Effects of weight cycling and aging on body protein content in female wistar rats

Nutrition Research, Vol. 19, No. 5, pp. 741-748, 1999 Copyright 0 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 027 l-53 17/99/S-see front matter ELSEVIER

PI1 SO271-5317(!99)00036-6

EFFECTS OF WEIGHT CYCLING AND AGING ON BODY PROTEIN CONTENT IN FEMALE WISTAR RATS K-L. Catherine Jen, Ph.D., Elizabeth S. LeClair, M.A., Anne Buison, M.S. and Michael A. Pellizzon, M.S. Department of Nutrition and Food Science, Wayne State University, Detroit, MI 48202

ABSTRACT The effects of body weight cycling (WC) on body protein content are inconclusive. This study was designed to investigate the interactive effects of WC and aging on body protein content in female Wistar rats. Young (2.5 months old at the beginning of the study) and old rats (11 months at the beginning of the study) were divided, within each age category, into: 1) low fat (LF) ad-lib fed control group (LFA); 2) LF restricted fed group (LFR); 3) high fat (HF) ad-lib fed group (HFA); 4) HF restricted fed group (HFR); 5) HF fed, weight cycled group by gaining weight first (HFG); and 6) HF fed, weight cycled group by losing weight first (HFL). WC was achieved by ad-lib and restricted feeding of the HF diet so rats’ body weight was cycling between baseline and 20% above or below the baseline level. This WC was repeated 3 to 5 times. At sacrifice, rats in restricted groups and cycling groups weighed the same. Old rats weighed significantly more than the young rats. There was no age effect on total and percent body fat, and on total protein content, although young rats tended to have higher percent protein than old rats. In the young groups, the protein percent was significantly lower in HFA, HFG and HFL groups as compared to all other groups within that age. In old rats, the protein percent of cycling groups was not different from other groups, with the exception that HFL group had significantly lower protein percent than that of the HFR group. Within each age category, the HFA group had significantly higher body weight and fat content than all other groups. Therefore, weight cycling starting at a young age, when accumulation of body protein is not yet complete, may deprive the body of the opportunity to lay down the normal amount of protein. This may have significant health implications for human teenagers engaging in weight cycling practice. 0 1999 Elsens Sc,cnceInc. KEYWORDS: Young, Old

Weight cycling, Protein percent, Fat percent, High fat feeding,

Corresponding Author: Dr. K-L. Catherine Jen, Department of Nutrition and Food Science, 3009 Science Hall, Wayne State University, Detroit, MI 48202. Phone: (3 13) 5771948; Fax: (313) 577-8616; e-mail: [email protected]

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INTRODUCTION

Weight cycling (WC), defined as repeated weight gain/loss, is a common practice in the obese population. The effects of WC on health and body weight regulation are debatable. It has been reported that WC increases the rate of weight regain and/or reduces the rate of weight loss (l-3), while others did not observe these alterations (4-9). Whether body composition is changed by WC is also controversial. Several studies have reported increased adipose tissue mass after WC (10, 1 l), while others have observed no change in fat mass (1, 6, 8, 12, 13). These inconsistent results may be due to the body weight at the time when animals were sacrificed. If rats were sacrificed when their body weight was at the lean control levels, then WC rats were more likely to have higher body fat content (10, 14). On the other hand, if rats were sacrificed when their body weight was at the level of obese, non-cycled rats, then WC rats had a similar body fat content as that of the obese rats (1, 6, 8, 12, 13). As for the effects of WC on body protein content, not much data have been reported. Brownell et al did not observe any change in body protein in WC rats (1). However, Lim et al (15) reported an increase in body protein content in WC rats while Jen et al reported significantly less protein content in WC rats (9). William and Senior have reported that WC rats gained less protein and more fat content during the refeeding phase as compared to control rats (16). Therefore, the effects of WC on body protein content deserves further exploration. In humans and animals, those who have undergone more WC cycles are more likely to be older than those with fewer WC cycles. It is well known that aging is associated with increased body fat and reduced lean body mass (17). Body protein content in elderly adults is significantly lower than that in their younger counterparts, as measured by body potassium content (18). Thus, the physiological changes associated with aging should be considered when examining the effects of WC on body composition. The present study was designed to investigate the effects of WC on body protein content in young and old rats.

METHODS AND MATERIALS

Animals Female Wistar rats (Harlan Sprague-Dawley, Indianapolis, IN) of 2 age groups were the subjects of this study. The young rats (Y) were 2.5 months old (300-325 g) while the old rats (0) were 11 months old (350-375 g) at the beginning of the study. Diets Three diets were used in this study: low fat control diet (LF), high fat diet (HF), and modified high fat diet (MHF). The LF diet was Purina Rodent Diet (#5001, PM1 Feeds, Richmond, IN). The HF diet was composed of Purina Rodent diet powder (#5001), vegetable shortening, casein (Dyets Inc., Bethlehem, PA), sucrose (Domino, New York, NY), AIN vitamin mixture (Dyets Inc., Bethlehem, PA) and AIN mineral mixture (Dyets Inc., Bethlehem, PA). The MHF diet had similar composition as the HF diet except that protein, vitamin and mineral contents were almost doubled at the expense of carbohydrate in order to assure adequate intake during restricted feeding period. The caloric content of carbohydrate, protein and fat of these 3 diets are as follows: LF diet: 60%, 28% and 12%; HF diet: 20%, 15%, and 65%; MHF diet: 8%, 30% and 62%. The caloric content of these diets are 13KJ/g, 23KJlg and 23KJ/g, respectively. The HF and MHF diets were stored in a cold room until use. These 2 diets were offered to the rats in a double-jar setup to prevent spillage.

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Procedure After a one week adaptation period, rats were divided into the following groups: (1) Young (YLFA, n=lO) and old rats (OLFA, n=12) were fed the LF diet ad-lib for the entire study period to serve as controls; (2) Young (YLFR, n=lO) and old rats (OLFR, n=12) were fed the LF diet in restricted amounts to maintain their body weight at the baseline level; (3) Young (YHFA, n=lO) and old rats (OHFA, s12) were fed the HF diet ad-lib for the entire experimental period, (4) Young (YHFG, n=lO) and old rats (OHFG, n=12) were fed the HF diet ad-lib until their body weight was 20% above the baseline level, and then they were fed the MHF diet at 50% of the ad-lib level until their body weight reached the baseline level. This was considered as one WC cycle. Young rats went through 4 WC cycles while old rats went through 5 cycles; (5) Young (YHFL, n=lO) and old rats (OHFL, n=12) were fed the MHF diet in restricted amount (SOO/,of ad-lib level) until their body weight was 20% below the baseline level. They were then fed the HF diet ad-lib until their body weight reached the baseline level. This was considered as one WC cycle. YHFL rats went through 3 such weight loss/regain cycles while OHFL group went through 4 such cycles; (6) Young (YHFR, n=lO) and old rats (OHFR, n=12) were fed the modified HF diet in restricted amount so their body weight did not change during the entire experimental period. All rats had water ad-lib. Food intake and body weight were measured 3 times per week. Except the rats in HFA and LFA groups, all other rats were sacrificed when their body weight was at the baseline level (young rats = 320f5 g, old rats = 370f8g). At sacrifice, all rats were exposed to CO, briefly before being decapitated. Liver and retroperitoneal fat pads were dissected, weighed and frozen in liquid nitrogen and stored in a -7O’C freezer for further analyses. All visible fat in the internal cavity was removed and weighed. This fat, together with retroperitoneal fat pads, was designated as internal fat. The remaining carcass was stored frozen until body composition analysis at a later date. Body composition analysis was performed according to the procedure described by Jen et al (19). In brief, eviscerated carcass was shaved, autoclaved and homogenized in a polytron homogenizer (Brinkmann, Westbury, NY). Triplicate samples of the homogenate were taken for body fat, protein and moisture determinations. Body fat content was measured by the method of Folch et al (20). Body protein was estimated using the homogenized carcass according to the method of Lowry et al (21). All data were expressed as mean + SEM. Analysis of variance was conducted with age and diet treatment as independent variables. The significant level was set at ~~0.05. When a significant result was obtained, post-hoc paired t-tests were performed to identify the groups that contributed to this difference.

RESULTS

Body weight Body weight changes for the young (upper panel) and old rats (lower panel) during the entire experimental period are depicted in Figure 1. The two ad-lib groups gained weight continuously while the two restricted groups maintained a similar body weight throughout the entire study period. The HFA groups weighed significantly more than the LFA groups. For the rest of the groups, their body weight cycled between 20% above or below baseline and baseline level. At sacrifice, there was no difference in body weight among these groups (Table 1). Body protein

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Age had no effect on total protein content, although young rats as a group tended to have higher percent protein than the old rats (~~0.065). When comparing each dietary treatment, young rats in the two WC groups had the lowest protein content as compared to all other young groups. In old rats, only WC below the baseline weight and OLFR groups had the lowest protein content. Young rats had higher percent body protein (YLFA and YLFR, pdO.05, Table 1) or had similar amounts (YHFA, YHFL and YHFR) as compared to their old counterparts. The YHFG group had significantly lower protein percent than their old counterparts. In young rats, YHFR, YLFA and YLFR groups had significantly higher percent body protein than the YHFA, YHFG and YHFL groups. In the old rats, OHFA, OHFL, OLFA and OLFR groups had similar percent body protein which was significantly lower than that of the OHFR group. The percent body protein of the OHFG group was not different from all other groups.

III

0 650 600 550

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10

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1’1”“““”

IIIII

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1 -.-

OHFA OHFG -.- OHFL

500 450 400 350 300

0

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Weeks FIG. 1. Body weight changes of all the rats during the entire experimental period. Upper panel depicts the young rats and lower pane1 represents the old rats. (Y: Young; 0: Old; HFA: High fat ad-lib; HFG: High fat cycling by gaining weight first; HFL: High fat cycling by losing weight first; HFR: High fat restricted feeding; LFA: Low fat ad-lib; LFR: Low fat restricted feeding.)

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Body fat

Table 1 shows the body fat content and percent at sacrifice for all the groups. HFA groups had significantly more fat mass than the other groups regardless of age. The difference in total fat and percent body fat between old and young rats failed to reach statistical significance. For the young rats, YHFA had significantly more percent fat than the rest of the groups while YHFR and YLFR groups had less percent fat as compared to other groups. For the old rats, the OHFA rats had significantly more percent fat than all other groups while there were no differences among the remaining groups.

TABLE 1 Body Weight, Protein%, Fat% and Protein%/fat% HFA

HFL

HFG

Ratio of All Groups of Rats at Sacrifice HFR

LFA

LFR

400f17d

3 18+4’

426MC

371+3b

Body weight (g) bc

bc

Young

557k27a

328k6

363fl lb

324f5

Old

586k7’

372f5b

348k23b

367tib

Total protein (g)

bd

bc

Young

133*18a

7OIkl3

Old

120f8a

93+1 7ac

acd lOlf8

8Ozkl3 bc 73f6

102+11

21.8+1.1a

31.2f1.0b

ab

abc

121kl la

99k8

107+1 la

90f5

bc

Protein% Young

22.6k0.8a

bc

29.6k0.8b

31.0;t1.2b

25.2f1.0a

24.3k0.8ac

19.8f5.7a

20.4f0.6a

29.2kl.l

Young

189k23a

64+6b

42k4b

66f8b

40f6b

Old

223fl 6a

63tib

65+6b

82tib

49f7b

Young

33.2+1.0a

18.5f1.0b

12.9k0.4c

16.5k0.6b

12.3kO.8’

Old

36.4k0.7a

15.0fl.9b

17.8k0.8b

19.0k0.6b

12.8k0.6b

Old Total fat (g)

Fat%

Protein %/fat % Young Old

.58+.02a .55+.02a

1.23f. 14ac

2.51f.lb

2.01f.l lbC

2.80f.33b

1.34+.05b

bc 1.58zk.07

1.46f.llb

2.04k.16

Values are expressed as means zk SEM. Values with different superscripts in each row are significantly different from each other (~~0.05 or 0.01).

cd

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K.-L.C. JEN et al.

Ratio of percent protein to percent fat In order to examine the relationship between body protein percent and fat percent, a protein percent/fat percent ratio was calculated and presented in Table 1. For young rats, YHFR, YLFA and YLFR rats had more than double the amount of protein as compared to fat. The two young cycling groups had similar amounts of protein and fat while the YHFA group had significantly more fat than protein. For the old rats, OLFR rats had twice the amount of protein as compared to fat. Old rats in the 2 cycling groups and OHFR as well as OLFA group had similar amounts of fat and protein. The OHFA rats had only half the amount of the protein as that of fat.

The purpose of this study was to examine the percent of protein in young and old rats subjected to WC. We have reported previously that during WC, both fat free mass and fat content are altered, although fat mass accounts more for the body weight changes (9). It was expected that in old rats, the increased body fat and reduced body protein content with aging will make old rats lose body protein more readily than young rats when exposed to WC. Results from this study demonstrated that young non-cycling rats tended to have higher percent body protein than old rats at sacrifice. However, WC affected percent protein differently between young and old rats. In old rats with a WC history of losing weight below the baseline level was associated with a loss of percent body protein. With WC between obese level and baseline weight, there was no reduction in percent body protein. In young rats, regardless whether WC occurred above or below the baseline weight, there was a significant reduction of total and percent body protein. This may be explained by the normal growth pattern of rats. It is known that rats fed standard laboratory rodent diet will continuously grow well into adulthood, although adult female rats grow at a slower rate compared to adult male rats (22). The growth rate is higher at a younger age than that at an older age (9,22). In the present study, the young noncycled rats grew at a rate of 10.3f1.2 g/wk, which tended to be higher than that of the old rats (7.75-0.8 g/wk, ~~0.06). During normal growth, both lean mass and fat mass are deposited in the body. It is also reasonable to speculate that young rats will accumulate more protein than the old rats. With the WC paradigm used in this study, repeated weight loss, presumably due to loss of protein and fat mass, deprived young rats the chance to accumulate an adequate amount of body protein. During a refeeding period of l-2 weeks, there may not be an adequate amount of time for rats to lay down a similar amount of protein as that of the non-cycling rats. This would result in a lower body protein content in young WC rats as compared to the old rats. In the old rats, only when WC occurred below the baseline level was a decrease in total body protein content observed. The difference in the age of rats used in WC studies may partially explain some of the controversies reported in the literature (1,9,. 15). Percent protein to percent fat ratio is another way to express the relationship between body fat and lean mass. Rats fed the HF diet ad-lib, regardless of the age, had nearly twice amount of fat as compared to protein content. The general pattern of this ratio among the other groups is that in old rats, the OLFR group had higher protein to fat ratio. This indicates that there is more protein than fat in the body. This implies that as rats grow old, their body fat would increase and/or body protein would reduce naturally, unless these rats were fed a low fat diet in restricted amount. In young rats, WC reduces protein percent to fat percent ratio to a level similar to that of the old WC rats. Thus, even at a young age, WC will alter body composition to the level as seen in old rats.

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In summary, WC in young rats reduced protein content in the body while in old rats, WC did not further reduce protein content beyond that was produced by aging. Even though extrapolation into human population deserves caution, this finding has significant health implications. In the young human population, many female teenagers or children are trying to lose weight, even though their body weight is in the normal range (23,24). It is known that in humans, body protein continuously accumulates in adolescents until they reach the age about 1% 20 years (25). Before their body protein content reaches peak level, dieting or WC may hinder their body’s ability to accumulate a normal amount of protein. This may reduce the basal metabolism and further predispose these young adults to obesity in the future. Considering the fact that obese children are the fastest growing population (26), WC may become more common in this population. The result of reduced body protein content due to WC in this population stresses the importance of nutrition education in preventing excess weight gain and obesity in adolescents.

REFERENCES

Brownell J, Greenwood M, Stellar E, Shrager E. The effects of repeated cycles of weight 1. loss and regain in rats. Physiol Behav 1986;38:459-464. 2. Archambault C, Czyzewski D, Cordua y Cruz G, Foreyt J, Mariotto M. Effects of weight cycling in female rats. Physiol Behav 1989;46:4 17-42 1. 3. Gerardo-Gettens T, Miller G, Horwitz B, et al. Exercise decreases fat selection in female rats during weight cycling. Am J Physiol 1991;260:R518-R524. 4. Contreras R, Williams A. High fat/sucrose feeding attenuates the hypertension spontaneously hypertensive rats. Physiol Behav 1989;46:285-291. 5. Cleat-y M. Consequences of restricted feeding/refeeding Zucker rats. J Nutr 1986;116:290-303.

of

cycles in lean and obese female

6. Graham B, Chang S, Lin D, Yakubu F, Hill J. Effect of weight cycling on susceptibility dietary obesity. Am J Physiol 1990;259:R1096-R1102.

to

7. Hill J, Thacker S, Newby D, Nickel M, DiGirolamo M. A comparison of constant feeding with bouts of fasting-refeeding at three levels of nutrition in the rats. Int. J. Obesity 1987;11:201-212. 8. Wheeler J, Martin R, Yakubu F, Lin D, Hill J. Weight cycling in female rats subjected to varying meal patterns. Am J Physiol 1990;258:R124-R139. 9. Jen K-L, Lu H, Savona L, Watkins A, Shaw M. Long-term weight cycling reduces body weight and fat free mass, but not fat mass in female Wistar rats. Int J Obesity 1995; 19:699-708. 10. Uhley V, Jen K-L. Changes in feeding efficiency and carcass composition repeated high-fat feedings. Int. J. Obesity 1989;13:849-856. 11. Rodin J, Radke-Sharpe N, Rebuffe-Strive distribution. Int J Obes 1990;14:303-310.

in rats on

M, Greenwood M. Weight cycling and fat

748

K.-L.C. JEN et al.

12. Hill J, Latiff H, DiGirolamo M. Influence of food restriction coupled with weight cycling on carcass energy restoration during ad-lib refeeding. Int J Obes 1988;12:547-555.

13. Reed G, Cox G, Yakubu F, Ding L, Hill J. Effects of weight cycling in rats allowed a choice of diet. Am J Physiol 1993;264:R35-R40. 14. Bell R, McGill T. Body composition in mice maintained restriction and refeeding. Nutr Res 1987;7: 173-182.

with cyclic periods of food

15. Lim k, Murakami E, Lee S, Shimonura Y, Suzuki M. Effects of intermittent food restriction and refeeding on energy efficiency and body fat deposition in sedentary and exercised rats. J Nutr Sci Vitamin 1996;42:449-468. 16. Williams VJ, Senior, W. Changes in body composition and efficiency of food utilization for growth in young adult female rats before, during and after a period of food restriction. Aust J Biol Sci 1979;32:41-50. 17. Bokan G, Norris A. Fat redistribution male. Human Biol 1977;49:495-5 14.

and the changing body composition

of the adult

18. Flynn M, Nolph G, Baker A, Martin W, Krause G. Total body potassium in aging humans: a longitudinal study. Am J Clin Nutr 1989;50:713-717. 19. Jen K-L, Greenwood M, Brasel J. Sex differences in the effects of high-fat feeding on behavior and carcass composition. Physiol Behav 1981;27:161-166. 20. Folch J, Lees M, Sloane-Stanley G. A simple method for the isolation and purification total lipids from animal tissues. J Biol Chem. 1957;226:1861-1869. 21. Lowry 0, Rosebrough N, Fat-r A, Randoll R. Protein measurement reagent. J Biol Chem 1951;193:265-275.

of

with the Folin phenol

22. Coscina D, Dixon L. Body weight regulation in anorexia nervosa: Insights from an animal model. In: Darby P, Garfinkel P, Gamer D, Coscina D, eds. Anorexia Nervosa: Recent Developments in Research. NY: Alan R. Liss, 1983:207-219. 23. Frank R, Serdula M, Adame D. Weight loss and bulimic eating behavior: changing patterns within a population of young adult women. Southern Med J 1991;84:457-460. 24. Maloney M, McGuire J, Daniels S, Specker B. Dieting behavior and eating attitudes in children. Pediatrics 1989;84:482-489. 25. Guo SS, Chumlea WC, Roche AF, Siervogel RM. Age- and maturity-related changes in body composition during adolescence into adulthood: The Fels Longitudinal Study. Int J Obes 1997;21:1167-1175. 26.

Rossner S. Childhood obesity and adulthood consequences. Accepted

for

publication

September 28,

1998.

Acta Paediatrica

1998;87:1-5.