Skeletal muscle insulin resistance after trauma: insulin
signaling and glucose transport
Lisa
Strömmer1,
Johan
Permert1,
Urban
Arnelo1,
Camilla
Koehler1,
Bengt
Isaksson1,
Jörgen
Larsson1,
Inger
Lundkvist1,
Marie
Björnholm2,
Yuichi
Kawano2,
Harriet
Wallberg-Henriksson2, and
Juleen
R.
Zierath2
1 Arvid Wretlinds Laboratory
for Metabolic and Nutritional Research, Department of Surgery,
Karolinska Institute at Huddinge University Hospital, 141 86 Huddinge;
and 2 Department of Clinical
Physiology, Karolinska Institute at Karolinska Hospital, 171 77 Stockholm, Sweden
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ABSTRACT |
Surgical trauma induces peripheral insulin
resistance; however, the cellular mechanism has not been fully
elucidated. We examined the effects of surgical trauma on insulin
receptor signaling and glucose transport in skeletal muscle, a tissue
that plays a predominant role in maintaining glucose homeostasis.
Surgical trauma was induced by intestinal resection in the rat.
Receptor phosphorylation was not altered with surgical trauma.
Phosphotyrosine-associated phosphatidylinositol (PI) 3-kinase
association was increased by 60 and 82% compared with fasted and fed
controls, respectively (P < 0.05).
Similar results were observed for insulin receptor
substrate-1-associated PI 3-kinase activity. Insulin-stimulated protein
kinase B (Akt kinase) phosphorylation was increased by
2.2-fold after surgical trauma (P < 0.05). The hyperphosphorylation of Akt is likely to reflect
amplification of PI 3-kinase after insulin stimulation. Submaximal
rates of insulin-stimulated
3-O-methylglucose transport were
reduced in trauma vs. fasted rats by 51 and 38% for 100 and 200 µU/ml of insulin, respectively (P < 0.05). In conclusion, insulin resistance in skeletal muscle after
surgical trauma is associated with reduced glucose transport but not
with impaired insulin signaling to PI 3-kinase or its downstream
target, Akt. The surgical trauma model presented in this report
provides a useful tool to further elucidate the molecular mechanism(s)
underlying the development of insulin resistance after surgical trauma.
insulin receptor; phosphatidylinositol 3-kinase; Akt/protein kinase
B; surgical stress
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INTRODUCTION |
TRAUMA-INDUCED METABOLIC STRESS from surgery or injury
is associated with alterations in carbohydrate metabolism (13, 20), including increased glucose production (6) and decreased peripheral glucose utilization (5, 41). The most common form of posttraumatic insulin resistance is iatrogenic in nature, developing in the postoperative state. Several lines of evidence suggest that peripheral tissues are the major site of the trauma-induced insulin resistance (3,
31). Glucose clamp studies in postoperative patients revealed that
impaired glucose disposal develops in peripheral tissues (3, 17, 41).
Interestingly, the magnitude of surgically induced insulin resistance
is related to the degree of surgical trauma, and this persists for
2-3 wk after uncomplicated abdominal surgery (32, 40-42).
Despite the considerable progress in understanding the development of
trauma-induced insulin resistance at the whole body level, the cellular
mechanism remains unclear.
Peripheral insulin resistance after posttraumatic stress may occur in
response to defects in early or intermediate components of the insulin
signal transduction pathway and/or in response to defects at
the level of glucose transport, a rate-limiting step in glucose
utilization (10). One of the earliest postreceptor events leading to
the metabolic effects of insulin includes the phosphorylation of the
insulin receptor substrate-1 (IRS-1) on tyrosine residues (46). IRS-1
serves as a multiple docking site for proteins containing Src-homology
2 domains, such as phosphatidylinositol (PI) 3-kinase
(46). Multiple lines of evidence suggest that PI 3-kinase plays a
central role in mediating insulin signaling to glucose transport (8,
37). Recently, we have reported that reduced insulin-stimulated PI
3-kinase activity is coupled to reduced glucose transport in skeletal
muscle from patients with non-insulin-dependent diabetes mellitus (2).
Thus defects at the level of the insulin signal transduction pathway
may contribute to impaired glucose transport (2, 14, 18, 19) and
glucose transporter isoform GLUT-4 translocation (49), and this may result in reduced whole body glucose homeostasis. In adipocytes, insulin resistance after trauma appears to develop from a postreceptor lesion, since glucose uptake is decreased despite normal insulin binding to the insulin receptor (34). Whether the reduced glucose transport with surgical trauma is a primary defect due to altered GLUT-4 traffic from an intracellular pool to the plasma membrane or a
secondary defect due to alterations in the insulin signal transduction
cascade remains unknown.
In the present study, we have adopted a small intestinal bowel
resection model in fasted rats to delineate the underlying mechanism
for the apparent peripheral insulin resistance that arises from
surgical trauma. We have assessed early, intermediate, and final
components of the insulin signal transduction pathway to glucose
transport in skeletal muscle. Specifically, we have assessed insulin
receptor binding and autophosphorylation, PI 3-kinase activity and
association to phosphotyrosines, protein kinase B (Akt kinase)
phosphorylation, and glucose transport to determine whether defects in
insulin signaling contribute to impaired glucose homeostasis after
postoperative stress.
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METHODS |
Animals.
Male Wistar rats (320-350 g; B&K Universal, Stockholm, Sweden)
were housed under controlled conditions with a 12:12-h light-dark cycle
(lights-on 7 AM to 7 PM). Rats were provided standard laboratory chow
and water ad libitum for 1 wk before experimentation. Rats were divided
into three groups: fed (n = 25),
fasted controls (n = 22), and trauma
(n = 30). Food was withdrawn 16 h
before experimentation from rats in the fasted and trauma groups and immediately before experimentation from rats in the fed group.
Operative procedure.
All procedures were conducted with approval from the local ethical
committee. Rats were anesthetized by an intraperitoneal injection of
ketamine and xylazine (70 and 10 mg/kg, respectively). Blood samples
were obtained for preoperative determination of glucose levels from the
tail vein. An 8-cm incision was made along the abdomen. Five
centimeters distal to the Treitz's ligament, a 5-cm small bowel
resection was performed. Thereafter, the bowel was sutured with
absorbable sutures (6/0, Vicryl), and the abdominal cavity was closed
using eight sutures (4/0, Vicryl). The animals were returned to
individual cages and had free access to water. Two hours later, rats
were reanesthetized, and a plastic catheter (Venflon, 0.8/25 mm) was
inserted into the internal jugular vein. Blood samples were obtained
for determination of glucose and insulin levels. Thereafter, a bolus
injection of 1 ml of 0.9% NaCl was administered under 1 min (iv), and
a muscle biopsy was obtained from the right gastrocnemius muscle.
Insulin was injected intravenously (10 U/kg, in 1 ml of 0.9% NaCl and
0.01% BSA) as a bolus delivered under 1 min. Four minutes after the
injection, a second biopsy was obtained from the left gastrocnemius
muscle. The muscle specimens were immediately frozen on excision and
stored in liquid nitrogen until analysis.
Blood chemistry.
In a subgroup of rats, a catheter was inserted into the jugular vein
immediately after the second anesthesia, and blood samples were
collected for determination of blood glucose and plasma levels of
insulin, nonesterified free fatty acids (NEFAs), epinephrine, cortisol,
and lactate. Blood glucose and plasma lactate were determined enzymatically (YSI 2000 system; Kebo, Stockholm, Sweden). Plasma insulin was analyzed using a commercially available kit (Diagnostica, Falkenberg, Sweden). Plasma NEFAs were analyzed using a kit from Wako
Chemicals (Neuss, Germany). Plasma epinephrine was determined by HPLC
with electrochemical detection, with 3,4-dihydroxybenzylamine hydrochloride as an internal standard (16). The size of the chromatography column was 150 × 4.6 mm (Catecholamine column 5 SA; Machery-Nagel, Düren, Germany), and an EC 2000 electrochemical detector was utilized (Therm Separation Products,
Riviera Beach, FL). Serum cortisol was measured in a solid-phase
time-resolved fluoroimmunoassay (9), based on a competitive reaction
between europium-labeled cortisol and sample cortisol (Wallac Oy,
Turku, Finland).
Insulin receptor binding and
phosphorylation.
Gastrocnemius muscle biopsies were ground to a fine powder with a
mortar and a pestle in liquid nitrogen and immediately homogenized in
ice-cold buffer as previously described (15). Homogenates were
solubilized by gentle mixing for 30 min at 4°C. After
centrifugation (35,000 g for 45 min at
4°C), the supernatant was removed, protein was determined (BCA
protein assay kit, Pierce, Rockford, IL), and aliquots were stored at
70°C. Insulin receptor binding and phosphorylation were
determined as described previously (15). A portion of the supernatant
was immunoprecipitated overnight at 4°C with a monoclonal antibody
directed against the
-subunit of the insulin receptor (IR 29B4,
Santa Cruz Biotechnology, Santa Cruz, CA). The precipitate was further
incubated for 30 min at 4°C with a secondary monoclonal antibody
(anti-IgG1; DAKO, Copenhagen, Denmark), followed by incubation for
1 h at 4°C with 125 µl of protein A (Pansorbin cells).
The immunoprecipitates were divided into four equal aliquots, washed,
and resuspended in Tris buffer containing 1% Triton X-100 and 1 mM
phenylmethylsulfonyl fluoride. A second aliquot of the supernatant (4 mg of protein) was used to immunoprecipitate tyrosine-phosphorylated
proteins (overnight at 4°C) with the use of an anti-phosphotyrosine
antibody (PY20; Signal Transduction Laboratories, Lexington, KY). The
immunocomplex was incubated with secondary antibody (anti-IgG2b, DAKO)
and protein G. The complex was washed three times and resuspended in
buffer (50 mM Tris, 1% Triton X-100, 1 mM Pervanadate) and divided
into four aliquots. In three aliquots of the insulin receptor
immunoprecipitate, insulin receptors were assessed using
125I-labeled insulin [25,000
counts/min (cpm)] overnight. Nonspecific binding was
determined in one of the aliquots by addition of unlabeled insulin (60 µM). To detect insulin receptor tyrosine phosphorylation, the
anti-phosphotyrosine immunoprecipitates were incubated with 125I-insulin (25,000 cpm)
overnight. The pellets were washed three times, and binding of labeled
insulin in anti-insulin receptor (
-IR) and anti-phosphotyrosine
(
-PY) immunoprecipitates was determined in a gamma counter.
Bound-free quotients per milligram protein were calculated for the two
different immunoprecipitated samples. Quotients of immunoprecipitates
were used to calculate fraction of phosphorylated receptors, corrected
for receptor binding under basal and insulin-stimulated conditions.
Tyrosine phosphorylation of IRS-1.
Equal amounts of protein (2 mg) were immunoprecipitated overnight with
IRS-1 antibody (gift from Dr. Morris White, Joslin Diabetes Center,
Boston, MA) coupled to protein A-Sepharose. The immunoprecipitates were
washed as described (14), resuspended in Laemmli sample buffer with 100 mM dithiothreitol, and heated (95°C) for 6 min. The proteins were
separated by SDS-PAGE, transferred to nitrocellulose membranes, and
blocked in Tris-buffered saline (TBS)-T (10 mM Tris, 0.01% Tween, pH
7.8) containing 3% milk. The membranes were immunoblotted with
horseradish peroxidase-conjugated anti-phosphotyrosine antibody (RC-20;
Signal Transduction) and washed thereafter with TBS-T. IRS-1 was
visualized by enhanced chemiluminescence (Amersham, Arlington Heights,
IL) and quantified by densitometric scanning (Imagemaster, Pharmacia
Biotech, Uppsala, Sweden).
Phosphotyrosine and IRS-1 association with the
85-kDa subunit of PI 3-kinase.
Phosphotyrosine- and IRS-1-associated PI 3-kinase was determined by
Western blot analysis. Equal amounts of protein (1.5 mg) were
immunoprecipitated with anti-phosphotyrosine or anti-IRS-1 antibody as
described in Insulin receptor binding and
phosphorylation. The immune complexes were
washed and solubilized in Laemmli sample buffer. Proteins were
separated by SDS-PAGE, transferred to polyvinylidene difluoride
membranes (Immobilon-P, Millipore, Bedford, MA), and blocked as
described above for tyrosine phosphorylation of IRS-1. The membranes
were incubated with a polyclonal anti-PI 3-kinase antibody (1:500;
Upstate Biotechnology, Lake Placid, NY) in TBS-T containing 5% milk.
The membranes were subsequently washed and incubated with a horseradish
peroxidase-labeled anti-rabbit antibody (1:5,000) in TBS-T containing
5% milk. After additional washing, the bound antibodies were detected
by enhanced chemiluminescence, and PI 3-kinase was quantitated by
densitometric scanning.
Phosphotyrosine- and IRS-1-associated PI 3-kinase
activity.
Equal amounts of protein (2 mg) were immunoprecipitated overnight
(4°C) with anti-phosphotyrosine or anti-IRS-1 antibody coupled to
protein A-Sepharose. The immune complexes were washed as described above and resuspended in 40 µl of buffer (20 mM HEPES,
pH 7.5, 180 mM NaCl). PI 3-kinase activity was assessed
directly on the protein A-Sepharose beads as previously
reported (19, 49). The bands corresponding to PI 3-phosphate were
quantitated using a phosphorimager (Fujix 2000, Fuji Photo Film, Fuji,
Japan).
Akt phosphorylation.
Aliquots of muscle homogenate (40 µg) were solubilized in Laemmli
sample buffer with 100 mM dithiothreitol and heated (95°C) for 6 min. Proteins were separated by SDS-PAGE, transferred to nitrocellulose
membranes, and immunoblotted as described in Tyrosine phosphorylation of IRS-1 with the use of a
phosphospecific antibody that recognizes Akt when phosphorylated at Ser
473 (New England Biolabs, Beverly, MA). Phosphorylated Akt was
visualized by enhanced chemiluminescence and quantified by
densitometric scanning.
Glucose transport.
Media were prepared from oxygenated (95%
O2-5%
CO2) Krebs-Henseleit buffer
(KHB) containing 5 mM HEPES and 0.1% BSA (RIA grade). Immediately after the second anesthesia, isolated soleus muscles were
removed from a subgroup of rats. The muscle was divided into three
equal portions, and the two outer portions (20 mg each) were used to
assess glucose transport (45). The muscle specimens were initially
incubated (30°C) for 15 min in KHB containing 2 mM pyruvate and 18 mM mannitol. Thereafter, muscles were incubated in the presence of
insulin (0, 100, 200, and 1,000 µU/ml) for 10 min in KHB containing
20 mM mannitol. Glucose transport was assessed under basal or
insulin-stimulated conditions using 8 mM
3-O-[methyl-3H]glucose
(2.5 µCi/mmol) and 12 mM
[14C]mannitol (26.3 µU/mmol). The muscles were processed as described (45). Glucose
transport activity is expressed as micromoles of glucose analog
accumulated per milliliter of intracellular water per hour.
Chemicals.
Human insulin (Actrapid) was purchased from Novo Nordisk (Bagsværd,
Denmark). Protein G-Sepharose-4 fast flow was from Pharmacia (Uppsala,
Sweden), and protein A (Pansorbin cells) was from
Calbiochem-Novabiochem (La Jolla, CA). All reagents for SDS-PAGE were
from Novex (San Diego, CA), and reagents for the protein assay were
from Pierce. 125I-insulin was from
Amersham (Buckinghamshire, UK). All other radioisotopes were purchased
from ICN Biomedical (Costa Mesa, CA). Phosphatidylinositol was from
Avanti-Polar Lipids (Alabaster, AL). Aluminum-backed silica gel-60
thin-layer chromatographic plates were from Merck (Darmstadt, Germany).
Other standard chemicals and reagents, including BSA, were purchased
from Sigma (St. Louis, MO).
Statistical analysis.
Data are presented as means ± SE. Statistical analysis was
performed using the Student's unpaired
t-test for pre- and postglucose values
and for Akt phosphorylation. Comparisons between the groups were
performed with ANOVA two-way multivariance analysis followed by the
Bonferroni correction (PI 3-kinase) or Dannet one-tailed post hoc test
(glucose transport). P < 0.05 was
considered statistically significant.
 |
RESULTS |
Blood glucose and plasma insulin levels.
Basal glucose was significantly lower in fasted control and traumatized
rats compared with fed control rats (4.5 ± 0.3 and 4.3 ± 0.2 vs. 7.0 ± 0.4 mM, respectively; Table
1). After insertion of a catheter in the
jugular vein, bloodglucose was significantly increased in fed rats
(7.0 ± 0.4 vs. 12.0 ± 0.5 mM), whereas no change was
observed in fasted controls (4.5 ± 0.3 vs. 5.3 ± 0.4 mM). In
traumatized rats, glucose levels were significantly elevated compared
with fasted controls (4.3 ± 0.3 vs. 6.5 ± 0.4 mM,
P < 0.05). Plasma insulin was
significantly higher in fed rats compared with traumatized and fasted
control rats (398 ± 94 vs. 56 ± 11 and 68 ± 7 pM,
respectively; P < 0.01). The insulin
values in fasted and traumatized rats were similar.
Plasma epinephrine and cortisol levels.
In fed and fasted controls, plasma epinephrine concentrations were
<0.3 nM, whereas in traumatized rats, all values were >0.3 nM
(range 0.88-4.07 nM) (Table 1). Statistical analysis was not performed because of the wide variation in the data. Serum cortisol values in traumatized rats were time matched to fasted and fed control
rats to correct for diurnal variation. Serum cortisol levels were
similar among all animals (310 ± 29, 271 ± 43, and 331 ± 64 mM in fed, fasted control, and traumatized rats,
respectively). In fasted control and trauma groups, serum cortisol
levels varied from 76 to 446 mM and from 87 to 497 mM, respectively.
Plasma NEFAs and lactate levels.
Plasma NEFA was significantly higher in fed rats compared with fasted
control and traumatized rats (1.9 ± 0.3 vs. 0.7 ± 0.2 and
0.3 ± 0.1 mM, respectively;
P < 0.01; Table 1). Plasma NEFA levels in traumatized rats tended to be lower than in fasted control rats (P < 0.08). Plasma lactate
concentration was comparable among all groups (1.1 ± 0.2, 0.9 ± 0.1, and 0.8 ± 0.1 mM for fed, fasted controls, and traumatized
rats, respectively).
Insulin receptor binding and
autophosphorylation.
The bolus injection of insulin (10 U/kg) resulted in high circulating
levels of insulin concentrations (>3,500 pM), which were maintained
throughout the muscle biopsy procedure (data not shown) and resulted in
a marked increase in insulin receptor phosphorylation (Table
2). The increase in insulin receptor
phosphorylation in response to insulin was approximately twofold
greater in fasted and traumatized rats compared with fed rats. In
fasted and traumatized rats, insulin receptor binding was not altered
after insulin stimulation. However, in fed rats, insulin receptor
binding decreased significantly (P < 0.05) with insulin stimulation (Table 2). Anti-insulin receptor immunoblots of insulin receptor precipitates failed to reveal any
reduction in insulin receptor content before and after insulin stimulation (data not shown). Thus the decrease in insulin receptor binding in fed control rats was not due to an actual reduction in the
number of receptors in the insulin-stimulated state. The ratio of the
bound-free quotients of
125I-insulin bound in
immunoprecipitates was determined before (
-PY/
-IR) and after insulin stimulation (+
-PY/+
-IR) as shown in Table 2.
When insulin receptor phosphorylation was corrected for the insulin
binding capacity under basal and insulin-stimulated conditions, this
fraction was increased to the same extent in all rats. The insulin-stimulated increase in phosphorylation was greater in fasted
(P < 0.05) and traumatized rats
(P < 0.05) compared with fed
controls (Table 2).
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Table 2.
Insulin receptor autophosphorylation and binding in phosphotyrosine
and insulin receptor immunoprecipitates per mg protein
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IRS-1 tyrosine phosphorylation.
We assessed the effects of surgical trauma on basal and in vivo
insulin-stimulated tyrosine phosphorylation of IRS-1 in skeletal muscle
(Fig. 1). Insulin-stimulated tyrosine
phosphorylation of IRS-1, expressed as percentage of the insulin effect
for control rats, was not altered by surgical trauma (100 ± 9 vs.
104 ± 20% for n = 8 control vs.
n = 8 traumatized rats, respectively).

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Fig. 1.
Representative immunoblot of basal and insulin-stimulated tyrosine
phosphorylation of insulin receptor substrate (IRS)-1 in skeletal
muscle from fed (n = 8) or fasted
(n = 8) traumatized rats. Hindlimb
muscle biopsies were homogenized, and equal amounts of solubilized
protein (2 mg) were immunoprecipitated with anti-IRS-1. Tyrosine
phosphorylation of IRS-1 was assessed under basal (B) or
insulin-stimulated (I) conditions as described in
METHODS by immunoblotting with
anti-phosphotyrosine antibody. Nos. at
left are molecular
mass.
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Phosphotyrosine- and IRS-1-associated PI
3-kinase.
An insulin-induced appearance of the p85 subunit of PI 3-kinase was
assessed by Western blot analysis of phosphotyrosine or IRS-1
immunoprecipitates. The presence of PI 3-kinase was confirmed with a
positive control and PI 3-kinase immunoblots with the use of an
antibody to recognize an 85-kDa form of PI 3-kinase. An internal
standard was included on all blots to correct for interassay variability. In the basal state, PI 3-kinase association to
tyrosine-phosphorylated proteins was similar among the three groups
(Fig. 2). However, under insulin-stimulated
conditions, PI 3-kinase association to tyrosine-phosphorylated proteins
was increased 175 ± 10, 200 ± 19, and 320 ± 38% for fed
control, fasted control, and traumatized rats, respectively. In
traumatized rats, there was a significant increase in PI 3-kinase
association compared with fasted (P < 0.05) and fed (P < 0.005) rats.
Similar results were observed in IRS-1 immunoprecipitates; surgical
trauma led to a 60% increase in insulin-stimulated PI 3-kinase
association to IRS-1 (226 ± 26 vs. 362 ± 36% for fed control
vs. traumatized rats, respectively; P < 0.05).

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Fig. 2.
Basal and insulin-stimulated phosphotyrosine-associated
phosphatidylinositol (PI) 3-kinase content in fed
(n = 10), fasted
(n = 8), and traumatized
(n = 8) rats. Hindlimb muscle biopsies
were homogenized, and equal amounts of solubilized proteins were
immunoprecipitated with anti-phosphotyrosine ( -PY) as described in
METHODS. Samples were resolved by
SDS-PAGE and immunoblotted with -PI 3-kinase. Results are expressed
as the mean ± SE percent increases over fed control rats.
* P < 0.05 and
P < 0.005 vs. control
fed rats.
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Phosphotyrosine- and IRS-1-associated PI 3-kinase
activity.
Recent studies provide evidence for several PI 3-kinase adapter subunit
variants, which exhibit different insulin-induced PI 3-kinase elevating
responses (1, 21, 22, 38). Thus the increase in p85 bound to
tyrosine-phosphorylated proteins or to IRS-1 noted in muscle from
surgically traumatized rats may not translate to increased activity of
PI 3-kinase. Consequently, we assessed phosphotyrosine- or
IRS-1-associated PI 3-kinase activity in muscle from control or
traumatized rats. Basal PI 3-kinase activity was similar among all
groups. Insulin-stimulated IRS-1-associated PI 3-kinase activity (Fig.
3) was significantly greater in traumatized vs. fed control rats (417 ± 41 vs. 716 ± 50%, respectively;
P < 0.05). PI 3-kinase activity was
also greater in traumatized rats compared with fasted control rats (417 ± 41 vs. 558 ± 44%); however, with Bonferroni correction, the
latter difference did not reach statistical significance. A similar
trend was observed in anti-phosphotyrosine immunoprecipitates;
insulin-stimulated phosphotyrosine-associated PI 3-kinase activity
tended to be increased in skeletal muscle after surgical trauma (244 ± 46 vs. 335 ± 63% for fed control vs. traumatized rats,
respectively; not significant).

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Fig. 3.
Basal and insulin-stimulated IRS-1-associated PI 3-kinase activity in
fed, fasted, and traumatized rats (n = 6 in all groups). Hindlimb muscle biopsies were homogenized, and equal
amounts of solubilized protein were immunoprecipitated with anti-IRS-1.
IRS-1-associated PI 3-kinase activity was assessed as described in
METHODS. PI 3-kinase activity was
calculated from quantitation of phosphorimage of
[32P]PI 3-kinase
products. Results are expressed as the mean ± SE percent increases
over fed control rats. * P < 0.05 vs. control fed rats.
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Akt phosphorylation.
The serine/threonine kinase Akt (protein kinase B/Rac), a downstream
target of PI 3-kinase, has been implicated to play a role in growth
factor signaling to glucose transport and glycogen synthesis (4, 7, 12,
25-27, 39, 44). Thus we assessed basal and insulin-stimulated Akt
phosphorylation in skeletal muscle from control or traumatized rats
(Fig. 4). Insulin induced a marked phosphorylation of Akt kinase in skeletal muscle from both control and
traumatized rats. Multiples of insulin stimulation could not be
calculated because of the complete lack of Akt phosphorylation under
basal conditions (Fig. 4). Surgical trauma resulted in a 2.2-fold
increase (P < 0.05) in
insulin-stimulated Akt phosphorylation (100 ± 9 vs. 221 ± 51%
for control vs. traumatized rats, respectively).

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Fig. 4.
Basal and insulin-stimulated protein kinase B (Akt) phosphorylation in
skeletal muscle from fed control or fasted traumatized rats was
determined in hindlimb skeletal muscle as described in
METHODS.
Top: representative immunoblot of
basal (B) and insulin-stimulated (I) Akt phosphorylation in control or
surgically traumatized rats. Bottom:
quantification of Akt phosphorylation in control
(n = 8) or surgically traumatized
(n = 8) rats. Results are expressed as
means ± SE. For each experiment, densitometric units of
each control insulin-stimulated sample were set to 100%.
* P < 0.05 vs. control fed
rats.
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Glucose transport in isolated soleus
muscle.
Glucose transport was studied in fasted controls
(n = 7) and traumatized
(n = 7) rats (Fig.
5). Basal (no insulin) glucose transport
was similar between the groups (0.77 ± 0.12 vs. 0.73 ± 0.13 µmol · ml
1 · h
1
for traumatized vs. fasted control rats, respectively). Furthermore, the response of glucose transport to a supraphysiological concentration of insulin was similar between the groups (2.87 ± 0.32 vs. 3.70 ± 0.41 µmol · ml
1 · h
1
for traumatized vs. fasted control rats, respectively). The insulin dose response for 3-O-methylglucose
transport was significantly different between fasted control and
traumatized rats (2-way ANOVA, P < 0.005). Post hoc analysis revealed that glucose transport activity
after submaximal insulin stimulation (100 and 200 µU/ml) was
significantly lower (P < 0.05) in
traumatized vs. fasted rats.

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Fig. 5.
3-O-methylglucose transport in soleus
muscle from fasted control and traumatized rats was determined as
described in METHODS. Basal glucose
transport was normalized to 0% and maximal insulin-stimulated rate of
glucose transport to 100%. Results are expressed as mean ± SE
percentages. * P < 0.05 vs.
control fed rats (2-way ANOVA).
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DISCUSSION |
Here we show that surgical trauma in combination with fasting leads to
increased insulin receptor phosphorylation and PI 3-kinase activity and
reduced insulin-stimulated glucose transport. Furthermore, surgical
trauma results in a moderate increase in the plasma concentrations of
glucose and epinephrine, with no change in cortisol levels. The reduced
submaximal insulin-stimulated glucose transport in skeletal muscle from
the traumatized rats suggests that the small bowel resection employed
in the present study was sufficient to induce altered glucose
homeostasis, a hallmark characteristic of surgical stress. Our finding
of reduced insulin-stimulated glucose transport in skeletal muscle is
consistent with previous reports in which marked peripheral insulin
resistance has been observed after different forms of catabolic stress,
including burns, sepsis, accidental trauma, and surgery in humans (3, 17, 23, 30, 41, 47, 48).
Fasting increases insulin signaling in skeletal muscle at the level of
the insulin receptor, IRS-1 (37) and PI 3-kinase (19). The increase in
insulin receptor phosphorylation after surgical stress in combination
with fasting was accompanied with an increase in PI 3-kinase activity
and an increased association of p85 to phosphotyrosines. Despite these
changes, insulin-stimulated glucose transport was reduced. Such a
dissociation between intermediate and final components of insulin
signaling has been observed previously. With streptozotocin-induced
diabetes, tyrosine phosphorylation of IRS-1 and IRS-1-associated PI
3-kinase activity is markedly increased in skeletal muscle (11) despite
severe insulin resistance (45). Thus trauma-induced insulin resistance
in skeletal muscle is neither at the level of the insulin receptor nor
at PI 3-kinase but may lie downstream from PI 3-kinase. Recently, the
serine/threonine kinase Akt (protein kinase B/Rac) has been suggested
to play a role in the signaling pathway to glucose transport (27, 39, 44). We have recently provided evidence that reduced insulin-stimulated Akt activity is associated with reduced glucose transport in muscle from diabetic Goto-Kakizaki rats (28). Interestingly, restoration of
glycemia completely normalized insulin action on Akt kinase (28).
Consequently, we hypothesized that reduced Akt kinase activity may lead
to decreased glucose transport in muscle after surgical trauma. Our
finding that surgical stress potentiates the effect of insulin on PI
3-kinase and Akt kinase in skeletal muscle does not support the
hypothesis that early or intermediate signaling defects contribute to
reduced glucose transport after surgical trauma. Alternatively, the
exocytotic machinery for GLUT-4 translocation may be altered by
surgical stress such that GLUT-4 vesicles fail to fuse or dock to the
plasma membrane, and thus glucose entry into the cell may be impaired.
Furthermore, hyperphosphorylation of early components of the insulin
signal transduction pathway may lead to negative feedback that inhibits
downstream signaling to glucose transport (11).
In the present study, insertion of a venous catheter in fed (i.e., a
minor trauma) but not in fasted rats resulted in a marked increase in
blood glucose. When fasted rats were subjected to a more severe trauma
(small bowel resection), blood glucose was significantly elevated. Thus
the degree of posttraumatic hyperglycemia appears to be dependent on
both the magnitude of trauma and on substrate availability. This
finding is consistent with previous findings observed in an animal
model of trauma and starvation (31).
Increased levels of NEFAs may reduce insulin action in skeletal muscle
(33). However, in the present study, NEFA levels were lower in
traumatized rats compared with fasted controls. Our finding is
consistent with previous observations in hyperglycemic postoperative
patients (24) and with reports of unchanged NEFA levels in septic
patients (47). Furthermore, we found no significant differences in
plasma lactate concentrations. Severe trauma may alter the circulatory
levels of insulin and insulin-antagonistic hormones such as epinephrine
and cortisol, which can affect glucose metabolism (29).
However, plasma insulin concentrations were similar between the fasted
and traumatized rats. Thus the defect in glucose transport was not due
to reduced insulin levels. After intestinal resection, plasma
concentrations of epinephrine were moderately increased, whereas
cortisol levels were not altered compared with time-matched controls.
The endocrine response found in the present study is similar to that
observed in postoperative patients, in which epinephrine and cortisol
levels are unaltered or moderately increased (17, 32, 41). Thus
increased levels of these hormones may be of minor importance for the
development of insulin resistance associated with sepsis or moderate
surgical stress in humans (32, 47).
In conclusion, insulin resistance in skeletal muscle after surgical
trauma is associated with reduced glucose transport but not with
impaired insulin signaling to PI 3-kinase. The surgical trauma model
presented in this report will be a useful tool to further elucidate the
molecular mechanism(s) underlying the development of insulin resistance
after surgical trauma.
 |
ACKNOWLEDGEMENTS |
This study was supported by grants from the Swedish Medical
Research Council (9517, 10402, 11823, 12211), the Swedish Cancer Society (2870-B96-06XCC), the Novo-Nordisk Foundation, and the Swedish
Diabetes Association. J. R. Zierath was the recipient of a Junior
Individual Grant from the Foundation for Strategic Research.
 |
FOOTNOTES |
Address for reprint requests: J. Permert, Dept. of Surgery, Huddinge
Hospital, 141 86 Huddinge, Sweden.
Received 29 October 1997; accepted in final form 10 April 1998.
 |
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