We hypothesized that insulin stimulates phosphorylation of CEACAM1 which in turn leads to upregulation of receptor-mediated insulin endocytosis and degradation in the hepatocyte. We have generated transgenic mice over-expressing in liver a dominant-negative, phosphorylation-defective S503A-CEACAM1 mutant. Supporting our hypothesis, we found that S503A-CEACAM1 transgenic mice developed hyperinsulinemia resulting from impaired insulin clearance. The hyperinsulinemia caused secondary insulin resistance with impaired glucose tolerance and random, but not fasting, hyperglycemia. Transgenic mice developed visceral adiposity with increased amounts of plasma free fatty acids and plasma and hepatic triglycerides. These findings suggest a mechanism through which insulin signaling regulates insulin sensitivity by modulating hepatic insulin clearance.

Insulin is viewed as a positive regulator of fatty acid synthesis by increasing fatty acid synthase (FAS) mRNA transcription. We uncover a new mechanism by which insulin acutely reduces hepatic FAS activity by inducing phosphorylation of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and its interaction with FAS. Ceacam1 null mice (Cc1(-/-)) show loss of insulin's ability to acutely decrease hepatic FAS activity. Moreover, adenoviral delivery of wild-type, but not the phosphorylation-defective Ceacam1 mutant, restores the acute effect of insulin on FAS activity in Cc1(-/-) primary hepatocytes. Failure of insulin to acutely reduce hepatic FAS activity in hyperinsulinemic mice, including L-SACC1 transgenics with liver inactivation of CEACAM1, and Ob/Ob obese mice, suggests that the acute effect of insulin on FAS activity depends on the prior insulinemic state. We propose that this mechanism acts to reduce hepatic lipogenesis incurred by insulin pulses during refeeding.

CEACAM1, a tumor suppressor (previously known as pp120), is a plasma membrane protein that undergoes phosphorylation on Tyr 488 in its cytoplasmic tail by the insulin receptor tyrosine kinase. Co-expression of CEACAM1 with insulin receptors decreased cell growth in response to insulin. Co-immunoprecipitation experiments in intact NIH 3T3 cells and glutathione S-transferase pull-down assays revealed that phosphorylated Tyr 488 in CEACAM1 binds to the SH2 domain of Shc, another substrate of the insulin receptor. Overexpressing Shc SH2 domain relieved endogenous Shc from binding to CEACAM1 and restored MAP kinase activity, growth of cells in response to insulin, and their colonization in soft agar. Thus, by binding to Shc, CEACAM1 sequesters this major coupler of Grb2 to the insulin receptor and down-regulates the Ras/MAP kinase mitogenesis pathway. Additionally, CEACAM1 binding to Shc enhances its ability to compete with IRS-1 for phosphorylation by the insulin receptor. This leads to a decrease in IRS-1 binding to phosphoinositide 3-kinase and to the down-regulation of the phosphoinositide 3-kinase/Akt pathway that mediates cell proliferation and survival. Thus, binding to Shc appears to constitute a major mechanism for the down-regulatory effect of CEACAM1 on cell proliferation.Insulin binding to its receptor activates its tyrosine kinase to cause phosphorylation of the receptor and of endogenous substrates, including CEACAM1 (previously known as pp120) (1), insulin receptor substrate proteins (IRS-1-4), 1 Shc (2, 3), and others. Phosphorylation of different substrates is required to mediate the diverse effects of hormones on metabolism and growth (4 -6). Shc is a Src homology 2 (SH2)-containing cytoplasmic adaptor protein that undergoes phosphorylation by receptors of the tyrosine kinase family (7). Activation of receptors causes redistribution of Shc from the perinuclear region to the cytosolic site of the plasma membrane (8). The tyrosine kinase receptors that phosphorylate Shc include insulin and insulin-like growth factor 1 (IGF-1) receptors (2, 9). The Shc family of proteins consists of three isoforms. p46/p52 that are ubiquitously expressed are the products of the same transcript and result from alternative usage of two in-frame ATGs. In contrast, p66 that is mostly expressed in epithelial cells is translated from a different transcript (10, 11). The three isoforms have overlapping domains as follows: an SH2 domain at the C terminus, an adjacent glycine/proline-rich collagen homology (CH1) domain, and a phosphotyrosine binding (PTB) domain in the N terminus of p46/p52. p66Shc contains an additional collagen homology (CH2) domain at its N terminus end.By binding to other signaling proteins, Shc exerts many effects on the cell. Upon its phosphorylation on Tyr 317 in the CH1 domain by the insulin receptor, Shc binds to the SH2 domain of Grb2 and couples it to the receptor (12, 13). This leads to the association of Grb2 with the Son of Sevenless Ras GDP/GTP exchanger, causing translocation of Son of Sevenless...

Incubation of uterine explants from immature rats with 0.01-100 ng of 17 beta-estradiol/mL resulted in approximately a fivefold increase in the number of oxytocin receptors per milligram of protein in 48 h. This increase was maintained for at least an additional 48 h in the presence of estrogen. When the explants were incubated with 1 microgram progesterone/mL from the outset, the concentration of oxytocin receptors was the same as initial (0 time) levels. The estrogen-induced increase in oxytocin receptor concentration was blocked by incubation with cycloheximide, an inhibitor of protein synthesis. Once increased, however, the concentration of oxytocin receptors exhibited no turnover for at least a 48-h period in the presence of estrogen. The addition of progesterone and estrogen to explants with elevated receptor levels resulted in almost a 60% reduction in oxytocin receptor concentration by 24 h, with no change in affinity of the receptor for oxytocin. The reduction in receptor concentration by progesterone was not prevented by cycloheximide. The progesterone effect may involve inactivation or degradation of oxytocin receptors or activation of substances that are inhibitory to oxytocin binding. The effects of estradiol and progesterone on oxytocin receptor concentration in uterine explants are similar to those seen when the steroids are administered in vivo. The explant system, therefore, should prove useful in clarifying factors and processes that are involved in regulation of oxytocin receptor concentration in the uterus and in the initiation of parturition in the rat.

pp120 (Ceacam 1) undergoes ligand-stimulated phosphorylation by the insulin receptor, but not by the insulin-like growth factor 1 receptor (IGF-1R). This differential phosphorylation is regulated by the C terminus of the ␤-subunit of the insulin receptor, the least conserved domain of the two receptors. In the present studies, deletion and site-directed mutagenesis in stably transfected hepatocytes derived from insulin receptor knockout mice (IR ؊/؊ ) revealed that Tyr 1316 , which is replaced by the nonphosphorylatable phenylalanine in IGF-1R, regulated the differential phosphorylation of pp120 by the insulin receptor. Similarly, the nonconserved Tyr 1316 residue also regulated the differential effect of pp120 on IGF-1 and insulin mitogenesis, with pp120 downregulating the growth-promoting action of insulin, but not that of IGF-1. Thus, it appears that pp120 phosphorylation by the insulin receptor is required and sufficient to mediate its downregulatory effect on the mitogenic action of insulin. Furthermore, the current studies revealed that the C terminus of the ␤-subunit of the insulin receptor contains elements that suppress the mitogenic action of insulin. Because IR ؊/؊ hepatocytes are derived from liver, an insulin-targeted tissue, our observations have finally resolved the controversy about the role of the least-conserved domain of insulin and IGF-1Rs in mediating the difference in the mitogenic action of their ligands, with IGF-1 being more mitogenic than insulin.The insulin receptor is essential to mediate insulin action on target cells (1,27). It is a cell surface glycoprotein of a heterotetrameric structure that consists of two ␣-and two ␤-subunits. The extracellular ␣-subunits contain the insulin binding domains, and the transmembrane ␤-subunits contain the tyrosine kinase and the phosphorylation sites. Insulin binding to its receptor activates the tyrosine kinase to phosphorylate the receptor and other endogenous substrates, such as pp120 (Ceacam 1) (5a, 44), insulin receptor substrate proteins (IRS-1, -2, -3, and -4), Shc, and others (reviewed in references 65 and 66). Phosphorylation of different substrates is required to mediate the diverse effects of hormones on metabolism and growth (3,60,68).Insulin and insulin-like growth factor 1 (IGF-1) receptors are structurally related, and all conserved tyrosine residues that are phosphorylated in the insulin receptor in response to insulin are also phosphorylated in the IGF-1 receptor in response to 17,23,48,71). Moreover, these receptors share many substrates, such as Shc and members of the IRS family, phosphorylation of which is regulated by the conserved Tyr 960 in the juxtamembrane domain of the insulin receptor (18, 22, 67) and its corresponding residue in the IGF-1 receptor (8). Phosphorylated IRS-1 engages, in turn, in the formation of signaling complexes via phosphotyrosine-containing binding motifs with Src homology 2 (SH2) found in molecules like growth factor receptor binding protein (GRB2) (32, 56), Syp (SH PTP2) phosphotyrosine phosphatase...