0021-972X/79/4805-0887$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1979 by The Endocrine Society
Vol. 48, No. 5 Printed in U.S.A.
REGULATORY ROLE OF TRIIODOTHYRONINE IN THE DEGRADATION OF LOW DENSITY LIPOPROTEIN BY CULTURED HUMAN SKIN FIBROBLASTS ALAN CHAIT, EDWIN L. BIERMAN, AND JOHN J. ALBERS Division of Metabolism and Endocrinology, and the Northwest Lipid Research Clinic, Department of Medicine, University of Washington, Seattle, WA 98195 125 ABSTRACT: Degradation of [ I]-labeled low density lipoprotein (LDL) by cultured normal human skin fibroblasts was enhanced by exposure to concentrations of L-triiodothyronine (T3) spanning the physiological range. Analysis of LDL concentration curves suggests that To may increase LDL receptor number rather than alter the affinity of LDL for its receptor. No effect of T3 on LDL degradation was observed in receptor negative cells. Thus thyroid hormone appears to regulate receptor-mediated LDL degradation by increasing the binding of the lipoprotein to its cell-surface receptor.
To determine whether the LDL receptor was necessary for an effect of T3 on LDL degradation, an additional experiment was performed using receptor negative cells.
INTRODUCTION Elevation of plasma cholesterol concentration is a hallmark of untreated hypothyroidism in man (1). The catabolic rate of low density lipoprotein (LDL), the major transport form of plasma cholesterol, has been shown to be delayed in hypothyroidism (2). Since LDL catabolism ±n vivo is believed to be mediated largely through the LDL cell-surface receptor (3), the role of thyroid hormones in this receptormediated degradation process was studied in human cells in culture. The LDL receptor in cultured skin fibroblasts is regulated by conditions which alter the supply of available cholesterol for the cell, such as the cholesterol concentration in the incubation medium (3). Little is known of potential hormonal regulation of these events. Using this in vitro system, we have already reported that receptormediated LDL degradation can be modulated by insulin (4). Results in this study demonstrate that L-triodothyronine (T3) also is capable of regulating the catabolism of LDL ^n vitro and suggest that thyroid hormones may play an important regulatory role in cellular cholesterol metabolism.
RESULTS When compared with cells exposed for the same period to medium from which thyroid hormones had been adsorbed, 7 day exposure to T3 resulted in stimulation of [125i]-LDL binding and degradation in a dose dependent fashion at concentrations spanning the physiological range (Fig. 1 ) . No enhancement of LDL degradation was observed in the presence of T3 (10~?M) with receptor negative cells in which the amount of LDL degraded was exceedingly small. In addition to being dose dependent, this stimulatory effect of T3 on LDL metabolism was also time dependent (Fig. 2). When [125I]-LDL and T3 were added simultaneously, T3 failed to stimulate LDL binding at 4 h. LDL uptake and degradation (measured 24 and 48h later respectively) were however stimulated under these conditions. Addition of the [-*-2->I]LDL one or 7 days after the addition of T3 magnified the stimulatory effect of T3 on LDL binding and degradation in a time dependent fashion (Fig. 2). Although free T3 levels were not measured, they would be expected to be of the order of 1-5% of total T3 in medium containing 10% PHS (6). To circumvent any potential influence of binding proteins and to enable precise free hormone concentrations to be known, further experiments were performed in serum-free medium.
METHODS Skin biopsies from the thighs of normal volunteers were explanted and trypsinized as previously described (4). 10^ cells were transferred to Petri dishes in medium containing 10% pooled human serum (PHS). Cells were grown to confluence in PHS, after which they were switched either to medium containing 10% PHS from which thyroid hormones had been removed by adsorption to Amberlite IRA 400 resin (5) or to serum-free medium. More than 99.5% of the thyroid hormones were removed, as assessed by the prior addition of [125i]-iabeled hormones. T3 in ethanol was added to give the final concentrations indicated. Control replicate dishes to which no T3 was added received equivalent amounts of ethanol. No difference in cell number was noted (at any time point) between dishes exposed to or not exposed to T3, suggesting that cells were still proliferating normally in tha T3 deficient state. After exposure of cells to T3 for the times stated, [125i]-LDL, prepared as previously described (4), was added at the concentrations indicated. To induce LDL receptors when PHS was used (3), 24h before adding [^-^I]-LISL t ^ e me dium was changed to one containing 10% lipoprotein deficient serum (LDS; d> 1.25) with or without added T3. LDS was prepared from the thyroid hormone-free serum pool by ultracentrifugation (4). After [-*-^^I]-LDL was added, the cells were incubated for 4h at 37CC for the determination of its binding, 24 h for uptake, and 24-48h for measurement of LDL degradation by methods previously described (4). T3 was present in the test dishes throughout. The coefficient of variation for replicate analysis of LDL degradation was
Vol. 48, No. 5 Printed in U.S.A.
REGULATORY ROLE OF TRIIODOTHYRONINE IN THE DEGRADATION OF LOW DENSITY LIPOPROTEIN BY CULTURED HUMAN SKIN FIBROBLASTS ALAN CHAIT, EDWIN L. BIERMAN, AND JOHN J. ALBERS Division of Metabolism and Endocrinology, and the Northwest Lipid Research Clinic, Department of Medicine, University of Washington, Seattle, WA 98195 125 ABSTRACT: Degradation of [ I]-labeled low density lipoprotein (LDL) by cultured normal human skin fibroblasts was enhanced by exposure to concentrations of L-triiodothyronine (T3) spanning the physiological range. Analysis of LDL concentration curves suggests that To may increase LDL receptor number rather than alter the affinity of LDL for its receptor. No effect of T3 on LDL degradation was observed in receptor negative cells. Thus thyroid hormone appears to regulate receptor-mediated LDL degradation by increasing the binding of the lipoprotein to its cell-surface receptor.
To determine whether the LDL receptor was necessary for an effect of T3 on LDL degradation, an additional experiment was performed using receptor negative cells.
INTRODUCTION Elevation of plasma cholesterol concentration is a hallmark of untreated hypothyroidism in man (1). The catabolic rate of low density lipoprotein (LDL), the major transport form of plasma cholesterol, has been shown to be delayed in hypothyroidism (2). Since LDL catabolism ±n vivo is believed to be mediated largely through the LDL cell-surface receptor (3), the role of thyroid hormones in this receptormediated degradation process was studied in human cells in culture. The LDL receptor in cultured skin fibroblasts is regulated by conditions which alter the supply of available cholesterol for the cell, such as the cholesterol concentration in the incubation medium (3). Little is known of potential hormonal regulation of these events. Using this in vitro system, we have already reported that receptormediated LDL degradation can be modulated by insulin (4). Results in this study demonstrate that L-triodothyronine (T3) also is capable of regulating the catabolism of LDL ^n vitro and suggest that thyroid hormones may play an important regulatory role in cellular cholesterol metabolism.
RESULTS When compared with cells exposed for the same period to medium from which thyroid hormones had been adsorbed, 7 day exposure to T3 resulted in stimulation of [125i]-LDL binding and degradation in a dose dependent fashion at concentrations spanning the physiological range (Fig. 1 ) . No enhancement of LDL degradation was observed in the presence of T3 (10~?M) with receptor negative cells in which the amount of LDL degraded was exceedingly small. In addition to being dose dependent, this stimulatory effect of T3 on LDL metabolism was also time dependent (Fig. 2). When [125I]-LDL and T3 were added simultaneously, T3 failed to stimulate LDL binding at 4 h. LDL uptake and degradation (measured 24 and 48h later respectively) were however stimulated under these conditions. Addition of the [-*-2->I]LDL one or 7 days after the addition of T3 magnified the stimulatory effect of T3 on LDL binding and degradation in a time dependent fashion (Fig. 2). Although free T3 levels were not measured, they would be expected to be of the order of 1-5% of total T3 in medium containing 10% PHS (6). To circumvent any potential influence of binding proteins and to enable precise free hormone concentrations to be known, further experiments were performed in serum-free medium.
METHODS Skin biopsies from the thighs of normal volunteers were explanted and trypsinized as previously described (4). 10^ cells were transferred to Petri dishes in medium containing 10% pooled human serum (PHS). Cells were grown to confluence in PHS, after which they were switched either to medium containing 10% PHS from which thyroid hormones had been removed by adsorption to Amberlite IRA 400 resin (5) or to serum-free medium. More than 99.5% of the thyroid hormones were removed, as assessed by the prior addition of [125i]-iabeled hormones. T3 in ethanol was added to give the final concentrations indicated. Control replicate dishes to which no T3 was added received equivalent amounts of ethanol. No difference in cell number was noted (at any time point) between dishes exposed to or not exposed to T3, suggesting that cells were still proliferating normally in tha T3 deficient state. After exposure of cells to T3 for the times stated, [125i]-LDL, prepared as previously described (4), was added at the concentrations indicated. To induce LDL receptors when PHS was used (3), 24h before adding [^-^I]-LISL t ^ e me dium was changed to one containing 10% lipoprotein deficient serum (LDS; d> 1.25) with or without added T3. LDS was prepared from the thyroid hormone-free serum pool by ultracentrifugation (4). After [-*-^^I]-LDL was added, the cells were incubated for 4h at 37CC for the determination of its binding, 24 h for uptake, and 24-48h for measurement of LDL degradation by methods previously described (4). T3 was present in the test dishes throughout. The coefficient of variation for replicate analysis of LDL degradation was
