Acylcarnitines: Nomenclature, Biomarkers, Therapeutic Potential, Drug Targets, and Clinical Trials

1. Carnitine Acetyltransferase

Mammalian carnitine acetyltransferase, or CrAT, is found in the mitochondrial matrix, the peroxisomal matrix, the endoplasmic reticulum, and the nucleus (Markwell et al., 1973; Ramsay et al., 2001). Recently, its activity has also been reported in the cytosol of mouse cardiomyocytes (Altamimi et al., 2018). In the mitochondria CrAT regulates the acetyl-CoA/free CoA ratio to prevent depletion of free CoA. For example, during intensive physical exercise when overproduction of acetyl-CoA occurs in muscle tissue, this process ensures replenishment of free CoA to facilitate pyruvate metabolism, but when the physical load ends, transacetylation of acetylcarnitine provides acetyl-CoA for the Krebs cycle (Stephens et al., 2007). The main function of CrAT in peroxisomes is to aid in the export of products of peroxisomal β-oxidation (Westin et al., 2008). In the murine cardiomyocyte cytosol, the reverse activity of CrAT might be related to providing acetyl-CoA for the biosynthesis of malonyl-CoA to inhibit fatty acid oxidation (Altamimi et al., 2018). It is also known that CrAT, by decreasing the availability of acetyl-CoA in the presence of carnitine overload (or when overexpressed), can alleviate inactivation of pyruvate dehydrogenase and increase glucose utilization in primary human skeletal myocytes (Noland et al., 2009). This can facilitate insulin secretion in β cells (Berdous et al., 2020). During ischemia, the lack of oxygen limits mitochondrial β-oxidation and respiration in isolated rat hearts, leading to the accumulation of both short-/long-chain acylcarnitines and acyl-CoA intermediates along with the corresponding decrease in free CoA (Whitmer et al., 1978). Although limited reserves of cytosolic CoA are likely converted to acyl-CoAs under acute stress conditions, preserving CrAT activity is crucial to ensure repletion of free CoA inside the mitochondria to support energy metabolism as long as possible.

2. Carnitine Octanoyltransferase

Carnitine octanoyltransferase, or CrOT, is found only in peroxisomes, and similar to CrAT, this enzyme aids the export of products of peroxisomal β-oxidation (Westin et al., 2008). The highest activity of CrOT is toward C4–C12 containing acylcarnitines as indicated by studies using rat and mouse liver enzymes (Fig. 4), whereas the activity rapidly decreases for chains with more than 14 carbons (Miyazawa et al., 1983; Farrell et al., 1984). It is interesting to note that although CrOT can use C10-CoA and 4,8-dimethylnonanoyl-CoA (a C11-CoA product of peroxisomal oxidation of pristanic acid) to produce the respective acylcarnitines, the human enzyme is inactive against 2,6-dimethylheptanoyl-CoA (C9-CoA), which is produced when C11-CoA is further β-oxidized (Ferdinandusse et al., 1999). Peroxisomes, via the help of CrOT, can partially contribute to medium-chain acylcarnitine production from the metabolism of medium- and long-chain fatty acids. Peroxisomes can also contribute to the oxidation of these fatty acids under normoxic conditions in isolated perfused rat hearts (Bian et al., 2005), particularly when mitochondrial fatty acid oxidation is impaired or overloaded as shown in a study using human skin fibroblasts (Violante et al., 2013b). Studies in isolated rat hearts indicate that peroxisomal fatty acid oxidation can provide more than 50% of the acetyl groups that are used to produce malonyl-CoA (Reszko et al., 2004). Moreover, the study of intact isolated rat hearts indicates that acetyl-CoA molecules formed from fatty acids that undergo the first steps of oxidation only in peroxisomes are used for malonyl-CoA biosynthesis but are not transferred to the mitochondria in detectable amounts (Bian et al., 2005). Similar to CPT1, CrOT contains a malonyl-CoA-sensitive domain and can be inhibited by malonyl-CoA at physiologic concentrations (A'Bhaird and Ramsay, 1992; Morillas et al., 2002). More than 30 years ago, it was suggested that the inhibition of rat liver peroxisomal long- and medium-chain acyltransferase (i.e., CrOT) by malonyl-CoA could be important for the regulation of mitochondrial fatty acid oxidation (Derrick and Ramsay, 1989). This is likely true for peroxisomal fatty acid oxidation itself, as the overexpression of the CrOT gene in human hepatic cancer cell lines results in an increase in CrOT activity and subsequent upregulation of enzymes that encode peroxisomal fatty acid oxidation. Conversely, small interfering RNA-induced knockdown of CrOT has been shown to result in decreased mRNA levels of key proteins of fatty acid metabolism in peroxisomes (Le Borgne et al., 2011). Overexpression of CrOT leads to a decrease in medium- and very long-chain fatty acid levels, whereas long-chain fatty acid levels are not affected. Furthermore, hepatic cancer cells with CrOT knockdown demonstrate an increase in medium-chain fatty acid levels (Le Borgne et al., 2011). Moreover, CrOT overexpression can result in an increase in the mRNA levels of key proteins involved in peroxisomal fatty acid metabolism but not mitochondrial CPT1 and acyl-CoA thioesterase. Notably, the redirection of myocardial fatty acid oxidation from the mitochondria to peroxisomes decreases ischemic damage in isolated rat hearts and improves recovery during reperfusion (Liepinsh et al., 2013).

3. Carnitine Palmitoyltransferases

Carnitine palmitoyltransferase 1 (CPT1) is the only carnitine acyltransferase with three isoforms (CPT1A, liver isoform; CPT1B, muscle isoform; and CPT1C, brain isoform). Each of these isoforms is specific for different tissue types. The heart is one of the tissues where both CPT1A and CPT1B are present. Although liver and muscle isoforms share a high degree of sequence homology, they differ significantly in their physiologic response to and regulation by malonyl-CoA and carnitine. A seminal paper by McGarry and colleagues (1983) describes, in great detail, the differences between the liver and muscle isoforms of CPT1 from rats, dogs, and humans. The Michaelis–Menten constant (K_m) of carnitine for CPT1 ranges from 32 and 39 μM in rat and human liver tissues to 500–600 μM in human, rat, and dog skeletal muscles to 197 and 695 μM in rat and dog cardiac tissues, respectively. The muscle isoform has a low affinity for carnitine, and the opposite is true for the liver isoform (McGarry et al., 1983; Distler et al., 2009). The IC₅₀ for malonyl-CoA of CPT1 in human and rat liver tissue is in the low micromolar range (1.6–2.7 μM), whereas it is in the low to medium nanomolar range in rat and dog cardiac tissues (25–100 nM) and rat, dog, and human skeletal muscle (17–34 nM) (McGarry et al., 1983). This high affinity toward the physiologic inhibitor malonyl-CoA means that muscle and cardiac tissues are more sensitive to malonyl-CoA than liver tissues (Bird and Saggerson, 1984). During fasting, when the malonyl-CoA tissue content decreases by factors of 4.4-, 3.3-, and 3-fold in liver, cardiac, and muscle tissues, respectively (McGarry et al., 1983), the activity of CPT1 increases only in the liver and not in cardiac tissues (Paulson et al., 1984; Bonnefont et al., 2004). On the other hand, a diet rich in carbohydrates activates insulin signaling. As a result, adenosine monophosphate-activated protein kinase (AMPK) is inhibited, and the production of malonyl-CoA by acetyl-CoA carboxylase (ACC, EC:6.4.1.2) is increased, leading to the inhibition of liver CPT1 activity, which facilitates liponeogenesis. Unlike heart and muscle tissues that predominantly express the ACC2 isoform, liver tissue expresses both ACC1 and ACC2, with the latter being responsible for malonyl-CoA synthesis (Munday, 2002). In a fasted state, the activity of ACC is inhibited via phosphorylation by AMPK. When the inhibitory activity of malonyl-CoA is averted, hepatic fatty acid β-oxidation is increased and triacylglycerol accumulation is prevented (Akkaoui et al., 2009). Within cardiac tissues, when the sensitivity of CPT1B toward malonyl-CoA is reduced, cardiac fatty acid utilization is increased (van Weeghel et al., 2018). Together, the interplay between malonyl-CoA and CPT1 is crucial for regulating long-chain fatty acid oxidation rates in liver, heart, and muscle tissues. Missense mutations may result in a deficiency of CPT1A that leads to partial or even complete loss of enzymatic activity (Gobin et al., 2003). This deficiency is characterized by life-threatening hypoketotic hypoglycemia and fasting intolerance (Bennett et al., 2004; Stoler et al., 2004).

The brain isoform of rat CPT1C has a 2-fold lower K_m than CPT1A for carnitine and a 5-fold higher K_m for palmitoyl-CoA, but its catalytic activity is up to 320-fold lower (Sierra et al., 2008). Although the exact role of CPT1C in brain tissue remains to be elucidated, recent studies have demonstrated that CPT1C and the availability of malonyl-CoA are required for proper axon growth by regulating anterograde transport of late endosomes and lysosomes (Palomo-Guerrero et al., 2019). Moreover, CPT1C is involved in the regulation of ceramide metabolism. Thus, the overexpression of this enzyme results in increased ceramide levels. Furthermore, it has been shown that a deficit of CPT1C diminishes ceramide levels and results in altered dendritic spine morphology and impaired spatial learning in mice (Carrasco et al., 2012). Overall, CPT1 enzymes are key regulators of long-chain acylcarnitine availability and thus have served as the primary target for altering long-chain acylcarnitine levels. Strategies to treat these CPT1-related conditions include increasing malonyl-CoA levels via inhibition of MCD (Fillmore and Lopaschuk, 2014) using CPT1 inhibitors, which have been proven effective in treating ischemic heart disease in an in vivo pig model (Cheng et al., 2006). Direct inhibition of CPT1 with pharmacological agents such as etomoxir (etomoxiryl-CoA) (Lopaschuk et al., 1988) or decreasing the availability of substrate (carnitine) by using OCTN2 inhibitors such as meldonium or methyl-GBB (Liepinsh et al., 2015) have also been explored as cardioprotective strategies in rat (in vivo) and isolated heart ischemia-reperfusion injury models.

CPT2 is another isoform of carnitine palmitoyltransferase that is located on the matrix side of the inner mitochondrial membrane and is tasked with converting long-chain acylcarnitines back to the respective long-chain acyl-CoAs. CPT2 is not sensitive to malonyl-CoA and catalyzes the transfer of the acyl group of long-chain acylcarnitines onto CoA. Data obtained from rat liver mitochondria suggest that CPT2 aids in the export of long-chain acyl-CoA esters from the mitochondria (Ventura et al., 1998) and can synthesize C8–C20 acylcarnitines (Fig. 4). On the other hand, human CPT2 has virtually no activity in handling shorter acyl-CoAs or very long-chain acyl-CoAs or acyl-CoAs derived from branched-chain amino acid metabolism (Violante et al., 2010). Although this activity could potentially remove toxic long-chain acyl-CoA esters from the mitochondrial matrix, it could also lead to the accumulation of long-chain acylcarnitines in the mitochondrial intermembrane space.

4. Acylcarnitine Transporters

The transporter CACT catalyzes the equimolar exchange of long-chain acylcarnitines for carnitine to ensure long-chain acylcarnitine transfer across the inner mitochondrial membrane (Murthy and Pande, 1984) via a ping-pong mechanism with one binding site alternately exposed to each side of the membrane. Moreover, carnitine uniport is also possible (Palmieri, 1994). Another transporter, human OCTN2 or SLC22A5, is involved not only in the transport of carnitine but also in the transport of acetylcarnitine (Wu et al., 1999). In rodents, OCTN2 also ensures the transport of acetylcarnitine across the blood-brain barrier (Kido et al., 2001). More recently, in a study using a mouse acylcarnitine transporter, it was shown that organic cation transporter 1 (OCT1, SLC22A1) is involved in the hepatic efflux of short-chain (C2–C6) acylcarnitines (Fig. 4) and participates in the regulation of plasma short-chain acylcarnitine levels (Kim et al., 2017). It remains to be determined which transport proteins (Table 8), if any, are involved in the efflux/transport of medium- and long-chain acylcarnitines.

1. Acylcarnitines for the Diagnosis of Rare Inherited Diseases and Conditions

Inherited fatty acid oxidation disorders (FAODs) may be diagnosed if any part of the mitochondrial fatty acid transport, or the short-, medium- or long-chain fatty acid β-oxidation pathways have been adversely affected. The FAODs related to the transport of long-chain acylcarnitines across the mitochondrial inner membrane (CACT deficiency) and conversion of acylcarnitines back to the respective acyl-CoAs (CPT2 deficiency) have been previously described (Houten et al., 2016; Vishwanath, 2016; Marsden et al., 2021). Defects in enzymes involved in mitochondrial β-oxidation such as very long-chain acyl-CoA dehydrogenase (VLCAD), long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), and mitochondrial trifunctional protein deficiency are often characterized by elevated long- and very long-chain acylcarnitine levels. These conditions have been extensively reviewed before, and readers are referred to the papers of Houten et al. (2016), Vishwanath (2016), and Marsden et al. (2021) for an in-depth understanding.

CACT-mediated transport is an essential step in the long-chain fatty acid β-oxidation pathway. Carnitine-acylcarnitine translocase deficiency is a rare autosomal recessive disease characterized by mutations in the CACT gene (SLC25A20). This condition may result in hypoketotic hypoglycemia, elevated creatine kinase and transaminases, dicarboxylic aciduria, very low free carnitine, and marked elevation of long-chain acylcarnitines (Rubio-Gozalbo et al., 2004). This can lead to cardiac complications (cardiac arrhythmia, cardiomyopathy, and heart block), muscle weakness, and seizures (Vitoria et al., 2015). Another condition called CPT2 deficiency is the most common of the rare disorders related to the metabolism of long-chain fatty acid oxidation. Particularly rare are lethal neonatal and severe infantile forms of CPT2 deficiency. On the other hand, the more common and milder version of this disease is the muscle form of CPT2 deficiency (Joshi and Zierz, 2020). It should be noted that severe carnitine deficiency and increased long-chain acylcarnitine levels are characteristic of this disorder. Long- and medium-chain acylcarnitine levels are used for the diagnosis of not only CACT disorders but also CPT2 and several β-oxidation enzyme deficiencies (Fig. 6 and Table 3).

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Fig. 6

Enzymes and transporters related to the development of FAOD and the accumulation of acylcarnitines. Red arrows indicate reverse flux of acyl moieties and efflux of acylcarnitines. The main enzymes and transporters related to FAOD are marked in blue. ACAD9, acyl-CoA dehydrogenase family member 9; ECH, enoyl-CoA hydratase; ETFQO, electron transfer flavoprotein-ubiquinone oxidoreductase; LCKAT/MCKAT, long-/medium-chain 3-ketothiolase, MCHAD/SCHAD, medium-/short-chain 3-hydroxyacyl-CoA dehydrogenase.

2. Long-Chain Acylcarnitine Measurements for the Diagnosis of Insulin Resistance

Although acylcarnitines have long been used to diagnose inherited metabolic disorders, they are also being studied for the diagnosis of acquired metabolic disorders such as diabetes and insulin resistance. Currently, glucose and glycated hemoglobin A1c are the main diagnostic markers of type 2 diabetes used in clinical practice (Inzucchi et al., 2015; American Diabetes Association, 2017). Recently, assessments of triglycerides and total cholesterol have been included as markers of diabetes progression and therapeutic efficacy (American Diabetes Association, 2017). Other circulating lipid metabolites have not yet been accepted into clinical practice as markers of insulin resistance. Given the role of insulin in the suppression of lipid metabolism, it would be reasonable to evaluate insulin sensitivity by measuring the levels of fatty acid metabolism intermediates. In this regard, long-chain acylcarnitines appear to be promising biomarkers.

Energy metabolism is significantly changed during fed and fasted cycles, as it depends on the availability of energy substrates in the circulation. The utilization of fatty acids dominates in a fasted state, whereas insulin-sensitive tissues switch to carbohydrate metabolism in a fed state in response to the increased availability of glucose. Long-chain acylcarnitines are produced from long-chain fatty acids in skeletal muscles and the heart. However, only the heart is thought to be the main contributor to the plasma long-chain acylcarnitine pool (Koves et al., 2008; Makrecka-Kuka et al., 2017). Activation of insulin signaling decreases CPT1 activity and thus reduces the long-chain acylcarnitine content in muscle and cardiac tissues (Mihalik et al., 2010). The inability to reduce fatty acid metabolism in a fed state is a metabolic inflexibility caused by insulin resistance (Goodpaster and Sparks, 2017; Smith et al., 2018). The plasma concentrations of long-chain acylcarnitines reflect the changes in acylcarnitine tissue contents in the fed and fasted states and thus can serve as a valuable marker of tissue-specific insulin sensitivity (Makarova et al., 2019). Importantly, insulin levels in a fasted state are substantially lower than those in a fed state. Therefore, the measurement of plasma acylcarnitine concentrations in the fasted state does not characterize insulin sensitivity. Accordingly, high concentrations of long-chain acylcarnitines in the postprandial or fed state are markers of insulin resistance. This is a result of insulin’s inability to inhibit CPT1-dependent fatty acid metabolism in the heart (Ngu et al., 2008; Ramos-Roman et al., 2012). In patients with type 2 diabetes, insulin resistance usually presents in the heart and skeletal muscle concurrently. Thus, long-chain acylcarnitine measurements might be useful for the diagnosis of insulin resistance in skeletal muscles (Paternostro et al., 1996; Luong et al., 2021).

In the clinical setting, the postprandial state is not well defined. Therefore, the oral glucose tolerance test (GTT) or a controlled high carbohydrate-containing meal is better suited for diagnosing diabetes, prediabetes, or insulin sensitivity. The GTT is widely accepted as a diagnostic method, and measurements of acylcarnitines before and after glucose administration are feasible in clinical situations (Jagannathan et al., 2020). GTT measurements characterize either mitochondrial energy status in the fasted state or in the insulin-mediated transition from a fasted to a postprandial state. The concept that insulin-resistant subjects are not able to adequately decrease long-chain acylcarnitine plasma concentrations during an insulin clamp or a standard meal has been validated in several clinical studies (Mihalik et al., 2010; Bouchouirab et al., 2018). Thus, measurements of plasma long-chain acylcarnitine levels during GTT and insulin clamp tests are suitable for evaluating insulin sensitivity in both muscle tissues and the heart.

3. Incomplete Long-Chain Acylcarnitine Metabolism as a Marker of Heart Failure and a Predictor of Major Cardiovascular Events

Oxidative phosphorylation (OXPHOS) in the mitochondria is the central energy production pathway of the heart. As a result, mitochondrial dysfunction has been considered to be an important reason for contractile failure in the heart. Long-chain fatty acids are the main energy substrates for oxidative metabolism in the heart, and fatty acid metabolism is primarily affected when there is reduced mitochondrial OXPHOS capacity. Insufficient fatty acid metabolism in the mitochondria results in a decreased ATP generation rate and persistent energy deficiency. AMPK can sense an increased AMP/ATP ratio and stimulate CPT1 activity. Since acylcarnitine accumulation does not induce any feedback inhibition of CPT1, acylcarnitine levels can increase significantly over time as the pathology progresses. During the progression of heart failure, cardiac metabolism switches to other available energy substrates, but these derangements do not affect acylcarnitine production. Heart tissues are suggested to be the main contributors to the plasma long-chain acylcarnitine pool (Makrecka-Kuka et al., 2017); therefore, long-chain acylcarnitine measurements in plasma are particularly suitable for the diagnosis of cardiac diseases related to mitochondrial dysfunction.

During fasting, plasma concentrations of medium- and long-chain acylcarnitines are continuously increasing in tissues and plasma as a result of stimulated intracellular fatty acid metabolism, which is proportional to the duration of the fast (Elizondo et al., 2020; Hung et al., 2020). This is a result of the low concentrations of insulin in the circulation, the limited availability of glucose as a metabolic substrate, and the stimulation of intracellular fatty acid metabolism. Increased fatty acid availability and facilitated CPT1 activity are appropriate metabolic conditions for the evaluation of mitochondrial capacity to metabolize fatty acids. In situations where β-oxidation and OXPHOS capacity are not capable of utilizing high amounts of synthesized acylcarnitines, elevated levels of medium- and long-chain acylcarnitines will appear in the plasma (Adams et al., 2009; Elizondo et al., 2020). Overall, a fasted state (or being on a ketogenic diet) is the most appropriate condition for the diagnosis of mitochondrial dysfunction in the heart.

Several studies have measured increased levels of circulating long-chain acylcarnitines in patients with chronic heart failure (Ueland et al., 2013; Ahmad et al., 2016; Hunter et al., 2016; Ruiz et al., 2017). In all studies with heart failure patients, elevated circulating levels of long-chain acylcarnitines were independently associated with adverse clinical outcomes. In the clinical trial HF-ACTION, higher plasma concentrations of long-chain acylcarnitines were independently associated with lower peak Vo₂ as well as both the primary and secondary clinical endpoints of the parent trial (Ahmad et al., 2016). In particular, plasma levels of C16, C18:1, and C18:2 acylcarnitines were significantly higher in patients with end-stage heart failure. In patients who underwent left ventricular assist device placement, plasma levels of long-chain acylcarnitines were decreased. Increased long-chain acylcarnitine concentrations reflect dysfunctional fatty acid metabolism in mitochondria and probably in peroxisomes (Houten et al., 2016). This suggests an important mechanism contributing to global lipid perturbations in humans with heart failure. Overall, long-chain acylcarnitines appear to be promising biomarkers for diagnosis or therapeutic interventions in patients with heart failure.

Multiple metabolomic profiling studies have been performed to identify biomarkers that predict major cardiovascular events (Shah et al., 2012; Rizza et al., 2014). A metabolomic profiling study identified medium- and long-chain acylcarnitines as biomarkers that could predict major cardiovascular events in elderly people (Shah et al., 2012). In another study, aging, mitochondrial dysfunction, and increased levels of long-chain acylcarnitines (evaluated by metabolomic profiling) were associated with major cardiovascular events, independent of standard predictors (Shah et al., 2012). Therefore, higher levels of plasma long-chain acylcarnitines can be a predictor for an increased risk of major cardiovascular events.

1. Enzymes, Ion Channels, and (Signaling) Pathways Affected by Acylcarnitines

Certain pathologic states are linked to inappropriate regulation of long-chain acylcarnitine synthesis or incomplete metabolism (McCoin et al., 2015a). This leads to an accumulation of long-chain acylcarnitines that may interact with several cytosolic and mitochondrial enzymes, ion channels, and signaling pathways (Fig. 7). The long-chain acylcarnitine palmitoylcarnitine has been shown to decrease OXPHOS-dependent mitochondrial respiration with complex I/II and complex II substrates in a dose-dependent manner. It has also been shown to induce mitochondrial membrane hyperpolarization and the subsequent production of reactive oxygen species (ROS) (Liepinsh et al., 2016). Palmitoylcarnitine can also induce mitochondrial damage leading to apoptotic cell death via activation of the caspase pathway (Mutomba et al., 2000; McCoin et al., 2015b). At physiologically relevant concentrations, palmitoylcarnitine was able to significantly decrease mitochondrial pyruvate and lactate oxidation (Makrecka et al., 2014) and to impact cellular energy metabolism pathways through dephosphorylation of the insulin receptor and protein kinase B (Akt) (Vilks et al., 2021).

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Fig. 7

Enzymes, ion channels, and (signaling) pathways affected by long-chain acylcarnitines in myocytes. Arrows indicate enzymatic reaction or transport; T-shaped arrows indicate inhibition. FA, fatty acids; FATP1, long-chain fatty acid transport protein 1; IRS, insulin receptor substrate; LC, long chain; mt-β-ox, mitochondrial fatty acid β oxidation; PDC, pyruvate dehydrogenase complex; TCA cycle, tricarboxylic acid cycle.

The sodium-potassium exchanger [(Na⁺/K⁺)-ATPase, EC 7.2.2.13, multiple isoforms] is inhibited by long-chain acylcarnitines (Adams et al., 1979; Lamers et al., 1984; Mészàros and Pappano, 1990). Moreover, the activity of the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA, EC 7.2.2.10, multiple isoforms) is increased in the presence of long-chain acylcarnitine concentrations below 20 μM and decreased when long-chain acylcarnitine concentrations are above 20 μM. This means that long-chain acylcarnitines facilitate Ca²⁺ release from the sarcoplasmic reticulum (Adams et al., 1979; Dumonteil et al., 1994; Yamada et al., 2000; McCoin et al., 2015b). In addition, it has been shown that extracellular long-chain acylcarnitines interact with the extracellular site of the voltage-gated potassium channel ionic current carried by hERG channels (Iherg) (Kv11.1, KCNH2) to increase the I_hERG amplitude (Ferro et al., 2012). Although it is clear that long-chain acylcarnitines affect cardiac excitation-contraction coupling (Aitken-Buck et al., 2020), it remains to be determined whether they have a relevant pathologic meaning (e.g., leading to a proarrhythmic action). These effects are further discussed in the following sections as risk factors for cardiometabolic diseases.

2. Detrimental Effects of Acylcarnitines in Ischemic Heart Disease and Heart Failure

The high energy demand and limited glucose availability in the heart ensure that fatty acid metabolism is the predominant energy production mechanism for this organ (Stanley et al., 2005). In the postprandial and fasted states, 60%–95% of the heart’s ATP is produced from fatty acid oxidation, respectively (Liepinsh et al., 2014). The content of acylcarnitines fully depends on their production and utilization rates. Therefore, if stimulated fatty acid flux and accelerated synthesis of acylcarnitines are not fully coupled with oxidation, then acylcarnitines will accumulate in cardiac mitochondria and other intracellular compartments. Acylcarnitine accumulation to relatively high levels can be considered a normal physiologic response to fasting or starving. For example, during the fasted state, the acylcarnitine content in the heart can be elevated by 5-fold compared with the fed state (Makrecka et al., 2014). The content of acylcarnitines increases even further in the case of prolonged fasting or starvation (Soeters et al., 2009). In a healthy heart, an increase in long-chain acylcarnitines does not lead to mitochondrial or cardiac damage; however, in the fasted state, cardiac oxygen utilization efficiency is significantly reduced (Liepinsh et al., 2014; Makrecka-Kuka et al., 2020c). In patients, fasting- and starvation-induced accumulation of long-chain acylcarnitines might lead to more pronounced cardiac damage in cases of ischemia (Liepinsh et al., 2014). Likewise, in patients with insulin resistance, the increased risk of heart disease (Peters et al., 2015) might be linked to higher levels of long-chain acylcarnitines in the heart.

During ischemia, the low oxygen availability in the heart limits mitochondrial β-oxidation (Fillmore et al., 2014), and incompletely metabolized fatty acids, along with respective acyl-CoAs and acylcarnitines, start to accumulate. Energetic deficiencies in the ischemic heart stimulate a number of energy production pathways, including long-chain fatty acid uptake and the corresponding long-chain acylcarnitine synthesis in mitochondria by CPT1. Accordingly, high CPT1 (A and B isoforms) activity in cardiac tissues results in an increased long-chain acylcarnitine production rate (Liepinsh et al., 2016). At the same time, within the ischemic mitochondrial matrix, the long-chain acylcarnitine utilization rate is substantially reduced because of limited CPT2 activity and subsequent β-oxidation. For their proper functioning, CPT2 and β-oxidation enzymes need a sufficient amount of free CoA as a cofactor; however, during ischemia, the acyl-CoA/CoA ratio is significantly increased, leading to a deficiency of free CoA (Whitmer et al., 1978). Additionally, oxygen deficiency induces the accumulation of NADH and FADH₂, which leads to inhibition of β-oxidation enzymes (Neely and Feuvray, 1981; Jaswal et al., 2011). Altogether, long-chain acylcarnitines accumulate in ischemic mitochondria because of stimulated synthesis and a limited utilization rate.

Multiple studies have noted the accumulation of fatty acid metabolites in the infarcted heart (Idell-Wenger et al., 1978; Whitmer et al., 1978; Corr et al., 1984; Ford et al., 1996). Moreover, there is direct evidence that long-chain acylcarnitine accumulation reaches levels in cardiac mitochondria that induced detrimental effects in an isolated rat heart infarction study (Liepinsh et al., 2016). The mechanisms of mitochondrial dysfunction induced by long-chain acylcarnitines have been described in several studies (Siliprandi et al., 1992; Korge et al., 2003; Tominaga et al., 2008). The main detrimental effects of long-chain acylcarnitine in mitochondria are related to the inhibition of OXPHOS, further resulting in inhibited ATP synthase activity and increased ROS production (Tominaga et al., 2008; Dambrova et al., 2021). This is supported by the finding that the highest measured levels of long-chain acylcarnitines are bound to the mitochondrial membranes and localized in the intermembrane space (Liepinsh et al., 2016). In addition to the direct effects on mitochondrial OXPHOS, long-chain acylcarnitines inhibit pyruvate and lactate metabolism in cardiac mitochondria (Makrecka et al., 2014). Overall, long-chain acylcarnitine accumulation is very harmful to mitochondrial functioning and is likely a factor that contributes to the energetic crisis in the myocardium during ischemia-reperfusion.

The amphiphilic nature of long-chain acylcarnitines and their nonspecific interaction with membranes have been widely studied (McCoin et al., 2015a). However, the presumptive nonspecific actions ascribed to long-chain acylcarnitines are somewhat doubtful because fatty acid-binding proteins are present in various intracellular and extracellular compartments and protect cardiomyocytes and mitochondria against the amphiphilic effects of long-chain acylcarnitines (Liepinsh et al., 2016). However, for in vitro studies, the role of fatty acid-binding proteins as factors limiting long-chain acylcarnitine toxicity effects has to be taken into account. Fatty acid-binding proteins scavenge fatty acids and metabolic intermediates to reduce their free fraction and deliver intermediates only for specific reactions in the appropriate target proteins, thus preventing nonspecific toxic reactions (Glatz and vanderVusse, 1996; Neess et al., 2015). In the circulation, the free fraction of long-chain acylcarnitines is very low because they are tightly bound to serum albumin. In the cell cytosol, the high abundance of fatty acid-binding proteins could be responsible for the handling of fatty acids and long-chain acylcarnitines (Liepinsh et al., 2016). In myoglobin-rich tissues, long-chain (C12–C18) acylcarnitines can also bind to oxymyoglobin and participate in the regulation of long-chain acylcarnitine pools (Chintapalli et al., 2016; 2018). Currently, the protective effects of binding proteins have not been studied in detail, and such assumptions are based on indirect evidence. Depending on whether an individual is in the fed or fasted state, long-chain acylcarnitine concentrations in the heart vary between 10 and 50 μM. The effects of long-chain acylcarnitines on ion channels, insulin signaling enzymes, pyruvate metabolism, and OXPHOS in mitochondria are typically found to occur between 5 and 20 μM if tested in the absence of cytosol or serum albumin. The addition of cytosolic fractions significantly limits the detrimental action of long-chain acylcarnitines on mitochondrial functionality (Liepinsh et al., 2016), which suggests the presence of protective proteins in the cytosol. Importantly, if fatty acids and long-chain acylcarnitines compete for the same binding protein, an increased level of fatty acids during starving and ischemia results in an increased free fraction of long-chain acylcarnitines. Since deoxymyoglobin has limited acylcarnitine binding compared with oxymyoglobin, it has also been hypothesized that this could contribute to the higher free acylcarnitine levels during hypoxia (Chintapalli et al., 2016; 2018). Therefore, conditions with increased fatty acid flux and hypoxia can be harmful because of the higher possibility of nonspecific actions of long-chain acylcarnitines.

3. Proarrhythmic Effects of Acylcarnitines

Most sudden cardiac deaths (SCDs) are caused by arrhythmias, particularly ventricular fibrillation. The majority of cases of SCD occur in patients who do not have traditional risk factors for arrhythmia (Srinivasan and Schilling, 2018). During cardiac ischemia and reperfusion, the concentration of long-chain acylcarnitines is markedly elevated and therefore associated with life-threatening arrhythmias (Aitken-Buck et al., 2020). Long-chain acylcarnitines can interfere with excitation-contraction coupling, cause arrhythmias, and reduce the conductivity between cardiac cells; reviews are available in McCoin et al. (2015a) and Aitken-Buck et al. (2020). It has been demonstrated that attenuation of increasing long-chain acylcarnitine levels during hypoxia through CPT1 inhibition is able to protect against hypoxia-mediated alterations in cardiomyocyte action potentials (Knabb et al., 1986). Several mechanisms of long-chain acylcarnitine interference have been proposed, including direct channel interaction (Yamada et al., 1994), nonselective permeabilization of the sarcolemma (Liu et al., 1991), reduction in gap junction permeability, and interference with calcium release from the sarcoplasmic reticulum (Liu et al., 1991). Furthermore, an extracellular increase in long-chain acylcarnitine levels has been shown to induce arrhythmias at the single-cell level (Wu and Corr, 1992). It must be noted that all of these observed effects of long-chain acylcarnitines strongly depend on the employed concentrations. It is therefore not well defined which of the long-chain acylcarnitine effects achieved at certain concentrations in vitro and ex vivo can be attributed to effects during ischemia in vivo.

Despite the strong indications that long-chain acylcarnitines interfere with cellular electrophysiology and excitation-contraction coupling and even though long-chain acylcarnitines promote arrhythmias in animal models, the targeting of long-chain acylcarnitine concentrations as an antiarrhythmic treatment strategy has not been tested in the clinic. Taking into account that there are clinically approved compounds that can be used for reducing long-chain acylcarnitine concentrations in humans, it would be reasonable to explore whether these compounds could find a new use in a patient group at high risk of cardiac arrhythmia during ischemia.

4. Acylcarnitine Accumulation-Induced Complications in Rare Inherited Diseases and Conditions

Several inborn errors of metabolism involve fatty acid metabolism defects and the accumulation of acylcarnitines (McCoin et al., 2015a). Altered energy metabolism and marked accumulation of long-chain acylcarnitines in the blood and many tissues have been observed in a number of rare inborn disorders, such as CACT, CPT2, VLCAD, LCHAD, and mitochondrial trifunctional protein deficiency. The enzymes associated with these deficiencies are involved in the transport and metabolism of long-chain acylcarnitines and facilitate mitochondrial fatty acid β-oxidation. Long-chain acylcarnitine accumulation is not present in all FAODs; it is present only when there is an enzyme deficiency in the fatty acid mitochondrial metabolism pathway downstream of CPT1 (A and B isoforms).

The clinical phenotypes seen in FAODs are heterogeneous and of different severity even when the same fatty acid oxidation gene is deficient (Baruteau et al., 2013; Joshi et al., 2014). Considerable effort has been devoted to identifying discriminating biochemical factors determining the more severe phenotypes and to identifying pathologic mechanisms leading to disease complications. Energy deficiency in the heart and muscles is considered the main reason for acute and long-term complications of fatty acid oxidation deficiencies. Often, acute onset of the metabolic crisis and clinical symptoms appear when patients are in a fasted state or during exercise (Joshi et al., 2014). Thus, certain adaptation mechanisms can compensate for a single gene deficiency and exhibit a normal metabolic phenotype despite fatty acid metabolism having an essential role in energy production in the heart and skeletal muscle. To a large extent, compensation for deficiencies in fatty acid mitochondrial metabolism is possible because of the switch to carbohydrate oxidation, which replaces fatty acids as energetic substrates. Additionally, during carbohydrate intake, activation of insulin signaling limits long-chain acylcarnitine synthesis (Elizondo et al., 2020). Instead, during prolonged fasting and exercise, the limited availability of glucose cannot match the energy demand and fully compensate for fatty acid metabolism deficiency (Houten et al., 2016). During fasting and exercise, a marked increase in the content of long-chain acylcarnitines is a physiologic response, and cells and mitochondria can usually tolerate elevated levels of acylcarnitines (Xu et al., 2016). In patients with inherited deficiencies in mitochondrial fatty acid metabolism, increased production is not coupled with acylcarnitine metabolism, and long-chain acylcarnitines accumulate, often substantially exceeding tolerable baseline levels (Chace et al., 2001; McHugh et al., 2011). Since the high content of long-chain acylcarnitines induces harmful effects on the mitochondria and energy metabolism pathways, long-chain acylcarnitines are involved in the induction of an energetic crisis (McCoin et al., 2015a).

More than 50% of patients with these conditions exhibit cardiac complications, including cardiomyopathy, arrhythmias, and SCD (Bonnet et al., 1999). The same cardiovascular events are linked to excessive accumulation of acylcarnitines in experimental and clinical studies in subjects without an inherited deficiency. In patients with deficiencies of primary carnitine carriers, CPT1, and medium-chain acyl-CoA dehydrogenase, long-chain acylcarnitines do not accumulate and arrhythmias are not observed (Bonnet et al., 1999). In the postmortem blood specimen samples from deceased patients with mitochondrial fatty acid oxidation deficiencies, the level of long-chain acylcarnitines was up to 100 times greater than the levels observed in acquired cardiac disorders (Chace et al., 2001; McHugh et al., 2011). In comparison, during myocardial infarction, mitochondrial long-chain acylcarnitine levels are increased 2- to 4-fold (Liepinsh et al., 2016). Patients with heart failure were found to have 2-fold higher concentrations of circulating long-chain acylcarnitine than control subjects (Ruiz et al., 2017). This evidence confirms that the long-chain acylcarnitine accumulation has a distinct role in the pathologic mechanism of cardiovascular complications.

The main treatment currently available for FAODs is a dietary intervention to limit long-chain fatty acids and increase consumption of carbohydrates to ensure adequate energy production (Spiekerkoetter et al., 2009). This treatment strategy could also affect long-chain acylcarnitine production because of reduced long-chain fatty acid flux and activated insulin signaling. Additionally, these dietary interventions might be based on diet supplementation with medium-chain fatty acid triglycerides, which are known to improve cardiac parameters but do not reduce rhabdomyolytic crises in FAOD patients (Gillingham et al., 2017; Vockley et al., 2021). Fatty acids with an uneven number of carbons might be beneficial because they replenish the Krebs cycle.

A less studied compensatory mechanism is the redistribution of fatty acid metabolism from mitochondria to peroxisomes. Long- and very long-chain fatty acids can be metabolized in peroxisomes, and in case of limited mitochondrial metabolism, peroxisomal proliferation and fatty acid oxidation are stimulated via activation of the peroxisome proliferator-activated receptor gamma coactivator 1/peroxisome proliferator-activated receptor α (PGC1/PPAR-α) pathway (Liepinsh et al., 2013; Wicks et al., 2015). Peroxisomes shorten (very) long-chain fatty acids down to chain lengths C6, thereby generating acetyl-CoA and acetylcarnitine, which increases acetyl group availability for metabolism in the Krebs cycle. At the same time, this process reduces the mitochondrial load of long-chain acylcarnitines. Additionally, in patients with at least some residual fatty acid oxidation enzyme activity, stimulation of fatty acid metabolism by PPAR- agonists might be beneficial. Indeed, bezafibrate treatment in CPT2-deficient patients reduced the average C16+C18:1 level from 38.39 to 7.73 M with a concomitant decrease in creatine kinase levels (Yamada et al., 2018). However, PPAR-α agonists are not effective in all patients because stimulation of fatty acid flux via CPT1 might induce additional acylcarnitine accumulation, especially in patients with a lack of residual mitochondrial enzyme activity (Yamada et al., 2018).

Despite early diagnosis and dietary therapy, a significant number of patients with FAODs still develop symptoms, which underlines the need for additional individualized treatment strategies. Targeted long-chain acylcarnitine-lowering strategies are currently not used for the treatment of mitochondrial FAODs. Promising results have been obtained in a recent in vitro study using the drug etomoxir. The reduced long-chain acylcarnitine production by etomoxir rescues the proarrhythmia defects in human induced pluripotent stem cells derived from fibroblasts of patients with VLCAD deficiency (Knottnerus et al., 2020). In patients with blocked mitochondrial fatty acid oxidation, the reduction in long-chain acylcarnitine synthesis appears to ensure certain benefit. Novel diagnostic methods using detailed functional studies in fibroblasts would help in the design of individualized treatments for patients with mitochondrial fatty acid oxidation deficiencies.

5. Prodiabetic Effects of Acylcarnitines

Insulin resistance, which is the main feature of type 2 diabetes, involves inappropriate glucose and fatty acid metabolism. Disturbances in glucose metabolism can be induced by a high fatty acid load in the diet, which results in the accumulation of lipid metabolites. Among the fatty acid intermediates involved in the development of insulin resistance are long-chain acylcarnitines. However, this is not widely acknowledged (Schooneman et al., 2013; McCoin et al., 2015a). Generally, the increased levels of long-chain acylcarnitines have been viewed as a marker of incomplete mitochondrial metabolism of fatty acids that activate proinflammatory pathways implicated in insulin resistance (Adams et al., 2009). However, because long-chain acylcarnitine-induced effects on insulin signaling were previously demonstrated (Aguer et al., 2015; Liepinsh et al., 2017; Blackburn et al., 2020; Vilks et al., 2021), an increased intracellular content of long-chain acylcarnitines results in feedback inhibition of insulin action (Fig. 7). Thus, insulin- and AMPK-mediated regulation of CPT1 activity has significant physiological impacts, and long-chain acylcarnitines are beginning to emerge as important metabolites involved in the regulation of energy metabolism. Long-chain acylcarnitines are also very active intermediates and effectively inhibit pyruvate and lactate oxidation in mitochondria. This compromises glucose metabolism in models of isolated rat cardiac mitochondria (Makrecka et al., 2014), cells in culture (Aguer et al., 2015), and ex vivo isolated rat hearts (Makrecka et al., 2014). It has been hypothesized that in the fasted state, long-chain acylcarnitines inhibit glucose uptake and metabolism to reduce the risk of hypoglycemia and generate energy from existing lipid stores (Liepinsh et al., 2017). Thus, long-chain acylcarnitines are important players in metabolism with important roles in the development of insulin resistance.

In several studies, it has been confirmed that long-chain acylcarnitines are not only markers for incomplete fatty acid oxidation but are also actively involved in the regulation of carbohydrate and lipid metabolism (Makrecka et al., 2014; Aguer et al., 2015; Liepinsh et al., 2017; Blackburn et al., 2020). The acute and chronic administration of palmitoylcarnitine in vivo inhibits the activation of insulin signaling and insulin-dependent glucose uptake in murine muscles (Liepinsh et al., 2017). Increased levels of long-chain acylcarnitines in muscle-specific CPT2 KO mice (SK^−/−) were shown to significantly reduce Akt Ser473 phosphorylation in liver and adipose tissue, whereas in muscles from high-fat diet (HFD)-fed mice, a marked decrease in Akt Ser473 phosphorylation in Sk^−/− mice compared with wild-type mice (Sk^+/+) was observed (Pereyra et al., 2020). Recently, it was discovered that long-chain acylcarnitines induce dephosphorylation of the insulin receptor through increased activity of protein tyrosine phosphatase 1B (Vilks et al., 2021). The same study reported that palmitoylcarnitine decreases Akt phosphorylation independent of the insulin receptor phosphorylation level. Thus, the mechanism behind long-chain acylcarnitine action in muscles appears to involve the activated dephosphorylation of the insulin receptor and Akt. Importantly, increased levels of insulin in vitro and in vivo can suppress the long-chain acylcarnitine effects on Akt phosphorylation (Liepinsh et al., 2017). This explains how, in the transition from a fasted to a fed state, increased levels of insulin overcome the effects of long-chain acylcarnitine and enable insulin signaling, which enhances glucose metabolism and inhibits fatty acid metabolism (Soeters et al., 2009; Consitt et al., 2016). In addition, to support the transition from fatty acid metabolism to glucose metabolism and to overcome transient insulin insensitivity in the muscles and heart, long-chain acylcarnitines appear to stimulate insulin release (Soni et al., 2014; Liepinsh et al., 2017) via suppression of Akt signaling in insulin-releasing cells (Vilks et al., 2021).

In healthy subjects, the increased concentration of insulin effectively inhibits long-chain acylcarnitine production via the increased tissue content of malonyl-CoA (Koves et al., 2008; Ussher et al., 2009). If insulin fails to inhibit long-chain acylcarnitine production in the fed state, it results in disturbances in glucose uptake and oxidation. In the early stage of insulin resistance, elevated levels of insulin can compensate for insulin resistance and thereby overcome the long-chain acylcarnitine-induced effects. In the later stages of insulin resistance, the inability of insulin to inhibit long-chain acylcarnitine production is accompanied by an increased concentration of long-chain acylcarnitines, which further stimulates the progression of glucose intolerance. Thus, the increased tissue content of long-chain acylcarnitines accelerates the progression of insulin resistance.

6. Acylcarnitine Effects on Inflammation

Inflammation is one of the key pathologies for many diseases and disorders, including insulin resistance, cardiomyopathy, organ rejection, and central nervous system (CNS)-related disorders. It has been previously demonstrated that when present in sufficiently high concentrations, palmitoylcarnitine (≥10–25 μM) promotes inflammation and oxidative stress in C2C12 myotubes and murine monocytes (Adams et al., 2009; Aguer et al., 2015; McCoin et al., 2015b). More recently, it was shown that the loss of CPT2 and subsequent endogenous accumulation of long-chain acylcarnitines does not affect palmitate-induced inflammation in C2C12 myotubes (Blackburn et al., 2020), suggesting that skeletal muscle cell inflammation cannot be attributed to the accumulation of long-chain acylcarnitine alone. However, the inhibition of CPT2 in vivo by aminocarnitine exacerbates inflammation in cardiac tissues in classic models of lipopolysaccharide-induced endotoxemia in mice (Makrecka-Kuka et al., 2020b). This suggests that further upregulation of inflammation in the case of endotoxemia or sepsis could be related to the accumulation of long-chain acylcarnitines.

In murine monocyte/macrophage cell lines, acylcarnitines activate proinflammatory signaling pathways in an acyl-chain length (C10–C18)-dependent and a concentration-dependent (5–25 μM) manner (Adams et al., 2009; Rutkowsky et al., 2014). In addition, it has been suggested that the long-chain acylcarnitine-induced proinflammatory response is mediated not through the Toll-like receptor but downstream of the myeloid differentiation factor 88 (MyD88) component (Rutkowsky et al., 2014). In FAOD patients with increased levels of long-chain acylcarnitines several changes in inflammatory proteins were observed. These included modestly increased plasma concentrations of IFN-γ and IL-8 and a decreased concentration of IL-10, an inflammation-dampening cytokine (McCoin et al., 2019). These findings suggest that long-chain acylcarnitines could take part in the activation of inflammatory signaling. It has also been shown that an excess of long-chain acylcarnitines, in combination with insufficient β-oxidation, promotes Th17 inflammation in patients with type 2 diabetes (Nicholas et al., 2019). Overall, these data suggest that the accumulation of long-chain acylcarnitines induces inflammation, thus potentiating the progression of diseases such as diabetes. Likewise, a drug-induced reduction in acylcarnitine availability has been shown to decrease the accumulation of macrophages and monocytes in atherosclerotic lesions. This also decreases the level of circulating inflammatory cytokines in an experimental model of atherosclerosis (Vilskersts et al., 2015). Moreover, enforced expression of CPT1A and increased fatty acid oxidation in murine macrophage cell lines have been shown to result in attenuated proinflammatory processes (Malandrino et al., 2015). This is likely related to the reduced accumulation of acylcarnitines in cells. Taken together, these findings suggest that the enzymes and proteins involved in long-chain acylcarnitine transport and production could serve as potential drug targets to attenuate inflammatory processes in chronic disorders.

Taking into account the growing significance of immunometabolism, it is intriguing to consider the role of acylcarnitines in the immune cell response. It should be noted that to date, studies on immune cells have mainly been focused on the investigation of fatty acid oxidation per se but not on the possible role of fatty acid metabolites, such as acylcarnitines, in immune cell functioning. Historically, it has been suggested that fatty acid oxidation is necessary for macrophage anti-inflammatory (M2) phenotype regulation (Vats et al., 2006). Follow up studies in primary rodent cells and human T cells and cultured human cells using etomoxir, an irreversible CPT1A inhibitor, demonstrated interesting off-target effects (Divakaruni et al., 2018; Raud et al., 2018) and brought into question whether fatty acid oxidation is essential for human macrophage M2 polarization (Namgaladze and Brune, 2014). Moreover, later studies have shown that CPT2 deletion in bone marrow-derived macrophages derived from mice does not affect polarization toward an M2 (anti-inflammatory) phenotype (Nomura et al., 2016). Overall, the current thinking in the immunometabolism field suggests that the anti-inflammatory macrophage response is more robustly supported by mitochondrial OXPHOS than by fatty acid oxidation. Interestingly, none of the abovementioned studies measured acylcarnitine profiles. High concentrations of etomoxir are known to induce the accumulation of long-chain acylcarnitines in the rat myocardium (Lopaschuk et al., 1988), which could further inhibit mitochondrial OXPHOS (Liepinsh et al., 2016). Thus, it could be hypothesized that etomoxir-induced mitochondrial dysfunction in macrophages could be secondary to the accumulation of long-chain acylcarnitines and that the accumulation of fatty acid metabolites could attenuate macrophage polarization to the M2 phenotype. However, this idea needs to be tested.

Interestingly, higher concentrations of circulating long- and medium-chain acylcarnitines have been found in sepsis nonsurvivors (Langley et al., 2013; Puskarich et al., 2018). This suggests the possible role of acylcarnitines in immune cell proinflammatory activation. However, data on the roles of fatty acid oxidation and long-chain acylcarnitines in the proinflammatory response are lacking. Given that the accumulation of long-chain acylcarnitines inhibits pyruvate metabolism and phosphorylation of Akt in rodent mitochondria and murine myoblasts (Makrecka et al., 2014; Liepinsh et al., 2017), it might be suggested that the availability of acylcarnitines could determine the energy metabolism pattern in immune cells and thus direct immune cell activation toward a proinflammatory phenotype.

7. Possible Effects of Acylcarnitines in Neurodegenerative and Neuropsychiatric Disorders

Fatty acid oxidation is not the primary energy production pathway in the brain (Schonfeld and Reiser, 2013). However, the role of fatty acid oxidation and acylcarnitines, especially long- and medium-chain acylcarnitines, in the progression of CNS-related disorders is increasingly being studied. A growing number of studies have observed unique acylcarnitine signatures in patients with neurodegenerative and neuropsychiatric disorders. In clinical studies, higher levels of circulating long- and medium-chain acylcarnitines have been found in patients with Alzheimer’s disease (van der Velpen et al., 2019). Moreover, higher levels of long-chain acylcarnitines are associated with disease progression (Chatterjee et al., 2021) and with cognitive impairment (Toledo et al., 2017). However, in larger cohorts, it has been shown that the higher serum levels of medium- and long-chain acylcarnitines actually predicted a lower risk of Alzheimer’s disease incidence (Huo et al., 2020). Furthermore, clinical trials of patients with Parkinson’s disease have consistently shown that circulating levels of long-chain acylcarnitines are decreased (Saiki et al., 2017; Chang et al., 2018; Molsberry et al., 2020). In both schizophrenia and psychosis patients, the levels of C3-carnitine and palmitoylcarnitine in the circulation were decreased (Kriisa et al., 2017; Cao et al., 2019). The levels of individual long-chain acylcarnitines in those patients do not follow the same pattern. Thus, for patients with schizophrenia, the levels of the majority of long- and medium-chain acylcarnitines are decreased (Cao et al., 2019), whereas for patients with psychosis, an increase in long-chain acylcarnitines was found (Kriisa et al., 2017). Overall, the metabolomic analysis of acylcarnitines in patients with CNS-related disorders has consistently identified alterations in circulating acylcarnitines. However, it is not clear whether acylcarnitine levels in the circulation could correspond to acylcarnitine levels in brain tissues or if an altered acylcarnitine profile in the plasma indicates possible crosstalk in neurodegenerative and neuropsychiatric disorders.

Interestingly, although fatty acid oxidation is not the main energy source for neuronal tissues, key enzymes involved in acylcarnitine synthesis, specifically CPT1, ACC, and MCD, are highly expressed in the hypothalamus (Sorensen et al., 2002; Lopez et al., 2006; Santos et al., 2013; Jernberg et al., 2017). This result suggests a possible involvement of acylcarnitine availability in the hypothalamic regulation of energy homeostasis. Previous studies have shown that the inhibition or genetic ablation of the acylcarnitine synthesizing enzyme CPT1 induces a reduction in food intake (Obici et al., 2003; Wolfgang et al., 2006; 2008). This finding implies that acylcarnitines could be involved in the regulation of nutrient sensing. Overall, these data suggest that acylcarnitine availability or content in the brain could alter energy metabolism in peripheral tissues (e.g., muscle and liver) by regulating hypothalamic nutrient sensing. Thus, it is more likely that changes in the circulating acylcarnitine profile are not directly driven by changes in the acylcarnitine profile in neuronal tissues; rather, these changes represent energy metabolism alterations in peripheral tissues.

In brain tissues, the acylcarnitine synthesis and turnover rate is extremely low compared with other tissues (Schonfeld and Reiser, 2013). Thus, disturbances in acylcarnitine metabolism might induce changes in the neuronal acylcarnitine content/profile and subsequent alterations in signaling pathways only over the long term. In general, the pathogenesis of many CNS-related disorders involves mitochondrial dysfunction and inflammation (Lucas et al., 2006; Golpich et al., 2017; Skaper et al., 2018). It has been shown that the accumulation of long-chain acylcarnitines in mitochondria induces disturbances in OXPHOS, stimulates ROS production in the heart (Tominaga et al., 2008; Liepinsh et al., 2016), and potentially promotes inflammation (Rutkowsky et al., 2014; Makrecka-Kuka et al., 2020b). Similarly, in the brain, the long-term accumulation of long-chain acylcarnitines could promote or enhance mitochondrial dysfunction along with inflammation-related disturbances.