The amplification was conducted according to the instructions provided by the manufacturer. Bone remodeling consists of resorption by osteoclasts followed by formation by osteoblasts, and osteoclasts are a source of bone formation-stimulating factors. Here we utilize osteoclast ablation by denosumab (DMAb) and RNA-sequencing of bone biopsies from postmenopausal women to identify osteoclast-secreted factors suppressed by DMAb. Based on these analyses, are likely osteoclast-derived coupling factors in humans. Given the role of Dipeptidyl Peptidase-4 (DPP4) in glucose homeostasis, we further demonstrate that DMAb-treated participants have a significant reduction in circulating DPP4 and increase in Glucagon-like peptide (GLP)-1 levels as compared to the placebo-treated group, 10074-G5 and also?that type 2 diabetic patients treated with DMAb show significant reductions in HbA1c as compared to patients treated either with bisphosphonates or calcium and vitamin D. Thus, our results identify several coupling factors in humans and uncover osteoclast-derived DPP4 as a potential link between bone remodeling and energy metabolism. or and did not pass the 10074-G5 limit of detection in this RNA-seq dataset, possibly because osteoclast genes could be underrepresented in the absence of centrifugation to remove marrow elements. However, similar to the centrifuged bone, the whole bone gene analysis showed a significant correlation between DMAb-suppressed osteoblast and osteoclast genes in untreated postmenopausal women (Supplementary Fig.?2), providing 10074-G5 further confirmation of the validity of these findings. Open in a separate window Fig. 2 Correlation of osteoclast and osteoblast genes and secreted factors altered TNFAIP3 by DMAb. a Heat maps showing the osteoclast and osteoblast normalized gene expression in placebo and DMAb-treated participant bone biopsies. Normalized gene expression (CQN values) were ranked for each gene across the placebo and DMAb participant biopsies (are most likely to be osteoclast-specific factors downregulated by DMAb, and to be potentially involved in coupling osteoclasts and bone resorption to bone formation in humans. Open in a separate window Fig. 3 Identification of osteoclast-derived secreted factors involved in coupling.a Flow chart describing processing of the bone biopsy samples to select for osteocyte- and osteoblast-enriched fractions. b Overlap of DMAb-suppressed secreted factor genes in centrifuged bone vs. the osteocyte-enriched fraction (value was calculated by the KruskalCWallis test. Individual values are plotted with mean and error bars represent SD. b Bone marrow plasma DPP4 levels (assessed by the Olink Proteomics) correlate with osteoblast and osteoclast gene sets in the placebo participants; Spearmans correlation coefficient was used to determine strength of relationships 10074-G5 (mRNA in osteoclasts, but not other cell types. Staining for mRNA (red stain) was abundant in osteoclasts (OC) on eroded surfaces (ES) of cancellous bone and intracortical canals. Osteoblasts on osteoid surfaces (OS) and bone lining cells on quiescent surfaces (QS) showed no staining. Dotted lines represent separation of bone surfaces. Scale bars?=?50?m. e Changes in serum GLP-1 (top) and glucose and insulin (bottom) levels in the placebo- and DMAb-treated participants (% change from baseline, mRNA expression in bone. Consistent with our mRNA expression approach, transcript was evident in osteoclasts on the bone surface, but not in lining cells, osteoblasts, or osteocytes in human cancellous and cortical bone (Fig.?4d). We next sought to determine if the reduction in DPP4 by DMAb (Fig.?4c) had a functional effect to increase GLP-1 levels and impact glucose homeostasis. Plasma taken before and after treatment revealed a significant increase in total GLP-1 in DMAb participants (Fig.?4e). However, DMAb did not significantly alter plasma glucose-dependent insulinotropic peptide (GIP) (Supplementary Table?5), glucose, or insulin levels in this healthy (non-diabetic) cohort of postmenopausal women (Fig.?4e). In addition, changes in blood lipids (total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides) did not differ between the control and DMAb groups nor did changes in Homeostatic Model Assessment?of?Insulin Resistance (HOMA-IR) or HOMA-beta-cell function (HOMA-) (Supplementary Table?5). DMAb improves glucose homeostasis in a diabetic cohort Because the noted lack of effect on glucose 10074-G5 homeostasis may reflect a lack of impaired glucose metabolism (i.e., lack of metabolic syndrome or diabetes) in these healthy participants, we next assessed a group of diabetic patients treated clinically with DMAb vs. bisphosphonate or calcium plus vitamin D for.