Effect of the ovarian hormones on GLUT4 expression and contraction-stimulated glucose uptake

S. E. Campbell and M. A. Febbraio

Department of Physiology, The University of Melbourne, Parkville, Victoria 3010, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study examined the roles of the female sex steroids, 17beta -estradiol (E2) and progesterone (Prog), on glucose uptake and GLUT4 protein expression. Female Sprague-Dawley rats were either sham operated (C) or ovariectomized and treated with placebo (O), E2 (E), Prog (P), or both hormones at physiological doses (P + E) or the same dose of Prog with a high dose of E2 (P + HiE) via timed-release pellets inserted at the time of surgery, 15 days before metabolic testing. On the morning of day 15, animals received a 300-µCi injection (ip) of 2-deoxy-[14C]glucose and then either exercised on a motorized treadmill for 30 min at 0.35 m/s or remained sedentary in their cages for the same period. Basal glucose uptake was not different between the treatment groups in either the red or white quadriceps. However, glucose uptake was decreased (P < 0.05) in O, P, and P + E rats during exercise in the red quadriceps compared with C rats, whereas E and P + HiE treatment restored glucose uptake. Glycogen content in skeletal muscle followed similar trends, with no differences seen in resting animals. Postexercise red quadriceps glycogen levels were higher (P < 0.05) in the E and P + HiE rats compared with O and P. Treatment of ovariectomized rats with progesterone (P rats) decreased (P < 0.05) GLUT4 content in the red quadriceps by 21% compared with C rats. These data demonstrate that estrogen-deficient animals have a decreased ability for contraction-stimulated glucose uptake and increased glycogen use during aerobic exercise. However, changes in contraction-stimulated glucose uptake could not be explained by altered transporter protein content, since the absence of E2 had no effect on GLUT4 protein.

ovariectomy; estrogen; progesterone; carbohydrate metabolism


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ALTHOUGH THE OVARIAN HORMONES operate primarily in reproduction, they are also known to influence glucose homeostasis. Altered postprandial glucose tolerance has been correlated with natural fluctuations in the ovarian hormones throughout the menstrual cycle (7, 9), during pregnancy (31), and after menopause (3). In addition, insulin-stimulated glucose uptake is often (6, 35) but not always (34, 40) impaired during the luteal phase of the menstrual cycle. Progesterone is thought to be at least partially responsible for insulin resistance during pregnancy and could possibly contribute to the onset of gestational diabetes mellitus (21, 32). Although the association between insulin resistance and the expression of the insulin-responsive glucose transporter (GLUT4) is well established (1, 18, 20, 33), it is somewhat surprising that no studies have examined the effect of the ovarian hormones on GLUT4 protein expression in insulin-responsive tissue.

Muscle contraction is a far more potent stimulus for glucose uptake than insulin (28). Although it is well known that the mechanisms for insulin- and contraction-mediated glucose uptake are quite different (16), exercise may, nonetheless, provide an excellent model for studying the effects of ovarian hormones on glucose disposal, because it places a major metabolic stress on the contracting skeletal muscle. Surprisingly, few studies have investigated such an effect. Zderic et al. (41) recently demonstrated that glucose disposal is decreased during exercise in the luteal compared with the follicular phase in healthy women. However, because of changes in both the absolute and relative concentrations of the ovarian hormones throughout the menstrual cycle, it is difficult to ascertain the individual effects of estrogen and progesterone from human studies. In rodents, in vitro electrical stimulation of glucose uptake was decreased by ovariectomy compared with intact animals (13), but because the sex steroids were not replaced in this study, it is unknown whether the effect was due to the absence of estrogen or progesterone.

Therefore, the purpose of this study was twofold. First, we sought to examine whether the ovarian hormones alter GLUT4 protein content in insulin-sensitive tissue. Second, we sought to determine whether basal and contraction-stimulated glucose uptake in skeletal muscle was influenced by the ovarian hormones. We hypothesized that GLUT4 protein content would be decreased by ovariectomy, resulting in a concomitant attenuation in glucose uptake in contracting muscle. Furthermore, we hypothesized that estrogen, but not progesterone, treatment would restore GLUT4 and glucose uptake to normal levels.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Female Sprague-Dawley rats 12-15 wk old, weighing 203.1 ± 4.5 (SE) g, were used in these experiments. All animals were housed in a temperature-controlled room (21 ± 2°C) with a 12:12-h light-dark cycle. Water was available ad libitum, and rats were given 20 g standard rat chow/day to control food intake. The amount of rat chow administered daily was selected based on pilot work from our laboratory, which demonstrated that food intake of 10% body weight was sufficient to maintain body weight over the duration of the experiment. This experiment was approved by the Animal Research Ethics Committee of the University of Melbourne.

Experimental design. Rats were bilaterally ovariectomized or sham operated under sodium brietal anesthesia (60 mg/kg ip). Groups of ovariectomized rats were treated immediately with timed-release hormone pellets (Innovative Research of America) inserted subcutaneously with either 17beta -estradiol (E2; 2.5 µg/day; E group), progesterone (Prog; 1.5 mg/day; P group), both hormones at the same doses (P + E), or both hormones with the same dose of Prog but pharmacological levels of E2 (25 µg/day; P + HiE). Groups of intact and ovariectomized rats were treated with vehicle-only placebo pellets (C and O, respectively). Animals were treated for 14 days postoperation before metabolic testing, which occurred on day 15. Efficacy of the ovariectomies and sex steroid treatments were confirmed by plasma E2 and Prog RIA kits (Diagnostic Products) at the end of the treatment period. Animals were randomly subdivided into either exercise or resting groups (n = 5 in each group).

Metabolic testing. Animals remained sedentary during the 14-day treatment period. Rats were fasted for 12 h before commencement of metabolic testing. On the morning of day 15, all animals received a 300-µCi injection (ip) of 2-deoxy-[14C]glucose (2-DG). Exercising rats were then run at 0.35 m/s for 30 min on a custom-made motorized treadmill, and resting rats remained inactive in their cages for 30 min. Immediately after removal from the treadmill or cage, animals were suffocated with CO2 (80:20 CO2-O2), rendering unconsciousness in <20 s. A midline incision was made, and the diaphragm was cut to ensure death. A cardiac puncture was performed, the blood was spun at 7,000 g for 2 min, and the plasma was removed and stored at -80°C for analysis of ovarian hormones, insulin, glucose, and lactate. The muscles of the hindlimb were exposed rapidly, the quadriceps was removed and dissected into white and red portions, and all visible connective tissue was removed. The liver and white adipose tissue (WAT) were also removed. All tissues were then snap-frozen in liquid nitrogen and stored at -80°C for later analysis.

GLUT4 protein. Samples of red and white quadriceps and WAT were thawed in homogenizing buffer (pH 7, 0.5 ml/20 mg tissue) containing 10 mM Tris buffer, 1 mM EDTA, 250 mM sucrose, and 1 M phenylmethylsulfonyl fluoride and were then minced and homogenized with a Polytron PT 3100 (Kinematica, Lucerne, Switzerland) at a setting of 12 for 2 × 15 s. Homogenates were spun at 1,000 g for 5 min at 4°C, and the supernatant was stored on ice. The pellet was resuspended in homogenizing buffer and was subjected to repeated homogenization and centrifugation as described above. The supernatant from this second spin was combined with that of the previous spin and spun again at 150,000 g for 60 min at 4°C. The resulting crude membrane pellet was resuspended in 50-100 µl of homogenizing buffer and stored at -80°C. The total protein content of the crude membrane suspension was determined using a commercially available protein assay kit (Bio-Rad, Richmond, CA). Crude membrane suspensions containing 10 µg of protein were then solubolized with an equal volume of Laemmli sample buffer and incubated for 1 h at 37°C. Samples were separated by SDS-PAGE on 7.5% polyacrylamide gels and were eletrophoretically transferred to polyvinylidene difluoride (PVDF) filter membranes for 1.5 h. PVDF membranes were blocked for 1 h at room temperature with 5% skim milk in Tris-buffered saline (50 mM Tris · HCl, 150 mM NaCl, 0.04% Igepal Ca 630, and 0.02% Tween 20; TBS) containing 3% BSA (fatty acid-free BSA; pH 7.4; Sigma-Aldrich) and were then incubated overnight at 4°C with the polyclonal antibody sc-1608 (Santa Cruz) diluted 1:2,000 with TBS containing 1% BSA. Membranes were then washed (3 × 15 min, 37°C) followed by a 1-h incubation with anti-goat IgG conjugated to horseradish peroxidase (sc-2020; Santa Cruz) in TBS containing 1% BSA and then washed again (3 × 15 min, 37°C). Immunoreactive bands were detected using enhanced chemiluminescence. A molecular weight marker, used to determine the relative molecular weight of labeled bands, and a muscle standard (an unrelated crude membrane suspension), for comparison between different immunoblots, was run on every gel. The resulting autoradiographs were analyzed by laser-scanning densitometry (Bio-Rad GS710 Calibrated Imaging Densitometer) and quantitated with QuantityOne (version 4.1).

Glucose uptake. Portions of red and white quadriceps muscle were weighed and digested in 1 ml of 1 M NaOH in a shaking water bath at 60°C for 1 h, followed by neutralization with 1 ml of 1 M HCl. The digest was then separated for two different treatments. In the first treatment, 500 µl of digest were deproteinized in equal volumes of Ba(OH)2 and ZnSO4 and then mixed and spun at 8,500 g for 5 min. The supernatant (1 ml) was recovered and added to 3 ml of water and 10 ml of liquid scintillation cocktail. In the second treatment, 500 µl of digest were deproteinized in 2 ml of 6% perchloric acid, mixed, and spun at 8,500 g for 10 min. The supernatant (2 ml) was recovered and added to 2 ml of water and 10 ml of liquid scintillation cocktail. Radioactivity of both treatments was measured by a beta -scintillation counter. The first treatment yielded unphosphorylated 2-DG, and the second treatment measured total 2-DG; hence, the difference gave phosphorylated 2-DG.

Tissue glycogen. Muscle samples to be analyzed for glycogen content were freeze-dried, weighed, powdered, and extracted in 250 µl of 2 M HCl at 100°C for 2 h and then neutralized with 750 µl of 0.667 M NaOH. The extract was then analyzed for glucose, as has been described previously (26). Liver samples to be analyzed for glycogen content were weighed and extracted in 70% perchloric acid and then assayed for glycogen by the amyloglucosidase method (26). Previous work from our laboratory has demonstrated that this latter method is a more sensitive and specific analysis for liver glycogen compared with the former, which is specific for skeletal muscle.

Blood hormone and metabolite assays. Plasma E2, Prog, and insulin were measured by commercially available double-antibody RIA kits (Diagnostic Products and Pharmacia & Upjohn). Plasma glucose and lactate were analyzed using an automated method (Electrolyte Metabolite Laboratory; Radiometer, Copenhagen, Denmark).

Statistics. All statistical comparisons were made using one- or two-way ANOVA, as appropriate, with significance set at the P < 0.05 level. Specific differences were located with a Student-Newman-Keul's F-test post hoc comparison. All data statistics were compared using the Statistica software package (Statsoft, Tulsa, OK), and data are reported as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The animal characteristics are presented in Table 1. Plasma E2 and Prog concentrations confirmed the efficacy of the ovariectomies and sex steroid treatments. No significant differences were found among the mean values of either initial or final body weight. Because no differences were found in either the sex steroid levels or weights between resting and exercised animals, these data were pooled.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Animal characteristics

The plasma glucose, lactate, and insulin data are presented in Fig. 1. There were no differences in plasma glucose or lactate values among the six groups of rats at either rest or after exercise. As expected, however, both glucose and lactate were elevated (P < 0.05) when comparing exercised with rested animals. Plasma insulin values were elevated (P < 0.05) in the P rats, both at rest and during exercise, compared with C, E, P + E, and P + HiE rats. As expected, there was also a decrease (P < 0.05) when exercised and rested animals were compared.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   Plasma glucose (A), lactate (B), and insulin (C) levels in intact and ovariectomized animals treated with sex steroids after 30 min of rest or exercise. * Main effect (P < 0.05) of exercise compared with rest. P < 0.05 compared with intact placebo (C; a), ovariectomized placebo (O; b), ovariectomized 17beta -estradiol (E2; E group; c); ovariectomized progesterone (Prog; P group; d), ovariectomized E2 + Prog (P + E; e), and ovariectomized E2 (pharmacological) + Prog (P + HiE; f).

Glucose uptake (Fig. 2) in the red quadriceps was similar among all treatment groups at rest. During exercise, however, there was a decrease (P < 0.05) in the O, P, and P + E rats compared with C rats. Treatment with E2 (E rats) returned glucose uptake to normal, and treatment with a high dose of E2 was able to compensate for the inhibitory effect of Prog on physiological concentrations of E2 (P + HiE rats), as these values were also similar to those in C. There were no differences in glucose uptake in the white quadriceps either at rest or during exercise.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   2-Deoxy-[14C]glucose (2-DG) uptake in red (A) and white (B) quadriceps muscles after 30 min of rest or exercise. * Main effect (P < 0.05) of exercise compared with rest. P < 0.05 compared with C (a), O (b), E (c); P (d),P + E (e), and P + HiE (f). dpm, Disintegrations/min.

Tissue glycogen concentrations are presented in Fig. 3. Glycogen content at rest was not different among the treatment groups in either red or white quadriceps. As expected, exercise decreased (P < 0.05) glycogen levels in both tissues in all treatment groups. Although ovariectomy did not decrease postexercise glycogen content compared with C rats, it did decrease (P < 0.05) glycogen content compared with E and P + HiE animals. Prog-treated rats also had less (P < 0.05) glycogen compared with E and P + HiE animals after exercise. Although P + E was not different from E, there was a trend (P = 0.055) for glycogen content to be lower after exercise. There were no differences among any of the treatment groups in glycogen content in the white quadriceps muscle. Correlations performed on glucose uptake and glycogen content in the red quadriceps, postexercise, demonstrate a positive correlation (P < 0.05) in each group. Trend lines illustrate that most groups had either a significant or a borderline significant relation between glucose uptake and glycogen content postexercise; r and P values for individual trend lines are presented in Table 2.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Glycogen content in red (A) and white (B) quadriceps muscles and liver (C) after 30 min of rest or exercise. dm, Dry mass; wm, wet mass. * Main effect (P < 0.05) of exercise compared with rest. P < 0.05 compared with C (a), O (b), E (c); P (d), P + E (e), and P + HiE (f).


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Trendline r and P values

There was a mean treatment effect for resting liver glycogen (Fig. 3), with E and P + HiE having higher (P < 0.05) glycogen stores than O and P. This effect was no longer seen postexercise, when hepatic glycogen stores were significantly reduced in all treatment groups.

Ovariectomy (O rats) did not decrease total GLUT4 protein content compared with C rats in any of the tissues. In contrast, treatment of ovariectomized rats with Prog (P rats) decreased GLUT4 content by 28 and 26% in WAT compared with C and O rats, respectively (P < 0.01). P rats also had decreased GLUT4 content in the red quadriceps (21%, P < 0.05) compared with C rats. There was a similar decrease compared with O rats (18%); however, this did not reach statistical significance (P = 0.054). These effects were not seen in any other treatment groups in these tissues, although there were trends for P + E rats to be decreased compared with C rats (WAT P = 0.07; red quadriceps P = 0.06). No differences were detected in white quadriceps between any treatment groups (Fig. 4).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   GLUT4 protein content in crude membranes (2 representative blots/treatment group) from red (A) and white (B) quadriceps skeletal muscle and white adipose tissue (C) of experimental animals; n = 10 experiments/group. P < 0.05 compared with C (a), O (b), E (c); P (d), P + E (e), and P + HiE (f).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first study to examine the effects of ovariectomy and hormone replacement therapies on GLUT4 protein and contraction-stimulated glucose uptake in vivo. The results demonstrate that the ovarian hormones exert a significant effect on carbohydrate metabolism in contracting skeletal muscle. Estrogen-deficient animals demonstrated impaired contraction-mediated glucose uptake and increased glycogen depletion compared with both control and E2-treated animals. Of note, Prog inhibited the beneficial effect of E2 at physiological concentrations, since P + E rats also demonstrated impaired contraction-mediated glucose uptake and increased glycogen depletion compared with both control and E2-treated animals. The data also demonstrate that Prog treatment decreases GLUT4 protein in insulin-responsive tissue. However, changes in contraction-stimulated glucose uptake could not be explained by altered transporter protein content, since the absence of E2 had no effect on crude membrane GLUT4.

Previous studies using an ovariectomized model have observed that ovariectomy usually results in a relative body weight gain and that this is reversed by treatment with E2 (25, 36). This is thought to be a result of neuroendocrine responses that increase food intake and reduce physical activity (36). Although previous studies have found that either ovariectomy or hormone replacement can adversely effect glucose and/or insulin homeostasis (5, 25), neither study controlled for food intake. Hence, it was not possible to directly attribute the observed metabolic dysfunction to alterations in the ovarian hormone levels. To our knowledge, this is the first study that has controlled food intake for the duration of the treatment to eliminate the effect of weight gain on metabolic dysfunction. Despite the maintenance of body weight, Prog-treated animals still developed insulin resistance, because their plasma insulin concentration was higher in the presence of normal plasma glucose concentrations (Fig. 1), whereas ovariectomized animals also had slightly elevated insulin levels (P = 0.11 compared with C rats), indicating that the ovarian hormones have direct effects on insulin sensitivity.

Contraction-stimulated glucose uptake in red skeletal muscle was decreased in the absence of estrogen. This observation is consistent with previous studies that have observed that estrogen increases basal glucose uptake in rat skeletal muscle in vivo (25) and insulin-stimulated mouse skeletal muscle in vitro (29). Of note, in these previous studies, Prog treatment had no effect. In contrast, in the present study, Prog treatment not only impaired contraction-mediated glucose uptake when solely administered but impaired glucose uptake in rats treated with physiological concentrations of both E2 and Prog (P + E). However, this inhibition could be overridden by the presence of pharmacological concentrations of E2, as demonstrated with the normal glucose uptake in P + HiE rats. The results demonstrate, therefore, that the concerted effects of physiological concentrations of estrogen and Prog on contraction-stimulated glucose uptake are indeed complex, since E2 appeared to have a positive effect, only in the absence of Prog. Of note was the difference in the glucose uptake between the P + E group, which received physiological concentrations of both E2 and Prog, and the intact C rats. This may best be explained by the fact that the intact rats were cycling normally and were therefore exposed to elevated Prog levels (40-50 ng/ml) for only ~12 h during the proestrous phase of their 4- to 5-day cycle. Furthermore, control rats were killed at various times throughout the estrous cycle so that a more representative collection of data points could be obtained; therefore, these pooled results incorporate the changing levels of the ovarian hormones. Conversely, the P + E rats were exposed continuously to high levels of Prog; therefore, the P + E rats would have a greater tendency to exhibit any inhibitory characteristics of Prog.

The mechanism(s) for the actions of the ovarian hormones on glucose uptake is not readily apparent from the present data. By measuring the effects of the ovarian hormones on GLUT4, we sought to determine whether any alterations in glucose transport were attributable to transporter protein content. We observed that Prog treatment suppressed GLUT4 expression in red quadriceps and WAT, whereas a similar trend (WAT P = 0.07; red quadriceps P = 0.06) was observed in rats receiving both hormones at physiological levels (P + E). It is possible that the decrease in GLUT4 protein content in skeletal muscle could be the result of relative inactivity of P and P + E rats compared with E rats, since a decrease in physical activity has previously been demonstrated to negatively affect GLUT4 protein content (24). However, the decrease in GLUT4 content in WAT was similar, and often greater, indicating that the effect of Prog is independent of the relative physical activities of the rats (Fig. 4). Although our observations with respect to GLUT4 are novel and important, we cannot attribute alterations in glucose transport to changes in GLUT4 protein levels, since ovariectomy markedly decreased contraction-mediated glucose uptake but had no effect on GLUT4 protein content in red quadriceps muscle. It is possible, therefore, that the ovarian hormones may influence signaling molecules and/or GLUT4 trafficking. In the present study, we were not able to measure GLUT4 protein content in subcellular and/or plasma membrane fractions of the muscle samples because of insufficient muscle sample size to complete all analyses. Our GLUT4 measures made on total crude membrane fractions can neither confirm nor refute the possibility that the ovarian hormones may affect GLUT4 translocation from the subcellular pools to the plasma membrane. Indeed, there a paucity of data that have examined such an effect, and further research in this area is warranted. Of note, however, Weiner et al. (37) have demonstrated that estrogen increases calcium-dependent nitric oxide synthase activity in skeletal muscle. This may offer a possible mechanism for the effects of the ovarian hormones on contraction-mediated glucose uptake, since nitric oxide has been implicated in augmenting glucose uptake in both rat (8) and human (2) skeletal muscle. Further research that examines the effect of the ovarian hormones on GLUT4 translocation during muscle contraction may shed further light on the current observations.

It is also possible that the ovarian hormones primarily affected lipid metabolism and that the changes we observed in carbohydrate metabolism were secondary to changes in fat metabolism. If this was the case, one would expect lipid metabolism to be increased with ovariectomy, since glucose uptake was reduced in O compared with C animals. To the contrary, we have recently observed that carnitine palmitoyltransferase I and beta -3-hyroxyacyl-CoA dehydrogenase activity, enzymes representative of fatty acid flux into the mitochondria and beta -oxidation, respectively, are also reduced with overiectomy (unpublished observation). Therefore, it appears that the effect of the ovarian hormones on carbohydrate metabolism is unlikely to be secondary to altered fat metabolism.

Glycogen levels in the red quadriceps were also different postexercise between the treatment groups, with E and P + HiE rats having higher glycogen content postexercise compared with O and P rats. Interestingly, these two groups also had higher glucose uptake, suggesting that the increase in glucose uptake was enough to result in a glycogen-sparing effect. There was a trend for a positive relation between glucose uptake and glycogen content in all groups (Fig. 5). If these data were pooled across groups, this relation was statistically significant (r = 0.876; P < 0.01), suggesting that the differences in glucose uptake between the animals might explain a significant portion (76%) of the variability in postexercise glycogen concentrations. It is, however, interesting to observe that P rats, the group with the weakest correlation, were also the group that exhibited insulin resistance. This suggests that these animals were perhaps less able to match glucose uptake to cellular carbohydrate metabolism. It is important to note that an increase in glucose uptake does not necessarily result in glycogen sparing in humans (10). This may be because rats can oxidize more glucose compared with humans (15, 38), an obvious interspecies difference.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5.   Correlation between 2-DG uptake and glycogen content in the red quadriceps muscle after 30 min of exercise.

The observation of differing hepatic glycogen content when comparing the treatment groups in the rested animals is consistent with previous studies that have found increased glycogen storage in E2-treated animals, E and P + HiE groups (4, 23). This is likely because of increased glycogen synthase activity (29). The results do, however, differ in that the P and P + E rats had lower hepatic glycogen, where previously Prog has been found to increase glycogen storage (19, 23). This is perhaps because we controlled for food intake and therefore limited the opportunity for these animals to supercompensate their glycogen stores. The increase in glycogen content previously seen with Prog treatment was thought to be the result of hyperinsulinemia (19, 23). Insulin levels in this study were not as elevated as in previous studies, because we controlled for food intake, and this could also result in less glycogen storage. Interestingly, there was no longer a treatment effect on hepatic glycogen concentrations postexercise, suggesting an increase hepatic glycogenolysis in E and P + HiE rats compared with the other groups. Because there was a significant increase in glucose uptake in the E and P + HiE rats, with no corresponding change in plasma glucose values, it is likely that the hepatic glucose production was increased in response to increased peripheral demand.

Considerable evidence exists linking Prog to insulin resistance (7, 22, 31), but the precise mechanisms involved in this peripheral insulin resistance are not fully understood. Of note, however, insulin resistance has previously been linked to abnormalities in glucose uptake, often via alterations in GLUT4 (12, 20, 39). Reduced basal and insulin-stimulated glucose transport rates in isolated adipocytes have been associated with a marked depletion of GLUT4 protein in insulin-resistant patients (12). This was not seen in skeletal muscle (11, 27), since only the vastus lateralis and rectus abdominis have been studied. Differential regulation of GLUT4 in individual muscles has demonstrated that the vastus lateralis and rectus abdominis respond minimally to diabetes in rats, whereas soleus and heart muscles have a marked suppression in glucose disposal in the diabetic compared with baseline condition (14). Thus it remains possible that GLUT4 depletion could cause insulin resistance in muscles. Indeed, a recent study has demonstrated that, in the absence of muscle, GLUT4 results in severe insulin resistance (42). Although in the present study we observed a statistically significant suppression in the quadricep muscle, it was only in the red portion. When red and white muscles were pooled, the decrease in GLUT4 content was no longer significant. Type I fibers are more insulin sensitive, with a greater capacity for stimulated glucose uptake (17, 20, 32) and, hence, are perhaps more susceptible to insulin-resistant pathologies.

Of particular interest in these results is the inhibition of the beneficial effects of E2 by Prog at physiological concentrations, seen in glucose uptake. This inhibition was abolished with pharmacological concentrations of E2, demonstrating that, in high enough concentrations, E2 can override the inhibitory effect of Prog. This could have significant clinical effects when considering long-term treatment of amenorrheic or postmenopausal women with oral contraceptive or hormone replacement therapy. In these situations, it may be prudent to treat with an estrogen-based therapy vs. either Prog or combination therapies. Conversely, if a combination hormone replacement therapy is being considered, our data suggest the utilization of elevated levels of E2 to offset any inhibition by Prog. It is interesting to note that similar results have been found in clinical studies. Women using combination therapy demonstrated poorer glucose homeostasis and insulin sensitivity than those using estrogen alone (7, 22).

In conclusion, the absence of the ovarian hormones in female rats resulted in a decrease in glucose uptake and glycogen content postexercise in red skeletal muscle. Treatment with E2 restored these parameters to control levels, whereas treatment with Prog alone had no effect. These data suggest that estrogen deficiency results in a limited capacity for glucose uptake in times of metabolic stress, forcing the cell to seek an alternative fuel source. If ovariectomized animals are treated with both E2 and Prog in physiological concentrations, Prog inhibits the oxidative effect of E2, and this is only restored with pharmacological concentrations of E2. The data also demonstrate that Prog treatment decreases GLUT4 protein in insulin-responsive tissue but that changes in contraction-stimulated glucose uptake could not be explained by altered transporter protein content, since the absence of E2 had no effect on GLUT4 protein.


    ACKNOWLEDGEMENTS

We thank Dr. Anne Thorburn, Dr. Kirsten Howlett, Dr. Sofianos Andrikopolous, and Tamara Konopka for valuable advice and technical expertise.


    FOOTNOTES

Address for reprint requests and other correspondence: M. A. Febbraio, Dept. of Physiology, The Univ. of Melbourne, Parkville 3010, Australia (E-mail: m.febbraio{at}physiology.unimelb.edu.au).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpendo.00184.2001

Received 24 April 2001; accepted in final form 8 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Berger, J, Biswas C, Vicario PP, Strout HV, Saperstein R, and Pilch PF. Decreased expression of the insulin-responsive glucose transporter in diabetes and fasting. Nature 340: 70-72, 1989[ISI][Medline].

2.   Bradley, SJ, Kingwell BA, and McConell GK. Nitric oxide synthase inhibition reduces leg glucose uptake but not blood flow during dynamic exercise in humans. Diabetes 48: 1815-1821, 1999[Abstract].

3.   Cagnacci, A, Soldani R, Carriero PL, Paoletti AM, Fioretti P, and Melis GB. Effects of low doses of transdermal 17 beta-estradiol on carbohydrate metabolism in postmenopausal women. J Clin Endocrinol Metab 74: 1396-1400, 1992[Abstract].

4.   Carrington, LJ, and Bailey CJ. Effects of natural and synthetic estrogens and progestins on glycogen deposition in female mice. Horm Res 21: 199-203, 1985[ISI][Medline].

5.   Costrini, NV, and Kalkhoff RK. Relative effects of pregnancy, estradiol, and progesterone on plasma insulin and pancreatic islet insulin secretion. J Clin Invest 50: 992-999, 1971[ISI][Medline].

6.   Diamond, MP, Simonson DC, and DeFronzo RA. Menstrual cyclicity has a profound effect on glucose homeostasis. Fertil Steril 52: 204-208, 1989[ISI][Medline].

7.   Elkind Hirsch, KE, Sherman LD, and Malinak R. Hormone replacement therapy alters insulin sensitivity in young women with premature ovarian failure. J Clin Endocrinol Metab 76: 472-475, 1993[Abstract].

8.   Etgen, GJ, Jr, Fryburg DA, and Gibbs EM. Nitric oxide stimulates skeletal muscle glucose transport through a calcium/contraction and phosphatidylinositol-3-kinase-independent pathway. Diabetes 46: 1915-1919, 1997[Abstract].

9.   Ezenwaka, EC, Akanji AO, Adejuwon CA, Abbiyesuku FM, and Akinlade KS. Insulin responses following glucose administration in menstruating women. Int J Gynaecol Obstet 42: 155-159, 1993[ISI][Medline].

10.   Febbraio, MA, Chiu A, Angus DJ, Arkinstall MJ, and Hawley JA. Effects of carbohydrate ingestion before and during exercise on glucose kinetics and performance. J Appl Physiol 89: 2220-2226, 2000[Abstract/Free Full Text].

11.   Garvey, WT, Maianu L, Hancock JA, Golichowski AM, and Baron A. Gene expression of GLUT4 in skeletal muscle from insulin-resistant patients with obesity, IGT, GDM, and NIDDM. Diabetes 41: 465-475, 1992[Abstract].

12.   Garvey, WT, Maianu L, Huecksteadt TP, Birnbaum MJ, Molina JM, and Ciaraldi TP. Pretranslational suppression of a glucose transporter protein causes insulin resistance in adipocytes from patients with noninsulin-dependent diabetes mellitus and obesity. J Clin Invest 87: 1072-1081, 1991[ISI][Medline].

13.   Hansen, PA, McCarthy TJ, Pasia EN, Spina RJ, and Gulve EA. Effects of ovariectomy and exercise training on muscle GLUT-4 content and glucose metabolism in rats. J Appl Physiol 80: 1605-1611, 1996[Abstract/Free Full Text].

14.   Hardin, DS, Dominguez JH, and Garvey WT. Muscle group-specific regulation of GLUT 4 glucose transporters in control, diabetic, and insulin-treated diabetic rats. Metabolism 42: 1310-1315, 1993[ISI][Medline].

15.   Hargreaves, M, and Briggs CA. Effect of carbohydrate ingestion on exercise metabolism. J Appl Physiol 65: 1553-1555, 1988[Abstract/Free Full Text].

16.   Hayashi, T, Dufresne SD, Aronson D, Sherwood DJ, Hirshman MF, Boppart MD, Fielding RA, and Goodyear LJ. Intracellular signaling pathways in contracting skeletal muscle. In: Biochemistry of Exercise X, edited by Hargreaves M, and Thompson M.. Champaign, IL: Human Kinetics, 1999, p. 19-34.

17.   Henriksen, EJ, Bourey RE, Rodnick KJ, Koranyi L, Permutt MA, and Holloszy JO. Glucose transporter protein content and glucose transport capacity in rat skeletal muscles. Am J Physiol Endocrinol Metab 259: E593-E598, 1990[Abstract/Free Full Text].

18.   Kahn, BB, Charron MJ, Lodish HF, Cushman SW, and Flier JS. Differential regulation of two glucose transporters in adipose cells from diabetic and insulin-treated diabetic rats. J Clin Invest 84: 404-411, 1989[ISI][Medline].

19.   Kalkhoff, RK. Metabolic effects of progesterone. Am J Obstet Gynecol 142: 735-738, 1982[ISI][Medline].

20.   Kern, M, Wells JA, Stephens JM, Elton CW, Friedman JE, Tapscott EB, Pekala PH, and Dohm GL. Insulin responsiveness in skeletal muscle is determined by glucose transporter (Glut4) protein level. Biochem J 270: 397-400, 1990[ISI][Medline].

21.   Kuhl, C. Insulin secretion and insulin resistance in pregnancy and GDM. Implications for diagnosis and management. Diabetes 2: 18-24, 1991.

22.   Lindheim, SR, Presser SC, Ditkoff EC, Vijod MA, Stanczyk FZ, and Lobo RA. A possible bimodal effect of estrogen on insulin sensitivity in postmenopausal women and the attenuating effect of added progestin. Fertil Steril 60: 664-6677, 1993[ISI][Medline].

23.   Matute, ML, and Kalkhoff RK. Sex steroid influence on hepatic gluconeogenesis and glucogen formation. Endocrinology 92: 762-768, 1973[ISI][Medline].

24.   McCoy, M, Proietto J, and Hargreaves M. Effect of detraining on GLUT-4 protein in human skeletal muscle. J Appl Physiol 77: 1532-1536, 1994[Abstract/Free Full Text].

25.   Nolan, C, and Proietto J. The effects of oophorectomy and female sex steroids on glucose kinetics in the rat. Diabetes Res Clin Pract 30: 181-188, 1995[ISI][Medline].

26.   Passonneau, JV, and Lauderdale VR. A comparison of three methods of glycogen measurement in tissues. Anal Biochem 60: 405-412, 1974[ISI][Medline].

27.   Pedersen, O, Bak JF, Andersen PH, Lund S, Moller DE, Flier JS, and Kahn BB. Evidence against altered expression of GLUT1 or GLUT4 in skeletal muscle of patients with obesity or NIDDM. Diabetes 39: 865-870, 1990[Abstract].

28.   Ploug, T, Galbo H, Vinten J, Jorgensen M, and Richter EA. Kinetics of glucose transport in rat muscle: effects of insulin and contractions. Am J Physiol Endocrinol Metab 253: E12-E20, 1987[Abstract/Free Full Text].

29.   Puah, JA, and Bailey CJ. Effect of ovarian hormones on glucose metabolism in mouse skeletal muscle. Endocrinology 117: 1336-1340, 1985[Abstract].

30.   Richardson, JM, Balon TW, Treadway JL, and Pessin JE. Differential regulation of glucose transporter activity and expression in red and white skeletal muscle. J Biol Chem 266: 12690-12694, 1991[Abstract/Free Full Text].

31.   Rushakoff, RJ, and Kalkhoff RK. Effects of pregnancy and sex steroid administration on skeletal muscle metabolism in the rat. Diabetes 30: 545-550, 1981[ISI][Medline].

32.   Ryan, EA, and Enns L. Role of gestational hormones in the induction of insulin resistance. J Clin Endocrinol Metab 67: 341-347, 1988[Abstract].

33.   Sivitz, WI, DeSautel SL, Kayano T, Bell GI, and Pessin JE. Regulation of glucose transporter messenger RNA in insulin-deficient states. Nature 340: 72-74, 1989[ISI][Medline].

34.   Toth, EL, Suthijumroon A, Crockford PM, and Ryan EA. Insulin action does not change during the menstrual cycle in normal women. J Clin Endocrinol Metab 64: 74-80, 1987[Abstract].

35.   Valdes, CT, and Elkind-Hirsch KE. Intravenous glucose tolerance test-derived insulin sensitivity changes during the menstrual cycle. J Clin Endocrinol Metab 72: 642-646, 1991[Abstract].

36.   Wade, GN. Gonadal hormones and behavioral regulation of body weight. Physiol Behav 8: 523-534, 1972[ISI][Medline].

37.   Weiner, CP, Lizasoain I, Baylis SA, Knowles RG, Charles IG, and Moncada S. Induction of calcium-dependent nitric oxide synthases by sex hormones. Proc Natl Acad Sci USA 91: 5212-5216, 1994[Abstract].

38.   Winder, WW, Arogyasami J, Yang HT, Thompson KG, Nelson LA, Kelly KP, and Han DH. Effects of glucose infusion in exercising rats. J Appl Physiol 64: 2300-2305, 1988[Abstract/Free Full Text].

39.   Yamada, K, Yamakawa K, Terada Y, Kawaguchi K, Sugaya A, Sugiyama T, and Toyoda N. Expression of GLUT4 glucose transporter protein in adipose tissue and skeletal muscle from streptozotocin-induced diabetic pregnant rats. Horm Metab Res 31: 508-513, 1999[ISI][Medline].

40.   Yki-Jarvinen, H. Insulin sensitivity during the menstrual cycle. J Clin Endocrinol Metab 59: 350-353, 1984[Abstract].

41.   Zderic, TW, Coggan AR, and Ruby BC. Glucose kinetics and substrate oxidation during exercise in the follicular and luteal phases. J Appl Physiol 90: 447-453, 2001[Abstract/Free Full Text].

42.   Zisman, A, Peroni OD, Abel ED, Michael MD, Mauvais-Jarvis F, Lowell BB, Wojtaszewski JF, Hirshman MF, Virkamaki A, Goodyear LJ, Kahn CR, and Kahn BB. Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance. Nat Med 6: 924-928, 2000[ISI][Medline].


Am J Physiol Endocrinol Metab 282(5):E1139-E1146
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society