Peripheral Effects of Nesfatin-1 on Glucose Homeostasis

<div><p>Aims/hypothesis</p><p>The actions of peripherally administered nesfatin-1 on glucose homeostasis remain controversial. The aim of this study was to characterize the mechanisms by which peripheral nesfatin-1 regulates glucose metabolism.</p><p>Methods</p><p>The effects of nesfatin-1 on glucose metabolism were examined in mice by continuous infusion of the peptide via osmotic pumps. Changes in AKT phosphorylation and Glut4 were investigated by Western blotting and immnuofluorescent staining. Primary myocytes, adipocytes and hepatocytes were isolated from male mice.</p><p>Results</p><p>Continuous peripheral infusion of nesfatin-1 altered glucose tolerance and insulin sensitivity in mice fed either normal or high fat diet<b>,</b> while central administration of nesfatin-1 demonstrated no effect. Nesfatin-1 increases insulin secretion in vivo, and in vitro in cultured min6 cells. In addition, nesfatin-1 up-regulates the phosphorylation of AKT in pancreas and min6 islet cells. In mice fed normal diet, peripheral nesfatin-1 significantly increased insulin-stimulated phosphorylation of AKT in skeletal muscle, adipose tissue and liver; similar effects were observed in skeletal muscle and adipose tissue in mice fed high fat diet. At basal conditions and after insulin stimulation, peripheral nesfatin-1 markedly increased GLUT4 membrane translocation in skeletal muscle and adipose tissue in mice fed either diet. In vitro studies showed that nesfatin-1 increased both basal and insulin-stimulated levels of AKT phosphorylation in cells derived from skeletal muscle, adipose tissue and liver.</p><p>Conclusions</p><p>Our studies demonstrate that nesfatin-1 alters glucose metabolism by mechanisms which increase insulin secretion and insulin sensitivity via altering AKT phosphorylation and GLUT 4 membrane translocation in the skeletal muscle, adipose tissue and liver.</p></div

Effects of peripheral nesfatin-1 infusion on AKT phosphorylation in mice fed control diet.

<p>Mice received subcutaneous infusion of saline (control) or nesfatin-1 (2.5 pmol/mouse/hour). Insulin at a dose of 2 IU/kg was injected intraperitoneally 10 min before animals were sacrificed. Phosphorylation of AKT (ser473) was detected by Western blotting using specific antibody and normalized to total AKT. Levels of AKT phosphorylation were examined in skeletal muscle (A), adipose tissue (B) and liver (C) in mice fed normal chow diet. Intensity of phosphor-AKT signals were quantified by NIH Image J software and expressed as mean±SEM. *<i>P</i><0.05 vs. insulin stimulation without nesfatin-1 treatment. Six samples were examined for each condition.</p

Effects of peripheral nesfatin-1 infusion on GLUT 4 in NCD mice.

<p>(A, C) Immunofluorescent staining for GLUT4. Immunofluorescent staining was performed using specific antibody against GLUT4 (red) in skeletal muscle (A) and adipose tissue (C). Nuclei were stained with Hoechst dye. Controls include substituting primary antibodies with mouse IgG. Signal intensity of cytoplasm membrane GLUT4 immunoreactivity was quantified and normalized to the basal control, Data are expressed as mean±SEM. *<i>P</i><0.05 vs. control, # <i>P</i><0.05 vs. insulin stimulation without nesfatin-1 treatment. (B, D) Western blotting for GLUT4. Western blot was performed using specific antibody against GLUT4 in skeletal muscle (B) and adipose tissue (D). GAPDH was used as the internal control. Shown were representative results from six individual experiments.</p

Effects of peripheral nesfatin-1 infusion on phosphorylation of AKT in mice fed high fat diet.

<p>Mice were fed high fat diet for 8 weeks, and then received subcutaneous infusion of saline (control) or nesfatin-1 for 2 weeks. Insulin at a dose of 2 IU/kg was injected intraperitoneally 10 min before animals were sacrificed. Phospho AKT (ser473) and AKT were detected by Western blotting using specific antibodies, in which AKT was used as internal controls. Shown are representative results from saline (control) and nesfatin-1 infusion mice with or without insulin injection. Levels of AKT phosphorylation were examined in skeletal muscle (A), adipose tissue (B) and liver (C) in mice fed high fat diet. Intensity of phosphor-AKT signals were quantified by NIH Image J software and expressed as mean±SEM. *<i>P</i><0.05 vs. insulin stimulation without nesfatin-1 treatment. Six samples were examined for each condition.</p

Effects of nesfatin-1 infusion on glucose homeostasis.

<p>Oral glucose tolerance tests and insulin sensitivity tests are shown in left and right panels, respectively. Results shown are for oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT) in C57BL/6J mice fed normal chow diet (A, B) or high fat diet (C, D) receiving saline (□ line) or nesfatin-1 infusion (▪ line) at a dose of 2.5 pmol/mouse/hour. Areas under curves were analyzed by Prism software. Eleven mice were examined for each condition. *<i>P</i><0.05 vs. control mice. Data expressed as mean±SEM. Intraperitoneal glucose tolerance tests (IGTT) and ITT were performed in light cycle (07∶00–19∶00) (E) and dark cycle (19∶00–07∶00) (G) in rats 10 min after 3^rd ICV injection of nesfatin-1 or vehicle. Shown are data on IGTT and ITT obtained in light cycle (E, F) and dark cycle (G, H). Areas under curves were analyzed by Prism software. Six rats were examined for each condition. Data expressed as mean±SEM.</p

Effects of nesfatin-1 on AKT phosphorylation in pancreas (A) and min6 islet cells (B).

<p>(A) Mice were fed high fat diet for 8 weeks, then received subcutaneous infusion of saline (control) or nesfatin-1 for 2 weeks. Levels of AKT phosphorylation were examined in pancreas by Western blotting using specific antibody. Shown are representative results from 6 individual experiments. B. Cultured min6 islet cells were treated with nesfatin-1 at a dose of 10⁻⁸ M for the time indicated. Shown is the representative western blot from 3 separate experiments.</p

Effects of peripheral nesfatin-1 infusion on GLUT4 in mice fed high fat diet.

<p>Immunofluorescent staining was performed using specific antibody against GLUT4 (red) in skeletal muscle and adipose tissue derived from mice consuming high fat diet and either saline (control) or nesfatin-1. Nuclei were stained with Hoechst dye. Controls include substituting primary antibodies with mouse IgG. Shown were representative results from six individual experiments. Images depict immunofluorescent staining for GLUT4 (red) in skeletal muscle (A) and adipose tissue (B). Data are expressed as mean±SEM as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071513#pone-0071513-g004" target="_blank">Figure 4</a>. *<i>P</i><0.05 vs. control, # <i>P</i><0.05 vs. insulin stimulation without nesfatin-1 treatment. Shown were representative results from six individual experiments.</p