scholarly journals Regulation of carnitine palmitoyltransferase activity by malonyl-CoA in mitochondria from sheep liver, a tissue with a low capacity for fatty acid synthesis

1985 ◽  
Vol 232 (1) ◽  
pp. 177-182 ◽  
Author(s):  
N P Brindle ◽  
V A Zammit ◽  
C I Pogson
Keyword(s):  
Fatty Acid ◽  
Rat Liver ◽  
Fatty Acid Synthesis ◽  
Acid Synthesis ◽  
Liver Mitochondria ◽  
Sheep Liver ◽  
Malonyl Coa ◽  
Cpt I ◽  
Guinea Pig Liver

The characteristics of inhibition of carnitine palmitoyltransferase (CPT) I by malonyl-CoA were studied for the enzyme in mitochondria isolated from sheep liver, a tissue with a very low rate of fatty acid synthesis. Malonyl-CoA was as potent in inhibiting the sheep liver enzyme as in inhibiting the enzyme in rat liver mitochondria. CPT I in guinea-pig liver mitochondria was also similarly inhibited. The inhibition showed the same time-dependent characteristics previously established for the rat liver enzyme. Methylmalonyl-CoA was as effective an inhibitor of CPT I as malonyl-CoA in sheep liver mitochondria, but did not affect CPT I activity in mitochondria from rat or guinea-pig liver. The concentrations of malonyl-CoA required to inhibit CPT I in sheep liver mitochondria in vitro were similar to those found in freeze-clamped sheep liver samples (about 7 nmol of malonyl-CoA/g wet wt.). In sheep liver cells the content of malonyl-CoA was only one-tenth of that observed in vivo when glucose only was added to the incubation medium. Inclusion of acetate and/or insulin increased the malonyl-CoA content about 10-fold, to values similar to those observed in vivo. The rate of fatty acid synthesis in sheep liver cells was about 1% of that observed in rat liver, but was correlated with the concentrations of malonyl-CoA in the cells under various incubation conditions. These observations are discussed in relation to (i) the regulatory role of malonyl-CoA in tissues that have a low capacity for fatty acid synthesis, and (ii) the utilization by sheep liver of propionate as a gluconeogenic precursor.

scholarly journals Monitoring of changes in hepatic fatty acid and glycerolipid metabolism during the starved-to-fed transition in vivo. Studies on awake, unrestrained rats

1993 ◽  
Vol 289 (1) ◽  
pp. 49-55 ◽  
Author(s):  
A M B Moir ◽  
V A Zammit
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Liver Mitochondria ◽  
In Vivo Studies ◽  
Maximal Effect ◽  
Jugular Veins ◽  
Selective Labelling ◽  
Malonyl Coa ◽  
Cpt I

1. The technique of selective labelling of hepatic fatty acids in vivo [Moir and Zammit (1992) Biochem. J. 283, 145-149] has been used to monitor non-invasively the metabolism of fatty acids in the livers of awake unrestrained rats during the starved-to-refed transition. Values for the incorporation of labelled fatty acid into liver and plasma glycerolipids and into exhaled carbon dioxide after injection of labelled lipoprotein and Triton WR 1339 into rats with chronically cannulated jugular veins were obtained for successive 1 h periods from the start of refeeding of 24 h-starved rats. 2. Starvation for 24 h resulted in marked and reciprocal changes in the incorporation of label into glycerolipids and exhaled 14CO2, such that a 4-fold higher value was obtained for the oxidation/esterification ratio in livers of starved rats compared with fed animals. 3. Refeeding of starved rats did not return this ratio to the value observed for fed animals for at least 7 h; during the first 3 h of refeeding the ratio was at least as high as that for starved rats. Between 4 h and 6 h of refeeding the ratio was still approx. 70% of that in starved animals, and 2.5-fold higher than in fed rats. 4. These data support the hypothesis that the capacity of the liver to oxidize fatty acids is maintained at a high level during the initial stages of refeeding [Grantham and Zammit (1986) Biochem. J. 239, 485-488] and that control of the flux of hepatic fatty acids into the oxidative pathway is largely lost from the reaction catalysed by mitochondrial overt carnitine palmitoyltransferase (CPT I) during this phase of recovery from the starved state. 5. Refeeding also resulted in a rapid (< 1 h) increase in hepatic malonyl-CoA concentrations to values intermediate between those in livers of fed and starved animals. The sensitivity of CPT I to malonyl-CoA inhibition in isolated liver mitochondria was only partially reversed even after 5 h of refeeding. 6. Refeeding resulted in an acute 35% inhibition of the fraction of synthesized triacylglycerol that was secreted into the plasma; the maximal effect occurred 2-3 h after the start of refeeding. The inhibition of the fractional secretion rate was fully reversed after 5 h of refeeding. 7. The amount of 14C label that was incorporated into phospholipids as a fraction of total glycerolipid synthesis was doubled within 2 h of the start of refeeding.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Effects of incubation at physiological temperatures on the concentration-dependence of [2-14C]malonyl-CoA binding to rat liver mitochondria

1985 ◽  
Vol 231 (2) ◽  
pp. 343-347 ◽  
Author(s):  
V A Zammit ◽  
C G Corstorphine
Keyword(s):  
Rat Liver ◽  
Specific Binding ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Temperature Dependent ◽  
Malonyl Coa ◽  
Saturation Kinetics ◽  
Different Temperatures ◽  
Cpt I

Specific binding of [2-14C] malonyl-CoA to rat liver mitochondria was measured at different temperatures and after various periods of time of exposure of the mitochondria to the ligand. Incubation of mitochondria at 37 degrees C in the absence of malonyl-CoA resulted in a decrease in their ability to bind malonyl-CoA at all concentrations tested (up to 55 microM). However, incubation of mitochondria in the presence of malonyl-CoA resulted in the loss of the binding only by a low-affinity component. By contrast, there was an increase in the binding that occurred at low, physiological, concentrations of malonyl-CoA. These differences in the response of the two binding components to incubation conditions were used to obtain quantitative data about their respective saturation kinetics. Evidence was obtained that, whereas the high-affinity component approached saturation hyperbolically with respect to malonyl-CoA concentration, the low-affinity component had sigmoidal characteristics. The concentrations of malonyl-CoA required to half-saturate the two components were 2-3 microM and 30 microM for the high- and low-affinity components respectively. Evidence was also obtained for the involvement of a temperature-dependent transition, that occurred at around 25 degrees C, in the modulation of malonyl-CoA binding to the mitochondria. The possible physiological roles of the two components of malonyl-CoA binding in relation to the regulation of overt carnitine palmitoyltransferase (CPT I) activity in vivo are discussed.

scholarly journals Ethanol administration and the relationship of malonyl-coenzyme A concentrations to the rate of fatty acid synthesis in rat liver

1973 ◽  
Vol 136 (3) ◽  
pp. 639-647 ◽  
Author(s):  
Robert W. Guynn ◽  
Dulce Veloso ◽  
Raymond L. Harris ◽  
J. W. Randolph Lawson ◽  
Richard L. Veech
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
De Novo ◽  
Fatty Acid Synthetase ◽  
Acid Synthesis ◽  
Ethanol Administration ◽  
Liver Fatty Acid ◽  
Malonyl Coa ◽  
Significant Difference

1. The effect of ethanol on liver fatty acid synthesis was studied in vivo in 24h-starved and ‘meal-fed’ rats (i.e. fed for 3h per day and not ad libitum). 2. In the fed animal3H2O was incorporated into fat at a rate of 0.46μmol of C2 units/min per g wet wt. of liver. Administration of either ethanol (3.2g/kg) or equicaloric amounts of glucose had no effect on the rate of3H2O incorporation into lipid. 3. In the 24h-starved animal, administration of the same dose of ethanol produced an increase in the rate of3H2O incorporation from 0.06 to 0.12μmol of C2 units/min per g fresh wt. after 3h whereas [malonyl-CoA] increased from 0.006 to 0.009μmol/g. Glucose given in amounts equicaloric to ethanol was significantly more lipogenic, increasing both the3H2O incorporation from 0.06 to 0.20μmol of C2 units/min per g and the malonyl-CoA content from 0.006 to 0.013 μmol/g wet wt. at 3h. 4. The decrease in the redox state of free cytoplasm NAD or NADP couples or the changes in content of citrate, glucose 6-phosphate and pyruvate of liver after ethanol administration had no measurable effect on the rate of fatty acid synthesis in vivo. 5. Under the conditions of the experiments there was no significant difference, among any of the groups, in the activity of liver fatty acid synthetase measured in vitro. A double-reciprocal plot of the rate of3H2O incorporation and the total tissue malonyl-CoA concentrations showed a striking relationship. It has been concluded that the rate of fatty acid synthesis in vivo is determined principally by the Vmax. of fatty acid synthetase and the concentration of free malonyl-CoA. 6. It has also been concluded that under the conditions of the present study, the synthesis of fatty acids de novo is unlikely to be an important factor in the increased liver lipid content associated with ethanol administration.

scholarly journals Sensitivity of carnitine acyltransferase I to malonyl-CoA inhibition in isolated rat liver mitochondria is quantitatively related to hepatic malonyl-CoA concentration in vivo

1982 ◽  
Vol 206 (1) ◽  
pp. 177-179 ◽  
Author(s):  
Ian N. Robinson ◽  
Victor A. Zammit
Keyword(s):  
Rat Liver ◽  
Acid Synthesis ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Reciprocal Regulation ◽  
Carnitine Acyltransferase ◽  
Malonyl Coa ◽  
Amplification Mechanism ◽  
Two Parameters

The sensitivity of carnitine acyltransferase I (EC 2.3.1.21) activity to malonyl-CoA inhibition in rat liver mitochondria isolated from animals in various physiological states was quantitatively proportional to the hepatic malonyl-CoA concentration in vivo. It is suggested that this relationship between the two parameters could result in a potent amplification mechanism for the reciprocal regulation of fatty acid synthesis and oxidation.

Reconstitution of purified, active and malonyl-CoA-sensitive rat liver carnitine palmitoyltransferase I: relationship between membrane environment and malonyl-CoA sensitivity

2000 ◽  
Vol 349 (1) ◽  
pp. 179-187 ◽  
Author(s):  
J. Denis MCGARRY ◽  
Nicholas F. BROWN
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Kinetic Properties ◽  
Suitable Model ◽  
Malonyl Coa ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I

Carnitine palmitoyltransferase I (CPT I) catalyses the initial step of fatty acid import into the mitochondrial matrix, the site of β-oxidation, and its inhibition by malonyl-CoA is a primary control point for this process. The enzyme exists in at least two isoforms, denoted L-CPT I (liver type) and M-CPT I (skeletal-muscle type), which differ in their kinetic characteristics and tissue distributions. A property apparently unique to L-CPT I is that its sensitivity to malonyl-CoA decreases in vivo with fasting or experimentally induced diabetes. The mechanism of this important regulatory effect is unknown and has aroused much interest. CPT I is an integral outer-membrane protein and displays little activity after removal from the membrane by detergents, precluding direct purification of active protein by conventional means. Here we describe the expression of a 6×His-tagged rat L-CPT I in Pichia pastoris and purification of the detergent-solubilized enzyme in milligram quantities. Reconstitution of the purified product into a liposomal environment yielded a 200-400-fold increase in enzymic activity and restored malonyl-CoA sensitivity. This is the first time that a CPT I protein has been available for study in a form that is both pure and active. Comparison of the kinetic properties of the reconstituted material with those of L-CPT I as it exists in mitochondria prepared from yeast over-expressing the enzyme and in livers from fed or fasted rats permitted novel insight into several aspects of the enzyme's behaviour. The malonyl-CoA response of the liposomal enzyme was found to be greater when the reconstitution procedure was carried out at 22 °C compared with 4 °C (IC50 ≈ 11 μM versus 30 μM, respectively). When the sensitivities of L-CPT I in each of the different environments were compared, they were found to decrease in the following order: fed liver > fasted liver≈ liposomes prepared at 22 °C≈ P. pastoris mitochondria > liposomes prepared at 4 °C. In addition, pre-treatment of L-CPT I liposomes with the membrane-fluidizing reagent benzyl alcohol caused densensitization to the inhibitor. In contrast with the variable response to malonyl-CoA, the liposomal L-CPT I displayed a pH profile and kinetics with regard to the carnitine and acyl-CoA substrates similar to those of the enzyme in fed or fasted liver mitochondria. However, despite a normal sensitivity to malonyl-CoA, L-CPT I in P. pastoris mitochondria displayed aberrant behaviour with regard to each of these other parameters. The kinetic data establish several novel points. First, even after stringent purification procedures in the presence of detergent, recombinant L-CPT I could be reconstituted in active, malonyl-CoA sensitive form. Second, the kinetics of the reconstituted, 6×His-tagged L-CPT I with regard to substrate and pH responses were similar to what is observed with rat liver mitochondria (whereas in P. pastoris mitochondria the enzyme behaved anomalously), confirming that the purified preparation is a suitable model for studying the functional properties of the enzyme. Third, wide variation in the response to the inhibitor, malonyl-CoA, was observed depending only on the enzyme's membrane environment and independent of interaction with other proteins. In particular, the fluidity of the membrane had a direct influence on this parameter. These observations may help to explain the mechanism of the physiological changes in the properties of L-CPT I that occur in vivo and are consistent with the current topographical model of the enzyme.

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