unesterified long-chain fatty acids inhibit thyroid hormone binding to the nuclear receptor :...

Eur. J. Biochem. 183, 565-572 (1989) 0 FEBS 1989

Unesterified long-chain fatty acids inhibit thyroid hormone binding to the nuclear receptor Solubilized receptor and the receptor in cultured cells

Akira INOUE, Naoki YAMAMOTO, Yuji MORISAWA, Toshiko UCHIMOTO, Munehiko YUKIOKA and Seiji MORISAWA Department of Biochemistry, Osaka City University Medical School

(Received February 6/April 10, 1989) - EJB 89 0138

Unesterified long-chain fatty acids strongly inhibited thyroid hormone (T3) binding to nuclear receptors extracted from rat liver, kidney, spleen, brain, testis and heart. Oleic acid was the most potent inhibitor, attaining 50% inhibition at 2.8 pM. Oleic acid similarly inhibited the partially purified receptor and enhanced dissociation of the preformed T3-receptor complex. The fatty acid acted in a soluble form and in a competitive manner for the T,-binding sites, thereby reducing the affinity of the receptor for T,. The affinity of the receptor for oleic acid (Ki) was 1.0 pM. In HTC rat hepatoma cells in culture, fatty acids added to the medium reached the nucleus and inhibited nuclear T3 binding; oleic acid being the most potent. T3 binding of the cells was reversibly restored in fresh medium free of added fatty acids. Oleic acid did not affect all the T3-binding sites in the HTC cells: one form (80%) was inhibited and the other was not and these two forms were commonly present in all rat tissues examined. Thus, fatty acids inhibited the solubilized nuclear receptor as well as a class of nuclear T3-binding sites in cells in culture.

Specific nuclear receptors for thyroid hormone (triiodothyronine; T3) exist in association with chromatin and partici- pate in the regulation of transcription [l]. T,-responsive el- ements have been identified in the 5’4anking regions of rat and human growth hormone genes [2 - 51. Other examples are the induction by T3 of the or-myosin heavy-chain gene [6, 71, and repression of thyrotropin a- and p-chain genes [8]. The hormone acts also at post-transcriptional steps [9 - 111, such as in the stabilization of nuclear mRNA precursors [12]. In addition to the chromatin-associated receptor, other T3-binding sites are present in the soluble cytoplasm, plasma membrane, mitochondria, endoplasmic reticulum and nuclear envelope [33, 141. The chromatin receptor has been separated from other T3 binders, essentially by isolation of nuclei, fol- lowed by repeated washing in a solution containing a detergent such as 3-[3-(chloramidopropy1)-dimethylammonio]-1- propane sulfate (Chaps) or Triton X-100, to remove cyto- plasmic contaminants and nuclear-envelope-associated binders [14-171.

In early work we found that when the solubilized nuclear T3 receptor was treated with a lipase, the T,-binding activity of the receptor was significantly reduced, These events proved to be linked to fatty acids, presumably generated by hydrolysis of lipids present in the solubilized receptor preparation (unpublished observation).

In the present study, we show that unesterified long-chain fatty acids inhibited T3 binding by the nuclear receptor

Correspondence to A. Inoue, Department of Biochemistry, Osaka City University Medical School, 1-4-54 Asahi-machi, Abeno-ku, Osaka-shi, Osaka-fu, Japan 545

Abbreviations. T3, 3,5,3’-triiodothyroninc; buffer A, 0.25 M sucrosc/2 mM MgC12/20 mM Tris/HCl, pH 7.6; buffer T, 50 mM NaC1/10% (by vol.) glycerol/2 mM EDTA/0.2 mM dithiothreitol/ 20 mM Tris/HCl, pH 8.2; HTC cells, Morris rat hepatoma 7288C cells.

solubilized from the chromatin of various rat organs, and also T3 binding in HTC rat hepatoma cells in culture. There were two forms of binding sites, in terms of sensitivity to inhibition in the cultured cells as well as in the isolated nuclei: one was inhibited by fatty acids and designated here as the responder, and the other was not inhibited and was designated the nonresponder. During preparation of this manuscript, inhibition of T3 binding to isolated nuclei by fatty acids was reported by Wiersinga et al. [18].

EXPERIMENTAL PROCEDURES

MuteriuO

[‘25T]Thyroid hormone (~-3,5,3’-triiodothyronine, 3000 Ci/ g) and [’H]oleic acid (9,lO-[,H], 8.9 mCi/rnol) were purchased from New England Nuclear. The radioimmunoassay kit for T3 (T3 RIA beads) was supplied by Dinabot. Boron trifluoride methanol complex (14%) and unlabelled fatty acids were purchased from Wako Pure Chemicals; monoolein, diolein, triolein, and phosphatidylserine were from Sigma. Analytical grade anion-exchange resin AG 1x8, 200-400 mesh (chlo- ride form) was from Bio-Rad Laboratories, and nitrocellulose membranes type HAWP 02500 were from Millipore Corpora- tion.

Concentrated fatty ucid solutions

Unsaturated fatty acids (30-25 mM) were suspended in H 2 0 by sonication. Saturated fatty acids were sonicated in H 2 0 in the presence of equimolar KOH. Glycerol esters of oleic acid were dissolved in CHCI3; the solvent was evaporated under a stream of nitrogen, and the lipids were sonicated in H,O at a concentration of 10 mM oleate. Phosphatidyl- serine in CHCl,/methanol (95 : 5) was similarly treated.

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Extraction of T3 receptor

Rat liver was homogenized in cold 0.25 M sucrose/5 mM MgCI2/20 mM Tris/HCl, pH 7.6, and centrifuged as described in [19]. The pelleted crude nuclei were washed three times in 0.25 M sucrose/2 mM MgCI2/20 mM Tris/HCl, pH 7.6 (buffer A) containing 0.5% (by vol.) Triton X-100, and once in buffer A, including filtration through cheesecloth at the third washing. The nuclear pellet was mixed with 0.4 M KCljl. 1 mM MgCI2/0.2 mM dithiothreitol/20 mM Tris/HCl, pH 7.9, left to stand for 20 min and centrifuged at 30000 x g for 20 min. The supernatant (1.3 mg protein/ml) was used as a nuclear receptor preparation.

Assay f o r solubilized receptor

Receptor extract (50 pl, 65 pg protein) and [lZ5I]T3 0.1 nM) were incubated at 0°C for the indicated time with various concentrations of fatty acids in a final volume of 0.5 ml in 50 mM NaCI/10% (by vol.) glycerol/2 mM EDTA/ 0.2 mM dithiothreitol/20 mM Tris/HCl, pH 8.2 (buffer T). Protein-bound [lZ5I]T3 was determined by filtration of the mixtures on nitrocellulose membranes, under suction at 2 "C. The procedure was described in detail under Standard assay procedure of membrane filtration for thyroid hormone receptors in [19]. Alternatively, bound ['251]T3 was determined in a similar manner to that described in [20]: the reaction mixture was vortexed with AG 1x8 resin (24 mg) suspension in cold buffer T (1.0 ml) for 4 s, and left in an ice bath for 4 min. After repeating the suspension/standing cycle five times, the mixture was centrifuged at 1500 x g for 4 min. A portion of the supernatant was counted for radioactivity. Data were corrected for non-specific binding, determined in the presence of a 500-fold molar excess of unlabelled hormone. Each determination was carried out in duplicate, to give a mean * range.

Thyroid-hormone-binding in cultured HTC cells

HTC (Morris rat hepatoma 7288C) cells obtained from Dainippon Pharmaceutical co. were grown in Eagle's minimum essential medium containing 7% calf serum at 37°C in an atmosphere of 95% air/5% C02. At confluency, the cells were incubated for the indicated time with [1251]T3 (0.2 - 0.5 nM) in the presence or absence of fatty acids. The cells were chilled on ice, rinsed with cold buffer A, and homogenized in 3 mM MgC12/buffer A in a glass/Teflon homogenizer. The nuclei, pelleted by centrifugation, were washed three times with 0.5% (by vol.) Triton X-lOO/buffer A by vortex/suspension and centrifugation. The final nuclear pellet was dissolved in 0.25 M NaOH (2 ml), sonicated briefly, and 12'1 radioactivity and A260 were measured. Data were corrected for nonspecific T3 binding, determined with a 50000-fold molar excess of unlabelled T3. All determinations were performed in duplicate. Since the same results were obtained with nuclei purified by centrifugation through a 2.3 M sucrose solution (see below), this step was omitted from the routine assays. Incubation with 0.4 mM oleic acid for 24 h produced no detectable change in cell viability.

Isolation ojnuclei f rom rat tissues

Purified nuclei were prepared from tissues of various rat organs, as described in [21], by centrifugation at 50000 x g for 90 min through 2.3 M sucrose/3 mM MgCI2/0.2 mM

phenylmethylsulfonyl fluoride, in 4.5 (brain), 7 (heart), 8 (spleen and testis), and 9 (liver and kidney) final tissue vol- umes. The pelleted nuclei were washed three times in buffer A and used for the T3-binding assay, or for solubilization of T3- receptor protein. The amount of ['251]T3 bound to the nuclei was determined after the nuclei had been washed with 0.5% Triton X-lOO/buffer A as above. The nuclear content was estimated from the A260 in 0.25 M NaOH, assuming 1 mg DNA to have 30 A260 units.

Determination of thyroid hormone in HTC cells

HTC cells were incubated with T3 (0.2 nM) for 1 h, and then with oleic acid (0.5 mM) for an additional 1 h. The cells were rinsed thoroughly and homogenized in 10 mM NaCIj 3 mM MgCI2/10 mM Tris/HCl, pH 7.4, in a glass/Teflon homogenizer. The homogenate was mixed with 2 vol. ethanol, left on ice for 20 min and centrifuged. Under these conditions, 91 - 93% T3 in the homogenate was recovered in the ethanolic supernatant, which was subsequently evaporated to dryness. The residues were suspended in H20, centrifuged to remove insoluble material, and assayed for T3 using a radioimmunoassay kit.

Determination of oleic acid in HTC cell nuclei

HTC cells, incubated with oleic acid, were rinsed and homogenized in buffer A. The pelleted nuclei were purified through 1.8 M sucrose/3 mM MgCI2/0.2 mM phenylmethylsulfonyl fluoride as above, suspended in H 2 0 by brief sonication, and made 4% Na2C03 and 0.15 M NaCl with concentrated stock solutions. Fatty acids were extracted three times with ethyl ether. Isolation and esterification of fatty acids with 14% (mass/vol.) BF3 in methanol were carried out as described in [22]. The final acyl esters were dissolved in hexane/CHCl, (2:1), and analyzed using a Hitachi gas chromatography apparatus, model 263-50, equipped with an OV-1 column.

RESULTS

Inhibition of the solubilized receptor by fa t ty acids

T3 binding by the solubilized nuclear thyroid hormone receptor was inhibited by various saturated long-chain fatty acids; myristic acid being the most potent. With deviations in the number of carbon atoms from 14, the inhibitory potency of fatty acids progressively decreased (Table 1).

Unsaturated fatty acids (c16:1, Cls : l , C18:2, ClSz3 , Cz0 : 4 ) exhibited higher inhibitory activities than did the saturated fatty acids, attaining 50% inhibition at 4.5 pM, 2.8 pM, 3.5 pM, 4.2 pM and 3.6 pM, respectively. Oleic acid (Cis: 1)

was the most potent; it inhibited the receptor by 84% and 100% at 10 pM and 30 pM, respectively, and was used in the following studies.

The unesterified form of oleic acid had higher inhbitory potency than did the esterified forms. Thus, much higher concentrations of the glycerol esters (mono-, di- and triolein) were needed to yield inhibition comparable to that obtained with oleic acid (Fig. 1). Phosphatidylserine, which contains two fatty acids (usually an unsaturated c16 or C18 fatty acid is esterified to the 2-hydroxyl of the glycerol), inhibited the receptor to the extent seen with triolein (not shown).

567

Table 1. Inhibition of the solubilized nuclear T3 receptor by saturated fatty acid.7 The receptor (65 pg protein) and [I2'I]T3 (0.1 nM) were incubated with fatty acids at the concentrations indicated for 20 h at 0°C. [125J]T3 specifically bound to the receptor was determined by the AG 1 X8 resin method described in Experimental Procedures. Duplicate determinations were averaged (mean f range) and expressed as a percentage of the control

Fatty acid Conc" T3 bound Activity

PM 10-3xcpm %

Control (minus fatty acid) 25 4.71 f0.04 100 n-Capric acid (Clo: o) 25 3.29 kO.02 70 Lauric acid (C12:o) 25 2 . 2 0 i 0.03 47 Myristic acid (CI4:o) 25 0.84k0.01 18 Palmitic acid (C16: o) 25 1.69 5 0.0 36 Stearic acid 25 3.39 kO.04 72 Oleic acid (C18: 1) 25 0.36 0.01 8

Control (minus fatty acid) 5 4.20 k 0.01 100 Palmitic acid (C16:o) 5 3.40i0.02 81 Stearic acid 5 3.99 k 0.03 95 Arachidic acid (Czo. o) 5 4.22 k 0.01 100 Oleic acid (C, 8 : 5 1.26 k 0.02 30

0,3 30 300 Concn of oleoyl res idue OJM)

Fig. 1 . Inhibitory activity ofoleic acid and glycerol esters. The receptor (65 pg protein) and ['251]T3 (0.1 nM) were incubated with oleic acid (0), monoolein (O) , diolein ( A ) or triolein ( 7 ) at various oleoyl residue concentrations for 24 ha t 0°C. [1251]T3 specifically bound was determined by the AG 1x8 resin method as described in Experimental Procedures. Results represent mean values of duplicate determinations k range. Specific and nonspecific binding of the control (minus additives) were 4840 f 62 cpm and 838 f 20 cpm, respectively

Action of oleic acid in a soluble form

The inhibition was not due to incorporation of [1251]T3 into the micelles of oleic acid. Here, we took advantage of the specific gravity of the fatty acid (at 25°C d = 0.895), which is lighter than aqueous solutions. Mixtures of ['251]T3 and [3H]oleic acid (50 pM) were incubated for 20 h and then centrifuged at 80000 xg for 2.5 h; fatty acid micelles floated on the top. T3 was not concentrated much in the micelles and was evenly distributed in the liquid phase, while 55% of the oleic acid floated up as micelle pellets, with the remainder forming a gradient concentrated in the upper part of the aqueous phase. Below 10 pM, however, oleic acid existed in

0'02 r P E g 0.01 -f Q

0 2 5 50 Oleic a c i d (pMf

Fig.2. Eifects of various solvents on oleic acid micelles. Oleic acid at 25 mM in HzO was sonicated, and diluted to various concentrations with ethanol (0), H 2 0 ( O ) , or buffer T (0). The turbidity of fatty acid micelles was measured at 400 nm

a soluble form. When the A400 was measured for fatty acid micelles, oleic acid exhibited the turbidity typical of micelles in H20, giving a straight line of absorption starting at the origin; however, no absorbance at 400 nm was measured in ethanol, where it exists completely in a soluble form (Fig. 2). In the assay medium, buffer T, there was no absorption up to 10 pM; then it began to rise, giving a straight line with a steeper slope above 30 pM. We thus concluded that below 10 pM, oleic acid existed in a soluble form in buffer T. In fact, in the centrifugation study described above oleic acid at 10 pM was evenly distributed in the aqueous phase. At 10 pM, the fatty acid inhibited T3 binding by the receptor by as much as 84%.

Enhanced dissociation of T3-receptor complex

Oleic acid also affected dissociation of the preformed ['2sI]T3-receptor complex. Dissociation half-time was pre- viously estimated at 36 h at pH 7.6 (0°C) [23]; in the present work at pH 8.2, it became greater than 100 h, and was remark- ably shortened by the fatty acid (10 pM), to 30 h.

On the other hand unlabelled T3 added to the reaction at a wide range of concentrations (1 nM - 10 pM) had essentially no effect on the oleic-acid-promoted dissociation reaction (Fig. 3).

Mechanism of inhibit ion

Scatchard analysis was performed under the conditions where 68% of T3-binding by the receptor was inhibited by oleic acid ( 5 pM). To obtain accurate data, bound [12'I]T3 was determined by filtration using nitrocellulose membranes [19]. The curves demonstrated that, although oleic acid had little effect on the maximum binding capacity of the receptor ( z 0.32 pmol/mg protein), it did alter the dissociation con- stant from 55 pM to 155 pM; thus, the fatty acid reduced the affinity of the receptor for hormone (Fig.4).

Similar binding studies were carried out with and without oleic acid, but for a short incubation time (3 h), to obtain kinetically the initial T3-binding activities of the receptor. Double-reciprocal plots for [' 251]T3 concentration versus bound [1251]T3 showed that the curves intersected the ordinate

568

f I

L- 0 -9 -7 - 5

log (Concn of unlabeled T3), ( M )

Fig. 3. Oleic-acid-enhunced dissociation of T3-receptor complex is unuf ,fitted by free T3. After the receptor (65 pg protein) and ['Z51]T3 (0.3 nM) were incubated at 0°C for 24 h, various amounts of unlabelled T3 (1 nM- 10 pM) were added, together with (0 ) or without (0) oleic acid (10 pM), and incubation was continued at 0°C for 27 h. [1251]T3 remaining bound to the receptor was determined by the AG 1x8 resin method, and expressed as a percentage of the control (zero h i e ; the start of dissociation). Specific and nonspecific binding at zero time was 4140 5 34 cpm, and 690 38 cpm, respectively. Duplicate determinations were averaged (mean & range)

.3 - 0

X

'0 C

r

3 0 P m

t-

0 0.5 1.0 1 0 3 x T3 boundltree T3 (mol/M)

Fig.4. Scutchard anulysis of the nleic acid effect. Receptor extract (100 pg protein) and various concentrations of [1251]T3 (0.02- 0.40 nM) were incubated with (8) and without (0) 5 pM sodium oleate, in the presence or absence of 0.2 pM unlabelled T3. After incubation at 0°C for 20 h, specific T3 binding was determined by the filtration method as described and the means of duplicate determinations were examined by Scatchard plot analysis

axis at almost the same points (Fig.5A), thereby indicating that oleic acid acted in a competitive manner with respect to T3 for the binding sites.

Displacement of T3 (0.1 nM) by oleic acid at various concentrations is shown in Fig. 5B. The curve demonstrates 50% displacement at 2.8 pM oleic acid, and this typical sigmoidal graph supports the notion that oleic acid binds competitively to the T,-binding sites and displaces T3. From the data, together with Kd (55 pM) obtained above, the affinity of the receptor for oleic acid (Ki) was calculated to be 1.0 pM.

0 1 2 3 lo-'/ 112 5 1 1 T3 concn (Me')

'1 I \

0.1 1.0 10 100 Ole ic acid concn ( p M )

Fig. 5. (A) Double-reciprocul plot unalysis. Receptor extract (65 pg protein) and various concentrations of [1251]T3 (0.3 - 1 .O nM) were incubated at 0°C for 3 h with ( 0 ) and without (0) oleic acid (3 pM), in the presence or absence of an excess of unlabelled T3 (2 pM). [1Z51]T3 specifically bound to the receptor was determincd, and analyzed by double-reciprocal plots. (B) Displucernent curve. Incu- bation of the receptor extract (65 pg protein) was performed for 24 h with 0.1 nM [lZ51]T3 and oleic acid at various concentrations, with or without excess unlabellcd T3. ['251]T3 spccifically bound was determined, and expressed as a percentage of the control (without oleic acid). The thin vertical line at each point indicates deviation from the mean of duplicate determinations

Analysis on a sucrose density gradient

When the receptor was partially purified by centrifugation on a sucrose density gradient, T3-binding activity sedimented as a peak at 3.5 S, and oleic acid, added to the fractionated samples at 30 pM, completely abolished the receptor activity (not shown). This result after partial purification supports the idea of a direct action on the receptor.

Fatty acid effect on the receptors from various tissues

All of the receptors extracted from purified nuclei of various tissues of the rat were similarly inhibited by oleic acid. In the presence of 3 FM oleic acid, T,-binding activities of the receptors from liver, kidney, spleen, brain, testis and heart were reduced to 43 f 1%, 50 f 1%, 49 f 3%, 48 & 3%, 60 f 7%, and 46 f 1 % of the controls, respectively.

569

100

- 2 c

50 V

0

2

c

0

i

'1 t

Fig. 6. l$j'iJcts .f:futty ucids on T3 binding in cultured cells. HTC cells in 50cm2 dishes were incubated for 60min at 37°C with ['251]T3 (0.2 nM) in the presence of various unesterified fatty acids (0.5 mM). 1 2 5 1 radioactivity bound to the nuclei was detcrmined and corrccted for nonspecific T3 binding, as described in the text. The results, representing mean values of duplicate detcrminations range, werc expressed as a percentage of the control (without fatty acid). Control binding was determined in quadruplicate and others in duplicate. Symbols are as in Table 1 and C I 6 : linoleic acid; C1 8 : 3, linolenic acid

palmitoleic acid;

' O ° K

V

5 0

i

0 0.5 1.0 O l e i c a c i d ( m M )

Fig. 7. Effect qf oleic acid on T3 binding in HTC cells. HTC cells were incubated for 1 h at 37°C with [1251]T3 (0.2 nM), in the presence of various concentrations of oleic acid; ['251]T3 bound to the nuclei was determined, and corrected for nonspecific binding. Specific and nonspecific binding in the absence of oleic acid was 351 -t 17 cpm and 40 & 3 cpm, respectively. Results represent mean values of duplicate determinations f range

Inhibition of nuclear T3 binding in cultured cells

In the following experiments, we examined whether the fatty acid could exert inhibitory effects on cells in culture. HTC rat hepatoma cells were incubated at 37°C with ['251]T3, and the amount of labelled hormone specifically bound to the nuclei was determined. As in the case with the solubilized receptor, all of the fatty acids tested exhibited inhibitory activities, and among them oleic acid was the most potent (Fig. 6). However, due to a fraction insensitive to fatty acids (designated here as the 'nonresponder'), fatty acids did not completely inhibit the nuclear hormone-binding activity. Oleic acid inhibited the sensitive fraction ('responder') by 50% at 0.16 mM; the inhibition reached a plateau above 0.4 mM (Fig. 7).

I h 0.5 0.25 0.1 0 Oleic a c i d (mM)

Fig. 8. Oleic ucid stirnulutes the cellular uptakes of T3. Following incubation with ['ZSI]T3 (0.2 nM) for 1 h at 37"C in the presence of oleic acid (0, 0.1, 0.25 or 0.5 mM) HTC cells were rinsed three times with buffer A and harvested. After the cells were again subjected to suspension and ccntrifugation, the associated radioactivity was counted. Results represent mean values of duplicatc determinations

range

In the time course, nuclear T3 binding proceeded with a lag in the first 15 min, then linearly up to 1 h, reaching a plateau level within 2 h; and the nonresponder was again observed throughout in the presence of oleic acid (not shown). The ratio of the responder and nonresponder fractions was approximately 80:20 as seen with 1 mM oleic acid (Fig.7). Both fractions seemed to be of nuclear origin, because, as seen later, they were also present in the highly purified nuclei.

Since serum albumin binds fatty acids, it might buffer the inhibitory activity. However, when the inhibition was examined as in Fig. 7 in medium free of added serum, the results were similar to those shown in Fig. 7: 71 YO and 78% of the nuclear T3 binding was inhibited by oleic acid at 0.5 mM and 1 .O mM. Therefore, albumin in the medium had no appreci- able buffering effect on the effective fatty acid concentration.

Increased cellular T3 content

The inhibition did not result from suppressed T3 entry into the cells nor from an increased degradation of thyroid hormone. Actually, cellular incorporation of [1251]T3 increased in proportion to the added oleic acid. For example, in the presence of 0.5 mM oleic acid, 2.7 times more T3 was taken up in a I-h incubations (Fig. 8). Radioimmunoassay confirmed this result: the treated cells contained 2.1 times more T3 than the control cells (data not shown) indicating that oleic acid stimulated T3 uptake by the cells. Therefore, the inhibitory effect of the fatty acid on nuclear T3 binding seemed to overcome the elevated concentrations of intracellular T3, which would otherwise augment hormone binding to the nucleus.

Enhanced dissociation of T3-receptor complex and reciprocal nuclear association of oleic acid

Dissociation of the hormone-receptor complex in cultured cells is shown in Fig. 9. After preincubation with ['251]T3 for 2 h, HTC cells were further incubated with oleic acid (0.5 mM). While the bound [1251]T3 maintained a plateau level in the control, it dissociated rapidly in the presence of oleic acid: 50% in 30 min and 75% in 60 min.

Conversely, [3H]oleic acid increasingly bound to the nuclei, reaching values as high as 35 6 nmol/ 3 nmo1/100 pl, 52

570

1.4

- Q 2 n

F E . v

U

'0 0.7 X

U c 3 0 P

m I- I

LI) N

I

-

0

I Y -4

+ r + I I I

10 30 60 Time (min)

Fig.9. Enhanced dissociation of T3-receptor complexes. After HTC cells were incubated with [lZ5I]T3 (0.2 nM) for 2 h at 37"C, oleic acid (0.5 mM) was added, and the incubation was continued for additional periods. [1251]T3 specifically bound to the cell nuclei was determined and corrected for nonspecific binding (8 - 36% of the total binding), as described. Symbols are: ['ZSI]T, remaining bound to the nuclei in the presence (0 ) and absence (0) of oleic acid. In a separate exper- iment, HTC cells werre incubated with [3H]oleic acid (5 WCi) at 0.5 mM; the nuclei were iosolated and washed in buffer A with ( W ) or without (0) Triton X-100, for determination of 3H radioactivity. Results represent mean values of duplicate determinations f range

100 p1 and 68 & 3 nmo1/100 pl packed isolated nuclei in 10 min, 30 min and 60 min, respectively (estimated from the incorporated radioactivity in the nuclei). When the nuclei were washed in 0.5% Triton X-100/0.25 M sucrose/% mM MgC12/20 mM Tris/HCI, pH 7.6, and the outer and parts of the inner nuclear membranes were removed [24-261, 34%, 27% and 20%, respectively, of the bound [3H]oleic acid re- mained in the nuclear samples (Fig. 9).

To obtain evidence for the unmetabolized oleic acid in the nucleus, the fatty acid associated with cultured cell nuclei was extracted and identified by gas chromatography. The HTC cells originally contained 9 nmol oleic acid/100 p1 packed nuclei as well as lesser amounts of other fatty acids such as palmitic acid and stearic acid. The cells incubated with 0.5 mM oleic acid augmented the nuclear content of unesterified oleic acid to 25 nmol/l00 p1 and 33 nmo1/100 p1 packed nuclei, or 2.8 times and 3.7 times the control value in 30 min and 60 min, respectively. These results suggest that the fatty acid had accumulated in the nucleus and acted on nuclear T3-binding sites.

Reversibility of fa t ty acid effect

To examine the possibility that the inhibition might have resulted from receptor degradation, HTC cells pretreated with oleic acid were washed and examined for their T3-binding activity. Irrespective of preincubation with oleic acid, nuclear T3 binding occurred in the washed cells to the same extent, as seen in Fig. 10. Thus, receptor degradation was not the cause of the inhibition.

0 1 2 Time ( h )

Fig. 10. Reversibilzty ofthe inhibition. HTC cells, incubated for 1 h in the presence (0 ) or absence (0) of 0.5 mM oleic acid, were rinsed thoroughly with Eagle's minimum essential medium, and successively incubated in fresh medium containing 7% calf serum, [1ZSI]T3 (0.2 nM) and cycloheximide (100 pg/ml). The cells were harvested at the indicated times and [lZ5I]T3 specifically bound to the nuclei was determined. Results represent mean values of duplicate determinations & range. Cycloheximide was included to prevent de novo synthesis of receptor protein, thereby allowing estimation of [Iz5I]T3- binding activity of the cells that have been treated with oleic acid

In addition, the fatty acid effect was reversible: T3 binding to the cultured cells was restored in fresh medium. This reversibility was also confirmed with the isolated nuclei (not shown).

Isolated nuc2ei

Fatty acid inhibition of nuclear T3 binding was next demonstrated directly with isolated nuclei. When highly purified rat liver nuclei were incubated at 0°C with ['251]T3, together with increasing concentrations of oleic acid, an inhibition curve very similar to that obtained with cultured HTC cells was obtained (Fig. 7), revealing 50% inhibition at 0.12 mM oleic acid (not shown). Again, there were the responder and nonresponder fractions, in a ratio (T3-binding activity) of 76:24. As shown above, incorporation cf [lZ5I]T3 into fatty acid micelles was not the cause of the inhibition (not shown).

The inhibition did not result from increased liberation of the nuclear receptor; although, irrespective of the presence of fatty acid, some binding activity was released from the nuclei into the medium (15% in a 24-h incubation).

Scatchard analysis of nuclear T3 binding

Scatchard analysis of the nuclear T3 binding was performed with rat liver nuclei, in the absence and presence of 1.0 mM oleic acid (at which fatty acid concentration, only the nonresponder binding sites could be measured). The two parallel lines (Fig. 11) demonstrated that in accord with the almost complete inhibition of the responder fraction, the maximum binding capacity decreased from 0.78 pmol/mg DNA to 0.25 pmol/mg DNA, and that the binding sites re- vealed similar Kd values (0.85 nM and 0.80 nM), in the absence and presence of oleic acid. The latter indicates that the nonresponder and the uninhibited responder had very close affinities for T3.

Fatty acid effect on nuclei of various tissue

Fatty acid inhibition of the nuclear T3 binding was also observed with all cell types examined, including brain, spleen,

571

0.8 r

0 5.0 10 4 -1 -1

Fig. 11. Scatchard analysis of the oleic acid effect on nuclear T3 binding. Washed nuclei (2.7 ,4260 units) from rat liver were incubated in duplicate for 24 h at 0°C with various concentrations of ['251]T3 (0.05- 0.8 nM) in the presence (0 ) and absence (0) of 1 mM oleic acid in buffer A/0.2 mM dithiothreitol (100 pl). After incubation, the nuclear-bound [1ZSI]T3 was determined, and corrected for nonspecific binding, as described. Duplicate determinations were averaged and subjected to Scatchard plot analysis

10xBoundT3/Free T3 , mo l -M .(mg DNA)

Brain Spleen Testis L i v e r Kidney

ll I 1 I; O,O.Z,l 0.0.2.1 0,02.1 0,OS.l 0,O.Z.l

O l e i c a c i d (mM)

Fig. 12. Oleic acid effects on T3-binding to nuclei of various tissues. Nuclei were isolated from various tissues of the rat and incubated for 20 h at 0°C with [12'I]T3 (0.5 nM), in the absence and presence of oleic acid; [12sI]T3 bound to the nuclei was determined, as described in the text. [1251]T3 specifically bound to the nuclei (8 ,4260 units) in the absence of the fatty acid was 8700, 2800, 3300, 10000 and 6000 cpm for brain, spleen, testis, liver and kidney tissues, respectively. Results representing mean values of duplicate determinations i range were expressed as a percentage of these controls

testis, liver and kidney of the rat (Fig.12). In addition, the nonresponder fraction was similarly observed in these nuclei. Thus, the fatty acid inhibition and the occurrence of two forms of binding sites seem general characteristics of these cells. However, the population of the nonresponders varied; it was highest in the testis and lowest in the liver.

DISCUSSION

Fatty acid action on the extracted receptor

Unesterified, long-chain fatty acids were shown to inhibit T3 binding by the thyroid hormone receptor extracted from rat liver nuclei. Among them, oleic acid was the most potent inhibitor. Oleic acid at 10 pM was soluble in the assay medium, and inhibited 84% of T3-binding to the receptor. This indicates that oleic acid acted on the receptor in a soluble form rather than in micelles. Fatty acid micelles did not incorporate [ '251]T3, therefore, they did not lower the effective concentration of T3.

Nonspecific detergent effects of fatty acids could cause the inhibition. However, the action of oleic acid seems specific, because a concentration as low as 2.8 pM was sufficient to inhibit 50% of the receptor activity, and because the affinity of the receptor for oleic acid (Ki) was as low as 1.0 pM. For example, the specific fatty-acid-binding proteins in cytoplasm have affinities (Kd) of 0.4-0.9 pM for long-chain fatty acids

The double-reciprocal plot and displacement analyses indicated that oleic acid acted competitively for the hormone- binding sites. On the other hand, oleic-acid-promoted dissociation of the T3-receptor complex occurred independently of unlabelled T3 present in the dissociation medium. This may suggest that T3 and oleic acid do not bind to the same sites in the receptor. The sites are most likely to overlap each other.

In the inhibition by fatty acids, there was a size preference (C,,) of the receptor for saturated fatty acids and a preference for unesterified fatty acids. In addition, the receptor was inhibited to a greater extent by unsaturated fatty acids. It is likely that the receptor has a structure at the fatty-acid-binding site that interacts with n electrons of fatty acids, as well as with a hydrophobic moiety, and probably with the terminal carboxyl group; and that oleic acid is structurally best fitted for the interaction. That thyroid hormone possesses a strong hydrophobicity and n electrons supports the notion that the T3- and fatty-acid-binding sites may be shared.

~ 7 1 .

Fatty acid action on cultured cells

When HTC cells were incubated with oleic acid, the fatty acid reached the cell nucleus and inhibition of the nuclear T3- binding activity occurred. Various control experiments and the results obtained with isolated nuclei and the solubilized receptor support this conclusion, The inhibition was reversible and, as with the solubilized receptor, oleic acid was the most potent inhibitor among the fatty acids tested.

To achieve 50% inhibition, various concentrations of oleic acid were needed, depending on the receptor forms: 2.8 pM for the solubilized receptor, and 0.12 mM and 0.16 mM for the receptors in the isolated nuclei and in HTC cells, respectively. This difference arose mainly from the presence of other cell structures such as nuclear membranes and chromatin (unpublished results). Another reason may be the metabolism that lowers intracellular concentrations of fatty acids.

There were two classes of nuclear T3-binding sites: the responder and the nonresponder, representing 80% and 20%, respectively, of the binding sites in HTC cells, and the population of the two sites varied with the tissues. The nonresponder may represent a basal activity of the responder whose affinity for T3 was maximally reduced by fatty acids. However, Scatchard plots showed that the affinities for T3 of the responder and the nonresponder were almost identical. Therefore, these receptor sites seem to represent different

512

entities. Since the solubilized receptor behaved totally as a responder, the difference between these sites seems to reside in distinct nuclear structure(s) with which the receptor proteins are associated. They may have different functions in the cells.

Possibility

The present study demonstrated that unesterified long- chain fatty acids competitively inhibited the nuclear T,-receptor solubilized from the chromatin from various tissues, and that the inhibition occurred in cells in culture, as well as in isolated nuclei.

While the physiological significance of our observations is unknown, it is interesting to note, that (a) T3 induces various enzymes involved in the synthesis of fatty acids [28, 291, and (b) that the levels of these lipogenic enzymes, such as fatty acid synthetase, malic enzyme, glucose-6-phosphate dehydro- genase and acetyl-CoA carboxylase, are reduced in rat [30] and chick embryo [31] hepatocytes grown in media with added unesterified fatty acids, and in animals fed unsaturateds fatty acids [32] or a fat diet 1331. The possibility that this suppression is induced by an inhibitory action of the fatty acids seems to warrant attention.

We thank Prof. I. Yano, Dept. of Bacteriology, Osaka City Uni- versity Medical School, for instruction and use of the gas chromatography apparatus and M. Ohara for helpful comments.

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unesterified long-chain fatty acids inhibit thyroid hormone binding to the nuclear receptor :...

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