Sibyl N. Swift

Cancellous bone formation response to simulated resistance training during disuse is blunted by concurrent alendronate treatment

The purpose of this study was to assess the effectiveness of simulated resistance training (SRT) exercise combined with alendronate (ALEN) in mitigating or preventing disuse‐associated losses in cancellous bone microarchitecture and formation. Sixty male Sprague‐Dawley rats (6 months old) were randomly assigned to either cage control (CC), hind limb unloading (HU), HU plus either ALEN (HU + ALEN), SRT (HU + SRT), or a combination of ALEN and SRT (HU + SRT/ALEN) for 28 days. HU + SRT and HU + SRT/ALEN rats were anesthetized and subjected to muscle contractions once every 3 days during HU (four sets of five repetitions, 1000 ms isometric + 1000 ms eccentric). Additionally, HU + ALEN and HU + SRT/ALEN rats received 10 µg/kg of body weight of ALEN three times per week. HU reduced cancellous bone‐formation rate (BFR) by 80%, with no effect of ALEN treatment (−85% versus CC). SRT during HU significantly increased cancellous BFR by 123% versus CC, whereas HU + SRT/ALEN inhibited the anabolic effect of SRT (−70% versus HU + SRT). SRT increased bone volume and trabecular thickness by 19% and 9%, respectively, compared with CC. Additionally, osteoid surface (OS/BS) was significantly greater in HU + SRT rats versus CC (+32%). Adding ALEN to SRT during HU reduced Oc.S/BS (−75%), Ob.S/BS (−72%), OS/BS (−61%), and serum TRACP5b (−36%) versus CC. SRT and ALEN each independently suppressed a nearly twofold increase in adipocyte number evidenced with HU and inhibited increases in osteocyte apoptosis. These results demonstrate the anabolic effect of a low volume of high‐intensity muscle contractions during disuse and suggest that both bone resorption and bone formation are suppressed when SRT is combined with bisphosphonate treatment.

Restriction of Dietary Energy Intake Has a Greater Impact on Bone Integrity Than Does Restriction of Calcium in Exercising Female Rats

We sought to elucidate the effects of restricting calcium, energy, or food on the skeletal integrity of exercising female rats. Female Sprague-Dawley rats (4 mo old) were randomly assigned to 5 groups (n = 10/group): ad libitum intake of an AIN-93M diet (Research Diets D10012M, Research Diets, Inc.) with no exercise (AL-S) or with exercise (AL-EX) or to 1 of 3 exercising restriction groups [40% restriction of calcium only (CAR-EX), energy only (ER-EX), or food (FR-EX)]. All EX rats were treadmill trained 3 d/wk, 45 min/d for 12 wk at ~60% maximal oxygen consumption. After 12 wk, total body bone mineral content (by DXA) and body mass, but not lean mass, were lower in ER-EX (-17%) and FR-EX rats (-13%) compared with the AL-EX group. CAR-EX had few negative effects on bone geometry (by peripheral quantitative computed tomography) or histomorphometry. However, declines in total volumetric bone mineral density at the proximal tibia metaphysic (PTM) were observed in ER-EX (-6%) and FR-EX (-8%) groups; only FR-EX rats exhibited increased osteoclast surface and decreased mineral apposition rate in PTM cancellous bone. Decrements in serum estradiol, uterine weights, or both in these 2 groups implicate altered estrogen status as contributory. Urine pH declined significantly by 12 wk in all restricted groups, but net acid excretion increased only in CAR-EX rats. These findings, when compared with published data on sedentary rats, suggest that treadmill running exercise may mitigate some, but not all, deleterious effects on bone after chronic energy or food restriction but is more protective during calcium restriction.

Increased training loads do not magnify cancellous bone gains with rodent jump resistance exercise

This study sought to elucidate the effects of a low- and high-load jump resistance exercise (RE) training protocol on cancellous bone of the proximal tibia metaphysis (PTM) and femoral neck (FN). Sprague-Dawley rats (male, 6 mo old) were randomly assigned to high-load RE (HRE; n = 16), low-load RE (LRE; n = 15), or sedentary cage control (CC; n = 11) groups. Animals in the HRE and LRE groups performed 15 sessions of jump RE during 5 wk of training. PTM cancellous volumetric bone mineral density (vBMD), assessed by in vivo peripheral quantitative computed tomography scans, significantly increased in both exercise groups (+9%; P < 0.001), resulting in part from 130% (HRE; P = 0.003) and 213% (LRE; P < 0.0001) greater bone formation (measured by standard histomorphometry) vs. CC. Additionally, mineralizing surface (%MS/BS) and mineral apposition rate were higher (50-90%) in HRE and LRE animals compared with controls. PTM bone microarchitecture was enhanced with LRE, resulting in greater trabecular thickness (P = 0.03) and bone volume fraction (BV/TV; P = 0.04) vs. CC. Resorption surface was reduced by nearly 50% in both exercise paradigms. Increased PTM bone mass in the LRE group translated into a 161% greater elastic modulus (P = 0.04) vs. CC. LRE and HRE increased FN vBMD (10%; P < 0.0001) and bone mineral content (∼ 20%; P < 0.0001) and resulted in significantly greater FN strength vs. CC. For the vast majority of variables, there was no difference in the cancellous bone response between the two exercise groups, although LRE resulted in significantly greater body mass accrual and bone formation response. These results suggest that jumping at minimal resistance provides a similar anabolic stimulus to cancellous bone as jumping at loads exceeding body mass.

The Androgen Metabolite, 5α-androstane-3β,17β-diol, Decreases Cytokine-Induced Cyclooxygenase-2, Vascular Cell Adhesion Molecule-1 Expression, and P-Glycoprotein Expression in Male Human Brain Microvascular Endothelial Cells

P-glycoprotein (Pgp), a multiple drug resistance transporter expressed by vascular endothelial cells, is a key component of the blood-brain barrier and has been shown to increase after inflammation. The nonaromatizable androgen, dihydrotestosterone (DHT), decreases inflammatory markers in vascular smooth muscle cells, independent of androgen receptor (AR) stimulation. The principal metabolite of DHT, 5α-androstane-3β,17β-diol (3β-diol), activates estrogen receptor (ER)β and similarly decreases inflammatory markers in vascular cells. Therefore, we tested the hypothesis that either DHT or 3β-diol decrease cytokine-induced proinflammatory mediators, vascular cell adhesion molecule-1 (VCAM-1) and cyclooxygenase-2 (COX-2), to regulate Pgp expression in male primary human brain microvascular endothelial cells (HBMECs). Using RT-qPCR, the mRNAs for AR, ERα, and ERβ and steroid metabolizing enzymes necessary for DHT conversion to 3β-diol were detected in male HBMECs demonstrating that the enzymes and receptors for production of and responsiveness to 3β-diol are present. Western analysis showed that 3β-diol reduced COX-2 and Pgp expression; the effect on Pgp was inhibited by the ER antagonist, ICI-182,780. IL-1β-caused an increase in COX-2 and VCAM-1 that was reduced by either DHT or 3β-diol. 3β-diol also decreased cytokine-induced Pgp expression. ICI-182,780 blocked the effect of 3β-diol on COX-2 and VCAM-1, but not Pgp expression. Therefore, in cytokine-stimulated male HBMECs, the effect of 3β-diol on proinflammatory mediator expression is ER dependent, whereas its effect on Pgp expression is ER independent. These studies suggest a novel role of 3β-diol in regulating blood-brain barrier function and support the concept that 3β-diol can be protective against proinflammatory mediator stimulation.

Beta-1 Adrenergic Agonist Treatment Mitigates Negative Changes in Cancellous Bone Microarchitecture and Inhibits Osteocyte Apoptosis during Disuse

The sympathetic nervous system (SNS) plays an important role in mediating bone remodeling. However, the exact role that beta-1 adrenergic receptors (beta1AR) have in this process has not been elucidated. We have previously demonstrated the ability of dobutamine (DOB), primarily a beta1AR agonist, to inhibit reductions in cancellous bone formation and mitigate disuse-induced loss of bone mass. The purpose of this study was to characterize the independent and combined effects of DOB and hindlimb unloading (HU) on cancellous bone microarchitecture, tissue-level bone cell activity, and osteocyte apoptosis. Male Sprague-Dawley rats, aged 6-mos, were assigned to either normal cage activity (CC) or HU (n = 18/group) for 28 days. Animals were administered either daily DOB (4 mg/kg BW/d) or an equal volume of saline (VEH) (n = 9/gp). Unloading resulted in significantly lower distal femur cancellous BV/TV (−33%), Tb.Th (−11%), and Tb.N (−25%) compared to ambulatory controls (CC-VEH). DOB treatment during HU attenuated these changes in cancellous bone microarchitecture, resulting in greater BV/TV (+29%), Tb.Th (+7%), and Tb.N (+21%) vs. HU-VEH. Distal femur cancellous vBMD (+11%) and total BMC (+8%) were significantly greater in DOB- vs. VEH-treated unloaded rats. Administration of DOB during HU resulted in significantly greater osteoid surface (+158%) and osteoblast surface (+110%) vs. HU-VEH group. Furthermore, Oc.S/BS was significantly greater in HU-DOB (+55%) vs. CC-DOB group. DOB treatment during unloading fully restored bone formation, resulting in significantly greater bone formation rate (+200%) than in HU-VEH rats. HU resulted in an increased percentage of apoptotic cancellous osteocytes (+85%), reduced osteocyte number (−16%), lower percentage of occupied osteocytic lacunae (−30%) as compared to CC-VEH, these parameters were all normalized with DOB treatment. Altogether, these data indicate that beta1AR agonist treatment during disuse mitigates negative changes in cancellous bone microarchitecture and inhibits increases in osteocyte apoptosis.

Acute toxicity of subcutaneously administered vitamin E isomers delta- and gamma-tocotrienol in mice.

The toxicity of parenterally administered vitamin E isomers, delta-tocotrienol (DT3) and gamma-tocotrienol (GT3), was evaluated in male and female CD2F1 mice. In an acute toxicity study, a single dose of DT3 or GT3 was administered subcutaneously in a dose range of 200 to 800 mg/kg. A mild to moderately severe dermatitis was observed clinically and microscopically in animals at the injection site at doses above 200 mg/kg. The severity of the reaction was reduced when the drug concentration was lowered. Neither drug produced detectable toxic effects in any other tissue at the doses tested. Based on histopathological analysis for both DT3 and GT3, and macroscopic observations of inflammation at the injection site, a dose of 300 mg/kg was selected as the lowest toxic dose in a 30-day toxicity study performed in male mice. At this dose, a mild skin irritation occurred at the injection site that recovered completely by the end of the experimental period. At a dose of 300 mg/kg of DT3 or GT3, no adverse effects were observed in any tissues or organs.

Skin wound trauma, following high-dose radiation exposure, amplifies and prolongs skeletal tissue loss

The present study investigated the detrimental effects of non-lethal, high-dose (whole body) γ-irradiation on bone, and the impact that radiation combined with skin trauma (i.e. combined injury) has on long-term skeletal tissue health. Recovery of bone after an acute dose of radiation (RI; 8 Gy), skin wounding (15-20% of total body skin surface), or combined injury (RI+Wound; CI) was determined 3, 7, 30, and 120 days post-irradiation in female B6D2F1 mice and compared to non-irradiated mice (SHAM) at each time-point. CI mice demonstrated long-term (day 120) elevations in serum TRAP 5b (osteoclast number) and sclerostin (bone formation inhibitor), and suppression of osteocalcin levels through 30 days as compared to SHAM (p<0.05). Radiation-induced reductions in distal femur trabecular bone volume fraction and trabecular number through 120 days post-exposure were significantly greater than non-irradiated mice (p<0.05) and were exacerbated in CI mice by day 30 (p<0.05). Negative alterations in trabecular bone microarchitecture were coupled with extended reductions in cancellous bone formation rate in both RI and CI mice as compared to Sham (p<0.05). Increased osteoclast surface in CI animals was observed for 3 days after irradiation and remained elevated through 120 days (p<0.01). These results demonstrate a long-term, exacerbated response of bone to radiation when coupled with non-lethal wound trauma. Changes in cancellous bone after combined trauma were derived from extended reductions in osteoblast-driven bone formation and increases in osteoclast activity.

Mechanical loading increases detection of estrogen receptor-alpha in osteocytes and osteoblasts despite chronic energy restriction

Estrogen receptor-α (ER-α) is an important mediator of the bone response to mechanical loading. We sought to determine whether restricting dietary energy intake by 40% limits the bone formation rate (BFR) response to mechanical loading (LOAD) by downregulating ER-α-expressing osteocytes, or osteoblasts, or both. Female rats (n = 48, 7 mo old) were randomized to ADLIB-SHAM and ADLIB-LOAD groups fed AIN-93M purified diet ad libitum or to ER40-SHAM and ER40-LOAD groups fed modified AIN-93M with 40% less energy (100% of all other nutrients). After 12 wk, LOAD rats were subjected to a muscle contraction protocol three times every third day. ER40 produced lower proximal tibia bone volume (-22%), trabecular thickness (-14%), and higher trabecular separation (+127%) in SHAM but not LOAD rats. ER40 rats exhibited reductions in mineral apposition rate, but not percent mineralizing surface or BFR. LOAD induced similar relative increases in these kinetic measures of osteoblast activity/recruitment in both diet groups., but absolute values for ER40 LOAD rats were lower vs. ADLIB-LOAD. There were fourfold and eightfold increases in proportion of estrogen receptor-α protein-positive osteoblast and osteocytes, respectively, in LOAD vs. SHAM rats, with no effect of ER40. These data suggest that a brief period of mechanical loading significantly affects estrogen receptor-α in cancellous bone osteoblasts and osteocytes. Chronic energy restriction does result in lower absolute values in indices of osteoblast activity after mechanical loading, but not by a smaller increment relative to unloaded bones; this change is not explained by an associated downregulation of ER-α in osteoblasts or osteocytes.

Fat and Lean Mass Predict Bone Mass During Energy Restriction in Sedentary and Exercising Rodents

Energy restriction (ER) causes bone loss, but the impact of exercise during ER is less understood. In this study, we examined the impact of metabolic hormones and body composition on both total body bone mineral content (BMC) and local (proximal tibia) volumetric bone mineral density (vBMD) during short- (4 weeks) and long-term (12 weeks) ER with and without exercise in adult female rats. Our first goal was to balance energy between sedentary and exercising groups to determine the impact of exercise during ER. Second, we aimed to determine the strongest predictors of bone outcomes during ER with energy-matched exercising groups. Methods: Female Sprague–Dawley rats were divided into three sedentary groups (ad libitum, –20% ER, and –40% ER) and three exercising groups (ad libitum, –10% ER, and –30% ER). Approximately a 10% increase in energy expenditure was achieved via moderate treadmill running (∼60–100 min 4 days/week) in EX groups. n per group = 25–35. Data were analyzed as a 2 × 3 ANOVA with multiple linear regression to predict bone mass outcomes. Results: At 4 weeks, fat and lean mass and serum insulin-like growth factor-I (IGF-I) predicted total body BMC (R2 = 0.538). Fat mass decreased with ER at all levels, while lean mass was not altered. Serum IGF-I declined in the most severe ER groups (–40 and –30%). At 12 weeks, only fat and lean mass predicted total body BMC (R2 = 0.718). Fat mass declined with ER level regardless of exercise status and lean mass increased due to exercise (+5.6–6.7% vs. energy-matched sedentary groups). At the same time point, BMC declined with ER, but increased with exercise (+7.0–12.5% vs. energy-matched sedentary groups). None of our models predicted vBMD at the proximal tibia at either time point. Conclusion: Both fat and lean mass statistically predicted total body BMC during both short- and long-term ER. Fat and lean mass decreased with ER, while lean mass increased with EX at each energy level. Measures that predicted total body skeletal changes did not predict site-specific changes. These data highlight the importance of maintaining lean mass through exercise during periods of ER.

Short-Term Reduction in Energy Availability Does Not Impair Exercise-Induced Gains in Bone Formation Rate: 677

Reduced energy availability (EA, defined as total energy intake minus exercise energy expenditure) can induce significant bone loss in humans and in rodents, but this effect may vary with exercise status. Purpose: This study focused on the effects of graded reductions in EA achieved with and without exercise on mid-shaft tibia cortical bone. We hypothesized that markers of bone formation would be reduced with 4 weeks of decreased EA, but these reductions would be mitigated in exercising animals. Methods: After 8 weeks of acclimation to AIN-93M purified diet, 5 mo-old virgin female SpragueDawley rats (n=72) were randomized in to sedentary (SED) and exercise (EX) groups, each of which was divided into 3 energy status groups: -12% EA/g body mass (-12EA) and -25% EA/g body mass (-25EA) vs. ad lib-fed controls. EX rats were restricted on diet intake slightly less than SED to account for the energy cost (+10%/day) of treadmill running (80-90 min/d, 4 d/wk, ~60% VO2max); custom versions of AIN-93M were used to assure restriction of only kcal (other nutrients at 100%). Calcein injections 9 and 2 days prior to euthanasia labeled mineralizing surfaces for histomorphometric analyses 2 mm proximal to the tibio-fibular junction for mineralizing surface (%MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR) on periosteal and endocortical surfaces. Results: After 4 weeks, only -25EA SED lost body mass (-11%); virtually all of this was fat mass. Periosteal BFR was 2 to 4 fold higher in EX rats vs. SED animals within each energy status group. The impact of EX on endocortical BFR was even greater (~5-fold increase) for all groups except in the -25 EA cohorts (-25 EA EX BFR ~ equal to -25EA SED BFR). Increases in BFR with EX were achieved by increases in both %MS/BS and MAR on both cortical surfaces. Conclusions: These data suggest that short-term graded reductions in EA do not inhibit BFR in SED animals nor the robust stimulation of BFR by moderately vigorous exercise training. The one exception observed was a suppression of the EX induced gain in endocortical BFR with the more stringent level of reduced EA. Whether this lack of effect of EA on BFR gains with EX persists with more chronic reductions in EA has yet to be determined. Funded by the Department of Defense #WSIXWH-06-1-0479