The effects of time-restricted eating and weight loss on bone metabolism and health: a 6-month randomized controlled trial.

OBJECTIVE: This study explored the impact of time-restricted eating (TRE) versus standard dietary advice (SDA) on bone health. METHODS: Adults with ≥1 component of metabolic syndrome were randomized to TRE (ad libitum eating within 12 hours) or SDA (food pyramid brochure). Bone turnover markers and bone mineral content/density by dual energy x-ray absorptiometry were assessed at baseline and 6-month follow-up. Statistical analyses were performed in the total population and by weight loss response. RESULTS: In the total population (n = 42, 76% women, median age 47 years [IQR: 31-52]), there were no between-group differences (TRE vs. SDA) in any bone parameter. Among weight loss responders (≥0.6 kg weight loss), the bone resorption marker ß-carboxyterminal telopeptide of type I collagen tended to decrease after TRE but increase after SDA (between-group differences p = 0.041), whereas changes in the bone formation marker procollagen type I N-propeptide did not differ between groups. Total body bone mineral content decreased after SDA (p = 0.028) but remained unchanged after TRE (p = 0.31) in weight loss responders (between-group differences p = 0.028). Among nonresponders (<0.6 kg weight loss), there were no between-group differences in bone outcomes. CONCLUSIONS: TRE had no detrimental impact on bone health, whereas, when weight loss occurred, it was associated with some bone-sparing effects compared with SDA.

Regeneration of Craniofacial Bone Induced by Periosteal Pumping.

A variety of surgical techniques and tissue engineering strategies utilizing osteogenic potential of the periosteum have been developed for the repair of extended bone deficiencies. The aim of the present study was to assess the impact of an alternating protocol of periosteal distraction osteogenesis (PDO) on bone regeneration in an intraoral model. Eight adult, male Beagle dogs were used for the study. Two distraction devices were placed on each side of the mandible. After a 7-day latency period, distraction devices in all animals were manipulated at the rate of 0.5 mm for a total of 8 days. The pumping protocol in two test groups proceeded twice daily by alternating activation with relaxation. In the periosteal pumping/distraction (PPDO) group, the distraction screws were activated two times (at 12 and 24 h) and then turned back (at 36 h), and in the periosteal pumping (PP) group repeatedly activated and turned back (at 12 h). In the PDO group, only activation was performed once daily (positive control). Devices were left inactivated in the negative control (NC) group. The samples were harvested after 8 weeks of consolidation period and investigated by micro-CT and histological analysis. New mature, lamellar bone was formed over the pristine bone in all groups. PPDO and PDO groups showed more new bone area (NBA) compared to the PP (p < 0.001 and p < 0.001, respectively) and to the NC group (p = 0.032 and p = 0.031, respectively). Furthermore, greater NBA was found in the PP group than the NC group (p = 0.006). PDO demonstrated higher relative connective tissue area than the PPDO group (p = 0.005) and lower relative new bone volume than the NC group (p = 0.025). Pumping protocol of periosteal distraction may successfully induce the endogenous regeneration of the mandibular bone in dogs. Impact Statement Repair of extended bone defects impose a significant challenge to oral and maxillofacial surgeons. In this article, a principle of distraction osteogenesis was applied to stimulate bone regeneration in the mandible. A periosteum-based regeneration approach may represent a valuable step toward creating a significant volume of hard and soft tissues, without need for autogenous bone harvesting or application of biomaterials.

Pathophysiology of Vascular Calcification and Bone Loss: Linked Disorders of Ageing?

Vascular Calcification (VC), low bone mass and fragility fractures are frequently observed in ageing subjects. Although this clinical observation could be the mere coincidence of frequent age-dependent disorders, clinical and experimental data suggest that VC and bone loss could share pathophysiological mechanisms. Indeed, VC is an active process of calcium and phosphate precipitation that involves the transition of the vascular smooth muscle cells (VSMCs) into osteoblast-like cells. Among the molecules involved in this process, parathyroid hormone (PTH) plays a key role acting through several mechanisms which includes the regulation of the RANK/RANKL/OPG system and the Wnt/ß-catenin pathway, the main pathways for bone resorption and bone formation, respectively. Furthermore, some microRNAs have been implicated as common regulators of bone metabolism, VC, left ventricle hypertrophy and myocardial fibrosis. Elucidating the common mechanisms between ageing; VC and bone loss could help to better understand the potential effects of osteoporosis drugs on the CV system.

ß-Klotho deficiency shifts the gut-liver bile acid axis and induces hepatic alterations in mice.

ß-Klotho (encoded by Klb) is an obligate coreceptor, mediating both fibroblast growth factor (FGF)15 and FGF21 signaling. Klb-/- mice are refractory to metabolic FGF15 and FGF21 action and exhibit derepressed (increased) bile acid (BA) synthesis. Here, we deeply phenotyped male Klb-/- mice on a pure C57BL/6J genetic background, fed a chow diet focusing on metabolic aspects. This aims to better understand the physiological consequences of concomitant FGF15 and FGF21 signaling deficiency, in particular on the gut-liver axis. Klb-/- mice present permanent growth restriction independent of adiposity and energy balance. Klb-/- mice also exhibit few changes in carbohydrate metabolism, combining normal gluco-tolerance, insulin sensitivity, and fasting response with increased gluconeogenic capacity and decreased glycogen mobilization. Livers of Klb-/- mice reveal pathologic features, including a proinflammatory status and initiation of fibrosis. These defects are associated to a massive shift in BA composition in the enterohepatic system and blood circulation featured by a large excess of microbiota-derived deoxycholic acid, classically known for its genotoxicity in the gastrointestinal tract. In conclusion, ß-Klotho is a gatekeeper of hepatic integrity through direct action (mediating FGF21 anti-inflammatory signaling) and indirect mechanisms (mediating FGF15 signaling that maintains BA level and composition).

Serum Levels of a Cathepsin-K Generated Periostin Fragment Predict Incident Low-Trauma Fractures in Postmenopausal Women Independently of BMD and FRAX.

Periostin is a matricellular protein involved in bone formation and bone matrix organization, but it is also produced by other tissues. Its circulating levels have been weakly associated with bone microstructure and prevalent fractures, possibly because periostin measured by the current commercial assays does not specifically reflect bone metabolism. In this context, we developed a new ELISA for a periostin fragment resulting from cathepsin K digestion (K-Postn). We hypothesized that circulating K-Postn levels could be associated with bone fragility. A total of 695 women (age 65.0 ± 1.5 years), enrolled in the Geneva Retirees Cohort (GERICO), were prospectively evaluated over 4.7 ± 1.9 years for the occurrence of low-trauma fractures. At baseline, we measured serum periostin, K-Postn, and bone turnover markers (BTMs), distal radius and tibia microstructure by HR-pQCT, hip and lumbar spine aBMD by DXA, and estimated fracture probability using the Fracture Risk Assessment Tool (FRAX). Sixty-six women sustained a low-trauma clinical fracture during the follow-up. Total periostin was not associated with fractures (HR [95% CI] per SD: 1.19 [0.89 to 1.59], p = 0.24). In contrast, K-Postn was significantly higher in the fracture versus nonfracture group (57.5 ± 36.6 ng/mL versus 42.5 ± 23.4 ng/mL, p < 0.001) and associated with fracture risk (HR [95%CI] per SD: 2.14 [1.54 to 2.97], p < 0.001). After adjustment for aBMD, FRAX, bone microstructure, or BTMs, K-Postn remained significantly associated with fracture risk. The performance of the fracture prediction models was improved by adding K-Postn to aBMD or FRAX (Harrell C index for fracture: 0.70 for aBMD + K-Post versus 0.58 for aBMD alone, p = 0.001; 0.73 for FRAX + K-Postn versus 0.65 for FRAX alone, p = 0.005). Circulating K-Postn predicts incident fractures independently of BMD, BTMs, and FRAX in postmenopausal women. Hence measurement of a periostin fragment resulting from in vivo cathepsin K digestion may help to identify subjects at high risk of fracture. © 2017 American Society for Bone and Mineral Research.

Development of a New Immunoassay for Human Cathepsin K-Generated Periostin Fragments as a Serum Biomarker for Cortical Bone.

Periostin is a matricellular protein mainly expressed by periosteal cells and osteocytes in bone, but is also present in several other tissues. Available immunoassays use antibodies of unclear specificity. The aim of the study was to develop a bone-specific periostin ELISA based on the detection of fragments generated by the osteoclastic and osteocytic protease cathepsin K. In vitro digestion of human recombinant intact periostin by cathepsin K leads to the generation of multiple fragments. Using LS-MS/MS, it was found that the GSLQPIIK peptide was the most efficiently and abundantly generated periostin fragment. A rabbit polyclonal antibody directed against the synthetic GSLQPIIK sequence was produced. Immunohistochemistry experiments of the tibia showed that the GSLQPIIK fragments localized at the periosteal surface and within the osteocytes. Using the same antibody, we developed an ELISA for the measurement of GSLQPIIK in the serum. This ELISA demonstrated intra- and interassay variability below 14% with a sensitivity allowing accurate determinations in the serum of healthy individuals. Serum GSLQPIIK was measured in 160 healthy postmenopausal women (mean age 65 year) participating in the Geneva Retiree Cohort. Serum GSLQPIIK levels did not correlate with total periostin, hip BMD, and the bone markers PINP and CTX. However, GSLQPIIK was negatively correlated (p values ranging from 0.007 to 0.03) with Hr-pQCT measures of tibia and radius cortical bone, but not with trabecular parameters. We have developed the first assay for the detection of periostin fragments generated by cathepsin K. Because serum levels of this new marker significantly correlated with cortical bone measurements in postmenopausal women, it may prove to be useful for the clinical investigation of patients with osteoporosis.

Structural Basis of Bone Fragility in Young Subjects with Inflammatory Bowel Disease: A High-resolution pQCT Study of the SWISS IBD Cohort (SIBDC).

BACKGROUND: The onset of inflammatory bowel disease (IBD) during childhood/adolescence compromises peak bone mass acquisition and predisposes to fractures later in life. However, the structural basis for bone fragility in young adults with IBD remains unknown. METHODS: One hundred two young subjects from the Swiss IBD cohort were included. Areal bone mineral density (aBMD) at distal radius, hip, and spine as well as morphometric vertebral fractures were assessed using dual-energy x-ray absorptiometry technique. Volumetric (v)BMD, trabecular, and cortical bone microstructure at the distal radius and tibia were assessed by high-resolution peripheral quantitative computed tomography. Areal, vBMD, and microstructure were compared between patients with IBD and healthy matched controls (n = 389). Multiple regression analysis was used to evaluate variables associated with bone microarchitecture and fractures. RESULTS: Clinical fractures were reported in 37 IBD subjects (mean age 23 yrs), mostly of the forearm; 5 subjects had morphometric vertebral fractures. After adjusting for age, sex, and height, tibia trabecular (Tb)vBMD, thickness, and distribution were significantly associated with fractures, whereas aBMD was not. After adjusting for aBMD, radius Tb distribution and tibia (Tb)vBMD and trabecular thickness still remained associated with fractures. Compared with healthy controls, patients with IBD had significantly lower aBMD at all sites, as well as alteration in (Tb)vBMD and trabecular microstructure at the distal radius and tibia, and these alterations were correlated with disease severity. CONCLUSIONS: Young patients with IBD have low aBMD and altered trabecular bone microarchitecture compared with healthy controls. The latter is independently associated with fractures and may predispose increased susceptibility to fragility fractures throughout life.

Cathepsin K Controls Cortical Bone Formation by Degrading Periostin.

Although inhibitors of bone resorption concomitantly reduce bone formation because of the coupling between osteoclasts and osteoblasts, inhibition or deletion of cathepsin k (CatK) stimulates bone formation despite decreasing resorption. The molecular mechanisms responsible for this increase in bone formation, particularly at periosteal surfaces where osteoclasts are relatively poor, remain unclear. Here we show that CatK pharmacological inhibition or deletion (Ctsk-/- mice) potentiates mechanotransduction signals mediating cortical bone formation. We identify periostin (Postn) as a direct molecular target for degradation by CatK and show that CatK deletion increases Postn and ß-catenin expression in vivo, particularly at the periosteum. In turn, Postn deletion selectively abolishes cortical, but not trabecular, bone formation in CatK-deficient mice. Taken together, these data indicate that CatK not only plays a major role in bone remodeling but also modulates modeling-based cortical bone formation by degrading periostin and thereby moderating Wnt-ß-catenin signaling. These findings provide novel insights into the role of CatK on bone homeostasis and the mechanisms of increased cortical bone volume with CatK mutations and pharmacological inhibitors. © 2017 American Society for Bone and Mineral Research.

Peripheral skeleton bone strength is positively correlated with total and dairy protein intakes in healthy postmenopausal women.

BACKGROUND: Bone mineral content (BMC) and bone mineral density (BMD) are positively correlated with dietary protein intakes, which account for 1-8% of BMC and BMD variances. However, the relation between bone strength and microstructure, which are variables that are not captured by areal bone mineral density (aBMD), and dietary protein intakes, particularly from specific dietary sources, has not been clearly established. OBJECTIVE: We investigated the association between the peripheral skeleton-predicted failure load and stiffness, bone microstructure, and dietary protein intakes from various origins (animal, divided into dairy and nondairy, and vegetable origins) in healthy postmenopausal women. DESIGN: In a cross-sectional study in 746 Caucasian women aged 65.0 ± 1.4 y, we measured the aBMD with the use of dual-energy X-ray absorptiometry, the distal radius and tibia bone microstructures with the use of high-resolution peripheral quantitative computerized tomography, and bone strength with the use of a finite element analysis, and we evaluated dietary protein and calcium with the use of a validated food-frequency questionnaire. RESULTS: Mean dietary calcium and protein intakes were greater than recommended amounts for this class of age. The predicted failure load and stiffness at the distal radius and tibia were positively associated with total, animal, and dairy protein intakes but not with vegetable protein intake. Failure load differences were accompanied by modifications of the aBMD and of cortical and trabecular bone microstructures. The associations remained statistically significant after adjustment for weight, height, physical activity, menopause duration, calcium intake, and the interaction between calcium and protein intake. A principal component analysis of the volumetric BMD and bone microstructure indicated that trabecular bone mainly contributed to the positive association between protein intakes and bone strength. CONCLUSIONS: These results, which were recorded in a very homogeneous population of healthy postmenopausal women, indicate that there is a beneficial effect of animal and dairy protein intakes on bone strength and microstructure. Specifically, there is a positive association between the bone failure load and stiffness of the peripheral skeleton and dietary protein intake, which is mainly related to changes in the trabecular microstructure. This trial was registered at www.controlled-trials.com as ISRCTN11865958.

Mechanisms of diabetes mellitus-induced bone fragility.

The risk of fragility fractures is increased in patients with either type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM). Although BMD is decreased in T1DM, BMD in T2DM is often normal or even slightly elevated compared with an age-matched control population. However, in both T1DM and T2DM, bone turnover is decreased and the bone material properties and microstructure of bone are altered; the latter particularly so when microvascular complications are present. The pathophysiological mechanisms underlying bone fragility in diabetes mellitus are complex, and include hyperglycaemia, oxidative stress and the accumulation of advanced glycation endproducts that compromise collagen properties, increase marrow adiposity, release inflammatory factors and adipokines from visceral fat, and potentially alter the function of osteocytes. Additional factors including treatment-induced hypoglycaemia, certain antidiabetic medications with a direct effect on bone and mineral metabolism (such as thiazolidinediones), as well as an increased propensity for falls, all contribute to the increased fracture risk in patients with diabetes mellitus.

Influence of Fatigue Loading and Bone Turnover on Bone Strength and Pattern of Experimental Fractures of the Tibia in Mice.

Bone fragility depends on bone mass, structure, and material properties, including damage. The relationship between bone turnover, fatigue damage, and the pattern and location of fractures, however, remains poorly understood. We examined these factors and their integrated effects on fracture strength and patterns in tibia. Adult male mice received RANKL (2 mg/kg/day), OPG-Fc (5 mg/kg 2×/week), or vehicle (Veh) 2 days prior to fatigue loading of one tibia by in vivo axial compression, with treatments continuing up to 28 more days. One day post fatigue, crack density was similarly increased in fatigued tibiae from all treatment groups. After 28 days, the RANKL group exhibited reduced bone mass and increased crack density, resulting in reduced bone strength, while the OPG-Fc group had greater bone mass and bone strength. Injury repair altered the pattern and location of fractures created by ex vivo destructive testing, with fractures occurring more proximally and obliquely relative to non-fatigued tibia. A similar pattern was observed in both non-fatigued and fatigued tibia of RANKL. In contrast, OPG-Fc prevented this fatigue-related shift in fracture pattern by maintaining fractures more distal and transverse. Correlation analysis showed that bone strength was predominantly determined by aBMD with minor contributions from structure and intrinsic strength as measured by nanoindentation and cracks density. In contrast, fracture location was predicted equally by aBMD, crack density and intrinsic modulus. The data suggest that not only bone strength but also the fracture pattern depends on previous damage and the effects of bone turnover on bone mass and structure. These observations may be relevant to further understand the mechanisms contributing to fracture pattern in long bone with different levels of bone remodeling, including atypical femur fracture.

Myostatin antibody (LY2495655) in older weak fallers: a proof-of-concept, randomised, phase 2 trial.

BACKGROUND: Myostatin inhibits skeletal muscle growth. The humanised monoclonal antibody LY2495655 (LY) binds and neutralises myostatin. We aimed to test whether LY increases appendicular lean body mass (aLBM) and improves physical performance in older individuals who have had recent falls and low muscle strength and power. METHODS: In this proof-of-concept, randomised, placebo-controlled, double-blind, parallel, multicentre, phase 2 study, we recruited patients aged 75 years or older who had fallen in the past year from 21 investigator sites across Argentina, Australia, France, Germany, Sweden, and the USA. Eligible patients had low performance on hand grip strength and chair rise tests, tested with the procedure described by Guralnik and colleagues. Participants were stratified by country, age, hand grip strength, and performance on the chair rise test, and were randomly assigned (1:1) by a computer-generated random sequence to receive subcutaneous injections of placebo or 315 mg LY at weeks 0 (randomisation visit), 4, 8, 12, 16, and 20, followed by 16 weeks observation. The primary outcome was change in aLBM from baseline to 24 weeks. We measured physical performance as secondary outcomes (four-step stair climbing time, usual gait speed, and time to rise five times from a chair without arms, or with arms for participants unable to do it without arms) and exploratory outcomes (12-step stair climbing test, 6-min walking distance, fast gait speed, hand grip strength, and isometric leg extension strength). Efficacy analyses included all randomly assigned patients who received at least one dose and had a baseline and at least one subsequent measure. The primary analysis and all other tests of treatment effect (except physical performance tests) were done at a two-sided alpha level of 0·05. Tests of treatment effect on physical performance tests were done at a pre-specified two-sided alpha level of 0·1. This trial is registered with ClinicalTrials.gov, number NCT01604408. FINDINGS: Between June 19, 2012, and Dec 12, 2013, we screened 365 patients. 99 were randomly assigned to receive placebo and 102 to receive LY. Treatment was completed in 85 (86%) of patients given placebo and in 82 (80%) given LY. At 24 weeks, the least-squares mean change in aLBM was -0·123 kg (95% CI -0·287 to 0·040) in the placebo group and 0·303 kg (0·135 to 0·470) in the LY group, a difference of 0·43 kg (95% CI 0·192 to 0·660; p<0·0001). Stair climbing time (four-step and 12-step tests), chair rise with arms, and fast gait speed improved significantly from baseline to week 24 with differences between LY and placebo of respectively -0·46 s (p=0·093), -1·28 s (p=0·011), -4·15 s (p=0·054), and 0·05 m/s (p=0·088). No effect was detected for other performance-based measures. Injection site reactions were recorded in nine (9%) patients given placebo and in 31 (30%) patients given LY (p<0·0001), and were generally mild, and led to treatment discontinuation in two patients given LY. INTERPRETATION: Our findings show LY treatment increases lean mass and might improve functional measures of muscle power. Although additional studies are needed to confirm these results, our data suggest LY should be tested for its potential ability to reduce the risk of falls or physical dependency in older weak fallers. FUNDING: Eli Lilly and Company.

Periostin expression contributes to cortical bone loss during unloading.

Periostin (a product of Postn gene) is a matricellular protein which is increased in periosteal osteoblasts and osteocytes upon mechanical stimulation. We previously reported that periostin-deficient mice (Postn(-/-)) have low bone mass and a diminished response to physical activity due to a lack of sclerostin (a product of Sost gene) inhibition by mechanical loading. Here we hypothesized that periostin could play a central role in the control of bone loss during unloading induced by hindlimb suspension (HU). In Postn(+/+) mice (wildtype littermate), HU significantly decreased femur BMD, as well as trabecular BV/TV and thickness (Tb.Th). Cortical bone volume and thickness at the femoral midshaft, also significantly decreased. These changes were explained by an inhibition of endocortical and periosteal bone formation activity and correlated with a decrease of Postn expression and a consecutive increase in Sost early after HU. Whereas trabecular bone loss in Postn(-/-) mice was comparable to Postn(+/+) mice, HU did not significantly alter cortical bone microstructure and strength in Postn(-/-) mice. Bone formation remained unchanged in these mice, as Sost did not increase in the absence of periostin. In contrast, changes in Dkk1, Rankl and Opg expression in response to HU were similar to Postn(+/+) mice, indicating that changes in periostin expression were quite specifically related to changes in Sost. In conclusion, HU inhibits periostin expression, which in turn plays an important role in cortical bone loss through an increase in Sost. These results further indicate that periostin is an essential mediator of cortical bone response to mechanical forces (loading and unloading).

Effects of an interleukin-15 antagonist on systemic and skeletal alterations in mice with DSS-induced colitis.

Inflammatory bowel diseases are commonly complicated by weight and bone loss. We hypothesized that IL-15, a pro-inflammatory cytokine expressed in colitis and an osteoclastogenic factor, could play a central role in systemic and skeletal complications of inflammatory bowel diseases. We evaluated the effects of an IL-15 antagonist, CRB-15, in mice with chronic colitis induced by oral 2% dextran sulfate sodium for 1 week, followed by another 1% for 2 weeks. During the last 2 weeks, mice were treated daily with CRB-15 or an IgG2a control antibody. Intestinal inflammation, disease severity, and bone parameters were evaluated at days 14 and 21. CRB-15 improved survival, early weight loss, and colitis clinical score, although colon damage and inflammation were prevented in only half the survivors. CRB-15 also delayed loss of femur bone mineral density and trabecular microarchitecture. Bone loss was characterized by decreased bone formation, but increased bone marrow osteoclast progenitors and osteoclast numbers on bone surfaces. CRB-15 prevented the suppression of osteoblastic markers of bone formation, and reduced osteoclast progenitors at day 14, but not later. However, by day 21, CRB-15 decreased tumor necrosis factor α and increased IL-10 expression in bone, paralleling a reduction of osteoclasts. These results delineate the role of IL-15 on the systemic and skeletal manifestations of chronic colitis and provide a proof-of-concept for future therapeutic developments.

Regulation of beta catenin signaling and parathyroid hormone anabolic effects in bone by the matricellular protein periostin.

Periostin (Postn) is a matricellular protein preferentially expressed by osteocytes and periosteal osteoblasts in response to mechanical stimulation and parathyroid hormone (PTH). Whether and how periostin expression influences bone anabolism, however, remains unknown. We investigated the skeletal response of adult Postn(-/-) and Postn(+/+) mice to intermittent PTH. Compared with Postn(+/+), Postn(-/-) mice had a lower bone mass, cortical bone volume, and strength response to PTH. PTH-stimulated bone-forming indices were all significantly lower in Postn(-/-) mice, particularly at the periosteum. Furthermore, in vivo stimulation of Wnt-ß-catenin signaling by PTH, as evaluated in TOPGAL reporter mice, was inhibited in the absence of periostin (TOPGAL;Postn(-/-) mice). PTH stimulated periostin and inhibited MEF2C and sclerostin (Sost) expression in bone and osteoblasts in vitro. Recombinant periostin also suppressed Sost expression, which was mediated through the integrin αVß3 receptor, whereas periostin-blocking antibody prevented inhibition of MEF2C and Sost by PTH. In turn, administration of a Sost-blocking antiboby partially restored the PTH-mediated increase in bone mass in Postn(-/-) mice. In addition, primary osteoblasts from Postn(-/-) mice showed a lower proliferation, mineralization, and migration, both spontaneously and in response to PTH. Osteoblastic gene expression levels confirmed a defect of Postn(-/-) osteoblast differentiation with and without PTH, as well as an increased osteoblast apoptosis in the absence of periostin. These data elucidate the complex role of periostin on bone anabolism, through the regulation of Sost, Wnt-ß-catenin signaling, and osteoblast differentiation.

A botulinum toxin-derived targeted secretion inhibitor downregulates the GH/IGF1 axis.

Botulinum neurotoxins (BoNTs) are zinc endopeptidases that block release of the neurotransmitter acetylcholine in neuromuscular synapses through cleavage of soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein receptor (SNARE) proteins, which promote fusion of synaptic vesicles to the plasma membrane. We designed and tested a BoNT-derived targeted secretion inhibitor (TSI) targeting pituitary somatotroph cells to suppress growth hormone (GH) secretion and treat acromegaly. This recombinant protein, called SXN101742, contains a modified GH-releasing hormone (GHRH) domain and the endopeptidase domain of botulinum toxin serotype D (GHRH-LHN/D, where HN/D indicates endopeptidase and translocation domain type D). In vitro, SXN101742 targeted the GHRH receptor and depleted a SNARE protein involved in GH exocytosis, vesicle-associated membrane protein 2 (VAMP2). In vivo, administering SXN101742 to growing rats produced a dose-dependent inhibition of GH synthesis, storage, and secretion. Consequently, hepatic IGF1 production and resultant circulating IGF1 levels were reduced. Accordingly, body weight, body length, organ weight, and bone mass acquisition were all decreased, reflecting the biological impact of SXN101742 on the GH/IGF1 axis. An inactivating 2-amino acid substitution within the zinc coordination site of the endopeptidase domain completely abolished SXN101742 inhibitory actions on GH and IGF1. Thus, genetically reengineered BoNTs can be targeted to nonneural cells to selectively inhibit hormone secretion, representing a new approach to treating hormonal excess.

Deletion of ß-adrenergic receptor 1, 2, or both leads to different bone phenotypes and response to mechanical stimulation.

As they age, mice deficient for the ß2-adrenergic receptor (Adrb2(-/-) ) maintain greater trabecular bone microarchitecture, as a result of lower bone resorption and increased bone formation. The role of ß1-adrenergic receptor signaling and its interaction with ß2-adrenergic receptor on bone mass regulation, however, remains poorly understood. We first investigated the skeletal response to mechanical stimulation in mice deficient for ß1-adrenergic receptors and/or ß2-adrenergic receptors. Upon axial compression loading of the tibia, bone density, cancellous and cortical microarchitecture, as well as histomorphometric bone forming indices, were increased in both Adrb2(-/-) and wild-type (WT) mice, but not in Adrb1(-/-) nor in Adrb1b2(-/-) mice. Moreover, in the unstimulated femur and vertebra, bone mass and microarchitecture were increased in Adrb2(-/-) mice, whereas in Adrb1(-/-) and Adrb1b2(-/-) double knockout mice, femur bone mineral density (BMD), cancellous bone volume/total volume (BV/TV), cortical size, and cortical thickness were lower compared to WT. Bone histomorphometry and biochemical markers showed markedly decreased bone formation in Adrb1b2(-/-) mice during growth, which paralleled a significant decline in circulating insulin-like growth factor 1 (IGF-1) and IGF-binding protein 3 (IGF-BP3). Finally, administration of the ß-adrenergic agonist isoproterenol increased bone resorption and receptor activator of NF-κB ligand (RANKL) and decreased bone mass and microarchitecture in WT but not in Adrb1b2(-/-) mice. Altogether, these results demonstrate that ß1- and ß2-adrenergic signaling exert opposite effects on bone, with ß1 exerting a predominant anabolic stimulus in response to mechanical stimulation and during growth, whereas ß2-adrenergic receptor signaling mainly regulates bone resorption during aging.

Early post-transplantation hypophosphatemia is associated with elevated FGF-23 levels.

BACKGROUND: We examined the hypothesis that high FGF-23 levels early after transplantation contribute to the onset of hypophosphatemia, independently of parathyroid hormone (PTH) and other factors regulating phosphate metabolism. METHODS: We measured serum phosphate levels (sPi), renal tubular reabsorption of Pi (TmPi/GFR), estimated GFR (eGFR), intact PTH (iPTH), calcitriol, intact (int) and C-terminal (Cter) FGF-23, dietary Pi intake and cumulative doses of glucocorticoids in 69 patients 12 days (95% confidence interval, 10-13) after renal transplantation. RESULTS: Hypophosphatemia was observed in 43 (62%) of the patients 12 days after transplantation. Compared with non-hypophosphatemic subjects, their post-transplantation levels of intact and CterFGF-23 were higher (195 (108-288) vs 48 (40-64) ng/l, P<0.002 for intFGF-23; 205 (116-384) vs 81 (55-124) U/ml, P<0.002, for CterFGF-23). In all subjects, Cter and intFGF-23 correlated inversely with sPi (r=-0.35, P<0.003; -0.35, P<0.003, respectively), and TmPi/GFR (r=-0.50, P<0.001; -0.54, P<0.001, respectively). In multivariate models, sPi and TmPi/GFR were independently associated with FGF-23, iPTH and eGFR. Pre-transplant iPTH levels were significantly higher in patients developing hypophosphatemia after renal transplantation. Pre-transplant levels of FGF-23 were not associated with sPi at the time of transplantation. CONCLUSION: In addition to PTH, elevated FGF-23 may contribute to hypophosphatemia during the early post-renal transplant period.

Effects of long-term supplementation with omega-3 fatty acids on longitudinal changes in bone mass and microstructure in mice.

A diet rich in omega-3s has previously been suggested to prevent bone loss. However, evidence for this has been limited by short exposure to omega-3 fatty acids (FAs). We investigated whether a diet enriched in eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) for the entire adult life of mice could improve bone microstructure and strength. Thirty female mice received a diet enriched in DHA or EPA or an isocaloric control diet from 3 to 17 months of age. Changes in bone microstructure were analyzed longitudinally and biomechanical properties were analysed by a three-point bending test. Bone remodelling was evaluated by markers of bone turnover and histomorphometry. Trabecular bone volume in caudal vertebrae was improved by EPA or DHA at 8 months (+26.6% and +17.2%, respectively, compared to +3.8% in controls, P=.01), but not thereafter. Trabecular bone loss in the tibia was not prevented by omega-3 FAs (BV/TV -94%, -93% and -97% in EPA, DHA and controls, respectively). EPA improved femur cortical bone volume (+8.1%, P<.05) and thickness (+4.4%, P<.05) compared to controls. EPA, but not DHA, reduced age-related decline of osteocalcin (-70% vs. -83% in controls, P<.05). EPA and DHA increased leptin levels (7.3±0.7 and 8.5±0.5 ng ml⁻¹, respectively, compared to 4.5±0.9 ng ml⁻¹ in controls, P=.001); however, only EPA further increased IGF-1 levels (739±108 ng ml⁻¹, compared to 417±58 ng ml⁻¹ in controls, P=.04). These data suggest that long-term intake of omega-3 FA, particularly EPA, may modestly improve the structural and mechanical properties of cortical bone by an increase in leptin and IGF-1 levels, without affecting trabecular bone loss.