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.

Previous studies demonstrated that intermittent treatment with PTH increases osteoblast number and bone formation in growing and adult rats. The cellular mechanism for this increase in osteoblast number was investigated in 16-month-old female rats. Continuous [3H]thymidine infusion over a 1-week intermittent PTH [human PTH-(1-34)] treatment period was performed to determine the percentage of newly formed osteoblasts that originate from progenitor cells. To verify increases in bone formation, we performed histomorphometry and Northern blot analysis of selected bone matrix proteins. PTH treatment resulted in dramatic increases in fluorochrome-labeled perimeter (727%), osteoid perimeter (735%), osteoblast number (626%), and steady state mRNA levels of osteocalcin (946%) and type 1 collagen (> 1000%). Autoradiographic analysis of metaphyseal sections revealed no difference in the percentage of [3H]thymidine-labeled osteoblasts between PTH- and vehicle-treated groups (4.3 +/- 1.3% vs. 5.7 +/- 2.7%, respectively). Similar changes were observed in PTH-treated ovariectomized rats. As the PTH-induced increase in osteoblast number did not require proliferation of progenitor cells we carried out an additional experiment in adult ovariectomized rats to determine the onset of PTH action. Incorporation of [3H]proline in the distal femoral epiphysis of PTH-treated adult ovariectomized rats was increased within 24 h. We conclude that the rapid PTH-induced rise in bone formation did not require cell proliferation and was most likely due to activation of preexisting bone lining cells to osteoblasts.

Bone loss occurs as a consequence of exposure to microgravity. Using the hindlimb-unloaded rat to model spaceflight, this study had as its purpose to determine whether skeletal unloading and cephalic fluid shifts alter bone blood flow. We hypothesized that perfusion would be diminished in the hindlimb bones and increased in skeletal structures of the forelimbs and head. Using radiolabeled microspheres, we measured skeletal perfusion during control standing and after 10 min, 7 days, and 28 days of hindlimb unloading (HU). Femoral and tibial perfusion were reduced with 10 min of HU, and blood flow to the femoral shaft and marrow were further diminished with 28 days of HU. Correspondingly, the mass of femora (-11%, P < 0. 05) and tibiae (-6%, P < 0.1) was lowered with 28 days of HU. In contrast, blood flow to the skull, mandible, clavicle, and humerus was increased with 10 min HU but returned to control levels with 7 days HU. Mandibular (+10%, P < 0.05), clavicular (+18%, P < 0.05), and humeral (+8%, P < 0.1) mass was increased with chronic HU. The data demonstrate that simulated microgravity alters bone perfusion and that such alterations correspond to unloading-induced changes in bone mass. These results support the hypothesis that alterations in bone blood flow provide a stimulus for bone remodeling during periods of microgravity.

Estrogen deficiency induced bone loss is associated with increased bone turnover in rats and humans. The respective roles of increased bone turnover and altered balance between bone formation and bone resorption in mediating estrogen deficiency-induced cancellous bone loss was investigated in ovariectomized rats. Ovariectomy resulted in increased bone turnover in the distal femur. However, cancellous bone was preferentially lost in the metaphysis, a site that normally experiences low strain energy. No bone loss was observed in the epiphysis, a site experiencing higher strain energy. The role of mechanical strain in maintaining bone balance was investigated by altering the strain history. Mechanical strain was increased and decreased in long bones of ovariectomized rats by treadmill exercise and functional unloading, respectively. Functional unloading was achieved during orbital spacef light and following unilateral sciatic neurotomy. Increasing mechanical loading reduced bone loss in the metaphysis. In contrast, decreasing loading accentuated bone loss in the metaphysis and resulted in bone loss in the epiphysis. Finally, administration of estrogen to ovariectomized rats reduced bone loss in the unloaded and prevented loss in the loaded limb following unilateral sciatic neurotomy in part by reducing indices of bone turnover. These results suggest that estrogen regulates the rate of bone turnover, but the overall balance between bone formation and bone resorption is inf luenced by prevailing levels of mechanical strain.Ovarian hormone deficiency is the most important risk factor for postmenopausal osteoporosis (1, 2). Bone loss also occurs in premenopausal women following ovariectomy (OVX) (3) or treatment with gonadotrophin-releasing hormone agonists (4). Estrogen replacement therapy prevents bone loss in postmenopausal and ovariectomized women, suggesting that 17␤-estradiol is the gonadal hormone that is essential for normal bone balance.The mechanism for the skeletal effects of estrogen are incompletely understood but have been the subject of intense study in laboratory animal models (5). The rat has proven to be especially useful. OVX and gonadotrophin-releasing hormone agonists result in bone loss in rats, and these changes are prevented by estrogen treatment (6-9). These observations suggest similar skeletal mechanisms of action of estrogen in rats and humans. Furthermore, the skeletal changes in rats in response to partial estrogen agonists have accurately predicted the differential responses of pre-and postmenopausal women to tamoxifen treatment (10, 11).The bone loss in postmenopausal women and ovariectomized women and rats is associated with elevated bone turnover (6,(12)(13)(14). However, the bone loss is not uniform; cancellous bone is at a greater risk than cortical bone (7,14,15). In addition, there is site specificity in the loss of cancellous bone. For example, cancellous bone is lost more rapidly from the proximal tibial metaphysis than from vertebral bodies (16). Also, bone is preferenti...