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

Syntaxin4 interacting protein (Synip) binds phosphatidylinositol (3,4,5) triphosphate.

The insulin responsive Glut4 transport vesicles contain the v-SNARE protein Vamp2 that associate with the plasma membrane t-SNARE protein Syntaxin 4 to drive insulin-stimulated Glut4 translocation in skeletal muscle and adipocytes. The syntaxin 4 interacting protein (Synip) binds to syntaxin 4 in the basal state and dissociates in the insulin-stimulated state allowing for the subsequent binding of Vamp2 containing Glut4 vesicles and fusion with the plasma membrane. In this study, we have found that Synip binds phosphatidylinositol 3,4,5-triphosphate (PIP3), but not phosphatidylinositol 3 phosphate (PIP) or phosphatidylinositol 3,4-biphosphate (PIP2) through the Synip WW domain as deletion of this domain (Synip ΔWW) failed to bind PIP3. Over-expressed Synip ΔWW in 3T3L1 adipocytes reduced the basal levels of Glut4 at the plasma membrane with no effect on the binding to syntaxin 4 in vitro. Subcellular fractionation demonstrated that the amount of Synip ΔWW at the PM was decreased in response to insulin in 3T3L1 adipocytes whereas the amount of Synip WT increased. These data suggest that in the presence of insulin, the dissociated Synip remains anchored to the plasma membrane by binding to PIP3

Synip binds phosphatidylinositol 3,4,5-triphosphate.

<p>(A) 96-well plates were coated with 3 µg/ml of phosphatidylinositol 3,4,5-triphosphate (PIP3) for 1 hr, followed by incubation with various amount of purified GST, GST-GRP-PH, or GST-Synip WT proteins. Bound fusion protein was detected using a polyclonal GST antibody and horseradish peroxidase-conjugated secondary anti-Rabbit IgG. Absorbance was measured at 450 nm. Data represent as means ±SE (n = 3 independent experiments), each samples run in triplicate. *, P<0.05. (B) 96-well plates were coated with 3 µg/ml of phosphatidylinositol (PI), phosphatidylinositol 3-phospahte (PIP), phosphatidylinositol 3,4-biphosphate (PIP2), or phosphatidylinositol 3,4,5-triphosphate (PIP3) and incubated with 16 nM of purified GST, or GST-Synip WT. The amount of bound GST or GST-Synip WT was determined as in (A). Data represent as means ±SE (n = 5 independent experiments), each samples run in triplicate. *, P<0.05. (C) 96-well plates were coated with 3 µg/ml of phosphatidylinositol 3,4,5-triphosphate (PIP3), followed by incubation with 16 nM of purified GST, GST-Synip WT, GST-Synip ΔPDZ, GST-Synip ΔCC, or GST-Synip ΔWW. Data represent as means ±SE (n = 3 independent experiments), each samples run in triplicate. GST, GST only; WT, GST-Synip WT; ΔPDZ, GST-Synip ΔPDZ; ΔWW, GST-Synip ΔWW; ΔCC, GST-Synip ΔCC ; *, P<0.05.</p

Insulin stimulation decreases the binding of Synip WT and ΔWW to syntaxin 4 in vitro.

<p>(A) Chinese Hamster Ovary (CHO) cells were transfected with Flag-Synip WT or Flag-Synip ΔWW by electroporation. After transfection, cells were serum starved for 4 hours and stimulated with or without 100 nM insulin for 15 min at 37°C. These cells were lysed with RIPA buffer and mixed glutathione sepharose beads coupled with the purified GST-syntaxin 4 protein for two hours at 4°C with rotation. After washing, elutes were resolved by SDS-PAGE followed by immunoblot assays probing with a mouse anti-Flag antibody. (B) 3T3L1 adipocytes were transfected with Flag-Synip WT or Flag-Synip ΔWW by electroporation. After transfection, cells were serum starved for 4 hours and lysed with RIPA buffer and mixed glutathione sepharose beads coupled with the purified GST-syntaxin 4 protein for two hours at 4°C with rotation. After washing, elutes were resolved by SDS-PAGE followed by immunoblot assays probing with a mouse anti-Flag antibody or anti-syntaxin 4 antibody as a control. Results are expressed as means ± SE (n = 3 independent experiments).</p

Synip delta WW domain suppressed Glut4 translocation.

<p>(A) 3T3-L1 adipocytes were transfected with Myc-Glut4-EGFP and pcDNA (vector only), Synip WT (WT), or Synip ΔWW (ΔWW) by electroporation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042782#s2" target="_blank">Material and Methods</a>. After 48 h, the cells were serum starved for 2 hours and treated with or without 100 nM insulin for 10 min, fixed and incubated with anti-Myc monoclonal antibody following peroxidase-conjugated goat anti-mouse IgG antibody. The specific exofacial exposure of the Myc epitope (Myc-Glut4-GFP fusion) was detected by mixing with o-phenylenediamine dihydrochloride peroxidase and the absorbance of the mixture was read at 420 nm. Results are expressed as means ± SE (n = 3 independent experiments). Open bar, basal; closed bar, 100 nM insulin; *, P<0.05. (B) 3T3L1 adipocytes were transfected with pcDNA (vector only), Flag-Synip WT or Flag-Synip ΔWW by electroporation. After transfection, cells were serum starved for 4 hours and stimulated with or without 100 nM insulin for 15 min at 37°C. After insulin stimulation, cells were serum starved again for two hours. Then, the PM enrich fraction of cells were purified and resolved by SDS-PAGE followed by immunoblot assays probing with a mouse anti-Glut4 antibody. Evaluation of band densities was measured using NIH-Image software. Data represent as means ±SE (n = 3 independent experiments). Open bar and C, basal; closed bar and Ins, 100 nM Insulin; slash bar and recover, 2^nd serum starvation *, P<0.05.</p

Synip membrane association following insulin stimulation is dependent on the WW domain.

<p>(A) Differentiated 3T3L1 adipocytes were serum starved for six hours and treated with 100 nM insulin for 15 min in the presence or absence of 25 µM LY294002 for 30 min. The cells were then fractionated using different centrifugation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042782#s2" target="_blank">Material and Methods</a>. The fractions enriched PM (10 µg) were resolved on a 10% SDS-PAGE and subjected to Western blotting using anti-Synip antibody, anti-Glut4 antibody or EGF receptor antibody as a loading control. Bottom panel, quantification of plasma membrane Synip protein. (B) 3T3L1 adipocytes were transfected with Flag-Synip WT or Flag-Synip ΔWW by electroporation. After twenty-four hours, the cells were serum starved for six hours and treated with or without 100 nM insulin for 15 min. The cells were then fractionated using ProteoExtract Subcellular Proteome Extraction kit according to the suppliers' protocols. The membrane fractions (10 µg) were resolved on a 10% SDS-PAGE and subjected to Western blotting using anti-mouse Flag antibody or EGF receptor antibody as a loading control. Evaluation of band densities was measured using NIH-Image software. Data are expressed as means ± SE (n = 3 independent experiments). Open bar and C, basal; closed bar and Ins, 100 nM Insulin.</p