GC-treated Mice Research Articles

RF21 | PSAT78 The beta3-Adrenergic Receptor as a Target for the Prevention of Glucocorticoid-induced Adipose Tissue Dysfunction in Rodents

Abstract Glucocorticoids (GC) are an integral part of anti-inflammatory therapy. However, their therapeutic effectiveness is limited by their deleterious adverse effects such as the development of obesity, diabetes, and cardiometabolic disease. GC-induced obesity arises from excessive fat accumulation in white adipose tissue (WAT) as well as suppressed thermogenic function of brown adipose tissue (BAT). The sympathetic nervous system (SNS) plays a key role in the regulation of both WAT as well as BAT biology. As part of this project, we investigate the interaction between SNS and GC signaling in the adipose organ. To that end, we modulated the SNS activity in adipose tissues of mice by altering the housing temperature of corticosterone-treated mice and respective controls to either 29°C, 22°C, or 13°C. Following the four-week treatment course, GC-treated mice housed at 29°C and 22°C gained more weight and accumulated more visceral and subcutaneous white fat than their temperature-matched controls. Interestingly, maintaining mice in the 13°C environment protected animals from GC-induced weight gain and obesity. Additionally, cold adaptation effectively prevented the development of GC-driven hyperglycemia, hyperinsulinemia and hyperleptinemia, all of which were readily observed at 29°C and 22°C. Furthermore, the sympathetic innervation as well as the thermogenic capacity of BAT were preserved in cold-acclimated mice in spite of corticosterone treatment, whereas mice housed at higher temperatures showed marked suppression of the thermogenic function in BAT following GC exposure. To further test whether sympathetic activation is responsible for the prevention of GC-induced dysmetabolism by prolonged adaption to cold, we investigated the effect of a pharmacological β3-AR agonist, CL316,243 during GC excess. Similar to cold exposure, selective activation of the β3-AR not only averted the development of GC-induced fat accumulation and hyperinsulinemia, but also preserved the thermogenic capacity in BAT despite exposure to supra-physiological levels of corticosterone. Taken together, our data indicate that both cold-acclimation as well as β3-AR activation counteract GC-induced obesity, adipose tissues dysfunction, and related metabolic comorbidities. Thus, targeting the β3-AR may represent a potential therapeutic approach to alleviate GC-induced metabolic abnormalities. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 12:42 p.m. - 12:47 p.m.

The critical role of T cells in glucocorticoid-induced osteoporosis

Glucocorticoids (GC) are widely used clinically, despite the presence of significant side effects, including glucocorticoid-induced osteoporosis (GIOP). While GC are believed to act directly on osteoblasts and osteoclasts to promote osteoporosis, the detailed underlying molecular mechanism of GC-induced osteoporosis is still not fully elucidated. Here, we show that lymphocytes play a pivotal role in regulating GC-induced osteoporosis. We show that GIOP could not be induced in SCID mice that lack T cells, but it could be re-established by adoptive transfer of splenic T cells from wild-type mice. As expected, T cells in the periphery are greatly reduced by GC; instead, they accumulate in the bone marrow where they are protected from GC-induced apoptosis. These bone marrow T cells in GC-treated mice express high steady-state levels of NF-κB receptor activator ligand (RANKL), which promotes the formation and maturation of osteoclasts and induces osteoporosis. Taken together, these findings reveal a critical role for T cells in GIOP.

Glucocorticoids impair HDL-mediated cholesterol efflux besides increased HDL cholesterol concentration: a proof of concept.

Glucocorticoids (GC) are associated with increased cardiovascular morbidity despite increased HDL-C concentration. HDL-mediated cholesterol efflux, a major anti-atherogenic property of HDL particles, is negatively associated with CVD risk. We aimed to determine whether HDL-mediated cholesterol efflux was influenced by GC. Prospective, observational study. Lipid parameters, HDL composition, HDL-mediated cholesterol efflux, cholesteryl ester transfer protein, phospholipid transfer protein and lecithin cholesterol acyl-transferase (LCAT) activities were determined in ten patients with giant cell arteritis before and 3 months after GC introduction and in seven control subjects. HDL concentration and composition, HDL-mediated cholesterol efflux and LCAT activity were determined in GC-treated mice. In patients, HDL-C concentration was higher after than before treatment GC-treatment (P = 0.002), while HDL-mediated cholesterol efflux was decreased (P = 0.008) and negatively associated with the proportion of cholesteryl ester in HDL (P = 0.04), independently of CRP. As well, in mice, HDL-C level was increased after GC exposure (P = 0.04) and HDL-mediated cholesterol efflux decreased (P = 0.04). GC-treated patients had higher cholesteryl ester content in HDL, higher HDL2-to-HDL3 ratio and higher LCAT activity than before treatment (P = 0.008, P = 0.02 and P = 0.004, respectively). We report, for the first time, that in patients with giant cell arteritis and mice treated with GC, HDL-mediated cholesterol efflux was impaired by GC besides an increased HDL-C level. This impaired HDL functionality, possibly related to HDL enrichment in cholesteryl ester, could contribute to the increased CVD risk observed in GC-treated patients. Further studies are needed in larger populations, to further decipher the effect of GC on HDL.

SAT-586 Cold-Acclimation Prevents the Onset of Glucocorticoid-Induced Adipose Dysfunction in Male Mice

Glucocorticoid (GC) excess, either endogenously or exogenously, has been causally linked to the development of chronic diseases such as obesity and type-2 diabetes mellitus. Continuously elevated GC levels result in an expansion of white adipose tissue (WAT) depots and a dramatic decrease in the thermogenic capacity in brown and beige adipose tissue (BAT and BeAT, respectively). Herein we aimed to examine the interaction between GCs and the sympathetic nervous system (SNS) in the regulation of WAT, BAT, and BeAT. To this end, we utilized an altered environmental temperature as a non-invasive method for the modulation of sympathetic activity - cold is an activator of SNS-mediated non-shivering thermogenesis. Thus, during a 4-week treatment with either corticosterone or placebo, 10-week-old male C57BL/6NRj mice were maintained at two different temperature levels: 29°C (thermoneutrality) or 13°C (cold temperature). Body weight, as well as energy and water intake, were monitored throughout the study. At sacrifice, serum and adipose tissues were collected for analysis. GC-exposed mice showed a marked increase in circulating corticosterone concentrations compared to placebo controls with no appreciable difference between temperature levels. At thermoneutrality, GC-treated mice gained more weight and consumed more food than their placebo-treated littermates. This GC-induced body weight gain was accompanied by increased (visceral & subcutaneous) WAT weight as well as adipocyte hypertrophy. Interestingly, cold-acclimatized mice showed a marked reduction in GC-induced weight gain, hyperphagia, and fat accumulation. Moreover, the GC-induced rise in blood glucose and serum insulin concentrations - readily observed when mice were maintained at thermoneutrality - was absent in cold-exposed animals. In addition, GC treatment at thermoneutrality resulted in increased lipid deposition and decreased UCP1 expression in BAT, the ‘whitening’ of BAT. This GC-induced loss of thermogenic capacity was profoundly reduced in cold-adapted mice. Across all groups, UCP-1 protein levels in BAT closely correlated (r2 = 0.70, p < 0.0001) with those of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis. These results indicate that cold-acclimation prevents the development of GC-induced metabolic dysfunction in mice. Thus, environmental temperature is a potent modulator of GC-induced adiposity and body weight gain, potentially via an interaction between SNS and GC signaling.

The adiponectin receptor agonist AdipoRon normalizes glucose metabolism and prevents obesity but not growth retardation induced by glucocorticoids in young mice

ObjectiveGlucocorticoids (GCs) are highly effective anti-inflammatory and immunosuppressive drugs. However, prolonged GC therapy may cause numerous adverse effects leading to diabetes and obesity, as well as bone disorders such as osteoporosis in adults and growth retardation in children and adolescents. Prevention and care of the GC-induced adverse effects remain challenging. We have previously demonstrated the efficacy of a treatment with a non-peptidic agonist of adiponectin receptors, AdipoRon, to reverse behaviour disorders and fat mass gain induced by long-term GC treatment. In this work, we have established a relevant model of GC-induced growth and metabolic disorders and determined that AdipoRon is a potential therapeutic tool to reverse these metabolic disturbances. Methods5-Week-old mice were treated continuously with or without corticosterone (35 mg/L) in drinking water for seven consecutive weeks. Taking advantage of this mouse model displaying various growth and metabolic disorders, we assayed whether AdipoRon (daily intraperitoneal injection of 1 mg/kg/day for the last 20 days) might prevent the GC-induced adverse effects. The control group was treated with vehicle only. Nutritional behaviors and metabolic parameters were followed-up throughout the treatment. Serum insulin and leptin levels were measured by ELISA. Computed tomography and histological analysis of adipose tissue were assessed at the end of the experimental procedure. ResultsWe found that GC treatment in young mice resulted in continuously increased body weight gain associated with a food intake increase. Compared to vehicle-, GC-treated mice displayed early major hyperleptinemia (up to 6-fold more) and hyperinsulinemia (up to 20-fold more) maintained throughout the treatment. At the end of the experimental procedure, GC-treated mice displayed bone growth retardation (e.g. femur length 15.1 versus 14.0 mm, P < 0.01), higher abdominal adipose tissue volume (4.1 versus 2.3, P < 0.01) and altered glucose metabolism compared to control mice. Interestingly, AdipoRon prevented GC-induced effects on energy metabolism such as abdominal adiposity, insulinemia and leptinemia. However, AdipoRon failed to counteract bone growth retardation. ConclusionWe characterized the very early pathological steps induced by long-term GC in young mice in a relevant model, including growth retardation, fat mass gain and glucose homeostasis dysregulation. The adiponectin system stimulation enabled normalization of the adipose tissue and metabolic features of GC-treated mice. Adiponectin receptor agonists such as AdipoRon might constitute a novel way to counteract some GC-induced adverse effects.

Prevalence of glucocorticoid induced osteonecrosis in the mouse is not affected by treatments that maintain bone vascularity

ObjectiveDetermine if LLP2A-Ale or PTH (1–34) affects the prevalence of glucocorticoid-induced osteonecrosis (ON) in a mouse model. MethodsEight-week-old young adult male BALB/cJ mice were weight-randomized into Control (Con), glucocorticoid (GC)-only, or concurrent treatments with GC and LLP2A-Ale (250 μg/kg or 500 μg/kg, IV, Days 1, 14, 28) or parathyroid hormone hPTH (1–34) (40 μg/kg, 5×/week). Mice were necropsied after 45 days for qualitative evaluation of prevalent ON and quantitative evaluation of vascularity in the distal femoral epiphysis (DFE); and quantitative evaluation of bone mass, microarchitecture, and strength in the distal femoral metaphysis and lumbar vertebral body. ResultsThe prevalence of ON was 14% in the Con group and 36% in the GC-only group (P = 0.07). The prevalence of ON did not differ among GC-only, GC + LLP2A-Ale, and GC + PTH groups. GC-only mice had significantly lower trabecular and cortical bone strength than Con, while GC + LLP2A-Ale (500 μg/kg) and GC + PTH (1–34) groups had significantly greater trabecular bone strength than the GC-only group. GC + LLP2A-Ale (250 μg/kg and 500 μg/kg) and GC + PTH had significantly higher trabecular bone volume than GC-only mice at the vertebrae, distal femoral epiphyses and distal femoral metaphyses. DFE vascularity was lower in GC-only mice than in all other groups. ConclusionNeither LLP2A-Ale nor hPTH (1–34) reduced the prevalence of GC-induced ON, compared to GC-only mice. However, GC-treated mice given LLP2A-Ale or hPTH (1–34) had better bone mass, microarchitecture, and strength in trabecular-rich regions, and higher levels of vascularity than GC-only mice.

Glucocorticoids decrease the numbers and activation of mast cells by inducing the transactivation receptors of AGEs.

Glucocorticoids (GCs) are potent anti-allergic compounds that function, at least in part, by inhibiting signaling pathways in mast cells. We hypothesized that the GC-induced mastocytopenia and suppression of mast cell activation are mediated by the advanced glycation end products (AGEs)/receptors of AGEs (RAGEs) signaling axis. We evaluated the role of AGEs in GC-mediated mastocytopenia and impaired mast cell degranulation in male Wistar rats and Swiss-Webster mice subcutaneously injected with dexamethasone or prednisolone (0.1mg/kg) once a day for 21 consecutive days. The animals were treated with either the AGE inhibitor aminoguanidine (250mg/kg), the RAGE antagonist FPS-ZM1 (1mg/kg) or the galectin-3 antagonist GSC-100 (1mg/kg) daily for 18 days, starting 3 days following GC treatment. Aminoguanidine inhibited GC-induced mast cell apoptosis and restored mast cell numbers in the pleural cavity of GC-treated rats. Aminoguanidine also reversed the GC-induced reduction in histamine release triggered by allergens or compound 48/80 in vitro. GC treatment induced RAGE and galectin expression in mast cells, and blocking these agents by FPS-ZM1 or GSC-100 significantly reversed mast cell numbers in the peritoneal cavity and mesenteric tissue of GC-treated mice. In addition, the combination of GC and AGE-induced mast cell apoptosis in vitro was inhibited by both FPS-ZM1 and GSC-100. We concluded that the GC-induced mastocytopenia and suppression of mast cell stimulation are associated with the gene transactivation of RAGE and galectin-3.

NMR Metabolomics Show Evidence for Mitochondrial Oxidative Stress in a Mouse Model of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is associated with metabolic and endocrine disorders in women of reproductive age. The etiology of PCOS is still unknown. Mice prenatally treated with glucocorticoids exhibit metabolic disturbances that are similar to those seen in women with PCOS. We used an untargeted nuclear magnetic resonance (NMR)-based metabolomics approach to understand the metabolic changes occurring in the plasma and kidney over time in female glucocorticoid-treated (GC-treated) mice. There are significant changes in plasma amino acid levels (valine, tyrosine, and proline) and their intermediates (2-hydroxybutyrate, 4-aminobutyrate, and taurine), whereas in kidneys, the TCA cycle metabolism (citrate, fumarate, and succinate) and the pentose phosphate (PP) pathway products (inosine and uracil) are significantly altered (p < 0.05) from 8 to 16 weeks of age. Levels of NADH, NAD(+), NAD(+)/NADH, and NADH redox in kidneys indicate increased mitochondrial oxidative stress from 8 to 16 weeks in GC-treated mice. These results indicate that altered metabolic substrates in the plasma and kidneys of treated mice are associated with altered amino acid metabolism, increased cytoplasmic PP, and increased mitochondrial activity, leading to a more oxidized state. This study identifies biomarkers associated with metabolic dysfunction in kidney mitochondria of a prenatal gluococorticoid-treated mouse model of PCOS that may be used as early predictive biomarkers of oxidative stress in the PCOS metabolic disorder in women.

Galla Chinensis attenuates cigarette smoke-associated lung injury by inhibiting recruitment of inflammatory cells into the lung.

Inflammation is a common feature in the pathogenesis of cigarette smoke (CS)-associated diseases. In this study, we investigated the effects of Galla Chinensis (GC) extract on pulmonary inflammatory responses in a CS-exposed mouse model. In vitro studies showed that GC extract reduced MCP-1 production in a dose-dependent manner. In addition, the recruitment of inflammatory cells into the lung was significantly inhibited in the bronchoalveolar lavage fluid (BALF) of the GC-treated mice after 3 weeks of daily CS exposure. GC treatment down-regulated TNF-α, IL-6 and MCP-1 mRNA expression levels in lung tissue. Finally, GC-treated mice showed less emphysematous change of alveolar compared to mice only exposed to CS. Our results show that GC extract reduces lung inflammation and emphysematous change by inhibiting the infiltration of inflammatory cells to the lung. These data indicate that GC extract is a therapeutic candidate for CS-induced lung injury.

β-cell adaptation in a mouse model of glucocorticoid-induced metabolic syndrome

Glucocorticoids (GCs) are stress hormones primarily responsible for mobilizing glucose to the circulation. Due to this effect, insulin resistance and glucose intolerance are concerns in patients with endogenous overproduction of GCs and in patients prescribed GC-based therapy. In addition, hypercortisolemic conditions share many characteristics with the metabolic syndrome. This study reports on a thorough characterization, in terms of glucose control and lipid handling, of a mouse model where corticosterone is given via the drinking water. C57BL/6J mice were treated with corticosterone (100 or 25 μg/ml) or vehicle in their drinking water for 5 weeks after which they were subjected to insulin or glucose tolerance tests. GC-treated mice displayed increased food intake, body weight gain, and central fat deposit accumulations. In addition, the GC treatment led to dyslipidemia as well as accumulation of ectopic fat in the liver and skeletal muscle, having a substantial negative effect on insulin sensitivity. Also glucose intolerance and hypertension, both part of the metabolic syndrome, were evident in the GC-treated mice. However, the observed effects of corticosterone were reversed after drug removal. Furthermore, this study reveals insights into β-cell adaptation to the GC-induced insulin resistance. Increased pancreatic islet volume due to cell proliferation, increased insulin secretion capacity, and increased islet chaperone expression were found in GC-treated animals. This model mimics the human metabolic syndrome. It could be a valuable model for studying the complex mechanisms behind the development of the metabolic syndrome and type 2 diabetes, as well as the multifaceted relations between GC excess and disease.

Interleukin-21 Accelerates Thymic Recovery from Glucocorticoïd-Induced Atrophy

Both physiological and psychological stress cause thymic atrophy via glucocorticoïd (GC)-dependent apoptosis of double-positive (DP) thymocytes. Given the pervasiveness of stress, GC-induced thymic atrophy is arguably the most common type of acquired immunodeficiency. We recently reported that interleukin-21 (IL-21) has a unique ability to expand the small subset of DP thymocytes (CD69+) which are ongoing positive selection, and that administration of IL-21 increases thymic output in aged mice. The goal of this study was to evaluate whether IL-21 could mitigate GC-induced thymic atrophy. In contrast to double-negative (DN) and single-positive (SP) thymocytes, most DP thymocytes (CD69−) do not constitutively express the IL-21 receptor (IL-21R). Accordingly, CD69− DP thymocytes from PBS-treated mice were unresponsive to IL-21 administration. However, following GC injection, surviving CD69− DP thymocytes up-regulated IL-21R and responded to IL-21 treatment as evidenced by enhancement of Bcl6 expression and phosphorylation of STAT1, STAT3 and STAT5. Consequently, IL-21 administration to GC-treated mice accelerated thymic recovery by expanding considerably DP thymocytes and, to a lesser extent, DN thymocytes. However, IL-21-induced expansion of DN/DP thymocytes did not alter the diversity of the intrathymic or peripheral T-cell receptor (TCR) repertoire. We conclude that IL-21 dramatically accelerates recovery from GC-induced thymic atrophy.

Impaired liver regeneration by β‐glucosylceramide is associated with decreased fat accumulation

To investigate the effect of β-glucosylceramide (GC), a natural glycolipid, on hepatic fat accumulation and regenerative response after partial hepatectomy (PH). Male C57Bl/6 mice were assigned to either 70% PH or sham surgery after receiving daily intraperitoneal injection of GC or vehicle for 3 days. Hepatic fat accumulation, cytokines, cell cycle proteins and adipogenic genes expression were assessed at various time points after PH. The administration of GC delayed hepatic triglyceride accumulation during hepatic regeneration. This observation was closely correlated with alterations in the expression of four major adipogenic genes during the course of liver regeneration, with reduced expression 3 h after PH and increased expression 48 h post-surgery. GC significantly reduced hepatocellular proliferation 48 h after PH. In GC-treated mice, both tumor necrosis factor-α and interleukin-6 levels were lower 3, 48 and 72 h after PH compared with the control group. Administration of GC delayed hepatic triglyceride accumulation and suppressed early adipogenic gene expression during the hepatic regenerative response. These changes are closely associated with early inhibition of liver regeneration and temporal alteration of cytokine secretion.

Glucocorticoid-Treated Mice Are an Inappropriate Positive Control for Long-Term Preclinical Studies in the mdx Mouse

Background Dmdmdx (mdx) mice are used as a genetic and biochemical model of dystrophin deficiency. The long-term consequences of glucocorticoid (GC) treatment on dystrophin-deficient skeletal and heart muscle are not yet known. Here we used systematic phenotyping to assess the long-term consequences of GC treatment in mdx mice. Our investigation addressed not only the effects of GC on the disease phenotype but also the question of whether GCs can be used as a positive control for preclinical drug evaluations.Methods and FindingsWe performed nine pre-clinical efficacy trials (treated N = 129, untreated N = 106) of different durations in 9-to-50-week-old dystrophic mdx mice over a 3-year time period using standardized methods. In all these trials, we used either 1 mg/kg body weight of prednisone or 5 mg/kg body weight of prednisolone as positive controls to compare the efficacy of various test drugs. Data from untreated controls and GC-treated mice in the various trials have been pooled and analyzed to assess the effects of GCs on dystrophin-deficient skeletal and cardiac muscles of mdx mice. Our results indicate that continuous GC treatment results in early (e.g., at 50 days) improvements in normalized parameters such as grip strength, motor coordination and maximal in vitro force contractions on isolated EDL muscle, but these initial benefits are followed by a progressive loss of muscle strength after 100 days. We also found a significant increase in heart fibrosis that is reflected in a significant deterioration in cardiac systolic function after 100 days of treatment.ConclusionContinuous administration of prednisone to mdx mice initially improves skeletal muscle strength, but further therapy result in deterioration of muscle strength and cardiac function associated with enhanced cardiac fibrosis. These results suggest that GCs may not serve as an appropriate positive control for long-term mdx mouse preclinical trials.

Concomitant administration of nitric oxide and glucocorticoids improves protection against bronchoconstriction in a murine model of asthma

Glucocorticoids (GC) remain the first choice of treatment in asthma, but GC therapy is not always effective and is associated with side effects. In a porcine study in our laboratory, simultaneous administration of GC and nitric oxide (NO) attenuated the endotoxin-induced inflammatory response and made GC treatment more effective than inhaled NO or steroids alone. In the present study, we aimed to further investigate the interactions between NO and GC treatment in two murine models of asthma. Inflammation was induced by endotoxin, ovalbumin, or a combination of both. With an animal ventilator and a forced oscillation method (FlexiVent), lung mechanics and airway reactivity to methacholine in response to various treatments were assessed. We also describe histology and glucocorticoid receptor (GR) protein expression in response to inhaled NO treatment [40 ppm NO gas or NO donors sodium nitroprusside (SNP) or diethylamine NONOate (DEA/NO)]. SNP and GC provided protection against bronchoconstriction to a similar degree in the model of severe asthma. When GC-treated mice were given SNP, maximum airway reactivity was further reduced. Similar effects were seen after DEA/NO delivery to GC-treated animals. Using 1-H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ), a soluble guanylate cyclase inhibitor, we found this effect of NO donors to be mediated through a cGMP-independent mechanism. In the severe model, prolonged NO treatment restored or even increased the nuclear levels of GR. In conclusion, in our murine model of severe asthma GC treatment provided protection to only a limited degree against bronchoconstriction, while concomitant treatment with a NO donor was markedly more potent than the use of either NO or GC alone.

Glucocorticoid Attenuates the Anabolic Effects of Parathyroid Hormone on Fracture Repair

Long-term use of glucocorticoid (GC) not only reduces bone mass and strength, which leads to a greater risk of fracture, but also hinders fracture repair. In this study, we produced open fractures in GC-treated mice and investigated the effects of human parathyroid hormone 1-34 (hPTH) on fracture repair. Swiss-Webster mice were randomly divided into five groups. Three groups of GC-treated mice were given prednisolone, which was slowly released from subcutaneously implanted pellets at the rate of 1.4 mg/kg/day. Placebo pellets were implanted into the animals in two placebo groups. Three weeks later, osteotomies at the midshaft femora were performed and intramedullary pins were inserted to stabilize the fracture site under general anesthesia. Following fracture surgery, three GC groups were treated subcutaneously with vehicle, PTH at a low dose (40 ug/kg/day), and PTH at a high dose (80 ug/kg/day), respectively. Two placebo groups were given vehicle and PTH at a dose of 40 ug/kg/day, respectively. Radiographs, dual-energy X-ray absorptiometry, and mechanical testing (four-point bending) were used to evaluate fracture repair at 4 weeks after fracture surgery. Callus development, endochondral ossification, and recovery of mechanical strength at the fracture sites in GC animals treated with vehicle were significantly suppressed compared to placebo animals. Normally, PTH accelerates fracture repair. In GC-treated mice, PTH fails to improve endochondral ossification and mechanical properties compared to vehicle treatment, suggesting that the anabolic effect of PTH on fracture healing can be attenuated by GC administration in mice.

Role of the N- and C-terminal Fragments of Parathyroid-Hormone-Related Protein as Putative Therapies to Improve Bone Regeneration Under High Glucocorticoid Treatment

The parathyroid-hormone-related protein (PTHrP) is an important modulator of bone formation and bone remodeling. High and/or prolonged glucocorticoid (GC) treatments inhibit PTHrP expression in osteoblastic cells and bone formation and repair. We assessed the ability of the N- and C-terminal PTHrP fragments to restore the GC-altered bone regeneration after bone marrow ablation in mice. Animals were administered 3-methylprednisolone or vehicle and PTHrP (1-36) or PTHrP (107-139) every other day, beginning 4 days before marrow ablation in the tibia, and euthanized 12 days later. GC-treated mice showed in the ablated tibia a decrease in bone formation and in osteoblast and sclerostin-positive osteocyte numbers, reduced expression of osteoblastic factors, decreased osteogenesis of bone-marrow-derived cells, an increase in the numbers of multinucleated osteoclasts and adipocytes, and decreased cortical vascularization, as well as altered bone structure (measured by microcomputerized tomography) in the intact femur. These effects were reversed at least in part by either PTHrP peptide. The present novel findings support the use of both PTHrP peptides tested as putative bone regenerative therapies in GC-related bone diseases.

Activators of PPARs and LXR decrease the adverse effects of exogenous glucocorticoids on the epidermis

While glucocorticoids (GC) exert beneficial effects (anti-inflammatory), they also have adverse effects on the epidermis including decreased epidermal differentiation, decreased keratinocyte proliferation, and decreased cutaneous permeability barrier homeostasis. Thus, the purpose of this study was to develop strategies to prevent these GC toxicities using simultaneous topical treatments in clobetasol-treated mice. While a triple-lipid mixture of stratum corneum lipids (ceramide, free fatty acid and cholesterol) was previously shown to reverse the GC-induced abnormality in cutaneous barrier function [J Invest Dermatol, 120 (2003) 456], this lipid mixture did not prevent the GC-induced abnormalities in either keratinocyte proliferation or differentiation. As activators of PPARalpha, beta/delta, gamma and LXR, regulate keratinocyte proliferation and differentiation and improve permeability barrier homeostasis, we next assessed the effects of these activators during concurrent GC treatment. Co-application of either ciglitazone (PPARgamma activator), clofibrate (PPARalpha activator) or 22R (OH) cholesterol (LXR activator) with clobetasol prevented the decrease in involucrin, filaggrin and loricrin expression. By contrast, a PPARbeta/delta activator (GW501516) normalized only the expression of involucrin and filaggrin but not loricrin. Moreover, topical application of PPARalpha, beta/delta or LXR activators partially prevented the decrease in keratinocyte proliferation in GC-treated murine skin, as measured using PCNA, while no effect was seen after co-treatment with PPARgamma activators. Finally, PPARgamma and PPARbeta/delta activators but not PPARalpha and LXR activators improved permeability barrier homeostasis in GC-treated mice. Together, these studies demonstrate that PPAR and LXR activators can prevent several of the adverse effects of topical GC on the epidermis.

β-Glucosylceramide ameliorates liver inflammation in murine autoimmune cholangitis

We have demonstrated spontaneous development of autoimmune cholangitis, similar to human primary biliary cirrhosis, in mice expressing a dominant negative form of the transforming growth factor-beta receptor (dnTGF-betaRII) restricted to T cells. The autoimmune cholangitis appears to be mediated by autoreactive CD8(+) T lymphocytes that home to the portal tracts and biliary system. Because the liver pathology is primarily secondary to CD8(+) T cells, we have determined herein whether administration of beta-glucosylceramide (GC), a naturally occurring plant glycosphingolipid, alters the natural history of disease in this model. We chose GC because previous work has demonstrated its ability to alter CD8(+) T cell responses and to down-regulate tissue inflammation. Accordingly, dnTGF-betaRII mice were treated with either GC or control for a period of 18 weeks beginning at 6 weeks of age. Importantly, in mice that received GC, there was a significant decrease in the frequency and absolute number of autoreactive liver-infiltrating CD8(+) T cells, accompanied by a significant decrease in activated CD44(high) CD8(+) T cell populations. Further, there was a significant reduction in portal inflammation in GC-treated mice. Interestingly, there were no changes in anti-mitochondrial antibodies, CD4(+) T cells, CD19(+) B cells or natural killer (NK) T cell populations, indicating further that the beneficial effects of GC on liver inflammation were targeted specifically to liver-infiltrating CD8(+) T cells. These data suggest that further work on GC in models of CD8(+) T-mediated inflammation are needed and point to a new therapeutic venue for potentially treating and/or modulating autoimmune disease.

Glucocorticoid excess in mice results in early activation of osteoclastogenesis and adipogenesis and prolonged suppression of osteogenesis: A longitudinal study of gene expression in bone tissue from glucocorticoid‐treated mice

Glucocorticoid (GC) excess induces alterations in bone metabolism that weaken bone structure and increase fracture risk. The aim of this study was to identify genes associated with bone metabolism in GC-treated mice, by performing a microarray analysis. Long bones from mice exposed to GC excess were collected after 0, 7, 28, and 56 days of treatment, to measure bone microarchitecture and extract RNA for microarray analyses. Bone loss in this animal model was confirmed by changes in bone turnover markers as well as bone architecture, as measured by microfocal computed tomography. GC excess induced an early up-regulation of genes involved in osteoclast activation, function, and adipogenesis, which peaked on day 7. The expression of genes associated with osteoclast cytoskeletal reorganization and genes associated with matrix degradation peaked on day 28. On day 28 and day 56, the expression of genes associated with osteoblast activation and maturation was decreased from baseline, while the expression of Wnt antagonists was increased. In addition, the expression of genes expressed in osteocytes associated with bone mineralization was significantly higher at the later time points, day 28 and day 56. Reverse transcription-polymerase chain reaction confirmed the results of microarray analysis in selected genes. GC excess is associated with early activation of genes associated with osteoclastogenesis and adipogenesis and a later suppression of genes associated with osteogenesis and mineralization. Novel interventions with agents that modulate either Wnt signaling or mineralization may be effective in GC-induced osteoporosis.

Natural glucocorticoids induce expansion of all developmental stages of murine bone marrow granulocytes without inhibiting function

Natural glucocorticoids (Gc) produced during stress have profound effects on the immune system. It is well known that Gc induce apoptosis in precursor T and B cells, markedly altering lymphopoiesis. However, it has been noted that marrow myeloid cells expanded both in proportion and absolute numbers in the mouse after Gc exposure. Mice were implanted with a corticosterone (CS) tablet that increased serum Gc and caused atrophied thymuses, both classic signs of activation of the stress axis. Blood neutrophil counts were elevated (4.8x), whereas lymphocyte counts declined. Flow cytometric analysis of the marrow revealed that the phenotypic distribution of the various major classes of cells was shifted by Gc exposure. As expected, marrow lymphocyte numbers declined >40% after 3 days of exposure to Gc. Conversely, in the myeloid compartment, both monocytes and granulocytes increased in number by >40%. Further, all granulocyte developmental stages showed large increases in both total number and percentage of cells. To investigate the functional capacity of mature granulocytes from Gc-treated mice, an improved granulocyte isolation method was developed. Gc exposure had little effect on the ability of granulocytes to produce superoxide or undergo chemotaxis or phagocytose bacteria. These results indicate that Gc treatment shifts bone marrow composition and provides evidence that granulocytes and their progenitors are selectively preserved under stressful conditions without losing function.