Opposing Roles of Sphingosine 1-Phosphate Receptors 1 and 2 in Fat Deposition and Glucose Tolerance in Obese Male Mice

Abstract

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that regulates fundamental cellular processes such as proliferation, migration, apoptosis, and differentiation through 5 cognate G protein-coupled receptors (S1P1-S1P5). We previously demonstrated that blockade of S1P2 signaling in S1P2-deficient mice attenuates high-fat diet-induced adipocyte hypertrophy and glucose intolerance and an S1P2-specific antagonist JTE-013 inhibits, whereas an S1P1/S1P3 dual antagonist (VPC23019) activates, adipogenic differentiation of preadipocytes. Based on those observations, this study examined whether an S1P1-specific agonist, SEW-2871, VPC23019, or their combination acts on obesity and glucose intolerance in leptin-deficient ob/ob mice. The oral administration of SEW-2871 or JTE-013 induced significant reductions in body/epididymal fat weight gains and epididymal/inguinal fat adipocyte sizes and improved glucose intolerance and adipocyte inflammation in ob/ob mice but not in their control C57BL/6J mice. Both SEW-2871 and JTE-013 decreased messenger RNA levels of tumor necrosis factor-α and CD11c, whereas they increased those of CD206 and adiponectin in the epididymal fats isolated from ob/ob mice with no changes in the levels of peroxisome proliferator activated receptor γ and its regulated genes. By contrast, VPC23019 did not cause any such alterations but counteracted with all those SEW-2871 actions in these mice. In conclusion, the S1P1 agonist SEW-2871 acted like the S1P2 antagonist JTE-013 to reduce body/epididymal fats and improve glucose tolerance in obese mice. Therefore, this study raises the possibility that endogenous S1P could promote obesity/type 2 diabetes through the S1P2, whereas exogenous S1P could act against them through the S1P1.

Keywords: S1P receptor; adipocyte; glucose intolerance; obesity; sphingosine 1-phosphate.

Figure 1.

Effects of JTE-013 (S1P₂ antagonist) and SEW-2871 (S1P₁ agonist) on body weight, food intake, and epididymal fat weight in wild-type (C57BL/6J) and ob/ob mice. A, Body weight change in wild-type (open symbols) and ob/ob (closed symbols) male mice fed the control diet (Con), JTE-013 (40 mg/kg)-formulated diet (JTE), or SEW-2871 (40 mg/kg)-formulated diet (SEW) after age 10 weeks. B, Daily food intake (g) at week 4, 8, and 12. Daily food intake was measured 3 times a week (Monday, Wednesday, and Friday) and then averaged. C, Epididymal fat weights at week 12. Data are mean ± SD (sample numbers [n] in parentheses) and the suppressive effects by JTE-013 or SEW-2871 are statistically significant at *P less than .05, **P less than .01, and ***P less than .001 in t test.

Figure 2.

Effects of JTE-013 and SEW-2871 on glucose tolerance and serum biochemical parameters in wild-type and ob/ob mice. A and B, Intraperitoneal glucose tolerance test (ip-GTT) in A, wild-type and B, ob/ob mice fed the control diet (Con), JTE-013 diet (JTE), or SEW-2871 diet (SEW) for 12 weeks. Areas under the curve (AUC) are presented in inset graphs. C to H, Serum levels of C, fasting blood glucose; D, fasting blood (immunoreactive) insulin; E, triacylglycerol (TAG); F, total cholesterol (T-Cho); G, aspartate transaminase (AST); and H, alanine transaminase (ALT) in wild-type and ob/ob mice fed the aforementioned diets for 12 weeks. Data are mean ± SD (n in parentheses), and the suppressive effects by JTE-013 or SEW-2871 are statistically significant at *P less than .05 and **P less than .01 in t test.

Figure 3.

Effects of JTE-013 and SEW-2871 on adipocyte hypertrophy and inflammation in epididymal and inguinal fats of wild-type and ob/ob mice. A to D, Representative hematoxylin/eosin (HE)-stained image of cross-sectional A, epididymal and C, inguinal adipocytes and their cell area sizes (B and D, respectively) from wild-type and ob/ob mice fed the control diet (Con), JTE-013 diet (JTE), or SEW-2871 diet (SEW) for 12 weeks. The suppressive effects by JTE-013 or SEW-2871 are statistically significant at *P less than .05, **P less than .01, ***P less than .001 in t test. Bars: 100 µm. E, Immunochemical CD11c and CD206 staining of epididymal adipocytes isolated from ob/ob mice. The nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). Bars: 50 µm. F, HE-stained hepatic sections isolated from ob/ob mice. Bars: 100 µm.

Figure 4.

Systemic effects of VPC23019 (S1P₁/S1P₃ antagonist) on obesity, glucose intolerance, and adipocyte hypertrophy/inflammation. A, Body weight change in wild-type (open symbols) and ob/ob (closed symbols) male mice fed the control diet (Con or C) or VPC23019 (40 mg/kg)-formulated diet (VPC or V) after age 10 weeks. B, Daily food intake calculated from 4 cycles of the sum values from 3 consecutive days between weeks 2 and 4 after food change. C, Epididymal fat weights at week 12. D, Intraperitoneal glucose tolerance test (ip-GTT) test in wild-type (left) and ob/ob (right) mice fed Con or VPC diet for 12 weeks. Areas under the curve (AUC) are presented in inset graphs. E to J, Serum levels of E, fasting blood glucose; F, fasting blood (immunoreactive) insulin; G, triacylglycerol; H, total cholesterol; I, aspartate transaminase (AST); and J, alanine transaminase (ALT) in wild-type and ob/ob mice fed the aforementioned diets for 12 weeks. K to N, Representative hematoxylin/eosin (HE)-stained image of K, epididymal and M, inguinal adipocytes and their cell area sizes (L and N, respectively) from wild-type and ob/ob mice. Bars: 100 µm. O, Immunochemical CD11c and CD206 staining of epididymal adipocyte sections prepared from ob/ob mice. The nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). Bars: 50 µm. P, HE-stained hepatic sections prepared from ob/ob mice. Bars: 100 µm. Data are mean ± SD (n in parentheses) and the effects of VPC23019 were not statistically significant in any parameters.

Figure 5.

Preventive effects of VPC23019 (S1P₁/S1P₃ antagonist) on SEW-2871 (S1P1 agonist)-induced alterations in obesity, glucose intolerance, and adipocyte hypertrophy/inflammation. A, Body weight change in wild-type (open symbols) and ob/ob (closed symbols) male mice fed with the control diet (Con or C), SEW-2871 (40 mg/kg)-formulated diet (SEW or S), SEW-2871 and VPC23019 (40 mg/kg each)-formulated diet (SEW + VPC or SV) after age 10 weeks. B, Daily food intake at week 12. C, Epididymal fat weights at week 12. D, Intraperitoneal glucose tolerance test (ip-GTT) test in wild-type (left) and ob/ob (right) mice fed Con, SEW, or SEW + VPC diet for 12 weeks. Areas under the curve (AUC) are presented in inset graphs. E to J, Serum levels of E, fasting blood glucose; F, fasting blood (immunoreactive) insulin; G, triacylglycerol; H, total cholesterol; I, aspartate transaminase (AST); and J, alanine transaminase (ALT) in wild-type and ob/ob mice fed the aforementioned diets for 12 weeks. K and L, Representative cross-sectional hematoxylin/eosin (HE)-stained images and cell area sizes of K, epididymal and L, inguinal adipocytes from wild-type and ob/ob mice. Bars: 100 µm. Data are mean ± SD (n in parentheses) and the VPC cotreatments mostly prevented the actions by SEW.

Figure 6.

Expression of adipocyte inflammation marker genes in epididymal and inguinal fats from wild-type and ob/ob mice fed the control, JTE-013, SEW-2871, or VPC23019 diet for 12 weeks. Messenger RNA levels of A and B, tumor necrosis factor α (Tnfa); C and D, CD11c (Cd11c); E and F, CD206 (Cd206); and G and H, adiponectin (Adipoq) in the A, C, E, and G, epididymal and B, D, F, and H, inguinal fats of wild-type and ob/ob mice were normalized by that of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and expressed with that of wild-type mice fed the same control diet as 1. Data are mean ± SD (n in parentheses); *P less than .05 and **P less than .01 vs wild-type samples (black asterisks) or control samples (red and blue asterisks for JTE-013 and SEW-2871 samples, respectively).

Figure 7.

Expression of Pparg and its regulated genes in epididymal and inguinal fats from wild-type and ob/ob mice fed the control, JTE-013, SEW-2871, or VPC23019 diet for 12 weeks. Messenger RNA levels of A and B, PPARγ (Pparg); C and D, fatty acid binding protein 4 (Fabp4); and E and F, lipoprotein lipase (Lpl) in the A, C, and E, epididymal and B, D, and F, inguinal (fats of wild-type and ob/ob mice were normalized by that of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and expressed with that of wild-type mice fed the same control diet as 1. Data are mean ± SD (n in parentheses) and no statistically significant differences were observed.

Figure 8.

Expression of uncoupling protein-1 (UCP1) in inguinal and epididymal fats. A, Immunochemical UPC1 (Ucp1) staining of inguinal adipocyte sections isolated from wild-type and ob/ob mice fed the control, JTE-013, SEW-2871, or VPC23019 diet for 12 weeks. The nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). B and C, Messenger RNA levels of UCP1 in the B, inguinal and C, epididymal fats of wild-type and ob/ob mice were normalized by that of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and expressed with that of wild-type mice fed the same control diet as 1. Data are mean ± SD (n in parentheses) and no statistically significant differences were observed.

Figure 9.

Proposed illustration of the actions of SEW-2871, JTE-013, and VPC23019 on obesity and glucose intolerance. Endogenously produced S1P acts mainly through S1P₂ to promote adipocyte hypertrophy and glucose intolerance in ob/ob mice, and the S1P₂ antagonist JTE-013 dampens its actions. By contrast, exogenously applied the S1P₁ agonist SEW-2871 activates only S1P₁ to improve adipocyte hypertrophy and glucose intolerance in ob/ob mice, which is prevented in the presence of the S1P1/S1P3 dual antagonist VPC23019. SEW-2871 could be a better option since it does not affect other S1P receptor subtypes and is not affected by endogenous S1P levels.