Download gender determines the igf-i sensitivity of muscle protein anabolism

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
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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
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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