Peter Kobelt

A pivotal role for galectin-1 in fetomaternal tolerance

A successful pregnancy requires synchronized adaptation of maternal immune-endocrine mechanisms to the fetus. Here we show that galectin-1 (Gal-1), an immunoregulatory glycan-binding protein, has a pivotal role in conferring fetomaternal tolerance. Consistently with a marked decrease in Gal-1 expression during failing pregnancies, Gal-1–deficient (Lgals1−/−) mice showed higher rates of fetal loss compared to wild-type mice in allogeneic matings, whereas fetal survival was unaffected in syngeneic matings. Treatment with recombinant Gal-1 prevented fetal loss and restored tolerance through multiple mechanisms, including the induction of tolerogenic dendritic cells, which in turn promoted the expansion of interleukin-10 (IL-10)–secreting regulatory T cells in vivo. Accordingly, Gal-1s protective effects were abrogated in mice depleted of regulatory T cells or deficient in IL-10. In addition, we provide evidence for synergy between Gal-1 and progesterone in the maintenance of pregnancy. Thus, Gal-1 is a pivotal regulator of fetomaternal tolerance that has potential therapeutic implications in threatened pregnancies.

Circulating levels of irisin in patients with anorexia nervosa and different stages of obesity--correlation with body mass index.

Irisin was recently identified as cleavage product of fibronectin type III domain containing 5 (FNDC5) and shown to increase energy expenditure in mice and humans and therefore was discussed as potential treatment option in obesity. However, the regulation of irisin under conditions of severely altered body weight such as anorexia nervosa and obesity remains to be investigated. We analyzed circulating irisin levels over a broad spectrum of body weight in 40 patients with anorexia nervosa (mean body mass index, BMI 12.6±0.7 kg/m(2)), normal weight controls (22.6±0.9 kg/m(2)) and obese patients with BMI of 30-40 (36.9±1.2 kg/m(2)), 40-50 (44.9±1.1 kg/m(2)) and >50 (70.1±2.7 kg/m(2), n=8/group). Correlation analyses were performed between irisin and different body indices, parameters of body composition and hormones involved in various homeostatic processes. Obese patients showed higher circulating irisin levels compared to normal weight and anorexic patients (p<0.05) resulting in a correlation of irisin with body weight (r=0.47, p<0.01) and BMI (r=0.50, p<0.001). Plasma irisin was also positively correlated with fat mass (r=0.48, p<0.01), body cell mass (r=0.45, p<0.01) and fat free mass (r=0.40, p<0.05). Insulin levels were positively correlated with irisin (r=0.45, p<0.01), whereas circulating ghrelin, cortisol, thyroid-stimulating hormone or C-reactive protein were not (p>0.05). These data indicate that circulating irisin is affected under conditions of altered BMI with highest levels in severely obese patients. The increase of irisin under conditions of obesity may indicate a physiological function to improve glucose tolerance which is often impaired in obese subjects.

Central Nesfatin-1 Reduces Dark-Phase Food Intake and Gastric Emptying in Rats: Differential Role of Corticotropin-Releasing Factor2 Receptor

Nesfatin-1, derived from nucleobindin2, is expressed in the hypothalamus and reported in one study to reduce food intake (FI) in rats. To characterize the central anorexigenic action of nesfatin-1 and whether gastric emptying (GE) is altered, we injected nesfatin-1 into the lateral brain ventricle (intracerebroventricular, icv) or fourth ventricle (4v) in chronically cannulated rats or into the cisterna magna (intracisternal, ic) under short anesthesia and compared with ip injection. Nesfatin-1 (0.05 microg/rat, icv) decreased 2-3 h and 3-6 h dark-phase FI by 87 and 45%, respectively, whereas ip administration (2 microg/rat) had no effect. The corticotropin-releasing factor (CRF)(1)/CRF(2) antagonist astressin-B or the CRF(2) antagonist astressin(2)-B abolished icv nesfatin-1s anorexigenic action, whereas an astressin(2)-B analog, devoid of CRF-receptor binding affinity, did not. Nesfatin-1 icv induced a dose-dependent reduction of GE by 26 and 43% that was not modified by icv astressin(2)-B. Nesfatin-1 into the 4v (0.05 microg/rat) or ic (0.5 microg/rat) decreased cumulative dark-phase FI by 29 and 60% at 1 h and by 41 and 37% between 3 and 5 h, respectively. This effect was neither altered by ic astressin(2)-B nor associated with changes in GE. Cholecystokinin (ip) induced Fos expression in 43% of nesfatin-1 neurons in the paraventricular hypothalamic nucleus and 24% of those in the nucleus tractus solitarius. These data indicate that nesfatin-1 acts centrally to reduce dark phase FI through CRF(2)-receptor-dependent pathways after forebrain injection and CRF(2)-receptor-independent pathways after hindbrain injection. Activation of nesfatin-1 neurons by cholecystokinin at sites regulating food intake may suggest a role in gut peptide satiation effect.

Desacyl ghrelin inhibits the orexigenic effect of peripherally injected ghrelin in rats

Studies showed that the metabolic unlike the neuroendocrine effects of ghrelin could be abrogated by co-administered unacylated ghrelin. The aim was to investigate the interaction between ghrelin and desacyl ghrelin administered intraperitoneally on food intake and neuronal activity (c-Fos) in the arcuate nucleus in non-fasted rats. Ghrelin (13 microg/kg) significantly increased food intake within the first 30 min post-injection. Desacyl ghrelin at 64 and 127 microg/kg injected simultaneously with ghrelin abolished the stimulatory effect of ghrelin on food intake. Desacyl ghrelin alone at both doses did not alter food intake. Both doses of desacyl ghrelin injected separately in the light phase had no effects on food intake when rats were fasted for 12h. Ghrelin and desacyl ghrelin (64 microg/kg) injected alone increased the number of Fos positive neurons in the arcuate nucleus compared to vehicle. The effect on neuronal activity induced by ghrelin was significantly reduced when injected simultaneously with desacyl ghrelin. Double labeling revealed that nesfatin-1 immunoreactive neurons in the arcuate nucleus are activated by simultaneous injection of ghrelin and desacyl ghrelin. These results suggest that desacyl ghrelin suppresses ghrelin-induced food intake by curbing ghrelin-induced increased neuronal activity in the arcuate nucleus and recruiting nesfatin-1 immunopositive neurons.

Peripheral injection of ghrelin induces Fos expression in the dorsomedial hypothalamic nucleus in rats.

Peripheral ghrelin has been shown to act as a gut-brain peptide exerting a potent orexigenic effect on food intake. The dorsomedial nucleus of the hypothalamus (DMH) is innervated by projections from other brain areas being part of the network of nuclei controlling energy homeostasis, among others NPY/AgRP-positive fibers arising from the arcuate nucleus (ARC). The aim of the study was to determine if peripherally administered ghrelin affects neuronal activity in the DMH, as assessed by Fos expression. The number of Fos positive neurons was determined in the DMH, paraventricular nucleus of the hypothalamus (PVN), ARC, ventromedial hypothalamic nucleus (VMH), nucleus of the solitary tract (NTS) and in the area postrema (AP) in non-fasted Sprague-Dawley rats in response to intraperitoneally (ip) injected ghrelin (3 nmol/rat) or vehicle (0.15 M NaCl). Peripheral ghrelin induced a significant increase in the number of Fos-ir positive neurons/section compared with vehicle in the ARC (mean+/-SEM: 49+/-2 vs. 23+/-2 neurons/section, p=0.001), PVN (69+/-5 vs. 34+/-3, p=0.001), and DMH (142+/-5 vs. 83+/-5, p<0.001). Fos-ir positive neurons were mainly localized within the ventral part of the DMH. No change in Fos expression was observed in the VMH (53+/-8 vs. 48+/-6, p=0.581), NTS (42+/-2 vs. 40+/-3, p=0.603), and in the AP (7+/-1 vs. 5+/-1, p=0.096). Additional double-labelling with anti-Fos and anti-AgRP revealed that Fos positive neurons in the DMH were encircled by a network of AgRP-ir positive fibers. These data indicate that peripheral ghrelin activates DMH neurons and that NPY-/AgRP-positive fibers may be involved in the response.

Anti-ghrelin Spiegelmer NOX-B11 inhibits neurostimulatory and orexigenic effects of peripheral ghrelin in rats

Background and aims: Ghrelin, the natural ligand of the growth hormone secretagogue receptor 1a, is the most powerful peripherally active orexigenic agent known. In rodents, ghrelin administration stimulates growth hormone release, food intake, and adiposity. Because of these effects, blocking of ghrelin has been widely discussed as a potential treatment for obesity. Spiegelmer NOX-B11 is a synthetic l-oligonucleotide, which was previously shown to bind ghrelin. We examined the effects of NOX-B11 on ghrelin induced neuronal activation and food intake in non-fasted rats. Methods: Animals received various doses of NOX-B11, inactive control Spiegelmer, or vehicle intravenously. Ghrelin or vehicle was administered intraperitoneally 12 hours later and food intake was measured over four hours. Neuronal activation was assessed as c-Fos-like immunoreactivity in the arcuate nucleus. Results: Treatment with NOX-B11 30 nmol suppressed ghrelin induced c-Fos-like immunoreactivity in the arcuate nucleus and blocked the ghrelin induced increase in food intake within the first half hour after ghrelin injection (mean 1.13 (SEM 0.59) g/kg body weight; 4.94 (0.63) g/kg body weight versus 0.58 (0.58) g/kg body weight; p<0.0001). Treatment with NOX-B11 1 nmol or control Spiegelmer had no effect whereas treatment with NOX-B11 10 nmol showed an intermediate effect on ghrelin induced food intake. Conclusions: Spiegelmer NOX-B11 suppresses ghrelin induced food intake and c-Fos induction in the arcuate nucleus in rats. The use of an anti-ghrelin Spiegelmer could be an innovative new approach to inhibit the biological action of circulating ghrelin. This may be of particular relevance to conditions associated with elevated plasma ghrelin, such as the Prader-Willi syndrome.

Novel insight in distribution of nesfatin-1 and phospho-mTOR in the arcuate nucleus of the hypothalamus of rats

Recently, two proteins have been localized in the arcuate nucleus (ARC) and implicated in the regulation of food intake: the serine-threonine-kinase mammalian target of rapamycin (mTOR) as part of the TOR signaling complex 1 (TORC1), and nesfatin-1 derived from the precursor protein nucleobindin2. However, the exact cell types are not well described. Therefore, we performed double-labeling studies for NPY, CART, nesfatin-1 and pmTOR in the ARC. In this study, we showed that nesfatin-1 is not only intracellularly co-localized with cocaine- and amphetamine-regulated transcript (CART) peptide as reported before, but also with phospho-mTOR (pmTOR) and neuropeptide Y (NPY) in ARC neurons. Quantification revealed that 59+/-5% of the pmTOR-immunoreactive (ir) neurons were immunoreactive for nesfatin-1. Moreover, double labeling for nesfatin-1 and NPY exhibited that 19+/-5% of the NPY positive cells were also immunoreactive for nesfatin-1. Furthermore, we could also confirm results from previous studies, showing that the majority of nesfatin-1 neurons are also positive for CART peptide, whereas most of the pmTOR is co-localized with NPY and only to a lesser extent with CART.

CCK-8S activates c-Fos in a dose-dependent manner in nesfatin-1 immunoreactive neurons in the paraventricular nucleus of the hypothalamus and in the nucleus of the solitary tract of the brainstem.

Recently, a new neuropeptide, named nesfatin-1, was discovered. It has been reported that nesfatin-1 inhibits food intake after injection into the third ventricle as well as intraperitoneal (ip) injection. Cholecystokinin (CCK) is well established to play a role in the regulation of food intake. The aim of the study was to examine whether CCK-8S injected ip modulates neuronal activity in nesfatin-1 immunoreactive (ir) neurons localized in the PVN and in the nucleus of the solitary tract (NTS). Additionally, tyrosine hydroxylase-immunoreactivity (TH-ir) in the PVN was determined to assess the distribution of TH-ir fibers in relation to nesfatin-1-ir. Non-fasted male Sprague-Dawley rats received 6 or 10 microg CCK-8S/kg or vehicle solution (0.15M NaCl; n=4 all groups) ip. The number of c-Fos-ir neurons was determined in the PVN, arcuate nucleus (ARC), and NTS. Double staining procedure for nesfatin-1 and c-Fos revealed that CCK-8S increased significantly and in a dose-dependent manner the number of c-Fos positive nesfatin-1-ir neurons in the PVN ( approximately 4-fold and approximately 7-fold) and NTS ( approximately 9-fold and approximately 26-fold). Triple staining in the PVN showed a dose-dependent neuronal activation of nesfatin-1 neurons that were colocalized with CRF and oxytocin. Double labeling against nesfatin-1 and TH revealed that nefatin-1-ir neurons were encircled in a network of TH-ir fibers in the PVN. No effect on the number of c-Fos-ir neurons was observed in the ARC. These results suggest that the effects of CCK on the HPA axis and on food intake may, at least in part, be mediated by nesfatin-1-ir neurons in the PVN.

Is desacyl ghrelin a modulator of food intake

Desacyl ghrelin is produced in the gastric mucosa and plasma by deacylation of ghrelin. It occurs in considerably larger amounts than ghrelin in various regions in the organisms of rats and mice. It exerts biological activities in vitro as different as stimulating adipogenesis or inhibiting glucose output in hepatocytes. In fasted rats, desacyl ghrelin levels decreased under catabolic metabolic conditions and in mice, high desacyl ghrelin concentrations went along with decreased food intake. These observations suggest an influence of the peptide on food intake and energy homeostasis. Behavioral studies led to controversial results, but several suggest an anorexigenic effect. Studies on desacyl ghrelin-induced modulation of food intake indicate the involvement of central nervous pathways, since it is said to cross the blood-brain barrier and to induce increased neuronal activity hypothalamic nuclei. It is likely to be involved in the regulation of the synthesis of anorexigenic hypothalamic mediators. Quite possibly, there might be means of interaction between desacyl ghrelin and its supposable precursor ghrelin.

The ghrelin activating enzyme ghrelin-O-acyltransferase (GOAT) is present in human plasma and expressed dependent on body mass index

Ghrelin is the only known peripherally produced and centrally acting peptide hormone stimulating food intake. The acylation of ghrelin is essential for binding to its receptor. Recently, the ghrelin activating enzyme ghrelin-O-acyltransferase (GOAT) was identified in mice, rats and humans. In addition to gastric mucosal expression, GOAT was also detected in the circulation of rodents and its expression was dependent on metabolic status. We investigated whether GOAT is also present in human plasma and whether expression levels are affected under different conditions of body weight. Normal weight, anorexic and obese subjects with body mass index (BMI) 30-40, 40-50 and >50 were recruited (n=9/group). In overnight fasted subjects GOAT protein expression was assessed by Western blot and ghrelin measured by ELISA. GOAT protein was detectable in human plasma. Anorexic patients showed reduced GOAT protein levels (-42%, p<0.01) whereas obese patients with BMI>50 had increased concentrations (+34%) compared to normal weight controls. Ghrelin levels were higher in anorexic patients compared to all other groups (+62-78%, p<0.001). Plasma GOAT protein expression showed a positive correlation with BMI (r=0.71, p<0.001) and a negative correlation with ghrelin (r=-0.60, p<0.001). Summarized, GOAT is also present in human plasma and GOAT protein levels depend on the metabolic environment with decreased levels in anorexic and increased levels in morbidly obese patients. These data may indicate that GOAT counteracts the adaptive changes of ghrelin observed under these conditions and ultimately contributes to the development or maintenance of anorexia and obesity as it is the only enzyme acylating ghrelin.