Identification of Nesfatin-1 in Human and Murine Adipose Tissue: A Novel Depot-Specific Adipokine with Increased Levels in Obesity

Nesfatin-1 is a recently identified anorexigenic peptide derived from its precursor protein, nonesterified fatty acid/nucleobindin 2 (NUCB2). Although the hypothalamus is pivotal for the maintenance of energy homeostasis, adipose tissue plays an important role in the integration of metabolic activity and energy balance by communicating with peripheral organs and the brain via adipokines. Currently no data exist on nesfatin-1 expression, regulation, and secretion in adipose tissue. We therefore investigated NUCB2/nesfatin-1 gene and protein expression in human and murine adipose tissue depots. Additionally, the effects of insulin, dexamethasone, and inflammatory cytokines and the impact of food deprivation and obesity on nesfatin-1 expression were studied by quantitative RT-PCR and Western blotting. We present data showing NUCB2 mRNA (P < 0.001), nesfatin-1 intracellular protein (P < 0.001), and secretion (P < 0.01) were significantly higher in sc adipose tissue compared with other depots. Also, nesfatin-1 protein expression was significantly increased in high-fat-fed mice (P < 0.01) and reduced under food deprivation (P < 0.01) compared with controls. Stimulation of sc adipose tissue explants with inflammatory cytokines (TNFalpha and IL-6), insulin, and dexamethasone resulted in a marked increase in intracellular nesfatin-1 levels. Furthermore, we present evidence that the secretion of nesfatin-1 into the culture media was dramatically increased during the differentiation of 3T3-L1 preadipocytes into adipocytes (P < 0.001) and after treatments with TNF-alpha, IL-6, insulin, and dexamethasone (P < 0.01). In addition, circulating nesfatin-1 levels were higher in high-fat-fed mice (P < 0.05) and showed positive correlation with body mass index in human. We report that nesfatin-1 is a novel depot specific adipokine preferentially produced by sc tissue, with obesity- and food deprivation-regulated expression.
Ramanjaneya et al. Endocrinology. 2010 Apr 28. [Epub ahead of print]

Ghrelin, des-acyl ghrelin and nesfatin-1 in gastric X/A-like cells: role as regulators of food intake and body weight.

Numerous peptides released from endocrine cells in the intestinal mucosa were established early on to be involved in the physiological regulation of food intake with a prominent role in termination of food ingestion when nutrients pass along the intestinal tract. Recently, peptides released from X/A-like endocrine cells of the gastric oxyntic mucosa were recognized as additional key players in the regulation of feeding and energy expenditure. Gastric X/A-like cells release the octanoylated peptide, ghrelin, the only known peripherally produced hormone stimulating food intake through interaction with growth hormone secretagogue 1a receptor (GHS-R1a). Additionally, non-octanoylated (des-acyl) ghrelin present in the circulation at higher levels than ghrelin is currently discussed as potential modulator of food intake by opposing ghrelin's action independent from GHS-R1a although the functional significance remains to be established. Obestatin, a ghrelin-associated peptide was initially reported as anorexigenic modulator of ghrelin's orexigenic action. However, subsequent reports did not support this contention. Interesting is the recent identification of nesfatin-1, a peptide derived from the nucleobindin2 gene prominently expressed in gastric X/A-like cells in different vesicles than ghrelin. Circulating nesfatin-1 levels vary with metabolic state and peripheral or central injection inhibits dark phase feeding in rodents. Overall, these data point to an important role of gastric X/A-like cells in food intake regulation through the expression of the orexigenic peptide ghrelin along with des-acyl ghrelin and nesfatin-1 capable of reducing food intake upon exogenous injection although their mechanisms of action and functional significance remain to be established.
Peptides. 2010 Feb;31(2):357-69.

Identification of nesfatin-1 as a satiety molecule in the hypothalamus.

The brain hypothalamus contains certain secreted molecules that are important in regulating feeding behaviour   Here we show that nesfatin, corresponding to NEFA/nucleobindin2 (NUCB2), a secreted protein of unknown function, is expressed in the appetite-control hypothalamic nuclei in rats. Intracerebroventricular (i.c.v.) injection of NUCB2 reduces feeding. Rat cerebrospinal fluid contains nesfatin-1, an amino-terminal fragment derived from NUCB2, and its expression is decreased in the hypothalamic paraventricular nucleus under starved conditions. I.c.v. injection of nesfatin-1 decreases food intake in a dose-dependent manner, whereas injection of an antibody neutralizing nesfatin-1 stimulates appetite. In contrast, i.c.v. injection of other possible fragments processed from NUCB2 does not promote satiety, and conversion of NUCB2 to nesfatin-1 is necessary to induce feeding suppression. Chronic i.c.v. injection of nesfatin-1 reduces body weight, whereas rats gain body weight after chronic i.c.v. injection of antisense morpholino oligonucleotide against the gene encoding NUCB2. Nesfatin-1-induced anorexia occurs in Zucker rats with a leptin receptor mutation, and an anti-nesfatin-1 antibody does not block leptin-induced anorexia. In contrast, central injection of alpha-melanocyte-stimulating hormone elevates NUCB2 gene expression in the paraventricular nucleus, and satiety by nesfatin-1 is abolished by an antagonist of the melanocortin-3/4 receptor. We identify nesfatin-1 as a satiety molecule that is associated with melanocortin signalling in the hypothalamus.
Oh-I et al. Nature. 2006 Oct 12;443(7112):709-12.

Nesfatin-1 secreted from the peripheral endocrine cells

Mapping in Rat Hypothalamus with Nesfatin-1 N-Terminal (Human) Antiserum (H-003-97)

Mapping in Human brain tissue by Rabbit Anti-Nesfatin-3, C-Terminal (Human) Antibody(H-003-27)

Protocol for antibody staining

Human Nesfatin-1 (1-82) / NUCB2 (25-106) ELISA Kit (EK-003-26)
Standard Range: 0.78-50 (ng/ml)

Rat Nesfatin-1 (1-82) EIA Kit (EK-003-22)
Linear Range: 1.26-17.7 ng/ml

Rat Nesfatin-1 (1-82) RIA Kit (RK-003-22)

Detection of NUCB2/Prepro Nesfatin-1 in Rat Brain by Nesfatin-1 (1-82) (Rat) Western Blot Kit (WBK-003-22)

Detection of NUCB2 in Rat Brain by Nesfatin-1 (1-82) (Rat) Western Blot Kit (WBK-003-96)

Amino acid sequence of human NEFA/nucleobindin2 (NUCN2)

Comparison of amino acid sequence between human and rat NUCB2 protein

Comparison of amino acid sequence between mouse and rat NUCB2 protein

Purification of Rat Nesfatin-1 (82 amino acid peptide) by RP-HPLC

Purification of human Nesfatin-1 (82 amino acid peptide) by RP-HPLC

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Expression of NUCB2 and nesfatin-1 in the rat hypothalamus

a–d, Immunohistochemical analysis., arcuate nucleus (Arc); b, PVN; c, supraoptic nucleus (SON); d, lateral hypothalamic area (LHA). 3V (third ventricle); f, (fornix); opt, (optic nerve). e, In situ hybridization with NUCB2 antisense cRNA. Zi, zona incerta. f, g, Food intake (n = 6–7) after i.c.v. injection of NUCB2: f, dose–response; g, time course (open bars, 0 pmol NUCB2; filled bars, 4 pmol NUCB2). h, Double immunostaining with nesfatin Ab24 (4-Cl-naphthol in grey) and PC antibodies (Alexa594 in red) in the hypothalamus. i,j, Hypothalamic NUCB2 mRNA levels (i, n = 6–7) and the PVN nesfatin-1 concentrations (j, n = 4) in rats fed ad libitum (open bars) or starved for 24 h (filled bars). Data are means s.e.m. Asterisk, P < 0.05; two asterisks, P < 0.01 compared with 0 pmol or ad libitum (Student's t-test).
Oh-I et al. Nature. 2006 Oct 12;443(7112):709-12.

Nesfatin-1-induced satiety in rats.

a, b, Food intake (n = 5–8) after i.c.v. injection of nesfatin-1;a, dose–response; b, time course (open bars, 0 pmol nesfatin-1; filled bars, 5 pmol nesfatin-1).c, d, Food intake (n = 5–10) after i.c.v. injection of each fragment (25 pmol) derived from NUCB2: c, 0–1 h; d, 1–3 h. e, Food intake (n = 5–7) after i.c.v. injection of 8 mg IgG (open bars, control rabbit IgG; grey bars, nesfatin Ab24 IgG; black bars, nesfatin Ab301 IgG. f–h, Food intake (n = 5–6) after i.c.v. injection of each intermediate (5 pmol) with wild-type Lys 83-Arg 84 (WT) or mutant Ala 83-Ala 84 (Mut). f, 0–1 h; g, 1–3 h; h, 3–6 h. Data are means s.e.m. Asterisk, P < 0.05; two asterisks, P < 0.01 compared with 0 pmol, vehicle (V) or control IgG.
Oh-I et al. Nature. 2006 Oct 12;443(7112):709-12.

Body weight changes after continuous i.c.v. injection of substances.

a, b, Daily food intake (a) and body weight gain (b; increment from day 0) in rats (n = 4–5) after nesfatin-1 injection (5 pmol daily). Open squares, vehicle; filled squares, nesfatin-1. c, d, Daily food intake (c) and body weight gain (d, increment from day 0) in rats (n = 4-5) after injection with NUCB2 missense (open squares) or antisense (filled squares) morpholino oligonucleotide (MON; 40 mg per day). Antisense (5'-ATGGTCCTCCACCTCATCTTCAGAG-3') and 5-missense (5'-ATCGTGCTCCACGTCATCTACACAG-3') MONs were purchased from Gene Tools. An Alzet osmotic mini-infusion pump was used for continuous injection. Data are means s.e.m. Asterisk, P < 0.05; two asterisks, P < 0.01 compared with vehicle or missense MON.
Oh-I et al. Nature. 2006 Oct 12;443(7112):709-12.

Nesfatin-1-induced satiety associated with leptin or melanocortin signalling.

a, b, Food intake in lean (a) and Zucker (b) rats (n = 5) after i.c.v. injection of 5 pmol nesfatin-1 (open bars, vehicle; filled bars, nesfatin-1). c, Effects of nesfatin Ab24 on leptin-induced anorexia (n = 6). Vehicle, 5 pmol nesfatin-1 or 5 pmol leptin was centrally administered 15 min after i.c.v. injection of Ab24 IgG (8  mg) during the dark phase. d, e, Effects of SHU9119 on nesfatin-1-induced anorexia (n = 6). Vehicle or 5 pmol nesfatin-1 was centrally administered 15 min after i.c.v. injection of 20 pmol SHU9119. Data are means s.e.m. Asterisk, P < 0.05; two asterisks, P < 0.01 compared with vehicle.
Oh-I et al. Nature. 2006 Oct 12;443(7112):709-12.

003-24 and 003-25 are two peptide fragments of human Nesfatin-1