Fig 1: The effects of TDBP-TAZTO (5 and 50 mg/kg) on plasma ACTH (A) and serum CORT (B).The results are presented as mean ± SE (n = 10/group). *p < 0.05, vs. control group.
Fig 2: Dietary supplementation with icosapent ethyl reduces corticoid levels in obese mice.(A) Schematic representation of the experimental setup. (B to D) Acyl chains of phospholipids (B), FFA (C), and lipid mediators (D) were measured by HPLC-MS in the adrenal glands of mice fed a HFD with or without icosapent ethyl (n = 7 mice per group). (E) Volcano plot based on RNA-seq data in the adrenal cortex of mice, which received HFD supplemented with icosapent ethyl versus mice fed a HFD without icosapent ethyl (n = 4 mice per group). (F to I) GSEA analysis for genes related to fatty acid metabolism, cholesterol homeostasis, steroid biosynthesis, and mitochondrial envelope formation (n = 4 mice per group). (J and K) Corticosterone and aldosterone plasma levels in mice, which received HFD without and with icosapent ethyl, measured by LC-MS/MS (n = 7 mice per group). (L and M) Primary CD31-CD45- adrenocortical cells (L) and NCI-H295R cells (M) were treated with 66 µM EPA for 18 or 48 hours, respectively, and Fads2 expression was determined by qPCR using 18S expression as a housekeeping gene [n = 7 in (L) and n = 8 in (M)]. (N) NCI-H295R cells were treated for 48 hours with 66 µM EPA and stimulated or not with forskolin (FSK;1 µM) in the last 24 hours. Steroids were measured in the culture supernatant by LC-MS/MS (n = 6). (O) Primary adrenocortical cells were treated for 18 hours with 66 µM EPA and stimulated or not with ACTH (10 ng/ml). Steroids were measured in the culture supernatant by LC-MS/MS (n = 10). Data in (B) to (D) and (L) and (M) are presented as mean ± SEM, data in (N) and (O) as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. N.D., not determined.
Fig 3: FADS2 determines the mitochondrial lipidome and is required for steroidogenesis in adrenocortical cells.(A) Relative Fads2 expression in different tissues of 8-week-old WT C57BL6J mice determined by qPCR using Tbp as a housekeeping gene. Data are shown as mean 2?Ct (n = 8). BAT, brown adipose tissue; SAT, subcutaneous adipose tissue; GAT, gonadal adipose tissue. (B) Relative Fads2 expression in CD31-CD45-, CD31+, and CD45+ cell populations of the mouse adrenal cortex. Expression of Fads2 in CD31-CD45- cells was set to 1. 18S was used as an internal control (n = 6). (C and D) PCA of the phospholipidome (C) and heatmap showing the differentially regulated lipid species (D) of mitochondrial fractions of NCI-H295R cells treated for 18 hours with SC-26196 (10 µM) or equal amount of DMSO (n = 3, one of two experiments). (E and F) Mitogreen (E) and TMRE (F) staining in primary adrenocortical cells treated for 18 hours with SC-26196 (10 µM) or DMSO (n = 6). MFI, mean fluorescence intensity. (G) OCR measurement of primary adrenocortical cells treated for 18 hours with SC-26196 or DMSO (n = 4). AUC, area under curve (H) Cholesterol levels determined by LC-MS/MS in mitochondrial fractions of NCI-H295R cells treated for 18 hours with SC-26196 or DMSO and stimulated or not with forskolin (FSK; 1 µM) for 15 min. Results of three independent experiments are shown here. (I) Progesterone, 11-deoxycorticosterone, corticosterone, and aldosterone levels were determined by LC-MS/MS in supernatants of primary adrenal cell cultures treated for 18 hours with SC-26196 or DMSO and then stimulated or not for 1 hour with ACTH (10 ng/ml) in the presence of SC-26196 or DMSO. The cell culture medium was changed before ACTH treatment (n = 12). Data in (B), (H), and (I) are shown as mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Supplier Page from Abnova Corporation for ACTH (Mouse/Rat) ELISA Kit
Stealth Micro Spotting Pins (Max.):Store the kit at 4°C.