Membrane transport of fatty acylcarnitine and free l‐carnitine by rat liver microsomes

Abstract

Recent studies have suggested that parts of the hepatic activities of diacylglycerol acyltransferase and acyl cholesterol acyltransferase are expressed in the lumen of the endoplasmic reticulum (ER). However the ER membrane is impermeable to the long-chain fatty acyl-CoA substrates of these enzymes. Liver microsomal vesicles that were shown to be at least 95% impermeable to palmitoyl-CoA were used to demonstrate the membrane transport of palmitoylcarnitine and free L-carnitine -processes that are necessary for an indirect route of provision of ER luminal fatty acyl-CoA through a luminal carnitine acyltransferase (CAT). Experimental conditions and precautions were established to permit measurement of the transport of [ ]carnitine by microsomes was measured directly. This process, mediated either by a channel or a carrier, was inhibited by mersalyl but not by N-ethylmaleimide or sulfobetaine -properties that differentiate it from the mitochondrial inner membrane carnitine/acylcarnitine exchange carrier. These findings are relevant to the understanding of processes for the reassembly of triacylglycerols that lipidate very low density lipoprotein particles as part of a hepatic triacylglycerol lipolysis/ re-esterification cycle.Keywords: acylcarnitine; carnitine; microsomes; liver; transport.Although mammalian intracellular membranes are impermeable to the Coenzyme A thioesters of long-chain fatty acids, these activated derivatives, which are synthesized from nonesterified fatty acids by fatty acyl-CoA synthetase on the cytosolic aspect of organelle membranes, are the substrates for metabolic processes within at least three cellular organelles. In mitochondria, it is well established that CPT 1 , a carnitine acyltransferase associated with the outer membrane, generates fatty acylcarnitine derivatives which can then traverse the inner membrane via a carnitine/acylcarnitine exchange carrier (CAC). Within the mitochondrial interior, the latent carnitine acyltransferase CPT 2 then facilitates the re-formation of fatty acylCoAs which are then substrates for b-oxidation within the mitochondrial matrix [1]. Fatty acyl-CoAs are also substrates for chain-shortening by b-oxidation within the matrix of peroxisomes. As peroxisomes also contain overt and latent carnitine acyltransferase activities [2,3] and express the CAC protein [4], it has been concluded that activated fatty acids access the peroxisomal matrix through a system that is closely analogous to the mitochondrial one. Enzymes within the lumen of the endoplasmic reticulum (ER) also require fatty acyl-CoA thioesters as substrate. A latent form of diacylglycerol acyltransferase (DGAT), assigned to the luminal surface of the ER membrane and which can be differentiated from a cytosolically oriented DGAT, has been described [5][6][7]. This latent DGAT may be involved in the reassembly of triacylglycerols which lipidate very low density lipoprotein (VLDL) particles as part of a hepatic triacylglycerol lipolysis/re-esterification cycle [2,3,[8][9][10][11][12]. Two forms of a...

Hhat Is a Palmitoylacyltransferase with Specificity for N-Palmitoylation of Sonic Hedgehog

“…Free fatty acids can be specifically transported into the ER lumen, where they are converted by luminal acyltransferases into palmitoyl-CoA (49). Moreover, the ER contains a carnitine palmitoyltransferase that generates palmitoylcarnitine, which is then transported across the ER membrane into the lumen and converted to palmitoyl-CoA (48,50). Palmitoyl-CoA hydrolase activities have also been detected in the ER lumen, consistent with the presence of luminal palmitoyl-CoA (51,52).…”

“…5), yet long chain acyl-CoAs are not permeable across the ER membrane (48). There are, however, several mechanisms that would allow palmitoyl-CoA to gain access to the lumen of the ER and/or Golgi.…”

Malonyl-CoA, a Key Signaling Molecule in Mammalian Cells

“…The lumen of the hepatic endoplasmic reticulum (ER) and the peroxisomal matrix contain latent, malonyl-CoA-insensitive CPT activities. Also, both the ER membrane and the peroxisomal membrane have the capability to transport fatty acylcarnitine and l-carnitine (43,49). Therefore, it has been suggested that liver CPT1A might be targeted to become an integral protein of both the ER and the peroxisomal membrane and thereby be part of a system for the transport of long-chain fatty acyl moieties into these organelles that is similar to the CPT1/CPT2 system of the mitochondria.…”

Carnitine acyltransferases and their influence on CoA pools in health and disease

“…Isolated ER has malonyl-CoA-sensitive CPT activity but immunoblotting failed to detect CPT 1. Recently, lumenal palmitoyl-CoA formation from added palmitoylcarnitine was demonstrated (Gooding et al, 2004). The synthesis of complex lipids (via, e.g., diacylglycerol acyltransferase and acyl cholesterol acyltransferase) inside the lumen of ER means that active acyl groups must be delivered there but the flux rate may be less critical than in mitochondria where the demands for fat breakdown and ketone body synthesis must be rapid and tightly regulated.…”