cAMP increases mitochondrial cholesterol transport through the induction of arachidonic acid release inside this organelle in Leydig cells

Abstract

Arachidonic acid (AA) is a fatty acid with 20 carbons and four cis double bonds that are the source of its flexibility and its reactivity with molecular oxygen. The oxidation can happen nonenzymatically or through the action of three types of oxygenases: cyclooxygenase, lipoxygenase and cytochrome P450. Most of the effects of AA are attributable to its conversion by those enzymes to prostaglandins, leukotrienes and other bioactive products [1]. AA itself also has biological activity; however, the number of its described actions is reduced compared to the effects described for the AA metabolites. Moreover, it is not very well documented whether nonmetabolized AA is released and elicits special functions in a specific cellular compartment [2].Transport of long-chain fatty acids in cells definitely occurs when they are tightly linked to CoA by esterification catalyzed by acyl-CoA synthetases [3]. In mammalian and yeast cells [4] it appears that the acyl-CoA synthetases merely enhance uptake indirectly. Thus, formation of the polar CoA-ester effectively traps the fatty acid in the cell and functions as part of a facilitated distribution in different cellular compartments.The mechanisms involved in the compartmentalization of long-chain acyl-CoA esters and free fatty acids We have investigated the direct effect of arachidonic acid on cholesterol transport in intact cells or isolated mitochondria from steroidogenic cells and the effect of cyclic-AMP on the specific release of this fatty acid inside the mitochondria. We show for the first time that cyclic-AMP can regulate the release of arachidonic acid in a specialized compartment of MA-10 Leydig cells, e.g. the mitochondria, and that the fatty acid induces cholesterol transport through a mechanism different from the classical pathway. Arachidonic acid and arachidonoyl-CoA can stimulate cholesterol transport in isolated mitochondria from nonstimulated cells. The effect of arachidonoyl-CoA is inhibited by the reduction in the expression or in the activity of a mitochondrial thioesterase that uses arachidonoyl-CoA as a substrate to release arachidonic acid. cAMP-induced arachidonic acid accumulation into the mitochondria is also reduced when the mitochondrial thioesterase activity or expression is blocked. This new feature in the regulation of cholesterol transport by arachidonic acid and the release of arachidonic acid in specialized compartment of the cells could offer novel means for understanding the regulation of steroid synthesis but also would be important in other situations such as neuropathological disorders or oncology disorders, where cholesterol transport plays an important role.Abbreviations AA, arachidonic acid; AA-CoA, arachidonoyl-CoA; Acot2, mitochondrial acyl-CoA thioesterase; ACS4, acyl-CoA synthetase 4; BPB, 4-bromophenacyl bromide; 8Br-cAMP, 8-bromo-cAMP; CHX, cycloheximide; CPT1, carnitine-palmitoyl transferase 1; DBI, diazepam-binding inhibitor; NDGA, nordihydroguaiaretic acid; P450scc, cholesterol side-chain cleavage cytochrome P-450 enzyme; PBR...

Functional Interaction between Acyl-CoA Synthetase 4, Lipooxygenases and Cyclooxygenase-2 in the Aggressive Phenotype of Breast Cancer Cells

“…ACSL4 provides arachidonoyl-CoA to the acyl-CoA thioesterase, ACOT2, which releases AA in the mitochondrion and directs this fatty acid to the LOX enzyme for its subsequent conversion to lipooxygenase metabolites [11], [12]. To investigate whether a similar mechanism operates in breast cancer cells, we studied ACOT2 expression and intramitochondrial AA and lipooxygenase metabolites levels in the MDA-MB-231 and MCF-7 cell lines.…”

“…[1- 14 C]-AA (New England Nuclear, Boston, MA, USA; specific activity 53.0 mCi.mmol-1) was added to the cultures in a concentration of 1 µCi.mmol-1per well (1×10 6 cells) in serum-free DMEM containing 0.5% fatty acid-free bovine serum albumin. The mitochondria were isolated, sonicated and lipids were extracted and analyzed by thin layer chromatography (TLC) on silica gel plates, as previously described [12]. The autoradiographs were quantified by densitometry.…”

“…However, an alternative pathway that releases AA in specific compartments of the cell, e.g. mitochondria, has been described in steroidogenic tissues [11], [12], [13], [14]. This pathway, in which the rate-limiting enzyme is ACSL4, provides arachidonoyl-CoA to a mitochondrial acyl-CoA thioesterase (ACOT2) that releases AA in mitochondria and directs this fatty acid to the LOX enzyme for its subsequent conversion to lipooxygenase metabolites [15], [16].…”

Mitochondrial Fusion and ERK Activity Regulate Steroidogenic Acute Regulatory Protein Localization in Mitochondria

“…In this regard, a previous report shows that over-expression or knockout of Mfn2 modulates and affects mRNA levels of several mitochondrial proteins [51], [52]. We have described an arachidonic acid [55] generation/exportation system that includes an Acyl-CoA synthetase 4 (Acsl4) [38], [40], [56]. Acsl4 is anchored at the MAM (mitochondrial associated-membrane) structures [57] and its activity determines the production rate of AA which is necessary for StAR gene expression [22].…”

Steroidogenesis in Leydig cells: effects of aging and environmental factors

“…Reduced numbers of LH receptors or their reduced coupling efficiency to G-protein affects cAMP production, and also affects other signaling cascades including arachidonic acid/cyclooxygenase-2 (COX-2) (Wang et al . 2005, Castillo et al . 2006) and mitogen-activated protein kinase (MAPK) (Tai & Ascoli 2011).…”