help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carrascosa, J. M.
Right arrow Articles by Herrera, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carrascosa, J. M.
Right arrow Articles by Herrera, E.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Endocrinology Vol. 139, No. 2 520-526
Copyright © 1998 by The Endocrine Society

ARTICLES

Changes in the Kinase Activity of the Insulin Receptor Account for an Increased Insulin Sensitivity of Mammary Gland in Late Pregnancy1

J. M. Carrascosa, P. Ramos, J. C. Molero and E. Herrera

Department of Biochemistry (J.M.C., J.C.M.), Centro Biología Molecular-Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, E-28049 Madrid, Spain; and Faculty of Experimental and Technical Sciences (P.R., E.H.), Universidad San Pablo-CEU, E-28668 Madrid, Spain

Address all correspondence and requests for reprints to: Emilio Herrera, Facultad de Ciencias Experimentales, Universidad San Pablo-CEU, Ctra. de Boadilla del Monte km 5.3, E-28668 Madrid, Spain. E-mail: e.herrera{at}offcampus.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mammary gland is an organ that undergoes cycles of growth, differentiation, and function during pregnancy and lactation. Although it is known that the gland enhances its sensitivity to insulin during lactation, it remains to be investigated whether this increased sensitivity develops during pregnancy and which are the molecular mechanisms underlying such a change. To address this issue, virgin and late-pregnant rats were subjected to a continuous infusion with 50% glucose for 72 h to produce a prolonged hyperinsulinemic-euglycemic condition. Insulin sensitivity in mammary gland was determined as the glucose utilization index by using 2-[3H]-deoxyglucose. Furthermore, binding characteristics and kinase activity were studied by means of both [125I]insulin binding and in vitro phosphorylation studies with insulin receptors partially purified from mammary gland.

Whereas the glucose utilization index in mammary gland from nonpregnant rats remained unaffected by hyperinsulinemia, glands from pregnant rats displayed a high insulin-dependent glucose uptake. This effect was not paralleled by changes in the binding characteristics of insulin to the high-affinity receptor, suggesting that the high insulin sensitivity of mammary gland in pregnancy is not accounted for by changes at the level of hormone-receptor interaction. Autophosphorylation studies showed that insulin-stimulated kinase activity of insulin receptors from mammary gland was 6- and 20-fold higher in pregnant than in virgin animals under normo- and hyperinsulinemic conditions, respectively. Moreover, insulin dose-response curves revealed that the efficacy of insulin to stimulate kinase activity of the insulin receptor was markedly higher in pregnant than in virgin rats, whereas its potency (ED50 ~ 15 nM) was not changed. These data, therefore, show that mammary glands develop increased insulin sensitivity during late pregnancy, caused by an augmented kinase activity of the insulin receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LATE pregnancy is characterized by the development of insulin resistance in both humans (1, 2, 3) and rats (4, 5, 6). Among the tissues that most actively contribute to the reduced insulin sensitivity are adipose tissue (4, 7) and skeletal muscle (8, 9). Whereas adipose tissue remains insulin-resistant during lactation (10), lactating mammary gland becomes highly sensitive to insulin (11, 12, 13). This enhanced mammary gland insulin sensitivity is rapidly abolished on weaning (12), but nothing is known about insulin responsiveness in the late-pregnancy mammary tissue.

The mammary gland in late pregnancy is characterized by alveolar morphogenesis, disappearance of adipose cells, and structural and functional differentiation of alveolar epithelial cells, enabling them to secrete milk fat and protein during lactation (14, 15). This development and differentiation of the rat mammary gland are further accompanied by changes in the expression of glucose transporters. Whereas in mammary gland of virgin rats, the insulin sensitive isoform of the glucose transporter Glut4 predominates (16), during late pregnancy, expression of Glut4 decreases and is abolished in the lactating mammary tissue (14, 16). Furthermore, lipoprotein lipase activity increases during late pregnancy (17), reaching a maximum during lactation (18). Thus, the morphological and functional changes characteristic for the lactating and mature mammary gland apparently start to develop already during pregnancy. Therefore, the present study was designed to clarify whether the increased insulin sensitivity of mammary gland observed in lactation does develop during pregnancy. To address this issue, virgin and pregnant rats were exposed to a continuous iv infusion with glucose that has been already shown to induce hyperinsulinemia (HI) unaccompanied by hypoglycemia (19, 20). In these animals, insulin sensitivity was assessed using the euglycemic-hyperinsulinemic clamp, and glucose utilization in the mammary gland was quantified by means of the 2-deoxyglucose (2-DOG) technique. These experiments revealed that mammary gland tissue becomes insulin sensitive during pregnancy. To further investigate the molecular mechanism underlying the increased responsiveness of the tissue, the study was extended to determine whether the interaction of insulin with its receptor and/or the insulin receptor kinase activity are affected during pregnancy under basal or hyperinsulinemic conditions, by means of radioligand binding and in vitro phosphorylation studies. Finally, insulin-dependent autophosphorylation of the receptors was correlated with the insulin responsiveness of the mammary gland, providing evidence for the involvement of increased receptor kinase activity in the enhanced insulin sensitivity of mammary gland during late pregnancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female Wistar rats were housed at 22–24 C, with light cycles from 0800 to 2000 h. They had free access to water and to a chow diet (Letica, Barcelona, Spain). Some animals were mated when they weighed 170–180 g. The beginning of pregnancy was determined by the presence of spermatozoids in vaginal smears. In pregnant rats at day 17 of gestation and in age-matched virgin rats, a SILASTIC brand catheter (Dow Corning, Midland, MI, 0.02 inch ID, 0.037 inch OD) was placed into the right jugular vein and another one into the right femoral vein, under ketamine cocktail anesthesia (ketamine, 50 mg/ml; diazepan, 5 mg/ml; and atropine, 1 mg/ml; 5/4/1, vol/vol/vol). After recovery from anesthesia, animals were housed in individual cages and continuously infused for 72 h with either bidistilled water or 50% glucose, through the catheter placed into the jugular vein, at the rate of 35 ml/day. Other methodological details have been previously described (19, 20). At the end of the 72-h infusion period, some animals from each group were decapitated, blood was collected from the neck wound, and mammary glands rapidly dissected and placed in liquid nitrogen, to be stored at -80 C until processed for insulin receptor studies, as described below. The experimental protocol was approved by the Animal Research Committee of the Faculty of Experimental Sciences, University San Pablo-CEU.

Studies of insulin resistance
Euglycemic clamp studies in the conscious rat. A number of rats from each group were subjected to an euglycemic- hyperinsulinemic clamp to test the insulin sensitivity state of the animals, as described before (20). In brief, after the 72-h infusion, blood samples were obtained from the tail tips for determination of blood glucose (21) and plasma insulin (22). The catheter placed in the jugular vein was connected to a two-way interconnector that received flow from two different infusion pumps (Precidor Infusion Pump Type 5003, Infors HT, Denkendorf, Germany). Human insulin (Actrapid monocomponent, Novo, Copenhagen, Denmark) was infused, by means of one pump at a constant rate of 16 µl/min (0.8 IU x h-1 x kg -1), for 60 min. Glucose infusion (20%) was given at a variable rate through the other pump via the same catheter, to maintain the blood glucose concentration constant at basal levels. A steady-state glucose infusion was normally achieved within 30 min after starting the clamp experiment. A few additional blood samples (200 µl) were collected to determine the insulin concentration (22) at the steady-state. The glucose disposal rate (M) was estimated as the rate of glucose infusion at the steady-state normalized to the body weight. The insulin sensitivity index (Sip), proposed by Ader and Bergman (23), was measured as described (19, 20).

Estimate of glucose utilization index (GUI) in mammary gland. The method of Sokoloff et al. (24), developed for the brain, was adapted for the unrestrained rat (19). Briefly, 2-deoxy-D-[1-3H]glucose (2-DOG; 18.3 Ci/mmol, from Amersham, Buckinghamshire, UK) was administered, as a bolus (30 µCi) through the catheter placed in the jugular vein, to virgin and pregnant rats: 1) at the end of a 72-h infusion with bidistilled water (basal group); 2) after 1 h of an euglycemic-hyperinsulinemic clamp, subsequent to a 72-h infusion with bidistilled water (1-h hyperinsulinemic group); or 3) at the end of a 72-h infusion with 50% glucose (72-h hyperinsulinemic group). In the 1-h hyperinsulinemic group, the clamp, i.e. insulin and 20% glucose infusions, was maintained until death. Blood samples (50 µl) were collected from the catheter placed in the femoral vein at 1, 2, 5, 7, 10, 20, 30, 45, and 60 min after the 2-DOG administration. Blood was deproteinized (25), and glucose concentrations (21) and 2-DOG radioactivity were determined in the supernatants. After the last blood collection, animals were decapitated, and blood was collected from the neck wound for plasma insulin determination (22). Mammary gland tissue was immediately dissected and immersed in 0.5 ml of 1 M NaOH to determine its content of 2-DOG-6-phosphate (26) and to estimate the GUI, as previously described (19).

Studies with insulin receptors
Preparation of partially purified insulin receptors. Pools of mammary gland from each group (7–11 g) were homogenized in 3 vol of 25 mM Tris-HCl (pH 7.4) buffer, containing 0.3 M sucrose, 7 mM EDTA, 50 mM NaF, 10 mM sodium pyrophosphate, 1.3 mM phenylmethylsulfonyl fluoride (PMSF), 2 mg/ml bacitracin, and 0.3 mg/ml trypsin inhibitor. After centrifugation at 3,000 x g for 15 min, the supernatant was centrifuged at 50,000 x g for 1 h. The microsomal pellet was resuspended in 50 mM Tris-HCl (pH 7.4) buffer, containing 150 mM NaCl and 1 mM PMSF, and stored at -70 C. Microsomes (500 µl) were solubilized for 1 h in 2 ml of 25 mM Tris-HCl (pH 7.4) buffer, containing 1% Triton-X-100, 4 mM EDTA, 7 mM sodium pyrophosphate, 30 mM NaF, 0.6 mg/ml benzamidine, 0.3 mg/ml trypsin inhibitor, 2.5 mg/ml bacitracin, and 1.3 mM PMSF. Insoluble material was removed by centrifugation at 150,000 x g for 1 h, and the clear supernatant was diluted 1:5 with 50 mM Tris-HCl (pH 7.4), containing 0.05% Triton-X-100, 100 mM NaCl, 2.5 mM KCl, and 1 mM CaCl2, added to 0.5 ml of wheat germ agglutinin-Sepharose (WGA-Sepharose, Pharmacia, Uppsala, Sweden) and rotated end-over-end for 2 h at room temperature. The WGA-Sepharose was then washed five times with 20 vol of the same buffer, and receptors were eluted by the addition of 500 µl of 0.3 M N-acetyl-glucosamine in the same buffer. The protein concentration was measured (27) in these fractions and always ranged between 0.2 and 0.4 mg/ml.

Binding to solubilized receptors. Aliquots (10 µl) of the WGA-Sepharose eluates were incubated overnight at 4 C, with [125I-TyrA14]insulin (100 pM, from Amersham) and various concentrations of human monocomponent insulin (0.16–1600 nM) in a final vol of 500 µl. After incubation, receptor-hormone complexes were separated from free insulin by 10% polyethylene glycol precipitation, and measurement of the bound hormone was done as previously described (28). Insulin binding constants [equilibrium dissociation constants (Kd) and maximal binding constants (Bmax)] were estimated by Scatchard analysis.

Autophosphorylation of solubilized receptors. Aliquots (10–15 µg of protein) of WGA-Sepharose purified insulin receptors were incubated at room temperature for 2 h in the N-acetyl-glucosamine buffer supplemented with increasing amounts of porcine insulin (Sigma, 0–160 nM). Phosphorylation assays were carried out at 0 C in the presence of [{gamma}-32P]ATP, as previously described (29). Briefly, the phosphorylation reaction was initiated by adding 50 µM [{gamma}-32P]ATP (specific radioactivity ~3300 cpm/pmol), 12 mM MgCl2, 6 mM MnCl2, and 1 mM sodium orthovanadate (final concentrations), to give a final vol of 80 µl. After a 15-min incubation, the reaction was ended by applying 80 µl of a 50 mM Tris-HCl (pH 7.4), containing 20 mM EDTA, 20 mM sodium pyrophosphate, 1 mM ATP, 1 mM sodium orthovanadate, and 1 mM dithiothreitol. Insulin receptors were quantitatively immunoprecipitated with antiphosphotyrosine antibodies prepared as in (30). After immobilization on Protein A (Sigma Chemical Co., St. Louis, MO), the pellets were washed three times with 50 mM TripH 7.4 (containing 0.1% SDS, 0.1% Triton-X-100, and 0.15 M NaCl), and proteins were separated by SDS-PAGE (7% acrylamide). Radioactive proteins were identified by autoradiography, and the 32P-incorporation into the insulin receptor ß-subunit was quantified by densitometric scanning.

Statistical analysis
Statistical comparisons were made with ANOVA, followed by the Tuckey test, or by a multiple linear-regression analysis with a 95% confidence interval, using the Systat program (Systat, Inc., Evanston, IL). Results are expressed as means ± SEM.

The Kd and Bmax were calculated using the nonlinear regression fitting option of the Sigma Plot Program (Jandel Scientific Corp., San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Model of the euglycemic-hyperinsulinemic rat
Body weight, circulating components. In the present study, to investigate the response of mammary glands to insulin in late pregnancy, an animal model of prolonged iv glucose infusion in the rat, to attain hyperinsulinemia under euglycemic conditions, was used (19, 20). Virgin and pregnant rats (17 day of gestation) were subjected to a continuous infusion with bidistilled water (control) or 50% glucose for 72 h. Animals within each group were of equal weight at the beginning of the infusion and, as shown in Table 1Go, 50% glucose treatment for 72 h did not affect the body weight of either virgin or pregnant rats. Blood glucose levels were lower in pregnant than in virgin rats and remained the same upon glucose infusion in both groups. Plasma insulin levels were higher in pregnant than in virgin rats, but both groups responded similarly to glucose treatment with a 2- to 3-fold increase in plasma insulin concentration (Table 1Go). These data, therefore, show that the model of glucose infusion for 3 days is suitable to generate hyperinsulinemic-euglycemic conditions, both in virgin and pregnant rats.


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of 50% glucose infusion (35 ml/day) for 3 days in pregnant (day 17–20 of gestation) and virgin rats on body weight and circulating components

 
Euglycemic-hyperinsulinemic clamp. To determine how the glucose infusion affected insulin sensitivity, an euglycemic-hyperinsulinemic clamp (0.8 IU insulin x h-1 x kg -1) was performed for 60 min in unrestrained virgin and pregnant rats at the end of the 72-h infusion with either 50% glucose or bidistilled water. Under these conditions, blood glucose remained stable, whereas plasma insulin increased in the four groups (Table 2Go) when compared with the basal values before the clamp (Table 1Go). Plasma insulin concentrations during the clamp were higher in the groups receiving the 50% glucose infusion than in their respective controls, and in pregnant than in virgin rats (Table 2Go). In virgin animals, 72 h of 50% glucose infusion resulted in a significant decrease in both the M and the Sip, by 39% and 43%, respectively, showing the development of insulin resistance under hyperinsulinemic-euglycemic conditions (Table 2Go). Pregnant control animals clearly exhibited insulin resistance, as evidenced by their low M and Sip-values (41% and 55% of virgin controls, respectively). In contrast to virgin animals, in pregnant rats, 72 h of 50% glucose infusion markedly increased the M by 64% and completely restored the impaired Sip to values that did not differ from those of the virgin control rats, thus indicating the full reversion of the insulin-resistant condition.


View this table:
[in this window]
[in a new window]
  		Table 2. Effect of sustained hyperinsulinemia-euglycemia on insulin sensitivity in virgin and pregnant rats

 
Insulin responsiveness of mammary gland in late pregnancy: GUI
As shown above, insulin resistance that normally develops during late pregnancy disappears in response to a prolonged hyperinsulinemia caused by the 72 h of 50% glucose infusion. To investigate insulin responsiveness of mammary gland during late pregnancy and how this tissue responds to such prolonged hyperinsulinemic condition, glucose utilization was quantified by measuring the GUI. GUI was assessed by the administration of an iv bolus of 2-DOG and the subsequent analysis of phosphorylated 2-DOG in the mammary tissue. These experiments were performed in pregnant and nonpregnant rats under normoinsulinemia (basal) and under both short- and long-term hyperinsulinemia, generated by means of the euglycemic-hyperinsulinemic clamp (1-h HI) and by the 72-h continuous glucose infusion (72-h HI), respectively. As shown in Fig. 1Go, in mammary gland of normoglycemic virgin animals (basal), the GUI value was 1.60 ± 0.15 mg x min-1 x kg-1, and neither short-term (1-h HI) nor prolonged (72-h HI) exposure to hyperinsulinemia affected the GUI in mammary glands of these animals (1.56 ± 0.34 and 1.91 ± 0.34 mg x min-1 x kg -1, respectively). In pregnant rats, under basal conditions, the GUI of mammary gland was 1.31 ± 0.26 mg x min-1 x kg -1 and, thus, did not differ from the value found in the virgin basal group. However, in contrast to virgin rats, mammary glands of pregnant animals responded to hyperinsulinemia with increased glucose utilization (Fig. 1Go). This effect seemed to be dependent on the duration of hyperinsulinemia, with GUI values increasing to 2.08 ± 0.15 mg x min-1 x kg-1 (1.6-fold) and to 3.08 ± 0.35 mg x min-1 x kg-1 (2.4-fold) upon exposure to hyperinsulinemic conditions for 1 and 72 h, respectively.



View larger version (59K):
[in this window]
[in a new window]
  		Figure 1. Effect of hyperinsulinemia (HI) on glucose utilization in the mammary gland of virgin and pregnant rats. GUI in mammary gland of virgin and 20-day-pregnant rats was measured as described in Materials and Methods under normoglycemic, short-term, and long-term hyperinsulinemic conditions, i.e. in rats at the end of a 72-h infusion with bidistilled water (basal group), after 1 h of an euglycemic-hyperinsulinemic clamp (0.8 IU insulin x h-1 x kg-1), subsequent to a 72-h infusion with bidistilled water (1-h HI) or at the end of a 72-h infusion with 50% glucose [72 h hyperinsulinemic group (72-h HI)], respectively. Statistical comparisons were made by ANOVA, followed by a Tuckey test with 95% confidence limits. Significance is shown by letters: different letters indicate significant difference (P < 0.05). Capital letters are used for the pregnant rats.

 
Insulin binding characteristics of insulin receptors from mammary gland
To investigate whether the increased insulin sensitivity of mammary gland in late pregnancy can be accounted for by changes in binding of insulin to its receptor, we performed competition binding studies with 125I-labeled insulin and insulin receptors partially purified from mammary glands of virgin and pregnant rats exposed to normo- and hyperinsulinemic conditions (i.e. infusion of bidistilled water or 50% glucose for 72 h, respectively). Scatchard analysis of competition binding curves (not shown) revealed that virgin and pregnant rats exhibited both low- and high-affinity sites for insulin. As shown in Table 3Go, both binding capacity and affinity of insulin receptors were similar in mammary gland of pregnant and virgin rats. The binding data also show that in virgin and pregnant animals, hyperinsulinemia caused by the 50% glucose infusion did not significantly modify either maximal insulin binding to both the high-(Bmax-1, ANOVA P = 0.68) and low-affinity (B max-2, ANOVA P = 0.08) receptor or the affinity of insulin for the high-(Kd-1, ANOVA P = 0.761) or for the low-affinity binding sites (Kd-2, ANOVA P = 0.203) (Table 3Go). Furthermore, the number of receptors per mg of tissue was not significantly affected either by pregnancy or by sustained hyperinsulinemia (ANOVA P = 0.177).


View this table:
[in this window]
[in a new window]
  		Table 3. Effect of 50% glucose infusion (35 ml/day) for 3 days on insulin binding to the mammary gland insulin receptor of virgin and pregnant rats

 
Kinase activity of insulin receptor from mammary gland
Because the binding data did not provide an explanation for the development of increased insulin sensitivity in the mammary gland during late pregnancy, we speculated that pregnancy might modify the kinase activity of the mammary gland insulin receptor. To address this issue, in vitro phosphorylation studies, with insulin receptors partially purified from mammary glands of control and glucose-infused virgin and pregnant rats, were performed; and the kinase activity of the receptor was determined as insulin-dependent autophosphorylation of its 95 kDa {alpha}-subunit. Representative autoradiographs of the phosphorylated and immunoprecipitated mammary gland insulin receptors, in the presence (160 nM) or the absence of insulin, are depicted in the upper part of Fig. 2Go. To quantify the kinase activity of the insulin receptor under the different experimental conditions in each preparation, 32P-incorporation into the ß-subunit was determined by scanning densitometry and normalized to its insulin binding activity (IBA). As shown in Fig. 2Go, insulin receptors from mammary glands displayed basal autophosphorylation that was approximately 20-fold higher in pregnant than in virgin rats. Although, in both groups, autophosphorylation was markedly increased upon incubation with 160 nM insulin, phosphorylation levels were always higher in the pregnant than in the virgin group. The effect of glucose infusion also was different in virgin and pregnant animals. The quantitative analysis shown in Fig. 2Go revealed that in receptors from mammary gland of glucose-infused virgin rats (i.e. rats kept hyperinsulinemic for 72 h), insulin (160 nM)-stimulated kinase activity was only 38% of that found in control animals. Hence, whereas in virgin rats the glucose infusion reduced the insulin-dependent phosphorylation even more, in pregnant rats it led to a slight (although not significant) increase in insulin-stimulated autophosphorylation. Thus, in the insulin receptors of mammary glands from pregnant rats, the insulin-stimulated kinase activity was 6- and 20-fold higher under normo- and hyperinsulinemic conditions, respectively, as compared with virgin animals.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. Insulin receptor (IR) ß-subunit autophosphorylation. Insulin receptors, partially purified from mammary gland, were phosphorylated after incubation in the absence or presence (160 nM) of insulin. Phosphorylated receptors were immunoprecipitated with antiphosphotyrosine specific antibodies and separated by SDS-PAGE. The autoradiographs shown in the upper part of the figure identify a 95-kDa band corresponding to the receptor ß-subunit. Equal amounts of WGA-purified protein were used in the four groups. The lower part of the figure shows the absolute 32P-incorporation into the insulin receptor. Autoradiographs were quantified by scanning densitometry, and data were corrected according to the IBA of each preparation. 32Pi-incorporation was expressed as percentage of maximal phosphorylation, which was always observed in the presence of 160 nM insulin, with receptors obtained from glucose-infused pregnant rats. Data are mean ± SEM of three experiments.

 
To determine whether pregnancy and/or hyperinsulinemia are accompanied by changes in the insulin-responsiveness of the kinase activity of the mammary gland insulin receptor, phosphorylation experiments were carried out in the presence of increasing concentrations of insulin (0–160 nM), and the degree of autophosphorylation of the receptor was quantified (by scanning densitometry of the obtained autoradiographs) and normalized to IBA. The dose-response curves (Fig. 3Go) show that the insulin concentrations required for half-maximal stimulation of receptor autophosphorylation were virtually identical (ED50 ~ 15 nM) under the various experimental conditions, i.e. virgin and pregnant rats under control or glucose-infused conditions. These curves clearly show that the efficacy of insulin to stimulate the kinase activity of the mammary gland insulin receptor is markedly higher in pregnant than in virgin rats and that the highest insulin-dependent phosphorylation was detected in the glucose-infused pregnant rat group (Fig. 3Go).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 3. Insulin dose-response curves for stimulation of receptor ß-subunit autophosphorylation. Autoradiographs like those in Fig. 2Go, but using different insulin concentrations, were quantified by scanning densitometry, and data were corrected according to the IBA of each preparation. Basal values of phosphorylation are considered 0%. Curves show the insulin-dependent phosphorylation (above basal), and data are represented as a percentage of the maximum insulin effect observed with glucose-infused pregnant-rat receptors. Insulin concentrations required for half-maximal stimulation (ED50) are shown by arrows. Values are mean ± SEM of three experiments. {triangleup}, Glucose infused pregnant rats; {blacktriangleup}, pregnant control rats; {circ}, glucose-infused virgin rats; •, virgin control rats.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The model of the euglycemic-hyperinsulinemic rat, obtained by 50% glucose infusion for 3 days, was used to study mammary gland insulin sensitivity during pregnancy and to shed light on the molecular mechanism underlying the responsiveness of this tissue to insulin. It was found here, in agreement with previous reports (19), that sustained euglycemia-hyperinsulinemia decreases overall insulin sensitivity in virgin rats and reverts the insulin resistance condition (19) normally present in pregnant animals (5, 6, 31, 32). Furthermore, the present study shows, for the first time, that in pregnant rats, mammary gland displays high insulin-dependent glucose uptake under hyperinsulinemia, caused by either the euglycemic-hyperinsulinemic clamp or by the continuous glucose infusion. In contrast, glucose utilization by the glands from nonpregnant rats is not affected under the same conditions. The increased insulin responsiveness in mammary glands of pregnant rats may be accounted for by changes in the binding characteristics of the insulin receptor, by an altered kinase activity and/or phosphorylation status of the receptor, or by postreceptor events. The data obtained from radioligand binding studies argue against changes in the hormone-receptor interaction in late pregnancy or in response to hyperinsulinemia, because both maximal insulin binding and affinity to both high- and low-affinity sites remain unaffected. These data agree with previous reports (33, 34), showing that the number of insulin receptors expressed per amount of protein in wheat-germ agglutinin eluates is similar in mammary gland tissue of untreated virgin and pregnant rats, without relevant differences in affinity values between the partially purified insulin receptors from both groups. Thus, similar to the situation found in liver (29), the moderate hyperinsulinemia characteristic of pregnancy apparently does not result in a down-regulation of insulin binding in mammary gland.

In various physiological and pathological situations, insulin resistance has been associated with changes in insulin receptor function (for review, see Ref.35) and, more specifically, to an impaired tyrosine kinase activity of the receptor (for review, see Refs. 36, 28, and 29) or with the existence of postreceptor defects (12). Supporting the hypothesis that changes in the insulin receptor kinase activity also may account for the increased insulin sensitivity of mammary gland during late pregnancy, it was found here that insulin-induced autophosphorylation of the receptor ß-subunit of pregnant rats was approximately 6-fold higher than in virgin animals. In addition, basal autophosphorylation of the insulin receptor, determined in the absence of insulin, was also markedly higher in mammary glands from pregnant rats than in those from virgin animals. These differences in basal autophosphorylation may be caused by changes in the receptor structure or in phosphatase levels. However, this increased basal autophosphorylation does not modify the glucose uptake in the pregnant mammary gland, as compared with the nonpregnant tissue. Thus, it seems that the differences in the basal phosphorylation are physiologically irrelevant. Dose-response curves confirmed that the concentration of insulin giving the half-maximal stimulation of autophosphorylation (ED50 ~15 nM) of the insulin receptor in mammary gland was similar in the virgin and pregnant groups. These results indicate that the increased kinase activity in pregnant rats is mainly caused by an enhanced responsiveness to insulin and not to an altered sensitivity to the hormone.

The hyperinsulinemic condition caused by the prolonged glucose infusion further impairs insulin stimulation of receptor ß-subunit phosphorylation in virgin rats. Incubation of adipocytes with high concentrations of glucose has been reported to inhibit insulin receptor kinase activity (37). Because adipocytes are the predominant cells in the mammary gland of virgin rats (14, 15), it may be proposed that, although glucose homeostasis is maintained during the glucose infusion, the increased availability of glucose might induce an inhibitory mechanism, impairing insulin receptor autophosphorylation in the mammary gland of virgin rats.

It could be argued that the observed differences in insulin sensitivity of mammary gland between pregnant and virgin rats are mainly caused by the insulin-sensitive epithelial cells, which are the major cellular type in the mature gland of the pregnant rat. However, it is known that lipoprotein lipase activity is associated with adipose cells in the mature mammary gland (15, 18) and becomes extremely sensitive to hyperinsulinemia in both pregnant (17) and lactating rats (10, 38). This, therefore, suggests that the enhanced insulin sensitivity of mammary gland tissue seen in late pregnancy results from the increased activity of insulin receptors from both adipose and epithelial cells. Furthermore, because adipose tissue is one of the most insulin-sensitive tissues, the absence of insulin response in virgin rat mammary gland (which is formed mainly by adipose cells) can be explained only by postulating the existence of mammary gland-specific mechanisms that inhibit the kinase activity of insulin receptors in virgin, but not in pregnant or lactating, animals.

In conclusion, present findings indicate that, opposite to the insulin resistance present in most tissues of late-pregnant rats, mammary glands exhibit increased insulin sensitivity. Furthermore, we provide evidence that the increased response to insulin of the mature mammary gland in late pregnancy is not accounted for by changes at the level of the hormone-receptor interaction but by up-regulation of insulin receptor kinase activity.


   Acknowledgments
 
The authors thank Beatriz Ramos for her editorial help.


   Footnotes
 
1 This study was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (PM 95–0044-002–01). The CBM-CSIC is recipient of an institutional grant from Fundación Ramón Areces. Back

Received June 19, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ryan EA, O’Sullivan MJ, Skyler JS 1985 Insulin action during pregnancy. Studies with the euglycemic clamp technique. Diabetes 34:380–389[Abstract]
  2. Freinkel N 1980 Banting lecture 1980. Of pregnancy and progeny. Diabetes 29:1023–1035[Abstract]
  3. Catalano PM, Tyzbir ED, Wolfe RR, Calles J, Roman NM, Amini SB, Sims EAH 1993 Carbohydrate metabolism during pregnancy in control subjects and women with gestational diabetes. Am J Physiol 264:E60–E67
  4. Leturque A, Ferré P, Burnol A-F, Kande J, Maulard P, Girard J 1986 Glucose utilization rates and insulin sensitivity in vivo in tissues of virgin and pregnant rats. Diabetes 35:172–177[Abstract]
  5. Leturque A, Burnol A-F, Ferré P, Girard J 1984 Pregnancy-induced insulin resistance in the rat: assessment by glucose clamp technique. Am J Physiol 246:E25–E31
  6. Knopp RH, Ruder HJ, Herrera E, Freinkel N 1970 Carbohydrate metabolism in pregnancy VII. Insulin tolerance during late pregnancy in the fed and fasted rat. Acta Endocrinol (Copenh) 65:352–360[Abstract/Free Full Text]
  7. Ciaraldi TP, Kettel M, El-Roeiy A, Madar Z, Reichart D, Yen SSC, Olefsky JM 1994 Mechanisms of cellular insulin resistance in human pregnancy. Am J Obstet Gynecol 170:635–641[Medline]
  8. Camps M, Gumá A, Testar X, Palacín M, Zorzano A 1990 Insulin resistance of skeletal muscle during pregnancy is not a consequence of intrinsic modifications of insulin receptor binding or kinase activities. Endocrinology 127:2561–2570[Abstract]
  9. Rushakoff RJ, Kalkhoff RK 1981 Effects of pregnancy and sex steroid administration on skeletal muscle metabolism in the rat. Diabetes 30:545–550[Medline]
  10. Da Costa THM, Williamson DH 1993 Effects of exogenous insulin or vanadate on disposal of dietary triacylglycerols between mammary gland and adipose tissue in the lactating rat: insulin resistance in white adipose tissue. Biochem J 290:557–561
  11. Burnol A-F, Ferré P, Leturque A, Girard J 1987 Effect of insulin on in vivo glucose utilization in individual tissues of anesthetized lactating rats. Am J Physiol 252:E183–E188
  12. Burnol A-F, Loizeau M, Girard J 1990 Insulin receptor activity and insulin sensitivity in mammary gland of lactating rats. Am J Physiol 259:E828–E834
  13. Vonderhaar BK, Ziska SE 1989 Hormonal regulation of milk protein gene expression. Annu Rev Physiol 51:641–652[CrossRef][Medline]
  14. Camps M, Vilaró S, Testar X, Palacín M, Zorzano A 1994 High and polarized expression of GLUT1 glucose transporters in epithelial cells from mammary gland: acute down-regulation of GLUT1 carriers by weaning. Endocrinology 134:924–934[Abstract]
  15. Jensen DR, Bessesen DH, Etienne J, Eckel RH, Neville MC 1991 Distribution and source of lipoprotein lipase in mouse mammary gland. J Lipid Res 32:733–742[Abstract]
  16. Burnol A-F, Leturque A, Loizeau M, Postic C, Girard J 1990 Glucose transporter expression in rat mammary gland. Biochem J 270:277–279[Medline]
  17. Ramos P, Herrera E 1996 Comparative responsiveness to prolonged hyperinsulinemia between adipose-tissue and mammary gland lipoprotein lipase activities in pregnant rats. Early Pregnancy. Biol Med 2:29–35
  18. Jensen DR, Gavigan S, Sawicki V, Witsell DL, Eckel RH, Neville MC 1994 Regulation of lipoprotein lipase activity and mRNA in the mammary gland of the lactating mouse. Biochem J 298:321–327
  19. Ramos P, Herrera E 1995 Reversion of insulin resistance in the rat during late pregnancy by 72-h glucose infusion. Am J Physiol 269:E858–E863
  20. Ramos P, Herrera E 1995 Effect of prolonged glucose infusion on insulin sensitivity in the conscious normal rat. Horm Metab Res 27:197–200[Medline]
  21. Hugget ASG, Nixon DA 1957 Use of glucose oxidase, peroxidase and O-dianisidine in determination of blood and urinary glucose. Lancet 1:368–370
  22. Heding LG 1972 Determination of total serum insulin (IRI) in insulin-treated diabetic patients. Diabetologia 8:260–266[CrossRef][Medline]
  23. Ader M, Bergman RN 1987 Insulin sensitivity in the intact organism. Baillieres Clin Endocrinol Metab 1:879–910[CrossRef][Medline]
  24. Sokoloff L, Reivich M, Kennedy C, Des Rosiers MH, Patlak CS, Pettigrew KD, Sakurada O, Shinohara M 1977 The [14C]Deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized rat. J Neurochem 28:897–916[Medline]
  25. Somogyi M 1945 Determination of blood sugar. J Biol Chem 160:69–73[Free Full Text]
  26. Ferré P, Leturque A, Burnol A-F, Penicaud L, Girard J 1985 A method to quantify glucose utilization in vivo in skeletal muscle and white adipose tissue of the anaesthetized rat. Biochem J 228:103–110[Medline]
  27. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  28. Carrascosa JM, Ruiz P, Martinez C, Pulido JA, Satrustegui J, Andrés A 1989 Insulin receptor kinase activity in rat adipocytes is decreased during aging. Biochem Biophys Res Commun 160:303–309[CrossRef][Medline]
  29. Martinez C, Ruiz P, Andrés A, Satrustegui J, Carrascosa JM 1989 Tyrosine kinase activity of liver insulin receptor is inhibited in rats at term gestation. Biochem J 263:267–272[Medline]
  30. Carrascosa JM, Schleicher E, Maier R, Hackeuberg C, Wieland OH 1988 Separation of the protein-tyrosine kinase and phosphatidylinositol kinase activities of the human placental insulin receptor. Biochim Biophys Acta 971:170–178[Medline]
  31. Leturque A, Ferré P, Satabin P, Kervran A, Girard J 1980 In vivo insulin resistance during pregnancy in the rat. Diabetologia 19:521–528[Medline]
  32. Martín A, Zorzano A, Caruncho I, Herrera E 1986 Glucose tolerance tests and "in vivo" response to intravenous insulin in the unanaesthesized late pregnant rat and their consequences to the fetus. Diabete Metab 12:302–307[Medline]
  33. O’Keefe E, Cuatrecasas P 1974 Insulin receptors in murine mammary cells: comparison in pregnant and nonpregnant animals. Biochim Biophys Acta 343:64–77[Medline]
  34. Inagaki Y, Kohmoto K 1982 Changes in Scatchard plots for insulin binding to mammary epithelial cells from cycling, pregnant, and lactating mice. Endocrinology 110:176–182[Abstract]
  35. Zick Y 1989 The insulin receptor: structure and function. Crit Rev Biochem Mol Biol 24:217–269
  36. Klip A, Douen AG 1989 Role of kinases in insulin stimulation of glucose transport. J Membr Biol 111:1–23[Medline]
  37. Müller HK, Kellerer M, Ermel B, Muhlhofer A, Obermaier-Kusser B, Vogt B, Häring HU 1991 Prevention by protein kinase C inhibitors of glucose-induced insulin-receptor tyrosine kinase resistance in rat fat cells. Diabetes 40:1440–1448[Abstract]
  38. Da Costa THM, Williamson DH 1994 Regulation of rat mammary-gland uptake of orally administered [1-14C]triolein by insulin and prolactin: evidence for bihormonal control of lipoprotein lipase activity. Biochem J 300:257–262



This article has been cited by other articles:


Home page
 EndocrinologyHome page
J. Sevillano, J. de Castro, C. Bocos, E. Herrera, and M. P. Ramos
Role of Insulin Receptor Substrate-1 Serine 307 Phosphorylation and Adiponectin in Adipose Tissue Insulin Resistance in Late Pregnancy
Endocrinology, December 1, 2007; 148(12): 5933 - 5942.
[Abstract] [Full Text] [PDF]

Home page
 J ANIM SCIHome page
C. M. Murrieta, B. W. Hess, E. J. Scholljegerdes, T. E. Engle, K. L. Hossner, G. E. Moss, and D. C. Rule
Evaluation of milk somatic cells as a source of mRNA for study of lipogenesis in the mammary gland of lactating beef cows supplemented with dietary high-linoleate safflower seeds
J Anim Sci, September 1, 2006; 84(9): 2399 - 2405.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
A. Martin-Hidalgo, L. Huerta, N. Alvarez, G. Alegria, M. del Val Toledo, and E. Herrera
Expression, activity, and localization of hormone-sensitive lipase in rat mammary gland during pregnancy and lactation
J. Lipid Res., April 1, 2005; 46(4): 658 - 668.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. G. Bonnette and D. L. Hadsell
Targeted Disruption of the IGF-I Receptor Gene Decreases Cellular Proliferation in Mammary Terminal End Buds
Endocrinology, November 1, 2001; 142(11): 4937 - 4945.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
P. Ramos, A. MartÍn-Hidalgo, and E. Herrera
Insulin-Induced Up-Regulation of Lipoprotein Lipase Messenger Ribonucleic Acid and Activity in Mammary Gland
Endocrinology, March 1, 1999; 140(3): 1089 - 1093.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carrascosa, J. M.
Right arrow Articles by Herrera, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carrascosa, J. M.
Right arrow Articles by Herrera, E.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals