Rapid method for the separation and detection of tissue short-chain coenzyme A esters by reversed-phase high-performance liquid chromatography

Demoz, Abraham; Garras, Alexis; Asiedu, Daniel K.; Netteland, Bjørn; Berge, Rolf K.

doi:10.1016/0378-4347(94)00595-v

Cited by 65 publications

(34 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Hepatic triglycerides and fatty acid content were quantified using a TAG kit (Sigma) and NEFA kit (Wako), respectively, in 3 mol/l KOH, 65% ethanol extracts, based on the method of Salmon and Flatt for liver saponification. Hepatic short chain acyl-CoAs (acetyl-CoA, malonyl-CoA, propionyl-CoA, and succinyl-CoA) were analyzed by high-performance liquid chromatography (HPLC) as described previously (14) in the Research Support Services from the University of Barcelona. Nucleotides (ATP/ADP/AMP) were determined in neutralized 10% perchloric acid extracts by HPLC.…”

Section: Methodsmentioning

confidence: 99%

Pck1 Gene Silencing in the Liver Improves Glycemia Control, Insulin Sensitivity, and Dyslipidemia in db/db Mice

Méndez-Lucas²,

et al. 2008

View full text Add to dashboard Cite

OBJECTIVE-Cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; encoded by Pck1) catalyzes the first committed step in gluconeogenesis. Extensive evidence demonstrates a direct correlation between PEPCK-C activity and glycemia control. Therefore, we aimed to evaluate the metabolic impact and their underlying mechanisms of knocking down hepatic PEPCK-C in a type 2 diabetic model. RESEARCH DESIGN AND METHODS-PEPCK-C gene targeting was achieved using adenovirus-transduced RNAi. The study assessed several clinical symptoms of diabetes and insulin signaling in peripheral tissues, in addition to changes in gene expression, protein, and metabolites in the liver. Liver bioenergetics was also evaluated.RESULTS-Treatment resulted in reduced PEPCK-C mRNA and protein. After treatment, improved glycemia and insulinemia, lower triglyceride, and higher total and HDL cholesterol were measured. Unsterified fatty acid accumulation was observed in the liver, in the absence of de novo lipogenesis. Despite hepatic lipidosis, treatment resulted in improved insulin signaling in the liver, muscle, and adipose tissue. O 2 consumption measurements in isolated hepatocytes demonstrated unaltered mitochondrial function and a consequent increased cellular energy charge. Key regulatory factors (FOXO1, hepatocyte nuclear factor-4␣, and peroxisome proliferator-activated receptor-␥ coactivator [PGC]-1␣) and enzymes (G6Pase) implicated in gluconeogenesis were downregulated after treatment. Finally, the levels of Sirt1, a redox-state sensor that modulates gluconeogenesis through PGC-1␣, were diminished. CONCLUSIONS-Our observations indicate that silencing PEPCK-C has direct impact on glycemia control and energy metabolism and provides new insights into the potential significance of the enzyme as a therapeutic target for the treatment of diabetes.

show abstract

Section: Methodsmentioning

confidence: 99%

Pck1 Gene Silencing in the Liver Improves Glycemia Control, Insulin Sensitivity, and Dyslipidemia in db/db Mice

Méndez-Lucas²,

et al. 2008

View full text Add to dashboard Cite

show abstract

“…The MCD inhibitor, bromopyruvic acid (Sigma), 1 mM, was used as a positive control. CoA derivatives and free CoA were measured using a modified high-pressure liquid chromatography procedure (Demoz et al, 1995). After transfection at times indicated or drug treatment for 6 h, 4 Â 10 4 MCF7 cells were homogenized in 120 ml of ice-cold 5% sulfosalicylic acid in 50 mM dithioerythritol and centrifuged at 600 g for 10 min.…”

Section: Sirna Preparation and Transfectionmentioning

confidence: 99%

Malonyl-CoA decarboxylase inhibition is selectively cytotoxic to human breast cancer cells

Zhou

Simpson

et al. 2009

Oncogene

View full text Add to dashboard Cite

Fatty acid synthase (FAS) inhibition initiates selective apoptosis of cancer cells both in vivo and in vitro, which may involve malonyl-CoA metabolism. These findings have led to the exploration of malonyl-CoA decarboxylase (MCD) as a potential novel target for cancer treatment. MCD regulates the levels of cellular malonyl-CoA through the decarboxylation of malonyl-CoA to acetylCoA. Malonyl-CoA is both a substrate for FAS and an inhibitor of fatty acid oxidation acting as a metabolic switch between anabolic fatty acid synthesis and catabolic fatty acid oxidation. We now report that the treatment of human breast cancer (MCF7) cells with MCD small interference RNA (siRNA) reduces MCD expression and activity, reduces adenosine triphosphate levels, and is cytotoxic to MCF7 cells, but not to human fibroblasts. In addition, we synthesized a small-molecule inhibitor of MCD, 5-{(Morpholine-4-carbonyl)-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-amino}-pentanoic acid methyl ester (MPA). Similar to MCD siRNA, MPA inhibits MCD activity in MCF7 cells, increases cellular malonyl-CoA levels and is cytotoxic to a number of human breast cancer cell lines in vitro. Taken together, these data indicate that MCD-induced cytotoxicity is likely mediated through malonyl-CoA metabolism. These findings support the hypothesis that MCD is a potential therapeutic target for cancer therapy.

show abstract

“…The cellular levels of acetyl-CoA and malonyl-CoA were quantified by RP-HPLC on Supelcosil LC-18 columns (SigmaAldrich), as previously described (29).…”

Section: Hplc Analysis Of Acetyl-coa and Malonyl-coamentioning

confidence: 99%

Enhanced expression of hepatic lipogenic enzymes in an animal model of sedentariness

Vecchini

Ceccarelli

Orvietani

et al. 2003

Journal of Lipid Research

View full text Add to dashboard Cite

The hindlimb-suspended rat was used as animal model to investigate the effects induced by immobilization of the skeletal muscle in the expression of the genes encoding hepatic lipogenic enzymes. Following a 14-day period of immobilization, rats were injected intraperitoneally with radioactive acetate, and the labeling of hepatic lipids and cholesterol was evaluated 15 min after the isotope injection. The incorporation of labeled acetate in lipids and cholesterol was almost three times higher in the liver of immobilized rats than in control animals as a consequence of the enhanced transcription of the genes encoding acetyl-CoA synthase, acetyl-CoA carboxylase, fatty acid synthase, and 3-hydroxy-3-methylglutaryl-CoA reductase. The high expression of the key enzymes for fatty acid and cholesterol synthesis induced by immobilization was not paralleled by an increase of the hepatic sterol-regulatory element binding protein (SREBP)-1 and SREBP-2 mRNA content. However, the expression of the mature form of SREBP-1 and SREBP-2 was higher in the nuclear fraction of immobilized rat liver than in controls due to a significant increase of the cleavage of the native proteins. Immobilization also affected the expression of proteins involved in lipid degradation. In fact, the hepatic content of peroxisome proliferator-activated receptor-␣ (PPAR ␣ ) mRNA and of PPAR ␣ target genes encoding carnitine palmitoyl transferase-1 and acyl-CoA oxidase were significantly increased upon immobilization. -Vecchini, A., V. Ceccarelli, P. Orvietani, P. Caligiana, F. Susta, L. Binaglia, G. Nocentini, C. Riccardi, and P. Di Nardo.

show abstract

Rapid method for the separation and detection of tissue short-chain coenzyme A esters by reversed-phase high-performance liquid chromatography

Cited by 65 publications

References 4 publications

Pck1 Gene Silencing in the Liver Improves Glycemia Control, Insulin Sensitivity, and Dyslipidemia in db/db Mice

Pck1 Gene Silencing in the Liver Improves Glycemia Control, Insulin Sensitivity, and Dyslipidemia in db/db Mice

Malonyl-CoA decarboxylase inhibition is selectively cytotoxic to human breast cancer cells

Enhanced expression of hepatic lipogenic enzymes in an animal model of sedentariness

Contact Info

Product

Resources

About