Effects of voluntary resistance exercise and pulse patterned
high-protein snack on bone mass composition and
,
strength in rats given glucocorticoid-injections
ATSUOTatsuhiro M
Abstract
We examined the effects of a voluntary resistance exercise (climbing together with high-protein snacks) pulse pattern on bone mass and strength in rats given glucocorticoid-injections mg/kg/day as a model of
( ) ( 2 )
age-related osteopenia Sixty-four male Wistar rats. , 10 weeks of age were assigned to low protein, (15% of total food intake or normal protein) (20 )% diet groups Half of the rats were exercised daily during dark period. - h These groups were further divided into groups that received no snack or a pulse pattern (20：00 8：00 ).
high-protein snack(75% of dietary protein during resting period All groups were meal-fed at) . 8：30 9：30- h and 20：30 21：30- h and the snack was fed at 12：30 13：30- h for 8 weeks Energy intake was. approximately equal in all groups The exercise groups were forced to climb a wire-mesh tower cage. ( 20 cm diameter× 200 cm height to drink water from a bottle set at the top Bone weight and calcium content were) . increased by climbing with pulse pattern high-protein snacks. Climbing significantly increased bone maximum load and structural stiffness However no differences in bone mass or strength were observed between low and. , normal protein feeding groups These results suggest that resistance exercise protects against the development of.
. .
osteoporosis associated with aging Pulse pattern high-protein snacks may enhance the effects of exercise resistance exercise pulse protein feeding bone mass bone strength glucocorticoid
Key words： , , , ,
Introduction
Osteoporosis a serious problem in elderly people is, , characterized by bone loss leading to fractures and high bone turnover.1,2） With age more amino acids are, absorbed from the digestive tracts and extracted by splanchnic tissues which can result in a lower availability, of dietary amino acids to the peripheral tissues.3） It would be reasonable to hypothesize that in cases of low protein intake or increased protein requirement this limited sys-, temic availability of dietary amino acids could contribute to decreased bone protein synthesis which could result in,
. osteoporosis in elderly persons
, In studies of bone protein synthesis and osteoporosis glucocorticoid-injected rats are commonly used as a model of aging because glucocorticoid hormones are involved in the aging process.4－6） Protein supplementation with a high insulinogenic carbohydrate after meals should increase amino acid supply to peripheral tissues We previously. reported that high protein snack feeding 3 h after regular meals increased total blood amino acid flow calculated by the area under the curve of diurnal amino acid concentration
in glucocorticoid-injected rats.6） In addition high protein, snack together with resistance exercise (voluntary tower climbing showed significant preventive effect on glucoco-)
. ,
rticoid-induced sarcopenia5 ,6） and osteopenia 4） However these studies did not investigate detailed effects of exercise
. on bone mass and strength
On the other hand Arnal, et al. proposed to modulate , . ., protein feeding pattern in humans7 ,8） and old rats9) i e
. without changing the total amount of protein given each day They found that consuming 80% of daily protein intake during one meal (pulse protein feeding pattern leads to) more efficient protein utilization In deed they showed. , that the pulse protein feeding pattern was more efficient in improving nitrogen balance than a spread pattern composed of 4 meals that equalized the daily protein intake over the
7 ,8）
feeding period in elderly women
The purpose of this study was to examine the preventive effects of voluntary climbing together with a pulse pattern high-protein snack on bone mass and strength in glucoco-rticoid-induced aging model rats Moreover we examined. , the effects of daily protein intake amounts because elderly person in Japan tend to decrease the intake of high protein
, . foods such as meats eggs or dairy products
Materials and Methods
Sixty-four male Wistar rats (5 weeks old were pur-) chased from Japan SLC Inc, . (Shizuoka and were accli-) ( 22 matized for a week under standard laboratory conditions
% humidity The light/dark cycle was h
±2℃, 60 ). 12
with lights on from 8：00 h to 20：00 h Rats were. ( φ20 × housed in metal cages with a wire mesh tower cm
cm that had two water bottles set at the top to adjust 200 )
21：30 8：00 5 climbing exercise4－6） from h to h for
. 10 ( ：178
weeks At the age of weeks mean initial weight g), rats were assigned to low protein (15% of total food intake or normal protein) (20 )% diet groups The half of. rats were exercised daily during dark period. These groups were further divided into groups that received no snack or
(75 )
pulse pattern high-protein snack % of dietary protein during resting period These groups were normal-protein-.
( ), ( ),
sedentary group N low-protein-sedentary group L normal-protein-sedentary with pulse pattern high-protein snack group NS( ), low-protein-sedentary with pulse pattern high-protein snack group (LS), normal-protein-exercise group (NE), low-protein-exercise group (LE), normal-protein-exercise with pulse pattern high protein snack group NSE and low-protein-exercise with pulse pattern high-( ),
( ). 2
protein snack group LSE All groups were given
( ,
mg/kg/day of prednisolon Wako Pure Chemical Industries Ltd., Osaka intraperitoneally at) 9：30 h Composition. of experimental diets were shown in Table 1 . The N and
NE groups were fed 6.5 g of normal protein diet the L, and LE groups were fed6.5 g of low protein diet twice a day(8：30 9：30, 20：30 21：30- - h for) 8 weeks from( 10 to18 weeks old). The NS and NSE groups were fed5 g of normal protein diet the LS and LSE groups were fed, g of low protein diet twice a day with g of a high
pro-5 3
tein snack at 12：30 13：30- h All rats were fed the. experimental diets isoenergetically during 8 weeks of ex-perimental period The NE LE NSE and LSE groups. , ,
8 .
exercised continuously in tower climbing cages for weeks The N L NS and LS groups were sedentary At the end, , . of the experiment the rats were killed by decapitation at, h after overnight fasting Blood was collected to
10：00 .
obtain serum Bilateral tibia were quickly removed freed. , of connective tissues and measured for length, mid shaft
. width and wet weight
Bone protein and calcium contents were determined by the methods described previously.4） A three-point bending test was performed as previously described4 ,10,11） using a
( , 33005, , .
load tester Rheoner Model RE- Yamaden Co Ltd., Tokyo). Each left tibia was placed on a holding device with supports located at a distance of1 2 mm with, the lesser trochanter proximal to and in contact with the, , proximal transverse bar The mid point served as the ante-. rior upper loading point A bending force was applied by( ) . the crosshead at a speed of 0.1 mm/sec until fracture occurred The breaking load. ( )N and structural stiffness N/mm were obtained directly from the load-deformation ( )
curves that were recorded continually in a computerized .
monitor linked to the load tester Table1 Composition of experimental diets
N NS L LS
Groups
Meal Meal Snack Meal Meal Snack
Ingredients 582.0 771.8 － 632.7 792.1 152.2 Cornstarch 200.0 80.0 600.0 150.0 60.0 450.0 Casein 68.0 － 294.0 68.0 － 294.0 Sucrose 50.0 50.0 － 50.0 50.0 － Cellulose 50.0 50.0 50.0 50.0 50.0 50.0 Soybean oil 35.0 35.0 35.0 35.0 35.0 35.0 Mineral mixture１ 10.0 10.0 10.0 10.0 10.0 10.0 Vitamine mixture１ 3.0 1.2 9.0 2.3 0.9 6.8 DL-Methionine 2.0 2.0 2.0 2.0 2.0 2.0 Choline chloride 0.01 0.01 0.01 0.01 0.01 0.01 Butylhydroxytoluene 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 Total
N normal protein diet L low protein diet S snack, ； , ； ,
76 .
１
Table2 Tibial structual measurements mechanical parameters and composition from each group of rats,
Dry weight Length Midshaft Width Maximum load Structural stiffness Protein Calcium
Group (mg) (mm) (mm) ( )N (N/mm) (mg) (mg) ｃ ｃ ｂｃ ｂ ｂｃ ｂ N 257±14 36.7±0.4 2.40±0.07 56.0±2.5 86.9±6.2 55.9±3.5 63.4±4.2 ｃ ｃ ｃ ｂ ｃ ｂ L 249±16 36.3±0.5 2.41±0.13 52.6±3.7 81.2±9.0 54.6±4.7 60.5±3.2 ｃ ｃ ｂｃ ｂ ｂｃ ｂ NS 251±10 36.5±0.4 2.43±0.03 55.7±2.2 83.0±5.2 54.6±2.4 62.9±2.8 ｂｃ ｂｃ ｂ ｂ ｂｃ ｂ LS 262± 9 36.4±0.3 2.43±0.08 57.1±2.5 83.8±4.8 56.6±2.6 63.9±3.4 ａｂ ａｂｃ ａ ａ ｂ ａ NE 276±18 36.5±0.4 2.51±0.08 62.0±5.3 93.5±9.8 58.8±4.6 68.4±4.6 ａ ａｂ ａ ａ ａ ａ LE 287±12 36.7±0.3 2.52±0.07 61.4±4.9 94.8±9.7 62.8±4.1 69.9±4.1 ａ ａ ａ ａ ａ ａ NSE 289±15 36.8±0.4 2.54±0.09 64.6±1.9 97.5±4.7 63.2±4.8 70.9±4.1 ａ ａ ａ ａ ａ ａ LSE 287±12 36.4±0.5 2.53±0.07 64.5±4.2 97.7±5.3 63.7±3.8 70.1±3.6 ± 6 8 . , ； , ； , ； , .
Values are means SD for - rats in each group N normal protein diet L low protein diet S snack E climbing exercise
Means with different superscripts within a column are significantly different at p＜0.05 calculated by three-way ANOVA and Fisher's PLSD .
tests
All values are expressed as mean ± SD Data were. . assessed by three-way ANOVA and Fisher's PLSD test Statistical significance was set at p value of ＜ 0.05. All analyses were performed with a commercially available
( 5.0, .,
statistical package StatView J- SAS Institute Inc , ).
Cary NC
Results and Discussion
Final body weight was not different among the all groups range of mean weights - g Chronic climbing
( ：207 217 ).
, ,
significantly enhanced tibial bone weight midshaft width maximum load structural stiffness protein and calcium, , contents but did not alter tibial length p( ＜0.05, three-way ANOVA) (Table 2). Pulse pattern high-protein snack sig-( ＜0.05, nificantly increased tibial mechanical parameters p three-way ANOVA) (Table 2). Tibial maximum load and structural stiffness were significantly increased by climbing with pulse pattern high-protein snacks. However no, differences in any of the parameters were observed between
( )
low and normal protein feeding three-way ANOVA Table Tibial bone weight midshaft width maximum
( 2). , ,
load structural stiffness protein and calcium contents, , were greatest in the NSE and LSE groups among all groups (Table2).
These results suggest that chronic voluntary climbing with pulse pattern high-protein snacks is more effective than amount of dietary protein in rats given glucocorticoid-injection. We previously demonstrated that voluntary climbing together with spread pattern high-protein snacks % of dietary protein showed significant preventive
(42 )
effects on glucocorticoid-induced osteopenia.4） In our previous study climbing with spread pattern high-protein, snacks increased 5.1 6.8, 9.5 16.1, 15.7 22.2- - - and 9.4
-% for bone protein calcium maximum load and
13.3 , ,
structural stiffness respectively On the other hand the, . , present study found that climbing with pulse pattern
high-(75 ) 13.1 16.6,
protein snacks % of dietary protein increased -11.8 15.9, 15.4 20.3- - and 12.3 20.3- % for bone protein, calcium maximum load and structural stiffness respec-, , tively Pulse pattern high-protein snack might be more. effective than spread pattern high-protein snack although a simple comparison of previous and present results is difficult
. because experimental conditions were different
We have reported that high-protein snacks increased serum essential amino acid.6） These results suggest that an abrupt increase in serum amino acid might stimulate bone protein synthesis However few studies have examined. , the relationship between serum amino acid and bone formation Arnal. et al.7,8） demonstrated that pulse pattern protein feeding (protein consumed mainly (80 )% in one meal restored stimulation of muscle protein synthesis) during the feeding period in old rats and elderly women better than a spread protein feeding pattern spreading daily( protein intake over four meals). Bohe et al.12） reported
－1 －1
that 162 mg of mixed amino acid kg body weight( ) h infused intravenuously for 6 h in healthy human caused muscle protein synthesis to respond rapidly to the increased
( 2 )
availability of amino acids but was then after hours inhibited despite continued amino acid availability, . Our
, present findings do not contradict these previous results though the mechanisms of bone protein synthesis may differ
. from those of muscle protein synthesis
In conclusion we show in this study that voluntary, resistance exercise together with pulse pattern high-protein snacks increases bone mass and strength in rats given glucocorticoid-injection while no effect was observed for, dietary protein These results suggest that resistance exer-.
cise and pulse pattern high protein supplementation may be an effective preventive therapy for osteoporosis associated
with aging However further studies will be required to. , .
address several unsolved problems
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自発的レジスタンス運動と高タンパク質パルスフィーディングがグルココルチコイド
投与ラットの骨重量，骨組成および骨強度に及ぼす影響
松尾達博 要 旨 自発的クライミング運動とパルスフィーディングによる高蛋白質間食が，グルココルチコイド投与ラット（骨減弱モ デル）の骨重量，骨組成および骨強度に及ぼす影響について検討した．10週齢のWistar系雄ラット64匹を低蛋白質食群 （総摂取量の15％）と普通蛋白質食群（同20％）に半数ずつ分け，続いて各々を安静群とクライミング運動群に分類した．さらにそれらを高蛋白質間食群（摂取蛋白質量の75％）と非間食群のサブグループに分類した．全てのラットに実 験食を８：30～９：30および20：30～21：30に与え，間食群には12：30～13：30に高蛋白質間食を与え，摂取エネル ギー量が各群で等しくなるように調節した．運動群のラットにはクライミング運動用タワー内を昇降させた．骨重量お よびカルシウム含量はクライミング運動とパルスフィーディングによる高蛋白質間食によって増加した．クライミング 運動は骨強度を有意に増加させた．しかしながら，低蛋白質食群と普通蛋白質食群の間には骨量および骨強度のいずれ も有意な差は見られなかった．以上の結果からクライミング運動とパルスフィーディングによる高蛋白質間食が老化に 伴う骨減弱症の予防に有効である可能性が示唆された．
