The primary goals of this study were to distinguish the acute effects of sucralose ingestion from the perception of the sweet taste of sucralose per se on the metabolic response to glucose ingestion, and to compare such acute effects of sucralose in people who are normal weight and those who are obese. We hypothesized that, compared with the ingestion of water before a glucose load, the ingestion of sucralose before a glucose load, but not sucralose taste alone (i.e., sucralose tasted and expectorated, hereafter sham-fed), would cause a greater increase in glucose-stimulated insulin response (as we found previously [ 18 ]) and impair the suppression of endogenous glucose production (EGP) in participants with obesity but not in those who are normal weight. A secondary goal of the study was to assess plasma sucralose concentrations achieved after sucralose ingestion. We hypothesized that people with obesity would be more vulnerable to the adverse metabolic effects of sucralose ingestion than those with a normal weight, in part because the increased intestinal permeability associated with obesity [ 22 ] would enhance the absorption of ingested sucralose and increase plasma sucralose concentrations.

We recently found that the ingestion of sucralose before an oral glucose tolerance test (OGTT) increased the plasma insulin response and decreased whole-body insulin sensitivity in people with obesity [ 18 ]. However, it is unknown whether this effect was mediated by the perception of sucralose sweet taste in the mouth or by sucralose post-oral effects. Further, data from most studies conducted in people who are normal weight show that acute sucralose ingestion does not affect the plasma glucose or insulin response to carbohydrate ingestion [ 15 21 ]. These findings suggest that acute sucralose ingestion has different effects in people who are obese than in those who are normal weight.

Even though LCSs have a negligible calorie content, data from animal models and human cell lines suggest that they are metabolically active, presumably by interaction with sweet taste receptors [ 4 12 ]. The sweet taste receptor T1R2 + T1R3 is a heterodimer that detects sugars and LCSs and is expressed in many tissues beyond the mouth, including the intestine and the pancreas [ 4 ]. Activation of these sweet taste receptors in the intestine results in a faster rate of glucose absorption [ 5 13 ] and in the secretion of incretins [ 9 ], although data from studies in vivo suggest that the extent of incretin secretion varies with different sweet taste ligands [ 14 15 ]. In addition, activation of these receptors in the pancreas can result in increased insulin secretion [ 10 12 ] and in the mouth, it can elicit pre-ingestive cephalic-phase responses, including a cephalic-phase insulin response (CPIR), which are thought to prime the body to better absorb and use ingested nutrients [ 16 17 ].

The primary outcome measure of this study was the comparison of the differences on insulin responses to glucose on the sucralose treatment versus water treatment between weight groups. Based on data from our previous sucralose study [ 18 ], we estimated that 10 participants in each weight group would allow us to detect a large effect (0.9) on insulin response between groups with a β-value of 0.20 (i.e., 80% power) and a α-value of 0.05. For the power calculation, we used a multivariate linear model for repeated measures with PROC GLMPower procedure using the Hotelling-Lawley Trace statistics and LEAR correlation for the structure of the covariance (SAS 9.3).

To determine the statistical significance of the effect of tasting or tasting and ingesting sucralose on plasma glucose, insulin, C-peptide, and GIP concentrations, as well as glucose kinetics (Total Ra, oral glucose rate of appearance, and EGP), ISR, S I , and insulin clearance during the OGTT, and to determine the effect of obesity on plasma sucralose concentrations, we used the general linear mixed model (PROC MIXED) analysis with subject-level random intercepts. Treatment (water, sucralose, and sham-fed), time (when applicable), and weight group (normal weight and obese), as well as all interactions (when applicable) were included in the model and treated as fixed effects. When differences in values were statistically significant, post hoc Fisher least significant difference analyses were conducted. Because the PROC MIXED analysis can handle unbalanced designs, data from two participants in the group with obesity who did not complete the sucralose sham-fed visits were also included (therefore, for the group with obesity PROC MIXED analyses included data on 11 participants for sucralose and water treatment and 9 participants for sham-fed). Data in the tables and figures are presented as the mean ± SEM unless otherwise stated. All analyses were performed with SAS 9.3 (SAS Institute, Cary, NC, USA), and criterion for statistical significance was p ≤ 0.05.

We used the oral C-peptide minimal model as described previously [ 27 ] to estimate the insulin secretion rate (ISR) in response to the oral glucose load and the sensitivity of the β-cell response to changes in plasma glucose. This model provides an estimate of the total amount of insulin secreted in response to plasma glucose as a function of time (i.e., total ISR, in pmol/min). This model also partitions the total ISR response into a dynamic component (ISRdynamic), representing the rapid release of a “readily releasable pool” of insulin secretory granules in response to the rate of increasing plasma glucose concentration, and a static component (ISRstatic), representing the slower release of a “reserve pool” of insulin secretory granules in response to the ambient plasma glucose concentration [ 27 ]. We also determined the β-cell response sensitivity parameters (Φtotal, Φdynamic, Φstatic) corresponding to the total, dynamic, and static ISR in response to changes in plasma glucose. We estimated the insulin clearance rate (pools/min) from plasma by dividing the iAUC of ISR by the iAUC of plasma insulin concentration. We then multiplied the insulin clearance rate by the volume of distribution for C-peptide from the ISR modeling (used as the volume of distribution for insulin) to express insulin clearance rate as L/min.

At all time points, plasma glucose was measured immediately after collection using an automated glucose analyzer (YSI 2300 STAT Plus; Yellow Springs Instruments, Yellow Springs, OH, USA). Blood samples were also collected in chilled EDTA tubes containing a protease inhibitor cocktail (Millipore, Billerica, MA, USA). These samples were placed on ice and centrifuged at 4 °C, and the plasma was stored at −80 °C for subsequent analyses. Plasma insulin and C-peptide concentrations were measured at the same time points as plasma glucose up to 60 min post-glucose ingestion and then every 40 min until the end of the test. Plasma insulin concentrations were determined using a two-site immunoenzymatic assay (DxI 800; Beckman Instruments, Chaska, MN, USA), and C-peptide by using a solid-phase two-site chemiluminescent immunometric assay (Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA). Plasma glucose-dependent insulinotropic peptide (GIP) was measured at 10 and 2 min before and at 0, 10, 30, 50, 60, 100, 140, and 180 min after glucose consumption using commercially available immunoassay kits from Millipore. The glucose tracer-to-tracee ratio in plasma was determined using GC-MS (Hewlett-Packard MSD 5973 system with a capillary column) after derivatization with acetic anhydride. Plasma sucralose concentrations were measured 10 min prior, immediately before, and at 10, 40, 70, 130, 190, and 310 min after sucralose consumption using liquid chromatography-mass spectrometry, as described previously [ 24 ].

Participants were enrolled in the study between April 2015 and August 2016. They completed a screening visit that included a standard 75 g 2 h OGTT and a questionnaire used in previous studies that inquired about typical LCSs use [ 18 23 ]. Participants with a plasma glucose concentration ≥7 mmol/L at fasting or ≥11.1 mmol/L at 2 h post-OGTT, or with significant insulin resistance (based on a homeostatic model assessment of insulin resistance score (HOMA-IR2)) > three were excluded from participation. Also, those who were regular consumers of LCSs (i.e., reported consuming more than one diet beverage per week, one spoonful of LCSs per week, or an equivalent amount of LCSs in foods); smoked tobacco cigarettes in the past six months; were pregnant or breastfeeding; had a history of malabsorptive syndromes, bariatric surgery, or inflammatory intestinal disease; or were taking any medication that may affect glucose metabolism were excluded. The study conformed to the revised Declaration of Helsinki and was approved by the institutional review board at Washington University School of Medicine in St. Louis, MO, USA. All screened participants gave informed written consent before participation. The study was registered at ClinicalTrials.gov, with the identifier: NCT02413424.

Using a randomized crossover design, participants were evaluated on three separate study visits. We chose this type of study design, in which each subject acts as his or her own control, to reduce the potential influence of confounding factors, such as dietary variations and baseline glucose metabolic characteristics. The study visits took place at the same times, approximately one week apart. For each study visit, participants were admitted at ~07:00 to the Clinical Research Unit at Washington University School of Medicine. Participants were instructed to fast overnight (12 h) at home and to avoid physical exercise for 3 days before each study visit. After vital signs were obtained, one catheter was inserted into a forearm vein for infusion, and a second catheter was inserted into a radial artery to obtain blood samples. A primed, continuous infusion of (6,6-2H2) glucose (priming dose, 22 µmol/kg; infusion rate of 0.22 µmol/kg·min) was started and maintained until the end of the study. After 3.5 h of tracer infusion, participants ingested a drink containing 75 g glucose mixed with 1.5 g [U-13C6] glucose. Blood samples were obtained at 40, 30, 20, 10, 8, 6, 4, and 2 min before and at 0, 10, 20, 30, 40, 50, and 60 min after ingestion of glucose, and then every 20 min for an additional 4 h to determine glucose kinetics, and plasma hormone and sucralose concentrations. In a randomized order, 10 min prior to glucose ingestion, participants drank 60 mL of 2 mmol/L sucralose (i.e., 48 mg sucralose, as in our previous study [ 18 ]) or an equivalent volume of distilled water, or they sham-fed sucralose. The sham-feeding procedure consisted of swishing the same amount of sucralose in their mouth for ~5 s and then spitting it out.

Participants with obesity had a higher oral glucose rate of appearance during the first 40 min of the OGTT than normal-weight participants (Weight Group × Time: F= 1.88,= 0.04; Figure 3 ); however, the iAUC of total glucose rate of appearance and of oral glucose rate of appearance were similar between weight groups ( Table 2 ). Glucose ingestion suppressed EGP rapidly in both groups (< 0.0001), but more so in participants with obesity (= 0.018; Figure 3 ). Sucralose, ingested or sham-fed, did not affect the total glucose rate of appearance ( Figure 1 ) or the oral glucose rate of appearance in either weight group ( Figure A5 in Appendix B ). For all treatments, and in both weight groups, the amount of ingested glucose reaching systemic circulation over 5 h was ~70% of the 75 g given ( Table 2 ).

Total, dynamic, and static ISR and the sensitivity of insulin secretion to plasma glucose in response to the OGTT were significantly higher in participants with obesity than in normal-weight participants across all treatments (all< 0.05; Table 3 and Figure A4 in Appendix B ). Treatment did not significantly affect the sensitivity of insulin secretion to plasma glucose or total or static ISR responses during the OGTT in either weight group. Sucralose modified the ISR dynamic response to the glucose load only in the group with obesity (Treatment × Weight Group × Time: F= 2.19;< 0.02; Figure A4 in Appendix B ). Compared with water, the sucralose treatment resulted in higher dynamic ISR at time 0 but lower at 20–30 min post-glucose ingestion. The dynamic ISR for sham-fed treatment was similar to that of sucralose but different from water treatment only at 10 min post-glucose load.

4. Discussion

The primary finding of this study is that the ingestion of sucralose, in a quantity equivalent to that in a commercial can of soda, has different effects on postprandial glucose metabolism in participants with obesity and in normal-weight participants—none of whom regularly consume LCSs. Sucralose ingestion similarly increased glucose iAUC by ~30% in both weight groups but had opposite effects on plasma insulin concentrations. That is, in normal-weight participants, sucralose ingestion or sham-fed sucralose modestly decreased plasma insulin concentrations within the first hour after a glucose load, whereas in participants with obesity, ingested sucralose caused significantly higher insulin concentrations than sham-fed sucralose. While participants with obesity achieved higher plasma sucralose concentrations than normal-weight participants 4–5 h post-sucralose ingestion, it is unlikely that such difference could explain the divergent insulin responses between weight groups observed 1 h post-sucralose ingestion. The mechanisms responsible for the sucralose effect on glucose iAUC are unclear. We hypothesized that sucralose could affect plasma glucose excursions after the OGTT by altering endogenous glucose production (EGP), but we found that sucralose, neither ingested nor sham-fed, did not affect EGP or the rate of glucose appearance derived from ingested glucose in either weight group.

In addition, we found that the extra activation of sweet taste sensory stimulation, by sham-fed sucralose before ingesting a glucose drink, dampened plasma insulin concentrations in both groups. The mechanism underlying this sucralose sham-fed associated dampened insulin response is unknown. Also unknown is whether solutions of other isointense non-sweet taste stimuli or other sweeteners would also cause this effect. However, findings from previous studies suggest that oral sensory stimulation, by triggering a cephalic phase insulin response (CPIR), can alter plasma glucose and insulin concentrations after intragastric glucose infusions to improve glucose metabolism in healthy lean men [ 30 ]. Further, brief intravenous infusion of insulin in a pattern that mimics a CPIR during food ingestion improves glucose tolerance in people with obesity [ 31 ]. Although sucralose did not trigger a measurable CPIR during the first 10 min post ingestion (neither in this study nor in our previous study [ 18 ]), a possibility that we did not evaluate is that tasting intense sweetness 10 min before an OGTT sensitized a CPIR, or other cephalic hormonal responses, to the ingestion of glucose. Interestingly, recent findings from clinical studies that used neuroimaging and indirect calorimetry also suggest that oral perception of sweetness plays a pivotal role in the regulation of carbohydrate metabolism [ 32 ]. Through a series of elegant studies, Veldhuizen et al. [ 32 ] manipulated sweetness levels independently from caloric load to demonstrate that the optimal metabolic response to a carbohydrate load (and its rewarding value) depends on the perfect match between the intensity of its sweetness and its energy content. Finally, our finding that sweetness perception before a glucose load decreased insulin response after an OGTT, complements findings from pre-clinical and clinical studies that show the opposite insulin response when subjects are deprived of sweet taste perception [ 33 34 ]. That is, the inhibition of sweet taste perception, either by direct administration of glucose to the stomach (i.e., bypassing oral taste perception) in rats [ 33 ], or by adding lactisole (a broad antagonist of the sweet taste receptor) to a glucose load in normal-weight participants [ 34 ] heightened insulin responses to an OGTT. However, it should be noticed that, as in this last study [ 34 ], lactisole was added to the oral glucose load, and it is unclear whether inhibition of sweetness signaling in the mouth and/or the gut altered postprandial insulin concentrations. For example, findings from a study in healthy lean subjects who received intragastric infusions of glucose with and without lactisole suggest that sweet taste receptors expressed in the gut can affect glucose metabolism, independently of sweetness signaling in the mouth [ 35 ]. In this study, lactisole, despite attenuating glucose-stimulated glucagon-like peptide-1 secretion, increased glucose AUC without changing insulin AUC, which suggest that post-oral sweetness signaling can affect insulin sensitivity [ 35 ]. Although more studies are needed to disentangle the effects of activating oral versus intestinal sweet taste signaling in glucose metabolism, collectively, these findings offer an innovative mechanism by which LCSs might upset metabolic fate of carbohydrates. We hypothesize that overly sweetened diets alter sweet taste signaling systems that play a role in the regulation of postprandial glucose metabolism, helping explain, at least in part, why high consumption of LCSs is associated with the same detrimental health effects as high consumption of added sugars, including an increased risk of developing type 2 diabetes [ 4 ].

I (by 23 ± 20%) in participants with obesity, here, we found that sucralose reduced S I in this weight group by only 12 ± 8%, which was not statistically significant. Remarkably, acute sucralose ingestion significantly increased S I by 52 ± 20% in normal-weight participants. The mechanism responsible for this acute effect of sucralose is unknown, but our data suggest that it is not likely to be explained by changes in hepatic insulin sensitivity. Neither ingested nor sham-fed sucralose affected the rate of EGP in either weight group. However, contrary to our finding of an increased S I after acute consumption of sucralose in normal-weight participants, two independent groups recently reported that chronic consumption of sucralose (i.e., daily for 2 weeks) in such a group decreased S I [36, I are different from chronic effects due to the development of compensatory responses (i.e., tolerance) to the consumption of sucralose. Typically, once tolerance to a substance has been developed, exposure to a smaller dose of such a substance, or to stimuli conditioned to the presence of the substance (such as its “taste”) triggers the compensatory response, which is commonly opposite to the initial acute effects of the substance [ Compared with water treatment, sucralose ingestion increased glucose-stimulated insulin responses only in participants with obesity. However, the increase in plasma insulin concentrations was of a smaller magnitude than in our previous study [ 18 ] (which may also explain why plasma glucose concentrations did not reach as low of a nadir). We hypothesize that differences in the magnitude of the insulin response may be related to the inclusion of a new treatment condition in this current study design (i.e., sucralose sham-fed), which had a significant (and opposite) effect in both weight groups. Inconsistent with our previous findings that sucralose ingestion before an OGTT significantly reduced S(by 23 ± 20%) in participants with obesity, here, we found that sucralose reduced Sin this weight group by only 12 ± 8%, which was not statistically significant. Remarkably, acute sucralose ingestion significantly increased Sby 52 ± 20% in normal-weight participants. The mechanism responsible for this acute effect of sucralose is unknown, but our data suggest that it is not likely to be explained by changes in hepatic insulin sensitivity. Neither ingested nor sham-fed sucralose affected the rate of EGP in either weight group. However, contrary to our finding of an increased Safter acute consumption of sucralose in normal-weight participants, two independent groups recently reported that chronic consumption of sucralose (i.e., daily for 2 weeks) in such a group decreased S 37 ]. Importantly, all these studies (including our own) included participants who were non-habitual consumers of LCSs at baseline. It is therefore plausible to hypothesize that acute effects of sucralose on Sare different from chronic effects due to the development of compensatory responses (i.e., tolerance) to the consumption of sucralose. Typically, once tolerance to a substance has been developed, exposure to a smaller dose of such a substance, or to stimuli conditioned to the presence of the substance (such as its “taste”) triggers the compensatory response, which is commonly opposite to the initial acute effects of the substance [ 38 ].

Sucralose did not significantly affect total glucose rate of appearance or oral glucose rate of appearance in either weight group. Interestingly, although total glucose rate of appearance was similar between groups, in participants with obesity the oral glucose rate of appearance was increased, and the rate of EGP was decreased during the first 40 min of the OGTT. The finding of a higher oral glucose rate of appearance in participants with obesity is consistent with results of previous studies that demonstrated increased expression of the sodium glucose transporter isoform 1 (SGLT1) in the duodenum of people with severe obesity [ 39 ]. Such an increased expression of SGLT1 has been associated with a faster rate of intestinal glucose absorption [ 39 40 ] and is likely to account, at least partially, for the observed insulin hypersecretion. In turn, the observed hyperinsulinemia after consuming the glucose drink in the group with obesity might explain the remarkable difference in EGP between groups, because EGP is very sensitive to small increases in circulating insulin [ 41 ]. That the group with obesity, which is more insulin resistant, suppressed EGP more than the group with normal weight, which is more insulin sensitive, may sound paradoxical. However, data suggest that, in participants without diabetes, EGP decreases (not increases) with increases in Body Mass Index) [ 42 ].