Fig 1.
AMPK expression in MLTC-1 cells.
(A) Identification of AMPK in MLTC-1. Cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose membrane and then, probed with an anti-AMPKα antibody. Cell lysates from mouse spleen were used as positive controls (A: right). Negative control (Ctrl): primary antibody is missing (A: left). (B), (C) Immunolocalization of AMPK in MLTC-1 cells. Subcellular distribution of AMPK in Leydig cells was observed by confocal microscopy. AMPKα (C, green) immunoreactivity is detected in both the cell membrane and the cytoplasm. Nuclei were stained with DAPI (B, C, blue). Negative control (Ctrl): primary antibody is missing (B).
Fig 2.
FLX enhances AMPK phosphorylation in MLTC-1 cells.
After a 1-hour incubation in the presence of FLX (A), MET (B) or A-769662 (C), MLTC-1 lysates were prepared and resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-phospho-Thr172-AMPKα, anti-AMPKα and anti-GADPH antibody. Bands for phospho-Thr172-AMPKα were detected at 62 kDa (top bands). Total AMPKα (62 kDa middle bands) and GADPH (37 kDa bottom bands) were used as loading controls and the phosphorylated protein AMPKα (Thr172)/total AMPKα ratio is shown at the bottom. Data represent mean ± SEM of 6 independent experiments (n = 6). Results were analyzed by one-way ANOVA, followed by the Dunnett’s test posttest). *: significant difference (* P< 0.05; ** P<0.01), ns: no significant difference.
Fig 3.
Effect of FLX on intracellular cAMP response of MLTC-1 cells to hLH or FSK/sub-hLH.
(A), (C) Real-time recording of luminescence under stimulation of MLTC-1 cells by hLH and FSK/sub-hLH in the presence of 0 to 100μM FLX. (B), (D) Dose-dependent effects of FLX on hLH and FSK/sub-hLH responses respectively, determined by the Area Under Curve (AUC) of individual kinetics in Fig 3A and 3C. Data represent mean ± SEM of 3 independent experiments (n = 3). Results were analyzed by one-way ANOVA, followed by the Dunnett’s test posttest). *: significant difference (** p < 0.01; *** p < 0.001), ns: no significant difference.
Fig 4.
Effect of A-769662 on intracellular cAMP response of MLTC-1 cells to hLH or FSK/sub-hLH.
(A), (C) Real-time recording of luminescence under stimulation of MLTC-1 cells by hLH and FSK/sub-hLH respectively in presence of 0 to 1000μM A-769662. (B), (D) Dose-dependent effects of A-769662 on hLH and FSK/sub-hLH responses respectively, determined by the Area Under Curve (AUC) of individual kinetics in Fig 4A and 4C. Data represent mean ± SEM of 3 independent experiments (n = 3). Results were analyzed by one-way ANOVA, followed by the Dunnett’s test posttest). *: significant difference (** p < 0.01; *** p < 0.001), ns: no significant difference.
Fig 5.
Effect of MET on cAMP response of MLTC-1 cells to hLH or FSK/sub-hLH.
(A), (C) Real-time recording of luminescence under stimulation of MLTC-1 cells by hLH or FSK/sub-hLH in the presence of 0 to 1000μM MET. (B), (D) Dose-dependent effects of MET determined on hLH and FSK/sub-hLH responses respectively, by the Area Under Curve (AUC) of individual kinetics in Fig 5A and 5C. Data represent mean ± SEM of 3 independent experiments (n = 3). Results were analyzed by one-way ANOVA, followed by the Dunnett’s test posttest). *: significant difference (* P< 0.05; ** P<0.01).
Fig 6.
Effect of Compound C on FLX, MET, or A-769662 induced AMPK phosphorylation.
MLTC-1 cells were preincubated with or without compound C (5μM) for 30 min before the addition of 100μM FLX or 12μM MET or 37μM A-769662, and incubated for an additional 60 min. The experiment was performed 4 times independent experiments (n = 4). Ratio values are means ± SEM. Results were analyzed by one-way ANOVA, followed by the Dunnett’s test posttest). *: significant difference (* P< 0.05; ** P<0.01), ns: no significant difference.
Fig 7.
Effect of Compound C on FLX, MET, or A-769662 on intracellular cAMP response of MLTC-1 cells to hLH.
(A1), (B1), (C1) Real-time recording of luminescence under stimulation of MLTC-1 cells by hLH in presence of FLX, MET, or A-769662. (A2), (B2), (C2) Dose-dependent response to FLX, MET, or A-769662 determined by the Area Under Curve (AUC) of individual kinetics in figs A1, B1 and C1 respectively. Data represent mean ± SEM of 3 independent experiments (n = 3). Results were analyzed by one-way ANOVA, followed by the Dunnett’s test posttest). *: significant difference (** p< 0.01; *** P<0.001), ns: no significant difference.
Fig 8.
Effect of FLX, MET, or A-769662 on MLTC-1 cells viability.
Cells were incubated at 37°C for 1 hour in the presence or absence of FLX (A) or MET (B) or A-769662 (C). The experiments were repeated 6 times, values (%) are mean ± SEM (n = 6). Results were analyzed by one-way ANOVA, followed by the Dunnett’s test posttest). ns = no significant difference.
Fig 9.
Effect of FLX or MET or A-769662 on ATP concentration in MLTC-1 cells.
ATP concentration in living cells was monitored using the Cell-Titer-Glo Assay. Cells were incubated at 37°C in the presence or absence of FLX (A) or MET (B) or A-769662 (C). The experiments were repeated 6 times independent experiments, values (%) are mean ± SEM (n = 6). Results were analyzed by one-way ANOVA, followed by the Dunnett’s test posttest). *: significant difference (* P< 0.05), ns: no significant difference.
Fig 10.
Effect of fluoxetine in hLH or FSK/sub-hLH -promoted steroid production in MLTC-1 and primary Leydig cells.
(A), (B) MLTC-1 cells were preincubated with or without FLX (or MET, or A-769662) for 1 hour and then the cells were stimulated for 3 hours with hLH or FSK/sub-hLH before progesterone productions were measured. (C) Primary Leydig cells were preincubated with or without FLX for 1 hour and then the cells were stimulated for 3 hours with hLH before testosterone productions were measured. Data are means ± SEM of 3 independent experiments performed in duplicate (n = 3). Results were analyzed by one-way ANOVA, followed by the Dunnett’s test posttest). *: significant difference (* P< 0.05; ** P<0.01), ns: no significant difference.
Fig 11.
Proposed mechanisms of FLX inhibition of cAMP accumulation in MLTC-1 cells under hLH stimulation.
Up or down arrows next to a metabolite or a protein show how the concentration or the activity changes in response to FLX, A-769662, or MET. After uptake into MLTC-1 cells MET and FLX accumulate in mitochondria where they inhibit the respiratory chain, lowering cytoplasmic ATP and increasing ADP and AMP. Increase of the AMP/ATP ratio activates AMPK, which in some way inhibits AC activity. Moreover, the decrease in ATP (AC substrate) also slowers cAMP synthesis. Unlike MET and FLX, treatment with A-769662 activates AMPK without inhibiting cellular ATP levels. As a consequence, the effect on the LH-stimulated cAMP pathway is decreased in MLTC-1 cells incubated with FLX, A-769662 or MET, the effect being AMPK-dependent and/or AMPK-independent; dashed black arrows mark are used for hypotheses.