, Department of Medicine, Harvard Medical School, One Joslin Place, Boston Massachusetts 02215, USA. 2 Inserm U515, Croissance, Différenciation et Processus Tumoraux, Hôpital Saint-Antoine, Paris, France. 3 Laboratory of Metabolic Diseases, The Rockefeller University, New York, New York, USA. 4 Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA. Correspondence should be addressed to R.N.K. (e-mail: rohit.kulkarni@joslin.harvard.edu). Regulation of glucose homeostasis by insulin depends on the maintenance of normal β-cell mass and function. Insulin-like growth factor 1 (Igf1) has been implicated in islet development and differentiated function 1,2 , but the factors controlling this process are poorly understood. Pancreatic islets produce Igf1 and Igf2, which bind to specific receptors on β-cells 3–6 . Igf1 has been shown to influence β-cell apoptosis 7 , and both Igf1 and Igf2 increase islet growth 8,9 ; Igf2 does so in a manner additive with fibroblast growth factor 2 (ref. 10). When mice deficient for the Igf1 receptor (Igf1r +/- ) are bred with mice lacking insulin receptor substrate 2 (Irs2 -/- ), the resulting compound knockout mice show a reduction in mass of β-cells 11 similar to that observed in pancreas of Igf1r -/- mice (ref. 11), suggesting a role for Igf1r in growth of β-cells. It is possible, however, that the effects in these mice occur secondary to changes in vascular endothelium 12 or in the pancreatic ductal cells, or because of a decrease in the effects of other hormones implicated in islet growth. To directly define the role of Igf1, we have created a mouse with a β-cell–specific knockout of Igf1r (βIgf1r -/- ). These mice show normal growth and development of β-cells, but have reduced expression of Slc2a2 (also known as Glut2) and Gck (encoding glucokinase) in β-cells, which results in defective glucose-stimulated insulin secretion and impaired glucose tol- erance. Thus, Igf1r is not crucial for islet β-cell development, but participates in control of differentiated function. We created mice with a β-cell specific knockout of Igf1r by breeding animals carrying Igf1r in which exon 3 was flanked with loxP sites (Igf1r tm1.1Mhz , hereafter referred to as Igf1r lox/lox ) 13 with mice expressing cre driven by the rat insulin promoter (TgN(ins2-cre)25Mgn, hereafter referred to as cre +/– ; Fig. 1a) 14 . Control (Igf1r lox/lox and cre +/– ) and knockout mice were born normally and survived equally into adulthood. Homozygous knockout of Igf1r in β-cells but not other islet cells was confirmed by RT–PCR using RNA from βIgf1r -/- islet cells separated by flow cytometry 14 (Fig. 1b), and by immunostaining of pancreas sections for Igf1 receptors (Fig. 1c). Pancreas sec- tions from six-month-old control and βIgf1r -/- mice showed no significant differences in β-cell mass (βIgf1r -/- , 1.1 ± 0.3 mg per Published online: 1 April 2002, DOI: 10.1038/ng872 Igf1r after exon 3 replacement and Neo excision genomic map of the wildtype Igf1r exon 3 E3 E4 5.4 kb 2.4 kb Neo E3 Hc Hc 4.6 kb 4.3 kb H H B I S I Igf1r lox/lox Hc S B Hc E3 H 2.5 kb Igf1r-neo - lox βIgf1r non-β-cells β-cells non-β-cells β-cells lox/lox allele Igf1r allele 574 bp 518 bp control βIgf1r Igf1r lox/lox –/– –/– –/– H H S E E Hc E B BHc E r Fig. 1 Conditional Igf1r targeting allows β-cell–specific Igf1r knock- out. a, The upper panel shows a partial genomic map of wildtype Igf1r. Individual letters represent restriction sites. The lower panel shows the targeted locus before and after excision of the neomycin selection cassette (Neo) that is flanked by one loxP site upstream and two loxP sites downstream of exon (E) 3. b, RT–PCR analysis of RNA obtained from islet cells sorted by flow cytometry into β- and non–β-cells. Amplification from the knockout transcript (518 bp) is evident in the lane correspond- ing to the βIgf1r -/- β-cells, indi- cating absence of exon 3. All other lanes show the intact lox/lox allele (574 bp). c, Repre- sentative islets from pancreas sec- tions of a Igf1r lox/lox control and a βIgf1r -/- mouse stained for Igf1 receptors. Scale bar, 25 μm. a b c © 2002 Nature Publishing Group http://genetics.nature.com