Biochimica et Biophysics Acta, 1047 (1990)
131
131-134
Elsevier BBALIP 53522
ambition
of nuclear T3 binding via PLA,-induced acids from nuclear membranes
release of fatty
Fiona R.M. van der Klis and Wilmar M. Wiersinga University of Amsterdam, Amsterdam (The Netherlandr)
(Received 8 June 1990)
Key words: Phospholipase A,; Nuclear membrane, Fatty acid; Triiodothyronine; Nuclear T3 receptor; (Rat liver)
This study was undertaken to explore pntative regulatory m~isms involved in the i~ibition of nuclear T3 binding (INB) by fatty acids. Ether extracts of intact rat liver nuclei contained INB-activity. Removement of the nuclear membrane resulted in the loss of INB-activity of the nuclei. Incubation of intact nuclei with phospholipase A, increased nuclear INB-activity in a time- and dose-dependent mauner; this was correlated with a rise of free fatty acid concentration in the ether extract. We conchrde that fatty acids present in the nuclear membrane can be released by phospholipase A,, and are capable of inhibiting nuclear I3 binding.
Introduction
Fatty acids, in particular those with unsaturated bonds, inhibit the specific binding of T3 to rat liver nuclear T3 receptors [I]. We have recently demonstrated the competitive nature of this inhibition and the ability of fatty acid binding agents like albumin to reverse this i~bition 121. The present studies were undertaken to explore putative regulatory mechanisms involved in the inhibition of nuclear T3 binding (INB) by fatty acids. We investigated the ability of nuclear lipids and fatty acids to inhibit nuclear T3 binding, and also if nuclei would be amenable to a phospholipase A, (PLA,)-induced release of fatty acids from nuclear phospholipids. For this purpose, rat liver nuclei were isolated with or without a nuclear membrane and incubated with PLA,. Ether extracts of nuclear preparations were subsequently tested for INB-activity. This work has been presented at the 18th meeting of the European Thyroid Association (1989) and is reported in Annales d’Endocrinologie (1989) 50, 150. Materials and Methods Chemicals
Phospholipase A, (EC 3.1.1.4) from the snake venom N&z mocam~i~ (spec. act. 1580 U/mg protein) was purchased from Sigma Chemical (St. Louis, U.S.A.).
Correspondence: F.R.M. van der Klis, Department of Endocrinology, Academic Medical Centre, F5 - 258, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. ~5-2740/~/$03.50
0 1990 Elsevier Science Publishers B.V. (Biorn~~
Triiodothyronine (T3) was obtained from Henning GmbH (Berlin, F.R.G.) and [1251]T3 (spec. act. 2200 Ci/mmol) from New England Nuclear (Boston, U.S.A.). Isolation of rat liver nuclei
Livers of male Wistar rats were excised and stored in liquid nitrogen until further processing. Nuclei were isolated at 4” C in two different ways. The first isolation method includes Triton X-100 washes which results in a loss of the nuclear outer membrane [2,3]. In short, the liver was homogenized in solution A (20 mM Tricine, 2 mM CaCl,, 1 mM MgCl,, 5% (v/v) glycerol, 0.25 M sucrose (pH 7.6)). After three washes (7 ruin, 800 X g) in solution A containing 0.5% Triton X-100, the remaining pellet was purified by ~nt~fugation (45 min, 45 000 x g) in solution A containing 2.4 M sucrose. After two further washes (7 min, 800 X g) in solution A f 0.5% Triton X-100, the liver nuclei were suspended in 0.5 vol (v/w) solution B (20 mM Tris-HQ, 25 mM KCl, 0.25 M sucrose, 1 mM EDTA, 50 mM NaCl and 5% (v/v) glycerol (pH 7.6)) (Nuclei A). Nuclei with an intact nuclear membrane were isolated according to Bloble and Potter [3]. The liver was homogenized in TKM-buffer (50 mM Tris-HCl, 5 mM MgCl,, 0.25 M sucrose (pH 7.5). The nuclei were pelleted (7 min, 800 x g) and washed twice in the TKMbuffer. The pellet was then suspended in TKM-buffer containing 1.62 M sucrose, carefully put on a cushion of 2.3 M sucrose in TKM-buffer and centrifugated for 35 ruin at 124000 x g,,. The pellet was washed twice (7 min, 800 X g) in TKM-0.25 M sucrose buffer and finally suspended in 0.3 vol (v/w) solution B (Nuclei B). Division)
132 Electron microscopy demonstrated an intact nuclear membrane in nuclei B; the nuclear outer membrane was absent and the inner membrane severely disrupted in nuclei A. DNA content of nuclei was measured by the fluorochrome Hoechst 33258 [4].
Phospholipase A, incubation 100 ~1 of nuclear suspension (see legends to Fig. 2 and Fig. 3 for DNA concentrations) in 0.5 vol. (v/w) solution A (pH 8.5) were incubated with 20 ~1 of various concentrations of PLA, dissolved in 0.1 M Tris, 0.075 M CaCl, buffer (pH 8.2) for 10 min at 22O C. The incubation was terminated by adding 50 ~1 EDTA (0.25 M) and putting the samples on ice. Control tubes (nuclei in the absence of PLA,) were treated identically.
Ether extracts of nuclei Extraction of nuclear lipids and fatty acids was done as follows: 0.1 ml of the nuclear suspension was thoroughly vortexed with 4 volumes of diethyl ether for 30 s and pelleted (5 min, 2000 rpm). After freezing the tubes in a dry ice-methanol bath the ether phase was decanted in another tube and evaporated to dryness at room temperature under a stream of nitrogen. The dried extracts were dissolved either in saline or in ether for fatty acid analysis and in solution B for assay of INBactivity.
Free fatty acid analysis Non-esterified fatty acids were assayed by a enzymatic calorimetric method (NEFAc, Wako, GmbH, Neuss, F.R.G.). Concentrations of individual fatty acids were determined by gas chromatography. After methylation of the fatty acids by the diazomethane method [5], they were analysed by a Hewlett Packard 5890 gas chromotograph equipped with a splitless capillary injection system, a flame ionisation detector and a HP-FFAP capillary column (25 m x 0.2 mm x 0.33 pm).
Nuclear T3 binding assay 0.1 ml suspension of freshly prepared nuclei A were incubated in solution B with [‘251]T3 and 5 mM dithiothreitol for 2 h at 22” C. The incubation was stopped by chilling the tubes on ice. After two washes with solution B containing 0.5% Triton X-100, the specific nuclear [‘251]T3 binding was calculated by subtracting the radioactivity remaining with the pellet of parallel incubations containing an excess (lop7 M) of nonradioactive T3 [2]. INB-activity is calculated by subtracting the amount of specifically bound T3 in the test tube from that in the control tube, and expressed as percentage of specific T3 binding of controls.
Results INB-activity of nuclei limited to the nuclear membrane Specific binding of [‘251]T3 to isolated nuclei was 30.1 f 2.1% (mean f S.E., n = 16); it was slightly lower in the presence of an ether extract of the incubation buffer (28.7 + 2.0%, n = 16) (Fig. 1). Nuclear T3 binding in the presence of ether extracts of nuclei with a disrupted nuclear membrane (nuclei A, 2.55 + 0.18 mg DNA/ml) was lower related to controls (24.8 k 2.1%, n = 16); it was much more decreased (17.8 5 2.0%, n = 16) in the presence of ether extracts of intact nuclei (nuclei B, 2.80 f 0.54 mg DNA/ml). INBactivity of nuclei A was 14.7 & 3.1%, that of nuclei B was 38.3 _t 4.5%; it increased with a higher DNA content of the extraction sample. INB-activity of nuclei increased by PLA, Nuclei A and nuclei B were treated for O-10 min with PLA,, 0.05 U/tube. Nuclear T3 binding decreased progressively with time in the presence of evaporated ether extracts of nuclei B (but not of nuclei A) (Fig. 2); after 10 min of incubation with PLA, nuclear T3 binding was 39.2% of the value obtained with nuclei B not treated with PLA,. A clear dose-response relationship was observed for the effect of PLA, on nuclei B, but not on nuclei A. INB-activity of nuclei B, treated with 0.15 U PLA,, was 61.7%. INB-activity of nuclei related to free fatty acids In the experiment of Fig. 3 the ether extract of nuclei A contained 0.7 nmol FFA; PLA, treatment did not increase FFA concentration. In contrast, the ether extract of nuclei B contained 4.1 nmol FFA and PLA,
30
20 1
10 c
0’ A
0
Fig. 1. Effect of ether extracts of buffer and of nuclei A (disrupted nuclear membrane) and nuclei B (intact nuclear membrane) on the specific binding of [‘*‘IIT to rat liver nuclear T3 receptors. Statistical analysis by paired r-test (n = 16): buffer vs. control, P < 0.001; buffer vs. nuclei A, P -c0.0001; buffer vs. nuclei B, P x 0.0001; nuclei A vs. nuclei B, P -C0.0001.
133 Discussion ..
0
nuclei A
,
L
,
I
2
4
6
6
,
10 time (min)
Fig. 2. Effect of ether extracts of nuclei A (disrupted nuclear membrane, 1.85 mg DNA/ml) and of nuclei B (intact nuclear membrane, 1.49 mg DNA/ml) after pretreatment with 0.05 U PLA,/tube for O-10 min on specific nuclear T3 binding (expressed as percentage of control tubes containing ether extracts of nuclei A or B without PLA *).
treatment increased FFA concentration dose-dependently, up to 20.1 nmol with 1.5 U PLA,. Table I depicts the concentrations of individual fatty acids found in a representative experiment. The ether extract of nuclei A contained predominately saturated fatty acids (80%) and treatment with PLA, had little effect. The ether extract of nuclei B contained much higher concentrations of fatty acids, composed of 50% saturated and 50% unsaturated ones; treatment with 0.05 U PLA, increased FFA 2-fold due to an abundant rise of unsaturated (not of saturated) fatty acids.
This study was undertaken to explore putative regulatory mechanisms involved in the inhibition of nuclear T3 binding (INB) by fatty acids. First, the question was addressed whether rat liver nuclei themselves contain INB-activity. Nuclei indeed possessed INB-activity which was confined to the nuclear membrane: removal of the outer and most of the inner nuclear membrane results in a clear loss of INB-activity of the nuclear ether extracts. These results are in agreement with previous findings that by far the major part of the nuclear lipids is located in the membrane of the nuclear envelope [6]. The fatty acid content of the nuclei with a disrupted membrane was also 6-9-fold lower than of intact nuclei. The relative contribution of individual fatty acids to the total fatty acid content of intact nuclei was as follows: 16:0 (26.4%), 18:0 (22.8X), 18:l (12.1%), 18:2 (17.2%), 20:4 (15.7%), 22:6 (5.8%). These figures are very close to those previously reported [7,8]. From the IC,, values of the individual fatty acids [9], it can be calculated that the actual concentrations of the various fatty acids can account for the observed INB-activity. The data suggest that there is no need to implicate other lipids present in the nuclear ether extracts in the observed INB-activity. The second question to be addressed was whether nuclear phospholipids are amenable to the action of PLA,. PLA, activity results in the liberation of (unsaturated) fatty acids located in the 2 position of phospholipids. PLA, treatment of nuclei with disrupted membrane did not result in any INB-activity of these nuclei. In contrast, the INB-activity of intact nuclei increased greatly after PLA, treatment in a time- and
TABLE I
Nuclei A
Gas chromatographic analysis of the fatty acids present in the ether extract of nuclei A (disrupted nuclear membrane, I.04 mg DNA/ml) and of nuclei B (intact nuclear membrane, 1.40 mg DNA/ml) before and after incubation with 0.05 Units PLAl for I5 min. Nuclei B
(FFA nmol) Nuclei A -
+
-
0.38 0.33 0.13 _
(6.5)
0.36 0.29 0.16 _
(l.5)
I
0.06 -
1.86 1.61 0.85 1.21 1.11 0.41
1.48 1.09 1.27 3.88 5.96 0.24 1.30
0.81
0.90
7.05
15.22
PLA, incubation
0.1
0.01
PI+
1.0
u/tubed
Fig. 3. Effect of treating nuclei A (disrupted nuclear membrane, 3.40 mg DNA/ml) and nuclei B (intact nuclear membrane, 3.80 mg DNA/ml) with various doses of PLA, for 10 min on the free. fatty acid concentration (interrupted lines) and INB-activity (nuclear T3 binding expressed as percentage of controls without PLA,; continuous lines) contained in the ether extracts of these nuclei.
16:0 18:0 18:l 18:2 20:4 22~5 22:6 Total
Nuclei B
(15.5) * (70.0) (6.0) (6.5)
+
* Values between brackets indicate the amount of fatty acid (nmol) that reduces specific nuclear T3 binding to 50% of controls (IC,, values) [9].
134 dose-dependent manner. Again, the rise in the concentrations of the individual fatty acids could completely account for the observed increase of INB-activity induced by PLA,. The quantity of fatty acids which in theory could be released from nuclear phospholipids can also be calculated. In the experiment of Fig. 3, the absolute protein content of incubation vessels containing 0.1 ml suspension of nuclei B in a volume of 0.5 ml was 0.233 mg. Assuming 200 nmol phospholipid/mg protein the maximal release of fatty acids from the nuclear phospholipids is approx. 47 nmol (figures of the same order of magnitude are reached in calculations using slightly different data for the ratio of phospholipids to protein or to DNA [G-S]. The observed amount of FFA in these incubation vessels ranges from 4.1 nmol in the absence of PLA, to 20 nmol in the presence of 1.5 U PLA,. These figures are in the same order of magnitude as the amount of fatty acids required in an incubation volume of 0.5 ml for 50% inhibition of nuclear T3 binding (12-13 nmol as indicated by the IC,, values of unsaturated fatty acids). We conclude that nuclear phospholipids present in the nuclear membrane are amenable to PLA, activity. It is conceivable that the PLA, induced local release of fatty acids from the nuclear membrane might inhibit the binding of T3 to its nuclear receptors in vivo. Recent reports indicate the presence of PLA, in the cytosol, the nuclear membrane and the nuclear matrix [lo-131. Modulation of PLA, activity occurs in various disease states, and it might well be that increased PLAz activity during illness contributes to a decreased nuclear T3
binding via a local release of nuclear fatty acids. This hypothesis fits in the well-known adaptation of thyroid hormone metabolism during illness, but remains to be proven. Acknowledgements The authors are grateful to M. Platvoet and M. van Beeren for experimental assistance and to A. Nijenhuis for gas chromatographic analysis. References 1 Wiersinga, W.M.. Chopra. LJ. and Chua Teco. G.N. (1988) Metabolism 37, 99661002. 2 Van der KIis, F.R.M. and Wiersinga, W.M. (1989) FEBS Lett. 246. 6612. 3 Bloble. G. and Potter, V.R. (1966) Science 154. 1662-1665. 4 Labarca. C. and Paigen, K. (1980) Anal. Biochem. 102, 344352. 5 Fales. H.M. (1973) Anal. Chem. 45, 2302-2303. 6 Kleinig. H. (1970) J. Cell. Biol. 46, 3966402. 7 Frederiks, W.M., James, J.. Arnouts, C., Broekhoven, S. and Morreau, J. (1978) Cytobiologie 18. 254-271. 8 Khandwala. AS. and Kasper. C.B. (1971) J. Biol. Chem. 246, 6242-6246. 9 Wiersinga, W.M. and Platvoet- ter Schiphorst. M. (1989) Int. J. Biochem., 22. 269-273. 10 Pierik, A.J.. Nijssen, J.G., Aarsman, A.J. and Van den Bosch. H. (1988) B&him. Biophys. Acta 962. 345-353. 11 Nikitin, V.N., Babenko, N.A. and Popova. L.-Ya. (1985) Doklady Akademii Nauk. SSSR. 281, 731-734. 12 Karagezyan, K.G. and Tadevosgyan, Yu-V. (1986) Doklady Akademii Nauk SSSR. 287. 1248-1250. 13 Tamiya-Koizumi. K., Umekawa. H., Yoshida, S.. Ishihara, H. and Kojima, K. (1989) B&him. Biophys. Acta 1002, 182-188.
131
131-134
Elsevier BBALIP 53522
ambition
of nuclear T3 binding via PLA,-induced acids from nuclear membranes
release of fatty
Fiona R.M. van der Klis and Wilmar M. Wiersinga University of Amsterdam, Amsterdam (The Netherlandr)
(Received 8 June 1990)
Key words: Phospholipase A,; Nuclear membrane, Fatty acid; Triiodothyronine; Nuclear T3 receptor; (Rat liver)
This study was undertaken to explore pntative regulatory m~isms involved in the i~ibition of nuclear T3 binding (INB) by fatty acids. Ether extracts of intact rat liver nuclei contained INB-activity. Removement of the nuclear membrane resulted in the loss of INB-activity of the nuclei. Incubation of intact nuclei with phospholipase A, increased nuclear INB-activity in a time- and dose-dependent mauner; this was correlated with a rise of free fatty acid concentration in the ether extract. We conchrde that fatty acids present in the nuclear membrane can be released by phospholipase A,, and are capable of inhibiting nuclear I3 binding.
Introduction
Fatty acids, in particular those with unsaturated bonds, inhibit the specific binding of T3 to rat liver nuclear T3 receptors [I]. We have recently demonstrated the competitive nature of this inhibition and the ability of fatty acid binding agents like albumin to reverse this i~bition 121. The present studies were undertaken to explore putative regulatory mechanisms involved in the inhibition of nuclear T3 binding (INB) by fatty acids. We investigated the ability of nuclear lipids and fatty acids to inhibit nuclear T3 binding, and also if nuclei would be amenable to a phospholipase A, (PLA,)-induced release of fatty acids from nuclear phospholipids. For this purpose, rat liver nuclei were isolated with or without a nuclear membrane and incubated with PLA,. Ether extracts of nuclear preparations were subsequently tested for INB-activity. This work has been presented at the 18th meeting of the European Thyroid Association (1989) and is reported in Annales d’Endocrinologie (1989) 50, 150. Materials and Methods Chemicals
Phospholipase A, (EC 3.1.1.4) from the snake venom N&z mocam~i~ (spec. act. 1580 U/mg protein) was purchased from Sigma Chemical (St. Louis, U.S.A.).
Correspondence: F.R.M. van der Klis, Department of Endocrinology, Academic Medical Centre, F5 - 258, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. ~5-2740/~/$03.50
0 1990 Elsevier Science Publishers B.V. (Biorn~~
Triiodothyronine (T3) was obtained from Henning GmbH (Berlin, F.R.G.) and [1251]T3 (spec. act. 2200 Ci/mmol) from New England Nuclear (Boston, U.S.A.). Isolation of rat liver nuclei
Livers of male Wistar rats were excised and stored in liquid nitrogen until further processing. Nuclei were isolated at 4” C in two different ways. The first isolation method includes Triton X-100 washes which results in a loss of the nuclear outer membrane [2,3]. In short, the liver was homogenized in solution A (20 mM Tricine, 2 mM CaCl,, 1 mM MgCl,, 5% (v/v) glycerol, 0.25 M sucrose (pH 7.6)). After three washes (7 ruin, 800 X g) in solution A containing 0.5% Triton X-100, the remaining pellet was purified by ~nt~fugation (45 min, 45 000 x g) in solution A containing 2.4 M sucrose. After two further washes (7 min, 800 X g) in solution A f 0.5% Triton X-100, the liver nuclei were suspended in 0.5 vol (v/w) solution B (20 mM Tris-HQ, 25 mM KCl, 0.25 M sucrose, 1 mM EDTA, 50 mM NaCl and 5% (v/v) glycerol (pH 7.6)) (Nuclei A). Nuclei with an intact nuclear membrane were isolated according to Bloble and Potter [3]. The liver was homogenized in TKM-buffer (50 mM Tris-HCl, 5 mM MgCl,, 0.25 M sucrose (pH 7.5). The nuclei were pelleted (7 min, 800 x g) and washed twice in the TKMbuffer. The pellet was then suspended in TKM-buffer containing 1.62 M sucrose, carefully put on a cushion of 2.3 M sucrose in TKM-buffer and centrifugated for 35 ruin at 124000 x g,,. The pellet was washed twice (7 min, 800 X g) in TKM-0.25 M sucrose buffer and finally suspended in 0.3 vol (v/w) solution B (Nuclei B). Division)
132 Electron microscopy demonstrated an intact nuclear membrane in nuclei B; the nuclear outer membrane was absent and the inner membrane severely disrupted in nuclei A. DNA content of nuclei was measured by the fluorochrome Hoechst 33258 [4].
Phospholipase A, incubation 100 ~1 of nuclear suspension (see legends to Fig. 2 and Fig. 3 for DNA concentrations) in 0.5 vol. (v/w) solution A (pH 8.5) were incubated with 20 ~1 of various concentrations of PLA, dissolved in 0.1 M Tris, 0.075 M CaCl, buffer (pH 8.2) for 10 min at 22O C. The incubation was terminated by adding 50 ~1 EDTA (0.25 M) and putting the samples on ice. Control tubes (nuclei in the absence of PLA,) were treated identically.
Ether extracts of nuclei Extraction of nuclear lipids and fatty acids was done as follows: 0.1 ml of the nuclear suspension was thoroughly vortexed with 4 volumes of diethyl ether for 30 s and pelleted (5 min, 2000 rpm). After freezing the tubes in a dry ice-methanol bath the ether phase was decanted in another tube and evaporated to dryness at room temperature under a stream of nitrogen. The dried extracts were dissolved either in saline or in ether for fatty acid analysis and in solution B for assay of INBactivity.
Free fatty acid analysis Non-esterified fatty acids were assayed by a enzymatic calorimetric method (NEFAc, Wako, GmbH, Neuss, F.R.G.). Concentrations of individual fatty acids were determined by gas chromatography. After methylation of the fatty acids by the diazomethane method [5], they were analysed by a Hewlett Packard 5890 gas chromotograph equipped with a splitless capillary injection system, a flame ionisation detector and a HP-FFAP capillary column (25 m x 0.2 mm x 0.33 pm).
Nuclear T3 binding assay 0.1 ml suspension of freshly prepared nuclei A were incubated in solution B with [‘251]T3 and 5 mM dithiothreitol for 2 h at 22” C. The incubation was stopped by chilling the tubes on ice. After two washes with solution B containing 0.5% Triton X-100, the specific nuclear [‘251]T3 binding was calculated by subtracting the radioactivity remaining with the pellet of parallel incubations containing an excess (lop7 M) of nonradioactive T3 [2]. INB-activity is calculated by subtracting the amount of specifically bound T3 in the test tube from that in the control tube, and expressed as percentage of specific T3 binding of controls.
Results INB-activity of nuclei limited to the nuclear membrane Specific binding of [‘251]T3 to isolated nuclei was 30.1 f 2.1% (mean f S.E., n = 16); it was slightly lower in the presence of an ether extract of the incubation buffer (28.7 + 2.0%, n = 16) (Fig. 1). Nuclear T3 binding in the presence of ether extracts of nuclei with a disrupted nuclear membrane (nuclei A, 2.55 + 0.18 mg DNA/ml) was lower related to controls (24.8 k 2.1%, n = 16); it was much more decreased (17.8 5 2.0%, n = 16) in the presence of ether extracts of intact nuclei (nuclei B, 2.80 f 0.54 mg DNA/ml). INBactivity of nuclei A was 14.7 & 3.1%, that of nuclei B was 38.3 _t 4.5%; it increased with a higher DNA content of the extraction sample. INB-activity of nuclei increased by PLA, Nuclei A and nuclei B were treated for O-10 min with PLA,, 0.05 U/tube. Nuclear T3 binding decreased progressively with time in the presence of evaporated ether extracts of nuclei B (but not of nuclei A) (Fig. 2); after 10 min of incubation with PLA, nuclear T3 binding was 39.2% of the value obtained with nuclei B not treated with PLA,. A clear dose-response relationship was observed for the effect of PLA, on nuclei B, but not on nuclei A. INB-activity of nuclei B, treated with 0.15 U PLA,, was 61.7%. INB-activity of nuclei related to free fatty acids In the experiment of Fig. 3 the ether extract of nuclei A contained 0.7 nmol FFA; PLA, treatment did not increase FFA concentration. In contrast, the ether extract of nuclei B contained 4.1 nmol FFA and PLA,
30
20 1
10 c
0’ A
0
Fig. 1. Effect of ether extracts of buffer and of nuclei A (disrupted nuclear membrane) and nuclei B (intact nuclear membrane) on the specific binding of [‘*‘IIT to rat liver nuclear T3 receptors. Statistical analysis by paired r-test (n = 16): buffer vs. control, P < 0.001; buffer vs. nuclei A, P -c0.0001; buffer vs. nuclei B, P x 0.0001; nuclei A vs. nuclei B, P -C0.0001.
133 Discussion ..
0
nuclei A
,
L
,
I
2
4
6
6
,
10 time (min)
Fig. 2. Effect of ether extracts of nuclei A (disrupted nuclear membrane, 1.85 mg DNA/ml) and of nuclei B (intact nuclear membrane, 1.49 mg DNA/ml) after pretreatment with 0.05 U PLA,/tube for O-10 min on specific nuclear T3 binding (expressed as percentage of control tubes containing ether extracts of nuclei A or B without PLA *).
treatment increased FFA concentration dose-dependently, up to 20.1 nmol with 1.5 U PLA,. Table I depicts the concentrations of individual fatty acids found in a representative experiment. The ether extract of nuclei A contained predominately saturated fatty acids (80%) and treatment with PLA, had little effect. The ether extract of nuclei B contained much higher concentrations of fatty acids, composed of 50% saturated and 50% unsaturated ones; treatment with 0.05 U PLA, increased FFA 2-fold due to an abundant rise of unsaturated (not of saturated) fatty acids.
This study was undertaken to explore putative regulatory mechanisms involved in the inhibition of nuclear T3 binding (INB) by fatty acids. First, the question was addressed whether rat liver nuclei themselves contain INB-activity. Nuclei indeed possessed INB-activity which was confined to the nuclear membrane: removal of the outer and most of the inner nuclear membrane results in a clear loss of INB-activity of the nuclear ether extracts. These results are in agreement with previous findings that by far the major part of the nuclear lipids is located in the membrane of the nuclear envelope [6]. The fatty acid content of the nuclei with a disrupted membrane was also 6-9-fold lower than of intact nuclei. The relative contribution of individual fatty acids to the total fatty acid content of intact nuclei was as follows: 16:0 (26.4%), 18:0 (22.8X), 18:l (12.1%), 18:2 (17.2%), 20:4 (15.7%), 22:6 (5.8%). These figures are very close to those previously reported [7,8]. From the IC,, values of the individual fatty acids [9], it can be calculated that the actual concentrations of the various fatty acids can account for the observed INB-activity. The data suggest that there is no need to implicate other lipids present in the nuclear ether extracts in the observed INB-activity. The second question to be addressed was whether nuclear phospholipids are amenable to the action of PLA,. PLA, activity results in the liberation of (unsaturated) fatty acids located in the 2 position of phospholipids. PLA, treatment of nuclei with disrupted membrane did not result in any INB-activity of these nuclei. In contrast, the INB-activity of intact nuclei increased greatly after PLA, treatment in a time- and
TABLE I
Nuclei A
Gas chromatographic analysis of the fatty acids present in the ether extract of nuclei A (disrupted nuclear membrane, I.04 mg DNA/ml) and of nuclei B (intact nuclear membrane, 1.40 mg DNA/ml) before and after incubation with 0.05 Units PLAl for I5 min. Nuclei B
(FFA nmol) Nuclei A -
+
-
0.38 0.33 0.13 _
(6.5)
0.36 0.29 0.16 _
(l.5)
I
0.06 -
1.86 1.61 0.85 1.21 1.11 0.41
1.48 1.09 1.27 3.88 5.96 0.24 1.30
0.81
0.90
7.05
15.22
PLA, incubation
0.1
0.01
PI+
1.0
u/tubed
Fig. 3. Effect of treating nuclei A (disrupted nuclear membrane, 3.40 mg DNA/ml) and nuclei B (intact nuclear membrane, 3.80 mg DNA/ml) with various doses of PLA, for 10 min on the free. fatty acid concentration (interrupted lines) and INB-activity (nuclear T3 binding expressed as percentage of controls without PLA,; continuous lines) contained in the ether extracts of these nuclei.
16:0 18:0 18:l 18:2 20:4 22~5 22:6 Total
Nuclei B
(15.5) * (70.0) (6.0) (6.5)
+
* Values between brackets indicate the amount of fatty acid (nmol) that reduces specific nuclear T3 binding to 50% of controls (IC,, values) [9].
134 dose-dependent manner. Again, the rise in the concentrations of the individual fatty acids could completely account for the observed increase of INB-activity induced by PLA,. The quantity of fatty acids which in theory could be released from nuclear phospholipids can also be calculated. In the experiment of Fig. 3, the absolute protein content of incubation vessels containing 0.1 ml suspension of nuclei B in a volume of 0.5 ml was 0.233 mg. Assuming 200 nmol phospholipid/mg protein the maximal release of fatty acids from the nuclear phospholipids is approx. 47 nmol (figures of the same order of magnitude are reached in calculations using slightly different data for the ratio of phospholipids to protein or to DNA [G-S]. The observed amount of FFA in these incubation vessels ranges from 4.1 nmol in the absence of PLA, to 20 nmol in the presence of 1.5 U PLA,. These figures are in the same order of magnitude as the amount of fatty acids required in an incubation volume of 0.5 ml for 50% inhibition of nuclear T3 binding (12-13 nmol as indicated by the IC,, values of unsaturated fatty acids). We conclude that nuclear phospholipids present in the nuclear membrane are amenable to PLA, activity. It is conceivable that the PLA, induced local release of fatty acids from the nuclear membrane might inhibit the binding of T3 to its nuclear receptors in vivo. Recent reports indicate the presence of PLA, in the cytosol, the nuclear membrane and the nuclear matrix [lo-131. Modulation of PLA, activity occurs in various disease states, and it might well be that increased PLAz activity during illness contributes to a decreased nuclear T3
binding via a local release of nuclear fatty acids. This hypothesis fits in the well-known adaptation of thyroid hormone metabolism during illness, but remains to be proven. Acknowledgements The authors are grateful to M. Platvoet and M. van Beeren for experimental assistance and to A. Nijenhuis for gas chromatographic analysis. References 1 Wiersinga, W.M.. Chopra. LJ. and Chua Teco. G.N. (1988) Metabolism 37, 99661002. 2 Van der KIis, F.R.M. and Wiersinga, W.M. (1989) FEBS Lett. 246. 6612. 3 Bloble. G. and Potter, V.R. (1966) Science 154. 1662-1665. 4 Labarca. C. and Paigen, K. (1980) Anal. Biochem. 102, 344352. 5 Fales. H.M. (1973) Anal. Chem. 45, 2302-2303. 6 Kleinig. H. (1970) J. Cell. Biol. 46, 3966402. 7 Frederiks, W.M., James, J.. Arnouts, C., Broekhoven, S. and Morreau, J. (1978) Cytobiologie 18. 254-271. 8 Khandwala. AS. and Kasper. C.B. (1971) J. Biol. Chem. 246, 6242-6246. 9 Wiersinga, W.M. and Platvoet- ter Schiphorst. M. (1989) Int. J. Biochem., 22. 269-273. 10 Pierik, A.J.. Nijssen, J.G., Aarsman, A.J. and Van den Bosch. H. (1988) B&him. Biophys. Acta 962. 345-353. 11 Nikitin, V.N., Babenko, N.A. and Popova. L.-Ya. (1985) Doklady Akademii Nauk. SSSR. 281, 731-734. 12 Karagezyan, K.G. and Tadevosgyan, Yu-V. (1986) Doklady Akademii Nauk SSSR. 287. 1248-1250. 13 Tamiya-Koizumi. K., Umekawa. H., Yoshida, S.. Ishihara, H. and Kojima, K. (1989) B&him. Biophys. Acta 1002, 182-188.
