Table 1.
Primers sequences used in the study.
Fig 1.
Effects of Liraglutide on the bone micro-architecture of the vertebra in OVX rats.
(A) Representative micro-CT images of the 5th lumbar vertebra. The upper row presents the coronal view, axial view and sagittal view in the different groups, while the lower row provides a three-dimensional visualization of the trabecular micro-architecture. (B) The trabecular micro-architecture parameters of the 5th lumbar vertebra were shown as: BV/TV, Tb.Th, Tb.N, trabecular BMD, and Tb.Sp. Eight-week-old female Wistar rats were subjected to a sham ovariectomy (SHAM, black bar) or ovariectomy surgery (OVX); then, 3 months after surgery when the rats had established osteoporosis, vehicle (OVX, white bar) or Liraglutide (OVX+L, red bar) were administered for 8 weeks. Values are expressed as the mean±SE; n = 6 rats per group. *, P< 0.05; **, P< 0.01, ***, P<0.001.
Fig 2.
Effects of Liraglutide treatment on the bone micro-architecture of the femur in OVX rats.
(A) Representative micro-CT images of the right femur. (B) The trabecular micro-architecture parameters of the distal end of the femur were shown as: BV/TV, Tb.Th, Tb.N, Tb.Sp, and trabecular BMD. The cortical parameters were also exhibited as the cortical BMD and cortical wall thickness. Eight-week-old female Wistar rats were subjected to a sham ovariectomy (SHAM, black bar) or ovariectomy surgery (OVX); then, 3 months after surgery when the rats had established osteoporosis, vehicle (OVX, white bar) or Liraglutide (OVX+L, red bar) were administered for 8 weeks. Values are expressed as the mean±SE; n = 6 rats per group.*, P<0.05; **, P<0.01; ***, P<0.001.
Fig 3.
Effects of Liraglutide on body weight and blood glucose levels in OVX rats.
The body weight (A) and random fed state blood glucose levels (B) were monitored weekly in vehicle-treated sham-operated Wistar rats (SHAM), vehicle-treated OVX rats (OVX) and Liraglutide-treated OVX rats (OVX+L) during 8 weeks of treatment with Liraglutide. Prior to euthanasia, an intraperitoneal glucose tolerance test (IPGTT) was performed. The glucose level and the area under curve (AUC) of the glucose tolerance in IPGTT (C, D) were also assessed. Values are expressed as the mean±SE; n = 6 rats per group.*, P<0.05; **, P< 0.01, ***, P<0.001; OVX vs. OVX+L.
Fig 4.
Effects of Liraglutide on adipogenesis and osteoblastogenesis of rat BMSCs.
Primary rat BMSCs were harvested from the femur and tibia bone marrow from 4-week-old male Wistar rats. After 3 passages, the rat BMSCs were induced to differentiate into adipocytes or osteoblasts in the presence or absence of 10 nM Liraglutide. (A) Following 14 days of treatment after adipogenic inducement, the cultures were fixed and stained with Oil red O, then visualized and photographed. (B) The amount of Oil Red O in the cells was quantified. (C) ALP staining, which indicates the early stage of osteoblastogenesis, and (D) Alizarin red staining, which represents the mineralization levels of later stages of osteoblastogenesis, was performed at the indicated time-points. (E) Alizarin red dye was analyzed. (F) PPARγ, a crucial gene promoting adipogenesis, was quantified using real-time PCR after 14 days of adipogenesis. (G. H. I) The expression patterns of osteoblastogenic differentiation markers (Runx2, ALP, Col-1) of rat BMSCs in response to 10 nM Liraglutide were assessed using real-time PCR. Relative expression levels of each gene were calculated using the 2-ΔΔCt method. The expression of GAPDH mRNA was used for reference. Values are expressed as the mean ±SE. *, P<0.05; **, P<0.01; ***, P<0.001.
Fig 5.
Effects of Liraglutide on adipogenesis and osteoblastogenesis of human BMSCs.
Primary human BMSCs were induced to differentiate into adipocytes or osteoblasts in the presence or absence of 10 nM Liraglutide. (A) Oil red O adipocyte staining was performed after 17 days of adipogenesis induction. (B) Total RNA was extracted and subjected to real-time PCR. The graph shows the mRNA expression levels of PPARγ at the indicated time-points. (C) ALP staining was performed after 7 days of osteogenesis induction. (D) Real-time PCR analysis was conducted to compare the expression levels of osteoblastogenesis-related genes in the vehicle-treated culture and Liraglutide-treated culture at the indicated time-points during osteoblastogenesis differentiation. Values are expressed as the mean±SE.*, P<0.05; **, P<0.01; ***, P<0.001.