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To directly assess c-myc function in cellular proliferation, differentiation, and embryogenesis, we have used homologous recombination in embryonic stem cells to generate both heterozygous and homozygous c-myc mutant ES cell lines. The mutation is a null allele at the protein level. Mouse chimeras from seven heterozygous cell lines transmitted the mutant allele to their offspring. The analysis of embryos from two clones has shown that the mutation is lethal in homozygotes between 9.5 and 10.5 days of gestation. The embryos are generally smaller and retarded in development compared with their littermates. Pathologic abnormalities include the heart, pericardium, neural tube, and delay or failure in turning of the embryo. Heterozygous females have reduced fertility owing to embryonic resorption before 9.5 days of gestation in 14% of implanted embryos, c-Myc protein is necessary for embryonic survival beyond 10.5 days of gestation; however, it appears to be dispensable for cell division both in ES cell lines and in the the embryo before that time.

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The insulin-regulated aminopeptidase (IRAP) is a zincdependent membrane aminopeptidase. It is the homologue of the human placental leucine aminopeptidase. In fat and muscle cells, IRAP colocalizes with the insulin-responsive glucose transporter GLUT4 in intracellular vesicles and redistributes to the cell surface in response to insulin, as GLUT4 does. To address the question of the physiological function of IRAP, we generated mice with a targeted disruption of the IRAP gene (IRAP؊/؊). Herein, we describe the characterization of these mice with regard to glucose homeostasis and regulation of GLUT4. Fed and fasted blood glucose and insulin levels in the IRAP؊/؊ mice were normal. Whereas IRAP؊/؊ mice responded to glucose administration like control mice, they exhibited an impaired response to insulin. Basal and insulin-stimulated glucose uptake in extensor digitorum longus muscle, and adipocytes isolated from IRAP؊/؊ mice were decreased by 30 -60% but were normal for soleus muscle from male IRAP؊/؊ mice. Total GLUT4 levels were diminished by 40 -85% in the IRAP؊/؊ mice in the different muscles and in adipocytes. The relative distribution of GLUT4 in subcellular fractions of basal and insulin-stimulated IRAP؊/؊ adipocytes was the same as in control cells. We conclude that IRAP؊/؊ mice maintain normal glucose homeostasis despite decreased glucose uptake into muscle and fat cells. The absence of IRAP does not affect the subcellular distribution of GLUT4 in adipocytes. However, it leads to substantial decreases in GLUT4 expression.The insulin-regulated membrane aminopeptidase (IRAP) 1 was first identified as a major protein in intracellular vesicles isolated from the low density microsomes of fat and muscle cells that also harbor the insulin-responsive glucose transporter isotype GLUT4 (1-3). IRAP was earlier referred to as vp165 (vesicle protein of 165 kDa) (1) or gp160 (glycoprotein of 160 kDa) (2). IRAP has been well characterized in muscle and fat cells with regard to its subcellular localization and the regulation of its trafficking by insulin (1, 2, 4 -8). The results from these studies showed that IRAP behaves almost identically to GLUT4. Under basal conditions, IRAP and GLUT4 are efficiently sequestered in intracellular membrane compartments. When cells are treated with insulin, IRAP and GLUT4 translocate to the plasma membrane. In adipocytes, this results in an 8-and 10 -20-fold increase of IRAP and GLUT4, respectively, at the cell surface (4, 9, 10). GLUT4 and IRAP are the only known molecules in fat and muscle cells that exhibit such marked translocation in response to insulin.The physiological role of GLUT4 and the significance of its differential subcellular distribution under basal and insulinstimulated conditions are well established. The number of GLUT4 at the cell surface is the major determinant for the amount of glucose transported into muscle and fat tissues (10, 11). Since skeletal muscle accounts for 80% of glucose disposal after feeding (12), and glucose uptake is the rate-limiting step in glucos...

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