Desensitization of cyclic GMP-mediated regulation of fatty acid metabolism in hepatocytes from ethanol-fed rats

Desensitization of cyclic GMP-mediated regulation of fatty acid metabolism in hepatocytes from ethanol-fed rats

The International Journal of Biochemistry & Cell Biology 37 (2005) 655–664 Desensitization of cyclic GMP-mediated regulation of fatty acid metabolism...

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The International Journal of Biochemistry & Cell Biology 37 (2005) 655–664

Desensitization of cyclic GMP-mediated regulation of fatty acid metabolism in hepatocytes from ethanol-fed rats Javier Garc´ıa-Villafranca, Alberto Guill´en, Jos´e Castro∗ Departamento de Bioqu´ımica y Biolog´ıa Molecular I, Facultad de Biolog´ıa, Universidad Complutense, 28040 Madrid, Spain Received 5 April 2004; received in revised form 20 July 2004; accepted 7 September 2004

Abstract The mechanisms by which ethanol causes accumulation of hepatic triacylglycerols are complex. It has been proposed that nitric oxide/cyclic GMP signaling pathway may be involved in regulation of fatty acid metabolism in the liver. Here, we investigated if this mechanism may have a role in adaptation to ethanol consumption. Hepatocytes were isolated from rats fed with an ethanolcontaining liquid diet and pair-fed control rats, and incubated with a range of concentrations of 8-bromo-cyclic GMP. In both types of cells, this cyclic GMP analog inhibited in parallel fatty acid synthesis de novo and acetyl-CoA carboxylase activity. Addition of 8-bromo-cyclic GMP also decreased the rate of palmitate esterification to triacylglycerols and phospholipids, whereas palmitate oxidation was increased. However, in all these metabolic effects, hepatocytes from ethanol-fed rats were significantly less sensitive to the addition of 8-bromo-cyclic GMP. In order to know if this may be a more general mechanism of adaptation to ethanol, we also studied the effects on glucose metabolism. Similarly, hepatocytes from ethanol-fed rats showed a decreased sensitivity in the inhibition by 8-bromo-cyclic GMP of glycogen synthesis, fatty acid synthesis and the synthesis of glycerol backbone of hepatic triacylglycerols. These data suggest that ethanol consumption induces a desensitization of the regulatory effects mediated by cyclic GMP in fatty acid metabolism, contributing to triacylglycerol accumulation in the liver. © 2004 Elsevier Ltd. All rights reserved. Keywords: Cyclic GMP; Fatty acid metabolism; Acetyl-CoA carboxylase; Hepatocyte; Ethanol

1. Introduction Chronic ethanol consumption produces marked alterations in lipid metabolism, one of the most significant being accumulation of hepatic triacylglycerols, resulting in fatty liver (Baraona & Lieber, 1998; Day ∗ Corresponding author. Tel.: +34 913944157; fax: +34 913944672. E-mail address: [email protected] (J. Castro).

1357-2725/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2004.09.004

& Yeaman, 1994). Various causes for this steatosis have been proposed. In early stages of ethanol administration, these mechanisms are related to ethanol oxidation by alcohol dehydrogenase, generating acetaldehyde and increasing NADH/NAD+ ratio (Lieber, 1994). After chronic alcohol consumption, the development of fatty liver persists despite the attenuation of the redox change (Salaspuro, Shaw, Jayatilleke, Ross, & Lieber, 1981), indicating the participation of other mechanisms, such as the stimulation of triacylglycerol

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synthesis, by increasing the levels of substrates glycerol 3-phosphate and fatty acids and by stimulating the activities of triacylglycerol-synthesizing enzymes (Savolainen, Baraona, Pikkarainen, & Lieber, 1984), as well as the impairment of mitochondrial fatty acid oxidation (Guzm´an & Castro, 1990; Lieber, 1994), concomitant to a decreased activity of carnitine palmitoyltransferase I (Guzm´an & Castro, 1990; Guzm´an, Castro, & Maquedano, 1987). Cyclic GMP (cGMP) is a second messenger in a large number of biological actions in various cell types (Robbins & Grisham, 1997; Schmidt, Lohmann, & Walter, 1993). Signaling pathways leading to these responses are mediated by the activation of any of multiple types of soluble and particulate guanylyl cyclases which catalyze the synthesis of cGMP (Lucas et al., 2000), and this may modulate cGMP-dependent protein kinases, cGMP-gated ion channels, cGMPregulated phosphodiesterases and, under certain conditions, cyclic AMP-dependent protein kinases. It is well known that many of the biological actions of nitric oxide (NO), but not all, are mediated by the direct activation of soluble guanylyl cyclase and the consequent increase in intracellular cGMP levels (Robbins & Grisham, 1997). We have recently reported that NO/cGMP signaling pathway is endowed with regulatory properties in fatty acid metabolism in rat hepatocytes (Garc´ıa-Villafranca, Guill´en, & Castro, 2003). Stimulation of this pathway inhibits in parallel fatty acid synthesis and acetyl-CoA carboxylase (ACC) activity, and these effects seem to be mediated by activation of cGMP-dependent protein kinase. In addition, cGMP analogs increase fatty acid oxidation activity at the expense of lipogenesis, suggesting a physiological role in the control of this metabolism in the liver (Garc´ıa-Villafranca et al., 2003). This is interesting, because these are opposite effects to those of ethanol administration. Therefore, in this study we went further to investigate if this cGMP-mediated mechanism may be altered in adaptation to ethanol consumption. We studied the effects of 8-bromo-cyclic GMP (8-Br-cGMP) on the main fatty acid-metabolizing pathways and ACC activity in hepatocytes from control and ethanol-fed rats. Our data show that ethanol administration leads to a desensitization of the effects mediated by this cGMP analog, which may facilitate lipid accumulation in hepatocytes, suggesting a physiological role of cGMP in metabolic adaptation to ethanol consumption.

2. Materials and methods 2.1. Materials Radiochemicals were all supplied by Amersham Biosciences. Collagenase (type I), digitonin, bovine serum albumin (BSA), NAD-ADH assay for enzymatic determination of ethanol, 8-Br-cGMP, and all chemicals and components of animal diets were purchased from Sigma-Aldrich. 2.2. Animal care and feeding Male Sprague–Dawley rats (150–170 g initial body weight) were individually housed, kept at controlled temperature, and humidity with a 12 h light–dark cycle (light on at 8:00 a.m.), and pair-fed daily either the ethanol or control Lieber–DeCarli liquid diets (Lieber & DeCarli, 1989). In these formulae, 18% of the calories are derived from protein, 35% from fat, and 47% from carbohydrate. The ethanol-fed group received a diet in which carbohydrate was partially replaced by ethanol (36% of the calories). Ethanol was introduced progressively, with 3% of the liquid diet for 2 days, 4% for the subsequent 2 days, followed by the final formula containing 5% (w/v). A mean intake of 14.6 ± 0.9 g ethanol/kg/day was achieved. The pair-fed control groups received the same diet except that dextrin isocalorically replaced ethanol. After 6 weeks of treatment, rats were sacrificed between 8:00 and 9:00 a.m. (Guzm´an & Castro, 1990). Animals were anaesthetized with ether, dissected and blood samples were obtained from abdominal vena cava for determination of blood ethanol concentration, using the enzymatic NAD-ADH assay kit (Sigma Diagnostics). All animal use procedures were in accordance with the guidelines of the Spanish Ministry of Health. Animals in the two groups of rats gained weight throughout the 6-week period. Despite isocaloric feeding (the average intake of liquid diet was 289 ± 19 ml/kg day), the weight gain of control rats (303 ± 15 g final weight) was somewhat higher, but it was not significantly different from that of ethanol-fed rats (281 ± 21 g final weight). The blood ethanol level for a representative ethanol-treated group was found to be 187 ± 47 mg/100 ml (n = 8) in the morning on the day of the killing.

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2.3. Isolation and incubation of hepatocytes Hepatocytes were isolated by the collagenase perfusion method in Krebs–Henseleit bicarbonate buffer as described (Beynen, Vaartjes, & Geelen, 1979). To minimize glycogenolysis, all the buffers used in the isolation procedure contained 10 mM glucose (Bijleveld & Geelen, 1987). Because it has been reported that lipogenesis is markedly depressed in hepatocytes immediately after isolation, cells were preincubated as recommended (Holland, Witters, & Hardie, 1984), during 15 min at 37 ◦ C in a gyratory metabolic shaker, to allow recovery of lipogenic activity before incubations with additions. Cell viability always exceeded 90%, as determined by trypan blue exclusion. Two milliliters of hepatocyte suspension (4–6 mg cellular protein/ml) in Krebs-Henseleit bicarbonate buffer, supplemented with 10 mM glucose and 1% (w/v) defatted and dialyzed BSA, were incubated with the additions indicated in experiments, at 37 ◦ C for 30 min with constant shaking under an atmosphere of O2 /CO2 (19:1). Additions used were: (±)-S-nitroso-N-acetylpenicillamine (SNAP); 8-Br-cGMP; KT5823, and rat atrial natriuretic peptide (rANP). The cellular protein was determined according to Lowry, Rosebrough, Farr, and Randall (1951). 2.4. Rates of fatty acid and glucose metabolism For the determination of the rate of fatty acid synthesis, reactions were started by the addition of [114 C]acetate (0.1 Ci/mol; 3 mM final concentration) to hepatocyte incubations. After 30 min reactions were stopped and total fatty acids were extracted (Bijleveld & Geelen, 1987). The rate of fatty acid oxidation was determined by adding to hepatocyte incubations [114 C]palmitate (0.05 Ci/mol; 0.4 mM final concentration) bound to albumin. After 20 min, reactions were stopped and oxidation products were extracted as acidsoluble fraction and quantified as previously described (Guzm´an & Castro, 1989). For the determination of the rate of fatty acid esterification, reactions were started by the addition to cell incubations of albumin-bound [1-14 C]palmitate (0.05 Ci/mol; 0.4 mM final concentration), and carried out for 30 min. Triacylglycerols and phospholipids were isolated by thin-layer chromatography and quantified (Guzm´an & Castro, 1989). In order to correct for losses in recovery of labeled

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metabolites, all extractions were performed regularly in parallel to extractions of samples containing a known amount of radioactive product. Rates of glucose metabolism were determined in hepatocyte suspensions by the addition of d-[U14 C]glucose (0.15 Ci/mol; 10 mM final concentration). After 30 min, reactions were stopped and radioactive products analysed. Glycogen was isolated by ethanol precipitation (Schmoll, F¨uhrmann, Gebhardt, & Hamprecht, 1995), and after additional washes with 75% (by volume) ice-cold ethanol, the glycogen pellet was resuspended in 1 ml of water and analysed for radioactive content. The rate of fatty acid synthesis from glucose was determined by extracting total fatty acids exactly as described (Bijleveld & Geelen, 1987). The rate of incorporation of carbon from radiolabeled glucose into triacylglycerols was determined by isolating this lipid fraction by thin-layer chromatography. Then, triacylglycerol fraction was analysed for the determination of radioactive content in both fatty acyl groups and glycerol backbone moieties as previously described (Maquedano, Guzm´an, & Castro, 1988). 2.5. Acetyl-CoA carboxylase activity ACC activity was determined in digitoninpermeabilized hepatocytes, as the incorporation of radiolabeled acetyl-CoA to fatty acids in an assay coupled to the reaction of fatty acid synthase (Bijleveld & Geelen, 1987). To measure enzyme activity, 0.1 ml of hepatocyte suspension was added to 0.1 ml of prewarmed digitonin-containing assay medium, and the reaction was carried out for 2 min at 37 ◦ C. The final mixture in 63 mM HEPES buffer (pH 7.5) was exactly as described (Bijleveld & Geelen, 1987), containing 0.062 mM [1-14 C]acetyl-CoA (4 Ci/mol) and 3.2 mU of rat liver fatty acid synthase purified as previously reported (Linn, 1981). 2.6. Statistical analysis Determinations of the rates of metabolic pathways and enzymatic assays were carried out in triplicate, with hepatocyte incubations also done in triplicate. Results in figures and tables are the mean ± S.E.M. of the number of rats indicated in every case. Statistical analysis was performed by one-way ANOVA using the software package Statgraphics.

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bound guanylyl cyclase A, was ineffective to inhibit either ACC activity or fatty acid synthesis.

3. Results 3.1. Effects of cGMP-related modulators on fatty acid synthesis Synthetic donors that slowly release nitric oxide are currently used in the investigation of NO-mediated biological actions. In the present work, hepatocytes from rats fed with the liquid control diet were used to study the effects of NO donor SNAP and cGMP-related modulators, on both the rate of fatty acid synthesis and the activity of acetyl-CoA carboxylase, because this key enzyme has been shown to play an important role in cGMP-mediated regulation of fatty acid metabolism (Garc´ıa-Villafranca et al., 2003). In hepatocytes treated with SNAP, ACC activity and fatty acid synthesis were inhibited in parallel (Table 1). Incubation with 8-BrcGMP, a phosphodiesterase-resistant cGMP analog, produced parallel but more potent decreases in both ACC activity and fatty acid synthesis. Moreover, the maximal effects of SNAP and 8-Br-cGMP were nonadditive (Table 1). In both activities, the inhibition produced by SNAP was completely abolished by KT5823, a highly specific inhibitor of cGMP-dependent protein kinase. However, rANP, an activator of membraneTable 1 Effects of SNAP and cGMP-related modulators on acetyl-CoA carboxylase activity and fatty acid synthesis Additions

None 0.5 mM SNAP 0.1 mM 8-Br-cGMP 0.5 mM SNAP + 0.1 mM 8-Br-cGMP 0.1 mM KT5823 0.5 mM SNAP + 0.1 mM KT5823 0.4 ␮M rANP

Parameter Acetyl-CoA carboxylase activity (% of control)

Fatty acid synthesis (% of control)

100 65.2 ± 3.5* 47.6 ± 5.7* 51.5 ± 6.3*

100 68.3 ± 5.5* 37.2 ± 4.3** 40.6 ± 5.7**

101.6 ± 5.2 98.3 ± 3.6

103.2 ± 7.1 99.1 ± 5.3

102.5 ± 7.2

97.3 ± 4.5

Hepatocytes were incubated in the presence of indicated additions. After 30 min, samples were removed to determine ACC activity and the rate of fatty acid synthesis. Results are expressed as percent of controls and represent mean ± S.E.M. of six different animals. Control values (100%) from incubations without additions were: ACC activity; 0.50 ± 0.05 nmol product/min mg cellular protein; rate of fatty acid synthesis 20.3 ± 1.4 nmol acetyl units/h mg cellular protein. * P < 0.01; ** P < 0.001, relative to incubations with no additions.

3.2. Effects of ethanol consumption on cGMP-dependent regulation of fatty acid metabolism Hepatocytes from both ethanol-fed and pair fed control rats were incubated with a range of concentrations of 8-Br-cGMP. The effects on fatty acid synthesis, esterification and oxidation were studied in intact cells. In addition, ACC activity was determined in digitoninpermeabilized hepatocytes. The first interesting result is that the rate of fatty acid synthesis de novo in hepatocytes from ethanol-fed rats (33.2 ± 4.3 nmol acetyl units/h mg cellular protein) is markedly enhanced compared to that of controls (19.5 ± 1.8, same units). In addition, when hepatocytes from both ethanol-fed and pair-fed controls were incubated with 8-Br-cGMP, ACC activity and the rate of fatty acid synthesis were inhibited in parallel (Fig. 1). However, metabolic responses in hepatocytes from ethanol-fed rats were less sensitive to varying concentrations of the cGMP analog. Thus, maximum inhibition of fatty acid synthesis was about 70% in control rats, whereas only about 32% in hepatocytes from ethanol-fed rats. Also in these cells IC50 value was two times higher than in control cells. Similarly, IC50 value for inhibition of ACC activity was significantly higher in cells from ethanol-fed rats, although maximum inhibition by 8-Br-cGMP was not significantly different in cells from these two groups of animals. After chronic ethanol consumption, two important mechanisms involved in lipid accumulation in hepatocytes are the enhanced synthesis of triacylglycerols and the inhibition of fatty acid oxidation. In this study, the rate of palmitate esterification to triacylglycerols was increased in hepatocytes from ethanolfed rats (27.2 ± 3.8 nmol palmitate/h mg cellular protein), compared to controls (18.5 ± 2.2, same units), whereas rates of phospholipid synthesis were similar in cells from both groups of treated animals (Fig. 2). In addition, our results indicate that incubation with 8-BrcGMP inhibited fatty acid esterification to both glycerolipid fractions (Fig. 2). However, the rates of both syntheses in hepatocytes from ethanol-fed rats showed a decreased sensitivity to varying concentrations of 8Br-cGMP, both in maximum inhibition and IC50 values.

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Fig. 1. Effect of 8-Br-cGMP on ACC activity (䊉, ) and fatty acid synthesis (, ) in hepatocytes from pair-fed control (filled symbols) and ethanol-fed (open symbols) rats. Hepatocytes were incubated in the presence of increasing concentrations of 8-Br-cGMP. After 30 min, samples were removed to determine the activity of ACC and the rate of fatty acid synthesis. Results are expressed as percent of control incubations, and represent mean ± S.E.M. of eight animals in each group. Control values (100%) in the absence of 8-Br-cGMP were: ACC activity, 0.46 ± 0.06 (control rats) and 0.55 ± 0.08 (ethanol-fed rats) nmol product/min mg cellular protein; fatty acid synthesis, 19.5 ± 1.8 (control rats) and 33.2 ± 4.3 (ethanol-fed rats) nmol acetyl units/h mg cellular protein. ** P < 0.01 and *** P < 0.001, relative to pair-fed controls.

Fig. 2. Effect of 8-Br-cGMP on palmitate esterification to triacylglycerols (䊉, ) and phospholipids (, ) in hepatocytes from pair-fed control (filled symbols) and ethanol-fed (open symbols) rats. Hepatocytes from the same animal groups were incubated and results are expressed as in Fig. 1. Control values (100%) in the absence of 8-Br-cGMP were: palmitate esterification to triacylglycerols, 18.5 ± 2.2 (control rats) and 27.2 ± 3.8 (ethanol-fed rats), and phospholipids, 13.6 ± 2.0 (control rats) and 13.4 ± 2.6 (ethanol-fed rats), all in nmol palmitate/h mg cellular protein. ** P < 0.01 and *** P < 0.001, relative to pair-fed controls.

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Fig. 3. Effect of 8-Br-cGMP on ketogenesis from palmitate in hepatocytes from pair-fed control (䊉) and ethanol-fed () rats. Hepatocytes from the same animal groups were incubated and results are expressed as in Fig. 1. Control values (100%) in the absence of 8-Br-cGMP were 21.2 ± 2.9 (control rats) and 19.6 ± 3.4 (ethanol-fed rats) nmol palmitate/h mg cellular protein. * P < 0.05 and ** P < 0.01, relative to pair-fed controls.

Inhibition of ACC by 8-Br-cGMP may be important for the control of fatty acid oxidation in hepatocytes because malonyl-CoA is a physiological inhibitor of carnitine palmitoyltransferase I, a key enzyme in the regulation of fatty acid oxidation (McGarry & Brown, 1997). Thus, fatty acid oxidation was modulated by 8Br-cGMP inversely to lipogenesis. Incubation with this cGMP analog increased the rate of ketogenesis from palmitate in both types of cells (Fig. 3), but this increase was significantly lower in hepatocytes from ethanolfed rats (maximum about 125%) than in control rats (maximum about 170%). In summary, desensitization of cGMP-mediated response in the regulation of fatty acid metabolism seems to be a result of chronic ethanol consumption, contributing to lipid accumulation in the liver. In order to gain an insight into the physiological significance of these effects of cGMP, we studied the recovery from ethanol-induced blunting of 8-Br-cGMP inhibition of triacylglycerol synthesis and stimulation of ketogenesis, at different times after eliminating ethanol in the diet (Table 2). This desensitization was found to be a reversible effect, with different characteristics in each pathway. Thus, the stimulating effect of 8-Br-cGMP on ketogenesis recovered the level of controls 2 days after withdrawal of ethanol, whereas

about 14 days were required to restore sensitivity of 8-Br-cGMP induced inhibition of triacylglycerol synthesis. 3.3. Effects of ethanol consumption on cGMP-dependent regulation of glucose metabolism With the aim to study if this desensitization effect is a more general mechanism of ethanol-induced response, three main pathways of glucose metabolism were analysed (Table 3). Glycogen synthesis was significantly decreased in hepatocytes from ethanol-fed rats, in agreement with previous reports (Van Horn, Ivester, & Cunningham, 2001). In addition, 8-Br-cGMP inhibited glycogen synthesis in cells from both animal groups, but this effect was greater in hepatocytes from pair-fed controls. Rates of total fatty acid synthesis from glucose were similar in both types of cells, but cGMP-mediated inhibition of this pathway was lower in hepatocytes from ethanol-fed rats. On the other hand, radiolabeled glucose was almost completely incorporated into the glycerol backbone of triacylglycerols (Table 3), in agreement with other reported data (Maquedano et al., 1988). This incorporation of glucose was strongly decreased by 8-Br-cGMP in control

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Table 2 Effects of ethanol feeding and withdrawal on the rates of triacylglycerol synthesis and ketogenesis Type of diet

Triacylglycerol synthesis Basal

C (42) E (42) W (2) W (6) W (14)

20.2 29.8 28.4 24.9 22.2

± ± ± ± ±

Ketogenesis +0.1 mM 8-Br-cGMP

1.8 3.1†† 3.5† 2.4 3.2

11.1 27.5 25.6 20.3 13.9

± ± ± ± ±

1.3**

(55.0%) 2.9 (92.3%) 2.7 (90.1%) 1.8 (81.5%) 1.6* (62.6%)

Basal

+0.1 mM 8-Br-cGMP

22.8 ± 2.1 19.5 ± 1.6 21.6 ± 2.3 20.8 ± 2.4 n.d.

36.5 ± 4.1** (160.1%) 24.1 ± 2.2* (123.6%) 35.6 ± 3.8** (164.8%) 35.2 ± 4.2** (169.2%) n.d.

Animals were fed either the control (C) or the ethanol-containing diet (E) for 42 days, or the ethanol-containing diet for 42 days followed by control diet – ethanol withdrawal (W) – for the number of days in parentheses. Then hepatocytes were isolated for determination of rates of triacylglycerol synthesis and ketogenesis. These rates are expressed all in nmol palmitate/h mg cellular protein, and represent mean ± S.E.M. of six different animals in each group. In parentheses are percent of values after 8-Br-cGMP addition relative to basal activities (n.d.: not determined). Relative to incubations in the absence of 8-Br-cGMP (basal): * P < 0.05 and ** P < 0.01. Relative to C (42) controls in basal incubations: † P < 0.05 and †† P < 0.01.

hepatocytes, whereas this inhibition was significantly lower in cells from ethanol-fed rats. Taken together, these data suggest that chronic ethanol consumption results in a decreased sensitivity of cGMP-mediated regulation affecting a number of important metabolic pathways in hepatocytes.

4. Discussion We have previously shown that fatty acid metabolism in rat hepatocytes may be regulated by a cGMP-mediated mechanism likely involving the activation of cGMP-dependent protein kinase (Garc´ıaVillafranca et al., 2003). The present study was designed to ascertain if this mechanism may have a physiological role in adaptation to ethanol consumption. Our data provide the first evidence that ethanol feeding to rats induces a desensitization of the regulatory effects mediated by cGMP in fatty acid

metabolism, which may contribute to lipid accumulation in the liver. Metabolic effects of this desensitization are not restricted to fatty acid metabolism, because they are also evident in important pathways of glucose metabolism. Chronic ethanol consumption induces accumulation of fatty acids of different sources as triacylglycerols in the liver because of different metabolic disturbances, including enhanced hepatic lipogenesis and decreased fatty acid oxidation (Baraona & Lieber, 1998; Day & Yeaman, 1994; Lieber, 1994). Impaired secretion of triacylglycerol-carrying lipoproteins could also contribute to hepatic fat accumulation, but this seems to be a complex effect (Baraona & Lieber, 1998). In our experimental conditions, we have obtained preliminary results in which the secretion of VLDL-triacylglycerols by hepatocytes of ethanol-fed rats showed a 16% decrease, compared to cells from pair-fed controls (results not shown). There is consensus that the availability of fatty acids for the synthesis of triacylglycerols in

Table 3 Effects of ethanol feeding on glucose metabolism and sensitivity to 8-Br-cGMP addition Parameter

Glycogen synthesis Fatty acid synthesis Triacylglycerol synthesis (glycerol backbone)

Control

Ethanol-fed

Basal

+0.1 mM 8-Br-cGMP

27.0 ± 3.1 8.4 ± 1.2 2.5 ± 0.4

7.7 ± (28.5%) 3.3 ± 0.5** (39.3%) 0.61 ± 0.1** (24.4%) 0.5***

Basal

+0.1 mM 8-Br-cGMP 1.0††

17.5 ± 7.7 ± 0.6 1.6 ± 0.3†

10.4 ± 0.6*** (59.4%) 5.8 ± 0.4* (75.3%) 1.2 ± 0.1 (75.0%)

Hepatocytes from either ethanol-fed or pair-fed control rats, were incubated in the absence (basal) or in the presence of 0.1 mM 8-Br-cGMP. Results are expressed as nmol glucosyl units/h mg cellular protein and represent mean ± S.E.M. of eight animals in each group. In parentheses are percent of values after 8-Br-cGMP addition relative to basal activities. Relative to incubations in the absence of 8-Br-cGMP (basal): * P < 0.05, ** P < 0.01 and *** P < 0.001. Relative to pair-fed controls in basal incubations: † P < 0.05 and †† P < 0.01.

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the liver is markedly enhanced, despite the fact that ethanol-fed rats show no changes in the activity of enzymes for de novo fatty acid synthesis (Tijburg, Maquedano, Bijleveld, Guzm´an, & Geelen, 1988). Our data are in agreement with this idea, showing a marked increase in the rate of fatty acid synthesis, whereas ACC activity was not significantly changed after ethanol feeding. Moreover, 8-Br-cGMP inhibited in parallel fatty acid synthesis and ACC activity in hepatocytes from these two animal groups, suggesting the functionality in both animal models of the recently proposed regulatory pathway, in which stimulation of NO/cGMP signaling inhibits ACC activity and fatty acid synthesis by a mechanism mediated by cGMP-dependent protein kinase (Garc´ıa-Villafranca et al., 2003). However, two considerations should be made. Firstly, inhibition of these two activities was less sensitive to varying concentrations of 8-Br-cGMP in hepatocytes from ethanolfed rats. In addition, the different profiles shown in the inhibition of fatty acid synthesis, compared to ACC activity, in cells obtained from these two groups of rats suggest that, even though ACC is a key regulatory enzyme of fatty acid synthesis, additional regulatory effects may contribute to partially counteract the inhibition of ACC in hepatocytes from ethanol-fed rats, which could be related to the excess of NADH, which may promote fatty acid synthesis (Baraona & Lieber, 1998; Lieber, 1994), and/or to a differentiated regulation of ACC activity by other factors in cells from ethanol-treated animals, an interesting point actually being investigated. From our data, it is evident that chronic ethanol feeding increased the rate of fatty acid esterification to triacylglycerols, but not to phospholipids. Ethanol administration may increase the rate of triacylglycerol synthesis by stimulating the activities of the enzymes involved, including diacylglycerol acyltransferase, which branches the pathway to the synthesis of triacylglycerols, responsible for a much higher increase in triacylglycerol than in phospholipid synthesis (Baraona & Lieber, 1998; Day & Yeaman, 1994). In addition, 8-Br-cGMP inhibited the synthesis of both classes of glycerolipids. However, by comparing cells of these two groups, hepatocytes from ethanol-fed rats showed a clear desensitization of this 8-Br-cGMPinduced inhibition. We suggest that this desensitization effect may contribute to triacylglycerol accumulation in liver in conditions of an increased NO production

and high levels of cGMP in hepatocytes, such as in chronic ethanol feeding (Nanji et al., 1995; Zima et al., 2001). This suggestion is also consistent with the finding that 8-Br-cGMP-induced stimulation of ketogenesis is blunted in hepatocytes from ethanol-fed rats. In conclusion, lipogenesis and fatty acid oxidation are inversely regulated by NO/cGMP signaling in hepatocytes, but these regulatory effects mediated by cGMP are significantly reduced after chronic ethanol consumption. In addition, the fact that this desensitization is a reversible effect, with clear differences in recovery from ethanol-induced blunting, suggests the possible implication in an adaptive mechanism acting directly on both triacylglycerol synthesis and ketogenesis, instead of reflecting a primary effect on either process, with the other as a passive consequence. The study of glucose metabolism lead us to suggest that this desensitization of cGMP-dependent regulation is a more general adaptive mechanism of ethanol consumption, with physiological implications in hepatocyte metabolism. The rate of the main pathway of glucose incorporation, glycogen synthesis, was decreased by 8-Br-cGMP, and this could result from the inhibition of glycogen synthase activity, caused by a decreased conversion of glycogen synthase b into glycogen synthase a (Klover & Mooney, 2004; Sprangers, Sauerwein, Romijn, van Woerkom, & Meijer, 1998). Addition of 8-Br-cGMP also inhibited fatty acid synthesis from glucose, and this effect seems to be related to the inhibition of ACC activity (Fig. 1), by the mechanism recently proposed (Garc´ıa-Villafranca et al., 2003). The effect of 8-Br-cGMP on the incorporation of glucose to glycerol moiety of triacylglycerols is also interesting, but because of the complexity of the pathway from glucose uptake, it remains to be demonstrated which are the steps inhibited by cGMP. It has been reported that ethanol consumption affects the activity of different signal transduction processes in the nervous system, and these changes are probably involved in neurological alterations in alcoholism (Crews et al., 2004; Diamond & Gordon, 1997; Mailliard & Diamond, 2004). The liver is also a major target for actions of ethanol, and signal transduction systems seem to be involved in short-term changes in cellular regulation and long-term changes in gene expression (Aroor & Shukla, 2004; Hoek, Thomas, Rooney, Higashi, & Rubin, 1992; Zhang & Farrell, 1997). Our data suggest that ethanol consumption induces

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a desensitization of NO/cGMP-mediated regulation of fatty acid metabolism. This impaired control by NO/cGMP signaling may be important in the response of liver in different pathophysiological conditions, such as chronic ethanol consumption, resulting in enhanced expression of inducible nitric oxide synthase and increased NO production in most types of cells in the liver, including Kupffer cells and hepatocytes (Li & Billiar, 1999; Sell, 2003; Tilg & Diehl, 2000). Conversely, an increase in sensitivity to NO-dependent inhibition of mitochondrial respiration may also be involved in alcohol-induced hepatotoxicity (Venkatraman et al., 2003). In conclusion, our results show that cGMP is involved in the control of metabolic fluxes in the liver, affecting fatty acid and glucose metabolism, and this regulation is endowed with physiological properties in adaptation to chronic ethanol consumption, in which desensitization of this cGMP-mediated signaling may contribute to triacylglycerol accumulation in the liver. However, further research is required to elucidate fully the molecular mechanisms involved in this adaptive response. Acknowledgements This work was supported by Grants from Spanish Comisi´on Interministerial de Ciencia y Tecnolog´ıa (SAF97-0142) and Universidad Complutense (PR181/96-6760). Javier Garc´ıa-Villafranca was the recipient of a fellowship from Universidad Complutense.

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