Sweet taste receptors play roles in artificial sweetener-induced enhanced urine output in mice

Artificial sweeteners (ASs) provide intense sweetness with minimal calories but have been associated with adverse metabolic outcomes, including weight gain, adiposity, type 2 diabetes, and cardiovascular disease. Sweet taste is mediated by T1R2/T1R3 receptors that trigger calcium-mediated signaling via gustducin. Components of sweet taste signaling are present in many non-taste organs, including kidney and bladder, suggesting extraoral roles. Prior work links AS consumption—especially saccharin—to increased urine volume and altered urinary electrolytes, and shows T1R2/T1R3 expression in bladder urothelium. However, whether sweet taste receptors mechanistically contribute to AS-induced changes in urine output remains unresolved. This study tests the hypothesis that AS exposure at ADI and higher doses alters urine output via modulation of T1R2/T1R3 expression and signaling in bladder.

Background studies show: (1) ASs activate T1R2/T1R3 and gustducin, initiating Ca2+-dependent pathways central to physiology; (2) T1R receptors are expressed in multiple non-taste organs, including bladder umbrella cells; (3) Saccharin is absorbed, not metabolized, and rapidly excreted in urine; (4) ASs can enhance bladder contraction in vitro via altered extracellular ions (K+, Cl−, Ca2+); (5) Overactive bladder can increase frequency and potentially urine volume. Acceptable Daily Intakes (ADIs) are 5 mg/kg/day for saccharin, 15 mg/kg/day for sucralose, and 9 mg/kg/day for acesulfame K. These findings motivated examining whether bladder sweet taste receptors mediate AS-induced changes in urinary output.

Study design: Male C57BL/6J mice and tas1r3 knockout (T1R3KO) mice were used. After 1-week acclimation, C57BL/6J mice (n=70) were randomized into 7 groups (n=10/group) receiving water (control) or sucralose, acesulfame K (AceK), or saccharin at ADI-based low dose or higher dose for 4 weeks. An additional cohort (n=30; 15 WT and 15 T1R3KO) was assigned to water or AceK at ADI or high dose (n=5/group). Housing: 25 °C, 40% humidity, 12 h light/dark cycle, standard chow. Sweetener dosing: Aqueous solutions prepared in deionized water. High-dose sweetness targets selected to equalize drinking preference across sweeteners (sucralose sweetness 24; AceK 20; saccharin 30). ADI doses calculated from regulatory ADI values and expected daily intake. Designed concentrations: sucralose ADI 0.18 mM; high 2 mM. Saccharin ADI 0.12 mM; high 20 mM. AceK ADI 0.20 mM; high 10 mM. Measurements: Body weight and drinking volume recorded every 2–3 days; AS intake derived from drinking volume. Urine output: Prior to collection, AS solutions were replaced with water for 48 h. Twenty-four-hour urine was collected in metabolic cages with ice-bath collectors to minimize evaporation/contamination. Relative urine output (RUO) was calculated as urine volume mL divided by (water intake mL × body weight g). Urinary electrolytes (Na+, K+, Cl−, Ca2+) were measured with a Synchron CX9 analyzer per manufacturer’s protocol. Tissue collection: After euthanasia, kidney, stomach, bladder, and testes were harvested, rinsed, snap-frozen, and stored at −80 °C. Western blotting: Protein (50 µg) from bladder (and kidney, stomach, testis for specific assays) was separated by SDS-PAGE, transferred to PVDF, blocked, and probed with primary antibodies against T1R2 (sc-50306), T1R3 (sc-50352), and β-actin (R1207), followed by HRP-conjugated secondary antibody. Bands were visualized by ECL and quantified by densitometry (ImageJ), normalized to β-actin. RT-qPCR: Total RNA from bladder was extracted (TRIzol), reverse-transcribed, and analyzed by SYBR Green qPCR. Primers: tas1r3 (forward 5'-CAGTCAAAGCATTGCTGCCT-3'; reverse 5'-ATAGCTGACCTGTGGCATGGA-3'); tas1r2 primer sets tested but yielded CT >37–38; GAPDH used as reference. ΔΔCt method for relative expression. Statistics: Data expressed as mean ± SD. One-way ANOVA with Dunnett’s post hoc test or Student’s t-test where appropriate; significance thresholds as reported (*P<0.05; **P<0.01; ***P<0.001). Ethical approval obtained (Zhejiang Gongshang University IACUC No. 2018R06).

• Body weight: No change at ADI doses; significant reductions in AceK 10 mM and saccharin 20 mM groups.
• Drinking volume: Increased significantly in all high-dose groups; ADI groups showed similar consumption to water.
• Relative urine output (RUO): Increased in both ADI and 10 mM AceK groups versus water. Saccharin increased RUO only at high dose (20 mM). Sucralose showed no significant RUO change at ADI or 2 mM. Among sweeteners, saccharin high-dose had the largest RUO effect; low-dose AceK produced higher RUO than high-dose sucralose.
• Urinary ions: Na+ significantly altered only in saccharin group (decrease vs water). K+ significantly reduced in all AceK groups and in high-dose sucralose and saccharin; K+ reduction positively correlated with increased RUO. Cl− showed no significant change in AceK groups; saccharin decreased Na+ and Cl−. Ca2+ showed no significant differences among groups.
• Tissue T1R3 protein expression (AceK 10 mM, 4 weeks): Bladder T1R3 increased approximately two-fold vs control (P<0.0001). Kidney T1R3 slightly down-regulated; stomach and testis slightly increased, not significant.
• Genotype effect (AceK exposure): WT C57BL/6J recapitulated RUO increases. In T1R3KO mice, ADI AceK produced no significant RUO change; high-dose AceK increased RUO vs KO controls but was attenuated compared with WT at the same dose, implicating T1R3 in the response.
• Bladder receptor expression by sweetener and dose (4 weeks): • T1R3 protein increased significantly in all high-dose groups (sucralose, AceK, saccharin); low-dose groups showed non-significant upward trends. • T1R2 protein: No significant change with sucralose (both doses). Increased significantly with AceK at both doses (high-dose >2× control). Increased with high-dose saccharin; no change at low-dose saccharin. • tas1r3 mRNA: Significantly increased at high-dose saccharin and high-dose AceK (>2-fold for saccharin vs control). Increases in sucralose (both doses) and low-dose saccharin were not significant. tas1r2 mRNA was not reliably quantifiable (CT >37–38).

Four weeks of oral exposure to artificial sweeteners differentially affected urine output in mice. AceK increased RUO at both ADI and high dose, while saccharin required high dose and sucralose had no detectable effect despite higher consumption at preference-matched sweetness. The lack of sucralose effect is consistent with its lower gastrointestinal absorption relative to AceK and saccharin, indicating that systemic availability may determine bladder exposure and physiological impact. Mechanistically, AceK and saccharin upregulated T1R3 (and, dose-dependently, T1R2) in bladder, with minimal or no upregulation in kidney, and AceK-induced RUO increases were blunted in T1R3KO mice. Together, these findings implicate extraoral sweet taste receptors—particularly T1R3 in bladder—in modulating urinary excretion in response to ASs. Changes in urinary K+ correlated with RUO, suggesting that AS-triggered sweet taste signaling may influence ion handling and/or bladder contractility, consistent with prior evidence that ASs can enhance bladder contraction via altered extracellular Ca2+ influx. The partial preservation of RUO increases in T1R3KO mice at high AceK dose suggests additional pathways, possibly renal effects or non-T1R3 chemosensory mechanisms, contribute to the phenotype. Overall, the data support a model in which AS exposure, especially AceK, enhances bladder sweet receptor signaling, leading to increased urine output and altered urinary electrolyte composition.

This study demonstrates that artificial sweeteners, particularly acesulfame K and high-dose saccharin, increase urine output in mice, and that this effect is associated with upregulation of sweet taste receptors (T1R2/T1R3) in the bladder. Genetic disruption of T1R3 attenuates AceK-induced RUO increases, supporting a causal role for sweet taste signaling in extraoral tissues. Urinary K+ changes track with RUO alterations, suggesting a link between sweet receptor activation, ion homeostasis, and bladder function. These findings identify bladder sweet taste receptors as contributors to AS-induced changes in urinary physiology and highlight differential effects among sweeteners, with AceK exerting effects even at ADI-equivalent low doses. Future work should employ bladder-specific T1R3 knockout models, perform comprehensive renal pathology and function assessments, and investigate gut–bladder signaling and hormonal mediators to delineate systemic versus local mechanisms.

• tas1r2 mRNA could not be reliably quantified in bladder (qPCR CT >37–38), limiting gene-level interpretation for T1R2.
• T1R3 knockout was global; tissue-specific (e.g., bladder-specific) knockout models are needed to ascribe causality to bladder receptors.
• Possible involvement of other organs (gut, stomach, kidney) and systemic factors (e.g., GLP-1/2) was not directly tested.
• Renal histopathology and detailed renal function parameters were not assessed; thus, contributions from kidney ion handling remain unresolved.
• Sample sizes for genotype comparison (n=5/group) were modest, potentially limiting power for some endpoints.
• Sucralose systemic exposure was not quantified; pharmacokinetics were inferred from literature rather than measured.