The authors have declared that no competing interests exist.
Conceived and designed the experiments: RB FM FC GM GZ FF VDF. Performed the experiments: RB FM PC AMG LR PM GLG TS. Analyzed the data: RB FM PC AMG LR PM GLG TS. Contributed reagents/materials/analysis tools: FC GM GZ FF VDF. Wrote the manuscript: RB FM AMG FC GM GZ FF VDF.
A new role for fat supplements, in particular conjugated linoleic acid (CLA), has been delineated in steroidogenesis, although the underlying molecular mechanisms have not yet been elucidated. The aims of the present study were to identify the pathway stimulated by CLA supplementation using a cell culture model and to determine whether this same pathway is also stimulated
Fat supplements are nutritional ergogenic aids used by elite and recreational athletes; many sport magazines advise that their intake can improve endurance capacity, increase VO2max, reduce fat body mass, increase lean body mass, reduce muscle glycogen breakdown, improve metabolism, and prevent or reduce muscle damage and inflammatory responses [
CLA, one of the commercially available fat supplements, refers to a group of positional and geometrical isomers of linoleic acid (cis-9, cis-12-octadecadienoic acid), an omega-6 essential fatty acid that exhibits various physiological effects including anti-adipogenic, anti-carcinogenic, and immune modulation effects [
To our knowledge, currently only two pathways have been shown to be responsible for the possible effect of CLA on testosterone biosynthesis. First, in adipocytes, perilipin and hormone-sensitive lipase (HSL) create a protective coat on the lipid droplet surface. Under stimulation, both proteins become hyperphosphorylated, and perilipin is displaced from the lipid droplet, allowing HSL to convert cholesteryl ester to free cholesterol. In Leydig cells, the same pathway may stimulate testosterone production under CLA treatment. Second, CLA may alter steroidogenesis by up-regulating specific genes encoding enzymes and transport proteins involved in the synthesis of testosterone, such as 17α-hydroxylase/17,20-lyase (CYP17A1), which converts progesterone into androstenedione. It has been demonstrated that a change in CYP17A1 expression may directly affect the level of testosterone [
We hypothesised that CLA supplementation may stimulate testosterone biosynthesis by two possible pathways: i) perilipin and HSL should become hyperphosphorylated, thereby allowing HSL to convert cholesteryl ester into free cholesterol to increase the hormone production; or ii) specific genes encoding enzymes and transport proteins involved in steroidogenesis should be up-regulated to promote testosterone production. The aims of the present study were, therefore, to identify the pathway stimulated by CLA supplementation using the rat Leydig tumour cell line and to determine whether the same pathway is affected by CLA supplementation in mice, as well as determine its association with exercise.
R2C cells (cat. No. 89031606, ECACC, Health Protection Agency Culture Collections, Salisbury, United Kingdom) were cultured in M-199 medium (Invitrogen Corp., Carlsbad, CA, USA) supplemented with 15% horse serum (Invitrogen Corp), 2.5% foetal bovine serum (FBS; Invitrogen Corp.), and an antibiotic and antimycotic solution (100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B; Invitrogen Corp). Cells were incubated at 37°C in a humidified atmosphere with 5% CO2 and maintained in culture using standard techniques as previously described [
All animal experiments were approved by the committee on the ethics of animal experiments at the University of Palermo and adhered to the recommendations in the guide for the care and use of laboratory animals set by the NIH. Moreover, all experiments were performed in the Human Physiology Laboratory of the Department of Experimental Biomedicine and Clinical Neuroscience at the University of Palermo, which was formally authorised by Ministero della Sanità (Roma, Italy). Thirty-two male mice (BALB/cAnNHsd) were obtained from Harlan laboratories S.r.l. (Udine, Italy). The animals were kept at a constant 12:12 h light-dark cycle and had free access to food and water. Mice were randomly assigned to one of four groups (n = 8 per group): placebo sedentary (PLA-SED); CLA sedentary (CLA-SED); placebo trained (PLA-TR); or CLA trained (CLA-TR). The CLA groups (CLA-SED; CLA-TR) were gavaged with 35 µL per day (corresponding to the 0.5% of food ingested, approximately 4 g) Tonalin® FFA 80 food supplement containing CLA throughout the 6 week experimental period, while the placebo groups (PLA-SED; PLA-TR) were gavaged with 35 µL per day sunflower oil [
Testosterone measurements were performed in the laboratory of “Azienda Ospedaliera Villa Sofia–Cervello Palermo”. The total testosterone was measured using an Immulite 2000 (Siemens, Milan, Italy), according to the manufacturer’s instructions for the specific kit (Immunolite 2000 testosterone total), as described previously [
A. Total testosterone biosynthesis following treatment with different CLA concentrations (w/o, 0.5, 1.5 and 7.5 µM CLA). B. Representative western blots showing HSL (84 kDa), perilipin A/C (58 and 42 kDa, respectively), and ß-actin (42 kDa) expression following electrophoretic separation of protein extracts obtained from R2C cells treated with different concentrations of CLA. C. Relative amounts of HSL, ß-actin, perilipin A and C. D. Representative immunofluorescent images of perilipin A/C expression in R2C cells treated with different concentrations of CLA. E. Representative immunofluorescent images of HSL expression in R2C cells treated with different concentrations of CLA. † Significant difference compared to w/o and 0.5 µM CLA (P<0.01).
The primary antibodies used in for the
Cells and half of the snap frozen testicles were lysed with lysis buffer (200 mM HEPES, 5 M NaCl, 10% Triton X-100, 0.5 M EDTA, 1 M DTT, 0.25 g Na-deoxycholate, 0.05 g SDS) supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich, USA). Equal amounts of proteins (60 µg/lane) were measured using the Quant-iT TM protein assay kit (Invitrogen Molecular Probes, Italy) and a Qubit fluorometer according to manufacturer’s instructions and were separated using a 12% SDS-PAGE gel. The separated proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (Hybond-PVDF, Amersham Biosciences, GE Healthcare, Little Chalfont, England) and incubated with a blocking solution containing 5% BSA (Sigma-Aldrich, USA) in Tris-Buffered Saline (20 mM Tris, 137 mM NaCl, pH 7.6) with 0.05% Tween-20 (T-TBS) for 1 h at RT. Blots were incubated in 0.5% BSA in T-TBS with primary antibodies diluted 1:1000 overnight at 4°C. Blots were washed in T-TBS and incubated for 1 h in 0.5% BSA in T-TBS with a secondary antibody (ECL™ anti-mouse/rabbit IgG, Horseradish Peroxidase linked whole antibody from sheep, GE Healthcare, UK). The final detection was performed using ECL Western Blotting Detection Reagent (Amersham Biosciences), according to the manufacturer’s instructions. Band analysis was performed with the ImageJ 1.41 software (National Institutes of Health, USA, http://rsb.info.nih.gov/ij). The phosphorylated state of the perilipin proteins was determined by visualisation of a delayed band in the western blotting analysis, as described by Servetnick et al. [
This technique was performed as described previously [
The qPCR technique was previously described in Di Felice et al. [
|
|
||
---|---|---|---|
To assess the consistency of the results, each
No differences in perilipin A/C and HSL expression were observed in the immunofluorescence experiments on R2C cells treated with different CLA concentrations compared to the control (
To investigate the effects of different concentrations of CLA on the testosterone biosynthesis pathway in R2C cells, the mRNA expression of specific genes encoding enzymes involved in the conversion of cholesterol to testosterone were measured using qPCR. Expression of STAR, CYP11A1, and HSD3B1 mRNA did not change following treatment with different concentrations of CLA. CYP17A1 mRNA expression in the R2C cells treated with 7.5 µM CLA working solution increased significantly compared to the control (P<0.01). The qPCR results of the R2C cells are summarised in
A. Real-time PCR analysis of genes encoding steroidogenic enzymes from
Lysates of R2C cells treated with different CLA concentrations were analysed by western blotting analysis to examine if the increase in CYP17A1 mRNA expression translated into an increase in protein levels. The protein expression of CYP17A1 was significantly higher in R2C cells treated with 7.5 µM CLA working solution compared to the control (P<0.01) (
Two days after the last exercise session, the mice were sacrificed, and plasma collected to measure the levels of free testosterone. The endurance training induced a significant increase in free testosterone (PLA-SED: 0.35±0.02 pg/mL; PLA-TR: 20.50±3.54 pg/mL; P<0.01). The CLA supplementation did not induce any significant changes in free testosterone level in the sedentary mice (PLA-SED: 0.35±0.02 pg/ml; CLA-SED: 0.31±0.04 pg/mL), but in the trained mice, it stimulated a further increase in free testosterone (PLA-TR: 20.50±3.54 pg/mL; CLA-TR: 29.20±0.71 pg/mL; P<0.04).
The mRNA extracted from the testicles of mice under the four different conditions were analysed using real-time quantitative qPCR to detect whether specific genes encoding enzymes involved in the conversion of cholesterol to testosterone were up-regulated (
A. Real-time PCR analysis of genes encoding steroidogenic enzymes from
The protein expression of CYP17A1 was significantly higher in both the trained groups (PLA-TR and CLA-TR) compared to the sedentary groups (PLA-SED and CLA-SED) (P<0.01). Moreover, CLA supplementation induced a further increase in CYP17A1 protein in the CLA-TR group compared to the PLA-TR group (P<0.01) (
Body weight, strength, and endurance performance were measured to study the functional effects of endogenous testosterone stimulation by CLA supplementation and exercise. Moreover, to evaluate hypertrophy, the anterior muscle groups of the hindlimb were excised and weighed following euthanasia. Due to normal growth during the 6 weeks of experimentation, the mice had a physiological increase in body weight. The increased body weight was significant in all groups (P<0.05) except in the CLA-TR group. No significant difference was observed among the groups (
A. Changes in body weight over time. B. Weight of the hindlimb anterior muscle groups (tibialis anterior, extensor hallucis longus, and extensor digitorum longus) normalised for body weight. C. Strength of the forelimb. D. Strength of the forelimb normalised to the body weight. E. Distance ran during the endurance test. SED, sedentary; TR, trained; PLA, placebo; CLA, conjugated linoleic acid. # Significant difference compared to time point “0” (P<0.05). ◊ Significant difference compared to the CLA-SED group (P<0.05). $ Significant difference compared to the PLA-SED group (P<0.05).
The main finding of this study was the identification of a novel pathway that stimulates the biosynthesis of testosterone both
At the beginning of the study, two possible pathways were identified as possible targets of CLA. The first pathway identified was based on the lipolytic effect of CLA on adipocytes, which appears to be mediated by an increase in perilipin A protein levels [
The second pathway investigated was based on the hypothesis that CLA may alter steroidogenesis by up- and down-regulating specific genes encoding enzymes and transport proteins involved in the conversion of cholesterol into testosterone. There are many steroidogenic enzymes involved in testosterone biosynthesis, including steroidogenic acute regulatory protein (STAR), which transfers cholesterol to the inner membrane of mitochondria, cholesterol side chain cleavage enzyme (CYP11A1), which converts cholesterol into pregnenolone within the mitochondria, 3ß-hydroxysteroid dehydrogenase (HSD3B), which converts pregnenolone into progesterone, CYP17A1, which converts progesterone into androstenedione, and 17ß-hydroxysteroid dehydrogenase (HSD17B3), which converts androstenedione into testosterone [
In the literature, CLA supplementation appears to be associated with a reduction in body weight, an increase in lean body mass, and a reduction in body fat mass [
The effects of exogenous testosterone on skeletal muscle are well known [
In conclusion, the results of the present study show that Leydig cells respond to CLA treatment or supplementation, enhancing testosterone biosynthesis by increasing the expression of CYP17A1. Because of the endogenous effect of CLA supplementation on testosterone production, this food supplement, in association with physical activity, may be used as an ergogenic aid in different fields of interest, including in sport science to enhance the positive effect of training on muscle mass and performance, in cachexia and anti-aging therapy to reduce the muscle wasting, in the alimentary industry to increase the number of progeny for butchery butcheries, and in human reproductive medicine to help aid in spontaneous conception or to increase the chance of conception with assisted reproductive treatment. Because this study was performed in mice, the results reveal its limitation on species generalisation. Thus, future research in other animal models and humans are needed to confirm these findings.