Selective inhibition of sweetness by the sodium salt of +/-2-(4-methoxyphenoxy)propanoic acid.

The purpose of this study was to determine the degree to which the sodium salt of +/-2-(4-methoxyphenoxy)propanoic acid (Na-PMP) reduced sweet intensity ratings of 15 sweeteners in mixtures. Na-PMP has been approved for use in confectionary/frostings, soft candy and snack products in the USA at concentrations up to 150 p.p.m. A trained panel evaluated the effect of Na-PMP on the intensity of the following 15 sweeteners: three sugars (fructose, glucose, sucrose), three terpenoid glycosides (monoammonium glycyrrhizinate, rebaudioside-A, stevioside), two dipeptide derivatives (alitame, aspartame), two N-sulfonylamides (acesulfame-K, sodium saccharin), two polyhydric alcohols (mannitol, sorbitol), 1 dihydrochalcone (neohesperidin dihydrochalcone), one protein (thaumatin) and one sulfamate (sodium cyclamate). Sweeteners were tested at concentrations isosweet with 2.5, 5, 7.5 and 10% sucrose in mixtures with two levels of Na-PMP: 250 and 500 p.p.m. In addition, the 15 sweeteners were tested either immediately or 30 s after a pre-rinse with 500 p.p.m. Na-PMP. In mixtures, Na-PMP at both the 250 and 500 p.p.m. levels significantly blocked sweetness intensity for 12 of the 15 sweeteners. However, when Na-PMP was mixed with three of the 15 sweeteners (monoammonium glycyrrhizinate, neohesperidin dihydrochalcone and thaumatin), there was little reduction in sweetness intensity. Pre-rinsing with Na-PMP both inhibited and enhanced sweetness with the greatest enhancements found for monoammonium glycyrrhizinate, neohesperidin dihydrochalcone and thaumatin, which were not suppressed by Na-PMP in mixtures. The mixture data suggest that Na-PMP is a selective competitive inhibitor of sweet taste. The finding that pre-treatment can produce enhancement may be due to sensitization of sweetener receptors by Na-PMP.     

AN OBJECTIVE NUMERICAL METHOD OF ASSESSING THE RELIABILITY OF TIME-INTENSITY PANELISTS

A measure of the reliability (T-IR) of time-intensity measurements was developed based on the concept of standard deviation as a measure of panelist variability. The T-IR measure was applied to time-intensity data collected from 10 panelists evaluating the sweetness of 4 model sweetener solutions on horizontal and vertical time-intensity line orientations. T-IR scores showed that the panelists were similarly reliable across the sweeteners and orientations. As well, independent of scale orientation, responses to sweeteners were similarly reliable. The T-IR measure can be used to maintain a high level of performance by monitoring time-intensity panelists. T-IR also provides an objective method of selecting panelists for time-intensity panels.     

Effects of Artificial Sweeteners on Insulin Release and Cationic Fluxes in Rat Pancreatic Islets

β-l-Glucose pentaacetate, but not -d-galactose pentaacetate, was recently reported to taste bitter and to stimulate insulin release. This finding led, in the present study, to the investigation of the effects of both bitter and non-bitter artificial sweeteners on insulin release and cationic fluxes in isolated rat pancreatic islets. Sodium saccharin (1.0–10.0 mM), sodium cyclamate (5.0–10.0 mM), stevioside (1.0 mM) and acesulfame-K (1.0–15.0 mM), all of which display a bitter taste, augmented insulin release from islets incubated in the presence of 7.0 mM d-glucose. In contrast, aspartame (1.0–10.0 mM), which is devoid of bitter taste, failed to affect insulin secretion. A positive secretory response to acesulfame-K was still observed when the extracellular K⁺ concentration was adjusted to the same value as that in control media. No major changes in ⁸⁶Rb and ⁴⁵Ca outflow from pre-labelled perifused islets could be attributed to the saccharin, cyclamic or acesulfame anions. It is proposed that the insulinotropic action of some artificial sweeteners and, possibly, that of selected hexose pentaacetate esters may require G-protein-coupled receptors similar to those operative in the recognition of bitter compounds by taste buds.     

Taste responses to sweet stimuli in alpha-gustducin knockout and wild-type mice

The importance of alpha-gustducin in sweet taste transduction is based on data obtained with sucrose and the artificial sweetener SC45647. Here we studied the role of alpha-gustducin in sweet taste. We compared the behavioral and electrophysiological responses of alpha-gustducin knockout (KO) and wild-type (WT) mice to 11 different sweeteners, representing carbohydrates, artificial sweeteners, and sweet amino acids. In behavioral experiments, over 48-h preference ratios were measured in two-bottle preference tests. In electrophysiological experiments, integrated responses of chorda tympani (CT) and glossopharyngeal (NG) nerves were recorded. We found that preference ratios of the KO mice were significantly lower than those of WT for acesulfame-K, dulcin, fructose, NC00174, D-phenylalanine, L-proline, D-tryptophan, saccharin, SC45647, sucrose, but not neotame. The nerve responses to all sweeteners, except neotame, were smaller in the KO mice than in the WT mice. The differences between the responses in WT and KO mice were more pronounced in the CT than in the NG. These data indicate that alpha-gustducin participates in the transduction of the sweet taste in general.     

Effects of artificial sweeteners on body weight, food and drink intake

Artificial sweeteners are widely used all over the world. They may assist in weight management, prevention of dental caries, control of blood glucose of diabetics, and also can be used to replace sugar in foods. In the animal experimentation mice were given oral doses of water solutions of table top artificial sweeteners (saccharin, cyclamate based, acesulfame-K based, and aspartame) the amount of maximum Acceptable Daily Intake (ADI) ad libitum. The controls received only tap water with the same drinking conditions as the treated groups. The mice were fed chow ad libitum.We measured food intake and body weight once a week, water and solutions of artificial sweeteners intake twice a week. The data were analysed by statistical methods (T-probe, regression analysis).Consumption of sweeteners resulted in significantly increased body weight; however, the food intake did not change.These results question the effect of non-caloric artificial sweeteners on weight-maintenance or body weight decrease.     

Whole nerve chorda tympani responses to sweeteners in C57BL/6ByJ and 129P3/J mice

The C57BL/6ByJ (B6) strain of mice exhibits higher preferences than does the 129P3/J (129) strain for a variety of sweet tasting compounds. We measured gustatory afferent responses of the whole chorda tympani nerve in these two strains using a broad array of sweeteners and other taste stimuli. Neural responses were greater in B6 than in 129 mice to the sugars sucrose and maltose, the polyol D-sorbitol and the non-caloric sweeteners Na saccharin, acesulfame-K, SC-45647 and sucralose. Lower neural response thresholds were also observed in the B6 strain for most of these stimuli. The strains did not differ in their neural responses to amino acids that are thought to taste sweet to mice, with the exception of L-proline, which evoked larger responses in the B6 strain. Aspartame and thaumatin, which taste sweet to humans but are not strongly preferred by B6 or 129 mice, did not evoke neural responses that exceeded threshold in either strain. The strains generally did not differ in their neural responses to NaCl, quinine and HCl. Thus, variation between the B6 and 129 strains in the peripheral gustatory system may contribute to differences in their consumption of many sweeteners.     

A microcomputer (PC 9801/MS mouse) system to record and analyze time-intensity curves of sweetness

The sweetness of sugar, glucose, xylose, saccharin, aspartame,etc. was assessed by magnitude estimation, with sensory intensitiesof 5, 10 and 20 defined as being equivalent to 2.5, 5.0 and10.0% concentrations of sugar in tap water. Sensory magnitudewas determined as being the input through a ‘mouse’to a microcomputer, PC 9801F2, which digitized the time intoseconds, while intensity was expressed in terms of tenths ofthe sensory magnitude 1.0. The two-dimensional information wasregistered on floppy disc memory and retrieved to calculatethe average time-intensity curves for each subject, or the averagefor several subjects. With this technique, it is easy to measure:(i) time of maximum intensity; (ii) height of maximum intensity;(iii) amount of adaptation; (iv) amount of after-taste; or (v)total amplitude (the sum of areas under the curve during andafter stimulation). The subjects were seven male students. Witha little training, they produced highly reliable curves forthe replicates of a sweetener at one concentration. Multidimensionalscaling of these curves did not disclose any clusterings ofnatural versus synthetic sweeteners.     

Effect of repeated presentation on sweetness intensity of binary and ternary mixtures of sweeteners

The purpose of the present study was to determine the effect of repeated presentation of the same sweet stimulus on sweetness intensity ratings. The sweet stimuli tested in this study were binary and ternary blends of 14 sweeteners that varied widely in chemical structure. A trained panel evaluated the sweetness intensity over four sips of a given mixture presented at 30 s intervals. The individual components in the binary sweetener combinations were intensity-anchored with 5% sucrose, while the individual sweeteners in the ternary mixtures were intensity-anchored with 3% sucrose (according to formulae developed previously). Each self-mixture was also evaluated (e.g. acesulfame-K-acesulfame-K). The main finding of this study was that mixtures consisting of two or three different sweeteners exhibited less reduction in sweetness intensity over four repeated sips than a single sweetener at an equivalent sweetness level. Furthermore, ternary combinations tended to be slightly more effective than binary combinations at lessening the effect of repeated exposure to a given sweet stimulus. These findings suggest that the decline in sweetness intensity experienced over repeated exposure to a sweet stimulus could be reduced by the blending of sweeteners.     

Allelic variation of the Tas1r3 taste receptor gene selectively affects taste responses to sweeteners: evidence from 129.B6-Tas1r3 congenic mice.

The Tas1r3 gene encodes the T1R3 receptor protein, which is involved in sweet taste transduction. To characterize ligand specificity of the T1R3 receptor and the genetic architecture of sweet taste responsiveness, we analyzed taste responses of 129.B6-Tas1r3 congenic mice to a variety of chemically diverse sweeteners and glucose polymers with three different measures: consumption in 48-h two-bottle preference tests, initial licking responses, and responses of the chorda tympani nerve. The results were generally consistent across the three measures. Allelic variation of the Tas1r3 gene influenced taste responsiveness to nonnutritive sweeteners (saccharin, acesulfame-K, sucralose, SC-45647), sugars (sucrose, maltose, glucose, fructose), sugar alcohols (erythritol, sorbitol), and some amino acids (D-tryptophan, D-phenylalanine, L-proline). Tas1r3 genotype did not affect taste responses to several sweet-tasting amino acids (L-glutamine, L-threonine, L-alanine, glycine), glucose polymers (Polycose, maltooligosaccharide), and nonsweet NaCl, HCl, quinine, monosodium glutamate, and inosine 5'-monophosphate. Thus Tas1r3 polymorphisms affect taste responses to many nutritive and nonnutritive sweeteners (all of which must interact with a taste receptor involving T1R3), but not to all carbohydrates and amino acids. In addition, we found that the genetic architecture of sweet taste responsiveness changes depending on the measure of taste response and the intensity of the sweet taste stimulus. Variation in the T1R3 receptor influenced peripheral taste responsiveness over a wide range of sweetener concentrations, but behavioral responses to higher concentrations of some sweeteners increasingly depended on mechanisms that could override input from the peripheral taste system.     

Sweetener preference of C57BL/6ByJ and 129P3/J mice

Previous studies have shown large differences in taste responses to several sweeteners between mice of the C57BL/6ByJ (B6) and 129P3/J (129) inbred strains. The goal of this study was to compare behavioral responses of B6 and 129 mice to a wider variety of sweeteners. Seventeen sweeteners were tested using two-bottle preference tests with water. Three main patterns of strain differences were evident. First, sucrose, maltose, saccharin, acesulfame-K, sucralose and SC-45647 were preferred by both strains, but the B6 mice had lower preference thresholds and higher solution intakes. Second, the amino acids D-phenylalanine, D-tryptophan, L-proline and glycine were highly preferred by B6 mice, but not by 129 mice. Third, glycyrrhizic acid, neohesperidin dihydrochalcone, thaumatin and cyclamate did not evoke strong preferences in either strain. Aspartame was neutral to all 129 and some B6 mice, but other B6 mice strongly preferred it. Thus, compared with the 129 mice the B6 mice had higher preferences for sugars, sweet tasting amino acids and several but not all non-caloric sweeteners. Glycyrrhizic acid, neohesperidin, thaumatin and cyclamate are not palatable to B6 or 129 mice.     

Oral zinc sulfate solutions inhibit sweet taste perception

We investigated the ability of zinc sulfate (5, 25, 50 mM) to inhibit the sweetness of 12 chemically diverse sweeteners, which were all intensity matched to 300 mM sucrose [800 mM glucose, 475 mM fructose, 3.25 mM aspartame, 3.5 mM saccharin, 12 mM sodium cyclamate, 14 mM acesulfame-K, 1.04 M sorbitol, 0.629 mM sucralose, 0.375 mM neohesperidin dihydrochalcone (NHDC), 1.5 mM stevioside and 0.0163 mM thaumatin]. Zinc sulfate inhibited the sweetness of most compounds in a concentration dependent manner, peaking with 80% inhibition by 50 mM. Curiously, zinc sulfate never inhibited the sweetness of Na-cyclamate. This suggests that Na-cyclamate may access a sweet taste mechanism that is different from the other sweeteners, which were inhibited uniformly (except thaumatin) at every concentration of zinc sulfate. We hypothesize that this set of compounds either accesses a single receptor or multiple receptors that are inhibited equally by zinc sulfate at each concentration.     

Artificial Sweeteners Stimulate Adipogenesis and Suppress Lipolysis Independently of Sweet Taste Receptors

G protein-coupled receptors mediate responses to a myriad of ligands, some of which regulate adipocyte differentiation and metabolism. The sweet taste receptors T1R2 and T1R3 are G protein-coupled receptors that function as carbohydrate sensors in taste buds, gut, and pancreas. Here we report that sweet taste receptors T1R2 and T1R3 are expressed throughout adipogenesis and in adipose tissues. Treatment of mouse and human precursor cells with artificial sweeteners, saccharin and acesulfame potassium, enhanced adipogenesis. Saccharin treatment of 3T3-L1 cells and primary mesenchymal stem cells rapidly stimulated phosphorylation of Akt and downstream targets with functions in adipogenesis such as cAMP-response element-binding protein and FOXO1; however, increased expression of peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α was not observed until relatively late in differentiation. Saccharin-stimulated Akt phosphorylation at Thr-308 occurred within 5 min, was phosphatidylinositol 3-kinase-dependent, and occurred in the presence of high concentrations of insulin and dexamethasone; phosphorylation of Ser-473 occurred more gradually. Surprisingly, neither saccharin-stimulated adipogenesis nor Thr-308 phosphorylation was dependent on expression of T1R2 and/or T1R3, although Ser-473 phosphorylation was impaired in T1R2/T1R3 double knock-out precursors. In mature adipocytes, artificial sweetener treatment suppressed lipolysis even in the presence of forskolin, and lipolytic responses were correlated with phosphorylation of hormone-sensitive lipase. Suppression of lipolysis by saccharin in adipocytes was also independent of T1R2 and T1R3. These results suggest that some artificial sweeteners have previously uncharacterized metabolic effects on adipocyte differentiation and metabolism and that effects of artificial sweeteners on adipose tissue biology may be largely independent of the classical sweet taste receptors, T1R2 and T1R3.     

Artificial sweeteners and salts producing a metallic taste sensation activate TRPV1 receptors

Throughout the world many people use artificial sweeteners (AS) for the purpose of reducing caloric intake. The most prominently used of these molecules include saccharin, aspartame (Nutrasweet), acesulfame-K, and cyclamate. Despite the caloric advantage they provide, one key concern in their use is their aversive aftertaste that has been characterized on a sensory level as bitter and/or metallic. Recently, it has been shown that the activation of particular T2R bitter taste receptors is partially involved with the bitter aftertaste sensation of saccharin and acesulfame-K. To more fully understand the biology behind these phenomena we have addressed the question of whether AS could stimulate transient receptor potential vanilloid-1 (TRPV1) receptors, as these receptors are activated by a large range of structurally different chemicals. Moreover, TRPV1 receptors and/or their variants are found in taste receptor cells and in nerve terminals throughout the oral cavity. Hence, TRPV1 activation could be involved in the AS aftertaste or even contribute to the poorly understood metallic taste sensation. Using Ca(2+) imaging on TRPV1 receptors heterologously expressed in the human embryonic kidney (HEK) 293 cells and on dissociated primary sensory neurons, we find that in both systems, AS activate TRPV1 receptors, and, moreover, they sensitize these channels to acid and heat. We also found that TRPV1 receptors are activated by CuSO(4), ZnSO(4), and FeSO(4), three salts known to produce a metallic taste sensation. In summary, our results identify a novel group of compounds that activate TRPV1 and, consequently, provide a molecular mechanism that may account for off tastes of sweeteners and metallic tasting salts.     

Female rats show a bimodal preference response to the artificial sweetener sucralose

The preference of female Sprague-Dawley rats for sucralose, a non-nutritive sweetener derived from sucrose, was evaluated in 23 h two-bottle tests with water or saccharin. Overall, the rats displayed weak or no preferences for sucralose (0.25-4 g/l) over water but strong preferences for saccharin (0.5-8 g/l) over water and saccharin (1 g/l) over sucralose (0.5 g/l). The rats also preferred a saccharin + sucrose mixture to sucrose, but sucrose to a sucralose + sucrose mixture. There were marked individual differences in sucralose preferences: about half the rats preferred sucralose to water at some concentrations while most remaining rats avoided sucralose. Both subgroups preferred saccharin to sucralose. Sucralose appears to have an aversive off-taste that reduces its palatability to rats.     

FORMULATING A NEW PASSION FRUIT JUICE BEVERAGE WITH DIFFERENT SWEETENER SYSTEMS

The aim of this work was to optimize the acceptability of a natural passion fruit beverage using different levels of passion fruit pulp and sucrose, and to determine the equi-sweet concentrations of aspartame, sucralose, and a blend of 80% aspartame  +  20% acesulfame-K for the optimized formula. A 2² central composite design was used to optimize the acceptability of the sucrose-sweetened beverage, which was accessed using a 9-point structured hedonic scale. Acceptability data were fitted to a second-order model equation provided in the design. The selected pulp content and sucrose concentration were, respectively, 2.5 ° Brix and 10%. Measurements of sweetness equivalence were accomplished using two types of sensory methods: magnitude estimation and difference-from-control tests. The concentrations of aspartame, sucralose and a blend of 80% aspartame  +  20% acesulfame-K found as equi-sweet to 10% sucrose in the studied passion fruit beverage were, respectively, 0.043, 0.016 and 0.026%.

PRACTICAL APPLICATIONS

This article deals with formulating a new passion fruit juice beverage with different sweetener systems. The research makes a very important contribution to the literature on sensory science used on product development by demonstrating the necessity to study the substitution of sucrose by high-intensity sweeteners every time a formulation is changed or a new product is developed.     

Artificial sweeteners and cancer risk: Results from the NutriNet-Santé population-based cohort study

BackgroundThe food industry uses artificial sweeteners in a wide range of foods and beverages as alternatives to added sugars, for which deleterious effects on several chronic diseases are now well established. The safety of these food additives is debated, with conflicting findings regarding their role in the aetiology of various diseases. In particular, their carcinogenicity has been suggested by several experimental studies, but robust epidemiological evidence is lacking. Thus, our objective was to investigate the associations between artificial sweetener intakes (total from all dietary sources, and most frequently consumed ones: aspartame [E951], acesulfame-K [E950], and sucralose [E955]) and cancer risk (overall and by site).Methods and findingsOverall, 102,865 adults from the French population-based cohort NutriNet-Santé (2009–2021) were included (median follow-up time = 7.8 years). Dietary intakes and consumption of sweeteners were obtained by repeated 24-hour dietary records including brand names of industrial products. Associations between sweeteners and cancer incidence were assessed by Cox proportional hazards models, adjusted for age, sex, education, physical activity, smoking, body mass index, height, weight gain during follow-up, diabetes, family history of cancer, number of 24-hour dietary records, and baseline intakes of energy, alcohol, sodium, saturated fatty acids, fibre, sugar, fruit and vegetables, whole-grain foods, and dairy products. Compared to non-consumers, higher consumers of total artificial sweeteners (i.e., above the median exposure in consumers) had higher risk of overall cancer (n = 3,358 cases, hazard ratio [HR] = 1.13 [95% CI 1.03 to 1.25], P-trend = 0.002). In particular, aspartame (HR = 1.15 [95% CI 1.03 to 1.28], P = 0.002) and acesulfame-K (HR = 1.13 [95% CI 1.01 to 1.26], P = 0.007) were associated with increased cancer risk. Higher risks were also observed for breast cancer (n = 979 cases, HR = 1.22 [95% CI 1.01 to 1.48], P = 0.036, for aspartame) and obesity-related cancers (n = 2,023 cases, HR = 1.13 [95% CI 1.00 to 1.28], P = 0.036, for total artificial sweeteners, and HR = 1.15 [95% CI 1.01 to 1.32], P = 0.026, for aspartame). Limitations of this study include potential selection bias, residual confounding, and reverse causality, though sensitivity analyses were performed to address these concerns.ConclusionsIn this large cohort study, artificial sweeteners (especially aspartame and acesulfame-K), which are used in many food and beverage brands worldwide, were associated with increased cancer risk. These findings provide important and novel insights for the ongoing re-evaluation of food additive sweeteners by the European Food Safety Authority and other health agencies globally.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03335644","term_id":"NCT03335644"}}NCT03335644.

Charlotte Debras and colleagues investigate investigate associations between artificial sweetener intakes and cancer risk in adults from a French population-based cohort.     

Larvicidal potential of the polyol sweeteners erythritol and xylitol in two filth fly species

The house fly, Musca domestica (L.) (Diptera: Muscidae), and the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), are two filth flies responsible for significant economic losses in animal production. Although some chemical control products target adults of both species, differences in mouthpart morphology and behavior necessitates distinct modalities for each. For these reasons, larvicides are an attractive means of chemical control. We assessed the potential of the polyol sweeteners erythritol and xylitol as larvicides to the house fly and stable fly. LC₅₀ values of erythritol against 2^nd instar larvae were 34.94 mg/g media (house fly) and 22.10 mg/g media (stable fly). For xylitol, LC₅₀ values were 74.91 mg/g media (house fly) and 41.58 mg/g media (stable fly). When given a choice, neither species showed a preference for ovipositing in media treated with either sweetener at various concentrations or in media without sweetener. Significantly lower development from egg to adult was observed when the 2^nd instar LC₅₀ equivalent of each sweetener was present in the media compared to controls. Erythritol and xylitol both have larvicidal qualities, however their effective concentrations would necessitate creative product formulation and deployment methods to control all stages of developing flies.     

Uptake of U-14C-glucose by Streptococcus mutans in the presence of saccharin

The uptake of U-14C-glucose by resting cells of Streptococcus mutans OMZ-176 was studied in the presence of the artificial sweetener saccharin as well as sodium chloride. Glucose grown cells were resuspended in phosphate buffer (0.05 M, pH 7.8), and the uptake of U-14C-glucose was observed for 150 min in time intervals of 30 min, in the presence of 0.02 and 2.00 mg/ml of sodium saccharin as well as sodium chloride. As compared to the control and the sodium chloride treatments, sodium saccharin at the highest concentration range more than doubled the accumulation of radioactive labelled carbon within the cells.     

Individual differences in perceived bitterness predict liking of sweeteners

Although recent molecular studies suggest that only one receptorand one signaling pathway are involved in the perception ofsweetness, this seems to contradict everyday experience thatpeople not only have different likes and dislikes of certainsweeteners but also perceive the sweeteners differently. Onepossible explanation is that variation in liking of sweetenersis due, in part, to variation across individuals in sensitivityto nonsweet tastes, such as bitterness, which are transducedby a variety of receptors. Fifty individuals were asked to rateintensities of several taste attributes of 10 sweeteners andto give hedonic assessments of each sweetener. Additionally,their sensitivity to 6-n-propyl-3-thiouracil (PROP) was determined.Results indicated that when matched for sweetness, the perceptionof bitterness and the sweetener compound were the 2 largestfactors contributing to overall liking of a sweetener. Sensitivityto PROP did not contribute significantly to the model.     

Saccharin induces morphological changes and enhances prolactin production in GH4C1 cells

Summary The artificial sweetener saccharin inhibits binding of epidermal growth factor (EGF) to cultured rat pituitary tumor cells (GH₄C₁ cells). Saccharin also causes morphological alterations in these cells, resulting in pronounced elongation, stretching, and firmer attachment of cells to the culture dishes. These alterations in cell shape are similar to those observed after treatment of GH₄C₁ cells with EGF and with thyrotropin-releasing hormone (TRH), both of which enhance prolactin (PRL) production in these cells. After assaying for PRL in saccharin-treated cultures, it was observed that this sweetener is also capable of stimulating PRL production two-to sixfold in a dose-dependent manner. Enhancement of PRL production can be observed at 0.5 mM saccharin, yet this is 10 times less than the saccharin concentration required to alter cell shape. These effects of saccharin on cell morphology and on PRL production are reversible in GH₄C₁ cell cultures. When added to cultures along with maximal concentrations of EGF or TRH, the effects of saccharin on PRL production are additive, suggesting that the actions of saccharin are mediated by a somewhat different pathway from that of the peptide hormones. Pulse labeling studies indicate that the enhancement of PRL production is highly specific inasmuch as saccharin was found to decrease the overall rate of protein synthesis in these cells. Saccharin also causes a decrease in the rate of DNA synthesis under these treatment conditions. Mitomycin C, which similarly inhibited DNA synthesis, had no effect on cell morphology or PRL production. This investigation was supported by a Faculty Research Grant from Wheaton College