Artificial sweeteners stimulate adipogenesis and suppress lipolysis independently of sweet taste receptors

Abstract

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.

Keywords: Acesulfame K; Adipogenesis; Akt; G Protein-coupled Receptors (GPCR); Lipolysis; Metabolism; Saccharin; T1R2; T1R3.

Figures

FIGURE 1.

Sweet taste receptors T1R2 and T1R3 are expressed constitutively throughout adipogenesis and in adipose tissues.A, T1R2 and T1R3 expression throughout 8 days of adipogenesis in 3T3-L1 cells. B, T1R2 and T1R3 expression over 12 days of adipogenesis in eMSCs. C, the indicated tissues were collected from control and 13–15-week-old female db/db mice maintained on a chow diet, and expression of T1R3 mRNA was evaluated by quantitative PCR. Data are expressed as mean ± S.D. (error bars) with n = 4–6/tissue. Analysis by two-way analysis of variance indicates a significant effect of genotype (p < 0.05). gWat, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; pWAT, perirenal white adipose tissue.

FIGURE 2.

Saccharin stimulates adipogenesis of mouse and human precursor cells.A, 3T3-L1 cells were differentiated with DI in the presence of the indicated concentrations of saccharin. Seven days after induction, cells were stained for neutral lipid with Oil Red-O (upper panel), and lysates were evaluated for expression of FABP4 (lower panel). ERK1/2 was used as a loading control. B, quantification of saccharin-stimulated FABP4 expression from four independent experiments in DI-treated 3T3-L1 cells. Data are expressed as mean ± S.D. (error bars). p < 0.01 is indicated with **, and p < 0.005 is indicated with ***. C, 3T3-L1 cells were differentiated with components of the MDI mixture as indicated in the presence or absence of 4.5 mm saccharin. Adipogenesis was then evaluated after 8 days by Western blotting for FABP4. D, eMSCs were incubated in the presence of MDI, DI, or FBS with or without 2 mm saccharin supplementation. At day 16 of differentiation, the degree of differentiation was evaluated with photomicrographs (upper panels) and by expression of FABP4 (lower panels). E, human SVCs were induced with MDI for 14 days in the absence or presence of 4.5 mm saccharin. Adipogenesis was assessed with photomicrographs and by expression of FABP4. M, methylisobutylxanthine; D, dexamethasone; I, insulin.

FIGURE 3.

AceK stimulates adipogenesis of mouse and human precursor cells.A, 3T3-L1 cells were induced with DI and treated for 8 days with the indicated concentrations of AceK. After 8 days, cells were stained for neutral lipid with Oil Red-O. Lysates were evaluated for expression of FABP4. B, eMSCs were incubated in the presence of MDI, DI, or FBS with or without 2 mm AceK. After 16 days, the degree of differentiation was evaluated with photomicrographs (upper panels) and by expression of FABP4 (lower panels). C, human SVCs were induced with MDI for 14 days in the absence or presence of 4.5 mm AceK. Adipogenesis was determined with photomicrographs (upper panel) and by expression of FABP4 (lower panel).

FIGURE 4.

Saccharin stimulates expression of PPARγ and C/EBPα late during differentiation.A, 3T3-L1 cells were induced with DI and supplemented with 4.5 mm Sacc at the indicated time points. Cells were stained with Oil Red-O (upper panel), and lysates were collected for immunoblotting after 8 days (lower panels). C/EBPα (B) and PPARγ (C) expression was measured over a time course of adipogenesis in 3T3-L1 cells induced with DI and treated with vehicle, 4.5 mm Sacc, or 4.5 mm AceK for 4 days. Expression of C/EBPβ (D), C/EBPδ (E), and PREF1 (F) was measured over the first 5 days of differentiation in 3T3-L1 cells treated with Sacc or AceK. Significance was determined using Student's t test. Significant differences (p < 0.05) between vehicle and Sacc are denoted with *, and those between vehicle and AceK are denoted with #. Data are expressed as mean ± S.D. (error bars).

FIGURE 5.

Saccharin activates Akt and ERK1/2 signaling pathways in preadipocytes.A, 3T3-L1 preadipocytes were serum-starved for 2 h in HBSS and then treated with 0.45 or 4.5 mm saccharin for 30 min before lysis and immunoblot analyses with the indicated antibodies. B, 3T3-L1 cells were serum-starved in HBSS for 2 h and pretreated with 50 μm LY294002 for 1 h. Cells were then treated with the indicated concentrations of saccharin in the absence or presence of LY294002 for 30 min. After lysis, samples were probed by immunoblot with the indicated antibodies. Insulin at 25 nm was included as a control. C, 3T3-L1 cells were serum-starved in HBSS for 2 h and pretreated with 1 μm U0216, 5 μm U73122, or 2 μm wortmannin for 1 h. Cells were then treated with 4.5 mm saccharin in the absence or presence of each inhibitor for 30 min before immunoblotting for the indicated proteins. D, 3T3-L1 preadipocytes were serum-starved in HBSS for 2 h and treated with 4.5 mm saccharin for the indicated time periods before lysis and immunoblotting. E, 3T3-L1 cells were stimulated with DI in the presence or absence of 4.5 mm saccharin. Lysates were prepared at the indicated time points and probed by immunoblot for the indicated proteins. T308, threonine 308; S473, serine 473; T Akt, total Akt; T CREB, total CREB; T FOXO1, total FOXO1; T ERK1/2, total ERK1/2; WM, wortmannin.

FIGURE 6.

T1R3 and T1R2 are not required for saccharin-stimulated adipogenesis or Akt phosphorylation.A, WT and T1R3 KO eMSCs were differentiated in FBS supplemented with 0.45 mm saccharin. After 12 days, lipid accumulation was evaluated with Oil Red-O. B, WT and T1R3 KO eMSCs were serum-starved for 2 h in HBSS and treated for 30 min with increasing concentrations of saccharin (0, 0.02, 0.045, 0.2, 0.45, 2, and 4.5 mm) before collecting lysates for immunoblotting (upper panel). WT and T1R3 KO eMSCs were serum-starved for 2 h in HBSS before being treated with saccharin for the indicated time intervals (lower panel). C, WT and T1R2 KO eMSCs were differentiated in FBS supplemented with 0.45 mm saccharin. After 12 days, lipid accumulation was evaluated with Oil Red-O. D, WT and T1R2 KO eMSCs were serum-starved for 2 h in HBSS and treated for 30 min with increasing concentrations of saccharin (0, 0.02, 0.045, 0.2, 0.45, 2, and 4.5 mm) before lysis and immunoblotting (upper panel). WT and T1R2 KO eMSCs were maintained in calf serum (−) or treated with DI in the absence (−) or presence (+) of 4.5 mm saccharin for 30 min (lower panel). E, WT and T1R2/T1R3 KO (DKO) eMSCs were differentiated in FBS supplemented with 0.45 mm saccharin. After 12 days, lipid accumulation was evaluated with Oil Red-O. F, WT and DKO eMSCs were serum-starved for 2 h in HBSS and treated for 30 min with 4.5 mm saccharin before lysis and immunoblotting. T Akt, total Akt.

FIGURE 7.

Artificial sweeteners suppress lipolysis.A, following 1-h Sacc or AceK treatment, 3T3-L1 adipocytes were flash frozen, and intracellular metabolites were evaluated on an LC-MS platform by the University of Michigan Molecular Phenotyping Core. Of the metabolites that were significantly altered by the treatment, 69 were unique to Sacc, three were unique to AceK, and 14 were shared between both sweeteners. B, distribution of metabolites altered by Sacc alone (blue) or Sacc and AceK (yellow) following 1-h treatment in 3T3-L1 adipocytes. Long-chain fatty acids decreased by both saccharin and AceK are shown in red. The significance level on the y axis extends to 10e−16. C, glucose uptake was measured in 3T3-L1 adipocytes as described under “Materials and Methods.” Significant differences (p < 0.05) between 0 and 4 nm insulin are denoted with #. D, incorporation of [13C]palmitate into dipalmitoyl diacylglycerol (DAG) with 1 or 4 h of 4.5 mm Sacc treatment in 3T3-L1 adipocytes. E, 3T3-L1 adipocytes were serum-starved for 2 h in HBSS before treatment with 4.5 mm Sacc, 4.5 mm mannitol, or 10 μm Fsk. Glycerol content of the medium was assayed at the indicated time points. F, non-esterified fatty acid (NEFA) (μm) was measured from assay medium after 60 min of 4.5 mm Sacc treatment. G, glycerol content of assay medium was measured following 1-h treatment with Sacc, AceK, or sucralose (Sucr) at 0.45 and 4.5 mm. H, epididymal white adipose tissue (eWAT) explants were collected from C57BL/6J mice 20 min following injection of Sacc. Glycerol content of the medium was then measured following a 4-h incubation with vehicle, Fsk, or Sacc. Significant differences from vehicle treatment p < 0.05, p < 0.01, and p < 0.005 are denoted with *, **, and ***, respectively. Data are expressed as mean ± S.D. (error bars). Significance was determined using Student's t test. 2DOG, 2-deoxyglucose.

FIGURE 8.

T1R2/T1R3 are dispensable for saccharin-suppressed lipolysis.A, 3T3-L1 adipocytes were treated for 1 h with Sacc, Fsk, or both. Glycerol concentration in the medium was measured before collection of protein lysates from control and treated cells. Lysates were then probed for pHSL by Western blotting (left panel). HSL phosphorylation was then quantified by densitometry (right panel). B, 3T3-L1 adipocytes were serum-starved for 2 h and pretreated with LY294002 (LY) for 1 h before a 2-h Sacc treatment. Glycerol secretion was then measured in medium. C, 3T3-L1 adipocytes were serum-starved for 2 h and then treated for 45 min with Fsk or Sacc. cAMP concentration was quantified by ELISA. T1R3 KO (D), T1R2 KO (E), or DKO (F) eMSC adipocytes were treated with Sacc, Fsk, or both for 4 h before measuring glycerol accumulation in medium. Significant differences p < 0.05, p < 0.01, and p < 0.005 are denoted *, **, and ***, respectively. Data are expressed as mean ± S.D. (error bars). Significance was determined using Student's t test. tHSL, total HSL.

FIGURE 9.

Saccharin and neotame bind similarly to membranes of control and T1R2/T1R3 DKO eMSC progenitors. WT and DKO membranes were incubated with the indicated concentrations of saccharin (A) or neotame (B), and binding of artificial sweeteners to membranes was evaluated by saturation transfer difference NMR.