Sensitivity of inhibition of rat liver mitochondrial outer-membrane carnitine palmitoyltransferase by malonyl-CoA to chemical- and temperature-induced changes in membrane fluidity

Abstract

We have tested the possibility that alterations in the fluidity of the outer membrane of rat liver mitochondria could result in changes in the sensitivity of overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA [Zammit (1986) Biochem. Soc. Trans. 14,[676][677][678][679]. The sensitivity of CPT I to malonyl-CoA inhibition was measured by using highly purified mitochondrial outer membranes prepared from fed or 48 h-starved rats in the presence and absence of agents that increase membrane fluidity by perturbing membrane lipid order [benzyl alcohol, isoamyl alcohol (3-methylbutan-1-ol) and 2-(2-methoxyethoxy)ethyl-8-(cis-2-n-octylpropyl)octanoate (A2C)]. All these agents resulted in marked decreases in the ability of malonyl-CoA to inhibit CPT I. This effect was accompanied by a modest increase in the absolute activity of CPT I in the absence ofmalonyl-CoA when the short-chain alcohols were used, but not when A2C was used, suggesting that the effect of increased membrane fluidity to decrease the malonyl-CoA sensitivity of CPT I may occur independently from other actions that may affect more directly the active site of the enzyme. In confirmation of the potential importance of fluidity changes, we showed that a marked increase in sensitivity of CPT I to malonyl-CoA could be produced when assays were performed at lower temperatures than those normally employed. These observations are discussed in the context of the slowness of the changes in CPT I sensitivity to malonyl-CoA inhibition that are induced by physiological perturbations. INTRODUCTIONThe sensitivity of mitochondrial outer-membrane carnitine palmitoyltransferase (CPT I) to malonyl-CoA inhibition changes with physiological state in rat liver (see [1] for review). Under conditions of increased fatty acid oxidation, e.g. starvation, diabetes, the enzyme becomes less sensitive to malonyl-CoA inhibition. This adaptation amplifies the effect of changes in hepatic malonyl-CoA concentration, as decreases in malonylCoA content of the liver are accompanied by decreased sensitivity [2]. Moreover, the two parameters appear to be positively related

The malonyl-CoA–long-chain acyl-CoA axis in the maintenance of mammalian cell function

“…It is to be emphasized that the sensitivity of L-CPTo to malonyl-CoA, as well as the K m for palmitoyl-CoA, are both modulated by physiological state [45][46][47][48] (but not in cardiac muscle [49]) and that these changes (i) can be mimicked by experimentally induced changes in the fluidity of the mitochondrial outer membrane in itro [50] and (ii) are correlated with changes in the molecular order of the lipids of outer membranes prepared from mitochondria isolated from livers of rats maintained under different physiological conditions [51]. It is inferred that protein-membrane interactions are likely to be important in determining the malonyl-CoA sensitivity of CPTo, and that they are likely to be due to interaction between the TM segments as well as between them and the annular membrane lipids [34].…”

“…Such interactions are also anticipated to modulate the degree of interaction between the N-and C-domains and may determine the kinetic characteristics of the protein under different physiological conditions. Therefore, it may be relevant that there are important structural differences between the predicted TM segments of the L-and M-isoforms of CPTo that may contribute towards their distinctive kinetic characteristics and their responses to membrane-fluidity changes in i o [51] and in itro [50,52].…”

“…In view of the dependence of CPTo activity on the physical state of the membrane [50,51], this may explain why ceramide appears to have a direct stimulatory effect on the activity of CPTo [138]. Such activation may, in turn, provide a feedback mechanism limiting the availability of acylCoA for further ceramide synthesis.…”

Role of insulin in hepatic fatty acid partitioning: emerging concepts

“…increasing the fluidity of the lipid bilayer) in purified outer-membrane preparations, whether this is induced chemically [56] or thermally [56][57][58]. In addition, studies on the overall lipid composition of outer membrane isolated from normal fed, starved and streptozotocin-diabetic rats indicate that there is a correlation between the mobility of a hydrophobic molecular probe (diphenylhexatriene) within the membrane (a measure of ' fluidity '), the cholesterol\phospholipid ratio in the membrane and the degree of desensitization of CPT I to malonyl-CoA (C. G. Corstorphine and V. A. Zammit, unpublished work).…”

Regulation of Carnitine Palmitoyltransferase (CPT) I during Fasting in Rainbow Trout (Oncorhynchus mykiss) Promotes Increased Mitochondrial Fatty Acid Oxidation

“…Regulation of fatty acid oxidation is dependent on the sensitivity of CPT I to this modulator. Most notably, changes in sensitivity can occur by changes in mitochondrial membrane fluidity (Kolodziej and Zammit 1990). This has been theorized to occur because the active site for malonyl-CoA is located on the cytosolic side of the transmembrane CPT I protein, and this area of the protein is adjacent to the mitochondrial membrane (Jackson et al 2000).…”