Endocrine Abstracts

Gastric inhibitory polypeptide (GIP) is a post-prandially secreted gut hormone that acts upon the GIP-receptor (GIPR), to stimulate insulin secretion. Animal studies indicate that GIP influences bone remodelling, and in humans, GIP administration decreases levels of bone resorption markers. However, the mechanisms by which GIP influences resorption remain to be elucidated. Therefore, we investigated how GIP (10nM) affects bone cell activity using primary human osteoclasts, human osteoblasts, and osteoclast-osteoblast co-cultures. We confirmed that GIP reduces bone resorptive activity in osteoclast (P < 0.01) and osteoclast-osteoblast cultures (P < 0.01), and showed reduced tartrate-resistant acid phosphatase (TRAcP) activity in mature osteoclasts, demonstrating that GIP reduces osteoclast differentiation. Signalling was then assessed in cells (5-9 donors) stimulated with GIP for 30 minutes, and compared to vehicle-treated cells. Direct stimulation of GIPR on osteoclasts and osteoblasts was shown by GIP-mediated elevations in cAMP (P < 0.01), which was reversed by pre-treatment with a GIPR antagonist, GIP(3-30)NH₂. AlphaLISA assays showed phosphorylation of c-Src, Akt1/2/3 and NFκB p65, which regulate cytoskeletal reorganisation necessary for resorption, osteoclast survival and differentiation, were all reduced by GIP in osteoclasts (P < 0.01). Furthermore, confocal microscopy revealed that GIP-treated osteoclasts had reduced levels of nuclear phosphorylated-NFATc1 (P < 0.0001), a key modulator of osteoclast differentiation and osteoclast-specific gene expression. Pre-exposure of cells to inhibitors of GIPR and the cAMP-induced protein kinase A (PKA) prevented these GIP-mediated effects on p-Akt1/2/3, p-p65 and NFATc1. RNA-sequencing revealed downregulation of >30 genes involved in osteoclast function, including genes for cathepsin K, TRAcP5 and carbonic anhydrase-2, known regulators of osteoclast resorption. Moreover, >70 cell survival genes were differentially expressed by GIP treatment. Assessment of caspase-3/7 activity showed that GIP also increases osteoclast apoptosis (P < 0.01). In summary, GIPR activation on osteoclasts suppresses c-Src, Akt1/2/3, NFκB and NFATc1 signalling, leading to decreased bone resorption, likely by reduced expression of osteoclast-specific genes and increased apoptosis.