Summary
Resistin, a recently cloned adipose-secreted factor, is primarily involved in the modulation of insulin sensitivity and adipocyte differentiation. However, additional metabolic or endocrine functions of this molecule remain largely unexplored. In this study, a series of experiments were undertaken to explore the potential expression, regulation and functional role of this novel adipocytokine in rat testis. Resistin gene expression was demonstrated in rat testis throughout postnatal development, with maximum mRNA levels in adult specimens. At this age, resistin peptide was immunodetected in interstitial Leydig cells and Sertoli cells within seminiferous tubules. Testicular expression of resistin was under hormonal regulation of pituitary gonadotropins and showed stage-specificity, with peak expression values at stages II-VI of the seminiferous epithelial cycle. In addition, testicular resistin mRNA was down-regulated by the selective agonist of PPARγ, rosiglitazone, in vivo and in vitro. Similarly, fasting and central administration of the adipocyte-derived factor, leptin, evoked a significant reduction in testicular resistin mRNA levels, whereas they remained unaltered in a model of diet-induced obesity. From a functional standpoint, resistin, in a dose-dependent manner, significantly increased both basal and choriogonadotropin-stimulated testosterone secretion in vitro. Overall, our present results provide the first evidence for the expression, regulation and functional role of resistin in rat testis. These data underscore a reproductive facet of this recently cloned molecule, which may operate as a novel endocrine integrator linking energy homeostasis and reproduction.
- Resistin
- Luteinizing hormone
- Follicle-stimulating hormone
- PPARγ
- Leptin
- Fasting
- Seminiferous tubules
- Testis
- Rat
Introduction
The adipose tissue is an active endocrine organ directly involved in the control of metabolism, energy balance and reproductive function through a large number of secreted cytokines and hormones, including leptin (Ahima and Flier, 2001). In this context, a novel adipocytokine, termed resistin, was recently cloned (Steppan et al., 2001a). Resistin, also known as found in inflammatory zone 3 (FIZZ3) or adipocyte-specific secretory factor (ADSF), belongs to the family of resistin-like molecules defined by a cystein-rich region in the C-terminal domain (Steppan et al., 2001b). Resistin was originally described as a factor expressed in the fat tissue with the ability to impair insulin sensitivity and glucose tolerance in rodents (Steppan et al., 2001a). Plasma resistin levels were found significantly elevated in genetically susceptible and diet-induced obese mice, where immunoneutralization of endogenous resistin improved hyperglycemia and insulin resistance. Conversely, administration of recombinant resistin disturbed glucose tolerance and insulin action in normal mice (Steppan et al., 2001a). Thus, resistin was initially proposed as a causative link between obesity and type 2 diabetes, although considerable controversy in the actual role of resistin in obesity-associated insulin resistance has emerged subsequently (Ukkola, 2002). In addition, resistin has also been reported to be involved in the control of adipocyte differentiation (Kim et al., 2001). The receptor(s) conveying the biological actions of resistin in target tissues remains, so far, uncloned.
Compelling evidence demonstrates a close link between energy status and reproductive function (Frisch, 1984; Spicer, 2001). The integrated control of those systems is probably a multi-faceted phenomenon conducted by an array of signals acting at different levels of the neuroendocrine axes, governing food intake, energy homeostasis, metabolism and fertility. For example, the adipocyte-derived hormone, leptin, operates as a pleiotropic regulator of several metabolic and neuroendocrine systems, including the reproductive axis, acting mainly at central hypothalamic levels (Casanueva and Dieguez, 1999; Spicer, 2001). However, expression of leptin receptors and direct actions of leptin in male and female gonads have been also reported (Tena-Sempere et al., 1999; Spicer, 2001; Tena-Sempere and Barreiro, 2002). It is tempting to propose that additional signals with relevant roles in the control of metabolism and adipose function may also be provided with specific reproductive and/or gonadal actions.
An association between adiposity and impairment of reproductive function has been previously reported. Thus, subnormal plasma testosterone concentrations and reduced sex hormone binding globulin (SHBG) levels are detected in obese men, with an inverse relationship between plasma testosterone and body weight (Seidell et al., 1990; Abate et al., 2002). Interestingly, it has also been shown that insulin resistance is frequently associated with low plasma testosterone concentrations in men, and patients with non-insulin dependent diabetes mellitus seem to have lower testosterone and SHBG concentrations (Barrett-Connor, 1992). The precise etiology of these alterations is unclear, although they are reversed after weight loss, thus suggesting a link to adipocyte cell dysfunction (Kopelman, 1992).
On the above basis, we aimed at assessing the potential role of the adipocyte-derived hormone, resistin, in testicular function. Thus, molecular and immunohistochemical approaches were undertaken to evaluate the expression of resistin in rat testis. As our initial evidence demonstrated testicular expression of this novel adipocytokine, different experimental settings were used to define the pattern of cellular location and hormonal regulation of resistin expression in rat testis, as well as the functional role of this molecule in the direct control of testicular testosterone secretion.
Materials and Methods
Animals and drugs
Sprague-Dawley male rats bred in the vivarium of our Institution were used, unless otherwise stated. The day the litters were born was considered day 1 of age. The animals were maintained under constant light intensity (14 hours of light; from 7:00 a.m.) and temperature (22°C), and had free access to standard pellet rat chow and tap water. Experimental procedures were approved by local Ethical Committees and conducted in accordance with the European Union normative for care and use of experimental animals. In all experiments, the animals were killed by decapitation and testes were immediately removed, decapsulated (free of surrounding epididymal fat), frozen in liquid nitrogen and stored at –80°C until processing. Highly purified human choriogonadotropin (hCG) and human recombinant follicle stimulating hormone (FSH) were purchased from Serono (Madrid, Spain). The selective agonist of peroxisome proliferator activated receptor-γ (PPARγ), rosiglitazone maleate, was obtained from Calbiochem (La Jolla, CA, USA), and metformin, an insulin-sensitizer unrelated to PPARγ, was provided by Sigma (St Louis, MI, USA). The active fragment of the resistin molecule, resistin (23-42), was purchased from Phoenix Peptides (Belmont, CA, USA). Human recombinant leptin was kindly supplied by Eli Lilly (Indianapolis, IN, USA).
Experimental designs
In Experiment 1, analysis of testicular expression of resistin mRNA was conducted at different stages of postnatal development. Thus, testicular samples were obtained from 5-, 15-, 30-, 60- and 90-day-old rats (n=5-10), as well as from 17-month-old rats. These correspond to the neonatal-infantile (5-day), prepubertal (15-day), pubertal (30-day), early adult (60-day), and adult (90-day) stages of postnatal maturation, and ageing (17-month old). In addition, testicular samples were taken from 90-day-old animals, and processed for immunohistochemical detection of resistin peptide, as described below.
The ability of pituitary gonadotropins to regulate testicular expression of resistin was monitored in Experiment 2. To this end, testicular resistin mRNA levels and peptide expression were analyzed in control and long-term hypophysectomized (HPX) rats, i.e. 4-weeks after pituitary removal, with or without gonadotropin replacement: human chorionic gonadotropin (hCG; 10 IU/rat/24 hours) or recombinant folicle stimulating hormone (FSH; 7.5 IU/rat/24 hours) for 7 days before sampling. In addition, acute regulation of testicular resistin gene expression by gonadotropins was explored in Experiment 3. Expression levels of resistin mRNA were assayed in testes from intact adult rats injected at 10:00 hours with hCG (25 IU/rat) or FSH (12.5 IU/rat) and sampled 2, 4, 8 and 24 hours after administration. Paired vehicle-injected animals served as controls.
In Experiment 4, expression of resistin mRNA was assessed in seminiferous tubule preparations at different stages of the epithelial cycle. Microdissection of seminiferous tubule segments was carried out as described in detail elsewhere (Suominen et al., 2001). Briefly, testes from adult rats were decapsulated and 5 mm seminiferous tubule segments were isolated under a transilluminating stereomicroscope. Specific stages of the seminiferous epithelial cycle were identified and pooled in four major groups corresponding to stages II-VI, stages VII-VIII, stages IX-XII and stages XIII-I of the cycle. After exhaustive washing, tubular tissue was processed for RNA analysis as described below. In addition, cultures of staged tubule preparations (twenty 5 mm segments per well) were conducted after stimulation with FSH (10 ng/ml) for 24 hours. Samples incubated in the presence of medium alone served as controls.
In Experiment 5, the ability of the selective agonist of PPARγ, rosiglitazone, to modulate testicular resistin mRNA expression was evaluated in vivo. Adult male rats were treated with rosiglitazone (5 mg/kg) by daily i.p. injection for 1 week, as described previously (Way et al., 2001), and testes were collected for RNA analysis. For comparative purposes, a similar setting was used to evaluate the effects of metformin (320 mg/kg daily by oral gavage for 1, 2 and 3 weeks), an insulin-sensitizer unrelated to PPARγ. In addition, in Experiment 6, the effects of rosiglitazone upon testicular resistin mRNA expression were assessed in vitro. Slices of testicular tissue were obtained from adult rats and incubated for 180 minutes in the presence of increasing concentrations (10–10-10–4 M) of rosiglitazone. At the end of the incubation period, testis samples were processed for RNA analysis.
In Experiment 7, the effects of fasting upon testicular expression of resistin were analyzed. Adult males were subjected to food deprivation for 48 hours, and testes were collected at the end of fasting period. In addition, the effects of central leptin administration upon resistin mRNA levels were evaluated in testes from rats fasted for 48 hours and animals fed ad libitum. In this setting, rats were infused with recombinant leptin (15 μg/day) or vehicle, for 7 days, into the lateral ventricle, using an osmotic minipump (Alza Corp., Palo Alto, CA, USA). Leptin-treated animals were subjected or not to food deprivation for the last 48 hours of treatment. Conversely, in Experiment 8, testicular resistin mRNA levels were monitored in a rat model of diet-induced obesity (DIO), as well as in obese ob/ob mice. Five-week-old male Sprague-Dawley rats, selectively bred for the diet-induced obesity (DIO) or diet-resistant (DR) traits, were obtained from the Rowett Research Institute (Aberdeen, UK), and subsequently fed a high fat diet for 14 weeks, as described in detail elsewhere (Archer et al., 2003). The high-energy diet (31% fat; 4.5 kcal/g) was composed of 8% corn oil, 44% sweetened condensed milk and 48% Purina rat chow (Research Diets no. C11024, New Brunswick, NJ, USA). Rats fed a standard rat chow diet served as controls. Adult obese ob/ob and lean (+/?) mice (Aston strain) were obtained from a colony maintained at the Rowett Research Institute.
Finally, in Experiment 9, the effects of resistin upon basal and stimulated testosterone secretion in vitro were assessed using static incubations of adult rat tissue, as described elsewhere (Tena-Sempere et al., 1999; Tena-Sempere et al., 2002). Tissue samples were incubated in the presence of increasing doses of resistin (10–10-10–6 M), under basal or stimulated (co-incubation with 10 IU/ml hCG) conditions. In addition to secretory responses, the effects of resistin on the mRNA levels of several key factors in the steroidogenic route were evaluated, following a previously published protocol (Tena-Sempere et al., 2002).
RNA analysis by semi-quantitative RT-PCR
Total RNA was isolated from testicular samples using the single-step, acid guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski and Sacchi, 1987). Testicular expression of resistin mRNA was assessed by RT-PCR, optimized for semi-quantitative detection, using a specific primer pair [forward (5′-ACTTCAGCTCCCTACTGCCA-3′) and reverse (5′-GCTCAGTTCTCAATCAACCGTCC-3′)] flanking a 253-bp coding area of rat resistin cDNA (GenBank Acc. no. AF378366). In addition, in selected experimental designs, semi-quantitative RT-PCR amplification of StAR, P450scc, 3β-HSD and 17β-HSD type III mRNAs was conducted, using primer pairs and conditions described in detail elsewhere (Tena-Sempere et al., 2002). As internal control, amplification of a 149 bp fragment of L19 ribosomal protein mRNA was carried out in parallel in each sample, using the primer pair: L19 forward (5′-GAA ATC GCC AAT GCC AAC TC-3′) and L19 reverse (5′-TCT TAG ACC TGC GAG CCT CA-3′).
For amplification of the targets, 2 μg of total RNA were used to perform RT-PCR. Complementary DNA was synthesized using 200 U MoML-reverse transcriptase (Invitrogen, Paisley, UK), 20 U ribonuclease inhibitor RNase-Out and 1 nM random hexamer primers, in a total volume of 30 μl. RT reactions were incubated at 37°C for 1 hour and at 42°C for 10 minutes, and were terminated by heating at 95°C for 5 minutes. PCR amplification of the generated cDNAs was carried out in 50 μl of 1×PCR buffer in the presence of 1.25 U Taq-DNA polymerase (Invitrogen) and 1 nM forward and reverse primers. The amplification profile for rat resistin was: denaturation at 98°C for 15 seconds, annealing at 60°C for 30 seconds and extension at 72°C for 1 minute. Different numbers of cycles (ranging between 20-40) were tested to optimize amplification in the exponential phase of PCR (see Fig. 1). On this basis, 33 PCR cycles were chosen for analysis of resistin mRNA in the experimental groups.
(A,B) Analysis of resistin gene expression in random testis specimens, as evaluated by RT-PCR and Southern blot (A), and optimization of RT-PCR conditions for semi-quantitative evaluation of relative expression levels of resistin mRNA in testis samples (B). For comparative purposes, expression of resistin gene in white adipose tissue (WAT) is presented. (C) The developmental profile of expression of the resistin gene in rat testis throughout postnatal maturation. A representative semi-quantitative RT-PCR assay of expression levels of resistin mRNA in testicular samples from 5-, 15-, 30-, 60- and 90-day-old rats. Parallel amplification of L19 ribosomal protein mRNA served as internal control. Semi-quantitative values are the mean±s.e.m. of at least four independent determinatio ns. a,b and c denote groups that are statistically different (P<0.01; ANOVA followed by Tukey's test).
PCR-generated DNA fragments were resolved in Tris-borate buffered 1.5% agarose gels and visualized by ethidium bromide staining. Specificity of PCR products was confirmed by Southern blot using a 32P cDNA specific probe for rat resistin. Quantitative evaluation of RT-PCR signals was carried out by densitometric scanning using an image analysis system (Gel Doc 1000 Documentation System; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and values of the specific targets were normalized to those of internal controls to express arbitrary units of relative expression. In all assays, liquid controls and reactions without RT resulted in no amplification.
Real-time quantitative RT-PCR
To verify changes in gene expression observed by final-time RT-PCR, real-time RT-PCR was performed in selected experimental groups using the ABI 7700 sequence detection system (Perkin-Elmer Applied Biosystems, Foster City, CA). Reactions were performed, at least, in quadruplicate. Reverse transcription of total RNA was conducted as described above. The synthesized cDNAs were further amplified by PCR using the fluorescent dye SYBR green I and 1× PCR Master Mix (Applied Biosystems) containing 300 nmol/l of forward and reverse primers, in a final volume of 25 μl. All reactions were carried out using the following cycling parameters: 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Product purity was confirmed by dissociation curves and random agarose gel electrophoresis. No-template controls were included in all assays, yielding no consistent amplification. Standard curves were constructed for resistin (specific target) and RP-L19 (internal control) by plotting values of CT (the cycle at which the fluorescence signal exceeds background) versus log cDNA input (in nanograms). Accordingly, CT values from each experimental sample were then used to calculate the amount of resistin and RP-L19 mRNAs relative to the standard. For each sample, results in terms of resistin expression levels were normalized to those of the internal control RP-L19.
Resistin immunohistochemistry
Immunohistochemical detection of resistin peptide was carried out in 4% paraformaldehyde-fixed sections of rat testes from adult rats using a guinea pig anti-mouse resistin antibody (Linco Research, St Charles, MI, USA). For immunolabeling, testicular sections (5 μm thick) were submitted to antigen retrieval in a microwave oven and incubated overnight with the primary antibody (diluted 1:200). The sections were then processed according to the avidin-biotin-peroxidase complex (ABC) technique, as described elsewhere (Gaytan et al., 2003). Resistin immunoreactivity was identified as brown cytoplasmic staining in testicular sections counterstained with Hematoxylin. Different testicular cell types were identified based in morphological criteria, in keeping with previous references (Gaytan et al., 1994). Negative controls were run routinely in parallel by replacing the primary antibody with pre-immune serum. In addition, as control for antibody specificity, immunohistochemical reactions were carried out following pre-absorption of the antiserum overnight at 4°C with resistin (51-108) amide (Phoenix Peptides).
Testosterone measurement by specific RIA
T levels in static incubation media were measured using a commercial kit from ICN Biomedicals (Costa Mesa, CA, USA). All medium samples were measured in the same assay. The sensitivity of the assay was 0.1 ng/tube and intra-assay coefficient of variation was 4.5%.
Presentation of data and statistics
Semi-quantitative and real-time RT-PCR analyses were carried out, at least in quadruplicate, using independent RNA samples. For presentation, in each experimental design the expression levels in control/reference groups were assigned to a values of 100, and the others were normalized accordingly. Tissue incubations were conducted in duplicate, with a total number of 10-12 samples/determinations per group. Quantitative data are presented as mean±s.e.m. Results were analyzed for statistically significant differences using ANOVA, followed by Tukey's test. P<0.05 was considered significant.
Results
Testicular expression of resistin mRNA during postnatal development and cellular distribution of resistin peptide in adult testis tissue
Initial RT-PCR and Southern blot analyses revealed that the resistin gene is expressed in random testicular specimens. As anticipated, similar amplicons were obtained from white adipose tissue (WAT) (Fig. 1A). Consequently, assessment of resistin gene expression in rat testis was conducted in different experimental settings by means of semi-quantitative RT-PCR. To obtain optimal conditions for amplification, i.e. in the exponential phase of PCR, different numbers of cycles were tested. As shown in Fig. 1B, plotting of intensity of PCR signals against the number of amplification cycles revealed a strong linear relationship between cycles 20 and 40 (correlation coefficient r2=0.97). Thus, PCR amplification of resistin transcript in the experimental samples was carried out using 33 cycles.
Resistin mRNA levels were first evaluated in testicular samples throughout postnatal development, from the neonatal period to adulthood. Persistent expression of resistin transcript in rat testis was detected at all ages studied (5-, 15-, 30-, 60- and 90-day-old rats) by means of semi-quantitative RT-PCR. However, testicular levels of resistin mRNA were low at birth and progressively increased thereafter, with maximum expression in early adult (60-day-old) samples (Fig. 1C). Such an expression profile was confirmed by real-time RT-PCR analysis of representative testicular samples that showed maximum expression levels in 60-day-old rat testis (Fig. 2). In addition, real-time RT-PCR assays revealed that resistin mRNA levels are similar in testes from adult (90-day-old) and aged (17-month-old) animals. Moreover, the pattern of cellular expression of resistin peptide in adult testis was evaluated by immunohistochemistry. This analysis demonstrated a scattered distribution of resistin immunoreactivity, with specific signals in Leydig cells (identified as clusters of cuboidal cells in the interstitium) and, at low intensity, the seminiferous tubules, where Sertoli cells were weakly immunolabeled (Fig. 3). In addition, in some testicular sections, resistin immunoreactivity was uneven in testicular macrophages, identified by a number of morphological criteria including the typical pattern of chromatin organization, the presence of cytoplasmic projections, the exocentric location of the nucleus, and their presentation as isolated cells in the interstitium (see Gaytan et al., 1994). Specificity of resistin immunostaining was confirmed by pre-absorption of the primary antibody; a procedure that completely blocked labeling of testis sections (data not shown).
Real-time RT-PCR analysis of testicular resistin mRNA levels throughout post-natal development and in aging. Total RNA samples were obtained from 5-day, 15-day, 30-day, 60-day, 90-day, and 17-month-old rats. Quantitative data in terms of resistin expression levels were normalized to those of the internal control RP-L19. Values are the mean±s.e.m. of at least four determinations. a,b and c denote groups that are statistically different (P<0.01; ANOVA followed by Tukey's test).
Immunolocalization of resistin protein in adult rat testis sections. (A) A representative reaction after immunolabeling with the specific anti-resistin primary antibody. At higher magnification (B), specific resistin immunoreactivity was clearly evident on interstitial Leydig cells and, at a lower intensity, in Sertoli cells within the seminiferous tubules. In addition, in some tissue sections, resistin peptide was also detected in macrophages present in the testicular interstitium. Scale bars: (A) 50 μm; (B) 20 μm.
Hormonal regulation of testicular expression of resistin by pituitary gonadotropins
Hormonal regulation of testicular expression of resistin was first assessed using the hypophysectomized (HPX) rat as an experimental model. Long-term (4 week) HPX, a procedure that resulted in the atrophy of all testicular compartments and apparent arrest of spermatogenesis at early meiosis, induced a decrease in resistin mRNA expression levels that was prevented by replacement with the super-agonist of LH, hCG (10 IU/rat/24 hours for 7 days), but not with FSH (Fig. 4). In keeping with RT-PCR data, our immunohistochemical assays demonstrated clear-cut suppression of resistin immunoreactivity in testes from HPX rats that was partially restored by hCG replacement (Fig. 4). In addition, resistin mRNA levels were monitored after acute administration of gonadotropins to adult intact rats, using a protocol similar to that used recently by our group to evaluate the regulation of testicular expression of leptin receptor and ghrelin genes (Tena-Sempere et al., 2001; Barreiro et al., 2002). Injection of a bolus of hCG (25 IU/rat) only induced a moderate transient increase in resistin mRNA levels 8 hours after administration (Fig. 5A). In contrast, a bolus of FSH (12.5 IU/rat) evoked a persistent increase in resistin mRNA levels throughout the study period (2-24 hours), with peak values at 24 hours after administration (Fig. 5B).
Effects of hypophysectomy (HPX) and gonadotropin replacement on testicular expression of resistin. (Left panels) A representative RT-PCR assay of resistin mRNA levels in testes from long-term (4-week) HPX rats, with or without replacement of hCG (10 IU/rat/24 hours for 7 days) or FSH (7.5 IU/rat/24 hours for 7 days). Semi-quantitative values are the mean±s.e.m. of at least four independent determ inations. a,b and c denote groups that are statistically different (P<0.01; ANOVA followed by Tukey's test). (Right panels) Representative photomicrographs of resistin immunoreactivity in testis tissue from HPX rats, with or without replacement of hCG. (A) A representative control section. (B,C) Long-term HPX resulted in clear-cut suppression of resistin immunoreactivity (B), whereas hCG replacement partially restored this signal (C). Scale bar: 20 μm.
Acute regulation of testicular resistin mRNA expression by gonadotropins. (A) A representative RT-PCR assay of the testicular expression levels of resistin mRNA at different time-points (2-24 h) after exposure to 25 IU hCG in vivo. (B) A representative RT-PCR assay is shown of testicular resistin mRNA levels at different times (2-24 hours) after exposure to 12.5 IU recombinant FSH in vivo. Semi-quantitative values are the mean±s.e.m. of at least four independent determinations. a,b and c denote groups that ar e statistically different (P<0.01; ANOVA followed by Tukey's test).
Stage-specific expression of resistin mRNA in seminiferous tubule preparations and regulation by pituitary FSH
Analysis of resistin mRNA expression, in basal and FSH-stimulated conditions, was undertaken in preparations of seminiferous tubule fragments isolated at different stages of the epithelial cycle. Tubule segments were separated into four major groups corresponding to stages II-VI, VII-VIII, IX-XII and XIII-I of the cycle. Positive amplification of resistin mRNA was obtained in seminiferous tubule preparations at all stages of the cycle in basal conditions. However, the relative levels of resistin mRNA varied throughout the cycle, with maximum values in stages II-VI. A similar pattern of staged expression of the resistin gene was detected in seminiferous tubule fragments after 24 hours incubation in medium alone. Stimulation of tubule preparations with FSH (10 ng/ml) for 24 hours significantly increased the expression levels of resistin mRNA at all stages of the seminiferous epithelial cycle, except stages XIII-I (Fig. 6).
Expression profile of resistin gene in seminiferous tubules segments at different stages of the seminiferous epithelial cycle, in basal conditions and after stimulation with FSH. A representative RT-PCR assay of the expression levels of resistin mRNA in tubule preparations at stages II-VI, VII-VIII, IX-XII, and XIII-I of the cycle, in three experimental situations: immediately after isolation (C-0), after 24-hour culture in the presence of medium alone (C-24), or after 24-hour culture in the presence of FSH (10 ng/ml) (FSH-24). Semi-quantitative values are the mean±s.e.m. of at least four independent determinations. a,b and c denote groups that are statistically different (P<0.01; ANOVA followed by Tukey's test).
Regulation of testicular expression of resistin mRNA by the agonist of PPARγ rosiglitazone
In a next step, we analyzed the ability of the selective ligand of PPARγ, rosiglitazone, to regulate testicular expression of resistin mRNA. Expression of PPARs, including the γ-type, has been detected in the testis (Elbrecht et al., 1996), and thiazolidinediones, such as rosiglitazone, acting through PPARγ, modulate the adipose expression of resistin (Ukkola, 2002). Administration of rosiglitazone to adult rats induced a significant reduction in testicular resistin mRNA levels (Fig. 7A), without affecting body weight (329±6.5 g in rats treated with rosiglitazone for 1-wk versus 333±7.5 g in controls). Specificity of this effect, as mediated through PPARγ, is suggested by the fact that metformin, an insulin-sensitizer unrelated to PPARγ, failed to modify the levels of resistin mRNA in rat testis (data not shown). In line with in the vivo data, incubation of testicular tissue with increasing concentrations of rosiglitazone (10–10-10–4 M) induced a clear-cut decrease in resistin expression levels. Interestingly, responsiveness to rosiglitazone showed a biphasic pattern; maximum suppression was detected between 10–10 and 10–8 M, with a trend to normalization of expression values for doses ≥10–6 M (Fig. 7B).
Regulation of resistin mRNA expression in rat testis by the selective ligand of PPARγ, rosiglitazone. (A) A representative RT-PCR assay of the testicular expression levels of resistin mRNA after a 1-week exposure to rosiglitazone (TZD; 5 mg/kg/day), in vivo. (B) A representative RT-PCR assay of testicular expression levels of resistin mRNA after in vitro exposure to 10–10-10–4 M rosiglitazone (TZD). Semi-quantitative values are the mean±s.e.m. of at least four independent determinations. In A, *P<0.05 versus the vehicle-treated (VEH) group; in B, a,b and c denote groups that are statistically different (P<0.01; ANOVA followed by Tukey's test).
Regulation of testicular resistin mRNA expression by the nutritional status and leptin
Expression of testicular resistin was monitored in the rats after 48 hours of food restriction. This fasting regimen induced an approximate 20% reduction in body weight (Table 1). In this setting, a significant decrease in relative expression levels of resistin mRNA was observed after the 48-hour fast (Fig. 8). To address the relative contribution of the adipocyte-derived hormone, leptin, to this phenomenon, the effects of central infusion of leptin upon testicular resistin mRNA expression were also assessed, under normal feeding and 48-hour fasting conditions. Central administration of recombinant leptin to males fed ad libitum for 7 days induced a significant reduction in daily food intake and an approximate 14% reduction in body weight (Table 1). These were associated to a significant decrease in testicular resistin mRNA levels (Fig. 8). Conversely, central treatment with leptin for 7 days did not evoke further reduction in resistin mRNA levels in testes from rats subjected to food deprivation for the last 48 hours before sampling. Similar responses were obtained by semi-quantitative and real-time RT-PCR analyses. Finally, testicular expression of the resistin gene was also monitored in a rat model of diet-induced obesity and in genetically obese ob/ob mice. Diet-induced obese (DIO) rats showed significantly increased body weights compared with diet restricted (DR) rats and control rats fed with a standard chow diet (Table 2). In this model, real-time RT-PCR assays demonstrated that testicular levels of resistin mRNA in DIO rats are similar to those of DR and control animals (Table 2). Similarly, real-time RT-PCR assays were conducted in testis samples from lean and genetically obese ob/ob mice. Obese mice showed higher body weights than controls, while testicular resistin mRNA levels were significantly lower than those of lean mice (Table 2).
Body weight and daily food intake in rats after 48 hours fasting and/or a 7-day central infusion of recombinant leptin (15 μg/day)
Regulation of resistin mRNA expression in rat testis by the nutritional status and the adipocyte-derived hormone, leptin. (A) A representative RT-PCR assay is presented of the testicular expression levels of resistin mRNA in male rats fed ad libitum and after short-term (48 hours) fasting. Animals were infused centrally with recombinant human leptin (Lep; 15 μg/day) or vehicle (C), for 7 days. (B) Quantitative values of resistin mRNA levels in the experimental groups, determined by real-time RT-PCR, are presented as the mean±s.e.m. of at least four independent determinations. a,b and c denote groups that are statistically different (P<0.01; ANOVA followed by Tukey's test).
Body weight and testicular mRNA levels of resistin in a rat model of diet-induced obesity and genetically obese ob/ob mice
Effects of resistin stimulation upon testicular testosterone secretion in vitro
Finally, the involvement of resistin in the modulation of testicular steroidogenesis was evaluated using an in vitro system. Analysis of testosterone responses to different doses of resistin was conducted, at 90 and 180 minutes incubation, in basal and hCG-stimulated conditions. For in vitro stimulation, we used an active fragment of the resistin molecule, resistin (23-42), that was previously reported to induce significant hypothalamic responses in terms of c-fos expression after central administration (Tung and Dickson, 2002). Resistin, in a dose-dependent manner, was able to stimulate basal testosterone secretion by incubated testicular tissue, with significant increases being detected after challenge with 10–8 and 10–6 M doses, but not 10–10 M resistin. Likewise, hCG-stimulated testosterone secretion was enhanced by co-incubation with resistin, with a similar dose-dependent profile (Fig. 9). These secretory effects were correlated with mRNA expression of several steroidogenic key factors, in basal conditions. The targets to be analyzed were selected on the basis of our previous studies on the effects of leptin and ghrelin upon the steroidogenic pathway (Tena-Sempere and Barreiro, 2002; Tena-Sempere et al., 2002) and included steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage (P450scc), 3β-hydroxy steroid dehydrogenase (3β-HSD), and testis-specific 17β-HSD type III. Semi-quantitative RT-PCR analyses showed that none of the selected targets was modified by a 180-minute incubation with 10–10-10–6 M resistin (data not shown).
Regulation of in vitro testicular testosterone secretion by resistin. Testicular slices were incubated with increasing doses of resistin-(23-42) (10–10-10–6 M) in the absence (–hCG; basal conditions) or the presence (+hCG; stimulated conditions) of 10 IU/ml hCG. The concentration of testosterone in the media was monitored after 90 and 180 minutes incubation; only data from the 90-minute incubation are presented. Values are normalized per gram of incubated tissue. Data are expressed as mean±s.e.m. (n=10-12 samples/group). **P<0.01 vs. corresponding controls (ANOVA followed by Tukey's test).
Discussion
Resistin has recently emerged as a novel adipocytokine with potential implications in the modulation of insulin sensitivity and adipocyte differentiation (Kim et al., 2001; Steppan et al., 2001a). However, a strong debate on the actual role of resistin in obesity-associated insulin resistance has arisen (Ukkola, 2002), and in addition additional physiological effects of resistin remain largely unknown. Expression of resistin has been reported in tissues and cell types other than the adipose, including endothelial and vascular smooth cells, peripheral mononuclear cells and macrophages in humans (Savage et al., 2001; Patel et al., 2003), as well as hypothalamus, pituitary, gastrointestinal tract and adrenal gland in rodents (Morash et al., 2002; Nogueiras et al., 2003a). Thus, endocrine and non-endocrine actions of resistin other than control of glucose tolerance are possible. In this context, our study provides novel evidence on the expression, regulation and functional role of resistin in rat testis. Testicular expression of resistin was assessed by molecular and immunological approaches. These demonstrated that the resistin gene is expressed in rat testis throughout postnatal development, with low levels in the neonatal period and increasing expression thereafter that peaked in early adult age. An apparent decline in the relative mRNA levels of resistin was detected in adult testes. The possibility, however, that this may be due to a genuine decrease in testicular resistin gene expression with ageing seems unlikely as no further reduction was observed in testes from aged (17-month-old) rats. Alternatively, as the major expression of resistin within the testis occurs in Leydig cells, it is possible that further enlargement of the spermatogenic fraction of the testis with age may have lowered the relative levels of the specific resistin signal.
In good agreement with RT-PCR data, our immunohistochemical assays demonstrated that resistin protein is detectable in the adult testis, with specific resistin signals being located in interstitial Leydig cells and, to a lesser extent, in Sertoli cells within seminiferous tubules. In addition, resistin immunoreactivity was unevenly detected in some macrophages of the testicular interstitium (see Figs 2 and 3). It is noteworthy that expression of resistin has been previously demonstrated in human macrophages (Patel et al., 2003). However, the possibility that this may solely account for the testicular expression of resistin reported herein can be ruled out, as expression of the resistin gene was under the regulation of pituitary gonadotropins and detected also in isolated seminiferous tubule preparations. Moreover, resistin immunoreactivity was clearly located in Leydig and Sertoli cells. The physiological role, if any, of resistin expression in testicular macrophages is presently under investigation in our laboratory.
The regulation of testicular resistin expression by gonadotropins was assessed, given their major role in the control of testis development and function (Tena-Sempere and Huhtaniemi, 2003). Our results showed that both luteinizing hormone (LH) and FSH participate in the tuning of resistin expression in rat testis, with partially different roles. In a gonadotropin deficient background (i.e. the HPX model), a significant decrease in resistin mRNA levels was noted, whereas treatment with hCG (as superagonist of LH), but not FSH, was able to restore resistin mRNA expression. In contrast, in a normal gonadotropin background (i.e. the intact rat), FSH stimulation evoked a persistent elevation of resistin levels up to 24 hours. The ability of FSH to acutely stimulate resistin mRNA was confirmed using a culture system of isolated seminiferous tubule fragments at different stages of the seminiferous epithelial cycle. This approach demonstrated also that expression of the resistin gene in seminiferous tubules is stage-specific, with peak values at stages II-VI. The latter may be due to the highest FSH receptor expression and FSH responsiveness in terms of cAMP production detected at these stages (Parvinen, 1993). Overall, our current results strongly suggest that expression of testicular resistin is subjected to precise regulation, involving stimulatory effects of pituitary gonadotropins.
Resistin gene expression in the adipose tissue is under the control of a plethora of hormones and mediators that includes the ligand-activated transcription factor PPARγ (Song et al., 2002). In fact, several studies indicate that the synthetic agonist of PPARγ, rosiglitazone, is able to decrease adipose expression of resistin (Steppan et al., 2001a; Ukkola, 2002; Song et al., 2002). In our experiments, testicular levels of resistin mRNA were inhibited by exposure to rosiglitazone, both in vivo and in vitro, suggesting the involvement of testicular PPARγ in local control of resistin gene expression. In this sense, expression of PPARγ has been previously detected in human testis (Elbrecht et al., 1996), yet the physiological relevance of this system in testicular function remains largely unknown. Of particular interest, in vitro exposure to increasing doses of rosiglitazone (10–10-10–4 M) evoked a clear-cut biphasic inhibitory effect upon resistin mRNA expression, with maximum suppression detected between 10–10 and 10–8 M, and a trend to normalization of expression values for doses ≥10–6 M. Such a biphasic response curve is strikingly similar to that reported for the effects of rosiglitazone upon resistin mRNA expression in human macrophages (Patel et al., 2003), and probably reflects the complex interaction of PPARγ with other transcription factors in the control of resistin gene expression in different cellular systems. Specificity of the inhibitory effects of rosiglitazone upon testicular expression of resistin is indirectly shown by the fact that metformin, an insulin sensitizer unrelated to PPARγ, was unable to induce similar inhibitory responses. We are currently investigating the role of the PPARγ-resistin system in the control of relevant testicular functions such as androgen secretion and spermatogenesis.
A putative regulator of testicular resistin gene expression is the nutritional status, as a protocol of 48-hour starvation evoked a significant decrease in resistin mRNA levels in rat testis. Several mechanisms may account for this effect, including mobilization of fatty acids (as endogenous ligands of PPARγ) and/or decreased gonadotropin secretion, which have been reported after extended fasting (Bergendahl et al., 1991). To note, however, in our fasting conditions, the decrease in mean LH levels was shortly below the limit of statistical significance (0.248±0.17 ng/ml in 48-hour fasted rats vs. 0.291±0.09 ng/ml in rats fed ad libitum). One tempting possibility is that reduced leptin secretion following food deprivation might be involved in such a phenomenon, as leptin receptors are expressed in rat testis (Tena-Sempere et al., 2001). In fact, central administration of leptin, which induced a significant weight loss (see Table 1), and probably lowered peripheral leptin levels, also evoked a significant reduction in testicular resistin mRNA expression in animals fed ad libitum. In contrast, however, resistin mRNA levels remained unaltered in diet-induced obese rats, which are expected to have high leptin levels. Thus, it appears probable that threshold leptin input is needed to maintain testicular expression of the resistin gene. In good agreement, obese ob/ob mice, lacking circulating leptin despite large fat stores, showed decreased testicular levels of resistin mRNA (see Table 2). Further experimental work, including systemic administration of leptin to fasting animals, is needed to fully prove this hypothesis.
Further evidence for a functional role of resistin in the control of testicular function is provided by its ability to moderately but significantly elicit basal and hCG-stimulated testosterone secretion in vitro. As the resistin receptor(s) remains, so far, uncloned, the primary site of action and mechanism for the steroidogenic effects of resistin were not directly evaluated. Recently, we have demonstrated that leptin and ghrelin, relevant signals in the control of energy homeostasis, are direct inhibitory factors in the regulation of testicular steroidogenic function; a phenomenon that involves decreased expression of the genes encoding relevant up-stream steps in the steroidogenic route as StAR protein and P450scc (Tena-Sempere and Barreiro, 2002; Tena-Sempere et al., 2002). In contrast, our present data indicate that resistin-induced stimulation of testosterone secretion is not linked to enhanced expression of the mRNAs coding for key steroidogenic factors such as StAR protein, P450scc, 3β-HSD and 17β-HSD. This suggests either that other steps within the steroidogenic cascade are involved or a relevant role for post-transcriptional events in this acute stimulatory response. Interestingly, a reciprocal stimulatory loop between androgen and resistin is likely to exist, as androgens elicit adipose expression of resistin (Ling et al., 2001; Nogueiras et al., 2003b), whereas resistin stimulates testicular testosterone secretion (Fig. 9). It is interesting that a strikingly opposite system is operative for leptin, as androgens inhibit adipose expression of leptin (Caprio et al., 2001), and leptin inhibits testosterone secretion in vitro (Tena-Sempere et al., 1999). The physiological relevance of the cross-talk between such stimulatory and inhibitory signals of the steroidogenic function is presently under evaluation in our laboratory.
In conclusion, our data demonstrate that the novel adipocytokine, resistin, is expressed in the rat testis throughout postnatal development under the precise control of an array of hormones and mediators that include gonadotropins, PPARγ transcription factor, leptin and the nutritional status. Moreover, our results are the first to provide evidence for a direct role of resistin in the control of testicular testosterone secretion. Based on similarities with other factors involved in the control of energy status and/or adipose function, such as leptin and ghrelin, it is tempting to hypothesize that resistin may operate as a novel endocrine integrator linking energy homeostasis and reproduction. In addition, the potential contribution of resistin to the inverse relationship between gonadal function, obesity and insulin resistance, such as that observed in type 2 diabetes, merits further investigation.
Acknowledgements
The authors are indebted to L. M. Williams (Rowett Research Institute, Aberdeen, UK) for her support in conducting the experimental studies in obese models. R.N. is the recipient of a Research Training Grant (FP-5; Contract HPMT-CT-2001-00410) from the European Commission. This work was supported by grants from DGESIC (Ministerio de Ciencia y Tecnología, Spain), the Academy of Finland and Turku University Central Hospital.
- Accepted March 4, 2004.
- © The Company of Biologists Limited 2004
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