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Saccharin and aspartame, compared with sucrose, induce greater weight gain in adult Wistar rats, at similar total caloric intake levels
Appetite 60 (2013) 203–207
Contents lists available at SciVerse ScienceDirect
Appetite journal homepage: www.elsevier.com/locate/appet
Research report
Saccharin and aspartame, compared with sucrose, induce greater weight gain in adult Wistar rats, at similar total caloric intake levels q Fernanda de Matos Feijó a, Cíntia Reis Ballard a, Kelly Carraro Foletto a, Bruna Aparecida Melo Batista b, Alice Magagnin Neves b, Maria Flávia Marques Ribeiro b, Marcello Casaccia Bertoluci a,c,⇑ a
Programa de Pós-Graduação em Medicina: Ciências Médicas, Faculdade de Medicina da Universidade Federal do Rio Grande do Sul, Ramiro Barcelos 2400, CEP 90035-003, Porto Alegre, Brazil Departamento de Fisiologia, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Sarmento Leite 500, CEP 90050-170, Porto Alegre, Brazil c Serviço de Medicina Interna, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos 2350, CEP 90035-903, Sala 700, 7° andar, Porto Alegre, Brazil b
a r t i c l e
i n f o
Article history: Received 19 March 2012 Received in revised form 3 October 2012 Accepted 10 October 2012 Available online 23 October 2012 Keywords: Nonnutritive sweeteners Saccharin Aspartame Sucrose Caloric intake Weight gain
a b s t r a c t It has been suggested that the use of nonnutritive sweeteners (NNSs) can lead to weight gain, but evidence regarding their real effect in body weight and satiety is still inconclusive. Using a rat model, the present study compares the effect of saccharin and aspartame to sucrose in body weight gain and in caloric intake. Twenty-nine male Wistar rats received plain yogurt sweetened with 20% sucrose, 0.3% sodium saccharin or 0.4% aspartame, in addition to chow and water ad libitum, while physical activity was restrained. Measurements of cumulative body weight gain, total caloric intake, caloric intake of chow and caloric intake of sweetened yogurt were performed weekly for 12 weeks. Results showed that addition of either saccharin or aspartame to yogurt resulted in increased weight gain compared to addition of sucrose, however total caloric intake was similar among groups. In conclusion, greater weight gain was promoted by the use of saccharin or aspartame, compared with sucrose, and this weight gain was unrelated to caloric intake. We speculate that a decrease in energy expenditure or increase in fluid retention might be involved. Ó 2012 Elsevier Ltd. All rights reserved.
Introduction Increased NNS consumption has been historically associated with the increasing prevalence of obesity (Fowler et al., 2008; Popkin & Nielsen, 2003; Yang, 2010). It has also been suggested that either the use of saccharin or aspartame is associated with an increased feeling of hunger (Appleton & Blundell, 2007; Blundell & Green, 1996; Lavin, French, & Read, 1997; Rogers, Carlyle, Hill, & Blundell, 1988). Rats whose diet was sweetened with saccharin for over 5 weeks, presented greater weight gain and adiposity as well as a decrease in the central body temperature when compared to glucose supplementation (Swithers & Davidson, 2008). On the other hand, some studies show that exposure to NNS may help control body weight (Blackburn, Kanders, Lavin, Keller, & Whatley, 1997; Phelan, Lang, Jordan, & Wing, 2009; Raben, Vasilaras, Møller,
q Acknowledgments: We thank the Fundo de Incentivo a Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) that supported this research. The funding agencies were not involved in the study design, collection, analysis or interpretation of the results, writing of the report or decision to submit the paper for publication. ⇑ Corresponding author. E-mail address: [email protected] (M.C. Bertoluci).
0195-6663/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.appet.2012.10.009
& Astrup, 2002) and have no effect on satiety (Anton et al., 2010; Canty & Chan, 1991; Drewnowski et al., 1994; Van Wymelbeke, Béridot-Thérond, de La Guéronnière, & Fantino, 2004). However, the effects of NNS on body weight and satiety are still inconclusive. The aim of the present study as to examine the long-term effects in weight gain and caloric intake of supplementing the diet of Wistar rats with NNS (either saccharin or aspartame), compared with sucrose, in the context of ad libitum ingestion of chow.
Methods Animals Adult male Wistar rats (n = 30), weighing 200–300 g at the start of testing, were randomly divided into three groups (n = 10 per group): saccharin (SAC), aspartame (ASP) and sucrose (SUC), according to the type of sweetener added to yogurt supplement. Standard chow and water were offered daily ad libitum for all groups. The animals were housed in individual transparent acrylic cages, with controlled humidity (65–70%) and temperature (22 ± 1 °C), maintained through a 12 h light and dark cycle. In order to minimize the impact of physical activity in weight changes we restrained the rats’ movements by confining them in
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44 34 16 cm cages during the 12 weeks of the experiment. Procedures were conducted in accordance with the Principles of Laboratory Animal Care (NIH publication No. 86-23, revised 1985) and guidelines of the National Research Council for the Care and Use of Laboratory Animals (Council, 2010) in accordance with the Brazilian Law for the Scientific Use of Animals. Study protocols were approved by the Ethics Committee for Experimental Procedures of Hospital de Clínicas de Porto Alegre, Brazil. Dietary manipulation Based on the Swithers & Davidson protocol (2008), the preparation of diets included 20 ml of plain yogurt (Piá™) to which 20% sucrose (União™), 0.3% sodium saccharin (Finn™) or 0.4% aspartame (Gold™) were added. During preparation, 10 ml pure water was added in all yogurt diets to adjust the viscosity. Yogurt diets were offered 5 days a week, in special bottles with beaks adapted for possible leakage. Yogurt bottles were offered at 6 pm and remained available for approximately 22 h. The exclusion criterion was ingesting less than 70% of sweetened yogurt offered. The caloric density (CD) of watered down yogurts was 0.9 kcal/g for yogurt sweetened with sucrose and 0.5 kcal/g for yogurt sweetened with saccharin or aspartame. Throughout each week of testing, the SUC group received a total of 139 kcal per week from the yogurt diet, while the SAC and ASP groups received 75 kcal per week. In addition to yogurt diet, standard chow pellet (CD: 2.93 kcal/g, Nuvital CR-1, Nuvilab™) and water were offered ad libitum 7 days a week, at 9 am and removed after 24 h. The large solid pellets were deposited in grid feeders, with a bottom crumb collector, on the outside of the cage where the rats were kept. Measurements of food intake and weight gain The control of food intake (chow and yogurt diets) was conducted daily by subtraction of the quantity remaining (g) from the quantity supplied (g). Cages were carefully monitored for any evidence of chow spillage and crumbs were considered for the control of chow intake. The yogurt bottles were also checked for any sign of leaking or clogging. The rats were weighed weekly at the same time in the morning. An electronic precision balance (AS 5500, Marte™) was used for all these measures and data collectors were blinded to group assignments. Determination of variables Variables over time Cumulative weight gain was calculated by the subtraction of the basal weight from the weight obtained every week, and expressed in grams. Cumulative total caloric intake (including yogurt and chow), cumulative caloric intake of yogurt and cumulative caloric intake of chow were calculated by the sum of calories ingested along each week, and corrected by the corresponding rat weight at the end of each week. These data were calculated in the cumulative mode for the 12-week period and were expressed as kcal/g of rat. Single variables The total weight gain was defined by the difference between the basal weight and the final weight, in the 12th week. Mean total caloric intake per week, mean caloric intake from yogurt per week, and mean caloric intake from chow per week were calculated as the mean of 12 periods of week caloric ingestion corrected by rat weight (g) at the end of each week and expressed as kcal/g/wk.
Statistical analysis To evaluate the effect over time, Linear Mixed Model (Cleophas, Zwinderman, & van Ouwerkerk, 2010; Shek & Ma, 2011; West, 2009) with random slopes and weekly measurement as a repeated effect were applied to these variables. In these analyses, basal weight was used as a covariate, the fixed effects in the model were the groups and the interaction groupweeks, while rats and weeks were treated as a random effect. First-order autoregressive covariance structure [AR(1)] were used in all these analysis. For single variables, one-way analysis of variance (ANOVA) with the Dunnett’s test was used to identify differences between groups, and was also used to compare the basal weight, and percentage of intake of yogurt diets. Normality assumptions were verified by Kolmogorov–Smirnov test, with Lilliefors significance correction, and Shapiro–Wilk statistic. Reported values were mean ± SE, and p < 0.05 was set for all analyses. We used the SPSS™ software version 19 (IBM Corporation, Somers, NY) for statistical analyses. Results Cumulative weight gain Body weight was similar among groups in the beginning of the experiment, F (2, 26) = 0.09, p = 0.92(263.49 ± 10.60) (Table 1). Weight increased considerably in all groups. In the linear mixed model analysis, SAC and ASP presented greater weight gain than the SUC group (Fig. 1A): SAC (b = 3.82, SE = 1.31, p = 0.005) and ASP (b = 2.70, SE = 1.34, p = 0.048), F (2, 73.47) = 4.51, p = 0.01. In the ANOVA analysis, total weight gain was 28% greater in SAC (p < 0.003) and 20% in ASP (p < 0.04) in relation to SUC, F (2, 26) = 6.73, p = 0.004 (Table 1). Total caloric intake Cumulative total caloric intake (yogurt plus chow) was similar among all groups over the 12 weeks of the experiment, F (2, 27.17) = 0.12, p = 0.89 (Fig. 1B). Mean total week caloric intake corrected by rat weight was also similar in all groups, F (2, 26) = 0.04, p = 0.96 (Table 1). Calories from yogurt In the mixed model analysis, the cumulative caloric intake of sweetened yogurt during the study was lower in both SAC and ASP compared to SUC (b = 0.16, SE = 0.01, p < 0.001) but did not differ between SAC and ASP (Fig. 1D). Likewise, the weekly average intake of yogurt corrected by weekly weight was confirmed to be lower in ASP and SAC groups, F (2, 26) = 60.64, p < 0.001 after the ANOVA analysis (Table 1). One rat from the ASP group was excluded from the study due to an ingestion of less than 70% of yogurt, the remaining rats presented an average intake of 88.52% (±1.68), F (2, 26) = 2.23, p = 0.13 (Table 1). Calories from chow The cumulative caloric intake from chow during the study was significantly increased in SAC vs. SUC (b = 0.17, SE = 0.03, p < 0.001) and in ASP vs. SUC (b = 0.18, SE = 0.03, p < 0.001), F (2, 35.07) = 27.07, p < 0.001 (Fig. 1C). The mean weekly intake of chow corrected by weekly weight was 16% higher in both the SAC and ASP groups in relation to the SUC group, F (2, 26) = 40.85, p < 0.001 (Table 1). This decrease in calories from chow in SAC
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F. M. Feijó et al. / Appetite 60 (2013) 203–207 Table 1 Weight and caloric intake parameters.
Yogurt intake (% of total offered) Basal weight (g) Total weight gain (g) Mean total caloric intake per week (kcal/g/wk)a Mean calories from yogurt per week (kcal/g/wk)a Mean calories from chow per week (kcal/g/wk)a
SAC (n = 10)
ASP (n = 9)
SUC (n = 10)
88.21 ± 2.21 260.83 ± 21.97 175.31 ± 6.47** 1.45 ± 0.02 0.18 ± 0.01*** 1.27 ± 0.01***
84.28 ± 4.39 259.41 ± 18.81 164.28 ± 10.65* 1.45 ± 0.03 0.18 ± 0.01*** 1.27 ± 0.02***
92.66 ± 1.15 269.82 ± 15.54 137.37 ± 5.51 1.44 ± 0.02 0.35 ± 0.01 1.10 ± 0.02
Group labels: saccharin (SAC), aspartame (ASP) and sucrose (SUC). Data are Mean ± SE. Analysis by ANOVA with Dunnett test. Asterisks indicate comparison with SUC. p < 0.05. ** p = 0.005. *** p < 0.001. a Mean weekly intake throughout the 12 week study period. *
Fig. 1. Cumulative effect of the sweetened supplements [saccharin (SAC), aspartame (ASP) or sucrose (SUC)] on feeding pattern and body weight gain during 12 weeks of consumption. The groups that received nonnutritive sweeteners (NNSs) consumed more chow than the group offered sucrose (D), compensating the caloric deficit of these sweeteners (B). Thus, the total calorie consumption (yogurt plus chow) did not differ among the experimental groups (C). However, the NNS groups gained more weight than the group supplemented with sucrose (A). Analysis by linear mixed model, n = 10 (9 for ASP). Asterisks indicate comparison with SUC: p < 0.001; p = 0.005 and p < 0.05. Error bars indicate SE.
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and ASP compensated the increased in calories from sweetened yogurt resulting in a similar total cumulative caloric intake among all groups (Fig. 1B).
Discussion In the present study, Wistar rats receiving yogurt sweetened with either saccharin or aspartame and free chow diet during 12 weeks presented increased weight gain compared to animals receiving the same free chow diet plus yogurt sweetened with sucrose. Although saccharin and aspartame promoted relatively fewer calories from yogurt intake when compared to sucrose, increases in calories from chow intake effectively compensated for decreases in calories from yogurt, in such a way that there was a similar total caloric intake among all groups after the 12-week period of the experiment. These data are consistent with the hypothesis that animals adjust for calories consumed on one occasion by reducing their caloric intake on subsequent opportunities to eat (Booth, 1972; Foltin, Fischman, Moran, Rolls, & Kelly, 1990; Mazlan, Horgan, & Stubbs, 2006; Rowland, Nasrallah, & Robertson, 2005). However, it was surprising to find that the NNS were able to induce weight gain without an increase in total caloric intake, suggesting that other mechanisms such as decreased caloric expenditure may occur after NNS use. Our data are in accordance with the study of Swithers and Davidson (2008) who, using a similar protocol in Sprague–Dawley rats, observed that rats did gain more weight when using saccharin compared to glucose, despite a similar total energy intake after 5 weeks of experimenting. Our results are also in accordance with the Polyák et al. study (2010), in which CBA/CA inbred mice with free chow were given saccharin or aspartame added to the water and compared to controls using pure water, during 25 weeks. They observed that animals exposed to NNS in water gained more weight than those drinking non-sweetened water, despite a similar total caloric intake. These studies taken together are consistent in demonstrating that positive caloric compensation in chow intake occurs among NNS-exposed rats, but the increased weight gain observed in these animals was not explained by caloric intake, which was similar in all three groups. Possible explanations for weight-gain in saccharin and aspartame groups without increasing energy intake are still widely speculative. We could not attribute it to technical bias because we had strict dally control of chow and yogurt intake. A possible cause could be the reduction of energy expenditure induced by NNS. Swithers and Davidson (2008) demonstrated that body core temperature can be increased acutely minutes after the ingestion of glucose compared to non-sweetened fluid, an effect that was not observed when saccharin is used. The authors consider that food intake elicits a reflexive cephalic phase thermogenic response, and the heat production may be mediated by orosensory stimuli that signal the absorption of nutrients in the gut. However, there has not yet been any conclusion regarding a more prolonged effect induced by saccharin. A second possibility would be that hyperinsulinemia could be induced by saccharin, with consequent weight gain (Corkey, 2012). In vitro studies, using islet cells from Wistar rats were able to demonstrate an insulinotropic effect due to saccharin and other NNS, although not with aspartame. Insulin secretion increased three times from basal levels when cells were exposed to increased saccharin concentrations. The authors speculated that the bitter taste of saccharin, due to hexose pentaacetate esters could act through cellular mechanisms similar to that involved in bittertaste by taste buds. This effect is supported by the finding that islet cells are equipped with GUST27 receptors also expressed in taste
buds which are mediated by G proteins activated with calcium release. These mechanisms are linked to the blockage of potassium channels and activation of cAMP breakdown which regulates insulin secretion (Malaisse, Vanonderbergen, Louchami, Jijakli, & Malaisse-Lagae, 1998). Increased insulin secretion induced by NNS however still remains to be established in animal and human studies. We also considered the possibility of increased fluid intake and retention due to sodium content in saccharin and also to the sweet taste per se. However, in our study, yogurt caloric intake was decreased both in saccharin and in aspartame groups compared to sucrose, making this hypothesis less likely. Likewise, since weight gain was similar in saccharin and in aspartame groups, we believe that the impact of sodium (from saccharin) may have been of minor significance. Conclusion We observed significantly greater weight gain among Wistar rats fed diets supplemented with NNS – whether saccharin or aspartame – compared with sucrose. This increase was not the result of increased total caloric intake. This discrepancy raises the possibility that weight gain in NNS-fed rats might result from decreases in energy expenditure, which have been observed in other studies. Further studies are necessary to address energy expenditure after NNS exposure in rats as well as long-term clinical trials to evaluate weight gain increases in humans. References Anton, S. D., Martin, C. K., Han, H., Coulon, S., Cefalu, W. T., Geiselman, P., et al. (2010). Effects of stevia, aspartame, and sucrose on food intake, satiety, and postprandial glucose and insulin levels. Appetite, 55(1), 37–43. Appleton, K. M., & Blundell, J. E. (2007). Habitual high and low consumers of artificially-sweetened beverages. Effects of sweet taste and energy on shortterm appetite. Physiology and Behavior, 92(3), 479–486. Blackburn, G. L., Kanders, B. S., Lavin, P. T., Keller, S. D., & Whatley, J. (1997). The effect of aspartame as part of a multidisciplinary weight-control program on short- and long-term control of body weight. American Journal of Clinical Nutrition, 65(2), 409–418. Blundell, J. E., & Green, S. M. (1996). Effect of sucrose and sweeteners on appetite and energy intake. International Journal of Obesity and Related Metabolic Disorders, 20(Suppl 2), S12–17. Booth, D. A. (1972). Conditioned satiety in the rat. Journal of Comparative and Physiological Psychology, 81(3), 457–471. Canty, D. J., & Chan, M. M. (1991). Effects of consumption of caloric vs noncaloric sweet drinks on indices of hunger and food consumption in normal adults. American Journal of Clinical Nutrition, 53(5), 1159–1164. Cleophas, T. J., Zwinderman, A. H., & van Ouwerkerk, B. (2010). Clinical Research. A Novel Approach to the Analysis of Repeated Measures. American Journal of Therapeutics, 1, 2. Corkey, B. E. (2012). Banting lecture 2011. Hyperinsulinemia. Cause or consequence? Diabetes, 61(1), 4–13. Council, N. R. (2010). Guide for the care and use of laboratory animals (8 ed.). Washington, DC: National Academies Press. Drewnowski, A., Massien, C., Louis-Sylvestre, J., Fricker, J., Chapelot, D., & Apfelbaum, M. (1994). Comparing the effects of aspartame and sucrose on motivational ratings, taste preferences, and energy intakes in humans. American Journal of Clinical Nutrition, 59(2), 338–345. Foltin, R. W., Fischman, M. W., Moran, T. H., Rolls, B. J., & Kelly, T. H. (1990). Caloric compensation for lunches varying in fat and carbohydrate content by humans in a residential laboratory. American Journal of Clinical Nutrition, 52(6), 969–980. Fowler, S. P., Williams, K., Resendez, R. G., Hunt, K. J., Hazuda, H. P., & Stern, M. P. (2008). Fueling the obesity epidemic? Artificially sweetened beverage use and long-term weight gain. Obesity (Silver Spring), 16(8), 1894–1900. Lavin, J. H., French, S. J., & Read, N. W. (1997). The effect of sucrose- and aspartamesweetened drinks on energy intake, hunger and food choice of female, moderately restrained eaters. International Journal of Obesity and Related Metabolic Disorders, 21(1), 37–42. Malaisse, W. J., Vanonderbergen, A., Louchami, K., Jijakli, H., & Malaisse-Lagae, F. (1998). Effects of artificial sweeteners on insulin release and cationic fluxes in rat pancreatic islets. Cell Signal, 10(10), 727–733. Mazlan, N., Horgan, G., & Stubbs, R. J. (2006). Energy density and weight of food effect short-term caloric compensation in men. Physiology and Behavior, 87(4), 679–686. Phelan, S., Lang, W., Jordan, D., & Wing, R. R. (2009). Use of artificial sweeteners and fat-modified foods in weight loss maintainers and always-normal
F. M. Feijó et al. / Appetite 60 (2013) 203–207 weight individuals. International Journal of Obesity (London), 33(10), 1183–1190. Polyák, E., Gombos, K., Hajnal, B., Bonyár-Müller, K., Szabó, S., Gubicskó-Kisbenedek, A., et al. (2010). Effects of artificial sweeteners on body weight, food and drink intake. Acta Physiologica Hungarica, 97(4), 401–407. Popkin, B. M., & Nielsen, S. J. (2003). The sweetening of the world’s diet. Obesity Research, 11(11), 1325–1332. Raben, A., Vasilaras, T. H., Møller, A. C., & Astrup, A. (2002). Sucrose compared with artificial sweeteners. Different effects on ad libitum food intake and body weight after 10 wk of supplementation in overweight subjects. American Journal of Clinical Nutrition, 76(4), 721–729. Rogers, P. J., Carlyle, J. A., Hill, A. J., & Blundell, J. E. (1988). Uncoupling sweet taste and calories. Comparison of the effects of glucose and three intense sweeteners on hunger and food intake. Physiology and Behavior, 43(5), 547–552. Rowland, N. E., Nasrallah, N., & Robertson, K. L. (2005). Accurate caloric compensation in rats for electively consumed ethanol-beer or ethanolpolycose mixtures. Pharmacology Biochemistry and Behavior, 80(1), 109–114.
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Shek, D. T., & Ma, C. M. (2011). Longitudinal data analyses using linear mixed models in SPSS. Concepts, procedures and illustrations. Scientific World Journal, 11, 42–76. Swithers, S. E., & Davidson, T. L. (2008). A role for sweet taste. Calorie predictive relations in energy regulation by rats. Behavioral Neuroscience, 122(1), 161–173. Van Wymelbeke, V., Béridot-Thérond, M. E., de La Guéronnière, V., & Fantino, M. (2004). Influence of repeated consumption of beverages containing sucrose or intense sweeteners on food intake. European Journal Clinical Nutrition, 58(1), 154–161. West, B. T. (2009). Analyzing longitudinal data with the linear mixed models procedure in SPSS. Evaluation and the Health Professions, 32(3), 207–228. Yang, Q. (2010). Gain weight by ‘‘going diet?’’ Artificial sweeteners and the neurobiology of sugar cravings. Neuroscience 2010. Yale Journal of Biology Medicine, 83(2), 101–108.
Contents lists available at SciVerse ScienceDirect
Appetite journal homepage: www.elsevier.com/locate/appet
Research report
Saccharin and aspartame, compared with sucrose, induce greater weight gain in adult Wistar rats, at similar total caloric intake levels q Fernanda de Matos Feijó a, Cíntia Reis Ballard a, Kelly Carraro Foletto a, Bruna Aparecida Melo Batista b, Alice Magagnin Neves b, Maria Flávia Marques Ribeiro b, Marcello Casaccia Bertoluci a,c,⇑ a
Programa de Pós-Graduação em Medicina: Ciências Médicas, Faculdade de Medicina da Universidade Federal do Rio Grande do Sul, Ramiro Barcelos 2400, CEP 90035-003, Porto Alegre, Brazil Departamento de Fisiologia, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Sarmento Leite 500, CEP 90050-170, Porto Alegre, Brazil c Serviço de Medicina Interna, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos 2350, CEP 90035-903, Sala 700, 7° andar, Porto Alegre, Brazil b
a r t i c l e
i n f o
Article history: Received 19 March 2012 Received in revised form 3 October 2012 Accepted 10 October 2012 Available online 23 October 2012 Keywords: Nonnutritive sweeteners Saccharin Aspartame Sucrose Caloric intake Weight gain
a b s t r a c t It has been suggested that the use of nonnutritive sweeteners (NNSs) can lead to weight gain, but evidence regarding their real effect in body weight and satiety is still inconclusive. Using a rat model, the present study compares the effect of saccharin and aspartame to sucrose in body weight gain and in caloric intake. Twenty-nine male Wistar rats received plain yogurt sweetened with 20% sucrose, 0.3% sodium saccharin or 0.4% aspartame, in addition to chow and water ad libitum, while physical activity was restrained. Measurements of cumulative body weight gain, total caloric intake, caloric intake of chow and caloric intake of sweetened yogurt were performed weekly for 12 weeks. Results showed that addition of either saccharin or aspartame to yogurt resulted in increased weight gain compared to addition of sucrose, however total caloric intake was similar among groups. In conclusion, greater weight gain was promoted by the use of saccharin or aspartame, compared with sucrose, and this weight gain was unrelated to caloric intake. We speculate that a decrease in energy expenditure or increase in fluid retention might be involved. Ó 2012 Elsevier Ltd. All rights reserved.
Introduction Increased NNS consumption has been historically associated with the increasing prevalence of obesity (Fowler et al., 2008; Popkin & Nielsen, 2003; Yang, 2010). It has also been suggested that either the use of saccharin or aspartame is associated with an increased feeling of hunger (Appleton & Blundell, 2007; Blundell & Green, 1996; Lavin, French, & Read, 1997; Rogers, Carlyle, Hill, & Blundell, 1988). Rats whose diet was sweetened with saccharin for over 5 weeks, presented greater weight gain and adiposity as well as a decrease in the central body temperature when compared to glucose supplementation (Swithers & Davidson, 2008). On the other hand, some studies show that exposure to NNS may help control body weight (Blackburn, Kanders, Lavin, Keller, & Whatley, 1997; Phelan, Lang, Jordan, & Wing, 2009; Raben, Vasilaras, Møller,
q Acknowledgments: We thank the Fundo de Incentivo a Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) that supported this research. The funding agencies were not involved in the study design, collection, analysis or interpretation of the results, writing of the report or decision to submit the paper for publication. ⇑ Corresponding author. E-mail address: [email protected] (M.C. Bertoluci).
0195-6663/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.appet.2012.10.009
& Astrup, 2002) and have no effect on satiety (Anton et al., 2010; Canty & Chan, 1991; Drewnowski et al., 1994; Van Wymelbeke, Béridot-Thérond, de La Guéronnière, & Fantino, 2004). However, the effects of NNS on body weight and satiety are still inconclusive. The aim of the present study as to examine the long-term effects in weight gain and caloric intake of supplementing the diet of Wistar rats with NNS (either saccharin or aspartame), compared with sucrose, in the context of ad libitum ingestion of chow.
Methods Animals Adult male Wistar rats (n = 30), weighing 200–300 g at the start of testing, were randomly divided into three groups (n = 10 per group): saccharin (SAC), aspartame (ASP) and sucrose (SUC), according to the type of sweetener added to yogurt supplement. Standard chow and water were offered daily ad libitum for all groups. The animals were housed in individual transparent acrylic cages, with controlled humidity (65–70%) and temperature (22 ± 1 °C), maintained through a 12 h light and dark cycle. In order to minimize the impact of physical activity in weight changes we restrained the rats’ movements by confining them in
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44 34 16 cm cages during the 12 weeks of the experiment. Procedures were conducted in accordance with the Principles of Laboratory Animal Care (NIH publication No. 86-23, revised 1985) and guidelines of the National Research Council for the Care and Use of Laboratory Animals (Council, 2010) in accordance with the Brazilian Law for the Scientific Use of Animals. Study protocols were approved by the Ethics Committee for Experimental Procedures of Hospital de Clínicas de Porto Alegre, Brazil. Dietary manipulation Based on the Swithers & Davidson protocol (2008), the preparation of diets included 20 ml of plain yogurt (Piá™) to which 20% sucrose (União™), 0.3% sodium saccharin (Finn™) or 0.4% aspartame (Gold™) were added. During preparation, 10 ml pure water was added in all yogurt diets to adjust the viscosity. Yogurt diets were offered 5 days a week, in special bottles with beaks adapted for possible leakage. Yogurt bottles were offered at 6 pm and remained available for approximately 22 h. The exclusion criterion was ingesting less than 70% of sweetened yogurt offered. The caloric density (CD) of watered down yogurts was 0.9 kcal/g for yogurt sweetened with sucrose and 0.5 kcal/g for yogurt sweetened with saccharin or aspartame. Throughout each week of testing, the SUC group received a total of 139 kcal per week from the yogurt diet, while the SAC and ASP groups received 75 kcal per week. In addition to yogurt diet, standard chow pellet (CD: 2.93 kcal/g, Nuvital CR-1, Nuvilab™) and water were offered ad libitum 7 days a week, at 9 am and removed after 24 h. The large solid pellets were deposited in grid feeders, with a bottom crumb collector, on the outside of the cage where the rats were kept. Measurements of food intake and weight gain The control of food intake (chow and yogurt diets) was conducted daily by subtraction of the quantity remaining (g) from the quantity supplied (g). Cages were carefully monitored for any evidence of chow spillage and crumbs were considered for the control of chow intake. The yogurt bottles were also checked for any sign of leaking or clogging. The rats were weighed weekly at the same time in the morning. An electronic precision balance (AS 5500, Marte™) was used for all these measures and data collectors were blinded to group assignments. Determination of variables Variables over time Cumulative weight gain was calculated by the subtraction of the basal weight from the weight obtained every week, and expressed in grams. Cumulative total caloric intake (including yogurt and chow), cumulative caloric intake of yogurt and cumulative caloric intake of chow were calculated by the sum of calories ingested along each week, and corrected by the corresponding rat weight at the end of each week. These data were calculated in the cumulative mode for the 12-week period and were expressed as kcal/g of rat. Single variables The total weight gain was defined by the difference between the basal weight and the final weight, in the 12th week. Mean total caloric intake per week, mean caloric intake from yogurt per week, and mean caloric intake from chow per week were calculated as the mean of 12 periods of week caloric ingestion corrected by rat weight (g) at the end of each week and expressed as kcal/g/wk.
Statistical analysis To evaluate the effect over time, Linear Mixed Model (Cleophas, Zwinderman, & van Ouwerkerk, 2010; Shek & Ma, 2011; West, 2009) with random slopes and weekly measurement as a repeated effect were applied to these variables. In these analyses, basal weight was used as a covariate, the fixed effects in the model were the groups and the interaction groupweeks, while rats and weeks were treated as a random effect. First-order autoregressive covariance structure [AR(1)] were used in all these analysis. For single variables, one-way analysis of variance (ANOVA) with the Dunnett’s test was used to identify differences between groups, and was also used to compare the basal weight, and percentage of intake of yogurt diets. Normality assumptions were verified by Kolmogorov–Smirnov test, with Lilliefors significance correction, and Shapiro–Wilk statistic. Reported values were mean ± SE, and p < 0.05 was set for all analyses. We used the SPSS™ software version 19 (IBM Corporation, Somers, NY) for statistical analyses. Results Cumulative weight gain Body weight was similar among groups in the beginning of the experiment, F (2, 26) = 0.09, p = 0.92(263.49 ± 10.60) (Table 1). Weight increased considerably in all groups. In the linear mixed model analysis, SAC and ASP presented greater weight gain than the SUC group (Fig. 1A): SAC (b = 3.82, SE = 1.31, p = 0.005) and ASP (b = 2.70, SE = 1.34, p = 0.048), F (2, 73.47) = 4.51, p = 0.01. In the ANOVA analysis, total weight gain was 28% greater in SAC (p < 0.003) and 20% in ASP (p < 0.04) in relation to SUC, F (2, 26) = 6.73, p = 0.004 (Table 1). Total caloric intake Cumulative total caloric intake (yogurt plus chow) was similar among all groups over the 12 weeks of the experiment, F (2, 27.17) = 0.12, p = 0.89 (Fig. 1B). Mean total week caloric intake corrected by rat weight was also similar in all groups, F (2, 26) = 0.04, p = 0.96 (Table 1). Calories from yogurt In the mixed model analysis, the cumulative caloric intake of sweetened yogurt during the study was lower in both SAC and ASP compared to SUC (b = 0.16, SE = 0.01, p < 0.001) but did not differ between SAC and ASP (Fig. 1D). Likewise, the weekly average intake of yogurt corrected by weekly weight was confirmed to be lower in ASP and SAC groups, F (2, 26) = 60.64, p < 0.001 after the ANOVA analysis (Table 1). One rat from the ASP group was excluded from the study due to an ingestion of less than 70% of yogurt, the remaining rats presented an average intake of 88.52% (±1.68), F (2, 26) = 2.23, p = 0.13 (Table 1). Calories from chow The cumulative caloric intake from chow during the study was significantly increased in SAC vs. SUC (b = 0.17, SE = 0.03, p < 0.001) and in ASP vs. SUC (b = 0.18, SE = 0.03, p < 0.001), F (2, 35.07) = 27.07, p < 0.001 (Fig. 1C). The mean weekly intake of chow corrected by weekly weight was 16% higher in both the SAC and ASP groups in relation to the SUC group, F (2, 26) = 40.85, p < 0.001 (Table 1). This decrease in calories from chow in SAC
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F. M. Feijó et al. / Appetite 60 (2013) 203–207 Table 1 Weight and caloric intake parameters.
Yogurt intake (% of total offered) Basal weight (g) Total weight gain (g) Mean total caloric intake per week (kcal/g/wk)a Mean calories from yogurt per week (kcal/g/wk)a Mean calories from chow per week (kcal/g/wk)a
SAC (n = 10)
ASP (n = 9)
SUC (n = 10)
88.21 ± 2.21 260.83 ± 21.97 175.31 ± 6.47** 1.45 ± 0.02 0.18 ± 0.01*** 1.27 ± 0.01***
84.28 ± 4.39 259.41 ± 18.81 164.28 ± 10.65* 1.45 ± 0.03 0.18 ± 0.01*** 1.27 ± 0.02***
92.66 ± 1.15 269.82 ± 15.54 137.37 ± 5.51 1.44 ± 0.02 0.35 ± 0.01 1.10 ± 0.02
Group labels: saccharin (SAC), aspartame (ASP) and sucrose (SUC). Data are Mean ± SE. Analysis by ANOVA with Dunnett test. Asterisks indicate comparison with SUC. p < 0.05. ** p = 0.005. *** p < 0.001. a Mean weekly intake throughout the 12 week study period. *
Fig. 1. Cumulative effect of the sweetened supplements [saccharin (SAC), aspartame (ASP) or sucrose (SUC)] on feeding pattern and body weight gain during 12 weeks of consumption. The groups that received nonnutritive sweeteners (NNSs) consumed more chow than the group offered sucrose (D), compensating the caloric deficit of these sweeteners (B). Thus, the total calorie consumption (yogurt plus chow) did not differ among the experimental groups (C). However, the NNS groups gained more weight than the group supplemented with sucrose (A). Analysis by linear mixed model, n = 10 (9 for ASP). Asterisks indicate comparison with SUC: p < 0.001; p = 0.005 and p < 0.05. Error bars indicate SE.
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and ASP compensated the increased in calories from sweetened yogurt resulting in a similar total cumulative caloric intake among all groups (Fig. 1B).
Discussion In the present study, Wistar rats receiving yogurt sweetened with either saccharin or aspartame and free chow diet during 12 weeks presented increased weight gain compared to animals receiving the same free chow diet plus yogurt sweetened with sucrose. Although saccharin and aspartame promoted relatively fewer calories from yogurt intake when compared to sucrose, increases in calories from chow intake effectively compensated for decreases in calories from yogurt, in such a way that there was a similar total caloric intake among all groups after the 12-week period of the experiment. These data are consistent with the hypothesis that animals adjust for calories consumed on one occasion by reducing their caloric intake on subsequent opportunities to eat (Booth, 1972; Foltin, Fischman, Moran, Rolls, & Kelly, 1990; Mazlan, Horgan, & Stubbs, 2006; Rowland, Nasrallah, & Robertson, 2005). However, it was surprising to find that the NNS were able to induce weight gain without an increase in total caloric intake, suggesting that other mechanisms such as decreased caloric expenditure may occur after NNS use. Our data are in accordance with the study of Swithers and Davidson (2008) who, using a similar protocol in Sprague–Dawley rats, observed that rats did gain more weight when using saccharin compared to glucose, despite a similar total energy intake after 5 weeks of experimenting. Our results are also in accordance with the Polyák et al. study (2010), in which CBA/CA inbred mice with free chow were given saccharin or aspartame added to the water and compared to controls using pure water, during 25 weeks. They observed that animals exposed to NNS in water gained more weight than those drinking non-sweetened water, despite a similar total caloric intake. These studies taken together are consistent in demonstrating that positive caloric compensation in chow intake occurs among NNS-exposed rats, but the increased weight gain observed in these animals was not explained by caloric intake, which was similar in all three groups. Possible explanations for weight-gain in saccharin and aspartame groups without increasing energy intake are still widely speculative. We could not attribute it to technical bias because we had strict dally control of chow and yogurt intake. A possible cause could be the reduction of energy expenditure induced by NNS. Swithers and Davidson (2008) demonstrated that body core temperature can be increased acutely minutes after the ingestion of glucose compared to non-sweetened fluid, an effect that was not observed when saccharin is used. The authors consider that food intake elicits a reflexive cephalic phase thermogenic response, and the heat production may be mediated by orosensory stimuli that signal the absorption of nutrients in the gut. However, there has not yet been any conclusion regarding a more prolonged effect induced by saccharin. A second possibility would be that hyperinsulinemia could be induced by saccharin, with consequent weight gain (Corkey, 2012). In vitro studies, using islet cells from Wistar rats were able to demonstrate an insulinotropic effect due to saccharin and other NNS, although not with aspartame. Insulin secretion increased three times from basal levels when cells were exposed to increased saccharin concentrations. The authors speculated that the bitter taste of saccharin, due to hexose pentaacetate esters could act through cellular mechanisms similar to that involved in bittertaste by taste buds. This effect is supported by the finding that islet cells are equipped with GUST27 receptors also expressed in taste
buds which are mediated by G proteins activated with calcium release. These mechanisms are linked to the blockage of potassium channels and activation of cAMP breakdown which regulates insulin secretion (Malaisse, Vanonderbergen, Louchami, Jijakli, & Malaisse-Lagae, 1998). Increased insulin secretion induced by NNS however still remains to be established in animal and human studies. We also considered the possibility of increased fluid intake and retention due to sodium content in saccharin and also to the sweet taste per se. However, in our study, yogurt caloric intake was decreased both in saccharin and in aspartame groups compared to sucrose, making this hypothesis less likely. Likewise, since weight gain was similar in saccharin and in aspartame groups, we believe that the impact of sodium (from saccharin) may have been of minor significance. Conclusion We observed significantly greater weight gain among Wistar rats fed diets supplemented with NNS – whether saccharin or aspartame – compared with sucrose. This increase was not the result of increased total caloric intake. This discrepancy raises the possibility that weight gain in NNS-fed rats might result from decreases in energy expenditure, which have been observed in other studies. Further studies are necessary to address energy expenditure after NNS exposure in rats as well as long-term clinical trials to evaluate weight gain increases in humans. References Anton, S. D., Martin, C. K., Han, H., Coulon, S., Cefalu, W. T., Geiselman, P., et al. (2010). Effects of stevia, aspartame, and sucrose on food intake, satiety, and postprandial glucose and insulin levels. Appetite, 55(1), 37–43. Appleton, K. M., & Blundell, J. E. (2007). Habitual high and low consumers of artificially-sweetened beverages. Effects of sweet taste and energy on shortterm appetite. Physiology and Behavior, 92(3), 479–486. Blackburn, G. L., Kanders, B. S., Lavin, P. T., Keller, S. D., & Whatley, J. (1997). The effect of aspartame as part of a multidisciplinary weight-control program on short- and long-term control of body weight. American Journal of Clinical Nutrition, 65(2), 409–418. Blundell, J. E., & Green, S. M. (1996). Effect of sucrose and sweeteners on appetite and energy intake. International Journal of Obesity and Related Metabolic Disorders, 20(Suppl 2), S12–17. Booth, D. A. (1972). Conditioned satiety in the rat. Journal of Comparative and Physiological Psychology, 81(3), 457–471. Canty, D. J., & Chan, M. M. (1991). Effects of consumption of caloric vs noncaloric sweet drinks on indices of hunger and food consumption in normal adults. American Journal of Clinical Nutrition, 53(5), 1159–1164. Cleophas, T. J., Zwinderman, A. H., & van Ouwerkerk, B. (2010). Clinical Research. A Novel Approach to the Analysis of Repeated Measures. American Journal of Therapeutics, 1, 2. Corkey, B. E. (2012). Banting lecture 2011. Hyperinsulinemia. Cause or consequence? Diabetes, 61(1), 4–13. Council, N. R. (2010). Guide for the care and use of laboratory animals (8 ed.). Washington, DC: National Academies Press. Drewnowski, A., Massien, C., Louis-Sylvestre, J., Fricker, J., Chapelot, D., & Apfelbaum, M. (1994). Comparing the effects of aspartame and sucrose on motivational ratings, taste preferences, and energy intakes in humans. American Journal of Clinical Nutrition, 59(2), 338–345. Foltin, R. W., Fischman, M. W., Moran, T. H., Rolls, B. J., & Kelly, T. H. (1990). Caloric compensation for lunches varying in fat and carbohydrate content by humans in a residential laboratory. American Journal of Clinical Nutrition, 52(6), 969–980. Fowler, S. P., Williams, K., Resendez, R. G., Hunt, K. J., Hazuda, H. P., & Stern, M. P. (2008). Fueling the obesity epidemic? Artificially sweetened beverage use and long-term weight gain. Obesity (Silver Spring), 16(8), 1894–1900. Lavin, J. H., French, S. J., & Read, N. W. (1997). The effect of sucrose- and aspartamesweetened drinks on energy intake, hunger and food choice of female, moderately restrained eaters. International Journal of Obesity and Related Metabolic Disorders, 21(1), 37–42. Malaisse, W. J., Vanonderbergen, A., Louchami, K., Jijakli, H., & Malaisse-Lagae, F. (1998). Effects of artificial sweeteners on insulin release and cationic fluxes in rat pancreatic islets. Cell Signal, 10(10), 727–733. Mazlan, N., Horgan, G., & Stubbs, R. J. (2006). Energy density and weight of food effect short-term caloric compensation in men. Physiology and Behavior, 87(4), 679–686. Phelan, S., Lang, W., Jordan, D., & Wing, R. R. (2009). Use of artificial sweeteners and fat-modified foods in weight loss maintainers and always-normal
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