Long-term intake of saccharin decreases post-absortive energy expenditure at rest and is associated to greater weight gain relative to sucrose in wistar rats

Background Non-nutritive sweeteners (NNS) have been associated with increased prevalence of obesity. In previous studies, we demonstrated that saccharin could induce an increase in weight gain either when compared to sucrose or to a non-sweetened control at a similar total caloric intake. These data raised the hypothesis that reduced energy expenditure (EE) could be a potential mechanism explaining greater weight gain with saccharin use in rats. The aim of the present study was to compare long-term energy expenditure at rest between rats using saccharin or sucrose and correlate it with weight gain. ﻿ Methods In the present study, we examine the potential impact of saccharin compared to sucrose in the EE of Wistar rats. In a controlled experiment of 17 weeks, 24 Wistar rats were divided into 2 groups: saccharin-sweetened yogurt (SAC) or sucrose-sweetened yogurt (SUC), plus a free chow diet. Only rats that consumed at least 70% of the offered yogurt were included. EE (kcal/day) was determined at rest through open circuit indirect calorimetry system in the early post-absorptive period with determinations of both VO2 consumption and CO2 production. Measurements were evaluated at baseline, 5 and 12 weeks of dietary intervention. Weight gain, caloric intake (from yogurt, from chow and total) were determined weekly. Results Body weight and EE were similar between groups at baseline: (p = .35) and (p = .67) respectively. At the end of the study, SAC increased total weight gain significantly more in relation to SUC (p = .03). Cumulative total caloric intake (yogurt plus chow) was similar between groups during the whole period (p = .54). At 12 weeks, the EE was smaller in SAC compared to SUC (p = .009). Considering both groups, there was a strong negative correlation between total weight gain and change in EE observed [r(20) = −.61, p = .003]. However, when analyzing the groups separately we found that SUC maintained this inverse correlation [r(8) = −.68, p = .03], while SAC did not [r(10) = −.33, p = .29]. Conclusion These data support the hypothesis that long-term use of saccharin may blunt post-absorptive EE at rest in Wistar rats, which is related to weight gain. On the other hand, long-term sucrose intake can increase energy expenditure in rats. This effect combined can explain, at least partially, the weight gain increases associated to saccharin in relation to sucrose in these animals.


Background
Non-nutritive sweeteners (NNS) have been associated with an increase in prevalence of obesity [1]. Epidemiological studies have shown that the use of NNS containing products correlates with the incidence of adiposity, metabolic syndrome (MS) [2,3], type 2 diabetes mellitus (DM2) and cardiovascular disease [4,5]. A clear cause and effect relationship, however, cannot be established.
The use of NNS, including saccharin, has been shown to interfere in the regulation of appetite and weight gain, which was demonstrated in some experimental studies and in randomized clinical trials [6][7][8]. Swithers and Davidson [9] conducted a series of experiments demonstrating that the use of saccharin resulted in reduced ability to compensate calories, providing a higher total caloric intake, greater weight gain and adiposity, when compared to the use of glucose.
In previous studies, we demonstrated that, in Wistar rats, saccharin can induce greater weight gain both in relation to sucrose [10] and to non-sweetened supplements [11], in spite of a similar total caloric intake. These experiments informed that, when rats receive saccharin-sweetened yogurt along with free chow intake, a compensatory increase in chow intake occurs. Nevertheless, the caloric deficit was perfectly compensated in a way that the total caloric intake (the sum of chow plus supplemented yogurt) was similar between groups. Despite this fact, significant increases in weight gain in the saccharin group could not be explained by increases in total caloric intake.
Mechanisms to explain why saccharin is associated with weight gain in rats are still not clear. One natural inference is to speculate that energy expenditure might be reduced in rats taking saccharin in relation to sucrose. Sprague-Dawley rats that were previously exposed to saccharin presented a smaller rise in the coretemperature after a standard meal when compared to rats pre-exposed to glucose, indicating that saccharin may interfere in heat production after meals and, therefore, in caloric expenditure [9].
In the present study, we hypothesized that the increase in long-term weight gain due to saccharin relatively to sucrose may be related to energy expenditure reduction induced by saccharin. We conducted a 17-week experiment with Wistar rats consuming either saccharin or sucrose along with a free chow diet, and compared the energy expenditure (EE) between groups at rest, in the post-absortive period at baseline, 5 and 12 weeks.

Study design and animals
In a 17-week controlled experiment, 24 adult male Wistar rats weighing~200 g were randomly divided equally into 2 groups according to the type of sweetener: saccharin-sweetened yogurt (SAC group) or sucrosesweetened yogurt (SUC group), besides standard chow and water ad lib. Total weight gain and caloric intake were measured weekly. EE was measured at baseline, 5 weeks and 12 weeks. The researchers were blinded to the assigned groups.
Animals were individually housed in translucent polypropylene plastic cages, with controlled humidity (65-70%) at room temperature (22 ± 1°C), maintained through a 12 h-light and dark cycle.
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 Committee for the Care and Use of Laboratory Animals [12] in the same degree as the Brazilian law for the scientific use of animals. Study protocol was approved by the ethics committee for experimental procedures of Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) and by the ethics committee for experimental procedures of Hospital de Clínicas de Porto Alegre (HCPA). This project was carried out in the Laboratory of Experimental Physiology at UFCSPA.

Dietary manipulation
All rats received standard chow pellets ad lib containing 2.93 kcal/g, (Nuvital CR-1, Nuvilab™). Additional chow was added to the top of the cages every 24 h as needed, and chow rest was weighed and registered once a week. For weighing, all chow was removed and weighed with an electronic precision scale (AS 5500, Marte™, SP, Brazil). The intake was calculated and registered weekly. The largest solid pellets were allocated in the grid feeders. A crumb collector was installed on the outside of the cage to minimize losses. Cages were carefully monitored for any evidence of chow spillage and crumbs were considered for the control of chow intake.
Sweetened yogurt supplements were prepared according to our former protocol [10,11]. In short, it included 20 ml of standardized low-fat yogurt (Nestlé™, SP, Brazil containing per unit of 160 g 57 kcal, 7.8 grams of carbohydrate, 5.8 grams of protein and 0 grams of total fat) to which it was added either dry sucrose (União™, SP, Brazil) or sodium-saccharin (Zero-Cal™, SP, Brazil) to include 20% sucrose solution and .3% saccharin solutions respectively. Yogurt supplements were offered 22 h daily, from 11 AM to 9 AM, 7 days a week during the whole experiment. Yogurt was offered in special bottles with adapted beaks to avoid leakage. Additionally, 15 ml of pure water was added into yogurt to dilute and adjust viscosity in order to allow easier drinking. Caloric densities of sucrose-sweetened yogurt and saccharinsweetened yogurt were: .63 kcal/ml (~170 kcal/wk) and .24 kcal/ml (~60 kcal/wk), respectively. Rats that drank less than 70% of sweetened yogurt were excluded from the study. The yogurt bottles were also checked for any sign of leaking or clogging.

Caloric intake determination
Mean caloric intake was determined by the week difference between the weight of offered chow and the weight of the chow left in the grid, corrected by the rat weight at the end of the week using the following formula: 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 17-week period and were expressed as kcal/g of rat.

Body weight
Rats were weighed weekly at the same time in the morning, using an electronic precision scale, suitable for weighing animals in motion (AS 5500, Marte™, SP, Brazil). The absolute weight gain after 17 weeks of dietary intervention was calculated as follows: Total weight gain g ð Þ ¼ body weight 17wkbody weight baseline Cumulative weight gain was calculated by the subtraction of the basal weight from the weight obtained every week, and expressed in grams.

Indirect calorimetric
The protocol of indirect calorimetric was adapted from Rodrigues et al. [13]. Measurements were made using an open-system oxygen gas chamber, at controlled room temperature of 21°C. The metabolic chamber was connected to a vacuum pump that generates 2.5 L/min O 2 flow. Oxygen flow was determined using O 2 and CO 2 analyzer (AVS Projects TM , São Carlos, SP, Brazil). Animals were placed into a gas chamber for 15 min to allow stabilization. After that, data started to be recorded for 5 min.
Tests were performed 2-3 h in the post-absorptive period at rest in the baseline, week 5 and week 12. All chow and yogurt were removed 2 h before experiments. All tests were performed in duplicate, being repeated in the immediate next following day. The mean of 2 results was considered for statistical analysis.

Determination of respiratory variables
Oxygen Consumption (VO 2 ) VO 2 was calculated according to the flow of environment air pumped into the metabolic chamber using the formula: "PF" is the measured flow through the metabolic chamber (2500 ml/min); "A" is the fraction of oxygen entering the chamber (environment air); "E" is the fraction of effluent oxygen; "BW" is the animal body weight in grams.

Carbone Dioxide Production (VCO 2 )
The VCO 2 production determination was obtained from the carbon dioxide removal detected by a sensor at the chamber output expressed in ml/kg/min. Both VO 2 and VCO 2 were calculated according to ACQUAD software.

Respiratory Exchange Ratio (RER)
The RER, also known as respiratory quotient, was calculated according to the formula: EE was estimated using the Weir equation [14] extrapolated to 24-h: Where VO 2 and VCO 2 were expressed in L/min and 1440 is the number of minutes in a day.
The change in EE was determined as the difference in EE at the end of 12 week and at baseline, calculated according to the formula:

Statistical analysis
The variables: total body weight gain, the area under the curve (AUC) , mean caloric intake of chow, yogurt, and total caloric intake (chow plus sweetened-yogurt) were compared using t-tests. ANOVA of repeated measures was used to compare the effect over time of cumulative weight gain, cumulative caloric intake (of sweetened-yogurt, of chow and total), RER, VO 2 , VCO 2 and EE.
We used Pearson's correlation coefficient (r) to evaluate the association between weight gain and change in energy expenditure over the entire experiment and it was presented graphically and numerically with r and p values. Reported values are mean and standard error ± SE, and p ˂ .05 was taken as significant for all analysis. Statistical analyses were performed using SPSS™ 21 (IBM Coorporation, Armonk, NY, USA). AUCs were obtained through software NCSS™ 2007 (Number Cruncher Statistical Systems, Kaysville, UT, USA), calculated by the trapezoidal method.

Caloric intake Sweetened-yogurt intake
Calories derived from sweetened-yogurt in SUC were 2.8 times greater than in SAC due to the higher caloric density in sucrose-sweetened yogurt; t(9.87) = 18.10, p < .001.

Chow intake
The mean caloric intake from chow tended to be greater in SAC than in SUC, corresponding to a 10% compensatory increment, although not statistically significant; t(20) = −1.56, p = .13. In the repeated measure analysis, the time x group interaction was not significant [F(1.42, 28.44) = 1.70, p = .21. (Fig. 2b).

Total caloric intake
Mean and Cumulative total caloric intake (chow intake plus sweetened-yogurt intake) was similar between groups in both analysis; t(20) = .63, p = .54 and F(1.38, 27.64) = 1.05, p = .34, respectively. This result indicates that chow intake in SAC was perfectly compensatory to the lack of calories in saccharin-sweetened yogurt (Fig. 2c).

Respiratory Exchange Ratio (RER)
At baseline, RER was similar between groups. At weeks 5 and 12 it was significantly lower in SAC than in SUC (p = .036 and p < .001, respectively); it was also confirmed when we analyzed AUC (0-12wk) ; t(20) = 2.82, p = .011.
When we compared week 12 from baseline there was an increase in 29% on SUC and a decrease in 15% on SAC, but without statistical difference (p = .06 and p = .67, respectively) (Fig. 3c).

Correlation between Total Weight Gain and Change in Energy Expenditure (EE)
Considering both groups together, there was a strong and negative correlation [r(20) = −.61, p = .003] between total weight gain and the change (delta) in energy expenditure from baseline to 12 weeks. However when analyzing the groups separately, we found that SUC maintained a strong and significant inverse correlation [r(8) = −.68, p = .03], while SAC did not [r(10) = −.33, p = .29] (Fig. 4).

Discussion
The present study confirmed our former observations [10,11] that rats consuming saccharin-sweetened yogurt had greater cumulative weight gain compared to rats eating sucrose-sweetened yogurt, when chow intake is not limited. Nonetheless, this increase was not related to differences in caloric intake, but rather to differences in EE between groups.
We observed that EE increased significantly (p < .001) from baseline in rats eating SUC, and decreased slightly (p = .04) in rats eating SAC, causing a significant difference between groups at 12 week (p = .009). This difference was primarily determined by a significant increase in carbon dioxide production (VCO 2 ) in SUC group at weeks 5 and 12 from baseline, combined with a stable oxygen consumption (VO 2 ) in both groups. The change in EE (difference from week 12 to week 0), presented as a strong and inverse correlation with the rate of weight gain, which was observed in SUC group (p = .03) but not in SAC group (p = .29). This suggests that increases in EE could explain the observed differences in weight gain between groups.
As rats were tested in the early post-absorptive period, an increase in EE was expected to be due to the nutrient absorption and digestive metabolism. So, it was interesting that, in contrast to SUC, EE in SAC group did not rise. This effect could be explained to either a lack of carbohydrate intake in SAC group or to a possible direct blunting effect in EE due to saccharin after food intake. As these differences were progressively seen along time, we inferred that they were likely to be a chronic-adaptive mechanism rather than an acute effect simply determined by a single meal deprived of sugar. It is also reasonable to speculate that metabolism adjustments might have occurred in rats using saccharin due to long-term sugar restriction. Future studies, however, are needed to clarify this hypothesis.
We were not able to find similar studies comparing EE between saccharin and sucrose in experimental models in literature. In an interesting study of Swithers et al., Sprague-Dawley rats fed for a long time with artificialsweetened yogurt presented smaller increases in body temperature after an acute single standard meal compared with rats that were fed with glucose-sweetened yogurt during the same period. These authors observed a possible blunting thermal response to food in rats eating saccharin, suggesting that metabolic adjustments of energy expenditure could occur, which is completely in agreement to the data in the present study [9]. Plausible biological mechanisms for explaining EE differences between groups are speculative. During aerobic glucose oxidation, oxygen consumption usually rises proportionally to carbon dioxide production [15] and a parallel rise between VO 2 and VCO 2 curves is expected. Nevertheless, as in the present study, VCO 2 production curve rose more steeply than the VO 2 curve, suggesting that the anaerobic glycolysis could be the predominant metabolic pathway in SUC rats. This is supported by the fact that RER was near 1 in SUC group and near 0.7 in SAC group, suggesting that carbohydrates were the main fuel used by SUC rats, while fat was predominantly used by SAC rats.
This study has some important strengths. We conducted the experiment in the early post-absorptive state, which is important to minimize the animal stress and the ketogenesis due to the prolonged fasting state, which might interfere in results. It is also important that we measured carbon dioxide directly, and all respiratory variables were measured in duplicate, obtained in the following day, which considerably reduced EE determination variability. Finally, rats were kept in individual cages, which allowed accurate control of caloric intake. One potential limitation in this study was that the Weir formula was originally designed to calculate energy expenditure in humans, although it was later popularized for experimental research [14].

Conclusion
We concluded that long-term saccharin use may decrease energy expenditure at rest in the post-absorptive, period relative to sucrose in Wistar rats. On the other hand, long-term sucrose intake can increase energy expenditure in rats. This combined effect could explain, at least partially, the weight gain associated to saccharin in relation to sucrose in these animals.