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Metabolic status in growing rats fed isocaloric diets with increased carbohydrate-to-fat ratio
Nutrition 21 (2005) 249 –254 www.elsevier.com/locate/nut
Basic nutritional investigation
Metabolic status in growing rats fed isocaloric diets with increased carbohydrate-to-fat ratio Carlota A. Gamba, M.S.a, Silvia M. Friedman, Ph.D.a,*, Patricia N. Rodriguez, Ph.D.a, Elisa V. Macria, Maria I. Vacas, Ph.D.a, Fima Lifshitz, M.D.b a
Department of Biochemistry, Faculty of Dentistry, University of Buenos Aires, Buenos Aires, Argentina b Miami Children’s Hospital, Miami, Florida, USA Manuscript received September 16, 2003; accepted April 24, 2004.
Abstract
Objective: A low-fat diet is hypothesized to be associated with significant weight loss. However, most previous studies have been limited to low-fat, low-calorie restrictive diets. This study evaluated the effect of isocaloric diets given “ad libitum” but different in relative amounts of fat and carbohydrate on body size, energy metabolism, body composition, insulin-like growth factor-1, and leptin serum levels in growing Wistar rats. Methods: Weanling male rats were fed with one of three diets that contained a ratio of carbohydrate to fat of 1:1, 2:1, or 3:1. Food intake, body weight, body length, oxygen consumption, and body composition were measured at ages 21 to 50 d. Serum levels of insulin-like growth factor-1 and leptin were also determined. Results: Energy intake was similar across groups. The ratio of body weight to body length remained adequate throughout the experimental period. However, groups that received 3:1 and 2:1 showed increased weight and progressive decreases in energy expenditure, body fat composition, and serum level of leptin, but the ratio of insulin-like growth factor-1 to body length was not affected. Conclusions: Dietary substitution of fat with carbohydrates contributes to weight gain by decreasing energy expenditure and possibly by decreasing leptin secretion. © 2005 Elsevier Inc. All rights reserved.
Keywords:
Weanling rats; Leptin; Body composition; Growth; Low-fat diets
Introduction Recent reports have described the high prevalence of inappropriate eating behaviors by decreasing fat intake and substituting lipids with carbohydrates in children and adolescents [1]. Regardless of the etiology, the most frequent complications are high prevalence of bone alterations [2], decreased energy balance, and growth retardation. Fat is generally regarded as a macronutrient in the diet. Fat not only provides energy but also facilitates absorption of fat-soluble vitamins and plays an important role in skelThis work was supported by grants TO-05 and O-017 from the University of Buenos Aires. * Corresponding author: Tel.:⫹54-11-4784-1111; fax: ⫹54-11-45083958. E-mail address: [email protected] (S.M. Friedman). 0899-9007/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2004.04.026
etal biology and bone health [3]. We previously studied the effect of total calorie-restricted diets on the growth of lean children and rats [4 –7]. However, the metabolic effect of long-term low-fat diets with adequate energy intake in children and adolescents are not known. The description and prediction of the physiologic processes that occur with low-fat diets cannot be evaluated in children. Fortunately, our laboratory has demonstrated that the growing rat can be used as an adequate experimental model in this respect [7]. Therefore, this study investigated, in an experimental model of weanling rats, the effect of long-term, isocaloric, low-fat and moderate-fat restricted diets on body growth, body composition, energy metabolism, and biochemical growth regulating factors (insulin-like growth factor-1 [IGF-1] and leptin).
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Materials and methods
Energy metabolism
Animals
Energy expenditure was measured every 7 d by indirect calorimetry with an OXYMAX system (model O2-ECO, Columbus Instruments, Columbus, OH, USA) that monitors oxygen consumption in experimental animals. The system requires the removal of water vapor to prevent an interaction between oxygen and CO2 analyzers. A glass column with anhydrous SO4 (Dried Rite, Hammond Corporation) and NaOH-CaO was used. Room air was circulated through the chamber, with one channel serving as the reference (room air) and the other measuring oxygen concentration within the chamber. This allowed the continuous connection of changes in oxygen concentration within the room. Rats were monitored by one investigator for the entire period (60 min). Oxygen consumption was continuously recorded during the study while the animal was awake and not engaged in physical activity and was calculated according to the following equation: VO2 (L · kg⫺1 · h⫺1) ⫽ 600 ⫻ Vi (L/min) ⫻ (%Xi ⫺ %Xo)/Wt (kg) ⫻ (100 ⫺ %Xo), where VO2 represents volume of oxygen consumed, Vi represents air flux input, %Xi represents oxygen fraction input, and %Xo represents oxygen fraction output. Energy expenditure was monitored as oxygen consumption in liters of oxygen per hour per rat and as daily calories per 100 g of rat Wt. The conversion factor for each calorie per oxygen liter was taken into account according to each diet (diet 1:1, RQ 0.91 ⫽ 4.936; diet 2:1, RQ 0.94 ⫽ 4.973; and diet 3:1, RQ 0.96) ⫽ 4.998 cal/L O2) [10].
The protocols for these experiments were approved by the University of Buenos Aires (Buenos Aires, Argentina). Fifty male weanling Wistar rats (age 21 d), with an initial body weight of 41.45 ⫾ 5.95 (mean ⫾ standard deviation), were obtained from the animal laboratory of the Department of Biochemistry, Faculty of Dentistry, University of Buenos Aires. Animals were housed in galvanized cages with meshed floors to maintain hygienic conditions and avoid coprophagia. They were exposed to a 12-h light, 12-h dark cycle. Room temperature was maintained at 21 ⫾ 1°C with a humidity of 50% to 60%. Animals were randomized to a baseline control group (n ⫽ 8) and three experimental groups (n ⫽ 14/group). Diets Animals were fed with one of three isocaloric balanced diets that had a ratio of carbohydrate to fat of 1:1, 2:1, or 3:1 during an experimental period of 28 d. The diet composition (per 100 g of diet) was 25 g of protein (lactic casein, 87% protein, mesh 90), 15 g (1:1), 10 g (2:1), or 7.5 (3:1) of lipids (corn oil), 34 g (1:1), 45 g (2:1), or 51 g (3:1) of carbohydrates (corn dextrin from corn refinery), 5 g of minerals (mixture of minerals manufactured by the Department of Food Science, School of Biochemistry, University of Buenos Aires) [8], 0.5 g of fat-soluble vitamins, 0.25 g of hydrosoluble vitamins (mixture by the Department of Food Science), 0.15 g of choline chloride salt (C-1879, Sigma), and up to 100 g of fiber (oatmeal). Nutritional status Zoometry was used to measure body weight (Wt) every 2 d and body length every 4 d. Both measurements were performed after a fasting period of 2 to 4 h. A Mettler PC 4000 scale was used to measure Wt with an accuracy of ⫾1 mg. Body length was determined in slightly anesthetized rats with a scaled ruler in millimeters from the nose tip to the hairline of the tail. Zoometric data were plotted against normal male rat growth patterns, i.e., Wt versus body length. The reference distribution used in this study was the normal rat growth pattern for age and sex as previously published [9], with unpublished adjustments due to improved growth over the years [7]. Daily weight gain velocity was calculated as grams of Wt per 100 g of rat Wt. Diet intake Food cups were refilled three times each week, and food consumption was measured with a Mettler scale (accuracy ⫾1 mg). Daily food intake was expressed as grams per 100 g of rat Wt.
Body composition At the end of the experimental period, animals were killed under ether anesthesia and carcasses were dried at 100°C until a constant weight was achieved. The amount of body water was calculated as the difference in weight before and after drying. Body fat was determined by Soxhlet’s method [11]. Total body water and fat were expressed as percentages of total weight (g/100 g of rat Wt).
Biochemical determinations On experimental days 0 and 28, blood was obtained by cardiac puncture and serum was removed and stored at ⫺20°C to determine serum levels of IGF-1 and leptin. Serum concentrations of IGF-1 were measured in duplicate by a rat double antibody radioimmunoassay (Diagnos Med Kit). To remove interfering IGF binding proteins, samples were previously subjected to acid-ethanol extraction followed by microcentrifugation. The sensitivity of this radioimmunoassay was equal to 50 ng/mL. Serum levels of leptin were analyzed in duplicate by using a rat immunometric enzyme-linked immunosorbent assay kit (Assay Designs, Inc., USA). The sensitivity was
C.A. Gamba et al. / Nutrition 21 (2005) 249 –254
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Table 1 Statistical analysis of data after 28-d consumption of experimental diets* Parameter
Diet
Mean ⫾ SD
Statistics
Comparisons
Wt (g)
1:1 2:1 3:1 1:1 2:1 3:1 1:1 2:1 3:1 1:1 2:1 3:1 1:1 2:1 3:1 1:1 2:1 3:1
165.62 ⫾ 10.96 190.86 ⫾ 20.36 187.75 ⫾ 17.10 4.19 ⫾ 0.30 4.43 ⫾ 0.23 4.44 ⫾ 0.14 18.83 ⫾ 0.44 19.49 ⫾ 0.74 19.94 ⫾ 0.72 67.75 ⫾ 13.11 68.50 ⫾ 14.51 57.68 ⫾ 9.75 13.31 ⫾ 1.16 10.96 ⫾ 1.04 9.81 ⫾ 1.82 65.89 ⫾ 0.43 66.91 ⫾ 1.37 69.20 ⫾ 0.90
a d/b c/b a c c a c/b d/b a a a a c d/c a a d
a versus c, P ⬍ 0.01 a versus d, P ⬍ 0.001 Same letters, NS
WtGV (daily g/100 g Wt)
L (cm)
IGF-1 (ng/ml)/body length (cm)
Body fat (g/100 g Wt)
Body water (g/100 g Wt)
a versus c, P ⬍ 0.01 Same letters, NS a versus c, P ⬍ 0.01 a versus d, P ⬍ 0.001 Same letters, NS Same letters, NS a versus c, P ⬍ 0.01 a versus d, P ⬍ 0.001 Same letters, NS a versus d, P ⬍ 0.001 Same letters, NS
IGF-1, insulin-like growth factor-1; L, body length; NS, not significant; SD, standard deviation; Wt, rat body weight; WtGV, gain velocity in rat body weight * Parameters analyzed as percentages of total body weight (see MATERIALS AND METHODS).
equal to 42 ng/mL. Leptin was expressed as serum leptin (ng/mL) and as a ratio of leptin to body fat (grams of fat/100 g of rat Wt). Statistical analysis All data were expressed as mean ⫾ standard deviation. For statistical analysis, data were tested for normality with Wilk-Shapiro test and Bartlett’s test (chi-square) for homogeneous variances with Statistix 2.1 for Windows (Analytical software 1998). When Wilk-Shapiro test and chi-square test produced P values greater than 0.05, data were analyzed by one-way analysis of variance; when significant differences were encountered, a Student-Newman-Keuls multiple comparisons test was performed. When Wilk-Shapiro test and/or chi-square test produced P values less than 0.05, data were analyzed by Kruskal-Wallis non-parametric analysis of variance and analysis of variance of ranks (F statistic). In all comparisons P ⬎ 0.05 was considered not significant [12].
Serum IGF-1 levels related to body length increased in all three groups after 28 d (baseline value at day 0 ⫽ 14.43 ⫾ 5.16 ng/mL of IGF-1/centimeter of body length; P ⬍ 0.001 versus day 28) but did not differ between groups (P ⬎ 0.05), so it was independent of dietary fat content (Table 1). We examined the effects of decreasing dietary fat from 1:1 to 2:1 and to 3:1. Although the diets remained isocaloric, there was a significant increase in daily food intake in the first 2 wk of the experiment (P ⬍ 0.05) that was not
Results No changes in Wt and body length (P ⬎ 0.05) were seen after decreasing dietary fat from 10% corn oil (2:1) to 7.5% corn oil (3:1); however, when the level of dietary fat was 15% (1:1), there was a progressive decrease in Wt and body length (P ⬍ 0.01 and P ⬍ 0.001, respectively; Table 1). The velocity of daily weight gain showed a similar pattern (Table 1). Moreover, the ratio of Wt to length remained adequate throughout the experimental period (Fig. 1).
Fig. 1. Profiles of body weight for length of experimental rats fed 1:1, 2:1, and 3:1 diets (see MATERIALS AND METHODS) were plotted against normal male rat growth pattern percentiles for body weight for length [9] (Friedman SM, Gamba CA, unpublished observations). Zoometric categories by percentile ranges of body weight to length: obese, greater than 95.0; overweight, 95.0 to 85.1; adequate, 85.0 to 15.1; lean, 15.0 to 5.1; undernourished, less than 5.0. All diets were adequate for profiles of body weight to length.
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Fig. 2. Daily intake (g diet/100 g of rat Wt, mean ⫾ standard deviation) of experimental rats fed 1:1, 2:1, and 3:1 diets ad libitum (see MATERIALS AND METHODS). For statistical analysis, data were pooled in two 14-d periods (0 to 2 wk and 2 to 4 wk). P ⬍ 0.01, a versus d; P ⬍ 0.05, a versus c and c versus d; P ⬍ 0.001, a versus b and b versus d. Same letters indicate non-significance (P ⬎ 0.05). Wt, body weight.
observed in the remaining 2 wk (P ⬎ 0.05; Fig. 2). When total energy intake was analyzed, it did not differ across groups after 28 d (total daily diet intakes: 1:1, 11.77 ⫾ 2.56 g/100 g of rat Wt; 2:1, 11.58 ⫾ 0.61 g/100 g of rat Wt; 3:1, 12.13 ⫾ 1.45 g diet/100 g of rat Wt; P ⬎ 0.05). In rats fed the 1:1, 2:1 or 3:1 diet, body lipid decreased as the level of dietary fat decreased (P ⬍ 0.01 and P ⬍ 0.001, respectively; Table 1). Body water did not tend to increase, except in the 3:1 group, in which it was significantly greater than in the other two groups (P ⬍ 0.001; Table 1). Serum concentrations of leptin were 44% lower in animals fed the low-fat diet (P ⬍ 0.001) than in animals fed the 1:1 or 2:1 diet, with no significant differences between these diets (P ⬎ 0.05; Fig. 3). Moreover, serum concentration of leptin per body fat ratio was significantly lower in rats that received the low-fat diet (3:1) after 4 wk of treatment (P ⬍ 0.01; Fig. 3, inbox). Oxygen consumption, measured as liters per hour per rat, at 28 d of treatment showed no significant difference between the 1:1 and 2:1 groups (P ⬎ 0.05) and a significant decrease between the 1:1 and 3:1 groups (P ⬍ 0.01; Fig. 4). Energy expenditures, calculated as daily calories per 100 g of rat Wt in addition to calorie per oxygen liter conversion factor of each diet (Fig. 4, inset), at day 28 were 15% and 19% lower in the 2:1 and 3:1 groups, respectively, when compared with the 1:1 group (P ⬍ 0.01) and showed no significant differences between them (P ⬎ 0.05).
Discussion Several studies have examined the effect of hypocaloric diets with different levels of fat and carbohydrate on weight loss [13–15]. However, most animal studies have not inves-
Fig. 3. Leptin serum levels (ng/mL, mean ⫾ standard deviation) in rats after 28 d of consuming the fed 1:1, 2:1, and 3:1 diets (see MATERIALS AND METHODS). P ⬍ 0.001, a versus d. (Inset) Ratio of leptin to body fat (ng/mL/fat body composition as percentage of total body weight, mean ⫾ standard deviation). P ⬍ 0.01, a versus c. Same letters indicate nonsignificant (P ⬎ 0.05). Wt, body weight.
tigated the metabolic effects of unrestricted isocaloric diets with different compositions. The 2:1 and 3:1 ratios of carbohydrate to fat resemble low-fat diets (26% and 19% of energy from fat, respectively) consumed by human populations [5]. This study was of practical significance because it compared Wt with body length, body composition, and how the dietary ratio of fat to carbohydrate affects some growth factors in weanling rats. The diets were prepared with a fat content similar in proportion to that observed in the pediatric population with failure to thrive [4,5]. Some investigators have reported greater weight loss when using a high-fat diet versus a high-carbohydrate one [13,16]. However, other investigators have found no effect of diet composition on weight loss [17]. The difference may be associated with the amount of fat used in those studies. With regard to IGF-1, it has been reported to be sensitive to protein, energy, and specific micronutrients for its optimal synthesis and anabolic effects [18] and as a mediator of growth, showing a similar ratio of IGF-1 to body length. Our results suggest that circulating IGF-1 concentrations were not affected by modifying dietary fat content from 1:1 to 3:1 when young rats were fed isocaloric unrestricted diets with respect to protein, energy, and micronutrients requirements. Our data showed that spontaneous total energy intake was identical across the three groups. A possible explanation of this finding is that decreasing fat content of the diet, from 1:1 to 2:1 and to 3:1, although holding total energy constant, does not affect energy intake, whereas replacing a large portion of fat with carbohydrate leads to a change in energy intake. Comparing a sequence of 2-wk dietary treatments has shown that the 1:1 group had decreased energy intake over 0 to 2 wk, resulting in corresponding low Wt, body length, and Wt velocity. The increased energy intake
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Fig. 4. Energy expenditure of rats fed the 1:1, 2:1, and 3:1 diets (see MATERIALS AND METHODS) was monitored weekly as VO2 (mean ⫾ SD) in liters of oxygen per hour per rat. Statistical analysis is shown at each period when differences were encountered: P ⬍ 0.05, a versus b; P ⬍ 0.01, a versus c; P ⬍ 0.001, a versus d. Same letters indicate non-significance (P ⬎ 0.05). (Inset) Daily energy expenditure after 28 d of treatment (cal/100 g of rat Wt). P ⬍ 0.01, a versus c. Same letters indicate nonsignificance (P ⬎ 0.05). SD, standard deviation; VO2, oxygen consumption.
in the other groups may have been due to the decreased plasma level of satiating hormones or changes in glycogen stores. Nutrient intake and utilization under eucaloric conditions suggest that fats are metabolized more efficiently than carbohydrates [16]. Our study showed that the 2:1 and 3:1 groups reached the highest weight gain velocities. Moreover, both groups achieved a larger body (weight and body length), possibly due to the source of fat and carbohydrate used in the diets, such as saturated fat (medium and long chain), monounsaturated and polyunsaturated fats (-3 and -6), and simple or complex carbohydrates. Human body fat burn is stimulated by an isocaloric, low-fat, high-carbohydrate diet. However, the type of sugar may also be important. A simple sugar has direct uptake and rapid phosphorylation in the liver, but this should not be generalized to starch, dextrin, or different amounts and types of carbohydrates [19,20]. Weight gain velocity may be achieved more easily with high-carbohydrate diets. Our finding is consistent with those of other investigators who fed rats under unrestricted conditions [21]. Our results showed that the pattern of dietary fat is similar to that of carcass fat gain. Gains in body energy through stored lipids and lipid intake were almost equivalent due to the age of the animals; although starting from about 55 d of age, lipid gain would represent only a part of lipid intake [14]. The slight increase in body water seen in the 3:1 group was not dependent on decreased body fat content. Moreover, all three groups remained within the normal range expected for age, sex, and strain [14,22]. However, the change in weight and water in the 3:1 group may have been the result of carbohydrate overfeeding accompanied by an
253
increase in glycogen stores. Studies of longer duration will lead to saturate glycogen stores and convert carbohydrate to fat [13]. When dietary fat was decreased in 3:1 group, rats had a lower percentage of lipid weight compared with the 1:1 and 2:1 groups, despite the decrease in the amount of dietary fat and the proportional increase in carbohydrate content. This indicates that body fat was affected to a greater degree by decreased dietary fat. Leptin is secreted from adipose tissue in proportion to the amount of body fat mass and can act on the brain to regulate the amount of adipose tissue stores [23–25]. Therefore, leptin may be a long-term regulator of body fat. Circulating leptin was directly related to body fat content. This is in agreement with the finding that serum concentration of leptin is not a direct effect of the diet, but rather due to a secondary increase in body lipid content [26]. Further, leptin may act in the hypothalamus by decreasing food intake, and, interestingly, in the 1:1 group, leptin was significantly higher than in the other groups and was related to decreased food intake and, hence, a smaller body. Although decreased fat intake was associated with body composition and leptin concentration, energy and nutrient intakes were not significantly different across groups, with adequate intake amounts for normal growth, and no evidence of growth deficiency was observed. However, further studies are needed to elucidate the effect of decreased leptin serum concentration on bone metabolism. Differences in leptin levels might influence bone development and growth without affecting body weight [27]. The lack of a significant difference in energy expenditure between the 2:1 and 3:1 groups may have been due to the small difference in energy provided by fat in the two diets. The effect of a much wider variation in energy from fat in the 1:1 group showed a strong influence of diet composition. The decrease in adipose tissue mass in response to dietary fat content produced a decrease in cellular activity [28]. Most animal studies have not investigated the metabolic effects of unrestricted isocaloric diets with different compositions. In summary, these studies demonstrated that, although energy, protein, vitamin, and micronutrient intakes were constant and adequate, when calorie ratios of carbohydrate to fat were progressively increased from 1:1 to 2:1 and to 3:1, the diet contributed to weight gain by decreasing energy metabolism and leptin secretion through adipose tissue. Further studies are required to demonstrate the importance of these findings in relation to metabolic changes.
Acknowledgments This paper is part of a doctoral thesis by Carlota A. Gamba to fulfill the academic requirements of the University of Buenos Aires. Partial results were presented at the
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XIII Argentine Nutrition Congress, Mar del Plata, Buenos Aires, 1999; the I International Symposium of Metabolic Unit, Bahia, Brasil, 1999; and at the annual conference of the Federation of American Societies for Experimental Biology, Orlando, Florida, 2001; and was published in abstract form (abstract 759.21, FASEB J 2001;15:A998).
References [1] Ricketts CD. Fat preferences, dietary fat intake and body composition in children. Eur J Clin Nutr 1997;51:443– 8. [2] Watkins BA, Li Y, Seifert MF. Nutraceutical fatty acids as biochemical and molecular modulators of skeletal biology. J Am Coll Nutr 2001;20(suppl):410S– 6. [3] Watkins BA, Lippman HE, Bouteiller LL, Li Y, Seifert MF. Bioactive fatty acids: role in bone biology and bone cell function. Prog Lipid Res 2001;40:125– 48. [4] Lifshitz F, Friedman S, Smith MM, et al. Nutritional dwarfing: a growth abnormality associated with reduced erythrocyte Na⫹, K⫹ATPase activity. Am J Clin Nutr 1991;54:997–1004. [5] Tarim O, Chaslow F, Murphy J, Rising R, Carrillo A, Lifshitz F. Evaluation of differential effects of carbohydrate and fat intake on weight gain, serum IGF-I and erythrocyte Na⫹K⫹ ATPase activity in suboptimal nutrition in rats. J Am Coll Nutr 1997;16:159 – 65. [6] Friedman SM, Rodriguez PN, Olivera MI, et al. Enanismo por desnutrición: cronodinamia de los procesos metabólicos en ratas. Medicina (B Aires) 1998;58:282– 6. [7] Friedman SM, Gamba CA, Boyer PM, et al. Growth deceleration and bone metabolism in nutritional dwarfing rats. Int J Food Sc Nutr 2001;52:225–33. [8] RDA and NRC. The nutrients requirements of laboratory animals. Number 10. Washington, DC: National Research Council; 1978. [9] Friedman SM, Boyer PM, Barrio Rendo ME, et al. Evaluación del crecimiento normal en ratas a través del z score. Arch Latinoam Nutr 1999;49:142– 8. [10] Burztein S, Elwyn D, Ashanazi J, Kinney J. The theoretical framework of indirect calorimetry and energy balance. In: Williams, Williams editors. Energy metabolism, indirect calorimetry and nutrition. 1987; p. 27– 83. [11] Association of Official Agricultural Chemists. Official methods for analysis. 15th ed. Washington, DC: US Government Printing Office; 1990.
[12] Sokal R, Rohlf J. Biometry. The principles and practice of statistics in biological research. San Francisco: WH Freeman; 1969. [13] Shah M, Garg A. High-fat and high-carbohydrate diets and energy balance. Diabetes Care 1996;19:1142–52. [14] Iossa S, Lionetti L, Mollica MP, Barletta A, Liverini G. Energy intake and utilization vary during development in rats. J Nutr 1999;129: 1593– 6. [15] Franz MJ. The answer to weight loss is easy. Doing it is hard! Clin Diabetes 2001;19:105–9. [16] Hudgins Cooper L, Hellerstein M, Seidman C, et al. Human fatty acid synthesis is stimulated by a eucaloric low fat, high carbohydrate diet. J Clin Invest 1996;97:2081–91. [17] Golay A, Allaz AF, Morel Y, et al. Similar weight loss with low or high carbohydrate diets. Am J Clin Nutr 1996;63:174 – 8. [18] Estivariz CF, Ziegler TR. Nutrition and the insulin-like growth factor system. Endocrine 1997;7:65–71. [19] Watkins BA, Li Y, Allen KGD, Hoffmann W, Seifert M. Dietary ratio of (n-6)/(n-3) polyunsaturated fatty acids alters the fatty acid composition of bone compartments and biomarkers of bone formation in rats. J Nutr 2000;130:2274 – 84. [20] Azian MJ, Hausman DB, Sisk MB, Flatt WP, Jewell DE. Dietary conjugated linoleic acid reduces rat adipose tissue cell size rather than cell number. J Nutr 2000;130:1548 –54. [21] Mac Cargar IJ, Baracos VE, Clandimin MT. Influence of dietary carbohydrate to fat ratio on whole body nitrogen retention and body composition in adults rats. J Nutr 1989;119:1240 –5. [22] Pullar JD, Webster JF. The energy cost of fat and protein deposition in the rat. Br J Nutr 1977;37:355– 63. [23] Campfield LA, Smith FJ, Guisez Y, Revos R, Burn P. Recombinant mouse OB protein: evidence of peripheral signal linking adiposity and central networks. Science 1995;269:546 –9. [24] Halaas JL, Gajiwaka KS, Maffei M, et al. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 1995;269: 543– 6. [25] Iritani N, Sugimoto T, Fukuda H. Gene expressions of leptin, insulin receptors and lipogenic enzymes are coordinately regulated by insulin and dietary fat in rats. J Nutr 2000;130:1183– 8. [26] Du F, Higginbotham A, White D. Food intake, energy balance and serum leptin concentrations in rats fed low-protein diets. J Nutr 2000;130:514 –21. [27] Cock TA, Auwerx J. Leptin:cutting the fat off the bone. Lancet 2003;362(9395):1572– 4. [28] Ainslie DA, Proietto J, Fam BC, Thorburn AW. Short-term, high-fat diets lower circulating leptin concentrations in rats. Am J Clin Nutr 2000;71:438 – 42.
Basic nutritional investigation
Metabolic status in growing rats fed isocaloric diets with increased carbohydrate-to-fat ratio Carlota A. Gamba, M.S.a, Silvia M. Friedman, Ph.D.a,*, Patricia N. Rodriguez, Ph.D.a, Elisa V. Macria, Maria I. Vacas, Ph.D.a, Fima Lifshitz, M.D.b a
Department of Biochemistry, Faculty of Dentistry, University of Buenos Aires, Buenos Aires, Argentina b Miami Children’s Hospital, Miami, Florida, USA Manuscript received September 16, 2003; accepted April 24, 2004.
Abstract
Objective: A low-fat diet is hypothesized to be associated with significant weight loss. However, most previous studies have been limited to low-fat, low-calorie restrictive diets. This study evaluated the effect of isocaloric diets given “ad libitum” but different in relative amounts of fat and carbohydrate on body size, energy metabolism, body composition, insulin-like growth factor-1, and leptin serum levels in growing Wistar rats. Methods: Weanling male rats were fed with one of three diets that contained a ratio of carbohydrate to fat of 1:1, 2:1, or 3:1. Food intake, body weight, body length, oxygen consumption, and body composition were measured at ages 21 to 50 d. Serum levels of insulin-like growth factor-1 and leptin were also determined. Results: Energy intake was similar across groups. The ratio of body weight to body length remained adequate throughout the experimental period. However, groups that received 3:1 and 2:1 showed increased weight and progressive decreases in energy expenditure, body fat composition, and serum level of leptin, but the ratio of insulin-like growth factor-1 to body length was not affected. Conclusions: Dietary substitution of fat with carbohydrates contributes to weight gain by decreasing energy expenditure and possibly by decreasing leptin secretion. © 2005 Elsevier Inc. All rights reserved.
Keywords:
Weanling rats; Leptin; Body composition; Growth; Low-fat diets
Introduction Recent reports have described the high prevalence of inappropriate eating behaviors by decreasing fat intake and substituting lipids with carbohydrates in children and adolescents [1]. Regardless of the etiology, the most frequent complications are high prevalence of bone alterations [2], decreased energy balance, and growth retardation. Fat is generally regarded as a macronutrient in the diet. Fat not only provides energy but also facilitates absorption of fat-soluble vitamins and plays an important role in skelThis work was supported by grants TO-05 and O-017 from the University of Buenos Aires. * Corresponding author: Tel.:⫹54-11-4784-1111; fax: ⫹54-11-45083958. E-mail address: [email protected] (S.M. Friedman). 0899-9007/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2004.04.026
etal biology and bone health [3]. We previously studied the effect of total calorie-restricted diets on the growth of lean children and rats [4 –7]. However, the metabolic effect of long-term low-fat diets with adequate energy intake in children and adolescents are not known. The description and prediction of the physiologic processes that occur with low-fat diets cannot be evaluated in children. Fortunately, our laboratory has demonstrated that the growing rat can be used as an adequate experimental model in this respect [7]. Therefore, this study investigated, in an experimental model of weanling rats, the effect of long-term, isocaloric, low-fat and moderate-fat restricted diets on body growth, body composition, energy metabolism, and biochemical growth regulating factors (insulin-like growth factor-1 [IGF-1] and leptin).
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Materials and methods
Energy metabolism
Animals
Energy expenditure was measured every 7 d by indirect calorimetry with an OXYMAX system (model O2-ECO, Columbus Instruments, Columbus, OH, USA) that monitors oxygen consumption in experimental animals. The system requires the removal of water vapor to prevent an interaction between oxygen and CO2 analyzers. A glass column with anhydrous SO4 (Dried Rite, Hammond Corporation) and NaOH-CaO was used. Room air was circulated through the chamber, with one channel serving as the reference (room air) and the other measuring oxygen concentration within the chamber. This allowed the continuous connection of changes in oxygen concentration within the room. Rats were monitored by one investigator for the entire period (60 min). Oxygen consumption was continuously recorded during the study while the animal was awake and not engaged in physical activity and was calculated according to the following equation: VO2 (L · kg⫺1 · h⫺1) ⫽ 600 ⫻ Vi (L/min) ⫻ (%Xi ⫺ %Xo)/Wt (kg) ⫻ (100 ⫺ %Xo), where VO2 represents volume of oxygen consumed, Vi represents air flux input, %Xi represents oxygen fraction input, and %Xo represents oxygen fraction output. Energy expenditure was monitored as oxygen consumption in liters of oxygen per hour per rat and as daily calories per 100 g of rat Wt. The conversion factor for each calorie per oxygen liter was taken into account according to each diet (diet 1:1, RQ 0.91 ⫽ 4.936; diet 2:1, RQ 0.94 ⫽ 4.973; and diet 3:1, RQ 0.96) ⫽ 4.998 cal/L O2) [10].
The protocols for these experiments were approved by the University of Buenos Aires (Buenos Aires, Argentina). Fifty male weanling Wistar rats (age 21 d), with an initial body weight of 41.45 ⫾ 5.95 (mean ⫾ standard deviation), were obtained from the animal laboratory of the Department of Biochemistry, Faculty of Dentistry, University of Buenos Aires. Animals were housed in galvanized cages with meshed floors to maintain hygienic conditions and avoid coprophagia. They were exposed to a 12-h light, 12-h dark cycle. Room temperature was maintained at 21 ⫾ 1°C with a humidity of 50% to 60%. Animals were randomized to a baseline control group (n ⫽ 8) and three experimental groups (n ⫽ 14/group). Diets Animals were fed with one of three isocaloric balanced diets that had a ratio of carbohydrate to fat of 1:1, 2:1, or 3:1 during an experimental period of 28 d. The diet composition (per 100 g of diet) was 25 g of protein (lactic casein, 87% protein, mesh 90), 15 g (1:1), 10 g (2:1), or 7.5 (3:1) of lipids (corn oil), 34 g (1:1), 45 g (2:1), or 51 g (3:1) of carbohydrates (corn dextrin from corn refinery), 5 g of minerals (mixture of minerals manufactured by the Department of Food Science, School of Biochemistry, University of Buenos Aires) [8], 0.5 g of fat-soluble vitamins, 0.25 g of hydrosoluble vitamins (mixture by the Department of Food Science), 0.15 g of choline chloride salt (C-1879, Sigma), and up to 100 g of fiber (oatmeal). Nutritional status Zoometry was used to measure body weight (Wt) every 2 d and body length every 4 d. Both measurements were performed after a fasting period of 2 to 4 h. A Mettler PC 4000 scale was used to measure Wt with an accuracy of ⫾1 mg. Body length was determined in slightly anesthetized rats with a scaled ruler in millimeters from the nose tip to the hairline of the tail. Zoometric data were plotted against normal male rat growth patterns, i.e., Wt versus body length. The reference distribution used in this study was the normal rat growth pattern for age and sex as previously published [9], with unpublished adjustments due to improved growth over the years [7]. Daily weight gain velocity was calculated as grams of Wt per 100 g of rat Wt. Diet intake Food cups were refilled three times each week, and food consumption was measured with a Mettler scale (accuracy ⫾1 mg). Daily food intake was expressed as grams per 100 g of rat Wt.
Body composition At the end of the experimental period, animals were killed under ether anesthesia and carcasses were dried at 100°C until a constant weight was achieved. The amount of body water was calculated as the difference in weight before and after drying. Body fat was determined by Soxhlet’s method [11]. Total body water and fat were expressed as percentages of total weight (g/100 g of rat Wt).
Biochemical determinations On experimental days 0 and 28, blood was obtained by cardiac puncture and serum was removed and stored at ⫺20°C to determine serum levels of IGF-1 and leptin. Serum concentrations of IGF-1 were measured in duplicate by a rat double antibody radioimmunoassay (Diagnos Med Kit). To remove interfering IGF binding proteins, samples were previously subjected to acid-ethanol extraction followed by microcentrifugation. The sensitivity of this radioimmunoassay was equal to 50 ng/mL. Serum levels of leptin were analyzed in duplicate by using a rat immunometric enzyme-linked immunosorbent assay kit (Assay Designs, Inc., USA). The sensitivity was
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Table 1 Statistical analysis of data after 28-d consumption of experimental diets* Parameter
Diet
Mean ⫾ SD
Statistics
Comparisons
Wt (g)
1:1 2:1 3:1 1:1 2:1 3:1 1:1 2:1 3:1 1:1 2:1 3:1 1:1 2:1 3:1 1:1 2:1 3:1
165.62 ⫾ 10.96 190.86 ⫾ 20.36 187.75 ⫾ 17.10 4.19 ⫾ 0.30 4.43 ⫾ 0.23 4.44 ⫾ 0.14 18.83 ⫾ 0.44 19.49 ⫾ 0.74 19.94 ⫾ 0.72 67.75 ⫾ 13.11 68.50 ⫾ 14.51 57.68 ⫾ 9.75 13.31 ⫾ 1.16 10.96 ⫾ 1.04 9.81 ⫾ 1.82 65.89 ⫾ 0.43 66.91 ⫾ 1.37 69.20 ⫾ 0.90
a d/b c/b a c c a c/b d/b a a a a c d/c a a d
a versus c, P ⬍ 0.01 a versus d, P ⬍ 0.001 Same letters, NS
WtGV (daily g/100 g Wt)
L (cm)
IGF-1 (ng/ml)/body length (cm)
Body fat (g/100 g Wt)
Body water (g/100 g Wt)
a versus c, P ⬍ 0.01 Same letters, NS a versus c, P ⬍ 0.01 a versus d, P ⬍ 0.001 Same letters, NS Same letters, NS a versus c, P ⬍ 0.01 a versus d, P ⬍ 0.001 Same letters, NS a versus d, P ⬍ 0.001 Same letters, NS
IGF-1, insulin-like growth factor-1; L, body length; NS, not significant; SD, standard deviation; Wt, rat body weight; WtGV, gain velocity in rat body weight * Parameters analyzed as percentages of total body weight (see MATERIALS AND METHODS).
equal to 42 ng/mL. Leptin was expressed as serum leptin (ng/mL) and as a ratio of leptin to body fat (grams of fat/100 g of rat Wt). Statistical analysis All data were expressed as mean ⫾ standard deviation. For statistical analysis, data were tested for normality with Wilk-Shapiro test and Bartlett’s test (chi-square) for homogeneous variances with Statistix 2.1 for Windows (Analytical software 1998). When Wilk-Shapiro test and chi-square test produced P values greater than 0.05, data were analyzed by one-way analysis of variance; when significant differences were encountered, a Student-Newman-Keuls multiple comparisons test was performed. When Wilk-Shapiro test and/or chi-square test produced P values less than 0.05, data were analyzed by Kruskal-Wallis non-parametric analysis of variance and analysis of variance of ranks (F statistic). In all comparisons P ⬎ 0.05 was considered not significant [12].
Serum IGF-1 levels related to body length increased in all three groups after 28 d (baseline value at day 0 ⫽ 14.43 ⫾ 5.16 ng/mL of IGF-1/centimeter of body length; P ⬍ 0.001 versus day 28) but did not differ between groups (P ⬎ 0.05), so it was independent of dietary fat content (Table 1). We examined the effects of decreasing dietary fat from 1:1 to 2:1 and to 3:1. Although the diets remained isocaloric, there was a significant increase in daily food intake in the first 2 wk of the experiment (P ⬍ 0.05) that was not
Results No changes in Wt and body length (P ⬎ 0.05) were seen after decreasing dietary fat from 10% corn oil (2:1) to 7.5% corn oil (3:1); however, when the level of dietary fat was 15% (1:1), there was a progressive decrease in Wt and body length (P ⬍ 0.01 and P ⬍ 0.001, respectively; Table 1). The velocity of daily weight gain showed a similar pattern (Table 1). Moreover, the ratio of Wt to length remained adequate throughout the experimental period (Fig. 1).
Fig. 1. Profiles of body weight for length of experimental rats fed 1:1, 2:1, and 3:1 diets (see MATERIALS AND METHODS) were plotted against normal male rat growth pattern percentiles for body weight for length [9] (Friedman SM, Gamba CA, unpublished observations). Zoometric categories by percentile ranges of body weight to length: obese, greater than 95.0; overweight, 95.0 to 85.1; adequate, 85.0 to 15.1; lean, 15.0 to 5.1; undernourished, less than 5.0. All diets were adequate for profiles of body weight to length.
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Fig. 2. Daily intake (g diet/100 g of rat Wt, mean ⫾ standard deviation) of experimental rats fed 1:1, 2:1, and 3:1 diets ad libitum (see MATERIALS AND METHODS). For statistical analysis, data were pooled in two 14-d periods (0 to 2 wk and 2 to 4 wk). P ⬍ 0.01, a versus d; P ⬍ 0.05, a versus c and c versus d; P ⬍ 0.001, a versus b and b versus d. Same letters indicate non-significance (P ⬎ 0.05). Wt, body weight.
observed in the remaining 2 wk (P ⬎ 0.05; Fig. 2). When total energy intake was analyzed, it did not differ across groups after 28 d (total daily diet intakes: 1:1, 11.77 ⫾ 2.56 g/100 g of rat Wt; 2:1, 11.58 ⫾ 0.61 g/100 g of rat Wt; 3:1, 12.13 ⫾ 1.45 g diet/100 g of rat Wt; P ⬎ 0.05). In rats fed the 1:1, 2:1 or 3:1 diet, body lipid decreased as the level of dietary fat decreased (P ⬍ 0.01 and P ⬍ 0.001, respectively; Table 1). Body water did not tend to increase, except in the 3:1 group, in which it was significantly greater than in the other two groups (P ⬍ 0.001; Table 1). Serum concentrations of leptin were 44% lower in animals fed the low-fat diet (P ⬍ 0.001) than in animals fed the 1:1 or 2:1 diet, with no significant differences between these diets (P ⬎ 0.05; Fig. 3). Moreover, serum concentration of leptin per body fat ratio was significantly lower in rats that received the low-fat diet (3:1) after 4 wk of treatment (P ⬍ 0.01; Fig. 3, inbox). Oxygen consumption, measured as liters per hour per rat, at 28 d of treatment showed no significant difference between the 1:1 and 2:1 groups (P ⬎ 0.05) and a significant decrease between the 1:1 and 3:1 groups (P ⬍ 0.01; Fig. 4). Energy expenditures, calculated as daily calories per 100 g of rat Wt in addition to calorie per oxygen liter conversion factor of each diet (Fig. 4, inset), at day 28 were 15% and 19% lower in the 2:1 and 3:1 groups, respectively, when compared with the 1:1 group (P ⬍ 0.01) and showed no significant differences between them (P ⬎ 0.05).
Discussion Several studies have examined the effect of hypocaloric diets with different levels of fat and carbohydrate on weight loss [13–15]. However, most animal studies have not inves-
Fig. 3. Leptin serum levels (ng/mL, mean ⫾ standard deviation) in rats after 28 d of consuming the fed 1:1, 2:1, and 3:1 diets (see MATERIALS AND METHODS). P ⬍ 0.001, a versus d. (Inset) Ratio of leptin to body fat (ng/mL/fat body composition as percentage of total body weight, mean ⫾ standard deviation). P ⬍ 0.01, a versus c. Same letters indicate nonsignificant (P ⬎ 0.05). Wt, body weight.
tigated the metabolic effects of unrestricted isocaloric diets with different compositions. The 2:1 and 3:1 ratios of carbohydrate to fat resemble low-fat diets (26% and 19% of energy from fat, respectively) consumed by human populations [5]. This study was of practical significance because it compared Wt with body length, body composition, and how the dietary ratio of fat to carbohydrate affects some growth factors in weanling rats. The diets were prepared with a fat content similar in proportion to that observed in the pediatric population with failure to thrive [4,5]. Some investigators have reported greater weight loss when using a high-fat diet versus a high-carbohydrate one [13,16]. However, other investigators have found no effect of diet composition on weight loss [17]. The difference may be associated with the amount of fat used in those studies. With regard to IGF-1, it has been reported to be sensitive to protein, energy, and specific micronutrients for its optimal synthesis and anabolic effects [18] and as a mediator of growth, showing a similar ratio of IGF-1 to body length. Our results suggest that circulating IGF-1 concentrations were not affected by modifying dietary fat content from 1:1 to 3:1 when young rats were fed isocaloric unrestricted diets with respect to protein, energy, and micronutrients requirements. Our data showed that spontaneous total energy intake was identical across the three groups. A possible explanation of this finding is that decreasing fat content of the diet, from 1:1 to 2:1 and to 3:1, although holding total energy constant, does not affect energy intake, whereas replacing a large portion of fat with carbohydrate leads to a change in energy intake. Comparing a sequence of 2-wk dietary treatments has shown that the 1:1 group had decreased energy intake over 0 to 2 wk, resulting in corresponding low Wt, body length, and Wt velocity. The increased energy intake
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Fig. 4. Energy expenditure of rats fed the 1:1, 2:1, and 3:1 diets (see MATERIALS AND METHODS) was monitored weekly as VO2 (mean ⫾ SD) in liters of oxygen per hour per rat. Statistical analysis is shown at each period when differences were encountered: P ⬍ 0.05, a versus b; P ⬍ 0.01, a versus c; P ⬍ 0.001, a versus d. Same letters indicate non-significance (P ⬎ 0.05). (Inset) Daily energy expenditure after 28 d of treatment (cal/100 g of rat Wt). P ⬍ 0.01, a versus c. Same letters indicate nonsignificance (P ⬎ 0.05). SD, standard deviation; VO2, oxygen consumption.
in the other groups may have been due to the decreased plasma level of satiating hormones or changes in glycogen stores. Nutrient intake and utilization under eucaloric conditions suggest that fats are metabolized more efficiently than carbohydrates [16]. Our study showed that the 2:1 and 3:1 groups reached the highest weight gain velocities. Moreover, both groups achieved a larger body (weight and body length), possibly due to the source of fat and carbohydrate used in the diets, such as saturated fat (medium and long chain), monounsaturated and polyunsaturated fats (-3 and -6), and simple or complex carbohydrates. Human body fat burn is stimulated by an isocaloric, low-fat, high-carbohydrate diet. However, the type of sugar may also be important. A simple sugar has direct uptake and rapid phosphorylation in the liver, but this should not be generalized to starch, dextrin, or different amounts and types of carbohydrates [19,20]. Weight gain velocity may be achieved more easily with high-carbohydrate diets. Our finding is consistent with those of other investigators who fed rats under unrestricted conditions [21]. Our results showed that the pattern of dietary fat is similar to that of carcass fat gain. Gains in body energy through stored lipids and lipid intake were almost equivalent due to the age of the animals; although starting from about 55 d of age, lipid gain would represent only a part of lipid intake [14]. The slight increase in body water seen in the 3:1 group was not dependent on decreased body fat content. Moreover, all three groups remained within the normal range expected for age, sex, and strain [14,22]. However, the change in weight and water in the 3:1 group may have been the result of carbohydrate overfeeding accompanied by an
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increase in glycogen stores. Studies of longer duration will lead to saturate glycogen stores and convert carbohydrate to fat [13]. When dietary fat was decreased in 3:1 group, rats had a lower percentage of lipid weight compared with the 1:1 and 2:1 groups, despite the decrease in the amount of dietary fat and the proportional increase in carbohydrate content. This indicates that body fat was affected to a greater degree by decreased dietary fat. Leptin is secreted from adipose tissue in proportion to the amount of body fat mass and can act on the brain to regulate the amount of adipose tissue stores [23–25]. Therefore, leptin may be a long-term regulator of body fat. Circulating leptin was directly related to body fat content. This is in agreement with the finding that serum concentration of leptin is not a direct effect of the diet, but rather due to a secondary increase in body lipid content [26]. Further, leptin may act in the hypothalamus by decreasing food intake, and, interestingly, in the 1:1 group, leptin was significantly higher than in the other groups and was related to decreased food intake and, hence, a smaller body. Although decreased fat intake was associated with body composition and leptin concentration, energy and nutrient intakes were not significantly different across groups, with adequate intake amounts for normal growth, and no evidence of growth deficiency was observed. However, further studies are needed to elucidate the effect of decreased leptin serum concentration on bone metabolism. Differences in leptin levels might influence bone development and growth without affecting body weight [27]. The lack of a significant difference in energy expenditure between the 2:1 and 3:1 groups may have been due to the small difference in energy provided by fat in the two diets. The effect of a much wider variation in energy from fat in the 1:1 group showed a strong influence of diet composition. The decrease in adipose tissue mass in response to dietary fat content produced a decrease in cellular activity [28]. Most animal studies have not investigated the metabolic effects of unrestricted isocaloric diets with different compositions. In summary, these studies demonstrated that, although energy, protein, vitamin, and micronutrient intakes were constant and adequate, when calorie ratios of carbohydrate to fat were progressively increased from 1:1 to 2:1 and to 3:1, the diet contributed to weight gain by decreasing energy metabolism and leptin secretion through adipose tissue. Further studies are required to demonstrate the importance of these findings in relation to metabolic changes.
Acknowledgments This paper is part of a doctoral thesis by Carlota A. Gamba to fulfill the academic requirements of the University of Buenos Aires. Partial results were presented at the
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XIII Argentine Nutrition Congress, Mar del Plata, Buenos Aires, 1999; the I International Symposium of Metabolic Unit, Bahia, Brasil, 1999; and at the annual conference of the Federation of American Societies for Experimental Biology, Orlando, Florida, 2001; and was published in abstract form (abstract 759.21, FASEB J 2001;15:A998).
References [1] Ricketts CD. Fat preferences, dietary fat intake and body composition in children. Eur J Clin Nutr 1997;51:443– 8. [2] Watkins BA, Li Y, Seifert MF. Nutraceutical fatty acids as biochemical and molecular modulators of skeletal biology. J Am Coll Nutr 2001;20(suppl):410S– 6. [3] Watkins BA, Lippman HE, Bouteiller LL, Li Y, Seifert MF. Bioactive fatty acids: role in bone biology and bone cell function. Prog Lipid Res 2001;40:125– 48. [4] Lifshitz F, Friedman S, Smith MM, et al. Nutritional dwarfing: a growth abnormality associated with reduced erythrocyte Na⫹, K⫹ATPase activity. Am J Clin Nutr 1991;54:997–1004. [5] Tarim O, Chaslow F, Murphy J, Rising R, Carrillo A, Lifshitz F. Evaluation of differential effects of carbohydrate and fat intake on weight gain, serum IGF-I and erythrocyte Na⫹K⫹ ATPase activity in suboptimal nutrition in rats. J Am Coll Nutr 1997;16:159 – 65. [6] Friedman SM, Rodriguez PN, Olivera MI, et al. Enanismo por desnutrición: cronodinamia de los procesos metabólicos en ratas. Medicina (B Aires) 1998;58:282– 6. [7] Friedman SM, Gamba CA, Boyer PM, et al. Growth deceleration and bone metabolism in nutritional dwarfing rats. Int J Food Sc Nutr 2001;52:225–33. [8] RDA and NRC. The nutrients requirements of laboratory animals. Number 10. Washington, DC: National Research Council; 1978. [9] Friedman SM, Boyer PM, Barrio Rendo ME, et al. Evaluación del crecimiento normal en ratas a través del z score. Arch Latinoam Nutr 1999;49:142– 8. [10] Burztein S, Elwyn D, Ashanazi J, Kinney J. The theoretical framework of indirect calorimetry and energy balance. In: Williams, Williams editors. Energy metabolism, indirect calorimetry and nutrition. 1987; p. 27– 83. [11] Association of Official Agricultural Chemists. Official methods for analysis. 15th ed. Washington, DC: US Government Printing Office; 1990.
[12] Sokal R, Rohlf J. Biometry. The principles and practice of statistics in biological research. San Francisco: WH Freeman; 1969. [13] Shah M, Garg A. High-fat and high-carbohydrate diets and energy balance. Diabetes Care 1996;19:1142–52. [14] Iossa S, Lionetti L, Mollica MP, Barletta A, Liverini G. Energy intake and utilization vary during development in rats. J Nutr 1999;129: 1593– 6. [15] Franz MJ. The answer to weight loss is easy. Doing it is hard! Clin Diabetes 2001;19:105–9. [16] Hudgins Cooper L, Hellerstein M, Seidman C, et al. Human fatty acid synthesis is stimulated by a eucaloric low fat, high carbohydrate diet. J Clin Invest 1996;97:2081–91. [17] Golay A, Allaz AF, Morel Y, et al. Similar weight loss with low or high carbohydrate diets. Am J Clin Nutr 1996;63:174 – 8. [18] Estivariz CF, Ziegler TR. Nutrition and the insulin-like growth factor system. Endocrine 1997;7:65–71. [19] Watkins BA, Li Y, Allen KGD, Hoffmann W, Seifert M. Dietary ratio of (n-6)/(n-3) polyunsaturated fatty acids alters the fatty acid composition of bone compartments and biomarkers of bone formation in rats. J Nutr 2000;130:2274 – 84. [20] Azian MJ, Hausman DB, Sisk MB, Flatt WP, Jewell DE. Dietary conjugated linoleic acid reduces rat adipose tissue cell size rather than cell number. J Nutr 2000;130:1548 –54. [21] Mac Cargar IJ, Baracos VE, Clandimin MT. Influence of dietary carbohydrate to fat ratio on whole body nitrogen retention and body composition in adults rats. J Nutr 1989;119:1240 –5. [22] Pullar JD, Webster JF. The energy cost of fat and protein deposition in the rat. Br J Nutr 1977;37:355– 63. [23] Campfield LA, Smith FJ, Guisez Y, Revos R, Burn P. Recombinant mouse OB protein: evidence of peripheral signal linking adiposity and central networks. Science 1995;269:546 –9. [24] Halaas JL, Gajiwaka KS, Maffei M, et al. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 1995;269: 543– 6. [25] Iritani N, Sugimoto T, Fukuda H. Gene expressions of leptin, insulin receptors and lipogenic enzymes are coordinately regulated by insulin and dietary fat in rats. J Nutr 2000;130:1183– 8. [26] Du F, Higginbotham A, White D. Food intake, energy balance and serum leptin concentrations in rats fed low-protein diets. J Nutr 2000;130:514 –21. [27] Cock TA, Auwerx J. Leptin:cutting the fat off the bone. Lancet 2003;362(9395):1572– 4. [28] Ainslie DA, Proietto J, Fam BC, Thorburn AW. Short-term, high-fat diets lower circulating leptin concentrations in rats. Am J Clin Nutr 2000;71:438 – 42.