Alterations in cardiac contractility and gene expression during low-T3 syndrome: prevention with T3

Harvey L. Katzeff, Saul R. Powell, and Kaie Ojamaa

Departments of Medicine and Surgery, North Shore University Hospital, Manhasset 10030; and Cornell University Medical College, New York, New York 10021

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The low-T3 syndrome is a metabolic response resulting in a decreased serum triiodothyronine (T3) concentration that has uncertain effects on thyroid hormone-responsive gene expression and function. We measured cardiac myocyte gene expression and cardiac contractility in young adult female rats using chronic calorie deprivation as a model of the low-T3 syndrome. Sarcoplasmic reticulum calcium adenosinetriphosphatase (SERCA2) and myosin heavy chain (MHC) isoform mRNA content were measured after 28 days on a 50% calorie-restricted diet (low T3) with or without T3 treatment (6 µg · kg body wt-1 · day-1). The low-T3 animals had decreased maximal rates of contraction (-13%; P < 0.05) and relaxation (-18%; P < 0.05) compared with the control and the T3-treated groups. There was a 21% (P < 0.05) increase in left ventricular (LV) relaxation time in the low-T3 animals vs. both control and T3-treated groups. The LV content of the SERCA2 mRNA was decreased significantly (37%) in the low-T3 rats and was increased (P < 0.05) with T3 treatment vs. controls. The alpha -MHC mRNA isoform decreased in the low-T3 animals but was unchanged in the T3-treated animals. T3 supplementation normalized both cardiac function and phenotype of calorie-restricted animals, suggesting a role for the low-T3 syndrome in the pathophysiological response to calorie restriction.

triiodothyronine; energy expenditure; sarcoplasmic reticulum calcium-binding protein; myosin heavy chain; euthyroid sick syndrome; left ventricular function

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE EUTHYROID SICK or low-T3 syndrome is a series of alterations in the pituitary-thyroidal axis resulting in a decreased serum and nonthyroidal tissue concentration of the active thyroid hormone triiodothyronine (T3) (31). The severity of this syndrome increases with the severity of the underlying illness and is associated with an increase in mortality in hospitalized patients (25). Although the low-T3 syndrome has been thought to enhance survival during illness by decreasing whole body energy expenditure and rates of protein catabolism and by reducing the loss of lean body mass, this adaptation may also be associated with impaired cardiac and skeletal muscle function (28, 31). Thyroxine therapy has no significant effect on biochemical or physiological parameters in this disorder. An important unanswered question is whether the low-T3 syndrome directly or indirectly impairs the effects of thyroid hormones on thyroid hormone-responsive gene expression and compromises organ-specific function.

Weight loss due to chronic caloric restriction and acute or chronic illness is associated with a low serum T3 concentration due, in part, to an inhibition of the type I 5'-iodothyronine deiodinase, which converts the prohormone L-thyroxine (T4) to its active metabolite, T3 (29). Impaired transport of T4 from the blood into hepatocytes may also be a significant factor, decreasing availability of substrate to be converted to T3 (30). Despite low serum levels of T3, serum thyrotropin [thyroid-stimulating hormone (TSH)] levels are within the normal range. These characteristics define the low-T3 syndrome, and potential contributing factors to this syndrome during acute stress include elevations in serum cortisol, tumor necrosis factor, and specific cytokines (9, 21, 31).

Chronic food restriction in experimental animals has been associated with altered cardiac function, including decreased maximum rate of contraction, decreased cardiac index, and a prolonged isovolumic relaxation time (1, 15). These functional changes may, in part, be the result of changes in mitochondrial oxidative enzyme activity and myosin adenosinetriphosphatase (ATPase) activity, which is determined by the relative expression of alpha - and beta -myosin heavy chain (MHC) genes (12, 14). Diastolic relaxation time is partially determined by the rate of calcium uptake into the sarcoplasmic reticulum (SR) by an SR membrane calcium ATPase (2). The alterations in MHC isoform expression in heart during undernutrition are similar to those observed in experimental hypothyroidism (13). Thyroid hormone response elements (TREs) have been identified within the promoter regions of several cardiac genes, including alpha - and beta -MHC, alpha -skeletal actin, and SR calcium ATPase (SERCA2) (13, 18). Thyroid hormone deficiency in the adult rodent heart is characterized by a shift in expression of the alpha -MHC to beta -MHC isoform and a decrease in SERCA2 enzyme activity (13).

Recent data suggest that the observed changes in cardiac function in undernutrition may be the result of the accompanying low serum T3 levels. Dillmann and co-workers (8) were able to prevent acute starvation-induced alterations in cardiac myosin ATPase activity by T3 supplementation. We undertook the present study to measure cardiac contractile function during nutrition-induced weight loss, using the isolated perfused heart protocol, and to correlate function with changes in the expression of the SERCA and the MHC genes. Furthermore, we studied whether supplementation with T3 could prevent the functional and phenotypic changes associated with the nutritionally induced low-T3 syndrome.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animal protocols. Sprague-Dawley female rats (200-225 g) obtained from Harlan Sprague Dawley Labs (Indianapolis, IN) were individually housed in polycarbonate cages and separated into five groups of six animals. The control group was fed ad libitum with standard rat chow (Purina Rat Chow; Ralston Purina, St. Louis, MO) and given free access to water. Two energy-restricted (ER) groups were fed diets at 75 and 50% (-25% ER and -50% ER) of the calorie intake of the control group. Two additional groups of calorie-restricted rats were supplemented with T3 (-25% ER + T3 and -50% ER + T3) by infusion of 3,5,3'-triiodo-L-thyronine (Sigma, St. Louis, MO) at a rate of 600 ng · day-1 · 100 g body wt-1, using a subcutaneously placed osmotic minipump (model 2002, Alza, Altec, CA). All four groups were treated for 28 days. On completion of the study, body weights were recorded, animals were anesthetized, and the hearts were rapidly removed and the great vessels and atria resected. The ventricles were frozen in liquid nitrogen and weighed before extraction of RNA. Blood was collected from the open chest cavity for thyroid hormone analysis. A separate cohort of female Sprague-Dawley rats (200-225 g) was used for analysis of left ventricular (LV) function by the isolated heart perfusion procedure. This cohort consisted of four groups of eight rats: control (fed ad libitum), -50% ER, -50% ER + T3, and thyroidectomized (hypothyroid). The hypothyroid rats were surgically thyroidectomized 2 wk before the start of the study protocol, and the adequacy of the procedure was determined by measuring serum thyroid hormone levels at 1 wk postsurgery.

Analysis of serum T3 and T4. Serum concentrations of T3 and T4 were measured by sensitive and specific radioimmunoassays, using thyroid hormone-depleted rat sera for standards.

Measurements of LV function. After the 28-day study protocol, rats received an injection of heparin sodium (500 U) intraperitoneally, 30 min before anesthesia with pentobarbital sodium (60 mg/kg ip). Hearts were removed and immediately placed in ice-cold heparinized saline. The hearts were then perfused in an orthograde fashion through the coronary arteries, as previously described (22), according to method of Langendorff, at a constant pressure of 95 cmH2O. A latex balloon (Hugo-Sacks Elecktronik, Hamburg, Germany) connected to a pressure transducer was inserted through the mitral valve into the left ventricle. The volume of the balloon was 0.06 ml for hearts <450 mg and was 0.10 ml for hearts >450 mg.

The perfusate was a modified Krebs-Henseleit buffer consisting of (in mM) 118 NaCl, 6.1 KCl, 2.5 CaCl2, 1.2 MgSO4, 25 NaHCO3, 1.0 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 11.1 glucose. Complete buffer was prepared on the day of the experiment by mixing the proper amounts of concentrated stock solutions, to which the appropriate quantities of glucose and calcium chloride were added. All concentrated solutions, with the exception of the magnesium sulfate, were treated with chelating resin beads (Chelex 100, iminodiacetic acid; Sigma) to minimize trace heavy metal contamination.

To develop a relationship between left ventricular end-diastolic pressure (LVEDP) and the maximal rates of contraction (+dP/dtmax) and relaxation (-dP/dtmax) (where P is pressure and t is time in seconds), the hearts were initially equilibrated for 10 min with the LVEDP fixed at 5 mmHg. A baseline measurement was recorded, and the LVEDP was then sequentially increased to 10, 20, and 40 mmHg by expansion of the balloon through injection of water. The LVEDP was fixed at each pressure for 5 min before a measurement was recorded. Measurements of +dP/dt and -dP/dt were derived directly from the pressure tracings. Measurement of LV relaxation time was performed as described by Gay et al. (11). The LV pressure decay recording from the point of -dP/dtmax to end-diastolic pressure was fit to the exponential equation P = Poe-lambda t, where Po is the pressure at -dP/dtmax, lambda  is the calculated slope, and t is time in seconds. The time to one-half maximal relaxation, tau 1/2, is the time at which P = 1/2Po.

Total RNA isolation and Northern blot analysis. Total cellular RNA was extracted from the left ventricle, using the acidic-phenol method with modifications as previously published (5). Five micrograms of total RNA were denatured in the presence of 2.2 M formaldehyde, 33% formamide, 3-(N-morpholino)propanesulfonic acid [19.5 mM 3-(N-morpholino)propanesulfonic acid], pH 7.0, 5 mM sodium acetate, and 1 mM EDTA at 65°C for 5 min and separated by electrophoresis on formaldehyde-1% agarose gels. The ribosomal RNA bands were visualized by ethidium bromide staining, and the RNA was transferred to Duralon ultraviolet (UV) membrane (Stratagene, San Diego, CA) by capillary blotting and then cross-linked to the membrane by exposure to UV light. Membranes were prehybridized at 42°C for 6-8 h in a solution containing 5× SSPE (0.75 M NaCl, 50 mM sodium phosphate, pH 7.4, 3,335 mM EDTA), 5× Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 5% bovine serum albumin), 50% formamide, 1% sodium dodecyl sulfate (SDS), and 100 µg/ml denatured salmon sperm DNA. Hybridization with a radiolabeled SERCA2 cDNA probe (kindly provided by W. H. Dillmann, University of California San Diego, La Jolla, CA), added at 106 disintegrations · min-1 (dpm) · ml hybridization solution-1, was carried out at 42°C for 18 h. Membranes were washed at high stringency in 0.2× SSPE, 1% SDS at 65°C for 30 min and were then exposed to X-ray film with intensifying screens at -80°C. To normalize for sample loading variability, Northern blots were washed and reprobed with a glyceraldehyde-3-phosphate dehydrogenase oligodeoxynucleotide probe (Oncogene Science, Uniondale, NY). Hybridization conditions were similar to the cDNA probe except that the stringent washes used 2× SSPE, 1% SDS at 65°C for 30 min. The mRNA was quantified by laser scanning densitometry of the autoradiograms at various exposure times to assure that the values were not saturating.

S1 nuclease protection analysis. The alpha - and beta -MHC mRNAs were quantified in the same sample of total RNA by using a single oligonucleotide probe in an S1 nuclease mapping assay as previously described (20). The end-labeled 61-base oligodeoxynucleotide probe was complementary to a 38-base coding sequence common to both alpha - and beta -MHC mRNAs and to an additional 8 bases unique to beta -MHC mRNA, thereby allowing the resolution of the two protected mRNAs by polyacrylamide electrophoresis. The MHC mRNA species were quantified by laser densitometric scanning of the autoradiograms, and the amount of each MHC mRNA was expressed as a fraction of the sum of the two isoforms.

Statistical analysis. The results are reported as means ± SE. Comparisons of body weight, serum thyroid hormone levels, and measures of contractility and gene expression in the calorie-restricted groups vs. control and T3-treatment groups were performed, using analysis of variance techniques and the Student-Newman-Keuls test (3). Standard linear regression techniques were used to analyze the relationship between thyroid hormones and body weight.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effects of calorie restriction on serum thyroid hormones. Calorie restriction for 28 days produced a low-T3 state as measured by serum levels of T3 and T4. The serum concentration of T3 in the 25% calorie-restricted group was significantly lower than controls (24.8 ± 3.3 and 61.7 ± 5 ng/dl, respectively), whereas serum T4 levels were not significantly different (3.9 ± 0.5 and 4.3 ± 0.8 µg/dl, respectively), as is characteristic of the low-T3 syndrome (Table 1). The 50% calorie-restricted group showed an 82% decrease in serum T3 levels compared with controls (P < 0.02), with a significant (P < 0.05) decrease in serum T4 concentration (2.9 ± 0.6 vs. 4.3 ± 0.8 µg/dl) (Table 1). The decrease in the serum T3 level in the two groups of calorie-restricted rats was directly proportional to the nutritionally induced decrease in body weight gain (r = 0.83; P < 0.001) (Fig. 1). T3 supplementation maintained serum T3 concentrations in both groups of calorie-restricted rats in the normal range of T3 serum values (45-85 ng/dl), with only the 25% ER group exhibiting a higher mean concentration of T3 (P < 0.01) (Table 1). Serum T4 levels were suppressed to undetectable levels in both T3 replacement groups.

                              
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Table 1.   Effects of energy restriction and T3 supplementation on body weight and heart mass


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Fig. 1.   Relationship between serum triiodothyronine (T3) level and change in body weight during control (solid circles), -25% (crosshatched circles), and -50% (open circles) calorie-restricted groups. There is a direct linear relationship between serum T3 level and body weight achieved after 28 days of underfeeding (r = 0.83; P < 0.005).

Effects of calorie restriction on heart and body weight changes. The heart weights of the -25% and -50% underfed groups were decreased 5.6% (P < 0.05) and 18.7% (P < 0.01) vs. control hearts, whereas total body weight decreased 8.9% (P < 0.05) and 21.4% (P < 0.01) compared with controls, as reported in Table 1. This resulted in maintenance of the heart weight-to-body weight ratios in both the 25 and 50% food-restricted groups compared with the control animals. Animals that were T3 treated and calorie restricted exhibited greater declines in body weight for the -25% (235 ± 12 vs. 256 ± 3 g; P < 0.05) and -50% calorie-restricted animals (201 ± 4 vs. 221 ± 6 g; P < 0.05). The heart weights for the T3-treated plus calorie-restricted groups were similar to those of food-matched groups, resulting in an increase in the heart weight-to-body weight ratios by 9.0% (P < 0.01) and 3.3% (P = not significant) in the 25% ER + T3 and 50% ER + T3 groups vs. their food-matched controls, respectively.

LV function in hypothyroid and calorie-restricted rats. Measurements of LV contractility and relaxation were obtained from analysis of the LV pressure waveform of isolated perfused hearts, as depicted in Fig. 2. The LV +dP/dt at 5 mmHg of end-diastolic pressure was decreased 13.0% (P < 0.05) in the hearts of the 50% calorie-restricted group and was normalized with T3 supplementation (-50% ER + T3) as reported in Table 2. Results were similar for +dP/dt measured at 10 and 20 mmHg of end-diastolic pressure (data not shown). The ventricular -dP/dt was decreased 19.3% (P < 0.05) compared with results from hearts of control rodents at 5 mmHg of end-diastolic pressure, with similar findings at 10 and 20 mmHg. Supplementation with T3 during 50% food restriction maintained normal ventricular -dP/dt. The mean LV relaxation time (tau 1/2) was increased in the 50% food-restricted group by 21.6% (P < 0.05) and was normal in the T3-treated group (Table 2). The relaxation time was not altered by increasing the end-diastolic pressure to 10 or 20 mmHg (data not shown).


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Fig. 2.   Left ventricular (LV) pressure tracings from representative hearts from control (Con), hypothyroid (Hypo), energy-restricted (ER), and ER + T3 treatment (ER + T3) groups. Heart rates for Hypo and ER groups are decreased vs. Con and ER + T3 groups. Lambda values are depicted for slopes of pressure (P) tracing lines at 1/2Pmax to calculate ventricular relaxation time (tau 1/2).

                              
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Table 2.   Parameters of left ventricular function

The hypothyroid animals showed similar measures of ventricular contractility to the calorie-restricted animals. Compared with control hearts, hypothyroid hearts exhibited a 15.6% (P < 0.05) lower rate of maximal pressure generation (+dP/dtmax) and a 13.7% (P < 0.05) lower ventricular -dP/dt. The isovolumic relaxation time was increased 33.6% (P < 0.01) at 5 mmHg for the hypothyroid group. Unlike the food-restricted animals, the hypothyroid animals had no decrease in the maximal systolic pressure.

Effects of calorie restriction on cardiac-specific gene expression. To understand a mechanism by which calorie restriction and T3 modulate cardiac contractility, we measured the mRNA content of the cardiac alpha - and beta -MHC and SERCA2 genes. Chronic underfeeding decreased SERCA2 mRNA content in a graded fashion in both the 25% and 50% food-restricted groups compared with controls (79.8 ± 5.6 and 66.1 ± 5.9 vs. 100 ± 8.5%; P < 0.05). The T3-supplemented groups had increased levels of SERCA2 mRNA content vs. underfeeding alone (155 ± 23 and 175 ± 14% for -25 and -50% groups, respectively; P < 0.01). The relative mRNA content of the SERCA2 gene in the heart for the -50% ER and T3-treated groups is depicted in Fig. 3.


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Fig. 3.   Relative content of sarcoplasmic reticulum calcium ATPase (SERCA2) mRNA in group fed ad libitum (control) compared with 50% calorie restriction (-50%) and calorie restriction + T3 treatment (-50% + T3) groups. Quantification was performed with the use of laser densitometry of Northern blots corrected for total RNA loading, using mRNA content of glyceraldehyde-3-phosphate dehydrogenase. Calorie restriction alone decreased SERCA2 isoform content, and -50% + T3 increased SERCA2 mRNA content vs. controls (* P < 0.05).

The alpha -MHC mRNA content was expressed as a percentage of the total MHC mRNA extracted. There was a 46.4 ± 7.7% decrease in the quantity of alpha -MHC mRNA in the hearts of the calorie-restricted groups compared with ad libitum-fed controls as shown in Fig. 4. In the 50% food-restricted group, the alpha -MHC mRNA content was similar to the amount previously observed in hypothyroid animals. The decline in alpha -MHC mRNA content was linearly related to the decrease in serum T3 observed (r = 0.71; P < 0.001). Supplementation with T3 normalized the alpha -MHC isoform content, despite food restriction.


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Fig. 4.   Percentage of alpha -myosin heavy chain (MHC) mRNA/total MHC mRNA during ad libitum feeding, -50%, and -50% + T3. The -50% group had a 52% reduction in alpha -MHC content (** P < 0.01) that was reversed with T3 treatment.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The cardiac-specific SERCA2 gene regulates cardiac myocyte relaxation during diastole via control of the rate of SR calcium uptake (2). Transcription of the gene encoding the SERCA2 protein is regulated in vivo by thyroid hormone, and TREs have been identified as the 5'-flanking region of this gene (23, 32). Both experimental hyperthyroidism and hypothyroidism in rodents have been shown to produce alterations in both SERCA2 mRNA content and protein content (24). This study is the first to describe a significant decrease in the level of expression of the cardiac SERCA2 gene in response to the low-T3 syndrome and to show that parameters of cardiac relaxation (-dP/dt and tau 1/2) were significantly altered compared with controls and were similar to values obtained in hypothyroid animals. We also observed that maintenance of physiological levels of T3 prevented the fall in SERCA2 mRNA content and maintained normal measures of cardiac muscle relaxation, despite a relative weight loss.

Further evidence for an effect of the low-T3 syndrome on both cardiac gene expression and function is the observed alterations in MHC isoform content and +dP/dtmax. A decrease in both +dP/dtmax and mRNA content of the alpha -MHC in the calorie-restricted low-T3 group of animals was measured, with values equivalent to those of hypothyroid hearts (2, 17). Treatment with T3 normalized both the mRNA expression and physiological measure of pressure generation. The ATPase activity of the two isoforms of the MHC proteins of the rat heart determines, in part, the rate of myofibrillar contraction, with the beta -MHC isoform exhibiting a slower rate of ATP hydrolysis compared with the alpha -MHC isoform (19, 27). These alterations in both SERCA2 and MHC gene expression and the pathophysiological alterations in both cardiac contractility and relaxation are consistent with the hypothesis that the low-T3 syndrome is associated with alterations in thyroid hormone-responsive gene expression and physiological function similar to those observed in hypothyroidism. Supplementation with T3 to maintain normal serum T3 levels during chronic undernutrition was able to both normalize parameters of cardiac contractility and maintain levels of expression of both the SERCA2 and MHC genes.

In addition to their effects on MHC and SERCA gene expression, thyroid hormones influence gene expression and function of other muscle-specific proteins and intermediary metabolism of the myocyte. These effects include a direct modulation of the responsiveness and density of beta -adrenergic receptors, mitochondrial oxidative capacity, and oxidative phosphorylation as well as total cellular protein synthesis (5). Thyroid hormones also regulate the gene expression of cytochrome c, malic enzyme, and possibly other regulators of oxidative metabolism within the myocyte. Maintenance of normal thyroid hormone action on the myocyte is thus critical for both muscle mass and function.

The metabolic response to chronic energy restriction is an integrated series of hormonal responses that are time dependent. Initially, there is an acute rise in stress hormones, including cortisol and catecholamines, and then a reduction in both anabolic and catabolic hormones associated with a decrease in protein turnover and metabolic rate. It is uncertain whether treatment with T3 during energy restriction alters either serum levels or responsiveness to other hormones, including insulin or growth hormone. However, treatment with T3 during acute starvation did not alter the decrease in serum insulin or protein synthesis in a rodent model. Additionally, streptozocin-induced diabetes mellitus lowers both serum insulin and T3 as well as myosin ATPase activity (16). Treatment with T3 normalized myosin ATPase activity despite insulin deficiency, suggesting that at least a portion of the effects of T3 is independent of insulin. Dillmann et al. (7, 8) initially reported that acute starvation decreased myosin ATPase activity in the rat heart and that T3 supplementation normalized enzymatic activity. They also noted that supplementation with carbohydrate and inhibition of cardiac fatty acid metabolism during acute starvation prevented the decrease in myosin ATPase activity independent of thyroid hormones, suggesting that both metabolic fuel composition and thyroid hormones were independent regulators of cardiac myosin gene expression (6). The finding that these two distinct conditions produced both a low-T3 state and an alteration in a thyroid hormone-responsive gene function that is responsive to T3 treatment provides further evidence for a direct effect of the low-T3 state on thyroid hormonespecific gene expression in cardiac tissue.

In summary, decreases in the serum T3 concentration associated with chronic calorie restriction were associated with impaired cardiac contractility due, in part, to alterations in cardiac SERCA2 and MHC gene expression. Supplementation with T3 during nutritionally induced weight loss completely prevented the phenotypic and physiological changes in the heart, despite cardiac atrophy. These data support further research into the pathophysiological role of the low-T3 syndrome on thyroid hormone action in chronic disease states.

    ACKNOWLEDGEMENTS

We thank Catherine Demaso, Deborah Carman, Jeanmarie Finnerty, and Ellen Gurzenda for technical expertise.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38337 (to H. L. Katzeff), National Heart, Lung, and Blood Institute Grant HL-45534, and American Heart Association Grant-in-Aids [to S. R. Powell (New York State) and to K. Ojamaa].

This work was presented in part at the 5th International Thyroid Congress, September 10-15, 1995, Toronto, Canada.

Address for reprint requests: H. L. Katzeff, Long Island Jewish Medical Center, New Hyde Park, NY 11040.

Received 13 December 1996; accepted in final form 7 July 1997.

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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Endocrinol Metab 273(5):E951-E956
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