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.

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.

(±)-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.

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.

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.

High-Dietary Alpha-Tocopherol or Mixed Tocotrienols Have No Effect on Bone Mass, Density, or Turnover in Male Rats During Skeletal Maturation.

High levels of alpha-tocopherol, the usual vitamin E supplement, are reported to decrease bone mass in rodents; however, the effects of other vitamin E forms on the skeleton are unknown. To test the hypothesis that high intakes of various vitamin E forms or the vitamin E metabolite, carboxyethyl hydroxy chromanol, were detrimental to bone status, Sprague-Dawley rats (n = 6 per group, 11-week males) for 18 weeks consumed semipurified diets that contained adequate alpha-tocopherol, high alpha-tocopherol (500 mg/kg diet), or 50% Tocomin (250 mg mixed tocopherols and tocotrienols/kg diet). Vitamin E status was evaluated by measuring plasma, liver, and bone marrow vitamin E concentrations. Bone density, microarchitecture (cross-sectional volume, cortical volume, marrow volume, cortical thickness, and cancellous bone volume fraction, trabecular number, thickness, and spacing), and cancellous bone formation were assessed in the tibia using dual-energy X-ray absorptiometry, microcomputed tomography, and histomorphometry, respectively. In addition, serum osteocalcin was assessed as a global marker of bone turnover; gene expression in response to treatment was evaluated in the femur using targeted (osteogenesis related) gene profiling. No significant differences were detected between treatment groups for any of the bone endpoints measured. Vitamin E supplementation, either as alpha-tocopherol or mixed tocotrienols, while increasing vitamin E concentrations both in plasma and tissues, had no effect on the skeleton in rats.

Increasing dietary nitrate has no effect on cancellous bone loss or fecal microbiome in ovariectomized rats.

SCOPE: Studies suggest diets rich in fruit and vegetables reduce bone loss, although the specific compounds responsible are unknown. Substrates for endogenous nitric oxide (NO) production, including organic nitrates and dietary nitrate, may support NO production in age-related conditions, including osteoporosis. We investigated the capability of dietary nitrate to improve NO bioavailability, reduce bone turnover and loss. METHODS AND RESULTS: Six-month-old Sprague Dawley rats [30 ovariectomized (OVX) and 10 sham-operated (sham)] were randomized into three groups: (i) vehicle (water) control, (ii) low-dose nitrate (LDN, 0.1 mmol nitrate/kg bw/day), or (iii) high-dose nitrate (HDN, 1.0 mmol nitrate/kg bw/day) for three weeks. The sham received vehicle. Serum bone turnover markers; bone mass, mineral density, and quality; histomorphometric parameters; and fecal microbiome were examined. Three weeks of LDN or HDN improved NO bioavailability in a dose-dependent manner. OVX resulted in cancellous bone loss, increased bone turnover, and fecal microbiome changes. OVX increased relative abundances of Firmicutes and decreased Bacteroideceae and Alcaligenaceae. Nitrate did not affect the skeleton or fecal microbiome. CONCLUSION: These data indicate that OVX affects the fecal microbiome and that the gut microbiome is associated with bone mass. Three weeks of nitrate supplementation does not slow bone loss or alter the fecal microbiome in OVX.

Leptin stimulates bone formation in ob/ob mice at doses having minimal impact on energy metabolism.

Leptin, the protein product of the ob gene, is essential for normal bone growth, maturation and turnover. Peripheral actions of leptin occur at lower serum levels of the hormone than central actions because entry of leptin into the central nervous system (CNS) is limited due to its saturable transport across the blood-brain barrier (BBB). We performed a study in mice to model the impact of leptin production associated with different levels of adiposity on bone formation and compared the response with well-established centrally mediated actions of the hormone on energy metabolism. Leptin was infused (0, 4, 12, 40, 140 or 400 ng/h) for 12 days into 6-week-old female ob/ob mice (n = 8/group) using sc-implanted osmotic pumps. Treatment resulted in a dose-associated increase in serum leptin. Bone formation parameters were increased at EC50 infusion rates of 7-17 ng/h, whereas higher levels (EC50, 40-80 ng/h) were required to similarly influence indices of energy metabolism. We then analyzed gene expression in tibia and hypothalamus at dose rates of 0, 12 and 140 ng/h; the latter dose resulted in serum leptin levels similar to WT mice. Infusion with 12 ng/h leptin increased the expression of genes associated with Jak/Stat signaling and bone formation in tibia with minimal effect on Jak/Stat signaling and neurotransmitters in hypothalamus. The results suggest that leptin acts peripherally to couple bone acquisition to energy availability and that limited transport across the BBB insures that the growth-promoting actions of peripheral leptin are not curtailed by the hormone's CNS-mediated anorexigenic actions.

Dietary dried plum increases bone mass, suppresses proinflammatory cytokines and promotes attainment of peak bone mass in male mice.

Nutrition is an important determinant of bone health and attainment of peak bone mass. Diets containing dried plum (DP) have been shown to increase bone volume and strength. These effects may be linked to the immune system and DP-specific polyphenols. To better understand these relationships, we studied DP in skeletally mature (6-month-old) and growing (1- and 2-month-old) C57Bl/6 male mice. In adult mice, DP rapidly (<2 weeks) increased bone volume (+32%) and trabecular thickness (+24%). These changes were associated with decreased osteoclast surface (Oc.S/BS) and decreased serum CTX, a marker of bone resorption. The reduction in Oc.S/BS was associated with a reduction in the osteoclast precursor pool. Osteoblast surface (Ob.S/BS) and bone formation rate were also decreased suggesting that the gain in bone in adult mice is a consequence of diminished bone resorption and formation, but resorption is reduced more than formation. The effects of DP on bone were accompanied by a decline in interleukins, TNF and MCP-1, suggesting that DP is acting in part through the immune system to suppress inflammatory activity and reduce the size of the osteoclast precursor pool. Feeding DP was accompanied by an increase in plasma phenolics, some of which have been shown to stimulate bone accrual. In growing and young adult mice DP at levels as low as 5% of diet (w/w) increased bone volume. At higher levels (DP 25%), bone volume was increased by as much as 94%. These data demonstrate that DP feeding dramatically increases peak bone mass during growth.

Dietary licorice root supplementation reduces diet-induced weight gain, lipid deposition, and hepatic steatosis in ovariectomized mice without stimulating reproductive tissues and mammary gland.

SCOPE: We studied the impact of dietary supplementation with licorice root components on diet-induced obesity, fat accumulation, and hepatic steatosis in ovariectomized C57BL/6 mice as a menopause model. MATERIALS AND METHODS: We evaluated the molecular and physiological effects of dietary licorice root administered to ovariectomized C57BL/6 mice as root powder (LRP), extracts (LRE), or isolated isoliquiritigenin (ILQ) on reproductive (uterus and mammary gland) and nonreproductive tissues important in regulating metabolism (liver, perigonadal, perirenal, mesenteric, and subcutaneous fat). Quantitative outcome measures including body weight, fat distribution (magnetic resonance imaging), food consumption, bone density and weight (Dual-energy X-ray absorptiometry), and gene expression were assessed by the degree of restoration to the preovariectomized health state. We characterized histological (H&E and oil red O staining) and molecular properties (expression of certain disease markers) of these tissues, and correlated these with metabolic phenotype as well as blood levels of bioactives. CONCLUSION: Although LRE and ILQ provided some benefit, LRP was the most effective in reducing body weight gain, overall fat deposition, liver steatosis, and expression of hepatic lipid synthesis genes following ovariectomy. Our data demonstrate that licorice root provided improvement of multiple metabolic parameters under conditions of low estrogen and high-fat diets without stimulating reproductive tissues.

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.

Genistein administered as a once-daily oral supplement had no beneficial effect on the tibia in rat models for postmenopausal bone loss.

OBJECTIVE: Estrogen deficiency after menopause results in rapid bone loss, predisposing women to osteoporotic fractures. Genistein, a phytoestrogen present in high concentrations in soy, is an ingredient in dietary supplements aggressively marketed for bone health. However, in a recent long-duration clinical trial in postmenopausal women, the efficacy of soy extracts in reducing bone loss was disappointing. To better understand the failure of soy extracts to consistently induce a robust skeletal response in women, we investigated the long-term (5 mo) efficacy of genistein, administered as a daily oral supplement, (1) in preventing cancellous bone loss in skeletally mature virgin Long-Evans rats ovariectomized at 7 months of age and (2) in improving cancellous bone mass and architecture in aged retired-breeder rats ovariectomized at 16 or 22 months of age. METHODS: Rats within each age group were randomly assigned into one of three treatment groups (n = 7-12 rats/group): (1) vehicle control, (2) genistein 485 µg/day, or (3) genistein 970 µg/day, resulting in mean (SE) serum genistein levels of 0.18 (0.10), 0.76 (0.15), and 1.48 (0.31) µM, respectively. Total tibia bone mass and density were evaluated using dual-energy x-ray absorptiometry, whereas cancellous bone mass and architecture in the tibial metaphysis, as well as cortical bone mass and architecture in the tibial diaphysis, were evaluated by micro-CT. RESULTS: Oral genistein administered as a dietary supplement did not influence the cumulative effects of ovariectomy, aging, and/or reproductive history on cancellous and cortical bone mass and architecture. CONCLUSIONS: Serum levels of genistein similar to those in women consuming a high-soy diet are ineffective in preventing or treating bone loss in rat models for postmenopausal osteoporosis.

Peripheral leptin regulates bone formation.

Substantial evidence does not support the prevailing view that leptin, acting through a hypothalamic relay, decreases bone accrual by inhibiting bone formation. To clarify the mechanisms underlying regulation of bone architecture by leptin, we evaluated bone growth and turnover in wild-type (WT) mice, leptin receptor-deficient db/db mice, leptin-deficient ob/ob mice, and ob/ob mice treated with leptin. We also performed hypothalamic leptin gene therapy to determine the effect of elevated hypothalamic leptin levels on osteoblasts. Finally, to determine the effects of loss of peripheral leptin signaling on bone formation and energy metabolism, we used bone marrow (BM) from WT or db/db donor mice to reconstitute the hematopoietic and mesenchymal stem cell compartments in lethally irradiated WT recipient mice. Decreases in bone growth, osteoblast-lined bone perimeter and bone formation rate were observed in ob/ob mice and greatly increased in ob/ob mice following subcutaneous administration of leptin. Similarly, hypothalamic leptin gene therapy increased osteoblast-lined bone perimeter in ob/ob mice. In spite of normal osteoclast-lined bone perimeter, db/db mice exhibited a mild but generalized osteopetrotic-like (calcified cartilage encased by bone) skeletal phenotype and greatly reduced serum markers of bone turnover. Tracking studies and histology revealed quantitative replacement of BM cells following BM transplantation. WT mice engrafted with db/db BM did not differ in energy homeostasis from untreated WT mice or WT mice engrafted with WT BM. Bone formation in WT mice engrafted with WT BM did not differ from WT mice, whereas bone formation in WT mice engrafted with db/db cells did not differ from the low rates observed in untreated db/db mice. In summary, our results indicate that leptin, acting primarily through peripheral pathways, increases osteoblast number and activity.

Hypothalamic leptin gene therapy prevents weight gain without long-term detrimental effects on bone in growing and skeletally mature female rats.

Hypothalamic leptin gene therapy normalizes the mosaic skeletal phenotype of leptin-deficient ob/ob mice. However, it is not clear whether increased hypothalamic leptin alters bone metabolism in animals already producing the hormone. The objective of this study was to evaluate the long duration effects of recombinant adeno-associated virus-rat leptin (rAAV-Lep) hypothalamic gene therapy on weight gain and bone metabolism in growing and skeletally mature leptin-replete female Sprague-Dawley rats. Rats were either unoperated or implanted with cannulas in the third ventricle of the hypothalamus and injected with either rAAV-Lep or rAAV-GFP (control vector encoding green fluorescent protein) and maintained on standard rat chow fed ad libitum for either 5 or 10 weeks (starting at 3 months of age) or 18 weeks (starting at 9 months of age). Tibias, femurs, or lumbar vertebrae were analyzed by micro-computed tomography and/or histomorphometry. In comparison with age-matched rAAV-GFP rats, rAAV-Lep rats maintained a lower body weight for the duration of studies. At 5 weeks after vector administration, rAAV-Lep rats had lower cancellous bone volume and bone marrow adiposity but higher osteoblast perimeter compared with nonoperated controls. However, these values did not differ between the two groups at 10 weeks after vector administration. Differences in cancellous bone volume and architecture were not detected between the rAAV-Lep and rAAV-GFP groups at either time point. Also, rAAV-Lep had no negative effects on bone in the 9-month-old skeletally mature rats at 18 weeks after vector administration. We hypothesize that the transient reductions in bone mass and bone marrow adiposity at 5 weeks after vector administration were due to hypothalamic surgery. We conclude that increased hypothalamic leptin, sufficient to prevent weight gain, has minimal specific effects (rAAV-Lep versus rAAV-GFP) on bone metabolism in normal female rats.

Consumption of green tea extract results in osteopenia in growing male mice.

Consumption of green tea may reduce body weight gain. Although many disorders are related to obesity, bone mass is positively correlated with body mass. Therefore, our purpose in this study was to determine the effects of green tea extract (GTE) on bone mass and architecture in rapidly growing lean [C57BL/6 wild type (WT)] and genetically obese, leptin-deficient (ob/ob) male mice. Five-week-old lean and ob/ob mice were assigned to diets containing GTE at 0, 1, or 2% for 6 wk. Femoral and lumbar vertebral bone volume and architecture were evaluated by micro-computed tomography (muCT). Following muCT analysis, femora were ashed to determine bone mineral content and density. Compared with WT mice, ob/ob mice had shorter femora (P < 0.001), lower femoral bone volume (P < 0.001), and lower femoral bone mineral content (P < 0.001), but higher cancellous bone volume in lumbar vertebrae (P < 001). Neither genotype nor treatment affected femoral bone mineral density, indicating normal mineralization. GTE consumption resulted in lower femur length, volume, mineral content, cortical volume, and cortical thickness (P < 0.001), as well as lower cancellous bone volume/tissue volume (P < 0.008) and trabecular thickness (P < 0.004) in lumbar vertebrae. The results indicate that leptin is not essential for the reduced gains in body weight and bone mass due to GTE in growing mice and suggest that consumption of large quantities of green tea may reduce the rate of bone accumulation during growth.

Leptin increases osteoblast-specific osteocalcin release through a hypothalamic relay.

Enhanced long-term expression of leptin by gene therapy selectively in the hypothalamus, without leakage to the systemic circulation, abrogated skeletal abnormalities and reinstated weight and insulin-glucose homeostasis in leptin-deficient ob/ob mice. Whether increases in osteocalcin, a hormone produced by osteoblasts and known to play a role in bone growth and recently in glucose-insulin homeostasis, may link these benefits of central leptin was assessed. The effects of a single intraventricular injection of non-immunogenic, non-pathogenic recombinant adeno-associated virus vector encoding leptin gene (rAAV-lep) or green fluorescent protein gene (rAAV-GFP, control) were studied in three genotypes, wild type (wt), obese diabetic, hyperinsulinemic ob/ob and non-obese, diabetic insulinopenic Akita mice. Selective hypothalamic leptin expression with central rAAV-lep treatment decreased weight, fat mass, food intake, suppressed insulin levels in ob/ob and wt mice, and conferred euglycemia by suppressing blood glucose in all three genotypes. Contemporaneously, rAAV-lep treatment also augmented blood osteocalcin levels. In wt mice, osteocalcin rose by 51% and, whereas, basal osteocalcin levels in ob/ob and Akita mice were significantly lower as compared to those in wt mice (26% and 55%, respectively), gene therapy reinstated levels to the control range in ob/ob mice, and raised 40% above the wt range even in the absence of insulin in Akita mice. These findings demonstrate that the central beneficial effects of leptin on bone growth involve increased hypothalamic relay of signals that augment osteocalcin efflux from osteoblasts into the general circulation, a response that, in turn, may also modulate glucose-insulin and weight homeostasis.

Central leptin gene therapy corrects skeletal abnormalities in leptin-deficient ob/ob mice.

Skeletal growth is tightly coupled to energy balance via complex and incompletely understood mechanisms. Leptin-deficient ob/ob mice are obese and develop multiple pathologies associated with the metabolic syndrome. Additionally, ob/ob mice have skeletal abnormalities. The objective of this study was to evaluate the effects of leptin deficiency and long duration selective central leptin repletion via recombinant adeno-associated virus-leptin (rAAV-lep) gene therapy on bone in growing ob/ob mice. The ob/ob mice were injected in the hypothalamus with either rAAV-lep or rAAV-GFP (control vector). Treated ob/ob and untreated wild-type (WT) mice were then maintained on a normal diet for 15 weeks. In a second experiment, similarly treated mice along with a group of pair-fed mice were maintained for 30 weeks. Leptin was not detected in blood of either rAAV-lep- or rAAV-GFP-treated mice although rAAV-lep-treated mice displayed leptin transgene expression in the hypothalamus. As expected, rAAV-lep normalized body weight and food intake. Compared to WT mice, rAAV-GFP-treated ob/ob mice had decreased femoral length (by 1.6 mm or 10%, P<0.001), decreased total femur bone volume (by 3.3 mm(3) or 19%, P<0.001), but increased cancellous bone volume in the distal femur (by 0.04 mm(3) or 60%, P<0.09) and lumbar vertebrae (by 0.26 mm(3) or 118%, P<0.001). Treatment with rAAV-lep rescued the ob/ob skeletal phenotype by increasing femoral length and total bone volume, and decreasing femoral and vertebral cancellous bone volume, so that at 15 weeks post-rAAV-lep injection the ob/ob mice no longer differed from WT mice. No further skeletal changes in either the femur or lumbar vertebra were observed at 30 weeks post-rAAV-lep administration. The results suggest that hypothalamic leptin functions as an essential permissive factor for normal bone growth.

Effects of parathyroid hormone (1-34) on tibia in an adult rat model for chronic alcohol abuse.

Chronic alcohol abuse is a risk factor for osteoporosis in men. Human recombinant parathyroid hormone (1-34) (PTH) therapy increases bone mass in patients with osteoporosis. The purpose of the present study was to determine whether PTH is effective in increasing bone formation and bone mass in a rat model for established osteopenia caused by chronic alcohol abuse. Eight-month-old male Sprague Dawley rats were fed the Lieber-DeCarli liquid diet in which 35% of the calories were derived from either maltose-dextran or ethanol. Measurements were performed 16 weeks later to establish the magnitude of bone changes in the rats fed alcohol. High dose PTH (80 microg/kg/day) was administered 5 days/week for 6 weeks to establish the differential efficacy of hormone therapy on bone formation in alcohol consuming and alcohol withdrawn rats. The effects of alcohol and PTH on cancellous and cortical bone mass, architecture and turnover were determined by densitometry and histomorphometry. Rats fed alcohol had reduced bone mineral contents and densities, cancellous and cortical bone areas and cancellous bone formation rates compared to pair-fed controls. Following the withdrawal of alcohol, indices of bone formation increased compared to baseline values. PTH treatment increased bone mineral content and density, bone formation rates, cortical bone area, cancellous bone area and trabecular number and thickness, but several indices of bone formation were reduced in the presence of continued alcohol consumption. These results suggest that alcohol consumption, in addition to inducing bone loss, may reduce the efficacy of PTH therapy to reverse osteoporosis.

Dose-response effects of intermittent PTH on cancellous bone in hindlimb unloaded rats.

UNLABELLED: HLU suppressed bone formation and resulted in bone loss in the tibial metaphysis of 6-month-old male rats. A human therapeutic dose of intermittent PTH (1 microg/kg/day) prevented the skeletal changes associated with HLU. INTRODUCTION: Skeletal unloading of skeletally mature rats results in trabecular thinning in the proximal tibial metaphysis, which is in part caused by a decrease in bone formation. We examined the efficacy of PTH in preventing the detrimental skeletal effects that occur with hindlimb unloading (HLU). MATERIALS AND METHODS: Six-month-old male Fisher 344 rats were HLU and treated with vehicle or recombinant human PTH(1-34) at 1, 5, 20, or 80 microg/kg/day for 2 weeks. The bone response was measured by microCT analysis of bone structure, histomorphometric analysis of static and dynamic bone parameters, and Northern blot analysis of mRNA levels for bone matrix proteins. The PTH-treated HLU animals were compared with vehicle-treated HLU and pair-fed normal weight-bearing controls. RESULTS: Unloading resulted in a decrease in cancellous bone volume that was caused in part by a dramatic 83% decrease in bone formation. All dose rates (1-80 microg/kg/day) of human PTH(1-34) significantly increased bone formation rates compared with vehicle-treated HLU controls. There was a dose response, and the highest dose rate of the hormone increased bone formation compared with normal weight-bearing rats by 708% (p < 0.0001). The increases in bone formation were accompanied by increases in mRNA levels for type 1 collagen, osteocalcin, and osteonectin. Also, treatment with PTH resulted in increases in mineral apposition rate and double-labeled perimeter, but the latter was disproportionally increased at high dose rates. A therapeutic dose of PTH (1 microg/kg/day) prevented disuse-induced trabecular thinning, whereas high-dose PTH (80 microg/kg/day) increased trabecular thickness compared with normal weight-bearing rats. CONCLUSIONS: These findings reveal that administration of a therapeutic dose of PTH to HLU rats prevents the decrease in bone formation and trabecular thinning, whereas high dose rates of the hormone increase bone formation and trabecular thickness to values that exceed normal values.

Strain-dependent variations in the response of cancellous bone to ovariectomy in mice.

UNLABELLED: The goal of this study was to characterize the skeletal response to ovariectomy in mice (129P3, C57BL/6, and B6129PF2) commonly used in gene manipulation studies to evaluate their potential as preclinical models of postmenopausal osteoporosis. The magnitude of cancellous bone loss and cellular indices of increased bone turnover in response to ovariectomy varied with mouse type and skeletal site, but in general, were less pronounced and less consistent than in Sprague-Dawley rats, the established preclinical model for postmenopausal bone loss. INTRODUCTION: The ovariectomized (OVX) rat is the most widely used preclinical rodent model for postmenopausal osteoporosis. However, the underlying mechanisms of bone disorders, including osteoporosis, have been explored predominantly in the mouse. The purpose of this study was to evaluate mice (129P3 and C57BL/6 inbred strains and their F2 hybrid offspring, B6129PF2), commonly used for gene knockout and overexpression studies, for their potential as preclinical models of postmenopausal bone loss. MATERIALS AND METHODS: The mice were OVX or sham-operated at 4 months of age and killed at 1 or 3 months after surgery. Lumbar vertebrae and distal femora were subjected to histomorphometric assessment. RESULTS: Mice in the two strains and the F2 hybrids (will be referred to as strain for the remainder of the abstract) lost vertebral cancellous bone after OVX; bone volume (BV/TV) was 20% and 27% lower at 1 and 3 months after surgery, respectively. The decreased cancellous BV/TV was associated with an increase in osteoclast surface at 1 month after OVX in the 129P3 strain only. Osteoblast surface was increased by 20% with OVX at both 1 and 3 months after surgery, irrespective of mouse strain. However, bone formation rate was not altered by OVX in any of the mouse strains. In contrast to the lumbar vertebrae, cancellous bone loss in response to OVX differed in the distal femur among the three mouse strains. OVX had no significant effect on distal femur BV/TV in the B6129PF2 mouse strain. In the C57BL/6 strain, cancellous BV/TV was reduced by OVX at 1 month after surgery but not at 3 months after surgery, whereas distal femur BV/TV in 129P3 mice was reduced at 3 months after surgery. Osteoclast surface was not affected by OVX at either time-point in the C57BL/6 strain, but was increased by 116% at 1 month after surgery in the 129P3 strain. Osteoblast surface was increased with OVX at 1 month after surgery, irrespective of strain, whereas bone formation rate was not altered by OVX at either time-point in any of the strains. CONCLUSIONS: The magnitude of cancellous bone loss and cellular indices of increased bone turnover in response to OVX varied with mouse strain and skeletal site, but in general, were less pronounced and less consistent than in the Sprague-Dawley rat. Although mouse models will continue to provide insights into genetic influences on bone mass and turnover, caution should be exercised when using 129P3 and C57BL/6 mice, and their F2 hybrids, as models for postmenopausal bone loss and preclinical testing of potential therapies for osteoporosis.