Holger Henneicke

Osteoblasts mediate the adverse effects of glucocorticoids on fuel metabolism

Long-term glucocorticoid treatment is associated with numerous adverse outcomes, including weight gain, insulin resistance, and diabetes; however, the pathogenesis of these side effects remains obscure. Glucocorticoids also suppress osteoblast function, including osteocalcin synthesis. Osteocalcin is an osteoblast-specific peptide that is reported to be involved in normal murine fuel metabolism. We now demonstrate that osteoblasts play a pivotal role in the pathogenesis of glucocorticoid-induced dysmetabolism. Osteoblast-targeted disruption of glucocorticoid signaling significantly attenuated the suppression of osteocalcin synthesis and prevented the development of insulin resistance, glucose intolerance, and abnormal weight gain in corticosterone-treated mice. Nearly identical effects were observed in glucocorticoid-treated animals following heterotopic (hepatic) expression of both carboxylated and uncarboxylated osteocalcin through gene therapy, which additionally led to a reduction in hepatic lipid deposition and improved phosphorylation of the insulin receptor. These data suggest that the effects of exogenous high-dose glucocorticoids on insulin target tissues and systemic energy metabolism are mediated, at least in part, through the skeleton.

Glucocorticoids and bone: local effects and systemic implications

Glucocorticoids (GCs) are highly effective in the treatment of inflammatory and autoimmune conditions but their therapeutic use is limited by numerous adverse effects. Recent insights into the mechanisms of action of both endogenous and exogenous GCs on bone cells have unlocked new approaches to the development of effective strategies for the prevention and treatment of GC-induced osteoporosis. Furthermore, topical studies in rodents indicate that the osteoblast-derived peptide, osteocalcin, plays a central role in the pathogenesis of GC-induced diabetes and obesity. These exciting findings mechanistically link the detrimental effects of GCs on bone and energy metabolism. In this article we review the physiology and pathophysiology of GC action on bone cells, and discuss current and emerging concepts regarding the molecular mechanisms underlying adverse effects of GCs such as osteoporosis and diabetes.

Corticosterone selectively targets endo-cortical surfaces by an osteoblast-dependent mechanism

BACKGROUND The pathogenesis of glucocorticoid-induced osteoporosis remains ill defined. In this study, we examined the role of the osteoblast in mediating the effects of exogenous glucocorticoids on cortical and trabecular bone, employing the Col2.3-11βHSD2 transgenic mouse model of osteoblast-targeted disruption of glucocorticoid signalling. METHODS Eight week-old male transgenic (tg) and wild-type (WT) mice (n=20-23/group) were treated with either 1.5 mg corticosterone (CS) or placebo for 4 weeks. Serum tartrate-resistant acid phosphatase 5b (TRAP5b) and osteocalcin (OCN) were measured throughout the study. Tibiae and lumbar vertebrae were analysed by micro-CT and histomorphometry at endpoint. RESULTS CS suppressed serum OCN levels in WT and tg mice, although they remained higher in tg animals at all time points (p<0.05). Serum TRAP5b levels increased in WT mice only. The effect of CS on cortical bone differed by site: At the endosteal surface, exposure to CS significantly increased bone resorption and reduced bone formation, resulting in a larger bone marrow cavity cross-sectional area (p<0.01). In contrast, at the pericortical surface bone resorption was significantly decreased accompanied with a significant increase in pericortical cross-sectional area (p<0.05) while bone formation remained unaffected. Vertebral cortical thickness and area were reduced in CS treatment mice. Tg mice were partially protected from the effects of exogenous CS, both on a cellular and structural level. At the CS doses used in this study, trabecular bone remained largely unaffected. CONCLUSION Endocortical osteoblasts appear to be particularly sensitive to the detrimental actions of exogenous glucocorticoids. The increase in tibial pericortical cross-sectional area and the according changes in pericortical circumference suggest an anabolic bone response to GC treatment at this site. The protection of tg mice from these effects indicates that both catabolic and anabolic action of glucocorticoids are, at least in part, mediated by osteoblasts.

The challenge of continuous exogenous glucocorticoid administration in mice.

BACKGROUND Chronic administration of exogenous glucocorticoids is often required in experimental research. We compared the efficacy and reliability of three different methods of continuous glucocorticoid administration in mice. MATERIALS AND METHODS Male CD1 Swiss White mice aged 7-9 weeks received corticosterone (CS) or carrier by either subcutaneous (s.c.) injection (n=15), s.c. implantation of micro-osmotic pumps (n=20) or s.c. implantation of slow-release pellets (n=20). Serial blood samples were taken for the measurement of plasma CS and osteocalcin (OC). Bone structural parameters were analysed by micro-computed tomography (micro-CT) in animals treated via slow-release pellets for 4 weeks. RESULTS Injection of CS (10 mg/kg) resulted in peak plasma CS levels of up to 2600 microg/L after 1 h, with levels returning to baseline within 4 h post-injection. Micro-osmotic pumps failed to consistently alter plasma CS levels and had variable effects on plasma OC levels. Implantation of 10 mg CS pellets induced hypercorticosteronemia within 24 h but levels returned to baseline within 7 days. Plasma OC levels fell rapidly on day 1 and remained suppressed until day 7. Weekly replacement of pellets maintained elevated plasma CS and suppressed plasma OC concentrations, and resulted in significant bone loss at the tibia and spine after 28 days. CONCLUSION Once-weekly s.c. implantation of slow-release pellets to mice appears to result in relatively consistent plasma CS and OC levels with significant biological effects. However, at least in our hands, no method delivered CS at a constant rate and variability in plasma CS levels was pronounced.

Disruption of glucocorticoid signaling in chondrocytes delays metaphyseal fracture healing but does not affect normal cartilage and bone development

States of glucocorticoid excess are associated with defects in chondrocyte function. Most prominently there is a reduction in linear growth but delayed healing of fractures that require endochondral ossification to also occur. In contrast, little is known about the role of endogenous glucocorticoids in chondrocyte function. As glucocorticoids exert their cellular actions through the glucocorticoid receptor (GR), we aimed to elucidate the role of endogenous glucocorticoids in chondrocyte function in vivo through characterization of tamoxifen-inducible chondrocyte-specific GR knockout (chGRKO) mice in which the GR was deleted at various post-natal ages. Knee joint architecture, cartilage structure, growth plates, intervertebral discs, long bone length and bone micro-architecture were similar in chGRKO and control mice at all ages. Analysis of fracture healing in chGRKO and control mice demonstrated that in metaphyseal fractures, chGRKO mice formed a larger cartilaginous callus at 1 and 2 week post-surgery, as well as a smaller amount of well-mineralized bony callus at the fracture site 4 week post-surgery, when compared to control mice. In contrast, chondrocyte-specific GR knockout did not affect diaphyseal fracture healing. We conclude that endogenous GC signaling in chondrocytes plays an important role during metaphyseal fracture healing but is not essential for normal long bone growth.

Association of adipokines and joint biomarkers with cartilage-modifying effects of weight loss in obese subjects

OBJECTIVES To determine (1) the effects of weight loss in obese subjects on six adipokines and joint biomarkers; and (2) the relationship between changes in these markers with changes in cartilage outcomes. DESIGN Plasma levels of adiponectin, leptin, IL-6, COMP, MMP-3 and urine levels of CTX-II were measured at baseline and 12 months from 75 obese subjects enrolled in two weight-loss programs. Magnetic resonance imaging (MRI) was used to assess cartilage volume and thickness. Associations between weight loss, cartilage outcomes and markers were adjusted for age, gender, baseline BMI, presence of clinical knee OA, with and without weight loss percent. RESULTS Mean weight loss was 13.0 ± 9.5%. Greater weight loss percentage was associated with an increase in adiponectin (β = 0.019, 95% CI 0.012 to 0.026,) and a decrease in leptin (β = -1.09, 95% CI -1.37 to -0.82). Multiple regression analysis saw an increase in adiponectin associated with reduced loss of medial tibial cartilage volume (β = 14.4, CI 2.6 to 26.3) and medial femoral cartilage volume (β = 18.1, 95% CI 4.4 to 31.8). Decrease in leptin was associated with reduced loss of medial femoral volume (β = -4.1, 95% CI -6.8 to -1.4) and lateral femoral volume (β = -1.8, 95% CI -3.7 to 0.0). When weight loss percent was included in the model, only the relationships between COMP and cartilage volume remained statistically significant. CONCLUSIONS Adiponectin and leptin may be associated with cartilage loss. Further work will determine the relative contributions of metabolic and mechanical factors in the obesity-related joint changes.

Long-Term Corticosterone Treatment Induced Lobe-Specific Pathology in Mouse Prostate

Glucocorticoids influence prostate development and pathology, yet the underlying mechanisms including possible direct glucocorticoid effect on the prostate are not well characterized.

Ubiquitous expression of the Pik3caH1047R mutation promotes hypoglycemia, hypoinsulinemia, and organomegaly

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K, are among the most common mutations found in human cancer and have also recently been implicated in a range of overgrowth syndromes in humans. We have used a novel inducible “exonswitch” approach to knock in the constitutively active Pik3caH1047R mutation into the endogenous Pik3ca gene of the mouse. Ubiquitous expression of the Pik3caH1047R mutation throughout the body resulted in a dramatic increase in body weight within 3 weeks of induction (mutant 150 ± 5%; wild‐type 117 ± 3%, mean ± sem), which was associated with increased organ size rather than adiposity. Severe metabolic effects, including a reduction in blood glucose levels to 59 ± 4% of baseline (11 days postinduction) and undetectable insulin levels, were also observed. Pik3caH1047R mutant mice died earlier (median survival 46.5 d post‐mutation induction) than wild‐type control mice (100% survival > 250 days). Although deletion of Akt2 increased median survival by 44%, neither organ overgrowth, nor hypoglycemia were rescued, indicating that both the growth and metabolic functions of constitutive PI3K activity can be Akt2 independent. This mouse model demonstrates the critical role of PI3K in the regulation of both organ size and glucose metabolism at the whole animal level.—Kinross, K. M., Montgomery, K. G., Mangiafico, S. P., Hare, L. M., Kleinschmidt, M., Bywater, M. J., Poulton, I. J., Vrahnas, C., Henneicke, H., Malaterre, J., Waring, P. M., Cullinane, C., Sims, N. A., McArthur, G. A., Andrikopoulos, S., Phillips, W. A. Ubiquitous expression of the Pik3caH1047R mutation promotes hypoglycemia, hypoinsulinemia, and organomegaly. FASEB J. 29, 1426‐1434 (2015). www.fasebj.org

Chronic Mild Stress Causes Bone Loss via an Osteoblast-Specific Glucocorticoid-Dependent Mechanism

Chronic stress and depression are associated with alterations in the hypothalamic-pituitary-adrenal signaling cascade and considered a risk factor for bone loss and fractures. However, the mechanisms underlying the association between stress and poor bone health are unclear. Using a transgenic (tg) mouse model in which glucocorticoid signaling is selectively disrupted in mature osteoblasts and osteocytes [11β-hydroxysteroid-dehydrogenase type 2 (HSD2)OB-tg mice], the present study examines the impact of chronic stress on skeletal metabolism and structure. Eight-week-old male and female HSD2OB-tg mice and their wild-type (WT) littermates were exposed to chronic mild stress (CMS) for the duration of 4 weeks. At the endpoint, L3 vertebrae and tibiae were analyzed by micro-computed tomography and histomorphometry, and bone turnover was measured biochemically. Compared with nonstressed controls, exposure to CMS caused an approximately threefold increase in serum corticosterone concentrations in WT and HSD2OB-tg mice of both genders. Compared with controls, CMS resulted in loss of vertebral trabecular bone mass in male WT mice but not in male HSD2OB-tg littermates. Furthermore, both tibial cortical area and area fraction were reduced in stressed WT but not in stressed HSD2OB-tg male mice. Osteoclast activity and bone resorption marker were increased in WT males following CMS, features absent in HSD2OB-tg males. Interestingly, CMS had little effect on vertebral and long-bone structural parameters in female mice. We conclude that in male mice, bone loss during CMS is mediated via enhanced glucocorticoid signaling in osteoblasts (and osteocytes) and subsequent activation of osteoclasts. Female mice appear resistant to the skeletal effects of CMS.

Continuous corticosterone delivery via the drinking water or pellet implantation: A comparative study in mice

In order to investigate the effects of glucocorticoid excess in rodent models, reliable methods of continuous glucocorticoid delivery are essential. The current study compares two methods of corticosterone (CS) delivery in regards to their ability to induce typical adverse outcomes such as fat accrual, insulin resistance, sarcopenia and bone loss. Eight-week-old mice received CS for 4weeks either via the drinking water (25-100μgCS/mL) or through weekly surgical implantation of slow release pellets containing 1.5mg CS. Both methods induced abnormal fat mass accrual, inhibited lean mass accretion and bone expansion, suppressed serum osteocalcin levels and induced severe insulin resistance. There was a clear dose dependant relationship between the CS concentrations in the drinking water and the severity of the phenotype, with a concentration of 50μg CS/mL drinking water most closely matching the metabolic changes induced by weekly pellet implantations. In contrast to pellets, however, delivery of CS via the drinking water resulted in a consistent diurnal exposure pattern, closely mimicking the kinetics of clinical glucocorticoid therapy. In addition, the method is safe, inexpensive, easily adjustable, non-invasive and avoids operative stress to the animals. Our data demonstrate that delivery of CS via the drinking water has advantages over weekly implantations of slow-release pellets. A dose of 50μg CS/mL drinking water is appropriate for the investigation of chronic glucocorticoid excess in mice.