Stabilization of the open conformation οf insulin-regulated aminopeptidase by a novel substrate-selective small-molecule inhibitor.

Insulin-regulated aminopeptidase (IRAP) is an enzyme with important biological functions and the target of drug-discovery efforts. We combined in silico screening with a medicinal chemistry optimization campaign to discover a nanomolar inhibitor of IRAP based on a pyrazolylpyrimidine scaffold. This compound displays an excellent selectivity profile versus homologous aminopeptidases, and kinetic analysis suggests it utilizes an uncompetitive mechanism of action when inhibiting the cleavage of a typical dipeptidic substrate. Surprisingly, the compound is a poor inhibitor of the processing of the physiological cyclic peptide substrate oxytocin and a 10mer antigenic epitope precursor but displays a biphasic inhibition profile for the trimming of a 9mer antigenic peptide. While the compound reduces IRAP-dependent cross-presentation of an 8mer epitope in a cellular assay, it fails to block in vitro trimming of select epitope precursors. To gain insight into the mechanism and basis of this unusual selectivity for this inhibitor, we solved the crystal structure of its complex with IRAP. The structure indicated direct zinc(II) engagement by the pyrazolylpyrimidine scaffold and revealed that the compound binds to an open conformation of the enzyme in a pose that should block the conformational transition to the enzymatically active closed conformation previously observed for other low-molecular-weight inhibitors. This compound constitutes the first IRAP inhibitor targeting the active site that utilizes a conformation-specific mechanism of action, provides insight into the intricacies of the IRAP catalytic cycle, and highlights a novel approach to regulating IRAP activity by blocking its conformational rearrangements.

Social regulation of arginine vasopressin and oxytocin systems in a wild group-living fish.

The neuropeptides arginine vasopressin (AVP) and oxytocin (OXT) are key regulators of social behaviour across vertebrates. However, much of our understanding of how these neuropeptide systems interact with social behaviour is centred around laboratory studies which fail to capture the social and physiological challenges of living in the wild. To evaluate relationships between these neuropeptide systems and social behaviour in the wild, we studied social groups of the cichlid fish Neolamprologus pulcher in Lake Tanganyika, Africa. We first used SCUBA to observe the behaviour of focal group members and then measured transcript abundance of key components of the AVP and OXT systems across different brain regions. While AVP is often associated with male-typical behaviours, we found that dominant females had higher expression of avp and its receptor (avpr1a2) in the preoptic area of the brain compared to either dominant males or subordinates of either sex. Dominant females also generally had the highest levels of leucyl-cystinyl aminopeptidase (lnpep)-which inactivates AVP and OXT-throughout the brain, potentially indicating greater overall activity (i.e., production, release, and turnover) of the AVP system in dominant females. Expression of OXT and its receptors did not differ across social ranks. However, dominant males that visited the brood chamber more often had lower preoptic expression of OXT receptor a (oxtra) suggesting a negative relationship between OXT signalling and parental care in males of this species. Overall, these results advance our understanding of the relationships between complex social behaviours and neuroendocrine systems under natural settings.

Intrathecal injections of angiotensin IV and oxytocin conjugates induce antihyperalgesia and antiallodynia in both sexes of rats.

Our previous studies have established that intrathecal oxytocin (OT) and angiotensin IV (Ang IV) injections induce antihyperalgesia and antiallodynia in rodents. Ang IV, a renin-angiotensin system hexapeptide, acts as an endogenous inhibitor that inhibits the oxytocin-degrading enzyme insulin-regulated aminopeptidase (IRAP). The pain inhibitory effects by Ang IV were found to be through its inhibition on IRAP to potentiate the effect of OT. However, these effects were found to be with a significant sex difference, which could be partially due to the higher expression of IRAP at the spinal cords of female. Therefore, we synthesized Ang IV and OT conjugates connected with a peptide bond and tested for their effects on hyperalgesia and allodynia. Carrageenan-induced hyperalgesia and partial sciatic nerve ligation (PSNL) were performed using rat models. Conjugates Ang IV-OT (Ang IV at the N-terminal) and OT-Ang IV (OT at the N-terminal) were synthesized and intrathecally injected into male and female rats. Our results showed that Ang IV-OT exhibited prominent antihyperalgesia in male rats, particularly during hyperalgesia recovery, whereas OT-Ang IV was more effective during development stage. Ang IV-OT showed clear antihyperalgesia in female rats, but OT-Ang IV had no significant effect. Notably, both conjugates alleviated neuropathic allodynia in male rats; however, OT-Ang IV had no effect in female rats, whereas Ang IV-OT induced significant antiallodynia. In conclusion, Ang IV-OT has greater therapeutic potential for treating hyperalgesia and allodynia than OT-Ang IV. Its effects were not affected by sex, unlike those of OT and OT-Ang IV, extending its possible clinical applications.

Development of a sandwich ELISA to detect circulating, soluble IRAP as a potential disease biomarker.

There is growing interest in the use of the enzyme, insulin regulated aminopeptidase (IRAP), as a biomarker for conditions such as cardio-metabolic diseases and ischemic stroke, with upregulation in its tissue expression in these conditions. However, quantification of circulating IRAP has been hampered by difficulties in detecting release of the truncated, soluble form of this enzyme into the blood stream. The current study aimed to develop a sandwich ELISA using novel antibodies directed towards the soluble portion of IRAP (sIRAP), to improve accuracy in detection and quantification of low levels of sIRAP in plasma. A series of novel anti-IRAP antibodies were developed and found to be highly specific for sIRAP in Western blots. A sandwich ELISA was then optimised using two distinct antibody combinations to detect sIRAP in the low nanogram range (16-500 ng/ml) with a sensitivity of 9 ng/ml and intra-assay variability < 10%. Importantly, the clinical validity of the ELISA was verified by the detection of significant increases in the levels of sIRAP throughout gestation in plasma samples from pregnant women. The specific and sensitive sandwich ELISA described in this study has the potential to advance the development of IRAP as a biomarker for certain diseases.

Inhibition of insulin-regulated aminopeptidase confers neuroprotection in a conscious model of ischemic stroke.

Stroke is a leading cause of mortality and morbidity with a paucity of effective pharmacological treatments. We have previously identified insulin-regulated aminopeptidase (IRAP) as a potential target for the development of a new class of drugs for the treatment of stroke, as global deletion of this gene in mice significantly protected against ischemic damage. In the current study, we demonstrate that small molecular weight IRAP inhibitors reduce infarct volume and improve neurological outcome in a hypertensive animal model of ischemic stroke. The effects of two structurally distinct IRAP inhibitors (HFI419 or SJM164) were investigated in a model of stroke where the middle cerebral artery was transiently occluded with endothelin-1 in the conscious spontaneously hypertensive rat. IRAP inhibitor was administered into the lateral ventricle at 2 or 6 h after stroke, with subsequent doses delivered at 24, 48 and 70 h post-stroke. Functional outcomes were assessed prior to drug treatment, and on day 1 and 3 post-stroke. Histological analyses and neuroinflammatory cytokine profiling were conducted at 72 and 24 h post-stroke respectively. IRAP inhibitor treatment following stroke significantly reduced infarct volume and improved neurological and motor deficits. These protective effects were maintained even when the therapeutic window was extended to 6 h. Examination of the cellular architecture at 72 h post-stroke demonstrated that IRAP expression was upregulated in CD11b positive cells and activated astrocytes. Furthermore, IRAP inhibitor treatment significantly increased gene expression for interleukin 6 and C-C motif chemokine ligand 2 in the ischemic core. This study provides proof-of-principle that selective inhibition of IRAP activity with two structurally distinct IRAP inhibitors reduces infarct volume and improves functional outcome even when the first dose is administered 6 h post-stroke. This is the first direct evidence that IRAP inhibitors are a class of drug with potential use in the treatment of ischemic stroke.

Characterization of serum and tissue oxytocinase and tissue oxytocin in the pregnant and non-pregnant mare.

Oxytocin is a hormone with functions in: reproduction, maternal bonding, milk ejection, and feeding/social behavior, and is reported to be present in a variety of tissues. Our goal is to characterize oxytocin and leucyl and cystinyl aminopeptidase (LNPEP/oxytocinase), a key regulator of oxytocin in mares. We measured serum and tissue LNPEP by ELISA from ovulation (D0) until D21-22 in non-pregnant (n = 5) and pregnant mares (n = 6); and in periparturient and postpartum mares (n = 18). Placenta (n = 7) and homogenized tissue of diestrus mares (n = 6) were evaluated using protein determinations and LNPEP ELISAs. Identification of LNPEP and OXT protein in tissues was also performed via western blot, immunohistochemistry and liquid chromatography-mass spectrometry (LC-MS/MS). Furthermore, in situ hybridization was performed for LNPEP and OXT on endometrium, myometrium, pituitary and corpus luteum (CL). Serum LNPEP concentration were similar. Placental LNPEP U/mg protein was highest in the body and pregnant horn. The highest to lowest LNPEP U/mg protein by tissue were: myometrium > follicle wall > endometrium > kidney > CL > liver. Oxytocin was identified in the equine pituitary, CL and placenta and is likely to act in autocrine or paracrine manner, while LNPEP may act systemically and locally to regulate the availability of OXT.

Serum progesterone and oxytocinase, and endometrial and luteal gene expression in pregnant, nonpregnant, oxytocin, carbetocin and meclofenamic acid treated mares.

Our objectives were to examine changes in endometrial and luteal gene expression during estrus, diestrus, pregnancy and treatments to induce luteolysis and putatively induce luteostasis. Groups were: Diestrus (DIEST), Estrus (ESTR), Pregnant (PREG), Oxytocin (OXY), Carbetocin (CARB), and Meclofenamic acid (MFA). Blood was obtained from day (D)12 to D15 for measurement of oxytocinase, also referred to as leucyl-cysteinyl aminopeptidase (LNPEP) and progesterone. Luteal biopsies were obtained on D12 and D15 and an endometrial biopsy on D15. Real-time RT-PCR was performed for the following genes: PGR, ESR1, OXTR,OXT, LNPEP, PTGS2, PTGFR, PLA2G2C, PTGES, SLC2A4, and SLC2A1. Regarding serum LNPEP, PREG and OXY (p-value<0.001) had higher concentrations than DIEST mares. Endometrial PTGES expression was higher (p-value <0.04) in DIEST, PREG and OXY than other groups. Endometrium from ESTR had increased expression of OXT (p-value < 0.02) compared to MFA and OXY mares. Carbetocin treatment: decreased serum progesterone and LNPEP; increased endometrial PLA2G2C; decreased endometrial PTGES; and decreased luteal aromatase and PTGES. Treatment with MFA: decreased endometrial PLA2G2C, increased endometrial PTGES; and resulted in less OXTR and OXT luteal abundance on D12 compared to D15. Endometrial and luteal expression of LNPEP is affected by physiologic stage and treatment and is involved in luteal function and pregnancy recognition pathways through effects on oxytocin and prostaglandin synthesis in the horse.

Endometrial and luteal gene expression of putative gene regulators of the equine maternal recognition of pregnancy.

Our understanding of the temporal changes in endometrial and luteal gene transcripts related to the actions of oxytocin and prostaglandin during early equine pregnancy is incomplete. Additionally, the role of oxytocinase, also known as Leucyl-cystinyl aminopeptidase (LNPEP), during early pregnancy in mares has not been previously investigated. Luteal and endometrial biopsies were obtained on Day (D)8, D10, D12 and D15 post-ovulation in pregnant (PREG) and diestrus (DIEST) mares for real-time qPCR. Differences in endometrial gene expression occurred over time in: SLC2A4, SLC2A1, PTGES, OXTR and LNPEP. PTGFR and PLA2G2C had lower relative abundance in PREG D15 endometrium compared to D10. OXT and OXTR were increased on D10 and 15 PREG, respectively. Regarding luteal mRNA relative abundance, ESR1, PTGS2, PTGFR, and PTGES had higher relative abundance in D12 of DIEST and PREG. Luteal expression of OXTR and OXT had higher relative abundance in D15 compared to D8, and LNPEP had higher relative abundance in D10 and 12. Endometrial and luteal PTGES had an increased mRNA abundance in both D12 DIEST and PREG mares, which may lead to additional luteoprotective prostaglandin E2 (PGE2) secretion. Furthermore, luteal SLC2A1 had higher relative abundance in pregnancy, and likely supports the high metabolic activity of luteal tissue by increasing glucose uptake. Oxytocinase is present in endometrial and luteal tissue and its role in oxytocin induced prostaglandin secretion is uncertain.

Control of IFN-I responses by the aminopeptidase IRAP in neonatal C57BL/6 alveolar macrophages during RSV infection.

Respiratory Syncytial Virus (RSV) is the major cause of lower respiratory tract infection in infants, in whom, the sensing of RSV by innate immune receptors and its regulation are still poorly described. However, the severe bronchiolitis following RSV infection in neonates has been associated with a defect in type I interferons (IFN-I) production, a cytokine produced mainly by alveolar macrophages (AMs) upon RSV infection in adults. In the present study, neonatal C57BL/6 AMs mobilized very weakly the IFN-I pathway upon RSV infection in vitro and failed to restrain virus replication. However, IFN-I productions by neonatal AMs were substantially increased by the deletion of Insulin-Responsive AminoPeptidase (IRAP), a protein previously involved in the regulation of IFN-I production by dendritic cells. Moreover, neonatal IRAPKO AMs showed a higher expression of IFN-stimulated genes than their wild-type C57BL/6 counterpart. Interestingly, depletion of IRAP did not affect adult AM responses. Finally, we demonstrated that newborn IRAPKO mice infected with RSV had more IFN-I in their lungs and eliminated the virus more efficiently than WT neonates. Taken together, early-life susceptibility to RSV infection may be related to an original age-dependent suppressive function of IRAP on the IFN-I driven-antiviral responses in neonatal AMs.

Molecular and functional diversity of the oxytocinase subfamily of M1 aminopeptidases.

The placental leucine aminopeptidase/insulin-regulated aminopeptidase, endoplasmic reticulum aminopeptidase 1 and endoplasmic reticulum aminopeptidase 2 are part of a distinct subfamily of M1 aminopeptidases termed the 'oxytocinase subfamily'. The subfamily members show molecular diversity due to differential usage of translation initiation sites, alternative splicing and multiple single nucleotide polymorphisms. It is becoming evident that, depending on their intracellular or extracellular location, members of the oxytocinase subfamily play important roles in the maintenance of homeostasis, including the regulation of blood pressure, maintenance of normal pregnancy, retention of memory and trimming of antigenic peptides presented to major histocompatibility complex class I molecules, by acting as either aminopeptidases or binding partners of specific functional proteins in the cells. Based on their molecular diversity and moonlighting protein-like properties, it is conceivable that the subfamily members exert pleiotropic effects during evolution, to become important players in the regulation of homeostasis.

Insulin-regulated aminopeptidase deficiency impairs cardiovascular adaptations and placental development during pregnancy.

Insulin-regulated aminopeptidase (IRAP), an enzyme that cleaves vasoactive peptides including oxytocin and vasopressin, is suggested to play a role in pregnancy and the onset of preeclampsia. Our aim was to examine the contribution of IRAP to arterial pressure regulation and placental development during pregnancy in mice. Mean arterial pressure and heart rate were measured via radiotelemetry in 12-week-old female wild-type and IRAP knockout mice. Females were time-mated with males of the same genotype. Placentae were collected at embryonic day 18.5 for histological analysis. Basal heart rate was ∼40 bpm lower in IRAP knockout females compared with wild-type females. The increase in heart rate across gestation was greater in IRAP knockout females than wild-type females. Neither basal nor gestational mean arterial pressure was different between wildtype and IRAP knockout females. Urine output and water intake of IRAP knockout mice were ∼45% less than wild-type mice at late gestation. IRAP deficiency had no effect on fetal weight. Morphological assessment of placentae revealed that IRAP deficiency was associated with reduced labyrinth surface area and accumulation of glycogen in the junctional zone. Our data demonstrate that IRAP deficiency alters maternal fluid handling and impairs placental labyrinth expansion at late gestation, indicating that IRAP contributes to the normal adaptions to pregnancy.

IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses.

T cell receptor (TCR) activation is modulated by mechanisms such as TCR endocytosis, which is thought to terminate TCR signalling. Here we show that, upon internalization, TCR continues to signal from a set of specialized endosomes that are crucial for T cell functions. Mechanistically, TCR ligation leads to clathrin-mediated internalization of the TCR-CD3ζ complex, while maintaining CD3ζ signalling, in endosomal vesicles that contain the insulin responsive aminopeptidase (IRAP) and the SNARE protein Syntaxin 6. Destabilization of this compartment through IRAP deletion enhances plasma membrane expression of the TCR-CD3ζ complex, yet compromises overall CD3ζ signalling; moreover, the integrity of this compartment is also crucial for T cell activation and survival after suboptimal TCR activation, as mice engineered with a T cell-specific deletion of IRAP fail to develop efficient polyclonal anti-tumour responses. Our results thus reveal a previously unappreciated function of IRAP-dependent endosomal TCR signalling in T cell activation.

Insulin-regulated aminopeptidase inhibitor-mediated increases in dendritic spine density are facilitated by glucose uptake.

Ethyl2-acetylamino-7-hydroxy-4-pyridin-3-yl-4H-chromene-3-carboxylate (HFI-419), the benzopyran-based inhibitor of insulin-regulated aminopeptidase (IRAP), has previously been shown to improve spatial working and recognition memory in rodents. However, the mechanism of its cognitive-enhancing effect remains unknown. There is a close correlation between dendritic spine density and learning in vivo and several studies suggest that increases in neuronal glucose uptake and/or alterations to the activity of matrix metalloproteinases (MMPs) may improve memory and increase dendritic spine density. We aimed to identify the potential mechanism by which HFI-419 enhances memory by utilizing rat primary cultures of hippocampal cells. Alterations to dendritic spine density were assessed in the presence of varying concentrations of HFI-419 at different stages of hippocampal cell development. In addition, glucose uptake and changes to spine density were assessed in the presence of indinavir, an inhibitor of the glucose transporter 4 (GLUT4 ), or the matrix metalloprotease inhibitor CAS 204140-01-2. We confirmed that inhibition of IRAP activity with HFI-419 enhanced spatial working memory in rats, and determined that this enhancement may be driven by GLUT4 -mediated changes to dendritic spine density. We observed that IRAP inhibition increased dendritic spine density prior to peak dendritic growth in hippocampal neurons, and that spine formation was inhibited when GLUT4 -mediated glucose uptake was blocked. In addition, during the peak phase of dendritic spine growth, the effect of IRAP inhibition on enhancement of dendritic spine density resulted specifically in an increase in the proportion of mushroom/stubby-like spines, a morphology associated with memory and learning. Moreover, these spines were deemed to be functional based on their expression of the pre-synaptic markers vesicular glutamate transporter 1 and synapsin. Overall, or findings suggest that IRAP inhibitors may facilitate memory by increasing hippocampal dendritic spine density via a GLUT4 -mediated mechanism. Cover Image for this issue: doi: 10.1111/jnc.14745.

A Mathematical Model Relating Pitocin Use during Labor with Offspring Autism Development in terms of Oxytocin Receptor Desensitization in the Fetal Brain.

This paper develops a mathematical model describing the potential buildup of high oxytocin concentrations in the maternal circulation during labor in terms of continuous Pitocin infusion rate, half-life, and maternal weight. Oxytocin override of the degradation of oxytocin by placental oxytocinase is introduced to model the potential transfer of oxytocin from the maternal circulation across the placenta into the fetal circulation and from there into the brain of the fetus. The desensitization unit D equal to 1.8E6 (pg·min)/ml is employed to establish a desensitization threshold and by extension, a downregulation threshold as a function of oxytocin override concentration and continuous Pitocin infusion time, that could be a factor in the subsequent development of autism among offspring. Epidemiological studies by Duke University [1], Yale University [2], and Harvard University [3] are discussed regarding Pitocin use and offspring autism development for an explanation of the weak correlations they identified. The findings of the Harvard epidemiological study are reinterpreted regarding Pitocin use and its conclusion questioned. Further evaluations of the findings of these three epidemiological studies are called for to incorporate medical information on quantity of Pitocin used, continuous Pitocin infusion rate, length of labor, and maternal weight to determine if a correlation can be established with offspring autism development above an empirically determined desensitization threshold for Pitocin use. Suggestions for research are discussed, including an alternative to continuous Pitocin infusion, pulsatile infusion of Pitocin during labor induction, which may mitigate possible offspring autism development.

Social behaviour is altered in the insulin-regulated aminopeptidase knockout mouse.

Oxytocin, and the closely related neuropeptide, vasopressin, are both known to modulate social behaviours. The pro-social effects of oxytocin are well-documented and have generated much interest into its suitability as a therapeutic for disorders characterised by social dysfunction. This study investigated the social phenotype of mice with a targeted deletion of the gene for insulin-regulated aminopeptidase, an enzyme involved in the degradation of oxytocin and vasopressin. In the 3-chamber sociability test, a genotype effect was observed and subsequent post hoc analysis revealed that male, but not female, insulin-regulated aminopeptidase knockout mice made significantly more approaches to the enclosure holding a stranger mouse than did wildtype mice (p = 0.0039). Male insulin-regulated aminopeptidase knockout mice also displayed decreased rearing (t = 2.309, df = 24, p = 0.0299) and locomotor activity (t = 2.134, df = 24, p = 0.043) in the open field test, suggestive of a reduced stress response to a novel environment. Our findings provide support for the role of insulin-regulated aminopeptidase in influencing social behaviour, possibly via modulation of oxytocin and vasopressin levels. The increase in social interaction observed in the male, but not female, insulin-regulated aminopeptidase knockout mice is in agreement with reports of sex differences in effects of oxytocin and vasopressin on social behaviours and should be explored further.

Decreased behavioural and neurochemical effects of angiotensin IV following prenatal alcohol exposure in the mouse.

Angiotensin IV (ang IV) is known to improve learning and memory in animal models but the mechanism is unclear. We have previously demonstrated sex differences in the pro-cognitive effects of ang IV, and that prenatal alcohol exposure (PAE) abolishes these effects. This study aimed to explore a possible mechanism underlying the sex differences and the effects of PAE in male mice. Mouse breeding harems received 5% ethanol in drinking water throughout pregnancy and lactation in a two-bottle schedule. The effects of ang IV were assessed in offspring at 4 months of age using the open field test, novel object recognition test and elevated plus maze. Aminopeptidase activity of brain insulin-regulated aminopeptidase (IRAP), a putative target of ang IV, was determined. As seen in a previous similar study, ang IV administered immediately after the second training trial significantly improved novel object recognition 24 h later in male mice but not female. PAE abolished this pro-cognitive effect in males. PAE also increased anxiety-like behaviour in male but not female offspring. Ang IV decreased the aminopeptidase activity of brain IRAP in control male, but not female, mice; PAE abolished this inhibitory effect. Ang IV improved memory consolidation in male but not female mice and PAE abolished this effect in the males. While the effects of PAE may be related to increased anxiety; ang IV decreased the aminopeptidase activity in male but not female mice and PAE abolished this inhibitory effect. The results therefore suggest that improvements in learning and memory induced by peripheral administration of ang IV correlate with a reduction of the enzyme activity of IRAP. This is the first demonstration that ang IV administered peripherally can induce long-term (24 h) changes in IRAP function which are probably not simple competitive inhibition and the first demonstration that PAE alters IRAP activity.

Insulin-responsive amino peptidase follows the Glut4 pathway but is dispensable for the formation and translocation of insulin-responsive vesicles.

In fat and skeletal muscle cells, insulin-responsive amino peptidase (IRAP) along with glucose transporter 4 (Glut4) and sortilin, represents a major component protein of the insulin-responsive vesicles (IRVs). Here, we show that IRAP, similar to Glut4 and sortilin, is retrieved from endosomes to the trans-Golgi network by retromer. Unlike Glut4, retrograde transport of IRAP does not require sortilin, as retromer can directly bind to the cytoplasmic tail of IRAP. Ablation of IRAP in 3T3-L1 adipocytes shifts the endosomal pool of Glut4 to more acidic endosomes, but does not affect IRV targeting, stability, and insulin responsiveness of Glut4.

Evidence for a role of arginine vasotocin receptors in the gill during salinity acclimation by a euryhaline teleost fish.

The nonapeptide arginine vasotocin (AVT) regulates osmotic balance in teleost fishes, but its mechanisms of action are not fully understood. Recently, it was discovered that nonapeptide receptors in teleost fishes are differentiated into two V1a-type, several V2-type, and two isotocin (IT) receptors, but it remains unclear which receptors mediate AVT's effects on gill osmoregulation. Here, we examined the role of nonapeptide receptors in the gill of the euryhaline Amargosa pupfish (Cyprinodon nevadensis amargosae) during osmotic acclimation. Transcripts for the teleost V1a-type receptor v1a2 were upregulated over fourfold in gill 24 h after transferring pupfish from 7.5 ppt to seawater (35 ppt) or hypersaline (55 ppt) conditions and downregulated after transfer to freshwater (0.3 ppt). Gill transcripts for the nonapeptide degradation enzyme leucyl-cystinyl aminopeptidase (LNPEP) also increased in fish acclimating to 35 ppt. To test whether the effects of AVT on the gill might be mediated by a V1a-type receptor, we administered AVT or a V1-type receptor antagonist (Manning compound) intraperitoneally to pupfish before transfer to 0.4 ppt or 35 ppt. Pupfish transferred to 35 ppt exhibited elevated gill mRNA abundance for cystic fibrosis transmembrane conductance regulator (cftr), but that upregulation diminished under V1-receptor inhibition. AVT inhibited the increase in gill Na+/Cl- cotransporter 2 (ncc2) transcript abundance that occurs following transfer to hypoosmotic environments, whereas V1-type receptor antagonism increased ncc2 mRNAs even without a change in salinity. These findings indicate that AVT acts via a V1-type receptor to regulate gill Cl- transport by inhibiting Cl- uptake and facilitating Cl- secretion during seawater acclimation.

The deubiquitinating enzyme USP25 binds tankyrase and regulates trafficking of the facilitative glucose transporter GLUT4 in adipocytes.

Key to whole body glucose homeostasis is the ability of fat and muscle cells to sequester the facilitative glucose transporter GLUT4 in an intracellular compartment from where it can be mobilized in response to insulin. We have previously demonstrated that this process requires ubiquitination of GLUT4 while numerous other studies have identified several molecules that are also required, including the insulin-responsive aminopeptidase IRAP and its binding partner, the scaffolding protein tankyrase. In addition to binding IRAP, Tankyrase has also been shown to bind the deubiquinating enzyme USP25. Here we demonstrate that USP25 and Tankyrase interact, and colocalise with GLUT4 in insulin-sensitive cells. Furthermore depletion of USP25 from adipocytes reduces cellular levels of GLUT4 and concomitantly blunts the ability of insulin to stimulate glucose transport. Collectively, these data support our model that sorting of GLUT4 into its insulin-sensitive store involves a cycle of ubiquitination and subsequent deubiquitination.

Identification of insulin-regulated aminopeptidase (IRAP) in the rat pineal gland and the modulation of melatonin synthesis by angiotensin IV.

A local renin-angiotensin system (RAS) has been postulated in the pineal gland. In addition to angiotensin II (Ang II), other active metabolites have been described. In this study, we aimed to investigate a role for Ang IV in melatonin synthesis and the presence of its proposed (IRAP)/AT4 receptor (insulin-regulated aminopeptidase) in the pineal gland. The effect of Ang IV on melatonin synthesis was investigated in vitro using isolated pinealocytes. IRAP protein expression and activity were evaluated by Western blot and fluorimetry using Leu-4Me-ß-naphthylamide as a substrate. Melatonin was analyzed by HPLC, calcium content by confocal microscopy and cAMP by immunoassay. Ang IV significantly augmented the NE-induced melatonin synthesis to a similar degree as that achieved by Ang II. This Ang IV effect in pinealocytes appears to be mediated by an increase in the intracellular calcium content but not by cAMP. The (IRAP)/AT4 expression and activity were identified in the pineal gland, which were significantly higher in membrane fractions than in soluble fractions. Ang IV significantly reduced IRAP activity in the pineal membrane fractions. The main findings of the present study are as follows: (1) Ang IV potentiates NE-stimulated melatonin production in pinealocytes, (2) the (IRAP)/AT4 receptor is present in the rat pineal gland, and (3) Ang IV inhibits IRAP activity and increases pinealocytes [Ca2+]i. We conclude that Ang IV is an important component of RAS and modulates melatonin synthesis in the rat pineal gland.