Search

Numerous nutritional approaches aimed at reducing body weight have been devel- oped as a strategy to reduce obesity. Most of these interventions rely on reducing caloric intake or limiting calories access to a few hours per day. In this work, we analyzed the effects of the extended (24 hours/day) or restricted (1 hour/day) access to a cafeteria-style (CAF) diet, on rat body weight and hepatic lipid metabolism, with respect to control rats (CTR) fed with a standard chow diet. The body weight gain of restricted-fed rats was not different from CTR, despite the slightly higher total caloric intake, but resulted significantly lower than extended-fed rats, which showed a CAF diet-induced obesity and a dramatically higher total caloric intake. However, both CAF-fed groups of rats showed, compared to CTR, unhealthy serum and hepatic parameters such as higher serum glucose level, lower HDL values, and increased hepatic triacylglycerol and cholesterol amount. The hepatic expression and activity of key enzymes of fatty acid synthesis, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS), was similarly reduced in both CAF-fed groups of rats with respect to CTR. Anyway, while in extended-fed rats this reduction was associ- ated to a long-term mechanism involving sterol regulatory element-binding protein-1 (SREBP-1), in restricted-fed animals a short-term mechanism based on PKA and AMPK activation occurred in the liver. Furthermore, hepatic fatty acid oxidation (FAO) and oxidative stress resulted significantly increased in extended, but not in restricted-fed rats, as compared to CTR. Overall, these results demonstrate that al- though limiting the total caloric intake might successfully fight obesity development, the nutritional content of the diet is the major determinant for the health status.

Binge eating disorder (BED) is the most frequent eating disorder, for which current pharmacotherapies show poor response rates and safety concerns, thus highlighting the need for novel treatment options. The lipid-derived messenger oleoylethanolamide (OEA) acts as a satiety signal inhibiting food intake through the involvement of central noradrenergic and oxytocinergic neurons. We investigated the anti-binge effects of OEA in a rat model of binge-like eating, in which, after cycles of intermittent food restrictions/refeeding and palatable food consumptions, female rats show a binge-like intake of palatable food, following a 15-min exposure to their sight and smell (“frustration stress”). Systemically administered OEA dose-dependently (2.5, 5, and 10 mg kg−1) prevented binge-like eating. This behavioral effect was associated with a decreased activation (measured by mapping the expression of c-fos, an early gene widely used as a marker of cellular activation) of brain areas responding to stress (such as the nucleus accumbens and amygdala) and to a stimulation of areas involved in the control of food intake, such as the VTA and the PVN. These effects were paralleled, also, to the modulation of monoamine transmission in key brain areas involved in both homeostatic and hedonic control of eating. In particular, a decreased dopaminergic response to stress was observed by measuring dopamine extracellular concentrations in microdialysates from the nucleus accumbens shell, whereas an increased serotonergic and noradrenergic tone was detected in tissue homogenates of selected brain areas. Finally, a decrease in corticotropin-releasing factor (CRF) mRNA levels was induced by OEA in the central amygdala, while an increase in oxytocin mRNA levels was induced in the PVN. The restoration of a normal oxytocin receptor density in the striatum paralleled the oxytocinergic stimulation produced by OEA. In conclusion, we provide evidence suggesting that OEA might represent a novel potential pharmacological target for the treatment of binge-like eating behavior.

Oleoylethanolamide (OEA) is a naturally occurring bioactive lipid belonging to the family of N-acylethanolamides. A variety of beneficial effects have been attributed to OEA, although the greater interest is due to its potential role in the treatment of obesity, fatty liver, and eating-related disorders. To better clarify the mechanism of the antiadipogenic effect of OEA in the liver, using a lipidomic study performed by 1H-NMR, LC-MS/MS and thin-layer chromatography analyses we evaluated the whole lipid composition of rat liver, following a two-week daily treatment of OEA (10 mg kg&minus, 1 i.p.). We found that OEA induced a significant reduction in hepatic triacylglycerol (TAG) content and significant changes in sphingolipid composition and ceramidase activity. We associated the antiadipogenic effect of OEA to decreased activity and expression of key enzymes involved in fatty acid and TAG syntheses, such as acetyl-CoA carboxylase, fatty acid synthase, diacylglycerol acyltransferase, and stearoyl-CoA desaturase 1. Moreover, we found that both SREBP-1 and PPAR&gamma, protein expression were significantly reduced in the liver of OEA-treated rats. Our findings add significant and important insights into the molecular mechanism of OEA on hepatic adipogenesis, and suggest a possible link between the OEA-induced changes in sphingolipid metabolism and suppression of hepatic TAG level.

Hepatic de novo lipogenesis (DNL), the process by which carbohydrates are converted into lipids, is strictly controlled by nutritional and hormonal status. 3,5-Diiodo-L-thyronine (T2), a product of the 3,5,3'-triiodo-L-thyronine (T3) peripheral metabolism, has been shown to mimic some T3 effects on lipid metabolism by a short-term mechanism independent of protein synthesis. Here, we report that T2, administered for 1 week to hypothyroid rats, increases total fatty acid synthesis from acetate in isolated hepatocytes. Studies carried out on liver subcellular fractions demonstrated that T2 not only increases the activity and the expression of acetyl-CoA carboxylase and fatty acid synthase but also of other proteins linked to DNL such as the mitochondrial citrate carrier and the cytosolic ATP citrate lyase. Parallelly, T2 stimulates the activities of enzymes supplying cytosolic NADPH needed for the reductive steps of DNL. With respect to both euthyroid and hypothyroid rats, T2 administration decreases the hepatic mRNA level of SREBP-1, a transcription factor which represents a master regulator of DNL. However, when compared to hypothyroid rats T2 significantly increases, without bringing to the euthyroid value, the content of both mature (nSREBP-1), and precursor (pSREBP-1) forms of the SREBP-1 protein as well as their ratio. Moreover, T2 administration strongly augmented the nuclear content of ChREBP, another crucial transcription factor involved in the regulation of lipogenic genes. Based on these results, we can conclude that in the liver of hypothyroid rats the transcriptional activation by T2 of DNL genes could depend, at least in part, on SREBP-1- and ChREBP-dependent mechanisms. © 2019 IUBMB Life, 2019.

International audience; Peripheral nerve regeneration is regulated through the coordinated spatio-temporal activation of multiple cellular pathways. In this work, an integrated proteomics and bioinformatics approach was employed to identify differentially expressed proteins at the injury-site of rat sciatic nerve at 20 days after damage. By a label-free liquid chromatography mass-spectrometry (LC-MS/MS) approach, we identified 201 differentially proteins that were assigned to specific canonical and disease and function pathways. These include proteins involved in cytoskeleton signaling and remodeling, acute phase response, and cellular metabolism. Metabolic proteins were significantly modulated after nerve injury to support a specific metabolic demand. In particular, we identified a group of proteins involved in lipid uptake and lipid storage metabolism. Immunofluorescent staining for acyl-CoA diacylglycerol acyltransferase 1 (DGAT1) and DAGT2 expression provided evidence for the expression and localization of these two isoforms in Schwann cells at the injury site in the sciatic nerve. This further supports a specific local regulation of lipid metabolism in peripheral nerve after damage.

Cholestasis is one of the major causes of liver diseases. A chronic accumulation of toxic bile acids in the liver, which occurs in this condition, can induce fibrosis and cirrhosis. Inflammation is a fundamental component of acute and chronic cholestatic liver injury.Platelet-activating factor (PAF) is a proinflammatory lipid which may be generated by two independent pathways called the de novo and remodeling pathway being the last responsible for the synthesis of PAF during inflammation. In recent years a key role in PAF remodeling has been attributed to lysophosphatidylcholine acyltransferase (LPCAT) enzymes. Although the knowledge on their characteristic is growing, the exact mechanism of LPCAT in pathological conditions remains still unknown.Here, we reported that the level of lyso-PAF and PAF significantly increased in the liver of cirrhotic vs. control rats together with a significant decrease in both mRNA abundance and protein level of both LPCAT1 and LPCAT2. Acyltransferase activities of both LPCAT1 and LPCAT2 were parallel decreased in the liver of cirrhotic animals. Interestingly, treatment with silybin strongly decreased the level of both pro-inflammatory lipids and restored the activity and expression of both LPCAT1 and LPCAT2 of cirrhotic liver. Silybin effect was specific for LPCAT1 and LPCAT2 since it did not affect LPCAT3 mRNA abundance of cirrhotic liver.

Although H2O2 is traditionally known to have cytotoxic effects, recent studies argue about its regulatory role on lipid metabolism. However, the mechanism underlying the induction of lipid biosynthesis by oxidative stress still remains unknown. To shed light on this aspect we investigated the H2O2-induced lipogenesis in rat liver BRL-3A cells. We found that a short-term exposition of cells to 35μM H2O2 didn't cause any significant sign of cell damage measured by following diene formation and lactate dehydrogenase release from cells. However, in this stressful condition, a significant increase of [1-(14)C]acetate incorporation into fatty acids and cholesterol, associated to an increase in the activity and expression of key enzymes of fatty acid and cholesterol synthesis, were measured. mRNA and protein contents of the transcription factors SREBP-1 and SREBP-2, involved in the activation of lipid synthesis, increased as well. The analysis of molecular mechanism of SREBP-1 activation revealed, in treated compared to control cells, a higher SREBP-1a mRNA translation involving an internal ribosome entry side (IRES), present in the leader region of its mRNA. Longer exposition to the pro-oxidant induced a progressive loss of cell viability together with an increase of cell triacylglycerol content.

Citrate carrier (CiC), an integral protein of the mitochondrial inner membrane, plays an important role in hepatic intermediary metabolism, supplying the cytosol with acetyl-coenzyme A for fatty acid and cholesterol synthesis. Here, the effect of streptozotocin-induced diabetes on CiC activity and expression in rat liver was investigated. The rate of citrate transport was reduced by about 35% in mitochondria from diabetic vs. control rats. Kinetic studies in mitochondria from diabetic rats showed a reduction in maximum velocity and almost unchanged Michaelis-Menten constant of the CiC protein. Mitochondrial phospholipid amount was not significantly affected, whereas an increase in the cholesterol content and in the cholesterol/phospholipid ratio was observed. To thoroughly investigate the mechanism responsible for the reduced CiC activity in the diabetic state, molecular studies were performed. Ribonuclease protection assays and Western blotting analysis indicated that both hepatic CiC mRNA accumulation and protein level decreased similarly to the CiC activity. The reduced mRNA level and the lower content of the mitochondrial CiC protein, might account for the decline of CiC activity in diabetic animals. To discriminate between the role played by hyperglycemia from that of hypoinsulinemia in the reduction of CiC activity and expression, studies were conducted administrating phlorizin or insulin to streptozotocin-diabetic rats. Our data indicated that both insulin and glucose affect CiC activity and expression in diabetic rats, although they act at different regulatory steps.

The mitochondrial tricarboxylate carrier (TCC) plays an important role in lipogenesis being TCC-responsible for the efflux from the mitochondria to the cytosol of acetyl-CoA, the primer for fatty acid synthesis. In this study, we investigated the effects of two high-fat diets with different fatty acid composition on the hepatic TCC activity. Rats were fed for 3 weeks on a basal diet supplemented with 15% of either coconut oil (CO), abundant in medium-chain saturated fatty acids, or fish oil (FO), rich in n-3 polyunsaturated fatty acids. Mitochondrial fatty acid composition was differently influenced by the dietary treatments, while no appreciable change in phospholipid composition and cholesterol level was observed. Compared with CO, the TCC activity was markedly decreased in liver mitochondria from FO-fed rats; kinetic analysis of the carrier revealed a decrease of the Vmax, with no change of the Km. No difference in the Arrhenius plot between the two groups was observed. Interestingly, the carrier protein level and the corresponding mRNA abundance decreased following FO treatment. These data indicate that FO administration markedly decreased the TCC activity as compared with CO. This effect is most likely due to a reduced gene expression of the carrier protein.

The accumulation of toxic hydrophobic bile acids in hepatocytes, observed during chronic cholestasis, induces substantial modification in the redox state and in mitochondrial functions. Recent reports have suggested a significant role of impaired lipid metabolism in the progression of chronic cholestasis. In this work we report that changes observed in the expression of the lipogenic enzymes acetyl-CoA carboxylase and fatty acid synthase were associated with a decrease in the activity of citrate carrier (CIC), a protein of the inner mitochondrial membrane closely related to hepatic lipogenesis. We also verified that the impairment of citrate transport was dependent on modification of the phospholipid composition of the mitochondrial membrane and on cardiolipin oxidation. Silybin, an extract of silymarin with antioxidant and anti-inflammatory properties, prevented mitochondrial reactive oxygen species (ROS) production, cardiolipin oxidation, and CIC failure in cirrhotic livers but did not affect the expression of lipogenic enzymes. Moreover, supplementation of silybin was also associated with mitochondrial biogenesis. In conclusion, we demonstrate that chronic cholestasis induces cardiolipin oxidation that in turn impairs mitochondrial function and further promotes ROS production. The capacity of silybin to limit mitochondrial failure is part of its hepatoprotective property.

An increased mitochondrial proton leak occurs in aging, but the origin of such modification remains unclear. This study defined the cause of mitochondrial uncoupling in mitotic (liver) and postmitotic (heart) rat tissues during aging and its effects on energy homeostasis and free radical production. Proton leak in old heart mitochondria was dependent on uncoupling proteins' upregulation, whereas it was caused by alterations in the mitochondrial membrane composition in old liver. ATP homeostasis was impaired in both tissues from old animals and was associated to disrupted F0F1-ATPase activity. H2O2 production rate and 4-hydroxy-2-nonenalprotein adducts were higher in old liver mitochondria compared with young liver mitochondria, but they were similar in heart mitochondria from both groups. Moreover, key mitochondrial biogenesis regulators were upregulated in old liver but downregulated in old heart. In conclusion, uncoupling proteins mediate proton leak and avoid oxidative damage in heart, acting as a protective mechanism. This does not occur in liver, where ATP depletion and oxidative stress may stimulate mitochondrial biogenesis and eliminate damaged cells.

The role played by oxidative stress in amiodarone-induced mitochondrial toxicity is debated. Dronedarone shows pharmacological properties similar to those of amiodarone but several differences in terms of toxicity. In this study, we analyzed the effects of the two drugs on liver mitochondrial function by administering an equivalent human dose to a rat model. Amiodarone increased mitochondrial H(2)O(2) synthesis, which in turn induced cardiolipin peroxidation. Moreover, amiodarone inhibited Complex I activity and uncoupled oxidative phosphorylation, leading to a reduction in the hepatic ATP content. We also observed a modification of membrane phospholipid composition after amiodarone administration. N-acetylcysteine completely prevented such effects. Although dronedarone shares with amiodarone the capacity to induce uncoupling of oxidative phosphorylation, it did not show any of the oxidative effects and did not impair mitochondrial bioenergetics. Our data provide important insights into the mechanism of mitochondrial toxicity induced by amiodarone. These results may greatly influence the clinical application and toxicity management of these two antiarrhythmic drugs.

There is growing evidence that mitochondrial dysfunction, and more specifically fatty acid β-oxidation impairment, is involved in the pathophysiology of non-alcoholic steatohepatitis (NASH). The goal of the present study was to achieve more understanding on the modification/s of carnitinepalmitoyltransferase-I (CPT-I), the rate-limiting enzyme of the mitochondrial fatty acid β-oxidation, during steatohepatitis. A high fat/methionine-choline deficient (MCD) diet, administered for 4 weeks, was used to induce NASH in rats.We demonstrated that CPT-I activity decreased, to the same extent, both in isolated liver mitochondria and in digitonin-permeabilized hepatocytes from MCD-diet fed rats.At the same time, the rate of total fatty acid oxidation to CO(2) and ketone bodies, measured in isolated hepatocytes, was significantly lowered in treated animals when compared to controls. Finally, an increase in CPT-I mRNA abundance and protein content, together with a high level of CPT-I protein oxidation was observed in treated rats. A posttranslational modification of rat CPT-I during steatohepatitis has been here discussed.

Mitochondrial dysfunction and oxidative stress are determinant events in the pathogenesis of nonalcoholic steatohepatitis. Silybin has shown antioxidant, anti-inflammatory, and antifibrotic effects in chronic liver disease. We aimed to study the effect of the silybin-phospholipid complex (SILIPHOS) on liver redox balance and mitochondrial function in a dietary model of nonalcoholic steatohepatitis. To accomplish this, glutathione oxidation, mitochondrial oxygen uptake, proton leak, ATP homeostasis, and H(2)O(2) production rate were evaluated in isolated liver mitochondria from rats fed a methionine- and choline-deficient (MCD) diet and the MCD diet plus SILIPHOS for 7 and 14 weeks. Oxidative proteins, hydroxynonenal (HNE)- and malondialdehyde (MDA)-protein adducts, and mitochondrial membrane lipid composition were also measured. Treatment with SILIPHOS limited glutathione depletion and mitochondrial H(2)O(2) production. Moreover, SILIPHOS preserved mitochondrial bioenergetics and prevented mitochondrial proton leak and ATP reduction. Finally, SILIPHOS limited the formation of HNE- and MDA-protein adducts. In conclusion, SILIPHOS is effective in preventing severe oxidative stress and preserving hepatic mitochondrial bioenergetics in nonalcoholic steatohepatitis induced by the MCD diet. The modifications of mitochondrial membrane fatty acid composition induced by the MCD diet are partially prevented by SILIPHOS, conferring anti-inflammatory and antifibrotic effects. The increased vulnerability of lipid membranes to oxidative damage is limited by SILIPHOS through preserved mitochondrial function.

Background Mitochondrial dysfunction is considered a key player in non-alcoholic steatohepatitis (NASH) but no data are available on the mitochondrial function and ATP homeostasis in the liver during NASH progression. In the present paper we evaluated the hepatic mitochondrial respiratory chain activity and ATP synthesis in a rodent model of NASH development. Materials and methods Male Wistar rats fed a High Fat/Methionine-Choline Deficient (MCD) diet to induce NASH or a control diet (SHAM), and sacrificed after 3, 7 and 11 weeks. The oxidative phosphorylation, the F(0)F(1)ATPase (ATP synthase) and the ATP content were assessed in liver mitochondria. Results NASH mitochondria exhibited an increased rate of substrate oxidation at 3 weeks, which returned to below the normal level at 7 and 11 weeks, concomitantly with the coupling between the phosphorylation activity and the mitochondrial respiration (ADP/O). Uncoupling of NASH liver mitochondria did not allow the recovery of the maximal respiration rate at 7 and 11 weeks. The ATPase (ATP synthase) activity was similar in NASH and SHAM rats, but the mitochondrial ATP content was significantly lower in NASH livers. Conclusions The loss of hepatic ATP stores is not dependent on the F(0)F(1)-ATPase but resides in the respiratory chain. Dysfunction of both Complex I and II of the mitochondrial respiratory chain during NASH development implies a mitochondrial adaptive mechanism occurring in the early stages of NASH.

Metabolic fate and short-term effects of a 1:1 mixture of cis-9,trans-11 and trans-10,cis-12-conjugated linoleic acids (CLA), compared to linoleic acid (LA), on lipid metabolism was investigated in rat liver. In isolated mitochondria CLA-CoA were poorer substrates than LA-CoA for carnitine palmitoyltransferase-I (CPT-I) activity. However, in digitonin-permeabilized hepatocytes, where interactions among different metabolic pathways can be simultaneously investigated, CLA induced a remarkable stimulatory effect on CPT-I activity. This stimulation can be ascribed to a reduced malonyl-CoA level in turn due to inhibition of acetyl-CoA carboxylase (ACC) activity. The ACC/malonyl-CoA/CPT-I system can therefore represent a coordinate control by which CLA may exert effects on the partitioning of fatty acids between esterification and oxidation. Moreover, the rate of oxidation to CO2 and ketone bodies was significantly higher from CLA; peroxisomes rather than mitochondria were responsible for this difference. Interestingly, peroxisomal acyl-CoA oxidase (AOX) activity strongly increased by CLA-CoA compared to LA-CoA. CLA, metabolized by hepatocytes at a higher rate than LA, were poorer substrates for cellular and VLDL-triacylglycerol (TAG) synthesis. Overall, our results suggest that increased fatty acid oxidation with consequent decreased fatty acid availability for TAG synthesis is a potential mechanism by which CLA reduce TAG level in rat liver.

All-trans-retinoic acid (atRA) is incorporated covalently into proteins of rat testes mitochondria. In this study, the effect of three diets with different fatty acid composition on the retinoylation of proteins of rat testes mitochondria has been investigated. Different groups of rats were fed on a basal diet supplemented with 15% of either coconut oil (CO), olive oil (OO) or fish oil (FO). We found that, when compared with CO, the binding of retinoic acid was decreased in FO- and OO-fed rats. Mitochondrial phospholipids composition was differently influenced by dietary treatments; minor changes were observed in fatty acid composition of phospholipids. Few differences were observed in the Arrhenius plots among the three groups of rats. Kinetic analysis revealed a decrease in the V (max) value in FO- and OO- as compared with CO-fed rats. No difference among the three groups were observed in the K ( M ) value. The retinoylation reaction was inhibited by 13-cis-RA and 9-cis-RA.

The effect of hypothyroidism on citrate carrier (CiC) activity has been investigated in rat-liver mitochondria. The rate of citrate transport was reduced by approximately 50% in mitochondria from hypothyroid as compared with euthyroid rats. In parallel, a decrease in the rate of de novo fatty acid synthesis was observed in the cytosol of the former animals. Kinetic analysis of citrate transport revealed that only the Vmax was reduced by hypothyroidism, while Km was almost unaffected. Hypothyroidism increased the mitochondrial percentage of phosphatidylcholine while decreased that of phosphatidylethanolamine; an altered fatty acid pattern but no significant difference in the sum of saturated and unsaturated fatty acids as well as in the unsaturation index was observed. The CiC Arrhenius plot did not show appreciable difference between the two groups of rats. However, Western blot analysis associated with mRNA quantitation indicated that both protein level and mRNA accumulation of hepatic CiC were noticeably decreased in hypothyroid state. Therefore, a reduced content of the carrier protein can represent a plausible mechanism to explain the decline in the CiC activity observed in rat liver mitochondria of hypothyroid rats.

Short-term effects of 3,5-l-diiodothyronine (T2) on lipid biosynthesis were studied in cultured hepatocytes from hypothyroid rats. A comparison with the effects of T3 was routinely carried out. After T2 addition to cell cultures, a distinct stimulation of fatty acid and cholesterol syntheses, measured as incorporation of [1-14C]acetate into these lipid fractions, was observed. The T2 dose-dependent effect on both metabolic pathways, already detectable at 10−8-10−9m, reached a 2-fold stimulation at 10−5m T2. At this concentration, the stimulatory effect was evident within 1 h of T2 addition to the hepatocytes and increased with time up to the length of the experimental period of 4 h. T2 stimulation of lipogenesis was also confirmed by incubating hepatocytes with [3H]H2O, used as an independent index of lipogenic activity. The effects of T2 are rather specific as 3,3′,5,5′-tetraiodo-d-thyronine and 3,5-diiodo-l-tyrosine were practically ineffective on both fatty acid and cholesterol synthesis. Analysis of various lipid fractions showed that T2 addition to the cells produced a significant stimulation of the incorporation of newly synthesized fatty acids into both neutral and polar lipids. By comparing the effects induced by T2 with those seen in the presence of T3, it appeared that T2 was able to mimic T3 effects. Experiments conducted in the presence of cycloheximide, a protein synthesis inhibitor, indicated that the T2 stimulatory effect on fatty acid and cholesterol synthesis was essentially independent of protein synthesis.

In the present study the effects of some C18fatty acids on hepatic fatty acid metabolism have been compared. Male rats were fed cholesterol-free diets containing either C18:0, C18:1cisor C18:1transisomers as the variables. In accordance with previous work, oleic acid in the diet caused an increase in cholesterol concentration in the liver and in the lipoprotein fraction of density (d; kg/l)18:1transisomers diet induced a decrease in the amount of cholesterol in total plasma as well as in the 1·019

Starvation has been associated with a reduced citrate carrier (CTP) activity in rat liver mitochondria. In the present study the molecular mechanism responsible for this reduction was investigated. Northern blot analysis performed with hepatic total RNA showed a decrease of about 40% in the CTP mRNA abundance in starved rats, when compared to fed animals. Nuclear run-on assay did not reveal any appreciable difference in the rate of CTP mRNA synthesis between the two groups of animals, while the apparent half-life of CTP mRNA in hepatocytes from fed and starved rats was 11 and 6 h, respectively. Therefore, these results suggest that in starved rats the regulation of hepatic CTP expression occurs at posttranscriptional level. Moreover, the reduced CTP activity in starved animals gradually increased by refeeding. The carrier activity reached fed rat values 6–9 h following refeeding. Interestingly, the accumulation of CTP mRNA raised in parallel with the transport activity.

The activity of the tricarboxylate (citrate) carrier has been assayed in intact liver mitochondria from yellow eel (Anguilla anguilla) and compared to that from rat. The eel-citrate carrier specific activity was approximately 1.7-fold higher than that assayed in rat-liver mitochondria. The content of the main mitochondrial phospholipids, phosphatidylethanolamine and phosphatidylcholine, did not show a significant difference between the two species, while in eel a higher cardiolipin level was observed. Fatty acid composition of eel-liver mitochondrial phospholipids was characterised by a large amount of unsaturated fatty acids, dominated by octadecaenoic acid (C(18:1) (n-9)) and docosahexaenoic acid (C(22:6) (n-3)). The cardiolipin fatty acid pattern of eel-liver mitochondria showed, with respect to the rat, a higher C(20:5) (n-3) and C(22:6) (n-3) content and a lower amount of C(18:2) (n-6) and C(20:4) (n-6). A noticeable activity of lipogenic enzymes was also detected in eel liver cytosol. The results of this study suggest that the remarkable activity of the citrate carrier in eel-liver mitochondria can most likely be ascribed to a considerable cardiolipin level. A covariance of citrate carrier and lipogenic enzyme activities was observed.

After in vivo administration of lead nitrate, functional changes of the mitochondrial tricarboxylate carrier and of the cytosolic lipogenic enzymes acetyl-CoA carboxylase and fatty acid synthetase have been detected in rat liver. The rate of citrate transport was greatly reduced in rats during both the proliferative phase (3 days after the lead nitrate administration) and the involutive phase (5 days after the metal injection), which follows hepatic hyperplasia and corresponds to the peak of hepatocyte apoptosis. In both phases, a decrease of the lipogenic enzyme activities has been detected. In treated animals, an alteration of mitochondrial lipid composition has also been found. The modified lipid microenvironment could be responsible for the decreased carrier activity which, in turn, may account for the reduced activities of the lipogenic enzymes.

The short-term effect of dexamethasone, a synthetic glucocorticoid, on both fatty acid and cholesterol synthesis has been investigated in rat hepatocyte cultures. Within 4h following hormone addition to the cultures, a noticeable stimulation of labelled acetate incorporation into fatty acids was observed. Similar behaviour was noticed when [3H]H2O was used as an independent index of the lipogenic activity. In the same cultures, however, cholesterol synthesis from both [14C]acetate or [3H]H2O was significantly reduced by dexamethasone addition. In these conditions, no significative variation of cholesterol synthesis starting from labelled mevalonate was observed.

The tricarboxylate carrier (TCC), an integral protein of the mitochondrial inner membrane, transports mitochondrial acetyl-CoA into the cytosol, where lipogenesis occurs. We investigated in rat liver mitochondria the effect of diets enriched with saturated fatty acids (beef tallow, BT), monounsaturated fatty acids (olive oil, OO) or n−3 polyunsaturated fatty acids (fish oil, FO), respectively, on the activity and expression of TCC. TCC activity decreased, in parallel with TCC mRNA abundance, only upon FO-feeding. The TCC transcription rate, mRNA turnover and RNA processing indicated that FO administration regulates TCC gene at transcriptional and post-transcriptional steps, whereas BT- and OO-feeding do not seem to affect either TCC activity or gene expression.