Anabolic action of insulin on skin wound protein is augmented
by exogenous amino acids
Xiao-Jun
Zhang,
David L.
Chinkes,
Øivind
Irtun, and
Robert R.
Wolfe
Metabolism Unit, Shriners Burns Hospital for Children and
Departments of Surgery and Anesthesiology, The University of Texas
Medical Branch, Galveston, Texas 77550
 |
ABSTRACT |
To
investigate the metabolic basis of skin wound healing, we measured in
anesthetized rabbits the responses of protein kinetics in scalded skin
to insulin and amino acids.
L-[ring-13C6]Phe was
infused on the 7th day after the ear was scalded, and the scalded ear
was used as an arteriovenous unit to reflect protein kinetics in skin
wound. The ipsilateral carotid artery was clamped to control the wound
blood flow within four- to fivefold the normal skin rate to measure the
enrichment difference in the scalded ear during hyperaminoacidemia.
Neither insulin (2.5 mU · kg
1 · min
1) nor
amino acid (2.5 mg · kg
1 · min
1) infusion
alone improved net protein balance in the skin wound. In contrast,
combined infusion of insulin and amino acids increased the net protein
balance in skin wound from
6.5 ± 4.5 to 1.4 ± 5.2 µmol · 100 g
1 · h
1
(P < 0.01, control vs. insulin plus amino acids). We
conclude that there is an interactive effect of insulin and sufficient amino acid supply on protein metabolism in skin wound, meaning that
their combined anabolic effect is greater than the sum of their
individual effects.
stable isotopes; gas chromatograph-mass spectrometer; rabbit ear; arteriovenous balance
 |
INTRODUCTION |
INSULIN IS A KEY ANABOLIC
HORMONE and not only plays an important role in substrate
metabolism but also is a regulator of protein synthesis and breakdown.
The effect of insulin on muscle protein has been investigated
extensively in the normal state (2, 6, 7, 9, 11-13, 18,
21) and also in catabolic states such as after severe burns
(5, 17). However, although the general role of insulin in
wound healing is well known (10, 14, 16), the effect of
insulin on protein metabolism in skin wound has not been assessed sufficiently.
The lack of information regarding the effect of insulin on protein
metabolism in skin wound is largely because of the lack of a method to
quantify both protein synthesis and breakdown in vivo. To this end, we
developed a rabbit ear model for measurement of protein metabolism in
skin wound (24, 26). Using this animal model, we
demonstrated in our previous experiment (24) that insulin
had an anabolic effect on wound protein resulting from the inhibition
of protein breakdown. It is possible that the failure of insulin to
stimulate protein synthesis was because of an insufficient supply of
amino acids (AAs). Although AAs were infused during the insulin
infusion, the plasma AA concentrations were only maintained at levels
that were either comparable to the postabsorptive state (during insulin
infusion at 0.6 mU · kg
1 · min
1) or even
lower (during insulin infusion at 2.3-3.4
mU · kg
1 · min
1).
Consequently, the potential role of sufficient AA supply in conjunction
with insulin on wound protein metabolism is not clear.
The present study was designed to investigate the potential interactive
or additive effect of insulin and abundant AA supply on protein
metabolism in skin wound. We selected a dose of insulin of 2.5 mU · kg
1 · min
1, which
is comparable to the higher-dose insulin (2.3-3.4
mU · kg
1 · min
1) in a
previous experiment (24). We used the same AA
solution (i.e., 10% Travasol) that we used in the previous experiment, but at a higher rate of infusion (2.5 mg · kg
1 · min
1). This dose
of AA infusion delivered AA nitrogen at a rate close to the average
rate of oral nitrogen intake of normal rabbits and was known from pilot
studies to increase plasma AA concentrations to more than two times the
postabsorptive levels.
 |
METHODS |
Animals.
Male New Zealand White rabbits (Myrtle's Rabbitry, Tompson Station,
TN), weighing ~4.5 kg, were used for this study. The rabbits were
housed in individual cages and were given 150 g of Lab Rabbit Chow
HF 5326 (Purina Mills, St. Louis, MO) per day for weight maintenance.
This feeding regime provided 4.7 g of crude protein and 38 kcal · kg
1 · day
1. This
study was approved by the Animal Care and Use Committee of The
University of Texas Medical Branch at Galveston.
Isotopes.
L-[ring-13C6]Phe (99%
enriched),
L-[ring-2H5]Phe (98%
enriched), and L-[2H7]proline
(97-98% enriched) were purchased from Cambridge Isotope Laboratories (Woburn, MA).
L-[ring-13C6]Phe was
used as the tracer for intravenous infusion.
L-[ring-2H5]Phe and
L-[2H7]proline were used as
internal standards for measurement of Phe and proline concentrations in
blood and in the free AA pool in skin wound.
Partial-thickness skin wound.
A partial-thickness thermal injury was created on the ear skin by
submerging the ear in 72°C water for 3 s, as we described in the
previous publications (24, 26). Immediately after the scald, a single dose of antibiotic (Bicillin; 50,000 U/kg; Wyeth Laboratories, Philadelphia, PA) was injected intramuscularly. When the
animals had awakened from anesthesia, they were given intramusclular
injections of buprenorphrine (0.03 mg/kg) two times a day for 3 days as
an analgesic.
Control of wound blood flow rate.
Our previous data showed that on the 7th day after injury the rate of
blood flow in the scalded skin was 9- to 15-fold greater than that in
the normal skin, and the arteriovenous (a-v) difference of tracer
enrichment was only 3-6% of the arterial enrichment (24). In a pilot study, we found that the infusion of a
large dose of AAs caused the a-v difference to become too small to
reliably measure. Consequently, we reduced the wound blood flow rate to within four- to fivefold the normal skin rate by clamping the ipsilateral carotid artery in the present study. Our previous experiments showed that the rates of protein synthesis in normal skin
and 7-day scalded skin were 12.8 ± 2.9 and 26.6 ± 8.7 µmol · 100 g
1 · h
1,
respectively (24, 26). We considered that a four- to
fivefold increase in wound blood flow should be sufficient to supply
nutrients for a twofold increase in synthesis. The clamping procedure
increased the a-v difference of tracer enrichment, thereby enabling
measurement of protein and AA kinetics in the skin wound under the
hyperaminoacidemic condition.
Experimental design.
There were five groups as follows: control group (n = 10), insulin group (n = 8), AA group (n = 7), insulin-AA group (n = 7), and microsphere group
(n = 4). The scald injury was created on the left ear
in the control, insulin, AA, and insulin-AA groups and on the right ear
in the microsphere group. After the scald injury, the rabbits were
given the same amount of food as before the injury, and daily food
consumption was recorded. The food was removed at 1700 on the 6th day,
and water was available all the time. After an overnight fast, the
isotope infusion or microsphere injection studies started at 0800 on
the 7th day. The rabbits, except in the microsphere group, received
L-[ring-13C6]Phe
infusion under different regimens of acute AAs and/or insulin infusion.
In the control group, neither insulin nor AAs were infused. In the
insulin and AA groups, either insulin (Humulin; Eli Lilly, Indianapolis, IN) or AAs (10% Travasol; Baxter Healthcare, Deerfield, IL) were infused. In the insulin-AA group, both insulin and AAs (i.e.,
10% Travasol) were infused. Insulin was infused at 2.5 mU · kg
1 · min
1 in 0.5%
albumin solution (10 ml/h), and the Travasol solution was infused at
2.5 mg · kg
1 · min
1 (1.5 ml · kg
1 · h
1). One hundred
milliliters of the Travasol solution contain 730 mg leucine, 600 mg
isoleucine, 580 mg lysine hydrochloride, 580 mg valine, 560 mg
phenylalanine, 480 mg histidine, 420 mg threonine, 400 mg methionine,
180 mg tryptophan, 2.07 g alanine, 1.15 g arginine, 1.03 g glycine, 680 mg proline, 500 mg serine, and 40 mg tyrosine.
The anesthetic and surgical procedures were described in our previous
publications (23-27). In brief, the rabbits were
anesthetized with ketamine and xylazine. Polyethylene catheters (PE-90;
Becton-Dickinson, Parsippany, NJ) were inserted in the right femoral
artery and vein through an incision on the groin. The arterial line was
used for blood collection and monitoring of heart rate and mean
arterial blood pressure; the venous line was used for infusion. A
tracheal tube was placed via tracheotomy. The free end of the tracheal tube was placed in an open hood that was connected to an oxygen supply
line so that the rabbits spontaneously breathed oxygen-rich room air.
The central artery of the scalded ear was isolated for placement of a
flow probe (model 1RB; Transonic Systems, Ithaca, NY). The flow probe
was connected to a small animal blood flowmeter (model T106; Transonic
Systems) for measurement of blood flow rate. The ipsilateral carotid
artery was isolated, and a bulldog clamp was placed on the artery to
control the blood flow rate in the scalded skin.
In the microsphere group, additional procedures were performed to
insert a 4.0-Fr polyurethane catheter (Cook Critical Care, Bloomington,
IN) in the left ventricle via the left carotid artery to inject
microspheres (26). Heparin (50 U/kg) was injected intravenously to prevent coagulation in the catheter placed in the left ventricle.
The isotope infusion protocol is shown in Fig.
1. After completion of the surgical
procedures, a blood sample was taken from the arterial line, and a skin
sample was taken from the groin incision for measurement of background
enrichment. The infusion of
L-[ring-13C6]Phe was
started after collection of the background samples in the control
group. In the insulin, AA, and insulin-AA groups, the infusion of
insulin (prime: 20 mU/kg; infusion rate: 2.5 mU · kg
1 · min
1) and/or
Travasol (prime: 100 mg/kg; infusion rate: 2.5 mg · kg
1 · min
1) was
started 1 h before the start of the tracer infusion. The dose of
L-[ring-13C6]Phe was
0.12 µmol · kg
1 · min
1
(prime: 4.8 µmol/ml) in the insulin group and 0.15 µmol · kg
1 · min
1 (prime:
6.0 µmol/ml) in the other three groups. In the insulin and insulin-AA
groups, a 25% glucose solution was infused at various rates to
maintain euglycemia. Arterial glucose concentration was measured every
10 min to estimate the rate of glucose replacement. Throughout the
experimental period, the position of the bulldog clamp was adjusted to
either totally or partially block the carotid artery to control the
wound blood flow rate within four- to fivefold the normal skin rate.
During the 150-240 min of the tracer infusion, four paired
arterial and ear-venous blood samples were drawn at intervals of ~20
min. The arterial blood was collected from the femoral artery catheter,
and the venous blood was obtained by directly puncturing the marginal
ear vein. The blood flow rate in the ear was recorded from the blood
flowmeter at each a-v blood sampling. At 240 min, a skin specimen was
taken from the ventral side of the scalded ear. The blood samples were
kept in an ice-water bath until the end of the infusion study. The skin
samples were immediately frozen in liquid nitrogen and stored at
80°C for later analysis. Additional blood was taken for measurement
of plasma concentrations of insulin and AAs. At the end of the
experiment, both ears were cut off at the skin fold between the base
and auricle to measure the ear weight.
In the microsphere group, the blood flow rate in the scalded skin was
recorded every 10 min in the first 40-60 min to obtain a baseline
value. When the blood flow rate reached a constant value, the
measurement of capillary flow was performed by injection of
microspheres into the left ventricle. The injections were performed when the ipsilateral carotid artery was either clamped or unclamped in
a random order. During the clamp periods, the blood flow rate in the
right scalded ear was reduced to four- to fivefold the normal skin rate
by adjusting the position of a bulldog clamp that was placed on the
ipsilateral carotid artery.
The colored dye-extraction microspheres (15 µm DIA Fluorescent
Microspheres; Interactive Medical Technology, Los Angeles, CA) were
injected into the left ventricle to measure capillary flow in the skin
wound. The injection procedure was described in detail in our previous
publication (26). Each injection contained 1.25 × 106 spheres of randomized colors. Each rabbit received
three injections of microspheres, which has been reported to be
acceptable in the rabbit (4). There was an interval of 15 min between injections. The rate of blood flow in the scalded ear was
recorded from the blood flowmeter at the time of each microsphere
injection. The animals were killed by intravenous injection of 5 ml of
saturated KCl under anesthesia. Skin samples were taken from the
scalded ears. Both kidneys were taken via laparotomy to ascertain if
the injected microspheres were uniformly distributed in the blood. The
tissues were kept in individual tubes, and the tissue weight was
recorded. The samples were shipped to Interactive Medical Technology
for analysis.
Heart rate, mean arterial blood pressure, and rectal temperature were
maintained stable by adjusting the infusion rates of anesthetics and
physiological saline and by use of heating lamps. These vital signs
were continuously monitored throughout the experiment and recorded
every 30 min. The surface temperature of the scalded skin was
maintained at 37°C by means of a heating lamp.
Sample analysis.
After completion of the isotope infusion study, an internal standard
solution, which contained 30 µmol/l of
L-[ring-2H5]Phe, was
added to the blood samples (50-75 µl to 0.25 ml blood), and the
samples were deproteinized by sulfosalicylic acid. The supernatant was
processed to make the t-butyldimethylsilyl (TBDMS) derivatives of AAs (20). Skin samples of ~30 mg were
homogenized in 5% perchloric acid three times at 4°C. The
supernatant was processed to make the TBDMS derivatives for measurement
of Phe enrichment in the free pool in skin wound (20).
To determine 19 AA concentrations (except proline) on an HPLC, 50 µl
of arterial plasma or 20 mg of tissue were mixed with 100 µl of
acetonitrile and 100 µl of standard solution that contained norvaline
(10 nmol/ml) and
-aminobutyric acid (30 nmol/ml). After centrifugation, the supernatant was transferred to centrifugal filtration vials (5000 NMWL filter unit; Millipore, Bedford, MA) and
centrifuged at 3,000 g for 5 h. The clear solution that
had passed through the filter was used for HPLC analysis. The protein precipitate was dried in an oven at 80°C to determine the dry weight
of the tissue. The weight difference between wet tissue and dry
precipitate was regarded as water content, which was used to calculate
the concentration of AAs in the free pool in skin wound.
Because the HPLC analysis did not include proline, we used the internal
standard method with mass spectrometry to measure proline
concentrations in biological fluids. Thus 50 µl of blood internal
standard solution (L-[2H7]proline
concentration: 60 µmol/l) were added to 0.25 ml of arterial blood,
and 30 µl of the tissue internal standard
(L-[2H7]proline concentration: 12 µmol/l) were added to 30 mg of tissue. The blood samples were
deproteinized by sulfosalicylic acid, and the tissue samples were
homogenized in 5% perchloric acid. After centrifugation, the
supernatant was processed for the
N-acetyl,n-propyl ester derivative of proline
(20).
The isotopic enrichments in the blood and tissue supernatants were
determined on a Hewlett-Packard 5980/5989B gas chromatograph-mass spectrometer; ions were selectively monitored at mass-to-charge ratios
of 234, 235, 239, and 240 for Phe enrichment and at mass-to-charge ratios of 200 and 207 for proline enrichment.
L-[ring-13C6]Phe
enrichment was corrected for the contribution of the abundance of
isotopomers of lower weight to the apparent enrichment of isotopomers with larger weight, and a skew correction factor was applied
(15). Isotope enrichment was expressed as the
tracer-to-tracee ratio for the internal standard method and as mole
percent excess for calculation of protein kinetics and AA transport.
Plasma insulin concentration was determined by the microparticle enzyme
immunoassay technique (1). AA concentrations (except proline) in plasma and in the free pool in skin wound were determined on an HPLC system (Waters 2690 HPLC system; Waters, Milford, MA) equipped with a Zorbax SB-C18 column. Blood glucose concentration was
measured on a glucose/lactate analyzer (model 2300; Yellow Springs
Instrument, Yellow Springs, OH). Blood hemoglobin (Hb) concentration
was measured on an automated hematology analyzer (model JT3; Coulter,
Hialeah, FL).
Calculations.
Protein and Phe kinetics in the scalded skin were calculated from the
following equations
|
(1)
|
|
(2)
|
|
(3)
|
|
(4)
|
|
(5)
|
|
(6)
|
|
(7)
|
|
(8)
|
|
(9)
|
|
(10)
|
|
(11)
|
where EA, EV, and ESK are
Phe enrichment in the arterial blood, venous blood, and free pool in
skin wound; CA and CV are Phe concentration in
the arterial and venous blood calculated by the internal standard
method (20); BF is blood flow rate in the scalded ear; and
NB is net balance. Equations 1-7 and 10 are published in our previous publications (27). Inflow is the
rate of AA entering the ear via the artery; inward transport is the rate of delivery from the artery to the free pool in skin wound; a-v
shunting is the rate of delivery directly from artery to vein; outward
transport is the rate of delivery from the free pool in skin wound to
ear vein; and outflow is the rate of AA exit via vein. Total synthesis
is the rate of protein synthesis using AAs from blood and endogenous
protein breakdown; total breakdown is the rate of AAs entering the free
pool in skin wound from endogenous protein breakdown. Thus the total
rate of appearance (total Ra) in the free pool in skin
wound is the sum of inward transport and total breakdown.
Equations 8 and 11 are derived from Galim et al.
(8). Because the arterial enrichment is used as precursor enrichment, Eq. 8 reflects the rate of synthesis from
blood-derived AAs (Sblood), which does not include
synthesis from breakdown-derived AAs (Sbreakdown);
Eq. 11 reflects the rate of AA release from breakdown into
the venous blood (Bblood), which does not include
reincorporation into protein. Consequently, Sbreakdown is
the difference between Stotal and Sblood (i.e.,
Eq. 9), which is also referred to as intracellular cycling.
The capillary flow measured from the colored microsphere method
was calculated by the following equation: flow rate = [(total tissue spheres)/(tissue weight in g) × (reference
spheres · ml
1 · min
1)]
(4).
Statistics.
Data are expressed as means ± SD. Differences between two
groups were evaluated by Student's t-test. Differences
among the four groups were evaluated using one-way ANOVA. Post hoc
testing was accomplished using the nonpaired Student's
t-test combined with Bonferroni's inequalities. A
P value <0.05 was considered statistically significant.
 |
RESULTS |
The rabbits ate little food during the first 48 h after
injury. Thereafter, the food intake increased gradually. On the 5th and
6th days, they all consumed 150 g rabbit chow/day as before the
injury. Table 1 lists the general
characteristics of the animals. There were no significant differences
in body weight, scald time, rectal temperature, arterial blood
O2 saturation, or blood flow rate in scalded skin among
groups. In the AA group, the heart rate was significantly
(P = 0.004) slower than in the insulin and insulin-AA
groups. The mean arterial blood pressure was significantly
(P = 0.02-0.04) lower in the insulin group than in
the control and AA groups. The physiological conditions in individual
rabbits remained stable during the tracer infusion, as demonstrated by
the small percentages of coefficients of variation (CV) of the measured
vital parameters in individual rabbits (n = 32):
CV = 0.6 ± 0.4% for rectal temperature; CV = 5.7 ± 3.0% for heart rate; CV = 5.3 ± 2.6% for mean arterial
blood pressure. The general characteristics of the four rabbits in the
microsphere group were not included in the statistical analysis, but
this group had characteristics similar to those of the other groups (Table 1).
Because of variable wound edema, the weight of scalded skin did not
entirely reflect skin mass. We dissected five pairs of normal and
scalded ears on the 7th day after injury. The wet weights of normal and
scalded ear skin were 13.1 ± 0.8 and 20.1 ± 3.5 g,
respectively (P = 0.0025). After baking in an oven at
80°C for 72 h, the dry weights were not significantly different
(4.14 ± 0.42 g for normal skin and 4.36 ± 0.66 g
for scalded skin; P = 0.15). Thus we used the
contralateral normal ear to estimate skin weight in the scalded ear to
avoid the complication of edema. The ear weight was multiplied by 0.78 because we previously determined that the skin accounts for 78% of ear
weight, with the remaining being ear cartilage (23).
Blood glucose concentrations were 9.9 ± 1.6 and 11.9 ± 0.9 mmol/l in the control and AA groups, respectively. These values represented a hyperglycemic response, because we reported in our previous publication (25) that the blood glucose
concentration in normal conscious rabbits after an overnight fast was
3.8 ± 0.3 mmol/l. In the insulin and insulin-AA groups,
euglycemia (5.3 ± 0.5 and 5.6 ± 0.8 mmol/l,
P > 0.05) was maintained by glucose infusion at
7.9 ± 3.0 and 7.6 ± 1.9 mg · kg
1 · min
1
(P > 0.05), respectively. Plasma insulin concentration
was not significantly different between control and AA groups (26 ± 26 and 40 ± 21 pmol/l in control and AA groups, respectively,
P > 0.05). Plasma insulin concentrations were raised
to 1,613 ± 158 and 1,680 ± 368 pmol/l (P > 0.05) in the insulin and insulin-AA groups, respectively.
Plasma AA concentrations are presented in Table
2. Compared with the control group,
infusion of insulin significantly (P < 0.05 or
P < 0.01) decreased plasma essential amino acid (EAA) concentrations (except tryptophan and methionine). Although the concentrations of some nonessential amino acids (NEAA) were also decreased in the insulin group (i.e., aspartic acid/asparagine, serine,
alanine, and proline), the total amount of NEAA was not significantly
(P = 0.47) different from that in the control group. In
contrast, the infusion of AAs significantly (P < 0.01)
increased almost all of the plasma AA concentrations (except tyrosine
and aspartic acid/asparagine). In the insulin-AA group, the total EAA
concentration was significantly (P < 0.01) greater
than that in the control group but less than in the AA group. AA
concentrations in the free pool in skin wound were not significantly
(P = 0.09 for EAA and P = 0.25 for
NEAA) different among groups. The AA concentrations in skin wound were
consistently higher (P < 0.01) than those in plasma
(Fig. 2, A and B).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 2.
A: concentrations of essential amino acids
(EAA) in the free pool in skin wound were not significantly different
between groups. Concentrations of EAA in plasma (open bars) were
significantly (* P < 0.01) lower than the
corresponding values in the free pool in skin wound (filled bars).
B: concentrations of nonessential amino acids (NEAA) in the
free pool in skin wound were not significantly different between
groups. Concentrations of NEAA in plasma (open bars) were significantly
(* P < 0.01) lower than the corresponding values in
the free pool in skin wound (filled bars). AA, amino acid.
|
|
Arterial Phe enrichment was at an isotopic plateau throughout the
150-240 min of the tracer infusion. Phe enrichment and
concentration in the arterial and venous blood and Phe enrichment in
the free pool in skin wound are presented in Table
3. The Phe concentrations in the
ear-venous blood have been corrected for water loss from the wound
surface. Because Hb remains in the vascular compartment when water
evaporates, the a-v difference of Hb reflects the extent of water loss.
We measured 25 pairs of arterial and ear-venous blood samples for Hb
concentration in the present study. The Hb concentrations were
118.6 ± 2.6 and 123.9 ± 2.8 g/l (P < 10
6 by paired t-test) in the arterial and
venous blood, respectively, and the corresponding blood flow rate was
32.7 ± 0.8 ml · 100 g
1 · min
1. These data mean that, to
account for the water loss from the wound surface, the measured venous
Phe concentration should be multiplied by 95.08%. Because the water
loss from the 7-day scalded skin can be considered constant under the
same experimental conditions, the a-v difference in Hb concentration
was inversely related to the blood flow rate. Therefore, we corrected
the venous Phe concentration for the blood flow rate in each rabbit as
follows: correction factor = [1
(32.7 × 4.92%/BF)], where 32.7 is the average blood flow rate
(ml · 100 g
1 · min
1) at the
time of collection of the a-v blood for Hb measurement; the percent
increase in Hb concentration from the artery to vein was 4.92%.
View this table:
[in this window]
[in a new window]
|
Table 3.
Phenylalanine enrichment and concentration in blood and phenylalanine
enrichment in the free amino acid pool in skin wound
|
|
Protein kinetics in the skin wound calculated from Eqs.
6-11 are presented in Table 4.
The insulin-AA group had the greatest value of net balance (1.4 ± 5.2 µmol · 100 g
1 · h
1),
which was significantly greater (P = 0.02 and
P = 0.001) than those in the control and insulin groups
(
6.5 ± 4.5 and
10.6 ± 6.6 µmol · 100 g
1 · h
1, respectively). The
difference in net balance between the control and insulin groups was
not significant (P = 0.27). The differences in total
protein synthesis (Stotal) did not reach statistical significance (P = 0.07) between groups. The rate of
total protein breakdown (Btotal) in the insulin group was
significantly (P = 0.01-0.02) greater than in the
control and insulin-AA groups. In the AA group, the rate of protein
synthesis from blood-derived AAs (Sblood) was significantly
(P = 0.04) greater than in the control and insulin
groups. The difference between the control and insulin-AA groups did
not reach significance (P = 0.09). The changes of the
rates of AAs release into blood from protein breakdown (Bblood) did not reach significance between groups
(P = 0.07). The rate of intracellular cycling
(Sbreakdown) in the insulin group was significantly greater
than that in other groups (P = 0.004 vs. control,
P = 0.02 vs. AA, and P = 0.03 vs.
insulin-AA).
Table 5 presents the values of Phe
transport that were calculated from Eqs. 1-5. Both the
Phe inflow into the ear via the artery and the rate of inward transport
from blood to the free pool in skin wound were significantly
(P = 0.01-0.005) greater in the AA group than the
corresponding rates in the control and insulin groups. There was a
significant difference between the rates of outward transport in the AA
and insulin-AA groups (P = 0.049) but not between the
control and AA groups (P = 0.07). In the insulin group,
the contribution of inward transport to total Ra in the free pool in skin wound (inward transport/total Ra) was
significantly (P = 0.001) less than in other groups. In
contrast, the contribution of breakdown (breakdown/total
Ra) in the insulin group was significantly (P = 0.001) greater than in the other groups. In the AA
group, the ratio inward transport/total Ra was
significantly (P = 0.03) greater than in the control
group and the ratio breakdown/total Ra was significantly
(P = 0.03) smaller.
In the microsphere group, the measured capillary flow rates in the
right and left kidneys were 4.57 ± 1.06 and 4.57 ± 1.30 ml · g
1 · min
1
(P = 0.15), indicating that the injected microspheres
were uniformly distributed in the blood stream. Six measurements were
performed when the carotid artery was not clamped, and the blood flow
rate measured by the ultrasonic blood flowmeter was 99.1 ± 25.9 ml · 100 g
1 · min
1. Another
six measurements were performed during the arterial clamp, and the
recorded blood flow rate from the flowmeter was 32.2 ± 5.6 ml · 100 g
1 · min
1. The
corresponding rates of capillary flow measured from the microsphere
technique were 29.9 ± 5.9 and 16.5 ± 3.4 ml · 100 g
1 · min
1 without and with
clamping, respectively. Because we placed a catheter in the left
carotid artery, the blood flow rate in the normal skin of the left ear
was reduced. Thus we used the capillary flow of normal ear skin of
3.4 ± 0.8 ml · 100 g
1 · min
1 from our previous study
(26) for comparison. The capillary flow rates in the
scalded skin were 8.8- and 4.9-fold the normal skin rate without the
arterial clamp and during the arterial clamp, respectively.
 |
DISCUSSION |
To repair skin defects, new proteins have to be deposited in the
wounded area to restore skin integrity. Therefore, the most important
parameter of wound protein kinetics is the net protein balance. In the
present study, the infusion of either insulin or AAs alone failed to
increase net protein balance in the skin wound. Only combined infusion
of insulin and AAs significantly increased net protein balance,
indicating an anabolic effect on protein in the skin wound. These data
support an interactive effect rather than an additive effect of insulin
and abundant AA supply on protein metabolism in the skin wound, because
the combined effect was greater than the sum of their individual effects.
Whereas insulin alone was not sufficient to induce an anabolic effect
on protein metabolism, it stimulated protein turnover in the skin wound
(Table 4). To obtain an insight into insulin's effect on wound protein
synthesis, we can consider protein synthesis as being composed of two
components (3). One component involves incorporation of
AAs from the arterial blood, and the other component involves
incorporation of AAs from endogenous protein breakdown within the
tissue (i.e., intracellular cycling). Similarly, the breakdown of
protein can also be divided into the following two components: one
component releases AAs into venous blood, and the other component
releases AAs for reincorporation into protein. An increase in
reutilization of AAs from protein breakdown will never lead to an
anabolic state, since 100% reutilization would yield a zero balance.
Only an increase in the use of blood-derived AA for synthesis could
lead to an improved net balance, provided that the component of
breakdown that releases AAs in blood is not concomitantly increased to
the same extent. In the insulin group, the component of synthesis using
blood-derived AAs was not increased, but the component of synthesis
using breakdown-derived AAs was significantly increased. Thus, although
insulin infusion alone increased the synthetic capacity, it was not
able to reduce net protein loss in the skin wound.
The protein kinetic data in the insulin group were supported by the Phe
transport data and AA concentrations in the free pool in skin wound.
Insulin infusion alone did not stimulate the inward transport of Phe
from arterial blood into the free pool in skin wound (Table 5).
Furthermore, in the free pool in skin wound, more Phe came from
endogenous breakdown, and less Phe came from inward transport in the
insulin group than in other groups. These findings are consistent with
the increased intracellular cycling, which uses breakdown-derived AAs
for protein synthesis. The concentrations of both EAA and NEAA in the
free pool in skin wound were not lower in the insulin group than in the
other groups (Fig. 2, A and B), which also
supports the absence of an increase in the net deposit of AAs into
wound protein. On the basis of the above findings, we propose that the
infusion of insulin stimulates the process of wound protein synthesis.
However, in the absence of exogenous AAs, insulin infusion causes a
systemic hypoaminoacidemia, so that, as a result, the inward transport
of AAs from artery to the free pool in skin wound is limited. The
increased demand for AAs for synthesis and limited availability of AAs
from blood trigger the acceleration of protein breakdown to maintain
the intracellular concentrations of AAs, which would otherwise be
reduced by the stimulation of synthesis. The outcome is that the rate
of protein turnover is accelerated and the net balance is not increased.
In the AA group, the plasma AA concentrations increased to over twofold
the control value (Table 2), which is consistent with increases in both
inflow via the artery and inward transport from the artery to the free
pool in skin wound (Table 5). When the contributions of Phe to protein
synthesis from the two sources are compared, AA infusion increased the
contribution of Phe from inward transport and decreased the
contribution of Phe from endogenous protein breakdown. However, the net
balance did not increase. The most likely explanation is that the
component of protein breakdown that releases AAs into blood was
concomitantly increased. In fact, the rate of Bblood was
greatest in the AA group, although the difference did not reach
significance (P = 0.07, see Table 4). The rate of
outward transport was also greatest in the AA group (Table 5). Thus it
seems that the changes in synthesis and breakdown in the AA group were
basically parallel, which may explain why there was no improvement of
net balance.
The combined infusion of insulin and abundant AA supply increased net
protein balance in skin wound. In general, an anabolic effect could be
induced by an increase in synthesis, decrease in breakdown, or both.
Because neither the increase in total synthesis nor the decrease in
total breakdown reached statistical significance in the insulin-AA
group, we cannot be certain of the mechanism responsible for the
increase in net balance. Most likely, there were additive effects of
changes in synthesis and breakdown that accounted for the improved net balance.
When comparison is made between the insulin-AA and insulin groups, the
insulin-AA group had significantly lower rates of total protein
breakdown and intracellular cycling. On the other hand, when comparison
is made between the insulin-AA and AA groups, the insulin-AA group had
a significantly lower rate of outward transport. These differences
suggest that the combined infusion of insulin and abundant AAs might
enable more efficient use of blood-derived AAs for synthesis while
simultaneously inhibiting the loss of AAs into blood.
Although the exact mechanism is not straightforward, the net anabolic
effect of insulin and exogenous AA supply on protein metabolism in skin
wound is conclusive. This is consistent with the notion that exogenous
AA supply plays an important role in insulin's anabolic effect on
protein metabolism in muscle (17, 19, 21, 22). In the
present experiment, we also determined the protein kinetics in the
skeletal muscle by using the hindlimb as an a-v unit. The responses of
muscle protein were basically the same as those of wound protein: only
combined infusion of insulin and AAs significantly increased the net
protein balance in muscle (data not shown).
In the present study, we reduced the rate of wound blood flow to within
four- to fivefold the normal skin rate by clamping the carotid artery.
This procedure increased the a-v difference of Phe enrichment to
10-20% of the arterial value (see Table 3), which enabled an
accurate determination of the a-v difference of Phe enrichment under
the hyperaminoacidemic condition. Even when the flow was clamped,
however, a-v shunting still accounted for 83-88% of Phe inflow
(estimated from Table 5). Thus at least some of the arterial inflow
likely did not provide nutritive flow to the tissues. We used the
microsphere technique to better assess the rate of capillary flow in
the skin wound. The results showed that, during the arterial clamping,
the capillary flow in the skin wound was approximately fivefold the
normal skin rate, so that there was sufficient blood perfusion in the
skin wound to enable the delivery of nutrients. This notion was
confirmed by the rate of protein synthesis. In the control group, the
rate of wound protein synthesis (32.7 ± 4.3 µmol · 100 g
1 · h
1) was 23% greater
(P > 0.05) than the control value in our previous experiment (26.6 ± 2.8 µmol · 100 g
1 · h
1) in which the wound blood
flow rate was not restricted (24). It is interesting that
the rate of the net protein loss in skin wound of the controls was
significantly (P < 0.05) lower than the previous value
(
23.1 ± 21.4 µmol · 100 g
1 · h
1) when blood flow rate was
not restricted (24). In the previous experiment, we also
found that the improvement of protein net balance in skin wound during
infusion of insulin and low-dose AAs was accompanied by a 41-44%
decrease of wound blood flow rate (24). It may be that a
rapid rate of blood flow increases the outward transport of AAs
resulting from a more extensive equilibration between the high
intracellular concentrations and the lower extracellular concentrations
(see Fig. 2, A and B). Thus, although our
experimental design did not enable us to assess the effect of blood
flow on wound protein metabolism, the experimental conditions provided adequate nutritive flow. Furthermore, the blood flow was similar in all
groups, so comparison of the metabolic effects of insulin and AAs was reasonable.
In summary, insulin's anabolic effect on protein metabolism in skin
wound required sufficient supply of exogenous AAs. Without exogenous AA
supply, insulin stimulated only the rate of intracellular cycling of
AAs. On the other hand, without insulin administration, infusion of a
large dose of a balanced AA solution (10% Travasol) was insufficient
to induce an anabolic response in the skin wound. Only the combined
infusion of insulin and abundant AAs significantly increased net
protein balance. These findings indicate an interactive rather than an
additive effect of insulin and exogenous AA supply.
 |
ACKNOWLEDGEMENTS |
We are grateful to the Animal Resource Center of The University of
Texas Medical Branch at Galveston for professional care of experimental
animals and to the Clinical Laboratory of the Shriners Hospital for
Children at Galveston, TX, for measurement of blood hemoglobin and
blood gas. We thank Yunxia Lin, Guy Jones, and Zhanpin Wu for technical assistance.
 |
FOOTNOTES |
This work was supported by Grants 8630 and 8490 from Shriners Hospital.
Address for reprint requests and other correspondence:
R. R. Wolfe, Shriners Burns Hospital, 815 Market St.,
Galveston, TX 77550 (E-mail:
rwolfe{at}utmb.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00361.2001
Received 7 August 2001; accepted in final form 4 February 2002.
 |
REFERENCES |
1.
Anderson, L,
Dinesen B,
Jørgensen PN,
Poulsen F,
and
Røder ME.
Enzyme immunoassay for intact human insulin in serium or plasma.
Clin Chem
39:
578-582,
1993[Abstract/Free Full Text].
2.
Biolo, G,
Declan Fleming RY,
and
Wolfe RR.
Physiological hyperinsulinemia stimulates protein synthesis and enhances transport of selected amino acids in human skeletal muscle.
J Clin Invest
95:
811-819,
1995[ISI][Medline].
3.
Biolo, G,
and
Wolfe RR.
Relationship between plasma amino acid kinetics and tissue protein synthesis and breakdown.
In: Modeling and Control Biomedical Systems. Proceedings of the IFAC Symposium, edited by Patterson BW.. Madison, WI: Omni-press, 1994, p. 358-359.
4.
Degens, H,
Craven AJ,
Jarvis JC,
and
Salmons S.
The use of colored dye-extraction microspheres to measure blood flow in rabbit skeletal muscle: a validation study with special emphasis on repeated measurements.
Exp Physiol
81:
239-249,
1996[Abstract].
5.
Ferrendo, AA,
Chinkes DL,
Wolf SE,
Martin S,
Herndon DN,
and
Wolfe RR.
A submaximal dose of insulin promotes net skeletal muscle protein synthesis in patients with severe burns.
Ann Surg
229:
11-18,
1999[ISI][Medline].
6.
Fryburg, DA,
Barrett EJ,
Louard RJ,
and
Gelfand RA.
Effect of starvation on human muscle protein metabolism and its response to insulin.
Am J Physiol Endocrinol Metab
259:
E477-E482,
1990[Abstract/Free Full Text].
7.
Fryburg, DA,
Jahn LA,
Hill SA,
Oliveras DM,
and
Barrett EJ.
Insulin and insulin-like growth factor-1 enhance human skeletal muscle protein anabolism during hyperaminoacidemia by different mechanisms.
J Clin Invest
96:
1722-1729,
1995[ISI][Medline].
8.
Galim, EB,
Hruska K,
Bier DM,
Matthews DE,
and
Haymond MW.
Branched-chain amino acid nitrogen transfer to alanine in vivo in dogs. Direct isotopic determination with [15N]leucine.
J Clin Invest
66:
1295-1304,
1980[ISI][Medline].
9.
Gelfand, RA,
and
Barrett EJ.
Effect of physiological hyperinsulinemia on skeletal muscle protein synthesis and breakdown in man.
J Clin Invest
80:
1-6,
1987[ISI][Medline].
10.
Greenway, SE,
and
Filler LE.
Wound healing in relation to insulin.
Int Surg
57:
229-232,
1972[Medline].
11.
Heslin, MJ,
Newman E,
Wolf RF,
Pisters PW,
and
Brennan MF.
Effect of hyperinsulinemia on whole body and skeletal muscle leucine carbon kinetics in humans.
Am J Physiol Endocrinol Metab
262:
E911-E918,
1992[Abstract/Free Full Text].
12.
Möller-Loswick, AC,
Zachrisson H,
Hyltander A,
Körner U,
Matthews DE,
and
Lundholm L.
Insulin selectively attenuates breakdown of nonmyofibrillar proteins in peripheral tissues of normal men.
Am J Physiol Endocrinol Metab
266:
E645-E652,
1994[Abstract/Free Full Text].
13.
Newman, E,
Heslin MJ,
Wolf RF,
Pisters PW,
and
Brennan MF.
The effect of systemic hyperinsulinemia with concomittant amino acid infusion on skeletal muscle protein turnover in the human forearm.
Metabolism
43:
70-78,
1994[ISI][Medline].
14.
Pierre, EJ,
Barrow RE,
Hawkins HK,
Nguyen TT,
Sakurai Y,
Desai M,
Wolfe RR,
and
Herndon DN.
Effects of insulin on wound healing.
J Trauma
44:
342-345,
1998[ISI][Medline].
15.
Rosenblatt, J,
Chinkes D,
Wolfe MH,
and
Wolfe RR.
Stable isotope tracer analysis by GC-MS, including quantification of isotopomer effects.
Am J Physiol Endocrinol Metab
263:
E584-E596,
1992[Abstract/Free Full Text].
16.
Rosenthal, SP.
Acceleration of primary wound healing by insulin.
Arch Surg
96:
53-55,
1968[ISI][Medline].
17.
Sakurai, Y,
Aarsland A,
Herndon DN,
Chinkes DL,
Pierre E,
Nguyen TT,
Patterson BW,
and
Wolfe RR.
Stimulation of muscle protein synthesis by long-term insulin infusion in severely burned patients.
Ann Surg
222:
283-297,
1995[ISI][Medline].
18.
Wolf, RF,
Heslin MJ,
Newman E,
Pearl-stone DB,
Gonenne A,
and
Brennan MF.
Growth hormone and insulin combine to improve whole body and skeletal muscle protein kinetics.
Surgery
112:
284-292,
1992[ISI][Medline].
19.
Wolfe, RR.
Nutrition and metabolism in burns.
In: Critical Care, edited by Shoemaker W.. Fullerton, CA: Soc Crit Care Med, 1986, vol. 7, p. 19-63.
20.
Wolfe, RR.
Radioactive and Stable Isotope Tracers in Biomedicine: Principles and Practice of Kinetic Analysis. New York: Wiley-Liss, 1992.
21.
Wolfe, RR.
Effects of insulin on muscle tissue.
Curr Opin Clin Nutr Metab Care
3:
67-71,
2000[Medline].
22.
Wolfe, RR,
and
Miller SL.
Amino acid availability controls muscle protein Metabolism.
Dial Nutr Metab
12:
322-328,
1999.
23.
Zhang, XJ,
Chinkes DL,
Doyle D, Jr,
and
Wolfe RR.
Metabolism of skin and muscle protein is regulated differently in response to nutrition.
Am J Physiol Endocrinol Metab
274:
E484-E492,
1998[Abstract/Free Full Text].
24.
Zhang, XJ,
Chinkes DL,
Wolf SE,
and
Wolfe RR.
Insulin but not growth hormone stimulates protein anabolism in skin wound and muscle.
Am J Physiol Endocrinol Metab
276:
E712-E720,
1999[Abstract/Free Full Text].
25.
Zhang, XJ,
Cortiella J,
Doyle D, Jr,
and
Wolfe RR.
Ketamine anesthesia causes greater muscle catabolism in rabbits than does propofol.
J Nutr Biochem
8:
133-139,
1997[ISI].
26.
Zhang, XJ,
Irtun Ø,
Zheng Y,
and
Wolfe RR.
Methysergide reduces nonnutritive blood flow in normal and scalded skin.
Am J Physiol Endocrinol Metab
278:
E452-E461,
2000[Abstract/Free Full Text].
27.
Zhang, XJ,
Sakurai Y,
and
Wolfe RR.
An animal model for measurement of protein metabolism in the skin.
Surgery
119:
326-332,
1996[ISI][Medline].
Am J Physiol Endocrinol Metab 282(6):E1308-E1315
0193-1849/02 $5.00
Copyright © 2002 the American Physiological Society