The mammalian hypothalamus strongly influences ingestive behaviour through several different signalling molecules and receptor systems. Here we show that CART (cocaine- and amphetamine-regulated transcript), a brain-located peptide, is a satiety factor and is closely associated with the actions of two important regulators of food intake, leptin and neuropeptide Y. Food-deprived animals show a pronounced decrease in expression of CART messenger RNA in the arcuate nucleus. In animal models of obesity with disrupted leptin signalling, CART mRNA is almost absent from the arcuate nucleus. Peripheral administration of leptin to obese mice stimulates CART mRNA expression. When injected intracerebroventricularly into rats, recombinant CART peptide inhibits both normal and starvation-induced feeding, and completely blocks the feeding response induced by neuropeptide Y. An antiserum against CART increases feeding in normal rats, indicating that CART may be an endogenous inhibitor of food intake in normal animals.

The novel neuropeptide cocaine-amphetamine-regulated transcript (CART) is expressed in several hypothalamic regions and has recently been shown to be involved in the central control of food intake. To characterize the hypothalamic CART neurons and understand the physiological functions they might serve, we undertook an in situ hybridization and immunohistochemical study to examine distribution and neurochemical phenotype of these neurons. In situ hybridization studies showed abundant CART mRNA in the periventricular nucleus (PeV), the paraventricular nucleus of the hypothalamus (PVN), the supraoptic nucleus (SON), the arcuate nucleus (Arc), the zona incerta, and the lateral hypothalamic area. The distribution of CART-immunoreactive neurons as revealed by a monoclonal antibody raised against CART(41-89) displayed complete overlap with CART mRNA. Double immunohistochemistry showed co-existence of CART immunoreactivity (CART-IR) and somatostatin in some neurons of the PeV. In the magnocellular division of the PVN as well as the SON, CART-IR was demonstrated in both oxytocinergic and vasopressinergic perikarya. In the medial parvicellular region of the PVN a few CART-IR neurons co-localized galanin, but none was found to co-localize corticotropin-releasing hormone. In the Arc, almost all pro-opiomelanocortinergic neurons were shown to contain CART, whereas no co-localization of CART with NPY was found. In the lateral hypothalamic area nearly all CART neurons were found to contain melanin-concentrating hormone. The present data support a role for CART in neuroendocrine regulation. Most interestingly, CART is co-stored with neurotransmitters having both positive (melanin-concentrating hormone) as well as a negative (pro-opiomelanocortin) effect on food intake and energy balance.

AbstractHemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (KD = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

Administration of ghrelin, the endogenous ligand for the GH secretagogue receptor, stimulates not only GH secretion but also appetite and food intake in humans. Endogenous ghrelin levels display a distinct circadian rhythm, which is reciprocal to that of insulin and presumed to be meal dependent and not associated with GH secretion. We tested the hypothesis that food deprivation could impact circadian serum ghrelin levels and unmask meal-independent regulatory mechanisms. Thirty-three young adults, subdivided according to gender and level of obesity, were studied with blood sampling every 3 h from 12-84 h of fasting. Serum ghrelin levels showed a marked diurnal rhythm with a nadir in the morning (0800 h), peak levels in the afternoon, and a gradual decline during the night. This pattern was preserved during the entire fasting period and was independent of gender and obesity. Mean 24-h ghrelin levels exhibited a small but significant decline during the fast (P < 0.001). As expected, GH secretion increased with fasting in lean subjects, and a gradual decline in insulin concentrations was observed in all subjects. Neither GH nor insulin showed any significant relationship to ghrelin. In contrast, serum cortisol exhibited a strong inverse temporal association with ghrelin (r = -0.79; P < 0.0001). In conclusion, our study yields no evidence that ghrelin stimulates GH release during fasting. As a novel finding, ghrelin appears to be related to cortisol. However, further studies are needed to elucidate the physiological mechanisms behind this relationship.

Because cocaine-and amphetamine-regulated transcript (CART) coexists with ␣-melanocyte stimulating hormone (␣-MSH) in the arcuate nucleus neurons and we have recently demonstrated that ␣-MSH innervates TRH-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN), we raised the possibility that CART may also be contained in fibers that innervate hypophysiotropic thyrotropin-releasing hormone (TRH) neurons and modulate TRH gene expression. Triple-labeling fluorescent in situ hybridization and immunofluorescence were performed to reveal the morphological relationships between pro-TRH mRNAcontaining neurons and CART-and ␣-MSH-immunoreactive (IR) axons. CART-IR axons densely innervated the majority of pro-TRH mRNA-containing neurons in all parvocellular subdivisions of the PVN and established asymmetric synaptic specializations with pro-TRH neurons. However, whereas all ␣-MSH-IR axons in the PVN contained CART-IR, only a portion of CART-IR axons in contact with pro-TRH neurons were immunoreactive for ␣-MSH.In the medial and periventricular parvocellular subdivisions of the PVN, CART was co-contained in ϳ80% of pro-TRH neuronal perikarya, whereas colocalization with pro-TRH was found in Ͻ10% of the anterior parvocellular subdivision neurons. In addition, Ͼ80% of TRH/CART neurons in the periventricular and medial parvocellular subdivisions accumulated Fluoro-Gold after systemic administration, suggesting that CART may serve as a marker for hypophysiotropic TRH neurons. CART prevented fasting-induced suppression of pro-TRH in the PVN when administered intracerebroventricularly and increased the content of TRH in hypothalamic cell cultures. These studies establish an anatomical association between CART and pro-TRH-producing neurons in the PVN and demonstrate that CART has a stimulatory effect on hypophysiotropic TRH neurons by increasing pro-TRH gene expression and the biosynthesis of TRH.