Axial Spondyloarthritis: Does Magnetic Resonance Imaging Classification Improve Report Interpretation.

OBJECTIVE: The interpretation of magnetic resonance imaging (MRI) reports is crucial for the diagnosis of axial spondyloarthritis, but the subjective nature of narrative reports can lead to varying interpretations. This study presents a validation of a novel MRI reporting system for the sacroiliac joint in clinical practice. METHODS: A historical review was conducted on 130 consecutive patients referred by 2 rheumatologists for initial MRI assessment of possible axial spondyloarthritis. The original MRI reports were interpreted by the rheumatologists and the radiologist who originally read the images and then categorized according to the novel system. Two musculoskeletal radiologists then reinterpreted the original MRI scans using the new system, and the resulting reports were interpreted and categorized by the same rheumatologists. The quality of the new framework was assessed by comparing the interpretations of both reports. RESULTS: Ninety-two patients met the study criteria. The rheumatologists disagreed on the categorization of the original MRI reports in 12% of cases. The rheumatologists and original radiologists disagreed on the categorization of the initial report in 23.4% of cases. In contrast, there was 100% agreement between the rheumatologists and radiologists on the categorization of the new MRI report. CONCLUSION: The new MRI categorization system significantly improved the agreement between the clinician and radiologist in report interpretation. The system provided a standard vocabulary for reporting, reduced variability in report interpretation, and may therefore improve clinical decision-making.

A Survey of Older Adults' Self-Managing Cancer.

BACKGROUND: Older adults living with cancer can experience significant challenges in managing their cancer treatment[s], care, and health. Cancer self-management is much discussed in the research literature, but less is known about the perceptions and experiences of older adults', including their self-management capacities and challenges. This study explored the factors that supported and hindered cancer self-management for older Canadian adults living with cancer. METHODS: We conducted a 17-item population-based telephone survey in the Canadian province of British Columbia among older adults (age ≥ 65) living with cancer. Descriptive and inferential statistics were used to analyze quantitative data and thematic analysis for open-text responses. RESULTS: 129 older adults participated in the study (median age 76, range: 65-93), of which 51% were living with at least one other chronic illness. 20% reported challenges managing their cancer treatment and appointments, while only ~4% reported financial barriers to managing cancer. We organized the findings around enabling and encumbering factors to older adults cancer self-management. The main encumbering factors to self-management included health system and personal factors (physical and emotional challenges + travel). Whereas enablers included: access to interpersonal support, helpful care team, interpersonal support and individual mindset. CONCLUSIONS: Considering factors which enable or encumber older adults' cancer self-management is critical to supporting the growing aging population in the work required to manage cancer treatment and navigate cancer services. Our findings may guide the development of tailored resources for bolstering effectual self-management for older Canadians living with cancer.

Digital interventions for depression and anxiety in older adults: a systematic review of randomised controlled trials.

One in five older adults experience symptoms of depression and anxiety. Digital mental health interventions are promising in their ability to provide researchers, mental health professionals, clinicians, and patients with personalised tools for assessing their behaviour and seeking consultation, treatment, and peer support. This systematic review looks at existing randomised controlled trial studies on digital mental health interventions for older adults. Four factors have been found that contributed to the success of digital mental health interventions: (1) ease of use; (2) opportunities for social interactions; (3) having human support; and (4) having the digital mental health interventions tailored to the participants' needs. The findings also resulted in methodological considerations for future randomised controlled trials on digital mental health interventions: (1) having a healthy control group and an intervention group with clinical diagnoses of mental illness; (2) collecting data on the support given throughout the duration of the interventions; (3) obtaining qualitative and quantitative data to measure the success of the interventions; and (4) conducting follow-up interviews and surveys up to 1 year post-intervention to determine the long-term outcomes. The factors that were identified in this systematic review can provide future digital mental health interventions researchers, health professionals, clinicians, and patients with the tools to design, develop, and use successful interventions for older users.

The sociocultural relationships of older immigrant Punjabi women living in Nova Scotia: Implications for well-being.

With the increase of South Asian immigrants in Canada, the relationship between older immigrant Punjabi women's sociocultural relationships and their mental health and well-being needs to be understood. Guided by the social determinants of health and intersectional feminist frameworks, five Punjabi women living in Nova Scotia, Canada were interviewed. Three themes were identified: (a) having freedom yet being dependent on families, (b) conflating having a happy family with having good mental health, and (c) needing ways to connect with other older Punjabi women. These findings articulate how Punjabi relationships affect women's views on mental health and well-being.

High-Versus Low-Dose Warfarin-Related Teratogenicity: A Case Report and Systematic Review.

OBJECTIVE: The optimal anticoagulant therapy during pregnancy in women with mechanical heart valves remains controversial. This study highlights a case of high-dose warfarin ingestion throughout pregnancy and performed a systematic review to assess rates of teratogenicity with high versus low warfarin dosing (≤5 mg daily). METHODS: A literature search for all case reports and available literature was conducted in PubMed, Medline, and EMBASE up to December 2016 using medical subject heading terms "mechanical prosthetic valves," "pregnancy," "oral anticoagulants," "warfarin," "coumarins," "heparin, low-molecular-weight," and "thromboembolism." To be included, warfarin had to be administered anytime between 6 and 12 weeks of gestation with the dose being specified. The Newcastle-Ottawa Scale was used to assess quality of the cohort data. RESULTS: The woman in the studied case received the highest reported warfarin doses throughout pregnancy (14.5-16.5 mg daily) and delivered a baby with no evidence of teratogenicity to the current age of 5 years. The study identified 23 case reports, with all demonstrating warfarin teratogenicity regardless of high-dose (n = 12) or low-dose (n = 11) warfarin. Twelve cohort studies identified a warfarin teratogenicity rate of 5.0%, with rates of 2.4% and 10.5% with low- and high-dose warfarin, respectively. Risk of bias was moderate (median Newcastle-Ottawa Scale score of 6) for all of the cohort studies. CONCLUSION: Although a lower prevalence of warfarin-induced teratogenicity is reported with low-dose warfarin, a safe "cut-off" dose is misleading. Teratogenic risk with warfarin is unpredictable, mandating individual decisions regardless of the dose.

Rates of Screening for Breast, Colorectal, and Cervical Cancers in Older People With Cognitive Impairment or Dementia: A Meta-Analysis.

Purpose: Cancer screening may not be appropriate for some older people. We compare the likelihood of screening for colorectal, breast, and cervical cancers in older people with versus without cognitive impairment or dementia. Method: Systematic search of MEDLINE, Embase, and PsycINFO (to March 9, 2018) for articles reporting screening for colon, breast, and cervical cancers in patients with and without cognitive impairment or dementia. Studies were summarized quantitatively (random effects meta-analysis), according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Results: Studies reported data 1989-2008. The rate of screening for breast cancer by mammography was lower in women with cognitive impairment or dementia compared with those without (pooled odds ratio [OR] = 0.81, 95% confidence interval [CI] = [0.71, 0.91], p = .0007, six studies, N = 18,562). The rates of screening for cervical cancer by Pap smear (pooled OR = 0.88, 95% CI = [0.71, 1.08], p = 0.22, five studies, N = 409,131) and colorectal cancer by fecal occult blood test (pooled OR = 0.87, 95% CI = [0.55, 1.38], p = .55, two studies, N = 2,718) were not significantly lower in people with cognitive impairment or dementia. Conclusion: These historical rates provide a baseline for discussions around the need for more specific guidance to assist with decisions to discontinue screening. The study also identifies a gap in reported knowledge with respect to screening under current guidelines.

Defects in NADPH Oxidase Genes NOX1 and DUOX2 in Very Early Onset Inflammatory Bowel Disease.

BACKGROUND & AIMS: Defects in intestinal innate defense systems predispose patients to inflammatory bowel disease (IBD). Reactive oxygen species (ROS) generated by nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases in the mucosal barrier maintain gut homeostasis and defend against pathogenic attack. We hypothesized that molecular genetic defects in intestinal NADPH oxidases might be present in children with IBD. METHODS: After targeted exome sequencing of epithelial NADPH oxidases NOX1 and DUOX2 on 209 children with very early onset inflammatory bowel disease (VEOIBD), the identified mutations were validated using Sanger Sequencing. A structural analysis of NOX1 and DUOX2 variants was performed by homology in silico modeling. The functional characterization included ROS generation in model cell lines and in in vivo transduced murine crypts, protein expression, intracellular localization, and cell-based infection studies with the enteric pathogens Campylobacter jejuni and enteropathogenic Escherichia coli. RESULTS: We identified missense mutations in NOX1 (c.988G>A, p.Pro330Ser; c.967G>A, p.Asp360Asn) and DUOX2 (c.4474G>A, p.Arg1211Cys; c.3631C>T, p.Arg1492Cys) in 5 of 209 VEOIBD patients. The NOX1 p.Asp360Asn variant was replicated in a male Ashkenazi Jewish ulcerative colitis cohort. All NOX1 and DUOX2 variants showed reduced ROS production compared with wild-type enzymes. Despite appropriate cellular localization and comparable pathogen-stimulated translocation of altered oxidases, cells harboring NOX1 or DUOX2 variants had defective host resistance to infection with C. jejuni. CONCLUSIONS: This study identifies the first inactivating missense variants in NOX1 and DUOX2 associated with VEOIBD. Defective ROS production from intestinal epithelial cells constitutes a risk factor for developing VEOIBD.

The Disconnect Between Novel Oral Anticoagulant Eligibility and Provincial Drug Coverage: An Albertan Anticoagulation Clinic Audit.

Canadian practice guidelines for nonvalvular atrial fibrillation (NVAF) recommend that most patients receive a novel oral anticoagulant (NOAC) in preference to warfarin to prevent stroke, but not all patients have insurance that covers NOACs. The gap between optimal therapy and drug coverage is unknown. We retrospectively assessed eligibility for NOACs in patients with NVAF at our single-centre anticoagulation clinic and ascertained whether provincial drug coverage would be in place. Most patients (89%-95%) were eligible, but only 39%-41% qualified for drug coverage. Our findings suggest most Albertans with NVAF might not have drug coverage for optimal medical therapy for stroke prevention.

Visualizing the engram: learning stabilizes odor representations in the olfactory network.

The nature of memory is a central issue in neuroscience. How does our representation of the world change with learning and experience? Here we use the transcription of Arc mRNA, which permits probing the neural representations of temporally separated events, to address this in a well characterized odor learning model. Rat pups readily associate odor with maternal care. In pups, the lateralized olfactory networks are independent, permitting separate training and within-subject control. We use multiday training to create an enduring memory of peppermint odor. Training stabilized rewarded, but not nonrewarded, odor representations in both mitral cells and associated granule cells of the olfactory bulb and in the pyramidal cells of the anterior piriform cortex. An enlarged core of stable, likely highly active neurons represent rewarded odor at both stages of the olfactory network. Odor representations in anterior piriform cortex were sparser than typical in adult rat and did not enlarge with learning. This sparser representation of odor is congruent with the maturation of lateral olfactory tract input in rat pups. Cortical representations elsewhere have been shown to be highly variable in electrophysiological experiments, suggesting brains operate normally using dynamic and network-modulated representations. The olfactory cortical representations here are consistent with the generalized associative model of sparse variable cortical representation, as normal responses to repeated odors were highly variable (∼70% of the cells change as indexed by Arc). Learning and memory modified rewarded odor ensembles to increase stability in a core representational component.

Variants in nicotinamide adenine dinucleotide phosphate oxidase complex components determine susceptibility to very early onset inflammatory bowel disease.

BACKGROUND & AIMS: The colitis observed in patients with very early onset inflammatory bowel disease (VEOIBD; defined as onset of disease at younger than 6 years of age) often resembles that of chronic granulomatous disease (CGD) in extent and features of colonic inflammation observed by endoscopy and histology. CGD is a severe immunodeficiency caused by defects in the genes that encode components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. We investigated whether variants in genes that encode NADPH oxidase components affect susceptibility to VEOIBD using independent approaches. METHODS: We performed targeted exome sequencing of genes that encode components of NADPH oxidases (cytochrome b light chain and encodes p22(phox) protein; cytochrome b-245 or NADPH oxidase 2, and encodes Nox2 or gp91(phox); neutrophil cytosol factor 1 and encodes p47 (phox) protein; neutrophil cytosol factor 2 and encodes p67 (phox) protein; neutrophil cytosol factor 4 and encodes p40 (phox) protein; and Ras-related C3 botulinum toxin substrate 1 and 2) in 122 patients with VEOIBD diagnosed at The Hospital for Sick Children, University of Toronto, from 1994 through 2012. Gene variants were validated in an independent International Early Onset Pediatric IBD Cohort Study cohort of patients with VEOIBD. In a second approach, we examined Tag single nucleotide polymorphisms in a subset of patients with VEOIBD in which the NOX2 NADPH oxidase genes sequence had been previously analyzed. We then looked for single nucleotide polymorphisms associated with the disease in an independent International Early Onset Pediatric IBD Cohort Study cohort of patients. We analyzed the functional effects of variants associated with VEOIBD. RESULTS: Targeted exome sequencing and Tag single nucleotide polymorphism genotyping identified 11 variants associated with VEOIBD; the majority of patients were heterozygous for these variants. Expression of these variants in cells either reduced oxidative burst or altered interactions among proteins in the NADPH oxidase complex. Variants in the noncoding regulatory and splicing elements resulted in reduced levels of proteins, or expression of altered forms of the proteins, in blood cells from VEOIBD patients. CONCLUSIONS: We found that VEOIBD patients carry heterozygous functional hypomorphic variants in components of the NOX2 NADPH oxidase complex. These do not cause overt immunodeficiency, but instead determine susceptibility to VEOIBD. Specific approaches might be developed to treat individual patients based on their genetic variant.

Mutations in tetratricopeptide repeat domain 7A result in a severe form of very early onset inflammatory bowel disease.

BACKGROUND & AIMS: Very early onset inflammatory bowel diseases (VEOIBD), including infant disorders, are a diverse group of diseases found in children younger than 6 years of age. They have been associated with several gene variants. Our aim was to identify the genes that cause VEOIBD. METHODS: We performed whole exome sequencing of DNA from 1 infant with severe enterocolitis and her parents. Candidate gene mutations were validated in 40 pediatric patients and functional studies were carried out using intestinal samples and human intestinal cell lines. RESULTS: We identified compound heterozygote mutations in the Tetratricopeptide repeat domain 7 (TTC7A) gene in an infant from non-consanguineous parents with severe exfoliative apoptotic enterocolitis; we also detected TTC7A mutations in 2 unrelated families, each with 2 affected siblings. TTC7A interacts with EFR3 homolog B to regulate phosphatidylinositol 4-kinase at the plasma membrane. Functional studies demonstrated that TTC7A is expressed in human enterocytes. The mutations we identified in TTC7A result in either mislocalization or reduced expression of TTC7A. Phosphatidylinositol 4-kinase was found to co-immunoprecipitate with TTC7A; the identified TTC7A mutations reduced this binding. Knockdown of TTC7A in human intestinal-like cell lines reduced their adhesion, increased apoptosis, and decreased production of phosphatidylinositol 4-phosphate. CONCLUSIONS: In a genetic analysis, we identified loss of function mutations in TTC7A in 5 infants with VEOIBD. Functional studies demonstrated that the mutations cause defects in enterocytes and T cells that lead to severe apoptotic enterocolitis. Defects in the phosphatidylinositol 4-kinase-TTC7A-EFR3 homolog B pathway are involved in the pathogenesis of VEOIBD.

Higher activity of the inducible nitric oxide synthase contributes to very early onset inflammatory bowel disease.

OBJECTIVES: The NOS2 gene encodes for the inducible nitric oxide synthase (iNOS), responsible for nitric oxide (NO) production, which contributes to antimicrobial and antipathogenic activities. Higher levels of both iNOS and NO-induced damage have been observed in inflammatory bowel disease (IBD) patients. NOS2 may have a role in a specific subset of IBD patients with severe and/or extensive colitis. Therefore, the aim of this study is to examine the role of NOS2 in such a subset, very early onset IBD (VEO-IBD). METHODS: Seventeen tag single nucleotide polymorphisms (SNPs) in the NOS2 gene were successfully genotyped in VEO-IBD patients. Genetic associations were replicated in an independent VEO-IBD cohort. Functional analysis for iNOS activity was performed on the most significantly associated functional variant. RESULTS: The NOS2 rs2297518 SNP was found to be associated in VEO-IBD in two independent cohorts. Upon combined analysis, a coding variant (S608L) showed the strongest association with VEO-IBD (Pcombined=1.13 × 10(-6), OR (odds ratio)=3.398 (95% CI (confidence interval) 2.02-5.717)) as well as associations with VEO-Crohn's disease and VEO-ulcerative colitis (UC). This variant also showed an association with UC diagnosed between 11 and 17 years of age but not with adult-onset IBD (>17 years). B-cell lymphoblastoid cell lines genotyped for the risk variant as well as Henle-407 cells transfected with a plasmid construct with the risk variant showed higher NO production. Colonic biopsies of VEO-IBD patients showed higher immunohistochemical staining of nitrotyrosine, indicating more nitrosative stress and tissue damage. CONCLUSIONS: These studies suggest the importance of iNOS in genetic susceptibility to younger IBD presentation due to higher NO production.

R-spondin 2 signalling mediates susceptibility to fatal infectious diarrhoea.

Citrobacter rodentium is a natural mouse pathogen widely used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections in humans. While C. rodentium causes self-limiting colitis in most inbred mouse strains, it induces fatal diarrhoea in susceptible strains. The physiological pathways as well as the genetic determinants leading to susceptibility have remained largely uncharacterized. Here we use a forward genetic approach to identify the R-spondin2 gene as a major determinant of susceptibility to C. rodentium infection. Robust induction of R-spondin2 expression during infection in susceptible mouse strains causes a potent Wnt-mediated proliferative response of colonic crypt cells, leading to the generation of an immature and poorly differentiated colonic epithelium with deficiencies in ion-transport components. Our data demonstrate a previously unknown role of R-spondins and Wnt signalling in susceptibility to infectious diarrhoea and identify R-spondin2 as a key molecular link between infection and intestinal homoeostasis.

Juvenile Psoriatic Arthritis (JPsA): juvenile arthritis with psoriasis?

BACKGROUND: Following the introduction of the ILAR criteria for juvenile idiopathic arthritis, juvenile psoriatic arthritis (JPsA) has become a better recognized category within the inflammatory arthritides of childhood. There are fewer reports describing the characteristics and long-term outcome of patients with JPsA than other subtypes of JIA.The aim of our study was to determine the long-term outcome and clinical course of patients with juvenile psoriatic arthritis (JPsA) and to define subgroups of JPsA. METHODS: Clinical records of all patients meeting criteria for JPsA were reviewed and divided into 4 groups depending on their clinical features and onset type. Patient characteristics and clinical features at onset and during follow-up were determined. RESULTS: The cohort consisted of 119 patients: 65 with oligoarticular-onset (55%; persistent 44 and extended 21), 34 (29%) with RF(-) and 4 (3%) RF(+) polyarticular and 16 (13%) enthesitis-related arthritis (ERA). At diagnosis patients with ERA were oldest and more commonly male (p=0.001 and =0.01 respectively). Patients with a polyarticular course had more involvement of small joints of the hands and wrist when compared to patients with persistent oligoarticular and ERA (p<0.001) while patients with ERA had more hip and sacroiliac arthritis (p<0.001 for both). Nail changes were seen in 66 patients (57%) and were associated with DIP involvement (p=0.0034). OUTCOME: Time to first inactive disease on, but not off, therapy was significantly longer among patients with polyarticular course when compared to oligoarticular and ERA (p=0.016 and p=0.48 respectively). Patients with polyarticular course more frequently had contractures during follow-up than other groups (p=0.01). CONCLUSION: The long-term outcome of with JPsA was generally good. Patients with JPsA did not appear to form distinct sub-group of patients but rather resembled JIA patients with onset types without psoriasis.

Gene array analysis of embryonic- versus adult-derived hypothalamic NPY-expressing cell lines.

Few studies have utilized microarray analysis to understand the genome wide changes involved in the development of the hypothalamus despite its overall importance to basic physiology. Gene expression profiling of immortalized, clonal hypothalamic neurons, embryonic-derived mHypoE-46 and adult-derived mHypoA-2/12, reveals that the expression of 1225 probes was significantly changed between the two neuronal models. Further comparison of the gene expression profiles identified two categories of genes that were confirmed with qRT-PCR: (i) genes implicated in the Wnt signaling pathway; and (ii) transcription factors previously implicated in the development of the central nervous system. Yet, functional analysis of the two cell lines, including hormonal responses and secretion, indicate that they are comparable despite their developmental origin. This study provides a comprehensive analysis of embryonic- and adult-derived hypothalamic neuronal cell models that both express neuropeptide Y, and identifies novel genes as candidates for mediating the development of specific hypothalamic neurons.

Effectiveness of Telemetry Guidelines in Predicting Clinically Significant Arrhythmias in Hospitalized Patients.

BACKGROUND: Cardiac rhythm monitoring is widely applied on hospitalized patients. However, its value has not been evaluated systematically. METHODS: This study considered the utility of our institutional telemetry guidelines in predicting clinically significant arrhythmias. A retrospective analysis was performed of 562 patients admitted to the telemetry unit. A total of 1932 monitoring days were evaluated. Patients were divided into 2 groups based on telemetry guidelines: "telemetry indicated" and "telemetry not indicated". RESULTS: Differences in arrhythmia event rates and pre-defined clinical significance were determined. One hundred and forty-four (34%) vs. 16 (11%) patients had at least one arrhythmic event in the "telemetry indicated" group compared with the "telemetry not indicated" group, respectively (P = 0.001). No patient in the "telemetry not indicated" group had a clinically significant arrhythmia. In contrast, of patients in the "telemetry indicated" group who had at least one arrhythmic event, 36% were considered clinically significant (P < 0.05). CONCLUSION: In conclusion, this study validates and supports the use of our institutional telemetry guidelines to allocate this resource appropriately and predict clinically significant arrhythmias.

Generation of immortal cell lines from the adult pituitary: role of cAMP on differentiation of SOX2-expressing progenitor cells to mature gonadotropes.

The pituitary is a complex endocrine tissue composed of a number of unique cell types distinguished by the expression and secretion of specific hormones, which in turn control critical components of overall physiology. The basic function of these cells is understood; however, the molecular events involved in their hormonal regulation are not yet fully defined. While previously established cell lines have provided much insight into these regulatory mechanisms, the availability of representative cell lines from each cell lineage is limited, and currently none are derived from adult pituitary. We have therefore used retroviral transfer of SV40 T-antigen to mass immortalize primary pituitary cell culture from an adult mouse. We have generated 19 mixed cell cultures that contain cells from pituitary cell lineages, as determined by RT-PCR analysis and immunocytochemistry for specific hormones. Some lines expressed markers associated with multipotent adult progenitor cells or transit-amplifying cells, including SOX2, nestin, S100, and SOX9. The progenitor lines were exposed to an adenylate cyclase activator, forskolin, over 7 days and were induced to differentiate to a more mature gonadotrope cell, expressing significant levels of α-subunit, LHß, and FSHß mRNAs. Additionally, clonal populations of differentiated gonadotropes were exposed to 30 nM gonadotropin-releasing hormone and responded appropriately with a significant increase in α-subunit and LHß transcription. Further, exposure of the lines to a pulse paradigm of GnRH, in combination with 17ß-estradiol and dexamethasone, significantly increased GnRH receptor mRNA levels. This array of adult-derived pituitary cell models will be valuable for both studies of progenitor cell characteristics and modulation, and the molecular analysis of individual pituitary cell lineages.

Cellular leptin resistance impairs the leptin-mediated suppression of neuropeptide Y secretion in hypothalamic neurons.

Evidence shows that neuropeptide Y (NPY) neurons are involved in mediating the anorexigenic action of leptin via neuronal circuits in the hypothalamus. However, studies have produced limited data on the cellular processes involved and whether hypothalamic NPY neurons are susceptible to cellular leptin resistance. To investigate the direct regulation of NPY secretion by leptin, we used novel NPY-synthesizing, immortalized mHypoA-NPY/green fluorescent protein and mHypoA-59 hypothalamic cell lines derived from adult hypothalamic primary cultures. We report that leptin treatment significantly suppressed NPY secretion in the cells by approximately 20%. We found a decrease in c-fos expression upon leptin exposure, indicating deactivation or hyperpolarization of the neurons. Protein analysis indicated that leptin inhibits AMP-activated protein kinase (AMPK) activity and activates acetyl-coenzyme A carboxylase in NPY neurons, supporting the hypothesis of an AMPK-dependent mechanism. Inhibiting both AMPK with Compound C or phosphatidylinositol 3 kinase (PI3K) with 2-(4-morpholinyl)-8-phenyl-1(4H)-1-benzopyran-4-one hydrochloride prevented the leptin-mediated decrease in NPY secretion, indicating both AMPK- and PI3K-mediated mechanisms. Further, NPY secretion was stimulated by 30% by the AMPK activator, aminoimidazole carboxamide ribonucleotide. Importantly, prolonged leptin exposure in the mHypoA-NPY/green fluorescent protein cells prevented leptin-induced changes in AMPK phosphorylation and suppression of NPY secretion, indicating that NPY neurons are susceptible to leptin resistance. Our studies indicate that AMPK and PI3K pathways are involved in leptin action in NPY neurons and that leptin resistance blocks the feedback response likely required to maintain energy homeostasis.

Neuronal suppressor of cytokine signaling-3 deficiency enhances hypothalamic leptin-dependent phosphatidylinositol 3-kinase signaling.

Suppressor of cytokine signaling-3 (SOCS3) is thought to be involved in the development of central leptin resistance and obesity by inhibiting STAT3 pathway. Because phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in transducing leptin action in the hypothalamus, we examined whether SOCS3 exerted an inhibition on this pathway. We first determined whether leptin sensitivity in the hypothalamic PI3K pathway was increased in brain-specific Socs3-deficient (NesKO) mice. In NesKO mice, hypothalamic insulin receptor substrate-1 (IRS1)-associated PI3K activity was significantly increased at 30 min and remained elevated up to 2 h after leptin intraperitoneal injection, but in wild-type (WT) littermates, the significant increase was only at 30 min. Hypothalamic p-STAT3 levels were increased up to 5 h in NesKO as opposed to 2 h in WT mice. In food-restricted WT mice with reduced body weight, leptin increased hypothalamic PI3K activity only at 30 min, and p-STAT3 levels at 30-120 min postinjection. These results suggest increased leptin sensitivity in both PI3K and STAT3 pathways in the hypothalamus of NesKO mice, which was not due to a lean phenotype. In the next experiment with a clonal hypothalamic neuronal cell line expressing proopiomelanocortin, we observed that whereas leptin significantly increased IRS1-associated PI3K activity and p-JAK2 levels in cells transfected with control vector, it failed to do so in SOCS3-overexpressed cells. Altogether, these results imply a SOCS3 inhibition of the PI3K pathway of leptin signaling in the hypothalamus, which may be one of the mechanisms behind the development of central leptin resistance and obesity.

Leptin differentially regulates NPY secretion in hypothalamic cell lines through distinct intracellular signal transduction pathways.

Leptin acts as a key peripheral hormone in distinct neurons in the hypothalamus to modulate both reproductive function and energy homeostasis. The control of neuropeptide Y (NPY) secretion is an example of a process that can be differentially regulated by leptin. In order to further understand these distinct modulatory effects, we have used immortalized, neuronal hypothalamic cell lines expressing NPY, mHypoE-38 and mHypoE-46. We found that these cell lines express the endogenous leptin receptor, ObRb, and secrete detectable levels of NPY. We exposed the neurons to 100nM leptin for 1h and determined that the basal levels of NPY in the cell lines were differentially regulated: NPY secretion was inhibited in mHypoE-46 neurons, whereas NPY secretion was induced in the mHypoE-38 neurons. In order to determine the mechanisms involved in the divergent regulation of NPY release, we analyzed the activity of a number of signaling components using phospho-specific antibodies directed towards specific proteins in the MAP kinase, PI3K, and AMPK pathways, among others. We found that leptin activated a different combination of second messengers in each cell line. Importantly, we could link the regulation of NPY secretion to different signaling pathways, AMPK in the mHypoE-46 and both MAPK and PI3K in the mHypoE-38 neurons. This is the first demonstration that leptin can specifically regulate individual NPY neuron secretory responses through distinct signaling pathways.