Obesity Research & Clinical Practice (2010) 4, e91—e100
ORIGINAL ARTICLE
Differential effects of rapid and slow body mass reduction on body composition during an equivalent weight loss in rats Shinji Tai, Yachiyo Harada, Yukari Yokota, Yasukimi Tsurumi, Mitsuhiko Masuhara, Koji Okamura ∗ Graduate School of Sport Sciences, Osaka University of Health and Sport Sciences, 1-1 Asashirodai, Kumatori, Sen-nan, Osaka 590-0496, Japan Received 17 March 2009 ; received in revised form 16 July 2009; accepted 28 September 2009
KEYWORDS Intensity of body mass reduction; Weight loss speed; Energy restriction; Splanchnic tissues
Summary It is unclear whether the rate of body mass (BM) reduction affects the body composition with an equivalent BM reduction and whether this is influenced by the intensity of BM reduction. To elucidate this, two experiments (Exp.) were conducted. In Exp. 1, the rats fasted for 3 days to decrease BM rapidly (R3); energy was restricted at 85% of their estimated basal metabolic rate to decrease BM slowly, until it reached the same BM as R3 (S20). In Exp. 2, the rats fasted for 7 days (R7); received a restricted diet as in Exp. 1 (S50). The BM decreased 11% in R3 and S20 showed a BM equivalent to R3 on Day 20. In Exp. 2, the BM decreased 18% in R7 and S50 reached the BM of R7 on Day 50. The mass and water and protein in the skeletal muscle and adipose tissue mass did not differ between the groups in both experiments. In contrast, the stomach mass was 12.9% lower in R3 than S20 (P < 0.05) and the liver mass was 9.9% lower in R3 (P = 0.078). In Exp. 2, the stomach and liver masses were 13.2% and 18.2% lower in R7 than S50 (P < 0.05), respectively. The differences in the rate of BM reduction were seen in splanchnic tissues than in skeletal muscles and adipose tissues regardless of BM intensity. The larger BM reduction appeared to be related to a greater difference in the liver mass between the rapid and slow BM reduction. © 2009 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Introduction
∗ Corresponding author. Tel.: +81 724 53 8839; fax: +81 724 53 8818. E-mail address: [email protected] (K. Okamura).
There are many advertisements and commercial copies for various body mass reduction methods in Japan. However, some of the information includes the method of decreasing body mass rapidly during the short period. Although the slow body mass
1871-403X/$ — see front matter © 2009 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.orcp.2009.09.006
e92 reduction has been recommended to decrease body mass safely, the rapid body mass reduction has not been recommended to the general population due to the several adverse effects [1,2]. Therefore, it is important to indicate the information for the body mass loss speed and it would promote awareness about the suitable method of body mass reduction. In addition, the understanding of various energy restrictions on the body composition may be relevant to clinics in obese human with metabolic syndrome. Research investigating the influence of the rate of body mass reduction on the body composition during the equivalent amount of body mass reduction in humans is limited. Kukidome et al. [2] showed that a rapid body mass reduction for 10 days decreased body mass by 7.8 kg and fat free mass by 73% of the total body mass loss while slow body mass reduction for 28 days reduced body mass by 6.9 kg and fat free mass by 53% of the total body mass loss in wrestlers. That study suggested that a rapid body mass reduction caused a greater loss of fat free mass in comparison to a slow body mass reduction. However, the mass and composition of both skeletal muscles and splanchnic tissues were not determined. Furthermore, there was a possibility that the results could not be applied to ordinary people since dehydrating by sweating and severe water restrictions were performed during the rapid body mass reduction experiment. Other studies regarding the rate of body mass reduction focused on the body fluid status [3] or exercise performance [4] in wrestlers and whole body protein kinetics [5] or glutathione synthesis [6] in lean and obese men. These studies also did not determine whether the mass and composition of skeletal muscles, adipose tissues and splanchnic tissues are influenced by the rate of body mass reduction. Afolabi et al. [5] showed the whole body protein breakdown and the oxidation rate to increase during rapid body mass reduction, whereas it either remained unchanged or decreased during slow body mass reduction when the body mass decreased 5% in humans. Furthermore, both the whole body protein breakdown and the oxidation rate decreased more when the intensity of body mass reduction increased from 5% to 7% during slow body mass reduction. Therefore, these results may indicate that the difference between rapid and slow body mass reduction on body composition increases as the intensity of body mass reduction increases. In animals, although the changes in the body composition after fasting or energy restriction have been described in many animal studies [7—12], no previous study has directly compared the effects of rapid vs. slow body mass reduction on body compo-
S. Tai et al. sition during an equivalent reduction in the amount of body mass. The purpose of the present study was to investigate whether the rate of body mass reduction influences the body composition, including the skeletal muscles, adipose tissues and splanchnic tissues in rats. In addition, this study investigated whether the influence of the rate of body mass reduction on body composition was altered by increasing the intensity of body mass reduction in rats. The hypothesis of this study is that a rapid body mass reduction might induce a greater decrease in the amount of the fat free mass such as the skeletal muscle, in comparison to a slow body mass reduction. Furthermore, the differences in the fat free mass between a rapid and slow body mass reduction are considered to increase as the intensity of body mass reduction increases.
Materials and methods Animals and study design The design of experiments (Exp.) 1 and 2 is summarized in Fig. 1A and B, respectively. Male Sprague—Dawley rats were obtained from Japan Clea, Inc. (Tokyo, Japan) and kept individually in metabolic cages. The reason why a rat model was used was because it is possible to isolate single muscle, adipose tissues and splanchnic tissues and perform an analysis on whole tissue preparations but the use of human subjects is limited to small tissue biopsies and indirect methods of determining tissue mass to assess tissue composition. The animal room was maintained at 23 ◦ C with a 12-h light—dark cycle (lights on from 2100 to 0900 h). The rats were fed ad libitum a standard rat chow (CE-2, Japan Clea, Inc.) before the commencement of body mass reduction and were allowed free access to water throughout the experiment. All study procedures were conducted in conformity with the ‘‘Guide for the Care and Use of Laboratory Animals’’ publication no. 85—23, revised 1985 of the NIH. In Exp. 1, the rats (23 weeks old, n = 14) were divided into two groups; one group fasted for 3 days to decrease body mass rapidly (Rapid, R3; n = 7); a second group received the restricted energy which was 15% lower than their estimated basal metabolic rate [13], to slowly reach a body mass comparable to that of the R group (Slow, S20; n = 7). In Exp. 2, rats (23 weeks old, n = 13) were separated into two groups; one group fasted for 7 days to rapidly decrease body mass (Rapid, R7; n = 7); second group received a restricted energy intake to gradually
Body mass reduction rate on body composition
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Figure 1 Outline of the protocol of experiment 1 (A) and experiment 2 (B). Ad-lib indicates ad libitum feeding.
reach a body mass comparable to that of R7 as well as the protocol of Exp. 1 (Slow, S50; n = 6). The fasting period was selected to produce a clear loss of body energy without causing emaciation [8,14]. In addition, this degree of energy restriction was used because the amount of energy intake gradually decreases the body mass in rats of this age (unpublished data from our laboratory). The body mass, food intake, water consumption and weight of urine and feces were recorded daily at 0900 h. Urine and feces from all rats were collected before the body mass reduction and the sacrifice in each group. Blood was taken from the tail vein at 0830 h before the beginning of experiment and on the final day of each group. Blood was immediately centrifuged at 3000 rpm for 15 min at 4 ◦ C. On the last days of body mass reduction in each group, the rats were euthanized under ether anesthesia. The skeletal muscles (gastrocnemius, soleus, plantaris, extensor digitorum longus and tibialis anterior), abdominal adipose tissues (perirenal and epididymal) and splanchnic tissues (liver, stomach and small intestine) were excised and the masses of these tissues were determined. The masses of the stomach and small intestine were measured after removing the contents of these organs with saline.
Nitrogen balance The nitrogen content in urine and feces was measured with the Kjeldahl method and the nitrogen balance was calculated as the difference between ingested nitrogen and nitrogen excreted in urine and feces.
Water balance The water balance was calculated as difference between water consumption and urine volume.
Blood analysis The plasma glucose concentration was measured with the mutarotase-glucose oxidase method (Glucose CII-test Wako, Wako Pure Chemical Industries, Ltd., Osaka, Japan).
Statistical analysis All values are expressed as the mean and SD. Data were assessed by unpaired t-test. Statistical significance was assigned if P < 0.05. All analyses were performed using a statistical software package (Stat View J-5.0, SAS Institute Inc., Cary, NC).
Measurement of tissue composition The tissues and collected feces samples were lyophilized. The sample water was determined as the difference of the mass before and after lyophilization. The nitrogen content in the samples was measured by the Kjeldahl method and converted to protein content by multiplying 6.25.
Results Body mass Exp. 1. The body masses during the experiment 1 are shown in Fig. 2. The body mass did not differ between R3 and S20 on the day before (Day −1)
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Figure 2 Change in the body mass for rapid body mass reduction (R3) and slow body mass reduction (S20) during the study. Values represent the means (SD) for 7 rats. *P < 0.05 vs. R3.
the experiment (R3 646.4 g (SD 44.1), S20 643.6 (35.1)). The body mass decreased by 11% after 3 days of fasting in R3 and S20 reached a body mass comparable to that of the R3 on the 20th day of energy restriction and the body mass was comparable between groups on the last day of each body mass reduction (R3 573.1 g (39.5), S20 570.1 (35.3)). Exp. 2. The body masses during experiment 2 are shown in Fig. 3. The body mass did not differ between the groups on the day before (Day −1) the experiment (R7 640.6 g (41.3), S50 640.5 (44.3)). The body mass decreased 18% by 7 days of fasting in R7 and S50 reached a body mass comparable to that of the R7 on the 50th day of energy restriction and it was similar between R7 and S50 after each body mass reduction (R7 527.1 g (41.5), S50 528.0 (47.0)).
Tissue mass and tissue compositions Exp. 1. After the body mass reductions, no differences were seen in the mass of all excised skeletal muscles between R3 and S20 (Table 1). In addition, the sum of all excised skeletal muscle masses was not significantly different between the groups.
S. Tai et al. The adipose tissues mass did not differ significantly between R3 and S20. As shown in Table 2, the water and protein content in the gastrocnemius and soleus were similar in the two groups, regardless of whether it was expressed in absolute or relative value. In contrast to the skeletal muscles and adipose tissues, the stomach mass was 12.9% lower in R3 than S20 (P < 0.05) and the liver mass tended to be 9.9% lower in R3 than S20 (P = 0.078, Table 1). The water content of the liver was significantly lower in R3 than S20 when expressed per the whole organ and it tended to be lower in R3 when expressed per gram of tissue (P = 0.060, Table 2). However, the protein content in the liver was not significantly different between the groups. The mass (Table 1) and content of water and protein (Table 2) in the small intestine did not differ between R3 and S20. Exp. 2. There was no difference in the excised skeletal muscle mass and the sum of these skeletal muscle masses between R7 and S50 (Table 3). The adipose tissues mass showed no difference in the two groups. The content of water and protein in the tibialis anterior did not differ between the groups (Table 4). In contrast, the liver and stomach mass were 18.2% and 13.2% lower in R7 than S50 (P < 0.05), respectively (Table 3) and the content of water and protein in liver and stomach were also significantly lower in R7 (Table 4) when it was expressed as an absolute or relative value, except for the relative water content in the stomach. The small intestine mass did not differ between the groups, while the water content tended to be lower in R7 than S50 (P = 0.077), when it was expressed per gram of tissue.
Nitrogen intake, urinary and fecal nitrogen excretion and nitrogen balance Exp. 1. As presented in Fig. 4D, the nitrogen balance did not differ between groups before the experi-
Figure 3 Change in the body mass with rapid body mass reduction (R7, n = 7) and slow body mass reduction (S50, n = 6) during the study. Values represent the means (SD). *P < 0.05 vs. R7; † P < 0.05 vs. Day 7 of R7.
Body mass reduction rate on body composition Table 1
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Tissue mass after the 3 days fasting or 20 days energy restriction. R3
S20
Skeletal muscles Gastrocnemius Soleus Plantaris Extensor digitorum longus Tibialis anterior Sum of skeletal muscles
6.022 0.428 1.199 0.679 2.381 10.708
Adipose tissues Perirenal Epididymal Sum of adipose tissues
10.860 (3.499) 9.355 (3.036) 20.214 (6.199)
9.995 (4.601) 9.129 (3.040) 19.124 (7.494)
Splanchnic tissues Liver Stomach Small intestine
11.986 (0.726) 2.176 (0.182) 9.165 (1.427)
13.310 (1.577) 2.497 (0.192)* 8.660 (1.672)
(0.376) (0.047) (0.123) (0.051) (0.161) (0.662)
6.050 0.416 1.206 0.635 2.219 10.526
(0.486) (0.038) (0.096) (0.090) (0.178) (0.712)
Values are means (SD) for 7 rats. R3, rapid weight reduction; S20, slow weight reduction. * P < 0.05 vs. R3.
ment (Days −3 to −1) and was significantly lower in R3 than S20 during 3 days of fasting (Days 1—3). However, it approached a positive value due to decreasing the urinary (Fig. 4B) and fecal (Fig. 4C) nitrogen excretion in R3 during the fasting. In S20, the nitrogen balance was positive throughout the experiment in spite of an approximately 47% decrease in the nitrogen intake (Fig. 4A) in comparison to that before body mass reduction. The nitrogen balance on the last day in each group was significantly lower in R3 than S20. Exp. 2. As shown in Fig. 5D, the nitrogen balance did not differ between groups before the experiment except on Day −4. Although the nitrogen balance in R7 was significantly lower than S50 Table 2
during 7 days of fasting, it approached a positive value, due to decreasing nitrogen excretion in urine (Fig. 5B) and feces (Fig. 5C) as well as R3 in Exp. 1. In S50, the nitrogen balance was negative during the first 2 days of energy restriction but it turned positive thereafter and it thereafter remained positive until Day 50. This was due to a decrease in excretion of urinary and fecal nitrogen during the energy restriction. The nitrogen balance on the last day in each group was lower in R7 than S50.
Water balance Exp. 1. The water balance did not differ between R3 and S20 before the body mass reduction (Days
Tissue water, protein content after 3 days fasting or 20 days energy restriction. Water
Protein
R3
S20
R3
S20
Gastrocnemius mg/g g
758 (5) 4.568 (0.300)
761 (11) 4.608 (0.394)
214 (5) 1.290 (0.069)
212 (10) 1.280 (0.109)
Soleus mg/g g
746 (9) 0.319 (0.037)
731 (16) 0.304 (0.031)
233 (13) 0.100 (0.013)
250 (18) 0.103 (0.007)
Liver mg/g g
685 (11) 8.204 (0.404)
695 (5) 9.253 (1.112)*
234 (5) 2.806 (0.211)
230 (10) 3.055 (0.324)
Small intestine mg/g g
738 (39) 6.764 (1.115)
758 (20) 6.565 (1.309)
128 (8) 1.171 (0.155)
136 (11) 1.172 (0.209)
Values are the means (SD) for 7 rats. * P < 0.05 vs. R3.
e96 Table 3
S. Tai et al. Tissue mass after the 7 days fasting or 50 days energy restriction. R7
S50
Skeletal muscles Gastrocnemius Soleus Plantaris Extensor digitorum longus Tibialis anterior Sum of skeletal muscles
5.999 0.420 1.138 0.634 2.255 10.446
Adipose tissues Perirenal Epididymal Sum of adipose tissues
10.778 (4.015) 6.802 (1.229) 17.580 (5.040)
8.273 (3.716) 8.285 (2.841) 16.558 (6.543)
Splanchnic tissues Liver Stomach Small intestine
10.731 (0.587) 2.111 (0.265) 4.618 (0.477)
13.122 (1.202)* 2.433 (0.231)* 4.877 (0.868)
(0.521) (0.052) (0.173) (0.059) (0.307) (1.052)
6.125 0.435 1.262 0.604 2.276 10.702
(0.903) (0.063) (0.213) (0.149) (0.393) (1.670)
Values represent the means (SD) for 7 rats. R7, rapid weight reduction; S50, slow weight reduction. * P < 0.05 vs. R7.
−3 to −1) (R3 13 g/day (3), S20 12 (4)). The water balance the last 3 days of each body mass reduction was −2 g/day (7) and 11 g/day (2) in R3 and S20, respectively, and it was significantly lower in R3. Exp. 2. The water balance did not differ between the groups before the body mass reduction (R7 12 g/day (5), S50 13 (5)). The water balance during last week of each body mass reduction was 1 g/day (6) and 11 g/day (2) in R7 and S50, respectively and it was significantly lower in R7.
Plasma glucose concentration Exp. 1. The plasma glucose concentration did not differ between the R3 and S20 before body mass Table 4
reduction (R3 123.8 mg/dl (10.8), S20 121.2 (16.3)). After each body mass reduction, the plasma glucose concentration decreased by 13% in R3 whereas it remained unchanged in S20 (R3 108.3 mg/dl (10.1), S20 119.1 (15.5)). However, the glucose concentration between the groups did not reach a statistical difference. Exp. 2. The plasma glucose concentration did not differ between R7 and S50 before body mass reduction (R7 143.2 mg/dl (16.0), S50 148.9 (7.8)). On the last day of each body mass reduction, the plasma glucose concentration decreased by 21% in R7, whereas it did not change in S50 (R7 113.0 mg/dl (17.0), S50 152.2 mg/dl (16.1)). The glucose concentration was significantly lower in R7 than in S50.
Tissue water, protein content after 7 days fasting or 50 days of energy restriction. Water
Protein
R7
S50
R7
S50
Tibialis anterior mg/g g
724 (44) 1.641 (0.310)
734 (25) 1.687 (0.343)
242 (38) 0.538 (0.066)
234 (21) 0.527 (0.059)
Liver mg/g g
681 (13) 7.308 (0.386)
698 (6)* 9.157 (0.821)*
226 (7) 2.418 (0.120)
236 (9)* 3.094 (0.264)*
Stomach mg/g g
776 (21) 1.616 (0.198)
782 (8) 1.903 (0.178)*
148 (9) 0.310 (0.034)
159 (8)* 0.385 (0.030)*
Small intestine mg/g g
750 (44) 3.456 (0.319)
786 (7) 3.832 (0.672)
155 (10) 0.716 (0.080)
161 (6) 0.783 (0.139)
Values represent the means (SD). R7, rapid weight reduction (n = 7); S50, slow weight reduction (n = 6). * P < 0.05 vs. R7.
Body mass reduction rate on body composition
Figure 4 Nitrogen (N) intake (A), urinary (B) and fecal (C) N excretion and N balance (D) in the rapid body mass reduction (R3) and slow body mass reduction (S20) during the study. 1 N balance = (N intake) − (urinary and fecal N excretion). Values are the means (SD) for 7 rats. *P < 0.05 vs. R3; † P < 0.05 vs. Day 3 of R3.
Discussion This is the first study to investigate the effects of the rate of body mass reduction on the body composition during an equivalent body mass reduction in rats. The primary finding is that differences in the rate of body mass reduction were observed in the liver and stomach but not in either skeletal muscles or adipose tissues and the splanchnic tissues mass was lower in the rapid than the slow body mass reduction in both experiments, which reduced the body mass 11% or 18% in the rats. In addition, the increasing intensity of the body mass reduction appeared to further widen the difference in the liver between the rapid and slow body mass reduction.
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Figure 5 Nitrogen (N) intake (A), urinary (B) and fecal (C) N excretion and N balance (D) in the rapid body mass reduction (R7, n = 7) and slow body mass reduction (S50, n = 6) during the study. 1 N balance = (N intake) − (urinary and fecal N excretion). Values are the means (SD). *P < 0.05 vs. R7; † P < 0.05 vs. Day 7 of R7.
Contrary to the initial hypothesis, the skeletal muscle mass did not differ between the rapid and slow body mass reduction in either Exp. 1 and 2. The calculation of the specific daily rate of body mass loss dM/M dt (g 100 g body mass−1 day−1 ) for each animal (dM represents the loss of body mass during dt = t1 − t0 and M is the rat body mass at t0 ) enabled the determination of the three fasting phases [15—17]. According to previous studies, the phase II fasting period is characterized by dropping the dM/M dt [15—17], decreasing the urinary nitrogen excretion [14,15,18] and sparing the skeletal muscle protein [14,15,18,19]. Within 2 days fasting, the calculated dM/M dt values declined by one-half and reached 2.7 g 100 g body mass−1 day−1 (SD 0.5) and 1.4 (0.2) in Exp. 1 and 2, respectively, on the last day of the fasting. The values are similar to those previously published for rat in the phase II
e98 fasting period [17]. In addition, the urinary nitrogen excretion decreased and low values were obtained even if body mass reduction was prolonged in Exp. 2. Therefore, these results indicate that the fasting period of the present study was categorized as phase II and consisted with the report that the phase II fasting period lasted between 2 and 8 days in 16-week-old rats [14]. Based on the daily rate of body mass loss and urinary nitrogen excretion, it was suggested that the skeletal muscles protein was maintained during the fasting period in either experiment in the present study. Therefore, it is possible that no difference was observed in the skeletal muscle mass between the rapid and slow body mass reduction because the skeletal muscle mass did not decrease in either. In any event, the present study showed clearly that the rate of body mass reduction might have less influence on skeletal muscles during the phase II fasting periods. The mechanism by which the skeletal muscle protein is spared, is considered to be related to the body fat stores and/or the contribution of metabolites from adipose tissues [14,15,18—22]. Unfortunately, the exact mechanism of skeletal muscle protein conservation could not be addressed in this study since the body fat stores before body mass reduction and the metabolites from adipose tissue during body mass reduction were not measured. However, since the isolated adipose tissue masses were similar between the rapid and slow body mass reductions after each body mass reduction, the sparing of the skeletal muscle protein by using the fat as fuel thus does not seem to be specific to either rapid or slow body mass reduction. The mass and water and protein in the liver were lower in the rapid than slow body mass reduction in the present study. The sum of the mean differences in the water and protein of the liver between rapid and slow body mass reductions was 1.298 g and 2.525 g in Exp. 1 and 2, respectively. The difference represents 98% and 105% (including slightly analysis error) of the mean difference of liver mass between the groups in Exp. 1 and Exp. 2, respectively. This suggests that the difference of the liver mass between groups was almost accounted for by the difference of water and protein. Furthermore, the contribution of water to the difference of liver mass between groups was 79% and 77% in Exp. 1 and 2, respectively. A large part of the difference in the liver mass between the groups was suggested to be due to the greater water loss by the fasting. Actually, it has been shown that water loss in the liver accounted for 67% of liver mass loss in phase II fasted rats [17] and it may be attributable to the depletion of the glycogen with water [23,24].
S. Tai et al. The notion that the absence of food in the upper gastrointestinal tract stimulates the atrophy of the gut has been shown in previous studies [25—29]. However, the mass of the small intestine in the food deprived rats (rapid group) showed no difference in comparison to the food restricted rats (slow group) in either experiment in the present study. Therefore, the result suggests that even if a food passes in the lumen of the intestine, atrophy of small intestine would be caused if the meal is restricted. The suggestion was supported by the report that the mass and protein in the small intestine decreased during the very low energy diet in rats [12]. In contrast, the mass and protein of the stomach did not decrease in the slow body mass reduction in association with energy restriction in the preliminary study (unpublished data). The stomach mass was lower in the rapid than slow body mass reduction in both Exp. 1 and 2 in the present study. Therefore, in contrast to the small intestine, the atrophy of the stomach appeared to be induced when the food was completely absent such as the fasting but not restriction. In addition, with the digestive system being the first line of organs to experience food deprivation, it makes sense that atrophy would start there. The implication was that a rapid body mass reduction using the fasting is more stressful on the digestive tract in comparison to the slow body mass reduction, regardless of the intensity of the body mass reduction. The nitrogen balance during the fasting periods of rapid group was higher in the slow than rapid body mass reduction and the difference of calculated cumulative nitrogen balance between groups was 1.0 g and 1.9 g in Exp. 1 and 2, respectively. Assuming that the nitrogen had been retained as protein in the body, it was estimated that difference of protein content in the whole body between the groups after the fasting periods was 6.2 g and 11.8 g in Exp. 1 and 2, respectively. However, the greater retention of the protein in the slow body mass reduction was not able to detect in the analyzed tissue specimens of the present study. Therefore, it is possible that the difference in the rate of body mass reduction on the protein content of the tissues is observed in not only the liver and stomach but also in other tissues. In Exp.1 and 2, the influence of the rate of body mass reduction was similar and it was observed in the liver and stomach, but not in skeletal muscles and adipose tissues in both experiments. These results suggest that the difference in the body composition between the rapid and slow body mass reduction is independent of the intensity of the body mass reduction. However, the difference in the mean values in the liver mass between the rapid
Body mass reduction rate on body composition and slow body mass reduction seemed to markedly increase to 18.2% (2.391 g) in Exp. 2 from 9.9% (1.324 g) in Exp. 1. Therefore, the increase in the intensity of body mass reduction appeared to be related to the further widening difference in the liver between the rapid and slow body mass reduction. The widening difference would be supported by the report that the mass and protein in the liver decreased dramatically with days of the fasting [8,17,28]. In contrast to the liver, the difference in the stomach mass between the groups did not change in Exp. 1 (12.9%) and Exp. 2 (13.2%), indicating the difference in the stomach between the rapid and slow body mass reduction is less influenced by the intensity of body mass reduction. Whether the differences between the rapid and slow body mass reductions on the skeletal muscles and adipose tissues appear when the intensity of body mass reduction further increased were unclear in the present study. However, in human, the severe body mass reduction which results in phase III may be limited to extremely malnourished conditions, such as anorexic or cancer patients [30] and it would not be commonly used in healthy humans. Therefore, no comparison of the rate of body mass reduction was conducted in the critical stage (phase III). Although the body imaging technique such as MRI may be available to determine the body composition [31], it has been indicated that this analysis was obscure to determine the masses of digestive tracts due to residues meal in the stomach or small intestine [32]. We selected the animal study because the obtained data from animals would be clearly able to indicate the influence of body mass loss method on digestive tract as well as the skeletal muscles and adipose tissues. This study has several limitations. First, it was not possible to determine the amount of tissue mass loss due to the lack of sufficient data of before body mass reduction. Second, the analysis of stomach was not conducted in Exp. 1 due to logistical reasons. Third, the time point of sacrifice was slightly different between the rapid and slow body mass reduction. For example, the age of R3 was 23 weeks old whereas the S20 was 25 weeks old at the sacrifice in Exp. 1. In this regard, there is concern that the growth per se had a protective effect against tissue mass loss in regard to slow body mass reduction. However, as the rats examined in the present study were 23 weeks old, it could be assumed that the influence of the skeletal muscle growth was minor [33]. In addition, since the body mass in S decreased gradually, it is unlikely that the skeletal muscles, visceral and adipose tissues grow dramatically in this period. Furthermore, the protocol of
e99 comparing about the body composition between different ages during the body mass loss has been accepted in the previous report [17]. Therefore, we considered that the influence of the growth on the body composition following slow body mass loss was minimal and that the comparison between R and S would be permissible in the present study. In summary, the present study suggested that the body composition is different if the rate of body mass reduction differs even if the weight loss is equivalent. The differences of rate of body mass reduction were shown in the liver and stomach rather than in skeletal muscles and adipose tissues and the splanchnic tissues were lower in the rapid reduction than in the slow in both experiments which reduced the body mass by 11% or 18% in rats. Therefore, the difference in the body composition due to the rate of body mass reduction appears to be seen in splanchnic tissue independently of intensity of the body mass reduction. The increase in the intensity was related to the further widening difference in the liver between the rapid and slow body mass reduction.
Conflict of interest S. Tai, Y. Harada, Y. Yokota, Y. Tsurumi, M. Masuhara, and K. Okamura, declare that they have no conflicts of interest.
References [1] Fryburg DA, Barrett EJ, Louard RJ, Gelfand RA. Effect of starvation on human muscle protein metabolism and its response to insulin. Am J Physiol 1990;259:E477—82. [2] Kukidome T, Sato M, Suzuki M. The effect of methods of weight reduction on body composition and subjective physical condition in college wrestlers (in Japanese). Health Sci 2001;17:26—31. [3] Yankanich J, Kenney WL, Fleck SJ, Kraemer WJ. Precompetition weight loss and changes in vascular fluid volume in NCAA Division I college wrestlers. J Strength Cond Res 1998;12:138—45. [4] Fogelholm GM, Koskinen R, Laakso J, Rankinen T, Ruokonen I. Gradual and rapid weight loss: effects on nutrition and performance in male athletes. Med Sci Sports Exerc 1993;25:371—7. [5] Afolabi PR, Jahoor F, Jackson AA, Stubbs J, Johnstone AM, Faber P, et al. The effect of total starvation and very low energy diet in lean men on kinetics of whole body protein and five hepatic secretory proteins. Am J Physiol 2007;293:E1580—9. [6] Faber P, Johnstone AM, Gibney ER, Elia M, Stubbs RJ, Duthie GG, et al. The effect of rate of weight loss on erythrocyte glutathione concentration and synthesis in healthy obese men. Clin Sci (Lond) 2002;102:569—77. [7] Dunn MA, Houtz SK, Hartsook EW. Effects of fasting on muscle protein turnover, the composition of weight loss, and
e100
[8]
[9]
[10]
[11] [12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
S. Tai et al.
energy balance of obese and nonobese Zucker rats. J Nutr 1982;112:1862—75. Goodman MN, Ruderman NB. Starvation in the rat. I. Effect of age and obesity on organ weights, RNA, DNA, and protein. Am J Physiol 1980;239:E269—76. Johnson G, Roussel D, Dumas JF, Douay O, Malthiery Y, Simard G, et al. Influence of intensity of food restriction on skeletal muscle mitochondrial energy metabolism in rats. Am J Physiol 2006;291:E460—7. Schemmel RA, Stone M, Warren MJ, Stoddart KA. Nitrogen and protein losses in rats during weight reduction with a high protein, very low energy diet or fasting. J Nutr 1983;113:727—34. Tsai RY, Cole JW, Ramsey CB. Effects of fasting on tissue shrinkage in rats. J Anim Sci 1972;35:345—50. Young EA, Ramos Jr RG, Harris MM. Gastrointestinal and cardiac response to low-calorie semistarvation diets. Am J Clin Nutr 1988;47:981—8. Terpstra AH. Differences between humans and mice in efficacy of the body fat lowering effect of conjugated linoleic acid: role of metabolic rate. J Nutr 2001;31:2067—8. Goodman MN, Larsen PR, Kaplan MM, Aoki TT, Young VR, Ruderman NB. Starvation in the rat. II. Effect of age and obesity on protein sparing and fuel metabolism. Am J Physiol 1980;239:E277—86. Cherel Y, Robin JP, Heitz A, Calgari C, Le Maho Y. Relationships between lipid availability and protein utilization during prolonged fasting. J Comp Physiol B 1992;162:305—13. Habold C, Foltzer-Jourdainne C, Le Maho Y, Lignot JH, Oudart H. Intestinal gluconeogenesis and glucose transport according to body fuel availability in rats. J Physiol 2005;566:575—86. Robin JP, Decrock F, Herzberg G, Mioskowski E, Le Maho Y, Bach A, et al. Restoration of body energy reserves during refeeding in rats is dependent on both the intensity of energy restriction and the metabolic status at the onset of starvation. J Nutr 2008;138:861—6. Goodman MN, Lowell B, Belur E, Ruderman NB. Sites of protein conservation and loss during starvation: influence of adiposity. Am J Physiol 1984;246:E383—90. Goodman MN, McElaney MA, Ruderman NB. Adaptation to prolonged starvation in the rat: curtailment of skeletal muscle proteolysis. Am J Physiol 1981;241:E321—7. Gormsen LC, Gjedsted J, Gjedde S, Nørrelund H, Christiansen JS, Schmitz O, et al. Dose—response effects of free fatty acids on amino acid metabolism and ureagenesis. Acta Physiol 2008;192:369—79.
[21] Nair KS, Welle SL, Halliday D, Campbell RG. Effect of beta-hydroxybutyrate on whole-body leucine kinetics and fractional mixed skeletal muscle protein synthesis in humans. J Clin Invest 1988;82:198—205. [22] Tessari P, Nissen SL, Miles JM, Haymond MW. Inverse relationship of leucine flux and oxidation to free fatty acid availability in vivo. J Clin Invest 1986;77:575—81. [23] Bergström J, Hultman E. Determination of water and electrolytes in muscle biopsies in the nutritional assessment of clinical disorders. JPEN 1987;11, 51S—S54. [24] Olsson KE, Saltin B. Variation in total body water with muscle glycogen changes in man. Acta Physiol Scand 1970;80:11—8. [25] Boza JJ, Möennoz D, Vuichoud J, Jarret AR, Gaudard-deWeck D, Fritsché R, et al. Food deprivation and refeeding influence growth, nutrient retention and functional recovery of rats. J Nutr 1999;129:1340—6. [26] Ekelund KM, Ekblad E. Structural, neuronal, and functional adaptive changes in atrophic rat ileum. Gut 1999;45:236—45. [27] Ekelund M, Kristensson E, Ekelund M, Ekblad E. Total parenteral nutrition causes circumferential intestinal atrophy, remodeling of the intestinal wall, and redistribution of eosinophils in the rat gastrointestinal tract. Dig Dis Sci 2007;52:1833—9. [28] Ju JS, Nasset ES. Changes in total nitrogen content of some abdominal viscera in fasting and realimentation. J Nutr 1959;68:633—45. [29] Sakamoto K, Hirose H, Onizuka A, Hayashi M, Futamura N, Kawamura Y, et al. Quantitative study of changes in intestinal morphology and mucus gel on total parenteral nutrition in rats. J Surg Res 2000;94:99—106. [30] Rigaud D, Hassid J, Meulemans A, Poupard AT, Boulier A. A paradoxical increase in resting energy expenditure in malnourished patients near death: the king penguin syndrome. Am J Clin Nutr 2000;2:355—60. [31] Tang H, Vasselli JR, Wu EX, Boozer CN, Gallagher D. High-resolution magnetic resonance imaging tracks changes in organ and tissue mass in obese and aging rats. Am J Physiol Regul Integr Comp Physiol 2002;282: R890—9. [32] Kukidome T, Shirai K, Kubo J, Matsushima Y, Yanagisawa O, Homma T, et al. MRI evaluation of body composition changes in wrestlers undergoing rapid weight loss. Br J Sports Med 2008;42:514—8. [33] Tamaki T, Uchiyama S. Absolute and relative growth of rat skeletal muscle. Physiol Behav 1995;57:913—9.
Available online at www.sciencedirect.com
ORIGINAL ARTICLE
Differential effects of rapid and slow body mass reduction on body composition during an equivalent weight loss in rats Shinji Tai, Yachiyo Harada, Yukari Yokota, Yasukimi Tsurumi, Mitsuhiko Masuhara, Koji Okamura ∗ Graduate School of Sport Sciences, Osaka University of Health and Sport Sciences, 1-1 Asashirodai, Kumatori, Sen-nan, Osaka 590-0496, Japan Received 17 March 2009 ; received in revised form 16 July 2009; accepted 28 September 2009
KEYWORDS Intensity of body mass reduction; Weight loss speed; Energy restriction; Splanchnic tissues
Summary It is unclear whether the rate of body mass (BM) reduction affects the body composition with an equivalent BM reduction and whether this is influenced by the intensity of BM reduction. To elucidate this, two experiments (Exp.) were conducted. In Exp. 1, the rats fasted for 3 days to decrease BM rapidly (R3); energy was restricted at 85% of their estimated basal metabolic rate to decrease BM slowly, until it reached the same BM as R3 (S20). In Exp. 2, the rats fasted for 7 days (R7); received a restricted diet as in Exp. 1 (S50). The BM decreased 11% in R3 and S20 showed a BM equivalent to R3 on Day 20. In Exp. 2, the BM decreased 18% in R7 and S50 reached the BM of R7 on Day 50. The mass and water and protein in the skeletal muscle and adipose tissue mass did not differ between the groups in both experiments. In contrast, the stomach mass was 12.9% lower in R3 than S20 (P < 0.05) and the liver mass was 9.9% lower in R3 (P = 0.078). In Exp. 2, the stomach and liver masses were 13.2% and 18.2% lower in R7 than S50 (P < 0.05), respectively. The differences in the rate of BM reduction were seen in splanchnic tissues than in skeletal muscles and adipose tissues regardless of BM intensity. The larger BM reduction appeared to be related to a greater difference in the liver mass between the rapid and slow BM reduction. © 2009 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Introduction
∗ Corresponding author. Tel.: +81 724 53 8839; fax: +81 724 53 8818. E-mail address: [email protected] (K. Okamura).
There are many advertisements and commercial copies for various body mass reduction methods in Japan. However, some of the information includes the method of decreasing body mass rapidly during the short period. Although the slow body mass
1871-403X/$ — see front matter © 2009 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.orcp.2009.09.006
e92 reduction has been recommended to decrease body mass safely, the rapid body mass reduction has not been recommended to the general population due to the several adverse effects [1,2]. Therefore, it is important to indicate the information for the body mass loss speed and it would promote awareness about the suitable method of body mass reduction. In addition, the understanding of various energy restrictions on the body composition may be relevant to clinics in obese human with metabolic syndrome. Research investigating the influence of the rate of body mass reduction on the body composition during the equivalent amount of body mass reduction in humans is limited. Kukidome et al. [2] showed that a rapid body mass reduction for 10 days decreased body mass by 7.8 kg and fat free mass by 73% of the total body mass loss while slow body mass reduction for 28 days reduced body mass by 6.9 kg and fat free mass by 53% of the total body mass loss in wrestlers. That study suggested that a rapid body mass reduction caused a greater loss of fat free mass in comparison to a slow body mass reduction. However, the mass and composition of both skeletal muscles and splanchnic tissues were not determined. Furthermore, there was a possibility that the results could not be applied to ordinary people since dehydrating by sweating and severe water restrictions were performed during the rapid body mass reduction experiment. Other studies regarding the rate of body mass reduction focused on the body fluid status [3] or exercise performance [4] in wrestlers and whole body protein kinetics [5] or glutathione synthesis [6] in lean and obese men. These studies also did not determine whether the mass and composition of skeletal muscles, adipose tissues and splanchnic tissues are influenced by the rate of body mass reduction. Afolabi et al. [5] showed the whole body protein breakdown and the oxidation rate to increase during rapid body mass reduction, whereas it either remained unchanged or decreased during slow body mass reduction when the body mass decreased 5% in humans. Furthermore, both the whole body protein breakdown and the oxidation rate decreased more when the intensity of body mass reduction increased from 5% to 7% during slow body mass reduction. Therefore, these results may indicate that the difference between rapid and slow body mass reduction on body composition increases as the intensity of body mass reduction increases. In animals, although the changes in the body composition after fasting or energy restriction have been described in many animal studies [7—12], no previous study has directly compared the effects of rapid vs. slow body mass reduction on body compo-
S. Tai et al. sition during an equivalent reduction in the amount of body mass. The purpose of the present study was to investigate whether the rate of body mass reduction influences the body composition, including the skeletal muscles, adipose tissues and splanchnic tissues in rats. In addition, this study investigated whether the influence of the rate of body mass reduction on body composition was altered by increasing the intensity of body mass reduction in rats. The hypothesis of this study is that a rapid body mass reduction might induce a greater decrease in the amount of the fat free mass such as the skeletal muscle, in comparison to a slow body mass reduction. Furthermore, the differences in the fat free mass between a rapid and slow body mass reduction are considered to increase as the intensity of body mass reduction increases.
Materials and methods Animals and study design The design of experiments (Exp.) 1 and 2 is summarized in Fig. 1A and B, respectively. Male Sprague—Dawley rats were obtained from Japan Clea, Inc. (Tokyo, Japan) and kept individually in metabolic cages. The reason why a rat model was used was because it is possible to isolate single muscle, adipose tissues and splanchnic tissues and perform an analysis on whole tissue preparations but the use of human subjects is limited to small tissue biopsies and indirect methods of determining tissue mass to assess tissue composition. The animal room was maintained at 23 ◦ C with a 12-h light—dark cycle (lights on from 2100 to 0900 h). The rats were fed ad libitum a standard rat chow (CE-2, Japan Clea, Inc.) before the commencement of body mass reduction and were allowed free access to water throughout the experiment. All study procedures were conducted in conformity with the ‘‘Guide for the Care and Use of Laboratory Animals’’ publication no. 85—23, revised 1985 of the NIH. In Exp. 1, the rats (23 weeks old, n = 14) were divided into two groups; one group fasted for 3 days to decrease body mass rapidly (Rapid, R3; n = 7); a second group received the restricted energy which was 15% lower than their estimated basal metabolic rate [13], to slowly reach a body mass comparable to that of the R group (Slow, S20; n = 7). In Exp. 2, rats (23 weeks old, n = 13) were separated into two groups; one group fasted for 7 days to rapidly decrease body mass (Rapid, R7; n = 7); second group received a restricted energy intake to gradually
Body mass reduction rate on body composition
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Figure 1 Outline of the protocol of experiment 1 (A) and experiment 2 (B). Ad-lib indicates ad libitum feeding.
reach a body mass comparable to that of R7 as well as the protocol of Exp. 1 (Slow, S50; n = 6). The fasting period was selected to produce a clear loss of body energy without causing emaciation [8,14]. In addition, this degree of energy restriction was used because the amount of energy intake gradually decreases the body mass in rats of this age (unpublished data from our laboratory). The body mass, food intake, water consumption and weight of urine and feces were recorded daily at 0900 h. Urine and feces from all rats were collected before the body mass reduction and the sacrifice in each group. Blood was taken from the tail vein at 0830 h before the beginning of experiment and on the final day of each group. Blood was immediately centrifuged at 3000 rpm for 15 min at 4 ◦ C. On the last days of body mass reduction in each group, the rats were euthanized under ether anesthesia. The skeletal muscles (gastrocnemius, soleus, plantaris, extensor digitorum longus and tibialis anterior), abdominal adipose tissues (perirenal and epididymal) and splanchnic tissues (liver, stomach and small intestine) were excised and the masses of these tissues were determined. The masses of the stomach and small intestine were measured after removing the contents of these organs with saline.
Nitrogen balance The nitrogen content in urine and feces was measured with the Kjeldahl method and the nitrogen balance was calculated as the difference between ingested nitrogen and nitrogen excreted in urine and feces.
Water balance The water balance was calculated as difference between water consumption and urine volume.
Blood analysis The plasma glucose concentration was measured with the mutarotase-glucose oxidase method (Glucose CII-test Wako, Wako Pure Chemical Industries, Ltd., Osaka, Japan).
Statistical analysis All values are expressed as the mean and SD. Data were assessed by unpaired t-test. Statistical significance was assigned if P < 0.05. All analyses were performed using a statistical software package (Stat View J-5.0, SAS Institute Inc., Cary, NC).
Measurement of tissue composition The tissues and collected feces samples were lyophilized. The sample water was determined as the difference of the mass before and after lyophilization. The nitrogen content in the samples was measured by the Kjeldahl method and converted to protein content by multiplying 6.25.
Results Body mass Exp. 1. The body masses during the experiment 1 are shown in Fig. 2. The body mass did not differ between R3 and S20 on the day before (Day −1)
e94
Figure 2 Change in the body mass for rapid body mass reduction (R3) and slow body mass reduction (S20) during the study. Values represent the means (SD) for 7 rats. *P < 0.05 vs. R3.
the experiment (R3 646.4 g (SD 44.1), S20 643.6 (35.1)). The body mass decreased by 11% after 3 days of fasting in R3 and S20 reached a body mass comparable to that of the R3 on the 20th day of energy restriction and the body mass was comparable between groups on the last day of each body mass reduction (R3 573.1 g (39.5), S20 570.1 (35.3)). Exp. 2. The body masses during experiment 2 are shown in Fig. 3. The body mass did not differ between the groups on the day before (Day −1) the experiment (R7 640.6 g (41.3), S50 640.5 (44.3)). The body mass decreased 18% by 7 days of fasting in R7 and S50 reached a body mass comparable to that of the R7 on the 50th day of energy restriction and it was similar between R7 and S50 after each body mass reduction (R7 527.1 g (41.5), S50 528.0 (47.0)).
Tissue mass and tissue compositions Exp. 1. After the body mass reductions, no differences were seen in the mass of all excised skeletal muscles between R3 and S20 (Table 1). In addition, the sum of all excised skeletal muscle masses was not significantly different between the groups.
S. Tai et al. The adipose tissues mass did not differ significantly between R3 and S20. As shown in Table 2, the water and protein content in the gastrocnemius and soleus were similar in the two groups, regardless of whether it was expressed in absolute or relative value. In contrast to the skeletal muscles and adipose tissues, the stomach mass was 12.9% lower in R3 than S20 (P < 0.05) and the liver mass tended to be 9.9% lower in R3 than S20 (P = 0.078, Table 1). The water content of the liver was significantly lower in R3 than S20 when expressed per the whole organ and it tended to be lower in R3 when expressed per gram of tissue (P = 0.060, Table 2). However, the protein content in the liver was not significantly different between the groups. The mass (Table 1) and content of water and protein (Table 2) in the small intestine did not differ between R3 and S20. Exp. 2. There was no difference in the excised skeletal muscle mass and the sum of these skeletal muscle masses between R7 and S50 (Table 3). The adipose tissues mass showed no difference in the two groups. The content of water and protein in the tibialis anterior did not differ between the groups (Table 4). In contrast, the liver and stomach mass were 18.2% and 13.2% lower in R7 than S50 (P < 0.05), respectively (Table 3) and the content of water and protein in liver and stomach were also significantly lower in R7 (Table 4) when it was expressed as an absolute or relative value, except for the relative water content in the stomach. The small intestine mass did not differ between the groups, while the water content tended to be lower in R7 than S50 (P = 0.077), when it was expressed per gram of tissue.
Nitrogen intake, urinary and fecal nitrogen excretion and nitrogen balance Exp. 1. As presented in Fig. 4D, the nitrogen balance did not differ between groups before the experi-
Figure 3 Change in the body mass with rapid body mass reduction (R7, n = 7) and slow body mass reduction (S50, n = 6) during the study. Values represent the means (SD). *P < 0.05 vs. R7; † P < 0.05 vs. Day 7 of R7.
Body mass reduction rate on body composition Table 1
e95
Tissue mass after the 3 days fasting or 20 days energy restriction. R3
S20
Skeletal muscles Gastrocnemius Soleus Plantaris Extensor digitorum longus Tibialis anterior Sum of skeletal muscles
6.022 0.428 1.199 0.679 2.381 10.708
Adipose tissues Perirenal Epididymal Sum of adipose tissues
10.860 (3.499) 9.355 (3.036) 20.214 (6.199)
9.995 (4.601) 9.129 (3.040) 19.124 (7.494)
Splanchnic tissues Liver Stomach Small intestine
11.986 (0.726) 2.176 (0.182) 9.165 (1.427)
13.310 (1.577) 2.497 (0.192)* 8.660 (1.672)
(0.376) (0.047) (0.123) (0.051) (0.161) (0.662)
6.050 0.416 1.206 0.635 2.219 10.526
(0.486) (0.038) (0.096) (0.090) (0.178) (0.712)
Values are means (SD) for 7 rats. R3, rapid weight reduction; S20, slow weight reduction. * P < 0.05 vs. R3.
ment (Days −3 to −1) and was significantly lower in R3 than S20 during 3 days of fasting (Days 1—3). However, it approached a positive value due to decreasing the urinary (Fig. 4B) and fecal (Fig. 4C) nitrogen excretion in R3 during the fasting. In S20, the nitrogen balance was positive throughout the experiment in spite of an approximately 47% decrease in the nitrogen intake (Fig. 4A) in comparison to that before body mass reduction. The nitrogen balance on the last day in each group was significantly lower in R3 than S20. Exp. 2. As shown in Fig. 5D, the nitrogen balance did not differ between groups before the experiment except on Day −4. Although the nitrogen balance in R7 was significantly lower than S50 Table 2
during 7 days of fasting, it approached a positive value, due to decreasing nitrogen excretion in urine (Fig. 5B) and feces (Fig. 5C) as well as R3 in Exp. 1. In S50, the nitrogen balance was negative during the first 2 days of energy restriction but it turned positive thereafter and it thereafter remained positive until Day 50. This was due to a decrease in excretion of urinary and fecal nitrogen during the energy restriction. The nitrogen balance on the last day in each group was lower in R7 than S50.
Water balance Exp. 1. The water balance did not differ between R3 and S20 before the body mass reduction (Days
Tissue water, protein content after 3 days fasting or 20 days energy restriction. Water
Protein
R3
S20
R3
S20
Gastrocnemius mg/g g
758 (5) 4.568 (0.300)
761 (11) 4.608 (0.394)
214 (5) 1.290 (0.069)
212 (10) 1.280 (0.109)
Soleus mg/g g
746 (9) 0.319 (0.037)
731 (16) 0.304 (0.031)
233 (13) 0.100 (0.013)
250 (18) 0.103 (0.007)
Liver mg/g g
685 (11) 8.204 (0.404)
695 (5) 9.253 (1.112)*
234 (5) 2.806 (0.211)
230 (10) 3.055 (0.324)
Small intestine mg/g g
738 (39) 6.764 (1.115)
758 (20) 6.565 (1.309)
128 (8) 1.171 (0.155)
136 (11) 1.172 (0.209)
Values are the means (SD) for 7 rats. * P < 0.05 vs. R3.
e96 Table 3
S. Tai et al. Tissue mass after the 7 days fasting or 50 days energy restriction. R7
S50
Skeletal muscles Gastrocnemius Soleus Plantaris Extensor digitorum longus Tibialis anterior Sum of skeletal muscles
5.999 0.420 1.138 0.634 2.255 10.446
Adipose tissues Perirenal Epididymal Sum of adipose tissues
10.778 (4.015) 6.802 (1.229) 17.580 (5.040)
8.273 (3.716) 8.285 (2.841) 16.558 (6.543)
Splanchnic tissues Liver Stomach Small intestine
10.731 (0.587) 2.111 (0.265) 4.618 (0.477)
13.122 (1.202)* 2.433 (0.231)* 4.877 (0.868)
(0.521) (0.052) (0.173) (0.059) (0.307) (1.052)
6.125 0.435 1.262 0.604 2.276 10.702
(0.903) (0.063) (0.213) (0.149) (0.393) (1.670)
Values represent the means (SD) for 7 rats. R7, rapid weight reduction; S50, slow weight reduction. * P < 0.05 vs. R7.
−3 to −1) (R3 13 g/day (3), S20 12 (4)). The water balance the last 3 days of each body mass reduction was −2 g/day (7) and 11 g/day (2) in R3 and S20, respectively, and it was significantly lower in R3. Exp. 2. The water balance did not differ between the groups before the body mass reduction (R7 12 g/day (5), S50 13 (5)). The water balance during last week of each body mass reduction was 1 g/day (6) and 11 g/day (2) in R7 and S50, respectively and it was significantly lower in R7.
Plasma glucose concentration Exp. 1. The plasma glucose concentration did not differ between the R3 and S20 before body mass Table 4
reduction (R3 123.8 mg/dl (10.8), S20 121.2 (16.3)). After each body mass reduction, the plasma glucose concentration decreased by 13% in R3 whereas it remained unchanged in S20 (R3 108.3 mg/dl (10.1), S20 119.1 (15.5)). However, the glucose concentration between the groups did not reach a statistical difference. Exp. 2. The plasma glucose concentration did not differ between R7 and S50 before body mass reduction (R7 143.2 mg/dl (16.0), S50 148.9 (7.8)). On the last day of each body mass reduction, the plasma glucose concentration decreased by 21% in R7, whereas it did not change in S50 (R7 113.0 mg/dl (17.0), S50 152.2 mg/dl (16.1)). The glucose concentration was significantly lower in R7 than in S50.
Tissue water, protein content after 7 days fasting or 50 days of energy restriction. Water
Protein
R7
S50
R7
S50
Tibialis anterior mg/g g
724 (44) 1.641 (0.310)
734 (25) 1.687 (0.343)
242 (38) 0.538 (0.066)
234 (21) 0.527 (0.059)
Liver mg/g g
681 (13) 7.308 (0.386)
698 (6)* 9.157 (0.821)*
226 (7) 2.418 (0.120)
236 (9)* 3.094 (0.264)*
Stomach mg/g g
776 (21) 1.616 (0.198)
782 (8) 1.903 (0.178)*
148 (9) 0.310 (0.034)
159 (8)* 0.385 (0.030)*
Small intestine mg/g g
750 (44) 3.456 (0.319)
786 (7) 3.832 (0.672)
155 (10) 0.716 (0.080)
161 (6) 0.783 (0.139)
Values represent the means (SD). R7, rapid weight reduction (n = 7); S50, slow weight reduction (n = 6). * P < 0.05 vs. R7.
Body mass reduction rate on body composition
Figure 4 Nitrogen (N) intake (A), urinary (B) and fecal (C) N excretion and N balance (D) in the rapid body mass reduction (R3) and slow body mass reduction (S20) during the study. 1 N balance = (N intake) − (urinary and fecal N excretion). Values are the means (SD) for 7 rats. *P < 0.05 vs. R3; † P < 0.05 vs. Day 3 of R3.
Discussion This is the first study to investigate the effects of the rate of body mass reduction on the body composition during an equivalent body mass reduction in rats. The primary finding is that differences in the rate of body mass reduction were observed in the liver and stomach but not in either skeletal muscles or adipose tissues and the splanchnic tissues mass was lower in the rapid than the slow body mass reduction in both experiments, which reduced the body mass 11% or 18% in the rats. In addition, the increasing intensity of the body mass reduction appeared to further widen the difference in the liver between the rapid and slow body mass reduction.
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Figure 5 Nitrogen (N) intake (A), urinary (B) and fecal (C) N excretion and N balance (D) in the rapid body mass reduction (R7, n = 7) and slow body mass reduction (S50, n = 6) during the study. 1 N balance = (N intake) − (urinary and fecal N excretion). Values are the means (SD). *P < 0.05 vs. R7; † P < 0.05 vs. Day 7 of R7.
Contrary to the initial hypothesis, the skeletal muscle mass did not differ between the rapid and slow body mass reduction in either Exp. 1 and 2. The calculation of the specific daily rate of body mass loss dM/M dt (g 100 g body mass−1 day−1 ) for each animal (dM represents the loss of body mass during dt = t1 − t0 and M is the rat body mass at t0 ) enabled the determination of the three fasting phases [15—17]. According to previous studies, the phase II fasting period is characterized by dropping the dM/M dt [15—17], decreasing the urinary nitrogen excretion [14,15,18] and sparing the skeletal muscle protein [14,15,18,19]. Within 2 days fasting, the calculated dM/M dt values declined by one-half and reached 2.7 g 100 g body mass−1 day−1 (SD 0.5) and 1.4 (0.2) in Exp. 1 and 2, respectively, on the last day of the fasting. The values are similar to those previously published for rat in the phase II
e98 fasting period [17]. In addition, the urinary nitrogen excretion decreased and low values were obtained even if body mass reduction was prolonged in Exp. 2. Therefore, these results indicate that the fasting period of the present study was categorized as phase II and consisted with the report that the phase II fasting period lasted between 2 and 8 days in 16-week-old rats [14]. Based on the daily rate of body mass loss and urinary nitrogen excretion, it was suggested that the skeletal muscles protein was maintained during the fasting period in either experiment in the present study. Therefore, it is possible that no difference was observed in the skeletal muscle mass between the rapid and slow body mass reduction because the skeletal muscle mass did not decrease in either. In any event, the present study showed clearly that the rate of body mass reduction might have less influence on skeletal muscles during the phase II fasting periods. The mechanism by which the skeletal muscle protein is spared, is considered to be related to the body fat stores and/or the contribution of metabolites from adipose tissues [14,15,18—22]. Unfortunately, the exact mechanism of skeletal muscle protein conservation could not be addressed in this study since the body fat stores before body mass reduction and the metabolites from adipose tissue during body mass reduction were not measured. However, since the isolated adipose tissue masses were similar between the rapid and slow body mass reductions after each body mass reduction, the sparing of the skeletal muscle protein by using the fat as fuel thus does not seem to be specific to either rapid or slow body mass reduction. The mass and water and protein in the liver were lower in the rapid than slow body mass reduction in the present study. The sum of the mean differences in the water and protein of the liver between rapid and slow body mass reductions was 1.298 g and 2.525 g in Exp. 1 and 2, respectively. The difference represents 98% and 105% (including slightly analysis error) of the mean difference of liver mass between the groups in Exp. 1 and Exp. 2, respectively. This suggests that the difference of the liver mass between groups was almost accounted for by the difference of water and protein. Furthermore, the contribution of water to the difference of liver mass between groups was 79% and 77% in Exp. 1 and 2, respectively. A large part of the difference in the liver mass between the groups was suggested to be due to the greater water loss by the fasting. Actually, it has been shown that water loss in the liver accounted for 67% of liver mass loss in phase II fasted rats [17] and it may be attributable to the depletion of the glycogen with water [23,24].
S. Tai et al. The notion that the absence of food in the upper gastrointestinal tract stimulates the atrophy of the gut has been shown in previous studies [25—29]. However, the mass of the small intestine in the food deprived rats (rapid group) showed no difference in comparison to the food restricted rats (slow group) in either experiment in the present study. Therefore, the result suggests that even if a food passes in the lumen of the intestine, atrophy of small intestine would be caused if the meal is restricted. The suggestion was supported by the report that the mass and protein in the small intestine decreased during the very low energy diet in rats [12]. In contrast, the mass and protein of the stomach did not decrease in the slow body mass reduction in association with energy restriction in the preliminary study (unpublished data). The stomach mass was lower in the rapid than slow body mass reduction in both Exp. 1 and 2 in the present study. Therefore, in contrast to the small intestine, the atrophy of the stomach appeared to be induced when the food was completely absent such as the fasting but not restriction. In addition, with the digestive system being the first line of organs to experience food deprivation, it makes sense that atrophy would start there. The implication was that a rapid body mass reduction using the fasting is more stressful on the digestive tract in comparison to the slow body mass reduction, regardless of the intensity of the body mass reduction. The nitrogen balance during the fasting periods of rapid group was higher in the slow than rapid body mass reduction and the difference of calculated cumulative nitrogen balance between groups was 1.0 g and 1.9 g in Exp. 1 and 2, respectively. Assuming that the nitrogen had been retained as protein in the body, it was estimated that difference of protein content in the whole body between the groups after the fasting periods was 6.2 g and 11.8 g in Exp. 1 and 2, respectively. However, the greater retention of the protein in the slow body mass reduction was not able to detect in the analyzed tissue specimens of the present study. Therefore, it is possible that the difference in the rate of body mass reduction on the protein content of the tissues is observed in not only the liver and stomach but also in other tissues. In Exp.1 and 2, the influence of the rate of body mass reduction was similar and it was observed in the liver and stomach, but not in skeletal muscles and adipose tissues in both experiments. These results suggest that the difference in the body composition between the rapid and slow body mass reduction is independent of the intensity of the body mass reduction. However, the difference in the mean values in the liver mass between the rapid
Body mass reduction rate on body composition and slow body mass reduction seemed to markedly increase to 18.2% (2.391 g) in Exp. 2 from 9.9% (1.324 g) in Exp. 1. Therefore, the increase in the intensity of body mass reduction appeared to be related to the further widening difference in the liver between the rapid and slow body mass reduction. The widening difference would be supported by the report that the mass and protein in the liver decreased dramatically with days of the fasting [8,17,28]. In contrast to the liver, the difference in the stomach mass between the groups did not change in Exp. 1 (12.9%) and Exp. 2 (13.2%), indicating the difference in the stomach between the rapid and slow body mass reduction is less influenced by the intensity of body mass reduction. Whether the differences between the rapid and slow body mass reductions on the skeletal muscles and adipose tissues appear when the intensity of body mass reduction further increased were unclear in the present study. However, in human, the severe body mass reduction which results in phase III may be limited to extremely malnourished conditions, such as anorexic or cancer patients [30] and it would not be commonly used in healthy humans. Therefore, no comparison of the rate of body mass reduction was conducted in the critical stage (phase III). Although the body imaging technique such as MRI may be available to determine the body composition [31], it has been indicated that this analysis was obscure to determine the masses of digestive tracts due to residues meal in the stomach or small intestine [32]. We selected the animal study because the obtained data from animals would be clearly able to indicate the influence of body mass loss method on digestive tract as well as the skeletal muscles and adipose tissues. This study has several limitations. First, it was not possible to determine the amount of tissue mass loss due to the lack of sufficient data of before body mass reduction. Second, the analysis of stomach was not conducted in Exp. 1 due to logistical reasons. Third, the time point of sacrifice was slightly different between the rapid and slow body mass reduction. For example, the age of R3 was 23 weeks old whereas the S20 was 25 weeks old at the sacrifice in Exp. 1. In this regard, there is concern that the growth per se had a protective effect against tissue mass loss in regard to slow body mass reduction. However, as the rats examined in the present study were 23 weeks old, it could be assumed that the influence of the skeletal muscle growth was minor [33]. In addition, since the body mass in S decreased gradually, it is unlikely that the skeletal muscles, visceral and adipose tissues grow dramatically in this period. Furthermore, the protocol of
e99 comparing about the body composition between different ages during the body mass loss has been accepted in the previous report [17]. Therefore, we considered that the influence of the growth on the body composition following slow body mass loss was minimal and that the comparison between R and S would be permissible in the present study. In summary, the present study suggested that the body composition is different if the rate of body mass reduction differs even if the weight loss is equivalent. The differences of rate of body mass reduction were shown in the liver and stomach rather than in skeletal muscles and adipose tissues and the splanchnic tissues were lower in the rapid reduction than in the slow in both experiments which reduced the body mass by 11% or 18% in rats. Therefore, the difference in the body composition due to the rate of body mass reduction appears to be seen in splanchnic tissue independently of intensity of the body mass reduction. The increase in the intensity was related to the further widening difference in the liver between the rapid and slow body mass reduction.
Conflict of interest S. Tai, Y. Harada, Y. Yokota, Y. Tsurumi, M. Masuhara, and K. Okamura, declare that they have no conflicts of interest.
References [1] Fryburg DA, Barrett EJ, Louard RJ, Gelfand RA. Effect of starvation on human muscle protein metabolism and its response to insulin. Am J Physiol 1990;259:E477—82. [2] Kukidome T, Sato M, Suzuki M. The effect of methods of weight reduction on body composition and subjective physical condition in college wrestlers (in Japanese). Health Sci 2001;17:26—31. [3] Yankanich J, Kenney WL, Fleck SJ, Kraemer WJ. Precompetition weight loss and changes in vascular fluid volume in NCAA Division I college wrestlers. J Strength Cond Res 1998;12:138—45. [4] Fogelholm GM, Koskinen R, Laakso J, Rankinen T, Ruokonen I. Gradual and rapid weight loss: effects on nutrition and performance in male athletes. Med Sci Sports Exerc 1993;25:371—7. [5] Afolabi PR, Jahoor F, Jackson AA, Stubbs J, Johnstone AM, Faber P, et al. The effect of total starvation and very low energy diet in lean men on kinetics of whole body protein and five hepatic secretory proteins. Am J Physiol 2007;293:E1580—9. [6] Faber P, Johnstone AM, Gibney ER, Elia M, Stubbs RJ, Duthie GG, et al. The effect of rate of weight loss on erythrocyte glutathione concentration and synthesis in healthy obese men. Clin Sci (Lond) 2002;102:569—77. [7] Dunn MA, Houtz SK, Hartsook EW. Effects of fasting on muscle protein turnover, the composition of weight loss, and
e100
[8]
[9]
[10]
[11] [12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
S. Tai et al.
energy balance of obese and nonobese Zucker rats. J Nutr 1982;112:1862—75. Goodman MN, Ruderman NB. Starvation in the rat. I. Effect of age and obesity on organ weights, RNA, DNA, and protein. Am J Physiol 1980;239:E269—76. Johnson G, Roussel D, Dumas JF, Douay O, Malthiery Y, Simard G, et al. Influence of intensity of food restriction on skeletal muscle mitochondrial energy metabolism in rats. Am J Physiol 2006;291:E460—7. Schemmel RA, Stone M, Warren MJ, Stoddart KA. Nitrogen and protein losses in rats during weight reduction with a high protein, very low energy diet or fasting. J Nutr 1983;113:727—34. Tsai RY, Cole JW, Ramsey CB. Effects of fasting on tissue shrinkage in rats. J Anim Sci 1972;35:345—50. Young EA, Ramos Jr RG, Harris MM. Gastrointestinal and cardiac response to low-calorie semistarvation diets. Am J Clin Nutr 1988;47:981—8. Terpstra AH. Differences between humans and mice in efficacy of the body fat lowering effect of conjugated linoleic acid: role of metabolic rate. J Nutr 2001;31:2067—8. Goodman MN, Larsen PR, Kaplan MM, Aoki TT, Young VR, Ruderman NB. Starvation in the rat. II. Effect of age and obesity on protein sparing and fuel metabolism. Am J Physiol 1980;239:E277—86. Cherel Y, Robin JP, Heitz A, Calgari C, Le Maho Y. Relationships between lipid availability and protein utilization during prolonged fasting. J Comp Physiol B 1992;162:305—13. Habold C, Foltzer-Jourdainne C, Le Maho Y, Lignot JH, Oudart H. Intestinal gluconeogenesis and glucose transport according to body fuel availability in rats. J Physiol 2005;566:575—86. Robin JP, Decrock F, Herzberg G, Mioskowski E, Le Maho Y, Bach A, et al. Restoration of body energy reserves during refeeding in rats is dependent on both the intensity of energy restriction and the metabolic status at the onset of starvation. J Nutr 2008;138:861—6. Goodman MN, Lowell B, Belur E, Ruderman NB. Sites of protein conservation and loss during starvation: influence of adiposity. Am J Physiol 1984;246:E383—90. Goodman MN, McElaney MA, Ruderman NB. Adaptation to prolonged starvation in the rat: curtailment of skeletal muscle proteolysis. Am J Physiol 1981;241:E321—7. Gormsen LC, Gjedsted J, Gjedde S, Nørrelund H, Christiansen JS, Schmitz O, et al. Dose—response effects of free fatty acids on amino acid metabolism and ureagenesis. Acta Physiol 2008;192:369—79.
[21] Nair KS, Welle SL, Halliday D, Campbell RG. Effect of beta-hydroxybutyrate on whole-body leucine kinetics and fractional mixed skeletal muscle protein synthesis in humans. J Clin Invest 1988;82:198—205. [22] Tessari P, Nissen SL, Miles JM, Haymond MW. Inverse relationship of leucine flux and oxidation to free fatty acid availability in vivo. J Clin Invest 1986;77:575—81. [23] Bergström J, Hultman E. Determination of water and electrolytes in muscle biopsies in the nutritional assessment of clinical disorders. JPEN 1987;11, 51S—S54. [24] Olsson KE, Saltin B. Variation in total body water with muscle glycogen changes in man. Acta Physiol Scand 1970;80:11—8. [25] Boza JJ, Möennoz D, Vuichoud J, Jarret AR, Gaudard-deWeck D, Fritsché R, et al. Food deprivation and refeeding influence growth, nutrient retention and functional recovery of rats. J Nutr 1999;129:1340—6. [26] Ekelund KM, Ekblad E. Structural, neuronal, and functional adaptive changes in atrophic rat ileum. Gut 1999;45:236—45. [27] Ekelund M, Kristensson E, Ekelund M, Ekblad E. Total parenteral nutrition causes circumferential intestinal atrophy, remodeling of the intestinal wall, and redistribution of eosinophils in the rat gastrointestinal tract. Dig Dis Sci 2007;52:1833—9. [28] Ju JS, Nasset ES. Changes in total nitrogen content of some abdominal viscera in fasting and realimentation. J Nutr 1959;68:633—45. [29] Sakamoto K, Hirose H, Onizuka A, Hayashi M, Futamura N, Kawamura Y, et al. Quantitative study of changes in intestinal morphology and mucus gel on total parenteral nutrition in rats. J Surg Res 2000;94:99—106. [30] Rigaud D, Hassid J, Meulemans A, Poupard AT, Boulier A. A paradoxical increase in resting energy expenditure in malnourished patients near death: the king penguin syndrome. Am J Clin Nutr 2000;2:355—60. [31] Tang H, Vasselli JR, Wu EX, Boozer CN, Gallagher D. High-resolution magnetic resonance imaging tracks changes in organ and tissue mass in obese and aging rats. Am J Physiol Regul Integr Comp Physiol 2002;282: R890—9. [32] Kukidome T, Shirai K, Kubo J, Matsushima Y, Yanagisawa O, Homma T, et al. MRI evaluation of body composition changes in wrestlers undergoing rapid weight loss. Br J Sports Med 2008;42:514—8. [33] Tamaki T, Uchiyama S. Absolute and relative growth of rat skeletal muscle. Physiol Behav 1995;57:913—9.
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