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1 Department of Physiology and Biophysics and
2 Department of Cellular and Development Biology, Institute of Biomedical Sciences, University of Sao Paulo (USP),
3 Department of Nursing and
4 Department of Internal Medicine, Medical Sciences Faculty, University of Campinas (UNICAMP), Brazil
(Requests for offprints should be addressed to C R O Carvalho, Departamento de Fisiologiae Biofísica, ICB-USP, Rua Prof. Lineu Prestes, 1524, sala 121, ICB1, Cidade Universitária, Butantã, São Paulo, SP, Brazil, 055096-900; Email: croc{at}icb.usp.br)
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Insulin has a stimulatory effect on steroidogenesis by granulosa cells from cow, rats, and women and interacts with luteinizing hormone (LH) in a synergistic manner, enhancing human chorionic gonadotropin (hCG)-induced ovarian growth and cyst formation (Franks et al. 1999). Evidence indicates that the insulin effect is mediated through the insulin receptor and not the type I insulin-like growth factor (IGF) receptor (Willis & Franks 1995, Nestler et al. 1998). We previously demonstrated a new intracellular pathway for LH/ hCG, showing that there is a positive crosstalk between insulin and the LH/hCG signaling pathway at the level of the PI 3-kinase/Akt pathway in the ovary of non-pregnant rats (Carvalho et al. 2003). In addition, it is well established that both insulin resistance and consequent hyperinsulinemia have an important role in the pathogenesis of polycystic ovary syndrome (PCOS) (Dunaif et al. 1989, Dunaif 1997). PCOS manifests as chronic anovulation and ovarian hyperandrogenism, both of which are amplified by insulin, in addition to the inability of the hormone to stimulate glucose uptake in classic target tissues such as muscle and fat (Dunaif 1997, Legro et al. 1999, Poretsky et al. 1999). However, the regulation of insulin signaling in ovaries of rats with PCOS has not yet been investigated.
In the present study, we assessed the regulation of the insulin-signaling pathways using immunoprecipitation and immunoblotting in whole extracts of ovaries from hCG-treated rats, hyperinsulinemic-induced rats, and hyperinsulinemic-induced rats treated with hCG. Basal and acute insulin-induced phosphorylation of insulin receptor (IR), IRS-1, IRS-2, Akt, and ERK and the association of IRS proteins with PI 3-kinase were examined.
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Materials and Methods |
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The reagents for SDS-PAGE, immunoprecipitation and immunoblotting were from Bio-Rad. High-purified hCG was from two sources: NIDDK’s National Hormone and Pituitary Program (Torrance, CA, USA) and Calbiochem (San Diego, CA, USA). Human recombinant insulin (Biohulin and Iolin R) was from Biobras (MG, Brazil). Anti-IR, anti-IRS-1, anti-IRS-2, anti-SHP2, antiphosphotyrosine, and phosphothreonine Akt (Thr308) antibodies were from Santa Cruz Technology (Santa Cruz, CA, USA). Anti-PI 3-kinase was from Upstate Biotechnology Incorporated (Lake Placid, NY, USA). Both phospho-Akt (Ser473) and phospho-ERK (Thr202/Tyr204) antibodies were from Cell Signaling (Beverly, MA, USA). The enhanced chemiluminescence reagent kit, ECL, and protein A Sepharose 6MB were from Amersham-Pharmacia Biotech.
Animal tissue extracts
Female Wistar rats (200–220 g) were housed with access to standard rodent chow and water ad libitum. All procedures with animals were conducted in accordance with the principles and procedures described by the NIH Guidelines for Care and Use of Experimental Animals and approved by the Ethics Committee for Animal Handling of the Instituto de Ciencias Biomedicas/USP. The rats were divided into four groups: control, hCG-treated, insulin-treated and hCG plus insulin-treated. The treatment protocol was composed of S.C. injections of saline (control animals), daily 3 IU hCG and insulin in order to obtain a hyperinsulinemic state. Briefly, saline and the hormones were administered twice a day, insulin injections started with 0.5 IU/day and gradually increased to 6 IU/day from the 11th day until the 22nd day (Poretsky et al. 1992). On the 23rd day, 14 h after the last hormone administration, the rats were anesthetized with sodium thiopental (25 mg/kg, i.p.; Cristalia, São Paulo, Brazil) and used 10–15 min later, as soon as anesthesia was assured by the loss of pedal and corneal reflexes.
Morphology of the ovaries
The ovaries from anesthetized rats were excised and dissected free from the surrounding tissues and fixed by immersion in 4% formaldehyde–PBS solution for 4–6 h and transferred to a 30% sucrose solution in PBS for cryoprotection. The paraformaldehyde-fixed and paraffin-embedded ovaries sections (5 µm) were counterstained with hematoxylin–eosin for morphological analysis. The ovary morphology was analyzed using a conventional light microscope.
Immunoblotting analysis
The abdominal cavity of anesthetized rats was opened, the cava vein exposed, and 0.5 ml saline (0.9% NaCl) with or without 6 µg insulin was injected as a bolus infusion. The ovaries were removed 5 min after the injection, which corresponded to the maximal insulin-induced IR, IRS-1 and IRS-2 tyrosyl phosphorylation (data not shown), and homogenized in ice-cold extraction buffer containing 100 mM Tris (pH: 7.4), 10 mM EDTA, 1% Triton-X-100, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM sodium vanadate, 2 mM phenyl-methylsulfonylfluoride, and 0.01 mg aprotinin/ml (Sigma). Pooled ovary extracts from rats were centrifuged at 15 000 rpm, 4 °C, for 15 min to remove insoluble material; the supernatant was then used for the assay. The hind limb gastrocnemius muscle was also removed 90 s after insulin injection and processed as described above for the ovary. Protein determination was performed by the Bradford dye binding method using the Bio-Rad reagent and BSA as the standard. Two or three milligrams protein from the supernatant was used for immunoprecipitation with anti-IR, anti-IRS-1, anti-IRS-2, and protein A-Sepharose 6MB before Laemmli sample buffer treatment and electrophoresis in SDS-PAGE (Bio-Rad), as described elsewhere (Carvalho et al. 2003). For whole tissue extracts, similar-sized aliquots (100 µg protein) were subjected to SDS-PAGE and subsequently to typical immunoblotting. Electrotransfer of proteins from the gel to nitrocellulose was performed for 90 min at 120 V (constant). To reduce non-specific protein binding to the nitrocellulose, the filter was pre-incubated overnight at 4 °C in blocking buffer (5% non-fat dry milk, 10 mM Tris, 150 mM NaCl and 0.02% Tween 20; Sigma). The nitrocellulose blots were incubated overnight at 22 °C with antibodies against phosphotyrosine, the p85 subunit of PI 3-kinase, the p95 subunit of IR, IRS-1, IRS-2, pAkt (pSer-Akt and pThr-Akt), and pERK diluted in blocking buffer with 3% non-fat dry milk followed by washing for 30 min in blocking buffer without milk. To visualize the autoradiogram, commercial enhanced chemiluminescence reagents exposed to photographic film were used. Quantitative analysis of the blots was performed using Scion Image software (Frederick, MD, USA).
Radioimmunoassay
Insulin, luteinizing hormone (LH), androstenedione, and estradiol were determined by standard RIA.
Statistical analysis
The results were expressed as the mean ± S.E.M. ANOVA factorial test was used in all statistical comparisons with P < 0.05 indicating significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Table 1
shows the effect of various treatments on mean serum levels of insulin, LH, androstenedione, and estradiol. Insulin concentrations were sevenfold increased in groups that received insulin injections. Serum LH concentrations were significantly increased in all treated groups, compared with controls (C: 0.40 ± 0.02; hCG: 1.02 ± 0.36; hCG+insulin: 4.46 ± 2.91; insulin: 0.87 ± 0.02 ng/ml, P < 0.05).
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Ovarian morphology
Figure 1 illustrates the histological examination of representative ovaries from all groups. In the control (Fig. 1A), the ovarian parenchyma consists of follicles in different developmental phases, which comprise primordial follicles until mature, or Graafian follicles. Follicular cells, zona pellucida, theca cells, and interstitial cells exhibit normal characteristics particular to the ovarian follicular phase in a cyclic young female.
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). Some of these cysts appeared to arise from differentiating follicles but others from corpus luteum. In the follicles containing a cystic center, the layer of lining of follicular cells frequently exhibited an abnormal morphology and organization.
Follicles in the final developmental phases share the ovarian parenchyma with numerous corpora lutea in the group of females that received insulin (Fig. 1D). The morphological analysis revealed two distinct characteristics resulting from this treatment; the corpora lutea did not reach the size of those found in the controls and the persistent interstitial cells were numerous and unusually arranged in cord-like clusters (Fig. 1E). The optically clear cytoplasm, however, suggested steroidogenic activity.
With the exception of extremely cystic structures, the morphology of the ovaries from LH–insulin-treated females seemed quite normal. Follicles in different phases of maturation formed the ovarian parenchyma (Fig. 1F). The cell layers and organization of these structures exhibited no changes. Cysts were still present similar to those found in LH-treated animals and were similarly also derived from differentiating follicles and corpus luteum. The lining wall of the cystic fluid was usually deeply altered and typical follicular cells were hardly/barely recognized (Fig. 1G).
Insulin receptor, IRS-1, IRS-2, PI 3-kinase, Akt, ERK1, and ERK2 protein levels and phosphorylation degrees in ovarian tissue homogenates
Figure 2A shows the effect of the treatment protocol on the insulin receptor protein level and tyrosine phosphorylation in the ovaries of intact rats. There was no change in the insulin receptor levels, as determined by immunoblotting with a specific antibody. In ovary samples immunoprecipitated previously with anti-insulin receptor antibody and immunoblotted with antiphosphotyrosine antibody, there was a statistically significant twofold increase (P < 0.05) in the insulin-stimulated phosphorylation of the insulin receptor in hCG plus insulin-treated rats when compared with the controls, hCG alone, and insulin-alone treatments (Fig. 2A, bar graph).
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), the levels of this protein were similar in all four groups. All conditions were similar in the basal tyrosine phosphorylation. After acute insulin infusion, all groups demonstrated an increase in the IRS-1 tyrosine phosphorylation above each basal. Comparisons between groups revealed that the extent of IRS-1 phosphorylation was a 1.3-fold increase (P < 0.05) in both hCG alone and insulin alone-treated animals, compared with the controls. The simultaneous treatment with hCG and insulin resulted in a significantly higher insulin-induced IRS-1 phosphorylation compared with the control (Fig. 2B, bar graph) (2.3-fold increase).
The protein level of IRS-2 was similar in ovaries from all groups (Fig. 2C). In contrast, the simultaneous treatment with hCG and insulin induced an up-regulation of IRS-2 tyrosine phosphorylation, compared with the control and to each treatment alone. However, similar enhancement in IRS-2 tyrosine phosphorylation were achieved following control, hCG, and hCG plus insulin treatment. The insulin-treated group demonstrated the highest acute insulin-induced IRS-2 tyrosine phosphorylation, with a twofold increase above each group (P < 0.05) (Fig. 2C, bar graph).
Strong and extensive evidence in the literature indicates that a stable high-affinity interaction exists between IRS-1 and IRS-2 and the 85 kDa subunit of the PI 3-kinase. These interactions allow each one of these proteins to be co-precipitated by antibodies of either protein. When blots that had been previously immunoprecipitated with antibody against IRS-1 were subsequently incubated with anti-PI 3-kinase antibody (Fig. 3A), there was some PI 3-kinase immunoreactivity in the basal state of control, hCG, and insulin alone-treated rats. However, this band was significantly increased by 1.4-fold above each distinct condition in hCG plus insulin-treated rats, suggesting a greater association between IRS-1/PI 3-kinase under basal conditions in this group. Comparison of the bands stimulated by acute insulin injection revealed that the amount of PI 3-kinase associated with IRS-1 had the highest increase in the hCG plus insulin-treated group to approximately 1.5-fold above the insulin-stimulated control, hCG, and insulin-treated rats (Fig. 3A, bar graph). The IRS-2 association with PI 3-kinase was analyzed in the samples previously immunoprecipitated with anti-IRS-2 antibody (Fig. 3B), as performed for IRS-1/PI 3-kinase association. There was a faint band corresponding to basal PI 3-kinase immunoreactivity associated with IRS-2 in the control group. However, in the three treated groups, this band was significantly increased by fourfold above control, suggesting a greater association between IRS-2/PI 3-kinase, induced by each treatment, with no further increase when simultaneous treatment was imposed. In contrast to our findings for acute insulin-induced IRS-1/PI 3-kinase association, the insulin-induced association of PI 3-kinase with IRS-2 demonstrated a further increase above the basal condition only in the ovaries of the control rats (Fig. 3B, bar graph). These treatments had no effect on the protein level of the 85 kDa subunit of PI 3-kinase (Fig. 3C).
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) and ERK1 and ERK2 (Fig. 5), were assessed by specific phospho-antibodies in the same ovary samples. Despite the similar degree of Akt serine phosphorylation induced by each condition, the acute insulin infusion led to a greater Akt serine phosphorylation in the ovary of rats that received both hormones together (~20-fold increase above basal; P < 0.05). On the other hand, both treatments with hCG alone or with insulin led to a greater Akt threonine phosphorylation compared with the control and insulin-treated alone rats (Fig. 4B). Despite these changes in the phosphorylation status, the PKB/Akt protein level was similar in all groups (Fig. 4C).
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). Insulin-induced IRS-1 and Akt serine phosphorylation in the hind limb skeletal muscle of rats
The skeletal muscle gastrocnemius was used as a classical insulin target (Fig. 6). The IRS-1 tyrosine phosphorylation status after insulin acute infusion was increased in the hCG-treated group by 1.5-fold (P < 0.05), compared with the control. In the hyperinsulinemic rats, there was a significant decrease in insulin-induced IRS-1 tyrosine phosphorylation compared to the control (P < 0.05). The hyperinsulinemic hCG-treated rats had a similar insulin-induced tyrosine phosphorylation of IRS-1 to the control animals (Fig. 6A). The skeletal muscle Akt serine phosphorylation, induced by insulin, showed a significant reduction in the hyperinsulinemic group compared with the control, while the other two groups presented similar patterns of phosphorylation to that detected in control animals (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results showed that simultaneous treatment with hCG and insulin, which induced more pronounced changes in ovarian morphology, also induced an up-regulation in insulin receptor and IRS-1 tyrosine phosphorylation, and in IRS-1 association with PI 3-kinase stimulated by acute insulin infusion. Conversely, insulin-induced IRS-2 tyrosine phosphorylation was increased in the ovary of hyperinsulinemic rats, suggesting that this pathway is up-regulated in this situation. These IRS-1 and IRS-2 docking proteins can bind and activate PI 3-kinase; one target of this phospholipid enzyme is PKB/Akt and its upstream activator, PDK. The serine phosphorylation of PKB/Akt was markedly increased in ovaries of rats treated with hCG plus insulin, in a pattern similar to that detected for IRS-1 phosphorylation and association with PI3-kinase. However, the hCG treatment, alone or with insulin, induced an enhancement in Akt threonine phosphorylation similar to that detected for IRS-2/PI 3-kinase association. Full activation of Akt requires the two phosphorylation events, and both IRS-1 and IRS-2 associations with PI 3-kinase may mediate the modulation of Akt in ovaries.
Akt activation is involved in cell growth, inhibition of apoptosis, and granulosa cell survival (Lawlor & Alessi 2001, Johnson et al. 2001). Our data demonstrated that chronic treatment with hCG, alone or plus insulin, enhanced Akt phosphorylation and may indicate a role for this intracellular mechanism in the development of follicular cysts in this animal model.
Previous data from our group demonstrated an acute positive crosstalk between LH and insulin in Akt serine phosphorylation without additive effects on ERK phosphorylation (Carvalho et al. 2003). In the present study, we show that chronic treatment with hCG and insulin induced a similar crosstalk in the Akt phosphorylation and ERK pathway. Recent evidence indicates that stimulation of the ERK cascade inhibits steroidogenesis in granulosa cells. ERK pathways were also found to inhibit the expression of the CYP17 gene in an adrenocortical cell line (Sewer & Waterman 2003, Munir et al. 2004, Nelson-Degrave et al. 2005).
Despite the insulin-induced enhancement in IRS-1 tyrosine phosphorylation in skeletal muscle from hCG-treated rats, insulin treatment alone induced insulin resistance, as demonstrated by decreased IRS-1 and Akt phosphorylation. Conversely, the associated hCG plus insulin treatment had no effect on these intracellular pathways in skeletal muscle, suggesting that non-redundant pathways are involved in these modifications. Interestingly, despite the reduced insulin signaling in muscle, hyperinsulinemia alone had no effect on the IRS-1/PI 3-kinase pathway in ovary. This tissue-specific modulation of insulin signaling has also been previously described by comparing muscle and adipose tissues in other animal models of insulin resistance presenting as hyperinsulinemia (Prada et al. 2005, Ueno et al. 2005). However, to our knowledge, these are the first data to demonstrate such a tissue-specific modulation of the IRS-1/ PI 3-kinase/Akt pathway in muscle and ovary. In summary, our data show that chronic treatment with hCG or hCG plus insulin can induce changes in ovaries that simulate PCOS. In these situations, increases occur in the insulin-induced IRS/ PI 3-kinase/Akt pathway in the ovary, suggesting that the activation of this pathway may have a role in the development of PCOS.
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Acknowledgements |
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References |
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Received in final form 12 March 2006
Accepted 7 April 2006
Made available online as an Accepted Preprint 9 May 2006
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