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Endocrinology Vol. 145, No. 3 1161-1166
Copyright © 2004 by The Endocrine Society

Glucocorticoids Differentially Modulate Insulin-Mediated Protein and Glycogen Synthetic Signaling Downstream of Protein Kinase B in Rat Myocardium

Yangsong Wu, Eugene J. Barrett, Wen Long and Zhenqi Liu

Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health System, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Zhenqi Liu, M.D., Department of Internal Medicine, Division of Endocrinology and Metabolism, P.O. Box 801410, University of Virginia Health System, Charlottesville, Virginia 22908-1410. E-mail: zl3e{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin and protein kinase B (or Akt) play critical roles in cardiomyocytic growth and survival. High concentrations of glucocorticoids antagonize insulin’s action. To examine whether endogenous glucocorticoids modulate insulin’s effect on Akt signaling in the protein and glycogen synthetic pathways in myocardium, we studied three groups of rats (n = 12 each) 4 d after either a bilateral adrenalectomy (ADX), ADX with physiological stress dose dexamethasone treatment (ADX + DEX), or a sham operation. Rats received either a saline infusion or a 3 mU/kg·min euglycemic insulin clamp for 3 h. ADX had no effect on myocardial Akt or GSK-3 [glycogen synthase (GS) kinase 3] phosphorylation, but it decreased the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (p70S6K) (P < 0.003 for both). Insulin enhanced the phosphorylation of Akt (P < 0.04), 4E-BP1 (P < 0.002), and p70S6K (P < 0.0001) in ADX, but not in sham rats. Dexamethasone restored the levels of 4E-BP1 and p70S6K phosphorylation and abrogated the insulin-stimulated Akt, 4E-BP1, and p70S6K phosphorylation. ADX rats had higher GS activity (P = 0.058) and lower glycogen content (P < 0.0001) than sham rats. GSK-3 phosphorylation after insulin infusion was greater in ADX rats. Insulin did not alter GS activity. Although insulin did not change the glycogen content in sham or ADX rats, it increased glycogen content by approximately 50% in ADX + DEX rats (P < 0.02). We conclude that endogenous glucocorticoids differentially modulate the regulation of Akt-4E-BP1/p70S6K and Akt-GSK-3-GS signaling pathways in heart by physiologic hyperinsulinemia over a range from deficiency to physiological stress concentrations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADULT CARDIOMYOCYTIC GROWTH and survival is a dynamic and delicate process involving many factors and several signaling pathways. Among them, insulin and the phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (Akt) signaling pathway each play critical roles. Insulin via PI3-kinase activates mRNA translation initiation, glucose transport, protein synthesis, glycogen synthesis, and cellular growth/survival.

Insulin’s signaling to glycogen synthesis and protein metabolism bifurcates downstream of Akt (1), a key threonine/serine protein kinase that promotes the phosphorylation and activation of mammalian target of rapamycin (mTOR) (1, 2, 3, 4). Activation of mTOR then increases the phosphorylation of eukaryotic initiation factor (eIF) 4E binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (p70S6K). Unphosphorylated 4E-BP1 functions as an mRNA translation initiation repressor by binding to eIF4E, whereas phosphorylation of 4E-BP1 frees eIF4E, which then associates with eIF4G to form the preinitiation complex and initiate translation (5, 6, 7). On the other hand, phosphorylation of p70S6K facilitates 5'-terminal oligopyrimidine mRNA translation and increases the synthesis of some ribosomal proteins, initiation factors and elongation factors that play important roles in protein synthesis (8, 9). Akt activation inhibits GSK-3 [glycogen synthase (GS) kinase 3] via phosphorylation at Ser21 (GSK-3{alpha}) and Ser9 (GSK-3ß) (1, 10, 11, 12, 13) and thereby increases the activity of GS.

Whereas insulin exerts many of its growth-promoting anabolic effects through the activation of the PI3-kinase/Akt signaling pathway, glucocorticoids antagonize multiple actions of insulin in this pathway. Previous evidence has demonstrated that excessive glucocorticoids decrease: 1) tyrosine phosphorylation of the insulin receptor and insulin receptor substrate 1; 2) the amount of insulin receptor substrate 1 (14); 3) the phosphorylation of both 4E-BP1 and p70S6k (15); 4) insulin-stimulated phosphorylation of 4E-BP1 and p70S6K (16); and 5) GS activity in human skeletal (17) and rat heart muscle (18). We have recently shown that glucocorticoids at physiological stress concentrations, either due to surgical stress or glucocorticoid replacement in ADX rats, impair insulin-stimulated activation of Akt, 4E-BP1, and p70S6K in skeletal muscle (19).

Although these observations have clarified the potential interactions between insulin and glucocorticoids on protein and glycogen metabolism, most studies have used pharmacological doses of insulin and/or glucocorticoids and have examined their interactions in skeletal muscle or in cultured cells. Consequently, the physiological significance of these findings to the heart is uncertain.

In the present study, we hypothesize that endogenous glucocorticoids modulate insulin’s action on Akt and its downstream regulatory proteins involved in the protein and glycogen synthesis in vivo in rat myocardium. Our data indicate that glucocorticoid deficiency and physiological stress concentrations of glucocorticoids differentially modulate insulin-mediated glycogen and protein synthetic signaling downstream of Akt in rat myocardium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal preparation and experimental protocols
The study protocol was approved by the University of Virginia Animal Care and Use Committee. Three groups of male Sprague Dawley rats (Harlan, Indianapolis, IN) (n = 12 each), initially weighing 225–250 g, were studied 4 d after either a bilateral adrenalectomy (ADX) or a sham operation through a midabdominal incision under generalized anesthesia using pentobarbital sodium (50 mg/kg ip; Abbott Laboratories, North Chicago, IL). One group of rats had the sham operation (sham) with both adrenal glands being isolated and left in place. The second group of rats had a bilateral ADX. The third group had a bilateral ADX and was placed on dexamethasone treatment at a dose of 5 µg/100 g body weight given sc twice daily (ADX + DEX). This dose was considered a physiological stress dose selected to approximate the glucocorticoid concentrations found in stressed rats (20, 21) (e.g. the sham-operated animals). After surgery, rats were maintained on a 12-h light, 12-h dark cycle with food and water provided ad libitum. Normal saline was provided instead of water to all ADX rats to prevent dehydration.

Four days after surgery, each group of rats was further divided into two subgroups, with one subgroup receiving a saline infusion and the other subgroup an insulin infusion (n = 6 for each subgroup). After an overnight fast, rats were again anesthetized with ip pentobarbital sodium (50 mg/kg). The external jugular vein, internal carotid artery, and trachea were exposed and cannulated through a midline neck incision. The arterial catheter was connected through a three-way stopcock to a pressure probe. Heart rate and mean arterial pressure were monitored throughout the study (Transonic Systems, Ithaca, NY). Pentobarbital sodium was infused at a variable rate to maintain a steady level of anesthesia throughout the study. After a 30- to 45-min baseline period to ensure hemodynamic stability and a stable level of anesthesia, rats in the saline subgroups received a 3-h infusion of normal saline, whereas rats in the insulin subgroups received a 3 mU/kg·min euglycemic insulin clamp for 3 h. Whole blood glucose was monitored every 10 min throughout the insulin infusion, and 30% dextrose was infused at a variable rate to maintain blood glucose within 10% of basal (22). The heart was quickly excised at the end of the infusion period and was freeze-clamped in liquid nitrogen. All heart samples were stored at -70 C until analysis.

Western immunoblotting technique
Pieces (~40 mg) of frozen heart muscle were powdered in frozen 25 mM Tris·HCl buffer [26 mM potassium fluoride and 5 mM EDTA (pH 7.5)] and then disrupted by sonication using a Fisher XL2020 sonicator (Fisher Scientific, Pittsburgh, PA). The homogenate was centrifuged at 2000 rpm for 2 min, and the protein content of the supernatant was determined using the Bradford method (23). Aliquots of the supernatant containing approximately 60 µg of protein were diluted with an equal volume of sodium dodecyl sulfate sample buffer and electrophoresed on a 10% polyacrylamide gel for Akt and GSK-3, 15% gel for 4E-BP1 and 8% gel for p70S6K. After being blocked with 5% low-fat milk in Tris-buffered saline plus Tween 20, membranes were incubated with either rabbit polyclonal Akt antibody or phospho-Akt (Ser473) antibody (New England Biolabs, Beverly, MA) overnight at 4–8 C, or rabbit anti-4E-BP1 or p70S6K (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature, or rabbit antirat GSK-3ß or phospho-GSK-3ß (Ser9) antibodies (New England Biolabs) overnight at 4 C. This was followed by a donkey antirabbit IgG coupled to horseradish peroxidase, and the blots were developed using an enhanced chemiluminescence Western blotting kit (Amersham Life Sciences, Piscataway, NJ).

Quantitation of Akt, 4E-BP1, p70S6K, and GSK-3 phosphorylation status
Autoradiographic films were scanned densitometrically (Molecular Dynamics, Piscataway, NJ) and quantitated using Imagequant 3.3. Figure 1Go illustrates the Akt, 4E-BP1, p70S6K, and GSK-3 phosphorylation status based on Western blot analysis. For Akt and GSK-3, both the total and phospho-specific densities were quantitated and the ratios of phosphospecific density to total density were calculated. Because there is a good correlation between phosphorylation and electrophoretic mobility for both 4E-BP1 (24, 25, 26) and p70S6K (27), and we have previously observed a good reproducibility of the fractional phosphorylation ratios for 4E-BP1 and p70S6K after loading gels with different amounts of protein (16), we calculated the ratio of the intensity of the most slowly migrating species ({gamma}) to that of the total intensity ({alpha} + ß + {gamma}) as the index of phosphorylation for 4E-BP1 and the ratio of the more slowly migrating forms (ß + {gamma}) to the total ({alpha} + ß + {gamma}) for p70S6K. This method exploits the different electrophoretic behaviors of proteins with various amount of phosphorylation on SDS-PAGE that allows the simultaneous quantification of multiple forms of both proteins, as well as internal normalization for both the recovery of target proteins from tissue and loading of gels.



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  		FIG. 1. Gel patterns of rat myocardial phospho-Akt (Ser473) (A), total Akt (B), 4E-BP1 (C), p70S6K (D), phospho-GSK-3ß (Ser9) (E), and total GSK-3ß (F) on SDS-PAGE. For 4E-BP1 and p70S6K, the {alpha}-band is the least phosphorylated portion and the {gamma}-band has the highest degree of phosphorylation, with the ß-band in between in both the extent of phosphorylation and the mobility on SDS-PAGE. The Western blot shows the pattern from sham (lanes 1 and 2), ADX (lanes 3 and 4), and ADX + DEX (lanes 5 and 6) rats. Lanes 1, 3 and 5, Saline infusion. Lanes 2, 4, and 6, Insulin infusion.

 
Measurement of GS activity
Heart muscle GS activity was measured in cell-free homogenates using previously established method (28). The active form (glucose-6-phosphate-independent form; GS-I) of the enzyme is defined as the rate (µmol/g·min) of incorporation of [U-14C]uridine diphosphoglucose (Amersham Biosciences, Piscataway, NJ) into glycogen at a physiological concentration (0.17 mM) of glucose-6-phosphate. Total GS (glucose-6-phosphate-dependent form; GS-D) is defined as that activity observed in the presence of 7.2 mM glucose-6-phosphate. The ratios of GS-I to GS-D were calculated.

Determination of heart muscle glycogen content
Approximately 30–40 mg of heart muscle was powdered and dissolved in 30% (wt/vol) potassium hydroxide. Glycogen was precipitated and washed three times with ice-cold ethanol. After being digested with amyloglucosidase (Sigma, St. Louis, MO), the glucose concentrations were measured using glucose oxidase method (29). Final glycogen contents were expressed as milligram glycogen per gram wet tissue weight.

Statistical analysis
All data are presented as mean ± SEM. Statistical comparisons between two different groups were made using a two-tailed, unpaired t test. All statistical analyses were performed using SigmaStat 3.0 software (SPSS Inc., Chicago, IL). A statistical significance is defined as P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of experimental animals
Table 1Go detailed the basal characteristics of all three groups of study animals. ADX rats, with or without stress dose glucocorticoid replacement, consumed less food and lost more weight than sham-operated rats. The mean arterial blood pressures and blood glucose concentrations were significantly lower in ADX rats than in their sham or ADX + DEX counterparts, consistent with glucocorticoid deficiency in those rats. Despite these differences, the heart weights were comparable among all groups. Plasma corticosterone concentrations declined modestly in sham rats after insulin infusion (298 ± 72 ng/ml vs. 150 ± 36 ng/ml during the 3-h insulin clamp), although this difference was not statistically significant (P = 0.18). In either ADX or ADX + DEX rats, plasma corticosterone concentrations were undetectable.


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  		TABLE 1. Characteristics of experimental animals

 
Effect of ADX and insulin infusion on Akt phosphorylation
ADX per se appeared to have no effect on basal Akt phosphorylation (0.37 ± 0.02 vs. 0.33 ± 0.05, sham saline vs. ADX saline rats, P = 0.58). Glucocorticoid replacement may have slightly increased the phosphorylation state of Akt to 0.51 ± 0.10, although this was not statistically different from the sham saline or ADX saline groups (P = 0.24 and 0.18, respectively). Physiologic hyperinsulinemia did not significantly increase the phosphorylation of Akt in sham-operated rats, but caused a more than 2-fold increase in ADX rats (0.33 ± 0.05 vs. 0.68 ± 0.01, P < 0.04). Dexamethasone treatment abrogated this insulin-stimulated Akt phosphorylation in ADX rats (Fig. 2Go).



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  		FIG. 2. Effect of ADX and insulin infusion on Akt phosphorylation in rat myocardium. Insulin infusion did not significantly stimulate Akt phosphorylation in sham rats, but it dramatically increased the phosphorylation of Akt in ADX rats. Dexamethasone treatment attenuated the stimulatory effect of insulin on Akt phosphorylation. *, P < 0.04 vs. ADX saline rats.

 
Effect of ADX and insulin infusion on phosphorylation of 4E-BP1 and p70S6K
For 4E-BP1, the {gamma}-band is the highly phosphorylated form and an increase in the quantity of this form corresponds to a greater amount of eIF4E available to initiate translation. The highly phosphorylated portion ({gamma}-band) of 4E-BP1 was significantly lower in ADX saline rats than that in sham saline rats (0.44 ± 0.02 vs. 0.58 ± 0.03, P < 0.003) and dexamethasone treatment restored the phosphorylation level back to that comparable to the sham saline rats (0.58 ± 0.08, P = 0.99). Physiologic hyperinsulinemia did not stimulate 4E-BP1 phosphorylation in sham-operated rats, but did significantly increase the phosphorylation state of 4E-BP1 in ADX rats (P < 0.002). Dexamethasone treatment blunted this increase in 4E-BP1 phosphorylation in ADX rats (Fig. 3AGo).



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  		FIG. 3. Effect of ADX and insulin infusion on rat myocardial 4E-BP1 and p70S6K phosphorylation. ADX significantly reduced the amount of phosphorylation of both 4E-BP1 and p70S6K. Insulin significantly increased the phosphorylated portion of 4E-BP1 and p70S6K in ADX rats, but not in sham rats. Dexamethasone treatment abrogated the stimulatory effect of insulin on the phosphorylation of both proteins. * and #, P < 0.003 vs. respective sham saline rats. **, P < 0.002; and ##, P < 0.0001 vs. respective ADX saline rats.

 
For p70S6K, the more slowly migrating ß- and {gamma}-bands represent the more highly phosphorylated forms of p70S6K and correspond to species with greater kinase activity. Similar to the effect of ADX on 4E-BP1, ADX significantly decreased p70S6K phosphorylation (0.64 ± 0.02 vs. 0.51 ± 0.02, sham saline vs. ADX saline rats, P < 0.003) and dexamethasone treatment of ADX rats abrogated this decrease in p70S6K phosphorylation (0.65 ± 0.09, P = 0.86 vs. sham saline rats). Insulin treatment stimulated p70S6K phosphorylation only in ADX rats (0.51 ± 0.02 vs. 0.83 ± 0.03, saline vs. insulin, P < 0.0001), but not in sham-operated rats or dexamethasone-treated ADX rats (P = 0.15 and 0.72, respectively) (Fig. 3BGo).

Effect of ADX and insulin infusion on GSK-3 phosphorylation
As shown in Fig. 4Go, the phosphorylation status of GSK-3 was comparable among all three subgroups of rats that received saline infusions, suggesting that ADX per se did not significantly affect basal GSK-3 phosphorylation (P = 0.9, ANOVA). However, 3 h of physiologic hyperinsulinemic clamp resulted in significantly higher GSK-3 phosphorylation in ADX rats, when compared with sham-operated rats and dexamethasone-treated ADX rats (P < 0.02, ANOVA).



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  		FIG. 4. Effect of ADX and insulin infusion on myocardial GSK-3 phosphorylation state. ADX with or without dexamethasone treatment had no significant impact on the phosphorylation of GSK-3. Insulin-stimulated GSK-3 phosphorylation was significantly higher in ADX rats (*, P < 0.02, ANOVA).

 
Effect of ADX and insulin infusion on GS activity and glycogen content
ADX per se appeared to have no impact on total myocardial GS activity (0.86 ± 0.04, 0.82 ± 0.06, and 0.76 ± 0.09 µmol/min·g, sham saline, ADX saline, and ADX + DEX saline, respectively, P = 0.597, ANOVA). However, the portions of GS in the active form (GS-I/GS-D ratio) tended to be higher (0.23 ± 0.02 vs. 0.34 ± 0.04, sham saline vs. ADX saline, P = 0.058) 4 d after bilateral ADX, whereas the glycogen content (5.47 ± 0.37 vs. 2.64 ± 0.10 mg/g wet tissue, P < 0.0001) was significantly lower in ADX saline rats when compared with sham saline rats (Fig. 5Go, A and B). Dexamethasone treatment did not increase the basal glycogen content in ADX + DEX saline rats. Three hours of insulin infusion did not alter the GS-I/GS-D ratios in any of the three groups of rats studied and did not increase the myocardial glycogen content in sham or ADX rats. However, in ADX rats treated with stress dose dexamethasone, we observed an approximately 50% increase in glycogen content after insulin infusion (2.58 ± 0.47 vs. 4.03 ± 0.24 mg/g wet weight, P < 0.02). There was a statistically significant negative correlation between the GS-I/GS-D ratios and the glycogen contents (r = -0.4483, P < 0.01, Fig. 5CGo).



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  		FIG. 5. Effect of ADX and insulin infusion on GS and glycogen content in rat myocardium. A, ADX enhanced the activity of GS, whereas insulin infusion had no significant impact on the GS-I/GS-D ratios in all groups of rats studied. *, P = 0.058 vs. sham saline rats. **, P < 0.02 vs. sham insulin rats. B, ADX significantly decreased the glycogen content and insulin infusion increased the amount of glycogen in myocardium only in ADX rats with dexamethasone treatment. *, P < 0.00001; and **, P < 0.0004 vs. sham saline rats. #, P < 0.02 vs. ADX + DEX saline rats. C, The GS-I/GS-D ratios correlated negatively with the glycogen contents in rat myocardium.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using an ADX rat model with or without physiological stress dose glucocorticoid replacement, the current study examined the interactive effects of glucocorticoids and physiological concentrations of insulin on Akt-mediated protein and glycogen synthetic signaling pathways in rat myocardium. The results showed that ADX per se decreased the phosphorylation of 4E-BP1 and p70S6K, increased the activity of GS, and decreased the glycogen content in rat myocardium. Insulin at physiologic concentrations significantly stimulated Akt, GSK-3, 4E-BP1, and p70S6K phosphorylation only in ADX rats and increased the glycogen content in ADX rats with stress dose glucocorticoid replacement. These findings indicate that endogenous glucocorticoids differentially modulate insulin’s effects on the regulatory proteins involved in the protein and glycogen synthetic signaling pathways over a range from deficiency to physiologic stress concentrations in rat myocardium.

Akt is a key enzyme linking PI3-kinase activation to many of insulin’s biological effects. Insulin has been shown to phosphorylate and activate Akt in various tissues and cells in a PI-3 kinase-dependent fashion (2, 3, 30). We have observed that insulin at the physiological concentrations stimulated Akt phosphorylation in skeletal muscle in anesthetized adult rats (data not shown) and glucocorticoids at physiological stress concentrations, induced by either transabdominal surgery or by glucocorticoid replacement in ADX rats, blunted this stimulatory effect of insulin on Akt (19). In the present study, 3 h of physiological hyperinsulinemia significantly stimulated Akt phosphorylation in ADX rats, but not in sham-operated rats or ADX rats treated with dexamethasone. Consistent with these findings, we also demonstrated that physiological concentrations of insulin stimulated the phosphorylation of 4E-BP1 and p70S6K only in hearts of ADX rats, but not sham-operated or glucocorticoid-treated rats. As discussed above, the phosphorylation of both 4E-BP1 and p70S6K, two key signal intermediates regulating mRNA translation initiation, is modulated by Akt/mTOR. That dexamethasone treatment alone, without mineralocorticoid or catecholamine replacement, blunted insulin-induced phosphorylation of Akt, 4E-BP1, and p70S6K in hearts of ADX rats suggests the greater insulin sensitivity of Akt-4E-BP1/p70S6K signaling pathway observed in ADX rat hearts is secondary to endogenous glucocorticoid deficiency, not mineralocorticoid or catecholamine deficiency. Taken together, our data suggest that glucocorticoid deficiency enhances, whereas glucocorticoids at physiological stress concentrations (induced either by abdominal surgery or dexamethasone injection) reduces, insulin sensitivity of Akt activation and translation initiation in rat myocardium.

It is of interest that ADX per se did not affect the phosphorylation status of Akt and GSK-3, but significantly decreased the amount of phosphorylated 4E-BP1 and p70S6K. Because glucocorticoid replacement alone abolished the suppressive effect of ADX on 4E-BP1 and p70S6K phosphorylation, it appears that glucocorticoid deficiency is responsible for the decreased 4E-BP1 and p70S6K phosphorylation in ADX rats seen in our study. This is different from our previous observation in the skeletal muscle in which ADX per se did not affect the basal phosphorylation of either 4E-BP1 or p70S6K (19). This is of no surprise because glucocorticoids have positive inotropic effects on heart muscle and we and others have previously observed that the protein turnover rates and the phosphorylation of 4E-BP1 and p70S6K were severalfold higher in myocardium when compared with skeletal muscle (31, 32, 33) (Long, W., and E. J. Barrett, unpublished observation). Our finding is also consistent with previous report that total RNA content in heart muscle cells decreased by about 32% 7 d after ADX (34). Glucocorticoid deficiency also significantly reduces the development of pressure-induced myocardial hypertrophy because coarctation of the abdominal aorta fails to increase ventricular weight, RNA content, and RNA-to-DNA ratio in rats without an adrenal cortex, but not in rats without an adrenal medulla (35). Although the adrenal cortex also produces appreciable amount of mineralocorticoid hormones and these hormones induce synthesis of specific proteins, they have no known effect on bulk protein metabolism.

Among all the hormones produced by the adrenal glands, glucocorticoids and catecholamines play important regulatory roles in glycogen synthesis and degradation in muscle cells. ADX has been shown to decrease glycogen content, glycogenolytic response to catecholamines, and total glycogen phosphorylase activity in myocardium (36). Although we did not measure glycogen content in liver and skeletal muscle in the current study, it has been well documented that ADX, fasted rats lack the ability to synthesize and accumulate hepatic glycogen, have a lesser activation of hepatic GS in response to acute glucose administration and have diminished insulin-stimulated glucose uptake and GS activation in perfused hindlimb muscle (37, 38). Our current study revealed that 4 d of ADX decreased the glycogen content and enhanced the GS activity in myocardium. The increase in GS activity is likely secondary to low glycogen content because we have observed a strong negative correlation between the glycogen content and the GS-I/GS-D ratio in heart in our study animals and a previous study has also reported a similar finding in skeletal muscle (38). An inverse relationship between GS-I/GS-D and glycogen content has been repeatedly demonstrated in cardiac muscle under various experimental conditions other than ADX (39, 40, 41, 42, 43). Three hours of insulin infusion did not increase GS activity or glycogen content in sham-operated rats, consistent with our findings that Akt or GSK-3 phosphorylation were unaffected by insulin infusion in this group of rats. Despite the fact that in ADX rats, dexamethasone treatment did not increase glycogen content and insulin infusion did not increase the GS-I/GS-D ratio, we did observe a significant increase in glycogen content after insulin infusion to nearly the level we observed in sham-operated rats with or without insulin infusion, suggesting that glucocorticoids are required for insulin-mediated myocardial glycogen repletion.

In summary, our data indicate that glucocorticoid deficiency suppresses basal phosphorylation of 4E-BP1 and p70S6K, decreases glycogen content, abolishes insulin-stimulated glycogen accumulation, but enhances insulin sensitivity in phosphorylation of Akt, 4E-BP1 and p70S6K, three key signal intermediates regulating mRNA translation whereas physiological stress concentrations of glucocorticoids counterpoise the effect of insulin on Akt, 4E-BP1, and p70S6K phosphorylation but facilitate insulin-mediated glycogen repletion in myocardium. We conclude that endogenous glucocorticoids play an important modulatory role in the regulation of Akt-4E-BP1/p70S6K and Akt-GSK-3-GS signaling pathways in heart by physiologic hyperinsulinemia over a range from deficiency to physiological stress concentrations.


   Footnotes
 
This work was supported by a research grant from the American Diabetes Association (to Z.L.) and NIH Grants RR15540 (to Z.L.) and DK-38578 and DK-54058 (to E.J.B.).

Abbreviations: ADX, Adrenalectomy/adrenalectomized; ADX + DEX, ADX with physiological stress dose dexamethasone treatment; Akt, protein kinase B; eIF, eukaryotic initiation factor; 4E-BP1, 4E binding protein 1; GS, glycogen synthase; GS-D, glucose-6-phosphate-dependent form of GS; GS-I, glucose-6-phosphate-independent form of GS; GSK-3, GS kinase-3; mTOR, mammalian target of rapamycin; PI3-kinase, phosphatidylinositol 3-kinase; p70S6K, ribosomal protein S6 kinase.

Received October 23, 2003.

Accepted for publication November 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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