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.

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.

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.

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.

Intact PTH combined with the PTH ratio for diagnosis of bone turnover in dialysis patients: a diagnostic test study.

BACKGROUND: Determination of parathyroid hormone (PTH) level is the most commonly used surrogate marker for bone turnover in patients with stage 5 chronic kidney disease on dialysis therapy (CKD-5D). The objective of this study is to evaluate the predictive value of various PTH measurements for identifying low or high bone turnover rate. STUDY DESIGN: Diagnostic test study. SETTINGS & PARTICIPANTS: 141 patients with CKD-5D from 15 US hemodialysis centers. INDEX TESTS: Intact PTH, PTH 1-84, and PTH ratio (ratio of level of PTH 1-84 to level of large carboxy-terminal PTH fragments). REFERENCE TEST OR OUTCOME: Bone turnover determined using bone histomorphometry. OTHER MEASUREMENTS: Demographic and treatment-related factors, serum calcium and phosphorus. RESULTS: Patients presented histologically with a broad range of bone turnover abnormalities. In white patients with CKD-5D (n = 70), PTH ratio <1.0 added to intact PTH level <420 pg/mL increased the positive predictive value for low bone turnover from 74% to 90%. In black patients (n = 71), adding PTH ratio <1.2 to intact PTH level <340 pg/mL increased the positive predictive value for low bone turnover from 48% to 90%. Adding PTH ratio >1.6 to intact PTH level of 340-790 pg/mL increased the positive predictive value for high bone turnover from 56% to 71%. LIMITATIONS: Because the research protocol called for carefully controlled blood specimen handling, blood drawing and routine specimen handling might be less stringent in clinical practice. By limiting study participation to black and white patients with CKD-5D, we cannot comment on the roles of intact PTH, PTH 1-84, and PTH ratio in other racial/ethnic groups. CONCLUSION: In black patients with CKD-5D, the addition of PTH ratio to intact PTH measurements is helpful for diagnosing low and high bone turnover. In white patients with CKD-5D, it aids in the diagnosis of low bone turnover.