The hepatocyte nuclear factor 4 (HNF-4) represses the mitochondrial HMG-CoA synthase gene

We have recently shown that the gene for the mitochondrial HMG-CoA synthase is a target for PPAR and that this receptor mediates the induction of this gene by fatty acids. With the aim of gaining further insight into the function and regulation of this gene we examined the effect of other members of the nuclear hormone receptor superfamily on its expression. We previously identified a regulatory element in the mitochondrial HMG-CoA synthase gene promoter that confers transcriptional regulation by PPAR, RXR and the orphan nuclear receptor COUP-TF, In this study we demonstrate a trans-repressing regulatory function for HNF-4 at this same nuclear receptor response element (NRRE). HNF-4 binds to the mitochondrial HMG-CoA synthase NRRE, and, in cotransfection assays in HepG2 cells, it represses PPAR-dependent activation of a reporter gene linked to the mitochondrial HMG-CoA synthase gene promoter. These results suggest that the mitochondrial HMG-CoA synthase gene is subject to differential regulation by the interplay of multiple members of the nuclear hormone receptor superfamily

Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import into mitochondria, and it is believed to be rate limiting for beta-oxidation of fatty acids. However, in muscle, other proteins may collaborate with CPT1. Fatty acid translocase/CD36 (FAT/CD36) may interact with CPT1 and contribute to fatty acid import into mitochondria in muscle. Here, we demonstrate that another membrane-bound fatty acid binding protein, fatty acid transport protein 1 (FATP1), collaborates with CPT1 for fatty acid import into mitochondria. Overexpression of FATP1 using adenovirus in L6E9 myotubes increased both fatty acid oxidation and palmitate esterification into triacylglycerides. Moreover, immunocytochemistry assays in transfected L6E9 myotubes showed that FATP1 was present in mitochondria and coimmunoprecipitated with CPT1 in L6E9 myotubes and rat skeletal muscle in vivo. The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1. However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects. These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids

Convenient synthesis of C75, an inhibitor of FAS and CPT1

C75 is a synthetic racemic α-methylene-γ-butyrolactone exhibiting anti-tumoral properties in vitro and in vivo as well as inducing hypophagia and weight loss in rodents. These interesting properties are thought to be a consequence of the inhibition of the key enzymes FAS and CPT1 involved in lipid metabolism. The need for larger amounts of this compound for biological evaluation prompted us to develop a convenient and reliable route to multigram quantities of C75 from easily available ethyl penta-3,4-dienoate 6. A recently described protocol for the addition of 6 to a mixture of dicyclohexylborane and nonanal followed by acidic treatment of the crude afforded lactone 8, as a mixture of cis and trans isomers, in good yield. The DBU-catalyzed isomerization of the methyl esters 9 arising from 8 gave a 10:1 trans/cis mixture from which the trans isomer was isolated and easily transformed into C75. The temporary transformation of C75 into a phenylseleno ether derivative makes its purification, manipulation and storage easier

(−)-UB006: A new fatty acid synthase inhibitor and cytotoxic agent without anorexic side effects

C75 is a synthetic anticancer drug that inhibits fatty acid synthase (FAS) and shows a potent anorexigenic side effect. In order to find new cytotoxic compounds that do not impact food intake, we synthesized a new family of C75 derivatives. The most promising anticancer compound among them was UB006 ((4SR,5SR)-4-(hydroxymethyl)-3-methylene-5-octyldihydrofuran-2(3H)-one). The effects of this compound on cytotoxicity, food intake and body weight were studied in UB006 racemic mixture and in both its enantiomers separately. The results showed that both enantiomers inhibit FAS activity and have potent cytotoxic effects in several tumour cell lines, such as the ovarian cell cancer line OVCAR-3. The (−)-UB006 enantiomer's cytotoxic effect on OVCAR-3 was 40-fold higher than that of racemic C75, and 2- and 38-fold higher than that of the racemic mixture and its opposite enantiomer, respectively. This cytotoxic effect on the OVCAR-3 cell line involves mechanisms that reduce mitochondrial respiratory capacity and ATP production, DDIT4/REDD1 upregulation, mTOR activity inhibition, and caspase-3 activation, resulting in apoptosis. In addition, central and peripheral administration of (+)-UB006 or (−)-UB006 into rats and mice did not affect food intake or body weight. Altogether, our data support the discovery of a new potential anticancer compound (−)-UB006 that has no anorexigenic side effects

Long-Term Increased Carnitine Palmitoyltransferase 1A Expression in Ventromedial Hypotalamus Causes Hyperphagia and Alters the Hypothalamic Lipidomic Profile

Lipid metabolism in the ventromedial hypothalamus (VMH) has emerged as a crucial pathway in the regulation of feeding and energy homeostasis. Carnitine palmitoyltransferase (CPT) 1A is the rate-limiting enzyme in mitochondrial fatty acid boxidation and it has been proposed as a crucial mediator of fasting and ghrelin orexigenic signalling. However, the relationship between changes in CPT1A activity and the intracellular downstream effectors in the VMH that contribute to appetite modulation is not fully understood. To this end, we examined the effect of long-term expression of a permanently activated CPT1A isoform by using an adeno-associated viral vector injected into the VMH of rats. Peripherally, this procedure provoked hyperghrelinemia and hyperphagia, which led to overweight, hyperglycemia and insulin resistance. In the mediobasal hypothalamus (MBH), long-term CPT1AM expression in the VMH did not modify acyl-CoA or malonyl-CoA levels. However, it altered the MBH lipidomic profile since ceramides and sphingolipids increased and phospholipids decreased. Furthermore, we detected increased vesicular c-aminobutyric acid transporter (VGAT) and reduced vesicular glutamate transporter 2 (VGLUT2) expressions, both transporters involved in this orexigenic signal. Taken together, these observations indicate that CPT1A contributes to the regulation of feeding by modulating the expression of neurotransmitter transporters and lipid components that influence the orexigenic pathways in VMH

Molecular interplay betweeen cáncer cell fatty acid metabolism and oncogenic signaling as resource for novel treatment strategies against ovarian cancer

The metabolic oncogene fatty acid synthase (FASN) is overexpressed in 80% of ovarian cancers (OC) and indicates poor prognosis. Exposure of OC to inhibitors of FASN elicits a complex stress response that interferes with receptor-PI3K-mTORC1 signaling (briefly designated 'PI3K pathway'). Here we demonstrate that FASN inhibitors capitalize on multiple mechanisms to interfere with the PI3K pathway, and that silencing this cascade is crucial for the anticancer action of the drugs

La acidúria 3-hidroxi 3-metilglutàrica: Una malatia molecular per deficiència de 3-hidroxi 3-metilglutaril coenzim A liasa

L'acidúria 3-hidroxi-3-metilglutàrica és una malaltia genètica autosòmica recessiva que es manifesta per una sèrie de símptomes que, en conjunt, s'assemblen a la síndrome de Reye. En aquesta revisió realitzada des de la presentació del primer cas, el 1976, s'ha tractat de recollir la informació sobre els aspectes clínics, els metabòlits anormals que apareixen a l'orina, la deficiència enzimàtica de la HMG-CoA liasa, el diagnòstic, el tractament i la genètica de la malaltia. Si bé són més de cinquanta els casos reportats d'acidúria 3-hidroxi-3-metilglutàrica, solament quinze pacients s'han estudiat des del punt de vista molecular des que el 1993 es va reportar el clonatge del cDNA de la HMG-CoA liasa humana. Es coneixen nou mutacions al·lèliques diferents fins ara. La major incidència de totes les reportades és la mutació puntual en el nucleòtid 109, que condueix a la producció de tres transcrits diferents, un amb un codó d'aturada, un altre en el qual s'han produït un splicing alternatiu amb eliminació de l'exó 2 i un tercer amb un splicing diferent i l'eliminació dels exons 2 i 3. Aquesta mutació és prevalent al sud d'Europa i representa el 37% de tots els casos reportats

La acidúria 3-hidroxi 3-metilglutàrica: Una malatia molecular per deficiència de 3-hidroxi 3-metilglutaril coenzim A liasa

L'acidúria 3-hidroxi-3-metilglutàrica és una malaltia genètica autosòmica recessiva que es manifesta per una sèrie de símptomes que, en conjunt, s'assemblen a la síndrome de Reye. En aquesta revisió realitzada des de la presentació del primer cas, el 1976, s'ha tractat de recollir la informació sobre els aspectes clínics, els metabòlits anormals que apareixen a l'orina, la deficiència enzimàtica de la HMG-CoA liasa, el diagnòstic, el tractament i la genètica de la malaltia. Si bé són més de cinquanta els casos reportats d'acidúria 3-hidroxi-3-metilglutàrica, solament quinze pacients s'han estudiat des del punt de vista molecular des que el 1993 es va reportar el clonatge del cDNA de la HMG-CoA liasa humana. Es coneixen nou mutacions al·lèliques diferents fins ara. La major incidència de totes les reportades és la mutació puntual en el nucleòtid 109, que condueix a la producció de tres transcrits diferents, un amb un codó d'aturada, un altre en el qual s'han produït un splicing alternatiu amb eliminació de l'exó 2 i un tercer amb un splicing diferent i l'eliminació dels exons 2 i 3. Aquesta mutació és prevalent al sud d'Europa i representa el 37% de tots els casos reportats

The peroxisome proliferator-activated receptor mediates the induction of the mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase gene by fatty acids

Fatty acids induce an increase in the transcription of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene, which encodes an enzyme that has been proposed as a control site of ketogenesis. We studied whether the peroxisome proliferator-activated receptor (PPAR) is involved in the mechanism of this transcriptional induction. We found that cotransfection of a rat mitochondrial HMG-CoA synthase promoter- chloramphenicol acetyltransferase reporter plasmid and a PPAR expression plasmid in the presence of the peroxisome proliferator clofibrate led to a more than 30-fold increase in chloramphenicol acetyltransferase activity, relative to the activity in the absence of both PPAR and inducer. Linoleic acid, a polyunsaturated fatty acid, increased this activity as potently as does clofibrate and more effectively than does monounsaturated oleic acid. We have identified, by deletional analysis, an element located 104 base pairs upstream of the mitochondrial HMG-CoA synthase gene, which confers PPAR responsiveness to homologous and heterologous promoters. This is the first example of a peroxisome proliferator-responsive element (PPRE)in a gene encoding a mitochondrial protein. This element contains an imperfect direct repeat that is similar to those described in theP PREs of other genes. Furthermore, gel retardation and cotransfection assays revealed that, as for other genes, PPAR heterodimerizes with retinoid X receptor and that both receptors cooperate for binding to the mitochondrial HMG-CoA synthase PPRE and subsequent activation of the gene. In conclusion, our data demonstrate that regulation of mitochondrial HMG-CoA synthase gene expression by fatty acids is mediated by PPAR, supporting the hypothesis that PPAR has an important role at the transcriptional level in the regulation of lipid metabolism

The peroxisome proliferator-activated receptor mediates the induction of the mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase gene by fatty acids

Fatty acids induce an increase in the transcription of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene, which encodes an enzyme that has been proposed as a control site of ketogenesis. We studied whether the peroxisome proliferator-activated receptor (PPAR) is involved in the mechanism of this transcriptional induction. We found that cotransfection of a rat mitochondrial HMG-CoA synthase promoter- chloramphenicol acetyltransferase reporter plasmid and a PPAR expression plasmid in the presence of the peroxisome proliferator clofibrate led to a more than 30-fold increase in chloramphenicol acetyltransferase activity, relative to the activity in the absence of both PPAR and inducer. Linoleic acid, a polyunsaturated fatty acid, increased this activity as potently as does clofibrate and more effectively than does monounsaturated oleic acid. We have identified, by deletional analysis, an element located 104 base pairs upstream of the mitochondrial HMG-CoA synthase gene, which confers PPAR responsiveness to homologous and heterologous promoters. This is the first example of a peroxisome proliferator-responsive element (PPRE)in a gene encoding a mitochondrial protein. This element contains an imperfect direct repeat that is similar to those described in theP PREs of other genes. Furthermore, gel retardation and cotransfection assays revealed that, as for other genes, PPAR heterodimerizes with retinoid X receptor and that both receptors cooperate for binding to the mitochondrial HMG-CoA synthase PPRE and subsequent activation of the gene. In conclusion, our data demonstrate that regulation of mitochondrial HMG-CoA synthase gene expression by fatty acids is mediated by PPAR, supporting the hypothesis that PPAR has an important role at the transcriptional level in the regulation of lipid metabolism