Glucagon-Like Peptide-1 (GLP-1) Receptor Agonist Liraglutide Alters Bone Marrow Exosome-Mediated miRNA Signal Pathways in Ovariectomized Rats with Type 2 Diabetes

Abstract

BACKGROUND Compared with normal postmenopausal women, estrogen deficiency and hyperglycemia in postmenopausal women with type 2 diabetes (T2DM) lead to more severe bone property degradation. Liraglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist, has been reported to improve bone condition among people with T2DM but the precise mechanisms remain unclear. Exosomes work as mediators in cell-to-cell communication, delivering functional miRNAs between cells. We aimed to explore the role of exosomes in T2DM-related bone metabolic disorders and the bone protective mechanisms of liraglutide. MATERIAL AND METHODS We made comparative analyses of bone marrow-derived exosomal miRNAs from ovariectomized (OVX) control rats, OVX + T2DM rats, and OVX + T2DM + liraglutide-treated rats. miRNA profiles were generated using high-throughput sequencing. Target gene prediction and pathway analysis were performed to investigate the signal pathway alterations. Three miRNAs were randomly chosen to validate their absolute expression levels by real-time quantitative PCR. RESULTS Bone marrow-derived exosomal miRNAs were different with respect to miRNA numbers, species, and expression levels. miRNA spectra varied under T2DM condition and after liraglutide treatment. By bioinformatics analysis, we found T2DM and liraglutide administration lead to significant changes in exosomal miRNAs which targeted to insulin secretion and insulin-signaling pathway. Wnt signaling pathway alteration was the critical point regarding bone metabolism. CONCLUSIONS Our findings show the selective packaging of functional miRNA cargoes into exosomes due to T2DM and liraglutide treatment. Bone marrow exosome-mediated Wnt signaling pathway alteration may play a part in the bone protective effect of liraglutide.

Figure 1

Blood glucose measurement during this study. ** Means p<0.01 vs. OVX group, ^&& means p<0.01 vs. LIR group. OVX – ovariectomized; DM – diabetes mellitus; LIR – liraglutide.

Figure 2

Characterization of exosomes. (A) Membrane markers CD63 and CD9 by Western blot. (B) Typical cup-rounded phenotype under transmission electron microscope. Scale bar=100 nm.

Figure 3

Exosomes images observed by atomic force microscopy. The topography of a 5×5 μm sample area was depicted. (A) Exosome images in 2-dimension. (B) Exosome images in 3-dimension. The maximum altitude of exosomes in this microscopic field was about 23.5 nm. (C) Peak force error images. In peak force error images, microstructure and nanomechanical images of the exosomes can be obtained simultaneously at the same area. Scale bar=5 μm.

Figure 4

Number of overlapping miRNA species by pairwise comparison (A–C). Ratio of overlapping miRNA reads (D–F). Ratio of overlapping miRNA reads=number of overlapping miRNA reads/total number of miRNA reads.

Figure 5

Wnt/β-catenin pathway. Three differentially expressed miRNAs (including let-7c-2-3p, let-7a-1-3p, and miR-322-3p) were predicted to participate in the Wnt/β-catenin pathway by regulating their potential target genes.