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. 2020 Sep 10;58(6):2349–2359. doi: 10.1007/s13197-020-04746-2

Effects on weaned male Wistar rats after 104, 197, and 288 days of chronic consumption of nutritive and non-nutritive additives in water

Samuel Mendoza-Pérez 1, Mauricia Betzabeth Guzmán-Gómez 1, Rolando Salvador García-Gómez 1, Guillermo Ordaz-Nava 2, María Isabel Gracia-Mora 3, Lucía Macías-Rosales 3, Héctor Morales-Rico 3, Gerardo Salas-Garrido 4, María del Carmen Durán-Domínguez-de-Bazúa 1,✉
PMCID: PMC8076370  PMID: 33967331

Abstract

Abstract

It has been suggested that the consumption of artificial sweeteners is related to greater body mass gain and diverse metabolic alterations. In this study, the effect of chronic consumption of nutritive sweeteners (fructose 7% and sucrose 10%) and non-nutritive or low-calorie sweeteners (acesulfame 0.015%, aspartame 0.3%, aspartame:acesulfame mixture 0.04%, saccharin 0.3%, and sucralose 0.19%), in drinking water, as well as a control group with no sweeteners, was evaluated. Body mass gain and glucose, insulin, triglycerides, and total cholesterol levels in blood were the parameters considered. For this purpose, 120 weaned male Wistar rats of the HsdHan:WIST line were used, 15 per group in first stage, then 10 and 5 per group for 2nd and 3rd stages, respectively. Body mass gain, food intake, and beverage consumption were daily quantified. After 104, 197, and 288 days of experimentation the concentrations of glucose, triglycerides, cholesterol, and insulin were determined. Only in the first stage there were significant differences in the body mass gain. In the three stages there were significant differences in the patterns of beverage intake and food consumption. The trend was the same in all 3 stages: rats drank more in the groups of drinks sweetened with nutritive sweeteners and ate more in the groups that drank non-nutritive artificial sweeteners. Regarding the biochemical profile, no sweetener either nutritive or non-nutritive caused that the serum levels of glucose, triglycerides, and cholesterol were at pathological levels. It is concluded that the sweeteners by themselves can modify certain biochemical parameters but not at a pathological level. Furthermore, by themselves they are not capable of triggering excess of body mass or obesity in the early and medium stages of life when consumed together with a balanced diet.

Graphic abstract

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Keywords: Body mass gain, Non-nutritive sweeteners, Serum glucose, Triglycerides, Total cholesterol, Insulin

Introduction

The prevalence of excess of body mass and obesity has increased alarmingly in recent decades. As a counter measure, the use of artificial non-nutritive sweeteners has been suggested. Nevertheless, non-nutritive sweeteners have been recently introduced into the human diet, so there is still controversy about its use. It has been suggested that the consumption of products containing high-intensity sweeteners is related to a higher prevalence of obesity (Fowler et al. 2015; Swithers et al. 2013). Several studies indicate that the consumption of beverages sweetened with high-intensity sweeteners make people prone to greater risk of the development of the metabolic syndrome, including type II diabetes and cardiovascular diseases (Bhupathiraju et al. 2013; Cohen et al. 2012; Fagherazzi et al. 2013; Gardener et al. 2012; Nettleton et al. 2009; Sakurai et al. 2013). Likewise, chronic consumption of a sweet/caloric imbalance triggers diverse responses such as: (a) increased motivation to eat, (b) positive energy balance, (c) weak predictive relationship between sweet taste and caloric consequences, and (d) greater body mass gain (Davidson et al. 2011; Swithers and Davidson 2008; Swithers 2015; Wang et al. 2016).

In a series of experiments, it was shown that saccharin could reduce the learning effectiveness associated between sweet taste and post-ingestion energy release, resulting in an increase body mass (Davidson et al. 2011). Similar studies performed by de Feijó et al. (2013) showed that saccharin and aspartame increased body mass gain compared to sucrose, but without differences in energy intake. Later, this was corroborated by Foletto et al. (2016), who demonstrated that saccharin induced greater body mass gain but did not increase energy intake. Additionally, it has been linked the consumption of artificial sweeteners with alterations in intestinal microbiota. The experiments of Suez et al. (2014) showed that the consumption of artificial sweeteners promoted the development of glucose intolerance through the induction of alterations in the composition and function of the intestinal microbiota. On the other hand, Palmnäs et al. (2014) showed that the consumption of aspartame modified the composition of the microbiota, increasing the abundance of Enterobacteriaceae and Clostridium.

Some studies conclude that the consumption of artificial sweeteners does not have any impact on body mass gain (Boakes et al. 2016; Markey et al. 2016), and has no effect on saciety (Sylveltsky et al. 2011). Other research indicates that the predictive interference between sweet taste and caloric prediction only occurs in sweet diets (Davidson et al. 2011). However, despite the increase in the use of artificial sweeteners and their supposed benefits in controlling energy intake, the prevalence of excess of body mass and obesity continue to increase (Pearlman et al. 2017). So, the effect of artificial sweeteners on appetite and body mass is still inconclusive.

Therefore, the aim of the present study was to examine the effects of chronic consumption of nutritive (sucrose, glucose, and fructose) and hypocaloric sweeteners (acesulfame K, aspartame, aspartame and acesulfame mixture, saccharin, and sucralose) in drinking water on body mass gain, beverage intake, food intake, basal levels of serum glucose, triglycerides, total cholesterol, and insulin; using male Wistar rats in their first stages of life after weaning. There were 3 experimental stages corresponding to days 104, 197, and 288 after weaning.

Materials and methods

Model animals

Male rats (n = 120) just weaned (body mass between 35 and 45 g) of the HsdHan:WIST line from the supplier Harlan Laboratories were used to perform the experiment for 288 days. The rats were randomly divided into 8 groups (n = 15 per group): Acesulfame K, aspartame, mixture of aspartame and acesulfame, saccharin, sucralose, fructose, sucrose, and control (drinking water without sweetener). The animals were housed in individual transparent polysulfone cages, with controlled humidity (65–70%), ventilation and temperature (22 ± 1 °C) and maintained during 12 h light-dark cycles. Procedures were conducted in accordance with guidelines of the National Research Council for the Care and Use of Laboratory Animals (National Research Council 2011). This study’s protocols were approved by the Institutional Committee for the Care and Use of Laboratory Animals. Biochemical parameters were determined for the 3 experimental stages (104, 197, and 288 days after weaning). In order to obtain the blood samples, five specimens were taken at the end of the 15 weeks, first stage, for euthanasia. Then, after the second stage, other five specimens were separated. Five specimens remained for the last stage. These exemplars were randomly taken. The rats were daily weighed at the same time and in the same order, using a precision electronic balance (SPE601, OHAUS™) throughout the experimental period. Cumulative body mass gain was calculated by the subtraction of the basal body mass from the mass obtained every day. The cumulative food intake and the cumulative water intake were calculated by the sum of the daily consumptions of each item. For the groups that ingested nutritive sweeteners the total energy input calculation was carried out by adding the energy provided by food as well as by the drinks.

Sweetened beverages

Supplied drinks had the following concentrations: Acesulfame 0.015%, aspartame 0.3%, mixture of aspartame 0.04% and acesulfame 0.04%, saccharin 0.3%, sucralose 0.19%, sucrose 10%, and fructose 7%. The concentrations of solutions of artificial sweeteners were determined as isosweet to sucrose 10% which was taken as a reference. All values are below the acceptable daily intakes established for each of them for humans. The drinks were supplied without time restriction. Every 24 h it the amount of food and drinks consumed were quantified. Drinks were replenished daily with freshly prepared solutions in clean bottles. The water intake was determined by subtracting the remaining amount of the daily supplied amount (250 mL). The presence of any possible spill that could affect the results was also daily verified. The energy contribution of nutritive beverages (fructose and sucrose) was 1.16 kJ/mL for fructose beverage and 1.67 kJ/mL for sucrose beverage (Belitz et al. 2009). For the non-nutritive sweeteners its caloric contribution was considered negligible (Jürgens et al. 2005).

Diet supply

The diet supplied to the 8 groups during the 288 days of experimentation was the large solid pellets Teklad Global 2018S© diet (Envigo™ Company 2016). The diet was supplied without time restrictions during the 228 days of this research. Grid feeders were located on the outside of the cages of the rats. The energy contribution of Teklad Global 2018S diet was 13 kJ/g. The control of food intake was daily conducted by subtraction of the quantity remaining (g) from the quantity daily supplied (100 g) using an electronic precision balance (SPE601, OHAUS™).

Euthanasia

Humanitarian euthanasia of a third of the specimens was performed on days 104, 197, and 288. Euthanasia was carried out after a 12 h fasting. To do this, 5 specimens per group, with each rat individually placed in a chamber with air rich in CO2 (70% minimum), were put to sleep. Then, they were decapitated with a guillotine for rodents. All experimental procedures were conducted in accordance with the guidelines of the National Research Council for the Care and Use of Laboratory Animals (National Research Council 2011).

Biochemical serum measurements

At the end of the experiment and 12 h fasting, blood samples were collected in specific tubes with a separator gel (SST 368159, BD Vacutainer) with the aid of individual polypropylene funnels. The tubes containing the samples were left to allow clotting for 30 min. Centrifugation was then used (3000 rpm for 15 min) collecting serum in 2 mL Eppendorf tubes to be stored and immediately frozen at − 70 °C for later analysis.

Insulin was determined by fluoroimmunoassays quantifying the mean fluorescence emitted by Luminex XMAP technology (MAGPIX®, LUMINEX), with kits provided by Millipore™, using Milliplex MAP Kit Rat Metabolic Magnetic Bead Panel Cat. #RMHMAG-84K.

Serum glucose, triglyceride and total cholesterol levels were determined by means of the automatic analyzer Cobas C111 (Roche™ brand).

All determinations were performed in triplicate.

Statistical analyses

The software StatGraphics Centurion XVI (StatPoint Technologie, Inc, Warrengton, VA, US) was used for the data statistical analyses. GrandPad Prism 6 software (GraphPad Software Inc., La Jolla, CA, US) produced the graphs. Analysis of variance (ANOVA) of one way or two ways was applied to the data gathered. The experimental design was multifactorial involving two categorical factors: (a) sweetener factor and (b) Age factor. To identify the differences between groups the Duncan test was used as a “post hoc” test. The assumptions of normality and homoscedasticity were verified with the Shapiro–Wilk test and the Levene test, respectively. Reported values are means and standard deviation (SD). The p < 0.05 was taken as significant for all analyses.

Results and discussion

Body mass gain

Figure 1a shows the cumulative mass gain curve over the 288 days and Table 1 shows the body mass gains from weaning to performed euthanasia. For each period, 104, 197, and 288 days, the statistical analysis of the body mass gain was done. The results obtained are discussed below. In the first stage (104 days) there were statistically significant differences (p < 0.05). The group that presented the greatest body mass gain was the group that ingested fructose (422.9 ± 47.7 g) and the group that had the lowest body mass gain was the group that ingested sucrose (378.2 ± 45.1 g). The above agrees with what was reported by Martínez et al. (2010). Additionally, it has been reported in the literature that the consumption of fructose favors a greater accumulation of adipose tissue and the development of non-alcoholic fatty liver (Abdelmalek et al. 2010; Bocarsly et al. 2010; Nomura and Yamanouchi 2012; Pollock et al. 2011; Tappy and Lê 2012).

Fig. 1.

Fig. 1

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a Average daily body mass of each sweetener during the experimentation stages (to see the graph more clearly here are shown every 5-day average data). b Average of the daily food intake in the three experimental stages. Mean ± SD. Different letters indicate significant differences a, b, c; A, B, C; A′, B′, C′. Duncan test (p < 0.05), n = 15 at 104 days, n = 10 at 197 days and n = 5 at 288 days. c Average of the daily beverage intake in the three experimental stages. Mean ± SD. Different letters indicate significant differences a, b, c, d; A, B, C, D, E; A′, B′, C′. Duncan test (p < 0.05), n = 15 at 104 days, n = 10 at 197 days and n = 5 at 288 days

Table 1.

Average body mass of the specimens groups for each period of experimentation

Sweetener 0 days (n = 15) 104 days (n = 15) 197 days (n = 10) 288 days (n = 5)
Mean ± SD Mean ± SD Mean ± SD Mean ± SD
Control 37.3 ± 6.4 406.6 ± 30.9 bc 465.5 ± 51.1 467.5 ± 18.3
Acesulfame K 38.2 ± 6.3 405.9 ± 43.7 abc 480.7 ± 55.0 483.9 ± 69.2
Ace:Asp mix 36.4 ± 6.9 415.5 ± 46.1 c 482.3 ± 53.0 470.2 ± 46.6
Aspartame 37.6 ± 4.5 402.7 ± 38.7 ab 482.0 ± 74.9 450.9 ± 32.4
Saccharin 38.0 ± 7.0 411.7 ± 45.8 bc 486.3 ± 71.5 485.1 ± 36.2
Sucralose 37.5 ± 5.8 391.5 ± 41.3 a 477.8 ± 41.9 486.2 ± 30.7
Fructose 37.8 ± 5.0 422.9 ± 47.7 c 463.5 ± 55.8 476.3 ± 54.4
Sucrose 37.1 ± 5.4 378.2 ± 45.1 a 451.9 ± 83.4 441.0 ± 39.8
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n = number of specimens. SD standard deviation. Means with the different letter within a column (a, b, c) are significantly different. In second stage (197 days) and third stage (288 days) there were no significant intergroup differences, Duncan test (p < 0.05). The bold numbers indicate the lowest parameter values in each period. The italic numbers indicate the highest value in each period

In contrast, in the second stage (197 days) the statistical analysis indicated that there were no significant differences in body mass gain. However, the group with the highest body mass gain was the saccharin group (486.3 ± 71.5 g). Additionally, the group with the lowest body mass gain at this stage was the sucrose group (451.9 ± 83.4 g). This agrees with the results of the research by de Feijó et al. (2013) and Foletto et al (2016) who, when comparing saccharin and glucose, found that the rats that ingested saccharin had a greater body mass gain compared to those that ingested glucose.

Finally, in the third stage (288), as in the second stage, the statistical analysis indicated that there were no significant differences in body mass gain. In this third stage, the group that presented the highest body mass gain was the group that drank sucralose (486.2 ± 30.7 g). In contrast, the group with the lowest body mass gain was the group that drank sucrose (441.0 ± 39.8 g), a trend observed in the previous two stages.

When the mass gain data is correlated with the energy intake data (Table 4), it is interesting to note that the groups of caloric sweeteners, despite ingesting more energy, did not produce an increase in body mass. These results agree with those found by Sheludiakova et al. (2012). As mentioned by these authors (Sheludiakova et al. 2012) it is necessary to perform a body composition analysis because probably despite the absence of a greater body mass gain, there may be an increase in the amount of adipose tissue in the specimens. Also, a previous experiment gave the same results (Martínez et al. 2010), and in that paper there were some references that mentioned that glucose is not metabolized in the same manner as fructose.

Table 4.

Average daily energy intake (kJ/rat) of the groups for each stage of experimentation

Sweetener 104 days (n = 15) 197 days (n = 10) 288 days (n = 5)
Mean ± SD Mean ± SD Mean ± SD
Control 317.1 ± 30.8 abc 302.4 ± 13.5 a 321.3 ± 17.0 ab
Acesulfame K 298.6 ± 19.6 c 304.9 ± 19.1 a 309.5 ± 25.3 ab
Ace:Asp mix 340.5 ± 28.9 bcd 301.9 ± 11.4 a 298.8 ± 39.1 a
Aspartame 287.7 ± 23.5 a 305.9 ± 28.7 ab 321.7 ± 50.8 ab
Saccharin 304.4 ± 39.7 ab 313.5 ± 23.1 ab 304.3 ± 23.1 ab
Sucralose 281.9 ± 46.0 a 292.1 ± 14.3 a 305.9 ± 13.8 ab
Fructose 361.7 ± 29.5 d 330.9 ± 14.0 b 315.9 ± 28.9 ab
Sucrose 354.4 ± 21.5 cd 360.2 ± 29.7 c 339.6 ± 23.1 b
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n = number of specimens. SD standard deviation. Means with the different letter within a column are significantly different Duncan test (p < 0.05). The bold numbers indicate the lowest parameter values in each period. The italic numbers indicate the highest value in each period

Food intake

Table 2 shows the average of the amount of food daily consumed per group in the three experimental stages according to the type of sweetener. In each of the three stages, the analysis of variance indicated the existence of significant differences (p < 0.05). In general, the trend observed in the three periods was as follows: The group that drank sucrose ingested the least amount of food, followed by the fructose group; the rest of the groups ingested the same amount as the control group. This general trend is like the one reported by Martínez et al. (2010).

Table 2.

Average of the daily food intake (g) in the three experimental stages

Sweetener 104 days (n = 15) 197 days (n = 10) 288 days (n = 5)
Mean ± SD Mean ± SD Mean ± SD
Control 22.28 ± 2.17 21.24 ± 0.94 22.58 ± 1.21
Acesulfame K 20.98 ± 1.39 21.44 ± 1.34 21.76 ± 1.77
Aspartame 20.22 ± 1.64 21.50 ± 2.03 22.62 ± 3.57
Mix Ace:Asp 23.94 ± 2.02 21.22 ± 0.78 21.06 ± 2.65
Saccharin 21.42 ± 2.81 22.02 ± 1.62 21.42 ± 1.24
Sucralose 19.8 ± 3.25 20.52 ± 1.01 21.50 ± 0.96
Fructose 19.84 ± 2.00 16.46 ± 0.69 16.46 ± 1.41
Sucrose 14.34 ± 1.28 14.34 ± 2.66 11.78 ± 1.55
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n = number of specimens. SD standard deviation. The bold numbers indicate the lowest parameter values in each period. The italic numbers indicate the highest value in each period

The results of the multiple comparisons by means of the Duncan test are illustrated in Fig. 1b. For the first stage (104 days), Duncan's test indicated the existence of three homogeneous groups designated by the letters a, b and c. Groups that do not share the same letters differ statistically. In the first stage, the control group (bc) only differed from the sucrose group (a). In the second stage there were also three homogeneous groups designated by the letters A, B, and C. Only the fructose (B) and sucrose (A) groups differed from the control (C). Also, in the third stage, the Duncan test found three homogeneous groups A′, B', and C′. As in the second stage, only the sucrose (A′) and fructose (B′) groups differed from the control (C′). As previously mentioned, in the 3 stages, only the groups of rats that ingested nutritive sweeteners (fructose and sucrose) presented significant differences with respect to the control group. No group of non-nutritive sweeteners differed in the amount of food ingested with respect to the control group. The above agrees with the reported by Boakes et al. (2016), de Feijó et al. (2013), Foletto et al. (2016), and Polyák et al. (2010). Hence, only nutritive sweeteners cause a feeling of satiety. The rats try to regulate their energy intake by reducing the food eaten when finding a source of energy easier to digest. As described below, the trend for beverage consumption was opposite to food intake.

Beverage intake

As already mentioned, the amount of beverage intake was higher in the nutritive sweetener groups (sucrose and fructose) (Fig. 1c). Of the two groups of nutritive sweeteners, preferred first sucrose and in second preference was fructose in the 3 stages. Table 3 shows the average of the food daily consumed in the three experimental stages according to the type of sweetener. In each of the three stages, the analysis of variance indicated the existence of significant differences (p < 0.05). The results of the multiple comparisons by means of the Duncan test are illustrated in Fig. 1c. Duncan test indicated the existence of homogeneous groups designated by letters. The homogeneous groups of the first stage were designated by the letters a, b, c, d. For the second stage the homogenous groups were designated with the letters A, B, C, D, E. Finally, for the third stage they were designated as A′, B′, C′.

Table 3.

Average of the daily beverage intake (mL) in the three experimental stages

Sweetener 104 days (n = 15) 197 days (n = 10) 288 days (n = 5)
Mean ± SD Mean ± SD Mean ± SD
Control 42.56 ± 4.42 45.78 ± 5.19 49.32 ± 10.23
Acesulfame K 53.2 ± 5.72 60.48 ± 12.18 51.24 ± 13.03
Aspartame 40.5 ± 7.54 56.76 ± 26.55 33.88 ± 6.79
Mix Ace:Asp 58.28 ± 7.90 50.96 ± 15.5 44.36 ± 7.59
Saccharin 48.10 ± 4.96 65.22 ± 6.35 51.3 ± 11.4
Sucralose 44.64 ± 11.98 39.6 ± 1.65 49.56 ± 10.93
Fructose 72.52 ± 5.97 88.24 ± 9.62 73.5 ± 9.29
Sucrose 90.06 ± 9.52 93.24 ± 19.92 103.02 ± 10.8
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n = number of specimens. SD standard deviation. The bold numbers indicate the lowest parameter values in each period. The italic numbers indicate the highest value in each period

In the first stage (104 days) the groups that differed significantly from the control (42.56 ± 4.42 mL) were sucrose (90.06 ± 9.52 mL), fructose (72.52 ± 5.97 mL) and the mixture of aspartame with acesulfame (58.28 ± 7.90 mL). On the other hand, in the second stage (197 days) the groups that ingested sucrose, fructose, acesulfame K, and saccharin were different from the control group. Finally, in the third stage it was observed that 2 groups ingested significantly more drink and one ingested less. The groups that ingested the most were, as in the previous two stages, the sucrose (103.02 ± 10.8 mL) and fructose (73.5 ± 9.29 mL) groups. In contrast, the group that drank the least was the aspartame group (33.88 ± 6.79 mL). Rats showed a low preference for aspartame which has already been reported by Sclafani and Abrams (1986). The greater preference for sucrose agrees with that reported by Martínez et al. (2010) as mentioned above.

Energy

Table 4 shows the Average daily energy intake (kJ/rat) of the groups for each period of experimentation according to the type of sweetener. In each of the three stages, the analysis of variance indicated the existence of significant differences (p < 0.05). The results of the multiple comparisons by means of the Duncan test indicated the existence of homogeneous groups designated by letters a, b, c, etc. In the first stage, no group differed from the amount of energy ingested with respect to the control except the fructose group. In the second stage, only the sucrose group ingested more energy than the control. Finally, in the third stage, no group differed significantly with respect to the control. In general, in all three stages, nutritive sweeteners groups had the highest energy intake. However, over time these differences were reduced and even in the third experimental stage there were no significant differences in energy intake between the groups of nutritive and non-nutritive sweeteners. This suggests that rats were probably able to regulate energy intake after a certain time and despite drinking caloric beverages. These findings are very relevant since the consumption of nutritive sweeteners has been associated with a higher energy intake. This can be seen when reviewing the data on the food eaten (Table 2); the food eaten by groups of nutritive sweeteners decreased in the last two stages, thus compensating for the energy coming from the drink (Stanhope et al. 2011).

Biochemical serum measurements

Fasting (12 h) serum concentrations of glucose, triglycerides, total cholesterol and insulin were determined after 120 days of experimentation.

Serum glucose levels

Regarding serum fasting glucose levels, significant differences were found in the three stages (Fig. 2a). Duncan test indicated the existence of homogeneous groups designated by the letters. The homogeneous groups of the first stage were designated by the letters a, b, c, d. For the second stage the homogenous groups were designated with the letters A, B, C. Finally, for the third stage they were designated as A′, B′, C′, D′. It is emphasized that in none of the 3 stages were these serum glucose values in a level that could be considered pathological (Envigo™ Company 2019). However, in the first stage the groups that ingested saccharin, sucralose, and aspartame differed from the control (p < 0.05). In the first stage, only the aspartame group had serum glucose levels higher than the control. The above agrees with that reported by Helal et al. (2019); they indicate that high blood glucose levels in rats that consumed aspartame can be attributed to the formation of amino acids from aspartame, where phenylalanine is considered to be both glucokinetic and ketone, while aspartic acid aspartic acid is a glycogenic amino acid and therefore can be converted to glucose in gluconeogenesis. Helal et al. (2019) also indicate that aspartame could stimulate glycogenolysis. According to the authors, these mechanisms are still speculative and should be further studied.

Fig. 2.

Fig. 2

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a Average of fasting blood glucose in the three experimental stages. Mean ± SD. Different letters indicate significant differences a, b, c, d; A, B, C; A′, B′, C′, D′. Duncan test (p < 0.05), n = 15 at 104 days, n = 10 at 197 days and n = 5 at 288 days. b Average of triglycerides levels in the three experimental stages. Mean ± SD. Different letters indicate significant differences a, b, c; A, B, ns no statistical difference. Duncan test (p < 0.05), n = 15 at 104 days, n = 10 at 197 days and n = 5 at 288 days. c Average of total cholesterol levels in the three experimental stages. Mean ± SD. Different letters indicate significant differences a, b, ns no statistical difference. Duncan test (p < 0.05), n = 15 at 104 days, n = 10 at 197 days, and n = 5 at 288 days

In the second experimental stage, all groups differed from the control except for the saccharin group. The above agrees with the observed by Foletto et al. (2016) who did not find differences in serum glucose levels between the groups of saccharin and control.

In the third stage, despite significant differences, no group differed from the control group. These results contrast with those obtained by Suez et al. (2014) who found that saccharin induced glucose intolerance due to alterations in the intestinal microbiota. As mentioned, the effect of sweeteners on serum glucose levels has not yet been fully elucidated, and further research on this topic should be conducted.

Triglycerides levels

Figure 2b shows the serum triglyceride levels in the 3 experimental stages. Analysis of variance and Duncan's test indicated that there were intergroup differences in stage 1 and stage 3. In the first stage, the homogeneous groups were designated with the letters a, b, c. While in the third stage they were designated with the letters A, B. In the first stage (104 days), the group that drank sucralose had levels of triglycerides significantly lower than the control. No group presented levels higher than the normal limits (< 200 mg/dL) nor were they significantly higher than the control group. In contrast, in the second stage (197 days) there were no significant intergroup differences. In the research by Saada et al. (2013) both healthy and 10-month-old diabetic rats showed a significant decrease in triglyceride levels. They suggest that the decrease in triglycerides could be attributed to the effect of sucralose on peroxisome proliferator-activated alpha receptors (PPAR-α), thus increasing the expression of lipoprotein lipase. Likewise, no group reached levels that could be considered pathological. The aforementioned shows that the consumption of sweeteners together with a balanced diet did not significantly alter triglyceride levels. On the other hand, in the third stage (288 days), only the groups that ingested sucrose, fructose and acesulfame had significantly higher triglyceride levels compared to the control group. Consumption of simple carbohydrates is associated with alterations in energy, glucose, and triglyceride homeostasis. Disproportionate glucose, fructose, and sucrose intake promotes de novo lipogenesis in the liver. This situation might lead to the development of hypertriglyceridemia and the development of a non-alcoholic fatty liver (Bocarsly et al. 2010; Lowndes et al. 2014; Sheludiakova et al. 2012; Tappy and Lê 2012). Also, various investigations have indicated that fructose over-stimulates hepatic lipogenesis (Abdelmalek et al. 2010; Bocarsly et al. 2010; Dirlewanger et al. 2000; Nomura and Yamanouchi 2012), which would explain that despite being consumed to a lesser extent that sucrose raised the levels to the same degree as this carbohydrate on stage 2 and 3. According to Tappy and Lê (2012), “the excessive consumption of fructose leads to the development of hepatic steatosis, favors visceral accumulation of fat and develops hypertriglyceridemia”. These findings encourage the continuation of this research to investigate the possible synergistic effects that exist between the consumption of unbalanced diets and the intake of caloric and non-caloric sweeteners as well as to elucidate the possible mechanisms that cause the observed effects.

Total cholesterol

Figure 2c shows the average of total cholesterol levels in the three experimental stages. The analysis of variance indicated that there were no significant differences in stages 1 and 3 (p > 0.05). In contrast, in stage 2, the groups that presented significantly higher levels than the control group were acesulfame K, aspartame, fructose, saccharin, and sucralose. However, in no case were normal levels exceeded (< 200 mg/dL). The above shows that the consumption of sweeteners, nutritive or hypocaloric, by themselves are not capable of altering the levels of total cholesterol to a pathological level. Research in humans has indicated that fructose consumption increases total cholesterol and LDL cholesterol levels in men and women (Jameel et al. 2014; Stanhope et al. 2011; Zhang et al. 2013). This trend appears to be the same as that observed in the present study for rats, since rats which consumed fructose, had the highest total cholesterol levels. On the other hand, Prokić et al. (2015) concluded that “the intake of aspartame induces oxidative stress in rat erythrocytes by altering the glutathione redox status. Together with the changes in biochemical and lipid parameters and indicators of liver damage, our findings suggest that chronic use of aspartame can lead to the development hyperglycemia, hypercholesterolemia”. This research showed that in the second stage aspartame significantly increased cholesterol levels. However, no pathological levels were reached. The mechanisms pointed out by Prokić et al. (2015) should be investigated in further research for aspartame and the rest of the sweeteners that increased cholesterol levels (acesulfame K, fructose, saccharin, and sucralose).

Insulin levels

Insulin is vital for regulating blood glucose levels and has many effects on the body. Figure 3 shows the average serum insulin levels that the groups had in each of the 3 experimental stages. The results of the Duncan test indicated the existence of two homogeneous groups in the first stage (a, b), three in the second stage (A, B, C), and two in the third stage (A', B'). In the 3 experimental stages there were significant differences in insulin levels (p < 0.05). However, in the first stage, no group differed in insulin levels with respect to the control. Regarding the trend of the group that ingested saccharin the results agree with those reported by Foletto et al. (2016), who found that although saccharin induced greater body mass gain in male Wistar rats, it did not alter insulin levels. The trend changes regarding the second stage. In this period, the groups that ingested sucrose and the mixture of aspartame and acesulfame had statistically higher levels compared to the control. However, this is consistent with glucose levels. In the second period, the groups of sucrose and mixture of aspartame with acesulfame were the groups that presented the highest serum glucose levels. Finally, in the third stage, the trend is like the first. That is, despite the existence of significant differences, no group differed statistically from the control group. However, now the groups that ingested sucrose and sucralose were the groups with the highest insulin levels. This is in accordance with the serum glucose levels observed in the third stage, where it is observed that no group differed from the control. Additionally, an analysis of variance indicated that the general insulin levels were similar between stages 1 and 3; in stage 2 the levels were, in general, significantly higher (p < 0.05). The results agree with that reported by Ford et al. (2011). They reported that oral stimulation with sucralose in humans had no effect on GLP-1, insulin, or appetite. Nevertheless, an in vitro study revealed that sucralose induces insulin secretion by Ca2+ and cAMP-dependent mechanisms (Nakagawa et al. 2009). As it is appreciated, there is still no consensus between the effects of sweeteners on insulin release and the possible biochemical mechanisms. The clinical significance of the results of this research needs to be investigated in longer follow-up studies.

Fig. 3.

Fig. 3

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Average insulin levels in the three experimental stages. Mean ± SD. Different letters indicate significant differences a, b; A, B, C; A′, B′. Duncan test (p < 0.05), n = 15 at 104 days, n = 10 at 197 days and n = 5 at 288 days

Conclusion

Experimental data indicated that, in terms of body mass gain in male rats, only in the first stage there were significant differences. There were significant differences in the serum levels of glucose, triglycerides, cholesterol and insulin. However, neither of the cases exceeded normal ranges nor did these metabolic alterations lead to the development of any pathology in this study period.

Regarding food and drink intake patterns, it was found that the groups of nutritive sweeteners consumed the least amount of food due to the higher intake of energy from drink, as expected.

Correlating the gain in mass with the energy ingested, it is concluded that a greater intake of energy does not necessarily reflect a greater body mass gain. But there is a positive correlation between energy intake and serum triglyceride levels. In general, the groups with the highest energy intake had the highest serum levels of triglycerides. No correlation was observed between triglyceride levels and cholesterol levels. Also, no clear correlation was observed between drink intake and serum glucose and insulin levels. In other words, the groups that drank the most calories did not have higher glucose levels. Likewise, there was also no clear correlation between insulin levels and triglyceride and cholesterol levels.

So, results obtained indicate that artificial sweeteners are not metabolically inert. They caused significant differences in glucose, triglycerides, cholesterol, and insulin levels, but the changes they cause, if and only if ingested together with a balanced diet, do not lead to the development of excess of body mass, obesity, and pathologies in the early stages of life.

Finally, there is still much to elucidate about the effect of chronic sweetener consumption on body mass gain, biochemical profile, hormone profile as well as the role of other additives added together with these sweeteners such artificial flavorings and colorants, and chemical antioxidants and preservatives in the nonalcoholic beverages and in the solid diet.

Acknowledgements

Authors recognize the financial support granted by Mexico’s National Council of Science and Technology (CONACYT, in Spanish) for the purchase of materials, reagents, equipment, and specimens throughout the Project CONACYT 82788. First author also received a scholarship from CONACYT for graduate studies. Authors dedicate this paper to the memory of Miss Mauricia Betzabeth Guzmán-Gómez, B.S. Chem., who died in the line of duty. She was an outstanding student and a very dedicated professional. Her family should be proud of her and her contribution to the well-being of humanity through her contribution to this manuscript (Los autores dedican este artículo a la memoria de la señorita QA Mauricia Betsabeth Guzmán-Gómez, fallecida en el cumplimiento del deber. Fue una estudiante sobresaliente y una profesional muy dedicada. Su familia debe estar orgullosa de ella y de su contribución al bienestar de la humanidad a través de su aportación a este manuscrito, in Spanish).

Compliance with ethical standards

Conflict of interest

The authors declare not having any personal or financial support or involvement with organizations with financial interest in the subject matter or any actual or potential conflict of interest.

Footnotes

Mauricia Betzabeth Guzmán-Gómez: Deceased

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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