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1 Centro de Investigaciones Endocrinológicas (CEDIE),, Hospital de Niños Ricardo Gutiérrez, Gallo 1330, 1425 Buenos Aires, Argentina 2 Departamento de Bioquímica Humana,, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155 5th floor, 1121 Buenos Aires, Argentina
(Correspondence should be addressed to S B Meroni; Email: smeroni{at}cedie.org.ar)
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Abstract |
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-subunit of AMPK showed that high levels of AMPK are present in Sertoli cells. Treatment of the cultures with AICAR resulted in a dose- and time-dependent increase of P-AMPK levels indicating activation of the enzyme. A possible effect of AICAR on Sertoli cell lactate production was then analyzed. A dose- and time-dependent increment in lactate secretion was observed. The participation of AMPK activation in different biochemical processes that may be implicated in the regulation of lactate production was also analyzed. AICAR stimulated glucose uptake in a dose- and time-dependent manner. Additionally, AICAR increased the glucose transporter 1 (GLUT1) and decreased the glucose transporter 3 (GLUT3) mRNA levels. As for the role of AMPK in the regulation of the monocarboxylate transporters 1 and 4 (MCT1 and MCT4), it has been observed that AICAR treatment decreased MCT1 and increased MCT4 mRNA levels. In summary, the results presented herein show that AMPK is present in Sertoli cells and that its activation by AICAR increases lactate production as a result, at least in part, of a) an increase in glucose uptake, b) an increase in GLUT1 expression, and c) a decrease in MCT1 and an increase in MCT4 levels. Altogether, these results suggest an important role of AMPK in modulating the nutritional function of Sertoli cells.
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Introduction |
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Cells change their metabolism by activating specific signal transduction pathways. The AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis (Hardie 2003). It is a trimeric enzyme consisting of a catalytic subunit , and two regulatory subunits ß and 
which is activated by an increase in the AMP:ATP ratio. Activation of AMPK involves several mechanisms. Firstly, AMP activates (5-fold) AMPK by direct allosteric regulation of the enzyme. Secondly, binding of AMP induces a conformational change that makes the protein a better substrate for the phosphorylation of the -subunit at a specific Threonine residue, Thr 172, by the upstream kinase LKB1. Phosphorylation by LKB1 constitutes the primary mechanism for the stimulation of AMPK activity (50- to 100-fold). Thirdly, the conformational modification of the protein induced by AMP binding also provokes a decrease in dephosphorylation by phosphatases and contributes to a sustained activation of the enzyme (Hardie 2004). Altogether, these mechanisms make the AMPK system ultrasensitive to energy changes, an important fact for the ability of the enzyme to maintain the energy status of the cell within narrow limits. The adenosine analog, 5-aminoimidazole-4-carboxamide-1-b-D-ribonucleoside (AICAR), is a potent activator of AMPK system (Corton et al. 1995). AICAR has been extremely useful to analyze the metabolic effects resulting from AMPK activation in skeletal muscle, adipose tissue, and liver (Xi et al. 2001, Fryer et al. 2002, Gaidhu et al. 2006).
Several reviews have dealt with the downstream targets and processes regulated by AMPK (Hardie & Pan 2002, Hardie 2005, Hardie & Sakamoto 2006). In general, activation of AMPK downregulates biosynthetic pathways such as fatty acid and cholesterol biosynthesis, yet switches on catabolic pathways that generate ATP, such as fatty acid oxidation, glucose uptake, and glycolysis. AMPK regulates the above-mentioned phenomena not only through direct phosphorylation of metabolic enzymes, but also through effects on the expression of genes that are important to these metabolic pathways.
Recently, different subunits of AMPK have been demonstrated to be present in the testis (Cheung et al. 2000). However, no studies are available on the downstream metabolic processes that may be activated in this organ. The aim of the present study was to investigate whether AMPK is present in Sertoli cells and its activation by AICAR results in the regulation of cell metabolism in order to ensure the supply of lactate for germ cell development.
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Materials and methods |
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Tissue culture media was purchased from GIBCO BRL (Life Technologies Ltd). AICAR was purchased from Calbiochem. [2,6-3H]-2-deoxy-D-glucose (2-DOG) was purchased from NEN (Boston, MA, USA). All other drugs and reagents were purchased from Sigma Chemical Co.
Sertoli cell isolation and culture
Sertoli cells from 20-day-old Sprague–Dawley rats were isolated as previously described (Meroni et al. 2002). Briefly, decapsulated testes were digested with 0.1% collagenase and 0.006% soybean trypsin inhibitor in Hanks’ balanced salt solution for 5 min at room temperature. Seminiferous tubules were saved, cut, and submitted to 1 M glycine–2 mM EDTA (pH 7.4) treatment to remove peritubular cells. The washed tubular pellet was then digested again with collagenase for 10 min at room temperature to remove germinal cells. The Sertoli cell suspension, collected by sedimentation, was resuspended in culture medium which consisted of modified Eagle's medium, supplemented with 20 mM HEPES, 100 IU/ml penicillin, 2.5 µg/ml amphotericin B, 1.2 mg/ml sodium bicarbonate, 10 µg/ml transferrin, 5 µg/ml insulin, 5 µg/ml vitamin E, and 4 ng/ml hydrocortisone. Sertoli cells were cultured in 25 cm2 flask, 6- or 24-multiwell plates (5 µg DNA/cm2) at 34 °C in a mixture of 5% CO2:95% air.
No myoid cell contamination was revealed in the cultures when an immunoperoxidase technique was applied to Sertoli cell cultures using a specific antiserum to smooth muscle actin. Remaining cell contaminants were of germ cell origin and this contamination was below 5% after 48 h in culture as examined by phase contrast microscopy.
Culture conditions
Sertoli cells were allowed to attach for 48 h in the presence of insulin and medium was replaced at this time with fresh medium without insulin. Stimulation with AICAR was performed with variable doses and for variable periods of time. The conditioned media and the cells pretreated for 48 h with variable doses of AICAR (0.3, 0.6, and 1 mM) harvested on day 5 were used to evaluate dose-dependent regulation of lactate and LDH activity respectively. The 12-, 24 -, and 48-h conditioned media of cells pretreated with 1 mM AICAR harvested on day 5 were used to evaluate time-dependent regulation of lactate production. Cells incubated for 12-, 24 -, and 48-h with 1 mM AICAR were used to evaluate time-dependent regulation of LDH activity and GLUT1, GLUT3, MCT1, MCT4, and LDHA mRNA levels. For 2-DOG uptake studies, cells cultured for 4 days under basal conditions and pretreated for 1, 2, or 4 h with 1 mM AICAR or for 2 h with 0.3, 0.6, and 1 mM AICAR were used.
Western blot analysis
Testicular homogenates were obtained from 20-day-old rats. Two testes were weighed, decapsulated, and homogenized with six gentle strokes of a glass-Teflon pestle homogenizer in 8 ml PBS containing 80 µl a protease inhibitor cocktail (P-8340, Sigma–Aldrich) and 2 mM phenylmethylsulfonylfluoride (PMSF) per gram of tissue. The homogenate was centrifuged at 200 g for 2 min to eliminate undisrupted tissue. The supernatant was used for electrophoretic studies.
Sertoli cells cultured in 6-multiwell plates were washed once with PBS at room temperature. Then, 200 µl PBS containing 2 µl protease inhibitor cocktail and 2 mM PMSF were added to each well. Cells were then placed on ice and disrupted by ultrasonic irradiation.
Protein content in testis homogenates and Sertoli cell lysates was determined by Lowry's assay (Lowry et al. 1951). For western blot analysis, 2x Laemmli buffer (4% w/v SDS, 20% v/v glycerol, 10% v/v 2-mercaptoethanol, 0.004% w/v bromophenol blue, and 0.125 M Tris–HCl, pH 6.8) was added and thoroughly mixed (Laemmli 1970). Samples were immersed in a boiling water bath for 5 min and then immediately settled on ice. In each lane, 30 µg protein aliquots were seeded. A biotinylated protein marker provided by Cell Signaling was also run. Proteins were resolved in 10% SDS-PAGE (10% acrylamide/bisacrylamide for the resolving gel and 4.3% acrylamide/bisacrylamide for the stacking gel) in a Mini Protean 3 Cell (Bio-Rad). After SDS-PAGE, gels were equilibrated in transfer buffer for 10 min and electrotransferred at 100 V for 60 min onto polyvinylidene difluoride membranes (Hybond-P, Amersham Pharmacia Biotech) using mini trans-blot cell (Bio-Rad). Membranes were probed with commercial antibodies for Phospho-AMPK- (Thr172) and AMPK- (New England Biolabs Inc., Beverley, MA, USA) which allow specific recognition of phosphorylated-AMPK (P-AMPK) and total-AMPK (T-AMPK) protein respectively, and for ß-tubulin (Sigma Chemical Co). The intensities of autoradiographic bands were estimated by densitometric scanning using NIH Image (Scion Corporation, Frederick, MD, USA) software.
Northern blot analysis
Total RNA from decapsulated 20-day-old rat testis was prepared. Extraction was performed using TRI reagent according to the manufacturer's recomendations. RNA from Sertoli cells cultured in 25 cm2 tissue culture flasks was obtained by the guanidinium isothiocyanate method (Chomczynski & Sacchi 1987). The amount of RNA was estimated by determining absorbance at 260 nm. For northern blot analysis, 20 µg total RNA was electrophoresed on a 1% agarose–10% formaldehyde gel. After migration, RNAs were transferred to Hybond-N nylon membrane (Amersham Pharmacia Biotech) by capillary transfer with 20xSSC (20xstock solution: 3 M NaCl and 0.3 M sodium citrate, pH 7.4) and fixed with u.v. Stratalinker (Stratagene Cloning Systems, La Jolla, CA, USA). cDNA probes (rat GLUT1 2.6 kb insert, EcoRI; mouse GLUT3 0.6 kb insert, EcoRI-HindIII; rat MCT1 1.9 kb insert, PstI-EcoRI; rat MCT4 1.7 kb insert, HindIII-BamHI; rat LDH-A 3'UTR 0.4 kb insert, Pst I-Bgl II and 18S oligonucleotide) were labeled with [-32P]deoxy-CTP (Amersham Pharmacia Biotech) using a random-primed labeling kit (Prime-a-Gene Labeling System, Promega Corporation). Blots were prehybridized for 5 h at 42 °C in 50% formamide, NaCl/Pi/EDTA (0.75 M NaCl, 20 mM sodium phosphate (pH 7.5) and 1 mM EDTA), 5xDenhart's solution, 10% dextran sulfate, 0.5% SDS, and 100 µg/ml herring sperm DNA. Hybridization was then performed overnight at 42 °C in the same hybridization buffer containing 1–4x106 c.p.m./ml 32P-labeled probe. Membranes were washed utilizing different astringency conditions depending on the probe utilized. Membranes were exposed to Kodak X-Omat S films (Eastman Kodak) software. The 18S signal was used to standardize mRNA contents.
Measurement of 2-DOG
Glucose transport was studied using the uptake of the labeled non-metabolizable glucose analogue 2-DOG. Cells were washed three times with glucose-free PBS at room temperature. Sertoli cells were then incubated at 34 °C in 0.5 ml glucose-free PBS containing [2,6-3H]-2-DOG (0.5 µCi/ml) for 30 min. Unspecific uptake was determined in incubations performed in the presence of a 10 000-fold higher concentration of unlabeled 2-DOG. At the end of the incubation period, dishes were placed on ice and washed extensively with ice-cold PBS until no radioactivity was present in the washings. Cells were then dissolved with 0.5 M sodium hydroxide and 0.4% sodium deoxycholate and counted in a liquid scintillation spectrophotometer. Parallel cultures receiving identical treatments to those performed before the glucose uptake assay were destined for DNA determinations. Results were expressed on a per microgram DNA basis.
LDH activity measurement
After incubation of Sertoli cells in the absence or presence of AICAR, culture medium was discarded and cells were disrupted by ultrasonic irradiation in 0.9% NaCl and centrifuged at 15 800 g for 10 min. The supernatant was used to measure LDH activity. Total LDH activity was determined by a routinely used spectrophotometric method (Randox Laboratories, Crumlin, UK). Results were expressed on a per microgram DNA basis.
Lactate determination
Lactate was measured by a standard method involving conversion of NAD+ to NADH determined as the rate of increase of absorbance at 340 nm. A commercial kit from Sigma–Aldrich was used. Results were expressed on a per microgram DNA basis.
Other assays
DNA content in Sertoli cell monolayers was determined by the method of Labarca & Paigen (1980).
Statistical analysis
All experiments were run in triplicate and repeated three to four times. Results are expressed as means±S.D. One-way ANOVA with Tukey–Kramer post test was performed using GraphPad InStat version 3.00 for Windows 95 (GraphPad Software, San Diego, CA, USA). Probabilities <0.05 were considered as statistically significant.
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Results |
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In order to determine whether AMPK is present in Sertoli cells, western blot analysis for AMPK-subunit was performed in Sertoli cell lysates and compared with that of muscle and testicular homogenates. Figure 1 shows that high levels of AMPK were present in Sertoli cells. The compound AICAR, a selective cell-permeable activator of AMPK, was utilized to study the participation of the enzyme in the regulation of Sertoli cell metabolism. Treatment of the cultures with 1 mM AICAR for 1, 2, and 4 h resulted in an increase of phospho-Thr172-AMPK levels indicating activation of the enzyme (Fig. 2A). In addition, a dose–response curve in 2-h incubations with AICAR was observed (Fig. 2B).
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Glucose uptake and GLUTs expression
As glucose availability for glycolysis is essential to lactate production, we decided to analyze whether AMPK activation modifies glucose uptake in Sertoli cells. Figure 4A shows the results obtained for 2-DOG uptake in cells stimulated with 1 mM AICAR for variable periods of time (1, 2, and 4 h). A significant increase in glucose incorporation into the cell in 2- and 4-h incubation periods with the riboside was observed. Figure 4B shows that treatment of Sertoli cell cultures with 0.3, 0.6, and 1 mM AICAR for 2 h promoted dose-dependent increments in 2-DOG incorporation.
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Figure 6A and B shows that AICAR did not promote time- or dose-dependent regulation of LDH activity. A slight decrease in LDH A mRNA levels in Sertoli cells treated for 48 h with 1 mM AICAR was observed. (Fig. 6C).
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Discussion |
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AMPK, a sensor of energy status of the cells, is a key enzyme in regulating glucose oxidation to obtain ATP. It has been shown that AMPK is present in the testis (Cheung et al. 2000). However, no reports are available for the presence of this enzyme specifically in Sertoli cells. The present study shows that in 20-day-old rats, when Sertoli cells in the testis are in a process of terminal differentiation, there are high levels of AMPK in these cells.
Stimulation of AMPK leads to metabolic changes responsible for obtaining maximal energy yield from glucose. In this context, AMPK activation by AICAR results in glucose oxidation and an increase in the activity of pyruvate dehydrogenase that favors the conversion of pyruvate in Acetyl-CoA in rat soleus muscle (Smith et al. 2005). In addition, Ceddia & Sweeney (2004) showed that AMPK activation promotes an increase in glucose oxidation with a reduction in lactate production and Putman et al. (2003) showed increases in citrate synthase activity and decreases in LDH activity in skeletal muscle cells. Altogether, the latter reports are consistent with a decrease in lactate production in response to AMPK activation that is destined to increase energy yield in the mitochondria. The present study shows that in Sertoli cells, AMPK activation by AICAR promotes a dose- and time-dependent increment in lactate secretion. These results suggest that under those conditions when there is an increase in the AMP:ATP ratio and in the activation of AMPK, Sertoli cells privilege lactate secretion in order to maintain the energy supply to germ cells. It is worth mentioning that knock-out male mice for LKB1 – the major upstream activator of the AMPK – are infertile (Sakamoto et al. 2005). Based on this observation, Sakamoto and collaborators have suggested a role for LKB1 in regulating spermatogenesis. Considering that our results show that AMPK activation leads to an increase in lactate production, it is tempting to speculate that one of the mechanisms underlying sterility may be related to the inability in the above-mentioned animal model to fully activate AMPK in Sertoli cells and consequently to produce sufficient amounts of lactate to maintain germ cell development.
Several biochemical mechanisms may contribute to an increase in Sertoli cell lactate secretion. Glucose transport mediated by GLUTs – the rate-limiting step for glucose metabolism – and LDH activities play important roles respectively, at the beginning of the process, providing the substrate, and at the end of the process, interconverting pyruvate and lactate. In addition, the modulation of lactate release from Sertoli cells, regulating the expression of those MCTs responsible for lactate export, may also contribute to improve lactate supply to germ cells.
Previous reports have shown an increase in glucose uptake following AMPK activation in a variety of tissues (Xi et al. 2001, Chen et al. 2002, Fryer et al. 2002, Lemieux et al. 2003, Pelletier et al. 2005). The present study shows that AMPK activation by AICAR increases glucose transport in rat Sertoli cells as well. In Sertoli cells, glucose transporters so far described include GLUT1, GLUT3 and GLUT8 (Ulisse et al. 1992, Kokk et al. 2004, Carosa et al. 2005). Glucose transport in Sertoli cells is probably mediated by the glucose transporters GLUT1 and GLUT3. Even though GLUT8 is present in rat Sertoli cells, this carrier protein is not present at the plasma membrane and probably is not involved in glucose transport from the extra cellular milieu.
Many of the known short-term effects of AMPK activation can be explained by direct phosphorylation and regulation of a variety of substrates (Hardie & Hawley 2001). However, long-term activation of the kinase has also effects on the pattern of gene expression in a cell (Leclerc et al. 2002, Leff 2003). Protein expression, as a whole, is downregulated following AMPK activation. Yet, some proteins, which are essential for metabolic adaptation, will bypass the condition of energy saving associated with the activation of this kinase and will increase their expression (Hardie & Hawley 2001). In relation to the latter assumption, we have observed that long-term activation of AMPK in rat Sertoli cells increases GLUT1 expression suggesting that this protein is essential for Sertoli cell functioning. The close relationship between AMPK and GLUT1 expression has been previously documented in skeletal muscle cells transfected with a constitutively active mutant form of the catalytic -subunit of AMPK and in DU145 prostate carcinoma cells stimulated with AICAR (Fryer et al. 2002, Yun et al. 2005). On the other hand, the decrease in GLUT3 mRNA levels following long-term activation of AMPK can be readily explained by the general inhibitory effect of AMPK activation on anabolic processes (Corton et al. 1994).
As for LDH isoenzyme system, Long et al. (2005) have demonstrated a role of AMPK in the expression of LDH-B subunit in white skeletal muscle cells in mice. On the other hand, Putman et al. (2003) have demonstrated that the long-term stimulation with AICAR reduces total LDH activity in skeletal muscle cells. The present study shows that in Sertoli cells, different from what Putman and collaborators observed in skeletal muscle cells, LDH activity is maintained after the long-term incubations with AICAR. Although a slight decrease in LDHA mRNA levels was observed in 48-h incubations, this decrease was not evident in total LDH activity. This sustained LDH activity in Sertoli cells may be the result of cell adaptation to ensure a continuous lactate offer to meiotic germ cells.
Finally, regulation of lactate exit from Sertoli cells may be also a mechanism involved in lactate supply to germ cells. The transport of lactate across the plasma membrane is mediated by a family of proton-linked MCTs (Halestrap & Price 1999). In the testis, the study of MCTs expression has been mainly analyzed in spermatogenic cells. RT-PCR, in situ mRNA hybridization, and immunocyto- and immunohistochemistry data show that pachytene spermatocytes express mainly MCT1 and MCT4 isoforms, while round spermatides also show expression of the MCT2 isoform (Goddard et al. 2003, Brauchi et al. 2005). We have looked in Sertoli cells for those MCTs that are expressed in the majority of the cells such as MCT1, and for those that are strongly expressed in glycolytic cells, which must export large amounts of lactate, such as MCT4 (Juel & Halestrap 1999, Dimmer et al. 2000). We have confirmed that MCT1 and MCT4 are expressed in testicular tissue and in addition we have demonstrated that these transporters are expressed in Sertoli cells. As for the regulation of these transporters by AMPK activation, we have observed that while MCT1 mRNA levels are downregulated by AICAR treatment, MCT4 mRNA levels are upregulated. It has been postulated that MCT1 has a role in lactate import from the extracellular milieu. On the other hand, MCT4 which has a much lower affinity for lactate than MCT1 has been proposed to serve as lactate exporter. The manner in which both transporters are regulated in Sertoli cells by long-term AMPK activation may reflect a situation of increased lactate export and decreased lactate recapture from the extracellular milieu leading to adequate lactate levels at the disposal of germ cells.
In conclusion, the results presented herein show that AMPK is present in Sertoli cells and that its activation by AICAR increases lactate production as a result, at least in part, of a) an increase in glucose uptake, b) an increase in GLUT1 expression, and c) a decrease in MCT1 and an increase in MCT4 levels. Altogether, these results suggest an important role of AMPK in modulating the nutritional function of Sertoli cells.
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Acknowledgements |
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References |
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Received in final form 6 July 2007
Accepted 7 August 2007
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