Body weight homeostat that regulates fat mass independently of leptin in rats and mice.

Subjects spending much time sitting have increased risk of obesity but the mechanism for the antiobesity effect of standing is unknown. We hypothesized that there is a homeostatic regulation of body weight. We demonstrate that increased loading of rodents, achieved using capsules with different weights implanted in the abdomen or s.c. on the back, reversibly decreases the biological body weight via reduced food intake. Importantly, loading relieves diet-induced obesity and improves glucose tolerance. The identified homeostat for body weight regulates body fat mass independently of fat-derived leptin, revealing two independent negative feedback systems for fat mass regulation. It is known that osteocytes can sense changes in bone strain. In this study, the body weight-reducing effect of increased loading was lost in mice depleted of osteocytes. We propose that increased body weight activates a sensor dependent on osteocytes of the weight-bearing bones. This induces an afferent signal, which reduces body weight. These findings demonstrate a leptin-independent body weight homeostat ("gravitostat") that regulates fat mass.

The effects of estradiol are modulated in a tissue-specific manner in mice with inducible inactivation of ERα after sexual maturation.

Mouse models with lifelong inactivation of estrogen receptor-α (ERα) show that ERα is the main mediator of estrogenic effects in bone, thymus, uterus, and fat. However, ERα inactivation early in life may cause developmental effects that confound the adult phenotypes. To address the specific role of adult ERα expression for estrogenic effects in bone and other nonskeletal tissues, we established a tamoxifen-inducible ERα-inactivated model by crossing CAGG-Cre-ER and ERαflox/flox mice. Tamoxifen-induced ERα inactivation after sexual maturation substantially reduced ERα mRNA levels in cortical bone, trabecular bone, thymus, uterus, gonadal fat, and hypothalamus, in CAGG-Cre-ERαflox/flox (inducible ERαKO) compared with ERαflox/flox (control) mice. 17ß-estradiol (E2) treatment increased trabecular bone volume fraction (BV/TV), cortical bone area, and uterine weight, while it reduced thymus weight and fat mass in ovariectomized control mice. The estrogenic responses were substantially reduced in inducible ERαKO mice compared with control mice on BV/TV (-67%), uterine weight (-94%), thymus weight (-70%), and gonadal fat mass (-94%). In contrast, the estrogenic response on cortical bone area was unaffected in inducible ERαKO compared with control mice. In conclusion, using an inducible ERαKO model, not confounded by lack of ERα during development, we demonstrate that ERα expression in sexually mature female mice is required for normal E2 responses in most, but not all, tissues. The finding that cortical, but not trabecular bone, responds normally to E2 treatment in inducible ERαKO mice strengthens the idea of cortical and trabecular bone being regulated by estrogen via different mechanisms.

Vitamin A decreases the anabolic bone response to mechanical loading by suppressing bone formation.

Increased vitamin A consumption is associated with decreased cortical bone mass and increased fracture risk in humans. Rodent studies have demonstrated that hypervitaminosis A increases cortical bone resorption, whereas the importance of the effects on bone formation is less well defined. We used an experimental model of increased bone formation by loading of the tibiae to investigate the effect of vitamin A on bone formation. Control [retinol activity equivalents (RAE) 4.5 µg/g chow] or vitamin A (RAE 60 µg/g chow) diets were given to female C57BL/6N mice for 4 wk, after which the tibiae were subjected to axial loading on alternate days for 2 wk, while the diets were continued. Vitamin A inhibited the loading-induced increase in trabecular and cortical bone volume. This was attributed to inhibition of loading-induced increase in osteoblast number and activity, and expression of osteoblastic genes Sp7, Alpl, and Col1a1 in cortical bone. Vitamin A, loading, and combination thereof also resulted in site-specific effects on bone composition measured by Raman spectroscopy. In summary, a clinically relevant dose of vitamin A suppresses the loading-induced gain of bone mass by decreasing bone formation. These observations may have implications for regulation of bone mass caused by physical activity and the risk of osteoporosis in humans.-Lionikaite, V., Henning, P., Drevinge, C., Shah, F. A., Palmquist, A., Wikström, P., Windahl, S. H., Lerner, U. H. Vitamin A decreases the anabolic bone response to mechanical loading by suppressing bone formation.

The bone-sparing effects of estrogen and WNT16 are independent of each other.

Wingless-type MMTV integration site family (WNT)16 is a key regulator of bone mass with high expression in cortical bone, and Wnt16(-/-) mice have reduced cortical bone mass. As Wnt16 expression is enhanced by estradiol treatment, we hypothesized that the bone-sparing effect of estrogen in females is WNT16-dependent. This hypothesis was tested in mechanistic studies using two genetically modified mouse models with either constantly high osteoblastic Wnt16 expression or no Wnt16 expression. We developed a mouse model with osteoblast-specific Wnt16 overexpression (Obl-Wnt16). These mice had several-fold elevated Wnt16 expression in both trabecular and cortical bone compared with wild type (WT) mice. Obl-Wnt16 mice displayed increased total body bone mineral density (BMD), surprisingly caused mainly by a substantial increase in trabecular bone mass, resulting in improved bone strength of vertebrae L3. Ovariectomy (ovx) reduced the total body BMD and the trabecular bone mass to the same degree in Obl-Wnt16 mice and WT mice, suggesting that the bone-sparing effect of estrogen is WNT16-independent. However, these bone parameters were similar in ovx Obl-Wnt16 mice and sham operated WT mice. The role of WNT16 for the bone-sparing effect of estrogen was also evaluated in Wnt16(-/-) mice. Treatment with estradiol increased the trabecular and cortical bone mass to a similar extent in both Wnt16(-/-) and WT mice. In conclusion, the bone-sparing effects of estrogen and WNT16 are independent of each other. Furthermore, loss of endogenous WNT16 results specifically in cortical bone loss, whereas overexpression of WNT16 surprisingly increases mainly trabecular bone mass. WNT16-targeted therapies might be useful for treatment of postmenopausal trabecular bone loss.

Increased adipose tissue aromatase activity improves insulin sensitivity and reduces adipose tissue inflammation in male mice.

Females are, in general, more insulin sensitive than males. To investigate whether this is a direct effect of sex-steroids (SS) in white adipose tissue (WAT), we developed a male mouse model overexpressing the aromatase enzyme, converting testosterone (T) to estradiol (E2), specifically in WAT (Ap2-arom mice). Adipose tissue E2 levels were increased while circulating SS levels were unaffected in male Ap2-arom mice. Importantly, male Ap2-arom mice were more insulin sensitive compared with WT mice and exhibited increased serum adiponectin levels and upregulated expression of Glut4 and Irs1 in WAT. The expression of markers of macrophages and immune cell infiltration was markedly decreased in WAT of male Ap2-arom mice. The adipogenesis was enhanced in male Ap2-arom mice, supported by elevated Pparg expression in WAT and enhanced differentiation of preadipocyte into mature adipocytes. In summary, increased adipose tissue aromatase activity reduces adipose tissue inflammation and improves insulin sensitivity in male mice. We propose that estrogen increases insulin sensitivity via a local effect in WAT on adiponectin expression, adipose tissue inflammation, and adipogenesis.

The estrogen receptor antagonist ICI 182,780 can act both as an agonist and an inverse agonist when estrogen receptor α AF-2 is modified.

The bone-sparing effect of estrogen is primarily mediated via estrogen receptor (ER) α, which stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal and AF-2 in the ligand-binding domain. It was recently demonstrated that the ER antagonist ICI 182,780 (ICI) acts as an ER agonist in uterus of mice with mutations in the ERα AF-2. To evaluate the estrogen-like effects of ICI in different tissues, ovariectomized wild-type mice and mice with mutations in the ERα AF-2 (ERαAF-2(0)) were treated with ICI, estradiol, or vehicle for 3 wk. Estradiol increased the trabecular and cortical bone mass as well as the uterine weight, whereas it reduced fat mass, thymus weight, and the growth plate height in wild-type but not in ERαAF-2(0) mice. Although ICI had no effect in wild-type mice, it exerted tissue-specific effects in ERαAF-2(0) mice. It acted as an ERα agonist on trabecular bone mass and uterine weight, whereas no effect was seen on cortical bone mass, fat mass, or thymus weight. Surprisingly, a pronounced inverse agonistic activity was seen on the growth plate height, resulting in enhanced longitudinal bone growth. In conclusion, ICI uses ERα AF-1 in a tissue-dependent manner in mice lacking ERαAF-2, resulting in no effect, agonistic activity, or inverse agonistic activity. We propose that ERα lacking AF-2 is constitutively active in the absence of ligand in the growth plate, enabling ICI to act as an inverse agonist.

Liver-derived IGF-I regulates cortical bone mass but is dispensable for the osteogenic response to mechanical loading in female mice.

Low circulating IGF-I is associated with increased fracture risk. Conditional depletion of IGF-I produced in osteoblasts or osteocytes inhibits the bone anabolic effect of mechanical loading. Here, we determined the role of endocrine IGF-I for the osteogenic response to mechanical loading in young adult and old female mice with adult, liver-specific IGF-I inactivation (LI-IGF-I(-/-) mice, serum IGF-I reduced by ≈70%) and control mice. The right tibia was subjected to short periods of axial cyclic compressive loading three times/wk for 2 wk, and measurements were performed using microcomputed tomography and mechanical testing by three-point bending. In the nonloaded left tibia, the LI-IGF-I(-/-) mice had lower cortical bone area and increased cortical porosity, resulting in reduced bone mechanical strength compared with the controls. Mechanical loading induced a similar response in LI-IGF-I(-/-) and control mice in terms of cortical bone area and trabecular bone volume fraction. In fact, mechanical loading produced a more marked increase in cortical bone mechanical strength, which was associated with a less marked increase in cortical porosity, in the LI-IGF-I(-/-) mice compared with the control mice. In conclusion, liver-derived IGF-I regulates cortical bone mass, cortical porosity, and mechanical strength under normal (nonloaded) conditions. However, despite an ∼70% reduction in circulating IGF-I, the osteogenic response to mechanical loading was not attenuated in the LI-IGF-I(-/-) mice.

SERMs have substance-specific effects on bone, and these effects are mediated via ERαAF-1 in female mice.

The bone-sparing effect of estrogens is mediated primarily via estrogen receptor (ER)α, which stimulates gene transcription through activation function (AF)-1 and AF-2. The role of ERαAF-1 for the estradiol (E2) effects is tissue specific. The selective ER modulators (SERMs) raloxifene (Ral), lasofoxifene (Las), and bazedoxifene (Bza) can be used to treat postmenopausal osteoporosis. They all reduce the risk for vertebral fractures, whereas Las and partly Bza, but not Ral, reduce the risk for nonvertebral fractures. Here, we have compared the tissue specificity of Ral, Las, and Bza and evaluated the role of ERαAF-1 for the effects of these SERMs, with an emphasis on bone parameters. We treated ovariectomized (OVX) wild-type (WT) mice and OVX mice lacking ERαAF-1 (ERαAF-1(0)) with E2, Ral, Las, or Bza. All three SERMs increased trabecular bone mass in the axial skeleton. In the appendicular skeleton, only Las increased the trabecular bone volume/tissue volume and trabecular number, whereas both Ral and Las increased the cortical bone thickness and strength. However, Ral also increased cortical porosity. The three SERMs had only a minor effect on uterine weight. Notably, all evaluated effects of these SERMs were absent in ovx ERαAF-1(0) mice. In conclusion, all SERMs had similar effects on axial bone mass. However, the SERMs had slightly different effects on the appendicular skeleton since only Las increased the trabecular bone mass and only Ral increased the cortical porosity. Importantly, all SERM effects require a functional ERαAF-1 in female mice. These results could lead to development of more specific treatments for osteoporosis.

Estrogen receptor-α in osteocytes is important for trabecular bone formation in male mice.

The bone-sparing effect of estrogen in both males and females is primarily mediated via estrogen receptor-α (ERα), encoded by the Esr1 gene. ERα in osteoclasts is crucial for the trabecular bone-sparing effect of estrogen in females, but it is dispensable for trabecular bone in male mice and for cortical bone in both genders. We hypothesized that ERα in osteocytes is important for trabecular bone in male mice and for cortical bone in both males and females. Dmp1-Cre mice were crossed with ERα(flox/flox) mice to generate mice lacking ERα protein expression specifically in osteocytes (Dmp1-ERα(-/-)). Male Dmp1-ERα(-/-) mice displayed a substantial reduction in trabecular bone volume (-20%, P < 0.01) compared with controls. Dynamic histomorphometry revealed reduced bone formation rate (-45%, P < 0.01) but the number of osteoclasts per bone surface was unaffected in the male Dmp1-ERα(-/-) mice. The male Dmp1-ERα(-/-) mice had reduced expression of several osteoblast/osteocyte markers in bone, including Runx2, Sp7, and Dmp1 (P < 0.05). Gonadal intact Dmp1-ERα(-/-) female mice had no significant reduction in trabecular bone volume but ovariectomized Dmp1-ERα(-/-) female mice displayed an attenuated trabecular bone response to supraphysiological E2 treatment. Dmp1-ERα(-/-) mice of both genders had unaffected cortical bone. In conclusion, ERα in osteocytes regulates trabecular bone formation and thereby trabecular bone volume in male mice but it is dispensable for the trabecular bone in female mice and the cortical bone in both genders. We propose that the physiological trabecular bone-sparing effect of estrogen is mediated via ERα in osteocytes in males, but via ERα in osteoclasts in females.

Identification of proteins highly expressed in uterine fluid from mice with hydrometra.

Estrogen receptor alpha (ERα) is an important regulator of the estrous cycle and mice with global ERα deletion, as well as some conditional knockout mouse lines, have an interruption in the estrous cycle. In this study we observed that conditional ERα knockout mice where the Cre gene is regulated by the rat insulin promoter (RIP), RIP-Cre/ERα(KO) mice, have a 3.7-fold increase in serum 17ß-estradiol levels, blocked estrous cycle, and develop a fluid-filled uterus (hydrometra). Using a proteomics approach, we identified three proteins, lactoferrin, complement C3 and chitinase 3-like protein 1 (CHI3L1), as highly expressed proteins in hydrometra fluid. The mRNA levels of the corresponding genes were more than 50-fold higher in RIP-Cre/ERα(KO) uterus compared to controls. High expression of CHI3L1 in the uterine fluid was not reflected as elevated levels in the serum. The high expression of lactoferrin, complement C3 and CHI3L1 in the uterine fluid, in association with elevated estrogen levels, prompted us to address if the expression of these genes is related to reproduction. However, gonadotropin treatment of mice reduced the uterine expression of these genes in a model of in vitro fertilization. Our findings identify lactoferrin, complement C3 and CHI3L1 as highly expressed proteins in hydrometra fluid in association with chronically elevated serum estradiol levels.

Estrogen receptor-α expression in neuronal cells affects bone mass.

It has generally been assumed that bone mass is controlled by endocrine mechanisms and the local bone environment. Recent findings demonstrate that central pathways are involved in the regulation of bone mass. Estrogen is involved in the regulation of bone homeostasis and the CNS is also a target for estrogen actions. The aim of this study was to investigate in vivo the role of central estrogen receptor-α (ERα) expression for bone mass. Nestin-Cre mice were crossed with ERα(flox) mice to generate mice lacking ERα expression specifically in nervous tissue (nestin-ERα(-/-)). Bone mineral density was increased in both the trabecular and cortical bone compartments in nestin-ERα(-/-) mice compared with controls. Femoral bone strength was increased in nestin-ERα(-/-) mice, as demonstrated by increased stiffness and maximal load of failure. The high bone mass phenotype in nestin-ERα(-/-) mice was mainly caused by increased bone formation. Serum leptin levels were elevated as a result of increased leptin expression in white adipose tissue (WAT) and slightly increased amount of WAT in nestin-ERα(-/-) mice. Leptin receptor mRNA levels were reduced in the hypothalamus but not in bone. In conclusion, inactivation of central ERα signaling results in increased bone mass, demonstrating that the balance between peripheral stimulatory and central inhibitory ERα actions is important for the regulation of bone mass. We propose that the increased bone mass in nestin-ERα(-/-) mice is mediated via decreased central leptin sensitivity and thereby increased secretion of leptin from WAT, which, in turn, results in increased peripheral leptin-induced bone formation.

The effect of estrogen on bone requires ERα in nonhematopoietic cells but is enhanced by ERα in hematopoietic cells.

The effects of estrogen on bone are mediated mainly via estrogen receptor (ER)α. ERα in osteoclasts (hematopoietic origin) is involved in the trabecular bone-sparing effects of estrogen, but conflicting data are reported on the role of ERα in osteoblast lineage cells (nonhematopoietic origin) for bone metabolism. Because Cre-mediated cell-specific gene inactivation used in previous studies might be confounded by nonspecific and/or incomplete cell-specific ERα deletion, we herein used an alternative approach to determine the relative importance of ERα in hematopoietic (HC) and nonhematopoietic cells (NHC) for bone mass. Chimeric mice with selective inactivation of ERα in HC or NHC were created by bone marrow transplantations of wild-type (WT) and ERα-knockout (ERα(-/-)) mice. Estradiol treatment increased both trabecular and cortical bone mass in ovariectomized WT/WT (defined as recipient/donor) and WT/ERα(-/-) mice but not in ERα(-/-)/WT or ERα(-/-)/ERα(-/-) mice. However, estradiol effects on both bone compartments were reduced (∼50%) in WT/ERα(-/-) mice compared with WT/WT mice. The effects of estradiol on fat mass and B lymphopoiesis required ERα specifically in NHC and HC, respectively. In conclusion, ERα in NHC is required for the effects of estrogen on both trabecular and cortical bone, but these effects are enhanced by ERα in HC.

RANKL-dependent osteoclast differentiation and gene expression in bone marrow-derived cells from adult mice is sexually dimorphic.

Sex-specific differences in bone integrity and properties are associated with age as well as the number and activity of cells involved in bone remodeling. The aim of this study was to investigate sex-specific differences in adhesion, proliferation, and differentiation of mouse bone marrow derived cells into osteoclasts. The adherent fraction of bone marrow- derived cells from 12-week-old male and female C57BL/6J mice were assessed for their adhesion, proliferation, and receptor activator of nuclear factor κB (RANKL)-induced differentiation into osteoclasts. Female bone marrow derived macrophages (BMDMs) displayed higher adhesion and proliferation ratio upon macrophage colony stimulating factor (M-CSF) (day 0) and M-CSF + RANKL (day 4) treatment, respectively. On the contrary, male BMDMs differentiated more efficiently into osteoclasts upon RANKL-treatment compared to females (day 5). To further understand these sex-specific differences at the gene expression level, BMDMs treated with M-CSF (day 0) and M-CSF + RANKL (day 4), were assessed for their differential expression of genes through RNA sequencing. M-CSF treatment resulted in 1106 differentially expressed genes, while RANKL-treatment gave 473 differentially expressed genes. Integrin, adhesion, and proliferation-associated genes were elevated in the M-CSF-treated female BMDMs. RANKL-treatment further enhanced the expression of the proliferation- associated genes, and of genes associated with inhibition of osteoclast differentiation in the females, while RANK-signaling-associated genes were upregulated in males. In conclusion, BMDM adhesion, proliferation and differentiation into osteoclasts are sex-specific and may be directed by the PI3K-Akt signaling pathway for proliferation, and the colony stimulating factor 1-receptor and the RANKLsignaling pathway for the differentiation.

A COX-2 Inhibitor Does Not Interfere With the Bone-Protective Effects of Loading in Male Mice With Arthritis.

Mechanical loading enhances bone strength and counteracts arthritis-induced inflammation-mediated bone loss in female mice. It is unknown whether nonsteroidal anti-inflammatory drugs (NSAIDs; eg, COX-2 inhibitors) can reduce inflammation without affecting the loading-associated bone formation in male mice. The aim of this study was to investigate if loading combined with a COX-2 inhibitor (NS-398) could prevent arthritis-induced bone loss and inflammation in male mice. Four-month-old male C57BL/6J mice were subjected to axial tibial mechanical loading three times/week for 2 weeks. Local mono-arthritis was induced with a systemic injection of methylated bovine serum albumin on the first day of loading, followed by a local injection in one knee 1 week later. The arthritis induction, knee swelling, bone architecture, and osteoclast number were evaluated in the hind limbs. C-terminal cross-links as a marker for osteoclast activity was measured in serum. Compared with loading and arthritis alone, loading of the arthritic joint enhanced swelling that was partly counteracted by NS-398. Loading of the arthritic joint enhanced synovitis and articular cartilage damage compared with loading alone. Loading increased cortical bone and counteracted the arthritis-induced decrease in epiphyseal bone. NS-398 did not alter the bone-protective effects of loading. C-terminal cross-links, a bone resorption marker, was increased by arthritis but not loading. In conclusion, loading prevented arthritis-induced epiphyseal and metaphyseal bone loss, and NS-398 reduced knee swelling without affecting the bone-protective effects of loading. If our results can be extrapolated to the human situation, specific COX-2 inhibitors could be used in combination with loading exercise to prevent pain and swelling of the joint without influencing the bone-protective effects of loading. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

STAT3 Hyperactivation Due to SOCS3 Deletion in Murine Osteocytes Accentuates Responses to Exercise- and Load-Induced Bone Formation.

Cortical bone develops and changes in response to mechanical load, which is sensed by bone-embedded osteocytes. The bone formation response to load depends on STAT3 intracellular signals, which are upregulated after loading and are subject to negative feedback from Suppressor of Cytokine Signaling 3 (Socs3). Mice with Dmp1Cre-targeted knockout of Socs3 have elevated STAT3 signaling in osteocytes and display delayed cortical bone maturation characterized by impaired accrual of high-density lamellar bone. This study aimed to determine whether these mice exhibit an altered response to mechanical load. The approach used was to test both treadmill running and tibial compression in female Dmp1Cre.Socs3f/f mice. Treadmill running for 5 days per week from 6 to 11 weeks of age did not change cortical bone mass in control mice, but further delayed cortical bone maturation in Dmp1Cre.Socs3f/f mice; accrual of high-density bone was suppressed, and cortical thickness was less than in genetically-matched sedentary controls. When strain-matched anabolic tibial loading was tested, both control and Dmp1Cre.Socs3f/f mice exhibited a significantly greater cortical thickness and periosteal perimeter in loaded tibia compared with the contralateral non-loaded bone. At the site of greatest compressive strain, the loaded Dmp1Cre.Socs3f/f tibias showed a significantly greater response than controls, indicated by a greater increase in cortical thickness. This was due to a greater bone formation response on both periosteal and endocortical surfaces, including formation of abundant woven bone on the periosteum. This suggests a greater sensitivity to mechanical load in Dmp1Cre.Socs3f/f bone. In summary, mice with targeted SOCS3 deletion and immature cortical bone have an exaggerated response to both physiological and experimental mechanical loads. We conclude that there is an optimal level of osteocytic response to mechanical load required for cortical bone maturation and that load-induced bone formation may be increased by augmenting STAT3 signaling within osteocytes. © 2021 American Society for Bone and Mineral Research (ASBMR).

Phosphorylation of S122 in ERα is important for the skeletal response to estrogen treatment in male mice.

Estrogen receptor alpha (ERα) signaling has beneficial skeletal effects in males. ERα signaling also affects other tissues, and to find bone-specific treatments, more knowledge regarding tissue-specific ERα signaling is needed. ERα is subjected to posttranslational modifications, including phosphorylation, which can influence ERα function in a tissue-specific manner. To determine the importance of phosphorylation site S122 (corresponding to human ERα site S118) for the skeleton and other tissues, male mice with a S122A mutation were used. Total areal bone mineral density was similar between gonadal intact S122A and WT littermates followed up to 12 months of age, and weights of estrogen-responsive organs normalized for body weight were unchanged between S122A and WT males at both 3 and 12 months of age. Interestingly, 12-month-old S122A males had decreased body weight compared to WT. To investigate if site S122 affects the estrogen response in bone and other tissues, 12-week-old S122A and WT males were orchidectomized (orx) and treated with estradiol (E2) or placebo pellets for four weeks. E2 increased cortical thickness in tibia in both orx WT (+ 60%, p < 0.001) and S122A (+ 45%, p < 0.001) males. However, the E2 effect on cortical thickness was significantly decreased in orx S122A compared to WT mice (- 24%, p < 0.05). In contrast, E2 affected trabecular bone and organ weights similarly in orx S122A and WT males. Thus, ERα phosphorylation site S122 is required for a normal E2 response specifically in cortical bone in male mice, a finding that may have implications for development of future treatments against male osteoporosis.

Comprehensive Sex Steroid Profiling in Multiple Tissues Reveals Novel Insights in Sex Steroid Distribution in Male Mice.

A comprehensive atlas of sex steroid distribution in multiple tissues is currently lacking, and how circulating and tissue sex steroid levels correlate remains unknown. Here, we adapted and validated a gas chromatography tandem mass spectrometry method for simultaneous measurement of testosterone (T), dihydrotestosterone (DHT), androstenedione, progesterone (Prog), estradiol, and estrone in mouse tissues. We then mapped the sex steroid pattern in 10 different endocrine, reproductive, and major body compartment tissues and serum of gonadal intact and orchiectomized (ORX) male mice. In gonadal intact males, high levels of DHT were observed in reproductive tissues, but also in white adipose tissue (WAT). A major part of the total body reservoir of androgens (T and DHT) and Prog was found in WAT. Serum levels of androgens and Prog were strongly correlated with corresponding levels in the brain while only modestly correlated with corresponding levels in WAT. After orchiectomy, the levels of the active androgens T and DHT decreased markedly while Prog levels in male reproductive tissues increased slightly. In ORX mice, Prog was by far the most abundant sex steroid, and, again, WAT constituted the major reservoir of Prog in the body. In conclusion, we present a comprehensive atlas of tissue and serum concentrations of sex hormones in male mice, revealing novel insights in sex steroid distribution. Brain sex steroid levels are well reflected by serum levels and WAT constitutes a large reservoir of sex steroids in male mice. In addition, Prog is the most abundant sex hormone in ORX mice.

Dehydroepiandrosterone Supplementation Results in Varying Tissue-specific Levels of Dihydrotestosterone in Male Mice.

Dehydroepiandrosterone (DHEA), an adrenal androgen precursor, can be metabolized in target tissues into active sex steroids. It has been proposed that DHEA supplementation might result in restoration of physiological local sex steroid levels, but knowledge on the effect of DHEA treatment on local sex steroid levels in multiple tissues is lacking. To determine the effects of DHEA on tissue-specific levels of sex steroids, we treated orchiectomized (ORX) male mice with DHEA for 3 weeks and compared them with vehicle-treated ORX mice and gonadal intact mice. Intra-tissue levels of sex steroids were analyzed in reproductive organs (seminal vesicles, prostate, m. levator ani), major body compartments (white adipose tissue, skeletal muscle, and brain), adrenals, liver, and serum using a sensitive and validated gas chromatography-mass spectrometry method. DHEA treatment restored levels of both testosterone (T) and dihydrotestosterone (DHT) to approximately physiological levels in male reproductive organs. In contrast, this treatment did not increase DHT levels in skeletal muscle or brain. In the liver, DHEA treatment substantially increased levels of T (at least 4-fold) and DHT (+536%, P < 0.01) compared with vehicle-treated ORX mice. In conclusion, we provide a comprehensive map of the effect of DHEA treatment on intra-tissue sex steroid levels in ORX mice with a restoration of physiological levels of androgens in male reproductive organs while DHT levels were not restored in the skeletal muscle or brain. This, and the unexpected supraphysiological androgen levels in the liver, may be a cause for concern considering the uncontrolled use of DHEA.

Mapping Regional Cortical Bone Responses to Local Changes in Loading and Systemic Stimuli.

Quantification of cortical bone mass and architecture using µCT is commonplace in osteoporosis and osteoarthritis research. Different groups often report substantially divergent mouse cortical bone responses to nominally comparable interventions. In the case of studies assessing bones' responses to externally applied loading, these differences are commonly associated with methodological differences in the loading regime. This chapter describes a widely published, standardized method of in vivo mouse tibia axial loading to produce lamellar bone formation. Despite uniform application of axial loading, changes in bone mass are highly site-specific within individual bones. For example, the mouse proximal tibia rapidly accrues new bone following axial loading, but this osteogenic response tapers to produce undetectable differences distally. Consequently, the bone sites selected for comparisons substantially influence the magnitude of differences observed. Application of the freely available Site Specificity software allows site-specific responses to be identified by rapidly quantifying cortical bone mass at each 1% site along the bone's length. This high-content screening tool has been informatively applied to study the local effects of changes in loading as well as systemic interventions including hormonal treatment and aging. Automated multisite analyses of cortical mass is increasingly identifying site-specific effects of "systemic" interventions such as global gene deletions. Biological mechanisms underlying this apparent regionalization of cortical responses are largely unknown but may start to be elucidated by increasingly widespread application of Site Specificity methods.

Bone gain following loading is site-specifically enhanced by prior and concurrent disuse in aged male mice.

The primary aim of osteoanabolic therapies is to strategically increase bone mass in skeletal regions likely to experience high strains. In the young healthy skeleton, this is primarily achieved by bone's adaptation to loading. This adaptation appears to fail with age, resulting in osteoporosis and fractures. We previously demonstrated that prior and concurrent disuse enhances bone gain following loading in old female mice. Here, we applied site specificity micro-computed tomography analysis to map regional differences in bone anabolic responses to axial loading of the tibia between young (19-week-old) and aged (19-month-old), male and female mice. Loading increased bone mass specifically in the proximal tibia in both sexes and ages. Young female mice gained more cortical bone than young males in specific regions of the tibia. However, these site-specific sex differences were lost with age such that bone gain following loading was not significantly different between old males and females. To test whether disuse enhances functional adaption in old male mice as it does in females, old males were subjected to sciatic neurectomy or sham surgery, and loading was initiated four days after surgery. Disuse augmented tibial cortical bone gain in response to loading in old males, but only in regions which were load-responsive in the young. Prior and concurrent disuse also increased loading-induced trabecular thickening in the proximal tibia of old males. Understanding how diminished background loading rejuvenates the osteogenic loading response in the old may improve osteogenic exercise regimes and lead to novel osteoanabolic therapies.