[Gly²]-GLP-2, But Not Glucagon or [D-Ala²]-GLP-1, Controls Collagen Crosslinking in Murine Osteoblast Cultures

Abstract

Bone tissue is organized at the molecular level to resist fracture with the minimum of bone material. This implies that several modifications of the extracellular matrix, including enzymatic collagen crosslinking, take place. We previously highlighted the role of several gut hormones in enhancing collagen maturity and bone strength. The present study investigated the effect of proglucagon-derived peptides on osteoblast-mediated collagen post-processing. Briefly, MC3T3-E1 murine osteoblasts were cultured in the presence of glucagon (GCG), [D-Ala²]-glucagon-like peptide-1 ([D-Ala²]-GLP-1), and [Gly²]-glucagon-like peptide-2 ([Gly²]-GLP-2). Gut hormone receptor expression at the mRNA and protein levels were investigated by qPCR and Western blot. Extent of collagen postprocessing was examined by Fourier transform infrared microspectroscopy. GCG and GLP-1 receptors were not evidenced in osteoblast cells at the mRNA and protein levels. However, it is not clear whether the known GLP-2 receptor is expressed. Nevertheless, administration of [Gly²]-GLP-2, but not GCG or [D-Ala²]-GLP-1, led to a dose-dependent increase in collagen maturity and an acceleration of collagen post-processing. This mechanism was dependent on adenylyl cyclase activation. In conclusion, the present study highlighted a direct effect of [Gly²]-GLP-2 to enhance collagen post-processing and crosslinking maturation in murine osteoblast cultures. Whether this effect is translatable to human osteoblasts remains to be elucidated.

Development of a first-in-class unimolecular dual GIP/GLP-2 analogue, GL-0001, for the treatment of bone fragility

“…As a proof of concept, and to follow our previous work on first generation analogues (14,15) , we co-administered the GIP and GLP-2 analogues, [D-Ala 2 ]GIP 1-30 and [Gly 2 ]GLP-2, in murine MC3T3-E1 osteoblast cultures at a concentration of 100 pM. This concentration was previously found to exhibit a pronounced biological response (14) .…”

Development of a First-in-Class Unimolecular Dual GIP/GLP-2 Analogue, GL-0001, for the Treatment of Bone Fragility

“…Fourteen days later, osteoblast cultures were either fixed in absolute ethanol, scrapped off the culture dish and transferred onto BaF2 windows for evaluation of collagen maturity or prepared for gene expression. For mechanistic investigations, administration of 50 µM 2',5'-DDA (15) and 0.25 mM bAPN (26) were added in the differentiation medium and replenished every second day. For silencing experiments, at day 7 of differentiation studies, osteoblasts were transfected with 50 nM of siRNA targeting murine GIPr (assay ID s233873), murine GLP-2r (assay ID s211996), murine lysyl oxidase (assay ID s69290) or a control scrambled siRNA (ref 4390843) using lipofectamine RNAimax.…”

“…For collagen crosslink analysis in osteoblast cultures, extracellular matrix was prepared as already reported (15) . Spectral analysis was performed using a Bruker Hyperion 3000 infrared microscope coupled to a Bruker Vertex 70 spectrometer and the standard single element MCT detector.…”

“…As a proof of concept, and to follow our previous work on first generation analogues (14,15) , we co-administered the GIP and GLP-2 analogues, [D-Ala²]GIP1-30 and [Gly²]GLP-2, in murine MC3T3-E1 osteoblast cultures at a concentration of 100 pM. This concentration was previously found to exhibit a pronounced biological response (14) .…”

“…The idea of selectively targeting bone ECM material properties to improve bone strength, rather than directly addressing low bone mass, has emerged as a result of pioneering works with two endogenous gut hormones, named GIP and GLP-2. Indeed, these peptides maintain optimal bone ECM material properties, through a cAMPdependent pathway (13)(14)(15)(16) . This concept was also reinforced by the use of first generation GIPr-and GLP-2r-specific analogues in several mice models of bone fragility (17)(18)(19)(20)(21) .…”