The Fate of Angiotensin II in Placental Tissue and Blood.

The existence of a non-canonical pathway of renin-angiotensin system in the blood pressure control system has been highlighted over the past three decades. The enzymes involved in this pathway include a series of angiotensinases such as neprilysin (NEP), aminopeptidase A (APA), carboxypeptidase, and angiotensin converting enzyme 2. The physiological roles of these peptidases have been reconsidered in this study, based on the publications of other research groups and the results from our previous study, regarding the liberation of constituent amino acids from angiotensin II by placenta tissue and blood serum, respectively.

Recent Insights into Human Endometrial Peptidases in Blastocyst Implantation via Shedding of Microvesicles.

Blastocyst implantation involves multiple interactions with numerous molecules expressed in endometrial epithelial cells (EECs) during the implantation window; however, there is limited information regarding the molecular mechanism underlying the crosstalk. In blastocysts, fibronectin plays a major role in the adhesion of various types of cells by binding to extracellular matrix proteins via the Arg-Gly-Asp (RGD) motif. In EECs, RGD-recognizing integrins are important bridging receptors for fibronectin, whereas the non-RGD binding of fibronectin includes interactions with dipeptidyl peptidase IV (DPPIV)/cluster of differentiation (CD) 26. Fibronectin may also bind to aminopeptidase N (APN)/CD13, and in the endometrium, these peptidases are present in plasma membranes and lysosomal membranes. Blastocyst implantation is accompanied by lysosome exocytosis, which transports various peptidases and nutrients into the endometrial cavity to facilitate blastocyst implantation. Both DPPIV and APN are released into the uterine cavity via shedding of microvesicles (MVs) from EECs. Recently, extracellular vesicles derived from endometrial cells have been proposed to act on trophectoderm cells to promote implantation. MVs are also secreted from embryonal stem cells and may play an active role in implantation. Thus, crosstalk between the blastocyst and endometrium via extracellular vesicles is a new insight into the fundamental molecular basis of blastocyst implantation.

The balance between fetal oxytocin and placental leucine aminopeptidase (P-LAP) controls human uterine contraction around labor onset.

A fetal pituitary hormone, oxytocin which causes uterine contractions, increases throughout gestation, and its increase reaches 10-fold from week 32 afterward. Oxytocin is, on the other hand, degraded by placental leucine aminopeptidase (P-LAP) which exists in both terminal villi and maternal blood. Maternal blood P-LAP increases with advancing gestation under the control of non-genomic effects of progesterone, which is also produced from the placenta. Progesterone is converted to estrogen by CYP17A1 localized in the fetal adrenal gland and placenta at term. The higher oxytocin concentrations in the fetus than in the mother demonstrate not only fetal oxytocin production but also its degradation and/or inhibition of leakage from fetus to mother by P-LAP. Until labor onset, the pregnant uterus is quiescent possibly due to the balance between increasing fetal oxytocin and P-LAP under control of progesterone. A close correlation exists between the feto-placental and maternal units in the placental circulation, although the blood in the two circulations does not necessarily mix. Fetal maturation results in progesterone withdrawal via the CYP17A1 activation accompanied with fetal oxytocin increase. Contribution of fetal oxytocin to labor onset has been acknowledged through the recognition that the effect of fetal oxytocin in the maternal blood is strictly regulated by its degradation by P-LAP under the control of non-genomic effects of progesterone. In all senses, the fetus necessarily takes the initiative in labor onset.

Crosstalk between foetal vasoactive peptide hormones and placental aminopeptidases regulates placental blood flow: Its significance in preeclampsia.

In pregnancy, placental circulation occurs through two independent circulation systems: foetoplacental and uterine (spiral artery)-placental lake. Crosstalk between the foetal peptide hormones, angiotensin II (A-II) and vasopressin (AVP), and their degrading placental aminopeptidases (APs), aminopeptidase A for A-II and placental leucine aminopeptidase for both AVP and oxytocin, primarily regulate placental circulation. On the other hand, placental circulation represents an arteriovenous shunt. In normal pregnancy, the blood pressure decreases, despite increased cardiac output and plasma volume, probably due to the arteriovenous shunt in the growing placenta. Actually, the foetal vasoactive hormones in the foetoplacental circulation are much higher than those in the maternal circulation throughout pregnancy. In normal pregnancy, AP activity derived from the placenta in maternal blood increases with gestation and placental growth. Foetal hypoxia increases the secretion of foetal both AVP and A-II. Although there is an increase in both AP activities in the maternal blood in normal pregnancy, their activities increase more than those in normal pregnancy during mild preeclampsia. However, both AP activities decline significantly compared than those in severe preeclampsia. This suggests that AP prevents leakage of increased foetal vasoactive hormones into the maternal blood in mild preeclampsia, and its protective role breaks down in severe preeclampsia, leading to a massive leak of the hormones into maternal circulation and consequent marked contraction of both the maternal vessels and the uterus. Consequently, AP activity in both placenta and maternal blood acts as the foeto-maternal barrier for foetal vasoactive hormones and thus contributes to the onset of preeclampsia.

Placental leucine aminopeptidase- and aminopeptidase A- deficient mice offer insight concerning the mechanisms underlying preterm labor and preeclampsia.

Preeclampsia and preterm delivery are important potential complications in pregnancy and represent the leading causes for maternal and perinatal morbidity and mortality. The mechanisms underlying both diseases remain unknown, thus available treatments (beta2-stimulants and magnesium sulfate) are essentially symptomatic. Both molecules have molecular weights less than 5-8 kDa, cross the placental barrier, and thus exert their effects on the fetus. The fetus produces peptides that are highly vasoactive and uterotonic and increase in response to maternal stress and with continued development. Fetal peptides are also small molecules that inevitably leak across into the maternal circulation. Aminopeptidases such as placental leucine aminopeptidase (P-LAP) and aminopeptidase A (APA) are large molecules that do not cross the placental barrier. We have shown that APA acts as an antihypertensive agent in the pregnant spontaneously hypertensive rat by degrading vasoactive peptides and as a result returns the animal to a normotensive state. P-LAP also acts as an antiuterotonic agent by degrading uterotonic peptides and thus prolongs gestation in the pregnant mouse. Given the ever increasing worldwide incidences of preeclampsia and preterm labor, it is imperative that new agents be developed to safely prolong gestation. We believe that the use of aminopeptidases hold promise in this regard.

Placental Leucine Aminopeptidase as a Potential Specific Urine Biomarker for Invasive Ovarian Cancer.

BACKGROUND: A non-invasive and sensitive biomarker for the detection of ovarian cancer (OvCa) is lacking. We aim to investigate if urinary placental leucine aminopeptidase (P-LAP) can serve as a reliable biomarker for OvCa. METHODS: P-LAP activity was measured using a LAP assay kit (Serotech Co., Ltd., Sapporo, Japan) in the urine of 22 patients with benign or borderline malignant ovarian tumors and 18 patients with OvCa. In this assay, L-methionine was added at 20 mM because P-LAP is functional, but other aminopeptidases are inhibited at this dose of L-methionine. RESULTS: The mean urinary P-LAP activity was significantly higher in the OvCa group than in the benign or borderline malignant tumor group. When the cut-off value of P-LAP was determined as 11.00 U/L, its sensitivity and specificity for differentiating invasive cancer were 77.8% and 95.5%, respectively. CONCLUSION: Although the usefulness of this test should be confirmed in a larger cohort of cases and controls, our study is the first to highlight the importance of urinary P-LAP as a biomarker for OvCa.

The expression of dipeptidyl peptidase IV (DPPIV/CD26) is associated with enhanced chemosensitivity to paclitaxel in epithelial ovarian carcinoma cells.

Dipeptidyl peptidase IV (DPPIV/CD26) is a multifunctional cell surface aminopeptidase that is widely expressed in different cell types. Recent studies have suggested that DPPIV plays an important role in tumor progression in several human malignancies. In the current study, we investigated the role of DPPIV in paclitaxel resistance in epithelial ovarian carcinoma (EOC) cells. We first examined the correlation between expression levels of DPPIV and sensitivity to paclitaxel in various EOC cell lines. Subsequently, to clarify the cellular functions of DPPIV, we investigated the role of this molecule in the sensitivity of EOC to paclitaxel in vitro and in vivo using stably DPPIV-transfected EOC cells. We identified a positive correlation between DPPIV expression and paclitaxel sensitivity in various EOC cell lines. In addition, we observed a significant increase in the paclitaxel sensitivity of DPPIV-overexpressing EOC cells. Furthermore, no apparent alteration in paclitaxel sensitivity was noted by the addition of a specific inhibitor of DPPIV activity in DPPIV-transfected or natively DPPIV-overexpressing EOC cells. In a subcutaneous murine model treated with paclitaxel, on Day 39, the tumor size of the DPPIV-transfected cell-inoculated group was as large as that of the vector-transfected cell-inoculated group. In contrast, on Day 61, the former was smaller than the latter. The present findings show that DPPIV may be involved in the increased sensitivity to paclitaxel of EOC cells regardless of the involvement of DPPIV activity.

New insights into human endometrial aminopeptidases in both implantation and menstruation.

The endometrium cycle involves proliferation of endometrial epithelial cells in preparation for implantation of fertilized ovum. With ovulation, the endometrium secretes nutrients such as peptides and amino acids into the endometrial cavity. The histological evidence of ovulation in normal menstrual cycle includes subnuclear glycogen vacuoles surrounded by placental leucine aminopeptidase (P-LAP) in endometrial epithelial cells. P-LAP is an essentially involved in intracellular trafficking of glucose transporter (GLUT) 4 which is primarily important for glucose uptake in skeletal muscles and fat tissues. On the other hand, glucose influx from blood into endometrial epithelial cells is not mainly mediated by GLUTs, but by coincident appearing progesterone just after ovulation. Progesterone increases permeability of not only plasma membranes, but also lysosomal membranes, and this may be primarily involved in glucose influx. Progesterone also expands the exocytosis in the endometrium after ovulation, and endometrial secretion after ovulation is possibly apocrine and holocrine, which is augmented and exaggerated exocytosis of the lysosomal contents. The endometrial spiral arteries/arterioles are surrounded by endometrial stromal cells which are differentiated into decidual/pre-decidual cells. Decidual cells are devoid of aminopeptidase A (APA), possibly leading to enhancement of Angiotensin-II action in decidual cell area due to loss of its degradation by APA. Angiotensin-II is thought to exert growth-factor-like effects in post-implantation embryos in decidual cells, thereby contributing to implantation. Without implantation, angiotensin-II constricts the endometrial spiral arteries/arterioles to promote menstruation. Thus, P-LAP and APA may be involved in homeostasis in uterus via regulating glucose transport and vasoconstrictive peptides.

Inhibition of APN/CD13 leads to suppressed progressive potential in ovarian carcinoma cells.

BACKGROUND: Aminopeptidase N (APN/CD13), a 150-kDa metalloprotease, is a multifunctional cell surface aminopeptidase with ubiquitous expression. Recent studies have suggested that APN/CD13 plays an important role in tumor progression of several human malignancies. In the current study, we investigated the role of APN/CD13 in ovarian carcinoma (OVCA) progression. METHODS: We first examined the expression of APN/CD13 at the protein level in a variety of OVCA cell lines and tissues. We subsequently investigated whether there was a correlation between APN/CD13 expression and invasive potential of various OVCA cell lines. Moreover, we investigated the function of APN/CD13 in OVCA cells using bestatin, an APN/CD13 inhibitor, or transfection of siRNA for APN/CD13. RESULTS: We confirmed that APN/CD13 was expressed in OVCA tissues and cell lines to various extents. There was a positive correlation between APN/CD13 expression and migratory potential in various OVCA cell lines with accordingly enhanced secretion of endogenous MMP-2. Subsequently, we found a significant decrease in the proliferative and migratory abilities of OVCA cells after the addition of bestatin or the inhibition of APN/CD13 expression by siRNA. Furthermore, in an animal model, daily intraperitoneal administration of bestatin after inoculation of OVCA cells resulted in a decrease of peritoneal dissemination and in prolonged survival of nude mice. CONCLUSION: The current data indicate the possible involvement of APN/CD13 in the development of OVCA, and suggest that clinical use of bestatin may contribute to better prognosis for ovarian carcinoma patients.

P-LAP/IRAP-induced cell proliferation and glucose uptake in endometrial carcinoma cells via insulin receptor signaling.

BACKGROUND: Hyperglycemia or hyperinsulinemia contributes to poorer endometrial cancer survival. It was shown that P-LAP/IRAP translocates to the plasma membrane in response to insulin stimulation. Recently, we demonstrated that P-LAP/IRAP is associated with a poor prognosis in endometrial adenocarcinoma patients. The aim of this study was to examine whether the malignant potential of endometrial cancer enhanced by P-LAP/IRAP is due to increased glucose uptake via the P-LAP/IRAP-mediated activation of insulin signaling. METHODS: We transfected P-LAP/IRAP cDNA into A-MEC cells (endometrial adenocarcinoma cell line), and A-MEC-LAP cells expressed a remarkably high level of GLUT4 proteins. RESULTS: 3H-2-deoxyglucose uptake which responds to insulin in A-MEC-LAP cells was significantly higher than that of A-MEC-pc cells. A-MEC-LAP cells exhibited a significant growth-stimulatory effect compared to A-MEC-pc cells. A-MEC-LAP cells expressed a remarkably high level of p85PI3K protein compared to A-MEC-pc cells, and showed a higher degree of AKT phosphorylation by insulin stimulation. CONCLUSION: In summary, P-LAP/IRAP was involved in the increasing malignant potential of endometrial cancer mediated by insulin. P-LAP/IRAP was suggested to be a potential new target of molecular-targeted therapy for endometrial cancer.

Involvement of placental peptidases associated with renin-angiotensin systems in preeclampsia.

Preeclampsia is characterized by pregnancy-induced hypertension accompanied with protein urea and generalized edema. Preeclampsia develops during the second half of pregnancy and resolves postpartum promptly, implicating the placenta as a primary cause in the disorder. Normal pregnancy is associated with reductions in arterial pressure and attenuated pressor response to exogenous infused angiotensin II (ANG II). In contrast, women with preeclampsia show the similar sensitivity to the pressor effect of ANG II as do non-pregnant women. To elucidate the involvement of placental peptidases associated with renin-angiotensin systems, we determined the localization of angiotensin-converting enzyme (ACE) and aminopeptidase A (AP-A), ANG II degrading enzyme, in the placenta and compared the expression of mRNA and protein in uncomplicated and preeclamptic placenta. In addition, AP-A expression in trophoblastic cells treated with ANG II and ACE expression in HUVECs under hypoxic condition were analyzed, respectively. The expression of both peptidases in the preeclamptic placenta was significantly higher than those from uncomplicated. ACE was primarily localized to venous endothelial cells of stem villous whereas AP-A expression was recognized in the trophoblast and pericytes of fetal arterioles and venules within stem villous. Hypoxia induced ACE expression in HUVECs while both hypoxia and ANG II evoked AP-A expression in trophoblast. These results suggested that hypoxic condition in preeclampsia induces ACE activation in feto-placental unit to maintain the fetal hemodynamics and placental AP-A plays a role as a component of the barrier of ANG II between mother and fetus.

Dipeptidyl peptidase IV in tumor progression.

Dipeptidyl peptidase IV (DPPIV) is a 110-kDa glycoprotein with ubiquitous expression. Several recent studies have shown that DPPIV affects tumor progression in several human malignancies. We found that ovarian carcinoma cell lines with higher DPPIV expression showed less invasive potential. Furthermore, introduction of DPPIV cDNA into SKOV3 cells (SKDPIV), derived from serous cystadenocarcinoma showing little DPPIV expression, caused a significant decrease in both migration and invasive potential. In addition, nude mice inoculated with SKDPIV cells showed significantly less peritoneal dissemination and longer survival time than those inoculated with parental or vector-transfected cells. We further examined the mechanisms of anti-invasive ability of DPPIV. The expression of E-cadherin was positively correlated with DPPIV expression among five independent ovarian carcinoma cell lines. The SKDPIV cells showed enhanced expression of E-cadherin with a cellular morphological change from a fibroblastic and motile phenotype to an epithelial phenotype compared to parental and MOCK cells. In addition, matrix metalloproteinase 2 (MMP-2) and membrane type 1 matrix metalloprotease (MT1-MMP), which are important markers associated with invasive and metastatic potential, were remarkably reduced in SKDPIV cells. In contrast, tissue inhibitors of matrix metalloproteinases (TIMPs) were enhanced by DPPIV transfection. These findings imply that DPPIV may functionally suppress peritoneal dissemination and progression of ovarian carcinoma by regulating the expression levels of several molecules associated with carcinoma cell invasion and progression.

Gene regulation and physiological function of placental leucine aminopeptidase/oxytocinase during pregnancy.

Human pregnancy serum and placenta have the ability to degrade uterotonic peptide oxytocin (OT). Placental leucine aminopeptidase (P-LAP), which is also called cystine aminopeptidase, is the only membrane aminopeptidase known to functionally degrade OT as oxytocinase (OTase). P-LAP/OTase hydrolyzes several peptides other than OT including vasopressin and angiotensin III. P-LAP/OTase predicted from cDNA sequence is a type II integral membrane protein, which is converted to a soluble form existing in maternal serum by metalloproteases, possibly ADAM (a disintegrin and metalloproteinase) members. P-LAP/OTase activity increases with normal gestation, while decreases in the patients with preterm delivery and severe preeclampsia. In placenta, P-LAP/OTase is predominantly expressed in differentiated trophoblasts, syncytiotrophoblasts. Activator protein-2 (AP-2) and Ikaros transcription factors play significant roles in exerting high promoter activity of P-LAP/OTase in the trophoblastic cells. Moreover, P-LAP/OTase is transcriptionally regulated in a trophoblast-differentiation-dependent fashion via up-regulation of AP-2, putatively AP-2alpha. P-LAP/OTase may be involved in maintaining pregnancy homeostasis via metabolizing peptides such as OT and vasopressin.

Endoplasmic reticulum aminopeptidase-1 mediates leukemia inhibitory factor-induced cell surface human leukocyte antigen-G expression in JEG-3 choriocarcinoma cells.

Maternal immune tolerance is required for extravillous trophoblasts (EVTs) to invade the decidua without rejection. Endoplasmic reticulum aminopeptidase-1 (ERAP1) generates human leukocyte antigen (HLA) class I-adapted antigenic peptides, but its function in trophoblasts lacking classical HLA class I molecules remains undetermined. Leukemia inhibitory factor (LIF) is produced from decidua during the implantation period and plays a necessary role in establishing pregnancy. This study is intended to investigate the location and the function of ERAP1 in trophoblastic cells, focusing on LIF. Immunohistochemistry showed strong ERAP1 expression in cultured EVTs. In choriocarcinoma cell lines used as a model for trophoblasts, ERAP1 was expressed more intensively in JEG-3 than BeWo cells. Immunoblot analysis and immunocytochemistry localized ERAP1 to the endoplasmic reticulum (ER) in JEG-3 cells. Flow cytometry with HLA-G antibody to monitor the supply of antigenic peptides presenting to HLA-G in the ER showed that reducing ERAP1 transcripts by RNA interference did not affect cell surface expression of membrane HLA-G1 (mHLA-G1) in JEG-3 cells under basal conditions. In LIF-treated JEG-3 cells, cell surface mHLA-G1 expression was increased along with ERAP1 protein and promoter activities. In contrast to nonstimulated cells, eliminating ERAP1 from LIF-treated JEG-3 cells reduced the cell surface mHLA-G1 expression and soluble HLA-G1 secretion. This study provides the first evidence showing that ERAP1 is localized in the ER of trophoblasts and is involved in regulating cell surface HLA-G expression in the presence of LIF. Consequently, ERAP1 would function to present antigenic peptides to HLA-G in trophoblasts.

Lack of angiotensin II conversion to angiotensin III increases water but not alcohol consumption in aminopeptidase A-deficient mice.

Elevated central concentrations of the vasopressor octapeptide angiotensin (Ang) II increase the water intake in mammals. Recently, we showed that central AngII is also crucial in alcohol-consuming behavior. Since the heptapeptide AngIII, an AngII metabolite, is discussed to mediate AngII-related effects, we investigated water and alcohol consumption in mice, genetically deficient in aminopeptidase A (APA), a peptidase responsible for AngII conversion to AngIII. Sixteen male APA-deficient mice and their age matched wild-type controls were monitored on their water intake under basal conditions and total fluid and alcohol intake before and after social stress in a two-bottle free-choice paradigm. Alterations were connected to the regulation in activity of Ang-related peptidases (APA, ACE; ACE2) in brain regions involved in alcohol intake and peripheral organs. In comparison to their wild-type controls, APA-deficient mice drank significantly more water but not more alcohol at all investigated time points. A reduction in water intake, as observed in wild-type animals after social stress, did not occur in knockout mice. However, the reduction in alcohol consumption after social stress was significantly reduced in both strains. Alcohol consumption upregulated all three peptidases in the kidney, but not in lung. Notable, renal ACE2 activity was significantly higher in APA-deficient mice under basal condition. While the inhibition of AngII metabolism to AngIII does not influence the alcohol intake, water consumption in mice deficient for APA was significantly elevated. These differences induced by an altered AngII/AngIII ratio oppose the hypothesis that central AngII and AngIII act in a congruent pattern.

Functional expression of the angiotensin II type 1 receptor in human ovarian carcinoma cells and its blockade therapy resulting in suppression of tumor invasion, angiogenesis, and peritoneal dissemination.

PURPOSE: Angiotensin II is a bioactive peptide of the renin-angiotensin system, acting not only as a vasoconstrictor but also as a growth promoter via angiotensin II type 1 receptors (AT1R). The present study examined AT1R expression in human ovarian carcinoma and attempted to determine whether AT1R blocker could suppress the tumor progression. EXPERIMENTAL DESIGN: Expression of AT1R, vascular endothelial growth factor (VEGF), and CD34 was immunohistochemically analyzed in ovarian tumor tissues (n=99). Effects of AT1R blocker on invasive potential and VEGF secretion in ovarian cancer cells were examined in vitro. Effects of AT1R blocker in vivo were evaluated in a mouse model of peritoneal carcinomatosis. RESULTS: AT1R was expressed in 57 of 67 (85%) invasive ovarian adenocarcinomas and 12 of 18 (66%) borderline malignant tumors but in only 2 of 14 (14%) benign cystadenomas. In invasive carcinomas, VEGF expression intensity and intratumor microvessel density were significantly higher in cases that were strongly positive for AT1R (n = 37) compared with those in cases weakly positive (n = 20) or negative (n = 10) for AT1R. Angiotensin II significantly enhanced the invasive potential and VEGF secretion in AT1R-positive SKOV-3 ovarian cancer cells, both of which were completely inhibited by the AT1R blocker candesartan. Administration of candesartan into SKOV-3-transplanted athymic mice resulted in the reduction of peritoneal dissemination, decreased ascitic VEGF concentration, and suppression of tumor angiogenesis. CONCLUSIONS: AT1R is functionally expressed in ovarian carcinoma and involved in tumor progression and angiogenesis. AT1R blockade therapy may become a novel and promising strategy for ovarian cancer treatment.

Neutral endopeptidase 24.11/CD10 suppresses progressive potential in ovarian carcinoma in vitro and in vivo.

Recently, numerous studies have shown that endothelin-1 (ET-1) is expressed in ovarian carcinoma and that ET-1 selectively acts as an autocrine or paracrine growth factor through the endothelin A receptor (ET(A)R), and is involved in cell proliferation, invasiveness, neovascularization, and prevention of apoptosis. Neutral endopeptidase 24.11 (NEP) is a cell surface aminopeptidase with a ubiquitous expression and is capable of degrading a number of bioactive peptides including ET-1. Our previous report showed that stromal NEP expression in ovarian carcinoma was down-regulated as the histologic grade advanced. Here, we confirmed that NEP was expressed in tumor cells as well as stromal tissues in ovarian carcinoma, and investigated the functions of NEP in this carcinoma. We showed that there was a significant decrease in cell proliferation and invasiveness with a reduction in the concentration of ET-1 in the conditioned medium on the NEP overexpression of NEP in ovarian carcinoma cells. In addition, the overexpression of NEP enhanced susceptibility to paclitaxel, resulting in an increased occurrence of apoptotic morphologic change. Furthermore, tumorigenesis was reduced in vivo with the overexpression of NEP, down-regulation of both matrix metalloproteinase-2, and vascular endothelial growth factor expression. This evidence suggests that NEP functionally suppresses the progression of ovarian carcinoma and further study of this enzyme may reveal an effective way to target ET-1 for the treatment of this carcinoma.

Impaired insulin-regulated membrane aminopeptidase translocation to the plasma membrane in adipocytes of Otsuka Long Evans Tokushima Fatty rats.

Insulin-regulated membrane aminopeptidase (IRAP) translocates to the plasma membrane with glucose transporter-4 (GLUT4) on insulin stimulation. However, this may be impaired in patients at risk of diabetes. Recently a novel technique has been developed to assess cell surface IRAP activity dynamically using a fluorogenic membrane impermeable substrate. In this study we measured the cell surface IRAP activity and 3-O-[methyl-14C]-D-Glucose uptake in adipocytes isolated from Otsuka Long Evans Tokushima Fatty rats (OLETF), developed as a model of type 2 diabetes mellitus, to evaluate whether the translocation of GLUT4/IRAP vesicles is affected. On the addition of insulin, the cell surface IRAP activity promptly increased to reach equilibrium in a hormone dose-dependent manner. OLETF rats showed significantly lower equilibrium activity than control rats (P<0.01). Time to reach the equilibrium was also significantly longer in the OLETF case, and adipocytes isolated from OLETF rats demonstrated both a delay and a reduction in 3-O-[methyl-14C]-D-Glucose uptake. This impairment in all parameters was alleviated by treatment with pioglitazone. Continuous measurement of cell surface IRAP activity allowed accurate evaluations of GLUT4/IRAP vesicle translocation and of the establishment of defects in OLETF rats.

Prolonged survival and decreased invasive activity attributable to dipeptidyl peptidase IV overexpression in ovarian carcinoma.

Dipeptidyl peptidase IV (DPPIV) is a multifunctional cell surface aminopeptidase with ubiquitous expression. Recent studies have suggested that DPPIV plays an important role in tumor progression in several human malignancies. In the present study, we investigated the correlation between DPPIV expression and progressive potential in ovarian carcinoma. We demonstrated that ovarian carcinoma cell lines with higher DPPIV expression were less invasive. Furthermore, DPPIV overexpression in SKOV3 cells, derived from serous cystadenocarcinoma, with little DPPIV expression induced a dramatic change in cellular morphology and a significant decrease in the abilities of both migration and invasion. In addition, we have also shown that nude mice inoculated with DPPIV-transfected SKOV3 cells showed significantly less peritoneal dissemination and longer survival time than those receiving the parental or vector-only transfected cells (mean survival time, 64.9 +/- 4.7, 35.7 +/- 2.8, and 36.6 +/- 1.8 days, respectively). This evidence implies that DPPIV may functionally suppress peritoneal dissemination in ovarian carcinoma.

Dipeptidyl peptidase IV overexpression induces up-regulation of E-cadherin and tissue inhibitors of matrix metalloproteinases, resulting in decreased invasive potential in ovarian carcinoma cells.

Dipeptidyl peptidase IV (DPPIV/CD26) is a multifunctional cell surface aminopeptidase that is widely expressed in different cell types. Our previous study demonstrated a possible link between DPPIV expression and decreased i.p. dissemination and loss of invasive potential of ovarian carcinoma. In this report, we examined the mechanisms of the anti-invasive ability of DPPIV in greater detail. Expression of E-cadherin and beta-catenin was positively correlated with DPPIV expression among five independent ovarian carcinoma cell lines. The introduction of DPPIV cDNA into an ovarian carcinoma cell line (SKOV3) with low DPPIV expression enhanced the expression of E-cadherin and beta-catenin, with a cellular morphological change from a fibroblastic and motile phenotype to an epithelial phenotype. In addition, matrix metalloproteinase 2 and membrane type 1 matrix metalloproteinase, important markers associated with invasive and metastatic potential, were remarkably reduced. In contrast, tissue inhibitors of matrix metalloproteinases were up-regulated by DPPIV transfection. Furthermore, suppression of the phosphorylation levels of mitogen-activated protein kinase isoform, extracellular signal-regulated kinase, was observed in DPPIV-overexpressing cells. To our knowledge, this is the first evidence that increasing DPPIV expression may contribute to prolonged survival by up-regulation of E-cadherin and tissue inhibitors of matrix metalloproteinases.