Melatonin suppresses autophagy in type 2 diabetic osteoporosis

Abstract

Type 2 diabetes mellitus is often complicated by osteoporosis, a process which may involve osteoblast autophagy. As melatonin suppresses autophagy under certain conditions, we its investigated the effects on bone autophagy during diabetes. We first assessed different body parameters in a diabetic rat model treated with various concentrations of melatonin. Dynamic biomechanicalmeasurements, bone organization hard slice dyeing and micro-CT were used to observe the rat bone microstructure, and immunohistochemistry was used to determine levels of autophagy biomarkers. We also performed in vitro experiments on human fetal osteoblastic (hFOB1.19) cells cultured with high glucose, different concentrations of melatonin, and ERK pathway inhibitors. And we used Western blotting and immunofluorescence to measure the extent of osteogenesis and autophagy. We found that melatonin improved the bone microstructure in our rat diabetes model and reduced the level of autophagy(50 mg/kg was better than 100 mg/kg). Melatonin also enhanced osteogenesis and suppressed autophagy in osteoblasts cultured at high glucose levels (10 μM was better than 1 mM). This suggests melatonin may reduce the level of autophagy in osteoblasts and delay diabetes-induced osteoporosis by inhibiting the ERK signaling pathway.

Keywords: ERK; autophagy; melatonin; osteoblast; type 2 diabetes osteoporosis.

Figure 1. Model evaluation: in vivo experiments

Forty-five SD rats were used to establish a diabetes model group, and were further divided into the HMT group (n=15, 100 mg/kg melatonin), LMT group (n=15, 50 mg/kg melatonin), and T2DM group (n=15). In addition,15 non-diabetic SD rats were given an intraperitoneal injection of melatonin (75 mg/kg) as the MT group, and 15 non-diabetic SD rats were included in the control group. A. Weight analysis indicated that the model animals' weights were lower than those of normal animals at 4,8, and 12 weeks. There was no significant difference between the control and MT groups. B. The FBG levels of the model animals were always higher than those of normal animals. There was no significant difference between the control and MT groups. C. The ISI levels of the model animals were always lower than those of normal animals. There was no significant difference between the control and MT groups. n=15 per group. Data are means ± SD. *P < 0.05.

Figure 2. Effect of melatonin on bone microstructure

The results of the double-fluorescent labeling method at 12 weeks are shown. The BFR/BV values of the model animals were always lower than those of the normal animals. The BFR/BV values of the LMT and HMT groups were always higher than those of the T2DM group. The BFR/BV values of the LMT group were higher than those of the HMT group at 8 and 12 weeks, although the statistical significance was stronger at 12 weeks. There was no significant difference between the control and MT groups. The MAR values of the model animals were always lower than those of the normal animals. The MAR values of the LMT and HMT groups were always higher than those of the T2DM group. The MAR values of the LMT group were higher than those of the HMT group at 8 and 12 weeks, although the statistical significance was stronger rat 12 weeks. There was no significant difference between the control and MT groups. n=15 per group. Data are means ± SD. *P < 0.05 vs. control, #P<0.05 vs. T2DM group, !P<0.05 vs. HMT group.

Figure 3. Effect of melatonin on bone microstructure

A. Masson-Goldnertrichrome staining at 12 weeks. The Tb.Th and Tb.N were significantly lower in the T2DM group than in the control group. Tb.Th and Tb.N were significantly greater in the HMT and LMT groups than in the T2DM group, although greater improvement was seen in the LMT group than in the HMT Group. B. Micro-CT scanning at 12 weeks. The BMD values of the LMT and HMT groups were always higher than those of the T2DM group. The BMD values of the LMT group were higher than those of the HMT group at 8 and12 weeks, although the statistical significance was stronger at 12 weeks. There was no significant difference between the control and MT groups. The Tb.N of the LMT and HMT group was always higher than that of the T2DM group. LMT group was always higher than that of the HMT group at 8W, 12W. There was always no significant difference between control group and MT group. The Tb.Th of the LMT and HMT group was always higher than that of the T2DM group. LMT group was always higher than that of the HMT group at 8W, 12W. There was always no significant difference between control group and MT group. n=15 per group. Data are means ± SD. *P < 0.05 vs. control, #P<0.05 vs. T2DM group, !P<0.05 vs. HMT group.

Figure 4. Effect of melatonin on bone tissue autophagy

Through IHC staining, we observed the expression of LC3 and Beclin-1 at 12 weeks. LC3 and Beclin-1 protein expression was significantly higher in theT2DM, LMT and HMT groups than in the control group; the expression was lower in the HMT and LMT groups than in the T2DM group, and was lower in the LMT group than in the HMT group. There was no significant difference between the control and MT groups. Scale bars, 20μm. n=15 per group. Data are means ± SD. *P < 0.05 vs. control, #P<0.05 vs. T2DM group, !P<0.05 vs. HMT group.

Figure 5. High glucose treatment of hFOB 1.19 cells induced autophagy, which was blocked by 3-MA

A. Western blot showing Beclin-1 and LC3 protein levels in hFOB 1.19 cells treated with high glucose (HG, 4500mg/L) for 0, 6, 12, 24, 48, 72 or 96 h. n=10 per group. Data are means ± SD. *P < 0.05 vs. 0 h. B. TEM results at 72 h. The number of autophagic bodies was significantly greater in the HG group than in the control and 3-MA + HG groups, while there was no significant difference between the control and 3-MA + HG groups. Scale bars, 2μm. n=10 per group. Data are means ± SD. *P < 0.05 vs. control. C. IHC results of LC3 at 72 h. The number of LC3 puncta was significantly greater in the HG group than in the control and 3-MA + HG groups, while there was no significant difference between the control and 3-MA + HG groups. Scale bars, 2μm. n=10 per group. Data are means ± SD. *P < 0.05 vs. control.

Figure 6. Effect of HG on the expression of the autophagy-related proteinsATG5, LC3, and p62 in hFOB 1.19 cells

A. Western blot showing ATG5 protein levels in hFOB 1.19 cells. The expression of ATG5 was significantly lower in cells treated with ATG5 siRNA than in control cells. B. Western blot showing LC3-II protein levels in hFOB 1.19 cells treated with HG, rapamycin, ATG5 siRNA and 3-MA. C. Western blot showing p62 protein levels inhFOB1.19 cells treated with HG, rapamycin and bafilomycin. n=10 per group. Data are means ± SD. *P < 0.05.

Figure 7. Effects of melatonin on osteoblast autophagy and osteogenesis

A. Western blotting results at 72 h. The protein expression of LC3-II, Beclin-1 and p-ERK1/2 was always higher in the HG, 1 mM + HG, and 10 μM + HG groups than in the other groups. There were no significant differences between the control, 3-MA + HG and PD98059 + HG groups. The expression of these three proteins was lower in the 1 mM + HG and 10 μM + HG groups than in the HG group, and was lower in the 10 μM + HG group than in the 1 mM + HG group. The protein expression of p62, OCN and OPG was always lower in the HG, 1 mM + HG, and 10 μM + HG groups than in the other groups. There were no significant differences between the control, 3-MA + HG and PD98059 + HG groups. The expression of these three proteins was higher in the1 mM + HG and 10 μM + HG groups than in the HG group, and was higher in the 10 μM + HG group than in the 1 mM + HG group. n=10 per group. Data are means ± SD. *P < 0.05 vs. control, #P<0.05 vs. HG group, !P<0.05 vs. 1mM + HG group. B. Real-time PCR results at 72 h. The mRNA expression of OCN, OPG and ALP was always lower in the HG, 1 mM + HG, and 10 μM + HG groups than in the other groups. There were no significant differences between the control, 3-MA + HG and PD98059 + HG groups. The expression of these three genes was higher in the 1 mM + HG and 10 μM + HG groups than in the HG group, and was higher in the10 μM + HG group than in the 1 mM + HG group. n=10 per group. Data are means ± SD. *P < 0.05 vs. control, #P<0.05 vs. HG group.