Long-chain Acylcarnitine Content Research Articles

Description of the fasted serum metabolomic signature of lean and obese cats at maintenance and of obese cats under energy restriction.

This study aimed to investigate the serum metabolomic profile of obese and lean cats as well as obese cats before and after energy restriction for weight loss. Thirty cats, 16 obese (body condition score 8 to 9/9) and 14 lean (body condition score 4 to 5/9), were fed a veterinary weight loss food during a 4-week period of weight maintenance (L-MAINT and O-MAINT). The 16 obese cats were then energy restricted by a 60% energy intake reduction with the same food for a 10-week period (O-RESTRICT). Fasted serum metabolites were measured using nuclear magnetic resonance and direct infusion mass spectrometry after the maintenance period for L-MAINT and O-MAINT cats and after the energy restriction period for O-RESTRICT and compared between groups using a two-sided t-test. Obese cats lost 672 g ± 303 g over the 10-week restriction period, representing a weight loss rate of 0.94 ± 0.28% per week. Glycine, l-alanine, l-histidine, l-glutamine, 2-hydroxybutyrate, isobutryric acid, citric acid, creatine, and methanol were greater in O-RESTRICT compared to O-MAINT. There was a greater concentration of long-chain acylcarnitines in O-RESTRICT compared to both O-MAINT and L-MAINT, and greater total amino acids compared to O-MAINT. Glycerol and 3-hydroxybutyric acid were greater in O-MAINT compared to L-MAINT, as were several lysophosphatidylcholines. Thus, energy restriction resulted in increased dispensable amino acids in feline serum which could indicate alterations in amino acid partitioning. An increase in lipolysis was not evident, though greater circulating acylcarnitines were observed, suggesting that fatty acid oxidation rates may have been greater under calorie restriction. More research is needed to elucidate energy metabolism and substrate utilization, specifically fatty acid oxidation and methyl status, during energy restriction in strict carnivorous cats to optimize weight loss.

Decreased long-chain acylcarnitine content increases mitochondrial coupling efficiency and prevents ischemia-induced brain damage in rats

Long-chain acylcarnitines (LCACs) are intermediates of fatty acid oxidation and are known to exert detrimental effects on mitochondria. This study aimed to test whether lowering LCAC levels with the anti-ischemia compound 4-[ethyl(dimethyl)ammonio]butanoate (methyl-GBB) protects brain mitochondrial function and improves neurological outcomes after transient middle cerebral artery occlusion (MCAO). The effects of 14 days of pretreatment with methyl-GBB (5 mg/kg, p.o.) on brain acylcarnitine (short-, long- and medium-chain) concentrations and brain mitochondrial function were evaluated in Wistar rats. Additionally, the mitochondrial respiration and reactive oxygen species (ROS) production rates were determined using ex vivo high-resolution fluorespirometry under normal conditions, in models of ischemia-reperfusion injury (reverse electron transfer and anoxia-reoxygenation) and 24 h after MCAO. MCAO model rats underwent vibrissae-evoked forelimb-placing and limb-placing tests to assess neurological function. The infarct volume was measured on day 7 after MCAO using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Treatment with methyl-GBB significantly reduced the LCAC content in brain tissue, which decreased the ROS production rate without affecting the respiration rate, indicating an increase in mitochondrial coupling. Furthermore, methyl-GBB treatment protected brain mitochondria against anoxia–reoxygenation injury. In addition, treatment with methyl-GBB significantly reduced the infarct size and improved neurological outcomes after MCAO. Increased mitochondrial coupling efficiency may be the basis for the neuroprotective effects of methyl-GBB. This study provides evidence that maintaining brain energy metabolism by lowering the levels of LCACs protects against ischemia-induced brain damage in experimental stroke models.

Increased acylcarnitine ratio indices in newborn screening for carnitine-acylcarnitine translocase deficiency shows increased sensitivity and reduced false-positivity.

Carnitine-acylcarnitine translocase (CACT) deficiency is a rare autosomal recessive metabolic disorder of mitochondrial long-chain fatty acid oxidation. Newborn screening via tandem mass spectrometry (MS/MS) technology enables early diagnosis. However, previous analyses of MS/MS data of patients showed that some results were misdiagnosed because they did not show typical acylcarnitine profiles of CACT deficiency. This study aimed to identify additional indices to assist the diagnosis of CACT deficiency. To evaluate the acylcarnitine profile and the acylcarnitine ratios of individuals with CACT deficiency, the MS/MS data of 15 patients diagnosed via genetic testing were retrospectively analysed. The sensitivity and false-positive rates of primary acylcarnitine markers and ratio indices were validated using the data from 28,261 newborns and 53 false-positive cases. Additionally, the MS/MS data of 20 newborns carrying the c.199-10T>G mutation in SLC25A20 and 40 normal controls were compared to verify whether the carriers had abnormal acylcarnitine concentrations. The acylcarnitine profiles from 15 patients were classified into three categories using C12, C14, C16, C18, C16:1, C18:1, and C18:2 as the primary diagnostic markers. The first category represented a typical profile (P1-P6). The second category for patients P7 and P8 showed a significant decrease in the C0 level and a normal concentration of long-chain acylcarnitines. The third category for patients P9-P15 showed the presence of interfering acylcarnitines. The second and third categories may have been misdiagnosed. An acylcarnitine ratio analysis showed that C14/C3, C16/C2, C16/C3, C18/C3, C16:1/C3, and C16:1-OH/C3 were significantly increased in all 15 patients. The verification of 28,261 newborn screening results showed that the false-positive rate of ratios, except for (C16 + C18)/C0, was lower than that of acylcarnitine indices (0.02-0.08% vs. 0.16-0.88%). None of the single long-chain acylcarnitines could separate patients from the false-positive cases; however, all ratios produced good discrimination between the two groups. Based on the primary acylcarnitine markers alone, CACT deficiency can be misdiagnosed in newborn screening. The ratios of the primary markers (C16 + C18:1)/C2, C16/C2, C16:1/C3, and C16:1-OH/C3 can facilitate the diagnosis of CACT deficiency, thereby increasing sensitivity and reducing false-positivity.

Circulating long-chain acylcarnitine concentrations are not affected by exercise training in pregnant women with obesity.

The purpose of this study was to determine the effect of exercise during pregnancy in sedentary women with obesity on longitudinal changes in long-chain acylcarnitine (LC-AC) concentrations. We hypothesized that exercise training would significantly decrease circulating LC-ACs throughout gestation compared with a nonexercise control group. Pregnant women with obesity considered otherwise healthy [n = 80, means ± SD; body mass index (BMI): 36.9 ± 5.7 kg/m2] were randomized into an exercise (n = 40, aerobic/resistance 3 times/wk, ∼13th gestation week until birth) or a nonexercise control (n = 40) group. At gestation week 12.2 ± 0.5 and 36.0 ± 0.4, a submaximal exercise test was conducted, and indirect calorimetry was used to measure relative resting energy expenditure (REE), as well as respiratory exchange ratio (RER) at rest. Fasting blood samples were collected and analyzed for LC-AC concentrations. Fitness improved with prenatal exercise training; however, exercise training did not affect circulating LC-AC. When groups were collapsed, LC-ACs decreased during gestation (combined groups, P < 0.001), whereas REE (kcal/kg/day, P = 0.008) increased. However, average REE relative to fat-free mass (FFM) (kcal/kg FFM/day) and RER did not change. There was an inverse relationship between the change in RER and all LC-ACs (except C18:2) throughout gestation (C14: r = -0.26, P = 0.04; C16: r = -0.27, P = 0.03; C18:1: r = -0.28, P = 0.02). In summary, a moderate-intensity exercise intervention during pregnancy in women with obesity did not alter LC-ACs concentrations versus control, indicating that the balance between long-chain fatty acid availability and oxidation neither improved nor worsened with an exercise intervention.NEW & NOTEWORTHY This research showed that a moderate-intensity prenatal exercise program, consisting of aerobic and resistance training, did not negatively impact normal alterations in substrate supply and demand for the mother and the offspring throughout gestation. Findings provide support for metabolic safety of exercise during pregnancy.

Muscle metabolome and adipose tissue mRNA expression of lipid metabolism-related genes in over-conditioned dairy cows differing in serum-metabotype

Over-conditioned dairy cows, classified by body condition score (BCS) and backfat thickness (BFT) are less able to metabolically adapt to the rapidly increasing milk yield after parturition. Based on serum metabolome and cluster analyses, high BCS cows (HBCS) could be classified into metabotypes that are more similar to normal (NBCS) cows, i.e., HBCS predicted normal (HBCS-PN) than the HBCS predicted high (HBCS-PH) cows—similar to the concept of obese but metabolically healthy humans. Our objective was to compare muscle metabolome and mRNA abundance of genes related to lipogenesis and lipolysis in adipose tissue between HBCS-PH (n = 13), HBCS-PN (n = 6), and NBCS-PN (n = 15). Tail-head subcutaneous fat was biopsied on d −49, 3, 21, and 84 relative to parturition. Potential differences in the oxidative capacity of skeletal muscle were assessed by targeted metabolomics in M. semitendinosus from d 21. Besides characteristic changes with time, differences in the mRNA abundance were limited to lipogenesis-related genes on d −49 (HBCS-PH > HBCS-PN). The HBCS-PH had more than two-fold higher muscle concentrations of short (C2, C4-OH, C6-OH) and long-chain acylcarnitines (C16, C18, and C18:1) than HBCS-PN, indicating a greater oxidative capacity for fatty acids (and utilization of ketones) in muscle of HBCS-PN than HBCS-PH cows.

Electrophysiological Abnormalities in VLCAD Deficient hiPSC-Cardiomyocytes Do not Improve with Carnitine Supplementation.

Patients with a deficiency in very long-chain acyl-CoA dehydrogenase (VLCAD), an enzyme that is involved in the mitochondrial beta-oxidation of long-chain fatty acids, are at risk for developing cardiac arrhythmias. In human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), VLCAD deficiency (VLCADD) results in a series of abnormalities, including: 1) accumulation of long-chain acylcarnitines, 2) action potential shortening, 3) higher systolic and diastolic intracellular Ca2+ concentrations, and 4) development of delayed afterdepolarizations. In the fatty acid oxidation process, carnitine is required for bidirectional transport of acyl groups across the mitochondrial membrane. Supplementation has been suggested as potential therapeutic approach in VLCADD, but its benefits are debated. Here, we studied the effects of carnitine supplementation on the long-chain acylcarnitine levels and performed electrophysiological analyses in VLCADD patient-derived hiPSC-CMs with a ACADVL gene mutation (p.Val283Ala/p.Glu381del). Under standard culture conditions, VLCADD hiPSC-CMs showed high concentrations of long-chain acylcarnitines, short action potentials, and high delayed afterdepolarizations occurrence. Incubation of the hiPSC-CMs with 400 µM L-carnitine for 48 h led to increased long-chain acylcarnitine levels both in medium and cells. In addition, carnitine supplementation neither restored abnormal action potential parameters nor the increased occurrence of delayed afterdepolarizations in VLCADD hiPSC-CMs. We conclude that long-chain acylcarnitine accumulation and electrophysiological abnormalities in VLCADD hiPSC-CMs are not normalized by carnitine supplementation, indicating that this treatment is unlikely to be beneficial against cardiac arrhythmias in VLCADD patients.

Decreases in Circulating Concentrations of Long-Chain Acylcarnitines and Free Fatty Acids During the Glucose Tolerance Test Represent Tissue-Specific Insulin Sensitivity.

Background: Insulin plays a pivotal role in the regulation of both carbohydrate and lipid intermediate turnover and metabolism. In the transition from a fasted to fed state, insulin action inhibits lipolysis in adipocytes, and acylcarnitine synthesis in the muscles and heart. The aim of this study was to measure free fatty acid (FFA) and acylcarnitine levels during the glucose tolerance test as indicators of tissue-specific insulin resistance.Results: Insulin release in response to glucose administration decreased both FFA and long-chain acylcarnitine levels in plasma in healthy control animals by 30% (120 min). The glucose tolerance test and [3H]-deoxy-D-glucose uptake in tissues revealed that high fat diet-induced lipid overload in C57bl/6N mice evoked only adipose tissue insulin resistance, and plasma levels of FFAs did not decrease after glucose administration. In comparison, db/db mice developed type 2 diabetes with severely impaired insulin sensitivity and up to 70% lower glucose uptake in both adipose tissues and muscles (skeletal muscle and heart), and both plasma concentrations of FFAs and long-chain acylcarnitines did not decrease in response to glucose administration.Conclusions: These results link impaired adipose tissue insulin sensitivity with continuous FFA release in the transition from a fasted to postprandial state, while a blunted decrease in long-chain acylcarnitine levels is associated with muscle and heart insulin resistance.

Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency: a case report

Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) is an autosomal recessive mitochondrial fatty acid beta-oxidation disorder with variable presentation including lack of energy (lethargy), low blood sugar (hypoglycemia), weak muscle tone (hypotonia), hepatic steatosis, and hypocarnitinemia. In this report, we describe a 9-month-old male patient who suffered from recurrent hypoglycemia with hypoglycemic convulsions, vomiting, and neurological regression since the age of 4 months. The patient presented with hypotonia, motor delay, hepatomegaly, protein-energy malnutrition (BMI SDS — 2.8). Biochemical tests demonstrated hypoglycemia (2.5 mmol/l), elevated lactate, creatine phosphokinase, and aminotransferases. There were also increased concentrations of long-chain acylcarnitine and 3-hydroxyacylcarnitine as well as a dramatic decrease in the carnitine level. Digestive tract malformations, endocrinopathies, and degenerative diseases of the nervous system were excluded hydroxyacyl-CoA dehydrogenase (HADHA) gene)testing revealed a homozygous mutation p.Glu474Gln. This confirmed the diagnosis of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. Diet correction by adding medium chain triglycerides, compensation of carnitine deficiency, and symptomatic therapy made it possible to avoid fatal metabolic crises and manage neurological regression. Early detection, diagnosis, and treatment of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency may improve clinical outcomes.

Pharmacological decrease in long-chain acylcarnitine content is an effective strategy to treat cardiometabolic diseases: proof-of-concept studies

Pharmacological decrease in long-chain acylcarnitine content is an effective strategy to treat cardiometabolic diseases: proof-of-concept studies

Altered gene expression and metabolism in fetal umbilical cord mesenchymal stem cells correspond with differences in 5-month-old infant adiposity gain

The intrauterine period is a critical time wherein developmental exposure can influence risk for chronic disease including childhood obesity. Using umbilical cord-derived mesenchymal stem cells (uMSC) from offspring born to normal-weight and obese mothers, we tested the hypothesis that changes in infant body composition over the first 5 months of life correspond with differences in cellular metabolism and transcriptomic profiles at birth. Higher long-chain acylcarnitine concentrations, lipid transport gene expression, and indicators of oxidative stress in uMSC-adipocytes were related to higher adiposity at 5 months of age. In uMSC-myocytes, lower amino acid concentrations and global differential gene expression for myocyte growth, amino acid biosynthesis, and oxidative stress were related to lower infant percent fat-free mass at 5 months of age, particularly in offspring of obese mothers. This is the first evidence of human infant adipocyte- or myocyte-related alterations in cellular metabolic pathways that correspond with increased adiposity and lower fat-free mass in early infancy. These pathways might reflect the effects of an adverse maternal metabolic environment on the fetal metabolome and genome. Our findings suggest that programmed differences in infant stem cell metabolism correspond with differences in body composition in early life, a known contributor to obesity risk.

Abstract 2504: Melatonin decreases plasma arginine, its precursors and acylcarnitines in breast cancer xenograft model at specific time point during circadian rhythm

Abstract The circadian rhythm regulates the whole metabolism modulating the concentration of various circulating substances through the clear and dark variation. Melatonin is the main mediator of this flow and breast tumors development is linked to imbalance of this rhythm. This hormone has several anti-cancer effects extensively proved by our group. Currently metabolomics analysis have been used in cancer to provide a comprehensive profile of the metabolic changes. Important tumor features are regulated by amino acids likewise fatty acids metabolism. Therefore the comprehension of specific metabolic pathways modulated by melatonin is essencial to verify its useful as adjuvant treatment. The aim of this study was to evaluate the metabolic profile in breast cancer xenograft model treated with melatonin and according the circadian cycle. Were used 160 female Balb/c nude athimic mice divided into four groups: Tumor / Melatonin, Tumor / Vehicle, Non-tumor / Melatonin and Non-tumor / Vehicle. The tumor-bearing animals were developed by implantation of 3x106 triple negative human breast cancer cells (MDA-MB-231) in the mammary fat pad. All the animals received 100 ul of solution by intraperitoneal injection (IP) for five days a week during 21 days. The treated groups received 40 mg/kg of melatonina. Blood sample were collected every 3 hours during 24 hours (eight time points) and the plasma was extracted for analysis by liquid chromatography-mass spectrometry (LC-MS) using the Absolute IDQ p180 Kit (BioCrates). Orthogonal Partial Least Squares (OPLS) analysis showed evident separation of the animals metabolic profile not only because the circadian cycle, but also because the melatonin treatment. We found a significant increasing in arginine and precursors concentration in blood of the tumor-bearing animals specificaly at 6am and 9am. Beside these data we also found alterations in the acylcarnitines levels. The concentrations of medium and long-chain acylcarnitines increased significantly at 3pm. Tumorbearing animals which have been treated with melatonin showed reduced levels of these amino acids and acylcarnitines in blood achieving the same levels of non-tumour bearing animals. In summary ourt work reveals a strong relationship between the metabolic profile and the entire circadian variation as well as the exogenous melatonin can regulates the metabolism. This pre-clinical trial so far unpublished has shown an increase of arginine, its precursors and acylcarnitines at specific time points in the plasma of breast tumor-bearing animals. The treatment with melatonin is capable to deacrese the concentration of these amino acids and acylcarnitines achievning basal levels. Our results corroborate the literature which characterizes these metabolites as biomarkers for cancer and increasingly allow the use of melatonin as an adjuvant therapy for breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Debora Zuccari, Rubens Paula Junior, Nathália Martins Sonehara, Roger Chammas, Florence Raynaud. Melatonin decreases plasma arginine, its precursors and acylcarnitines in breast cancer xenograft model at specific time point during circadian rhythm [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2504. doi:10.1158/1538-7445.AM2017-2504

Plasma acylcarnitines are associated with pulmonary hypertension

Quantifying metabolic derangements in pulmonary hypertension (PH) by plasma metabolomics could identify biomarkers useful for diagnosis and treatment. The objective of this paper is to test the hypotheses that circulating metabolites are differentially expressed in PH patients compared with controls and among different hemodynamic subtypes of PH associated with left heart disease. We studied patients enrolled in the CATHGEN biorepository with PH (right heart catheterization mPAP ≥ 25 mmHg; n = 280). Of these, 133 met criteria for postcapillary PH, 82 for combined precapillary and postcapillary PH (CpcPH), and 65 for precapillary PH. Targeted profiling of 63 metabolites (acylcarnitines, amino acids, and ketones) was performed using tandem flow injection mass spectrometry. Multivariable linear regression was used to determine differences in metabolite factors derived from a principal components analysis between PH cases, PH subtypes, and non-PH controls. In adjusted models, the metabolite factor loaded with long-chain acylcarnitines was higher in all PH cases versus non-PH controls (P = 0.00008), but did not discriminate between CpcPH and postcapillary PH (P = 0.56). In analyses of subtypes, CpcPH patients had lower levels of factors loaded with urea cycle amino acids and short chain acylcarnitines as compared to controls (P = 0.002 and P = 0.01, respectively) and as compared to postcapillary PH (P = 0.04 and P = 0.02, respectively). Compared to controls, PH was strongly associated with greater concentrations of long-chain acylcarnitines. Postcapillary PH and CpcPH were weakly associated with distinct metabolomic profiles. These findings suggest the presence of unique metabolic abnormalities in subtypes of PH and may reflect underlying pathophysiology.

Long-chain acylcarnitines determine ischaemia/reperfusion-induced damage in heart mitochondria.

The accumulation of long-chain fatty acids (FAs) and their CoA and carnitine esters is observed in the ischaemic myocardium after acute ischaemia/reperfusion. The aim of the present study was to identify harmful FA intermediates and their detrimental mechanisms of action in mitochondria and the ischaemic myocardium. In the present study, we found that the long-chain acyl-CoA and acylcarnitine content is increased in mitochondria isolated from an ischaemic area of the myocardium. In analysing the FA derivative content, we discovered that long-chain acylcarnitines, but not acyl-CoAs, accumulate at concentrations that are harmful to mitochondria. Acylcarnitine accumulation in the mitochondrial intermembrane space is a result of increased carnitine palmitoyltransferase 1 (CPT1) and decreased carnitine palmitoyltransferase 2 (CPT2) activity in ischaemic myocardium and it leads to inhibition of oxidative phosphorylation, which in turn induces mitochondrial membrane hyperpolarization and stimulates the production of reactive oxygen species (ROS) in cardiac mitochondria. Thanks to protection mediated by acyl-CoA-binding protein (ACBP), the heart is much better guarded against the damaging effects of acyl-CoAs than against acylcarnitines. Supplementation of perfusion buffer with palmitoylcarnitine (PC) before occlusion resulted in a 2-fold increase in the acylcarnitine content of the heart and increased the infarct size (IS) by 33%. A pharmacologically induced decrease in the mitochondrial acylcarnitine content reduced the IS by 44%. Long-chain acylcarnitines are harmful FA intermediates, accumulating in ischaemic heart mitochondria and inducing inhibition of oxidative phosphorylation. Therefore, decreasing the acylcarnitine content via cardioprotective drugs may represent a novel treatment strategy.

Decreased acylcarnitine content improves insulin sensitivity in experimental mice models of insulin resistance

The important pathological consequences of insulin resistance arise from the detrimental effects of accumulated long-chain fatty acids and their respective acylcarnitines. The aim of this study was to test whether exercise combined with decreasing the content of long-chain acylcarnitines represents an effective strategy to improve insulin sensitivity in diabetes.We used a novel compound, 4-[ethyl(dimethyl)ammonio]butanoate (methyl-GBB), treatment and exercise to decrease acylcarnitine contents in the plasma and muscles in the insulin resistance models of high fat diet (HFD) fed C57BL/6 mice and db/db mice.The methyl-GBB treatment induced a substantial decrease in all acylcarnitine concentrations in both fed and fasted states as well as when it was combined with exercise. In the HFD fed mice methyl-GBB treatment improved both glucose and insulin tolerance. Methyl-GBB administration, exercise and the combination of both improved insulin sensitivity and reduced blood glucose levels in db/db mice. Methyl-GBB administration and the combination of the drug and exercise activated the PPARα/PGC1α signaling pathway and stimulated the corresponding target gene expression. Insulin insensitivity in db/db mice was not induced by significantly increased fatty acid metabolism, while increased insulin sensitivity by both treatments was not related to decreased fatty acid metabolism in muscles.The pharmacologically reduced long-chain acylcarnitine content represents an effective strategy to improve insulin sensitivity. The methyl-GBB treatment and lifestyle changes via increased physical activity for one hour a day have additive insulin sensitizing effects in db/db mice.

Acylcarnitines--old actors auditioning for new roles in metabolic physiology.

Perturbations in metabolic pathways can cause substantial increases in plasma and tissue concentrations of long-chain acylcarnitines (LCACs). For example, the levels of LCACs and other acylcarnitines rise in the blood and muscle during exercise, as changes in tissue pools of acyl-coenzyme A reflect accelerated fuel flux that is incompletely coupled to mitochondrial energy demand and capacity of the tricarboxylic acid cycle. This natural ebb and flow of acylcarnitine generation and accumulation contrasts with that of inherited fatty acid oxidation disorders (FAODs), cardiac ischaemia or type 2 diabetes mellitus. These conditions are characterized by very high (FAODs, ischaemia) or modestly increased (type 2 diabetes mellitus) tissue and blood levels of LCACs. Although specific plasma concentrations of LCACs and chain-lengths are widely used as diagnostic markers of FAODs, research into the potential effects of excessive LCAC accumulation or the roles of acylcarnitines as physiological modulators of cell metabolism is lacking. Nevertheless, a growing body of evidence has highlighted possible effects of LCACs on disparate aspects of pathophysiology, such as cardiac ischaemia outcomes, insulin sensitivity and inflammation. This Review, therefore, aims to provide a theoretical framework for the potential consequences of tissue build-up of LCACs among individuals with metabolic disorders.

Dual effects of fibroblast growth factor 21 on hepatic energy metabolism.

The aim of this study was to investigate the mechanisms by which fibroblast growth factor 21 (FGF21) affects hepatic integration of carbohydrate and fat metabolism in Siberian hamsters, a natural model of adiposity. Twelve aged matched adult male Siberian hamsters maintained in their long-day fat state since birth were randomly assigned to one of two treatment groups and were continuously infused with either vehicle (saline; n=6) or recombinant human FGF21 protein (1 mg/kg per day; n=6) for 14 days. FGF21 administration caused a 40% suppression (P<0.05) of hepatic pyruvate dehydrogenase complex (PDC), the rate-limiting step in glucose oxidation, a 34% decrease (P<0.05) in hepatic acetylcarnitine accumulation, an index of reduced PDC flux, a 35% increase (P<0.05) in long-chain acylcarnitine content (an index of flux through β-oxidation) and a 47% reduction (P<0.05) in hepatic lipid content. These effects were underpinned by increased protein abundance of PD kinase-4 (PDK4, a negative regulator of PDC), the phosphorylated (inhibited) form of acetyl-CoA carboxylase (ACC, a negative regulator of delivery of fatty acids into the mitochondria) and the transcriptional co-regulators of energy metabolism peroxisome proliferator activated receptor gamma co-activator alpha (PGC1α) and sirtuin-1. These findings provide novel mechanistic basis to support the notion that FGF21 exerts profound metabolic benefits in the liver by modulating nutrient flux through both carbohydrate (mediated by a PDK4-mediated suppression of PDC activity) and fat (mediated by deactivation of ACC) metabolism, and therefore may be an attractive target for protection from increased hepatic lipid content and insulin resistance that frequently accompany obesity and diabetes.

The brain acylcarnitine profile depends on nutritional state

In addition to glucose, also fatty acids are important as an energy source in the brain where about 20% of the energy is gained from mitochondrial oxidation of fatty acids. Fatty acids are transported into mitochondria in the form of acylcarnitine. It is known that acylcarnitine concentration in the heart and blood plasma varies depending on the nutritional state, fed or fasted. The aim of the present study was to compare the concentration of short-chain (C2-C5), medium-chain (C6-C12) and long-chain (C14-C18) acylcarnitines in fed and fasted states in rat brain structures: cerebellum, cortex and hypothalamus. The total concentration of acylcarnitines was not affected by nutrient state in none of brain structures studied, but we found that cortex contained less acylcarnitines than cerebellum and hypothalamus. The nutritional state did not affect the overall concentration of short-chain acylcarnitines in the brain structures. In contrary, the nutritional state significantly affected the concentration of total medium chain acylcarnitines in the cortex: 0.33 and 0.46nmol/g tissues in fed and fasted state, respectively. The highest concentration of medium-chain acylcarnitines was found in hypothalamus in fed and fasted state compared to cortex and cerebellum. The nutritional state significantly affected the concentration of total long-chain acylcarnitines in the cortex: 4.92 and 5.88nmol/g tissues in fed and fasted state, respectively. In fed state the highest concentrations of long-chain acylcarnitines was found in hypothalamus compared to cortex and cerebellum. The results demonstrate that the nutrition state affects brain acylcarnitine concentration in different brain structures, and cortex is the most affected brain structure. Further studies are needed to investigate the role of changes in acylcarnitine profile in the brain signalling pathways.

Long-chain acylcarnitines activate cell stress and myokine release in C2C12 myotubes: calcium-dependent and -independent effects.

Acylcarnitines, important lipid biomarkers reflective of acyl-CoA status, are metabolites that possess bioactive and inflammatory properties. This study examined the potential for long-chain acylcarnitines to activate cellular inflammatory, stress, and death pathways in a skeletal muscle model. Differentiated C2C12 myotubes treated with l-C14, C16, C18, and C18:1 carnitine displayed dose-dependent increases in IL-6 production with a concomitant rise in markers of cell permeability and death, which was not observed for shorter chain lengths. l-C16 carnitine, used as a representative long-chain acylcarnitine at initial extracellular concentrations ≥25 μM, increased IL-6 production 4.1-, 14.9-, and 31.4-fold over vehicle at 25, 50, and 100 μM. Additionally, l-C16 carnitine activated c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase between 2.5- and 11-fold and induced cell injury and death within 6 h with modest activation of the apoptotic caspase-3 protein. l-C16 carnitine rapidly increased intracellular calcium, most clearly by 10 μM, implicating calcium as a potential mechanism for some activities of long-chain acylcarnitines. The intracellular calcium chelator BAPTA-AM blunted l-C16 carnitine-mediated IL-6 production by >65%. However, BAPTA-AM did not attenuate cell permeability and death responses, indicating that these outcomes are calcium independent. The 16-carbon zwitterionic compound amidosulfobetaine-16 qualitatively mimicked the l-C16 carnitine-associated cell stress outcomes, suggesting that the effects of high experimental concentrations of long-chain acylcarnitines are through membrane disruption. Herein, a model is proposed in which acylcarnitine cell membrane interactions take place along a spectrum of cellular concentrations encountered in physiological-to-pathophysiological conditions, thus regulating function of membrane-based systems and impacting cell biology.

Inhibition of L-carnitine biosynthesis and transport by methyl-γ-butyrobetaine decreases fatty acid oxidation and protects against myocardial infarction.

The important pathological consequences of ischaemic heart disease arise from the detrimental effects of the accumulation of long-chain acylcarnitines in the case of acute ischaemia-reperfusion. The aim of this study is to test whether decreasing the L-carnitine content represents an effective strategy to decrease accumulation of long-chain acylcarnitines and to reduce fatty acid oxidation in order to protect the heart against acute ischaemia-reperfusion injury. In this study, we used a novel compound, 4-[ethyl(dimethyl)ammonio]butanoate (Methyl-GBB), which inhibits γ-butyrobetaine dioxygenase (IC₅₀ 3 μM) and organic cation transporter 2 (OCTN2, IC₅₀ 3 μM), and, in turn, decreases levels of L-carnitine and acylcarnitines in heart tissue. Methyl-GBB reduced both mitochondrial and peroxisomal palmitate oxidation rates by 44 and 53% respectively. In isolated hearts treated with Methyl-GBB, uptake and oxidation rates of labelled palmitate were decreased by 40%, while glucose oxidation was increased twofold. Methyl-GBB (5 or 20 mg·kg(-1)) decreased the infarct size by 45-48%. In vivo pretreatment with Methyl-GBB (20 mg·kg(-1)) attenuated the infarct size by 45% and improved 24 h survival of rats by 20-30%. Reduction of L-carnitine and long-chain acylcarnitine content by the inhibition of OCTN2 represents an effective strategy to protect the heart against ischaemia-reperfusion-induced damage. Methyl-GBB treatment exerted cardioprotective effects and increased survival by limiting long-chain fatty acid oxidation and facilitating glucose metabolism.

Fiber-type-specific sensitivities and phenotypic adaptations to dietary fat overload differentially impact fast- versus slow-twitch muscle contractile function in C57BL/6J mice

High-fat diets (HFDs) have been shown to interfere with skeletal muscle energy metabolism and cause peripheral insulin resistance. However, understanding of HFD impact on skeletal muscle primary function, i.e., contractile performance, is limited. Male C57BL/6J mice were fed HFD containing lard (HFL) or palm oil (HFP), or low-fat diet (LFD) for 5weeks. Fast-twitch (FT) extensor digitorum longus (EDL) and slow-twitch (ST) soleus muscles were characterized with respect to contractile function and selected biochemical features. In FT EDL muscle, a 30%–50% increase in fatty acid (FA) content and doubling of long-chain acylcarnitine (C14–C18) content in response to HFL and HFP feeding were accompanied by increase in protein levels of peroxisome proliferator-activated receptor-γ coactivator-1α, mitochondrial oxidative phosphorylation complexes and acyl-CoA dehydrogenases involved in mitochondrial FA β-oxidation. Peak force of FT EDL twitch and tetanic contractions was unaltered, but the relaxation time (RT) of twitch contractions was 30% slower compared to LFD controls. The latter was caused by accumulation of lipid intermediates rather than changes in the expression levels of proteins involved in calcium handling. In ST soleus muscle, no evidence for lipid overload was found in any HFD group. However, particularly in HFP group, the peak force of twitch and tetanic contractions was reduced, but RT was faster than LFD controls. The latter was associated with a fast-to-slow shift in troponin T isoform expression. Taken together, these data highlight fiber-type-specific sensitivities and phenotypic adaptations to dietary lipid overload that differentially impact fast- versus slow-twitch skeletal muscle contractile function.