- Email: [email protected]
Inhibition of nuclear T3 binding by fatty acids
Inhibition
of Nuclear
T, Binding
by Fatty
Acids
Wilmar M. Wiersinga, lnder J. Chopra, and Guadalupe N. Chua Teco Studies were performed to evaluate a possible modulatory role of lipids on the binding of T, to rat liver nuclear receptors in vitro. Unsaturated fatty acids were potent inhibitors of the binding of [‘261] T, to isolated rat liver nuclei. Doses (in pmol/L) causing a 50% inhibition of nuclear T, binding were 10 for palmitoleic acid, 11 for linoleic acid, 22 for oleic acid, 24 for arachidonic acid, and 37 for linolenic acid. Other lipids had less or no inhibitory activity. Unsaturated fatty acids reduced the affinity constant (K,) of the binding of T, to nuclear receptors to 57.4% f 11 .O% that of controls (mean + SE 1.04 f 0.14 v 1.97 + 0.23 lo9 L/M, n = 5: P < .02) but did not affect the maximal binding capacity (MBCI (1.47 f 0.20 v 1.55 + 0.10 lo-” M/L; NS). Evaporated ether extracts of rat liver homogenate pretreated with phospholipase A, for five to 20 minutes (that liberates unsaturated fatty acids from phospholipidsl demonstrated a progressive inhibition of nuclear T, binding with time when compared with ether extracts of untreated rat liver homogenate (F = 16.1; P -c -01). Evaporated, fatty-acid-rich ether extracts of human sera caused a dose-dependent inhibition in the binding of [‘261] T, to nuclear T, receptors. Nuclear T, binding in the presence of extracts of 22 sera (0.1 mL-Eq aliquots) of patients with nonthyroid& illness (NTI) was 58.9% ? 4.9% (mean ? SE; range 17% to 107%) that of normal sera (100% + 6.2%. n = 9; P =z .0005); it was more that 2 SD below the normal mean in 12 (55%) patients. Inhibition of nuclear T, binding by NTI sera was correlated with the presence of a thyroid hormone-binding inhibititor (THBI) in these sera (r = .89; P < .0005). Evaporated ether extracts of NTI sera reduced the K, values of the binding of T, to nuclear receptors to 47.5% + 10.8% that of normal sera (n = 4: P < .Ol 1,without an effect on MBC. These various data suggest that unsaturated fatty acids can modulate thyroid hormone binding to its nuclear receptor in vitro. Whether or not fatty acids exert a similar effect in vivo is presently unknown. m 198i by Grune & Stratton, Inc.
U
FATTY ACIDS are important modulators of cellular metabolism. The impact of various derivatives (like prostaglandins and leukotrienes) of arachidonic acid that may be liberated from phospholipids in the cell membrane by the action of phospholipases is well known. Recent reports indicate that unsaturated fatty acids interfere with the binding of some ligands to their receptors. Thus, fatty acids inhibit specific binding of angiotensin to receptors in adrenal glomerulosa and fasciculata cells.’ Unsaturated fatty acids also act as endogenous inhibitors of tamoxifen binding to antiestrogen binding sites.’ Additionally, it has been shown that modification of the phospholipid component of the nuclear membrane in vitro by phospholipase modulated the activity of Sa-reductase in a highly specific and structure-related manner.’ Unsaturated fatty acids have also been shown able to influence thyroid hormone physiology substantially. Thus, they inhibit the binding of T, to serum proteins4-’ and extrathyroidal conversion of T, to more potent T,.* In this study, we have questioned whether or not fatty acids affect the binding of T, to its specific nuclear receptors. NSATURATED
MATERIALS
AND METHODS
Male Sprague-Dawley rats were killed by cervical dislocation, and livers were excised and processed immediately thereafter at 0 to 4°C
From the Departments of Medicine. UCLA Centerfor the Health Sciences, Lxx Angeles, and Academisch Medisch Centrum, University of Amsterdam, The Netherlands. Supported by a travel grant from the Netherlands Organization for the advancement of Pure Research (ZWO) and by USPHS Grant No. AM-16155from the National institutes of Health. Address reprint requests to Inder J. Chopra, MD. Department of Medicine, UCLA Center for the Health Sciences, Los Angeles, California 90024. o 1988 by Grune & Stratton, inc. 0026-0495/88/371 O-001 6%03.00/O 996
for the preparation of isolated nuclei.’ In short, after homogenization of the liver in solution A (20 mmol/L tricine, 0.25 mol/L sucrose, 2 nimol/L CaCl,, 1 mmol/L MgCl,, and 5% (vol/vol) glycerol, pH 7.6) by a motor-driven Teflon pestle in a Potter-Elvehjem-type Wheaton tube, the nuclei were pelleted and washed two times in solution A containing 0.5% Triton X-100. Thereafter, the pellet was thoroughly suspended in solution A containing 2.4 mol/L sucrose, and the suspension was centrifuged for 45 minutes at 53,000 x g. The remaining pellet was washed again three times in solution A containing 0.5% Triton X-100, and finally suspended in 0.5 vol (wt/vol) solution B (20 mmol/L TRIS, 0.25 mol/L sucrose, 1 mmol/L EDTA, 50 mmol/L NaCl and 5% (vol/vol) glycerol, pH 7.6). The DNA concentration of the nuclear suspensions was determined according to the method of Webb and Levy,“’ and protein measurements were performed by the Bio-Rad assay”; the mean ratio of DNA to protein was 3.71 + 0.55. The binding of ‘*51-labeled T, (specific activity 1,200 pCi/rg, New England Nuclear, Boston) to isolated nuclei was studied by the addition of approximately 20 to 40 femtomoles [‘251]T, to 0.1 mL nuclear suspension (representing 200 mg of starting tissue) in the presence of 5 mmol/L dithiothreitol (DTT). The total incubation volume was 0.5 mL; all reagents were dissolved in solution B, unless stated otherwise. Incubations were for two hours in a shaking waterbath at 22OC. Incubations were stopped by chilling tubes in ice-cold water. Two milliliters of solution B containing 0.5% Triton X-100 was then added to each tube. After vortexing, the tubes were centrifuged at 4OCfor 15 minutes at 1,250 x g. The supernatant was aspirated, and the radioactivity in the pellet (ie, the hormonal function bound to the nuclei) was counted. Specific binding was 24.6% f 0.9%, calculated as the difference between the counts bound to the nuclei in the absence and in the presence of an excess of nonradioactive T, ( 10m6mol/L); the nonspecific binding was 2.4% k 0.1% of total radioactivity (mean * SE; n = 23). Scatchard plots’* were prepared from data on bound and free T, in the absence and in the presence of increasing quantities of nonradioactive T, ranging in concentration from 1.0 to 33 x lo-“’ M. All measurements were performed in duplicate. Studies
With Lipids
Various lipids were purchased from Sigma Chemical Co, St Louis. They were dissolved in ethanol, and concentrations for use in Metabolism, Vol 37, No 10
(October), 1988: pp 996-1002
EFFECT OF FATI-Y ACIDS ON NUCLEAR T, BINDING
_
the nuclear Ts binding assay were prepared in buffer B. Arachidonic acid (sodium salt) was in clear solution in aqueous media. Other fats were in fine suspension. The final concentration of ethanol in the assay (C 1%) did not affect nuclear T, binding.
unsaturated -
fatty acids
-‘K--__-~__---n__-__
Studies With Phospholipase A2
a-_ \ \
When the effect of phospholipase A2 (PLAI) was to be studied, rat liver homogenate was treated with PLA2 by a modification of the method of Roelofsen et al.” Highly purified PLA2 from the snake venom of Naja Mozambique Mozambique (SA 1,490 U/mL protein; Sigma Chemical Co, St. Louis) was dissolved in 0.1 mol/L TRIS buffer (pH 8.2) containing 0.075 mol/L calcium chloride. Five units of PLA, in 0.05 mL buffer were incubated with 0.25 mL rat liver homogenate (-15 mg protein) for 0 to 20 minutes at 22’Y in a shaking water bath, and the reaction was stopped by the addition of 0.15 mL 0.25 mol/L EDTA. Control tubes contained 0.05 mL buffer without PLA, and 0.25 mL homogenate, and were treated identically. Preparation of ether extracts was as follows: Two volumes anhydrous diethyl ether (0.9 mL) were added to each tube, and the mixture was swirled on a vortex mixer for 30 seconds and centrifuged at 1,000 x g for ten minutes at 4°C. The tubes were put on dry ice, and the ether phase was decanted into separate tubes. The ether was evaporated to dryness at room temperature under a stream of nitrogen, and the residue was suspended in 10 mL of buffer B; a measured volume (0.025 mL) was assayed for nuclear T, bindinginhibiting activity.
- saturated
\
. linolanicacid P arachidonic acid 0 oleic acid l palmitoleicacid l finofeicacid
Ao
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o arachidic acid G stearic acid l palmitic acid - myristic acid
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0.3
mf4
cholesterol and mono-, di-, triglycerides E *
1
120.
8
4
0
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‘
Studies With Human Sera When sera were to be tested for inhibition of nuclear T, binding (INB), ether extracts were prepared using 4 vol diethyl ether for each volume of serum. The mixture was swirled on a vortex mixer for 30 seconds and put on dry ice. After freezing of the serum phase the supernatant ether phase was decanted into another tube and evaporated to dryness at room temperature under a stream of nitrogen. The residue was suspended in solution B of the nuclear Ts binding assay. After initial studies with pooled sera from hospitalized patients (stored for two days at 4°C and subsequently frozen at - 20°C), individual serum samples were prospectively obtained from nine normal subjects (five men and four women, 18 to 57 years of age) and 22 patients (12 men and ten women, 17 to 87 years of age); patients were admitted to intensive care units with a variety of systemic illnesses. Individual serum samples were frozen within 30 to 40 minutes after collection, and stored at -20°C until assay. The major disorders included postsurgical state (radical cystectomy, liver resection, excision of cerebellar tumor, aneurysmectomy, tricusip valve replacement, hysterectomy; seven patients), infectious diseases (herpes encephalitis, sepsis, pneumonia; six patients), comatose state (multiple injuries, subdural hematoma, unknown cause; three patients), cardiac disease (ventricular septum rupture, congestive heart failure; two patients), pulmonary disease (respiratory insufficiency; two patients), renal disease (renal failure; one patient), and gastrointestinal disease (pancreatic abscess with fistula; one patient). Serum T, and T, were determined by specific radioimmunoassays,‘4~‘5 and TSH by immunoradiometric assay.16 Thyroid hormone-binding inhibitor (THBI) was measured in diethyl ether extracts of sera by a competitive ligand-binding assay as described previously.” RESULTS
Lipids as Inhibitors of Nuclear T3 Binding The effects of various lipids on nuclear Ts binding in vitro was studied. Nonspecific binding (mean + SE) of [‘*‘I]T, in the presence of lipids was 2.1% + O.l%, not different from
\
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cholesterol triofain tripalmitin dilaurin dilonolain l monolinolein . monofinolenin 0 monolaurin
4 b 0 v
B
01
h.
0.011
0.033
0.1
-&3
rnM
Fig 1. Effects of various unsaturated and saturated fatty acids (A) and of cholesterol and various mono-, di-. and triglycerides (B) on the binding of [“‘I] T, to isolated rat liver nuclei. Nuclear T, binding in the absence of lipids (control) was arbitrarily assigned a value of 100. and nuclear T, binding in the presence of lipids was expressed as a percentage of the control value.
that of 2.2% f 0.1% in the absence of lipids. Nuclear T, binding was strongly inhibited by all unsaturated fatty acids so studied, and moderately inihibited by some saturated fatty acids (Fig 1A). The lipid concentrations (pmol/L) that caused a 50% inhibition of nuclear TS binding were 10 for palmitoleic acid, 11 for linoleic acid, 22 for oleic acid, 24 for arachidonic acid, 37 for linolenic acid, 60 for myristic acid, 300 for palmitic acid, and >l,OOO for stearic acid and arachidonic acid (the lipid concentration in Gmol/L divided by 2,000 will give the amount of lipid present in the incubation mixture, in terms of micromoles/tube). The mean concentration of the unsaturated fatty acids required for a 50% inhibition was 20 pmol/L, equivalent to 0.01 micromoles per tube. The monoglyceride monolinolein had no inhibitory effect on nuclear T, binding, but monolaurin and
WIERSINGA, CHOPRA, AND CHUA TECO
monolinolenin had a moderate inhibitory activity (50% inhibition at 85 and 390 pmol/L, respectively) (Fig 1B). Diglycerides (dilaurin, dilinolein) and triglycerides (tripalmitin, triolein) in concentrations up to 300 Kmol/L and cholesterol in concentrations up to 1 mmol/L had no effect on nuclear T, binding. To examine the nature of inhibition of nuclear T, binding by lipids, we performed Scatchard analysis of the binding of [‘*‘I] T, to isolated rat liver nuclei in the absence and the presence of unsaturated fatty acids (at concentrations that caused 50% inhibition of nuclear T, binding) (Fig 2). Maximal binding capacity was not affected, but the affinity constant decreased: K, values in the presence of unsaturated fatty acids were 57.4% k 11.O% of K, values in the control experiments (P < .Ol). Since fatty acids possess detergent-like properties, we examined the effects of some detergents on nuclear T, binding. Sodium cholate and CHAPS (3-[(3-cholamidopropyl) dimethylammonio] I-propanesulfonate) had no appreciable inhibitory effect on nuclear T, binding in concentrations up to 1 mmol/L. The potent detergent sodium deoxycholate caused a 50% inhibition of nuclear T, binding at a concentration of 0.33 mmol/L, a l&fold higher dose than that of 0.02 mmol/L for unsaturated fatty acids. It is conceivable that fatty acids inhibit nuclear T, binding not by interference with the binding sites on the nuclear T, receptor, but by an interaction with T, that would leave less T, available for nuclear binding. To explore this possibility, we preincubated 0.5 mL [“‘I] T, in solution B with either 0.5 B/F
mL 0.35 mmol/L linolenic acid in solution B (tube 1) or with 0.5 mL solution B (tube 2, control) at room temperature for 30 minutes. Four volumes of ether (4 mL) were then added to each tube, and the tubes were swirled on a vortex mixer for 30 seconds and centrifuged at 1,000 x g for ten minutes at 4OC. The aqueous and ether phases were separated. In one study, 50 PL of the aqueous phase was substituted for [lz51] T, in the nuclear T, binding assay; specifically bound T, was 31.2% using the aqueous phase of tube 1, not different from that of 30.3% for control tube 2. In another study, 400 rL of the ether phase was evaporated to dryness, and the dissolved residue was assayed in the nuclear T, binding assay; specifically bound T, was 8.2% in the presence of ether extract of tube 1, which contained fatty acid, compared with that of 29.3% in the presence of the extract of control tube 2. Eflect of PLA, Treatment of Tissue The effect of PLA, treatment was studied to determine whether fatty acids liberated as a result of activation of tissue PLA, might affect nuclear T, binding. After treatment of rat liver homogenate with PLA, for 5, 10, 15, or 20 minutes, the nuclear T, binding in the presence of evaporated ether extracts of the liver homogenate decreased progressively (P < .Ol); all these values were more than 2 SD below the mean of control incubations (Fig 3). At 20 minutes, the nuclear T, binding-inhibiting activity of the PLA,-treated liver homogenate was 62% that of the control mixture. PLA, treatment of tissue phospholipids results in genera-
0.35
1 effect of phospholipase
4
%
treatment
. contmt n unsaturated
fatty
acids
1100
1
80
60 10 0 control l
PLA*
5 Scatchard plots of the binding of [“g T, to isolated rat Fig 2. liver nuclei in the absence (control) and in the presence of unsaturated fatty acids (at concentrations that caused approximately 50% inhibition of nuclear T, binding: palmitoleic acid 0.01 mmol/L, linoleic acid 0.01 mmol/L, oleic acid 0.02 mmol/L. araohidonic acid 0.026 mmol/L and linolenic acid O.OS6 mmol/Ll. Each point represents the mean of five experiments; each of the experiments included a Scatchard plot without lipids and one plot with a particular unsaturated fatty acid. Statistical analyses of changes in maximal binding capacity (MBC) and affinity constant (K,) (values as mean f SE) were made by the paired t test. MBC: control 1.56 t 0.10, unsaturated fatty acids 1.47 -t 0.20, lo-” M/L; NS. K,: control 1.97 f 0.23, unsaturated fatty acids 1.04 f 0.14, 10’ L/M; P < .025.
1
0
I
5
10
15
I
20 time ( min
1
Effect of PLA, treatment. Rat liver homogenate was Fig 3. incubated with PLA,, and the reaction was stopped by addition of EDTA. Evaporated ether extracts of incubation mixtures were assayed for inhibiting activity on the binding of [“v] T, to isolated rat liver nuclei. Each result is the mean of duplicate determinations. Nuclear T, binding in the PLA,-treated incubation mixtures was lower than in controls as indicated by one-way ANOVA IF- 16.1:Pc .Ol).
EFFECT OF FAll-Y ACIDS ON NUCLEAR T, BINDING
999
tion of lysolecithin (L-a-phospatidylcholine) in addition to fatty acids. Some of the liberated fatty acids are amenable to oxidation and may have been converted in part to prostaglandins. Therefore, we tested the effect of phospholipids and prostaglandins on nuclear T, binding. Phosphatidylcholine (lecithin) and phosphatidylcholine dioleyl in concentrations up to 300 rmol/L had no nuclear T, binding-inhibiting activity. Lysophosphatidylcholine (lysolecithin) and lysophosphatidylcholine oleyl had some effect: 50% inhibition of nuclear T, binding was observed at concentrations of 840 pmol/L and 740 pmol/L, respectively, a 40-fold higher dose than that of 20 pmol/L for unsaturated fatty acids. The naturally occurring prostaglandins E,, E, and F,, had no nuclear T, binding-inhibiting activity in concentrations up to 1 mmol/L; 50% inhibition of nuclear T, binding was caused by 1.25 mmol/L prostaglandin A,. Studies
With Human Serum Samples
Figure 4 compares the dose-response curves for the effect of ether extracts of three sera of normal subjects and three pools of sera of nonthyroidal illness (NTI) patients on the binding of T, to isolated rat liver nuclei. It is clear that the
o normal subjects l
2 A! s
nonthyroidal illness patients
60
C
:
1 I
0
, 0.05
, 0.1
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0.3 ml-equivalent
IO* serum
Fig 4. EHsct of evaporated ether extracts of tern of normal subjects fn = 3) and pooled sera of hospitalized patients with NTI (n = 3) on T, binding by rat liver nuclei. Nuclear T, binding in the absence of serum extraot (control) was arbitrarily assigned a value of 100, and nudear T, binding in the presence of serum extract was expressed as a percentage of the control value. The data are expressed as the mean + SE and statistical analyses indicated were made using Student’s t tests. tP < .Ol: lP < .005; .*P < 9025 v normal subjects. Nuclear T, binding in the presence of serum axtracts of patients was also significantly different from that of normal subjects when data were analyzed by one-way ANOVA IF = 10.9: P < .Ol j.
extracts of sera of NT1 patients had greater nuclear T, binding-inhibiting activity than the extracts of sera of normal subjects. We next sought nuclear T, binding-inhibiting activity in nine sera of normal subjects and in 22 sera of NT1 patients. For convenience, we used an ether extract of 0.1 mL serum in each case. Nonspecific binding in the presence of evaporated ether extracts of human sera (2.5% 5 0.1%) was not different from that of controls without serum extracts. The mean specific nuclear T, binding in the presence of extracts of sera of nine normal subjects was assigned a value of 100, and the results in all normal subjects and patients were expressed as a percentage of this. Nuclear T, binding in the presence of extracts of sera from NT1 patients ranged between 17% and 107% of the mean control value. The mean + SE value of 58.9% + 4.9% in the 22 NT1 patients was significantly (P-C .OOOS)lower than that of 100% + 6.2% in the nine normal subjects. Nuclear T, binding in the presence of extracts of patients’ sera was more than 2 SD below the normal mean in 12 of 22 (55%) NT1 patients. The mean serum (*SE) T, concentration of 32 * 9 ng/dL (cf normal value of 106 + 8) in these 12 patients did not differ from that of 27 2 6 ng/dL in the other 10 patients; serum T, (4.6 t 1.0 v 4.3 * 1.0 rg/dL) and serum TSH (1.9 r 0.6 v 1.2 + 0.5 U/mL) were also similar. Since THBI, like nuclear T, binding-inhibiting activity, is extractable from serum by ether treatment, we studied the relationship between the two activities in the abovementioned sera. Sixteen of the 22 individual sera of NT1 patients were THBI positive (THBI value > 1.60”). and six were THBI-negative. The mean nuclear T, binding in the presence of extracts of THBI-positive sera was 48% + 3.7% that of 100% + 6.2% in the presence of extracts of normal sera (P < .OOOS). The mean nuclear T, binding in the presence of extracts of THBI-negative sera of 86.7% + 7.6% was not significantly different from the corresponding value in normal subjects (Fig 5). Five patients were receiving low-dose heparin administered subcutaneously; all had THBI-positive sera. However, THBI and INB values in these five patients were not different from those in the other 11 THBI-positive patients who did not receive any heparin (THBI, 2.80 k 0.30 v 2.71 i 0.35, NS; INB, 44.6% + 5.3% v 50.8% 2 4.9%, NS). Serum activities of THBI and the INB were correlated highly significantly (y = 95.4 x -“.76, r = .89, P -z .0005; the closest agreement with the experimental data was found using a program that fits a power curve y = axb to a set of data points {(xi, y,), i = 1, 2, . . n)). To examine the nature of inhibition of nuclear T, binding by human serum extracts, we performed Scatchard analysis of the binding of [“‘I] T, to isolated rat liver nuclei in the absence and in the presence of ether extracts (Fig 6). Maximal binding capacity was not affected, but the affinity constant decreased: K, values in the presence of evaporated ether extracts of NT1 sera were 47.5% f 10.8% of K, values obtained in the presence of ether extracts of normal sera (P < .Ol). Reproducibility
The reproducibility of the assay system for nuclear T, binding-inhibiting activity in human sera was tested by
WIERSINGA. CHOPRA, AND CHUA TECO
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Fig 8. Scatchard plots of the binding [“‘I] T, to isolated rat liver nuclei in the absence of serum (control) and in the presence of evaporated ether extracts of sera of normal subjects (nuclear T, binding, 96.9% + 16.4%) and of patients with NTI lnuclear T, binding, 47.1% -c 9.2%). Each point represents the mean of four experiments. Statistical analyses of changes in MBC and K, were made by the paired t test leech of the four experiments included a Scatchard plot without serum, with normal serum, and with NTI serum: values are given as mean + SE).
t
MBC (10~” M/Lj Control
40
K, (10’ L/Ml 2.88 + 0.34
1.26 f 0.25 NS
l Normal sera 1.44 f 0.33
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0.36
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:
NTI sers
20
1.71 f 0.54
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variation was 7.26% (73.5% k 5.3%) 9.17 (68.9% r 6.3%), 8.91% (29.5% + 2.6%), and 3.04% (15.0% + 0.5%). Role of Assay Conditions in the Activity of INB
0 normal subjects
(9)
THBl+ve NTI-pat. (16)
THBI-ve NTI-pat. (6)
Fig 5. Nuclear T, binding-inhibiting activity in ether axtracts of sera of NTI patients and normal subjects. The mean binding of “‘11 T, during incubation of isolated rat liver nuclei with evapoI rated ether extracts of sera of normal subjects was arbitrarily assigned a control value of 100, and the results in all normal subjects and NTI patients were expressed as a percentage of the mean control value.
assaying each of four serum samples five times in one assay and also in duplicates in five consecutive assays. Intra-assay coefficient of variation (SD/mean x 100%) was (mean + SD of nuclear T, binding in parantheses) 5.04% (70.2% + 3.5%), 4.06% (35.8% + l.S%), 9.04% (32.3% f 2.9%), and 10.42% (15.6% + 1.6%); inter-assay
The nuclear T, binding-inhibiting activity of human sera was demonstrable both in the presence and the absence of DTT. The ratio of specifically bound [‘*‘I] T, in the absence of DTT to that in the presence of DTT was 0.45 (12.8%/ 28.2% without serum extract), 0.40 (7.9%/19.8% in the presence of ether extract of normal serum), and 0.33 (1.5%/ 4.4% in the presence of extract of NT1 serum). In other studies, we examined the effects of ether extracts of human sera on the release of T, receptors from the nuclear pellet into the supernatant during the incubation period.18 The incubation mixture at the end of the incubation period was centrifuged and the supernatant was aspirated and passed through a small Sephadex G-25 (medium) column prepared in a Pasteur pipette; the radioactivity in five 0.8-mL fractions was counted using solution B as the eluant. Radioactivity in the remaining nuclear pellet was counted after washing with 2 mL of solution B containing 0.5% Triton X-100 as described above. The ratio of specifically bound [‘25I] T, in the pellet to that in the supernatant was 1.49 (no serum present), 1.49 (normal serum), and 1.91 (NT1 serum). These data provided no support for the possi-
EFFECT OF FAllY
ACIDS ON NUCLEAR T, BINDING
bility that serum extracts inhibit binding of ‘251-T~to nuclei by causing a release of T, nuclear receptors in the medium.
DISCUSSION
In the present study, we have demonstrated that several lipids, especially unsaturated fatty acids, are potent inhibitors of nuclear T, binding in vitro. The decreased nuclear T, binding was caused by a reduction in the affinity of the nuclear receptors for T,. The mechanism by which unsaturated fatty acids inhibit nuclear T, binding remains unclear, but several possibilities may be considered. First, fatty acids may affect the T, binding site by altering its hydrophobic environment; fatty acids have been shown to alter the fluidity and the surface charges of cell membranes.‘9-2’ Second, the inhibitory effect of fatty acids on nuclear T, binding may be due to oxidation products that may compromise the stimulatory effect of DTT in the nuclear T, binding assay.** However, this appears unlikely because of the following: (1) Inhibition of nuclear T, binding is demonstrable equally well in the presence as in the absence of DTT. (2) Some fatty acids poorly amenable to oxidation, eg, myristic acid and oleic acid, are potent INBs. (3) Some products of fatty acid oxidation, eg, prostaglandins, had no or very weak INB activity. Third, it is conceivable that the detergent-like action of fatty acids contributes importantly to their inhibitory effect on nuclear T, binding. This, too, appears unlikely because potent detergents like CHAPS, sodium cholate, and deoxycholate had either no or weak INB activity; and the binding of T, to receptors remaining in the nuclear pellet was depressed to a similar extent as the binding of T, to receptors released into the supernatant. Additionally, our studies provided no support for the possibility that the inhibitory effect of fatty acids on nuclear T, binding was a result of release of receptors from the nucleus to the supernatant of the incubation medium, as would be expected if fatty acids were potent detergents in the low concentrations present in incubation medium. Fourth, it is possible that fatty acids reduce nuclear T, binding by their interaction with T, in a manner that leaves less T, available for receptor binding. However, our studies on preincubation of [‘*‘I] T, with fatty acids did not support this possibility. Therefore, we presently consider an allosteric hindrance of the binding of T, to its nuclear receptor by fatty acids as a likely mechanism of their action as INBs. A similar mechanism of action of fatty acids has been suggested previously for their inhibition of hepatic T,-5’-monodeiodinase,8 T, binding to serum proteins,” and T, binding to anti-T,-antibodies. ‘*” Interestingly, the inhibitory effect of unsaturated fatty acids is greater in the case of hydrophobic binding sites, eg, nuclear T, receptor, T, 5’monodeiodinase than in the case of hydrophilic binding sites of T,-binding T, antiserum, This is illustrated in an interesting manner by the concentration of oleic acid required for a 50% inhibition of 0.022 mmol/L for nuclear T, binding (vide supra); 0.8 mmol/L for T, 5’-monodeiodination,* and 1.32 mmol/L for T, binding by anti-T, antiserum.4 A similar pattern is observed with the compound 8-anilino-l-naphthalene sulphonic acid (ANS), which interferes strongly with the binding of thyroid hormones to the hydrophobic binding
1001
sites on serum proteins23 and with nuclear T, binding,24 but only weakly with the binding to thyroid hormone-binding antibodies.14 A direct comparison between the concentrations of oleic acid or ANS in various assays may not be entirely appropriate, however, because of differences in various assay conditions, especially concentration of proteins in the incubation mixture. We had studied the effect of PLA, on tissue INB activity and serum INB activity in a hope to gather some insight into potential in vivo application of INB. Activation of PLA, results in the liberation of unsaturated fatty acids, located mainly at the no. 2 position of membrane phospholipids. Incubation with PLA, increased the INB activity in rat liver homogenates significantly. Since effective stimuli for activation of PLA, (like catecholamines, bradykinin, angiotensin II, vasopressin, thrombin, collagen, chemotactic peptides, immunoglobulin, complement CSa and histamine)25-27 are present in many circumstances of an NTI, it is conceivable that activation of phospholipases of the A, type occurs during NTI. This might result in the release of some liberated fatty acids into the circulation, which together with the lipolysis of depot fat frequently causes an increase in serum free fatty acids in NTL4 Our studies have shown that, relative to serum samples of normal subjects, many NT1 sera demonstrate significant INB activity. The nature of INB in sera of NT1 patients is unclear. However, available data suggest that it is a lipid, more specifically, unsaturated fatty acids. Thus, we find that: (1) INB is extracted from serum by lipid solvents like diethyl ether; (2) unsaturated fatty acids are the most potent INBs of all lipids, so studied; (3) NT1 sera and unsaturated fatty acids both inhibit nuclear T, binding by affecting a decrease in the affinity constant of the binding interaction; (4) serum concentrations of unsaturated fatty acids are frequently increased in NT14; and (5) the serum concentration of INB is correlated highly signficantly with that of THBI, a lipidic substance detected in sera of many NT1 patients4 While it is possible that circulating INB in human serum affects the binding of T, to its nuclear receptors in vivo, our studies have not addressed this possibility directly. Similarly, it is conceivable that unsaturated fatty acids liberated in the plasma membrane are transported to the nucleus or, alternatively, that phospholipases are present in the nuclear membrane and are activated during an illness. A recent study has indeed demonstrated the presence of PLA, activity in nuclear membranes of rat liver.*’ It seems important to carefully examine the various above-mentioned issues in future studies to evaluate the importance of fatty acids in modulation of nuclear-bound T, and thereby, thyroid hormone effects in the tissues. Previous studies in experimental animals with various forms of NT1 have demonstrated, in general, a reduced binding capacity of hepatic nuclear T, receptors without changes in the binding affinity.22129-36 These findings do not necessarily argue against an involvement of unsaturated fatty acids in alterations of nuclear T, binding in NTI, since the studies were conducted in vitro to isolated nuclei and since fatty acids exerting allosteric hindrance of the binding of T, are likely to be removed in the experimental procedure requiring purification of nuclei by repeated washes with
1002
WIERSINGA,
detergents such as Triton X-100. Moreover, the effect of prolonged exposure to high concentrations of unsaturated fatty acids on nuclear binding of T, is not known at this time. It would be interesting to examine this and other abovementioned issues in future studies of the role of unsaturated fatty acids in altered thyroid hormone economy in NTI.
CHOPRA, AND CHUA TECO
ACKNOWLEDGMENT
We appreciate the helpful advice and comments of Dr David H. Solomon and Dr Ruben Boado, the secretarial assistance of Martha Komende Vries and Judy Sirand, the technical help of Julie Smick, and the support of Dr Hans Ramijn in the collection of patients’ samples.
REFERENCES
1. Goodfriend TL, Ball DL: Fatty acid effects on angiotensin receptors. J Cardiovasc Pharmacol 8: 1276-l 283, 1986 2. Hwang PLH: Unsaturated fatty acids as endogenous inhibitors of tamoxifen binding to anti-oestrogen-binding sites. Biochem J 237:749-755,1986 3. Cooke GM, Robaire B: Modulation of epididymal A4-steroid 5,-reductase activity in vitro by the phospholipid environment. J Biol Chem 260:7489-7495, 1986 4. Chopra IJ, Chua Teco GN. Mead JF, et al: Relationship between serum free fatty acids and thyroid hormone binding inhibitor in nonthyroid illnesses. J Clin Endocrinol Metab 60:980-984, 1985 5. Chopra IJ, Huang TS, Solomon DH, et al: The role of thyroxine (T&binding serum proteins in oleic acid-induced increase in free T, in nonthyroidal illnesses. J Clin Endocrinol Metab 63~776-779, 1986 6. Chopra IJ, Huang TS, Beredo A, et al: Serum thyroid hormone binding inhibitor in nonthyroidal illnesses. Metabolism 35:152-159, 1986 7. Mendel CM, Frost PH, Cavalieri RR: Effect of free fatty acids on the concentration of free thyroxine in human serum: The role of albumin. J Clin Endocrinol Metab 63:1394-1399, 1986 8. Chopra IJ, Huang TS, Beredo A, et al: Evidence for an inhibitor of extrathyroidal conversion of thyroxine to 3,5,3’-triicdothyronine in sera of patients with nonthyroidal illnesses. J Clin Endocrinol Metab 60:666-672, 1985 9. Spindler BJ, McLeod KM, Ring J, et al: Thyroid hormone receptors. Binding characteristics and lack of hormonal dependency for nuclear localization. J Biol Chem 250:4113-4119, 1975 10. Webb JM, Levy HB: A sensitive method for the determination of deoxyribonucleic acid in tissues and microorganisms. J Biol Chem 213:107-l 17, 1955 11. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254, 1976 12. Scatchard G: The attraction of proteins for small molecules and ions. Ann NY Acad Sci 51:660-672,1949 13. Roelofsen B, Zeal1 FRA, Woodward CB: The action of pure phospolipases on native and ghost red cell membranes, in Colowick SP, Kaplan NO (eds): Methods in Enzymology, vol 32. San Diego, Academic, 1974, p 13 1 14. Chopra IJ: A radioimmunoassay for measurement of thyroxine in unextracted serum. J Clin Endocrinol Metab 34938-947, 1972 15. Chopra IJ: An improved radioimmunoassay of triiodothyronine in serum: Its application to clinical and physiological studies. J Lab Clin Med 80:729-739, 1972 16. Semple CG, Slater SD, Reid AM, et al: A sensitive immunoradiometric assay for serum thyroid stimulating hormone. Br Med J 290:69-70, 1985 17. Chopra IJ, Huang TS, Hurd RE, et al: A competitive ligand binding assay for measurement of thyroid hormone-binding inhibitor in serum and tissues. J Clin Endocrinol Metab 58:619-628, 1984 18. Bernal J, DeGroot LJ: Thyroid hormone receptors: Release of receptor to the medium during in vitro incubation of isolated rat liver nuclei. Endocrinology 100:648-655, 1977
19. Ahrens ML: Electrostatic control by lipids upon the membrane bound (Na+-K+) ATP ase. Biochim Biophys Acta 642:252266, 1981 20. Schmalizing G, Kutschera P: Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A,. J Membr Biol69:65-76, 1982 21. Wojtczak L, Nalecz M: Surface charge of biological membranes as a possible regulator of membrane-bound enzymes. Eur J Biochem 94:99-107, 1979 22. Wiersinga WM, Frank HJL, Chopra IJ, et al: Alterations in hepatic nuclear binding of triiodothyronine in experimental diabetes mellitus in rats. Acta Endocrinol99:79-85, 1982 23. Wartofsky L, Burman KD: Alterations in thyroid function with systemic illness: The “euthyroid sick syndrome.” Endocr Rev 3:164-217, 1983 24. Burman KD, Lukes UG, Latham KR, et al: Ipodate and %anilino-1-naphthalene sulfonc acid block receptor binding of T, in rat liver. Horm Metab Res 12:685-687, 1980 25. Lapetina EG: Regulation of arachidonic acid production: Role of phospholipase C and A,. Trends Pharm Sci 3:115-118, 1982 26. Nishizuka Y: Phospholipid degradation and signal translation for protein phosphorylation. Trends Biochem Sci 8: 13- 16, 1983 27. Abdel-Latif AA: Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers. Pharmacol Rev 38:227-272, 1986 28. Karagezyan KG, Tadevosyan Yu V: Enzyme systems of phospholipid metabolism in the nuclear membranes of albino rat livers. Dokl Akad Nauk SSSR Proc 287: 1248- 1250,1986 29. DeGroot LJ, Coleoni AH, Rue PA, et al: Reduced nuclear triiodothyronine receptors in starvation-induced hypothyroidism. Biochem Biophys Res Commun 79:173-178,1977 30. Burman KD, Lukes Y, Wright FD, et al: Reduction in hepatic triiodothyronine binding capacity induced by fasting. Endocrinology 101:1331-1334,1977 3 1. Schussler GC, Orlando J: Fasting decreases triiodothyronine receptor capacity. Science 199:686-688, 1978 32. Surks MI, Grajower MM, Tai M, et al: Decreased nuclear L-triiodothyronine receptors in rat and mice bearing transplantable neoplasms. Endocrinology 103:2234-2239, 1978 33. Das DK, Ganguly M: Diabetes, hypophysectomy, or thyroidectomy reduces nuclear L-triiodothyronine-binding capacity of rat lung. Endocrinology 109:296-300, 1981 34. Lim VS, Passo C, Murata Y, et al: Reduced triiodothyronine content in liver but not pituitary of the uremic rat model: Demonstration of changes compatible with thyroid hormone deficiency in liver only. Endocrinology 114:280-286, 1984 35. Little JS: The effect of Streptococcus pneumoniue infection on the binding of triiodothyronine to nuclei isolated from rat liver. Endocrinology 117: 180- 186, 1985 36. Nakamura H, Yokota T, Imura H: Different alterations of nuclear triiodothyronine receptor capacity in liver and kidney induced by starvation and triiodothyronine administration. Acta Endocrinol 108:206-210, 1985
of Nuclear
T, Binding
by Fatty
Acids
Wilmar M. Wiersinga, lnder J. Chopra, and Guadalupe N. Chua Teco Studies were performed to evaluate a possible modulatory role of lipids on the binding of T, to rat liver nuclear receptors in vitro. Unsaturated fatty acids were potent inhibitors of the binding of [‘261] T, to isolated rat liver nuclei. Doses (in pmol/L) causing a 50% inhibition of nuclear T, binding were 10 for palmitoleic acid, 11 for linoleic acid, 22 for oleic acid, 24 for arachidonic acid, and 37 for linolenic acid. Other lipids had less or no inhibitory activity. Unsaturated fatty acids reduced the affinity constant (K,) of the binding of T, to nuclear receptors to 57.4% f 11 .O% that of controls (mean + SE 1.04 f 0.14 v 1.97 + 0.23 lo9 L/M, n = 5: P < .02) but did not affect the maximal binding capacity (MBCI (1.47 f 0.20 v 1.55 + 0.10 lo-” M/L; NS). Evaporated ether extracts of rat liver homogenate pretreated with phospholipase A, for five to 20 minutes (that liberates unsaturated fatty acids from phospholipidsl demonstrated a progressive inhibition of nuclear T, binding with time when compared with ether extracts of untreated rat liver homogenate (F = 16.1; P -c -01). Evaporated, fatty-acid-rich ether extracts of human sera caused a dose-dependent inhibition in the binding of [‘261] T, to nuclear T, receptors. Nuclear T, binding in the presence of extracts of 22 sera (0.1 mL-Eq aliquots) of patients with nonthyroid& illness (NTI) was 58.9% ? 4.9% (mean ? SE; range 17% to 107%) that of normal sera (100% + 6.2%. n = 9; P =z .0005); it was more that 2 SD below the normal mean in 12 (55%) patients. Inhibition of nuclear T, binding by NTI sera was correlated with the presence of a thyroid hormone-binding inhibititor (THBI) in these sera (r = .89; P < .0005). Evaporated ether extracts of NTI sera reduced the K, values of the binding of T, to nuclear receptors to 47.5% + 10.8% that of normal sera (n = 4: P < .Ol 1,without an effect on MBC. These various data suggest that unsaturated fatty acids can modulate thyroid hormone binding to its nuclear receptor in vitro. Whether or not fatty acids exert a similar effect in vivo is presently unknown. m 198i by Grune & Stratton, Inc.
U
FATTY ACIDS are important modulators of cellular metabolism. The impact of various derivatives (like prostaglandins and leukotrienes) of arachidonic acid that may be liberated from phospholipids in the cell membrane by the action of phospholipases is well known. Recent reports indicate that unsaturated fatty acids interfere with the binding of some ligands to their receptors. Thus, fatty acids inhibit specific binding of angiotensin to receptors in adrenal glomerulosa and fasciculata cells.’ Unsaturated fatty acids also act as endogenous inhibitors of tamoxifen binding to antiestrogen binding sites.’ Additionally, it has been shown that modification of the phospholipid component of the nuclear membrane in vitro by phospholipase modulated the activity of Sa-reductase in a highly specific and structure-related manner.’ Unsaturated fatty acids have also been shown able to influence thyroid hormone physiology substantially. Thus, they inhibit the binding of T, to serum proteins4-’ and extrathyroidal conversion of T, to more potent T,.* In this study, we have questioned whether or not fatty acids affect the binding of T, to its specific nuclear receptors. NSATURATED
MATERIALS
AND METHODS
Male Sprague-Dawley rats were killed by cervical dislocation, and livers were excised and processed immediately thereafter at 0 to 4°C
From the Departments of Medicine. UCLA Centerfor the Health Sciences, Lxx Angeles, and Academisch Medisch Centrum, University of Amsterdam, The Netherlands. Supported by a travel grant from the Netherlands Organization for the advancement of Pure Research (ZWO) and by USPHS Grant No. AM-16155from the National institutes of Health. Address reprint requests to Inder J. Chopra, MD. Department of Medicine, UCLA Center for the Health Sciences, Los Angeles, California 90024. o 1988 by Grune & Stratton, inc. 0026-0495/88/371 O-001 6%03.00/O 996
for the preparation of isolated nuclei.’ In short, after homogenization of the liver in solution A (20 mmol/L tricine, 0.25 mol/L sucrose, 2 nimol/L CaCl,, 1 mmol/L MgCl,, and 5% (vol/vol) glycerol, pH 7.6) by a motor-driven Teflon pestle in a Potter-Elvehjem-type Wheaton tube, the nuclei were pelleted and washed two times in solution A containing 0.5% Triton X-100. Thereafter, the pellet was thoroughly suspended in solution A containing 2.4 mol/L sucrose, and the suspension was centrifuged for 45 minutes at 53,000 x g. The remaining pellet was washed again three times in solution A containing 0.5% Triton X-100, and finally suspended in 0.5 vol (wt/vol) solution B (20 mmol/L TRIS, 0.25 mol/L sucrose, 1 mmol/L EDTA, 50 mmol/L NaCl and 5% (vol/vol) glycerol, pH 7.6). The DNA concentration of the nuclear suspensions was determined according to the method of Webb and Levy,“’ and protein measurements were performed by the Bio-Rad assay”; the mean ratio of DNA to protein was 3.71 + 0.55. The binding of ‘*51-labeled T, (specific activity 1,200 pCi/rg, New England Nuclear, Boston) to isolated nuclei was studied by the addition of approximately 20 to 40 femtomoles [‘251]T, to 0.1 mL nuclear suspension (representing 200 mg of starting tissue) in the presence of 5 mmol/L dithiothreitol (DTT). The total incubation volume was 0.5 mL; all reagents were dissolved in solution B, unless stated otherwise. Incubations were for two hours in a shaking waterbath at 22OC. Incubations were stopped by chilling tubes in ice-cold water. Two milliliters of solution B containing 0.5% Triton X-100 was then added to each tube. After vortexing, the tubes were centrifuged at 4OCfor 15 minutes at 1,250 x g. The supernatant was aspirated, and the radioactivity in the pellet (ie, the hormonal function bound to the nuclei) was counted. Specific binding was 24.6% f 0.9%, calculated as the difference between the counts bound to the nuclei in the absence and in the presence of an excess of nonradioactive T, ( 10m6mol/L); the nonspecific binding was 2.4% k 0.1% of total radioactivity (mean * SE; n = 23). Scatchard plots’* were prepared from data on bound and free T, in the absence and in the presence of increasing quantities of nonradioactive T, ranging in concentration from 1.0 to 33 x lo-“’ M. All measurements were performed in duplicate. Studies
With Lipids
Various lipids were purchased from Sigma Chemical Co, St Louis. They were dissolved in ethanol, and concentrations for use in Metabolism, Vol 37, No 10
(October), 1988: pp 996-1002
EFFECT OF FATI-Y ACIDS ON NUCLEAR T, BINDING
_
the nuclear Ts binding assay were prepared in buffer B. Arachidonic acid (sodium salt) was in clear solution in aqueous media. Other fats were in fine suspension. The final concentration of ethanol in the assay (C 1%) did not affect nuclear T, binding.
unsaturated -
fatty acids
-‘K--__-~__---n__-__
Studies With Phospholipase A2
a-_ \ \
When the effect of phospholipase A2 (PLAI) was to be studied, rat liver homogenate was treated with PLA2 by a modification of the method of Roelofsen et al.” Highly purified PLA2 from the snake venom of Naja Mozambique Mozambique (SA 1,490 U/mL protein; Sigma Chemical Co, St. Louis) was dissolved in 0.1 mol/L TRIS buffer (pH 8.2) containing 0.075 mol/L calcium chloride. Five units of PLA, in 0.05 mL buffer were incubated with 0.25 mL rat liver homogenate (-15 mg protein) for 0 to 20 minutes at 22’Y in a shaking water bath, and the reaction was stopped by the addition of 0.15 mL 0.25 mol/L EDTA. Control tubes contained 0.05 mL buffer without PLA, and 0.25 mL homogenate, and were treated identically. Preparation of ether extracts was as follows: Two volumes anhydrous diethyl ether (0.9 mL) were added to each tube, and the mixture was swirled on a vortex mixer for 30 seconds and centrifuged at 1,000 x g for ten minutes at 4°C. The tubes were put on dry ice, and the ether phase was decanted into separate tubes. The ether was evaporated to dryness at room temperature under a stream of nitrogen, and the residue was suspended in 10 mL of buffer B; a measured volume (0.025 mL) was assayed for nuclear T, bindinginhibiting activity.
- saturated
\
. linolanicacid P arachidonic acid 0 oleic acid l palmitoleicacid l finofeicacid
Ao
1
0.011
o arachidic acid G stearic acid l palmitic acid - myristic acid
.._
0.033
0.1
0.3
mf4
cholesterol and mono-, di-, triglycerides E *
1
120.
8
4
0
Y
‘
Studies With Human Sera When sera were to be tested for inhibition of nuclear T, binding (INB), ether extracts were prepared using 4 vol diethyl ether for each volume of serum. The mixture was swirled on a vortex mixer for 30 seconds and put on dry ice. After freezing of the serum phase the supernatant ether phase was decanted into another tube and evaporated to dryness at room temperature under a stream of nitrogen. The residue was suspended in solution B of the nuclear Ts binding assay. After initial studies with pooled sera from hospitalized patients (stored for two days at 4°C and subsequently frozen at - 20°C), individual serum samples were prospectively obtained from nine normal subjects (five men and four women, 18 to 57 years of age) and 22 patients (12 men and ten women, 17 to 87 years of age); patients were admitted to intensive care units with a variety of systemic illnesses. Individual serum samples were frozen within 30 to 40 minutes after collection, and stored at -20°C until assay. The major disorders included postsurgical state (radical cystectomy, liver resection, excision of cerebellar tumor, aneurysmectomy, tricusip valve replacement, hysterectomy; seven patients), infectious diseases (herpes encephalitis, sepsis, pneumonia; six patients), comatose state (multiple injuries, subdural hematoma, unknown cause; three patients), cardiac disease (ventricular septum rupture, congestive heart failure; two patients), pulmonary disease (respiratory insufficiency; two patients), renal disease (renal failure; one patient), and gastrointestinal disease (pancreatic abscess with fistula; one patient). Serum T, and T, were determined by specific radioimmunoassays,‘4~‘5 and TSH by immunoradiometric assay.16 Thyroid hormone-binding inhibitor (THBI) was measured in diethyl ether extracts of sera by a competitive ligand-binding assay as described previously.” RESULTS
Lipids as Inhibitors of Nuclear T3 Binding The effects of various lipids on nuclear Ts binding in vitro was studied. Nonspecific binding (mean + SE) of [‘*‘I]T, in the presence of lipids was 2.1% + O.l%, not different from
\
>
cholesterol triofain tripalmitin dilaurin dilonolain l monolinolein . monofinolenin 0 monolaurin
4 b 0 v
B
01
h.
0.011
0.033
0.1
-&3
rnM
Fig 1. Effects of various unsaturated and saturated fatty acids (A) and of cholesterol and various mono-, di-. and triglycerides (B) on the binding of [“‘I] T, to isolated rat liver nuclei. Nuclear T, binding in the absence of lipids (control) was arbitrarily assigned a value of 100. and nuclear T, binding in the presence of lipids was expressed as a percentage of the control value.
that of 2.2% f 0.1% in the absence of lipids. Nuclear T, binding was strongly inhibited by all unsaturated fatty acids so studied, and moderately inihibited by some saturated fatty acids (Fig 1A). The lipid concentrations (pmol/L) that caused a 50% inhibition of nuclear TS binding were 10 for palmitoleic acid, 11 for linoleic acid, 22 for oleic acid, 24 for arachidonic acid, 37 for linolenic acid, 60 for myristic acid, 300 for palmitic acid, and >l,OOO for stearic acid and arachidonic acid (the lipid concentration in Gmol/L divided by 2,000 will give the amount of lipid present in the incubation mixture, in terms of micromoles/tube). The mean concentration of the unsaturated fatty acids required for a 50% inhibition was 20 pmol/L, equivalent to 0.01 micromoles per tube. The monoglyceride monolinolein had no inhibitory effect on nuclear T, binding, but monolaurin and
WIERSINGA, CHOPRA, AND CHUA TECO
monolinolenin had a moderate inhibitory activity (50% inhibition at 85 and 390 pmol/L, respectively) (Fig 1B). Diglycerides (dilaurin, dilinolein) and triglycerides (tripalmitin, triolein) in concentrations up to 300 Kmol/L and cholesterol in concentrations up to 1 mmol/L had no effect on nuclear T, binding. To examine the nature of inhibition of nuclear T, binding by lipids, we performed Scatchard analysis of the binding of [‘*‘I] T, to isolated rat liver nuclei in the absence and the presence of unsaturated fatty acids (at concentrations that caused 50% inhibition of nuclear T, binding) (Fig 2). Maximal binding capacity was not affected, but the affinity constant decreased: K, values in the presence of unsaturated fatty acids were 57.4% k 11.O% of K, values in the control experiments (P < .Ol). Since fatty acids possess detergent-like properties, we examined the effects of some detergents on nuclear T, binding. Sodium cholate and CHAPS (3-[(3-cholamidopropyl) dimethylammonio] I-propanesulfonate) had no appreciable inhibitory effect on nuclear T, binding in concentrations up to 1 mmol/L. The potent detergent sodium deoxycholate caused a 50% inhibition of nuclear T, binding at a concentration of 0.33 mmol/L, a l&fold higher dose than that of 0.02 mmol/L for unsaturated fatty acids. It is conceivable that fatty acids inhibit nuclear T, binding not by interference with the binding sites on the nuclear T, receptor, but by an interaction with T, that would leave less T, available for nuclear binding. To explore this possibility, we preincubated 0.5 mL [“‘I] T, in solution B with either 0.5 B/F
mL 0.35 mmol/L linolenic acid in solution B (tube 1) or with 0.5 mL solution B (tube 2, control) at room temperature for 30 minutes. Four volumes of ether (4 mL) were then added to each tube, and the tubes were swirled on a vortex mixer for 30 seconds and centrifuged at 1,000 x g for ten minutes at 4OC. The aqueous and ether phases were separated. In one study, 50 PL of the aqueous phase was substituted for [lz51] T, in the nuclear T, binding assay; specifically bound T, was 31.2% using the aqueous phase of tube 1, not different from that of 30.3% for control tube 2. In another study, 400 rL of the ether phase was evaporated to dryness, and the dissolved residue was assayed in the nuclear T, binding assay; specifically bound T, was 8.2% in the presence of ether extract of tube 1, which contained fatty acid, compared with that of 29.3% in the presence of the extract of control tube 2. Eflect of PLA, Treatment of Tissue The effect of PLA, treatment was studied to determine whether fatty acids liberated as a result of activation of tissue PLA, might affect nuclear T, binding. After treatment of rat liver homogenate with PLA, for 5, 10, 15, or 20 minutes, the nuclear T, binding in the presence of evaporated ether extracts of the liver homogenate decreased progressively (P < .Ol); all these values were more than 2 SD below the mean of control incubations (Fig 3). At 20 minutes, the nuclear T, binding-inhibiting activity of the PLA,-treated liver homogenate was 62% that of the control mixture. PLA, treatment of tissue phospholipids results in genera-
0.35
1 effect of phospholipase
4
%
treatment
. contmt n unsaturated
fatty
acids
1100
1
80
60 10 0 control l
PLA*
5 Scatchard plots of the binding of [“g T, to isolated rat Fig 2. liver nuclei in the absence (control) and in the presence of unsaturated fatty acids (at concentrations that caused approximately 50% inhibition of nuclear T, binding: palmitoleic acid 0.01 mmol/L, linoleic acid 0.01 mmol/L, oleic acid 0.02 mmol/L. araohidonic acid 0.026 mmol/L and linolenic acid O.OS6 mmol/Ll. Each point represents the mean of five experiments; each of the experiments included a Scatchard plot without lipids and one plot with a particular unsaturated fatty acid. Statistical analyses of changes in maximal binding capacity (MBC) and affinity constant (K,) (values as mean f SE) were made by the paired t test. MBC: control 1.56 t 0.10, unsaturated fatty acids 1.47 -t 0.20, lo-” M/L; NS. K,: control 1.97 f 0.23, unsaturated fatty acids 1.04 f 0.14, 10’ L/M; P < .025.
1
0
I
5
10
15
I
20 time ( min
1
Effect of PLA, treatment. Rat liver homogenate was Fig 3. incubated with PLA,, and the reaction was stopped by addition of EDTA. Evaporated ether extracts of incubation mixtures were assayed for inhibiting activity on the binding of [“v] T, to isolated rat liver nuclei. Each result is the mean of duplicate determinations. Nuclear T, binding in the PLA,-treated incubation mixtures was lower than in controls as indicated by one-way ANOVA IF- 16.1:Pc .Ol).
EFFECT OF FAll-Y ACIDS ON NUCLEAR T, BINDING
999
tion of lysolecithin (L-a-phospatidylcholine) in addition to fatty acids. Some of the liberated fatty acids are amenable to oxidation and may have been converted in part to prostaglandins. Therefore, we tested the effect of phospholipids and prostaglandins on nuclear T, binding. Phosphatidylcholine (lecithin) and phosphatidylcholine dioleyl in concentrations up to 300 rmol/L had no nuclear T, binding-inhibiting activity. Lysophosphatidylcholine (lysolecithin) and lysophosphatidylcholine oleyl had some effect: 50% inhibition of nuclear T, binding was observed at concentrations of 840 pmol/L and 740 pmol/L, respectively, a 40-fold higher dose than that of 20 pmol/L for unsaturated fatty acids. The naturally occurring prostaglandins E,, E, and F,, had no nuclear T, binding-inhibiting activity in concentrations up to 1 mmol/L; 50% inhibition of nuclear T, binding was caused by 1.25 mmol/L prostaglandin A,. Studies
With Human Serum Samples
Figure 4 compares the dose-response curves for the effect of ether extracts of three sera of normal subjects and three pools of sera of nonthyroidal illness (NTI) patients on the binding of T, to isolated rat liver nuclei. It is clear that the
o normal subjects l
2 A! s
nonthyroidal illness patients
60
C
:
1 I
0
, 0.05
, 0.1
‘Y\,
0.3 ml-equivalent
IO* serum
Fig 4. EHsct of evaporated ether extracts of tern of normal subjects fn = 3) and pooled sera of hospitalized patients with NTI (n = 3) on T, binding by rat liver nuclei. Nuclear T, binding in the absence of serum extraot (control) was arbitrarily assigned a value of 100, and nudear T, binding in the presence of serum extract was expressed as a percentage of the control value. The data are expressed as the mean + SE and statistical analyses indicated were made using Student’s t tests. tP < .Ol: lP < .005; .*P < 9025 v normal subjects. Nuclear T, binding in the presence of serum axtracts of patients was also significantly different from that of normal subjects when data were analyzed by one-way ANOVA IF = 10.9: P < .Ol j.
extracts of sera of NT1 patients had greater nuclear T, binding-inhibiting activity than the extracts of sera of normal subjects. We next sought nuclear T, binding-inhibiting activity in nine sera of normal subjects and in 22 sera of NT1 patients. For convenience, we used an ether extract of 0.1 mL serum in each case. Nonspecific binding in the presence of evaporated ether extracts of human sera (2.5% 5 0.1%) was not different from that of controls without serum extracts. The mean specific nuclear T, binding in the presence of extracts of sera of nine normal subjects was assigned a value of 100, and the results in all normal subjects and patients were expressed as a percentage of this. Nuclear T, binding in the presence of extracts of sera from NT1 patients ranged between 17% and 107% of the mean control value. The mean + SE value of 58.9% + 4.9% in the 22 NT1 patients was significantly (P-C .OOOS)lower than that of 100% + 6.2% in the nine normal subjects. Nuclear T, binding in the presence of extracts of patients’ sera was more than 2 SD below the normal mean in 12 of 22 (55%) NT1 patients. The mean serum (*SE) T, concentration of 32 * 9 ng/dL (cf normal value of 106 + 8) in these 12 patients did not differ from that of 27 2 6 ng/dL in the other 10 patients; serum T, (4.6 t 1.0 v 4.3 * 1.0 rg/dL) and serum TSH (1.9 r 0.6 v 1.2 + 0.5 U/mL) were also similar. Since THBI, like nuclear T, binding-inhibiting activity, is extractable from serum by ether treatment, we studied the relationship between the two activities in the abovementioned sera. Sixteen of the 22 individual sera of NT1 patients were THBI positive (THBI value > 1.60”). and six were THBI-negative. The mean nuclear T, binding in the presence of extracts of THBI-positive sera was 48% + 3.7% that of 100% + 6.2% in the presence of extracts of normal sera (P < .OOOS). The mean nuclear T, binding in the presence of extracts of THBI-negative sera of 86.7% + 7.6% was not significantly different from the corresponding value in normal subjects (Fig 5). Five patients were receiving low-dose heparin administered subcutaneously; all had THBI-positive sera. However, THBI and INB values in these five patients were not different from those in the other 11 THBI-positive patients who did not receive any heparin (THBI, 2.80 k 0.30 v 2.71 i 0.35, NS; INB, 44.6% + 5.3% v 50.8% 2 4.9%, NS). Serum activities of THBI and the INB were correlated highly significantly (y = 95.4 x -“.76, r = .89, P -z .0005; the closest agreement with the experimental data was found using a program that fits a power curve y = axb to a set of data points {(xi, y,), i = 1, 2, . . n)). To examine the nature of inhibition of nuclear T, binding by human serum extracts, we performed Scatchard analysis of the binding of [“‘I] T, to isolated rat liver nuclei in the absence and in the presence of ether extracts (Fig 6). Maximal binding capacity was not affected, but the affinity constant decreased: K, values in the presence of evaporated ether extracts of NT1 sera were 47.5% f 10.8% of K, values obtained in the presence of ether extracts of normal sera (P < .Ol). Reproducibility
The reproducibility of the assay system for nuclear T, binding-inhibiting activity in human sera was tested by
WIERSINGA. CHOPRA, AND CHUA TECO
S/F
N.S.
$~D.ooo5 , l
p
g
F 2 3
1
~O.ooo5
I
6
o.35
I
1
120 :
.I
l
100
-i-l
P
z
P
2
bound
T, ( IO-*M/L
I
80 : 00
60
l l
l 0
+
Fig 8. Scatchard plots of the binding [“‘I] T, to isolated rat liver nuclei in the absence of serum (control) and in the presence of evaporated ether extracts of sera of normal subjects (nuclear T, binding, 96.9% + 16.4%) and of patients with NTI lnuclear T, binding, 47.1% -c 9.2%). Each point represents the mean of four experiments. Statistical analyses of changes in MBC and K, were made by the paired t test leech of the four experiments included a Scatchard plot without serum, with normal serum, and with NTI serum: values are given as mean + SE).
t
MBC (10~” M/Lj Control
40
K, (10’ L/Ml 2.88 + 0.34
1.26 f 0.25 NS
l Normal sera 1.44 f 0.33
l
NB
PC 1.46 +
0.36
NS
:
NTI sers
20
1.71 f 0.54
.06 PC P < .Ol
a05
0.72 f 0.29
variation was 7.26% (73.5% k 5.3%) 9.17 (68.9% r 6.3%), 8.91% (29.5% + 2.6%), and 3.04% (15.0% + 0.5%). Role of Assay Conditions in the Activity of INB
0 normal subjects
(9)
THBl+ve NTI-pat. (16)
THBI-ve NTI-pat. (6)
Fig 5. Nuclear T, binding-inhibiting activity in ether axtracts of sera of NTI patients and normal subjects. The mean binding of “‘11 T, during incubation of isolated rat liver nuclei with evapoI rated ether extracts of sera of normal subjects was arbitrarily assigned a control value of 100, and the results in all normal subjects and NTI patients were expressed as a percentage of the mean control value.
assaying each of four serum samples five times in one assay and also in duplicates in five consecutive assays. Intra-assay coefficient of variation (SD/mean x 100%) was (mean + SD of nuclear T, binding in parantheses) 5.04% (70.2% + 3.5%), 4.06% (35.8% + l.S%), 9.04% (32.3% f 2.9%), and 10.42% (15.6% + 1.6%); inter-assay
The nuclear T, binding-inhibiting activity of human sera was demonstrable both in the presence and the absence of DTT. The ratio of specifically bound [‘*‘I] T, in the absence of DTT to that in the presence of DTT was 0.45 (12.8%/ 28.2% without serum extract), 0.40 (7.9%/19.8% in the presence of ether extract of normal serum), and 0.33 (1.5%/ 4.4% in the presence of extract of NT1 serum). In other studies, we examined the effects of ether extracts of human sera on the release of T, receptors from the nuclear pellet into the supernatant during the incubation period.18 The incubation mixture at the end of the incubation period was centrifuged and the supernatant was aspirated and passed through a small Sephadex G-25 (medium) column prepared in a Pasteur pipette; the radioactivity in five 0.8-mL fractions was counted using solution B as the eluant. Radioactivity in the remaining nuclear pellet was counted after washing with 2 mL of solution B containing 0.5% Triton X-100 as described above. The ratio of specifically bound [‘25I] T, in the pellet to that in the supernatant was 1.49 (no serum present), 1.49 (normal serum), and 1.91 (NT1 serum). These data provided no support for the possi-
EFFECT OF FAllY
ACIDS ON NUCLEAR T, BINDING
bility that serum extracts inhibit binding of ‘251-T~to nuclei by causing a release of T, nuclear receptors in the medium.
DISCUSSION
In the present study, we have demonstrated that several lipids, especially unsaturated fatty acids, are potent inhibitors of nuclear T, binding in vitro. The decreased nuclear T, binding was caused by a reduction in the affinity of the nuclear receptors for T,. The mechanism by which unsaturated fatty acids inhibit nuclear T, binding remains unclear, but several possibilities may be considered. First, fatty acids may affect the T, binding site by altering its hydrophobic environment; fatty acids have been shown to alter the fluidity and the surface charges of cell membranes.‘9-2’ Second, the inhibitory effect of fatty acids on nuclear T, binding may be due to oxidation products that may compromise the stimulatory effect of DTT in the nuclear T, binding assay.** However, this appears unlikely because of the following: (1) Inhibition of nuclear T, binding is demonstrable equally well in the presence as in the absence of DTT. (2) Some fatty acids poorly amenable to oxidation, eg, myristic acid and oleic acid, are potent INBs. (3) Some products of fatty acid oxidation, eg, prostaglandins, had no or very weak INB activity. Third, it is conceivable that the detergent-like action of fatty acids contributes importantly to their inhibitory effect on nuclear T, binding. This, too, appears unlikely because potent detergents like CHAPS, sodium cholate, and deoxycholate had either no or weak INB activity; and the binding of T, to receptors remaining in the nuclear pellet was depressed to a similar extent as the binding of T, to receptors released into the supernatant. Additionally, our studies provided no support for the possibility that the inhibitory effect of fatty acids on nuclear T, binding was a result of release of receptors from the nucleus to the supernatant of the incubation medium, as would be expected if fatty acids were potent detergents in the low concentrations present in incubation medium. Fourth, it is possible that fatty acids reduce nuclear T, binding by their interaction with T, in a manner that leaves less T, available for receptor binding. However, our studies on preincubation of [‘*‘I] T, with fatty acids did not support this possibility. Therefore, we presently consider an allosteric hindrance of the binding of T, to its nuclear receptor by fatty acids as a likely mechanism of their action as INBs. A similar mechanism of action of fatty acids has been suggested previously for their inhibition of hepatic T,-5’-monodeiodinase,8 T, binding to serum proteins,” and T, binding to anti-T,-antibodies. ‘*” Interestingly, the inhibitory effect of unsaturated fatty acids is greater in the case of hydrophobic binding sites, eg, nuclear T, receptor, T, 5’monodeiodinase than in the case of hydrophilic binding sites of T,-binding T, antiserum, This is illustrated in an interesting manner by the concentration of oleic acid required for a 50% inhibition of 0.022 mmol/L for nuclear T, binding (vide supra); 0.8 mmol/L for T, 5’-monodeiodination,* and 1.32 mmol/L for T, binding by anti-T, antiserum.4 A similar pattern is observed with the compound 8-anilino-l-naphthalene sulphonic acid (ANS), which interferes strongly with the binding of thyroid hormones to the hydrophobic binding
1001
sites on serum proteins23 and with nuclear T, binding,24 but only weakly with the binding to thyroid hormone-binding antibodies.14 A direct comparison between the concentrations of oleic acid or ANS in various assays may not be entirely appropriate, however, because of differences in various assay conditions, especially concentration of proteins in the incubation mixture. We had studied the effect of PLA, on tissue INB activity and serum INB activity in a hope to gather some insight into potential in vivo application of INB. Activation of PLA, results in the liberation of unsaturated fatty acids, located mainly at the no. 2 position of membrane phospholipids. Incubation with PLA, increased the INB activity in rat liver homogenates significantly. Since effective stimuli for activation of PLA, (like catecholamines, bradykinin, angiotensin II, vasopressin, thrombin, collagen, chemotactic peptides, immunoglobulin, complement CSa and histamine)25-27 are present in many circumstances of an NTI, it is conceivable that activation of phospholipases of the A, type occurs during NTI. This might result in the release of some liberated fatty acids into the circulation, which together with the lipolysis of depot fat frequently causes an increase in serum free fatty acids in NTL4 Our studies have shown that, relative to serum samples of normal subjects, many NT1 sera demonstrate significant INB activity. The nature of INB in sera of NT1 patients is unclear. However, available data suggest that it is a lipid, more specifically, unsaturated fatty acids. Thus, we find that: (1) INB is extracted from serum by lipid solvents like diethyl ether; (2) unsaturated fatty acids are the most potent INBs of all lipids, so studied; (3) NT1 sera and unsaturated fatty acids both inhibit nuclear T, binding by affecting a decrease in the affinity constant of the binding interaction; (4) serum concentrations of unsaturated fatty acids are frequently increased in NT14; and (5) the serum concentration of INB is correlated highly signficantly with that of THBI, a lipidic substance detected in sera of many NT1 patients4 While it is possible that circulating INB in human serum affects the binding of T, to its nuclear receptors in vivo, our studies have not addressed this possibility directly. Similarly, it is conceivable that unsaturated fatty acids liberated in the plasma membrane are transported to the nucleus or, alternatively, that phospholipases are present in the nuclear membrane and are activated during an illness. A recent study has indeed demonstrated the presence of PLA, activity in nuclear membranes of rat liver.*’ It seems important to carefully examine the various above-mentioned issues in future studies to evaluate the importance of fatty acids in modulation of nuclear-bound T, and thereby, thyroid hormone effects in the tissues. Previous studies in experimental animals with various forms of NT1 have demonstrated, in general, a reduced binding capacity of hepatic nuclear T, receptors without changes in the binding affinity.22129-36 These findings do not necessarily argue against an involvement of unsaturated fatty acids in alterations of nuclear T, binding in NTI, since the studies were conducted in vitro to isolated nuclei and since fatty acids exerting allosteric hindrance of the binding of T, are likely to be removed in the experimental procedure requiring purification of nuclei by repeated washes with
1002
WIERSINGA,
detergents such as Triton X-100. Moreover, the effect of prolonged exposure to high concentrations of unsaturated fatty acids on nuclear binding of T, is not known at this time. It would be interesting to examine this and other abovementioned issues in future studies of the role of unsaturated fatty acids in altered thyroid hormone economy in NTI.
CHOPRA, AND CHUA TECO
ACKNOWLEDGMENT
We appreciate the helpful advice and comments of Dr David H. Solomon and Dr Ruben Boado, the secretarial assistance of Martha Komende Vries and Judy Sirand, the technical help of Julie Smick, and the support of Dr Hans Ramijn in the collection of patients’ samples.
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19. Ahrens ML: Electrostatic control by lipids upon the membrane bound (Na+-K+) ATP ase. Biochim Biophys Acta 642:252266, 1981 20. Schmalizing G, Kutschera P: Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A,. J Membr Biol69:65-76, 1982 21. Wojtczak L, Nalecz M: Surface charge of biological membranes as a possible regulator of membrane-bound enzymes. Eur J Biochem 94:99-107, 1979 22. Wiersinga WM, Frank HJL, Chopra IJ, et al: Alterations in hepatic nuclear binding of triiodothyronine in experimental diabetes mellitus in rats. Acta Endocrinol99:79-85, 1982 23. Wartofsky L, Burman KD: Alterations in thyroid function with systemic illness: The “euthyroid sick syndrome.” Endocr Rev 3:164-217, 1983 24. Burman KD, Lukes UG, Latham KR, et al: Ipodate and %anilino-1-naphthalene sulfonc acid block receptor binding of T, in rat liver. Horm Metab Res 12:685-687, 1980 25. Lapetina EG: Regulation of arachidonic acid production: Role of phospholipase C and A,. Trends Pharm Sci 3:115-118, 1982 26. Nishizuka Y: Phospholipid degradation and signal translation for protein phosphorylation. Trends Biochem Sci 8: 13- 16, 1983 27. Abdel-Latif AA: Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers. Pharmacol Rev 38:227-272, 1986 28. Karagezyan KG, Tadevosyan Yu V: Enzyme systems of phospholipid metabolism in the nuclear membranes of albino rat livers. Dokl Akad Nauk SSSR Proc 287: 1248- 1250,1986 29. DeGroot LJ, Coleoni AH, Rue PA, et al: Reduced nuclear triiodothyronine receptors in starvation-induced hypothyroidism. Biochem Biophys Res Commun 79:173-178,1977 30. Burman KD, Lukes Y, Wright FD, et al: Reduction in hepatic triiodothyronine binding capacity induced by fasting. Endocrinology 101:1331-1334,1977 3 1. Schussler GC, Orlando J: Fasting decreases triiodothyronine receptor capacity. Science 199:686-688, 1978 32. Surks MI, Grajower MM, Tai M, et al: Decreased nuclear L-triiodothyronine receptors in rat and mice bearing transplantable neoplasms. Endocrinology 103:2234-2239, 1978 33. Das DK, Ganguly M: Diabetes, hypophysectomy, or thyroidectomy reduces nuclear L-triiodothyronine-binding capacity of rat lung. Endocrinology 109:296-300, 1981 34. Lim VS, Passo C, Murata Y, et al: Reduced triiodothyronine content in liver but not pituitary of the uremic rat model: Demonstration of changes compatible with thyroid hormone deficiency in liver only. Endocrinology 114:280-286, 1984 35. Little JS: The effect of Streptococcus pneumoniue infection on the binding of triiodothyronine to nuclei isolated from rat liver. Endocrinology 117: 180- 186, 1985 36. Nakamura H, Yokota T, Imura H: Different alterations of nuclear triiodothyronine receptor capacity in liver and kidney induced by starvation and triiodothyronine administration. Acta Endocrinol 108:206-210, 1985