Carlos F. Mendez

Silencing the expression of mitochondrial acyl-CoA thioesterase I and acyl-CoA synthetase 4 inhibits hormone-induced steroidogenesis

Arachidonic acid and its lypoxygenated metabolites play a fundamental role in the hormonal regulation of steroidogenesis. Reduction in the expression of the mitochondrial acyl‐CoA thioesterase (MTE‐I) by antisense or small interfering RNA (siRNA) and of the arachidonic acid‐preferring acyl‐CoA synthetase (ACS4) by siRNA produced a marked reduction in steroid output of cAMP‐stimulated Leydig cells. This effect was blunted by a permeable analog of cholesterol that bypasses the rate‐limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The inhibition of steroidogenesis was overcome by addition of exogenous arachidonic acid, indicating that the enzymes are part of the mechanism responsible for arachidonic acid release involved in steroidogenesis. Knocking down the expression of MTE‐I leads to a significant reduction in the expression of steroidogenic acute regulatory protein. This protein is induced by arachidonic acid and controls the rate‐limiting step. Overexpression of MTE‐I resulted in an increase in cAMP‐induced steroidogenesis. In summary, our results demonstrate a critical role for ACS4 and MTE‐I in the hormonal regulation of steroidogenesis as a new pathway of arachidonic acid release different from the classical phospholipase A2 cascade.

EFFECTS OF L-ARGININE IN RAT ADRENAL CELLS : INVOLVEMENT OF NITRIC OXIDE SYNTHASE

The effects of l-arginine on corticosterone production, cGMP, and nitrite levels were examined in zona fasciculata adrenal cells. l-Arginine significantly decreased both basal and ACTH-stimulated corticosterone production. This effect was still evident when steroidogenesis was induced by 8-bromo-cAMP and 22(R)-hydroxycholesterol, but not in the presence of exogenously added pregnenolone. l-Arginine increased cGMP and nitrite levels,; these effects were blocked by the nitric oxide synthase inhibitor, NG-nitro-l-arginine methyl-ester. Transport of l-[3H]arginine was rapid, saturable, and monophasic, with an apparent Km of 163 ± 14 μm and a maximum velocity of 53 ± 6 pmol/min·105 cells. The basic amino acids l-lysine and l-ornithine, but not d-arginine or the nitric oxide synthase inhibitors NG-nitro-l-arginine methyl-ester and NG-nitro-l-arginine, impaired l-arginine uptake. Taken together, these results suggest that steroidogenesis in zona fasciculata adrenal cells may be negatively modulated by l-arginine...

Involvement of arachidonic acid and the lipoxygenase pathway in mediating luteinizing hormone-induced testosterone synthesis in rat leydig cells

Evidence has been introduced linking the lipoxygenase products and steroidogenesis in Leydig cells, thereby supporting that this pathway may be a common event in the hormonal control of steroid synthesis. On the other hand, it has also been reported that lipoxygenase products of arachidonic acid (AA) may not be involved in Leydig cells steroidogenesis. In this paper, we investigated the effects of PLA2 and lipoxygenase pathway inhibitors on steroidogenesis in rat testis Leydig cells. The effects of two structurally unrelated PLA2 inhibitors (4-bromophenacyl bromide (BPB) and quinacrine) were determined. BPB blocked the LH- and Bt2cAMP-stimulated testosterone production but had no effect on 22(4)-OH-cholesterol conversion to testosterone. Quinacrine caused a dose-dependent inhibition of LH- and Bt2cAMP-induced steroidogenesis. The effects of different lipoxygenase pathway inhibitors (nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), caffeic acid and esculetin) have also been determined. Both NDGA and ETYA inhibited LH- and Bt2cAMP-stimulated steroid synthesis in a dose-related manner. Furthermore caffeic acid and esculetin also blocked the LH-stimulated testosterone production. Moreover, exogenous AA induced a dose-dependent increase of testosterone secretion which was inhibited by NDGA. Our results strongly support the previous concept that the lipoxygenase pathway is involved in the mechanism of action of LH on testis Leydig cells.

Control of muscle mitochondria by insulin entails activation of Akt2-mtNOS pathway: implications for the metabolic syndrome.

Background In the metabolic syndrome with hyperinsulinemia, mitochondrial inhibition facilitates muscle fat and glycogen accumulation and accelerates its progression. In the last decade, nitric oxide (NO) emerged as a typical mitochondrial modulator by reversibly inhibiting citochrome oxidase and oxygen utilization. We wondered whether insulin-operated signaling pathways modulate mitochondrial respiration via NO, to alternatively release complete glucose oxidation to CO2 and H2O or to drive glucose storage to glycogen. Methodology/Principal Findings We illustrate here that NO produced by translocated nNOS (mtNOS) is the insulin-signaling molecule that controls mitochondrial oxygen utilization. We evoke a hyperinsulinemic-normoglycemic non-invasive clamp by subcutaneously injecting adult male rats with long-lasting human insulin glargine that remains stable in plasma by several hours. At a precise concentration, insulin increased phospho-Akt2 that translocates to mitochondria and determines in situ phosphorylation and substantial cooperative mtNOS activation (+4–8 fold, P<.05), high NO, and a lowering of mitochondrial oxygen uptake and resting metabolic rate (−25 to −60%, P<.05). Comparing in vivo insulin metabolic effects on gastrocnemius muscles by direct electroporation of siRNA nNOS or empty vector in the two legs of the same animal, confirmed that in the silenced muscles disrupted mtNOS allows higher oxygen uptake and complete (U-14C)-glucose utilization respect to normal mtNOS in the vector-treated ones (respectively 37±3 vs 10±1 µmolO2/h.g tissue and 13±1 vs 7.2±1 µmol 3H2O/h.g tissue, P<.05), which reciprocally restricted glycogen-synthesis by a half. Conclusions/Significance These evidences show that after energy replenishment, insulin depresses mitochondrial respiration in skeletal muscle via NO which permits substrates to be deposited as macromolecules; at discrete hyperinsulinemia, persistent mtNOS activation could contribute to mitochondrial dysfunction with insulin resistance and obesity and therefore, to the progression of the metabolic syndrome.

MAPK Phosphatase-1 (MKP-1) Expression Is Up-Regulated by hCG/cAMP and Modulates Steroidogenesis in MA-10 Leydig Cells

MAP kinases (MAPKs), such as ERK1/2, exert profound effects on a variety of physiological processes. In steroidogenic cells, ERK1/2 are involved in the expression and activation of steroidogenic acute regulatory protein, which plays a central role in the regulation of steroidogenesis. In MA-10 Leydig cells, LH and chorionic gonadotropin (CG) trigger transient ERK1/2 activation via protein kinase A, although the events that lead to ERK1/2 inactivation are not fully described. Here, we describe the hormonal regulation of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates MAPKs, in MA-10 cells. In our experiments, human CG (hCG)/cAMP stimulation rapidly and transiently increased MKP-1 mRNA levels by a transcriptional action. This effect was accompanied by an increase in protein levels in both nuclear and mitochondrial compartments. In cells transiently expressing flag-MKP-1 protein, hCG/cAMP promoted the accumulation of the recombinant protein in a time-dependent manner (10-fold at 1 h). Moreover, hCG/cAMP triggered ERK1/2-dependent MKP-1 phosphorylation. The blockade of cAMP-induced MAPK kinase/ERK activation abated MKP-1 phosphorylation but only partially reduced flag-MKP-1 protein accumulation. Together, these results suggest that hCG regulates MKP-1 at transcriptional and posttranslational level, protein phosphorylation being one of the mechanisms involved in this regulation. Our study also demonstrates that MKP-1 overexpression reduces the effects of cAMP on ERK1/2 phosphorylation, steroidogenic acute regulatory gene promoter activity, mRNA levels, and steroidogenesis, whereas MKP-1 down-regulation by small interfering RNA produces opposite effects. In summary, our data demonstrate that hCG regulates MKP-1 expression at multiple stages as a negative feedback regulatory mechanism to modulate the hormonal action on ERK1/2 activity and steroidogenesis.

Activation of the alternative pathway of complement during the acute phase of typical haemolytic uraemic syndrome

Haemolytic uraemic syndrome (HUS) is characterized by haemolytic anaemia, thrombocytopenia and acute renal failure. We studied the activation state of classical and alternative pathways of complement during the acute phase of Shiga toxin‐associated HUS by performing a prospective study of 18 patients and 17 age‐matched healthy controls to evaluate C3, C3c, C4, C4d, Bb and SC5b‐9 levels. SC5b‐9 levels were increased significantly in all patients at admission compared to healthy and end‐stage renal disease controls, but were significantly higher in patients presenting with oliguria compared to those with preserved diuresis. C3 and C4 levels were elevated significantly at admission in the non‐oliguric group when compared to controls. No significant differences were found for C4d values, whereas factor Bb was elevated in all patients and significantly higher in oliguric patients when compared to both controls and non‐oliguric individuals. A positive and significant association was detected when Bb formation was plotted as a function of plasma SC5b‐9 at admission. Bb levels declined rapidly during the first week, with values not significantly different from controls by days 3 and 5 for non‐oligurics and oligurics, respectively. Our data demonstrate the activation of the alternative pathway of complement during the acute phase of Stx‐associated HUS. This finding suggests that complement activation may represent an important trigger for the cell damage that occurs during the syndrome.

Cisplatin inhibits testosterone synthesis by a mechanism that includes the action of reactive oxygen species (ROS) at the level of P450scc

Cisplatin (Cs) is a chemotherapeutic agent able to generate reactive oxygen species (ROS) which are linked to several side effects of the drug. Even when it is known that Cs produces Leydig cell dysfunction, it is unknown whether this particular side effect is mediated by ROS. The aim of this study was to evaluate the in vitro effects of Cs on testosterone production and the participation of ROS in this effect. We demonstrate that Cs promotes the generation of ROS in a time-, and concentration-dependent fashion, not only in mouse testicular interstitial cells but also in MA-10 Leydig cells. Also, Cs inhibits testosterone synthesis in a concentration-dependent fashion (5-50 μM for 4 h) and to a similar extent, in cells exposed to human chorionic gondadotropin hormone (hCG), to an analog of the second messenger cAMP (8Br-cAMP) or to a freely diffusible cholesterol analog (22R-hydroxycholesterol). However, this treatment does not inhibit the conversion of pregnenolone to testosterone. These data suggest that Cs exerts its inhibitory action on testosterone synthesis by an action at the level of P450scc. We also demonstrated that an antioxidant impairs the inhibitory effect of Cs on the conversion of the cholesterol analog into pregnenolone and that Cs does not change the expression level of P450scc mRNA. Therefore, it is concluded that Cs inhibits testosterone synthesis by a mechanism that includes the inhibition of P450scc by ROS.

Arachidonic acid regulation of steroid synthesis: new partners in the signaling pathway of steroidogenic hormones.

Although the role of arachidonic acid (AA) in trophic hormone‐stimulated steroid production in various steroidogenic cells is well documented 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, the mechanism responsible for AA release remains unknown. We have previously shown evidence of an alternative pathway of AA generation in steroidogenic tissues. Our results are consistent with the hypothesis that, in steroidogenic cells, AA is released by the action of a mitochondrial acyl‐CoA thioesterase (MTE‐I). We have shown that recombinant MTE‐I hydrolyses arachidonoyl‐CoA to release free AA. An acyl‐CoA synthetase specific for AA, acyl‐CoA synthetase 4, has also been described in steroidogenic tissues. In the present study we investigate the new concept in the regulation of intracellular levels of AA, in which trophic hormones can release AA by mechanisms different from the classical PLA2‐mediated pathway. Inhibition of ACS4 and MTE‐I activity by triacsin C and NDGA, respectively results in a reduction of StAR mRNA and protein abundance. When both inhibitors are added together there is a synergistic effect in the inhibition of StAR mRNA, StAR protein levels and ACTH‐stimulated steroid synthesis. The inhibition of steroidogenesis produced by the NDGA and triacsin C can be overcome by the addition of exogenous AA. In summary, results shown here demonstrate a critical role of the acyl‐CoA synthetase and the acyl‐CoA thioesterase in the regulation of AA release, StAR induction, and steroidogenesis. This further suggests a new concept in the regulation of intracellular distribution of AA through a mechanism different from the classical PLA2‐mediated pathway that involves a hormone‐induced acyl‐CoA synthetase and a hormone‐regulated acyl‐CoA thioesterase.

Oxidative stress status during the acute phase of haemolytic uraemic syndrome

BACKGROUND Haemolytic uraemic syndrome (HUS) is characterized by haemolytic anaemia, thrombocytopaenia and acute renal failure. The aim of this study was to investigate the levels of oxidative stress (OS) during the acute phase of HUS. METHODS This prospective study included 18 patients diagnosed with D + HUS, 6 age-matched healthy controls and 29 children with end-stage renal disease (ESRD) not caused by HUS under regular haemodialysis. Plasma lipid peroxidation and non-enzymatic antioxidant defences were measured as thiobarbituric acid-reactive substances (TBARs) and total reactive antioxidant potential (TRAP), respectively, during hospitalization and in control individuals. RESULTS TBARs were significantly higher in both oliguric and non-oliguric patients at admission (1.8 ± 0.1; 1.7 ± 0.2 μM) and discharge (1.5 ± 0.1; 1.0 ± 0.1 μM) vs controls (0.5 ± 0.1 μM, P < 0.01) following disease progression. Maximal TBARs values differed significantly between oliguric and non-oliguric groups (4.5 ± 0.9 vs 2.4 ± 0.3 μM, P < 0.01) and were significantly higher (P < 0.05) than those found in ESRD patients (1.63 ± 0.1). TRAP values were significantly higher at admission and when the disease was fully established (measured here as highest TBARs record) vs controls (675 ± 51, 657 ± 60 and 317 ± 30 μM Trolox, P < 0.01), and were similar to control values at discharge (325 ± 33 μM Trolox). CONCLUSIONS We demonstrate here increased levels of OS during the acute phase of HUS, with peak plasma lipid peroxidation values well above those registered in ESRD individuals, and suggest a connection between OS and the clinical course of HUS.

DETECTION OF A MITOCHONDRIAL KINASE COMPLEX THAT MEDIATES PKA-MEK- ERK-DEPENDENT PHOSPHORYLATION OF MITOCHONDRIAL PROTEINS INVOLVED IN THE REGULATION OF STEROID BIOSYNTHESIS

In order to achieve the goal of this article, as an example we will describe the strategies followed to analyze the presence of the multi-kinase complex at the mitochondria and the posttranslational modification of two key mitochondrial proteins, which participate in the regulation of cholesterol transport across the mitochondrial membranes and in the regulation of steroid biosynthesis. Hormones, ions or growth factors modulate steroid biosynthesis by the posttranslational phosphorylation of proteins. The question still remains on how phosphorylation events transmit a specific signal to its mitochondrial site of action. Cholesterol transport requires specific interactions in mitochondria between several proteins including a multi-kinase complex. The presence of this multi-kinase complex at the mitochondria reveals the importance of the posttranslational modification of mitochondrial proteins for its activity and functions. The activation of PKA triggers the posttranslational modification of the mitochondrial acyl-CoA thioesterase (Acot2), which releases arachidonic acid (AA) in the mitochondria, and the activation of a kinase cascade that leads to the phoshorylation of the steroidogenic acute regulatory (StAR) protein. The function of StAR is to facilitate the access of cholesterol to the first enzyme of the biosynthesis process and its induction is dependent on Acot2 and intramitochondrial AA release. Truncation of the StAR protein is associated with the steroid deficiency disease, congenital lipoid adrenal hyperplasia.