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Interleukin 1 Stimulates Lactate Dehydrogenase A Expression and Lactate Production in Cultured Porcine Sertoli Cells¹

^a Institut National de la Santé et de la Recherche Médicale, INSERM U407, Communications Cellulaires en Biologie de la Reproduction, Centre Hospitalier Lyon-Sud, 69 495 Pierre-Bénite, France

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
By using cultured porcine Sertoli cells as a model, the action of interleukin 1 (IL-1) on lactate production and the site of this action were studied. IL-1 stimulated Sertoli cell lactate production in a time- and dose-dependent manner (with a half-maximal effect [ED₅₀] of 6 pM). Two major sites involved in IL-1 action were identified. First, IL-1 was shown to increase the uptake of glucose substrate in a time- and dose-dependent manner. The maximal effect, with an ED₅₀ of 10 pM, was observed after 24 h of treatment. Second, IL-1 increased the activity of the lactate dehydrogenase (LDH) A4 isoform, which is involved in the conversion of pyruvate into lactate. This increase in LDH A4 activity was detected at 12 h and was maximal, with an ED₅₀ of 9 pM, after 24-h treatment with IL-1. The increase was related to an increase in LDH A4 expression, since IL-1 stimulated LDH A mRNA (size: 1.5 kilobases, evidenced through Northern blotting analysis) in a dose- and time-dependent manner. Assuming that IL-1 might be produced in the seminiferous tubules by both Sertoli and germ cells, which utilize lactate for their energy metabolism, we suggest that these results together show 1) that the cytokine may represent a signal in the metabolic cooperation existing between Sertoli cells and germ cells, and 2) that a redistribution of LDH isoforms in favor of LDH A4 under IL-1 control is a key mechanism(s) in such cooperation used by germ cells to enhance lactate production in Sertoli cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Spermatogenesis is a complex process that takes place in the testicular seminiferous tubules, during which undifferentiated spermatogonia divide and differentiate into mature spermatozoa [1]. Such a process is highly dependent upon Sertoli cells [1–3]. Under endocrine control (FSH, testosterone), Sertoli cells develop and reorganize to generate the hematotesticular barrier through their tight junctional complexes and to provide nutrients and regulatory factors to the germ cells [4]. Together, this tissue remodelling process and the production of Sertoli cell factors lead to the constitution of a specific biochemical and cytoarchitectural microenvironment in the adluminal compartment, in which germ cells will proliferate and differentiate. Among the Sertoli cell products are binding and transport proteins [5], extracellular matrix and junctional proteins [6, 7], proteases and protease inhibitors [8], growth factors [9–12], and energy substrates such as lactate [13].

Several observations have indicated that lactate may represent a preferential energetic substrate for germ cells [14, 15]. Indeed, 1) the inability of germ cells (particularly postmeiotic germ cells) to use glucose to energize their metabolism, 2) their preference for lactate as an energy source, and 3) the capacity of Sertoli cells to produce high amounts of lactate have generated a concept related to Sertoli cell-germ cell metabolic cooperation with lactate playing a pivotal role [14–16]. Lactate production in Sertoli cells has been shown to be predominantly under the control of the endocrine system including FSH [17–19], insulin [18, 19], and insulin-like growth factor I (IGF-I) [18, 20]. Although several biochemical steps involved in lactate production (including, at least, glucose substrate uptake, glycolysis, and the conversion of pyruvate into lactate) might represent potential targets for the hormone action, up to now only glucose uptake has been reported to be targeted by the endocrine system in Sertoli cells [18–21].

In the present study, by using cultured Sertoli cells as an experimental model, we demonstrate that lactate production might also be under a local (testicular) control in addition to the endocrine control. Specifically, we show that Sertoli cell lactate is stimulated by interleukin 1 (IL-1), a cytokine previously reported to be produced in the seminiferous tubules [22] by Sertoli [23, 24] and germ [25] cells. Additionally, we show that the mechanisms of IL-1 action on lactate production are novel in that they involve a redistribution of lactate dehydrogenase (LDH) isoforms and particularly an increase in the expression and activity of LDH A, which is known to favor the conversion of pyruvate into lactate.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials

Dulbecco's modified Eagle's (DME)/Ham's F-12 medium and TRIzol were obtained from Life Technologies (Eragny, France). Collagenase/dispase, human recombinant IL-1 (spec. act. > 5.0 x 10⁷ U/mg, determined by measurement of [³H]thymidine incorporation into mouse C3H/HeJ thymocytes), and human recombinant IL-1ß (spec. act. > 1.0 x 10⁷ U/mg, determined by measurement of [³H]thymidine incorporation into mouse thymocytes in the presence of low amounts of phytohemagglutinin) were obtained from Boehringer (Mannheim, Germany). Human recombinant IL-6 was purchased from R&D Systems (Minneapolis, MN). Porcine LDH A and rat GAPDH probes were kindly provided by Dr. S.S.L. Li (Laboratory of Genetics, Research Triangle Park, NC, USA) and Dr. J.M. Blanchard (Faculté des Sciences, Montpellier, France), respectively. Sigma Chemical Co. (St. Louis, MO) was the source for transferrin, insulin, tocopherol, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), deoxyribonuclease type I (DNase), LDH (from rabbit muscle), 2-deoxy-D-glucose (2-DOG), and NAD. 2-Deoxy-D-[2,6-³H]glucose (17 Ci/mmol) and [-³²P]dCTP were purchased from Amersham (Aylesbury, UK).

Sertoli Cell Isolation and Culture

Isolated Sertoli cells were prepared from immature porcine testes (2–3 wk old) by collagenase treatment as previously described [26]. Briefly, decapsulated testes were minced and washed in DME/Ham's F-12 medium (1:1). After collagenase dissociation (0.5 mg/ml, 90 min at 32°C), cells were washed by centrifugation (200 x g for 10 min). The resulting pellet was then resuspended, and after a sedimentation of 5 min the sedimented tubules were recovered and washed three times by gravity in DME/F-12 medium. These tubules were then incubated for 10 min (room temperature) in 20 ml of 1 M glycine, 2 mM EDTA, and 20 IU/ml DNase in Ca²⁺/Mg²⁺-free PBS solution, pH 7.2. This treatment led to the release of contaminating interstitial (Leydig) cells. The glycine-treated tubules were then washed three times (in DME/F-12 medium) by gravity and incubated in 100 ml DME/F-12 medium containing collagenase (0.5 mg/ml), DNase (0.05 mg/ml), and soybean trypsin inhibitor (0.05 mg/ml) for 15 min at 32°C. The supernatants containing the peritubular myoid cell fraction were removed, and the sedimented tubules were treated again as described above with collagenase (0.5 mg/ml, 20 min, 32°C) until small clumps resulted. Clumps were left to settle, and the supernatants were discarded. This procedure led to a purified Sertoli cell population not contaminated by Leydig cells or germ cells and containing between 2% and 5% peritubular myoid cells as evaluated by using desmin and fibronectin immunostaining [27].

Sertoli cells were plated in Falcon (Los Angeles, CA) 24-multiwell plates (0.5 x 10⁶ cells/dish) and cultured at 32°C in a humidified atmosphere of 5% CO₂:95% air in DME/Ham's F-12 medium (1:1) containing 1.2 mg/ml sodium bicarbonate, 15 mM HEPES, and 20 µg/ml gentamycin. This medium was supplemented with transferrin (5 µg/ml) and -tocopherol (10 µg/ml).

Measurement of Lactate Production

The amounts of lactate present in Sertoli cell-conditioned media were estimated by an enzymatic method [28] using a Kontron fluorimeter (Kontron Instruments, Zurich, Switzerland) at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The cell number was determined by using a Coulter counter (Coultronics, Margency, France) once the cells were removed from the culture dishes with trypsin-EDTA.

Measurement of 2-DOG Transport

Glucose transport was studied using the uptake of the labeled nonmetabolizable glucose analogue 2-deoxy-D-[2,6-³H]glucose ([2,6-³H]-2-DOG) as previously described [19].

Two different protocols were used to characterize both the short-term and the long-term effects of IL-1 on glucose uptake in cultured Sertoli cells. For the short-term action (0.5–2.5 h) of the cytokine on glucose uptake, the culture medium of Sertoli cells was discarded, and cells were washed three times with glucose-free PBS. The cells were then incubated at 32°C in 0.3 ml glucose-free PBS containing [2,6-³H]-2-DOG (0.5 µCi/ml) in the absence or presence of the cytokine for 0.5–2.5 h. At the end of the incubation, dishes were placed on ice and extensively washed with ice-cold buffer until no radioactivity was present in the washings. The cells were then dissolved in 0.5 N sodium hydroxide, 0.4% deoxycholate buffer. Aliquots were taken for liquid scintillation spectrophotometry. To characterize the long-term action (6–48 h) of IL-1 on glucose uptake, Sertoli cells were preincubated with the cytokine. At the end of incubation, the cells were washed three times with glucose-free PBS. After a 10-min incubation at 32°C (transport is linear under these conditions) with [2,6-³H]-2-DOG (0.5 µCi/ml), Sertoli cells were washed rapidly with ice-cold buffer and solubilized by the addition of 0.5 N sodium hydroxide and 0.4% deoxycholate buffer. Aliquots of the solubilized extract were assayed for radioactivity.

All the results were corrected for extracellular trapping and passive diffusion of [2,6-³H]-2-DOG, both of which were measured in the presence of unlabeled 2-DOG at a 1000-fold higher concentration. The nonspecific radioactivity was lower than 5% of the total radioactivity.

LDH Activity Measurement

After incubation of Sertoli cells in the absence or presence of the cytokine, the culture medium was discarded and the cells were sonicated (3 x 5 sec) in 500 µl NaCl 0.9% and centrifuged (15 800 x g, 10 min). The supernatant containing the cell extracts was collected and stored at -70°C for measurement of total activity of LDH as well as of the different LDH isozymes after electrophoresis.

Total LDH activity was determined by a spectrophotometric method using the Enzyline LDH-Kit (BioMerieux, Lyon, France). One hundred fifty microliters of supernatant was used for estimation of the activity of the LDH isoenzyme by measuring the oxidation of NADH at 340 nm using a OPEN 30 (BioMerieux). The extinction was recorded at 340 nm for 2 min. The results were expressed as international units (UI) of enzyme activity per 10⁶ cells.

Measurement of the Activity of LDH Isozymes

A sensitive agarose gel electrophoresis system in nondenaturing buffer, according to the method used in the serum test, was adapted to separate Sertoli cell LDH isozymes [29]. LDH isozyme activities were visualized by nitro blue tetrazolium reduction to formazan (Titan Gel LD-Kit isozyme procedure; Helena Laboratories, Beaumont, TX). Activity bands were visualized using lactate and NAD as substrates and phenazine methosulfate as the final hydrogen acceptor. The different bands were quantified with an integrating densitometer at 870 nm (Cellosystem 2; Sebia, Issy-les-Moulineaux, France). LDH isozyme activity was calculated as the percentage of total LDH activity and expressed as mUI.

Analysis of mRNA Levels

Total RNA was extracted from Sertoli cells cultured in Petri dishes with TRIzol reagent, a mono-phasic solution of phenol and guanidine isothiocyanate. This reagent is an improvement on the single-step RNA isolation developed by Chomczynski and Sacchi [30]. The amount of RNA was estimated by spectrophotometry at 260 nm. About 20 µg of total RNA (denatured 15 min at 65°C in the presence of 2.2 M formaldehyde, 12.5 M formamide, single-strength 3(N-morpholino)propanesulfonic acid [MOPS]) were electrophoresed on a 1.2% agarose/2.2 M formaldehyde gel. After migration in 0.02 M MOPS running buffer, RNAs were transferred to nitrocellulose membrane Hybond-C extra (Amersham, UK) in 10-strength SSC (1.5 M NaCl, 0.15 M sodium citrate) and fixed at 80°C for 2 h. Complementary DNA probes (LDH-A, 1.5 kilobases [kb] Xho-EcoRI and GAPDH, 1.3 kb PstI) were labeled with 40 µCi of [-³²P]dCTP (SA, 10⁹ dpm/µg DNA) using a random-primed labeling kit (Promega, Madison, WI). Labeled probe was separated from free nucleotides by filtration through a DEAE-cellulose column. After 5 h of prehybridization at 42°C, filters were hybridized with labeled probe (1–4 x 10⁶ cpm/ml) overnight at 42°C in 50% formamide, 5-strength SSPE (0.9 M NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7.4), 5-strength Denhardt's solution (1 g Ficoll, 1 g polyvinylpyrrolidone, 1 g BSA/L), 1% SDS, and 100 µg/ml herring sperm DNA. Afterwards, membranes were washed four times in double-strength SSC, 0.1% SDS (20 min at room temperature) then 40 min at 55°C. Filters were exposed to Kodak X-OMAT S films (Eastman Kodak, Rochester, NY) at -70°C for 1–2 days. Intensities of autoradiographic bands were estimated by densitometric scanning using the Bioimage scanner (Millipore SA, Saint Quentin, France). The data were expressed as the LDH A:GAPDH mRNA ratio.

Data Analysis

All experimental data are presented as the mean ± SD of triplicate determinations of three replicate cultures within each treatment group. All experiments reported here were repeated a least three times with independent cell preparations. A representative experiment from each series of experiments is presented. Statistical significance between groups was determined by Student's t-test using the StatWorks (Hyden and Son Ltd., London, UK) package on a Macintosh computer. Differences are accepted as significant at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effect of IL-1 on Lactate Production in Cultured Sertoli Cells

IL-1 stimulated lactate accumulation in Sertoli cell culture medium in a time- and dose-dependent manner (Fig. 1). Lactate accumulation in culture medium increased between 3 and 24 h in both untreated and IL-1-treated Sertoli cells. IL-1 (50 UI/ml, 1 ng/ml) exerted a stimulatory effect on lactate accumulation at 6 h (p < 0.006), 12 h (p < 0.001), and 24 h (p < 0.001) (Fig. 1A). The stimulating action of IL-1 on lactate production was dose-dependent with a half-maximal (ED₅₀) effect observed with 5 UI/ml (0.1 ng/ml, 6 pM, p < 0.001). The maximal effect was observed with 50 UI/ml (1 ng/ml, 0.06 nM) (Fig. 1B).

View larger version (15K): [in this window] [in a new window]   FIG. 1. Effect of IL-1 on lactate production. Sertoli cells were incubated A) for different times (3-24 h) in the absence or presence of IL-1 (50 UI/ml) and B) with increasing concentrations of IL-1 (0.8-200 UI/ml, 12 h). The results represent the mean ± SD of triplicate incubations.

To clarify whether the IL-1 effect was specific, we compared the action of this cytokine with other regulators of Sertoli cell functions such as FSH, IL-1ß, and IL-6. As expected, FSH enhanced lactate accumulation in Sertoli cell medium (Table 1). IL-1 had an additive effect on lactate production in the presence of FSH (Table 1). These data suggest that the cytokine and the hormone may use different intracellular signaling pathways to enhance lactate production. The data shown in Table 2 indicate that the stimulatory effects of IL-1 and IL-1ß on lactate production were comparable if not similar. As IL-6 has been suggested to mediate some IL-1 effects in different cellular systems and particularly in Sertoli cells [31], we tested the effects of IL-6 on lactate production. As shown in Table 3, IL-6 (1–10 ng/ml) had no significant effect on lactate production, suggesting that IL-6 is probably not the mediator of IL-1 effects on lactate production.

View this table: [in this window] [in a new window]   TABLE 1. Effects of IL-1 and FSH on lactate production by Sertoli cells following 12 h culture (mean ± SD; triplicate incubations).

View this table: [in this window] [in a new window]   TABLE 2. Effects of IL-1 and IL-1ß on lactate production by Sertoli cells following 12 h culture (mean ± SD; triplicate incubations).

Effect of IL-1 on Glucose Uptake in Cultured Sertoli Cells

In these experiments, we tested the possibility that the cytokine may exert an acute and/or a long-term effect on glucose uptake in Sertoli cells. The acute (0.5–2.5 h) effect of IL-1 on glucose uptake was evaluated in parallel with a positive control, insulin. As expected, insulin (1 µg/ml) enhanced the hexose uptake at different times tested, whereas, under similar experimental conditions, IL-1 (50 UI/ml, 1 ng/ml, 0.06 nM) exerted no significant action on glucose uptake (Table 4).

By contrast, when the cytokine (50 UI/ml) was added to cultured Sertoli cells for a longer time (6–48 h), a stimulating effect was detected at 6 h (p < 0.02) and was maximal at 24 h (p < 0.001) (Fig. 2A). Such a time-delayed effect of the cytokine was dose-dependent with, again, half-maximal (ED₅₀) and maximal effects observed with 12.5 UI/ml (0.25 ng/ml, 0.01 nM) and 50 UI/ml (1 ng/ml, 0.06 nM), respectively (Fig. 2B).

View larger version (21K): [in this window] [in a new window]   FIG. 2. Effect of IL-1 on [2,6-3H]-2-DOG uptake in porcine Sertoli cells. Sertoli cells were treated A) for different times (6-48 h) in the absence or presence of IL-1 (50 UI/ml) and B) for 24 h in the presence of increasing concentrations (0.8-200 UI/ml) of IL-1. After the cells were washed with PBS, [2,6-3H]-2-DOG (0.5 µCi/ml) was added in glucose-free PBS for 10 min. The results represent the mean ± SD of triplicate incubations.

Effect of IL-1 on LDH Activity in Cultured Sertoli Cells

IL-1 (1 ng/ml) enhanced LDH activity in Sertoli cells after at least a 12-h incubation (p < 0.001; Fig. 3A). The maximal stimulatory effect was observed after 48 h of treatment (p < 0.001). This stimulatory effect of IL-1 on LDH activity was dose-dependent; half-maximal and maximal effects were observed with 7.8 UI/ml (0.16 ng/ml, 9 pM) and 50 UI/ml (1 ng/ml, 0.06 nM), respectively (Fig. 3B).

View larger version (21K): [in this window] [in a new window]   FIG. 3. Effect of IL-1 on LDH activity in cultured Sertoli cells. Sertoli cells were treated A) for different times (6-48 h) in the absence or presence of IL-1 (50 UI/ml) and B) in the presence of increasing concentrations (0.8-200 UI/ml, 48 h) of IL-1. The results represent the mean ± SD of triplicate incubations.

In control experiments, we confirmed that this LDH activity was exclusively detected in Sertoli cells but not in their conditioned media whether the cells were treated with the cytokine or not (data not shown). This observation therefore excludes any cytotoxic effect of the cytokine on cultured Sertoli cells.

Effect of IL-1 on LDH Isozyme Distribution

To characterize further the action of IL-1 on Sertoli cell LDH activity, the electrophoretic pattern of LDH isozymes in cultured Sertoli cells was studied in the absence or presence of the cytokine. LDH isozymes were separated on an agarose slab gel, and their relative activities were compared as indicated in Materials and Methods. In the presence of IL-1 (1 ng/ml, 48 h), the pattern of LDH isozyme activities dramatically changed in that the activities of LDH isoforms rich in A subunits (LDH-5 [A4] and, to a lesser extent, LDH-4 [A3B1]) were increased (Fig. 4A). The most significant (p < 0.001) increase (about 6-fold) after IL-1 treatment was observed on the activity of LDH-5 (A4) (Fig. 4B).

View larger version (32K): [in this window] [in a new window]   FIG. 4. Effect of IL-1 on the activity of the different LDH isozymes in cultured Sertoli cells. Sertoli cells were treated in the absence or presence of IL-1 (50 UI/ml). Then LDH isozyme activities were measured as described in Materials and Methods. A) Pattern of LDH isozymes; a representative electrophoresis is shown. B) Relative activities of the different isozymes. The results represent the mean ± SD of triplicate incubations.

Effect of IL-1 on LDH-5 (A4) Activity

IL-1 increased LDH-A4 activity in cultured porcine Sertoli cells in a time-dependent manner. This increase was significant (p < 0.003) after 12-h exposure to the cytokine and was maximal after 24-h exposure (Fig. 5A).

View larger version (17K): [in this window] [in a new window]   FIG. 5. Effect of IL-1 on the LDH-A4 activity in cultured Sertoli cells. Sertoli cells were incubated A) for different times (6-48 h) in the absence or presence of IL-1 (50 UI/ml) and B) in the presence of increasing concentrations (0.8-200 UI/ml, 24 h) of IL-1. The results represent the mean ± SD of triplicate incubations.

The increase in LDH-A4 activity was dependent upon IL-1 concentration in the culture medium since half-maximal and maximal effects were observed with 8 UI/ml (0.16 ng/ml, 9 pM) and 50 UI/ml (1 ng/ml), respectively (Fig. 5B).

Effect of IL-1 on LDH A mRNA Expression

IL-1 treatment resulted in a dose- and time-dependent increase in LDH-A mRNA (size 1.5 kb) expression. A significant (p < 0.02) effect was detected after 12 h of treatment, and the maximal stimulatory effect was observed after 24 h (p < 0.008) of treatment (Fig. 6) with IL-1. The dose effect experiments showed that the maximal (p < 0.02) effect was observed with 100 UI/ml (2 ng/ml, 0.1 nM) of IL-1 (Fig. 7).

View larger version (59K): [in this window] [in a new window]   FIG. 6. Kinetic study of IL-1 action on LDH-A mRNA levels. Sertoli cells were incubated for 0-48 h in the presence of IL-1 (100 UI/ml). Total cellular RNAs were then extracted, and Northern blotting analysis was performed using 20 µg total RNA per lane. Membranes were successively hybridized with the LDH-A and GAPDH cDNA. A) Data yielded by scanning three autoradiographs were expressed as LDH-A:GAPDH mRNA ratios. Values represent mean ± SD (n = 3). B) A representative Northern blot is shown.

View larger version (56K): [in this window] [in a new window]   FIG. 7. Dose effect of IL-1 on LDH-A mRNA levels. Sertoli cells were incubated for 24 h in the presence of increasing concentrations of IL-1 (0-500 UI/ml). Total cellular RNAs were then extracted, and Northern blotting analysis was performed using 20 µg of total RNA per lane. Membranes were successively hybridized with the LDH-A and GAPDH cDNAs. A) Data yielded by scanning three autoradiographs were expressed as LDH-A:GAPDH ratios; values represent mean ± SD (n = 3). B) A representative Northern blot is shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The aim of the present study was to investigate the effect and the mechanisms of action of IL-1 on lactate production in Sertoli cells. For this purpose, the experimental model used was purified porcine Sertoli cells cultured in defined medium. The study demonstrated that IL-1 stimulates lactate production, probably by an increase in glucose uptake and also a redistribution of LDH isoforms, mainly through an increase in LDH A expression and the activity of LDH A4, which favors the transformation of pyruvate into lactate.

Among the probable steps involved in the production of lactate in Sertoli cells, the present study focused on two major steps: the glucose substrate transport into the cell and the LDH isoenzyme system (which reversibly catalyzes the interconversion of lactate and pyruvate). With regard to glucose uptake in Sertoli cells, while different hormones such as insulin [18, 19], IGF-I [18, 20], and FSH [18–20] stimulate glucose uptake rapidly (i.e., in terms of minutes), the effects of IL-1 were observed after a long-term treatment: the stimulatory action of the cytokine on glucose uptake was detected at 6 h and was maximal at 24 h. This observation suggests that IL-1 may affect Sertoli cell glucose transport through different mechanisms. First, it is possible that the cytokine may use Sertoli cell intermediates to enhance glucose uptake. Among the potential candidates are IGF-I and epidermal growth factor (EGF)/transforming growth factor (TGF), since these two factors 1) are produced in Sertoli cells (for reviews see [9–12]) and 2) enhance (rapidly) glucose uptake [18, 20, 32], probably through specific receptors identified in Sertoli cells [33, 34]. However, our recent observations indicating that IL-1 did not affect IGF-I production and IGF-I receptor mRNA and protein in Sertoli cells (unpublished data) suggest that IGF-I is probably not the appropriate Sertoli cell intermediate involved in IL-1 action on glucose uptake. On the other hand, at the present time we do not known whether IL-1 affects EGF/TGF and EGF receptor expression in Sertoli cells. Earlier studies [31] have shown that in rat Sertoli cells, IL-1 stimulated IL-6 production. In the present study, we have shown that the stimulatory effect of IL-1 on lactate production is not due to IL-6 since IL-6 has no effect on lactate production. Second, IL-1 may increase glucose uptake through a direct action on Sertoli cell glucose transporters. Such a hypothesis is supported by data showing that 1) IL-1 increased the transcript for glucose transporters GLUT3 and GLUT1 in granulosa ovarian cells (female counterpart of Sertoli cells) [35], and 2) Sertoli cells expressed GLUT1 [36].

In the present study, we show that the expression of LDH A in terms of protein and mRNAs is another potential site of action of IL-1 in stimulating lactate production. However, we cannot exclude that IL-1 enhanced both LDH A4 protein content and LDH A4 activity. This is, to our knowledge, the first report indicating that LDH A is a target for IL-1 action. Biochemical and genetic studies of LDH have shown that its isozymes are encoded by three different genes: ldh a (muscle type), ldh b (heart type), and ldh c (testicular germ cell type) [29, 37]. The ldh c gene product corresponds to the homotetrameric LDH C4 isozyme present only in mature testis and spermatozoa [29]. The ldh a and ldh b genes give rise to various combinations of the LDH A and LDH B proteins and particularly to five tetrameric LDH isozymes: LDH-1 (B4), LDH-2 (A1B3), LDH-3 (A2B2), LDH-4 (A3B1), and LDH-5 (A4). These five isozymes are found in various proportions in different somatic tissues including Sertoli cells [29]. The LDH-5 (A4) isozyme exhibits a higher K_m value for pyruvate than for lactate [38]; an increase in the activity of this isozyme after IL-1 treatment would therefore favor the conversion of pyruvate into lactate. The mechanisms involved in such a selective increase in the activity of LDH A4 are yet unknown, and it remains to be clarified whether the positive effect of IL-1 on LDH A mRNA results from an enhancement in the gene transcription and/or the mRNA stabilization.

IL-1 enhanced lactate production, glucose uptake, and LDH A expression and activity in a nanomolar concentration range. Such a concentration is compatible with the presence of IL-1 receptors (type I) detected in mouse [39] and rat [40] Sertoli cells, indicating that IL-1 action on lactate production in Sertoli cells might be exerted in a physiological context. IL-1 intracellular transducing pathways involved in the cytokine action to stimulate lactate production remains to be identified. There is now a general agreement that IL-1, on binding to its receptors, activates different intracellular signalling pathways including protein kinase C, protein kinase A, and sphingomyelinase, which probably results in the activation of different transcription factors including activating protein-1 (AP-1) and nuclear factor (NF) B (for a review, see [41]). Although which of the transducing system(s) and transcription factors operate to increase LDH A levels in Sertoli cells after IL-1 treatment remains to be investigated, an AP-1 enhancer module has been identified in the ldh a promoter [42].

Although there are now several reports indicating that, in addition to endocrine control, Sertoli cell activity is regulated by germ cells (for reviews see [2, 43, 44]), the germ cell signalling molecules involved in such a control of Sertoli cells remain largely unknown. Our present findings, demonstrating that IL-1 (produced in germ cells [25, 40]) stimulates lactate production in Sertoli cells, make this cytokine a good candidate for involvement in the control exerted by germ cells on Sertoli cell activity. IL-1 originates from germ cells [25, 40] but also from Sertoli cells [24, 25], suggesting an autocrine control of lactate production via IL-1. Sertoli cell IL-1 production has been shown to be not only under the control of different signalling molecules including different hormones and growth factors but also under that of the residual bodies [40]. Thus, it is possible that residual bodies (via the production of Sertoli cell IL-1) and germ cells (via their own biosynthesis of the cytokine) may control and direct glucose metabolism in the Sertoli cell toward the formation of lactate, a metabolite that postmeiotic germ cells utilize as a preferential substrate. Sertoli cell lactate production appears to be under endocrine system (FSH, IGF-I) control as well as local (germ cell) control. Local control, which is probably specific to the different steps of the spermatogenic cycle, as IL-1 is produced at stages VIII and IX–X in the rat [45, 46], is probably more appropriate than systemic (endocrine) control in that it better takes into account the specific germ cell metabolic requirements. It is also possible that hormones such as FSH may act during the onset of puberty and that the growth factors act later in the more mature testes. Indeed, growth factor action is probably more specific in that it is more dependent upon the seminiferous epithelium stages. Finally, in addition to its regulatory action on different Sertoli cell functions (for reviews, see [10, 11]) and to its action as a spermatogonial growth factor [22, 45–47], IL-1 may play a critical role in postmeiotic germ cell development through the control of energetic (glucose) metabolism (the present study).

In conclusion, by using cultured Sertoli cells as a model, we report that IL-1 stimulates lactate production, probably through an increase in LDH A expression and activity. Assuming that the cytokine is produced in the seminiferous tubules, it is suggested that IL-1 may represent one of the signalling molecules involved in germ cell-Sertoli cell metabolic cooperation.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. S.S.L. Li (Laboratory of Genetics, Research Triangle Park, NC, USA) and Dr. J.M. Blanchard (Faculté des Sciences, Montpellier, France) for providing us with porcine LDH-A and GAPDH cDNA, respectively.

	FOOTNOTES

 
¹ This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM U407), and Ministère de l'Enseignement Supérieur et de la Recherche Scientifique (MESRS).

² Correspondence: M. Benahmed, INSERM U407, Communications Cellulaires en Biologie de la Reproduction, BP12, Faculté de Médecine Lyon-Sud, 69 921 Oullins cedex, France. FAX: 33 4 78 86 59 22; benahmed{at}lsgrisn1.univ-lyon1.fr

Accepted: July 30, 1998.

Received: April 7, 1998.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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