Glucagon-like peptide-1 attenuates diabetes-associated osteoporosis in ZDF rat, possibly through the RAGE pathway

Abstract

Background: Diabetes-associated osteoporosis are partly caused by accumulation of advanced glycation endproducts (AGEs). Glucagon-like peptide-1 (GLP-1) has been shown to regulate bone turnover. Here we explore whether GLP-1 receptor agonist (GLP1RA) can have a beneficial effect on bone in diabetes by ameliorating AGEs.

Methods: In the present study, we evaluated the effects of the GLP-1 receptor agonist liraglutide, insulin and dipeptidyl peptidase-4 inhibitor saxagliptin on Zucker diabetic fatty rats. Meanwhile, we observed the effect of GLP-1 on AGEs-mediated osteoblast proliferation and differentiation and the signal pathway.

Results: Liraglutide prevented the deterioration of trabecular microarchitecture and enhanced bone strength. Moreover, it increased serum Alpl, Ocn and P1NP levels and decreased serum CTX. In vitro we confirmed that GLP-1 could attenuate AGEs-mediated damage in osteogenic proliferation and differentiation. Besides, GLP-1 down-regulated the ROS that caused by AGEs and the mRNA and protein expression of Rage .

Conclusions: Altogether, our findings suggest that GLP-1 receptor agonist promotes osteoblastogenesis and suppresses bone resorption on obese type 2 diabetic rats to a certain degree. The mechanism of these effects may be partly mediated by AGEs-RAGE-ROS pathway via the interaction with GLP-1 receptor.

Keywords: Advanced glycation endproducts; Diabetes; Glucagon-like peptide-1; Liraglutide; Osteoporosis.

Fig. 1

Metabolic parameters in ZDF rats. A Foodtake throughout the study period; B Body weight throughout the study period; C Body weight changes (at Week 21 from Week 11); D Blood glucose throughout the study period; E Blood glucose profiles during the OGTT at the end of 9 weeks treatment; F AUC of the blood glucose profiles during the OGTT at the end of 9 weeks treatment; G Blood glucose changes (at Week 21 from Week 11); H HbA1c at the end of 9 weeks treatment. 11-week-old ZDF rats were randomly assigned into four subgroups: treated with vehicle (ZDF); treated with insulin (INS); treated with saxagliptin (SAXA); treated with liraglutide (LIRA). OGTT, oral glucose tolerance test; HbA1c, glycated hemoglobin; AUC, area under curve. The Newman-Keuls Multiple Comprison Test was used, and (mean, SD) was displayed above the bar. ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001 vs. ZLC group; *P < 0.05, ** P < 0.01, *** P < 0.001 vs. ZDF group; ^#P < 0.05, ^## P < 0.01, ^### P < 0.001 vs. LIRA group

Fig. 2

Effects of 9 weeks treatment on serum and bone remodeling parameters. A-D Serum bone remodeling parameters; E-H mRNA levels of bone remodeling parameters in femur; (I) mRNA levels of Rage in femur. Ocn, osteocalcin; Alpl, bone alkaline phosphatase; P1NP, procollagen type 1 N-terminal peptide; CTX, C-terminal telopeptide of collagen type 1; Opn, osteopontin; Osx, Osterix; Rage, receptor for AGEs. Values are means ± SD (n = 7). The Newman-Keuls Multiple Comprison Test was used. ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001 vs. ZLC group; *P < 0.05, ** P < 0.01, *** P < 0.001 vs. ZDF group; ^#P < 0.05, ^## P < 0.01, ^### P < 0.001 vs. LIRA group

Fig. 3

Bone effect of ZDF rats after treatment of Liraglutide. A-I The distal femur of structural bone parameters analyzed by micro-CT; B Total bone mineral density (BMD); C trabecular BMD; D cortical BMD; E Bone volume per total volume (BV/TV); F Trabecular number (Tb.N); G Trabecular thickness (Tb.Th.); H Trabecular spacing (Tp.Sp.); I Structure model index; J Cortical thickness (Ct.Th.); K-P Femoral biomechanical structural properties in ZDF rats via three-point bending test, including max load, fracture load, ultimate displacement, yield displacement, stiffness, and total absorbed energy. Values are means ± SD (n = 7); The Newman-Keuls Multiple Comprison Test was used. ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001 vs. ZLC group; *P < 0.05, ** P < 0.01, *** P < 0.001 vs. ZDF group; ^#P < 0.05, ^## P < 0.01, ^### P < 0.001 vs. LIRA group

Fig. 4

Effect of GLP-1 on the AGEs-mediated damage in proliferation and differentiation of osteoblasts. A, B Glp1r protein was detected by immunofluorescence and western blot; C Alpl staining, which indicates the early stage of osteoblastogenesis was performed at the indicated time points; D Cell proliferation was measured by the absorbance at 570 mm with a MTT assay; E-H. The mRNA expression of osteogenic differentiation markers (Ocn, Alpl, Opg, Runx2) of osteoblasts assessed by real-time PCR. Ocn, osteocalcin; Alpl, Alkaline phosphatase, liver/bone/kidney; Opg, osteoprotegerin; Runx2, Runt-related transcription factor 2. Values are means ± SD from three independent experiments. The Newman-Keuls Multiple Comprison Test was used. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. AGEs group

Fig. 5

Effect of GLP-1 on the AGEs-mediated ROS and Rage expression. A, B Intracellular ROS generation was measured with the probe DCFH-DA, and visualized using a fluorescent microscope; C Immunofluorescence was used to detect Rage antibody (red fluorescence) coupled to secondary antibodies. Nuclei were stained with DAPI (blue fluorescence); D Rage Protein and mRNA relative levels in osteoblasts were detected by western blot analysis and PCR. Values are means ± SD from three independent experiments. The Newman-Keuls Multiple Comprison Test was used. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. AGEs group