Endotoxin inhibition of glucocorticoid enzyme induction and in vivo3H-dexamethasone labelling of rat liver nuclei

hf. J. Biochem. Vol. 21, No. 6, pp. 701-705, Printed in Great Britain. All rights reserved

1989 Copyright

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0020-711X/89 $3.00 + 0.00 1989 Perpmon Press plc

ENDOTOXIN INHIBITION OF GLUCOCORTICOID ENZYME INDUCTION AND IN VW0 3H-DEXAMETHASONE LABELLING OF RAT LIVER NUCLEI KALINA I. VAPTZAROVA,’ EUGENIA N. BARAMOVA’and PANTELEYG. Popov’ ‘Regeneration Research Laboratory, Bulgarian Academy of Sciences, ul. Zdrave 2, 1431 Sofia, Bulgaria *State Institute of Drugs, boul. Zaimov 26, 1040 Sofia, Bulgaria (Received 14 November 1988)

Abstract-I. The effect of endotoxin on glucocorticoid (GC) induction of liver TO and TAT was investigated. 2. It was found that endotoxin inhibited not only TO GC induction, but also that of TAT, though to a lesser extent (17.41%). 3. Endotoxin did not influence the binding capacity of liver cytosol for ‘H-dexamethasone at the second hour after the toxin administration. 4. In in vivo experiments endotoxin inhibited with 57.2% the binding of 3H-dexamethasone to hepatic nuclei. 5. It is suggested that the lower extent of endotoxin inhibition of GC induction of TAT may be due to the counteracting action of some inductor(s) for TAT only.

INTRODUCTION Endotoxin, the lipopolysaccharide component of the cell wall of Gram negative bacteria, causes a

pronounced hypoglycemia in which hepatic gluconeogenesis play a major role in experimental animal8 (McCallum and Berry, 1972). The administration of glucose results in a temporary relief but has little effect on mortality rate of endotoxin treated animals (Berry, 1971). In this case the application of glucocorticoid hormones (GCH), have a pronounced lethality-preventing action (White et al., 1978) and stimulate gluconeogenesis by induction its key enzymes (Goldstein et al., 1962; Shrago et al., 1963). At present it is generally accepted that glucocorticoid (GC) induction of liver enzymes involves the following steps: penetration of the hormone into the cell, binding to the specific receptors, activation of the hormone-receptor complexes (HRC), interaction of the latter with so-called GC responsive elements in the regulatory enhancer sequences of the inducible structural gene of the respective enzyme (Gluecksohn-Waelsh, 1987). Berry et al. (1980) have demonstrated that endotoxin suppresses the GC induction of the following enzymes: phosphoenolpyruvate carboxykinase (EC 4.1.1.32.) (PEPCK); glucose-6-phosphatase (EC 3.1.3.9.); fructose-1,ddiphosphatase (EC 3.1.3.11.); glycogen synthase (EC 2.4.2.11.) and tryptophan oxygenase (EC 1.13.11.11.; TO). The precise mechanism of the inhibiting effect of endotoxin remains unknown. Neither it is clear why endotoxin does not block the GC induction of tyrosine aminotransferase (EC 2.6.1.5.; TAT). GCH penetration into the cell is not affected in endotoxemia (Berry and Schackleford, 1984). The binding capacity of GC receptors (GCR) after endotoxin administration, however, is a subject

of controversy. According to the data of Stith and McCallum (1983), the binding of ‘H-dexamethasone (3HDM) to the cytosol receptors declines 6 hr after endotoxin administration. Berry and Schackleford (1984) however, fail to demonstrate any changes in the binding capacity of receptors 4 hr after injection of endotoxin. The effect of endotoxin on the HRCchromatin interaction is even less clear. Tavakoli and Moon (1981) in in vitro experiments incubated chromatin with activated HRC in presence of native or alkaline-detoxified endotoxin, the latter acting as a control since it does not suppress PEPCK induction by GCs. The results obtained demonstrate, that both types of endotoxin reduce the binding of activated HRC to chromatin. Berry (1971) suggests, that the effect of endotoxin as an inhibitor of GC induction of liver enzymes is not a direct one but is mediated by a proteinacious factor named by Berry “glucocorticoid antagonizing factor” (GAF), released from the cells of the reticuloendothelial system. Taking into account the data suggesting a possible intranuclear localization of steroid receptors (Gase and Baulieu, 1986) and the results demonstrating that steroid hormones are required for receptors to bind DNA in vivo, but not in vitro (Groyer et al., 1987) we investigated the in vivo 3HDM distribution in hepatocytes from adrenalectomized endotoxin-treated rats. We have also attempted to elucidate the lack of effect of endotoxin on TAT GC induction. MATERIALSAND METHODS Endotoxin (phenol-extracted from Salmonella typhimurium), L-tryptophan, hemin, a-ketoglutarate and dexamethasone were purchased from Sigma. ‘HDM with sp. act. 40 Ci/mmol was obtained from Amersham. 701

KALINA I. VAPTZAROVAet al.

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Table 2. Effect of endotoxin on TAT GC induction

Treatment of animals All experiments were carried out on male albino Wistar rats weighing 120-15Og. The animals were adrenalectomized translumbarly and used in experiments on the 4th day after the operation. During this period the rats were given food and saline ad libitum. The animals were killed between 9 and 12 a.m. in order to minimize the influence of diurnal variation of enzyme activity. 6-methylprednisolone microcrystalline suspension (“Urbasone”(6-MP), Hoechst) was administered i.p. at a dose of 20 mg/kg body weight. Endotoxin, dissolved in saline was injected i.p. at a dose of 0.1 mg/kg which was determined to be approximately LD,, for adrenalectomized rats. To animals receiving both substances, endotoxin was administered first and 6-MP followed after 2 hr. The TO and TAT activities were determined 4 hr after the injection of 6-MP.

Treatment 1. 2. 3. 4.

Controls Endotoxin 6-MP Endotoxin & CLMP

Enzyme activity pmol p-hydroxyphenyl pyruvate/hr per g wet liver (X + SD) 257.08 + 244.14 f 1052.63 f 870.75 +

13.68 28.35 37.64 67.06

No. of animals IO IO 16 12

P (P > 0.05) (P < 0.001)’ (P < 0.02)b

Enzyme activity was determined 4 hr after endotoxin (0.1 mg/kg) and 6-MP (20mg/kg) administration. To animals receiving both substances endotoxin was administered first and 6-MP followed after 2 hr; %ompand with controls; %ompand with 6-MP treated rats.

TO activity assay The activity of the enzyme was determined according to Knox et al. (1966). In short the livers were quickly excised, chilled on ice, minced and homogenized with 3~01 (w/v) of cold buffer (2mmol/l L-tryptiphan, 14Ommol/l KCl, 0.2 mol/l sodium nhosuhate. DH 7.0) and centrifuged 30 min at 12,odo g at 4°C. Thi up,; lipid iayer was dis&ded and supematant was used as enzyme preparation. Enzyme activity was calculated using an extinction coefficient for kynurenin of 4500 cm*. mmol- I.

In vivo ‘HDM labeiling of hepatic nuclei and cytosols

TAT activity assay The livers were processed as above and homogenized with 9 vol (w/v) of ice-cold buffer (0.125 mol/l K,HPO,, 0.125 mol/l KH,PO,, pH 7.6). Homogenates were centrifuged at 12,000 g for 15 min. The supematant was used for TAT activity assay according to Diamondstone (1966). Enzyme activity was calculated using an extinction coefficient of 19,900 cm2.mmol-’ for p-hydroxybenzaldehyde formed under the assay conditions. The results obtained for both enzymes are expressed as units per gram wet weight of liver, 1U being defined as the amount of enzyme, catalysing the formation of 1pmol product per 1 g wet weight of liver for 1hr at 37”C, after activation of the latent forms of these enzymes by preincubation with the cofactors of TO and TAT. In vitro GC receptor binding capacity assay The rat livers were homogenized in two volumes of cold buffer (20 mmol/l Tris, 3 mmol/l MgCl,, 0.15 mmol/l EDTA, 1mmol/l 2-mercaptoethanol, pH 7.6) in a PotterElvehjem glass-teflon homogenizer. Homogenates were centrifuged at 10,OOOgfor 15 min. The supematants were centrifuged in 4°C at 105,OOOgfor 6Omin. The resultant supernatants are referred to as cytosols. Protein concentration was determined by the method of Gomall et al. (1949). Cytosol protein concentration was adjusted to 23 mg/ml with buffer. The steroid binding capacity was determined by the method of Baxter and Tomkins (1970). Free 3HDM in incubation mixture was separated from

Table

I. Effect of endotoxin on TO nlucocorticoid induction Enzyme activity ymol kynureninei hr/g wet liver

Treatment I. Controls 2. Endotoxin 3. 6.MP 4. Endotoxin

bound by dextran-coated charcoal (0.1% Dextran T-500, Pharmacia, and 1% activated charcoal, Merck) suspended in buffer. Scatchard (1949) plots were constructed with 5 dilutions of ‘HDM and experimental points were fitted according to a linear regression equation. Nonspecific binding was determined by adding nonlabelled dexamethasone in IOOO-fold excess. Specific steroid receptor binding of liver cytosols was determined 2 hr after endotoxin administration.

No. of

(x f SD)

animals

6.85 * 0.47 6.00 5 0.40 30.19 * 1.88 13.19 +_2.39

5 6 6 6

P (P > 0.05) (P < 0.001) (P < 0.001)b

+6-MP Enzymeactivitywas determined 4 hr after cndotoxin (0.1mg/kg)and 6-MP (20mg/kg) administration. To animals receiving both substancesendotoxin was administeredtint and 6-MP aher 2 hr; ‘compared with controls; %ompared with 6.MP treated rats.

‘HDM was injected i.p. at a dose of 38 PCi per 100 g body weight 2 hr after endotoxin administration and the animals were killed 30min later. The isolation of hepatic nuclei was performed according to the method of Dabeva et al. (1978). In brief, livers were homogenized 1:2 (w/v) in cold buffer (20 mmol/l Tris, 6 mmol/l I&$&, 0.25 mol/l sucrose, 1 mol/l 2-mercautoethanol. DH 7.6) in a Potter-Elvehiem gla&eflon homogenizer 4th a lbose pestle. The hornogenates were centrifuged for 10min at lO,OOOgat 4°C. The supematants were collected, recentrifuged at 105,000 g, treated with dextran-charcoal to remove the unbound 3HDM, and radioactivity was counted for determination of cytosol in viva labelling. The sediments were resuspended with cold buffer (62Ommol/l Tris, 2.3 mol/l sucrose, 1 mmol/l MgCl,, 2 mmol/l CaCl,, pH 7.6) and carefully layered on 2.3 mol/l sucrose. After 60 min centrifugation at 20,000 rev/min in a MSE 6 x 16.5 swing-out rotor (700001 at the bottom of the tubes), the nuclei were resuspended and twice washed with ice-cold buffer (20 mmol/l MnCl,, 0.25 mol/l sucrose, pH 7.6). The last ‘sediment was r& suspended in distilled water, the DNA content determined according to the method of Burton (1956) and the radioactivity of an aliquote was counted in a Triton X-lOOtoluene (1:3)-based scintillation cocktail with 38% efficiency in a Beckman LS 9000 counter. The purity of nuclear fraction was checked by light microscopy as follows: the nuclear suspension war tied in 4% f&rnaldehyde for 10-15 min. Samnles were taken. stained with Toluidine Blue and differentiated in 96% ethanol. Statistical analysis of the data was performed using the Student’s r-test for two means, P < 0.05 was adopted as the level for significant difference. RESULTS

GCH had a pronounced inducing effect on rat liver TAT and TO activity. The data in Tables 1 and 2 demonstrate that 4 hr after 6-MP administration in adrenalectomized rats there was 4.40-fold increase in TO (P < 0.001) and 4.19-fold increase in TAT activity (P < 0.001). Endotoxin did not alter the basal activity of either enzyme. The simultaneous application of 6-MP and endotoxin resulted in a 56.61%

703

Endotoxin inhibition of GC enzyme induction (P < 0.001) inhibition of TO induction and 17.41% (P < 0.02) inhibition of the GC induction of TAT. These results suggest that endotoxin exerts a less pronounced but still measurable inhibitory effect on TAT GC induction. It is also possible, however, that an endotoxin suppression of GC TAT induction similar in degree to that of TO, is masked by stimulation of TAT by a specific inducer. TO and TAT GC induction follows after a latent period of about 90min after the hormone injection (Granner et al., 1970). An increase in enzyme activity is noted on the 2nd hr and a peak is reached between the 4th and 6th hr after the hormone application. Penetration of the hormone into the cell, receptor binding and stimulation of gene expression take place during the latent period. The most pronounced effect of various factors interfering with GC induction could be expected during this period. On the other hand, Berry and Schackleford (1984) point out that 2 hr after endotoxin administration, the plasma GAF level is sufficient to inhibit the GC induction of PEPCK. Taking into account these data, we studied the in vitro binding of ‘HDM to the cytosol of control and endotoxin-treated animals, 2 hr after the lipopolysaccharide (LPS) injection. No difference has been found in the 3HDM binding capacity of cytosol from control and experimental animals: 580 pmol/mg Pr, Kd = 11.63 x 10-9mol/1 for the control and 540 pmol/mg Pr, K,, = 11.49 x 10e9 mol/l for the endotoxin treated animals (Fig. 1). In the in vivo experiments, ‘HDM was injected 2 hr after the endotoxin administration. The animals were killed 30 min later. There is no significant difference (P > 0.05) in the in oivo binding in liver cytosol in control and treated animals, 2 hr after endotoxin application (Fig. 2). Surprisingly, the binding of 3HDM-receptor complexes to hepatic nuclei in oivo is inhibited in endotoxin-treated rats by 57.2% (P < 0.001). The data obtained for controls (2.26 fmol ‘HDM/pg DNA) are in a good agreement with the results of Tulp and Sluycer (1977) and

0’F 0.06

I 100 200

300 LOO 500

B f mollmg

600

Prohin

Fig. 1. Endotoxin influence on steroid binding capacity of glucocorticoid receptors. In parentheses: the number of experimental animals; 1 and (0) control rats; 2 and (0) endotoxin treated (0.1 mg/kg).

A

h w.1(19) 1

2

Fig. 2. Endotoxin influence on binding of ‘H-DM in oiuoto hepatic cytosol (A) and nuclei (B). In parentheses: the number of experimental animals; l-control rats; 2-endotoxin treated (0.1 mg/kg).

suggest that the radioactivity found in hepatocytes’ nuclei corresponds to the bound HRC. In in vivo experiments, GC induction may be expected to be maximal and the same is true with the endotoxin effect. A significant inhibition of nuclear ‘HDM labelling is observed in these experiments. In its nature and time of occurrence this phenomenon could be related to the inhibitory effect of endotoxin on TO and TAT GC induction. DISCUSSION

The results presented raise the question: why the ‘HDM label in hepatocytes’ nuclei is so strongly reduced in endotoxemia? There are two possibilities worthy to be discussed: (1) in in vivo experiments endotoxin could interfere with the GCH penetration in the cells or (2) it could interfere directly or indirectly with the binding of HRC to chromatin. Shackleford et al. (1986) have demonstrated that endotoxin has no effect on the GCH penetration in the cells, receptor binding capacity or activation of HRC. We have also found that there is no difference in cytosol labelling in control and endotoxin-treated animals. Some recent results of Ser8ing et al. (1985) corroborated the idea of direct binding of LPS to the metallothionein gene. It could be supposed a direct binding of endotoxin to the genes of enzymes inducible by GC. In such a case endotoxin could prevent at least partially the presence of 3HDM in the nucleus. A lowered level of labelled corticosterone binding in hepatocyte’s nuclei of endotoxin-treated mice has been noted by Agarwal(1972). The interpretation of these results remained difficult due to the vast excess of unlabelled cortisone. In our experiments the interference of endogenous corticosterone was precluded by adrenalectomy of the rats. Goodrum and Berry (1978) demonstrated that endotoxin does not block GC induction when added to hepatoma cell culture. GC induction of hepatic enzymes was suppressed by the addition of serum from endotoxin-treated mice. These and a number of other data led Berry et al. (1984) to hypothesize the

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existence of GAF. In a recent publication Sakaguchi and Jokota (1987) revealed that the injection of 100 pg of purified GAF (a glycoprotein, IU, 90,ooO) inhibited entirely the TO GC induction, whereas TAT activity remains little or not affected. It is well known that endotoxin induces the action of a number of biologically active mediators such as insulin-like mediator (Filkins, 1980), interleukin I, interferon, prostaglandins etc. (Mathison and Ulevitch, 1983). For this reason it is difficult to decide whether the nature of endotoxin effect on HRC-chromatin interaction, is direct or mediated. If one considers the possibility that the direct LPS binding to hepatocytes’ nuclei in endotoxemia is related to the inhibition of gluconeogenesis key enzymes, the question arises as to why TAT induction solely remains unaffected? Our data obtained from large number of animals (12), demonstrate, that GC induction of TAT is suppressed by endotoxin, although to a lesser extent than that of TO (Tables 1 and 2). This could be due to a selectively weaker

interaction of endotoxin with TAT gene, for reasons which remain unknown. It is even more difficult to understand this difference because according to the latest results of Beato et al. (1986) the binding sites for GC receptors on GC regulated genes are the same or, have nearly identical nucleotide sequence. The following possibility should also be taken into account: endotoxin may have a similar inhibitory effect on the induction of all the enzymes inducible by GC, but could activate an inducer specific for TAT only. In this case the suppression on GC induction of TAT would be masked. GCH inducible enzymes are subjected to multihormonal control. On the other hand, endotoxin causes an enhanced activity of many endocrine glands by increasing the secretion of ACTH (Ando et al., 1964), insulin (Jones and Yelich, 1987), glucagon (Ishida, 1985), and catecholamines (Benedeczky and Bertok, 1968). The increased insulin secretion in endotoxemia is of special interest, since this hormone totally inhibits the inducing effect of DM and CAMP on PEPCK (Chu et al., 1987). At the same time the simultaneous application of insulin and GCH leads to a pronounced additive induction of TAT (Holten and Kenney, 1967). It have to be noted, that endotoxin inhibited GC induction of TO, whereas that of TAT progressed unaltered in viva even in isolated, perfused rat liver (Agarwal, 1975), which should eliminate the action of secreted insulin. The action of insulin-like mediator, however, and that of intracellular CAMP cannot be eliminated. The mechanism of interaction of these factors (synergists as well as antagonists) on the binding of HRC to the GC responsive elements in the regulatory sequences of the GC inducible structural gene in endotoxemia remain to be clarified.

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