Gabriele V. Gnoni

Oleic acid is a potent inhibitor of fatty acid and cholesterol synthesis in C6 glioma cells

Glial cells play a pivotal role in brain fatty acid metabolism and membrane biogenesis. However, the potential regulation of lipogenesis and cholesterologenesis by fatty acids in glial cells has been barely investigated. Here, we show that physiologically relevant concentrations of various saturated, monounsaturated, and polyunsaturated fatty acids significantly reduce [1-14C]acetate incorporation into fatty acids and cholesterol in C6 cells. Oleic acid was the most effective at depressing lipogenesis and cholesterologenesis; a decreased label incorporation into cellular palmitic, stearic, and oleic acids was detected, suggesting that an enzymatic step(s) of de novo fatty acid biosynthesis was affected. To clarify this issue, the activities of acetyl-coenzyme A carboxylase (ACC) and FAS were determined with an in situ digitonin-permeabilized cell assay after incubation of C6 cells with fatty acids. ACC activity was strongly reduced (∼80%) by oleic acid, whereas no significant change in FAS activity was observed. Oleic acid also reduced the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). The inhibition of ACC and HMGCR activities is corroborated by the decreases in ACC and HMGCR mRNA abundance and protein levels. The downregulation of ACC and HMGCR activities and expression by oleic acid could contribute to the reduced lipogenesis and cholesterologenesis.

Structural and functional characterization of Fof1-ATP synthase on the extracellular surface of rat hepatocytes

Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-F(o)F(1)-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F(1) (alpha/beta and gamma) and F(o) (F(o)I-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF(1) is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H(+) and are inhibited by F(1) and F(o)-targeting inhibitors. We hypothesise that ecto-F(o)F(1)-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.

Quercetin inhibits fatty acid and triacylglycerol synthesis in rat-liver cells.

Background  Quercetin plays a cardiovascular protective role because of its antioxidant capacity and ability to modulate dyslipidemia. As alterations in hepatic lipid synthesis are crucial to the regulation of serum lipid levels, we investigated the quercetin effect on lipogenesis in rat liver cells.

Oxidation of Hepatic Carnitine Palmitoyl Transferase-I (CPT-I) Impairs Fatty Acid Beta-Oxidation in Rats Fed a Methionine-Choline Deficient Diet

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-Iactivity 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 CO2 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.

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.

Resveratrol inhibits fatty acid and triacylglycerol synthesis in rat hepatocytes

Background  The putative role of resveratrol, a polyphenol present in grapes and other plants, in modulating dislypidemia, thus preventing cardiovascular diseases, is generally based on proliferating cell lines and in vivo studies in different pathological conditions. The aim of the present study was to investigate whether resveratrol plays a role on lipid biosynthesis in rat hepatocytes.

Mitochondrial oxidative stress and respiratory chain dysfunction account for liver toxicity during amiodarone but not dronedarone administration.

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.

A Silybin-Phospholipid Complex Prevents Mitochondrial Dysfunction in a Rodent Model of Nonalcoholic Steatohepatitis

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 H2O2 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 H2O2 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.

Natural merodiploidy involving duplicated rpoB alleles affects secondary metabolism in a producer actinomycete

Actinomadura sp. ATCC 39727 produces the glycopeptide antibiotic A40926, structurally similar to teicoplanin. Production of A40926 is governed by the stringent response at the transcriptional level. In fact, addition of an amino acid pool prevented the transcription of dbv cluster genes involved in the A40926 biosynthesis and the antibiotic production in chemically defined media, and a thiostrepton‐resistant relaxed mutant was severely impaired in its ability to produce the antibiotic. The derivative strain rif19, highly resistant to rifampicin (minimal inhibitory concentration, MIC > 200 µg ml−1), was isolated from the wild type strain that exhibited low resistance to rifampicin (MIC < 25 µg ml−1). In this strain A40926 production started earlier than in the wild type, and reached higher final levels. Moreover, the antibiotic production was not subjected to the stringent control. Molecular analysis led to the identification of two distinct rpoB alleles, rpoBS and rpoBR, in both the wild type and the rif19. rpoBR harboured the H426N missense which is responsible for rifampicin‐resistance in bacteria, in addition to other nucleotide substitutions affecting the primary structure of the RNA polymerase β‐chain. Transcript analysis revealed that rpoBR was expressed at a very low level in the wild type strain during the pseudo‐exponential growth phase, and that the amount of rpoBR mRNA increased during the transition to the stationary phase. In contrast, expression of rpoBR was constitutive in the rif19. The results of mRNA half‐life analysis did not support the hypothesis that post‐transcriptional events are responsible for the different rpoB expression patterns in the two strains, suggesting a role of transcriptional mechanisms.

Effect of starvation on the activity of the mitochondrial tricarboxylate carrier

The effect of starvation on the activity of the tricarboxylate carrier has been investigated in intact rat liver mitochondria and in a reconstituted system. In both experimental conditions, the rate of citrate transport, when compared to control, is greatly reduced (35-40%) in starved rats. Similar behaviour is shown by the cytosolic lipogenic enzymes. Kinetic analysis of the carrier activity in intact mitochondria and in the proteoliposomal system has showed that during starvation only the Vmax of this process decreases while there is no change in the Km. No difference in the Arrhenius plot and in the lipid composition has been detected, which indicates that the reduced transport activity in fasted animals is not due to a change in the carrier lipid microenvironment. In starved rats, a reduction of the carrier activity has occurred even after the addition of increasing cardiolipin concentrations to proteoliposomes. These findings thus suggest that starvation-induced decrease of citrate carrier activity could be due to a change of the intrinsic properties of the transport protein.