EFFECTS OF ACUTE AND CHRONIC ETHANOL ADMINISTRATION ...

Alcohol & Alcoholism Vol. 36, No. 5, pp. 381–387, 2001

EFFECTS OF ACUTE AND CHRONIC ETHANOL ADMINISTRATION ON THE RESPONSE OF MOUSE ADIPOSE TISSUE HORMONE-SENSITIVE LIPASE TO α2-ADRENOCEPTOR ACTIVATION BY UK 14304 MEI-FEN SHIH1 and PETER V. TABERNER* Department of Pharmacology, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK (Received 8 November 2000; in revised form 7 March 2001; accepted 18 April 2001) Abstract — Untreated (control) obese CBA mice had lower hormone-sensitive lipase (HSL) activity and cAMP levels in brown adipose tissue than normal lean mice, but white adipose tissue HSL activity and cAMP were similar in obese and lean mice. In the obese mice, chronic ethanol treatment increased HSL activity and cAMP levels in both brown and white adipose tissue to above the levels in lean mice. In the lean mice, chronic ethanol only stimulated white adipose tissue. UK 14304 [5-bromo-6-(2-imidazolin2-ylamino)-quinoxaline: 2 mg/kg] inhibited HSL activity in both brown and white adipose tissues in lean mice, but a higher dose (3 mg/kg) was required to produce the same inhibition in obese mice. After chronic ethanol adipose tissues were more sensitive to UK 14304; only half the dose being required to produce the same level of lipase inhibition. We propose that, although chronic ethanol consumption increases cAMP levels in adipose tissue, particularly in obese mice, it also sensitizes the tissues to α2-adrenoceptor stimulation. These effects may explain the increased sympathetic nervous system activity observed in alcohol withdrawal.

α2-adrenoceptors; the balance between activation of the various adrenoceptor subtypes determines the final adipocyte response to circulating glucose, lipids or insulin (Lafontan and Berlan, 1993). Obese CBA mice appear to have lower Giα protein expression in white adipose tissue than lean mice, but show normal expression of Gsα protein (Palmer et al., 1992). Since the alcohol-withdrawal syndrome is associated with overactivity of the sympathetic nervous system, and many of the observable symptoms of withdrawal can be ascribed to the post-synaptic actions of noradrenaline (Linnoila et al., 1987; Glue et al., 1989), it might be expected that adrenoceptor activity will be affected by chronic ethanol consumption. Recently, Berggren et al. (2000) have reported that α2-adrenoceptor function (assessed by the growth hormone response to clonidine) is down-regulated in patients in acute alcohol withdrawal. Although it has been shown that chronic ethanol administration has little effect on adipose tissue β-adrenoceptor activity (Al-Qatari, 1993), α2-adrenoceptor activity in adipose tissue from either lean or obese CBA/Ca mice has not been investigated. Preliminary studies have indicated that the selective α2-agonist UK 14304 [5-bromo-6-(2-imidazolin-2ylamino)-quinoxaline] suppresses lipogenesis in both brown and white adipose tissue and that this effect is increased after chronic ethanol consumption (Williams et al., 1998). We have therefore used the same ethanol treatment protocol as previously to investigate whether chronic ethanol consumption has any effect on the response of brown and white adipose tissue hormone-sensitive lipase and cAMP levels to acute administration of UK 14304 in lean and obese CBA mice.

INTRODUCTION A proportion of male inbred CBA/Ca mice develop a spontaneous maturity-onset diabetic obese syndrome which is characterized by hyperphagia, hyperglycaemia, hyperinsulinaemia, islet hypertrophy, hypertriglyceridaemia, impaired glucose tolerance and insulin resistance (Connelly and Taberner, 1989; Figueroa and Taberner, 1994), but, unlike homozygous obese-diabetic rodent models (e.g. ob/ob, db/db, fa/fa), the male obese CBA mice have a normal life expectancy. This enables them to be used in chronic treatment studies. Previous work in this laboratory has shown that chronic ethanol drinking in these mice ameliorates the diabetes and obesity syndrome in terms of glucose tolerance and insulin resistance (Al-Qatari et al., 1996). Light to moderate ethanol consumption has also been shown to enhance insulin sensitivity in diabetics (Facchini et al., 1994). More recently we have demonstrated that chronic ethanol administration dose-dependently increases brown adipose tissue lipoprotein lipase and decreases hormone-sensitive lipase activities in normal CBA/Ca mice, whilst simultaneously increasing hormone-sensitive lipase activity in white adipose tissue (Shih and Taberner, 1997). In this respect, chronic ethanol treatment is having an insulin-like antilipolytic effect in brown fat, but a lipid-mobilizing effect in white fat. Following abstinence, there is a rebound increase in brown adipose tissue hormone-sensitive lipase activity (Shih and Taberner, 2000) which peaks at 9 h into withdrawal. Five adrenoceptor subtypes are present in adipose tissues: β1-, β2- and β3-adrenoceptors increase adenylyl cyclase activity, stimulating cAMP accumulation, which, in turn, activates hormone-sensitive lipase activity and, consequently, lipolysis. This cascade is inhibited by activation of postsynaptic α2-adrenoceptors. The release of noradrenaline from sympathetic nerves is regulated by presynaptic

MATERIALS AND METHODS Animals and diet Adult male CBA/Ca mice, aged 14–20 weeks, were bred within the University of Bristol School of Medical Sciences. They were housed eight mice per cage, at 20–22°C with controlled humidity on a 12 h light–12 h dark cycle. The dark

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Present address: Department of Pharmacy, Chai-Nan University of Pharmacy & Sciences, Tainan, Taiwan. *Author to whom correspondence should be addressed. 381

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period was between 21.00 and 09.00. The mice were provided with tap water and pelleted CRM high carbohydrate pellet diet ad libitum. At 16 weeks of age the mice were divided into two groups: normal (lean) males, defined as those mice which weighed 40 g with blood glucose >15 mM. Mice which did not meet these criteria were not used. All the animal studies described were conducted in accordance with the conditions approved under a Home Office Project Licence. Ethanol and drug treatments Ethanol was administered acutely intraperitoneally (i.p.) at a dose of 2.5 g/kg in a volume of 0.1 ml of saline per 10 g body weight. The assays for hormone-sensitive lipase and cAMP were performed 1 h later. For the chronic ethanol treatment, mice received 4% (w/v) ethanol solution as their sole drinking fluid (diluted from 95% ethanol; Hayman, Witham, UK) for 2 days, then 8% (w/v) ethanol solution for another 2 days, followed by 12% (w/v) ethanol solution for 3 days. Subsequently, a 20% (w/v) ethanol solution was given for 4 weeks to complete the chronic ethanol drinking schedule. The average daily intake of ethanol at this stage was 14.4 ± 1.5 g/kg body wt. UK 14304 was made up in sterile physiological saline and injected i.p. in a volume of 0.1 ml per 10 g body wt. In all groups of mice, hormone-sensitive lipase and cAMP were measured in both brown and white adipose tissues. Hormone-sensitive lipase preparation and assay Bilateral interscapular brown adipose tissue pads (BAT) were dissected out, weighed, minced, then homogenized in 10 volumes of medium containing 0.25 M sucrose, 1 mM EDTA, 4 mg/ml leupeptin, 1 mg/ml pepstatin A, and 1 mM dithiothreitol (pH 7.0). This was centrifuged at 105 000 g for 45 min at 4°C. The top ‘fat cake’ was removed and the clear infranatant faction was decanted and used for the enzyme assay by standard procedures (Shih and Taberner, 1997). Epididymal white fat pads (WAT) were removed and prepared as for BAT, except that 2 volumes of medium were added. Dried acetone powder extracts of brown and white adipose tissue were prepared as described by Carnheim et al. (1984) and stored at –20°C for use within 1 week. The protein concentrations of acetone powder extracts were determined using Coomassie Blue (Bradford, 1976). The triolein emulsion for the assay substrate was prepared freshly by the method described by Nilsson-Ehle and Schotz (1976) to give final concentrations in the 0.2 ml assay volume as follows: 100 mmol/l Tris–HCl, pH 7.0, 5 g/l of bovine serum albumin (fatty acid free fraction V), 250 mmol/l NaCl to inhibit lipoprotein lipase activity, and 4.58 mmol/l triolein emulsion (Nilsson-Ehle and Schotz, 1976). The reaction was initiated by adding 100 ml of the assay substrate. After a 15-min incubation at 37°C, the reaction was stopped by adding 3.25 ml of methanol:chloroform:heptane (ratio 14:12.5:10 by vol). The results were expressed as nmol fatty acids (FFA) released/min/mg of protein. cAMP assay Adipocytes were prepared according to Rodbell (1964), but incubated for 30 min in Krebs–Ringer phosphate buffer

(pH 7.4), containing 128 mM NaCl, 1.4 mM CaCl2, 1.4 mM MgSO4, 5.2 mM KCl, and 10 mM Na2HPO4, plus collagenase at either 3 mg/ml (BAT) or 1 mg/ml (WAT). The cell suspensions were washed three times in collagenase-free Krebs– Ringer phosphate buffer containing 4% bovine serum albumin (BSA), then 20 µl of 2 M HCl were added to a 200 µl volume of cell suspension. This mixture was vortexed, then centrifuged at 3000 g for 5 min at 4°C. The supernatant (150 µl) was collected and frozen at –20°C for subsequent cAMP measurement by a competition binding assay based on that described by Gilman (1970). Standard cAMP was diluted over the range 0.125–10 pmol/100 µl, and 100 µl of 0.5 mM standard cAMP used for estimation of the non-specific binding. The reaction was started by adding 100 µl binding protein (prepared from bovine adrenal cortex) into the assay tubes. After 90 min incubation at 4°C, 200 µl of ‘Charcoal’ (2.5 g charcoal/g of BSA in 50 µl of 50 mM Tris–HCl buffer containing 4 mM EDTA) were added and the tubes incubated for a further 20 min at 4°C. The samples were centrifuged at 3000 g for15 min at 4°C and the supernatant decanted into scintillation vials for counting. Sample values were determined from standard curves fitted to a logistic expression. The protein content of the cell suspensions was determined using Coomassie Blue and the results expressed as pmol of cAMP/mg protein. Reagents Triolein [C18:1,(cis)-9] [1,2,3-tri(cis-9-octadecenoyl) glycerol], oleic acid (cis-9-octadecenoic acid), phosphatidylcholine (L-αlecithin), leupeptin (acetyl-Leu-Leu-Arg-al) hemisulphate, pepstatin A (iso-valeryl-Val-Sta-Ala-Sta), DL-dithiothreitol, collagenase Type I, and cAMP were obtained from Sigma, Poole, Dorset, UK; sodium heparin (5000 units/ 5 ml) from Leo Laboratories Ltd, Princes Risborough, UK; glycerol tri-[9,10(n)-3H] oleate (185 MBq), [1-14C] oleic acid (1.85 MBq) and [8-3H] adenosine 3′,5′-cyclic phosphate, ammonium salt (9.25 MBq) from Amersham, Little Chalfont, UK. Data analysis The assays, conducted in triplicate unless otherwise stated, have been presented as means ± SEM. Data were combined from experiments conducted on four or five different days with the mice randomized between treatment groups to avoid bias. Statistical comparisons were made by unpaired Student’s t-test at a significance level of P < 0.05. RESULTS Effects of ethanol on hormone-sensitive lipase and cAMP Hormone-sensitive lipase activity in brown adipose tissue was lower in obese mice compared to lean controls (P < 0.05, Fig. 1a). Similarly, the cAMP level was also lower in the obese mice (Fig. 1b). Acute ethanol administration significantly decreased hormone-sensitive lipase activity in lean mice (P < 0.01) but not in obese mice. After chronic ethanol administration, lipase activity was significantly decreased in lean mice (P < 0.05), but, in contrast, was significantly increased in obese mice (P < 0.005), compared to their respective controls (Fig. 1a). Brown adipose tissue therefore responds in a qualitatively different manner in lean and

ETHANOL AND ADIPOSE TISSUE α2-ADRENOCEPTORS

Fig. 1. Effects of ethanol on hormone-sensitive lipase (HSL) activity and cAMP accumulation in brown adipose tissue. Hormone-sensitive lipase activity (a) is expressed as nmol free fatty acids released per min per mg of tissue protein. cAMP accumulation (b) is expressed as nmol/mg of tissue protein. Acute ethanol treatment was a single dose of 2.5 g/kg i.p. 60 min prior to the assays. Chronic ethanol treatment consisted of 20% ethanol as sole drinking fluid for 4 weeks. The results are shown as means ± SEM from 20–24 mice for lean groups and 8–10 mice for obese groups. *P < 0.05, **P < 0.01 ethanol group < control; ¶P < 0.05, ethanol group > control; †P < 0.05, lean versus obese control.

obese mice to the same schedule of ethanol treatment. These changes in brown adipose tissue lipase activity after the ethanol treatments were mirrored in the changes observed in cAMP accumulation (Fig. 1b). In contrast, hormone-sensitive lipase activity (Fig. 2a) and cAMP production (Fig. 2b) in white adipose tissue were almost identical in the lean and the obese mice, and neither parameter was affected by acute ethanol in both lean and obese mice. Chronic ethanol treatment, on the other hand, stimulated lipase activity (Fig. 2a) and increased cAMP production (Fig. 2b) in both lean and obese mice. Thus, the changes in hormone-sensitive lipase activity in brown and white adipose tissues observed after ethanol treatments in lean and obese mice may be mediated by alterations in cAMP level. Obese mice possessed a significantly greater mass of interscapular brown adipose tissue (Fig. 3a) and epididymal white adipose tissue (Fig. 4a) than lean mice, although the brown adipose tissue protein concentration in the obese mice was very much lower than that in the lean mice (Fig. 3b), suggesting that the greater tissue wet weight represented an increase in stored triglyceride content, rather than an increase

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Fig. 2. Effects of ethanol on hormone-sensitive lipase (HSL) activity and cAMP accumulation in white adipose tissue. Hormone-sensitive lipase activity (a) is expressed as nmol of free fatty acids released per min per mg of tissue protein. cAMP accumulation (b) is expressed as nmol/mg of tissue protein. Acute ethanol treatment was a single dose of 2.5 g/kg i.p. 60 min prior to the assays. Chronic ethanol treatment consisted of 20% ethanol as sole drinking fluid for 4 weeks. The results are shown as means ± SEM from 20–24 mice for lean groups and 8–10 mice for obese groups. ¶P < 0.05, ¶¶P < 0.01 ethanol > control.

in cell number. Despite the heavier tissue weight of white adipose tissue observed in the obese mice compared to the lean mice, the protein concentration in the tissues was very similar (Fig. 4b). Effects of UK 14304 on adipose tissue hormone-sensitive lipase activity In order to find the lowest effective dose of UK 14304 in vivo, the dose–response effect of this compound on hormonesensitive lipase activity was determined in brown and white adipose tissue from control mice which had received no alcohol; the results are shown in Fig. 5. The lowest dose of UK 14304 (1 mg/kg) had no significant effect in either brown (Fig. 5a) or white (Fig. 5b) adipose tissue in lean mice. Doubling the dose to 2 mg/kg significantly suppressed lipase activity (P < 0.01) in both tissues in lean mice but this dose did not alter the enzyme activity in obese mice. This suggests that the obese mice are less sensitive to the action of this α2-adrenoceptor agonist. On the basis of these findings, the effects of the lower dose (1 mg/kg) of UK 14304 were examined in chronic ethanol-treated mice in order to determine whether the

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Fig. 3. Effects of chronic ethanol drinking (CED) on tissue wet weight (a) and protein content (b) of brown adipose tissue in lean and obese mice. The results are shown as means ± SEM from 20–24 mice per group. ¶P < 0.05 obese > lean mice; **P < 0.01 obese < lean mice.

ethanol treatment restored the sensitivity of the lipase to α2-adrenoceptor activation. The results from brown and white adipose tissues are shown in Figs 6 and 7 respectively. In brown adipose tissue, UK 14304 (1 mg/kg) did not significantly affect the chronic ethanol-induced changes in lipase activity (Fig. 6a) or cAMP production (Fig. 6b) in either lean or obese mice. However, at the same time and in the same animals, hormone-sensitive lipase activity and cAMP production in white adipose tissue, which had been significantly increased (+43%) by the chronic ethanol treatment (Fig. 7a, b) compared to control untreated mice, were significantly decreased (–52%) by UK 14304. This effect was greater in the lean than in the obese mice, although the difference was not statistically significant. The decrease in cAMP production induced by UK 14304 had tended to match closely the decrease in hormone-sensitive lipase activity (Fig. 5); however, in ethanol-treated obese mice, there was a disproportionate decrease in hormone-sensitive lipase activity in white adipose tissue (–58%, Fig. 7a) compared to cAMP production (–40%, Fig. 7b) by this α2-adrenoceptor agonist. DISCUSSION With the aim of investigating further the selective ability of chronic ethanol to affect lipolysis and ameliorate the diabetic

Fig. 4. Effects of chronic ethanol drinking (CED) on tissue wet weight (a) and protein content (b) of white adipose tissue in lean and obese CBA mice. The results are shown as means ± SEM from 20–24 mice per group. ¶P < 0.05 experimental group > control lean.

condition, we have compared the sensitivity of adipose tissue α2-adrenoceptors to agonist stimulation in normal (lean) and obese–diabetic CBA/Ca mice. Although we have previously demonstrated that chronic ethanol consumption can produce changes in brown and white adipose tissue lipoprotein lipase and hormone-sensitive lipase activity in normal lean mice (Shih and Taberner, 1997), this is the first time that changes in hormone-sensitive lipase activity in brown and white adipose tissue have been measured in obese diabetic mice. Our previous finding, that white adipose tissue hormone-sensitive lipase activity in lean mice increased after chronic ethanol treatment (Shih and Taberner, 1997), was confirmed in the present studies and was shown also to occur in the obese–diabetic mice. The changes observed in hormonesensitive lipase activity paralleled those in cAMP production in obese mice after ethanol treatments, strongly suggesting that the effects of ethanol on hormone-sensitive lipase activity are mediated via cAMP accumulation. Ethanol and adipose tissue The enlarged brown and white fat pads observed in obese mice could be due either to increased adipocyte size or cell number. However, the lower protein content of brown (Fig. 3)

ETHANOL AND ADIPOSE TISSUE α2-ADRENOCEPTORS

Fig. 5. Dose–response effects of UK 14304 on hormone-sensitive lipase (HSL) activity in brown and white adipose tissue. Hormone-sensitive lipase activity in brown (a) or white (b) adipose tissue is expressed as nmol of free fatty acids released per min per mg tissue protein. The results are shown as means ± SEM from groups of six mice each and were combined from different experimental days. Mice received saline or UK 14304 60 min prior to the assay. **P < 0.01 versus saline control; ¶P < 0.05, ¶¶P < 0.01 obese saline versus lean saline.

and white (Fig. 4) adipose tissues in obese mice indicates that the increased tissue wet weight is more likely a consequence of cell enlargement. Chronic ethanol treatment restored the adipose tissue mass to normal in the obese mice, suggesting that this drinking regime may either be inhibiting fat storage or stimulating fat breakdown. The stimulation of hormonesensitive lipase activity in white fat by chronic ethanol administration indicates that it is fat breakdown (lipolysis) which is being increased. It has been shown that long-term

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Fig. 6. Effects of UK 14304 on hormone-sensitive lipase (HSL) activity and cAMP accumulation in brown adipose tissue. Hormone-sensitive lipase activity (a) is expressed as nmol of free fatty acids released per min per mg of tissue protein. cAMP accumulation (b) is expressed as nmol/mg of tissue protein. Control (untreated) and chronic ethanol drinking (CED) groups received saline or UK 14304 (1 mg/kg) 60 min prior to the assay. The results are shown as means ± SEM from 20–24 mice for lean groups, 16–20 mice for obese groups, and 8–10 mice for UK 14304-treated groups. †P < 0.05 obese versus lean; *P < 0.05 chronic ethanol < control; ¶P < 0.05 chronic ethanol > control.

ethanol consumption in man affects the development of brown adipose tissue (Huttunen and Kortelainen, 1990); the protein content of adipose tissue samples from alcoholic patients was observed to be twice that of controls. These latter authors concluded that chronic ethanol intake may induce a change of the white fat, particularly around the thoracic aorta and common carotid arteries of human adults, into brown fat. Thus, chronic ethanol intake could also facilitate fat cell differentiation from white to brown in both lean and obese CBA mice. Ethanol and adrenoceptor function Obese Zucker rats are known to exhibit relatively lower sympathetic nervous system tone in brown adipose tissue (York et al., 1985), and lower lipogenic rates in brown fat have

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Fig. 7. Effects of UK 14304 on hormone-sensitive lipase (HSL) activity and cAMP accumulation in white adipose tissue. Hormone-sensitive lipase activity (a) is expressed as nmol of free fatty acids released per min per mg of tissue protein. cAMP accumulation (b) is expressed as nmol/mg of tissue protein. Control (untreated) and chronic ethanol drinking (CED) groups received saline or UK 14304 60 min prior to the assay. The results are shown as means ± SEM from 20–24 mice for lean groups, 16–20 for obese groups, and 8–10 mice for UK 14304-treated groups. ¶¶P < 0.01 chronic ethanol > control; **P < 0.01 UK 14304 versus control.

also been shown in obese CBA mice (Mercer et al., 1992). Low levels of β-adrenoceptor activation in brown adipose tissue might therefore be responsible for the low level of brown fat hormone-sensitive lipase activity in obese CBA mice. Chronic ethanol intake has been shown to stimulate sympathetic activation of brown adipose tissue in both animals and man (Nicholls, 1979; Huttunen and Kortelainen, 1988, 1990). Therefore, we might expect the reduced brown adipose tissue lipase activity observed in obese mice to be restored after chronic ethanol consumption. The different effects of acute and chronic ethanol treatments we have observed are more likely to be due to long-term adaptive changes in enzyme activity than a difference in dose, since the acute dose of ethanol was selected to produce similar plasma ethanol levels (between 5 and 10 mM) after 60 min to those previously

observed at 09.00 with the same chronic ethanol drinking schedule (Jelic et al., 1998). Untreated (control) obese mice were markedly less sensitive to the α2-adrenoceptor agonist, UK 14304, than the lean mice (see Fig. 5a, b) although a dose-dependent effect was observed in both groups. Higher doses of UK 14304 than those used here could not be used since the drug tends to reduce body temperature, which would affect brown adipose tissue activation by stimulating thermogenesis. In addition, α2-agonists have marked sedative properties and some (e.g. xylazine), are employed as veterinary sedatives. One possible explanation for the loss of receptor sensitivity could be that obese CBA mice express less Giα protein in white fat than lean mice (Palmer et al., 1992). Alternatively, increasing fat cell size is associated with decreasing α2-adrenoceptor activity (Arner et al., 1987). Catecholamine resistance in obese patients has been linked to α2-adrenoceptor sensitivity, which is increased during diet-induced weight loss (Hellstrom et al., 1997). However, it has not previously been reported whether chronic ethanol has any effect on adipose tissue α2-adrenoceptor activity. At low concentrations, noradrenaline interacts mainly with the α2-adrenoceptor, its activation exerting a tonic inhibitory effect on adipose tissue lipolysis. Conversely, when high concentrations of noradrenaline exist in the fat cell environment, β-adrenoceptors are maximally stimulated, and their activation largely masks the modulatory inhibitory action linked to α2-adrenoceptor stimulation. Therefore, the increased α2-adrenoceptor sensitivity and increased hormone-sensitive lipase activity seen in white adipose tissue after chronic ethanol treatment can be reconciled, since ethanol increases sympathetic activity in adipose tissue (Huttunen and Kortelainen, 1988) and the inhibitory effect of α2-adrenoceptor activation would be overcome by the greater activating effect mediated via β-adrenoceptors. Chronic ethanol treatment has been shown to increase Giα protein expression in mouse brain (Wand et al., 1993). Thus, sensitization of α2-adrenoceptor activity by chronic ethanol may be mediated through an increase in Gi protein expression. Future binding studies in adipocytes using selective adrenoceptor ligands, together with the measurement of Gi protein expression, should answer this question. Acknowledgements — We are grateful to Dr Eamonn Kelly at the University of Bristol for providing the binding protein and Tocris Cookson Ltd, Bristol, for a gift of UK 14304.

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