The mitochondrial citrate carrier: metabolic role and regulation of its activity and expression.

The citrate carrier (CiC), a nuclear-encoded protein located in the mitochondrial inner membrane, is a member of the mitochondrial carrier family. CiC plays an important role in hepatic lipogenesis, which is responsible for the efflux of acetyl-CoA from the mitochondria to the cytosol in the form of citrate, the primer for fatty acid and cholesterol synthesis. In addition, CiC is a key component of the isocitrate-oxoglutarate and the citrate-malate shuttles. CiC has been purified from various species and its reconstituted function characterized as well as its cDNA isolated and sequenced. CiC mRNA and/or CiC protein levels are high in liver, pancreas, and kidney, but are low or absent in brain, heart, skeletal muscle, placenta, and lungs. A reduction of CiC activity was found in diabetic, hypothyroid, starved rats, and in rats fed on a polyunsaturated fatty acid (PUFA)-enriched diet. Molecular analysis suggested that the regulation of CiC activity occurs mainly through transcriptional and post-transcriptional mechanisms. This review begins with an assessment of the current understanding of CiC structural and biochemical characteristics, underlying the structure-function relationship. Emphasis will be placed on the molecular basis of the regulation of CiC activity in coordination with fatty acid synthesis.

Enigmatic effect of cellular ATP on fatty acid biosynthesis. Stimulation by moderate decrease and inhibition by increase of cellular ATP.

The cellular ATP concentration was tested for its effect on fatty acid biosynthesis from glucose in hepatocytes. ATP was manipulated by adding increasing concentrations of cycloheximide, amytal, atractyloside, 2,4-dinitrophenol or adenosine. A slight decrease in cellular ATP coincided with a stimulation of fatty acid biosynthesis whereas a further lowering of cellular ATP resulted in a gradual inhibition. Increasing the cellular ATP level by titration with adenosine had the opposite effect. These results are in line with the suggestion that fatty acid biosynthesis from glucose is an energy-yielding process which is stimulated by a moderate drop in cellular ATP.

Metabolism and short-term metabolic effects of conjugated linoleic acids in rat hepatocytes.

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.

Effect of dietary conjugated linoleic acid on body composition and energy balance in broiler chickens.

The effect of dietary conjugated linoleic acid (CLA) on body composition and energy metabolism was investigated in broiler chickens. Male broiler chicks were assigned to receive either a control diet (1 % sunflower oil) or a diet containing CLA (1 % of a 1:1 mixture of trans-10, cis-12 and cis-9, trans-11 isomers of octadecadienoic acid). The diets were fed ad libitum for 3 weeks and there were eight replicates per diet, each replicate including four chickens so that each treatment had thirty-two animals. The proportion of body fat was lower in the control group than in the CLA group. No significant differences as to the proportions of body water, ash and protein were observed. Feed and energy intake were significantly lower in CLA-fed birds. The percentage of ingested energy lost in excreta was higher after CLA feeding and heat expenditure as a percentage of ingested energy was lower in the CLA-fed group. The CLA-fed group showed a higher percentage of SFA and lower percentages of MUFA and PUFA in carcass fat. It is concluded that CLA stimulated de novo fatty acid synthesis and lowered desaturase activity.

Short-term stimulation of lipogenesis by 3,5-L-diiodothyronine in cultured rat hepatocytes.

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(-9) M, reached a 2-fold stimulation at 10(-5) M 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.

Fatty liver in dairy cows post partum is associated with decreased concentration of plasma triacylglycerols and decreased activity of lipoprotein lipase in adipocytes.

Cholesterol and phospholipid concentrations in serum lipoproteins, plasma activities of lecithin:cholesterol acyltransferase (LCAT) and phospholipid transfer protein (PLTP) and lipoprotein lipase (LPL) activity in adipose tissue biopsies were measured ante and post partum in dairy cows given either free or restricted access to feed during the dry period. After parturition, all cows were fed ad libitum. The purpose of this study was to try to understand the earlier observed marked drop post partum in plasma triacylglycerol (TAG) in terms of lipoprotein metabolism in cows developing fatty liver post partum. As would be expected, free access to feed during the dry period induced a rise of hepatic TAG concentrations post partum associated with a decrease in plasma TAG levels. Total and free cholesterol concentrations in the VLDL, IDL, LDL and HDL2 fractions fell immediately after parturition. VLDL and IDL cholesterol concentrations remained at a constant, low level during the entire sampling period post partum, whereas the drop in LDL and HDL2 cholesterol post partum was followed by a rebound rise. Plasma LCAT and PLTP activities decreased by on average 19% and 33%, respectively, after parturition and then rose to values seen before parturition, but there was no effect of feeding regimen during the dry period. Activities of LCAT and PLTP were significantly correlated with cholesterol and phospholipid concentrations in LDL and HDL2. Plasma LCAT activity, as measured with exogenous substrate, and PLTP activity were both positively correlated with HDL3 phospholipid levels. LPL activity in adipose tissue dropped after parturition, the drop being smaller after feeding ad libitum during the dry period. It is concluded that the drop in adipose tissue LPL activity post partum is at variance with the simultaneous fall in plasma TAG. Possibly, the decrease in adipose tissue LPL activity helps to channel fatty acids away from adipose tissue into the udder. The post-partum changes in lipid transfer proteins in the blood are in line with the changes observed in the levels of the lipoproteins.

Hepatic lipid and carbohydrate metabolism in rats fed a commercial mixture of conjugated linoleic acids (Clarinol G-80).

BACKGROUND: Conjugated linoleic acids (CLAs) exert numerous effects in animal models as well as in humans. Among other things, CLAs decrease plasma lipid levels and bring about hepatic steatosis. The latter effects are attributed to an agonistic action of CLAs on the peroxisome-proliferator-activated receptor family primarily responsible for activating genes involved in lipid metabolism and are related to changes in mRNA levels. Such changes are not necessarily reflected in changes in activity of controlling enzymes. AIM OF THE STUDY: To investigate the effects of CLAs treatment on lipid metabolism, we determined lipid concentrations in plasma, lipoproteins and liver and measured the activity of a number of key enzymes in hepatic lipid metabolism as differences in lipid concentrations should be related to changes in enzyme activities. These variables were determined with the rat as a model. METHODS: Rats were fed a control diet or a diet containing 1.15% trans-10, cis-12 isomer and 1.11% cis-9, trans-11 isomer as part of a commercial mixture of CLAs. After 2 w the animals were killed, and plasma and liver fractions isolated. Subsequently, lipid concentrations of cholesterol, triacylglycerols and phospholipids were determined in the isolated lipoproteins. In livers homogenates, the concentrations of glycogen, cholesterol, triacylglycerol and phospholipids and the activities of enzymes catalyzing pacesetting steps of metabolism were determined, i. e. acetyl-CoA carboxylase, fatty acid synthase, diacylglycerol acyltransferase, 3-hydroxy- acyl-CoA dehydrogenase, citrate synthase and phosphofructokinase. RESULTS: CLAs induced a lowering of the cholesterol levels in total plasma and in LDL and HDL lipoproteins and of phospholipid concentrations in LDL and HDL. CLAs treatment decreased the hepatic activity of diacylglycerol acyltransferase and had no effect on any of the other enzyme activities. CONCLUSIONS: In other studies enhanced specific activities of ACC and FAS were found in livers of mice using the same or similar methods and experimental protocol as in the present study. The lack of effect of CLAs treatment on hepatic key enzymes of fatty acid synthesis and oxidation in Wistar rats questions the use of this strain for studying the mechanism(s) underlying CLA's effects on these parameters. However, in the rat model we observed reduced levels of cholesterol in total plasma and in LDL and HDL. Therefore, some aspects like loss of body fat are better studied in mice; for other aspects like reduction in serum cholesterol level the rat may be the model of choice.

Prolonged feeding of mice with conjugated linoleic acid increases hepatic fatty acid synthesis relative to oxidation.

Feeding mice conjugated linoleic acid (9 cis,11 trans/9 trans,11 cis-and 10 trans,12 cis-CLA in equal amounts) resulted in triacylglycerol accumulation in the liver. The objective of this study was to examine whether this steatosis is associated with changes in hepatic fatty acid synthesis and oxidation. Therefore, we measured the activities of key enzymes of fatty acid synthesis, i.e., acetyl-CoA carboxylase and fatty acid synthase and of fatty acid oxidation, i.e., 3-hydroxy-acyl-CoA dehydrogenase and citrate synthase in livers of mice fed a diet with 0.5% (w/w) CLA. CLA (a 1:1 mixture of the 10 trans, 12 cis and 9 cis, 11 trans isomers of octadecadenoic acid) was administered for 3 and 12 weeks with high-oleic sunflower oil fed as control. The proportion of body fat was significantly lower on the CLA than on the control diet and this effect was already significant after 3 weeks. The specific activites of 3-hydroxy-acyl-CoA dehydrogenase and citrate synthase were unaffected by CLA both after 3 and 12 weeks. The specific activity of fatty acid synthase was nonsignificantly raised (by 12%) after 3 weeks on the CLA diet but had increased significantly (by 34%) after 12 weeks of feeding. The specific activity of acetyl-CoA carboxylase had also increased both after 3 weeks (by 53%) and 12 weeks (by 23%) on the CLA diet, but this effect did not reach statistical significance. Due to CLA-induced hepatomegaly, the overall capacity for both fatty acid oxidation and synthesis-as evidenced by the total hepatic activities of 3-hydroxy-acyl-CoA dehydrogenase, citrate synthase, acetyl-CoA carboxylase, and fatty acid synthase-was significantly greater in the CLA-fed group after 12 weeks, although the overall capacity for fatty acid synthesis had increased more than that for fatty acid oxidation. Thus, this study indicates that prolonged, but not short-term, feeding mice with CLA increased hepatic fatty acid synthesis relative to oxidation, despite the decrease in body fat and the increase in liver weight seen earlier. It is concluded that the observed CLA-induced changes in hepatic fatty acid synthesis and oxidation are the result, rather than the cause, of the lowering of body fat.

Activities of the enzymes of hepatic gluconeogenesis in periparturient dairy cows with induced fatty liver.

The objective was to measure the activities of all the enzymes essential for hepatic gluconeogenesis in dairy cows with induced fatty liver. We aimed to induce severe fatty liver in ten experimental cows by overfeeding them during the dry period while seven control cows were maintained on a restricted diet. To induce a marked negative energy balance, the experimental cows were deprived of feed for 8 h immediately after parturition. In addition, the experimental cows were given a restricted amount of diet during the first 5 d of lactation. Liver samples were collected 1 week before and 1, 2 and 4 weeks after parturition. Before parturition, liver triacylglycerol concentrations did not differ between the two groups. After parturition, the experimental cows developed marked fatty liver as indicated by a higher level of triacylglycerols in the liver compared with the control cows. Before parturition, all gluconeogenic enzymes in the liver were lower in experimental cows than in control cows. Phosphoenolpyruvate carboxykinase, pyruvate carboxylase and propionyl-CoA carboxylase were significantly lower and fructose 1,6-bisphosphatase and glucose 6-phosphatase tended to be lower in the experimental cows. The activities of two crucial enzymes for gluconeogenesis in ruminants, i.e., phosphoenolpyruvate carboxykinase and propionyl-CoA carboxylase, remained low throughout the sampling period post partum. Activities of pyruvate carboxylase and glucose 6-phosphatase in the experimental cows post partum were upgraded to values similar to those of the control cows. The results showed that the capacity for hepatic gluconeogenesis before parturition was lower in cows with induced fatty liver than in control cows. After parturition, the low activities of crucial gluconeogenic enzymes indicated insufficient production of glucose. It is suggested that the low gluconeogenic capacity leads successively to low blood glucose concentrations, low insulin levels and high rates of mobilization of fatty acid, causing severe hepatic lipidosis.

Ceramide sensitizes astrocytes to oxidative stress: protective role of cannabinoids.

Cannabinoids induce apoptosis on glioma cells via stimulation of ceramide synthesis de novo, whereas they do not affect viability of primary astrocytes. In the present study, we show that incubation with Delta9-tetrahydrocannabinol did not induce accumulation of ceramide on astrocytes, although incubation of these cells in a serum-free medium (with or without cannabinoids) led to stimulation of ceramide synthesis de novo and sensitization to oxidative stress. Thus treatment with H2O2 induced apoptosis of 5-day-serum-deprived astrocytes and this effect was abrogated by pharmacological blockade of ceramide synthesis de novo. The sensitizing effect of ceramide accumulation may depend on p38 mitogen-activated protein kinase activation rather than on other ceramide targets. Finally, a protective role of cannabinoids on astrocytes is shown as a long-term incubation with cannabinoids prevented H2O2-induced loss of viability in a CB1 receptor-dependent manner. In summary, our results show that whereas challenge of glioma cells with cannabinoids induces accumulation of de novo -synthesized ceramide and apoptosis, long-term treatment of astrocytes with these compounds does not stimulate this pathway and also abrogates the sensitizing effects of ceramide accumulation.

Hepatic fatty acid metabolism in rats fed diets with different contents of C18:0, C18:1 cis and C18:1 trans isomers.

In the present study the effects of some C18 fatty acids on hepatic fatty acid metabolism have been compared. Male rats were fed cholesterol-free diets containing either C18:0, C18:1 cis or C18:1 trans isomers 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) < 1.006. Oleic acid also reduced the triacylglycerol:cholesterol value in this fraction. Surprisingly, the C18:1 trans isomers diet induced a decrease in the amount of cholesterol in total plasma as well as in the 1.019 < d < 1.063 lipoprotein fraction. Both oleic acid and C18:1 trans isomers increased the concentration of triacylglycerols in the liver. The two C18:1 fatty acids differently influenced the hepatic activities of carnitine palmitoyltransferase-I and 3-hydroxy-acyl-CoA dehydrogenase; both enzymes were inhibited by C18:1 trans isomers, while no change was induced by oleic acid. The activity of the citrate carrier was lower in the oleic acid- and C18:1 trans isomers-fed rats, when compared with the rats fed stearic acid. No diet effects were seen for the activities of acetyl-CoA carboxylase, fatty acid synthase, diacylglycerol acyltransferase, citrate synthase and phosphofructokinase. The results are interpreted in that oleic acid raised liver triacylglycerol by reducing the secretion of it with the d < 1.006 lipoprotein fraction whereas the C18:1 trans isomers enhanced liver triacylglycerol by lowering the hepatic oxidation of fatty acids.

Measurement of diacylglycerol acyltransferase activity in isolated hepatocytes.

An assay procedure for diacylglycerol acyltransferase that allows rapid measurement of the activity of this enzyme in isolated hepatocytes is described. The one-step procedure involves permeabilization of the plasma membrane with digitonin and simultaneous measurement of diacylglycerol acyltransferase activity. Digitonin at a concentration of 64 microg/mg of cellular protein was found to be optimal for exposing microsomal diacylglycerol acyltransferase to the components of the assay. The enzyme assay is linear with time up to 4 min and with protein concentrations in the range 0.25-2.4 mg of cellular protein/assay. It is shown that there is a good correlation of cellular enzyme activity as determined in digitonin-permeabilized hepatocytes with the rate of triacylglycerol synthesis in intact hepatocytes.

The effect of postpartum rumen undegradable protein supplementation on hepatic gluconeogenic enzyme activities in dairy cows with fatty liver.

The effect of postpartum supplementation with rumen undegradable protein on the activities of gluconeogenic enzymes was studied in cows with induced fatty liver. Prepartum liver and blood samples were collected at about one week before the expected date of calving and postpartum samples were collected at 10 and 20 days (d) postpartum. At 10 d postpartum, concentrations of serum nonesterified fatty acids and hepatic triacylglycerol levels were higher than at one wk before parturition. The postpartum increases in nonesterified fatty acids and hepatic triacylglycerols were significantly higher in the cows that were fed extra protein than in the control cows. There were no differences between the groups with regard to postpartum changes in the concentrations of plasma glucose, liver glycogen, and serum insulin. The postpartum increase in the activity of fructose 1-6-bisphosphatase was higher in the test group than in the control group, but the increase in the activity of glucose-6-phosphatase was lower. There were no group differences in the postpartum activities of phosphoenolpyruvate carboxykinase, pyruvate carboxylase, and propionyl-CoA carboxylase. Our results suggest that intense lipolysis released more glycerol in the protein-supplemented cows, which stimulated the activity of fructose 1-6-bisphosphatase. However, postpartum rumen undegradable protein supplementation did not affect the activities of the other enzymes of gluconeogenesis, and fatty liver was even exacerbated.

Regulation of phosphatidylcholine and phosphatidylethanolamine synthesis in rat hepatocytes by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR).

The present study was undertaken to study the role of AMP-activated kinase (AMPK) in the biosynthesis of two major membrane phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an activator of AMPK, produced dose-dependent inhibition of the incorporation of [(3)H]choline and [(3)H]ethanolamine into PC and PE, respectively. Determination of the cellular uptake of choline and ethanolamine showed that the reduced synthesis of PC and PE did not result from impaired uptake of these two precursors. The decreased synthesis of PC was not mirrored by a reduction in the activities of the enzymes of the CDP-choline pathway. The diminution of PE biosynthesis, however, was paralleled by a depressed activity of CTP:phosphoethanolamine cytidylyltransferase (ET), the pace-setting enzyme of the CDP-ethanolamine pathway. AICAR treatment of hepatocytes stimulated the conversion of choline into betaine, indicating that reduced PC synthesis most probably resulted from a decrease in the availability of choline. In addition, AICAR induced a 50% reduction in the cellular level of diacylglycerols, which may further impair the synthesis of PC and PE. The results thus indicate that AICAR inhibits the biosynthesis of PC and PE and that the effect is exerted at different sites in the two pathways. Increased oxidation of choline to betaine is the main target of AICAR in the PC pathway, whereas inhibition of ET activity is the locus of AICAR action in the PE pathway.