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. 2018 Dec 20;3(24):e97293. doi: 10.1172/jci.insight.97293

Figure 2. LPA4 selectively activates Gα12/13 protein in C3H10T1/2- and mouse embryonic fibroblast–derived (MEF-derived) adipocytes.

Figure 2

(A) Top, schematic of the assay system for predominant activation of LPA4 with octadecenyl phosphate (ODP) in the presence of Ki16425. Middle and bottom, ethidium bromide–stained agarose gels of reverse transcription PCR (RT-PCR) products of expressed LPA receptors in C3H10T1/2-derived (middle) and MEF-derived (bottom) adipocytes. (B and C) Effects of ODP (2.5 μM) on intracellular cAMP levels in C3H10T1/2-derived adipocytes (n = 4). Isoproterenol (ISOP, 10 μM) and lactic acid (30 mM) were used as positive controls for Gαs (B) and Gαi (C) protein activation, respectively. (D) A representative chart recording of intracellular Ca2+ level in C3H10T1/2-derived adipocytes upon stimulation with ODP (1 μM). Endothelin-1 (ET-1, 100 nM) was used as a positive control for Gαq protein activation. (E) Effects of ODP and siRNA against LPA4 or Gα12/13 on serum response factor–responsive element (SRF-RE) luciferase activity in C3H10T1/2-derived adipocytes (n = 4). (F) Effects of Rho and ROCK inhibition by Rho inhibitor I (2 μg/ml) and Y27632 (10 μM), respectively, on ODP-induced SRF-RE luciferase activity in C3H10T1/2-derived adipocytes (n = 4). (G and H) Effects of ODP (1 μM) on intracellular cAMP levels in MEF-derived adipocytes (n = 4). Isoproterenol (ISOP, 10 μM) and lactic acid (30 mM) were used as positive controls for Gαs (G) and Gαi (H) protein activation, respectively. (I) A representative chart recording of intracellular Ca2+ level in MEF-derived adipocytes upon stimulation with ODP (1 μM). Endothelin-1 (ET-1, 100 nM) was used as a positive control for Gαq protein activation. (J) Effects of ODP (1 μM) on SRF-RE luciferase activity in MEF-derived adipocytes isolated from WT and Lpar4-KO mice (n = 4). Results are representative of at least 3 independent experiments. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; 1-way ANOVA with Bonferroni’s (E and F) or Dunnett’s (B,C,G,H) post hoc test, and unpaired Student’s t test (J) were used.