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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2011 Aug;92(4):272–280. doi: 10.1111/j.1365-2613.2011.00772.x

Effect of dexamethasone and testosterone treatment on the regulation of insulin-degrading enzyme and cellular changes in ventral rat prostate after castration

Juliany S B César Vieira *, Karina L A Saraiva †, Maria C L Barbosa *, Regina C C Porto *, Juan C Cresto ‡, Christina A Peixoto †, Maria I Wanderley *, Daniel P Udrisar *
PMCID: PMC3144516  PMID: 21507087

Abstract

Insulin-degrading enzyme (IDE) has been shown to enhance the binding of androgen and glucocorticoid receptors to DNA in the nuclear compartment. Glucocorticoids cause hyperglycaemia, peripheral resistance to insulin and compensatory hyperinsulinaemia. The aim of the present study was to investigate the effect of dexamethasone (D), testosterone (T) and dexamethasone plus testosterone (D + T) on the regulation of IDE and on the remodelling of rat ventral prostate after castration (C). Castration led to a marked reduction in prostate weight (PW). Body weight was significantly decreased in the castrated animals treated with dexamethasone, and the relative PW was 2.6-fold (±0.2) higher in the D group, 2.8-fold (±0.3) higher in the T group and 6.6-fold (±0.6) higher in the D + T group in comparison with the castrated rats. Ultrastructural alterations in the ventral prostate in response to androgen deprivation were restored after testosterone and dexamethasone plus testosterone treatments and partially restored with dexamethasone alone. The nuclear IDE protein level indicated a 4.3-fold (±0.4) increase in castrated rats treated with D + T when compared with castration alone. Whole-cell IDE protein levels increased approximately 1.5-fold (±0.1), 1.5-fold (±0.1) and 2.9-fold (±0.2) in the D, T and D + T groups, respectively, when compared with castration alone. In conclusion, the present study reports that dexamethasone-induced hyperinsulinaemic condition plus exogenous testosterone treatment leads to synergistic effects of insulin and testosterone in the prostatic growth and in the amount of IDE in the nucleus and whole epithelial cell.

Keywords: castration, glucocorticoid, insulin resistance, insulin-degrading enzyme, prostate, testosterone, ultrastructure

Insulin-degrading enzyme (IDE), a zinc metalloprotease with a molecular weight of approximately 110 kDa, was first discovered by Mirsky as ‘insulinase’ (Mirsky & Broh-Kahn 1949). Insulin-degrading enzyme degrades insulin, amyloid-β (A-β) peptide (Qiu et al. 1998), peptides that are critically important in the pathogenesis of type 2 diabetes mellitus (DM2) and Alzheimer's disease (AD), respectively, and also insulin-like growth factors I and II transforming growth factor-α and other bioactive peptide substrates (Duckworth et al. 1998; Guo et al. 2010). Studies on insulin processing (Udrisar et al. 1984), proteasome modulation (Duckworth et al. 1998), β-amyloid peptide clearance regulation (Kurochkin 1998) and interaction with androgen receptor and glucocorticoid receptor (Kupfer et al. 1993) suggest that IDE is a multifunctional protein with broad and relevant roles in several basic cellular processes. The ability of IDE to interact with and enhance the DNA binding of AR and GR suggests that IDE may be important for transcriptional activity of these receptors (Kupfer et al. 1993). In a previous work, we showed that testosterone and oestrogen regulate the expression and activity of IDE in male and female reproductive system respectively, where IDE is involved in cellular growth and differentiation (Udrisar et al. 2005). However, the precise mechanism of the physiological role of IDE is yet to be established. It is well known that the administration of glucocorticoids leads to an increased production of hepatic glucose, peripheral resistance to insulin action and compensatory hyperinsulinaemia as well as an increase in pancreatic islet mass (Rafacho et al. 2008). Many risk factors for benign prostatic hyperplasia (BPH) such as insulin, insulin-like growth factors (IGFs) and dyslipidaemia might act through androgen-independent mechanism, and it appears that prostate cancer may be an additional aspect of the insulin resistance syndrome (Barnard et al. 2002). Vikram et al. (2010) have recently reported a correlation between hyperinsulinaemia and prostate growth linking hyperinsulinaemia with BPH in insulin-resistant rats. After castration, the rat ventral prostate undergoes regression because of the induction of apoptosis in the epithelial cells of the gland. However, treatment with testosterone stimulates the re-growth of the prostate gland (Cunha et al. 1987). The aim of the present study was to investigate the effect of dexamethasone, testosterone and dexamethasone plus testosterone on the regulation of IDE and the remodelling of rat ventral prostate following castration. The results reveal that the level of IDE highly increased in the nucleus as well as the whole cell during prostatic re-growth promoted by dexamethasone plus testosterone after rats’ castration. Therefore, the increased expression of insulin-degrading enzyme could be important to tissue remodelling after dexamethasone and testosterone treatment in castrated rats.

Materials and methods

Animals

Adult male Wistar rats weighing 250–300 g, bred in our animal facilities, were housed under a controlled conditions (temperature: 25–29 °C; light from 6 am to 6 pm) with free access to standard laboratory chow and tap water. Castration was performed via a scrotal incision under CO2 followed by ether anaesthesia. After 1 day, two groups of animals began to drink water containing dexamethasone (Decadron; Lab. Merck Sharp and Dohme, São Paulo, Brazil) at doses calculated based on the water intake of each rat to represent approximately 1 mg/Kg dexamethasone (D) per rat per day (Saad et al. 1993). Oral D administration was continued until the rats were sacrificed. Three days after castration, two groups [castrated + testosterone (T) and castrated + D + T] received 300 μg/100 g body weight (BW) of testosterone propionate (Sigma Chemical Co. St. Louis. MO, USA) in corn oil injected subcutaneously daily for 3 days (Udrisar et al. 2005). The other group (castrated alone) received a vehicle instead of testosterone propionate. This yielded four experimental groups: castrated (C), castrated plus dexamethasone (D), castrated plus testosterone (T) and castrated plus dexamethasone plus testosterone (D + T). Rats were sacrificed 24 h after the final injection and 6 days after castration by exposure to CO2 followed by decapitation. The ventral prostate was immediately removed, weighed and placed in the corresponding buffers for ultrastructural and immunocytochemical studies or stored in liquid nitrogen for the insulin degradation study. The relative weight is given as the percentage of ventral prostate weight (PW) divided by whole BW; the value obtained for intact rat was taken as 100% (de Carvalho et al. 1997). Blood from fasted (12–14 h) rats was obtained from the tail tip, and whole-blood glucose concentrations were measured using a glucometer (Accu-Chek Advantage, Roche, Brazil). The local ethics committee for animal experimentation at the institution approved all treatments.

Preparation of cytosolic fractions from rat ventral prostate and degradation assay

The preparation of the cytosolic fraction and 125I-insulin degradation were performed in separate groups, as described in detail elsewhere (Udrisar et al. 2005). 125I-insulin control (precipitation with TCA without the cytosolic fraction) contained more than 92% intact insulin; hence, the experimental results were corrected for this value. Values are given as percentage of 125I-insulin degradation per 60 μg/ml of cytosolic protein. Protein concentrations were determined based on Bradford (1976), using bovine serum albumin as the standard.

Electron transmission microscopy

For routine procedures, fragments of the ventral prostate from the rats of the different groups (intact, C, D, T and D + T) were fixed overnight in a solution containing 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer. After fixation, the samples were washed twice in the same buffer, postfixed in a solution containing 1% osmium tetroxide, 2 mM calcium chloride and 0.8% potassium ferricyanide in 0.1 M cacodylate buffer, pH 7.2, dehydrated in acetone and embedded in Embed 812. (Electron Microscopy Science, Washington, PA, USA) Polymerization was carried out at 60 °C for 3 days (Saraiva et al. 2006, 2009). Ultrathin sections were collected on 300-mesh nickel grids, counterstained with 5% uranyl acetate and lead citrate and examined with a FEI Morgani 268D (North America NanoPort, Hillsboro, OR, USA) transmission electron microscope. For the immunocytochemical study, the samples from the castrated, dexamethasone, testosterone and dexamethasone plus testosterone groups were fixed overnight in a solution containing 0.1% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer. After fixation, the samples were washed three times in the same buffer, incubated with 50 mM ammonium chloride for 40 min, dehydrated in ethanol and embedded in LR-White resin. Polymerization was carried out at 30 °C for 5 days. This procedure was carried out as described by Peixoto et al. (1999).

Immunocytochemistry

Ultrathin sections of ventral prostate from the castrated, dexamethasone, testosterone and dexamethasone plus testosterone groups were cut with a diamond knife, collected on nickel grids and incubated for 30 min at room temperature in 0.02 M PBS, pH 7.2, containing 1% BSA and 0.01% Tween 20 (PBS-BT). The sections were then incubated for one hour with primary antibodies against IDE (mouse monoclonal antibody, MMS-282R; Covance Research Products, Berkeley, CA, USA) or against AR (mouse monoclonal antibody AR441:SC-7305; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:50 in PBS-BT. The sections were then washed in PBS-BT and incubated with a secondary antibody – 10-nm colloidal gold-labelled goat anti-mouse IgG (Sigma Chemical Co., St. Louis, MO, USA). As the antibody control, sections were incubated only in the presence of the gold-labelled marker. Following the immunostaining procedures, the sections were counterstained with 5% uranyl acetate and lead citrate (Peixoto et al. 1999). Quantitative analysis was performed on photomicrographs at a final magnification of 28,000× taken from 10 different, randomly chosen, portions of nucleus and cytoplasm for the comparison of the numbers of gold-labelled particles among the different groups using the Student's t-test. As the samples were processed in an identical manner, no correction of tissue shrinkage was performed.

Statistics

Results are expressed as the mean ± SEM (n = 3–9 experimental determinations) of at least two different experiments. The data were analysed by one-way analysis of variance (anova, Dunnett multiple comparison test). Statistical comparisons between groups were made using the Student's t-test. Linear regression was calculated between the nuclear IDE and the relative PW. The level of significance was set at P < 0.05.

Results

Body weight was significantly decreased (Figure 1a), and glucose was increased (Figure 1b) in animals treated with dexamethasone. Castration led to a marked reduction in absolute and relative PW (Figure 2a,b). Five days after dexamethasone treatment and 3 days after the administration of testosterone or both dexamethasone plus testosterone to castrated rats, the regression of absolute and relative PW was significantly inhibited in castrated plus D group and significantly restored in castrated plus T and castrated plus D plus T groups (Figure 2a,b). The relative PW was 2.6-fold (±0.2) higher in the D group, 2.8-fold (±0.3) higher in the T group and 6.6-fold (±0.6) higher in the D + T group in comparison with the castrated rats (Figure 2b). In intact rats, the ultrastructure of the acinar epithelium of the ventral prostate was characterized by columnar cells with a basal nucleus at the periphery, rough endoplasmic reticulum (RER) intensively arranged in the perinuclear surface, Golgi complex scattered throughout the cytoplasm and mitochondria well distributed in the cell; secretory vacuoles were mainly in the apical region, and homogenous secretion was observed in the lumen of prostate acini (Figure 3a,b). In castrated rats, the acinar epithelium of ventral prostate was characterized by marked atrophy of the organelles; the RER was scarce and disintegrated; residual bodies were encountered in the cytoplasm; and a heterogeneous electron-dense secretion was observed in the lumen of prostate acini (Figure 3c,d). In castrated rats treated with dexamethasone, the epithelial cells exhibited dilated organelles, including RER, Golgi and mitochondria, and the luminal space exhibited homogenous secretion (Figure 3e,f). The castrated rats treated with testosterone (Figure 3g,h) and dexamethasone plus testosterone (Figure 3i,j) had similar characteristics to those of the control group (intact rats); abundant RER, Golgi complexes, mitochondria scattered throughout the cytoplasm and homogenous secretion in the lumen of prostate acini were observed. Ultrastructural changes in the ventral prostate occurred in response to androgen deprivation. The alterations were restored following treatment with testosterone and dexamethasone plus testosterone and partially restored with dexamethasone alone. To study the effect of dexamethasone, testosterone and dexamethasone plus testosterone on the activity and amount of the IDE in rat ventral prostate after castration, the degradation of 125I-insulin in cytosolic fraction was measured (60 μg/ml of prostate homogenate at 100,000 g for 60 min for supernatant), along with the immunodetection of IDE by immunocytochemistry. Figure 4 illustrates the effect of castration and castration following the administration of dexamethasone, testosterone or both treatments on cytosolic IDE activity. The insulin degradation analysis indicated a 1.4-fold (±0.1), 2.1-fold (±0.1) and 1.7-fold (±0.1) increase in IDE activity 6 days following castration in the D, T and D + T groups, respectively, in comparison with the castrated group. Mouse monoclonal antibody against IDE was used to detect the presence of IDE in the nucleus and whole epithelial cell in all experimental groups (Figure 5). The immunocytochemistry analysis revealed the presence of gold-labelled particles corresponding to the IDE protein in all groups and in both the nucleus (a) and whole cells (b) (Figure 6). The estimation of nuclear IDE protein level after immunocytochemistry indicated a 4.3-fold (±0.4) increase in IDE protein levels 6 days after castration in the rats treated with dexamethasone plus testosterone in comparison with castrated rats. Whole-cell IDE protein levels increased approximately 1.5-fold (±0.1), 1.5-fold (±0.1) and 2.9-fold (±0.2) in the D, T and D + T groups, respectively, in comparison with the castrated rats. Figure 7(a,b) displays the results of the immunocytochemistry analysis when the antibody against AR was used. Nuclear AR (a) protein level indicated a 4.6-fold (±0.4), 10.5-fold (±2.2) and 3.6-fold (±0.6) increase in the D, T and D + T groups, respectively, in comparison with the castrated rats. Following the same pattern, the whole AR (b) protein level indicated a 3.1-fold (±0.3), 5.1-fold (±0.7) and 3.0-fold (±0.3) increase in the D, T and D + T groups, respectively, in comparison with the castrated rats. Figure 8 shows that nuclear IDE plotted against the relative PW in all four groups (data derived from Figures 2b and 6a) results in a linear correlation (r2 = 0.944), suggesting that the altered relative PW observed in all four groups is related to the nuclear IDE level.

Figure 1.

Figure 1

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Body weight changes (panel a) and basal glycaemia (panel b) in intact rats, castrated (c) rats, castrated rats treated with dexamethasone (D), castrated rats treated with testosterone (T) and castrated rats treated with dexamethasone plus testosterone (D + T); data are expressed as mean ± SEM; n = 3; *P < 0.001 vs. intact (one-way analysis of variance anova, Dunnett multiple comparison test); for conditions, see Materials and methods.

Figure 2.

Figure 2

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Absolute and relative prostate weight (a and b) in castrated rats after 5 days, castrated rats treated with dexamethasone (1 mg/Kg per day for 5 days), castrated rats after 3 days plus 3 days of testosterone administration and castrated rats treated with dexamethasone plus testosterone; data were obtained 6 days after castration; columns represent mean ± SEM of four to eight rats; *P < 0.01 vs. castrated group, (one-way analysis of variance anova, Dunnett multiple comparison test); for conditions, see Materials and methods.

Figure 3.

Figure 3

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Ultrastructural analysis of epithelium of ventral lobe prostate; (a and b) control columnar cells with basal nucleus (N), RER, mitochondria, well-developed Golgi complex (arrow) and secretory vacuoles; (c and d) cells from castrated rats with atrophied RER, mitochondria, Golgi complex (arrows) and residual body (full star); luminal surface with heterogeneous electron-dense secretion (empty star); (e and f) cells from castrated rats treated with dexamethasone presenting dilated RER, mitochondria and Golgi (arrow); secretory vacuoles in apical region and luminal space with homogenous secretion (empty star); (g and h) cells from castrated rats treated with testosterone showing abundant RER, Golgi complex (arrows), mitochondria and presence of secretory vacuoles; (i and j) cells from castrated rats treated with dexamethasone plus testosterone presenting well-distributed RER, Golgi complex (arrow) and mitochondria; RER, rough endoplasmic reticulum; M, mitochondria; SV, secretory vacuoles; L, lipid droplet; Bar = 1 μm.

Figure 4.

Figure 4

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Cytosolic 125I-insulin degradation in ventral prostates six days after castration in castrated rats, castrated rats treated with dexamethasone (1 mg/Kg per day for 5 days), castrated rats after 3 days plus 3 days of testosterone administration and castrated rats treated with dexamethasone plus testosterone; Cytosolic proteins (60 μg/ml of prostate homogenate at 100,000 g for 60 mins for supernatant) were incubated with 125I-insulin (20 000 cpm) in 500 μl of 20 mM Tris–HCl, with 10 mM MgCl2, pH 7.4, for 15 mins at 32 °C. The reaction was terminated by the addition of 0.4 vol TCA (50% v/v) and 0.6 vol bovine albumin (1% w/v) at 4 °C. After centrifugation, the pellet and supernatant were separated; the pellet was washed, and radioactivity was measured (radioactivity total = cpm pellet + cpm supernatant). The radioactivity in the supernatant was considered degraded 125I-insulin. The degradation was linear in relation to incubation time and cytosolic protein concentration. Values are expressed as percentage of degraded 125I-insulin (see Materials and methods) per 60 μg/ml of cytosolic protein. Results are expressed as the mean ± SEM of three determinations; *P < 0.001 vs. castrated group, unpaired Student's t-test.

Figure 5.

Figure 5

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Insulin-degrading enzyme (IDE) immunocytochemistry. (a and b) Few labelling was observed in castrated samples for IDE in nucleus (a) and cytoplasm portion (b) (arrows). (c and d) An increased labelling to IDE in nucleus (b) and cytoplasm (d) was detected in cells from castrated rats treated with dexamethasone plus testosterone (arrows). M, mitochondria; N, nucleus; RER, rough endoplasmic reticulum. Bars = 500 nm.

Figure 6.

Figure 6

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Quantitative analysis of gold-labelled particle distribution (anti-insulin-degrading enzyme as first antibody) in nucleus (panel a) and whole cell (panel b) of the epithelium of the ventral prostate lobe in C, D, T and D + T groups;*P < 0.0001 vs. castrated group, unpaired Student's t-test; for conditions, see Materials and methods.

Figure 7.

Figure 7

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Quantitative analysis of gold-labelled particle distribution (anti-AR as first antibody) in nucleus (panel a) and whole cell (panel b) of the epithelium of the ventral prostate lobe in C, D, T and D + T groups;*P < 0.0001, unpaired Student's t-test; for conditions, see Materials and methods.

Figure 8.

Figure 8

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Correlation between nuclear insulin-degrading enzyme (gold-labelled particle; panel a of Figure 6) and relative ventral prostate weight (panel b of Figure 2); Y-intercept when X = 0.0: 0.116 ± 2.36, X-intercept when Y = 0.0: −0.7585, r2 = 0.944; Pearson r: 0.972; total number of values: 40.

Discussion

Following castration, the rat ventral prostate undergoes regression because of the induction of apoptosis in the epithelial cells of the gland. However, treatment with testosterone stimulates the re-growth of the prostate gland (Cunha et al. 1987). It is established that the administration of dexamethasone leads to an increased production of hepatic glucose, peripheral resistance to insulin and compensatory hyperinsulinaemia (Saad et al. 1993). Experimental hypoinsulinaemia and castration causes decrease in the weight of prostate, and diet-induced hyperinsulinaemia causes increase in the cellular proliferation and the enlargement of the prostate gland (Vikram et al. 2010). It was showed that insulin delays castration-associated increased apoptosis in the prostate of diabetic rats and that the survival and anti-apoptotic effect of insulin on ventral prostate epithelial cells were in both the presence and absence of androgen stimulation (Damas-Souza et al.2010). Thus, chronic change in the systemic insulin level affects the growth of the prostate gland. It has been shown that IDE is regulated during development and is differentially expressed in various rat tissues, supporting the possibility that IDE plays a physiological role in the regulation of cell growth and development (Kuo et al. 1993; Udrisar et al. 2005). In the present study, the effect of dexamethasone-induced hyperinsulinaemia, testosterone and dexamethasone plus testosterone on the activity and quantity of IDE in ventral prostate after castration was investigated, along with the remodelling of rat ventral prostate. The prostate was used as a suitable model of castration-induced apoptosis and testosterone-induced cell proliferation (re-growth of the prostate gland after castration) with the addition of dexamethasone-induced hyperinsulinaemia in both experimental conditions. Castration, as expected, produced a marked reduction in absolute PW. The regression of PW was significantly inhibited in castrated plus D group and significantly restored in castrated plus T and castrated plus D plus T groups. These results are magnified when expressed as relative PW because BW was significantly decreased in animals treated with dexamethasone. The reduced regression in castrated plus D group and the re-growth of prostate gland after dexamethasone plus testosterone treatment in castrated rats can be attributed to high dose of dexamethasone-induced hyperinsulinaemia. Yono et al. (2008) addressed that the higher prostatic weight in genetically diabetic rats as compared with streptozotocin-treated (STZ) hypoinsulinaemic rats was because of the difference in the level of insulin. In addition, the survival and anti-apoptotic effect of insulin on ventral prostate epithelial cells was recently reported by Damas-Souza et al.(2010). In contrast, it was showed that dexamethasone treatment in intact rats resulted in atrophy and decreased proliferative activity of prostatic epithelial cells (Ribeiro et al. 2008). Under the condition of castration plus D, plus T and plus D + T, there was an increase in IDE activity in the cytosolic fraction of the prostate homogenate as well as the amount of IDE in the whole cell when compared with castration alone. The androgen receptor (AR) level also increased in the proliferative state in the nucleus and whole cell, but to a lower degree when in the presence of dexamethasone. The increase in the amount of IDE in the nucleus and whole cell in the D + T group suggests an additive effect of hyperinsulinaemic state induced by dexamethasone and testosterone treatment. The same additive effect between insulin and testosterone was also found in the PW and in the relative PW. Thus, the present study demonstrates a positive correlation between nuclear IDE and the relative PW. Recently, the synergistic action of insulin and testosterone in the prostatic growth was reported (Fan et al. 2007). The importance of hyperinsulinaemia and testosterone in the activity and amount of IDE was also previously reported by us (Udrisar et al. 1984, 2005). Further, literature also supports molecular basis for the interaction between insulin and androgen signalling (Fan et al. 2007; Srinivasan & Nawaz 2010). Ultrastructural changes occurred in the acinar epithelium of ventral prostate in response to androgen deprivation. These alterations were restored following treatment with testosterone and dexamethasone plus testosterone and were partially restored with dexamethasone alone. The development, growth and function of the prostate are known to be androgen dependent (Cunha et al. 1987), and AR levels are regulated by androgens (autoregulation). While androgen stabilizes AR levels, glucocorticoids block this effect and predominate, as demonstrated by Burnstein et al. (1995) and the present study. Androgen action is mainly indirect, occurring through the prostatic production of certain growth factors (Steiner 1993; Lopaczynski et al. 2001; Cunha 1996). These locally produced growth factors are considered autocrine and/or paracrine mediators of the stromal–epithelial interaction (Culig et al. 1996). A number of studies have indicated that IGFs, epidermal growth factor (EGF), keratinocyte growth factor, TGF-α and basic fibroblastic growth factor are mitogenic in prostate tumour cells and normal prostate cells (Steiner 1993; Culig et al. 1996; Cohen et al. 1991; Byrne et al. 1996). A link between hyperinsulinaemia and benign prostate hyperplasia has recently been demonstrated in insulin-resistant rats (Vikram et al. 2010). Some of these growth factors are IDE substrates, such as insulin, IGFs and TGF-α, or bind to IDE, such as EGF (Gehm & Rosner 1991; Misbin & Almira 1989). Moreover, there is evidence that IDE and the receptors for insulin and IGFs share a common anatomical distribution (Bondy et al. 1994). As IDE is a protease that degrades insulin and growth factors, it is feasible to presume that the increase in growth factors that occurs in the prostate in situations of cell proliferation may lead to an increase in the growth factor receptors and, consequently, to an increase in IDE to terminate the growth signal. On the other hand, a reduction in the expression and/or bioavailability of these growth factors may lead to apoptosis. Castration is known to significantly reduce the local expression and concentration or bioavailability of certain prostate-derived growth factors that affect prostate cell proliferation (Nishi et al. 1996; Nickerson et al. 1998). This reduction in the expression and/or bioavailability of growth factors leading to apoptosis must consequently lead to a reduction in IDE. A previous study demonstrated a reduction in both the level and activity of IDE in the prostate gland of castrated animals and revealed IDE in the nuclear fraction of the ventral prostate with no degradation activity, thereby suggesting that proteolytic activity is not the sole physiological function of this enzyme (Udrisar et al. 2005). Furthermore, the presence of IDE in the nucleus and its lack of degradation activity may suggest an interaction between IDE and AR, as has been reported previously (Kupfer et al. 1993, 1994; Udrisar et al. 2005). The inhibition of IDE activity has been shown to be necessary for the accumulation of insulin or insulin–cytosolic protein complexes in nuclei (Cresto et al. 1984; Harada et al. 1999). It has been demonstrated that IDE activity is inhibited by a phosphorylation reaction (Udrisar & Wanderley 1992) and that ATP induces conformational changes in IDE and inhibits insulin degradation in vitro (Camberos et al. 2001). Furthermore, recent studies (Pivovarova et al. 2009; Fawcett & Duckworth 2009) suggest that the increase in intracellular ATP under conditions of high glucose may result in the inhibition of IDE activity. It was recently demonstrated in rats fed a high-fat diet that hyperinsulinaemia per se can result in enhanced mitogenic signalling in the prostate gland and consequent cell proliferation and prostate hyperplasia (Vikram et al. 2010). The regulatory protein function of IDE has also been observed in different subcellular fractions, with different intracellular effectors related to intracellular insulin action (Kupfer et al. 1994; Duckworth et al. 1997; Harada et al. 1999). It has also been demonstrated that oestradiol upregulates IDE in the rat uterus (Udrisar et al. 2005). Insulin-degrading enzyme degrades insulin and amyloid β-protein (Aβ), and alterations in the metabolism of these substrates are critically important in the pathogenesis of type II diabetes mellitus and Alzheimer's disease (AD) respectively (Fawcett & Duckworth 2009; Perez et al. 2000; Farris et al. 2003). It has been suggested that testosterone and 17β-oestradiol reduce the neuronal secretion of β-amyloid peptides in AD (Gouras et al. 2000; Xu et al. 1998). It has also been demonstrated that ovariectomy and 17β-oestradiol modulate the levels of Aβ peptides in the brain (Petanceska et al. 2000). A decrease in PW has been described as a consequence of hypoinsulinaemia induced by STZ or alloxan, and a greater PW has been reported in genetically diabetic rats (Yono et al. 2008). These findings, together with those of the present study, demonstrate the important role of insulin in the development and differentiation of the prostate gland. Thus, physiological conditions or compounds that upregulate or disinhibit IDE would be expected to lower hyperinsulinism or Aβ levels in vivo. The data presented here suggest that IDE may participate in prostatic growth and that insulin plus testosterone may be an important factor for the expression and regulation of IDE in this tissue. The possibility of a protein–protein interaction (IDE-insulin, IDE–AR, IDE–GR) for IDE regulation exists (Kupfer et al.1993, 1994; Udrisar et al. 2005) and may be important for various biological phenomena, such as insulin resistance, AD and benign prostate hyperplasia.

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