Guillermina Asins - Academia.edu (original) (raw)

Papers by Guillermina Asins

Research paper thumbnail of et al. Expression of GLUT-2 antisense RNA in B cells of transgenic mice leads to diabetes

Journal of Biological Chemistry

ABSTRACT

Research paper thumbnail of Purification and properties of a protein inhibitor that inhibits phosphatase 2A activity when hydroxymethylglutaryl coenzyme A reductase is the substrate

Journal of Biological Chemistry

A heat-stable protein inhibitor of the hydroxymethylglutaryl-CoA reductase phosphatase 2A activit... more A heat-stable protein inhibitor of the hydroxymethylglutaryl-CoA reductase phosphatase 2A activity has been identified and purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The apparent molecular mass was 20,000 Da. The protein lost its inhibitory properties when incubated with trypsin or treated with ethanol. The inhibitor protein does not inhibit type 1 phosphatase when either phosphorylase or hydroxymethylglutaryl-CoA reductase is the substrate. In contrast, this protein inhibitor inhibits the rat liver type 2A phosphatase activity when hydroxymethylglutaryl-CoA reductase is the substrate but not when phosphorylase a is the substrate. The inhibitor protein is not activated by incubation with ATP and cyclic AMP-dependent protein kinase and it is not phosphorylated by glycogen synthase kinase-3. These results, together with those of the kinetic experiments, suggest that the reductase phosphatase inhibitor is distinct from protein phosphatase inhibitor-1 and inhibitor-2.

Research paper thumbnail of Inactivation of rat liver HMG-CoA reductase phosphatases by nucleotides

Revista española de fisiología

Incubation of four purified rat liver HMG-CoA reductase phosphatases, with ATP, ADP and AMP cause... more Incubation of four purified rat liver HMG-CoA reductase phosphatases, with ATP, ADP and AMP caused a concentration-dependent inactivation of enzyme activities. The nucleotides of guanine, cytosine and uracil produced similar effects to those by the nucleotides of adenine for the same number of phosphates present in the molecules. The greater the number of phosphate groups in nucleotides, the higher was the inhibition in reductase phosphatases observed. Preincubation of phosphatases with ATP and subsequent dilution did not diminish the inactivation effect, showing that nucleotides inhibit the enzyme prior to their binding to the substrate. A relationship was observed between those concentrations of nucleotides which produce 50% inactivation and the logarithm stability constant of Mg or Mn salts of nucleotides. ATP-inactivated enzymes were reactivated by Mn++ and to a lesser proportion by Mg++, the conclusion being that HMG-CoA reductase phosphatases have the characteristics of metalloenzymes.

Research paper thumbnail of cis-Acting elements controlling transcription from rat serine protease inhibitor 2.1 gene promoter. Characterization of two growth hormone response sites and a dominant purine-rich element

Journal of Biological Chemistry

The cis-acting elements that are functionally important for the basal, the growth hormone (GH), a... more The cis-acting elements that are functionally important for the basal, the growth hormone (GH), and the glucocorticoid hormone (GC) regulation of expression of the rat serine protease inhibitor 2.1 gene (spi 2.1) were mapped. Normal rat hepatocytes were transiently transfected with constructs harboring deleted or mutated versions of the spi 2.1 proximal promoter region fused to the chloramphenicol acetyltransferase gene. A purinerich sequence (GAGAbox, nucleotides -57 to -46), whose mutation or deletion almost completely knocks out both basal and hormone-stimulated promoter activities, plays the role of a key control element. A positive GC response element, spanning nucleotides -88 to -74, confers GC responsiveness to a heterologous promoter. Two structurally unrelated GH-response elements (GHRE) were identified. GHRE-I1 (nucleotides -136 to -104) contains a CCAAT enhancer binding protein binding site whose mutation completely abolishes its GH-dependent enhancer function. G I " , which spans nucleotides -61 to +8, is not an enhancer element. Its GH-dependent activity depends on the preservation of the distance separating the GAGA box and elements of the basic transcriptional machinery.

Research paper thumbnail of Purification and characterization of a protein inhibitor from rat liver that inhibits type 1 protein phosphatase when 3-hydroxy-3-methylglutaryl CoA reductase is the substrate

The Journal of Lipid Research

Research paper thumbnail of Overexpression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in transgenic mice causes hepatic hyperketogenesis

Journal of Biological Chemistry

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCoAsynthase) is a key enzyme in the ket... more Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCoAsynthase) is a key enzyme in the ketone body pathway. To determine its role in the regulation of liver ketogenesis, transgenic mice expressing a P-enolpyruvate carboxykinase/HMGCoA synthase chimeric gene have been obtained. An increase in the concentration of mitochondrial HMG-CoA synthase mRNA was detected in these mice, which was associated with a %fold increase in HMG-CoA synthase activity in liver mitochondrial extracts. Transgenic mice were normoglycemic and had normal levels of plasma triglycerides and lower free fatty acids. However, the plasma concentration of ketone bodies was about three times higher in transgenic mice than in control animals. Hepatocytes in primary culture from transgenic mice expressed the chimeric gene in a regulated manner and showed a 3-fold increase in P-hydroxybutyrate and acetoacetate concentrations in the medium. This animal model thus shows that the overexpression of mitochondrial HMG-CoA synthase causes ketone body overproduction, suggesting that this enzyme may be a regulatory step in liver ketogenesis.

Research paper thumbnail of Gene expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in a poorly ketogenic mammal: effect of starvation during the neonatal period of the piglet

The Biochemical journal, Jan 15, 1997

The low ketogenic capacity of pigs correlates with a low activity of mitochondrial 3-hydroxy-3-me... more The low ketogenic capacity of pigs correlates with a low activity of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. To identify the molecular mechanism controlling such activity, we isolated the pig cDNA encoding this enzyme and analysed changes in mRNA levels and mitochondrial specific activity induced during development and starvation. Pig mitochondrial synthase showed a tissue-specific expression pattern. As with rat and human, the gene is expressed in liver and large intestine; however, the pig differs in that mRNA was not detected in testis, kidney or small intestine. During development, pig mitochondrial HMG-CoA synthase gene expression showed interesting differences from that in the rat: (1) there was a 2-3 week lag in the postnatal induction; (2) the mRNA levels remained relatively abundant through the suckling-weaning transition and at maturity, in contrast with the fall observed in rats at similar stages of development; and (3) the gene expression was hig...

Research paper thumbnail of The effect of fasting and insulin treatment on carnitine palmitoyl transferase I and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase mRNA levels in liver from suckling rats

Biochemical Society transactions, 1995

Research paper thumbnail of Developmental changes in carnitine palmitoyltransferases I and II gene expression in intestine and liver of suckling rats

The Biochemical journal, 1995

Carnitine palmitoyltransferase (CPT) I is expressed in the intestine of suckling rats; its mRNA i... more Carnitine palmitoyltransferase (CPT) I is expressed in the intestine of suckling rats; its mRNA increases very rapidly after birth, remains on a plateau until day 18 and decreases until weaning, when basal (adult) values are reached, which remain unchanged thereafter. CPT II mRNA values do not show any appreciable change in this period. CPT I and CPT II are expressed mainly in mucosa and, to a lesser extent, in the muscular part of the intestine. Intestinal expression of CPT I is maximal in duodenum and jejunum, whereas CPT II is expressed in a similar pattern throughout the whole intestine. Dam's milk may influence the intestinal expression of CPT I, since mRNA levels at birth are low but increase after the first lactation. Moreover, rats weaned at either day 18 or 21 decrease their mRNA levels. Apparently, CPT II gene expression is not influenced by the mother's milk. CPT I and CPT II are also expressed in the liver of suckling rats. Hepatic CPT I is maximal at day 3, and ...

Research paper thumbnail of Isolation and partial characterization of a protein with HMG-CoA reductase phosphatase activity associated with rat liver microsomal membranes

Several rat liver HMG-CoA-reductase (HMG-CoA- Rd) phosphatase activities have been shown to be as... more Several rat liver HMG-CoA-reductase (HMG-CoA- Rd) phosphatase activities have been shown to be associated with the endoplasmic reticulum. These activities were not due to glycogen contamination, as judged not only from different patterns of solubilization of the microsomal membranes and the glycogen pellet but also by differential centrifugation behavior under standard conditions and in a sucrose gradient. We present evidence

Research paper thumbnail of The effect of dexamethasone treatment on the expression of the regulatory genes of ketogenesis in intestine and liver of suckling rats

Molecular and Cellular Biochemistry, 1998

The influence of the injection of dexamethasone on ketogenesis in 12 day old suckling rats was st... more The influence of the injection of dexamethasone on ketogenesis in 12 day old suckling rats was studied in intestine and liver by determining mRNA levels and enzyme activity of the two genes responsible for regulation of ketogenesis: carnitine palmitoyl transferase I (CPT I) and mitochondrial HMG-CoA synthase. Dexamethasone produced a 2 fold increase in mRNA and activity of CPT I in intestine, but led to a decrease in mit. HMG-CoA synthase. In liver the mRNA levels and activity of both CPT I and mit. HMG-CoA synthase decreased. Comparison of these values with the ketogenic rate of both tissues following dexamethasone treatment suggests that mit. HMG-CoA synthase could be the main gene responsible for the regulation of ketogenesis in suckling rats. The changes produced in serum ketone bodies by dexamethasone, with a profile that is more similar to the ketogenic rate in the liver than that in the intestine, indicate that liver contributes more to ketone body synthesis in suckling rats. Two day treatment with dexamethasone produced no change in mRNA or activity levels for CPT I in liver or intestine. While mRNA levels for mit. HMG-CoA synthase changed little, the enzyme activity is decreased in both tissues.

Research paper thumbnail of Processing of Carnitine Octanoyl Transferase pre-mRNAs by cis and trans-splicing

Advances in Experimental Medicine and Biology, 2002

Trans-splicing is a mechanism by which two pre-mRNAs are processed to produce a mature transcript... more Trans-splicing is a mechanism by which two pre-mRNAs are processed to produce a mature transcript that contains exons from both precursors. This process has been described mostly in trypanosoma, nematodes, plant/algal chloroplasts and plant mitochondria [Bonen et al. (1993) FASEB J. 7, 40-46]. Our studies clearly demonstrate that a trans-splicing reaction occurs in the processing of the carnitine octanoyltransferase (COT) gene in rat liver. Three different mature transcripts of COT have been found in vivo, the canonical cis-spliced mRNA and two trans-spliced transcripts, in which either exon 2 or exons 2 and 3 are repeated. Splicing experiments in vitro also indicate the capacity of exon 2 to act either as a donor or as an acceptor of splicing, allowing the trans-splicing reactions to occur.

Research paper thumbnail of Influence of etomoxir on the expression of several genes in liver, testis and heart

General Pharmacology: The Vascular System, 1995

1. The effect of ethyl-2-[6-(4-chlorophenoxy)hexyl) oxirane-2-carboxylate (etomoxir) and its oxir... more 1. The effect of ethyl-2-[6-(4-chlorophenoxy)hexyl) oxirane-2-carboxylate (etomoxir) and its oxirane analogues on the expression of several genes from liver and testis as well as the beneficial effect of etomoxir on heart performance and myosin isozyme expression is reviewed. 2. In liver, the effect of etomoxir, alone or in combination with fat or di-(2-ethylhexyl)phthalate (DEHP) on the expression of several genes related to lipid metabolism has been studied. The simultaneous addition of etomoxir and a fat diet produces an increase in the expression of carnitine palmitoyl transferase (CPT) I, cytochrome P-450 4A1 omega-hydroxylase and fatty acid binding protein (L-FABP). The mRNA levels of other genes such as CPT II, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, and fatty acid synthase (FAS) are increased by etomoxir alone. Neither cytosolic nor mitochondrial HMG-CoA synthase have any significant effect on the mRNA levels induced by etomoxir. A probably frequent mechanism for the action of etomoxir may involve the overload of non-metabolized fatty acids produced after the inhibition of CPT I by the oxirane compounds. There is some speculation as to whether the peroxisome proliferator activated receptor (PPAR) increases its participation in the expression, under the action of etomoxir. 3. In testis, the changes in several genes related to cholesterogenesis, ketogenesis, fatty acid synthesis and transport of fatty acids into mitochondria have also been reviewed. Etomoxir in testis does not appear to produce any effect either alone or in combination with DEHP or a fat diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Research paper thumbnail of The Expression of Mitochondrial 3-Hydroxy-3-Methylglutaryl-Coenzyme-A Synthase in Neonatal Rat Intestine and Liver is Under Transcriptional Control

European Journal of Biochemistry, 1996

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HOMeGlt-CoA) synthase regulates ketogenesis in the ... more Mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HOMeGlt-CoA) synthase regulates ketogenesis in the liver of adult rat and in the intestine and liver of neonatal animals but whose mechanisms of regulation have not been fully defined. To investigate transcriptional control of this gene in intestine and liver of suckling rats a quantitative PCR amplification of the pre-mRNA (heteronuclear RNA), compose of part of the first exon and of the first intron, was carried out. Results show that the intestinal pre-mRNA for mitochondrial HOMeGlt-CoA synthase from suckling rats follows a pattern that is nearly identical to that of mature mRNA, with maximum levels on the ninth postnatal day then decreasing smoothly so that at weaning there is no transcriptional activity. Mitochondrial HOMeGlt-CoA synthase protein follows a pattern that is identical to the pre-mRNA and mature mRNA, suggesting no translational regulation. The changes in transcriptional activity are not produced by the presence of an alternative promoter, since the transcription-initiation site is identical in several tissues assayed, including intestine and liver. Enterocytes are the only intestinal cells that express this ketogenic enzyme, as deduced from immunolocalization experiments. The mature intestinal protein is located in mitochondria and not in the cytosol, which coincides with what is found in liver. By using analogous techniques we conclude that hepatic pre-mRNA of mitochondrial HOMeGlt-CoA synthase from suckling rats follows a pattern of expression identical to that of mature hepatic mRNA, which also suggests a transcriptional modulation of this gene in the liver of neonatal rats.

Research paper thumbnail of Natural trans-splicing in carnitine octanoyltransferase pre-mRNAs in rat liver

Proceedings of the National Academy of Sciences, 1998

Carnitine octanoyltransferase (COT) transports medium-chain fatty acids through the peroxisome. D... more Carnitine octanoyltransferase (COT) transports medium-chain fatty acids through the peroxisome. During isolation of a COT clone from a rat liver library, a cDNA in which exon 2 was repeated, was characterized. Reverse transcription-PCR amplifications of total RNAs from rat liver showed a three-band pattern. Sequencing of the fragments revealed that, in addition to the canonical exon organization, previously reported [Choi, S. J. et al. (1995) Biochim. Biophys. Acta 1264, 215-222], there were two other forms in which exon 2 or exons 2 and 3 were repeated. The possibility of this exonic repetition in the COT gene was ruled out by genomic Southern blot. To study the gene expression, we analyzed RNA transcripts by Northern blot after RNase H digestion of total RNA. Three different transcripts were observed. Splicing experiments also were carried out in vitro with different constructs that contain exon 2 plus the 5' or the 3' adjacent intron sequences. Our results indicate that accurate joining of two exons 2 occurs by a trans-splicing mechanism, confirming the potential of these structures for this process in nature. The trans-splicing can be explained by the presence of three exon-enhancer sequences in exon 2. Analysis by Western blot of the COT proteins by using specific antibodies showed that two proteins corresponding to the expected Mr are present in rat peroxisomes. This is the first time that a natural trans-splicing reaction has been demonstrated in mammalian cells.

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

PLoS ONE, 2014

Lipid metabolism in the ventromedial hypothalamus (VMH) has emerged as a crucial pathway in the r... more 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.

Research paper thumbnail of Modulation in vitro of H-ras oncogene expression by trans-splicing

Oncogene, 2001

In man, activated N-, K- and H-ras oncogenes have been found in around 30% of the solid tumours t... more In man, activated N-, K- and H-ras oncogenes have been found in around 30% of the solid tumours tested. An exon known as IDX, which has been described previously and is located between exon 3 and exon 4A of the c-H-ras pre-mRNA, allows an alternative splicing process that results in the synthesis of the mRNA of a putative protein named p19. It has been suggested that this alternative pathway is less tumorigenic than that which results in the activation of p21. We have used the mammalian trans-splicing mechanism as a tool with which to modulate this particular pre-mRNA processing to produce mRNA similar to that of mature p19 RNA. The E4A exon of the activated H-ras gene was found to be a good target for external trans-splicing. We reprogrammed the rat carnitine octanoyltransferase exon 2 to specifically invade the terminal region of H-ras. Assays performed with this reprogrammed trans-exon showed that the trans-splicing product was obtained in competition with cis-splicing of the D intron of the H-ras gene, and was associated with concomitant down-modulation of D intron cis-splicing. We also found that the exon 4A of the human c-H-ras gene underwent successive trans-splicing rounds with an external exon.

Research paper thumbnail of Localization of an exonic splicing enhancer responsible for mammalian natural trans-splicing

Nucleic Acids Research, 2001

Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and ... more Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which may lead to the synthesis of two proteins. Generation of the three COT transcripts in rat does not depend on sex, development, fat feeding, the inclusion of the peroxisome proliferator diethylhexyl phthalate in the diet or hyperinsulinemia. In addition, trans-splicing was not detected in COT of other mammals, such as human, pig, cow and mouse, or in Cos7 cells from monkey. Rat COT exon 2 contains two purine-rich sequences. Mutation of the rat COT exon 2 upstream box does not affect the trans-splicing in vitro between two truncated constructs containing exon 2 and its adjacent intron boundaries. In contrast, mutation of the downstream box from the rat sequence (GAAGAAG) to a random sequence or the sequence observed in the other mammals (AAAAAAA) decreased trans-splicing in vitro. In contrast, mutation of the AAAAAAA box of human COT exon 2 to GAAGAAG increases trans-splicing. Heterologous reactions between COT exon 2 from rat and human do not produce trans-splicing. HeLa cells transfected with minigenes of rat COT sequences produced cis- and trans-spliced bands. Mutation of the GAAGAAG box to AAAAAAA abolished trans-splicing and decreased cis-splicing in vivo. We conclude that GAAGAAG is an exonic splicing enhancer that could induce natural trans-splicing in rat COT.

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

The Journal of Lipid Research, 2009

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import ... more 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.

Research paper thumbnail of Preparation of α-labeled aldehydes by base-catalyzed exchange reactions

Journal of Labelled Compounds and Radiopharmaceuticals, 2010

A simple, efficient protocol for the preparation of a-labeled aldehydes based on H/D exchange cat... more A simple, efficient protocol for the preparation of a-labeled aldehydes based on H/D exchange catalyzed by 4-(N,Ndimethylamino)pyridine or Et 3 N is described. High chemical yields and ratios of isotope incorporation were obtained even when small amounts (1 mmol) of aldehyde were used.

Research paper thumbnail of et al. Expression of GLUT-2 antisense RNA in B cells of transgenic mice leads to diabetes

Journal of Biological Chemistry

ABSTRACT

Research paper thumbnail of Purification and properties of a protein inhibitor that inhibits phosphatase 2A activity when hydroxymethylglutaryl coenzyme A reductase is the substrate

Journal of Biological Chemistry

A heat-stable protein inhibitor of the hydroxymethylglutaryl-CoA reductase phosphatase 2A activit... more A heat-stable protein inhibitor of the hydroxymethylglutaryl-CoA reductase phosphatase 2A activity has been identified and purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The apparent molecular mass was 20,000 Da. The protein lost its inhibitory properties when incubated with trypsin or treated with ethanol. The inhibitor protein does not inhibit type 1 phosphatase when either phosphorylase or hydroxymethylglutaryl-CoA reductase is the substrate. In contrast, this protein inhibitor inhibits the rat liver type 2A phosphatase activity when hydroxymethylglutaryl-CoA reductase is the substrate but not when phosphorylase a is the substrate. The inhibitor protein is not activated by incubation with ATP and cyclic AMP-dependent protein kinase and it is not phosphorylated by glycogen synthase kinase-3. These results, together with those of the kinetic experiments, suggest that the reductase phosphatase inhibitor is distinct from protein phosphatase inhibitor-1 and inhibitor-2.

Research paper thumbnail of Inactivation of rat liver HMG-CoA reductase phosphatases by nucleotides

Revista española de fisiología

Incubation of four purified rat liver HMG-CoA reductase phosphatases, with ATP, ADP and AMP cause... more Incubation of four purified rat liver HMG-CoA reductase phosphatases, with ATP, ADP and AMP caused a concentration-dependent inactivation of enzyme activities. The nucleotides of guanine, cytosine and uracil produced similar effects to those by the nucleotides of adenine for the same number of phosphates present in the molecules. The greater the number of phosphate groups in nucleotides, the higher was the inhibition in reductase phosphatases observed. Preincubation of phosphatases with ATP and subsequent dilution did not diminish the inactivation effect, showing that nucleotides inhibit the enzyme prior to their binding to the substrate. A relationship was observed between those concentrations of nucleotides which produce 50% inactivation and the logarithm stability constant of Mg or Mn salts of nucleotides. ATP-inactivated enzymes were reactivated by Mn++ and to a lesser proportion by Mg++, the conclusion being that HMG-CoA reductase phosphatases have the characteristics of metalloenzymes.

Research paper thumbnail of cis-Acting elements controlling transcription from rat serine protease inhibitor 2.1 gene promoter. Characterization of two growth hormone response sites and a dominant purine-rich element

Journal of Biological Chemistry

The cis-acting elements that are functionally important for the basal, the growth hormone (GH), a... more The cis-acting elements that are functionally important for the basal, the growth hormone (GH), and the glucocorticoid hormone (GC) regulation of expression of the rat serine protease inhibitor 2.1 gene (spi 2.1) were mapped. Normal rat hepatocytes were transiently transfected with constructs harboring deleted or mutated versions of the spi 2.1 proximal promoter region fused to the chloramphenicol acetyltransferase gene. A purinerich sequence (GAGAbox, nucleotides -57 to -46), whose mutation or deletion almost completely knocks out both basal and hormone-stimulated promoter activities, plays the role of a key control element. A positive GC response element, spanning nucleotides -88 to -74, confers GC responsiveness to a heterologous promoter. Two structurally unrelated GH-response elements (GHRE) were identified. GHRE-I1 (nucleotides -136 to -104) contains a CCAAT enhancer binding protein binding site whose mutation completely abolishes its GH-dependent enhancer function. G I " , which spans nucleotides -61 to +8, is not an enhancer element. Its GH-dependent activity depends on the preservation of the distance separating the GAGA box and elements of the basic transcriptional machinery.

Research paper thumbnail of Purification and characterization of a protein inhibitor from rat liver that inhibits type 1 protein phosphatase when 3-hydroxy-3-methylglutaryl CoA reductase is the substrate

The Journal of Lipid Research

Research paper thumbnail of Overexpression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in transgenic mice causes hepatic hyperketogenesis

Journal of Biological Chemistry

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCoAsynthase) is a key enzyme in the ket... more Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCoAsynthase) is a key enzyme in the ketone body pathway. To determine its role in the regulation of liver ketogenesis, transgenic mice expressing a P-enolpyruvate carboxykinase/HMGCoA synthase chimeric gene have been obtained. An increase in the concentration of mitochondrial HMG-CoA synthase mRNA was detected in these mice, which was associated with a %fold increase in HMG-CoA synthase activity in liver mitochondrial extracts. Transgenic mice were normoglycemic and had normal levels of plasma triglycerides and lower free fatty acids. However, the plasma concentration of ketone bodies was about three times higher in transgenic mice than in control animals. Hepatocytes in primary culture from transgenic mice expressed the chimeric gene in a regulated manner and showed a 3-fold increase in P-hydroxybutyrate and acetoacetate concentrations in the medium. This animal model thus shows that the overexpression of mitochondrial HMG-CoA synthase causes ketone body overproduction, suggesting that this enzyme may be a regulatory step in liver ketogenesis.

Research paper thumbnail of Gene expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in a poorly ketogenic mammal: effect of starvation during the neonatal period of the piglet

The Biochemical journal, Jan 15, 1997

The low ketogenic capacity of pigs correlates with a low activity of mitochondrial 3-hydroxy-3-me... more The low ketogenic capacity of pigs correlates with a low activity of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. To identify the molecular mechanism controlling such activity, we isolated the pig cDNA encoding this enzyme and analysed changes in mRNA levels and mitochondrial specific activity induced during development and starvation. Pig mitochondrial synthase showed a tissue-specific expression pattern. As with rat and human, the gene is expressed in liver and large intestine; however, the pig differs in that mRNA was not detected in testis, kidney or small intestine. During development, pig mitochondrial HMG-CoA synthase gene expression showed interesting differences from that in the rat: (1) there was a 2-3 week lag in the postnatal induction; (2) the mRNA levels remained relatively abundant through the suckling-weaning transition and at maturity, in contrast with the fall observed in rats at similar stages of development; and (3) the gene expression was hig...

Research paper thumbnail of The effect of fasting and insulin treatment on carnitine palmitoyl transferase I and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase mRNA levels in liver from suckling rats

Biochemical Society transactions, 1995

Research paper thumbnail of Developmental changes in carnitine palmitoyltransferases I and II gene expression in intestine and liver of suckling rats

The Biochemical journal, 1995

Carnitine palmitoyltransferase (CPT) I is expressed in the intestine of suckling rats; its mRNA i... more Carnitine palmitoyltransferase (CPT) I is expressed in the intestine of suckling rats; its mRNA increases very rapidly after birth, remains on a plateau until day 18 and decreases until weaning, when basal (adult) values are reached, which remain unchanged thereafter. CPT II mRNA values do not show any appreciable change in this period. CPT I and CPT II are expressed mainly in mucosa and, to a lesser extent, in the muscular part of the intestine. Intestinal expression of CPT I is maximal in duodenum and jejunum, whereas CPT II is expressed in a similar pattern throughout the whole intestine. Dam's milk may influence the intestinal expression of CPT I, since mRNA levels at birth are low but increase after the first lactation. Moreover, rats weaned at either day 18 or 21 decrease their mRNA levels. Apparently, CPT II gene expression is not influenced by the mother's milk. CPT I and CPT II are also expressed in the liver of suckling rats. Hepatic CPT I is maximal at day 3, and ...

Research paper thumbnail of Isolation and partial characterization of a protein with HMG-CoA reductase phosphatase activity associated with rat liver microsomal membranes

Several rat liver HMG-CoA-reductase (HMG-CoA- Rd) phosphatase activities have been shown to be as... more Several rat liver HMG-CoA-reductase (HMG-CoA- Rd) phosphatase activities have been shown to be associated with the endoplasmic reticulum. These activities were not due to glycogen contamination, as judged not only from different patterns of solubilization of the microsomal membranes and the glycogen pellet but also by differential centrifugation behavior under standard conditions and in a sucrose gradient. We present evidence

Research paper thumbnail of The effect of dexamethasone treatment on the expression of the regulatory genes of ketogenesis in intestine and liver of suckling rats

Molecular and Cellular Biochemistry, 1998

The influence of the injection of dexamethasone on ketogenesis in 12 day old suckling rats was st... more The influence of the injection of dexamethasone on ketogenesis in 12 day old suckling rats was studied in intestine and liver by determining mRNA levels and enzyme activity of the two genes responsible for regulation of ketogenesis: carnitine palmitoyl transferase I (CPT I) and mitochondrial HMG-CoA synthase. Dexamethasone produced a 2 fold increase in mRNA and activity of CPT I in intestine, but led to a decrease in mit. HMG-CoA synthase. In liver the mRNA levels and activity of both CPT I and mit. HMG-CoA synthase decreased. Comparison of these values with the ketogenic rate of both tissues following dexamethasone treatment suggests that mit. HMG-CoA synthase could be the main gene responsible for the regulation of ketogenesis in suckling rats. The changes produced in serum ketone bodies by dexamethasone, with a profile that is more similar to the ketogenic rate in the liver than that in the intestine, indicate that liver contributes more to ketone body synthesis in suckling rats. Two day treatment with dexamethasone produced no change in mRNA or activity levels for CPT I in liver or intestine. While mRNA levels for mit. HMG-CoA synthase changed little, the enzyme activity is decreased in both tissues.

Research paper thumbnail of Processing of Carnitine Octanoyl Transferase pre-mRNAs by cis and trans-splicing

Advances in Experimental Medicine and Biology, 2002

Trans-splicing is a mechanism by which two pre-mRNAs are processed to produce a mature transcript... more Trans-splicing is a mechanism by which two pre-mRNAs are processed to produce a mature transcript that contains exons from both precursors. This process has been described mostly in trypanosoma, nematodes, plant/algal chloroplasts and plant mitochondria [Bonen et al. (1993) FASEB J. 7, 40-46]. Our studies clearly demonstrate that a trans-splicing reaction occurs in the processing of the carnitine octanoyltransferase (COT) gene in rat liver. Three different mature transcripts of COT have been found in vivo, the canonical cis-spliced mRNA and two trans-spliced transcripts, in which either exon 2 or exons 2 and 3 are repeated. Splicing experiments in vitro also indicate the capacity of exon 2 to act either as a donor or as an acceptor of splicing, allowing the trans-splicing reactions to occur.

Research paper thumbnail of Influence of etomoxir on the expression of several genes in liver, testis and heart

General Pharmacology: The Vascular System, 1995

1. The effect of ethyl-2-[6-(4-chlorophenoxy)hexyl) oxirane-2-carboxylate (etomoxir) and its oxir... more 1. The effect of ethyl-2-[6-(4-chlorophenoxy)hexyl) oxirane-2-carboxylate (etomoxir) and its oxirane analogues on the expression of several genes from liver and testis as well as the beneficial effect of etomoxir on heart performance and myosin isozyme expression is reviewed. 2. In liver, the effect of etomoxir, alone or in combination with fat or di-(2-ethylhexyl)phthalate (DEHP) on the expression of several genes related to lipid metabolism has been studied. The simultaneous addition of etomoxir and a fat diet produces an increase in the expression of carnitine palmitoyl transferase (CPT) I, cytochrome P-450 4A1 omega-hydroxylase and fatty acid binding protein (L-FABP). The mRNA levels of other genes such as CPT II, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, and fatty acid synthase (FAS) are increased by etomoxir alone. Neither cytosolic nor mitochondrial HMG-CoA synthase have any significant effect on the mRNA levels induced by etomoxir. A probably frequent mechanism for the action of etomoxir may involve the overload of non-metabolized fatty acids produced after the inhibition of CPT I by the oxirane compounds. There is some speculation as to whether the peroxisome proliferator activated receptor (PPAR) increases its participation in the expression, under the action of etomoxir. 3. In testis, the changes in several genes related to cholesterogenesis, ketogenesis, fatty acid synthesis and transport of fatty acids into mitochondria have also been reviewed. Etomoxir in testis does not appear to produce any effect either alone or in combination with DEHP or a fat diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Research paper thumbnail of The Expression of Mitochondrial 3-Hydroxy-3-Methylglutaryl-Coenzyme-A Synthase in Neonatal Rat Intestine and Liver is Under Transcriptional Control

European Journal of Biochemistry, 1996

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HOMeGlt-CoA) synthase regulates ketogenesis in the ... more Mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HOMeGlt-CoA) synthase regulates ketogenesis in the liver of adult rat and in the intestine and liver of neonatal animals but whose mechanisms of regulation have not been fully defined. To investigate transcriptional control of this gene in intestine and liver of suckling rats a quantitative PCR amplification of the pre-mRNA (heteronuclear RNA), compose of part of the first exon and of the first intron, was carried out. Results show that the intestinal pre-mRNA for mitochondrial HOMeGlt-CoA synthase from suckling rats follows a pattern that is nearly identical to that of mature mRNA, with maximum levels on the ninth postnatal day then decreasing smoothly so that at weaning there is no transcriptional activity. Mitochondrial HOMeGlt-CoA synthase protein follows a pattern that is identical to the pre-mRNA and mature mRNA, suggesting no translational regulation. The changes in transcriptional activity are not produced by the presence of an alternative promoter, since the transcription-initiation site is identical in several tissues assayed, including intestine and liver. Enterocytes are the only intestinal cells that express this ketogenic enzyme, as deduced from immunolocalization experiments. The mature intestinal protein is located in mitochondria and not in the cytosol, which coincides with what is found in liver. By using analogous techniques we conclude that hepatic pre-mRNA of mitochondrial HOMeGlt-CoA synthase from suckling rats follows a pattern of expression identical to that of mature hepatic mRNA, which also suggests a transcriptional modulation of this gene in the liver of neonatal rats.

Research paper thumbnail of Natural trans-splicing in carnitine octanoyltransferase pre-mRNAs in rat liver

Proceedings of the National Academy of Sciences, 1998

Carnitine octanoyltransferase (COT) transports medium-chain fatty acids through the peroxisome. D... more Carnitine octanoyltransferase (COT) transports medium-chain fatty acids through the peroxisome. During isolation of a COT clone from a rat liver library, a cDNA in which exon 2 was repeated, was characterized. Reverse transcription-PCR amplifications of total RNAs from rat liver showed a three-band pattern. Sequencing of the fragments revealed that, in addition to the canonical exon organization, previously reported [Choi, S. J. et al. (1995) Biochim. Biophys. Acta 1264, 215-222], there were two other forms in which exon 2 or exons 2 and 3 were repeated. The possibility of this exonic repetition in the COT gene was ruled out by genomic Southern blot. To study the gene expression, we analyzed RNA transcripts by Northern blot after RNase H digestion of total RNA. Three different transcripts were observed. Splicing experiments also were carried out in vitro with different constructs that contain exon 2 plus the 5' or the 3' adjacent intron sequences. Our results indicate that accurate joining of two exons 2 occurs by a trans-splicing mechanism, confirming the potential of these structures for this process in nature. The trans-splicing can be explained by the presence of three exon-enhancer sequences in exon 2. Analysis by Western blot of the COT proteins by using specific antibodies showed that two proteins corresponding to the expected Mr are present in rat peroxisomes. This is the first time that a natural trans-splicing reaction has been demonstrated in mammalian cells.

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

PLoS ONE, 2014

Lipid metabolism in the ventromedial hypothalamus (VMH) has emerged as a crucial pathway in the r... more 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.

Research paper thumbnail of Modulation in vitro of H-ras oncogene expression by trans-splicing

Oncogene, 2001

In man, activated N-, K- and H-ras oncogenes have been found in around 30% of the solid tumours t... more In man, activated N-, K- and H-ras oncogenes have been found in around 30% of the solid tumours tested. An exon known as IDX, which has been described previously and is located between exon 3 and exon 4A of the c-H-ras pre-mRNA, allows an alternative splicing process that results in the synthesis of the mRNA of a putative protein named p19. It has been suggested that this alternative pathway is less tumorigenic than that which results in the activation of p21. We have used the mammalian trans-splicing mechanism as a tool with which to modulate this particular pre-mRNA processing to produce mRNA similar to that of mature p19 RNA. The E4A exon of the activated H-ras gene was found to be a good target for external trans-splicing. We reprogrammed the rat carnitine octanoyltransferase exon 2 to specifically invade the terminal region of H-ras. Assays performed with this reprogrammed trans-exon showed that the trans-splicing product was obtained in competition with cis-splicing of the D intron of the H-ras gene, and was associated with concomitant down-modulation of D intron cis-splicing. We also found that the exon 4A of the human c-H-ras gene underwent successive trans-splicing rounds with an external exon.

Research paper thumbnail of Localization of an exonic splicing enhancer responsible for mammalian natural trans-splicing

Nucleic Acids Research, 2001

Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and ... more Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which may lead to the synthesis of two proteins. Generation of the three COT transcripts in rat does not depend on sex, development, fat feeding, the inclusion of the peroxisome proliferator diethylhexyl phthalate in the diet or hyperinsulinemia. In addition, trans-splicing was not detected in COT of other mammals, such as human, pig, cow and mouse, or in Cos7 cells from monkey. Rat COT exon 2 contains two purine-rich sequences. Mutation of the rat COT exon 2 upstream box does not affect the trans-splicing in vitro between two truncated constructs containing exon 2 and its adjacent intron boundaries. In contrast, mutation of the downstream box from the rat sequence (GAAGAAG) to a random sequence or the sequence observed in the other mammals (AAAAAAA) decreased trans-splicing in vitro. In contrast, mutation of the AAAAAAA box of human COT exon 2 to GAAGAAG increases trans-splicing. Heterologous reactions between COT exon 2 from rat and human do not produce trans-splicing. HeLa cells transfected with minigenes of rat COT sequences produced cis- and trans-spliced bands. Mutation of the GAAGAAG box to AAAAAAA abolished trans-splicing and decreased cis-splicing in vivo. We conclude that GAAGAAG is an exonic splicing enhancer that could induce natural trans-splicing in rat COT.

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

The Journal of Lipid Research, 2009

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import ... more 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.

Research paper thumbnail of Preparation of α-labeled aldehydes by base-catalyzed exchange reactions

Journal of Labelled Compounds and Radiopharmaceuticals, 2010

A simple, efficient protocol for the preparation of a-labeled aldehydes based on H/D exchange cat... more A simple, efficient protocol for the preparation of a-labeled aldehydes based on H/D exchange catalyzed by 4-(N,Ndimethylamino)pyridine or Et 3 N is described. High chemical yields and ratios of isotope incorporation were obtained even when small amounts (1 mmol) of aldehyde were used.