Previous work from our lab indicated that following exposure to dietary supplements of saccharin-sweetened yogurt, rats showed both impaired ability to compensate for calories and a diminished thermic effect of food following consumption of a novel, sweet-tasting test meal of thickened Chocolate Ensure Plus [2
]. Therefore, the goal of Experiment 1 was to determine whether exposure to saccharin-sweetened yogurt altered glycemic responses to this novel test meal compared to exposure to glucose-sweetened yogurt. The results of this experiment indicated that animals with experience consuming saccharin-sweetened yogurts showed hyperglycemic responses when given a novel, sweet-tasting test meal compared to animals that had experience consuming glucose-sweetened yogurts, and that these differences appeared during the first blood sample, collected 15 min following delivery of the test meal. These data suggested that exposure to saccharin-sweetened diets may have resulted in altered cephalic phase responses, such as a diminished cephalic phase insulin release (CPIR), when a novel, sweet-tasting diet that delivered energy was consumed.
The goals of Experiment 2 were to examine how rapidly these changes in glycemic responses were observed and to measure whether in fact differences in the CPIR contributed to differences in the glycemic response; data were collected during a standard glucose tolerance test rather than during consumption of a novel, complex test meal as was done in Experiment 1. To collect samples rapidly, indwelling jugular catheters were implanted following exposure to saccharin-sweetened or glucose-sweetened yogurt, and animals were then given access to an oral glucose solution while samples were collected every 4 min. The results demonstrated that prior exposure to saccharin-sweetened yogurt resulted in hyperglycemic responses to oral consumption of a glucose solution compared to prior exposure to glucose-sweetened yogurt, and that this hyperglycemic response appeared during the early stages of ingestion. Further, the increase in blood glucose did not appear to result from an impaired release of insulin, since insulin levels did not differ between the two groups during testing at any time point, even during the earliest sample collected at 4 min.
In Experiment 3, we compared glycemic responses to oral ingestion of a glucose solution to the same glucose solution delivered by gavage to bypass oral stimulation in animals exposed to saccharin-sweetened yogurt versus glucose-sweetened yogurt. The results indicated that differences in glucose tolerance following exposure to saccharin-sweetened yogurt compared to glucose-sweetened yogurt do not occur when glucose is delivered directly into the stomach, bypassing the oral cavity. This finding is consistent with the idea that saccharin consumption weakens the ability of a sweet taste in the mouth to evoke cephalic phase responses involved with controlling blood glucose levels.
Experiment 4 assessed the effects of oral taste stimulation and delivery rate on glycemic and insulin responses to glucose test solutions by rats that had previously consumed yogurt sweetened with saccharin or glucose. These responses were compared for rats that consumed the glucose test solution orally (Oral), had the solution administered by gavage (Gavage) or had the solution administered by gavage in conjunction with having the tongue rinsed with glucose solution. These results indicated that blood glucose responses were different in both conditions when animals tasted the solution (Oral and Taste Gavage), but not when the solution was delivered directly by gavage (Gavage). The use of the Taste Gavage meant that animals in both groups received the taste stimuli over a similar period of time, ruling out differences in the rate of consumption of the glucose solutions during the oral tests as a necessary condition for differences between groups. As in Experiments 2 and 3, changes in oral glucose tolerance following exposure to saccharin were not accompanied by changes in insulin levels in Experiment 4.
In the final two experiments, animals were maintained on a high-fat, glucose-sweetened diet that mimicked characteristics of the diet widely-consumed by humans in the U.S. Along with increased food intake, body weight gain, and impaired oral glucose tolerance, decreased release of GLP-1 during the oral glucose test was associated with prior consumption of saccharin (relative to glucose) in solution (Experiment 5) or mixed in yogurt (Experiment 6). Given findings that implicate GLP-1 in both satiety and glucose homeostasis, a reduction in GLP-1 response to glucose intake could contribute to increased intake and impaired glucoregulation by the rats that had consumed saccharin.
The results of this series of experiments also confirm [2
] that animals given access to dietary supplements containing the high-intensity sweetener saccharin show increased body weight gain relative to animals given access to the same supplements containing the caloric sweetener glucose. In 5 of the 6 experiments, such differences in weight gain were statistically significant, whether the sweetener was mixed in a yogurt supplement (3.1.1, 3.2.1, 3.4.1, 3.6.1) or provided in liquid form (3.5.1). In Experiment 3, animals did not show significant differences in weight gain after two weeks exposure to saccharin-sweetened yogurt compared to glucose-sweetened yogurt. This lack of differential weight gain may be related to the fact that animals were smaller at the start of this experiment, and were still in a rapid growth phase. The high rate of growth in both groups in this experiment, which appear to be greater than in all of the other experiments, may have obscured differences in weight gain based on exposure to the differential relations between sweet tastes and calories. Nevertheless, when taken together with previous results [2
], the data are consistent with the hypothesis that exposure to a relation in which sweet taste reliably predicts the delivery of calories, such as that provided by glucose-sweetened diets, results in reduced weight gain relative to a diet in which sweet taste does not reliably predict calories, such as that provided by saccharin-sweetened diets. As previously demonstrated [2
] differences in body weight gain are related to differences in food intake; animals given saccharin-sweetened solutions consumed more total calories than animals given glucose-sweetened solutions in Experiment 5 (3.5.2) as did animals given saccharin-sweetened yogurt supplements in Experiment 6 (3.6.2).
In addition to these differences in body weight gain and food intake, oral glucose tolerance tests consistently revealed that animals given experience with saccharin-sweetened yogurt or liquid showed higher levels of blood glucose within the first 8 – 16 minutes of consuming either a glucose solution or a novel sweet-tasting caloric test meal. Thus, compared to experience with glucose-sweetened diets, experience with saccharin-sweetened diet appeared to impair glycemic responses. This impairment appeared to be dependent on stimulation of the oral cavity, since delivery of an identical glucose solution directly into the stomach by gavage did not produce differential glycemic responses (3.3.2, 3.4.2, 3.5.3). Differential glycemic responses were observed following gavage only after the tongue was first stimulated with the glucose solution (3.4.2). Thus, experience with a saccharin-sweetened diet led to exaggerated blood glucose levels when animals ate sweet-tasting foods that actually delivered energy and calories. This effect appeared early in testing (within the first 8 –16 minutes), and was transient in nature, supporting the idea that alterations were related to an impaired ability to predict the arrival of energy in the saccharin-exposed animals, rather than a general impairment in glucose utilization.
The mechanism underlying changes in blood glucose regulation in saccharin-exposed animals does not appear to be related to decreased release of insulin since no significant differences in insulin release were observed in any experiments. However, given that the earliest time sample collected was 4 min (Experiment 2) or 8 min (Experiments 4–6) following presentation of the glucose solution it is possible that differences in CPIR were missed, and that samples collected more rapidly might reveal differences between the groups. Nevertheless, the current data do not provide evidence for altered insulin release in the observed effects on glycemic responses.
The results from Experiments 5 and 6 suggest that a primary deficit resulting from exposure to saccharin-sweetened diets may be a decreased secretion of GLP-1 in response to sweet tastes in the mouth. GLP-1 levels were similar at baseline, but secretion of GLP-1 was significantly lower, and blood glucose levels were significantly higher, in animals previously exposed to saccharin-sweetened liquids or saccharin-sweetened yogurts during the first two samples (8 and 16 min) after consumption of an oral glucose load (see 3.5.3, 3.5.4, 3.6.4 and 3.6.5).
Based on principles of associative learning, experience with consuming a sweet taste that is not followed by the anticipated energetic consequence could cause the sweet taste to become less effective at eliciting release of GLP-1 over time. Release of GLP-1 by sweet taste in the mouth would then become blunted even when caloric sweeteners are subsequently consumed. This diminished ability of sweet taste to release GLP-1 could underlie increased food intake, as both peripheral and central actions of GLP-1 during meals have been directly implicated in satiety (e.g. [20
]). A reduction in the release of GLP-1 could also lead to increased blood glucose levels by a variety of mechanisms. For example, GLP-1 can contribute to glucose homeostasis independent of effects on insulin release, by enhancing glucose metabolism in skeletal muscle, liver and adipose tissue, regulating of glucose transporter expression, suppressing of glucagon release and slowing of gastric emptying (e.g. [23
]). Thus, for example, diminished release GLP-1 in response to a sweet-tasting food or glucose solution could promote more rapid gastric emptying which would then lead to more rapid delivery of glucose to the intestines, and more rapid elevations of blood glucose levels (e.g. [23
]). Increased gastric emptying would also lead to diminished gastric distension, reducing another potential source of satiety signals that could contribute to increased food intake. Further, decreased glucose utilization in muscle, liver or adipose tissue related to decreased levels of GLP-1 would lead to higher blood glucose levels (e.g. [23
]). Lower GLP-1 release could also result in increased blood glucose levels due to diminished suppression of glucagon release (e.g. [40
These data are consistent with the hypothesis that dysregulation of energy balance and glucose homeostasis can occur following exposure to high-intensity sweeteners. One might question this conclusion based on the facts that final sample sizes were relatively small, and in each experiment a number of animals were excluded, either due to technical difficulties with gavage or blood collection, or due the animals’ failure to consume the diets or liquids during training or the glucose or test meal during testing. However, there did not appear to be systematic differences between groups in consumption of the assigned diets during training or in consumption of the glucose solution or Ensure test meal during glucose tolerance testing, thus differences between animals that fail to eat the assigned diets during exposure or testing and those that did eat do not appear to be directly related to sweetener exposure. There also could be differences in the intensity of the sweet taste arising resulting from the sweeteners used. For example, rats may perceive the sweetness of the 0.3% saccharin to be higher or lower in intensity than the 20% glucose added to the yogurts used in Experiments 1–4 and 6, or the 10% glucose solution used in Experiment 5. However, even if differences in perceived intensity existed, it would be difficult to see how they could account for the current findings.
Thus, the results from these experiments suggest that exposure to sweet tastes that do not reliably predict the delivery of energy or glucose produces alterations in glycemic responses to orally ingested glucose, but not to glucose delivered to the gut directly. These differences in glucose homeostasis were associated with decreased release of GLP-1 early in the meal, which may have contributed to the increases in food intake and body weight gain in saccharin-exposed animals. Such results are consistent with the hypothesis that rather than preventing or reversing overweight and obesity, consumption of foods or beverages manufactured with high-intensity sweeteners may contribute to dysregulation of body weight by altering cephalic phase responses. This could occur because high-intensity sweeteners interfere with conditioned cephalic phase responses, because glucose-sweetened foods enhance conditioned cephalic phase responses, or both. In any of those cases, consuming diets prepared with high-intensity sweeteners results in augmented food intake, body weight gain, altered glucose homeostasis and diminished release of GLP-1 compared to the same diets prepared with glucose.
To date, little attention has been paid to the possibility that such experiences affect glycemic or other responses in humans. Recent studies examining glycemic responses to delivery of high-intensity sweeteners alone, for example, demonstrate that human subjects consuming these sweeteners orally do not show increases in plasma GLP-1, PYY, insulin or glucose [60
] or that delivery of high-intensity sweeteners directly into the gut do not affect gastric emptying, blood glucose, insulin or release of GLP-1 (e.g. [62
]). These results have been used to argue that there are fundamental differences between humans and rodents, and that high-intensity sweeteners cannot or do not alter glucose homeostasis, satiety or energy balance in humans. However, this conclusion can be challenged on several grounds.
First, studies that only deliver sweeteners directly into the stomach or intestines do not test the hypothesis that glucose homeostasis and energy balance depend on the validity of the predictive relationship between sweet taste and caloric outcomes. Thus results showing that humans do not release hormones such as GLP-1 or insulin in response to delivery of sweeteners directly into the gut [62
] do not speak to the hypothesis, and are consistent with some data in rats showing a similar lack of effect in rats when sweeteners are delivered directly into the gut in rats [65
]. The critical role of taste is demonstrated in the present data. When animals consume a glucose solution oral during glucose tolerance testing, previous exposure to saccharin resulted in hyperglycemia relative to previous exposure to glucose. In contrast, no effects of previous exposure to saccharin on glucose homeostasis were observed in glucose tolerance tests where the glucose solution is delivered directly into the stomach.
Second, measuring acute metabolic or hormonal responses evoked by oral intake of non-caloric sweeteners consumed in isolation, does not address the more important hypothesis, from our perspective, that exposure to non-caloric sweeteners impairs the ability of consuming sweet, high-calorie, substances to evoke those responses. Third, most studies in humans fail to consider the possibility that the effects repeated or chronic exposure to high-intensity sweeteners and their consequences may be different compared to acute exposure. If consumption of high-intensity sweeteners interferes with conditioned responses, then previous exposure to the high-intensity sweeteners could affect responses during tests and a single exposure to a high-intensity sweetener might be insufficient to elicit any altered responses. Consistent with this hypothesis is recent data documenting differences in brain activity in response to an oral sucrose solution in human subjects with histories of high versus low consumption of high-intensity sweeteners [66
]. Examining the effects of a single exposure to a sweetener, or failing to account for the amount of previous exposure to the sweeteners therefore complicates interpretation of human studies attempting to examine how high-intensity sweeteners might influence food intake, body weight and/or glucose homeostasis.
Within our theoretical framework, the critical questions are not about the acute effects of high intensity sweeteners when they are consumed alone or when they are delivered directly to the gut. The real issue is how does chronic, oral consumption of non-caloric sweeteners impact energy regulation when sweet caloric substances are orally consumed. This is the question we have tried to address in our animal studies. And the question is important because, in the current human food environment, high-intensity sweeteners are typically (a) consumed chronically, not acutely; (b) consumed not in isolation, but by people who also consume sweet high-calorie foods and beverages; (c) consumed and tasted orally, not in ways that bypass taste and the oral cavity.
There are clear differences between rats and humans that prescribe caution when applying the results of studies such as ours to regulation of human body weight. However, it is also important to avoid dismissing the potential implication of studies conducted in rats, such as those described above, on the basis of human studies that fail to adequately evaluate the hypothesis.