The structural components of the skeleton develop and are maintained in a 1 g environment, shaped by the mechanical load to which they are constantly exposed. Altering such a mechanical load by reducing the gravitational force imposed on the system, as in space flight, has profound effects on the skeleton and permits an exploration of the molecular events which regulate normal skeletal homeostasis. The objective was to determine whether simulated weightlessness reduced intestinal calcium transport, and if so, to determine the molecular mechanisms for such an effect. A nonstressful tail suspension in which the rats gained weight normally while suspended was used to simulate weightlessness. A significant change in intestinal calcium transport was not demonstrated. However, a cyclic change in bone formation with suspension was shown. Based on these observations, the objective changed to determination of the hormonal regulation of bone formation during simulated weightlessness

Bikle, D. D.

English

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ALTERATIONS IN GUT TRANSPORT OF MINERALS AND IN BINDING PROTEINS
DURING SIMULATED WEIGHTLESSNESS: NAGW-236
Final Technical Report
(Sisk-C8-175637) IITFFATIC ,S Ir GUT
TBINSFOAT CF mjhffAIS Ahr Ib EIbDING
P3CT$INS rUBING SIMULATED Nr1Gb:LESSNSSS
Final Technical nEFcrt, 1 auq. 1981 - 1 bov.
1934 (California Univ.)
	 13 F HC 102/!!F A01 G3/52
NE5-24709
Unclas
14829
dPrincipal Investigator: Daniel D. Bikle, M.D., Ph.D.
Period Covered: 08/01/81 - 11/01/84
Grantee Institution:	 University of California, San Francisco
145 Irving St.
San Francisco, CA 94143
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The structural components of the skeleton have developed and are maintained in
a 1 g environment, shaped in part by the mechanical load to which they are constantly
exposed. Altering such a mechanical load by reducing the gravitational force imposed
on the system, as in space flight, has profound effects on the skeleton and permits an
exploration of the molecular events which regulate normal skeletal homeostasis.
Objectives
At the onset of this project, the primary objective was to determine whether
simulated weightlessness reduced intestinal calcium transport, and if so, to determine
the molecular mechanisms for such an effect. A nonstressful tail suspension in which
the rats gained weight normally while suspended was used to simulate weightlessness.
Using the model, we could not demonstrate a significant change in intestinal calcium
transport. However, we did demonstrate a cyclic change in bone formation with
suspension. Based on these observations, our objective changed to determining the
hormonal regulation of bone formation during simulated weightlessness. This work is
continuing in our new grant with NASA-Ames Research Center (NCC 2 328).
Accomplishments
We have suspended growing rats (160-200g) by their tails for at least 4 weeks
without altering their growth patterns. No difference in weight gain between such
animals and their pair fed cont-ols is observed. The suspended rats in comparison to
the pair fed controls show a decrease in fat free weight, ash weight and calcium
content of the unloaded bones (tibia, lumbar vertebra) but not of the normally loaded
bones (humerus and mandible) (fig. 1) Most of the difference in bone mass between
suspended and pair fed controls occurs progressively between days 2 to 10, after which
no further difference between suspended and pair fed controls is observed. In the
experiment depicted in figure 1 all rats were killed at the same age; the differences
observed were the result of suspending rats at different times (and, thus, age) prior to
sacrifice. We also suspended rats of the same age for differing periods of time (up to
4 weeks) and compared their bones with those of pair fed controls. These results
(fig. 2) demonstrate that tibial bone mass increases linearly with time in the pair fed
controls. In contrast, tibial bone mass did not increase between the 1st and 2nd week
of suspension, but resumed a rate of growth parallel to that in controls between the
2nd and 4th weeks. The humerus of the suspended rats did not show this cessation of
bone growth.
We examined the effect of suspension on two biochemical measures of bone
formation: 45calcium uptake presumably into mineral and 3H-proline uptake
presumably into matrix. In the experiment depicted in figure 3 and table 1 we
separated the long bones into the proximal third (labelled metaphysis) and the shaft
(labelled diaphysis) to determine whether suspension altered these regions of bone
differently. As anticipated, the proximal portion of the long bones, which contains a
higher percentage of endosteal bone than the shaft (which is primarily cortical bone
;nd has a h4Sher per 
3 
ntage of calcium/mg fat free weight) had a three fold greater
amount of Ca and H-proline incorporation (table 0. With suspension both portions
of the tibia showed a decrement in calcium content (fig 3A), calcium uptake (fig. 3B),
and proline inco r poration (fig. 3C) by 5 to 7 days when compared to the tibia from pair
fed controls. After 10 days no further decrement in calcium content was seen and
-.
0
45 Ca and 3H-proline uptake rebounded to control or above control levels. The bone
formation rate was determined at the tibia-fibula junction (cortical bone) in suspended
rats and pair fed controls using three tetracycline labels administered on days 3, 9, and
14 to mark two periods of bone formation: period 1 = days 3 - 9, period 2 = days 9 - 14.
The results (table 2) indicate that suspension leads to a 50% reduction in bone
formation in the tibia during period 1, but with continued suspension these
measurements of bone growth return toward normal.
Our initial approach to determining whether the calciotropic hormones were
involved in signaling the unloaded bone to stop growing was to measure calcium,
phosphorus, parathyroid hormone and 1,25(OH)2D in the serum of suspended and pair
fed control rats (table 3). The results indicate no differences in the serum levels of
any of these ions or hormones at 15 days. However, small ^ncreases in serum calcium
were observed at days 5 to 7 of suspension which return to lair fed control values by
day 15 (fig 4). The rise in serum calcium was associated with a fall in serum
1,25(OH)2D values (fig. 5). Furthermore, a small and usually .nsignificant reduction in
intestinal calcium transport (a process regulated by 1,25(OH)2D) occurs between
5-10 days of suspension, but calcium transport returns to control levels by day .5
(fig. 6). These observations suggest that changes in the serum levels of the
calciotropic hormones during skeletal unloading may provide at least part of the
mechanism by which bone formation is inhibited by day 5 and is restored to normal by
day 10.
In an initial effort to prevent the deleterious effect of suspension on bone mass
we determined whether increments in dietary calcium could prevent or lessen the
inhibition of bone formation caused by suspension. As seen in figure 7, increasing
dietary calcium from 0.1 to 2.4% marked:y increased the calcium content of all bones,
weighted or unweighted. However, the unweighted bones (tibia, L-1) in the suspended
animals had a lower calcium content at all levels of dietary calcium abo- ^ 0.1%.
Histomorphometric analysis of these bones suggests that increasing dietary calcium
reduces bone resorption with little effect or. bone formation; the unweighted bones
from suspended animals have a lower rate of bone formation than pair fed controls
regardles° of dietary calcium.
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Table 1
Calcium Uptake, Proline Uptake and Bone Mass in Different Regions of Bone
f
Tibia	 Humerus
I
Metaphysis	 Diaphysis	 Meta h sis	 Diaphysis
I
45Ca/bone 1	215,800 + 7300 59,900 + 3000	 138,200 + 5200 46,200 + 2000
3H-pro/bone l 116,000 + 8700 33,600 + 2300	 69,800 + 4900 23,500 + 1700
mg Ca/bone 2	20.4+0.4	 24.7+0.2	 21.5+0.6	 15.5+0.2
mgbone3	124+4	 116+3	 11.1+3	 69+4
1 = dpm per total bone fragment + SEM
2 = mg calcium per total bone fragment + SEM
3 = fat free weight of bone fragment + SEM
27 (h)
Table 2
Bone Formation Rate at the Tibia - Fibula Junction during the
Initial (Days 3-9) and Later (Days 9-14) Periods of Suspension
Days 3 - 9	 Da, s 9 - 14
Suspended	 0.019 + 0.003 1*	0.030 + 0.005*+
Control	 0.038 + 0.003	 0.041 + 0.005
1 = mean mm 2 + SD
* = p < 0.05 compared to control
+ = p < 0.05 compared to initial period
27 (i)
Table 3
Serum Calcium, Phosphorus, PTH and 1,25(0i-i)2D
in Rats Suspended for Two Weeks and in their Pair-fed Controls
Suspended Control
•	 Calcium l 10.2 + 0.2 10.2 + 0.1
Phosphorus l 6.2 + 0.2 6.1 + 0.1
PTH 2 0.65+0.18 0.65+O.1
1,25(OH)2D 3 168 + 22 173 + 28
1 = mg/dl
2 = ng eq/ml
3 = pg/ml
27 (j)
PUBLICATIONS
1. Bikle, D.D., R.K. Globus anti E.R. Morey. Calcium transport from the intestine
and into bone rti a rat model simulating weightlessness. Physiologist 25:5143-
144, 1982.
2. Simmons, D.J., B. Gr2 z — an, J.E. Russell, W.V. Wall(-,-, D.D. Bikle, E.R. Morey.
Simulatir,g certain aspects of hypogravity: effects on bone maturation in the
non-weight bearing skeleton. Aviation. Space Environ Med 54:1080-1084, 1983.
3. Globus, R.K., Bikle, D.D., Morey-Holton, E. Effects of simulated we:ghtlessriess
on bone mineral metabolism. Endo-- rinology 114:2264-2270, '984.
Globus R.K., Bikle, D.D., Holton, E.M. The temporal response of bone to
unloading. Submitted to Endocrinology.
ABSTRACTS
1. Bikle, D.D., R.K. G:obus and E.R. Mor--y. CALCIUM TRANSPORT FROM THE
INTESTINE INTO BONE IN A RAT MODEL SIMULATING WEIGHTLESSNESS.
Amer Physiol Soc 33rd Annual Meeting, 1982.
2. Bikle, D.D., R. Globus, and E. Morey-Holton. BONE LOSS IN TAIL-SUSPENDED
RATS IS RESTRICTED TC THE L I NWEIGHTED LIMBS. Presented at the 1983
Meeting of the NASA Spa,.e Biology Symposium, 1983.
3. Pa t terson-Allen, P., R. Globus, B. Halloran, D. Elkle, C. Cann, .and E. Morey.
RELATION OF SERUM OSTEOCALCIN AND 1,25-DIHYDFOX 'J!TAMIN D 3 TO
BONE FORMATION DURING SKELETAL UNLOADING. Calcif Tissue Int
36:468, 1984.
Wronski, T.J., B.P. Halloran, U.U. Bikle, R.K. Globus, ar„-1 E.R. Morey.
SKELETAL RESPONSE TO SIMULATED WEIGHTLESSNESS: A COMPARISON
OF ANIMAL MODELS. Presented at the 6th Annual Meeting of the IUPS
Commission on Gravitational Physiology, Lausanne, Switzerland, 1984.
5. Bikle, D.D., R.K. Globus, B.P. slloran, E.M. Ho''on. THE SALUTARY EFFECT
OF DIETARY CALCIUM ON BONE MASS IN A RAT MODEL. OF SIMULATED
WEIGHTLESSNESS. Presented at the 1984 Meeting of the NASA Space Biology
Symposium, 1984.
6. Halloran, B.P., D.D. Bikle, E.M. Holton, M. Levers, R.K. Clobus. THE ROLE OF
VITAMIN D IN THE BONE CHANGES ASSOCIATED WITH SIMULATED
WEIGHTLESSNESS. Presented at the 1984 Meeting of the Space Biology
Symposium, 1984.
7. Globus, R.K., D.D. Bikle, B.P. Halloran, and E.M. Holton. EFFECTS OF
DIETARY CALCIUM ON SKELETAL RESPONSE TO UNWEIGHTING. Clin Res
33:102A, 1985.
8. Halloran, B.P., D.D. Bikle, T.J. Wronski, R.K. Globus, M.J. Levins, and E.M.
Holton. CHANGES IN VITAMIN D METABOLISM AND BONE HISTOLOGY
ASSOCIATED WITH SKELETAL UNLOADING. Amer Soc Bone Min Res, 1985.
E
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Alterations in gut transport of minerals and in binding proteins during simulated weightlessness

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