Imaging flow cytometry of tumoroids: A new method for studying GPCR expression.

Fluorescence confocal microscopy is commonly used to analyze the regulation membrane proteins expression such as G protein-coupled receptors (GPCRs). With this approach, the internal movement of GPCRs within the cell can be observed with a high degree of resolution. However, these microscopy techniques led to complex and time-consuming analysis and did not allow a large population of events to be sampled. A recent approach termed imaging flow cytometry (IFC), which combines flow cytometry and fluorescence microscopy, had two main advantages to study the regulation of GPCRs expression such as orexins receptors (OXRs): the ability (1) to analyze large numbers of cells and; (2) to visualize cell integrity and fluorescent markers localization. Here, we compare these two technologies using the orexin A (OxA) ligand coupled to rhodamine (OxA-rho) to investigate anti-tumoral OX1R expression in human digestive cancers. IFC has been adapted for cancer epithelial adherent cells and also to 3D cell culture tumoroids which partially mimic tumoral structures. In the absence of specific antibody, expression of OX1R is examined in the presence of OxA-rho. 2D-culture of colon cancer cells HT-29 exhibits a maximum level of OX1R internalization induced by OxA with 19% ± 3% colocalizing to early endosomes. In 3D-culture of HT-29 cells, internalization of OX1R/OxA-rho reached its maximum at 60 min, with 30.7% ± 6.4% of OX1R colocalizing with early endosomes. This is the first application of IFC to the analysis of the expression of a native GPCR, OX1R, in both 2D and 3D cultures of adherent cancer cells.

Cognitive and functional patterns of nondemented subjects with equivocal visual amyloid PET findings.

PURPOSE: Despite good to excellent inter-reader agreement in the evaluation of amyloid load on PET scans in subjects with Alzheimer's disease, some equivocal findings have been reported in the literature. We aimed to describe the clinical characteristics of subjects with equivocal PET images. METHODS: Nondemented subjects aged 70 years or more were enrolled from the MAPT trial. Cognitive and functional assessments were conducted at baseline, at 6 months, and annually for 3 years. During the follow-up period, 271 subjects had (18)F-AV45 PET scans. Images were visually assessed by three observers and classified as positive, negative or equivocal (if one observer disagreed). After debate, equivocal images were reclassified as positive (EP+) or negative (EP-). Scans were also classified by semiautomated quantitative analysis using mean amyloid uptake of cortical regions. We evaluated agreement among the observers, and between visual and quantitative assessments using kappa coefficients, and compared the clinical characteristics of the subjects according to their PET results. RESULTS: In 158 subjects (58.30 %) the PET scan was negative for amyloid, in 77 (28.41 %) the scan was positive and in 36 (13.28 %) the scan was equivocal. Agreement among the three observers was excellent (kappa 0.80). Subjects with equivocal images were more frequently men (58 % vs. 37 %) and exhibited intermediate scores on cognitive and functional scales between those of subjects with positive and negative scans. Amyloid load differed between the EP- and negative groups and between the EP+ and positive groups after reclassification. CONCLUSION: Equivocal amyloid PET images could represent a neuroimaging entity with intermediate amyloid load but without a specific neuropsychological pattern. Clinical follow-up to assess cognitive evolution in subjects with equivocal scans is needed.

Amyloid beta peptide immunotherapy in Alzheimer disease.

Recent advances in the understanding of Alzheimer's disease pathogenesis have led to the development of numerous compounds that might modify the disease process. Amyloid ß peptide represents an important molecular target for intervention in Alzheimer's disease. The main purpose of this work is to review immunotherapy studies in relation to the Alzheimer's disease. Several types of amyloid ß peptide immunotherapy for Alzheimer's disease are under investigation, active immunization and passive administration with monoclonal antibodies directed against amyloid ß peptide. Although immunotherapy approaches resulted in clearance of amyloid plaques in patients with Alzheimer's disease, this clearance did not show significant cognitive effect for the moment. Currently, several amyloid ß peptide immunotherapy approaches are under investigation but also against tau pathology. Results from amyloid-based immunotherapy studies in clinical trials indicate that intervention appears to be more effective in early stages of amyloid accumulation in particular solanezumab with a potential impact at mild Alzheimer's disease, highlighting the importance of diagnosing Alzheimer's disease as early as possible and undertaking clinical trials at this stage. In both phase III solanezumab and bapineuzumab trials, PET imaging revealed that about a quarter of patients lacked fibrillar amyloid pathology at baseline, suggesting that they did not have Alzheimer's disease in the first place. So a new third phase 3 clinical trial for solanezumab, called Expedition 3, in patients with mild Alzheimer's disease and evidence of amyloid burden has been started. Thus, currently, amyloid intervention is realized at early stage of the Alzheimer's disease in clinical trials, at prodromal Alzheimer's disease, or at asymptomatic subjects or at risk to develop Alzheimer's disease and or at asymptomatic subjects with autosomal dominant mutation.

Using PET with 18F-AV-45 (florbetapir) to quantify brain amyloid load in a clinical environment.

PURPOSE: Positron emission tomography (PET) imaging of brain amyloid load has been suggested as a core biomarker for Alzheimer's disease (AD). The aim of this study was to test the feasibility of using PET imaging with (18)F-AV-45 (florbetapir) in a routine clinical environment to differentiate between patients with mild to moderate AD and mild cognitive impairment (MCI) from normal healthy controls (HC). METHODS: In this study, 46 subjects (20 men and 26 women, mean age of 69.0 ± 7.6 years), including 13 with AD, 12 with MCI and 21 HC subjects, were enrolled from three academic memory clinics. PET images were acquired over a 10-min period 50 min after injection of florbetapir (mean ± SD of radioactivity injected, 259 ± 57 MBq). PET images were assessed visually by two individuals blinded to any clinical information and quantitatively via the standard uptake value ratio (SUVr) in the specific regions of interest, which were defined in relation to the cerebellum as the reference region. RESULTS: The mean values of SUVr were higher in AD patients (median 1.20, Q1-Q3 1.16-1.30) than in HC subjects (median 1.05, Q1-Q3 1.04-1.08; p = 0.0001) in the overall cortex and all cortical regions (precuneus, anterior and posterior cingulate, and frontal median, temporal, parietal and occipital cortex). The MCI subjects also showed a higher uptake of florbetapir in the posterior cingulate cortex (median 1.06, Q1-Q3 0.97-1.28) compared with HC subjects (median 0.95, Q1-Q3 0.82-1.02; p = 0.03). Qualitative visual assessment of the PET scans showed a sensitivity of 84.6% (95% CI 0.55-0.98) and a specificity of 38.1% (95% CI 0.18-0.62) for discriminating AD patients from HC subjects; however, the quantitative assessment of the global cortex SUVr showed a sensitivity of 92.3% and specificity of 90.5% with a cut-off value of 1.122 (area under the curve 0.894). CONCLUSION: These preliminary results suggest that PET with florbetapir is a safe and suitable biomarker for AD that can be used routinely in a clinical environment. However, the low specificity of the visual PET scan assessment could be improved by the use of specific training and automatic or semiautomatic quantification tools.

Six-month outcome of elderly people hospitalized via the emergency department: the SAFES cohort.

BACKGROUND: The objective of the study was to identify factors predictive of 6-month institutionalization or mortality in frail elderly patients after acute hospitalization. METHODS: A prospective cohort of elderly subjects 75 years and older was set up in nine French teaching hospitals. Data obtained from a comprehensive geriatric assessment were used in a Cox model to predict 6-month institutionalization or mortality. Institutionalization was defined as incident admission either to a nursing home or other long-term care facility during the follow-up period. RESULTS: Crude institutionalization and death rates after 6 months of follow-up were 18% and 24%, respectively. Independent predictors of institutionalization were: living alone (HR=1.83; 95% CI=1.27-2.62) or a higher number of children (HR=0.86; 95% CI=0.78-0.96), balance problems (HR=1.72; 95% CI=1.19-2.47), malnutrition or risk thereof (HR=1.93; 95% CI=1.24-3.01), and dementia syndrome (HR=1.88; 95% CI=1.32-2.67). Factors found to be independently related to 6-month mortality were exclusively medical factors: malnutrition or risk thereof (HR=1.92; 95% CI=1.17-3.16), delirium (HR=1.80; 95% CI=1.24-2.62), and a high level of comorbidity (HR=1.62; 95% CI=1.09-2.40). Institutionalization (HR=1.92; 95% CI=1.37-2.71) and unplanned readmission (HR=4.47; 95% CI=3.16-2.71) within the follow-up period were also found as independent predictors. CONCLUSION: The main factors predictive of 6-month outcome identified in this study are modifiable by global and multidisciplinary interventions. Their early identification and management would make it possible to modify frail elderly subjects' prognosis favorably.

Framework Implementation of the INSPIRE ICOPE-CARE Program in Collaboration with the World Health Organization (WHO) in the Occitania Region.

INTRODUCTION: Limiting the number of dependent older people in coming years will be a major economic and human challenge. In response, the World Health Organization (WHO) has developed the «Integrated Care for Older People (ICOPE)¼ approach. The aim of the ICOPE program is to enable as many people as possible to age in good health. To reach this objective, the WHO proposes to follow the trajectory of an individual's intrinsic capacity, which is the composite of all their physical and mental capacities and comprised of multiple domains including mobility, cognition, vitality / nutrition, psychological state, vision, hearing. OBJECTIVE: The main objective of the INSPIRE ICOPE-CARE program is to implement, in clinical practice at a large scale, the WHO ICOPE program in the Occitania region, in France, to promote healthy aging and maintain the autonomy of seniors using digital medicine. METHOD: The target population is independent seniors aged 60 years and over. To follow this population, the 6 domains of intrinsic capacity are systematically monitored with pre-established tools proposed by WHO especially STEP 1 which has been adapted in digital form to make remote and large-scale monitoring possible. Two tools were developed: the ICOPE MONITOR, an application, and the BOTFRAIL, a conversational robot. Both are connected to the Gerontopole frailty database. STEP 1 is performed every 4-6 months by professionals or seniors themselves. If a deterioration in one or more domains of intrinsic capacity is identified, an alert is generated by an algorithm which allows health professionals to quickly intervene. The operational implementation of the INSPIRE ICOPE-CARE program in Occitania is done by the network of Territorial Teams of Aging and Prevention of Dependency (ETVPD) which have more than 2,200 members composed of professionals in the medical, medico-social and social sectors. Targeted actions have started to deploy the use of STEP 1 by healthcare professionals (physicians, nurses, pharmacists,…) or different institutions like French National old age insurance fund (CNAV), complementary pension funds (CEDIP), Departmental Council of Haute Garonne, etc. Perspective: The INSPIRE ICOPE-CARE program draws significantly on numeric tools, e-health and digital medicine to facilitate communication and coordination between professionals and seniors. It seeks to screen and monitor 200,000 older people in Occitania region within 3 to 5 years and promote preventive actions. The French Presidential Plan Grand Age aims to largely implement the WHO ICOPE program in France following the experience of the INSPIRE ICOPE-CARE program in Occitania.

A new solvent system: Hydrothermal molten salt.

This work proposes a new solvent system composed of a molten salt in pressurized water, so-called hydrothermal molten salt (HyMoS). This system changes the paradigm of the solubility of inorganics in supercritical water. Using as an example NaOH, a low melting temperature salt, we show the possibility to precipitate it at a temperature above its melting one, leading to the instantaneous formation of the HyMoS. The molten salt is then capable of dissolving a large amount of inorganic salt, as exemplified with Na2SO4. This solvent system opens innovative ways with a potential to impact applications in many fields including materials synthesis, biomass conversion, recycling, green chemistry, catalysis, sustainable manufacturing and others. Beyond the impact on the hydrothermal community, this work also offers previously unexplored opportunities for the molten salt field with access to flow chemistry and insights regarding salt precipitation mechanism.

Derivation and validation of a mortality-risk index from a cohort of frail elderly patients hospitalised in medical wards via emergencies: the SAFES study.

To identify predictive factors for 2-year mortality in frail elderly patients after acute hospitalisation, and from these to derive and validate a Mortality Risk Index (MRI). A prospective cohort of elderly patients was set up in nine teaching hospitals. This cohort was randomly split up into a derivation cohort (DC) of 870 subjects and a validation cohort (VC) of 436 subjects. Data obtained from a Comprehensive Geriatric Assessment were used in a Cox model to predict 2-year mortality and to identify risk groups for mortality. A ROC analysis was performed to explore the validity of the MRI. Five factors were identified and weighted using hazard ratios to construct the MRI: age 85 or over (1 point), dependence for the ADL (1 point), delirium (2 points), malnutrition risk (2 points), and co-morbidity level (2 points for medium level, 3 points for high level). Three risk groups were identified according to the MRI. Mortality rates increased significantly across risk groups in both cohorts. In the DC, mortality rates were: 20.8% in the low-risk group, 49.6% in the medium-risk group, and 62.1% in the high-risk group. In the VC, mortality rates were respectively 21.7, 48.5, and 65.4%. The area under the ROC curve for overall score was statistically the same in the DC (0.72) as in the VC (0.71). The proposed MRI appears as a simple and easy-to-use tool developed from relevant geriatric variables. Its accuracy is good and the validation procedure gives a good stability of results.

Predicting early mortality among elderly patients hospitalised in medical wards via emergency department: the SAFES cohort study.

OBJECTIVES: The aim of the study was, by early identification of deleterious prognostic factors that are open to remediation, to be in a position to assign elderly patients to different mortality risk groups to improve management. DESIGN: Prospective multicentre cohort. SETTING: Nine French teaching hospitals. PARTICIPANTS: One thousand three hundred and six (1 306) patients aged 75 and over, hospitalised after having passed through Emergency Department (ED). MEASUREMENTS: Patients were assessed using Comprehensive Geriatric Assessment (CGA) tools. A Cox survival analysis was performed to identify prognostic variables for six-week mortality. Receiver Operating Characteristics analysis was used to study the discriminant power of the model. A mortality risk score is proposed to define three risk groups for six-week mortality. RESULTS: Crude mortality rate after a six week follow-up was 10.6% (n=135). Prognostic factors identified were: malnutrition risk (HR=2.1; 95% CI: 1.1-3.8; p=.02), delirium (HR=1.7; 95% CI: 1.2-2.5; p=.006), and dependency: moderate dependency (HR=4.9; 95% CI: 1.5-16.5; p=.01) or severe dependency (HR=10.3; 95% CI: 3.2-33.1; p < .001). The discriminant power of the model was good: the c-statistic representing the area under the curve was 0.71 (95% IC: 0.67 - 0.75; p < .001). The six-week mortality rate increased significantly (p < .001) across the three risk groups: 1.1% (n=269; 95% CI=0.5-1.7) in the lowest risk group, 11.1% (n=854; 95% CI=9.4-12.9) in the intermediate risk group, and 22.4% (n=125; 95% CI=20.1-24.7) in the highest risk group. CONCLUSIONS: A simple score has been calculated (using only three variables from the CGA) and a practical schedule proposed to characterise patients according to the degree of mortality risk. Each of these three variables (malnutrition risk, delirium, and dependency) identified as independent prognostic factors can lead to a targeted therapeutic option to prevent early mortality.

Ectopic expression of OX1R in ulcerative colitis mediates anti-inflammatory effect of orexin-A.

Orexins (orexin-A and orexin-B) are hypothalamic peptides that are produced by the same precursor and are involved in sleep/wake control, which is mediated by two G protein-coupled receptor subtypes, OX1R and OX2R. Ulcerative colitis (UC) is an inflammatory bowel disease, (IBD) which is characterized by long-lasting inflammation and ulcers that affect the colon and rectum mucosa and is known to be a significant risk factor for colon cancer development. Based on our recent studies showing that OX1R is aberrantly expressed in colon cancer, we wondered whether orexin-A could play a role in UC. Immunohistochemistry studies revealed that OX1R is highly expressed in the affected colonic epithelium of most UC patients, but not in the non-affected colonic mucosa. Injection of exogenous orexin-A specifically improved the inflammatory symptoms in the two colitis murine models. Conversely, injection of inactive orexin-A analog, OxB7-28 or OX1R specific antagonist SB-408124 did not have anti-inflammatory effect. Moreover, treatment with orexin-A in DSS-colitis induced OX1R-/- knockout mice did not have any protective effect. The orexin-A anti-inflammatory effect was due to the decreased expression of pro-inflammatory cytokines in immune cells and specifically in T-cells isolated from colonic mucosa. Moreover, orexin-A inhibited canonical NFκB activation in an immune cell line and in intestinal epithelial cell line. These results suggest that orexin-A might represent a promising alternative to current UC therapies.

Characterization and distribution of alpha 2-adrenergic receptors in the human intestinal mucosa.

The subtype and the expression of the alpha 2-adrenergic receptor were investigated in the normal mucosa from human intestine by means of radioligand binding, RNase mapping, and measurement of adenylate cyclase activity. The study of the binding of the alpha 2-adrenergic antagonist, [3H]RX821002, to epithelial cell membranes indicated the existence of a single class of noninteracting sites displaying a high affinity for the radioligand (Kd = 1.1 +/- 0.5 nM). The rank order of potency of antagonists to inhibit [3H]RX821002 binding (RX821002 > yohimbine = rauwolscine > phentolamine approximately idazoxan >> chlorpromazine > prazosin) suggested that the receptor is of the alpha 2A subtype. A conclusion which is confirmed by the fact that only alpha 2C10 transcripts were found in the human intestine mucosa. Competition curves with (-)-norepinephrine demonstrated that 60% of the receptor population exhibited high affinity for agonists. This high-affinity state was abolished by the addition of GTP plus Na+ or by prior treatment of the membranes with pertussis toxin indicating it corresponded to G protein-coupled receptors. [32P]ADP-ribosylation and immunoblotting experiments identified two pertussis toxin-sensitive G proteins corresponding to Gi2 and Gi3. The study of the distribution of the receptor indicated that (a) the proximal colon is the intestine segment exhibiting the highest receptor density and (b) the receptor is predominantly expressed in crypts and is preferentially located in the basolateral membrane of the polarized cell. The distribution of the receptor along the crypt-surface axis of the colon mucosa can be correlated with a higher level of alpha 2C10-specific mRNA and a higher efficiency of UK14304 to inhibit adenylate cyclase in crypt cells.

PepT1-mediated epithelial transport of dipeptides and cephalexin is enhanced by luminal leptin in the small intestine.

Dietary proteins are mostly absorbed as di- and tripeptides by the intestinal proton-dependent transporter PepT1. We have examined the effects of leptin on PepT1 function in rat jejunum and in monolayers of the human enterocyte-like 2 cell Caco-2. Leptin is produced by the stomach and secreted in the gut lumen. We show here that PepT1 and leptin receptors are expressed in Caco-2 and rat intestinal mucosal cells. Apical (but not basolateral) leptin increased Caco-2 cell transport of cephalexin (CFX) and glycylsarcosine (Gly-Sar), an effect that was associated with increased Gly-Sar uptake, increased membrane PepT1 protein, decreased intracellular PepT1 content, and no change in PepT1 mRNA levels. The maximal velocity (Vmax) for Gly-Sar transport was significantly increased by leptin, whereas the apparent Michaelis-Menten constant (Km) did not change. Furthermore, leptin-stimulated Gly-Sar transport was completely suppressed by colchicine, which disrupts cellular translocation of proteins to plasma membranes. Intrajejunal leptin also induced a rapid twofold increase in plasma CFX after jejunal perfusion with CFX in the rat, indicating enhanced intestinal absorption of CFX. These data revealed an unexpected action of gastric leptin in controlling ingestion of dietary proteins.

Neuropsychological Profile of "Cognitive Frailty" Subjects in MAPT Study.

OBJECTIVES: An international group proposed the existence of "cognitive frailty", a condition defined by simultaneous presence of physical frailty and cognitive impairment in the absence of dementia. The objective was to compare the neuropsychological profiles in subgroups of elders differentiated across their physical frailty (Fried phenotype) and cognitive status (Clinical Dementia Rating score) to characterize the "cognitive frailty" entity. METHOD: We studied baseline characteristics of 1,617 subjects enrolled in Multidomain Alzheimer Disease Preventive Trial (MAPT). Included subjects were aged 70 years or older and presented at least 1 of the 3 following clinical criteria: (1) Memory complaint spontaneously reported to a general practitioner, (2) limitation in one instrumental activity of daily living, (3) slow gait speed. Subjects with dementia were not included in the trial. RESULTS: "Cognitive frailty individuals" significantly differed from "individuals with cognitive impairment and without physical frailty", scoring worse at executive, and attention tests. They presented subcortico-frontal cognitive pattern different of Alzheimer Disease. Cognitive performance of subjects with 3 criteria or more of the frailty phenotype are cognitively more impaired than subjects with only one. DISCUSION: The characterization of "cognitive frailty" must be done in frail subjects to set up specific preventive clinical trials for this population.

Predictive factors of emergency hospitalisation in Alzheimer's patients: results of one-year follow-up in the REAL.FR Cohort.

OBJECTIVE: To determine the predictive factors of hospitalisation in patients with Alzheimer's disease followed in the REAL.FR cohort. METHODS: A French multicentre prospective study (REAL.FR) following 516 patients who had mild to moderate AD at inclusion. Analysis of the data after one year of follow-up. RESULTS: 139 (26,9%) of the 516 initial AD patients were hospitalized during the 1 year of follow-up. After bivariate analysis, the principal predictive factors of hospitalisation were high scores on the Reisberg scale (> or = 5: P = 0.0149) and the CDR (1: P = 0.0289; 2 or 3: P = 0.0078); > or = 2 intercurrent diseases (P = 0.00104); > or = 3 other treatments (not including specific treatments for AD) (P = 0.0026); BMI (kg/m2) between 25 and 30 (P = 0.0147); impossibility of single-leg stance (P = 0.02); > or = 1 disabilities on the ADL (P = 0.0009) and > or = 2 disabilities on the IADL (P = 0.0017); use of medical services (P = 0.0236) and of non-medical services (P = 0.0403); delirium or hallucinations (P = 0.0135), depression (P = 0.0014), or disinhibited behaviour (P = 0.0030); the gravity and frequency of behavioural symptoms (NPI freq x grav > or = 11 (median), P = 0.0012); and lastly, a score of > or = 20 for subjective caregiver burden on the Zarit scale (P < 0.0001). Multivariate analysis revealed an association between the risk of hospitalisation and the following variables : the type of centre to which the patient was admitted (neurological, psychiatric or geriatric), impaired orientation on the MMS, BMI, the number of disabilities on the ADL, and caregiver burden as evaluated by the Zarit scale. CONCLUSION: At inclusion, patients with more severe cognitive disorders, poor nutritional status and those who were the most dependent for basic activities of daily living were already at greater risk of hospitalisation. Exhaustion of the informal caregiver was an independent and supplementary predictive factor of hospitalisation.

Predictive factors for rapid loss on the mini-mental state examination in Alzheimer's disease.

OBJECTIVES: To seek predictors of rapid loss of cognition and to evaluate their frequency in a prospective study of patients suffering from Alzheimer's disease (AD). DESIGN: A one-year prospective study. METHODS: 312 AD patients from the memory clinic at Toulouse University Hospital, participants in the ELSA study, were enrolled. Rapid cognitive decline was defined as a 4-point or greater loss on the Mini-Mental State Examination (MMSE) in 6 months. Comprehensive geriatric and neuropsychological assessment was conducted at baseline, 6 months and one year. RESULTS: Seventy-nine (24.8%) patients presented rapid cognitive loss over 6 months. The majority were stable at one year whereas 15 experienced continued rapid cognitive loss. Multivariate analysis showed that only the Mini Nutritional Assessment score (MNA) was correlated with rate of decline. Patients with rapid cognitive decline were also significantly more dependent at 6 months. CONCLUSIONS: Our findings demonstrate that rapid cognitive loss is frequent in AD patients, probably indicating underlying frailty. Future studies should lead to a practical approach to detecting these frail patients and to increasing preventive interventions.

Stable Freestanding Thin Films of Copolymer Melts Far from the Glass Transition.

Thin polymer films have attracted attention because of both their broad range of applications and of the fundamental questions they raise regarding the dynamic response of confined polymers. These films are unstable if the temperature is above their glass transition temperature Tg. Here, we describe freestanding thin films of centimetric dimensions made of a comb copolymer melt far from its glass transition that are stable for more than a day. These long lifetimes allowed us to characterize the drainage dynamics and the thickness profile of the films. Stratified regions appear as the film drains. We have evidence that the stability, thinning dynamics, and thickness profile of the films result from structural forces in the melt. Understanding the key mechanisms behind our observations may lead to new developments in polymeric thin films, foams, and emulsions without the use of stabilizing agents.

Identification and functional studies of a specific peptide YY-preferring receptor in dog adipocytes.

Specific binding sites for peptide YY (PYY) and neuropeptide Y (NPY) as well as functional responses were identified in dog adipocytes. Studies were carried out using the radioligand [125I-Tyr1]monoiodo-PYY on crude adipocyte membranes. [125I]PYY bound to dog adipocyte membranes with a high affinity (156 +/- 24 pM) and binding capacity of 314 +/- 48 fmol/mg protein. Competition studies revealed a higher affinity of the binding sites for PYY than NPY (inhibition constants were 118 +/- 17 pM and 300 +/- 53 pM, respectively, P < or = 0.001). NPY analogs displaced [125I]PYY specific binding with the following order of potency: NPY-(13-36) > NPY-(18-36) > NPY-(22-36) >> [Leu31-Pro34]NPY. Neither adrenergic nor adenosine agents (activating or inhibiting other antilipolytic systems) interacted with [125I]PYY binding sites. So [125I]PYY binding was specific, saturable, and reversible. Lipolysis experiments performed with PYY, NPY, and NPY analogs confirm the relative order of potency found in competition experiments. The data agree with the definition of PYY-preferring receptor which resembles a Y2 receptor subtype since NPY-(13-36), a specific Y2 receptor agonist, inhibited binding and lipolysis in a similar way to PYY, whereas [Leu31-Pro34]NPY did not. No difference was observed in the antilipolytic response between IC50 values measured on omental, perirenal, and subcutaneous fat deposits. Moreover, PYY and NPY (10(-6) M) significantly attenuated forskolin-stimulated cAMP levels, involving inhibition of adenylyl cyclase as a transmembrane signaling mechanism. Cross-linking of bound [125I]PYY to membranes indicated that the mol wt of the receptor was 62K. The relative importance of such a receptor on fat cells alongside another powerful antilipolytic receptor--the alpha 2-adrenoceptor--is discussed.

Structure-activity studies including a Psi(CH(2)-NH) scan of peptide YY (PYY) active site, PYY(22-36), for interaction with rat intestinal PYY receptors: development of analogues with potent in vivo activity in the intestine.

Peptide YY (PYY) is a gut hormone that inhibits secretion and promotes absorption and growth in the intestinal epithelium. We have performed structure-activity studies with the active site, N-alpha-Ac-PYY(22-36)-NH(2), for interaction with intestinal PYY receptors. Investigation of aromatic substitutions at position 27 resulted in analogues that exhibited potent in vitro antisecretory potencies with N-alpha-Ac-[Trp(27)]PYY(22-36)-NH(2) exhibiting even greater potency than intact PYY. In vivo studies in dogs revealed that this analogue also promoted intestinal absorption of water and electrolytes during continuous intravenous and intraluminal infusion. Investigations carried out to identify features that would enhance stability revealed that incorporation of Trp(30) increased affinity for PYY receptors. A "CH(2)-NH" scan revealed that incorporation of reduced bonds at position 28-29 or 35-36 imparted greater receptor affinity. In general, disubstituted analogues designed based on the results of single substitutions exhibited good receptor affinity with N-alpha-Ac-[Trp(27),CH(2)-NH(35-36)]PYY(22-36)-NH(2) having the greatest affinity (IC(50) = 0.28 nM). Conservative multiple substitutions with Nle-->Leu and Nva-->Val also imparted good affinity. An analogue designed to encompass most of the favored substitutions, N-alpha-Ac-[Nle(24,28),Trp(30),Nva(31), CH(2)-NH(35-36)]PYY(22-36)-NH(2), exhibited a proabsorptive effect in dogs comparable to, but longer lasting than, that of intact hormone. Selected analogues also exhibited good antisecretory potencies in rats with N-alpha-Ac-[Trp(30)]PYY(22-36)-NH(2) being even more potent than PYY. However, the potencies did not correlate well with the PYY receptor affinity or the proabsorptive potencies in dogs. These differences could be due to species effects and/or the involvement of multiple receptors and neuronal elements in controlling the in vivo activity of PYY compounds. PYY(22-36) analogues exhibited good affinity for neuronal Y2 receptors but poor affinity for Y1 receptors. Also, crucial analogues in this series hardly bound to Y4 and Y5 receptors. In summary, we have developed PYY(22-36) analogues which, via interacting with intestinal PYY receptors, promoted potent and long-lasting proabsorptive and antisecretory effects in in vivo models. These compounds or analogues based on them may have useful clinical application in treating malabsorptive disorders observed under a variety of conditions.

Comparison of the antisecretory effect of endogenous forms of peptide YY on fed and fasted rat jejunum.

It is intriguing that the antisecretory peptide YY is present in plasma in two forms: PYY1-36 and PYY3-36. PYY3-36 has been found in human and rabbit blood within 30 min of the beginning of the meal, when the peak of water and electrolyte secretion occurs in the duodeno-jejunum. The aim of this study was therefore to compare the antisecretory effect of PYY1-36 and PYY3-36 in fed and fasted rat jejunum. The variations in electrolyte secretion were assessed by measuring the variations in short-circuit current (delta Isc) and transepithelial isotopic chloride fluxes in jejunal mucosa isolated from fed and fasted animal, and mounted in Ussing Chambers. In fasted animals, 2 x 10(-7) M PYY3-36 induced a reduction in Isc of -0.50 +/- 0.01 microEq/hr.cm2, which was not statistically different from that induced by 2 x 10(-7) M PYY1-36 (-0.60 +/- 0.01 microEq/h cm2). In contrast, in fed animals, 2 x 10(-7) M PYY3-36 did not trigger a significant response on Isc and net chloride flux, while the response to PYY1-36 was present but blunted. The absence of response was probably not related to the presence of secretory peptides because PYY3-36 was still able to induce a reduction in Isc after stimulation by a series of 10 different secretory peptides. After 10(-8) M PYY3-36 addition to an epithelium from the fasted animal, response to 10(-7) M PYY3-36 was blunted for 30 min and returned to control value after 60 min. Plasma concentration of PYY was higher in the fed rats compared to fasted (213.78 +/- 38 vs. 53.62 +/- 11.47 pg/ml p < 0.01). After incubation of crypt cells with or without 0.1 microM of unlabeled PYY for 60 min, Scatchard analysis of equilibrium binding data show that binding capacity (Bmax) of receptors was reduced when crypt cells were previously incubated with unlabeled PYY without significant modification of dissociation constants. Bmax were 183 +/- 27 in control vs. 56 +/- 11 fmol/mg protein. These results confirm the antisecretory activity of PYY1-36 in the jejunum of fasted and fed rats. They further indicate that PYY3-36 displays similar activity to PYY1-36 in fasted animals, but lack of activity in fed animals. These results suggest that the two circulating forms of PYY act as antisecretory peptides by two different mechanisms, implying a C-terminal specificity.

Structure-activity studies of peptide YY(22-36): N-alpha-Ac-[Phe27]PYY(22-36), a potent antisecretory peptide in rat jejunum.

Peptide YY (PYY) and its homologous peptide, neuropeptide Y (NPY), are known to exhibit potent antisecretory effects in the intestine. To determine the structural requirements to elicit antisecretory effects, we have synthesized several analogs of the PYY active site, PYY(22-36), and compared their binding affinities and antisecretory potencies in rat jejunum. These investigations revealed that the hydroxyl groups of Ser23 and Thr32, as well as the imidazole group of His26, are important for activity in the intestine. N-alpha-acetylation of PYY(22-36) increased both the binding affinity and antisecretory potency. Structure-activity studies with N-alpha-Ac-PYY(22-36) showed that substitution of His26 with parachlorophenylalanine (pCl-Phe) or Tyr36 with N-Me-Tyr reduced receptor affinity, while replacement of Tyr27 with Phe increased the activity substantially. Furthermore, acylation of the alpha-NH2 group with hydrophobic groups, myristic and naphthaleneacetic acids, substantially reduced the antisecretory potencies but not the binding affinities. Further modification of N-alpha-Ac-[Phe27]PYY(22-36) may lead to the development of more potent agonist compounds, which may provide a framework for the design of a new class of antidiarrheal drugs.