J Appl Physiol 95: 1083–1089, 2003;
10.1152/japplphysiol.01148.2002.
Differential responses of IGF-I molecular complexes to
military operational field training
Bradley C. Nindl,1 John W. Castellani,2 Andrew J. Young,3 John F. Patton,1
M. Javad Khosravi,4,5 Anastasia Diamandi,4 and Scott J. Montain3
1
Military Performance Division, 2Thermal and Mountain Medicine Division, 3Military Nutrition Division,
United States Army Research Institute of Environmental Medicine, Natick, Massachussetts 01760;
4
Diagnostic Systems Laboratories Canada, and 5Department of Laboratory Medicine and
Pathobiology, University of Toronto and Mount Sinai Hospital, Toronto, Canada M5G 1X5
Submitted 12 December 2002; accepted in final form 19 May 2003
somatotropic hormones; nutritional status; binary concentrations; magnetic exclusion separation; fasting
(IGF) I system plays a
pivotal role in mediating metabolic, mitogenic, and
anabolic cellular responses during altered energy
states (10, 16, 31, 34). The regulatory complexity of the
IGF-I system is demonstrated by its family of six binding proteins (BPs) that differentially modulate (i.e.,
either stimulate or inhibit) IGF-I bioavailability (2, 16,
18, 26). Given that some BPs potentiate and other BPs
inhibit IGF-I action, IGF-I and its family of BPs must
be viewed concurrently to glean greater spatial and
temporal resolution of underlying IGF-I physiology
comparing normal vs. perturbed conditions.
More than 75% of IGF-I circulates in a 150-kDa
ternary complex comprised of IGF-I, IGFBP-3, and an
acid labile subunit (ALS), with smaller proportions
circulating in a 30- to 50-kDa binary (IGF-I bound to
one of its smaller molecular mass BPs) or free forms
(16, 37, 40). The physiological relevance of assessing
nonternary concentrations resides in the fact that
IGF-I complexed in ternary form cannot pass out of the
vascular space, whereas the binary and free IGF-I
forms can pass through the capillary fenestrations and
bind to cell surface receptors. IGF-I possesses substantial hypoglycemic effects (5), but the ternary complex
affects the amount of IGF-I available to alter glucose
uptake. Changes in IGFBPs within the hormonal milieu are presumed to induce a shift in the relative
proportion of ternary vs. binary forms of IGF-I, yet few
investigations have directly evaluated this hypothesis.
A major environmental factor influencing IGF-I concentrations is nutritional status (2, 4, 9, 19, 20, 36, 40).
Caloric and protein restriction can reduce IGF-I gene
expression and circulating IGF-I and IGFBP concentrations (8, 13, 33). In studies that characterized the
long-term (i.e., 8 wk) physiological responses to military operational stress (prolonged physical exertion,
and caloric and sleep restriction) (11), IGF-I declined
abruptly and remained low until the combined stressors were removed, and the magnitude of IGF-I decline
was indirectly related to the magnitude of body mass
loss. Accompanying the decline in IGF-I during mili-
Address for reprint requests and other correspondence: B. C.
Nindl, Military Performance Division, United States Army Research
Institute of Environmental Medicine, Natick, MA 01760 (E-mail:
[email protected]).
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.
http://www.jap.org
THE INSULIN-LIKE GROWTH FACTOR
1083
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 17, 2017
Nindl, Bradley C., John W. Castellani, Andrew J.
Young, John F. Patton, M. Javad Khosravi, Anastasia
Diamandi, and Scott J. Montain. Differential responses of
IGF-I molecular complexes to military operational field training. J Appl Physiol 95: 1083–1089, 2003; 10.1152/japplphysiol.
01148.2002.—Insulin-like growth factor (IGF) I and IGF
binding proteins (IGFBPs) modulate metabolic activity and
tissue repair and are influenced by nutritional status. IGF-I
circulates in free, ternary [IGF-I ⫹ IGFBP-3 ⫹ acid labile
subunit (ALS)], and binary (IGF-I ⫹ IGFBP) molecular complexes, and the relative proportions regulate IGF-I extravascular shifting and bioavailability. This study examined the
hypothesis that sustained physical activity and sleep deprivation superimposed on a short-term energy deficit would
alter the IGFBP concentrations and alter the proportions of
IGF-I circulating in ternary vs. binary molecular complexes.
Components of the IGF-I system (total and free IGF-I;
IGFBP-1, -3, and ALS; nonternary IGF-I and IGFBP-3),
biomarkers of metabolic and nutritional status (transferrin,
ferritin, prealbumin, glucose, free fatty acids, glycerol, ␤-hydroxybutyrate), and body composition were measured in 12
men (22 ⫾ 3 yr, 87 ⫾ 8 kg, 183 ⫾ 7 cm, 20 ⫾ 5% body fat) on
days 1, 3, and 4 during a control and experimental (Exp)
period. During Exp, subjects performed prolonged work (energy expenditure of ⬃4,500 kcal/day) with caloric (1,600
kcal/day) and sleep (6.2 h total) restriction. IGF-I and
IGFBP-3 were measured by immunoassay before and after
immunoaffinity depletion of ALS-based complexes (i.e., ternary complex removal). Exp produced losses in body mass
(⫺3.0%), lowered total IGF-I (⫺24%), free IGF-I (⫺42%),
IGFBP-3 (⫺6%), nonternary IGF-I (⫺27%), and IGFBP-3
(⫺16%), and increased IGFBP-1 (256%). No Exp effects were
observed for ALS. No changes were observed in the proportion of IGF-I circulating in free (⬃1.2%), ternary (⬃87.4%), or
nonternary (⬃11.4%) molecular complexes. During Exp, glucose concentrations were lower on day 3, but days 1 and 4
were statistically similar. In conclusion, during a short-term
energy deficit in young, healthy men, 1) IGF-I system components differentially respond (both in direction and magnitude) to a given metabolic perturbation and 2) the relative
proportion of IGF-I sequestered in ternary vs. nonternary
molecular complexes appears to be well maintained.
1084
DIFFERENTIAL RESPONSES OF IGF-I MOLECULAR COMPLEXES
tary operational field training are losses of lean body
mass, decreased lymphocyte proliferation, and greater
insulin resistance (11, 27). How IGFBPs respond to
this multistressor environment and its impact on IGF-I
availability have not been studied.
This study examined the hypothesis that military
operational field training (sustained physical activity
and sleep deprivation superimposed on a short-term
energy deficit) would alter IGFBPs and alter the proportion of IGF-I circulating in ternary vs. binary molecular complexes. The specific objectives of this study
were to 1) assess IGF-I and IGFBP temporal responses
during 3 days of sustained physical exertion combined
with food and sleep restriction, 2) employ a novel magnetic separation procedure to measure nonternary (i.e.,
binary) IGF-I and IGFBP-3 concentrations, and 3) examine the relationship between changes in the IGF-I
system and other traditional markers of metabolic status.
Subjects. Twelve young, healthy male soldiers (23 ⫾ 1 yr,
85 ⫾ 4 kg, 180 ⫾ 3 cm, 19 ⫾ 1% body fat; means ⫾ SE)
volunteered for this investigation, which was approved by
United States Army Research Institute of Environmental
Medicine and Medical Research and Materiel Command human use review boards. Before study participation, subjects
listened to a thorough study briefing detailing study requirements and associated risks. Subjects then read and signed
volunteer affidavit agreements. All subjects were required to
pass a medical exam before entry into the study.
Experimental approach. A repeated-measures within-subject design was used to study alterations in IGF-I system
components and biomarkers of nutritional status during 3
days of military operational stress. The study was conducted
over a 2-wk period. During week 1 (control), morning (0630)
fasted blood was obtained on days 1, 3, and 4. Week 2 was the
experimental week (Exp) during which morning fasted blood
was also sampled on days 1 (before the subjects were exposed
to the experiment), 3, and 4 during sustained operations
(SusOps). During Exp, the soldiers completed field training,
which consisted of a variety of military-relevant tasks and
skills training. Physical activity and movement were recorded every minute by a wrist-worn actigraph (Mini Mitter,
Bend, OR, and Precision Control Designs, Ft. Walton Beach,
FL) to quantify amount of sleep obtained during Exp. Time
sleeping was calculated by summing the number of minutes
whose signal was zero or when at background levels. Food
and sleep restriction and sustained physical exertion were
deliberate stressors during the training. Caloric intake was
restricted to one meal ready to eat plus either a small breakfast (bagel, juice, and fruit) or a snack each day. The caloric
and nutritional composition of the daily rations were calculated from the meal ready to eat version and menu consumed,
manufacturer supplied labels, and American Diabetes Association Exchange values (see Table 1). Sleep was restricted
by only scheduling two 1-h blocks of sleep a day and keeping
the soldiers busy performing mental and physical tasks for
the majority of each day. The level of caloric restriction, sleep
deprivation, and prolonged work were chosen to model “realworld” scenarios expected by the US military in combat
situations. The physical activity of the military training
could be regarded as very light (map reading, etc.) to very
high (confidence course, repetitive box lifting, etc.). The maJ Appl Physiol • VOL
Total energy, kcal
Carbohydrate, g
Fat, g
Protein, g
Protein, g/kg BM
SusOps Day 1
SusOps Day 2
SusOps Day 3
1,680 ⫾ 28
226 ⫾ 11
53 ⫾ 4
77 ⫾ 2
0.9 ⫾ 0.1
1,507 ⫾ 17
203 ⫾ 8
62 ⫾ 2
43 ⫾ 3
0.5 ⫾ 0.1
1,788 ⫾ 24
263 ⫾ 8
50 ⫾ 2
80 ⫾ 2
0.9 ⫾ 0.1
Values are means ⫾ SE. SusOps, sustained operations; BM, body
mass.
jor contributor to energy expenditure was sustained aerobic
exercise associated with nightly road marches, land navigation, and patrolling activities (estimated exercise intensity of
30–40% maximal oxygen consumption). Other results from
this research study have been published elsewhere (7, 30).
Dual-energy X-ray absorptiometry. Body composition was
assessed by whole body dual-energy X-ray absorptiometry
(DXA). Total body estimates of %body fat, bone mineral
density, and bodily content of bone, fat, and nonbone lean
tissue were determined by using manufacturer-described
procedures and supplied algorithms (Total Body Analysis,
version 3.6, Lunar, Madison, WI). Precision of this measurement is better than ⫾0.5% body fat (27). Scanning was in
1-cm slices from head to toe by using the 20-min scanning
speed. Subjects had DXA scans performed before and the
morning after the study.
Blood collection. At 0630 on days 1, 3, and 4 of control and
Exp, morning fasting blood samples were obtained by venipuncture. Blood was allowed to clot at room temperature and
was centrifuged for 30 min at 3,000 g at 4°C. After centrifugation, serum was aliquoted into an appropriate storage vial,
flash frozen in liquid nitrogen, and stored at ⫺80°C for later
analysis. To eliminate interassay variances, all samples were
assayed within the same batch.
IGF-I system assays. Total IGF-I, free IGF-I, IGFBP-1, and
IGFBP-3 concentrations were determined by a two-site immunoradiometric assay (Diagnostic System Laboratories,
Webster, TX). The sensitivity of these assays were 2.06, 0.03,
0.33, and 0.05 ng/ml for total IGF-I, free IGF-I, IGFBP-1, and
IGFBP-3, respectively. The intra-assay variances for the
immunoradiometric assay were all ⬍5.3%. IGFBP-2 concentrations were determined by a radioimmunoassay (Diagnostic Systems Laboratories). The sensitivity of this assay
was 0.05 ng/ml. The intra-assay variance was ⬍6.2%.
To determine the extent of the nonternary (i.e., binary)
association, IGF-I and IGFBP-3 were also measured by the
Diagnostic Systems Laboratory (DSL) IGF-I and IGFBP-3
methods after immunoaffinity depletion of the ALS-based
complexes, as previously described (21). Briefly, a predetermined amount of anti-ALS antibody (DSL) was coupled to a
streptavidin-coated magnetic particle and used for immunodepletion of the ALS-based complexes. An optimized
amount of the antibody-coupled particles (300 ␮l of 1 mg/ml
particle suspension) was added to polystyrene tubes (12 ⫻ 75
mm), and the tubes were placed onto the magnetic separation
rack to side-separate the particles and completely remove the
particles’ suspension buffer. To each tube was then added 20
␮l of the serum samples and 20 ␮l of the assay buffer, which
was allowed to incubate for 2 h with continuous shaking. The
tubes were then placed on the magnetic separation rack, and
the ALS-depleted samples were assayed for IGF-I and
IGFBP-3, as well as ALS, to ensure complete removal of the
ALS immunoreactivity. In addition, the efficiency of ALS
removal was assessed by analyzing acromegalic samples
95 • SEPTEMBER 2003 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 17, 2017
METHODS
Table 1. Caloric intake over the course
of the SusOps week
DIFFERENTIAL RESPONSES OF IGF-I MOLECULAR COMPLEXES
1085
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 17, 2017
with high ALS content and showing the efficiency of the
method for removing ⬎99% of the ALS immunoreactivity
from 25-␮l aliquots of the samples (data not shown).
Nutritional and metabolic biomarker analyses. Serum glucose concentrations (BioChem, Lakewood, NJ), free fatty
acids (Wako Chemicals, Richmond, VA), glycerol (Sigma
Diagnostics, St. Louis, MO) and ␤-hydroxybutyrate (Sigma
Diagnostics) were measured according to manufacturer’s
instructions on an ATAC 8000 (Elan Diagnostics, Smithfield, RI). Ferritin and transferrin were determined with
solid-phase enzyme-linked immunoabsorbent assays (Bethyl
Laboratories, Montgomery, TX; American Laboratory Products, Windham, NH). Prealbumin concentrations were determined by using a nephelometric method (Minineph, The
Binding Site, San Diego, CA). The intra-assay variances
were all ⬍7%.
Statistical analyses. All data are presented as means ⫾
SE. A two (i.e., control vs. Exp) by three (i.e., days 1, 3, and
4) analysis of variance with repeated measures was employed
for statistical analyses for the study. Where appropriate, a
Duncan’s post hoc follow-up test was used. A P value of ⱕ0.05
was used for all statistical tests. Pearson product moment
correlations were calculated between the changes in IGF-I
measures and changes in nutritional and metabolic biomarkers and body composition between days 1 and 4 of the
SusOps week. All statistical analyses were performed with
Statistica software packages (StatSoft, Tulsa, OK).
RESULTS
Body composition. Subjects consumed an average of
1,653 ⫾ 25 kcal/day (225 ⫾ 6 g of carbohydrate, 54 ⫾
2 g of fat, 69 ⫾ 3 g of protein) and slept a total of 6.2 ⫾
0.4 h during the entire 72-h study period, as assessed
by actigraphy. Energy expenditure, estimated from
distance traversed and predicted energy cost of the
activities during the course, was ⬃4,500 kcal/day. Body
mass (control: 84.8 ⫾ 3.7 kg; Exp: 82.2 ⫾ 3.5 kg), fat
free mass (control: 69.0 ⫾ 2.7 kg; Exp: 67.7 ⫾ 2.5 kg),
fat mass (control: 15.8 ⫾ 1.7 kg; Exp: 14.5 ⫾ 1.8 kg),
and %body fat (control: 18.4 ⫾ 1.3; Exp: 17.4 ⫾ 1.5) all
significantly declined (P ⬍ 0.05) during Exp.
IGF-I system responses. The temporal responses of
the IGF-I system components are shown in Figs. 1 and
2. Most IGF-I system components were stable during
the control week, with the exceptions being nonternary
IGFBP-3 (decreased) and ALS (decreased). Total
IGF-I, nonternary IGF-I, and free IGF-I are shown in
Fig. 1. Compared with Exp day 1 concentrations, lower
values were observed for total IGF-I (Exp day 1: 289 ⫾
36 ng/ml ⬎ Exp day 3: 197 ⫾ 35 ng/ml ⫽ Exp day 4:
221 ⫾ 29 ng/ml), nonternary IGF-I (Exp day 1: 25.6 ⫾
1.6 ng/ml ⬎ Exp day 3: 18.2 ⫾ 1.5 ng/ml ⫽ Exp day 4:
18.7 ⫾ 1.3 ng/ml), and free IGF-I (Exp day 1: 3.3 ⫾ 0.5
ng/ml ⬎ Exp day 3: 1.7 ⫾ 0.3 ng/ml ⫽ Exp day 4: 1.9 ⫾
0.3 ng/ml). Figure 2 shows the responses of the IGF
system BPs. IGFBP-1 (Fig. 2A) increased (Exp day 1:
16 ⫾ 6 ng/ml ⬎ Exp day 3: 110 ⫾ 7 ng/ml ⫽ Exp day 4:
57 ⫾ 14 ng/ml) during Exp. IGFBP-3 (Fig. 2B; Exp day
1: 3,769 ⫾ 19.4 ng/ml ⬎ Exp day 3: 3,560 ⫾ 25.2
ng/ml ⫽ Exp day 4: 3,547 ⫾ 18.2 ng/ml) and nonternary
IGFBP-3 (Fig. 2C; Exp day 1: 451 ⫾ 19 ng/ml ⬎ Exp
day 3: 392 ⫾ 25 ng/ml ⫽ Exp day 4: 377 ⫾ 18 ng/ml)
both declined during Exp. Day effects, but not experiJ Appl Physiol • VOL
Fig. 1. Total insulin-like growth factor (IGF)-I (A), nonternary IGF-I
(B), and free IGF-I (C) responses on days 1, 3, and 4 during the
control (F) and sustained operations (SusOps; E) weeks. Means ⫾ SE
are shown. With the exception of nonternary IGF-I, all values were
stable during the control week. * Significantly different (P ⱕ 0.05)
from day 1 of the SusOps week.
mental effects, were observed for ALS (Fig. 2D; day 1 ⬎
day 3 ⫽ day 4).
Table 2 illustrates the proportion of IGF-I and
IGFBP-3 existing as nonternary molecular complexes.
The proportion of IGF-I (⬃11%) circulating in nonternary molecular complexes was apparently not affected
by the SusOps stress, whereas the proportion of IGFBP-3
circulating in nonternary molecular complexes exhibited
a small but significant decrease (12.1 vs. 11.6%).
95 • SEPTEMBER 2003 •
www.jap.org
1086
DIFFERENTIAL RESPONSES OF IGF-I MOLECULAR COMPLEXES
Fig. 2. IGF binding protein (IGFBP)-1 (A),
IGFBP-3 (C), nonternary IGFBP-3 (B), and acid
labile subunit (ALS; D) responses during days 1,
3, and 4 (x-axes) of the control and SusOps
weeks. Means ⫾ SE are shown. * Significant
difference (P ⱕ 0.05) from day 1 of the control or
SusOps week, respectively. Day effects, but not
experimental effects, were observed for ALS
(day 1 ⬎ day 3 ⫽ day 4).
DISCUSSION
Although energy restriction is known to reduce circulating IGF-I and alter IGFBP concentrations, the
impact of the combined stress of sustained physical
activity superimposed on an energy deficit and sleep
deprivation on the relative distribution of IGF-I circulating in free, ternary, and nonternary molecular forms
is unknown. Such information has physiological importance because IGF-I extravascular shifting, half-life,
and bioavailability are all regulated by the relative
proportion of IGF-I circulating in free form, or in binary or ternary complexes (14, 37, 38). The practical
significance of understanding how stress affects the
circulating forms of IGF-I resides in the fact that IGF-I
can exert metabolic control, and circulating levels may
be useful biomarkers of tissue remodeling, fitness, and
nutritional status (28). IGF-I shares many of insulin’s
actions and can affect glucose uptake and glycogen
synthesis (5, 25). As such, it might be expected that,
J Appl Physiol • VOL
during periods of metabolic strain, IGF-I and its family
of BPs would serve a counterregulatory role to sustain
glucose levels. The outcomes of this study provide a
comprehensive characterization of the IGF-I system in
response to several days of near-continuous work, underfeeding, and sleep deprivation.
Our data indicate that the military operational
stress scenario was physiologically challenging, as evidenced by the estimated daily caloric deficit (⬃3,000
kcal), the loss in body mass (⬃3.0%), and the limited
amount of sleep attained (total of 6.2 h). The 1.8% or
1.2-kg decline in DXA-assessed lean mass should be
interpreted with caution because of potential artifacts
introduced by short-term alterations in hydration status, although they do suggest that the subjects were in
a negative nitrogen balance. Total IGF-I, nonternary
IGF-I, free IGF-I, IGFBP-3, and nonternary IGFBP-3
all declined during Exp, whereas IGFBP-1 increased
and ALS was not altered. Contrary to our original
hypothesis, the reduction in IGF-I was proportionally
similar across all of the various molecular complexes
(i.e., ternary vs. nonternary), whereas the proportion of
IGFBP-3 circulating in ternary complexes exhibited a
small but significant increase. This study demonstrates that IGF-I system components differentially
respond (both in direction and magnitude) to the
unique metabolic perturbation of this study. These
dissimilar alterations presumably influence IGF-I extravascular shifting, half-life, and subsequent bioavailability.
IGF-I and IGFBP temporal responses. Total IGF-I
concentrations declined during SusOps and were 32
95 • SEPTEMBER 2003 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 17, 2017
Nutritional and metabolic biomarker responses. Table 3 displays changes in other measures of nutritional
and metabolic biomarkers during the control weeks
and Exp. Ferritin, free fatty acids, glycerol, and ␤-hydroxybutyrate increased during SusOps, whereas
transferrin was unchanged and prealbumin decreased.
Glucose concentrations were increased over SusOps
day 1 values on day 3 but not day 4. Correlational
analyses did not reveal any statistically significant
relationships between changes in any of the IGF-I
system components with the nutritional and metabolic
markers or body composition variables.
1087
DIFFERENTIAL RESPONSES OF IGF-I MOLECULAR COMPLEXES
Table 2. Proportion of IGF-I and IGFBP-3
circulating in various molecular complexes
during the control and SusOps weeks
IGF-I molecular complexes
Free IGF-I
Nonternary IGF-I
Ternary IGF-I
IGFBP-3 molecular complexes
Nonternary IGFBP-3
Ternary IGFBP-3
Control, %
SusOps, %
1.3 ⫾ 0.1
11.2 ⫾ 0.9
87.5 ⫾ 0.9
1.1 ⫾ 0.1
11.6 ⫾ 1.4
87.3 ⫾ 1.5
12.1 ⫾ 0.8
87.9 ⫾ 0.8
11.6 ⫾ 0.7*
88.4 ⫾ 0.7*
Values are means ⫾ SE and represent the marginal main effects
means from ANOVA. IGF, insulin-like growth factor; IGFBP, IGF
binding protein. * Significantly different from control (P ⬍ 0.05).
Table 3. Nutritional and metabolic biomarker changes during the control and SusOps weeks
Control Week
Ancillary Biomarkers
Nutritional biomarkers
Transferrin, ng/ml
Ferritin, ng/ml
Prealbumin, mg/dl
Metabolic biomarkers
Glucose, mg 䡠 dl⫺1 䡠 l⫺1
Free fatty acids, mEq/l
Glycerol, mM
␤-Hydroxybutyrate, mg/dl
SusOps Week
Day 1
Day 3
Day 4
Day 1
Day 3
Day 4
41.9 ⫾ 6.3
54.5 ⫾ 12.0
0.21 ⫾ 0.01
38.4 ⫾ 4.8
56.1 ⫾ 11.8
0.20 ⫾ 0.01
43.9 ⫾ 5.1
58.4 ⫾ 11.2
0.21 ⫾ 0.01
41.6 ⫾ 5.5
50.6 ⫾ 11.8
0.20 ⫾ 0.01
46.3 ⫾ 6.0
80.2 ⫾ 16.1*
0.20 ⫾ 0.01
44.4 ⫾ 6.1
88.7 ⫾ 17.2*
0.17 ⫾ 0.01*
96.5 ⫾ 2.5
0.24 ⫾ 0.05
0.14 ⫾ 0.01
2.40 ⫾ 0.13
96.6 ⫾ 2.0
0.27 ⫾ 0.05
0.14 ⫾ 0.01
2.31 ⫾ 0.13
96.3 ⫾ 1.7
0.31 ⫾ 0.06
0.13 ⫾ 0.01
2.34 ⫾ 0.15
99.3 ⫾ 1.9
0.20 ⫾ 0.02
0.14 ⫾ 0.02
2.43 ⫾ 0.11
86.1 ⫾ 6.1*
1.06 ⫾ 0.11*
0.17 ⫾ 0.018
4.63 ⫾ 0.61*
89.4 ⫾ 1.8
0.95 ⫾ 0.12*
0.17 ⫾ 0.01*
3.27 ⫾ 0.32*
Values are means ⫾ SE. All values were stable during control week. * Significantly different from SusOps day 1 value (P ⬍ 0.05).
J Appl Physiol • VOL
95 • SEPTEMBER 2003 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 17, 2017
and 23% lower at 48 and 72 h, respectively. IGF-I has
been shown to decline by 67 and 70% after 5 and 10
days of complete fasting in healthy adults (8, 15, 40)
and by ⬃50% after 14 days of military operational
stress in Army Ranger students (11). That nearly onehalf the magnitude of these reductions was achieved
after only 48 h in the present study underscores the
severity of the caloric imbalance and metabolic strain
imposed by our SusOps model. Previous studies suggest that, during negative energy balance, reductions
in circulating IGF-I are a direct result of lowered production from the liver (39, 40). These reductions occur
even in the presence of an amplification of growth
hormone profiles and may be influenced by other counterregulatory hormones (i.e., insulin and cortisol) (19,
20). During a 10-day fast in obese men, Clemmons et
al. (8) reported that IGF-I exhibited a linear and progressive fall, and Isley et al. (15) reported that this fall
was accelerated during a similar fast in normal-weight
subjects. These results are in contrast to the present
findings where the temporal response pattern exhibited a marked decline after 48 h followed by a slight
rebound over the next 24 h. Using a rat model, Frystyk
et al. (13) also demonstrated that the drop in IGF-I
after 2 days of fasting had plateaued by day 3. Thus the
temporal response pattern for IGF-I for the present
paradigm can be best characterized as declining, then
plateauing (i.e., nonlinear). One possible explanation
for this response could be the fact that our subjects
were ingesting daily calories and that the level of
energy provided may have been sufficient to support
the maintenance of IGF-I concentrations after an initial drop. Kupfer et al. (23) reported that the levels of
energy and protein that support normal IGF-I concentrations are ⬃20 kcal/kg body mass and 1 g protein 䡠 kg
body mass⫺1 䡠 day⫺1, respectively. Our subjects received ⬃18.8 kcal/kg body mass and 0.80 g protein 䡠 kg
body mass⫺1 䡠 day⫺1 (range ⫽ 0.6–1.1 g 䡠 kg body
mass⫺1 䡠 day⫺1). However, the protein content of the
diet was lower preceding the day 3 blood samples (see
Table 1). It might be possible that the acute changes in
protein intake influenced the observed responses of the
IGF-I system from day 3 to day 4.
Of the BPs measured, IGFBP-1 and IGFBP-3 were
most affected, with IGFBP-1 rising ⬃250% and
IGFBP-3 falling 6%. The striking increase in IGFBP-1
is consistent with previous literature on fasting (13,
16). IGFBP-1 is regulated by insulin and cortisol, and
is involved in glucose counterregulation (1). IGFBP-1 is
thought to exhibit an inhibitory effect on IGF-I action
by interfering with IGF-I ligand-receptor interactions,
(18) and increases in IGFBP-1 and -2 are associated
with decreases in free IGF-I (3, 16, 26). Frystyk et al.
(13) have speculated that the IGFBP-1 increase may be
viewed as a protective mechanism by neutralizing the
insulin-like hypoglycemic activity of IGF-I and serving
to mobilize energy from fat and protein. Compared
with total and free IGF-I and IGFBP-1 changes,
IGFBP-3 reductions were smaller in magnitude (⬃5%
decline from the control week to Exp). Day effects were
evident for ALS concentrations, although ALS was
seemingly unaffected by SusOps. ALS is also a component of the ternary complex and has been shown to be
reduced with fasting in many but not all studies. The
failure of the ALS to be influenced by the Exp was
surprising and suggests ALS responses lag behind
IGF-I and IGFBP-3. The modest decline in IGFBP-3
and the lack of change in the ALS ensure that IGF-I
would remain sequestered in the ternary complex and
would restrain the insulin-like effects of IGF-I on peripheral tissues (24). It would appear from our data
that the ternary complex is a stable reservoir not easily
disassembled, even during short-term metabolic stress
(6). Our findings also help to delineate a hierarchy with
regard to the magnitude of reduction because IGF-I ⬎
IGFBP-3 ⬎ ALS.
1088
DIFFERENTIAL RESPONSES OF IGF-I MOLECULAR COMPLEXES
J Appl Physiol • VOL
changes in the IGF-I system observed in this study, it
is likely that the major influence was the energy deficit.
Older et al. (32) have reported that 3 consecutive days
of delta-wave sleep interruption had no effect on IGF-I,
and acute physical exercise has either shown no
change or increases in circulating IGF-I (29).
In summary, our results indicate that short-term
military operational stress (i.e., caloric and sleep restriction superimposed on sustained physical exertion)
lowers IGF-I and IGFBP-3, as well as nonternary
IGF-I and nonternary IGFBP-3, and increases
IGFBP-1. These alterations were concomitant with
losses in body mass and lean mass but were not significantly correlated with changes in other traditional
biomarkers of nutritional status. The decline in IGF-I
was proportional among its various molecular complexes. In conclusion, during a short-term energy deficit associated with prolonged physical work and sleep
disruption in young healthy men, 1) IGF-I system
components differentially respond (both in direction
and magnitude) and 2) the relative proportion of IGF-I
sequestered in ternary vs. nonternary molecular complexes appears to be well maintained.
The authors gratefully acknowledge the dedicated and motivated
group of warfighters who participated in this arduous study. We also
are indebted to the many people who offered technical and logistical
support, without which successful completion of this study would not
have been possible. Special recognition goes to Cpt. Mark Kellogg,
Sgt. Dean Stulz, Sgt. David Degroot, Ssg. James Moulton, Spc.
Michele Ward, Scott Robinson, Janet Staab, Doreen Hafeman, Cara
D. Leone, Robert P. Mello, Louis Marchitelli, and Joe Alemany.
DISCLOSURES
The views, opinions and/or findings contained in this publication
are those of the authors and should not be construed as an official
Department of the Army position, policy, or decision unless so designated by other documentation. The investigators have adhered to
the policies of human subjects as prescribed in Army Regulation
70-25, and the research was conducted in adherence with the provisions of 45 CFR Part 46.
REFERENCES
1. Baxter RC. Insulin-like growth factor binding proteins as glucoregulators. Metabolism 44: 12–17, 1995.
2. Baxter RC. The binding protein’s binding protein—clinical applications of the acid-labile subunit (ALS) measurement. J Clin
Endocrinol Metab 82: 3941–3943, 1997.
3. Baxter RC. Insulin-like growth factor (IGF)-binding proteins:
interactions with IGFs and intrinsic bioactivities. Am J Physiol
Endocrinol Metab 278: E967–E976, 2000.
4. Baxter RC, Hawker FH, To C, Stewart PM, and Holman
SR. Thirty-day monitoring of insulin-like growth factors and
their binding proteins in intensive care units patients. Growth
Horm IGF Res 8: 455–463, 1998.
5. Binoux M. The IGF system in metabolism regulation. Diabetes
Metab 21: 330–337, 1995.
6. Boislair YR, Rhoads RP, Ueki I, Wang J, and Ooi GT. The
acid-labile subunit (ALS) of the 150 kDa IGF-binding protein
complex: an important but forgotten component of the circulating IGF system. J Endocrinol 170: 63–70, 2001.
7. Castellani JW, Stulz DA, Degroot DW, Blanchard LA, Cadarette BS, Nindl BC, and Montain SJ. 84 hours of sustained
operations alter thermoregulation during cold exposure. Med Sci
Sports Exerc 35: 175–181, 2003.
8. Clemmons DR, Klibanski A, Underwood LE, McArthur
JW, Ridgway EC, and Van Wyk JJ. Reduction of plasma
95 • SEPTEMBER 2003 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 17, 2017
Nonternary IGF-I and IGFBP-3 temporal responses.
The reduction in IGF-I after 48–72 h of negative energy balance was proportionally similar across all of
the various IGF-I molecular complexes. Thissen et al.
(38) reported that, in protein-restricted rats, the proportion of IGF-I in smaller molecular mass complexes
(⬃30 kDa), as measured by size-exclusion HPLC, increased from ⬃33% during control to 65% after protein
restriction. Certain pathophysiological conditions (i.e.,
growth hormone deficiency, starvation, tumor hypoglycemia) have also shown that the ratio of IGF-I bound to
the ternary vs. binary complexes is decreased (12, 16,
17). Furthermore, recent studies (29, 35) have demonstrated that both acute exercise and chronic physical
training can increase circulating concentrations of low
molecular mass IGFBPs.
Conceptually, a caloric deficit might lead to a preferential binding of IGF-I in nonternary complexes due
to a destabilization of the ternary complex (as evidenced by declines in either IGFBP-3 or ALS) and/or
an increase in other smaller molecular mass BPs (i.e.,
BP-1 and -2). Redistribution of IGF-I from ternary
complexes to lower molecular mass forms could facilitate IGF-I transport out of the vascular compartment
and toward tissues (29, 40). The present study did not
demonstrate that IGF-I was differentially partitioned
among its various molecular forms during 3–4 days of
underfeeding. Using protein malnutrition in a rat
model, Oster et al. (33) also failed to observe any
alterations in the ternary molecular complex. ALS is
an integral component of the ternary complex, and
because ALS is in 50–60% molar excess (1, 22), a
precipitous decline may be necessary to significantly
destabilize the ternary complex and alter IGF-I distribution between ternary and nonternary molecular
complexes. ALS disassociation from the ternary complex may be the important regulatory step in shifting
IGF-I from ternary to binary molecular complexes. It
may also be that a more severe energy deficit (either in
duration or magnitude) or a greater degree of protein
restriction than the present study (as occurs in critical
illness and other clinical conditions) is required before
there is a decrease in the proportion of IGF-I circulating in ternary complexes.
A question that the present study could not address
was whether the partitioning of IGF-I among the nonternary complexes was altered. There are six different
BPs for IGF-I, and the magnetic separation procedures
used in this study were not able to separate the relative
concentrations of the BPs in the IGF-I binary pool.
Because the IGFBPs are known to respond differentially to nutritional stress, it is possible that IGF-I was
preferentially complexed with BPs that increase with
fasting. The small but significant decrease in IGFBP-3
circulating in binary complexes and the increase in
IGFBP-1 after SusOps might suggest that there was a
shift in the distribution of IGF-I sequestered among
the BPs.
Although it is difficult to ascertain the precise contributions for the singular effects of physical exertion,
sleep deprivation, and energy restriction on the
DIFFERENTIAL RESPONSES OF IGF-I MOLECULAR COMPLEXES
9.
10.
11.
12.
13.
14.
15.
17.
18.
19.
20.
21.
22.
23.
J Appl Physiol • VOL
24. Lamson G, Giudice LC, Cohen P, Liu F, Gargosky S, Muller HL, Oh Y, Wilson KF, Hintz RL, and Rosenfeld RG.
Proteolysis of IGFBP-3 may be a common regulatory mechanism
of IGF action in vivo. Growth Regul 3: 91–94, 1993.
25. Le Roith D, Scavo L, and Butler A. What is the role of
circulating IGF-I? Trends Endocrinol Metab 12: 48–52, 2001.
26. Mohan S and Baylink DJ. Editorial: insulin-like growth factor
(IGF)-binding proteins in serum— do they have additional roles
besides modulating the endocrine IGF actions? J Clin Endocrinol Metab 81: 3817–3820, 1996.
27. Nindl BC, Friedl KE, Frykman PN, Marchitelli LJ, Shippee RL, and Patton JF. Physical performance and metabolic
recovery among lean, healthy men following a prolonged energy
deficit. Int J Sports Med 18: 317–324, 1997.
28. Nindl BC, Kellogg MD, Khosravi MJ, Diamandi A, Alemany JA, Pietila DM, Young AJ, and Montain SJ. Measurement of IGF-I during military operational stress via a filter
paper blood spot assay. Diabet Tech Therapeut 5: 455–461, 2003.
29. Nindl BC, Kraemer WJ, Marx JO, Arciero PJ, Dohi K,
Kellogg MD, and Loomis GA. Overnight responses of the
circulating IGF-I system after acute, heavy-resistance exercise.
J Appl Physiol 90: 1319–1326, 2001.
30. Nindl BC, Leone CD, Tharion WJ, Johnson RF, Castellani
JW, Patton JF, and Montain SJ. Physical performance responses during 72 hrs of military operational stress. Med Sci
Sports Exerc 34: 1814–1822, 2003.
31. Nindl BC, Scoville CR, Sheehan KM, Leone CD, and Mello
RP. Gender differences in regional body composition and somatotrophic influences of IGF-I and leptin. J Appl Physiol 92:
1611–1616, 2001.
32. Older SA, Battafarano DF, Danning CL, Ward JA, Grady
EP, Derman S, and Russel IJ. The effects of delta wave sleep
interruption on pain thresholds and fibromylagia-like symptoms
in healthy subjects; correlations with insulin-like growth factor-I. J Rheumatol 25: 1180–1186, 1998.
33. Oster MH, Fielder PJ, Levin N, and Cronin MJ. Adaptation
of the growth hormone and insulin-like growth factor-I axis to
chronic and severe calorie or protein malnutrition. J Clin Invest
95: 2258–2265, 1995.
34. Rajaram S, Baylink DJ, and Mohan S. Insulin-like growth
factor-binding proteins in serum and other biological fluids:
regulation and functions. Endocr Rev 18: 801–831, 1997.
35. Rosendal L, Langberg H, Flyvbjerg A, Frystyk J, Orskov
H, and Kjaer M. Physical capacity influences the response of
insulin-like growth factor and its binding proteins to training.
J Appl Physiol 93: 1669–1675, 2002.
36. Ross RJM. GH, IGF-I and binding proteins in altered nutritional states. Int J Obes 24: S92–S95, 2000.
37. Sara VR and Hall K. Insulin-like growth factors and their
binding proteins. Physiol Rev 70: 591–614, 1990.
38. Thissen J, Davenport ML, Pucilowska JB, Miles MV, and
Underwood LE. Increased serum clearance and degradation of
125
I-labeled IGF-I in protein-restricted rats. Am J Physiol Endocrinol Metab 262: E406–E411, 1992.
39. Thissen J, Underwood LE, and Ketelsegers J. Regulation of
insulin-like growth factor-I in starvation and injury. Nutr Rev
57: 167–176, 1999.
40. Thissen JP, Ketelslegers JM, and Underwood LE. Nutritional regulation of the insulin-like growth factors. Endocr Rev
15: 80–101, 1994.
95 • SEPTEMBER 2003 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 17, 2017
16.
immunoreactive somatomedin-C during fasting. J Clin Endocrinol Metab 53: 1247–1250, 1991.
Estivariz CF and Ziegler TR. Nutrition and the insulin-like
growth factor system. Endocrine 7: 65–71, 1997.
Florini JR, Ewton DZ, and Coolican SA. Growth hormone
and the insulin-like growth factor system in myogenesis. Endocr
Rev 17: 481–517, 1996.
Friedl KE, Moore RJ, Hoyt RW, Marchitelli LJ, MartinezLopez LE, and Askew EW. Endocrine markers of semistarvation in healthy lean men in a multistressor environment. J Appl
Physiol 88: 1820–1830, 2000.
Frost RA, Fuhrer J, Steigbige R, Mariuz P, Lang CH, and
Gelato MC. Wasting in the acquired immune deficiency syndrome is associated with multiple defects in the serum insulinlike growth factor system. Clin Endocrinol (Oxf) 44: 501–514,
1996.
Frystyk J, Delhanty PJD, Skjaerbaek C, and Baxter RC.
Changes in the circulating IGF system during short-term fasting
and refeeding in rats. Am J Physiol Endocrinol Metab 277:
E245–E252, 1999.
Holman SR and Baxter RC. Insulin-like growth factor binding
protein-3: factors affecting binary and ternary complex formation. Growth Regul 6: 42–47, 1996.
Isley WL, Underwood LE, and Clemmons DR. Dietary components that regulate serum somatomedin-C in humans. J Clin
Invest 71: 175–182, 1983.
Jones JI and Clemmons DR. Insulin-like growth factors and
their binding proteins: biological actions. Endocr Rev 16: 3–34,
1995.
Juhl A, Moller S, Mosfeldt-Laursen E, Rasmussen MH,
Scheike T, Pedersen SA, Kastrup KW, Yu H, Mistry J,
Rasmussen S, Muller J, Henriksen J, and Skakkebaek NE.
The acid-labile subunit of human ternary insulin-like growth
factor binding protein complex in serum: hepatosphlantic release, diurnal variation, circulating concentrations in healthy
subjects, and diagnostic use in patients with growth hormone
deficiency. J Clin Endocrinol Metab 83: 4408–4415, 1998.
Kelley KM, Oh Y, Gargosky SE, Gucev Z, Matsumato T,
Hwa V, Ng L, Simpson DM, and Rosenfeld RG. Insulin-like
growth factor-binding proteins (IGFBPs) and their regulatory
dynamics. Int J Biochem Cell Biol 28: 619–637, 1996.
Ketelslegers J, Maiter D, Maes M, Underwood LE, and
Thissen J. Nutritional regulation of the growth hormone and
insulin-like growth factor-binding proteins. Horm Res 45: 252–
257, 1996.
Ketelslegers J, Maiter D, Maes M, Underwood LE, and
Thissen J. Nutritional regulation of insulin-like growth factor-I.
Metabolism 10: 50–57, 1997.
Khosravi MJ, Diamandi A, and Mistry J. Nonternary complex IGF-I, IGF-II, and IGFBP-3 in normal, growth hormone
deficient (GHD) and acromegalic subjects. In: The Endocrine
Society 82nd Annual Meeting. Chevy Chase, MD: The Endocrine
Society, 2000, p. 483.
Khosravi MJ, Diamandi A, Mistry J, Ktrshna RG, and
Khare A. Acid-labile subunit of human insulin-like growth factor-binding protein complex: measurement, molecular, and clinical evaluation. J Clin Endocrinol Metab 82: 3944–3951, 1997.
Kupfer SR, Underwood LE, Baxter RC, and Clemmons
DR. Enhancement of the anabolic effects of growth hormone and
insulin-like growth factor-I by use of both agents simultaneously. J Clin Invest 91: 391–396, 1993.
1089