From intestines to skeleton: The impact of incretin hormones on human bone

The human skeleton is a dynamic tissue that constantly undergoes remodeling through a coordinated microscopical process to adapt to changing mechanical and physiological needs and maintain strength. This process takes place in millions of small units at different skeletal sites, and include removal of old bone by osteoclasts, and formation and mineralization of new bone by osteoblasts. The whole-body activity of resorption and formation (bone turnover) can be measured by circulating biomarkers, referred to as bone turnover markers.
   Food intake is a potent regulator of bone turnover. When we eat, bone resorption is immediately inhibited while bone formation is sustained. This has led to the assumption that there exists an endocrine crosstalk between the intestines and skeleton, in which gastrointestinal hormones secreted during food intake mediate effects on bone tissue. Of particular interest are the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). While the main function of GLP-1 and GIP is to stimulate insulin secretion from the pancreas via interaction with their respective G-protein coupled receptors (GPCRs) in response to food intake, preclinical and clinical data indicate that GLP-1 and GIP acutely impair bone resorption and maintain or increase bone formation. Therefore, drugs mimicking GLP-1 (GLP-1 receptor agonists) and GIP (dual GIP-GLP-1 agonist), which are extensively used in the treatments of type 2 diabetes and obesity, may exert positive effects on bone. Therefore, the overall aim of this thesis was to evaluate the effects of GIP, GLP-1, and GLP-1 receptor agonist treatment on human bone resorption and formation in vitro and in vivo.

In Paper I, the aim was to identify knowledge gaps in the field of incretin hormones and human bone based on preclinical and clinical data on the effects of GIP, GLP-1, and GLP-1 receptor agonists on bone.

Based on findings in Paper I, the regulatory effects of incretin hormones seemed more profound on bone resorption than formation although data on incretin hormones and human osteoclasts in general were lacking. Therefore, the primary focus of the in vitro studies conducted in this thesis was aimed at human osteoclast differentiation and activity. In line with this, the aim of Paper II was to characterize the in vitro genetics of human osteoclast biology. This was done by analyzing the transcriptional changes that occur at different time points of osteoclast differentiation using RNA sequencing (RNAseq). A secondary aim of the paper was to identify novel regulators of osteoclast differentiation and activity. The study showed that the GIP receptor is expressed at early and late stages of human osteoclast differentiation. Furthermore, three GPCRs were identified as potential regulators of osteoclast differentiation and activity.

The primary aim of Paper III was to investigate if GIP inhibits human osteoclast activity. Extending the findings in Paper II, this study showed that human osteoclasts express a functional GIP receptor that activates multiple signaling pathways, which result in inhibition of human bone resorption. In addition, GIP did not alter osteoblast activity despite decreasing osteoclast activity, indicating that GIP uncouples human bone resorption from formation.

The aim of Paper IV was to investigate the effects of GLP-1 on human bone cell activity in vitro, specifically if GLP-1 inhibits human osteoclast activity. The study showed that GLP-1 increased osteoclast activity after 72 hours of exposure in vitro. This effect was reversed by pretreatment with a specific GLP-1 receptor antagonist, indicating direct effects of GLP-1 on osteoclasts. Based on confocal microscopy and RNAseq, GLP-1 receptor expression was not detected in osteoclasts. In addition, GLP-1 enhanced osteoblast differentiation and indirectly increased osteoblast activity via activation of osteoclasts. Combined, this study indicates that sustained exposure of GLP-1 has a differential effect on bone resorption compared to the acute effect.

In Paper V, the aim was to investigate if the GLP-1 receptor agonist semaglutide increases bone formation in individuals with increased fracture risk. This was a randomized clinical trial including 64 participants randomized to either semaglutide or placebo once weekly for 52 weeks. Based on analyses of changes in plasma levels of a circulating biomarker for bone formation (primary endpoint), this study did not support that semaglutide treatment increases bone formation. Rather, semaglutide associated with increased plasma levels of a circulating biomarker for bone resorption and decreased bone mineral density at the lumbar spine and hip. As semaglutide also induced a 9% weight loss, the skeletal effects were likely mediated by biological adaptation mechanisms to reduced load.

Collectively, the work in this thesis adds new insights to the preclinical effects of GIP and GLP-1 on human osteoclasts and osteoblasts in vitro, and the clinical effects of GLP-1 receptor agonist treatment on human bone in vivo. The work in this thesis has established procedures and ideas for future studies aimed to expand our knowledge on the crosstalk between incretin hormones and bone, and the clinical impact of treatment with drugs targeting incretin hormone receptors on skeletal health.