Low-dose T3 improves the bed rest model of simulated weightlessness in men and women

Jennifer C. Lovejoy1, Steven R. Smith1, Jeffrey J. Zachwieja1, George A. Bray1, Marlene M. Windhauser1, Peter J. Wickersham1, Johannes D. Veldhuis3, Richard Tulley1, and Jacques A. de la Bretonne2

1 Pennington Biomedical Research Center, Louisiana State University, 2 Baton Rouge General Health Center, Baton Rouge, Louisiana 70808; and 3 University of Virginia Health Sciences Center and National Science Foundation Center for Biological Timing, Charlottesville, Virginia 22908


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study tested the hypothesis that low-dose 3,5,3'-triiodothyronine (T3) administration during prolonged bed rest improves the ground-based model of spaceflight. Nine men (36.4 ± 1.3 yr) and five women (34.2 ± 2.1 yr) were studied. After a 5-day inpatient baseline period, subjects were placed at total bed rest with 6° head-down tilt for 28 days followed by 5-day recovery. Fifty micrograms per day of T3 (n = 8) or placebo (n = 6) were given during bed rest. Serum T3 concentrations increased twofold, whereas thyroid-stimulating hormone was suppressed in treated subjects. T3-treated subjects showed significantly greater negative nitrogen balance and lost more weight (P = 0.02) and lean mass (P < 0.0001) than placebo subjects. Protein breakdown (whole body [13C]leucine kinetics) increased 31% in the T3 group but only 8% in the placebo group. T3-treated women experienced greater changes in leucine turnover than men, despite equivalent weight loss. Insulin sensitivity fell by 50% during bed rest in all subjects (P = 0.005), but growth hormone release and insulin release were largely unaffected. In conclusion, addition of low-dose T3 to the bed rest model of muscle unloading improves the ground-based simulation of spaceflight and unmasks several important gender differences.

thyroid hormones; spaceflight; insulin resistance; growth hormone; gender differences


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SPACEFLIGHT results in negative nitrogen and calcium balance associated with reduced gravity (9, 21, 32). After a period of time, these adaptive changes in the musculoskeletal system can produce decreases in lean body mass, posing a significant clinical concern for long-duration spaceflights. To study the effects of microgravity more conveniently, a ground-based model of the effects of spaceflight has been developed utilizing complete bed rest with a 6° head-down tilt (16). Although this model elicits many of the effects observed in actual spaceflight, alterations in bone metabolism are less striking and long-duration bed rest is required to obtain comparable changes in lean body mass (16). Thus there is a need to improve this model so that the physiological changes that occur are more comparable to those of spaceflight and, particularly, to shorten the length of time subjects need to be studied. If such a modification is successfully achieved, it could also benefit other studies of the physiological effects of inactivity.

We hypothesized that the ground-based model of microgravity effects could be enhanced by introducing a catabolic agent such as thyroid hormone to accelerate muscle and bone catabolism during bed rest. It is well known that elevations in thyroid hormone levels are associated with increased catabolism, negative nitrogen and calcium balance, loss of body protein stores, and loss of body fat (11). If thyroid hormone is to be useful as a modification of the spaceflight model, it must be shown to 1) rapidly produce changes in lean body mass that are more comparable to those of spaceflight per se, 2) be safe for use in healthy volunteers, and 3) result in changes in other physiological systems that are comparable to spaceflight.

In the present study, the effects of bed rest alone vs. bed rest with low-dose 3,5,3'-triiodothyronine (T3) treatment on body composition, protein metabolism, metabolic rate, insulin sensitivity and secretion, and growth hormone (GH) secretion were compared. These outcome measures were selected because previous studies have shown them to be altered by spaceflight. Calcium metabolism and markers of bone turnover were also measured and will be reported elsewhere.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. Fourteen healthy volunteers (9 men and 5 women) were recruited for the study with the use of newspaper, radio, and television advertisements. Seven of the volunteers were Caucasian (5 men and 2 women), and seven were African-American (4 men and 3 women). To be eligible, subjects were nonsmokers with a normal chemistry panel, blood count, urinalysis, and psychological profile at screening. Only premenopausal women were studied. All female volunteers received a continuous dosage of monophasic oral contraceptives throughout the study to avoid potential confounding effects of menstrual cycle phase on study variables. The oral contraceptives were started a minimum of 4 wk before the study. All subjects also underwent thorough psychological testing and a drug screen to ensure their suitability for the study. The volunteers provided written informed consent before participation, and the protocol and consent form were approved by the Louisiana State University Institutional Review Board.

Experimental design. The study consisted of a 38-day stay in an inpatient Metabolic Research Unit (Fig. 1). Before the bed rest with or without the T3-experimental period, subjects were adapted to the inpatient unit and followed a controlled diet for 5 days. Baseline nitrogen balance and measures of study variables were performed during this 5-day run-in period. Thereafter, subjects began total bed rest with 6° head-down tilt, which continued for 28 days. Except for being allowed to raise themselves on one elbow to eat meals and shower on a horizontal platform, the head-down bed rest was continuously maintained. Repeat assessment of study variables was performed during the last week of bed rest, with the exception of dual energy X-ray absorptiometry (DEXA) and computerized tomography (CT) scans which were performed early during the recovery period. Nitrogen balance was determined daily. After the 28-day bed rest, a 5-day recovery period in the unit allowed subjects to regain their strength and mobility before being discharged.


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Fig. 1.   Outline of experimental design showing days when endpoints were measured. Pl, placebo treatment; DEXA, dual energy X-ray absorptiometry; CT, computed tomography; RMR, resting metabolic rate; GH, growth hormone; T3, 3,5,3'-triiodothyronine; T4, L-thyroxine; R, recovery; TSH, thyroid-stimulating hormone.

Subjects were randomly assigned to receive either T3 (n = 8) or placebo (n = 6) during the 28-day bed rest period. Treatment was balanced across gender with five men receiving T3 and four men placebo and three women receiving T3 and two women placebo. T3 was given orally as a loading dose of 100 µg for men and 75 µg for women, followed by a treatment dose of 50 µg/day given in five divided doses throughout the day. The dose was selected on the basis of our previous data on ambulatory men, which showed that this dose increases serum T3 concentrations approximately twofold and suppresses thyroid-stimulating hormone (TSH) to below detectable levels (12).

During the study, the diet was designed to approximate the diet consumed by space shuttle astronauts (7) and consisted of standard foods. Calorie requirements were individualized for each subject based on 130% of his/her measured metabolic rate to account for low activity levels. Calcium, magnesium, sodium, and potassium intakes were controlled. A calcium supplement was provided that brought total daily calcium intake to 1,000 mg/day. Sodium was targeted to 3,600 mg/day, magnesium to 300 mg/day, and potassium to 4,000 mg/day. The diet was 30% energy as fat (ratio of saturated to polyunsaturated to monounsaturated fat = 10:10:10). High quality protein foods (e.g., seafood, meats, eggs, and cheese) were provided in amounts of ~1.0 g protein · kg body weight-1 · day-1. For each subject, the level of protein was determined as (lean body mass + bone mineral content) × 1.3. The average intakes of dietary protein for the men and women were 78.5 and 47.1 g, respectively. Additional calories needed to meet energy requirements above the basal diet were given as refined carbohydrates (i.e., sugar) and fats. All foods were weighed to within 0.1 g. Distilled water was used in all food preparation and was provided for drinking. Complete consumption was encouraged and generally achieved, but foods not consumed were weighed so that total daily consumption could be determined for balance calculations.

Nitrogen balance. All food and fluids consumed were measured, and all urine and stools were collected for balance studies. Analysis of creatinine excretion was used to verify the accuracy of daily urine collections. Fecal collection periods were 7 days, separated by administration of an indigestible fecal marker (carmine red dye), with the exception of the run-in period, which was only a 5-day fecal collection. Corrections for fecal losses were carried out by quantitating the nonabsorbable marker polyethylene glycol (10% solution), which was given (10 ml) with each meal. Blood nitrogen loss (experimental or menstrual) was calculated, skin and sweat losses were estimated (4), and net nitrogen balance values were corrected for these figures. Values reported are based on creatinine-corrected urine and polyethylene glycol-corrected stool nitrogen. Because the recovery period was only 5 days, fecal nitrogen losses were estimated from the last full week of the study.

Body composition. Body composition (lean, fat, and bone) was quantitated during the run-in period and immediately on getting out of bed at the end of the study by DEXA (Hologic QDR2000; Waltham, MA). Additionally, CT was performed during run-in and recovery to determine changes in intra-abdominal fat stores (General Electric High-Speed Advantage; Milwaukee, WI). A single abdominal CT scan was obtained to measure visceral and subcutaneous abdominal fat areas at the level of the L4-L5 intervertebral space. Calculation of fat areas from the 1-cm scans was accomplished with software on the CT scanner with a range of -190 to -30 HU for adipose tissue and 30-80 HU for muscle tissue.

Resting metabolic rate. Resting metabolic rate (RMR) was determined from indirect calorimetry with Sensormedics 2900Z portable metabolic carts (Yorba Linda, CA). RMR was measured for 30 min after a 30-min rest period in a supine (during run-in) or 6° head-down position (in the last week of bed rest).

Protein turnover (leucine kinetics). After an overnight fast, a forearm vein was cannulated for infusion of solutions and a superficial hand vein in the contralateral arm was cannulated in a retrograde direction and kept open by normal saline infusion. The hand with the sampling vein was kept warm with a heating pad to provide arterialized venous blood samples (1). After baseline blood and breath samples were obtained, a primed (4.8 µmol/kg) continuous (3.6 µmol · kg-1 · h-1) infusion of L-[1-13C]leucine was given. In addition, a 0.087 mg/kg NaH13CO3 bolus was given to prime the bicarbonate pool (2). Blood and breath samples were obtained every 15 min from 120-180 min, and urine was collected to measure nitrogen excretion. O2 consumption and CO2 production were measured with a Sensormedics 2900Z metabolic cart (Yorba Linda, CA).

Plasma alpha -ketoisocaproic acid (KIC) enriched with 13C was analyzed by gas chromatography coupled to a mass spectrometer, with electron impact ionization and selected ion monitoring at mass-to-charge ratios of 232 and 233. The 13CO2 enrichment in expired air was measured with an automated trapping box and a Finnigan MAT 252 gas isotope ratio mass spectrometer. Leucine rate of appearance (Ra; i.e., protein breakdown), oxidation, and nonoxidative leucine disposal (i.e., protein synthesis) were calculated as described previously (14). The rate of 13CO2 released by oxidation of labeled leucine was calculated from the CO2 production rate, the 13CO2 enrichment in expired air at isotopic steady state (with 0.81 as a correction for the fraction of 13CO2 retained in the bicarbonate pool), and from the enrichment of KIC, the immediate precursor for the oxidative decarboxylation of leucine (14, 15). Nonoxidative leucine disposal was calculated from the difference between leucine Ra and leucine oxidation rate.

Insulin sensitivity. Insulin sensitivity was assessed with the minimal model method (3). This method utilizes a mathematical model to fit the temporal pattern of glucose and insulin levels in blood during a frequently sampled intravenous glucose tolerance test. Subjects were studied after an overnight fast. Briefly, 300 mg/kg glucose was injected at time 0, followed by collection of blood samples (4 cc) at 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, and 19 min. At 20 min, 0.03 U/kg regular human insulin (Humulin, Eli Lilly, Indianapolis, IN) was given as a bolus injection and blood sampling continued at 22, 23, 24, 25, 27, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, and 180 min. Each blood sample was analyzed for glucose and insulin. The glucose and insulin data are used for calculation of the insulin sensitivity index (SI) and glucose effectiveness (SG). SI reflects the effect of an incremental change in plasma insulin to increase fractional glucose clearance independent of glycemia. SG is the fractional glucose disappearance rate at basal insulin. The minimal model analysis was accomplished with the MINMOD-PC program (copyright R. Bergman).

Pulsatile hormone secretion. Episodic secretion of GH was estimated via a waveform-specific deconvolution technique (6, 30) applied to serum GH samples (3 cc) collected every 10 min from 2100 to 0900. This technique estimates all measured plasma GH concentrations in relation to 1) the number, 2) the amplitudes, and 3) the duration of significant GH secretory bursts, as well as the subject-specific hormone half-life. Additionally, an approximate entropy statistic (ApEn) was used to evaluate the relative degree of serial orderliness of the GH concentration profiles. A larger ApEn value reflects greater apparent randomness (or less consistent repetition of subpatterns; Ref. 19).

Short-term insulin pulsatility was evaluated as described by Peiris et al. (18). After subjects were fasted overnight, an intravenous cannula was inserted into their antecubital vein. A heating pad was used to obtain "arterialized" venous samples. Beginning at 0730, blood samples (3 cc) were withdrawn every 2 min for 90 min for determination of peripheral serum insulin concentration. Deconvolution analysis was used to determine frequency, amplitude, pulse area, interpulse interval, and short-term insulin oscillatory peaks as previously validated (20).

Analytical methods. Nitrogen was determined in daily urinary composites, 7-day fecal composites, and 7-day food composites (except during run-in when 5-day composites were used). Urine and fecal nitrogen were measured by chemiluminescence with a Model 703C pyrochemiluminescent system (Antek Instruments, Houston, TX) equipped with an automatic sample injector, and a Spectra Physics computing integrator, a method that correlates well with the Kjeldahl method for total nitrogen content. Food nitrogen was determined with a Perkin Elmer Model 2410 nitrogen analyzer (Norwalk, CT).

Serum insulin concentrations were measured on an Abbott IMx analyzer (Abbott Laboratories, Abbott Park, IL) with a microparticle enzyme immunoassay. Serum thyroid hormones [T3, L-thyroxine (T4), TSH, T-uptake, and the free thyroxine index] were measured by paramagnetic-particle, chemiluminescence assay (Access Immunoassay System; Sanofi Diagnostics-Pasteur, Chaska, MN). The Access T-uptake assay indirectly assesses the activity of thyroid binding globulin. When used in conjunction with measurements of T3 and T4, it allows estimation of the free (unbound) fractions of those hormones. Because of gender differences in thyroid binding, both free T3 and T4 estimates are reported. Serum GH was measured with an ultrasensitive chemiluminescent assay (Nichols; San Juan Capistrano, CA) with a sensitivity of 0.02 ng/l and within-assay coefficient of variation of 4-5%.

Statistical methods. Data were analyzed with SAS for Windows, version 6.12 (SAS, Cary, NC). Univariate statistics were calculated to determine means and standard errors and to assess normality of the data. All data are shown as the mean ± SE. Changes with treatment for measures performed at baseline and 4 wk were performed by calculating the difference from baseline for each subject and assessing whether the deltas were significantly different from zero with a paired, two-tailed Student's t-test. Changes in nitrogen balance over time were calculated by subtracting baseline values from each pooled 7-day period of bed rest. These values were then analyzed with a repeated-measures ANOVA with compound symmetry covariance structure. Reported nitrogen balance data were corrected for urine, fecal, menstrual blood, and insensible losses but not nonphysiological blood losses (i.e., blood sampling). The primary statistical comparisons were 1) T3 vs. placebo (across both genders), 2) men vs. women (both treatments combined), and 3) time effects (baseline vs. posttreatment across genders and treatment). Tukey's adjustments were used in post hoc multiple comparisons. An alpha  < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The subjects were selected to be of comparable age to astronauts (30-50 yr) and averaged 36.4 ± 1.3 yr for the men and 34.2 ± 2.1 yr for the women. At baseline, the mean body weight in the men was 81.2 ± 2.2 kg and in the women was 57.7 ± 1.4 kg, corresponding to body mass index (BMI) values of 24.8 ± 0.7 and 22.8 ± 0.5 kg/m2, respectively.

Serum concentrations of T3 in men and women are shown in Fig. 2. During treatment, serum T3 concentrations increased to a peak of 119% over baseline in men and 183% over baseline in women receiving the active drug. Serum T3 remained unchanged in individuals receiving placebo. There was a significant gender-time interaction (P = 0.01), because serum levels of total T3 were higher in women than in men, possibly due to the effect of estrogens (both exogenous and endogenous) on thyroid hormone-binding proteins and/or the larger T3 dose per unit body mass administered to the women. Because of the gender difference in total T3, a calculated "free T3 index" was determined as the product of total T3 and T-uptake. These adjusted T3 values were not statistically different in men and women, although the values in women were still higher (peak adjusted T3: 105.1 ± 24.8 for men and 149.5 ± 14.9 for women).


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Fig. 2.   Serum concentrations of T3 in women (A) and men (B) treated with either 50 µg/day T3 () or placebo (down-triangle) during 4 wk of bed rest.

There was a main effect of treatment on T4 concentrations (P = 0.002), which declined by week 4 to 54% of baseline in men and 61% in women receiving T3 while remaining unchanged by placebo (Fig. 3). The failure of euthyroid individuals to completely suppress T4 during T3 administration is expected and was originally reported by Duick et al. (17) in the 1970s. There were also significant gender-time (P = 0.002) and treatment-time (P = 0.009) interactions for T4. The treatment-time interaction was due to T4 being higher in men and women receiving placebo than those receiving T3 at each time point. The gender-time interaction was due to the fact that, overall, the T4 response to T3 treatment was less in the women than in the men (Fig. 3). A free thyroxine index was calculated as (total T4 × %T-uptake)/40. This value did not differ significantly between men and women or by treatment (data not shown).


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Fig. 3.   Serum concentrations of T4 in women (A) and men (B) treated with either 50 µg/day T3 () or placebo (down-triangle) during 4 wk of bed rest.

TSH was suppressed below detectable levels in all T3-treated subjects, while remaining unchanged from baseline in subjects receiving placebo (data not shown). There was no gender difference in TSH response.

Figure 4 shows nitrogen balance values during the study. Nitrogen balance was near zero during the baseline run-in period before bed rest. During bed rest, nitrogen losses were significantly greater in the T3-treated group than the placebo-treated group. Nitrogen balance during the 4 wk of bed rest in T3-treated subjects was also significantly lower than baseline. Nitrogen balance was slightly positive in both groups during recovery, differing significantly from weeks 1-4 in the T3-treated group. There was a significant gender-treatment interaction (P = 0.03) due to the fact that T3-treated men showed a greater decrease in nitrogen balance (adjusted for lean body mass) relative to baseline than did T3-treated women. Although both genders reached similar nadirs in nitrogen balance (approximately -3.0 g/day), the women were, on average, in greater negative nitrogen balance during the run-in period (-2.25 g/day) than were the males (-0.5 g/day) in the T3-treated group. Thus, when the difference from baseline was calculated, T3-treated men achieved a greater relative decrease in nitrogen balance.


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Fig. 4.   Nitrogen balance (intake - output) during 4 wk of bed rest in healthy volunteers treated with either 50 µg/d T3 or placebo. Average nitrogen intake during bed rest phase of study was 12.6 g/day in men and 7.5 g/day in women. * Significantly different from placebo and from baseline values within group, P < 0.05.

Changes in body weight and composition tend to parallel the nitrogen balance data (Table 1). All subjects lost weight during the period of bed rest (main effect of time; P < 0.0001), and there was a significant time-treatment interaction (P = 0.02) because subjects receiving T3 lost more weight than those receiving placebo. There was no difference between men and women in the amount of weight lost. The weight loss was due to a decrease in lean body mass in both genders, because total body fat was largely unchanged after the 28 days of bed rest. Lean body mass was significantly decreased from baseline to end of study (P < 0.0001), and there was a significant time-treatment interaction (P = 0.04), again due to greater losses of lean mass in T3-treated subjects. Despite the lack of change in total body fat by DEXA, total abdominal fat area by CT scan declined in all subjects (main effect of time: P = 0.02).

                              
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Table 1.   Changes in body composition variables in men and women as a result of bed rest with or without T3 treatment

Whole body leucine turnover data are shown in Table 2. Leucine turnover increased in both groups as a result of bed rest, with significantly greater effects in subjects who received T3. Across both genders, leucine Ra increased by 31% in the T3-treated subjects but only 8% in the placebo-treated subjects (main effect of treatment: P = 0.01). Nonoxidative leucine disposal also increased to a greater extent in the group treated with T3, with significant main effects of both time (P = 0.001) and treatment (P = 0.04). Leucine oxidation was not significantly affected by treatment, but increased from baseline to week 4 (main effect of time: P = 0.03). Bed rest-T3 treatment appeared to have a greater effect on leucine turnover in women than in men, in that the change in both leucine Ra and nonoxidative leucine disposal from baseline to week 4 of treatment was significant in women receiving T3 but not in those receiving placebo (Table 2). There were main effects of gender on both leucine oxidation (P = 0.04) and leucine balance (synthesis minus breakdown; P = 0.04). Three-way interactions (gender-time-treatment) were found for nonoxidative leucine disposal (P = 0.02) and leucine Ra (P = 0.07).

                              
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Table 2.   Leucine turnover results before and after 28 days of bed rest with or without T3 treatment

RMR (adjusted for lean body mass) tended to increase from baseline to week 4, particularly in the group treated with T3 [placebo: 1,526 ± 56.4 to 1,549 ± 56.4 kcal/day; T3: 1,552 ± 47.6 to 1,658 ± 47.6 kcal/day; nonsignificant (NS)]. The changes in RMR with treatment were similar in both genders. Average RMR tended to be slightly higher in men than in women, even after adjusting for lean body mass as a covariate (1,799 ± 79 vs. 1,356 ± 135 kcal/day; main effect of gender: P = 0.06).

Insulin sensitivity (SI from the Minimal Model) was substantially reduced by bed rest across all subjects (43% decrease) (main effect of time: P = 0.005). In subjects treated with placebo, SI dropped from 3.66 ± 0.74 to 2.26 ± 0.74 ×10-4 min · µU-1 · ml-1 (NS), whereas in the group treated with T3, SI dropped from 4.24 ± 0.68 to 2.24 ± 0.68 ×10-4 min · µU-1 · ml-1 (P = 0.06). Because of difficulties with venous access, the insulin sensitivity assessment could only be completed in three women; therefore, analysis by gender was not performed. SI did not correlate significantly with leucine Ra, explaining only 6% of the variance in this variable (r = 0.43, NS). Noninsulin-mediated glucose disposal, or SG, was not significantly changed by bed rest or T3 treatment.

There was a significant main effect of time on fasting glucose levels (P = 0.04), which increased from baseline to after bed rest with no difference between T3- and placebo-treated groups (5.08 ± 0.13 to 5.26 ± 0.14 and 5.12 ± 0.14 to 5.26 ± 0.14 mmol/l, respectively). Fasting insulin levels also increased significantly with treatment (P = 0.004 for main effect). The insulin increase in the placebo group was not statistically significant (from 26.3 ± 6.6 to 37.2 ± 6.6 pmol/l; NS) but that in the T3 group was (from 34.3 ± 6.0 to 52.7 ± 6.0 pmol/l; P = 0.04).

Insulin pulsatility data are shown in Table 3. Bed rest produced a significant increase in the absolute pulse increment of insulin release independent of treatment (2.19 ± 0.23 at baseline vs. 2.91 ± 0.24 after bed rest across both genders and treatments; P = 0.035), a finding that is consistent with higher fasting insulin levels observed after bed rest. There was an unexpected main effect of gender on absolute peak insulin increment with women having larger insulin peaks than men (P = 0.04). Insulin half-life in blood tended to decrease during bed rest (from 13.6 ± 1.0 to 10.1 ± 1.0 min; P = 0.07), but there was no effect of treatment. Insulin half-life was significantly greater in women than in men, irrespective of treatment (P = 0.04). There was no effect of bed rest or T3 on insulin peak duration or relative increment above baseline.

                              
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Table 3.   Insulin pulsatility data before and after bed rest with or without T3 treatment in 14 subjects

GH secretion data are shown in Table 4. Spaceflight and bed rest alter GH secretion patterns, and GH may modulate changes in body composition during bed rest. Basal GH secretion was significantly decreased by bed rest (7.2 ± 0.5 vs. 5.4 ± 0.5 × 10-3 µg · l-1 · min-1 across gender and treatments; P = 0.04). There was a significant main effect of treatment on basal GH secretion, with subjects in the T3 group showing a lesser decrease in GH secretion than those in the placebo group. Similarly, there were significant main effects of treatment on 24-h GH secretion, which was greater in placebo than in T3-treated subjects (2,892 ± 331 vs. 1,195 ± 320 µg/l; P = 0.004). Women had significantly greater basal and 24-h integrated GH secretion than did men (main effect of gender P < 0.0002 for both variables), and there was a significant gender-treatment interaction for 24-h integrated secretion (P = 0.002), due to the greater difference between placebo and T3 groups in women than in men (Table 4). Finally, there was a significant time-treatment interaction for GH burst half-duration (the duration of the calculated secretory event at half-maximal amplitude; P = 0.03). This was due to the fact that GH half-duration decreased from baseline to posttreatment in the placebo group (30.1 ± 2.3 to 21.2 ± 2.3 min) but increased in the T3-treated group (23.2 ± 2.3 to 27.3 ± 2.3 min). Similarly, there was a significant time-treatment interaction for the GH ApEn statistic (P = 0.02), which increased from pre- to posttreatment in the group receiving T3 during bed rest but decreased in the placebo group. There were no significant time or treatment effects on the other parameters of GH secretion (Table 4).

                              
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Table 4.   GH pulsatility data before and after bed rest with or without T3 treatment in 14 subjects

Side effects observed during the study were primarily those previously reported with bed rest, including headache, sinus congestion, constipation, gastroesophageal reflux, and backache. Two T3-treated subjects and four placebo-treated subjects reported nausea, and a rash occurred in two T3-treated subjects and one placebo-treated subject. One female subject developed a urinary tract infection, treated with ciprofloxacin, and another woman developed significant vertigo, which was treated with meclizine. The side effects were generally considered to be mild by the participants. When needed, acetaminophen, decongestants, and laxatives were given to the subjects. Medications were selected that would not interfere with any endpoint measures. There were no significant differences between subjects receiving T3 and those receiving placebo in the frequency or severity of side effects (data not shown).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Prolonged unloading of major muscle groups, as occurs during enforced bed rest and spaceflight, poses considerable problems for human health and welfare. Because of the importance of clinical research on the physiological sequelae of muscle unloading, the present study evaluated the utility of supplementation with low-dose exogenous T3 in the existing ground-based model of spaceflight. A comparison of the magnitude of physiological effects of spaceflight reported in the literature and the effects observed in the present study with bed rest alone or bed rest plus T3 is shown in Table 5. Although it is obvious that the bed rest plus T3 model does not truly emulate spaceflight, it is clear that the physiological changes in body composition and protein turnover in this model are closer to the magnitude of changes seen with actual spaceflight than the standard model of bed rest alone.

                              
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Table 5.   Comparison of physiological effects of spaceflight, bed rest alone, and bed rest plus T3

Comparison of bed rest alone vs. bed rest plus T3. Subjects in the bed rest-T3 condition had a significantly more negative nitrogen balance throughout the study compared with bed rest alone, losing on average 2.5-3.0 g/day, whereas the placebo group remained in near-zero nitrogen balance. There are limited data available on nitrogen losses during actual spaceflight, which vary depending on diet and exercise regimens followed by the crew. Data from Skylab showed that nitrogen excretion was increased by ~3 g/day after 28 days of spaceflight (10), a value very similar to our bed rest model with addition of T3.

Leucine turnover was also significantly greater in subjects who received T3 during bed rest compared with bed rest alone. Previous studies in ambulatory men given T3 have also shown increases in leucine turnover (13). There are limited data on protein turnover in spaceflight, although Stein et al. (25) recently reported that whole body protein synthesis assessed by the [15N]glycine method increases by ~24% in space shuttle astronauts. This change is similar to the 20% increase observed in subjects receiving T3 during bed rest, whereas the increase in those receiving placebo was significantly less (~6%).

In short-term space missions, alterations in protein metabolism are probably mainly the result of a stress response. Stein et al. (24) reported that a typical metabolic stress response occurs in spaceflight; i.e., whole body protein synthesis increases, acute phase protein synthesis increases, and urinary cortisol is elevated. Stress is clearly not a major factor in bed rest models, and others have shown that whole body protein synthesis rates are actually decreased with short-term bed rest (26). Ideally, measures of urinary or serum cortisol would have been made in the present study. Unfortunately, with the large numbers of other measures being performed on each sample, this was not feasible.

Changes in body composition paralleled the losses of body nitrogen and protein. Subjects receiving T3 treatment lost significantly more total weight and lean mass than those receiving placebo. The apparent decrease in total abdominal fat, despite the lack of decrease in total body fat, is likely to be due to differences in the methodology used to assess these variables (i.e., DEXA vs. CT scan). Stein and Gaprindashvili (23) have reported that prolonged space missions are associated with decreases in both lean and fat mass, largely as a result of muscle deconditioning and atrophy.

One potential criticism of the present model is that the changes in body composition and protein turnover that occur during weightlessness are not mediated by hyperthyroidism. Although this is true, spaceflight may be, in fact, associated with some changes in the thyroid axis. In Apollo astronauts, free T4 concentrations were increased after spaceflight (22). Similar findings were reported in Skylab astronauts; however, T3 levels were unchanged from pre- to postflight, whereas TSH was increased postflight (8). Concentrations of thyroid hormones during spaceflight have not been well documented, so it is not clear how the changes in thyroid hormone in the present model relate to actual spaceflight-associated changes. During bed rest, thyroid hormone rhythmicity has been reported to be altered, suggesting that thyroid rhythms are posture dependent (28). Clearly, the imposed T3 treatment alters this expected influence of bed rest.

Bed rest may induce transient glucose intolerance and insulin resistance (10, 26). The limited data available from spaceflight suggest that glucose and insulin decrease during flight, but insulin increases postflight (8). The present study is the first to our knowledge to directly measure peripheral insulin sensitivity and non-insulin-mediated glucose disposal (SG) by Minimal Model during bed rest. We observed a pronounced decrease in SI with bed rest, independent of treatment. There was no effect of bed rest or T3 treatment on SG, suggesting the defect is mainly in the insulin-mediated component of glucose disposal. Others have reported that the insulin resistance of bed rest occurs mainly in muscle, rather than liver or other tissues (26). The observed changes in insulin secretion (increased incremental peaks and amplitude and decreased half-life) are consistent with the hyperinsulinemia that typically accompanies insulin-resistant states, although our design did not allow us to address whether insulin resistance or hyperinsulinemia is primary. The relevance of these changes to nitrogen loss is unclear because there was not a significant correlation between SI and protein breakdown.

Basal and integrated GH secretion were decreased by bed rest in both T3- and placebo-treated subjects, although no other parameter of GH secretion showed significant changes during the protocol. There were, however, significant main effects of treatment on these two GH-secretion parameters, with greater decreases being observed in subjects in the placebo group. The only spaceflight data available showed that serum GH concentrations were increased during the first few days of Skylab (8). During bed rest, Vernikos-Danellis et al. (27) report that GH concentrations drop after 10 days, followed by an increase by 20 days and gradual decline up to 54 days. These authors further observed a change in the diurnal rhythm of GH profiles, although deconvolution analyses were not performed. We performed our analyses of GH secretion after ~24-26 days of bed rest, and thus, according to the data of Vernikos-Danellis et al., anticipated an increase, rather than a decrease, in GH secretion. However, our method of assessing GH secretion was quite different from that of the previous studies. Additionally, our study included women, who are known to have different patterns of GH secretion than men (31). Although gender differences in GH secretion were observed, the response of GH secretion to bed rest or T3 treatment did not differ between men and women. Finally, with the small number of subjects in our study, it is possible that we did not have adequate statistical power to detect small changes in GH secretion. In this regard, it would be of interest to have plasma insulin-like growth factor I concentrations in future studies.

Comparison of effects in men vs. women. To our knowledge, only one study has previously examined the effects of head-down bed rest in women (29). Vernikos et al. (29) compared the effects of 7 days of head-down bed rest in eight male and eight female volunteers. In their study, women exhibited lower ACTH levels and urinary cortisol excretion and greater changes in the renin-aldosterone system than did men.

In the present study, women had greater increases in leucine turnover in response to T3 plus bed rest, although leucine turnover during bed rest in the placebo group did not differ between genders. The difference may have been due to differences in the absolute elevation in total T3 between genders. Total serum T3 levels were higher in the women, possibly due in part to the effects of estrogens on thyroid binding because all the women were taking oral contraceptives during the study. Additionally, because T3 was administered in a constant dose rather than per kilogram body mass, the women received, effectively, a higher dose of the hormone. Nevertheless, the calculated free T3 index did not differ significantly between men and women; thus biologically active T3 was not likely substantially higher in women. The correlation between the calculated free T3 index and protein breakdown was 0.60 (P < 0.05). To our knowledge, the effects of T3 on leucine turnover have not been compared in healthy men and women, but it is possible that T3 may have a greater effect on leucine turnover in women than men.

In contrast to the leucine turnover results, T3-treated women had a smaller change from baseline in nitrogen balance than did T3-treated men under conditions of strict bed rest. It is possible that this difference is related to the difference in relative adiposity between men and women. Previous studies have suggested that initial levels of body fat predict changes in nitrogen or lean body mass loss during fasting or other catabolic conditions (5). Because women have greater percentage body fat than men (34.5 ± 1.9 vs. 23.5 ± 2.2% in our study), this may explain why they lost less nitrogen during prolonged bed rest. The correlation between baseline percent body fat and nitrogen balance during bed rest week 4 was -0.39, which, although weak, suggests that this relationship may at least partially explain the data. Despite the gender differences during T3-bed rest in protein turnover and nitrogen balance, there was no gender difference in the body composition response.

Women receiving either placebo or T3 developed a more pronounced increase in insulin secretion, as assessed by insulin pulsatility, than did men. Unfortunately, because of technical problems obtaining adequate venous access in women, we were unable to perform the insulin sensitivity test before and after bed rest in the majority of female volunteers. It is therefore not possible at present to assess whether the gender differences in insulin secretion also reflect gender differences in peripheral insulin sensitivity.

In summary, we have investigated a new clinical research model of simulated weightlessness that includes the addition of low-dose T3 administration to the conventional head-down bed rest paradigm in both men and women. Our studies suggest that bed rest with T3 results in greater loss of body protein stores, more similar to that of spaceflight per se. Furthermore, there were no significant differences in the adverse event profiles of bed rest alone vs. bed rest with T3. Although there were some significant differences between the placebo and T3-treated groups in their endocrine (insulin and GH) responses to bed rest, overall the results indicate that similar changes in these parameters occurred. Women exhibited greater responses to the bed rest with T3 condition than men for leucine turnover; however, it is unclear whether this result was due to the higher serum levels of T3 achieved in the women. Further research is thus needed to understand the applicability of this model to female astronauts. We postulate that combined bed rest and T3 administration may have utility in evoking greater structural-catabolic changes over a shorter interval in clinical studies of both the impact of musculoskeletal unloading and the effectiveness of countermeasures for resultant physical deconditioning that accompanies microgravity or enforced physical inactivity.


    ACKNOWLEDGEMENTS

We thank the volunteers who gave time and effort to ensure the success of this project. We also thank the nurses and the technical and kitchen staff of the Inpatient Metabolic Research Unit; particularly, Ricky Brock, Mark Klemperer, and Helena Duplantis. The valuable suggestions of the advisory board, consisting of Drs. David Clemmons, Helen Lane, John Nicoloff, and Frank Svec, regarding the design and conduct of this study are gratefully acknowledged.


    FOOTNOTES

This research was supported by a grant from the National Aeronautics and Space Agency (NAG 9-714 to G. A. Bray), the National Science Foundation Center for Biological Timing, and National Institute on Aging (R01-AG-147991 to J. D. Veldhuis).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. C. Lovejoy, Pennington Biomedical Research Center, 6400 Perkins Rd., Baton Rouge, LA 70808-4124 (E-mail: lovejoj{at}mhs.pbrc.edu).

Received 17 September 1998; accepted in final form 20 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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