Sergueï O. Fetissov

Important role of hypothalamic Y2 receptors in body weight regulation revealed in conditional knockout mice

Neuropeptide Y is implicated in energy homeostasis, and contributes to obesity when hypothalamic levels remain chronically elevated. To investigate the specific role of hypothalamic Y2 receptors in this process, we used a conditional Y2 knockout model, using the Cre-lox system and adenoviral delivery of Cre-recombinase. Hypothalamus-specific Y2-deleted mice showed a significant decrease in body weight and a significant increase in food intake that was associated with increased mRNA levels for the orexigenic NPY and AgRP, as well as the anorexic proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) in the arcuate nucleus. These hypothalamic changes persisted until at least 34 days after Y2 deletion, yet the effect on body weight and food intake subsided within this time. Plasma concentrations of pancreatic polypeptide and corticosterone were 3- to 5-fold increased in hypothalamus-specific Y2 knockout mice. Germ-line Y2 receptor knockout also produced a significant increase in plasma levels of pancreatic polypeptide. However, these mice differed from conditional knockout mice in that they showed a sustained reduction in body weight and adiposity associated with increased NPY and AgRP but decreased POMC and CART mRNA levels in the arcuate nucleus. The transience of the observed effects on food intake and body weight in the hypothalamus-specific Y2 knockout mice, and the difference of this model from germ-line Y2 knockout mice, underline the importance of conditional models of gene deletion, because developmental, secondary, or extrahypothalamic mechanisms may mask such effects in germ-line knockouts.

Autoantibodies against appetite-regulating peptide hormones and neuropeptides: Putative modulation by gut microflora

Abstract Objective Peptide hormones synthesized in gastrointestinal and adipose tissues in addition to neuropeptides regulate appetite and body weight. Previously, autoantibodies directed against melanocortin peptides were found in patients with eating disorders; however, it remains unknown whether autoantibodies directed against other appetite-regulating peptides are present in human sera and whether their levels are influenced by gut-related antigens. Methods Healthy women were studied for the presence of immunoglobulin (Ig) G and IgA autoantibodies directed against 14 key appetite-regulating peptides. The concept of molecular mimicry was applied to search in silico whether bacteria, viruses, or fungi contain proteins with amino acid sequences identical to appetite-regulating peptides. In addition, autoantibodies serum levels were studied in germ-free and specific pathogen-free rats. Results We found these IgG and IgA autoantibodies directed against leptin, ghrelin, peptide YY, neuropeptide Y, and other appetite-regulating peptides are present in human sera at levels of 100–900 ng/mL. Numerous cases of sequence homology with these peptides were identified among commensal and pathogenic micro-organisms including Lactobacilli, bacteroides, Helicobacter pylori, Escherichia coli, and Candida species. Decreased levels of IgA autoantibodies directed against several appetite-regulating peptides and increased levels of antighrelin IgG were found in germ-free rats compared with specific pathogen-free rats. Conclusion Healthy humans and rats display autoantibodies directed against appetite-regulating peptide hormones and neuropeptides, suggesting that these autoantibodies may have physiologic implications in hunger and satiety pathways. Gut-related antigens including the intestinal microflora may influence production of theses autoantibodies, suggesting a new link between the gut and appetite control.

Autoantibodies against α-MSH, ACTH, and LHRH in anorexia and bulimia nervosa patients

The hypothalamic arcuate nucleus is involved in the control of energy intake and expenditure and may participate in the pathogenesis of eating disorders such as anorexia nervosa (AN) and bulimia nervosa (BN). Two systems are of particular interest in this respect, synthesizing α-melanocyte-stimulating hormone (α-MSH) and synthesizing neuropeptide Y, respectively. We report here that 42 of 57 (74%) AN and/or BN patients studied had in their plasma Abs that bind to melanotropes and/or corticotropes in the rat pituitary. Among these sera, 8 were found to bind selectively to α-MSH-positive neurons and their hypothalamic and extrahypothalamic projections as revealed with immunostaining on rat brain sections. Adsorption of these sera with α-MSH peptide abolished this immunostaining. In the pituitary, the immunostaining was blocked by adsorption with α-MSH or adrenocorticotropic hormone. Additionally, 3 AN/BN sera bound to luteinizing hormone-releasing hormone (LHRH)-positive terminals in the rat median eminence, but only 2 of them were adsorbed with LHRH. In the control subjects, 2 of 13 sera (16%) displayed similar to AN/BN staining. These data provide evidence that a significant subpopulation of AN/BN patients have autoantibodies that bind to α-MSH or adrenocorticotropic hormone, a finding pointing also to involvement of the stress axis. It remains to be established whether these Abs interfere with normal signal transduction in the brain melanocortin circuitry/LHRH system and/or in other central and peripheral sites relevant to food intake regulation, to what extent such effects are related to and/or could be involved in the pathophysiology or clinical presentation of AN/BN, and to what extent increased stress is an important factor for production of these autoantibodies.

Characterization of neuropeptide Y2 receptor protein expression in the mouse brain. I. Distribution in cell bodies and nerve terminals.

Neuropeptide Y (NPY), a 36‐amino‐acid peptide, mediates biological effects by activating Y1, Y2, Y5, and y6 receptors. NPY neurons innervate many brain regions, including the hypothalamus, where NPY is involved in regulation of a broad range of homeostatic functions. We examined, by immunohistochemistry with tyramide signal amplification, the expression of the NPY Y2 receptor (Y2R) in the mouse brain with a newly developed rabbit polyclonal antibody. Y2R immunoreactivity was specific with its absence in Y2R knockout (KO) mice and in adjacent sections following preadsorption with the immunogenic peptide (10−5 M). Y2R‐positive processes were located in many brain regions, including the olfactory bulb, some cortical areas, septum, basal forebrain, nucleus accumbens, amygdala, hippocampus, hypothalamus, substantia nigra compacta, locus coeruleus, and solitary tract nucleus. However, colchicine treatment was needed to detect Y2R‐like immunoreactivity in cell bodies in many, but not all, areas. The densest distributions of cell bodies were located in the septum basal forebrain, including the bed nucleus, and amygdala, with lower density in the anterior olfactory nucleus, nucleus accumbens, caudal striatum, CA1, CA2, and CA3 hippocampal fields, preoptic nuclei lateral hypothalamus, and A13 DA cells. The widespread distribution of Y2R‐positive cell bodies and fibers suggests that NPY signaling through the Y2R is common in the mouse brain. Localization of the Y2R suggests that it is mostly presynaptic, a view supported by its frequent absence in cell bodies in the normal mouse and its dramatic increase in cell bodies of colchicine‐treated mice. J. Comp. Neurol. 499:357–390, 2006.

Neuropeptide expression in rats exposed to chronic mild stresses.

To investigate a possible link between some neuropeptides and depression, we analyzed their mRNA levels in brains of rats exposed to chronic mild stresses (CMS; a stress-induced anhedonia model), a commonly used model of depression. Rats exposed for 3 weeks to repeated, unpredictable, mild stressors exhibited an increased self-stimulation threshold, reflecting the development of an anhedonic state, which is regarded as an animal model of major depression. In situ hybridization was employed to monitor mRNA levels of neuropeptide Y (NPY), substance P and galanin in several brain regions. In the CMS rats, NPY mRNA expression levels were significantly decreased in the hippocampal dentate gyrus but increased in the arcuate nucleus. The substance P mRNA levels were increased in the anterodorsal part of the medial amygdaloid nucleus, in the ventromedial and dorsomedial hypothalamic nuclei and the lateral hypothalamic area, whereas galanin mRNA levels were decreased in the latter two regions. These findings suggest a possible involvement of these three peptides in mechanisms underlying depressive disorders and show that similar peptide changes previously demonstrated in genetic rat models also occur in the present stress-induced anhedonia model.

Altered hippocampal expression of neuropeptides in seizure-prone GALR1 knockout mice.

Summary:  Purpose: Mice carrying a deletion of the GALR1 galanin receptor have recently showed spontaneous seizure phenotype with 25% penetrance. To better understand the role of neuropeptides, which are known to undergo complex plasticity changes with development of epileptic seizures, we characterized their expression in the hippocampal formation in GALR1‐ knockout (‐KO) mice with or without seizures and in wild‐type (WT) mice.

Bacterial ClpB heat-shock protein, an antigen-mimetic of the anorexigenic peptide α-MSH, at the origin of eating disorders

The molecular mechanisms at the origin of eating disorders (EDs), including anorexia nervosa (AN), bulimia and binge-eating disorder (BED), are currently unknown. Previous data indicated that immunoglobulins (Igs) or autoantibodies (auto-Abs) reactive with α-melanocyte-stimulating hormone (α-MSH) are involved in regulation of feeding and emotion; however, the origin of such auto-Abs is unknown. Here, using proteomics, we identified ClpB heat-shock disaggregation chaperone protein of commensal gut bacteria Escherichia coli as a conformational antigen mimetic of α-MSH. We show that ClpB-immunized mice produce anti-ClpB IgG crossreactive with α-MSH, influencing food intake, body weight, anxiety and melanocortin receptor 4 signaling. Furthermore, chronic intragastric delivery of E. coli in mice decreased food intake and stimulated formation of ClpB- and α-MSH-reactive antibodies, while ClpB-deficient E. coli did not affect food intake or antibody levels. Finally, we show that plasma levels of anti-ClpB IgG crossreactive with α-MSH are increased in patients with AN, bulimia and BED, and that the ED Inventory-2 scores in ED patients correlate with anti-ClpB IgG and IgM, which is similar to our previous findings for α-MSH auto-Abs. In conclusion, this work shows that the bacterial ClpB protein, which is present in several commensal and pathogenic microorganisms, can be responsible for the production of auto-Abs crossreactive with α-MSH, associated with altered feeding and emotion in humans with ED. Our data suggest that ClpB-expressing gut microorganisms might be involved in the etiology of EDs.

Characterization of neuropeptide Y Y2 and Y5 receptor expression in the mouse hypothalamus

Neuropeptide Y (NPY) neurons abundantly innervate the hypothalamus, where NPY is involved in the regulation of a broad range of homeostatic functions. In the present work we studied NPY Y2 and Y5 receptor (R) gene expression in the mouse hypothalamus by using immunohistochemical detection of β‐galactosidase (β‐gal), a gene reporter molecule for Y2R and Y5R in Y2R‐knockout (KO) and Y5R‐KO mice, respectively. With this approach, cells normally expressing Y2R or Y5R are immunopositive for β‐gal. In the hypothalamus of the Y2R‐KO mouse, β‐gal immunoreactivity (‐ir) was found in numerous neurons of the medial preoptic nucleus as well as in the lateral anterior, periventricular, dorsomedial, tuberal, perifornical, and arcuate nuclei. Most of the dopaminergic neurons in the A13 dorsal hypothalamic group were β‐gal positive, whereas other hypothalamic dopaminergic neurons rarely displayed β‐gal‐ir. In the arcuate nucleus, most of the β‐gal‐positive neurons expressed NPY, but colocalizations with β‐endorphin were also found; in the tuberal and perifornical nuclei, many β‐gal‐positive neurons contained nitric oxide synthase. β‐Gal‐ir was also found in other forebrain regions of the Y2R‐KO mouse, including the amygdala, thalamic nuclei, hippocampal CA3 area, and cortex. In the hypothalamus of the Y5R‐KO mouse, β‐gal‐positive neurons were found mainly in the arcuate nucleus and contained β‐endorphin. The present data show that Y2R and Y5R are expressed in distinct groups of hypothalamic neurons. High levels of Y2R expression in the preoptic nuclei suggest an involvement of Y2R in the regulation of reproductive behavior, whereas Y2R expression in the arcuate, dorsomedial, and perifornical nuclei may be relevant to feeding and body weight control. The finding that A13 dopaminergic neurons express Y2R suggests a new mechanism putatively involved in the central control of feeding, in which NPY can modulate dopamine secretion. The distribution of Y5R expression supports earlier evidence for involvement of this receptor in control of feeding and body weight via NPYs action on proopiomelanocortin‐expressing neurons. J. Comp. Neurol. 470:256–265, 2004.

Dopamine and serotonin VMN release is related to feeding status in obese and lean Zucker rats.

Study of neurotransmitter role in food intake regulation in a leptin signaling deficient model, such as the Zucker rat, would benefit in the understanding of mechanisms of human obesity, in which leptin resistance is a common syndrome. We studied dopamine (DA) and serotonin (5-HT) concentrations in vivo in the ventromedial nucleus (VMN) of the hypothalamus, as they relate to eating after food deprivation in obese and lean 9-week-old male Zucker rats. DA and 5-HT concentrations were measured by HPLC via microdialysis before and during refeeding in 24-h food-deprived rats. Before food was provided, mean baseline DA and 5-HT levels were lower in obese than in lean rats (9.2 ± 0.9 vs 15.1 ± 1.9 pg/10 μl, p < 0.01, and 0.68 ± 0.05 vs 1.17 ± 0.02 pg/10 μl, p < 0.001, respectively). Food intake was accompanied by a decrease in DA levels in both obese and lean rats to 64% (p < 0.01) and 65% (p < 0.02) of their baseline levels respectively. 5-HT levels were significantly increased during eating by 41% in obese and 35% in lean rats (p < 0.01) from the baseline levels. Thus in obese rats with altered leptin signaling we found an unaltered pattern of DA and 5-HT release associated with food deprivation and refeeding, but with presence of their low levels. This points to an impaired postsynaptic monoaminergic action to produce an adequate metabolic response in obese Zucker rats in response to feeding state.

Aggressive Behavior Linked to Corticotropin-Reactive Autoantibodies

BACKGROUND Altered stress response is characteristic for subjects with abnormal aggressive and antisocial behavior, but the underlying biological mechanisms are unclear. We hypothesized that autoantibodies (autoAbs) directed against several stress-related neurohormones may exist in aggressive subjects. METHODS Using enzyme-linked immunosorbent assay, we studied whether autoAbs directed against corticotropin (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), oxytocin, and vasopressin are present in serum of male subjects with conduct disorder and prisoners with history of violence. Healthy blood donors served as control subjects. RESULTS Both conduct disorder and prisoners groups displayed strongly increased levels of ACTH-reactive immunoglobulin G (IgG) and immunoglobulin M (IgM) autoAbs compared with control subjects. Levels of oxytocin-reactive IgM autoAbs were slightly increased in both groups of aggressive subjects, whereas levels of vasopressin-reactive IgG and IgM autoAbs were lower only in conduct disorder. No differences in the levels of alpha-MSH-reactive autoAbs were found between aggressive and control subjects. CONCLUSIONS High levels of ACTH-reactive autoAbs as well as altered levels of oxytocin- and vasopressin-reactive autoAbs found in aggressive subjects may interfere with the neuroendocrine mechanisms of stress and motivated behavior. Our data suggest a new biological mechanism of human aggressive behavior that involves autoAbs directed against several stress-related neurohormones.