Long-duration leptin transgene expression in dorsal vagal complex does not alter bone parameters in female Sprague Dawley rats.

The hypothalamus and dorsal vagal complex (DVC) are both important for integration of signals that regulate energy balance. Increased leptin transgene expression in either the hypothalamus or DVC of female rats was shown to decrease white adipose tissue and circulating levels of leptin and adiponectin. However, in contrast to hypothalamus, leptin transgene expression in the DVC had no effect on food intake, circulating insulin, ghrelin and glucose, nor on thermogenic energy expenditure. These findings imply different roles for hypothalamus and DVC in leptin signaling. Leptin signaling is required for normal bone accrual and turnover. Leptin transgene expression in the hypothalamus normalized the skeletal phenotype of leptin-deficient ob/ob mice but had no long-duration (≥10 weeks) effects on the skeleton of leptin-replete rats. The goal of this investigation was to determine the long-duration effects of leptin transgene expression in the DVC on the skeleton of leptin-replete rats. To accomplish this goal, we analyzed bone from three-month-old female rats that were microinjected with recombinant adeno-associated virus encoding either rat leptin (rAAV-Leptin, n = 6) or green fluorescent protein (rAAV-GFP, control, n = 5) gene. Representative bones from the appendicular (femur) and axial (3rd lumbar vertebra) skeleton were evaluated following 10 weeks of treatment. Selectively increasing leptin transgene expression in the DVC had no effect on femur cortical or cancellous bone microarchitecture. Additionally, increasing leptin transgene expression had no effect on vertebral osteoblast-lined or osteoclast-lined bone perimeter or marrow adiposity. Taken together, the findings suggest that activation of leptin receptors in the DVC has minimal specific effects on the skeleton of leptin-replete female rats.

Bone Marrow Adipose Tissue Is Not Required for Reconstitution of the Immune System Following Irradiation in Male Mice.

Bone marrow adipose tissue (BMAT) is hypothesized to serve as an expandable/contractible fat depot which functions, in part, to minimize energy requirements for sustaining optimal hematopoiesis. We investigated whether BMAT is required for immune reconstitution following injury. Male wild type (WBB6F1, WT) and BMAT-deficient WBB6F1/J-KitW/KitW-v/J (KitW/W-v) mice were lethally irradiated. Irradiation was followed by adoptive transfer of 1000 purified WT hematopoietic stem cells (HSCs). The extent of immune reconstitution in blood, bone marrow, and lymph nodes in the irradiated mice was determined using HSCs from green fluorescent protein (GFP)-expressing mice. We also evaluated skeletal response to treatment. Detection of GFP-positive B and T cells in peripheral blood at 4 and 9 weeks following adoptive transfer and in bone marrow and lymph nodes following necropsy revealed excellent immune reconstitution in both WT and BMAT-deficient mice. Adipocytes were numerous in the distal femur of WT mice but absent or rare in KitW/W-v mice. Bone parameters, including length, mass, density, bone volume, microarchitecture, and turnover balance, exhibited few differences between WT and BMAT-deficient mice. The minimal differences suggest that BMAT is not required for reconstitution of the immune system following lethal radiation and is not a major contributor to the skeletal phenotypes of kit signaling-deficient mice.

Diet composition influences the effect of high fat diets on bone in growing male mice.

The effect of diet-induced obesity on bone in rodents is variable, with bone mass increases, decreases, and no impact reported. The goal of this study was to evaluate whether the composition of obesogenic diet may influence bone independent of its effect on body weight. As proof-of-principle, we used a mouse model to compare the skeletal effects of a commonly used high fat 'Western' diet and a modified high fat diet. The modified high fat diet included ground English walnut and was isocaloric for macronutrients, but differed in fatty acid composition and contained nutrients (e.g. polyphenols) not present in the standard 'Western' diet. Eight-week-old mice were randomized into 1 of 3 dietary treatments (n = 8/group): (1) low fat control diet (LF; 10 % kcal fat); (2) high fat 'Western' diet (HF; 46 % kcal fat as soybean oil and lard); or (3) modified high fat diet supplemented with ground walnuts (HF + walnut; 46 % kcal fat as soybean oil, lard, and walnut) and maintained on their respective diets for 9 weeks. Bone response in femur was then evaluated using dual energy x-ray absorptiometry, microcomputed tomography, and histomorphometry. Consumption of both obesogenic diets resulted in increased weight gain but differed in impact on bone and bone marrow adiposity in distal femur metaphysis. Mice consuming the high fat 'Western' diet exhibited a tendency for lower cancellous bone volume fraction and connectivity density, and had lower osteoblast-lined bone perimeter (an index of bone formation) and higher bone marrow adiposity than low fat controls. Mice fed the modified high fat diet did not differ from mice fed control (low fat) diet in cancellous bone microarchitecture, or osteoblast-lined bone perimeter, and exhibited lower bone marrow adiposity compared to mice fed the 'Western' diet. This proof-of-principal study demonstrates that two obesogenic diets, similar in macronutrient distribution and induction of weight gain, can have different effects on cancellous bone in distal femur metaphysis. Because the composition of the diets used to induce obesity in rodents does not recapitulate a common human diet, our finding challenges the translatability of rodent studies evaluating the impact of diet-induced obesity on bone.

Estrogen-regulated miRs in bone enhance osteoblast differentiation and matrix mineralization.

Estrogen signaling is critical for the development and maintenance of healthy bone, and age-related decline in estrogen levels contributes to the development of post-menopausal osteoporosis. Most bones consist of a dense cortical shell and an internal mesh-like network of trabecular bone that respond differently to internal and external cues such as hormonal signaling. To date, no study has assessed the transcriptomic differences that occur specifically in cortical and trabecular bone compartments in response to hormonal changes. To investigate this, we employed a mouse model of post-menopausal osteoporosis (ovariectomy, OVX) and estrogen replacement therapy (ERT). mRNA and miR sequencing revealed distinct transcriptomic profiles between cortical and trabecular bone in the setting of OVX and ERT. Seven miRs were identified as likely contributors to the observed estrogen-mediated mRNA expression changes. Of these, four miRs were prioritized for further study and decreased predicted target gene expression in bone cells, enhanced the expression of osteoblast differentiation markers, and altered the mineralization capacity of primary osteoblasts. As such, candidate miRs and miR mimics may have therapeutic relevance for bone loss resulting from estrogen depletion without the unwanted side effects of hormone replacement therapy and therefore represent novel therapeutic approaches to combat diseases of bone loss.

Leptin and environmental temperature as determinants of bone marrow adiposity in female mice.

Bone marrow adipose tissue (BMAT) levels are higher in distal femur metaphysis of female mice housed at thermoneutral (32°C) than in mice housed at 22°C, as are abdominal white adipose tissue (WAT) mass, and serum leptin levels. We performed two experiments to explore the role of increased leptin in temperature-enhanced accrual of BMAT. First, we supplemented 6-week-old female C57BL/6J (B6) mice with leptin for 2 weeks at 10 µg/d using a subcutaneously implanted osmotic pump. Controls consisted of ad libitum (ad lib) fed mice and mice pair fed to match food intake of leptin-supplemented mice. The mice were maintained at 32°C for the duration of treatment. At necropsy, serum leptin in leptin-supplemented mice did not differ from ad lib mice, suggesting suppression of endogenous leptin production. In support, Ucp1 expression in BAT, percent body fat, and abdominal WAT mass were lower in leptin-supplemented mice. Leptin-supplemented mice also had lower BMAT and higher bone formation in distal femur metaphysis compared to the ad lib group, changes not replicated by pair-feeding. In the second experiment, BMAT response was evaluated in 6-week-old female B6 wild type (WT), leptin-deficient ob/ob and leptin-treated (0.3 µg/d) ob/ob mice housed at 32°C for the 2-week duration of the treatment. Compared to mice sacrificed at baseline (22°C), BMAT increased in ob/ob mice as well as WT mice, indicating a leptin independent response to increased temperature. However, infusion of ob/ob mice with leptin, at a dose rate having negligible effects on either energy metabolism or serum leptin levels, attenuated the increase in BMAT. In summary, increased housing temperature and increased leptin have independent but opposing effects on BMAT in mice.

Isoliquiritigenin Decreases Bone Resorption and Osteoclast Differentiation.

SCOPE: A dose-ranging study is performed using young estrogen-depleted rats to determine whether dietary isoliquiritigenin (ILQ) alters bone metabolism and if the effects are associated with estrogen receptor signaling. METHODS AND RESULTS: Six-week-old rats (ovariectomized at 4 weeks of age) are fed diets containing 0, 100, 250, or 750 ppm ILQ (n = 5/treatment) for 7 days. Gene expression in femur and uterus, blood markers of bone turnover, body composition, and uterine weight and epithelial cell height are determined. Because ILQ lowers bone resorption, the effect of ILQ on in vitro differentiation of osteoclasts from bone marrow of mice is assessed. Treatment resulted in a dose-dependent increases in serum ILQ but no changes in serum osteocalcin, a marker of global bone formation. Contrastingly, ILQ administration results in reduced serum CTX-1, a marker of global bone resorption, and reduces tartrate resistant acid phosphatase expression in osteoclast culture. ILQ treatment and endogenous estrogen production had limited overlap on gene expression in femur and uterus. However, uterine epithelial cell hyperplasia is observed in two of five animals treated with 750 ppm. CONCLUSIONS: In conclusion, dietary ILQ reduces bone resorption in vivo and osteoclast differentiation in vitro, by mechanisms likely differing from actions of ovarian hormones.

Caloric Restriction and Hypothalamic Leptin Gene Therapy Have Differential Effects on Energy Partitioning in Adult Female Rats.

Dieting is a common but often ineffective long-term strategy for preventing weight gain. Similar to humans, adult rats exhibit progressive weight gain. The adipokine leptin regulates appetite and energy expenditure but hyperleptinemia is associated with leptin resistance. Here, we compared the effects of increasing leptin levels in the hypothalamus using gene therapy with conventional caloric restriction on weight gain, food consumption, serum leptin and adiponectin levels, white adipose tissue, marrow adipose tissue, and bone in nine-month-old female Sprague-Dawley rats. Rats (n = 16) were implanted with a cannula in the 3rd ventricle of the hypothalamus and injected with a recombinant adeno-associated virus, encoding the rat gene for leptin (rAAV-Lep), and maintained on standard rat chow for 18 weeks. A second group (n = 15) was calorically-restricted to match the weight of the rAAV-Lep group. Both approaches prevented weight gain, and no differences in bone were detected. However, calorically-restricted rats consumed 15% less food and had lower brown adipose tissue Ucp-1 mRNA expression than rAAV-Lep rats. Additionally, calorically-restricted rats had higher abdominal white adipose tissue mass, higher serum leptin and adiponectin levels, and higher marrow adiposity. Caloric restriction and hypothalamic leptin gene therapy, while equally effective in preventing weight gain, differ in their effects on energy intake, energy expenditure, adipokine levels, and body composition.

Effects of graded increases in ethanol consumption on biochemical markers of bone turnover in young adult male cynomolgus macaques.

Chronic heavy alcohol use is often associated with reduced bone mineral density and altered bone turnover. However, the dose response effects of ethanol on bone turnover have not been established. This study examined the effects of graded increases of ethanol consumption on biochemical markers of bone turnover in young adult male cynomolgus macaques (Macaca fascicularis). For this study, 6.6-year-old (95% CI: 6.5, 6.7) male macaques were subjected to three 30-day sessions of increased ethanol intake over a 90-day interval. During the first 30 days, the monkeys drank a predetermined volume of ethanol corresponding to 0.5 g/kg/day, followed by 1.0 g/kg/day and 1.5 g/kg/day. Osteocalcin, a marker of bone formation, and carboxyterminal cross-linking telopeptide of type 1 collagen (CTX), a marker of resorption, were measured during each 30-day session. In addition, the ratio of osteocalcin to CTX was determined as a surrogate measure of global turnover balance. Mean osteocalcin decreased by 2.6 ng/mL (1.8, 3.5) for each one-half unit (0.5 g/kg/day) increase in dose (p < 0.001). Mean CTX decreased by 0.13 ng/mL (0.06, 0.20) for each one-half unit increase in dose (p < 0.001). Furthermore, there was an inverse relationship between dose and the ratio of osteocalcin to CTX, such that the mean ratio decreased by 0.9 (0.3, 1.5) for each one-half unit increase in dose (p = 0.01). In summary, male cynomolgus macaques had decreased blood osteocalcin and CTX, and osteocalcin to CTX ratio during the 90-day interval of graded increases in ethanol consumption, indicative of reduced bone turnover and negative turnover balance, respectively. These findings suggest that over the range ingested, ethanol resulted in a linear decrease in bone turnover. Furthermore, the negative bone turnover balance observed is consistent with reported effects of chronic alcohol intake on the skeleton.

17α-Estradiol prevents ovariectomy-mediated obesity and bone loss.

Menopause is a natural physiological process in older women that is associated with reduced estrogen production and results in increased risk for obesity, diabetes, and osteoporosis. 17α-estradiol (17α-E2) treatment in males, but not females, reverses several metabolic conditions associated with advancing age, highlighting sexually dimorphic actions on age-related pathologies. In this study we sought to determine if 17α-E2 could prevent ovariectomy (OVX)-mediated detriments on adiposity and bone parameters in females. Eight-week-old female C57BL/6J mice were subjected to SHAM or OVX surgery and received dietary 17α-E2 during a six-week intervention period. We observed that 17α-E2 prevented OVX-induced increases in body weight and adiposity. Similarly, uterine weight and luminal cell thickness were decreased by OVX and prevented by 17α-E2 treatment. Interestingly, 17α-E2 prevented OVX-induced declines in tibial metaphysis cancellous bone. And similarly, 17α-E2 improved bone density parameters in both tibia and femur cancellous bone, primarily in OVX mice. In contrast, to the effects on cancellous bone, cortical bone parameters were largely unaffected by OVX or 17α-E2. In the non-weight bearing lumbar vertebrae, OVX reduced trabecular thickness but not spacing, while 17α-E2 increased trabecular thickness and reduced spacing. Despite this, 17α-E2 did improve bone volume/tissue volume in lumbar vertebrae. Overall, we found that 17α-E2 prevented OVX-induced increases in adiposity and changes in bone mass and architecture, with minimal effects in SHAM-operated mice. We also observed that 17α-E2 rescued uterine tissue mass and lining morphology to control levels without inducing hypertrophy, suggesting that 17α-E2 could be considered as an adjunct to traditional hormone replacement therapies.

Effects of Propranolol on Bone, White Adipose Tissue, and Bone Marrow Adipose Tissue in Mice Housed at Room Temperature or Thermoneutral Temperature.

Growing female mice housed at room temperature (22°C) weigh the same but differ in body composition compared to mice housed at thermoneutrality (32°C). Specifically, mice housed at room temperature have lower levels of white adipose tissue (WAT). Additionally, bone marrow adipose tissue (bMAT) and cancellous bone volume fraction in distal femur metaphysis are lower in room temperature-housed mice. The metabolic changes induced by sub-thermoneutral housing are associated with lower leptin levels in serum and higher levels of Ucp1 gene expression in brown adipose tissue. Although the precise mechanisms mediating adaptation to sub-thermoneutral temperature stress remain to be elucidated, there is evidence that increased sympathetic nervous system activity acting via ß-adrenergic receptors plays an important role. We therefore evaluated the effect of the non-specific ß-blocker propranolol (primarily ß1 and ß2 antagonist) on body composition, femur microarchitecture, and bMAT in growing female C57BL/6 mice housed at either room temperature or thermoneutral temperature. As anticipated, cancellous bone volume fraction, WAT and bMAT were lower in mice housed at room temperature. Propranolol had small but significant effects on bone microarchitecture (increased trabecular number and decreased trabecular spacing), but did not attenuate premature bone loss induced by room temperature housing. In contrast, propranolol treatment prevented housing temperature-associated differences in WAT and bMAT. To gain additional insight, we evaluated a panel of genes in tibia, using an adipogenesis PCR array. Housing temperature and treatment with propranolol had exclusive as well as shared effects on gene expression. Of particular interest was the finding that room temperature housing reduced, whereas propranolol increased, expression of the gene for acetyl-CoA carboxylase (Acacb), the rate-limiting step for fatty acid synthesis and a key regulator of ß-oxidation. Taken together, these findings provide evidence that increased activation of ß1 and/or ß2 receptors contributes to reduced bMAT by regulating adipocyte metabolism, but that this pathway is unlikely to be responsible for premature cancellous bone loss in room temperature-housed mice.

(±)-Equol does not interact with genistein on estrogen-dependent breast tumor growth.

Equol (EQ) is a prominent microbial metabolite of the soy isoflavone, daidzein, with estrogen-like properties. The major soy isoflavone, genistein (GEN), stimulated growth of estrogen-dependent breast cancer (EDBC) cells in vitro and tumor growth in vivo but EQ did not. To understand possible interactions of EQ and GEN on EDBC, EQ was used with GEN in combination in vitro and in vivo. Effects of EQ, GEN and EQ + GEN were evaluated using MCF-7 and T47D EDBC. Ovariectomized athymic mice were used as a model for in vivo tumor growth. Dietary EQ had no effect on MCF-7 tumor growth and the absence of effect was confirmed using a T47D EDBC in vivo model. EQ alone or in combination with GEN increased EDBC cell proliferation in vitro. EQ alone neither stimulated EDBC tumor growth in vivo at various doses nor suppressed tumor growth induced by dietary GEN. In summary, EQ has similar estrogenic effect as GEN in vitro but does not interact with GEN on EDBC tumor growth. Based on the evidence presented here, dietary EQ is unlikely to have estrogenic effects in vivo.

Effects of Alcohol and Estrogen Receptor Blockade Using ICI 182,780 on Bone in Ovariectomized Rats.

BACKGROUND: Estrogen signaling is essential for the sexual dimorphism of the skeleton, is required for normal bone remodeling balance in adults, and may influence the skeletal response to alcohol. High levels of alcohol consumption lower bone mass in ovary-intact but not ovariectomized (ovx) rats. However, the extremely rapid rate of bone loss immediately following ovx may obscure the effects of alcohol. We therefore determined (i) whether heavy alcohol consumption (35% caloric intake) influences bone in sexually mature ovx rats with established cancellous osteopenia and (ii) whether ICI 182,780 (ICI), a potent estrogen receptor signaling antagonist, alters the skeletal response to alcohol. METHODS: Three weeks following ovx, rats were randomized into 5 groups, (i) baseline, (ii) control + vehicle, (iii) control + ICI, (iv) ethanol (EtOH) + vehicle, or (v) EtOH + ICI, and treated accordingly for 4 weeks. Dual-energy X-ray absorptiometry, microcomputed tomography, blood measurements of markers of bone turnover, and gene expression in femur and uterus were used to evaluate response to alcohol and ICI. RESULTS: Rats consuming alcohol had lower bone mass and increased fat mass. Bone microarchitecture of the tibia and gene expression in femur were altered; specifically, there was reduced accrual of cortical bone, net loss of cancellous bone, and differential expression of 19/84 genes related to bone turnover. Furthermore, osteocalcin, a marker of bone turnover, was lower in alcohol-fed rats. ICI had no effect on weight gain, body composition, or cortical bone. ICI reduced cancellous bone loss and serum CTX-1, a biochemical marker of bone resorption; alcohol antagonized the latter 2 responses. Neither alcohol nor ICI affected uterine weight or gene expression. CONCLUSIONS: Alcohol exaggerated bone loss in ovx rats in the presence or absence of estrogen receptor blockade with ICI. The negligible effect of alcohol on uterus and limited effects of ICI on bone in alcohol-fed ovx rats suggest that estrogen receptor signaling plays a limited role in the action of alcohol on bone in a rat model for chronic alcohol abuse.

Voluntary Chronic Heavy Alcohol Consumption in Male Rhesus Macaques Suppresses Cancellous Bone Formation and Increases Bone Marrow Adiposity.

BACKGROUND: Chronic heavy alcohol consumption is an established risk factor for bone fracture, but comorbidities associated with alcohol intake may contribute to increased fracture rates in alcohol abusers. To address the specific effects of alcohol on bone, we used a nonhuman primate model and evaluated voluntary alcohol consumption on: (i) global markers of bone turnover in blood and (ii) cancellous bone mass, density, microarchitecture, turnover, and microdamage in lumbar vertebra. METHODS: Following a 4-month induction period, 6-year-old male rhesus macaques (Macaca mulatta, n = 13) voluntarily self-administered water or ethanol (EtOH; 4% w/v) for 22 h/d, 7 d/wk, for a total of 12 months. Control animals (n = 9) consumed an isocaloric maltose-dextrin solution. Tetracycline hydrochloride was administered orally 17 and 3 days prior to sacrifice to label mineralizing bone surfaces. Global skeletal response to EtOH was evaluated by measuring plasma osteocalcin and carboxyterminal collagen cross-links (CTX). Local response was evaluated in lumbar vertebra using dual-energy X-ray absorptiometry, microcomputed tomography, static and dynamic histomorphometry, and histological assessment of microdamage. RESULTS: Monkeys in the EtOH group consumed an average of 2.8 ± 0.2 (mean ± SE) g/kg/d of EtOH (30 ± 2% of total calories), resulting in an average blood EtOH concentration of 88.3 ± 8.8 mg/dl 7 hours after the session onset. Plasma CTX and osteocalcin tended to be lower in EtOH-consuming monkeys compared to controls. Significant differences in bone mineral density in lumbar vertebrae 1 to 4 were not detected with treatment. However, cancellous bone volume fraction (in cores biopsied from the central region of the third vertebral body) was lower in EtOH-consuming monkeys compared to controls. Furthermore, EtOH-consuming monkeys had lower osteoblast perimeter and mineralizing perimeter, no significant difference in osteoclast perimeter, and higher bone marrow adiposity than controls. No significant differences between groups were detected in microcrack density (2nd lumbar vertebra). CONCLUSIONS: Voluntary chronic heavy EtOH consumption reduces cancellous bone formation in lumbar vertebra by decreasing osteoblast-lined bone perimeter, a response associated with an increase in bone marrow adiposity.

A de novo substitution in BCL11B leads to loss of interaction with transcriptional complexes and craniosynostosis.

Craniosynostosis, the premature ossification of cranial sutures, is a developmental disorder of the skull vault, occurring in approximately 1 in 2250 births. The causes are heterogeneous, with a monogenic basis identified in ~25% of patients. Using whole-genome sequencing, we identified a novel, de novo variant in BCL11B, c.7C>A, encoding an R3S substitution (p.R3S), in a male patient with coronal suture synostosis. BCL11B is a transcription factor that interacts directly with the nucleosome remodelling and deacetylation complex (NuRD) and polycomb-related complex 2 (PRC2) through the invariant proteins RBBP4 and RBBP7. The p.R3S substitution occurs within a conserved amino-terminal motif (RRKQxxP) of BCL11B and reduces interaction with both transcriptional complexes. Equilibrium binding studies and molecular dynamics simulations show that the p.R3S substitution disrupts ionic coordination between BCL11B and the RBBP4-MTA1 complex, a subassembly of the NuRD complex, and increases the conformational flexibility of Arg-4, Lys-5 and Gln-6 of BCL11B. These alterations collectively reduce the affinity of BCL11B p.R3S for the RBBP4-MTA1 complex by nearly an order of magnitude. We generated a mouse model of the BCL11B p.R3S substitution using a CRISPR-Cas9-based approach, and we report herein that these mice exhibit craniosynostosis of the coronal suture, as well as other cranial sutures. This finding provides strong evidence that the BCL11B p.R3S substitution is causally associated with craniosynostosis and confirms an important role for BCL11B in the maintenance of cranial suture patency.

Maintenance of Near Normal Bone Mass and Architecture in Lethally Irradiated Female Mice following Adoptive Transfer with as few as 750 Purified Hematopoietic Stem Cells.

Total-body irradiation (TBI) followed by transfer of bone marrow cells from donors is routinely performed in immunology research and can be used to manipulate differentiation and/or function of bone cells. However, exposure to high-dose radiation can result in irreversible osteopenia, and transfer of heterogeneous cell populations can complicate interpretation of results. The goal of this research was to establish an approach for reconstituting bone marrow using small numbers of purified donor-derived hematopoietic stem cells (HSCs) without negatively affecting bone metabolism. Gamma-irradiated (9 Gy) WBB6F1 mice were engrafted with bone marrow cells (5 × 106 cells) or purified HSCs (3,000 cells) obtained from GFP transgenic mice. In vivo analysis and in vitro differentiation assays performed two months later established that both methods were effective in reconstituting the hematopoietic compartment with donor-derived cells. We confirmed these findings by engrafting C57Bl/6 (B6) mice with bone marrow cells or purified HSCs from CD45.1 B6 congenic mice. We next performed adoptive transfer of purified HSCs (750 cells) into WBB6F1 and radiosensitive KitW/W-v mice and evaluated the skeleton two months later. Minimal differences were observed between controls and WBB6F1-engrafted mice that received fractionated doses of 2 × 5 Gy. Kitw/wv mice lost weight and became osteopenic after 2 × 5 Gy irradiations but these abnormalities were negligible after 5 Gy irradiation. Importantly, adoptive transfer of wild-type cells into Kitw/wv mice restored normal Kit expression in bone marrow. Together, these findings provide strong evidence for efficient engraftment with purified HSCs after lethal TBI with minimal collateral damage to bone. This approach will be useful for investigating mechanisms by which hematopoietic lineage cells regulate bone metabolism.

Thermally Abused Frying Oil Potentiates Metastasis to Lung in a Murine Model of Late-Stage Breast Cancer.

Deep-frying is a popular form of food preparation used globally and throughout in the United States. Each time dietary oils are heated to deep-frying temperatures, they undergo chemical alterations that result in a new matrix of lipid structures. These lipid products include triglyceride dimers, polymers, oxidized triglycerides, and cyclic monomers, which raises nutritional concerns about associations between these lipid products and heightened health risks. Reports of associations between thermally abused frying oil and deleterious health outcomes currently exist, yet there is little information concerning the effects of thermally abused frying oil consumption and the progression of breast cancer. This study used a late-stage breast cancer murine model and in vivo bioluminescent imaging to monitor progression of metastasis of 4T1 tumor cells in animals consuming fresh soybean oil (SBO) and a thermally abused frying oil (TAFO). Bioluminescent and histologic examinations demonstrated that TAFO consumption resulted in a marked increase of metastatic lung tumor formation compared to SBO consumption. Further, in animals consuming the TAFO treatment diet, metastatic tumors in the lung displayed a 1.4-fold increase in the Ki-67 marker of cellular proliferation and RNA-sequencing analysis of the hepatic tissue revealed a dietary-induced modulation of gene expression in the liver.

Metabolic Coupling Between Bone Marrow Adipose Tissue and Hematopoiesis.

PURPOSE OF REVIEW: Mesenchymal stem cells (MSCs) located in the bone marrow have the capacity to differentiate into multiple cell lineages, including osteoblast and adipocyte. Adipocyte density within marrow is inversely associated with bone mass during aging and in some pathological conditions, contributing to the prevailing view that marrow adipocytes play a largely negative role in bone metabolism. However, a negative association between marrow adipocytes and bone balance is not universal. Although MAT levels appear tightly regulated, establishing the precise physiological significance of MAT has proven elusive. Here, we review recent literature aimed at delineating the function of MAT. RECENT FINDINGS: An important physiological function of MAT may be to provide an expandable/contractible fat depot, which is critical for minimization of energy requirements for sustaining optimal hematopoiesis. Because the energy requirements for storing fat are negligible compared to those required to maintain hematopoiesis, even small reductions in hematopoietic tissue volume to match a reduced requirement for hematopoiesis could represent an important reduction in energy cost. Such a physiological function would require tight coupling between hematopoietic stem cells and MSCs to regulate the balance between MAT and hematopoiesis. Kit-ligand, an important regulator of proliferation, differentiation, and survival of hematopoietic cells, may function as a prototypic factor coupling MAT and hematopoiesis. Crosstalk between hematopoietic and mesenchymal cells in the bone marrow may contribute to establishing the balance between MAT levels and hematopoiesis.

Non-estrogenic Xanthohumol Derivatives Mitigate Insulin Resistance and Cognitive Impairment in High-Fat Diet-induced Obese Mice.

Xanthohumol (XN), a prenylated flavonoid from hops, improves dysfunctional glucose and lipid metabolism in animal models of metabolic syndrome (MetS). However, its metabolic transformation into the estrogenic metabolite, 8-prenylnaringenin (8-PN), poses a potential health concern for its use in humans. To address this concern, we evaluated two hydrogenated derivatives, α,ß-dihydro-XN (DXN) and tetrahydro-XN (TXN), which showed negligible affinity for estrogen receptors α and ß, and which cannot be metabolically converted into 8-PN. We compared their effects to those of XN by feeding C57BL/6J mice a high-fat diet (HFD) containing XN, DXN, or TXN for 13 weeks. DXN and TXN were present at higher concentrations than XN in plasma, liver and muscle. Mice administered XN, DXN or TXN showed improvements of impaired glucose tolerance compared to the controls. DXN and TXN treatment resulted in a decrease of HOMA-IR and plasma leptin. C2C12 embryonic muscle cells treated with DXN or TXN exhibited higher rates of uncoupled mitochondrial respiration compared to XN and the control. Finally, XN, DXN, or TXN treatment ameliorated HFD-induced deficits in spatial learning and memory. Taken together, DXN and TXN could ameliorate the neurocognitive-metabolic impairments associated with HFD-induced obesity without risk of liver injury and adverse estrogenic effects.

Effects of hypothalamic leptin gene therapy on osteopetrosis in leptin-deficient mice.

Impaired resorption of cartilage matrix deposited during endochondral ossification is a defining feature of juvenile osteopetrosis. Growing, leptin-deficient ob/ob mice exhibit a mild form of osteopetrosis. However, the extent to which the disease is (1) self-limiting and (2) reversible by leptin treatment is unknown. We addressed the first question by performing histomorphometric analysis of femurs in rapidly growing (2-month-old), slowly growing (4-month-old) and skeletally mature (6-month-old) wild-type (WT) and ob/ob male mice. Absent by 6 months of age in WT mice, cartilage matrix persisted to varying extents in distal femur epiphysis, metaphysis and diaphysis in ob/ob mice, suggesting that the osteopetrotic phenotype is not entirely self-limiting. To address the second question, we employed hypothalamic recombinant adeno-associated virus (rAAV) gene therapy to restore leptin signaling in ob/ob mice. Two-month-old mice were randomized to one of the three groups: (1) untreated control, (2) rAAV-Leptin or (3) control vector rAAV-green fluorescent protein and vectors injected intracerebroventricularly. Seven months later, rAAV-leptin-treated mice exhibited no cartilage in the metaphysis and greatly reduced cartilage in the epiphysis and diaphysis. At the cellular level, the reduction in cartilage was associated with increased bone turnover. These findings (1) support the concept that leptin is important for normal replacement of cartilage by bone, and (2) demonstrate that osteopetrosis in ob/ob mice is bone-compartment-specific and reversible by leptin at skeletal sites capable of undergoing robust bone turnover.

Skeletal and Uterotrophic Effects of Endoxifen in Female Rats.

Endoxifen, the primary active metabolite of tamoxifen, is currently being investigated as a novel endocrine therapy for the treatment of breast cancer. Tamoxifen is a selective estrogen receptor modulator that elicits potent anti-breast cancer effects. However, long-term use of tamoxifen also induces bone loss in premenopausal women and is associated with an increased risk of endometrial cancer in postmenopausal women. For these reasons, we have used a rat model system to comprehensively characterize the impact of endoxifen on the skeleton and uterus. Our results demonstrate that endoxifen elicits beneficial effects on bone in ovary-intact rats and protects against bone loss following ovariectomy. Endoxifen is also shown to reduce bone turnover in both ovary-intact and ovariectomized rats at the cellular and biochemical levels. With regard to the uterus, endoxifen decreased uterine weight but maintained luminal epithelial cell height in ovariectomized animals. Within luminal epithelial cells, endoxifen resulted in differential effects on the expression levels of estrogen receptors α and ß as well as multiple other genes previously implicated in regulating epithelial cell proliferation and hypertrophy. These studies analyze the impact of extended endoxifen exposure on both bone and uterus using a Food and Drug Administration-recommended animal model. Although endoxifen is a more potent breast cancer agent than tamoxifen, the results of the present study demonstrate that endoxifen does not induce bone loss in ovary-intact rats and that it elicits partial agonistic effects on the uterus and skeleton in ovariectomized animals.