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
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ABSTRACT |
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INTRODUCTION |
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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 anti-lipolytic 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: ß1-, ß2- and ß3-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 2-adrenoceptors. The release of noradrenaline from sympathetic nerves is regulated by presynaptic
2-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 Gi
protein expression in white adipose tissue than lean mice, but show normal expression of Gs
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
2-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 ß-adrenoceptor activity (Al-Qatari, 1993),
2-adrenoceptor activity in adipose tissue from either lean or obese CBA/Ca mice has not been investigated. Preliminary studies have indicated that the selective
2-agonist UK 14304 [5-bromo-6-(2-imidazolin-2-ylamino)-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.
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MATERIALS AND METHODS |
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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 TrisHCl, 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 KrebsRinger 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 KrebsRinger 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 0.12510 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 TrisHCl 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-lecithin), 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.
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RESULTS |
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DISCUSSION |
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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) 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 hormone-sensitive 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 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 also been shown in obese CBA mice (Mercer et al., 1992
). Low levels of ß-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 2-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,
2-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 Gi
protein in white fat than lean mice (Palmer et al., 1992
). Alternatively, increasing fat cell size is associated with decreasing
2-adrenoceptor activity (Arner et al., 1987
). Catecholamine resistance in obese patients has been linked to
2-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
2-adrenoceptor activity.
At low concentrations, noradrenaline interacts mainly with the 2-adrenoceptor, its activation exerting a tonic inhibitory effect on adipose tissue lipolysis. Conversely, when high concentrations of noradrenaline exist in the fat cell environment, ß-adrenoceptors are maximally stimulated, and their activation largely masks the modulatory inhibitory action linked to
2-adrenoceptor stimulation. Therefore, the increased
2-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
2-adrenoceptor activation would be overcome by the greater activating effect mediated via ß-adrenoceptors.
Chronic ethanol treatment has been shown to increase Gi protein expression in mouse brain (Wand et al., 1993
). Thus, sensitization of
2-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.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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* Author to whom correspondence should be addressed.
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