Journal: The Journal of Biological Chemistry
Article Title: Metabolic control of luteinizing hormone-responsive ovarian steroidogenesis
doi: 10.1016/j.jbc.2024.108042
Figure Lengend Snippet: LHCGR/PKA pathway triggers phosphorylation of enzymes involved in de novo lipogenesis. A , phosphorylation of ACC1 (ACACA) Ser79 and ACLY Ser455 in the small luteal cells treated with different concentrations of LH (1–100 ng/ml) for 30 min. Data are presented as fold change and mean ± SD (n = 3–5). B , phosphorylation of ACC1 Ser79 and ACLY Ser455 in the small luteal cells treated with cAMP/PKA activator-forskolin (FSK; 10 μM) for 2 to 30 min. Data are presented as a fold change (FC) and mean ± SD (n = 3). Data normalized to total protein loaded on each lane. C , ATP production in the small luteal cells pretreated with ACLY inhibitor (BMS303141; 10–50 μm) and then treated with LH (10 ng/ml) for 240 min. Data are presented as fold change (FC) and mean ± SD (n = 3). D , cyclic AMP (cAMP) production by the small luteal cells pretreated with ACLY inhibitor (BMS303141; 25 μm) and then treated with LH (10 ng/ml) for 240 min. Data are presented as fold change (FC) and mean ± SD (n = 3). E and F , representative blots showing phosphorylation of PKA substrates and content of steroidogenic proteins (STAR, CYP11A1), electron transport chain proteins or marker of mitochondria (TOM20) and PKA catalytic subunits in the small luteal cells pretreated with ACLY inhibitor (BMS303141; 25 μM) and then treated with LH (10 ng/ml) for 240 min. G , cyclic AMP (cAMP) production by the small luteal cells with knockdown CPT1A (si CPT1A ; 50 nM) and then treated with LH (10 ng/ml) for 240 min. Data are presented as fold change (FC) and mean ± SD (n = 3). H , representative blots showing phosphorylation of PKA substrates and content of PKA catalytic subunits in the small luteal cells with knockdown CPT1A (si CPT1A ; 50 nM) and then treated with LH (10 ng/ml) for 240 min. For all graphs ∗, ∗∗, ∗∗∗ and ∗∗∗∗ mean significant change with p < 0.05, p < 0.01, p < 0.001, and p < 0.0001. Data were analyzed by one-way ANOVA followed by the Bonferroni post hoc test. ACACA, acetyl-CoA carboxylase alpha; ACLY, ATP citrate lyase; CPT1A, carnitine palmitoyltransferase 1A; LH, luteinizing hormone; PKA, protein kinase A.
Article Snippet: Phospho-ACLY (Ser455; #4331), phospho-ACACA (Ser79; #3661), CYP11A1 (#14217), phospho-PKA substrates (#9624), and TOM20 (#42406) antibodies were purchased from Cell Signaling Technology, Inc. Total OXPHOS Antibody Cocktail (ab110413), STAR (ab96637), ATP assay, and L-lactate assay were purchased from Abcam.
Techniques: Phospho-proteomics, Marker, Knockdown
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