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.

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.

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.

Hypothalamic leptin gene therapy reduces body weight without accelerating age-related bone loss.

Excessive weight gain in adults is associated with a variety of negative health outcomes. Unfortunately, dieting, exercise, and pharmacological interventions have had limited long-term success in weight control and can result in detrimental side effects, including accelerating age-related cancellous bone loss. We investigated the efficacy of using hypothalamic leptin gene therapy as an alternative method for reducing weight in skeletally-mature (9 months old) female rats and determined the impact of leptin-induced weight loss on bone mass, density, and microarchitecture, and serum biomarkers of bone turnover (CTx and osteocalcin). Rats were implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either recombinant adeno-associated virus encoding the gene for rat leptin (rAAV-Leptin, n=7) or a control vector encoding green fluorescent protein (rAAV-GFP, n=10) and sacrificed 18 weeks later. A baseline control group (n=7) was sacrificed at vector administration. rAAV-Leptin-treated rats lost weight (-4±2%) while rAAV-GFP-treated rats gained weight (14±2%) during the study. At study termination, rAAV-Leptin-treated rats weighed 17% less than rAAV-GFP-treated rats and had lower abdominal white adipose tissue weight (-80%), serum leptin (-77%), and serum IGF1 (-34%). Cancellous bone volume fraction in distal femur metaphysis and epiphysis, and in lumbar vertebra tended to be lower (P<0.1) in rAAV-GFP-treated rats (13.5 months old) compared to baseline control rats (9 months old). Significant differences in cancellous bone or biomarkers of bone turnover were not detected between rAAV-Leptin and rAAV-GFP rats. In summary, rAAV-Leptin-treated rats maintained a lower body weight compared to baseline and rAAV-GFP-treated rats with minimal effects on bone mass, density, microarchitecture, or biochemical markers of bone turnover.

Morbid obesity attenuates the skeletal abnormalities associated with leptin deficiency in mice.

Leptin-deficient ob/ob mice are morbidly obese and exhibit low total bone mass and mild osteopetrosis. In order to disassociate the skeletal effects of leptin deficiency from those associated with morbid obesity, we evaluated bone mass, architecture, gene expression, and indices of bone turnover in WT mice, ob/ob mice allowed to feed ad libitum (ob/ob), and ob/ob mice pair-fed equivalent to WT mice (pair-fed ob/ob). Mice were maintained at 32 °C (thermoneutral) from 6 to 18 weeks of age to minimize differences in resting energy expenditure. ob/ob mice were heavier, had more abdominal white adipose tissue (WAT), and were hyperglycemic compared with WT mice. Femur length, bone mineral content (BMC) and bone mineral density, and midshaft femur cortical thickness were lower in ob/ob mice than in WT mice. Cancellous bone volume (BV) fraction was higher but indices of bone formation and resorption were lower in ob/ob mice compared with WT mice; reduced bone resorption in ob/ob mice resulted in pathological retention of calcified cartilage. Pair-fed ob/ob mice were lighter and had lower WAT, uterine weight, and serum glucose than ob/ob mice. Similarly, femoral length, BMC, and cortical thickness were lower in pair-fed ob/ob mice compared with ob/ob mice, as were indices of cancellous bone formation and resorption. In contrast, bone marrow adiposity, calcified cartilage, and cancellous BV fraction were higher at one or more cancellous sites in pair-fed ob/ob mice compared with ob/ob mice. These findings indicate that the skeletal abnormalities caused by leptin deficiency are markedly attenuated in morbidly obese ob/ob mice.