Type I beta-turn conformation is important for biological activity of the melanocyte-stimulating hormone analogues

In order to define which structure of alpha-melanocyte-stimulating hormone (MSH) analogues plays a critical role for ligand-receptor interaction and selectivity, we analysed receptor-binding and cAMP-generating activity in Chinese hamster ovary cell lines stably transfected with rMC3R and hMC4R, as well as the NMR structures of chemically synthesized alpha-MSH analogues. Compared with [Ahx4]alpha-MSH, the linear MTII designated as alpha-MSH-ND revealed a preference for the MC4R, whereas its IC50 and EC50 values were comparable to those of MTII reported previously. Truncation of Ahx4 and Asp5 of alpha-MSH-ND remarkably decreased the receptor-binding and cAMP-generating activity. Meanwhile, maximum cAMP-generating activity was observed at a higher concentration (10(-5) M) of alpha-MSH-ND(6-10), and MC4R preference was changed into MC3R preference. In contrast, [Gln6]alpha-MSH-ND(6-10) lost its cAMP-generating activity almost completely, even though it bound to both receptors. Whereas the solution conformation of alpha-MSH-ND revealed a stable type I beta-turn structure, [Gln6]alpha-MSH-ND(6-10) revealed a tight gamma-turn composed of Gln6-D-Phe7-Arg8. Replacement of the His6 residue of alpha-MSH-ND by Gln, Asn, Arg or Lys decreased not only the receptor binding, but also the cAMP-generating activity in both the MC3R and the MC4R. The structure of [Gln6]alpha-MSH-ND exhibited a stable type I' beta-turn comprising Asp5, Gln6, D-Phe7 and Arg8. [Lys6]alpha-MSH-ND showed a greatly reduced binding affinity and cAMP-generating activity with the loss of MC4R selectivity. In NMR studies, [Lys6]alpha-MSH-ND also demonstrated a gamma-turn conformation around Lys6-DPhe7-Arg8. From the above results, we conclude that a type I beta-turn conformation comprising the residues Asp5-His6-(D-Phe7)-Arg8 was important for receptor binding and activation, as well as the selectivity of MSH analogues.ope

Romosozumab in Postmenopausal Korean Women with Osteoporosis: A Randomized, Double-Blind, Placebo-Controlled Efficacy and Safety Study

Background: This phase 3 study evaluated the efficacy and safety of 6-month treatment with romosozumab in Korean postmenopausal women with osteoporosis. Methods: Sixty-seven postmenopausal women with osteoporosis (bone mineral density [BMD] T-scores ≤-2.5 at the lumbar spine, total hip, or femoral neck) were randomized (1:1) to receive monthly subcutaneous injections of romosozumab (210 mg; n=34) or placebo (n=33) for 6 months. Results: At month 6, the difference in the least square (LS) mean percent change from baseline in lumbar spine BMD (primary efficacy endpoint) between the romosozumab (9.5%) and placebo (-0.1%) groups was significant (9.6%; 95% confidence interval, 7.6 to 11.5; P<0.001). The difference in the LS mean percent change from baseline was also significant for total hip and femoral neck BMD (secondary efficacy endpoints). After treatment with romosozumab, the percent change from baseline in procollagen type 1 N-terminal propeptide transiently increased at months 1 and 3, while that in C-terminal telopeptide of type 1 collagen showed a sustained decrease. No events of cancer, hypocalcemia, injection site reaction, positively adjudicated atypical femoral fracture or osteonecrosis of the jaw, or positively adjudicated serious cardiovascular adverse events were observed. At month 9, 17.6% and 2.9% of patients in the romosozumab group developed binding and neutralizing antibodies, respectively. Conclusion: Treatment with romosozumab for 6 months was well tolerated and significantly increased lumbar spine, total hip, and femoral neck BMD compared with placebo in Korean postmenopausal women with osteoporosis (ClinicalTrials.gov identifier NCT02791516).ope

The Role of Nuclear Factor-E2-Related Factor 1 in the Oxidative Stress Response in MC3T3-E1 Osteoblastic Cells

BACKGROUND: Reactive oxygen species (ROS) and antioxidants are associated with maintenance of cellular function and metabolism. Nuclear factor-E2-related factor 1 (NFE2L1, Nrf1) is known to regulate the expression of a number of genes involved in oxidative stress and inflammation. The purpose of this study was to examine the effects of NFE2L1 on the response to oxidative stress in osteoblastic MC3T3-E1 cells. METHODS: The murine calvaria-derived MC3T3-E1 cell line was exposed to lipopolysaccharide (LPS) for oxidative stress induction. NFE2L1 effects were evaluated using small interfering RNA (siRNA) for NFE2L1 mRNA. ROS generation and the levels of known antioxidant enzyme genes were assayed. RESULTS: NFE2L1 expression was significantly increased 2.4-fold compared to the control group at 10 μg/mL LPS in MC3T3-E1 cells (P<0.05). LPS increased formation of intracellular ROS in MC3T3-E1 cells. NFE2L1 knockdown led to an additional increase of ROS (20%) in the group transfected with NFE2L1 siRNA compared with the control group under LPS stimulation (P<0.05). RNA interference of NFE2L1 suppressed the expression of antioxidant genes including metallothionein 2, glutamatecysteine ligase catalytic subunit, and glutathione peroxidase 1 in LPS-treated MC3T3-E1 cells. CONCLUSION: Our results suggest that NFE2L1 may have a distinct role in the regulation of antioxidant enzymes under inflammation-induced oxidative stress in MC3T3-E1 osteoblastic cells.ope

The Effects of a High Fat Diet Containing Diacylglycerol on Bone in C57BL/6J Mice

PURPOSE: In epidemiologic and animal studies, a high fat diet (HFD) has been shown to be associated with lower bone mineral density (BMD) and a higher risk of osteoporotic fractures. Meanwhile, consuming a HFD containing diacylglycerol (DAG) instead of triacylglycerol (TAG) is known to offer metabolically beneficial effects of reductions in body weight and abdominal fat. The purpose of this study was to investigate the effects of a HFD containing DAG (HFD-DAG) on bone in mice. MATERIALS AND METHODS: Four-week-old male C57BL/6J mice (n=39) were divided into three weight-matched groups based on diet type: a chow diet group, a HFD containing TAG (HFD-TAG) group, and a HFD-DAG group. After 20 weeks, body composition and bone microstructure were analyzed using dual energy X-ray absorptiometry and micro-computed tomography. Reverse transcription-polymerase chain reaction (PCR) and real-time PCR of bone marrow cells were performed to investigate the expressions of transcription factors for osteogenesis or adipogenesis. RESULTS: The HFD-DAG group exhibited lower body weight, higher BMD, and superior microstructural bone parameters, compared to the HFD-TAG group. The HFD-DAG group showed increased expression of Runx2 and decreased expression of PPARgamma in bone marrow cells, compared to the HFD-TAG group. The HFD-DAG group also had lower levels of plasma glucose, insulin, total cholesterol, and triglyceride than the HFD-TAG group. CONCLUSION: Compared to HFD-TAG, HFD-DAG showed beneficial effects on bone and bone metabolism in C57BL/6J mice.ope

Identification of domains directing specificity of coupling to G-proteins for the melanocortin MC3 and MC4 receptors

The melanocortin receptors, MC3R and MC4R, are G protein-coupled receptors that are involved in regulating energy homeostasis. Using a luciferase reporter gene under the transcriptional control of a cAMP- responsive element (CRE), the coupling efficiency of the MC4R and MC3R to G-proteins was previously shown to be different. MC4R exhibited only 30–50% of the maximum activity induced by MC3R. To assess the role of the different MC3R and MC4R domains in G-protein coupling, several chimeric MC3R/MC4R receptors were constructed. The relative luciferase activities, which were assessed after transfecting the chimeric receptors into HEK 293T cells, showed that the i3 (3rd intracellular) loop domain has an essential role in the differential signaling of MC3R and MC4R. To reveal which amino acid residue was involved in the MC4R-specific signaling in the i3 loop, a series of mutant MC4Rs was constructed. Reporter gene analysis showed that single mutations of Arg220 to Ala and Thr232 to either Val or Ala increased the relative luciferase activities, which suggests that these specific amino acids, Arg220 and Thr232, in the i3 loop of MC4R play crucial roles in G-protein coupling and the subtype-specific signaling pathways. An examination of the inositol phosphate (IP) levels in the cells transfected with either MC3R or MC4R after being exposed to the melanocortin peptides revealed significant stimulation of IP production by MC3R but no detectable increase in IP production was observed by MC4R. Furthermore, none of the MC4R mutants displayed melanocortin peptide-stimulated IP production. Overall, this study demonstrated that MC3R and MC4R have distinct signaling in either the cAMP- or the inositol phospholipid-mediated pathway with different conformational requirements. Melanocortins are peptide hormones that are derived from the precursor peptide pro-opiomelanocortin, by a series of proteolytic cleavages (1). The melanocortins are known to have a broad spectrum of physiological actions, which include the regulation of melanocyte pigmentation (2), thermoregulation (3), obesity (4), control of the cardiovascular system (5), and learning and memory (6), and have also been found to have immunomodulatory effects (7). These hormones mediate their effects through G protein-coupled receptors by stimulating adenylate cyclase (8). To date five melanocortin receptor subtypes, with different patterns of tissue expression in the brain and peripheral body, have been cloned and characterized (8-12). It has been reported that the activation of melanocortin 4 receptor (MC4R)1 by α-melanocyte-stimulating hormone (MSH) increases energy expenditure and decreases food intake. Moreover, the genetic disruption of MC4R was found to cause obesity in mice (13). Recent experiments in MC3R-null mice indicate that the inactivation of MC3R results in increased fat mass and reduced body mass, despite the fact that the animals were hypophagic and maintained normal metabolic rates (14, 15). These results suggest the nonredundancy of the MC3R and MC4R melanocortin receptors in the regulation of energy homeostasis (14, 15). In previous studies, we and others have demonstrated that heterologously expressed MC3R and MC4R are coupled to the cAMP pathway. We analyzed several α-MSH analogues upon stimulation of MC3R and MC4R using a CRE (cAMP responsive element)-mediated reporter gene transcription activity assay (16), and were able to show that both MC3R and MC4R, expressed in human cell line HEK 293T, stimulate transcription when stimulated using different analogues of melanocortin at different levels. Our previous studies have shown that MC3R and MC4R may have differential efficiencies and/or modes of signaling in terms of G-protein coupling, in addition to their specific ligand-receptor interactions, which can specify subtype-specific signaling pathwaysin vivo (16). The role of the third intracellular (i3) loop in G-protein coupling specificity has been investigated extensively for many seven-transmembrane domain receptors, including adrenergic, serotonergic, muscarinic, and dopaminergic receptors (17-21). For example, swapping experiments performed upon two different G protein-coupled receptors demonstrated the importance of this loop in selective coupling to specific G-protein/effector systems (22, 23). To identify the role(s) of the third intracellular loop of the MC3R and MC4R receptors in terms of G-protein coupling specificity and receptor activation, several chimeras were constructed and characterized. We used the CRE-luciferase reporter gene assay to score the efficacy of receptor-G proteins coupling (24, 25). In parallel, amino acid mutations were generated in the third intracytoplasmic loop of MC4R to identify the residues that play a role in G-protein coupling. These mutant receptors were examined in terms of their abilities to bind melanocortin receptor-specific ligands and with respect to signal transduction at the cAMP level.ope

Determinants of Bone Mass and Insulin Resistance in Korean Postmenopausal Women: Muscle Area, Strength, or Composition?

PURPOSE: Muscle mass, strength, and composition determine muscle quantity and quality. However, data on muscle properties in relation to bone mass or insulin resistance are limited in Asian populations. This study aimed to investigate the relative importance of muscle measurements in regards to their relationship with lower bone mass and insulin resistance. MATERIALS AND METHODS: In this study, 192 postmenopausal women (age, 72.39±6.07 years) were enrolled. We measured muscle cross-sectional area (CSA) and attenuation at the gluteus maximus and quadriceps muscles through quantitative computed tomography. Muscle strength and physical performance were evaluated with the hand grip test and Short Physical Performance Battery (SPPB). Pearson correlation analysis and linear regression were performed to evaluate the relationship between muscle properties and homeostatic model assessment-insulin resistance (HOMA-IR) or bone mineral density (BMD). RESULTS: Muscle CSA, hand grip strength, and SPPB score held positive correlations with spine and hip BMDs, but not with insulin resistance. In contrast, muscle attenuation of the gluteus maximus or quadriceps was inversely related to HOMA-IR (r=-0.194, p=0.018 and r=-0.292, p<0.001, respectively), but not BMD. Compared with the control group, muscle CSA was significantly decreased in patients with osteoporosis; however, decreased muscle attenuation, indicating high fat infiltration, was found only in patients with diabetes. CONCLUSION: Muscle mass, strength, and physical performance were associated with low bone mass, and accumulation of intramuscular fat, a histological hallmark of persistently damaged muscles, may play a major role in the development of insulin resistance in Korean postmenopausal women.ope

Efficacy and safety of vitamin D3 B.O.N intramuscular injection in Korean adults with vitamin D deficiency

Objective : There has been no prospective study that examined intramuscular injection of high-dose vitamin D in Korean adults. The aim of this study was to assess the efficacy and safety of high-dose vitamin D3 after intramuscular injection in Korean adults with vitamin D deficiency. Method : This study was a 24-week, prospective, multicenter, randomized, double-blind, placebo-controlled trial. A total of 84 subjects ≥19 and <65 years of age were randomly allocated to either the vitamin D3 or placebo group in a 2:1 ratio. After randomization, a single injection of plain vitamin D3 200,000 IU or placebo was intramuscularly administered. If serum 25-hydroxyvitamin D (25[OH]D) concentrations were <30 ng/mL on week 12 or thereafter, a repeat injection was administered. Results : After a single intramuscular injection of vitamin D3 to adults with vitamin D deficiency, the proportion of subjects with serum 25(OH)D concentrations ≥30 ng/mL within 12 weeks was 46.4% in the vitamin D3 group and 3.6% in the placebo group (p < 0.0001). The proportion of subjects with serum 25(OH)D concentrations ≥30 ng/mL within 24 weeks was 73.2% in the vitamin D3 group and 3.6% in the placebo group (p < 0.0001). Mean change in serum 25(OH)D concentrations at weeks 12 and 24 after vitamin D3 injection was 12.8 ± 8.1 and 21.5 ± 8.1 ng/mL, respectively, in the vitamin D3 group, with no significant changes in the placebo group. Serum parathyroid hormone concentrations showed a significant decrease in the vitamin D3 group but no change in the placebo group. Conclusion : Intramuscular injection of vitamin D3 200,000 IU was superior to placebo in terms of its impact on serum 25(OH)D concentrations, and is considered to be safe and effective in Korean adults with vitamin D deficiency.ope

Reversible extraskeletal uptake of bone scanning in primary hyperparathyroidism

Metastatic calcification within soft tissue, such as the lung and stomach, is associated with hyperparathyroidism, chronic renal failure, hemodialysis, metastatic neoplasm and hypervitaminosis D. Bone scanning agents variably accumulate within these extraskeletal metastatic calcifications. We report a patient with primary hyperparathyroidism whose bone scan revealed abnormal uptake in the liver, lung, stomach and parathyroid gland followed by complete resolution of extraskeletal uptake less than 1 wk after parathyroidectomy.ope

Cyclized Oligopeptide Targeting LRP5/6-DKK1 Interaction Reduces the Growth of Tumor Burden in a Multiple Myeloma Mouse Model

PURPOSE: Dickkopf 1 (DKK1) has been extensively investigated in mouse models of multiple myeloma, which results in osteolytic bone lesions. Elevated DKK1 levels in bone marrow plasma and serum inhibit the differentiation of osteoblast precursors. Present pharmaceutical approaches to target bone lesions are limited to antiresorptive agents. In this study, we developed a cyclized oligopeptide against DKK1-low density lipoprotein receptor-related protein (LRP) 5/6 interaction and tested the effects of the oligopeptide on tumor burden. MATERIALS AND METHODS: A cyclized oligopeptide based on DKK1-LRP5/6 interactions was synthesized chemically, and its nuclear magnetic resonance structure was assessed. Luciferase reporter assay and mRNA expressions of osteoblast markers were evaluated after oligopeptide treatment. MOPC315.BM.Luc cells were injected into the tail vein of mice, after which cyclized oligopeptide was delivered subcutaneously 6 days a week for 4 weeks. RESULTS: The cyclized oligopeptide containing NXI motif bound to the E1 domain of LRP5/6 effectively on surface plasmon resonance analysis. It abrogated the Wnt-β-catenin signaling inhibited by DKK1, but not by sclerostin, dose dependently. RT-PCR and alkaline phosphatase staining showed increased expressions of osteoblast markers according to the treatment concentrations. Bioluminescence images showed that the treatment of cyclized oligopeptide reduced tumor burden more in oligopeptide treated group than in the vehicle group. CONCLUSION: The cyclized oligopeptide reported here may be another option for the treatment of tumor burden in multiple myeloma.ope

Identification and Validation of the Relationship of the Anabolic Effect of Parathyroid Hormone with the Wnt/β-catenin Canonical Pathway

Background It has been well established that daily injections of low dose parathyroid hormone (PTH) increase bone mass in animals and humans. However, the precise mechanisms by which PTH exerts its anabolic action on bone are incompletely understood. The canonical Wnt-β-catenin signaling pathway has recently been demonstrated to have an important role in bone cell function. In the present study, we have examined the interaction between the PTH and Wnt signaling pathways in mouse osteoblastic MC3T3-E1 cells. Methods & Results MC3T3-E1 cells were treated with 0.01?0.84 µM recombinant PTH. β-catenin expression was significantly increased after 30 minutes of exposure to PTH and reached a maximum 2.7 fold increase at 1 hr and expression then faded at 6 hrs. In addition, treatment with PTH increased nuclear accumulation of activated β-catenin; the ratio between the nuclear to cytoplasmic protein was more than three fold at 30 minutes and beyond. Moreover, PTH stimulated T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter gene activity in MC3T3-E1 cells. Confocal microscopy revealed nuclear translocation of β-catenin by PTH as compared with a glycogen synthase kinase-3β (GSK-3β) inhibitor. Conclusion These results suggest that the anabolic mechanism of PTH might be partially associated with the Wnt-canonical pathway. The appropriate target of another anabolic agent should be determined through further studies of this pathway.ope