Journal of Applied Physiology AJP: Endocrinology and Metabolism
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J Appl Physiol 83: 172-178, 1997;
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Journal of Applied Physiology
Vol. 83, No. 1, pp. 172-178, July 1997
METABOLISM

Effects of vigorous exercise training and beta -agonist administration on bone response to hindlimb suspension

Susan A. Bloomfield, Beverly E. Girten, and Steven E. Weisbrode

School of Health, Physical Education, and Recreation and Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210-1290

ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES

ABSTRACT

Bloomfield, Susan A., Beverly E. Girten, and Steven E. Weisbrode. Effects of vigorous exercise training and beta -agonist administration on bone response to hindlimb suspension. J. Appl. Physiol. 83(1): 172-178, 1997.---The effectiveness of dobutamine (Dob) in preventing bone loss during 14 days of hindlimb suspension (Sus) was tested in exercise-trained (Ex; n = 25) and sedentary (Sed; n = 22) rats (age 155 days). One-half of each group was given Dob (2 mg · kg-1 · day-1) or saline (Sal). Histomorphometric measurements at midfemur revealed a 17% smaller cortical bone area (CBA) and a 32% lower periosteal mineral apposition rate (MAR) in suspended vs. nonsuspended Sed/Sal rats. Dob abolished this decline in CBA in Sed/Sus rats, probably via an attenuation of the decrease in periosteal MAR; similar but nonsignificant effects on cross-sectional moment of inertia were observed. Nonsuspended Ex rats had no change in bone CBA when CBA is indexed to body weight. Sus appeared to uncouple the relationship between soleus weight and CBA. Dob attenuated the 43% decline in soleus weight after Sus in Ex but not in Sed rats. In summary, vigorous Ex before Sus does not affect loss of bone mass due to unloading; Dob effectively maintains CBA in Sed rats subjected to suspension.

dobutamine; rat; unloading; histomorphometry; cortical bone

INTRODUCTION

SIGNIFICANT DECREASES in mechanical loading invariably produce large and rapid decrements in bone mass in the affected limbs. Prolonged exposure to bed rest (16) or to microgravity (23) results in a 3-6% decrease in bone mass per month. The physiological mechanisms for bone loss incurred with immobilization or during spaceflight are not yet clearly defined nor are the relative roles of weight bearing and muscle contraction. Hindlimb suspension of rats produces changes in muscle and bone very similar to those seen after similar periods of spaceflight (15, 20, 30). Changes in bone cell activity are noted after 7 days of suspension (29), with decrements in cancellous bone mass noted within 14 days (30). Significant loss of cortical bone mass requires up to 90 days suspension (15), although region-specific changes in cortical bone geometry are noted within 28 days (25).

A number of interventions have been tested for minimizing the loss of bone mass with unloading. Antiresorptive agents such as bisphosphonates (2) appear to effectively minimize loss of bone mass and strength during periods of disuse. However, resorption rates return to normal during the second week of suspension; after this time point, continued bone loss is due primarily to reductions in bone formation activity (29). Clearly, it is desirable to find an anabolic agent to stimulate osteoblastic activity to counteract this suppression of bone formation during unloading.

beta -Adrenergic agonists minimize the reduction in femoral and tibial ash weight after sciatic denervation, primarily because of their effects on maintaining muscle mass and contractile tension (31). Dobutamine, a synthetic catecholamine used clinically, can attenuate the decrements in maximal O2 consumption (VO2 max) and skeletal muscle oxidative enzyme activity observed during bed rest in healthy men (26). If dobutamine has an anabolic effect on myofibrillar protein similar to that exerted by other adrenergic agonists (4, 31), skeletal muscle atrophy with suspension might be minimized. Increased mechanical loading might provide an alternative anabolic stimulus, given the increased bone formation activity noted in numerous experimental models (10, 28). However, daily intensive treadmill running during a period of suspension does not prevent changes in cortical bone morphology after 28 days of hindlimb suspension and actually appears to exacerbate some decrements in mechanical strength of femoral bone (25).

Our working hypotheses, therefore, were 1) improved maintenance of skeletal muscle mass in the hindlimb of dobutamine-treated rats will minimize loss of bone mass with suspension and 2) increasing bone mass before suspension with vigorous exercise training previous to suspension will diminish the magnitude of bone loss incurred during the period of unloading.

METHODS

Animals and study design. Forty-eight male Sprague-Dawley rats were randomized into exercise-trained or sedentary groups. All rats were housed in light- and temperature-controlled quarters and given food and water ad libitum. Beginning at 2 mo of age, 25 rats were treadmill trained for 11 wk, 5 days/wk, with progressive increases in running speed and duration. By week 7, rats were running at 31 m/min for 70-80 min at an 8% grade, or ~78 ml · kg-1 · min-1 (estimated from data in Ref. 3). Reported values for VO2 max in male Sprague-Dawley rats range from 80 to 110 ml · kg-1 · min-1 (3), so this exercise intensity is equivalent to >= 70% VO2 max. At the end of this 11-wk training, rats were randomly assigned to hindlimb suspension or cage-activity weight-bearing control treatments for 14 days and individually housed in identical cages. All animals were 135 ± 10 days old at the beginning of this suspension or cage-activity control period.

Rats were suspended at 30° head-down tilt, with the hindlimbs free to move but completely non-weight bearing by using the x-y axis pulley system of Wronski and Morey-Holton (30). Body weights and food consumption were monitored daily. Exercise-trained rats that were not suspended were placed on a lower intensity program of 40-min treadmill running, 3 days/wk at 20 m/min, to avoid significant loss of training adaptations over the 14-day experimental period. One-half of each group was given twice daily intraperitoneal injections of 1 mg/kg dobutamine (Dobutrex; Lilly, Indianapolis, IN) over the 14 days; matched controls were given intraperitoneal saline injections. Pilot studies verified that 1 mg/kg dobutamine produced moderate increases (280 ± 18 to 424 ± 22 beats/min in unconscious rats) in heart rate for 35-55 min after each injection, similar to what might be seen during exercise. All rats were injected intraperitoneally as follows with fluorochrome labels during the experimental period: day 0 = 10 mg/kg tetracycline; day 6 = 10 mg/kg xylenol orange; day 12 = 10 mg/kg calcein (1 day on, 5 days off-1 day on; 5 days off-1 day on; 2 days off schedule).

Tissue processing. On day 15 of the experimental period, anesthetized animals were killed by decapitation. Suspended rats were off suspension for no more than 1 h before time of death. Soleus muscles were dissected out rapidly, blotted dry, and weighed. Left hindlimb bones were stored in 70% ethanol until processing for quantitative histomorphometry. Femurs were cleaned of soft tissue and then dehydrated through increasing concentrations of ethanol, defatted in acetone, and embedded in methylmethacrylate. Thick (200-µm) cross sections of undecalcified femur were cut just distal to the third trochanter with a low-speed diamond wheel saw (Isomet 11-1180, Buehler, Lake Bluff, IL). One-half of the prepared sections were stained by use of a modification of the toluidine blue technique (24), and the others were left unstained for measurement of fluorochrome-labeled surfaces.

Cortical bone histomorphometry. Undecalcified femoral cross sections were analyzed by using a Zeiss IIRS microscope equipped with a Zeiss Videoplan2 digitizing system. The same investigator performed all measurements on numbered slides blinded to treatment. Transmitted light was used to measure cortical bone area, cortical width in four orthogonal quadrants, and periosteal and endosteal (marrow) diameters in the anteroposterior and mediolateral axes at ×4 magnification. Maximal cross-sectional moment of inertia (CSMImax) was estimated from these diameters by using the following equation for CSMImax of an ellipse
CSMI<SUB>max</SUB> = &pgr;/64 [(OD<SUB>min</SUB> ∗ OD<SUP>3</SUP><SUB>max</SUB>) − (ID<SUB>min</SUB> ∗ ID<SUP>3</SUP><SUB>max</SUB>)]
where ODmax and ODmin are mediolateral and anteroposterior periosteal diameters, respectively, and IDmax and IDmin are mediolateral and anteroposterior endosteal diameters, respectively. Interlabel distance between fluorochrome labels was measured at ×200 magnification with epifluorescent light. The xylenol orange label could not be seen on these cortical bone cross sections; therefore, mineral apposition rate (MAR) was calculated as interlabel distance (between tetracycline and calcein labels) divided by 12 (days between delivery of these two labels).

Statistical analyses. All data were analyzed by using Statistical Analysis Software on a university mainframe VAX computer. Group differences were tested by three-way analysis of variance with Tukey post hoc testing; results from least squares means analyses are reported as means ± SE. Pearson correlation coefficients were calculated to test for relationships between cortical bone area and muscle or body weight. Significance level was set at P < 0.05.

RESULTS

Body and muscle weights. One sedentary and five exercise-trained animals died of respiratory tract infections before euthanasia day; technical error resulted in the loss of one sedentary animal's bone morphometry data. Mortality in the exercise group was evenly distributed among the four treatment groups. Reported group sizes (Table 1) reflect these losses. At the start of the experimental period, all exercise-trained rats weighed significantly less than the sedentary nonsuspended (control) saline (Sed/Con/Sal) rats (354 ± 25 vs. 426 ± 28 g, respectively). Sedentary and exercise-trained animals ate 7-9 and 6 g/day, respectively, less rat chow the first 3 days of suspension vs. consumption during the pretreatment period. Food consumption returned to pretreatment levels by 8 days of suspension. Weight loss during the 14-day experimental period averaged 45 and 7 g in sedentary and exercise-trained rats, respectively. Nonsuspended rats gained small amounts of weight during this period (10 and 15 g in sedentary and exercise-trained rats, respectively). Body weights in all suspended animals at decapitation were 20-50 g lower than those of nonsuspended animals (Table 1); all trained rats weighed less than sedentary rats within treatment groups.

Table  1.   Body and muscle weights at euthanasia
Group n BW, g Sol Wt, mg Sol Wt Index, mg/100 g BW
Sedentary rats
  Con/Sal 6 436 ± 9  185 ± 5  42.4 ± 0.9 
  Con/Dob 4 417 ± 9  178 ± 6  42.8 ± 1.4 
  Sus/Sal 6 381 ± 6* 106 ± 4* 27.8 ± 1.2*
  Sus/Dob 6 397 ± 7* 123 ± 8* 31.0 ± 1.6*
Exercised rats
  Con/Sal 6 374 ± 8* 154 ± 8* 40.9 ± 1.3 
  Con/Dob 6 366 ± 8* 148 ± 10* 40.3 ± 2.0 
  Sus/Sal 6 346 ± 7*, Dagger 88 ± 2*, Dagger 25.4 ± 0.6*, Dagger
  Sus/Dob 7 348 ± 6*, Dagger 116 ± 4*, dagger , Dagger 33.3 ± 1.0*, dagger , Dagger
Values are means ± SE; n, no. of animals. BW, body weight; Sol Wt, soleus weight; Con, nonsuspended controls; Sus, suspended rats; Sal, saline treated; Dob, dobutamine treated. * P < 0.05 vs. Sed/Con/Sal (baseline controls). dagger P < 0.05, Dob vs. Sal rats within treatment pair. Dagger P < 0.05 vs. exercise-trained Con/Sal rats.

Suspension produced a mean 43% decline in absolute soleus weight in all suspended rats compared with their respective control groups (Table 1), confirming previous observations (27). This effect is still apparent when changes in total body weight are accounted for; soleus weight index (mg/100 g body wt) declined 33 and 39% in sedentary and exercised-trained rats, respectively, on suspension. Soleus weight indexes in nonsuspended exercise-trained rats (Ex/Con/Sal) were identical to those in sedentary control rats (i.e., Sed/Con/Sal). Dobutamine injections effectively attenuated the loss of soleus weight in the exercise-trained, but not sedentary, suspended rats.

Cortical bone area. We confirmed the typically observed decrease in cortical bone area in sedentary rats subjected to suspension (Fig. 1A). This 17% decline in bone area (vs. Sed/Con/Sal rats) was totally abolished by dobutamine administration. Paradoxically, the reverse situation was true in the exercised groups: cortical bone area in the exercised suspended saline (Ex/Sus/Sal) rats was 15% greater, on average, than that in all other exercise-trained groups and was not significantly different from that of the Sed/Con/Sal rats. Interestingly, cortical bone area of exercised suspended dobutamine-treated (Ex/Sus/Dob) animals was 16% less than that observed in Ex/Sus/Sal rats. When given to sedentary and exercise-trained control rats, dobutamine had no effect on cortical bone area.
Fig. 1. A: variations in cortical bone area at middiaphysis of femur in adult male rats after 14 days of hindlimb suspension or cage activity, with or without dobutamine (Dob) injections. B: variations in cortical bone area indexed to total body weight (BW; CBA index) for same group of animals. Con, control; Sus, suspension. * Significantly different from sedentary Con rats given saline, P < 0.05. + Significantly different from exercised Con rats given saline, P < 0.05. ++ Significantly different from saline-treated rats in same treatment pair, P < 0.05.
[View Larger Version of this Image (26K GIF file)]

All exercised rats, except those on Sus/Sal treatments, had 13-16% lower cortical bone areas than the Sed/Con/Sal rats. When cortical bone area is expressed relative to body weight, however, most of these differences disappear (Fig. 1B), as does that in sedentary suspended rats vs. sedentary controls. Ex/Sus/Sal rats, however, appear to have a relative excess of bone mass for their (reduced) body weight, exhibiting a 23% larger cortical bone index than Ex/Con/Sal rats (P < 0.05). Dobutamine treatments given to suspended rats resulted in significantly larger (17%) and significantly smaller (16.5%) cortical bone indexes vs. those in sedentary and exercise-trained Sus/Sal rats, respectively. Total body weight was strongly related (r = 0.93) to cortical bone cross-sectional area in nonsuspended rats but much less so in suspended rats (r = 0.49) (Fig. 3A). Soleus weight and bone cross-sectional area are significantly correlated in weight-bearing control animals (r = 0.78) but not in suspended animals (Fig. 3B).
Fig. 3. Correlations between BW (A) and soleus weight (B) and cortical bone area in adult male rats after 14 days of hindlimb Sus (n = 25) or weight-bearing cage activity (Con; n = 22).
[View Larger Version of this Image (19K GIF file)]

Histomorphometric variables (Table 2). Decreases in cortical bone area were not due to uniform reductions in cortical width but to site-specific reductions in the width of cortical bone, as noted previously (25). In sedentary rats subjected to suspension, the anterior and medial cortical widths were 10 and 26% smaller, respectively, than the matched widths in the Sed/Con/Sal group. The dobutamine-treated sedentary rats subjected to hindlimb suspension (Sed/Sus/Dob) exhibited no declines in cortical width relative to Sed/Con/Sal rats at any site. Exercise alone (Ex/Con/Sal rats) appeared to result in smaller cortical widths at the posterior and medial quadrants (29 and 20%, respectively) vs. those in Sed/Con/Sal rats.

Table  2.   Histomorphometric measurements of femoral middiaphyseal bone after 14 days of hindlimb suspension and/or dobutamine injections
Sedentary Rats
Exercised Rats
Con/Sal Con/Dob Sus/Sal Sus/Dob Con/Sal Con/Dob Sus/Sal Sus/Dob
Ct.Wi, mm
  Anterior 0.61 ± 0.02  0.58 ± 0.02  0.55 ± 0.02* 0.59 ± 0.02  0.58 ± 0.02  0.57 ± 0.02  0.58 ± 0.02  0.56 ± 0.02 
  Posterior 0.97 ± 0.04  0.89 ± 0.05  0.87 ± 0.04  1.01 ± 0.04dagger 0.69 ± 0.04* 0.74 ± 0.04* 0.73 ± 0.04* 0.70 ± 0.03*
  Medial 0.72 ± 0.03  0.67 ± 0.03  0.53 ± 0.03* 0.69 ± 0.03dagger 0.58 ± 0.03* 0.59 ± 0.03* 0.70 ± 0.03Dagger 0.57 ± 0.03*, dagger
  Lateral 1.14 ± 0.06  1.11 ± 0.07  1.06 ± 0.06  1.16 ± 0.06  1.06 ± 0.06  1.00 ± 0.06  1.04 ± 0.06  1.00 ± 0.05 
  Mean 0.85 ± 0.02  0.82 ± 0.03  0.75 ± 0.02* 0.86 ± 0.02dagger 0.73 ± 0.02* 0.72 ± 0.02* 0.76 ± 0.02* 0.71 ± 0.02*
B.Dm, mm
  Anteroposterior 3.40 ± 0.05  3.51 ± 0.06  3.21 ± 0.05* 3.50 ± 0.05dagger 3.19 ± 0.05* 3.13 ± 0.05* 3.33 ± 0.05  3.09 ± 0.05*, dagger
  Mediolateral 4.24 ± 0.11  4.50 ± 0.14  4.02 ± 0.11  4.42 ± 0.11dagger 4.02 ± 0.11  4.02 ± 0.11  4.46 ± 0.11Dagger 4.08 ± 0.10dagger
Ma.Dm, mm
  Anteroposterior 1.78 ± 0.06  1.98 ± 0.08  1.77 ± 0.06  1.84 ± 0.06  2.02 ± 0.06* 1.81 ± 0.06dagger , Dagger 1.99 ± 0.06* 1.83 ± 0.06Dagger
  Mediolateral 2.53 ± 0.07  2.71 ± 0.09  2.46 ± 0.07  2.56 ± 0.07  2.56 ± 0.07  2.44 ± 0.07  2.70 ± 0.07  2.51 ± 0.06dagger
MAR, µm/day
  n 6 3 5 6 6 4 4 7
  Anterior 1.42 ± 0.12  1.63 ± 0.17  1.00 ± 0.13* 1.38 ± 0.12dagger 1.38 ± 0.12  1.47 ± 0.15  1.17 ± 0.15  1.18 ± 0.11 
  Posterior 2.17 ± 0.22  2.06 ± 0.31  1.47 ± 0.24* 1.82 ± 0.22  2.01 ± 0.22  1.98 ± 0.27  1.02 ± 0.27*, Dagger 1.25 ± 0.20*, Dagger
  Medial 1.06 ± 0.18  1.11 ± 0.26  0.89 ± 0.20  0.90 ± 0.18  1.16 ± 0.18  1.20 ± 0.22  0.96 ± 0.22  1.04 ± 0.17 
  Lateral 2.64 ± 0.19  3.08 ± 0.26  1.75 ± 0.20* 1.87 ± 0.19* 2.40 ± 0.19  1.96 ± 0.23* 1.51 ± 0.23*, Dagger 1.56 ± 0.17*, Dagger
  Mean 1.83 ± 0.12  1.97 ± 0.18  1.28 ± 0.14* 1.49 ± 0.12  1.74 ± 0.12  1.65 ± 0.15  1.14 ± 0.15*, Dagger 1.26 ± 0.12*, Dagger
Values are means ± SE; n, no. of animals. Ct.Wi, cortical width; B.Dm, bone diameter; Ma.Dm, marrow diameter; MAR, mineral apposition rate at periosteal surface. * P < 0.05 vs. Sed/Con/Sal (baseline controls). dagger P < 0.05, Dob rats vs. Sal rats within treatment pair. Dagger P < 0.05 vs. exercise-trained Con/Sal rats.

Total bone diameter (B.Dm) was measured to estimate net changes in bone formation at the periosteal surface. B.Dm in the anteroposterior plane was smaller in Sed/Sus/Sal rats (5.6%) and in three of the four exercised rat groups (6-9%) (Table 2). Exercise-trained rats on suspension had B.Dm values similar to those in Sed/Sus/Sal rats. Mediolateral B.Dm changed less over the treatment period, although in this plane B.Dm in Ex/Sus/Sal rats was significantly greater (11%) than that of the nonsuspended exercise-trained rats (Ex/Con/Sal). In both sedentary and exercise-trained rats, dobutamine treatments appeared to reverse the changes noted in B.Dm in both planes after suspension.

Exercise alone and exercise plus suspension appeared to stimulate increased endosteal resorption, as indicated by significantly larger marrow diameters (Ma.Dm) in the anteroposterior plane than were seen in Sed/Con/Sal rats. Ma.Dm in the mediolateral plane appeared largely unaffected. Dobutamine injections effected decreases in the anteroposterior Ma.Dm in exercised, nonsuspended rats and the mediolateral Ma.Dm in exercised, suspended rats (10 and 8%, respectively, vs. within-group saline controls), indicating a suppression of endosteal resorption. No changes in Ma.Dm with suspension or dobutamine injections were noted among the sedentary groups, suggesting no change in endosteal resorption rate with these treatments. These site-specific changes in orthogonal dimensions produced changes in cross-sectional geometry, as indicated by changes in CSMImax (Fig. 2). Sed/Sus/Sal rats exhibited a 24% reduction in CSMImax, and three of four exercise-trained groups had a mean 25% reduction in CSMImax vs. Sed/Con/Sal rats. Because of the large variance in the CSMI values in the Sed/Con/Sal group, none of these differences was statistically significant.
Fig. 2. Variations in maximal cross-sectional moment of inertia (CSMImax) at middiaphysis of femur in adult male rats after 14 days of hindlimb Sus or cage activity, with or without Dob injections. There were no significant differences in CSMImax for any group vs. sedentary saline-treated Con rats nor within Dob-saline-treatment pairs.
[View Larger Version of this Image (28K GIF file)]

Region-specific MAR, as determined from interlabel distances on double-labeled surfaces, was dramatically lower (30-34%) in anterior, posterior, and medial quadrants of femoral cortical bone in sedentary suspended rats vs. controls. Dobutamine injections effectively restored MAR to normal in the anterior quadrant only. Exercise alone did not appear to affect MAR, but rats subjected to exercise training and then suspension exhibited 43 and 53% lower MAR (compared with Sed/Con/Sal values) in the lateral and posterior quadrants of femoral bone, respectively. Suspension resulted in lower MARs within exercise-trained groups in posterior and lateral quadrants (49 and 37%, respectively, vs. Ex/Con/Sal values); dobutamine did not alter this suppression of MAR.

DISCUSSION

Effect of exercise training before hindlimb suspension period. We could not confirm our original working hypothesis that exercise training would increase bone mass and therefore reduce the magnitude or rate of bone loss during suspension because our training protocol produced an apparent decrease in cortical bone area. Three of the four exercise groups had lower bone areas than those of the Sed/Con/Sal rats, with smaller cortical widths in two of four quadrants. Previous studies in mice (13), rats (18), dogs (22), and rhesus monkeys (5) have demonstrated reduced growth rate and/or bone mass in those animals subjected to vigorous exercise training, particularly in skeletally immature animals. Furthermore, reduced structural strength (e.g., bending stiffness, energy to fracture) has been observed in young (4-mo-old) female rats subjected to 10 wk of a training protocol similar to ours (12, 18). Our training protocol may have exceeded the intensity threshold above which normal skeletal growth and modeling activity are suppressed (9). In the present study femoral lengths were not measured, so we cannot confirm what effect this training protocol had on longitudinal growth in young adult rat femurs.

When cortical bone area is indexed to total body weight, the apparent reduction in bone area with training disappears (Fig. 1B), suggesting that the predominant effect of this exercise training was to produce smaller rats with proportionately smaller femoral bone size. This is further supported by the significant correlations observed between body weight and cortical bone area (Fig. 3). The reduced cortical widths in at least two quadrants coupled with smaller B.Dm imply slower periosteal bone formation rates and, presumably, reduced modeling activity in these exercise-trained rats. Large (but statistically nonsignificant) reductions in CSMI in all exercise-trained rats except Ex/Sus/Sal rats suggest that bone strength (strongly correlated to CSMI) might be compromised by this type of training protocol to a degree similar to that seen with unloading in sedentary animals (Sed/Sus/Sal). MAR, i.e., mineralization of previously synthesized bone matrix, was uninfluenced by exercise alone. Male rats (4 mo old) trained with a similar treadmill running protocol during 28 days of hindlimb suspension exhibit no change in longitudinal growth of femurs or tibias but experience some decrements in bone strength measurements (25).

Effect of hindlimb suspension on bone morphology and mineralization. This 14-day period of suspension produced an apparent decrease in body weight, presumably due to the decreased food consumption observed during the first 3 days as well as some stress to the animal. Given that male rats are still growing, albeit slowly, at 5 mo of age, these lower body weights may reflect some attenuation of growth as well as absolute decreases in body mass. The declines in soleus weight, however, are far greater than would be expected by a mere slowing of growth. The slow-twitch ankle extensors are preferentially affected by unloading; atrophy of the soleus, in particular, is nearly maximal after 14 days of hindlimb suspension (15). Decreases in type I and type II fiber area are also observed in the vastus medialis of rats after 14 days of spaceflight (20) and are qualitatively similar to those seen in ankle extensors. Hence, muscles acting on the femur experience similar changes with unloading as does the soleus, the muscle studied in this investigation.

With the removal of weight bearing, the presumed change in strain distribution experienced by hindlimb bones produces significant decrements in bone formation at the periosteal surface (19), primarily due to a deficit of osteoblast activity (8). Most previous data have been collected on young (1- to 3-mo-old) rats, which are experiencing rapid bone growth. Our rats were 5 mo old at time of death and closer to skeletal maturity. After 14 days of hindlimb suspension, our sedentary rats did exhibit a smaller bone area than that observed in nonsuspended controls, primarily due to decreased periosteal formation rate. When indexed to body weight, however, the suspended rats' bone area is not significantly different from that of Sed/Con/Sal rats, suggesting that the smaller bone size in the former group remains appropriately scaled to body size. Body weight is normally a major determinant of the daily mechanical loading a limb bone receives; the strength of the correlation in weight-bearing rats was halved in those animals experiencing unloading of the hindlimbs (Fig. 3A). The loading the hindlimbs experience during suspension is minimal without ground reaction forces, even though the hindlimb muscles are free to contract; hence, the contribution of body weight to daily loading of the femur becomes irrelevant during suspension, and the correlation between body weight and bone area is weakened.

Bone mineralization rate (i.e., MAR) was significantly lower in the suspended rats. We have no direct measurement of bone resorption, but smaller bone areas coupled with smaller B.Dm and decreased MAR imply that resorption activity at the periosteal surface did not decrease during suspension or at least not as much as did bone formation activity. No apparent changes were noted in resorption activites at the cortical endosteum. We did note region-specific changes in cortical width as previously observed (25) in female rats after 28 days of suspension, which yielded a (nonsignificant) decrease in CSMI.

Paradoxically, exercise-trained rats subjected to 14 days of suspension (Ex/Sus/Sal) had a larger mean cortical bone area and cortical bone index than did Ex/Con/Sal rats. In fact, all morphometric variables measured in Ex/Sus/Sal rats were identical to those in Sed/Con/Sal rats except for a decrease in posterior cortical width and an increase in anteroposterior Ma.Dm in the Ex/Sus/Sal rats, suggesting focal changes in remodeling at the posterior edge of the femur. We cannot explain these results. It should be noted that total body weight of Ex/Sus/Sal rats was significantly lower than that of Ex/Con/Sal rats, an effect that exaggerates the difference in cortical bone areas when indexed to body weight (Fig. 1B).

Most of this larger bone area in Ex/Sus/Sal rats appears to be due to periosteal expansion in the mediolateral plane compared with that in Ex/Con/Sal rats. Resorptive activity at the cortical endosteum appeared to be unaffected, with no change in Ma.Dm. Interestingly, there was a significant decline (43-53%) in MAR in exercised, suspended rats in two of four cortical bone quadrants relative to Sed/Con/Sal rats. No change was noted in these variables in exercised rats not subjected to suspension and maintained on training. These data suggest that, were suspension continued for significantly longer than 14 days, mineralized bone area would eventually be reduced even in these exercise-trained rats.

Effect of dobutamine on cortical bone changes with suspension. Clenbuterol, a beta 2-agonist, can retard the bone loss seen with immobilization of a limb caused by denervation, apparently mediated by stimulating an increase in frequency of spontaneous contractions in denervated muscle (6). It is ineffective, however, in preventing loss of tibial ash weight when administered after ablation of muscles loading the tibia (31). Therefore, some minimum level of contractile activity in intact musculature appears to be required for clenbuterol to have this bone-maintaining effect. Dobutamine is a synthetic catecholamine commonly administered to congestive heart failure patients for its positive inotropic effects, mediated by strong beta 1-adrenergic-receptor effects. It also has mild beta 2- and alpha -adrenergic effects (17). Administration of dobutamine (3.6 mg · kg-1 · day-1) in young female rats prevents the decrease in VO2 max observed during 5 wk of hindlimb suspension but has no effect on atrophy of the soleus (7).

Daily dobutamine injections in this study (2 mg · kg-1 · day-1) had no measurable effects on bone morphometry in sedentary and exercise-trained control rats, except for a 10% decrease in anteroposterior Ma.Dm in exercised rats (Ex/Con/Dob). Dobutamine treatments given to sedentary, suspended rats virtually abolished the decreases in cortical bone area, cortical bone index, and cortical width observed in saline-injected suspended rats (Sed/Sus/Sal). Preliminary evidence indicates that bone quality (as indicated by mineral content of proximal and midshaft tibia in rats) is maintained by dobutamine treatments during suspension (21). These results contrast markedly with those of Zeman et al. (31), who found no attenuation of loss of bone ash weight after 4 wk of hindlimb suspension in rats that were given the beta 2-agonist clenbuterol. The effect of dobutamine in the present study does not appear to be due to the prevention of weight loss during suspension because the body weight of dobutamine-treated suspended rats was still lower than that in control animals (Sed/Con/Sal).

In vitro evidence from work with cloned bone cell lines MC3T3-E1 or UMR-106 suggests that beta -agonists can increase adenylate cyclase activity or intracellular adenosine 3',5'-cyclic monophosphate levels in these bone cells (11, 14). Whether these agents have similar in vivo effects on bone growth and metabolism via direct effects on bone cells or indirectly via calciotropic hormones (parathyroid hormone, 1alpha ,25-dihdroxyvitamin D3, calcitonin) is unknown. Because soleus weight and soleus weight index were still significantly lower in dobutamine-treated suspended rats than in Sed/Con/Sal rats, and only minimally different in Ex/Sus/Dob vs. Ex/Sus/Sal rats, this beta -agonist does not appear to effectively maintain muscle mass per se. Even though electromyographic activity of the soleus returns to normal weight-bearing patterns after 7 days of hindlimb suspension, one can observe continued loss of muscle and bone (1). Even if an adrenergic agonist were to elevate basal contractile activity of suspended muscle or maintain muscle mass, it is unlikely that either factor would help maintain bone mass in the suspended limbs without the loading provided by ground reaction forces incurred during weight-bearing activity. The strong positive relationship between muscle weight and bone area observed in control animals virtually disappeared in suspended rats (Fig. 3B).

Interestingly, dobutamine injections were somewhat effective in ameliorating muscle mass loss in the exercised rats subjected to suspension. The 18% decrease in soleus weight index in these rats was significantly less than the 39% loss observed in the Ex/Sus/Sal group. Despite this, Ex/Sus/Dob rats had smaller bone areas than Ex/Sus/Sal animals. Because MAR did not vary significantly between these two group, whereas B.Dm values were significantly lower in the dobutamine-treated group, we surmise that the Ex/Sus/Dob rats experienced some larger reduction in bone matrix formation rates at periosteal surfaces during suspension relative to that in Ex/Sus/Sal rats.

In summary, the most significant finding of this study is the prevention of suspension-induced loss of bone mass in sedentary rats by the administration of dobutamine, a synthetic adrenergic agonist, mediated in part by the maintenance of normal mineralization rates at the periosteum during the period of unweighting. Further studies are needed to confirm the mechanism for this response, be it mediated by some indirect effect of dobutamine on limb muscle or via some direct effect on bone cell activities. Vigorous endurance exercise training preceding suspension is not an effective countermeasure for the deleterious effects of unweighting on bone.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the facilitation of this project by Dr. Leon Kazarian, who was Director of the Armstrong Aerospace Medical Research Laboratory (AAMRL) at Wright-Patterson Air Force Base, Dayton, Ohio, during the period of this project. Clarence Oloff and Ed Eveland of the AAMRL and Kathy Bailey of the Veterinary Pathobiology Bone Histology Laboratory of The Ohio State University lent invaluable technical assistance. We gratefully acknowledge Dr. Emily Morey-Holton for loaning a number of her x-y axis pulley systems for the suspension cages and Dr. A. J. Merola (Dept. of Physiological Chemistry, The Ohio State Univ.) for the use of his laboratory facilities for animal training and euthanasia.

FOOTNOTES

   This work was supported by funding from The Air Force Office of Scientific Research (project no. 2312V6), a Johnston Graduate Fellowship (Oberlin College), and by the School of Health, Physical Education, and Recreation (The Ohio State Univ.).

Address for reprint requests: S. A. Bloomfield, Dept. of Health and Kinesiology, Texas A&M Univ., College Station, TX 77843-4243 (E-mail: sbloom{at}acs.tamu.edu).

Received 10 July 1996; accepted in final form 12 March 1997.

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0161-7567/97 $5.00 Copyright © 1997 the American Physiological Society


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