Tissue-Specific Ablation of ACSL4 Results in Disturbed Steroidogenesis

Abstract

ACSL4 is a member of the ACSL family that catalyzes the conversion of long-chain fatty acids to acyl-coenzyme As, which are essential for fatty-acid incorporation and utilization in diverse metabolic pathways, including cholesteryl ester synthesis. Steroidogenic tissues such as the adrenal gland are particularly enriched in cholesteryl esters of long-chain polyunsaturated fatty acids, which constitute an important pool supplying cholesterol for steroid synthesis. The current studies addressed whether ACSL4 is required for normal steroidogenesis. CYP11A1 promoter‒mediated Cre was used to generate steroid tissue‒specific ACSL4 knockout (KO) mice. Results demonstrated that ACSL4 plays an important role in adrenal cholesteryl ester formation, as well as in determining the fatty acyl composition of adrenal cholesteryl esters, with ACSL4 deficiency leading to reductions in cholesteryl ester storage and alterations in cholesteryl ester composition. Statistically significant reductions in corticosterone and testosterone production, but not progesterone production, were observed in vivo, and these deficits were accentuated in ex vivo and in vitro studies of isolated steroid tissues and cells from ACSL4-deficient mice. However, these effects on steroid production appear to be due to reductions in cholesteryl ester stores rather than disturbances in signaling pathways. We conclude that ACSL4 is dispensable for normal steroidogenesis.

Figure 1.

ACSL4 mRNA expression in various tissues under different physiological conditions. (A) mRNA was isolated from various tissues of 16- to 24-wk-old male and female mice (n = 3 to 6), and levels of ACSL4 mRNA were analyzed using RT-PCR. (B) Male mice that were 16 to 24 wk old were injected with ACTH or carrier, and tissues were collected 1 h later for analysis of ACSL4 mRNA levels (n = 3 to 6). (C) Female mice that were 16 to 24 wk old were injected with ACTH or carrier, and various tissues were collected 1 h later for analysis of ACSL4 mRNA levels (n = 3 to 6). Data are expressed as means ± SEM. *P < 0.05. BAT, brown adipose tissue; Epi, epididymal adipose tissue; Gastro-R; gastrocnemius muscle-red; Retro, retroperitoneal adipose tissue; SubQ, subcutaneous adipose tissue.

Figure 2:

Generation of tissue-specific ACSL4 KO mice. (A) Levels of ACSL4 mRNA in control and ACSL4 KO mice. mRNA was isolated from adrenals and other tissues of 16- to 24-week-old mice (n = 3), and levels of ACSL4 mRNA were analyzed using RT-PCR. Experiments were repeated three times. (B). Western blot analysis of ACSL4 protein in various tissues and isolated cells from control and ACSL4 KO mice. Total cell extracts were prepared from tissues of 16- to 24-week-old control and ACSL4 KO mice (n = 3 to 5). Leydig and granulosa cells were isolated from male and female mice, respectively, following the protocol described in the Materials and Methods. Protein levels of ACSL4 were analyzed by immunoblotting with ACSL4 antibody. (C) Analysis of expression of various ACSL genes and selected genes that are involved in steroidogenesis in the adrenal using TaqMan quantitative PCR. (D) Analysis of expression of various genes involved in steroidogenesis in the adrenal using TaqMan quantitative PCR. (E) Histochemical analysis of ACSL4 in the adrenal, ovary, and testis of control and ACSL4 KO mice. Data are expressed as means ± SEM. **P < 0.01; ***P < 0.005. BAT, brown adipose tissue; Ctrl, control; ND, not detected; WAT, white adipose tissue.

Figure 3.

Total cholesterol and cholesteryl esters in control and ACSL4 KO mice adrenals. Total lipids were extracted from the adrenals of 12- to 24-week-old female control and ACSL4 KO mice (n = 7 or 8) using the Folch method. (A) Total cholesterol and (B) cholesteryl esters were assayed. (C) Cholesteryl ester compositions were analyzed using mass spectrometry (n = 5 or 6). Data are expressed as means ± SEM. *P < 0.05; ***P < 0.005.

Figure 4.

Serum corticosterone in control and ACSL4 KO mice. (A) Serum corticosterone levels in control and ACSL4 KO male mice. Male control and ACSL4 KO mice that were 16 to 24 wk old (n = 10) were treated with cosyntropin (2.5 IU per mouse, 1 h), and serum was collected before and 1 h after the treatment. (B) Serum corticosterone levels in control and ACSL4 KO female animals: 12- to 24-wk-old female control and ACSL4 KO mice (n = 9) were treated with cosyntropin (2.5 IU per mouse, 1 h), and serum was collected before and 1 h after the treatment. Serum corticosterone level was assayed using an ELISA kit. Data are presented as means ± SEM. *P < 0.05. -, without; +, with.

Figure 5.

Decreased ex vivo adrenal steroidogenesis in ACSL4 KO mice. (A) Ex vivo corticosterone production. Adrenals from 12- to 24-wk-old female mice (n = 9) were cut in half and cultured ex vivo. After 0.5-h preincubation, media were changed with fresh media with or without 2.5 mM of Bt₂cAMP and incubated for another 1 h. Corticosterone levels in the media were measured. (B) Total adrenal protein. Protein content in tissue extracts of adrenals was assayed using bicinchoninic acid reagent. Total protein content of the adrenals was assayed and expressed as a percentage of the animal’s body weight. (C) Effects of HDL on ex vivo corticosterone production. Adrenals from 12- to 24-wk-old female mice (n = 9) were cut in half and cultured ex vivo. After 0.5-h preincubation, media were changed with fresh media with 2.5 mM of Bt₂cAMP and with or without HDL (500 µg CE/mL) and incubated for another 1 h. Corticosterone levels in the media were measured. Data are expressed as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005. -, without; +, with.

Figure 6.

Gonadal steroid production in ACSL4 KO mice. (A) Serum testosterone in control and ACSL4 KO male mice. Male control and ACSL4 KO mice that were 16 to 24 wk old (n = 10) were treated with hCG (2.5 IU per mouse), and serum was collected before and 4 h after the treatment for measurement of testosterone. (B) Testosterone production in isolated Leydig cells. Leydig cells were isolated from 16- to 24-wk-old male mice (n = 6) using Percoll gradients after the procedures described in “Materials and Methods.” Freshly purified Leydig cells were incubated without (basal) or with Bt₂cAMP (2.5 mM) and/or HDL (500 µg CE/mL) for 5 h, and incubation media were collected and frozen until assayed by RIA for secreted testosterone. Three separate experiments were performed, with triplicate samples for each treatment. (C) Serum progesterone in control and ACSL4 KO female mice. Female control and ACSL4 KO mice that were 16 to 24 wk old (n = 10) were treated with PMSG/hCG (2.5 IU per mouse), and serum was collected before and 5 h after the treatment for measurement of progesterone. (D) Progesterone production in isolated granulosa cells. Age-matched female control and ACSL4 KO mice (n = 3 to 5) were injected twice with 5 IU of PMSG for 48 h. Granulosa cells were isolated after the procedures described in the Materials and Methods. Freshly purified granulosa cells were incubated without (basal) or with Bt₂cAMP (2.5 mM) and/or human apoE-free HDL (500 µg CE/mL) for 5 h. After incubation, media were collected and frozen until assayed by ELISA for secreted progesterone. Three separate experiments were performed, with triplicate samples for each treatment. Data are expressed as means ± SEM. *P < 0.05; **P < 0.01. -, without; +, with.