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. 2021 Jan 9;296:100274. doi: 10.1016/j.jbc.2021.100274

Figure 7.

Figure 7

Activation of GPRC6A signaling upregulates ATGL expression in 3T3-L1 adipocytes.A, immunoblot analysis of adipose triglyceride lipase (ATGL) and forkhead box O1 (FoxO1) in 3T3-L1 adipocytes stimulated with the indicated concentrations of ornithine or uncarboxylated osteocalcin (GluOC) for 8 h. B and D, cells were incubated with the indicated concentrations of ornithine for 8 h, after which the amount of free fatty acids released into the culture medium was measured (D). Cell lysates were subjected to immunoblot analysis of acyl-CoA oxidase-1 (ACOX1), medium-chain acyl-CoA dehydrogenase (MCAD), and peroxisome proliferator–activated receptor α (PPARα) (B). C, immunoblot analysis of GPRC6A and ATGL in 3T3-L1 adipocytes transfected with control or GPRC6A siRNAs and stimulated with the indicated concentrations of ornithine, GluOC, or dibutyryl-cAMP (db-cAMP) for 8 h. E, immunoblot analysis of ATGL in 3T3-L1 adipocytes stimulated with the indicated concentrations of ornithine, GluOC, or db-cAMP in the absence or the presence of protein kinase A (PKA) inhibitor (PKI) 14-22 for 8 h. F, immunoblot analysis of ATGL in explants of the adipose tissue from control and adG6AKO stimulated with the indicated concentrations of ornithine or GluOC for 24 h. G, immunoblot analysis of ATGL in explants of the adipose tissue from control mice stimulated with the indicated concentrations of ornithine, GluOC, or db-cAMP in the absence or the presence of PKI 14-22 for 24 h. Representative blots and quantitative data for relative protein abundance normalized to that of β-actin from multiple blots are shown. All quantitative data are the mean ± SEM from at least three independent experiments. ∗p < 0.05, ∗∗p < 0.01 for the indicated comparisons or #p < 0.05 versus the corresponding value for its corresponding control (one-way ANOVA).