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Background: Germline pathogenic variants in the breast cancer type 1 susceptibility gene BRCA1 are associated with a 60% lifetime risk for breast and ovarian cancer. This overall risk estimate is for all BRCA1 variants; obviously, not all variants confer the same risk of developing a disease. In cancer patients, loss of BRCA1 function in tumor tissue has been associated with an increased sensitivity to platinum agents and to poly-(ADP-ribose) polymerase (PARP) inhibitors. For clinical management of both at-risk individuals and cancer patients, it would be important that each identified genetic variant be associated with clinical significance. Unfortunately for the vast majority of variants, the clinical impact is unknown. The availability of results from studies assessing the impact of variants on protein function may provide insight of crucial importance., Results and Conclusion: We have collected, curated, and structured the molecular and cellular phenotypic impact of 3654 distinct BRCA1 variants. The data was modeled in triple format, using the variant as a subject, the studied function as the object, and a predicate describing the relation between the two. Each annotation is supported by a fully traceable evidence. The data was captured using standard ontologies to ensure consistency, and enhance searchability and interoperability. We have assessed the extent to which functional defects at the molecular and cellular levels correlate with the clinical interpretation of variants by ClinVar submitters. Approximately 30% of the ClinVar BRCA1 missense variants have some molecular or cellular assay available in the literature. Pathogenic variants (as assigned by ClinVar) have at least some significant functional defect in 94% of testable cases. For benign variants, 77% of ClinVar benign variants, for which neXtProt Cancer variant portal has data, shows either no or mild experimental functional defects. While this does not provide evidence for clinical interpretation of variants, it may provide some guidance for variants of unknown significance, in the absence of more reliable data. The neXtProt Cancer variant portal ( https://www.nextprot.org/portals/breast-cancer ) contains over 6300 observations at the molecular and/or cellular level for BRCA1 variants.

The development of efficient text-mining tools promises to boost the curation workflow by significantly reducing the time needed to process the literature into biological databases. We have developed a curation support tool, neXtA5, that provides a search engine coupled with an annotation system directly integrated into a biocuration workflow. neXtA5 assists curation with modules optimized for the thevarious curation tasks: document triage, entity recognition and information extraction.Here, we describe the evaluation of neXtA5 by expert curators. We first assessed the annotations of two independent curators to provide a baseline for comparison. To evaluate the performance of neXtA5, we submitted requests and compared the neXtA5 results with the manual curation. The analysis focuses on the usability of neXtA5 to support the curation of two types of data: biological processes (BPs) and diseases (Ds). We evaluated the relevance of the papers proposed as well as the recall and precision of the suggested annotations.The evaluation of document triage by neXtA5 precision showed that both curators agree with neXtA5 for 67 (BP) and 63% (D) of abstracts, while curators agree on accepting or rejecting an abstract ~80% of the time. Hence, the precision of the triage system is satisfactory.For concept extraction, curators approved 35 (BP) and 25% (D) of the neXtA5 annotations. Conversely, neXtA5 successfully annotated up to 36 (BP) and 68% (D) of the terms identified by curators. The user feedback obtained in these tests highlighted the need for improvement in the ranking function of neXtA5 annotations. Therefore, we transformed the information extraction component into an annotation ranking system. This improvement results in a top precision (precision at first rank) of 59 (D) and 63% (BP). These results suggest that when considering only the first extracted entity, the current system achieves a precision comparable with expert biocurators.

The neXtProt human protein knowledgebase (https://www.nextprot.org) continues to add new content and tools, with a focus on proteomics and genetic variation data. neXtProt now has proteomics data for over 85% of the human proteins, as well as new tools tailored to the proteomics community.Moreover, the neXtProt release 2016-08-25 includes over 8000 phenotypic observations for over 4000 variations in a number of genes involved in hereditary cancers and channelopathies. These changes are presented in the current neXtProt update. All of the neXtProt data are available via our user interface and FTP site. We also provide an API access and a SPARQL endpoint for more technical applications., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The rapid increase in the number of published articles poses a challenge for curated databases to remain up-to-date. To help the scientific community and database curators deal with this issue, we have developed an application, neXtA5, which prioritizes the literature for specific curation requirements. Our system, neXtA5, is a curation service composed of three main elements. The first component is a named-entity recognition module, which annotates MEDLINE over some predefined axes. This report focuses on three axes: Diseases, the Molecular Function and Biological Process sub-ontologies of the Gene Ontology (GO). The automatic annotations are then stored in a local database, BioMed, for each annotation axis. Additional entities such as species and chemical compounds are also identified. The second component is an existing search engine, which retrieves the most relevant MEDLINE records for any given query. The third component uses the content of BioMed to generate an axis-specific ranking, which takes into account the density of named-entities as stored in the Biomed database. The two ranked lists are ultimately merged using a linear combination, which has been specifically tuned to support the annotation of each axis. The fine-tuning of the coefficients is formally reported for each axis-driven search. Compared with PubMed, which is the system used by most curators, the improvement is the following: +231% for Diseases, +236% for Molecular Functions and +3153% for Biological Process when measuring the precision of the top-returned PMID (P0 or mean reciprocal rank). The current search methods significantly improve the search effectiveness of curators for three important curation axes. Further experiments are being performed to extend the curation types, in particular protein-protein interactions, which require specific relationship extraction capabilities. In parallel, user-friendly interfaces powered with a set of JSON web services are currently being implemented into the neXtProt annotation pipeline.Available on: http://babar.unige.ch:8082/neXtA5Database URL: http://babar.unige.ch:8082/neXtA5/fetcher.jsp., (© The Author(s) 2016. Published by Oxford University Press.)

neXtProt (http://www.nextprot.org) is a human protein-centric knowledgebase developed at the SIB Swiss Institute of Bioinformatics. Focused solely on human proteins, neXtProt aims to provide a state of the art resource for the representation of human biology by capturing a wide range of data, precise annotations, fully traceable data provenance and a web interface which enables researchers to find and view information in a comprehensive manner. Since the introductory neXtProt publication, significant advances have been made on three main aspects: the representation of proteomics data, an extended representation of human variants and the development of an advanced search capability built around semantic technologies. These changes are presented in the current neXtProt update., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

About 5000 (25%) of the ~20400 human protein-coding genes currently lack any experimental evidence at the protein level. For many others, there is only little information relative to their abundance, distribution, subcellular localization, interactions, or cellular functions. The aim of the HUPO Human Proteome Project (HPP, www.thehpp.org ) is to collect this information for every human protein. HPP is based on three major pillars: mass spectrometry (MS), antibody/affinity capture reagents (Ab), and bioinformatics-driven knowledge base (KB). To meet this objective, the Chromosome-Centric Human Proteome Project (C-HPP) proposes to build this catalog chromosome-by-chromosome ( www.c-hpp.org ) by focusing primarily on proteins that currently lack MS evidence or Ab detection. These are termed "missing proteins" by the HPP consortium. The lack of observation of a protein can be due to various factors including incorrect and incomplete gene annotation, low or restricted expression, or instability. neXtProt ( www.nextprot.org ) is a new web-based knowledge platform specific for human proteins that aims to complement UniProtKB/Swiss-Prot ( www.uniprot.org ) with detailed information obtained from carefully selected high-throughput experiments on genomic variation, post-translational modifications, as well as protein expression in tissues and cells. This article describes how neXtProt contributes to prioritize C-HPP efforts and integrates C-HPP results with other research efforts to create a complete human proteome catalog.

neXtProt (http://www.nextprot.org/) is a new human protein-centric knowledge platform. Developed at the Swiss Institute of Bioinformatics (SIB), it aims to help researchers answer questions relevant to human proteins. To achieve this goal, neXtProt is built on a corpus containing both curated knowledge originating from the UniProtKB/Swiss-Prot knowledgebase and carefully selected and filtered high-throughput data pertinent to human proteins. This article presents an overview of the database and the data integration process. We also lay out the key future directions of neXtProt that we consider the necessary steps to make neXtProt the one-stop-shop for all research projects focusing on human proteins.

The GO annotation dataset provided by the UniProt Consortium (GOA: http://www.ebi.ac.uk/GOA) is a comprehensive set of evidenced-based associations between terms from the Gene Ontology resource and UniProtKB proteins. Currently supplying over 100 million annotations to 11 million proteins in more than 360,000 taxa, this resource has increased 2-fold over the last 2 years and has benefited from a wealth of checks to improve annotation correctness and consistency as well as now supplying a greater information content enabled by GO Consortium annotation format developments. Detailed, manual GO annotations obtained from the curation of peer-reviewed papers are directly contributed by all UniProt curators and supplemented with manual and electronic annotations from 36 model organism and domain-focused scientific resources. The inclusion of high-quality, automatic annotation predictions ensures the UniProt GO annotation dataset supplies functional information to a wide range of proteins, including those from poorly characterized, non-model organism species. UniProt GO annotations are freely available in a range of formats accessible by both file downloads and web-based views. In addition, the introduction of a new, normalized file format in 2010 has made for easier handling of the complete UniProt-GOA data set.

There is growing interest to use mass spectrometry data to search genome sequences directly. Previous work by other authors demonstrated that this approach is able to correct and complement available genome annotations. We discuss the practical difficulty of searching large eukaryotic genomes with peptide ion trap tandem mass spectra of small proteins (<40 kDa). The challenging problem of automatically identifying peptides that span across exon/intron boundaries is explored for the first time by using experimental data. In a human genome search, we find that roughly 30% of the peptides are missed, due to various reasons, compared to a Swiss-Prot search. We show that this percentage is significantly reduced with improved parent mass accuracy. We finally provide several examples of predicted gene structures that could be improved by proteomics data, in particular by peptides spanning across exon/intron boundaries.

We present an integrated proteomics platform designed for performing differential analyses. Since reproducible results are essential for comparative studies, we explain how we improved reproducibility at every step of our laboratory processes, e.g. by taking advantage of the powerful laboratory information management system we developed. The differential capacity of our platform is validated by detecting known markers in a real sample and by a spiking experiment. We introduce an innovative two-dimensional (2-D) plot for displaying identification results combined with chromatographic data. This 2-D plot is very convenient for detecting differential proteins. We also adapt standard multivariate statistical techniques to show that peptide identification scores can be used for reliable and sensitive differential studies. The interest of the protein separation approach we generally apply is justified by numerous statistics, complemented by a comparison with a simple shotgun analysis performed on a small volume sample. By introducing an automatic integration step after mass spectrometry data identification, we are able to search numerous databases systematically, including the human genome and expressed sequence tags. Finally, we explain how rigorous data processing can be combined with the work of human experts to set high quality standards, and hence obtain reliable (false positive < 0.35%) and nonredundant protein identifications.

Human blood plasma is a useful source of proteins associated with both health and disease. Analysis of human blood plasma is a challenge due to the large number of peptides and proteins present and the very wide range of concentrations. In order to identify as many proteins as possible for subsequent comparative studies, we developed an industrial-scale (2.5 liter) approach involving sample pooling for the analysis of smaller proteins (M(r) generally < ca. 40 000 and some fragments of very large proteins). Plasma from healthy males was depleted of abundant proteins (albumin and IgG), then smaller proteins and polypeptides were separated into 12 960 fractions by chromatographic techniques. Analysis of proteins and polypeptides was performed by mass spectrometry prior to and after enzymatic digestion. Thousands of peptide identifications were made, permitting the identification of 502 different proteins and polypeptides from a single pool, 405 of which are listed here. The numbers refer to chromatographically separable polypeptide entities present prior to digestion. Combining results from studies with other plasma pools we have identified over 700 different proteins and polypeptides in plasma. Relatively low abundance proteins such as leptin and ghrelin and peptides such as bradykinin, all invisible to two-dimensional gel technology, were clearly identified. Proteins of interest were synthesized by chemical methods for bioassays. We believe that this is the first time that the small proteins in human blood plasma have been separated and analyzed so extensively.

In a previous paper we introduced a novel model-based approach (OLAV) to the problem of identifying peptides via tandem mass spectrometry, for which early implementations showed promising performance. We recently further improved this performance to a remarkable level (1-2% false positive rate at 95% true positive rate) and characterized key properties of OLAV like robustness and training set size. We present these results in a synthetic and coherent way along with detailed performance comparisons, a new scoring component making use of peptide amino acidic composition, and new developments like automatic parameter learning. Finally, we discuss the impact of OLAV on the automation of proteomics projects.

Objective: The aims of the present study were to determine whether increased body weight resulting from intracerebroventricular (ICV) glucocorticoid (dexamethasone) infusion in normal rats is associated, as in obesity, with changes in glucose metabolism and to investigate whether the parasympathetic nervous system is involved in the glucocorticoid-induced effects., Research Methods and Procedures: Male Sprague-Dawley rats were infused with ICV dexamethasone (2.5 microg/d) or its vehicle for 2 days during which food intake, body weight, and basal insulinemia were measured. Euglycemic-hyperinsulinemic clamps associated with the labeled 2-deoxyglucose technique were then performed to determine the total rate of glucose disappearance and the tissue glucose use indices. Similar experiments were carried out in vagotomized rats., Results: Two days of ICV glucocorticoid infusion in normal rats resulted in increases in food intake, body weight, basal insulinemia, and produced decreases in the insulin-stimulated total rate of glucose disappearance, as well as in glucose use indices of all muscle types studied. None of these alterations was observed when glucocorticoid infusion was carried out in vagotomized rats., Discussion: These data show that central glucocorticoid infusion favors anabolic processes, such as feeding behavior, body weight gain, and insulin output, while promoting muscle insulin resistance. These effects seem to be mediated by an activation of the parasympathetic nervous system, because they all disappear when tested in vagotomized rats.

The preferential channeling of different fuels to fat and changes in the transcription profile of adipose tissue and skeletal muscle are poorly understood processes involved in the pathogenesis of obesity and insulin resistance. Carbohydrate and lipid metabolism may play relevant roles in this context. Freely moving lean Zucker rats received 3- and 24-h infusions of Intralipid (Pharmacia and Upjohn, Milan, Italy) plus heparin, or saline plus heparin, to evaluate how an increase in free fatty acids (nonesterified fatty acid [NEFA]) modulates fat tissue and skeletal muscle gene expression and thus influences fuel partitioning. Glucose uptake was determined in various tissues at the end of the infusion period by means of the 2-deoxy-[1-3H]-D-glucose technique after a euglycemic-hyperinsulinemic clamp: high NEFA levels markedly decreased insulin-mediated glucose uptake in red fiber-type muscles but enhanced glucose utilization in visceral fat. Using reverse transcriptase-polymerase chain reaction and Northern blotting analyses, the mRNA expression of fatty acid translocase (FAT)/CD36, GLUT4, tumor necrosis factor (TNF)-alpha, peroxisome proliferator-activated receptor (PPAR)-gamma, leptin, uncoupling protein (UCP)-2, and UCP-3 was investigated in different fat depots and skeletal muscles before and after the study infusions. GLUT4 mRNA levels significantly decreased (by approximately 25%) in red fiber-type muscle (soleus) and increased (by approximately 45%) in visceral adipose tissue. Furthermore, there were marked increases in FAT/CD36, TNF-alpha, PPAR-gamma, leptin, UCP2, and UCP3 mRNA levels in the visceral fat and muscle of the treated animals in comparison with those measured in the saline-treated animals. These data suggest that the in vivo gene expression of FAT/CD36, GLUT4, TNF-alpha, PPAR-gamma, leptin, UCP2, and UCP3 in visceral fat and red fiber-type muscle are differently regulated by circulating lipids and that selective insulin resistance seems to favor, at least in part, a prevention of fat accumulation in tissues not primarily destined for fat storage, thus contributing to increased adiposity and the development of a prediabetic syndrome.

Leptin acts as a satiety factor within the central nervous system by binding to its receptor located in the hypothalamus. A missense mutation of the leptin receptor induces hyperphagia and obesity in the obese Zucker fa/fa rat. Since the CNS is an important target of leptin action, we hypothesized that leptin gene transfer into the lateral cerebral ventricle could efficiently lead to inhibition of food intake and reduction of body weight in obese fa/fa rats as well as in lean animals. A single intracerebroventricular injection of an adenoviral vector containing a cDNA encoding leptin resulted in the expression of leptin in the ependymal cells lining the ventricle and the secretion of leptin into the cerebrospinal fluid (CSF). During the first week after injection, when high concentrations of leptin were produced in the CSF, the reducing effects of leptin on food intake and body weight were comparable in lean and in obese fa/fa rats. The subsequent decline in CSF leptin levels, that was similar in lean and obese fa/fa rats, resulted in the faster resumption of food intake and body weight gain in obese than in lean animals, confirming a reduced sensitivity to leptin in the obese group. The results of this study show that leptin gene delivery into the cerebral ventricles allows for the production of elevated leptin concentrations in CSF, and they support the hypothesis that the impaired sensitivity to leptin may be overcome in obese fa/fa rats.

We have shown previously that continuous (6 days) intracerebroventricular (ICV) leptin infusion in normal rats resulted in decreases in food intake and body weight. A reduction of food intake imposed on control rats (pair-feeding), aimed at mimicking leptin-induced hyperphagia, produced a marked decrease in the expression of muscle uncoupling protein-3 (UCP-3), whereas ICV infusion of leptin prevented such a decrease in UCP-3. To investigate an involvement of thyroid hormones in this effect of leptin, plasma levels of these hormones were determined in ICV leptin-infused, ICV vehicle-infused ad libitum fed or pair-fed controls. ICV leptin infusion and pair-feeding resulted in decreased plasma thyroid-stimulating hormone (TSH) and T4 levels relative to ad libitum fed controls. ICV leptin infusion maintained plasma levels of T3, but the levels were decreased by pair-feeding. The activity of the enzyme (hepatic 5'-monodeiodinase) responsible for T4/T3 conversion was measured. In the leptin-infused group, the activity of 5'-monodeiodinase was maintained at the values measured in ad libitum fed rats; in pair-fed rats, activity was reduced. Thus, conversion of T4 to T3 is decreased by pair-feeding, whereas such is not the case during leptin infusion. To further substantiate an involvement of thyroid hormones in the effect of leptin on muscle UCP-3 expression, hypothyroid rats were ICV infused with leptin or vehicle. It was observed that in hypothyroid rats, ICV leptin was unable to maintain muscle UCP-3 expression at values measured in ad libitum fed controls. These results suggest that central leptin stimulates T3 production via an activation of T4 to T3 conversion, and that this stimulation could be responsible for the effect of leptin on muscle UCP-3 expression. Thyroid hormones could thus be important mediators of the effect of leptin on energy expenditure.

We demonstrate that chronic intracerebroventricular infusion of leptin dramatically decreases the number of [(3)H]paroxetine binding sites in the frontal cortex of the rat brain. In contrast, the density in paroxetine binding sites estimated in the region containing raphe projecting cell bodies (i.e., the dorsal and median raphe nuclei) remains unchanged. Since leptin treatment significantly decreases food intake, [(3)H]paroxetine binding parameters were also estimated in the frontal cortex of pair-fed control rats. No significant difference in [(3)H]paroxetine binding was observed between pair-fed and ad libitum fed control rats. These data indicate that leptin treatment could regionally down-regulate serotonin transporter binding sites in the brain. Although the cellular and molecular mechanisms underlying such an effect of leptin need further investigation, our observations support the notion of a possible interaction between leptin and the serotonergic system of potential interest in the pathophysiology of depression.

Centrally administered leptin has been shown to increase insulin-stimulated glucose utilization and to favor the expression of uncoupling proteins (UCPs). To study if leptin also has direct peripherally mediated effects on these processes, this hormone (1 mg/day) or its vehicle was infused i.v. for 4 days to lean rats and insulin-stimulated glucose utilization in skeletal muscle and adipose tissue as well as the expression of UCP messenger RNAs (mRNAs) in brown adipose tissue were measured. I.v. leptin administration resulted in decreases in food intake (31%), body weight gain, and plasma insulin levels (45%), in increases in overall (23%) as well as brown adipose tissue and muscle glucose utilization, and in decreases in white adipose tissue glucose uptake. Most of these changes were mimicked, in control rats, by giving them the same amount of food as that consumed by the leptin-infused group (pair-feeding). I.v. leptin infusion also favored the expression of UCPs in brown adipose tissue, either by increasing their expression or preventing the fall occurring during the pair-feeding regimen. Relative UCP expression levels were 100, 104, and 33 for UCP1, 100, 191, and 125 for UCP2 and 100, 107, and 29 for UCP3 in ad libitum fed control rats, in leptin-treated rats and in pair-fed control rats, respectively. These results suggest that the overall effect of leptin on glucose utilization and on the expression of UCPs may be mediated through central mechanism.

It has been reported that hyperphagia and excessive body weight gain of genetically obese rodents were abolished by adrenalectomy. High hypothalamic levels of neuropeptide Y (NPY) were found in obese rodents. A chronic intracerebroventricular (icv) infusion of NPY in normal rats was shown to produce most hormono-metabolic abnormalities of genetically obese animals, and to be inefficient in doing so in adrenalectomized (ADX) rats. The combined presence of NPY and of glucocorticoids thus appeared to be necessary for inducing obesity. This study, therefore, was aimed at determining the consequences of a chronic i.c.v. NPY infusion in ADX rats receiving or not i.c.v. glucocorticoids. It was found that the combined i.c.v. infusion of NPY and dexamethasone in ADX rats increased food intake, body weight, plasma insulin, leptin, and triglyceride levels relative to vehicle-infused ADX controls. The infusion of NPY alone, or of dexamethasone alone in ADX rats failed to produce these effects. In contrast, the icv infusion of NPY alone greatly decreased the expression of brown adipose tissue uncoupling protein-1 and -3. This was not modified by the superimposed infusion of dexamethasone. It is concluded that, although many of centrally elicited NPY effects require the central presence of glucocorticoids, those bearing on the inhibition of uncoupling proteins expression (energy dissipation) do not require central glucocorticoids.

It has been claimed that factors favoring the development or maintenance of animal or human obesity may include increases in glucocorticoid production or hyperresponsiveness of the hypothalamic-pituitary-adrenal axis. In normal rats, glucocorticoids have been shown to be necessary for chronic intracerebroventricular infusion of neuropeptide Y to produce obesity and related abnormalities. Conversely, glucocorticoids inhibited the body weight-lowering effect of leptin. Such dual action of glucocorticoids may occur within the central nervous system, since both neuropeptide Y and leptin act within the hypothalamus. The aim of this study was to determine the effects of glucocorticoids (dexamethasone) given intracerebroventricularly to normal rats on body weight homeostasis and hypothalamic levels of neuropeptide Y and corticotropin-releasing hormone. Continuous central glucocorticoid infusion for 3 days resulted in marked sustained increases in food intake and body weight relative to saline-infused controls. The infusion abolished endogenous corticosterone output and produced hyperinsulinemia, hypertriglyceridemia, and hyperleptinemia, three salient abnormalities of obesity syndromes. Central glucocorticoid infusion also produced a marked decrease in the expression of uncoupling protein (UCP)-1 and UCP-3 in brown adipose tissue and UCP-3 in muscle. Finally, chronic central glucocorticoid administration increased the hypothalamic levels of neuropeptide Y and decreased those of corticotropin-releasing hormone. When the same dose of glucocorticoids was administered peripherally, it resulted in decreases in food intake and body weight, in keeping with the decrease in hypothalamic neuropeptide Y levels. These results suggest that glucocorticoids induce an obesity syndrome in rodents by acting centrally and not peripherally.

The concept of interrelationships between the central nervous system and the periphery aimed at maintaining normal body weight homeostasis has been strengthened by the discovery of hypothalamic neuropeptide Y (NPY) and adipose tissue leptin. NPY, when infused intracerebroventricularly in normal animals produces hyperphagia and hormono-metabolic changes (hyperinsulinemia, hypercorticism) channeling nutrients preferentially toward lipogenesis and storage in adipose tissue and away from their utilization by muscles (muscle insulin resistance). Storage in NPY-infused rats is further favored by the observed decrease in the expression of uncoupling proteins. NPY-induced hyperinsulinemia and hypercorticosteronemia also promote leptin over-secretion. Released leptin, acting within the hypothalamus, decreases hypothalamic NPY levels (probably those of other hypothalamic neuropeptides as well), food intake, insulinemia, insulin sensitivity of white adipose tissue, while increasing that of muscles. Leptin acting centrally additionally favors the expression of uncoupling protein 1, 2, and 3, in keeping with an eflect on energy dissipating mechanisms. The respective hormono-metabolic eflects of NPY and leptin maintain a normal body homeostasis. In most obesity syndromes, the functional relationships between NPY and leptin are altered. Due to hypothalamic leptin receptor mutations or dysfunctions, leptin cannot exert its eflects: NPY levels (possibly those of other neuropeptides) remain elevated, maintaining excess storage, insulin as well as leptin resistance.

Neuropeptide Y in the hypothalamus is a potent physiological stimulator of feeding, and may contribute to the characteristic metabolic defects of obesity when hypothalamic levels remain chronically elevated. Since corticosterone and insulin are important regulators of fuel metabolism, the longitudinal effects of chronic (6 days) intracerebroventricular infusion of neuropeptide Y in normal rats on the hypothalamo-pituitary-adrenal axis and on insulin secretion were studied. Neuropeptide Y-infused rats were either allowed to eat ad libitum, or were pair-fed with normophagic control rats. Neuropeptide Y increased the basal plasma concentrations of adrenocorticotropic hormone and corticosterone during the first 2 days of its intracerebroventricular infusion and increased cold stress-induced plasma adrenocorticotropic hormone concentrations. After 4-6 days of central neuropeptide Y infusion, however, basal plasma adrenocorticotropic hormone and corticosterone concentrations were no different from control values (except in ad libitum-fed rats in which corticosteronaemia remained elevated), they were unaffected by the stress of cold exposure, and the hypothalamic content of corticotropin-releasing factor immunoreactivity was significantly decreased. A state of hyperinsulinaemia was present throughout the 6 days of intracerebroventricular neuropeptide Y infusion, being more marked in the ad libitum-fed than in the pair-fed group. The proportions of insulin, proinsulin, and conversion intermediates in plasma and pancreas were unchanged. Hyperinsulinaemia of the pair-fed neuropeptide Y-infused rats was accompanied by muscle insulin resistance and white adipose tissue insulin hyperresponsiveness, as assessed by the in vivo uptake of 2-deoxyglucose. Finally, bilateral subdiaphragmatic vagotomy prevented both the basal and the marked glucose-induced hyperinsulinaemia of animals chronically infused with neuropeptide Y, demonstrating that central neuropeptide Y-induced hyperinsulinaemia is mediated by the parasympathetic nervous system.

The product of the ob gene, leptin, is a hormone secreted by adipose tissue that acts in the hypothalamus to regulate the size of the body fat depot. Its central administration has been shown to decrease food intake and body weight, while favoring energy dissipation. As glucocorticoids are known to play a permissive role in the establishment and maintenance of obesity syndromes in rodents, it was hypothesized that they do so by restraining the effect of leptin. Leptin injected intracerebroventricularly as a bolus of 3 microg in normal rats induced modest reductions in body weight and food intake. In marked contrast, the same dose of leptin had very potent and long-lasting effects in decreasing both body weight and food intake when administered to adrenalectomized rats. Further, glucocorticoid supplementation of adrenalectomized rats dose-dependently inhibited these potent effects of leptin. These data suggest that glucocorticoids play a key inhibitory role in the action of leptin. Under normal conditions, this inhibitory influence of glucocorticoids may prevent lasting hypophagia. In obesity with degrees of hypercorticism, it may contribute to "leptin resistance," whose etiology is still little understood.

Neuropeptide Y (NPY) in the hypothalamus plays an important role in the regulation of food intake and body weight and seems to be implicated in the etiology of obesity. When intracerebroventricularly (ICV) infused for 6 days in normal rats, NPY resulted in hyperphagia, increased body weight gain, hyperinsulinemia, hypercorticosteronemia, and hypertriglyceridemia compared with vehicle-infused control rats. NPY infusion also resulted in an insulin-resistant state in muscles and in a state of insulin hyperresponsiveness in white adipose tissue, as assessed by the measurement of the in vivo glucose utilization index of these tissues during euglycemic-hyperinsulinemic clamps. All of these hormono-metabolic effects produced by chronic central NPY infusion were completely prevented when rats were adrenalectomized before NPY administration. Adrenalectomy per se had no effect on any of the parameters mentioned above. The levels of mRNA for the obese gene were increased in white adipose tissue after 6 days of ICV NPY infusion in normal rats, and white adipose tissue weight was also increased. These effects of ICV NPY infusion were markedly decreased by prior adrenalectomy, although NPY infusion was able to somewhat enhance the low white adipose tissue obese mRNA levels and tissue weight of adrenalectomized rats. In conclusion, intact adrenal glands, and probably circulating corticosterone in particular, are necessary for the establishment of most of the hormonal and metabolic effects induced by chronic ICV infusion of NPY in normal rats.

Over the years, the work of research laboratories in Baton Rouge (USA), Seattle (USA) and Geneva (Switzerland) have reached analogous conclusions regarding the main etiology of obesity as studied in animals: it largely lies within the brain, notably within the hypothalamus. The hypothalamus is indeed known to modulate food intake and energy partitioning, while the periphery has also been proposed to feed-back on the central nervous system (CNS) to provide information on the state of body energy stores, the two together constituting a loop system connecting the brain to the periphery (1,2,3). This etiologic viewpoint of a pivotal role of the hypothalamus in obesity syndromes has been strengthened by the discovery of one hypothalamic neuropeptide and one peripheral (adipose tissue) hormone, respectively neuropeptide Y (4), and quite particularly, leptin (5). As neuropeptide Y produces hyperphagia (6, 7) and as leptin produces hypophagia in normal animals (8,9,10), the loop system just mentioned was thought to comprise functional relationships, at least between these two factors. Other evidence also suggested that such a loop system was altered in obese animals.

The effect of different doses of leptin, given as an intracerebroventricular (ICV) bolus, on body weight gain and food intake was investigated during refeeding, following a 24-h fast in lean (FA/fa) rats. It was observed that ICV leptin resulted in a dose-dependent decrease in body weight gain, compared with vehicle injection, a difference that persisted for at least 6 days. This was associated with a transient reduction in food intake over the first 2 days after leptin injection. More importantly, the effect of leptin was also observed in genetically obese fa/fa rats but at the expense of two to ten times higher leptin concentrations, indicating the presence of decreased leptin sensitivity. Furthermore, ICV leptin injections were able to decrease neuropeptide Y (NPY) levels in the arcuate and paraventricular hypothalamic nuclei in both lean and genetically obese fa/fa rats, although a higher leptin dose was again needed in the obese group. These observations provide further evidence for the implication of NPY and leptin in a regulatory loop controlling body homeostasis. This loop is functional in lean and genetically obese fa/fa rats, provided that leptin levels in the central nervous system are high enough in the obese group, in particular. Since human obesity is frequently associated with elevated circulating leptin levels, a state of decreased leptin sensitivity (i.e., leptin resistance), similar to that described here in fa/fa rats, could possibly occur in human syndromes as well.

Obesity and non-insulin-dependent diabetes are estimated to affect millions of people in the world. This pathology is multifactorial, comprising complex interactions of genetic and environmental factors and lacking a specific therapy. Great interest arose from the recent discovery of the ob gene expressed only in adipose tissue and coding for a protein that appears to regulate adiposity, potentially by acting as a satiety factor. We report here that in normal rats, ob mRNA is respectively up- or downregulated by a rise in insulinemia (induced by 2-day insulin infusion while maintaining euglycemia) or a decrease in insulinemia (induced by a 3-day fast). Our results also show that in genetically obese fa/fa rats studied longitudinally, white adipose tissue ob mRNA levels increase in parallel with early occurring and steadily increasing hyperinsulinemia. This results in adult obese animals having markedly higher ob mRNA levels than age-matched normoinsulinemic lean rats. Furthermore, in adult obese rats, ob mRNA escapes down-regulation as normalization of hyperinsulinemia due to fasting fails to reduce the high ob mRNA levels.