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Experimental Biology and Medicine 230:479-486 (2005)
© 2005 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Androgen- and Estrogen-Dependent Regulation of Insulin-Degrading Enzyme in Subcellular Fractions of Rat Prostate and Uterus

Daniel P. Udrisar*,1, Maria I. Wanderley*, Regina C. C. Porto*, Carla L. P. Cardoso*, Maria C. L. Barbosa*, Maria C. Camberos{dagger} and Juan C. Cresto{dagger}

* Laboratory of Endocrinology and Metabolism, Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife, Pernambuco, Brazil; and {dagger} CEDIE–Endocrinology, Htal. de Niños "R. Gutiérrez", Buenos Aires, Argentina

1To whom requests for reprints should be addressed at Departamento de Fisiologia e Farmacologia, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901 Recife, PE, Brazil. E-mail: dpu{at}ufpe.br


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Innumerous data support the fact that insulin-degrading enzyme (IDE) is the primary enzymatic mechanism for initiating and controlling cellular insulin degradation. Nevertheless, insulin degradation is unlikely to be the only cellular function of IDE, because it appears that some cellular effects of insulin are mediated by IDE as a regulatory protein. Insulin-degrading enzyme shows a significant correlation with various cellular functions, such as cellular growth and differentiation, and the expression of IDE is developmentally regulated. Besides insulin, other substrates are also degraded by IDE, including various growth-promoting peptides. It has also been shown that IDE enhances the binding of androgen to DNA in the nuclear compartment. It is also known that the androgen hormones have a stimulatory effect on prostate growth, and that estradiol stimulates uterine growth. To establish whether IDE is regulated by a cellular prostate/uterine growth stimulus, the present study assessed whether IDE was modified in quantity and activity during proliferative conditions (castration + testosterone in the male rat, or castration + estradiol or the proestrus phase of the estrous cycle in the female rat) and autolysis (castration or the metestrus phase of the estrous cycle) using cytosolic and nuclear fractions of rat prostate and cytosolic fractions of rat uterus. The activity and amount of IDE decreased in the cytosolic fraction with castration and during metestrus, and increased with testosterone or estradiol treatment and during proestrus. In the nuclear fraction, the quantity of the IDE followed the same pattern observed in the cytosolic fraction, although without degradative activity. The data presented here suggest that IDE may participate in prostatic and uterine growth and that the testosterone or estradiol and/or prostate and uterus insulin-like growth factors may be important factors for the expression and regulation of IDE in the prostate and uterus.

Key Words: insulin-degrading enzyme • testosterone • estradiol • castration • prostate • uterus • estrous cycle


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Insulin-degrading enzyme (IDE) is an intracellular protein that degrades insulin and other peptides and hormones (1). Insulin degradation is a regulated process and is considered an integral part of the hormone interaction with its target tissues (2, 3). Insulin-degrading enzyme also degrades insulin-like growth factor-I and -II (IGF-I and -II; Ref. 4) and transforming growth factor-{alpha} (TGF-{alpha}; Refs. 5, 6) that, together with insulin, produce biological effects that induce growth and differentiation (7). In addition to degradation, IDE has multiple cellular functions, including binding and regulatory functions, evidencing a more direct role of this enzyme in generating insulin effects (2). For example, it has been demonstrated that IDE interacts with and enhances the DNA binding of androgen receptor (AR) and glucocorticoid receptor (GR), suggesting that IDE may be important for the transcriptional activity of steroid receptors (8). Further, Kuo et al. (9) have demonstrated that IDE is differentially expressed and regulated during development and growth in various tissues, particularly those of the male reproductive system. All of these observations suggest that IDE is involved in the regulation of cell growth and development. However, the physiological role of IDE is yet to be established. To evaluate the regulation of IDE by stimulus of cell proliferation and apoptosis, we used the androgenic regulation of rat prostate gland and the estrogenic regulation of rat uterus as experimental models. The objective of the present study was to examine whether IDE is modified in quantity and activity in the cytosolic and nuclear fractions of a rat prostate homogenate and in the cytosolic fraction of a rat uterus homogenate in conditions of cell proliferation (castration + testosterone for male rats, and castration + estradiol or during the proestrus phase of the estrous cycle for female rats) or apoptosis (castration or the metestrus phase of the estrous cycle). The results demonstrated that the level and activity of IDE increased in the cytosolic fraction during the induction of cell proliferation and decreased in conditions of apoptosis in both tissues. In the nuclear fraction of the prostate homogenate, the quantity of the IDE followed the same pattern observed in the cytosolic fraction, although, interestingly, without degradative activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials.
Crystalline pork insulin was kindly provided by Bio-Bras (Belo Horizonte, Brazil). Carrier-free 125I was obtained from Amersham International (Buckinghamshire, UK). Sephadex G-50 Fine was obtained from Pharmacia (Uppsala, Sweden). Peptide synthesis was performed by the Instituto de Quimica, USP (São Paulo, Brazil). Hemocyanin and activated agarose (Sulfolink Coupling Gel) were purchased from Pierce Chemical Co. (Rockford, IL). Reagents for polyacrylamide gel electrophoresis (SDS-PAGE), DEAE-Bio-Gel A Agarose and Affi-Gel Hz were obtained from Bio-Rad (Hercules, CA). Testosterone propionate was obtained from Sigma Chemical Co. (St. Louis, MO). Estradiol benzoate was obtained from Hoechst Marion Roussel S/A (São Paulo, Brazil). All drugs were of analytical grade.

Animals, Treatments, and Preparation of Tissue Extracts.
Adult male and female Wistar rats weighing 250–300 g, bred in our animal facilities, were housed under a controlled environment (temperature, 25°–29°C; lights on from 0600 to 1800 hrs) with free access to standard laboratory chow and tap water. Castration in the male rat was performed via a scrotal incision under ether anesthesia. After castration, the rats were divided into two groups. Three days after castration, one group (Castrated + T; n = 5) received 300 µg/100 g body weight of testosterone propionate in corn oil injected subcutaneously daily for 3 days. The other group (Castrated) received vehicle instead of testosterone propionate. All female rats used in the experiments showed regular 4–5 day estrous cycles monitored by vaginal cytology for 2 weeks before the initiation of the experiment. The animals were ovariectomized and, 3 days later, treatment was begun with 2 µg/100 g body weight of estradiol benzoate (OVX + EB; n = 5) in corn oil injected subcutaneously daily for 3 days. One group of ovariectomized (OVX; n = 5) rats was used as a control and received vehicle instead of estradiol benzoate. Rats were sacrificed 24 hrs after the final injection and 6 days after castration. Additionally, intact female rats were sacrificed on the day of proestrus (n = 5) or during metestrus (n = 5). After the treatment period previously described, the animals were sacrificed using ether anesthesia, and their ventral prostates or uteri were collected quickly, weighed and placed immediately, and either stored in liquid nitrogen for posterior Western blot analysis and insulin degradation study or were used for determination of dry weight. Our institution’s animal care committee approved all treatments.

Preparation of Cytosolic and Nuclear Fractions From Rat Ventral Prostates.
All operations were performed at 0°–4°C. Ventral prostates or uteri (500 mg, pooled from three to four rats for each experiment) were obtained from intact Wistar rats, from castrated rats, and from castrated plus testosterone- or estradiol-treated rats; cut into small pieces, and homogenized in 2 ml of buffer A (1.5 mM EDTA, 2 mM mercaptoethanol, 15% glycerol [vol/vol], 3 mM MgCl2, 3 mM CaCl2, and 10 mM Tris-HCl, pH 7.4), by 10 strokes of a motor-driven Teflon-glass homogenizer (7 ml; Kontes, Vineland, NJ). The homogenate was filtered through nylon gauze and centrifuged at 700 g for 10 mins. The supernatant was centrifuged at 100,000 g for 60 mins. The resultant supernatant was considered a cytosolic fraction. The nuclear pellets were resuspended once in 3 ml of buffer B (buffer A plus 1% Triton X-100 and 10% ethanol; Ref. 10), stirred, and resedimented. This washing procedure was repeated twice with buffer A. After centrifugation (2000 g for 3 mins), the pellet was resuspended in buffer C (buffer A plus 0.5% Triton X-100 and 400 mM NaCl) and kept under agitation (every 15 mins) for 2 hrs at 4°C, before centrifugation at 10,000 g for 5 mins. The supernatant was kept as the nuclear fraction. Proteins from nuclear and cytosolic fractions were precipitated with ammonium sulfate (0.42 g/ml of supernatant, 60% saturation; Ref. 11). The suspension was centrifuged at 17,000 g for 20 mins. The supernatant was discarded and the precipitate was dissolved in buffer D (1.5 mM EDTA, 10% glycerol [vol/vol], 10 mM MgCl2, and 10 mM Tris-HCl, pH 7.4). This material was used for Western blot and 125I-insulin degradation studies.

SDS-PAGE and Immunoblotting.
Electrophoresis and immunoblotting were carried out with minigels (7 cm x 8 cm x 1 mm) containing a 10% gradient of polyacrylamide, following established procedures (12, 13). Samples (25–30 µg of protein) were subjected to electrophoresis for 45 mins at 200 V. After electrophoresis, the gels were soaked in transfer buffer (25 mM Tris-HCl, 192 mM glycine, and 20% [vol/vol] methanol, pH 8.3) for 10 mins, sandwiched between a sheet of nitrocellulose paper and two sheets of blotting paper, and assembled in an immunoblotting apparatus (Bio-Rad), in which proteins were electrotransferred to nitrocellulose paper at 70 V (350 mA) in transfer buffer. The membrane was blocked for 2 hrs at room temperature with 5% of albumin in 20 mM Tris-HC1, pH 7.5, and 0.154 M NaCl. Then, the nitrocellulose was incubated overnight at 4°C with anti-p15 (1:500 dilution), washed, and the antibody was bound and detected with alkaline phosphatase–conjugated goat anti-rabbit IgG (Bio-Rad), according to manufacturers’ protocols.

Preparation of IDE Antibody.
The synthetic peptide, YKEMLAVDAPRRHK (p15), corresponding to residues 940 to 953 of rat IDE sequence (14) plus a cysteine added to the N-terminal, was prepared by the Instituto de Quimica, USP. The peptide was coupled to keyhole limpet hemocyanin (KLH) using maleimide (inject-activated immunogen conjugation kit; Pierce), mixed with complete Freund’s adjuvant, and injected into two Norfolk-Californian female rabbits (LD or LM). The coupled-peptide preparation was 175 µg of p15 per 1 mg of KLH. The immunization scheme was as follows: 150 µg of coupled p15 was injected into rabbits in the first and the second injection (first booster). The second and the third boosters were 50 µg of coupled p15. All boosters were performed monthly using incomplete Freund’s adjuvant. Blood was collected between 15 and 20 days after each booster, and the antibody was tested for specific reactivity with the labeled peptide (p15). Antibody titer was controlled by radioimmunoassay (RIA; 5 pg/ml of 125I-p15) and variable serum dilution. When an adequate titer of anti-p15 was reached, specificity and sensitivity was controlled with cytosol from different rat tissues, using a 1:30,000 dilution of anti-p15. The specificity was assumed as the superposition (parallelism) of the competitive curves obtained with 50–1000 pg/ml of p15 pure peptide as standard and different cytosolic protein concentrations (0.33–4 mg/ml). Anti-p15 was immunopurified by affinity chromatography using an agarose-bound peptide (p15) column (Sulfo-link Coupling gel; Pierce). After elution with 0.1 mM glycine, pH 2.5, the eluate containing anti-p15 was dialyzed and concentrated. Both IgG antibody and immunopurified IgG antibody were used. Immunoblot comparison of LD antibody (450 µg/ml) suggests a 700 times purification of IgG anti-p15.

Degradation Assay.
125I-insulin was predominantly labeled in tyrosine A-14, with a specific activity between 250 and 300 mCi/mg and conserved biological activity (15). 125I-insulin degradation was measured by trichloroacetic acid (TCA) precipitation (16). Cytosolic and nuclear fractions (protein concentrations ranging from 25 mg/ml to 125 mg/ml) in 500 µl of 20 mM Tris-HCI, pH 7.4, and 10 mM MgCl2 with 125I-insulin were incubated at 32°C during 15 mins. The reaction was stopped by the addition of 200 µl of 50% (v/v) TCA and 300 µl of 1% (w/v) bovine serum albumin (BSA) at 4°C, kept for 120 mins at room temperature or overnight at 4°C, then centrifuged in a Sorvall RC-2B at 4000 rpm for 15 mins at 4°C, after which the pellets and supernatants were separated. Degradation was calculated as the increase in 125I-insulin fragments (TCA soluble) in the supernatant. This reaction was linear in relation to protein concentrations (25 mg/ml to 125 mg/ml) and time of incubation (5 mins to 60 mins) at 32°C.

Other Analysis.
Protein concentrations were determined according to Bradford (17) using bovine serum albumin as standard. 125I-insulin control (precipitation with TCA without cytosolic fraction) contained more than 92% of intact insulin; hence, the experimental results were corrected for this value. Values are given as percent of 125I-insulin degradation per milligram of cytosolic proteins/ml x 10–1 (3). Data are expressed as the mean ± SEM (n = 3 to 9 experimental determinations) of at least two different experiments. Statistical comparisons between groups were performed using the Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To study whether IDE is modified in quantity and activity in the cytosolic and nuclear fractions of a rat prostate homogenate in conditions of cell proliferation (castration + testosterone) or apoptosis (castration), we measured the quantity of IDE separately in cytoplasmic and nuclear fractions by Western blot. The same subcellular fractions were also used to study insulin-degrading activity. Supernatant (100,000 g for 60 mins) and nuclear (800 g for 10 mins, pellet) protein extracts from ventral prostate homogenates were considered as cytosolic and nuclear fractions, respectively. These fractions were used to study insulin-degrading activity and relative IDE protein levels from ventral prostates of intact, 6-day castrated, and 3-day castrated rats after 3 days of testosterone administration. Immunoblotting with antisera obtained against the carboxyl-terminal (p15 peptide) region of IDE along with the TCA method were used for detecting the presence and the activity of IDE at a nuclear and cytosolic fraction level in all of the experimental groups. The Western blot analyses reveal (Fig. 1A and BGo, upper panel) a 110-kDa band corresponding to the IDE protein in all groups and in both subcellular fractions (nuclear and cytosolic). The relative IDE protein level, as determined by densitometric measurement of Western blotting, indicated a 2.4- and 2.7-fold reduction of IDE protein levels from 6-day castrated rats compared with intact rats in nuclear and cytosolic fraction, respectively. Three days after the administration of testosterone to castrated rats, IDE protein levels increased approximately 4.4- and 4.0-fold compared with castrated rats in nuclear and cytosolic fractions, respectively. Figure 1CGo shows that there is a linear correlation (r 2 = 0.97) between the amount of IDE in nuclear and cytosolic fractions, as determined by comparative densitometric analysis of IDE Western blot (data derived from Fig. 1A and BGo). These results suggest that the alteration observed in IDE protein level relative to the different androgenic status is similar in both subcellular fractions. To assess whether the alteration observed in the IDE level is associated with IDE activity, we determined the 125I-insulin degradation in the same protein extracts from the three experimental groups. The insulin degradation was not detectable in the nuclear fraction of any group. In the cytosolic fraction, the insulin degradation was linear with the amount of proteins and incubation time at 32°C in the three experimental groups. Figure 2Go illustrates the effect of castration and of castration following the administration of testosterone on cytosolic IDE activity. The insulin degradation analysis, Figure 2AGo, indicated a 1.6-fold reduction of IDE activity in 6-day castrated rats compared with intact rats, and a 2.3-fold increase of IDE activity in the testosterone-treated group compared with the castrated group. Insulin degradation plotted against IDE amount in all three groups (Fig. 2BGo; data derived from Figs. 1BGo and 2AGo) results in a linear correlation (r 2 = 0.995), suggesting that the altered insulin degradation observed in the cytosolic fraction in all three groups is related to the amount of IDE expressed as a consequence of the different androgenic status. In another series of experiments, we explored whether IDE is also modified in quantity and activity in cytosolic fractions of a rat uterus homogenate in conditions of cell proliferation (OVX + estradiol and proestrus) or apoptosis (OVX and metestrus). Immunoblot and activity analysis were performed on cytoplasmic fractions of rat uterus, as described in Materials and Methods. An immunoreactive band (with antisera obtained against the carboxyl-terminal, p15 peptide) at 110 kDa was observed in cytoplasmic fractions from uteri of OVX and OVX + EB rats and from uteri of rats in proestrus or metestrus, as shown in Figures 3AGo and 4AGo (upper panel), respectively. Similarly, in cytosolic fractions from uteri of female rats, the relative IDE protein, insulin degradation, and uterine weight increased by 62.5%, 38.0%, and 50.1%, respectively, concomitant with estradiol-induced cell proliferation (as shown in Fig. 3A and BGo). These parameters were also studied in the physiological conditions of a high estradiol level (proestrus) and a low estradiol level (metestrus). Figure 4A and BGo illustrates the increase of 66.9%, 17.4%, and 35.9% of the relative IDE protein, insulin degradation, and uterine weight, respectively, in proestrus as compared with metestrus.



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  		Figure 1. Insulin-degrading enzyme protein levels as determined by Western blot analysis. Densitometric measurement of relative IDE protein levels in nuclear (A) and cytosolic fractions (B) from ventral prostates of intact rats, 6-day castrated rats, and 3-day castrated rats, after 3 days of testosterone administration. (C) Correlation between IDE protein levels in nuclear and cytosolic fractions; r 2 = 0.9724. Data are expressed as means ± SEM; n = 3. **P < 0.005 vs. intact; ***P < 0.001 vs. intact; for conditions, see Materials and Methods.

 


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  		Figure 2. (A) Cytosolic 125I-insulin degradation from ventral prostates of 6-day castrated rats and castrated rats after administrations of testosterone. Different concentrations of cytosolic proteins (60, 90, 120 mg/ml from prostate; 100,000 g for 60 mins, supernatant) were incubated with 125I-insulin (20,000 cpm) in 10 mM MgCl2 in 20 mM Tris-HCl, pH 7.4, for 15 mins at 32°C. The reaction was terminated by addition of 0.4 vol TCA (50% v/v) and 0.6 vol bovine albumin (1% w/v) at 4°C. After centrifugation, pellet and supernatant were separated, and the pellet was washed and radioactivity measured (radioactivity total = cpm pellet + cpm supernatant). The radioactivity in the supernatant was considered degraded 125I-insulin. The degradation was linear in relationship to the incubation time and cytosolic protein concentrations. Values are given as percent of degraded 125I-insulin (see Materials and Methods) per mg/ml x 10–1 of cytosolic proteins. Results are the means ± SEM of nine determinations. ***P < 0.001 vs. intact, unpaired t test, 2-tailed. (B) Correlation between percent of insulin degradation and IDE protein levels in cytosolic fractions; r 2 = 0.9950.

 


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  		Figure 3. (A) Insulin-degrading enzyme protein levels as determined by Western blot analysis. Densitometric measurement of relative IDE protein levels in cytosolic fractions from uteri of 6-day castrated rats (OVX) and 3-day castrated rats after 3 days of estradiol administration (OVX + EB). Data are expressed as means ± SEM (n = 5). **P < 0.001 vs. OVX + EB. (B) Cytosolic 125I-insulin degradation (light gray column) and uterine weight/body weight (mid gray column) from uteri of 6-day ovariectomized rats and ovariectomized rats after administrations of estradiol. Values of 125I-insulin degradation are given as percent of degraded 125I-insulin per mg/ml x 10–1 of cytosolic proteins. Results are the means ± SEM of four determinations. Light gray column: *P < 0.001 vs. OVX + EB; unpaired t test, 2-tailed. Values of uterine weight/body weight are given as mg/g; mid gray column: **P < 0.001 vs. OVX + EB, unpaired t test, 2-tailed. For experimental conditions, see Materials and Methods and Figure 2AGo.

 


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  		Figure 4. (A) Insulin-degrading enzyme protein levels as determined by Western blot analysis. Densitometric measurement of relative IDE protein levels in cytosolic fractions from uteri of rats in the proestrus and metestrus phases of the estrous cycle. Data are expressed as means ± SEM (n = 5), *P < 0.001 vs. proestrus, unpaired t test, 2-tailed. (B) Cytosolic 125I-insulin degradation (light gray column) and uterine weight/body weight (mid gray column) from uteri of rats in the proestrus and metestrus phases of the estrous cycle. Values of 125I-insulin degradation are given as percent of degraded 125I-insulin per mg/ml x 10–1 of cytosolic proteins. Results are the means ± SEM of four determinations; light gray column, *P < 0.001 vs. OVX + EB, unpaired t test, 2-tailed. Values of uterine weight/body weight are given as mg/g; mid gray column: **P < 0.001 vs. OVX + EB, unpaired t test, 2-tailed. For experimental conditions, see Materials and Methods and Figure 2AGo.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 cellular growth and development (9). In the present study, we evaluated the activity and quantity of the IDE during situations of cell proliferation and apoptosis in vivo in the adult rat. We used the prostate and the uterus as suitable models of induced apoptosis by castration or of cell proliferation by testosterone/estradiol treatment of castrated rats. In the present study, we found that, under the condition of cell proliferation, the activity and quantity of the IDE increases in the cytosolic fraction of the prostate homogenate, whereas, under the condition of apoptosis, there is a decrease in these two parameters. We also detected the presence of IDE in the nuclear fraction of the prostate homogenate. In this fraction, the quantity of the IDE followed the same pattern observed in the cytosolic fraction, although no degradative activity of this enzyme was found. Our study demonstrated a positive correlation between 125I-insulin degradation and the relative quantification of IDE in the cytosolic fraction, indicating that the activity of the IDE is dependent on the quantity present, and that both parameters are dependent on the androgenic state of the animal. It is known that the development, growth, and function of the prostate are androgen dependent (18). Androgen action is mainly indirect, through prostatic production of certain growth factors (19–21). These locally produced growth factors are considered autocrine and/or paracrine mediators of the stromal–epithelial interaction (22). Several studies have indicated that insulin-like growth factors (IGFs), epidermal growth factor (EGF), keratinocyte growth factor, TGF-{alpha}, and basic fibroblastic growth factor are mitogenic in prostate tumor cells and normal prostate cells (19, 22–24). Some of these growth factors are IDE substrates, such as IGFs and TGF-{alpha}, or bind to IDE, such as EGF (6, 25). Additionally, there is evidence that IDE and the receptors for insulin and IGFs share a common anatomic distribution (26). Being a protease that degrades insulin and growth factors, it is feasible to presume that the increase in growth factors that occur in the prostate in situations of cellular proliferation may lead to an increase in the receptors for growth factors 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. It is known that castration significantly reduces the local expression and concentration or bioavailability of certain prostate-derived growth factors that are known to affect prostate cell proliferation (27, 28). This reduction in the expression and/or bioavailability of growth factors leading to apoptosis should consequently lead to a reduction in IDE. Indeed, we observed a reduction in both the levels and the activity of IDE in the prostate gland of castrated animals. The results obtained in the nuclear fraction were interesting because they showed the presence of IDE with no degradative activity, demonstrating that the proteolytic activity is not the sole physiological function of the enzyme. The possibility of contamination of the nuclear fraction by the cytosolic IDE was ruled out because no degradative activity was found in the nuclear fraction. The presence of IDE in the nucleus and the absence of its degradative activity suggest an interaction between IDE and AR, as has been previously shown (3, 8, 29). The inhibition of IDE activity was shown to be necessary for the accumulation of insulin or insulin–cytosolic protein complexes in nuclei (30). We have demonstrated that IDE activity is inhibited by a phosphorylation reaction (3). Furthermore, recent studies1 suggest that inhibition of IDE activity in the cytosolic fraction mediated by a phosphorylation reaction increases the binding of IDE to DNA-cellulose; this could have significant effects on the interaction of IDE with AR, and nuclear translocation. The regulatory protein function of IDE has also been observed in different subcellular fractions, with different intracellular effectors related to intracellular insulin action (29, 31). Insulin acts intracellularly on proteasomes through IDE (31), and variation in the amounts of IDE induced by androgen or estrogen status could be important for the regulation of protein content and turnover. We have also demonstrated that estradiol upregulates IDE in the uteri of rats. The quantity and activity of IDE were increased in cytosolic fractions of a homogenate of rat uterus in the proestrus phase of the estrous cycle or when ovariectomized and treated with estradiol, and were decreased in rat uterus in the metaestrus phase of the estrous cycle or when ovariectomized. In the first experimental situation, the endometrium proliferates in response to the cyclic increase in plasma estradiol during proestrus or to the exogenous administration of estradiol. In the second experimental situation, the endometrium atrophies as a consequence of the cyclic decrease in plasma estradiol during metestrus or after ovariectomy. It is well established that the cyclic pattern of cellular proliferation that occurs in the endometrium during the estrous cycle reflects changes in the systemic levels of estradiol and progesterone (32). During the estrous cycle, estradiol levels begin to increase late in metestrus, reaching peak levels at the height of proestrus, and returning to baseline at estrus (33). An elevation of total uterine DNA and increase in cell proliferation also characterize proestrus (34, 35). Our results suggest that IDE may play a significant role in uterine cell proliferation during the estrous cycle of adult female rats and in response to estradiol. These results also suggest that the activity of IDE is dependant on quantity, and that both parameters are dependent on the estrogenic status of the animals. Further, the conditions of cell proliferation and apoptosis may regulate the activity and quantity of IDE, and estradiol may be an important factor for the gene expression of IDE in the uterus. The mechanisms through which estradiol affects the expression of IDE are yet to be determined. Estradiol stimulates uterine proliferation and differentiation by acting through the estrogen receptor (ER). Several studies have demonstrated that the effect of estradiol on uterine growth is mediated by locally produced growth factors (36–38), where IGFs appear to play major roles in the action of estradiol (39, 40). Insulin-degrading enzyme degrades insulin and amyloid ß-protein (Aß), and alterations in the metabolism of these substrates are critically important in the pathogenesis of Type II diabetes mellitus and Alzheimer’s disease, respectively (2, 41, 42). Recent studies suggest that testosterone and 17ß-estradiol reduce neuronal secretion of Alzheimer’s ß-amyloid peptides (43, 44). Additionally, it has been demonstrated that ovariectomy and 17ß-estradiol modulate the levels of Aß peptides in the brain (45). Thus, physiological conditions or compounds that upregulate or disinhibit IDE would be expected to lower hyperinsulinism or Aß levels in vivo. The data presented here suggest that IDE may participate in prostatic and uterine growth and that testosterone/estradiol may be an important factor for the expression and regulation of IDE in both tissues. The possibility of a protein–protein interaction (IDE–AR, IDE–ER) for IDE regulation exists (3, 8, 29, 46), and may be important for various biological phenomena, such as insulin resistance and Alzheimer’s disease.


   Footnotes
 
1 Udrisar DP, Wanderley MI, Cresto JC, unpublished results, 1999. Back

Received for publication February 15, 2005. Accepted for publication April 14, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Burghen GA, Kitabchi AE, Brush JS. Characterization of a rat liver protease with specificity for insulin. Endocrinology 91:633–642, 1972.[Medline]
  2. Duckworth WC, Bennett RG, Hamel FG. Insulin degradation: progress and potential. Endocr Ver 19:608–624, 1998.
  3. Udrisar DP, Wanderley MI. Fluoride and phosphatidylserine-induced inhibition of cytosolic insulin-degrading activity. Acta Physiol Pharmacol Latinoam 42:183–193, 1992.
  4. Misbin RI, Almira EC, Duckworth WC, Mehl TD. Inhibition of insulin degradation by insulin-like growth factors. Endocrinology 113:1525–1527, 1983.[Abstract]
  5. Garcia JV, Gehm BD, Rosner MR. An evolutionarily conserved enzyme degrades transforming growth factor-alpha as well as insulin. J Cell Biol 109:1301–1307, 1989.[Abstract/Free Full Text]
  6. Gehm BD, Rosner MR. Regulation of insulin, epidermal growth factor, and transforming growth factor-{alpha} levels by growth factor degrading enzymes. Endocrinology 128:1603–1609, 1991.[Abstract]
  7. Cross M, Dexter TM. Growth factors in development, transformation, and tumorigenesis. Cell 64:271–280, 1991.[Medline]
  8. Kupfer S, Marschke K, Wilson EM, French FS. Receptor accessory factor enhances specific DNA binding of androgen and glucocorticoid receptors J Biol Chem 268:17519–17527, 1993.[Abstract/Free Full Text]
  9. Kuo WL, Montag AG, Rosner MR. Insulin-degrading enzyme is differentially expressed and developmentally regulated in various rats tissues. Endocrinology 132:604–611, 1993.[Abstract]
  10. Blondeau JP, Baulieu EE, Robel P. Androgen-dependent regulation of androgen nuclear receptor in the rat ventral prostate. Endocrinology 110:1926–1932, 1982.[Abstract]
  11. Duckworth WC, Heinemann MA, Kitabchi AE. Purification of insulin-specific protease by affinity chromatography. Proc Nat Acad Sci U S A 69:3698–3702, 1972.[Abstract/Free Full Text]
  12. Udrisar D, Rodbell M. Microsomal and cytosolic fractions of guinea pig hepatocytes contain 100-kilodalton GTP-binding proteins reactive with antisera against alpha subunits of stimulatory and inhibitory heterotrimeric GTP-binding proteins Proc Natl Acad Sci U S A 87:6321–6325, 1990.[Abstract/Free Full Text]
  13. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685, 1970.[Medline]
  14. Baumeister H, Muller D, Rebbein M, Ritcher D. The rat insulin-degrading enzyme: molecular cloning and characterization of tissue-specific transcripts. FEBS Lett 317:250–254, 1993.[Medline]
  15. Cresto JC, Udrisar DP, Camberos MC, Basabe JC, Gomez Acuña P, de Majo SF. Preparation of biologically active mouo-l251-insulin of high specific activity. Acta Physiol Latinoam 31:13–24, 1981.[Medline]
  16. Udrisar DP, Camberos MC, Basabe JC, Cresto JC. Insulin processing. Its correlation with glucose conversion to CO2. Acta Physiol Pharmacol Latinoam 34:427–440, 1984.[Medline]
  17. Bradford MM. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254, 1976.[Medline]
  18. Cunha GR, Donjacour AA, Cooke PS, Mee S, Bigsby RM, Higgins SJ, Sugimura Y. The endocrinology and developmental biology of the prostate. Endocr Rev 8:338–362, 1987.[Medline]
  19. Steiner MS. Role of peptide growth factors in the prostate: a review. Urology 42:99–110, 1993.[Medline]
  20. Lopaczynski W, Hruszkewycz AM, Lieberman R. Preprostatectomy: a clinical model to study stromal-epithelial interactions. Urology 57(Suppl 4A):194–199, 2001.[Medline]
  21. Cunha GR. Growth factors as mediators of androgen action during male urogenital development. Prostate 6:22–25, 1996.[Medline]
  22. Culig Z, Hobisch A, Cronauer MV, Radmayr C, Hittmair A, Zhang J, Thurnher M, Bartsch G, Klocke H. Regulation of prostatic growth and function by peptide growth factors. Prostate 28:392–405, 1996.[Medline]
  23. Cohen P, Peehl DM, Lamson G, Rosenfeld RG. Insulin-like growth factors IGFs, IGF receptors, and IGF-binding proteins in primary cultures of prostate epithelial cells. J Clin Endocrinol Metab 73:401–407, 1991.[Abstract]
  24. Byrne RL, Leung H, Neal DE. Peptide growth factors in the prostate as mediators of stromal epithelial interaction. Br J Urol 77:627–633, 1996.[Medline]
  25. Misbin RI, Almira EC. Degradation of insulin and insulin-like growth factors by enzyme purified from human erythrocytes. Comparison of degradation products observed with A14- and B26-[125I]monoiodoinsulin. Diabetes 38:152–157, 1989.[Abstract]
  26. Bondy CA, Zhou J, Chin E, Reinhardt RR, Roth RA. Cellular distribution of insulin-degrading enzyme gene expression: comparison with insulin and insulin-like growth factor receptors. J Clin Invest 93:966–973, 1994.
  27. Nishi N, Oya H, Matsumoto K, Nakamura T, Miyanka H, Wada F. Changes in gene expression of growth factors and their receptors during castration-induced involution and androgen-induced regrowth of rat prostate. Prostate 28:139–152, 1996.[Medline]
  28. Nickerson T, Pollak M, Huynh H. Castration-induced apoptosis in the rat ventral prostate is associated with increased expression of genes encoding insulin-like growth factor binding proteins 2, 3, 4 and 5. Endocrinology 139:807–810, 1998.[Abstract/Free Full Text]
  29. Kupfer S, Wilson EM, French FS. Androgen and glucocorticoid receptors interact with insulin degrading enzyme. J Biol Chem 269:20622–20628, 1994.[Abstract/Free Full Text]
  30. Harada S, Smith RM, Jarett L. Mechanisms of nuclear translocation of insulin. Cell Biochem Biophys 313:307–219, 1999.
  31. Duckworth WC, Bennett RG, Hamel FG. The significance of intracellular insulin to insulin action. J Invest Med 45:20–27, 1997.[Medline]
  32. Sato T, Fukazawa Y, Kojima H, Enari M, Iguchi T, Ohta Y. Apoptotic cell death during the estrous cycle in the rat uterus and vagina. Anat Rec 248:76–83, 1997.[Medline]
  33. Butcher RL, Collins WE, Fugo NW. Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17beta throughout the 4-day estrous cycle of the rat. Endocrinology 946:1704–1708, 1974.
  34. Shelesnyak MC, Tic L. Studies on the mechanism of nidation. V. Suppression of synthetic processes of the uterus DNA, RNA e protein following inhibition of decidualization by na antiestrogen, ethamoxytriphetol MER-25. Acta Endocrinol 43:462–468, 1963.
  35. Marcus GJ. Mitosis in the rat uterus during the estrous cycle, early pregnancy, and early pseudopregnancy. Biol Reprod 10:447–452, 1974.[Abstract]
  36. Murphy LJ, Ghahary A. Uterine insulin-like growth factor-1: regulation of expression and its role in estrogen-induced uterine proliferation. Endocr Rev 11:443–453, 1990.[Medline]
  37. Brigstock DR. Growth factors in the uterus: steroidal regulation and biological actions. Baillieres Clin Endocrinol Metab 5:791–808, 1991.[Medline]
  38. Richards RG, DiAugustine RP, Petrusz P, Clark GC, Sebastian J. Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus. Proc Natl Acad Sci U S A 9321:12002–12007, 1996.
  39. Giudice LC, Mark SP, Irwin JC. Paracrine actions of insulin-like growth factors and IGF binding protein-1 in non-pregnant human endometrium and at the decidual-trophoblast interface. J Reprod Immunol 39:133–148, 1998.[Medline]
  40. Collins BM, McLachlan JA, Arnold SF. The estrogenic and antiestrogenic activities of phytochemicals with the human estrogen receptor expressed in yeast. Steroids 62:365–372, 1997.[Medline]
  41. Perez A, Morelli L, Cresto JC, Castano EM. Degradation of soluble amyloid beta-peptides 1–40, 1–42, and the Dutch variant 1-40Q by insulin degrading enzyme from Alzheimer disease and control brains. Neurochem Res 252:247–255, 2000.
  42. Farris W, Mansourian S, Chang Y, Lindsley L, Eckman EA, Frosch MP, Eckman CB, Tanzi RE, Selkoe DJ, Guenette S. Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo. Proc Natl Acad Sci U S A 1007:4162–4167, 2003.
  43. Gouras GK, Xu H, Gross RS, Greenfield JP, Hai B, Wang R, Greengard P. Testosterone reduces neuronal secretion of Alzheimer’s ß-amyloid peptides. Proc Natl Acad Sci U S A 97:1202–1205, 2000.[Abstract/Free Full Text]
  44. Xu H, Gouras GK, Greenfield JP, Vincent B, Naslund J, Mazzarelli L, Fried G, Jovanovic JN, Seeger M, Relkin NR, Liao F, Checler F, Buxbaum JD, Chait BT, Thinakaran G, Sisodia SS, Wang R, Greengard P, Gandy S. Estrogen reduces neuronal generation of Alzheimer beta-amyloid peptides. Nat Med 44:447–451, 1998.
  45. Petanceska SS, Nagy V, Frail D, Gandy S. Ovariectomy and 17beta-estradiol modulate the levels of Alzheimer’s amyloid beta peptides in brain. Neurology 5412:2212–2217, 2000.
  46. Camberos MC, Perez AA, Udrisar DP, Wanderley MI, Cresto JC. ATP inhibits insulin-degrading enzyme activity. Exp Biol Med (Maywood) 2264:334–341, 2001.



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