Dcc haploinsufficiency regulates dopamine receptor expression across postnatal lifespan.

Adolescence is a period during which the medial prefrontal cortex (mPFC) undergoes significant remodeling. The netrin-1 receptor, deleted in colorectal cancer (DCC), controls the extent and organization of mPFC dopamine connectivity during adolescence and in turn directs mPFC functional and structural maturation. Dcc haploinsufficiency leads to increased mPFC dopamine input, which causes improved cognitive processing and resilience to behavioral effects of stimulant drugs of abuse. Here we examine the effects of Dcc haploinsufficiency on the dynamic expression of dopamine receptors in forebrain targets of C57BL6 mice. We conducted quantitative receptor autoradiography experiments with [3H]SCH-23390 or [3H]raclopride to characterize D1 and D2 receptor expression in mPFC and striatal regions in male Dcc haploinsufficient and wild-type mice. We generated autoradiograms at early adolescence (PND21±1), mid-adolescence (PND35±2), and adulthood (PND75±15). C57BL6 mice exhibit overexpression and pruning of D1, but not D2, receptors in striatal regions, and a lack of dopamine receptor pruning in the mPFC. We observed age- and region-specific differences in D1 and D2 receptor density between Dcc haploinsufficient and wild-type mice. Notably, neither group shows the typical pattern of mPFC dopamine receptor pruning in adolescence, but adult haploinsufficient mice show increased D2 receptor density in the mPFC. These results show that DCC receptors contribute to the dynamic refinement of D1 and D2 receptor expression in striatal regions across adolescence. The age-dependent expression of dopamine receptor in C57BL6 mice shows marked differences from previous characterizations in rats.

Immunohistochemical distribution of neuropeptide Y, peptide YY, pancreatic polypeptide-like immunoreactivity and their receptors in the epidermal skin of healthy women.

Few studies have suggested that neuropeptide Y (NPY) could play an important role in skin functions. However, the expression of NPY, the related peptides, peptide YY (PYY) and pancreatic polypeptide (PP) and their receptors have not been investigated in human skin. Using specific antisera directed against NPY, PYY, PP and the Y1, Y2, Y4 and Y5 receptor subtypes, we investigated here the expression of these markers. NPY-like immunoreactivity (ir) in the epidermal skin could not be detected. For the first time we report the presence of positive PP-like ir immunofluorescent signals in epidermal cells, i.e. keratinocytes of skin from three areas (abdomen, breast and face) obtained as surgical left-overs. The immunofluorescent signal of PP-like ir varies from very low to high level in all three areas. In contrast, PYY-like ir is only expressed in some cells and with varied level of intensity. Furthermore and for the first time we observed specific Y1 and Y4 receptor-like ir in all epidermal layers, while the Y2 and Y5 subtypes were absent. Interestingly, as seen in human epidermis, in Episkin, a reconstituted human epidermal layer, we detected the presence of PP-like as well as Y1-like and Y4-like ir. These data have shown the presence and distribution of PYY, PP and Y1 and Y4 receptors in the human skin and Episkin, suggesting possible novel roles of NPY related peptides and their receptors in skin homeostasis.

Aging and long-term caloric restriction regulate neuropeptide Y receptor subtype densities in the rat brain.

The effects of aging and long-term caloric restriction (LTCR), on the regulation of neuropeptide Y (NPY) Y1, Y2 and Y5 receptors subtypes, was studied in 20-month-old male rats fed ad libitum (AL) or submitted to a 40% caloric restriction for 12 months. [(125)I]GR231118, a Y1 antagonist was used as Y1 receptor radioligand. [(125)I][Leu(31), Pro(34)]PYY, a high affinity agonist of Y1 and Y5 subtypes was used in the absence or presence of 100 nM BIBO3304 (a highly selective Y1 receptor antagonist) to assess the apparent levels of [(125)I][Leu(31), Pro(34)]PYY/BIBO3304 insensitive sites (Y5-like) from [(125)I][Leu(31), Pro(34)]PYY/BIBO3304 sensitive sites (Y1). [(125)I]PYY(3-36) was used to label the Y2 receptor. In the brain of 3-month-old AL rats, the distribution and densities of Y1, Y2 and Y5 receptors were in agreement with previous reports. In the brain of 20AL rats, a decrease of NPY receptor subtype densities in regions having important physiological functions such as the cingulate cortex, hippocampus and dentate gyrus, thalamus and hypothalamus was observed. In contrast, LTCR had multiple effects. It induced specific decreases of Y1-receptor densities in the dentate gyrus, thalamic and hypothalamic nuclei and lateral hypothalamic area and Y2-receptor densities in the suprachiasmatic nucleus of hypothalamus. Moreover, it prevented the age-induced increase in Y1-receptor densities in the ventromedial hypothalamic nucleus and decrease in the mediodorsal thalamic nucleus, and increased Y2-receptor densities in the CA2 subfield of the hippocampus. These results indicate that LTCR not only counteracts some of the deleterious effects of aging on NPY receptor subtype densities but exerts specific effects of its own. The overall impact of the regulation of NPY receptor subtypes in the brain of old calorie-restricted rats may protect the neural circuits involved in pain, emotions, feeding and memory functions.

Mood and memory-associated behaviors in neuropeptide Y5 knockout mice.

Recent data led to suggest that in addition to Y1 and Y2 subtypes, Y5 receptors may be involved in mood-related behaviors (Morales-Medina et al., 2010). In the present study, using a battery of behavioral tests to assess anxiety and depression-like paradigms, as well as memory function, we evaluated the potential behavioral changes induced in mice devoid of Y5 receptors. Those paradigms were assessed using the open field (OF), elevated plus maze (EPM), forced swim test (FST), social interaction test (SI), object recognition test (ORT) and Morris water maze (MWM) in Y5 knockout (KO) mice and wild type (WT) animals. In the tests associated to anxiety related behaviors (OF, EPM and SI), no difference for locomotion and time spent in the lateral area of open field were observed between Y5 KO and WT mice. Similar results were observed for time and number of entries in open arms in EPM. Additionally, in SI test, Y5 KO mice spent same amount of time and number of entries in the stranger chamber as compared to WT animals. In the FST, as compared to WT mice, Y5 KO mice had similar immobility time on day 1. No memory dysfunction was observed in the MWM and ORT in Y5 KO mice, as compared to WT. Altogether these data suggest that under basal conditions Y5 KO and WT mice display similar mood behaviors and memory functions. However, as compared to WT, Y5 KO mice display increased grooming and rearing in the OF, lower ratio entries in open arms in the EPM and increased immobility time on the second day of the FST.

Role of neuropeptide Y Y1 and Y2 receptors on behavioral despair in a rat model of depression with co-morbid anxiety.

Accumulated evidence suggests that neuropeptide Y (NPY) is involved in emotional disorders by acting on Y(1) and Y(2) receptors. This hypothesis is based on animal studies carried out in naïve normal animals but not in animal models of depression, including the olfactory bulbectomized (OBX) rat. The OBX rat produces a wide array of symptoms that mimic several aspects of human depression and anxiety disorders. In the present study, we aimed to investigate the effects of sustained (2 weeks) intracerebroventricular administration of NPY Y(1) and Y(2) agonists and antagonists in a battery of behavioral tests including the open field, forced swim test (FST) and social interaction (SI) tests in OBX rats. The levels of Y(1) and Y(2) receptors in the hippocampus and basolateral amygdala (BLA) were also evaluated. Treatment with the Y(1)-like receptor agonist, [Leu(31)Pro(34)]PYY, decreased both depressive- and anxiogenic-like behaviors. The Y(2) receptor antagonist, BIIE0246, decreased the immobility time in the FST in OBX animals and increased active contacts in the SI test in sham rats. The Y(2) agonist, PYY3-36, increased the immobility time in the FST in OBX rats. Additionally, increased levels of Y(2) receptor binding were quantified in the dorsal hippocampus and BLA in OBX rats. Taken together, the autoradiographic results add further evidence that the NPYergic system is altered in disturbed emotional states. Moreover, we demonstrate a differential role for NPY Y(1) and Y(2) receptors in emotional processes under control and challenged conditions. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Protective action of resveratrol in human skin: possible involvement of specific receptor binding sites.

BACKGROUND: Resveratrol is a plant-derived polyphenol with purported protecting action on various disorders associated with aging. It has been suggested that resveratrol could exert its protective action by acting on specific plasma membrane polyphenol binding sites (Han Y.S., et al. (2006) J Pharmacol Exp Ther 318:238-245). The purpose of this study was to investigate, in human skin, the possible existence of specific binding sites that mediate the protective action of resveratrol. METHODS AND FINDINGS: Using human skin tissue, we report here the presence of specific [(3)H]-resveratrol binding sites (K(D)  =  180 nM) that are mainly located in the epidermis. Exposure of HaCaT cells to the nitric oxide free radical donor sodium nitroprusside (SNP; 0.3-3 mM) resulted in cell death which was reduced by resveratrol (EC(50)  =  14.7 µM), and to a much lesser extent by the resveratrol analogue piceatannol (EC(50)  =  95 µM) and epigallocatechin gallate (EC(50)  =  200 µM), a green-tea derived polyphenol. The protective action of resveratrol likely relates to its anti-apoptotic effect since at the same range of concentration it was able to reduce both the number of apoptotic cells as well as mitochondrial apoptotic events triggered by SNP. CONCLUSION: Taken together, these findings suggest that resveratrol, by acting on specific polyphenol binding sites in epidermis, may be useful to prevent skin disorders associated with aging.

Tyrosine-based rivastigmine-loaded organogels in the treatment of Alzheimer's disease.

Organogels can be prepared by immobilizing an organic phase into a three-dimensional network coming from the self-assembly of a low molecular weight gelator molecule. In this work, an injectable subcutaneous organogel system based on safflower oil and a modified-tyrosine organogelator was evaluated in vivo for the delivery of rivastigmine, an acetylcholinesterase (AChE) inhibitor used in the treatment of Alzheimer's disease. Different implant formulations were injected and the plasmatic drug concentration was assayed for up to 35 days. In parallel, the inhibition of AChE in different brain sections and the biocompatibility of the implants were monitored. The pharmacokinetic profiles were found to be influenced by the gel composition, injected dose and volume of the implant. The sustained delivery of rivastigmine was accompanied by a significant prolonged inhibition of AChE in the hippocampus, a brain structure involved in memory. The implant induced only a minimal to mild chronic inflammation and fibrosis, which was comparable to poly(D,L-lactide-co-glycolide) in situ-forming implants. These findings suggest that tyrosine-based organogels could represent an alternative approach to current formulations for the sustained delivery of cholinesterase inhibitors.

Lipopolysaccharide induces calcitonin gene-related peptide in the RAW264.7 macrophage cell line.

SUMMARY: Calcitonin gene-related peptide (CGRP) is widely distributed and plays important roles in a wide array of biological functions. It is enriched in primary sensory neurons and hence involved in nociception and neurogenic inflammation. Recent studies have shown that CGRP can be produced by immune cells such as monocytes/macrophages following inflammatory stimulation, suggesting a role in innate immunity. However, it is unclear how CGRP is up-regulated in macrophages and if it plays a role in macrophage functions such as the production of cytokines and chemokines. Using enzyme-linked immunosorbent assay (ELISA) and multiplex ELISA, lipopolysaccharide (LPS) was found to induce CGRP in the RAW 264.7 macrophage cell line. LPS-induced inflammatory mediators such as nerve growth factor (NGF), interleukin-1beta (IL-1beta), IL-6, prostaglandin E(2) (PGE(2)) and nuclear factor-kappaB (NF-kappaB) signalling are involved in inducing CGRP, whereas the NGF receptor trkA and CGRP receptor signalling pathways are unexpectedly involved in suppressing LPS-induced CGRP, which leads to the fine-tune regulation of CGRP release. Exogenous CGRP and CGRP receptor antagonists, in a concentration-dependent manner, stimulated, inhibited or had no effect on basal or LPS-induced release of monocyte chemoattractant protein-1, IL-1beta, IL-6, tumour necrosis factor-alpha and IL-10 in RAW macrophages. The ligand-concentration-dependent regulation of the production of inflammatory mediators by CGRP receptor signalling is a novel mechanism underlying the stimulating and suppressing role of CGRP in immune and inflammatory responses. Together, our data suggest that monocytes/macrophages are an important source of CGRP. Inflammation-induced CGRP has a positive or negative reciprocal effect on the production of other pro- and anti-inflammatory mediators. Thereby CGRP plays both facilitating and suppressing roles in immune and inflammatory responses.

Neuropeptide Y receptor subtypes in the dorsal vagal complex under acute feeding adaptation in the adult rat.

Neuropeptide Y (NPY), Peptide YY (PYY) and pancreatic polypeptides (PPs) belong to the same peptide family called the Y or NPY family. Central and peripheral injections of these peptides are implicated in the regulation of food intake at the level of the hypothalamus (central effects; increased food intake) and dorsal vagal complex (DVC) (peripheral effects; decreased food intake). The DVC of the brainstem is a satiety reflex key region, which includes the nucleus tractus solitarius (NTS), area postrema (AP) and dorso motor nucleus of the vagus (DMX). NPY binding sites were quantified on serial DVC sections using in vitro receptor autoradiography in two feeding adaptation models: fasting and inflammatory anorexia. Receptor autoradiography revealed that Y(1), Y(2), Y(4) and Y(5) receptor subtypes are present in all nuclei of the DVC. Additionally, we also observed significant amount of specific labelling remaining even after having blocked all known NPY receptor subtypes targeted by radioligands such as [(125)I][Leu(31), Pro(34)]PYY, [(125)I]PYY3-36 and [(125)I]hPP. This binding is referred as an atypical NPY site. Lipopolysaccharide (LPS) injection and food deprivation (24-48h) did not induce any change in the expression of NPY Y(1), Y(2,) Y(4) and Y(5) receptors at the level of the NTS and DMX. However, a significant decrease in [(125)I]PYY3-36/Y(2) and [(125)I]hPP/Y(4)- and Y(5)-insensitive binding sites (residual or atypical site) was observed in the AP. Together, these data could suggest that residual or atypical NPY binding site in the AP is modulated by food deprivation and may be physiologically relevant and implicated in feeding behaviors.

A possible role of neuropeptide Y in depression and stress.

Neuropeptide Y (NPY) mediates its physiological effects through at least four receptors known as Y(1), Y(2), Y(4), and Y(5). This peptide is one of the most abundant peptides in the central nervous system and is highly conserved throughout evolution. The most abundant receptors of the NPY family, the Y(1) and Y(2) receptors, are densely expressed in the cortex, hippocampus, and amygdala. These brain regions are particularly associated with mood disorders, stress responses, and memory processing. With this in mind, researchers suggested the involvement of NPY as well as the Y(1) and Y(2) receptors in affective disorders. Earlier studies showed that NPY and the Y(1) and Y(2) receptors mediate some aspects of depression-like disorders and stress responses in rodents. Recent research also suggests the involvement of the Y(4) and Y(5) receptors in emotion-related processes in rodents. In addition, human studies have consistently suggested a role for NPY in stress responses, whereas conflicting data have been obtained in relation to the role of NPY in depression-related illnesses. However, novel evidence from polymorphisms in the prepro-NPY gene has shed new light on the potential clinical relevance of NPY in depression. In this article, we review the literature from both animal and human studies regarding the contribution of NPY and its receptors in depression and stress.

Morphological reorganization after repeated corticosterone administration in the hippocampus, nucleus accumbens and amygdala in the rat.

Elevated levels of corticosteroids and stress play key roles in the pathophysiology of affective disorders. Corticosterone (CORT)-treated rats have emerged as a pharmacological model of depression-like behaviors. Previous studies have shown that CORT administration induces neuronal atrophy in the CA3 subfield of the hippocampus and laminae II/III of the prefrontal cortex. However, little attention has been given to other limbic structures such as the amygdala and the nucleus accumbens (NAcc). We investigated here whether 3 weeks of CORT administration in rats causes dendritic remodeling and spine density reorganization in the basolateral amygdala and pyramidal neurons of the CA1 subfield of the hippocampus as well as in spiny medium neurons of NAcc. Quantitative morphological analysis revealed retracted neuronal arborizations and modified configuration of length depending on branch order in medium spiny neurons of the NAcc of CORT-treated animals. Moreover, distal dendritic sections of the NAcc showed massive reductions in the number of spines caused by the CORT treatment. This treatment also induced a reduction in total dendritic length specific to fourth and sixth branch orders of pyramidal CA1 hippocampal neurons. These neurons also showed decreased branching and diminished number of spines. Finally, pyramidal neurons of the basolateral amygdala were apparently not significantly affected by the CORT treatment. Taken together, these data show for the first time neuronal morphological alterations in the NAcc in the CORT model of depression-like behaviors. Our results also add further information about the morphological reorganization occurring in CORT-sensitive regions of the limbic system.

Comparative neuroprotective properties of stilbene and catechin analogs: action via a plasma membrane receptor site?

Various studies have reported on the neuroprotective effects of polyphenols, widely present in food, beverages, and natural products. For example, we have shown that resveratrol, a polyphenol enriched in red wine and other foods such as peanuts, protects hippocampal cells against beta-amyloid (Abeta)-induced toxicity, a key protein involved in the neuropathology of Alzheimer disease. This effect involves, at least in part, the capacity of resveratrol to activate the phosphorylation of delta isoform of protein kinase C (PKC-delta). The neuroprotective action of resveratrol is shared by piceatannol, a stilbene derivative, as well as by tea-derived catechin gallate esters. The thioflavin T assay indicated that all these polyphenols inhibited the formation of Abeta fibrils, suggesting that this action likely also contributes to their neuroprotective effects. Binding and autoradiographic studies revealed that the effects of polyphenols might involve specific binding sites that are particularly enriched in the choroid plexus in the rat brain. Interestingly, the choroid plexus secretes transthyretin, a protein that has been shown to modulate Abeta aggregation and that may be critical to the maintenance of normal learning capacities in aging. Taken together, these data suggest that polyphenols target multiple enzymes/proteins, leading to their neuroprotective actions, possibly through action via specific plasma membrane binding sites.

Adrenomedullin-2/intermedin induces cAMP accumulation in dissociated rat spinal cord cells: evidence for the existence of a distinct class of sites of action.

Adrenomedullin-2/intermedin is structurally related to the calcitonin family of peptides, which includes calcitonin gene-related peptide (CGRP), adrenomedullin, and amylin. We recently reported that CGRP and adrenomedullin act through distinct receptors to induce cyclic adenosine monophosphate (cAMP) accumulation in dispersed cells from embryonic rat spinal cord. Here, we investigated the apparent affinity and efficacy of adrenomedullin-2/intermedin for these receptors. Adrenomedullin-2/intermedin competed with [(125)I]-CGRP for binding to specific embryonic spinal cord cells with a pIC(50) of 9.73 +/- 0.06. Interestingly, adrenomedullin-2/intermedin competed for specific [(125)I]-adrenomedullin binding in a biphasic manner with pIC(50) of 9.03 +/- 0.22 and 6.45 +/- 0.24, respectively. Cellular levels of cAMP were increased by adrenomedullin-2/intermedin (pEC(50) 7.84 +/- 0.08) when cells were exposed to this peptide for 10 min at 37 degrees C. This effect was partially inhibited by the non-peptide antagonist BIBN4096BS (pA(2) 6.56 +/- 0.12), the adrenomedullin antagonist hAM(22-52) (pA(2) 6.36 +/- 0.30), and the adrenomedullin/CGRP antagonist CGRP(8-37) (pA(2) 7.24 +/- 0.60). More interestingly, a highly significant effect of adrenomedullin-2/intermedin on cAMP accumulation (pEC(50) 7.3 +/- 0.14) was still observed even in the presence of a mixture of saturating concentrations of BIBN4096BS, hAM(22-52), and the amylin antagonist AC187. Taken together, these data provide evidence for the possible existence of a distinct class of receptor sites for adrenomedullin-2/intermedin in embryonic rat spinal cord cells.

Distribution of peripherally injected peptide YY ([125I] PYY (3-36)) and pancreatic polypeptide ([125I] hPP) in the CNS: enrichment in the area postrema.

The mechanism by which blood-borne peptide YY (3-36) (PYY(3-36)) and pancreatic polypeptide (PP) inhibit food intake is not clear and could implicate peripheral (vagal afferent pathways) and/or central (direct action on specific brain nuclei) mechanisms. To identify the primary brain structure(s) that could be activated after a peripheral injection of neuropeptide Y-related peptides, we investigated the distribution of radioactive materials using whole body autoradiography and coronal brain sections. Rats were injected with [125I] porcine (p) PYY(3-36) (i.p., 10 microCi) and killed after 30 min, 1, 2, or 4 h. After i.p. administration, significant amounts of radioactive materials were rapidly (<30 min) detected in the blood circulation and various tissues including the kidneys, liver, lung, heart, bone marrow, gastrointestinal tract, and thyroid gland, whereas in the brain, low but significant amounts of radioactive materials were detected at the level of the area postrema. Next, we investigated the distribution of radioactive labeling in the brain after i.v. injections of [125I]pPYY(3-36) (Y2 and Y5 subtypes), [125I] human (h) PP (Y4 and Y5 receptors), and [125I][Leu(31), Pro(34)] pPYY (Y1, Y4 and Y5 classes) in the rat brain. Fifteen minutes post injection, autoradiograms revealed positive signals only in the area postrema after the injection of [125I]-hPP and [125I][Leu(31), Pro(34)]pPYY. Whereas the presence of [125I]pPYY(3-36)-related labeling was detected in the area postrema, subfornical organ, and median eminence. In all other brain structures, including all hypothalamic nuclei and other circumventricular organs, near background level signals were detected. These data suggest that the inhibition of food intake observed after peripheral injections of pPYY(3-36) and hPP could involve receptor activation preferentially located at the level of the area postrema, a structure well-known to be involved in the modulation of food intake.

Emotional behavior in aged neuropeptide Y (NPY) Y2 knockout mice.

Neuropeptide Y (NPY) was shown to modulate anxiety- and depression-related behaviors in various animal models. Previous studies demonstrated that NPY Y2 receptor knockout (KO) mice display an anxiolytic- and antidepressant-like phenotype compared with control animals. However, the long-term effect of the deletion of this receptor in aged animals is unknown. Thus, anxiety- and depression-related behaviors were investigated in 2-yr-old NPY Y2 KO mice. Aged NPY Y2 KO mice display an anxiolytic-like profile as assessed in the elevated plus-maze and open field, providing further support for a role for Y2 receptors in anxiety-related behaviors. Furthermore, aged NPY Y2 KO mice have significantly lower immobility scores in the forced swim test; supporting the role for this receptor in antidepressant-like behaviors. These data provide further evidence that modulators of the NPY Y2 receptor subtype are drug targets for the treatment of anxiety and mood disorders in human subjects.

Specific plasma membrane binding sites for polyphenols, including resveratrol, in the rat brain.

Using [3H]resveratrol (3,5,4'-trihydroxy-trans-stilbene) as radioligand, we investigated the possible existence of specific polyphenol binding sites at the level of the cellular plasma membrane in rat brain. Specific [3H]resveratrol binding sites were found to be enriched in the plasma membrane pellet with lower levels in the nuclear and cell debris fraction. Specific [3H]resveratrol binding to the plasma membrane fraction was sensitive to trypsin digestion and protein denaturation but not to DNase and RNase treatment. Saturation binding experiments revealed that specific [3H]resveratrol recognized a single class of sites with an apparent affinity (KD) of 220+/-45 nM and a maximal capacity (Bmax) of 1060+/-120 fmol/mg protein. Various polyphenols and resveratrol derivatives competed against specific [3H]resveratrol binding in rat brain plasma membrane homogenates with the tea catechin gallates (epigallocatechin gallate and epicatechin gallate) displaying the highest affinities (Ki=25-45 nM) followed by resveratrol (Ki=102 nM). Quantitative autoradiographic studies revealed that specific [3H]resveratrol binding sites are broadly distributed in the rat brain, with highest levels of labeling seen in the choroid plexus and subfornical organ. Finally, the potency of various polyphenols and resveratrol analogs in protecting hippocampal cells against beta-amyloid-induced toxicity correlated well (r=0.74) with their apparent affinity in the [3H]resveratrol binding assay. Taken together, these results suggest that the neuroprotective action of various polyphenols and resveratrol analogs could be mediated by the activation of common "receptor" binding sites particularly enriched at the level of the cellular plasma membrane in the rat brain.

BODIPY-conjugated neuropeptide Y ligands: new fluorescent tools to tag Y1, Y2, Y4 and Y5 receptor subtypes.

N-terminal labelled fluorescent BODIPY-NPY peptide analogues were tested in Y1, Y2, Y4 and Y5 receptor-binding assays performed in rat brain membrane preparations and HEK293 cells expressing the rat Y1, Y2, Y4 and Y5 receptors. BODIPY TMR/FL-[Leu31, Pro34]NPY/PYY were able to compete for specific [125][Leu31, Pro34]PYY-binding sites with an affinity similar to that observed for the native peptide at the Y1 (Ki=1-6 nM), Y2 (Ki>1000 nM), Y4 (Ki=10 nM) and Y5 (Ki=1-4 nM) receptor subtypes. BODIPY FL-PYY(3-36) was able to compete for specific Y2 (Ki=10 nM) and Y5 (Ki=30 nM) binding sites, but had almost no affinity in Y1 and Y4 assays. BODIPY FL-hPP was able to compete with high affinity (Ki; 1 and 15 nM) only in Y4 and Y5 receptor-binding assays. BODIPY TMR-[cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP and BODIPY TMR-[hPP(1-17), Ala31, Aib32]NPY were potent competitors only on specific Y5-binding sites (Ki=0.1-0.6 nM). As expected, these fluorescent peptides inhibited forskolin-induced cAMP accumulation, demonstrating that they retained their agonist properties. When tested in confocal microscopy imaging, fluorescent Y1 and Y5 agonists internalized in a time-dependent manner in Y1 and Y5 transfected cells, respectively. These results demonstrate that BODIPY-conjugated NPY analogues retain their selectivity, affinity and agonist properties for the Y1, Y2, Y4 and Y5 receptor subtypes, respectively. Thus, they represent novel tools to study and visualize NPY receptors in living cells.

FoxO-dependent and -independent mechanisms mediate SirT1 effects on IGFBP-1 gene expression.

Sirtuin 1 (SirT1), an NAD-dependent deacetylase that is important for promoting longevity during caloric restriction, can deacetylate and enhance the function of forkhead box transcription factors, O subfamily (FoxO). We examined the effect of SirT1 on the regulation of insulin-like growth factor-binding protein 1 (IGFBP-1), a known target of FoxO proteins that is increased in fasting. Co-transfection with a SirT1 expression vector dose-dependently stimulated IGFBP-1 promoter activity and a heterologous reporter gene construct containing three FoxO-binding sites linked to a minimal promoter. This effect is mimicked by 20muM resveratrol, a potent SirT1 activator, and immunoprecipitation and Western blotting confirm that SirT1 and FoxO1 interact in cells. Interestingly, mutation of FoxO-binding sites in the IGFBP-1 promoter reduces, but does not completely disrupt, the stimulatory effect of SirT1 on promoter activity. We found that overexpression of SirT1 is accompanied by enhanced mitogen-activated protein kinase (MAPK) activation. Treatment of SirT1-cotransfected cells with PD98059, which inhibits MAPK activation, decreased IGFBP-1 promoter activity by approximately 50%, in a FoxO-binding site-independent manner, and disrupts the residual effect of SirT1. These results indicate that SirT1 stimulates IGFBP-1 promoter activity through FoxO-dependent and -independent mechanisms, and provides the first evidence that activation of MAPK contributes to effects of SirT1 on gene expression.