Real-Time Observation and Analysis of Magnetomechanical Actuation of Magnetic Nanoparticles in Cells.

The origin of cell death in the magnetomechanical actuation of cells induced by magnetic nanoparticle motion under low-frequency magnetic fields is still elusive. Here, a miniaturized electromagnet fitted under a confocal microscope is used to observe in real time cells specifically targeted by superparamagnetic nanoparticles and exposed to a low-frequency rotating magnetic field. Our analysis reveals that the lysosome membrane is permeabilized in only a few minutes after the start of magnetic field application, concomitant with lysosome movements toward the nucleus. Those events are associated with disorganization of the tubulin microtubule network and a change in cell morphology. This miniaturized electromagnet will allow a deeper insight into the physical, molecular, and biological process occurring during the magnetomechanical actuation of magnetic nanoparticles.

Prescription of benzodiazepines and Z-drugs among older patients in primary care: a French, national, cohort study.

BACKGROUND: In France, general practitioners (GPs) prescribe benzodiazepines and Z-drugs (BZD/ZDs) widely, and especially to older adults. Several characteristics of patients and/or GPs linked to BZD/ZD overprescription have been described in the general population but not among older patients in primary care. OBJECTIVES: To estimate the proportion of GP consultations by patients aged 65 and over that resulted in a BZD/ZD prescription, and determine whether any GP-related factors predicted BZD/ZD overprescription in this setting. METHODS: We analyzed sociodemographic and practice-related GP characteristics, and aggregated data on consultations recorded prospectively by 117 GPs in a database between 2000 and 2010. Next, we used logistic regression models to look for factors potentially associated with BZD/ZD overprescription (defined as an above-median prescription rate). RESULTS: The GPs' mean age at inclusion was 47.4 (7.1), and 87.9% were male. During the study period, the median (95% confidence interval) proportion of consultations with patients aged 65 and over resulting in a BZD/ZD prescription was 21.8% (18.1-26.1) (range per GP: 5-34.1%). In a multivariable analysis, a greater number of chronic disease (OR [95% CI] = 2.10 [1.22-3.64]), a greater number of drugs prescribed per consultation (5.29 [2.72-10.28]), and shorter study participation were independently associated with BZD/ZD overprescription. CONCLUSIONS: BZD/ZD overprescription was associated with a greater chronic disease burden and the number of drugs prescribed per consultation but not with any sociodemographic or practice-related GP characteristics. Targeted actions are needed to help GPs limit their prescription of BZD/ZDs to older patients with multiple comorbidities and polypharmacy.

In France, general practitioners (GPs) prescribe benzodiazepines and Z-drugs (BZD/ZDs) widely, and especially to older adults. Even though BZD/ZDs may not have a favorable risk­benefit ratio in older patients, we lack data on GP-related factors that might influence BZD/ZD overprescription in our population. The objectives of the present study were to (i) estimate the proportion of GP consultations by patients aged 65 and over that resulted in a BZD/ZD prescription and (ii) identify GP-related factors that were predictive of overprescription. To achieve this goal, we analyzed consultation notes registered by 117 GPs in a database curated by the French Society of General Practice between 2000 and 2010. About 22% of consultations by patients aged 65 and over resulted in a BZD/ZD prescription. With regard to the GPs, we did not find any sociodemographic or practice-related characteristics associated with overprescription. A greater chronic disease burden and the number of drug prescriptions (other than BZD/ZDs) per consultation was independently associated with overprescription. Targeted actions are therefore needed to help GPs limit their prescription of BZD/ZDs in older patients with multimorbidity and polypharmacy.

Upregulated Apelin Signaling in Pancreatic Cancer Activates Oncogenic Signaling Pathways to Promote Tumor Development.

Despite decades of effort in understanding pancreatic ductal adenocarcinoma (PDAC), there is still a lack of innovative targeted therapies for this devastating disease. Herein, we report the expression of apelin and its receptor, APJ, in human pancreatic adenocarcinoma and its protumoral function. Apelin and APJ protein expression in tumor tissues from patients with PDAC and their spatiotemporal pattern of expression in engineered mouse models of PDAC were investigated by immunohistochemistry. Apelin signaling function in tumor cells was characterized in pancreatic tumor cell lines by Western blot as well as proliferation, migration assays and in murine orthotopic xenograft experiments. In premalignant lesions, apelin was expressed in epithelial lesions whereas APJ was found in isolated cells tightly attached to premalignant lesions. However, in the invasive stage, apelin and APJ were co-expressed by tumor cells. In human tumor cells, apelin induced a long-lasting activation of PI3K/Akt, upregulated ß-catenin and the oncogenes c-myc and cyclin D1 and promoted proliferation, migration and glucose uptake. Apelin receptor blockades reduced cancer cell proliferation along with a reduction in pancreatic tumor burden. These findings identify the apelin signaling pathway as a new actor for PDAC development and a novel therapeutic target for this incurable disease.

Predictive factors for non-participation or partial participation in breast, cervical and colorectal cancer screening programmes†.

BACKGROUND: No study has investigated factors associated with non-participation or partial participation in the different combination patterns of screening programmes for all three cancers, that is, breast, colorectal and cervical cancer. METHODS: In a retrospective cohort study, we sought to describe combinations of cancer screening participation rates among women in the Val-de-Marne area of France and to identify individual and contextual factors associated with non-participation or partial participation. RESULTS: Women aged between 50 and 65 and who were eligible for all three screening programmes (n = 102 219) were analysed in multilevel logistic models, with the individual as the Level 1 variable and the place of residence as the Level 2 variable. The women who did not participate in any of the screening programmes were 34.4%, whereas 30.1%, 24% and 11.5% participated in one, two or all three screening programmes, respectively. Age below 55, a previous false-positive mammography, prior opportunistic mammography only, no previous mammography, membership of certain health insurance schemes (all P < 0.05) and residence in a deprived area (P < 0.001) were independently associated with non-participation or partial participation. We observed a stronger effect of deprivation on non-participation in all three cancers than in combinations of screening programmes. CONCLUSION: Our findings suggest that the health authorities should focus on improving cancer screenings in general rather than screenings for specific types of cancer, especially among younger women and those living in the most socially deprived areas.

Real-Time Analysis of Magnetic Hyperthermia Experiments on Living Cells under a Confocal Microscope.

Combining high-frequency alternating magnetic fields (AMF) and magnetic nanoparticles (MNPs) is an efficient way to induce biological responses through several approaches: magnetic hyperthermia, drug release, controls of gene expression and neurons, or activation of chemical reactions. So far, these experiments cannot be analyzed in real-time during the AMF application. A miniaturized electromagnet fitting under a confocal microscope is built, which produces an AMF of frequency and amplitude similar to the ones used in magnetic hyperthermia. AMF application induces massive damages to tumoral cells having incorporated nanoparticles into their lysosomes without affecting the others. Using this setup, real-time analyses of molecular events occurring during AMF application are performed. Lysosome membrane permeabilization and reactive oxygen species production are detected after only 30 min of AMF application, demonstrating they occur at an early stage in the cascade of events leading eventually to cell death. Additionally, lysosomes self-assembling into needle-shaped organization under the influence of AMF is observed in real-time. This experimental approach will permit to get a deeper insight into the physical, molecular, and biological process occurring in several innovative techniques used in nanomedecine based on the combined use of MNPs and high-frequency magnetic fields.

[Restructuring of ambulatory care in France: proposals for the management of hypertensive patients]. / Restructuration des soins ambulatoires en France: propositions de gestion des patients hypertendus.

One of the factors responsible for the creation of multidisciplinary health centres is the growth of outpatient management of multiple chronic conditions. Based on a classification of hypertensive patients into eight groups, the authors discuss the interrelations between health care organization and modification of management. They discuss the effects of modification of health care structures and the need to create new job positions for the purposes of coordination, support of patients in the form of therapeutic education and support ofyoung professionals in multidisciplinary practice. External effects are improvement of office-hospital flows, especially with the development of second-line consultant roles and improved management of patient admissions and discharges. However, to ensure sustainable changes, there must be a change of mentalities with new modalities of remuneration of private practitioners and development of the health information system.

Oxidative stress induced by inactivation of TP53INP1 cooperates with KrasG12D to initiate and promote pancreatic carcinogenesis in the murine pancreas.

Tumor protein p53-induced nuclear protein 1 (TP53INP1) is involved in cell stress response. Its expression is lost at the pancreatic intraepithelial neoplasia 1b (PanIN1b)/PanIN2 stage of pancreatic carcinogenesis. Our objective was to determine whether TP53INP1 loss of expression contributes to pancreatic cancer formation in a conditional KrasG12D mouse model. We generated Kras-INP1KO mice using LSL-Kras(G12D/+);Pdx1-Cre(+/-) mice (Kras mice) and TP53INP1(-/-) mice. Analysis of pancreases during ageing shows that in the presence of activated Kras, TP53INP1 loss of expression accelerated PanIN formation and increased pancreatic injury and the number of high-grade lesions as compared with what occurs in Kras mice. Moreover, cystic lesions resembling intraductal papillary mucinous neoplasm (IPMN) were observed as early as 2 months of age. Remarkably, TP53INP1 is down-regulated in human IPMN. Activation of the small GTPase Rac1 shows that more oxidative stress is generated in Kras-INP1KO than in Kras mice pancreas despite elevated levels of the Nrf2 antioxidant regulator. We firmly establish the link between Kras-INP1KO pancreatic phenotype and oxidative stress with rescue of the phenotype by the antioxidant action of N-acetylcysteine. Our data provide in vivo functional demonstration that TP53INP1 deficiency accelerates progression of pancreatic cancer, underlining its role in the occurrence of IPMN and highlighting the importance of TP53INP1 in the control of oxidative status during development of pancreatic cancer.

Distinct CCK-2 receptor conformations associated with ß-arrestin-2 recruitment or phospholipase-C activation revealed by a biased antagonist.

Seven-transmembrane receptors (7TMRs), also termed G protein-coupled receptors (GPCRs), form the largest class of cell surface membrane receptors, involving several hundred members in the human genome. Nearly 30% of marketed pharmacological agents target 7TMRs. 7TMRs adopt multiple conformations upon agonist binding. Biased agonists, in contrast to non-biased agonists, are believed to stabilize conformations preferentially activating either G-protein- or ß-arrestin-dependent signaling pathways. However, proof that cognate conformations of receptors display structural differences within their binding site where biased agonism initiates, are still lacking. Here, we show that a non-biased agonist, cholecystokinin (CCK) induces conformational states of the CCK2R activating Gq-protein-dependent pathway (CCK2R(G)) or recruiting ß-arrestin2 (CCK2R(ß)) that are pharmacologically and structurally distinct. Two structurally unrelated antagonists competitively inhibited both pathways. A third ligand (GV150013X) acted as a high affinity competitive antagonist on CCK2R(G) but was nearly inefficient as inhibitor of CCK2R(ß). Several structural elements on both GV150013X and in CCK2R binding cavity, which hinder binding of GV150013X only to the CCK2R(ß) were identified. At last, proximity between two conserved amino acids from transmembrane helices 3 and 7 interacting through sulfur-aromatic interaction was shown to be crucial for selective stabilization of the CCK2R(ß) state. These data establish structural evidence for distinct conformations of a 7TMR associated with ß-arrestin-2 recruitment or G-protein coupling and validate relevance of the design of biased ligands able to selectively target each functional conformation of 7TMRs.

Quality of Life in COVID-19 Outpatients: A Long-Term Follow-Up Study.

Background: The long-term issues faced by COVID-19 survivors remain unclear. Symptoms may persist for several months, even in non-hospitalized patients, probably impacting the quality of life. Objective: To assess the health-related quality of life of outpatients one year after SARS-CoV-2 infection. Design, Settings, and Participants: This prospective multicentre study, conducted in France from February 2020 to February 2022, compared 150 COVID-19 cases (PCR+ and/or CT scan+) and 260 controls (PCR-) selected from a database of four COVID centres. Main outcomes: Health-related quality of life assessed using the EQ-5D-5L scale. Results: COVID-19 outpatients (n = 96) had significantly lower health-related quality of life than controls (n = 81) one year after SARS-CoV-2 infection: the EQ-5D-5L index averaged 0.87 in cases and 0.95 in controls (p = 0.002); the EQ- VAS averaged 78 in cases and 86.7 in controls (p < 0.001). This alteration in quality of life was more intense in the areas of pain or discomfort and daily activities. Conclusions: This study is the first to show an alteration in the quality of life of COVID-19 outpatients after one year. Appropriate guidance and community rehabilitation programs are required for outpatients with persistent symptoms of COVID-19. Research must continue to confirm these results in larger cohorts.

Magneto-mechanical destruction of cancer-associated fibroblasts using ultra-small iron oxide nanoparticles and low frequency rotating magnetic fields.

The destruction of cells using the mechanical activation of magnetic nanoparticles with low-frequency magnetic fields constitutes a recent and interesting approach in cancer therapy. Here, we showed that superparamagnetic iron oxide nanoparticles as small as 6 nm were able to induce the death of pancreatic cancer-associated fibroblasts, chosen as a model. An exhaustive screening of the amplitude, frequency, and type (alternating vs. rotating) of magnetic field demonstrated that the best efficacy was obtained for a rotating low-amplitude low-frequency magnetic field (1 Hz and 40 mT), reaching a 34% ratio in cell death induction; interestingly, the cell death was not maximized for the largest amplitudes of the magnetic field. State-of-the-art kinetic Monte-Carlo simulations able to calculate the torque undergone by assemblies of magnetic nanoparticles explained these features and were in agreement with cell death experiments. Simulations showed that the force generated by the nanoparticles once internalized inside the lysosome was around 3 pN, which is in principle not large enough to induce direct membrane disruption. Other biological mechanisms were explored to explain cell death: the mechanical activation of magnetic nanoparticles induced lysosome membrane permeabilization and the release of the lysosome content and cell death was mediated through a lysosomal pathway depending on cathepsin-B activity. Finally, we showed that repeated rotating magnetic field exposure halted drastically the cell proliferation. This study established a proof-of-concept that ultra-small nanoparticles can disrupt the tumor microenvironment through mechanical forces generated by mechanical activation of magnetic nanoparticles upon low-frequency rotating magnetic field exposure, opening new opportunities for cancer therapy.

Id3 modulates cellular localization of bHLH Ptf1-p48 protein.

Ptf1-p48 is a pancreas-specific bHLH transcriptional protein, which, in the normal adult pancreas, shows a restricted expression in acinar cells where it is predominantly localized in the nucleus and activates the transcription of exocrine-specific genes. Ptf1-p48 partners with two proteins to form the PTF1 active complex: a bHLH E-protein and suppressor of hairless RBP-J. Cytoplasmic mislocalization of Ptf1-p48 has been reported in pancreatic pathologies, suggesting its contribution in the early steps of pancreatic carcinogenesis. The aim of the our work was to elucidate the mechanisms regulating Ptf1-p48 subcellular localization. We hypothesized a role of Id proteins acting in a dominant-negative fashion by heterodimerizing with bHLH proteins. We reproduced Ptf1-p48 cytoplasmic mislocalization in acinar AR4-2J cells and demonstrated that a proliferative signal elicited by gastrin leads to increases in Id3 protein expression and levels of Id3/E47 and Id3/Ptf1-p48 interactions, and a decrease in the level of E47/Ptf1-p48 interaction. By contrast, Id3 silencing reversed the cytoplasmic mislocalization of Ptf1-p48 induced by gastrin. As E47 is responsible for the nuclear import of the PTF1 complex, disruption of this complex via Id3 interactions with both E47 and Ptf1-p48 appears to induce cytoplasmic mislocalization of Ptf1-p48. We then found that Ptf1-p48 is either absent or mislocalized in the cytoplasm and Id3 is overexpressed in human and murine pancreatic preneoplastic lesions. Our data provide novel insight into the regulation of Ptf1-p48 function and provide evidence that Ptf1-p48 cytoplasmic mislocalization and Id3 overexpression are early events in pancreatic cancer progression.

Continuity of care in general practice at cancer diagnosis (COOC-GP study): a national cohort study of 2853 patients.

BACKGROUND: At cancer diagnosis, it is unclear whether continuity of care (COC) between the patient and GP is safeguarded. AIM: To identify patient-GP loss of COC around the time of, and in the year after, a cancer diagnosis, together with its determinants. DESIGN AND SETTING: A post-hoc analysis of data from a prospective cohort of GPs in France, taken from a survey by the Observatoire de la Médecine Générale. METHOD: A prospective GP cohort (n = 96) filed data on patients who were diagnosed with incident cancer between 1 January 2000 and 31 December 2010. COC was assessed by ascertaining the frequency of consultations and the maximal interval between them. (In France, patients see their referring/named GP in most cases.) A loss of COC was measured during the trimester before and the year after the cancer diagnosis, and the results compared with those from a 1-year baseline period before cancer had been diagnosed. A loss of COC was defined as a longer interval (that is, the maximum number of days) between consultations in the measurement periods than at baseline. Determinants of the loss in COC were assessed with univariate and multivariate logistic regression models. RESULTS: In total, 2853 patients were included; the mean age was 66.1 years. Of these, 1440 (50.5%) were women, 389 (13.6%) had metastatic cancer, and 769 (27.0%) had a comorbidity. The mean number of consultations increased up to, and including, the first trimester after diagnosis. Overall, 26.9% (95% confidence interval [CI] = 25.3 to 28.6) of patients had a loss of COC in the trimester before the diagnosis, and 22.3% (95% CI = 20.7 to 23.9) in the year after. Increasing comorbidity score was independently associated with a reduction in the loss of COC during the year after diagnosis (adjusted odds ratio [OR] comorbidity versus no comorbidity 0.61, 95% CI = 0.48 to 0.79); the same was true for metastatic status (adjusted OR metastasis versus no metastasis 0.49, 95% CI = 0.35 to 0.70). CONCLUSION: As COC is a core value for GPs and for most patients, special care should be taken to prevent a loss of COC around the time of a cancer diagnosis, and in the year after.

Prescribing patterns for upper respiratory tract infections in general practice in France and in the Netherlands.

BACKGROUND: France and the Netherlands are often presented as two contrasting countries with regard to drug prescriptions and consumption. This study aimed to analyse general practitioners' (GP's) prescription patterns for upper respiratory tract infections (URTI). METHODS: Data on diagnoses and prescriptions were derived from two databases recording daily electronic medical patient files: the 'Société Française de Médecine Générale' database (SFMG-DB) and the Dutch Landelijk Informatie Netwerk Huisatsenzorg database (LINH-DB). Logit regression models were developed to estimate and compare prescription patterns in both countries. We carried out a study including all the patients consulting for URTI in 2003. RESULTS: French GPs had more URTI patients than their Dutch counterparts (372.1 URTI patients/GP versus 181.3). They prescribed higher volumes of URTI medications (3.55 per patient/year versus 0.82). Striking differences were observed in analgesic and symptomatic prescriptions (0.84 per patient/year versus 0.12 and 1.01 per patient/year versus 0.21, respectively). We did not observe important discrepancies in volume of antibiotic prescriptions (0.29 per patient/year in France versus 0.32). After adjustment for patient characteristics, the logit model showed that prescription patterns for antibiotic were quite similar and associated with a diagnosis of acute tonsillitis. CONCLUSION: The analysis per consultation in this study did not highlight important differences in antibiotic prescribing volumes and patterns. But symptomatic and analgesic prescriptions were significantly higher in the French database. This can be explained by differences in help-seeking behaviour, medication perception, status of OTC medications and remuneration system.

Combined Treatments of Magnetic Intra-Lysosomal Hyperthermia with Doxorubicin Promotes Synergistic Anti-Tumoral Activity.

Doxorubicin is a cytotoxic drug used for the treatment of many cancer types. However, its significant dose-related adverse effects including cardiotoxicity may hamper its efficiency. Moreover, the multidrug resistance that appears during treatments limits anti-cancer therapies. Hyperthermia has been introduced as an adjuvant anti-cancer therapy and presents promising opportunities especially in combination with chemotherapy. However, hyperthermia methods including standard magnetic hyperthermia do not discriminate between the target and the surrounding normal tissues and can lead to side effects. In this context, a Magnetic Intra-Lysosomal Hyperthermia (MILH) approach, which occurs without perceptible temperature rise, has been developed. We previously showed that minute amounts of iron oxide magnetic nanoparticles targeting the gastrin receptor (CCK2R) are internalized by cancer cells through a CCK2R-dependent physiological process, accumulated into their lysosomes and kill cancer cells upon high frequency alternating magnetic field (AMF) application through lysosomal cell death. Here, we show that the combination of MILH with doxorubicin increases the efficiency of the eradication of endocrine tumor cells with synergism. We also demonstrate that these two treatments activate two different cell death pathways that are respectively dependent on Caspase-1 and Caspase-3 activation. These findings will result in the development of new anti-tumoral, intra-lysosomal-thermo/chemotherapy with better curative effects than chemotherapy alone and that are devoid of adverse effects linked to standard hyperthermia approaches.

Targeted Magnetic Intra-Lysosomal Hyperthermia produces lysosomal reactive oxygen species and causes Caspase-1 dependent cell death.

Therapeutic strategies using drugs which cause Lysosomal Cell Death have been proposed for eradication of resistant cancer cells. In this context, nanotherapy based on Magnetic Intra-Lysosomal Hyperthermia (MILH) generated by magnetic nanoparticles (MNPs) that are grafted with ligands of receptors overexpressed in tumors appears to be a very promising therapeutic option. However, mechanisms whereby MILH induces cell death are still elusive. Herein, using Gastrin-grafted MNPs specifically delivered to lysosomes of tumor cells from different cancers, we provide evidences that MILH causes cell death through a non-apoptotic signaling pathway. The mechanism of cell death involves a local temperature elevation at the nanoparticle periphery which enhances the production of reactive oxygen species through the lysosomal Fenton reaction. Subsequently, MILH induces lipid peroxidation, lysosomal membrane permeabilization and leakage of lysosomal enzymes into the cytosol, including Cathepsin-B which activates Caspase-1 but not apoptotic Caspase-3. These data highlight the clear potential of MILH for the eradication of tumors overexpressing receptors.

Efficacy of nurse-led and general practitioner-led comprehensive geriatric assessment in primary care: protocol of a pragmatic three-arm cluster randomised controlled trial (CEpiA study).

INTRODUCTION: Older patients raise therapeutic challenges, because they constitute a heterogeneous population with multimorbidity. To appraise this complexity, geriatricians have developed a multidimensional comprehensive geriatric assessment (CGA), which may be difficult to apply in primary care settings. Our primary objective was to compare the effect on morbimortality of usual care compared with two complex interventions combining educational seminars about CGA: a dedicated geriatric hotline for general practitioners (GPs) and CGA by trained nurses or GPs. METHODS AND ANALYSIS: The Clinical Epidemiology and Ageing study is an open-label, pragmatic, multicentre, three-arm, cluster randomised controlled trial comparing two intervention groups and one control group. Patients must be 70 years or older with a long-term illness or with unscheduled hospitalisation in the past 3 months (750 patients planned). This study involves volunteering GPs practising in French primary care centres, with randomisation at the practice level. The multifaceted interventions for interventional arms comprise an educational interactive multiprofessional seminar for GPs and nurses, a geriatric hotline dedicated to GPs in case of difficulties and the performance of a CGA updated to primary care. The CGA is systematically performed by a nurse in arm 1 but is GP-led on a case-by-case basis in arm 2. The primary endpoint is a composite criterion comprising overall death, unscheduled hospitalisations, emergency admissions and institutionalisation within 12 months after inclusion. Intention-to-treat analysis will be performed using mixed-effects logistic regression models, with adjustment for potential confounders. ETHICS AND DISSEMINATION: The protocol was approved by an appropriate ethics committee (CPP Ile-de-France IV, Paris, France, approval April 2015;15 664). This study is conducted according to principles of good clinical practice in the context of current care and will provide useful knowledge on the clinical benefits achievable by CGA in primary care. TRIAL REGISTRATION NUMBER: NCT02664454; Pre-results.

Involvement of cholecystokinin 2 receptor in food intake regulation: hyperphagia and increased fat deposition in cholecystokinin 2 receptor-deficient mice.

The role of cholecystokinin (CCK) as a satiety factor has been extensively documented. Although most work implies that CCK1 receptor mediates the control of food intake, a contributing role for CCK2 receptor (CCK2R) in the CCK-induced satiety cannot be totally excluded. The hypothesis that CCK2R invalidation disrupts regulatory pathways with impact on feeding behavior was examined in CCK2R(-/-) mice. CCK2R(-/-) mice developed obesity that was associated with hyperphagia. Obesity was related with increased fat deposition resulting from adipocyte hypertrophy. Expression of several adipokines was dysregulated consistently with obesity. Moreover, obesity was associated with disturbed glucose homeostasis as revealed by increased fasting glycemia and insulinemia, impaired glucose tolerance, and hepatic insulin resistance in CCK2R(-/-) mice. In vitro analysis of isolated adipocytes metabolism was consistent with increased storage but preserved insulin sensitivity. Suppression of feeding and concomitant increased expression of hypothalamic proopiomelanocortin after intracerebroventricular injection of gastrin into control mice demonstrates that hypothalamic CCK2 receptors mediate inhibition of food intake. Comparative analysis of hypothalamic mediator gene expression in fed knockout and control mice demonstrated overexpression of ghrelin receptors in CCK2R(-/-) mice, indicating up-regulation of orexigenic pathways. This effect was also observed after body weight normalization, indicating a causative role in the development of hyperphagia and obesity of CCK2R(-/-) mice. Our results give evidence that CCK2 receptor activity plays a contributing regulatory role in the control of food intake.

Internalized Receptor for Glucose-dependent Insulinotropic Peptide stimulates adenylyl cyclase on early endosomes.

Until very recently, G-protein dependent signal of GPCRs was thought to originate exclusively from the plasma membrane and internalized GPCRs were considered silent. Here, we demonstrated that, once internalized and located in the membrane of early endosomes, glucose-dependent Insulinotropic receptor (GIPR) continues to trigger production of cAMP and PKA activation. Direct evidence is based on identification of the active form of Gαs in early endosomes containing GIPR using a genetically encoded GFP tagged nanobody, and on detection of a distinct FRET signal accounting for cAMP production at the surface of endosomes containing GIP, compared to endosomes without GIP. Furthermore, decrease of the sustained phase of cAMP production and PKA activation kinetics as well as reversibility of cAMP production and PKA activity following GIP washout in cells treated with a pharmacological inhibitor of GIPR internalization, and continuous increase of cAMP level over time in the presence of dominant-negative Rab7, which causes accumulation of early endosomes in cells, were noticed. Hence the GIPR joins the few GPCRs which signal through G-proteins both at plasma membrane and on endosomes.

Internalization and desensitization of the human glucose-dependent-insulinotropic receptor is affected by N-terminal acetylation of the agonist.

How incretins regulate presence of their receptors at the cell surface and their activity is of paramount importance for the development of therapeutic strategies targeting these receptors. We have studied internalization of the human Glucose-Insulinotropic Polypeptide receptor (GIPR). GIP stimulated rapid robust internalization of the GIPR, the major part being directed to lysosomes. GIPR internalization involved mainly clathrin-coated pits, AP-2 and dynamin. However, neither GIPR C-terminal region nor ß-arrestin1/2 was required. Finally, N-acetyl-GIP recognized as a dipeptidyl-IV resistant analogue, fully stimulated cAMP production with a ∼15-fold lower potency than GIP and weakly stimulated GIPR internalization and desensitization of cAMP response. Furthermore, docking N-acetyl-GIP in the binding site of modeled GIPR showed slighter interactions with residues of helices 6 and 7 of GIPR compared to GIP. Therefore, incomplete or partial activity of N-acetyl-GIP on signaling involved in GIPR desensitization and internalization contributes to the enhanced incretin activity of this peptide.