Insulin resistance occurs frequently in patients with chronic kidney disease. However, the mechanisms of insulin resistance associated with chronic kidney disease are unclear. It is known that an increase in the mitochondrial acetyl-CoA (AcCoA)/CoA ratio causes insulin resistance in skeletal muscle, and this ratio is regulated by carnitine acetyltransferase that exchanges acetyl moiety between CoA and carnitine. Because excess acetyl moiety of AcCoA is excreted in urine as acetylcarnitine, we hypothesized that retention of acetylcarnitine might be a cause of insulin resistance in chronic kidney disease patients. Serum acetylcarnitine concentrations were measured in chronic kidney disease patients, and were significantly increased with reduction of renal function. The effects of excess extracellular acetylcarnitine on insulin resistance were studied in cultured skeletal muscle cells (C2C12 and human myotubes), and insulin-dependent glucose uptake was significantly and dose-dependently inhibited by addition of acetylcarnitine. The added acetylcarnitine was converted to carnitine via reverse carnitine acetyltransferase reaction, and thus the AcCoA concentration and AcCoA/CoA ratio in mitochondria were significantly elevated. The results suggest that increased serum acetylcarnitine in CKD patients causes AcCoA accumulation in mitochondria by stimulating reverse carnitine acetyltransferase reaction, which leads to insulin resistance in skeletal muscle.

Fabry disease (FD) is a X-linked lysosomal storage disorder caused by deficient function of the alpha-galactosidase A (α-GalA) enzyme. α-GalA deficiency leads to multisystemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide (Gb3). A hallmark symptom of FD patients is neuropathic pain that appears in the early stage of the disease as a result of peripheral small fiber damage. Previous studies have shown that Acetyl-L-carnitine (ALC) has neuroprotective, neurotrophic, and analgesic activity in animal models of neuropathic pain. To study the action of ALC on neuropathic pain associated with FD, we treated α-GalA gene null mice (α-GalA(-/0)) with ALC for 30 days. In α-Gal KO mice, ALC treatment induced acute and long-lasting analgesia, which persisted 1 month after drug withdrawal. This effect was antagonized by single administration of LY341495, an orthosteric antagonist of mGlu2/3 metabotropic glutamate receptors. We also found an up-regulation of mGlu2 receptors in cultured DRG neurons isolated from 30-day ALC-treated α-GalA KO mice. However, the up-regulation of mGlu2 receptors was no longer present in DRG neurons isolated 30 days after the end of treatment. Taken together, these findings suggest that ALC induces analgesia in an animal model of FD by up-regulating mGlu2 receptors, and that analgesia is maintained by additional mechanisms after ALC withdrawal. ALC might represent a valuable pharmacological strategy to reduce pain in FD patients.

Nicotinamide riboside (NR) is an NAD+ precursor that boosts cellular NAD+ concentrations. Preclinical studies have shown profound metabolic health effects after NR supplementation.

To cover increasing energy demands during exercise, tricarboxylic cycle (TCA) flux in skeletal muscle is markedly increased, resulting in the increased formation of intramyocellular acetylcarnitine (AcCtn). We hypothesized that reduced substrate availability within the exercising muscle, reflected by a diminished increase of intramyocellular AcCtn concentration during exercise, might be an underlying mechanism for the impaired exercise performance observed in adult patients with growth hormone deficiency (GHD). We aimed at assessing the effect of 2 hours of moderately intense exercise on intramyocellular AcCtn concentrations, measured by proton magnetic resonance spectroscopy (1H-MRS), in seven adults with GHD compared to seven matched control subjects (CS). Compared to baseline levels AcCtn concentrations significantly increased after 2 hours of exercise, and significantly decreased over the following 24 hours (ANOVA p for effect of time = 0.0023 for all study participants; p = 0.067 for GHD only, p = 0.045 for CS only). AcCtn concentrations at baseline, as well as changes in AcCtn concentrations over time were similar between GHD patients and CS (ANOVA p for group effect = 0.45). There was no interaction between group and time (p = 0.53). Our study suggests that during moderately intense exercise the availability of energy substrate within the exercising muscle is not significantly different in GHD patients compared to CS.

Maternal-fetal HIV-1 transmission can be prevented by administration of AZT, alone or in combination with other antiretroviral drugs to pregnant HIV-1-infected women and their newborns. In spite of the benefits deriving from this life-saving prophylactic therapy, there is still considerable uncertainty on the potential long-term adverse effects of antiretroviral drugs on exposed children. Clinical and experimental studies have consistently shown the occurrence of mitochondrial dysfunction and increased oxidative stress following prenatal treatment with antiretroviral drugs, and clinical evidence suggests that the developing brain is one of the targets of the toxic action of these compounds possibly resulting in behavioral problems. We intended to verify the effects on brain and behavior of mice exposed during gestation to AZT, the backbone of antiretroviral therapy during human pregnancy. We hypothesized that glutamate, a neurotransmitter involved in excitotoxicity and behavioral plasticity, could be one of the major actors in AZT-induced neurochemical and behavioral alterations. We also assessed the antioxidant and neuroprotective effect of L-acetylcarnitine, a compound that improves mitochondrial function and is successfully used to treat antiretroviral-induced polyneuropathy in HIV-1 patients. We found that transplacental exposure to AZT given per os to pregnant mice from day 10 of pregnancy to delivery impaired in the adult offspring spatial learning and memory, enhanced corticosterone release in response to acute stress, increased brain oxidative stress also at birth and markedly reduced expression of mGluR1 and mGluR5 subtypes and GluR1 subunit of AMPA receptors in the hippocampus. Notably, administration during the entire pregnancy of L-acetylcarnitine was effective in preventing/ameliorating the neurochemical, neuroendocrine and behavioral adverse effects induced by AZT in the offspring. The present preclinical findings provide a mechanistic hypothesis for the neurobehavioral effects of AZT and strongly suggest that preventive administration of L-acetylcarnitine might be effective in reducing the neurological side-effects of antiretroviral therapy in fetus/newborn.

Carnitine is well recognized as a key regulator of long-chain fatty acyl group translocation into the mitochondria. In addition, carnitine, as acetylcarnitine, acts as an acceptor of excess acetyl-CoA, a potent inhibitor of pyruvate dehydrogenase. Here, we provide a new methodology for accurate quantification of acetylcarnitine content and determination of its localization in skeletal muscles. We used matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) to visualize acetylcarnitine distribution in rat skeletal muscles. MALDI-IMS and immunohistochemistry of serial cross-sections showed that acetylcarnitine was enriched in the slow-type muscle fibers. The concentration of ATP was lower in muscle regions with abundant acetylcarnitine, suggesting a relationship between acetylcarnitine and metabolic activity. Using our novel method, we detected an increase in acetylcarnitine content after muscle contraction. Importantly, this increase was not detected using traditional biochemical assays of homogenized muscles. We also demonstrated that acetylation of carnitine during muscle contraction was concomitant with glycogen depletion. Our methodology would be useful for the quantification of acetylcarnitine and its contraction-induced kinetics in skeletal muscles.

Acetylcarnitine plays an important role in fat metabolism and can be detected in proton magnetic resonance spectra in skeletal muscle. An inverse relationship of acetylcarnitine to intramyocellular lipids and metabolic markers of chronic hyperglycemia has been suggested. This study aimed to compare the acetylcarnitine concentrations and intramyocellular lipids measured noninvasively by proton magnetic resonance spectroscopy (1H MRS) in the tibialis anterior and the soleus of three different groups of volunteers with a broad range of glycemic control.

The epigenetic agents, L-acetylcarnitine (LAC) and L-methylfolate (MF) are putative candidates as add-on drugs in depression. We evaluated the effect of a combined treatment with LAC and MF in two different paradigms of chronic stress in mice and in human inducible pluripotent stem cells (hiPSCs) differentiated into dopaminergic neurons. Two groups of mice were exposed to chronic unpredictable stress (CUS) for 28 days or chronic restraint stress (CRS) for 21 day, and LAC (30 or 100 mg/kg) and/or MF (0.75 or 3 mg/kg) were administered i.p. once a day for 14 days, starting from the last week of stress. In both stress paradigms, LAC and MF acted synergistically in reducing the immobility time in the forced swim test and enhancing BDNF protein levels in the frontal cortex and hippocampus. In addition, LAC and MF acted synergistically in enhancing type-2 metabotropic glutamate receptor (mGlu2) protein levels in the hippocampus of mice exposed to CRS. Interestingly, CRS mice treated with MF showed an up-regulation of NFκB p65, which is a substrate for LAC-induced acetylation. We could also demonstrate a synergism between LAC and MF in cultured hiPSCs differentiated into dopamine neurons, by measuring dendrite length and number, and area of the cell soma after 3 days of drug exposure. These findings support the combined use of LAC and MF in the treatment of MDD and other stress-related disorders.

Acetylcarnitine is an essential metabolite for maintaining metabolic flexibility and glucose homeostasis. The in vivo behavior of muscle acetylcarnitine content during exercise has not been shown with magnetic resonance spectroscopy. Therefore, this study aimed to explore the behavior of skeletal muscle acetylcarnitine during rest, plantar flexion exercise, and recovery in the human gastrocnemius muscle under aerobic conditions. Ten lean volunteers and nine overweight volunteers participated in the study. A 7 T whole-body MR system with a double-tuned surface coil was used to acquire spectra from the gastrocnemius medialis. An MR-compatible ergometer was used for the plantar flexion exercise. Semi-LASER-localized 1H MR spectra and slab-localized 31P MR spectra were acquired simultaneously in one interleaved exercise/recovery session. The time-resolved interleaved 1H/31P MRS acquisition yielded excellent data quality. A between-group difference in acetylcarnitine metabolism over time was detected. Significantly slower τPCr recovery, τPCr on-kinetics, and lower Qmax in the overweight group, compared to the lean group was found. Linear relations between τPCr on-kinetics, τPCr recovery, VO2max and acetylcarnitine content were identified. In conclusion, we are the first to show in vivo changes of skeletal muscle acetylcarnitine during acute exercise and immediate exercise recovery with a submaximal aerobic workload using interleaved 1H/31P MRS at 7 T.

Type 2 diabetes patients and individuals at risk of developing diabetes are characterized by metabolic inflexibility and disturbed glucose homeostasis. Low carnitine availability may contribute to metabolic inflexibility and impaired glucose tolerance. Here, we investigated whether carnitine supplementation improves metabolic flexibility and insulin sensitivity in impaired glucose tolerant (IGT) volunteers.

Background L-acetylcarnitine, a drug marketed for the treatment of chronic pain, causes analgesia by epigenetically up-regulating type-2 metabotropic glutamate (mGlu2) receptors in the spinal cord. Because the epigenetic mechanisms are typically long-lasting, we hypothesized that analgesia could outlast the duration of L-acetylcarnitine treatment in models of inflammatory and neuropathic pain. Results A seven-day treatment with L-acetylcarnitine (100 mg/kg, once a day, i.p.) produced an antiallodynic effect in the complete Freund adjuvant mouse model of chronic inflammatory pain. L-Acetylcarnitine-induced analgesia persisted for at least 14 days after drug withdrawal. In contrast, the analgesic effect of pregabalin, amitryptiline, ceftriaxone, and N-acetylcysteine disappeared seven days after drug withdrawal. L-acetylcarnitine treatment enhanced mGlu2/3 receptor protein levels in the dorsal region of the spinal cord. This effect also persisted for two weeks after drug withdrawal and was associated with increased levels of acetylated histone H3 bound to the Grm2 gene promoter in the dorsal root ganglia. A long-lasting analgesic effect of L-acetylcarnitine was also observed in mice subjected to chronic constriction injury of the sciatic nerve. In these animals, a 14-day treatment with pregabalin, amitryptiline, tramadol, or L-acetylcarnitine produced a significant antiallodynic effect, with pregabalin displaying the greatest efficacy. In mice treated with pregabalin, tramadol or L-acetylcarnitine the analgesic effect was still visible 15 days after the end of drug treatment. However, only in mice treated with L-acetylcarnitine analgesia persisted 37 days after drug withdrawal. This effect was associated with an increase in mGlu2/3 receptor protein levels in the dorsal horns of the spinal cord. Conclusions Our findings suggest that L-acetylcarnitine has the unique property to cause a long-lasting analgesic effect that might reduce relapses in patients suffering from chronic pain.

Intramyocellular acetylcarnitine (IMAC) is involved in exercise-related fuel metabolism. It is not known whether levels of systemic glucose influence IMAC levels in type 1 diabetes.

Biomarker identification is becoming increasingly important for the development of personalized or stratified therapies. Metabolomics yields biomarkers indicative of phenotype that can be used to characterize transitions between health and disease, disease progression and therapeutic responses. The desire to reproducibly detect ever greater numbers of metabolites at ever diminishing levels has naturally nurtured advances in best practice for sample procurement, storage and analysis. Reciprocally, since many of the available extensive clinical archives were established prior to the metabolomics era and were not processed in such an 'ideal' fashion, considerable scepticism has arisen as to their value for metabolomic analysis. Here we have challenged that paradigm.

Acylcarnitine metabolites have gained attention as biomarkers of nutrient stress, but their physiological relevance and metabolic purpose remain poorly understood. Short-chain carnitine conjugates, including acetylcarnitine, derive from their corresponding acyl-CoA precursors via the action of carnitine acetyltransferase (CrAT), a bidirectional mitochondrial matrix enzyme. We show here that contractile activity reverses acetylcarnitine flux in muscle, from net production and efflux at rest to net uptake and consumption during exercise. Disruption of this switch in mice with muscle-specific CrAT deficiency resulted in acetyl-CoA deficit, perturbed energy charge, and diminished exercise tolerance, whereas acetylcarnitine supplementation produced opposite outcomes in a CrAT-dependent manner. Likewise, in exercise-trained compared to untrained humans, post-exercise phosphocreatine recovery rates were positively associated with CrAT activity and coincided with dramatic shifts in muscle acetylcarnitine dynamics. These findings show acetylcarnitine serves as a critical acetyl buffer for working muscles and provide insight into potential therapeutic strategies for combatting exercise intolerance.

A new and fully automated newborn screening method for mass spectrometry was introduced in this paper. Pathological relevant amino acids, acylcarnitines, and certain steroids are detected within 4 min per sample. Each sample is treated in an automated and standardized workflow, where a mixture of deuterated internal standards is sprayed onto the sample before extraction. All compounds showed good linearity, and intra- and inter-day variation lies within the acceptance criteria (except for aspartic acid). The described workflow decreases analysis cost and labor while improving the sample traceability towards good laboratory practice.

In the metabolism of acetate several enzymes are involved, which play an important role in free fatty acid oxidation. Fatty acid metabolism is altered in diabetes patients and therefore acetate might serve as a marker for pathological changes in the fuel selection of cells, as these changes occur in diabetes patients. Acetylcarnitine is a metabolic product of acetate, which enables its transport into the mitochondria for energy production. This study investigates whether the ratio of acetylcarnitine to acetate, measured by noninvasive hyperpolarized [1-(13)C]acetate magnetic resonance spectroscopy, could serve as a marker for myocardial, hepatic, and renal metabolic changes in rats with Streptozotocin (STZ)-induced diabetes in vivo. We demonstrate that the conversion of acetate to acetylcarnitine could be detected and quantified in all three organs of interest. More interestingly, we found that the hyperpolarized acetylcarnitine to acetate ratio was independent of blood glucose levels and prolonged hyperglycemia following diabetes induction in a type-1 diabetes model.

Adverse drug reactions (ADRs) are considered an inherent risk of medication use, and some ADRs have been associated with off-target drug interactions with mitochondria. Metabolites that reflect mitochondrial function may help identify patients at risk of mitochondrial toxicity. We employed a database strategy to identify candidate mitochondrial metabolites that could be clinically useful to identify individuals at increased risk of mitochondrial-related ADRs. This led to L-carnitine being identified as the candidate mitochondrial metabolite. L-carnitine, its acetylated metabolite, acetylcarnitine and other acylcarnitines are mitochondrial biomarkers used to detect inborn errors of metabolism. We hypothesized that changes in L-carnitine disposition, induced by a "challenge test" of intravenous L-carnitine, could identify mitochondrial-related ADRs by provoking variation in L-carnitine and/or acetylcarnitine blood levels. To test this hypothesis, we induced mitochondrial drug toxicity with clofazimine (CFZ) in a mouse model. Following CFZ treatment, mice received an L-carnitine "challenge test". CFZ-induced changes in weight were consistent with previous work and reflect CFZ-induced catabolism. L-carnitine induced differences in whole blood acetylcarnitine concentrations in a manner that was dependent on CFZ treatment. This supports the usefulness of a database strategy for the discovery of candidate metabolite biomarkers of drug toxicity and substantiates the potential of the L-carnitine "challenge test" as a "probe" to identify drug-related toxicological manifestations.

Mitochondrial health declines with age, and older patients can demonstrate dysfunction in mitochondrial-rich tissues, such as cardiac and skeletal muscle. Aged mitochondria may make older adults more susceptible to adverse drug reactions (ADRs). We assessed mitochondrial metabolic function by measuring two metabolites, l-carnitine and acetylcarnitine, to determine their effectiveness as candidate clinical biomarkers for age-related, drug-induced alterations in mitochondrial metabolism. To study age- and medication-related changes in mitochondrial metabolism, we administered the FDA-approved mitochondriotropic drug, clofazimine (CFZ), or vehicle for 8 weeks to young (4-week-old) and old (61-week-old) male C57BL/6J mice. At the end of treatment, whole blood and cardiac and skeletal muscle were analyzed for l-carnitine, acetylcarnitine, and CFZ levels; muscle function was measured via a treadmill test. No differences were found in blood or cardiac carnitine levels of CFZ-treated mice, but CFZ-treated mice displayed lost body mass and alterations in endurance and levels of skeletal muscle mitochondrial metabolites. These findings demonstrate the age-related susceptibility of the skeletal muscle to mitochondria drug toxicity. Since drug-induced alterations in mitochondrial metabolism in skeletal muscle were not reflected in the blood by l-carnitine or acetylcarnitine levels, drug-induced catabolism and changes in muscle function appear more relevant to stratifying individuals at increased risk for ADRs.

Calorie restriction (CR), an age delaying diet, affects fat oxidation through poorly understood mechanisms. We investigated the effect of CR on fat metabolism gene expression and intermediate metabolites of fatty acid oxidation in the liver. We found that CR changed the liver acylcarnitine profile: acetylcarnitine, short-chain acylcarnitines, and long-chain 3-hydroxy-acylcarnitines increased, and several long-chain acylcarnitines decreased. Acetyl-CoA and short-chain acyl-CoAs were also increased in CR. CR did not affect the expression of CPT1 and upregulated the expression of long-chain and very-long-chain Acyl-CoA dehydrogenases (LCAD and VLCAD, respectively). The expression of downstream enzymes such as mitochondrial trifunctional protein and enzymes in medium- and short-chain acyl-CoAs oxidation was not affected in CR. CR shifted the balance of fatty acid oxidation enzymes and fatty acid metabolites in the liver. Acetyl-CoA generated through beta-oxidation can be used for ketogenesis or energy production. In agreement, blood ketone bodies increased under CR in a time of the day-dependent manner. Carnitine acetyltransferase (CrAT) is a bidirectional enzyme that interconverts short-chain acyl-CoAs and their corresponding acylcarnitines. CrAT expression was induced in CR liver supporting the increased acetylcarnitine and short-chain acylcarnitine production. Acetylcarnitine can freely travel between cellular sub-compartments. Supporting this CR increased protein acetylation in the mitochondria, cytoplasm, and nucleus. We hypothesize that changes in acyl-CoA and acylcarnitine levels help to control energy metabolism and contribute to metabolic flexibility under CR.

Ionizing radiation may cause cardiotoxicity not only at high, but even at low (considered as harmless) doses, yet the molecular mechanisms of the heart's response to low doses are not clear. In this work, we used high-resolution nuclear magnetic resonance (NMR) spectroscopy to detect the early and late effects of radiation on the metabolism of murine hearts. The hearts of C57Bl/6NCrl female mice were irradiated in vivo with single 0.2 Gy or 2 Gy doses using 6 MV photons, then tissues were collected 48 h and 20 weeks after exposure. The most distinct changes in the profile of polar metabolites were detected 48 h after irradiation with 2 Gy, and included increased levels of pantothenate and glutamate as well as decreased levels of alanine, malonate, acetylcarnitine, glycine and adenosine. Significant effects of the 2 Gy dose were also observed 20 weeks after irradiation and included decreased levels of glutamine and acetylcarnitine when compared with age-matched controls. Moreover, several differences were observed between hearts irradiated with 2 Gy and analyzed either 48 h or 20 weeks after the exposure, which included changes in levels of acetylcarnitine, alanine, glycine, glutamate, glutamine, formate, myo-inositol and trimethylamine. No statistically significant effects induced by the 0.2 Gy dose were observed 20 weeks after irradiation. In general, radiation-affected compounds were associated with energy metabolism, fatty acid beta-oxidation, oxidative stress and damage to cell structures. At the same time, radiation-related effects were not detected at the level of tissue histology, which indicated a higher sensitivity of metabolomics-based tests for cardiac tissue response to radiation.