[Show abstract][Hide abstract] ABSTRACT: Regular consumption of extra virgin olive oil (EVOO) is associated with a low incidence of atherosclerotic diseases. The phenolic component contributes to the hypolipidemic action of EVOO, although the biochemical mechanisms leading this beneficial outcome are not fully understood. Since liver plays a pivotal role in the whole body lipid homeostasis, we investigated the short-term effects of EVOO extract, with a high phenol content (HPE), on lipid synthesis in primary rat hepatocytes. Refined olive oil extract, with a low phenol content, was used throughout this study as a control.
European Journal of Nutrition 10/2014; DOI:10.1007/s00394-014-0761-5 · 3.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hydroxytyrosol, tyrosol, and oleuropein, the main phenols present in extra virgin olive oil, have been reported to exert several biochemical and pharmacological effects.
Here, we investigated the short-term effects of these compounds on lipid synthesis in primary-cultured rat-liver cells. Hydroxytyrosol, tyrosol and oleuropein inhibited both de novo fatty acid and cholesterol syntheses without an effect on cell viability. The inhibitory effect of individual compounds was already evident within 2 h of 25 μM phenol addition to the hepatocytes. The degree of cholesterogenesis reduction was similar for all phenol treatments (−25/30%), while fatty acid synthesis showed the following order of inhibition: hydroxytyrosol (−49%) = oleuropein (−48%) > tyrosol (−30%). A phenol-induced reduction of triglyceride synthesis was also detected.
To clarify the lipid-lowering mechanism of these compounds, their influence on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase and fatty acid synthase) triglyceride synthesis (diacylglycerol acyltransferase) and cholesterogenesis (3-hydroxy-3-methyl-glutaryl-CoA reductase) was investigated in situ by using digitonin-permeabilized hepatocytes. Acetyl-CoA carboxylase, diacylglycerol acyltransferase and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities were reduced after 2 h of 25 μM phenol treatment. No change in fatty acid synthase activity was observed. Acetyl-CoA carboxylase inhibition (hydroxytyrosol, −41%, = oleuropein, −38%, > tyrosol, −17%) appears to be mediated by phosphorylation of AMP-activated protein kinase. These findings suggest that a decrease in hepatic lipid synthesis may represent a potential mechanism underlying the reported hypolipidemic effect of phenols of extra virgin olive oil.
The Journal of nutritional biochemistry 07/2014; 25(7). DOI:10.1016/j.jnutbio.2014.01.009 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Extra virgin olive oil (EVOO), the principal source of fat in the Mediterranean diet, has been associated with a low incidence of coronary heart disease and certain cancers. The beneficial effects of EVOO have historically been attributed to its elevated oleic acid content; more recently, converging evidence indicates that hydroxytyrosol (HTyr), tyrosol (Tyr), and oleuropein (Ole) the main phenols present in EVOO non-saponifiable fraction, significantly promotes human health. In the present study, we investigated the short-term effects of these compounds on lipid synthesis in primary cultures of rat liver cells. HTyr, Tyr and Ole, dose-dependently (2.5-100 M) inhibited both de novo fatty acid and cholesterol syntheses, without effect on cell viability. Within 2 h of 25 µM phenol addition to the hepatocytes, the degree of cholesterogenesis reduction was similar for all phenol treatments, while fatty acid synthesis showed the following order of inhibition: HTyr = Ole > Tyr. A phenol-induced reduction of triglyceride synthesis was also detected.
To clarify the lipid-lowering mechanism of these compounds, their influence on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase, ACC), triglycerides synthesis (diacylglycerol acyltransferase, DGAT) and cholesterogenesis (3-hydroxy-3-methyl-glutaryl-CoA reductase, HMGCR), was investigated in situ by using digitonin-permeabilized hepatocytes. Within 2 h of treatment, the three tested compounds were able to inhibit ACC, DGAT and HMGCR activities. ACC inhibition appears to be mediated by AMP-activated protein kinase phosphorylation. These findings suggest that a reduction in hepatic lipid synthesis may be envisaged when assessing the potential benefits of EVOO phenols.
7th International Immunonutrition Workshop: Eating For Preventing, Carovigno (BR); 05/2014
[Show abstract][Hide abstract] ABSTRACT: The lipid-lowering effect of resveratrol (RSV), a polyphenol present in grapes, is generally investigated in proliferating cell lines and in vivo studies in different pathological conditions. The aim of the present study was to investigate whether RSV plays a role on lipid metabolism per se in normal cells. To this end isolated hepatocyte suspensions were chosen as experimental tool as close as possible to an in vivo, non pathological, conditions. The effect of RSV on total rate of fatty acid, cholesterol and complex lipid synthesis, assayed by the incorporation of [1-14C]acetate into these lipid fractions, showed that RSV-treated hepatocytes exhibited a short-term (30 min) inhibition (IC50 ~ 25 μM) of total fatty acid and triacylglycerol syntheses; conversely, cholesterol synthesis was almost unaffected.
Among neosynthesized fatty acids, palmitic acid formation was mainly reduced, thus suggesting that enzymatic step(s) of de novo fatty acid synthesis was influenced by RSV. In order to ascertain this hypothesis, the activity of acetyl-CoA carboxylase (ACC), the pace-setting enzyme of the above-mentioned lipogenic pathway, was then investigated in digitonin-permeabilized hepatocytes, showing a RSV-mediated reduction of ACC-specific activity. Under the same experimental condition (30 min, 25 μM), RSV did not show any appreciable effect on the enzymatic activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase, regulatory enzyme of cholesterol synthesis.
Results here reported show that in isolated hepatocytes from normal rats a RSV -induced short-term inhibition of fatty acid and triacylglycerol synthesis occurs. This finding may represent a potential mechanism contributing to the reported hypolipidemic effect of RSV.
7th International Immunonutrition Workshop: Eating for Preventing, Carovigno (Br); 05/2014
[Show abstract][Hide abstract] ABSTRACT: Growing evidence shows that, among triiodothyronine derivatives, 3,5 diiodo-L-thyronine (T(2)) plays an important role in energy metabolism and fat storage. In the present study, short-term effects of T(2) administration to hypothyroid rats on fatty acid oxidation rate and bioenergetic parameters were investigated. Within 1 h following T(2) injection, state 3 and state 4 respiration rates, which were reduced in hypothyroid mitochondria, were noticeably increased particularly in succinate- with respect to glutamate/malate-energized mitochondria. Maximal respiratory activity, observed when glutamate/malate/succinate were simultaneously present in the respiratory medium, was significantly stimulated by T(2) treatment. A T(2)-induced increase in respiratory rates was also observed when palmitoyl-CoA or L-palmitoylcarnitine were used as substrates. No significant change in respiratory control index and ADP/O ratio was observed. The activities of the mitochondrial respiratory chain complexes, especially Complex II, were increased in T(2)-treated rats. In the latter, Complex V activities, assayed in both ATP synthesis and hydrolysis direction, were enhanced. The rate of fatty acid oxidation, followed by conversion of [(14)C]palmitate to CO(2) and ketone bodies, was higher in hepatocytes isolated from T(2)-treated rats. This increase occurs in parallel with the raise in the activity of carnitine palmitoyltransferase-I, the rate limiting enzyme of fatty acid β-oxidation, assayed in situ in digitonin-permeabilized hepatocytes. Overall, these results indicate that T(2) rapidly increases the ability of mitochondria to import and oxidize fatty acids. An emerging idea in the literature is the ability of T(2) to reduce adiposity and dyslipidemia and to prevent the development in liver steatosis. The results of the present study, showing a rapid T(2)-induced increase in the ability of mitochondria to import and oxidize fatty acids, may contribute to understand the biochemical mechanisms of T(2)-metabolic effects.
PLoS ONE 01/2013; 8(1):e52328. DOI:10.1371/journal.pone.0052328 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 3,5-diiodo-L-thyronine (T2), previously considered only a catabolic product of thyroid hormone metabolism, has been recently shown to play an important role on energy metabolism (1-2). In this study, we investigated the effect of acute administration of T2 to hypothyroid rats on hepatic fatty acid oxidation rate and bioenergetic parameters. Coupled (state 3), uncoupled (state 4) and FCCP-stimulated respiration, which, as compared to control, were reduced in hypothyroid mitochondria, were noticeably increased within 1h following i.p. injection of T2. The hormone effect was more pronounced in succinate- with respect to glutamate/malate-energized mitochondria. T2 enhancement of fatty acid oxidation rate was assessed by using palmitoyl-CoA (+carnitine) or palmitoylcarnitine as respiratory substrate. No significant change in respiratory control index and ADP/O ratio was observed. The activities of the mitochondrial respiratory chain complexes, especially Complex II, were increased in T2-treated rats. In the latter, Complex V activities, assayed in both ATP synthesis and hydrolysis direction, were enhanced. The total rate of fatty acid oxidation, followed by conversion of [14C] palmitate to 14CO2 and ketone bodies, was higher in hepatocytes isolated from T2-treated rats. This increase occurs in parallel with the enhanced activity of carnitine palmitoyltransferase-I (CPT-I), the rate limiting enzyme of fatty acid β-oxidation, thus suggesting CPT-I activity as a possible target of T2 action. Taken together, these results indicate that T2 rapidly promotes the ability of mitochondria to import and oxidize fatty acids, and contribute to explain recent findings showing that T2 may powerfully reduce adiposity and body weight gain (2), and reverse hepatic steatosis without inducing thyrotoxicity.
National Meeting of the Italian Society of Biochemistry and Molecular Biology - Chieti 26-29 Sept 2012, Chieti; 09/2012
[Show abstract][Hide abstract] ABSTRACT: The carnitine/acylcarnitine translocase (CACT), an integral protein of the mitochondrial inner membrane, belongs to the carnitine-dependent system of fatty acid transport into mitochondria, where beta-oxidation occurs. CACT exchanges cytosolic acylcarnitine or free carnitine for carnitine in the mitochondrial matrix. The object of this study was to investigate in rat liver the effect, if any, of diets enriched with saturated fatty acids (beef tallow, BT, the control), n-3 polyunsaturated fatty acids (PUFA) (fish oil, FO), n-6 PUFA (safflower oil, SO), and mono-unsaturated fatty acids (MUFA) (olive oil, OO) on the activity and expression of CACT. Translocase exchange rates increased, in parallel with CACT mRNA abundance, upon FO-feeding, whereas OO-dietary treatment induced a decrease in both CACT activity and expression. No changes were observed upon SO-feeding. Nuclear run-on assay revealed that FO-treatment increased the transcriptional rate of CACT mRNA. On the other hand, only in the nuclei of hepatocytes from OO-fed rats splicing of the last intron of CACT pre-mRNA and the rate of formation of the 3'-end were affected. Overall, these findings suggest that compared to the BT-enriched diet, the SO-enriched diet did not influence CACT activity and expression, whereas FO- and OO-feeding alters CACT activity in an opposite fashion, i.e. modulating its expression at transcriptional and post-transcriptional levels, respectively.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 09/2011; 6(9):e24084. DOI:10.1371/journal.pone.0024084 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
International Union of Biochemistry and Molecular Biology Life 10/2009; 61(10):987-94. DOI:10.1002/iub.249 · 2.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.