Neuregulin 4 Downregulation Induces Insulin Resistance in 3T3-L1 Adipocytes through Inflammation and Autophagic Degradation of GLUT4 Vesicles.

The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy.

Conventional dendritic cells require IRAP-Rab14 endosomes for efficient cross-presentation.

Dendritic cells (DCs) use cellular pathways collectively referred to as cross-presentation to stimulate CD8(+) T cells with peptide Ags derived from internalized, exogenous Ags. We have recently reported that DCs rely on aminoterminal trimming of cross-presented peptides by insulin-responsive aminopeptidase (IRAP), an enzyme localized in a regulated endosomal storage compartment. Considering a report contending that this role is limited to inflammatory DCs (Segura et al. 2009. Proc. Natl. Acad. Sci. USA 106: 20377-20381), in this study, we examined the role of IRAP in steady-state DC subpopulations. Steady-state conventional DCs (cDCs) and plasmacytoid DCs expressed similar amounts of IRAP. IRAP colocalized with the endosomal markers Rab14 and syntaxin 6, both known to be associated with regulated endosomal storage compartments, in CD8(+) and CD8(-) cDCs-however, to a greater extent in the former population. Likewise, IRAP recruitment to phagosomes was significantly stronger in CD8(+) DCs. IRAP deficiency compromised cross-presentation of soluble and particulate Ag by both CD8(+) and CD8(-) cDCs, again with a stronger effect in the former population. Thus, the requirement of IRAP in cross-presentation extends to steady-state cDCs. Moreover, these data suggest that increased recruitment of an IRAP(+)/Rab14(+) compartment to Ag-containing vesicles contributes to the superior cross-presentation efficacy of CD8(+) cDCs.

Placental leucine aminopeptidase efficiently generates mature antigenic peptides in vitro but in patterns distinct from endoplasmic reticulum aminopeptidase 1.

All three members of the oxytocinase subfamily of M1 aminopeptidases, endoplasmic reticulum aminopeptidase 1 (ERAP1), ERAP2, and placental leucine aminopeptidase (PLAP), also known as insulin-regulated aminopeptidase, have been implicated in the generation of MHC class I-presented peptides. ERAP1 and 2 trim peptides in the endoplasmic reticulum for direct presentation, whereas PLAP has been recently implicated in cross-presentation. The best characterized member of the family, ERAP1, has unique enzymatic properties that fit well with its role in Ag processing. ERAP1 can trim a large variety of long peptide sequences and efficiently accumulate mature antigenic epitopes of 8-9 aa long. In this study, we evaluate the ability of PLAP to process antigenic peptide precursors in vitro and compare it with ERAP1. We find that, similar to ERAP1, PLAP can trim a variety of long peptide sequences efficiently and, in most cases, accumulates appreciable amounts of correct length mature antigenic epitope. Again, similar to ERAP1, PLAP continued trimming some of the epitopes tested and accumulated smaller products effectively destroying the epitope. However, the intermediate accumulation properties of ERAP1 and PLAP are distinct and epitope dependent, suggesting that these two enzymes may impose different selective pressures on epitope generation. Overall, although PLAP has the necessary enzymatic properties to participate in generating or destroying MHC class I-presented peptides, its trimming behavior is distinct from that of ERAP1, something that supports a separate role for these two enzymes in Ag processing.

Placental leucine aminopeptidase might regulate the effects of oxytocin with resolution in endothelial cells.

BACKGROUND: Oxytocin (OT) is known to cause vascular relaxation via nitric oxide (NO) production and proliferation of endothelial cells. Previously we revealed that human umbilical vascular endothelial cells (HUVECs) expresses placental leucine aminopeptidase (P-LAP)/insulin regulated aminopeptidase (IRAP), which becomes translocation to the cell surface on activation of oxytocin receptor (OTR). However, the physiological roles of P-LAP in HUVECs have not been elucidated. MATERIAL/METHODS: Actions of OT on HUVECs transfected with a copy of the human P-LAP gene were therefore examined with a focus on changes in parameters linked to OTR such as calcium mobilization, NO production and cell proliferation. RESULTS: Cell surface P-LAP activity was significantly elevated (approximately 3 fold) in P-LAP overexpressing-HUVECs and overexpression of P-LAP resulted in remarkable inhibition of OT effects on HUVECs such as cell proliferation, [Ca2+]i, and NO production. CONCLUSIONS: These findings suggested that P-LAP on the plasma membrane in HUVECs regulates the effects of OT with resolution around OTR.

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.

Placental leucine aminopeptidase (P-LAP) and glucose transporter 4 (GLUT4) expression in benign, borderline, and malignant ovarian epithelia.

OBJECTIVE: Increased glucose consumption is a characteristic of malignant cells. Glucose is transported into the cell via facilitative glucose transporters, which are known to be members of a supergene family. The insulin-responsive GLUT4 isoform is expressed almost exclusively in insulin target tissues. P-LAP is a cell surface aminopeptidase, and is a synonym for oxytocinase. P-LAP is also referred to as insulin-regulated membrane aminopeptidase (IRAP) associated with GLUT4-containing vesicle. The authors evaluated P-LAP and GLUT4 expression in benign, borderline, and malignant ovarian epithelia. METHODS: Histologic sections of formalin-fixed, paraffin-embedded specimens from 11 patients with benign serous or mucinous cystadenomas, 14 patients with serous or mucinous borderline tumors, and 80 patients with epithelial-ovarian adenocarcinomas (29 serous, 17 endometrioid, 14 mucinous, and 20 clear cell adenocarcinomas) were stained for P-LAP and GLUT4 using each polyclonal antibody. Expressions of P-LAP and GLUT-4 in ovarian cancer cells were detected by Western blotting. RESULTS: P-LAP immunoreactivity was detected in 2 of 11 benign cystadenomas. None of the 11 benign ovarian tumors showed any immunoreactivity for GLUT4. Seven of 14 borderline tumors demonstrated P-LAP immunoreactivity, while 5 of 14 borderline tumors demonstrated GLUT4 immunoreactivity. P-LAP was expressed in 23 of 29 in serous, 15 of 17 endometrioid, 13 of 14 mucinous, and all clear-cell adenocarcinomas. The tendency toward increased P-LAP expression with advancing grade was observed in serous adenocarcinomas. GLUT4 was expressed in 13 of 29 serous, 13 of 17 endometrioid, 13 of 14 mucinous, and 18 of 20 clear-cell adenocarcinomas. In invasive carcinomas, there was a direct correlation between P-LAP immunoreactivity and GLUT4 immunoreactivity (correlation coefficient [r] = 0.58; P < 0.01). Furthermore, P-LAP overexpression in SKOV3 cells induced the GLUT4 expression. CONCLUSIONS: P-LAP and GLUT4 are available not only for the evaluation of ovarian epithelial malignancy, but also as targets for molecular therapy. Further study to investigate the roles of P-LAP and GLUT4 in ovarian carcinoma is needed.

Placental leucine aminopeptidase (P-LAP) expression is associated with chemosensitivity in human endometrial carcinoma.

OBJECTIVE: Although treatment for advanced or recurrent endometrial carcinoma has improved over recent years with the introduction of paclitaxel- and platinum-based chemotherapy, in most, the disease remains incurable because of resistance to chemotherapy. In the previous study, we have shown that placental leucine aminopeptidase (P-LAP) is associated with poor prognosis. The objective of this study was to determine whether P-LAP expression affects the chemosensitivity in endometrial carcinoma patients. METHODS: Here, we investigated the effect of P-LAP to response for paclitaxel and carboplatin in advanced and recurrence endometrial carcinoma. Furthermore, we transfected P-LAP cDNA into endometrial carcinoma cells (AMEC) and investigated cell growth and apoptosis by paclitaxel or carboplatin. RESULTS: In 15 of 17 patients, P-LAP was positive. Twelve of seventeen patients were evaluable for response. Among the eight patients strongly positive for P-LAP, only two patients (25%) showed PR. However, all four patients who were weakly positive for P-LAP showed either complete response (CR) or partial response (PR). P-LAP overexpressor (P-LAP2 and P-LAP8) and a vector control were used to assay chemosensitivity. P-LAP2 clone displayed a 1.7-fold increase in IC(50) against paclitaxel and carboplatin when compared with the vector control, and P-LAP8 clone displayed a 1.6-fold increased in IC(50) against paclitaxel and carboplatin when compared with V1. Compared to vector control cells, apoptotic effect by carboplatin treatment was clearly inhibited in P-LAP2 and P-LAP8 cells. Carboplatin, 10(-6) M, induced the 12.5-fold rate of apoptosis compared to that without treatment at 48 h in vector control cells. However, in P-LAP2 and P-LAP8 clones, 10(-6) M carboplatin induced only 3.2- and 5.1-fold rates of apoptosis, respectively, compared to that of without treatment. CONCLUSIONS: P-LAP was suggested to be involved in reducing chemosensitivity and may be a therapeutic target in endometrial carcinoma.

Possible role of placental leucine aminopeptidase in the antiproliferative effect of oxytocin in human endometrial adenocarcinoma.

PURPOSE: Oxytocin (OT) was reported to inhibit the proliferation of various neoplastic tissues and cells, however, the regulation system remains unclear. This study examined the role of OT and its regulatory ability in endometrial adenocarcinoma. EXPERIMENTAL DESIGN: To investigate the possible function of placental leucine aminopeptidase (P-LAP) in endometrial adenocarcinoma, we transfected P-LAP cDNA into A-MEC cells, showing the lowest enzyme activity of P-LAP. Also we examined P-LAP protein expression in human endometrial adenocarcinoma. RESULTS: We demonstrated the presence of P-LAP, which is identical to cystine aminopeptidase as oxytocinase, in human endometrial adenocarcinoma tissues and found that the expression of P-LAP increase with advances in the grade. Exposure of endometrial adenocarcinoma cell lines to OT caused dose- and time-dependent inhibition of growth. Treatment with 10(-7) M OT for 72 h reduced cell growth by 62, 25, and 30% in A-MEC, HEC1A, and Ishikawa cells, respectively. P-LAP-transfectant cells not only partially recovered from OT-induced growth inhibition but also showed a higher growth rate than parental cells under condition without OT. An OT receptor antagonist and a protein kinase A inhibitor blocked OT-induced growth inhibition in A-MEC and A-MEC-pc cells but not in A-MEC-LAP cells. CONCLUSIONS: These findings suggested that P-LAP might be functionally positive on carcinoma cell growth by degrading suppressive peptides such as OT.

Cloning, expression, and regulation by androgens of a putative member of the oxytocinase family of proteins in the rat prostate.

BACKGROUND: Proteases are relevant in the physiology of the prostate, and its expression is regulated by androgens. METHODS: Isolation of a novel cDNA from the rat prostate was done by reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends. By Northern blot, we analyzed the RNA expression in different tissues and in the prostate after orchidectomy and androgen treatment. By using in situ hybridization, we studied the cellular localization of the RNA. RESULTS: The cDNA codes a putative novel form of the vp-165 aminopeptidase family of proteins that we named short-vp. The short-vp probe labels one mRNA of 1.3 kb in the prostate, brain, testis, heart, and kidney. In the prostate, the levels of short-vp mRNA decrease after orchidectomy and increase with testosterone treatment. The short-vp mRNA is expressed by the prostatic epithelial cells. CONCLUSION: We isolated one putative member of the oxytocinase family of proteins that is expressed in various tissues and by the epithelial cells of the prostate. The expression of short-vp mRNA in the prostate depends on androgen levels.

Expression of placental leucine aminopeptidase/oxytocinase in neuronal cells and its action on neuronal peptides.

The placental leucine aminopeptidase (P-LAP)/oxytocinase whose serum level increases with gestation is thought to contribute to the maintenance of normal pregnancy. P-LAP mRNAs are expressed in various tissues other than the placenta. In this study, we identified P-LAP protein in the brain. In contrast with the placenta where a significant portion of P-LAP is released, the enzyme was localized in the membrane fraction in brain and PC12 cells and no soluble form of the enzyme was detected. When PC12 cells were differentiated into neuronal cells by nerve growth factor (NGF), a significant increase in the expression level of P-LAP in the cell was observed. As in the case of insulin treatment of 3T3-L1 adipocytes, treatment of PC12 cells with forskolin caused the translocation of the enzyme from intracellular vesicle to the cell surface plasma membrane. In addition, P-LAP was shown to degrade several bioactive neuropeptides such as Met-enkephalin and dynorphin A (1-8). These results suggest that P-LAP plays an important role in the regulation of neuronal cell function in the brain.

Oxytocin secretion and human parturition: pulse frequency and duration increase during spontaneous labor in women.

The secretory pattern of oxytocin was determined in blood samples taken at 1-minute intervals for 30 minutes from 32 parturient women. The samples were collected in a manner that minimized degradation by plasma oxytocinase, and a highly specific antibody was used for the radioimmunoassay. The results indicated that oxytocin is secreted in discrete pulses of short duration. The frequency of the pulses was significantly higher during spontaneous labor than before the onset of labor. The mean pulse frequencies per 30 minutes were 1.2 +/- 0.54 before labor, 4.2 +/- 0.45 during the first stage, and 6.7 +/- 0.49 during the second and third stages of labor. The mean pulse durations in these three groups were 1.2 +/- 0.20, 1.9 +/- 0.28, and 2.0 +/- 0.26 minutes, respectively. The amplitude of the pulses was variable with no significant differences between the groups, the majority being around 1.0 microU/ml. The spontaneous pulses were of similar magnitude as those measured in 18 women after intravenous injections of 4 to 16 mU of oxytocin, which doses stimulated uterine contractions. We therefore conclude that the pulses of oxytocin observed at increasing frequency during spontaneous labor are of physiologic significance and provide evidence for the participation of oxytocin in the onset and maintenance of spontaneous labor.