Experiment 1 was designed to determine whether a high level of fat within bone marrow is associated with accelerated bone loss during simulated spaceflight. Weanling (1-month-old) WT and leptin-deficient ob/ob mice were maintained for 3 months under thermoneutral (32ºC) housing conditions to equalize resting energy expenditure between WT and ob/ob mice. The ob/ob mice were pair-fed to the WT mice to prevent development of morbid obesity in the ob/ob mice. Additionally: serum glucose, P1NP (bone formation marker), osteocalcin (bone turnover marker), and corticosterone (stress hormone) levels were measured at necropsy.
Aim 1 Results: We evaluated the impact of a 14d spaceflight (in slowly growing ovariectomized rats) and a 14d simulated spaceflight (hindlimb unloading (HU) in adult male mice) on bone marrow adiposity. As described in detail in Keune et al., 2016, bone marrow adipose tissue (MAT) increased in the distal femur metaphysis during spaceflight. During the same interval there was a reduction in cancellous bone volume fraction. The observed bone loss was due to increased bone resorption; osteoblast number, osteoblast activity and osteoblast turnover were normal. In agreement, HU resulted in bone loss in male mice and the bone loss was due to increased bone resorption. However, we were surprised to find that HU did not result in an increase in MAT (publication under review) in WBB6F1/J mice. It should be noted that this unexpected finding was replicated in HU C57BL6 mice (publication in preparation). We conclude from these results that increased MAT is not a prerequisite for bone loss in HU mice.
We originally hypothesized that mice incapable of producing MAT would be protected from HU-induced cancellous bone loss whereas mice with excessive MAT would exhibit increased bone loss. We tested these hypotheses using kitw/w-v (MAT-deficient) mice and ob/ob (excessive MAT) mice. As expected, decreased MAT was associated with increased bone formation and increased MAT was associated with decreased bone formation. However, HU-induced cancellous bone loss was exaggerated in MAT- mice and not impacted by excessive MAT (publication in preparation).
Taken together, our studies demonstrate that spaceflight-induced cancellous bone loss in ovariectomized rats and HU-induced cancellous bone loss in male mice each occur as a result of increased bone turnover, where the increase in resorption is inadequately compensated for by bone formation. While lineage decision (mesenchymal stem cells to osteoblasts or adipocytes) was shown to influence bone formation and MAT levels, our findings do not support a causal relationship between MAT infiltration and bone loss during spaceflight. Indeed, our studies in MAT-deficient mice suggest that MAT may have a protective role in limiting bone loss. Further research will be required to verify these findings and determine the underlying mechanisms.
Aim 2 Results:
We performed a series of studies to test this hypothesis. Initial studies were focused on establishing the effects of leptin on MAT and bone turnover in normal rats and leptin-deficient ob/ob mice (Turner et al., 2014; Turner et al., 2015; Philbrick KA et al., 2015; Lindenmaier et al., 2016). We also addressed the controversy as to whether the physiological actions of leptin on bone are mediated through peripheral or central nervous system (CNS)-mediated signaling (publication under review). This question is important because leptin regulates energy metabolism through CNS signaling. Subsequent studies focused on how leptin impacts bone and energy metabolism during HU (publication in preparation).
Taken together, our studies provide strong evidence that leptin administered subcutaneously and hypothalamic leptin gene therapy have surprisingly similar effects on bone metabolism in ob/ob mice; specifically, leptin decreased MAT, increased osteoblast number and activity, and increased osteoclast activity. Dose response studies revealed that the skeletal effects of leptin occur at very low circulating levels of the hormone (levels having minimal effects on food intake or body weight), suggesting that previously published reports demonstrating skeletal effects of leptin delivered into the hypothalamus were due to transport of leptin into peripheral circulation and not due to CNS mediated actions. If confirmed, our finding that the actions of peripheral leptin on bone and central actions of leptin on energy metabolism are dissociable from one another would represent a paradigm shift.
It is well established that there is a close association between the magnitude of mechanical loading of the skeleton and bone mass, and that bone loss often accompanies weight loss induced by dieting (Iwaniec and Turner, 2016). Potentially, this is due to combined decreases in peripheral and CNS leptin levels. In support, we have shown that increasing hypothalamic levels in adult rats can reduce body weight but, in contrast to caloric restriction, promote weight loss without bone loss (Turner et al., 2015). It is well established that weight loss due to reduced food intake contributes to bone loss during HU and for this reason, weight bearing controls are normally pair-fed to HU rodents. However, as shown for weight bearing rodents (Turner et al., 2015), leptin treatment of HU ob/ob mice, although inducing weight loss, did not result in further bone loss (publication in preparation).
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