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1 Department of Functional Biology, Physiology Area, University of Oviedo, Oviedo, Spain
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(Received 11 July 2006;
accepted after revision 23 October 2006; first published online 26 October 2006)
Corresponding author C. González: Department of Functional Biology, Physiology Area, University of Oviedo, C/ Julián Clavería s/n 3306, Oviedo, Spain. Email: tinog{at}uniovi.es
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Introduction |
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In contrast, variations in oestradiol and progesterone concentrations have been proposed to be two of the aetiological factors in the development of insulin resistance. Insulin sensitivity is modulated under different physiological conditions, such as puberty, menstrual cycle, pregnancy and menopause. In this way, insulin resistance during pregnancy may result from disruption of the first step of the insulin signalling cascade (Gonzalez et al. 2002b). Moreover, the loss of ovarian function associated with the menopause is associated with a reduction in whole body insulin-mediated glucose uptake (Proudler et al. 1992), and the changes that accompany the menopause may further reduce glycaemic control and insulin sensitivity in women with type 1 DM (Strotmeyer et al. 2003). Therefore, the beneficial effects on insulin sensitivity conferred by low-dose oestradiol replacement therapy in healthy postmenopausal women could be of particular benefit to postmenopausal women with diabetes (Andersson et al. 1997).
In contrast, artificially increased levels of the sex steroid hormones oestrogen and progesterone (e.g. in the case of women taking the combined oral contraceptive pill or receiving hormone replacement therapy) are also involved in the development of insulin resistance. In women receiving the oral contraceptive pill, the reduction in insulin sensitivity was attributed to progesterone (Shoupe, 1993). The experimental findings of many authors (Leturque et al. 1987; Cordoba et al. 1991; Lindheim et al. 1993, 1994) indicate that progesterone administration leads to insulin resistance, whereas oestrogen administration seems to maintain insulin sensitivity in female rats. However, it is not clear whether oestrogen alone or progesterone alone can cause insulin resistance, and the role of sex hormones in type 1 DM remains to be elucidated.
Taken together, these ideas indicate a relationship between plasma concentrations of sex steroids and the insulin resistance associated with type 1 DM. Therefore, the first aim of this study was to determine the effects of hyperglycaemia on insulin sensitivity and insulin signalling in ovariectomized diabetic rats. The second aim was to asses the possible role of 17ß-oestradiol and progesterone on the insulin sensitivity in ovariectomized diabetic rats, focusing on the effects on skeletal muscle IR and glucose transporter-4 (Glut-4) protein concentrations.
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Methods |
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Twelve-week-old virgin female Wistar rats (from the Biotery of the University of Oviedo, Spain), weighing 250–280 g, were used in this study and were kept under standard conditions: temperature 23 ± 3°C, humidity 65 ± 1%, and a regular lighting schedule of 12 h light–12 h dark cycle (lights on 08.00–20.00 h). The animals were fed with standard diet (Panlab A04, Barcelona, Spain) and they were allowed food and water ad libitum.
The experimental manipulations were performed between 09.30 and 12.30 h. The animal experiments were approved by the University of Oviedo Ethics Committee following the ‘Guiding Principles for Research Involving Animals and Human Beings: Recommendations from the Declaration of Helsinki’ and the Guide for the Care and Use of Laboratory Animals approved by the Council of The American Physiological Society (Institute for Laboratory Animal Research, 1996).
Experimental design
Three days before initiating the hormone treatment, the rats were ovariectomized through a midl-ine incision under halothane anaesthesia. Immediately after surgery, type 1 DM was induced by injection of streptozotocin (55 mg (kg body weight)–1; Sigma, St Louis, MO, USA) in 0.9% NaCl, through the tail vein. Rats were considered diabetics when, their plasma glucose levels were greater than 300 mg dl–1.
Animals were separated randomly into four groups: control (V) and diabetic rats (SV) treated with vehicle (olive oil and ethanol 3:2, v/v), diabetic rats treated with 17ß-oestradiol (SE) and diabetic rats treated with progesterone (SP). All animals were housed individually throughout the experiment.
The hormone treatment was applied according to the temporal diagram shown in Fig. 1. In order to allow recovery from the stress of surgery and to allow the decrease of endogenous steroid hormone levels, experimental treatment started 4 days after ovariectomy. The rats were injected subcutaneously every 12 h (09.00 and 21.00 h) for 20 days with 0.1 ml of a suspension in olive oil and ethanol (3:2, v/v) of 17ß-oestradiol (Sigma; SE group) or progesterone (Sigma; SP group). The control (V) and diabetic control (SV) groups injected with vehicle were followed in parallel.
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Euglycaemic–hyperinsulinaemic clamp studies
Clamp experiments were performed in anaesthetized rats using a procedure previously described (Gonzalez et al. 2000). Briefly, after 12 h of fasting on days 6, 11 and 16, the animals were anaesthetized by intraperitoneal sodium pentobarbitone administration (50 mg kg–1) and, after a stabilization period of 30 min, the left saphenous vein was catheterized for insulin and glucose infusion. Biosynthetic human insulin (Actrapid, Novo Nordisk, Copenhagen, Denmark) was infused at a constant rate of 20 µl min–1 (0.4 i.u. kg–1 h–1), and the blood glucose level was clamped at the level measured in the basal state using a variable infusion of glucose dissolved in 0.9% NaCl.
Hormone levels
After the clamp study, blood samples (4 ml), for the determination of plasma concentrations of 17ß-oestradiol and progesterone, were collected from the jugular vein into heparinized tubes, centrifuged at 1200 g for 20 min at 4°C, and plasma was immediately harvested and stored frozen at –20°C until assayed. Plasma 17ß-oestradiol was measured by RIA using Immuchen kits of cover tubes (ICN Pharmaceuticals Inc., Costa Mesa, USA). The assay sensitivity was 10 pg ml–1, and the intra-assay coefficient of variation was 12.26%. Plasma progesterone was measured by radioimmunoassay (RIA) using Immuchen kits of cover tubes (ICN Pharmaceuticals Inc., Costa Mesa, CA, USA). The sensitivity of the assay was 0.15 ng ml–1, and the intra-assay coefficient of variation was 11.48%. The samples were assayed in duplicate. All samples were assayed on the same day.
Finally, samples of skeletal muscle (flexor digitorum superficialis, extensor digitorum longus, soleus and extensor digitorum lateralis) were collected and immediately frozen in liquid nitrogen for use in future experiments. The animals were killed by exsanguination.
Immunoprecipitation and Western blot analysis
The samples of skeletal muscle (100 mg) were prepared as previously described (Gonzalez et al. 2002a, 2003). Briefly, the samples were washed with ice-cold phosphate-buffered saline and homogenized immediately in 3 ml of lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet®P40 (Roche Diagnostics, Barcelona, Spain), 0.05% sodium deoxycholate and 1 mM sodium orthovanadate] at 4°C. The extracts were centrifuged at 12 000g at 4°C for 10 min, and the protein content was determined using the Bradford dye-binding method (Bradford, 1976). To ensure that the proteins were in a linear range of detection, preliminary experiments were conducted to determine that the amount of homogenate protein load was within a range that resulted in a proportionate change in signal intensity as the amount of protein loaded was varied. The aqueous fraction, containing 250 µg of protein, was used for immunoprecipitation (IP) with 0.25 µg of polyclonal antibody against the insulin receptor ß-subunit (B-IR; sc-711, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immune complexes were precipitated with protein G–agarose beads (Roche Diagnostics) overnight at 4°C in a rocking platform and were washed several times in wash buffer [50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 0.1% Nonidet®P40, 0.05% sodium deoxycholate, 0.1% ortho vanadate 1 M]. After washing, the pellet was suspended in protein loading buffer [250 mM Tris-HCl (pH 6.8), 8% SDS, 8 mM EDTA, 35% glycerol, 2.5% ß-mercapto-ethanol, Bromophenol Blue] and heated in a boiling water bath for 5 min. Similarly sized aliquots were subjected to SDS-PAGE (8% Tris-Acrilamide-Bisacrilamide), and the proteins were electrotransferred from the gel onto nitrocellulose membranes (Hybond-ECL, Amersham Pharmacia Biotechnology, Barcelona, Spain) as described by Towbin et al. (1979). Non-specific protein binding to the nitrocellulose membranes was reduced by pre-incubating the filter in blocking buffer (Tris-NaCl-Tween 20 TNT, 7% bovine serum albumin), and probing was carried out using a 1:75 000 dilution of antiphosphotyrosine antibody (sc-7020, Santa Cruz Biotechnology) conjugated to horseradish peroxidase diluted in blocking buffer. The membranes were rinsed several times with blocking buffer without BSA. The protein bands were visualized using enzyme chemiluminescense (sc-2048, Santa Cruz Biotechnology).
Next, the membranes were incubated in stripping buffer [50 ml 62.5 mM Tris-HCl (pH 6.8), 1 g SDS and 0.34 ml ß-mercaptoethanol] at 60°C. Subsequently, another Western blot analysis was performed using a 1:2000 dilution of polyclonal antibody against the insulin receptor ß-subunit (B-IR; sc-711, Santa Cruz Biotechnology) as the primary antibody.
To determine the level of total Glut-4, similar sized aliquots (20 µg total protein) were subjected to 7% SDS-PAGE and transferred to nitrocellulose membranes, and Western blot analysis was performed using a 1:1000 dilution of anti-Glut-4 antibody (sc-7938, Santa Cruz Biotechnology).
Adult rat skeletal muscle protein was used as a positive control. To verify equal protein load, all the membranes were routinely stained with Ponceau Red. Insulin receptor tyrosine phosphorylation was expressed relative to the protein levels as the phosphorylation rate. All membranes were stripped and probed with monoclonal anti-ß-actin antibody (dilution 1:2500; sc-1615, Santa Cruz Biotechnology) to ensure equal protein loading. Scanning densitometric analysis was normalized to ß-actin analysis to ensure that the differences observed were not the result of differences in protein load. To facilitate intergel comparisons and to avoid artefacts due to differences on film exposure during analysis, standards prepared from pooled cardiac muscle were also run in each gel. In this sample, the quotient between protein scanning densitometric analysis and ß-actin analysis should be similar in all the membranes processed.
The bands were quantified using a digital scanner (Nikon AX-110) and NIH Image 1.57 software (Scion Corporation, Frederick, MA, USA).
Statistics
Data are expressed as means ± S.E.M. Previously, we evaluated the Gaussian distribution of each variable. Thereafter, the comparisons were made using an analysis of variance and the Student–Newman–Keuls test. A P value of 0.05 was considered significant. Statistical analysis was performed using SPSS v.6.01 for Windows.
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Results |
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Plasma 17ß-oestradiol and progesterone values observed throughout the study are shown in Fig. 2. In non-hormone-treated groups (V and SV), plasma concentrations of 17ß-oestradiol and progesterone were significantly lower than in treated groups (SE and SP; Fig. 2A and B). The plasma levels of 17ß-oestradiol in the SE group (Fig. 2A) and those of progesterone in the SP group (Fig. 2B) were similar to those described during rat pregnancy (Gonzalez et al. 1997, 1998).
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To investigate insulin sensitivity in rats at different stages of the treatment, clamp experiments were carried out under euglycaemic and hyperinsulinaemic conditions. Figure 3 shows the comparison of glucose infusion rates in the studied groups.
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Protein content and tyrosine phosphorylation of IR in skeletal muscle
Firstly, we investigated whether our experimental model of type 1 DM has effects on the amount of IR and/or in the insulin-induced tyrosine phosphorylation of IR. The amount of IR (Fig. 4A) of the V group did not change during the experiment, while in the SV and SE groups the pattern of variation in this protein was similar: a significant increase between days 6 and 11 and a significant decrease between days 11 and 16. In the SP group, we found a significant decrease at day 16. Intergroup comparisons showed that at day 6, IR protein content was lower in the SV than in the V and SP groups. However, at day 16, the V group had the greatest amount of this protein.
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The studies with antiphosphotyrosine antibody provide information about tyrosine phosphorylation of IR, but not the number of IR or the stoichiometry of phosphorylation. For this reason, we have calculated IR relative tyrosine phosphorylation (Fig. 4C) as the ratio between IR tyrosine phosphorylation and IR protein content. In non-hormone-treated groups (V and SV), this parameter decreased progressively throughout the treatment; however, only in the SV group were these differences significant. In contrast, in the SE group, the duration of the experimental period was associated with a significant increase in this parameter. In SP rats, IR relative tyrosine phosphorylation did not change throughout the treatment. When we compared this parameter between groups, we could see that the V group had the highest relative tyrosine phosphorylation of IR at days 6 and 11 of the experiment. At day 16, this parameter was higher in the V and SE than in the SV and SP groups.
Glut-4 content in skeletal muscle
Figure 5 shows the Glut-4 content in our experimental groups. In the V group, the amount of this protein decreased significantly during the experimental period. In the SV and SE groups, this protein reached a maximum level at day 11, but only in the SV group were these differences significant. In the SP group, no significant differences were noted. Intragroup comparisons showed that in the SE group the lowest content of Glut-4 was reached at days 6 and 16; however, these differences were not significant. At day 11, in the V group, this parameter was lower than in the SV and SE groups.
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Discussion |
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To confirm whether oestrogen or progesterone affects insulin sensitivity in ovariectomized rats compared with ovariectomized streptozotocin-induced diabetic rats, we have developed a euglycaemic–hyperinsulinaemic clamp (Fig. 3), which has become the standard reference method for the study of glucose metabolism (DeFronzo et al. 1979; Radziuk, 2000). In non-hormone-treated groups (V and SV), there was a progressive reduction in insulin sensitivity during the course of treatment. These observations are in agreement with those of other authors (Kumagai et al. 1993; Gonzalez et al. 1997), who have shown that the absence of female sex steroids results in a reduction in insulin sensitivity. Taken together, our results in ovariectomized type 1 diabetic rats (SV) show that STZ treatment caused a reduction in insulin sensitivity during the experimental treatment. We believe that this could be explained by the hyperglycaemic state, responsible for reducing tyrosine phosphorylation of IR in the SV group, thereby reducing signal transduction (Figs 3 and 4C). Indeed, several studies have proposed that hyperglycaemia can modulate IR tyrosine phosphorylation by stimulation of the activity of serine/threonine kinase and/or tyrosine phosphatases, depending on the exposure time (Hauguel-de Mouzon et al. 1993; Pillay et al. 1996; Tang et al. 2001). Our results regarding Glut-4 concentration in the SV group (Fig. 5) are in agreement with those of Kahn et al. (1991), who previously observed that hyperglycaemia has different effects on Glut-4 according to the exposure time. Indeed, it has been reported that the absence of female sex steroids (as in the SV group) impairs Glut-4 translocation from intracellular storage vesicles to the plasma membrane (Rincon et al. 1996), which could explain the apparent discrepancy between Glut-4 protein levels and insulin sensitivity (Figs 3 and 5). Moreover, in this group, the reduction in Glut-4 at the end of the experimental period (day 16) may explain the insulin resistance described above.
Our results for the SE and SP groups show that during the early period of treatment (days 6–11), when the plasma concentrations of 17ß-oestradiol and progesterone are low and similar to those found during early pregnancy (Gonzalez et al. 2000), insulin sensitivity is higher, even higher than in non-hormone-treated groups (V and SV). However, during the late period of treatment (days 11–16), when plasma levels of 17ß-oestradiol and progesterone in the SE and SP groups, respectively, are high and similar to those of late pregnancy (Gonzalez et al. 2000), the role of both hormones is different. Although 17ß-oestradiol improves insulin sensitivity, progesterone reduces insulin sensitivity (Fig. 3). These results are in line with studies reporting that oestrogens increase sensitivity to the actions of insulin (Kumagai et al. 1993) and others showing that progesterone administration reduces insulin sensitivity in rats (Gonzalez et al. 2000). In conclusion, we suggest that, although progesterone could oppose the effects of hyperglycaemia during the early period of treatment (days 6–11) when its plasma levels are low, 17ß-oestradiol treatment always improves the insulin sensitivity independently of its plasma concentration. In contrast, we suspect that the combined administration of oestradiol and progesterone could have different effects on insulin sensitivity, because in previous work (Gonzalez et al. 2000) we have shown that when non-diabetic ovariectomized rats were treated with low doses of combined oestradiol and progesterone their insulin sensitivity was higher that of rats treated with oestradiol alone or progesterone alone. However, in the present experimental design, we have not included an intact control group, so it will be necessary for further investigations in order to assign a specific role to each hormone.
Most previous studies have shown that, in rodents, STZ-induced type 1 DM results in a reduced response to insulin, despite increased numbers of IR, owing to hyperglycaemia (Kadowaki et al. 1984). In light of our results (Fig. 4A), we propose that hyperglycaemia has different effects on the number of IR depending on the exposure time: short exposure times (up to day 11) could increase the number of IR in the SV and SE groups, and prolonged exposures to high blood glucose concentrations (day 16) could reduce the number of IR. Moreover, it is established that increased blood glucose levels can modulate the downstream targets of IR (Giorgino et al. 1992; Oku et al. 2001). Considering the results in the SE and SP groups (Figs 3, 4A and C and 5), we postulated that at the beginning of treatment (days 6–11) low levels of 17ß-oestradiol and progesterone, respectively, accompanied by a short exposure to hyperglycaemia, might reverse the negative effects of hyperglycaemia by modulating insulin signalling downstream of IR and improving insulin sensitivity. By contrast, at the end of treatment (day 16), our results in the SE group indicate that high concentrations of 17ß-oestradiol accompanied by a long exposure to hyperglycaemia may oppose the negative effects of high glucose concentrations earlier in the signalling pathway (IR tyrosine phosphorylation), thereby improving insulin sensitivity (Figs 3 and 4C). In a recent study, Barros et al. (2006) have shown that the effects of oestradiol on Glut-4 are dependent on the oestrogen receptor isoform involved: the 
isoform is a positive regulator of Glut-4 expression, while the ß isoform plays a suppressive role. The physiological implication of this divergent regulation remains to be elucidated. However, in the SP group at day 16, when progesterone plasma levels were increased, analysis of the results for IR indicated a close relationship between the level of IR tyrosine phosphorylation and Glut-4 protein and the development of insulin resistance (Figs 3, 4A and C and 5).
It is important to emphasize that hyperglycaemia not only represents the manifestation of DM owing to the development of insulin resistance in peripheral tissues, but that it is also a self-perpetuating factor in the diabetic state, known as ‘glucose toxicity’ (Nawano et al. 2000; Oku et al. 2000). When exposed to chronic hyperglycaemia, body tissues (especially skeletal muscle) seem to protect themselves against excessive glucose utilization, at least in part. These protective mechanisms include a reduction in insulin-stimulated glucose disposal (Rossetti et al. 1990). Therefore, in light of our results in the SP group with respect to IR and Glut-4 during the early period of treatment (days 6–11; Figs 4A and 5), we believe that progesterone could initiate a protective mechanism to combat the effects of glucose toxicity, which might begin at day 11. However, in the SE group, this protection might appear later (day 16), owing to a longer exposure to hyperglycaemia.
In summary, this study shows that 17ß-oestradiol and progesterone have important roles in the regulation of insulin sensitivity in diabetic rats. Our results indicate that 17ß-oestradiol improves insulin sensitivity throughout the course of treatment. However, the present experimental design does not permit us to elucidate clearly the influence of doses and effect of exposure time. Therefore, this question requires further investigation. By contrast, progesterone only improves insulin sensitivity during the early period of treatment (days 6–11), and the effects are not associated with changes in IR and Glut-4 levels. Both female sex steroids exert a protective role in skeletal muscle against glucose toxicity, but the effects begin at different stages of treatment. Moreover, we think that these novel findings are important for our understanding of insulin resistance in type 1 DM and of the risk/benefit ratio when 17ß-oestradiol and progesterone are used in oral contraceptives or hormone replacement therapy taken by menopausal women with controlled type 1 DM.
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Footnotes |
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P < 0.05 versus day 11. Intergroup comparisons: aP < 0.05 versus V; bP < 0.05 versus SV; cP < 0.05 versus SE; and dP < 0.05 versus SP.
