Carnitine transport and fatty acid oxidation

Abstract

Carnitine is essential for the transfer of long-chain fatty acids across the inner mitochondrial membrane for subsequent β-oxidation. It can be synthesized by the body or assumed with the diet from meat and dairy products. Defects in carnitine biosynthesis do not routinely result in low plasma carnitine levels. Carnitine is accumulated by the cells and retained by kidneys using OCTN2, a high affinity organic cation transporter specific for carnitine. Defects in the OCTN2 carnitine transporter results in autosomal recessive primary carnitine deficiency characterized by decreased intracellular carnitine accumulation, increased losses of carnitine in the urine, and low serum carnitine levels. Patients can present early in life with hypoketotic hypoglycemia and hepatic encephalopathy, or later in life with skeletal and cardiac myopathy or sudden death from cardiac arrhythmia, usually triggered by fasting or catabolic state. This disease responds to oral carnitine that, in pharmacological doses, enters cells using the amino acid transporter B(0,+). Primary carnitine deficiency can be suspected from the clinical presentation or identified by low levels of free carnitine (C0) in the newborn screening. Some adult patients have been diagnosed following the birth of an unaffected child with very low carnitine levels in the newborn screening. The diagnosis is confirmed by measuring low carnitine uptake in the patients' fibroblasts or by DNA sequencing of the SLC22A5 gene encoding the OCTN2 carnitine transporter. Some mutations are specific for certain ethnic backgrounds, but the majority are private and identified only in individual families. Although the genotype usually does not correlate with metabolic or cardiac involvement in primary carnitine deficiency, patients presenting as adults tend to have at least one missense mutation retaining residual activity. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

Keywords: Arrhythmia; Autism; Carnitine; Newborn screening; OCTN2; SLC22A5.

Copyright © 2016 Elsevier B.V. All rights reserved.

Figures

FIGURE 1. Fatty acids oxidation during fasting

During periods of fasting, fatty acids released from the adipose tissues are oxidized in the liver, skeletal muscle, and cardiac muscle for energy production. The brain does not directly utilize fatty acids, but oxidizes ketone bodies derived from β-oxidation of fatty acids in the liver. When fatty acid oxidation is defective, fats released from the adipose tissue cannot be oxidized, and accumulate in organs such as the skeletal and cardiac muscles, impairing their function. Moreover, the liver is unable to produce ketones bodies resulting in energy deficiency.

FIGURE 2. The carnitine cycle in fatty acid oxidation

Fatty acids bound to albumin are transferred across the plasma membrane by the action of fatty acid transport proteins (FATP), fatty acid translocase (FAT/CD36), caveolins and plasma membrane fatty acid binding proteins (FABPpm). Inside the cell, fatty acids undergo vectorial acylation, a process catalyzed by acyl-CoA synthases (ACS), that traps them in the cytoplasm as acyl-CoA thioesters. The acyl-CoA thioesters are then conveyed through different metabolic pathways in mitochondria, peroxisomes and microsomes based on the cell energy status. ACS: acyl-CoA synthases; CACT: carnitine acyl carnitine translocase; CPT-1: carnitine palmitoyl transferase-1; CPT-2: carnitine palmitoyl transferase-2; FA: fatty acid; FABPpm: plasma membrane fatty acid binding proteins; FAT/CD36: fatty acid translocase; FATP: fatty acid transport proteins; OCTN2: organic cation transporter novel 2.

FIGURE 3. The human carnitine transporter

The SLC22A5 gene composed of 10 exons on the long arm of chromosome 5 encodes for the high-affinity carnitine transporter OCTN2 mRNA that has an open reading frame (ORF) of 1,674 nt. The transporter has 12 predicted transmembrane domains, 3 extracellular glycosylation sites, a glucose-transporter signature motif between transmembrane domains 2 and 3, a nucleotide binding motif in the intracellular loop between transmembrane domains 4 and 5, and several intracellular motifs that might be involved in regulatory mechanisms through the action of protein kinase A (PK-A) and C (PK-C).

FIGURE 4. Confocal imaging of the OCTN2 transporter tagged with the green fluorescent protein expressed in Chinese Hamster Ovary cells

The OCTN2 transporter localizes to the plasma membrane and operates a sodium/carnitine co-transport. The cytoplasm (with the Golgi apparatus stained in red by Bodipy-ceramide) has a low concentration of sodium (about 20 mM) and membrane potential is about -65mV [96]. The resulting sodium electrochemical gradient energizes a more than 80-fold intracellular carnitine accumulation [97].

FIGURE 5. Carnitine and acylcarnitine levels in infants with primary carnitine deficiency (patients) and in infants whose mothers are affected by primary carnitine deficiency (mothers)

Carnitine and acylcarnitines were measured in newborn screening blood spots by tandem mass spectrometry using standard methods. The first newborn screening (NBS1) was collected between 12 and 36 hours of life; the second between 7 and 21 days of life (NBS2). Data are averages of values from 4 patients and 4 mothers and from 150,000 unaffected controls, with the standard deviations indicated. Values for carnitine (C0), other acylcarnitine species (C3-, C16-, C18-, C18:1-carnitine) in patients and mothers were significantly (p

FIGURE 6

Missense mutations in the OCTN2 carnitine transporter in patients with primary carnitine deficiency.

FIGURE 7. Carnitine biosynthesis in humans

Carnitine is synthetized in mammals from lysine residues of certain proteins that are post-translationally N-methylated by S-adenosyl-methionine: ε-N-lysine methyltransferase (1), with formation of protein-6-N-trimethyllysine that directs the protein to the lysosome for further degradation. Hydrolysis of protein-6-N-trimethyllysine in the lysosomes by a peptidase (2) releases ε-N-trimethyllysine that is transferred to mitochondria and hydroxylated to β-hydroxy-N-ε-trimethyllysine by ε-N-trimethyllysine hydroxylase (3). In the cytosol, β-hydroxy-N-ε-trimethyllysine is cleaved by a β-hydroxy-N-ε-trimethyllysine aldolase (4) to produce glycine and 4-N-trimethylaminobutyraldehyde that is converted by 4-N-trimethylaminobutyraldehyde dehydrogenase (5) to γ-butyrobetaine. In the last step of carnitine endogenous biosynthesis, γ-butyrobetaine is hydrolyzed by a cytosolic γ−butyrobetaine hydroxylase (6) to carnitine. Brain, liver and kidney are capable of full carnitine biosynthesis, while other tissues, such as cardiac and skeletal muscle, can only synthetize γ−butyrobetaine, and obtain carnitine from the circulation using the OCTN2 carnitine transporter.