Acetylcarnitine Concentrations Research Articles

Correlation between skeletal muscle acetylcarnitine and phosphocreatine metabolism during submaximal exercise and recovery: interleaved 1H/31P MRS 7 T study

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.

Database screening as a strategy to identify endogenous candidate metabolites to probe and assess mitochondrial drug toxicity

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.

Evaluation of free and bound carnitine in young athletes

The study of the characteristics of carnitine metabolism in representatives of certain sports makes it possible to study the effect of physical loads of various nature on the body of a young athlete, and also makes it possible to assess the competitive potential and predict the tolerance of long-term and intense loads. Purpose. This study is to investigate the characteristics of carnitine metabolism in young athletes of various specializations. Material and methods. The study included athletes aged 14 to 18 who had been engaged in intense physical exercise for at least 12 hours per week forthe past 12 months or more. The control group consisted of healthy children aged 14 to 18 who were not involved in sports. In the blood serum of all study participants, the concentration of free carnitine, acetylcarnitine and acylcarnitines was determined. Based on the data obtained, ratios were calculated reflecting the activity of lipid beta-oxidation enzymes. Results. We found that the content of free carnitine in the representatives of all the studied groups was within the normal range. At the same time, the level of free carnitine in the blood of athletes involved in field hockey was significantly lower than that of athletes-swimmers and representatives of the control group (p<0.001). It was also studied that, compared with athletes-swimmers and the control group, athletes-hockey players have significantly higher functional activity of carnitine palmitoyltransferase 1 (p<0.05) and lipid beta-oxidation enzymes (p<0.001). Conclusion. In our work we have demonstrated that athletes involved in team sports (field hockey) have a more pronounced shift towards fatty acid oxidation compared to aerobic sports (swimming). A possible reason for the identified changes may be a more significant effect of alternating aerobic and anaerobic loads on lipid metabolism compared to exclusively aerobic activity.

The influence of a maternal vegan diet on carnitine and vitamin B2 concentrations in human milk.

The maternal diet greatly influences the nutritional composition of human milk. With the rise of vegan diets by lactating mothers, there are concerns about the nutritional adequacy of their milk. Two important nutrients, vitamin B2 and carnitine, are mostly ingested via animal products. We investigated the influence of a vegan diet on the vitamin B2 and carnitine concentrations in milk and serum of lactating women. In this case-control study, 25 lactating mothers following an exclusive vegan diet were comparted to 25 healthy lactating mothers with an omnivorous diet without use of supplements. High-performance liquid chromatography and liquid chromatography-tandem mass spectrometry were used to measure vitamin B2 and carnitine concentrations, respectively. A linear regression model was used to determine differences in human milk and serum concentrations between study groups. Vitamin B2 concentrations in human milk and serum did not differ between study groups. While the human milk free carnitine (C0) and acetyl carnitine (C2) concentrations did not differ between study groups, serum carnitine concentrations were lower in participants following a vegan diet than in omnivorous women (p < 0.0001). A maternal vegan diet did not affect human milk concentration of vitamin B2 and carnitine. Breastfed infants of mothers following an exclusive vegan diet therefore are likely not at increased risk of developing a vitamin B2 or carnitine deficiency.

Effects of dietary rumen-protected choline supplementation to periparturient dairy cattle on inflammation, metabolism, and performance during an intramammary lipopolysaccharide challenge

Recent studies have suggested that dietary rumen-protected choline (RPC) supplementation can modulate immune function, attenuate inflammation, and improve performance in periparturient dairy cattle; however, this has yet to be evaluated during a mastitis challenge. Therefore, the objective of this study was to examine the effects of supplementation and dose of RPC on metabolism, inflammation, and performance during an intramammary lipopolysaccharide (LPS) challenge. Parous Holstein cows (parity, mean ± SD, 1.9 ± 1.1 at enrollment) were blocked by calving month and randomly assigned within block to receive either 45 g/d of RPC (20.4 g/d of choline ions; CHOL45, n = 18), 30 g/d of RPC (13.6 g/d of choline ions; CHOL30, n = 21), or no RPC (CON, n = 19) as a top-dress starting 24 d before expected calving until 21 d postpartum. Cows were alternately assigned within treatment group to either receive an intramammary LPS challenge (200 μg in each rear quarter; Escherichia coli O111:B4) or not at 17 DIM. Before the challenge, CHOL45 and CHOL30 cows produced 3.4 and 3.8 (±1.2 SED) kg/d more milk than CON, respectively. Dietary RPC supplementation did not mitigate the milk loss associated with the intramammary LPS challenge; however, CHOL45 and CHOL30 cows produced 3.1 and 3.5 (±1.4 SED) kg/d more milk than CON, respectively in the carryover period (22 to 84 DIM). Dietary RPC supplementation enhanced plasma β-hydroxybutyrate (BHB) concentrations before the LPS challenge, and increased plasma nonesterified fatty acids (NEFA) and acetylcarnitine concentrations during the LPS challenge, potentially reflecting greater adipose tissue mobilization, fatty acid transport and oxidation. Aside from trimethylamine N-oxide and sarcosine, which were increased in CHOL45-LPS as compared with CON-LPS, most other choline metabolite concentrations in plasma were unaffected by treatment, likely because more choline was being secreted in milk. Plasma lactic acid concentrations were decreased in CHOL45-LPS and CHOL30-LPS as compared with CON-LPS, suggesting a reduction in glycolysis or an enhancement in the flux through the lactic acid cycle to support gluconeogenesis. Plasma concentrations of fumaric acid, a byproduct of AA catabolism and the urea cycle, were increased in both choline groups as compared with CON-LPS during the LPS challenge. Cows in the CHOL45 group had greater plasma antioxidant potential before the LPS challenge and reduced plasma methionine sulfoxide concentrations during the LPS challenge compared with CON-LPS, suggesting an improvement in oxidant status. Nevertheless, concentrations of inflammatory markers such as haptoglobin and tumor necrosis factor α (TNFα) were not affected by treatment. Taken together, our data suggest that the effects of dietary RPC supplementation on milk yield could be mediated through metabolic pathways and are unlikely to be related to the resolution of inflammation in periparturient dairy cattle. Lastly, dose responses to dietary RPC supplementation were not found for various economically important outcomes including milk yield, limiting the justification for feeding a greater dietary RPC dose in industry.

Cardiac Molecular Remodeling by Anticancer Drugs: Doxorubicin Affects More Metabolism While Mitoxantrone Impacts More Autophagy in Adult CD-1 Male Mice

Doxorubicin (DOX) and mitoxantrone (MTX) are classical chemotherapeutic agents used in cancer that induce similar clinical cardiotoxic effects, although it is not clear if they share similar underlying molecular mechanisms. We aimed to assess the effects of DOX and MTX on the cardiac remodeling, focusing mainly on metabolism and autophagy. Adult male CD-1 mice received pharmacologically relevant cumulative doses of DOX (18 mg/kg) and MTX (6 mg/kg). Both DOX and MTX disturbed cardiac metabolism, decreasing glycolysis, and increasing the dependency on fatty acids (FA) oxidation, namely, through decreased AMP-activated protein kinase (AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) content and decreased free carnitine (C0) and increased acetylcarnitine (C2) concentration. Additionally, DOX heavily influenced glycolysis, oxidative metabolism, and amino acids turnover by exclusively decreasing phosphofructokinase (PFKM) and electron transfer flavoprotein-ubiquinone oxidoreductase (ETFDH) content, and the concentration of several amino acids. Conversely, both drugs downregulated autophagy given by the decreased content of autophagy protein 5 (ATG5) and microtubule-associated protein light chain 3 (LC3B), with MTX having also an impact on Beclin1. These results emphasize that DOX and MTX modulate cardiac remodeling differently, despite their clinical similarities, which is of paramount importance for future treatments.

Carnitine affects Octanoate Oxidation to Carbon Dioxide and Dicarboxylic Acids in Colostrum-Deprived Piglets: In Vivo Analysis of Mechanisms Involved Based on CoA- and Carnitine-Ester Profiles

Newborn, colostrum-deprived piglets (n = 21) were used to study the effects of L-carnitine supplementation on the in vivo oxidation of [1-14C]octanoate to CO2 and dicarboxylic acids. Pigs were fitted with arterial and bladder catheters and were infused with octanoate (supplying 35-100% of piglets' energy expenditure) and with or without valproate for a period of 24 h. After achieving steady-state octanoate oxidation, carnitine was coinfused [50 mumol/kg 0.75 prime plus 20 mumol/h.kg 0.75)], and deviations in the octanoate oxidation rate, dicarboxylic acid excretion rate, and carnitine metabolism were monitored. At the end of the 24-h infusion, samples of liver and muscle were analyzed for carnitine- and CoA-esters by HPLC. Carnitine stimulated octanoate oxidation by 7% (P < 0.05) and decreased dicarboxylic acid excretion by 45% (P < 0.05). Carnitine supplementation increased (P < 0.05) concentrations of carnitine and acetyl carnitine in hepatic tissue (three- and 55-fold, respectively) and plasma (seven- and 11-fold); whereas, muscle-carnitine concentration doubled upon carnitine supplementation, but acetyl carnitine concentration remained unaltered. Urinary excretion of acetyl and free carnitine also increased with carnitine supplementation, but accounted for < 10% of carnitine infused. Hepatic total CoA and CoA esters increased with carnitine supplementation, whereas muscle acetyl-CoA decreased. Valproate had only marginal effects on octanoate metabolism. These data confirm the hypothesis that carnitine effects the in vivo oxidation of octanoate in colostrum-deprived piglets and suggest that the effects may be mediated by aiding the export of excess acetyl groups from muscle or by enhancing uptake of octanoate into liver mitochondria.

Skeletal muscle mitochondrial inertia is associated with carnitine acetyltransferase activity and physical function in humans.

BACKGROUNDAt the onset of exercise, the speed at which phosphocreatine (PCr) decreases toward a new steady state (PCr on-kinetics) reflects the readiness to activate mitochondrial ATP synthesis, which is secondary to Acetyl-CoA availability in skeletal muscle. We hypothesized that PCr on-kinetics are slower in metabolically compromised and older individuals and are associated with low carnitine acetyltransferase (CrAT) protein activity and compromised physical function.METHODSWe applied 31P-magnetic resonance spectroscopy (31P-MRS) to assess PCr on-kinetics in 2 cohorts of volunteers. Cohort 1 included patients who had type 2 diabetes, were obese, were lean trained (VO2max > 55 mL/kg/min), and were lean untrained (VO2max < 45 mL/kg/min). Cohort 2 included young (20-30 years) and older (65-80 years) individuals with normal physical activity and older, trained individuals. Previous results of CrAT protein activity and acetylcarnitine content in muscle tissue were used to explore the underlying mechanisms of PCr on-kinetics, along with various markers of physical function.RESULTSPCr on-kinetics were significantly slower in metabolically compromised and older individuals (indicating mitochondrial inertia) as compared with young and older trained volunteers, regardless of in vivo skeletal muscle oxidative capacity (P < 0.001). Mitochondrial inertia correlated with reduced CrAT protein activity, low acetylcarnitine content, and functional outcomes (P < 0.001).CONCLUSIONPCr on-kinetics are significantly slower in metabolically compromised and older individuals with normal physical activity compared with young and older trained individuals, regardless of in vivo skeletal muscle oxidative capacity, indicating greater mitochondrial inertia. Thus, PCr on-kinetics are a currently unexplored signature of skeletal muscle mitochondrial metabolism, tightly linked to functional outcomes. Skeletal muscle mitochondrial inertia might emerge as a target of intervention to improve physical function.TRIAL REGISTRATIONNCT01298375 and NCT03666013 (clinicaltrials.gov).FUNDINGRM and MH received an EFSD/Lilly grant from the European Foundation for the Study of Diabetes (EFSD). VS was supported by an ERC starting grant (grant 759161) "MRS in Diabetes."

Serum metabolomics analysis reveals increased lipid catabolism in mildly hyperbilirubinemic Gilbert's syndrome individuals

The protective role of mildly elevated bilirubin against CVD and diabetes mellitus type 2 (DMT2) is associated with a favorable lipid phenotype. As the mechanistic understanding of this protection in humans remains elusive, we aimed to assess the metabolomics profile of mildly hyperbilirubinemic (Gilbert's syndrome; GS) individuals especially targeting lipid catabolism. Using NMR serum metabolomics of 56 GS individuals and 56 age and gender-matched healthy controls, GS individuals demonstrated significantly greater concentrations of acetylcarnitine (+20%, p < 0.001) and the ketone bodies, 3-hydroxybutyric acid (+132%, p < 0.001), acetoacetic acid (+95%, p < 0.001) and acetone (+46%, p < 0.001). Metabolites associated with an increased mitochondrial lipid metabolism such as citrate (+15%, p < 0.001), anaplerotic amino acid intermediates and creatinine were significantly greater and creatine significantly reduced in GS individuals. Stimulators of lipid catabolism including AMPK (+59%, p < 0.001), pPPARα (+24%, p < 0.001) and T3 (+9%, p = 0.009) supported the metabolomics data while concomitantly blood glucose and insulin (-33%, p = 0.002) levels were significantly reduced. We further showed that the increased lipid catabolism partially mediates the favorable lipid phenotype (lower triglycerides) of GS individuals. Increased trimethylamine (+35%, p < 0.001) indicated changes in trimethylamine metabolism, an emerging predictor of metabolic health. We showed an enhanced lipid catabolism in mildly hyperbilirubinemic individuals, novel evidence as to why these individuals are leaner and protected against chronic metabolic diseases emphasizing bilirubin to be a promising future target in obese and dyslipidemia patients.

Limited Impact of Pivalate-Induced Secondary Carnitine Deficiency on Hepatic Transcriptome and Hepatic and Plasma Metabolome in Nursery Pigs.

Administration of pivalate has been demonstrated to be suitable for the induction of secondary carnitine deficiency (CD) in pigs, as model objects for humans. In order to comprehensively characterize the metabolic effects of secondary CD in the liver of pigs, the present study aimed to carry out comparative analysis of the hepatic transcriptome and hepatic and plasma metabolome of a total of 12 male 5-week-old pigs administered either pivalate (group PIV, n = 6) or vehicle (group CON, n = 6) for 28 days. Pigs of group PIV had approximately 40–60% lower concentrations of free carnitine and acetylcarnitine in plasma, liver and different skeletal muscles than pigs of group CON (p < 0.05). Transcript profiling of the liver revealed 140 differentially expressed genes (DEGs) between group PIV and group CON (fold change > 1.2 or <−1.2, p-value < 0.05). Biological process terms dealing with the innate immune response were found to be enriched with the DEGs (p < 0.05). Using a targeted metabolomics approach for the simultaneous quantification of 630 metabolites, 9 liver metabolites and 18 plasma metabolites were identified to be different between group PIV and group CON (p < 0.05). Considering the limited alterations of the hepatic transcriptome and of the liver and plasma metabolome, it can be concluded that pivalate-induced secondary CD is not associated with significant hepatic metabolism changes in pigs.

Branched-Chain Amino Acid Catabolism and Cardiopulmonary Function Following Acute Maximal Exercise Testing in Adolescents.

Background: To provide energy for cardiopulmonary function and maintenance of blood glucose, acute aerobic exercise induces lipolysis, fatty acid oxidation (FAO), glycolysis, and glycogenolysis/gluconeogenesis. These adaptations are mediated by increases in cortisol, growth hormone (GH), and catecholamines and facilitated by a decline in insulin. Branched-chain amino acids (BCAA) also undergo catabolism during intense exercise. Here, we investigated the relationship between BCAA catabolism and metrics of cardiopulmonary function in healthy, well-developed, mature adolescent athletes undergoing an acute bout of maximal aerobic exercise.Hypothesis: We hypothesized: (a) acute maximal exercise in adolescents induces lipolysis, FAO, and BCAA catabolism associated with increases in GH and cortisol and a reduction in insulin; (b) increases in GH are associated with increases in ghrelin; and (c) metrics of cardiopulmonary function (aVO2, rVO2, aVO2/HRmax) following maximal exercise correlate with increases in GH secretion, FAO, and BCAA catabolism.Methods: Blood samples before and after maximal cardiopulmonary exercise in 11 adolescent athletes were analyzed by tandem-mass spectrometry. Paired, two-tailed student's t-tests identified significant changes following exercise. Linear regression determined if pre-exercise metabolite levels, or changes in metabolite levels, were associated with aVO2, rVO2, and aVO2/HRmax. Sex and school of origin were included as covariates in all regression analyses.Results: Following exercise there were increases in GH and cortisol, and decreases in ghrelin, but no changes in glucose or insulin concentrations. Suggesting increased lipolysis and FAO, the levels of glycerol, ketones, β-hydroxybutyrate, and acetylcarnitine concentrations increased. Pyruvate, lactate, alanine, and glutamate concentrations also increased. Plasma concentrations of valine (a BCAA) declined (p = 0.002) while valine degradation byproducts increased in association with decreases in urea cycle amino acids arginine and ornithine. Metrics of cardiopulmonary function were associated with increases in propionylcarnitine (C3, p = 0.013) and Ci4-DC/C4-DC (p < 0.01), byproducts of BCAA catabolism.Conclusions: Induction of lipolysis, FAO, gluconeogenesis, and glycogenolysis provides critical substrates for cardiopulmonary function during exercise. However, none of those pathways were significantly associated with metrics of cardiopulmonary function. The associations between rVO2, and aVO2/HRmax and C3 and Ci4-DC/C4-DC suggest that the cardiopulmonary response to maximal exercise in adolescents is linked to BCAA utilization and catabolism.

Metabolomics analysis reveals altered metabolites in lean compared with obese adolescents and additional metabolic shifts associated with hyperinsulinaemia and insulin resistance in obese adolescents: a cross-sectional study

IntroductionHyperinsulinaemia and insulin resistance (IR) are strongly associated with obesity and are forerunners of type 2 diabetes. Little is known about metabolic alterations separately associated with obesity, hyperinsulinaemia/IR and impaired glucose tolerance (IGT) in adolescents.ObjectivesTo identify metabolic alterations associated with obesity, hyperinsulinaemia/IR and hyperinsulinaemia/IR combined with IGT in obese adolescents.Methods81 adolescents were stratified into four groups based on body mass index (lean vs. obese), insulin responses (normal insulin (NI) vs. high insulin (HI)) and glucose responses (normal glucose tolerance (NGT) vs. IGT) after an oral glucose tolerance test (OGTT). The groups comprised: (1) healthy lean with NI and NGT, (2) obese with NI and NGT, (3) obese with HI and NGT, and (4) obese with HI and IGT. Targeted nuclear magnetic resonance-based metabolomics analysis was performed on fasting and seven post-OGTT plasma samples, followed by univariate and multivariate statistical analyses.ResultsTwo groups of metabolites were identified: (1) Metabolites associated with insulin response level: adolescents with HI (groups 3–4) had higher concentrations of branched-chain amino acids and tyrosine, and lower concentrations of serine, glycine, myo-inositol and dimethylsulfone, than adolescents with NI (groups 1–2). (2) Metabolites associated with obesity status: obese adolescents (groups 2–4) had higher concentrations of acetylcarnitine, alanine, pyruvate and glutamate, and lower concentrations of acetate, than lean adolescents (group 1).ConclusionsObesity is associated with shifts in fat and energy metabolism. Hyperinsulinaemia/IR in obese adolescents is also associated with increased branched-chain and aromatic amino acids.

Effects of delivery and storage conditions on concentrations of amino acids and carnitines in neonatal dried blood spots

To explore effects of different delivery and storage conditions on concentrations of amino acids and carnitines in neonatal dried blood spots (DBS), so as to provide evidence for improving accurate and reliable detection by tandem mass spectrometry. A total of 1 254 616 newborn DBS samples in Newborn Screening Center of Zhejiang Province were delivered and stored at room temperature (group A, n=338 467), delivered by cold-chain logistics system and stored at low temperature (group B, n=480 021), or delivered by cold-chain logistics system and stored at low temperature and low humidity (group C, n= 436 128), respectively. The concentrations of amino acids and carnitines in DBS were detected by tandem mass spectrometry. Data analysis was performed by SPSS 24.0 to explore the influence of temperature and humidity on the concentrations of amino acids and carnitines. The concentrations of amino acids and carnitines in the three groups were skewed, and the differences in amino acid and carnitine concentrations among groups were statistically significant (all P<0.01). The median concentration of tyrosine was lower in group A than those in group B and group C by 18%and 16%respectively, while there was no significant difference between the last two groups. The median concentrations of methionine were lower in group A and group B than that in group C by 15%and 11%, respectively. The median concentrations of arginine were lower in group A and group B than that in group C by 12%and 25%, respectively. The median concentration of free carnitine (C0) was higher in group A than that in group C by 12%, while there was no significant difference between group A and group B. The median concentrations of acetylcarnitine (C2), propionyl carnitine (C3), C3DC+C4OH, C5DC+C6OH and hexadecanoyl carnitine (C16) were lower in group A than those in group B and group C by 21%-64%. The concentrations of other amino acids and acylcarnitines differed little among three groups. The monthly median coefficients of variation of other amino acids and carnitines in group A were higher than those in group B and group C except for citrulline, C4DC+C5OH and isovalerylcarnitine (C5). Cold-chain logistics system and storage in low temperature and low humidity can effectively reduce degradation of some amino acids and carnitines in DBS, improve the accuracy and reliability of detection, and thus ensures the quality of screening for neonatal metabolic diseases.

Nicotinamide riboside supplementation alters body composition and skeletal muscle acetylcarnitine concentrations in healthy obese humans

ABSTRACTBackgroundNicotinamide riboside (NR) is an NAD+ precursor that boosts cellular NAD+ concentrations. Preclinical studies have shown profound metabolic health effects after NR supplementation.ObjectivesWe aimed to investigate the effects of 6 wk NR supplementation on insulin sensitivity, mitochondrial function, and other metabolic health parameters in overweight and obese volunteers.MethodsA randomized, double-blinded, placebo-controlled, crossover intervention study was conducted in 13 healthy overweight or obese men and women. Participants received 6 wk NR (1000 mg/d) and placebo supplementation, followed by broad metabolic phenotyping, including hyperinsulinemic-euglycemic clamps, magnetic resonance spectroscopy, muscle biopsies, and assessment of ex vivo mitochondrial function and in vivo energy metabolism.ResultsMarkers of increased NAD+ synthesis—nicotinic acid adenine dinucleotide and methyl nicotinamide—were elevated in skeletal muscle after NR compared with placebo. NR increased body fat-free mass (62.65% ± 2.49% compared with 61.32% ± 2.58% in NR and placebo, respectively; change: 1.34% ± 0.50%, P = 0.02) and increased sleeping metabolic rate. Interestingly, acetylcarnitine concentrations in skeletal muscle were increased upon NR (4558 ± 749 compared with 3025 ± 316 pmol/mg dry weight in NR and placebo, respectively; change: 1533 ± 683 pmol/mg dry weight, P = 0.04) and the capacity to form acetylcarnitine upon exercise was higher in NR than in placebo (2.99 ± 0.30 compared with 2.40 ± 0.33 mmol/kg wet weight; change: 0.53 ± 0.21 mmol/kg wet weight, P = 0.01). However, no effects of NR were found on insulin sensitivity, mitochondrial function, hepatic and intramyocellular lipid accumulation, cardiac energy status, cardiac ejection fraction, ambulatory blood pressure, plasma markers of inflammation, or energy metabolism.ConclusionsNR supplementation of 1000 mg/d for 6 wk in healthy overweight or obese men and women increased skeletal muscle NAD+ metabolites, affected skeletal muscle acetylcarnitine metabolism, and induced minor changes in body composition and sleeping metabolic rate. However, no other metabolic health effects were observed.This trial was registered at clinicaltrials.gov as NCT02835664

Muscle-Specific Relation of Acetylcarnitine and Intramyocellular Lipids to Chronic Hyperglycemia: A Pilot 3-T 1H MRS Study.

ObjectiveAcetylcarnitine 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.MethodsAcetylcarnitine and intramyocellular lipid concentrations were measured in 35 individuals stratified into three groups according to glucose tolerance and/or manifestation of type 2 diabetes mellitus. All MRS measurements were performed on a 3‐T MR system.ResultsThe differences in patient phenotype were mirrored by increased intramyocellular lipids in the tibialis anterior and decreased acetylcarnitine concentrations in the soleus muscle of type 2 diabetes patients when compared with normal glucose‐tolerant individuals. Results suggest that intramyocellular lipids mirror whole‐body glucose tolerance better in the tibialis anterior muscle, whereas acetylcarnitine is a better discriminator in the soleus muscle.ConclusionsThis muscle‐specific behavior of metabolites could represent different fiber compositions in the examined muscles and should be considered when planning future metabolic studies.

Selection for Growth and Precocity Alters Muscle Metabolism in Nellore Cattle.

To clarify the relationship between beef genetic selection for growth and precocity with muscle metabolism and metabolites, we performed metabolomic analysis using Longissimus lumborum (LL) muscle from Nellore cattle with divergent selection for these traits (high growth, HG; low growth, LG; high precocity, HP; low precocity, LP). Genetic potential for growth affected muscle protein and energetic metabolism. HG animals had a high concentration of arginine, carnosine, and leucine compared to LG animals. HP animals presented a high concentration of glutamine, betaine, creatinine, isoleucine, carnitine, acetyl carnitine, and lower levels of glucose compared to LP animals, affecting protein and fatty acid metabolism. Intensity of selection (high or low) was correlated with changes in protein metabolism, and the type of selection (growth or precocity) affected fat metabolism. In conclusion, both HG and HP appear to be correlated with a high concentration of protein metabolites and changes in protein metabolic pathways, while selection for precocity is more correlated with changes in fat metabolism compared to animals selected for growth.

The effect of exercise on intramyocellular acetylcarnitine (AcCtn) concentration in adult growth hormone deficiency (GHD)

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.

Carnitine supplementation improves metabolic flexibility and skeletal muscle acetylcarnitine formation in volunteers with impaired glucose tolerance: A randomised controlled trial.

BackgroundType 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.MethodsEleven IGT- volunteers followed a 36-day placebo- and L-carnitine treatment (2 g/day) in a randomised, placebo-controlled, double blind crossover design. A hyperinsulinemic-euglycemic clamp (40 mU/m2/min), combined with indirect calorimetry (ventilated hood) was performed to determine insulin sensitivity and metabolic flexibility. Furthermore, metabolic flexibility was assessed in response to a high-energy meal. Skeletal muscle acetylcarnitine concentrations were measured in vivo using long echo time proton magnetic resonance spectroscopy (1H-MRS, TE=500 ms) in the resting state (7:00AM and 5:00PM) and after a 30-min cycling exercise. Twelve normal glucose tolerant (NGT) volunteers were included without any intervention as control group.ResultsMetabolic flexibility of IGT-subjects completely restored towards NGT control values upon carnitine supplementation, measured during a hyperinsulinemic-euglycemic clamp and meal test. In muscle, carnitine supplementation enhanced the increase in resting acetylcarnitine concentrations over the day (delta 7:00 AM versus 5:00 PM) in IGT-subjects. Furthermore, carnitine supplementation increased post-exercise acetylcarnitine concentrations and reduced long-chain acylcarnitine species in IGT-subjects, suggesting the stimulation of a more complete fat oxidation in muscle. Whole-body insulin sensitivity was not affected.ConclusionCarnitine supplementation improves acetylcarnitine formation and rescues metabolic flexibility in IGT-subjects. Future research should investigate the potential of carnitine in prevention/treatment of type 2 diabetes.

Effect of lifelong carnitine supplementation on plasma and tissue carnitine status, hepatic lipid metabolism and stress signalling pathways and skeletal muscle transcriptome in mice at advanced age

While strong evidence from clinical studies suggests beneficial effects of carnitine supplementation on metabolic health, serious safety concerns associated with carnitine supplementation have been raised from studies in mice. Considering that the carnitine doses in these mice studies were up to 100 times higher than those used in clinical studies, the present study aimed to address possible safety concerns associated with long-term supplementation of a carnitine dose used in clinical trials. Two groups of NMRI mice were fed either a control or a carnitine-supplemented diet (1 g/kg diet) from weaning to 19 months of age, and parameters of hepatic lipid metabolism and stress signalling and skeletal muscle gene expression were analysed in the mice at 19 months of age. Concentrations of free carnitine and acetylcarnitine in plasma and tissues were higher in the carnitine than in the control group (P&lt;0·05). Plasma concentrations of free carnitine and acetylcarnitine were higher in mice at adult age (10 and 15 months) than at advanced age (19 months) (P&lt;0·05). Hepatic mRNA and protein levels of genes involved in lipid metabolism and stress signalling and hepatic and plasma lipid concentrations did not differ between the carnitine and the control group. Skeletal muscle transcriptome analysis in 19-month-old mice revealed only a moderate regulation between carnitine and control group. Lifelong carnitine supplementation prevents an age-dependent impairment of plasma carnitine status, but safety concerns associated with long-term supplementation of carnitine at doses used in clinical trials can be considered as unfounded.

1861-P: Pioglitazone Modulates Hepatic Pyruvate Dehydrogenase and TCA Cycle Flux

Background: The insulin sensitizing actions of the antidiabetic drug Pioglitazone (PIO) have classically been ascribed to the reversal of ectopic lipid accumulation. We recently demonstrated acute, direct metabolic effects of PIO in hepatocytes, linking the suppression of hepatic glucose production to an inhibition of mitochondrial pyruvate metabolism. As the relevance of this mechanism in vivo is unclear, we studied the effects of PIO on hepatic pyruvate metabolism in a mouse model of insulin resistance. Methods: Eight-week-old male ob/ob mice were placed on a regular diet (10% kcal fat) with (n=10) or without (n=10) 300 mg/kg PIO for four weeks before study during euglycemic-hyperinsulinemic clamp (5 mU/kg min). Endogenous glucose production (EGP), hepatic glycogen synthesis and anabolic/oxidative pyruvate flux ratio (PC/PDH) were determined from [3-3H] glucose infusion and 13C NMR isotopomer analysis using [2,3-13C] pyruvate. Livers were freeze-clamped in situ for pyruvate dehydrogenase (PDH), metabolite and protein analysis. Results: PIO treatment lowered EGP by 62% (4.6 ± 1.0 vs. 12.1 ± 1.7 mg/kg min; P&amp;lt;0.01) and increased glycogen synthesis 10-fold (19.7 ± 4.8 vs. 1.9 ± 0.8 μ mol/kg liver min; P&amp;lt;0.005), whereas PC/PDH remained unchanged. PIO also suppressed liver PDH activation (14.6 ± 3.4 vs. 29.6 ± 4.3 µmol/kg liver min; P&amp;lt;0.05), as well as acetylcarnitine (140 ± 18 vs. 297 ± 40) and acetyl-CoA (17.7 ± 2.4 vs. 47.8 ± 8.8 µmol/kg liver) concentrations (P&amp;lt;0.01), whilst liver PDK4 protein expression was increased 46% compared to controls (P&amp;lt;0.05). Conclusions: Inhibition of PDH activation by Pioglitazone curtails mitochondrial acetyl-CoA delivery, relieving allosteric stimulation of pyruvate carboxylase and gluconeogenesis. Parallel reductions in PDH flux and EGP reconcile an unchanged PC/PDH ratio, suggesting an overall slowing of TCA cycle flux with PIO. These novel data highlight the therapeutic relevance of hepatic mitochondrial metabolism in the treatment of type 2 diabetes and liver disease. Disclosure C. Shannon: None. M. Ragavan: None. R.A. DeFronzo: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Elcelyx Therapeutics, Inc., Intarcia Therapeutics, Inc., Janssen Pharmaceuticals, Inc., Novo Nordisk Inc. Research Support; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Janssen Pharmaceuticals, Inc., Merck &amp; Co., Inc. Speaker's Bureau; Self; AstraZeneca, Novo Nordisk Inc. M. Merritt: None. L. Norton: None. Funding University of Florida’s Southeast Center for Integrated Metabolomics (U24DK097209); National Institutes of Health