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V A Zammit

Publications and source records attributed to V A Zammit.

At least 37 records · Page 2Linked to original sources
PubMed1999

Submitochondrial and subcellular distributions of the carnitine-acylcarnitine carrier.

The submitochondrial and subcellular distributions of the carnitine-acylcarnitine translocase (CAC) have been studied. CAC is enriched to a much lesser extent than the carnitine palmitoyltransferases within the contact sites of mitochondria. A high-abundance protein of identical molecular size as the mitochondrial CAC that is immunoreactive with an anti-peptide antibody raised against a linear epitope of mitochondrial CAC is present in peroxisomes but not in microsomes. This suggests that CAC is targeted to at least two different locations within the liver cell and that acylcarnitine transport into peroxisomes is CAC mediated.

Animals↗
PubMed1999

Insulin stimulates triacylglycerol secretion by perfused livers from fed rats but inhibits it in livers from fasted or insulin-deficient rats implications for the relationship between hyperinsulinaemia and hypertriglyceridaemia.

We determined whether the direction of the acute effect of insulin on hepatic triacylglycerol secretion is dependent on the prior physiological state or on the in vitro experimental system used. The effect of insulin on triacylglycerol secretion was studied using perfused livers isolated from rats under three metabolic conditions: fed normo-insulinaemic, 24-h fasted and fed, streptozotocin-diabetic (insulin-deficient). Insulin acutely activated triacylglycerol secretion (by 43%) in organs from fed, normo-insulinaemic animals, whereas it inhibited triacylglycerol secretion in livers isolated from fasted or insulin-deficient rats (by 30 and 33%, respectively). By contrast, in 24-h-cultured hepatocytes insulin invariably acutely inhibited triacylglycerol secretion irrespective of the metabolic state of the donor animals. It is concluded that the use of perfused livers enables the observation of a switch in the direction of insulin action on hepatic triacylglycerol secretion from stimulatory, in the normo-insulinaemic state, to inhibitory in the fasting or insulin-deficient state. The possible implications of this switch for the relationship between hyperinsulinaemia, increased hepatic very-low-density lipoprotein-triacylglycerol secretion and hypertriglyceridaemia observed in vivo are discussed.

Animals↗
PubMed1999

Subcellular distribution of mitochondrial carnitine palmitoyltransferase I in rat liver. Evidence for a distinctive N-terminal structure of the microsomal but not the peroxisomal enzyme.

Mitochondria, microsomes and peroxisomes all express overt (cytosol-facing) carnitine palmitoyltransferase activities that are inhibitable by malonyl-CoA and are collectively termed CPTo. In order to quantify the relative contribution of the different membrane systems towards overall hepatocyte activity, all three membrane fractions and a high-speed supernatant (soluble) fraction were prepared quantitatively from rat liver homogenates. The overt (malonyl-CoA-sensitive) carnitine palmitoyltransferase activity (CPTo) associated with the different fractions were measured. In parallel experiments, rat livers were perfused in situ with oxygenated medium containing dinitrophenyl (DNP)-etomoxir in order to label covalently (with DNP-etomoxiryl-CoA) and quantitatively the molecular species responsible for CPTo activity in each of the membrane systems under near-physiological conditions. Mitochondria accounted for only 65% of total cellular overt CPT activity, with the microsomal and peroxisomal contributions accounting for the remaining 25% and 10%, respectively. A single major protein with an identical molecular size (Mr 88,000) was labelled by DNP-etomoxir perfusion in all three membrane fractions. The abundance of this 88 kDa protein in each fraction was quantitatively positively related to the respective specific activities of overt CPT. The same protein was immunoreactive with three anti-peptide antibodies raised against linear epitopes within the N- and C-terminal and loop (L) domains of the mitochondrial outer membrane CPT I of the liver mitochondrial outer membrane (L-CPT I). However, whereas reaction with anti-L and anti-C antipeptide antibodies were proportional to the respective overt CPT activities and DNP-etomoxir labelling in all three membrane fractions, reaction with anti-N peptide antibody was much stronger for microsomal CPT. We conclude that in all three membrane systems overt CPT activity is associated with the same or highly similar molecular species to mitochondrial outer membrane CPT I, but that the protein expressed in microsomes has a modified N-terminal domain, which gives the microsomal enzyme its higher malonyl-CoA sensitivity and may target the protein to its microsomal location.

Animals↗
PubMed1998

Characteristics of L-carnitine transport by lactating rat mammary tissue.

The transport of L-carnitine by lactating rat mammary tissue has been examined. L-carnitine uptake by rat mammary tissue explants isolated from lactating rats, 3-4 days post partum, was via both Na+-dependent and Na+-independent pathways. The Na+-dependent pathway, the predominant route for L-carnitine uptake, was a saturable process: the Km and Vmax were, respectively, 132 microM and 201 pmol/2 h/mg of intracellular water. The Na+-independent pathway, which was non-saturable, had a coefficient of 0.26 microl/mg of intracellular water/2 h. The Na+-dependent component of L-carnitine uptake by mammary tissue explants was cis-inhibited by D-carnitine and acetyl-L-carnitine, but not by choline or taurine. In contrast, the Na+-independent component of L-carnitine uptake was not affected by any of these compounds. The uptake of L-carnitine by mammary tissue isolated from lactating rats, 10-12 days post partum, was qualitatively similar to that by mammary tissue taken from rats during the early stage of lactation. However, L-carnitine uptake was quantitatively lower: this was attributable to a reduction in the Na+-dependent component of L-carnitine uptake. L-Carnitine efflux from rat mammary tissue taken from animals 3-4 days post partum, consisted of at least two components; a fast extracellular component and a slow membrane-limited component. Reversing the trans-membrane Na+-gradient did not stimulate L-carnitine efflux suggesting that the Na+-dependent L-carnitine carrier operates with asymmetrical kinetics. A hyposmotic shock, hence cell-swelling, increased L-carnitine efflux from mammary tissue explants.

Animals↗
PubMed1998

Enrichment of carnitine palmitoyltransferases I and II in the contact sites of rat liver mitochondria.

The submitochondrial distribution of the overt and latent carnitine palmitoyltransferases (CPT I and II respectively) of rat liver mitochondria were studied. Separation of outer and inner membranes, as well as of a fraction of intermediate density consisting of contact sites between the two membranes, was achieved, as judged by the distribution of marker enzymes. Both CPT I and CPT II were found to be enriched within the contact- site fraction of mitochondria. These data show that the two carnitine acyltransferases are distributed non-uniformly within their respective membranes, and that subpopulations of the two enzymes occur in close proximity within the mitochondrial membrane structure, while retaining their different accessibilities to cytosolic and matrix pools of metabolites. As the number of contact sites is known to vary with changes in the energy status of mitochondria, the possibility that such changes may acutely affect the proportion of CPT I within the distinctive lipid environment of the contact sites, and thus its overall kinetic characteristics, is discussed.

Animals↗
PubMed1998

Lipid molecular order in liver mitochondrial outer membranes, and sensitivity of carnitine palmitoyltransferase I to malonyl-CoA.

Mitochondrial outer membranes were prepared from livers of rats that were in the normal fed state, starved for 48 h, or made diabetic by injection of streptozotocin. Membranes were also prepared from starved late-pregnant rats. The latter three conditions have previously been shown to induce varying degrees of desensitization of mitochondrial overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA inhibition. We measured the fluorescence polarization anisotropy of two probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluenes ulfonate (TMA-DPH) which, when incorporated into membranes, report on the hydrophobic core and on the peripheral regions of the bilayer, respectively. The corresponding polarization indices (rDPH and rTMA-DPH) were calculated. In membranes of all three conditions characterized by CPT I desensitization to malonyl-CoA, rDPH was decreased, whereas there was no change in rTMA-DPH, indicating that CPT I is sensitive to changes in membrane core, rather than peripheral, lipid order. The major lipid components of the membranes were analyzed. Although significant changes with physiological state were observed, there was no consistent pattern of changes in gross lipid composition accompanying the changes to membrane fluidity and CPT I sensitivity to malonyl-CoA. We conclude that CPT I kinetic characteristics are sensitive to changes in lipid composition that are localized to specific membrane microdomains.

Animals↗
PubMed1998

Use of cytosolic triacylglycerol hydrolysis products and of exogenous fatty acid for the synthesis of triacylglycerol secreted by cultured rat hepatocytes.

The fatty acyl moieties incorporated into triacylglycerol (TAG) secreted by rat hepatocytes are derived from diacylglycerol (DAG) that is synthesized de novo through the phosphatidate pathway or derived from endogenous, cytosolic TAG after hydrolysis to DAG and re-esterification. We have used a dual-labeling technique (overnight labeling of cell TAG with [3H]oleate, followed by 3 h incubation with [14C]oleate) to quantify the contributions of the two sources towards TAG secretion by cultured rat hepatocytes. A wide range of TAG secretion rates was achieved by short-term incubation of the cells under a variety of conditions. There was no correlation between the overall amount of exogenous 14C-labeled fatty acid metabolized and the rate of either [14C]- or [3H]TAG secretion. By contrast, there was a strong positive correlation between the fraction of newly synthesized [14C]TAG that was secreted (the fractional secretion rate, FSR) and the absolute rate of TAG secretion. This suggests that the partitioning of DAG between (re)synthesis of cytosolic TAG and synthesis of secreted TAG is an important locus for the control of the rate of TAG secretion. Comparison of the ratio: oxidation/TAG secretion for 3H- and 14C-labeled acyl moieties indicated that, for all conditions studied, approximately half the acyl moieties already esterified to the glyceroyl backbone within cytosolic TAG remain unavailable for oxidation when this pool of TAG is mobilized for the synthesis of secreted TAG. The data provide evidence that hydrolysis of cytosolic triacylglycerol (TAG) does not proceed fully to the constituent fatty acids and glycerol, but only to the level of diacylglycerol, followed by re-modelling of approximately half of its acyl chains, before re-esterification to form secretory TAG.

Animals↗
PubMed1997

Topology of carnitine palmitoyltransferase I in the mitochondrial outer membrane.

The topology of carnitine palmitoyltransferase I (CPT I) in the outer membrane of rat liver mitochondria was studied using several approaches. 1. The accessibility of the active site and malonyl-CoA-binding site of the enzyme from the cytosolic aspect of the membrane was investigated using preparations of octanoyl-CoA and malonyl-CoA immobilized on to agarose beads to render them impermeant through the outer membrane. Both immobilized ligands were fully able to interact effectively with CPT I. 2. The effects of proteinase K and trypsin on the activity and malonyl-CoA sensitivity of CPT I were studied using preparations of mitochondria that were either intact or had their outer membranes ruptured by hypo-osmotic swelling (OMRM). Proteinase K had a marked but similar effect on CPT I activity irrespective of whether only the cytosolic or both sides of the membrane were exposed to it. However, it affected sensitivity more rapidly in OMRM. By contrast, trypsin only reduced CPT I activity when incubated with OMRM. The sensitivity of the residual CPT I activity was unaffected by trypsin. 3. The proteolytic fragments generated by these treatments were studied by Western blotting using three anti-peptide antibodies raised against linear epitopes of CPT I. These showed that a proteinase K-sensitive site close to the N-terminus was accessible from the cytosolic side of the membrane. No trypsin-sensitive sites were accessible in intact mitochondria. In OMRM, both proteinase K and trypsin acted from the inter-membrane space side of the membrane. 4. The ability of intact mitochondria and OMRM to bind to each of the three anti-peptide antibodies was used to study the accessibility of the respective epitopes on the cytosolic and inter-membrane space sides of the membrane. 5. The results of all these approaches indicate that CPT I adopts a bitopic topology within the mitochondrial outer membrane; it has two transmembrane domains, and both the N- and C-termini are exposed on the cytosolic side of the membrane, whereas the linker region between the transmembrane domains protrudes into the intermembrane space.

Acyl Coenzyme A↗
PubMed1997

Overt and latent activities of diacylglycerol acytransferase in rat liver microsomes: possible roles in very-low-density lipoprotein triacylglycerol secretion.

The possibility that triacylglycerol (TAG) synthesis occurs on both aspects of the endoplasmic-reticular membrane during the process of incorporation of TAG into secreted very-low-density lipoprotein (VLDL) [Zammit (1996) Biochem. J. 314, 1-14] was investigated by measuring the latency of diacylglycerol acyltransferase (DGAT) in microsomal fractions obtained from rat liver homogenates. Permeabilization of microsomes with taurocholate resulted in the doubling of the activity, indicating that DGAT activities of approximately equal magnitude occur on either aspect of the microsomal membrane. The taurocholate concentrations required for exposure of the latent activity of DGAT were identical with those that resulted in the exposure of marker enzymes for the lumen of the endoplasmic reticulum. Fractionation of the microsomes into smooth and rough populations indicated that the distribution of overt and latent DGAT activities was the same throughout. The possibility that taurocholate effects may result from non-specific activation of the overt enzyme was excluded by employing the channel-forming peptide alamethicin to effect permeabilization, and by varying the mode of delivery of diacylglycerol substrate to the microsomal membranes. Permeabilization using alamethicin gave a slightly higher latent/overt ratio for DGAT. The possible roles of overt and latent DGAT activities in the synthesis and secretion of TAG by the liver are discussed.

Acyltransferases↗
PubMed1997

Involvement of Ca2+/calmodulin-dependent protein kinase II in the activation of carnitine palmitoyltransferase I by okadaic acid in rat hepatocytes.

The present work was undertaken to study the mechanism by which okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, stimulates carnitine palmitoyltransferase I (CPT-I) in isolated rat hepatocytes [Guzmán, Kolodziej, Caldwell, Costorphine and Zammit (1994) Biochem. J. 300, 693-699]. The OA-induced stimulation of CPT-I was abolished by the general protein kinase inhibitor K-252a as well as by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CM-PKII). However, neither the protein kinase C-specific inhibitor bisindolylmaleimide nor the protein kinase A/protein kinase C inhibitor H-7 was able to prevent the OA-induced stimulation of CPT-I. Hepatocyte-shrinkage-induced stimulation of CPT-I as well as OA-induced hepatocyte shrinkage was prevented by KN-62. KN-62 also antagonized the OA-enhanced release of lactate dehydrogenase from digitonin-permeabilized hepatocytes. Exposure of 32P-labelled hepatocytes to OA increased the degree of phosphorylation of Ca2+/CM-PKII, as immunoprecipitated by a monoclonal antibody raised against the alpha-subunit of rat brain kinase. This effect of OA was also antagonized by KN-62. The results thus indicate that the OA-dependent stimulation of CPT-I may be mediated (at least in part) by increased phosphorylation and subsequent activation of Ca2+/CM-PKII.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Regulation of mitochondrial outer-membrane carnitine palmitoyltransferase (CPT I): role of membrane-topology.

The topology of the outer membrane carnitine palmitoyltransferase (CPT I) of rat liver mitochondria was studied systematically using several experimental approaches. Studies with immobilized malonyl-CoA and octanoyl-CoA showed that functionally the active and regulatory sites of CPT I are exposed on the outer (cytosolic) surface of the mitochondrial outer membrane. Anti-peptide antibodies generated against three linear peptide sequences that occur in between and on either side of two hydrophobic, putative transmembrane domains were used to (a) ascertain which were bound by intact mitochondria and mitochondria in which the outer membrane was permeabilized to proteins; and (b) to determine the size of fragments generated by limited proteolysis (by trypsin or proteinase K) of CPT I in intact or outer membrane-ruptured mitochondria. The sizes and immunoreactivity of the proteolytic fragments generated were correlated with the effects of the proteases on CPT I activity and malonyl-CoA sensitivity. The results of all the different approaches suggested the following: (i) CPT I has two transmembrane domains; (ii) both the N- and C-termini are exposed on the cytosolic side of the membrane; (iii) the linker region between the two transmembrane domains protrudes into the intermembrane space; (iv) both the active site and the malonyl-CoA-binding site are exposed on the cytosolic side of the membrane; (v) the amino-terminus of the protein interacts with the C-terminal domain of the protein to maintain the optimal conformation required for activity of the enzyme and its sensitivity to malonyl-CoA.

Acyl Coenzyme A↗
PubMed1996

Flux control exerted by overt carnitine palmitoyltransferase over palmitoyl-CoA oxidation and ketogenesis is lower in suckling than in adult rats.

We examined the potential of overt carnitine palmitoyltransferase (CPT I) to control the hepatic catabolism of palmitoyl-CoA in suckling and adult rats, using a conceptually simplified model of fatty acid oxidation and ketogenesis. By applying top-down control analysis, we quantified the control exerted by CPT I over total carbon flux from palmitoyl-CoA to ketone bodies and carbon dioxide. Our results show that in both suckling and adult rat, CPT I exerts very significant control over the pathways under investigation. However, under the sets of conditions we studied, less control is exerted by CPT I over total carbon flux in mitochondria isolated from suckling rats than in those isolated from adult rats. Furthermore the flux control coefficient of CPT I changes with malonyl-CoA concentration and ATP turnover rate.

Animals↗
PubMed1996

The role of changes in the sensitivity of hepatic mitochondrial overt carnitine palmitoyltransferase in determining the onset of the ketosis of starvation in the rat.

The relationships between the increase in blood ketone-body concentrations and several parameters that can potentially influence the rate of hepatic fatty acid oxidation were studied during progressive starvation (up to 24 h) in the rat in order to discover whether the sensitivity of mitochondrial overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA plays an important part in determining the intrahepatic potential for fatty acid oxidation during the onset of ketogenic conditions. A rapid increase in blood ketone-body concentration occurred between 12 and 16 h of starvation, several hours after the marked fall in hepatic malonyl-CoA and in serum insulin concentrations and doubling of plasma non-esterfied fatty acid (NEFA) concentration. Consequently, both the changes in hepatic malonyl-CoA and serum NEFA preceded the increase in blood ketone-body concentration by several hours. The maximal activity of CPT I increased gradually throughout the 24 h period of starvation, but the increases did not become significant before 18 h of starvation. By contrast, the sensitivity of CPT I to malonyl-CoA and the increase in blood ketone-body concentration followed an identical time course, demonstrating the central importance of this parameter in determining the ketogenic response of the liver to the onset of the starved state.

Animals↗
PubMed1996

Flux control exerted by mitochondrial outer membrane carnitine palmitoyltransferase over beta-oxidation, ketogenesis and tricarboxylic acid cycle activity in hepatocytes isolated from rats in different metabolic states.

The Flux Control Coefficients of mitochondrial outer membrane carnitine palmitoyltransferase (CPT I) with respect to the overall rates of beta-oxidation, ketogenesis and tricarboxylic acid cycle activity were measured in hepatocytes isolated from rats in different metabolic states (fed, 24 h-starved, starved-refed and starved/insulin-treated). These conditions were chosen because there is controversy as to whether, when significant control ceases to be exerted by CPT I over the rate of fatty oxidation [Moir and Zammit (1994) Trends Biochem. Sci. 19, 313-317], this is transferred to one or more steps proximal to acylcarnitine synthesis (e.g. decreased delivery of fatty acids to the liver) or to the reaction catalysed by mitochondrial 3-hydroxy-3-methyl-glutaryl-CoA synthase [Hegardt (1995) Biochem. Soc. Trans. 23, 486-490]. Therefore isolated hepatocytes were used in the present study to exclude the involvement of changes in the rate of delivery of non-esterified fatty acids (NEFA) to the liver, such as occur in vivo, and to ascertain whether, under conditions of constant supply of NEFA, CPT I retains control over the relevant fluxes of fatty acid oxidation to ketones and carbon dioxide, or whether control is transferred to another (intrahepatocytic) site. The results clearly show that the Flux Control Coefficients of CPT I with respect to overall beta-oxidation and ketogenesis are very high under all conditions investigated, indicating that control is not lost to another intrahepatic site during the metabolic transitions studied. The control of CPT I over tricarboxylic acid cycle activity was always very low. The significance of these findings for the integration of fatty acid and carbohydrate metabolism in the liver is discussed.

Animals↗