Ghrelin Receptor Is Required for the Effect of Nesfatin-1 on Glucose Metabolism

Abstract

Studies of nesfatin-1 in glucose metabolism have become a topic of interest recently, however, the specific receptor for nesfatin-1 has not yet been identified. Some studies hinted at a connection between nesfatin-1 and the ghrelin receptor, growth hormone secretagogue receptor. Therefore, we aimed to study the role of GHSR in the glycemic effects of nesfatin-1 as well as its downstream pathways. We employed C57/BL6 mice (wild type and GHSR knockout mice) eating a normal chow diet and a high fat diet in this study, and the experimental technique included western blot, real-time PCR, immunofluorescence and ELISA. We found that in mice fed a normal chow diet (NCD), nesfatin-1 improved glucose tolerance, up-regulated AKT kinase (AKT) mRNA levels and phosphorylation and GLUT4 membrane translocation in skeletal muscle. These effects were blocked by co-injection of GHSR antagonist [D-Lys3]-GHRP-6 and were attenuated in GHSR knockout mice. In mice fed high-fat diet (HFD), nesfatin-1 not only exerted the effects observed in NCD mice, but also suppressed appetite and raised AKT levels in liver tissues that also required GHSR. Peripheral nesfatin-1 suppressed c-fos expression of GHSR immunoreactive neurons induced by fasting in hypothalamic nuclei, indicating that nesfatin-1 inhibited the activation of central GHSR. We concluded that the effects of nesfatin-1 on food intake and glucose metabolism were GHSR-dependent, and that the glycemic effect was associated with AKT and GLUT4. This study should stimulate further exploration of the nesfatin-1 receptor.

Keywords: AKT; ghrelin receptor; glucose metabolism; high fat diet; nesfatin-1.

Figure 1

The differences between GHSR^+/+ and GHSR^−/− mice in food intake and blood glucose. Eight-week-old GHSR^+/+ and GHSR^−/− mice were fed with NCD or HFD for 8 weeks: then mice were inspected respectively. (A,D) Cumulative food intake in GHSR^+/+ and GHSR^−/− mice fed with (A) NCD or HFD tested after mice fasting for 6 h. (B,E) Blood glucose in GHSR^+/+ and GHSR^−/− mice fed with (B) NCD or (E) HFD under ad libitum or 24 h fasting conditions. (C) Changes in body weight during 8 weeks of HFD. (F) Oral glucose tolerance tests (OGTT) in mice fed with NCD and HFD. (A,B,D,E) NCD-GHSR^+/+: n = 4; NCD-GHSR^−/−: n = 3; HFD-GHSR^+/+: n = 6; HFD-GHSR^−/−: n = 6. (C) n = 10/group. (F) n = 12/group. Data are expressed as mean ± SEM. (A–E) ^*P < 0.05, ^**P < 0.01 for the effect of GHSR^−/− mice vs. GHSR^+/+ mice; (F) ^**P < 0.01 for the effect of HFD-mice vs. NCD-mice. Student's t-test was applied to analyze the statistical difference.

Figure 2

Circulating Nesfatin-1 in GHSR^+/+ and GHSR^−/− Mice. GHSR^+/+ and GHSR^−/− mice were fasted for 12 h or fed ad libitum. We obtained blood samples from the end of the tail, after which we performed ELISA. Data are expressed as mean ± SEM (n = 6–7/group). ^**P < 0.01 for the effect of GHSR^−/− mice in fasting condition vs. GHSR^−/− mice in ad libitum. GHSR^−/− mice in fasting condition vs. GHSR^+/+ mice in fasting condition: P = 0.05. One-way ANOVA was applied to analyze the statistical difference.

Figure 3

[D-Lys3]-GHRP-6 attenuated nesfatin-1's effects on glucose metabolism. GHSR^+/+ mice fed NCD underwent chronic tail vein administration of vehicle, nesfatin-1 (Nes-1), [D-Lys3]-GHRP-6 (GHRP-6), and [D-Lys3]-GHRP-6+nesfatin-1 (GHRP-6+Nes-1) for 11 days. (A) Food intake and (C) blood glucose changes after one acute injection in mice fasting for 6 h. (B) Blood glucose changes after 11-day chronic injection compared with drug treatment prior. (D) OGTT after 8 days of injection in mice fasting for 16 h. Data are expressed as mean ± SEM. Vehicle: n = 6; Nes-1: n = 6; GHRP-6: n = 6; GHRP-6+Nes-1: n = 8. ^*P < 0.05 for the effect of nesfatin-1 vs. vehicle; ^#P < 0.05, ^##P < 0.01 for the effect of GHRP-6+nesfatin-1 vs. nesfatin-1. One-way ANOVA was applied to analyze the statistical difference.

Figure 4

Ghrelin receptor knockout blocked nesfatin-1's effects on glucose metabolism in NCD-fed mice. GHSR^−/− mice fed NCD underwent chronic tail vein administration of vehicle or nesfatin-1 (Nes-1) for 11 days. (A) Food intake and (C) blood glucose changes after one acute injection in mice fasting for 6 h. (B) Blood glucose changes after 11-day chronic injection compared with drug treatment prior. (D) OGTT after 8 days of injection in mice fasting for 16 h. Data are expressed as mean ± SEM. GHSR^−/−+Vehicle: n = 3; GHSR^−/−+Nes-1: n = 4. No statistical significance is detected. Student's t-test was applied to analyze the statistical difference.

Figure 5

Ghrelin receptor knockout blocked nesfatin-1's effects on food intake and glucose metabolism in HFD-fed mice. GHSR^+/+ and GHSR^−/− mice fed HFD underwent chronic tail vein administration of vehicle or nesfatin-1 (Nes-1) for 11 days, respectively. (A,E) Food intake and (B,F) blood glucose changes after one acute injection in two types of mice fasting for 6 h. (C,G) Blood glucose changes after 11-day chronic injection compared with drug treatment prior. (D,H) OGTT after 8 days of injection in mice fasting for 16 h. Data are expressed as mean ± SEM. GHSR^+/+ +Vehicle, GHSR^+/+ +Nes-1: n = 6/group; GHSR^−/− +Vehicle, GHSR^−/− +Nes-1: n = 5/group. ^*P < 0.05; ^**P < 0.01 for the effect of nesfatin-1 vs. vehicle in GHSR^+/+ mice. Student's t-test was applied to analyze the statistical difference.

Figure 6

GHSR mediated the effect of nesfatin-1 on AKT phosphorylation and GLUT4 membrane translocation in skeletal muscle. GHSR^+/+ mice fed NCD underwent chronic tail vein administration of vehicle, nesfatin-1 (Nes-1), [D-Lys3]-GHRP-6 (GHRP-6), and [D-Lys3]-GHRP-6+nesfatin-1 (GHRP-6+Nes-1) for 12 days. GHSR^−/− mice fed NCD underwent chronic tail vein administration of vehicle and nesfatin-1 for 12 days. Insulin (2 IU/kg) was intraperitoneally injected into mice 10 min before mice were sacrificed. (A,D) mRNA expression levels of AKT and GLUT4 detected by RT-PCR in skeletal muscle of (A) GHSR^+/+ and (D) GHSR^−/− mice. (B,E) P-AKT and (C,F) GLUT4 protein levels detected by Western blotting were normalized to total AKT and β-actin, respectively. (B,E) P-AKT/AKT level in skeletal muscle of (B) GHSR^+/+ and (E) GHSR^−/− mice. (C,F) GLUT4 level in skeletal muscle of (C) GHSR^+/+ and (F) GHSR^−/− mice. (G,H) GLUT4 membrane translocation detected by immunofluorescence in skeletal muscle of GHSR^+/+ mice treated with (G) vehicle and (H) nesfatin-1. (I,J) GLUT4 membrane translocation detected by immunofluorescence in skeletal muscle of GHSR^−/− mice treated with (I) vehicle and (J) nesfatin-1. (K) GLUT4 average staining value in skeletal muscle of GHSR^+/+ mice and GHSR^−/− mice. n = 5/group; (D) GHSR^−/− + Vehicle: n = 3, GHSR^−/− + Nes-1: n = 4; (E,F) n = 3/group; (G–K) n = 3/group. Data are expressed as mean ± SEM. ^*P < 0.05; ^**P < 0.01 for the effect of nesfatin-1 vs. vehicle; ^#P < 0.05, ^##P < 0.01 for the effect of GHRP-6+nesfatin-1 vs. nesfatin-1; ^∧∧P < 0.01 for the effect of GHRP-6 vs. vehicle. (A–C,K) One-way ANOVA was applied to analyze the statistical difference for multiple groups. (D–F) Student's t-test was applied to analyze the statistical difference for two groups.

Figure 7

GHSR mediated the effect of nesfatin-1 on AKT phosphorylation in liver of HFD-fed mice. GHSR^+/+ mice underwent chronic tail vein administration of vehicle, nesfatin-1 (Nes-1), [D-Lys3]-GHRP-6 (GHRP-6) and [D-Lys3]-GHRP-6+nesfatin-1 (GHRP-6+Nes-1) for 12 days. GHSR^−/− mice fed underwent chronic tail vein administration of vehicle and nesfatin-1 for 12 days. Insulin (2 IU/kg) was intraperitoneally injected into mice 10 min before mice were sacrificed. (A,C,E,G) mRNA expression levels of AKT detected by RT-PCR in liver of (A) GHSR^+/+, (C) GHSR^−/− mice fed NCD and (E) GHSR^+/+, (G) GHSR^−/− mice fed HFD. (B,D,F,H) P-AKT/AKT level in liver of (B) GHSR^+/+, (D) GHSR^−/− mice fed NCD and (F) GHSR^+/+, (H) GHSR^−/− mice fed HFD. (A,B) n = 5/group; (C,D) n = 3/group; (E–H) n = 5/group. Data are expressed as mean ± SEM. ^*P < 0.05; ^**P < 0.01 for the effect of nesfatin-1 vs. vehicle. (A,B) One-way ANOVA was applied to analyze the statistical difference for multiple groups. (C–H) Student's t-test was applied to analyze the statistical difference for two groups.

Figure 8

Nesfatin-1 inhibited c-fos expression induced by fasting in arcuate nucleus. GHSR^+/+ mice were treated with tail vein injection of vehicle and nesfatin-1 after 12 h fasting. (A) C-fos expression in hypothalamic ARC of mice injected with vehicle. (B) C-fos expression in hypothalamic ARC of mice injected with nesfatin-1. N = 3/group.