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AJP-Endo Articles in PresS. Published on October 1, 2002 as DOI 10.1152/ajpendo.00028.2002 Manuscript # E-00028-2002 Final Accepted Version 1 Adrenalectomy Enhances the Insulin Sensitivity of Muscle Protein Synthesis Wen Long, Eugene J. Barrett, Liping Wei, and Zhenqi Liu Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, VA 22908 Running Title: Glucocorticoids, insulin and protein synthesis Address correspondence to Zhenqi Liu, M.D. Department of Internal Medicine Division of Endocrinology and Metabolism PO Box 801410 University of Virginia Health Sciences Center Charlottesville, VA 22908-1410 Phone (434)- 924-1175 FAX (434)- 924-1284 Email [email protected] Copyright 2002 by the American Physiological Society. Manuscript # E-00028-2002 Final Accepted Version 2 ABSTRACT After confirming adrenalectomy per se does not affect skeletal muscle protein synthetic rates, we examined whether endogenously produced glucocorticoids modulate the effect of physiologic insulin concentrations on protein synthesis in overnight fasted rats 4 days after either a bilateral adrenalectomy (ADX), an ADX with dexamethasone treatment (ADX+DEX), or a sham-operation (Sham)(n = 6 each). Rats received a 3 hr euglycemic insulin clamp (3 mU/min/kg). Rectus muscle protein synthesis was measured at the end of the clamp and the phosphorylation states of protein kinase B (Akt), eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (p70S6K) were quantitated before and after the insulin clamp. The basal phosphorylation states of Akt, 4E-BP1, and p70S6K were similar between ADX and Sham rats. Insulin significantly enhanced the phosphorylation of Akt (p<0.03), 4E-BP1 (p=0.003), and p70S6K (p<0.002) in ADX, but not in Sham rats. Protein synthesis was significantly greater after insulin infusion in ADX than in Sham rats (p=0.01). Glucocorticoid replacement blunted the effect of insulin on Akt, 4E-BP1, and p70S6K phosphorylation and protein synthesis. In conclusion, glucocorticoid deficiency enhances the insulin sensitivity of muscle protein synthesis, which is mediated by increased phosphorylation of translation initiation regulatory proteins. Keywords: dexamethasone; translation initiation; protein kinase B; 4E-BP1; p70S6K 3 Manuscript # E-00028-2002 Final Accepted Version INTRODUCTION It is well recognized that insulin exerts anabolic actions and glucocorticoids catabolic effects on skeletal muscle protein metabolism. Both protein synthesis and degradation are affected by these two hormones. However, the cellular mechanisms underlying the interactions between these factors remain poorly defined. Insulin activates several key signal intermediates that regulate protein synthesis (47), including protein kinase B (PKB or Akt) (48), eukaryotic initiation factor 4Ebinding protein 1 (4E-BP1, or PHAS-I) (26, 27), and ribosomal protein S6 kinase (p70S6K) (42). Akt is a key element of the phosphatidylinositol 3-kinase (PI3kinase)/mammalian target of rapamycin (mTOR) pathway involved in the phosphorylation and activation of 4E-BP1 and p70S6K. It is downstream of PI3-kinase, and itself promotes the phosphorylation and activation of mTOR (4, 7, 48, 57). 4E-BP1, in its unphosphorylated form, functions as an mRNA translation initiation repressor by binding to eukaryotic initiation factor 4E (eIF4E). Phosphorylation of 4E-BP1 frees eIF4E, which can then associate with eukaryotic initiation factor 4G (eIF4G) to initiate mRNA translation. Phosphorylation of p70S6K increases the phosphorylation of ribosomal protein S6 (44), which facilitates 5’-terminal oligopyrimidine mRNA translation and increases the synthesis of some ribosomal proteins, translation initiation factors and elongation factors that play important roles in protein synthesis (28, 43). Glucocorticoid excess induces negative nitrogen balance in both experimental animals and humans by enhancing proteolysis, decreasing transport of amino acids into muscle, and inhibiting protein synthesis (10, 45, 54, 56). Glucocorticoid administration acutely inhibits muscle protein synthesis in rats as early as 4 hr (46, 49, 55). The Manuscript # E-00028-2002 Final Accepted Version 4 mechanism(s) underlying this effect on protein synthesis remains incompletely understood. Recent studies suggest that the major site for glucocorticoid’s negative action on protein synthesis is the inhibition of mRNA translation initiation (45, 49). In rats, high-dose dexamethasone treatment (100 µg/100 g body weight) acutely inhibits protein synthesis, decreases the phosphorylation of both 4E-BP1 and p70S6K, and lowers the amounts of eIF4E bound to eIF4G (49). Interestingly, more prolonged, but lower (though still pharmacologic) doses of dexamethasone (2mg every 6 hours for 3 days) did not affect postabsorptive protein synthesis or the phosphorylation status of either 4E-BP1 or p70S6K in humans (31, 34). However, insulin and amino acids induced activation of 4EBP1 and/or p70S6K was significantly impaired, both in rats (33) and in humans (31). These observations have shed some light on the potential mechanisms for the interactions between the stimulatory effect of insulin and inhibitory effect of glucocorticoids on protein synthesis. However, most studies have used pharmacological doses of insulin and/or glucocorticoids, making the physiological significance of these findings less certain. Considering insulin for example, many investigators have failed to demonstrate a stimulatory effect of physiological insulin concentrations on either whole body or skeletal muscle protein synthesis in adult rats or humans in vivo (1, 9, 16, 20, 36, 37, 39, 41, 59, 62). We have previously reported that physiological hyperinsulinemia stimulates the phosphorylation of p70S6K, but not 4E-BP1 in rat and human skeletal muscle (22, 32). Whether physiological concentrations of glucocorticoids modulate the activation of protein synthetic pathway by physiological concentrations of insulin has not been defined. The major purpose of this study was to examine whether endogenous Manuscript # E-00028-2002 Final Accepted Version 5 glucocorticoids modulate insulin-stimulated muscle protein synthesis at physiologic stress concentrations in vivo and whether the signaling pathway that regulates protein synthesis via phosphorylation of Akt, 4E-BP1 and p70S6K is affected by adrenalectomy. We used an adrenalectomized rat model with or without glucocorticoid replacement to study the effect of insulin on protein synthesis and the insulin signal transduction pathway. The results showed that insulin at physiologic concentrations significantly stimulated skeletal muscle protein synthesis and increased the phosphorylation of Akt, 4E-BP1 and p70S6K in adrenalectomized rats compared with sham-operated animals. Dexamethasone at a dose selected to replace endogenous glucocorticoids in adrenalectomized rats blunted the muscle’s response to insulin. These findings suggest that endogenous glucocorticoids play a significant role in blunting insulin’s stimulatory effect on protein synthesis at physiologic concentration in vivo. MATERIALS AND METHODS Animal preparation and experimental protocols. Male Sprague-Dawley rats, weighing 225-250 g, were studied 4 days after either a bilateral adrenalectomy or a sham operation under generalized anesthesia using pentobarbital sodium (50 mg/kg ip; Abbott Laboratories, North Chicago, IL). Two separate studies were conducted. In study one, as a preliminary study, we examined whether adrenalectomy per se affects the rates of skeletal muscle protein synthesis in two groups of rats (n=5 each). One group underwent adrenalectomy and the other group had sham operation. All surgeries were done through a mid-abdominal incision. In sham-operated rats, both adrenal glands were isolated, then left in place. After surgery, rats were maintained on a 12:12-hr lightdark cycle with food and water provided ad libitum. Normal saline was provided instead Manuscript # E-00028-2002 Final Accepted Version 6 of water to all adrenalectomized rats to prevent dehydration. Four days after surgery, rats were fasted overnight and then anesthetized with intraperitoneal 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 45-min baseline period to assure hemodynamic stability and a stable level of anesthesia, all rats received a three hour saline infusion and a phenylalanine bolus (150 µmols/100 g body weight) containing L-[ring-2,6-3H] phenylalanine (375 µCi) was injected through the jugular vein 10 minutes prior to the completion of saline infusion. Gastrocnemius muscle was removed at the end of the infusion period and was freeze-clamped in liquid nitrogen. All muscle samples were stored at -70°C until analysis. In study two, three groups of rats were studied to examine the effect of adrenalectomy on insulin stimulated translation initiation and protein synthesis. One group of rats (n=6) had a bilateral adrenalectomy (ADX), one group of animals (n=6) had a bilateral adrenalectomy and was placed on dexamethasone 5 µg per 100 g body weight subcutaneously twice daily (ADX+DEX), and another group of rats (n=6) had sham operation (Sham). In ADX+DEX rats, the dose of dexamethasone was considered a physiological stress dose that approximates the glucocorticoid level found in stressed rats (8, 35). Four days after surgery, all rats were prepared as described above for study one. After a 45-min baseline period to assure hemodynamic stability and a stable level of anesthesia, a piece of rectus muscle was biopsied and quickly freeze-clamped in liquid Manuscript # E-00028-2002 Final Accepted Version 7 nitrogen. After another 30-min stabilization period, rats received 3 mU/kg/min euglycemic clamp for 3 hours. Whole blood glucose was monitored every 10 min (AccuChek, Roche Diagnostics Corporation, Indianapolis, IN) throughout the insulin infusion, and 30% dextrose was infused at a variable rate to maintain blood glucose within 10% of basal (13). A phenylalanine bolus (150 µmols/100 g body weight) containing L-[ring-2,63 H] phenylalanine (375 µCi) was injected through the jugular vein 10 minutes prior to the completion of insulin infusion. Rectus muscle was again biopsied at the end of the infusion period and was freeze-clamped in liquid nitrogen. Both muscle biopsies were taken from lower abdominal rectus muscle that was not wounded by prior surgery. All muscle samples were stored at -70°C freezer until analysis. Plasma insulin concentrations were measured by radioimmunoassay in plasma obtained at the beginning and the completion of the study. Plasma corticosterone concentrations were measured by radioimmunoassay in plasma obtained at the beginning of the study to confirm the success of adrenalectomy. Measurement of skeletal muscle protein synthesis. Protein synthesis was estimated using [3H]phenylalanine flooding technique as described previously (19). In brief, the phenylalanine concentration and the specific radioactivity in plasma and the muscle protein were measured by HPLC and β-counting. The protein synthetic rate is estimated from the radioactivity incorporated into the muscle protein over the 10-min period divided by the tracer specific activity and is presented as a fractional turnover rate (%/day). This method uses the tissue free amino acid pool as the precursor pool for the calculations. The phenylalanine bolus results in a rapid rise in specific radioactivity of phenylalanine in tissues and two recent studies have demonstrated that the phenylalanine Manuscript # E-00028-2002 Final Accepted Version 8 specific activity of this pool is similar to that of the true precursor pool in skeletal muscle in neonatal pigs (12) and dogs (5). Western immunoblotting technique. Pieces (~30 mg) of frozen rectus muscle were weighed and powdered in frozen 25 mM Tris · HCl buffer (26 mM KF and 5 mM EDTA, pH 7.5). Tissue was then disrupted by sonication with the use of a microtip probe (0.5 s on and 0.5 s off for 45 s total) at a 3.0 power setting on the Fisher XL2020 sonicator (Fisher Scientific, Pittsburgh, PA). The homogenate was centrifuged at 2,000 rpm for 2 min, and the protein content of the supernatant was determined using the Bradford method (3). For Akt, one aliquot of the supernatant containing ~ 60 µg of protein was diluted with an equal volume of SDS sample buffer and electrophoresed on an 8% polyacrylamide gel. For 4E-BP1, one aliquot of the supernatant containing ~ 60 µg of protein was diluted with an equal volume of SDS sample buffer and electrophoresed on a 15% polyacrylamide gel. For p70S6K, another aliquot of supernatant containing ~ 50 µg of protein was diluted with an equal volume of SDS sample buffer and electrophoresed on an 8% polyacrylamide gel. Proteins were then electrophoretically transferred to nitrocellulose membranes. 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 4-8oC, or rabbit anti-4E-BP1 or p70S6K (Santa Cruz Biotechnology, Santa Cruz, CA) for one hour at room temperature. This was followed by a donkey anti-rabbit IgG coupled to horseradish peroxidase, and the blot was developed using an enhanced chemiluminescence Western blotting kit (Amersham Life Sciences, Piscataway, NJ). Quantitation of Akt, 4E-BP1, and p70S6K phosphorylation status. Autoradiographic films Manuscript # E-00028-2002 Final Accepted Version 9 were scanned densitometrically (Molecular Dynamics, Piscataway, NJ) and quantitated using Imagequant 3.3. Figure 1 illustrates the Akt, 4E-BP1 and p70S6K phosphorylation status based on Western blots analysis. For Akt, both the total and phospho-Akt (Ser473) densities were quantitated and the ratios of phosphospecific density to total density were calculated. To quantify the extent of phosphorylation of 4E-BP1, we measured the ratio of the intensity of the most slowly migrating species (γ) to that of the total intensity (α+β+γ). For p70S6K, we determined the ratio of the more slowly migrating forms (β+γ) to the total (α+β+γ). Exploiting the different electrophoretic behaviors of proteins with various amount of phosphorylation on SDS-PAGE 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. The available data support a good correlation between activity and electrophoretic mobility for both 4E-BP1 (15, 40, 42) and p70S6K (61). We have observed good reproducibility of the fractional phosphorylation ratios for 4E-BP1 and p70S6K after loading gels with different amounts of protein (33). Statistical analysis. All data are presented as the mean ± SEM. Statistical comparisons between the basal and insulin infusion periods were made using a two-tailed, paired t test. Data comparisons among different groups were based on one-way ANOVA with post hoc testing or two-tailed t test. All statistical analyses were performed using SigmaStat 3.0 software. RESULTS Manuscript # E-00028-2002 Final Accepted Version 10 Effect of adrenalectomy on basal protein synthesis. We first examined whether adrenalectomy affected the basal rate of skeletal muscle protein synthesis in animals in study one. In this study, saline was infused and the gastrocnemius muscle was used as this is distant from the site of the initial surgical incision used for adrenalectomy and as repeated samples were not being taken for Western blotting. The fractional rates of protein synthesis in the gastrocnemius muscle were 4.43±0.39 and 4.40±0.66 %/day in the adrenalectomized versus sham-operated animals (p>0.9). These findings suggest that adrenalectomy does not alter basal rates of skeletal muscle protein synthesis. Characterization of study two experimental animals (Table 1). After adrenalectomy, rats consumed less food and weighed less than sham-operated rats. The plasma corticosterone concentrations averaged 280±55 ng/mL for Sham rats, and were undetectable for both ADX and ADX+DEX rats, confirming the success of the adrenalectomy. The mean arterial blood pressures were not different among any groups either at baseline or during the insulin infusion period. Basal insulin concentrations were similar between Sham and ADX rats. Physiologic stress dose glucocorticoid replacement increased the basal insulin concentrations by nearly three-fold. After euglycemic hyperinsulinemic clamp, insulin concentrations were compatible among all three groups of rats. The basal glucose concentrations were significantly lower in ADX rats than in Sham rats and dexamethasone treatment eliminated this difference. During the insulin clamp, the steadystate (120-180 min) glucose infusion rates required to maintain their blood glucose at their respective baseline were not different among all groups of rats. Effect of adrenalectomy and insulin infusion on phosphorylation of Akt. The basal phosphorylation of Akt was comparable between sham-operated rats and ADX rats Manuscript # E-00028-2002 Final Accepted Version 11 (0.212 ± 0.04 vs. 0.162 ± 0.03, respectively, p=0.25). Glucocorticoid replacement slightly decreased basal phosphorylation state of Akt to 0.11 ± 0.02, though this was not statistically significantly different from sham-operated or ADX rats (p=0.11 and 0.47 respectively). Insulin treatment significantly increased the phosphorylation of Akt in adrenalectomized rats by 112% (0.162 ± 0.03 vs. 0.345 ± 0.08, p<0.03), but not in shamoperated rats. Dexamethasone treatment abolished this insulin-stimulated Akt phosphorylation in adrenalectomized rats (Fig. 2) Effect of adrenalectomy and insulin infusion on phosphorylation of 4E-BP1. To quantitate the phosphorylation status of 4E-BP1, we calculated the ratio of the intensity of the most slowly migrating γ band to that of the total integrated intensity (α+β+γ, Fig. 3). The γ-band is the highly phosphorylated form of 4E-BP1 and an increase in the quantity of this form would correspond to an increase in the phosphorylation of 4E-BP1 and a greater amount of eIF4E available to initiate translation. Basal phosphorylation status of 4E-BP1 was comparable among all three groups of rats, suggesting that adrenalectomy per se does not change the phosphorylation status of this protein. Physiologic hyperinsulinemia did not stimulate 4E-BP1 phosphorylation in shamoperated rats (0.26 ± 0.02 vs. 0.29 ± 0.04, basal vs. insulin respectively, p=0.24), but did significantly increase the phosphorylation status of 4E-BP1 in ADX rats (0.23 ± 0.02 vs. 0.39 ± 0.03, p=0.003). Dexamethasone treatment blunted this increase in 4E-BP1 phosphorylation in adrenalectomized rats (0.26 ± 0.02 vs. 0.34 ± 0.06, p=0.24). Effect of adrenalectomy and insulin infusion on phosphorylation of p70S6K. The extent of phosphorylation of p70S6K was quantitated by measuring the ratio of the intensity of the 12 Manuscript # E-00028-2002 Final Accepted Version more slowly migrating species (β+γ) to that of the total integrated intensity (α+β+γ, Fig. 4). The overall p70S6K kinase activity is dependent on the phosphorylation of at least seven Ser/Thr residues at three separate domains (44, 61). The more slowing migrating β and γ bands represent the more highly phosphorylated forms of p70S6K and generally correspond to species with greater kinase activity. Similar to the effect of adrenalectomy on 4E-BP1 phosphorylation, adrenalectomy appears to have no effect on the baseline phosphorylation of p70S6K which was comparable among all three groups of rats. Insulin treatment did not stimulate p70S6K phosphorylation in sham-operated rats (0.50 ± 0.06 vs. 0.46 ± 0.07, basal vs. insulin respectively, p=0.42), but did significantly increase the phosphorylation status of p70S6K in ADX rats (0.42 ± 0.05 vs. 0.63 ± 0.04, Fig. 4). Dexamethasone treatment again blunted this increase in p70S6K phosphorylation in adrenalectomized rats (0.40 ± 0.09 vs. 0.49 ± 0.07, p=0.24). Effect of adrenalectomy on insulin stimulated protein synthesis. Fig. 5 illustrates the rates of protein synthesis observed in sham-operated, ADX, and ADX+DEX rats infused with insulin. Following three hours of euglycemic hyperinsulinemia, the skeletal muscle fractional protein synthesis rate in ADX rats was 30% higher than that in sham-operated rats (8.8±0.9 vs. 6.1±0.4 %/day, p=0.01). Glucocorticoid replacement with dexamethasone blunted this increase in insulin-stimulated protein synthesis to 7.8±1.4 %/day. DISCUSSION The adrenal glands produce glucocorticoids, mineralocorticoids, catecholamines and small amounts of sex steroids. Both glucocorticoids and catecholamines antagonize Manuscript # E-00028-2002 Final Accepted Version 13 insulin’s hypoglycemic effect. Mineralocorticoid hormones induce synthesis of specific proteins but have no known effect on bulk muscle protein metabolism. Catecholamines acutely promote protein anabolism by decreasing proteolysis (17, 29). It appears that the major adrenal hormones with negative impact on protein synthesis are glucocorticoid hormones. Glucocorticoids at pharmacological concentrations attenuate translation initiation and protein synthesis (50-52) and blunt the anabolic action of insulin, mixed amino acids, and branch-chain amino acids by inhibiting the phosphorylation of 4E-BP1 and p70S6K (31, 33). Whether physiologic stress concentrations of glucocorticoids modulate the anabolic action of insulin is not clear. In the present study, three hours of physiological euglycemic hyperinsulinemia in adrenalectomized rats significantly increased the skeletal muscle protein synthesis, and the phosphorylation of Akt, 4E-BP1, and p70S6k, three key regulatory proteins in the insulin-stimulated protein synthesis signal pathway, but had no effect in sham-operated rats. Dexamethasone treatment to replace physiologic stress levels of glucocorticoids blunted insulin’s action on all parameters observed. These findings suggest that glucocorticoid insufficiency produced by adrenalectomy enhances the insulin sensitivity of muscle protein synthesis and that endogenously present glucocorticoids can modulate insulin’s anabolic action on muscle protein synthesis in vivo. At pharmacological doses, insulin has been consistently shown to stimulate protein synthesis, both in vivo and in vitro (23-25, 38). A similar effect of physiologic hyperinsulinemia is consistently seen in immature animals (11). Insulin activates the PI3 kinase/mTOR pathway and increases the phosphorylations of 4E-BP1 and p70S6K (26, 33, 53). Within this pathway, Akt, 4E-BP1 and p70S6K are three proteins with pivotal roles in Manuscript # E-00028-2002 Final Accepted Version 14 insulin-stimulated protein synthesis. Akt is activated in response to activation of PI3 kinase by phosphorylation at Thr308 and Ser473, and its activation is necessary for insulinstimulated mTOR protein kinase activity (48). Both 4E-BP1 and p70S6K are downstream of Akt/mTOR in the insulin signal transduction pathway and phosphorylation of these two proteins is critical in the initiation and maintenance of protein synthesis. Phosphorylation of 4E-BP1 increases the amount of eIF4E available for the formation of the translation initiation complex (30), a necessary step for the translation of mRNAs with m7GTP at the 5'-Cap and phosphorylation of p70S6K correlates strongly with increases in ribosomal S6 kinase activity (61), which appears to be necessary for maintaining the apparatus required for ongoing protein synthesis. In sham-operated rats, insulin did not stimulate protein synthesis, nor did it activate the phosphorylation of Akt, 4E-BP1, and p70S6K, confirming the absence of a stimulatory effect of physiological hyperinsulinemia on bulk protein synthesis in skeletal muscle in vivo. However, our results clearly demonstrated a significant increase in protein synthesis after insulin administration in adrenalectomized rats. In accord with this increased protein synthesis, insulin also increased the phosphorylation status of Akt, 4E-BP1, and p70S6K in adrenalectomized rats. These findings suggest that adrenalectomized rats have heightened sensitivity to the effect of physiological concentrations of insulin on protein metabolism. Our observation of 4-day adrenalectomy per se having no significant impact on the rates of protein synthesis in the gastrocnemius muscle in study one is entirely consistent with our findings of no significant difference in the basal phosphorylation of Akt, 4E-BP1 and p70S6K in the rectus among all treatment groups in study two. Previous report has also shown that the whole body protein synthesis rates are essentially the same in 15 Manuscript # E-00028-2002 Final Accepted Version adrenalectomized rats and adrenalectomized rats receiving stress dose dexamethasone (same dose as ours) (35). It is interesting that the glucose infusion rates were not different among three groups of rats studied. Akt activation is thought to play a critical role in insulin stimulated GLUT4 translocation and glucose uptake (57, 60). With significantly increased phosphorylation of Akt in ADX rats following insulin treatment, one would expect that the glucose infusion rates be higher in this group of rats than in Sham rats. However, multiple confounding factors may have contributed to the lack of higher glucose infusion rates in ADX rats in the current study. Firstly, the basal blood glucose concentrations were significantly lower in ADX rats than in Sham rats and the glucose was infused to maintain the blood glucose concentrations at their respective basal levels. Secondly, adrenalectomized rats have lower body weight and less muscle mass, the site for insulin stimulated glucose disposal. Also, the adrenalectomized rats being more stressed than sham-operated rats may have higher levels of growth hormone, which is a counterregulatory hormone and antagonistic towards insulin’s hypoglycemic effect. The elevated basal insulin concentrations and slightly lower, though not statistically significant, glucose infusion rate observed in ADX + DEX rats than in Sham and ADX rats are consistent with relative insulin resistance associated with physiologic stress concentrations of glucocorticoids, a well known clinical observation in patients with persistent and severe physical stress. That dexamethasone treatment alone, administered at doses selected to replace glucocorticoids to physiologic stress concentrations, without mineralocorticoid or catecholamine replacement, blunted insulin-induced protein synthesis, and Manuscript # E-00028-2002 Final Accepted Version 16 phosphorylation of Akt, 4E-BP1, and p70S6K in adrenalectomized rats suggests the increment in insulin sensitivity observed in adrenalectomized rats is secondary to endogenous glucocorticoid deficiency, not mineralocorticoid or catecholamine deficiency. Inasmuch as it has been previously observed that glucocorticoid given in excess blunt the action of amino acids (31, 33) and pharmacological doses of insulin (33) to stimulate protein synthesis, the current findings suggest that glucocorticoids modulate protein synthesis over the entire range of their availability. In addition, the current findings suggest that the presence of endogenous glucocorticoid hormones may be largely responsible for the lack of stimulatory effect of insulin on protein synthesis observed in most in vivo studies using physiologic concentrations of insulin (1, 2, 6, 14, 18, 36, 37, 41). We have previously observed that pharmacological doses of insulin enhance the phosphorylation of both 4E-BP1 and p70S6K, whereas physiological hyperinsulinemia differentially phosphorylated p70S6K, but not 4E-BP1 in both human and rat skeletal muscle (22, 32). In the present study, the physiologic hyperinsulinemic clamp did not increase the phosphorylation of p70S6K in the sham-operated rats. The lack of effect of insulin on downstream signaling pathways in the sham rats could be due in part to experimental conditions, including the involvement of increased concentrations of glucocorticoids. Abdominal surgery and prolonged anesthesia are associated with significant stress and increased endogenous production of glucocorticoid hormones, sufficient to cause insulin resistance and may have diminished the response to insulin in sham rats. Excessive glucocorticoids decrease total tyrosine phosphorylation of the insulin receptor and insulin receptor substrate 1 (IRS-1) as well as the protein content of 17 Manuscript # E-00028-2002 Final Accepted Version IRS-1 (21); decrease the phosphorylation of both 4E-BP1 and p70S6K (50); and impair insulin-induced phosphorylation of 4E-BP1 and p70S6K in rats (33). Shah and colleagues have demonstrated that p70S6K is more sensitive to inhibition by glucocorticoids under growth-promoting conditions than is 4E-BP1(51). In the present phosphorylation state selected as minimal trauma study, there study it of allows to Akt, used be other an the 4E-BP1 repeated the would we rectus additional of to p70S6K. Rectus and measurements portions muscle from the concern snip muscle. inasmuch assay muscle was biopsies with In as the the an current abdominal incision was used for the adrenalectomy four days prior to the infusion study. However, we believe that this surgery per se had little impact on the results observed for the following reasons: 1) each animal served as its own intermediates; control 2) to all examine animals the phosphorylation including the status sham of signaling operated animals received the same abdominal surgery and there were no differences in the basal aseptic phosphorylation inflammation, status as of might these be proteins expected among at the all site groups; of 3) abdominal incision, does not appear to affect the phosphorylation of 4E-BP1(58); and finally 4) the muscle biopsies from the rectus were made at sites in the lower abdominal rectus muscle, making every effort to avoid previously incised areas of muscle. In conclusion, the current results suggest an important action of physiologic stress concentrations of glucocorticoids in the regulation of protein synthesis by physiologic Manuscript # E-00028-2002 Final Accepted Version 18 hyperinsulinemia. In particular, our results suggest that low doses of glucocorticoids are able to specifically block the activation by insulin of three proteins that regulate mRNA translation, Akt, 4E-BP1 and p70S6k. This action of endogenous glucocorticoids to counterpoise effect of insulin on the phosphorylation of translation regulatory proteins may modulate the effect of physiologic doses of insulin to stimulate protein synthesis in vivo. ACKNOWLEDGEMENTS This work was supported by NIH grants RR15540 (ZL), DK-38578 and DK-54058 (EJB). Manuscript # E-00028-2002 Final Accepted Version 19 Reference: 1. Baillie, A. G. S., and P. J. Garlick. Attenuated responses of muscle protein synthesis to fasting and insulin in adult female rats. Am J Physiol 262: E1-E5, 1992. 2. Barrett, E. J., J. H. Revkin, L. H. Young, B. L. Zaret, R. Jacob, and R. A. Gelfand. An isotopic method for measurement of muscle protein synthesis and degradation in vivo. Biochem J 245: 223-228, 1987. 3. Bradford, M. 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Somwar, P. J. Bilan, Z. Liu, J. Jin, J. R. Woodgett, and A. Klip. Protein kinase B/Akt participates in GLUT4 translocation by insulin in L6 myoblasts. Mol Cell Biol 19: 4008-18, 1999. 61. Weng, Q.-P., M. Kozlowski, C. Belham, A. Zhang, M. Comb, and J. Avruch. Regulation of p70 S6 kinase by phosphorylation in vivo. J Biol Chem 273: 16621-16629, 1998. 62. Young, L. H., W. Stirewalt, P. H. McNulty, J. H. Revkin, and E. J. Barrett. Effect of insulin on rat heart and skeletal muscle phenylalanyl-tRNA labeling and protein synthesis in vivo. Am J Physiol 267: E337-E342, 1994. Manuscript # E-00028-2002 Final Accepted Version 27 FIGURES AND LEGENDS Fig. 1: Rat skeletal muscle phospho-Akt (Ser473) (panel A), total Akt (panel B), 4E-BP1 (panel C) and p70S6K (panel D) gel patterns on SDS-PAGE. For 4E-BP1 and p70S6K, the α band is the least phosphorylated portion and has the most rapid electrophoretic mobility. The γ band is the most phosphorylated form and moves the slowest. The β band is phosphorylated more than the α band and less than the γ band, and shows up in between 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 – basal period. Lanes 2, 4, and 6 – after insulin infusion. 28 Manuscript # E-00028-2002 Final Accepted Version 0.5 Basal * Insulin Phospho-Akt/total 0.4 0.3 0.2 0.1 0 Sham ADX ADX+DEX Fig. 2: Effect of adrenalectomy and insulin infusion on Akt phosphorylation. Insulin infusion did not stimulate Akt phosphorylation in sham control rats, but significantly increased the phosphorylation of Akt in ADX rats. Dexamethasone treatment abolished this effect. * p<0.03 vs. Basal. 0.5 Basal Insulin * γ/total 0.4 0.3 0.2 0.1 0 Sham ADX ADX+DEX Fig. 3: Effect of adrenalectomy and insulin infusion on 4E-BP1 phosphorylation. Insulin significantly increased the phosphorylated portion of 4E-BP1 in ADX rats, but not in Sham or ADX+DEX rats. * p=0.003 vs. Basal. 29 Manuscript # E-00028-2002 Final Accepted Version 0.8 Basal Insulin * β+γ/total 0.6 0.4 0.2 0 Sham ADX ADX+DEX Fig. 4: Effect of adrenalectomy and insulin infusion on p70S6K phosphorylation state. Insulin significantly stimulated p70S6K phosphorylation in ADX rats, but not in sham or ADX+DEX rats. * p<0.002 vs. Basal. * 8 (fractional %/day) Protein synthesis 10 6 4 2 0 Sham ADX ADX+DEX Fig. 5: Effect of adrenalectomy on insulin stimulated skeletal muscle protein synthesis. Protein synthesis was estimated using flooding bolus of [3H]-phenylalanine technique and expressed as fractional %/day. Insulin treatment significantly increased the protein synthesis rate in adrenalectomized rats. * p=0.01 vs. Sham. 30 Manuscript # E-00028-2002 Final Accepted Version Table 1. Characterization of Sham, ADX and ADX+DEX animals in study two Sham ADX ADX+DEX Body weight (g) 272±6 248±11 233±8a,b Food consumption (g/day) 38±4 24±2.5c 24.5±5 Basal blood pressure (mmHg) 117±3 105.±4 110±4 Steady-state blood pressure (mmHg) 106±6.0 99±3.4 103±2.3 Basal insulin concentration (pmol/L) 71±5 38±2 200±17e Steady-state insulin concentration (pmol/L) 520±26 548±63 412±31 Basal blood glucose (mM) 5.3±0.3 4.4±0.2d 5.1±0.1 Steady-state blood glucose (mM) 5.6±0.4 4.5±0.3 5.3±0.2 Glucose infusion rate (mg/kg/min) 9.9±2.1 7.9±0.9 6.0±0.9 a. b. c. d. e. P=0.0001 vs. Sham p=0.01 vs. ADX p=0.011 vs. Sham p=0.022 vs. Sham p<0.01 vs. Sham or ADX