The Effect of Exercise and Dietary Protein Levelson Somatic Growth, Body Composition, andSerum Lipid Levels in Adult Hamsters1

KATARINA T. BORER, JOHN HALLFRISCH,ALAN C. TSAI, CLAIRE HALLFRISCH ANDLAWRENCE R. KUHNSDepartment of Physical Education, Human NutritionProgram, and Department of Radiology, Universityof Michigan, Ann Arbor, Michigan 48109

ABSTRACT The role of increased protein intake in the acceleration ofgrowth in adult female hamsters during, and immediately following, voluntary exercise, was examined in a 2 X 2 factorial experiment with the variables of physical activity (exercise or sedentary condition) and dietaryprotein levels ( 18 or 39% ). Cornstarch and casein were added to a commercial powdered stock diet to produce 18 and 39%-protein diets, respectively.Two groups of seven hamsters (an 18 and 39%-protein group) were sedentary throughout 92 days, while the other two groups (n = 7) had access tovoluntary disc activity during the first 47 days and were retired from activity for the remaining 45 days. Protein intake of all hamsters was maintainedat the same level. Increased intake of energy during the 47 days of exerciseand first 29 days of retirement of the exercised group was supplied bycornstarch. On day 45 of retirement, prior exercise was associated with increased somatic growth, increase in serum concentrations of cholesterol, andno changes in the percentage of body fat and protein. On day 92, increasein the protein content of the diet was associated with increased percentageof body fat, decreased percentage of body protein, increased concentrationof serum cholesterol, and no acceleration of somatic growth. Serum triglycéridelevels were not changed. We conclude that the acceleration ofgrowth associated with voluntary exercise in adult hamsters does not resultfrom increased protein intake. J. Nutr. Õ09:222-228, 1979.INDEXING KEY WORDS dietary protein •exercise •growth •obesity •cholesterol

Voluntary exercise accelerates the rate of to slow sedentary level and the percentagesomatic growth in adult hamsters which of fat in the body is unchanged (1).have attained the slow, asymptotic rate of The mechanism of exercise-inducedgrowth (1, 2). In such slowly growing growth in adult hamsters has not been fullyanimals, the rate of weight gain (ponderal elucidated. It was recently shown (3) thatgrowth) is increased during exercise and exercise induced increases in serum con-for several days after its termination. Axialskeleton grOWS during exercise, while the Received for publication February 28, 1978.aDDCndicular skeleton grows during both >This research was supported by grants-ln-ald from

^r . IB ,o , The Weight Watchers Foundation, Inc., to K. T.exercise and early retirement. In tne nam- Borer, from the Honors council of the university of«rpr«\vhifVi havp hf>pn rfHred from exercise Michigan School of Literature, Arts, and Sciencessters wnicn nave it renreu irum exen-i»< to j HaUfrlgch and from Michigan Heart Agsociationfor several weeks, the growth rate returns to A. c. Tsai.

222

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DIETARY PROTEIN AND GROWTH IN EXERCISE 223

centrations of growth hormone (GH) andinsulin in adult hamsters. However, boththe endocrine changes and the somaticgrowth of exercising hamsters depend onthe quantities of ingested nutrients. In adlibitum fed animals, increased growth andincreased concentrations of GH in theserum of exercising and recently-retiredhamsters are associated with increased consumption of food. If exercising hamsters arematched in the amounts of food availableto sedentary hamsters, then the increase insomatic growth is prevented or delayeduntil such time when unlimited quantitiesof food are again made available (3). Inaddition, increases in the serum concentration of GH are seen in food restricted hamsters 4 hours after refeeding but not aftera 14-hour fast. Thus, exercise-induced GHsecretion and acceleration of somaticgrowth depend in some way on increasedconsumption of food.

In the present study we have tried toresolve the question of whether growth associated with voluntary exercise dependson increased ingestion of energy or increased intake of a specific nutrient contained in the diet. In particular, we wantedto test the hypothesis that exercising andrecently-retired hamsters grow faster because they ingest greater quantities of protein as a consequence of their increasedfood intake. It was previously shown thatrats fed a low protein diet will not increasetheir food consumption to levels that wouldensure adequate growth or survival (4)except under special circumstances such aspregnancy (5). It was suggested that inthose instances quantitative regulation ofenergy intake interfered with the adequateselection of dietary constituents. If, however, rats fed a low-protein diet were exposed to increased energy expenditure inthe form of exercise (4) or low ambienttemperature (4, 6) then their growth rateand survival improved as a result of increased protein consumption that accompanied increased food intake.

We tested the hypothesis of the dependence of growth associated with exercisein adult hamsters on dietary protein in twoways. First, we equalized the consumptionof basal diets and consequently of protein,in exercising and sedentary hamsters by

supplying the additional energy duringexercise in the form of carbohydrate(starch). And secondly, we performed theexperiment with diets containing 18 or39% crude protein. These levels are 5%less and 16% more protein than is available in the commercial diet previouslyshown to allow exercise-induced growth.

Our expectations were that the proteindependence of somatic growth during exercise and early retirement would be revealed in equivalent somatic growth ofexercising and sedentary hamsters consuming the same quantities of protein andin superior somatic growth of hamsterseating the high-protein diet as comparedto hamsters eating lower-protein diet.

MATERIALS AND METHODS

Animals and experimental design.Twenty-eight adult female hamsters (Meso-cricetus auratus, Waterhouse) 2 weighingabout 95 g and approximately 10 weeks oldwere allotted to four groups based onmatching of body weight and body lengthon the first day of the experiment. Animalswere maintained in a room with controlledtemperature (21°) and light-dark cycles

12L:12D). Hamsters were individuallyhoused in acrylic boxes 30 cm wide, 40 cmhigh, and 40 cm long with wire-mesh floorsduring the first 47 days of the experiment.A freely-turning horizontal disc, 25 cm indiameter (7 ) was mounted inside the cagesof exercising animals. During the remaining45 days of the experiment all of the animalswere individually housed in stainless steelstandard suspended cages with wire-meshfloors. Exposure to exercise lasted 47 days,and activity levels were electronically recorded as revolutions per day (RPD). Theexperiment was a 2 X 2 factorial designwith physical-activity (exercise or sedentary), and the protein-level (high protein(HP) or low-protein (LP) diets) as variables.

Diets and food consumption. A commercially available ground stock diet3 was con-

*Hamsters were obtained from Engle LaboratoryAnimals, Farmersburg, Indiana.

"Formulab Purina meal 5008 consisting of 23%protein, 6.5% (at, 3.8% crude fiber, and 49.9%nitrogen-free extract according to the manufacturer.

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224 BORER ET AL.

O o.O o 8u. Or-6

EXERCISE

O IO 20 30 40 50 60 70 80

DAYS

Fig. I Changes in the mean consumption offood, in g/100 g body weight/day, for four groupsof seven hamsters subjected to exercise and HPdiet (solid triangles), exercise and LP diet (solidcircles), sedentary condition and HP diet (opentriangles), sedentary condition and LP diet (opencircles) during the 76 days of the experiment.

verted to a LP basal diet containing 18%protein with the addition of cornstarch, andto HP basal diet containing 33% proteinwith the addition of casein. Diets were supplied to hamsters in glass jars 6 cm highand 6 cm in diameter attached to a wall ofthe cage. Food consumption was determined from the weight of food jars and ofspilled food every 3 days during the first76 days of the experiment. Cornstarch wasadded to diets of exercising hamsters inproportion to their increased food intake.

Growth measurements. Ponderal growthwas recorded every 3 days throughout theexperiment. Total body length and lengthsof selected bones were determined on day1 and day 90 of the experiment. For bodylength and skeletal measurements hamsterswere anesthetized with sodium pentobarbi-tal (75 mg/kg IP) and fully extended.Total body length was noted between thetip of the nose and the tip of the tail. Inaddition, lengths of skull, of vertebralcolumn of the body and tail, and of hu-merus and femur, were measured withdirect-reading point calipers from thewhole-body radiographs obtained with astandardized procedure ( 1).

Body composition and blood analyses.At the termination of the experiment, hamsters were killed by decapitation. Bloodwas collected from the neck and used for

determination of serum lipid levels. Thecarcass (without gut contents) of eachhamster was autoclaved, homogenized, andanalyzed for dry matter content and forlipid levels. The dry-matter content wasdetermined by freeze-drying, and totallipid content was determined in Goldfischfat extractor with petroleum ether.4 Thedefatted sample was then used for determination of crude protein with the Kjeldahlmethod.5 Total serum cholesterol concentration was determined according to theprocedure described by Kim and Goldberg(8) and triglycérideconcentration by themethod of Biggs et al. (9 ).

Statistical methods. Standard errors ofthe mean (SEM) are used to express datavariability. Analysis of variance (AOV)was employed to determine the effects ofexercise and protein intake on food intake,somatic growth, body composition, andserum lipid levels. Scheffe simultaneous inference procedure (10) was used for multiple comparisons, and Student's i-test wasused in two-group comparisons.

RESULTS

Activity levels. During the 47 days ofvoluntary exercise, HP and LP groups engaged in comparable levels of runningactivity (27,266 ±1,224 versus 28,389 ±1, 782 RPD, respectively).

Food consumption. Exercising and sedentary hamsters consumed equivalentquantities of protein throughout 76 days ofthe experiment. The cumulative total protein consumption was 316.3 ±3.9 and 305.7±9.1 g for the HP exercising and sedentary groups, respectively, and 155.9 ±3.0and 147.2 ±5.5 g for the LP exercising andsedentary groups, respectively. The changesin food consumption in the course of theexperiment are presented in figure 1. Exercise was associated with a significant increase in food consumption during 47 daysof exercise and 29 days of retirement

4 Meat and meat products (1970) 24.005, Crudefat or ether extract-Official final action. In : OfficialMethods of Analysis of AOAC (Horwitz, W., ed.),llth ed.. p. 392, AOAC, Washington, D.C.

6 Meat and meat products (1970) 24.010. Nitrogen-Official final action. In : Official Methods of Analysis of AOAC (Horwitz, W., ed.), llth ed., p. 393,AOAC. Washington, D.C.

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DIETARY PROTEIN AND GROWTH IN EXERCISE

whether one considered absolute food consumption values ( P < 0.001 ) or food consumption expressed per 100 g of hamsterbody weight (fig. l, P < 0.001). Duringthe first 76 days of the experiment, themean absolute food consumption of exercising HP and LP groups and of sedentaryHP and LP groups was 12.9 ±0.2, 13.1 ±0.3, 10.4 ±0.3, and 11.0 ±0.4 g/day, respectively. The corresponding mean foodconsumption values expressed per 100 gbody weight were 10.1 ±2, 10.5 ±0.2,8.7 ±0.1, and 8.9 ±0.1 g, respectively. Thelevel of protein in the diet had no influenceover food consumption.

Somatic growth. All hamster groupsgained weight at about the same rate during the exercise period (fig. 2). However,during the first 3 weeks after the termination of exercise, the retired hamsters gainedweight faster than the sedentary animals.The overall weight increments during theentire experimental period were significantly greater in exercising than in sedentary hamsters (table 1). Exercise was alsoassociated with significant linear growth ofthe total body, skull, vertebral column ofthe body, and humérus(table 1). Dietaryprotein levels had no effect on somaticgrowth.

irò

1603" 150

x 140—LU130S

120>-oO HOm

100<LU 90S

80EXERCISE

0 10 20 30 40 50 60 70 80 90DAYS

Fig. 2 Changes in the mean body weights ofthe four groups of seven hamsters as a function ofHP or LP diets, exercise, and sedentary conditions.The same symbols as in figure 1 are employed.

Body composition. Dietary protein levelshad a significant effect on the percentagesof body fat and of body protein ( table 1).HP groups contained significantly greaterpercentage of body fat ( P < 0.05 ) and significantly smaller percentage of body pro-

TABLE 1

Somatic growth (between days 1 and 92}, body composition, and serum concentrations ofcholesterol and triglycérides(on day 92 of the experiment)

High protein Low protein

Exercised Sedentary Exercised Sedentary AOV

Weight increments,gLengthincrements,mmTotal

bodySkullVertebral

columnTailHumérusFemurBody

water(%)Bodyfat(%)Bodyprotein(%)Serum

cholesterol(mg/100ml)Serum

triglycérides(mg/100ml)69.4

±5.9«18.4

±1.13.23±0.2013.50±

0.831.62±0.223.36±0.203.20±

0.1256.3±1.120.2±1.420.3±0.6194.0

+12.5150.0

±19.343.4

±4.312.4

±1.22.77±0.109.4

1±0.830.75±0.422.38±

0.242.24±0.2855.2±0.820.2±1.321.0±0.6162.0

±13.6141.0

±15.160.1

±3.518.0

±0.73.25±0.1213.06±

0.350.75±0.503.28±

0.252.78±0.4258.2

±1.116.1±1.422.6±0.5164.0

±6.4109.0

±17.742.6

+7.913.1

±1.22.90±0.1510.46±0.870.67±0.722.33±0.242.59

±0.2958.1±1.718.1±1.921.1±0.4143.0

±10.9132.0

±19.3EEEEEPPE,P

1Analysis of variance E designates significant effect due to exercise at P < 0.05. P designates significanteffect (P < 0.05) due to high protein level in the diet. * Mean±l SEMfor seven hamsters. The exercisedgroup had access to an exercise disc for days 1 to 47 of the experiment.

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226 BORER ET AL.

tein (P < 0.05) than the LP groups. Bodycomposition of exercising and sedentaryhamsters was similar on the last day of theexperiment.

Serum cholesterol and triglycérideconcentrations. Changes in serum cholesteroland triglycéridesare presented in table 1.High protein intake and exercise significantly elevated serum cholesterol levels.Changes in serum triglycéride concentration were not significant.

DISCUSSION

Forty five days after their retirementfrom exercise hamsters displayed significantly greater ponderal (fig. 2) and skeletal growth (table 1) than the sedentaryhamsters consuming the same quantity ofprotein. Conversely, increased protein intake had no effect on the rate of somaticgrowth whether the hamsters were sedentary or exercising. The increase (69%) inthe protein content of HP diet over thatavailable in unmodified stock diet is of thesame order of magnitude as the amount ofextra protein ingested by exercising hamsters when they voluntarily increase theconsumption of commercial diet (7 ). Theseresults rule out the increased consumptionof protein as the probable cause of growthacceleration associated with voluntary exercise in adult hamsters. Our results confirm the conclusions reached by others ( 11)that 20 to 25% of protein in the diet adequately supports the rapid growth rate inhamsters. In contrast to studies conductedby Andik et al. (6) and by Meyer andHargus (4 ), with diets containing very lowlevels of protein, results of the presentstudy suggest that increases in the proteincontent of the diet above the adequatelevel does not influence ponderal growthin hamsters. Instead, the reduction ingrowth rate which is seen in hamsters fedpelleted stock diet after they exceed theage of about 70 days and body weight ofabout 100 g (2) is related to maturationalchanges in the neuroendocrine controls ofgrowth. These controls are disturbed byvoluntary disc exercise which is accompanied by increased secretion of GH and

insulin (3). These controls can also be disturbed by direct manipulations of adulthamster limbic forebrain. Thus lesions ofmedial rostral septal nucleus in adult hamsters lead to striking acceleration in somaticgrowth, increased concentrations of GHand insulin in the serum, and decreasedcontent and concentration of GH in theanterior pituitary without an effect on thepercentage of body fat (13). In addition,transection of nerve pathways connectingthe rostral septum with the hippocampusand hypothalamus also leads to acceleration of ponderal and skeletal growth inadult hamsters.6

The present study supports our earlierconclusion (3) that hamsters have to increase their energy intake during and/orimmediately following voluntary exercisebefore they can display increased ponderalgrowth. In the absence of the additionalenergy intake to compensate for the energycost of running (3, 12), the stimulation ofsomatic growth in adult hamster by exercise is blocked. Further research is necessary to determine the relative contributionof increased energy intake, increased GHsecretion, and increased insulin secretion,all of which covary during and immediatelyfollowing voluntary exercise in adult hamsters, on the acceleration of ponderal andskeletal growth.

Manipulation of the protein content ofthe diet had a significant effect over twoparameters of hamsters body composition.Hamsters fed 39% protein diet had a significantly greater percentage of fat and asignificantly smaller percentage of proteinin their bodies than the hamsters fed a18% protein diet (table 1). It would, thus,appear that supplementation of protein inthe diet above the levels required for optimal growth stimulates the deposition of fatin the adult hamster. In other studies (7),increased deposition of fat was noted inhamsters offered sunflower seeds in addition to a stock diet. In this instance, accumulation of excess fat was accompaniedby a preference for sunflower seeds and ingestiónof fat in quantities greater than areavailable in stock diets. In accordance with

•Borer. K. T., Trulson, M. E. & Kulms. L. R.(1978) The role of llmbic system in the control ofhamster growth. Brain Research Bulletin (In Press).

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DIETARY PROTEIN AND GROWTH IN EXERCISE 227

our previous findings using indirect methods of estimation of body fat ( 14 ), resultsof the present study have confirmed thatexercise is not associated with change inthe percentage of body fat at several weeksafter the termination of activity (table 1).

An unanticipated finding of the presentstudy was the maintenance of high ratesof weight gain associated in adult sedentary hamsters fed powdered diets. When apelleted diet is fed, ponderal growth rateof hamsters weighing between 100 and 120g ranges between 0.0 and 0.5 g/day (2).Introduction of sunflower seeds is also associated with a short-lived acceleration ofponderal growth rate (14). In the presentstudy, all four hamster groups gained about0.85 g/day during the first 47 days of theexperiment following the introduction ofLP and HP powdered diets (fig. 2). Following the seventh week of the experimentthe rate of weight gain of sedentary hamsters subsided, and the effects of exerciseover ponderal growth became evident(fig. 2). Since exercise is effective in accelerating growth only in animals that haveattained the slow rate of somatic growth(2) and this condition did not occur in thepresent study during the first 47 days following the introduction of powdered diets,the stimulatory effect of exercise oversomatic growth was restricted to the periodof retirement. The superior weight gain ofhamsters fed the powdered diets stands incontrast to the experience of others (11).

Finally, the values for serum cholesterolconcentration obtained in the present studyare in agreement with the findings reportedby others ( 15, 16). Total cholesterol concentration in hamster serum is of the sameorder of magnitude as in the dog and monkey and substantially higher than thevalues obtained for cat, rat, rabbit, andguinea pig. We have found that both exercise and the increase in protein intake areassociated with significant elevations ofserum cholesterol concentration 6 weeksafter the termination of exercise.

Exercise has often been reported to havea hypoglyceridemic effect both in humans( 17) and in rats ( 18 ). However, its effecton serum cholesterol level is less clear, although small decreases have been noted

(17, 18). It should be noted that in thepresent study, serum lipid concentrationswere determined several weeks after thetermination of activity at the time the rateof weight gain was independent of exercise and dietary variables. Thus the observed effects are probably not comparableto those noted during voluntary or forcedexercise. The significance of these changesis currently under further investigation.

ACKNOWLEDGMENT

We thank E. S. Valenstein for the use ofsome laboratory facilities and equipment.

LITERATURE CITED

1. Borer, K. T. & Kuhns, L. R. (1977) Radio-graphic evidence for acceleration of skeletalgrowth in adult hamsters by exercise. Growth41, 1-13.

2. Borer, K. T. & Kaplan, L. R. (1977) Exercise-induced growth in golden hamsters: Effects of age, weight, and activity level. Physiol.Behav. 18, 29-34.

3. Borer, K. T. & Kelch, R. P. (1978) Increased serum growth hormone and somaticgrowth in exercising adult hamsters. Am. J.Physiol. 234, E611-E616.

4. Meyer, J. H. & Hargus, W. A. (1959)Factors influencing food intake of rats fedlow-protein ratios. Am. J. Physiol. 197, 1350-1352.

5. Menaker, L. & Navia, J. M. (1973) Appetite regulation in the rat under various physiological conditions: The role of dietary proteinand calories. J. Nutr. 103, 347-352.

6. Andik, I., Donhoffer, S., Farkas, M. & Schmidt,P. ( 1963 ) Ambient temperature and survival on a protein-deficient diet. Br. J. Nutr.17, 257-261.

7. Borer, K. T. (1974) Absence of weightregulation in exercising hamsters. Physiol.Behav. 12, 589-597.

8. Kim, E. & Goldberg, M. (1969) Serumcholesterol assay using a stable Liebermann-Burchold reagent. Clinical Chemistry 15,1171-1179.

9. Biggs, H. G., Erikson, J. M. & Moorehead,W. R. ( 1975 ) Manual colorimetrie assayof triglycérides in serum. Clin. Chem. 21,437-441.

10. Scheffe, H. The analysis of variance. J. Wiley& Sons, New York, 1959.

11. Banta, C. A., Warner, R. G. & Robertson, J. B.( 1975 ) Protein nutrition of the goldenhamster. J. Nutr. 105, 38-45.

12. Browne, S. A. H. & Borer, K. T. (1978)Basis for the exercise-induced hyperphagia inadult hamsters. Physiol. Behav. 20, 553-552.

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13. Borer, K. T., Kelch, R. P., White, M. P.,Dolson, L. & Kuhns, L. R. (1977) The roleof septal area in the neuroendocrine controlof growth in the adult golden hamster. Neuro-endocrinology 23, 133-150.

14. Borer, K. T. & Kooi, A. A. (1975) Regulatory defense of the exercise-induced weightelevation in hamsters. Behav. Biology 13,301-310.

15. Ho, K. J. ( 1975) Effect of cholesterol feeding in circadian rhythm of hepatic and intestinal biosynthesis in hamsters. Proceed.Soc. Exp. Biol. Med. 150, 271-277.

16. Lee, C.-C., Hermann, R. G. & Froman, R. O.(1959) Serum, bile, and liver total cholesterol of laboratory animals, toads, and frogs.Proc. Soc. Exp. Biol. Med. 102, 542-544.

17. Wood, P. D., Haskell, W. L., Stern, M. P.,Lewis, S. & Perry, C. (1977) Plasma lipo-protein distributions in male and female runners. Ann. N.Y. Acad. Sci. 301, 748-763.

18. Lampman, R. M., Foss, M. L., Block, W. D.& Flora, J. D. (1976) Metabolic adaptation with physical training: "C-acetate incorporation into tissue lipids. Metabolism 25,1601-1609.

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