palmitoyl-L-carnitine Research Articles

MiR-21-3p inhibitor exerts myocardial protective effects by altering macrophage polarization state and reducing excessive mitophagy

Chronic heart failure (CHF) is closely associated with inflammation and mitochondrial dysfunction in cardiomyocytes. This study attempts to investigate the effects of microRNA-21-3p (miR-21-3p) on macrophage polarization and mitophagy in CHF. Here we found miR-21-3p was upregulated in CHF and negatively correlated with carnitine palmitoyl transferase 1A (CPT1A). L-palmitoyl carnitine (L-PC) exacerbated isoproterenol (ISO)-induced myocardial structural disruption and fibrosis in rats, which was exacerbated by miR-21-3p. Mechanistically, miR-21-3p accelerated M1 macrophage polarization. Both miR-21-3p inhibitor and CPT1A overexpression suppressed mitophagy. The inhibition of CPT1A on mitophagy was reversed by miR-21-3p. MiR-21-3p targeted CPT1A mRNA and co-localized with CPT1A protein in cardiomyocytes. In the co-culture system of M1 macrophages and H9c2 cells, miR-21-3p mimics in H9c2 cells promoted M1 polarization, whereas miR-21-3p inhibitor reduced M1 phenotype. M1 macrophages exacerbated H9c2 cell damage. These findings support the potential therapeutic targeting of miR-21-3p to regulate inflammation and mitophagy by inducing CPT1A in CHF.

L-Palmitoylcarnitine potentiates plasmin and tPA to inhibit thrombosis

l-Palmitoylcarnitine (L-PC) is an important endogenous fatty acid metabolite. Its classical biological functions are involved in the regulations of membrane molecular dynamics and the β-oxidation of fatty acids. Decreased plasma long-chain acylcarnitines showed the association of venous thrombosis, implying anticoagulant activity of the metabolites and inspiring us to investigate if and how L-PC, a long-chain acylcarnitine, takes part in coagulation. Here we show that L-PC exerted anti-coagulant effects by potentiating the enzymatic activities of plasmin and tissue plasminogen activator (tPA). L-PC directly interacts with plasmin and tPA with an equilibrium dissociation constant (KD) of 6.47 × 10–9 and 4.46 × 10–9 M, respectively, showing high affinities. In mouse model, L-PC administration significantly inhibited FeCl3-induced arterial thrombosis. It also mitigated intracerebral thrombosis and inflammation in a transient middle cerebral artery occlusion (tMCAO) mouse model. L-PC induced little bleeding complications. The results show that L-PC has anti-thrombotic function by potentiating plasmin and tPA.Graphical

Beneficial effects of Lactobacillus rhamnosus hsryfm 1301 fermented milk on rats with nonalcoholic fatty liver disease

A growing stream of research suggests that probiotic fermented milk has a good effect on nonalcoholic fatty liver disease. This work aimed to study the beneficial effects of Lactobacillus rhamnosus hsryfm 1301 fermented milk (fermented milk) on rats with nonalcoholic fatty liver disease induced by a high-fat diet. The results showed that the body weight and the serum levels of total cholesterol, total glyceride, low-density lipoprotein, alanine transaminase, aspartate aminotransferase, free fatty acid, and reactive oxygen species were significantly increased in rats fed a high-fat diet (M) for 8 wk, whereas high-density lipoprotein cholesterol and superoxide dismutase were significantly decreased. However, the body weight and the serum levels of total cholesterol, total glyceride, alanine transaminase, aspartate aminotransferase, free fatty acid, reactive oxygen species, interleukin-8, tumor necrosis factor-α, and interleukin-6 were significantly decreased with fermented milk (T) for 8 wk, and the number of fat vacuoles in hepatocytes was lower than that in the M group. There were significant differences in 19 metabolites in serum between the M group and the C group (administration of nonfermented milk) and in 17 metabolites between the T group and the M group. The contents of 7 different metabolites, glycine, glycerophosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, thioetheramide-PC, d-aspartic acid, oleic acid, and l-glutamate, were significantly increased in the M group rat serum, and l-palmitoyl carnitine, N6-methyl-l-lysine, thymine, and 2-oxadipic acid were significantly decreased. In the T group rat serum, the contents of 8 different metabolites-1-O-(cis-9-octadecenyl)-2-O-acetyl-sn-glycero-3-phosphocholine, acetylcarnitine, glycine, glycerophosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, d-aspartic acid, oleic acid, and l-glutamate were significantly decreased, whereas creatinine and thymine were significantly increased. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that 50 metabolic pathways were enriched in the M/C group and T/M group rat serum, of which 12 metabolic pathways were significantly different, mainly distributed in lipid metabolism, amino acid, and endocrine system metabolic pathways. Fermented milk ameliorated inflammation, oxygenation, and hepatocyte injury by regulating lipid metabolism, amino acid metabolic pathways, and related metabolites in the serum of rats with nonalcoholic fatty liver disease.

Untargeted muscle tissue metabolites profiling in young, adult, and old rats supplemented with tocotrienol-rich fraction.

The greatest significant influence on human life span and health is inevitable ageing. One of the distinguishing characteristics of ageing is the gradual decrease of muscle mass and physical function. There has been growing evidence that tocotrienol can guard against age-associated chronic diseases and metabolic disorders. This study aimed to elucidate the effects of tocotrienol-rich fraction (TRF) on muscle metabolomes and metabolic pathways in ageing Sprague Dawley (SD) rats. Three months, 9 months, and 21 months old male SD rats were divided into control and treated groups with 10 rats per group. Rats in control and treated groups were given 60 mg/kg body weight/day of palm olein and 60 mg/kg body weight/day of TRF, respectively, via oral gavage for 3 months. Muscle performance was assessed at 0 and 3 months of treatment by measuring muscle strength and function. Our results showed that TRF treatment caused a significant increase in the swimming time of the young rats. Comparison in the control groups showed that metabolites involved in lipid metabolisms such as L-palmitoyl carnitine and decanoyl carnitine were increased in ageing. In contrast, several metabolites, such as 3-phosphoglyceric acid, aspartic acid and aspartyl phenylalanine were decreased. These findings indicated that muscle metabolomes involved in lipid metabolism were upregulated in aged rats. In contrast, the metabolites involved in energy and amino acid metabolism were significantly downregulated. Comparison in the TRF-supplemented groups showed an upregulation of metabolites involved in energy and amino acid metabolism. Metabolites such as N6-methyl adenosine, spermine, phenylalanine, tryptophan, aspartic acid, histidine, and N-acetyl neuraminic acid were up-regulated, indicating promotion of amino acid synthesis and muscle regeneration. Energy metabolism was also improved in adult and old rats with TRF supplementation as indicated by the upregulation of nicotinamide adenine dinucleotide and glycerol 3-phosphate compared to the control group. In conclusion, the mechanism underlying the changes in skeletal muscle mass and functions in ageing was related to carbohydrate, lipid and amino acid metabolism. Tocotrienol supplementation showed beneficial effects in alleviating energy and amino acid synthesis that may promote the regeneration and renewal of skeletal muscle in ageing rats.

An integrated network pharmacology and cell metabolomics approach to reveal the role of rhein, a novel PPARα agonist, against renal fibrosis by activating the PPARα–CPT1A axis

BackgroundRhein, an anthraquinone compound, displays extensive antifibrotic effects; however, its potential mechanisms are not fully understood. In this study, we explored the underlying molecular mechanism of action of rhein. MethodAn integrated network pharmacology and cell metabolomics approach was developed based on network pharmacology and bioinformatics method, and then successfully applied to speculate the potential targets of rhein and construct a rhein–target–metabolic enzyme–metabolite network. Thereafter, the antifibrotic mechanism of rhein was validated in TGF-β- and oleic acid- induced HK-2 and NRK-52E cells in vitro as well as a unilateral ischemia–reperfusion injury Sprague-Dawley rat model. ResultsBased on the construction of the rhein–target–metabolic enzyme–metabolite network, we found that rhein played an antifibrotic role through the PPAR–α–CPT1A–l-palmitoyl–carnitine axis. In vitro experiments demonstrated that rhein effectively activated the expression of PPARα and its downstream proteins (CPT1A and ACOX1) to alleviate lipid accumulation and fibrosis development. In vivo experiments indicated that rhein attenuated renal fibrosis mainly by activating the fatty acid oxidation pathway and improving lipid metabolism. ConclusionTaken together, our findings reveal that rhein is a novel agonist of PPARα, which contributes to its renoprotection through the regulation of the PPARα–CPT1A axis. Moreover, our study provides a novel insight into an integrated network pharmacology–metabolomics strategy for uncovering the pharmacological mechanisms of drugs from the system perspective.

Zhibaidihuang Decoction Ameliorates Cell Oxidative Stress by Regulating the Keap1-Nrf2-ARE Signalling Pathway.

Zhibaidihuang decoction (ZBDHD) is a Chinese herbal formula, which is used in Chinese traditional medicine to treat symptoms of Yinxuhuowang (Yin deficiency and high fire) syndrome. This study elucidates the mechanism of ZBDHD on oral ulcers, one Yinxuhuowang syndrome. Simultaneously, some ingredients in ZBDHD were found and identified by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). A Ganjiangfuzirougui decoction- (GJD-) induced Yinxuhuowang syndrome SD rat model was used to demonstrate the efficiency of ZBDHD treatment. The oral mucosa of rat in the GJD group, stained with hematoxylin and eosin (H&E), showed epidermal shedding and inflammatory cell infiltration. And an alleviation efficiency of ZBDHD in GJD-induced pathological changes in the oral mucosa could be obtained. ZBDHD treatment restored the GJD-induced imbalance of metabolites, which were choline, glycocholic acid, and palmitoyl-L-carnitine (PALC). GJD stimulated the expression of NF-κB. And the overexpressed of NF-κB in mucosa of rat in the GJD group could be inhibited by ZBDHD treatment. Simultaneously, the optimal efficiency of ZBDHD treatment on the cellular ATP content, oxygen consumption rate (OCR), and superoxide dismutase (SOD) concentration was evaluated, in vitro assay. Compared to the control cells, the ATP content, OCR, and SOD activity in the ZBDHD-treated cells were significantly higher. For the mechanisms study, seven cytokines were screened with a Dual-Luciferase Reporter gene assay. In the ARE assay, the luciferase signal was stimulated significantly by ZBDHD. In cells, the transcription of nrf2, maf, and keap1, which were related to the ARE pathway, was elevated by ZBDHD treatment. Our study demonstrated that high-dose GJD could lead to Yinxuhuowang syndrome, such as oral ulcers, and the imbalance in serum metabolites. And ZBDHD can improve oral cell inflammation and the imbalance of metabolism by inhibiting NF-κB and enhancing the activity of the ARE signalling pathway to ameliorate oxidative stress in the cell. This study provides a theoretical basis for the clinical application of ZBDHD.

Skeletal muscle mitochondria exhibit decreased pyruvate oxidation capacity and increased ROS emission during surgery-induced acute insulin resistance.

Development of acute insulin resistance represents a negative factor after surgery, but the underlying mechanisms are not fully understood. We investigated the postoperative changes in insulin sensitivity, mitochondrial function, enzyme activities, and release of reactive oxygen species (ROS) in skeletal muscle and liver in pigs on the 2nd postoperative day after major abdominal surgery. Peripheral and hepatic insulin sensitivity were assessed by D-[6,6-²H₂]glucose infusion and hyperinsulinemic euglycemic step clamping. Surgical trauma elicited a decline in peripheral insulin sensitivity (∼34%, P<0.01), whereas hepatic insulin sensitivity remained unchanged. Intramyofibrillar (IFM) and subsarcolemma mitochondria (SSM) isolated from skeletal muscle showed a postoperative decline in ADP-stimulated respiration (V(ADP)) for pyruvate (∼61%, P<0.05, and ∼40%, P<0.001, respectively), whereas V(ADP) for glutamate and palmitoyl-L-carnitine (PC) was unchanged. Mitochondrial leak respiration with PC was increased in SSM (1.9-fold, P<0.05) and IFM (2.5-fold, P<0.05), indicating FFA-induced uncoupling. The activity of the pyruvate dehydrogenase complex (PDC) was reduced (∼32%, P<0.01) and positively correlated to the decline in peripheral insulin sensitivity (r=0.748, P<0.05). All other mitochondrial enzyme activities were unchanged. No changes in mitochondrial function in liver were observed. Mitochondrial H₂O₂ and O₂·⁻ emission was measured spectrofluorometrically, and H₂O₂ was increased in SSM, IFM, and liver mitochondria (∼2.3-, ∼2.5-, and ∼2.3-fold, respectively, all P<0.05). We conclude that an impairment in skeletal muscle mitochondrial PDC activity and pyruvate oxidation capacity arises in the postoperative phase along with increased ROS emission, suggesting a link between mitochondrial function and development of acute postoperative insulin resistance.

Identification of Minuscule Inward Currents as Precursors to Membrane Electroporation-Induced Currents: Real-Time Prediction of Pore Appearance

Background/Aims: The objective of this study is to examine the current signals in response to large hyperpolarizations with the aid of principal component analysis (PCA) to search for or even predict current fluctuations related to membrane electroporation-induced current (I_MEP). Methods: The characteristics of principal eigenvalues generated for I_MEP and the current signals at 10 sec prior to the start of initial I_MEP (I_Pre) were examined. As membrane hyperpolarizations were applied at 0.1 Hz, the appearance of I_MEP coincided with the higher principal eigenvalues extracted in PCA. Results: Subsequent addition of LaCl₃ (100 µM) greatly reduced I_MEP and associated principal eigenvalues. In real-time analysis for a single frame (i.e, 300 msec), in response to large hyperpolarization, multiple runs of heralded minuscule inward currents (I_min) occurring before large rise in current amplitudes were detected. With PCA, such heralded I_min was noted to coincide with the extreme principal eigenvalues. The duration of I_min together with large principal eigenvalues was influenced by different levels of membrane hyperpolarization. In GH3 cells, palmitoyl-L-carnitine (PALCAR), a long-chain acylcarnitine, effectively increased the I_MEP amplitude with an EC₅₀ value of 2.4 µM. However, in PALCAR-treated cells, the I_min together with higher principal eigenvalues disappeared, while in isoflurane-treated cells, I_min occurring before large rise of current amplitude remained intact. Similarly, the PCA analysis from I_Pre in RAW 264.6 macrophages showed the presence of herald I_min accompanied by the extreme principal eigenvalues. Conclusion: It is clear from this study that these large principal eigenvalues are representative of MEP-associated formation of electropores. Therefore, different compositions around the surface membrane of cells may alter the appearance of I_min followed by I_MEP emergence.

Protective effects of dilazep and its derivative K-7259 on the haemolysis induced by amphiphiles in rat erythrocytes.

The effects of dilazep and K-7259, a dilazep derivative, on the haemolysis (as evidenced by release of haemoglobin) induced by palmitoyl-L-carnitine (PAL-CAR) or palmitoyl 1-alpha-lysophosphatidylcholine (PAL-LPC) have been determined in rat erythrocytes. At concentrations above the critical micelle concentration both PAL-CAR and PAL-LPC induced haemolysis; the concentrations of PAL-CAR and PAL-LPC producing 50% haemolysis were approximately 13 and 14 microM, respectively. The 50% haemolysis induced by PAL-CAR or PAL-LPC was attenuated by dilazep (1, 10 or 100 microM) but not at the highest concentration used (1 mM). K-7259 attenuated the 50% haemolysis induced by PAL-CAR or PAL-LPC at concentrations ranging from 1 microM to 1 mM. Similarly, dilazep (1 to 100 microM) and K-7259 (1 microM to 1 mM) significantly or insignificantly attenuated the 25% and 75% haemolysis induced by PAL-CAR or PAL-LPC. Neither dilazep nor K-7259 affected micelle formation by PAL-CAR or PAL-LPC, nor, at concentrations of 1 and 10 microM, did they attenuate the haemolysis induced by osmotic imbalance (hypotonic haemolysis). These results suggest that both dilazep and K-7259 protect the erythrocyte membrane from the damage induced by PAL-CAR or PAL-LPC. The protective effects of dilazep and K-7259 are mediated by some mechanism other than prevention of micelle formation or protection of the erythrocyte membrane against osmotic imbalance.

Pyruvate Sparing by Fatty Acid and Glutamate in Sparrow Skeletal Muscle Mitochondria

The ability of avian muscle mitochondria (MITO) to oxidize fatty acids is crucial as only lipid can provide the high energy density fuel required for flight. When exercising at ∼70-80% VO2max, a flying pigeon has an RQ of ∼.73, whereas a running rat has an RQ of ∼.96, indicating the bird is oxidizing primarily fat (FAT) for fuel while the rat is almost exclusively oxidizing carbohydrate (CHO). PURPOSE: To assess the control sensitivity of CHO and FAT oxidation in MITO isolated from avian skeletal muscle and examine fuel selection at the level of the mitochondrial matrix when multiple substrates are provided. METHODS: MITO were isolated from English sparrow pectoralis and rat mixed hindlimb muscles and the rate of oxygen consumption (Jo) at varying values of ΔGATP were assessed with either pyruvate (P) + malate (M) (CHO) or palmitoyl-l-carnitine (PC) + M (FAT) as substrates. ΔGATP was established by using the creatine kinase energetic clamp. The slope of ΔGATP:Jo was determined with each fuel. Next, sparrow MITO were incubated at ΔGATP = -13.0, -13.5, and -14.0 kcal/mol with P+M, P+ glutamate (G)+M, P+PC+M, G+PC+M, or P+G+PC+M as substrates. The steady state Jo and total O2 consumed were measured polarographically. Samples were taken just after MITO addition and ∼10.0 min later and fluorometrically assayed to determine P utilization. RESULTS: Sparrow MITO showed dramatic increases in Jo as ΔGATP decreased from -14.0 to -13.0 kcal/mol with both P+M and PC+M as substrates, whereas rat MITO only showed this relationship with P+M. Specifically, the slope of the ΔGATP:Jo relationship, a measure of the elasticity of oxidative flux to ΔGATP, is 7 fold greater with CHO than with FAT in rat MITO, whereas in sparrow MITO, CHO and FAT oxidation provide identical control sensitivity. This indicates sparrow MITO are able to fuel high ATP turnover rates with FAT or CHO as fuel, while in rat MITO only CHO oxidation can fuel high turnover rates, which is consistent with the mammalian requirement to oxidize CHO during exercise at and above moderate intensities. Substrate competition assays indicate, at the level of the mitochondrial matrix, either PC or G addition to P+M results in more than a 50% decrease in the rate of P utilization over the three values of ΔGATP examined, with more pronounced suppression as flux decreased and energy state increased. Adding G+PC to P+M oxidation decreased P utilization further. CONCLUSIONS: Avian MITO, at the level of the matrix, are able to maintain a high ATP turnover rate with FAT as fuel and preferentially oxidize PC (FAT) and/or G over P (CHO), possibly due to PDH inhibition by matrix redox potential and/or inhibition due to acetyl-CoA. Supported by NSF (IBN-0116997).

Role of phospholipases in cytosolic calcium overload and cardiomyocytes death in the presence of activated fatty acid derivatives

Ten to fifty micromoles of palmitoyl-L-carnitine (PC) or myristoyl-D,L-carnitine (MC) evoke a high-amplitude elevation of cytosolic calcium level ([Ca2+]i), hypercontraction and cell death in the primary culture of rat ventricular myocytes. The lag period of this effect varies within 2–8 min and depends on the mitochondrial capacity to accumulate Ca2+. Maximal level of Ca2+, attainable at the end of the lag period, depends on calcium concentration in the external medium and is mediated by plasma membrane nonspecific permeability. Preincubation of cardiomyocytes with the inhibitors of phospholipase C, cytosolic phospholipase A2 and/or Ca2+/calmodulin-dependent protein kinase II prevents cell death, increases lag period duration and reduces maximal [Ca2+]i. Both PC and MC, even at low concentrations (1–5 μM), dramatically increase the frequency of Ca2+-sparks and Ca2+-waves in cardiomyocytes and promote the formation of sustained microdomains with elevated calcium concentration. We discuss possible mechanisms of Ca2+-microdomain formation, where the “vicious circle” of Ca2+-dependent phospholipases activation may arise. The “vicious circle” with combined autocatalytic action of Ca2+-dependent phospholipases may be implicated in hydrolysis of membrane phosphatidylcholine and subsequent induction of nonselective permeability for Na+ and Ca2+ (lipid pore).

The Late Na+ Current (INa) Inhibitor Ranolazine Attenuates Effects of Palmitoyl-L-Carnitine to Increase Late INa and Cause Ventricular Diastolic Dysfunction

Palmitoyl-L-carnitine (PC), an ischemic metabolite, causes cellular Na(+) and Ca(2+) overload and cardiac dysfunction. This study determined whether ranolazine [(+/-)-1-piperazineacetamide, N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-] attenuates PC-induced Na(+) current and ventricular contractile dysfunction of the isolated heart. PC (4 microM, 30 min) increased late Na(+) current by 1034 +/- 349% in guinea pig isolated ventricular myocytes; ranolazine (10 microM) and tetrodotoxin (TTX, 3 microM) significantly attenuated this effect of PC. PC increased left ventricular end-diastolic pressure (LVEDP), coronary perfusion pressure (CPP), wall stiffness, and cardiac lactate and adenosine release from the isolated heart. Ranolazine (10 microM) significantly reduced the PC-induced increase in LVEDP by 72 +/- 6% (n = 6, p < 0.001), reduced left ventricular wall stiffness, and attenuated the PC-induced increase of CPP by 53 +/- 10% (n = 6-7, p < 0.05). Ranolazine (10 microM) reduced the PC-induced increases of lactate and adenosine release by 70 +/- 8 and 81 +/- 5%, respectively (n = 6, p <or= 0.05 for both). TTX (2 microM) significantly (p < 0.05) reduced PC-induced increases of CPP and LVEDP. Pretreatment of isolated myocytes or hearts with the free radical scavenger tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid, disodium salt) (1 mM) significantly reduced the effects of PC to cause increases of late Na(+) current and LVEDP, respectively, but unlike ranolazine or TTX, tiron did not reverse increases of late Na(+) current and LVEDP caused by PC. In summary, ranolazine and TTX, inhibitors of the late Na(+) current, attenuated the PC-induced ventricular contractile dysfunction and increase of coronary resistance in the guinea pig isolated heart.

Ranolazine Attenuates Palmitoyl‐L‐Carnitine Induced Ventricular Diastolic Dysfunction

Background: Palmitoyl‐L‐carnitine (PC), an ischemic metabolite, increases late Na+ channel current, causing cellular Na+ and Ca2+ overload and contractile dysfunction. Ranolazine (Ran) is a selective blocker of late sodium current. This study was conducted to determine whether Ran attenuates PC‐induced diastolic dysfunction.Methods: Guinea pig isolated hearts perfused at constant flow (Langendorff method) were used to study left ventricular end‐diastolic pressure (LVEDP), coronary perfusion pressure (CPP), ventricular wall stiffness, and cardiac lactate and adenosine release. Hearts were treated with 4 μM PC for 30 min followed by treatment with Ran or vehicle for 30 min.Results: PC significantly increased LVEDP, CPP, wall stiffness, and lactate and adenosine release. Ran (10 μM) significantly attenuated the PC‐induced increase in LVEDP by 61 ± 5 % (n=6, p&lt;0.001), the PC‐induced increase of CPP by 27 ± 3% (n=6–7, p&lt;0.05), and the PC‐induced increases of lactate and adenosine release by 50 ± 7 and 75 ± 4 %, respectively (n=6, p ≤0.05 for both). Ran reduced the PC‐induced increase of wall stiffness: LVEDP at all intraventricular volumes was lower in the presence of Ran + PC than in the presence of PC alone.Conclusion: Ran attenuated PC‐induced ventricular diastolic dysfunction, as indicated by reductions of LVEDP, ischemic metabolites, and CPP.

Mitochondrial efficiency in rat skeletal muscle: influence of respiration rate, substrate and muscle type

To investigate the hypothesis that mitochondrial efficiency (i.e. P/O ratio) is higher in type I than in type II fibres during submaximal rates of respiration. Mitochondria were isolated from rat soleus and extensor digitorum longus (EDL) muscles, representing type I and type II fibres, respectively. Mitochondrial efficiency (P/O ratio) was determined with pyruvate (Pyr) or palmitoyl-l-carnitine (PC) during submaximal (constant rate of adenosine diphosphate infusion) and maximal (V(max), state 3) rates of respiration and fitted to monoexponential functions. There was no difference in V(max) between PC and Pyr in soleus but in EDL V(max) with PC was only 58% of that with Pyr. The activity of 3-hydroxyacyl-CoA dehydrogenase was threefold higher in soleus than in EDL. P/O ratio at V(max) was 8-9% lower with PC [2.33 +/- 0.02 (soleus) and 2.30 +/- 0.02 (EDL)] than with Pyr [2.52 +/- 0.03 (soleus) and 2.54 +/- 0.03 (EDL)] but not different between the two muscles (P > 0.05). P/O ratio was low at low rates of respiration and increased exponentially when the rate of respiration increased. The asymptotes of the curves were similar to P/O ratio at V(max). P/O ratio at submaximal respirations was not different between soleus and EDL neither with Pyr nor with PC. Mitochondrial efficiency, as determined in vitro, was not significantly different in the two fibre types neither at V(max) nor at submaximal rates of respiration. The low V(max) for PC oxidation in EDL may relate to low activity of beta-oxidation.

Energy metabolism in orchid bee flight muscles: carbohydrate fuels all

The widely accepted idea that bees fuel flight through the oxidation of carbohydrate is based on studies of only a few species. We tested this hypothesis as part of our research program to investigate the size-dependence of flight energetics in Panamanian orchid bees. We succeeded in measuring rates of O(2) consumption and CO(2) production in vivo during hovering flight, as well as maximal activities (V(max) values) in vitro of key enzymes in flight muscle energy metabolism in nine species belonging to four genera. Respiratory quotients (ratios of rates of CO(2) production to O(2) consumption) in all nine species are close to 1.0. This indicates that carbohydrate is the main fuel used for flight. Trehalase, glycogen phosphorylase and hexokinase activities are sufficient to account for the glycolytic flux rates estimated from rates of CO(2) production. High activities of other glycolytic enzymes, as well as high activities of mitochondrial oxidative enzymes, are consistent with the estimated rates of carbohydrate-fueled oxidative metabolism. In contrast, hydroxyacylCoA dehydrogenase, an enzyme involved in fatty acid oxidation, was not detectable in any species. Thoracic homogenates displayed ADP-stimulated oxidition of pyruvate + proline, but did not oxidize palmitoyl l-carnitine + proline as substrates. A metabolic map, based on data reported herein and information from the literature, is presented. The evidence available supports the hypothesis that carbohydrate serves as the main fuel for flight in bees.

Endurance and Interval Training and Palmitoyl L-Carnitine Oxidation by Mitochondria from VLCAD Deficient Mice

2258 Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a fatty acid oxidation disorder that limits oxidation of fatty acids sixteen carbons and longer. However, even in VLCAD deficiency, some residual enzyme activity toward palmitate remains because of the overlap of specificity with LCAD. PURPOSE: This project examined the hypothesis that exercise training would enhance gastrocnemius subsarcolemmal (SS) and intermyoflbrillar (IMF) mitochondrial oxidation of palmitoyl l-carnitine plus malate and that high-intensity interval training would have a greater effect than endurance training. METHODS: The groups were sedentary control mice (C), endurance trained mice (E), and interval trained mice (I). Both groups of training mice exercised 5 dfiwk−1. During the first four weeks of training the durations and intensity of exercise were progressively increased. At the end of the four weeks, endurance training mice were running on the motor driven treadmill at a speed of 24 m fimin−1 for 60 min. Interval training mice ran for 2 min at 24 m fimin−1 followed by 2 min at 41 m. min−1. The intervals were then alternated for a total of 46 min. day−1. Beginning at the end of the 8th week, the gastrocnemius muscle was removed and the oxidation of palmitoyl-carnitine plus malate determined polarographically. RESULTS: Oxidation was significantly (p <0.05) depressed in IMF mitochondria from VLCAD deficient mice but not subsarcolemmal mitochondria. High-intensity interval training caused large increases in palmitoyl l-carnitine oxidation by both subsarcolemmal (p < 0.05) and intermyofibrillar mitochondria. Endurance training did not cause a significant increase in either subsarcolemmal or intermyofibrillar oxidation rates. CONCLUSIONS: Unlike previous observations in mice, these results show that high intensity, interval training is more effective than endurance training in improving fatty acid oxidation, at least in subsarcolemmal mitochondria. Further, the results show that mitochondrial oxidation of palmitoyl l-carnitine is reduced in VLCAD deficient mice relative to non-deficient mice.

Endurance and Interval Training and Palmitoyl L-Carnitine Oxidation by Mitochondria from VLCAD Deficient Mice

2258 Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a fatty acid oxidation disorder that limits oxidation of fatty acids sixteen carbons and longer. However, even in VLCAD deficiency, some residual enzyme activity toward palmitate remains because of the overlap of specificity with LCAD. PURPOSE: This project examined the hypothesis that exercise training would enhance gastrocnemius subsarcolemmal (SS) and intermyoflbrillar (IMF) mitochondrial oxidation of palmitoyl l-carnitine plus malate and that high-intensity interval training would have a greater effect than endurance training. METHODS: The groups were sedentary control mice (C), endurance trained mice (E), and interval trained mice (I). Both groups of training mice exercised 5 dfiwk−1. During the first four weeks of training the durations and intensity of exercise were progressively increased. At the end of the four weeks, endurance training mice were running on the motor driven treadmill at a speed of 24 m fimin−1 for 60 min. Interval training mice ran for 2 min at 24 m fimin−1 followed by 2 min at 41 m. min−1. The intervals were then alternated for a total of 46 min. day−1. Beginning at the end of the 8th week, the gastrocnemius muscle was removed and the oxidation of palmitoyl-carnitine plus malate determined polarographically. RESULTS: Oxidation was significantly (p <0.05) depressed in IMF mitochondria from VLCAD deficient mice but not subsarcolemmal mitochondria. High-intensity interval training caused large increases in palmitoyl l-carnitine oxidation by both subsarcolemmal (p < 0.05) and intermyofibrillar mitochondria. Endurance training did not cause a significant increase in either subsarcolemmal or intermyofibrillar oxidation rates. CONCLUSIONS: Unlike previous observations in mice, these results show that high intensity, interval training is more effective than endurance training in improving fatty acid oxidation, at least in subsarcolemmal mitochondria. Further, the results show that mitochondrial oxidation of palmitoyl l-carnitine is reduced in VLCAD deficient mice relative to non-deficient mice.

Alteration of the membrane lipid environment by L-palmitoylcarnitine modulates K(ATP) channels in guinea-pig ventricular myocytes.

Sarcolemmal adenosine 5'-triphosphate-sensitive K+ channels (K(ATP)) are dramatically up-regulated by a membrane phospholipid, phosphatidyl-inositol-4,5-bisphosphate (PIP2). During ischaemia, L-palmitoylcarnitine (L-PC), a fatty acid metabolite, accumulates in the sarcolemma and deranges the membrane lipid environment. We therefore investigated whether alteration of the membrane lipid environment by L-PC modulates the K(ATP) channel activity in inside-out patches from guinea-pig ventricular myocytes. L-PC (1 microM) inhibited KATP channel activity, without affecting the single channel conductance, through interaction with Kir6.2. L-PC simultaneously enhanced the ATP sensitivity of the channel [concentration for half-maximal inhibition (IC50) fell from 62.0+/-2.7 to 30.3+/-5.5 microM]. In contrast, PIP2 attenuated the ATP sensitivity (IC50 343.6+/-54.4 microM) and restored Ca2+-induced inactivation of KATP channels (94.1+/-13.7% of the control current immediately before the Ca2+-induced inactivation). Pretreatment of the patch membrane with 1 microM L-PC, however, reduced the magnitude of the PIP2-induced recovery to 22.7+/-6.3% of the control (P<0.01 vs. 94.1+/-13.7% in the absence of L-PC). Conversely, after the PIP2-induced recovery, L-PC's inhibitory action was attenuated, but L-PC partly reversed the PIP2-mediated decrease in the ATP sensitivity (IC50 fell from 310+/-19.2 to 93.1+/-9.8 microM). Thus, interaction between L-PC and PIP2 in the plasma membrane appears to regulate K(ATP) channels.

Multiple agents potentiate alpha1-adrenoceptor-induced conduction depression in canine cardiac purkinje fibers.

Halothane more so than isoflurane potentiates an alpha1-adrenoceptor (alpha1-AR)-mediated action of epinephrine that abnormally slows conduction in Purkinje fibers and may facilitate reentrant arrhythmias. This adverse drug interaction was further evaluated by examining conduction responses to epinephrine in combination with thiopental and propofol, which "sensitize" or reduce the dose of epinephrine required to induce arrhythmias in the heart, and with etomidate, which does not, and responses to epinephrine with verapamil, lidocaine, and l-palmitoyl carnitine, a potential ischemic metabolite. Action potentials and conduction times were measured in vitro using two microelectrodes in groups of canine Purkinje fibers stimulated at 150 pulses/min. Conduction was evaluated each minute after exposure to 5 microm epinephrine (or phenylephrine) alone or with the test drugs. Changes in the rate of phase 0 depolarization (Vmax) and the electrotonic spread of intracellular current were measured during exposure to epinephrine with octanol to evaluate the role of inhibition of active and passive (intercellular coupling) membrane properties in the transient depression of conduction velocity. Lidocaine (20 microm) and octanol (0.2 mm) potentiated alpha1-AR-induced conduction depression like halothane (0.4 mm), with maximum depression at 3-5 min of agonist exposure, no decrease of Vmax, and little accentuation at a rapid (250 vs. 150 pulses/min) stimulation rate. Thiopental (95 microm), propofol (50 microm), and verapamil (2 microm) similarly potentiated epinephrine responses, whereas etomidate (10 microm) did not. Between groups, the decrease of velocity induced by epinephrine in the presence of (10 microm) l-palmitoyl carnitine (-18%) was significantly greater than that resulting from epinephrine alone (-6%; 0.05 </= P </= 0.10). Current injection experiments were consistent with marked transient inhibition of cell-to-cell coupling correlating with alpha1-AR conduction depression in fibers exposed to octanol. Anesthetic "sensitization" to the arrhythmogenic effects of catecholamines may be a special case of a more general phenomenon by which not only some anesthetics and antiarrhythmic drugs but also possible ischemic fatty acid metabolites potentiate conduction depression due to acute alpha1-AR-mediated cell-to-cell uncoupling.

Effect of 5-(N,N-Dimethyl)-Amiloride, a Specific Inhibitor of Na+/H+ Exchanger, on the Palmitoyl-L-Carnitine-Induced Mechanical and Metabolic Derangements in the Isolated Perfused Rat Heart

The present study was carried out to determine the effect of 5-(N,N-dimethyl)-amiloride (DMA), a specific inhibitor of the Na⁺/H⁺ exchanger, on the cardiac mechanical and metabolic derangements induced by palmitoyl-L-carnitine (PALCAR). Rat hearts were perfused aerobically at a constant flow according to the Langendorff technique, while being paced electrically. PALCAR (5 μmol/l) increased the left-ventricular end-diastolic pressure, decreased the left ventricular developed pressure (i.e. mechanical dysfunction), decreased the tissue level of ATP and increased the tissue level of AMP (i.e. metabolic change). DMA (10 or 20 μmol/l) attenuated the mechanical and metabolic alterations induced by PALCAR in a concentration-dependent way. Nevertheless, DMA (10 or 20 μmol/l) did not affect the mechanical function or energy metabolism in the normal (PALCAR-untreated) heart. These results suggest that inhibition of the Na⁺/H⁺ exchanger with DMA is effective in attenuating the PALCAR-induced mechanical and metabolic derangements in the heart.