A novel regulatory function of sweet taste-sensing receptor in adipogenic differentiation of 3T3-L1 cells

Abstract

Background: Sweet taste receptor is expressed not only in taste buds but also in nongustatory organs such as enteroendocrine cells and pancreatic beta-cells, and may play more extensive physiological roles in energy metabolism. Here we examined the expression and function of the sweet taste receptor in 3T3-L1 cells.

Methodology/principal findings: In undifferentiated preadipocytes, both T1R2 and T1R3 were expressed very weakly, whereas the expression of T1R3 but not T1R2 was markedly up-regulated upon induction of differentiation (by 83.0 and 3.8-fold, respectively at Day 6). The α subunits of Gs (Gαs) and G14 (Gα14) but not gustducin were expressed throughout the differentiation process. The addition of sucralose or saccharin during the first 48 hours of differentiation considerably reduced the expression of peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer-binding protein α (C/EBPα at Day 2, the expression of aP2 at Day 4 and triglyceride accumulation at Day 6. These anti-adipogenic effects were attenuated by short hairpin RNA-mediated gene-silencing of T1R3. In addition, overexpression of the dominant-negative mutant of Gαs but not YM-254890, an inhibitor of Gα14, impeded the effects of sweeteners, suggesting a possible coupling of Gs with the putative sweet taste-sensing receptor. In agreement, sucralose and saccharin increased the cyclic AMP concentration in differentiating 3T3-L1 cells and also in HEK293 cells heterologously expressing T1R3. Furthermore, the anti-adipogenic effects of sweeteners were mimicked by Gs activation with cholera toxin but not by adenylate cyclase activation with forskolin, whereas small interfering RNA-mediated knockdown of Gαs had the opposite effects.

Conclusions: 3T3-L1 cells express a functional sweet taste-sensing receptor presumably as a T1R3 homomer, which mediates the anti-adipogenic signal by a Gs-dependent but cAMP-independent mechanism.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Expression of T1Rs during differentiation of 3T3-L1 cells.

A. The total RNAs prepared from 3T3-L1 cells at the indicated time points during differentiation were subjected to quantitative RT-PCR using mouse ribosomal protein S18 as an internal control as described in`Materials and Methods'. The mRNA levels of T1R2 and T1R3 are shown as the percentage of that of T1R3 at Day 6. Results are shown as the mean ± SE (n = 3–6). B. Immumoblot data for T1R3 and actin in undifferentiated (Day 0) and differentiated (Day 7) 3T3-L1 cells. C. Immunofluorescence staining images for T1R3 (a and b, red) and GLUT4 (c and d, green) in undifferentiated (Day 0, a and c) and differentiated (Day 7, b and d) 3T3-L1 cells. Nuclei were visualized with DAPI (blue). e, Subcellular distribution of T1R3 (red) in Day 7 cells. Arrowheads indicate peripheral localization of T1R3.

Figure 2. Effects of Sweeteners on Adipogenesis.

A. 3T3-L1 cells were differentiated in the presence of the indicated concentrations of sucralose or saccharin, and the expression levels of PPARγ and C/EBPα at Day 2 (48 hours) were examined by immunoblotting. Representative immunoblot data for PPARγ and C/EBPα (upper panel) and the relative amounts of the proteins normalized with β-tubulin (lower panel) are shown. Results are shown as the mean values of two independent experiments. B. 3T3-L1 cells were differentiated without (control) or with D-mannitol (20 mM), sucralose (20 mM), or saccharin (20 mM) during the first 48 hours of differentiation. The expression levels of PPARγ and C/EBPα at Day 2, and aP2 at Day 4 were examined by immunoblotting. Representative immunoblot data (upper panel) and the relative amounts of the proteins normalized with β-tubulin (lower panel) are shown. Results are shown as the mean ± SE (n = 3). ?, Pa), D-mannitol (20 mM) (b), sucralose (20 mM) (c) or saccharin (20 mM) (d) during the first 48 hours are shown (right panel). D. Adipose tissue-derived stem cells were differentiated to adipocytes as described in ‘Materials and Methods’ in the presence of none (control) (a), D-mannitol (20 mM) (b), sucralose (20 mM) (c) or saccharin (20 mM) (d) for 6 days, and were stained with Oil Red-O. The amounts of Oil Red-O (left panel) and microscopic images (right panel) are shown. Results are presented as the mean ± SE (n = 3). ?, P<0.001; ??, P<0.01 (vs. control). E. Undifferentiated 3T3-L1 cells were transfected with the pGIPz expression vectors containing non-silencing or T1R3-targeting shRNA sequences (30 μg each) by electroporation as described in ‘Materials and Methods’. Transfected cells were seeded on a 12-well dish and cultured to confluence before induction of differentiation without (control) or with sucralose (20 mM) or saccharin (20 mM). The expression level of T1R3 was measured by quantitative RT-PCR immediately before induction of differentiation (at Day 0) (left panel). The expression levels of PPARγ and C/EBPα at Day 2 (48 hours) were measured by immunoblotting. Representative immunoblot data and the relative amounts of the proteins normalized with β-tubulin are shown (middle panel). Results are presented as the mean ± SE (n = 3). ?, P<0.05 (vs. shControl). The amounts of Oil Red-O and the microscopic images of stained cells at Day 6 are shown (right panel). Cells transfected with non-silencing (a–c) or T1R3-targeting shRNA (d-f) were differentiated in the absence (a and d) or the presence of sucralose (20 mM) (b and e) or saccharin (20 mM) (c and f) during the first 48 hours. Results are presented as the mean ± SE (n = 3). ?, P<0.01; ??, P<0.05 (vs. shControl).

Figure 3. Roles for G proteins in Sweeteners Effects on Differentiation of 3T3-L1 cells.

A. Expression profiles of Gαgust, Gα14 and Gαs during differentiation of 3T3-L1 cells. The total RNAs were prepared from 3T3-L1 cells as described in Fig. 1 and the mRNA levels of Gαgust, Gα14 and Gαs were measured by quantitative RT-PCR using mouse ribosomal protein S18 as an internal control. Results are shown as the mean ± SE (n = 3–6). B. 3T3-L1 cells were differentiated without (control) or with sucralose (20 mM), saccharin (20 mM), or endothelin-1 (20 nM) in the absence (0.1% DMSO) or the presence of YM-254890 (10 µM). The expression levels of PPARγ and C/EBPα at Day 2 (48 hours) were measured by immunoblotting. Representative immunoblot data (upper panel) and the relative amounts of the proteins normalized with β-tubulin (lower panel) are shown. Gray and black bars show the control and the plus YM-254890 data, respectively. Results are shown as the mean values from two independent experiments. C. Undifferentiated 3T3-L1 cells were detached and transfected with the expression vectors containing wild-type or G226A mutant Gαs cDNAs (20 μg each) by electroporation as described in ‘Materials and Methods’. Transfected cells were seeded on a 6-well culture dish and cultured to confluence before induction of differentiation without (control) or with sucralose (20 mM) or saccharin (20 mM). The expression levels of PPARγ and C/EBPα were measured by immunoblotting at Day 2 (48 hours). Representative immunoblot data (upper panel) and the relative amounts of the proteins normalized with β-tubulin (lower panel) are shown. Gray and black bars show the control and the Gαs-G226A data, respectively. Results are shown as the mean ± SE (n = 3). P

Figure 4. Effects of Sweeteners on cAMP.

A. 3T3-L1 cells at Day 0 (undifferentiated), Day 2 and Day 6 of differentiation were stimulated without (control) or with sucralose (20 mM) or saccharin (20 mM) for 30 min at 37°C, and the cellular cAMP contents were measured as described in ‘Materials and Methods’. Results are shown as the mean ± SE (n = 3)., Pc). At the time point indicated with the arrow, cells were stimulated with 20 mM of sucralose. The [cAMP]c was shown as the reciprocal of the emission ratio of EYFP/ECFP (i.e. ECFP/EYFP). C. HEK293 cells were transfected with pcDNA3.1 vector (20 μg) alone (mock) or with the vector containing mouse T1R3 cDNA (20 μg) by electroporation and seeded on a 12-well culture plate. After incubation for 24 hours, cells were stimulated without (control) or with sucralose (20 mM) or saccharin (20 mM) for 30 minutes at 37°C and the cellular contents of cAMP were measured as described in ‘Materials and Methods’. Results are shown as the mean ± SE (n = 4–6)., P<0.05, P<0.01 (vs. control). D. HEK293 cells transfected by electroporation with pcDNA3.1 vector (mock), T1R2 (20 μg), T1R3 (20 μg), or both T1R2 and T1R3 (10 μg, each) were seeded on a 35 mm glass bottom dish, cultured for 24 hours and stimulated with 20 mM of sucralose at the time point indicated with the arrow. The [cAMP]c were monitored as described in B.

Figure 5. Role for Gαs in adipogenesis.

A. 3T3-L1 cells were differentiated for 48 hours in the absence (control) or the presence of cholera toxin (0.1 μg/ml) or forskolin (20 μM). Then the expression levels of PPARγ and C/EBPα were measured by immunoblotting. The representative immunoblot data (left panel) and the relative amounts of the proteins normalized with β-tubulin (right panel) are shown. Data are shown as the mean ± SE (n = 3)., P

Figure 6. A model for signal transduction mechanism downstream of the sweet taste-sensing receptors in taste cells and 3T3-L1 cells.

In taste cells (in the left side), T1R2+T1R3 heterodimeric sweet receptor activates PLCβ via gustducin (Ggust) or other G proteins, leading to [Ca2+]c elevation and membrane depolarization. In 3T3-L1 cells (in the right side), T1R3 homomeric receptor may activates Gs, which mediates the anti-adipogenic signal by a cAMP-independent mechanism. PLCβ: phospholipase C-β; DAG: diacylglycerol; IP3: inositol 1,4,5-trisphosphate; Px1: pannexin 1; AC: adenylate cyclase; PDE: cAMP phosphodiesterase.