Tissue Deiodinase Activity during Prolonged Critical Illness: Effects of Exogenous Thyrotropin-Releasing Hormone and Its Combination with Growth Hormo
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《内分泌学杂志》
Department of Intensive Care Medicine (Y.D., B.E., L.M., G.V.d.B.), Laboratory for Experimental Medicine and Endocrinology (E.V.H., W.C.)
Laboratory of Comparative Endocrinology (V.D.), Catholic University of Leuven, B-3000 Leuven, Belgium
Department of Anesthesiology and Intensive Care Medicine, University Hospital of Muenster (B.E.), D-48149 Muenster, Germany
Abstract
Prolonged critical illness is characterized by reduced pulsatile TSH secretion, causing reduced thyroid hormone release and profound changes in thyroid hormone metabolism, resulting in low circulating T3 and elevated rT3 levels. To further unravel the underlying mechanisms, we investigated the effects of exogenous TRH and GH-releasing peptide-2 (GHRP-2) in an in vivo model of prolonged critical illness. Burn-injured, parenterally fed rabbits were randomized to receive 4-d treatment with saline, 60 μg/kg·h GHRP-2, 60 μg/kg·h TRH, or 60 μg/kg·h TRH plus 60 μg/kg·h GHRP-2 started on d 4 of the illness (n = 8/group). The activities of the deiodinase 1 (D1), D2, and D3 in snap-frozen liver, kidney, and muscle as well as their impact on circulating thyroid hormone levels were studied. Compared with healthy controls, hepatic D1 activity in the saline-treated, ill animals was significantly down-regulated (P = 0.02), and D3 activity tended to be up-regulated (P = 0.06). Infusion of TRH and TRH plus GHRP-2 restored the catalytic activity of D1 (P = 0.02) and increased T3 levels back within physiological range (P = 0.008). D3 activity was normalized by all three interventions, but only addition of GHRP-2 to TRH prevented the rise in rT3 seen with TRH alone (P = 0.02). Liver D1 and D3 activity were correlated (respectively, positively and negatively) with the changes in circulating T3 (r = 0.84 and r = –0.65) and the T3/rT3 ratio (r = 0.71 and r = –0.60). We conclude that D1 activity during critical illness is suppressed and related to the alterations within the thyrotropic axis, whereas D3 activity tends to be increased and under the joint control of the somatotropic and thyrotropic axes.
Introduction
PROLONGED CRITICAL ILLNESS is characterized by a uniformly suppressed function of the thyrotropic and somatotropic axes, as revealed by reduced pulsatile TSH secretion with low T4 and T3 and elevated rT3 levels, a constellation commonly known as the low T3 syndrome, and a suppressed pulsatile GH release with low levels of IGF-I (1, 2). The hyposomatotropism of prolonged critical illness seems to be caused predominantly by altered hypothalamic control, because a continuous infusion of GH-releasing peptide-2 (GHRP-2) reactivates pulsatile GH secretion and normalizes IGF-I levels (3, 4). The pathophysiology of the low T3 syndrome is even more complex, with reduced TRH gene expression explaining reduced TSH secretion and thyroidal T4 release and, concomitantly, alterations in peripheral thyroid hormone metabolism playing a role in bringing about the low T3 concentrations (5). Indeed, continuous iv infusion of TRH is able to reactivate TSH secretion in prolonged critical illness, which increases circulating T4 and T3 back into the normal range, but also elevates circulating levels of rT3 (6). The latter is prevented by adding GHRP-2 to the TRH infusion (7, 8). Complex interactions between the somatotropic and thyrotropic axes are likely to be involved.
In normal physiology, the thyroid predominantly secretes the prohormone T4 and only a small amount of the active hormone T3 (9). The bulk of daily T3 production occurs in various extrathyroidal tissues via outer ring deiodination of T4 (10). This activation may be catalyzed by two different deiodinases, D1 and D2, which are distinguished by their kinetic properties and substrate specificity (11). In healthy humans, 15–81% of T3 is derived from the D1-containing tissues, liver and kidney, with the remaining originating from D2-containing tissues, such as skeletal muscle (10). Besides outer ring deiodination of T4 into T3, T4 is converted into rT3 by inner ring deiodination exerted by type 3 deiodinase (D3). T3 and rT3 undergo additional deiodination, predominantly to the common metabolite 3,3'-diiodothyronine, which is generated by, respectively, inner and outer ring deiodination of T3 and rT3 (10).
In prolonged critical illness, the low T3 and concomitantly elevated rT3 concentrations can be explained at least in part by reduced D1 and elevated D3 activity, whereby the conversion of T4 into active T3 is reduced and, instead, T4 is metabolized into inactive rT3 (12, 13). In an animal model of prolonged critical illness that has been shown to mimic the endocrine and metabolic changes observed in human critical illness, a combined infusion of TRH and GHRP-2 was found to increase the catalytic activity of D1 and depress that of D3 (14). Because suppression of D3, but not stimulation of D1, also occurred in response to administration of GH (14), these findings suggested that during critical illness, up-regulation of D3 activity is partly related to the lack of GH activity (15). Reactivation of D1 activity, on the contrary, was absent after GH administration and thus could be due to either a direct effect of GHRP-2 or TRH or the induced rise in circulating T4 and T3 concentrations (14). The study design, however, did not allow distinguishing among these possibilities.
We hypothesized that during critical illness, D1 activity is regulated predominantly by changes within the thyrotropic axis, and D3 activity is modulated by alterations within the somatotropic axis. To test this hypothesis, we studied the effects of TRH, GHRP-2, and TRH plus GHRP-2 on peripheral thyroid hormone metabolism in our animal model of prolonged critical illness.
Materials and Methods
Study set-up and protocol
All animals were treated according to the Principles of Laboratory Animal Care formulated by the U.S. National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Institutes of Health. The study protocol was approved by the Leuven University ethical review board for animal research.
The model has been described in detail previously (16). In brief, male New Zealand White rabbits were purchased from a local rabbitry, housed individually, and exposed to artificial light for 14 h/d. On d 1, the animals were anesthetized with 30 mg/kg ketamine, im (Merial, Lyon, France), and 0.15 ml/kg medetomidine (Orion Corp., Ospoo, Finland), im. After weighing and shaving neck and flanks, anesthesia was supplemented by isoflurane (Forene, Abbott Laboratories, Inc., Queensborough, UK) added to the breathing gas via regular vaporizer. Both the carotid artery and jugular vein were cannulated, and catheters were filled with heparin (5000 IU/ml; Heparine, Rhne Poulenc Rorer, Brussels, Belgium) to assure patency. Thereafter, a supplemental paravertebral block with 1% xylocaine (Astra Pharmaceuticals, Brussels, Belgium) was performed, and a full thickness burn injury equaling 15–20% of the total body surface area was imposed. At the end of the procedure, animals returned to their cages, and overnight fluid resuscitation was started with a continuous infusion of Ringer’s lactate at six drops per minute (±18 ml/h) through a volumetric infusion pomp (IVAC 531 infusion pump, IVAC Corp., San Diego, CA). To assure free movement in the cage and to safe-guard the arterial and venous lines, animals wore a homemade jacket, and infusions were connected to the vascular access via a swivel device. In the evening, a supplemental dose of a major analgesic (Dipidolor, Janssen-Cilag, Beerse, Belgium) was given. The next morning, parenteral nutrition (PN) was initiated in all rabbits at four drops per minute (±12 ml/h). Blood glucose levels were kept below 180 mg/dl by frequent blood glucose monitoring and titration of insulin infusion (100 IU/ml; Actrapid Novolet, Novo Nordisk, Bagsvaerd, Denmark; via an SE200B infusion pump, Vial Medical, Brezins, France) when necessary. Because GHRP-2 is known to induce insulin resistance (6), insulin therapy was started directly after randomization in rabbits allocated to receive GHRP-2 or TRH plus GHRP-2. All iv infusions were weighed before and after administration, allowing accurate determination of the infused quantity. PN was prepared daily in the hospital pharmacy under laminar air-flow conditions. The infusion bags contained 150 ml Clinomel N7 (Baxter, Clintec Parenteral, Maurepas, France) and 175 ml sterile water. Thus, bags with 325 ml solution contained 156 kcal nonprotein calories and 0.99 g nitrogen. Of all nonprotein calories, 61.5% were delivered as carbohydrates and 38.5% as fat. Protein intake equaled 1.49 g/kg·d. No additional vitamins or trace elements were added. PN was continuously administered, and the bags were changed daily. Animals had free access to water, but oral food intake was denied.
On the morning of d 4, surviving rabbits were randomly (by sealed envelopes) allocated to one of the following treatment groups (Fig. 1). Group 1 (saline group) received a 4-d continuous infusion of 0.9% NaCl. Group 2 (GHRP-2 group) received an initial bolus of 60 μg/kg GHRP-2 (Kaken Pharmaceutical Co. Ltd., Tokyo, Japan), followed by a continuous infusion of 60 μg/kg·h. Group 3 (TRH group) received an initial bolus of 60 μg/kg TRH (UCB Pharma, Brussels, Belgium), followed by a continuous infusion of 60 μg/kg·h. Group 4 (TRH+GHRP-2 group) received an initial bolus of 60 μg/kg TRH plus 60 μg/kg GHRP-2, followed by a continuous infusion of 60 μg/kg·h TRH and 60 μg/kg·h GHRP-2. The effective doses of TRH and GHRP-2 were defined in a previous study (16).
On d 8, the animals were weighed and then killed with sodium-pentobarbital (60 mg/ml; Nembutal, Sanofi-Winthrop, New York, NY). Tissue samples were taken from liver, kidney, and muscle and snap-frozen in liquid nitrogen, after which they were stored at –80 C until additional analysis of deiodinase activity.
To control blood glucose levels, two to four arterial blood samples were taken daily. On d 1 after catheter insertion, d 4 before randomization, and at 0900 h on d 5, 6, 7, and 8, an additional 4 ml blood was sampled. Blood for biochemical analyses was sampled in lithium citrate tubes (BD Biosciences, Temse, Belgium), immediately centrifuged, and then stored at –30 C until assay.
Assays
Plasma rabbit GH (rGH) concentrations were measured with a specific RIA (reagents provided by Dr. A. Parlow, National Pituitary Agency, Torrance, CA). The detection limit was 1 μg/liter, and the within-assay coefficient of variation (CV) was 2.3%. All samples contained detectable plasma concentrations. Because frequent blood sampling for determination of rGH concentration time series would evoke an intolerable amount of blood loss, we analyzed GH in a single sample.
Plasma IGF-I concentrations were measured by RIA, using a slightly modified version of that described previously (16, 17). After acidification of the plasma samples with formic acid, binding proteins were separated by acid gel filtration on an Econo-Pac column (Bio-Rad Laboratories, Richmond, CA). Des-IGF-I was used as tracer to avoid binding to residual small IGF-binding proteins. The intraassay CV was 4.6%, and the detection limit was 20 μg/liter.
Plasma concentrations of rTSH were measured by a specific RIA (reagents provided by Dr. A. Parlow, National Pituitary Agency). The detection limit was 1.2 mU/liter, and the intraassay CV was 5.3%. For samples with undetectable levels, a value representing half the detection limit was entered. As for rGH, rTSH was only analyzed in a single sample.
Total concentrations of plasma T4, T3, and rT3 were determined by in-house RIA (18, 19). The detection limit and intraassay CV were, respectively, 0.3, 0.03, and 0.05 nmol/liter and 8.5%, 6%, and 9%. All samples were measured in a single assay run. Measurement of total thyroid hormone levels was performed as catheters for blood sampling needed to be heparinized, which induces artifactual free hormone determinations (20).
Arterial blood was analyzed on an ABL 700 analyzer (Radiometer Medical A/S, Copenhagen, Denmark) to quantify pH, hemoglobin, lactate, and glucose.
Deiodinase activity
For determination of D1, D2, and D3 activities, tissue microsomal fractions were prepared and assayed as described by Darras et al. (21, 22). Final incubations were performed in a total volume of 200 μl. For D1 activity, final incubation mixtures contained 1 μM rT3, 50,000 cpm/tube [3',5'-125I]rT3 (labeled using 17 Ci/mg carrier-free 125I from NEN Life Science Products, Boston, MA), 0.2 mg microsomal protein/ml (liver and kidney) or 0.5 mg/ml (muscle), 2 mM EDTA, and 5 mM dithiothreitol (DTT) and were incubated for 30 min (120 min for muscle) at 37 C. For D2 activity, incubations contained 1 nM T4, 50,000 cpm/tube [3'-5'125I]T4 (labeled as described above), 1 mg microsomal protein/ml (liver and kidney) or 0.5 mg/ml (muscle), 100 nM T3 (to block interference from D3), 2 mM EDTA, and 25 mM DTT and were incubated for 120 min at 37 C. Samples were also tested using the same conditions, but 100 nM T4 was used to determine whether the activity was blocked at this high substrate concentration. Only in this case was the activity regarded as true D2 activity. For D3 activity, incubations contained 1 nM T3, 200,000 cpm/tube [3'-125I]T3 (labeled as described above), 1 mg microsomal protein/ml (liver and kidney) or 0.5 mg/ml (muscle), 1 μM rT3 and 0.1 mM propylthiouracil (to block D1 interference), 2 mM EDTA, and 50 mM DTT and were incubated for 120 min at 37 C.
Statistical analysis
The effects of interventions were analyzed using repeated measures and factorial ANOVA with Fisher’s protected least significant difference post hoc testing. Within-group changes were analyzed by Friedman, Mann-Whitney U, Wilcoxon, Kruskal-Wallis, z, and t test, as appropriate, with Bonferroni correction for multiple comparisons where indicated. Correlation analysis was performed with linear or logarithmic regression. The area under the curve (AUC), calculated by the trapezoid rule, was used as a marker of the total amount of hormone liberated in response to an intervention. All data are expressed as the mean ± SEM unless specified otherwise. A two-sided value of P < 0.05 was considered significant.
Results
The experiment was designed to obtain eight survivors on d 8 in all groups. In total, 70 male rabbits were used, of which 13 were excluded due to technical problems (catheter dislocation, cerebral embolism during blood withdrawal, failure of infusion pump, and death during anesthesia); 57% of the remaining animals survived the 8 d of the experiment. Mortality rate did not differ among the four groups. Four healthy animals, matched for gender, age, and body weight, were used as controls.
Body weight and PN
At the start of the study, rabbits weighed 2904 ± 52 g, and the weight decreased to 2732 ± 53 g at the end of the experiment (P < 0.0001 vs. baseline). Starting body weight and loss of body weight were similar in the four groups. PN was administered in equal amounts to rabbits in all groups.
Glucose control
At baseline, the mean blood glucose concentration was 130 ± 5 mg/dl, and it remained constant throughout the whole experiment. The mean total amount of insulin used per rabbit was 13 ± 1 IU in the GHRP-2 group, 22 ± 8 IU in the TRH plus GHRP-2 group, and minimal in the two other groups (2 ± 1 IU in the saline group; 1 ± 1 IU in the TRH group).
Blood gases and hemoglobin
As previously described, this model of sustained critical illness is characterized by anemia (14, 16). The baseline hemoglobin level was 12.2 ± 0.2 g/dl and decreased progressively to 7.9 ± 0.4 g/dl on d 8 (P < 0.0001 vs. baseline). Hemoglobin levels were similar in all groups throughout the experiment. Lactate levels remained constant throughout the experiment, and no differences in arterial pH among the four groups were observed (data not shown).
Somatotropic axis
Plasma IGF-I concentrations at baseline (135 ± 9 μg/liter) and on d 4 (103 ± 10 μg/liter) were comparable in all groups (Fig. 2). Plasma IGF-I decreased significantly during the first 4 d after injury (P = 0.01, d 4 vs. baseline). After randomization, plasma IGF-I levels remained stable in the saline and TRH groups, whereas they increased in animals receiving GHRP-2 and TRH plus GHRP-2 (Fig. 2).
Baseline rGH concentrations measured in a single sample were identical in the four groups. During the experiment, these single sample rGH levels increased in all groups (data not shown).
Thyrotropic axis
In all groups, plasma T4 levels decreased significantly between baseline (38.2 ± 2.0 nmol/liter) and d 4 (31.9 ± 1.6 nmol/liter; P = 0.006). After randomization, T4 levels increased in the TRH and TRH plus GHRP-2 groups, whereas they remained stable in the saline and GHRP-2 groups (Fig. 3).
Compared with healthy controls (1.76 ± 0.3 nmol/liter), plasma T3 levels in the critically ill animals were significantly lower on d 1 (0.84 ± 0.04 nmol/liter; P = 0.002) and d 4 (0.98 ± 0.06 nmol/liter; P = 0.004). In the saline and GHRP-2 groups, T3 remained low, whereas in the TRH and GHRP-2 plus TRH groups, there was a significant increase (Fig. 3).
rT3 levels were identical at baseline and before randomization in all groups (d 4, 0.50 ± 0.19 nmol/liter). After randomization, only administration of TRH significantly increased plasma rT3 (P = 0.02; Fig. 4).
rTSH levels, measured in a single sample, decreased significantly between baseline and d 4 (3.03 ± 0.13 vs. 2.64 ± 0.15 mU/liter; P = 0.008). After randomization, single-sample rTSH levels remained stable, and at no time was there a significant difference among the four groups (data not shown).
Measured in tissue harvested on d 8, hepatic D1 activity in the saline group was significantly lower than that in healthy controls (P = 0.02; Fig. 5). Hepatic D1 activity was significantly higher in the TRH-treated and TRH- plus GHRP-2-treated groups than in groups not receiving TRH (P = 0.02). Hepatic D3 activity in the saline group tended to be higher than that in healthy animals (P = 0.06), whereas in the other three groups, D3 activity was similar to that found in healthy controls (Fig. 5). Renal D1 and D3 activities were also similar in all groups. D2 deiodinase activity was undetectable in all examined tissues, as were D1 and D3 activities in skeletal muscle.
Hepatic D1 activity on d 8 showed a strong positive correlation with AUC of T4 (r = 0.64; P < 0.0001), AUC of T3 (r = 0.84; P < 0.0001), and d 8 plasma T3/rT3 (r = 0.71; P < 0.0001). Also, renal D1 activity correlated positively with AUC of T4 (r = 0.57; P = 0.0006), AUC of T3 (r = 0.70; P < 0.0001), and d 8 plasma T3/rT3 (r = 0.64; P = 0.0001). Liver D3 activity was negatively correlated with AUC of T4 (r = –0.47; P = 0.006), AUC of T3 (r = –0.65; P < 0.0001), and d 8 plasma T3/rT3 (r = –0.60; P = 0.0003; Fig. 6).
Discussion
In our rabbit model of prolonged critically illness, D1 activity in the liver was significantly down-regulated, whereas D3 activity tended to be up-regulated compared with healthy controls, which was related to the changes in circulating T3 and the T3/rT3 ratio. Infusion of TRH and TRH plus GHRP-2 restored the catalytic activity of hepatic D1 and increased T3 levels to within the physiological range. D3 activity was normalized by all three interventions, but only the addition of GHRP-2 to TRH prevented the rise of rT3 seen with TRH alone. Although both GHRP-2 and TRH plus GHRP-2 were able to reactivate the somatotropic axis, only combined infusion of TRH and GHRP-2 induced a significant increase in IGF-I levels into the range observed in healthy rabbits.
Our data confirm that in prolonged critical illness, the low T3 syndrome does not result solely from suppressed TSH secretion, which reduces T4 production (23, 24). Indeed, concomitant changes in peripheral thyroid hormone metabolism appear to play a role (25, 26). D1 and D3 activities have recently been measured in liver samples of critically ill patients who died in the intensive care unit (12, 13) and in liver of prolonged critically ill animals (14). Although these studies did not include a healthy control group, the results suggested that low D1 activity and induction of D3 activity may play a role in the pathogenesis of the low T3 syndrome during critical illness. In our current experiment, we demonstrated that hepatic D1 activity during prolonged critical illness is indeed significantly lower than that in healthy controls. Infusion of TRH and TRH plus GHRP-2 was able to restore the catalytic activity of hepatic D1 and, concomitantly, increase T3 levels to within the physiological range. These results indicate that a depressed hepatic D1 activity is a major mechanism bringing about the low T3 syndrome during critical illness. Although reactivation of pulsatile TSH secretion is thought to predominantly discharge thyroidal T4, and the majority of T3 is derived from peripheral T4 to T3 conversion (9, 10), direct TSH-dependent release of T3 from the thyroid gland may have contributed to the observed rise in T3 with TRH and GHRP-2 plus TRH infusions (27). However, in view of the short half-life of T3 and the increase still being present after 4 d of treatment without concomitant differences in TSH, we believe that this possible contribution of direct T3 release from the thyroid gland was minor. The elevated levels of rT3 during critical illness may also be explained predominantly by a reduced D1 activity, because D1 is the major pathway of rT3 clearance (10, 28).
D2 activity is regulated by substrate-induced enzyme inactivation, and there is reason to believe that D2-induced T3 production in skeletal muscle contributes to circulating T3, especially in the hypothyroid situation (10, 29, 30). Hence, in view of the low plasma T4 levels during prolonged critical illness, it is intriguing that we failed to detect any D2 activity in the skeletal muscle samples of critically ill rabbits, in line with what we previously observed in patients (12, 13). A possible explanation is the elevated plasma concentration of rT3 during critically illness, because rT3 is known to inactivate D2 (31). Hence, a complete absence of muscle D2 activity during prolonged critical illness may be at least partly due to elevated rT3 and may contribute to the low circulating and tissue T3 levels in critically ill patients (10, 29).
Apart from decreased D1 activity, the reciprocal changes in T3 and rT3 during critical illness can also be explained by induction of D3, resulting in enhanced degradation of T3 and augmented production of rT3 (10). Overexpression of D3 in hemangiomas has been shown to cause consumptive hypothyroidism, a syndrome characterized by very low levels of circulating T4 and T3 in combination with high levels of rT3 (32, 33). Hence, the previous observation that D3 activity is clearly detectable in the liver of critically ill patients who died in the intensive care unit suggests a potential role for this enzyme in the pathophysiology of the low T3 syndrome (12, 13). Our current experiments also support such a role, because hepatic D3 activity in saline-treated, prolonged critically ill rabbits appeared higher than that in healthy controls, and a clear negative correlation was found between the T3/rT3 ratio on d 8 and D3 activity in the liver. The expression and activity of D3 during health have only been shown in liver tissue during fetal development, not in adult tissue (10, 34). Hence, it was surprising to document hepatic D3 activity in healthy control rabbits, which may point to a certain degree of stress attributable to manipulation of the controls (12). This could also explain the borderline level of significance for the observed D3 elevation in the sick rabbits compared with the controls.
The regulation of deiodinase activities is a complex process. D1 is thought to be under the positive control of thyroid hormone status, presumably mediated by T3 (35, 36), and GH appears to suppress D3 activity (15, 37). However, little is known about the regulation of deiodinase activity during critical illness. The observation that hepatic D1 was only reactivated in animals receiving TRH, either alone or in combination with GHRP-2, clearly indicates that D1 activity during critical illness is mainly regulated via alterations within the thyroid axis. Because thyroid hormones are known potent inducers of hepatic D1 activity (35, 36), the increase in D1 activity most likely results from the increase in T3 and/or T4. In agreement with this, hepatic D1 activity was positively correlated with the amount of thyroid hormone liberated by the intervention and the T3/rT3 ratio on d 8. However, the present data do not allow us to distinguish between a role for T3 and T4 and a direct effect of TRH or TSH. Because GHRP-2-treated animals revealed levels of D1 activity similar to those in the saline group, a major role of the somatotropic axis in the regulation of D1 during critical illness can be excluded.
Normal D3 activity after TRH plus GHRP-2 infusion confirmed our previous observation of lowered D3 activity with this intervention in the same experimental model of critical illness (14). Administration of GH in that set of experiments similarly reduced D3 activity, which is in line with the normal D3 activity after GHRP-2 infusion in our current experiment. An effect of GH on D3 activity has also been shown to occur in healthy chickens (15, 37) and in both healthy and GH-deficient humans (38, 39). Such an effect can be mediated either by GH itself or by IGF-I. Our observation that the rise in IGF-I obtained with combined infusion of TRH plus GHRP-2 was higher than with GHRP-2 alone, probably explained by the permissive role of thyroid hormones on GH secretion (40, 41) and on the IGF-I response to GH (42, 43), and the observed prevention of the rT3 rise only seen with the combined GHRP-2 plus TRH infusion favors an effect of IGF-I on rT3 generation via D3. However, TRH infusion alone in the sick rabbits was also associated with normal D3 activity in the current experiment. This suggests that alterations within the somatotropic axis as well as those within the thyrotropic axis may explain the up-regulation of D3 activity during critical illness.
We conclude that during critical illness, D1 activity is suppressed, and D3 activity tends to be increased; both are related to the changes in circulating T3 and rT3. D1 activity is restored by TRH infusion, whereas both TRH and GHRP-2 may control D3 activity during critical illness. The extent to which the effects of TRH and GHRP-2 on deiodinase activity are explained by direct effects of the releasing factors or, alternatively, by the induced peripheral hormonal responses during critical illness, remains unclear.
Acknowledgments
We acknowledge the generosity of Dr. C. Y. Bowers, U.C.B. Pharma Belgium, Baxter Belgium, Fresenius-Kabi Belgium, Eddy Vanonckelen (Alaris Medical Systems, Belgium), and Wouter Diddens (Tyco Healthcare, Belgium) in providing, respectively, GHRP-2, TRH, Clinomel, total PN bags, infusion pumps, and arterial catheters for these experiments. We appreciate the technical support of Mrs. Veerle Leunens, Mrs. Magda Mathys, and Mr. Kristof Reyniers (Center for Experimental Surgery and Anesthesiology); Mr. Willy Van Ham, Mrs. Francine Voets, and Mrs. Lut Noterdaeme (Laboratory of Comparative Endocrinology); and Mark Denturck and Wilfried Frooninckx (Technical Department University Hospital Leuven).
Footnotes
This work was supported by the Fund for Scientific Research-Flanders, Belgium [Ph.D. scholarship, Aspirantenmandaat (to Y.D.) and G.0144.00, G.0278.03, and G.0258.05 (to G.V.d.B.)], a grant from Innovative Medizinische Forschung (EL 610304) and B. Braun Stiftung, Germany (to B.E.), and the Research Council of the University of Leuven (OT 03/56; to G.V.d.B.).
First Published Online September 8, 2005
1 Y.D. and B.E. contributed equally to this work.
Abbreviations: AUC, Area under the curve; CV, coefficient of variation; D1, deiodinase; DTT, dithiothreitol; GHRP-2, GH-releasing peptide-2; PN, parenteral nutrition; r, rabbit.
Accepted for publication September 1, 2005.
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Laboratory of Comparative Endocrinology (V.D.), Catholic University of Leuven, B-3000 Leuven, Belgium
Department of Anesthesiology and Intensive Care Medicine, University Hospital of Muenster (B.E.), D-48149 Muenster, Germany
Abstract
Prolonged critical illness is characterized by reduced pulsatile TSH secretion, causing reduced thyroid hormone release and profound changes in thyroid hormone metabolism, resulting in low circulating T3 and elevated rT3 levels. To further unravel the underlying mechanisms, we investigated the effects of exogenous TRH and GH-releasing peptide-2 (GHRP-2) in an in vivo model of prolonged critical illness. Burn-injured, parenterally fed rabbits were randomized to receive 4-d treatment with saline, 60 μg/kg·h GHRP-2, 60 μg/kg·h TRH, or 60 μg/kg·h TRH plus 60 μg/kg·h GHRP-2 started on d 4 of the illness (n = 8/group). The activities of the deiodinase 1 (D1), D2, and D3 in snap-frozen liver, kidney, and muscle as well as their impact on circulating thyroid hormone levels were studied. Compared with healthy controls, hepatic D1 activity in the saline-treated, ill animals was significantly down-regulated (P = 0.02), and D3 activity tended to be up-regulated (P = 0.06). Infusion of TRH and TRH plus GHRP-2 restored the catalytic activity of D1 (P = 0.02) and increased T3 levels back within physiological range (P = 0.008). D3 activity was normalized by all three interventions, but only addition of GHRP-2 to TRH prevented the rise in rT3 seen with TRH alone (P = 0.02). Liver D1 and D3 activity were correlated (respectively, positively and negatively) with the changes in circulating T3 (r = 0.84 and r = –0.65) and the T3/rT3 ratio (r = 0.71 and r = –0.60). We conclude that D1 activity during critical illness is suppressed and related to the alterations within the thyrotropic axis, whereas D3 activity tends to be increased and under the joint control of the somatotropic and thyrotropic axes.
Introduction
PROLONGED CRITICAL ILLNESS is characterized by a uniformly suppressed function of the thyrotropic and somatotropic axes, as revealed by reduced pulsatile TSH secretion with low T4 and T3 and elevated rT3 levels, a constellation commonly known as the low T3 syndrome, and a suppressed pulsatile GH release with low levels of IGF-I (1, 2). The hyposomatotropism of prolonged critical illness seems to be caused predominantly by altered hypothalamic control, because a continuous infusion of GH-releasing peptide-2 (GHRP-2) reactivates pulsatile GH secretion and normalizes IGF-I levels (3, 4). The pathophysiology of the low T3 syndrome is even more complex, with reduced TRH gene expression explaining reduced TSH secretion and thyroidal T4 release and, concomitantly, alterations in peripheral thyroid hormone metabolism playing a role in bringing about the low T3 concentrations (5). Indeed, continuous iv infusion of TRH is able to reactivate TSH secretion in prolonged critical illness, which increases circulating T4 and T3 back into the normal range, but also elevates circulating levels of rT3 (6). The latter is prevented by adding GHRP-2 to the TRH infusion (7, 8). Complex interactions between the somatotropic and thyrotropic axes are likely to be involved.
In normal physiology, the thyroid predominantly secretes the prohormone T4 and only a small amount of the active hormone T3 (9). The bulk of daily T3 production occurs in various extrathyroidal tissues via outer ring deiodination of T4 (10). This activation may be catalyzed by two different deiodinases, D1 and D2, which are distinguished by their kinetic properties and substrate specificity (11). In healthy humans, 15–81% of T3 is derived from the D1-containing tissues, liver and kidney, with the remaining originating from D2-containing tissues, such as skeletal muscle (10). Besides outer ring deiodination of T4 into T3, T4 is converted into rT3 by inner ring deiodination exerted by type 3 deiodinase (D3). T3 and rT3 undergo additional deiodination, predominantly to the common metabolite 3,3'-diiodothyronine, which is generated by, respectively, inner and outer ring deiodination of T3 and rT3 (10).
In prolonged critical illness, the low T3 and concomitantly elevated rT3 concentrations can be explained at least in part by reduced D1 and elevated D3 activity, whereby the conversion of T4 into active T3 is reduced and, instead, T4 is metabolized into inactive rT3 (12, 13). In an animal model of prolonged critical illness that has been shown to mimic the endocrine and metabolic changes observed in human critical illness, a combined infusion of TRH and GHRP-2 was found to increase the catalytic activity of D1 and depress that of D3 (14). Because suppression of D3, but not stimulation of D1, also occurred in response to administration of GH (14), these findings suggested that during critical illness, up-regulation of D3 activity is partly related to the lack of GH activity (15). Reactivation of D1 activity, on the contrary, was absent after GH administration and thus could be due to either a direct effect of GHRP-2 or TRH or the induced rise in circulating T4 and T3 concentrations (14). The study design, however, did not allow distinguishing among these possibilities.
We hypothesized that during critical illness, D1 activity is regulated predominantly by changes within the thyrotropic axis, and D3 activity is modulated by alterations within the somatotropic axis. To test this hypothesis, we studied the effects of TRH, GHRP-2, and TRH plus GHRP-2 on peripheral thyroid hormone metabolism in our animal model of prolonged critical illness.
Materials and Methods
Study set-up and protocol
All animals were treated according to the Principles of Laboratory Animal Care formulated by the U.S. National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Institutes of Health. The study protocol was approved by the Leuven University ethical review board for animal research.
The model has been described in detail previously (16). In brief, male New Zealand White rabbits were purchased from a local rabbitry, housed individually, and exposed to artificial light for 14 h/d. On d 1, the animals were anesthetized with 30 mg/kg ketamine, im (Merial, Lyon, France), and 0.15 ml/kg medetomidine (Orion Corp., Ospoo, Finland), im. After weighing and shaving neck and flanks, anesthesia was supplemented by isoflurane (Forene, Abbott Laboratories, Inc., Queensborough, UK) added to the breathing gas via regular vaporizer. Both the carotid artery and jugular vein were cannulated, and catheters were filled with heparin (5000 IU/ml; Heparine, Rhne Poulenc Rorer, Brussels, Belgium) to assure patency. Thereafter, a supplemental paravertebral block with 1% xylocaine (Astra Pharmaceuticals, Brussels, Belgium) was performed, and a full thickness burn injury equaling 15–20% of the total body surface area was imposed. At the end of the procedure, animals returned to their cages, and overnight fluid resuscitation was started with a continuous infusion of Ringer’s lactate at six drops per minute (±18 ml/h) through a volumetric infusion pomp (IVAC 531 infusion pump, IVAC Corp., San Diego, CA). To assure free movement in the cage and to safe-guard the arterial and venous lines, animals wore a homemade jacket, and infusions were connected to the vascular access via a swivel device. In the evening, a supplemental dose of a major analgesic (Dipidolor, Janssen-Cilag, Beerse, Belgium) was given. The next morning, parenteral nutrition (PN) was initiated in all rabbits at four drops per minute (±12 ml/h). Blood glucose levels were kept below 180 mg/dl by frequent blood glucose monitoring and titration of insulin infusion (100 IU/ml; Actrapid Novolet, Novo Nordisk, Bagsvaerd, Denmark; via an SE200B infusion pump, Vial Medical, Brezins, France) when necessary. Because GHRP-2 is known to induce insulin resistance (6), insulin therapy was started directly after randomization in rabbits allocated to receive GHRP-2 or TRH plus GHRP-2. All iv infusions were weighed before and after administration, allowing accurate determination of the infused quantity. PN was prepared daily in the hospital pharmacy under laminar air-flow conditions. The infusion bags contained 150 ml Clinomel N7 (Baxter, Clintec Parenteral, Maurepas, France) and 175 ml sterile water. Thus, bags with 325 ml solution contained 156 kcal nonprotein calories and 0.99 g nitrogen. Of all nonprotein calories, 61.5% were delivered as carbohydrates and 38.5% as fat. Protein intake equaled 1.49 g/kg·d. No additional vitamins or trace elements were added. PN was continuously administered, and the bags were changed daily. Animals had free access to water, but oral food intake was denied.
On the morning of d 4, surviving rabbits were randomly (by sealed envelopes) allocated to one of the following treatment groups (Fig. 1). Group 1 (saline group) received a 4-d continuous infusion of 0.9% NaCl. Group 2 (GHRP-2 group) received an initial bolus of 60 μg/kg GHRP-2 (Kaken Pharmaceutical Co. Ltd., Tokyo, Japan), followed by a continuous infusion of 60 μg/kg·h. Group 3 (TRH group) received an initial bolus of 60 μg/kg TRH (UCB Pharma, Brussels, Belgium), followed by a continuous infusion of 60 μg/kg·h. Group 4 (TRH+GHRP-2 group) received an initial bolus of 60 μg/kg TRH plus 60 μg/kg GHRP-2, followed by a continuous infusion of 60 μg/kg·h TRH and 60 μg/kg·h GHRP-2. The effective doses of TRH and GHRP-2 were defined in a previous study (16).
On d 8, the animals were weighed and then killed with sodium-pentobarbital (60 mg/ml; Nembutal, Sanofi-Winthrop, New York, NY). Tissue samples were taken from liver, kidney, and muscle and snap-frozen in liquid nitrogen, after which they were stored at –80 C until additional analysis of deiodinase activity.
To control blood glucose levels, two to four arterial blood samples were taken daily. On d 1 after catheter insertion, d 4 before randomization, and at 0900 h on d 5, 6, 7, and 8, an additional 4 ml blood was sampled. Blood for biochemical analyses was sampled in lithium citrate tubes (BD Biosciences, Temse, Belgium), immediately centrifuged, and then stored at –30 C until assay.
Assays
Plasma rabbit GH (rGH) concentrations were measured with a specific RIA (reagents provided by Dr. A. Parlow, National Pituitary Agency, Torrance, CA). The detection limit was 1 μg/liter, and the within-assay coefficient of variation (CV) was 2.3%. All samples contained detectable plasma concentrations. Because frequent blood sampling for determination of rGH concentration time series would evoke an intolerable amount of blood loss, we analyzed GH in a single sample.
Plasma IGF-I concentrations were measured by RIA, using a slightly modified version of that described previously (16, 17). After acidification of the plasma samples with formic acid, binding proteins were separated by acid gel filtration on an Econo-Pac column (Bio-Rad Laboratories, Richmond, CA). Des-IGF-I was used as tracer to avoid binding to residual small IGF-binding proteins. The intraassay CV was 4.6%, and the detection limit was 20 μg/liter.
Plasma concentrations of rTSH were measured by a specific RIA (reagents provided by Dr. A. Parlow, National Pituitary Agency). The detection limit was 1.2 mU/liter, and the intraassay CV was 5.3%. For samples with undetectable levels, a value representing half the detection limit was entered. As for rGH, rTSH was only analyzed in a single sample.
Total concentrations of plasma T4, T3, and rT3 were determined by in-house RIA (18, 19). The detection limit and intraassay CV were, respectively, 0.3, 0.03, and 0.05 nmol/liter and 8.5%, 6%, and 9%. All samples were measured in a single assay run. Measurement of total thyroid hormone levels was performed as catheters for blood sampling needed to be heparinized, which induces artifactual free hormone determinations (20).
Arterial blood was analyzed on an ABL 700 analyzer (Radiometer Medical A/S, Copenhagen, Denmark) to quantify pH, hemoglobin, lactate, and glucose.
Deiodinase activity
For determination of D1, D2, and D3 activities, tissue microsomal fractions were prepared and assayed as described by Darras et al. (21, 22). Final incubations were performed in a total volume of 200 μl. For D1 activity, final incubation mixtures contained 1 μM rT3, 50,000 cpm/tube [3',5'-125I]rT3 (labeled using 17 Ci/mg carrier-free 125I from NEN Life Science Products, Boston, MA), 0.2 mg microsomal protein/ml (liver and kidney) or 0.5 mg/ml (muscle), 2 mM EDTA, and 5 mM dithiothreitol (DTT) and were incubated for 30 min (120 min for muscle) at 37 C. For D2 activity, incubations contained 1 nM T4, 50,000 cpm/tube [3'-5'125I]T4 (labeled as described above), 1 mg microsomal protein/ml (liver and kidney) or 0.5 mg/ml (muscle), 100 nM T3 (to block interference from D3), 2 mM EDTA, and 25 mM DTT and were incubated for 120 min at 37 C. Samples were also tested using the same conditions, but 100 nM T4 was used to determine whether the activity was blocked at this high substrate concentration. Only in this case was the activity regarded as true D2 activity. For D3 activity, incubations contained 1 nM T3, 200,000 cpm/tube [3'-125I]T3 (labeled as described above), 1 mg microsomal protein/ml (liver and kidney) or 0.5 mg/ml (muscle), 1 μM rT3 and 0.1 mM propylthiouracil (to block D1 interference), 2 mM EDTA, and 50 mM DTT and were incubated for 120 min at 37 C.
Statistical analysis
The effects of interventions were analyzed using repeated measures and factorial ANOVA with Fisher’s protected least significant difference post hoc testing. Within-group changes were analyzed by Friedman, Mann-Whitney U, Wilcoxon, Kruskal-Wallis, z, and t test, as appropriate, with Bonferroni correction for multiple comparisons where indicated. Correlation analysis was performed with linear or logarithmic regression. The area under the curve (AUC), calculated by the trapezoid rule, was used as a marker of the total amount of hormone liberated in response to an intervention. All data are expressed as the mean ± SEM unless specified otherwise. A two-sided value of P < 0.05 was considered significant.
Results
The experiment was designed to obtain eight survivors on d 8 in all groups. In total, 70 male rabbits were used, of which 13 were excluded due to technical problems (catheter dislocation, cerebral embolism during blood withdrawal, failure of infusion pump, and death during anesthesia); 57% of the remaining animals survived the 8 d of the experiment. Mortality rate did not differ among the four groups. Four healthy animals, matched for gender, age, and body weight, were used as controls.
Body weight and PN
At the start of the study, rabbits weighed 2904 ± 52 g, and the weight decreased to 2732 ± 53 g at the end of the experiment (P < 0.0001 vs. baseline). Starting body weight and loss of body weight were similar in the four groups. PN was administered in equal amounts to rabbits in all groups.
Glucose control
At baseline, the mean blood glucose concentration was 130 ± 5 mg/dl, and it remained constant throughout the whole experiment. The mean total amount of insulin used per rabbit was 13 ± 1 IU in the GHRP-2 group, 22 ± 8 IU in the TRH plus GHRP-2 group, and minimal in the two other groups (2 ± 1 IU in the saline group; 1 ± 1 IU in the TRH group).
Blood gases and hemoglobin
As previously described, this model of sustained critical illness is characterized by anemia (14, 16). The baseline hemoglobin level was 12.2 ± 0.2 g/dl and decreased progressively to 7.9 ± 0.4 g/dl on d 8 (P < 0.0001 vs. baseline). Hemoglobin levels were similar in all groups throughout the experiment. Lactate levels remained constant throughout the experiment, and no differences in arterial pH among the four groups were observed (data not shown).
Somatotropic axis
Plasma IGF-I concentrations at baseline (135 ± 9 μg/liter) and on d 4 (103 ± 10 μg/liter) were comparable in all groups (Fig. 2). Plasma IGF-I decreased significantly during the first 4 d after injury (P = 0.01, d 4 vs. baseline). After randomization, plasma IGF-I levels remained stable in the saline and TRH groups, whereas they increased in animals receiving GHRP-2 and TRH plus GHRP-2 (Fig. 2).
Baseline rGH concentrations measured in a single sample were identical in the four groups. During the experiment, these single sample rGH levels increased in all groups (data not shown).
Thyrotropic axis
In all groups, plasma T4 levels decreased significantly between baseline (38.2 ± 2.0 nmol/liter) and d 4 (31.9 ± 1.6 nmol/liter; P = 0.006). After randomization, T4 levels increased in the TRH and TRH plus GHRP-2 groups, whereas they remained stable in the saline and GHRP-2 groups (Fig. 3).
Compared with healthy controls (1.76 ± 0.3 nmol/liter), plasma T3 levels in the critically ill animals were significantly lower on d 1 (0.84 ± 0.04 nmol/liter; P = 0.002) and d 4 (0.98 ± 0.06 nmol/liter; P = 0.004). In the saline and GHRP-2 groups, T3 remained low, whereas in the TRH and GHRP-2 plus TRH groups, there was a significant increase (Fig. 3).
rT3 levels were identical at baseline and before randomization in all groups (d 4, 0.50 ± 0.19 nmol/liter). After randomization, only administration of TRH significantly increased plasma rT3 (P = 0.02; Fig. 4).
rTSH levels, measured in a single sample, decreased significantly between baseline and d 4 (3.03 ± 0.13 vs. 2.64 ± 0.15 mU/liter; P = 0.008). After randomization, single-sample rTSH levels remained stable, and at no time was there a significant difference among the four groups (data not shown).
Measured in tissue harvested on d 8, hepatic D1 activity in the saline group was significantly lower than that in healthy controls (P = 0.02; Fig. 5). Hepatic D1 activity was significantly higher in the TRH-treated and TRH- plus GHRP-2-treated groups than in groups not receiving TRH (P = 0.02). Hepatic D3 activity in the saline group tended to be higher than that in healthy animals (P = 0.06), whereas in the other three groups, D3 activity was similar to that found in healthy controls (Fig. 5). Renal D1 and D3 activities were also similar in all groups. D2 deiodinase activity was undetectable in all examined tissues, as were D1 and D3 activities in skeletal muscle.
Hepatic D1 activity on d 8 showed a strong positive correlation with AUC of T4 (r = 0.64; P < 0.0001), AUC of T3 (r = 0.84; P < 0.0001), and d 8 plasma T3/rT3 (r = 0.71; P < 0.0001). Also, renal D1 activity correlated positively with AUC of T4 (r = 0.57; P = 0.0006), AUC of T3 (r = 0.70; P < 0.0001), and d 8 plasma T3/rT3 (r = 0.64; P = 0.0001). Liver D3 activity was negatively correlated with AUC of T4 (r = –0.47; P = 0.006), AUC of T3 (r = –0.65; P < 0.0001), and d 8 plasma T3/rT3 (r = –0.60; P = 0.0003; Fig. 6).
Discussion
In our rabbit model of prolonged critically illness, D1 activity in the liver was significantly down-regulated, whereas D3 activity tended to be up-regulated compared with healthy controls, which was related to the changes in circulating T3 and the T3/rT3 ratio. Infusion of TRH and TRH plus GHRP-2 restored the catalytic activity of hepatic D1 and increased T3 levels to within the physiological range. D3 activity was normalized by all three interventions, but only the addition of GHRP-2 to TRH prevented the rise of rT3 seen with TRH alone. Although both GHRP-2 and TRH plus GHRP-2 were able to reactivate the somatotropic axis, only combined infusion of TRH and GHRP-2 induced a significant increase in IGF-I levels into the range observed in healthy rabbits.
Our data confirm that in prolonged critical illness, the low T3 syndrome does not result solely from suppressed TSH secretion, which reduces T4 production (23, 24). Indeed, concomitant changes in peripheral thyroid hormone metabolism appear to play a role (25, 26). D1 and D3 activities have recently been measured in liver samples of critically ill patients who died in the intensive care unit (12, 13) and in liver of prolonged critically ill animals (14). Although these studies did not include a healthy control group, the results suggested that low D1 activity and induction of D3 activity may play a role in the pathogenesis of the low T3 syndrome during critical illness. In our current experiment, we demonstrated that hepatic D1 activity during prolonged critical illness is indeed significantly lower than that in healthy controls. Infusion of TRH and TRH plus GHRP-2 was able to restore the catalytic activity of hepatic D1 and, concomitantly, increase T3 levels to within the physiological range. These results indicate that a depressed hepatic D1 activity is a major mechanism bringing about the low T3 syndrome during critical illness. Although reactivation of pulsatile TSH secretion is thought to predominantly discharge thyroidal T4, and the majority of T3 is derived from peripheral T4 to T3 conversion (9, 10), direct TSH-dependent release of T3 from the thyroid gland may have contributed to the observed rise in T3 with TRH and GHRP-2 plus TRH infusions (27). However, in view of the short half-life of T3 and the increase still being present after 4 d of treatment without concomitant differences in TSH, we believe that this possible contribution of direct T3 release from the thyroid gland was minor. The elevated levels of rT3 during critical illness may also be explained predominantly by a reduced D1 activity, because D1 is the major pathway of rT3 clearance (10, 28).
D2 activity is regulated by substrate-induced enzyme inactivation, and there is reason to believe that D2-induced T3 production in skeletal muscle contributes to circulating T3, especially in the hypothyroid situation (10, 29, 30). Hence, in view of the low plasma T4 levels during prolonged critical illness, it is intriguing that we failed to detect any D2 activity in the skeletal muscle samples of critically ill rabbits, in line with what we previously observed in patients (12, 13). A possible explanation is the elevated plasma concentration of rT3 during critically illness, because rT3 is known to inactivate D2 (31). Hence, a complete absence of muscle D2 activity during prolonged critical illness may be at least partly due to elevated rT3 and may contribute to the low circulating and tissue T3 levels in critically ill patients (10, 29).
Apart from decreased D1 activity, the reciprocal changes in T3 and rT3 during critical illness can also be explained by induction of D3, resulting in enhanced degradation of T3 and augmented production of rT3 (10). Overexpression of D3 in hemangiomas has been shown to cause consumptive hypothyroidism, a syndrome characterized by very low levels of circulating T4 and T3 in combination with high levels of rT3 (32, 33). Hence, the previous observation that D3 activity is clearly detectable in the liver of critically ill patients who died in the intensive care unit suggests a potential role for this enzyme in the pathophysiology of the low T3 syndrome (12, 13). Our current experiments also support such a role, because hepatic D3 activity in saline-treated, prolonged critically ill rabbits appeared higher than that in healthy controls, and a clear negative correlation was found between the T3/rT3 ratio on d 8 and D3 activity in the liver. The expression and activity of D3 during health have only been shown in liver tissue during fetal development, not in adult tissue (10, 34). Hence, it was surprising to document hepatic D3 activity in healthy control rabbits, which may point to a certain degree of stress attributable to manipulation of the controls (12). This could also explain the borderline level of significance for the observed D3 elevation in the sick rabbits compared with the controls.
The regulation of deiodinase activities is a complex process. D1 is thought to be under the positive control of thyroid hormone status, presumably mediated by T3 (35, 36), and GH appears to suppress D3 activity (15, 37). However, little is known about the regulation of deiodinase activity during critical illness. The observation that hepatic D1 was only reactivated in animals receiving TRH, either alone or in combination with GHRP-2, clearly indicates that D1 activity during critical illness is mainly regulated via alterations within the thyroid axis. Because thyroid hormones are known potent inducers of hepatic D1 activity (35, 36), the increase in D1 activity most likely results from the increase in T3 and/or T4. In agreement with this, hepatic D1 activity was positively correlated with the amount of thyroid hormone liberated by the intervention and the T3/rT3 ratio on d 8. However, the present data do not allow us to distinguish between a role for T3 and T4 and a direct effect of TRH or TSH. Because GHRP-2-treated animals revealed levels of D1 activity similar to those in the saline group, a major role of the somatotropic axis in the regulation of D1 during critical illness can be excluded.
Normal D3 activity after TRH plus GHRP-2 infusion confirmed our previous observation of lowered D3 activity with this intervention in the same experimental model of critical illness (14). Administration of GH in that set of experiments similarly reduced D3 activity, which is in line with the normal D3 activity after GHRP-2 infusion in our current experiment. An effect of GH on D3 activity has also been shown to occur in healthy chickens (15, 37) and in both healthy and GH-deficient humans (38, 39). Such an effect can be mediated either by GH itself or by IGF-I. Our observation that the rise in IGF-I obtained with combined infusion of TRH plus GHRP-2 was higher than with GHRP-2 alone, probably explained by the permissive role of thyroid hormones on GH secretion (40, 41) and on the IGF-I response to GH (42, 43), and the observed prevention of the rT3 rise only seen with the combined GHRP-2 plus TRH infusion favors an effect of IGF-I on rT3 generation via D3. However, TRH infusion alone in the sick rabbits was also associated with normal D3 activity in the current experiment. This suggests that alterations within the somatotropic axis as well as those within the thyrotropic axis may explain the up-regulation of D3 activity during critical illness.
We conclude that during critical illness, D1 activity is suppressed, and D3 activity tends to be increased; both are related to the changes in circulating T3 and rT3. D1 activity is restored by TRH infusion, whereas both TRH and GHRP-2 may control D3 activity during critical illness. The extent to which the effects of TRH and GHRP-2 on deiodinase activity are explained by direct effects of the releasing factors or, alternatively, by the induced peripheral hormonal responses during critical illness, remains unclear.
Acknowledgments
We acknowledge the generosity of Dr. C. Y. Bowers, U.C.B. Pharma Belgium, Baxter Belgium, Fresenius-Kabi Belgium, Eddy Vanonckelen (Alaris Medical Systems, Belgium), and Wouter Diddens (Tyco Healthcare, Belgium) in providing, respectively, GHRP-2, TRH, Clinomel, total PN bags, infusion pumps, and arterial catheters for these experiments. We appreciate the technical support of Mrs. Veerle Leunens, Mrs. Magda Mathys, and Mr. Kristof Reyniers (Center for Experimental Surgery and Anesthesiology); Mr. Willy Van Ham, Mrs. Francine Voets, and Mrs. Lut Noterdaeme (Laboratory of Comparative Endocrinology); and Mark Denturck and Wilfried Frooninckx (Technical Department University Hospital Leuven).
Footnotes
This work was supported by the Fund for Scientific Research-Flanders, Belgium [Ph.D. scholarship, Aspirantenmandaat (to Y.D.) and G.0144.00, G.0278.03, and G.0258.05 (to G.V.d.B.)], a grant from Innovative Medizinische Forschung (EL 610304) and B. Braun Stiftung, Germany (to B.E.), and the Research Council of the University of Leuven (OT 03/56; to G.V.d.B.).
First Published Online September 8, 2005
1 Y.D. and B.E. contributed equally to this work.
Abbreviations: AUC, Area under the curve; CV, coefficient of variation; D1, deiodinase; DTT, dithiothreitol; GHRP-2, GH-releasing peptide-2; PN, parenteral nutrition; r, rabbit.
Accepted for publication September 1, 2005.
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