J Appl Physiol 117: 1380–1387, 2014.
First published September 25, 2014; doi:10.1152/japplphysiol.00175.2014.
Glucose and insulin administration while maintaining normoglycemia inhibits
whole body protein breakdown and synthesis after cardiac surgery
Roupen Hatzakorzian,1,2 Dominique Shum-Tim,3 Linda Wykes,4 Ansgar Hülshoff,1 Helen Bui,5
Evan Nitschmann,4 Ralph Lattermann,1 and Thomas Schricker1
1
Department of Anaesthesia, McGill University Health Center, Montreal, Canada; 2Department of Critical Care, McGill
University Health Center, Montreal, Canada; 3Department of Cardiovascular Surgery, McGill University Health Center,
Montreal, Canada; 4School of Dietetics and Human Nutrition, McGill University, Montreal, Canada; and 5Department
of Endocrinology and Metabolism, McGill University Health Center, Montreal, Canada
Submitted 25 February 2014; accepted in final form 24 September 2014
insulin; amino acids; protein breakdown; protein synthesis; cardiac
surgery
THE BODY’S METABOLIC RESPONSE
to surgical tissue trauma is
characterized by hyperglycemia, enhanced proteolysis, and
amino acid (AA) oxidation, resulting in nitrogen loss and
negative protein balance (33). This response to stress was first
described almost 80 years ago in four patients with lower limb
injuries (7). Since then the stress response to trauma and
surgery has been the focus of much attention in the medical
literature. The metabolic response to stress is mediated through
neural pathways and the neuroendocrine axis. Various horAddress for reprint requests and other correspondence: R. Hatzakorzian,
McGill Univ. Health Center, Royal Victoria Hospital, 687 Pine Ave. West
S5.05, Montreal, Quebec H3A 1A1, Canada (e-mail: roupen.hatzakorzian
@muhc.mcgill.ca).
1380
mones (catecholamines, cortisol) and cytokines [IL-1, IL-6,
and tumor necrosis factor (TNF)] are involved in the catabolic
response to the surgical tissue trauma. The main goal of this
response is restoration of adequate tissue perfusion, oxygenation, and release of substrates for the vital organs (8, 30). Over
the last few decades attempts have been made to find different
ways to modulate this exaggerated neurohormonal stress response to improve delivery and utilization of substrates. Some
strategies include changing surgical techniques, enhancing anesthetic strategies (neuraxial blockade), and introducing nutritional/metabolic modalities, for instance the hyperinsulinemicnormoglycemic clamp (HNC) technique (5, 21).
Stress hyperglycemia is the result of increased hepatic gluconeogenesis, glycogenolysis, and peripheral insulin resistance
(8). This rise in blood glucose levels is directly related to the
severity of the surgical stress. In patients undergoing cardiac
surgery, blood glucose concentrations can increase up to
10 –12 mmol/l for 24 h. The initial rise in blood glucose during
the surgical procedure is due to breakdown of hepatic glycogen. As the surgical stress continues and glycogen stores in the
liver get consumed and depleted, hepatic gluconeogenesis
becomes a significant contributor to glucose blood. AAs derived from muscle protein breakdown are one of the essential
substrates in hepatic gluconeogenesis along with lactate, pyruvate, and glycerol. As such, muscle proteolysis, the hallmark of
the catabolic response to surgical tissue trauma, is directly
related to glucose production (23). Because even moderately
increased blood glucose concentrations are associated with
poor clinical outcome after open heart surgery, insulin is being
used frequently to maintain normoglycemia (9, 12). Although
the pivotal role of insulin in the regulation of glucose homeostasis is well known, its effect on protein catabolism in this
patient population is not (28).
We recently demonstrated that insulin administered as part
of a hyperinsulinemic-normoglycemic clamp during open heart
surgery caused significant hypoaminoacidemia (15). Branchedchain amino acids decreased by almost 70%, while AAs linked
with the malate-aspartate cycle were reduced by 30%. As the
measurement of static plasma concentrations does not allow
any conclusion with regard to underlying dynamic metabolic
changes, it remained unclear whether hypoaminoacidemia was
a consequence of reduced proteolysis or increased incorporation of AA into newly synthesized proteins.
The purpose of the present study was to investigate the effect
of insulin on whole body protein kinetics in patients undergoing cardiac surgery. Our hypothesis was that hypoaminoacidemia in the presence of hyperinsulinemia and normoglycemia
8750-7587/14 Copyright © 2014 the American Physiological Society
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Hatzakorzian R, Shum-Tim D, Wykes L, Hülshoff A, Bui H,
Nitschmann E, Lattermann R, Schricker T. Glucose and insulin
administration while maintaining normoglycemia inhibits whole
body protein breakdown and synthesis after cardiac surgery. J Appl
Physiol 117: 1380 –1387, 2014. First published September 25,
2014; doi:10.1152/japplphysiol.00175.2014.—We investigated the
effect of insulin administered as part of a hyperinsulinemic-normoglycemic clamp on protein metabolism after coronary artery bypass
grafting (CABG) surgery. Eighteen patients were studied, with nine
patients in the control group receiving standard metabolic care and
nine patients receiving insulin (5 mU·kg⫺1·min⫺1). Whole body
glucose production, protein breakdown, synthesis, and oxidation were
determined using stable isotope tracer kinetics (L-[1-13C]leucine,
[6,6-2H2]
glucose) before and 6 h after the procedure. Plasma amino acids,
cortisol, and lactate were also measured. Endogenous glucose production (preoperatively 10.0 ⫾ 1.6, postoperatively 3.7 ⫾ 2.5
␮mol·kg⫺1·min⫺1; P ⫽ 0.0001), protein breakdown (preoperatively
105.3 ⫾ 9.8, postoperatively 85.2 ⫾ 9.2 mmol·kg⫺1·h⫺1; P ⫽ 0.0005)
and synthesis (preoperatively 88.7 ⫾ 8.7, postoperatively 72.4 ⫾ 8.4
mmol·kg⫺1·h⫺1; P ⫽ 0.0005) decreased in the presence of hyperinsulinemia, whereas both parameters remained unchanged in the control group. A positive correlation between endogenous glucose production and protein breakdown was observed in the insulin group
(r2 ⫽ 0.385). Whole body protein oxidation and balance decreased
after surgery in patients receiving insulin without reaching statistical
significance. In the insulin group the plasma concentrations of 13 of
20 essential and nonessential amino acids decreased to a significantly
greater extent than in the control group. In summary, supraphysiological hyperinsulinemia, while maintaining normoglycemia, decreased
whole body protein breakdown and synthesis in patients undergoing
CABG surgery. However, net protein balance remained negative.
Anticatabolic Effects of Insulin after Cardiac Surgery
is the result of suppressed protein breakdown rather than
stimulated protein synthesis.
METHODS
Study Population
Perioperative Care
All patients received standard surgical and anesthetic care as
established by the Departments of Anaesthesia and Cardiac Surgery at
the Royal Victoria Hospital, McGill University Health Centre. All
patients were operated on by the same surgeon (DST). General
anesthesia was induced with intravenous midazolam (0.1 mg/kg) and
sufentanil (1 ␮g/kg) and maintained with inhaled sevoflurane, intravenous sufentanil (0.5 ␮g·kg⫺1·h⫺1), and midazolam (40 ␮g·
kg⫺1·h⫺1). Hemodynamic monitoring was performed with catheters
placed in the right radial artery, the superior vena cava, and the
pulmonary artery. Tranexamic acid (2 g bolus followed by continuous
infusion of 5 mg·kg⫺1·h⫺1) was used as the antifibrinolytic agent
during surgery.
Prior to CPB heparin 400 U/kg was given intravenously to obtain
an activated clotting time ⬎ 500 s. The ascending aorta and the right
atrium were cannulated, the aorta was cross-clamped, and cardioplegia administered. Once coronary anastomoses were sutured and the
aortic cross-clamp removed, the patient was separated from CPB.
Intravenous dobutamine (2.5 ␮g·kg⫺1·min⫺1) was started if the cardiac index remained ⬍2.2 l·min⫺1·m⫺2 despite adequate fluid resuscitation. Intravenous norepinephrine (1–10 ␮g/min) was used if systolic blood pressure remained consistently ⬍100 mmHg.
One day before surgery (sx)
L-[1-13C]leucine-infusion
[6,6-2H2]glucose-infusion
[IC]
160
170 180
1381
After the administration of protamine (1 mg/100 U heparin) aortic
and venous cannulas were removed, homeostasis established, and the
pericardium and sternum closed. Patients were transferred to the
Intensive Care Unit (ICU) and ventilated to normocapnia. Sedation
was maintained using continuous infusions of propofol (10 –25
␮g·kg⫺1·min⫺1) until the end of the study. All subjects were maintained normothermic during the study period.
Study Protocol
Control group. Arterial blood glucose measurements were performed every 30 min using the Accu-chek glucose monitor (Roche
Diagnostics). If the blood glucose was ⱖ10.0 mmol/l, an insulin
(Humulin R regular insulin, Eli Lilly, Indianapolis, IN) bolus of 2 U
was given followed by the infusion of 2 U/h. The rate of insulin
infusion was adjusted according to the following sliding scale: 1)
⬍4.1 mmol/l, stop insulin infusion and administer 25 ml dextrose
50%; 2) 4.1– 6.0 mmol/l, stop insulin infusion; 3) 6.1–10.0 mmol/l,
maintain current infusion rate; and 4) ⬎10.0 mmol/l, increase infusion
by 2 U/h.
Insulin group. After obtaining a baseline blood glucose value, a 2
U priming bolus of insulin was given followed by an insulin infusion
of 5 mU·kg⫺1·min⫺1. Additional boluses of insulin were given if the
blood glucose remained ⬎6.0 mmol/l with incremental 2 U of insulin
for each 2 mmol/l increase in blood glucose. Ten minutes after
commencing the insulin infusion, and when the blood glucose was
ⱕ6.0 mmol/l, a variable continuous infusion of glucose (dextrose
20%) supplemented with potassium (40 meq/l) and phosphate (30
mmol/l) was administered to maintain the blood glucose between 4.0
and 6.0 mmol/l. Insulin infusions were continued until the end of the
study period. Arterial blood glucose was measured every 15–20 min.
Leucine Kinetics, Metabolic Substrates, and Hormones
Whole body leucine and glucose metabolism measurements were
made under postabsorptive conditions on the day before surgery and,
postoperatively, in the ICU (Fig. 1). Plasma kinetics of glucose and
leucine, i.e., the glucose and leucine rate of appearance (Ra), leucine
oxidation, and nonoxidative leucine disposal, were determined by a
primed constant infusion of tracer quantities of L-[1-13C]leucine and
[6,6-2H2]glucose. Blood and expired air samples were collected,
before the infusion, to analyze baseline enrichments. Priming doses of
NaH13CO3 (1 ␮mol/kg po), L-[1-13C]leucine (4 ␮mol/kg iv), and
[6,6-2H2]glucose (22 ␮mol/kg iv) were administered followed by the
infusion of L-[1-13C]leucine (0.06 ␮mol·kg⫺1·min⫺1) and [6,6-
Day of sx
During sx
0min 150
0800 am
Hatzakorzian R et al.
2 hours after sx
L-[1-13C]leucine-infusion
[6,6-2H2]glucose-infusion
Start of sx
End of sx
0min 150
2hrs post-sx
160
170
180
[IC]
Fig. 1. Study protocol. Whole body leucine and glucose
metabolism measurements made under postabsorptive
conditions on the day before surgery (sx) and, postoperatively, in the ICU.
HNC ------------------------------------------------------------
Isotopic enrichments of [1-13C]ketoisocaproate and [6,6-2H2]glucose in plasma and expired 13CO2
Hormones, metabolic substrates
[IC]
Indirect calorimetry
HNC
Hyperinsulinemic-normoglycemic clamp (insulin group only)
The control group did not receive HNC during and following surgery
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With the approval of the Research Ethics Board of the McGill
University Health Center we obtained written informed consent from
patients scheduled for elective coronary artery bypass grafting
(CABG) requiring cardiopulmonary bypass (CPB).
Exclusion criteria included severe malnutrition (weight loss ⬎20%
in the preceding 3 mo, albumin level ⬍35 g/l, and body mass index ⬍
20 kg/m2), morbid obesity (body mass index ⬎ 35 kg/m2), chronic
liver disease, severe left ventricular dysfunction (left ventricular
ejection fraction ⬍ 20%), cancer, and dialysis.
Using a computer program (Plan procedure; SAS software, Cary,
NC), consenting patients were randomly allocated to receive insulin or
standard metabolic care. The surgeon, nurses, and personnel performing the laboratory analyses were blinded to the patients’ group
assignment.
•
Anticatabolic Effects of Insulin after Cardiac Surgery
Table 1. Patient and surgical characteristics
Insulin
9
59.9 ⫾ 9.6
6/3
81.8 ⫾ 13.2
1.7 ⫾ 0.1
28.9 ⫾ 4.1
1
1
0
6.2 ⫾ 0.7
43.3 ⫾ 13.5
7
2
5
335 ⫾ 41
252 ⫾ 39
121 ⫾ 28
103 ⫾ 29
4.6 ⫾ 1.5
500 ⫾ 154
2.4 ⫾ 1.3
9
65.9 ⫾ 7.2
8/1
79.2 ⫾ 15.4
1.7 ⫾ 0.1
25.8 ⫾ 4.1
2
2
0
6.0 ⫾ 0.3
50.6 ⫾ 12.6
6
1
3
370 ⫾ 50
281 ⫾ 36
137 ⫾ 23
116 ⫾ 23
5.2 ⫾ 0.8
572 ⫾ 135
2.1 ⫾ 1.6
2
9
0
7
Values are means ⫾ SD or no. of patients. M, male; F, female; BMI, body
mass index; DM, diabetes mellitus; HbA1c, hemoglobin A1c; LVEF, left
ventricular ejection fraction; CPB, cardiopulmonary bypass; EBL, estimated
blood loss; PRBC, packed red blood cells. P values were determined by
unpaired t-test, ␹2 test, and Fisher’s exact test where appropriate.
H2]glucose (0.44 ␮mol·kg⫺1·min⫺1). For the determination of 13CO2
isotope enrichments four expired breath samples were taken after 150,
160, 170, and 180 min of isotope infusion. In addition four blood
samples were collected to determine whole body leucine and glucose
kinetics. Plasma concentrations of glucose, lactate, amino acids,
insulin, and cortisol were determined at 180 min of the pre- and
postoperative isotope infusion period.
Blood samples were immediately transferred to a heparinized tube,
centrifuged at 4°C, and the resulting plasma was stored at ⫺70°C until
analysis. Breath samples were collected through a mouthpiece in a
3-liter bag and transferred immediately to 10 ml vacutainers until
analysis. During the postoperative period, while the patients’ lungs
were ventilated, expired gases were collected by a one-way valve
connected to the ventilator.
Plasma ␣-[1-13C]ketoisocaproic acid (␣-[1-13C]KIC) enrichment
was analyzed by electron-impact selected-ion monitoring gas chromatography mass spectrometry (GC/MS) after derivatization to its
pentafluorobenzyl ester (Hewlett-Packard 5988A GC/MS, Palo Alto,
CA) (18).
Plasma glucose was derivatized to its penta-acetate compound, and
the [6,6-2H2]glucose enrichment determined by GC/MS using electron-impact ionization.
Isotopic enrichments were calculated as molecules percent excess
(MPE). Expired 13CO2 enrichment for the calculation of leucine
oxidation was analyzed by isotope ratio mass spectrometry (IRMS
Analytical Precision AP2, 003, Manchester, UK) (22).
Plasma glucose was measured by a glucose-oxide method using a
Glucose Analyzer 2 (Beckman Instruments, Fullerton, CA). Plasma
lactate was measured by an assay founded on lactate oxidase using the
Synchron CX system (Beckman Instruments, Fullerton, CA). Plasma
cortisol and insulin were analyzed by sensitive and specific doubleantibody radioimmunoassay (Amersham International, Amersham,
Bucks, UK).
The concentrations of the following 20 plasma AAs were measured
by high-performance liquid chromatography: isoleucine, leucine, va2
Hatzakorzian R et al.
line, lysine, methionine, phenylalanine, threonine, tryptophan, alanine, arginine, asparagine, citrulline, glutamate, glutamine, glycine,
histidine, ornithine, proline, serine, and tyrosine. Plasma samples were
deproteinized by the addition of 300 ␮l of methanol to 100 ␮l of
plasma followed by filtration with 0.2-␮m filters. All samples were
analyzed, in duplicate, using an Agilent 1290 UHPLC (Agilent
Technologies, Mississauga, Canada) on an Agilent Poroshell 120
EC-C18 4.6 ⫻ 150 mm 2.7 ␮m column. Plasma AAs were analyzed
by UHPLC. The AA were detected after precolumn derivatization
with o-phthalaldehyde (OPA) and 9-fluorenyl-methyl chloroformate
(FMOC). Separation was carried out at a flow rate of 1.5 ml/min under
gradient conditions. Mobile phase A was 10 mM Na2HPO4, 10 mM
Na2B4O7, pH 8.2, and mobile phase B was acetonitrile:methanol:
water (45:45:10). Initial gradient was 2% B for 0.5 min, then 2% to
57% B in 20 min followed by 5 min reequilibration before the next
injection. Primary and secondary AAs were measured by fluorescence
with excitation and emission wavelengths of 230 nm and 450 nm,
respectively. Data from standards and samples were analyzed using
MassHunter B.05 software.
Gaseous Exchange
Oxygen consumption and carbon dioxide production were measured by indirect calorimetry (Datex Instrumentation Deltatrac, Helsinki, Finland) over a period of 20 min toward the end of each isotope
infusion study. Average values were taken, with a coefficient of
variation (CV) ⬍10%.
Calculations
Whole body leucine and glucose kinetics were calculated by the
conventional isotope dilution technique using a two-pool random
model during steady-state conditions (19, 29). At isotopic steady state
the Ra of unlabeled substrate in plasma is derived from the plasma
isotope enrichment, expressed as MPE, according to the following
equation: Ra ⫽ I·(MPEinf/MPEpl ⫺ 1), where I is the infusion rate of
the tracer, MPEinf is the enrichment of the tracer in the infusate, and
MPEpl is the tracer enrichment in plasma. The final MPE values
represent the mean of all the MPE measurements during each isotopic
plateau. Isotopic steady-state conditions were regarded as valid when
the CV of the MPE values at isotopic plateau was ⬍5%.
At isotopic steady state, leucine flux (Q) is quantified by the
following formula: Q ⫽ S ⫹ O ⫽ B ⫹ I, where S is the rate of
synthesis of protein from leucine, O is the rate of oxidation, B is
Fig. 2. Perioperative blood glucose concentrations. CPB, cardiopulmonary
bypass. P value was determined by linear mixed models. *P ⬍ 0.05 vs. control.
Values are means ⫾ SD.
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N
Age, yr
Sex, M/F
Weight, kg
Height, m
BMI, kg/m2
DM
Oral agents
Insulin
HbA1c, %
LVEF, %
␤-Blockers
Calcium channel blockers
Renin-angiotensin inhibitors
Anesthesia time, min
Surgical time, min
CPB time, min
Aortic x-clamp time, min
Grafts, n
EBL, ml
PRBC transfusions, units
Inotropic and vasopressor therapy
Dobutamine (2.5–5.0
␮g·kg⫺1·min⫺1)
Norepinephrine (1–10 ␮g/min)
Control
•
Blood glucose (mmol(l)
1382
Anticatabolic Effects of Insulin after Cardiac Surgery
MPE [1-13C]α-KIC (%)
A
Time (min)
Hatzakorzian R et al.
1383
branched-chain amino acids, we expected a difference of at least 40%
between the two groups. To detect this difference with a type I error
of 5% and a power of at least 80%, nine patients in each group were
required.
Patients’ characteristics and perioperative data were compared
using the unpaired Student’s t-test, chi-square test, and Fisher’s exact
test where appropriate. Blood glucose levels at different time points
between the two groups were compared using linear mixed models.
Unpaired Student’s t-test was used to compare AA concentrations
between the two groups. Paired Student’s test was used to assess the
differences in the AA levels within the same group. The difference in
whole body leucine kinetics, gaseous exchange, and plasma concentrations of hormones and substrates between the two groups was
analyzed by using ANOVA for repeated measurements (before and
after surgery). The relationship between Ra leucine and endogenous
Ra glucose was evaluated by the correlation coefficient. All data are
presented as means ⫾ SD unless otherwise specified. Statistical
significance was set as P ⬍ 0.05. All P values presented are twotailed. All statistical analyses were performed using SAS 9.2 (SAS
Institute).
RESULTS
We enrolled and studied 18 patients with 9 in the control
group and 9 in the insulin group. Baseline characteristics and
surgical data were similar in the two groups (Table 1).
Blood glucose concentrations progressively rose in the control group without exceeding 10 mmol/l (Fig. 2). Hence no
protein breakdown, and I is the dietary intake. Furthermore Q is equal
to Ra (Ra ⫽ B ⫹ I) and the rate of disappearance (Rd; Rd ⫽ S ⫹ O).
When tracer studies are done in fasting states, leucine flux equals B.
The rate of protein synthesis is calculated by subtracting leucine
oxidation from leucine flux (S ⫽ Q ⫺ O). Protein balance is calculated as protein synthesis minus leucine Ra with positive values
indicating anabolism and negative values catabolism. Plasma ␣[1-13C]KIC is used to calculate the flux and oxidation of leucine. The
␣-KIC is formed intracellularly from leucine and is released into the
systemic circulation. It reflects the intracellular precursor pool enrichment more accurately than plasma leucine itself (20). In the calculation of leucine oxidation, a correction factor of 0.76 was used in the
fasted state to account for the fraction of 13CO2 released from leucine
but retained within slow turnover rate pools of the body (19).
Under postabsorptive conditions the Ra of glucose represents endogenous production of glucose. During glucose infusion, endogenous Ra of glucose was calculated by subtracting the glucose infusion
rate from the total Ra of glucose.
In the calculation of leucine oxidation in patients receiving the
hyperinsulinemic-normoglycemic clamp, correction was made for the
dilution effect of 13CO2 due to the low 13C content of the glucose
infusion. The correctional factor obtained in two additional clamp
studies without L-[1-13C]leucine was 10%.
Time (min)
B
MPE [6,6-2H2] glucose (%)
Time (min)
Fig. 3. A: isotopic enrichments of ␣-[1-13C]KIC before surgery. Values are
means ⫾ SD. B: isotopic enrichments of ␣-[1-13C]KIC after surgery. Values
are means ⫾ SD. MPE, molecules percent excess.
MPE [6,6-2H2] glucose (%)
A
Time (min)
Statistical Analysis
The primary end point of the study was the Ra of leucine on the first
postoperative day. Based on previously observed differences in
Fig. 4. A: isotopic enrichments of [6,6-2H2]glucose before surgery. Values are
means ⫾ SD. B: isotopic enrichments of [6,6-2H2]glucose after surgery.
Values are means ⫾ SD.
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MPE [1-13C]α-KIC (%)
B
•
1384
Anticatabolic Effects of Insulin after Cardiac Surgery
Hatzakorzian R et al.
MPE 13CO2 (%)
Endogenous Ra glucose (µmol·kg-1·min-1)
A
•
Time (min)
Ra leucine (µmol·kg-1·h-1)
B
MPE 13CO2 (%)
Time (min)
Fig. 5. A: isotopic enrichmentsof 13CO2 before surgery. Values are means ⫾
SD. B: isotopic enrichments of 13CO2 after surgery. Values are means ⫾ SD.
insulin was required. In the insulin group blood glucose levels
were kept in the normoglycemic range between 4.0 and 6.0
mmol/l (Fig. 2). No episode of hypoglycemia (blood glucose ⬍
3.5 mmol/l) was observed.
Isotopic plateaus of plasma ␣- [1-13C]KIC, [6,6-2H2]
glucose, and expired 13CO2 were obtained in all patients (Figs.
3, 4, and 5). There was no difference in preoperative leucine
and glucose kinetics between groups. Postoperative Ra leucine
and protein synthesis significantly decreased in patients receiving insulin and glucose, while protein kinetics did not change
in the control group (Table 2). There was a nonsignificant
decrease in leucine oxidation and balance after surgery in the
insulin group. Endogenous glucose production postoperatively
decreased in the insulin group and remained unchanged in the
control group (Table 2). A positive correlation between endogenous Ra glucose and Ra leucine was observed in patients
receiving insulin (Fig. 6, r2 ⫽ 0.385).
Whole body O2 consumption remained unchanged postoperatively in both groups. Whole body CO2 production and
respiratory quotient increased after surgery in the insulin group
indicating stimulated glucose oxidation (Table 3).
Lactate concentrations increased in both groups to a similar
extent after surgery (control group preoperatively 0.9 ⫾ 0.2,
postoperatively 1.6 ⫾ 0.8 mmol/l; insulin group preoperatively
0.7 ⫾ 0.3, postoperatively 1.3 ⫾ 0.3 mmol/l). Preoperative
plasma insulin (control group 51 ⫾ 24 pmol/l, insulin group
45 ⫾ 17 pmol/l) and cortisol (control group 173 ⫾ 74 nmol/l,
Table 2. Whole body leucine and glucose kinetics
P Value
Ra leucine, mmol·kg⫺1·h⫺1
Before surgery
After surgery
Leucine oxidation, mmol·kg⫺1·h⫺1
Before surgery
After surgery
Protein synthesis, mmol·kg⫺1·h⫺1
Before surgery
After surgery
Leucine balance, mmol·kg⫺1·h⫺1
Before surgery
After surgery
Endogenous Ra glucose, ␮mol·kg⫺1·min⫺1
Before surgery
After surgery
Control
Insulin
Time*
Group†
Interaction‡
95.0 ⫾ 15.1
97.5 ⫾ 12.9
105.3 ⫾ 9.8
85.2 ⫾ 9.2
0.0011
0.7594
0.0005
16.3 ⫾ 3.2
16.6 ⫾ 3.2
16.6 ⫾ 3.3
12.8 ⫾ 4.1
0.0656
0.1261
0.0811
78.6 ⫾ 12.3
80.9 ⫾ 10.3
88.7 ⫾ 8.7
72.4 ⫾ 8.4
0.0015
0.9273
0.0005
⫺16.3 ⫾ 3.2
⫺16.3 ⫾ 3.2
⫺16.6 ⫾ 3.3
⫺12.8 ⫾ 4.1
0.0656
0.1261
0.0811
9.3 ⫾ 1.2
12.7 ⫾ 1.2
10.0 ⫾ 1.6
3.7 ⫾ 2.5
0.0045
⬍0.0001
⬍0.0001
Values are means ⫾ SD. Ra, rate of appearance. P value was determined by ANOVA for repeated measures. *Probability that values change after surgery.
†Probability that values are different between the two groups. ‡Probability that postoperative changes are different between the two groups.
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Fig. 6. Relationship between rate of appearance (Ra) of leucine and endogenous rate of appearance of glucose in patients receiving insulin. Ra glucose ⫽
⫺9.3 ⫹ 0.17·Ra leucine. r2 ⫽ 0.385.
Anticatabolic Effects of Insulin after Cardiac Surgery
1385
Hatzakorzian R et al.
•
Table 3. Gaseous exchange
P Value
V̇O2, ml/min
Before surgery
After surgery
V̇CO2, ml/min
Before surgery
After surgery
RQ
Before surgery
After surgery
Control
Insulin
Time*
Group†
Interaction‡
224 ⫾ 40
241 ⫾ 43
235 ⫾ 45
233 ⫾ 43
0.4189
0.9308
0.3349
171 ⫾ 36
187 ⫾ 52
169 ⫾ 29
199 ⫾ 26
0.0076
0.7566
0.3464
0.73 ⫾ 0.04
0.73 ⫾ 0.05
0.73 ⫾ 0.02
0.86 ⫾ 0.10
0.0049
0.0100
0.0052
Values are means ⫾ SD. V̇O2, whole body oxygen consumption; V̇CO2, whole body carbon dioxide production; RQ, respiratory quotient. P value was
determined by ANOVA for repeated measures. *Probability that values change after surgery. †Probability that values are different between the two groups.
‡Probability that postoperative changes are different between the two groups.
DISCUSSION
The results of the present study demonstrate that insulin,
administered at 5 mU·kg⫺1·min⫺1 as part of a normoglycemic
clamp, reduces whole body protein breakdown by 20% and
synthesis by 19% after open heart surgery. This decrease in
proteolysis in the presence of supraphysiological hyperinsulinemia (plasma insulin at 3,600 pmol/l) was less pronounced
than that previously reported in healthy subjects. Fukagawa et
al. (11) showed a 25% lower leucine rate of appearance during
the infusion of 30 mU·m⫺2·min⫺1 insulin and a plasma insulin
level at 520 pmol/l, and a 30% inhibition of protein breakdown
with the infusion of 400 mU·m⫺2·min⫺1 insulin and a plasma
insulin concentration of 16,600 pmol/l. Tessari et al. (27)
Table 4. Plasma AA concentrations
Control
BL
ICU
Insulin
%Decrease
BL
ICU
P Value for Differences
%Decrease
Between groups
at BL*
Between groups
at ICU*
Between BL and
ICU in control
group†
Between BL and
ICU in Insulin
group†
0.4327
0.0217
0.0005
0.0386
0.7809
0.8033
⬍0.0001
0.0004
0.0132
0.0195
0.0033
0.4834
⬍0.0001
0.0001
0.0297
0.0002
0.0223
0.0025
0.0037
⬍0.0001
0.0019
⬍0.0001
⬍0.0001
0.0340
⬍0.0001
0.0002
⬍0.0001
0.0846
0.0289
0.0018
0.4195
0.9241
0.1164
0.0055
0.0318
0.0093
0.0034
0.0254
0.4775
⬍0.0001
0.0009
⬍0.0001
0.0151
0.0106
0.0032
0.0010
0.0268
⬍0.0001
0.0046
0.6074
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
0.0001
⬍0.0001
Plasma EAA concentrations, ␮mol/l
Histidine
Threonine
Valine
Methionine
Tryptophan
Phenylalanine
Isoleucine
Leucine
Lysine
67 ⫾ 6
122 ⫾ 40
237 ⫾ 30
19 ⫾ 5
48 ⫾ 7
82 ⫾ 10
73 ⫾ 9
143 ⫾ 15
168 ⫾ 19
58 ⫾ 10
75 ⫾ 26
230 ⫾ 42
8⫾4
28 ⫾ 7
67 ⫾ 12
40 ⫾ 13
116 ⫾ 27
120 ⫾ 18
14
38
3
60
42
18
45
19
28
Glutamate
Asparagine
Serine
Glutamine
Glycine
Citrulline
Arginine
Alanine
Tyrosine
Ornithine
Proline
100 ⫾ 69
38 ⫾ 7
85 ⫾ 16
627 ⫾ 93
168 ⫾ 51
34 ⫾ 10
75 ⫾ 21
368 ⫾ 91
68 ⫾ 14
74 ⫾ 20
306 ⫾ 140
111 ⫾ 50
20 ⫾ 6
52 ⫾ 8
420 ⫾ 80
118 ⫾ 16
22 ⫾ 3
44 ⫾ 9
250 ⫾ 84
57 ⫾ 17
42 ⫾ 7
176 ⫾ 87
⫺10
48
39
33
30
35
42
32
16
44
43
70 ⫾ 11
124 ⫾ 32
237 ⫾ 50
22 ⫾ 11
52 ⫾ 11
80 ⫾ 24
74 ⫾ 19
145 ⫾ 30
183 ⫾ 37
54 ⫾ 10
49 ⫾ 15
154 ⫾ 28
3⫾5
29 ⫾ 9
66 ⫾ 15
7⫾7
68 ⫾ 15
94 ⫾ 20
24
60
35
88
44
18
91
53
49
0.4354
0.9090
0.9991
0.4961
0.3770
0.8585
0.8530
0.8361
0.2679
Plasma NEAA concentrations, ␮mol/l
71 ⫾ 29
38 ⫾ 14
102 ⫾ 18
736 ⫾ 122
187 ⫾ 45
45 ⫾ 16
67 ⫾ 9
310 ⫾ 50
63 ⫾ 11
75 ⫾ 24
197 ⫾ 45
75 ⫾ 29
12 ⫾ 8
37 ⫾ 8
382 ⫾ 107
119 ⫾ 32
18 ⫾ 7
30 ⫾ 8
169 ⫾ 22
38 ⫾ 10
27 ⫾ 10
79 ⫾ 28
⫺6
67
64
48
36
61
55
45
40
65
60
0.2562
0.9942
0.0500
0.0500
0.4085
0.0994
0.3159
0.1125
0.4058
0.8977
0.0689
Values are means ⫾ SD. EAA, essential amino acids; NEAA, nonessential amino acids; BL, baseline. %Decreases are calculated from the mean values (%
of ICU values decrease from BL). *Unpaired t-test; †paired t-test.
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insulin group 131 ⫾ 57 nmol/l) concentrations were similar in
the two groups. In the insulin group plasma insulin levels were
significantly elevated compared with patients in the control
group (3,647 ⫾ 532 pmol/l vs. 52 ⫾ 35 pmol/l, P ⬍ 0.001).
Plasma cortisol levels increased postoperatively without showing any difference between the two groups (control group
793 ⫾ 381 nmol/l, insulin group 620 ⫾ 251 nmol/l), indicating
similar effects of anesthesia and analgesia on the endocrine
response to surgical stress.
Plasma AA concentrations were similar between the groups
at baseline. In the insulin group, the plasma concentrations of
13 of 20 essential (EAA) and nonessential AA (NEAA) decreased to a significantly greater extent than in the control
group (Table 4).
1386
Anticatabolic Effects of Insulin after Cardiac Surgery
Hatzakorzian R et al.
ability caused by the inhibition of leucine oxidation could,
therefore, directly stimulate protein synthesis (17).
In summary, we observed that achieving supraphysiological
levels of exogenous insulin while maintaining normoglycemia
after CABG surgery decreased whole body protein breakdown
and synthesis. However, net protein balance remained negative. It remains to be investigated whether anabolism can be
achieved in this patient population by the avoidance of hypoaminoacidemia, by simultaneous infusion of amino acids.
ACKNOWLEDGMENTS
We thank the nursing teams on the cardiac surgery ward and the Intensive
Care Unit for their valuable support. We also thank A. Wright for reviewing
the manuscript.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: R.H., D.S.-T., L.W., and T.S. conception and design
of research; R.H., A.H., E.N., and T.S. performed experiments; R.H., D.S.-T.,
L.W., A.H., H.B., E.N., R.L., and T.S. analyzed data; R.H., D.S.-T., L.W.,
A.H., H.B., E.N., R.L., and T.S. interpreted results of experiments; R.H., R.L.,
and T.S. prepared figures; R.H., L.W., H.B., and T.S. drafted manuscript; R.H.,
D.S.-T., L.W., A.H., H.B., E.N., R.L., and T.S. edited and revised manuscript;
R.H., D.S.-T., L.W., A.H., H.B., E.N., R.L., and T.S. approved final version of
manuscript.
REFERENCES
1. Barazzoni R, Short KR, Asmann Y, Coenen-Schimke JM, Robinson
MM, Nair KS. Insulin fails to enhance mTOR phosphorylation, mitochondrial protein synthesis and ATP production in human skeletal muscle
without amino acid replacement. Am J Physiol Endocrinol Metab 303:
E1117–E1125, 2012.
2. Bell JA, Fujita S, Volpi A, Gardenas JG, Rasmussen BB. Short-term
insulin and nutritional energy provision do not stimulate muscle protein
synthesis if blood amino acid availability decreases. Am J Physiol Endocrinol Metab 289: E999 –E1006, 2005.
3. Biolo G, De Cicco M, Lorenzon S, Dal Mas V, Fantin D, Paroni R,
Barazzoni R, Zanetti M, Iapichino G, Guarnieri G. Treating hyperglycemia improves skeletal muscle protein metabolism in cancer patients
after major surgery. Crit Care Med 36: 1768 –1775, 2008.
4. Castellino P, Luzi L, Simonson DC, Haymond M, DeFronzo RA.
Effect of insulin and plasma acid concentrations on leucine metabolism in
man. J Clin Invest 80: 1784 –1793, 1987.
5. Carvalho G, Pelletier P, Albacker T, Lachapelle K, Joanisse DR,
Hatzakorzian R, Lattermann R, Sato H, Marette A, Schricker T.
Cardioprotective effects of glucose and insulin administration while maintaining normoglycemia (GIN therapy) in patients undergoing coronary
artery bypass grafting. J Clin Endocrinol Metab 96: 1469 –1477, 2011.
6. Chevalier S, Gougeon R, Kreisman SH, Cassis C, Morais JA. The
hyperinsulinemic amino acid clamp increases whole-body protein synthesis in young subjects. Metabolism 53: 388 –936, 2004.
7. Cuthbertson DP. Observations on the disturbance of metabolism produced by injury to the limbs. Q J Med I: 233–246, 1932.
8. Desborough JP. The stress response to trauma and surgery. Br J Anaesth
85: 109 –117, 2000.
9. Estrada CA, Young JA, Nifong LW, Chitwood WR, Jr. Outcomes and
perioperative hyperglycemia in patients with or without diabetes mellitus
undergoing coronary artery bypass grafting. Ann Thorac Surg 75: 1392–
1399, 2003.
10. Flakoll PJ, Hill JO, Abumrad NN. Acute hyperglycemia enhances
proteolysis in normal man. Am J Physiol Endocrinol Metab 265: E715–
E721, 1993.
11. Fukagawa NK, Minaker KL, Rowe JW, Goodman MN, Matthews DE,
Bier DM, Young VR. Insulin-mediated reduction of whole body protein
breakdown. Dose-response effects on leucine metabolism in postabsorptive men. J Clin Invest 76: 2306 –2311, 1985.
12. Gandhi GY, Nuttall GA, Abel MD, Mullany CJ, Scaff HV, Williams
BA, Schrader LM, Rizza RA, McMahon MM. Intraoperative hypergly-
J Appl Physiol • doi:10.1152/japplphysiol.00175.2014 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on July 31, 2017
demonstrated comparable suppression of protein breakdown
with the administration of insulin at 500 mU·m⫺2·min⫺1 in
normal subjects after overnight fasting.
Similar to the observed change in protein breakdown, whole
body protein synthesis decreased in patients receiving insulin,
most likely a consequence of reduced substrate supply, i.e.,
hypoaminoacidemia. As demonstrated in a previous study in
cardiac surgery insulin administration led to a 30 – 60% decrease in the circulating concentrations of most AAs (15). By
contrast, patients with lung cancer and healthy young subjects
showed increased protein synthesis and positive protein balance if hypoaminoacidemia was avoided by exogenous provision of amino acids (6, 32) suggesting that the anabolic action
of insulin requires adequate substrate supply (1, 4). This
assumption is supported by studies in burn patients showing
anabolic effects of insulin on muscle protein synthesis when
iso- or hyperaminoacidemia was maintained (14).
Surgical stress induces transient insulin resistance, which
leads to an increase in hyperglycemia and an inability of
insulin to inhibit proteolysis via the phosphatidylinositol-3kinase (PI3K)/Akt pathway that activates two important ubiquitin protein ligases: Atrogen-1/MAFbx and MURF-1 (26, 28,
31). High-dose insulin, through its membrane receptors, overcomes insulin resistance and thereby decreases whole body protein wasting and lowers plasma AA levels. The lack of circulating
AAs is one of the main reasons why protein synthesis is inhibited
in the presence of insulin. However, this can be overcome by the
simultaneous provision of AAs (2, 6, 14).
Although the present study was not designed to dissect the
mechanisms responsible for the metabolic effects of insulin,
two additional factors may have contributed to the metabolic
effects of HNC: administration of glucose as part of the
normoglycemic clamp and strict maintenance of normoglycemia. Patients in the hyperinsulinemic-normoglycemic clamp
group received glucose at an average dose of 3.6 ⫾ 0.7
mg·kg⫺1·min⫺1. The administration of that amount of glucose
inhibits gluconeogenesis and, thus, lowers the need for glucoplastic AAs. In fact, similar to previous reports in surgical
patients a positive correlation between protein breakdown and
endogenous glucose production was observed in our patients
receiving insulin and glucose (24).
Hyperglycemia, per se, has been shown to exacerbate protein catabolism during critical illness and may blunt the anabolic response to feeding. In burn patients the extent of net
muscle protein catabolism increased proportionally with the
level of blood glucose (13). In contrast active glycemic control
was associated with less nitrogen loss in the critically ill (16).
After major cancer surgery, hyperglycemia induced by parenteral nutrition was accompanied by muscle protein catabolism,
while maintenance of normoglycemia restored a neutral protein
balance in these patients (3). The mechanism underlying the
anticatabolic influence of normoglycemia can be attributed to
the stimulation of skeletal muscle protein synthesis and the
decrease in extramuscular tissue proteolysis (3, 10). Hyperglycemia has been shown to be associated with a 50% greater
skeletal leucine oxidation rate when compared with normoglycemia (10). Moreover, hyperglycemia prompts the activation
of the enzyme branched-chain ␣-ketoacid dehydrogenase,
which catalyzes the initial irreversible step in the leucine
oxidation pathway (25). A greater intracellular leucine avail-
•
Anticatabolic Effects of Insulin after Cardiac Surgery
13.
14.
15.
16.
17.
18.
20.
21.
Hatzakorzian R et al.
1387
22. Schricker T, Wykes L, Carli F. Epidural blockade improves substrate
utilization after surgery. Am J Physiol Endocrinol Metab 279: E646 –
E653, 2000.
23. Schricker T, Wykes L, Carli F. Effect of epidural blockade on protein,
glucose and lipid metabolism in the fasted state and during dextrose
infusion in volunteers. Anesthesiology 92: 62–69, 2000.
24. Schricker T, Lattermann R, Fiset P, Wykes L, Carli F. Integrated
analysis of protein and glucose metabolism during surgery: effects of
anesthesia. J Appl Physiol 91: 2523–2530, 2001.
25. Shimomura Y, Obayashi M, Murakami T, Harris RA. Regulation of
branched-chain amino acid catabolism: Nutritional and hormonal regulation of activity and expression of the branched-chain alpha-keto acid
dehydrogenase kinase. Curr Opin Clin Nutr Metab Care 4: 419 –423,
2001.
26. Taniguchi CM, Emanuelli B, Kahn CR. Critical nodes in signaling
pathways: insights into insulin action. Nat Rev Mol Cell Biol 7: 85–96,
2006.
27. Tessari P, Trevisan R, Inchiostro S, Biolo G, Nosadini R, De Kreutzenberg SV, Inchiostro S, Duner E, Biolo G, Marescotti MC, Tiengo
A, Crepaldi G. Dose-response curves of effects of insulin on leucine
kinetics in humans. Am J Physiol Endocrinol Metab 251: E334 –E342,
1986.
28. Van den Berghe G. How does blood glucose control with insulin save
lives in the intensive care? J Clin Invest 114: 1187–1195, 2004.
29. Wagenmakers AJ. Tracers to investigate protein and amino acid metabolism in human subjects. Proc Nutr Soc 58: 987–1000, 1999.
30. Wilmore DW. Metabolic response to severe surgical stress: overview.
World J Surg 24: 705–711, 2000.
31. Wing SS. Control of ubiquitination in skeletal muscle wasting. Int J
Biochem Cell Biol 37: 2075–2087, 2005.
32. Winter A, MacAdams J, Chevalier S. Normal protein anabolic response
to hyperaminoacidemia in insulin-resistant patients with lung cancer
cachexia. Clin Nutr 31: 765–773, 2012.
33. Wolfe RR. Regulation of skeletal muscle protein metabolism in catabolic
states. Curr Opin Clin Nutr Metab Care 8: 61–65, 2005.
J Appl Physiol • doi:10.1152/japplphysiol.00175.2014 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on July 31, 2017
19.
cemia and perioperative outcomes in cardiac surgery patients. Mayo Clin
Proc 80: 862–866, 2005.
Gore DC, Chinkes DL, Hartl DW, Wolf SE, Herndon DN, Sanford
AP. Hyperglycemia exacerbates muscle protein catabolism in burn-injured
patients. Crit Care Med 30: 2438 –2442, 2002.
Gore DC, Wolf SE, Sanford AP, Herndon DN, Wolfe RR. Extremity
hyperinsulinemia stimulates muscle protein synthesis in severely injured
patients. Am J Physiol Endocrinol Metab 286: E529 –E534, 2004.
Hatzakorzian R, Carvalho G, Bui H, Sato T, Wykes L, Shum-Tim D,
Schricker T. High-dose insulin administration is associated with hypoaminocidemia during cardiac surgery. Metabolism 60: 1392–1397,
2011.
Hsu CW, Sun SF, Lin SL, Huang HH, Wong KF. Moderate glucose
control results in less negative nitrogen balances in medical intensive
care unit patients: a randomized, controlled study. Crit Care 16: R56,
2012.
Kimball SR, Jefferson LS. Signaling pathways and molecular mechanisms through which branched-chain amino acids mediate translational
control of protein synthesis. J Nutr 136: 227S–231S, 2006.
Mamer OA, Montgomery JA. Determination of branched-chain 2-hydroxy and 2-keto acids by mass spectrometry. Methods Enzymol 166:
27–38, 1988.
Matthews DE, Motil KJ, Rohrbaugh DK, Burke JF, Young VR, Bier
DM. Measurement of leucine metabolism in man from a primed, continuous infusion of L-[13C]leucine. Am J Physiol Endocrinol Metab 238:
E473–E479, 1980.
Matthews DE, Schwarz HP, Yang RD, Motil KJ, Young VR, Bier DM.
Relationship of plasma leucine and alpha-ketoisocaproate during a L-[113
C]leucine infusion in man: a method for measuring human intracellular
leucine tracer enrichment. Metabolism 31: 1105–1112, 1982.
Sato H, Carvalho G, Sato T, Bracco D, Codere-Maruyama T, Lattermann R, Hatzakorzian R, Takashi M, Schricker T. Perioperative tight
glucose control with hyperinsulinemic-normoglycemic clamp technique in
cardiac surgery. Nutrition 26: 1122–1129, 2010.
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