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NUCB2 /Nesfatin-1Brain hypothalamus expressed several secreted molecules that function in regulating feeding behavior [1]. NUCB2 (NUCB2 also called NEFA for DNA binding /EF-hand /acidic protein) is a hypothalamus-secreted protein containing 396 amino acids that is highly conserved in human, mice and rat [2, 3]. NUCB2 when injected directly into the brain of rats promotes anorexia and decreases body weight [3]. NUCB2 is cleaved posttranslationally by prohormone convertases into a N-terminus-fragment Nesfatin-1 and two C-terminal peptides, Nesfatin-2 and Nesfatin-3 [3]. Nesfatin-1 possesses all of the anorexigenic property of NUCB2 [4]. The conversion of NUCB2 into Nesfatin-1 is indispensable for its activity in vivo [3]. Nesfatin-1 is found in discrete nuclei of the hypothalamus where it probably activates a leptin-independent melanocortin pathway [3]. Nesfatin-1 crosses the Blood Brain Barrier (BBB) in both the blood-to-brain and brain-to-blood directions by a nonsaturable system [5]. NUCB2 is also expressed in the adipocyte cell line 3T3L1 suggesting other functions of Nesfatin-1 outside brain or peripheral source of Nesfatin-1 affecting brain function [6]. Nesfatin-1 in rat stimulates calcium influx and interacts with a G protein-coupled receptor still to be characterized [7]. Nesfatin-1 (m & r) Detection Set (APO-54N-036)Summary of features
 Principle of the KitThis assay is a sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA) developed for the direct measurement of mouse or rat Nesfatin-1 in biological fluids. A monoclonal antibody specific for mouse/ rat Nesfatin-1 (COAT) is coated onto the wells of the microtiter plate. Samples of rat or mouse and standards of rat Nesfatin-1 are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, Nesfatin-1 is recognized by the addition of a biotinylated monoclonal antibody specific for mouse/ rat Nesfatin-1 (DET). After removal of excess biotinylated antibody, streptavidine-peroxidase is added. Following a final washing, peroxidase activity is quantified using the substrate 3,3’,5,5’-tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of rat or mouse Nesfatin-1 in the samples. Mouse and Rat Nesfatin-1 have only 2 amino acids difference and are recognized with similar affinity by the antibodies used in the ELISA.
Literature Overview:1. Stanley, S., Wynne, K., McGowan, B., and Bloom, S. (2005). Hormonal regulation of food intake. Physiological reviews 85, 1131-1158. 2. Wendel, M., Sommarin, Y., Bergman, T., and Heinegard, D. (1995). Isolation, characterization, and primary structure of a calcium-binding 63-kDa bone protein. J. Biol. Chem. 270, 6125-6133. 3. Oh, I.S., Shimizu, H., Satoh, T., Okada, S., Adachi, S., Inoue, K., Eguchi, H., Yamamoto, M., Imaki, T., Hashimoto, K., et al. (2006). Identification of nesfatin-1 as a satiety molecule in the hypothalamus. Nature 443, 709-712. 4. Nonogaki, K., Ohba, Y., Sumii, M., and Oka, Y. (2008). Serotonin systems upregulate the expression of hypothalamic NUCB2 via 5-HT2C receptors and induce anorexia via a leptin-independent pathway in mice. Biochem. Biophys. Res. Com. 5. Price, T.O., Samson, W.K., Niehoff, M.L., and Banks, W.A. (2007). Permeability of the blood-brain barrier to a novel satiety molecule nesfatin-1. Peptides 28, 2372-2381. 6. Adachi, J., Kumar, C., Zhang, Y., and Mann, M. (2007). In-depth analysis of the adipocyte proteome by mass spectrometry and bioinformatics. Mol. Cell Proteomics 6, 1257-1273. 7. Brailoiu, G.C., Dun, S.L., Brailoiu, E., Inan, S., Yang, J., Chang, J.K., and Dun, N.J. (2007). Nesfatin-1: distribution and interaction with a G protein-coupled receptor in the rat brain. Endocrinology 148, 5088-5094.
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