Preeclampsia is a heterogeneous syndrome affecting 3–5% of all pregnancies. An imbalance of the anti and pro-angiogenic factors, soluble receptor fms-like tyrosine kinase 1 (sFLT1) and placental growth factor (PGF), are thought to contribute to the pathophysiology of preeclampsia. Maternal plasma PGF and sFLT1 were quantified by specific immunoassays in cross-sectional samples from 130 preeclamptic subjects and 342 normotensive controls at delivery, and longitudinally in samples from 50 women who developed preeclampsia and 250 normotensive controls. Among women who developed preeclampsia, 46% (n=23) evidenced a pattern of consistently low maternal PGF across pregnancy below the lower 95%CI of controls from 15 weeks gestation to term. In contrast, the remaining 54% (n=27) women who developed preeclampsia had maternal PGF concentrations similar to or above (n=7) those of normotensive controls. Subjects with low PGF across pregnancy who developed preeclampsia evidenced significantly higher blood pressure in early pregnancy (p<0.05), and after diagnosis, earlier gestational age at delivery (p<0.05), and more preterm birth (p<0.05) compared to preeclamptic patients with high PGF. A significant subset of women who develop preeclampsia evidence consistently low PGF across pregnancy. Low PGF with preeclampsia was associated with preterm delivery compared to preeclamptic patients with high PGF. Identifying women with consistently low plasma PGF during pregnancy may provide a greater understanding of preeclampsia pathophysiology, and may provide more focused research and clinical activities.

Insulin stimulates phosphorylation of a tyrosine residue(s) on a 15-kDa protein (p15), and the cytosolic phosphorylated protein (pplS) accumulates only when 3T3-L1 adipocytes are treated with phenylarsine oxide. It has been shown previously that phenylarsine oxide, an agent that complexes vicinal dithiols, interrupts signal transmission from the insulin receptor to the glucose transport system. Several lines of evidence presented here indicate the involvement of ppl5 in insulin receptor-initiated signal transduction to the glucose transport system. The reciprocal effects of phenylarsine oxide on the insulin-activated accumulation of pplS and on insulin-stimulated hexose uptake are reversed by the vicinal dithiol 2,3-dimercaptopropanol but not by the monothiol 2-mercaptoethanol. Thus, a cellular dithiol appears to function in the signal transmission pathway downstream from ppl5. Like the insulin-activated autophosphorylation of the receptor's j3 subunit (on tyrosine), activation of phosphorylation of p15 is specific, with insulin-like growth factors 1 and 2, epidermal growth factor, and platelet-derived growth factor being inactive. Moreover, both processes exhibit identical insulin concentration dependence. The temporal kinetic relationship of insulin-activated receptor ,B-subunit phosphorylation, followed by the phosphorylation of p15 and then increased hexose uptake rate, is consistent with an intermediary signaling role for ppl5 in insulin-stimulated glucose uptake.By binding to its specific receptors on the plasma membrane ofthe adipocyte, insulin activates energy storage processesnotably, the uptake of glucose and fatty acids, glycogenesis, and lipogenesis. Considerable progress has been made in understanding the apparent initial events by which the receptor triggers these processes. The insulin receptor is an oligomeric transmembrane allosteric enzyme with an insulin binding site located extracellularly on the a subunit (1-5) and a tyrosine-specific protein kinase catalytic site on the intracellular domain of the (3 subunit (4-13). Studies with cell-free receptor preparations (7-14) and intact cells (15,16) cells. The function of this protein is unknown. In the present paper we report the insulin-stimulated and phenylarsine oxide-dependent accumulation of a 15-kDa phosphoprotein (ppl5) that is phosphorylated on tyrosine residues and appears to function in the activation of glucose uptake in 3T3-L1 adipocytes. Our previous studies (29) showed that phenylarsine oxide reversibly blocks activation of hexose uptake by insulin without affecting hexose uptake per se. Evidence presented in this paper indicates that ppl5 functions specifically with the insulin receptor. EXPERIMENTAL PROCEDURESCells. 3T3-L1 preadipocytes were cultured and differentiated as described previously (30). Briefly, cells were grown to confluence, and 2 days later conversion into adipocytes was induced by feeding Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, isobutylmethylxanthine, dexamethasone, and ...

ABSTRACT[32P]ppl5, the [32P]phosphorylated form of a specific cytosolic substrate of the insulin receptor tyrosihe kinase, was purified to homogeneity from mouse 3T3-L1 adipocytes incubated with 32p;. Evidence The pleiotropic actions of insulin are initiated by binding of the hormone to its specific cell-surface receptors on target cells (1). This insulin-receptor interaction stimulates autophosphorylation of the receptor by its intrinsic tyrosine kinase (1)(2)(3)(4)(5), thereby activating phosphorylation of protein substrates (6-8). These findings prompted a search for authentic cellular protein substrates of the receptor that become phosphorylated on tyrosine and act as intermediates in signal transmission by the receptor. Although several protein candidates have been identified (9-15), linkage between their phosphorylation and defined metabolic responses to insulin has not been established.Recently, we identified a cellular "target" of the insulin receptor tyrosine kinase in 3T3-L1 adipocytes that appears to function in signal transmission to the glucose transport system as illustrated below.The phosphorylation of this 15-kDa protein (p15) on tyrosine is stimulated by insulin, and its cellular accumulation is dependent upon the presence of phenylarsine oxide (PAO), an agent that forms stable ring complexes with vicinal dithiols. Our previous results have shown that PAO blocks insulinstimulated glucose uptake in 3T3-L1 adipocytes but has no effect on insulin binding to its receptor, insulin receptor autophosphorylation, or the receptor's tyrosine kinase activity (16-18). These findings led to the conclusion that the site of PAO action lies beyond the insulin receptor. The phosphorylation of p15 is highly specific for insulin in that insulin-like growth factors 1 and 2, epidermal growth factor, and plateletderived growth factor are inactive despite the presence of receptors for these growth factors on 3T3-L1 adipocytes (16). The temporal relationship between insulin receptor autophosphorylation, p15 phosphorylation, and insulin-stimulated glucose uptake is consistent with an intermediary role for phosphorylated p15 (pp15 In this paper we report the purification of [32P]ppl5 from 3T3-L1 adipocytes after treatment with insulin, vanadate, and PAO. Amino acid sequence analysis of phosphotyrosinecontaining peptides generated by incomplete tryptic cleavage of ppl5, as well as other results, shows that pplS is the phosphorylation product of 442(aP2) protein, an adipocyte homologue of myelin P2. The cDNA encoding the full-length mRNA for 422(aP2) protein was previously cloned and sequenced in this laboratory (22).EXPERIMENTAL PROCEDURES PAO was from Aldrich, digitonin from Sigma, and Na3VO4 from Fisher. Tosylphenylethyl chloromethyl ketone (TPCK)-treated trypsin was obtained from Worthington. All other reagents were from sources previously described (16, 19) or of the highest quality available.Labeling of Cells with 32Pi and Preparation of Cytosolic Fraction. Mouse 3T3-L1 preadipocytes in monolayer culture were indu...