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 [1-14 C]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 acetylcoenzyme 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.-Natali, F., L. Siculella, S. Salvati, and G. V. Gnoni. Oleic acid is a potent inhibitor of fatty acid and cholesterol synthesis in C6 glioma cells. J. Lipid Res. 2007Res. . 48: 1966Res. -1975. After white adipose tissue, the brain is the organ with the highest lipid content of the body. The biosynthesis and deposition of lipids play an important role in maintaining brain structure and function, for example, during development-associated biogenesis of neural cell membranes. It is well established that alterations in lipid metabolism are the cause of or are associated with many neurological diseases (1-3).Astrocytes, the major class of glial cells in the mammalian brain, play an active role in brain metabolism. These cells surround intraparenchymal blood capillaries so that they represent the first cellular barrier for nutrients and other substances entering the brain system. A metabolic coupling between astrocytes and neurons to maintain energy metabolism homeostasis has been described (4, 5). Metabolic regulation in the brain has been investigated extensively, and those studies focused mostly on carbohydrate and amino acid metabolism (for review, see Ref. 6). During neuronal activity, glucose taken up by astrocytes is converted into lactate, which is then released into the extracellular space to be used by neurons (6). Regarding lipid metabolism, astroglial ketone body synthesis, showing characteristics strikingly similar to those of hepatic ketogenesis (7), may represent an important pathway for brain energy production and/or biosynthetic processes. The involvement of fatty acids in cell death pathways, particularly in the con...

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.

SummaryThe 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.

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.

In hepatocytes from normal rats, the quercetin-induced decrease in both de novo fatty acid and TAG synthesis, with a consequent reduction in VLDL-TAG formation, may represent a potential mechanism contributing to the reported hypotriacylglycerolemic effect of quercetin.

SummaryActinomadura 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 m m m m g ml), was isolated from the wild type strain that exhibited low resistance to rifampicin (MIC < 25 m m m m g ml). 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, rpoB S and rpoB R , in both the wild type and the rif19 . rpoB R 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 b b b b -chain. Transcript analysis revealed that rpoB R was expressed at a very low level in the wild type strain during the pseudo-exponential growth phase, and that the amount of rpoB R mRNA increased during the transition to the stationary phase. In contrast, expression of rpoB R 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.

SREBPs (sterol-regulatory-element-binding proteins) are a family of transcription factors that modulate the expression of several enzymes implicated in endogenous cholesterol, fatty acid, triacylglycerol and phospholipid synthesis. In the present study, evidence for SREBP-1 regulation at the translational level is reported. Using several experimental approaches, we have demonstrated that the 5'-UTR (untranslated region) of the SREBP-1a mRNA contains an IRES (internal ribosome entry site). Transfection experiments with the SREBP-1a 5'-UTR inserted in a dicistronic reporter vector showed a remarkable increase in the downstream cistron translation, through a cap-independent mechanism. Insertion of the SREBP-1c 5'-UTR in the same vector also stimulated the translation of the downstream cistron, but the observed effect can be ascribed, at least in part, to a cryptic promoter activity. Cellular stress conditions, such as serum starvation, caused an increase in the level of SREBP-1 precursor and mature form in both Hep G2 and HeLa cells, despite the overall reduction in protein synthesis, whereas mRNA levels for SREBP-1 were unaffected by serum starvation. Transfection experiments carried out with a dicistronic construct demonstrated that the cap-dependent translation was affected more than IRES-mediated translation by serum starvation. The thapsigargin- and tunicamycin-induced UPR (unfolded protein response) also increased SREBP-1 expression in Hep G2 cells, through the cap-independent translation mediated by IRES. Overall, these findings indicate that the presence of IRES in the SREBP-1a 5'-UTR allows translation to be maintained under conditions that are inhibitory to cap-dependent translation.

The mitochondrial tricarboxylate (citrate) carrier plays an important role in hepatic intermediary metabolism because, among other functions, it supplies the cytosol with acetyl units for fatty-acid synthesis. In this study, the effect of polyunsaturated fatty acids (PUFA, n-6) on the function of this mitochondrial transporter and on lipogenic enzyme activities was investigated by feeding rats for 4 weeks with a 15%-fat diet composed of high linoleic safflower oil. Citrate transport was strongly reduced in liver mitochondria isolated from PUFA-treated rats. A reduced transport activity was also observed when solubilized mitochondrial citrate carrier from PUFA-treated rats was reconstituted into liposomes. In the same animals, a decrease of cytosolic lipogenic enzyme activities was observed. These results indicate a coordinated modulation of citrate carrier and of lipogenic enzyme activities by PUFA feeding. Kinetic analysis of the carrier activity showed that only V max decreased, whereas K m was almost virtually unaffected. The PUFA-mediated effect is most likely due to the reduced mRNA level and lower content of the citrate carrier protein observed in the safflower oil-fed rats.