Figure 1.
Structures of P2Y6R ligands: nucleotide agonists.
Figure 2.
(A and B). Effects of P2Y6R agonist MRS2957 on glucose uptake in C2C12 cells (A) and 3T3-L1 cells (B).
MRS2957 was applied in the presence or absence of P2Y6R antagonist MRS2578 (1 µM). *P<0.05, when compared to controls (n = 3).
Figure 3.
(A and B). Effects of inhibitors of intracellular signaling pathways on P2Y6R-mediated glucose uptake.
C2C12 cells (A) or 3T3-L1 cells (B) were preincubated with inhibitors of MAPK (U0126, 10 µM) and AMPK (Compound C, 10 µM) followed by treatment with P2Y6R agonist MRS2957. * P<0.05, when compared to controls, #P<0.05, when compared to MRS2957, 100 nM (n = 3).
Figure 4.
Glucose uptake in C2C12 myotubes (A) and 3T3-L1 adipocytes (B) transfected with AMPK siRNA.
*P<0.05, when compared to controls, NS, non-significant when compared to controls (n = 3).
Figure 5.
Phosphorylation of AMPK in C2C12 myotubes (A and B) after treatment with MRS2957.
This is a representative blot from three individual experiments. The density of the bands were quantified and compared to the controls (B and D). *P<0.05, when compared to controls, NS, non-significant when compared to controls (n = 3). Lane 1, Control, 2, MRS2957 (100 nM), 3, MRS2578(1 µM), 4, MRS2578+ MRS2957, 5, AICAR (200 µM), 6, Suramin (20 µM)+MRS2957.
Figure 6.
Phosphorylation of AMPK in 3T3-L1 adipocytes (A and B) after treatment with MRS2957.
This is a representative blot from three individual experiments. The density of the bands were quantified and compared to the controls (B and D). *P<0.05, when compared to controls, NS, non-significant when compared to controls (n = 3). Lane 1, Control, 2, MRS2957 (100 nM), 3, MRS2578(1 µM), 4, MRS2578+ MRS2957, 5, AICAR (200 µM), 6, Suramin (20 µM)+MRS2957.
Figure 7.
Translocation of glucose transporters GLUT4 in the plasma membrane upon activation of P2Y6R.
(A) Immunoblotting is used to determine the changes in the membrane levels of the GLUT4 transporter. This is a representative blot from three individual experiments. Key to lanes: 1–3, C2C12 myotubes; 4–6, 3T3-L1 adipocytes. 1 and 4, controls; 2 and 5, MRS2957, 100 nM; 3 and 6, 200 nM insulin. (B) GLUT4/β-actin ratio based on the quantitation of the bands.
Figure 8.
Glucose uptake assay in primary adipocytes isolated from WT (A) and P2Y6R-KO mice (B).
*P<0.05, when compared to controls, NS, non-significant when compared to controls (n = 3).
Figure 9.
HDAC5 associated GLUT4 promoter DNA in the MEF2 binding region following treatment with MRS2957 (100 nM) or AICAR (200 µM) or MRS2578 (1 µM)+MRS2957 in C2C12 myotubes using ChIP assay.
*P<0.05, when compared to controls, #P<0.05, when compared to MRS2578+ MRS2957 (n = 3).