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1.
J Appl Toxicol ; 44(3): 400-414, 2024 Mar.
Article in English | MEDLINE | ID: mdl-37814191

ABSTRACT

Small intestinal epithelial vacuolation induced by a heteroaryldihydropyrimidine compound (HAP-1) was observed in rats but not in dogs at termination in screening toxicity studies, despite the plasma exposure being higher in dogs. To understand the species differences, investigational studies with multiple time points following single dose (SD) and 7-day repeated dose (RD) were conducted in both species at doses resulting in comparable plasma exposures. In rats, epithelial vacuolation in the duodenum and jejunum were observed at all time points. In dogs, transient vacuolation was noted at 8 h post-SD (SD_8h) and 4 h post-RD (RD_4 h), but not at termination (RD_24 h). Special stains demonstrated lipid accumulation within enterocytes in both species and intracytoplasmic inclusion bodies in rats. Transmission electron microscopy identified these inclusion bodies as endoplasmic reticulum (ER) membranous structures. Transcriptomic analysis on jejunal mucosa at SD_8 h and RD_24 h revealed perturbations of lipid metabolism-related genes at SD_8 h in both species, but not at RD_24 h in dogs. ER stress-related gene changes at both time points were observed in rats only. Despite comparable HAP-1 plasma exposures, the duodenum and jejunum tissue concentrations of HAP-1 and acyl glucuronide metabolite were >5- and >30-fold higher in rats than in dogs, respectively. In vitro, similar cytotoxicity was observed in rat and dog duodenal organoids treated with HAP-1. In conclusion, HAP-1-induced intestinal epithelial vacuolation was related to lipid metabolism dysregulation in both species and ER-related injuries in rats only. The species differences were likely related to the difference in intestinal exposure to HAP-1 and its reactive metabolite.


Subject(s)
Intestine, Small , Pyrimidines , Rats , Dogs , Animals , Species Specificity
2.
Elife ; 122023 Dec 08.
Article in English | MEDLINE | ID: mdl-38063302

ABSTRACT

The maintenance of the functional integrity of the intestinal epithelium requires a tight coordination between cell production, migration, and shedding along the crypt-villus axis. Dysregulation of these processes may result in loss of the intestinal barrier and disease. With the aim of generating a more complete and integrated understanding of how the epithelium maintains homeostasis and recovers after injury, we have built a multi-scale agent-based model (ABM) of the mouse intestinal epithelium. We demonstrate that stable, self-organizing behaviour in the crypt emerges from the dynamic interaction of multiple signalling pathways, such as Wnt, Notch, BMP, ZNRF3/RNF43, and YAP-Hippo pathways, which regulate proliferation and differentiation, respond to environmental mechanical cues, form feedback mechanisms, and modulate the dynamics of the cell cycle protein network. The model recapitulates the crypt phenotype reported after persistent stem cell ablation and after the inhibition of the CDK1 cycle protein. Moreover, we simulated 5-fluorouracil (5-FU)-induced toxicity at multiple scales starting from DNA and RNA damage, which disrupts the cell cycle, cell signalling, proliferation, differentiation, and migration and leads to loss of barrier integrity. During recovery, our in silico crypt regenerates its structure in a self-organizing, dynamic fashion driven by dedifferentiation and enhanced by negative feedback loops. Thus, the model enables the simulation of xenobiotic-, in particular chemotherapy-, induced mechanisms of intestinal toxicity and epithelial recovery. Overall, we present a systems model able to simulate the disruption of molecular events and its impact across multiple levels of epithelial organization and demonstrate its application to epithelial research and drug development.


Subject(s)
Intestinal Mucosa , Intestines , Mice , Animals , Cell Proliferation/physiology , Intestinal Mucosa/metabolism , Cell Differentiation/physiology , Homeostasis/physiology
3.
CPT Pharmacometrics Syst Pharmacol ; 12(10): 1511-1528, 2023 10.
Article in English | MEDLINE | ID: mdl-37621010

ABSTRACT

We have built a quantitative systems toxicology modeling framework focused on the early prediction of oncotherapeutic-induced clinical intestinal adverse effects. The model describes stem and progenitor cell dynamics in the small intestinal epithelium and integrates heterogeneous epithelial-related processes, such as transcriptional profiles, citrulline kinetics, and probability of diarrhea. We fitted a mouse-specific version of the model to quantify doxorubicin and 5-fluorouracil (5-FU)-induced toxicity, which included pharmacokinetics and 5-FU metabolism and assumed that both drugs led to cell cycle arrest and apoptosis in stem cells and proliferative progenitors. The model successfully recapitulated observations in mice regarding dose-dependent disruption of proliferation which could lead to villus shortening, decrease of circulating citrulline, increased diarrhea risk, and transcriptional induction of the p53 pathway. Using a human-specific epithelial model, we translated the cytotoxic activity of doxorubicin and 5-FU quantified in mice into human intestinal injury and predicted with accuracy clinical diarrhea incidence. However, for gefitinib, a specific-molecularly targeted therapy, the mice failed to reproduce epithelial toxicity at exposures much higher than those associated with clinical diarrhea. This indicates that, regardless of the translational modeling approach, preclinical experimental settings have to be suitable to quantify drug-induced clinical toxicity with precision at the structural scale of the model. Our work demonstrates the usefulness of translational models at early stages of the drug development pipeline to predict clinical toxicity and highlights the importance of understanding cross-settings differences in toxicity when building these approaches.


Subject(s)
Citrulline , Drug-Related Side Effects and Adverse Reactions , Mice , Humans , Animals , Fluorouracil/toxicity , Fluorouracil/metabolism , Intestinal Mucosa/metabolism , Diarrhea/chemically induced , Doxorubicin/toxicity
4.
Eur J Pharm Sci ; 188: 106481, 2023 Sep 01.
Article in English | MEDLINE | ID: mdl-37244450

ABSTRACT

Intestinal organoids derived from LGR5+ adult stem cells allow for long-term culturing, more closely resemble human physiology than traditional intestinal models, like Caco-2, and have been established for several species. Here we evaluated intestinal organoids for drug disposition, metabolism, and safety applications. Enterocyte-enriched human duodenal organoids were cultured as monolayers to enable bidirectional transport studies. 3D enterocyte-enriched human duodenal and colonic organoids were incubated with probe substrates of major intestinal drug metabolizing enzymes (DMEs). To distinguish human intestinal toxic (high incidence of diarrhea in clinical trials and/or black box warning related to intestinal side effects) from non-intestinal toxic compounds, ATP-based cell viability was used as a readout, and compounds were ranked based on their IC50 values in relation to their 30-times maximal total plasma concentration (Cmax). To assess if rat and dog organoids reproduced the respective in vivo intestinal safety profiles, ATP-based viability was assessed in rat and dog organoids and compared to in vivo intestinal findings when available. Human duodenal monolayers discriminated high and low permeable compounds and demonstrated functional activity for the main efflux transporters Multi drug resistant protein 1 (MDR1, P-glycoprotein P-gp) and Breast cancer resistant protein (BCRP). Human 3D duodenal and colonic organoids also showed metabolic activity for the main intestinal phase I and II DMEs. Organoids derived from specific intestinal segments showed activity differences in line with reported DMEs expression. Undifferentiated human organoids accurately distinguished all but one compound from the test set of non-toxic and toxic drugs. Cytotoxicity in rat and dog organoids correlated with preclinical toxicity findings and observed species sensitivity differences between human, rat, and dog organoids. In conclusion, the data suggest intestinal organoids are suitable in vitro tools for drug disposition, metabolism, and intestinal toxicity endpoints. The possibility to use organoids from different species, and intestinal segment holds great potential for cross-species and regional comparisons.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1 , Neoplasm Proteins , Adult , Humans , Animals , Dogs , Rats , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Caco-2 Cells , Organoids , Adenosine Triphosphate
5.
Mol Cell Endocrinol ; 514: 110891, 2020 08 20.
Article in English | MEDLINE | ID: mdl-32533994

ABSTRACT

Renal calcium and phosphate handling is an important contributor to mineral homeostasis and bone health and the androgen receptor (AR) is highly expressed in the kidney. We investigated the short term effects of androgen deprivation on renal calcium and phosphate reabsorption, independent of their effects on bone. Two weeks following orchidectomy (ORX) of adult mice, bone loss occurred along with hypercalciuria, which was similarly prevented by testosterone and dihydrotestosterone supplementation. Treatment with bisphosphonates prior to ORX also inhibited hypercalciuria, indicating that the calcium flux originated from the bone. Renal calcium and phosphate transporter expression was increased post-ORX, independent of bisphosphonates. Furthermore, androgen deprivation appeared to stimulate local synthesis of 1,25(OH)2D3. When bisphosphonate-treated mice were fed a low calcium diet, bone resorption was no longer blocked and secondary hyperparathyroidism developed, which was more pronounced in ORX mice than sham-operated mice. In conclusion, this study shows that androgen deprivation increased renal calcium and phosphate transporter expression, independent of bone, and underlines the importance of adequate intestinal calcium supply in circumstances of androgen deprivation and bisphosphonate treatment.


Subject(s)
Androgens/pharmacology , Calcium, Dietary/pharmacology , Calcium/metabolism , Diphosphonates/pharmacology , Kidney/drug effects , Phosphates/metabolism , Animals , Bone Density/drug effects , Bone Resorption/metabolism , Bone Resorption/pathology , Bone and Bones/drug effects , Bone and Bones/metabolism , Diet , Kidney/metabolism , Male , Mice , Mice, Inbred C57BL , Orchiectomy , Urinalysis
6.
FASEB J ; 34(5): 7118-7126, 2020 05.
Article in English | MEDLINE | ID: mdl-32239553

ABSTRACT

Sexually dimorphic bone structure emerges largely during puberty. Sex steroids are critical for peak bone mass acquisition in both genders. In particular, the biphasic effects of estrogens mediate the skeletal sexual dimorphism. However, so far the stimulatory vs inhibitory actions of estrogens on bone mass are not fully explained by direct effects on bone cells. Recently, it has become evident that there is possible neuroendocrine action of estrogen receptor alpha (ERα) on the skeleton. Based on these considerations, we hypothesized that neuronal ERα-signaling may contribute to the skeletal growth during puberty. Here, we generated mice with tamoxifen-inducible Thy1-Cre mediated ERα inactivation during late puberty specifically in extrahypothalamic neurons (N-ERαKO). Inactivation of neuronal ERα did not alter the body weight in males, whereas N-ERαKO females exhibited a higher body weight and increased body and bone length compared to their control littermates at 16 weeks of age. Ex vivo microCT analysis showed increased radial bone expansion of the midshaft femur in female N-ERαKO along with higher serum levels of insulin-like growth factor (IGF)-1 as well as IGF-binding protein (IGFBP)-3. Furthermore, the 3-point bending test revealed increased bone strength in female N-ERαKO. In contrast, inactivation of neuronal ERα had no major effect on bone growth in males. In conclusion, we demonstrate that central ERα-signaling limits longitudinal bone growth and radial bone expansion specifically in females potentially by interacting with the GH/IGF-1 axis.


Subject(s)
Bone Development/physiology , Estrogen Receptor alpha/metabolism , Neurons/metabolism , Sexual Maturation/physiology , Animals , Biomechanical Phenomena , Bone Density/genetics , Bone Density/physiology , Bone Development/genetics , Bone and Bones/anatomy & histology , Bone and Bones/diagnostic imaging , Bone and Bones/physiology , Estrogen Receptor alpha/deficiency , Estrogen Receptor alpha/genetics , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sex Characteristics , Sexual Maturation/genetics , Signal Transduction , X-Ray Microtomography
7.
Front Chem ; 7: 475, 2019.
Article in English | MEDLINE | ID: mdl-31334221

ABSTRACT

Novel antibiotic compounds have been prepared through a selective multicomponent reaction upon the known drug Trimethoprim. The Groebke-Blackburn-Bienaymé reaction involving this α-aminoazine, with a range of aldehydes and isocyanides afforded the desired adducts in one-step. The analogs display meaningful structural features of the initial drug together with relevant modifications at several points, keeping antibiotic potency and showing satisfactory antimicrobial profile (good activity levels and reduced growth rates), especially against methicillin-resistant Staphylococcus aureus. The new products may open new possibilities to fight bacterial infections.

8.
J Bone Miner Res ; 34(3): 508-519, 2019 03.
Article in English | MEDLINE | ID: mdl-30496619

ABSTRACT

Androgens via the androgen receptor (AR) are required for optimal male bone health. The target cell(s) for the effects of androgens on cortical bone remain(s) incompletely understood. In females, estrogen receptor alpha in neurons is a negative regulator of cortical and trabecular bone. Whether neuronal AR regulates bone mass in males remains unexplored. Here, we inactivated AR in neurons using a tamoxifen-inducible CreERT2 under the control of the neuronal promoter Thy1. Tamoxifen induced a 70% to 80% reduction of AR mRNA levels in Thy1-CreERT2-positive brain regions cerebral cortex and brainstem as well as in the peripheral nervous tissue of male neuronal AR knockout (N-ARKO) mice. Hypothalamic AR mRNA levels were only marginally reduced and the hypothalamic-pituitary-gonadal axis remained unaffected, as determined by normal levels of serum testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). In contrast to orchidectomy, deletion of neuronal AR did not alter body weight, body composition, hindlimb muscle mass, grip strength, or wheel running. MicroCT analysis of the femur revealed no changes in bone accrual during growth in N-ARKO mice. However, 36- and 46-week-old N-ARKO mice displayed an accelerated age-related cortical involution, namely a more pronounced loss of cortical thickness and strength, which occurred in the setting of androgen sufficiency. Neuronal AR inactivation decreased the cancellous bone volume fraction in L5 vertebra but not in the appendicular skeleton of aging mice. MicroCT findings were corroborated in the tibia and after normalization of hormonal levels. Serum markers of bone turnover and histomorphometry parameters were comparable between genotypes, except for a 30% increase in osteoclast surface in the trabecular compartment of 36-week-old N-ARKO mice. Cortical bone loss in N-ARKO mice was associated with an upregulation of Ucp1 expression in brown adipose tissue, a widely used readout for sympathetic tone. We conclude that androgens preserve cortical integrity in aging male mice via AR in neurons. © 2018 American Society for Bone and Mineral Research.


Subject(s)
Aging/pathology , Cortical Bone/pathology , Neurons/metabolism , Receptors, Androgen/metabolism , Animals , Body Composition , Body Weight , Bone Resorption/pathology , Cancellous Bone/pathology , Femur/pathology , Gene Deletion , Gonads/metabolism , Hypothalamo-Hypophyseal System/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Muscles/metabolism , Osteogenesis , Uncoupling Protein 1/metabolism
9.
J Endocrinol ; 238(1): R31-R52, 2018 07.
Article in English | MEDLINE | ID: mdl-29743340

ABSTRACT

Physical inactivity is a pandemic that contributes to several chronic diseases and poses a significant burden on health care systems worldwide. The search for effective strategies to combat sedentary behavior has led to an intensification of the research efforts to unravel the biological substrate controlling activity. A wide body of preclinical evidence makes a strong case for sex steroids regulating physical activity in both genders, albeit the mechanisms implicated remain unclear. The beneficial effects of androgens on muscle as well as on other peripheral functions might play a role in favoring adaptation to exercise. Alternatively or in addition, sex steroids could act on specific brain circuitries to boost physical activity. This review critically discusses the evidence supporting a role for androgens and estrogens stimulating male physical activity, with special emphasis on the possible role of peripheral and/or central mechanisms. Finally, the potential translation of these findings to humans is briefly discussed.


Subject(s)
Androgens/pharmacology , Brain/drug effects , Estrogens/pharmacology , Exercise/physiology , Muscles/drug effects , Sex Characteristics , Androgens/metabolism , Animals , Estrogens/metabolism , Female , Gonadal Steroid Hormones/metabolism , Gonadal Steroid Hormones/pharmacology , Humans , Male , Nerve Net/drug effects , Nerve Net/physiology
10.
Sci Rep ; 8(1): 957, 2018 01 17.
Article in English | MEDLINE | ID: mdl-29343749

ABSTRACT

Low testosterone (T) in men, especially its free fraction, has been associated with loss of energy. In accordance, orchidectomy (ORX) in rodents results in decreased physical activity. Still, the mechanisms through which T stimulates activity remain mostly obscure. Here, we studied voluntary wheel running behavior in three different mouse models of androgen deficiency: ORX, androgen receptor (AR) knock-out (ARKO) and sex hormone binding globulin (SHBG)-transgenic mice, a novel mouse model of "low free T". Our results clearly show a fast and dramatic action of T stimulating wheel running, which is not explained by its action on muscle, as evidenced by neuromuscular studies and in a muscle-specific conditional ARKO mouse model. The action of T occurs via its free fraction, as shown by the results in SHBG-transgenic mice, and it implies both androgenic and estrogenic pathways. Both gene expression and functional studies indicate that T modulates the in vivo sensitivity to dopamine (DA) agonists. Furthermore, the restoration of wheel running by T is inhibited by treatment with DA antagonists. These findings reveal that the free fraction of T, both via AR and indirectly through aromatization into estrogens, stimulates physical activity behavior in male mice by acting on central DA pathways.


Subject(s)
Dopamine/metabolism , Dopaminergic Neurons/metabolism , Physical Conditioning, Animal/physiology , Testosterone/metabolism , Androgens/metabolism , Animals , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic/metabolism , Motor Activity/physiology , Orchiectomy/methods , Receptors, Androgen/metabolism , Running/physiology
12.
Mol Cell Endocrinol ; 465: 61-72, 2018 04 15.
Article in English | MEDLINE | ID: mdl-29155307

ABSTRACT

Calcium and phosphate are vital for the organism and constitute essential components of the skeleton. Serum levels are tightly hormonally regulated and maintained by exchange with three major sources: the intestines, the kidney and the bone. The effects of sex steroids on the bone have been extensively studied and it is well known that sex steroid deficiency induces bone loss, indirectly influencing renal calcium and phosphate homeostasis. However, it is unknown whether sex steroids also directly regulate renal calcium and phosphate handling, hereby potentially indirectly impacting on bone. The presence of androgen receptors (AR) and estrogen receptors (ER) in both human and rodent kidney, although their exact localization within the kidney remains debated, supports direct effects. Estrogens stimulate renal calcium reabsorption as well as phosphate excretion, while the effects of androgens are less clear. Many of the studies performed with regard to renal calcium and/or phosphate homeostasis do not correct for the calcium and phosphate fluxes from the bone and intestines, which complicates the differentiation between the direct effects of sex steroids on renal calcium and phosphate handling and the indirect effects via the bone and intestines. The objective of this study is to review the literature and current insight of the role of sex steroids in calcium and phosphate handling in the kidney.


Subject(s)
Calcium/metabolism , Gonadal Steroid Hormones/metabolism , Kidney/metabolism , Phosphates/metabolism , Animals , Humans , Models, Biological
13.
Mol Cell Endocrinol ; 452: 57-63, 2017 09 05.
Article in English | MEDLINE | ID: mdl-28504114

ABSTRACT

The selective estrogen receptor modulator tamoxifen exerts estrogen agonistic or antagonistic actions on several tissues, including bone. The off-target effects of tamoxifen are one of the most widely recognized pitfalls of tamoxifen-inducible Cre recombinases (CreERs), potentially confounding the phenotypic findings. Still, the validation of tamoxifen induction schemes that minimize the side effects of the drug has not been addressed. Here, we compared the side effects on the skeleton and other androgen-responsive targets of a shortened tamoxifen regimen (2 doses of 190 mg/kg body weight by oral gavage) to a standard protocol (4 doses) and determined their efficiency in inducing CreER-mediated gene deletion. In addition, both a vehicle- and a 10-dose group, which served as a positive control for tamoxifen side effects, were also included. For this purpose, we generated male mice with a floxed androgen receptor (AR) and a neuron-specifically expressed CreER. Treatment with two doses of tamoxifen was the only regimen that did not diminish androgenic bioactivity, as assessed by both seminal vesicles and levator ani/bulbocavernosus muscle weights and serum testosterone concentrations. Similarly, trabecular and cortical femoral bone structure were dramatically altered by both the standard and high-dose protocols but not by the shortened version. Serum osteocalcin and bone-gene expression analyses confirmed the absence of effects on bone by 2 doses of tamoxifen. This protocol decreased AR mRNA levels efficiently and specifically in the nervous system. Thus, we optimized a protocol for tamoxifen-induced CreER gene deletion in mice without off-target effects on bone and male reproductive organs.


Subject(s)
Bone and Bones/drug effects , Gene Deletion , Gene Knockout Techniques , Integrases/metabolism , Recombination, Genetic/drug effects , Selective Estrogen Receptor Modulators/pharmacology , Tamoxifen/pharmacology , Analysis of Variance , Animals , Body Weight/drug effects , Cancellous Bone/drug effects , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Estrogens/agonists , Integrases/genetics , Male , Mice , Mice, Inbred C57BL , Osteocalcin/blood , Osteocalcin/genetics , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Selective Estrogen Receptor Modulators/administration & dosage , Selective Estrogen Receptor Modulators/adverse effects , Seminal Vesicles , Tamoxifen/administration & dosage , Tamoxifen/adverse effects , Testosterone/blood , Time Factors
14.
Sci Rep ; 6: 35539, 2016 10 17.
Article in English | MEDLINE | ID: mdl-27748448

ABSTRACT

Sex hormone-binding globulin (SHBG) is the high-affinity binding protein for androgens and estrogens. According to the free hormone hypothesis, SHBG modulates the bioactivity of sex steroids by limiting their diffusion into target tissues. Still, the in vivo physiological role of circulating SHBG remains unclear, especially since mice and rats lack circulating SHBG post-natally. To test the free hormone hypothesis in vivo, we examined total and free sex steroid concentrations and bioactivity on target organs in mice expressing a human SHBG transgene. SHBG increased total androgen and estrogen concentrations via hypothalamic-pituitary feedback regulation and prolonged ligand half-life. Despite markedly raised total sex steroid concentrations, free testosterone was unaffected while sex steroid bioactivity on male and female reproductive organs was attenuated. This occurred via a ligand-dependent, genotype-independent mechanism according to in vitro seminal vesicle organ cultures. These results provide compelling support for the determination of free or bioavailable sex steroid concentrations in medicine, and clarify important comparative differences between translational mouse models and human endocrinology.


Subject(s)
Androgens/metabolism , Models, Biological , Sex Hormone-Binding Globulin/metabolism , Animals , Estrogens/metabolism , Female , Half-Life , Humans , Hypertrophy , Hypogonadism/pathology , Ligands , Male , Mice, Inbred C57BL , Mice, Transgenic , Morphogenesis , Phenotype , Reproducibility of Results , Steroids/pharmacokinetics , Tissue Distribution
15.
Bone ; 93: 33-42, 2016 12.
Article in English | MEDLINE | ID: mdl-27622887

ABSTRACT

Aging hypogonadal men are at increased risk of osteoporosis and sarcopenia. Testosterone is a potentially appealing strategy to prevent simultaneous bone and muscle loss. The androgen receptor (AR) mediates antiresorptive effects on trabecular bone via osteoblast-lineage cells, as well as muscle-anabolic actions. Sex steroids also modify the skeletal response to mechanical loading. However, it is unclear whether the effects of androgens on bone remain effective independent of mechanical stimulation or rather require indirect androgen effects via muscle. This study aims to characterize the effects and underlying mechanisms of androgens on disuse osteosarcopenia. Adult male mice received a unilateral botulinum toxin (BTx) injection, and underwent sham surgery or orchidectomy (ORX) without or with testosterone (ORX+T) or dihydrotestosterone (ORX+DHT) replacement. Compared to the contralateral internal control hindlimb, acute trabecular number and bone volume loss was increased by ORX and partially prevented DHT. T was more efficient and increased BV/TV in both hindlimbs over sham values, although it did not reduce the detrimental effect of BTx. Both androgens and BTx regulated trabecular osteoclast surface as well as tartrate-resistant acid phosphatase expression. Androgens also prevented BTx-induced body weight loss but did not significantly influence paralysis or muscle atrophy. BTx and ORX both reduced cortical thickness via endosteal expansion, which was prevented by T but not DHT. In long-term follow-up, the residual trabecular bone volume deficit in sham-BTx hindlimbs was prevented by DHT but T restored it more efficiently to pre-treatment levels. Conditional AR deletion in late osteoblasts and osteocytes or in the satellite cell lineage increased age-related trabecular bone loss in both hindlimbs without influencing the effect of BTx on trabecular osteopenia. We conclude that androgens have antiresorptive effects on trabecular disuse osteopenia which do not require AR actions on bone via muscle or via osteocytes.


Subject(s)
Androgens/therapeutic use , Bone Diseases, Metabolic/drug therapy , Bone Resorption/drug therapy , Cancellous Bone/pathology , Muscular Atrophy/drug therapy , Muscular Disorders, Atrophic/drug therapy , Acute Disease , Androgens/pharmacology , Animals , Body Weight , Bone Diseases, Metabolic/complications , Bone Diseases, Metabolic/pathology , Bone Diseases, Metabolic/physiopathology , Bone Remodeling/drug effects , Bone Resorption/complications , Bone Resorption/pathology , Bone Resorption/physiopathology , Calcification, Physiologic , Cancellous Bone/diagnostic imaging , Cancellous Bone/drug effects , Cancellous Bone/physiopathology , Cortical Bone/diagnostic imaging , Cortical Bone/drug effects , Cortical Bone/pathology , Cortical Bone/physiopathology , Extracellular Matrix Proteins/metabolism , Female , Gene Deletion , Integrases/metabolism , Male , Mice, Inbred C57BL , Muscular Atrophy/complications , Muscular Atrophy/pathology , Muscular Atrophy/physiopathology , Muscular Disorders, Atrophic/complications , Muscular Disorders, Atrophic/pathology , Muscular Disorders, Atrophic/physiopathology , MyoD Protein/metabolism , Organ Size , Receptors, Androgen/metabolism , X-Ray Microtomography
16.
Endocrinology ; 157(2): 648-65, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26562264

ABSTRACT

Androgen deficiency is associated with obesity, metabolic syndrome, and type 2 diabetes mellitus in men, but the mechanisms behind these associations remain unclear. In this study, we investigated the combined effects of androgen deficiency and high-fat diet (HFD) on body composition and glucose homeostasis in C57BL/6J male mice. Two models of androgen deficiency were used: orchidectomy (ORX) and androgen receptor knockout mice. Both models displayed higher adiposity and serum leptin levels upon HFD, whereas no differences were seen on a regular diet. Fat accumulation in HFD ORX animals was accompanied by increased sedentary behavior and occurred in spite of reduced food intake. HFD ORX mice showed white adipocyte hypertrophy, correlated with decreased mitochondrial content but not function as well as increased lipogenesis and decreased lipolysis suggested by the up-regulation of fatty acid synthase and the down-regulation of hormone-sensitive lipase. Both ORX and androgen receptor knockout exacerbated HFD-induced glucose intolerance by impairing insulin action in liver and skeletal muscle, as evidenced by the increased triglyceride and decreased glycogen content in these tissues. In addition, serum IL-1ß levels were elevated, and pancreatic insulin secretion was impaired after ORX. Testosterone but not dihydrotestosterone supplementation restored the castration effects on body composition and glucose homeostasis. We conclude that sex steroid deficiency in combination with HFD exacerbates adiposity, insulin resistance, and ß-cell failure in 2 preclinical male mouse models. Our findings stress the importance of a healthy diet in a clinical context of androgen deficiency and may have implications for the prevention of metabolic alterations in hypogonadal men.


Subject(s)
Androgens/deficiency , Diet, High-Fat , Glucose Intolerance/etiology , Obesity/etiology , Receptors, Androgen/genetics , Adiposity/genetics , Animals , Body Composition/genetics , Disease Progression , Glucose Intolerance/genetics , Glucose Intolerance/metabolism , Glucose Intolerance/pathology , Hypogonadism/complications , Hypogonadism/genetics , Hypogonadism/pathology , Insulin Resistance/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Obesity/genetics , Obesity/metabolism , Obesity/pathology
17.
Mol Cell Endocrinol ; 432: 14-36, 2016 09 05.
Article in English | MEDLINE | ID: mdl-26506009

ABSTRACT

Bone is a biomechanical tissue shaped by forces from muscles and gravitation. Simultaneous bone and muscle decay and dysfunction (osteosarcopenia or sarco-osteoporosis) is seen in ageing, numerous clinical situations including after stroke or paralysis, in neuromuscular dystrophies, glucocorticoid excess, or in association with vitamin D, growth hormone/insulin like growth factor or sex steroid deficiency, as well as in spaceflight. Physical exercise may be beneficial in these situations, but further work is still needed to translate acceptable and effective biomechanical interventions like vibration therapy from animal models to humans. Novel antiresorptive and anabolic therapies are emerging for osteoporosis as well as drugs for sarcopenia, cancer cachexia or muscle wasting disorders, including antibodies against myostatin or activin receptor type IIA and IIB (e.g. bimagrumab). Ideally, increasing muscle mass would increase muscle strength and restore bone loss from disuse. However, the classical view that muscle is unidirectionally dominant over bone via mechanical loading is overly simplistic. Indeed, recent studies indicate a role for neuronal regulation of not only muscle but also bone metabolism, bone signaling pathways like receptor activator of nuclear factor kappa-B ligand (RANKL) implicated in muscle biology, myokines affecting bone and possible bone-to-muscle communication. Moreover, pharmacological strategies inducing isolated myocyte hypertrophy may not translate into increased muscle power because tendons, connective tissue, neurons and energy metabolism need to adapt as well. We aim here to critically review key musculoskeletal molecular pathways involved in mechanoregulation and their effect on the bone-muscle unit as a whole, as well as preclinical and emerging clinical evidence regarding the effects of sarcopenia therapies on osteoporosis and vice versa.


Subject(s)
Bone and Bones/physiology , Models, Animal , Muscles/physiology , Animals , Endocrine System/metabolism , Humans , Signal Transduction , Weight-Bearing
18.
Endocrinology ; 156(12): 4522-33, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26393303

ABSTRACT

Androgens increase skeletal muscle mass, but their clinical use is hampered by a lack of tissue selectivity and subsequent side effects. Selective androgen receptor modulators elicit muscle-anabolic effects while only sparingly affecting reproductive tissues. The selective androgen receptor modulator, GTx-024 (enobosarm), is being investigated for cancer cachexia, sarcopenia, and muscle wasting diseases. Here we investigate the role of muscle androgen receptor (AR) in the anabolic effect of GTx-024. In mice lacking AR in the satellite cell lineage (satARKO), the weight of the androgen-sensitive levator ani muscle was lower but was decreased further upon orchidectomy. GTx-024 was as effective as DHT in restoring levator ani weights to sham levels. Expression of the muscle-specific, androgen-responsive genes S-adenosylmethionine decarboxylase and myostatin was decreased by orchidectomy and restored by GTx-024 and DHT in control mice, whereas the expression was low and unaffected by androgen status in satARKO. In contrast, insulin-like growth factor 1Ea expression was not different between satARKO and control muscle, decreased upon castration, and was restored by DHT and GTx-024 in both genotypes. These data indicate that GTx-024 does not selectively modulate AR in the satellite cell lineage and that cells outside this lineage remain androgen responsive in satARKO muscle. Indeed, residual AR-positive cells were present in satARKO muscle, coexpressing the fibroblast-lineage marker vimentin. AR positive, muscle-resident fibroblasts could therefore be involved in the indirect effects of androgens on muscle. In conclusion, both DHT and GTx-024 target AR pathways in the satellite cell lineage, but cells outside this lineage also contribute to the anabolic effects of androgens.


Subject(s)
Amides/pharmacology , Anabolic Agents/pharmacology , Body Composition/drug effects , Muscle, Skeletal/drug effects , Receptors, Androgen/metabolism , Satellite Cells, Skeletal Muscle/drug effects , Androgens/pharmacology , Anilides , Animals , Cachexia/drug therapy , Cell Lineage , Dihydrotestosterone/pharmacology , Male , Mice , Mice, Knockout , Muscle, Skeletal/metabolism , Orchiectomy , Organ Size/drug effects , Receptors, Androgen/genetics , Sarcopenia/drug therapy , Satellite Cells, Skeletal Muscle/metabolism , Wasting Syndrome/drug therapy
19.
Mol Cell Endocrinol ; 411: 198-206, 2015 Aug 15.
Article in English | MEDLINE | ID: mdl-25958043

ABSTRACT

Androgen deficiency or androgen receptor knockout (ARKO) causes high-turnover osteopenia, but the target cells for this effect remain unclear. To examine whether AR in osteoclasts directly suppresses bone resorption, we crossed AR-floxed with cathepsin K-Cre mice. Osteoclast-specific ARKO (ocl-ARKO) mice showed no changes neither in osteoclast surface nor in bone microarchitecture nor in the response to orchidectomy and androgen replacement, indicating that the AR in osteoclasts is not critical for bone maintenance. In line with the lack of a bone phenotype, the levels of AR were very low in osteoclast-enriched cultures derived from bone marrow (BM) and undetectable in osteoclasts generated from spleen precursors. Since tibiae of ubiquitous ARKO mice displayed increased osteoclast counts, the role of AR was further explored using cell cultures from these animals. Osteoclast generation and activity in vitro were similar between ARKO and wildtype control (WT) mice. In co-culture experiments, BM stromal cells (BMSCs) were essential for the suppressive action of AR on osteoclastogenesis and osteoclast activity. Stimulation with 1,25(OH)2 vitamin D3 increased Rankl and decreased Tnfsf11 (osteoprotegerin, Opg) gene expression in BMSCs more than in osteoblasts. This increase in the Rankl/Opg ratio following 1,25(OH)2D3 stimulation was lower, not higher, in ARKO mice. Runx2 expression in BMSCs was however higher in ARKO vs. WT, suggesting that ARKO mice may more readily commit osteoprogenitor cells to osteoblastogenesis. In conclusion, the AR does not seem to suppress bone resorption through direct actions in osteoclasts. BMSCs may however represent an alternative AR target in the BM milieu.


Subject(s)
Bone Resorption/metabolism , Osteoclasts/metabolism , Receptors, Androgen/metabolism , Acid Phosphatase/blood , Animals , Cells, Cultured , Femur/cytology , Femur/metabolism , Isoenzymes/blood , Male , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mice , Mice, Knockout , Osteocalcin/blood , Osteoclasts/cytology , Receptors, Androgen/genetics , Tartrate-Resistant Acid Phosphatase , Tibia/cytology , Tibia/metabolism
20.
J Neurogastroenterol Motil ; 21(2): 222-35, 2015 Mar 30.
Article in English | MEDLINE | ID: mdl-25843075

ABSTRACT

BACKGROUND/AIMS: Enteric dysbiosis is a risk factor for dietary proteins-associated intestinal alterations, contributing to the development of food allergies and the symptomatology of functional gastrointestinal disorders, mainly irritable bowel syndrome (IBS). We explored if a dysbiotic-like state, simulated by intraperitoneal administration of bacterial lipopolysaccharides (LPS), facilitates the sensitiza-tion to a luminal antigen, ovalbumin (OVA), in rats. METHODS: Rats were exposed to oral OVA for 1 week, alone or with LPS. Thereafter, colonic histology, goblet cell density, mucosal eosi-nophils and mucosal mast cell (MMC) and connective tissue mast cell (CTMC) were evaluated. Colonic expression (real-time quantitative polymerase chain reaction) of interleukins, IFN-α1 and integrins was assessed to determine local immune responses. Luminal and wall adhered microbiota were characterized by fluorescence in situ hybridization. Colonic contractility (in vitro) served to assess functional changes associated to OVA and/or LPS. RESULTS: Neither OVA nor LPS, alone or combined, lead to structural alterations, except for a reduced goblet cell density in OVA-LPS- treated rats. MMC density was unaffected, while CTMC counts increased within the submucosa of OVA-LPS-treated animals. Marginal immune activation (IFN-α1 up-regulation) was observed in OVA-LPS-treated rats. LPS induced a dysbiotic-like state characterized by decreased luminal bacterial counts, with a specific loss of clostridia. LPS facilitated Clostridium spp. wall adherence, an effect prevented by OVA. Colonic contractility was altered in OVA-LPS-treated animals, showing increased basal activity and enhanced motor responses to OVA. CONCLUSIONS: Changes in gut microbiota and/or direct effects of LPS might enhance/facilitate local neuroimmune responses to food antigens leading to motor alterations similar to those observed in IBS.

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