Ghrelin, through action on its receptor, GH secretagogue receptor type 1a (GHS-R1a), exerts a variety of metabolic functions including stimulation of appetite and weight gain and suppression of insulin secretion. In the present study, we examined the effects of novel small-molecule GHS-R1a antagonists on insulin secretion, glucose tolerance, and weight loss. Ghrelin dose-dependently suppressed insulin secretion from dispersed rat islets. This effect was fully blocked by a GHS-R1a antagonist. Consistent with this observation, a single oral dose of a GHS-R1a antagonist improved glucose homeostasis in an ip glucose tolerance test in rat. Improvement in glucose tolerance was attributed to increased insulin secretion. Daily oral administration of a GHS-R1a antagonist to diet-induced obese mice led to reduced food intake and weight loss (up to 15%) due to selective loss of fat mass. Pair-feeding experiments indicated that weight loss was largely a consequence of reduced food intake. The impact of a GHS-R1a antagonist on gastric emptying was also examined. Although the GHS-R1a antagonist modestly delayed gastric emptying at the highest dose tested (10 mg/kg), delayed gastric emptying does not appear to be a requirement for weight loss because lower doses produced weight loss without an effect on gastric emptying. Consistent with the hypothesis that ghrelin regulates feeding centrally, the anorexigenic effects of potent GHS-R1a antagonists in mice appeared to correspond with their brain exposure. These observations demonstrate that GHS-R1a antagonists have the potential to improve the diabetic condition by promoting glucose-dependent insulin secretion and promoting weight loss.

Edited by Alex Toker Enhanced osteoclast-mediated bone resorption and diminished formation may promote bone loss. Pleckstrin homology (PH) domain and leucine-rich repeat protein phosphatase 1 (Phlpp1) regulates protein kinase C (PKC) and other proteins in the control of bone mass. Germline Phlpp1 deficiency reduces bone volume, but the mechanisms remain unknown. Here, we found that conditional Phlpp1 deletion in murine osteoclasts increases their numbers, but also enhances bone mass. Despite elevating osteoclasts, Phlpp1 deficiency did not increase serum markers of bone resorption, but elevated serum markers of bone formation. These results suggest that Phlpp1 suppresses osteoclast formation and production of paracrine factors controlling osteoblast activity. Phlpp1 deficiency elevated osteoclast numbers and size in ex vivo osteoclastogenesis assays, accompanied by enhanced expression of proto-oncogene C-Fms (C-Fms) and hyper-responsiveness to macrophage colony-stimulating factor (M-CSF) in bone marrow macrophages. Although Phlpp1 deficiency increased TRAP ؉ cell numbers, it suppressed actinring formation and bone resorption in these assays. We observed that Phlpp1 deficiency increases activity of PKC, a PKC isoform controlling cell polarity, and that addition of a PKC pseudosubstrate restores osteoclastogenesis and bone resorption of Phlpp1-deficient osteoclasts. Moreover, Phlpp1 deficiency increased expression of the bone-coupling factor collagen triple helix repeat-containing 1 (Cthrc1). Conditioned growth medium derived from Phlpp1-deficient osteoclasts enhanced mineralization of ex vivo osteoblast cultures, an effect that was abrogated by Cthrc1 knockdown. In summary, Phlpp1 critically regulates osteoclast numbers, and Phlpp1 deficiency enhances bone mass despite higher osteoclast numbers because it apparently disrupts PKC activity, cell polarity, and bone resorption and increases secretion of bone-forming Cthrc1.

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Prior studies demonstrated that deletion of the protein phosphatase Phlpp1 in Ctsk-Cre expressing cells enhances bone mass, characterized by diminished osteoclast activity and increased coupling to bone formation. Due to non-specific expression of Ctsk-Cre, the definitive mechanism for this observation was unclear. To further define the role of bone resorbing osteoclasts, we performed ovariectomy (Ovx) and Sham surgeries on Phlpp1 cKOCtsk and WT mice. Micro-CT analyses confirmed enhanced bone mass of Phlpp1 cKOCtsk Sham females. In contrast, Ovx induced bone loss in both groups, with no difference between Phlpp1 cKOCtsk and WT mice. Histomorphometry demonstrated that Ovx mice lacked differences in osteoclasts per bone surface, suggesting that estradiol (E2) is required for Phlpp1 deficiency to have an effect. We performed high throughput unbiased transcriptional profiling of Phlpp1 cKOCtsk osteoclasts and identified 290 differentially expressed genes. By cross-referencing these differentially expressed genes with all estrogen response element (ERE) containing genes, we identified IGFBP4 as potential estrogen-dependent target of Phlpp1. E2 induced PHLPP1 expression, but reduced IGFBP4 levels. Moreover, genetic deletion or chemical inhibition of Phlpp1 was correlated with IGFBP4 levels. We then assessed IGFBP4 expression by osteoclasts in vivo within intact 12-week-old females. Modest IGFBP4 immunohistochemical staining of TRAP+ osteoclasts within WT females was observed. In contrast, TRAP+ bone lining cells within intact Phlpp1 cKOCtsk females robustly expressed IGFBP4, but levels were diminished within TRAP+ bone lining cells following Ovx. These results demonstrate that effects of Phlpp1 conditional deficiency are lost following Ovx, potentially due to estrogen-dependent regulation of IGFBP4.

Prior work demonstrated that Phlpp1 deficiency alters trabecular bone mass and enhances M-CSF responsiveness, but the cell types and requirement of Phlpp1 for this effect were unclear. To understand the function of Phlpp1 within myeloid lineage cells, we crossed Phlpp1 floxed mice with mice harboring LysM-Cre. Micro-computed tomography of the distal femur of 12-week-old mice revealed a 30% increase in bone volume per total volume of Phlpp1 female conditional knockouts, but we did not observe significant changes within male Phlpp1 cKOLysM mice. Bone histomorphmetry of the proximal tibia further revealed that Phlpp1 cKOLysM females exhibited elevated osteoclast numbers, but conversely had reduced levels of serum markers of bone resorption as compared to littermate controls. Osteoblast number and serum markers of bone formation were unchanged. In vitro assays confirmed that Phlpp1 ablation enhanced osteoclast number and area, but limited bone resorption. Additionally, reconstitution with exogenous Phlpp1 suppressed osteoclast numbers. Dose response assays demonstrated that Phlpp1−/− cells are more responsive to M-CSF, but reconstitution with Phlpp1 abrogated this effect. Furthermore, small molecule-mediated Phlpp inhibition enhanced osteoclast numbers and size. Enhanced phosphorylation of Phlpp substrates—including Akt, ERK1/2, and PKCζ—accompanied these observations. In contrast, actin cytoskeleton disruption occurred within Phlpp inhibitor treated osteoclasts. Moreover, Phlpp inhibition reduced resorption of cells cultured on bovine bone slices in vitro. Our results demonstrate that Phlpp1 deficiency within myeloid lineage cells enhances bone mass by limiting bone resorption while leaving osteoclast numbers intact; moreover, we show that Phlpp1 represses osteoclastogenesis and controls responses to M-CSF.