Fig 1.
Effects of vaspin on blood glucose level and body weight.
The rats were randomly assigned into NC group (n = 10) and HFD group (n = 20). The body weight of the rats was measured weekly, fasting blood glucose (FBG) of the rats was measured biweekly. (A-B) The trends of blood glucose and body weight in rats fed with normal diet and high fat diet before intervention. The results were compared and analyzed with Student's unpaired and two-tailed t tests. (C-D) After 16 weeks of dietary manipulation, high-fat-diet rats were randomly divided into HF group (n = 10) and HF plus vaspin group (n = 10). At the end of intervention with vaspin (20 ng/ml, 3ml/kg) intraperitoneally once daily for 4 week, blood glucose level and body weight were compared between the groups. One-way analysis of variance followed by least significant difference (LSD) t-test was used to calculate differences among the various study groups. Data were shown as mean ± SD. * P < 0.05 as compared with NC group; # P < 0.05 as compared with HF group.
Fig 2.
Effects of vaspin on OGTT and ITT in different group rats, n = 10/group.
At the end of intervention with vaspin, the oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed in overnight-fasted rats. (A) oral glucose tolerance test (OGTT). (B) area under curve of OGTT. (C) insulin tolerance test (ITT). (D) ITT show as a percentage. (E) area under curve of ITT. Data were shown as mean ± SD. The results were analyzed with One-way analysis of variance followed by LSD test. * P < 0.05 as compared with NC group; # P < 0.05 as compared with HF group.
Fig 3.
Results of hyperglycemia clamps test in different group rats, n = 10/group.
At the end of intervention with vaspin, hyperglycemic clamp test was performed in overnight-fasted rats. The reaction of islet β cells to glucose and the islet secretion capacity were assessed according to the GIR. Data were shown as mean ± SD. The data obey normal distribution, but the variance was not uniform, thus Kruskal wallis test was used to analyze the difference in different groups rats. * P < 0.05 compared with NC group; # P < 0.05 compared with HF group.
Fig 4.
Effects of vaspin on IRS-2 mRNA and protein levels in INS-1 cells.
INS-1 cells were treated with 0 (control), 80, 160, 320 ng/ml vaspin for 24 h. (A) Effect of vaspin on IRS-2 mRNA in INS-1 cells. The level of IRS-2 mRNA was determined by Real-time PCR. (B-D) Effects of vaspin on the protein levels of total IRS-2, pIRS-2 and pIRS-2/IRS-2 ratio in INS-1 cells. Cell lysates were subjected to western blotting and incubated with antibodies against IRS-2 and pIRS-2. Data were shown as mean ± SD of at least three independent experiments. Differences among groups were compared with one-way analysis of variance followed by LSD test. * P < 0.05 compared with control group; # P < 0.05 compared with PA group; Φ P < 0.05 compared with PA plus 80 ng/ml vaspin group.
Fig 5.
Effects of vaspin on PI3K/Akt signaling pathway in INS-1 cells.
INS-1 cells were treated with 0 (control), 80, 160, 320 ng/ml vaspin for 24h. (A) Effect of vaspin on Akt mRNA in INS-1 cells. The levels of Akt mRNA were determined by Real-time PCR. (B-D) Effects of vaspin on the protein levels of total Akt, pAkt and pAkt/Akt in INS-1 cells. Cell lysates were subjected for western blotting and incubated with antibodies against Akt and pAkt. Data were shown as mean ± SD of at least three independent experiments. One way analysis of variance followed by LSD test was used compare differences between experimental groups and control group. * P < 0.05 compared with control group; # P < 0.05 compared with PA group; Φ P < 0.05 compared with PA plus 80 ng/ml vaspin group; θ P < 0.05 compared with PA plus 160 ng/ml vaspin group.
Fig 6.
Vaspin promotes the phosphorylation of Akt by activiting PI3K.
INS-1 cells of same amount (2.5×105 cell/well) were treated with serum-free medium for 24 h. The medium was then replaced by fresh serum-free medium containing 0.5 mmol/L palmitic acid alone or 0.5 mmol/L palmitic acid plus vaspin (320 ng/ml) in the absence or presence of 25 μmol/L ly294002 for 24 h. The protein levels of Akt, pAkt were determined by western blot analysis. Data were shown as mean ± SD of at least three independent experiments. One way analysis of variance followed by LSD test was used to calculate differences among the various study groups. * P < 0.05 compared with PA group; # P < 0.05 compared with PA plus vaspin group.
Fig 7.
Effect of vaspin on the function of INS-1 was mediated by PI3K/Akt signaling pathway.
INS-1 cells were seeded in 24-well culture dishes and treated with serum-free medium for 24 h. The medium was then replaced by fresh serum-free medium containing 0.5 mmol/L palmitic acid alone or 0.5 mmol/L palmitic acid plus vaspin (320 ng/ml) in the absence or presence of 25 μmol/L ly294002 for 24 h. After intervention, INS-1 cells were treated with glucose (16.7 mmol/L) for 1 h after sugar-free cultured 1 h. Data were shown as mean ± SD of at least three independent experiments. Differences among groups were compared with one way analysis of variance followed by LSD test. * P < 0.05 compared with PA group; # P < 0.05 compared with PA + vaspin group.
Fig 8.
Effect of vaspin on mTOR/p70S6K signaling pathway in INS-1 cells.
(A-B) INS-1 cells were treated with 0 (control), 80, 160, 320 ng/ml vaspin for 24 h. Cell lysates were subjected to western blotting and incubated with antibodies against mTOR, p-mTOR, p70S6K, p-p70S6K. (C-D) INS-1 cells of same amount (2.5×105 cell/well) were incubated in serum-free medium for 24 h. The medium was then replaced by fresh serum-free medium in the absence or presence of 0.5 mmol/L palmitic acid alone for 24 h. Then, a final concentration of 25 nmol/L rapamycin or equivalent volumes of DMSO (vehicle) added to medium over 60 min. Finally, the cells were incubated with or without vaspin for 24 h. The protein levels of mTOR, p-mTOR, p70S6K, p-p70S6K were determined by western blotting analysis. Data were shown as mean ± SD of at least three independent experiments. The results were analyzed with One-way analysis of variance followed by LSD test. * P < 0.05 compared with NC group; # P < 0.05 compared with PA group; Φ P < 0.05 compared with PA plus vaspin group; θ P < 0.05 compared with PA +vaspin +Rapamycin group.
Fig 9.
Effect of vaspin on INS-1 cell proliferation in vitro.
INS-1 cells were assigned into five groups: NC group, PA group, PA + vaspin group, PA + vaspin + Rapamycin group, PA + Rapamycin group. The effect of vaspin on INS-1 cell proliferation was determined by CCK-8 assays. Data were shown as mean ± SD of at least three independent experiments. Groups comparison using one-way analysis of variance followed by LSD test. * P < 0.05 compared with NC group; # P < 0.05 compared with PA group; Φ P < 0.05 compared with PA plus vaspin group; θ P < 0.05 compared with PA +vaspin +Rapamycin group.
Fig 10.
The effect of vaspin on NF-κB inflammation signaling pathway in INS-1 cells.
INS-1 cells of same amount (2.5×105 cell/well) in 24 well dishes were treated with 0 (control), 80, 160, 320 ng/ml vaspin for 24h. (A) Effect of vaspin on mRNA of NF-κB in INS-1 cells. The levels of NF-κB mRNA were determined by Real-time PCR. (B) Effects of vaspin on the protein levels of NF-κB p65 in INS-1 cells. Cell lysates were subjected to western blotting and incubated with antibodies against NF-κB p65. Data were shown as mean ± SD of at least three independent experiments. Multiple samples mean comparison was done using to one-way analysis of variance followed by LSD test. * P < 0.05 compared with control group; # P < 0.05 compared with PA group; Φ P < 0.05 compared with PA plus 80 ng/ml vaspin group; θ P < 0.05 compared with PA plus 160 ng/ml vaspin group.
Fig 11.
Effect of vaspin on INS-1 cell function was mediated by inhibiting NF-κB inflammation signaling pathway.
INS-1 cells were seeded in 24-well culture dishes and treated with PA, PA + vaspin (320 ng/ml) and PA + TPCK (NF-κB inhibitor) 20 μmol/L for 24 h. (A) The level of NF-κB mRNA was determined by Real-time PCR. (B) The protein level of NF-κB p65 was determined by western blot analysis. (C) After intervention, INS-1 cells were treated with glucose (16.7 mmol/L) for 1 h following cultured 1 h in sugar-free medium. Data were shown as mean ± SD of at least three independent experiments. One-way analysis of variance followed by LSD test was used to calculate differences among the various study groups. * P < 0.05 compared with PA group; # P < 0.05 compared with PA + vaspin group.