Altered secretion of growth hormone and luteinizing hormone after 84 ...

J Appl Physiol 100: 120 –128, 2006. First published September 1, 2005; doi:10.1152/japplphysiol.01415.2004.

Altered secretion of growth hormone and luteinizing hormone after 84 h of sustained physical exertion superimposed on caloric and sleep restriction Bradley C. Nindl,1 Kevin R. Rarick,1 John W. Castellani,2 Alexander P. Tuckow,1 John F. Patton,1 Andrew J. Young,3 and Scott J. Montain3 1

Military Performance Division, 2Thermal and Mountain Medicine Division, and 3Military Nutrition Division, United States Army Research Institute of Environmental Medicine, Natick, Massachusetts Submitted 23 December 2004; accepted in final form 24 August 2005

Nindl, Bradley C., Kevin R. Rarick, John W. Castellani, Alexander P. Tuckow, John F. Patton, Andrew J. Young, and Scott J. Montain. Altered secretion of growth hormone and luteinizing hormone after 84 h of sustained physical exertion superimposed on caloric and sleep restriction. J Appl Physiol 100: 120 –128, 2006. First published September 1, 2005; doi:10.1152/japplphysiol.01415.2004.—The pulsatile release of growth hormone (GH) and luteinizing hormone (LH) from the anterior pituitary gland is integral for signaling secretion of insulin-like growth factor (IGF)-I and testosterone, respectively. This study examined the hypothesis that 84 h of sustained physical exertion with caloric and sleep restriction alters the secretion of GH and LH. Ten male soldiers [22 yr (SD 3), 183 cm (SD 7), 87 kg (SD 8)] had blood drawn overnight from 1800 to 0600 every 20 min for GH, LH, and leptin and every 2 h for IGF-I (total and free), IGF binding proteins-1 and -3, testosterone (total and free), glucose, and free fatty acids during a control week and after 84 h of military operational stress. Time-series cluster and deconvolution analyses assessed the secretion parameters of GH and LH. Significant results (P ⱕ 0.05) were as follows: body mass (⫺3%), fat-free mass (⫺2.3%), and fat mass (⫺7.3%) declined after military operational stress. GH and LH secretion burst amplitude (⬃50%) and overnight pulsatile secretion (⬃50%), IGF binding protein-1 (⫹67%), and free fatty acids (⫹33%) increased, whereas leptin (⫺47%), total (⫺27%) and free IGF-I (⫺32%), total (⫺24%) and free testosterone (⫺30%), and IGF binding protein-3 (⫺6%) decreased. GH and LH pulse number were unaffected. Because GH and LH positively regulate IGF-I and testosterone, these data imply that the physiological strain induced a certain degree of peripheral resistance. During periods of energy deficiency, amplitude modulation of GH and LH pulses may precede alterations in pulse numbers.

THE ENDOCRINE RESPONSE TO sustained physical exertion superimposed on caloric and sleep restriction serves to provide adequate fuels to meet metabolic needs for such physiological processes as tissue repair, regeneration, and recovery (5, 7, 20). In particular, the somatotropic [i.e., growth hormone (GH)/ insulin-like growth factor (IGF)-I] and pituitary-testicular [i.e., luteinizing hormone (LH)/testosterone] axes mediate many metabolic and anabolic processes during altered energy states (15, 16, 24, 32). The pulsatile release of GH and LH from the anterior pituitary gland is integral for the trophic effects that these hormones exert on IGF-I and testosterone, respectively (35, 36). The manner in which anterior pituitary hormones are released and delivered to target tissues is more biologically

significant than the overall mean hormonal concentrations was well illustrated by Isgaard et al. (17), who demonstrated that GH administered in a pulsatile manner elicited greater increases in IGF-I than GH administered in a continuous manner. Because of the episodic release pattern of GH and LH, multiple time-point measures are necessary to fully characterize the trophic effects these hormones may have (18, 34, 35). Although alterations in IGF-I and testosterone are known to be mediated by fluctuations in GH and LH, few studies have concomitantly measured these hormones, especially in healthy subjects exposed to multiple physiological stressors. Additionally, recent work has suggested that leptin may also serve as an integrator of neuroendocrine secretion of GH and LH (5, 24, 25). Information concerning the pulse parameters (i.e., frequency, amplitude, etc.) of GH and LH is important to understand when delineating central vs. peripheral mechanisms underlying alterations in circulating endogenous hormones. Although prior pulsatility programs (i.e., Pulsar, etc.) were limited in that they provided only descriptive information about hormone concentration peaks, recent advances in time series software analysis now allow accurate determination of underlying glandular hormonal secretion rate and half-life from measurements obtained from the systemic circulation (34 –36). Military operational scenarios represent a unique paradigm in which to study endocrine responses to metabolic stress. Soldiers, analogous to emergency rescue personnel (i.e., wildlife firefighters, medical response teams, etc.) and ultra endurance athletes (i.e, cyclists during the Tour de France, participants in the Eco challenge, etc.), are often required to perform heavy sustained physical exertion in the face of nutritional and sleep deprivation. Prior work on military populations has shown that continued and prolonged exposure to such physiological strain can lead to a suppression of circulating anabolic growth factors, losses of lean body mass, increased susceptibility to disease and infections, and diminished physical and cognitive performance capabilities (4, 8, 9, 19, 27). For example, Friedl et al. (9) extensively evaluated the effects of 62 days of United States Army Ranger training during which young healthy soldiers average losses of 15% in body mass, 7% in fat-free mass, 65% in fat mass, and 20% decline in strength and power, with 65 and 86% declines in IGF-I and testosterone, respectively. The causal mechanisms underlying such overreaching and overtraining phenomena, particularly the shortterm endocrine and metabolic adaptations that may precede critical functional impairments (i.e., chronic fatigue syn-

Address for reprint requests and other correspondence: B. C. Nindl, Military Nutrition 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.

hormone pulsatility; insulin-like growth factor-I; soldiers; leptin; testosterone

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ALTERED GH AND LH SECRETION

drome), are still poorly understood. Establishing a greater insight into the fundamental origin of such processes could lead to the identification of suitable “biomarkers” that once validated could lead to early diagnostic and intervention strategies aimed at buffering and sustaining peak physical performance. Although much of the prior research on overtraining has concentrated on expedient, “snapshot” biochemical measures in the periphery, we hypothesized that alterations in the neuroendocrine release of GH and LH from the anterior pituitary gland would also potentially represent a regulatory mechanism in the early phase adaptational process after exposure to multiple physiological stressors. The purpose of the present study was to further characterize the physiological consequences of short-term military operational stress on the secretory patterns of GH and LH release as analyzed by deconvolution algorithms by obtaining serial blood samples (every 20 min) drawn for 12 h overnight from soldiers before and after 4 days of military operational stress. The level of caloric restriction, sleep deprivation, and prolonged work were chosen to model current “real-world” scenarios expected by the United States military in high-intensity combat situations. In addition, we concomitantly measured the downstream anabolic mediators of these signaling peptides: testosterone and IGF-I, IGF binding proteins (BPs)-1 and -3, and other metabolic markers (i.e., leptin, free fatty acids, and glucose). METHODS

Experimental design. This study utilized a within-subjects repeatedmeasures design consisting of two 96-h testing blocks (see Table 1). The results of the physical performance tests performed on the morning of day 1, day 3, and day 4 of each test block, as well as cold stress test on day 4, have been reported previously (4, 30). The initial block (control week) included the experimental tests but no other scheduled activities. Sleep and food intake were ad libitum during the

control week. The following week (military operational stress), the second 96-h block was performed. During the military operational stress week, the subjects performed nearly continuous physical work. Sleep was restricted to two 1-h sleep periods per day. Food was limited to one meal, ready-to-eat (1,200 –1,300 kcal/meal), and an additional small meal per day for a total caloric intake of ⬃1,600 kcal/day. Field training consisted of the performance of military relevant tasks and military basic skill training. It included basic patrolling, combat drills, road marches, land navigation, a litter obstacle course, and a confidence course (refer to Table 1) (31). 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). Sleep time was calculated by adding the time periods in which no activity signal was measured. Water during the field training was consumed ad libitum. Subjects. Fourteen healthy male soldiers volunteered to participate in the study. Three injuries and one illness prevented four of these test subjects from completing the study. The data from the remaining 10 volunteers [22 yr (SD 3), 183 cm (SD 7), 87 kg (SD 8)] are presented in this paper. The experiment was approved by the appropriate Institutional Scientific Review and Human Use Review Committees. The subjects were briefed on the study procedures, and written consent was obtained before study participation. Body composition. Body composition was assessed by whole body dual-energy X-ray absorptiometry. Total body estimates of percent fat, bone mineral density, and bodily content of bone, fat, and nonbone lean tissue were determined 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. Scanning was in 1-cm slices from head to toe using the 20-min scanning speed (8). Overnight serial blood sampling. On two separate occasions (see Table 1), subjects had blood drawn overnight in a metabolic research laboratory. The subjects reported to the laboratory at 1630, were served a dinner meal at 1700, and then had an indwelling catheter inserted at 1745. Beginning at 1800, blood was serially sampled every

Table 1. Timeline and activities for the experimental protocol Time

0100 0200 0300 0400 0500 0600 0700 0800 0900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Day 1

Day 2

Road march

Day 3

Day 4

Road march

Road march

Day 5

12-h hormonal profile analysis*

Travel/sleep Blood draw* Cognitive and marksmanship testing* Physical performance testing* Travel to field site Set up command post Land navigation course

Break Battle drills Sleep Road march

Sleep Break Confidence course Break Battle drills

Travel Litter carry Battle drills Pack command post Sleep Road march

Unload truck/break Blood draw* Cognitive and marksmanship testing* Physical performance testing* Break Hot walk*

Sleep Break Blood draw* Cognitive and marksmanship testing* Physical performance testing* Cold test*

Body composition

Debrief

Break Physical training Break Light training

Break 12-h hormonal profile analysis*

Sleep Road march

*Testing blocks that were performed during both the control and military operational stress weeks; all other areas were activities that were only performed during the military operational stress week. J Appl Physiol • VOL

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20 min for GH, LH, and leptin (36 samples total) and every 2 h for the IGF-I system components, testosterone, glucose, and free fatty acids (6 samples total) until 0600 the next morning. Blood sampling followed the same procedure following the military operational stress paradigm. To facilitate familiarization with the facility and minimize sleep disruptions, all subjects slept in the metabolic laboratory the night before the control overnight serial blood draw. Conditions were mimicked exactly to include the taping of a catheter near the antecubital vein for the night. During each overnight trial, subjects were allowed to move freely within the laboratory. Lights were turned off at 2200, and the TV was turned off at 2300. At bedside, a registered nurse and research technicians unobtrusively performed the serial blood draws throughout the night. Blood was collected in glass vacutainers, allowed to clot on ice, and centrifuged for 30 min at 1,500 g at 4°C. After centrifugation, serum was aliqoted into the appropriate Eppendorf tubes, flash frozen in liquid nitrogen, and stored at ⫺80°C until later analysis. Biochemical assays. GH, LH, and leptin concentrations were measured using a two-site immunoradiometric assays (Diagnostic Systems Laboratories, Webster, TX). The sensitivity and intra-assay variances for these assays were 0.01 ng/ml and 3.9%, 0.12 mIU/ml and 6.8%, and 0.10 ng/ml and 3.7% for GH, LH, and leptin, respectively. Total IGF-I, free IGF-I, IGFBP-1, and IGFBP-3 concentrations were determined by a two-site immunoradiometric assays (Diagnostic System Laboratories). The sensitivity of these assays was 2.06, 0.03, 0.33, and 0.05 ng/ml for total IGF-I, free IGF-I, IGFBP-1, and IGFBP-3, respectively. Total and free testosterone concentrations were determined by a radioimmunoassay (Diagnostic Systems Laboratories). The sensitivity of these assays was 0.08 ng/ml and 0.18 pg/ml, respectively. The intra-assay variances were both ⬍4.0%. Radioimmunoassays were performed on a Cobra gamma counter (Packard Instruments, Downers Grove, IL). Serum glucose concentrations (Bio-Chem Laboratory Systems, Lakewood, NJ) and free fatty acids (Wako Chemicals, Richmond, VA) were measured according to manufacturer’s instructions on an ATAC 8000 (Elan Diagnostics, Smithfield, RI). The intra-assay variances were all ⬍7%. Samples were run in duplicate for all assays. All samples for a given subject were run in the same assay batch to eliminate interassay variance. Cluster analysis. The cluster analysis program was used to analyze the overall mean GH, LH, and leptin concentrations, 12-h integrated area under the curve via trapezoidal rule, and characteristics of concentration “peaks” (18, 35, 36). The cluster algorithm searched for significant increases and decreases among data points in a series via pooled t-tests. Two points were used for the determination of a peak and one point to establish a nadir. A peak was defined as a series of concentrations that demonstrated an increase over time followed by a decrease, with the additional requirement that a nadir (i.e., a decrease followed by an increase) was present on each side of the peak. Cluster analysis was utilized to provide descriptive information regarding observed concentrations as a function of time, such as the number of peaks in 12 h, the mean interval between peaks, the mean peak width

(i.e., duration), the mean peak height (i.e., amplitude), and the area under peaks. Deconvolution analysis. Multiple-parameter analysis was used to estimate the secretion and elimination characteristics of the hormones based on the assay-measured concentrations. This method used a convolution integral that is solved by nonlinear least squares parameter estimation (18, 35, 36). For this analysis, we used the same data file that was configured and used for the Cluster program. The hormone concentration data was initially deconvolved using the Pulse2 program to estimate the number and position of secretion bursts. Variable basal secretion and an estimated half-life, chosen to produce a convolved curve that closely approximated the actual concentration data, were selected as the input parameters for Pulse2. Deconvolution analysis was further carried out with multiple-parameter fitting at 95% joint statistical confidence intervals for all calculated secretory burst variables. Half-life, secretion burst number, interval between bursts, burst area, burst amplitude, amplitude of largest burst, basal secretion per minute, overnight basal secretion (basal secretion ⫻ 720 min), overnight pulsatile secretion (burst area ⫻ secretion burst number), and total overnight secretion (overnight basal secretion ⫹ overnight pulsatile secretion) were determined for GH and LH. Statistical analyses. All data are presented as means (SD). A two-way repeated ANOVA with repeated measures was employed for statistical analyses. Where appropriate, a Tukey’s post hoc follow-up test was used. For the secretion characteristics, a dependent t-test was used to compare the two means. All statistical analyses were performed with Statistica software packages (StatSoft, Tulsa, OK). An alpha level of 0.05 was used for all statistical evaluations. RESULTS

Body composition. Subjects consumed an average of 1,653 kcal/day (SD 25) [225 g carbohydrate (SD 6), 54 g fat (SD 2), 69 g protein (SD 3)] and slept a total of 6.2 h (SD 0.4) during the entire 84-h experimental study period as assessed by actigraphy. Energy expenditure, estimated from distance traversed and predicted energy cost of the activities during the course, was ⬃4,000 – 4,500 kcal/day. Body mass [control: 84.8 (SD 3.7); military operational stress: 82.2 (SD 3.5)], fat free mass [control: 69.0 (SD 2.7); military operational stress: 67.7 (SD 2.5)], fat mass [control: 15.8 (SD 1.7); military operational stress: 14.5 (SD 1.8)], and percent body fat [control: 18.4 (SD 1.3); military operational stress: 17.4 (SD 1.5)] all significantly declined (P ⬍ 0.05) during the experimental condition. Cluster analysis of LH, GH, and leptin. Table 2 presents the LH, GH, and leptin concentration parameters estimated by cluster analysis. LH, GH, and leptin mean overnight concentration values and total area under the curve were significantly different (P ⬍ 0.05) between the control and military opera-

Table 2. Hormone concentration parameters from Cluster 7 program for LH, GH, and leptin during the control and military stress conditions LH, IU/ml

GH, ng/ml

Leptin, ng/ml

Parameter

Control

Military stress

P

Control

Military stress

P

Control

Military stress

P

Mean, min Total area, min Peak height, min Peak number Peak width, min Peak area, min

3.7 (1.0) 2,534.1 (795.7) 5.5 (1.8) 3.5 (1.2) 128.3 (27.0) 165.1 (79.1)

5.4 (1.8) 3,633.1 (1276.5) 8.6 (2.5) 3.2 (1.6) 136.8 (45.1) 284.0 (154.5)

0.014* 0.014* 0.029* 0.530 0.642 0.176

0.6 (0.5) 426.7 (365.4) 1.8 (1.7) 3.5 (1.1) 151.4 (60.4) 148.5 (187.8)

0.9 (0.4) 653.0 (284.2) 2.9 (1.7) 3.0 (0.9) 146.5 (46.0) 182.9 (114.3)

0.023* 0.027* 0.083 0.170 0.853 0.584

3.8 (2.6) 2,555.9 (1,771.2) 4.4 (2.9) 3.1 (1.6) 185.6 (121.3) 300.4 (554.9)

2.0 (1.4) 1,333.8 (958.2) 2.3 (1.5) 3.3 (2.2) 106.0 (75.3) 56.7 (53.9)

0.009* 0.009* 0.005* 0.931 0.107 0.262

Values are means (SD). GH, growth hormone; LH, luteinizing hormone. *Significant difference between conditions (P ⬍ 0.05). J Appl Physiol • VOL

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tional stress conditions, respectively. LH peak height was significantly higher after military operational stress, whereas leptin peak height was lower. There were no statistical differences found for LH, GH, or leptin between the control and military stress conditions for number of peaks, width of peaks, or area under a peak. GH and LH cluster analysis graphs for two representative subjects are presented in Figs. 1 and 2. Figure 3 displays the composite hormonal concentrations for all subjects in both the control and military stress conditions for GH, LH, and leptin. Deconvolution analysis of LH and GH. The LH and GH secretory dynamics as estimated by deconvolution analysis are given in Table 3. LH half-life, number of bursts, amplitude of largest burst, basal secretion rate, overnight basal secretion, or total overnight secretion (basal ⫹ pulsatile) was similar (P ⱖ 0.05) between conditions. However, LH overnight pulsatile secretion, area under bursts, amplitude of bursts, and interval between bursts were greater after the military operational stress condition. Thus the increase in mean LH concentration was accounted for by an increase in the amount of hormone secreted per burst and not an increase in the number of bursts or any change in the basal secretion rate.

Fig. 2. Cluster and deconvolution analyses of luteinizing hormone (LH) (subjects C and D; A and B, respectively) for the 12-h overnight sampling period. Top: hormone concentrations from cluster analysis for both control (left) and military stress (right) conditions. Bottom: corresponding secretory bursts from deconvolution analysis.

Fig. 1. Cluster and deconvolution analyses of growth hormone (GH) (subjects A and B; A and B, respectively) for the 12-h overnight sampling period. Top: hormone concentrations from cluster analysis for both control (left) and military stress (right) conditions. Bottom: corresponding secretory bursts from deconvolution analysis. J Appl Physiol • VOL

Mean GH concentration was significantly increased after military stress compared with the control condition. The increase in mean GH concentration was accompanied by an increase in the following secretion parameters: area under bursts, amplitude of bursts, basal secretion rate, overnight basal secretion, overnight pulsatile secretion, and total overnight secretion. GH half-life, number of bursts, interval between bursts, and amplitude of largest burst were similar between the control and military operational stress conditions. Representative GH and LH profiles of deconvolution analysis for two subjects are presented in Figs. 1 and 2. IGF-I system, testosterone, glucose and free fatty acids. Figures 4 – 6 display the overnight metabolic and hormonal responses sampled every 2 h overnight for the control and the military stress conditions. Table 4 presents a summary for the overall main effects means for these variables. Total IGF-I, free IGF-I, IGFBP-3, total testosterone, and free testosterone were significantly lower after military stress, whereas IGFBP-1 and free fatty acids were significantly elevated after military stress. Glucose concentrations were similar for both conditions.

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Fig. 3. Mean concentration of all subjects over the 12-h sampling period. Samples were taken every 20 min for a total of 36 blood draws as represented on the x-axis. Main effect mean concentrations of GH (A), LH (B), and leptin (C) after the military stress condition are statistically different (P ⱕ 0.05) from control for all hormones (also see Table 2). DISCUSSION

In an effort to yield novel insights into the early phase neuroendocrine responses to physiological strain, we modeled military operational field training (i.e., 4 days of sustained physical activity, caloric restriction, and sleep deprivation) to examine overnight somatotrophic hormonal responses. Our

data indicate that the experimental paradigm was metabolically challenging as evidenced by the estimated daily caloric deficit (⬃2,500 –2,900 kcal), the loss in body mass (⬃3.0%), and the limited amount of sleep attained (total of 6.2 h). Marked alterations in the secretory release patterns (i.e., amplifications) of GH and LH and the concentrations of other metabolic and hormonal modulators (i.e., IGF-I, testosterone, leptin, free fatty acids) as well as body composition parameters were observed. The fact that short-term (4 days) military operational stress can have striking effects on the neuroendocrine system raises questions for future studies concerning the potential adverse impact of long-term military operational stress (i.e., overseas deployments lasting 6 –12 mo) on soldier physiology and the subsequent health and fitness of our military personnel. This study used a rigorous serial blood sampling scheme and subsequent time-series deconvolution analyses to assess GH and LH secretion patterns. These data expand on prior research that relied on one time-point blood samples to glean information on the endocrine responses to stress that are subject to episodic and circadian release patterns (9, 13, 27). The physiological significance of studying the pulsatile release of these hormones lies in the fact that the episodic release pattern is thought to be integral in eliciting optimal agonist function at the cellular level (17, 34 –36). The salient finding emerging from this study was an amplification of GH and LH secretion with a concomitant decline in IGF-I and testosterone. Because GH and LH are known to positively regulate IGF-I and testosterone, these data imply that the physiological strain experienced in this study induced a certain degree of peripheral resistance. GH/IGF-I axis. The mean GH concentration and total area were 50% greater after military operational stress as assessed by cluster analysis. More detailed analysis of the secretory dynamics of GH indicated an amplification in the following pulsatility parameters: area (43%) and amplitude (50%) of bursts, basal secretion rate (63%), overnight basal (54%) and pulsatile (55%) secretion rate, as well as total overnight secretion (55%). GH half-life and the number of bursts were unaffected. Although the magnitude of these responses is substantial, they are considerably less than observed after similar periods of total fasting. For example, Ho et al. (16) reported a 100% increase in GH peak amplitude after 5 days of fasting in healthy young men, and Hartman et al. (15) reported a 400 –500% increase in GH production rate and a 200%

Table 3. Hormone secretion parameters from deconvolution analysis for LH and GH during the control and military stress conditions LH, IU/ml

GH, ng/ml

Parameters

Control

Military Stress

P

Control

Military Stress

P

Half-life, min Number of bursts/12 h Interval between bursts, min Area under bursts, min Amplitude of bursts, min Amplitude of largest burst, min Basal secretion rate, min Overnight basal secretion, 12 h Overnight pulsatile secretion, 12 h Total overnight secretion, 12 h

45.15 (8.66) 6.00 (0.89) 109.30 (15.54) 3.86 (2.00) 0.13 (0.07) 0.23 (0.11) 0.03 (0.01) 19.22 (6.64) 22.78 (11.85) 42.00 (17.62)

51.77 (9.96) 5.67 (1.03) 120.31 (15.27) 5.90 (2.65) 0.21 (0.08) 0.32 (0.10) 0.03 (0.01) 18.46 (7.71) 33.20 (15.78) 51.66 (20.94)

0.095 0.363 0.049* 0.028* 0.016* 0.100 0.797 0.797 0.021* 0.107

16.36 (4.33) 4.62 (1.51) 96.06 (37.99) 3.04 (1.47) 0.10 (0.05) 0.24 (0.15 0.001 (0.000 0.80 (0.19 15.14 (11.09 15.94 (11.13)

18.52 (4.58) 5.62 (1.30) 95.48 (28.92) 4.31 (2.16) 0.16 (0.09) 0.36 (0.22) 0.002 (0.001) 1.29 (0.55) 23.28 (10.16) 24.57 (10.59)

0.233 0.050 0.978 0.044* 0.040* 0.186 0.020* 0.020* 0.029* 0.027*

Values are means (SD). *Significant difference between conditions (P ⬍ 0.05). J Appl Physiol • VOL

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Fig. 4. Insulin-like growth factor (IGF)-I system responses during the control and military stress week measured bi-hourly from 1800 to 0600. A: total IGF-I. B: free IGF-I. C: IGF binding protein (BP)-1. D: IGFBP-3.

increase in GH mean burst mass in older subjects after a 48-h fast. More recently, Maccario et al. (24) sampled blood every 30 min from 0800 to 1600 after a 36-h fast and observed a mean increase in GH from 0.5 ng/ml in the fed condition to 6.2 ng/ml in the fasted condition, whereas Chan et al. (5) sampled blood every 15 min for 24 h after a 72-h fast in young, lean men and reported ⬃114% increases in mean GH concentration and integrated area. The obvious explanation for dissimilarity between our results and those reported above was that, although our subjects were exposed to a significant energy deficit, they were provided daily meals (⬃1,600 kcal/day). Friedl et al. (9) have previously reported that even small increases in caloric consumption can have significant effects on restoring hormonal concentrations toward baseline values in soldiers exposed to field training. The results from the present study also differed from the literature in that there were no significant differences in the number of pulses observed, whereas pure fasting studies have reported increases up to 130% in pulse frequency (5, 15, 24). These findings could perhaps be interpreted to suggest that, during periods of energy deficiency, amplitude modulation of GH pulses precedes alterations in pulse number. IGF-I and its family of BPs are well recognized as metabolic/ growth/maintenance modulators during altered energy states (1, 2, 26, 28, 29, 32). Our laboratory previously reported responses of IGF-I molecular complexes during this same J Appl Physiol • VOL

military operational field exercise as measured by morning blood draws taken throughout the study (26). The present results utilize a series of blood draws to expand on those observations by showing the sustained impact on the IGF-I system during the 12-h overnight period. Total, free IGF-I, and IGFBP-3 were reduced after the military operational stress by 27, 32, and 6%, respectively. In contrast, IGFBP-1 increased by 67%. IGF-I has been shown to decline by 67 and 70% after 5 and 10 days, respectively, of complete fasting in healthy adults (6) and by ⬃50% after 14 days of United States Army Ranger training (9). That nearly one-half the magnitude of these reductions was achieved in the present study underscores the severity of the caloric imbalance and metabolic strain imposed in the present study and how rapidly IGF-I can change. IGF-I secretion from the liver is the primary biological targeted outcome for GH. The fact that IGF-I decreased even in the presence of amplified GH release suggests that the liver was “GH resistant” (32). The underlying mechanism may be associated with an impairment of the JAK-STAT signaling pathway. Beauloye et al. (3) reported a decrease in GHstimulated tyrosine phosphoryaltion of JAK2, GHR, and STAT5, without significant changes in their protein content in rat liver after a 48-h fast. Elevated cortisol (not measured in the present study) could also have contributed to a decreased peripheral GH sensitivity. As pointed out by Chan et al. (5), the adaptive value of increased GH and decreased IGF-I would be

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Fig. 5. Testosterone responses during the control and military stress week measured bi-hourly from 1800 to 0600. A: total testosterone response. B: free testosterone response.

a diminishment of energy expenditure required for growthrelated processes while enabling GH to promote the mobilization of alternative fuels through lipolysis. Of the IGF-I system components measured, IGFBP-1 demonstrated the largest magnitude change. Frystyk et al. (10) 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 fats and protein. Furthermore, it has been suggested that low levels of liver glycogen are linked to an increased secretion of IGFBP-1 (21). IGFBP-1 remains an important regulatory BP to study relative to physiological stress as increased levels may be one mechanism whereby IGF-I bioactivity is adjusted to meet metabolic needs. LH/testosterone axis. The mean LH concentration increased 46% for the military stress condition. This is in contrast to J Appl Physiol • VOL

Fig. 6. Glucose (A) and free fatty acids (B) during the control and military stress week measured bi-hourly from 1800 to 0600.

Opstad (31), who reported a 33% decrease in LH after a 5-day military operational period as measured from a one-time morning blood sample. Consistent with the increase in overall mean LH concentration, deconvolution analysis of LH showed inTable 4. Means for main condition effects during the control and military stress conditions Variable

Control

Military Stress

P

Total IGF-I, ng/ml Free IGF-I, ng/ml IGFBP-I, ng/ml IGFBP-3, ng/ml Total testosterone, ng/ml Free testosterone, pg/ml Free fatty acid, mEq/l Glucose, mg/dl

156.8 (65.0) 2.2 (1.3) 22.1 (22.9) 35.5 (8.7) 5.5 (1.9) 17.9 (7.4) 0.4 (0.2) 90.4 (10.6)

114.1 (49.4) 1.5 (0.8) 37.0 (33.5) 33.6 (9.9) 4.2 (1.6) 12.5 (6.1) 0.6 (0.3) 93.2 (13.2)

0.004* 0.025* 0.007* 0.064 0.002* 0.001* 0.013* 0.406

Values are means (SD). *Significant difference between conditions (P ⬍ 0.05).

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creases in burst area (53%), burst amplitude (64%), and overnight pulsatile secretion (46%). Loucks et al. (22) have recently reported that in women LH pulsatility is disrupted when energy intake is below a threshold of 30 kcal䡠kg LBM⫺1 䡠day⫺1. Similar to our findings, Loucks and Thuma (22) reported an increase in burst amplitude (21%), but in contrast to our results, they reported a slowing of the LH burst frequency (⫺16%) and a maintenance of LH mean concentration. In our study, subjects consumed an average of 23.5 kcal䡠kg LBM⫺1 䡠day⫺1, placing them well into a negative energy balance with a deficit of ⬃2,500 –2,900 kcal/day and below the hypothesized threshold of 30 kcal䡠kg LBM⫺1 䡠day⫺1 necessary for the disruption of LH pulsatility in women (22). Deconvolution analysis revealed the military stress condition to have a significantly increased interval between bursts but a nonsignificant decline (⫺8.6%) in LH burst frequency. The increased burst amplitude and the maintained burst frequency resulted in an increased LH mean concentration. Despite enhanced release of LH, testosterone concentrations decreased. During the 12-h overnight sampling period, total and free testosterone concentrations were 24 and 30% lower after the military stress condition, respectively. Decreased levels of testosterone have also been observed in other studies that investigated the effects of short-term military stress (9, 27, 31). Opstad (31) found a 47% decrease in morning testosterone concentration after a 5-day military training course. Under similar conditions, Gomez-Merino et. al. (11) found a 35% decrease in morning testosterone concentrations. Both of these studies employed a single time-point analysis. By considering the circadian rhythm of testosterone, our study has extended these findings by demonstrating that testosterone concentrations are decreased and remain depressed overnight for a 12-h period following the conclusion of a multiple-day military stress condition. Conceptually, a mechanism to maintain normal testosterone concentrations could be an enhanced pulsatile release of LH. Decreased testosterone concentrations relieve the inhibition of the negative feedback loop at the hypothalamus/pituitary gland, resulting in an enhanced release of gonadotropin-releasing hormone and subsequently enhancing LH secretory dynamics. For example, compared with younger men, older men tend to possess greater LH secretion, which is hypothesized to represent a biological attempt to maintain normal testosterone concentrations (14). Endurance-trained athletes have also shown decreased testosterone and increased LH concentrations (11, 12). The literature with regard to acute exercise stress has been equivocal. Tanaka et al. (33) have reported decreased concentrations of testosterone and increased LH concentrations for up to 2 days after a marathon and suggested that elevated catecholamines may be partially responsible for the decreased testicular sensitivity. We have previously reported decreased LH secretion and decreased testosterone concentrations after heavy resistance exercise in men. The fact that all subjects were provided a small meal ⬃1 h before the overnight blood draws may be of consequence in terms of interpreting the LH data in that a single ad libitum meal has been reported to stimulate LH pulsatility within hours in a number of different species (25). Thus, although LH pulsatility is slowly restored in women (23), there is evidence to suggest that gonadotropinreleasing hormone neurons may rapidly respond to caloric intake. J Appl Physiol • VOL

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Leptin/glucose/free fatty acid responses. Leptin concentrations declined from 3.8 to 2.0 ng/ml (⬃47% decline) after military stress. These declines were coincident with marked reductions in fat mass (⬃8%) and increased concentrations of circulating free fatty acids (⬃33%). This magnitude decrease for leptin is consistent with the 44% decrease reported by Maccario et al. (24) after 36 h of fasting, but less than the ⬃66% decline observed by Gomez-Merino et al. (11) after a 5-day military combat course. These changes (leptin and free fatty acids) are reflective of substantial lipolytic activity (also supported by the amplification of GH release). Also of interest is the fact that the overall main effect mean for glucose concentrations was similar between the control (90.4 mg䡠dl⫺1 䡠l⫺1) and military stress (93.2 mg䡠dl⫺1 䡠l⫺1) condition, suggesting that glucose homeostasis was well defended in this paradigm. Although not measured in the present study, other studies have reported robust changes in cortisol after military stress (9), and it is also probable that both cortisol and catecholamine elevations played a contribution in maintaining glucose concentrations. The fact that the absolute losses in fat-free mass (1.3 kg) and fat mass (1.3) were similar suggests that amino acid fuel partially contributed to gluconeogenesis. Converging lines of evidence in sheep, rat, monkey, and human models suggest that leptin acts as a peripheral signal to mediate metabolic regulation of GH and LH secretion from the anterior pituitary gland (5, 25). Leptin appears to prevent the fasting-induced suppression of pulsatile release of LH secretion. In a recent study that evaluated the effects of differing doses of exogenously administered r-metHuLeptin in men during 73 h of fasting and employing 24-h blood sampling, Chan et al. (5) demonstrated a role for leptin in normalizing starvation-induced neuroendocrine changes, notably the hypothalamic-pituitary-gonadal axis. Chan et al. (5) hypothesized that a threshold leptin level between 0.5 and 2.0 ng/ml may be necessary for LH secretion. It is interesting to note that, in our study, the leptin concentrations after military stress were 2.0 ng/ml and that there was not a decline in LH pulse frequency. Although glucose concentrations have also been speculated to act as a peripheral signal relaying information concerning nutritional status and within the context of the dramatic hormonal changes observed (20), it is remarkable that glucose homeostasis was well maintained. The overall physiological and metabolic stress encountered in the present study appears to fall below a threshold necessary to disrupt the maintenance of normal glucose homeostasis. In conclusion, our results demonstrate that brief periods (i.e., 4 days) of intensive physical activity superimposed on energy and sleep restriction induced amplifications in GH and LH secretion (no change in pulse number, however), yet at the same time resulted in decreases in hormones that these signaling peptides are responsible for stimulating, namely IGF-I and testosterone. That short-term military operational stress can exert marked effects in neuroendocrine secretion patterns raises important questions concerning the potential adverse effects on longer-term military operational stress (i.e., 6- to 12-mo overseas deployments) on underlying physiology and metabolism and the health status of military personnel. REFERENCES 1. Baxter RC. Insulin-like growth factor binding proteins as glucoregulators. Metabolism 44: 12–17, 1995.

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